Administration with emulsion containing py-GnRH peptide into scallop gonad.

<p>A) Shells clamped with clothespin to expose the gonad for injection with emulsion. B) Schematic diagram indicating the position of injection. Gonad intestinal loop is indicated with dashed line in the gonad. AM, adductor muscle; DG, digestive gland; G, gonad; L, lips. C) The treated scallops were placed in net cages for rearing. D) Excised ovary with solidified emulsion taken from the scallop reared for 6 weeks. Arrowheads indicate the solidified emulsion remaining in the gonad.</p

Changes in gonad index (GI) in the scallops after py-GnRH administration during 6 weeks.

<p>The GIs for all specimens (A), females (B), males (C) and hermaphrodites (D) were assessed separately. Black and blue lines for the GnRH(-) and GnRH(+) groups, respectively. All data are presented as a mean ± standard error of the mean. Different superscript letters indicate significant differences within the same treatment group. The parenthetical references indicate specimen number for each group assessed. Asterisks (*) indicate significant differences in the GnRH(+) group compared to GnRH(-) group at a particular sampling point at p < 0.05.</p

Detection of apoptotic cells in the gonads of the scallops after py-GnRH administration in week 2 and 4.

<p>The female gonads of GnRH(+) group in week 2 (A, a, B, b) and hermaphrodite gonads of the GnRH(+) group in week 2 (C, c) and 4 (d, e). A-C) Adjacent sections of a-c were stained by H.E. a-e) The sections were immunostained with DAB substrate to detect apoptotic cells. A, a, B, b) The germinal acini containing abnormal spaces in the ovary are encircled with dashed lines. a-e) Apoptotic cells found in the gonads of GnRH(+) are indicated with arrowheads. GAS; germinal acinus containing spermatogenic cells. The inset in c was captured from another area in the same section indicating that both GAS and germinal acinus containing oocytes exist.</p

Estrogens disrupt serotonin receptor and cyclooxygenase mRNA expression in the gonads of mussels (Mytilus edulis)

No description supplie

Novel Matrix Proteins of <i>Pteria penguin</i> Pearl Oyster Shell Nacre Homologous to the Jacalin-Related β-Prism Fold Lectins

<div><p>Nacreous layers of pearl oyster are one of the major functional biominerals. By participating in organic compound-crystal interactions, they assemble into consecutive mineral lamellae-like photonic crystals. Their biomineralization mechanisms are controlled by macromolecules; however, they are largely unknown. Here, we report two novel lectins termed PPL2A and PPL2B, which were isolated from the mantle and the secreted fluid of <i>Pteria penguin</i> oyster. PPL2A is a hetero-dimer composed of α and γ subunits, and PPL2B is a homo-dimer of β subunit, all of which surprisingly shared sequence homology with the jacalin-related plant lectin. On the basis of knockdown experiments at the larval stage, the identification of PPLs in the shell matrix, and <i>in vitro</i> CaCO₃ crystallization analysis, we conclude that two novel jacalin-related lectins participate in the biomineralization of <i>P. penguin</i> nacre as matrix proteins. Furthermore, it was found that trehalose, which is specific recognizing carbohydrates for PPL2A and is abundant in the secreted fluid of <i>P. penguin</i> mantle, functions as a regulatory factor for biomineralization via PPL2A. These observations highlight the unique functions, diversity and molecular evolution of this lectin family involved in the mollusk shell formation.</p></div

Vascular Smooth Muscle Cells Stimulate Platelets and Facilitate Thrombus Formation through Platelet CLEC-2: Implications in Atherothrombosis

