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H Sasakawa

Publications and source records attributed to H Sasakawa.

18 recordsLinked to original sources
PubMed2000

Analysis of accumulated T cell clonotypes in patients with systemic lupus erythematosus.

OBJECTIVE: To compare the accumulated T cell clonotypes in peripheral blood (PB) samples obtained at various times, and the accumulated T cell clonotypes in a PB sample and in an affected kidney, from patients with systemic lupus erythematosus (SLE). METHODS: Peripheral blood mononuclear cells (PBMC) were obtained at 2-4 different times from each of 5 SLE patients, with or without flare-up of the disease; in addition, a biopsied kidney tissue sample was obtained from 1 of the patients. RNA was extracted from each sample and complementary DNA was prepared. Genes that encode the variable region of T cell receptor (TCR) B chains (BV) of 3 BV families, 5S1, 8, and 14, were amplified by reverse transcription-polymerase chain reaction (PCR), and the PCR products were cloned for sequencing. RESULTS: A total of 877 cloned TCR genes was detected in the PBMC samples and the kidney sample. Oligoclonal T cell expansion was detected in 34 of the 36 PCR-amplified BV samples from PBMC (amplification of 3 BV families in 2-4 samples from 5 patients). The composition of clonally expanded T cell clonotypes was relatively stable in the patients with inactive SLE. In contrast, the composition of clonotypes in the PB changed drastically after the patient experienced the active phase of the disease. T cell clonotypes that had accumulated in the kidney appeared to be restricted and distinct from those that had accumulated in the PB of the same patient. CONCLUSION: Different T cell clonotypes expand at different times and at different sites in patients with active SLE. The sensitizing antigens may change over the course of the disease and may be different at each site.

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↗
PubMed1999

Comparison of T-cell receptor Jbeta gene usage in spleen cells of different mouse strains.

To elucidate whether T-cell receptor Jbeta gene usage was affected by major histocompatibility complex haplotypes and other genetic backgrounds, we investigated such usage with Jbeta-specific probes in four different mouse strains. As a result, (a) frequent usage of Jbeta2.1 and Jbeta2.6, (b) infrequent usage of Jbeta1.3, Jbeta1.5 and Jbeta1.6, and (c) predominant usage of the Jbeta2 cluster compared to the Jbeta1 cluster were found. Importantly, these biases were common to almost all the tested Vbeta families of the four strains. Thus, TCR Jbeta usage would be independent of the major histocompatibility complex haplotypes and other genetic backgrounds.

Animals↗
PubMed1998

Amelioration of lymphoid hyperplasia and hypergammaglobulinemia in lupus-prone mice (gld) by Fas-ligand gene transfer.

We recently demonstrated that the transplantation of wild-type bone marrow cells into lupus-prone mice (gld), resulted in the normalization of autoimmune syndromes due to induction of direct elimination of pathogenic cells by apoptosis via Fas/Fas ligand (L) interactions. This finding supports the beneficial therapeutic effect of Fas-mediated apoptosis on autoimmunity in gld mice. To further establish the therapeutic effect of Fas-mediated apoptosis on autoimmunity, we investigated the effect of cells transfected with the FasL gene on autoimmune symptoms in gld mice. The FasL transfectants exhibited cytotoxic activity against gld splenocytes via the Fas/FasL system in vitro. In vivo administration of irradiated-FasL transfectants induced a reduction in hypergammaglobulinemia, the disappearance of lymphoid hyperplasia and of the accumulation of gld cells (B220+ T-cells). Furthermore, in situ nick end labelling analysis revealed that cells in the spleen and lymph nodes frequently underwent apoptosis. These results clearly indicate that FasL transfectants induce the apoptosis of the pathogenic cells responsible for hypergammaglobulinemia and lymphoid hyperplasia in gld mice by cell/cell interaction via the Fas/FasL system. Thus, ex vivo gene transfer of FasL may represent a new therapeutic strategy for autoimmunity caused by the FasL dysfunction.

