Amplification and overexpression of the cyclin D1 gene in aggressive human hepatocellular carcinoma.

We analyzed the genetic alterations of the cyclin D1 and INT-2 genes in hepatocellular carcinomas (HCCs) from 45 patients. Among these, expression of the cyclin D1 mRNA was also analyzed in 18 of them by Northern blotting. The cyclin D1 gene was amplified 3-16 fold in five HCCs (11%); among these, the INT-2 gene was also amplified 2-10 fold in four HCCs. We analyzed the mRNA of cyclin D1 in four HCCs with gene amplifications, and 6-10 fold overexpressions were detected in all of them. Because the cyclin D1 gene was amplified in patients at an advanced stage of HCC with rapid tumor growth, it appeared to be associated with the aggressive behavior of tumors. Studies on loss of heterozygosity on chromosome 13q, where the retinoblastoma (RB) gene is located, indicated that all HCCs with an amplified cyclin D1 gene retained heterozygosity on chromosome 13q. These results suggest that amplification and overexpression of the cyclin D1 gene result in the rapid growth of a subset of HCC, even though the function of the RB gene is retained.

Regulation and evaluation of five methanol-inducible promoters in the methylotrophic yeast Candida boidinii.

We isolated the promoter regions of five methanol-inducible genes (P(AOD1), alcohol oxidase; P(DAS1), dihydroxyacetone synthase; P(FDH1), formate dehydrogenase; P(PMP20), Pmp20; and P(PMP47), Pmp47) from the Candida boidinii genome, and evaluated their strength and studied their regulation using the acid phosphatase gene of Saccharomyces cerevisiae (ScPHO5) as the reporter. Of the five promoters, P(DAS1) was the strongest methanol-inducible promoter whose strength was approximately 1.5 times higher than that of the commonly used P(AOD1) in methanol-induced cells. Although the expression of P(AOD1) and P(DAS1) was completely repressed by the presence of glucose, formate-induced expression of P(FDH1) was not repressed by glucose. Expression under P(PMP47), another methanol-inducible promoter, was highly induced by oleate. The induction kinetics of P(PMP47) and P(DAS1) revealed that methanol induces the expression of peroxisome membrane protein Pmp47, earlier than the expression of matrix enzyme dihydroxyacetone synthase (Das1p), and that this information is contained in the promoter region of the respective gene. This is the first report which evaluates several methanol-inducible promoters in parallel in the methylotrophic yeast.

Crystal structure of glycosyltrehalose trehalohydrolase from the hyperthermophilic archaeum Sulfolobus solfataricus.

The crystal structure of glycosyltrehalose trehalohydrolase from the hyperthermophilic archaeum Sulfolobus solfataricus KM1 has been solved by multiple isomorphous replacement. The enzyme is an alpha-amylase (family 13) with unique exo-amylolytic activity for glycosyltrehalosides. It cleaves the alpha-1,4 glycosidic bond adjacent to the trehalose moiety to release trehalose and maltooligo saccharide. Unlike most other family 13 glycosidases, the enzyme does not require Ca(2+) for activity, and it contains an N-terminal extension of approximately 100 amino acid residues that is homologous to N-terminal domains found in many glycosidases that recognize branched oligosaccharides. Crystallography revealed the enzyme to exist as a homodimer covalently linked by an intermolecular disulfide bond at residue C298. The existence of the intermolecular disulfide bond was confirmed by biochemical analysis and mutagenesis. The N-terminal extension forms an independent domain connected to the catalytic domain by an extended linker. The functionally essential Ca(2+) binding site found in the B domain of alpha-amylases and many other family 13 glycosidases was found to be replaced by hydrophobic packing interactions. The enzyme also contains a very unusual excursion in the (beta/alpha)(8) barrel structure of the catalytic domain. This excursion originates from the bottom of the (beta/alpha)(8) barrel between helix 6 and strand 7, but folds upward in a distorted alpha-hairpin structure to form a part of the substrate binding cleft wall that is possibly critical for the enzyme's unique substrate selectivity. Participation of an alpha-beta loop in the formation of the substrate binding cleft is a novel feature that is not observed in other known (beta/alpha)(8) enzymes.

Italy-Japan international project-based learning for developing human resources using design of welfare equipment as a subject.

