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Biomedical subjects

S Fujimaki

Publications and source records attributed to S Fujimaki.

At least 19 recordsLinked to original sources
PubMed2001

Caspase-independent apoptosis induced by differentiation-inducing factor of Dicytostelium discoideum in INS-1 cells.

Differentiation-inducing factor (DIF) is a lipophilic hormone of Dicytostelium discoideum and has been shown to exert diverse effects in mammalian cells. We investigated the effect of DIF on cell viability in insulin-secreting INS-1 cells. DIF induced cell death in a dose-dependent manner. In DIF-treated cells, nuclear condensation and shrinkage of the cell body were observed. After 6 h of DIF treatment, cells became Tdt-mediated dUTP-biotin nick end-labeling-positive, and DNA ladder formation was detected, indicating that DIF induced apoptosis in these cells. DIF did not activate caspase-3, a key enzyme mediating apoptotic signals generated by various agents. Furthermore, DIF-induced cell death was not affected by Z-asp-2, 6-dichlorobenzoyloxymethylketone, a broad inhibitor of the caspases. As is the case in other types of cells, DIF increased cytoplasmic free calcium concentration in INS-1 cells. However, DIF-induced cell death was not affected by chelating intracellular free calcium by 1, 2-bis(2-aminoophenoxy)ethane-N, N, N, N-tetra acetic acid (BAPTA). These results indicate that DIF induces apoptosis in INS-1 cells by a mechanism independent of caspase-3. DIF-induced elevation of cytoplasmic calcium does not mediate the effect of DIF on cell death.

Animals↗
PubMed2001

Decreased expression of transcription factor GATA-2 in haematopoietic stem cells in patients with aplastic anaemia.

Aplastic anaemia is characterized by reduced haematopoiesis resulting in pancytopenia. It has been speculated that there is an injury in haematopoietic stem cells in the bone marrow; however, the precise nature of the injury has not been elucidated. In this study, the levels of expression of mRNAs for three transcription factors, GATA-2, SCL and AML1, which function in the early stages of haematopoiesis, were examined by quantitative polymerase chain reaction in patients with aplastic anaemia, idiopathic thrombocytopenic purpura (ITP) and normal subjects. Among these factors, expression of GATA-2 mRNA in purified CD34-positive cells was markedly decreased in aplastic anaemia compared with that in ITP and in normal subjects. The expression levels of SCL and AML1 mRNA in CD34-positive cells in aplastic anaemia were not different from those in normal subjects. When the expression of GATA-2 protein in CD34-positive cells was examined by immunocytochemical analysis, the percentage of GATA-2-positive cells in aplastic anaemia was lower than that in normal subjects. These findings strongly suggest that there is an aberrant expression of transcription factors in stem cells in aplastic anaemia, which may be responsible for the development of the disease.

Anemia, Aplastic↗
PubMed2001

Childhood T-cell acute lymphoblastic leukemia with four distinct immunophenotypes representing different stages of T-cell development.

The authors report on a 14-year-old boy who developed T-cell acute lymphoblastic leukemia (FAB:L1) displaying 4 immunophenotypically distinct leukemic cell populations by 3-color immunofluorescence staining. Cytogenetic analysis at diagnosis showed 46,XY,add(4)(p16)[12]/46,XY[2]. A single rearrangement of the T-cell antigen receptor beta- and gamma-chain genes in these cells indicated monoclonality of the leukemic cells. These findings suggest that leukemic blast cells of monoclonal origin in this case were divided into 4 immunophenotypic populations, representing various stages of differentiation.

Adolescent↗
PubMed2001

Increased sensitivity to cytosine arabinoside in human leukemia by c-raf-1 antisense oligonucleotides.

c-raf-1, a cytoplasmic serine/threonine protein kinase, plays an important role in mitogen- and damage-responsive cellular signal transduction pathways. Expression of c-raf-1 modifies cell growth, proliferation and survival. Although expression of c-raf-1 has been studied in several tumors, the role of c-raf-1 in leukemia is so far unclear. We examined the expression of c-raf-1 in the human leukemia cell lines U937 and K562, and in a cytosine arabinoside (Ara-C)-resistant cell line (K562AC) derived from K562. Expression of c-raf-1 was increased in U937 and in Ara-C-resistant K562AC cells compared with the parental cells. We then investigated whether inhibition of c-raf-1 expression by antisense oligonucleotides increases the sensitivity to Ara-C in U937 and K562AC cells. Antisense oligonucleotides for c-raf-1 inhibited expression of c-raf-1 mRNA, but did not affect cell growth and increased sensitivity to Ara-C but not to other drugs such as adriamycin, VP-16 or vincristine. These results suggest that c-raf-1 is one of the factors involved in Ara-C resistance in leukemia and lend weight to the case for development of anti-cancer therapeutics involving oncogene-targeted antisense oligonucleotides.

