Search PubMed⌕ Search

Biomedical subjects

J Uitto

Publications and source records attributed to J Uitto.

At least 289 records · Page 16Linked to original sources
PubMed1993

Recessive dystrophic epidermolysis bullosa keratinocytes synthesize type VII collagen alpha chains of normal molecular size but the anchoring fibril assembly is impaired.

Recessive dystrophic epidermolysis bullosa is a blistering skin disease characterized by diminished anchoring fibrils within the cutaneous basement membrane zone. Because anchoring fibrils are composed of type VII collagen, we compared the synthesis of type VII collagen by keratinocytes from patients and normal subjects. By Western blot analysis and immunoprecipitation of cell extracts, we found that both cell types express type VII collagen alpha chains of equivalent size (Mr = 290,000). Transforming growth factor-beta enhanced type VII collagen in both cell types. In contrast, the expression of type VII collagen within the culture medium was markedly diminished or absent in cultures of keratinocytes from patients. The patients' keratinocytes are capable of synthesizing type VII collagen of a normal molecular size, but they cannot assemble or secrete type VII collagen properly into the extracellular space, or they secrete a product that is abnormally sensitive to proteolytic digestion.

Basement Membrane↗
PubMed1993

Cloning of type XVII collagen. Complementary and genomic DNA sequences of mouse 180-kilodalton bullous pemphigoid antigen (BPAG2) predict an interrupted collagenous domain, a transmembrane segment, and unusual features in the 5'-end of the gene and the 3'-untranslated region of the mRNA.

Thus far, 16 distinct vertebrate collagens (types I-XVI) have been delineated. In this study, we have cloned a mouse collagenous protein, the 180-kDa bullous pemphigoid antigen (BPAG2). Isolation of over-lapping clones, together with 5' and 3' rapid amplification of cDNA ends, allowed delineation of the entire coding sequence. The 5' and 3' ends of the mRNA transcripts were confirmed by primer extension and anchored reverse transcription polymerase chain reaction analyses. The deduced polypeptide contained 1,433 amino acids, including a collagenous domain that consisted of 13 separate segments. Computer analysis of the deduced amino acid sequence demonstrated the presence of a membrane-associated segment. Examination of the 5' end of the BPAG2 gene revealed that the 295-base pair (bp) exon 1 contained two segments of (T)13AA and TT(A)11, whereas exon 2 was shown to contain the translation initiation codon. The 3' end of the mRNA transcript identified two 6-bp inverted repeat sequences that could form a stem for a 30-bp hairpin loop followed by a series of U residues. Comparison of mouse and human BPAG2 sequences demonstrated 86% homology and the unit of evolutionary period of 4.2 million years. In summary, we have cloned full-length mouse BPAG2 cDNA sequences that encode a collagenous polypeptide. We propose that this polypeptide be designated as the alpha 1-chain of type XVII collagen.

Amino Acid Sequence↗
PubMed1993

Human recombinant interleukin-1 beta up-regulates elastin gene expression in dermal fibroblasts. Evidence for transcriptional regulation in vitro and in vivo.

The effects of human recombinant interleukin (IL)-1 beta on elastin gene expression were studied in human skin fibroblast cultures by Northern hybridization and transient transfection experiments. Incubation of the cells with IL-1 beta elevated the elastin mRNA steady-state levels by approximately 3- to 4-fold. A similar increase was noted at the protein level, when estimated by indirect immunofluorescence of cultured cells. This effect was independent of the on-going protein synthesis, as tested by incubation with cycloheximide. Transient transfections of the dermal fibroblasts with a human elastin promoter/chloramphenicol acetyltransferase (CAT) reporter gene construct suggested transcriptional regulation, since the CAT activity in cells incubated with IL-1 beta was similarly increased approximately 3-fold. Enhancement of the human elastin promoter activity by IL-1 beta was also noted in fibroblast cultures established from the skin and lungs of transgenic mice which have integrated the human promoter/CAT construct into their genome and express it in a tissue-specific manner. Furthermore, subcutaneous injection of IL-1 beta to the mice resulted in a approximately 4-fold elevation of the CAT activity in the skin after a 30-h incubation, as compared to the CAT activity in the skin of control animals. Collectively, these data indicate that IL-1 beta up-regulates elastin gene expression in fibroblast cultures as well as in the skin in vivo, and the activation occurs at the transcriptional level.

