The impact of lesion length and vessel size on outcomes after sirolimus-eluting stent implantation for in-stent restenosis.

OBJECTIVES: We evaluated the predictors of recurrent restenosis and the impact of lesion length and vessel size on outcomes in patients treated with routine sirolimus-eluting stent (SES) implantation for in-stent restenosis (ISR) of bare-metal stent (BMS). METHODS: In this study, 250 consecutive patients with 275 lesions after SES implantation for ISR of BMS were enrolled. Follow-up angiogram was obtained in 239 patients with 258 lesions eight months after implantation (follow-up rate: 95.6%). We compared characteristics of patients and lesions between the two groups (the recurrent restenosis group and the no-restenosis group). RESULTS: Recurrent restenosis was angiographically documented in 43 lesions (16.7%). Recurrent restenosis was found in 30.4% with small vessel lesions (reference diameter of less than 2.5 mm, 92 lesions) and 23% with the diffuse type lesions (106 lesions). Seventy-two per cent of patients had a focal pattern of recurrent restenosis. Previously recurrent ISR lesions (odds ratio (OR) 1.94, 95% confidence interval (CI) 0.94 to 4.06, p = 0.05), reference diameter of less than 2.5 mm (OR 2.41, CI 1.05 to 5.41, p = 0.03), diffuse type restenosis (OR 4.48, CI 2.12 to 9.94, p = 0.0001) and dialysis patients (OR 4.72, CI 1.42 to 15.7, p = 0.01) were independent predictors of recurrent restenosis. CONCLUSIONS: Small vessels, diffuse type restenosis and dialysis patients were still the predictors of recurrent restenosis in patients treated with SES for ISR of BMS.

Molecular behavior in living mitotic cells of human centromere heterochromatin protein HPLalpha ectopically expressed as a fusion to red fluorescent protein.

We constructed stable mammalian cell lines in which human heterochromatin protein HP1alpha and kinetochore protein CENP-A were differentially expressed as fusions to red (RFP-HP1) and green fluorescent proteins (GFP-CENP-A). Heterochromatin localization of RFP-HP1 was clearly shown in mouse and Indian muntjac cells. By preparing mitotic chromosome spreads, the inner centromere localization of RFP-HP1 was observed in human and Indian muntjac cells. To characterize its molecular behavior in living mitotic cells, time-lapse images of RFP-HP1 were obtained by computer-assisted image analyzing system, mainly with mouse cells. In G2 phase, a significant portion of RFP-HP1 diffused homogeneously in the nucleus and further dispersed into the cytoplasm soon after the nuclear membrane breakdown, while some remained in the centromeric region. Simultaneous observations with GFP-CENP-A in human cells showed that RFP-HP1 was located just between the sister kinetochores and then aligned to the spindle midzone. With the onset of anaphase, once it was released from there, it moved to the centromeres of segregating chromosomes or returned to the spindle equator. As cytokinesis proceeded, HP1alpha was predominantly found in the newly formed daughter nuclei and again displayed a heterochromatin-like distribution. These results suggested that, although the majority of HP1alpha diffuses into the cytoplasm, some populations are retained in the centromeric region and involved in the association and segregation of sister kinetochores during mitosis.

The characteristics of mediastinal neuroblastoma.

The prognosis of mediastinal neuroblastoma has been reported to be better than for other neuroblastomas. The reason for this is however not clear. Furthermore, a comparison between mediastinal neuroblastoma and the other neuroblastomas has been rarely reported so far. In this study, the characteristics of mediastinal neuroblastoma (84 cases) are investigated and compared with those of other neuroblastomas (440 cases). Regarding clinical factors, the age distribution and the rate of cases detected at mass screening were similar in both groups. According to Evan's staging system, the rates of early stage (I, II) were 62% in the mediastinal neuroblastoma and 38% in the other neuroblastomas (p<0.001). Regarding the biological prognostic factors, a favorable histology based on Shimada's classification was found in 100% (35/35) of the mediastinal neuroblastoma cases and in 85% (112/132) of the other neuroblastoma cases (p<0.05). With regard to N-myc amplification, all of the examined 42 cases in mediastinal neuroblastoma had a N-copy number of less than 10 copies, while 32 of the examined 263 cases (12%) in the other neuroblastomas had an amplification of N-myc of more than 10 copies (p<0.05). The 5-year survival rates were 78% in the mediastinal neuroblastoma and 59% in the other neuroblastomas, respectively. Of the cases who underwent an incomplete resection of primary tumors in localized neuroblastoma, the 5-year survival rate of the mediastinal neuroblastoma cases was significantly more favorable than that of the other neuroblastomas. The majority of mediastinal neuroblastoma cases showed an early stage and favorable prognostic factors. It is likely that the clinical and biological prognostic factors of the tumor are therefore more closely correlated with the outcome of mediastinal neuroblastoma rather than the degree of the surgical resection. Regarding the treatment for mediastinal neuroblastoma, it is most important to evaluate the biology of the tumor after surgical resection.

