Globin's structure and function in vesicomyid bivalves from the Gulf of Guinea cold seeps as an adaptation to life in reduced sediments.

Vesicomyid bivalves form dense clam beds in both deep-sea cold seeps and hydrothermal vents. The species diversity within this family raises questions about niche separation and specific adaptations. To compare their abilities to withstand hypoxia, we have studied the structure and function of erythrocyte hemoglobin (Hb) and foot myoglobin (Mb) from two vesicomyid species, Christineconcha regab and Laubiericoncha chuni, collected from the Regab pockmark in the Gulf of Guinea at a depth of 3,000 m. Laubiericoncha chuni possesses three monomeric globins, G1 (15,361 Da), G2 (15,668 Da), and G3 (15,682 Da) in circulating erythrocytes (Hb), and also three globins, G1, G3, and G4 (14,786 Da) in foot muscle (Mb). Therefore, globins G2 and G4 appear to be specific for erythrocytes and muscle, respectively, but globins G1 and G3 are common. In contrast, C. regab lacks erythrocyte Hb completely and possesses only globin monomers G1' (14,941 Da), G2' (15,169 Da), and G3' (15,683 Da) in foot muscle. Thus, these two vesicomyid species, C. regab and L. chuni, show a remarkable diversity in globin expression when examined by electrospray ionization mass spectrometry. Oxygen-binding affinities reveal extremely high oxygen affinities (P50 < 1 Torr, from 5° to 15°C at pH 7.5), in particular L. chuni globins, which might be an advantage allowing L. chuni to dig deeply for sulfides and remain buried for long periods in reduced sediments.

Dissociation in a polymerization model of homochirality.

A fully self-contained model of homochirality is presented that contains the effects of both polymerization and dissociation. The dissociation fragments are assumed to replenish the substrate from which new monomers can grow and undergo new polymerization. The mean length of isotactic polymers is found to grow slowly with the normalized total number of corresponding building blocks. Alternatively, if one assumes that the dissociation fragments themselves can polymerize further, then this corresponds to a strong source of short polymers, and an unrealistically short average length of only 3. By contrast, without dissociation, isotactic polymers becomes infinitely long.

Homochiral growth through enantiomeric cross-inhibition.

The stability and conservation properties of a recently proposed polymerization model are studied. The achiral (racemic) solution is linearly unstable once the relevant control parameter (here the fidelity of the catalyst) exceeds a critical value. The growth rate is calculated for different fidelity parameters and cross-inhibition rates. A chirality parameter is defined and shown to be conserved by the nonlinear terms of the model. Finally, a truncated version of the model is used to derive a set of two ordinary differential equations and it is argued that these equations are more realistic than those used in earlier models of that form.

Protein tyrosine phosphorylation during meiotic divisions of starfish oocytes.

We have used an antibody specific for phosphotyrosine to investigate protein phosphorylation on tyrosine during hormone-induced maturation of starfish oocytes. Analysis of immunoprecipitates from cortices of in vivo labeled Marthasterias glacialis oocytes revealed the presence of labeled phosphotyrosine-containing proteins only after hormone addition. Six major phosphoproteins of 195, 155, 100, 85, 45, and 35 kDa were detected. Total activity in immunoprecipitates increased until first polar body emission and was greatly reduced upon completion of meiosis but some proteins exhibited different kinetics. The labeling of the 155-kDa protein reached a maximum at germinal vesicle breakdown, while the 35-kDa appeared later and disappeared after polar body emission. Similar results were obtained with Asterias rubens oocytes. In vitro phosphorylation of cortices showed that tyrosine kinase activity is a major protein kinase activity in this fraction, the main endogenous substrate being a 68-kDa protein. The proteins phosphorylated on tyrosine in vitro were almost similar in extracts from oocytes treated or not with the hormone.

Melanin-concentrating hormone (MCH) immunoreactivity in the brain and pituitary of the dogfish Scyliorhinus canicula. Colocalization with alpha-melanocyte-stimulating hormone (alpha-MSH) in hypothalamic neurons.

