An assay system for measuring the acute production of sphingosine 1-phosphate in intact monolayers

Sphingosine kinase (SK) is a signaling enzyme that phosphorylates sphingosine to produce sphingosine 1-phosphate. Sphingosine and sphingosine 1-phosphate (S1P) belong to a class of bioactive sphingolipid metabolites that are critical in a number of cellular processes, yet often have opposing biological functions. The intracellular localization of sphingosine kinase has been demonstrated in multiple studies to be a critical aspect of its signaling function. To date, assays of sphingosine kinase activity have been developed for measuring activity in lysates, where the effects of localization are lost. Here we outline a system in which the rate of production of S1P can be measured in intact cells using exogenously added radiolabeled ATP instead of tritiated sphingosine. The surprising ability of ATP to enter unpermeabilized monolayers is one aspect that makes this assay simple, efficient, and inexpensive, yet sensitive enough to measure endogenous enzyme activity. The assay is well behaved in terms of kinetics and substrate dependence. Overall, this assay is ideal for future studies to identify changes in S1P production in intact cells such as those that result from the differential intracellular targeting of sphingosine kinase.     

A comparison of the dynamics of pantothenate synthetase from M. tuberculosis and E. coli: computational studies

Pantothenate synthetase (PS) catalyzes the final step of the pantothenate pathway, in which pantothenate is formed from pantoate and β-alanine in an ATP-dependent reaction. Mycobacterium tuberculosis PS (MTB PS) is functionally a dimer and a potential target for novel antitubercular drugs. Molecular dynamics simulations show that the functional dynamics of the enzyme are dominated by motions of a flexible gate loop in the N-terminal domain and of the C-terminal domain. The gate loop motions dominate in MTB PS while the C-terminal domain motion dominates in Escherichia coli PS. Simulations also show that the correlated motions of the domains are severely compromised in the monomeric forms. Mutations that reduce the mobility of the gate loop in MTB PS and increased it in E. coli PS were designed and validated through simulations.     

The compartmentalization and translocation of the sphingosine kinases: mechanisms and functions in cell signaling and sphingolipid metabolism

Members of the sphingosine kinase (SK) family of lipid signaling enzymes, comprising SK1 and SK2 in humans, are receiving considerable attention for their roles in a number of physiological and pathophysiological processes. The SKs are considered signaling enzymes based on their production of the potent lipid second messenger sphingosine-1-phosphate, which is the ligand for a family of five G-protein-linked receptors. Both SK1 and SK2 are intracellular enzymes and do not possess obvious membrane anchor domains within their primary sequences. The native substrates (sphingosine and dihydrosphingosine) are lipids, as are the corresponding products, and therefore would have a propensity to be membrane associated, suggesting that specific membrane localization of the SKs could affect both access to substrate and localized production of product. Here, we consider the emerging picture of the SKs as enzymes localized to specific intracellular sites, sometimes by agonist-dependent translocation, the mechanism targeting these enzymes to those sites, and the functional consequence of that localization. Not only is the signaling output of the SKs affected by subcellular localization, but the role of these enzymes as metabolic regulators of sphingolipid metabolism may be impacted as well.     

Mycolactone diffuses into the peripheral blood of Buruli ulcer patients--implications for diagnosis and disease monitoring

Background

Mycobacterium ulcerans, the causative agent of Buruli ulcer (BU), is unique among human pathogens in its capacity to produce a polyketide-derived macrolide called mycolactone, making this molecule an attractive candidate target for diagnosis and disease monitoring. Whether mycolactone diffuses from ulcerated lesions in clinically accessible samples and is modulated by antibiotic therapy remained to be established.

Methodology/Principal Finding

Peripheral blood and ulcer exudates were sampled from patients at various stages of antibiotic therapy in Ghana and Ivory Coast. Total lipids were extracted from serum, white cell pellets and ulcer exudates with organic solvents. The presence of mycolactone in these extracts was then analyzed by a recently published, field-friendly method using thin layer chromatography and fluorescence detection. This approach did not allow us to detect mycolactone accurately, because of a high background due to co-extracted human lipids. We thus used a previously established approach based on high performance liquid chromatography coupled to mass spectrometry. By this means, we could identify structurally intact mycolactone in ulcer exudates and serum of patients, and evaluate the impact of antibiotic treatment on the concentration of mycolactone.

