Biomedical subjects

R Manjunatha Kini

Publications and source records attributed to R Manjunatha Kini.

At least 37 records · Page 2Linked to original sources

PubMed2005

Ohanin, a novel protein from king cobra venom, induces hypolocomotion and hyperalgesia in mice.

We have identified, purified, and determined the complete amino acid sequence of a novel protein, ohanin from Ophiophagus hannah (king cobra) venom. It is a small protein containing 107 amino acid residues with a molecular mass of 11951.47 +/- 0.67 Da as assessed by electrospray ionization-mass spectrometry. It does not show similarity to any known families of snake venom proteins and hence is the first member of a new family of snake venom proteins. It shows similarity to PRY and SPRY domain proteins. It is nontoxic up to 10 mg/kg when injected intraperitoneally in mice. Ohanin produced statistically significant and dose-dependent hypolocomotion in mice. In a pain threshold assay, it showed dose-dependent hyperalgesic effect. The ability of the protein to elicit a response at greatly reduced doses when injected intracerebroventricularly as compared with intraperitoneal administration in both the locomotion and hot plate experiments strongly suggests that ohanin acts on the central nervous system. Since the natural abundance of the protein in the venom is low (approximately 1 mg/g), a synthetic gene was constructed and expressed. The recombinant protein, which was obtained in the insoluble fraction in Escherichia coli, was purified under denaturing condition and was refolded. Recombinant ohanin is structurally and functionally similar to native protein as determined by circular dichroism and hot plate assay, suggesting that it will be useful in future structure-function relationship studies.

Amino Acid Sequence↗

PubMed2005

Pharmacological characterisation of a neurotoxin from the venom of Boiga dendrophila (mangrove catsnake).

In this study, we have pharmacologically characterised boigatoxin-A, a three finger toxin isolated from the venom of the colubrid, Boiga dendrophila (Mangrove catsnake). In the chick biventer cervicis nerve-muscle preparation boigatoxin-A (1 microM) displayed poorly reversible postsynaptic blockade as evidenced by the inhibition of indirect (0.1 Hz, 0.2 ms, supramaximal V) twitches and responses to exogenous acetylcholine (1 mM) and carbachol (20 microM). Boigatoxin-A (0.3-0.5 microM) caused a concentration-dependent depression of the maximum response of cumulative concentration response curves to CCh (0.6-80 microM). Boigatoxin-A (1 microM) induced readily reversible inhibition of electrically evoked (0.2 Hz, 0.3 ms, 70-100 V) twitches of the prostatic segment of the rat vas deferens. This inhibition was not significantly attenuated by 8-phenyltheophylline (20 microM) or idazoxan (1 microM). Boigatoxin-A (1 microM) did not affect alpha,beta-mATP (10 microM) or noradrenaline (25 microM) responses in unstimulated epididymal segments of the rat vas deferens. Our data suggests that this toxin has weak postsynaptic neurotoxicity in skeletal muscle and also prejunctional neurotoxic activity in the smooth muscle of the rat vas deferens to inhibit the release of neurotransmitter(s), but not via prejunctional purinergic or adrenergic receptors. This is the first report of such activity for a toxin isolated from snake venom and reinforces the largely untapped potential of colubrid venoms.

Animals↗

PubMed2005

Putting the brakes on snake venom evolution: the unique molecular evolutionary patterns of Aipysurus eydouxii (Marbled sea snake) phospholipase A2 toxins.

