Hiromi Kumon

Diabetic Cystopathy Correlates With a Long-Term Decrease in Nerve Growth Factor Levels in The Bladder and Lumbosacral Dorsal Root Ganglia

PURPOSE It has been proposed that a deficiency in the axonal transport of nerve growth factor (NGF) may have an important role in inducing diabetic neuropathy, which contributes to diabetic cystopathy. Therefore, in streptozotocin (Sigma Chemical Co., St. Louis, Missouri) induced diabetic rats we investigated the relationship of bladder function with NGF levels in the bladder and lumbosacral dorsal root ganglia, which contain afferent neurons innervating the bladder. MATERIALS AND METHODS At 6 and 12 weeks after the induction of diabetes with streptozotocin (65 mg./kg. intraperitoneally) the effects of diabetes on Adelta afferent fiber dependent, conscious voiding were evaluated by metabolic cage measurements and awake cystometry. The effects of diabetes on C-fiber mediated bladder nociceptive responses were also investigated by cystometry with intravesical instillation of 0.25% acetic acid in the rats under urethane anesthesia. NGF levels in the bladder and L6 to S1 dorsal root ganglia were measured by enzyme-linked immunosorbent assay 3, 6, 9 and 12 weeks after streptozotocin injection. RESULTS In diabetic rats NGF levels in the bladder and L6 to S1 dorsal root ganglia were significantly decreased 12 weeks after streptozotocin injection (p <0.01). In cystometry and metabolic cage studies bladder capacity and post-void residual volume were significantly increased 12 weeks after streptozotocin injection (p <0.01). Bladder nociceptive responses revealed by a reduction in inter-contraction intervals after acetic acid infusion were significantly decreased in a time dependent manner 12 weeks after streptozotocin injection.CONCLUSIONS Rats with streptozotocin induced diabetes mellitus showed a significant time dependent decrease in NGF levels in the bladder and L6 to S1 dorsal root ganglia that was associated with voiding dysfunction attributable to defects in Adelta and C-fiber bladder afferents. Therefore, reduced production of NGF in the bladder and/or impaired transport of NGF to L6 to S1 dorsal root ganglia, which contain bladder afferent neurons, may be an important mechanism inducing diabetic cystopathy.

Distribution of dynamins in testis and their possible relation to spermatogenesis

Dynamin 2 and dynamin 3 are highly expressed in testis. However, their functions in the tissue remain unclear. Considering that dynamin 1, neuron-specific isoform of dynamin, plays a pivotal role in endocytosis, functions of dynamin 2 and dynamin 3 in testis must be essential. Cellular expression and subcellular localization of dynamin 2 and dynamin 3 in testis were investigated. Dynamin 2 and dynamin 3 were highly expressed in germ cells and Sertoli cells, constituents of seminiferous tubules. By immunofluorescence it was revealed that dynamin 2 colocalizes with clathrin both at the plasmamembrane and at Golgi in a cell line of Sertoli cells. Immunoreactivity for dynamin 3, on the other hand, appeared as finer puncta, which did not colocalize with clathrin, suggesting that these two dynamins have distinct functions in Sertoli cells. In the klotho deficient mouse testis, which demonstrates disorder in spermatogenesis, expression of dynamin 2 and dynamin 3 was drastically reduced indicating possible association of these proteins with spermatogenesis.

Phosphatidylinositol 4,5-bisphosphate stimulates vesicle formation from liposomes by brain cytosol

As a step toward the elucidation of mechanisms in vesicle budding, a cell-free assay that measures cytosol-induced vesicle generation from liposomes was established. This assay then was used to explore the role of phosphoinositides in vesicle formation. Liposomes incubated with brain cytosol in the presence of ATP and GTP massively generated small vesicles, as assessed both quantitatively and qualitatively by a dynamic light-scattering assay. Both ATP and GTP were required. Vesicle formation was inhibited greatly by the immunodepletion of dynamin 1 from the cytosol, indicating a major contribution of this GTPase in this reaction and suggesting that it mimics endocytic vesicle fission. Increasing the concentration of l-α-phosphatidylinositol 4,5-bisphosphate [PtdIns(4,5)P2] but not of l-α-phosphatidylinositol 4-monophosphate or l-α-phosphatidylinositol in the lipid membranes enhanced vesicle formation. Lipid analysis revealed rapid degradation of PtdIns(4,5)P2 to l-α-phosphatidylinositol during the incubation with the reaction reaching a maximum within 5 sec, whereas vesicle formation proceeded with a longer time course. PtdIns(4,5)P2 degradation was independent of vesicle formation and occurred also in the presence of guanosine 5′-O-(thiotriphosphate), where few vesicle formations occurred. These results suggest that PtdIns(4,5)P2 plays a critical role in the early step of vesicle formation, possibly in the recruitment of coats and fission factors to membranes.

