Arg660Ser mutation in Thermus aquaticus DNA polymerase I suppresses T-->C transitions: implication of wobble base pair formation at the nucleotide incorporation step.

We examined the replication fidelity of an Arg660Ser (R660S) mutant of Thermus aquaticus DNA polymerase I (Taq pol I). In a forward mutation assay, R660S showed a marked reduction in T-->C transitions, one of the most frequent errors made by the wild-type enzyme. Steady-state kinetics showed that R660S discriminates against dGTP incorporation at a template T 13-fold better than the wild-type. R660S was also 3.2-fold less efficient than the wild-type at extending a T:dG mismatch. These results indicate that R660S has enhanced fidelity during incorporation and extension, which reduces its T-->C transition frequency. Interestingly, R660S also discriminated correct from incorrect nucleotides at the incorporation step of C:dATP, A:dATP, G:dATP and C:8-OH-dGTP mispairs 28-, 6.0-, 4.1- and 6.8-fold better, respectively, than the wild-type, although it may not always be as accurate as the wild-type at the extension step. A structural model suggests that Arg660 may participate in two interactions that influence fidelity; the guanidinium group of Arg660 might interact with the incoming guanine base at the major groove and it might compete for forming another interaction with the primer terminus. Substituting Arg with Ser may eliminate or alter these interactions and destabilize the closed complex with incorrect substrates. Our data also suggest that T:dGTP and C:dATP base pairs form 'wobble' structures at the incorporation step of Taq pol I.

Atypical multidrug resistance may be associated with catalytically active mutants of human DNA topoisomerase II alpha.

In human cells, atypical drug resistance was previously identified with reduced catalytic activity or nuclear localization efficiency of DNA topoisomerase II alpha (TOP2 alpha). We have shown two etoposide resistant hTOP2 alpha mutants, K798L and K798P confer resistance to etoposide. In this work, we showed these mutants are also resistant against doxorubicin and mAMSA in vivo in the yeast strain ISE2, rad52, top2-4 at the non-permissive temperature. We purified these mutants to characterize the drug resistant mechanism. Purified recombinant proteins were 8- to 12-fold more resistant to etoposide and doxorubicin than wild type TOP2 alpha, and 2-fold more resistant to amsacrine, as measured by accumulation of cleavable DNA. These data show that K798L and K798P may be intrinsically resistant against these drugs in vitro and that this character may confer atypical multidrug resistant phenotype in vivo in yeast.

Autohemicastration in a man without schizophrenia.

A 52-year-old unmarried bag craftsman was admitted to East Tokyo Metropolitan Hospital because of a large scrotal hematoma. He had accidentally hit his right testis against the edge of a desk early the previous morning. He had resected his right testis with scissors to release from severe pain 30 min after the accident. He had sutured the scrotal incision with two stitches of string by himself. At the emergency operation 36 h after the self-mutilation, we removed a hematoma weighing 283 g and ligated the cut end of the right spermatic cord after adequate debridement. He was diagnosed by a psychiatrist as having slightly low intelligence without psychotic disorder or drug abuse.

O-helix mutant T664P of Thermus aquaticus DNA polymerase I: altered catalytic properties for incorporation of incorrect nucleotides but not correct nucleotides.

Previous studies indicate that the O-helix of Thermus aquaticus (Taq) DNA polymerase I (pol I) plays an important role in the replication fidelity of the enzyme. This study examines the role of Thr-664, which lies in the middle of the O-helix of Taq pol I. A mutant of Taq Pol I with a proline substitution of Thr-664 (T664P) exhibits much lower replication fidelity than the wild type enzyme in a forward mutation assay. T664P produces base substitution, single-base deletion, and single-base insertion errors at 20-, 5, and 50-fold higher rates than wild type, respectively. In specific activity and steady-state kinetic experiments, T664P was catalytically robust for insertion of correct nucleotides. In contrast, it incorporated incorrect nucleotides 6.1- to 10-fold more efficiently than wild type at a template dC. Mismatched primer termini were extended by T664P 4.2- to 9.5-fold more efficiently than wild type. These data imply that the O-helix with a proline at position 664 functions like wild type Taq pol I for correct nucleotide incorporations, but bends and enlarges the catalytic pocket of the enzyme and increases the rate of nucleotide misincorporation.

