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Postprocedural mitral stenosis (MS), or increased transmitral mean pressure gradient (TMPG), is one of the limitations of transcatheter edge-to-edge mitral valve repair using MitraClip (Abbott Vascular Inc., Santa Clara, USA); however, the usefulness of three-dimensional transesophageal echocardiography (3D-TEE) for predicting postprocedural MS in functional mitral regurgitation (MR) has not been fully elucidated.Eighty-two consecutive functional MR patients who underwent transcatheter mitral valve repair using MitraClip were retrospectively studied. Postprocedural MS was defined as TMPG ≥ 5 mmHg by echocardiography.Ten patients had postprocedural MS, and 3D-TEE showed that patients with postprocedural MS had smaller preprocedural mitral valve orifice area (MVOA), anteroposterior and mediolateral diameter, leaflet area, and annulus area. Receiver operating characteristic analysis showed that leaflet area (area under the curve (AUC) 0.829), annulus area (AUC 0.813), anteroposterior diameter (AUC 0.797) and mediolateral diameter (AUC 0.803) evaluated using 3D-TEE were predictors of postprocedural MS, and their predictive abilities were higher than those of preprocedural MVOA (AUC 0.756) and preprocedural TMPG (AUC 0.716). Adding leaflet area to TMPG and MVOA resulted in higher C-statistics for predicting postprocedural MS (from 0.716 to 0.845 and from 0.756 to 0.853, respectively).In functional MR patients treated with MitraClip, leaflet area and annulus area evaluated using 3D-TEE had high predictive values for postprocedural MS, and their predictive abilities were higher than those of preprocedural TMPG or MVOA.

In asymptomatic patients with mitral regurgitation (MR), data of exercise-induced pulmonary hypertension (EIPH) are limited, and feasibility of evaluating EIPH is not high. We aimed to investigate prognostic impact of EIPH and its substitute parameters. Exercise stress echocardiography (ESE) were performed in 123 consecutive patients with moderate to severe degenerative MR. The endpoint was a composite of death, hospitalization for heart failure, and worsening of symptoms. EIPH [tricuspid regurgitation peak gradient (TRPG) at peak workload ≥ 50 mmHg] was shown in 57 patients (46%). TRPG at low workload was independently associated with TRPG at peak workload (β = 0.67, p

The aim of the study is to compare the utility and efficacy of the LigaSure system and standard surgical clips for robot-assisted radical prostatectomy. The medical records of 473 patients who underwent robot-assisted radical prostatectomy between May 2011 and 2018 were retrospectively reviewed and analyzed. The patients were divided into the LigaSure (n = 125) and Hem-o-lok (n = 348) groups, based on the device used to ligate the vascular pedicle. Owing to differences in the patient characteristics, a 1:2 propensity score matching was performed. In the final matched cohort, 375 patients were included for analysis. The median initial prostate-specific antigen level was not significantly different between the two groups. The median surgical and console times were significantly shorter for the LigaSure group than for the Hem-o-lok group (p

To assess the impact of interactions between kidney stone formation and conventional risk factors on incident chronic kidney disease (CKD). A total of 11,402 subjects (men 30–69 years of age, Japanese) without CKD at baseline were observed over an average period of 4 years. Cox proportional hazards regression models were used to calculate hazard ratios (HRs) with 95% confidence intervals (CIs) to determine the association between incident CKD, kidney stone formation, and conventional risk factors (diabetes mellitus, hypertension, overweight/obesity, dyslipidemia, and hyperuricemia/gout). We also examined the interactions of renal stones and the conventional risk factors for CKD. In total, 2301 men (20.2%) developed incident CKD during the follow-up period. After multivariable adjustment, kidney stones were found to increase the risk of incident CKD (HR 1.16; 95% CI 1.03–1.32). Kidney stone formers with hypertension, dyslipidemia, or hyperuricemia/gout presented a greater risk for incident CKD than those who had either kidney stones or other risk factors. However, no significant interactions between kidney stones and other risk factors were found to increase CKD risk. On the other hand, a negative interactive effect between kidney stones and overweight/obesity was observed, leading to reversed risk of incident CKD in coexistence of both factors. Kidney stones were linked to a higher risk for the development of CKD. However, no positive interactive effects were identified between renal stones and conventional risk factors on the risk of incident CKD.