Gene therapy for prostate cancer: toxicological profile of four HSV- tk transducing adenoviral vectors regulated by different promoters

Adenoviral vector delivery of the Herpes simplex virus thymidine kinase (HSV-tk) gene in combination with the prodrug ganciclovir (GCV) has been tested in phase I clinical trials for prostate cancer and found to exhibit a satisfactory toxicity profile. We have developed additional adenoviral vectors with differing promoters to optimize the expression profile and in the present study evaluate the potential systemic toxicity of these vectors. Four recombinant adenoviral vectors that express the HSV-tk gene were generated using three different promoters: CMV (leftward orientation); RSV (both rightward and leftward orientation); and the mouse caveolin-1 (cav-1) promoter (leftward orientation). Efficacy was determined in vitro by cytotoxicity assays in a mouse prostate cancer cell line, RM-9, and in vivo by treating orthotopic tumors. Potential toxicity was evaluated from liver histology and apoptotic cell counts and enzyme levels in the serum following intravenous adenoviral vector injection. Although there were differences in HSV-tk expression at the protein level among the four vectors there were no significant differences in in-vitro cytotoxicity studies with GCV or in vivo in tumor growth suppression of an orthotopic mouse prostate cancer model in GCV treated mice. Intravenous delivery of high doses of all adenoviral vectors lead to abnormalities in liver function as measured by specific serum markers and histological evaluation of liver tissue and increased levels of apoptosis in the liver. These abnormalities were most prevalent with the vector containing the CMV promoter and the rightward oriented RSV promoter. They were least prevalent in the vector regulated by the cav-1 promoter. Upregulation of specific chemokines, MIP-2 and MIP-1β was correlated with apoptotic counts. Our results demonstrate that comprehensive toxicological analysis of adenoviral vectors provides internally consistent information that can differentiate vectors with comparable efficacy based on toxicity. In these studies vectors with the cav-1 promoter-driven and leftward RSV-driven HSV-tk gene demonstrated minimal toxicities with cytotoxic effectiveness comparable to more toxic vectors. Our studies further suggest that promoter selection can influence the toxic effects of an adenoviral gene therapy vector.

Prostate specific antigen complexed to α-1-antichymotrypsin in patients with intermediate prostate specific antigen levels

The authors attempted to evaluate prospectively the usefulness of serum prostate specific antigen (PSA) complexed to α‐1‐antichymotrypsin (PSA‐ACT) in the early detection of prostate carcinoma and its ability to discriminate between prostate carcinoma and benign prostatic hyperplasia (BPH), especially among patients with intermediate PSA levels.

Evaluation of 15 Motility Media and a Direct Microscopic Method for Detection of Motility in Enterococci

ABSTRACT Isolation of motile, vanC enterococci has yet to be a major infection control concern; however, rapid detection still is important. We evaluated 15 motility media from three manufacturers and a 2-h direct microscopic method for accurate detection of 89 enterococcal strains, including 72 vanC enterococcal strains. Resistance genes were confirmed by a multiplex PCR method with the vanC gene detected in all motile enterococci. Motility in the 72 vanC enterococci was detected at 30°C within 72 h in BD Biosciences motility nitrate medium and in Remel motility B medium, motility B medium supplemented with methyl-α-d-glucopyranoside (investigational), motility S medium, motility test medium, and motility test medium with tetrazolium indicator. Motility was also observed for all vanC enterococci with the 2-h direct (30°C incubation) microscopic detection method. All Enterococcus faecalis and Enterococcus faecium isolates were observed to be nonmotile in all media and by the direct microscopic method. Since differences between the various motility media tested were observed, the medium used for detection of enterococcal motility must be selected carefully.

