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Biomedical subjects

K Kawakami

Publications and source records attributed to K Kawakami.

At least 91 records · Page 5Linked to original sources
PubMed2000

MethyLight: a high-throughput assay to measure DNA methylation.

Cytosine-5 DNA methylation occurs in the context of CpG dinucleotides in vertebrates. Aberrant methylation of CpG islands in human tumors has been shown to cause transcriptional silencing of tumor-suppressor genes. Most methods used to analyze cytosine-5 methylation patterns require cumbersome manual techniques that employ gel electrophoresis, restriction enzyme digestion, radiolabeled dNTPs or hybridization probes. The development of high-throughput technology for the analysis of DNA methylation would significantly expand our ability to derive molecular information from clinical specimens. This study describes a high-throughput quantitative methylation assay that utilizes fluorescence-based real-time PCR (TaqMan) technology that requires no further manipulations after the PCR step. MethyLight is a highly sensitive assay, capable of detecting methylated alleles in the presence of a 10,000-fold excess of unmethylated alleles. The assay is also highly quantitative and can very accurately determine the relative prevalence of a particular pattern of DNA methylation. We show that MethyLight can distinguish between mono-allelic and bi-allelic methylation of the MLH1 mismatch repair gene in human colorectal tumor specimens. The development of this technique should considerably enhance our ability to rapidly and accurately generate epigenetic profiles of tumor samples.

Adaptor Proteins, Signal Transducing↗
PubMed2000

IL-4, but not vitamin D(3), induces monoblastic cell line UG3 to differentiate into multinucleated giant cells on osteoclast lineage.

The formation of multinucleated giant cells (MGCs) from monocytes/macrophages is controlled by various cytokines, the roles of which are not fully understood. Both interleukin (IL)-4 and 1alpha,25(OH)(2) vitamin D(3) (D(3)) are known to induce MGC formation from monocytes/macrophages. D(3) is also known as a stimulator of osteoclast formation in the presence of stroma cells, and IL-4 as an inhibitor. Previously, we showed that IL-4-induced MGCs from monocytes/macrophages expressed tartrate resistant acid phosphatase (TRAP) activity and hydroxyapatite-resorptive activity in the presence of M-CSF without stroma cells. In this study, we examined the effects of D(3) and/or IL-4 on MGC formation and the characteristics of these MGCs using a monoblastic cell line (UG3), to elucidate the involvement of these factors in osteoclast development without stroma cells. D(3)-induced MGCs showed none of the markers of osteoclasts, such as TRAP activity, calcitonin receptor (cal-R) expression, hydroxyapatite-resorptive activity, and bone-resorptive activity. A low concentration of D(3) synergistically stimulated IL-4-induced TRAP-positive MGC formation, whereas a high concentration of D(3) inhibited it. When IL-4 was added on day 7 of the 2-week culture with D(3), TRAP positivity reached maximum. On the other hand, delayed addition of D(3) on day 7 of culture did not increase the TRAP positivity. Although the fusion rate increased during the first week of the 2-week culture in the presence of D(3), it increased further in the second week following the addition of IL-4 on day 7. Furthermore, IL-4-induced, or IL-4- and D(3)-induced MGCs differentiated into functional osteoclasts with bone-resorptive activity following coculture with osteoblastic cells, whereas D(3)-induced MGCs did not acquire bone-resorptive activity even after coculture with osteoblastic cells in the presence of D(3). These findings suggest that IL-4 initiates osteoclast development of UG3 cells, although stroma cells were necessary for development of functional osteoclasts. On the other hand, D(3) had only a "supportive" effect on this differentiation. IL-4 and direct contact with stroma cells may regulate different stages in the multistep process of osteoclastogenesis of UG3 cells.

3T3 Cells↗
PubMed2000

Six family genes--structure and function as transcription factors and their roles in development.

