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Biomedical subjects

M Amagai

Publications and source records attributed to M Amagai.

At least 145 records · Page 8Linked to original sources
PubMed1993

Desmoyokin, a 680 kDa keratinocyte plasma membrane-associated protein, is homologous to the protein encoded by human gene AHNAK.

We have obtained a monoclonal antibody (33A-3D) that specifically recognize desmoyokin, a 680 kDa desmosomal plaque protein that is well characterized in bovine muzzle epidermis. A cDNA clone (DY6, 3693 bp) was isolated by immunoscreening a mouse keratinocyte expression library with 33A-3D, and it was confirmed that DY6 has a partial coding sequence for desmoyokin. DY6 consists of highly homologous repeats about 128 residues long. Furthermore, the 128-residue repeats exhibit a quasi seven-residue substructure, which we believe will adopt an antiparallel beta-sheet structure. Surprisingly, the amino acid sequence showed a significant homology with AHNAK, a newly identified human gene encoding a 700 kDa protein, which was suggested to be down-regulated in neuroblastoma. From its extensive homology, the similarity in both size and structure, and the identical patterns on Southern blot analysis of genomic DNAs, desmoyokin and AHNAK protein are thought to be identical. Although the desmoyokin/AHNAK protein is detected in a variety of cell types at both protein and mRNA levels, its distribution in keratinocytes (associated closely with cell membrane) is quite different from that in cells other than keratinocytes (distributed diffusely in the cytoplasm). These findings suggest that the desmoyokin/AHNAK protein is a ubiquitous molecule with a unique structure and appears to have different distributions (and probably different functions) among different cells.

Amino Acid Sequence↗
PubMed1992

Association of ras p21 with differentiation of epidermal keratinocytes in proliferating skin diseases.

We examined the localization of DNA replicating cells and ras oncogene product p21 positive cells in proliferating skin diseases, such as psoriasis vulgaris, lichen planus, verruca vulgaris, verruca plana juvenilis and seborrheic keratosis. ras p21-positive cells were found rather in the differentiated layers than in the proliferating layers of the epidermis. We indicate that the expression of ras p21 can be associated with the differentiation of epidermal keratinocytes not only in tumor tissues but also in inflammatory skin disease.

Cell Differentiation↗
PubMed1992

Autoantibodies against the amino-terminal cadherin-like binding domain of pemphigus vulgaris antigen are pathogenic.

Complementary DNA cloning of the 130-kD pemphigus vulgaris (PV) autoantigen (PVA) has indicated that it is a member of the cadherin family of Ca(2+)-dependent cell adhesion molecules. By homology with typical cadherins, PVA has five extracellular domains (EC1 through EC5). To localize immunogenic domains and to determine whether antibodies against them might be pathogenic, we produced beta-galactosidase fusion proteins with cDNA encoding different portions of the extracellular domains of PVA (EC1-2, EC3-5, and each individual domain). Immunoblot analysis of these fusion proteins with 23 PV patients' sera demonstrated that major immunogenic regions of PVA are located on the EC1, EC2, and EC4 domains. IgG was affinity-purified from PV sera on fusion proteins representing the amino (EC1-2) and carboxy (EC3-5) terminus of the extracellular PVA, and injected into neonatal mice. PV IgG affinity-purified on the EC1-2 fusion protein caused suprabasilar acantholysis, the typical histological finding of PV, but IgG affinity-purified on the EC3-5 fusion protein or beta-galactosidase alone did not. These results indicate that at least one pathogenic epitope, which is sufficient to cause suprabasilar acantholysis in neonatal mice, is located on the amino-terminal region of PVA, an area thought to be important in cadherin homophilic adhesion.

Amino Acid Sequence↗
PubMed1992

[The role of bronchial responsiveness to leukotrienes in antigen-induced immediate and late asthmatic response].