Antimetabolites, Antineoplastic↗
PubMed2001

Induction of autoimmune hypothyroidism and subsequent hyperthyroidism by TSH receptor antibodies following subacute thyroiditis: a case report.

A 45 year-old man had a typical episode of subacute thyroiditis with tender goiter, depressed radioiodine uptake (RAIU) and elevated erythrocyte sedimentation rate. The titer of TSH binding inhibitor immunoglobulin (TBII), which had been 8.6% at initial presentation, rose to 14.9% in 2 weeks. TBII consisted of high titers (94%) of TSH stimulation-blocking antibodies (TBAb) and negative thyroid stimulating antibodies (TSAb). About 2 months after the first visit, TBII titers had risen to 48.9% and were persistently elevated for 5 months with high TBAb activity. The patient developed hypothyroidism with a maximum serum TSH level of 54.5 microU/ml. TBII and TBAb titers then gradually decreased, and the patient spontaneously recovered from hypothyroidism. Eighteen months after the episode of subacute thyroiditis, he became hyperthyroid with elevated TSAb and negative TBAb values. Doppler ultrasonography showed increased blood flow in the thyroid, and RAIU at 24 h was 53%. He was treated with antithyroid drugs, and soon became euthyroid. This case indicates that subacute thyroiditis can induce thyroid autoimmunity, and that the character of TSH receptor antibodies (TSHRAb) in these patients can change thereby modifying their thyroid function.

Antithyroid Agents↗
PubMed2001

Induction of apoptosis and necrosis by zinc in human thyroid cancer cell lines.

Zinc at concentrations of 150, microM or higher induced necrosis as well as apoptosis in thyroid cancer cell lines. Necrosis was induced by zinc in a dose-dependent manner, whereas apoptosis did not increase at higher concentrations of zinc. The expression of the antiapoptotic protein phosphorylated Bad was markedly increased, whereas the expression of the proapoptotic proteins Bax and Bad decreased following Zn(2+) exposure. Zn(2+) induced rapid degradation of IkappaB, and an increase in the binding of nuclear transcription factor-kappaB (NF-kappaB). These observations indicate that antiapoptotic pathways were activated in thyroid cancer cells following exposure to Zn(2+). This may be a self-defence mechanism against apoptosis and may underlie the general resistance of thyroid cancer cells to apoptotic stimuli. Zinc may be a potential cytotoxic agent for the treatment of thyroid cancer.

Animals↗
PubMed2000

Accurate mass determination by multiple sprayers nano-electrospray mass spectrometry on a magnetic sector instrument.

A new technique for accurate mass determination by using multiple sprayers nano-electrospray ionization mass spectrometry (nano-ESI-MS) on a magnetic sector instrument is described. Metal coated glass capillaries were used as nano-ESI sprayers. One of the sprayers was used for the reference compound solution, and others were used for the introduction of sample solutions. The spectra of the different compounds were obtained by shifting each sprayer's position relative to the sampling orifice. The accurate masses of several standard compounds were obtained with good accuracy, without problems arising from differences in ionization efficiency between the sample compounds and reference compound.

Enkephalin, Leucine↗
PubMed2000

In vitro leukemia cell models of Ara-C resistance.

Cells of the human leukemia line K562 were continuously exposed to cytosine arabinoside (Ara-C) at increasing concentrations for 3 months. The resulting cell line, termed K562/AC, showed 48-fold resistance to Ara-C, compared with the parental K562 cells. The sensitivities of K562/AC to adriamycin (ADR), vincristine (VCR) and etoposide (VP16) were similar to those of parental K562. Gene analysis revealed that this cell line lacked expression of the deoxycytidine kinase (dCK) gene, which was evident in Ara-C-sensitive cells. As in K562 cells, multidrug resistance (MDR-1) and multidrug resistance protein (MRP) genes were not expressed in K562/AC. We also established an in vitro model of Ara-C resistance using phosphorothioate antisense oligonucleotides to dCK (dCK-AS). Treatment of K562 with dCK-AS caused decreased dCK expression and 6- to 10-fold increases in resistance to Ara-C, compared with that in cells treated with sense oligonucleotides to dCK (dCK-S) or in non-transfected cells. The cells described here may contribute to the study of a novel mechanism associated with Ara-C resistance, in which reduced dCK activity may play an important role.

Antimetabolites, Antineoplastic↗
PubMed2000

A quantitative reverse transcriptase polymerase chain reaction method for the detection of leukaemic cells with t(8;21) in peripheral blood.

