Phospholipase Cε plays a crucial role in neutrophilic inflammation accompanying acute lung injury through augmentation of CXC chemokine production from alveolar epithelial cells.

BACKGROUND: We have shown that phospholipase Cε (PLCε), an effector of Ras and Rap1 small GTPases, plays pivotal roles in inflammation and inflammation-associated carcinogenesis by augmenting proinflammatory cytokine production from epithelial cells of various organs. The purpose of this study is to analyze its role in neutrophilic alveolar inflammation accompanying acute lung injury (ALI), focusing on that in alveolar epithelial cells (AECs), which are known to make a major contribution to the pathogenesis of ALI. METHODS: We examine the effect of the PLCε genotypes on the development of ALI induced by intratracheal administration of lipopolysaccharide (LPS) to PLCε wild-type (PLCε+/+) and knockout (PLCεΔX/ΔX) mice. Pathogenesis of ALI is analyzed by histological examination of lung inflammation and measurements of the levels of various cytokines, in particular neutrophil-attracting chemokines such as Cxcl5, by quantitative reverse transcription-polymerase chain reaction and immunostaining. Primary cultures of AECs, established from PLCε+/+ and PLCεΔX/ΔX mice, are used to analyze the roles of PLCε, protein kinase D (PKD) and nuclear factor-κB (NF-κB) in augmentation of LPS-induced Cxcl5 expression. RESULTS: Compared to PLCε+/+ mice, PLCεΔX/ΔX mice exhibit marked alleviation of lung inflammation as shown by great reduction in lung wet/dry weight ratios, accumulation of inflammatory cells in the alveolar space and thickening of alveolar walls as well as the number of neutrophils and the protein concentration in bronchoalveolar lavage fluid. Also, LPS-induced expression of the CXC family of chemokines, in particular Cxcl5, is substantially diminished in the total lung and AECs of PLCεΔX/ΔX mice. Moreover, LPS-induced Cxcl5 expression in primary cultured AECs is markedly suppressed on the PLCεΔX/ΔX background (p < 0.05 versus PLCε+/+ AECs), which is accompanied by the reduction in phosphorylation of inhibitor κB (IκB), PKD and nuclear translocation of NF-κB p65. Also, it is suppressed by the treatment with inhibitors of PKD and IκB kinase, suggesting the involvement of the PLCε-PKD-IκB-NF-κB pathway. CONCLUSIONS: PLCε-mediated augmentation of the production of the CXC family of chemokines, in particular Cxcl5, in AECs plays a crucial role in neutrophilic alveolar inflammation accompanying ALI, suggesting that PLCε may be a potential molecular target for the treatment of acute respiratory distress syndrome.

Molecular Basis for Allosteric Inhibition of GTP-Bound H-Ras Protein by a Small-Molecule Compound Carrying a Naphthalene Ring.

The ras oncogene products (H-Ras, K-Ras, and N-Ras) have been regarded as some of the most promising targets for anticancer drug discovery because their activating mutations are frequently found in human cancers. Nonetheless, molecular targeted therapy for them is currently unavailable. Here, we report the discovery of a small-molecule compound carrying a naphthalene ring, named KBFM123, which binds to the GTP-bound form of H-Ras. The solution structure of its complex with the guanosine 5'-(ß,γ-imide) triphosphate-bound form of H-RasT35S (H-RasT35S·GppNHp) indicates that the naphthalene ring of KBFM123 interacts directly with a hydrophobic pocket located between switch I and switch II and allosterically inhibits the effector interaction by inducing conformational changes in switch I and its flanking region in strand ß2, which are directly involved in recognition of the effector molecules, including c-Raf-1. In particular, Asp38 of H-Ras, a crucial residue for the interaction with c-Raf-1 via the formation of a salt bridge with Arg89 of the Ras-binding domain (RBD) of c-Raf-1, shows a drastic conformational change: its side chain orients toward the opposite direction. Consistent with these results, KBFM123 exhibits an activity to inhibit, albeit weakly, the association of H-RasG12V·GppNHp with the c-Raf-1 RBD. The binding of the naphthalene ring to the hydrophobic pocket of H-RasT35S·GppNHp is further supported by nuclear magnetic resonance analyses showing that two other naphthalene-containing compounds with distinct structures also exhibit similar binding properties with KBFM123. These results indicate that the naphthalene ring could become a promising scaffold for the development of Ras inhibitors.

