[Blood and plasma viscosity].
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Blood Viscosity↗
Biomedical subjects
M Higashihara
Publications and source records attributed to M Higashihara.
A novel domain in the CD30 cytoplasmic tail mediates NFkappaB activation.
About 100 amino acid residues in the C-terminal region are conserved among human, rat and murine CD30, a member of the tumor necrosis factor receptor (TNFR) superfamily, and can be separated into three subdomains with relatively higher conservation (D1, D2 and D3). Activation of NFkappaB by CD30 was shown to be mediated through interaction of TNFR-associated factor (TRAF) 1, 2 and 5 with the D2 and D3 subdomains. However, the function of the other conserved subdomain, D1, remained to be determined. Deletion of the D2 and D3 subdomains abolished interactions with TRAF2 and 5 but it did not affect NFkappaB activation. Reporter gene assays using deletion and mutant constructs of CD30 revealed that the D1 subdomain is sufficient for NFkappaB activation, without interaction with TRAF2 or 5, and that each subdomain alone can activate NFkappaB. Electrophoretic mobility shift assays revealed constitutive and CD30-induced NFkappaB activation in stable transformants of 293 cells expressing CD30 or a deletion mutant lacking D2 and D3 subdomains. Deletion of C-terminal 19 amino acid residues of the D1 subdomain abolished activation of NFkappaB. Substitution of alanine for one of the two threonine residues (amino acid position 524 and 529), one of which is a potential phosphorylation site in the D1 subdomain, also abolished the NFkappaB activation. Overexpression of the TRAF domain of TRAF2 or 5 had a dominant negative effect on the NFkappaB activation mediated by the D1 subdomain, thereby suggesting involvement of TRAF proteins in the signaling. Thus, the C-terminal 100 amino acid region of CD30 is composed of three independent functional subdomains, two of which contain binding sites for TRAF proteins. A novel domain in the cytoplasmic tail mediates NFkappaB activation, without direct interaction of TRAF2 or 5. Our observations suggest involvement of an unknown TRAF protein(s) in the signal transduction pathway of CD30.
Binding Sites↗
Evaluation of thrombopoiesis in thrombocytopenic disorders by simultaneous measurement of reticulated platelets of whole blood and serum thrombopoietin concentrations.
To evaluate thrombopoiesis in thrombocytopenic disorders, we simultaneously determined reticulated platelet counts in whole blood by FACScan flow cytometry and serum thrombopoietin (TPO) concentrations by a sensitive sandwich ELISA. The subjects were 40 healthy volunteers and 45 thrombocytopenic patients. In idiopathic thrombocytopenic purpura (ITP), the percentage of reticulated platelets was significantly elevated (5.61 +/- 2.02%: mean +/- SD) relative to normal controls (2.17 +/- 0.90%), but serum TPO concentrations (1.91 +/- 1.27 fmol/l) did not differ significantly from the normal range (1.43 +/- 0.62 fmol/l). The patients with aplastic anemia (AA) had decreased reticulated platelet counts and markedly increased serum TPO concentrations (13.65 +/- 10.64 fmol/l). In thrombocytopenic patients with liver cirrhosis (LC), the absolute number of reticulated platelets (1.65 +/- 1.11 x 10(9)/l) decreased similarly that in AA. However, serum TPO concentrations (1.38 +/- 0.50 fmol/l) did not increase in contrast to AA. Our findings suggested a possible dual mechanism of thrombocytopenia in LC; that is, thrombocytopenia in LC results from the decreased TPO production primarily in the liver adding to an increase in platelet sequestration in the spleen.
Anemia, Aplastic↗
[antiplatelet therapy of thrombosis].
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Aspirin↗
Thrombotic microangiopathy associated with alpha-interferon therapy for chronic myelocytic leukemia.
A 31-year-old man diagnosed as having chronic myelocytic leukemia (CML) developed renal insufficiency with nephrotic-range proteinuria during alpha-interferon (IFN) therapy for CML. A renal biopsy specimen showed remarkable thrombotic microangiopathic lesions resembling those of hemolytic-uremic syndrome. The patient had papules on both lower legs, and a cutaneous biopsy showed similar microangiopathic lesions in dermal and subcutaneous vessels. Although discontinuation of IFN and initiation of prednisolone therapy resulted in resolution of proteinuria, renal insufficiency persisted. These findings suggest that long-term IFN therapy can induce late-onset thrombotic microangiopathy in systemic microvessels.
