L-Lysine production independent of the oxidative pentose phosphate pathway by Corynebacterium glutamicum with the Streptococcus mutans gapN gene

Published online 29 March 2016We have recently developed a Corynebacterium glutamicum strain that generates NADPH via the glycolytic pathway by replacing endogenous NAD-dependent glyceraldehyde 3-phosphate dehydrogenase (GapA) with a nonphosphorylating NADP-dependent glyceraldehyde 3-phosphate dehydrogenase (GapN) from Streptococcus mutans. Strain RE2, a suppressor mutant spontaneously isolated for its improved growth on glucose from the engineered strain, was proven to be a high-potential host for l-lysine production (Takeno et al., 2010). In this study, the suppressor mutation was identified to be a point mutation in rho encoding the transcription termination factor Rho. Strain RE2 still showed retarded growth despite the mutation rho696. Our strategy for reconciling improved growth with a high level of l-lysine production was to use GapA together with GapN only in the early growth phase, and subsequently shift this combination-type glycolysis to one that depends only on GapN in the rest of the growth phase. To achieve this, we expressed gapA under the myo-inositol-inducible promoter of iolT1 encoding a myo-inositol transporter in strain RE2. The resulting strain RE2Aiol was engineered into an l-lysine producer by introduction of a plasmid carrying the desensitized lysC, followed by examination for culture conditions with myo-inositol supplementation. We found that as a higher concentration of myo-inositol was added to the seed culture, the following fermentation period became shorter while maintaining a high level of l-lysine production. This finally reached a fermentation period comparable to that of the control GapA strain, and yielded a 1.5-fold higher production rate compared with strain RE2. The transcript level of gapA, as well as the GapA activity, in the early growth phase increased in proportion to the myo-inositol concentration and then fell to low levels in the subsequent growth phase, indicating that improved growth was a result of increased GapA activity, especially in the early growth phase. Moreover, blockade of the pentose phosphate pathway through a defect in glucose 6-phosphate dehydrogenase did not significantly affect l-lysine production in the engineered GapN strains, while a drastic decrease in l-lysine production was observed for the control GapA strain. Determination of the intracellular NADPH/NADP+ ratios revealed that the ratios in the engineered strains were significantly higher than the ratio of the control GapA strain irrespective of the pentose phosphate pathway. These results demonstrate that our strain engineering strategy allows efficient l-lysine production independent of the oxidative pentose phosphate pathway.ArticleMETABOLIC ENGINEERING. 37:1-10 (2016)journal articl

Life sciences

Aims: To examine the circadian expression changes in bladder clock genes in Dahl salt-sensitive rats following high salt intake. Main methods: Eighteen rats were divided into three groups: the high-salt diet group (HS group), the normal-salt diet group (NS group), and the salt-load interruption group (from a 4 % salt diet to a normal diet; salt-load interruption group [SI group]). Each rat was placed in an individual metabolic cage for 24 h twice weekly. Water intake, urine production, voiding frequency, and voided volume per micturition were recorded. Furthermore, 108 control rats were prepared. Bladders were harvested every 4 h at six time points. Furthermore, the mRNA expression of clock genes and mechanosensors was analyzed. Key findings: In the HS group, the bladder clock genes showed lower mRNA levels than in the NS group. The amplitude of circadian expression changes in bladder clock genes in the HS group was lower than that in the NS group. However, after changing from a 4 % salt diet to a normal diet, the waveforms of the clock gene expression in the SI group were closer to those of the NS group. The 24-h water intake and urinary volume of the SI group decreased to levels comparable to those of the NS group. Significance: Reduced salt intake partially restored the circadian rhythms of bladder clock genes.博士（医学）・甲第857号・令和4年12月22日Copyright © 2022 Elsevier Inc. All rights reserved

ラット膀胱での水吸収におけるアクアポリン-2の役割

AIM: We investigated the role of the bladder wall in permeating water, focusing on aquaporins. METHODS: Female Sprague-Dawley rats weighing 300 g were used to investigate the role of the bladder wall in saline permeation. Changes in intravesical fluid volume and sodium concentration were measured in the desmopressin acetate hydrate-loaded and control groups 3 h after administration. Bladders were resected to measure aquaporin-1, 2, and 3 gene expression using qRT-PCR. Additionally, the change of aquaporin-2 expression was measured using Western blotting and immunohistochemistry in intravesical aquaporin-2 siRNA-treated and control groups. RESULTS: Although the intravesical fluid volume and sodium concentration significantly decreased from 0 to 3 h (1.00 ± 0.00 vs 0.83 ± 0.08 mL, 157.80 ± 1.30 vs 146.8 ± 1.92 mEq/mL, P < 0.01, respectively in the control group), administration of desmopressin did not affect the extent of volume change. Aquaporin-2 expression was significantly higher in the 3-h distended bladders than in the empty bladder. Aquaporin-2 siRNA treatment suppressed aquaporin-2 expression and the change of intravesical fluid volume from 0 to 3 h (1.00 ± 0.00 and 0.99 ± 0.02 mL), which was related to the suppression of sodium concentration change in comparison with control siRNA treatment (149.6 ± 2.4 vs 143.6 ± 3.67 mEq/mL, P < 0.05). CONCLUSIONS: The rat urinary bladder absorbs water and salts under the full-filled condition. Aquaporin-2 plays an important role in the transport of water, accompanied by sodium concentration change. We demonstrated a part of the bladder absorption mechanism, which may lead to development of a new method for regulating bladder storage function.博士（医学）・甲第697号・平成31年3月15日© 2018 Wiley Periodicals, Inc.This is the pre-peer reviewed version of the following article: [https://onlinelibrary.wiley.com/doi/full/10.1002/nau.23715], which has been published in final form at [http://dx.doi.org/10.1002/nau.23715]. This article may be used for non-commercial purposes in accordance with Wiley Terms and Conditions for Use of Self-Archived Versions