New type of febrifugine analogues, bearing a quinolizidine moiety, show potent antimalarial activity against Plasmodium malaria parasite.

Febrifugine (1) and isofebrifugine (2), isolated from the roots of Dichroa febrifuga Lour. (Chinese name: Cháng Shan), are active principles against malaria. Adducts of 1 and 2 with acetone, Df-1 (3) and Df-2 (4), respectively, were obtained using silica gel and acetone. They showed high activity against P. falciparum malaria in vitro. Compound 3 was found to be equally effective against P. berghei in vivo as the clinically used drug chloroquine, whereas 4 showed only 1/24 of the activity of 3. Metabolism studies of these compounds revealed that compound 4 is readily metabolized in mouse liver. Accordingly, the dose of 4 must be higher than that of 3 to attain blood levels sufficient for a favorable therapeutic effect.

Treatment of standard-risk acute lymphoblastic leukemia in children: the results of protocol AL841 from the Kyushu-Yamaguchi Children's Cancer Study Group in Japan.

A total of 62 patients with standard-risk acute lymphoblastic leukemia received three-drug induction consisting of vincristine, prednisolone, and L-asparaginase (l-Asp) followed by consolidation therapy with intermediate-dose methotrexate (MTX), intrathecal MTX, and 18 Gy of cranial irradiation. Maintenance therapy consisting of 6 drugs including daunorubicin (DNR, 450 mg/m2 in total) was continued for 3 years. Patients were randomized and half of them received weekly l-Asp during maintenance therapy as a late intensification. Complete remission (CR) was achieved in 61/62 (98.4%), and 11 of 61 patients relapsed. At 10 years, the event-free survival (EFS) was 80.6 +/- 5.0% and overall survival was 88.7 +/- 4.0%; median follow-up time was 9.3 years. The 10-year EFS of patients with additional l-Asp (84.8 +/- 6.2%) was superior to that without l-Asp (75.9 +/- 7.9%), although it was not statistically significant. No patients who received a full dose of DNR and maintained CR developed heart failure, although the shortening fraction decreased from 41.0% at diagnosis to 35.2% (median). The protocol AL841 provided good long-term disease control without severe late cardiac dysfunction.

Mass screening for neuroblastoma at 6 months of age: difficult to justify.

BACKGROUND/PURPOSE: A statistical analysis of the mass screening for neuroblastoma in Japan based on a population study rarely has been reported. This study aims to evaluate retrospectively the effectiveness of mass screening at 6 months of age using the available population data. METHODS: The data on the neuroblastoma cases registered by the Committee for Pediatric Solid Malignant Tumors in the Kyushu area were analyzed based on both screened and unscreened populations in the Kyushu area. RESULTS: From 1988 to 1992, the cumulative incidence of neuroblastoma in children less than 5 years of age was 82 in 484,599 for screened children, and 11 in 92,966 for unscreened children, respectively. Fourteen of the 82 screened patients had negative findings at 6 months of age (MS-negative cases). No significant difference was observed in the cumulative mortality rates from neuroblastoma in children younger than 5 years of age between the screened children and the unscreened children. Six of seven patients who died among the screened children were MS-negative cases with stage III or IV disease. In addition, no significant difference was found in the cumulative mortality rates from the neuroblastoma cases in patients less than 5 years of age between the children screened from 1988 to 1992 (7 of 484,599) and all children from 1980 to 1984 (14 of 668,084). CONCLUSIONS: These findings suggests that the majority of the patients detected by mass screening had a favorable prognosis, and, mass screening in Japan for children less than 6 months of age was not observed to reduce the incidence and mortality from neuroblastoma. Therefore, mass screening at 6 months of age was not found to improve substantially the prognosis of patients with unfavorable neuroblastoma identified over 1 year of age, which is the primary purpose of such mass screening for neuroblastoma.

Donor leukocyte infusion after allogeneic bone marrow transplantation was not effective for relapsed rhabdomyosarcoma.

