Musashi-1, an RNA-binding protein, is indispensable for survival of photoreceptors.

Musashi-1 (Msi1), an RNA-binding protein (RBP), has been postulated to play important roles in the maintenance of the stem-cell state, differentiation, and tumorigenesis. However, the expression and function of Msi1 in differentiated cells remain obscure. Here we show that Msi1 is expressed in mature photoreceptors and retinal pigment epithelium (RPE) cells, and is indispensable for the survival of photoreceptors. We found in the adult newt eye that Msi1 is expressed in all photoreceptors and RPE cells as well as in the retinal stem/progenitor cells in the ciliary marginal zone (CMZ). We found in the analyses of the newt normal and regenerating retinas that the expression profiles of the Msi1 transcripts and protein isoforms in the photoreceptors are different from those in the retinal stem/progenitor cells. Furthermore, we found that all photoreceptors and RPE cells of the adult mice also express Msi1, and that Msi1 knockout (Msi1-KO) results in degeneration of photoreceptors and a lack of a visual cycle protein RPE65 in the microvilli of RPE cells. Taken together, our current results demonstrate that the expression of Msi1 in mature photoreceptors and RPE cells is evolutionarily conserved, and that Msi1 bears essential functions for vision. Considering such an Msi1-KO phenotype in the retina, it is now reasonable to address whether defects of the Msi1 functions are responsible for inherited retinal diseases. Studying the regulation of Msi1 and the target RNAs of Msi1 in photoreceptors and RPE cells might contribute to fundamental and clinical studies of retinal degeneration.

A patient with adenocarcinoma of the jejunum successfully diagnosed by preoperative endoscopy for the small intestine.

In this study, the authors report the case of a 35-year-old man diagnosed preoperatively as having adenocarcinoma of the jejunum using a conventional endoscopy, usually used for the examination of the large intestine.

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.

Thrombomodulin alfa attenuates the procoagulant effect and cytotoxicity of extracellular histones through the promotion of protein C activation.

INTRODUCTION: Extracellular histones are reported to increase thrombin generation in the plasma and induce endothelial cell death in vitro. These effects of histones were suggested to involve histone-induced inhibition of TM-dependent activated protein C (APC) generation. Therefore, we hypothesized that TM alfa, a recombinant human soluble TM, attenuates these effects of histones by promoting the generation of APC. In the present study, we investigated the effects of TM alfa on the histone-induced decrease in APC generation, an increase in thrombin generation, and endothelial cell death in vitro. METHODS: APC generation was investigated using a chromogenic substrate based assay. Thrombin generation in plasma was studied by using a calibrated automated thrombogram method. Histone cleavage was detected by western blot analysis. Histone-induced endothelial cell death was evaluated by the trypan blue exclusion test. RESULTS: Histones decreased APC generation and increased thrombin generation in the presence of endothelial cells. TM alfa increased APC generation and decreased thrombin generation in the presence of histones and endothelial cells. TM alfa with thrombin and protein C cleaved histone H3, and attenuated histone-induced endothelial cell death. Antithrombin, an endogenous thrombin inhibitor, and gabexate mesilate, a synthetic protease inhibitor, inhibited thrombin generation, decreased APC generation, and did not have any effect on histone H3 cleavage or histone-induced endothelial cell death. CONCLUSIONS: TM alfa attenuated the histone-induced increase in thrombin generation and endothelial cell death by promoting APC generation in vitro.

Structural insights into the competitive inhibition of the ATP-gated P2X receptor channel.