In order to clarify the role of leukotriene in the antigen-induced asthmatic response, we carried out mite-antigen, leukotriene D4 (LTD4) and histamine inhalation tests in 21 mite-sensitive atopic asthma patients, and investigated the relationship between the bronchial responsiveness to mite (PD20-Mite) and that to LTD4 and histamine (PD20-LTD4, PD20-Hist). We then compared PD20-LTD4 and PD20-Hist between the groups with and without late asthmatic response (LAR), and investigated the relationship between the peak percentage fall of FEV1.0 in LAR (magnitude of LAR) and PD20-LTD4 and PD20-Hist. There were significant correlations between PD20-LTD4 and PD20-Mite (r = 0.590, p < 0.01), and between PD20-Hist and PD20-Mite (r = 0.669, p < 0.001). The bronchial responsiveness to LTD4 and histamine was significantly higher in the group with LAR. In addition, PD20-LTD4 showed a significant negative correlation with the magnitude of LAR (r = 0.724, p < 0.001). Furthermore we analyzed the relationship between PD20-LTD and the ratio of the magnitude of LAR to that of IAR (immediate asthmatic response), because there was a significant correlation between the magnitude of IAR and that of LAR (r = 0.842, p < 0.001) in 16 asthmatic patients with dual asthmatic response, and found that the correlation coefficient was also very high (r = -0.836, p < 0.001). These results suggest an important role of leukotriene in the antigen-induced asthmatic response.

Adolescent↗
PubMed1992

Eosinophil infiltration and enhancement of airway reactivity by leukocyte chemotactic factor, formyl-methionyl-leucyl-phenylalanine (fMLP), in guinea pigs.

N-formyl-L-methionyl-L-leucyl-L-phenylalanine (fMLP) is a bacterial-derived chemotactic factor for eosinophils and neutrophils. This study is aimed to examine whether or not eosinophil infiltration induced by intra-airway administration of fMLP causes the damage of the bronchial epithelium and results in airway hyperresponsiveness in normal non-sensitized guinea pigs. In normal guinea pigs fMLP administered by aerosol inhalation or intratracheal injection caused significant infiltration of eosinophils in the tracheal mucosa and enhanced bronchial reactivity to inhaled histamine 6 and 24 hours after exposure. Electron microscopic examination showed damage of the alignment of the epithelial cells in the bronchial mucosa in fMLP-treated guinea pigs. PAF antagonists CV3988 and WEB2086 and a 5-lipoxygenase inhibitor (AA-861) did not prevent fMLP induced eosinophil infiltration, which suggests that fMLP caused eosinophil infiltration mainly by its chemotactic activity, not by the release of platelet activating factor (PAF) or leukotrienes in this experimental condition. These results showed that in normal guinea pigs a bacteria-derived chemoattractant of fMLP could reproduce a sequence of eosinophil infiltration and airway hyperresponsiveness, similar to the inflammatory pathophysiology after antigen challenge in sensitized animals. We concluded that eosinophil infiltration induced by either immunological or non-immunological mechanisms can cause airway damage and airway hyperresponsiveness.

Administration, Inhalation↗
PubMed1991

Autoantibodies against a novel epithelial cadherin in pemphigus vulgaris, a disease of cell adhesion.

Pemphigus vulgaris (PV) is a life-threatening skin disease in which autoantibodies against a keratinocyte cell surface 130 kd glycoprotein, PV antigen (PVA), cause loss of cell-cell adhesion, with resultant epidermal blisters. We used affinity-purified PV IgG to isolate cDNA, containing the entire coding sequence for PVA, from human keratinocyte expression libraries. Northern blot analysis indicated PV mRNA expression only in stratified squamous epithelia. The deduced amino acid sequence of PVA was unique but showed significant homology with members of the cadherin family of Ca(2+)-dependent cell adhesion molecules, most markedly to desmoglein I. These findings demonstrate that a novel epithelial cadherin is the target of autoantibodies in PV.

Amino Acid Sequence↗
PubMed1991

Southern analysis of the 230-kD bullous pemphigoid antigen gene in normal humans, animals, and patients with junctional epidermolysis bullosa.