A 10-year-old boy with metastatic rhabdomyosarcoma had an HLA-identical sibling and received an allogeneic BMT. Recurrence was detected in the BM as the only site of treatment failure 12 months after BMT. Donor leukocyte infusion (DLI) was chosen as salvage therapy. Although sufficient cells (a total of 29.7 x 10(7)/kg) were infused, no signs of acute GVHD nor BM aplasia occurred and the patient died of disease progression 9 months after DLI.

Therapeutic trial of intensified conditioning regimen with high-dose cytosine arabinoside, cyclophosphamide and either total body irradiation or busulfan followed by allogeneic bone marrow transplantation for myelodysplastic syndrome in children.

Ten children with myelodysplastic syndrome underwent an allogeneic bone marrow transplantation (BMT) with an intensified conditioning regimen. The median age of the patients was 8 years (range 2-10), and included 6 males and 4 females. The subtype of the disease was refractory anemia (RA) in 4, RA with excess blasts (RAEB) in 4, RAEB in transformation (RAEB-T) in 1, and juvenile chronic myelogenous leukemia (JCML) in 1. All patients were conditioned with high-dose cytosine arabinoside (12000 mg/m2), cyclophosphamide (120 mg/kg) and either total body irradiation (10-13.2 Gy) or busulfan (16 mg/kg or 560 mg/m2). Cyclosporine A and/or methotrexate were used for the prophylaxis of graft-versus-host disease (GVHD). Engraftment was prompt in all but one patient. Severe acute GVHD (grade 3) (n = 1), interstitial pneumonitis (n = 1) and veno-occlusive disease of the liver (n = 1) occurred. The disease relapsed in one patient with RAEB-T. Seven of the 10 patients were alive and disease free 2-74 months after BMT. The disease-free survival rate at 4 years was 69 +/- 15%. All surviving patients were in the full performance status. The examined children with MDS tolerated this intensified conditioning regimen well.

Origin of the pulmonary venous orifice in the mouse and its relation to the morphogenesis of the sinus venosus, extracardiac mesenchyme (spina vestibuli), and atrium.

BACKGROUND: Human embryology textbooks indicate that the trunks of the pulmonary vein and artery originate from the left atrium and aortic sac, respectively, based on histological analyses of limited human specimens. However, our studies show that the pulmonary venous trunk in the mouse as in other nonhuman vertebrates originates from a vascular "sac" at the venous pole, the sinus venosus. METHODS: Mouse embryos of 9-11 days gestation were obtained and staged according to Theiler's criteria and fixed in Carnoy's solution. Samples were embedded in paraffin and serial sections were prepared. RESULTS: Histological analysis showed that at day 9.5 the pulmonary venous rudiment was initially observed along the left margin in the extracardiac mesenchyme that separated the venous pole of the heart from the lung buds. The endothelium of the pulmonary vein was continuous, with a vascular sac we identified as sinus venosus based on its location immediately posterior to the left sinoatrial fold. The sinus venosus became incorporated into the left atrium (days 10-10.5) to form part of the posterior atrial wall. Similarly, the pulmonary vein and associated extracardiac mesenchyme were "drawn" into the atrium. This extracardiac mesenchyme of the venous pole, also called "spina vestibuli" and containing the pulmonary vein at its left margin, formed a wedge-shaped invagination within the atrium that contributed nonmuscular tissue to the primary atrial septum. CONCLUSIONS: We propose that the orifice of the pulmonary vein establishes a link with the left side of the atrium as a consequence of a venous sac, the sinus venosus, and its associated mesenchyme (in which the root of the pulmonary vein is embedded) being incorporated into the atrium.

Mass screening for neuroblastoma: quo vadis? A 9-year experience from the Pediatric Oncology Study Group of the Kyushu area in Japan.

Since 1985, a nationwide program of mass screening for neuroblastoma has been available for 6-month-old infants throughout Japan. From 1985 to 1993, the authors studied 285 patients with neuroblastoma among their regional population of 15 million. There was an increase in the total number of patients per year in comparison to the previous 6-year period (1979 to 1984). However, no significant difference was noted in the number of patients older than 1 year or in the incidence of advanced-stage (stages III and IV) unscreened cases. The majority of neuroblastomas in the screened group showed favorable biological factors, even in the advanced stages. However, there was a small group with histologically and/or biologically unfavorable factors; five of 115 had amplified N-myc oncogene, four of 74 showed unfavorable Shimada histological findings, and three of 33 had an unfavorable DNA ploidy pattern. One case from this group with unfavorable factors died of the tumor. 3) Thirty-eight cases were negative at the time of mass screening, but later presented with neuroblastoma. Most of them were diagnosed between 1 and 3 years of age, and 30 of the 38 cases (78.9%) were advanced stage with unfavorable prognostic factors. Thus, the authors conclude that mass screening at 6 months can detect a selected population of infants with neuroblastoma; some of the tumors may represent subclinical masses destined for spontaneous regression. However, some tumors with unfavorable factors have been detected by mass screening before progression and/or dissemination. Infants in this group are considered to benefit most from early diagnosis and treatment.