The distribution of melanin-concentrating hormone (MCH) in the central nervous system of the dogfish Scyliorhinus canicula was determined by indirect immunofluorescence and peroxidase-anti-peroxidase techniques, using an antiserum raised against synthetic salmon MCH. Three groups of MCH-positive cell bodies were localized in the posterior hypothalamus. The most prominent cell group was detected in the nucleus sacci vasculosi. Scattered MCH-immunoreactive cells were observed in the nucleus tuberculi posterioris and in the nucleus lateralis tuberis. At the pituitary level, the caudal part of the median lobe of the pars distalis contained strongly MCH-positive perikarya. Some of these cells were liquor-contacting-type. Immunoreactive fibers originating from the hypothalamic perikarya projected throughout the dorsal wall of the posterior hypothalamus. Positive fibers were also detected within the thalamus and the central gray of the mesencephalon. The distribution of MCH-containing neurons was compared to that of alpha-MSH-immunoreactive elements using consecutive, 5-micron thick sections. Both MCH- and alpha-MSH-immunoreactive peptides were found in the same neurons of the nucleus sacci vasculosi. These data suggest that MCH and alpha-MSH, two neuropeptides which exert antagonistic activities on skin melanophores, may also act in a coordinate manner in the central nervous system of cartilaginous fish.

Localization and characterization of prolactin-like immunoreactivity in the pituitary of the frog Rana ridibunda.

Distribution and quantification of PRL in the pituitary gland of the frog Rana ridibunda were investigated using a high-affinity antiserum raised against bullfrog prolactin (PRL). The immunoreactive PRL-producing cells were distributed throughout the pars distalis, the highest density of cells being observed in the rostral region of the adenohypophysis facing the neurointermediate lobe. The dorsal region of the pars distalis contained only a few scattered PRL-immunoreactive cells. At the electron microscopic level, PRL-containing cells were visualized using the immunogold procedure. PRL-immunoreactive material was exclusively stored in secretory granules (size ranging from 200 to 700 nm in diameter). Neither the rough endoplasmic reticulum nor the Golgi apparatus were immunolabeled. Using a radioimmunoassay method we have compared the displacement curves obtained with bullfrog PRL and acetic extracts from Rana ridibunda pituitary. The two binding curves were not completely parallel, suggesting the existence of slight variations of the amino acid sequences of PRL in the two species. The concentration of PRL in the green frog adenohypophysis appeared somewhat higher (35.3 +/- 8.8 micrograms/mg protein) than that in the bullfrog pituitary. These results validate the use of an antiserum to bullfrog PRL to investigate the regulation of PRL secretion in Rana ridibunda.

Immunohistochemical localization of gonadotropin-releasing-hormone-associated peptide in the brain of the frog.

Gonadotropin-releasing-hormone (GnRH)-associated peptide (GnRH-AP) is a 56 amino acid neuropeptide derived from the GnRH prohormone. GnRH-AP corresponds to the C-terminal fragment flanking the GnRH peptide. Using an antiserum raised against human GnRH-AP [1-56], or against human GnRH, we have investigated the neuronal systems containing either peptide in the central nervous system of the frog Rana ridibunda by immunohistological techniques. A main group of GnRH-AP-containing perikarya was found in a dorsoventral orientation of the supra anterior preoptic area (SAPA) just in front of the preoptic recess. Fibers originating from these perikarya projected rostrally toward the medial septal nucleus and the diagonal band of Broca. A network of GnRH-AP-immunoreactive (ir) fibers runs caudally from the SAPA toward the ventral hypothalamus. A high density of GnRH-AP-ir terminals was found in the median eminence. A few positive fibers were detected in the neural lobe of the pituitary, particularly in the region bordering the pars intermedia. Labelling of consecutive sections by either GnRH-AP or GnRH antibodies showed that GnRH and GnRH-AP-like irs were contained in the same cells of the SAPA. The double-staining technique with electrophoretic elution confirmed the colocalization of GnRH and GnRH-AP within the same neurons. Such a coexistence indicates that frog GnRH originates from a high molecular weight precursor which is closely related to rat pro-GnRH. The relative preservation of the C-terminal sequence of the pro-GnRH during evolution suggests that GnRH-AP may possess intrinsic biological activity. The high density of GnRH-AP-containing neurons projecting through the external zone of the median eminence would support the concept that GnRH-AP is involved in the modulation of pituitary hormone secretion.

Coexistence of melanin-concentrating hormone and alpha-melanocyte-stimulating hormone immunoreactivities in the central nervous system of the locust, Locusta migratoria.