Animals↗
PubMed1998

Contribution of the T cell receptor BJ gene to recognition of the P91A tumor antigen in DBA/2 mice.

To understand specific immune responses against a tumor, it is important to characterize T cells that recognize the tumor antigen. The mouse P91A antigen is one of the well-defined tumor antigens that is expressed on the P911 cell line, and T cells responding to the antigen in DBA/2 mice were reported to be restricted to BV8S2/S3 families in their T cell receptor (TCR) BV gene usage. We have further characterized the P91A-responding T cells in DBA/2 mice, focusing on TCR BJ gene usage and using the polymerase chain reaction/enzyme-linked immunosorbent assay and DNA sequencing studies of their third complementarity-determining (CDR3) regions. As a result, T cells with cytotoxic activity to the P91A antigen, induced from murine spleen cells both in vivo and in vitro, showed predominant use of the BJ2S1 gene segment in both BV8S2 and BV8S3 T cells compared to unmanipulated murine spleen cells. Sequencing studies of the CDR3 regions in the BV8S3 T cells revealed clonal expansion of T cells with the BV8S3-BJ2S1 combination in two of three DBA/2 mice tested. In the remaining mouse, clonal expansion was not detected despite predominant use of the BJ2S1 segment by these T cells. These data suggest that P91A-recognizing T cells would predominantly use the BV8S2/S3-BJ2S1 combination. Analysis of T cells with these TCR BV-BJ gene combinations may contribute to the evaluation, monitoring and development of a T-cell-mediated immunotherapeutic strategy.

Animals↗
PubMed1997

T cell clonality in synovial fluid of a patient with rheumatoid arthritis: persistent but fluctuant oligoclonal T cell expansions.

Recently, oligoclonal T cell accumulation has been reported in affected joints of patients with rheumatoid arthritis. To characterize such clonally accumulating T cells, we quantitatively monitored their frequency by analyzing TCR B chains. As a result, despite a similar TCR BV usage between synovial fluid (SF) and PBL, obviously skewed TCR BJ usage was detected in SF. Subsequent DNA sequencing demonstrated that the skewed BJ gene usage in SF resulted from clonal T cell accumulation. The complementarity-determining region 3 of TCR B chains of the detected clones appeared to have homologous amino acid sequences. Further, one of the predominant TCR B chains of the clones was identical with that reported previously. In the follow-up study, most of the accumulated clones persisted; however, in some, proportions were drastically decreased or increased during the time course. In particular, one of the clones expanded sixfold, from 11 to 67%, in the BV8-BJ2S3 TCR population of SF, even though the clone was not detected in PBL. In summary, oligoclonal T cell accumulation in SF was persistent, but it appeared to fluctuate independently of the clonality in PBL. The expansion of the clones in SF may occur by antigenic stimuli within the joint.

Amino Acid Sequence↗
PubMed1996

Characterization of T cell receptor beta chains of accumulating T cells in skin allografts in mice.

The study of T cells involved in the immune reaction that occurs in engrafted organs should provide information that would be helpful in the regulation of allograft rejection in organ transplantation. Toward this end, we focused on detection and characterization of accumulating T cells in mouse skin allografts from B10.A(4R) to C57BL/6 mice in vivo. T cell receptor beta genes were amplified by reverse transcriptase-PCR from mRNA of the skin grafts, and accumulating T cell receptor beta gene clonotypes were identified by their single strand conformation polymorphism. Their joining region usage and the amino acid sequences of the complementarity-determining region-3 were then determined. The results were as follows: (1) Distinct oligoclonal accumulation of T cells was more prevalent in the skin allografts than in the syngenic skin grafts. (2) Although the accumulating T cell clonotypes appeared to use many different variable-region gene families, preferential combinations of variable region-joining region were found. (3) Several homologous amino acid sequences were found in these accumulating TCR beta genes in allografts, suggesting that these T cells are driven by the same or similar antigens. (4) In addition, little T cell accumulation was found in spleens from the mice with allografts or syngenic skin grafts. Taken together, accumulating T cells in the skin allografts were detected in vivo, and some appeared to have characteristics in common. This may lead to T cell clonotype-specific therapy in organ transplantation.

