Novel and potent inhibitors of stearoyl-CoA desaturase-1. Part I: Discovery of 3-(2-hydroxyethoxy)-4-methoxy-N-[5-(3-trifluoromethylbenzyl)thiazol-2-yl]benzamide

A series of structurally novel stearoyl-CoA desaturase-1 (SCD-1) inhibitors has been identified by optimizing a hit from our corporate library. Preliminary structure–activity relationship (SAR) studies led to the discovery of the highly potent and orally bioavailable thiazole-based SCD-1 inhibitor, 3-(2-hydroxyethoxy)-4-methoxy-N-[5-(3-trifluoromethylbenzyl)thiazol-2-yl]benzamide (23a).     

Infrared laser‐mediated local gene induction in medaka,zebrafish and Arabidopsis thaliana

Heat shock promoters are powerful tools for the precise control of exogenous gene induction in living organisms. In addition to the temporal control of gene expression, the analysis of gene function can also require spatial restriction. Recently, we reported a new method for in vivo, single‐cell gene induction using an infrared laser‐evoked gene operator (IR‐LEGO) system in living nematodes (Caenorhabditis elegans). It was demonstrated that infrared (IR) irradiation could induce gene expression in single cells without incurring cellular damage. Here, we report the application of IR‐LEGO to the small fish, medaka (Japanese killifish; Oryzias latipes) and zebrafish (Danio rerio), and a higher plant (Arabidopsis thaliana). Using easily observable reporter genes, we successfully induced gene expression in various tissues in these living organisms. IR‐LEGO has the potential to be a useful tool in extensive research fields for cell/tissue marking or targeted gene expression in local tissues of small fish and plants.     

Generation of medaka gene knockout models by target-selected mutagenesis

We have established a reverse genetics approach for the routine generation of medaka (Oryzias latipes) gene knockouts. A cryopreserved library of N-ethyl-N-nitrosourea (ENU) mutagenized fish was screened by high-throughput resequencing for induced point mutations. Nonsense and splice site mutations were retrieved for the Blm, Sirt1, Parkin and p53 genes and functional characterization of p53 mutants indicated a complete knockout of p53 function. The current cryopreserved resource is expected to contain knockouts for most medaka genes.     

Single nucleotide polymorphisms associated with aggressive periodontitis and severe chronic periodontitis in Japanese

Periodontitis is a common inflammatory disease causing destruction of periodontal tissues. It is a multifactor disease involving genetic factors and oral environmental factors. To determine genetic risk factors associated with aggressive periodontitis or severe chronic periodontitis, single nucleotide polymorphisms (SNPs) in multiple candidate genes were investigated in Japanese. We studied 134 patients with aggressive periodontitis, 117 patients with severe chronic periodontitis, and 125 healthy volunteers without periodontitis, under case-control setting, and 310 SNPs in 125 candidate genes were genotyped. Association evaluation by Fisher's exact test (p < 0.01) revealed statistically significant SNPs in multiple genes, not only in inflammatory mediators (IL6ST and PTGDS, associated with aggressive periodontitis; and CTSD, associated with severe chronic periodontitis), but also in structural factors of periodontal tissues (COL4A1, COL1A1, and KRT23, associated with aggressive periodontitis; and HSPG2, COL17A1, and EGF, associated with severe chronic periodontitis). These appear to be good candidates as genetic factors for future study.     

