RESUMEN

Coleoid cephalopods show remarkable evolutionary convergence with vertebrates in their neural organization, including (1) eyes and visual system with optic lobes, (2) specialized parts of the brain controlling learning and memory, such as vertical lobes, and (3) unique vasculature supporting such complexity of the central nervous system. We performed deep sequencing of eye transcriptomes of pygmy squids (Idiosepius paradoxus) and chambered nautiluses (Nautilus pompilius) to decipher the molecular basis of convergent evolution in cephalopods. RNA-seq was complemented by in situ hybridization to localize the expression of selected genes. We found three types of genomic innovations in the evolution of complex brains: (1) recruitment of novel genes into morphogenetic pathways, (2) recombination of various coding and regulatory regions of different genes, often called "evolutionary tinkering" or "co-option", and (3) duplication and divergence of genes. Massive recruitment of novel genes occurred in the evolution of the "camera" eye from nautilus' "pinhole" eye. We also showed that the type-2 co-option of transcription factors played important roles in the evolution of the lens and visual neurons. In summary, the cephalopod convergent morphological evolution of the camera eyes was driven by a mosaic of all types of gene recruitments. In addition, our analysis revealed unexpected variations of squids' opsins, retinochromes, and arrestins, providing more detailed information, valuable for further research on intra-ocular and extra-ocular photoreception of the cephalopods.

Asunto(s)

Encéfalo/anatomía & histología , Cefalópodos/anatomía & histología , Evolución Molecular , Regulación del Desarrollo de la Expresión Génica/fisiología , Fenómenos Fisiológicos Oculares/genética , Secuencia de Aminoácidos , Animales , Arrestina/genética , Arrestina/metabolismo , Cefalópodos/genética , Cristalino , Células Fotorreceptoras/fisiología , Filogenia , Isoformas de Proteínas

RESUMEN

Praziquantel, the drug of choice against schistosomiasis, disrupts calcium (Ca2+) homeostasis in schistosomes via an unknown mechanism. Voltage-gated Ca2+ channels are heteromultimeric transmembrane protein complexes that contribute to impulse propagation and also regulate intracellular Ca2+ levels. Beta subunits modulate the properties of the pore-forming alpha1 subunit of high voltage-activated Ca2+ channels. Unlike other Ca2+ channel beta subunits, which have current stimulatory effects, a beta subunit subtype found in S. mansoni (SmbetaA) and S. japonicum (Sjbeta) dramatically reduces current levels when co-expressed with Ca2+ channel alpha1 subunits in Xenopus oocytes. It also confers praziquantel sensitivity to the mammalian Cav2.3 alpha1 subunit. The Beta Interaction Domains (BIDs) of SmbetaA and Sjbeta lack 2 conserved serines that each constitute a consensus site for protein kinase C (PKC) phosphorylation. Here, we use site-directed mutagenesis of schistosome beta subunits to show that these unique functional properties are correlated with the absence of these consensus PKC sites in the BID. Furthermore, a second schistosome beta subunit subtype contains both serines in the BID, enhances currents through alpha1 subunits, and does not confer praziquantel sensitivity. Thus, phosphorylation sites in the BID may play important roles in defining the modulatory properties and pharmacological sensitivities of schistosome Ca2+ channel beta subunits.

Asunto(s)

Antihelmínticos/farmacología , Canales de Calcio/efectos de los fármacos , Praziquantel/farmacología , Schistosoma mansoni/efectos de los fármacos , Secuencia de Aminoácidos , Animales , Secuencia de Bases , Canales de Calcio/genética , Canales de Calcio/metabolismo , Clonación Molecular , ADN de Helmintos/química , ADN de Helmintos/genética , Potenciales de la Membrana/fisiología , Datos de Secuencia Molecular , Mutagénesis Sitio-Dirigida , Técnicas de Placa-Clamp , Filogenia , Schistosoma mansoni/genética , Schistosoma mansoni/metabolismo , Alineación de Secuencia , Análisis de Secuencia de ADN , Xenopus/genética

RESUMEN

Voltage-gated calcium (Ca2+) channels contribute to impulse propagation in excitable cells and also regulate intracellular levels of Ca2+. High voltage-activated (HVA) Ca2+ channels are heteromultimeric membrane proteins. The pore-forming, voltage-sensing subunit is the alpha1 subunit. We have cloned 3 HVA Ca2+ channel alpha1 subunit cDNAs from Schistosoma mansoni. One of these sequences most closely resembles the L-type class of HVA alpha1 subunits. The other two sequences are most closely related to non L-type alpha1 subunits. These schistosome alpha1 subunits have many of the features common to HVA Ca2+ channels, but also have distinct structural motifs. Analysis of the structural and functional properties of schistosome Ca2+ channel subunits may provide information about these critical components of excitable cells.