Enhanced ribonucleotide incorporation by an O-helix mutant of Thermus aquaticus DNA polymerase I.

The O-helix of DNA polymerases has been implicated in substrate discrimination and replication fidelity. In this study, wild-type Thermus aquaticus DNA polymerase I (Taq pol I) and an O-helix mutant A661E was examined for their ability to discriminate between ribonucleotides and deoxyribonucleotides. Steady-state nucleotide extension kinetics were carried out using a template cytidine and each nucleotide dNTP and rGTP. Wild-type Taq pol I and A661E demonstrated similar Vmax and Km values for the correct nucleotide dGTP. However, A661E discriminated between incorrect and correct nucleotide less well than wild-type; discrimination was reduced by factors of 9.5-, 5.6- and 15-fold for dATP, dTTP and rGTP, respectively. These data suggest that A661E is efficient polymerases in the presence of the correct deoxynucleotide, dGTP, but it is impaired in ability to discriminate between correct and incorrect deoxyribonucleotides or between ribo- and deoxyribonucleotides. A structural model of Taq pol I is described in which the mutation A661E alters the interactions between the O-helix and the terminal two phosphate groups in the primer strand.

Endocrinologically active retroperitoneal ganglioneuroma with positive iodine-131-metaiodobenzylguanidine scintigraphy.

BACKGROUND: A case of retroperitoneal ganglioneuroma incidentally found by ultrasonography in a 56-year old woman is presented. METHODS/RESULTS: Computed tomography revealed a solid round tumor 6 cm in diameter on the upper pole of the right kidney. Iodine-131-metaiodobenzylguanidine weakly accumulated in the tumor 24 and 48 h after the injection. Her serum catecholamines were within normal limits apart from slightly elevated norepinephrine at one of the two examinations. During the operation her serum epinephrine and dopamine levels were elevated to about 50 and 800 times higher than pre-operative values, respectively. CONCLUSION: This case is a rare ganglioneuroma in an adult patient that is endocrinologically active.

Advanced testicular germ cell tumor in a hemophilic patient with human immunodeficiency virus infection.

A stage IIIB anaplastic seminoma which occurred in an HIV-infected hemophilia is reported. The patient with hemophilia A was 36 years old and had been seropositive for HIV antibody for 3 years. Inguinal orchiectomy and subsequent chemoradiotherapy for retroperitoneal lymphadenopathy were performed and a marker negative partial response was obtained. In spite of a low initial CD4+ lymphocyte count (90/microliter), the patient tolerated the treatment well without life-threatening opportunistic infection. Although factor VIII supplement was performed, continuous bleeding from the operative wound made postoperative care difficult.

Sex differences in function and distribution of beta-adrenoceptors in rabbit urinary bladder.

PURPOSE: To examine whether there are sex-related differences in urinary bladder function, we evaluated the function and distribution of beta-adrenoceptor in the male and female rabbit urinary bladder. MATERIALS AND METHODS: Ten male and ten female rabbits were studied. The function and distribution of beta-adrenoceptors were studied by measuring changes in contractile forces from isoproterenol, a beta-adrenoceptor agonist, and forskolin, an adenylate cyclase activator, and by measuring beta-adrenoceptor densities using radioligand receptor binding techniques. RESULTS: Isoproterenol caused significantly greater relaxation in female trigonal muscles than in male trigonal muscles. It caused similar relaxation responses in the male and female detrusors. The Bmax values for [3H]dihydroalprenolol (DHA) binding in trigonal muscles were significantly higher in the female than in the male, whereas Bmax values for [3H]DHA binding in the male and the female detrusor were similar. In contrast, forskolin caused similarly marked relaxations in the male and the female detrusor and trigonal muscles. CONCLUSION: These findings demonstrate that there are sex-related differences in the function and the distribution of beta-adrenoceptors in rabbit trigonal smooth muscles.

