The diversity of shell matrix proteins: genome-wide investigation of the pearl oyster, Pinctada fucata.

In molluscs, shell matrix proteins are associated with biomineralization, a biologically controlled process that involves nucleation and growth of calcium carbonate crystals. Identification and characterization of shell matrix proteins are important for better understanding of the adaptive radiation of a large variety of molluscs. We searched the draft genome sequence of the pearl oyster Pinctada fucata and annotated 30 different kinds of shell matrix proteins. Of these, we could identified Perlucin, ependymin-related protein and SPARC as common genes shared by bivalves and gastropods; however, most gastropod shell matrix proteins were not found in the P. fucata genome. Glycinerich proteins were conserved in the genus Pinctada. Another important finding with regard to these annotated genes was that numerous shell matrix proteins are encoded by more than one gene; e.g., three ACCBP-like proteins, three CaLPs, five chitin synthase-like proteins, two N16 proteins (pearlins), 10 N19 proteins, two nacreins, four Pifs, nine shematrins, two prismalin-14 proteins, and 21 tyrosinases. This diversity of shell matrix proteins may be implicated in the morphological diversity of mollusc shells. The annotated genes reported here can be searched in P. fucata gene models version 1.1 and genome assembly version 1.0 ( http://marinegenomics.oist.jp/pinctada_fucata ). These genes should provide a useful resource for studies of the genetic basis of biomineralization and evaluation of the role of shell matrix proteins as an evolutionary toolkit among the molluscs.

Crystallographic characterization of the crossed lamellar structure in the bivalve Meretrix lamarckii using electron beam techniques.

To understand the formation mechanism of crossed lamellar structures in molluskan shells, the crystallographic structural features in the shell of a bivalve, Meretrix lamarckii, were investigated using scanning electron microscopy, electron backscattered diffraction, and transmission electron microscopy with a focused ion beam sample preparation technique. Approximately 0.5 µm-thick lamellae (the second-order units) are piled up obliquely toward the growth direction to form the first-order unit and the obliquity is inverted between adjacent units along the shell thickness direction. The first-order units originate around the center of the shell, initially growing parallel to the shell and subsequently curving toward the inner or outer surfaces. The lamellae consist of aragonite granular and columnar layers, which group together to adopt the same crystal orientation forming crystallographic units (crystallites). Multiple {110} twins are common both in the granular and columnar layers. The crystallite c-axis is parallel to the columns and is inclined at angles 0-50° from the lamellar normal (dispersing among individual lamellae), toward the shell growth direction. Probably, the directions of the a- and b-axes are random in the lamellae, showing no specific orientation.

Spontaneous necrosis of solid gallbladder adenocarcinoma accompanied with pancreaticobiliary maljunction.

A 71-year-old Japanese man with acute cholecystitis and an incarcerated gallbladder (GB) stone was admitted. Plain ultrasonography (US) incidentally detected a mass-like lesion in the fundus. Doppler US revealed that this elevated lesion had no blood flow. Computed tomography showed a relatively low-density mass, measuring 5 cm multiply 4 cm in diameter, with no positive enhancement. Magnetic resonance imaging showed a mass in the fundus with a slightly low intensity on T1-weighted images and a slightly high intensity on T2-weighted images. We were agonized in making the qualitative diagnosis of mass-like lesions of the fundus, such as a benign tumor, cancer, or debris. We performed laparoscopic cholecystectomy, because the incarcerated GB stone clearly caused acute cholecystitis. Intra-operative cholangiography clearly revealed pancreaticobiliary maljunction. Amylase levels in the common bile duct and gallbladder were quite high. The elevated lesion in the fundus clearly showed severe necrosis. Although this necrotic nodule included non-viable adenocarcinoma cells, viable cancer cell nests were located in the muscularis propria and subcutaneous layer. Histopathological examination confirmed a solid adenocarcinoma. Thus, we diagnosed it as a gallbladder cancer, based on histopathological analysis of the resected specimen. We therefore undertook radical surgery, including wedge resection of the liver, radical dissection of regional lymph nodes, and resection of the extrahepatic bile duct. Histopathological findings revealed no cancer, hyperplasia or dysplasia in the additionally resected specimens. The patient was finally staged as T2, N0, H0, P0, M(-), stage II. We present the first case of spontaneous necrosis of solid gallbladder adenocarcinoma, with a review of previous studies.

Repair of articular cartilage defect by autologous transplantation of basic fibroblast growth factor gene-transduced chondrocytes with adeno-associated virus vector.

