Biomedical subjects
Publications and source records attributed to Mitsuo Kaku.
We developed a decision support system that helps doctors select appropriate first-line drugs. The system classifies patients' abilities to protect themselves from infectious diseases as a risk level for infection. In an evaluation of the prototype system, the risk level it determined correlated with the decisions of specialists. The system is very effective and convenient for doctors to use.
Flt3 is a type III RTK and approximately 30% of AML patients harbor an internal tandem duplication (ITD) of the juxtamembrane region or a point mutation of the Flt3 protein leading to the constitutive activation of downstream signaling pathways and aberrant cell growth. The cyclin-dependent kinase inhibitor p21 inhibits cell growth when expressed at high levels and induces cell growth when expressed at lower levels. In this study, we have addressed the role of Flt3-ITD in the regulation of p21. Co-transfection of p21 promoter-luciferase constructs with Flt3-ITD plasmid into K562 and BaF3 cells results in the induction of p21 promoter activity and a -692/-684 STAT site is important for the induction. STAT5a binds specifically to this element and Flt3-ITD enhances the protein binding to this site. Overexpression of Flt3-ITD led to the induction of endogenous p21 expression in various cells. These results may implicate p21 in Flt3-ITD induced leukemogenesis.
Fms-like tyrosine kinase 3 (Flt3) is a type III receptor tyrosine kinase (RTK). Between 20% and 30% of acute myeloid leukemia (AML) patients have either an internal tandem duplication (ITD) of the juxtamembrane region or a point mutation of the Flt3 receptor leading to the constitutive activation of downstream signaling pathways and aberrant cell growth. The silencing mediator of retinoic and thyroid hormone receptors (SMRT) corepressor mediates transcriptional repression by interacting with transcription factors such as the promyelocytic leukemia zinc finger (PLZF) protein. Previous reports indicate that SMRT interaction with transcription factors can be disrupted by phosphorylation through activation of RTK pathways. We report here that the Flt3-ITD interferes with the transcriptional and biologic action of the PLZF transcriptional repressor. In the presence of Flt3-ITD, PLZF-SMRT interaction was reduced, transcriptional repression by PLZF was inhibited, and PLZF-mediated growth suppression of leukemia cells was partially blocked. Furthermore, overexpression of Flt3-ITD led to a partial relocalization of SMRT protein from the nucleus to the cytoplasm. Nuclear export was dependent on the SMRT receptor interaction domain (RID), and Flt3-ITD enhances the binding of nuclear-cytoplasm shuttling protein nuclear factor-kappaB-p65 (NFkappaB-p65) to this region. These data suggest that activating mutations of Flt3 may disrupt transcriptional repressor function resulting in aberrant gene regulation and abnormal leukemia cell growth.
BACKGROUND: It is sometimes difficult to detect the bone marrow infiltration of lymphoma cells, because lymphoma cells are not distinguishable from normal lymphocytes due to the similarity of their phenotype. METHODS: Bone marrow involvement of 17 samples of 15 patients with follicular lymphoma, whose lymphoma cells were confirmed to harbor the translocation of chromosome14q32, were examined by microscopic analysis of bone marrow smear and biopsy, flow cytometorical analysis (FCM), chromosomal analysis of G-banding and fluorescence in situ hybridization (FISH). FISH was performed using a probe, which detects the split of IGH gene on 14q32. RESULTS: The positivity of FISH was highest among these methods and FISH was able to detect the bone marrow involvement in one case who was defined as negative by bone marrow biopsy. CONCLUSIONS: FISH can be used for detection of bone marrow involvement of malignant lymphoma that carries chromosomal rearrangement involving 14q32.
We screened for mutations of the glucocorticoid receptor (GR) gene in patients with systemic lupus erythematosus (SLE), a typical autoimmune disease. Polymerase chain reaction-single-strand conformation polymorphism analysis (PCR-SSCP) revealed a single mutation in exon 9 of the GR gene in 11/132 (8.3%) among 66 patients with SLE. No mutations were detected in 52 healthy individuals (0/104, 0.0%), but the same mutation was detected in other autoimmne diseases (4/108, 3.7%). DNA sequencing showed a T to C substitution at codon 766 (position 2430) of the GR gene, which does not alter the amino acid sequence of the GR. Further analysis using a LightCycler generated different melting curves indicates that the pattern with this mutation is different from that of wild type. The identified mutation of the GR gene may represent a polymorphism associated with SLE.
