Identification of tyrosine residues involved in ligand recognition by the phosphatidylinositol 3-kinase Src homology 3 domain: circular dichroism and UV resonance Raman studies.

Src homology 3 (SH3) domains are small noncatalytic protein modules capable of mediating protein-protein interactions. We previously demonstrated that the association of a ligand peptide RLP1 (RKLPPRPSK) causes environmental and structural changes of Trp55 and some of seven Tyr residues in the phosphatidylinositol 3-kinase (PI3K) SH3 domain by circular dichroism (CD) and 235-nm excited UV resonance Raman (UVRR) spectroscopies [Okishio, N., et al. (2000) Biopolymers 57, 208-217]. In this work, the affected Tyr residues were identified as Tyr12, Tyr14, and Tyr73 by the CD analysis of a series of mutants, in which every single Tyr residue was replaced by a Phe residue. Among these three residues, Tyr14 was found to be a main contributor to the UVRR spectral change upon the RLP1 binding. Interestingly, CD and UVRR analyses revealed that RLP1 associates with the Y14F and Y14H mutants in different ways. These results suggest that Tyr14 plays a crucial role in the ligand recognition, and the amino acid substitution at Tyr14 affects the mode of PI3K SH3-ligand interaction. Our findings give an insight into how SH3 domains can produce diversity and specificity to transduce signaling within cells.

Role of the conserved acidic residue Asp21 in the structure of phosphatidylinositol 3-kinase Src homology 3 domain: circular dichroism and nuclear magnetic resonance studies.

To elucidate a role of the Src homology 3 (SH3)-conserved acidic residue Asp21 of the phosphatidylinositol 3-kinase (PI3K) SH3 domain, structural changes induced by the D21N mutation (Asp21 --> Asn) were examined by circular dichroism (CD) and nuclear magnetic resonance (NMR) spectroscopies. In the previous study, we demonstrated that environmental alterations occurred at the side chains of Trp55 and some Tyr residues from the comparison of the near-UV CD spectra of the PI3K SH3 domain with or without a D21N mutation [Okishio, N., et al. (2000) Biopolymers 57, 208-217]. In this work, the affected Tyr residues were identified as Tyr14 and Tyr73 by the CD analysis of a series of mutants, in which every single Tyr residue was replaced by a Phe residue with or without a D21N mutation. The (1)H and (15)N resonance assignments of the PI3K SH3 domain and its D21N mutant revealed that significant chemical shift changes occurred to the aromatic side-chain protons of Trp55 and Tyr14 upon the D21N mutation. All these aromatic residues are implicated in ligand recognition. In addition, the NMR analysis showed that the backbone conformations of Lys15-Asp23, Gly54-Trp55, Asn57-Gly58, and Gly67-Pro70 were affected by the D21N mutation. Furthermore, the (15)N[(1)H] nuclear Overhauser effect values of Tyr14, Glu19, and Glu20 were remarkably changed by the mutation. These results show that the D21N mutation causes structural deformation of more than half of the ligand binding cleft of the domain and provide evidence that Asp21 plays an important role in forming a well-ordered ligand binding cleft in cooperation with the RT loop (Lys15-Glu20).

Interactions of phosphatidylinositol 3-kinase Src homology 3 domain with its ligand peptide studied by absorption, circular dichroism, and UV resonance raman spectroscopies.

Absorption, circular dichroism (CD), and UV resonance Raman (UVRR) spectroscopies were applied to selectively examine the environmental and structural changes of Trp and Tyr residues in the phosphatidylinositol 3-kinase (PI3K) SH3 domain induced by ligand association. Comparison of the spectra of PI3K SH3 in the presence or absence of its ligand peptide RLP1 (RKLPPRPSK) indicated that RLP1 binding changed the environment of Trp55 of the SH3 to be more hydrophilic and its H bonding weaker and that of Tyr residues to be more hydrophobic. The D21N mutant (Asp21 --> Asn) of the SH3 yielded a UV CD distinct from that of the wild type, and its spectral changes induced by RLP1 binding were smaller and different from those of the wild type in absorption, CD, and UVRR spectra, suggesting that the mutation of conserved Asp21 affected the conformation of the ligand binding cleft and thus might lead to the decrease in the ligand affinity. These data provide direct evidence for the occurrence of environmental and structural changes of PI3K SH3 by the association of a ligand and the D21N mutation.

[Malignant lymphoma of the penis: report of two cases].

