Discovery of prolactin-like in lamprey: Role in osmoregulation and new insight into the evolution of the growth hormone/prolactin family.

We used a representative of one of the oldest extant vertebrate lineages (jawless fish or agnathans) to investigate the early evolution and function of the growth hormone (GH)/prolactin (PRL) family. We identified a second member of the GH/PRL family in an agnathan, the sea lamprey (Petromyzon marinus). Structural, phylogenetic, and synteny analyses supported the identification of this hormone as prolactin-like (PRL-L), which has led to added insight into the evolution of the GH/PRL family. At least two ancestral genes were present in early vertebrates, which gave rise to distinct GH and PRL-L genes in lamprey. A series of gene duplications, gene losses, and chromosomal rearrangements account for the diversity of GH/PRL-family members in jawed vertebrates. Lamprey PRL-L is produced in the proximal pars distalis of the pituitary and is preferentially bound by the lamprey PRL receptor, whereas lamprey GH is preferentially bound by the lamprey GH receptor. Pituitary PRL-L messenger RNA (mRNA) levels were low in larvae, then increased significantly in mid-metamorphic transformers (stage 3); thereafter, levels subsided in final-stage transformers and metamorphosed juveniles. The abundance of PRL-L mRNA and immunoreactive protein increased in the pituitary of juveniles under hypoosmotic conditions, and treatment with PRL-L blocked seawater-associated inhibition of freshwater ion transporters. These findings clarify the origin and divergence of GH/PRL family genes in early vertebrates and reveal a function of PRL-L in osmoregulation of sea lamprey, comparable to a role of PRLs that is conserved in jawed vertebrates.

Roles of leptin in initiation of acquired growth hormone resistance and control of metabolism in rainbow trout.

Catabolic conditions often induce concomitant changes in plasma leptin (Lep), growth hormone (GH), and insulin growth factor I (IGF-I) levels in teleost fish, but it is unclear whether these parts of the endocrine system are responding independently or functionally linked. In this study, fasted rainbow trout was used to study the effects of Lep on the GH-IGF-I system and metabolism. Fish were implanted intraperitoneally with recombinant rainbow trout Lep pellets and remained unfed. After 4 days, plasma GH levels were elevated in the Lep-treated fish in a dose-dependent manner; the expression of hepatic igf1 and plasma IGF-I levels were suppressed accordingly. In vitro Lep treatment reversed ovine GH (oGH)-stimulated expression of igf1 and igf2 in hepatocytes isolated from fasted fish, similar to the inhibitory effects of the MEK1/2 inhibitor U0126 treatment. However, Lep treatment alone had no effect on the expression of igfs or oGH-stimulated ghr2a expression in the hepatocytes. These results demonstrate an additive effect of Lep on suppression of IGF-I under catabolic conditions, indicating that Lep is likely involved in initiation of acquired GH resistance. Although the Lep-implant treatment had no effect on standard metabolic rate, it significantly suppressed gene expression of hepatic hydroxyacyl-CoA dehydrogenase, phosphoenolpyruvate carboxykinase, and glucose 6-phosphatase, which are key enzymes in lipid utilization and gluconeogenesis, in different patterns. Overall, this study indicates that the Lep increase in fasting salmonids is an important regulatory component for physiological adaptation during periods of food deprivation, involved in suppressing growth and hepatic metabolism to spare energy expenditure.

Impaired central leptin signaling and sensitivity in rainbow trout with high muscle adiposity.

The hormone leptin has been identified in all vertebrate classes, but its physiological roles in non-mammalian vertebrates are not well defined. To elucidate leptin regulation in energy homeostasis in a teleost fish species, this study compares hypothalamic and pituitary leptin signaling systems in energetically divergent rainbow trout lines selected for low (lean line, LL) and high (fat line, FL) muscle adiposity under feeding and starvation conditions. In fed fish, hypothalamic gene expression and protein density of the full-functional leptin receptor (LepRL), as well as a leptin binding protein (LepBP) expression, are lower in FL than LL fish. The FL fish have also lower activation of leptin-relevant signaling pathways involving protein kinase B (Akt) and extracellular signal-related kinase. These observations suggests impaired central leptin action in FL fish. During fasting, hypothalamic LepRL and LepBP expression, as well as active Akt levels are downregulated after one week, while pituitary LepRL expression is upregulated, in the LL fish only. After four weeks, hypothalamic LepRL protein levels return to normal levels in both fish lines and Akt is reactivated, although not to the same extent in FL as in LL fish, indicating that FL fish have low leptin sensitivity to nutritional changes. Neuropeptide Y and orexin expression is downregulated to similar levels in both fish lines after one-week fasting. The divergent leptin system profiles between the two fish lines demonstrate that phenotypic selection for high muscle adiposity affects leptin endocrinology, indicating regulatory roles for leptin in rainbow trout energy homeostasis.