Stanniocalcin is a glycoprotein hormone that regulates the calcium level in fish. We found that mRNA of human stanniocalcin 1 (STC-1) is detectable in phytohemagglutinin-stimulated T cells and in most human leukemia cell lines, suggesting a role of STC-1 for cell proliferation. This finding prompts us to study the usefulness of STC-1 for monitoring acute leukemia. The levels of STC-1 transcripts increased in patients with acute leukemia at diagnosis and relapse, as judged by quantitative real-time RT-PCR. Levels of transcripts rapidly decreased to within the cut-off levels, when the blast numbers decreased with chemotherapy. Prolonged elevation of STC-1 levels after treatment was associated with a poor prognosis. All of 7 patients relapsed 1 to 4 months after they showed an elevated level of the transcripts in clinical remission. These results indicate that STC-1 is a novel marker for minimal residual disease of acute leukemia, and for an early diagnosis of relapse.
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The antibacterial activity of meropenem (MEPM) and other parenteral antibiotics against clinical isolates of 899 strains of Gram-positive bacteria, 1500 strains of Gram-negative bacteria, and 158 strains of anaerobic bacteria obtained from 28 medical institutions during 2002 was measured. The results were as follows; 1. MEPM was more active than other carbapenem antibiotics against Gram-negative bacteria, especially against enterobacteriaceae and Haemophilus influenzae. MIC90 of MEPM against Pseudomonas aeruginosa was the lowest of the drugs tested. MEPM showed low cross-resistant rate against both imipenem-resistant P. aeruginosa and ciprofloxacin-resistant P. aeruginosa. MEPM was active against most of the species tested in Gram-positive and anaerobic bacteria, except for multi-drug resistant strains including methicillin-resistant Staphylococcus aureus (MRSA), methicillin-resistant Staphylococcus epidermidis (MRSE). 2. The proportion of extended-spectrum beta-lactamase (ESBL) strains was 3.1% (4 strains) in Escherichia coli and 1.9% (2 strains) in Klebsiella pneumoniae. Carbapenems including MEPM were active against these ESBL strains. In conclusion, the results from this surveillance study suggest that MEPM retains its potent and broad antibacterial activity and therefore is a clinically useful carbapenem; at present, 7 years after available for commercial use.
GATA-1 is a transcription factor essential for erythroid cell development, and knockdown of GATA-1 gene results in maturation arrest and transformation of erythroblasts. To clarify the mechanism that gives rise to this abnormal phenotype, genes that are aberrantly expressed in the spleen of heterozygous GATA-1 knockdown mutant mouse (referred to as GATA-1 mutant mouse) were identified by using cDNA array. One of these genes, YB-1, was found to be highly expressed in the spleen of GATA-1 mutant mouse. Reporter and electrophoretic mobility shift assays revealed that the proximal GATA element in 5'-UTR region of YB-1 gene functions positively in K562 cells. Furthermore, both GATA-1 and GATA-2, which were transiently expressed in COS-7 cells, bound to this element and activated the YB-1 promoter through this element. These results suggest that YB-1 functions under the regulation of GATA factors in erythroid differentiation and aberrant expression of YB-1 gene may result in dyserythropoiesis.
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BACKGROUND: Although GATA-1 and GATA-2 have been shown to play an important role in hematopoiesis, the expression levels of these GATA proteins in the targeted cell population of clinical samples have not been studied. We applied flow cytometry (FCM) to examine the expression levels of these GATA proteins in the selected subpopulation in heterogeneous blood cells. METHODS: Cells were treated with a fixing solution and methanol followed by staining with specific antibodies to GATA proteins in a permeabilizing solution. Immunofluorescence microscopy and Northern blot analysis using GATA-1 and GATA-2 transfected cell lines and various leukemic cell lines were used to confirm the specificity of this method. Subsequently, the method was applied in two-parameter studies combining GATA expression with surface marker expression in clinical samples. RESULTS: The positive signals were specifically detected in transfected cells and leukemic cell lines by FCM in agreement with the results of Northern blot and immunofluorescence microscopy. The expression of these GATA factors in the targeted cell population was easily detectable by gating with lineage-specific cell surface markers. When the expression of these GATA proteins was examined in glycophorin A-positive cells in clinical samples, the level of GATA-1 was markedly different among the samples. CONCLUSIONS: This detection system is useful to evaluate the relative expression level of each GATA protein in the targeted cell population among heterogeneous cells, and the results suggest an aberrant expression of GATA factors in hematological diseases.