Adult↗
Positive correlation between prolonged potentiation of binding of double-stranded oligonucleotide probe for the transcription factor AP1 and resistance to transient forebrain ischemia in gerbil hippocampus.
Gel retardation electrophoresis revealed that binding of a radiolabelled double-stranded oligonucleotide probe for the nuclear transcription factor activator protein-1 was markedly potentiated in the CA1 and CA3 subfields and the dentate gyrus of the hippocampus of the gerbils with transient forebrain ischemia for 5 min, which is known to induce delayed death of pyramidal neurons exclusively in the CA1 subfield. The potentiation was transient in the vulnerable CA1 subfield, but persistent up to 18 h in the resistant CA3 subfield and dentate gyrus. However, no significant alteration was detected in endogenous levels of cyclic AMP response element binding protein phosphorylated at serine133 in these three different hippocampal structures 3 h after the reperfusion. On the other hand, hypothermia during ischemia which is known to protect the CA1 subfield against ischemic damages, led to a prolonged elevation of the activator protein-1 binding up to 9 h after the reperfusion in this vulnerable subfield at least in part through expression of c-Fos protein. Moreover, activator protein-1 binding was significantly elevated in the CA1 subfield up to 12 h after forebrain ischemia for 2 min which is shown not to induce marked damages to the vulnerable subfield. These results suggest that prolonged elevation of DNA binding activity of activator protein-1 may be responsible for molecular mechanisms underlying the unique vulnerability and/or resistance of particular subfields to a transient ischemic insult in the gerbil hippocampus.
Adaptor Protein Complex 1↗
Early serodiagnosis of porcine reproductive and respiratory syndrome virus infection of pigs by detection of slow-reacting and complement-requiring neutralizing antibody.
In order to evaluate and enhance the sensitivity of the neutralization (NT) test for detecting antibody in pigs infected with porcine reproductive and respiratory syndrome (PRRS) virus, the effect of altered incubation conditions and complement use on neutralizing (NT) antibody titer were investigated. Higher NT antibody titers were consistently obtained by addition of 20% guinea pig fresh serum to virus-serum mixtures in NT tests. Furthermore, the complement-requiring NT antibody titer increased in many serum samples when the virus-serum mixtures, rather than being incubated at 37 degrees C for 60 min, were incubated first at 4 degrees C for 48 hr and then with a complement at 37 degrees C for 60 min. The slow-reacting and complement-requiring NT antibody was detected as early as 8 days post-inoculation. It was detected in sera collected at 8 to 28 days post-inoculation and was sensitive to 2-mercaptoethanol treatment. Sera collected at 35 to 44 days post-inoculation contained 2-mercaptoethanol resistant NT antibodies. These results indicate that the modified NT test is useful for early serodiagnosis of PRRS virus infection through detection of higher NT antibody titers, and in detecting them earlier.
Animals↗
Protein kinase C plays a key role in the cross-talk between intracellular signalings via prostanoid receptors in a megakaryoblastic cell line, MEG-01s.
In a previous study, we characterized prostanoid and thrombin receptors expressed on a megakaryoblastic cell line, MEG-01s (Blood 78, 2328-2336, 1991). In this study, we examines the mechanism of cross-talk between intracellular Ca2+ ([Ca2+]i) and cAMP signalings through prostanoid and thrombin receptors. Addition of a thromboxane (TX)A2 mimetic (U46619 or STA2) or thrombin stimulated the formation of inositol phosphates and dose-dependently augmented a prostaglandin (PG)I2 mimetic (iloprost)- or forskolin-induced cAMP formation. 12-O-tetradecanoylphorbol-13-acetate (TPA) and ionomycin, to lesser extent, also augmented iloprost-induced cAMP formation. The enhancing effect of U46619 or TPA on cAMP formation was inhibited by prolonged pretreatment of the cells with TPA (2.5 microM, 24 h), but not with calmodulin-antagonists; W-7, W-5, or KN-62. The elevation of [Ca2+]i induced by thrombin, STA2 or PGE2 was significantly suppressed by pretreatment of the cells with TPA (100 nM) as well as cAMP mimetics such as dibutyryl cAMP (5 mM), forskolin (5 microM) and iloprost (1 microM). These results suggest the key role of PKC on the cross-talk between [Ca2+]i and cAMP signalings through prostanoid and thrombin receptors; PKC, which is activated with TXA2 or thrombin, concomitantly suppress further [Ca2+]i elevation and enhances the PGI2 receptor-mediated cAMP formation, which, in turn, suppress [Ca2+]i elevation.