We report two cases of malignant lymphoma of the penis. A 64-year-old man presented with painful indurations of the penis. A computerized tomography (CT) scan showed a swollen retroperitoneal lymph node 5 cm in diameter. Penectomy confirmed the diagnosis of B cell malignant lymphoma, diffuse large type. Despite systemic chemotherapy with a CHOP regimen, he died of disease 7 months postoperatively. The second case was in a 63-year-old man presenting with multiple nodules in the penis and scrotum. Biopsy of a scrotal nodule revealed B cell malignant lymphoma, diffuse medium size type. A CT scan demonstrated widespread lesions (stage III E). He has been in complete remission for 10 months following, multidisciplinary treatments. We reviewed 21 cases of malignant lymphoma of the penis which have been reported in the literature.

Binding of the influenza virus NS1 protein to model genome RNAs.

We have previously shown that the influenza virus NS1 protein exhibits stable binding to a model mini vRNA which is stronger than its binding to dsRNA. In this study, we confirmed that the binding depended on a higher-order structure of the model RNA, probably the panhandle structure formed by pairing between the 5'- and 3'-terminal common sequences. The formation of the structure was enhanced by the NS1 protein itself. We propose that an A bulge in a stretch of double helix results in a binding site with greater affinity for the NS1 protein.

[A case report of primary renal carcinoid tumor].

A 54-year-old woman was referred to our institute because of abdominal pain and constipation. The computed tomography of the abdomen revealed a 3 x 3 cm of low density tumor at the upper pole of the left kidney and para-aortic lymphadenopathy. Gastrointestinal investigation revealed no particular findings suggesting a carcinoid syndrome associated with the left kidney tumor. The specimen of the resected tumor showed staining pattern specific to carcinoid tumor; positive staining for chromogranin A and neuron-specific-enolase. Electron microscopy confirmed the presence of dense-core granules in the tumor cells. No evidence of metastasis or recurrence was noted for 13 months after the operation. This was a very rare case of primary renal carcinoid, representing the 26th case in the literature.

[Clinical analysis of 683 prostatic cancer patients in the Tokai Urological Cancer Registry].

Of 815 patients with prostatic carcinoma registered in Tokai Urological Cancer Registry between 1989 and 1991, 683 patients followed up until April to July 1992 were analyzed. The patients were between 48 and 94 years old (mean 73.2). The clinical stage was A in 96 (14.1%), B in 118 (17.3%), C in 117 (17.1%) and D in 352 (51.5%) patients. The histological study well differentiated adenocarcinoma in 150 (22%) patients, moderately differentiated adenocarcinoma in 305 (44.6%), and poorly differentiated adenocarcinoma in 194 (28.4%) patients. There were 12 undifferentiated adenocarcinomas and 22 were not classified. The incidence of patients with poorly differentiated adenocarcinoma increased with the progression of clinical stage. Hormonal therapy was the main treatment (92.8%) and a variety of hormonal therapies with surgery or chemotherapy were attempted through out the clinical stages. Overall survival rate at 5 years was 48.6%. The 5-year survival rage for stage A, B, C and D carcinoma was 84.7, 93.1, 51.8 and 30.3%, respectively. Significant differences in the survival of patients were noted among stage A or B, stage C and stage D (Logrank test: p < 0.0001). The 5-year survival rate was 70.2% for well differentiated, 53.0% for moderately differentiated, and 32.6% for poorly differentiated adenocarcinoma (Logrank test: p < 0.0001). Disease-specific death was observed in 128 patients (66%), and cardio- or cerebrovascular death accounted for 16 (8%) deaths.

Fission yeast Nda1 and Nda4, MCM homologs required for DNA replication, are constitutive nuclear proteins.

The nda1+ and nda4+ genes of the fission yeast Schizosaccharomyces pombe encode proteins similar to budding yeast MCM2 and MCM5/CDC46, respectively, which are required for the early stages of DNA replication. The budding yeast Mcm proteins display cell-cycle dependent localization. They are present in the nucleus specifically from late M phase until the beginning of S phase, so that they were suggested to be components of a replication licensing factor, a positive factor for the onset of replication, which is thought to be inactivated after use, thus restricting replication to only once in a cell cycle. In the present study, we raised antibodies against Nda1 or Nda4 and identified 115 kDa and 80 kDa proteins, respectively. Their immunolocalization was examined in wild-type cells and in various cell-cycle mutants. Both Nda1 and Nda4 proteins remained primarily in the nucleus throughout the cell cycle. In mutants arrested in G1, S, and G2 phases, these proteins were also enriched in the nucleus. These results indicate that the dramatic change in subcellular localization as seen in budding yeast is not essential in fission yeast for the functions of Nda1 and Nda4 proteins to be executed. The histidine-tagged nda1+ gene was constructed and integrated into the chromosome to replace the wild-type nda1+ gene. The resulting His-tagged Nda1 protein was adsorbed to the Ni-affinity column, and co-eluted with the untagged Nda4 protein, suggesting that they formed a complex.