Surgical hand hygiene reduces the risk of surgical site infections (SSIs). SSIs are not considered an issue in endourological surgery, whereas febrile urinary tract infections (f-UTIs) and urological sepsis are becoming problematic. We wondered whether surgical hand hygiene is necessary for endourological surgery. Therefore, we aimed to evaluate the influence of surgical hand hygiene on f-UTI onset in endourological surgery by comparing procedures in which surgical hand hygiene with double gloving was used with those in which regular hand hygiene with double gloving was used between April 2016 and July 2020. In this prospective cohort study of 477 patients who underwent endourological surgeries, surgeons in the surgical hand hygiene and regular hygiene groups performed surgery on 259 and 218 patients, respectively. There was no significant difference in patient background, and multivariate analyses revealed no significant differences in f-UTI onset (odds ratio, 0.87; p = 0.74) between the two groups. In conclusion, regular hand hygiene with double gloving may be considered an alternative to surgical hand hygiene to prevent endourological f-UTIs, which could alter operational protocols for endourological surgery. Further studies are needed to validate our findings.

Risk assessment for urinary stones has been mainly based on urinary biochemistry. We attempted to identify the risk factors for urinary stones by statistically analyzing urinary biochemical and inflammation-related factors. Male participants (age, 20–79 years) who visited Nagoya City University Hospital were divided into three groups: a control group (n = 48) with no history of stones and two stone groups with calcium oxalate stone experience (first-time group, n = 22; recurring group, n = 40). Using 25-µL spot urine samples, we determined the concentrations of 18 candidate urinary proteins, using multiplex analysis on a MagPix® system. In univariate logistic regression models classifying the control and first-time groups, interleukin (IL)-1a and IL-4 were independent factors, with significantly high areas under the receiver operating characteristic curve (1.00 and 0.87, respectively, P

The standard treatment of prosthetic paravalvular leakage (PVL) accompanied by heart failure or hemolytic anemia is repeat open surgery. Although favorable outcomes for transcatheter prosthetic PVL closure in patients with a high surgical risk for repeat open surgery have been reported, procedural feasibility has not been examined in Japan. From March 2015 to November 2015, transcatheter PVL closure in mitral position was performed in four patients (age range 41–78, three females) with high surgical risk due to history of repeated surgeries, chest radiation and reconstruction of the mitral annulus after debridement of abscess owing to infective endocarditis. All procedures were performed via a transapical approach under general anesthesia. Of four patients, the indications for PVL closure of two patients were heart failure, and those of the others were hemolytic anemia. There were no major complications, mortalities, or prolonged intensive care unit or hospital stays. Technical success was achieved in two patients. Moderate paravalvular regurgitation persisted in one patient, although regurgitation reduction of one grade was obtained. In one patient, occluder devices were not deployed because the wire could not cross the defect. Improvement in New York Heart Association functional class compared with that at baseline was observed in two patients. The safety and acute technical success rates of transcatheter mitral PVL closure via a transapical approach were confirmed in Japanese patients. In Japan, transcatheter PVL closure may be an alternative option for patients with PVL who have a high surgical risk.

Background Serum Wisteria floribunda agglutinin positive Mac-2 binding protein (WFA+-M2BP) or Mac-2 Binding Protein Glycosylation Isomer (M2BPGi) is a novel biomarker currently applied for evaluating hepatic fibrosis. The aim of this study was to evaluate the utility of serum WFA+-M2BP level as a biomarker in chronic heart failure (HF) patients with abnormal liver function.

To compare the longitudinal health-related quality of life (HRQoL) after surgical intervention with ureteroscopic lithotripsy (URSL) and shock wave lithotripsy (SWL) and to evaluate the factors affecting HRQoL in urolithiasis patients. A total of 262 patients who underwent lithotripsy (SWL, n = 61; URSL, n = 201) for upper urinary tract calculi treatment between June 2012 and January 2015 were evaluated. All patients were administered the Short-Form 36-item survey (SF-36) to assess HRQoL at four timepoints: before surgery, on the day of discharge, and 1 and 6 months after lithotripsy. Stone-free rates, complications, and analgesic requirements were evaluated to compare the effects of the two procedures on HRQoL. At the day of discharge, patients in the URSL group had significantly lower mean scores on five different subscales of the SF-36 questionnaire, namely, physical functioning, role-physical, social functioning, role-emotional, and mental health. The stone-free rate at 3 months after lithotripsy was significantly lower in the SWL group (72.1% vs. URSL, 93.0%; p

