Scorpions are the oldest known arachnids and include some of the earliest invertebrates to have become fully terrestrial, with fossil records dating back to the Silurian period approximately 444–419 million years ago. Scorpion anatomy features two main segments: a frontal prosoma, to which eight legs are attached, and an opisthosoma, divided in mesosoma as the main body and metasoma as the tail. Taxonomically, scorpions belong to the class Arachnida which includes spiders (order: Araneae), ticks and mites (order: Acarina) among other orders, and form the order Scorpiones. Over 2200 species have been described and these are distributed all over the earth with the exception of Antarctica. Relatively few scorpion genera are considered of public health importance with regards to clinical consequences of their stings and these are mostly found in Africa, Middle East and Central-Southern America. Such genera of clinical relevance include Androctonus, Buthus, Centruroides, Hemiscorpius, Hottentotta, Leiurus, Odontobuthus, Parabuthus and Tityus and around 30 species. Scorpion venoms encode toxins, primarily acting on sodium and potassium ion channels which are responsible for clinical effects resulting in the depolarization of excitable nerve and muscle cells. In addition, scorpion venoms also contain non-toxic metalloproteinases, calcium channel and chloride channel toxins, bradykinin-potentiating peptides, serine protease inhibitors, phospholipase A2 enzymes as well as defensins and antimicrobial peptides. Scorpion envenomation and associated symptoms are usually classified as Class I-mild, Class II-moderate and Class III-severe. The clinical management of scorpion stings is described here within a protocol.

Scorpion venoms are rich sources of bioactive peptides and are widely known for their ion channel inhibiting properties. We have isolated, cloned and characterized a venom protein (Bumarsin) from the Chinese scorpion, Mesobuthus martensii Karsch. Bumarsin cDNA encodes a 8132 Da, 72 amino acid mature protein that most probably exists in its native form as a Cys-bridged homodimer. We have identified this novel protein to be an inhibitor of 3-hydroxy-3-methylglutaryl-coenzyme A (HMG-CoA) reductase activity. 0.6 μM of Bumarsin inhibits 32% of the HMG-CoA reductase activity, in comparison to 10 μM simvastatin which only inhibits 35% of the activity. RT-PCR and SELDI-TOF mass spectrometric studies demonstrate that bumarsin regulates the expression of both genes and proteins involved in cholesterol homeostasis. Our results suggest that bumarsin may provide a model for the design of novel drugs that can be used to modulate cholesterol homeostasis.

The biophysical properties of small conductance Ca(2+)-activated K(+) (SK) channels are well suited to underlie afterhyperpolarizations (AHPs) shaping the firing patterns of a conspicuous number of central and peripheral neurons. We have identified a new scorpion toxin (tamapin) that binds to SK channels with high affinity and inhibits SK channel-mediated currents in pyramidal neurons of the hippocampus as well as in cell lines expressing distinct SK channel subunits. This toxin distinguished between the SK channels underlying the apamin-sensitive I(AHP) and the Ca(2+)-activated K(+) channels mediating the slow I(AHP) (sI(AHP)) in hippocampal neurons. Compared with related scorpion toxins, tamapin displayed a unique, remarkable selectivity for SK2 versus SK1 ( approximately 1750-fold) and SK3 ( approximately 70-fold) channels and is the most potent SK2 channel blocker characterized so far (IC(50) for SK2 channels = 24 pm). Tamapin will facilitate the characterization of the subunit composition of native SK channels and help determine their involvement in electrical and biochemical signaling.

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.

OBJECTIVES:To describe a case of equine muscular dystrophy with myotonia.METHODS:A 5-year-old horse presented with hypertrophy and delayed relaxation of the muscles of the hindlimbs from age 2 months. Testicular atrophy developed from 2 years of age. Action and percussion myotonia was associated with weakness in these muscles, and EMG showed diffuse myotonic discharges and myopathic features. Biopsy of the gluteal muscle showed adipose and connective tissue infiltration, marked variation in muscle fibre size, and moth-eaten, ring and whorled fibres.RESULTS:Injection of apamin, a peptide blocker of calcium-activated potassium channels, which inhibits myotonia in human myotonic dystrophy, was ineffective in blocking myotonic discharges. Discharges promptly abated with 2% lidocaine injection.CONCLUSIONS:Myotonia in this horse is associated with dystrophic changes similar to human myotonic dystrophy, though there are some pharmacological differences.