OBJECTIVE: To examine the effects of basic fibroblast growth factor (bFGF) gene-transduced chondrocytes on the repair of articular cartilage defects. METHODS: LacZ gene or bFGF gene was transduced into primary isolated rabbit chondrocytes with the use of a recombinant adeno-associated virus (AAV) vector. These gene-transduced chondrocytes were embedded in collagen gel and transplanted into a full-thickness defect in the articular cartilage of the patellar groove of a rabbit. The efficiency of gene transduction was assessed according to the percentage of LacZ-positive cells among the total number of living cells. The concentration of bFGF in the culture supernatant was measured by enzyme-linked immunosorbent assay to confirm the production by bFGF gene-transduced chondrocytes. At 4, 8, and 12 weeks after transplantation, cartilage repair was evaluated histologically and graded semiquantitatively using a histologic scoring system ranging from 0 (complete regeneration) to 14 (no regeneration) points. RESULTS: LacZ gene expression by chondrocytes was maintained until 8 weeks in >85% of the in vitro population. LacZ-positive cells were found at the transplant sites for at least 4 weeks after surgery. The mean concentration of bFGF was significantly increased in bFGF gene-transduced cells compared with control cells (P < 0.01). Semiquantitative histologic scoring indicated that the total score was significantly lower in the bFGF-transduced group than in the control group throughout the observation period. CONCLUSION: These results demonstrated that gene transfer to chondrocytes by an ex vivo method was established with the AAV vector, and transplantation of bFGF gene-transduced chondrocytes had a clear beneficial effect on the repair of rabbit articular cartilage defects.

Repair of articular cartilage defect by intraarticular administration of basic fibroblast growth factor gene, using adeno-associated virus vector.

The objective of this study was to establish the potency of adeno-associated virus (AAV) as a viral vector to transport the basic fibroblast growth factor (bFGF) gene into synovial tissue, and to evaluate the consequent repair of articular cartilage defects. In the in vitro study, LacZ- and bFGF-encoding genes were transduced into rabbit synoviocytes by recombinant adeno-associated virus (AAV) vector, and the cells were cultured for 2 weeks. The percentage of successfully transduced LacZ-positive cells was assessed by 5-bromo-4-chloro-3-indolyl-beta-D-galactopyranoside staining, and the concentration of bFGF in the culture supernatant was confirmed by bFGF-specific enzyme-linked immunosorbent assay. In the in vivo study, 12- to 14-week-old Japanese white rabbits (all female) were used. AAV-bFGF was administered into an artificially created full-thickness defect (5 mm in diameter and 3 mm deep) in the patellar groove of the distal femur. Cartilage repair was subsequently monitored at 4, 8, and 12 weeks, by macroscopic and histological examination, and results were graded on the basis of semiquantitative scores. lacZ gene expression in synoviocytes reached more than 93% within the first 2 weeks, and the mean bFGF concentration in the culture supernatant of the bFGF gene-transduced group was significantly increased (p < 0.01). Semiquantitative macroscopic and histological assessment indicated that the average score was significantly better in the bFGF-transduced group throughout the observation period, suggesting better cartilage repair. These results demonstrate that gene transfer into synoviocytes, using the AAV vector, was a potent method of gene transduction. Moreover, after intraarticular administration of AAV-bFGF, constant expression of bFGF in the knee joints resulted in substantial cartilage regeneration that, with further long-term study, could possibly merit consideration for clinical application.

Gene marking in adeno-associated virus vector infected periosteum derived cells for cartilage repair.

OBJECTIVE: To evaluate both the potential for transferring genes to periosteal cells using an adeno-associated virus (AAV) vector and the potential for gene expression after transplantation of those cells to a cartilage defect in vivo. METHODS: Periosteum was obtained from the tibia of 6-week-old rabbits and enzymatically digested. The isolated periosteum derived cells were cultured and the subconfluence cells were infected with a recombinant AAV expressing the LacZ gene (AAV-LacZ). Collagen gel containing the LacZ transferred, periosteum derived cells was transplanted into a full thickness articular cartilage defect in 10 rabbits. RESULTS: Infected cells still growing on the plate continued to express LacZ at least 12 weeks after AAV infection, with the highest percentage of LacZ positive cells reaching 74.4%. The LacZ positive cells were recognized at the transplant sites in 8 out of 10 knees. CONCLUSION: Gene expression in periosteum derived cells was sustained in vitro for at least 12 weeks using the AAV vector, and for 2 weeks ex vivo after transplantation into a cartilage defect.