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Biomedical subjects

M Shima

Publications and source records attributed to M Shima.

At least 73 records · Page 4Linked to original sources
PubMed1999

Identification of the binding site for an alloantibody to von Willebrand factor which inhibits binding to glycoprotein Ib within the amino-terminal region flanking the A1 domain.

An alloantibody to von Willebrand factor (vWF) which developed in a Japanese boy with type 3 von Willebrand disease has been characterized. The antibody was non-precipitating IgG and the main subclasses were IgG2 and IgG4. The antibody inhibited completely ristocetin-induced platelet aggregation (RIPA) and high shear stress-induced platelet aggregation (SIPA). Its predominant inhibitory role was focused, therefore, on the interaction between vWF and platelet gycoprotein Ib. The antibody reacted with a 52/48 kDa tryptic fragment of vWF (residues 449-728). No reaction was seen, however, with either a 39/34 kDa dispase fragment (480-718) or a recombinant vWF fragment (residues 465-728). These findings suggested that the essential epitope resided in the amino-terminal flanking region of the Al domain. We synthesized overlapping peptides corresponding to the region containing D3/A1 boundary. A peptide, residues 458-472, bound to the antibody and dose-dependently blocked the antibody binding to the 52/48 kDa fragment. The same peptide neutralized the inhibitory effect of the alloantibody on SIPA. The data are consistent with the presence of an epitope within residues 458-472 which reacted with the 52/48 kDa fragment. Furthermore, the specific component of the antibody, directed against residues 458-472, blocked vWF binding to GPIb in absence of exogenous agonist. Our results suggest that the region flanking the Al domain plays an important role in regulating vWF binding to GPIb.

Antibody Specificity↗
PubMed1999

[Detection and genotype analysis of GB virus C/hepatitis G virus (GBV-C/HGV) in the sera of Japanese patients with hemophilia A].

The recently discovered GB virus C/hepatitis G virus (GBV-C/HGV) has been shown to be scarcely associated with hepatitis, though its possible pathogenesis remains unknown. In previous report, we designed primers for semi-nested reverse transcription-polymerase chain reaction (RT-PCR) to detect 5'-noncoding region of GBV-C/HGV, and screened the sera from 45 patients with liver diseases or on hemodialysis. In this study, we studied the prevalence of GBV-C/HGV infection in the sera from 35 patients with hemophilia A by this RT-PCR method, and also performed sequence analysis for all isolates including those from the non-hemophilic patients previously reported. GBV-C/HGV was detected in 8.57% (3/35) of the hemophilic patients. The isolates from hemophilic patients had several common mutations, and these mutations were also common in the "West African" genotype isolates represented by the prototype GBV-C. However, sequences in the isolates from other positive patients with liver diseases or on hemodialysis were quite different from those in the isolates from hemophilic patients, and those in the "European/American" genotype isolates represented by the prototype HGV. These sequences were consistent with "Asian" genotype. Simple restriction fragment length polymorphism (RFLP) analysis using the restriction enzyme HhaI successfully discriminated these genotypes. In the hemophilic patients, GBV-C/HGV might have been transmitted by transfusions of the imported plasma-derived clotting factor concentrates. Thus the difference in GBV-C/HGV genotype between the hemophilic patients and the others is suggested to be related to transmission route.

Adolescent↗
PubMed1998

Residues Glu2181-Val2243 contain a major determinant of the inhibitory epitope in the C2 domain of human factor VIII.

The human blood coagulation factor VIII C2 domain (Ser2173-Tyr2332) contains an epitope recognized by most polyclonal inhibitory anti-factor VIII alloantibodies and autoantibodies. We took advantage of the differential reactivity of inhibitory antibodies with human and porcine factor VIII and mapped a major determinant of the C2 epitope by using a series of active recombinant hybrid human/porcine factor VIII molecules. A series of five C2-specific human antibodies and a murine anti-factor VIII monoclonal antibody, NMC-VIII/5, inhibited a hybrid containing a substitution of porcine sequence for Glu2181-Val2243 significantly less than human factor VIII. In contrast, four of the five patient antibodies and NMC-VIII/5 inhibited a hybrid containing a substitution of porcine sequence for Thr2253-Tyr2332 equally well as human factor VIII. Thus, a major factor VIII inhibitor epitope determinant is bounded by Glu2181-Val2243 at the NH2-terminal end of the C2 domain. Because C2 inhibitors block the binding of factor VIII to phospholipid and von Willebrand factor, for which binding sites have been localized to Thr2303-Tyr2332, these results imply that the segment bounded by Glu2181-Val2243 also is involved in these macromolecular interactions.

