HIV-2 Infection in Malaysia: Current situation and the use of in-house real-time reverse transcription PCR for HIV-2.

HIV-2 surveillance has been carried out in Malaysia for more than 25 years ago. Tests to discriminate HIV-1 and HIV-2 are available but the options of test are limited and the need to develop a new in-house HIV-2 real-time reverse transcription PCR (RT-PCR) is crucial. A study was done on 29 samples from hospitals in Malaysia which were found to be positive screening for HIV-2 antibodies by the commercial Western Blot assay. These samples were further tested by a Western Blot assay that detects specific antibodies to HIV-2. Detection of HIV-2 genome was then performed by using a commercial kit. Fifteen samples were evaluated by using in-house real-time RT-PCR for HIV-2. Ninety-three percent (27/29) of samples have positive results for HIV-2 on HIV-2 Western Blot with only 2 samples showing indeterminate results. All samples showed negative results for HIV-2 genomes by using a PCR commercial kit and the 15 samples that were subjected to our in-house real-time RT-HIV-2 PCR were also tested negative for HIV-2 RNA. Results of HIV-2 Western Blot did not reflect the actual positivity as both HIV-1 and HIV-2 antibodies may cross-react with either viral proteins. None of the samples was confirmed positive for HIV-2 by the commercial and in-house real-time RTPCR. In-house real-time RT-HIV-2 PCR assay can be further used to confirm the presence of HIV-2 genome. Up to the year 2015, Malaysia is still free from HIV-2 infection.

Membrane reconstitution of ABC transporters and assays of translocator function.

In this protocol, we describe a procedure for incorporating ATP-binding cassette (ABC) transporters into large unilamellar vesicles (LUVs) and assays to determine ligand binding and solute translocation by these membrane-reconstituted systems. The reconstitution technique as described has been optimized for ABC transporters but can be readily adapted for other types of transport systems. Purified transporters are inserted into detergent-destabilized preformed liposomes and detergent is subsequently removed by adsorption onto polystyrene beads. Next, Mg-ATP or an ATP-regenerating system is incorporated into the vesicle lumen by one or more cycles of freezing-thawing, followed by extrusion through polycarbonate filters to obtain unilamellar vesicles. Binding and translocation of substrates are measured using isotope-labeled ligands and rapid filtration to separate the proteoliposomes from the surrounding medium. Quantitative information is obtained about dissociation constants (K(d)) for ligand binding, number of binding-sites, transport affinities (K(m)), rates of transport, and the activities of transporter molecules with opposite orientations in the membrane. The full protocol can be completed within 4-5 d.

Ion specificity and ionic strength dependence of the osmoregulatory ABC transporter OpuA.

The ATPase subunit of the osmoregulatory ATP-binding cassette transporter OpuA from Lactococcus lactis has a C-terminal extension, the tandem cystathionine beta-synthase (CBS) domain, which constitutes the sensor that allows the transporter to sense and respond to osmotic stress (Biemans-Oldehinkel, E., Mahmood, N. A. B. N., and Poolman, B. (2006) Proc. Natl. Acad. Sci. U. S. A. 103, 10624-10629). C-terminal of the tandem CBS domain is an 18-residue anionic tail (DIPDEDEVEEIEKEEENK). To investigate the ion specificity of the full transporter, we probed the activity of inside-out reconstituted wild-type OpuA and the anionic tail deletion mutant OpuADelta12; these molecules have the tandem CBS domains facing the external medium. At a mole fraction of 40% of anionic lipids in the membrane, the threshold ionic strength for activation of OpuA was approximately 0.15, irrespective of the electrolyte composition of the medium. At equivalent concentrations, bivalent cations (Mg(2+) and Ba(2+)) were more effective in activating OpuA than NH(4)(+), K(+), Na(+), or Li(+), consistent with an ionic strength-based sensing mechanism. Surprisingly, Rb(+) and Cs(+) were potent inhibitors of wild-type OpuA, and 0.1 mM RbCl was sufficient to completely inhibit the transporter even in the presence of 0.2 M KCl. Rb(+) and Cs(+) were no longer inhibitory in OpuADelta12, indicating that the anionic C-terminal tail participates in the formation of a binding site for large alkali metal ions. Compared with OpuADelta12, wild-type OpuA required substantially less potassium ions (the dominant ion under physiological conditions) for activation. Our data lend new support for the contention that the CBS module in OpuA constitutes the ionic strength sensor whose activity is modulated by the C-terminal anionic tail.

Comparative disposition and metabolism of 1,2,3-trichloropropane in rats and mice.

1,2,3-Trichloropropane (TCP) has been used as a solvent and degreasing agent and as an intermediate in pesticide manufacture. TCP is currently the subject of a National Toxicology Program chronic toxicity study. The present study is part of a larger effort to characterize the toxicity of TCP. Following acute oral exposure of male and female F344 rats (30 mg/kg) and male B6C3F1 mice (30 and 60 mg/kg), TCP was rapidly absorbed, metabolized, and excreted. The major route of excretion of TCP was in the urine. By 60 hr postdosing, rats had excreted 50% and mice 65% of the administered dose by this route. Exhalation as 14CO2 and excretion in the feces each accounted for 20% of the total dose in 60 hr rats and 20 and 15%, respectively, in mice. No apparent sex-related differences were observed in the ability of the rats to excrete TCP-derived radioactivity. At 60 hr, TCP-derived radioactivity was most concentrated in the liver, kidney, and forestomach in both rats and male mice. Male mice eliminated TCP-derived radioactivity more rapidly than rats and lower concentrations of radioactivity were found in tissues 60 hr after dosing in mice. Two urinary metabolites were isolated and identified by NMR, mass spectroscopy, and comparison with synthetic standards, as N-acetyl- and S-(3-chloro-2-hydroxypropyl)cysteine. Analyses of the early urine (0-6 hr) showed this mercapturic acid to be the major metabolite in rat urine and was only a minor component in mouse urine. 2-(S-Glutathionyl)malonic acid was identified by NMR and mass spectrometry and by chemical synthesis as the major biliary metabolite in rats.(ABSTRACT TRUNCATED AT 250 WORDS)

Fast atom bombardment and tandem mass spectrometry for structure determination of cysteine, N-acetylcysteine, and glutathione adducts of xenobiotics.

