ETB receptor protects the tubulointerstitium in experimental thrombotic microangiopathy

ETB receptor protects the tubulointerstitium in experimental thrombotic microangiopathy.BackgroundThe characteristic features of thrombotic microangiopathy (TMA) include glomerular and peritubular capillary endothelial cell injury with thrombus formation and subsequent ischemic tubulointerstitial damage. The endothelin ETB receptor has been shown to mediate both endothelial cell proliferation and vasodilation, and we therefore hypothesized that blockade of this receptor might promote more severe injury in this model.MethodsTMA was induced in recently established transgenic rats that lack expression of ETB receptor in the kidney; these animals were compared to control rats with TMA both in the short-term (days 1 and 3) when acute glomerular injury was most manifest, and the long-term (day 17) when glomeruli have recovered but tubulointerstitial injury is still present. Renal damage was assessed by histological analysis and blood urea nitrogen (BUN) measurements.ResultsNo difference in the TMA model was observed between rats with and without ETB receptor on days 1 or 3. At day 17, however, rats without the ETB receptor showed more severe tubulointerstitial injury compared with those with ETB receptor, which was associated with higher BUN levels. The tubulointerstitial damage was associated with a more severe loss of peritubular capillaries.ConclusionsThese findings suggest that the ETB receptor may protect peritubular capillaries under the ischemic insult, and serve a defensive role in the tubulointerstitium induced by renal microvascular injury

Isolation of non-urea producing candidates from Gunma KAZE3 yeast by ion-beam mutagenesis

Gunma KAZE yeasts, bred for ginjyo-sake brewing, are utilized in many sake-manufactures of Gunma prefecture. Urea is a main precursor in Japanese sake of ethyl carbamate which is classified in the group 2A “probably the cause of cancer” by International Agency for Research on Cancer. We previously bred non-urea producing Gunma KAZE1 yeast (KAZE1-Arg) by natural mutation and Gunma KAZE2 yeast (KAZE2-Arg) by ion-beam irradiation. However, the growth of Gunma KAZE3 yeast (KAZE3), the second most utilized strain in Gunma prefecture, could not be suppressed on modified CAO medium. On the other hand, we improved the viability of sake yeasts prepared for ion-beam mutagenesis. Thus, in this year, we attempted to improve the medium condition to suppress cell growth and isolate non-urea producing candidates from KAZE3. KAZE3 cells were irradiated with carbon ion beams (12C5+, 220 MeV, 200 - 300 Gy) accelerated by using the AVF cyclotron at TIARA, TARRI, QST. Twenty-two candidates were obtained after 5 weeks cultivation on the medium. From these 22 candidates, 17 arginase-inactivate mutants were selected as non-urea producing mutants. To investigate fermentation ability, ginjyo-aroma productivity and urea-productivity, laboratory scale sake brewing experiment was performed. All mutants did not produce urea. However, 11 mutants changed the brewing profiles. Six mutants were selected for future experiments

Development of Sake Yeasts Preparation Method with Improved Viability for Ion-Beam Mutagenesis

Japanese sake, called as “koku-syu” is a traditional liquor of Japan. It is made from steamed-rice, koji-rice, and water. The various characters of Japanese sake such as sweetness/dryness, rich/light, acidity, and ginjyo-aroma vary depending on sake yeasts. Therefore, various sake yeasts are isolated and bred at each region of Japan. Our group have succeeded in breeding and practical utilization of sake yeast Saccharomyces cerevisiae No. 227 by ion-beam mutagenesis for the first time in the world. Moreover, we also bred non-urea producing Gunma KAZE2 yeast (KAZE2-Arg) which is suitable for export by ion-beam irradiation. However, the viabilities of sake yeasts prepared for ion-beam mutagenesis were under 0.1%. Thus, it was considered that some mutagenesis stress was provided for yeast cells before ion-beam irradiation. Therefore, some improvement trials are needed for evaluating superiority of ion-beam breeding methods. In this study, we investigated conditions to prepare yeast cells for ion-beam mutagenesis. Consequently, improved conditions were follows. Yeast cells were cultivated in YPDS medium with shaking at 150 rpm, 30 °C for 24 h. Cells were harvested and washed with sterilized saline (0.9% NaCl) and adhered on cellulose acetate membrane (0.22 µm). After air-dried treatment, 20 ml YPD medium was added on the membrane and incubated at room temperature for 1-2 h. Yeast viabilities were reached at 90%

Effects of carbon ion beam-induced mutagenesis for the screening of RED production-deficient mutants of Streptomyces coelicolor JCM4020.