Several experimental and animal studies have demonstrated that substances rich in antioxidants can reduce the physicochemical and peroxidative risk factors for calcium oxalate (CaOx) renal stone formation in urine and blood. However, there are very few such investigations in humans. In the present pilot study, two varieties of tea, a green one from Japan (JGT) and a herbal one from South Africa (Rooibos) (RT), both rich in antioxidants, were administered to a group of CaOx stone formers (SF) (n = 8) for 30 days. Both teas were analysed for polyphenols by high-performance liquid chromatography and for minerals by plasma atomic and optical emission spectroscopy. 24 h urines (baseline and day 30) were analysed for lithogenic factors. CaOx metastable limits and crystal nucleation and growth kinetics were also determined in each urine sample. Deposited crystals were inspected by scanning electron microscopy. Blood samples were collected (baseline and day 30). Biomarkers of oxidative stress including plasma and urinary thiobarbituric acid reactive substances (TBARS) and urinary N-acetyl-β-D-glucosaminidase (NAG) were also determined. Urinary physicochemical risk factors were also investigated after ingestion of RT for 30 days in two control groups (CG1 and CG2), the latter one of which consisted of habitual JGT drinkers. Statistical analyses were performed using Wilcoxon signed rank tests and Mann-Whitney tests for paired and independent measurements, respectively. Several flavonoids and catechins were quantified in RT and JGT, respectively, confirming that both teas are rich sources of antioxidants. Mineral content was found to be far below dietary reference intakes. There were no significant changes in any of the urinary physicochemical or peroxidative risk factors in the control groups or in SF, except for the supersaturation (SS) of brushite (Bru) which decreased in the latter group after ingestion of JGT. Crystal morphology showed a tendency to change from mixed CaOx mono- and di-hydrate to monohydrate after ingestion of each tea. Since the latter form has a stronger binding affinity for epithelial cells, this effect is not protective. Analysis of the physicochemical and peroxidative risk factors in CG1 and CG2 did not reveal any evidence of a synergistic effect between the two teas. Paradoxically, baseline risk factors in the habitual JGT control group were significantly raised relative to those in CG1. Our preliminary results suggest that ingestion of RT and JGT does not reduce the risk factors for CaOx stone formation in humans, but these findings need to be tested in further studies involving much larger sample sizes.

Background Pheochromocytomas are rare neuroendocrine tumors, with a malignancy frequency of approximately 10%. The treatment of malignant pheochromocytoma is palliative, and the traditional management strategy has limited efficacy. Furthermore, no clear criteria exist for the treatment of metastatic pheochromocytoma, especially for unresectable lesions. We report a case of complete remission of metastatic pheochromocytoma in 123I-metaiodobenzylguanidine (MIBG) scintigraphy after a single session of 131I-MIBG therapy. Case presentation A 61-year-old woman had a right adrenal grand tumor and lymph node metastasis on the hilum of the right kidney, both of which incorporated MIBG. After surgery, immunostaining of a tumor specimen showed expression of the tumor makers chromogranin and synaptophysin. One year postoperatively, abdominal computed tomography revealed a local recurrence and retroperitoneal lymph node swelling. The local recurrence was positive for MIBG uptake, whereas the swollen retroperitoneal lymph nodes were negative. She underwent surgery again, but the local recurrence was unresectable because of rigid adhesion to the surrounding tissue. Immunostaining of an intraoperatively extracted swollen retroperitoneal lymph node showed expression of tumor markers. The patient then underwent a single session of 131I-MIBG therapy (7.4 GBq, 200 mCi), after which the residual lesions no longer incorporated MIBG, and a complete response in 123I- metaiodobenzylguanidine (MIBG) scintigraphy was achieved. The 131I-MIBG treatment was repeated 6 months later. None of the lesions were positive for MIBG uptake. Conclusions 131I-MIBG therapy efficaciously treats unresectable lesions that are positive for MIBG uptake.