Conclusions/Significance

Our study provides the proof of concept that assays based on mycolactone detection in serum and ulcer exudates can form the basis of BU diagnostic tests. However, the identification of mycolactone required a technology that is not compatible with field conditions and point-of-care assays for mycolactone detection remain to be worked out. Notably, we found mycolactone in ulcer exudates harvested at the end of antibiotic therapy, suggesting that the toxin is eliminated by BU patients at a slow rate. Our results also indicated that mycolactone titres in the serum may reflect a positive response to antibiotics, a possibility that it will be interesting to examine further through longitudinal studies.     

Oral treatment with γ-aminobutyric acid improves glucose tolerance and insulin sensitivity by inhibiting inflammation in high fat diet-fed mice

Adipocyte and β-cell dysfunction and macrophage-related chronic inflammation are critical for the development of obesity-related insulin resistance and type 2 diabetes mellitus (T2DM), which can be negatively regulated by Tregs. Our previous studies and those of others have shown that activation of γ-aminobutyric acid (GABA) receptors inhibits inflammation in mice. However, whether GABA could modulate high fat diet (HFD)-induced obesity, glucose intolerance and insulin resistance has not been explored. Here, we show that although oral treatment with GABA does not affect water and food consumption it inhibits the HFD-induced gain in body weights in C57BL/6 mice. Furthermore, oral treatment with GABA significantly reduced the concentrations of fasting blood glucose, and improved glucose tolerance and insulin sensitivity in the HFD-fed mice. More importantly, after the onset of obesity and T2DM, oral treatment with GABA inhibited the continual HFD-induced gain in body weights, reduced the concentrations of fasting blood glucose and improved glucose tolerance and insulin sensitivity in mice. In addition, oral treatment with GABA reduced the epididymal fat mass, adipocyte size, and the frequency of macrophage infiltrates in the adipose tissues of HFD-fed mice. Notably, oral treatment with GABA significantly increased the frequency of CD4(+)Foxp3(+) Tregs in mice. Collectively, our data indicated that activation of peripheral GABA receptors inhibited the HFD-induced glucose intolerance, insulin resistance, and obesity by inhibiting obesity-related inflammation and up-regulating Treg responses in vivo. Given that GABA is safe for human consumption, activators of GABA receptors may be valuable for the prevention of obesity and intervention of T2DM in the clinic.     

Heme oxygenase-1 gene transfer inhibits angiotensin II-mediated rat cardiac myocyte apoptosis but not hypertrophy

Cardiac myocyte apoptosis underlies the pathophysiology of cardiomyopathy, and plays a critical role in the transition from myocardial hypertrophy to heart failure. Angiotensin II (Ang II) induces cardiac myocyte apoptosis and hypertrophy which contribute to heart failure possibly through enhanced oxidative stress; however, the mechanisms underlying the activation of both pathways and their interactions remain unclear. In the present study, we have investigated whether overexpression of the antioxidant protein heme oxygenase-1 (HO-1) protects against apoptosis and hypertrophy in cultured rat cardiac myocytes treated with Ang II. Our findings demonstrate that Ang II (100 nM, 24 h) alone upregulates HO-1 expression and induces both myocyte hypertrophy and apoptosis, assessed by measuring terminal deoxynucleotidyltransferase dUTP nick-end labelling (TUNEL) staining, caspase-3 activity and mitochondrial membrane potential. Ang II elicited apoptosis was augmented in the presence of tin protoporphyrin, an inhibitor of HO activity, while HO-1 gene transfer to myocytes attenuated Ang II-mediated apoptosis but not hypertrophy. Adenoviral overexpression of HO-1 was accompanied by a significant increase in Ang II induced phosphorylation of Akt, however, Ang II-mediated p38 mitogen activated protein kinase (MAPK) phosphorylation was attenuated. Inhibition of phosphotidylinositol-3-kinase enhanced myocyte apoptosis elicited by Ang II, however, p38MAPK inhibition had no effect, suggesting that overexpression of HO-1 protects myocytes via augmented Akt activation and not through modulation of p38MAPK activation. Our findings identify the signalling pathways by which HO-1 gene transfer protects against apoptosis and suggest that overexpression of HO-1 in cardiomyopathies may delay the transition from myocyte hypertrophy to heart failure.     

