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Biomedical subjects

E Tatsumi

Publications and source records attributed to E Tatsumi.

At least 19 recordsLinked to original sources
PubMed1998

Conformational state of ovalbumin at acidic pH as evaluated by a novel approach utilizing intrachain sulfhydryl-mixed disulfide exchange reactions.

Ovalbumin contains four cysteine sulfhydryls (Cys11, Cys30, Cys367, and Cys382) and one cystine disulfide (Cys73-Cys120). A highly reactive aromatic disulfide, 2,2'-dipyridyl disulfide, reacts specifically with Cys367 of ovalbumin at pH 2.2 generating a mixed disulfide protein derivative [Tatsumi, E., and Hirose, M. (1997) J. Biochem. 122, 300-308]. The mode of conformational fluctuation in ovalbumin was investigated at pH 2.2 using the mixed disulfide derivatives of the cystine-intact and cystine-reduced protein forms. In the presence of a high concentration of urea, both the mixed disulfide derivatives underwent rapid cysteine sulfhydryl/mixed disulfide exchanges, thereby releasing the quantitative amount of 2-thiopyridone. A peptide mapping analysis for disulfide-forming cysteines revealed that this release was mostly accounted for by the nucleophile attack on the Cys367-mixed disulfide by the nearest cysteine residue in the primary structure, Cys382. At the acidic pH, the exchange reaction was practically restricted to the cysteine sulfhydryl/mixed disulfide exchanges; no other exchange reaction, such as the cysteine sulfhydryl/cystine disulfide exchange reaction, was detected. In the absence of urea, the cystine-reduced form, but not the cystine-intact form, underwent significant sulfhydryl/mixed disulfide exchange reactions at a physiological temperature, as determined by the release of 2-thiopyridone. A kinetic analysis for the generation of disulfide-forming cysteines with Cys367 at 37 degreesC revealed that the rate for the intrachain exchange reaction was quite different for the five cysteine sulfhydryls. The effective concentrations of the five cysteine sulfhydryls relative to the Cys367-mixed disulfide were determined by using three related model reactions: the obtained values were 11.4, 4.6, 15.2, 5.9, and 8.9 microM for Cys11, Cys30, Cys73, Cys120, and Cys382, respectively. Implications of the effective concentrations for the conformational state of acidic ovalbumin are discussed.

Acids↗
PubMed1998

Elevation of the serum Fas ligand in patients with hemophagocytic syndrome and Diamond-Blackfan anemia.

Fas ligand (FasL) is a membrane protein that is expressed in activated T cells and natural killer cells. FasL binds to Fas on target cells and induces apoptosis. There exists a soluble form of FasL (sFasL), and sFasL also induces apoptosis of Fas-bearing cells. The serum sFasL concentrations were reported to be elevated in patients with large granular lymphocytic leukemia and natural killer cell lymphoma. In this study, we have measured serum sFasL concentrations in other hematological disorders, including severe aplastic anemia (SAA), hemophagocytic lymphohistiocytosis (HLH), and Diamond-Blackfan anemia (DBA). The serum sFasL concentration of age-matched healthy controls was 0.16 +/- 0.11 ng/mL (mean +/- SD, n = 22). The serum sFasL levels in the patients with HLH and DBA were 3.75 +/- 3.82 (n = 19; P < .0001, HLH v control) and 2.76 +/- 2.43 ng/mL (n = 6; P = .012, DBA v control), respectively. Serum interferon-gamma concentration was elevated in the patients with HLH (1.61 +/- 2.62 ng/mL) but not in those with DBA (below the detectable level). These results suggest that the Fas-FasL system plays a role, at least in part, in the pathophysiology of HLH and DBA.

Adolescent↗
PubMed1998

Translocation (10;12)(q24;q15) in a T-cell lymphoblastic lymphoma with myeloid hyperplasia.

