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1.
Biochim Biophys Acta ; 1574(2): 145-51, 2002 Mar 19.
Artículo en Inglés | MEDLINE | ID: mdl-11955623

RESUMEN

PC2 prohormone convertases are enzymes involved in the proteolytic maturation of neuropeptide precursors. In the present work, a cDNA encoding a PC2-like enzyme (OrlPC2) was cloned from crayfish eyestalk ganglia (medulla terminalis) containing the X-organ, a major neuroendocrine center. The predicted 634 amino acid preproprotein exhibits highest sequence identity, especially in the catalytic domain, with PC2s from arthropods and nematodes, and less with mollusc and vertebrate enzymes. It was demonstrated by in situ hybridization on crayfish medulla terminalis sections that OrlPC2 is expressed in a large number of neuron perikarya, including those producing the well known crustacean hyperglycemic hormone.


Asunto(s)
Astacoidea/genética , Subtilisinas/genética , Secuencia de Aminoácidos , Animales , Secuencia de Bases , Clonación Molecular , Cartilla de ADN , ADN Complementario/biosíntesis , ADN Complementario/química , Hibridación in Situ , Datos de Secuencia Molecular , Proproteína Convertasa 2 , Alineación de Secuencia , Subtilisinas/biosíntesis , Subtilisinas/química
2.
Biol Bull ; 201(2): 167-74, 2001 Oct.
Artículo en Inglés | MEDLINE | ID: mdl-11687388

RESUMEN

This study investigates the salinity tolerance and the pattern of osmotic and ionic regulation of Bythograea thermydron Williams, 1980, a brachyuran crab endemic to the deep-sea hydrothermal vent habitat. Salinities of 33 per thousand-35 per thousand were measured in the seawater surrounding the captured specimens. B. thermydron is a marine stenohaline osmoconformer, which tolerates salinities ranging between about 31 per thousand and 42 per thousand. The time of osmotic adaptation after a sudden decrease in external salinity is about 15-24 h, which is relatively short for a brachyuran crab. In the range of tolerable salinities, it exhibits an iso-osmotic regulation, which is not affected by changes in hydrostatic pressure, and an iso-ionic regulation for Na(+) and Cl(-). The hemolymph Ca(2+) concentration is slightly hyper-regulated, K(+) concentration is slightly hyper-hypo-regulated, and Mg(2+) concentration is strongly hypo-regulated. These findings probably reflect a high permeability of the teguments to water and ions. In addition to limited information about salinity around hydrothermal vents, these results lead to the hypothesis that B. thermydron lives in a habitat of stable seawater salinity. The osmoconformity of this species is briefly discussed in relation to its potential phylogeny.


Asunto(s)
Braquiuros/fisiología , Cloruros/fisiología , Sodio/fisiología , Equilibrio Hidroelectrolítico/fisiología , Animales , Braquiuros/crecimiento & desarrollo , Braquiuros/metabolismo , Cloruros/metabolismo , Hemolinfa/química , Océano Pacífico , Sodio/metabolismo
3.
J Biol Chem ; 275(48): 37870-5, 2000 Dec 01.
Artículo en Inglés | MEDLINE | ID: mdl-10993902

RESUMEN

Modification of the chirality of a single amino acid residue within a peptide chain appears to be novel additional mechanism leading to structural and functional diversification of eukaryotic bioactive peptides. This phenomenon has been studied at the cellular level in a neuroendocrine organ which elaborates a mixture of diastereoisomers of a 72-residue neuropeptide, crustacean hyperglycemic hormone. For the first time, amino acid isomerization has been shown to occur in the perikarya of fully specialized neurosecretory cells, as a late step of the maturation of the hyperglycemic hormone precursor and after propeptide cleavage. The specificity and efficiency of this phenomenon indicates the existence of a new enzyme family involved in the biogenesis of peptide hormones.