Synthesis and X-ray structures of rhenium(I) carbonyl aminoalkoxide and aminocarboxylate complexes

The complexes [Re{MeN(CH₂CH₂O)(CH₂CH₂OH)-κ³N,O,O}(CO)₃] (1), [Re{N(CH₂CH₂O)(CH₂CH₂OH)₂-κ³N,O,O}(CO)₃] (2), [Me₃NH]₂[(OC)₃Re{N(CH₂CO₂)₂-κ³N,O,O}CH₂CH₂{N(CH₂CO₂)₂-κ³N,O,O}Re(CO)₃] (3), [Me₃NH]₂[Re₂{μ₂-2,6-(O₂C)₂(C₅H₃N)-κ³N,O,O}₂(CO)₆] (4) and [Re₂{μ₂-2,6-(OCH₂)(C₅H₃N)(CH₂OH)-κ²N,O}₂(CO)₆] (5) were synthesized in high yields via the reactions of [Re₂(CO)₁₀] and Me₃NO with MeN(CH₂CH₂OH)₂, N(CH₂CH₂OH)₃, EDTA, pyridine-2,6-dicarboxylic acid and pyridine-2,6-dimethanol, respectively. Complexes 1-5 were characterized by IR and ¹H NMR spectroscopy, elemental analysis and X-ray crystallography.     

DNA binding and nucleolytic properties of Cu(ii) complexes of salicylaldehyde semicarbazones

The copper(ii) complexes of two salicylaldehyde semicarbazones, HOC(6)H(4)CH[double bond, length as m-dash]N-NHCONR(2) [H(2)Bnz(2) (R = CH(2)Ph) and H(2)Bu(2) (R = Bu)], were evaluated for their DNA binding and cleavage properties by spectrophotometric DNA titration, ethidium bromide displacement assay and electrophoretic mobility shift assay. Results showed that the Cu(ii) complexes can bind to DNA via a partial intercalation mode with binding constants of 1.1 × 10(4) and 9.5 × 10(3) M(-1) for [Cu(HBnz(2))Cl] and [Cu(HBu(2))Cl], respectively. These complexes also cleave DNA in the presence of ascorbic acid, most likely through hydroxyl radicals that are generated via the reduction of a Cu(ii) to a Cu(i) species. The complexes show similar DNA cleavage activity, which is reflected in the similarity of their frontier molecular orbital energies calculated by density functional theory. These results are discussed in relation to the anticancer properties of the complexes.     

Gastrointestinal and urinary tract pathogenic infections among HIV seropositive patients at the Komfo Anokye Teaching Hospital in Ghana

ABSTRACT: BACKGROUND: Gastrointestinal and urinary tract pathogenic infections are aggravating the incidence and progression of the Human Immunodeficiency Virus (HIV) infection into Acquired Immune Deficiency Syndrome (AIDS) more especially in the developing countries. This study was conducted to assess the common gastrointestinal and urinary infections among HIV/AIDS patients at the Komfo Anokye Teaching Hospital (KATH) in Ghana between April and December 2008. FINDINGS: This work reports on gastrointestinal and urinary tract pathogenic infections among 500 HIV seropositive and 300 HIV seronegative patients. There was a 35% (175/500) prevalence of intestinal parasites among HIV seropositive patients compared to 4.3% (13/300) in HIV seronegative patients. Giardia lamblia and Cryptosporidium accounted for 19% (95/500) and 14% (70/500) respectively, while Schistosoma mansoni, Strongyloides stercoralis and hookworm together accounted for 2% (10/500) of intestinal parasitic infections among the HIV seropositive patients. There was no significant difference (p > 0.05) in urinary parasitic infection between HIV seropositive 1% (2/500) and seronegative patients 0.7% (2/300). Most, 60 (86%) out of 70, of the urinary tract infection among the HIV seropositive patients was due to bacteria with E. coli being the most predominant isolate, 28 (47%) out of 60. There was no significant difference in infections based on age and gender. CONCLUSION: G. lamblia and Cryptosporidium were the most common gastrointestinal parasites detected while bacteria accounted for majority of the urinary tract infections among the HIV seropositive patients at the hospital.