筋層浸潤性膀胱癌における壁浸潤長は予後予測因子であり、血清cell-free DNAと関連する

Background: We investigated the potential of the depth of invasion (DOI) as a prognostic factor in patients with muscle-invasive bladder cancer (MIBC) who underwent radical cystectomy (RC). Moreover, we examined the association between the preoperative levels of circulating cell-free DNA and DOI.博士（医学）・甲第876号・令和5年3月15

PDE5阻害薬であるタダラフィルは糖尿病ラットの膀胱血流を改善し、初期段階に低下する下部尿路機能を回復する

AIMS: To investigate the effect of tadalafil on bladder blood flow and lower urinary tract function in a rat model of diabetes. MATERIALS AND METHODS: We studied female Sprague-Dawley rats and induced diabetes in some using a single intraperitoneal injection of streptozotocin. We divided the rats into nondiabetes (ND), diabetes (D), and diabetes with tadalafil (DT) groups. The rats were raised for an additional 7 weeks after diabetes induction. The DT group received oral tadalafil (2 mg/kg/day) for 7 days before the experiments. At 7 weeks after diabetes induction, we performed cystometry, resected the bladders for immunohistochemistry (hypoxia-inducible factor-1α [HIF-1α] and 8-oxo-2'-deoxyguanosine [8-OHdG] staining), and measured bladder blood supply using a laser blood flow meter. RESULTS: The opening pressure, when the urethra opens and urine flow starts, was significantly lower in the DT group than in the D group (24.9 ± 5.9 vs 43.6 ± 12.3 cmH2 O). The inter-contraction interval was significantly longer in the D group than in the ND and DT groups (1566.2 ± 168.7 vs 702.9 ± 165.2 and 787.4 ± 148.8 s). Immunohistochemistry showed positive staining of the urothelial layer for both HIF-1α and 8-OHdG in the D group, but not in the ND or DT groups. Bladder blood flow was significantly lower in the D group than in the ND or DT groups. CONCLUSIONS: Tadalafil improves bladder blood supply and lower urinary tract function in diabetic rats. Tadalafil may be a promising drug that restores lower urinary tract dysfunction in the early phase of diabetes.博士（医学）・甲第677号・平成30年3月15日© 2017 Wiley Periodicals, Inc.This is the peer reviewed version of the following article: "http://dx.doi.org/10.1002/nau.23372", which has been published in final form at [Link to final article using the DOI]. This article may be used for non-commercial purposes in accordance with Wiley Terms and Conditions for Use of Self-Archived Versions

Ambient dose rate in Mikurashima Island after the Fukushima Daiichi Nuclear Power Plant accident

No detailed ambient dose rate has been reported, either before or after the Fukushima Daiichi Nuclear Power Plant (F1-NPP) accident, for Mikurashima Island, one of the islands within the authority of the Tokyo Metropolitan Government. Thus, the ambient dose rate on Mikurashima Island was observed 4.5 years after the F1-NPP accident. A car-borne survey and a foot patrol survey of the ambient dose rates were conducted over the entire island using a 3-in × 3-in NaI(Tl) scintillation spectrometer. The average ambient dose rate was 27 nGy h^-1 (12 – 45 nGy h^-1) and a higher dose distribution was observed for the northern and eastern areas of Mikurashima Island. However, the impact from the F1-NPP accident (i.e., the presence of artificial radionuclides) was mainly observed for the mountain area of the island (4 – 9 nGy h^-1; located about the center to south-southeast direction). Based on the measured ambient dose rate, the estimated annual effective dose after the accident was 0.09 mSv y^-1

Clinical impact of postoperative loss in psoas major muscle and nutrition index after radical cystectomy for patients with urothelial carcinoma of the bladder

Figure S3. Comparison of changes after radical cystectomy between neoadjuvant chemotherapy-treated group and non-treated group. Time-course changes in cross-section area of the psoas major muscle at the level of L3 (a), abdominal skeletal muscle area at the level of L3 (b), the PNI (c) and, the CONUT score (d). Data of neoadjuvant chemotherapy-treated group (red) and non-treated group (blue) are plotted by means ¹ SD. Scores of two groups compared in each time point by the Mann-Whitney U-test. ns, not significant. (TIFF 7424 kb