[Management of invasive carcinoma of bladder and abdominal aortic aneurysm (AAA); technique of radical total cystectomy and simultaneous AAA repair].

BACKGROUND: There is no consensus on the optimal surgical treatment for patients with concomitant invasive carcinoma of bladder and abdominal aortic aneurysm (AAA). We experienced two patients who were treated successfully with simultaneous radical cystectomy and AAA repair. The techniques required for the combined procedure and case reports are discussed. PROCEDURE: The goal of the one-stage operation was to minimize the risk of graft infection without compromising postoperative morbidity and mortality secondary to carcinoma of bladder. Initially pelvic lymph node dissection and radical cystectomy were performed. We preferred retrograde cyctoprostatectomy because most of the cystectomy procedure can be performed without opening the peritoneal cavity and the extent of the retroperitoneal dissection can be minimal. A single-stoma ureterocutaneostomy was preferable urinary diversion. Urinary diversions which utilize intestine such as ileal conduit or ileal urinary reservoir may cause contamination of a graft with bowel content and should be avoided. Before or after urinary diversion, aneurysmal resection and a bifurcated graft replacement were performed. The replaced graft was wrapped with the aneurysmal wall. The major omentum was mobilized and fixed in front of the graft, thereby serving as a protective barrier of the graft. A Dacron graft which was sealed with rifampicin-bonding gelatin was used to further reduce the risk of graft infection. RESULT: Two male patients were treated with the one stage radical cystectomy and AAA repair. Single-stoma ureterocutaneostomy and bilateral ureterocutaneostomy were selected as a urinary diversion. No major postoperative complications, except for paralytic ileus in one case, were observed. CONCLUSION: Our experience and reports of others indicate that radical cystectomy and simultaneous AAA repair can be safely performed with less morbidity than staged operations for the management of concomitant invasive carcinoma of bladder and AAA.

Evaluation of transrectal voiding ultrasonography in men with micturition difficulties without apparent organic obstruction of the lower urinary tract.

OBJECTIVE: To examine whether transrectal voiding ultrasonography (TRVUS) can evaluate voiding movement in men with dysfunctional voiding. METHODS: Ninety-nine consecutive men complaining of voiding difficulties without benign prostatic hyperplasia, prostatic cancer, severe bladder neck contracture and urethral stricture received uroflowmetry and TRVUS. Those who had abnormal findings on both uroflowmetry and TRVUS underwent subsequent cystometry combined with electromyography (EMG) to confirm the presence of dysfunctional voiding. RESULTS: Uroflowmetry indicated abnormal findings in 31 of the 99 patients, and TRVUS demonstrated abnormal movements of the posterior urethra during voiding in all of these 31 patients and 11 of the other 68 patients whose uroflowmetry did not indicate abnormality. TRVUS findings of the former 31 were divided into type E (the external urethral sphincter closed or intermittently opened while the bladder neck manifested an opening movement of > 7 mm during voiding in 20) and type I (both the bladder neck and external urethral sphincter manifested an intermittent movement of < 7 mm in 11). Subsequent cystometry combined with EMG in the 31 patients who had abnormal findings on both uroflowmetry and TRVUS revealed overactivity of the external urethral sphincter (OS) and underactivity of the detrusor (UD) in 85 and 35% of type-E group and 55 and 73% of type-I group, respectively. Type E included significantly more OS without UD than type I (65 vs. 18%; p = 0.0233). All of type-E (20/20) and 91% of type-I (10/11) patients had voiding difficulty which resulted from either OS or UD, while a very limited number of patients (4/31) manifested neurological symptoms such as paraplegia except for voiding difficulties. CONCLUSIONS: Both uroflowmetry and TRVUS are easy and useful methods to evaluate dysfunctional voiding in men, especially when neural disorders or organic obstruction of the lower urinary tract are not apparent.