Gene transfer by direct intramuscular injection of naked plasmid DNA has been shown to be a safe, simple but relatively inefficient method for gene delivery in vivo. Eukaryotic plasmid expression vectors incorporating the Epstein–Barr virus (EBV) origin of replication (oriP) and EBNA1 gene have been shown to act as autonomous episomally replicating gene transfer vectors which additionally provide nuclear matrix retention functions. Prolonged expression of a LacZ reporter gene and recombinant human dystrophin was shown using EBV-based plasmid vectors transfected into C2C12 mouse myoblast and myotube cultures. Intramuscular injection of EBV-based dystrophin expression plasmids into nude/mdx mice resulted in significant enhancement in the number of muscle fibres expressing recombinant dystrophin compared with a conventional vector. This effect was observed for over 10 weeks after a single administration. These results indicate the potential advantage of EBV-based expression vectors for focal plasmid-mediated gene augmentation therapy in Duchenne muscular dystrophy (DMD) and a range of other gene therapeutic applications.

The mutation in the myotonic dystrophy (DM) gene is an expansion in a triplet (CTG)n repeat in the 3′ untranslated region of a novel gene that partially encodes a serine-threonine protein kinase (DMPK), with closest sequence homology to a small subgroup of protein kinases involved in the control of proliferation and cell shape. Expansion of the repeat correlates reasonably well with disease severity and offers a plausible molecular explanation for the previously contentious issue of anticipation. There is considerable heterogeneity in CTG expansion size in different tissues of affected individuals. The consensus of data from many laboratories indicates that DMPK mRNA is most probably downregulated as a consequence of the repeat expansion. Two polypeptides (68/78 kDa) have been shown to be absent in mouse knockout mutants and therefore can be considered as bona fide gene products. Previous data suggesting that 52-55 kDa polypeptides were likely candidates, have been firmly ruled out at the same time. Further results from studies of knockout and overexpressing transgenic mice indicate that neither simple loss nor gain of DMPK expression is sufficient to account for the DM clinical phenotype. One of the most pressing questions now being addressed is how expansion of the CTG repeat within the DMPK gene affects gene expression, not only of DMPK, but of all genes at the 19q13.3 locus: is DMPK actually responsible for the clinical phenotype seen in DM? The identification of both immediate upstream and downstream human genes (59 and DMRHP, respectively) has been an important first step to answering these questions. Only when these matters have been dealt with can one reasonably expect to start to delineate the different metabolic and signalling pathways responsible for the diverse phenotypes that make up the complex clinical picture of DM.

Taicatoxin, isolated from the venom of the Australian taipan snake Oxyuranus scutellatus, has been previously regarded as a specific blocker of high threshold Ca2+ channels in heart. Here we show that taicatoxin (in contrast to a range of other Ca2+ channel blockers) interacts with apamin-sensitive, small conductance, Ca2+-activated potassium channels on both chromaffin cells and in the brain. Taicatoxin displays high affinity recognition of125I-apamin acceptor-binding sites, present on rat synaptosomal membranes (K i = 1.45 ± 0.22 nm) and also specifically blocks affinity-labeling of a 33-kDa 125I-apamin-binding polypeptide on rat brain membranes. Taicatoxin (50 nm) completely blocks apamin-sensitive after-hyperpolarizing slow tail K+currents generated in rat chromaffin cells (mean block 97 ± 3%,n = 12) while only partially reducing total voltage-dependent Ca2+ currents (mean block 12 ± 4%, n = 6). In view of these findings, the use of taicatoxin as a specific ligand for Ca2+ channels should now be reconsidered.

