[Leaflet extension technique in a congenital aortic valve regurgitation].

A 17-year-old boy who was diagnosed as congenital aortic valve regurgitation underwent the leaflet extension technique in 3 aortic leaflets using glutaraldehyde-preserved autologous pericardium. Intraoperative transesophageal echocardiography and postoperative cardiac catheter revealed grade I regurgitation and the patient has been doing well for more than 4 months after the surgery. The leaflet extension technique is considered to be an acceptable surgical treatment for aortic valve disease especially in young patients to whom valve replacement or Ross procedure might not be suitable. A careful long-term follow-up will be necessary to evaluate the durability of the leaflet extension technique with autologous pericardium.

Oral combination of cyclophosphamide, uracil plus tegafur and estramustine for hormone-refractory prostate cancer.

OBJECTIVE: To evaluate the clinical usefulness of an oral combination of cyclophosphamide, uracil plus tegafur (UFT) and estramustine in the treatment of patients with hormone-refractory prostate cancer (HRPC). METHODS: Twenty-one patients were treated with oral administration of cyclophosphamide (100 mg/day), UFT (400 mg/day) and estramustine phosphate (560 mg/day). The median age of the patients was 70 years. Twelve patients had symptomatic bone metastasis, 6 had asymptomatic bone metastasis, 5 had lymph node metastasis, while 2 had only biochemical progression evaluated by prostate-specific antigen (PSA). RESULTS: Twelve (57%) out of 21 patients showed a PSA decline of 50% or greater. The median response duration was 7 months (range 2-15 months). Among the 20 patients assessable for bone pain, 2 (10%) improved, 12 (60%) remained stable and 6 (30%) progressed. Among the 10 patients assessable for bone metastasis, 1 (10%) improved, 5 (50%) were stable and 4 (40%) progressed on bone scan. Among 3 patients assessable for measurable disease (lymph node metastasis), 2 (67%) showed partial response and 1 (33%) progression. Most toxicities were mild. CONCLUSIONS: The combination of cyclophosphamide, UFT and estramustine is an active and well-tolerated regimen for HRPC. To evaluate the survival benefit, further randomized studies are required.

[A case of ammonium urate urinary stones with anorexia nervosa].

A 27-year-old woman had been suffering from bulimia and habitual vomiting for about 7 years and was incidentally found to have right renal stones by computed tomography. She was referred to our hospital for the treatment of these caluculi. On admission, she presented with hypokalemia, hypochloremia and metabolic alkalosis and was diagnosed with anorexia nervosa. Following successful removal by percutaneous nephrolithotripsy and extracorporeal shockwave lithotripsy the stones were found to consist of pure ammonium urate. Since the urine of an anorexia nervosa patient tends to be rich in uric acid and ammonium, anorexia nervosa seems to be associated with ammonium urate urinary stones.

[A case of urinary stone in ureterocele extracted transurethrally after ESWL].

A 66-year-old man was referred to our hospital with chief complaints of difficulty in urination and terminal micturition pain. Ureteroceles were identified bilaterally, and a ureteral stone (19 x 12 mm) existed in the right ureterocele. After crushing the stone by extra corporeal shock wave lithotripsy (ESWL), we removed the stone transurethrally with a small incision in the right ureterocele. The vesicoureteral reflux (VUR) was not detected postoperatively. Now, we recommend the combination of ESWL and a small transurethral incision of the urelerocele for the treatment of ureteral stones in a ureterocele in order to prevent postoperative VUR.

Bcl-2/E1B 19 kDa-interacting protein 3-like protein (Bnip3L) interacts with bcl-2/Bcl-xL and induces apoptosis by altering mitochondrial membrane permeability.

We have previously reported on cloning of the human gene encoding Bcl-2/adenovirus E1B 19 kDa-interacting protein 3-like protein (Bnip3L) and its growth inhibitory effect on cancer cells. Here we show that Bnip3L contains a motif similar to the BH3 domain which is conserved in Bcl-2 family proteins as well as containing a membrane-anchoring domain, and that Bnip3L interacts with Bcl-2 and Bcl-xL. Immunofluorescence microscopy revealed that Bnip3L was localized in the mitochondria, when in the presence of the membrane-anchoring domain. Transient expression of Bnip3L induced apoptosis of Rat-1 and HeLa cells and mutational analysis revealed that the BH3 domain and the membrane-anchoring domain were required for Bnip3L to induce cell death. Addition of recombinant Bnip3L to isolated mitochondria induced membrane potential loss and cytochrome c release both of which have been suggested to be prerequisite for apoptotic cell death. These results suggest that Bnip3L is one of the BH3-containing pro-apoptotic proteins and that it targets the mitochondria when inducing apoptosis.