P2X receptors are non-selective cation channels gated by extracellular ATP, and the P2X7 receptor subtype plays a crucial role in the immune and nervous systems. Altered expression and dysfunctions of P2X7 receptors caused by genetic deletions, mutations, and polymorphic variations have been linked to various diseases, such as rheumatoid arthritis and hypertension. Despite the availability of crystal structures of P2X receptors, the mechanism of competitive antagonist action for P2X receptors remains controversial. Here, we determine the crystal structure of the chicken P2X7 receptor in complex with the competitive P2X antagonist, TNP-ATP. The structure reveals an expanded, incompletely activated conformation of the channel, and identified the unique recognition manner of TNP-ATP, which is distinct from that observed in the previously determined human P2X3 receptor structure. A structure-based computational analysis furnishes mechanistic insights into the TNP-ATP-dependent inhibition. Our work provides structural insights into the functional mechanism of the P2X competitive antagonist.P2X receptors are nonselective cation channels that are gated by extracellular ATP. Here the authors present the crystal structure of chicken P2X7 with its bound competitive antagonist TNP-ATP and give mechanistic insights into TNP-ATP dependent inhibition through further computational analysis and electrophysiology measurements.

[Ileovesical fistula caused by bladder cancer with bladder stone].

Enterovesical fistula is a very rare complication of primary urological malignancies. A case of ileovesical fistula caused by a bladder carcinoma is presented. A 66-year-old male was referred with complaints of urinary pain. On admission, fecaluria and urinary tract infection with bladder stone were detected. Cystography revealed the passage of contrast medium into the small bowel. Under the diagnosis ofileovesical fistula due to suspected inflammatory disease, sigmoidectomy and segmental small bowel resection with partial cystectomy were performed. Histological evaluation revealed a poorly differentiated urothelial carcinoma. Without further treatment, the patient died from cancer five months after operation. However, it is hard to assess the effect of fistulas on prognosis. Since it has been reported that about 40% of the patients with T4 bladder tumors could be potentially cured with radical resection, we recommend a thorough examination to confirm the diagnosis of primary disease to obtain the best results.

Anti-inflammatory and anti-fibrinolytic effects of thrombomodulin alfa through carboxypeptidase B2 in the presence of thrombin.

BACKGROUND: Thrombomodulin (TM) alfa, a recombinant human soluble TM, enhances activation of pro-carboxypeptidase B2 (pro-CPB2) by thrombin. Activated pro-CPB2 (CPB2) exerts anti-inflammatory and anti-fibrinolytic activities. Therefore, TM alfa may also have anti-inflammatory and anti-fibrinolytic effects through CPB2. However, these effects of TM alfa have not been elucidated. In the present study, we investigated the effects of TM alfa on inactivation of complement component C5a as an anti-inflammatory effect and prolongation of clot lysis time as an anti-fibrinolytic effect via CPB2 in vitro. METHODS: CPB2 activity and tissue factor-induced thrombin generation was examined by a chromogenic assay. C5a inactivation was evaluated by C-terminal cleavage of C5a and inhibition of C5a-induced human neutrophil migration. Clot lysis time prolongation was examined by a tissue-type plasminogen activator-induced clot lysis assay. RESULTS: CPB2 activity in human plasma was increased by TM alfa and thrombin in a concentration-dependent manner. TM alfa inhibited tissue factor-induced thrombin generation and enhanced pro-CPB2 activation in human plasma simultaneously. The mass spectrum of C5a treated with TM alfa, thrombin, and pro-CPB2 was decreased at 156m/z, indicating that TM alfa enhanced the processing of C5a to C-terminal-cleaved C5a, an inactive form of C5a. C5a-induced human neutrophil migration was decreased after C5a treatment with TM alfa, thrombin, and pro-CPB2. TM alfa prolonged the clot lysis time in human plasma, and this effect was completely abolished by addition of a CPB2 inhibitor. CONCLUSIONS: TM alfa exerts anti-inflammatory and anti-fibrinolytic effects through CPB2 in the presence of thrombin in vitro.

Piezo1 expression increases in rat bladder after partial bladder outlet obstruction.

