Biomedical subjects

K Owaribe

Publications and source records attributed to K Owaribe.

At least 37 records · Page 2Linked to original sources

PubMed1998

Internalization of the 180 kDa bullous pemphigoid antigen as immune complexes in basal keratinocytes: an important early event in blister formation in bullous pemphigoid.

We have previously shown that the 180 kDa bullous pemphigoid antigen (BPAG2) is distributed on the lateral-apical (as a pool) and ventral (as hemidesmosomes) cell membranes of monolayer cultured keratinocytes and that addition of IgG purified from bullous pemphigoid (BP) patients (BP-IgG) causes the internalization of immune complexes of BPAG2 and BP-IgG from the lateral-apical cell membrane. This internalization of BPAG2 is believed to inhibit the Ca2+ induced reformation of hemidesmosomes on the ventral cell membrane, possibly by inhibiting the supply of the antigen from the lateral-apical to the ventral membranes to form hemidesmosomes. The purpose of this paper is to examine, by using biopsy specimens from BP patients (12 cases), whether or not this internalization of BPAG2 is generated in situ. The fates of BPAG2, 230 kDa BPA (BPAG1) and bound BP-IgG were traced by immunofluorescence microscopy using monoclonal antibodies to BPAG1, BPAG2 and human IgG. In more than half of the lesional and perilesional biopsy specimens, internalization of BPAG2 into the basal cells was observed, while in normal skin BPAG2 was detected on the whole surface of the basal cells without its internalization. No internalization of BPAG1 was detected in BP and normal epidermis. These results suggest that binding of BP-IgG on the lateral-apical cell surface of basal cells causes internalization of BPAG2 in situ in the epidermis of BP patients similar to that seen in cultured cell systems, and that this internalization of immune complexes of BPAG2 and BP-IgG may play an important part in blister formation in BP.

Aged↗

PubMed1998

Successful prenatal exclusion of an unspecified subtype of severe epidermolysis bullosa.

BACKGROUND: In most cases of prenatal diagnosis of epidermolysis bullosa (EB), the subtype of severe EB from which the fetus is at risk is identified by studying the specimens of the proband. In this study, the parents of a child with an unspecified subtype of severe EB sought prenatal diagnosis for their second and third pregnancies. METHODS: The firstborn of a couple (the proband) suffered generalized blistering and erosions of the skin present from delivery, and died on the 11th postnatal day of severe EB of an unspecified type. The only diagnostic specimen available from the first infant was a conventionally stained skin section for light microscopy that showed the dermo-epidermal separation. For prenatal diagnosis in the second and third pregnancies, fetal skin biopsies were performed at 19 weeks of gestation. RESULTS: In both cases, fetal skin showed no ultrastructural abnormalities and no evidence of dermo-epidermal separation. Indirect immunofluorescence was positive for monoclonal antibodies against type VII collagen, laminin 5, uncein, alpha6 and beta4 integrins, BPAG2, and HD1/plectin, which are known to be reduced or absent in specific subsets of severe EB. The pregnancies were therefore continued, and normal healthy second and third children were delivered. CONCLUSIONS: Fetal skin biopsy, together with a panel of newly developed monoclonal antibodies, provided reliable prenatal diagnosis in the present family in which preliminary information of the EB proband was limited.

Biopsy↗

PubMed1998

Difference between dorsal and ventral iris in lens producing potency in normal lens regeneration is maintained after dissociation and reaggregation of cells from the adult newt, Cynops pyrrhogaster.

In Wolffian lens regeneration, lentectomized newt eye can produce a new lens from the dorsal marginal iris, but the ventral iris has never shown such capabilities. To investigate the difference of lens regenerating potency between dorsal and ventral iris epithelium at the cellular level, a transplantation system using cell reaggregates was developed. Two methods were devised for preparing the reaggregates from pigmented iris epithelial cells. One was rotating cells in an agar-coated multiplate on a gyratory shaker and the other was incubating cells in a microcentrifuge tube after slight centrifugation. Reaggregates made of dorsal iris cells that had been completely dissociated into single cells were phenotypically transformed into a lens when placed in the pupillary region of the lentectomized host eye. None of the ventral reaggregates produced a lens. Even dorsal reaggregates could not transdifferentiate into lens when they were placed away from the pupil. The produced lenses from the reaggregates were morphologically and immunohistochemically identified. To obtain evidence whether produced lenses really originated from singly dissociated cells, we labeled dissociated cells with a fluorescent dye (PKH26) before reaggregate formation and then traced it in the produced lens.

