Biomedical subjects

K Sakurada

Publications and source records attributed to K Sakurada.

At least 73 records · Page 4Linked to original sources

PubMed1994

Phosphorylation of calponin by PKC is blocked by F-actin in vitro.

Polyclonal antibody against phosphorylated calponin was raised in rabbits by application of the peptide corresponding to residues 183-195 of calponin phosphorylated by protein kinase C. When calponin was incubated with protein kinase C, only free calponin was recognized by this antibody and calponin of native thin filament or that binding to F-actin did not. In experiments done using [gamma-32P] ATP, no radioactivity was detected except for free calponin. Calponin phosphorylation was suppressed in an actin dose-dependent manner and the phosphorylation of calponin was completely blocked when the actin molar ratio to calponin exceeded 10. These data suggest that phosphorylation of calponin by protein kinase C was apparently blocked by F-actin.

Actins↗

PubMed1994

Structure and chromosomal locus of the mouse gene encoding a cerebellar Purkinje cell-specific helix-loop-helix factor Hes-3.

HES-3 is a cerebellar Purkinje cell-specific helix-loop-helix factor structurally related to the products of the Drosophila hairy and Enhancer of split genes. Here, we report the nucleotide sequence and chromosomal locus of the mouse Hes-3 gene. This gene consists of four exons and the exon-intron boundaries are well conserved when compared with those of the mouse Hes-1 and Drosophila hairy genes. Southern blot and interspecies backcross analyses show that the mouse Hes-3 gene is a single-copy gene and is located around position 80 on chromosome 4. Further analysis indicates that this locus is close to the Hes-5 locus, which is different from the Hes-1 locus (position 26 on chromosome 16). These results suggest that the Hes-3 and Hes-5 genes may be clustered on chromosome 4 while the Hes-1 gene is not.

Amino Acid Sequence↗

PubMed1994

Increased incidence of unpartnered single chromatids in metaphase II oocytes in 39,X(XO) mice.

Since rare cases of sex chromosome anomalies such as XXX and XXY were observed in the offspring of our XO breeder mice, we performed a cytogenetic analysis of metaphase II oocytes of XO mice to determine whether any changes in chromosomal configurations occur. We found a significantly increased incidence of unpartnered single chromatids in metaphase II oocytes of XO mice. Such single chromatids may contribute to embryonic aneuploidy. In addition, the tendency of the X-chromosome to segregate non-randomly to the oocyte rather than to the polar body was confirmed.

Animals↗

PubMed1994

Expression of adhesion molecules on human hematopoietic progenitor cells at different maturational stages.

In this report we examined the expression of several adhesion molecules on human hematopoietic progenitor cells at different maturational stages. Human hematopoietic progenitor cell-enriched fractions were prepared from bone marrow cells by depleting lymphocytes and monocytes (CD2+, CD14+ and CD19+ cells). These cells were separated into adhesion molecule-positive and -negative cell populations by immunomagnetic separation methods and then assessed for their ability to form various colony forming cells (CFC). CD44 and CD49d were expressed on multipotent hematopoietic progenitor cells, or mixed colony forming units (CFU-Mix), erythroid burst forming units (BFU-E), granulocyte-macrophage CFU (CFU-GM) and erythroid CFU (CFU-E). Leu8 was expressed on CFU-Mix, BFU-E and some populations of CFU-GM, but not CFU-E. CD11a was expressed on some populations of CFU-Mix, CFU-GM and BFU-E. CD54 was expressed only on some populations of CFU-GM. These results suggest that Leu8, CD44, CD49d and CD11a appear to play important roles in the differentiation and proliferation of human hematopoietic progenitor cells at different maturational stages in the bone marrow microenvironment.

Antigens, CD↗

PubMed1994

Evaluation of mixed chimerism by two-step polymerase chain reaction amplification of hypervariable region MCT118 after allogeneic bone marrow transplantation.

We assessed the origin of peripheral blood cells and bone marrow cells of 92 samples obtained from 19 patients after allo BMT by two-step polymerase chain reaction (PCR) amplification of MCT118, one of the variable number of tandem repeat regions (VNTR) which has a different length in 19 of 32 sibling pairs examined, that can detect the DNA pattern of a minor cell population of only 1% without using radioisotopes. Mixed chimerism (MC) was detected in the hematopoietic cells of four patients. Two patients who showed progressive MC developed relapse of leukemia 3 months and 4 months after the detection of MC, and two patients died with bone marrow hypoplasia 61 days and 7 months after BMT. These data suggest the clinical importance of analyzing the correlation of MC and clinical complications after BMT by this method, which can be used to monitor MC in about two thirds of allo BMT patients with an adequate sensitivity.

