The triglycerides and glucose index is associated with cardiovascular risk factors in metabolically obese normal-weight subjects.

PURPOSE: The aim of this study was to determine whether the triglycerides and glucose (TyG) index is associated with the presence of metabolically obese normal-weight (MONW) phenotype and related cardiovascular risk factors. METHODS: Apparently healthy men and non-pregnant women aged 20-65 years were enrolled in a population-based cross-sectional study. Overweight, obesity, smoking, alcohol consumption, pregnancy, diagnosis of hypertension, diabetes, cardiovascular disease, liver disease, renal disease, malignancy, and medical treatment were exclusion criteria. Subjects were allocated into the MONW or normal-weight groups. MONW phenotype was defined by normal weight and the presence of at least one of the following cardiovascular risk factors: elevated blood pressure, hyperglycemia, hypertriglyceridemia, and low HDL cholesterol. RESULTS: A total of 542 subjects were enrolled and allocated into the MONW (n = 354) and normal-weight (n = 188) groups. The adjusted logistic regression analysis showed that the elevated TyG index is significantly associated with the presence of MONW phenotype (OR = 11.14; 95% CI 6.04-20.57), hyperglycemia (OR = 3.18; 95% CI 1.95-5.21), hypertriglyceridemia (OR = 399.19; 95% CI 94.01-1694.98), and low HDL-C (OR = 2.60; 95% CI 1.74-3.87), but not with elevated blood pressure (OR = 1.55; 95% CI 0.93-2.60). CONCLUSION: Results of this study support that the TyG index may be a useful indicator to detect MONW phenotype and associated cardiovascular risk factors.

Biochemical characterization of the venom from the Mexican scorpion Centruroides ornatus, a dangerous species to humans.

Every year in Mexico, around 300,000 people suffer from accidents related to scorpion stings. Among the scorpion species dangerous to human is Centruroides ornatus, whose venom characterization is described here. From this venom, a total of 114 components were found using chromatographic separation and mass spectrometry analysis. The most abundant ones have molecular masses between 3000-4000 Da and 6000-8000 Da respectively, similar to other known K+ and Na+-channel specific scorpion peptides. Using intraperitoneal injections into CD1 mice, we were able to identify and fully sequenced three new lethal toxins. We propose to name them Co1, Co2 and Co3 toxins, which correspond to toxins 1 to 3 of the abbreviated species name (Co). Electrophysiology analysis of these peptides using heterologously expressed human Na+-channels revealed a typical ß-toxin effect. Peptide Co52 (the most abundant peptide in the venom) showed no activity in our in vivo and in vitro model assays. A phylogenetic analysis groups the Co1, Co2 and Co3 among other ß-toxins from Centruroides scorpions. Peptide Co52 segregates among peptides of unknown defined functions.

Cn29, a novel orphan peptide found in the venom of the scorpion Centruroides noxius: Structure and function.

A peptide (Cn29) from the venom of the scorpion Centruroides noxius (about 2% of the soluble venom) was purified and its primary and three-dimensional structures were determined. The peptide contains 27 amino acids with primary sequence: LCLSCRGGDYDCRVKGTCENGKCVCGS. The peptide is tightly packed by three disulfide linkages formed between C2-C23, C5-C18 and C12-C25. Since the native peptide was obtained in limited amounts, the full synthetic peptide was prepared using the standard F-moc-based solid phase synthesis method of Merrifield. The native and synthetic peptides were shown to be identical by sequencing, HPLC separation and mass spectrometry. The solution structure of the peptide solved from NMR data shows that it consists of a well-defined N-terminal region without regular secondary structure extending from Leu 1 to Asp 9, followed by a short helical fragment from Tyr10 to Val14 and two short ß strands (Thr17-Glu19 and Lys22-Val24). The primary and tertiary structures of Cn29 are different from all other scorpion peptides described in the literature. Transcriptome analysis of RNA obtained from C. noxius confirmed the expression of a gene coding for Cn29 in its venom gland. Initial experiments were conducted to identify its possible function: lethality tests in mice and insects as well as ion-channel binding using in vitro electrophysiological assays. None of the physiological or biological tests displayed any activity for this peptide, which at present is considered to be another orphan peptide found in scorpion venoms. The peptide is thus the first example of a novel structural component present in scorpion venoms.

Synthetic undecapeptide (NTX10-20) of noxiustoxin blocks completely the I(A) potassium currents of cerebellum granular cells.