Rapgef2, a guanine nucleotide exchange factor for Rap1 small GTPases, plays a crucial role in adherence junction (AJ) formation in radial glial cells through ERK-mediated upregulation of the AJ-constituent protein expression.

Rapgef2 and Rapgef6 define a subfamily of guanine nucleotide exchange factors for Rap1, characterized by possession of the Ras/Rap-associating domains and implicated in the etiology of schizophrenia. We previously found that dorsal telencephalon-specific Rapgef2 conditional knockout mice exhibits severe defects in formation of apical surface adherence junctions (AJs) and localization of radial glial cells (RGCs). In this study, we analyze the underlying molecular mechanism by using primary cultures of RGCs established from the developing cerebral cortex. The results show that Rapgef2-deficient RGCs exhibit a decreased ability of neurosphere formation, morphological changes represented by regression of radial glial (RG) fibers and reduced expression of AJ-constituent proteins such as N-cadherin, zonula occludens-1, E-cadherin and ß-catenin. Moreover, siRNA-mediated knockdown of Rapgef2 or Rap1A inhibits the AJ protein expression and RG fiber formation while overexpression of Rapgef2, Rapgef6, Rap1AG12V or Rap1BG12V in Rapgef2-deficient RGCs restores them. Furthermore, Rapgef2-deficient RGCs exhibit a reduction in phosphorylation of extracellular signal-regulated kinase (ERK) leading to downregulation of the expression of c-jun, which is implicated in the AJ protein expression. These results indicate a crucial role of the Rapgef2-Rap1A-ERK-c-jun pathway in regulation of the AJ formation in RGCs.

In silico discovery of small-molecule Ras inhibitors that display antitumor activity by blocking the Ras-effector interaction.

Mutational activation of the Ras oncogene products (H-Ras, K-Ras, and N-Ras) is frequently observed in human cancers, making them promising anticancer drug targets. Nonetheless, no effective strategy has been available for the development of Ras inhibitors, partly owing to the absence of well-defined surface pockets suitable for drug binding. Only recently, such pockets have been found in the crystal structures of a unique conformation of Ras⋅GTP. Here we report the successful development of small-molecule Ras inhibitors by an in silico screen targeting a pocket found in the crystal structure of M-Ras⋅GTP carrying an H-Ras-type substitution P40D. The selected compound Kobe0065 and its analog Kobe2602 exhibit inhibitory activity toward H-Ras⋅GTP-c-Raf-1 binding both in vivo and in vitro. They effectively inhibit both anchorage-dependent and -independent growth and induce apoptosis of H-ras(G12V)-transformed NIH 3T3 cells, which is accompanied by down-regulation of downstream molecules such as MEK/ERK, Akt, and RalA as well as an upstream molecule, Son of sevenless. Moreover, they exhibit antitumor activity on a xenograft of human colon carcinoma SW480 cells carrying the K-ras(G12V) gene by oral administration. The NMR structure of a complex of the compound with H-Ras⋅GTP(T35S), exclusively adopting the unique conformation, confirms its insertion into one of the surface pockets and provides a molecular basis for binding inhibition toward multiple Ras⋅GTP-interacting molecules. This study proves the effectiveness of our strategy for structure-based drug design to target Ras⋅GTP, and the resulting Kobe0065-family compounds may serve as a scaffold for the development of Ras inhibitors with higher potency and specificity.

Fatal pulmonary arterial hypertension in an infant girl with incontinentia pigmenti.

We report the case of an infant girl with incontinentia pigmenti (IP) complicated by fatal pulmonary arterial hypertension (PAH). She was diagnosed with IP, based on the presence of specific skin lesions, neonatal seizures, hypereosinophilia and a maternal family history of IP. At the age of 2 months, she was diagnosed with PAH on systolic heart murmur due to tricuspid valve regurgitation. Despite several treatments for PAH but not including epoprostenol, severe PAH persisted and she died of pulmonary hypertensive crisis at the age of 5 months. On postmortem histopathology the pulmonary artery had severe intimal thickening, with occlusion or stenosis of the vascular lumen of the small pulmonary arteries as well as partial plexiform lesions, all of which were compatible with PAH. Modulation of nuclear factor-κB signaling may be involved in the development of PAH in IP.

A new treatment for retroperitoneal fibrosis: initial experiences of using Seprafilm® to wrap the ureter.