Animals↗

PubMed1998

Novel homozygous and compound heterozygous COL17A1 mutations associated with junctional epidermolysis bullosa.

Junctional epidermolysis bullosa is a heritable, heterogeneous blistering skin disease with mechanically induced dermal-epidermal separation, mild skin atrophy, nail dystrophy, and alopecia. Four unrelated junctional epidermolysis bullosa families with different phenotypes were investigated here and four novel mutations associated with the disease were identified. Patients 1, 2, and 3 had generalized atrophic benign epidermolysis bullosa, with nonscarring blistering and varying degree of alopecia. Patient 4 had the localisata variant of junctional epidermolysis bullosa, with predominantly acral blistering and normal hair. All patients had mutations in the COL17A1 gene encoding collagen XVII, a hemidesmosomal transmembrane protein. Patients 1 and 2 carried homozygous deletions 520delAG and 2965delG, respectively. Patient 3 was compound heterozygous for a missense and a deletion mutation (G539E and 2666delTT), and patient 4 was heterozygous for a known mutation R1226X. The deletions led to premature termination codons and to drastically reduced collagen XVII mRNA and protein levels, consistent with the absence of the collagen in generalized atrophic benign epidermolysis bullosa skin. The missense mutation G539E allowed synthesis of immunoreactive collagen XVII in keratinocytes, but prevented its secretion, thus causing lack of the protein in the skin. The data suggest that different COL17A1 mutations and their combinations can result in a spectrum of biologic and clinical phenotypes of not only generalized atrophic benign epidermolysis bullosa, but also localized junctional epidermolysis bullosa.

Aged↗

PubMed1997

Polarized expression of HD1: relationship with the cytoskeleton in cultured human colonic carcinoma cells.

Hemidesmosomes (HDs) mediate adhesion of epithelial cells to the extracellular matrix and have morphological associations with intermediate-size filaments (IFs). Hemidesmosomal molecular components including HD1, the two bullous pemphigoid antigens, and the integrin alpha 6 beta 4 have been identified in HDs of stratified and complex epithelium. In this study, we report that HT29-Fu cells, a human colonic tumor cell line, express two hemidesmosomal components (HD1, alpha 6 beta 4) associated in an adhesion structure termed type II HDs. Immunofluorescence studies showed a colocalization of HD1 and alpha 6 beta 4 in basal patches between actin stress fibers. Using cytochalasin B or vinblastine, two drugs which disrupt the cytoskeleton, we demonstrate that the redistribution of HD1 was probably induced by the reorganization of the basal cytokeratin network. We also show that in vitro HD1 binds to polymerized cytokeratin intermediate filaments; this suggests that HD1 in intestinal epithelial cells functions as a linker protein connecting cytokeratin filaments to the basal plasma membrane, probably through the beta 4 subunit of the integrin alpha 6 beta 4.

Actins↗

PubMed1997

Revertant mosaicism in epidermolysis bullosa caused by mitotic gene conversion.

Mitotic gene conversion acting as reverse mutation has not been previously demonstrated in human. We report here that the revertant mosaicism of a compound heterozygous proband with an autosomal recessive genodermatosis, generalized atrophic benign epidermolysis bullosa, is caused by mitotic gene conversion of one of the two mutated COL17A1 alleles. Specifically, the maternal allele surrounding the mutation site on COL17A1 (1706delA) showed reversion of the mutation and loss of heterozygosity along a tract of at least 381 bp in revertant keratinocytes derived from clinically unaffected skin patches; the paternal mutation (R1226X) remained present in all cell samples. Revertant mosaicism represents a way of natural gene therapy.

Adult↗

PubMed1997

Three novel homozygous point mutations and a new polymorphism in the COL17A1 gene: relation to biological and clinical phenotypes of junctional epidermolysis bullosa.