Project-based learning (PBL) is effective for developing human resources of young students. The design of welfare equipment, such as wheelchairs and gait assistive devices, is taken as the subject in this study because these devices must be fit to their environment, users, and method of use; students must consider the circumstances of each country concerned. The program commenced in 2012 at L'Aquila, Italy, and the Shibaura Institute of Technology, Japan and has been continuing for three years. Students were divided into four groups and discussions were held on how to adapt the equipment to the user and environment. After discussion, they designed and simulated a model of the equipment using CAD. Finally, they presented their designs to each other. Through the program, students had fruitful discussions, exchanged ideas from different cultures, and learned from each other. Furthermore, friendships among the students were nurtured. It is believed that the objective of the program was satisfactorily accomplished.

Chromosomal instability and human hepatocarcinogenesis.

Recently, many studies have identified losses and gains of several chromosomal loci in human hepatocellular carcinoma (HCC) with fine microsatellite analysis and comparative genomic hybridization. Although distribution of aberrant chromosomal arms differs among HCCs, loss of 1p, 4q, 6q, 8p, 9p, 10q, 13q, 16q and 17p, and gain of 1q, 6p, 8q, 17q and 20q have been recurrently reported, and loss of 4q and 16q seems to occur preferentially in hepatitis B virus-related HCCs. Accumulation of these aberrant chromosomal regions is associated with tumor progression, and some chromosomal aberrations, such as loss of 1p, are frequently identified in well-differentiated HCCs and also detected even in dysplastic nodule and cirrhotic nodule. This evidence suggests that chromosomal instability (CIN) emerges at an early stage during hepatocarcinogenesis and is successively inherent to tumor cells, resulting in acquisition of malignant phenotype. The molecular basis of CIN is beginning to be explored; however, several mechanisms may be involved for CIN of HCC.

Treatment of extrinsic flexion deformity of the toes associated with previous removal of a vascularized fibular graft.

BACKGROUND: Complications from vascularized fibular bone-grafting are infrequent. We saw six patients who had a painful flexion deformity of the great and lesser toes after a free vascularized fibular graft had been obtained from the ipsilateral leg. In this report, we discuss our management of these patients. METHODS: Painful flexion deformity of the toes that had developed in six adults after removal of a free vascularized fibular graft was treated by cutting of the flexor hallucis longus alone in three patients, by lengthening of the flexor hallucis longus alone in one, and by cutting of both the flexor hallucis longus and the flexor digitorum longus in two. RESULTS: After an average duration of follow-up of six years and eleven months, the flexion deformity of the great and lesser toes had decreased or disappeared, leading to improved or full extension of the digits. Preoperative and postoperative measurements of muscle strength for plantar flexion of the interphalangeal joints did not change appreciably. CONCLUSIONS: Cutting or lengthening of the flexor hallucis longus behind the ankle provides an adequate release of digital flexion deformities that occur after removal of a vascularized fibular bone graft.

Incidence and characteristics of thyroid dysfunction following interferon therapy in patients with chronic hepatitis C.

Thyroid functions were analyzed before, during and after interferon (IFN) therapy in patients with chronic hepatitis C. According to the results of routine thyroid function tests and measurements of the levels of anti-thyroid autoantibody prior to the therapy, patients were divided into 2 groups; Group A (19 patients) had at least one abnormal finding related to the thyroid, and Group B (40 patients) did not show any abnormality. Five patients (26%) in Group A and 4 (10%) in Group B showed thyroid dysfunctions which were very clearly reflected by thyrotropin (TSH) measurements. Interestingly, the time of peak TSH elevation in Group A (mean +/- SD, 4.3 +/- 0.8 months) was significantly earlier than that in Group B (6.8 +/- 0.8). Most patients in Group B were diagnosed as having destructive thyroiditis. These findings may suggest that the pathogenesis of IFN-induced thyroid dysfunction consists not only of exacerbation of pre-existing thyroid autoimmunity but also of de novo destructive changes even in the intact thyroid before IFN therapy.

Development of a tool system to support endoscope.

Natural Orifice Translumenal Endoscopic Surgery (NOTES) is a type of minimally invasive surgery. This surgery needs working space for operating the endoscope, but there is no space for it in body cavity. Therefore, we have been developing a surgical tool system which can be inserted from the mouth and ensure space in body cavity. We use magnets and a tool to ensure space, like in abdominal wall lifting method, without use of wire. In this paper, we designed and realized a system which used a tool consisting of link mechanism and magnets. The link mechanism consists of four-bar linkage and a compression spring. It can be driven only by pulling wires, changing the diameter of the tool. We designed this surgical tool prototype, analyzed the stress and did basic experiments.