We established an experimental co-culture system for renal tubular cells and adipocytes to investigate kidney stone formation mechanisms under metabolic syndrome (MetS) conditions and examined the interaction between these cells morphologically and genetically. M-1s and 3T3-L1s were cultured individually (control, CON), with 24-h culture media from each cell type added to the other cell type (replacement, RP) in 2-layer co-culture dishes for 24 h (transwell, TW). M-1s were then exposed to calcium oxalate monohydrate (COM) crystals, and attached (14)C-labeled COM crystals were quantified. Expression of kidney stone- and adipocyte-related genes was analyzed. The radioactivity of adherent COM crystals significantly increased in TW and was relatively higher in RP compared to CON. M-1s demonstrated significant upregulation of adiponectin (Adipoq) in RP and secreted phosphoprotein 1 (Spp1) in TW compared to CON before COM crystal exposure, and significant downregulation of Spp1 in TW and upregulation of tumor necrosis factor (Tnf), interleukin 6 (Il-6), and chemokine (C-C motif) ligand 2 (Ccl2) compared to CON after COM crystal exposure. 3T3-L1s showed significant upregulation of Spp1, Adipoq, Tnf-α, and Ccl2 compared to CON. Enzyme-linked immunosorbent assays of co-culture medium revealed significantly increased TNF-α in TW. Our results highlight the potential for paracrine interactions between renal tubular cells and adipocytes and suggest that MetS conditions may lead to kidney stone formation.

To evaluate the efficacy of company-initiated training of urologists on shock wave lithotripsy (SWL) treatment results, we retrospectively assessed 602 patients who underwent SWL in Nagoya City University Hospital between January 2004 and June 2011 using Lithotripter S (Dornier MedTech, Japan). Training—provided by a training specialist of the company in June 2010—focused on the targeting of renal and proximal ureter stones with a combination of radiography and ultrasonography (US). The stretcher wedges were positioned in the semi-prone position or the semi-supine position for middle and distal ureter stones, respectively. Success rates between 519 pre-training treatments and 83 post-training treatments were compared. Patient age and stone location, burden, number, and composition did not significantly differ between pre- and post-training. Training improved the overall success rate from 66.3 to 87.2 % (P < 0.0001). The mean number of SWL treatments decreased from 1.8 ± 1.8 to 1.4 ± 1.3 (P = 0.01). The first SWL treatment success rate increased from 67.1 to 83.7 % (P = 0.002), and the need for multiple treatments decreased. The frequency of detection of renal and proximal ureter stones by both radiography and US increased from 10.5 % before training to 58.2 % after training (P < 0.0001). Significant factors for successful SWL were determined to be training and prone position for distal ureter stones by multivariate analysis and ultrasonic detection for renal and proximal ureter stones by univariate analysis. Skills in targeting stones using ultrasonography and selecting the proper therapeutic position are essential for improving the success rate of stone removal.

The objective of this study was to evaluate the efficacy of a gemcitabine and docetaxel (GD) combination as a second-line treatment for patients with metastatic urothelial carcinoma (UC) after failure of first-line treatment with platinum-based chemotherapy.From June 2006 to January 2012, 38 patients with metastatic UC previously treated with platinum-based chemotherapy received GD therapy. This consisted of gemcitabine 800 mg/m(2) and docetaxel 40 mg/m(2) on days 1 and 8 of each 21-day cycle as second-line chemotherapy. All the patients were evaluated for toxicity and assessed every cycle by imaging. We analyzed the efficacy of GD as second-line chemotherapy in the follow-up study.The median number of GD treatment cycles was 4 (range 2-9); the objective response rate was 47.4 %; and the median progression-free survival and median overall survival were 4.1 and 10.8 months, respectively. Univariate and multivariate analyses on the GD treated group showed that the existence of lung metastases was the only prognostic factor for tumor response. Grade 3 treatment-related toxicity included neutropenia (31.6 %) and thrombocytopenia (15.8 %), and only one patient with grade 4 toxicity had thrombocytopenia (2.6 %).The GD regimen as second-line chemotherapy was especially effective for lung metastatic UC and yielded favorable results in patients whose first-line platinum-based chemotherapy had failed. Given the safety and benefit profile seen in this study, a large prospective study is warranted to consider the potential utility of GD chemotherapy as a second-line for UC.

