Biochemical Characterisation of Human Transglutaminase 4.

Transglutaminases are protein-modifying enzymes involved in physiological and pathological processes with potent therapeutic possibilities. Human TG4, also called prostate transglutaminase, is involved in the development of autoimmune and tumour diseases. Although rodent TG4 is well characterised, biochemical characteristics of human TG4 that could help th e understanding of its way of action are not published. First, we analysed proteomics databases and found that TG4 protein is present in human tissues beyond the prostate. Then, we studied in vitro the transamidase activity of human TG4 and its regulation using the microtitre plate method. Human TG4 has low transamidase activity which prefers slightly acidic pH and a reducing environment. It is enhanced by submicellar concentrations of SDS suggesting that membrane proximity is an important regulatory event. Human TG4 does not bind GTP as tested by GTP-agarose and BODIPY-FL-GTPγS binding, and its proteolytic activation by dispase or when expressed in AD-293 cells was not observed either. We identified several potential human TG4 glutamine donor substrates in the AD-293 cell extract by biotin-pentylamine incorporation and mass spectrometry. Several of these potential substrates are involved in cell-cell interaction, adhesion and proliferation, suggesting that human TG4 could become an anticancer therapeutic target.

Expression and function of human ribonuclease 4 in the kidney and urinary tract.

Antimicrobial peptides are essential host defense mechanisms that prevent urinary tract infections. Recent studies have demonstrated that peptides in the ribonuclease A superfamily have antimicrobial activity against uropathogens and protect the urinary tract from uropathogenic Escherichia coli (UPEC). Little is known about the antibacterial function or expression of ribonuclease 4 (RNase 4) in the human urinary tract. Here, we show that full-length recombinant RNase 4 peptide and synthetic amino-terminal RNase 4 peptide fragment have antibacterial activity against UPEC and multidrug-resistant (MDR)-UPEC. RNASE4 transcript expression was detected in human kidney and bladder tissue using quantitative real-time PCR. Immunostaining or in situ hybridization localized RNase 4 expression to proximal tubules, principal and intercalated cells in the kidney's collecting duct, and the bladder urothelium. Urinary RNase 4 concentrations were quantified in healthy controls and females with a history of urinary tract infection. Compared with controls, urinary RNase 4 concentrations were significantly lower in females with a history of urinary tract infection. When RNase 4 was neutralized in human urine or silenced in vitro using siRNA, urinary UPEC replication or attachment to and invasion of urothelial and kidney medullary cells increased. These data show that RNase 4 has antibacterial activity against UPEC, is expressed in the human urinary tract, and can contribute to host defense against urinary tract infections.NEW & NOTEWORTHY Ribonuclease 4 (RNase 4) is a newly identified host defense peptide in the human kidney and bladder. RNase 4 kills uropathogenic Escherichia coli (UPEC) and multidrug-resistant UPEC. RNase 4 prevents invasive UPEC infection and suppressed RNase 4 expression may be a risk factor for more severe or recurrent urinary tract infection.

Cell proliferation of rat bladder urothelium induced by nicotine is suppressed by the NADPH oxidase inhibitor, apocynin.

Tobacco smoking is a major risk factor for human cancers including urinary bladder carcinoma. In a previous study, nicotine enhanced rat urinary bladder carcinogenesis in a two-stage carcinogenesis model. Nicotine also induced cytotoxicity in the bladder urothelium in a short-term study. In the present study, male rats were treated with nicotine (40 ppm) in drinking water co-administered with the NADPH oxidase inhibitor, apocynin (0, 250 or 750 mg/kg) in diet for 4 weeks. The apocynin treatment induced no clinical toxic effects. Reduction of reactive oxygen species (ROS) by apocynin was confirmed by immunohistochemistry of 8-OHdG in the bladder urothelium. Incidences of simple hyperplasia, cell proliferation and apoptosis were reduced by apocynin treatment in the bladder urothelium. However, despite reduction of cell proliferation (labeling index), apocynin did not affect the incidence of simple hyperplasia, apoptosis, or ROS generation in the kidney pelvis urothelium, in addition to 8-OHdG positivity induced by nicotine being lower. In vitro, apocynin (500 µM) reduced ROS generation, but induced cell proliferation in bladder cancer cell lines (T24 and UMUC3 cells). These data suggest that oxidative stress may play a role in the cell proliferation of the bladder urothelium induced by nicotine.