OBJECTIVE: To confirm the efficacy of using Seprafilm® (Genzyme Corp., Cambridge, MA, USA) for wrapping the ureter to treat the ureteric stenosis caused by retroperitoneal fibrosis (RPF). PATIENTS AND METHODS: Between August 2010 and September 2012, 11 ureters in eight patients with RPF (seven males and one female, mean age 65 years) were treated. The mean (range) length of the narrow segment of the ureter was 30 (10-90) mm. During surgery, after having been released from adhesive tissue, the stenotic segment of the ureter was wrapped with Seprafilm to isolate it from the surrounding tissue. A radiographic follow-up was performed every 6 months using computed tomography, i.v. pyelography and/or (99m) Tc-mercapto-acetylglycyl-glycyl-glycine ((99m) Tc-MAG3) renal scintigraphy. RESULTS: For the unilateral operations, the mean estimated blood loss was 39 mL, and the mean operating time was 154 min. All ureters were isolated from the fibrotic tissue and wrapped with Seprafilm successfully without major complications. During the mean follow-up period of 17 months, no ureteric restenoses were observed in the affected sides, but new stenosis occurred in the contralateral side of the ureter in one patient. CONCLUSIONS: Although the follow-up period is still limited, we believe that the use of Seprafilm has the potential to become an effective option in the treatment of ureteric stenosis caused by RPF, when the omentum cannot be used. To establish the relative advantages of using Seprafilm over performing a standard omental wrap, further experimentation will be required to compare the two techniques.

Hyperinsulinemic hypoglycemia of infancy in Sotos syndrome.

Sotos syndrome (OMIM #117550) is a congenital syndrome characterized by overgrowth with advanced bone age, macrocephaly, and learning difficulties. Endocrine complications of this syndrome have not yet been fully described in previous reports. We here investigated the clinical manifestations of Sotos syndrome in Japanese patients who presented with hyperinsulinemic hypoglycemia of infancy. We recruited patients diagnosed as having Sotos syndrome who presented with the complication of hyperinsulinemia during the neonatal period using a survey of the abstracts of Pediatric Meetings in domestic areas of Japan from 2007 to 2011. As a result, five patients (four females and one male) were recruited to evaluate the clinical presentation of Sotos syndrome by reference to the clinical record of each patient. A 5q35 deletion including the NSD1 gene was detected in all patients. Major anomalies in the central nervous, cardiovascular, and genito-urinary systems were frequently found. Hypoglycemia occurred between 0.5 and 3 hr after birth and high levels of insulin were initially found within 3 days of birth. The patients were treated with intravenous glucose infusion at a maximum rate of 4.6-11.0 mg/kg/min for 12-49 days. Three of the five patients required nasal tube feeding. One patient received medical treatment with diazoxide. This study shows that patients with Sotos syndrome may present with transient hyperinsulinemic hypoglycemia in the neonatal period.

Randomized controlled trial of pelvic floor muscle training with or without biofeedback for urinary incontinence.

INTRODUCTION AND HYPOTHESIS: To compare the effects of pelvic floor muscle training (PFMT), with or without biofeedback (BF), for stress urinary incontinence (SUI), focusing on condition-specific quality of life (QOL) outcomes. METHODS: Women with SUI were randomized to PFMT with BF (BF group, n = 23) or without BF (PFMT group, n = 23) for 12 weeks. As primary outcome measures, subjective symptoms and QOL were assessed by the King's Health Questionnaire (KHQ) and International Consultation on Incontinence Questionnaire-Short Form (ICIQ-SF). A voiding diary, 1-h pad test, and measurement of PFM strength were secondary outcome measures. Changes in the primary and secondary outcomes were assessed before and after 12 weeks' exercise training. RESULTS: Of the 9 domains of the KHQ, the scores of 5 significantly decreased in the PFMT group, and the scores of 7 significantly decreased in the BF group. All ICIQ-SF items and the total score significantly decreased in both groups after therapy. The number of incontinence episodes significantly decreased in the PFMT group, and tended to decrease in the BF group, but this was not significant (P = 0.054). The leakage volume in the 1-h pad test tended to decrease in both groups, but was not significant. Maximum vaginal squeeze pressure significantly increased in both groups. There were no significant inter-group differences in the changes in any of the parameters assessed. CONCLUSIONS: The results indicate that PFMT is effective for treating SUI. There is no apparent add-on effect of BF training in short-term follow-up.

[Assessing weight and other factors associated with the urinary incontinence of elderly women: a cross-sectional study].