We evaluated the usefulness of a recently developed real-time reverse transcriptase polymerase chain reaction (RT-PCR) system to detect minimal residual diseases (MRD) in patients with acute myelogenous leukaemia (AML) with chromosomal translocation t(8:21). The method was simple, rapid and reproducible for the quantity of chimeric AML1-ETO (MTG8) transcripts. The ratio of the absolute copy number of a target gene (AML1-ETO) to a control gene (glyceraldehyde-3-phosphate dehydrogenase, GAPDH) was calculated by using a fluorescence curve prepared from amplicons of serially diluted standard RNA. The relative points of MRD in bone marrow (BM) of 8 patients in the acute phase of the disease was from 0.85 to 3.0, whereas those of MRD in complete remission (CR) decreased to below 6.4 x 10(-3). This method was also applied to evaluate chimeric transcripts in peripheral blood (PB) samples. The values in patients with t(8;21) AML were from 0.97 to 2.0 in the acute phase, whereas those in CR showed less than 2.2 x 10(-4). There was 10(-5)-fold difference in AML1-ETO mRNA expression between PB samples in the acute phase and those in CR. The results suggest that we may easily monitor MRD in patients with t(8;21) AML through quantitative analysis of AML1-ETO transcripts in blood samples.

Chromosomes, Human, Pair 21↗
PubMed2000

Circulating interleukin-6 and interleukin-6 receptors in patients with acute and recent myocardial infarction.

Interleukin-6 (IL-6), a proinflammatory cytokine, plays a key role in the pathogenesis of coronary artery disease (CAD). We investigated circulating IL-6 and its receptors in patients with CAD. We evaluated 39 Japanese patients with CAD (30 males and 9 females aged 36-79 years), measuring their plasma levels of IL-6 and IL-6 receptors alpha and beta (IL-6R alpha, IL-6R beta). Circulating levels of IL-6, IL-6R alpha and IL-6R beta were measured by an enzyme-linked immunosorbent assay. Blood was sampled immediately after admission and again after 1, 2, 3, 6 and 9 h, then every 12 h for 5 days. Atrial natriuretic peptide (ANP) and B-type natriuretic peptide (BNP) were measured on day 3 after symptom onset. Plasma levels of IL-6 and IL-6Rs were significantly increased in 28 patients with acute myocardial infarction (AMI) compared with 15 normal controls. However, neither IL-6 nor IL-6Rs showed an increase in 6 patients with angina pectoris. We observed two peaks for circulating IL-6 in AMI, the first of which showed a significant correlation with ANP as well as BNP. These results may help to explain why the amount of IL-6 produced is closely related to the severity of myocardial dysfunction in patients with CAD.

Adult↗
PubMed2000

[The construction of a standard RNA synthesized for quantitative RT-PCR system].

We constructed the standard RNA synthesized for the chimeric AML1-MTG8 transcripts and the house-keeping gene, glyceraldehyde-3-phosphate dehydrogenase(GAPDH) transcripts in real-time quantitative RT-PCR system. AML-MTG8 transcripts was detectable in 10 fg of synthetic RNA(3.5 x 10(3) copies). Linearity was from 3.5 x 10(3) to 3.5 x 10(9) copies. Threshold cycle(CT) is defined as the fractional cycle number at which the reporter fluorescence generated by cleavage of the probe passes a fixed threshold above baseline. The standard curve, where the known amounts of RNAs were used, showed a good correlation between the copies of AML1-MTG8 RNA and CT(r = -0.995). The within-run and day-to-day coefficients of variation(CV) in AML1-MTG8 RNA by this system were 9.5-24.7%(n = 10) and 21.7-42.2% (n = 8), respectively. GAPDH transcripts was detectable in 10 fg of synthetic RNA(6.1 x 10(4) copies). Linearity was from 6.1 x 10(4) to 6.1 x 10(8) copies. The standard curve, where the known amounts of RNAs were used, showed a good correlation between the copies of GAPDH RNA and CT(r = -0.993). The within-run and day-to-day CV in GAPDH RNA by this system were 9.3-14.6%(n = 10) and 14.7-15.8% (n = 10), respectively. Thus, we suggested that synthesized RNA as a standard RNA may be useful in quantitative RT-PCR for clinical application.

Core Binding Factor Alpha 2 Subunit↗
PubMed2000

[Genetic diagnosis for drug resistance in cancers].

The failure of chemotherapy to eradicate tumor cells is often due to the development of drug resistance. MDR(multidrug resistance) whose one form of resistance results from a decreased intracellular accumulation of the drugs, most often mediated by the overexpression of P-glycoprotein. MRP also related to pump function of cell membrane in acute leukemia. We have developed the new quantitative assay based on real-time PCR to measure expression of drug-resistance related genes such as MDR-1 and MRP in clinical samples. These results indicates that real-time PCR system is a reliable method to quantitatively determine drug resistant genes expression, it may be to predict responsiveness to chemotherapy by using this technique.