Amino Acid Sequence↗
PubMed1995

Autoepitopes of the 52-kd SS-A/Ro molecule.

OBJECTIVE: This study was undertaken to clarify the mechanisms responsible for the generation of anti-52-kd SS-A/Ro autoantibodies and to elucidate why, as has recently been reported, anti-52-kd autoantibodies preferentially recognize the denatured form rather than the native 52-kd molecule. METHODS: Using a series of truncated 52-kd autoantigens, produced as beta-galactosidase fusion proteins in Escherichia coli, the B cell epitope distribution was probed with 18 anti-Ro-positive sera by immunoblotting and by enzyme-linked immunosorbent assay. RESULTS: Nearly all the antigenicity of the molecule was found to be linked to its leucine zipper region. In a further study using 9 of the 18 sera, the antigenicity of the molecule was found to be mainly formed by multiple conformational epitopes, and one of these epitopes appeared to be universally recognized by all the sera tested. CONCLUSION: The recognition of multiple epitopes indicates that the Ro 52-kd antigen itself drives the autoimmunity to this molecule. Further, the concentration of the antigenicity at the leucine zipper region may explain why anti-52-kd antibodies preferentially recognize the denatured protein rather than its native form.

Autoantibodies↗
PubMed1995

Mammalian expression of single chain variable region fragments dimerized by Fc regions.

To promote application of a single chain variable region fragment (sFv) in immunoglobulins, a sFv gene was connected to an IgGi Fc gene, designated as a sFvc gene, and used for transfection of Sp2/0. As a result, the sFvc protein was found to be secreted in a dimeric form. It is thus felt that the sFvc protein, which mimicks the shape of a naturally occurring antibody, can be simple and useful to reproduce divalency and Fc-associated effecter functions as seen in a natural antibody.

Animals↗
PubMed1994

Comparison of the J beta gene usage among different T cell receptor V beta families in spleens of C57BL/6 mice.

To clarify whether there is a bias in the V-D-J combination of T cell receptor (TcR) genes, J beta gene usage has been investigated in a total of 743 TcR beta genes of V beta 2, V beta 8.2, and V beta 14 families expressed in C57BL/6 mouse spleens. Genes of TcR beta chains, amplified by a reverse transcriptase-polymerase chain reaction, were individually cloned into plasmids. Cloned genes (61 to 106), randomly selected in each respective V beta family from three different mice, were tested by means of hybridization with 12 oligo DNA probes which were designed to differentiate 12 murine functional J beta gene segments. The results are enumerated below. (1) The J beta 2.6 gene segment was found to be most frequently used (V beta 2, 19.8%; V beta 8.2, 21.2%; and V beta 14, 19.2%). In contrast, usage of the J beta 1.6 gene segment was most infrequent (V beta 2, 1.9%; V beta 8.2, 2.9%; and V beta 14, 0.5%); (2) High frequency of the J beta 2.1 gene segment and low frequency of the J beta 1.3 and J beta 1.5 gene segments were also observed; (3) The J beta 2 cluster was used in preference to the J beta 1 cluster (usages of the J beta 2 cluster: V beta 2, 67.8%; V beta 8.2, 65.9%; and V beta 14, 70.4%); and (4) These biases were generally common to all three V beta families examined and differences among individual mice were mostly small. Considering these findings, we conclude that the TcR J beta gene segments in C57BL/6 mice splenocytes are selected with a bias, but are selected independently of the V beta families.

