First Complete Genome Sequence of Barley Virus G Identified from Proso Millet (Panicum miliaceum) in South Korea.

The complete genome sequence of an Uiseong isolate of barley virus G (BVG) on proso millet plants in a field in South Korea was determined by RNA sequencing and Sanger sequencing. To our knowledge, this is the first complete genome sequence report of BVG infecting proso millet in South Korea.

AMP-activated protein kinase is involved in the activation of the Fanconi anemia/BRCA pathway in response to DNA interstrand crosslinks.

Fanconi anemia complementation group (FANC) proteins constitute the Fanconi Anemia (FA)/BRCA pathway that is activated in response to DNA interstrand crosslinks (ICLs). We previously performed yeast two-hybrid screening to identify novel FANC-interacting proteins and discovered that the alpha subunit of AMP-activated protein kinase (AMPKα1) was a candidate binding partner of the FANCG protein, which is a component of the FA nuclear core complex. We confirmed the interaction between AMPKα and both FANCG using co-immunoprecipitation experiments. Additionally, we showed that AMPKα interacted with FANCA, another component of the FA nuclear core complex. AMPKα knockdown in U2OS cells decreased FANCD2 monoubiquitination and nuclear foci formation upon mitomycin C-induced ICLs. Furthermore, AMPKα knockdown enhanced cellular sensitivity to MMC. MMC treatment resulted in an increase in AMPKα phosphorylation/activation, indicating AMPK is involved in the cellular response to ICLs. FANCA was phosphorylated by AMPK at S347 and phosphorylation increased with MMC treatment. MMC-induced FANCD2 monoubiquitination and nuclear foci formation were compromised in a U2OS cell line that stably overexpressed the S347A mutant form of FANCA compared to wild-type FANCA-overexpressing cells, indicating a requirement for FANCA phosphorylation at S347 for proper activation of the FA/BRCA pathway. Our data suggest AMPK is involved in the activation of the FA/BRCA pathway.

Preventive effects of green tea (Camellia sinensis var. assamica) on diabetic nephropathy.

PURPOSE: This study aimed to evaluate the preventive effects of Camellia sinensis var. assamica (CSVA) on diabetic nephropathy in in vitro and in vivo models. MATERIALS AND METHODS: MDCK cells were incubated with 1 mM of oxalate with or without different concentrations of CSVA, then MTT and malondialdehyde (MDA) assays were performed to investigate the preventive effects of CSVA on oxalate-induced cytotoxicity and oxidative stress. Thirty male db/db mice were divided into three groups. Group 1 were fed AIN-93G ad libitum; group 2 were fed AIN-93G mixed with 10% fermented CSVA ad libitum; group 3 were fed AIN-93G mixed with 10% non-fermented CSVA ad libitum. The mice were sacrificed 14 weeks later, and the serum glucose level, 24-hour urine chemistry, and morphological changes in the kidneys were examined. RESULTS: As CSVA concentrations increased, viable MDCK cells increased in concentration. MDA production decreased over time in the CSVA treated group. The creatinine clearance of group 3 was lower than those of groups 1 and 2. The amount of urine microalbumin and protein in group 1 were higher than those in groups 2 and 3. Also, more glomerulus basement membrane foot processes were preserved in groups 2 and 3. CONCLUSION: In conclusion, CSVA has beneficial preventive tendencies towards diabetic nephropathy in both in vitro and in vivo models.

Reduction of oxidative stress in cultured renal tubular cells and preventive effects on renal stone formation by the bioflavonoid quercetin.

PURPOSE: We investigated the effects of quercetin on renal tubular cell injury induced by oxalate and the inhibitory effects of quercetin on urinary crystal deposit formation in an animal model. MATERIALS AND METHODS: MDCK cells (American Type Culture Collection, Manassas, Virginia) were incubated with different concentrations of oxalate with and without quercetin. MTT (Sigma) assays for cell viability, malondialdehyde and catalase activity were measured to investigate the antioxidant effect of quercetin. Male Sprague-Dawley rats were divided into 3 groups. Group 1 was fed standard rat chow. Groups 2 and 3 rats were fed standard chow supplemented with 3% sodium oxalate for 4 weeks. For the first 8 days in 4 weeks each rat in groups 2 and 3 also received gentamicin intramuscularly. Additionally, group 3 rats were administered quercetin for 4 weeks. Rats were sacrificed after 4 weeks, after which 24-hour urine collections and kidney removal were performed. In the renal tissue malondialdehyde, superoxide dismutase and catalase activity was measured. Bisected kidneys were examined under microscopy to determine the number of crystals. RESULTS: The viability of MDCK cells significantly decreased and malondialdehyde production increased in the presence of oxalate. However, co-exposure to quercetin inhibited the decrease in cell viability and inhibited the lipid peroxidation production induced by oxalate. In the animal study malondialdehyde production in group 3 significantly decreased compared to that in group 2. Catalase and superoxide dismutase activity was increased in group 3 compared to that in group 2. The number of crystals in kidneys in group 3 was decreased significantly compared to that in group 2. CONCLUSIONS: Quercetin has an inhibitory effect on urinary crystal deposit formation.

Effects of green tea on urinary stone formation: an in vivo and in vitro study.

