Yukio Yasukochi

MICA gene polymorphism in Takayasu's arteritis and Buerger's disease

To further clarify the HLA-linked genes susceptible to arterio-vasculitis of unknown etiology, Takayasus arteritis and Buergers disease, polymorphism in the MICA gene, a newly identified gene near the HLA-B gene and expressed in epithelial cell lineage, was investigated. Polymerase chain reaction (PCR)-DNA conformation polymorphism (DCP) analysis and subsequent sequencing of the MICA gene have revealed that there are 5 MICA alleles which are different in the number of a GCT repeat in exon 5: MICA alleles MICA-1.1, -1.2, -1.3 and -1.4 have 9, 6, 5 and 4 GCT repeats, respectively, and MICA-1.5 has 5 GCT repeats with a 1 bp frameshift insertion in the repeat. MICA genotyping data in 81 Japanese patients with Takayasus arteritis, 38 Japanese patients with Buergers disease, and 160 healthy Japanese controls showed that MICA-1.2 and -1.4 were significantly associated with Takayasus arteritis and Buergers disease, respectively. Because MICA-1.2 and -1.4 were in strong linkage disequilibria with HLA-B52 and -B54 in the Japanese populations, respectively, we have compared the odds ratio (OR) of the risk to the diseases for individuals having both or each of the disease-associated MICA and HLA-B alleles. It was found that MICA-1.2 gave a significantly high OR of risk to Takayasus arteritis in the absence of HLA-B52, suggesting that the HLA-linked gene susceptible to Takayasus arteritis is mapped near the MICA gene. In contrast, MICA-1.4 gave a significantly high OR of risk to Buergers disease only in the presence of HLA-B54, suggesting that the HLA-linked gene susceptible to Buergers disease is linked to the HLA-B54-MICA-1.4 haplotype, and may be differently mapped from that to Takayasus arteritis.

Transitional change in interaction between HIF-1 and HNF-4 in response to hypoxia.

AbstractThe roles of the erythropoietin (Epo) 3′ enhancer in the activation of gene expression in response to hypoxia were investigated. The enhancer contains hypoxia-inducible enhancer binding site 1(HIF-1 element) and two direct repeats of hexanucleotide consensus nuclear receptor half site (HNF-4 element). HIF-1, which is a heterodimeric complex of HIF-1α and aryl hydrocarbon receptor nuclear translecator (ARNT), binds to HIF-1 element. HNF-4 binds to HNF-4 element as a homodimeric complex. Studies on mutant reporter plasmids demonstrated that both HIF-1α and HNF-4 elements were necessary for augmentation of the enhancer activity, since mutation of either the HIF-1 or the HNF-4 element caused loss of inducibility under hypoxic conditions. Mammalian two-hybrid experiments in vivo revealed that transitional change took place from the interaction of HNF-4 with ARNT to that with HIF-1α in response to hypoxia. Such interactive domains were identified in amino acids 369–465 containing the C-terminal of HNF-4 and amino acids 1–458 containing basic helix-loop-helix (bHLH) and Per-ARNT-AHR-Sim (PAS) domains of ARNT in normoxia. Also, an extended sequence containing ligand and dimerization domains, and the C-terminal of HNF-4 (amino acids 135–465), and the PAS domain (amino acids 106–526) of HIF-1α were used for the interaction between the two transcription factors in hypoxia. From these data, the functional significance of the transitional change in the augmentation of gene expression by the Epo enhancer in hypoxia is discussed.

HLA class II DNA typing in Buerger's Disease

To analyze the genetic factors involved in the pathogenesis of Buergers disease, the polymorphisms of HLA class II genes (HLA-DP, -DQ and -DRB) were investigated in 36 patients using polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) method. HLA class I (B54 and B52) and HLA class II (DQ alleles) genes were also typed by polymerase chain reaction-sequence-specific oligonucleotide probe (PCR SSOP) method to confirm alleles. PCR-RFLP typing revealed that the frequencies of HLA class II genes, DRB1*0405, DQB1*0401, DQA1*03 and DPB1*0501, were significantly increased in the patients with Buergers disease compared with the healthy controls. Further, DNA typing confirmed that HLA-B54 was associated with Buergers disease. These results suggested that the haplotype of HLA-B54-DRB1*0405-DQB1*0401- DQA1*03-DPB1*0501 was associated with Buergers disease in Japanese population.