AIMS: For patients with benign prostatic hyperplasia (BPH), storage symptoms due to bladder dysfunction are bothersome, and that mechanism elucidation is needed. Piezo1, a mechanically activated ion channel, is believed to play a role in sensing bladder distension. To investigate the involvement of Piezo1 in bladder dysfunction, we examined the expression and distribution of Piezo1 and neurofilament (NF-L) to understand pathological alterations in rat bladders with partial bladder outlet obstruction (pBOO), an animal model of BPH. MAIN METHODS: Female Sprague-Dawley rats were subjected to sham or pBOO operations. On days 3, 7, and 14 after pBOO, Piezo1 mRNA levels in the bladder were examined by quantitative real-time PCR. The expression of light NF-L was also examined by western blotting. On day 7, the distributions of Piezo1 were examined by in situ hybridization. KEY FINDINGS: The expression levels of Piezo1 mRNA in whole bladder were significantly increased from days 3 to 14 after pBOO. On day 7 in pBOO rats, significant increases in Piezo1 mRNA were observed in the detrusor layer as well as the suburothelial layer, while the predominant distribution was observed in the urothelium of sham rats. Coinciding with the increase in Piezo1, the decreases in NF-L expression were observed in the bladder from pBOO rats. SIGNIFICANCE: The increase in Piezo1 in pBOO rat bladders might be involved in the compensatory mechanism associated with bladder denervation including the decrease in NF-L. Inhibition of Piezo-1 may be a new therapeutic approach to ameliorate the storage dysfunction shown in pBOO.

Large-scale identification and characterization of human genes that activate NF-kappaB and MAPK signaling pathways.

We have carried out a large-scale identification and characterization of human genes that activate the NF-kappaB and MARK signaling pathways. We constructed full-length cDNA libraries using the oligo-capping method and prepared an arrayed cDNA pool consisting of 150 000 cDNAs randomly isolated from the libraries. For analysis of the NF-kappaB signaling pathway, we introduced each of the cDNAs into human embryonic kidney 293 cells and examined whether it activated the transcription of a luciferase reporter gene driven by a promoter containing the consensus NF-kappaB binding sites. In total, we identified 299 cDNAs that activate the NF-kappaB pathway, and we classified them into 83 genes, including 30 characterized activator genes of the NF-kappaB pathway, 28 genes whose involvement in the NF-kappaB pathways have not been characterized and 25 novel genes. We then carried out a similar analysis for the identification of genes that activate the MARK pathway, utilizing the same cDNA resource. We assayed 145 000 cDNAs and identified 57 genes that activate the MARK pathway. Interestingly, 27 genes were overlapping between the NF-kappaB and the MAPK pathways, which may indicate that these genes play cross-talking roles between these two pathways.

A case of small cell carcinoma of the common bile duct.

Small cell carcinoma occasionally occurs in the gastrointestinal tract, but rarely in the biliary tract. We report a case of small cell carcinoma which occurred in the common bile duct. A 66-year-old female complained of epigastralgia and weight loss. Computed tomography and ultrasonography showed a mass near the pancreas head and dilatation of the intrahepatic bile ducts. Endoscopic nasobiliary drainage was undertaken, and it revealed obstruction of the common bile duct. The patient was diagnosed preoperatively as having extrahepatic bile duct cancer. Upon laparotomy, a tumor was found to be located in the middle common bile duct. Pylorus-preserving pancreaticoduodenectomy was performed. The main trunk of the portal vein and the right hepatic artery were resected concomitantly because of tumor involvement. Postoperative pathological examination revealed well-differentiated papillary adenocarcinoma on the surface of the bile duct lumen, but a large part of the extraductal component was small cell carcinoma. Upon immunohistochemical examination, synaptophysin and chromogranin A were found to be focally positive in small cell carcinoma, but negative for L-26 and CEA. The patient then underwent two postoperative courses of systemic chemotherapy. Nevertheless, she died of cancer recurrence eight months after the operation, which showed that the tumor had a highly lethal nature, with rapid and widespread dissemination. Further therapeutic trials are needed to improve survival in such cases.

Increased expression of 5-HT(2A) and 5-HT(2B) receptors in detrusor muscle after partial bladder outlet obstruction in rats.