Base Sequence↗

PubMed1994

Effect of herbimycin A, an inhibitor of tyrosine kinase, on protein tyrosine kinase activity and phosphotyrosyl proteins of Ph1-positive leukemia cells.

Herbimycin A, a benzoquinonoid anasamycin antibiotic, preferentially inhibited the in vitro growth of Ph1-positive leukemia cell lines. On the other hand, genistein, which was developed as an inhibitor of receptor-type tyrosine kinase, and other protein kinase inhibitors showed no selective inhibition of Ph1-positive leukemia cell growth. Herbimycin A also displayed an abrogative effect on the transformation of murine hematopoietic cells by transfection with a bcr/abl oncoprotein-expressing retroviral vector. The antitumor action of herbimycin A on Ph1-positive leukemia cells is related to an inhibition of activity of bcr/abl protein tyrosine kinase and a subsequent reduction of the constitutive phosphotyrosyl proteins, however, the antibiotic has no effect on the expression of bcr/abl mRNA and oncoprotein. Therefore, herbimycin A may provide an important insight into the oncogenic action of bcr/abl oncoprotein and the future development of oncoprotein-targeted therapeutic agents.

Antibiotics, Antineoplastic↗

PubMed1994

Recipient-mediated effect of a traditional Chinese herbal medicine, ren-shen-yang-rong-tang (Japanese name: ninjin-youei-to), on hematopoietic recovery following lethal irradiation and syngeneic bone marrow transplantation.

Ren-shen-yang-rong-tang (Japanese name: Ninjin-youei-to, NYT), a traditional Chinese herbal medicine, was evaluated for recipient-mediated effect on hematopoietic recovery in a murine model of syngeneic bone marrow transplantation (BMT). BALB/c recipient mice were preconditioned with a lethal total body irradiation (TBI) at a dose of 6.5 Gy and transplanted with syngeneic bone marrow (BM) cells. NYT treatments, given intraperitoneally (i.p.) once per day for 3 consecutive days in a dose of 0.625 mg, were performed either before or after TBI and BMT to assess any recipient-mediated effect of this compound. NYT pretreatment was as effective as NYT posttreatment in enhancing the total number of colony-forming unit erythroid (CFU-E) and colony-forming unit granulocyte-macrophage (CFU-GM) per marrow and spleen after TBI and BMT. NYT pretreatment caused a significant increase in marrow and splenic CFU-E and CFU-GM numbers over a prolonged period following TBI and BMT, and affected late-stage erythropoiesis (CFU-E) more profoundly than early-stage erythropoiesis (burst-forming unit erythroid, BFU-E). NYT pretreatment significantly accelerate recovery of not only erythrocyte and leukocyte counts but also platelet counts after transplantation with a limited number (1 x 10(5)) of BM cells. The same treatment, however, was significantly less effective in hematopoietic recovery after transplantation with a minimal number (1 x 10(4)) of BM cells, indicating that NYT accelerates recovery of donor-derived rather than recipient-derived cells.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗

PubMed1994

Effects of interleukin-6 on hematopoiesis in allogeneic and syngeneic bone marrow chimeras.

Effects of interleukin-6 (IL-6) on hematopoietic progenitor cells were analyzed in murine bone marrow chimeras. When IL-6 was injected into syngeneic [C3H/He-->C3H/He] bone marrow chimeras from day 1 to day 12, the numbers of highly proliferative potential colony-forming units (CFU-HPP) or colony-forming units mix (CFU-Mix) in spleen cells and bone marrow cells increased on day 14 although there was a marked increase in spleen cells but not in bone marrow cells on day 21. The numbers of CFU-HPP increased in spleen cells from allogeneic [BALB/c-->C3H/He] bone marrow chimeras injected with IL-6 on days 14 and 21. In syngeneic bone marrow chimeras, the numbers of colony-forming units granulocyte/macrophage (CFU-GM) and burst colony-forming units (BFU-E) increased similarly to those of CFU-HPP and CFU-Mix on day 14. On day 21, these were mainly increased in spleen cells. In allogeneic bone marrow chimeras, IL-6 decreased the numbers of CFU-GM and BFU-Mix dose-dependently on day 14. Only 10 micrograms of IL-6 increased the numbers of CFU-GM and BFU-E on day 21. In our previous work, we showed that platelet counts increased on day 14 in syngeneic bone marrow chimeras injected with IL-6, whereas platelet and leukocyte counts increased on days 14 and 24 in allogeneic bone marrow chimeras injected with IL-6, correlating inversely with the numbers of hematopoietic progenitor cells. Overall, primitive hematopoietic progenitors (i.e., CFU-HPP and CFU-Mix) existed primarily in spleen cells of allogeneic bone marrow chimeras on day 14, whereas those in spleen cells of syngeneic bone marrow chimeras were found on day 21. These findings indicate that the effect of IL-6 on hematopoiesis in allogeneic bone marrow chimeras is completely different from that in syngeneic bone marrow chimeras, probably via graft-versus-host reaction (GVHR) but not GVH disease (GVHD).

