The role of DNase and EDTA on DNA degradation in formaldehyde fixed tissues.

Degradation and extraction of high molecular weight DNA from formaldehyde fixed tissues suitable for gene analysis are presented. We previously reported that DNase might play an important role in the degradation of DNA extracted from formaldehyde fixed tissues (Tokuda et al. 1990). In the present study, DNase activity of the supernatant from rat tissues fixed in buffered formaldehyde at room temperature was negligible within 3 hr. Analysis of DNA extracted from reconstituted chromatin revealed that the degradation increased in the absence of DNase depending on the duration of the formaldehyde fixation. Furthermore, high molecular weight DNA could be extracted from tissues devoid of DNase activity fixed in buffered formaldehyde containing EDTA. These results demonstrated that DNA degradation was due mainly to a mechanism other than DNAse which was inhibited by EDTA. For clinical application, v-H-ras gene was successfully detected by Southern blotting from rat spleen tissues fixed in buffered formaldehyde especially at 4 C. Fixation at low temperature is useful for gene analysis.

In vitro and in vivo evaluation of ellagic acid on melanogenesis inhibition.

The efficacy of ellagic acid (EA), one of the naturally occurring polyphenols, in inhibiting melanogenesis was examined in vitro and in vivo. When mushroom-derived tyrosinase, a metaloprotein containing copper, was incubated with EA, enzymatic activity tended to decrease with decreasing copper concentration. Enzyme activity partially recovered when copper was added to the inactivated enzyme. Tyrosinase activity in the B16 melanoma cells was observed to recover in a dose-dependent manner when copper ions were added to the medium containing EA. Based on these results, EA is thought to react specifically with the copper located at the active centre of the tyrosinase molecule. Furthermore, when EA was applied for 6 weeks to brownish guinea-pigs, which have melanocytes in their skin, at the same time as irradiating for 2 weeks with ultra-violet light, skin pigmentation was clearly suppressed and the skin to which EA had been applied showed features similar to that of non-irradiated skin. These areas were irradiated again when the application of EA had been completed, and skin pigmentation occurred at the former site of EA application. In similar studies with hydroquinone, re-pigmentation did not occur on the sites at which hydroquinone (1%) had been applied. Based on the results reported here, EA is thought to suppress melanogenesis by reacting with activated melanocytes and without injuring cells.

[Uretero-external iliac artery fistula: a case report].

We report a case of uretero-external iliac artery fistula. A 60-year-old female was referred to our hospital complaining of intermittent gross macrohematuria. She had undergone radical hysterectomy, radiation therapy and chemotherapy for advanced cervical cancer 2 years ago. The patient had a 7 Fr ureteral double-J stent for left hydronephrosis. Retrograde urography showed a filling defect (8 mm in diameter) of the left ureter. A contrast-enhanced computed tomographic scan showed left hydronephrosis and hydroureter but no evidence of fistula formation or extravasation. A pelvic arteriography revealed a pseudoaneurysm of the left external iliac artery at the crosspoint between the left ureter and the iliac artery. Surgical repair of the left uretero-external arterial fistula was successfully performed as well as left nephroureterectomy. The possibility of fistula formation between ureter and artery should be kept in mind in patients with long-term indwelling ureteral stents and history of radiation therapy.

[Postoperative care of neobladder using a detubularized intestinal segment].

Neobladder using a detubularized intestinal segment was constructed in 74 cystectomized patients between October 1986 and July 1994. There were 65 males (87.8%) and 9 females (12.2%) with an average age of 63 years (range 36 to 77 years). The mean follow-up period was 35 months (range 7 to 85). Problems of postoperative care assessed were continence, renal function, metabolic consequences, neoplasms and other complications. Moreover, the impact of these problems on the quality of life was evaluated by a self-administered questionnaire. Continence in the daytime was achieved in 70/74 (94.6%) patients. Nocturnal incontinence was noted in 15/74 (20.3%) patients. Serum BUN and creatinine levels were maintained within normal limits. Metabolic acidosis (base excess < -5.0 occurred in 7/74 (9.5%) patients, 4 patients of whom needed alkalizing agents. Colon adenomas in neobladder were found and resected in 3 patients. The other postoperative complications were stone formation in neobladder in 4 (5.4%), urethral stricture in 7 (9.5%) and ureteral stenosis in 3 (4.1%) patients respectively, which were satisfactorily corrected by endourological procedures. As to quality of life assessment, the majority was satisfied, whereas 11% of the patients reported emotional distress and limitations in the usual physical activities by nocturnal incontinence. Our finding suggest that nocturnal incontinence is the most important problem and we need longer follow-up to evaluate the problems of metabolic consequences and neoplasms of the urinary tract.

[Statistics on operations at Hara Genitourinary Hospital (1971-1991)].

