RESUMEN
BACKGROUND: High levels of albumin in some biological fluids are generally associated with abnormal process of permeation during an inflammatory response. In some cases, the nasal albumin levels can be used as an indirect molecular marker of epithelial damage. METHODS: We carried out an evaluation study of nasal albumin from a population (14 volunteers), exposed for 15 days to high urban pollution (O(3) 10.644 ppm/h average 10.2 h/day) in Mexico City for the first time, and when they returned to their original non-polluted city (Veracruz, State of Veracruz, Mexico) 13 days later. The nasal albumin samples were fractionated by gel electrophoresis techniques, and albumin determination analyses were done by gel scanning. The densitometry values obtained from the albumin-stained bands were compared with an internal standard and the average values compared with other groups used as reference, under conditions of non-polluted and polluted cities. RESULTS: Our findings showed in the group exposed to pollution for the first time a significant increment 2 days after entering Mexico City urban pollution. They, subsequently, had a progressive recovery 4 days later up to day 13 in their original place of residence. The reference groups for comparison were from a non-polluted city (at sea level on the Pacific Ocean) Manzanillo, Colima, Mexico, and a very polluted urban metropolis (Mexico City). The group showed a significant difference of protein levels about 2.5 higher apparently at the expense of albumin. These populations were permanent residents of environmental conditions we wanted to evaluate. Our approach was to monitor quantitatively the time course of the change of a biochemical parameter in normal mucus from a population never exposed to Mexico City urban pollution. CONCLUSIONS: Our conclusions from this study point out that pollution causes diverse mucosal damage that can be followed by biochemical monitoring.
Asunto(s)
Contaminación del Aire/efectos adversos , Albúminas/metabolismo , Exposición por Inhalación/efectos adversos , Mucosa Nasal/metabolismo , Oxidantes Fotoquímicos/efectos adversos , Ozono/efectos adversos , Adulto , Biomarcadores , Humanos , Población UrbanaRESUMEN
Calmodulin, bound to the alpha(1) subunit of the cardiac L-type calcium channel, is required for calcium-dependent inactivation of this channel. Several laboratories have suggested that the site of interaction of calmodulin with the channel is an IQ-like motif in the carboxyl-terminal region of the alpha(1) subunit. Mutations in this IQ motif are linked to L-type Ca(2+) current (I(Ca)) facilitation and inactivation. IQ peptides from L, P/Q, N, and R channels all bind Ca(2+)calmodulin but not Ca(2+)-free calmodulin. Another peptide representing a carboxyl-terminal sequence found only in L-type channels (designated the CB domain) binds Ca(2+)calmodulin and enhances Ca(2+)-dependent I(Ca) facilitation in cardiac myocytes, suggesting the CB domain is functionally important. Calmodulin blocks the binding of an antibody specific for the CB sequence to the skeletal muscle L-type Ca(2+) channel, suggesting that this is a calmodulin binding site on the intact protein. The binding of the IQ and CB peptides to calmodulin appears to be competitive, signifying that the two sequences represent either independent or alternative binding sites for calmodulin rather than both sequences contributing to a single binding site.
Asunto(s)
Canales de Calcio Tipo L/química , Canales de Calcio Tipo L/metabolismo , Calmodulina/metabolismo , Secuencia de Aminoácidos , Animales , Sitios de Unión , Western Blotting , Calcio/metabolismo , Bovinos , Células Cultivadas , Relación Dosis-Respuesta a Droga , Electroforesis en Gel de Poliacrilamida , Electrofisiología , Ensayo de Inmunoadsorción Enzimática , Datos de Secuencia Molecular , Músculo Esquelético/metabolismo , Mutación , Miocardio/citología , Péptidos/química , Péptidos/metabolismo , Unión Proteica , Estructura Terciaria de Proteína , Conejos , Homología de Secuencia de Aminoácido , Espectrometría de FluorescenciaRESUMEN
Helothermine, a protein from the venom of the Mexican beaded lizard (Heloderma horridum horridum), was found to inhibit [3H]ryanodine binding to cardiac and skeletal sarcoplasmic reticulum, to block cardiac and skeletal ryanodine receptor channels incorporated into planar bilayers, and to block Ca(2+)-induced Ca2+ release triggered by photolysis of nitr-5 in saponin-permeabilized trabeculae from rat ventricle. Cloning of the helothermine cDNA revealed that the protein is composed of 223 amino acids with a molecular mass of 25,376 daltons, and apparently is stabilized by eight disulfide bridges. The peptide sequence showed significant homology with a family of cysteine-rich secretory proteins found in the male genital tract and in salivary glands. The interaction of helothermine and ryanodine receptors should serve to define functional domains within the channel structure involved in the control of Ca2+ release from sarcoplasmic reticulum.