Animals↗

PubMed1994

Effects of 15-deoxyspergualin on proliferative responses and cytokine gene expression in vitro.

15-deoxyspergualin (DSG)-treated BALB/c spleen cells showed increased spontaneous proliferation and increased alloreactive mixed lymphocyte reactions (MLRs) when a 3-h treatment was carried out. However, when spleen cells were treated with DSG for 5 days without washing out DSG, decreased spontaneous proliferation was observed, although alloreactive MLRs against C3H/He and C57BL/6 alloantigens were increased. In contrast, cyclosporin A (CsA) induced markedly decreased alloreactive MLRs. Decreased concanavalin A (Con A)- and pokeweed mitogen (PWM)-induced responses were observed in spleen cells treated with DSG for 3 h; whereas increased phytohemagglutinin (PHA)-induced responses were observed. On the other hand, increased Con A- and PHA-induced responses were observed in spleen cells treated with DSG for 2 days, whereas PWM-induced responses were decreased. CsA-treatment induced markedly decreased mitogen-induced responses. These results suggest that the immunosuppressive mechanism of DSG differs from that of CsA. Reverse transcriptase-polymerase chain reaction (RT-PCR) method showed that interleukin 1 beta (IL-1 beta), IL-2, IL-3, IL-4, IL-5, and leukemia inhibitory factor (LIF) mRNA expression in DSG-treated spleen cells were increased by Con A stimulation, thus indicating that DSG modulates cytokine gene expression and inducing immunosuppressive mechanisms different from CsA.

Animals↗

PubMed1994

An antibody for phosphorylated myosin light chain of smooth muscle: application to a biochemical study.

An antibody with specificity for the 20 kDa myosin light chain of smooth muscle phosphorylated by myosin light chain kinase was developed. The antibody was raised against the phosphorylated synthetic peptide, Lys-Lys-Arg-Pro-Gln-Arg-Ala-Thr-phospho-Ser-Asn-Val-Phe-Cys (residues 11-22 of the myosin light chain). This antibody only recognized the monophosphorylated myosin light chain at serine 19, i.e., with no detectable recognition of nonphosphorylated or diphosphorylated serine 19 or threonine 18. The specificity was tested by EIA assaying of myosin light chain kinase activity using a 96-well plate coated with the light chain. This EIA system was as sensitive as the radioisotopic method, and the newly developed non-radioisotopic method.

Amino Acid Sequence↗

PubMed1994

Cytokine gene expression in the mixed lymphocyte culture in allogenic bone marrow transplants as a predictive method for transplantation-related complications.

We have investigated cytokine gene expression in the two-way mixed lymphocyte cultures (MLC) enhanced by concanavalin A to assess whether this is a useful predictive method for severe graft-versus-host disease (GVHD) and graft failure in nine allogeneic bone marrow transplantation (allo-BMT) patients. Our present study revealed that increased IL-2, IL-5 and IFN-gamma mRNA expression and IL-2 and IFN-gamma production in the MLC in two cases with severe lethal transplantation-related complications (graft failure and grade III acute GVHD). These findings suggest that increased cytokine mRNA expression and cytokine products in this assay may be predictive of the development of transplantation-related complications.

Adolescent↗

PubMed1994

Establishment and characterization of a new Ph1-positive ALL cell line (ALL/MIK) presenting bcr gene rearrangement on bcr-2 and ALL-type bcr/abl transcript: suggestion of in vitro differentiation to monocytoid lineage.