Changes in the expression and function of the PDE5 pathway in the obstructed urinary bladder.

Our study aims to explore changes in bladder contractility and the phosphodiesterase type 5 (PDE5) signalling pathway in response to partial bladder outlet obstruction (PBOO). A surgically induced male rat PBOO model and human obstructed bladder tissues were used. Histological changes were examined by H&E and Masson's trichrome staining. Bladder strip contractility was measured via organ bath. The expressions of nitric oxide synthase (NOS) isoforms, PDE5, muscarinic cholinergic receptor (CHRM) isoforms and PDE4 isoforms in bladder were detected by RT-PCR and Western blotting. The immunolocalization of the PDE5 protein and its functional activity were also determined. PBOO bladder tissue exhibited significant SM hypertrophy and elevated responsiveness to KCl depolarization and the muscarinic receptor agonist carbachol. NOS isoforms, PDE5, CHRM2, CHRM3 and PDE4A were up-regulated in obstructed bladder tissue, whereas no change in PDE4B and PDE4D isoform expression was observed. With regard to PDE5, it was expressed in the SM bundles of bladder. Interestingly, obstructed bladder exhibited less relaxation responsiveness to sodium nitroprusside (SNP), but an exaggerated PDE5 inhibition effect. The up-regulation of PDE5 could contribute to the lack of effect on Qmax for benign prostatic hyperplasia/lower urinary tract symptom (BPH/LUTS) patients treated with PDE5 inhibitors. Moreover, PDE5 (with presence of NO) and PDE4 may serve as new therapeutic targets for bladder diseases such as BPH-induced LUTS and overactive bladder (OAB).

The STK16 inhibitor STK16-IN-1 inhibits non-adrenergic and non-neurogenic smooth muscle contractions in the human prostate and the human male detrusor.

Mixed lower urinary tract symptoms (LUTS) (voiding symptoms suggestive of benign prostatic hyperplasia plus storage symptoms, which can be caused by overactive bladder) are common in men. Unwanted contraction of prostate and/or bladder smooth muscle has been implied in the pathophysiology of male LUTS. Here, we examined effects of the serine/threonine kinase 16 (STK16) inhibitor STK16-IN-1 on contraction of human tissues from the prostate and male detrusor. Tissues were obtained from radical prostatectomy and radical cystectomy. Contractions were studied in an organ bath and STK16 expressions by Western blot analyses and fluorescence staining. In prostate tissues, STK16-IN-1 (1 µM) inhibited contractions induced by endothelin-1 and the thromboxane A2 analog U46619. Contractions of prostate tissues induced by noradrenaline, the α1-agonists phenylephrine and methoxamine, or electric field stimulation (EFS) were not changed by STK16-IN-1. In male detrusor tissues, STK16-IN-1 inhibited contractions induced by the cholinergic agonists carbachol and metacholine, and contractions induced by U46619. EFS-induced contractions of detrusor tissues were not changed by STK16-IN-1. Western blot analyses of prostate and detrusor tissues revealed bands matching the molecular weight of STK16. Fluorescence staining of prostate tissues using STK16 antibodies resulted in immunoreactivity in smooth muscle cells. STK16-IN-1 selectively inhibits non-adrenergic/non-neurogenic smooth muscle contractions in the male prostate and to limited extent in the bladder. Because non-adrenergic contractions in the male LUTS may account for limited efficacy of α1-blockers and for α1-blocker-resistant symptoms, studies assessing add-on of STK16-IN-1 to α1-blockers in mixed LUTS appear feasible.

Lipoxygenase-5 Expression in Canine Urinary Bladder: Normal Urothelium, Cystitis and Transitional Cell Carcinoma.

Transitional cell carcinoma (TCC) is the most common canine urinary tract tumour and mimics human invasive TCC. Human TCCs overexpress lipoxygenase (LOX)-5 and the use of target inhibitors has proven effective in inhibiting neoplastic growth. In this study, we investigated the immunohistochemical expression of LOX-5 in normal canine urinary bladder, cystitis and TCC. The comparative expression of LOX-5, cyclo-oxygenase (COX)-1 and COX-2 among the three tissue groups was also examined. Biopsy samples from cases of cystitis and TCC were reviewed from 2012 to 2016; samples of histologically normal bladder were used as controls. Dogs were excluded if they had received glucocorticoids, non-steroidal anti-inflammatory drugs (NSAIDs) and/or chemotherapy prior to tissue collection. LOX-5 was expressed in 95% of TCCs, 23% of cases of cystitis and 10% of controls. LOX-5 and COX-2 immunohistochemistry scores were significantly (P <0.01) higher in TCCs versus cystitis and normal bladders. Results of this study support the rationale for further investigation of the use of NSAIDs with dual anti COX-2 and LOX-5 effect for the treatment of canine TCC.