It has been shown that aberrant expression of a transcription factor, GATA-1, leads to maturation arrest and transformation of erythroid cells. We previously reported that a multifunctional protein, YB-1, was expressed strongly in the spleen of a GATA-1 mutant mouse, which was filled with transformed erythroblasts. This finding suggested that YB-1 has roles in erythropoiesis. In this study, we examined in vivo expression of YB-1 messenger RNA (mRNA) in bone marrow erythroid cells and erythroid leukemic cell lines. During erythroid differentiation of erythroid leukemic cell lines, the expression level of YB-1 mRNA was highest at the early phase of differentiation and then decreased. In human bone marrow cells, the in vivo expression level of YB-1 mRNA was higher in glycophorin A-positive cells than in glycophorin A-negative cells. An interesting finding was that expression of YB-1 was higher in erythroblasts in myelodysplastic syndrome-refractory anemia (MDS-RA) than in normal cells. The findings suggested that YB-1 functions in the early stage of erythropoiesis and that aberrant expression of this protein may induce hematological diseases such as MDS.
BACKGROUND: Umbilical cord blood (CB) is being used as a source of alternative HPCs for transplantation with increasing frequency. The goal of CB banks for unrelated transplantation is to provide good quality-controlled CB units that can be transplanted for HPCs into the largest possible number of patients. STUDY DESIGN AND METHODS: Large CB samples in freezing bags wrapped with insulators and small samples in cryotubes placed into double styrene-foam boxes were cryopreserved at -85 degrees C without a rate-controlled freezing machine, followed by storage in the liquid phase of nitrogen. After thawing these cells, the viability and recovery of cells, as well as the recovery rate of HPCs such as CD34+ cells, CFU-GM, and total CFU were evaluated. RESULTS: Measurement of the freezing rate in CB bags and cryotubes demonstrated that this simple method for cryopreservation of CB cells provided optimal conditions for both large-scale and small-scale cryopreservation. Recovery of CB progenitor cells after cryopreservation was also shown to be potentially acceptable when evaluated with CD34+ cells, CFU-GM, and total CFU. These results were comparable to the method using a rate-controlled programmed freezer. CONCLUSIONS: A simple method for cryopreservation of CB cells without a rate-controlled programmed freezer could provide a sufficient-enough potential for the transplantability of HPCs after thawing.
The newly developed Rapid Lumi Eiken/IS60 (RL/IS60) system automatically determines MICs by detecting chemiluminescence produced in the reaction of a chemiluminescent probe and oxygen metabolites from living microorganisms. The present study evaluated this system for accuracy in antimicrobial susceptibility testing. Chemiluminescence intensities after 4 h of cultivation of clinically important strains were plotted against various concentrations of antimicrobial agents, which resulted in curves reflecting the levels of susceptibility. Sixty-percent inhibitory concentrations based on the susceptibility curves agreed with MICs determined by the reference microdilution method. When the MICs of antimicrobial agents for four quality control (QC) strains (Staphylococcus aureus, Enterococcus faecalis, Escherichia coli, and Pseudomonas aeruginosa) were determined by the RL/IS60 system, most (91.1%) of them were within the QC limits proposed by the National Committee for Clinical Laboratory Standards. The system was further assessed for a total of 162 clinical isolates, including E. coli, Citrobacter freundii, Enterobacter cloacae, Klebsiella pneumoniae, Serratia marcescens, Proteus mirabilis, Morganella morganii, P. aeruginosa, Haemophilus influenzae, S. aureus, coagulase-negative staphylococci, Enterococcus faecalis, Enterococcus faecium, and Streptococcus pneumoniae. Overall, there was 90.6% agreement between the RL/IS60 system and the reference microdilution method. Our results suggest that the RL/IS60 system provides rapid and reliable MICs of a variety of antimicrobial agents for clinical isolates as well as QC strains.