A previous genome-wide association study (GWAS) reported three novel nephrolithiasis-susceptibility loci at 5q35.3, 7p14.3 and 13q14.1. Here, we investigated the association of these loci with nephrolithiasis by using an independent Japanese sample set. We performed case-control association analysis using 601 patients with nephrolithiasis and 201 control subjects. We selected seven single-nucleotide polymorphisms (SNPs): rs12654812 and rs11746443 from 5q35.3 (RGS14-SLC34A1-PFN3-F12); rs12669187 and rs1000597 from 7p14.3 (INMT-FAM188B-AQP1); and rs7981733, rs1170155, and rs4142110 from 13q14.1 (DGKH (diacylglycerol kinase)), which were previously reported to be significantly associated with nephrolithiasis. rs12654812, rs12669187 and rs7981733 were significantly associated with nephrolithiasis after Bonferroni's correction (P=3.12 × 10(-3), odds ratio (OR)=1.43; P=6.40 × 10(-3), OR=1.57; and P=5.00 × 10(-3), OR=1.41, respectively). Meta-analysis of current and previous GWAS results indicated a significant association with nephrolithiasis (P=7.65 × 10(-15), 7.86 × 10(-14) and 1.06 × 10(-9), respectively). We observed a cumulative effect with these three SNPs; individuals with three or more risk alleles had a 5.9-fold higher risk for nephrolithiasis development than those with only one risk allele. Our findings elucidated the significance of genetic variation at these three loci in nephrolithiasis in the Japanese population.

Urinary stones consist of two phases-an inorganic (mineral) phase and an organic (matrix) phase. Studies on the organic components of kidney stones have been undertaken later than those on the inorganic components. After osteopontin was identified as one of the matrix components, the biomolecular mechanism of urinary stone formation became clearer. It also triggered the development of new preventive treatments. Osteopontin expression is sporadically observed in normal distal tubular cells and is markedly increased in stone-forming kidneys. Calcium oxalate crystals adhering to renal tubular cells are incorporated into cells by the involvement of osteopontin. Stimulation of crystal-cell adhesion impairs the opening of mitochondrial permeability transition pores (mPTP) in tubular cells and produces oxidative stress, apoptosis, and osteopontin expression. Macrophages phagocytose and digest a small amount of crystals, but many crystals aggregate into a mass containing osteopontin and epithelial cell debris and are excreted into the renal tubular lumen, becoming nuclei of urinary stones. This biomolecular mechanism is similar to atherosclerotic calcification. Based on these findings, new preventive treatments have been developed. Dietary control such as low-cholesterol intake and the ingestion of antioxidative foods and vegetables have successfully reduced the 5-year recurrence rate. Osteopontin antibodies and cyclosporine A, which blocks the opening of mPTP, have markedly inhibited the expression of osteopontin and urinary stone formation in animal models.

Osteopontin (OPN) is an important matrix protein of renal calcium stone. However, the function of OPN in the early phase of renal crystal formation is not well defined. In this study, we examined OPN expression in the early phase of renal crystal formation with ultra-microstructural observations and immuno-TEM (transmission electron microscopy) in control and OPN knock-out (OPN-KO) mice. Glyoxylate (100 mg/kg) was intra-abdominally administered to male wild-type mice (C57BL/6, 8 weeks of age) and OPN-KO mice (C57BL/6, 8 weeks of age). Kidney was collected before and 6, 12, and 24 h after administration. We examined the relation between renal crystal formation and microstructural OPN location using TEM and immunohistochemical staining of OPN as well as western blotting and quantitative RT-PCR for OPN. OPN protein expression gradually increased in the renal cortex-medulla junction after glyoxylate administration, and OPN mRNA was increased until 12 h, but decreased at 24 h. In ultra-microstructural observation, OPN began to appear on the luminal side of renal distal tubular cells at 6 h and was gradually detected in the tubular lumen at 12 h. OPN was present in the crystal nuclei and collapsed mitochondria in the tubular lumen. In the OPN-KO mice, collapsed mitochondria were present, but no crystal nuclei formation were detected at 24 h. Based on the results this study proposed that the appearance of organelles, such as mitochondria and microvilli, in the tubular lumen after cell injury may be the starting point of crystal nucleus formation due to the aggregation ability of OPN.