The members of the Six gene family were identified as homologues of Drosophila sine oculis which is essential for compound-eye formation. The Six proteins are characterized by the Six domain and the Six-type homeodomain, both of which are essential for specific DNA binding and for cooperative interactions with Eya proteins. Mammals possess six Six genes which can be subdivided into three subclasses, and mutations of Six genes have been identified in human genetic disorders. Characterization of Six genes from various animal phyla revealed the antiquity of this gene family and roles of its members in several different developmental contexts. Some members retain conserved roles as components of the Pax-Six-Eya-Dach regulatory network, which may have been established in the common ancestor of all bilaterians as a toolbox controlling cell proliferation and cell movement during embryogenesis. Gene duplications and cis-regulatory changes may have provided a basis for diverse functions of Six genes in different animal lineages.

Amino Acid Sequence↗
PubMed2000

A malignant rhabdoid tumor of the kidney occurring concurrently with a brain tumor: report of a case.

Malignant rhabdoid tumor of the kidney (MRTK) is one of the most lethal neoplasms to occur in young infants. Cases of MRTK accompanying an embryonal tumor in the central nervous system have occasionally been described. We present herein an interesting case of MRTK that was clinically diagnosed preoperatively. A male infant aged 6 months with both a midline brain tumor and a renal neoplasm was transferred to our institution. Although roentgenographic evaluation suggested that the renal lesion was a Wilms' tumor, midkine (MK), a growth and differentiation factor characteristically present in the urine of patients with Wilms' tumor, was not detected. A preoperative diagnosis of MRTK was established based on the lack of urinary MK in addition to the typical clinical features of the young age and the concurrent brain tumor.

Brain Neoplasms↗
PubMed2000

Galloylglucoses and riccionidin A in Rhus javanica adventitious root cultures.

Adventitious root cultures of Rhus javanica L. produced large amounts of galloylglucoses (gallotannins) and an anthocyanidin, riccionidin A, formerly found only in liverworts. Production of both galloylglucoses and riccionidin A in the adventitious root culture system was suppressed by light. The Rhus root culture showed the highest productivity for those secondary metabolites in a modified Linsmaier-Skoog (LS) liquid medium containing 30 mM NH4+ and 30 mM NO3- as nitrogen sources in the presence of 10(-6) M 3-indoleacetic acid (IAA).

Anthocyanins↗
PubMed2000

Expression of three zebrafish Six4 genes in the cranial sensory placodes and the developing somites.

The homeobox gene Six4/AREC3 is a member of the vertebrate Six family of transcription factor genes. In this study we describe the cloning and expression of three zebrafish homologues of Six4/AREC3, six4.1, six4.2 and six4.3. These zebrafish Six4 proteins show high homology to the mammalian Six4/AREC3 proteins in the most C-terminal region in addition to the Six domain and homeodomain. Whole-mount in situ hybridization showed that six4.1 is expressed in the presumptive cranial placodal precursor cells, and later in the olfactory, otic and lateral line placodes. six4.2 is expressed in the presomitic mesoderm, somites and pectoral fin bud. six4.3 appears to be a unique member of the Six4 proteins and is expressed ubiquitously at gastrulation and later in the tectum.

Amino Acid Sequence↗
PubMed2000

Discovery of a muscarinic M3 receptor antagonist with high selectivity for M3 over M2 receptors among 2-[(1S,3S)-3-sulfonylaminocyclopentyl]phenylacetamide derivatives.

In the course of developing a metabolically stable M3 receptor antagonist from the prototype antagonist, J-104129 (1), introduction of certain substituents into the cyclopentane ring of 1 was found to be effective not only in improving metabolic stability but also in greatly enhancing the subtype selectivity. Among the cyclopentane analogues, sulfonamide derivatives (10f) and (10g) displayed 160- and 310-fold selectivity for M3 over M2 receptors, and both were significantly more selective than the prototype antagonist (120-fold). Subsequent derivatization of the sulfonamide series led to the highly selective M3 receptor antagonists (10h, 10i and 10j) with >490-fold selectivity for M3 over M2 receptors. Among them, p-nitrophenylsulfonamide (J-107320, 10h) exhibited 1100-fold selectivity for M3 receptors (Ki = 2.5 nM) over M2 receptors (Ki = 2800 nM) in the human muscarinic receptor binding assay using [3H]-NMS as a radio ligand.