Animals↗
PubMed1993

Identification of a leucine-to-proline mutation in the keratin 5 gene in a family with the generalized Köbner type of epidermolysis bullosa simplex.

We have previously reported linkage of a large Finnish family with the generalized (Köbner) type of epidermolysis bullosa simplex to chromosome 12q in the region containing the type II keratin gene cluster (Ryynänen et al., Am J Human Genet 49:978-984, 1991). In this study, we examined the possibility that keratin 5, the type II keratin expressed in the basal keratinocytes, harbors the mutation in this family. Nucleotide sequencing revealed a T-to-C transition within exon 7 of the keratin 5 gene in the affected individuals of the family, while the unaffected individuals showed no evidence of C. The presence of the T-to-C transition in the affected individuals was confirmed by restriction enzyme digestion analysis with NciI endonuclease, as well as with PCR amplification of specific alleles (PASA) analysis. The PASA analysis also indicated that the mutated allele was not found among the 100 alleles tested within the general Finnish population indicating that the mutated allele is not a common polymorphism. Furthermore, the mutated allele was not present in nine individuals representing three different EBS families of Finnish origin. The T-to-C transition at the nucleotide level resulted in substitution of a leucine by a proline at the amino acid level, and the substitution affected a leucine residue which was invariant among eight different human keratins in a highly conserved segment at the carboxy-terminal region of the keratin 5 polypeptide.(ABSTRACT TRUNCATED AT 250 WORDS)

Amino Acid Sequence↗
PubMed1993

Chromosomal localization of mouse bullous pemphigoid antigens. BPAG1 and BPAG2: identification of a new region of homology between mouse and human chromosomes.

Two bullous pemphigoid antigens, BPAG1 and BPAG2, have been recently cloned and mapped to human chromosomes 6p12-p11 and 10q24.3, respectively. In this study, we localized the corresponding mouse genes by interspecific backcross analysis. Bpag-1 mapped to the proximal region of mouse chromosome 1, identifying a new region of homology between human chromosome 6 and mouse chromosome 1. Bpag-2 mapped to the distal end of mouse chromosome 19 in a region of homology to human chromosome 10q. These assignments confirm and extend the relationships between the human and the mouse chromosomes.

Amino Acid Sequence↗
PubMed1993

cDNA cloning and chromosomal mapping of the mouse type VII collagen gene (Col7a1): evidence for rapid evolutionary divergence of the gene.

Type VII collagen is the major component of anchoring fibrils, critical attachment structures at the dermal-epidermal basement membrane zone. Genetic linkage analyses with recently cloned human type VII collagen cDNAs have indicated that the corresponding gene, COL7A1, is the candidate gene in the dystrophic forms of epidermolysis bullosa. To gain insight into the evolutionary conservation of COL7A1, in this study we have isolated mouse type VII collagen cDNAs by screening a mouse epidermal keratinocyte cDNA library with a human COL7A1 cDNA. Two overlapping mouse cDNAs were isolated, and Northern hybridization of mouse epidermal keratinocyte RNA with one of them revealed the presence of a mRNA transcript of approximately 9.5 kb, the approximate size of the human COL7A1 mRNA. Nucleotide sequencing of the mouse cDNAs revealed a 2760-bp open reading frame that encodes the 5' half of the collagenous domain and a segment of the NC-1, the noncollagenous amino-terminal domain of type VII collagen. Comparison of the mouse amino acid sequences with the corresponding human sequences deduced from cDNAs revealed 82.5% identity. The evolutionary divergence of the gene was relatively rapid in comparison to other collagen genes. Despite the high degree of sequence variation, several sequences, including the size and the position of noncollagenous imperfections and interruptions within the Gly-X-Y repeat sequence, were precisely conserved. Finally, the mouse Col7a1 gene was located by interspecific backcross mapping to mouse Chromosome 9, a region that corresponds to human chromosome 3p21, the position of human COL7A1. This assignment confirms and extends the relationship between the mouse and the human chromosomes in this region of the genome.