Serotonin (5-hydroxytryptamine; 5-HT)-induced bladder contraction is enhanced after partial bladder outlet obstruction (pBOO) in rats. We investigated time-dependent changes in bladder contraction and expression of 5-HT(2A) and 5-HT(2B) receptor mRNA in bladder tissue to elucidate the mechanism of this enhancement. On day 3 and 7 after pBOO, contractile responses of isolated rat bladder strips to 5-HT were increased compared with that in sham-operated rats; on day 14, the response had decreased to the same level as that in sham rat bladders. In contrast, carbacholinduced contraction was not enhanced by pBOO at any time point. In sham rats, 5-HT(2A) receptor mRNA was expressed in the urothelium, and 5-HT(2B) receptor mRNA was expressed in the detrusor muscle layer. In pBOO rats, both receptor mRNAs were increased in the detrusor muscle and subserosal layers, but not in the urothelium. The increase of 5-HT(2A) receptor mRNA was maintained from day 3 to day 14 after pBOO, and 5-HT(2B) receptor mRNA was increased on day 7 after pBOO. These results suggested that pBOO induced up-regulation of the 5-HT(2A) and 5-HT(2B) receptors in the detrusor muscle and subserosal layers of the bladder, and such up-regulation may be related to the enhanced bladder contractile response to 5-HT.

Inter-male mating-like behavior in the domesticated house musk shrew, Suncus murinus.

In the present study, inter-male interaction of the domesticated house musk shrew was observed in detail under laboratory conditions. In most cases, during inter-male interaction, male house musk shrews exhibited a sequence of behavior items including tail-wagging, following, mounting and thrusting. In the minority of cases, males did not progress beyond following. Offensive behavior was not sufficiently violent to cause injury. It appeared that role assignment was decided by contact manner and vocalization. One of fundamental characters of this animal made a start of following, in which one shrew followed another, who touched and then separated. Role assignment (i.e., which male led and which followed) was decided in status battle. Roles were often reversed during following. Following behavior was maintained by 'polite' mutual contact, and the interaction progressed to thrusting in the majority cases. After role assignment, the variation in contact manner decreased. The ratio of time spent in front-and-behind contact to that spent in multi-lateral contact increased when both males commenced following behavior simultaneously. This ratio was maintained until the following male snapped after he finished thrusting. Even if the following male did not reach thrusting, he mounted the preceding male. The pairs who did not reach thrusting repeated following behavior or mounting. In those cases, while one male concentrated on touching the other to maintain following, the other attempted to divert attention from the following behavior. Male shrews were able to reach thrusting irrespective of sex.

The force driving mating behavior in the house musk shrew (Suncus murinus).

The mating behavior of the domesticated house musk shrew was observed in detail under laboratory conditions. The observations revealed that the house musk shrew has a series of behaviors before copulation. Tactile, auditory and chemical senses appear to function as flags for the recognition of conspecifics and to promote the development of an interaction between the sexes. The tactile senses and the use of the snout were particularly important in the mating sequence, and mutual contact appeared to give rise to driving the sequence to completion. The two sexes contacted each other 'politely', came to mounting by continuous following, and the male finished with a series of post-ejaculatory offensive behaviors and scent markings. The variation in the contact reduced once the female commenced tail-wagging. The ratio of the time spent in front-and-behind contact to that spent in multi-lateral contact increased when both sexes commenced following formation. This ratio was maintained until the male's post-ejaculatory offensive behavior finished. The series of mating behavior was completed by the continuous touching of both sexes and by changes in the manner of contact.

[Stage A prostate cancer: comparison of subclassification between Japanese rule and TNM, and outcome].

