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. In this study, we examined the diversity, origin and evolution of chromoviruses in Eukaryota. Chromoviral diversity in plants, fungi and vertebrates, as shown by phylogenetic analyses, was found to be much greater than previously expected. A novel centromere-specific chromoviral lineage was found to be widespread and highly conserved in all seed plants. The age of chromoviruses has been significantly extended by finding their representatives in the most basal plant lineages (green and red algae), in Heterokonta (oomycetes) and in Cercozoa (plasmodiophorids). The evolutionary origin of chromoviruses has been found to be no earlier than in Cercozoa, since none can be found in the basal eukaryotic lineages, despite the extensive genome data. The evolutionary dynamics of chromoviruses can be explained by a strict vertical transmission in plants and fungi, while in Metazoa it is more complex. The currently available genome data clearly show that chromoviruses are the most widespread and one of the oldest Metaviridae clade.

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 0.5% of total cell protein. Analysis of the potential secondary structure of the mutant mRNA in the translation initiation region suggested that the Ala1 (GCC) and Leu2 (CUG) codons used are likely to be involved in a hairpin structure with the Thr13 (ACG) and Gly14 (GGG) codons, hindering effective translation at the ribosome. To weaken this structure (by DeltaG of about 20 kJ/mol) the same double mutant was prepared using another mutagenic oligonucleotide with silent mutations in the Ala1 (GCU) and Leu2 (UUG) codons. The mutant was successfully produced at a level of approximately 15% of total protein, with the initial Met completely removed in the bacterial cell. Such an approach could be important in solving similar problems in bacterial production of other toxic proteins.

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. However, as shown by immunological analysis and tandem mass spectrometry sequencing of several internal peptides derived from the purified receptors, both belong to secretory phospholipase A(2) receptors of the M type, which are Ca(2+)-dependent multilectins homologous to the macrophage mannose receptor. Based on Southern blot analysis of genomic DNA and deglycosylation of the receptors, the difference between the two proteins most likely stems from the different posttranscriptional and posttranslational modifications of a single gene product. Our findings raise the possibility that the M-type receptors for secretory phospholipases A(2) may display different physiological properties in different tissues.

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). Most of the reptilian LINE2s examined are inactive since they contain multiple stop codons, indels, or frameshift mutations that disrupt the RT. Analysis of reptilian LINE2s has shown a high degree of sequence divergence and an unexpectedly large number of deletions. The evolutionary dynamics of LINE2s in reptiles has been found to be complex. LINE2s are shown to form a novel clade of non-LTR retrotransposons that is well separated from the CR1 clade. This novel L2 clade is more widely distributed than previously thought, and new representatives have been discovered in echinoderms, insects, teleost fishes, Xenopus, Squamata, and marsupials. There is an apparent absence of LINE2s from different vertebrate classes, such as cartilaginous fishes, Archosauria (birds and crocodiles), and turtles. Whereas the LINE2s are present in echinoderms and teleost fishes in a conserved form, in most tetrapods only highly degenerated pseudogenes can be found. The predominance of inactive LINE2s in Tetrapoda indicates that, in the host genomes, only inactive copies are still present. The present data indicate that the vertical inactivation of LINE2s might have begun at the time of Tetrapoda origin, 400 MYA. The evolutionary dynamics of the L2 clade in Deuterostomia can be described as a gradual vertical inactivation in Tetrapoda, stochastic loss in Archosauria and turtles, and strict vertical transmission in echinoderms and teleost fishes.

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. The long-term evolution of Bov-B LINEs was studied in 10 species of Squamata by analysis of a PCR-amplified 1.8-kb fragment encoding part of apurinic/apyrimidinic endonuclease, the intervening domain, and the palm/fingers subdomain of reverse transcriptase. A very high level of conservation in Squamata Bov-B long interspersed nuclear elements has been found, reaching 86% identity in the nearly 600 amino acids of ORF2. The same level of conservation exists between the ancestral snake lineage and Ruminantia. Such a high level is exceptional when compared with the level of conservation observed in nuclear and mitochondrial proteins and in other transposable elements. The RTE clade has been found to be much more widely distributed than previously thought, and novel representatives have been discovered in plants, brown algae, annelids, crustaceans, mollusks, echinoderms, and teleost fishes. Evolutionary relationships in the RTE clade were deduced at the amino acid level from three separate regions of ORF2. By using different independent methods, including the divergence-versus-age analysis, several examples of horizontal transfer in the RTE clade were recognized, with important implications for the existence of HT in 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. Due to the strong sequence similarity, highly selective reagents are needed to elucidate the physiological functions of the two enzymes. Monoclonal antibodies (mAbs) have been prepared against recombinant human cathepsin L. Antibodies produced by five clones reacted with procathepsin L and mature cathepsin L. They also reacted with cathepsin L in complex with a peptide fragment, which is identical to the alternatively spliced segment of the p41 form of MHC Class II associated invariant chain. Two mAbs, (M105 and H102) were specific for cathepsin L, while three (N135, B145 and D24) cross-reacted with cathepsin V. None of the mAbs cross-reacted with cathepsins B, H and S. We have developed a sandwich enzyme-linked immunosorbent assay (ELISA) for quantifying cathepsin L. This sandwich ELISA uses a combination of two monoclonal antibodies which recognize different, non-overlapping epitopes on the cathepsin L molecule. The lower detection limit of the sandwich ELISA was 5 ng of cathepsin L per ml.