OBJECTIVES: Urination diseases--particularly urinary incontinence (UI)--strongly affect individuals' quality of life (QOL). It is important to maintain overall QOL in societies with an increasing number of long-living elderly people, such as in Japan. Thus, this study aims to clarify the risk factors concerning UI in elderly women. METHODS: For this study, we obtained the approval of the Ethical Committee of Sapporo Medical University. In October 2010, we randomly selected 1,600 women, aged between 65 and 74 years, from the registry of Sapporo city residents and mailed out a self-administered questionnaire. Our questionnaire consisted of five subsections (24 items in total), including fundamental attributes, health condition, past history, present illness, and the International Consultation on Incontinence Questionnaire-Short Form (ICIQ-SF). The self-reported prevalence of UI was defined as frequent UI being at least once a week. A logistic regression analysis was used to assess the risk factors for UI. RESULTS: Among those who received the questionnaire, 802 women (response rate: 50.1%) returned a completed questionnaire with written informed consent. Their mean age (+/- standard deviation) was 69.8 +/- 2.6 years, and the prevalence of UI was found to be 29.7%. The mean scores (+/- standard deviation) of ICIQ-SF were 1.7 +/- 2.9 for all participating subjects, and 5.55 +/- 2.50 and 0.09 +/- 0.53 for the groups with or without UI, respectively. The results of the multiple logistic regression analysis were as follows: the odds ratio (95% confidence interval) was 1.94 (1.32, 2.85) for a past maximum weight heavier than or equal to 60 kg; 1.98 (1.18, 3.32) for a smoking index more than or equal to 300; 2.54 (1.47, 4.39) for a poor self-perceived health status; 1.62 (1.09, 2.40) for having a past history of bladder diseases; 1.72 (1.11, 2.69) for having a past history of hemorrhoidal disease; and 2.05 (1.36, 3.10) for a history of UI in one's mother. CONCLUSION: The self-reported prevalence rate of UI was 29.7% in women aged 65-74 years. In the future, we plan to conduct a follow-up survey to further clarify the risk factors of UI that have been implicated in this cross-sectional study.

[Pre-evening meal administration of tacrolimus improved refractory ocular symptoms in two young children with latent general myasthenia gravis].

We report two patients with latent general myasthenia gravis (MG) with refractory ocular symptoms who were successfully treated with pre-evening meal administration of tacrolimus. Patient 1 was a 4-year-old girl with persistent ocular symptoms despite high-dose steroid therapy and thymectomy. Oral tacrolimus was initiated at the age of 3 years, which was resulted in complete resolution of symptoms. After one year, hemilateral ptosis recurred. The plasma consentration of tacrolimus was very low, probably due to sudden weight gain. Increasing the dose and a change from post- to pre-evening meal administration of tacrolimus enabled maintenance of its concentration and complete control of ocular symptoms. Patient 2 was a 2-year-old boy whose symptoms were refractory to steroid therapy after his first relapse. Since post-meal administration of tacrolimus provided partial benefit, the closing schedule was changed to pre-evening meal administration, with good results. Neither patient had adverse effects of tacrolimus. It is difficult to maintain an effective tacrolimus concentration in children due to marked growth and rapid metabolic rates. Pre-evening meal administration of tacrolimus is an easy, safe and useful method of treatment in MG young children.

Solution structure of the state 1 conformer of GTP-bound H-Ras protein and distinct dynamic properties between the state 1 and state 2 conformers.

Ras small GTPases undergo dynamic equilibrium of two interconverting conformations, state 1 and state 2, in the GTP-bound forms, where state 2 is recognized by effectors, whereas physiological functions of state 1 have been unknown. Limited information, such as static crystal structures and (31)P NMR spectra, was available for the study of the conformational dynamics. Here we determine the solution structure and dynamics of state 1 by multidimensional heteronuclear NMR analysis of an H-RasT35S mutant in complex with guanosine 5'-(ß, γ-imido)triphosphate (GppNHp). The state 1 structure shows that the switch I loop fluctuates extensively compared with that in state 2 or H-Ras-GDP. Also, backbone (1)H,(15)N signals for state 2 are identified, and their dynamics are studied by utilizing a complex with c-Raf-1. Furthermore, the signals for almost all the residues of H-Ras·GppNHp are identified by measurement at low temperature, and the signals for multiple residues are found split into two peaks corresponding to the signals for state 1 and state 2. Intriguingly, these residues are located not only in the switch regions and their neighbors but also in the rigidly structured regions, suggesting that global structural rearrangements occur during the state interconversion. The backbone dynamics of each state show that the switch loops in state 1 are dynamically mobile on the picosecond to nanosecond time scale, and these mobilities are significantly reduced in state 2. These results suggest that multiconformations existing in state 1 are mostly deselected upon the transition toward state 2 induced by the effector binding.