The distribution of melanin concentrating hormone (MCH) in the central nervous system of the locust Locusta migratoria was studied by the indirect immunofluorescence technique, using antibodies against salmon MCH. Most MCH-immunoreactive perikarya were found in the optic lobes at both sides of the brain, dorsally with respect to the lamina ganglionaris. The same neurons also contain alpha-melanocyte-stimulating hormone (alpha-MSH)-like material. In addition, a moderate number of MCH-like neurons, which were devoid of alpha-MSH-immunoreactive substances, was observed in the pars intercerebralis. Bright immunofluorescent fibers were visualized in various regions of the central nervous system of the locust: the optic lobes, the ocelli, the proto-and deuterocerebrum, the subesophageal connectives and the corpora cardiaca. In the ventral nerve cord and the subesophageal ganglion, where alpha-MSH-like cell bodies were encountered, MCH immunoreactive perikarya were absent and immunoreactive fibers were scarce. The coexistence of MCH and alpha-MSH-immunoreactive material within the same specific neurons might indicate an evolutionary relationship of both peptides.

Co-distribution of neuropeptide Y and its C-terminal flanking peptide in the brain and pituitary of the frog Rana ridibunda.

By means of the peroxidase-anti-peroxidase technique, the distribution of neuropeptide tyrosine (NPY) and its C-terminal flanking peptide (C-PON) has been studied on serial sections of the brain and pituitary of the frog Rana ridibunda. Throughout the brain, NPY and C-PON-immunoreactive perikarya exhibited a remarkable co-distribution. These two peptides were found to be co-located within the same cell bodies in various brain regions including the dorsal and ventral pallium, the dorsal and ventral infundibular nuclei and the preoptic nucleus. The distribution of NPY- and C-PON-containing fibers in the brain and pituitary was similar. Sequential double immunohistochemical staining using the indirect immunofluorescence method, showed that NPY and C-PON were actually located within the same nerve processes throughout the frog brain and in the intermediate lobe of the pituitary. These studies indicate that the deduced C-PON sequence is present within the frog precursor to NPY and is formed in vivo in the frog brain. Like NPY, C-PON is transported distally in nerve terminals and is likely released with NPY in various regions of the brain and in the intermediate lobe of the pituitary.

Coexistence of melanin-concentrating hormone (MCH) and alpha-melanocyte-stimulating hormone (alpha-MSH) in the preoptic nucleus of the frog brain.

Coexistence of MCH- and alpha-MSH-like peptides in specific neurons of the frog hypothalamus has been investigated on serial frozen sections using the indirect immunofluorescence method. In the anterior region of the preoptic nucleus, perikarya containing MCH- and alpha-MSH-immunoreactive materials were co-distributed and the two peptides were generally co-sequestered within the same neurons. In contrast, alpha-MSH immunoreactive neurons of the ventral infundibular nucleus did not contain any MCH-like peptide. These data suggest that MCH and alpha-MSH are transported by the same nerve fibers originating from preoptic perikarya and are likely released together by axon terminals. Since MCH and alpha-MSH exert antagonistic hormonal activities on dermal melanophores, our results suggest that the two regulatory peptides may also interact in the central nervous system.

Localization of melanin-concentrating hormone-like immunoreactivity in the brain and pituitary of the frog Rana ridibunda.

The distribution of melanin-concentrating hormone (MCH) in the central nervous system of the frog Rana ridibunda was determined by the indirect immunofluorescence technique using antibodies against synthetic salmon MCH, generated in rabbits. The most prominent group of MCH-like containing perikarya was detected in the preoptic nucleus. Comparatively, a moderate number of cell bodies was observed in the dorsal infundibular nucleus and in the ventral thalamic area. Brightly immunofluorescent nerve bundles were found in the preoptic nucleus and in the ventral infundibular nucleus, coursing towards the internal zone of the median eminence and the pituitary stalk. An intense network of immunofluorescent fibers was localized in the neural lobe of the pituitary. The subcellular localization of MCH-like material was studied in the neurohypophysis using the immunogold technique. It was demonstrated that MCH-like material was contained in dense core vesicles (80-90 mm in diameter) within specific nerve terminals. The present findings indicate that, in amphibians, MCH-like peptide is located in specific hypothalamic neurons. Our data suggest that MCH may be released by neurohypophyseal nerve endings as a typical neurohormone.