Ocean acidification and host-pathogen interactions: blue mussels, Mytilus edulis, encountering Vibrio tubiashii.

Ocean acidification (OA) can shift the ecological balance between interacting organisms. In this study, we have used a model system to illustrate the interaction between a calcifying host organism, the blue mussel Mytilus edulis and a common bivalve bacterial pathogen, Vibrio tubiashii, with organisms being exposed to a level of acidification projected to occur by the end of the 21st century. OA exposures of the mussels were carried out in relative long-term (4 months) and short-term (4 days) experiments. We found no effect of OA on the culturability of V. tubiashii, in broth or in seawater. OA inhibited mussel shell growth and impaired crystalline shell structures but did not appear to affect mussel immune parameters (i.e haemocyte counts and phagocytotic capacity). Despite no evident impact on host immunity or growth and virulence of the pathogen, V. tubiashii was clearly more successful in infecting mussels exposed to long-term OA compared to those maintained under ambient conditions. Moreover, OA exposed V. tubiashii increased their viability when exposed to haemocytes of OA-treated mussel. Our findings suggest that even though host organisms may have the capacity to cope with periods of OA, these conditions may alter the outcome of host-pathogen interactions, favouring the success of the latter.

Long-term fasting in the anadromous Arctic charr is associated with downregulation of metabolic enzyme activity and upregulation of leptin A1 and SOCS expression in the liver.

The life strategy of the anadromous Arctic charr (Salvelinus alpinus) includes several months of voluntary fasting during overwintering in freshwater, leading to emaciation prior to seawater migration in spring. In this study we compared changes in condition, substrate utilization and liver metabolism between captive anadromous charr subjected to food deprivation during late winter and spring, and conspecifics fed in excess. In March, nine out of the 10 sampled fed fish had not eaten, indicating that they were in a voluntary anorexic state. In June, the fed fish were eating and all had higher body mass, condition factor and adiposity than in March. In fasted fish there were only small decreases in body mass, condition factor and adiposity between March and May, but all these parameters decreased markedly from May to June. The fasted fish were depleted in fat and glycogen in June, had suppressed activity of hepatic enzymes involved in lipid metabolism (G6PDH and HOAD) and seemed to rely on protein-derived glucose as a major energy source. This was associated with upregulated liver gene expression of leptin A1, leptin A2, SOCS1, SOCS2 and SOCS3, and reduced IGF-I expression. In an in vitro study with liver slices it was shown that recombinant rainbow trout leptin stimulated SOCS1 and SOCS3 expression, but not SOCS2, IGF-I or genes of enzymes involved in lipid (G6PDH) and amino acid (AspAT) metabolism. It is concluded that liver leptin interacts with SOCS in a paracrine fashion to suppress lipolytic pathways and depress metabolism when fat stores are depleted.

Biosilicification of loricate choanoflagellate: organic composition of the nanotubular siliceous costal strips of Stephanoeca diplocostata.

Loricate choanoflagellates (unicellular, eukaryotic flagellates; phylum Choanozoa) synthesize a basket-like siliceous lorica reinforced by costal strips (diameter of approximately 100 nm and length of 3 µm). In the present study, the composition of these siliceous costal strips is described, using Stephanoeca diplocostata as a model. Analyses by energy-dispersive X-ray spectroscopy (EDX), coupled with transmission electron microscopy (TEM), indicate that the costal strips comprise inorganic and organic components. The organic, proteinaceous scaffold contained one major polypeptide of mass 14 kDa that reacted with wheat germ agglutinin. Polyclonal antibodies were raised that allowed mapping of the proteinaceous scaffold, the (glyco)proteins, within the costal strips. Subsequent in vitro studies revealed that the organic scaffold of the costal strips stimulates polycondensation of ortho-silicic acid in a concentration- and pH-dependent way. Taken together, the data gathered indicate that the siliceous costal strips are formed around a proteinaceous scaffold that supports and maintains biosilicification. A scheme is given that outlines that the organic template guides both the axial and the lateral growth of the strips.