Structural basis for conformational dynamics of GTP-bound Ras protein.

Ras family small GTPases assume two interconverting conformations, "inactive" state 1 and "active" state 2, in their GTP-bound forms. Here, to clarify the mechanism of state transition, we have carried out x-ray crystal structure analyses of a series of mutant H-Ras and M-Ras in complex with guanosine 5'-(beta,gamma-imido)triphosphate (GppNHp), representing various intermediate states of the transition. Crystallization of H-RasT35S-GppNHp enables us to solve the first complete tertiary structure of H-Ras state 1 possessing two surface pockets unseen in the state 2 or H-Ras-GDP structure. Moreover, determination of the two distinct crystal structures of H-RasT35S-GppNHp, showing prominent polysterism in the switch I and switch II regions, reveals a pivotal role of the guanine nucleotide-mediated interaction between the two switch regions and its rearrangement by a nucleotide positional change in the state 2 to state 1 transition. Furthermore, the (31)P NMR spectra and crystal structures of the GppNHp-bound forms of M-Ras mutants, carrying various H-Ras-type amino acid substitutions, also reveal the existence of a surface pocket in state 1 and support a similar mechanism based on the nucleotide-mediated interaction and its rearrangement in the state 1 to state 2 transition. Intriguingly, the conformational changes accompanying the state transition mimic those that occurred upon GDP/GTP exchange, indicating a common mechanistic basis inherent in the high flexibility of the switch regions. Collectively, these results clarify the structural features distinguishing the two states and provide new insights into the molecular basis for the state transition of Ras protein.

The M-Ras-RA-GEF-2-Rap1 pathway mediates tumor necrosis factor-alpha dependent regulation of integrin activation in splenocytes.

The Rap1 small GTPase has been implicated in regulation of integrin-mediated leukocyte adhesion downstream of various chemokines and cytokines in many aspects of inflammatory and immune responses. However, the mechanism for Rap1 regulation in the adhesion signaling remains unclear. RA-GEF-2 is a member of the multiple-member family of guanine nucleotide exchange factors (GEFs) for Rap1 and characterized by the possession of a Ras/Rap1-associating domain, interacting with M-Ras-GTP as an effector, in addition to the GEF catalytic domain. Here, we show that RA-GEF-2 is specifically responsible for the activation of Rap1 that mediates tumor necrosis factor-alpha (TNF-alpha)-triggered integrin activation. In BAF3 hematopoietic cells, activated M-Ras potently induced lymphocyte function-associated antigen 1 (LFA-1)-mediated cell aggregation. This activation was totally abrogated by knockdown of RA-GEF-2 or Rap1. TNF-alpha treatment activated LFA-1 in a manner dependent on M-Ras, RA-GEF-2, and Rap1 and induced activation of M-Ras and Rap1 in the plasma membrane, which was accompanied by recruitment of RA-GEF-2. Finally, we demonstrated that M-Ras and RA-GEF-2 were indeed involved in TNF-alpha-stimulated and Rap1-mediated LFA-1 activation in splenocytes by using mice deficient in RA-GEF-2. These findings proved a crucial role of the cross-talk between two Ras-family GTPases M-Ras and Rap1, mediated by RA-GEF-2, in adhesion signaling.

Impact of urinary incontinence on the psychological burden of family caregivers.

