Bacillus Calmette-Guérin instillation treatment for carcinoma in situ of the upper urinary tract.

OBJECTIVE: To analyse the efficacy and safety of bacillus Calmette-Guérin (BCG) perfusion treatment forcarcinoma in situ (CIS) of the upper urinary tract. PATIENTS AND METHODS: Six patients with cytologically diagnosed CIS of the upper urinary tract were treated by BCG instillation via retrograde catheterization using a 6 F ureteric catheter or an 8 F indwelling JJ ureteric stent between the ureter and bladder. BCG (Tokyo 172 strain, 80 mg in 100 mL normal saline) was instilled weekly for 4 or 8 weeks. The efficacy and safety of the treatment was assessed. RESULTS: The mean (range) follow-up was 22 (9-38) months; none of the patients died, and all were negative for cytology in urine collected from the upper urinary tracts. However, one patient had recurrent CIS in the prostatic urethra; the patient was treated by intravesical BCG instillation. In all patients, positive cytology became negative after one or two instillations of BCG. The ureter became stenotic in two patients. Although irritative symptoms occurred in all patients, such side-effects disappeared in a few months and were not clinically significant. CONCLUSION: In these six patients retrograde BCG instillation for CIS of the upper urinary tract was effective and safe. Based on the cytology results after only two BCG treatments, the dose or number of BCG instillations could possibly be reduced, but further study is needed.

Gene cloning, nucleotide sequencing, and purification and characterization of the D-stereospecific amino-acid amidase from Ochrobactrum anthropi SV3.

The gene encoding the D-stereospecific amino-acid amidase from Ochrobactrum anthropi SV3 was cloned and sequenced. Analysis of 7.3 kb of genomic DNA revealed the presence of six ORFs, one of which (daaA) encodes the D-amino-acid amidase. This enzyme, DaaA, is composed of 363 amino-acid residues (molecular mass 40 082 Da), and the deduced amino-acid sequence exhibits homology to alkaline D-peptidase from Bacillus cereus DF4-B (32% identity), DD-peptidase from Streptomyces R61 (29% identity), and other penicillin-recognizing proteins. The DaaA protein contains the typical SXXK, YXN, and H(K)XG active-site motifs identified in the penicillin-binding proteins and beta-lactamases. The daaA gene modified in the nucleotide sequence upstream from its start codon was overexpressed in Escherichia coli. The activity of the recombinant DaaA enzyme in cell-free extracts of E. coli was 33.6 U. mg-1 with D-phenylalaninamide as substrate, which is about 350-fold higher than in extracts of O. anthropi SV3. This enzyme was purified to electrophoretic homogeneity by ammonium sulfate fractionation and three column chromatography steps. On gel-filtration chromatography, DaaA appeared to be a monomer with a molecular mass of 40 kDa. It had maximal activity at 45 degrees C and pH 9.0, and was completely inactivated in the presence of phenylmethanesulfonyl fluoride or Zn2+. DaaA had hydrolyzing activity toward D-amino-acid amides with aromatic or hydrophobic side chains, but did not act on the substrates for the DD-peptidase and beta-lactamase, despite their sequence similarity to DaaA. The characteristics of the recombinant DaaA are similar to those found for the native enzyme partially purified from O. anthropi SV3.

[Bacillus Calmette-Guerin intravesical instillation treatment for carcinoma in situ of the bladder. Gifu BCG Instillation Therapy Research Group].

We performed a retrospective long-term study to evaluate the efficacy of intravesical instillation of Tokyo 172 strain Bacillus Calmette-Guerin (BCG) on carcinoma in situ (CIS) of the bladder. Between 1989 and 1998, 42 patients with CIS of bladder underwent intravesical instillation of BCG. In the follow-up period from 6 to 116 months (mean: 37.3 months), the efficacy rate of intravesical BCG instillation for CIS of the bladder was 81.0%. Two patients died from the bladder cancer. The non-recurrence rate in patients with grade 2 carcinoma (19 cases) was not significantly different from that in those with grade 3 carcinoma (23 cases). However, the recurrence rate in patients with secondary CIS (15 cases) was significantly higher than in those with primary CIS (27 cases). The recurrence of CIS was observed in 7 of 42 cases. In 6 of 7 patients, CIS recurred within one year after treatment. Total cystectomy was performed in 10 of 42 patients, and pathological findings of muscle layer invasion were detected in 8 patients. Although side effects occurred in 33 patients (77.5%), no clinically significant side effects were observed. Our results suggest that intravesical BCG therapy may be useful for the treatment of CIS of the bladder.