A new Ph1-positive acute lymphoblastic leukemia cell line, designated as ALL/MIK, has been developed from a patient with Ph1-positive acute leukemia. The ALL/MIK cells showed an immunophenotype of common ALL with rearranged JH and Jk genes. The ALL/MIK cells showed no M-bcr rearrangement using Southern blot analysis with either 3' or 5' M-bcr probes, but had the bcr gene rearrangement on bcr-2 within the first intron of the bcr gene. Consistent with this result, the reverse transcriptase-dependent polymerase chain reaction (RT-PCR) assay revealed that the ALL/MIK cells contained the transcript derived fusion of the first exon of bcr gene and the second exon of abl gene. Although the ALL/MIK cells were defined as early pre-B cells by immunophenotypical and genotypical analyses, they were capable of differentiating into monocytoid lineage by when cultured with TPA. Furthermore, another Ph1-positive ALL cell line, (TOM-1), was investigated for its ability to differentiate to monocytoid lineage. TOM-1 was also induced to monocytoid lineage by TPA. Thus, the present study suggested that the leukemic transformation in some Ph1-positive ALL may occur at the level of multipotential hematopoietic cells capable of differentiating towards lymphoid and myelo-monocytoid lineage.

Aged↗

PubMed1994

[Longitudinal assessment of hematological parameters in the elderly].

Red blood cell count (RBC), hemoglobin level (Hb), hematocrit value (Ht), and white blood cell count (WBC) were determined periodically in 499 subjects (274 males, 225 females) aged 60 and over. RBC, Hb, and Ht showed a significant decrease after 5 years in both younger (60-64) and older (65 and over) male groups, and in the female groups except for the Hb level in the older group. Comparison between the younger and older group (cross-sectional study), revealed that the older male group showed lower levels of RBC, Hb, Ht than the younger male group, but the situation was completely the reverse in the females. No significant age-related changes were observed in WBC, but it was significantly higher in both the younger and older male groups than in the female groups. A significant decline with age was observed in both male and female MCV values. On the contrary, from a cross-sectional standpoint, the MCV values in the older female group were higher than those in the younger group. These findings revealed a completely reverse outcome in some parameters, when studied longitudinally and cross-sectionally. Therefore, the data should be evaluated longitudinally to elucidate the real effect of aging. It is pertinent to apply the WHO criteria (male 13 g/dl, female 12 g/dl) to the diagnosis of anemia of elderly people.

Aged↗

PubMed1994

Cytokine gene expression by concanavalin A-stimulated peripheral mononuclear cells after bone marrow transplantation: an indicator of immunological abnormality due to chronic graft-versus-host disease.

The response of IFN-gamma, IL-2 and IL-5 mRNA expression to the stimulation of concanavalin A (Con A) in peripheral blood mononuclear cells (PBMC) after bone marrow transplantation (BMT) was analyzed using reverse-transcriptase polymerase chain reaction (RT-PCR) to assess the recovery of T cell function. The subjects were 23 patients undergoing allogeneic BMT, 1 syngeneic BMT, 1 autologous BMT and 2 normal individuals. IFN-gamma mRNA expression increased after Con A stimulation in 6 patients who had limited chronic graft versus host disease (GVHD), 14 patients who did not have chronic GVHD, each one patient receiving syngeneic and autologous BMT and 2 normal individuals. On the other hand, IFN-gamma mRNA expression was not increased by Con A stimulation in 4 patients who had extensive chronic GVHD. Also, the concentration of IFN-gamma in cultured medium in a patient with extensive chronic GVHD was not detectable. A similar low response of IL-2 and IL-5 mRNA expression to Con A was observed in these patients with extensive chronic GVHD. These findings indicate that the cytokine productive capacity of T cell (IFN-gamma and IL-2 could be produced by type 1 T helper (Th1) cells and IL-5 could be produced by type 2 T helper (Th2) cells) was suppressed in patients who had extensive chronic GVHD, while that capacity was almost normal in patients without chronic GVHD and with limited chronic GVHD. Therefore, the analysis of cytokine gene response to Con A stimulation may provide useful information regarding immune reconstitution after BMT.

Adolescent↗

PubMed1994

Hyperacute graft-versus-host disease accompanied by increased serum interleukin-6 levels.