We present a case of childhood T-cell lymphoblastic lymphoma (T-LBL) with a translocation (10;12)(q24;q15) as a main clonal abnormality, which to our knowledge is the first reported karyotype of this malignancy. The patient's peripheral blood and bone marrow showed marked leukocytosis mostly myeloid lineage cells, at diagnosis. The enlarged lymph node consisted of two different cell populations: CD2+/CD7+ prothymic lymphoblasts and a cluster of peroxidase-positive myeloid cells around vessels. This case might represent a rare but distinct clinical entity of LBL.

Adolescent↗
PubMed1998

Phenotypical heterogeneity of CD4+CD8+ double-positive chronic T lymphoid leukemia.

Chronic T lymphoid leukemias are defined as leukemias of post-thymic T cells. The CD4+CD8+ double-positive (DP) phenotype is seen in a few cases. Since DP generally occurs in thymic T cells, whether the DP T leukemia cells represent thymic or peripheral T cells has been a matter of controversy. To address this issue, we studied phenotypical features in eight cases of DP T cell leukemia. Thymic DP T cells and peripheral CD8+ T cells have CD8 of alphabeta subunit, while CD8alphaalpha is induced in CD4+ T cells on activation with IL-4. We found that two patients with DP T large granular lymphocyte leukemia (LGLL) showed dim expression of CD8alphaalpha, identical to the phenotype on IL-4-activated DP-T cells. The leukemic cells of these patients expressed IL-4 mRNA and produced high levels of IL-4. These findings suggest that they may be derived from peripheral CD4+ T cells. Three patients with adult T cell leukemia/lymphoma (ATLL) showed CD8alphaalpha, suggestive of an activated peripheral T cell origin. One case expressed CD8alphaalpha dim and IL-4 mRNA, while the other two cases expressed no IL-4 mRNA and showed CD8alphaalpha bright phenotype, features not found in normal T cell populations. Three patients with T-prolymphocytic leukemia (T-PLL) expressed CD8alphabeta. The DP phenotype is relatively common in T-PLL, and CD4+CD8alphabeta+ is characteristic of thymic T cells. The DP T-PLL cells did not express TdT,CD1 or recombination activating gene-1 (RAG-1), which is down-regulated at the late stage of thymic T cell development. On the basis of these findings, we propose a late thymic origin for DP T-PLL. The phenotype of DP T cells differed for each entity and appeared to correlate with minor normal DP T cell population.

Adult↗
PubMed1998

Transient appearance of CD3+CD8+ T lymphocytes with monoclonal gene rearrangement of T-cell receptor beta locus.

A benign, transient proliferation of atypical lymphocytes and a monoclonal rearrangement of the T-cell receptor beta (TRB) locus was found in a 60-year-old woman who presented with low-grade fever, anorexia and fatigue. A marked and transient atypical lymphocytosis (white blood cell count 90.5 x 10(9)/l) with CD8 surface antigen improved without specific treatment. Although tests for IgM antibodies to hepatitis A, varicella zoster, Epstein-Barr virus (EBV), and cytomegalovirus (CMV) were all negative, a monoclonal gene rearrangement of TRB locus was observed in the DNA of the proliferated atypical lymphocytes by Southern blotting. The clonal rearrangement and the atypical lymphocytes disappeared after 14 d, and the patient has remained well for 7 years. These results suggest that monoclonal proliferation of CD8 lymphocytes can occur based on a non-neoplastic aetiology.

Blotting, Southern↗
PubMed1998

Prolonged nonpulsatile left heart bypass with reduced systemic pulse pressure causes morphological changes in the aortic wall.

We investigated the morphological changes in the aorta due to reduced systemic pulse pressure in prolonged nonpulsatile left heart bypass (LHB). Nineteen adult goats were divided into 3 groups, the nonpulsatile group in which nonpulsatile LHB was conducted, the pulsatile group in which pulsatile LHB was conducted, and the control group used as the normal control. The average aortic pulse pressures were 12, 47, and 37 mm Hg, respectively. The descending aorta was subjected to morphological examination. In the nonpulsatile group, the wall was significantly thinner, and the volume ratio of smooth muscle cells (SMCs) was much lower. In terms of the SMC type classification, the proportion of SMCs with low activity and low contractility was higher, and the cell density of the SMCs was increased compared to those in the other groups. These results indicate that prolonged nonpulsatile LHB causes morphological atrophic changes in the aorta.