Identification of active sites in amidase: evolutionary relationship between amide bond- and peptide bond-cleaving enzymes.

Mainly based on various inhibitor studies previously performed, amidases came to be regarded as sulfhydryl enzymes. Not completely satisfied with this generally accepted interpretation, we performed a series of site-directed mutagenesis studies on one particular amidase of Rhodococcus rhodochrous J1 that was involved in its nitrile metabolism. For these experiments, the recombinant amidase was produced as the inclusion body in Escherichia coli to greatly facilitate its recovery and subsequent purification. With regard to the presumptive active site residue Cys203, a Cys203 --> Ala mutant enzyme still retained 11.5% of the original specific activity. In sharp contrast, substitutions in certain other positions in the neighborhood of Cys203 had a far more dramatic effect on the amidase. Glutamic acid substitution of Asp191 reduced the specific activity of the mutant enzyme to 1.33% of the wild-type activity. Furthermore, Asp191 --> Asn substitution as well as Ser195 --> Ala substitution completely abolished the specific activity. It would thus appear that, among various conserved residues residing within the so-called signature sequence common to all amidases, the real active site residues are Asp191 and Ser195 rather than Cys203. Inasmuch as an amide bond (CO-NH2) in the amide substrate is not too far structurally removed from a peptide bond (CO-NH-), the signature sequences of various amidases were compared with the active site sequences of various types of proteases. It was found that aspartic acid and serine residues corresponding to Asp191 and Ser195 of the Rhodococcus amidase are present within the active site sequences of aspartic proteinases, thus suggesting the evolutionary relationship between the two.

Mycoplasma genitalium in the cervices of Japanese women.

BACKGROUND: Mycoplasma genitalium is considered a cause of nongonococcal urethritis in men. This organism also is a cause of genital infections in women, and has been detected in women attending sexually transmitted disease clinics in the United Kingdom and Denmark, although its prevalence is unknown in Japanese women. GOALS: To determine the prevalence of M. genitalium in the cervices of women with cervicitis or adnexitis as well as in asymptomatic pregnant women in Japan. STUDY DESIGN: Two hundred women who attended obstetric and gynecologic clinics were recruited. Sixty-four women had cervicitis, 53 had adnexitis, and 3 had both. Eighty pregnant women were asymptomatic for infection. Cervical swab specimens were examined for M. genitalium using a polymerase chain reaction-based assay. RESULTS: Five (7.8%) of 64 women with cervicitis and 3 (5.7%) of 53 women with adnexitis were positive for M. genitalium. After exclusion of Chlamydia-positive women, 5 (8.8%) of 57 women with cervicitis, and 2 (4.1%) of 49 women with adnexitis were positive for M. genitalium. In none of 80 asymptomatic pregnant women, including a Chlamydia-positive woman, was M. genitalium detected. Overall, 7 (6.6%) of 106 women with Chlamydia-negative genital infections were positive for the M. genitalium. This prevalence was significantly greater than that in asymptomatic pregnant women (P < 0.05). CONCLUSIONS: A significantly greater prevalence of M. genitalium was demonstrated in Japanese women with Chlamydia-negative cervicitis or adnexitis, compared with that in asymptomatic pregnant women. This study suggests that M. genitalium may play a pathogenic role in a portion of cases with Chlamydia-negative genital infections.

Prevalence of Mycoplasma genitalium in asymptomatic men in Japan.