Accelerated evolution of toxins is a unique feature of venoms, with the toxins evolving via the birth-and-death mode of molecular evolution. The venoms of sea snakes, however, are remarkably simple in comparison to those of land snakes, which contain highly complex venoms. Aipysurus eydouxii (Marbled sea snake) is a particularly unique sea snake, feeding exclusively upon fish eggs. Secondary to this ecological change, the fangs have been lost and the venom glands greatly atrophied. We recently showed that the only neurotoxin (a three-finger toxin) gene found in the sea snake A. eydouxii has a dinucleotide deletion, resulting in the loss of neurotoxic activity. During these studies, we isolated and identified a number of cDNA clones encoding isozymes of phospholipase A(2) (PLA(2)) toxins from its venom gland. Sixteen unique PLA(2) clones were sequenced from the cDNA library and TA cloning of reverse transcription-polymerase chain reaction products. Phylogenetic analysis of these clones revealed that less diversification of the PLA(2) toxins has occurred in the A. eydouxii venom gland in comparison to equivalent terrestrial and other marine snakes. As there is no longer a positive selection pressure acting upon the venom, mutations have accumulated in the toxin-coding regions that would have otherwise had a deleterious effect upon the ability to use the venom for prey capture. Such mutations include substitutions of highly conserved residues; in one clone, the active site His(48) is replaced by Arg, and in two other clones, highly conserved cysteine residues are replaced. These mutations significantly affect the functional and structural properties of these PLA(2) enzymes, respectively. Thus, in A. eydouxii, the loss of the main neurotoxin is accompanied by a much slower rate of molecular evolution of the PLA(2) toxins as a consequence of the snake's shift in ecological niche. This is the first case of decelerated evolution of toxins in snake venom.

Amino Acid Sequence↗

PubMed2005

Eggs-only diet: its implications for the toxin profile changes and ecology of the marbled sea snake (Aipysurus eydouxii).

Studies so far have correlated the variation in the composition of snake venoms with the target prey population and snake's diet. Here we present the first example of an alternative evolutionary link between venom composition and dietary adaptation of snakes. We describe a dinucleotide deletion in the only three finger toxin gene expressed in the sea snake Aipysurus eydouxii (Marbled Sea Snake) venom and how it may have been the result of a significant change in dietary habits. The deletion leads to a frame shift and truncation with an accompanying loss of neurotoxicity. Due to the remarkable streamlining of sea snake venoms, a mutation of a single toxin can have dramatic effects on the whole venom, in this case likely explaining the 50- to 100-fold decrease in venom toxicity in comparison to that of other species in the same genus. This is a secondary result of the adaptation of A. eydouxii to a new dietary habit--feeding exclusively on fish eggs and, thus, the snake no longer using its venom for prey capture. This was parallel to greatly atrophied venom glands and loss of effective fangs. It is interesting to note that a potent venom was not maintained for use in defense, thus reinforcing that the primary use of snake venom is for prey capture.

Amino Acid Sequence↗

PubMed2005

Hemextin AB complex--a snake venom anticoagulant protein complex that inhibits factor VIIa activity.

Snake venom is a veritable gold mine of bioactive molecules, capable of binding to a wide variety of pharmacological targets, including the blood coagulation cascade. Here, we report the isolation and characterization of two synergistically acting anticoagulant three-finger proteins, hemextin A and hemextin B, from the venom of Hemachatus haemachatus (African Ringhals cobra). Hemextin A but not hemextin B exhibits mild anticoagulant activity. However, hemextin B interacts with hemextin A and forms a complex (hemextin AB complex), and synergistically enhances its anticoagulant potency. Prothrombin time assay showed that these two proteins form a 1:1 complex. Using a 'a dissection approach', we found that hemextins A and AB complex prolong clotting by inhibiting extrinsic tenase activity. Further studies showed that hemextin AB complex potently inhibits the proteolytic activity of factor VIIa (FVIIa) and its complexes. Kinetic studies showed that hemextin AB complex is a non-competitive inhibitor of FVIIa-soluble tissue factor proteolytic activity with a K(i) of 25 nM. Hemextin AB complex is the first reported natural inhibitor of FVIIa that does not require either tissue factor or factor Xa scaffold to mediate its inhibitory activity. Molecular interactions of hemextin AB complex with FVIIa/tissue factor-FVIIa may provide a new paradigm in the search for anticoagulants inhibiting the initiation of blood coagulation.

Animals↗

PubMed2005

Serine proteases affecting blood coagulation and fibrinolysis from snake venoms.