Ribonuclease 7 Shields the Kidney and Bladder from Invasive Uropathogenic Escherichia coli Infection.

BACKGROUND: Evidence suggests that antimicrobial peptides, components of the innate immune response, protect the kidneys and bladder from bacterial challenge. We previously identified ribonuclease 7 (RNase 7) as a human antimicrobial peptide that has bactericidal activity against uropathogenic Escherichia coli (UPEC). Functional studies assessing RNase 7's contributions to urinary tract defense are limited. METHODS: To investigate RNase 7's role in preventing urinary tract infection (UTI), we quantified urinary RNase 7 concentrations in 29 girls and adolescents with a UTI history and 29 healthy female human controls. To assess RNase 7's antimicrobial activity in vitro in human urothelial cells, we used siRNA to silence urothelial RNase 7 production and retroviral constructs to stably overexpress RNase 7; we then evaluated UPEC's ability to bind and invade these cells. For RNase 7 in vivo studies, we developed humanized RNase 7 transgenic mice, subjected them to experimental UTI, and enumerated UPEC burden in the urine, bladder, and kidneys. RESULTS: Compared with controls, study participants with a UTI history had 1.5-fold lower urinary RNase 7 concentrations. When RNase 7 was silenced in vitro, the percentage of UPEC binding or invading human urothelial cells increased; when cells overexpressed RNase 7, UPEC attachment and invasion decreased. In the transgenic mice, we detected RNase 7 expression in the kidney's intercalated cells and bladder urothelium. RNase 7 humanized mice exhibited marked protection from UPEC. CONCLUSIONS: These findings provide evidence that RNase 7 has a role in kidney and bladder host defense against UPEC and establish a foundation for investigating RNase 7 as a UTI prognostic marker or nonantibiotic-based therapy.

The dominant protein phosphatase PP1c isoform in smooth muscle cells, PP1cß, is essential for smooth muscle contraction.

Contractile force development of smooth muscle is controlled by balanced kinase and phosphatase activities toward the myosin regulatory light chain (RLC). Numerous biochemical and pharmacological studies have investigated the specificity and regulatory activity of smooth muscle myosin light-chain phosphatase (MLCP) bound to myosin filaments and comprised of the regulatory myosin phosphatase target subunit 1 (MYPT1) and catalytic protein phosphatase 1cß (PP1cß) subunits. Recent physiological and biochemical evidence obtained with smooth muscle tissues from a conditional MYPT1 knockout suggests that a soluble, MYPT1-unbound form of PP1cß may additionally contribute to myosin RLC dephosphorylation and relaxation of smooth muscle. Using a combination of isoelectric focusing and isoform-specific immunoblotting, we found here that more than 90% of the total PP1c in mouse smooth muscles is the ß isoform. Moreover, conditional knockout of PP1cα or PP1cγ in adult smooth muscles did not result in an apparent phenotype in mice up to 6 months of age and did not affect smooth muscle contractions ex vivo In contrast, smooth muscle-specific conditional PP1cß knockout decreased contractile force development in bladder, ileal, and aortic tissues and reduced mouse survival. Bladder smooth muscle tissue from WT mice was selectively permeabilized to remove soluble PP1cß to measure contributions of total (α-toxin treatment) and myosin-bound (Triton X-100 treatment) phosphatase activities toward phosphorylated RLC in myofilaments. Triton X-100 reduced PP1cß content by 60% and the rate of RLC dephosphorylation by 2-fold. These results are consistent with the selective dephosphorylation of RLC by both MYPT1-bound and -unbound PP1cß forms in smooth muscle.

Distribution of mast cell subtypes in interstitial cystitis: implications for novel diagnostic and therapeutic strategies?

