Phylogenomic analysis of the L1 retrotransposons in Deuterostomia.

L1 retrotransposons constitute the largest single component of mammalian genomes. In contrast to the single remaining lineage of L1 retrotransposons in mammalian genomes, some teleost fishes contain a highly diverse L1 retrotransposon repertoire. Major evolutionary changes in L1 retrotransposon repertoires have therefore taken place in the land vertebrates (Tetrapoda).

Phylogenomic analysis of chromoviruses.

Genome sequences of model organisms provide a unique opportunity to obtain insight into the complete diversity of any transposable element (TE) group. A limited number of chromoviruses, the chromodomain containing genus of Metaviridae, is known from plant, fungal and vertebrate genomes. By searching diverse eukaryotic genome databases, we have found a surprisingly large number of new, structurally intact and highly conserved chromoviral elements, greatly exceeding the number of previously known chromoviruses.

Ammodytoxin, a secretory phospholipase A2, inhibits G2 cell-cycle arrest in the yeast Saccharomyces cerevisiae.

Ammodytoxin (Atx), an sPLA2 (secretory phospholipase A2), binds to g and e isoforms of porcine 14-3-3 proteins in vitro. 14-3-3 proteins are evolutionarily conserved eukaryotic regulatory proteins involved in a variety of biological processes, including cell-cycle regulation. We have now shown that Atx binds to yeast 14-3-3 proteins with an affinity similar to that for the mammalian isoforms.

Basic amino acid residues in the beta-structure region contribute, but not critically, to presynaptic neurotoxicity of ammodytoxin A.

The molecular mechanism of action of presynaptically toxic secreted phospholipases A2 (sPLA2s) isolated from snake venoms is not completely understood. It has been proposed that the positive charge in the beta-structure region is important for their toxic activity. To test this hypothesis, we characterised several mutants of ammodytoxin A (AtxA) possessing substitution of all five basic residues in this region.

Ammodytoxin, a neurotoxic secreted phospholipase A(2), can act in the cytosol of the nerve cell.

Recent identification of intracellular proteins that bind ammodytoxin (calmodulin, 14-3-3 proteins, and R25) suggests that this snake venom presynaptically active phospholipase A(2) acts intracellularly. As these ammodytoxin acceptors are cytosolic and mitochondrial proteins, the toxin should be able to enter the cytosol of a target cell and remain stable there to interact with them. Using laser scanning confocal microscopy we show here that Alexa-labelled ammodytoxin entered the cytoplasm of the rat hippocampal neuron and subsequently also its nucleus.

Evolutionary genomics of chromoviruses in eukaryotes.

The diversity, origin, and evolution of chromoviruses in Eukaryota were examined using the massive amount of genome sequence data for different eukaryotic lineages. A surprisingly large number of novel full-length chromoviral elements were found, greatly exceeding the number of the known chromoviruses. These new elements are mostly structurally intact and highly conserved.

R25 is an intracellular membrane receptor for a snake venom secretory phospholipase A(2).

Ammodytoxin is a presynaptically neurotoxic (beta-neurotoxic) snake venom secretory phospholipase A(2) (sPLA(2)). We detected a 25 kDa protein which binds the toxin with very high affinity (R25) in porcine cerebral cortex. Here we show that R25 is an integral membrane protein with intracellular localisation.

Adaptive evolution in the snake venom Kunitz/BPTI protein family.

Snake venoms are rich sources of serine proteinase inhibitors that are members of the Kunitz/BPTI (bovine pancreatic trypsin inhibitor) family. However, only a few of their gene sequences have been determined from snakes. We therefore cloned the cDNAs for the trypsin and chymotrypsin inhibitors from a Vipera ammodytes venom gland cDNA library.

Identification of a novel binding site for calmodulin in ammodytoxin A, a neurotoxic group IIA phospholipase A2.