Identification of radiation-sensitive mutants in the Medaka, Oryzias latipes

We screened populations of N-ethyl-N-nitrosourea (ENU)-mutagenized Medaka, (Oryzias latipes) for radiation-sensitive mutants to investigate the mechanism of genome stability induced by ionizing radiation in developing embryos. F3 embryos derived from male founders that were homozygous for induced the mutations were irradiated with gamma-rays at the organogenesis stage (48hpf) at a dose that did not cause malformation in wild-type embryos. We screened 2130 F2 pairs and identified three types of mutants with high incidence of radiation-induced curly tailed (ric) malformations using a low dose of irradiation. The homozygous strain from one of these mutants, ric1, which is highly fertile and easy to breed, was established and characterized related to gamma-irradiation response. The ric1 strain also showed higher incidence of malformation and lower hatchability compared to the wild-type CAB strain after gamma-irradiation at the morula and pre-early gastrula stages. We found that the decrease in hatching success after gamma-irradiation, depends on the maternal genotype at the ric1 locus. Terminal deoxynucleotidyl transferase-mediated deoxy-UTP nick end-labeling assays showed a high frequency of apoptosis in the ric1 embryos immediately after gamma-irradiation at the pre-early gastrula stage but apoptotic cells were not observed before midblastula transition (MBT). The neutral comet assay revealed that the ric1 mutant has a defect in the rapid repair of DNA double-strand breaks induced by gamma-rays. These results suggest that RIC1 is involved in the DNA double strand break repair in embryos from morula to organogenesis stages, and unrepaired DNA double strand breaks in ric1 trigger apoptosis after MBT. These results support the use of the ric1 strain for investigating various biological consequences of DNA double strand breaks in vivo and for sensitive monitoring of genotoxicity related to low dose radiation.     

Genotoxicity of 2-[2-(acetylamino)-4-[bis(2-hydroxyethyl)amino]-5-methoxyphenyl]-5-amino-7-bromo-4-chloro-2H-benzotriazole (PBTA-6) and 4-amino-3,3'-dichloro-5,4'-dinitro-biphenyl (ADDB) in goldfish (Carassius auratus) using the micronucleus test and the comet assay

2-[2-(Acetylamino)-4-[bis(2-hydroxyethyl)amino]-5-methoxyphenyl]-5-amino-7-bromo-4-chloro-2H-benzotriazole (PBTA-6) and 4-amino-3,3'-dichloro-5,4'-dinitrobiphenyl (ADDB) are two compounds, which show strong mutagenicity toward bacteria, that have been identified as major mutagens in river water in Japan. In the present study, we examined the genotoxicity of PBTA-6 and ADDB in goldfish (Carassius auratus) by the micronucleus test and single-cell gel electrophoresis (comet assay). The frequencies of micronuclei in gill cells gradually increased until 96h after i.p. injection of PBTA-6 and ADDB at doses of 50mg/kg body weight, and then decreased 144h after injection. PBTA-6 induced micronuclei in gill cells dose-dependently at a dose range of 1-100mg/kg body weight, giving significantly high frequencies at doses of 50 and 100mg/kg body weight. On the other hand, no significant increase was observed in the peripheral erythrocytes of goldfish exposed to PBTA-6 or ADDB. In the comet assay, values of DNA tail moment and tail length in peripheral erythrocytes increased significantly until 6h after the i.p. injection of PBTA-6 (50mg/kg body weight), only to decrease by 9h after injection. Both the DNA tail moment and tail length were dose-dependently increased by injections of PBTA-6 at doses ranging from 1 to 50mg/kg. Significantly high values for tail moment and tail length were found in peripheral erythrocytes 3h after an i.p. injection of ADDB and persisted for up to 6h. These results show that both PBTA-6 and ADDB have genotoxic effects in goldfish.     

A chimeric mouse model of Gaucher disease

BACKGROUND: There is a major need for a mouse model of Gaucher disease, but the glucocerebrosidase knockout mouse is not viable; it dies shortly before or immediately after birth, apparently because of involvement of the central nervous system and/or skin. The most common form of Gaucher disease, type I, has a phenotype that is limited to the monocyte-macrophage system. MATERIALS AND METHODS: We have created a chimeric mouse by infusing hematopoietic stem cells from fetuses that are homozygous for the glucocerebrosidase knockout into irradiated mice. RESULTS: The chimeric mice manifested a severe deficiency of glucocerebrosidase activity in peripheral blood cells and spleen indicating a lack of cell-cell correction. Levels of glucocerebroside in spleen and liver are increased, and infusing the mice with exogenous glucocerebroside/albumin particles produced a marked increase in the amount of glucocerebroside stored in liver and spleen. Morphologically identifiable Gaucher cells were not present. CONCLUSIONS: The chimeric model reflects the increased glycolipid storage in the reticuloendothelial system that is characteristic of Gaucher disease, and could be useful as a model for studying treatment of Gaucher disease.     