[Molecular staging of testicular cancer using polymerase chain reaction of the testicular cancer-specific genes].

Most testicular germ cell tumors have serological tumor markers such as alpha-fetoprotein (AFP) and human chorionic gonadotropin beta-subunit (beta-HCG). On the other hand, molecular staging of these tumors has not been well established compared to other urogenital malignancies like prostate cancer. Recently, melanoma antigen (MAGE) which is one of the tumor-associated antigens recognized by cytotoxic T lymphocytes (CTL) has been found to be present in a variety of malignant tumor types and normal testis. In addition, Wilms' tumor-associated gene WT1 has been found to be expressed in some testicular cancers. Thus, we examined the expression of these genes in testicular cancer tissues and peripheral blood of cancer-bearing patients using reverse transcription-polymerase chain reaction (RT-PCR). The expression of the MAGE1-3 genes was examined in 34 testicular germ cell tumors (24 seminomas and 10 nonseminomas). Of the seminomas and nonseminomas, 87.5% and 40% were positive for MAGE1, 91.7% and 60% for MAGE2, and 83.3% and 30% for MAGE3, respectively. The expression of the MAGE genes was not correlated with the tumor stage. The expression of the WT1 gene was quantified in 26 testicular germ cell tumors. WT1 was highly expressed in 5 of the 7 high stage cases, but in only 4 of the 19 low stage cases (p < 0.01). The mRNA of these genes could not be detected from the peripheral blood of patients with high stage tumors. These results suggest that MAGE genes may be useful tumor markers for molecular staging of testicular cancer, especially seminoma, and that the WT1 gene may be a tumor marker for high stage testicular germ cell tumors. However, these genes cannot yet be used for molecular staging of testicular germ cell tumors.

Analysis of caspases that are activated during apoptosis in leukemia U937 cells in response to geranylgeraniol.

Affinity labeling showed that active caspases with molecular masses of 20 kDa, 19 kDa, and 17 kDa were formed upon treatment of human leukemia U937 cells with GGO. These caspases are quite similar to those activated by treatments with other apoptosis-inducers, such as VP16 and camptothecin, suggesting that similar caspases, such as caspases 3 and 6, are activated during apoptosis in U937 cells that is induced by a variety of apoptosis-inducing stimuli. An inhibitor of caspases, Z-Asp-CH2DCB, inhibited DNA fragmentation in response to GGO in vivo by blocking the cleavage of 20-kDa to 17-kDa peptides. This cleavage is catalyzed by caspase 3 itself or by a caspase-3-like protease. In contrast, other inhibitors of caspases such as Z-DEVD-FMK and Z-VAD-FMK, inhibited the processing of the caspase 3 precursor p32 to 20-kDa and 17-kDa peptides, a result which suggests that these inhibitors inhibited other upstream caspases. Treatment of U937 cells with GGO resulted in the release of cytochrome c from mitochondria prior to DNA fragmentation and the release of cytochrome c was inhibited by Zn2+ ions and by a chelator of Ca2+ ions but not by inhibitors of caspases such as Z-Asp-CH2DCB or Z-VAD-FMK. These results suggest that intracellular free Ca2+ ions, and some caspases that are inhibited by Zn2+ ions, but not by Z-Asp-CH2DCB or Z-VAD-FMK are necessary for the release of cytochrome c that is caused by the treatment with GGO.

Distribution of PNP 14 (beta-synuclein) in neuroendocrine tissues: localization in Sertoli cells.

Phosphoneuroprotein (PNP 14) is abundant in the central nervous system and is localized at nerve endings but not in synaptic vesicles. In this study, we examined the presence of PNP 14 in various endocrine tissues of the rat. PNP 14 was not detected in the endocrine cells of the intestine, testes, or adrenal gland, but it was present in axon terminals in both the medulla of the adrenal gland and the anterior pituitary gland. When testes were stained with PNP 14-specific antibodies by an indirect immunofluorescence method, PNP 14 was found in Sertoli cells of the testes, associated with fibrillar structures. PNP 14 was also detected in cultured Sertoli cells with a fibrillar pattern in the cytoplasm and around the nuclei. The fibrillar structure did not resemble actin stress fibers, microtubules, or intermediate filaments. The amount of PNP 14 in the testis changed with development. It increased markedly during the first 4 weeks after birth and then decreased. During the first 4 weeks after birth, spermatogonia undergo two rounds of meiosis. It is possible, therefore, that PNP 14 might be a factor related to meiosis.

