RESUMEN
Background: Skin, and epidermis, is innervated by sensory nerve fibres. Interactions between them and signal transduction are only partially elucidated in physiological/pathological conditions, especially in pruritus. Objectives: To study the mechanisms involved in pruritus in vitro, we developed a skin explant model re-innervated by sensory neurons. Methods: This model is based on the co-culture of human skin explants and sensory neurons from dorsal root ganglia of rats. Innervation and the expression of protease activated receptor 2 (PAR2), transient receptor potential vanilloid 1 (TRPV1) and transient receptor potential ankyrin one (TRPA1) was analysed by immunostaining. The response of the model to TRPV1, PAR2 and TRPA1 agonists was analysed by patch-clamp, qPCR and enzyme-linked immunosorbent assay. Results: After 5 days of re-innervating nerve fibres was evidenced in the epidermis. Re-innervation was correlated with decrease of epidermal thickness and the number of apoptotic cells in the tissue. The major actors of non-histaminergic itch (PAR-2, thymic stromal lymphopoietin [TSLP], TSLP-R, TRPA1 and TRPV1) were expressed in neurons and/or epidermal cells of skin explants. After topical exposure of TRPV1-(Capsaicin), TRPA1-(Polygodial) and PAR2-agonist (SLIGKV-NH2) activation of reinnervating neurons could be shown in patch-clamp analysis. The release of TSLP was increased with capsaicin or SLIGKV but decreased with polygodial. Release of CGRP was increased by capsaicin and polygodial but decreased with SLIGKV. Activation by SLIGKV showed a decrease of VEGF; polygodial induced an increase of TSLP, Tumour necrosis factor (TNF) and nerve growth factor and capsaicin lead to a decrease of sema3 and TNF expression. Conclusion: The present model is suitable for studying itch and neurogenic inflammation pathways in vitro. We observed that activation of TRPV1, TRPA1 and PAR-2 leads to different response profiles in re-innervated skin explants.
RESUMEN
Myocardial depression, frequently observed in septic shock, is mediated by circulating molecules such as cytokines. TNF-α appears to be the most important pro-inflammatory cytokine released during the early phase of a septic shock. It was previously shown that TNF-α had a negative inotropic effect on myocardium. Now, the aim of this study was to investigate the effects of the activation of PKC by TNF-α on heart function, and to determine if this cytokine could induce a decrease of membrane excitability. Isolated rat hearts (nâ¯=â¯6) were perfused with Tyrode solution containing TNF-α at 20â¯ng/ml during 30â¯min by using a Langendorff technique. Expressions of PKC-α and PKC-ε were analysed by western blot on membrane and cytosol proteins extracted from ventricular myocardium. Patch clamp was performed on freshly isolated cardiomyocytes (nâ¯=â¯8). Compared to control situation, 30â¯min of TNF-α perfusion led to cardiac dysfunction with a decrease of the heart rate (-83%), the force (-20%) and speed of relaxation (-18%) and the coronary flow (-25%). This is associated with an activation and a membrane targeting of both PKC-α and PKC-ε isoforms in ventricle with respectively +123% and +54% compared to control hearts. Nevertheless, TNF-α had no significant effect on voltage-gated sodium current (109.0%+/- 12.5) after addition of the cytokine when compared to control. These results showed that TNF-α had a negative inotropic effect on the isolated rat heart and can induce PKC activation leading to an impaired contractility of the heart. However the early heart dysfunction induced by the cytokine was not associated to a decrease of cardiomyocytes membrane excitability as it has been evidenced in skeletal muscle fibres.