[Relationship between pretreatment serum levels of prostate specific antigen and bone metastasis in prostate cancer].

We retrospectively reviewed pretreatment levels of serum prostate specific antigen (PSA) in 138 newly diagnosed, untreated patients with prostate adenocarcinoma to evaluate the usefulness of serum PSA levels to predict results of radionuclide bone scans. All of the 24 patients with serum PSA levels above 200 ng/ml showed positive bone scans whereas those with serum PSA levels below 10 ng/ml included only three cases (3/51, 5.1%) positive for bone metastasis. All of the three had poorly differentiated adenocarcinoma of the prostate. In conclusion, bone scans may not be necessary in the cases with serum PSA levels less than 10 ng/ml except for those with poorly differentiated adenocarcinoma.

[The experimental evaluation of the difference in closing dynamics of internal urethral ostium on the urinary continence and on the ejaculation induced by an administration of phenylephrine].

BACKGROUND: The internal urethral ostium is usually closed both on the ejaculation and on the urine collection, although the difference of the closing characteristics have not been known. METHODS: The difference in closing style of bladder neck (internal urethral ostium) between on the phenylephrine administration corresponding state to the ejaculation and on the filling of urinary bladder corresponding state to the urinary continence was evaluated by Stereo-UPP method using mongrel dogs. RESULTS: On phenylephrine administration, the length between verumontanum and bladder neck was lengthened, and a remarkable pressure increase was observed in the area. On the urinary continence filling physiological saline in the bladder, the length of the urethra on the UPP curve of the anterior wall of urethra was lengthened (10%) and the base of bladder was gradually become flat. The pressure in the bladder neck was not relatively increased. CONCLUSIONS: The internal urethral ostium was closed both on the phenylephrine administration and on the urine collection, although the closing characteristics of each was very different. It was demonstrated that on the phenylephrine administration the area between the verumontanum and the bladder neck was lengthened, together with the rise of pressure in the area, whereas on the urine collection bladder neck was closed in low pressure by flattening the base of bladder.

[Extracorporeal shock wave lithotripsy monotherapy using Lithostar--factors influencing actuarial residual stone rates].

For the evaluation of post treatment course after initial extracorporeal shock wave lithotripsy (ESWL) as a monotherapy using the Lithostar, the actuarial residual stone rate (ARSR) was calculated by the Kaplan-Meier formula in 713 calculi (255 renal and 458 ureteral calculi). ARSRs in all calculi at 3, 6, 12 and 48 months were 41.6, 30.5, 20.6 and 10.3%, respectively. Significantly higher ARSRs were observed in the following former group compared to the latter: large calculi to small or middle sized calculi, renal calculi to ureteral calculi (small or middle sized), and plural renal calculi (middle sized) to single renal calculi (middle sized). There was no significant difference either between large renal and large ureteral calculi, or overall single and plural calculi. These findings show that ARSRs calculated by the Kaplan-Meier formula clearly reveal factors influencing the ESWL effects.

[Study on prevention of flare-up phenomenon following initial LH-RH analogue administration: combination therapy with diethylstilbestrol].

To eliminate the initial testosterone (T) surge and prevent the risk of flare-up induced by the first administration of luteinizing hormone-releasing hormone (LH-RH) analogue, we examined the effectiveness of short-term combination therapy with diethylstilbestrol (DES). Sixteen previously untreated patients with prostate carcinoma (Stage C: 4 cases, Stage D2: 12 cases) were randomly assigned to 4 groups. Each group included 4 patients. Group 1 received DES (500 mg daily injection) for 7 days, staring before the first subcutaneous injection of luteinizing hormone releasing hormone (LH-RH) analogue (3.6 mg of Zoladex depot); Group 2 received 500 mg of DES injection for 7 days prior to the initiation of therapy with the LH-RH analogue, and moreover received a DES injection for 7 days; Group 3 received 500 mg of DES injection for 7 days simultaneously with the first injection of the LH-RH analogue; Group 4 was pretreated with 500 mg of DES injection for 7 days prior to the initiation of therapy with the LH-RH analogue, with additional DES injection for 3 days. In groups 1, 2 and 4 the level of T decreased during the 7 days of DES therapy, and at 3 days after the first injection of LH-RH analogue increased again. The mean T value in groups 1, 2 and 4 decreased by 33.2 +/- 27.6% (mean +/- SE), 20.5 +/- 20.8%, 13.2 +/- 9.8%, respectively, at the day of the first injection of LH-RH analogue, and increased by 75.2 +/- 21.6%, 70.7 +/- 63.4%, 56.7 +/- 46.4%, respectively, at 3 days after the first injection of LH-RH analogue.(ABSTRACT TRUNCATED AT 250 WORDS)