We aim to clarify the prevalence of Mycoplasma genitalium in asymptomatic men in Japan. First-catch urine specimens were obtained from 187 asymptomatic Japanese men and examined for the presence of M. genitalium using a polymerase chain reaction (PCR)-based assay. Two (1.1%) of 187 first-catch urine specimens were positive for M. genitalium. The prevalence of M. genitalium in urine specimens of asymptomatic men in Japan is lower than that in asymptomatic men in the UK (6%) and Denmark (9%).

A novel transporter involved in cobalt uptake.

Cobalt is an essential component of a low molecular-mass nitrile hydratase (L-NHase) from Rhodococcus rhodochrous J1. We have found a new gene, nhlF, in the DNA region sandwiched between nhlBA encoding L-NHase and amdA encoding amidase, which are involved in the degradation of nitriles. The product of nhlF, NhlF, shows a significant sequence similarity with those of hoxN from Alcaligenes eutrophus, hupN from Bradyrhizobium japonicum, nixA from Helicobacter pylori, and ureH from Bacillus sp., which are considered to be involved in nickel uptake into these cells. Sequence and hydropathy plot analyses have shown that NhlF encodes a 352-amino acid (aa) protein with eight hydrophobic putative membrane-spanning domains. nhlF expression in R. rhodochrous ATCC 12674 and Escherichia coli JM109 confers uptake of 57Co in their cells, but not of 63Ni. The expression of both nhlF and nhlBA in R. rhodochrous ATCC 12674 exhibited higher NHase activity than nhlBA expression. These findings together with the inhibitory effect by uncouplers (CCCP and SF6847) for the cobalt uptake suggest that NhlF mediates the cobalt transport into the cell energy-dependently finally to provide L-NHase.

Transcriptional regulation of the Rhodococcus rhodochrous J1 nitA gene encoding a nitrilase.

The 1.4-kb downstream region from a nitrilase gene (nitA) of an actinomycete Rhodococcus rhodochrous J1, which is industrially in use, was found to be required for the isovaleronitrile-dependent induction of nitrilase synthesis in experiments using a Rhodococcus-Escherichia coli shuttle vector pK4 in a Rhodococcus strain. Sequence analysis of the 1.4-kb region revealed the existence of an open reading frame (nitR) of 957 bp, which would encode a protein with a molecular mass of 35,100. Deletion of the central and 3'-terminal portion of nitR resulted in the complete loss of nitrilase activity, demonstrating that nitR codes for a transcriptional positive regulator in nitA expression. The deduced amino acid sequence of nitR showed similarity to a positive regulator family including XylS from Pseudomonas putida and AraC from E. coli. By Northern blot analysis, the 1.4-kb transcripts for nitA were detected in R. rhodochrous J1 cells cultured in the presence of isovaleronitrile, but not those cultured in the absence of isovaleronitrile. The transcriptional start site for nitA was mapped to a C residue located 26 bp upstream of its translational start site. Deletion analysis to define the nitA promoter region suggested the possible participation of an inverted repeat sequence, centered on base pair -52, in induction of nitA transcription.

Prevalence of Mycoplasma genitalium in men with gonococcal urethritis.

A significant association of Mycoplasma genitalium with non-gonococcal urethritis has been reported, but the prevalence of this mycoplasma in men with gonococcal urethritis has not been so well studied. In this study, we examined urethral swab specimens from 45 Japanese male patients with gonococcal urethritis for the presence of M. genitalium by using a polymerase chain reaction-based assay. We also sought Chlamydia trachomatis by an enzyme immunoassay (Chlamydiazyme). Of the 45 specimens, 2 (4.4%) were positive for the mycoplasma and 12 (26.7%) were positive for C. trachomatis. The findings suggest that M. genitalium may be a cause not only of non-gonococcal urethritis but also of postgonococcal urethritis.

Comparison among performances of a ligase chain reaction-based assay and two enzyme immunoassays in detecting Chlamydia trachomatis in urine specimens from men with nongonococcal urethritis.