Divergent genes encoding the putative receptors for growth hormone and prolactin in sea lamprey display distinct patterns of expression.

Growth hormone receptor (GHR) and prolactin receptor (PRLR) in jawed vertebrates were thought to arise after the divergence of gnathostomes from a basal vertebrate. In this study we have identified two genes encoding putative GHR and PRLR in sea lamprey (Petromyzon marinus) and Arctic lamprey (Lethenteron camtschaticum), extant members of one of the oldest vertebrate groups, agnathans. Phylogenetic analysis revealed that lamprey GHR and PRLR cluster at the base of gnathostome GHR and PRLR clades, respectively. This indicates that distinct GHR and PRLR arose prior to the emergence of the lamprey branch of agnathans. In the sea lamprey, GHR and PRLR displayed a differential but overlapping pattern of expression; GHR had high expression in liver and heart tissues, whereas PRLR was expressed highly in the brain and moderately in osmoregulatory tissues. Branchial PRLR mRNA levels were significantly elevated by stage 5 of metamorphosis and remained elevated through stage 7, whereas levels of GHR mRNA were only elevated in the final stage (7). Branchial expression of GHR increased following seawater (SW) exposure of juveniles, but expression of PRLR was not significantly altered. The results indicate that GHR and PRLR may both participate in metamorphosis and that GHR may mediate SW acclimation.

Immunolocalization of matrix proteins in nacre lamellae and their in vivo effects on aragonitic tablet growth.

How matrix proteins precisely control the growth of nacre lamellae is an open question in biomineralization research. Using the antibodies against matrix proteins for immunolabeling and in vivo experiments, we investigate the structural and functional roles of EDTA-soluble matrix (SM) and EDTA-insoluble matrix (ISM) proteins in nacre biomineralization of the pearl oyster Pinctada fucata. Immunolabeling reveals that a SM protein, nacrein, distributes within aragonitic tablets and intertabular matrix. An ISM protein, which we named P43, has been specifically recognized by polyclonal antibodies raised against the recombinant protein of P. fucata bone morphogenetic protein 2 in immunoblot analysis. Immunolabeling indicates that P43 is localized to interlamellar sheet, and also embedded within aragonitic tablets. Although nacrein and P43 both distribute within aragonitic tablets, they function differently in aragonitic tablet growth. When nacrein is suppressed by the antibodies against it in vivo, crystal overgrowth occurs, indicating that this SM protein is a negative regulator in aragonitic tablet growth. When P43 is suppressed in vivo, the organo-mineral assemblage is disrupted, suggesting that P43 is a framework matrix. Taken together, SM and ISM proteins are indispensable factors for the growth of nacre lamellae, controlling crystal growth and constructing the framework of aragonitic tablets.

Characterization of calcium deposition and shell matrix protein secretion in primary mantle tissue culture from the marine pearl oyster Pinctada fucata.

In this study, we established and characterized a long-term primary mantle tissue culture from the marine pearl oyster Pinctada fucata for in vitro investigation of nacre biomineralization. In this culture system, the viability of mantle tissue cells lasted up to 2 months. The tissue cells were demonstrated to express nacre matrix proteins by RT-PCR, and a soluble shell matrix protein, nacrein, was detected in the culture medium by Western blot analysis. On the other hand, 15 days after initiating culture, a large amount of calcium deposits with major elements, including calcium, carbon, and oxygen, were generated in the mantle explants and cell outgrowth area. The quantity and size of calcium deposits increased with the prolonged cultivation, and their location and nanogranular structure suggested their biogenic origin. These calcium deposits specifically appeared in mantle tissue cultures, but not in heart tissue cultures. Taken together, these results demonstrate that the mantle tissue culture functions similarly to mantle cells in vivo. This study provides a reliable approach for the further investigation on nacre biomineralization at the cellular level.

The Impact of Initial Energy Reserves on Growth Hormone Resistance and Plasma Growth Hormone-Binding Protein Levels in Rainbow Trout Under Feeding and Fasting Conditions.