Native noxiustoxin (NTX) and synthetic peptides corresponding to its primary sequence, from positions 1-9, 1-14, 1-20, 10-20, 21-39 and 30 39, were prepared and assayed on the K+ currents of cerebellum granular cells, using the patch-clamp technique in the whole-cell configuration system. Native toxin has a reversible inhibitory effect (IC50 = 360 nM), whereas synthetic peptides NTXI-20 and NTX1-9 had a half-effective dose IC50 of approximately 2 and 10 microM, respectively, which correlates with their biological effects in vivo. Synthetic peptide NTX10-20 was quite remarkable in having a preference for the IA current, which was completely inhibited at high peptide concentration. The effects of the other peptides (NTXI 14, NTX21-39 and NTX30-39), although positive and reversible, required higher concentrations (50 200 microM) to block both currents, suggesting no affinity or, at least, much lower specificity for the channels responsible for the potassium currents in the granular cells studied.

Hadrurin, a new antimicrobial peptide from the venom of the scorpion Hadrurus aztecus.

A new antimicrobial peptide, hadrurin, was isolated from the venom of the Mexican scorpion Hadrurus aztecus, by gel filtration on a Sephadex G-50 column, followed by high performance liquid chromatography. It is a basic peptide composed of 41 amino-acid residues with a molecular mass of 4436 Da, and contains no cysteines. A model of the three-dimensional folding of hadrurin is compatible with that of an amphipatic molecule with two alpha-helical segments. Hadrurin demonstrates antimicrobial activity at low micromolar concentration, inhibiting the growth of bacteria such as: Salmonella thyphi, Klebsiella pneumoniae, Enterococcus cloacae, Pseudomonas aeruginosa, Escherichia coli and Serratia marscences. It also shows cytolytic activity when tested in human erythrocytes. Hadrurin and two analogs (C-terminal amidated, and all D-enantiomer) were chemically synthesized. They were used to study the possible molecular mechanism of action by testing their ability to dissipate the diffusion potential of liposomes of different compositions. The results obtained indicate that there are no specific receptor molecules for the action of hadrurin, and the most probable mechanism is through a membrane destabilization activity. It is surmised that hadrurin is used by the scorpion as both an attack and defense element against its prey and putative invasive microorganisms. It is a unique peptide among all known antimicrobial peptides described, only partially similar to the N-terminal segment of gaegurin 4 and brevinin 2e, isolated from frog skin. It would certainly be a model molecule for studying new antibiotic activities and peptide-lipid interactions.

Conversion of an inactive cardiac dihydropyridine receptor II-III loop segment into forms that activate skeletal ryanodine receptors.

A 25 amino acid segment (Glu666-Pro691) of the II-III loop of the alpha1 subunit of the skeletal dihydropyridine receptor, but not the corresponding cardiac segment (Asp788-Pro814), activates skeletal ryanodine receptors. To identify the structural domains responsible for activation of skeletal ryanodine receptors, we systematically replaced amino acids of the cardiac II-III loop with their skeletal counterparts. A cluster of five basic residues of the skeletal II-III loop (681RKRRK685) was indispensable for activation of skeletal ryanodine receptors. In the cardiac segment, a negatively charged residue (Glu804) appears to diminish the electrostatic potential created by this basic cluster. In addition, Glu800 in the group of negatively charged residues 798EEEEE802 of the cardiac II-III loop may serve to prevent the binding of the activation domain.

A toxin to nervous, cardiac, and endocrine ERG K+ channels isolated from Centruroides noxius scorpion venom.

Toxins isolated from a variety of venoms are tools for probing the physiological function and structure of ion channels. The ether-a-go-go-related genes (erg) codify for the K+ channels (ERG), which are crucial in neurons and are impaired in human long-QT syndrome and Drosophila 'seizure' mutants. We have isolated a peptide from the scorpion Centruroides noxius Hoffmann that has no sequence homologies with other toxins, and demonstrate that it specifically inhibits (IC50=16+/-1 nM) only ERG channels of different species and distinct histogenesis. These results open up the possibility of investigating ERG channel structure-function relationships and novel pharmacological tools with potential therapeutic efficacy.

Site directed mutants of Noxiustoxin reveal specific interactions with potassium channels.

Several site directed mutations were introduced into a synthetic Noxiustoxin (NTX) gene. Alanine scanning of the nonapeptide at the N-terminal segment of NTX (threonine 1 (T1) to serine 9 (S9)) was constructed and the recombinant products were obtained in pure form. Additionally, lysine 28 (K28) was changed to arginine (R) or glutamic acid (E), cysteine 29 was changed to alanine, and residues 37-39 (Tyr-Asn-Asn) of the carboxyl end were deleted. The recombinant mutants were tested for their ability to displace 125I-NTX from rat brain synaptosome membranes, as well as for their efficiency in blocking the activity of Kv1.1 K+ channels expressed in Xenopus laevis oocytes. The main results indicate that residues K6, T8 at the amino end, and K28 and the tripeptide YNN at the carboxyl end are involved in specific interactions of NTX with rat brain and/or Kv1.1 K+ channels.

