Showing papers by "Ponnampalam Gopalakrishnakone published in 2003"

Practical integration of polymerase chain reaction amplification and electrophoretic analysis in microfluidic devices for genetic analysis.

Abstract: An integrated system of a silicon-based microfabricated polymerase chain reaction (microPCR) chamber and microfabricated electrophoretic glass chips have been developed. The PCR chamber was made of silicon and had aluminum heaters and temperature sensors integrated on the glass anodically bonded cover. Temperature uniformity in the reaction chamber was +/-0.3 degrees C using an improved novel "joint-heating" scheme. Thermal cycling was digitally controlled with a temperature accuracy of +/- 0.2 degrees C. Small operating volumes together with high thermal conductivity of silicon made the device well suited to rapid cycling; 16 s/cycle were demonstrated. For analysis of the PCR products, the chamber output was transferred to the glass microchip by pressure. Analysis time of PCR amplified genomic DNA was obtained in the microchip in less than 180 s. The analysis procedure employed was reproducible, simple and practical by using viscous sieving solutions of hydroxypropylmethylcellulose and dynamically coated microchip channels with poly(vinylpyrrolidone). DNA fragments that differ in size by 18 base pairs (bp) were resolved. Analysis of genomic male and female amplified DNA by microPCR was achieved in microchip, and application of the integrated microPCR-microchip for the identification of bird sex was tested. Genomic DNA samples from several bird species such as pigeon and chicken were analyzed. Hence, the system could be used as well to determine the sex of avian species.

Protective role of melatonin in domoic acid-induced neuronal damage in the hippocampus of adult rats

Abstract: Domoic acid (DA), a kainite-receptor agonist and potent inducer of neurotoxicity, has been administered intravenously in adult rats in the present study (0.75 mg/kg body weight) to demonstrate neuronal degeneration followed by glial activation and their involvement with inducible nitric oxide synthase (iNOS) in the hippocampus. An equal volume of normal saline was administered in control rats. The pineal hormone melatonin, which protects the neurons efficiently against excitotoxicity mediated by sensitive glutamate receptor, was administered intraperitoneally (10 mg/kg body weight), 20 min before, immediately after, and 1 h and 2 h after the DA administration, to demonstrate its role in therapeutic strategy. Histopathological analysis (Nissl staining) demonstrated extensive neuronal damage in the pyramidal neurons of CA1, CA3 subfields and hilus of the dentate gyrus (DG) in the hippocampus at 5 days after DA administration. Sparsely distributed glial fibrillary acidic protein (GFAP)-immunoreactive astrocytes were observed in the hippocampus at 4-24 h after DA administration and in the control rats. Astrogliosis was evidenced by increased GFAP immunoreactivity in the areas of severe neuronal degeneration at 5 days after DA administration. Along with this, microglial cells exhibited an intense immunoreaction with OX-42, indicating upregulation of complement type 3 receptors (CR3). Ultrastructural study revealed swollen or shrunken degenerating neurons in the CA1, CA3 subfields and hilus of the DG and hypertrophied astrocytes showing accumulation of intermediate filament bundles in the cytoplasm were observed after administration of DA. Although no significant change could be observed in the mRNA level of iNOS expression between the DA-treated rats and controls at 4-24 h and at 5-day time intervals, double immunofluorescense revealed co-expression of induced iNOS with GFAP immunoreactive astrocytes, but not in the microglial cells, and iNOS expression in the neurons of the hippocampal subfields at 5 days after DA administration. Expression of iNOS was not observed in the hippocampus of control rats. DA-induced neuronal death, glial activation, and iNOS protein expression were attenuated significantly by melatonin treatment and were comparable to the control groups. The results of the present study suggest that melatonin holds potential for the treatment of pathologies associated with DA-induced brain damage. It is speculated that astrogliosis and induction of iNOS protein expression in the neurons and astrocytes of the hippocampus may be in response to DA-induced neuronal degeneration.

Neuromuscular effects of candoxin, a novel toxin from the venom of the Malayan krait (Bungarus candidus).