Fission yeast genes nda1+ and nda4+, mutations of which lead to S-phase block, chromatin alteration and Ca2+ suppression, are members of the CDC46/MCM2 family.

Fission yeast cold-sensitive mutants nda1-376 and nda4-108 display a cell cycle block phenotype at the restrictive temperature (cell elongation with the single nucleus) accompanied by an alteration in the nuclear chromatin region. DNA content analysis shows that the onset of DNA synthesis is blocked or greatly delayed in both mutant cells, the block being reversible in nda4-108. Upon release to the permissive temperature, nda4-108 cells resumed replicating DNA, followed by mitosis and cytokinesis. The nda4 phenotype was partly rescued by the addition of Ca2+ to the medium; Ca2+ plays a positive role in the nda4+ function. The predicted protein sequences of nda1+ and nda4+ isolated by complementation are similar to each other and also, respectively, to those of the budding yeast, MCM2 and CDC46, both of which are members of the gene family required for the initiation of DNA replication. The central domains of these proteins are conserved, whereas the NH2- and COOH- domains are distinct. Results of the disruption of the nda1+ and nda4+ genes demonstrates that they are essential for viability.

[Clinical statistics of the patients admitted to the Department of Urology Shizuoka Red Cross Hospital between 1981 and 1990].

A statistic survey was made on the patients, diseases and operations experienced at the Urological ward of Shizuoka Red Cross Hospital between 1981 and 1991. The total number of inpatients was 2,830 and the male to female ratio was 3.5 to 1. The most frequent diseases among the inpatients were obstructive uropathy (25.5%), malignant neoplasia (23.7%), non-specific infection (16.4%) urolithiasis (16.1%). The number of operations was 1922. Endoscopic surgery was the most frequent form of operation (55.2%).

[Clinical study of sparfloxacin, a new quinolone derivative, on urinary tract infection].

Sparfloxacin (SPFX) was evaluated for its clinical efficacy in 22 patients with acute uncomplicated cystitis (AUC) and 51 with chronic complicated urinary tract infection (CC-UTI). SPFX was administered in a single oral daily dose of 100 mg for 3 days to the patients with AUC. According to the UTI criteria, the efficacy rate was 100% (excellent rate was 78.9%). SPFX was also administered in a single oral dose of 200 to 300 mg mostly for 5 days to patients with CC-UTI. The efficacy rate according to the UTI criteria in the evaluable 22 cases was 45.5%. The efficacy rate according to the physicians in charge, evaluated as either excellent or good for the 23 cases treated with SPFX for 7 and 14 days was 65.2%, the overall efficacy rate in 49 cases including the administration for 5 days being 55.1%. In safety profile, subjective side effects were observed in 9 cases (12.3%), in 2 of which the medication was discontinued (2.7%). These side effects, flowever, recovered without any treatment to normal at the completion or discontinuation of the medication. The clinical laboratory values showed a slight elevation of BUN in 2 cases. SPFX was the most potent antimicrobial brug with a long blood elimination half life among the existing current new quinolones (NQs), indicating a possibility of once-a-day treatment regimen. In safety, side effects of SPFX were similar in general to those of other current NQs. Thus, SPFX is regarded as a highly useful antimicrobial drug when the drug is administered with consideration for its unique characteristics.

[Survival rate of bladder tumors--an analysis of 2304 patients with bladder tumors in the Tokai Urological Cancer Registry].

During the 7 years from 1980 to 1986, 2860 cases of bladder tumors were registered in the Tokai Urological Cancer Registry. Among the 2860 cases, 2304 cases were selected from the registered cases for the present study. The 5-year relative (actual) survival rates were 73.8% (61.9%) of all patients; 48.9% (42.4%) in those with malignant neoplasma of urinary bladder excluding transitional cell carcinoma; 48.8% (41/3%) in those with mixed tumor. In patients with transitional cell carcinoma, the 5-year relative (actual survival rates were 93.7% (78.8%) for G1, 87.2% (74.1%) for G2 and 47.3% (38.9%) for G3. As to staging, the 5-year survival rates were 101.9% (88.0%), 87.6% (75.3%), 57.9% (47.8%), 33.7% (28.2%) and 6.1% (5.0%) in patients with stage of Ta, T1, T2, T3 and T4, respectively. The tumors with muscle infiltration and high grade malignancy obviously deteriorated patients' survival. The 5-year relative (actual) survival rate for patients treated with TUR was 98.1% (82.2%). As to grading, the 5-year survival rates were 102.2% (86.6%) for G1, 104.3% (88.3%) for G2 and 56.9% (48.3%) for G3. The 5-year survival rates of those with Ta, T1 and T2 were 103.9% (89.7%), 96.0% (82.6) and 61.1% (49.1%), respectively. The 5-year relative (actual) survival rate for patients undergoing total cystectomy was 62.4% (52.3%). In those patients, the 5-year survival rates were 96.7% (80.9%) for G1, 63.6% (55.7%) for G2 and 55.4% (47.1%) for G3. As to staging, the 5-year survival rates were 102.3% (90.6%), 77.8% (68.2%), 56.3% (47.9%), 41.8% (34.9%) and 15.2% (13.1%) in patients with stage of Ta, T1, T2, T3 and T4, respectively. The 3 and 5-year relative (actual) survival rates in patients with advanced bladder tumors were 5.3% (4.8%) and 0.87% (0.73%), respectively.