A selective immunoaffinity chromatography for determination of plasma 1 alpha, 25-dihydroxyvitamin D3: application of specific antibodies raised against a 1 alpha, 25-dihydroxyvitamin D3-bovine serum albumin conjugate linked through the 11 alpha-position.

A selective and simple immunoaffinity chromatography of 1 alpha, 25-dihydroxyvitamin D3 has been developed and found to be a useful pretreatment tool for determining the metabolite in human plasma. A reasonably designed, haptenic derivative, 11 alpha-hemiglutaryloxy-1 alpha, 25-dihydroxyvitamin D3, was linked to bovine serum albumin, and rabbits were immunized repeatedly with the conjugate. The resulting polyclonal antibodies were specific to 1 alpha, 25-dihydroxyvitamin D3, recognizing both the A-ring and the side-chain structures. The antibodies were then immobilized on agarose gel to produce an immunosorbent which was stable and repeatedly usable. A plasma extract prepared with a Chem Elut column was applied to an affinity column containing the immunosorbent. After adequate washing, the adsorbed 1 alpha, 25-dihydroxyvitamin D3 was eluted selectively with a satisfactory recovery rate. This immunoaffinity chromatography enabled a simple radioreceptor assay for human plasma 1 alpha, 25-dihydroxyvitamin D3 which does not require any preparative high-performance liquid chromatography. The mean (+/-SD) values for 30 normal subjects and 8 patients with chronic renal failure were 36.0 (10.2) and 13.1 (2.1) pg/ml, respectively. The present method also gave reliable assay values for the plasma specimens from 1 alpha-hydroxyvitamin D3-administered volunteers, which have conventionally been difficult to measure unless complicated pretreatment is used.

Efficient and beta-stereoselective synthesis of 4(5)-methyl-5(4)-(5-amino-5-deoxy-beta-D-ribofuranosyl)imidazole and related compounds exhibiting antiulcer activity.

The reaction of 2,3,5-tri-O-benzyl-D-ribose with the lithium salt of an imidazole derivative gave an adduct 17RS. Treatment of 17RS with 1.5N HCl in refluxing tetrahydrofuran gave the beta-4(5)-ribofuranosylimidazole 19 (35%) and the ribosylimidazole 18 (51%). The latter was converted into beta-19 in 86% yield by the Mitsunobu cyclization. This synthetic method produced only the desired beta-anomer. Protection of the imidazole nitrogen of 19 with an ethoxycarbonyl group followed by debenzylation gave 21, which was successively derived to the 5'-amino derivative 1 via the 5'-substituted phthalimide 23, followed by hydrazine degradation in excellent yield. Compound 1 was then converted into the 5'-cyanoguanidine 2 in 79% yield. The 5'-amino derivatives 3-9 lacking a methyl group were efficiently synthesized. Among them, the cyanoguanidine 5 and phenylthiourea 8 exhibited antiulcer activities with half the efficacy of cimetidine. The molecular conformation of 5 was determined by X-ray structure analysis.

Production and characterization of monoclonal antibodies against a novel 1alpha,25-dihydroxyvitamin D3-bovine serum albumin conjugate linked through the 11alpha-position.

To obtain an antibody which is useful for developing a selective immunoaffinity chromatography (IAC) of 1alpha,25-dihydroxyvitamin D3 [1,25(OH)2D3], which is an effective pretreatment tool for determining this metabolite in biological fluids, a variety of monoclonal antibodies have been produced against a novel haptenic derivative, 11alpha-hemiglutaryloxy-1alpha,25-dihydroxyvitamin D3, linked to bovine serum albumin. BALB/c, A/J and C57BL/6 mice, as well as Wistar and SD rats, were immunized with the hapten-carrier conjugate and the titer of the anti-1,25(OH)2D3 antibody in serum was examined. Spleen cells from A/J mice or SD rats, which showed a high titer, were fused with P3/NS1/1-Ag4-1 myeloma cells. After serial screening by enzyme-linked immunosorbent assay and radioimmunoassay, sixteen kinds of hybridoma clones secreting different anti-1,25(OH)2D3 antibodies were established. Scatchard analysis revealed that 12 antibodies out of 16 have enough affinity to 1,25(OH)2D3 (Ka>10(9) M[-1]). By a cross-reaction study using various related compounds, two antibodies, M114 and M129, both derived from A/J mice, were found to have suitable specificity. Although they showed relatively high cross-reactivities with the 1,25(OH)2D3 derivatives having 24-hydroxylated (both 20%) or lactonized (30%, 8.9%) side chain, all the tested 1alpha-deoxy-type metabolites, some of which interfere with the 1,25(OH)2D3 assays, were successfully discriminated [25(OH)D3: 0.67%, 0.40%; 24,25(OH)2D3: 0.27%, 0.24%; 25(OH)D3 3-sulfate: 0.11%, 0.14%]. These monoclonal antibodies will be applicable for developing not only IAC but also various immunoassay procedures.