The statistical analysis of the inpatients and operations in our department from April, 1971 to March, 1991 revealed a total of 9,170 operations. One of the biggest changes in treatment is a marked increase in closed surgery. This includes endourological surgeries; transurethral resections of prostate and bladder tumor (TUR-P, TUR-Bt), percutaneous nephrolithotripsy (PNL), transurethral uretero-lithotripsy (TUL) and extracorporeal shock-wave lithotripsy (ESWL). From 1976 to 1980 there were 721 open surgical procedures (71.0% of cases) and 295 closed procedures (21.0% of cases). This ratio has now been reversed completely. From 1986-1991, there were 342 open procedures (8.2% of cases) and 3808 closed procedures (91.8% of cases). The main reason for this change was the introduction of the new techniques PNL, TUL and ESWL. Other important observations include: The incidence of emergency operations was higher in this private institution than that seen in non-private hospitals. From 1986, there was an increase in the use of PNL and TUL for the management of urinary calculi, and from 1988, ESWL has overtaken PNL and TUL in the treatment of this condition. Recently the limitations of ESWL have been appreciated and the value of a combined approach to therapy, for selected cases, is being recognized. A total of 238 ileal or ileocecal conduits were created with good results. A low complication rate has been observed with preservation of renal function.

[Laparoscopic approach to idiopathic retroperitoneal fibrosis].

A 49-year-old male patient underwent an exploratory laparoscopy for a lumbosacral prevertebral mass which caused right hydronephrosis by extrinsic ureteral stricture. The diagnosis of idiopathic retroperitoneal fibrosis (IRPF) was made by the laparoscopic biopsy. We then proceeded to perform successfully laparoscopic ureterolysis and intraperitonealization of the ureter on the patient. Laparoscopic approach provides a less invasive alternative in diagnosis and treatment of the patient with IRPF.

[An urodynamic study on neobladder function].

OBJECTIVE: To analyze the urodynamic characteristics of neobladders, we conducted a pressure-flow study in patients with orthotopic urinary reservoirs. PATIENTS AND METHODS: From 1986 to 1996, 90 patients underwent bladder replacement following cystectomy, using a right colonic, ileocolic, ileal, or sigmoid colonic segment. The subjects were 38 patients (31 men and 7 women) with stable urination and no evidence of cancer recurrence, urethral stricture, urinary tract infection or vesicoureteral reflux. Their mean age was 60.5 years, with a range of 38 to 77 years. Information on neobladder function, such as desire to void, force of micturition, urinary incontinence and other complaints, was obtained by questionnaire. A pressure-flow study was performed in all patients 3 months to 103 months postoperatively to evaluate total reservoir pressure, abdominal pressure and subtracted reservoir pressure during filling and voiding phases. RESULTS: Ten of 38 patients (26.3%) were dissatisfied with their neobladder function, due to weakness of urinary sensation, loss of urinary force and enuresis. In 6 of the 7 patients with enuresis, the urinary reservoir had been created by Heineke-Mikulicz's procedure of detubularization; 4 of these patients had a high degree (over 40 cmH2O) of phasic contraction during the filling phase. In only 2 of the 38 patients, a pressure-flow study showed an almost same pattern as that obtained with a normal urinary bladder. Twelve patients had increased electromyogram of the external urethral sphincter during the voiding phase, while half of the 38 patients showed a flat electromyogram during both the filling and voiding phases. Thus, 31 of 38 patients revealed a sphincter dyssynergia pattern. Mean total reservoir pressure at maximum cystometric capacity was 65.5 +/- 42.1, 48.4 +/- 19.0, 66.0 +/- 61.0 and 107.0 +/- 43.3 cmH2O in ileal, ileocecal, right colonic and sigmoid neobladders, respectively. The value for sigmoid neobladder was statistically different from that for ileocecal neobladder (p < 0.05). Mean total reservoir pressure at maximal flow was 73.1 +/- 42.4, 56.4 +/- 22.6, 88.9 +/- 69.4 and 94.0 +/- 31.8 cmH2O in ileal, ileocecal, right colonic and sigmoid neobladders, respectively. There were no statistically significant differences among these values. The ratio of subtracted reservoir pressure to total reservoir pressure was lower at maximal flow than at onset. Subtracted reservoir pressure may contribute to total reservoir pressure to a greater extent in sigmoid neobladders than in other types of neobladder. CONCLUSIONS: 1. Enuresis may have various causes such as external sphincter dysfunction and involuntary contraction of the reservoir. 2. Urine is evacuated not only by abdominal pressure but also by subtracted reservoir pressure in neobladders. 3. Sphincter dyssynergia due to absence of the detrusor muscle may be one cause of dysuria.

[VAB-6 chemotherapy of advanced testicular cancers--its efficacy and limitation].

Fifteen patients with advanced testicular germ cell tumor were treated by VAB-6 chemotherapy after radical orchiectomy. CR was achieved in two (13.3%) and PR in nine (60.0%). The one, three and five year survival rates were 92.2, 64.2 and 51.4%, respectively. Nine patients (60.0%) remain with no evidence of disease (NED) after combined therapy (chemotherapy and surgery) with a median follow up of 4.3 months. We performed resection of the residual tumors and examined histologically. Out of 11 cases, four (36.3%) were with necrosis/fibrosis, two (18.2%) teratoma and five (45.5%) cancer. All of the six cases with non-cancerous tissues remain in NED. However, only two of the five cases with cancerous tissues remain in NED. We studied the NED rate based on four prognostic factors, (1) bulky abdominal diseases, (2) advanced lung diseases, (3) other metasteses, and (4) choriocarcinoma. In our analysis, advanced lung diseases and choriocarcinoma were poor prognostic factors. We should treat these poor prognostic cases initially with VP-16, ifosfamide, high doses cisplatin etc, which were used as salvaged drugs.