Amino Acid Sequence↗
PubMed1993

Basement membranes during development of human nerve: Schwann cells and perineurial cells display marked changes in their expression profiles for laminin subunits and beta 1 and beta 4 integrins.

The formation of the connective tissue compartments of human sciatic and tibial nerves was studied with special reference to the maturation of the basement membranes during foetal development (11-35 weeks of gestation). All Schwann cells were surrounded by continuous basement membranes as early as at week 11, while the perineurial cells became covered by basement membranes gradually between weeks 17 and 35, as estimated by electron microscopy. The first laminin subunits detectable within the nerve were the B1, B2 and M chains. These laminin subunits were present in Schwann cell basement membrane zone at week 11, and in the perineurium at week 17 and later. Laminin A and S chains were first detected at 26 weeks in the perineurium, and at a later stage (35 weeks) on Schwann cells. In mature nerves, all these five laminin chains could be demonstrated in both Schwann cell and perineurial cell basement membrane zones, although A, S and B2 chains predominated in the perineurium, and M, B1 and B2 were the predominant chains in Schwann cell basement membranes. Beta 1 and beta 4 integrins were expressed by all Schwann cells in samples from the youngest foetuses (11-17 weeks). At 22-35 weeks, however, only a subpopulation of Schwann cells stained positively for beta 1 and beta 4 integrins. Perineurial cells expressed beta 1 integrins at all ages studied. Staining for beta 4 integrin in perineurium became detectable and intensified concomitant with the formation of structural basement membranes. The results demonstrate that Schwann cells and perineurial cells change their laminin and integrin expression profiles during the maturation of peripheral nerve.

Antigens↗
PubMed1993

A missense mutation in type VII collagen in two affected siblings with recessive dystrophic epidermolysis bullosa.

Recessive dystrophic epidermolysis bullosa is a severe mutilating genodermatosis. Previous ultrastructural demonstrations of altered anchoring fibrils, and recent genetic linkage analyses have suggested that type VII collagen, the major component of anchoring fibrils, is a candidate gene. We have identified a homozygous methionine-to-lysine mutation in two affected siblings, while their unaffected mother and half-brother are heterozygous carriers. The mutation resides in a highly conserved region of the C-terminus of type VII collagen, strongly suggesting that it is the cause of the disease in this family.

Amino Acid Sequence↗
PubMed1993

A homozygous insertion-deletion in the type VII collagen gene (COL7A1) in Hallopeau-Siemens dystrophic epidermolysis bullosa.

The Hallopeau-Siemens type of recessive dystrophic epidermolysis bullosa (HS-RDEB) is a life-threatening autosomal disease characterized by loss of dermal-epidermal adherence with abnormal anchoring fibrils (AF). We recently linked HS-RDEB to the type VII collagen gene (COL7A1) which encodes the major component of AF. We describe a patient who is homozygous for an insertion-deletion in the FN-4A domain of the COL7A1 gene. This defect causes a frameshift mutation which leads to a premature stop codon in the FN-5A domain, resulting in a marked diminution in mutated mRNA levels, with no detectable type VII collagen polypeptide in the patient. Our data suggest strongly that this null allele prevents normal anchoring fibril formation in homozygotes and is the underlying cause of HS-RDEB in this patient.

Base Sequence↗
PubMed1993

L-tryptophan and the eosinophilia-myalgia syndrome: current understanding of the etiology and pathogenesis.