Animals↗
PubMed1998

Development of design methods of a centrifugal blood pump with in vitro tests, flow visualization, and computational fluid dynamics: results in hemolysis tests.

There are few established engineering guidelines aimed at reducing hemolysis for the design of centrifugal blood pumps. In this study, a fluid dynamic approach was applied to investigate hemolysis in centrifugal pumps. Three different strategies were integrated to examine the relationship between hemolysis and flow patterns. Hemolytic performances were evaluated in in vitro tests and compared with the flow patterns analyzed by flow visualization and computational fluid dynamic (CFD). Then our group tried to establish engineering guidelines to reduce hemolysis in the development of centrifugal blood pumps. The commercially available Nikkiso centrifugal blood pump (HPM-15) was used as a standard, and the dimensions of 2 types of gaps between the impeller and the casing, the axial and the radial gap, were varied. Four impellers with different vane outlet angles were also prepared and tested. Representative results of the hemolysis tests were as follows: The axial gaps of 0.5, 1.0, and 1.5 mm resulted in normalized index of hemolysis (NIH) values of 0.0028, 0.0013 and 0.0008 g/100 L, respectively. The radial gaps of 0.5 and 1.5 mm resulted in NIH values of 0.0012 and 0.0008 g/100 L, respectively. The backward type vane and the standard one resulted in NIH values of 0.0013 and 0.0002 g/100 L, respectively. These results revealed that small gaps led to more hemolysis and that the backward type vane caused more hemolysis. Therefore, the design parameters of centrifugal blood pumps could affect their hemolytic performances. In flow visualization tests, vortices around the impeller outer tip and tongue region were observed, and their patterns varied with the dimensions of the gaps. CFD analysis also predicted high shear stress consistent with the results of the hemolysis tests. Further investigation of the regional flow patterns is needed to discuss the cause of the hemolysis in centrifugal blood pumps.

Animals↗
PubMed1997

Refolding process of ovalbumin from urea-denatured state. Evidence for the involvement of nonproductive side chain interactions in an early intermediate.

Ovalbumin contains one cystine disulfide (Cys73-Cys120) and four cysteine sulfhydryls (Cys11, Cys30, Cys367, and Cys382) in a single polypeptide chain of 385 amino acid residues. The refolding mechanism of ovalbumin was investigated under disulfide-bonded and disulfide-reduced conditions using the denatured protein state, DA, as the starting protein sample. For the preparation of DA, the disulfide-intact and disulfide-reduced forms of ovalbumin were denatured by protein incubation in 9 M urea at pH 2.2. When DA was placed in a refolding buffer, pH 8.2, an intermediate state IN was produced in either the disulfide-bonded or the disulfide-reduced condition; IN showed about 60% of the native CD ellipticity at 222 nm and the intrinsic tryptophan fluorescence with the native spectrum peak but with decreased intensity. The formation of IN as detected by far UV CD ellipticity was quite rapid and finished within a mixing dead time of 20 ms. When DA was diluted with an acidic buffer, pH 2.2, a partially folded equilibrium intermediate IA with the structural characteristics equivalent to those of IN was formed. After the formations of IN and IA, the regains in CD ellipticity and tryptophan fluorescence at pH 8.2 followed biphasic kinetics in the disulfide-bonded condition but monophasic kinetics in the disulfide-reduced condition. As unexpected findings, the native disulfide in DA and IA underwent nonproductive disulfide rearrangements in the disulfide-bonded condition at an early refolding stage and then was recovered during the subsequent refolding. The integrity of overall refolding was confirmed by the observation that the proteins refolded for 20 h in the disulfide-bonded and disulfide-reduced conditions showed, on differential scanning calorimetry analyses, almost exactly the same denaturation temperatures as their native protein counterparts. These results were consistent with a refolding process for ovalbumin which includes nonproductive side chain-side chain interactions in the early intermediate IN, which requires subsequent reorganization for the correct refolding.