I-125-Apamin and photolabile derivatives of the toxin have been used to investigate the binding properties and subunit composition of small conductance Ca2+-activated potassium channels (SKCa, channels) expressed on plasma membranes from rat brain, rabbit liver, or rat pheochromocytoma (PC12) cells, On all preparations, I-125-apamin recognized single classes of acceptor binding sites with similar high affinity (Kd similar to 3-6 pM), Gallamine, however, was found to readily discriminate between I-125-apamin accepters present in these preparations, showing a maximal approx ninefold difference in affinity for accepters expressed by rabbit liver or PC 12 cells. Affinity-labeling patterns revealed the expression of different hetero-oligomeric combinations of high (86 or 59 kDa) and low (33 or 30 kDa) molecular mass I-125-apamin-binding polypeptides, consistent with pharmacological differences. Alternative expression of either 86- or 59-kDa polypeptides appeared to be the most important factor influencing gallamine's affinity for SKCa channel subtypes, Both high- and low-molecular-mass polypeptides are integral membrane proteins, the latter being glycosylated in a tissue-specific manner. (C) 1997 Academic Press.

Duchenne muscular dystrophy is a primary muscle disease that manifests itself in young boys as a result of a defect in a gene located on the X-chromosome. This gene codes for dystrophin, a normal muscle protein that is located beneath the sarcolemma of muscle fibres. Therapies to alleviate this disease have centred on implanting normal muscle precursor cells into dystrophic fibres to compensate for the lack of this gene and its product. To date, donor cells for implantation in such therapy have been of myogenic origin, derived from paternal biopsies. Success in human muscle, however, has been limited and may reflect immune rejection problems. To overcome this problem the patient's own myogenic cells, with the dystrophin gene inserted, could be used, but this could lead to other problems, since these cells are those that are functionally compromised by the disease. Here, we report the presence of high numbers of dystrophin-positive fibres after implanting dermal fibroblasts from normal mice into the muscle of the mdx mouse-the genetic homologue of Duchenne muscular dystrophy. Dystrophin-positive fibres were also abundant in mdx muscle following the implantation of cloned dermal fibroblasts from the normal mouse. Our results suggest the in vivo conversion of these non-myogenic cells to the myogenic pathway resulting in the formation of dystrophin-positive muscle fibres in the deficient host. The use of dermal fibroblasts may provide an alternative approach to the previously attempted myoblast transfer therapy, which in human trials has yielded disappointing results.

Small-conductance calcium-activated potassium channels (SK channels) can be operationally defined as those having single-channel conductance of less than 20 pS. These channels are found in a wide range of excitable and non-excitable cells. In nerve and muscle, the ionic currents that flow through these channels are responsible for maintaining the slow after-hyperpolarizing potential (AHP) that follows bursts of action potentials. Two venom toxins have been characterized that appear to interact specifically with SK channels: apamin and leiurotoxin. Apamin, a neurotoxin isolated from the venom of the European honey bee Apis mellifera, is the first potassium channel toxin to be isolated and characterized. It is a basic peptide with a molecular weight of 2000. It has been used as a pharmacological tool to characterize ionic currents flowing through SK channels and for the quantification of apamin-binding sites and the identification of putative SK channel polypeptides. Apamin-binding assays are used to monitor the solubilization and purification of SK channel proteins. The toxin is also used to provide evidence of a regulatory role for SK channels in cell metabolism.

Apamin, a peptide neurotoxin from bee venom, blocks small conductance Ca(2+)-activated K+ channels in central synapses and peripheral tissues. Using 125I-apamin, single classes of high affinity binding sites (Kd 1-3 pM) were identified on plasma membranes from rat, rabbit, guinea pig, and bovine brain and from rabbit, guinea pig, and bovine liver. Binding was sensitive to scyllatoxin, dequalinium, gallamine, and d-tubocurarine but not to charybdotoxin, toxin I, or mast cell degranulating peptide. In contrast, saturable binding of 125I-apamin to rat liver plasma membranes was virtually undetectable, thereby providing a correlation with the ability to measure apamin-sensitive Ca(2+)-activated potassium currents in rabbit and guinea pig hepatocytes but not in rat hepatocytes. In agreement with membrane binding studies, homobifunctional cross-linkers identified apparently identical 33-kDa 125I-apamin binding polypeptides on brain plasma membranes from all species and analogous but distinct polypeptides on plasma membranes from rabbit, guinea pig, and bovine liver. None of these affinity-labeled polypeptides were detectable on plasma membranes from rat liver. Affinity labeling was abolished on both liver and brain membranes by apamin, scyllatoxin, dequalinium, gallamine, and d-tubocurarine. These results indicate that comparable approximately 30-kDa polypeptides may fulfill equivalent functional roles within putative subtypes of apamin-sensitive small conductance Ca(2+)-activated K+ channels.