Asunto(s)
Corazón/efectos de los fármacos , Contracción Miocárdica/efectos de los fármacos , Miocardio/enzimología , Proteína Quinasa C-alfa/metabolismo , Proteína Quinasa C-epsilon/metabolismo , Factor de Necrosis Tumoral alfa/farmacología , Animales , Activación Enzimática/efectos de los fármacos , Femenino , Corazón/fisiología , Técnicas In Vitro , Contracción Miocárdica/fisiología , Miocitos Cardíacos/efectos de los fármacos , Miocitos Cardíacos/enzimología , Miocitos Cardíacos/fisiología , Técnicas de Placa-Clamp , Perfusión , Ratas WistarRESUMEN
ICU acquired neuromyopathy (IANM) is the most frequent neurological pathology observed in ICU. Nerve and muscle defects are merged with neuromuscular junction abnormalities. Its physiopathology is complex. The aim is probably the redistribution of nutriments and metabolism towards defense against sepsis. The main risk factors are sepsis, its severity and its duration of evolution. IANM is usually diagnosed in view of difficulties in weaning from mechanical ventilation, but electrophysiology may allow an earlier diagnosis. There is no curative therapy, but early treatment of sepsis, glycemic control as well as early physiotherapy may decrease its incidence. The outcomes of IANM are an increase in morbi-mortality and possibly long-lasting neuromuscular abnormalities as far as tetraplegia.
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Cuidados Críticos , Enfermedades Neuromusculares/diagnóstico , Enfermedades Neuromusculares/etiología , Enfermedades Neuromusculares/terapia , Atrofia , Glucemia/metabolismo , Diagnóstico Diferencial , Humanos , Incidencia , Enfermedades Neuromusculares/epidemiología , Enfermedades Neuromusculares/fisiopatología , Unión Neuromuscular/fisiología , Estrés Oxidativo , Pronóstico , Cuadriplejía/etiología , Factores de Riesgo , Sepsis/complicaciones , Sepsis/prevención & control , Desconexión del VentiladorRESUMEN
During muscle contraction, lactate production and translocation across the membrane increase. While it has recently been shown that lactate anion acts on chloride channel, less is known regarding a potential effect on the voltage-gated sodium channel (Na(v)) of skeletal muscle. The electrophysiological properties of muscle Na(v) were studied in the absence and presence of lactate (10 mM) by using the macropatch-clamp method in dissociated fibers from rat peroneus longus (PL). Lactate in the external medium (petri dish + pipette) increases the maximal sodium current, while the voltage dependence of activation and fast inactivation are shifted toward the hyperpolarized potentials. Lactate induces a leftward shift in the relationship between the kinetic parameters and the imposed potentials, resulting in an earlier recruitment of muscle Na(v). In addition, lactate significantly decreases the time constant of activation at voltages more negative than -10 mV, corresponding to an acceleration of Na(v) activation. The slow inactivation process is decreased by lactate, corresponding to an enhancement in the number of excitable Na(v). In an additional series of experiments, lactate (10 mM) was only added to the petri dish, while the pipette remained sealed on the membrane area. With this approach, the electrophysiological properties of Na(v) were unaffected by lactate compared with the control condition. Altogether, these data indicate that lactate modulates muscle Na(v) properties by an extracellular pathway. These effects are consistent with an enhancement in excitability, providing new insights into the role of lactate in muscle physiology.
Asunto(s)
Activación del Canal Iónico , Ácido Láctico/metabolismo , Contracción Muscular , Músculo Esquelético/metabolismo , Canales de Sodio/metabolismo , Sodio/metabolismo , Animales , Femenino , Cinética , Potenciales de la Membrana , Técnicas de Placa-Clamp , Ratas , Ratas WistarRESUMEN
Heart cells from the clam Ruditapes decussatus were routinely cultured with a high level of reproducibility in sea water based medium. Three cell types attached to the plastic after 2 days and could be maintained in vitro for at least 1 month: epithelial-like cells, round cells and fibroblastic cells. Fibroblastic cells were identified as functional cardiomyocytes due to their spontaneous beating, their ultrastructural characteristics and their reactivity with antibodies against sarcomeric α-actinin, sarcomeric tropomyosin, myosin and troponin T-C. Patch clamp measurements allowed the identification of ionic currents characteristic of cardiomyocytes: a delayed potassium current (I (K slow)) strongly suppressed (95%) by tetraethylammonium (1 mM), a fast inactivating potassium current (I (K fast)) inhibited (50%) by 4 amino-pyridine at 1 mM and, at a lower level (34%) by TEA, a calcium dependent potassium current (I (KCa)) activated by strong depolarization. Three inward voltage activated currents were also characterized in some cardiomyocytes: L-type calcium current (I (Ca)) inhibited by verapamil at 5 × 10(-4) M, T-type Ca(2+) current, rapidly activated and inactivated, and sodium current (I (Na)) observed in only a few cells after strong hyperpolarization. These two currents did not seem to be physiologically essential in the initiation of the beatings of cardiomyocytes. Potassium currents were partially inhibited by tributyltin (TBT) (1 µM) but not by okadaic acid (two marine pollutants). DNA synthesis was also demonstrated in few cultured cells using BrdU (bromo-2'-deoxyuridine). Observed effects of okadaic acid and TBT demonstrated that cultured heart cells from clam Ruditapes decussatus can be used as an experimental model in marine toxicology.