Junctional epidermolysis bullosa (JEB) is a clinically and biologically heterogeneous genodermatosis, characterized by trauma-induced blistering and healing without scarring but sometimes with skin atrophy. We investigated three unrelated patients with different JEB phenotypes. Patients 1 and 2 had generalized atrophic benign epidermolysis bullosa (GABEB), with features including skin atrophy and alopecia. Patient 3 had the localisata variant of JEB, with predominantly acral blistering and normal hair. All patients carried novel homozygous point mutations (Q1016X, R1226X, and R1303Q) in the COL17A1 gene encoding collagen XVII, a hemidesmosomal transmembrane component; and, therefore, not only GABEB but also the localisata JEB can be a collagen XVII disorder. The nonsense mutations led to drastically reduced collagen XVII mRNA and protein levels. In contrast, the missense mutation allowed expression of abnormal collagen XVII, and epidermal extracts from that patient contained polypeptides of normal size, as well as larger aggregates. The homozygous nonsense mutations in the COL17A1 gene were consistent with the absence of the collagen from the skin and with the GABEB phenotype, whereas homozygosity for the missense mutation resulted in expression of aberrant collagen XVII and, clinically, in localisata JEB.

Adolescent↗

PubMed1997

The subcellular distribution of the high molecular mass protein, HD1, is determined by the cytoplasmic domain of the integrin beta 4 subunit.

The high molecular mass protein, HD1, is a structural protein present in hemidesmosomes as well as in distinct adhesion structures termed type II hemidesmosomes. We have studied the distribution and expression of HD1 in the GD25 cells, derived from murine embryonal stem cells deficient for the beta 1 integrin subunit. We report here that these cells possess HD1 but not BP230 or BP180; two other hemidesmosomal constituents, and express only traces of the alpha 6 beta 4 integrin. By immunofluorescence and interference reflection microscopy HD1 was found together with vinculin at the end of actin filaments in focal contacts. In OVCAR-4 cells, derived from a human ovarian carcinoma which, like GD25 cells, only weakly express alpha 6 beta 4, HD1 was also localized in focal contacts. Upon transfection of both GD25 and OVCAR-4 cells with cDNA for the human beta 4 subunit the subcellular distribution of HD1 changed significantly. HD1 is then no longer present in focal contacts but in other structures at cell-substrate contacts, colocalized with alpha 6 beta 4. These junctional complexes are probably the equivalent of the type II hemidesmosomes. Transfection of GD25 cells with beta 1 cDNA did not affect the distribution of HD1, which indicates that the localization of HD1 in focal contacts was not due to the absence of beta 1. Moreover, in GD25 cells transfected with cDNA encoding a beta 4/beta 1 chimera, in which the cytoplasmic domain of beta 4 was replaced by that of beta 1, the distribution of HD1 was unaffected. Our findings indicate that the cytoplasmic domain of beta 4 determines the subcellular distribution of HD1 and emphasize the important role of alpha 6 beta 4 in the assembly of hemidesmosomes and other junctional adhesive complexes containing HD1.

Animals↗

PubMed1997

Altered distribution of plectin/HD1 in dystrophinopathies.

Plectin/HD1 is a high molecular weight protein (approximately 500 kDa) that has been proposed to act as an important and versatile cytoskeletal cross-linker molecule. Mutations of the human plectin gene have recently been associated with the autosomal recessive disorder epidermolysis bullosa simplex with muscular dystrophy. We studied the expression of plectin/HD1 in various neuromuscular disorders by indirect immunofluorescence. In cross sections of normal human muscle, plectin/HD1 showed a checkerboard-like distribution with moderate to intense cytoplasmic and sarcolemmal staining in type 1 fibers and a faint staining of the sarcolemma in type 2 fibers. In longitudinal sections of plectin/HD1-positive fibers a cross-striation staining pattern was noted. This fiber type-related expression was significantly altered in the group of dystrophinopathies, whereas it was maintained in all other myopathies and denervating disorders. In seven dystrophinopathies studied, a markedly increased plectin/HD1 immunoreactivity at the sarcolemmal level of type 2 fibers was observed. Confocal laser microscopy of normal skeletal muscle revealed a colocalization of desmin and plectin/HD1 at the level of the sarcolemma. This suggests that plectin/HD1- in analogy to its demonstrated involvement in cytokeratin-hemidesmosome linkage in epidermis-may mediate the anchorage of desmin to the sarcolemma (i.e. to costameres).

Blotting, Southern↗

PubMed1996

Loss of plectin causes epidermolysis bullosa with muscular dystrophy: cDNA cloning and genomic organization.