The growth hormone (GH)-insulin-like growth factor I (IGF-I) system regulates important physiological functions in salmonid fish, including hydromineral balance, growth, and metabolism. While major research efforts have been directed toward this complex endocrine system, understanding of some key aspects is lacking. The aim was to provide new insights into GH resistance and growth hormone-binding proteins (GHBPs). Fish frequently respond to catabolic conditions with elevated GH and depressed IGF-I plasma levels, a condition of acquired GH resistance. The underlying mechanisms or the functional significance of GH resistance are, however, not well understood. Although data suggest that a significant proportion of plasma GH is bound to specific GHBPs, the regulation of plasma GHBP levels as well as their role in modulating the GH-IGF-I system in fish is virtually unknown. Two in vivo studies were conducted on rainbow trout. In experiment I, fish were fasted for 4 weeks and then refed and sampled over 72 h. In experiment II, two lines of fish with different muscle adiposity were sampled after 1, 2, and 4 weeks of fasting. In both studies, plasma GH, IGF-I, and GHBP levels were assessed as well as the hepatic gene expression of the growth hormone receptor 2a (ghr2a) isoform. While most rainbow trout acquired GH resistance within 4 weeks of fasting, fish selected for high muscle adiposity did not. This suggests that GH resistance does not set in while fat reserves as still available for energy metabolism, and that GH resistance is permissive for protein catabolism. Plasma GHBP levels varied between 5 and 25 ng ml-1, with large fluctuations during both long-term (4 weeks) fasting and short-term (72 h) refeeding, indicating differentiated responses depending on prior energy status of the fish. The two opposing functions of GHBPs of prolonging the biological half-life of GH while decreasing GH availability to target tissues makes the data interpretation difficult, but nutritional regulatory mechanisms are suggested. The lack of correlation between hepatic ghr2a expression and plasma GHBP levels indicate that ghr2a assessment cannot be used as a proxy measure for GHBP levels, even if circulating GHBPs are derived from the GH receptor molecule.

Cloning, characterization and expression analysis of calcium channel beta subunit from pearl oyster (Pinctada fucata).

The absorption, transport and localization of calcium underlie the basis of biomineralization, and Ca(2+) entry into epithelial cell is the primary step in shell formation. However, the related mechanism of Ca(2+) transport is poorly documented at the gene or protein level. L-type voltage-dependent calcium channels may be involved in calcium transport for biomineralization in some marine invertebrates. In this study, a full-length cDNA of a voltage-dependent calcium channel beta subunit from Pinctada fucata (PCabeta) was cloned, and its amino acid sequence was deduced. PCabeta shared 51%-67% apparently sequence identity with voltage-dependent calcium channel beta subunits from other species. However, PCabeta was much shorter than other voltage-dependent calcium channel beta subunits particularly at the carboxyl terminus, indicating that it is likely a truncated beta subunit isoform. Semi-quantitative RT-PCR analysis showed that PCabeta was expressed in all the tested tissues and that it had a higher expression level in the gill tissue and hemolymph than in other tissues, suggesting that L-type voltage-dependent calcium channels are responsible for Ca(2+) absorption in the gill and Ca(2+) entry into hemocytes. In the mantle, PCabeta mRNA was predominantly expressed in the inner and middle folds of the mantle epithelium, suggesting that L-type voltage-dependent calcium channels are involved in Ca(2+) absorption from the ambient medium in the mantle. All these results suggest that voltage-dependent calcium channels are involved in Ca(2+) uptake and transport during oyster biomineralization.

Characterization of a thermostable alkaline phosphatase from a novel species Thermus yunnanensis sp. nov. and investigation of its cobalt activation at high temperature.

A thermostable alkaline phosphatase with high specific activity and thermal resistance was purified from a novel species of Thermus sp. named as Thermus yunnanensis sp. nov. The enzyme contains a single peptide with a molecular mass of about 52 kDa on SDS-PAGE analysis and appears to be a homodimer in solution with the molecular mass of 104 kDa. The optimal pH and temperature for its activities are pH 8.0-10.0 and 70-80 degrees C, respectively. The catalytic activities of the enzyme are metal ion dependent, and Mg2+, Zn2+ and Co2+ are the main activators. Among these, Co2+ is the most active stimulator and has unique activation effect at high temperature. Metal binding analysis showed the binding of Mg2+ at the metal binding site was easy to loss in the thermoinactivation, and Co2+ was apt to bind at that site and kept the favorable configuration of catalysis, which would result high activation in the incubation with Co2+ at high temperature. According to this study, a model was proposed for the explanation of the activation and the results of actual experiments demonstrated the validity of the model.