1-(5-Isoquinolinesulfonyl)-2-Methylpiperazine↗
A variant CD30 protein lacking extracellular and transmembrane domains is induced in HL-60 by tetradecanoylphorbol acetate and is expressed in alveolar macrophages.
We identified and cloned cDNAs for two novel CD30 mRNAs of 2.3 kb that are induced by 12-O-tetradecanoylphorbol 13-acetate (TPA) in the human myeloid leukemia cell line HL-60. These transcripts were transcribed from the intronic region just upstream of the exon coding for the transmembrane domain of the CD30 protein. The shorter cDNA had a deletion of 54 nucleotides corresponding to the 3' region of the transmembrane domain of the CD30 and which was probably caused by alternative splicing. Translation of these transcripts appeared to start from the internal methionine codon at nucleotide position 289 that corresponds to that of 1612 in the CD30 cDNA, and encode a protein of 132 amino acid residues which corresponds exactly to the C-terminal cytoplasmic domain of CD30 protein. The calculated molecular mass of this variant CD30 (CD30v) protein was 14,087. Thus, the predicted CD30v protein retains most of the cytoplasmic region, but lacks the extracellular and transmembrane domains. Northern blots detected the expression of CD30v transcripts only in the lung and the TPA-stimulated HL-60 cell line. Translation of this mRNA in vitro produced a protein of 25 kD. Immunoblotting analysis with HCD30C1, a rabbit polyclonal antibody raised against the cytoplasmic domain of CD30 protein, detected proteins with an apparent Mr 25 kD expressed in TPA-stimulated HL-60 and COS-7 cells that were transfected with both types of CD30v cDNAs. Constitutive phosphorylation of the CD30v protein was demonstrated by in vitro labeling with [32P]. Immunohistochemical studies demonstrated CD30v protein was in alveolar macrophages. Cotransfection experiments using a kappa B-site-dependent reporter construct showed that CD30v can transactivate gene expression through activation of NF kappa B, as was noted on the authentic CD30 protein. Overexpression of the CD30v induced differentiation of HL-60 cells as evidenced by an increased NBT reduction activity. These observations provided new insights into the molecular heterogeneity and biological function of CD30 in myeloid cells.
Animals↗
Primary structure of light and heavy chain variable regions of antibodies recognizing phosphorylated vimentins.
We determined the primary structure of three types of monoclonal antibodies against phosphorylated vimentin, 4A4, YT33, and MO82, which recognize phosphorylated Ser55, Ser33, and Ser82 on vimentin, respectively. The amino acid sequences between these antibodies and the anti-phosphotyrosine antibodies previously reported, (Asn/Gln)-X-(Gln/Tyr)-Ser-Tyr in the complimentarity determining region (CDR) 3 of the light chain of 4A4 and YT33, Asn-Pro-Asn-(Asn/Ser)-Gly-X-(Ser/Thr)-(Ser/Thr)-Tyr-Asn-Gln-(Arg/Lys)-Ph e-Lys in the heavy chain CDR2 of MO82, and Lys-X-Ser-(Ser/Asn) in the heavy chain CDR3 of YT33 and MO82, were highly conserved. These motifs may play a role in recognizing phosphate groups of phosphoserine and phosphotyrosine.
Amino Acid Sequence↗
Suppression of humoral immunity by monoclonal antibody to CD79b, an invariant component of antigen receptors on B lymphocytes.