Asunto(s)

Canales de Calcio/genética , ADN de Helmintos/genética , Schistosoma mansoni/fisiología , Secuencia de Aminoácidos , Animales , Secuencia de Bases , Northern Blotting , Canales de Calcio/química , ADN de Helmintos/química , Femenino , Ratones , Datos de Secuencia Molecular , Filogenia , ARN de Helminto/química , ARN de Helminto/genética , ARN de Helminto/aislamiento & purificación , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Schistosoma mansoni/química , Schistosoma mansoni/genética , Esquistosomiasis mansoni/sangre , Alineación de Secuencia , Análisis de Secuencia de ADN , Homología de Secuencia de Aminoácido

RESUMEN

Schistosomes are parasitic flatworms that cause schistosomiasis, a major tropical disease. The current drug of choice against schistosomiasis is praziquantel (PZQ), which has minimal side effects and is potent against all schistosome species. The mode of action of PZQ is unknown, though the drug clearly affects Ca(2+) homeostasis in worms, and there is indirect evidence for interaction of PZQ with schistosome voltage-gated Ca(2+) channels. We have cloned and expressed two Ca(2+) channel beta subunits, one from Schistosoma mansoni and one from Schistosoma japonicum. These two subunits (SmCa(v)beta A and SjCa(v)beta) have structural motifs that differ from those found in other known beta subunits. Surprisingly, coexpression of either SmCa(v)beta A or SjCa(v)beta with a cnidarian (CyCa(v)1) or mammalian (Ca(v)2.3) Ca(2+) channel alpha(1) subunit results in a striking reduction in current amplitude. In the case of Ca(v)2.3, this current reduction can be partially reversed by addition of 100 nm PZQ, which results in a significant increase in current amplitude. Thus, these unusual schistosome beta subunits can confer PZQ sensitivity to an otherwise PZQ-insensitive mammalian Ca(2+) channel, indicating that a possible target for PZQ action is the interaction between beta subunits and pore-forming alpha(1) subunits in schistosomes.

Asunto(s)

Canales de Calcio/genética , Proteínas del Helminto/genética , Schistosoma/genética , Secuencia de Aminoácidos , Animales , Antihelmínticos/farmacología , Canales de Calcio/química , Canales de Calcio/fisiología , Clonación Molecular , ADN Complementario/análisis , Electrofisiología , Proteínas del Helminto/química , Proteínas del Helminto/fisiología , Datos de Secuencia Molecular , Praziquantel/farmacología , Schistosoma/efectos de los fármacos , Schistosoma/metabolismo , Schistosoma/fisiología , Homología de Secuencia de Aminoácido , Transfección , Xenopus laevis

RESUMEN

The distribution of nitric oxide synthase (NOS) immunoreactivity and putative NOS activity in adult Schistosoma mansoni was analysed using 3 different types of NOS antibodies and NADPH-diaphorase histochemistry. Although potential involvement of the gaseous radical nitric oxide (NO) in host response to infection by schistosomes has been suggested, there is little or no information available regarding the role, or even the presence, of the NO pathway in schistosomes themselves. Here, we demonstrate that antibodies against neuronal NOS (nNOS) and inducible NOS (iNOS) isoforms stain adult worms with distinctive patterns; anti-endothelial NOS (eNOS) shows no selective labelling. nNOS-like immunoreactivity is found in the main nerve cords and the peripheral nervous system. Putative sensory neurons with apical neuronal processes leading to the tegument of male worms are also immunoreactive for nNOS. Anti-iNOS labels a variety of predominantly non-neuronal tissues, showing intense labelling at or near the surface of the worm and in components of the gastrointestinal tract. The distribution of NADPH-diaphorase reactivity (a histochemical marker of NOS), is generally similar to the pattern of NOS immunoreactivity, including labelling of neuronal-like cells as well as developing eggs. These results suggest that an NOS-like enzyme is present in S. mansoni, and indicate potential roles for the different NOS isoforms in neuronal signalling, reproduction and development.

Asunto(s)

Óxido Nítrico Sintasa/metabolismo , Schistosoma mansoni/enzimología , Animales , Femenino , Inmunohistoquímica , Estadios del Ciclo de Vida/fisiología , Masculino , Ratones , NADPH Deshidrogenasa/metabolismo , Sistema Nervioso/enzimología , Neuronas/enzimología , Óxido Nítrico Sintasa de Tipo I , Señales de Clasificación de Proteína/fisiología , Schistosoma mansoni/fisiología

RESUMEN

The preference of the 'prohormone thiol protease' (PTP), a candidate prohormone processing enzyme, for different peptide precursors was assessed in vitro with recombinant prohormones near estimated in vivo levels. Pro-neuropeptide Y (pro-NPY), proopiomelanocortin (POMC), and proenkephalin (PE) were expressed at high levels in E. coli. Purification of prohormones utilized a combination of DEAE-Sepharose, Mono Q, and preparative electrophoresis. PTP cleaved PE most readily, and also cleaved pro-NPY. The processing of POMC by PTP was minimal. These results demonstrate PTP's preference for certain prohormone substrates.

