[Short-term intravesical instillation of pirarubicin (THP) in prophylactic treatment after transurethral resection of superficial bladder tumor].

We conducted a prospective randomized controlled study on the prophylactic effects of short-term intravesical instillation of pirarubicin (THP) against recurrence to determine the effective administration schedule. All patients gave their informed consent. The subjects included bladder cancer patients who had pTa or pT1, and G1 or G2 cancer, and became tumor-free after transurethral resection of the bladder tumor (TUR-BT). After dissolving 30 mg of THP into 5 ml of distilled water, physiological saline was added to adjust the total volume to 50 ml, which was then instilled into the bladder, and was retained for 5 minutes. The schedule of instillation was for daily for 7 consecutive days from the day of TUR-BT and subsequently once a week for 10 weeks, 17 times in total for Group I, and once every two weeks for 6 months (12 times) starting 2 weeks after TUR and subsequently once a month until one year had passed after surgery (6 times), 18 times in total for Group II. The total number of cases was 69 (36 in Group I, 33 in Group II). The tumor-free ratios determined by the Kaplan-Meier analysis were 93.9% in Group I and 72.7% in Group II for one year, and 86.8% in Group I and 59.5% in Group II for two years. There was a statistically significant difference in the tumor-free ratios between the two groups by the generalized Wilcoxon test and the Log rank test (p = 0.0145 and 0.0107, respectively). Multivariated analysis using Cox's comparison hazard model produced p-values of 0.0002, 0.0007, 0.0009 and 0.0040 in the order of therapeutic mode, initial onset/recurrence, stage and number of tumor. Adverse events that forced discontinuation of the therapy for a while occurred in 4.3%. These results demonstrated that short-term intensive intravesical instillation of THP immediately after TUR-BT was a safe and effective therapy.

[Normal term delivery after adrenalectomy for Cushing syndrome in a pregnant patient--a case report].

This is a case of a 29-year-old female. She visited our hospital with the chief complaint of swelling of the legs and abdominal bloating at the 10th week of pregnancy. Edema and central obesity were observed in the lower extremities. Hematological and biochemical tests revealed hypokalemia. Gynecologically, she was normal. Elevated blood cortisol levels were identified on the hormonal tests, which suggested the existence of Cushing's syndrome. Ultrasound revealed the presence of a tumor in the left adrenal gland, and she was referred to our department for surgery. On September 13, 1994, left adrenalectomy was performed in a right lateral position under inhalation anesthesia and epidural anesthesia. Adjuvand steroid therapy was initiated during surgery and the blood cortisol levels were normalized within 2 months. She delivered a baby girl weighing 2,722 g at the 40th week of pregnancy via a normal transvaginal delivery. Pregnancy rarely occurs in patients with Cushing's syndrome. We think she was the first case in Japan who had normal 40th week transvaginal delivery after adrenalectomy during pregnancy. We present a description of our case here with reference to the relevant literature.

Is diabetic neuropathy responsible for diabetic impotence?

It is well known that diabetes mellitus is accompanied by complications of sexual dysfunction and it is believed that diabetic neuropathy may cause impotence. In our study, we found that not all the patients who visited our center with the chief complaint of diabetic impotence were suffering from organic impotence, and diabetes mellitus per se served as a means of psychological stress in a substantial number of cases. Probably because no method has been available to provide precise information on the state of the penile-controlling nerves, we found that a larger number of patients than expected were suffering from a vascular disorder.

The 5'-terminal region of the apocytochrome b transcript in Crithidia fasciculata is successively edited by two guide RNAs in the 3' to 5' direction.

We analyzed the chimeric guide RNA (gRNA)-mRNA molecules in Crithidia fasciculata that are predicted to transiently exist in editing of the 5'-terminal domain of apocytochrome b (CYb) mRNA, by polymerase chain reaction amplification and DNA sequencing, and obtained evidence suggesting that among the 14 editing sites numbered from 3' to 5', the sequence in the 3'-half of the sites (3' block) was specified by one guide RNA species (gRNA-I) and that in the remaining half of the sites (5' block) by the other guide RNA species (gRNA-II) and that the direction of editing in each block was 3' to 5'. The predicted transition site of editing by two gRNAs was between the first and second U residues from the 3' end within editing site 7. We found that a stretch of the edited sequence in the 3' block of mRNA could form a stable duplex with a stretch immediately upstream of the guide sequence in gRNA-II. The result leads to a successive editing model that the 3' block of pre-edited mRNA is first edited by gRNA-I, and after completion of editing, the 5' portion of gRNA-II pairs with the edited mRNA for editing of the 5' block.