Asunto(s)
Proteínas del Tejido Nervioso/metabolismo , Procesamiento Proteico-Postraduccional , Secuencia de Aminoácidos , Animales , Proteínas de Artrópodos , Astacoidea , Femenino , Hormonas de Invertebrados , Isomerismo , Masculino , Proteínas del Tejido Nervioso/química , Sistemas Neurosecretores/citología
4.
Methods Cell Sci ; 21(4): 193-8, 1999.
Artículo en Inglés | MEDLINE | ID: mdl-10627671

RESUMEN

Crustacean cell culture has gained attention as a potent model to assist in the development of diagnostic reagents and probes for use in the shrimp, crayfish and lobster industries. The availability of such cellular tools is especially important to industries which use intensive aquaculture methods and thus have increased risk of disease problems. Indeed, crustacean cell cultures offer potential for studying the effects of pathogens in vitro and for increasing our knowledge on developmental and sexual maturation processes, or endocrine regulation in crustaceans. Although numerous attempts have been undertaken, no established cell line of marine crustaceans has been reported to date. However, primary cultures obtained from various organ sources are reported with increasing frequency. They represent the first steps towards the establishment of cell lines and they provide useful information concerning the most suitable cell culture conditions involved in the survival and proliferative capacity of the various tissues.


Asunto(s)
Técnicas de Cultivo de Célula/métodos , Crustáceos/citología , Animales , Antibacterianos , Antifúngicos , Células Cultivadas , Técnicas de Cultivo de Órganos/métodos
5.
Anal Chem ; 70(9): 1805-11, 1998 May 01.
Artículo en Inglés | MEDLINE | ID: mdl-9599581

RESUMEN

The combination of two sensitive and powerful analytical techniques on the same biological sample was examined: (i) matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS), which gives informative peptide profiling on complex samples such as organs or cells; (ii) immunological tools such as enzyme-linked immunosorbent assay (ELISA) and immunocytochemistry to probe for specific peptides in biological extracts or cells. The cellular expression of the two precursors of the hyperglycemic hormone (cHH) was analyzed in neurosecretory cells (30-micron diameter) from the crayfish Orconectes limosus. Neurohemal organs were used to optimize the sample preparation and to demonstrate that, after peptide fingerprinting by MALDI-TOF MS, the sample can be recovered from the MALDI plate for further immunological analysis by ELISA. It was also established that, after immunocytochemistry following 4% paraformaldehyde fixation of the organ, the stained tissue could be recovered for further MALDI-TOF MS analysis. This dual characterization was successfully scaled down to the level of a single crayfish neurosecretory cell. Direct peptide profiling by MALDI-TOF MS on a single cHH-producing cell previously identified by immunocytochemistry demonstrated that both procHH isoforms were expressed in each cell analyzed.


Asunto(s)
Astacoidea/química , Células/química , Glándulas Endocrinas/química , Péptidos/análisis , Animales , Ensayo de Inmunoadsorción Enzimática , Espectrometría de Masa por Láser de Matriz Asistida de Ionización Desorción
6.
Mol Cell Endocrinol ; 128(1-2): 139-49, 1997 Apr 04.
Artículo en Inglés | MEDLINE | ID: mdl-9140085

RESUMEN

Ecdysteroid biosynthesis was analyzed in vitro using dissociated Y-organ cells from the shore crab Carcinus maenas. 3-Dehydroecdysone (3DE) was detected as a minor secretory product, in addition to the formerly identified end-products 25-deoxyecdysone and ecdysone (E). In conversion studies, 3DE was formed from tritiated 5beta-ketodiol (2,22,25-trideoxyecdysone), 2,22-deoxyecdysone and 2-deoxyecdysone but not from E. Further experiments were performed in order to understand the interconversions between 3-oxo and 3beta-OH compounds in the crab Y-organ. The enzyme involved in 3beta-dehydrogenation was not ecdysone oxidase, a soluble enzyme found in peripheral tissues of many arthropods but it presented strong similarities with 3beta-hydroxysteroid dehydrogenase enzymes from vertebrates: it was membrane-bound and NAD+-dependent. Moreover, a NADH-dependent 3beta-reduction of several 3-oxo-ecdysteroids was obtained using the same microsomal fraction (100,000 x g pellet) of Y-organs, indicating that the reaction might be reversible. As this activity was specific of molting glands, we hypothesize that there is at least one 3beta-hydroxysteroid dehydrogenase enzyme involved in the biosynthetic pathway of ecdysteroids.