We isolated five bacterial strains from patients diagnosed as having nocardiosis. Bacterial species were identified based on the similarities in the nucleotide sequences of 16S ribosomal RNAs. Three of the five strains were identified as Nocardia asteroids, but unexpectedly other two were Streptomyces hygroscopicus and Rothia dentocariosa. The latter two species are not members of the family Nocardiaceae. We investigated the susceptibilities of these five strains to the following nine antimicrobial agents: trimethoprim/sulfamethoxazole (TMP/SMX), minocycline (MINO), erythromycin (EM), amikacin (AMK), cefotaxime (CTX), faropenem (FRPM), imipenem (IPM), ciprofloxacin (CPFX), and sparfloxacin (SPFX). The minimum inhibitory concentration (MIC) ranges (mg/ml) were as follows: TMP-SMX, 4- > 32; MINO, 0.125-8; EM, < or = 0.016- > 32; AMK, 1-2; CTX, 0.063- > 32; FRPM, 0.063-16; IPM, 0.125-2; CPFX, 4-32; and SPFX, 0.5-16. Moreover, the synergistic effects of AMK in combination with each of TMP-SMX, MINO, EM, CTX, IPM, and SPFX were investigated by checkerboard synergy testing. No antagonism was recognized for the three N. asteroides strains. Synergistic and additive effects were observed for the combinations of AMK with CTX, IPM, or SPFX.
Autonomic nervous function was evaluated by means of power spectral analysis of heart rate variability in patients with essential hypertension who showed early morning rise of blood pressure (BP). We monitored ambulatory (BP) and physical activity in 80 untreated patients using TM 2425 (A&D Co.Ltd.). Early morning rise of BP was defined a patient's blood pressure obtained between 6:00 to 9:00am is 170mmHg or higher. Patients who showed early morning rise of BP were categorized into groups. Twenty patients, whose mean BP obtained between 6:00 to 9:00am were higher than those obtained between 3:00 to 600am by 30mmHg or more, were defined as a surge type. Sixty patients, in whom the difference in BP was less than 30mmHg, were defined as a sustained type. Power spectral analysis of hourly R-R intervals for 24 hours was performed to obtain the low frequency power (LF, 0.04 to 0.15 Hz) and high frequency power (HF, 0.15 to0.04 Hz). LFn and LF/HF ratio were considered to be indexes of sympathetic nervous activity, and HF parasympathetic nervous activity, respectively. Between 3:00 to 6:00am, LFn and LF/HF were significantly higher and HF was significantly lower in the sustained type patients than in the surge type patients. LF/HF and LFn in the surge type elevated significantly beween 6:00 to 9:00am. These results indicate that both the loss of nocturnal decline in BP in the sustained type and the morning rise in BP in the surge type to occurred in association with changes in autonomic nervous system.
The purpose of this paper is to give an insight into the medical education program of our division at Tohoku University. Laboratory medicine in medical education is a field of learned basic clinical tests. Students have to learn the clinical laboratory through early clinical exposure in the first grade and try clinical technology through small-group learning in the fifth grade. Finally, they learn laboratory medicine such as infection control in our or another clinical hospital. The objects of our course are to encourage and promote the highest standards of training and post-graduate education of physicians and scientists at our university.
A survey was conducted to determine the antimicrobial activity of fluoroquinolones and other antimicrobial agents against 8,474 clinical isolates obtained from 37 Japanese medical institutions in 2000. A total of 25 antimicrobial agents were used, comprising 4 fluoroquinolones, 13 beta-lactams, minocycline, chloramphenicol, clarithromycin, azithromycin, gentamicin, amikacin, sulfamethoxazole-trimethoprim, and vancomycin. A high resistance rate of over 85% against fluoroquinolones was exhibited by methicillin-resistant Staphylococcus aureus (MRSA) and Enterococcus faecium. Isolates showing resistance to fluoroquinolones among methicillin-resistant coagulase-negative Staphylococci, Enterococcus faecalis, and Pseudomonas aeruginosa from UTI accounted for 30-60%. However, many of the common pathogens were still susceptible to fluoroquinolones, such as Streptococcus pneumoniae (including penicillin-resistant isolates), Streptococcus pyogenes, methicillin-susceptible S. aureus (MSSA), methicillin-susceptible coagulase-negative Staphylococci, Moraxella catarrhalis, the Enterobacteriaceae family, and Haemophilus influenzae (including ampicillin-resistant isolates). About 85% of P. aeruginosa isolated from RTI were susceptible to fluoroquinolones. In conclusion, this survey of sensitivity to antimicrobial agents clearly indicated trend for increasing resistance to fluoroquinolones among MRSA, Enterococci, and P. aeruginosa isolated from UTI, although fluoroquinolones are still effective against other organisms and P. aeruginosa from RTI as has been demonstrated in previous studies.