An adolescent case of retroperitoneal pure choriocarcinoma: successful treatment with MCNU-containing high-dose chemotherapy followed by autologous bone marrow transplantation after multiple brain metastases.

Choriocarcinoma is a rare disease in pediatric neoplasms. The prognosis of the disease is extremely poor once when patients relapse or become refractory to cisplatin (CDDP). A 17-year-old male who had retroperitoneal pure choriocarcinoma of advanced stage was treated with CDDP-based intensive chemotherapy. In spite of the initial good response to CDDP-based intensive chemotherapy, the tumor metastasized to multiple areas of the brain during chemotherapy. Since the brain in this case was thought to be a sanctuary, after radiotherapy to the whole cranium, the patient was treated with high-dose chemotherapy consisting of etoposide, carboplatin, and ranimustine (MCNU), which can penetrate the blood-brain barrier, followed by autologous bone marrow transplantation (ABMT). Twenty-four months after ABMT, the patient had no sign of disease recurrence. MCNU-containing high-dose chemotherapy with ABMT appears to be quite effective in cases that present with relapsing multiple brain metastases during CDDP-based chemotherapy.

Semantide- and chemotaxonomy-based analyses of some problematic phenotypic clusters of slowly growing mycobacteria, a cooperative study of the International Working Group on Mycobacterial Taxonomy.

During previous cooperative numerical taxonomic studies of slowly growing mycobacteria, the International Working Group on Mycobacterial Taxonomy described a number of strains whose taxonomic status was ambiguous. A new study of DNA, RNA, and proteins from 66 of these organisms was performed to correlate their properties with phenotypic clustering behavior; the results of this study permitted 51 of the strains studied to be assigned to known species. The methods used to characterize the semantides included nucleotide sequencing and assessment of levels of semantide relatedness by affinity binding techniques, including whole DNA-DNA hybridization, probe hybridization, and antibody binding. There was good overall agreement between the phenotypic and chemotaxonomic clusters and the groups of organisms identified by semantide analyses. Our results supported the conclusion that we should continue to rely on polyphasic taxonomy to provide satisfactory systematic resolution of members of the genus Mycobacterium. We identified no single 16S rRNA interstrain nucleotide sequence difference value that unequivocally defined species boundaries. DNA-DNA hybridization remains the gold standard, but common resources are needed to permit DNA-DNA hybridization analyses to be made available to laboratories that are not prepared to use this technology. One of the large novel clusters which we studied corresponds to the recently described species Mycobacterium interjectum, a pathogen that resembles the nonpathogen Mycobacterium gordonae phenotypically. We also identified strains that appear to represent ribovars of Mycobacterium intracellulare which do not react with the commercial diagnostic probes that are currently used for identification of this species. Other branches or clusters consisted of too few strains to permit a decision about their taxonomic status to be made.

Protective immune responses induced by secretion of a chimeric soluble protein from a recombinant Mycobacterium bovis bacillus Calmette-Guérin vector candidate vaccine for human immunodeficiency virus type 1 in small animals.

A recombinant Mycobacterium bovis bacillus Calmette-Guérin (BCG) vector-based vaccine that secretes the V3 principal neutralizing epitope of human immunodeficiency virus (HIV) could induce immune response to the epitope and prevent the viral infection. By using the Japanese consensus sequence of HIV-1, we successfully constructed chimeric protein secretion vectors by selecting an appropriate insertion site of a carrier protein and established the principal neutralizing determinant (PND)-peptide secretion system in BCG. The recombinant BCG (rBCG)-inoculated guinea pigs were initially screened by delayed-type hypersensitivity (DTH) skin reactions to the PND peptide, followed by passive transfer of the DTH by the systemic route. Further, immunization of mice with the rBCG resulted in induction of cytotoxic T lymphocytes. The guinea pig immune antisera showed elevated titers to the PND peptide and neutralized HIVMN, and administration of serum IgG from the vaccinated guinea pigs was effective in completely blocking the HIV infection in thymus/liver transplanted severe combined immunodeficiency (SCID)/hu or SCID/PBL mice. In addition, the immune serum IgG was shown to neutralize primary field isolates of HIV that match the neutralizing sequence motif by a peripheral blood mononuclear cell-based virus neutralization assay. The data support the idea that the antigen-secreting rBCG system can be used as a tool for development of HIV vaccines.