Disturbance of a met-enkephalin-like hormone in the hepatopancreas of crabs contaminated by metals.

This study combines trace-metal analysis with an immunofluorescent detection of a methionine-enkephalin-like substance in the digestive gland of the shore crab, Carcinus maenas L. The crabs were taken from two sites: Saint Nazaire and Le Croisic, the first being polluted in comparison to the second. The experimental crabs were also taken in Le Croisic and contaminated with Cd, Pb, Cu, and Zn during 1 to 3 weeks in the laboratory. The immunohistological observations indicate a change in the localization of the immunofluorescent methionine-enkephalin-like substance in the cells of the tubules constituting the digestive gland. In crabs from the clean site, experimentally starved or not, the immunofluorescence appears mostly basal while it exhibits an apical localization in metal-contaminated crabs or crabs caught in the polluted area. The detected substance, the nature of which remains unknown, accompanies cytoplasmic secretory granules during their migration to the cellular apex preceding the apocrine secretion. This change of the immunoreactivity enables the detection of metal contamination but it is nonspecific and therefore, a general environment pollution could produce the same phenomenon. In the particular case of zinc, this alteration appears at a Zn concentration in seawater which does not disturb the natural level of this essential metal in the digestive gland of C. maenas.

Immunohistological detection of methionine-enkephalin-like and endorphin-like material in the digestive tract and in the nervous system of the mussel: Mytilus edulis L.

Using the indirect immunofluorescence technique, methionine-enkephaline-like, alpha- and beta-endorphin-like peptides were detected on whole body sections of Mytilus edulis L. Met-enkephalin-like immunoreactivity was localized in the epithelium of the digestive tract, in the hepatopancreas, and in the nervous system. The immunoreactive cell bodies were very abundant in the anterior gastric epithelium, but sparse in the terminal portion of the digestive tract. By their basal processes the immunoreactive cells were in contact with a plexus of immunoreactive cells and fibers located in the connective tissue underlying the digestive epithelium. In the principal hepatopancreatic ducts, isolated cells showing met-enkephalin-like immunoreactivity were detected between the epithelial cells and the basal lamina. A few immunoreactive cells and fibers were observed between the hepatopancreatic tubules. The three pairs of nervous ganglia contained in their cortical layer numerous met-enkephalin-like immunoreactive perikarya. Their central area possessed fluorescent immunoreactive bundles of fibers extending to the commissures, the connectives, and the nerves. Met-enkephalin-like immunoreactive fibers were detected between the smooth muscle cells. At the surface of these smooth muscle cells, immunopositive met-enkephalin-like tapered nervous endings were observed. The alpha- and beta-endorphin antisera produced a positive immunoreaction in some gastric epithelial cells, in some perikarya of the pedal ganglia, and in some nervous fibers. The endorphin-like structures were far less abundant than the met-enkephalin-like structures, but very close to them.

Detection of endocrine cells by immunofluorescence method in the gastroenteropancreatic system of the adult eel, glass-eel, and leptocephalic larva (Anguilla anguilla L.).

Five antisera against insulin (Ins), glucagon (Glu), somatostatin (SRIF), met-enkephalin (met-enk), and serotonin (5-HT) were used for immunofluorescence detection of endocrine cells in pancreas and gastrointestinal tract (GIT) of the European eel (Anguilla anguilla L.) at three stages of development (leptocephalic larva, glass-eel, and adult eel). Comparable distribution of endocrine cells was observed for adults and glass-eels. In their pancreatic islets, positive immunoreactions were obtained only for Ins, SRIF, and Glu; this later was also present in the pancreatic ducts. 5-HT cells were present throughout the GIT. SRIF cells were situated mostly in the stomach and less in the intestine. Met-enk cells were abundant in the pyloric cecum, but less frequent in the intestinal mucosa. Glu cells were present only in the intestine. No insulin-immunoreactive cells could be detected in the GIT. The pancreatic islets of leptocephalic larvae exhibited a strong reaction for SRIF, a weak reaction for Glu, and none at all for Ins, met-Enk, or 5-HT. The GIT of these larvae contained numerous met-enk cells, mainly in the foregut. In the fore- and midgut, cells exhibited a weak fluorescence after treatment with Glu antiserum. No positive immunoreactive cells were observed with 5-HT, SRIF, or Ins antisera.