RESUMEN
This study analyzes changes in the distribution, electrophysiological properties, and proteic composition of voltage-gated sodium channels (Na(V)) in cultured adult rat skeletal muscle fibers. Patch clamp and molecular biology techniques were carried out in flexor digitorum brevis (FDB) adult rat skeletal muscle fibers maintained in vitro after cell dissociation with collagenase. After 4 days of culture, an increase of the Na(V)1.5 channel type was observed. This was confirmed by an increase in TTX-resistant channels and by Western blot test. These channels exhibited increased activation time constant (tau(m)) and reduced conductance, similar to what has been observed in denervated muscles in vivo, where the density of Na(V)1.5 was increasing progressively after denervation. By real-time polymerase chain reaction, we found that the expression of beta subunits was also modified, but only after 7 days of culture: increase in beta(1) without beta(4) modifications. beta(1) subunit is known to induce a negative shift of the inactivation curve, thus reducing current amplitude and duration. At day 7, tau(h) was back to normal and tau(m) still increased, in agreement with a decrease in sodium current and conductance at day 4 and normalization at day 7. Our model is a useful tool to study the effects of denervation in adult muscle fibers in vitro and the expression of sodium channels. Our data evidenced an increase in Na(V)1.5 channels and the involvement of beta subunits in the regulation of sodium current and fiber excitability.
Asunto(s)
Fibras Musculares Esqueléticas/metabolismo , Canales de Sodio/metabolismo , Animales , Femenino , Inmunohistoquímica , Técnicas In Vitro , Canal de Sodio Activado por Voltaje NAV1.5 , Técnicas de Placa-Clamp , Ratas , Ratas Wistar , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Canales de Sodio/genéticaRESUMEN
BACKGROUND: Tacrolimus is an immunosuppressant drug currently used for the treatment of atopic dermatitis and pruritus. This topical therapy is effective and safe, but transient burning, stinging and itch are frequently reported. OBJECTIVES: To understand the mechanisms underlying these burning sensations. METHODS: We examined the impact of tacrolimus on substance P (SP) release in an in vitro model of cutaneous neurogenic inflammation. Because phosphorylation of TRPV1 (transient receptor potential subtype vanilloid 1) plays a role in the induction of pain, we investigated whether tacrolimus regulates the phosphorylation state of TRPV1. Finally, we used a macropatch to evaluate the impact of tacrolimus on voltage-gated calcium currents of sensory neurons. RESULTS: Tacrolimus was able to induce initial SP release by extracellular calcium influx and inhibited SP release induced by capsaicin after 1, 24 and 72 h of pretreatment. Analysis of TRPV1 phosphorylation by Western blot confirmed the capacity of tacrolimus to favour phosphorylation. An electrophysiological study showed inhibitory effects on calcium currents. CONCLUSIONS: The efficacy of tacrolimus in pruritus, as well as the sensory side-effects, could be explained by a direct effect on neurons through an effect on calcineurin, possibly by a desensitization of TRPV1 and calcium currents through the PIP(2) regulation pathway.