Drug Resistance, Multiple↗
PubMed2000

[Molecular diagnosis and new gene engineering in hematological malignancies].

Advancement molecular diagnostics in hematological malignancy has provided us with a broad menu of new assays and techniques. By integrating the data gleaned from these techniques we can formulate a more rational broad-based diagnosis. Hematological malignancies have traditionally been classified by morphological criteria. However, molecular advances have provided new insights in the genetic backbone of chimeric genes. These malignancies have shown chromosome abnormalities of translocation with chimeric genes, and revealed the rearrangement of chimeric genes by PCR analysis and quantitative PCR system. This review summarized the recent technology for detecting chimeric genes along with concepts of laboratory performance.

Adult↗
PubMed1999

Secondary structures and structural fluctuation in a dimeric protein, Streptomyces subtilisin inhibitor.

Based on the nuclear magnetic resonance assignments of a dimeric protein, Streptomyces subtilisin inhibitor (SSI), microscopic details of secondary structures in solution have been elucidated. The chemical shift index of C(alpha) signals, together with information on the hydrogen exchange rates of the backbone amide protons, were used to identify secondary structures. The locations of these secondary structures were found to be different in some critical points from those determined earlier by X-ray crystallography of the crystal. Notably, the beta3 strand is completely missing and the alpha2 helix is extended toward the C-terminus. Furthermore, hydrogen exchange experiments of individual peptide NH protons under strongly folding conditions revealed mechanisms of global and local structural fluctuation within the dimeric structure. It has been suggested that the global fluctuation of the monomeric unit occurs without affecting the accompanying monomer, in contrast to the equilibrium thermal unfolding, which is cooperative. Higher protection against hydrogen exchange for residues in part of the beta4 strand implies that this region might serve as a folding core.

Amino Acid Sequence↗
PubMed1993

[The detection of Helicobacter pylori by PCR in gastric diseases].

There are several methods for detecting Helicobacter pylori (H.pylori) infection, but all of them have both advantage and disadvantage. In this study, we investigated the usefulness of PCR as a routine screening method for H.pylori infection. We used PCR by identification of bacterial specific 16SrRNA gene. This test was performed on gastric biopsy specimens and gastric aspirate and stomatic mucus from 23 patients in gastric disease, and compared the sensitivity with other methods such as urease and culture test. We could detect H.pylori above 10(3)/ml of bacterial volume by PCR and H.pylori were positive in 96% of all gastric biopsy specimens. PCR was most sensitive and specific method for detecting H.pylori. Thus, PCR was considered to have a possibility of becoming an accurate routine screening method of H.pylori infection.

Adult↗
PubMed1993

[Risk factors for contrast nephropathy in diabetic patients undergoing cardioangiography].

Risk factors for contrast nephropathy were prospectively studied in 17 patients with non-insulin dependent diabetes mellitus undergoing cardioangiography. Contrast nephropathy, defined as a serum creatinine increase of greater than 25% at 3 day after angiography, occurred in 29.4% of diabetic patients. Patients who developed contrast nephropathy had significantly higher serum creatinine (Cr), fractional excretion of sodium (FENa), urinary albumin excretion rate (AER), and lower 24hr Ccr than patients who did not (Cr: 1.5 +/- 0.3 mg/dl vs. 0.8 +/- 0.1 mg/dl, FENa: 1.9 +/- 0.5% vs. 0.6 +/- 0.1%, AER: 522 +/- 335 micrograms/min vs. 27 +/- 13 micrograms/min, 24hr Ccr: 39.1 +/- 11.6 ml/min vs. 86.2 +/- 9.3 ml/min, P < 0.05). Contrast nephropathy developed in all of two patients with overt proteinuria (AER more than 200 micrograms/min), but none of eight patients with normoalbuminuria (AER below 15 micrograms/min). Three of seven patients with microalbuminuria developed contrast nephropathy, and two of them had advanced nephropathy. FENa obtained next day was significantly elevated over baseline in patients with contrast nephropathy (1.9 +/- 0.5% vs. 9.7 +/- 4.5%, P < 0.05), but unchanged in patients without contrast nephropathy. The rise in C beta 2-microglobulin/Ccr and enzymuria was noted in both group. Percentage decrease of Ccr on the next day was positively correlated with FENa before angiography (r = 0.645, p < 0.01). Of 24hr Ccr, AER, and FENa before angiography, FENa was revealed as a statistically significant discriminant factor for contrast nephropathy by stepwise discriminant analysis (p = 0.0008). These results suggest that contrast nephropathy develops predominantly in the stage not of incipient but of overt diabetic nephropathy indicated by a decline of glomerular filtration, overt proteinuria, and tubular dysfunction. Of them, tubular dysfunction may be the most important risk factor for contrast nephropathy.

Aged↗