To begin to characterize the 230-kD bullous pemphigoid antigen (BPA) gene, we performed Southern analysis on genomic DNA with probes derived from 7 kb of cDNA that spans most of the coding region of this hemidesmosomal plaque protein. When hybridized to a 1-kb fragment of this BPA cDNA, normal human genomic DNA digested with EcoRI, BamHI, PstI, HindIII, or EcoRV showed only a single band, which was unique for each enzyme, indicating a single human gene for BPA. To determine if a related gene exists in animals, we used probes covering the full 7 kb of cDNA for Southern analysis of genomic DNA from various vertebrates. A related gene was detected in other mammals (monkey, cow, dog, rabbit, mouse, and rat) but not in chicken, frog, or fish. Under these same hybridization conditions a probe for human beta-actin could detect an actin gene in all these species. Furthermore, immunofluorescence showed that an antibody raised against portions of the 230-kD BPA bound to the epidermal basement membrane of mammals but not that of a bird or amphibian. Finally, because most patients with junctional epidermolysis bullosa (JEB) have defective hemidesmosomes in ultrastructure, and probably function, we analyzed genomic DNA from these patients. No restriction fragment length polymorphisms (RFLP) were detected when the DNA from 11 normals and 8 JEB patients (representing 16 possible defective genes) was digested with BamHI, EcoRI, or PstI and hybridized to any part of the cDNA. These findings indicate that 1) there is a single BPA gene in humans; 2) a closely related gene exists in other mammals but not birds, amphibia, or fish; and 3) gross abnormalities of the BPA gene are not characteristic of JEB patients.

Animals↗
PubMed1991

Characterization of bullous pemphigoid antibodies by use of recombinant bullous pemphigoid antigen proteins.

The seroreactivity of patients with bullous pemphigoid (BP) to recombinant proteins representing sequences in the carboxyl domain of the murine 230-kD BP antigen (BPA) was determined. Sera from 133 patients with BP, 20 patients with pemphigus, and 21 normal subjects were examined by Western blotting by using two recombinant proteins: RP120 (MW = 120 kD), representing the C-terminal half of the 230-kD BPA, and RP60 (MW = 60 kD), representing the C-terminal quarter. These RP120 and RP60 were recognized by 84% and 61%, respectively, of the BP sera that reacted with the 230-kD BPA in epidermal extract, and not by any of pemphigus or normal sera. Furthermore, these RP120 and RP60 were not recognized by any BP sera that reacted only with the 170-kD BPA, which is known to be another major BPA. These findings indicate that one or more of the major antigenic regions localizes in the carboxyl-half domain of the 230-kD BPA, and also suggest that the 230-kD BPA may be distinct from the 170-kD BPA.

Animals↗
PubMed1991

Trichilemmal tumor arising in a seborrheic keratosis: analysis of cell kinetics by BrdU staining.

We report a case of a 74-year-old male with a trichilemmal tumor arising in a seborrheic keratosis on the buttock and the results of a cell kinetic study of this tumor using a BrdU staining method. The incidence of trichilemmal tumor arising in a seborrheic keratosis seems to be extremely rare. The labeling index of this tumor was 12.0%; this was a level intermediate between normal epidermis and a variety of hyperproliferative skin diseases such as squamous cell carcinoma, Bowen's disease, and psoriasis vulgaris. DNA replicating cells were present in the germinative layers in normal epidermis and the benign hyperproliferative skin diseases, psoriasis vulgaris. In contrast, DNA replicating cells were found throughout the entire epidermis in premalignant and malignant tumors such as in Bowen's disease and squamous cell carcinoma. In this case, DNA replicating cells were localized mainly in the basal and parabasal cell layers, but also seen in the upper squamous layers. These findings suggest that this trichilemmal tumor had a malignant tendency, though it was slow-growing and relatively benign in nature.

Aged↗
PubMed1991

Localization of the human gene for 230-kDal bullous pemphigoid autoantigen (BPAG1) to chromosome 6pter----q15.

Chromosome mapping of the human gene encoding the 230-kDal autoantigen of an autoimmune skin disease, bullous pemphigoid, was performed using flow-sorted human chromosomes of cells of normal karyotype and cells carrying a reciprocal translocation t(6;16)(q15;q24). The cDNA of the autoantigen hybridized with intact chromosome 6 and translocation chromosome 6p- (6pter----q15::16q24----qter). The gene (BPA230) was located to the chromosome region 6pter----q15.