Amino Acid Sequence↗
PubMed1998

24R,25-dihydroxyvitamin D3 increases cyclic GMP contents, leading to an enhancement of osteocalcin synthesis by 1,25-dihydroxyvitamin D3 in cultured human osteoblastic cells.

The effect of the physiological vitamin D metabolite 24R, 25-dihydroxyvitamin D3 [24R,25(OH)2D3] on human osteoblastic cells was assessed. Physiological concentrations (10(-9)-10(-8) M) of 24R, 25(OH)2D3 significantly increased the cyclic guanosine 5'-monophosphate (cGMP) content in the human osteoblastic cells by approximately 200% in 5 to 15 min. In contrast, 24S, 25-dihydroxyvitamin D3 had only a weak effect on the cGMP content, and 1,25-dihydroxyvitamin D3 [1,25(OH)2D3] did not affect the content. The production of osteocalcin was not induced by 10(-9)-10(-8) M of 24R,25(OH)2D3 in the absence of 1,25(OH)2D3. However, the same concentration of 24R,25(OH)2D3 showed stimulatory effects on osteocalcin synthesis in the presence of 10(-9) M 1, 25(OH)2D3. Rp-8Br-cyclic GMP, a specific inhibitor of cyclic GMP-dependent protein kinase, significantly inhibited the cooperative effect of 24R,25(OH)2D3 with 1,25(OH)2D3 on the osteocalcin synthesis, although Rp-8Br-cyclic AMP, a specific inhibitor of cyclic AMP-dependent protein kinase, did not affect the cooperative effect. In addition, okadaic acid enhanced the osteocalcin synthesis induced by 1,25(OH)2D3. These observations suggest that 24R,25(OH)2D3 has a unique activity of increasing cGMP contents in osteoblastic cells, and that the increase in cGMP contents may lead to the cooperative effect of 24R,25(OH)2D3 with 1, 25(OH)2D3 on osteocalcin synthesis. These data support the hypothesis that 24R,25(OH)2D3 has a physiological role in human bone and mineral metabolism.

24,25-Dihydroxyvitamin D 3↗
PubMed1998

Some human inhibitor antibodies interfere with factor VIII binding to factor IX.

Factor VIII (fVIII) functions as a cofactor of factor IXa in the intrinsic pathway of blood coagulation. Its absence or abnormality causes the bleeding disorder hemophilia A. About 23% of hemophiliacs who receive therapeutic fVIII infusions develop antibodies that inhibit its activity. We previously showed by inhibitor neutralization assays that the fVIII A2 and C2 domain polypeptides contain common inhibitor epitopes. Often hemophilic inhibitor plasmas were partially neutralized by C2 and more completely neutralized by fVIII light chain (A3-C1-C2), suggesting the presence of an additional major inhibitor epitope(s) within the A3-C1 domains. In immunoprecipitation assays, 17 of 18 inhibitor IgGs bound to recombinant 35S-A3-C1. Amino acids 1811-1818 of the A3 domain comprise a binding site for factors IX and IXa. Three inhibitor IgGs prevented binding of factor IXa to fVIII light chain, and the binding of each IgG to light chain was competed by A3 peptide 1804-1819. The generation of factor Xa by the fVIIIa/fIXa complex in a chromogenic assay was prevented by these inhibitors. Therefore, we propose that another important mechanism of fVIII inactivation by human inhibitors is the prevention of fVIIIa/fIXa association.

Antibodies, Blocking↗
PubMed1998

The role of platelet von Willebrand factor in the binding of factor VIII to activated platelets.

Factor VIII binds to activated platelets and contributes to the tenase complex assembled on the platelet membrane surface. We have examined the role of platelet von Willebrand factor in the binding of factor VIII to platelets using a platelet captured enzyme-linked immunosorbent assay. Purified factor VIII bound to activated normal platelets in a dose dependent manner. Factor VIII also bound to platelets obtained from a patient with Type 2N von Willebrand disease, although in this case the binding was reduced to approximately 50% of that seen with control platelets. Furthermore, factor VIII bound to Type 3 von Willebrand disease platelets in the absence of detectable von Willebrand factor. In this instance the binding reaction appeared to be approximately 30% of that seen with the same number of normal platelets. An anti-A3 domain monoclonal antibody, NMC-VIII/10, which recognizes the amino-terminal acidic region of the factor VIII light chain, and an anti-C2 domain monoclonal antibody, NMC-VIII/5, which also moderates the binding of factor VIII to phosphatidylserine, inhibited the association between factor VIII and platelets. Inhibition was more remarkable with NMC-VIII/5 than with NMC-VIII/ 10 but not complete. The findings suggest that the binding of factor VIII to activated platelets is not based on a single ligand-receptor relationship, although a predominant role exists for the platelet von Willebrand factor. Furthermore, both the amino-terminal acidic region of the A3 domain and the C2 domain participate in the binding of factor VIII to activated platelets.