Alkenes↗
PubMed2000

Just say no (to stereotyping): effects of training in the negation of stereotypic associations on stereotype activation.

The primary aim of the present research was to examine the effect of training in negating stereotype associations on stereotype activation. Across 3 studies, participants received practice in negating stereotypes related to skinhead and racial categories. The subsequent automatic activation of stereotypes was measured using either a primed Stroop task (Studies I and 2) or a person categorization task (Study 3). The results demonstrate that when receiving no training or training in a nontarget category stereotype, participants exhibited spontaneous stereotype activation. After receiving an extensive amount of training related to a specific category, however, participants demonstrated reduced stereotype activation. The results from the training task provide further evidence for the impact of practice on participants' proficiency in negating stereotypes.

Attitude↗
PubMed2000

Mechanisms of the in vitro fungicidal effects of human neutrophils against Penicillium marneffei induced by granulocyte-macrophage colony-stimulating factor (GM-CSF).

We examined the in vitro fungicidal activity of human neutrophils against conidia and yeast cells of Penicillium marneffei. Neutrophils showed a small but significant anti-fungal effect against the yeast form of P. marneffei. Treatment of neutrophils with GM-CSF significantly augmented their anti-fungal activity. In contrast, the conidia form resisted killing even by stimulated neutrophils. Neutrophil fungicidal effect was not inhibited by superoxide dismutase (SOD), while the same treatment significantly suppressed the killing of Candida albicans by GM-CSF-stimulated neutrophils. For effective killing of P. marneffei yeast cells by GM-CSF-stimulated neutrophils, direct contact between the two was essential; interference in such interaction by separation using a 0. 45-microm-pored membrane prevented such an effect. Addition of colchicine attenuated GM-CSF-stimulated neutrophil fungicidal activity in a dose-dependent manner. This effect did not appear to be mediated by interference with neutrophil mobility toward yeast cells, because similar results were obtained when the cultures were set in round-bottomed wells which facilitate their direct contact. Finally, granular extracts derived from unstimulated neutrophils significantly suppressed the growth of microorganisms. Pretreatment of neutrophils with GM-CSF markedly enhanced this effect. The fungicidal activity of granular lysates was strongly, but not completely, reduced by heat treatment. Considered together, our results indicate that GM-CSF-stimulated neutrophils killed the yeast form of P. marneffei present in close proximity, probably in a superoxide anion-independent mechanism, but through exocytosis of granular enzymes which were largely heat-labile.

Antifungal Agents↗
PubMed2000

Chemokine synthesis and cellular inflammatory changes in lungs of mice bearing p40tax of human T-lymphotropic virus type 1.

To elucidate the pathogenic mechanisms of human T-lymphotropic virus type 1 (HTLV-1)-associated lung inflammation, we conducted a histopathological and molecular analysis study using transgenic mice bearing pX region of this virus. In these mice, accumulations of inflammatory cells consisting mainly of lymphocytes were present in peribronchiolar and perivascular areas and alveolar septa, while control littermate mice did not show such changes. In situ hybridization showed that the anatomic distribution of p40tax mRNA was similar to that of inflammatory cells, typically in peribronchiolar areas and to a lesser extent in perivascular and alveolar septa. Inflammatory cytokines, including IL-1beta, tumour necrosis factor-alpha and interferon-gamma, and several chemokines, such as monocyte chemotactic protein-1 (MCP-1), RANTES, macrophage inflammatory protein-1alpha (MIP-1alpha) and IP-10, were detected in lungs of transgenic mice but not control mice. Semiquantitative analysis using reverse transcription-polymerase chain reaction showed a significant correlation between MCP-1 mRNA expression and p40tax mRNA, but not with other chemokines. The gene expression of the above chemokines, with the exception of MIP-1alpha, correlated with the severity of histopathological changes in the lung. Considered together, our results suggested that p40tax synthesis may be involved in the development of lung lesions caused by HTLV-1 through the induction of local production of chemokines.