Acute anorexigenic action of leptin in rainbow trout is mediated by the hypothalamic Pi3k pathway.

Leptin (Lep) is an anorexigenic hormone and regulates appetite-related neuropeptides in mammals. A number of neuropeptides have also been linked to appetite regulation in teleost fish, but Lep signaling activation and effects on appetite-regulating neurons are poorly elucidated in early vertebrates. This study uses cellular, tissue and organismal approaches to elucidate the acute, central Lep action in rainbow trout. The results demonstrate that Lep activates phosphorylation of protein kinase B (Akt) and signal transducer and activator of transcription 3 in rainbow trout hypothalamus-derived cells, and that the phosphatidylinositol-3-kinase (Pi3k) inhibitor LY294002 can suppress the Lep-induced Akt phosphorylation. Intracerebroventricular (ICV) Lep administration strongly suppresses food intake at the doses of 0.05 and 0.5 µg Lep fish(-1) At low dose, Lep stimulates hypothalamic transcription of anorexigenic cocaine- and amphetamine-regulated transcript (Cart) and orexigenic neuropeptide Y. At high dose, Lep stimulates hypothalamic transcription of anorexigenic proopiomelanocortin (Pomc) A1, A2, and B, while coinjection with LY294002 reverses this upregulation. The data suggest that the anorexigenic action of Lep in rainbow trout is mediated through stimulation of the anorexigenic neuropeptides Pomc and Cart. Furthermore, ICV Lep treatment increases phosphor-Akt-immunoreactive cells in the nucleus lateralis tuberis, periventricular zone along infundibulum, and lateral recess surrounded by nucleus anterior tuberis, while LY294002 inhibits this effect. Lep receptor-immunoreactive cells are also predominant in these regions. These results demonstrate that Lep activates the Pi3k-Akt pathway in the lateral tuberal hypothalamus of rainbow trout for acute appetite regulation, indicating the conservation of anorexigenic Lep action in the mediobasal hypothalamus.

Energy stores, lipid mobilization and leptin endocrinology of rainbow trout.

The physiological role of leptin in fish is not fully elucidated. In the present study, the involvement of the leptin system in lipid deposition and mobilization in rainbow trout during feeding and 1, 2 and 4 weeks of fasting was investigated in two lines of rainbow trout with different muscle and visceral adiposity: a fat line (FL) with high total energy reserves, high muscle adiposity, but low visceral adiposity and a lean line (LL) with lower total energy reserves and lower muscle adiposity, but higher visceral adiposity. During 4 weeks of fasting, muscle lipids decreased by 63 % in the FL fish, while no such energy mobilization from muscle occurred in the LL fish. On the other hand, lipid stores in liver and visceral adipose tissue was utilized to a similar extent by the two fish lines during fasting. Under normal feeding conditions, plasma leptin levels were higher in the LL than the FL fish, suggesting a possible contribution of visceral adipocytes to plasma leptin levels. Plasma leptin-binding protein levels did not differ between the lines and were not affected by fasting. After 4 weeks of fasting, the long leptin receptor and the leptin-binding protein isoforms 1 and 3 muscle expression increased in the LL fish, as well as hepatic expression of leptin A1 and the two binding protein isoforms. These responses were not seen in the FL fish. The data suggest that the Lep system in rainbow trout is involved in regulation of energy stores and their mobilization.

Leptin signaling in the rainbow trout central nervous system is modulated by a truncated leptin receptor isoform.