Binding Sites↗
PubMed1998

Indoor nitrogen dioxide in homes along trunk roads with heavy traffic.

OBJECTIVES: To assess the distribution of indoor nitrogen dioxide (NO2) concentrations in homes located in differing environments, and to investigate the influence of factors such as automobile exhaust on the indoor environment. METHODS: The concentrations of indoor NO2 over 24 hours were measured in both the heating and non-heating periods in homes of pupils from nine elementary schools in Chiba, Japan. Information on factors that could influence indoor environments was collected by questionnaire. RESULTS: Indoor NO2 concentrations during the heating period were higher in homes with unvented heaters than in homes with vented heaters, although the concentrations varied greatly among homes primarily because of the type of heating device used. During the non-heating period, indoor NO2 concentrations were significantly higher in homes adjacent to trunk roads than in homes located in other areas. Multiple regression analysis showed that indoor NO2 concentrations were associated with atmospheric NO2 in homes with vented heaters during the heating period, and in homes in areas other than on the roadside during the non-heating period. In areas other than the roadside, cigarette smoking in indoor environments also significantly contributed to indoor NO2. The average concentrations of indoor NO2 in the homes of pupils attending each school were significantly related to the atmospheric NO2 in areas other than the roadside. However, the relation between indoor and atmospheric NO2 concentrations was not significant in roadside areas. CONCLUSIONS: These findings suggest that indoor NO2 concentrations are related to the atmospheric NO2 and type of heating appliances, and are also affected by automobile exhaust in homes located in roadside areas.

Air Pollution↗
PubMed1998

Analysis of localization of mutated tissue-nonspecific alkaline phosphatase proteins associated with neonatal hypophosphatasia using green fluorescent protein chimeras.

Hypophosphatasia is associated with a defect of the tissue-nonspecific alkaline phosphatase (TNSALP) gene. The onset and clinical severity are usually correlated in hypophosphatasia; patients with perinatal hypophosphatasia die approximately at the time of birth. In contrast, we describe a male neonatal patient with hypophosphatasia who had no respiratory problems and survived. He was compound heterozygous for the conversion of Phe to Leu at codon 310 (F310L) and the deletion of a nucleotide T at 1735 (delT1735), causing the frame shift with the result of the addition of 80 amino acids at the C-terminal of the protein. Because the C-terminal portion of TNSALP is known to be important for TNSALP to bind to the plasma membrane, the localization of wild-type and mutated TNSALP proteins was analyzed using green fluorescent protein chimeras. The expression vectors containing the complementary DNA of fusion proteins consisting of signal peptide, green fluorescent protein, and wild-type or mutated TNSALP, caused by delT1735 or F310L mutation, were introduced transiently or stably in Saos-2 cells. The delT1735 mutant failed to localize at the cell surface membrane, whereas the wild-type and the F310L mutants were located in the plasma membrane and cytoplasm. The assay for enzymatic activity of TNSALP revealed that the delT1735 mutant lost the activity and that the F310L mutant exhibited an enzymatic activity level that was 72% of the normal level. The F310L mutation was also detected in another neonatal patient with relatively mild (nonlethal) hypophosphatasia (reported in J Clin Endocrinol Metab, 81:4458-4461, 1996), suggesting that residual ALP activity of the F310L mutant contributes to the less severe phenotype. The patient is unique, with respect to a discrepancy between onset and clinical severity in hypophosphatasia.

Alkaline Phosphatase↗
PubMed1998

Hypophosphatemic rickets accompanying congenital microvillous atrophy.

This report concerns an 11-year-old boy who manifested hypophosphatemic rickets associated with congenital microvillous atrophy (CMA). He had been suffering from vomiting and severe diarrhea from the first day of life and had been treated with total parenteral nutrition (TPN) since he was 67 days old. At 4 years of age, intestinal biopsy resulted in a diagnosis of CMA. He was admitted to our hospital complaining of leg pain at the age of 11. Laboratory data revealed hypophosphatemia, elevated serum 1, 25-dihydroxyvitamin D (1,25(OH)2D) levels, and hypercalciuria. A roentgenogram showed rickets in the extremities. A balance study of phosphate in urine and stool indicated that the amount of phosphate leaking into the stool was greater than that into the urine. Moreover, the total amount of phosphate leaking from both the intestine and kidney exceeded the amount of phosphate intake from TPN. The rickets was healed by increasing the phosphate concentration in TPN. This case is different from X-linked hypophosphatemic rickets but similar to hereditary hypophosphatemic rickets with hypercalciuria (HHRH) in terms of hypercalciuria and elevated serum 1,25(OH)2D levels. The effectiveness of phosphate treatments used here is also similar to that used for HHRH. However, this type of hypophosphatemic rickets is unique in that phosphate leaking into the intestine plays an important role in its pathogenesis.