Asunto(s)
Inmunosupresores/farmacología , Células Receptoras Sensoriales/efectos de los fármacos , Piel/efectos de los fármacos , Tacrolimus/farmacología , Animales , Calcineurina/metabolismo , Canales de Calcio/efectos de los fármacos , Capsaicina/metabolismo , Inmunosupresores/efectos adversos , Masculino , Modelos Animales , Fosforilación/efectos de los fármacos , Piel/inervación , Sustancia P/metabolismo , Porcinos , Canales Catiónicos TRPV/metabolismo , Tacrolimus/efectos adversosRESUMEN
The myosin heavy chain (MHC) isoform determines the characteristics and shortening velocity of muscle fibres. The functional properties of the muscle fibre are also conditioned by its membrane excitability through the electrophysiological properties of sodium voltage-gated channels. Macropatch-clamp is used to study sodium channels in fibres from peroneus longus (PL) and soleus (Sol) muscles (Wistar rats, n = 8). After patch-clamp recordings, single fibres are identified by SDS-PAGE electrophoresis according to their myosin heavy chain isoform (slow type I and the three fast types IIa, IIx, IIb). Characteristics of sodium currents are compared (Student's t test) between fibres exhibiting only one MHC isoform. Four MHC isoforms are identified in PL and only type I in Sol single fibres. In PL, maximal sodium current (I(max)), maximal sodium conductance (g(Na,max)) and time constants of activation and inactivation ((m) and (h)) increase according to the scheme I-->IIa-->IIx-->IIb (P < 0.05). (m) values related to sodium channel type and/or function, are similar in Sol I and PL IIb fibres (P = 0.97) despite different contractile properties. The voltage dependence of activation (V(a,1/2)) shows a shift towards positive potentials from Sol type I to IIa, IIx and finally IIb fibres from PL (P < 0.05). These data are consistent with the earlier recruitment of slow fibres in a fast-mixed muscle like PL, while slow fibres of postural muscle such as soleus could be recruited in the same ways as IIb fibres in a fast muscle.
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Activación del Canal Iónico/fisiología , Potenciales de la Membrana/fisiología , Fibras Musculares de Contracción Rápida/fisiología , Fibras Musculares de Contracción Lenta/fisiología , Cadenas Pesadas de Miosina/metabolismo , Canales de Sodio/fisiología , Sodio/metabolismo , Animales , Células Cultivadas , Femenino , Ratas , Ratas WistarRESUMEN
The ciliary neurotrophic factor (CNTF), known to exert long-term myotrophic effects, has not yet been shown to induce a rapid biological response in skeletal muscles. The present in vitro study gives rise to the possibility that CNTF could affect the sodium channel activity implied in the triggering of muscle fibre contraction. Therefore, we investigated the effects of an external CNTF application on macroscopic sodium current (I(Na)) in rat native fast-twitch skeletal muscle (flexor digitorum brevis, FDB) by using a cell-attached patch-clamp technique. The I(Na) peak amplitude measured at a depolarizing pulse from -100 to -10 mV is rapidly reduced in a time- and dose-dependent manner by CNTF (0.01-20 ng ml(-1)). The maximal decrease is 25% after 10 min incubation in 2 ng ml(-1) CNTF. There was no alteration in activation or inactivation kinetics, or in activation curves constructed from current-voltage relationships in the presence of CNTF. In contrast, the relative I(Na) inhibition induced by CNTF is accompanied by a hyperpolarizing shift in the midpoint of the inactivation curves: -6 and -10 mV for the steady-state fast and slow inactivation, respectively. Furthermore, CNTF induces a 5 mV hyperpolarization of the resting membrane potential of the fibres. The effects of CNTF are similar to those of 1-oleoyl-2-acetyl-sn-glycerol (OAG), a protein kinase C (PKC) activator, when no effect is observed in the presence of chelerythrine, a PKC inhibitor. These results suggest that, in skeletal muscle, CNTF can rapidly decrease sodium currents by altering inactivation gating, probably through an intracellular PKC-dependent mechanism that could lead to decreased membrane excitability. The present study contributes to a better understanding of the physiological role of endogenous CNTF.