CD79b is an invariant component of antigen receptors on B lymphocytes. Previous data have suggested that monoclonal antibody (mAb) to CD79b would introduce negative signals into B lymphocytes and suppress humoral immunity. We tested this hypothesis in this study using in vitro assay systems, and revealed that anti-CD79b mAb effectively suppressed the antibody response to a T-cell dependent antigen. The speculated mechanisms for this immunosuppression were: (i) down-modulation of antigen receptors, (ii) inhibition of B lymphocyte differentiation, and (iii) induction of B lymphocyte unresponsiveness. Of these three, we confirmed that the first two were actually induced by anti-CD79b mAb treatment, whereas the in vitro system could not induce the unresponsiveness of B lymphocytes.
Antibodies, Monoclonal↗
Signal transduction through mu kappa B-cell receptors expressed on pre-B cells is different from that through B-cell receptors on mature B cells.
We introduced kappa light chain genes into pre-B cells to increase the surface mu HC expression, and established transfectants expressing mature type of B-cell receptors (BCR) on pre-B-cell surfaces. Since the cytoplasmic conformations of the reconstituted BCR and intrinsic pre-B-cell receptor (pre-BCR) are identical, they would be connected with the identical signal transduction pathways in pre-B cells. By using the transfectants, we revealed that the reconstituted BCR on pre-B cells was functionally equivalent to BCR on mature B cells in terms of the induction of intracellular Ca++ mobilization. However, we found that the signal-transduction pathways through BCR on pre-B cells were quantitatively different from those mature B cells in two ways. First, cross-linkage of the reconstituted BCR on pre-B cells induced preferential tyrosine phosphorylation of p120 and p100, which was not observed when BCR on mature B cells was cross-linked. Second, BCR in pre-B cells was physically associated with a larger amount of phosphatidylinositol-3 kinase (PI-3K) than BCR in mature B cells in spite of the fact that both pre-B and B cells expressed a similar amount of PI-3K in cytoplasm. Signals through pre-BCR and BCR are known to cause distinct biological effects in B-cell development. The biochemical features in the downstream of reconstituted BCR on pre-B cells, which we revealed in this study, will be of help in understanding the mechanism of functional differences between pre-BCR and BCR.
B-Lymphocytes↗
[Fatal case of Crow-Fukase syndrome with kidney failure, acute consciousness disorder and thrombocytopenia].
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Acute Disease↗
Inhibition of 20-kDa myosin light chain exchange by monoclonal antibodies against 17-kDa myosin light chain.
Two anti-17,000 Da myosin light chain (LC17) monoclonal antibodies (MM2 and MM10), which increase the actin-activated Mg(2+)-ATPase activity of dephosphorylated smooth muscle myosin, inhibited the exchange of the 20,000 Da regulatory light chain of myosin (LC20). MM2, which shows higher potency of activation of ATPase activity, inhibited the exchange more extensively than MM10, suggesting that there is a correlation between the activation of ATPase activity and the inhibition of the LC20 exchange. The inhibition of the exchange was observed for intact myosin and heavy meromyosin but not subfragment 1, suggesting that the heavy chain at the head-rod junction is involved in the inhibition of LC20 exchange by anti-LC17 antibodies. Alternatively, the interaction between the two heads of the myosin molecule may influence the inhibition of LC20 exchange. These results suggest that LC20 interacts with both LC17 and the heavy chain, and the interaction between LC20 and LC17 is involved in the activation of actin-activated ATPase activity of smooth muscle myosin.
Actins↗
The novel variants of mb-1 and B29 transcripts generated by alternative mRNA splicing.
The Ig-alpha/Ig-beta heterodimers encoded by mb-1 and B29 genes, respectively, are crucial for the constitution of the B-cell receptor (BCR). We report here novel variants of mb-1 and B29 transcripts produced by alternative mRNA splicing. The proteins encoded by these variants are predicted to conserve transmembrane and cytoplasmic portions of Ig-alpha and Ig-beta but lack a part of the extracellular portions containing cysteine residues which are required for intramolecular and intermolecular S-S bonds. Transfection studies revealed that the variant mb-1 and B29 did not contribute to the BCR expression on cell surfaces. Although peripheral B cells contain small amounts of the variant mb-1 and B29 transcripts, treatment with an anti-IgM antibody, LPS or IL-4 induces a significant increase in amounts of the variant transcripts. These observations suggest that B-cell activation induces alternative splicing of mb-1 and B29 transcripts which encode proteins unable to constitute the BCR.
Alternative Splicing↗
Increased levels of soluble CD8 and CD4 in patients with infectious mononucleosis.