[Extracorporeal shock wave lithotripsy on patients with spinal cord injury with special reference to autonomic hyperreflexia].

We performed treatment of urolithiasis on 7 patients with spinal cord injury (6 males and one female, with a mean age of 41 years old) by extracorporeal shock wave lithotripsy (ESWL) using a Lithostar (Siemens). The level of injury was cervical in 3 patients and lower thoracic in 4. The treated stones were renal in 6 patients, including one staghorn caliculus, ureteral in 2, and bladder stones in 2 patients. ESWL was performed under general anesthesia in 2 patients, and in another patient, epidural anaesthesia was employed in the first several sessions, but thereafter treatment was safely continued with no anesthesia. The other 4 patients were treated without anesthesia. In most patients, the stones (9 stones in 6 cases) were easily disintegrated after treatment of from one to 14 sessions, except one case of staghorn caliculus, and the clearance of fragments was also satisfactory. In one patient, hypertension and bradycardia due to autonomic hyperreflexia were observed during ESWL, and the treatment was discontinued, but the stone was successfully disintegrated and the fragments were voided. In another patient, autonomic hyperreflexia was observed while the fragments passed through the ureter, although no signs of this reflexia were seen during the ESWL procedure.

Benign renal tumors detected among healthy adults by abdominal ultrasonography.

Renal ultrasonography was performed on 17,941 healthy adults without any signs suggestive of urinary tract malignancies. Diffusely hyperechoic renal masses indicative of angiomyolipoma were found in 41 (0.23%) of them and those with echogenic image suggestive of renal cell carcinoma in 45 (0.25%). Final diagnosis of the former group included 24 angiomyolipoma, 1 renal cell carcinoma, 9 calculus and 7 normal variants. The latter group included 19 renal cell carcinomas, 1 leiomyoma, 1 cavernous hemangioma and 24 normal variants or cysts. In total, 24 (0.13%) angiomyolipomas, 20 (0.11%) renal cell carcinomas, 1 leiomyoma, and 1 cavernous hemangioma were discovered. The results indicate that asymptomatic benign renal tumors mainly consisting of angiomyolipoma are not uncommon.

Epidermal growth factor binding by membranes of human renal cell carcinomas: establishment of an epidermal growth factor receptor assay for clinical use.

In order to quantitate epidermal growth factor receptors (EGFR) in human renal cell carcinoma (RCC) tissues, the binding of isotope-labeled epidermal growth factor (125I-EGF) to pooled membrane samples from human RCC was studied. Specific EGF binding to membranes at 4 degrees C reached a plateau after 2 h of incubation and remained constant up to 8 h; specific binding at 25 degrees C reached a plateau after 30 min of incubation, then decreased gradually. 125I-EGF binding to the membrane was displaced by both EGF and transforming growth factor-alpha, but not by insulin and basic fibroblast growth factor. Scatchard analysis of the specific EGF binding generated a straight line, indicating a single class of binding sites for EGF, with a dissociation constant (Kd) of 21.1 x 10(-10) M and a maximum number of binding sites of 57.2 fmol/mg membrane protein. When the protein concentration in the incubation medium was adjusted from 0.71 mg/ml to 2.84 mg/ml, Scatchard analysis revealed identical Kd values, and the maximum number of binding sites was proportional to the protein concentration. These results demonstrate the presence of EGFR on RCC membranes and indicate that these receptors can be studied quantitatively.