Asunto(s)
Bloqueadores de los Canales de Calcio/química , Canales de Calcio/fisiología , Proteínas Musculares/fisiología , Péptidos/química , Péptidos/farmacología , Secuencia de Aminoácidos , Animales , Secuencia de Bases , Unión Competitiva , Bloqueadores de los Canales de Calcio/farmacología , Canales de Calcio/efectos de los fármacos , Clonación Molecular , ADN Complementario , Membrana Dobles de Lípidos , Lagartos , Potenciales de la Membrana/efectos de los fármacos , Microsomas/metabolismo , Datos de Secuencia Molecular , Proteínas Musculares/efectos de los fármacos , Músculo Esquelético/metabolismo , Miocardio/metabolismo , Sondas de Oligonucleótidos , Biosíntesis de Péptidos , Conejos , Proteínas Recombinantes/biosíntesis , Proteínas Recombinantes/química , Proteínas Recombinantes/farmacología , Rianodina/metabolismo , Canal Liberador de Calcio Receptor de Rianodina , Retículo Sarcoplasmático/metabolismo , Homología de Secuencia de Aminoácido , Porcinos , Toxinas Biológicas/química , Toxinas Biológicas/farmacologíaRESUMEN
Helothermine, a recently isolated toxin from the venom of the Mexican beaded lizard Heloderma horridum horridum was tested on K+ currents of newborn rat cerebellar granule cells. In whole-cell voltage-clamp experiments, cerebellar granule neurons exhibited at least two different K+ current components: a first transient component which is similar to an IA-type current, is characterized by fast activating and inactivating kinetics and blocked by 4-aminopyridine; a second component which is characterized by noninactivating kinetics, is blocked by tetraetylammonium ions and resembles the classical delayed-rectifier current. When added to the standard external solution at concentrations ranging between 0.1 and 2 microM, helothermine reduced the pharmacologically isolated IA-type current component in a voltage- and dose-dependent way, with a half-maximal inhibitory concentration (IC50) of 0.52 microM. A comparison between control and helothermine-modified peak transient currents shows a slowdown of activation and inactivation kinetics. The delayed-rectifier component inhibition was concentration dependent (IC50 = 0.86 microM) but not voltage dependent. No frequency- or use-dependent block was observed on both K+ current types. Perfusing the cells with control solution resulted in quite a complete current recovery. We conclude that helothermine acts with different affinities on two types of K+ current present in central nervous system neurons.
Asunto(s)
Cerebelo/citología , Péptidos/farmacología , Canales de Potasio/fisiología , Ponzoñas/farmacología , 4-Aminopiridina/farmacología , Animales , Membrana Celular/fisiología , Membrana Celular/ultraestructura , Cerebelo/fisiología , Cerebelo/ultraestructura , Relación Dosis-Respuesta a Droga , Lagartos , Neuronas/fisiología , Neuronas/ultraestructura , Canales de Potasio/efectos de los fármacos , Ratas , Ratas Wistar , Compuestos de Tetraetilamonio/farmacologíaRESUMEN
Taicatoxin is a new complex oligomeric toxin that was isolated from the venom of the Australian taipan snake Oxyuranus scutellatus scutellatus. It is composed of three different molecular entities: an alpha-neurotoxin-like peptide of mol. wt 8000, a neurotoxic phospholipase of mol. wt of 16,000 and a serine protease inhibitor of mol. wt 7000, linked by non-covalent bonds, at an approximate stoichiometry of 1:1:4. The most active form of the complex was isolated by ion exchange chromatography through DE-Cellulose followed by two steps of CM-Cellulose chromatography at pH 4.7 and pH 6.0, respectively. At this stage the complex migrates as a single component in beta-alanine-acetate-urea gel electrophoresis and is very toxic to mice (1 or 2 micrograms of the complex protein kills a mouse of 20 g within 2 hr). It blocks the high threshold calcium channel current of excitable membranes in heart and does not affect the low threshold calcium channel current. The block occurs at a site that is accessible extracellularly but not intracellularly. The block is selective for calcium channels, reversible, does not affect single channel conductance but only changes channel gating, and is voltage dependent with higher affinity for inactivated channels. The phospholipase activity of the complex toxin can be separated by affinity-chromatography using a phospholipid analog (PC-Sepharose). The resulting complex contains only alpha-neurotoxin and protease inhibitor and is still capable of blocking calcium channels, although with less potency than the native oligomeric form. Sephadex G-50 gel filtration chromatography in the presence of high salt (1M NaCl) at alkaline pH (8.2), separates the alpha-neurotoxin-like peptide from the protease inhibitor, but at this stage the resulting peptides lose physiological activity towards the calcium channels. The amino acid sequence of the protease inhibitor was determined by automatic Edman degradation. The alpha-neurotoxin-like peptide and two isosubunits displaying phospholipase activity were sequenced at the N-terminal part of the molecule.