Asunto(s)

Cisteína Endopeptidasas/metabolismo , Encefalinas/metabolismo , Neuropéptido Y/metabolismo , Proopiomelanocortina/metabolismo , Precursores de Proteínas/metabolismo , Secuencia de Aminoácidos , Animales , Secuencia de Bases , Encefalinas/biosíntesis , Encefalinas/genética , Encefalinas/aislamiento & purificación , Escherichia coli/genética , Datos de Secuencia Molecular , Neuropéptido Y/biosíntesis , Neuropéptido Y/genética , Neuropéptido Y/aislamiento & purificación , Proopiomelanocortina/biosíntesis , Proopiomelanocortina/genética , Proopiomelanocortina/aislamiento & purificación , Precursores de Proteínas/biosíntesis , Precursores de Proteínas/genética , Precursores de Proteínas/aislamiento & purificación , Procesamiento Proteico-Postraduccional , Ratas , Proteínas Recombinantes de Fusión/biosíntesis , Proteínas Recombinantes de Fusión/aislamiento & purificación , Proteínas Recombinantes de Fusión/metabolismo , Especificidad por Sustrato , Porcinos

RESUMEN

Molecular cloning of bovine adrenal medulla (AM) and pituitary (Pit) alpha 1-antichymotrypsin cDNAs indicated novel isoforms of ACT. The deduced primary sequences indicated that the AM ACT and Pit ACT possess COOH-terminal reactive-site domains that are characteristic of serpins (serine protease inhibitors). Of high interest was the finding of unique reactive sites within AM ACT and Pit ACT which are predicted to possess Arg as P1 residue. Arginine as P1 residue parallels the cleavage specificity of neuroendocrine prohormone processing enzymes cleaving at basic residues. Furthermore, RT-PCR indicated tissue-specific expression of AM and Pit ACT mRNAs. The AM and Pit isoforms of ACT may regulate novel target proteases involved in neuroendocrine function.

Asunto(s)

Médula Suprarrenal/metabolismo , Isoenzimas/genética , Hipófisis/metabolismo , alfa 1-Antiquimotripsina/genética , Secuencia de Aminoácidos , Animales , Secuencia de Bases , Bovinos , Clonación Molecular , ADN Complementario , Isoenzimas/metabolismo , Hígado/metabolismo , Datos de Secuencia Molecular , Reacción en Cadena de la Polimerasa , ARN Mensajero/genética , Homología de Secuencia de Aminoácido , alfa 1-Antiquimotripsina/metabolismo

RESUMEN

Sulfate conjugation by sulfotransferases is important for the metabolism of drugs, xenobiotics, monoamines, and neuropeptides (1-3). In this study, molecular cloning resulted in the isolation of a 1.2-kb human brain hippocampal cDNA encoding a member of the phenol sulfotransferase (PST) family of enzymes. The hippocampal phenol sulfotransferase (H-PST) cDNA possesses differences in its deduced primary sequence compared to human liver monoamine (M form) (4,5) and phenol (P form) (6,7) isoforms of PST. The hippocampal PST contrasts with previous studies that identified two human brain PST cDNAs whose coding sequences are identical to the human liver M and P forms of PST. Importantly, this study provides evidence for a human hippocampal form of PST.

Asunto(s)

Arilsulfotransferasa/biosíntesis , Hipocampo/enzimología , Isoenzimas/biosíntesis , Secuencia de Aminoácidos , Arilsulfotransferasa/química , Arilsulfotransferasa/genética , Secuencia de Bases , Clonación Molecular/métodos , ADN Complementario/aislamiento & purificación , Humanos , Isoenzimas/química , Isoenzimas/genética , Hígado/enzimología , Datos de Secuencia Molecular , Peso Molecular , Proteínas Recombinantes/biosíntesis , Proteínas Recombinantes/química , Homología de Secuencia de Aminoácido