[A case of Fournier's gangrene].

A 59-year-old man was admitted to our hospital complaining of perineal pain and scrotal redness. He was diagnosed with diabetes mellitus 12-years ago but had not received treatment. The scrotum soon showed necrosis and pus discharge. A culture study revealed Clostridium, Bacteroides and many other bacteria. Antibiotics chemotherapy (CMZ, AMK, MINO) was done but because necrotizing fasciitis progressed, we performed surgical treatment of necrosis. The skin defect was closed naturally with wound irrigation. The patient is doing well and his diabetes is under control. We found 13 other cases of Fournier's gangrene in the Japanese literature and their age, culture study and complications are discussed.

[Clinical studies on Suncefal (cefpiramide) concentration in prostatic tissue and bladder wall].

The cefpiramide (CPM) concentration in prostatic tissue of 19 patients with benign prostatic hypertrophy and bladder wall of 2 patients with bladder tumors was measured, after 2 g of CPM was administered intravenously by bolus technique prior to operation. Prostatic tissue level was 28.4 to 131 micrograms/g and tissue to serum ratio 0.15 to 0.63 at 12 min. to 220 min. after administration. The T1/2 of CPM in prostatic tissue was 3.29 hours. The bladder wall level was high at both determinations. CPM level in prostatic tissue and bladder wall covered the minimum inhibitory concentration of many bacteria detected from the infected sites. Therefore, CPM which has a strong antimicrobial activity can be considered as a highly useful antibiotic for the treatment of postoperative infections.

[Urinary tract infection associated with benign prostatic hypertrophy and related diseases and transurethral surgery].

Three hundred and fifty five patients were treated with transurethral resection of the prostate for the relief of partial urinary obstruction. Of these, 188 had suffered from bacteriuria before surgery and the remaining 167 had been free of urinary infection before surgery. Urinary infection was caused by urethral instrumentation including indwelling catheter for various periods of time. A significant correlation between volume of residual urine and preoperative infection was demonstrated. No correlation between preoperative infection and postoperative voiding efficiency was however noticed though significant improvement in voiding was demonstrated by postoperative uroflowmetry.

[Malignant peritoneal mesothelioma of the prostate: a case report].

A case of malignant peritoneal mesothelioma of prostate in a 52-year-old male is reported. He was admitted to the hospital with the complaint of perineal pain. The prostate was enlarged markedly, elastic soft and severely tender. CT scan and some examinations suggested the possibility of prostatic malignancy. The histological findings of the needle biopsy specimen revealed peritoneal mesothelioma. Complete pelvic exenteration was performed, because of direct invasions to the urinary bladder and the rectal anterior wall. The final pathological diagnosis revealed malignant fibrous peritoneal mesothelioma with solitary type of the rectovesical recess. At three months after the operation, the patient died of local resurrence and multiple metastasis to liver and lung. Only three cases of peritoneal mesothelioma, with solitary type, of rectovesical recess or retrovesical space have so far been reported in Japan.

[Clinical results and safety of ceftazidime, a new injectable cephalosporin, in long-term administration of the treatment of infections in urology].

CAZ was administered to 34 patients in the field of urology for treatment or prevention of serious infections with many complicated factors. The duration of treatment ranged from 9 to 30 days, the most frequent being 14 days, which was the duration originally set as the standard. As the results CAZ was considered to be one of the drugs of choice in the cases requiring long-term treatment, from the viewpoint of both efficacy and usefulness.

[Hemangiopericytoma of the prostate: a case report].

A case of hemangiopericytoma arising in the prostate is presented. The patient, a 67-year-old male, was admitted to the hospital for difficulty in urination and underwent transurethral resection of the prostate. The pathological diagnosis was "well-differentiated adenocarcinoma and focal hemangiopericytoma". To the best of our knowledge, the only four cases of hemangiopericytoma of the prostate have been reported in the English literature so far and none in the Japanese literature.