Animals↗
PubMed1992

Cloning and sequence analysis of cDNA encoding aspartate aminotransferase isozymes from Panicum miliaceum L., a C4 plant.

The cytosolic and mitochondrial isozymes of aspartate aminotransferase (AspAT) function in the C4 dicarboxylate cycle of photosynthesis. We constructed a cDNA library from leaf tissues of Panicum miliaceum, an NAD-malic-enzyme-type C4 plant and screened the library for AspAT isozymes. A full-length cDNA clone for cytosolic AspAT was isolated. This clone contains an open reading frame that encodes 409 amino acids. We also isolated two cDNA clones for different precursors of mitochondrial AspAT. Comparing these two sequences in the coding regions, we found 12 amino acid substitutions out of 28 base substitutions. The encoded amino acid sequences predict that mitochondrial AspAT are synthesized as precursor proteins of 428 amino acid residues, which each consist of a mature enzyme of 400 amino acid residues and a 28-amino-acid presequence. This prediction coincides with the observation that the in vitro translation product of the mRNA for mitochondrial AspAT was substantially larger than the mature form. A comparison of the amino acid sequences of the AspAT isozymes from P. miliaceum with the published sequences for the enzymes from various animals and microorganisms reveals that functionally and/or structurally important residues are almost entirely conserved in all AspAT species.

Amino Acid Sequence↗
PubMed1990

Regulation of expression of carbon-assimilating enzymes by nitrogen in maize leaf.

We have utilized the cellular differentiation gradient of the developed, youngest leaf to examine the regulation by nitrogen of levels of phosphoenolpyruvate carboxylase (PEPCase), pyruvate orthophosphate dikinase (PPDK), and ribulose 1,5-bisphosphate carboxylase in maize (Zea mays L.). The protein whose level regulated most preferentially by N availability was PEPCase, followed by PPDK, and the changes in level occurred most conspicuously at the photosynthetically maturing cells. Pulse and pulse-chase experiments to analyze photosynthetic fixation of [(14)C]CO(2) indicate that maize leaf primarily exploited a C(4)-mode of photosynthetic fixation of carbon dioxide even under a selective reduction in levels of these proteins. The effects of N on the synthesis of these proteins and the accumulation of corresponding mRNAs during recovery from a deficiency were examined by pulse and pulse-chase labeling with [(35)S]Met and by hybridization, respectively. The rate of turnover of PPDK was substantially higher than that of the other proteins. Results also showed that the reduced accumulation of PEPCase, as well as PPDK, under N deficiency could largely be accounted for a reduced level of synthesis of protein with a concomitant reduction in level of their mRNAs. This indicates that the N-dependent selective accumulation of these enzymes is primarily a consequence of level of its mRNAs.

Journal Article↗
PubMed1989

deltaC Values in Maize Leaf Correlate with Phosphoenolpyruvate Carboxylase Levels.

Values of delta(13)C and levels of phosphoenolpyruvate carboxylase and ribulose 1,5-bisphosphate carboxylase/oxygenase were analyzed in segments from the fourth leaf of young maize (Zea mays L.) plants. The delta(13)C values became significantly more negative from the base to the tip of the leaves. Phosphoenolpyruvate carboxylase levels and ribulose bisphosphate carboxylase levels both increased from the base to the tip. The principal effect of phosphoenolpyruvate carboxylase levels or delta(13)C should arise through its effect on the carboxylation/diffusion balance in the mesophyll. In this case, delta(13)C values should become more negative as phosphoenolpyruvate carboxylase levels increase, unless there are offsetting changes in stomatal aperture. The principal effect of ribulose bisphosphate carboxylase/oxygenase on delta(13)C should occur through its effect on the extent of leakage of CO(2) from the bundle sheath cells. In this case, delta(13)C values should become more positive as ribulose bisphosphate carboxylase levels increase. Accordingly, the variation in delta(13)C values seen in maize leaves appears to be the result of variations in the level of phosphoenolpyruvate carboxylase.