AIMS: To identify the presence and geographical distribution of mast cell (MC) subtypes: MCT (tryptase positive-chymase negative) and MCTC (tryptase positive-chymase positive) in bladder tissue. METHODS: Bladder tissue was obtained from patients with painful bladder syndrome/interstitial cystitis (n=14) and normal histology from University Hospital Southampton tissue bank. Sequential tissue slices were immunohistochemically stained for MC subtypes using anti-MC tryptase (for MCT and MCTC) and anti-MC chymase (for MCTC). Stained sections were photographed, and positively stained MCs were quantified using ImageJ. Data were analysed using descriptive statistics and individual paired t-tests. RESULTS: There was a significant difference in the density of MCs between each layer of the disease bladder, with the greatest accumulation within the detrusor (p<0.001). There was a significant increase in MCTC subtype in the lamina (p=0.009) in painful bladder syndrome/interstitial cystitis. CONCLUSIONS: Our results suggest that mastocytosis is present within all layers of disease bladder, especially the muscle layer. The varying increase in MC subtypes in the lamina and mucosa may explain the variability in painful bladder syndrome/interstitial cystitis symptoms. A high influx of MCTC in the mucosa of individuals who also had ulceration noted within their diagnostic notes may be of the Hunner's ulcer subclassification. These findings suggest a relationship between the pathogenesis of MC subtypes and the clinical presentation of painful bladder syndrome/interstitial cystitis. A cohort study would further elucidate the diagnostic and/or therapeutic potential of MCs in patients with painful bladder syndrome/interstitial cystitis.

The extracellular matrix-degrading protein ADAMTS5 is expressed in the nuclei of urothelial cells in healthy rats.

OBJECTIVE: The aim of this study was to investigate whether protein expression of the extracellular matrix-degrading protease ADAMTS5 can be demonstrated in the urinary bladder of healthy rats, and, if so, to determine the localization of this enzyme. MATERIALS AND METHODS: The experiments were conducted with eight inbred male Sprague-Dawley rats. Immunohistochemistry was used to investigate the expression of ADAMTS5 in the urinary bladder. Negative controls were established by either excluding the primary antibody or applying the antibody after it had been preabsorbed with its immunogenic peptide. Confocal microscopy was used to visualize the distribution of ADAMTS5 in the urinary bladder tissue. RESULTS: Immunoreactivity for ADAMTS5 was demonstrated in the urothelium and in the detrusor. This expression was localized not only in the cytoplasm, but also in the nuclei. Confocal microscopy corroborated these findings. CONCLUSION: Expression of ADAMTS5 was demonstrated in the cytoplasm as well as in the nuclei of the urothelium and detrusor cells, suggesting that it may play a role at the transcriptional level.

RAB27B requirement for stretch-induced exocytosis in bladder umbrella cells.

Umbrella cells, which must maintain a tight barrier, modulate their apical surface area during bladder filling by exocytosis of an abundant, subapical pool of discoidal- and/or fusiform-shaped vesicles (DFVs). Despite the importance of this trafficking event for bladder function, the pathways that promote DFV exocytosis remain to be identified. We previously showed that DFV exocytosis depends in part on a RAB11A-RAB8A-MYO5B network, but RAB27B is also reported to be associated with DFVs, and knockout mice lacking RAB27B have fewer DFVs. However, the RAB27B requirements for DFV exocytosis and the relationship between RAB27B and the other umbrella cell-expressed RABs remains unclear. Using a whole bladder preparation, we observed that filling-induced exocytosis of human growth hormone-loaded DFVs was significantly inhibited when RAB27B expression was downregulated using shRNA. RAB27A was also expressed in rat urothelium; however, RAB27A-specific shRNAs did not inhibit exocytosis, and the combination of RAB27A and RAB27B shRNAs did not significantly affect DFV exocytosis more than treatment with RAB27B shRNA alone. RAB27B and RAB11A showed a small degree of overlap when quantified using Squassh segmentation software, and expression of dominant-active or dominant-negative mutants of RAB11A or RAB8A, or expression of a RAB11A-specific shRNA, had no significant effect on the size, number, or intensity of RAB27B-positive DFVs. Likewise, treatment with RAB27B-specific shRNA had no effect on RAB11A-positive DFV parameters. We conclude that RAB27B, but not RAB27A, regulates DFV exocytosis in bladder umbrella cells in a manner that may be parallel to the previously described RAB11A-RAB8A-MYO5B pathway.

FEATURES OF HISTOCHEMICAL CHANGES IN THE ACTIVITY OF SUCCINATE DEHYDROGENASE OF ARTIFICIAL BLADDER IN DYNAMICS (EXPERIMENTAL STUDY).