Snake venom proteases, in addition to their contribution to the digestion of the prey, affect various physiological functions. They affect platelet aggregation, blood coagulation, fibrinolysis, complement system, blood pressure and nervous system. This review provides a ready reference for serine proteases that interfere in blood coagulation and fibrinolysis. They exhibit their activity by activation of specific zymogens of coagulation factors. These serine proteases serve as tools to study molecular details in the activation of specific factors involved in coagulation and fibrinolytic cascades and are useful in treating various thrombotic and hemostatic conditions.

Animals↗

PubMed2005

Gene structures of trocarin D and coagulation factor X, two functionally diverse prothrombin activators from Australian rough scaled snake.

Activation of prothrombin to thrombin is the key reaction in blood coagulation cascade. We have recently shown that Australian rough scaled snake, Tropidechis carinatus, possesses two parallel prothrombin activator systems. Trocarin D, a venom prothrombin activator produced in the venom gland, plays an offensive role as a toxin, whereas factor X is produced in the liver and plays a role in the hemostatic mechanism. These two proteins are structurally similar and have identical domain architecture. Because of the differences in their physiological roles, and tissue-specific expression, we determined the gene structure of these closely related proteins. Both the genes have eight exons similar to all mammalian factor X genes. All the exon-intron boundaries of these two genes are at the same position and the splice junctions are almost identical. Partial sequencing of the introns shows that they share a very high degree of sequence identity indicating that the gene duplication is a recent event. Further studies on the characterization of these two genes particularly the promoter regions are in progress.

Animals↗

PubMed2005

Two parallel prothrombin activator systems in Australian rough-scaled snake, Tropidechis carinatus. Structural comparison of venom prothrombin activator with blood coagulation factor X.

It is uncommon for similar pathways/systems to be involved in highly divergent functions within single organisms. Earlier, we have shown that trocarin D, a venom prothrombin activator, from the Australian rough-scaled snake Tropidechis carinatus, is structurally and functionally similar to the blood coagulation factor Xa (FXa). The presence of a haemostatic system in these snakes implies that they have two parallel prothrombin activating systems: one in the plasma, that participates in the life saving process of blood clotting and the other in their venom, where it acts as a toxin. Here, we report the complete cDNA sequence encoding the blood coagulation factor X (FX) from the liver of T. carinatus. Deduced T. carinatus FX sequence shows approximately 80% identity with trocarin D but approximately 50% identity with the mammalian FX. Our present study confirms the presence of two separate genes--one each for FX and trocarin D, that code for similar proteins in T. carinatus snake. These two genes have different expression sites and divergent uses suggesting that snake venom prothrombin activators have probably evolved by the duplication of the liver FX gene and subsequently marked for tissue-specific expression in the venom gland.

Amino Acid Sequence↗

PubMed2005

Gene duplication of coagulation factor V and origin of venom prothrombin activator in Pseudonaja textilis snake.

The origin and evolution of venom toxins is a mystery that has evoked much interest. We have recently shown that pseutarin C, a prothrombin activator from Pseudonaja textilis venom, is structurally and functionally similar to mammalian coagulation factor Xa-factor Va complex. Its catalytic subunit is homologous to factor Xa while the nonenzymatic subunit is homologous to factor Va. P. textilis therefore has two parallel prothrombin activator systems: one expressed in its venom gland as a toxin and the other expressed in its liver and released into its plasma as a haemostatic factor. Here we report the complete amino acid sequence of factor V (FV) from its liver determined by cDNA cloning and sequencing. The liver FV shows 96% identity to pseutarin C nonenzymatic subunit. Most of the functional sites involved in its interaction with factor Xa and prothrombin are conserved. However, many potential sites of post-translational modifications and one critical cleavage site for activated protein C are different. The absence of the latter cleavage site makes pseutarin C nonenzymatic subunit resistant to inactivation and enhances its potential as an excellent toxin. By PCR and real-time quantitative analysis, we show that pseutarin C nonenzymatic subunit gene is expressed specifically in the venom gland at approximately 280 fold higher than that of FV gene in liver. These two are thus encoded by two separate genes that express in a highly tissue-specific manner. Our results imply that the gene encoding pseutarin C nonenzymatic subunit was derived by the duplication of plasma FV gene and they have evolved to perform distinct functions.