Secretion of MPB64 antigen by a recombinant clone of Mycobacterium smegmatis: characterization and application for the diagnosis of tuberculosis.

MPB64, a specific antigen to Mycobacterium tuberculosis complex (TB complex), was produced and secreted by a clone of M. smegmatis-MPB64 where the structural gene of MPB64 was inserted using a new mycobacteria, E. coli shuttle plasmid pNIS vector. Antibodies against the recombinant MPB64 (rMPB64) were used for the reverse particle latex agglutination (RPLA) test to detect the MPB64 antigen rapidly. RPLA tests were applied to the shock cultures and the clinical isolates of mycobacteria to identify TB complex. RPLA with anti-MPB64 antibody-coated latex beads completely distinguished TB complex from other mycobacteria. Thus, it is suggested that RPLA with anti-MPB64 antibody would be a new, easy and inexpensive method for the rapid diagnosis of tuberculosis.

Herpes zoster in children with bone marrow transplantation: Report from a single institution.

Herpes zoster (HZ) has been often observed after bone marrow transplantation (BMT) in childhood. The occurrence of HZ was reviewed in children who received BMT. The clinical features of HZ were reviewed in 44 children who underwent BMT at Kyushu Cancer Center. Among the 35 recipients with a history of varicella before BMT, several factors associated with BMT and the lymphocyte subsets were compared between the patients who developed HZ (HZ+ group) and those who did not (HZ- group). Twenty-two recipients (50%) developed HZ; in two-thirds of these cases (15/22: 68%), HZ occurred between 80 and 120 days after BMT (median 101 days). The recipients treated with busulfan had a higher occurrence of HZ than those treated without it. The patients with Grade II-IV acute graft-versus-host disease (GVHD) developed HZ more frequently. In the HZ+ group, the absolute number of lymphocytes, CD3+, CD4+ or CD8+ cells at 3 months was significantly lower than that observed at 12 months after BMT and the CD4/CD8 ratio was significantly lower at 1 month than after 3 months of BMT. In conclusion, recipients were susceptible to HZ at around 100 days after BMT. The development of HZ may be associated with unbalanced T lymphocytes at that time.

[Production of tumor necrosis factor alpha and interleukin-6 by peripheral monocytes from patients with atypical mycobacteriosis--relationship to clinical activity].

The production of tumor necrosis factor alpha (TNF alpha) and of interleukin-6 (IL-6) by peripheral blood monocytes (PBMs) from patients infected with Mycobacterium avium intracellular complex (MAC) were assessed. Spontaneous release of both TNF alpha and IL-6 were greater during the active stage than during the inactive stage and in healthy controls. When the cells were stimulated with MAC-derived purified protein derivative B (PPD-B). TNF alpha production by PBMs in the active stage increased and IL-6 production by cells in both the active and inactive stages decreased. Moreover, the in vitro increase in TNF alpha production after stimulation in the active stage seemed to be related to the persistent MAC infection, which resulted in an exhaustion of nutrition. These results suggest that the ability of PBMs to produce TNF alpha and IL-6 in vitro is closely related to the clinical stage of MAC infection.

An effective salvage regimen with aclarubicin for daunorubicin-resistant acute non-lymphocytic leukemia in children.

We evaluated the efficacy and toxicity of aclarubicin for acute non-lymphocytic leukemia (ANLL) refractory to daunorubicin in childhood. Twenty-four patients were treated with aclarubicin and prednisolone with or without 6-mercaptopurine and behenoyl-cytosine arabinoside daily for 5 to 14 days. Of 21 evaluable patients, 14 (67%) responded: 12 obtained complete remission and 2 partial remission. The median time to reach complete remission was 37 days (range, 16 to 60 days), and the median duration of complete remission was 5.5 months (range, 2 to 41 months). The cumulative dose of anthracycline administered before the study was not considered significant for the response. The only major complication was severe bone marrow suppression; infectious episodes occurred in 14 patients (58%) and three died of sepsis and/or bleeding. The observed non-hematologic toxicities included hematuria, an elevation of serum amylase, nausea/vomiting, and angitis. In addition, one patient showed abnormal cardiac function. Aclarubicin is therefore considered a highly active drug for remission reinduction of previously treated children suffering from ANLL with an acceptable toxicity.