Differentiation-Induced Transmigration of HL60 Cells across Activated HUVEC Monolayer Involves E-selectin-Dependent Mechanisma

Abstract: The leukocyte‐endothelial adhesive interaction is one of the key mechanisms during inflammation. The human promyelocytic cell line HL60 has been used in a number of studies to characterize leukocyte‐endothelial interactions, especially selectin‐mediated adhesion. HL60 also has been used in studies to characterize the myeloid cell function during differentiation. In this study, we investigated the adhesive interactions of HL60 to vascular endothelium, either in its undifferentiated state or after dimethylsulfoxide‐induced granulocytic differentiation. Granulocytic differentiation of HL60 cells significantly enhanced their transmigration across cytokine‐activated (IL‐1β 10 U/ml, 4 h) HUVEC monolayer. Interestingly, this enhanced transmigration of differentiated HL60 cells was inhibited by pretreatment of the monolayers with anti‐E‐selectin mAb as well as anti‐ICAM‐1 mAb or anti‐VE‐cadherin mAb, suggesting a potential role for E‐selectin in transendothelial migration. Further study of this enhanced transmigration mechanism may elucidate the regulation of selectin‐mediated leukocyte‐endothelial interactions.

Significance of carbohydrate antigen sialyl-Lewis X, sialyl-Lewis A, and possible unknown ligands to adhesion of human urothelial cancer cells to activated endothelium

Objective: To assess the contribution of the carbohydrate antigens, sialyl-Lewis X (sLex) and sialyl-Lewis A (sLea), which are known to be ligands for E-selectin, to the adhesion between human urothelial cancer cells and cytokine-activated human endothelial cells. Materials and Methods: We studied the expression of sLex and sLea antigens of three bladder cancer cell lines (JTC 30, JTC 32, and T24) by flow cytometry and the adherence to interleukin 1β-activated human umbilical vein endothelial cells (HUVEC). Results: JTC 30 and JTC 32 cells expressed both sLex and sLea antigens, and showed adhesion to activated HUVEC, which was completely abolished by anti-E-selectin antibody. T24 cells expressed neither sLex nor sLea antigen, and did not adhere to activated HUVEC. Each of anti-sLea or anti-sLex antibody partially blocked the attachment of JTC 30 cells to activated HUVEC, and combination of these antibodies almost completely blocked the adhesion. The combination of antibodies did not significantly influence the adhesion of JTC 32 cells. Conclusion: These results indicate that both sLea and sLex carbohydrate antigens are involved in E-selectin-mediated adhesion of some urothelial cancers, and that there might be unknown ligands for E-selectin on urothelial cancer cells.

Cell-cycle-dependent phosphorylation of the basal transcription factor RAP74.

In this report, cell‐cycle‐dependent effects of TFIID on other basal transcription factors were investigated. We purified TFIID fractions from HeLa cells synchronized in the S/G2 phases and in early G1 phase, and show that RAP74 is phosphorylated more highly by the S/G2 phase TFIID fraction than by the early G1 phase TFIID fraction. Further analyses using deletion mutants of RAP74 revealed that amino acid residues 206–256 are phosphorylated by the TFIID fraction. Reconstitution of in vitro transcription activity indicates that the cell‐cycle‐dependent phosphorylation of RAP74 increases TFIIF transcription activity.

Potential Role of Recombinant Annexin II in Diabetic Vascular Injury

Abstract: Hyperinsulinemia and hyperglycemia have been associated with vascular injury such as atherosclerosis in diabetes mellitus. Recently, annexin II, a member of annexin family proteins, has been found to work as co‐receptor on endothelial cells for plasminogen and tissue plasminogen activator, facilitating plasmin generation on the surface of vascular endothelium. In this review, we overviewed the effect of glucose and insulin on plasmin generation in endothelial cells and its potential modulation by recombinant annexin II (rAN II) based on our data.