The molecular mechanism of the presynaptic neurotoxicity of snake venom phospholipases A2 (PLA2s) is not yet fully elucidated. Recently, new high-affinity binding proteins for PLA2 toxins have been discovered, including the important intracellular Ca2+ sensor, calmodulin (CaM). In the present study, the mode of interaction of group IIA PLA2s with the Ca2+-bound form of CaM was investigated by mutational analysis of ammodytoxin A (AtxA) from the long-nosed viper (Vipera ammodytes ammodytes).

Quantification of binding of some thiol-reactive clenbuterol analogues to bovine serum albumin by electron paramagnetic resonance spectroscopy.

The only free thiol group of bovine serum albumin (BSA) was coupled in a high yield with some novel thiol-reactive clenbuterol analogues. The unreacted cysteines were probed with maleimide spin label to determine the yield of the coupling reaction. A novel approach to determining free thiol groups of BSA quantitatively by electron paramagnetic resonance spectroscopy and spectral decomposition without the usual gel-filtration step or extensive dialysis is presented.

Crotoxin acceptor protein isolated from Torpedo electric organ: binding properties to crotoxin by surface plasmon resonance.

Crotoxin, a potent neurotoxin from the South American rattlesnake Crotalus durissus terrificus, is a heterodimeric phospholipase A(2) (EC 3.1.1.

The neurotoxic phospholipase A2 associates, through a non-phosphorylated binding motif, with 14-3-3 protein gamma and epsilon isoforms.

Two novel acceptors for ammodytoxin C, a presynaptically neurotoxic phospholipase A(2) from snake venom, have been purified from porcine cerebral cortex by a toxin-affinity-based procedure. Using tandem mass spectrometry, the isolated acceptors were identified as 14-3-3 gamma and epsilon isoforms, highly conserved cytoplasmic proteins involved in the regulation of numerous physiological processes. The interaction between ammodytoxin C and 14-3-3 proteins is direct and not mediated by calmodulin, a high-affinity acceptor for both ammodytoxin C and 14-3-3 proteins, as demonstrated in pull-down experiments and by surface plasmon resonance.

The C-terminal region of ammodytoxins is important but not sufficient for neurotoxicity.

Ammodytoxins (Atxs) are presynaptically acting snake venom phospholipase A2 (PLA2) toxins the molecular mechanism of whose neurotoxicity is not completely understood. Two chimeric PLA2s were prepared by replacing the C-terminal part of a nontoxic venom PLA2, ammodytin I2, with that of AtxA(K108N). The chimeras were not toxic, but were able to bind strongly to an Atxs-specific neuronal receptor, R25.

Phenylalanine-24 in the N-terminal region of ammodytoxins is important for both enzymic activity and presynaptic toxicity.

Ammodytoxins (Atxs) are group II phospholipases A(2) (PLA(2)s) with presynaptic toxicity from venom of the snake Vipera ammodytes ammodytes. The molecular basis of their neurotoxicity, and that of similar PLA(2) toxins, is still to be explained. To address this problem, a surface-exposed aromatic residue, Phe(24), in the N-terminal region of the most potent Atx, AtxA, was replaced by other aromatic (tyrosine, tryptophan), hydrophobic (alanine) and polar uncharged (serine, asparagine) residues.

mRNA secondary structure can greatly affect production of recombinant phospholipase A(2) toxins in bacteria.

The neurotoxic activity of ammodytoxin A (AtxA), a phospholipase A(2) from Vipera ammodytes ammodytes venom, has been investigated by protein engineering. With the aim of obtaining AtxA as a non-fused protein in the bacterial cytoplasm and avoiding problems with incomplete cleavage in vivo of the initial Met preceding the first residue (Ser1), a double mutant (S1A/E4Q) was prepared and expressed in Escherichia coli. Immunoblotting of the bacterial lysate showed that the mutant was synthesized at a low level not exceeding 0.

Evolutionary dynamics in a novel L2 clade of non-LTR retrotransposons in Deuterostomia.

The evolution of the novel L2 clade of non-long terminal repeat (LTR) retrotransposons and their evolutionary dynamics in Deuterostomia has been examined. The short-term evolution of long interspersed nuclear element 2s (LINE2s) has been studied in 18 reptilian species by analysis of a PCR amplified 0.7-kb fragment encoding the palm/fingers subdomain of reverse transcriptase (RT).