Amino Acid Sequence↗

PubMed2004

In vitro neuromuscular activity of 'colubrid' venoms: clinical and evolutionary implications.

In this study, venoms from species in the Colubrinae, Homalopsinae, Natricinae, Pseudoxyrhophiinae and Psammophiinae snake families were assayed for activity in the chick biventer cervicis skeletal nerve muscle preparation. Boiga dendrophila, Boiga cynodon, Boiga dendrophila gemincincta, Boiga drapiezii, Boiga irregularis, Boiga nigriceps and Telescopus dhara venoms (10 microg/ml) displayed postsynaptic neuromuscular activity as evidenced by inhibition of indirect (0.1 Hz, 0.2 ms, supramaximal V) twitches. Neostigmine (5 microM) reversed the inhibition caused by B. cynodon venom (10 microg/ml) while the inhibitory effects of Psammophis mossambicus venom (10 microg/ml) spontaneously reversed, indicating a reversible mode of action for both venoms. Trimorphodon biscutatus (10 microg/ml) displayed irreversible presynaptic neurotoxic activity. Detectable levels of phospholipase A2 activity were found only in T. biscutatus, T. dhara and P. mossambicus venoms. The results demonstrate a hitherto unsuspected diversity of pharmacological actions in all lineages which may have implications ranging from clinical management of envenomings to venom evolution.

Analysis of Variance↗

PubMed2004

The catalytic subunit of pseutarin C, a group C prothrombin activator from the venom of Pseudonaja textilis, is structurally similar to mammalian blood coagulation factor Xa.

Pseutarin C, a group C prothrombin activator from Pseudonaja textilis venom, is a large protein complex consisting of catalytic and nonenzymatic subunits, which are functionally similar to the mammalian FXa-FVa complex. Here, we present the complete cDNA sequence of the catalytic subunit of pseutarin C. The cDNA of the catalytic subunit encodes a protein of 449 amino acids, which includes a 22-residue signal peptide, 18-residue propeptide and a mature protein of 409 amino acids. The deduced amino acid sequence shows 74-83% identity to group D prothrombin activators from snake venom and approximately 42% identity to mammalian FX and has identical domain structure. The precursor of the catalytic subunit of pseutarin C has several unique features. The activation peptide of the catalytic subunit of pseutarin C is significantly smaller (27 as compared to 52 residues in mammalian FX) and does not contain any glycosylation sites. Unlike coagulation FXa, Ser52 and Asn45 of the light and heavy chains are O- and N-glycosylated in pseutarin C catalytic sub-unit. There is a 12-residue insertion in pseutarin C catalytic sub-unit close to the region that is implicated in binding to FVa. This is the first sequence of the catalytic subunit of a group C prothrombin activator.

Animals↗

PubMed2004

Snake venom prothrombin activators similar to blood coagulation factor Xa.

Activation of prothrombin to mature thrombin in vivo occurs by the proteolytic action of the prothrombinase complex consisting of serine proteinase factor Xa, and cofactors that include factor Va, Ca(2+) ions and phospholipids. Several exogenous prothrombin activators are found in snake venom. Among these, Group C prothrombin activators resemble the factor Xa-factor Va complex, while Group D activators are structurally and functionally similar to factor Xa. This review provides a detailed description of current knowledge on Group D prothrombin activators and highlights the importance of studying this family of proteins in enhancing our understanding of structure-function relationships in the mammalian prothrombinase complex.

Animals↗

PubMed2004

Hypotensive agents from snake venoms.