The gold standard for the treatment of invasive bladder cancer recognized throughout the world is radical cystectomy with orthotopic ileocystoplasty using the ileal intestinal tract. The study of the effect of urine on the adaptation of the mucosa of the artificial bladder continues for the last twenty years. According to the researchers, the results are quite contradictory, as some scientists note the hypersecretion of sulphomucins, sialomucins, progressive atrophy of microvilli, adenomatous hyperplasia and dysplasia. The aim of investigation to study the features of the histochemically revealed activity of succinate dehydrogenase in the wall of the artificial bladder and ileum in experimental animals. The material of the present study were the results obtained from the study of 18 female mini-pigs aged 4-5 months and weighing 8-10 kg. The modeling of the artificial bladder was performed in experimental animals, by cystectomy and subsequent ileo-cystoplasty. Experimental animals with a bladder model in groups of 6 animals were withdrawn from the experiment 3, 6 and 12 months after operational modeling. As for the wall of the official bladder, the changes in the activity of the studied enzymes were significant and showed not only possible changes in the activity of the Krebs cycle, but also about periodic displacements of the accents of substrate maintenance. These changes, in our view, are related to the transformation processes in the structural elements of the ileum wall, from which an unproblem has been formed to fulfill new functional duties. Signs of a violation of energy metabolism indicate the processes of hypoxia in the tissue of the artificial bladder and require further study and observation.

Effect of lupeol on antioxidants and xenobiotic enzymes in N-Butyl-N-(4-hydroxybutyl) nitrosamine induced bladder carcinogenesis in experimental rats.

OBJECTIVE: Urothelial carcinoma of the bladder is a common malignancy ranked 9th with an estimated 356,600 new cases diagnosed annually worldwide. The study showed the protective effects of Lupeol in N-Butyl-N-(4-hydroxybutyl) nitrosamine induced bladder carcinogenesis in in vivo experimental model. Forty male healthy wistar rats were selected randomly divided into four groups. Group I rats served as healthy control. Group II rats were treated with BBN (150 mg/gavage/twice a week) for 8 weeks. Group III rats were treated with BBN + Lupeol [ Lupeol (50 mg/kg bw/day) treatment was started 1 week prior to the BBN treatment, and it was orally administered for 8 weeks]. Group IV rats were treated with Lupeol alone (50 mg/kg bw/day) for 8 weeks. All the experimental rats were maintained and euthanized at 32nd week. Serum and bladder tissues were collected and examined for biochemical parameters, serum markers and histopathological evaluation. Preventive (BBN + Lupeol) group modulates the activity of antioxidant enzymes such as Superoxide dismutase, Catalase, Reduced glutathione, Glutathione Peroxidase, Thiobarbituric acid reactive substances (TBARS) and drug metabolizing enzymes such as Cytochrome P450, Cytochrome b5, NADPH Cytochrome c reductase, NADPH- Quinone Oxidoreductase 1 and Glutathione-S-transferase when compared to BBN treated rats. Serological markers such as Aspartate aminotransferase (AST) and Alanine aminotransferase (ALT) were significantly (P&#60;0.05) decreased in preventive lupeol treated groups. Lupeol supplementation protects BBN induced bladder carcinogenesis in experimental rats by its antioxidant, anti-inflammatory and antiproliferative properties.

Collaboration between Distinct Rab Small GTPase Trafficking Circuits Mediates Bacterial Clearance from the Bladder Epithelium.

Rab small GTPases control membrane trafficking through effectors that recruit downstream mediators such as motor proteins. Subcellular trafficking typically involves multiple Rabs, with each specific step mediated by a distinct Rab protein. We describe a collaboration between two distinct Rab-protein-orchestrated trafficking circuits in bladder epithelial cells (BECs) that expels intracellular uropathogenic Escherichia coli (UPEC) from their intracellular niche. RAB11a and RAB27b and their trafficking circuitry are simultaneously involved in UPEC expulsion. While RAB11a recruits its effector RAB11FIP3 and cytoskeletal motor Dynein, RAB27b mobilizes the effector MyRIP and motor Myosin VIIa to mediate bacterial expulsion. This collaboration is coordinated by deposition of the exocyst complex on bacteria-containing vesicles, an event triggered by the innate receptor Toll-like receptor 4. Both RAB11a and RAB27b are recruited and activated by the exocyst complex components SEC6/SEC15. Thus, the cell autonomous defense system can mobilize and coalesce multiple subcellular trafficking circuitries to combat infections.