Renal stone formation and renal failure among Chinese infants administered melamine-containing formula were increasingly reported in 2008. We investigated the mechanism by which melamine and cyanuric acid induce renal stone formation and renal failure. Ten-week-old rats were administered either melamine [2.4, 24, or 240 mg/kg/day], both melamine and cyanuric acid [each at 1.2, 12, or 120 mg/kg/day], or water (controls). Blood and 24-h urine samples and kidney sections were evaluated on days 3, 7, and 14. In rats administered melamine alone or the low-dose melamine/cyanuric acid combination [1.2 mg/kg/day], crystals were not detected. On day 3, crystal formation was observed in the renal distal tubular lumens and collecting ducts of rats administered the intermediate-dose melamine/cyanuric acid [12 mg/kg/day], and the number of crystals increased during the course of the experiment. In rats administered the high-dose melamine/cyanuric acid [120 mg/kg/day], crystals were found in the proximal tubular lumens of the renal cortex on day 3, but acute renal failure resulted in death by day 7. Polarized light optical microphotography and scanning electron microscopy revealed tubular lumens occluded by a layer of axle-shaped crystals. X-ray diffraction findings revealed a nitrogen component but no calcium. The upper regions of occluded tubes were expanded, and the epithelium was thin. Melamine and cyanuric acid in combination, but not by melamine alone induce crystal formation and affected renal functioning. Renal failure due to melamine cyanurate crystals appears to occur via tubular occlusion.

Streptococcus pyogenes is indigenous to the human pharynx and causes acute pharyngitis. Balanoposthitis is an inflammatory disease of the glans and the foreskin. However, balanoposthitis caused by S. pyogenes is not widely recognized as a sexually transmitted disease. In addition, bacteriological features of the isolates causing balanoposthitis are unclear. The four S. pyogenes strains isolated from adult balanoposthitis were examined. We performed emm typing, T antigen typing, RAPD assay, PCR assay for the streptococcal pyrogenic exotoxin-related genes and antibiotic-resistant genes, and antibiotic susceptibility assay. All four strains were suspected to be transmitted by penile-oral sexual intercourse, were found to be different by genetic analysis, and also harbored some antibiotic-resistant factors. We propose that S. pyogenes should be considered as a causative agent of sexually transmitted disease. The drug resistant S. pyogenes must be taken into account when balanoposthitis patients are treated with antibiotic.

Background The purpose of this study was to assess the efficacy of Bacillus Calmette-Guerin (BCG) therapy after a second transurethral resection (TUR) in new onset high-grade T1 bladder cancer. Methods From January 2008 to September 2013, 207 patients with new onset high-grade T1 bladder cancer after an initial TUR were treated at our university and at affiliated hospitals. Residual cancer rate, intravesical recurrence-free survival (RFS), and risk factors for intravesical recurrence were analyzed. Results Among a total of 207 patients, 42 patients were treated with BCG therapy following a second TUR (group 1), 23 were treated with second TUR alone (group 2), 72 were treated with BCG alone (group 3), and 70 were treated without a second TUR or BCG. The median patients’ age was 72.0 years, and the median follow-up period was 33.5 months. The second TUR revealed that 34 patients (52 %) had residual cancer. Between groups 1 and 2 and groups 1 and 3, the differences in RFS were statistically significant (p = 0.002 and 0.045, respectively). In addition, BCG therapy was the most significant factor to predict RFS after the second TUR. Among the 31 patients whose pathology of the second TUR was pT0, only 1 of 12 patients (8 %) in group 1 and 11 of 19 patients (58 %) in group 2 had a recurrence. Conclusions BCG instillation following a second TUR decreases intravesical recurrence, even if the pathology of the second TUR is pT0.

Diterpenoid phytoalexins such as momilactones and phytocassanes are produced via geranylgeranyl diphosphate in suspension-cultured rice cells after treatment with a chitin elicitor. We have previously shown that the production of diterpene hydrocarbons leading to phytoalexins and the expression of related biosynthetic genes are activated in suspension-cultured rice cells upon elicitor treatment. To better understand the elicitor-induced activation of phytoalexin biosynthesis, we conducted microarray analysis using suspension-cultured rice cells collected at various times after treatment with chitin elicitor. Hierarchical cluster analysis revealed two types of early-induced expression (EIE-1, EIE-2) nodes and a late-induced expression (LIE) node that includes genes involved in phytoalexins biosynthesis. The LIE node contains genes that may be responsible for the methylerythritol phosphate (MEP) pathway, a plastidic biosynthetic pathway for isopentenyl diphosphate, an early precursor of phytoalexins. The elicitor-induced expression of these putative MEP pathway genes was confirmed by quantitative reverse-transcription PCR. 1-Deoxy-D: -xylulose 5-phosphate synthase (DXS), 1-deoxy-D: -xylulose 5-phosphate reductoisomerase (DXR), and 4-(cytidine 5'-diphospho)-2-C-methyl-D: -erythritol synthase (CMS), which catalyze the first three committed steps in the MEP pathway, were further shown to have enzymatic activities that complement the growth of E. coli mutants disrupted in the corresponding genes. Application of ketoclomazone and fosmidomycin, inhibitors of DXS and DXR, respectively, repressed the accumulation of diterpene-type phytoalexins in suspension cells treated with chitin elicitor. These results suggest that activation of the MEP pathway is required to supply sufficient terpenoid precursors for the production of phytoalexins in infected rice plants.