Neuroblastoma in infants aged less than 6 months: is more aggressive treatment necessary? A report from the Pediatric Oncology Study Group of the Kyushu area.

Infants with neuroblastoma are known to have a better prognosis than older children. In Japan in 1985, mass screening for neuroblastoma in infants aged 6 months was introduced. With this policy, there has been an increase in the number of patients seen with neuroblastoma between 6 and 11 months of age. In a previous report the authors described the management and prognosis of infants with disease detected by mass screening, but there is still little information regarding the strategies of management for infants with neuroblastoma aged less than 6 months. The authors analyzed the data regarding 27 patients aged less than 6 months registered in their region (population 15 million) from 1985 to 1992, and compared it with that of the previous 8-year period. In the younger age group, there was a significantly higher rate of advanced disease stages (III and IV). In spite of the variation in treatment related to the choice of individual institutions, infants with stages I, II, and III disease had a good outcome, suggesting that aggressive chemotherapy is not necessary unless poor prognostic factors are present. One patient with stage IV disease died of disseminated disease and one with stage IVs and 22 copies of N-myc oncogene also died of tumor relapse in spite of aggressive chemotherapy. It is therefore concluded that the prognosis in infants with stage IV and IVs neuroblastoma under the age of 6 months is not as good as had previously been believed, and that such patients, therefore, require special consideration.

[Sensitivity and specificity for detection of islet cell cytoplasmic antibodies using rat pancreatic sections].

We determined islet cell cytoplasmic antibodies (ICA) using rat pancreatic sections as a test substrate substitutive for human pancreatic sections by indirect immunofluorescent technique. ICA were measured in sera from 58 patients with insulin-dependent diabetes mellitus (IDDM), 456 with non-insulin-dependent diabetes mellitus (NIDDM), 50 patients with autoimmune diseases, and 110 healthy controls. Seventeen of 58 patients with IDDM showed recent-onset (within 3 months). ICA were also measured in some samples using blood group O human pancreatic sections, and the ICA titers were compared with those measured using rat pancreatic sections. The prevalence of ICA was 55.2% (32/58) in patients with IDDM, 1.5% (7/456) in those with NIDDM, 0% (0/50) in those with autoimmune diseases, and 0.9% (1/110) in the healthy controls. Of the 17 recent-onset IDDM ICA were positive in 14 (82.3%). In comparative study of titers for ICA using rat pancreatic sections or human pancreatic sections, rat pancreatic sections yielded ICA titers as high as human pancreatic sections did. These results demonstrate that ICA assay using rat pancreatic sections was disease-specific, and that antigenicity of the substrate was favorable to ICA. Rat pancreas presents the advantage of greater availability, while providing an identical substrate for ICA. In conclusion, rat pancreatic sections are useful substrate for detecting ICA.

Mutations in rpoB gene of rifampicin resistant clinical isolates of Mycobacterium tuberculosis in Japan.

In Mycobacterium tuberculosis, involvement of alterations of the RNA polymerase beta subunit in resistance to rifampicin has been described by Telenti et al. To determine if the same correlation could be observed between the mutation of the rpoB gene and clinically isolated M. tuberculosis of the rifampicin-resistant phenotype in Japan, 47 strains of M. tuberculosis of the rifampicin-resistant phenotype, 17 of the rifampicin-susceptible phenotype, and 4 type strains were examined. A 411-base pair (bp) rpoB fragment was amplified by the polymerase chain reaction and subjected to solid phase direct sequencing. By comparing the nucleotides, mutation involving 8 conserved amino acids were identified in 44 of the 47 (93.6%) rifampicin-resistant isolates, but in none of the 17 sensitive isolates and 4 type strains. All mutations found were clustered within a region of 23 amino acids. Thus, similar to the results reported by Telenti et al., substitution of a limited number of highly conserved amino acids encoded by the rpoB gene appears to be the molecular mechanism responsible for resistance to rifampicin in Japanese clinical isolates of M. tuberculosis. Our results suggest that direct DNA sequencing of the rpoB gene may be a reliable method for identifying rifampicin-resistant M. tuberculosis strains among Japanese clinical isolates.