<div><p>The platelet receptor CLEC-2 is involved in thrombosis/hemostasis, but its ligand, podoplanin, is expressed only in advanced atherosclerotic lesions. We investigated CLEC-2 ligands in vessel walls. Recombinant CLEC-2 bound to early atherosclerotic lesions and normal arterial walls, co-localizing with vascular smooth muscle cells (VSMCs). Flow cytometry and immunocytochemistry showed that recombinant CLEC-2, but not an anti-podoplanin antibody, bound to VSMCs, suggesting that CLEC-2 ligands other than podoplanin are present in VSMCs. VSMCs stimulated platelet granule release and supported thrombus formation under flow, dependent on CLEC-2. The time to occlusion in a FeCl₃-induced animal thrombosis model was significantly prolonged in the absence of CLEC-2. Because the internal elastic lamina was lacerated in our FeCl₃-induced model, we assume that the interaction between CLEC-2 and its ligands in VSMCs induces thrombus formation. Protein arrays and Biacore analysis were used to identify S100A13 as a CLEC-2 ligand in VSMCs. However, S100A13 is not responsible for the above-described VSMC-induced platelet activation, because S100A13 is not expressed on the surface of normal VSMCs. S100A13 was released upon oxidative stress and expressed in the luminal area of atherosclerotic lesions. Suspended S100A13 did not activate platelets, but immobilized S100A13 significantly increased thrombus formation on collagen-coated surfaces. Taken together, we proposed that VSMCs stimulate platelets through CLEC-2, possibly leading to thrombus formation after plaque erosion and stent implantation, where VSMCs are exposed to blood flow. Furthermore, we identified S100A13 as one of the ligands on VSMCs.</p></div

Coronary artery smooth muscle cells (CASMCs) stimulated release of α and dense granule contents through CLEC-2.

<p>Washed platelets obtained from the CLEC-2-deficient chimeras were incubated with buffer, CASMCs, CRP, rhodocytin, or lysis buffer for 10 min. The platelets were centrifuged. The amount of secreted 5-hydroxytryptamine (5-HT) (A) or platelet-derived growth factor (PDGF) (B) in the supernatants was measured by ELISA. Platelet lysates were used to measure the total amount of 5-HT or PDGF stored in platelets. The results were expressed as the percentage of secreted 5-HT or PDGF relative to the total amount stored in platelets ± SE (A. n = 9 from three independent experiments, B. n = 12 from four independent experiments). Three asterisks denote p < 0.005.</p

Purification of PPL2s.

<p>A, Affinity chromatography of the secreted fluid of <i>Pteria penguin</i> mantle on a trehalose-Sepharose 4B column. Arrows indicates elution buffer: 0.2 M trehalose in 10 mM Tris-HCl buffer (pH 7.5) containing 0.5 M NaCl and 5 mM CaCl₂. B, Cation-exchange chromatography of adsorbed fraction of trehalose-affinity chromatography of secreted fluid of <i>Pteria penguin</i> mantle on a Resource S column (6.4×30 mm). Flow rate was 1 ml/min. C, SDS-PAGE (15%) profiles of PPL2A and PPL2B under non-reducing (−ME) and reducing (+ME) conditions after ion-exchange chromatography. Lanes 1, 2 and 3 correspond to fractions Nos.1, 2 and 3 in Fig. 1B, respectively. D, 2D gel electrophoresis of PPL2s.</p

Identification of PPL2 in the <i>P. penguin</i> nacreous layer.

<p>A, Purification of matrix proteins from <i>P. penguin</i> nacreous layer by RP-HPLC. B, SDS-PAGE profiles and Western blot analysis of fractions of nacreous layer matrix proteins (F1 to F4) from <i>P. penguin</i> under reducing conditions. Proteins were detected by silver staining. C, Western blot analysis of nacreous layer matrix proteins and PPL2A using anti-PPL2A antibody. D, Western blot analysis of nacreous layer matrix proteins (MP), PPL2A and PPL2B using specific antibodies against PPL2 α, β, and γ subunits, respectively. Chemiluminescence signals were detected by CCD cameras with exposure time of 2∼3 sec. MP(En) indicated the experiments with enhanced exposure time of 30 sec. M: monomer, D: dimer, P: polymer. E, <i>In situ</i> localization of PPL2 as an organic matrix protein in the nacreous layer detected by combined anti-PPL2A antibody (mouse) and HRP-conjugated anti-mouse IgG 2ndary antibody. HRP was detected by DAB stain and enhanced by osmium tetroxide treatment. Arrowheads indicate the positive staining by anti-PPL2A antibody with HRP-conjugated anti-mouse IgG antibody and DAB-OsO₄. Control was treated with HRP-conjugated anti-mouse IgG antibody and DAB-OsO₄ staining without anti-PPL2A antibody.</p

Nucleotide sequences of cDNAs and the corresponding amino acid sequences of PPL2 subunits.

<p>Nucleotide residues and amino acid residues identical to those of PPL2α subunit are indicated by ‘dot’ symbol, respectively. Dash (–) symbol indicates the deletion of nucleotides and amino acids. Underline indicates the putative polyadenylation signal, AATAAA. Shaded region indicates the signal peptide region.</p