Animals↗
PubMed1997

Refolding of urea-denatured ovalbumin that comprises non-native disulfide isomers.

Ovalbumin, which contains one cystine disulfide (Cys73-Cys120) and four cysteine sulfhydryls (Cys11, Cys30, Cys367, and Cys382) in the native state, undergoes intrachain sulfhydryl-disulfide exchanges at high concentrations of urea, generating many non-native disulfide isomers [E. Tatsumi, N. Takahashi, and M. Hirose (1994) J. Biol. Chem. 269, 28062-28067]. The refolding of ovalbumin from the urea-denatured state was investigated. When the denatured protein was diluted 20-fold with a refolding buffer (pH 8.2), an initial burst intermediate I(N) was produced within the 20 ms instrumental dead time; I(N) showed about 60% of the native CD ellipticity at 222 nm. The intrinsic tryptophan fluorescence of I(N) showed the same peak (338 nm), but with decreased intensity (57%), as compared to the native protein. After the rapid formation of I(N), most of the ovalbumin molecules correctly refolded into the native state with slow biphasic kinetics, as evaluated by far-UV CD, tryptophan fluorescence, and trypsin-resistance analyses. Furthermore, a peptide-mapping analysis revealed that sulfhydryl/disulfide exchange reactions occurred during the refolding, thereby increasing the formation of the native disulfide. The integrity of overall refolding was confirmed by a differential scanning calorimetry analysis. These data were consistent with the view that most, if not all, of the mispaired disulfide isomers in the urea-denatured ovalbumin can correctly refold into the native state via intrachain disulfide rearrangements.

Calorimetry, Differential Scanning↗
PubMed1997

Highly ordered molten globule-like state of ovalbumin at acidic pH: native-like fragmentation by protease and selective modification of Cys367 with dithiodipyridine.

Structural characteristics of ovalbumin at acidic pH were investigated by a variety of analytical approaches. At pH 2.2, the protein appeared to assume a partially denatured, molten globule-like conformation as evaluated by the binding of a hydrophobic probe, anilino-1-naphthalene-8-sulfonate. The protein was, however, resistant to proteolysis with pepsin under conditions in which the urea-denatured form was extensively hydrolyzed. Furthermore, under more drastic proteolytic conditions, the acid ovalbumin was specifically proteolyzed at the N-terminal site of Ala351, which is located in close proximity to the canonical serpin cleavage site Ala352-Ser353 that is known to be the cleavage site at neutral pH with subtilisin and elastase in native ovalbumin. Among the four cysteine residues (Cys11, Cys30, Cys367, and Cys382), which are all known to be buried in the native ovalbumin molecule, only Cys367 was specifically modified with 2,2'-dithiodipyridine, generating a mixed-disulfide protein derivative. Upon incubation of the derivative with a high concentration of L-cysteine, the thiopyridine mixed disulfide did not undergo any bimolecular exchange reaction with the thiol in the absence of an added denaturant, indicating that the mixed disulfide group is inaccessible. The far-UV CD spectra indicated that the native secondary structure is retained in either the modified or non-modified protein; but as evaluated by the near-UV CD spectra, the asymmetric nature of aromatic side chains in the non-modified ovalbumin and of the mixed-disulfide group in the modified protein was almost lost at pH 2.2. These results are consistent with a highly ordered molten globule-like state for OVA at pH 2.2, in which side chains, but not the backbone chain, significantly fluctuate.

2,2'-Dipyridyl↗
PubMed1997

Long-term evaluation of a nonpulsatile mechanical circulatory support system.