AIMS: The present study was conducted to assess the impact of urinary incontinence on the psychological burden of family caregivers providing care to disabled family members with urinary incontinence. MATERIALS AND METHODS: This survey was conducted using an Internet panel for family caregivers in Japan, consisting of 7,316 members. We requested the members to respond to an online questionnaire to assess the impact of urinary incontinence on the burden of caregivers. The questionnaire included basic characteristics, and the Japanese version of the Zarit Caregiver Burden Interview (ZBI) comprising 22 questions related to the impact of the patient's disabilities on the burden of their caregivers. Additionally, the participants were asked to include the level of care need, ranging from 0 to 5 according to the grade of disability of the cared individuals. RESULTS: Relevant data obtained from 757 caregivers were analyzed. Of these caregivers, 452 provided care to family members with urinary incontinence (group 1), and 305 provided care to those without urinary incontinence (group 2). The mean total ZBI score was significantly higher in group 1 than in group 2 (mean, 40.7 vs. 34.7, P < 0.001). The difference in the scores for each item was also significant in 20 of the 22 items. With regard to the level of care need, irrespective of the level, the ZBI scores in group 1 tended to be higher than those in group 2. CONCLUSIONS: The present survey demonstrated a negative impact of urinary incontinence on the psychological burden of family caregivers.

Laparoscopic versus open radical prostatectomy: urodynamic evaluation of vesicourethral function.

OBJECTIVES: To assess the impact of laparoscopic radical prostatectomy on vesicourethral function and compare it to that of open radical prostatectomy. METHODS: Sixty-three patients undergoing laparoscopic radical prostatectomy for localized prostate cancer were included in this retrospective analysis. Urodynamic parameters, including maximum urethral closing pressure (MUCP), functional profile length (FPL), bladder compliance, maximum cystometric capacity (MCC) and detrusor overactivity, were considered. Continence status and changes in urodynamic findings before and after surgery were evaluated. In addition, postoperative urodynamic findings were compared with those in 58 patients undergoing open radical prostatectomy. RESULTS: After laparoscopic radical prostatectomy, MUCP and FPL showed a significant postoperative decrease. Continence rates after surgery were 82% in the laparoscopic and 78% in the open group. Comparison of postoperative data between continent and incontinent patients in both surgical groups showed significantly lower MUCP, shorter FPL, lower bladder compliance and higher incidence of detrusor overactivity in incontinent patients. Although there was no significant difference in postoperative MUCP and FPL between the two groups, bladder compliance was significantly lower and incidence of detrusor overactivity was significantly higher in the open prostatectomy group. CONCLUSIONS: Laparoscopic radical prostatectomy has a negative impact on storage function by impairing function of the urethral sphincter and decreasing bladder compliance. There is no difference in postoperative urethral function between open and laparoscopic radical prostatectomy. Laparoscopic surgery might be associated with less impairment of bladder function than open surgery.

Five-antituberculosis Drug-resistance Genes Detection Using Array System.

Detection of resistance to drugs for Mycobacterium tuberculosis takes about two months from the sample collection using culture-based methods. To test a rapid method for detection of resistance for five antituberculosis drugs using DNA microarray and to examine its potential for clinical use, we employed a DNA microarray for detection of seven mutations genes related to resistance of five kinds of antituberculous drugs using Mycobacterium tuberculosis DNA isolated from sputum. The results of microarray analysis were compared with the results of a standard culture method of Lowenstein-jensen drug sensitivity testing system. DNA microarray analysis showed a high sensitivity (>90%) for all five drugs. Specificity of rifampicin and ethambutol were nearly 90%, however specificity of isoniazid (60%) and kanamycin (67%) were not enough. The amount of Mycobacterium tuberculosis DNA required for microarray analysis corresponded to at least 1-9 Acid-Fast Bacilli per 10 fields by carbolfuchsin staining. DNA microarray analysis appears to be useful for estimation of drug resistances, nevertheless its limitations. To minimize misunderstanding, it is necessary to confirm the number of bacilli in the sputum, and culture method is needed for comparison when use the PCR-based array system.

[Detrusor underactivity following laparoscopic radical prostatectomy].

Strain voiding has been reported to be a frequent symptom following radical prostatectomy. However, pathophysiology of vesicourethral function underlying voiding difficulty has not been well studied. In the present study, we investigated detrusor underactivity following radical prostatectomy. The records on urodynamic study (pressure-flow study, urethral pressure profile) were retrospectively investigated in 80 patients undergoing laparoscopic radical prostatectomy and all urodynamic studies pre- and post-operatively. We extracted the cases with detrusor underactivity according to the criteria of overt strain voiding pattern on post-operative pressure flow study; detrusor pressure at the maximum flow rate (Pdet Q(max)) of less than 10 cmH2O in conjunction with an increase of abdominal pressure. Of the 80 patients, 6 (7.5%) were found to have detrusor underactivity. In all patients, good detrusor contraction was confirmed on the pre-operative urodynamic study performed before surgery. On the voiding phase of pressure-flow study in these patients, mean Pdet Q(max) showed a significant decrease postoperatively from 58.5 cmH2O to 3.0 cmH2O (p < 0.01), although mean abdominal pressure at Q(max) significantly increased from 24.2 cmH2O to 105.8 cmH2O (p < 0.05). Mean Q(max) on free uroflowmetry showed a significant increase from 12.8 ml/sec to 22.1 ml/sec (p < 0.05). No patient had significant post-void residual urine. On the storage phase of the study, however, maximum cystometric capacity, maximum urethral closing pressure showed no significant change between pre- and post-operative studies. Five patients acquired continence and one had mild urinary incontinence using one pad a day. The present study showed that detrusor contaractility could be impaired during radical prostatectomy, but, no apparent operative procedure related to detrusor dysfunction could be identified in the present patients.