Many snake venoms contain toxins which produce profound cardiovascular effects. The site of action of these toxins includes cardiac muscle, vascular smooth muscle and the capillary vascular bed. Some snake venoms, for example, contain peptides that inhibit angiotensin converting enzyme and potentiate the biological actions of bradykinin. Other snake venoms contain structural and functional equivalents of mammalian natriuretic peptides. Sarafotoxins are short peptide toxins found in the venoms of snakes from Atractaspis spp. which display potent vasoconstriction properties. These peptides, which share a high degree of sequence identity with endothelins, recognize and bind to endothelin receptors. Snakes have also evolved toxins which block L-type Ca(2+) currents (eg. calciseptine, FS2 toxins, C(10)S(2)C(2) and S(4)C(8)). Snake venom proteins have also been shown to increase vascular permeability. One such protein, increasing capillary permeability protein (ICPP) has recently been isolated from the venom of Vipera lebetina. ICPP is an extremely potent permeability factor with a structure similar to vascular endothelial growth factor (VEGF). Thus there is a vast array of snake toxins with potent cardiovascular activity. Some of these proteins and peptides have proven to be highly selective tools in the study of physiological processes. Others have been used as probes of potential therapeutic targets or as lead compounds in the development of therapeutic agents. Therefore these and other related snake venom proteins hold great promise in the future understanding and treatment of cardiovascular diseases.

Amino Acid Sequence↗

PubMed2004

Platelet aggregation and exogenous factors from animal sources.

Platelet aggregation plays a crucial role in thrombosis. This review describes exogenous factors isolated from various animal sources, including venoms and the salivary glands that interfere in platelet aggregation. Some of these factors induce platelet aggregation or agglutination, whereas others inhibit platelet aggregation. These proteins range from small molecular weight peptides to large proteins. Some of these proteins exhibit various enzymatic activities, while others are nonenzymatic. These exogenous factors affect platelet aggregation by various mechanisms and thus they have been classified based on their mechanism of action. Many of these proteins have evolved through both convergent and divergent evolution. For example, platelet aggregation inhibitors, which interfere in the interactions between fibrinogen and its receptor, the glycoprotein IIb/IIIa complex, show extreme structural diversity but they share the common functional site of Arg-Gly-Asp (RGD) tripeptide segment. On the other hand, C-type lectin related proteins exhibit diverse biological effects by interacting with different proteins, but share common structural scaffold. Thus the mechanistic and structure-function studies of these exogenous proteins have contributed significantly to the understanding of molecular mechanisms of platelet aggregation and to the development of potent antiplatelet agents, respectively. A number of new exogenous factors have been identified recently and the search is still on for novel factors that interfere with platelet aggregation. Further studies in this area will help in the development of novel strategies for treating cardiovascular and hematological disorders.

Animals↗

PubMed2003

Excitement ahead: structure, function and mechanism of snake venom phospholipase A2 enzymes.

Venom phospholipase A2 (PLA2) enzymes share similarity in structure and catalytic function with mammalian enzymes. However, in contrast to mammalian enzymes, many are toxic and induce a wide spectrum of pharmacological effects. Thus structure-function relationship of this group of small proteins is subtle, but complex puzzle to protein biochemists, molecular biologists, toxinologists, pharmacologists and physiologists. This review describes the present status of our understanding of their structure, function and mechanism. It was proposed that their unique ability to 'target' themselves to a specific organ or tissue is due to their high affinity binding to specific proteins which act as receptors (more precisely, acceptors). This specific binding of PLA2 is conferred by the presence of a 'pharmacological site' on its surface which is independent of the catalytic site. The high affinity interaction of PLA2 with its acceptor (or target protein) is probably due to the complementarity, in terms of charges, hydrophobicity and van der Waal's contact surfaces, between the pharmacological site and the binding site on the surface of the acceptor protein. Upon binding to the target, the PLA2 can induce its pharmacological effects by mechanisms either dependent on or independent of its catalytic activity. Because of the unprecedented wide spectrum of specific targeting to various tissues and organs, identification of the pharmacological sites has potential for exploitation in development of novel systems useful for 'delivering' specific proteins to a particular target tissue or organ. Thus research in this field will provide a lot of exciting opportunities.

Animals↗

PubMed2003

Identification of a novel family of proteins in snake venoms. Purification and structural characterization of nawaprin from Naja nigricollis snake venom.