Central leptin (Lep) signaling is important in control of appetite and energy balance in mammals, but information on Lep signaling and physiological roles in early vertebrates is still lacking. To elucidate fish Lep signaling activation and modulation, a long-form Lep receptor (LepRL) and a truncated LepR (LepRT) are functionally characterized from rainbow trout. The receptors generated in alternatively splicing events have identical extracellular and transmembrane domains but differ in the intracellular sequence, both in length and identity. Gene transfection experiments show that LepRL is expressed as a 125-kDa protein in rainbow trout hepatoma cell line RTH-149, whereas LepRT is 100 kDa; both receptors specifically bind Lep. Homogenous Lep induces tyrosine phosphorylation of Janus kinase 2 and signal transducer and activation of transcription 3 in LepRL-expressing RTH-149 cells. This response is diminished in cells coexpressing LepRL and LepRT, suggesting that the LepRT which lacks these kinase-associated motifs competes with the LepRL for Lep availability, thus attenuating the Lep signal. Both receptor genes are highly expressed in the central nervous system. The mRNA levels of LepRT in hypothalamus, but not LepRL, change postprandially, with decreased transcription at 2 hours postfeeding and then elevated at 8 hours, concomitant with changes in proopiomelanocortin-A1 transcription. However, both receptors have no change in mRNA levels during 3 weeks of fasting. These data indicate that LepRT transcription is more likely a mechanism for modulating Lep effects on short-term feed intake than in regulating energy balance in the long term. In vitro and physiological characterization of LepR isoforms indicates divergent Lep signaling modulation patterns among vertebrates with different life histories and metabolic profiles.

Alternative splice variants of the rainbow trout leptin receptor encode multiple circulating leptin-binding proteins.

In mammals, leptin (Lep) binding proteins (LepBPs) derived from Lep receptor (LepR) gene or protein bind most of the circulating Lep, but to date, information on LepBPs in nonmammalian vertebrate classes is lacking. This study details the characterization of multiple LepBPs in rainbow trout (Oncorhynchus mykiss), an early poikilothermic vertebrate, and presents the complete coding sequences for 3 of them. Size-exclusion chromatography and cross-linking assay identified plasma proteins bound to Lep ranging from 70 to 100 kDa. LepBPs were isolated from plasma by affinity chromatography, and their binding specificity was assessed by a competitive binding assay. A RIA for LepBPs indicates that plasma LepBP levels decline after fasting for 3 weeks. Immunoblotting of LepBPs using antibodies against different LepR epitopes shows that the LepBPs are indeed LepR isoforms. The alternatively spliced LepR transcripts (LepR(S1-3)) that include only the extracellular segment transcribe the 90-kDa LepBP1, the 80-kDa LepBP2, and the 70-kDa LepBP3, respectively. LepR(S1) generally has lower expression than the long-form LepR in most tissues. LepR(S2) is primarily expressed in adipose tissue, whereas LepR(S3) is expressed abundantly in brain and spleen, and moderately in liver and gills. The mRNA levels of hepatic LepR(S3) increase after 2 weeks of fasting. This study demonstrates a mechanism in fish for the generation of LepBPs that differs from that seen in mammals and indicates that the physiologic action of Lep in these poikilothermic vertebrates can be modulated, both centrally and peripherally, by the differentiated, tissue-specific expression of multiple LepBPs.

Cloning and characterization of the activin like receptor 1 homolog (Pf-ALR1) in the pearl oyster, Pinctada fucata.

The signal transduction mechanisms in mollusks are still elusive since the genome information is incomplete and cell lines are not available. In previous study, we cloned a highly conserved Smad3 homolog (designated as Pf-Smad3) from the pearl oyster, Pinctada fucata. It seems that transforming growth factor beta (TGFbeta) signaling may play similar roles in the oyster as in vertebrate. Here we report a cDNA encoding an activin like receptor 1 homolog (designated as Pf-ALR1) of the oyster, another kind of TGFbeta superfamily member. Compared to the activin receptor-like kinases (ALK) in human, the amino acid sequence of Pf-ALR1 is more similar to that of ALK1, especially the L45 loop. Reverse transcription-polymerase chain reaction results indicate that Pf-ALR1 mRNA is expressed ubiquitously in the adult oyster. Thus, Pf-ALR1 may be important for many physiological processes in the oyster. To lay a basis for further investigation of the TGFbeta signal pathway functions in the oyster shell formation, in this report, the Pf-ALR1 mRNA expression in the oyster mantle was detected by in situ hybridization. The results show that Pf-ALR1 in the oyster mantle is mainly expressed at the inner epithelial cells of the outer fold and the outer epithelial cells of the middle fold, similarly as Pf-Smad3. The mRNA levels of Pf-ALR1 and Pf-Smad3 are all changed after shell notching. These results indicate that both Pf-ALR1 and Pf-Smad3 may take part in shell formation and repair. The results of drug treatment experiments with in-vitro cultured oyster mantle tissue cells demonstrate that the mRNA expression levels of Pf-Smad3, Pf-ALR1 and two oyster nuclear factor-kappaB (NF-kB) members can be adjusted and correlated. All our observations suggest that there should be similar TGFbeta signal pathways in the oyster and vertebrate. However, the potential functions of Pf-ALR1 and the relations of TGFbeta and NF-kB members in the oyster all need to be thoroughly investigated.