High-molecular-mass receptors for ammodytoxin in pig are tissue-specific isoforms of M-type phospholipase A(2) receptor.

Studying the molecular basis of presynaptic neurotoxicity of ammodytoxin C, a secretory phospholipase A(2) from the venom of Vipera a. ammodytes snake, we demonstrated the existence of two high-molecular-mass ammodytoxin C-binding proteins in porcine tissues, one in cerebral cortex and the other in liver. These proteins differ considerably in stability and Western blotting properties.

Purification and characterisation of two hemorrhagic metalloproteinases from the venom of the long-nosed viper, Vipera ammodytes ammodytes.

Two hemorrhagic proteins, VaH1 and VaH2, have been purified from Vipera ammodytes ammodytes venom. They are monomeric glycoproteins of an apparent molecular mass of 70kDa and multiple isoelectric points around pH 5.5.

Evolutionary dynamics and evolutionary history in the RTE clade of non-LTR retrotransposons.

This study examined the evolutionary dynamics of Bov-B LINEs in vertebrates and the evolution of the RTE clade of non-LTR retrotransposons. The first full-length reptilian Bov-B LINE element is described; it is 3.2 kb in length, with a structural organization typical of the RTE clade of non-LTR retrotransposons.

Anti-cathepsin L monoclonal antibodies that distinguish cathepsin L from cathepsin V.

Cathepsin L is a lysosomal cysteine protease involved in intracellular protein degradation. Recently, several new cysteine proteases have been identified. Human cathepsin V, a thymus- and testis-specific human cysteine protease, shares 78% sequence identity with human cathepsin L.

A high affinity acceptor for phospholipase A2 with neurotoxic activity is a calmodulin.

One of the high affinity binding proteins for ammodytoxin C, a snake venom presynaptically neurotoxic phospholipase A(2), has been purified from porcine cerebral cortex and characterized. After extraction from the membranes, the toxin-binding protein was isolated in a homogenous form using wheat germ lectin-Sepharose, Q-Sepharose, and ammodytoxin-CH-Sepharose chromatography. The specific binding of (125)I-ammodytoxin C to the isolated acceptor was inhibited to different extents by some neurotoxic phospholipases A(2), ammodytoxins, bee venom phospholipase A(2), agkistrodotoxin, and crotoxin; but not by nontoxic phospholipases A(2), ammodytin I(2), porcine pancreatic phospholipase A(2), and human type IIA phospholipase A(2); suggesting the significance of the acceptor in the mechanism of phospholipase A(2) neurotoxicity.

Recombinant anti-stefin A Fab fragment: sequence analysis of the variable region and expression in Escherichia coli.

Human stefin A is an inhibitor of lysosomal cysteine proteinases cathepsin B, H, L and S. In the present report we describe the cloning and expression of anti-stefin A Fab fragment A22 in E. coli.

Adaptive evolution of animal toxin multigene families.

Animal toxins comprise a diverse array of proteins that have a variety of biochemical and pharmacological functions. A large number of animal toxins are encoded by multigene families. From studies of several toxin multigene families at the gene level the picture is emerging that most have been functionally diversified by gene duplication and adaptive evolution.

Neuronal receptors for phospholipases A(2 )and beta-neurotoxicity.

Some phospholipases A(2) interrupt neuromuscular communication by blocking the release of neurotransmitter into the synaptic cleft. Despite numerous studies, the molecular mechanism of their action is still largely obscure. In this review the best-characterized receptors for beta-neurotoxins are presented.

Charge reversal of ammodytoxin A, a phospholipase A2-toxin, does not abolish its neurotoxicity.

The positive charge concentrated at the C-terminal region of ammodytoxin (Atx) A, which is involved in presynaptic toxicity, has been reversed. A six-site mutant of AtxA (K108N/K111N/K127T/K128E/E129T/K132E , where K108N=Lys(108)-->Asn etc. ) was prepared, in which five out of seven C-terminal basic amino acid residues were substituted with neutral or acidic ones.