Animals↗
PubMed1990

Placental glutathione-S-transferase-pi mRNA is abundantly expressed in human skin.

Four overlapping cDNA clones were isolated from a lambda gt11 human placenta cDNA library using purified human IgG antibody, from a patient with bullous pemphigoid. The sequence was homologous to human placenta glutathione-S-transferase-pi (GST-pi). Using the placenta clone, epidermal cDNA clones were isolated from a human keratinocyte library. Expression of GST-pi mRNA in human skin, cultured keratinocytes and fibroblasts, and disorders of squamous hyperplasia was demonstrated by Northern blotting and in situ hybridization. Human epidermal and placental cDNA clones hybridized to the same genomic DNA fragments. Hybridization of placental cDNA to interspecific somatic cell hybrids showed retention of chromosome 11, confirming the assignment of GST 3 to the long arm of chromosome 11 by molecular means. Anti-GST-pi antibody did not give a basement membrane zone pattern, although some normal and BP sera contained antibodies to GST-pi. Human skin expresses glutathione-S-transferase-pi, which belongs to an enzyme family important for detoxification and carcinogenesis.

Amino Acid Sequence↗
PubMed1990

Partial cDNA cloning of the 230-kD mouse bullous pemphigoid antigen by use of a human monoclonal anti-basement membrane zone antibody.

A cDNA clone with the coding sequence for the 230-kD bullous pemphioid (BP) antigen was isolated from a mouse cDNA expression library by using an anti-basement membrane zone human monoclonal antibody (MoAb-5E). The lambda gt11 cDNA expression library was constructed from poly(A)+RNA from the mouse epidermal cell line, Pam cells, by random priming. 1.5 X 10(5) recombinant clones were screened by immunostaining with MoAb-5E and one positive clone (BPM1) was obtained. All of the ten BP sera but none of the five normal or seven pemphigus sera tested reacted with the fusion protein produced by BPM1. The size of the cDNA was 3.2 kb. Northern blot analysis indicated that BPM1 cDNA hybridized to a mRNA of about 9 kb, which is large enough to encode for a 230-kD protein. DNA sequencing demonstrated a 2,991-bp open reading frame encoding a peptide of 115 kD. Sequence comparison between mouse and human cDNA clones revealed that the 230-kD BP antigen was well conserved during evolution except for the carboxyl terminus. Highly conserved and hydrophilic regions in the molecule were considered to be good candidates for antigenic determinants. This cDNA clone will be useful not only for diagnosis of BP, e.g., enzyme-linked immunosorbent assay using recombinant proteins or synthetic peptides as antigens, but also for pathophysiologic study in which mouse models of BP might be used.

Amino Acid Sequence↗
PubMed1990

Association of EGF receptor expression with proliferating cells and of ras p21 expression with differentiating cells in various skin tumours.

The localization of DNA replicating cells, epidermal growth factor (EGF) receptor-expressing cells and ras oncogene product p21 (p-21ras) positive cells were examined in various skin tumours to elucidate the role of EGF receptor and p21ras in the epidermis. Normal skin, keratoacanthoma (KA), solar keratosis (SK), Bowen's disease (BD), squamous cell carcinoma (SCC), basal cell carcinoma (BCC) and extramammary Paget's disease (PD) were studied. EGF receptors were seen in proliferating layers, where DNA replicating cells localize, but p21ras was found in the more differentiated layers. We conclude that EGF receptor expression is closely associated with cellular proliferation, but p21ras may play a role in the differentiation of cells in various skin tumours.

Cell Differentiation↗
PubMed1990

T cell receptor gene rearrangement analysis in mycosis fungoides and disseminated lymphocytoma cutis.

