Jun Yasuda

Significance of the Grb2 and son of sevenless (Sos) proteins in human bladder cancer cell lines.

The epidermal growth factor (EGF) receptor has been suggested to have an important role in tumor initiation and progression of human bladder cancers. Grb2 protein, which is the downstream effector of the EGF receptor, acts as an adaptor protein between the EGF receptor and the Ras guanine‐nucleotide exchange factor, son of sevenless (Sos) protein. Sos protein regulates the action of Ras protein by promoting the exchange of GDP for GTP . However, the significance of Grb2 and Sos proteins, which is related to EGF‐triggered Ras activation, has not been elucidated in human bladder cancer. The aim of the present study is to clarify the significance of these proteins in human bladder cancer cell lines. In the present study, we used four human bladder cancer cell lines (T24, KU‐7, UMUC‐2, UMUC‐6) and two kinds of cultured normal urothelial cells (HMKU‐1, HMKU‐2) isolated from patients with no malignancy. We examined the expression of EGF receptor, Grb2, and Sos proteins in these cells by Western blot analysis. Furthermore, the bladder cancer cell lines were subjected to sequence analysis to identify a point mutation in the c‐H‐ras gene at codon 12. There was no marked difference in the expression of the EGF receptor between human bladder cancer cell lines and cultured normal urothelial cells. On the other hand, expression of Grb2 and Sos proteins was substantially increased in all human bladder cancer cell lines examined in comparison with cultured normal urothelial cells, whether codon 12 of H‐ras was mutated or not. These results suggest that the amplification of both Grb2 and SOS proteins plays an important role in the carcinogenesis of human bladder cancer.

Suppressive effects of dominant negative ras mutant N116Y on transformed phenotypes of human bladder cancer cells

To investigate the suppressive effect of dominant negative H-ras mutant N116Y on transformed phenotypes, we established two N116Y ras mutant stable transfectant clones (C5, C13) of human bladder cancer cell line, UMUC-2. These N116Y ras mutant transfectants, especially the C5 cells, showed a dramatic change of cellular morphology and significantly reduced growth in soft agar compared to their control. Furthermore, phosphorylation of the Jun NH2-terminal kinase (JNK) was significantly decreased in these transfectants compared to the control. These results suggest that the N116Y-induced suppression of transformed phenotypes in UMUC-2 cells is associated with inhibition of JNK phosphorylation.

Ultrasonographic and serologic studies of experimental cysticercosis in rats infected with Taenia taeniaeformis

Rats experimentally infected with Taenia taeniaeformis were followed‐up until 14 weeks post inoculation with eggs (PIE) by hepatic ultrasonographic (US) image and serum antibody response analyses. Parasitic cysts could be imaged as small (2 mm in diameter) anechoic areas with or without a parenthesis‐like echogenic small line from two weeks PIE. Immunoblot analysis using antigens from oncospheres (TtO), 30‐day‐old (TtM‐30) and 300‐day‐old metacestodes (TtM‐300) revealed that: (1) these three different developmental stages showed their own unique patterns suggesting the presence of stage‐specific antigens; (2) faint IgM antibody responses to some components of TtO and TtM‐30 or TtM‐300 could be detected from one and two weeks PIE, respectively, and (3) IgG responses to some major components of both TtO and TtM‐300, and TtM‐30 were easily detected from four and five weeks PIE onwards, respectively. Both TtO and TtM (especially TtM‐300) appeared to be highly useful for detection of antibody responses in experimentally infected rats. Due to the easiness in preparation of antigens, fully developed metacestodes may be the best candidate antigens for serodiagnosis. These results strongly suggest that both US image and antibody analyses using antigens from fully developed metacestodes are useful for detection of the early stage of cysticercosis in laboratory animal model.

Detection of Antibodies to Leptospiral Genus-Specific Antigen in Human and Animal Sera by Indirect Hemagglutination Test with a Partially Purified Genus-Specific Protein Antigen

Antibodies against leptospiral genus-specific antigen were detected in the sera from clinically diagnosed human leptospirosis and suspected animal leptospirosis by indirect hemagglutination (IHA) test with a partially purified genus-specific protein antigen (GP-Ag). The reaction was positive in the infected humans and animals irrespective of the leptospiral serovars. No significant correlation was found between IHA titer against GP-Ag and microscopic agglutination (MA) titer. IHA titer did not always develop in parallel with MA titers. Sera obtained from healthy individuals were negative in both IHA and MA tests.

Assay of Urinary Enzyme in a Dog

In a dog, gentamicin (10mg/kg) was given every 8 hours for 9 days to produce experimental renal failure. Then urinalysis showed an increase in urine volume, a decrease in urinary specific gravirty, proteinuria, glucosuria, celluria and enzymuria. Elevation in the activities of the urinary enzymes preceded a rise in levels of creatinine (Cr) and BUN in the serum. Activities of serum Cr rose by day 10 and of BUN by day 14. Urinary LDH, T-GTP, NAG and ALP began to rise steadily by day 7 and increased rapidly after that. Urinary LAP rose slightly by day 12 and rapidly after that. Urinary LDH5 increased with a rapid rise of total urinary LDH activity. Renal biopsy specimens on days 5 and 9 were examined histologically, showing granular degeneration of the proximal tubules. Necropsy was made on day 16. The renal lesions were characterized by hydropic degeneration and necrotic changes in the proximal tubules and occasional interstitial infiltration of macrophages. Accordingly, it was suggested that assays of urinary enzymes might be more sensitive indicators of renal damage than conventional renal function tests in the small animal practice.