Hyperacute graft-versus-host disease (GVHD), which progresses severely and rapidly, was observed in three patients with acute leukemia transplanted with bone marrow cells from human leukocyte antigens (HLA)-identical and mixed leukocyte reactions (MLR)-negative siblings. Hyperacute GVHD developed in these patients within 7 days after bone marrow transplantation (BMT) and was rapidly aggravated inspite of cyclosporin A (CyA) and short-term methotrexate (MTX) prophylaxis and treatment with bolus methylprednisolone (mPSL). These patients showed markedly increased serum interleukin-6 (IL-6) levels after BMT, whereas other cytokines such as interferon gamma (IFN gamma), tumor necrosis factor alpha (TNF alpha), and IL-2 were not increased compared with pretreatment levels. These findings suggest that markedly increased IL-6 levels may be related to hyperacute GVHD.

Acute Disease↗

PubMed1994

[Multiple myeloma with numb chin syndrome as the initial manifestation].

The numb chin syndrome (NCS) is characterized by chin or lower lip numbness restricted to the distribution of the mental nerve (the distal trigeminal nerve). A case of multiple myeloma with polycythemia vera was diagnosed with NCS as the initial manifestation. A 73-year-old Japanese male was admitted to our hospital in April, 1993, because of paresthesia around the chin and lower lip. X-ray film showed multiple osteolytic lesions. According to serum and urine immunoelectrophoresis, lambda type Bence Jones protein was demonstrated. The bone marrow aspiration showed the normocellular marrow with 14.1% myeloma cells. He was diagnosed a suffering from multiple myeloma and was treated with melphalan and prednisolone. He is alive at present with resolution of NCS. We discussed pathogenesis, diagnosis, and treatment of NCS.

Aged↗

PubMed1994

Cloning and characterization of human type 2 and type 3 inositol 1,4,5-trisphosphate receptors.

We have cloned cDNAs coding for human type 2 and type 3 and part of type 1 inositol 1,4,5-trisphosphate receptors (IP3Rs). The complete nucleotide sequences for type 2 and type 3 receptors were determined and the pharmacological properties of the latter were characterized. Human type 2 and type 3 IP3Rs are 2701 amino acids and 2671 amino acids long, respectively, and have significant sequence homologies as well as structural similarities including the six membrane-spanning regions near the C-termini when compared with the rat or mouse counterpart. COS-7 cells transfected with human type 3 IP3R showed characteristic inositol 1,4,5-trisphosphate (IP3)-binding properties with Kd values of 28.8 nM. The order of potency of competition with IP3 was Ins(1,4,5)P3 (IP3) > Ins(2,4,5)P3 > Ins(1,3,4,5)P4 > Ins(1,2,3,4,5,6)P6. Type 2 and type 3 IP3Rs were mapped to human chromosomes 12p11 and 6p21, respectively, by in situ hybridization. cDNA cloning of the human IP3Rs allowed us to identify the types of the receptor expressed in various human hematopoietic and lymphoma cell lines. The type 3 receptor was present in all of cell lines tested, while the type 1 or 2 receptor was expressed in only particular cell types. The differential expression of the IP3R types could confer the cell-specific regulation on the IP3/Ca2+ signalling.

Amino Acid Sequence↗

PubMed1994

[Clinical application of gene technology to monitor bone marrow transplantation].

We assessed the origin of peripheral blood cells and bone marrow cells obtained from 15 patients after allogeneic bone marrow transplantation (allo BMT) by sensitive two-step polymerase chain reaction (PCR) amplification of MCT118, a variable number of tandem repeats regions (VNTR), that can be used to detect the DNA pattern of a minor cell population of only 1% without using radioisotopes. Mixed chimerism(MC) was detected in the haematopoietic cells of 3 patients. Two patients developed relapse of leukaemia after the detection of MC and one patient died of bone marrow hypoplasia 7 months after BMT. These findings indicate the clinical usefulness of this method to monitor patients with MC. Also, we analyzed cytokine gene expression in peripheral blood mononuclear cells during the development of graft-versus-host disease (GVHD) in patients who underwent allo BMT using a semiquantitative reverse-transcriptase polymerase chain reaction (RT-PCR). The expression of interleukin(IL)-1 beta, IL-6, and tumor necrosis factor (TNF)-alpha mRNA was increased during the development of GVHD and the degree of this increment depended on the severity of the disease. These findings suggest that IL-1 beta, IL-6, and TNF-alpha produced by peripheral blood mononuclear cells play an important role in the development of GVHD. Therefore, analysis of MC and cytokine mRNA expression using the PCR technique after allogeneic bone marrow transplantation provide important information for treatment and monitoring of marrow transplant patients.

Adolescent↗