Site-specific dissection of E. coli chromosome by lambda terminase.

We have succeeded the targeted cleavage of chromosomes by lambda terminase that introduces double-strand cleavages in DNA recognizing the lambda cos sequence. When chromosomal DNAs of various Escherichia coli K-12 strains were subjected to terminase digestion, all were found to contain two common cleavage sites. Therefore, DNAs from lambda lysogens in which lambda DNA was inserted at different chromosomal sites were specifically cleaved at one more additional site. The two sites, termed ecos1 and ecos2, were mapped at approximately 35.1' and 12.7' of E. coli genetic map. The ecos1 and ecos2 sites were included in qin and qsr' regions, respectively. Therefore, the cleavage sites were associated with cryptic prophages. Sequences at the ecos1 and ecos2 sites showed 98% homology to the lambda cos sequence, indicating high fidelity of sequence recognition by the terminase. Since the strategy for integration of a DNA segment into chromosomal DNA through homologous recombination has been established, the dissection method that uses lambda terminase should be applicable for gene mapping as well as construction of macrophysical maps of larger genomes.

[Bilateral multilocular cystic renal cell carcinoma].

A case of bilateral renal cell carcinoma with multilocular cysts is described. A 42-year-old female was admitted to our hospital for the purpose of further examination or right renal cystic mass discovered incidentally by ultrasonography. After she was examined by CT-scan and angiography, right nephrectomy was performed under the diagnosis of right renal cancer. Histological diagnosis was multilocular cystic renal cell carcinoma. One year later a small cystic legion was found in the left kidney. Under the diagnosis of left renal cancer, left partial nephrectomy was performed. Histological diagnosis was multilocular cystic renal cell carcinoma same as the right kidney. Multilocular renal cyst is classified into several classes by its style of development. This case of bilateral renal cell carcinoma which grew to be multilocular cysts is the first case in Japan as far as we can search.

Transcription in vivo within the replication origin of the Escherichia coli chromosome: a mechanism for activating initiation of replication.

Within the replication origin, oriC, of the Escherichia coli chromosome, novel in vivo transcripts were detected which proceeded rightward and whose production was activated by DnaA protein. In contrast, DnaA protein repressed the previously described ori-L leftward transcription. The former should introduce negative supercoiling, and the latter positive supercoiling, into the 13-mers. The effects of transcription on the initiation of replication were also investigated by making constructs with promoters placed near oriC. Transcription was found to enhance the origin activity only when it was oriented in such a way as to introduce negative supercoiling into the 13-mers. From these results, we propose that transcription within oriC regulates replication initiation by altering the topology of the 13-mer region.

[A clinical evaluation of AMS Hydroflex in the treatment of impotence].

The AMS Hydroflex penile prosthesis was implanted in 34 organic impotence patients between June 20, 1986 and Aug. 6, 1990. Patients ages ranged between 26 and 71 with an average of 50.5. The causes of impotences were; 17 cases of post-radical pelvic surgery, 7 cases of injury, 6 cases of diabetes and 4 of others. First evaluation was made after 12 weeks of implantation in terms of patient satisfaction with sexual intercourse and post operative complication. Sixteen patients (47.1%) were highly satisfied with intercourse, 13 (38.2%) satisfied with intercourse, 2 (5.9%) slightly dissatisfied even with successful intercourse, 1 (2.9%) with no improvement and 2 (5.9%) could not follow. Utility of the prosthesis, e.g. effectiveness and safety was; high utility in 20 (58.8%), good utility in 11 (32.4%), slight utility in 1 (2.9%), no favorable in 1 (2.9%) and no judgement in 1 (2.9%). The overall effectiveness and safety of the Hydroflex was demonstrated based on the above findings. Long term evaluation was made from the initial implantation of the device up to the termination of the trial with average of 19.2 months and was mentioned altogether.

The HgaI restriction-modification system contains two cytosine methylase genes responsible for modification of different DNA strands.

