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Biomedical subjects

P Gopalakrishnakone

Publications and source records attributed to P Gopalakrishnakone.

At least 37 records · Page 2Linked to original sources
PubMed2002

Pharmacological characterization of mikatoxin, an alpha-neurotoxin isolated from the venom of the New-Guinean small-eyed snake Micropechis ikaheka.

Symptoms of envenomation by the New-Guinean small-eyed snake Micropechis ikaheka (Elapidae) include peripheral neurotoxicity and myotoxicity. We have now purified to homogeneity a long-chain neurotoxin, mikatoxin, from M. ikaheka venom by successive gel filtration and reverse-phase chromatography. Electrospray ionization mass spectrometry showed mikatoxin to be a homogenous peptide of MW 7775.6. Mikatoxin was devoid of any phospholipase A(2) activity associated with the crude venom and did not exhibit any intrinsic anticholinesterase activity. In the chick biventer cervicis muscle, it produced an irreversible, concentration-dependent block of responses to exogenously applied acetylcholine and carbachol as well as twitches evoked by nerve, but not by direct muscle stimulation. Moreover, mikatoxin, like alpha-bungarotoxin and erabutoxin-b, did not show significant fade response to train-of-four stimulation of the mouse phrenic nerve-hemi diaphragm muscle. It also failed to block ganglionic transmission in the guinea pig ileum and muscarinic responses in the rat anococcygeus muscle. Our study provides strong evidence for the presence of a neurotoxin (mikatoxin) in M. ikaheka venom that produces neuromuscular blockade in skeletal muscle attributable to selective and irreversible antagonism of postsynaptic nicotinic acetylcholine receptors of the neuromuscular junction and likely contributes to the peripheral neurotoxicity observed in M. ikaheka envenomation.

Acetylcholine↗
PubMed2002

An ISFET-based immunosensor for the detection of beta-Bungarotoxin.

An ion-sensitive field effect transistor (ISFET)-based immunosensor was developed to detect/quantitate beta-Bungarotoxin (beta-BuTx), a potent presynaptic neurotoxin from the venom of Bungarus multicinctus. A murine monoclonal antibody (mAb 15) specific to beta-BuTx was immobilized onto silicon nitride wafers after silanization and activation with glutaraldehyde. A chip based enzyme linked-immunosorbantassay (ELISA) was performed to ascertain antigen binding to the immobilized antibody. To develop an electrochemical immunosensing system for the detection/quantitation of beta-BuTx, an ISFET was used as a solid phase detector. MAb 15 was immobilized on the gate region of the ISFET. The antigen antibody reaction was monitored by the addition of urease conjugated rabbit anti-beta-BuTx antibodies. The sensor can detect toxin level as low as 15.6 ng/ml. The efficacy of the sensor for the determination of beta-BuTx from B. multicinctus venom was demonstrated in mouse model. Toxin concentration was highest at the site of injection (748.0+/-26 ng/ml) and moderate amount was found in the plasma (158.5+/-13 ng/ml).

Animals↗
PubMed2001

Domoic acid-induced neuronal damage in the rat hippocampus: changes in apoptosis related genes (bcl-2, bax, caspase-3) and microglial response.

Domoic acid (DA), a potent neurotoxin, administered intravenously (0.75 mg/kg body weight) in adult rats evoked seizures accompanied by nerve cell damage in the present study. Neuronal degeneration and microglial reaction in the hippocampus were investigated, and the temporal profile of bcl-2, bax, and caspase-3 genes in cell death or survival was assessed following the administration of DA. Nissl staining showed darkly stained degenerating neurons in the hippocampus following the administration of DA at 1-21 days, the degeneration being most severe at 5 days. Ultrastructural study in CA1 and CA3 regions of hippocampus revealed two types of neuronal degeneration, cells that exhibited swollen morphology and shrunken electron-dense cells. Immunoreactivity of Bcl-2 and Bax was increased considerably at 16 hr and 24 hr in the neurons of the hippocampus following DA administration. No significant change was observed in the immunoreactivity of caspase-3 in the controls and DA-treated rats at any time interval. Microglial cells in the hippocampus showed intense immunoreaction with the antibodies OX-42 and OX-6 at 1-21 days after DA administration, indicating the up-regulation of complement type 3 receptors and major histocompatibility complex type II antigens for increased phagocytic activity and antigen presentation, respectively. Terminal deoxynucleotidyl-transferase-mediated dUTP nick end labeling (TUNEL) showed occasional positive neurons in the CA1 and CA3 regions at 5 days after DA administration, with no positive cells in the controls. RT-PCR analysis revealed that bcl-2 and bax mRNA transcripts in the hippocampus were significantly increased at 16 hr and gradually decreased at 24 hr following the administration of DA. Although bax and bcl-2 mRNA expression is rapidly induced at early stages, in situ hybridization analysis revealed complete loss of bcl-2, bax, and caspase-3 mRNA at 24 hr after DA administration in the region of neuronal degeneration in the hippocampus. These results indicate that the pattern of neuronal degeneration observed during DA-induced excitotoxic damage is mostly necrotic.