Abstract: Candoxin (MW 7334.6), a novel toxin isolated from the venom of the Malayan krait Bungarus candidus, belongs to the poorly characterized subfamily of nonconventional three-finger toxins present in Elapid venoms. The current study details the pharmacological effects of candoxin at the neuromuscular junction. Candoxin produces a novel pattern of neuromuscular blockade in isolated nerve-muscle preparations and the tibialis anterior muscle of anaesthetized rats. In contrast to the virtually irreversible postsynaptic neuromuscular blockade produced by curaremimetic α-neurotoxins, the neuromuscular blockade produced by candoxin was rapidly and completely reversed by washing or by the addition of the anticholinesterase neostigmine. Candoxin also produced significant train-of-four fade during the onset of and recovery from neuromuscular blockade, both, in vitro and in vivo. The fade phenomenon has been attributed to a blockade of putative presynaptic nicotinic acetylcholine receptors (nAChRs) that mediate a positive feedback mechanism and maintain adequate transmitter release during rapid repetitive stimulation. In this respect, candoxin closely resembles the neuromuscular blocking effects of d-tubocurarine, and differs markedly from curaremimetic α-neurotoxins that produce little or no fade. Electrophysiological experiments confirmed that candoxin produced a readily reversible blockade (IC50∼10 nM) of oocyte-expressed muscle (αβγδ) nAChRs. Like α-conotoxin MI, well known for its preferential binding to the α/δ interface of the muscle (αβγδ) nAChR, candoxin also demonstrated a biphasic concentration–response inhibition curve with a high- (IC50∼2.2 nM) and a low- (IC50∼98 nM) affinity component, suggesting that it may exhibit differential affinities for the two binding sites on the muscle (αβγδ) receptor. In contrast, curaremimetic α-neurotoxins have been reported to antagonize both binding sites with equal affinity. Keywords: Bungarus candidus, postsynaptic neurotoxin, three-finger toxin, neuromuscular junction, nicotinic acetylcholine receptors, train-of-four fade Introduction Snake venoms are complex mixtures of protein and polypeptide toxins that encompass an arsenal of lethal neurotoxins. These include curaremimetic or α-neurotoxins which target muscle (αβγδ) nicotinic acetylcholine receptors (nAChRs) with high affinity (Kd 10−9–10−11 M) to produce postsynaptic neuromuscular blockade (Endo & Tamiya, 1991; Servent & Menez, 2001; Hodgson & Wickramaratna, 2002). Based on the length of their polypeptide chains, α-neurotoxins have been classified as short-chain neurotoxins (e.g. erabutoxin-b (Laticauda semifasciata); toxin-α (Naja nigricollis)) that have 60–62 residues and four conserved disulphide bonds and long-chain neurotoxins (e.g. α-bungarotoxin (Bungarus multicinctus); α-cobratoxin (Naja kaouthia)) with 66–75 residues and five disulphide bonds, with the additional disulphide bridge located in the middle loop (loop II) (Endo & Tamiya, 1991). In common, these neurotoxins belong to the superfamily of three-finger proteins that are characterized by a common tertiary structure consisting of three loops extending from a globular core crosslinked by four conserved disulphide bonds (Tsetlin, 1999; Servent & Menez, 2001; Kini, 2002). We have recently reported the isolation and purification of a novel three-finger toxin, candoxin, from the venom of the Malayan krait Bungarus candidus (Nirthanan et al., 2002a). Candoxin (MW 7334.6) consists of a single polypeptide chain of 66 amino acids with five disulphide bridges, including four that are conserved among all three-finger toxins (see Figure 6). The fifth disulphide bridge (Cys6-Cys11) in candoxin is located at the tip of loop I (N-terminus loop), instead of in loop II as present in conventional long-chain α-neurotoxins as well as in κ-neurotoxins that have a predilection for neuronal (α3β2) nAChRs. This cysteine motif is typical of the poorly characterized subfamily of nonconventional toxins, isolated exclusively from Elapid venoms (Servent & Menez, 2001; Nirthanan et al., 2003). This class of toxins is typically characterized by a lower order of toxicity (LD50 from ∼5 to 80 mg kg−1) as opposed to prototype α-neurotoxins (LD50∼0.04–0.3 mg kg−1) and, because of this, they have also been referred to as weak toxins (Utkin et al., 2001). Apart from toxicity studies, the nonconventional toxins have been poorly investigated in terms of their function or molecular targets. Recently, it has been reported that two nonconventional (‘weak') toxins from cobra venoms (WTX from Naja kaouthia and Wntx-5 from Naja sputatrix) produced a weak and irreversible inhibition of both, muscle (αβγδ) and α7 nAChRs, in micromolar inhibitory concentrations (Utkin et al., 2001; Poh et al., 2002). In contrast, candoxin has been shown to be a potent antagonist of muscle (αβγδ) (IC50∼10 nM) and α7 (IC50∼50 nM) nAChRs in electrophysiological experiments (Nirthanan et al., 2002a). Clearly therefore, nonconventional (weak) toxins do not appear to be a functionally homogeneous class of toxins. The present study provides a detailed account of the effects of a nonconventional toxin, candoxin, from krait venom at the neuromuscular junction. Figure 6 Comparison of the amino-acid sequence of candoxin with the sequences of other (a) neurotoxins and (b) ‘reversible' neurotoxins and neurotoxin homologues. The number of amino-acid residues in each sequence is indicated at the end of the respective ... Methods Animals Swiss Albino mice, Sprague–Dawley rats and Hartley guinea pigs were purchased from the Laboratory Animals Centre, National University of Singapore, (Sembawang, Singapore) and housed in the Animal Holding Unit of the Department of Pharmacology, National University of Singapore, before use. Water and food (Glen Forrest Stockfeeders, WA, Australia) were provided ad libitum and a 12 h light–12 h dark cycle was maintained. Paper pellet bedding was also purchased from Glen Forrest Stockfeeders, WA, Australia. Locally bred chicks were purchased from a farm. These were delivered on the day of the experiment. The animals were handled according to the Guidelines of the National Medical Ethics Committee (Singapore), which conform to the World Health Organization's International Guiding Principles for Animal Research (The WHO Chronicle (1985); 39(2) 51–56: International Guiding Principles for Biomedical Research involving Animals), adapted by the Council for International Organizations of Medical Sciences in 1985.