The three-dimensional structure of nawaprin has been determined by nuclear magnetic resonance spectroscopy. This 51-amino acid residue peptide was isolated from the venom of the spitting cobra, Naja nigricollis, and is the first member of a new family of snake venom proteins referred to as waprins. Nawaprin is relatively flat and disc-like in shape, characterized by a spiral backbone configuration that forms outer and inner circular segments. The two circular segments are held together by four disulfide bonds, three of which are clustered at the base of the molecule. The inner segment contains a short antiparallel beta-sheet, whereas the outer segment is devoid of secondary structures except for a small turn or 310 helix. The structure of nawaprin is very similar to elafin, a human leukocyte elastase-specific inhibitor. Although substantial parts of the nawaprin molecule are well defined, the tips of the outer and inner circular segments, which are hypothesized to be critical for binding interactions, are apparently disordered, similar to that found in elafin. The amino acid residues in these important regions in nawaprin are different from those in elafin, suggesting that nawaprin is not an elastase-specific inhibitor and therefore has a different function in the snake venom.

Amino Acid Sequence↗

PubMed2003

Characterization of the interaction of wheat HMGa with linear and four-way junction DNAs.

Wheat HMGa protein is a typical member of the plant HMGA family. It has four AT hooks and a histone H1-like region. A panel of deletion mutants of HMGa was generated to study the role of different regions of HMGa in its binding to 4H (a synthetic DNA that mimics the in vivo structure of intermediates of homologous recombination and DNA repair) and linear DNAs. Although the histone H1-like region of HMGa does not bind to 4H or linear DNAs, it does enhance the binding. Mutants with any two adjacent AT hooks show specific binding to both 4H and linear P268 (and P31) with different binding affinities, which is partly due to the flanking regions between AT hooks. Conformational studies indicate that the alpha-helical content of HMGa increases significantly when it binds to 4H compared to that after binding to P31, linear DNA. In contrast, linear DNA, but not 4H, undergoes substantial conformational change when it binds to HMGa, indicating that linear DNA is relatively more flexible than 4H. A more significant difference in the affinities of binding of the mutants of HMGa to 4H was observed compared to their affinities of binding to linear DNA, P31. These differences could be due to the rigidity of the DNA and the characters of the AT hook regions in the mutants.

Amino Acid Sequence↗

PubMed2003

The nonenzymatic subunit of pseutarin C, a prothrombin activator from eastern brown snake (Pseudonaja textilis) venom, shows structural similarity to mammalian coagulation factor V.

Pseutarin C is a group C prothrombin activator from the venom of the eastern brown snake Pseudonaja textilis. It is a multi-subunit protein complex consisting of catalytic and nonenzymatic subunits similar to coagulation factor Xa and factor Va, respectively. Here we describe the complete sequence of the nonenzymatic subunit. Based on the partial amino acid sequence of the nonenzymatic subunit, degenerate primers were designed. Using a "walking" strategy based on sequentially designed primers, we determined the complete cDNA sequence of the nonenzymatic subunit. The cDNA encodes a protein of 1461 amino acid residues, which includes a 30-residue signal peptide, a mature protein of 1430 amino acid residues, and a stop codon. cDNA blot analysis showed a single transcript of approximately 4.6 kb. The deduced amino acid sequence shows approximately 50% identity to mammalian factor V and by homology has a similar domain structure consisting of domains A1-A2-B-A3-C1-C2. Interestingly, the B domain of pseutarin C is shorter than that of mammalian factor V (FV). Although most of the proteolytic activation sites are conserved, 2 of 3 proteolytic sites cleaved by activated protein C are mutated, and thus activated protein C is not able to inactivate this procoagulant toxin. The predicted posttranslational modifications, including disulfide bonds, N-glycosylation, phosphorylation, and sulfation, in pseutarin C are significantly different compared with bovine factor V. Thus, our data demonstrate that the nonenzymatic subunit of group C prothrombin activators is structurally similar to mammalian FV.

Amino Acid Sequence↗