Journal Article↗
PubMed1986

Root respiration associated with nitrate assimilation by cowpea.

Nitrate uptake by roots of cowpea (Vigna unguiculata) was measured using (15)NO(3) (-), and the energy cost to the root was estimated by respirometry. Roots of 8-day-old cowpea seedlings respired 0.6 to 0.8 milligram CO(2) per plant per hour for growth and maintenance. Adding 10 millimolar NO(3) (-) to the root medium increased respiration by 20 to 30% during the following 6 hours. This increase was not observed if the shoots were in the dark. Removal of NO(3) (-) from the root medium slowed the increase of root respiration. The ratios of additional respiration to the total nitrogen uptake and reduced nitrogen content in roots were 0.4 gram C per gram N and 2.3 grams C per gram N, respectively. The latter value is close to theoretical estimates of nitrate assimilation, and is similar to estimates of 1 to 4 grams C per gram N for the respiratory cost of symbiotic N(2) fixation.

Journal Article↗
PubMed1979

Effects of red, far red, and blue light on enhancement of nitrate reductase activity and on nitrate uptake in etiolated rice seedlings.

The effects of red (R), far red (FR), or blue light (B) on the enhancement of nitrate reductase (NR) activity and on nitrate uptake in etiolated rice seedlings were examined. On 5-minute illumination followed by 12-hour dark, R caused marked increase of NR activity, but FR and B caused only slight increase. Illumination with 560 ergs per square centimeter per second of R for 5 minutes caused maximal increase. The effect of R was almost completely counteracted by subsequent illumination with 2,000 ergs per square centimeter per second of FR for 10 minutes, indicating that NR induction was mediated by phytochrome. Exogenous supply of inducer nitrate was not required during the 5-minute illumination and the R-FR cycles, if the seedlings were transferred to nitrate solution at the beginning of the dark incubation. NR activity in the shoots was found high when shoots were illuminated but was low when only roots were illuminated. On continuous illumination for 12 hours, B had more effect on NR increase than R.Nitrate uptake during 6-hour dark was not increased by exposure to R, FR, or B for 5 minutes at the beginning. On continuous illumination for 6 hours, R slightly increased nitrate uptake, whereas FR and B had no effect.

Journal Article↗
PubMed1978

Comparison of the uptake of nitrate and ammonium by rice seedlings: influences of light, temperature, oxygen concentration, exogenous sucrose, and metabolic inhibitors.

The uptake of nitrate and ammonium by rice (Oryza sativa) seedlings was compared under various conditions. Nitrate uptake showed a 1-hour lag phase and then a rapid absorption phase, whereas ammonium uptake showed passive absorption during the first hour, then a shoulder of absorption, followed by a rapid metabolism-dependent absorption phase. Light did not affect the uptake of nitrate or ammonium. The uptake of nitrate and ammonium was markedly suppressed by removal of the endosperm. After removal of the endosperm, the uptake was restored by exogenous supply of 30 mm sucrose. No appreciable nitrate uptake was detected at temperatures below 15 C, whereas appreciable ammonium uptake occurred at 15 C, although lowered passive absorption and complete inhibition of the rapid metabolism-dependent absorption phase were observed at 5 C.Nitrate uptake was decreased by aeration and also by bubbling the mixture with O(2). It was greatly depressed by bubbling the mixture with N(2). These results suggest that there is a low optimal O(2) concentration for nitrate uptake. In contrast, ammonium uptake was increased by aeration, not influenced by bubbling with O(2), and slightly decreased by bubbling the mixture with N(2).Nitrate uptake was almost entirely inhibited by addition of cycloheximide, KCN, or arsenate. These compounds had scarcely any effect on the passive absorption of ammonium, but entirely depressed its successive metabolism-dependent absorption. Ammonium uptake occurred before nitrate uptake in solution containing both nitrate and ammonium.

Journal Article↗