Plectin is a widely expressed high molecular weight protein that is involved in cytoskeleton-membrane attachment in epithelial cells, muscle, and other tissues. The human autosomal recessive disorder epidermolysis bullosa with muscular dystrophy (MD-EBS) shows epidermal blister formation at the level of the hemidesmosome and is associated with a myopathy of unknown etiology. Here, plectin was found to be absent in skin and cultured keratinocytes from an MD-EBS patient by immunofluorescence and immunoprecipitation, suggesting that plectin is a candidate gene/protein system for MD-EBS mutation. The 14800-bp human plectin cDNA was cloned and sequenced. The predicted 518-kD polypeptide has homology to the actin-binding domain of the dystrophin family at the amino terminus, a central rod domain, and homology to the intermediate filament-associated protein desmoplakin at the carboxyl terminus. The corresponding human gene (PLEC1), consisting of 33 exons spanning >26 kb of genomic DNA was cloned, sequenced, and mapped to chromosomal band 8q24. Homozygosity by descent was observed in the consanguineous MD-EBS family with intragenic plectin polymorphisms. Direct sequencing of PCR-amplified plectin cDNA from the patient's keratinocytes revealed a homozygous 8-bp deletion in exon 32 causing a frameshift and a premature termination codon 42 bp downstream. The clinically unaffected parents of the proband were found to be heterozygous carriers of the mutation. These results establish the molecular basis of MD-EBS in this family and clearly demonstrate the important structural role for plectin in cytoskeleton-membrane adherence in both skin and muscle.

Actins↗

PubMed1996

Demonstration of the molecular shape of BP180, a 180-kDa bullous pemphigoid antigen and its potential for trimer formation.

The 180-kDa bullous pemphigoid antigen (BP180) is a hemidesmosomal transmembrane glycoprotein comprising interrupted collagen domains in its extracellular part. BP180 is also termed type XVII collagen. But the question of whether it actually takes a collagen-like triple helical conformation in vivo has remained unanswered. Using a monoclonal antibody, we found that a subpopulation of BP180 localizes at the lateral surfaces of corneal basal cells and cultured cells, in addition to the basal surface. This subpopulation of BP180 could be solubilized by 0.5% Triton X-100 and, among examined cell lines, was found to be most abundant in BMGE+H, a bovine mammary gland epithelial cell line. The Triton-soluble fraction of BMGE+H cells was used for characterization. On sucrose gradient centrifugation, the soluble BP180 demonstrated a value of approximately 7 S, and chemical cross-linking experiments revealed a trimer form. The calculated frictional ratio, f/f0 = 2.8, suggests an asymmetric configuration. For further characterization, we purified the soluble BP180 by immunoaffinity column chromatography using an anti-BP180 monoclonal antibody. Rotary shadowing images of the purified BP180 showed a quaver-like molecule consisting of a globular head, a central rod, and a flexible tail. With regard to the primary structure and species comparisons, the central rod, 60-70 nm in length, probably corresponds to the largest collagenous region, forming a collagen-like triple helix, in human form. The globular head and the flexible tail seem to correspond to the cytoplasmic and the interrupted collagenous region, respectively, of the extracellular portions. In conclusion, the present demonstration of the entire configuration of BP180, with a collagen-like trimer in its extracellular part, suggests that BP180 is one of the major components of anchoring filaments.

Animals↗

PubMed1996

Defective expression of plectin/HD1 in epidermolysis bullosa simplex with muscular dystrophy.

Epidermolysis bullosa simplex with muscular dystrophy (MD-EBS) is a disease characterized by generalized blistering of the skin associated with muscular involvement. We report that the skin of three MD-EBS patients is not reactive with antibodies 6C6, 10F6, or 5B3 raised against the intermediate filament-associated protein plectin. Immunofluorescence and Western analysis of explanted MD-EBS keratinocytes confirmed a deficient expression of plectin, which, in involved skin, correlated with an impaired interaction of the keratin cytoskeleton with the hemidesmosomes. Consistent with lack of reactivity of MD-EBS skin to plectin antibodies, plectin was not detected in skeletal muscles of these patients. Impaired expression of plectin in muscle correlated with an altered labeling pattern of the muscle intermediate filament protein desmin. A deficient immunoreactivity was also observed with the monoclonal antibody HD121 raised against the hemidesmosomal protein HD1. Furthermore, immunofluorescence analysis showed that HD1 is expressed in Z-lines in normal skeletal muscle; whereas this expression is deficient in patient muscle. Colocalization of HD1 and plectin in normal skin and muscle, together with their impaired expression in MD-EBS tissues, strongly suggests that plectin and HD1 are closely related proteins. Our results therefore provide strong evidence that, in MD-EBS patients, the defective expression of plectin results in an aberrant anchorage of cytoskeletal structures in keratinocytes and muscular fibers leading to cell fragility.