Preoperative computerized tomography detection of extensive invasion of the inferior vena cava by renal cell carcinoma: possible indication for resection with partial cardiopulmonary bypass and patch grafting.

The relationship of the diameter of the inferior vena cava as measured by computerized tomography (CT) and tumor invasion of the inferior vena caval wall was determined in patients with renal cell carcinoma. In addition, the indications and usefulness of surgery using partial cardiopulmonary bypass and a polytetrafluoroethylene (Gore-Tex) patch graft are discussed. In all 7 patients with an inferior vena caval diameter of 40 mm. or larger on CT tumor had extensively invaded the vessel wall macroscopically and microscopically. Therefore, resection of the inferior vena caval wall and repair with a patch graft were necessary. Partial cardiopulmonary bypass was used in 6 of these 7 patients. On the other hand, of 11 patients with an inferior vena cava less than 40 mm. in diameter only 2 with extensive tumor invasion of the vessel wall underwent a patch graft procedure without partial cardiopulmonary bypass. One patient who had massive hemorrhage before bypass was started died while in a coma. The survival of the remaining patients ranged from 6 to 131 months (median 19 months). Blood loss in patients who underwent surgery with partial cardiopulmonary bypass was much less than that in patients without bypass. In our series, there were no complications related to the graft itself and graft patency was excellent. Our results indicate that an inferior vena caval diameter of 40 mm. or more on CT probably indicates extensive tumor invasion. Although further experience and observation are necessary to evaluate whether partial cardiopulmonary bypass and/or a patch graft improves the prognosis of patients with extensive inferior vena caval invasion by renal cell carcinoma, this method was relatively safe and decreased blood loss.

A new approach to orthotopic bladder replacement using the transplanted bowel segment.

PURPOSE: When constructing an orthotopic neobladder, a short colonic mesentery may make the operation difficult. We have developed a new technique to autotransplant a free segment of intestine into the pelvis to augment the bladder. METHODS AND MATERIALS: In this animal feasibility study, we used eight female dogs to perform orthotopic bladder replacement using a transplanted bowel segment. A segment of bowel was transected and revascularized between the ileal artery of the reservoir and the internal iliac artery in the end-to-end fashion. RESULTS: Follow-up studies revealed that the bladder autoaugmentation using the transplanted bowel segment was functional and free of leakage. CONCLUSIONS: Our work in dogs demonstrates that the use of an autotransplanted segment of intestine appears to be feasible for augmenting the bladder in those cases where mechanical difficulty arises in anastomosing the urinary reservoir to the urethra. This procedure merits clinical evaluation in cases that present difficulty for completing orthotopic neobladder.

Long-term experience with orthotopic reconstruction of the lower urinary tract in women.

PURPOSE: We investigated the long-term outcome of orthotopic neobladders in women. MATERIALS AND METHODS: At our institutions 8 women have undergone lower urinary tract reconstruction with an orthotopic neobladder and have been followed for more than 4 years. After urethral sparing cystectomy the neobladder was constructed and connected to the native urethra. Late complications were analyzed, and voiding function was evaluated by a pressure flow micturition study and bead-chain cystourethrography. RESULTS: No late complications related to the surgery were noted. Of the 8 patients 7 were completely dry day and night. Four patients with ileal neobladder required clean intermittent catheterization, and hypercontinence might have been caused by downward migration of the neobladder and reduced neobladder pressure at voiding. No recurrence in the native urethra was noted, and all patients were satisfied postoperatively. CONCLUSIONS: These results of orthotopic neobladder in women are encouraging. Overall patient satisfaction was excellent. An international collaborative study is necessary to achieve the number of patients required to determine the ideal orthotopic neobladder for women.

Purification and properties of a novel surface-active agent- and alkaline-resistant protease from Bacillus sp. Y.

In the course of a search for an alkaline stable protease for industrial use, an alkaline protease (protease BYA) was isolated from an alkalophilic Bacillus sp. Y, and its properties were characterized. Its optimum pH was pH 10.0-12.5, when casein was used as a substrate. In addition to the stability of protease BYA at pH 6.5-13.0, it was also very stable towards various surface-active agents, such as sodium dodecyl sulfate and sodium linear alkylbenzene sulfonate. Protease BYA was most active at 70 degrees C. The isoelectric point (pI) of protease BYA was about 10.1. Protease BYA was characterized as a serine protease because of its sensitivity to phenylmethanesulfonyl fluoride and diisopropyl fluorophosphate. The protease seems to be related to proteases of the subtilisin family, such as subtilisin BPN', subtilisin Carlsberg, and No. 221 protease.