Fast atom bombardment mass spectrometry (FAB/MS) and FAB combined with tandem mass spectrometry (MS/MS) were examined for their applicability to the structure determination of xenobiotics conjugated with the members of the glutathione family (glutathione, cysteine, and N-acetylcysteine). Comparisons between FAB/MS and thermospray MS data are made. FAB/MS is generally successful at generating molecular ion species under both positive and negative ion conditions. Upon collisional activation the adducts undergo characteristic cleavages around the sulfur providing structural information about the conjugate. The analysis of the N-acetylcysteine conjugate isolated from rat urine is presented as an example of the application of FAB/MS/MS to biological problems.

Anticholinesterase poisonings in dogs from a cyanobacterial (blue-green algae) bloom dominated by Anabaena flos-aquae.

Cyanobacteria (blue-green algae) implicated in the deaths of 9 dogs at Richmond Lake, SD, on Aug 26, 1985, were analyzed. The dominant cyanobacterial species from the water sample was Anabaena flos-aquae. The lyophilized bloom material or the high-performance liquid chromatography purified toxin peak, when administered to mice IP, induced clinical signs of salivation, lacrimation, urinary incontinence, defecation, convulsion, fasciculation, and respiratory arrest. Further comparison of the semipurified bloom toxin with an irreversible anticholinesterase anatoxin-a(s), produced by A flos-aquae strain NRC-525-17, revealed the bloom toxin and anatoxin-a(s) had similar properties on high-performance liquid chromatography and on the inhibition of electric eel acetylcholinesterase (EC 3.1.1.7).

Anatoxin-a(s), an anticholinesterase from the cyanobacterium Anabaena flos-aquae NRC-525-17.

Anatoxin a(s) [antx-a(s)] given intraperitoneally to Sprague-Dawley rats at different doses (0.1-1.0 mg/kg) caused signs of severe cholinergic overstimulation. Assays of rat blood acetylcholinesterase (AChE) revealed a dose-dependent inhibition. The in vitro inhibition of electric eel acetylcholinesterase (AChE, E.C. 3.1.1.7) and horse serum butyrylcholinesterase (BUChE, E.C. 3.1.1.8) by antx-a(s) was time- and concentration-dependent. The inhibition of electric eel AChE follows first order kinetics, indicative of irreversible inhibition. The irreversibility of electric eel AChE inhibition was confirmed by a plot of Vmax versus total enzyme concentration [ET]. The kinetics of inhibition of cholinesterase by antx-a(s) supports the previous pharmacological findings that antx-a(s) is an anticholinesterase and that signs of intoxication by it are primarily due to cholinesterase inhibition.

The pharmacology of anatoxin-a(s), a neurotoxin produced by the freshwater cyanobacterium Anabaena flos-aquae NRC 525-17.

Anatoxin-a(s) [antx-a(s)] is produced by Anabaena flos-aquae clone NRC 525-17 and is different from anatoxin-a, a known depolarizing agent produced by A. flos-aquae NRC 44-1. Purification of antx-a(s) from lyophilized cells involved extraction with 1.0 M acetic acid: ethanol (80:20), column chromatography (Sephadex G-15 and CM-Sephadex C-25) and high performance liquid chromatography. Purified toxin has an LD50 (i.p., mouse) of approximately 50 micrograms/kg. Gross pharmacological tests of antx-a(s) on isolated chick biventer cervicis and frog rectus abdominis muscles showed no direct agonistic effect. Instead, antx-a(s) augments the acetylcholine response and antagonizes the actions of d-tubocurarine. Twitch potentiation and tetanic fade were observed on isolated rat phrenic nerve--diaphragm muscle when stimulated indirectly at different frequencies. In acute toxicity tests with mice and rats the signs of poisoning were indicative of excessive cholinergic stimulation. Mice pretreated with atropine sulfate showed longer survival times and no parasympathomimetic signs of toxicity. The mice still died of respiratory arrest with convulsions, which indicated that toxicity is due to more than just the peripheral muscarinic action of antx-a(s). Assays of serum cholinesterase of rats in acute toxicity tests showed complete inactivation of the enzyme at doses of 350 and 600 micrograms/kg. It was concluded that antx-a(s) may be acting as an anticholinesterase, thereby causing toxicity.

Paralytic shellfish poisons produced by the freshwater cyanobacterium Aphanizomenon flos-aquae NH-5.

A single filament clonal isolate of Aphanizomenon flos-aquae was made from a water bloom sample taken at a small pond near Durham, New Hampshire, in 1980. When batch cultured the strain was toxic to mice and had an i.p. LD50 of about 5.0 mg/kg. Using an extraction procedure originally designed for paralytic shellfish poisons and other neurotoxins of freshwater cyanobacteria, a purification method was developed. The procedure involved acidified water/ethanol extraction of the cells followed by ultrafiltration, gel filtration, use of C18 cartridges to remove pigments, ion-exchange and high performance liquid chromatography using u.v. detection at 220 or 240 nm. Thin-layer chromatography and high performance liquid chromatography results indicate that Aphanizomenon flos-aquae NH-5 may produce paralytic shellfish poisons, mainly neo-saxitoxin and saxitoxin. Three labile toxins were also detected which were not similar to any of the known paralytic shellfish poisons.