A DNA fragment of about 3.4 kilobase pairs that expressed the HgaI modification activity was cloned from the chromosomal DNA of Haemophilus gallinarum, and its nucleotide sequence was determined. Two open reading frames (ORF) which could code for structurally similar proteins were identified in the upstream and middle regions and a truncated ORF in the downstream region in the same orientation. When the respective ORFs were separately cloned, the clones carrying the upstream and middle ORFs both expressed the modification activity, indicating that the two genes are involved in modification of the HgaI restriction-modification system. In order to determine the sites of modification precisely, the respective genes were recloned into an expression vector, from which gene products were purified. A short DNA fragment carrying the HgaI recognition site was treated with each of these enzymes, and, after separation of the two strands by duplex formation with M13 viral DNAs carrying the respective strands, the presence or absence of modification was judged from susceptibility to HgaI endonuclease. The results of analysis showed that different strands were modified in an asymmetric way by each gene product. Analysis of the species and positions of modified bases by the Maxam-Gilbert method further demonstrated that the gene products from the upstream and middle ORFs participated in methylation of the internal cytosine residues of the strands carrying 3'-CTGCG-5' and 5'-GACGC-3', respectively. We concluded that the HgaI modification system consisted of two cytosine methylase genes responsible for modification of different strands in the target DNA.

Cross-talk between the virulence and phosphate regulons of Agrobacterium tumefaciens caused by an unusual interaction of the transcriptional activator with a regulatory DNA element.

Transcription of a virulence gene on the hairy-root-inducing plasmid A4, which is induced by plant factors in Agrobacterium tumefaciens, was also activated by phosphate limitation in both A. tumefaciens and Escherichia coli. The starting site of RNA synthesized under the two inducing conditions was the same, and an identical promoter was responsible for both inducible expressions. The response of the virulence gene to phosphate limitation did not require the positive regulator VirG for the virulence regulon, but depended entirely on the presence of PhoB protein, the positive regulator for the phosphate regulon. The DNA signal upstream of the virulence gene, which is targeted by the VirG protein, was recognized by the E. coli PhoB protein in vitro. These results indicate that cross-talk between the two regulons occurred during the recognition of a DNA signal by the regulatory protein.

Combined cisplatin and radiation therapy for advanced bladder cancer.

Since September 1985, 44 patients with advanced urinary bladder cancer have been treated by combined cisplatin and full-dose radiotherapy. The patients were 32 males and 12 females, and their ages ranged from 33 to 83 years, with a median of 67.4 years. Radiotherapy consisting of a tumor dose of 50-60 Gy was administered with cobalt-60. Cisplatin was infused 5 days at a daily dose of 20 mg on the 1st and 4th weeks of treatment. Of the 39 evaluable patients 27 (69.2%) achieved a complete response. Toxicity was also evaluated for those 44 patients. Mainly gastrointestinal toxicity was noted: loss of appetite in 28 (64%), nausea and/or vomiting in 21 (48%), and diarrhea in 8 (18%). Leukocytopenia was noted in 16 (33%) and mild thrombocytopenia in 5 (11%). Mild dermatitis was noted in 8 (18%).

Clinical experience and successful impregnation using an artificial spermatocele.

We have to date installed artificial spermatoceles made of silicone in a total of 33 patients. Artificial insemination of husband (AIH) was performed in 1-9 times using semen collected from 11 of the 33 patients in whom spermatocele was installed, and pregnancy was achieved in 2 cases. Reported cases of pregnancy achieved using semen collected from artificial spermatoceles are few in number; indeed, our successful cases were the fifth and sixth reported so far. The key point for success in achieving pregnancy by means of the artificial spermatocele is considered to be attempting AIH as soon as possible after collecting the sperm, because collection of sperm soon becomes impossible, due to early obstruction of the incised region at the epididymis by degeneration of fibrous tissue. 3% HSA-TMPA medium was found to be useful for sperm collection.

The AT richness and gid transcription determine the left border of the replication origin of the E. coli chromosome.