Asunto(s)
3-Hidroxiesteroide Deshidrogenasas/metabolismo , Esteroides/biosíntesis , Animales , Braquiuros , Células Cultivadas , Cromatografía Líquida de Alta Presión , Ecdisteroides , Femenino , Cinética , Masculino , Muda , Especificidad de Órganos , Especificidad por Sustrato
7.
Gen Comp Endocrinol ; 86(3): 424-32, 1992 Jun.
Artículo en Inglés | MEDLINE | ID: mdl-1356879

RESUMEN

A molecular with a molecular weight, estimated by gel filtration, of approximately 22 kDa and immunoreactive to anti-human hypophysial growth hormone (hGH) has been identified by radioimmunoassay in the digestive gland and hemolymph of the mussel Mytilus edulis L. The dilution curve of this molecule was parallel to that of hGH, suggesting that the antigenic site of the Mytilus molecule is similar to that of hGH. Immunoreactive fractions resulting from gel filtration failed to stimulate protein synthesis in dispersed mantle-edge cells in vitro. No hGH-immunoreactive material was detected in the cerebral ganglia. It is thus clear that a small protein-synthesis-stimulating factor (PSSF), identified in the cerebral ganglia and hemolymph by its action in vitro on dispersed mantle-edge cells, is not analogous to the Mytilus hGH-immunoreactive molecule. Likewise, a somatostatin-immunoreactive molecule present in the hemolymph of Mytilus did not coelute with PSSF. Evidence is presented that PSSF is a hydrophilic peptide that stimulates DNA, RNA, and protein synthesis and that is not tissue specific. These characteristics suggest that PSSF is a growth hormone.


Asunto(s)
Bivalvos/crecimiento & desarrollo , Sustancias de Crecimiento/fisiología , Animales , Cromatografía en Gel , Glándulas Exocrinas/química , Ganglios/química , Hormona del Crecimiento/inmunología , Sustancias de Crecimiento/análisis , Hemolinfa/química , Hidrólisis , Leucina/metabolismo , Peso Molecular , Radioinmunoensayo , Somatostatina/inmunología , Tripsina
8.
Experientia ; 48(3): 272-7, 1992 Mar 15.
Artículo en Inglés | MEDLINE | ID: mdl-1547860

RESUMEN

In crustaceans, all the steps in the assimilation of food take place in the hepatopancreas. To facilitate the study of this organ, a method for the dissociation of cell types was developed. The hepatopancreas of the prawn Palaemon serratus was mechanically dissociated and the cells separated by Percoll density-gradient centrifugation. The E and R cells had similar densities of around 1.05 g/ml. The F cells were separated into two distinct fractions with densities of 1.075 and 1.082 g/ml. The B cells sedimented at a density of 1.12 g/ml. The ratio between the two populations of F cells was found to vary during the intermolt cycle while B cells disappeared after the molt. When the density gradient fractions were incubated with 3H-leucine, incorporation was highest in the F cell fractions. Measurements of alpha-amylase activity, indicated that the two populations of F cells may be derived from the same cell type.


Asunto(s)
Sistema Digestivo/metabolismo , Biosíntesis de Proteínas , alfa-Amilasas/metabolismo , Animales , Separación Celular , Centrifugación por Gradiente de Densidad , Leucina , Hígado/citología , Hígado/metabolismo , Palaemonidae , Páncreas/citología , Páncreas/metabolismo
9.
C R Acad Sci III ; 305(7): 265-9, 1987.
Artículo en Francés | MEDLINE | ID: mdl-3113687

RESUMEN

Growth hormone-like peptides cross-reacting with an antiserum human specific were detected in the haemolymph, the midgut gland and stomach extracts of the prawn Palaemon serratus using radioimmunoassay. Parallelism was observed between the dilution sample curves and the hGH standard curve. Moreover, at least one peptide exhibiting a similar apparent molecular weight (22,000 daltons) with hGH was detected. In the three samples different amounts were detected during intermolt cycle: between 1.3 and 2.2 ng/ml in the haemolymph, 23 and 84 ng/g wet weight for the midgut gland, 12 and 71 ng/g ww for the stomach. The stages with highest values were respectively: D 1" and D 1"', D 1"' and D 2, D 3 and AB.


Asunto(s)
Hormona del Crecimiento/análogos & derivados , Metamorfosis Biológica , Palaemonidae/química , Animales , Hormona del Crecimiento/análisis , Hormona del Crecimiento/sangre , Hemolinfa/química , Hígado/química , Peso Molecular , Palaemonidae/crecimiento & desarrollo , Páncreas/química , Estómago/química
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