Between 1980 and June, 2002, transurethral resection of prostate was performed against 3294 cases of benign prostatic hyperplasia, and 144 cases of stage A cancer were detected (4.4%). Among these cases, 136 cases which had complete records of examination were studied. Annual number of stage A and A1:A2 ratio were not influenced by introducing PSA determination from 1991, although the number of T1c has been increasing gradually. Since subclassification of stage A is different between Japanese rules (A1; 3 chips of cancer with well-differentiated adenocarcinoma, A2; others) and TNM (T1a; 5% of less number of chips with cancer, T1b; others), two criteria were compared. Coincidence was found with 93.7%, and disagreement was due to ratio of number of chips with cancer to whole number resected, or different grade. The former difference was caused by a larger or smaller prostate. Most cases of A1 and A2 were subjected to watchful waiting or subsequent therapy. PSA was elevated in 10 cases (7%), two of which died from progression of cancer. Other cases were disease-free. Individual pathological findings are important for subclassification of stage A.

Molecular and immunohistologic analyses cannot reliably solve diagnostic variation of flat intraepithelial lesions of the urinary bladder.

We examined diagnostic variation of flat intraurothelial lesions with comparison with immunohistochemical and fluorescence in situ hybridization (FISH) analyses. Nine uropathologists diagnosed 23 biopsy samples from the urinary bladder. The samples were analyzed by immunohistochemical expression of cytokeratin 20, high-molecular-weight cytokeratin, Ki-67, p53, and p16(INK4a), and multicolor FISH using the UroVysion probe set (Vysis, Abbott, Des Plaines, IL). Diagnostic agreement for each classification and for nonneoplastic or neoplastic lesions was obtained in 8 (35%) and 16 (70%) of 23 lesions, respectively. The preference ratio of neoplasia to nonneoplasia (0.9 to 4.8) or carcinoma in situ to dysplasia (0.2 to 4.0) also varied among the pathologists. In 6 ancillary analyses, the majority of neoplastic lesions with diagnostic agreement indicated more than 2 aberrant results, whereas the majority of lesions without diagnostic agreement showed no or only 1 aberrant result. The molecular and immunohistochemical analyses can discriminate between neoplastic and nonneoplastic lesions; however, they cannot reliably solve diagnostic variation of flat intraepithelial lesions.

Collecting duct carcinoma of the kidney: an immunohistochemical evaluation of the use of antibodies for differential diagnosis.

Collecting duct carcinoma is a highly aggressive renal epithelial malignancy, although it accounts for less than 1% of the incidence of renal epithelial neoplasms. Differential diagnoses between collecting duct carcinoma, pelvic urothelial carcinoma with marked invasion to the renal parenchyma (invasive urothelial carcinoma), and papillary renal cell carcinoma is often challenging. In our current study, we examined the utility of using commercially available antibodies, in conjunction with lectin histochemistry, for such differential diagnoses. We examined 17 cases of collecting duct carcinoma, 10 cases of invasive urothelial carcinoma and 15 cases of papillary renal cell carcinoma (type 1, 6 cases; type 2, 9 cases) in these evaluations. Our results indicated that Ulex europaeus agglutinin 1, E-cadherin, and c-KIT were frequently positive in collecting duct carcinoma and invasive urothelial carcinoma, in comparison with papillary renal cell carcinoma, which had negative results for CD10 and alpha-methylacyl CoA racemase. We found, however, that collecting duct carcinoma showed positivity for high-molecular-weight cytokeratin and low-molecular-weight cytokeratin at a low frequency compared with invasive urothelial carcinoma, and that these distinctions need further careful evaluation. In addition, high-molecular-weight cytokeratin positivity was not a reliable marker for collecting duct carcinoma. We conclude that Ulex europaeus agglutinin 1 reactivity and positivity for E-cadherin and c-KIT are effective in distinguishing collecting duct carcinoma from papillary renal cell carcinoma, and that negative results for alpha-methylacyl CoA racemase and CD10 are potentially useful hallmarks of this distinction also. In contrast, a differential diagnosis for collecting duct carcinoma and invasive urothelial carcinoma will require careful examination of multiple routinely stained specimens, particularly in cases of in situ neoplastic lesions in the pelvic mucosa.

Comparison of Gleason grade and score between preoperative biopsy and prostatectomy specimens in prostate cancer.