We have identified novel, cis-acting elements which enhance in vivo the replication activity of plasmids carrying the minimal oriC of Escherichia coli. These are (i) the AT rich sequence ('AT-cluster') which exists immediately left of the 13mer repeats and (ii) the gid transcriptional unit. The 'AT-cluster' was functionally replaced by an unrelated AT rich sequence. This was also the case for the left and middle 13mers; they were substituted by the AT rich fragment from mini-F plasmid. The left 13mer was replaced by the AT rich sequence which did not show the 'reduced helical stability' known as the important character of the 13mer region. In contrast to these results, the right 13mer sequence was strictly required. As to the effect of the transcription from the gid promoter, the minimal oriC was activated only when the transcription was directed away from the left side of it. mioC transcription proceeding toward the oriC had no effect on the activation. Mutations in the DnaA boxes were partially suppressed by gid transcription leaving oriC from the left side. From these results, we propose that the AT richness is a determinant to identify the left border of oriC. It is presumed that gid transcription introduces negative superhelicity at the AT rich region and facilitates DnaA dependent duplex opening.

Binding of the regulatory protein VirG to the phased signal sequences upstream from virulence genes on the hairy-root-inducing plasmid.

The VirG protein is a positive regulator for the virulence genes of which expression is induced by a plant factor, and is essential for Agrobacterium pathogenicity on dicotyledonous plants. The VirG protein of the hairy-root-inducing plasmid A4 was overproduced in Escherichia coli cells, and purified to homogeneity. DNase I footprinting experiments revealed that the purified VirG protein was bound to the upstream region of virulence genes including the phased vir box sequences, which had been presumed to be the VirG recognition signal from the sequence analysis. In dimethyl sulfate footprinting, the VirG protein specifically protected the guanine residues within every vir box sequence. It was concluded that the VirG protein was bound to the phased vir box sequences from the major groove along one side of double-helical DNA.

[Treatment using CDDP and radiation therapy in treating bladder tumor].

Combination therapy consisting of cis-diamine-dichloroplatinum (CDDP) and radiotherapy was used to treat bladder cancer. The subjects were 20 patients with bladder cancer we experienced since September, 1986. They were between 33 and 83 years old and consisted of 13 males and 7 females. Among the histotypes and grades, there were 3 cases of G2, 7 of G2-G3 and 10 of G3 metastatic epithelial carcinoma. The classification of primary tumors was T2 in six cases, T3a in 12 cases and T4 in two cases. In 12 cases where the bladder was preserved, this therapy was performed after TUR biopsy or TUR, and the effects were evaluated after observation periods of 5-17 months. Side effects caused by CDDP were anorexia in 15 cases, nausea and vomiting in 10 cases, leukopenia in 10 cases and thrombocytopenia in four cases. Those caused by the radiotherapy were diarrhea in seven cases and skin disorders in six cases. All of these side effects were mild and the patients recovered spontaneously.

Content and distribution of vasoactive intestinal polypeptide (VIP) in cavernous tissue of human penis.

Penile erection is controlled by a valvular structure in the helicine artery in humans. The opening and closing of this valve are believed to be regulated by the autonomic nervous system, especially through the release of vasoactive intestinal polypeptide (VIP). We determined the content of VIP in cavernous tissue in 18 impotent patients and in 5 normal controls by radioimmunoassay, and we examined the distribution of VIP-ergic nerve fibers in cavernous tissue by an immunohistochemical method. As a result, it was found that the lower penile VIP content was more frequent among patients with organic impotence than among the controls. Furthermore, VIP-ergic nerve fibers were seen to be diffusely and loosely distributed in a large number of organic impotence patients. These findings suggest that organic impotence in some patients may be due to decreases in the VIP content and in VIP-ergic nerve fibers.

Signal structure for transcriptional activation in the upstream regions of virulence genes on the hairy-root-inducing plasmid A4.

The inducibility of the vir genes (virA, -B, -C, -D, -E, and -G) on pRiA4 was examined at the transcriptional level, and the RNA-starting sites were determined by S1-nuclease mapping and primer-extension experiments. All of these genes were inducible, while virA, -E, and -G were transcribed even under noninducing conditions. Each transcription of virB, -C, -D, and -E was initiated at one particular site, but that of virA and -G occurred at two and three sites, respectively, depending on the conditions used. In the DNA region upstream from each inducible transcript, one or more blocks of six base-pairs, 5'TGATAACT3' (vir box), were found to be placed characteristically. These blocks were phasing with an interval of 11 base-pairs, and the most upstream one in each upstream region was preceded by an additional block in the inverted orientation. Although the distance between the block(s) and the promoter varied with the vir gene, every block was placed in a phase nearly opposite to the -35 and -10 regions of the promoter.