Animals↗
PubMed2001

Functional site of bukatoxin, an alpha-type sodium channel neurotoxin from the Chinese scorpion (Buthus martensi Karsch) venom: probable role of the (52)PDKVP(56) loop.

Alpha-toxins from scorpion venoms prolong the action potential of excitable cells by blocking sodium channel inactivation. We have purified bukatoxin, an alpha-toxin from scorpion (Buthus martensi Karsch) venom, to homogeneity. Bukatoxin produced marked relaxant responses in the carbachol-precontracted rat anococcygeus muscle (ACM), which were mediated through the L-arginine-nitric oxide synthase-nitric oxide pathway, consequent to a neuronal release of nitric oxide. Based on the presence of proline residues in the flanking segments of protein-protein interaction sites, we predicted the site between (52)PP(56) to be the potential interaction site of bukatoxin. A homology model of bukatoxin indicated the presence of this site on the surface. Buka11, a synthetic peptide designed based on this predicted site, produced a concentration-dependent nitric oxide-mediated relaxant response in ACM. Using alanine-substituted peptides, we have shown the importance (53)DKV(55) flanked by proline residues in the functional site of bukatoxin.

Amino Acid Motifs↗
PubMed2001

Purification and properties of three new phospholipase A2 isoenzymes from Micropechis ikaheka venom.

Three new phospholipase A2 (PLA2) isoenzymes were purified from the Micropechis ikaheka venom by successive chromatographies. The homogeneity of them was accessed by capillary zone electrophoresis and mass spectrometry. Their N-terminal sequences showed high identity (94, 88 and 90, respectively) with MiPLA-1, a group IB PLA2 also from this venom. In addition, strong immuno-cross-reaction with anti-MiPLA-1 serum was observed. These results suggested that three newly purified PLA2 belonged to group IB. Beside enzymatic activity, they induced various pharmacological effects, including myotoxic, anticoagulant effects and insulin secretion stimulating effects. Our results indicated that enzymatic activity is essential for their myotoxic and anticoagulant effects. On the other hand, no direct correlation between their insulin secretion stimulating effect and enzymatic activity was observed, suggesting that they may stimulate insulin secretion through a non-enzymatic mechanism.

Amino Acid Sequence↗
PubMed2001

Tamulustoxin: a novel potassium channel blocker from the venom of the Indian red scorpion Mesobuthus tamulus.

We have characterized tamulustoxin, a novel 35-amino-acid peptide found in the venom of the Indian red scorpion (Mesobuthus tamulus). Tamulustoxin was identified through a [125I]toxin I screen, designed to identify toxins that block voltage-activated potassium channels. Tamulustoxin has also been cloned by RT-PCR, using RNA extracted from scorpion venom glands. Tamulustoxin shares no homology with other scorpion venom toxins, although the positions of its six cysteine residues would suggest that it shares the same structural scaffold. Tamulustoxin rapidly inhibited both peak and steady-state currents (18.9 +/- 1.0 and 37 +/- 1.1%, respectively) produced by injecting CHO cells with mRNA encoding the hKv1.6 channel.

Amino Acid Sequence↗
PubMed2000

lambda-conotoxins, a new family of conotoxins with unique disulfide pattern and protein folding. Isolation and characterization from the venom of Conus marmoreus.