AIM: Although the histopathological findings obtained from biopsy specimens are important for choosing the appropriate management of prostate cancer, there have been some discrepancies in Gleason grade and consequently, score between biopsy and surgical specimens. A comparison of findings between these two kinds of specimens was performed. METHODS: Radical prostatectomy was performed at Asahi General Hospital on 223 cases of T1b-T3 without previous cancer treatment, and the Gleason grade and score of the biopsy and surgical specimens were compared. RESULTS: A 37% coincidence in Gleason score was obtained between biopsy and surgical specimens; coincidence including one digit difference in score was approximately 70%. Upgrading was more than downgrading. Disagreement in secondary grade was greater than that in primary grade. Disagreement in Gleason score was roughly similar among different score items and was not influenced by level of prostate-specific antigen, however, the small volume of the cancer tissues more affected the discrepancy in score. CONCLUSION: The use of biopsy findings is required to be taken into account regarding the discrepancy.

Collecting duct (Bellini duct) renal cell carcinoma: a nationwide survey in Japan.

PURPOSE: Collecting duct carcinoma, a rare type of renal cell carcinoma, remains poorly understood. To analyze the nature of collecting duct carcinoma a retrospective survey was performed in Japan. MATERIALS AND METHODS: This survey was done from August 2001 to April 2003. A total of 281 institutions throughout Japan were requested to document all cases of collecting duct carcinoma. All pertinent clinical information was compiled, including patient age, sex, mode of presentation, evaluation modality, preoperative laboratory data, surgery type, macroscopic and microscopic findings, and survival data. Two urological pathologists reviewed microscopic slides of all tumor specimens to confirm collecting duct carcinoma. RESULTS: Two pathologists confirmed collecting duct carcinoma in 81 of the 120 cases documented as collecting duct carcinoma. Mean patient age was 58.2 years and males comprised 71.6% of all patients. The mode of presentation was classified as symptomatic in 65.4% of cases, incidental in 24.7% and not available in 9.9%. Regional lymph node metastasis was histologically detected in 44.2% of patients who underwent lymph node dissection, while 32.1% of the population had distant metastasis at presentation. Although postoperative adjuvant therapy against metastasis or recurrence was performed in 25 patients, no obvious responses were identified except in 1 with lung metastases, who showed a partial response to combined gemcitabine and carboplatin therapy. At a median followup of 15 months 1, 3, 5 and 10-year disease specific survival was 69.0%, 45.3%, 34.3% and 13.7%, respectively. CONCLUSIONS: We report what is to our knowledge the largest known series of collecting duct carcinoma. Since advanced or recurrent collecting duct carcinoma is resistant to standard treatment modalities, new treatment strategies are needed for advanced collecting duct carcinoma.

Centaurin-alpha1 is a phosphatidylinositol 3-kinase-dependent activator of ERK1/2 mitogen-activated protein kinases.

Centaurin-alpha1 is known to be a phosphatidylinositol 3,4,5-triphosphate (PIP3)-binding protein that has two pleckstrin homology domains and a putative ADP ribosylation factor GTPase-activating protein domain. However, the physiological function of centaurin-alpha1 is still not understood. Here we have shown that transient expression of centaurin-alpha1 in COS-7 cells results in specific activation of ERK, and the activation is inhibited by co-expression of a dominant negative form of Ras. We have also found that a mutant form of centaurin-alpha1 that is unable to bind PIP3 fails to induce ERK activation and that a phosphatidylinositol 3-kinase inhibitor LY294002 inhibits centaurin-alpha1-dependent ERK activation. Furthermore, transient knockdown of centaurin-alpha1 by small interfering RNAs results in reduced ERK activation after epidermal growth factor stimulation in T-REx 293 cells. These results suggest that centaurin-alpha1 contributes to ERK activation in growth factor signaling, linking the PI3K pathway to the ERK mitogen-activated protein kinase pathway through its ability to interact with PIP3.