We evaluated the performances of a ligase chain reaction (LCR)-based assay and two enzyme immunoassays (Chlamydiazyme and IDEIA) in the detection of Chlamydia trachomatis in urine specimens. We compared the results of testing urine specimens by these assays with those of urethral swab culture by examining samples from 131 men with nongonococcal urethritis. Discrepant results were analyzed by testing urethral swab specimens for C. trachomatis by a PCR-based assay. After the resolution of discrepant results, the sensitivity of urethral swab culture was 85.3%, whereas those of the LCR assay, Chlamydiazyme, and IDEIA with urine specimens were 94.1, 82.4, and 94.1%, respectively. The LCR assay and IDEIA were more sensitive than was urethral swab culture. In addition, the LCR assay, with a sensitivity equal to that of IDEIA, was more specific. Overall, the LCR assay proved to be superior to the enzyme immunoassays in detecting C. trachomatis in urine specimens. Testing urine specimens by LCR assay should be a helpful alternative method for diagnosing C. trachomatis urethral infection in men with nongonococcal urethritis.

A novel gene cluster including the Rhodococcus rhodochrous J1 nhlBA genes encoding a low molecular mass nitrile hydratase (L-NHase) induced by its reaction product.

The 3.5 kilobases (kb) of the 5'-upstream region from nhlBA encoding a cobalt-containing low molecular mass nitrile hydratase (L-NHase) from Rhodococcus rhodochrous J1 was found to be required for the amide-dependent expression of nhlBA in experiments using a Rhodococcus transformation system. Sequence analysis of the 3.5-kb fragment revealed the presence of two open reading frames (nhlD and nhlC) in this fragment. NhlD has similarity to regulators MerR, CadC, and ArsR. NhlC has similarity to the regulators AmiC, for the expression of an aliphatic amidase from Pseudomonas aeruginosa, and NhhC, for the expression of a high molecular mass nitrile hydratase from R. rhodochrous J1. Assays of NHase activity of transformants carrying nhlD deletion or nhlC deletion mutations suggest a negative regulatory role for nhlD and a positive regulatory role for nhlC in the process of the L-NHase formation. Assays of NHase and amidase activities and Western blot analyses of each Rhodococcus transformant carrying various deletion plasmids, have shown that nhlBA and amdA encoding an amidase, which is located 1.9 kb downstream of nhlBA, were regulated in the same manner. These findings present the genetic evidence for a novel gene cluster controlling the expression of L-NHase, which is induced by the reaction product (amide) in the "practical microorganism" R. rhodochrous J1.

Characterization of the gene cluster of high-molecular-mass nitrile hydratase (H-NHase) induced by its reaction product in Rhodococcus rhodochrous J1.

The 4.6-kb region 5'-upstream from the gene encoding a cobalt-containing and amide-induced high molecular mass-nitrile hydratase (H-NHase) from Rhodococcus rhodochrous J1 was found to be required for the expression of the H-NHase gene with a host-vector system in a Rhodococcus strain. Sequence analysis has revealed that there are at least five open reading frames (H-ORF1 approximately 5) in addition to H-NHase alpha- and beta-subunit genes. Deletion of H-ORF1 and H-ORF2 resulted in decrease of NHase activity, suggesting a positive regulatory role of both ORFs in the expression of the H-NHase gene. H-ORF1 showed significant similarity to a regulatory protein, AmiC, which is involved in regulation of amidase expression by binding an inducer amide in Pseudomonas aeruginosa. H-ORF4, which has been found to be uninvolved in regulation of H-NHase expression by enzyme assay for its deletion transformant and Northern blot analysis for R. rhodochrous J1, showed high similarity to transposases from insertion sequences of several bacteria. Determination of H-NHase activity and H-NHase mRNA levels in R. rhodochrous J1 has indicated that the expression of the H-NHase gene is regulated by an amide at the transcriptional level. These findings suggest the participation of H-ORF4 (IS1164) in the organization of the H-NHase gene cluster and the involvement of H-ORF1 in unusual induction mechanism, in which H-NHase is formed by amides (the products in the NHase reaction), but not by nitriles (the substrates).