Adult↗

PubMed1996

Generalized atrophic benign epidermolysis bullosa. Either 180-kd bullous pemphigoid antigen or laminin-5 deficiency.

BACKGROUND: Generalized atrophic benign epidermolysis bullosa (GABEB) is a form of nonlethal junctional epidermolysis bullosa, clinically characterized by generalized blistering after birth, atrophic healing, and incomplete universal atrophic alopecia with onset in childhood. Recently, we discovered a deficiency of the 180-kd bullous pemphigoid antigen (BP180) and a reduced amount of BP180 messenger RNA in three patients with GABEB. It is not yet clear, however, whether GABEB is invariably caused by BP180 deficiency. RESULTS: We examined 18 patients with nonlethal junctional epidermolysis bullosa from unrelated families; nine of these individuals presented with the clinical characteristics of GABEB. Specimens of clinically normal skin obtained from the patients were stained by immunofluorescence with monoclonal antibodies to BP180 and laminin-5. The BP180 epitopes were not expressed in eight patients, all of whom were sharing the typical clinical features of GABEB. In one of the nine patients with GABEB, the BP180 level was sufficient, but the laminin-5 level was reduced. Among the nine patients with junctional epidermolysis bullosa without atrophic alopecia, laminin-5 level was not expressed in one patient, while in the other patients both antigens were normally expressed. CONCLUSIONS: Not all patients with GABEB are deficient in BP180, since some individuals with GABEB only exhibit reduction of the laminin-5 expression. The BP180 deficiency in the skin invariably seems to result in GABEB. Immunofluorescence analysis using monoclonal antibodies against BP180 (and laminin-5) may allow early subtyping, which is of prognostic significance, in children born with junctional epidermolysis bullosa.

Adolescent↗

PubMed1996

Plectin deficiency results in muscular dystrophy with epidermolysis bullosa.

We report that mutation in the gene for plectin, a cytoskeleton-membrane anchorage protein, is a cause of autosomal recessive muscular dystrophy associated with skin blistering (epidermolysis bullosa simplex). The evidence comes from absence of plectin by antibody staining in affected individuals from four families, supportive genetic analysis (localization of the human plectin gene to chromosome 8q24.13-qter and evidence for disease segregation with markers in this region) and finally the identification of a homozygous frameshift mutation detected in plectin cDNA. Absence of the large multifunctional cytoskeleton protein plectin can simultaneously account for structural failure in both muscle and skin.

Amino Acid Sequence↗

PubMed1996

Intracellular ionic changes induced by bullous pemphigoid IgG subclasses.