Asunto(s)
Factor Neurotrófico Ciliar/farmacología , Activación del Canal Iónico/efectos de los fármacos , Músculo Esquelético/fisiología , Proteína Quinasa C/metabolismo , Canales de Sodio/fisiología , Animales , Factor Neurotrófico Ciliar/fisiología , Relación Dosis-Respuesta a Droga , Técnicas In Vitro , Activación del Canal Iónico/fisiología , Masculino , Potenciales de la Membrana/efectos de los fármacos , Potenciales de la Membrana/fisiología , Técnicas de Placa-Clamp , Ratas , Ratas Wistar , Transducción de Señal/efectos de los fármacos , Transducción de Señal/fisiología , Sodio/metabolismoRESUMEN
Galectins are animal proteins which specifically bind beta-D-galactoside residues and their specific cellular function is not yet clearly established. However, these proteins seem to play a role in neoplastic transformations. Po66 is a murine monoclonal antibody directed against a protein from human lung carcinoma, Po66 Carbohydrate-Binding-Protein (Po66-CBP), which belongs to the galectin-8 family. Our results show that the Po66-CBP gene generates five transcripts by alternative splicing, which could give rise to five proteins: two proteins belong to the tandemly repeated galectin family and three belong to the single carbohydrate recognition domain galectins. All these proteins are encoded by a unique gene located in 1q42. Experiments carried out by reverse transcriptase-polymerase chain reaction show that the levels of expression of these five galectin-8 isoforms are variable during the culture time in SK-MES-1, a human lung squamous carcinoma cell line. Cancer Genome Anatomy Project database analysis confirms the presence of Po66-CBP in lung cancer and its absence in healthy lung.
Asunto(s)
Proteínas Portadoras/genética , Galectinas , Lectinas/genética , ARN Mensajero/genética , Secuencia de Aminoácidos , Secuencia de Bases , Northern Blotting , Mapeo Cromosómico , Cromosomas Humanos Par 1/genética , ADN Complementario/química , ADN Complementario/genética , ADN Complementario/aislamiento & purificación , Femenino , Expresión Génica , Humanos , Neoplasias Pulmonares/genética , Neoplasias Pulmonares/patología , Masculino , Datos de Secuencia Molecular , Isoformas de Proteínas/genética , ARN Mensajero/metabolismo , Mapeo de Híbrido por Radiación , Análisis de Secuencia de ADN , Homología de Secuencia de Aminoácido , Distribución Tisular , Células Tumorales CultivadasRESUMEN
Galectins are animal lectins, that can specifically bind beta-galactosides. Twelve galectins have been described in vertebrates, belonging to three different groups: prototype, tandem-repeat and chimeric. These proteins seem to be involved in cellular interactions and neoplastic transformations. We present an overview of a particular galectin member: galectin-8. This galectin, which has been intensively studied over the last six years, presents a particular type of gene regulation. It is widely expressed in tumoral tissues and seems to be involved in integrin-like cell interactions. Studies show that the LGALS8 gene encodes for almost seven mRNAs by alternative splicing pathways and various polyadenylation sites. These mRNAs could encode for six isoforms of galectin-8, of which three belong to the tandem-repeat galectin group (with two carbohydrate binding domains) and the three others to the prototype group (one carbohydrate binding domain). All these isoforms seem to be differentially expressed in various tumoral cells. This untypical galectin-8 subfamily seems to have a complex expression regulation, that could be involved in cancer phenomena.
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Galectinas , Lectinas/genética , Empalme Alternativo , Animales , Regulación de la Expresión Génica , Genes/genética , Humanos , Isoformas de Proteínas/genéticaRESUMEN
Two copies of IS1675, a novel lactococcal insertion element from the IS4 family, are present on a 70-kb plasmid, where they frame the lantibiotic lacticin 481 operon. The whole structure could be a composite transposon designated Tn5721. This study shows that the lacticin 481 operon does not include any regulatory gene and provides a new example of a transposon-associated bacteriocin determinant. We identified five other IS1675 copies not associated with the lacticin 481 operon. The conservation of IS1675 flanking sequences suggested a 24-bp target site.
Asunto(s)
Antibacterianos , Bacteriocinas , Elementos Transponibles de ADN , ADN Bacteriano , Genes Bacterianos , Lactococcus lactis/genética , Operón , Secuencia de Aminoácidos , Proteínas Bacterianas/genética , Secuencia de Bases , Datos de Secuencia Molecular , Análisis de Secuencia de ADNRESUMEN
Class AII and AIII lantibiotics and mersacidin are antibacterial peptides containing unusual residues obtained by posttranslational modifications of prepeptides, presumably catalyzed by LanM. LctM, the LanM for lacticin 481, is essential for the production of this class AII lantibiotic. Using the yeast two-hybrid system, we showed direct contact between the prelacticin 481 and LctM, supporting the proposed LctM function. Sixteen domains are conserved between the 10 known LanM proteins, whereas three additional domains were found only in class AII LanM proteins and in MrsM, the LanM for mersacidin. All the truncated LctM proteins that we tested presented impaired LctA-binding activity.