Identification of a Tn1546-Like (Type 2) Element in Vancomycin-Resistant Enterococcus faecium Isolated from Hospitalized Patients in Japan

Vancomycin-resistant enterococci (VRE) have been isolated from about 50 Japanese patients since the first incidence in 1996 (1, 6; N. Fujita, M. Yoshimura, T. Komori, K. Tanimoto, and Y. Ike, Letter, Antimicrob. Agents Chemother. 42:2150, 1998). A connection between Japanese outbreaks and imported chickens has been suggested (5; Y. Ozawa, T. Nomura, T. Murata, K. Tanimoto, S. Fujimoto, and Y. Ike, Abstr. 1st Intl. Am. Soc. Microbiol. Conf. Enterococci, abstr. 103, p. 62, 2000). The link between animal colonization with VRE and human infection has been established in Europe, where the glycopeptide avoparcin previously was used as a feed additive (13). Clones of VRE identical to VRE of animal origin have been found not only in hospitalized patients but also in nonhospitalized humans, indicating the dynamic transfer of vancomycin resistance affecting public health (8, 12). Vancomycin resistance disseminates not only clonally but also by transfer of resistant elements horizontally to different clones of enterococci in animals and humans (4). Therefore, molecular epidemiological studies of these resistant elements are very important for the understanding of possible dissemination and elucidation of the spread of VRE. In May 1998, a patient colonized with VanA-type Enterococcus faecium (VREF) was hospitalized at the Okayama University Hospital (Okayama, Japan). Fourteen isolates with indistinguishable pulsed-field gel electrophoresis (PFGE) patterns were collected from this patient, who was undergoing a bone marrow transplantation. The genetic background for vancomycin resistance was confirmed to be vanA (Tn1546) by using PCR methods (3, 9). A more thorough investigation of the Tn1546-like elements in these isolates was performed by using a combination of PCR, nucleotide sequencing, and hybridization (7). The results were compared to previously published variations in Tn1546 and indicated that all isolates possessed the unique molecular variations characteristic of a Tn1546-like (type 2) element, as defined by Jensen et al. (7; L. B. Jensen, Letter, Antimicrob. Agents Chemother. 42:2463–2464, 1998). The variations included the presence of an IS1216V-IS3-like element in the left end of Tn1546 and a point mutation (G to T) at position 8234 in the essential vanX gene of Tn1546. We have further characterized Tn1546-like elements from selected VanA-type VRE of human and poultry origin from Japan. VRE have not been isolated from domestic porcine origin and imported pork samples tested in Japan (Y. Ike [Gunma University School of Medicine, Japan], personal communication). No Tn1546-like (type 2) elements were detected in vanA-containing VRE (7 E. faecalis and 1 E. faecium isolates) isolated from chicken fecal samples from three poultry farms (H. Yoshimura, M. Ishimaru, Y. S. Endoh, M. Suginaka, and S. Yamatani, Letter, Antimicrob. Agents Chemother. 42:3333, 1998). The Tn1546-like (type 2) element was detected in only 3 isolates (all from one hospital) out of 17 vanA-containing VRE (16 E. faecium and 1 E. faecalis isolates) isolated from 17 patients in six hospitals. The Tn1546-like (type 1) element was detected in 12 isolates from human and poultry origin, and 10 isolates indicating new variations in the Tn1546-like elements were untypeable according to the typing method of Jensen et al. (Table ​(Table1).1). Four human isolates possessing Tn1546-like (type 2) elements had closely related PFGE patterns, indicating clonal relationship according to the criteria of Tenover et al. (11), but were unrelated to other VRE isolates of human and poultry origin from Japan. TABLE 1 VRE (vanA type) of human and animal origins from Japan, Europe, and the United States The Tn1546-like (type 2) element has previously been associated with VREF isolated from pig feces in Europe (2, 10, 14, 15) and has thus far been found in some hospitalized patients but not in feces from other food animals (Table ​(Table1).1). Genetic characterization of VRE isolates from patients in Japan revealed that Tn1546-like (type 2) elements were present in independent isolates from a small geographical area in Japan. Since Tn1546-like (type 2) elements have not been found in VRE of poultry origin either from Europe or from Japan, it is unlikely that the VRE containing Tn1546-like (type 2) elements originated from poultry. In Japan, no glycopeptides have been used as growth promoters for domestic pigs. Therefore, the Japanese VREF isolates containing Tn1546-like (type 2) elements could have come from porcine origin outside Japan. (This work was presented in part at the 1st International American Society for Microbiology Conference on Enterococci-Banff, Alberta, Canada, 27 February to 2 March 2000.) We thank H. Yoshimura, National Veterinary Assay Laboratory, Ministry of Agriculture, Forestry and Fisheries, Japan, for providing VRE isolated from chickens. We also thank many colleagues in Japan for providing clinical isolates for this study. We acknowledge J. W. Chow for reviewing the manuscript.