DNA gyrase mutations in quinolone-resistant clinical isolates of Neisseria gonorrhoeae.

Eight quinolone-resistant clinical isolates of Neisseria gonorrhoeae were shown to carry mutations in their GyrA proteins. Six isolates had a single amino acid change of serine to phenylalanine at the position corresponding to Ser-83 in Escherichia coli. In addition to the change of serine to phenylalanine, two isolates had another change of aspartic acid to asparagine at the position corresponding to Asp-87 in E. coli.

[Detection of Mycoplasma genitalium from male patients with non-gonococcal urethritis by polymerase chain reaction].

Mycoplasma genitalium causes urethritis in non-human primates, but studies on its pathogenicity in man have been hampered by the difficulty in isolating this oragnism in culture. We have used a specific polymerase chain reaction to examine the role of M. genitalium in non-gonococcal urethritis (NGU). Oligonucleotide primers were used to amplify a 281 bp of 140-KDa adhesin gene of M. genitalium. A characteristic PCR product was amplified, when M. genitalium DNA was template for the PCR. No amplified product was detected in Mycoplasma pneumoniae DNA, Mycoplasma hominis DNA or other bacterial DNAs. M. genitalium DNA was detected in urethral swabs from 17 (14.9%) of 114 men with NGU. Three (9.1%) of the 33 men with Chlamydia-positive NGU and 14 (17.3%) of the 81 with Chlamydia-negative NGU were positive for M. genitalium DNA, but 29 men without urethritis were negative. The prevalence of M. genitalium in NGU and in Chlamydia-negative NGU was significantly higher than that in the normal control. These findings suggest that M. genitalium would be a cause of NGU.

[Clinical investigation of clean intermittent catheterization].

We evaluated 120 patients with neurogenic bladder treated by clean intermittent self catheterization (CIC) in our department. These cases were divided into 2 groups: early treatment cases in which CIC started within 1 year after onset of dysuria, and late treatment cases in which CIC started after more than 1 year. Urinary tract infections (UTI) were recognized in 35% of the early treatment cases and 80% of the late treatment cases in the subsequent period. Pyelonephritis was experienced in 4% of the early treatment cases and 12% of the late treatment cases. Antibiotics therapy was considered unnecessary for asymptomatic UTIs. After CIC treatment, hydronephrosis detected by intravenous pyelography (IVP) and ultrasonography was improved in 18 of the 20 cases, and no cases showed deteriorated renal function. In the cases with neurogenic bladder after radical operations of the uterus or rectum, 45% of the early treatment cases have become free from CIC within 3 months postoperatively, and 84% eventually became free. Most of the late treatment cases have been continuing CIC. We considered that CIC was unnecessary when the residual urine was less than 100ml based on the periodical urinalysis and observation of renal function.

[Infection in genitourinary cancer patients during cancer chemotherapy].

To examine the frequency and the type of infection during cancer chemotherapy in the urological field, we studied the causes of fever in 67 patients with genitourinary cancer. Twenty-six patients had developed a fever of higher than 38 degrees C. Although fever without proven infection was seen in 11 patients (42.3%), fever caused by pyelonephritis was the most common infection. There was a relationship between fever and the presence of hydronephrosis. Fever was observed more often in patients with a leucocyte count of less than 2,000 white blood cell/mm3. In conclusion, we recommend the interruption of cancer chemotherapy or the use of granulocyte colony-stimulating factor for the prevention of infection, when the leukocyte count is less than 2,000 cell/mm3, especially in patients with hydronephrosis.

Detection of micrometastatic prostate cancer cells in lymph nodes by reverse transcriptase-polymerase chain reaction.