Conotoxins are multiple disulfide-bonded peptides isolated from marine cone snail venom. These toxins have been classified into several families based on their disulfide pattern and biological properties. Here, we report a new family of Conus peptides, which have a novel cysteine motif. Three peptides of this family (CMrVIA, CMrVIB, and CMrX) have been purified from Conus marmoreus venom, and their structures have been determined. Their amino acid sequences are VCCGYK-LCHOC (CMrVIA), NGVCCGYKLCHOC (CMrVIB), and GICCGVSFCYOC (CMrX), where O represents 4-trans-hydroxyproline. Two of these peptides (CMrVIA and CMrX) have been chemically synthesized. Using a selective protection and deprotection strategy during disulfide bond formation, peptides with both feasible cysteine-pairing combinations were generated. The disulfide pattern (C(1)-C(4), C(2)-C(3)) in native toxins was identified by their co-elution with the synthetic disulfide-isomeric peptides on reverse-phase high pressure liquid chromatography. Although cysteine residues were found in comparable positions with those of alpha-conotoxins, these toxins exhibited a distinctly different disulfide bonding pattern; we have named this new family "lambda -conotoxins." CMrVIA and CMrX induced different biological effects when injected intra-cerebroventricularly in mice; CMrVIA induces seizures, whereas CMrX induces flaccid paralysis. The synthetic peptide with lambda-conotoxin folding is about 1150-fold more potent in inducing seizures than the mispaired isomer with alpha-conotoxin folding. Thus it appears that the unique disulfide pattern, and hence the "ribbon" conformation, in lambda-conotoxins is important for their biological activity.

Amino Acid Sequence↗
PubMed2000

Recombinant antitoxic and antiinflammatory factor from the nonvenomous snake Python reticulatus: phospholipase A2 inhibition and venom neutralizing potential.

From the serum of the nonvenomous snake Python reticulatus, a new phospholipase A(2) (PLA(2)) inhibitor termed phospholipase inhibitor from python (PIP) was purified by sequential chromatography and cloned to elucidate its primary structure and fundamental biochemical characteristics. A cDNA clone encoding PIP was isolated from the liver total RNA by reverse transcriptase-polymerase chain reaction (RT-PCR). It contained a 603 bp open reading frame that encoded a 19-residue signal sequence and a 182-residue protein. PIP showed about 60% sequence homology with those PLA(2) inhibitors having a urokinase-type plasminogen activator receptor-like domain structure. PIP was also functionally expressed as a fusion protein in Escherichia coli to explore its potential therapeutic significance. The recombinant PIP was shown to be identical to the native form in chromatographic behavior and biochemical characteristics. Both the native and recombinant PIP appear to exist as a hexamer of 23-kDa subunits having an apparent molecular mass of approximately 140 kDa. PIP showed ability to bind to the major PLA(2) toxin (daboiatoxin, DbTx) of Daboia russelli siamensis at 1-2-fold molar excess of inhibitor to toxin. It exhibited broad spectra in neutralizing the toxicity of various snake venoms and toxins and inhibited the formation of edema in mice. Our data demonstrate the venom neutralizing potential of the recombinant PIP and suggest that the proline-rich hydrophobic core region may play a role in binding to PLA(2).

Amino Acid Sequence↗
PubMed2000

Purification, structure determination and synthesis of covalitoxin-II, a short insect-specific neurotoxic peptide from the venom of the Coremiocnemis validus (Singapore tarantula).

Spider venoms contain toxins that specifically immobilize and kill insects. We report the purification and characterization of a new insect-specific toxin named covalitoxin-II (Cvtx-II; mass, 3406. 24+/-0.64), from Coremiocnemis validus (Singapore tarantula) venom. The complete 31 amino acid sequence of Cvtx-II has been determined and it shows less than 40% identity with spider toxins. However, Cvtx-II has conserved cystine motif analogous to other spider and omega-conotoxins. Cvtx-II was chemically synthesized and identified with the native Cvtx-II. Synthetic Cvtx-II induced insect-specific non-lethal excitatory activity when injected into crickets, but not in cockroaches and mice.

Amino Acid Motifs↗
PubMed2000

Morbidity after a bite from a 'non-venomous' pet snake.

We report the first recorded case of morbidity from the bite of a red-neck keelback snake (Rhabdophis subminiatus) from South East Asia. This is a species of the Colubrid family which originated from South East Asia. Severe envenomation from this snake was reported as poisonous in the West as far back as 1978 but it is still being classified as non-venomous. This classification led our patient to keep this 'harmless' snake as a pet. We recommend that this snake be reclassified as 'venomous' or at least warnings be issued to the public not to keep it as a pet.

Adult↗
PubMed1999

Purification, properties, and amino acid sequence of a hemoglobinuria-inducing phospholipase A(2), MiPLA-1, from Micropechis ikaheka venom.