Animals↗
PubMed2000

Granzyme B-positive primary gastric T-cell lymphoma: gastric T-cell lymphoma with the possibility of extrathymic T cell origin.

A case of primary gastric T-cell lymphoma, which was positive for granzyme B, is reported. The patient was a 47-year-old Japanese female who complained of a dull upper abdominal pain. Radiographic and endoscopic examinations revealed an ulcerative infiltrative lesion in her stomach. Following the confirmation of a high-grade malignant lymphoma, a distal gastrectomy with regional lymph nodal dissection was performed. The histology of the gastric lesion revealed a malignant lymphoma of the diffuse pleomorphic type without lymph nodal involvement. Immunohistochemistry revealed that the tumor cells were positive for LCA, CD3, TIA-1 and granzyme B, but were negative for CD4, CD8, CD56, CD30, L-26, EMA, TCR alpha/beta and TCR gamma/delta. Because the tumor cells showed T cell nature with cytotoxic activity proved by TIA-1 and granzyme B, and without evidence of further maturation of T cell, a malignant lymphoma originating from extrathymic-derived T cells was suggested.

Biomarkers, Tumor↗
PubMed2000

A comparative clinical study of pneumonia by penicillin-resistant and -sensitive Streptococcus pneumoniae in a community hospital.

OBJECTIVE: This study aimed to determine the clinical difference of pneumonia between penicillin-resistant and penicillin-sensitive Streptococcus pneumoniae. METHODOLOGY: Forty-nine cases in 46 patients of pneumococcal pneumonia were studied from December 1992 to May 1997. There were 24 cases (in 22 patients) of penicillin-resistant pneumococci (PRSP) pneumonia which were compared with 25 cases (in 24 patients) with penicillin-sensitive pneumococci (PSSP). RESULTS: Both the mean age and the underlying disease states did not differ between the two groups. However, hospital-acquired pneumonia and previous use of antibiotics were observed in eight (33.3%) and 12 (50.0%) patients in PRSP compared with three (12.0%) and two (8.0%) in PSSP, respectively. The clinical efficacy rate and bacteriological eradication rates were 87.5 and 87.5% in PRSP compared with 87.5 and 87.0% in PSSP, respectively. Minimum inhibitory concentration (MIC) of antibiotics against 30 pneumococcal isolates was examined, and 10 strains ranged from 0.10-0.78 microg/mL and five strains were more than 1.56 microg/mL against penicillin G, while the MIC showed higher resistance to other antibiotics except for the carbapenems. Serotyping of the isolates by antiserum revealed differences in the predominant types PRSP (19F) and PSSP (6A,9V) [corrected]. CONCLUSIONS: We must care for not only community-acquired infection but also nosocomial transmission of PRSP pneumonia. Most patients with infections due to PRSP tended to have a milder illness with a good outcome (no patient died). As such it appears that empiric therapy for pneumococcal pneumonia does not require modification from what is recommended at present. However, in patients with infection due to highly resistant strains, and who are not responding to conventional therapy should have their treatment modified according to subsequent susceptibility testing.

Anti-Bacterial Agents↗
PubMed2000

Intracellular Na+ directly modulates Na+,K+-ATPase gene expression in normal rat kidney epithelial cells.