Cloning and characterization of Prisilkin-39, a novel matrix protein serving a dual role in the prismatic layer formation from the oyster Pinctada fucata.

Molluscs form their shells out of CaCO(3) and a matrix of biomacromolecules. Understanding the role of matrices may shed some light on the mechanism of biomineralization. Here, a 1401-bp full-length cDNA sequence encoding a novel matrix protein was cloned from the mantle of the bivalve oyster, Pinctada fucata. The deduced protein (Prisilkin-39), which has a molecular mass of 39.3 kDa and an isoelectric point of 8.83, was fully characterized, and its role in biomineralization was demonstrated using both in vivo and in vitro crystal growth assays. Prisilkin-39 is a highly repetitive protein with an unusual composition of Gly, Tyr, and Ser residues. Expression of Prisilkin-39 was localized to columnar epithelial cells of the mantle edge, corresponding to the calcitic prismatic layer formation. Immunostaining in situ and immunodetection in vitro revealed the presence of a characteristic pattern of Prisilkin-39 in the organic sheet and in sheaths around the prisms. Prisilkin-39 binds tightly with chitin, an insoluble polysaccharide that forms the highly structured framework of the shell. Antibody injection in vivo resulted in dramatic morphological deformities in the inner shell surface structure, where large amounts of CaCO(3) were deposited in an uncontrolled manner. Moreover, Prisilkin-39 strictly prohibited the precipitation of aragonite in vitro. Taken together, Prisilkin-39 is the first protein shown to have dual function, involved both in the chitinous framework building and in crystal growth regulation during the prismatic layer mineralization. These observations may extend our view on the rare group of basic matrices and their functions during elaboration of the molluscan shell.

Culture of outer epithelial cells from mantle tissue to study shell matrix protein secretion for biomineralization.

Mantle tissue plays an important role in shell biomineralization by secreting matrix proteins for shell formation. However, the mechanism by which it regulates matrix protein secretion is poorly understood, largely because of the lack of cellular tools for in vitro study and techniques to evaluate matrix protein secretion. We have isolated the outer epithelial cells of the mantle of the pearl oyster, Pinctada fucata, and evaluated cellular metabolism by measuring the secretion of the matrix protein, nacrein. A novel sensitive sandwich enzyme-linked immunosorbent assay (ELISA) was established to quantify nacrein. Mantle explant culture was demonstrated to provide dissociated tissue cells with high viability. Single dissociated cell types from explant culture were separated by density in a discontinuous Percoll gradient. The outer epithelial cells were isolated from other cell types by their higher density and identified by immunolabeling and ultrastructure analysis. ELISA assays revealed that the outer epithelial cells retained the ability to secrete nacrein in vitro. Moreover, increased nacrein secretion resulted from an increased Ca(2+) concentration in the culture media of the outer epithelial cells, in a concentration-dependent manner. These results confirm that outer epithelial cell culture and the ELISA method are useful tools for studying the regulatory mechanisms of shell biomineralization.

N40, a novel nonacidic matrix protein from pearl oyster nacre, facilitates nucleation of aragonite in vitro.

A novel nonacidic matrix protein from pearl oyster nacre has been purified by cation-exchange chromatography. It was designated N40 for the nacreous protein of approximately 40 kDa. On the basis of the extraction method (with Tris-buffered Milli-Q water) and amino acid compositions (Gly- and Ala-rich), N40 was inferred to be a conventional "insoluble matrix protein". Crystallization experiments showed that N40 could facilitate the nucleation of aragonite drastically. So far, among the macromolecules that have been purified from the shell, N40 is an exclusive protein that can nucleate aragonite by itself, without the need for adsorption to a substrate. Thus, the present study has proposed the possibility that the nonacidic shell protein (maybe a conventional "insoluble framework protein") can also directly participate in aragonite nucleation and even act as a nucleation site. It is a valuable supplement to the classic biomineralization theory, in which the soluble acidic proteins of the shell are generally believed to function as a nucleation site.