To ascertain whether membrane signal transduction is induced by bullous pemphigoid (BP) antibody and whether cell lysis is induced by its complement activation, we assessed the intracellular Ca2+ concentration ([Ca2+]i), intracellular pH, membrane potential and morphology of living cells by following the time course of fluorescence intensity of Fluo-3/AM, Snaff-1/AM, Dioc-5 and Luciffer yellow, respectively. A transient increase of Fluo-3 fluorescence intensity in DJM-1 cells (a squamous cell carcinoma line) was revealed when the cells were incubated with 2 of five IgG1 BP antibodies. However, no transient increase of Fluo-3 fluorescence intensity was revealed when the cells were incubated with IgG2 and IgG4 BP antibodies. A transient increase of Fluo-3 fluorescence intensity was revealed in DJM-1 cells incubated with 3 of seven IgG1 and 1 of four IgG2 BP antibodies in an EGTA-containing low-Ca2+ medium. On the other hand, the Dioc-5 fluorescence intensity did not change significantly, though the increase of Fluo-3 fluorescence intensity was observed. The increase of Snarf-1 fluorescence intensity was revealed in DJM-1 cells incubated with 2 of five IgG1 BP antibodies, but was not revealed in the cells incubated with IgG2 or IgG4 of BP antibodies. Study of complement activation by BP IgG1 showed a transient increase of Fluo-3 fluorescence intensity of with 3 of five IgG1 BP antibodies when DJM-1 cells were incubated with complement-supplemented normal-Ca2+ medium. At the same time, however, endocytosis and cell lysis were not observed with 2 IgG1 BP antibodies which did induce an increase of Fluo-3 fluorescence intensity when Lucifer-yellow-loaded DJM-1 cells were incubated with complement-supplemented normal-Ca2+ medium. We examined next whether anti-180 kD BP antigen monoclonal antibodies (mAbs R-223 and 233) induce an increase of Fluo-3 fluorescence intensity. MAb R-223 did not induce any increase of Fluo-3 fluorescence intensity in DJM-1 cells, when incubated with normal- and low-Ca2+ media However, mAb R-223 induced a transient increase of Fluo-3 fluorescence intensity in DJM-1 cells when incubated with complement-supplemented normal-Ca2+ medium. MAb 233 did not induced an increase of Fluo-3 fluorescence intensity in DJM-1 cells when incubated with normal- and low-Ca2+ media. These results suggest that the BP IgG1 induces Ca2+ release from intracellular storage sites, however, the complement activated by BP IgG1 does not induce cell lysis. It could not be confirmed that anti-180 kD BP antigen antibody induced Ca2+ release from intracellular storage sites.

Calcium↗

PubMed1995

Lichen planus pemphigoides: identification of 180 kd hemidesmosome antigen.

We describe a man with lichen planus pemphigoides. Direct immunofluorescence studies of peribullous skin showed linear deposition of IgG and C3 in the basement membrane zone. Indirect immunofluorescence studies disclosed circulating anti-basement membrane zone antibodies. Immunoelectron microscopy demonstrated binding of antibodies to the hemidesmosomes and lamina lucida. The patient's serum defined only the minor bullous pemphigoid antigen with a molecular weight of 180 kd. These findings suggest the coexistence of lichen planus and bullous pemphigoid in lichen planus pemphigoides.

Aged↗

PubMed1995

Mutations in the 180-kD bullous pemphigoid antigen (BPAG2), a hemidesmosomal transmembrane collagen (COL17A1), in generalized atrophic benign epidermolysis bullosa.

Junctional epidermolysis bullosa (JEB) is a heterogeneous autosomal recessively inherited blistering skin disorder associated with fragility at the dermal-epidermal junction. Characteristic ultrastructural findings in JEB are abnormalities in the hemidesmosome-anchoring filament complexes. These focal attachment structures, which extend from the intracellular compartment of the basal keratinocytes to the underlying basement membrane, have been shown to be hypoplastic or rudimentary in different forms of JEB. Previously, in different JEB phenotypes, mutations have been found in the three genes for the anchoring filament component laminin 5 (LAMA3, LAMB3, and LAMC2) and in the gene for the hemidesmosome-associated integrin beta 4 subunit. Here, we describe the first mutations in the gene encoding the 180-kD bullous pemphigoid antigen (BPAG2), a transmembranous hemidesmosomal collagen, also known as type XVII collagen (COL17A1). The patient is affected with generalized atrophic benign epidermolysis bullosa (GABEB), a rare variant of JEB, and is a compound heterozygote for premature termination codons on both alleles. These novel findings emphasize the molecular heterogeneity of this group of genodermatoses, and attest to the importance of BPAG2 in maintaining adhesion between the epidermis and the dermis.

Adolescent↗

PubMed1995

Pemphigoid nodularis: a case with 230 kDa hemidesmosomes antigen associated with bullous pemphigoid antigen.

We report a 73-year-old woman with typical clinical, histological and immunofluorescence features of pemphigoid nodularis. Direct immunofluorescence studies of prurigo nodularis-like lesions and peribullous skin showed the linear deposition of IgG and C3 at the basement membrane zone. Circulating IgG against the basement membrane was also detected by indirect immunofluorescence. The serum from the patient was shown to contain the autoantibody against 230 kDa hemidesmosomal antigen associated with bullous pemphigoid antigen.

Aged↗