BACKGROUND: In a wide variety of cell systems, increases in cell Na+ ([Na+]i) lead to an induction of N+,K+-ATPase mRNA expression. On the other hand, the increase in [Na+]i can also induce a rise in cell Ca2+ ([Ca2+]i) through a secondary inhibition of Na+/Ca2+ exchange and a decrease in cell pH (pHi) through a secondary inhibition of Na+/H+ exchange. It is not known whether [Na+]i, [Ca2+]i, and/or pHi directly modulate N+,K+-ATPase mRNA expression. METHODS: We used normal rat kidney epithelial cells (NRK) to examine the effects of ouabain on N+,K+-ATPase alpha1- and beta1-mRNA accumulation by Northern blot analysis and the relationship between the mRNA accumulation and [Na+]i, [Ca2+]i, or pHi. [Na+]i, [Ca2+]i, and pHi were measured using a Na+-sensitive fluorescent dye (SBFI), a Ca2+-sensitive fluorescent dye (Fura-2), and a pH-sensitive fluorescent dye (BCECF), respectively. RESULTS: Ouabain (1 mmol/L) significantly increased [Na+]i. Upon addition of ouabain, alpha1-mRNA levels increased to 2. 3 times the control level at three hours, with maximum 3.3-fold elevations at 12 hours. beta1-mRNA levels also increased to 2.4 times the control level at 3 hours, with a maximum 3.3-fold increase at 12 hours. The ouabain-mediated alpha1- and beta1-mRNA induction was inhibited by both the RNA transcription inhibitor (actinomycin D) and the protein synthesis inhibitor (cycloheximide). Ouabain at three hours caused an increase in [Ca2+]i. Similar increases in [Ca2+]i, which were elicited by the Ca2+ ionophore (ionomycin) in the presence of extracellular Ca2+, had no effect on alpha1- or beta1-mRNA levels. In Ca2+-free medium treated with EGTA, ouabain at three hours caused a significant increase in [Na+]i without any changes in [Ca2+]i, and also increased alpha1- and beta1-mRNA levels. Ouabain at three hours caused a significant decrease in pHi. Similar decreases in pHi, which were elicited by the specific inhibitor of Na+/H+ exchange (ethylisopropylamiloride), caused no effect on alpha1- or beta1-mRNA levels. Exposure of NRK to the Na+ ionophore (monensin) in the absence of extracellular Ca2+ increased [Na+]i and alpha1- and beta1-mRNA levels. The increases in alpha1- and beta1-mRNA levels upon addition of ouabain were associated with significant increases in alpha1- and beta1-subunit proteins. CONCLUSIONS: In NRK, ouabain causes an increase in [Na+]i, which directly modulates Na+,K+-ATPase alpha1- and beta1-mRNA accumulation.

Animals↗
PubMed2000

CD138-positive and Kaposi's sarcoma-associated herpesvirus (KSHV)-negative B-cell lymphoma with serosal spreading of the body cavity and lymphadenopathy: an autopsy case.

CD138-positive and Kaposi's sarcoma-associated herpes virus (KSHV)-negative B cell lymphoma with serosal spreading of the body cavity and lymphadenopathy is presented. Our lymphoma cells showed pleomorphic morphology and a clonal immunoglobulin gene rearrangement. Immunophenotypically, they lacked B- and T-cell-associated antigens but expressed strong membranous CD138 antigen along the serosa. Although our case was not conventional primary effusion lymphoma (PEL) because of the absence of KSHV and the presence of lymphadenopathy, its unique phenotype and serosal spreading were consistent with those of PEL. Our case suggests that, irrespective of KSHV infection, some pleomorphic B cell lymphomas with membranous CD138 expression show a peculiar serosal spreading.

Ascites↗
PubMed2000

Improved survival for patients with advanced neuroblastoma after high-dose combined chemotherapy based in part on N-myc amplification.