Plasma levels of soluble CD8 (sCD8) and soluble CD4 (sCD4) in 44 patients with infectious mononucleosis (IM) were studied. A marked increase in sCD8 (22366 +/- 2702 U/ml; control: 219 +/- 10 U/ml; P < 0.0001) and significant increase in sCD4 (19.3 +/- 0.9; control: 8.1 +/- 0.2, P < 0.0001) strongly suggest activation of both CD8+ and CD4+ lymphocytes, which is important in restraining Epstein-Barr virus-infected B lymphocytes. Levels of sCD8 strongly correlated with the percentage and the absolute number of both CD8+ and CD8(+)-HLA-DR+ lymphocytes. In addition, we showed increased release of sCD8 from lymphocytes in vitro and increased ratio between plasma sCD8 and the number of CD8+ lymphocytes in blood, indicating that elevation of plasma sCD8 is due to expansion of CD8+ subset as well as increased sCD8 release from each CD8+ cell. Increased sCD4 release from CD4+ lymphocytes, the number of which is not increased in the blood during IM, was also seen. Patients with more severe fever had higher levels of sCD8 and sCD4. During convalescence sCD8 and sCD4 levels showed progressive decrease; however, even at 60-119 d after onset the levels of sCD8 and sCD4 remained higher than normal, suggesting prolonged lymphocyte activation. These results suggest that sCD8 and sCD4 are useful in monitoring immune activation during IM.
Acute Disease↗
Establishment and characterization of a new human mesothelioma cell line (T-85) from malignant peritoneal mesothelioma with remarkable thrombocytosis.
A mesothelioma cell line, termed T-85, was established from a patient with malignant peritoneal mesothelioma and remarkable thrombocytosis (1.4 x 10(6)/mm3). Electron microscopically, two types of mesothelioma cells have been characterized; the major type of cells with dense-cored granules in the cytoplasm and the minor one with evenly dense granules. Immunologically, the cells showed staining for interleukin-6 (IL-6), cytokeratin, collagen type IV, vimentin, laminin, fibronectin and Factor VIII-related antigen. Quantitation by ELISA revealed a high concentration of IL-6 in T-85 cell culture supernatants. RT-polymerase chain reaction of T-85 cells showed two positive bands of cDNA at 628 and 251 base pairs indicating the constitutive expression of IL-6 and IL-6 receptor mRNA. Moreover, prominent pro-platelet process formation activity in T-85 cell culture supernatants indicated the presence of a thrombopoietic activity due mainly to IL-6 but not the c-Mpl ligand or erythropoietin. However, the fact that 15% of PPF activity remained in the supernatants treated with anti-IL-6 antibody indicated the presence of another thrombopoietic substance. T-85 is so far the first mesothelioma cell line derived from a case with remarkable thrombocytosis.
Base Sequence↗
[Soluble lymphocyte antigens in hematological diseases].
Plasma levels of soluble CD8 (sCD8) and soluble CD4 (sCD4) in patients with infectious mononucleosis (IM) and hematological disorders were studied. In IM patients, a marked increase in sCD8 (22, 366 +/- 2,702U/ml, control: 219 +/- 10U/ml, p < 0.0001) and significant increase in sCD4 (19.3 +/- 0.9, control: 8.1 +/- 0.2, p < 0.0001) strongly suggest activation of both CD8+ and CD4+ lymphocytes, which is important in restraining Epstein-Barr virus-infected B lymphocytes. We showed that the elevation of plasma sCD8 is due to expansion of CD8+ subset as well as increased sCD8 release from each CD8+ cell. Increased sCD4 release from CD4+ lymphocytes was also seen. During convalescence sCD8 and sCD4 levels showed progressive decrease; however, even at 60-120 days after onset the levels of sCD8 and sCD4 remained higher than normal, suggesting prolonged lymphocyte activation. In hematological malignancies, elevated serum levels of sCD4 and sCD8 were found in non-Hodgkin lymphoma (NHL), acute lymphocytic leukemia, multiple myeloma, acute non-lymphocytic leukemia and chronic myelogenous leukemia. Levels of sCD4 and sCD8 in patients with NHL reflect disease status and are useful in monitoring disease activity.
CD4 Antigens↗