Decreased Monoamine Oxidase A Expression and Activity in the Bladders of Rats With Partial Outlet Obstruction.

OBJECTIVE: To investigate changes in expression and activity of monoamine oxidase A (MAO-A) in rats with partial bladder outlet obstruction (pBOO). Previous studies suggested that monoamines, such as serotonin (5-hydroxytryptamine [5-HT]) and noradrenaline, are involved in bladder hyperactivity secondary to pBOO. However, little is known about the role of MAO-A, an enzyme oxidizing 5-hydroxytryptamine and noradrenalin, in the pathogenesis. MATERIALS AND METHODS: Female Sprague Dawley rats were subjected to sham or pBOO operations for 7 days, then their bladders were isolated. MAO-A protein levels in the bladder were examined by Western blotting. MAO-A activity was measured by the commercially available MAO-Glo Assay kit. In addition, MAO-A distribution in the bladder was examined by immunohistochemistry. RESULTS: Weights of the bladders from rats with pBOO were increased about 3.5-fold, compared with those from sham rats. Significant decreases in MAO-A protein and activity levels were observed in whole bladder of rats with pBOO compared with those of sham rats. By immunohistochemistry, it was firstly demonstrated that MAO-A was predominantly expressed in the detrusor layer of the sham rat bladders. The intensity of staining was decreased after pBOO operation. CONCLUSION: We demonstrated, for the first time, the distribution of MAO-A in the bladder and the pathologic changes in MAO-A protein and activity levels in rats with pBOO. This marked decrease in MAO-A potentially resulting in increased monoamine levels, especially in the detrusor of rat bladder, might be a key factor explaining the mechanism of bladder overactivity associated with pBOO.

Loss-of-Function Variants in MYLK Cause Recessive Megacystis Microcolon Intestinal Hypoperistalsis Syndrome.

Megacystis microcolon intestinal hypoperistalsis syndrome (MMIHS) is a congenital disorder characterized by loss of smooth muscle contraction in the bladder and intestine. To date, three genes are known to be involved in MMIHS pathogenesis: ACTG2, MYH11, and LMOD1. However, for approximately 10% of affected individuals, the genetic cause of the disease is unknown, suggesting that other loci are most likely involved. Here, we report on three MMIHS-affected subjects from two consanguineous families with no variants in the known MMIHS-associated genes. By performing homozygosity mapping and whole-exome sequencing, we found homozygous variants in myosin light chain kinase (MYLK) in both families. We identified a 7 bp duplication (c.3838_3844dupGAAAGCG [p.Glu1282_Glyfs∗51]) in one family and a putative splice-site variant (c.3985+5C>A) in the other. Expression studies and splicing assays indicated that both variants affect normal MYLK expression. Because MYLK encodes an important kinase required for myosin activation and subsequent interaction with actin filaments, it is likely that in its absence, contraction of smooth muscle cells is impaired. The existence of a conditional-Mylk-knockout mouse model with severe gut dysmotility and abnormal function of the bladder supports the involvement of this gene in MMIHS pathogenesis. In aggregate, our findings implicate MYLK as a gene involved in the recessive form of MMIHS, confirming that this disease of the visceral organs is heterogeneous with a myopathic origin.

Upregulated glutathione transferase omega-1 correlates with progression of urinary bladder carcinoma.

OBJECTIVES: Newly discovered glutathione transferase omega 1 (GSTO1-1) plays an important role in the glutathionylation cycle, a significant mechanism of protein function regulation. GSTO1-1 expression pattern has not been studied in transitional cell carcinoma (TCC), as yet. METHODS: A total of 56 TCC tumor and corresponding non-tumor specimens were investigated. Glutathione content and thioltransferase activity were measured spectrophotometrically. Protein-glutathione mixed disulfides were measured fluorimetrically. GSTO1-1 expression was determined by immunoblot and qPCR. Immunoprecipitation with GSTO1-1 antibody was followed by immunoblot using anti-GSTO1, GSTP1, c-Jun, JNK, Akt, phospho-Akt, and ASK1 antibody, while for the total S-glutathionylation levels non-reducing electrophoresis was performed. RESULTS: The contents of reduced glutathione and thioltransferase activity were significantly increased in tumor compared to non-tumor tissue. The increased GSTO1 expression in tumor tissue showed clear correlation with grade and stage. However, decreased total protein glutathionylation level in tumor compared to non-tumor samples was found. Immunoprecipitation has shown an association of GSTO1-1 with GSTP1, Akt, phospho-Akt, and ASK1 proteins. CONCLUSIONS: GSTO1 deglutathionylase activity suggests its potential important role in redox perturbations present in TCC. Increased GSTO1-1 expression might contribute to TCC development and/or progression supporting the notion that GSTO1-1 may be a promising novel cancer target.