We have developed a highly sensitive method for detecting prostate cancer cells using reverse transcriptase-polymerase chain reaction (RT-PCR) with primers specific for prostate-specific antigen gene. Forty-four lymph nodes obtained from 22 patients with prostate cancers were analyzed by RT-PCR to detect metastatic prostate cancer cells. RT-PCR could detect prostate-specific antigen mRNA in five lymph nodes with histologically and/or immunohistochemically identifiable metastases and in four lymph nodes with negative histological and immunohistochemical analyses for metastases. RT-PCR was a more sensitive method than histology and immunohistochemistry in detecting metastatic prostate cancer cells and could be applied for diagnosing micrometastases of prostate cancer to lymph nodes. This highly sensitive RT-PCR will be a relevant tool to allow a more accurate clinical assessment of lymph node metastases of prostate cancer and to understand lymphatic dissemination of prostate cancer biologically.

Amidase coupled with low-molecular-mass nitrile hydratase from Rhodococcus rhodochrous J1. Sequencing and expression of the gene and purification and characterization of the gene product.

The cloned 9.4-kb insert of plasmid pNHJ20L containing low-molecular-mass nitrile hydratase (L-NHase) gene from Rhodococcus rhodochrous J1 [Kobayashi, M. et al. (1991) Biochim. Biophys. Acta 1129, 23-33] was digested with various restriction enzymes, and the trimmed fragments were inserted into pUC18 or pUC19. A 1.96-kb EcoRI-SphI region located 1.9-kb downstream of the L-NHase gene was found to be essential for the expression of amidase activity in Escherichia coli; the gene arrangement of the amidase and the NHase in R. rhodochrous J1 differed from those in Rhodococcus species including N-774 and Pseudomonas chlororaphis B23. The nucleotide-determined sequence indicated that the amidase consists of 515 amino acids (54626 Da) and the deduced amino acid sequence of the amidase had high similarity to those of amidases from Rhodococcus species including N-774 and P. chlororaphis B23 and to indole-3-acetamide hydrolase from Pseudomonas savastanoi. The amidase gene modified in the nucleotide sequence upstream from its start codon expressed 8% of the total soluble protein in E. coli under the control of lac promoter. The level of amidase activity in cell-free extracts of E. coli was 0.468 unit/mg using benzamide as a substrate. This amidase was purified to homogeneity from extracts of the E. coli transformant with 30.4% overall recovery. The molecular mass of the enzyme estimated by HPLC was about 110 kDa and the enzyme consists of two subunits identical in molecular mass (55 kDa). The enzyme acted upon aliphatic amides such as propionamide and also upon aromatic amides such as benzamide. The apparent Km values for propionamide and benzamide were 0.48 mM and 0.15 mM, respectively. This amidase was highly specific for the S-enantiomer of 2-phenylpropionamide, but could not recognize the configuration of 2-chloropropionamide. It also catalyzed the transfer of an acyl group from an amide to hydroxylamine to produce the corresponding hydroxamate.

[A study of methicillin-resistant Staphylococcus aureus (MRSA) infection in urological field].

Out of 110 strains of Staphylococcus aureus isolated from 1985 to 1990, isolation rate of methicillin-resistant S. aureus (MRSA) was investigated. Nineteen strains of 59 S. aureus from outpatients and 20 strains of 51 S. aureus from inpatients were determined as MRSA. Isolation frequency of MRSA from inpatients was increasing in the recent two years. Coagulase type, enterotoxin type and production of toxic shock syndrome toxin-1 (TSST-1) were examined in 22 strains of MRSA. Coagulase type II (86%), enterotoxin type C (68%) and TSST-1 positive strain was most dominant. Susceptibility of MRSA to 4 antimicrobial agents were measured, MRSA were sensitive to vancomycin (VCM), arbekacin (ABK) and minocycline, but resistant to flomoxef. Thirty-four patients from whom MRSA was isolated including 20 patients from urine, 13 from pus and 1 from blood, were analyzed clinically. Pyuria was not recognized in some cases in whom MRSA was isolated from their urine. Concomitant polymicrobial infection was frequently noted in those patients with MRSA in their urinary tract. These facts show that the pathogenic role of MRSA in the urinary tract infection was not significant. On the other hand, when MRSA was isolated from pus or blood, serious infections could be caused by MRSA, especially in compromised host. Regarding the treatment in these cases, administration of VCM or ABK was though to be necessary.