Dark-colored urine is one of the clinical symptoms of envenomation by Micropechis ikaheka (New Guinea small-eyed snake). We have purified a phospholipase A(2), MiPLA-1, which induces dark-colored urine in experimental mice, to homogeneity. The analysis of the dark-colored urine by electrophoresis and N-terminal sequence determination indicated that the color of mouse urine is due to hemoglobin in the urine but not myoglobin. MiPLA-1 is the first hemoglobinuria-inducing toxin. Insignificant hemolytic activity of MiPLA-1 indicates that hemoglobinuria is not due to lysis of erythrocytes by MiPLA-1. This suggests that hemoglobinuria induced by MiPLA-1 may be due to kidney leakage caused by unknown mechanisms. MiPLA-1 also showed other biological effects, including myotoxicity as well as anticoagulant and antiplatelet effects. Structural studies show that MiPLA-1 is a basic protein with a molecular mass of 14041.60 +/- 1.78 as determined by electrospray mass spectrometry. We have determined the complete amino acid sequence of MiPLA-1. It is a 124-amino-acid protein with a "pancreatic loop" and belongs to group IB phospholipase A(2) enzymes. Two short segments flanked by proline brackets are found in the sequence of MiPLA-1. These segments are on the surface of the molecule and hence may be involved in protein-protein recognition.

Amino Acid Sequence↗
PubMed1999

Tests for detection of snake venoms, toxins and venom antibodies: review on recent trends (1987-1997).

Various methods developed for the detection of snake venoms, toxins and venom antibodies, during the last decade is reviewed. Radioimmunoassay, agglutination assay, enzyme-linked immunosorbent assay (ELISA), fluorescence immunoassay etc. have been used for detection of venoms and toxins. Important contributions have been made to improve the specificity, sensitivity, rapidity and simplicity of the ELISA method. Monoclonal antibodies and affinity-purified venom-specific antibodies were used to achieve species specificity of ELISA and the latter seems to be the ideal for venom detection. Incorporation of avidin-biotin system as well as the fluorogenic substrate in the enzyme immunoassay sufficiently increased the sensitivity of the assay to detect venom concentrations to picogram levels. The ability to use undiluted blood and other whole biological fluids reduce the assay time considerably. Although there have been several reports were on venom detection, so far only a few field kits have been developed. This implies that the experiments and design were only at the laboratory levels and still more work has to be carried out before it could be used in the field. Concerning the venom antibody detection, ELISA has been used extensively and the humoral response of patients envenomed by snake has been investigated in detail. Non-specific reactivity along with cross-reactivity still limits the use of ELISA for species identification in epidemiological studies. Overall, ELISA remains the suitable method for the detection of snake venoms, toxins and venom antibodies in body fluids. The possible use of a biosensor approach to solve some of the problems associated with the ELISA method are also discussed.

Agglutination Tests↗
PubMed1999

Histopathological studies of the acute inflammation in synovial tissue of rat knee joint following intra-articular injection of PLA2 from Chinese Cobra (Naja naja atra) venom.

A phospholipase A2 was purified from Chinese Cobra Naja naja atra by a two-step procedure: gel filtration on Superdex 75 and reverse-phase high-performance liquid chromatography (HPLC) on a NUCLEOSIL 5 C18 column. Purified phospholipase A2 was homogeneous, as indicated by capillary electrophoresis and electrospray mass spectrometry. It was a basic protein (pI = 8.2 +/- 0.03) with a molecular mass of 13,258 Da. Amino acid sequence analysis of the N-terminal demonstrated a high degree of homology with other related PLA2 from elapid venoms. The histopathological effects of the purified PLA2 on synovial tissue of knee joint were studied in Wistar rats. Rats were injected intra-articularly with 100 microl solution of PLA2 of different concentrations. Synovial tissue samples with patella were taken for light microscope study. Histopathological evaluation revealed a significant induction of acute inflammation in synovial tissue after injection, as indicated by synovial lining-cell hyperplasia, subsynovial cellular infiltration, and peri-articular soft-tissue cellular infiltration. Marked inflammatory and proliferative changes in synovial tissue were evident after repeated intra-articular injections of 100 microg PLA2. This study failed to show any significant histological changes in cartilage of patella as well as in the surrounding muscle tissue of the knee joints. These results suggest that PLA2 purified from Chinese Cobra venom induce time- and dose-dependent inflammatory changes in the synovial tissue of rat knee joint.

Acute Disease↗
PubMed1999

Nuclear microscopy of normal and necrotic skeletal muscle fibers: an intracellular elemental microanalysis.