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Antibacterianos/biosíntesis , Antibacterianos/metabolismo , Proteínas Bacterianas/metabolismo , Lactococcus lactis/genética , Lactococcus lactis/metabolismo , Operón , Péptidos , Precursores de Proteínas/metabolismo , Secuencia de Aminoácidos , Proteínas Bacterianas/biosíntesis , Proteínas Bacterianas/química , Proteínas Bacterianas/genética , Bacteriocinas/biosíntesis , Sitios de Unión , Clonación Molecular , Secuencia Conservada , Datos de Secuencia Molecular , Saccharomyces cerevisiae/metabolismo , Alineación de Secuencia , Homología de Secuencia de AminoácidoRESUMEN
Vitellogenins (Vtg) are the major yolk proteins in most oviparous organisms. They are encoded by a small number of genes--between one and four depending on the species. Characterization of the Vtg region in the genome of the rainbow trout reveals unusual features, however, in that this locus contains twenty complete genes and ten pseudogenes per haploid genome. The Vtg genes differ from each other by insertion, deletion and rearrangement events, although, at the sequence level, they show a high degree of similarity. Fluorescent in situ hybridization (FISH), pulsed-field gel electrophoresis (PFGE) and Southern analysis indicate that all gene copies are contained in a single 1,500-kb region, and that most of the genes form tandem arrays separated by a conserved 4.5-kb intergenic region. The presence of large reiterated fragments indicates that this region has been subjected to several amplification events. The presence of a retroposon element (called 19) in Vtg intron 9 appears to be responsible for the silencing of at least nine of the ten pseudogenes. Two other incomplete retrotransposons (one LTR- and one LINE-type) and sequences derived from a HIV-like retrovirus are inserted into the conserved intergenic region, very close to the transcription start site. Their presence in all Vtg 5'-flanking regions suggests a possible role in gene amplification at this locus.
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Amplificación de Genes , Oncorhynchus mykiss/genética , Retroelementos , Vitelogeninas/genética , Animales , Secuencia de Bases , ADN/genética , Evolución Molecular , Femenino , Genoma , Hibridación Fluorescente in Situ , Modelos Genéticos , Datos de Secuencia Molecular , Familia de Multigenes , Mapeo Restrictivo , Homología de Secuencia de Ácido Nucleico , Secuencias Repetidas en TándemRESUMEN
The lantibiotic lacticin 481 is a bacteriocin produced by Lactococcus lactis strains. The genetic determinants of lacticin 481 production are organized as an operon encoded by a 70-kb plasmid. We previously reported the first three genes of this operon, lctA, lctM, and lctT, which are involved in the bacteriocin biosynthesis and export (A. Rincé, A. Dufour, S. Le Pogam, D. Thuault, C. M. Bourgeois, and J.-P. Le Pennec, Appl. Environ. Microbiol. 60:1652-1657, 1994). The operon contains three additional open reading frames: lctF, lctE, and lctG. The hydrophobicity profiles and sequence similarities strongly suggest that the three gene products associate to form an ABC transporter. When the three genes were coexpressed into a lacticin 481-sensitive L. lactis strain, the strain became resistant to the bacteriocin. This protection could not be obtained when any of the three genes was deleted, confirming that lctF, lctE, and lctG are all necessary to provide immunity to lacticin 481. The quantification of the levels of immunity showed that lctF, lctE, and lctG could account for at least 6% and up to 100% of the immunity of the wild-type lacticin 481 producer strain, depending on the gene expression regulation. The lacticin 481 biosynthesis and immunity systems are discussed and compared to other lantibiotic systems.