Two similar peptides from the venom of the scorpion Pandinus imperator, one highly effective blocker and the other inactive on K+ channels.

Two novel peptides, named Pi4 and Pi7, were purified from the venom of the scorpion Pandinus imperator, and their primary structures were determined. These peptides have 38 amino acids residues, compacted by four disulfide bridges, instead of the normal three found in most K+-channel specific toxins. Both peptides contain 25 identical amino acid residues in equivalent positions (about 66% identity), including all eight half-cystines. Despite the fact that their C-terminal sequence comprising amino acid residues 27 to 37 are highly conserved (10 out of 11 amino acids are identical), Pi4 blocks completely and reversibly Shaker B K+ -channels (a Kv1.1 sub-family type of channel) at 100nM concentration, whereas Pi7 is absolutely inactive at this concentration. Similar effects were observed in binding and displacement experiments to rat brain synaptosomal membranes using 125I-Noxiustoxin, a well known K+-channel specific toxin. In this preparation Pi4 displaces the binding of radiolabeled Noxiustoxin with Ic50 in the order of 10 nM, whereas Pi7 is ineffective at same concentration. Comparative analysis of Pi4 and Pi7 sequences with those obtained by site directed mutagenesis of Charybdotoxin, another very well studied K -channel blocking toxin, shows that the substitution of lysine (in Pi4) for arginine (in Pi7) at position 26, might be one of the important 'point mutations' responsible for such impressive variation in blocking properties of both toxins, here described.

Synthesis and expression of the gene coding for noxiustoxin, a K+ channel-blocking peptide from the venom of the scorpion Centruroides noxius.

A set of six synthetic overlapping oligonucleotides coding for noxiustoxin were coupled into a continuous DNA fragment by means of recursive polymerase chain reaction. The polymerase chain reaction product was digested with SalI and HindIII, ligated into the E, coli vector pCSP 105 and expressed as a fusion protein. The fusion protein was purified and digested with trypsin and the hydrolysis products were separated by high-performance liquid chromatography. Approximately 1.3 mg of recombinant noxiustoxin per liter of culture was obtained. Amino acid analysis and N-terminal amino acid sequence of the recombinant noxiustoxin confirmed the nucleotide sequence of the cloned DNA. Binding experiments using rat brain synaptosomal membranes revealed that recombinant noxiustoxin displaced bound radioactive native NTX with a similar efficiency to cold native noxiustoxin.

A novel structural class of K+-channel blocking toxin from the scorpion Pandinus imperator.

A novel peptide was purified and characterized from the venom of the scorpion Pandinus imperator. Analysis of its primary structure reveals that it belongs to a new structural class of K+-channel blocking peptide, composed of only 35 amino acids, but cross-linked by four disulphide bridges. It is 40, 43 and 46% identical to noxiustoxin, margatoxin and toxin 1 of Centruroides limpidus respectively. However, it is less similar (26 to 37% identity) to toxins from scorpions of the geni Leiurus, Androctonus and Buthus. The disulphide pairing was determined by sequencing heterodimers produced by mild enzymic hydrolysis. They are formed between Cys-4-Cys-25, Cys-10-Cys-30, Cys-14-Cys-32 and Cys-20-Cys-35. Three-dimensional modelling, using the parameters determined for charybdotoxin, showed that is it possible to accommodate the four disulphide bridges in the same general structure of the other K+-channel blocking peptides. The new peptide (Pil) blocks Shaker B K+ channels reversibly. It also displaces the binding of a known K+-channel blocker, [125I]noxiustoxin, from rat brain synaptosomal membranes with an IC50 of about 10 nM.

Novel K(+)-channel-blocking toxins from the venom of the scorpion Centruroides limpidus limpidus Karsch.

Two novel toxins were purified from the venom of the Mexican scorpion Centruroides limpidus limpidus, using an immunoassay based on antibodies raised against noxiustoxin (NTX), a known K(+)-channel-blocker-peptide. The primary structure of C. l. limpidus toxin 1 was obtained by Edman degradation and was shown to be composed of 38 amino acid residues, containing six half-cystines. The first 36 residues of C. l. limpidus toxin 2 were also determined. Both toxins are capable of displacing the binding of radio-labelled NTX to rat brain synaptosomes with high affinity (about 100 pM). These toxins are capable of inhibiting transient K(+)-currents (resembling IA-type currents), in cultured rat cerebellar granule cells. About 50% of the peak currents are reduced by application of a 1.5 microM solution of toxins 1 and 2 The K+ current reduction is partially reversible, under washing but not voltage-dependent. Comparison of the primary structure of C. l. limpidus toxin 1 with other known toxins shows 74% identity with margatoxin, 64% with NTX, 51% with kaliotoxin, 39% with iberiotoxin, 37% with charybdotoxin and Lq2, and 29% with leirutoxin 1. The only invariant amino acids in all these toxins are the six cysteines, a glycine in position 26 and two lysines at positions 28 and 33, respectively. The relevance of these differences in terms of possible structure-function relationships is discussed.