Streptomyces lividans TK23 interacts with mycolic acid-containing bacteria (MACB), such as Tsukamurella pulmonis TP-B0596, and this direct cell contact activates its secondary metabolism (e.g., the production of undecylprodigiosin: RED). Here, we employed carbon (12C5+) ion beam-induced mutagenesis to investigate the signature of induced point mutations and further identify the gene(s) responsible for the production of secondary metabolites induced by T. pulmonis. We irradiated spores of the Streptomyces coelicolor strain JCM4020 with carbon ions to generate a mutant library. We screened the RED production-deficient mutants of S. coelicolor by mixing them with T. pulmonis TP-B0596 on agar plates, identifying the red/white phenotype of the growing colonies. Through this process, we selected 59 RED-deficient mutants from around 152,000 tested spores. We resequenced the genomes of 16 mutants and identified 44 point mutations, which revealed the signatures induced by 12C5+-irradiation. Via gene complementation experiments, we also revealed that two genes-glutamate synthase (gltB) and elongation factor G (fusA)-are responsible for the reduced production of RED

Characterization of TetR-type Repressor Which Cause the Delayed Contact-dependent RED Production in Streptomyces coelicolor

Actinomycetes, filamentous Gram-positive bacteria, are a major source of bioactive natural products which can be drug candidates. We have studied about the bacterial interaction induced production of secondary metabolites by actinomycetes. Tsukamurella pulmonis TP-B0596 (Tp) had been shown to possess ability to induce production of secondary metabolites by Streptomyces species, which are not detected or poorly produced in a mono-culture. Until now, 7 classes, total 29 new compounds had been isolated from the co-culture with various actinomycetes and Tp. Object of this study is to elucidate the gene(s) which are involved in the response for activation of secondary metabolism within actinomycetes. Elucidation of the mechanism can lead to the fundamental understanding of bacterial interaction and secondary metabolism, as well as application for genetic tools to discover novel bioactive natural products from untapped gene matters. Here we employed forward-genetic study using mutagenesis by heavy ion beam to investigated the gene(s) responsible for the responsive production of secondary metabolism induced by Tp. Through this screening, 118 mutants from around 152,000 tested spores were obtained. We further tested the phenotype of the 118 mutants by growth on minimum medium and formation of aerial mycelia, and finally selected 59 mutants. We then re-sequenced the genome of 26 mutants and further gene complementation study revealed that molybdopterin biosynthetic enzyme (moeA), and tetR-like transcriptional regulator are also responsible for the phenotype differences. We speculate that the TetR-like protein is a repressor for the expression of adjacent ABC transporter, thus efflux of the unknown small molecule is prevented and accumulated in the cell, which caused stress to cell that can lead to the undecylprodigiosin production

Identification of Genes Responsible for the Contact-dependent RED Response in Streptomyces coelicolor

Actinomycetes, filamentous Gram-positive bacteria, are a major source of bioactive natural products which can be drug candidates. We are studying about the bacterial-bacterial interaction induced secondary metabolism by filamentous actinomycetes e.g. Streptomyces species. Mycolic acid-containing bacteria, Tsukamurella pulmonis TP-B0596 (Tp) possess ability to induce production of secondary metabolites in a range of filamentous actinomycetes, which are not produced in their single culture. Object of this study is to elucidate the gene(s) which are involved in the response for activation of secondary metabolism within filamentous actinomycetes. Elucidation of the mechanism may lead for development of versatile genetic tools to discover novel bioactive natural products. S. lividans TK23 and S.s coelicolor JCM4020 were observed to produce red pigment compound (RED) in response to direct cell-cell contact interaction of Tp. We investigated the gene(s) responsible for the activation of RED which was induced by Tp using S. coelicolor JCM4020 as a model to explore the molecular basis of the response mechanism. We employed carbon ion beams (12C5+, 220 MeV), accelerated by the AVF cyclotron at TIARA, to induce mutagenesis on spores to generate mutant library of S. coelicolor. We screened the RED production deficient mutants of S. coelicolor by mixing them with Tp on agar plates. Using red / white color phenotype of the colonies as indication, we obtained 59 mutants which were deficient in induced RED production by Tp, but were normal in their apparent growth and morphological development. We re-sequenced the genome of 16 mutants using MiSeq and identified mutational points existed in CDS

Characterization of ABC transporter which cause the delayed contact-dependent RED production in Streptomyces coelicolor

We have studied about the bacterial interaction induced production of secondary metabolites by actinomycetes. Tsukamurella pulmonis TP-B0596 (Tp) had been shown to possess ability to induce production of secondary metabolites by Streptomyces species, which are not detected or poorly produced in a mono-culture. Until now, more than 30 new compounds had been isolated from the co-culture with various actinomycetes and Tp. Object of this study is to elucidate the gene(s) which are involved in the regulation of secondary metabolism caused by bacterial interaction. Elucidation of the mechanism can lead to the fundamental understanding of bacterial interaction induced secondary metabolism, as well as application for genetic tools to discover novel bioactive natural products from untapped genes. We had employed carbon ion beams induced mutation to investigated the gene(s) responsible for the responsive production of secondary metabolism induced by Tp. Using Streptomyces coelicolor JCM4020, random spore mutant library was generated. Undecylprodigiosin (RED) production deficient mutants was screened by mixing with Tp on agar plates. Through this screening, 59 mutants from around 152,000 tested spores were obtained. The genome re-sequence and gene complementation studies were revealed that molybdopterin biosynthetic enzyme, and TetR-like transcriptional regulator are also responsible for the phenotype. We further analyzed the involvement of TetR-like transcriptional regulator by targeted gene disruption in S. coelicolor A3(2) and confirmed the delayed production of RED