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Transportadoras de Casetes de Unión a ATP/genética , Bacteriocinas/genética , Genes Bacterianos , Lactococcus lactis/genética , Operón , Secuencia de Aminoácidos , Proteínas Bacterianas/genética , Proteínas Bacterianas/inmunología , Bacteriocinas/inmunología , Lactococcus lactis/inmunología , Datos de Secuencia MolecularRESUMEN
In this paper we describe the first complete structure of a fish vitellogenin gene. A 22 kb genomic region from rainbow trout (Oncorhynchus mykiss) was cloned and analysed. This region was shown to contain two tandemly arranged vitellogenin genes. Both genes are 98.7% similar, indicating that they result from a recent local duplication. The complete sequence encoding one of the two genes was determined and the gene organization was established. The gene is 10.3 kb long and has 34 exons, it lacks one exon compared to amphibian and avian vitellogenin genes. Exons 22 and 23 of the Xenopus and chicken genes were shown to be merged into a single exon in the trout genome. Other splicing sites appeared highly conserved between the three vertebrate genes. In contrast, little similarity between invertebrate and vertebrate vitellogenin genes was observed with respect to the number and organization of introns. The comparison of 17 independent invertebrate splicing sites with the 34 vertebrate sites indicated that a few sites are probably ancient. However, most of the splicing junctions compared appeared unrelated. Results suggest that vitellogenin genes have been reshaped through multiple insertions and deletions of intervening sequences during evolution.
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Evolución Molecular , Genes/genética , Oncorhynchus mykiss/genética , Vitelogeninas/genética , Secuencia de Aminoácidos , Animales , Clonación Molecular , Exones/genética , Intrones/genética , Invertebrados/genética , Datos de Secuencia Molecular , Mapeo Restrictivo , Análisis de Secuencia de ADN , Vertebrados/genéticaRESUMEN
The nucleotide sequence of the vitellogenin cDNA from the rainbow trout Oncorhynchus mykiss was determined. Analysis of the deduced amino acid sequence (1659 residues) places the lipovitellin I, phosvitin and lipovitellin II domains between amino acids 16 to 1088, 1089 to 1145 and 1146 to 1659, respectively. The general structure is similar to other vertebrate vitellogenins except for the serine rich phosvitin domain which is the shortest identified so far in vertebrates (57 amino acids), being 2 to 4 times smaller than in other species. Sequence comparisons between vertebrate and invertebrate vitellogenins as well as with distantly related proteins allowed to identify two short amino acid motifs particularly well conserved, RGILN and TCGLCG in lipovitellin I and II domains, respectively, and strongly suggest that the lipovitellin II domain is involved in protein interactions via disulfide bridge formation.
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Oncorhynchus mykiss/genética , Vitelogeninas/genética , Secuencia de Aminoácidos , Animales , Secuencia de Bases , ADN Complementario/genética , ADN Recombinante , Datos de Secuencia Molecular , Oncorhynchus mykiss/metabolismo , Alineación de Secuencia , Análisis de SecuenciaRESUMEN
Monoclonal antibody (MAb) Po66, a murine IgG1, was raised by immunization against human lung squamous cell carcinoma. When injected intravenously, Po66 showed prolonged retention in the tumor. It recognized an intracellular antigen. The human lung squamous carcinoma cell line SK-MES-1 expresses the antigen recognized by MAb Po66 and was used as a source of biological material for its purification. The SK-MES-1 cell line was labeled in culture with [35S]methionine and its lysate was immunoprecipitated with Po66 immobilized on Protein G-Sepharose. The precipitate contained three proteins (47, 50 and 69 kDa) absent in the controls. The 69 kDa polypeptide was further purified by anion exchange and immunoaffinity chromatographies. To date, no other tumor marker expressed in non-small cell lung cancer with these characteristics has been described and as such this marker is interesting for future use in immunotherapy and in diagnosis.
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Anticuerpos Monoclonales , Biomarcadores de Tumor/aislamiento & purificación , Carcinoma de Células Escamosas/química , Neoplasias Pulmonares/química , Animales , Cromatografía de Afinidad , Cromatografía por Intercambio Iónico , Electroforesis en Gel de Poliacrilamida , Humanos , Inmunoglobulina G/inmunología , Ratones , Peso MolecularRESUMEN
The concept of the ionic channel, as a protein with more discrete ionic conductance levels, is analyzed and a computer program, aiming to simulate its dynamics under the action of a pharmacological agent, is presented. The binding of a pharmacological agent modifies the set of the possible configurations and the channel energy spectrum. Therefore it modifies the transition probabilities and the dwelling times in the excited (generally conductive) states. The program creates a library of simulations of the electric activity of some user designated hypothetical models. The simulations are then statistically compared to the recorded single channel events to select the most suitable model.