PURPOSE: We evaluated whether epigallocatechin gallate (EGCG), a main constituent of green tea polyphenols, could protect against cellular toxicity by oxalate and whether green tea supplementation attenuates the development of nephrolithiasis in an animal model. MATERIALS AND METHODS: Cells of the NRK-52E line were incubated with different concentrations of oxalate with and without EGCG, and toxicity and malondialdehyde assays were done to investigate the cytotoxic effect of oxalate and the anti-oxalate effect of EGCG.. In a second series of experiments, male Sprague-Dawley rats were divided into three groups. Group 1 animals (controls) were fed regular chow and drank water ad libitum; group 2 animals were fed chow containing 3% sodium oxalate with the administration of gentamicin (40 mg/kg) and drank water ad libitum; group 3 animals were fed the same diet as group 2 with gentamicin administration and drank only green tea. Rats were killed 4 weeks later after a 24-hour urine collection, and the kidneys were removed for morphologic examination. RESULTS: As oxalate concentrations increased, the number of surviving cells decreased, and the formation of free radicals increased. The administration of EGCG inhibited free-radical production induced by oxalate. Green tea supplementation decreased the excretion of urinary oxalate and the activities of urinary gammaglutamyltranspeptidase and N-acetylglucosaminidase. The number of crystals within kidneys in group 3 was significantly lower than in group 2. CONCLUSIONS: Green tea has an inhibitory effect on urinary stone formation, and the antioxidative action of EGCG is considered to be involved.

An animal model of calcium oxalate urolithiasis based on a cyclooxygenase 2 selective inhibitor.

Our aim was to develop a stone-forming animal model involving renal tubular injury using a cyclooxygenase 2 selective inhibitor. Male Sprague-Dawley rats fed chow containing 3% sodium oxalate with or without 100 mg/kg celecoxib were compared to animals fed normal chow. Rats were killed after 2 or 4 weeks and the kidneys were harvested for morphological examination. Collections of 24-h urine were made before kidney harvest. After 2 weeks only a few crystals were observed in rats that received oxalate and celecoxib, but after 4 weeks more crystals were observed at the renal papilla than in rats that received only oxalate. Few crystals were found in rats fed normal chow with or without celecoxib. The urinary activities of gamma-glutamyl transpeptidase (GGT) were increased by celecoxib administration whereas creatinine clearance rates were unchanged. In rats fed oxalate, urinary oxalate excretion increased, but calcium excretion decreased. This model using a cyclooxygenase 2 selective inhibitor is a useful stone forming animal model involving mild renal tubular injury together with mild hyperoxaluria.

Apoptosis induced by oxalate in human renal tubular epithelial HK-2 cells.

Oxalate is not only considered to be one of the main constituents of urinary stones, but it also has toxic effects on renal tubular epithelial cells, affecting the pathogenesis of nephrolithiasis. We tried to elucidate the effects of oxalate on human renal tubular epithelial cells (HK-2 cells). In addition, we investigated whether the toxic effect of oxalate occurs by apoptosis, and determined the expression of Bcl-2 family and caspase 9 proteins known as apoptosis-related protein. HK-2 cells were incubated with different concentrations of oxalate, and the effect of oxalate on the growth of the cells was assessed by MTT assay. A caspase-3 activity assay and TUNEL assay were performed on HK-2 cells after oxalate exposure in order to evaluate apoptosis. Immunoblot analysis of Bax, Bcl-2, Bcl-xL, and caspase-9 were performed. Oxalate exposure resulted in significant growth inhibition of HK-2 cells as oxalate concentrations increased. The toxic effect of oxalate on HK-2 cells was considered to occur through apoptosis, as suggested by the increase of caspase-3 activity. The percentage of positive nuclei stained using the TUNEL method was 18+/-2.3 in oxalate-treated cells and 2.5+/-0.9 in untreated cells (P<0.05). Bax and caspase-9 protein expression increased significantly as oxalate concentrations increased, but Bcl-2 protein expression decreased. There was no difference in Bcl-xL protein expression among the various concentrations of oxalate. Our results show that oxalate has a toxic effect on HK-2 cells and that this effect is induced by apoptosis, which may be mediated by an intrinsic pathway.

Involvement of Rho-kinase in the contractile mechanism of human ureteral smooth muscle.

AIM: Even though many agents have been implicated as modulators of ureteral contractile activity, the exact mechanisms that control human ureteral smooth muscle contractility have yet to be clearly defined. Recently, Rho-kinase has been reported to be involved in the contractile mechanism of smooth muscles in various organs. In the present study, we sought to investigate whether or not Rho-kinase is expressed in the human ureteral smooth muscle, and to study its role regarding human ureteral smooth muscle contractility. METHODS: Ureteral samples were obtained from human adult subjects undergoing radical nephrectomy. Immunohistochemistry and Western blotting were performed to determine the presence of Rho-kinase in human ureter. Functional studies were performed with human ureteral strips suspended in organ bath, and the effects of Y-27632, a specific inhibitor of Rho-kinase, on baseline tensions, spontaneous contractions, and electrical field stimulation (EFS)-induced contractions were analyzed. RESULTS: The results of immunohistochemistry and immunoblotting study indicated that Rho-kinase is present in human ureteral smooth muscle. In functional analysis, Y-27632 was shown to decrease the baseline tension. And, both spontaneous and EFS-induced contractile responses of human ureteral strips were attenuated by Y-27632 in dose-dependent manners. CONCLUSIONS: For the first time, the results of the present study indicate that Rho-kinase is present in human ureteral smooth muscle and may play an important role in the intricate mechanism of human ureteral contractility and tone.