Yasuhiko Shibanaka

Coexpression of CYP11B2 or CYP11B1 with adrenodoxin and adrenodoxin reductase for assessing the potency and selectivity of aldosterone synthase inhibitors

Excessive production of aldosterone has been implicated in the pathogenesis of hypertension and heart failure. One approach to ameliorate the deleterious effects of aldosterone is to suppress its biosynthesis. The enzyme aldosterone synthase (CYP11B2) is responsible for the final step of aldosterone synthesis. It requires electron transfer from the adrenodoxin/adrenodoxin reductase system to catalyze the production of aldosterone. A stable cell line simultaneously overexpressing recombinant human CYP11B2 as well as human adrenodoxin and adrenodoxin reductase was established to help maximize the enzyme activity. The homogenate of these cells was used to develop an in vitro CYP11B2 assay using 11-deoxycorticosterone as a substrate. By the same strategy, another stable cell line simultaneously overexpressing human 11beta-hydroxylase (CYP11B1), an enzyme responsible for the final step of cortisol biosynthesis, and the two electron transfer proteins was also established, and an in vitro CYP11B1 assay using 11-deoxycortisol as a substrate was likewise developed to assess the selectivity of CYP11B2 inhibitors. FAD286, a reference CYP11B2 inhibitor, inhibited CYP11B2 and CYP11B1 activities with IC(50) values of 1.6+/-0.1 and 9.9+/-0.9 nM (mean+/-SEM, n=3-6), respectively. Kinetics studies revealed that the compound inhibited the activity of both enzymes competitively with respective K(i) values of 0.8+/-0.04 and 2.2+/-0.2 nM (n=3-4). These assays can be used for assessing the potency and selectivity of CYP11B2 inhibitors for the treatment of hypertension and heart failure.

Modified substrate specificity of pyrroloquinoline quinone glucose dehydrogenase by biased mutation assembling with optimized amino acid substitution

A biased mutation-assembling method—that is, a directed evolution strategy to facilitate an optimal accumulation of multiple mutations on the basis of additivity principles, was applied to the directed evolution of water-soluble PQQ glucose dehydrogenase (PQQGDH-B) to reduce its maltose oxidation activity, which can lead to errors in blood glucose determination. Mutations appropriate for the reduction without fatal deterioration of its glucose oxidation activity were developed by an error-prone PCR method coupled with a saturation mutagenesis method. Moreover, two types of incorporation frequency based on their contribution were assigned to the mutations: high (80%) and evens (50%), in constructing a multiple mutant library. The best mutant created showed a marked reduction in maltose oxidation activity, corresponding to 4% of that of the wild-type enzyme, with 35% retention of glucose oxidation activity. In addition, this mutant showed a reduction in galactose oxidation activity corresponding to 5% of that of the wild-type enzyme. In conclusion, we succeeded in developing the PQQGDH-B mutants with improved substrate specificity and validated our method coupled with optimized mutations and their contribution-based incorporation frequencies by applying it to the development.

A comprehensive analysis of loss of heterozygosity caused by hemizygous deletions in renal cell carcinoma using a subtraction library

Several new loci were identified by a comprehensive analysis of loss of heterozygosity (LOH) using a subtraction library between matched normal and renal cell carcinoma (RCC) tissues. A total of 187 clones from the library, with a complexity of 1×104, were mapped, and 44 clusters of the mapped loci were subjected to LOH analysis using microsatellite markers. A total of 27 loci, which exhibited frequencies of LOH of at least 10% among 44 tumors, mostly clear‐cell RCC, included several loci that were reported previously, such as, the von Hippel‐Lindau gene, adenomatous polyposis coli, and interferon regulatory factor‐1, as well as new loci, at 5q32‐q34, 6q21‐q22, 8p12, and others. These loci exhibited LOH among 11.8–93.8% of tumors, and most, if not all, were derived from the sites of hemizyous deletions. The minimum regions of LOH of chromosomes 5, 6, and 8 were 9.0, 10.3, and 0.775 Mb, respectively. The average distance between the cloned fragments on the chromosomes was 2.2 Mb in 187 clones, indicating that the minimum LOH size expected from this subtraction analysis was roughly 50 kb. Therefore, the strategy described here provides comprehensive analysis of LOH sites, which were mostly caused by hemizygous deletions.