A cDNA from the CT beta 2 region of the T cell receptor gene was used as a probe to investigate the clonal composition of T cells in the skin lesions of 3 patients with mycosis fungoides (MF) and 1 patient with disseminated lymphocytoma cutis (DLC). All of these skin lesions showed helper/inducer T cell dominant infiltration. Clonal T cell populations, as evidenced by rearranged DNA bands, were demonstrated in all the skin lesions of the 3 patients with MF, but not in the lesion of the patient with DLC. The patients with MF appeared to have the same gene rearrangement pattern in DNA obtained from separate lesions, providing evidence that the T cells in both sites were derived from the same clone. Our results show that this gene rearrangement analysis helps to diagnose difficult cases of lymphoproliferative disorders.

Adult↗
PubMed1990

[Antigen-induced biphasic eosinophil infiltration in the airways of actively sensitized guinea pigs and its inhibition by PAF antagonist and cyclosporin A].

Bronchial eosinophilia is a characteristic of asthma. To elucidate the mechanisms of eosinophil accumulation in the airways, the time course of eosinophil infiltration in the airway mucosa after antigen inhalation was examined in actively sensitized and passively sensitized guinea pig models of asthma. The lungs and tracheae were removed at intervals after antigen challenge, fixed and stained. The eosinophil infiltration was quantitated in the tracheal walls by counting the number of cells per square millimeter. Guinea pigs sensitized by intraperitoneal injection of ovalbumin (OA) responded to a single exposure to aerosolized OA with biphasic infiltration of eosinophils in the tracheal walls; a striking early-phase which peaked at 6 hr and a delayed-phase which peaked at 24 hr and persisted for as long as 5 days. These kinetics were different from those observed with passively sensitized animals which showed only early-phase infiltration. Administration of CV-6209, a specific PAF antagonist, before and 12 hrs after antigen challenge significantly (p less than 0.01) inhibited the early-phase but not the delayed-phase eosinophil infiltration in actively sensitized animals. In contrast, when guinea pigs were treated with Cyclosporin A, a T lymphocyte-selective immunosuppressive agent, throughout the immunization period, the delayed-phase but not the early-phase infiltration was significantly (p less than 0.01) inhibited. These results suggest that PAF may contribute to early-phase and T cell factor(s) may contribute to delayed-phase eosinophil infiltration of the airways.

Animals↗
PubMed1989

Cloning of TPA-inducible early (TIE) genes by differential hybridization using TPA-nonresponsive variant of mouse 3T3-L1 cells.

The tumor promoter 12-O-tetradecanoylphorbol-13-acetate (TPA) induces DNA synthesis in quiescent 3T3-L1 cells but not in its variant VT-1 cells. A lambda gt10 cDNA library was constructed using poly(A)+ RNA from 3T3-L1 cells that were stimulated by TPA for 20 min. Radioactive cDNA probes were prepared from mRNAs of TPA-treated 3T3-L1 and VT-1 cells and used for screening of the 3T3-L1 cDNA library by differential hybridization. Nine of 6000 phase plaques hybridized only to the 3T3-L1 cDNA probe. Analysis of the nucleotide sequence of five of these clones indicated a high degree of homology with human or mouse type I and type III collagen genes. Three other independent clones showed no homology with any known DNA sequences. These isolated clones of TPA-inducible early (TIE) genes may be useful to study the signal transduction pathway of phorbol esters.

Amino Acid Sequence↗
PubMed1989

Vesicular pemphigoid--ultrastructural and immunoelectron microscopic study.

We report a patient with vesicular pemphigoid who showed clinically numerous small vesicles and figurate erythema over the trunk and extremities. Although large blisters were absent, immunofluorescent study revealed IgG and C3 deposition at the basement membrane zone of the lesional skin. The patient responded well to the combined use of corticosteroid and dapsone. Immunoelectron microscopy showed IgG and C3 deposition at the undersurface membrane of the basal cells and the lamina lucida. Electron microscopy of the nonvesiculated early erythematous skin showed small vacuoles and lacunae along the uppermost portion of the dermis. Although the clinical features of this patient and the good therapeutic response to dapsone were atypical, the results of immunopathology and immunoelectron microscopy were consistent with typical pemphigoid.

Aged↗