[Case report of malignant sertoli cell tumor].

Malignant sertoli cell tumor is a rare disease and only a few cases have been described previously. We report a terminal case of malignant sertoli cell tumor. A 38-year-old male visited a hospital with a complaint of swelling his left testis. He underwent high left orchiectomy. His pathologic diagnosis was suspected seminoma, and all tumor markers (LDH, HCG, AFP) were negative, and CT imaging confirmed clinical stage 1 (pT1N0M0S0). One year later, a CT scan showed a small retroperitoneum lymph node swelling. Four months later, these lesions increased to 55 x 45 x 70 mm in diameter. He received 3 courses of chemotherapy with BEP (bleomycine, etoposide, cisplatin), but, lymph node size did not change. After he underwent a CT guided lymph node biopsy, his pathologic diagnosis was viable embryonal carcinoma. He then came to our hospital. We selected CPT-11 and nedaplatin for his salvage chemotherapy, but lymph node lesions did not change. After he received 3 courses of chemotherapy, we performed retroperitoneal lymphadenectomy. His pathologic diagnosis was viable sertoli cell tumor, malignant type. After 30 days, he had multiple liver metastases ane died 27 months after orchiectomy. All tumor markers were negative in his all clinical courses.

Ras inhibitors display an anti-metastatic effect by downregulation of lysyl oxidase through inhibition of the Ras-PI3K-Akt-HIF-1α pathway.

Metastasis stands as the major obstacle for the survival from cancers. Nonetheless most existing anti-cancer drugs inhibit only cell proliferation, and discovery of agents having both anti-proliferative and anti-metastatic properties would be more beneficial. We previously reported the discovery of small-molecule Ras inhibitors, represented by Kobe0065, that displayed anti-proliferative activity on xenografts of human colorectal cancer (CRC) cell line SW480 carrying the K-rasG12Vgene. Here we show that treatment of cancer cells carrying the activated ras genes with Kobe0065 or a siRNA targeting Ras downregulates the expression of lysyl oxidase (LOX), which has been implicated in metastasis. LOX expression is enhanced by co-expression of RasG12V through activation of phosphatidylinositol 3-kinase (PI3K)/Akt and concomitant accumulation of hypoxia-inducible factor (HIF)-1α. Furthermore, Kobe0065 effectively inhibits not only migration and invasion of cancer cells carrying the activated ras genes but also lung metastasis of human CRC cell line SW620 carrying the K-rasG12V gene. Collectively, these results indicate that Kobe0065 prevents metastasis through inhibition of the Ras-PI3K-Akt-HIF-1α-LOX signaling and suggest that Ras inhibitors in general might exhibit both anti-proliferative and anti-metastatic properties toward cancer cells carrying the activated ras genes.

Structural basis for intramolecular interaction of post-translationally modified H-Ras•GTP prepared by protein ligation.

Ras undergoes post-translational modifications including farnesylation, proteolysis, and carboxymethylation at the C terminus, which are necessary for membrane recruitment and effector recognition. Full activation of c-Raf-1 requires cooperative interaction of the farnesylated C terminus and the activator region of Ras with its cysteine-rich domain (CRD). However, the molecular basis for this interaction remains unclear because of difficulties in preparing modified Ras in amounts sufficient for structural studies. Here, we use Sortase A-catalyzed protein ligation to prepare modified Ras in sufficient amounts for NMR and X-ray crystallographic analyses. The results show that the farnesylated C terminus establishes an intramolecular interaction with the catalytic domain and brings the farnesyl moiety to the proximity of the activator region, which may be responsible for their cooperative recognition of c-Raf-1-CRD.