Potassium channels form a remarkably diverse group of ion channel structures.1 More subtypes of potassium channels, with different biophysical and/or pharmacologic properties, are known than of any other type of ion channel. One of the most important roles of these channels is the regulation of membrane potential and excitability in nerve and muscle cells. Many potassium channels in turn are regulated by second messengers and other molecules of intermediary metabolism, such as G proteins, cAMP, ATP, inositol trisphosphate, and calcium.2 In this manner potassium channels provide an important link between membrane potential and cell metabolism.

Although many Duchenne muscular dystrophy patients have a deletion in the dystrophin gene which disrupts the translational reading frame, they express dystrophin in a small proportion of skeletal muscle fibers ("revertant fibers"). Antibody studies have shown, indirectly, that dystrophin synthesis in revertant fibers is facilitated by a frame-restoring mechanism; in the present study, the feasibility of mRNA splicing was investigated. Dystrophin transcripts were analyzed in skeletal muscle from individuals possessing revertant fibers and a frameshift deletion in the dystrophin gene. In each case a minor in-frame transcript was detected, in which exons adjacent to those deleted from the genome had been skipped. There appeared to be some correlation between the levels of in-frame transcripts and the predicted translation products. Low levels of alternatively spliced transcripts were also present in normal muscle. The results provide further evidence of exon skipping in the dystrophin gene and indicate that this may be involved in the synthesis of dystrophin by revertant fibers.

Using high-resolution PAGE and Western-blotting techniques the lectin Ricinus communis agglutinin-1 (RCA-1) was tested for its ability to recognize laminin subunits from the mouse Engelbreth-Holm-Swarm (EHS) tumour and from bovine cardiac and skeletal muscle. Biotinylated RCA-1 recognized both the A and B chains of purified EHS-tumour laminin with a sensitivity comparable to anti-(EHS laminin) antibodies. In cardiac and skeletal muscle RCA-1 also recognized the B chains of laminin, together with a approximately 330 kDa RCA-1-binding glycoprotein that was undetectable in smooth muscle. This glycoprotein was not recognized by antibodies raised to laminin from the EHS tumour. Purification of the 330 kDa binding glycoprotein from skeletal muscle, using ion-exchange and lectin-affinity chromatography, revealed that in its native form, this glycoprotein is disulphide-bonded to the B chains of laminin. The demonstrated properties of the approximately 330 kDa RCA-1-binding glycoprotein are identical to those reported for the variant M chain of merosin which is known to replace the A chain in laminin from the extrasynaptic regions of skeletal muscle. These results establish that biotinylated RCA-1 can recognize A-, B- and M-chain subunits of laminin isoforms, and that, when used in conjunction with other techniques, they provide a useful method for the assignment of laminin heavy chains.

In order to identify the protein product of the recently characterised myotonic dystrophy gene, we have raised an antibody (DMAP1) to a peptide sequence of the predicted gene product. This antibody identifies a novel 52kDa protein in a range of mouse tissues, and in addition a related 42kDa protein in brain and heart. A second antibody raised to a different peptide from the same predicted sequence, also identities the 52kDa protein, which strongly implies that this 52kDa protein is a major translation product of the myotonic dystrophy gene.

Dystrophin is expressed only in muscle and brain, but is absent from all tissues of the adult mdx mouse, a mutant with a single base substitution in the dystrophin gene. The brains of both normal and mdx mice contain a protein of approximately 230 kDa that is recognised by anti-dystrophin antibodies raised to the N-terminal region of the rod-like domain. Although the N-terminal and central rod regions of dystrophin share structural homologies with spectrin, the 230-kDa protein represents neither of the presently described forms of brain spectrin by a variety of criteria (molecular weight, cerebellar localisation, and developmental regulation) and is distinct from the product of the dystrophin gene. Studies of mdx and normal mouse brain show different postnatal developmental regulation of the 230-kDa dystrophin-immunoreactive protein.