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. The isolated acceptor was identified as calmodulin by tandem mass spectrometry. Since calmodulin is generally considered as an intracellular protein, the identity of this acceptor supports the view that secretory phospholipase A(2) neurotoxins have to be internalized to exert their toxic effect. Moreover, since ammodytoxin is known to block synaptic transmission, its interaction with calmodulin as an acceptor may constitute a valuable probe for further investigation of the role of the latter in this Ca(2+)-regulated process.

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. The mutant was approximately 30-fold less lethal, but still neurotoxic. Consistent with this, its binding affinity for the neuronal receptors decreased by only a factor of five. Additionally, a single-site mutant of AtxA was prepared, with substitution at only one position (K127T) out of six mutated in the six-site mutant. Its toxicity indicated that most, if not all, of the six mutated residues partially contribute to the decreased lethality of the multiple-site mutant.

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. We propose a model which could be useful in investigating the apparent inconsistency between the observed heterogeneity in the neuronal binding of beta-neurotoxins and the very similar pathomorphological and electrophysiological effects which they produce in the intoxicated tissue. We assume that beta-neurotoxins enter the nerve ending to exert their toxic effect. The model involves different pathways for phospholipase A(2) neurotoxins to reach the site of action inside the neuron, their respective extra- and intracellular neuronal receptors being key features of the pathway. Once in the nerve cell, beta-neurotoxins impair the function of the synaptic vesicles by phospholipid hydrolysis of the inner leaflet of the vesicle bilayer. The proportion of the products of the phospholipid hydrolysis, lysophospholipids and phospholipids in the membrane, has been demonstrated to be very important for the shaping of the membrane, affecting its fusogenic properties. Due to the same final step in the action of beta-neurotoxins, phospholipid hydrolysis, the consequences of their poisoning are practically identical.

The amino acid region 115-119 of ammodytoxins plays an important role in neurotoxicity.

Quadruple (Y115K/I116K/R118M/N119L) and double (Y115K/I116K) mutants of ammodytoxin A, a presynaptically toxic phospholipase A(2) from Vipera ammodytes ammodytes venom, were prepared and characterized. The enzymatic activity of the quadruple mutant on phosphatidylcholine micelles was threefold higher than that of AtxA, presumably due to higher phospholipid-binding affinity, whereas the activity of the double mutant was twofold lower. The substantial decrease by more than two orders of magnitude in the lethal potency of both mutants, together with their decreased binding affinity for neuronal receptors, indicates involvement of the amino acid region 115-119 in neurotoxicity. The similar decrease of toxicity for the two mutants points to the importance of the residues Y115 and I116.

Effects of ammodytin L on miniature and endplate potentials in neuromuscular junction of frog m. cutaneus pectoris.

Neurotoxic effects of ammodytin L (AtnL), a potent phospholipase A2 homologue, was studied in frog neuromuscular preparation m. cutaneous pectoris by measuring the influence of the toxin on the amplitude and the frequency of miniature and endplate potentials (MEPPs, EPPs). AtnL, in 100 nM concentration, significantly increases spontaneous quantal acetylcholine release from the motor nerve endings, observed as the increase in MEPPs frequency. At 100 nM or higher concentration the toxin decreases EPPs amplitude and the membrane potential (MP) simultaneously. No significant effect of AtnL on EPPs was observed at any concentration bellow 100 nM. Our results indicate that in frog AtnL shows the typical myotoxic effects, but it also exerts presynaptic effects.

Polymerase chain reaction as a diagnostic tool for detecting Leishmania.