Fas/APO-1-mediated apoptosis of human renal cell carcinoma.

We have investigated the Fas-mediated apoptosis of cultured human renal cell carcinoma OUR-10. OUR-10 cells express Fas antigen on their cell surface and showed apoptotic cell death by stimulation with anti-Fas agonistic antibody. Cell proliferation of OUR-10 was reduced to 62% by the addition of anti-Fas antibody. The cytotoxic effect of anti-Fas antibody on OUR-10 was augmented by the pretreatment of these cells with interferon-gamma, not with interferon-alpha or with interleukin-2. Flow cytometry demonstrated that preculture of Our-10 cells with interferon-gamma increased the cell surface Fas antigen. Immunohistochemical analysis of resected tissues from the patients with renal cancer revealed stronger expression of Fas antigen on the surface of cancer cells than that of normal cells. These data suggest that Fas may be one of the possible targets for a novel approach to human kidney cancer, and the efficiency of this Fas-mediated treatment for kidney cancer could be augmented by the treatment with interferon-gamma.

Involvement of CPP32/Yama(-like) proteases in Fas-mediated apoptosis.

Fas (Apo-1/CD95) belongs to the tumor necrosis factor/nerve growth factor receptor family and transmits apoptotic signals by binding to its ligand. Interleukin-1beta-converting enzyme (ICE), which shows substantial homology to the product of the cell death gene, ced-3, of Caenorhabditis elegans, is reported to be involved in Fas-mediated apoptosis. Using two human carcinoma-derived cell lines with undetectable levels of ICE, we found that an agonistic antihuman Fas antibody induces the activation of CPP32/Yama(-like) proteases that are ICE(-like) protease family members, and that a tetrapeptide inhibitor of CPP32/Yama protease, DEVD-CHO, inhibits the Fas-mediated activation of the proteases, Fas-mediated apoptosis, and CPP32/Yama(-like) proteolytic activities in vitro. Fas-mediated apoptosis is inhibited by the CPP32/Yama inhibitor DEVD-CHO, but not by the ICE inhibitor YVAD-CHO, suggesting a dominant role for the CPP32/Yama(-like) proteases and not ICE itself in Fas-mediated apoptosis of the human carcinoma cell lines.

Tandem immunoaffinity chromatography for plasma 1 alpha,25-dihydroxyvitamin D3 utilizing two antibodies having different specificities: a novel and powerful pretreatment tool for 1 alpha,25-dihydroxyvitamin D3 radioreceptor assays.

We report here a novel and powerful pretreatment method for radioreceptor assays (RRAs) for human plasma 1 alpha,25-dihydroxyvitamin D3 (1,25(OH)2D3) based on "Tandem" immunoaffinity chromatography (Tandem IAC). Two antibodies having different specificities were each immobilized on agarose gel with cyanogen bromide to produce immunosorbents which were stable and repeatedly usable. An ethyl ether extract of plasma was applied to the first affinity column, from which 1,25(OH)2D3 could be preferentially eluted and separated from 1 alpha-deoxy type metabolites. The effluent was then submitted to the second column, and the 1,25(OH)2D3 retained was eluted after non- or weakly-absorbed interfering substances were washed out. This procedure allowed efficient purification without careful handling or strict time-management in the entire operation and enabled avoiding preparative high-performance liquid chromatography (HPLC) from RRA even with a conventional chick intestinal vitamin D receptor. Mean (+/- SD) plasma 1,25(OH)2D3 values of 56 normal subjects and 10 patients with chronic renal failure, obtained with this Tandem IAC/RRA system, were 36.4 (8.7) and 11.2 (4.0) pg/ml, respectively. The Tandem IAC will also be useful for developing immunoassays or gas chromatography-mass spectrometry of 1,25(OH)2D3.

[Clinical studies of chemotherapy for patients with a solitary kidney after nephroureterectomy for advanced upper urothelial transitional cell carcinoma].