RESUMEN

Comparison of bovine alpha 1-antichymotrypsin (ACT) protease inhibitor with that in human was achieved by cloning a nearly full-length bovine ACT cDNA of 1.5 kb, obtained by screening a bovine liver cDNA library with the human liver ACT cDNA. The deduced primary sequence indicated that the 1456-bp bovine ACT cDNA encodes a protein of 416 amino acids that contains the predicted full-length ACT with a 26-residue NH2-terminal signal sequence. Overall, the primary sequence of bovine ACT possesses a high degree of homology (55%) with human ACT; both bovine and human ACTs share common sequences in the reactive-site domains. Importantly, the reactive site of bovine ACT possesses serine as the predicted P1 position (residue at the NH2-terminal side of the cleaved peptide bond) of the reactive site, whereas human ACT contains leucine in the P1 position. Interestingly, further evidence for heterogeneity in P1 residues was provided by a second partial 0.9-kb bovine liver ACT cDNA clone (pHHK11) that contains isoleucine as P1 residue and shares only partial homology (68%) with the deduced primary sequence of the full-length bovine liver ACT cDNA clone (pHHK12). These findings suggest that isoforms of ACT in bovine liver vary in reactive-site P1 residues; the P1 position of the reactive site is often involved in protease inhibitor specificity. Consistent with the hypothesis of ACT isoforms was the demonstration of multiple copies of the bovine ACT gene by genomic blots.

Asunto(s)

Hígado/metabolismo , alfa 1-Antiquimotripsina/biosíntesis , Secuencia de Aminoácidos , Animales , Secuencia de Bases , Northern Blotting , Bovinos , Clonación Molecular , ADN/química , ADN/genética , ADN Complementario , Biblioteca de Genes , Humanos , Hibridación in Situ , Datos de Secuencia Molecular , ARN Mensajero/aislamiento & purificación , ARN Mensajero/metabolismo , Mapeo Restrictivo , Homología de Secuencia de Aminoácido

RESUMEN

The efficacy of abamectin 1%, when injected subcutaneously in cattle at a dose of 200 micrograms/kg body weight, against the larval stages (grubs) of the fly Dermatobia hominis was evaluated in two trials in endemic areas of Brazil and Argentina. Eighteen Holstein x Brahman castrated males and 16 Brahman-cross with natural infestations were used. Larvae were counted by instar in situ on both sides of each animal before treatment, and were expressed, identified as to stage and classified as live or dead 10 days after treatment. Further larval counts were made periodically until day 79 to evaluate the degree of reinfestation and the stage of larval development. Reinfestation was first detected in the abamectin-treated cattle on day 44. Live larvae were found on 6-8 (Argentina) and on all (Brazil) controls at each post-treatment examination. The difference in numbers of live larvae between treatment groups was statistically significant (P < 0.05) at all post-treatment examinations. These data show that abamectin at a dose of 200 micrograms/kg body weight is highly effective in the treatment and control of established parasitic stages of D. hominis in cattle. No adverse reactions were observed in any of the treated animals.

Asunto(s)

Enfermedades de los Bovinos/tratamiento farmacológico , Ivermectina/análogos & derivados , Miasis/veterinaria , Animales , Bovinos , Enfermedades de los Bovinos/parasitología , Femenino , Ivermectina/uso terapéutico , Masculino , Miasis/tratamiento farmacológico

RESUMEN

The phase diagram of dipalmitoylphosphatidylcholine (DPPC) and palmitic acid mixtures in excess D2O was studied by 13C-NMR. Phase boundaries were determined from plots of apparent spin-spin relaxation time T2 (for both choline methyl and fatty acid chain carbons) versus temperature. A peritectic transition in the 1-10 mol% region, whose existence has been theoretically inferred from the Gibbs phase rule but which was undetectable by differential thermal analysis (DTA) (S.E. Schullery et al. Biochemistry, 20 (1981) 6818-6824), was located by NMR at 41.6 degrees C. A second, nearby peritectic line at 44 degrees C, which had been shown by DTA to extend from about 3-25 mol% palmitic acid, was seen by NMR only above 10 mol%. The palmitic acid/DPPC complex (2:1), with a sharp melting point at 64 degrees C, reported in earlier studies, was also seen by NMR. A phase diagram including both NMR and DTA results is presented. Important general conclusions from this study are: (i) NMR and scanning thermal analysis are complementary techniques for phase studies; each can see transitions that are invisible to the other. (ii) The case for the applicability of the Gibbs phase rule to lipid bilayer systems has been strengthened by the observance of two predicted, close-spaced boundaries. (iii) Low concentrations of fatty acids and related molecules can not be assumed to disperse as simple ideal solutes in the bilayer matrix.

Asunto(s)

Ácidos Palmíticos , Surfactantes Pulmonares , Isótopos de Carbono , Espectroscopía de Resonancia Magnética/métodos , Modelos Biológicos , Conformación Molecular , Ácido Palmítico , Termodinámica