Polymerase chain reaction (PCR) has been used to identify a Leishmania parasite in a cutaneous ulcer from a 27-year-old patient infected during travel in Peru. The available classical diagnostic methods could not confirm the diagnosis in a sufficiently short time. Therefore, two sets of oligonucleotides were designed and with both of them fragments of the expected size were obtained. The sequence of the fragment derived from kinetoplast DNA corresponds to the Leishmania Viannia complex. Polymerase chain reaction has advantages over classical diagnostic methods, which makes it an important technique in those hospitals and clinical laboratories in Europe which lack standard diagnostic tests for Leishmania.

Molecular cloning and chimerisation of CDI 315B monoclonal antibody.

A chimeric mouse-human antibody has been created that recognizes an antigen found on breast cancer cells and melanoma cells. Immunoglobulin constant domains of mouse monoclonal antibody CDI 315B Cgamma1 and CK, were substituted by the human Cgamma1 and Ckappa. The CDI 315B variable heavy and light chain regions were PCR amplified from hybridoma RNA and sequenced. Mouse variable VH and VL regions were joint to human IgG1 and kappa constant regions and subcloned into pcDNA3 expression vectors. The Sp2/0 murine myeloma cells were transfected with expression vectors pcDNA3L and pcDNA3H and the reactivity of chimeric antibodies was tested by indirect ELISA using B16F1 murine melanoma cells as well as MCF7 human breast cancer cells, as antigen.

The effect of ammodytin L on frog neuromuscular junction.

Effects of myotoxic ammodytin L on frog neuromuscular junction were examined by morphological, enzymatic and electrophysiological studies. Electron micrographs of the frog neuromuscular junction revealed destruction of muscle fibers. Nerve terminals seemed to remain intact. The activity of the muscle specific enzymes in the bathing solution rose with time in a dose-dependent manner. Ammodytin L depressed the response of the neuromuscular preparation to acetylcholine. Measurements of electrically triggered isometric muscle contractions show that the toxin depresses the neuromuscular transmission both in the curarised and in the untreated frog neuromuscular preparation with marked difference between the time-courses of both effects, indicating that neurotoxic effect may also be involved.

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. The number of pharmacological activities in most toxin multigene families results from their adaptive evolution. The molecular evolution of animal toxins has been analysed in some multigene families, at both the intraspecies and interspecies levels. In most toxin multigene families, the rate of non-synonymous to synonymous substitutions (dN/dS) is higher than one. Thus natural selection has acted to diversify coding sequences and consequently the toxin functions. The selection pressure for the rapid adaptive evolution of animal toxins is the need for quick immobilization of the prey in classical predator and prey interactions. Currently available evidence for adaptive evolution in animal toxin multigene families will be considered in this review.

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. We have determined the nucleotide sequences of the antibody heavy and light chain and compared them to the murine immunoglobulin germ line sequences. Expression of the two antibody chains was achieved using a single vector with a PhoA promoter and coding regions placed after the signal sequences, directing them to the periplasmic space. The A22 Fab fragment was extracted from the periplasmic space and expression was confirmed by Western blot analysis. The recombinant A22 Fab fragment had an affinity for stefin A comparable to the original monoclonal antibody, as determined by ELISA.

Molecular evolution of Bov-B LINEs in vertebrates.

Since their discovery in family Bovidae (bovids), Bov-B LINEs, believed to be order-specific SINEs, have been found in all ruminants and recently also in Viperidae snakes. The distribution and the evolutionary relationships of Bov-B LINEs provide an indication of their origin and evolutionary dynamics in different species. The evolutionary origin of Bov-B LINE elements has been shown unequivocally to be in Squamata (squamates). The horizontal transfer of Bov-B LINE elements in vertebrates has been confirmed by their discontinuous phylogenetic distribution in Squamata (Serpentes and two lizard infra-orders) as well as in Ruminantia, by the high level of nucleotide identity, and by their phylogenetic relationships. The direction of horizontal transfer from Squamata to the ancestor of Ruminantia is evident from the genetic distances and discontinuous phylogenetic distribution of Bov-B LINE elements. The ancestor of Colubroidea snakes has been recognized as a possible donor of Bov-B LINE elements to Ruminantia. The timing of horizontal transfer has been estimated from the distribution of Bov-B LINE elements in Ruminantia and the fossil data of Ruminantia to be 40-50 My ago. The phylogenetic relationships of Bov-B LINE elements from the various Squamata species agrees with that of the species phylogeny, suggesting that Bov-B LINE elements have been stably maintained by vertical transmission since the origin of Squamata in the Mesozoic era.