The in situ total elemental composition and elemental concentrations present in mouse soleus (type I) and gastrocnemius (type IIA) muscle fibers were analyzed by using nuclear microscopy (NM). Elemental changes in necrotic fibers, induced by intramuscular injection with snake venom (Pseudechis australis), were also studied 3 h post-injection. Nuclear microscopy is a new method based on nuclear technology that utilizes the interaction between a million-electron-volt nuclear particle beam and the muscle sample (in the case of the present study). Elemental analysis was done at the parts per million (ppm) level of sensitivity on unfixed, rapidly frozen and unstained single fast- and slow-twitch muscle fibers with imaging capabilities of micron spatial resolution and in multi-elemental mode. In total, 12 different intracellular elements were mapped, co-localized and analyzed in single normal and necrotic skeletal muscle fibers from mice. Elements such as potassium, sulfur, phosphorus, chlorine and sodium were found in concentrations from 1000 to 18,000 ppm. Unlike conventional electron-probe X-ray microanalysis, NM also detected and analyzed the trace elements such as magnesium, calcium, iron and zinc that were found in concentrations of 50 to 1000 ppm. Other elements--copper, manganese and rubidium--were also detected in concentrations of less than 50 ppm. The trace elements calcium, iron and zinc were more abundant in the soleus than the gastrocnemius (the level of iron was statistically significant). Calcium, sodium and chlorine were significantly elevated in venom-induced necrotic soleus muscle fibers.

Animals↗
PubMed1999

In situ hybridization and immunohistochemical analysis of the expression of cardiotoxin and neurotoxin genes in Naja naja sputatrix.

Secretory processes and their regulation have been extensively studied in mammalian salivary parotid glands. However, little is known regarding the secretory mechanism in the venom glands of snakes, which invariably produce one of the most complex of all animal secretions. The pharmacologically important and toxic components of the Malayan spitting cobra (Naja naja sputatrix) venom include postsynaptic neurotoxins (NTX), presynaptic neurotoxins (phospholipase A2, PLA2), and cardiotoxins (CTX) which, for convenience, have been collectively referred to as "toxins." We report here for the first time the mechanism of toxin gene expression by studying the accumulated mRNA level and protein synthesis rates for the three toxins over a period of 8 days after stimulation of venom synthesis by manual "milking" of the venom gland. Immunofluorescence and in situ hybridization were used to localize the toxins and their mRNAs in venom gland sections. The rate of protein synthesis, as determined by immunofluorescence and liquid chromatography-mass spectrometry (LC-MS) techniques, increased in parallel with the increase in the toxin mRNA content in the secretory epithelial cells, suggesting that transcriptional regulation of the toxin genes is involved. (J Histochem Cytochem 47:551-560, 1999)

Animals↗
PubMed1999

Postsynaptic alpha-neurotoxin gene of the spitting cobra, Naja naja sputatrix: structure, organization, and phylogenetic analysis.

The venom of the spitting cobra, Naja naja sputatrix contains highly potent alpha-neurotoxins (NTXs) in addition to phospholipase A2 (PLA2) and cardiotoxin (CTX). In this study, we report the complete characterization of three genes that are responsible for the synthesis of three isoforms of alpha-NTX in the venom of a single spitting cobra. DNA amplification by long-distance polymerase chain reaction (LD-PCR) and genome walking have provided information on the gene structure including their promoter and 5' and 3' UTRs. Each NTX isoform is approximately 4 kb in size and contains three exons and two introns. The sequence homology among these isoforms was found to be 99%. Two possible transcription sites were identified by primer extension analysis and they corresponded to the adenine (A) nucleotide at positions +1 and -45. The promoter also contains two TATA boxes and a CCAAT box. Putative binding sites for transcriptional factors AP-2 and GATA are also present. The high percentage of similarity observed among the NTX gene isoforms of N. n. sputatrix as well as with the alpha-NTX and kappa-NTX genes from other land snakes suggests that the NTX gene has probably evolved from a common ancestral gene.

3' Untranslated Regions↗
PubMed1998

Cytopathic effect of Blastocystis hominis after intramuscular inoculation into laboratory mice.

The present study investigated the pathogenesis of Blastocystis hominis by intramuscular injection of the organism into experimental mice. A total of 27 naïve BALB/c mice aged 6-8 weeks were injected in the leg muscle with axenic culture isolate B of B. hominis. Histological examination at different times revealed that B. hominis could produce a severe inflammatory reaction and myonecrosis. Most changes were observed at 6 h after injection and for up to 2-3 days. By 2 weeks the muscle had regained normal histology. There was infiltration of polymorphonuclear leukocytes (PML) into the injection site, indicating that B. hominis had a strong chemoattractant activity for PML.

Animals↗