Asunto(s)
Bloqueadores de los Canales de Calcio/farmacología , Venenos Elapídicos/análisis , Venenos Elapídicos/farmacología , Alquilación , Secuencia de Aminoácidos , Animales , Bloqueadores de los Canales de Calcio/toxicidad , Cromatografía en Gel , Cromatografía por Intercambio Iónico , Venenos Elapídicos/enzimología , Venenos Elapídicos/aislamiento & purificación , Venenos Elapídicos/toxicidad , Electroforesis en Gel de Poliacrilamida , Endopeptidasas/metabolismo , Cobayas , Corazón/efectos de los fármacos , Hidrólisis , Técnicas In Vitro , Potenciales de la Membrana/efectos de los fármacos , Ratones , Ratones Endogámicos , Datos de Secuencia Molecular , Peso Molecular , Fosfolipasas/metabolismo , RatasRESUMEN
A neurotoxic phospholipase A2 was purified from the venom of the taipan snake Oxyuranus scutellatus scutellatus by three consecutive chromatographic steps on ion exchange resins, followed by an affinity column prepared with a phosphatidylcholine derivative attached to Sepharose. The phospholipase was shown to be of type A2 (specific activity of 85 units/mg protein), and an apparent molecular weight of 16,000. Amino acid analysis shows the presence of approx. 150 residues with the N-terminal amino acid sequence: NLAQFGFMIRCANGGSRSALDYADYGC, different from all the phospholipases described until now. This enzyme is lethal to experimental mice (LD50 = 10 micrograms/20 g mouse weight) and affects ionic currents in chick (Gallus domesticus) dorsal root ganglion cells, measured by the whole-cell clamp technique. In symmetrical external/internal ionic solutions, after suppression of Na+, K+ and Ca2+ currents, external application of phospholipase at a low concentration (30 nM) was shown to increase the baseline current in a reversible manner. The augmented response was voltage-dependent and the effect was much greater for negative currents. In the presence of a salt gradient across the membrane (out 40 mM NaCl/in 140 mM CsCl), the current reversal potential revealed a shift in the positive direction typically due to Cl- ion flux through the membrane. External application of a 50 microM concentration of picrotoxin caused a reversible reduction of the phospholipase-induced chloride current. Moreover, no appreciable current block was detected after addition of 50 microM DIDS.
Asunto(s)
Venenos Elapídicos/farmacología , Canales Iónicos/metabolismo , Neuronas Aferentes/fisiología , Fosfolipasas A/farmacología , Secuencia de Aminoácidos , Animales , Aniones/metabolismo , Células Cultivadas , Embrión de Pollo , Canales de Cloruro , Venenos Elapídicos/química , Venenos Elapídicos/aislamiento & purificación , Conductividad Eléctrica , Ganglios Espinales/citología , Ganglios Espinales/fisiología , Proteínas de la Membrana/metabolismo , Datos de Secuencia Molecular , Fosfolipasas A/química , Fosfolipasas A/aislamiento & purificación , Fosfolipasas A2 , Proteínas de ReptilesRESUMEN
A protein toxic to mice was isolated from the venom of the Mexican beaded lizard Heloderma horridum horridum by a combination of gel filtration (Sephadex G-75) and ion exchange chromatography (both diethylaminoethyl-cellulose [DE-cellulose] and carboxymethyl-cellulose [CM-cellulose]). The purified polypeptide component has an apparent mol. wt of 25,500 and is composed of approximately 220 amino acid residues. It has an isoelectric point (pI) of 6.8 and its N-terminal amino acid sequence was shown to be: Glu-Ala-Ser-Pro-Lys-Leu-Pro-Gly-Leu-Met-Thr-Ser-Asn-Pro-Asp-Gln-Gln-Thr- Glu-Ile. The sequence has no significant similarity with any other protein previously reported in the literature. Enzymatic activities such as phospholipase, hyaluronidase and proteinase, commonly present in venoms, could not be demonstrated in this protein. Patch-clamp experiments conducted with excitable membranes show no effects on Na+, K+ or Ca2+ ion channels. Among the constant physiological effects observed in mice injected with this toxin are lethargy, partial paralysis of rear limbs and lowering of body temperature, suggesting that it might be a hypothermic toxin. We propose calling this toxin Helothermine.