The incidence of urolithiasis has increased in the industrialized nations. However, both the pathogenesis and methods for its prevention remain to be clarified. We demonstrate that the antioxidative effect of green tea decreases the formation of calcium oxalate stones, OPN (osteopontin) expression, and apoptosis, and increases SOD (superoxide dismutase) activity in rat kidney tissues. The inhibitory effect of green tea on calcium oxalate urolithiasis is most likely due to its antioxidative effects. Therefore, we examined oxidative stress in vivo applied to Madin-Darby canine kidney (MDCK) cells, to which catechin, an antioxidant, was added. To evaluate the effects of oxidative stress on MDCK cells, we use a hypoxic condition because hypoxia is known to lead to oxidative stress. Confluent cultures of MDCK cells were exposed to (-)epigallocatechin 3 gallate (EGCG) (0, 0.1, 0.5, 5.0 mg/ml) for 2, 4, 8 or 16 h to determine changes in protein secretion and apoptosis. OPN protein expression was observed in MDCK cells of all 16 groups. The levels of expression of OPN protein were the same among all groups. In all groups, SOD protein expression was observed. In the groups exposed to EGCG 0.5, 5.0 mg/ml, SOD staining was more enhanced than in the EGCG 0 and 0.1 mg/ml groups. No deposits were detected in any of the 16 groups. RT-PCR was performed to detect sequences from OPN (979 bp) and SOD (447 bp). Quantitative analyses showed that SOD activity decreased gradually in all groups. Only in the EGCG 0 mg/ml 16 h group were TUNEL-positive cells observed. In the other groups, TUNEL-positive cells were not detected. EGCG used as an antioxidant protects renal tubular cell from cellular injury caused by oxidative stress through SOD protein expression.

Osteoporosis is associated with the pathogenesis of urinary stone formation. Urinary stones are similar to bone diseases such as osteoporosis and bone metabolism in terms of pathogenesis. Bisphosphonates are potent inhibitors of bone resorption, and are used in the management of bone disease. Furthermore, bisphosphonates have a strong affinity for calcium, and a reported inhibitory effect on calcium oxalate crystallization in vitro. Thus, bisphosphonates might also inhibit urinary stone formation. Madin-Darby canine kidney (MDCK) cells form calcium phosphate microliths at the basolateral side in vitro. We investigated the inhibitory effects of new generation bisphosphonates (alendronate and incadronate) on calcium phosphate microlith formation and on the expression of osteopontin, which is an important urinary stone matrix. MDCK cells formed two types of colonies in three-dimensional soft agar culture; dark colonies containing calcium phosphate microliths and clear colonies free from microliths. We applied purified alendronate and incadronate at concentrations of 10(-11), 10(-9), 10(-7) and 10(-5) M to MDCK cells cultured in three-dimensional soft agar and investigated the efficiency of colony formation and the dark colony ratio (number of dark colonies relative to the total number of colonies). The administration of 10(-9) and 10(-7) M alendronate decreased the dark colony ratio compared with controls, whereas incadronate did not significantly alter this colony ratio compared with controls. The expression of osteopontin in cultured cells was inhibited by the 10(-7) M alendronate administration. The present findings show that alendronate inhibits calcium stone formation, suggesting that it is effective in the prevention of urolithiasis.

Testing methods for crack-growth resistance-curve (R-curve) behavior were investigated and developed to analyze the toughened mechanism in polycry stalline ceramics. These methods are a biaxial-flexure method for small-scale disc-shaped specimens with micro-indentation cracks and a single-edged notched beam flexural method with crack stabilizers. In both methods, the growing crack length is measured directly as a function of applied stress, using the system that consist of a microscope and a CCD camera. Applying these testing methods, R-curve behavior of a toughened silicon nitride with a preferred orientation of elongated grains was evaluated to characterize the toughened mechanism, comparing with the behavior in a commercially available silicon nitride. The behavior having these rising R-curves is discussed with emphasis on the effects of microstructure such as grain-growth and grain-orientation, and resultant grain-bridgings behind the crack-tip.