Identification and purification of a novel receptor for secretory phospholipase A(2) in porcine cerebral cortex.

A specific phospholipase A(2) receptor from porcine cerebral cortex has been characterized (K(d) = 145 nM, B(max) = 0.4 pmol/mg membrane protein) by using a radioiodinated derivative of ammodytoxin C (AtxC), a snake venom presynaptically neurotoxic group IIA phospholipase A(2). After the receptor was solubilized in a ligand-binding form, it was approximately 14,000-fold enriched by chromatography on wheat germ lectin-Sepharose and AtxC-Affi-Gel 10. The receptor is a single chain glycoprotein with an apparent molecular mass of 180 kDa and binds toxic and non-toxic phospholipases A(2) of either group I or II. It also recognizes conjugates of bovine serum albumin with mannose, N-acetylglucosamine, and galactose. In its molecular mass and pharmacological profile, the AtxC receptor resembles the M-type receptor for secretory phospholipases A(2) from rabbit skeletal muscle (a C-type multilectin, homologous to macrophage mannose receptor), yet in terms of relative abundance in brain and antigenicity, these two receptors are completely different. A further AtxC receptor of approximately 200 kDa discovered in porcine liver was, however, recognized by anti-rabbit M-type phospholipase A(2) receptor antibodies. There are, therefore, two immunologically distinct secretory phospholipase A(2) receptors of about 200 kDa in the same species. Although the liver receptor is related to the M-type secretory phospholipase A(2) receptors, the brain receptor is not and belongs to a novel group of secretory phospholipase A(2) receptors.

Partial primary structure of a fibrinogenase from the venom of the snake Lachesis stenophrys.

The partial primary structure of an Mr 24,000 non-haemorrhagic metalloproteinase isolated from the venom of the snake Lachesis stenophrys has been determined. The native proteinase was resistant to Edman degradation exhibiting the N-terminal blockade. The pyridylethylated or native proteinase was chemically and enzymatically fragmented and the obtained peptides were separated by gel or reversed-phase chromatography, and sequenced. The metalloproteinase from Lachesis stenophrys contains a putative zinc-chelating sequence HELGHNLGMKH, characteristic for the reprolysin family of zinc-metalloproteinases. It contains six cysteine residues in the standard positions for this group of proteins suggesting the same disulfide bonding. Interestingly, it has almost identical sequence as the metalloproteinase from Lachesis muta muta, LHF-II, which is, however, haemorrhagic. The main structural differences between the two molecules were found in their N-terminal parts and in glycosylation. As the substrate-binding regions of both proteinases are practically identical, we suggest that the absence of haemorrhagicity in Lachesis stenophrys enzyme is due to its lower affinity for the matrix proteins and not due to different substrate specificity.

Cysteine-scanning mutagenesis of an eukaryotic pore-forming toxin from sea anemone: topology in lipid membranes.

Equinatoxin II is a cysteineless pore-forming protein from the sea anemone Actinia equina. It readily creates pores in membranes containing sphingomyelin. Its topology when bound in lipid membranes has been studied using cysteine-scanning mutagenesis. At approximately every tenth residue, a cysteine was introduced. Nineteen single cysteine mutants were produced in Escherichia coli and purified. The accessibility of the thiol groups in lipid-embedded cysteine mutants was studied by reaction with biotin maleimide. Most of the mutants were modified, except those with cysteines at positions 105 and 114. Mutants R144C and S160C were modified only at high concentrations of the probe. Similar results were obtained if membrane-bound biotinylated mutants were tested for avidin binding, but in this case three more mutants gave a negative result: S1C, S13C and K43C. Furthermore, mutants S1C, S13C, K20C, K43C and S95C reacted with biotin only after insertion into the lipid, suggesting that they were involved in major conformational changes occurring upon membrane binding. These results were further confirmed by labeling the mutants with acrylodan, a polarity-sensitive fluorescent probe. When labeled mutants were combined with vesicles, the following mutants exhibited blue-shifts, indicating the transfer of acrylodan into a hydrophobic environment: S13C, K20C, S105C, S114C, R120C, R144C and S160C. The overall results suggest that at least two regions are embedded within the lipid membrane: the N-terminal 13-20 region, probably forming an amphiphilic helix, and the tryptophan-rich 105-120 region. Arg144, Ser160 and residues nearby could be involved in making contacts with lipid headgroups. The association with the membrane appears to be unique and different from that of bacterial pore-forming proteins and therefore equinatoxin II may serve as a model for eukaryotic channel-forming toxins.