Antithrombogenicity of a centrifugal pump (CP) developed in our institute is provided by a central balancing hole (BH) in the impeller. A current CP, the National Cardiovascular Center (NCVC)-2, was ameliorated to improve antithrombogenicity, whereby the BH diameter was widened to improve self washout flow velocity, and an edge of the thrust bearing was rounded off to minimize flow separation. Effects of these modifications were assessed in a long-term in vivo experiment. The antithrombogenicity, hemolytic property, and mechanical durability of the NCVC-2 were investigated in 3 goats. The NCVC-2 was installed paracorporeally between the left atrium and the aorta and driven as long as possible at rotating speeds of about 2,800 rpm. The NCVC-2 ran for 50, 200, and 367+ days. The mean bypass flow rates were 6.8, 5.0, and 5.3 L/min, respectively. Creatinine, blood urea nitrogen (BUN), glutamic-oxaloacetic transaminase (GOT), and glutamic-pyruvic transaminase (GPT) did not increase until one week before termination. Plasma free hemoglobin was kept to a level less than 15 mg/dl, except for the last week of the second case. These results indicate that the NCVC-2 has excellent antithrombogenicity, an acceptable hemolytic property and the necessary durability for prolonged use.

Alanine Transaminase↗
PubMed1997

Noninvasive pump flow estimation of a centrifugal blood pump.

A flow rate estimating method was investigated for a centrifugal blood pump developed in our institute. The estimated flow rate was determined by the power consumption, the rotating speed of the motor, and the hematocrit value. The power consumption and the rotating speed of the motor were measured with a wattmeter. The examinations were performed in a closed mock loop filled with goat blood with hematocrit values of 21.5%, 28%, 34%, and 42%. Measured values of blood viscosity were 2.47, 3.09, 3.71, and 5.07 mPa.s at a share rate of 37.5/s, respectively. A linear correlation between the power consumption and the pump flow rate was observed in all hematocrit values. But variations in hematocrit caused a difference in the flow rate up to 1.1 L/min at the same power consumption and rotating speed. Effects of blood viscosity on the flow estimation were corrected by the hematocrit value. The value of the coefficient of determination, R2, between the estimated flow rate and the measured flow rate was 0.988. These results may indicate that the flow estimating method calculated by the power consumption of the motor, the rotating speed, and the hematocrit value is useful in the clinical situation.

Animals↗
PubMed1997

[Localization of 8q24 break-point of Burkitt lymphoma in Japan : relationship to EBV status].

It has been reported that the break point of 8q24 in t (8; 14) (q24; q32) is located far up-stream from c-myc gene locus in endemic EBV (Epstein-Barr virus)-positive BL, while the break-point is located close to the 1st intron of c-myc gene in sporadic EBV-negative BL. Considering that no statistical analysis is available regarding BL in Japan, the break-point of chromosome No.8 was investigated in 13 BL/L3 cell lines (having t(8; 14)) and 4 fresh samples derived from Japanese patients, including 3 EBV-positive BL cell lines, by using long-distance PCR. In this PCR, one primer was set in the 2nd intron of the c-myc gene, and the other primer in Ig constant region gene, mu, gamma, alpha and epsilon. This long distance PCR can cover up to 30 kb. Thus, this PCR does'nt generate product, if the 8q24 break-point is located far up-stream (more than 50 kb) fom c-myc gene. In 2 of the 3 t (8; 14) EBV-positive BL lines, no product was generated in two lines(N831 and Middle 91), while a product was synthesized in one line(Akata), indicating that the 8q24 break-point is near the c-myc gene in Akata. In all the other BL/L3 lines, a product was synthesized. A larger number of BL cases are necessary to investigate in order to know which 8q24 break-point pattern is exhibited by EBV-positive BL in Japan, while this method is suitable for testing a large number of case materials.

Burkitt Lymphoma↗
PubMed1996

Application of long-distance polymerase chain reaction to detection of junctional sequences created by chromosomal translocation in mature B-cell neoplasms.