The disulfide bridges of toxin 2 from the scorpion Centruroides noxius Hoffmann and its three-dimensional structure calculated using the coordinates of variant 3 from Centruroides sculpturatus.

The disulfide bridges of toxin 2 from the venom of the scorpion Centruroides noxius Hoffmann were found by amino acid sequence determination of fragments of native toxin, produced by enzymatic cleavage and separated by high-performance liquid chromatography (HPLC). They are: Cys12-Cys65, Cys16-Cys41, Cys25-Cys46 and Cys29-Cys48. The coordinates of the X-ray diffraction structure of toxin variant 3 of C. sculpturatus [(1980) Proc. Natl. Acad. Sci. USA 77, 6496-6500] were used to construct a three-dimensional model of toxin 2. All the amino acid replacements were easily accommodated, and the modeled structure reveals a clustered pattern of sequence variation, which may help to identify residues responsible for functional differences among toxins of mammals and insects.

Isolation and characterization of a novel toxin from the venom of the scorpion Centruroides limpidus limpidus Karsch.

A novel peptide, toxic to mice, was purified from the venom of the Mexican scorpion Centruroides limpidus limpidus, by means of gel filtration and ion exchange chromatography, followed by high performance liquid chromatography (HPLC). The complete amino acid sequence was determined by automatic Edman degradation of reduced and alkylated toxin, and by overlapping sequences of fragments of the toxin, generated by cleavage with proteinase V8 separated by HPLC. This toxin is composed of 66 amino acid residues, contains eight half-cystine residues, and is highly similar (91%) to the amino acid sequence deduced for toxin 1 of C. limpidus tecomanus and toxin 4 from C. noxius venom (89%). This peptide displaces the binding of radiolabeled toxin 2 of C. noxius from synaptosomal membranes of rat brain with superimposable kinetics, supporting the conclusion that it belongs to the beta-scorpion toxin class. Further characterization of C. l. limpidus toxin 1, as we have named it, was performed by means of competition experiments with monoclonal antibodies and various purified scorpion toxins, using an ELISA assay. A panel of six distinct monoclonal antibodies (mAB) against toxin 2 and 3 of C. noxius was used. From these, only three clones, originally named BCF1, BCF8 and BCF9, were able to recognize toxin 1 from C. l. limpidus.

Blockade of a KCa channel with synthetic peptides from noxiustoxin: a K+ channel blocker.

Using the outside-out configuration of the patch-clamp method, we studied the effect of several synthetic peptides corresponding to various segments from the N-terminal region of noxiustoxin (NTX) on single Ca(2+)-activated K+ (KCa) channels of small conductance obtained from cultured bovine aortic endothelial cells. These peptides induced diverse degrees of fast blockade in the endothelial KCa channel. The most effective blockers were the peptides NTX1-39 (IC50 = 0.5 microM) and NTX1-20 comprising the first 20 amino acids from the native toxin (IC50 approximately 5 microM), while less effective was the hexapeptide NTX1-6, from the first six amino acid residues of NTX (IC50 = 500 microM). This was the minimum sequence required to block the channel. By testing overlapping sequences from the entire molecule, specially those corresponding to the N-terminal region of NTX, we have been able to determine their different apparent affinities for the KCa channel. Synthetic peptides from the C-terminal region produced no effect on the KCa channel at the concentrations tested (up to 1 mM). These results confirm that in the N-terminal region of the NTX is located part of the sequence that may recognize K+ channels, as we have suggested previously from in vivo experiments. The blockade induced by native NTX was poorly affected by changes in membrane potential; however, the blockage induced by synthetic peptides lacking the C-terminal region was partially released by depolarization.

Neurotoxins that act selectively on voltage-dependent cardiac calcium channels.

We searched for and found a toxin that acts specifically on cardiac calcium channels. This toxin, which we have designated TaiCatoxin (TCX), was purified from the venom of the Australian Taipan snake (Oxyuranus s. scutellatus), one of the world's most poisonous snakes. TCX is a highly charged, basic polypeptide with a molecular weight of 8,000. It blocks high- but not low-threshold cardiac calcium channels in a voltage-dependent manner and has no effect on potassium or sodium channels. The block occurs at nanomolar concentrations, is reversible, and is due to binding at an extracellularly facing site on the channel itself.