Inhibition by (±)-indenestrol A of interferon gamma-stimulated nitric oxide formation in murine macrophage RAW 264.7 cells

We examined the effects of (+/-)-indenestrol A (IA), an antioxidative and superoxide-producing metabolite of diethylstilbestrol (DES), on the activation of murine macrophages (RAW 264.7 cells) in vitro, particularly with regard to interferon (IFN)-gamma-induced nitric oxide (NO) production. (+/-)-IA inhibited NO production more strongly than DES as assessed by a nitrite assay. The inhibitory effect of (+/-)-IA on IFN-gamma-induced intracellular NO production was confirmed by direct staining of intracellular NO with diaminofluorescein-2 diacetyl. Inhibition of NO production was confirmed by Western blot analysis of IFN-gamma-induced NO synthase. Under IFN-gamma-stimulated conditions, the IFN-gamma activation site (GAS), which was the most important transcription factor, was significantly inhibited by (+/-)-IA. (+/-)-IA also promoted the activation of NF-kappaB. (+/-)-IA at 1 and 3 microM delayed the onset of apoptosis. Our results suggest that (+/-)-IA inhibited the activation of macrophages, resulting in the suppression of NO-mediated apoptosis. These results suggest a novel mechanism for the carcinogenic promoting activity of DES via its metabolite, (+/-)-IA.

Construction and characterization of a Notl-BsuE linking library from the human X chromosome

Abstract We describe the construction and characterization of methylation-resistant sequence-tagged Not I linking clones specific for the X chromosome, referred to as Not I- Bsu E linking clones. The approach consists of methylating the X-chromosome-specific cloned DNA with Bsu E methylase (M. Bsu E), an enzyme that methylates the first C residue in the CGCG sequence, followed by selection of the methylation-resistant Not I sites by insertion of a kanamycin-resistance gene in the clones cleavable by Not I. The frequent occurrence of Not I sites in CpG islands is expected to cause methylation of a large number of Not I sites with Bsu E methylase, thereby rendering them resistant to Not I cleavage. Thus, the combination of M. Bsu E and Not I yields less frequent cutting than the Not I alone. We have isolated, partially squenced, and characterized 113 Not I- Bsu E linking clones, and mapped 50 clones to various regions along the chromosome.

Multiple hemoglobins in the golden hamster.

Abstract 1. 1. Seven distinct benzidine-positive bands were found on starch-gel electrophoresis at pH 8.6 of the hemolysates from fetal, newborn and adult golden hamsters. Four of the seven benzidine-positive bands were analyzed and named Hemoglobins I, II, III and IV according to their increasing electrophoretic mobility. Hemoglobins I and II were found as major components in the fetal stage. Hemoglobin I gradually decreased and disappeared by the 11th day after birth. Hemoglobin II, however, gradually decreased but remained as a minor component in the newborn and adult. Hemoglobins III and IV were found as minor components in the fetal stage, the former as a major component and the latter as a minor one in the newborn and adult stages. 2. 2. The amino acid compositions of the polypeptide chains from these hemoglobins were determined. Few differences among the αI, αIII and αIV chains were found in the amino acid compositions. There were apparent differences between the αII and other α chains involving a minimum of eleven amino acid residues. Among the non-α chains, the βII chain was very similar to the βIII chain and there were distinct differences among the γI, βIII and βIV chains. 3. 3. The tryptic peptide patterns of the aminoethylated (AE) αI, AE αII and AE αIV chains were closely similar to one another. In the patterns of the AE αIII and AE αII chains, one peak of the AE αIII was replaced by one acidic peak and two basic peaks of the AE αII chain. On the other hand the AE γI, AE βIII and AE βIV chains were quite different from one another in the tryptic pattern, while the pattern of the AE βII chain was similar to that of the AE βIII chain.