Secretory phospholipase A2 activity in the normal and kainate injected rat brain, and inhibition by a peptide derived from python serum.

Abstract: The present study aimed to elucidate sPLA2 activity in the normal and kainate-lesioned hippocampus using selective inhibitors of sPLA2. In normal rats the highest levels of sPLA2 were observed in the hippocampus, pons, and medulla, followed by the cerebral neocortex and caudate nucleus. After intracerebroventricular kainate injections an increase in total PLA2 activity was observed in the rat hippocampus. Using a selective sPLA2 inhibitor 12-epi-scalaradial, sPLA2 activity was found to be significantly increased by 2.5-fold on the side of the intracerebroventricular injection compared to the contralateral side. A peptide P-NT.II, derived from the amino acid sequence of "PLA2-inhibitory protein," discovered in the serum of the reticulated python, also showed potent sPLA2 inhibitory activity in homogenates from the kainate-injected hippocampus. These results show that there is a high level of sPLA2 activity in the normal hippocampus, pons, and medulla oblongata, and that the level increases further in the hippocampus after kainate-induced excitotoxic injury. The increased PLA2 activity was inhibited by P-NT.II, indicating a potential use of this peptide as a PLA2 inhibitory agent in the brain.

Distribution of NADPH-diaphorase and expression of nNOS, N-methyl-D-aspartate receptor (NMDAR1) and non-NMDA glutamate receptor (GlutR2) genes in the neurons of the hippocampus after domoic acid-induced lesions in adult rats.

Abstract: Neuronal degeneration followed by detection of nitric oxide (NO)-producing neurons of the hippocampus was investigated at 4 h, 16 h, 24 h, 2 days, 5 days, and 14 days after administration of domoic acid (DA), in the present study. Histopathological analysis (Nissl staining) displayed dark-stained degenerating neurons in the hippocampus at 24 h to 14 days after DA administration, with degeneration most severe at 5–14 days. NADPH-d-positive neurons were observed in different subfields of the hippocampus in control rats and DA treated rats at 4–24 h. Complete loss of NADPH-d-positive neurons in the CA1 and CA3 subfields and also in the hilus of dentate gyrus (DG) was observed at 5 days and 14 days after the administration of DA. In contrast, at 4–24 h, neuronal nitric oxide synthase (nNOS)-immunoreactive cells were absent from the hippocampal subfields in control and DA-treated animals but were observed at 5 days and 14 days after DA administration. N-methyl-D-aspartate receptor (NMDAR1) immunoreactivity was increased in the hippocampal neurons at 5 days after DA administration and double immunofluorescence demonstrated its coexpression with induced nNOS expression. No significant change could be observed in the immunoreactivity of non-NMDA receptor (GlutR2) as compared with the controls, while occasional immunoreactive neurons were colocalized with induced nNOS expression. Reverse transcription-polymerase chain reaction analysis showed the upregulated expression of nNOS and downregulated expression of NMDAR1 at 5 days after the administration of DA. Although nNOS mRNA expression was rapidly induced at 5 days after DA administration, in situ hybridization analysis revealed complete loss of nNOS mRNA expression in the region of neuronal degeneration in the hippocampus at 24 h and 5 days after DA administration. The present study has shown that NADPH-d and nNOS express differentially in the neurons of the hippocampus in DA-induced neurotoxicity. It is speculated that induction of nNOS and glutamate receptor genes in the neurons of the hippocampus in response to DA-induced neurotoxicity could have contributed to the neuronal degeneration. Hippocampus 2003;13:260–272. © 2003 Wiley-Liss, Inc.

Crystallization and preliminary x-ray analysis of candoxin, a novel reversible neurotoxin from the malayan krait bungarus candidus

Abstract: Candoxin, a novel three-finger toxin from Bungarus candidus, is a reversible antagonist of muscle (alphabetagammadelta) but a poorly reversible antagonist of neuronal alpha7 nicotinic acetylcholine receptors. It has a molecular weight of 7344 Da, with 66 amino-acid residues including ten half-cystines. The fifth disulfide bridge is located at the tip of loop I (Cys6-Cys11) instead of in loop II as found in other alpha-neurotoxins. Interestingly, candoxin lacks the segment cyclized by the fifth disulfide bridge at the tip of the middle loop of long-chain neurotoxins, which was reported to be critical for binding to alpha7 receptors. As a first step to determining its three-dimensional structure, candoxin was crystallized by the hanging-drop vapour-diffusion technique in conditions around 1.5 M sodium chloride, 10%(v/v) ethanol. The crystals formed belonged to the hexagonal system, space group P6(2)22, with unit-cell parameters a = 54.88, b = 54.88, c = 75.54 A, alpha = beta = 90, gamma = 120 degrees, and diffract to a resolution of 1.80 A. The crystallographic asymmetric unit contains one molecule of candoxin, with an estimated solvent content of 44.6%. Attempts to solve these structures by molecular-replacement methods have not been successful and a heavy-atom derivative search has been initiated.