BACKGROUND/PURPOSE: In spite of many different kinds of chemotherapy for neuroblastoma, the prognosis for advanced neuroblastoma remains unsatisfactory. In particular, the outcome of advanced neuroblastoma with high copies of the N-myc gene tend to be poor. Therefore, the new high-dosage combined chemotherapy regimens for advanced neuroblastoma based in part on the N-myc amplification status has been utilized in the Kyushu area of Japan since 1991. This study aims to investigate whether these new regimens based in part on N-myc amplification have improved the survival rate of stage III and stage IV patients in comparison with the old regimens. METHODS: Between 1983 and 1995, 77 patients over 1 year of age and with stage III or IV neuroblastoma were registered in the Kyushu Area. Between 1983 and 1990, 49 patients received 1 of 2 combined chemotherapy regimens consisting of cyclophosphamide, cisplatin plus VM-26, and Adriamycin plus DTIC. Since 1991, two new regimens (New A1 and A3) have been administered based on the N-myc amplification status in a total of 28 patients. The New A1 regimen, which consists of cyclophosphamide, cisplatin, Adriamycin, and VP-16 has been administered in cases of less than 10 copies of N-myc, whereas the A3 regimen, consisting of a higher dose of cyclophosphamide, cisplatin, Adriamycin, and VP-16, has been administered in cases of more than 10 copies of N-myc. The survival rate was then compared between the old regimens and the new regimens. RESULTS: The 3-year survival rate (61.5%) for patients treated by the new regimens was significantly higher than that (32.7%) for patients treated by the old regimens (P <.01). Regarding the 24 cases of more than 10 copies of N-myc, the 3-year survival rate (35.9%) of the 13 patients treated by the A3 regimen was higher than that (0%) of the 11 patients treated by the old regimens (P <.05). However, in the 19 stage IV patients treated by the new regimens, the 3-year survival rate (11.1%) of the 9 cases of more than 10 copies was significantly lower than that (77.8%) of the 10 cases of less than 10 copies of N-myc (P <.01). CONCLUSIONS: These results suggest that high-dose combined chemotherapy based in part on the N-myc amplification status significantly improved the prognosis of patients with advanced neuroblastoma. However, stage IV patients with N-myc amplification still require a more effective treatment modality.

Antineoplastic Combined Chemotherapy Protocols↗
PubMed2000

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.

Child, Preschool↗
PubMed2000

Feasibility study of autologous peripheral blood stem cell transplantation for the treatment of childhood acute myelogenous leukemia.

BACKGROUND: The primary object of this study was to identify treatment-related variables that may predict relapse of acute myelogenous leukemia (AML) after autologous peripheral blood stem cell transplantation (PBSCT), which will be critical for the development of a suitable protocol for wider application. METHODS: A total of 28 children (age 0-18 years) with AML underwent PBSCT and have had a minimum follow-up of 25 months; including 24 patients in their first complete remission (CR) and four in their second CR. The patients were divided into two cohorts according to the study phase: 16 patients were treated in an early phase pilot study and 12 patients in their first CR were treated in a prospective trial. Fifteen of the first-CR patients had any of the cited high-risk features of high WBC count (>100x10(9)/l; n = 5), FAB M0/M4/M5/M7 subtypes (n = 11) or delayed achievement of CR (n = 9). Except in one patient, cytoreductive regimens did not include total body irradiation (TBI). RESULTS: After PBSCT, one patient died of veno-occulsive disease (VOD) and another patient relapsed early on day 43, but the remaining patients showed engraftment. Leukemic relapse was observed 1-29 months after PBSCT (median, 8 months); in all of the 4 children treated in their second CR and in 11 of the 24 patients (46%) treated in their first CR. The remaining patients have been disease-free for 24 to 97 months (median, 53 months). Using a multivariate analysis, the timing of apheresis was the most significant prognostic factor for those treated in their first CR (p = 0.03); 12 of the 16 patients whose PBSC were collected beyond 2.5 months of CR continue to remain in CR, while seven of the eight patients whose PBSC were harvested within 2.5 months of CR relapsed. CONCLUSION: Although the small number of patients studied does not allow firm conclusions to be drawn regarding the relative effectiveness of this therapy, the results do suggest the feasibility of further studies of PBSCT for the treatment of childhood AML with high-risk features including the assessment of minimum residual disease.

Adolescent↗