Absorptiometry, Photon↗
PubMed1997

Detection of glycosaminoglycans as a copper(II) complex in high-performance liquid chromatography.

Glycosaminoglycans including heparin, heparan sulfate, chondroitin sulfate, dermatan sulfate and hyaluronan were analyzed by high-performance gel filtration chromatography. Detection was achieved at 240 nm based on the formation of copper(II) complex in copper sulfate solution at low pH. Detection of the copper(II)-heparin complex is sensitive, permitting the analysis of as little as 10(-7) g. This method was also successfully applied to the analysis of the chemically derivatized glycosaminoglycans (de-N-acetylated and/or oversulfated chondroitin sulfate) that cannot be depolymerized by enzymes such as heparin and chondroitin lyases.

Chelating Agents↗
PubMed1997

Crystal structure of NMC-4 fab anti-von Willebrand factor A1 domain.

We have solved the crystal structure of the Fab fragment of NMC-4, a mouse monoclonal antibody that binds to the A1 domain of von Willebrand factor (vWF). Two Asp and three Tyr residues in the complementarity determining regions 1 and 3 of the heavy chain exhibited a spatial orientation suggestive of a dominant role in establishing contact with the antigen. A cluster of Asp and Tyr residues occurs also in a region of the platelet glycoprotein (GP) Ib alpha amino terminal domain known to be critically involved in vWF binding. Thus, the structural information obtained with NMC-4 may prove relevant to understand the stereochemical bases of the GP Ib alpha-vWF interaction essential for thrombus formation at sites of vascular lesion.

Amino Acid Sequence↗
PubMed1997

Anti-factor VIII inhibitor alloantibodies recognizing the A2 domain in the human factor VIII heavy chain poorly bind to porcine factor VIII.

Anti-factor VIII (FVIII) inhibitor alloantibodies from 11 patients with hemophilia A, along with five anti-FVIII neutralizing monoclonal antibodies, were examined for differences in their reactivities with the A2 and C2 domains of human and porcine FVIII. None of the patients had been previously treated with porcine FVIII. Six inhibitors which specifically recognized the human FVIII C2 domain bound to both the 76-kDa porcine FVIII light chain and its 69-kDa proteolyzed fragments, showing cross-reactivity against porcine FVIII between 33 and 100%. Two A2-specific inhibitors did not react with porcine FVIII. The cross-reactivity was low (0-0.5%). The inhibitors recognizing both C2 and A2 reacted with the 76- and 69-kDa bands of porcine FVIII light chain, with cross-reactivity of between 11 and 33%. Monoclonal antibodies recognizing A1 (C-5) and A2 (JR8) did not react with the porcine FVIII. No anti-porcine FVIII neutralizing activity was detected in these antibodies. Monoclonal antibodies to the amino-terminal portion of A3 (NMC-VIII/10 and C-2) poorly reacted with the 76-kDa band, the cross-reactivities being 0 and 0.5%, respectively. NMC-VIII/5 recognizing C2 which competes with the C2-specific inhibitor, reacted with both the 76- and 69-kDa fragments showing cross-reactivity of 13%. These findings suggest that porcine A2 is antigenically different from human A2. The A2-specific inhibitor is a useful indicator for therapy with porcine FVIII.

Animals↗
PubMed1997

Factor VIII gene analysis in Japanese CRM-positive and CRM-reduced haemophilia A patients by single-strand conformation polymorphism.

Haemophilia A is the most common X-linked blood coagulation disorder; it is caused by deficiency of factor VIII activity (FVIII:C). Half of the affected patients do not have detectable levels of FVIII protein in their plasma, whereas about 5% have normal levels of the FVIII antigen (FVIII:Ag) (> 50 u/dl), and are called cross-reacting material (CRM) positive (CRM+ or A+). About 45% of patients have reduced levels of the FVIII:Ag (1-50 u/dl), classified as CRM reduced (CRM[R] or A[R]). We screened the FVIII gene of 13 Japanese patients (five CRM+ and eight CRM[R]) by single-strand conformation polymorphism, and identified 11 different mutations in 13 patients by analysing all 26 exons (Trp255Cys, Tyr473Cys, Gly479Arg, Arg531His, Thr667Arg, Arg1689Cys, Arg1941Gln, Arg2150His, Arg2159Cys, Thr2245Ala and Gly2285Val). Seven mutations were identified in the A domains (four in the A2 domain). All the mutations are point mutations resulting in missense codons. Four mutations (Trp255Cys, Thr667Arg, Thr2245Ala and Gly2285Val) have not been described previously.

Enzyme-Linked Immunosorbent Assay↗