Slowly cycling Rho kinase-dependent actomyosin cross-bridge "slippage" explains intrinsic high compliance of detrusor smooth muscle.

Biological soft tissues are viscoelastic because they display time-independent pseudoelasticity and time-dependent viscosity. However, there is evidence that the bladder may also display plasticity, defined as an increase in strain that is unrecoverable unless work is done by the muscle. In the present study, an electronic lever was used to induce controlled changes in stress and strain to determine whether rabbit detrusor smooth muscle (rDSM) is best described as viscoelastic or viscoelastic plastic. Using sequential ramp loading and unloading cycles, stress-strain and stiffness-stress analyses revealed that rDSM displayed reversible viscoelasticity, and that the viscous component was responsible for establishing a high stiffness at low stresses that increased only modestly with increasing stress compared with the large increase produced when the viscosity was absent and only pseudoelasticity governed tissue behavior. The study also revealed that rDSM underwent softening correlating with plastic deformation and creep that was reversed slowly when tissues were incubated in a Ca2+-containing solution. Together, the data support a model of DSM as a viscoelastic-plastic material, with the plasticity resulting from motor protein activation. This model explains the mechanism of intrinsic bladder compliance as "slipping" cross bridges, predicts that wall tension is dependent not only on vesicle pressure and radius but also on actomyosin cross-bridge activity, and identifies a novel molecular target for compliance regulation, both physiologically and therapeutically.

PAK5 mediates cell: cell adhesion integrity via interaction with E-cadherin in bladder cancer cells.

Urothelial bladder cancer is a major cause of morbidity and mortality worldwide, causing an estimated 150 000 deaths per year. Whilst non-muscle-invasive bladder tumours can be effectively treated, with high survival rates, many tumours recur, and some will progress to muscle-invasive disease with a much poorer long-term prognosis. Thus, there is a pressing need to understand the molecular transitions occurring within the progression of bladder cancer to an invasive disease. Tumour invasion is often associated with a down-regulation of E-cadherin expression concomitant with a suppression of cell:cell junctions, and decreased levels of E-cadherin expression have been reported in higher grade urothelial bladder tumours. We find that expression of E-cadherin in a panel of bladder cancer cell lines correlated with the presence of cell:cell junctions and the level of PAK5 expression. Interestingly, exogenous PAK5 has recently been described to be associated with cell:cell junctions and we now find that endogenous PAK5 is localised to cell junctions and interacts with an E-cadherin complex. Moreover, depletion of PAK5 expression significantly reduced junctional integrity. These data suggest a role for PAK5 in maintaining junctional stability and we find that, in both our own patient samples and a commercially available dataset, PAK5mRNA levels are reduced in human bladder cancer compared with normal controls. Taken together, the present study proposes that PAK5 expression levels could be used as a novel prognostic marker for bladder cancer progression.

Cigarette smoke induced urocystic epithelial mesenchymal transition via MAPK pathways.

Cigarette smoke has been shown to be a major risk factor for bladder cancer. Epithelial-mesenchymal transition (EMT) is a crucial process in cancer development. The role of MAPK pathways in regulating cigarette smoke-triggered urocystic EMT remains to be elucidated. Human normal urothelial cells and BALB/c mice were used as in vitro and in vivo cigarette smoke exposure models. Exposure of human normal urothelial cells to cigarette smoke induced morphological change, enhanced migratory and invasive capacities, reduced epithelial marker expression and increased mesenchymal marker expression, along with the activation of MAPK pathways. Moreover, we revealed that ERK1/2 and p38 inhibitors, but rather JNK inhibitor, effectively attenuated cigarette smoke-induced urocystic EMT. Importantly, the regulatory function of ERK1/2 and p38 pathways in cigarette smoke-triggered urocystic EMT was further confirmed in mice exposed to CS for 12 weeks. These findings could provide new insight into the molecular mechanisms of cigarette smoke-associated bladder cancer development as well as its potential intervention.