Between June, 1987 and December, 1993, ten patients with solitary kidney after total nephroureterectomy for advanced upper urothelial transitional cell carcinoma were treated with chemotherapy (M-VAC or modified M-VAC). This series comprised 6 males and 4 females between 27 and 81 years of age (mean age: 58.5 years). The site of primary lesions was the renal pelvis in one case, ureter in 5 and renal pelvis and ureter in 4. Histologically, these extripated tumors were all identified as transitional cell carcinoma, the stage being pT3 and pT4 in 9 and grade being G3 in 8 of the 10 patients. Among the 13 cases including the 3 cases of recurrence after first line chemotherapy, 7 had lesions suitable for the evaluation. Two of the 7 cases achieved complete response and four achieved partial response, resulting in an 86% response rate. Of the 10 patients, 4 died of metastasis of carcinoma and the others are still alive. The average period after operation among 10 patients was 25 months. Side effects related to this chemotherapy were as follows: general fatigue, nausea or vomiting and alopecia 100%, leucocytepenia (< or = 1,000/mm3) 23%, anemia (RBC < or = 250 x 10(4)/mm3) 62%, thrombocytopenia (< or = 5 x 10(4)/mm3) 46%. However, nephrotoxicity in spite of solitary kidney was not noticed in any patients. From our experience, we suggest that M-VAC or modified M-VAC chemotherapy are safe against patients with a solitary kidney after nephroureterectomy for advanced transitional cell carcinoma of the upper urinary tract.

[A case report of condyloma acuminatum in female urethra].

A case of condyloma acuminatum in female urethra is reported. A 53-year-old woman was referred to our clinic because of urethral bleeding. A tumor was noted around the external urethral meatus and the size of the tumor was 20 mm in diameter. Excision was performed on the tumor including the external urethral meatus. Pathological examination revealed condyloma acuminatum. Condyloma acuminatum in female urethra is very rare. We reviewed and discussed 6 cases of condyloma acuminatum in female urethra, including our case, in the Japanese literature.

[Cysts in the cavum tunica vaginalis testis: a case report].

A case of cysts in the cavum tunica vaginalis testis was reported. A 58-year-old man visited our clinic with the chief complaint of urethral bleeding. Urethro-cystogram revealed stricture of bulbous urethra. We palpated several pea-sized painless masses in the left scrotum. On ultrasonogram of the left scrotal contents several cystic lesions were detected at the cranial side of the left testis. At operation, cysts were found in the cavum tunica vaginalis testis, and were resected along with the tunica vaginalis. Fluid of the cysts was serous without sperm. Histopathologically mesothelial cyst was suspected. The incidence of cysts in the cavum tunica vaginalis testis is rare and only 17 cases have been reported in Japan. We reviewed 9 cases of cysts arising from lamina parietalis of tunica vaginalis.

[Leiomyoma of the kidney: a case report].

A case of leiomyoma of the kidney is reported. A 57-year-old man with the chief complaint of right back pain was consulted to our hospital. Abdominal computed tomography (CT) demonstrated a right renal tumor. Right renal angiography revealed a hypovascular lesion. Under the diagnosis of right renal tumor, right radical nephrectomy was performed. Gross evaluation revealed a well encapsulated solid mass. The tumor was 48 x 43 x 42 mm in diameter and a hemorrhagic region was present. Microscopically, the tumor was composed of interlacing bundles of spindle cells. Diagnosis was leiomyoma of the kidney. We reviewed 42 cases of renal leiomyoma reported in Japanese literature.

[A case of bilateral epididymal leiomyomas].

A case of bilateral epididymal leiomyomas is reported. A 62-year-old man was referred to our hospital under the suspicion of left testicular tumor. A small hen-egg sized painless mass was palpated in the left scrotum and a little-finger-tip-sized mass in the right scrotum. At the operation, bilateral epididymal tumors were revealed, left high orchiectomy and right epididymectomy were performed. Gross evaluation revealed elastic hard tumors at the tail of bilateral epididymis. The left tumor was 40mm in diameter and the right was 10mm. On the cut surface, the tumors were yellowish and solid. Histopathologically they were diagnosed as epididymal leiomyoma. We reviewed 75 cases of epididymal leiomyoma reported in the Japanese literature. Bilateral cases accounted for 21% of the 75 cases.

Ureteropelvic junction obstruction with renal pelvic calcification: a case report.

Calcification in the wall of the renal pelvis is rare. We report on a 65-year-old man with hydronephrosis secondary to ureteropelvic junction obstruction with renal pelvic calcification. Calcium deposit was found in the wall of the severely dilated renal pelvis. Pathological examination revealed a damaged and hyalinized fibrous renal pelvic wall and serum calcium level was normal. Thus, we speculated that this calcification was dystrophic. Chronic extensive dilatation with intermittent hemorrhage of the renal pelvic wall may have caused this dystrophic renal pelvic calcification.