The crucial role of cyclic GMP in the eclosion hormone mediated signal transduction in the silkworm metamorphoses

The signal transduction of the peptide, eclosion hormone, in the silkworm Bombyx mori appears to be mediated via the second messenger cyclic GMP throughout their life cycle. Injection of 8-bromo-cGMP induced the ecdysis behavior in pharate adults with similar latency to eclosion hormone-induced ecdysis; the moulting occurred 50-70 min after the injection. The potency of 8Br-cGMP was 10(2) fold higher than that of cGMP and the efficacy was increased by the co-injection of the phosphodiesterase inhibitor IBMX. On the other hand, in the silkworm pupal ecdysis the eclosion hormone and also 8Br-cGMP induced the moulting behavior in a dose-dependent manner. The adult development of the ability to respond to 8Br-cGMP took place concomitantly with the response to the eclosion hormone. Both the developmental time courses were shifted by a shift of light and dark cycles. Accordingly, the sensitivities to the peptide and cyclic nucleotide developed correspondently under the light and dark circadian rhythm. Thus throughout the silkworm life cycle, eclosion hormone is effective to trigger the ecdysis behavior and cGMP plays a crucial role as the second messenger in the eclosion hormone-mediated signal transduction.

Candidate regions of tumor suppressor locus on chromosome 9q31.1 in gastric cancer.

Loss of heterozygosity (LOH) is an important event of tumorigenesis. In gastric cancer, we found a novel region of LOH in chromosome 9q having about 800 kb deletions at 9q31.1. The microsatellite marker D9S938 in that region exhibiting the highest LOH frequency, 56.5%. In addition, the LOH at 9q31.1 did not show any relationship to either histologic types or stages of gastric cancers, and several genes were predicted in the remaining allele by in silico methods. These data suggest that the deletion at 9q31.1 would be common in both differentiated‐type and undifferentiated‐type gastric cancers. Furthermore, this deletion was found in the primary tumors of early‐stage gastric cancer, indicating that loss of function of predicted genes appears to be associated with the tumorigenesis of gastric cancer.

Expression of a silkworm eclosion hormone gene in yeast

Recombinant silkworm eclosion hormone was produced for the first time in yeast which was transformed with a shuttle plasmid containing a construct coding a signal peptide and the mature sequence of the silkworm eclosion hormone. Successfully transformed yeast processed recombinant silkworm eclosion hormone I (EH-I) and transported it to periplasm at the concentration of 60 micrograms per liter of culture. The biological activity of the purified recombinant silkworm eclosion hormone exhibited the ED50 value of 0.2 ng which is the same as that of the authentic hormone isolated from the silkworm brain.

Cloning of novel LERGU mRNAs in GPR30 3' untranslated region and detection of 2 bp-deletion polymorphism in gastric cancer

The improved IGCR (In–Gel Competitive Reassociation) method was applied to the analysis of human gastric cancer genomic DNA to identify its alterations, and it appeared that the IGCR library contained a fragment of 3′‐untranslated region (3′ UTR) of G‐protein coupled receptor 30 (GPR30) mRNA. When we searched genomic DNA pairs of gastric cancer patients with this IGCR clone, we found the deletion polymorphism with or without 2 bp (Cytosine and Thymine; CT). We confirmed the existence of a novel mRNA in GPR30 3′UTR by northern blotting, cloned this novel mRNA and named it Leucine Rich Protein in GPR30 3′UTR (LERGU). The EST database search gave one alternative splicing form in this 3′ UTR, which was named as LERGU‐1. A novel alternative splicing form of this mRNA was also identified from the stomach total RNA, which was named LERGU‐2. The LERGU mRNA was also detected in eight gastric cancer cell lines, but GPR30 mRNA scarcely existed. Furthermore, we detected the 2 bp‐deletion form in genomic DNAs and mRNAs derived from gastric cancers, but not in other type cancers. Since the 2 bp‐deletion position on LERGU corresponds to its alternative splicing site, this deletion may produce a frame‐shifted protein. Overall, our findings suggest that a mutation or disappearance of the normal LERGU protein may have a function in the development of gastric cancer. (Cancer Sci 2005; 96: 191–196)