Anti-proliferative effects of heating on the human prostatic carcinoma cells in culture

Prostatic cancers are well-known to be sensitive to heat stress. However, the mechanism by which the cancer cells are killed by high temperature remains poorly understood. The present study was undertaken to determine the anti-proliferative effects of heat stress on the prostatic cancer cells in culture. Heat shock at 43 degrees C inhibited the cell growth of three different prostatic cell lines. Flow cytometrical analysis using BrdU and PI showed a decrease in the proportion of cells in an S phase, accompanied by cell accumulation in G1 and G2, in both JCA-1 and PC-3 but not in LNcap. Both JCA-1 and PC-3 presented a strong expression of hsp70 at 37 degrees C. The heat shock caused apparent enhancement of the expression of hsp70 through the cell cycle. A treatment at 43 degrees C for 8 hours resulted in not only an apparent increment of positive hsp70 cells, but cells with subdiploid DNA content in LNcap. Flow cytometrical analysis by FITC-labeled Annexin V showed increment of apoptotic cells at 43 degrees C for 8 hours in LNcap cells. The results suggest that apoptosis is an important pathway of heat-induced killing of these cells. In conclusion, the cell growth of prostatic cancers may be affected by the temperature through relationship of the cell cycle and hsp70.     

Identification of Aeromonas Species Isolated from Freshwater Fish with the Microplate Hybridization Method

Aeromonas isolates were obtained from the intestinal tracts of six species of cultured freshwater fish and identified on the basis of their genotypic and phenotypic characters. The microplate hybridization method could differentiate type strains of Aeromonas species and related bacteria. DNA-DNA hybridization analysis showed that 65 aeromonad isolates were 72 to 100% related with either Aeromonas caviae, Aeromonas hydrophila, Aeromonas jandaei, Aeromonas sobria, or Aeromonas veronii. As many as 48% of the genotypically identified A. caviae, A. hydrophila, and A. sobria isolates differed from the type strains of corresponding species in one to three phenotypic characters. These results strongly suggest that not all aeromonad isolates from freshwater fish could be identified correctly on the basis of only the phenotypic characters, indicating the usefulness of the microplate hybridization method for the identification of aeromonads.     

ROLE OF ADENOSINE 3'', 5''-MONOPHOSPHATE IN THE REGULATION OF CIRCADIAN OSCILLATION OF SEROTONIN N-ACETYLTRANSFERASE ACTIVITY IN CULTURED CHICKEN PINEAL GLAND

—When pineal glands of 10–12-day-old chicks were organ-cultured in darkness, serotonin N-acetyltransferase activity was low during the daytime, increased at midnight and then decreased to the daytime level the next morning. The pattern of increase and decrease of enzyme activity in cultured pineal glands was comparable to the circadian rhythm of N-acetyltransferase activity in vivo. When pineal glands were kept at a low temperature for 5 h prior to culture, the phase of autonomous rhythm of enzyme activity was delayed. When chicken pineal glands were cultured during the daytime for 6 h, derivatives of adenosine 3′, 5′-monophosphate (cyclic AMP), cholera toxin, a high concentration of KCl and phosphodiesterase inhibitors increased N-acetyltransferase activity 3–7-fold, indicating an involvement of cyclic AMP in the regulation of N-acetyltransferase activity in chicken pineal gland as has been shown in rat pineal gland. When pineal glands were cultured at night in darkness, cholera toxin or a high KCl did not enhance the night-time increase of the enzyme activity. Derivatives of cyclic AMP or phosphodiesterase inhibitors enhanced the autonomous night-time increase of N-acetyltransferase activity in an additive or more than additive manner in cultured pineal glands. These observations suggest that adenylate cyclase of pinealocytes is inactive during daytime, but is activated at night in darkness, which is transduced to the synthesis of N-acetyltransferase molecules. Catecholamines suppressed the basal level and the nocturnal increase of N-acetyltransferase activity via α-adrenergic receptor. The nocturnal increase of enzyme activity was prevented by cycloheximide or actinomycin D. Cocaine, which stabilizes cell membrane potential or light exposure, blocked the nighttime increase of N-acetyltransferase activity in cultured chicken pineal glands.