Junctional sequences created by chromosomal translocations in mature B-cell neoplasms, which involve immunoglobulin gene loci (IG) and putative proto-oncogenes on reciprocal partner chromosomes, are unique to neoplastic cells characterized by particular histological and immunological phenotypes. To establish a rapid and sensitive method to detect neoplastic cells carrying a specific chromosomal translocation, we have developed a novel strategy based on long-distance polymerase chain reaction (LD-PCR) amplification. Genomic DNA was extracted from tumor cells carrying t(14;19)(q32;q13), t(8;14)(q24;q32), t(3;22)(q27;q11), t(2;3)(p12;q27), or t(3;14)(q27;q32). Thirty-two to 35-mer oligonucleotide primer pairs were designed to be complementary to exons or flanking sequences of the BCL3, c-MYC and BCL6 oncogenes, and to IG constant region genes. LD-PCR with a newly available Taq polymerase for longer product synthesis successfully amplified fragments representing BCL3/C alpha junctional sequences for t(14;19); c-MYC/C mu, c-MYC/C gamma, and c-MYC/C alpha for t(8;14); BCL6/C lambda for t(3;22); BCL6/C kappa for t(2;3); 5'-BCL6/C mu, and 5'-BCL6/C gamma for t(3;14). In Burkitt's lymphoma/leukemia, all materials in which c-MYC rearrangements were detectable by conventional Southern blot hybridization showed positive LD-PCR amplification. The sizes of the amplified fragments varied from 1.8 kb to 12 kb, and these were specific to each material. Serial dilution of tumor cells or DNA in negative materials demonstrated a single band on agarose gel electrophoresis stained with ethidium bromide at a level of sensitivity of 10(-3), and hybridization with radioactive probe improved the level by one order of magnitude (1 cell in 10(4)), indicating that this LD-PCR approach is a sensitive technique capable of detecting minimal residual disease. Thus, the present study provided a useful tool for diagnosis and subsequent management of B-cell neoplasms characterized by specific chromosomal translocations.

Base Sequence↗
PubMed1996

Impact of systemic depulsation on tissue perfusion and sympathetic nerve activity.

BACKGROUND: We postulated that pathophysiologic processes under nonpulsatile circulation are related to the behavior of the sympathetic nerve activity that regulates tissue perfusion. METHODS: Pulsatile and nonpulsatile pumps were installed in parallel in the left heart bypass circuit of anesthetized goats (n = 9) so that pulsatile circulation could be converted to nonpulsatile circulation instantly. At 5 minutes before and after systemic depulsation, we measured hemodynamic indices, renal nerve activity, and regional blood flow of the brain, heart, and renal cortex. RESULTS: Renal nerve activity was significantly elevated after systemic depulsation (15.6 +/- 9.3 versus 19.4 +/- 9.8 microV), when mean aortic pressure remained almost constant. The renal cortical flow was significantly reduced after depulsation (3.61 +/- 1.23 versus 2.93 +/- 1.19 mL.min-1.g-1), whereas no significant difference was found in the regional blood flow of the brain or the heart. CONCLUSIONS: The significant reduction of renal cortical blood flow after systemic depulsation is associated with a significant increase in renal nerve activity. Our results suggest that increased renal nerve activity plays an important role in the reduction of renal function after systemic depulsation.

Animals↗
PubMed1996

Influence of ventricular fibrillation on sympathetic nerve activity under biventricular bypass circulation.

Autonomic nerves play an important role in circulatory control. The characteristics of sympathetic nerve activity (SNA) during artificial circulation, however, are not well understood. In this study, we examined the influence of natural heart beating on the renal SNA (RSNA) using pulsatile 100% biventricular assistance and electrically induced ventricular fibrillation in anesthetized goats, whose heart rate and size are similar to the human heart's. Following induction of the ventricular fibrillation, the pulse-synchronous discharges in the RSNA did not change their periodicity and quantity significantly. It was concluded that the beating of the heart had little influence on the RSNA and was presumably not essential for maintaining nervous control of circulation as long as the circulation was maintained by an artificial heart.

Animals↗