[Dynamic study of the hormonal levels and tumor markers after the first administration of long-acting LH-RH analogue in patients with prostate cancer].

The dynamics of hormonal levels and tumor markers after the first administration of long-acting luteinizing hormone-releasing hormone (LH-RH) analogue were evaluated in patients with prostate cancer. Eight patients with histopathologically proved prostate cancer who were previously untreated were studied. The surge in plasma testosterone was recognized in 7 patients after the first administration of a long-acting LH-RH analogue, and reached the highest level after the 3rd day in 6 patients and 14th day in 1 patient. The onset of flare-up reaction due to a transient increase in plasma testosterone was recognized in 3 patients, whose clinical symptoms and signs were increased bone pain in 2 patients and acute urinary retention in 1 patient. An abnormal level of serum prostatic acid phosphatase (PAP) and prostate specific antigen (PSA) was observed in 7 of the 8 patients before treatment. The serum PAP and PSA levels slightly increased after treatment in 4 and 3 patients, respectively. These findings suggest that the combination of estrogen or antiandrogen would allow a safer use of long-acting LH-RH analogue to prevent the risk of a flare-up reaction associated with the first administration of long-acting LH-RH analogue.

Epidermal growth factor receptor content in human renal cell carcinomas.

BACKGROUND: The expression of epidermal growth factor receptor (EGFR) mRNA has been demonstrated in human renal cell carcinomas (RCC), but few reports quantitate EGFR in RCC and correlate EGFR content with clinicopathologic findings. METHODS: Using 125I-EGF as a ligand, the maximum binding of EGF in membrane preparations from RCC (EGFR content) was studied by Scatchard analysis. RESULTS: A single class of binding sites for EGF was observed in 74% of RCC and 50% of normal renal tissues. The EGFR content was increased significantly in RCC compared with normal tissues. In all cases in which EGFR was undetectable, there was no evidence of distant metastasis, venous invasion, or regional lymph node involvement, and these patients had a better clinical outcome than patients with detectable EGFR. The EGFR content was significantly lower in nuclear Grade 1 tumors than in tumors of higher nuclear grades. No significant difference between EGFR content and other clinicopathologic findings was detected. CONCLUSION: The determination of EGFR content in RCC may become an important prognostic factor for the biologic behavior of RCC.

[A report of two cases of multilocular cystic renal cell carcinoma: review of 51 cases reported and the results of a prognostic survey].

We report 2 cases of multilocular cystic renal cell carcinoma. One was in a 33-year-old male, presenting with ultrasonic abnormality of the left kidney at an annual employee health care examination. Computerized tomography (CT) demonstrated a 5 cm of multilocular cystic mass adjacent to the lower pole of the left kidney. Another was in a 44-year-old male, presenting with microscopic hematuria at an annual employee health care examination. CT of the abdomen revealed a 6.5 cm of multilocular cystic mass on the upper pole of the right kidney. Both were diagnosed as renal cell carcinoma by the angiography and underwent radical nephrectomy. Gross specimens showed typical multilocular cystic appearance and histopathology showed clear cell carcinoma infiltrating septa and replacing epithelium of the cyst walls. Both patients are alive without evidence of disease at, 21 months and 14 months after operation, respectively. Including our cases, 51 multilocular cystic renal cell carcinoma and multilocular cystic nephroma associated with renal cell carcinoma have been reported. From the review of the literatures and the answer of the questionnaires inquiring about the outcome of the patient to Japanese reporters, the outcome of 38 patients was ascertained. The 10-year survival rates and non-recurrence rate after operation calculated by the Kaphan-Meier formula were 97.3% and 90.3%, respectively. Because of the good prognosis of reported cases, we concluded that we should choose kidney-sparing surgery for the operation of multilocular cystic renal cell carcinoma.