The eosinophilia-myalgia syndrome (EMS) is a newly recognized illness that occurred in an epidemic form during the summer of 1989. The illness was characterized in the acute phase by myalgia and eosinophilia, followed in many patients by chronic cutaneous lesions, progressive neuropathy, and myopathy. EMS was associated with ingestion of L-tryptophan, an essential amino acid marketed as a nutritional supplement but widely used as a therapeutic agent. Evidence of abnormal L-tryptophan metabolism has been described in patients with EMS, and most likely reflects increased activity of indoleamine 2,3-dioxygenase, the rate-limiting enzyme of tryptophan metabolism. A contaminant identified in EMS-associated L-tryptophan preparations has been isolated and characterized, but its biologic effects and role as the etiologic agent in EMS remain to be established. Pathologic observations and experimental studies indicate that eosinophils, mononuclear inflammatory cells, and fibroblasts are potential effector cells, and interleukin-5 and transforming growth factor-beta are important mediators in the pathogenesis of the syndrome. Although few new cases of EMS occurred following the withdrawal of L-tryptophan, affected patients continue to manifest late sequelae of the disease, including dermal fibrotic conditions. This tragic outbreak of a newly recognized illness has focused interest on the role of chemical and environmental agents in the pathogenesis of various idiopathic illness characterized by tissue inflammation and fibrosis.

Animals↗
PubMed1993

Direct interaction of antifungal azole-derivatives with calmodulin: a possible mechanism for their therapeutic activity.

Azole derivatives, such as ketoconazole and bifonazole, are well-established antifungal drugs. Recently, these compounds have been reported to have therapeutic efficacy also in inflammatory skin disorders. There is increasing evidence that calmodulin is involved in fungal infections as well as in inflammatory skin diseases. Therefore, we investigated the effects of various antifungal drugs on calmodulin activity, using calmodulin-dependent phosphodiesterase as an indicator for the calmodulin activity. All azole derivatives tested competitively inhibited calmodulin activity with 50% inhibitory concentration values in the low micromolar range. In contrast, antifungal drugs belonging to other chemical classes did not display inhibitory activity. Thus, this study provides evidence that direct interaction with calmodulin might contribute to the therapeutic activity of azole derivatives, particularly to their efficacy in the treatment of inflammatory skin disorders.

Antifungal Agents↗
PubMed1993

Collagen gene expression and wound strength in normal and radiation-impaired wounds. A model of radiation-impaired wound healing.

BACKGROUND: Poorly healing wounds result in significant morbidity postoperatively. Numerous attempts have been made to study wound healing in vivo to understand better the normal healing process and factors that impair healing. Animal models of wound healing have been developed to evaluate wound healing in a systematic and controlled setting. Incisional wounds are created in animals to mimic the surgical patient. They may then be evaluated by a variety of methods for degree of healing. OBJECTIVE: To give insight into the mechanisms of wound healing impairment, we developed a model of impaired wound healing in guinea pigs using radiation applied to the skin surfaces only. METHODS: Wound bursting strength, a direct measure of the force required to burst apart healing linear incisions, was measured. Collagen content, measured indirectly as collagen gene expression, was measured. RESULTS: Significant reductions in wound bursting strength were noted after radiation administration. Collagen gene expression was decreased in wounds 7 days after radiation, but recovered to control levels 14 days after irradiation. Our model enables the inclusion of irradiated and unirradiated skin flaps within the same animal, thus eliminating intra-animal variation when comparing impaired and normal wounds. CONCLUSION: Wound bursting strength analysis, combined with techniques aimed at elucidating changes at the molecular level, provides a useful tool for the study of factors that impair healing and potential treatments for resulting healing deficits.

Animals↗
PubMed1993

Epidermolytic hyperkeratosis (bullous congenital ichthyosiform erythroderma). Genetic linkage to chromosome 12q in the region of the type II keratin gene cluster.

Epidermolytic hyperkeratosis (EHK) is an autosomal dominant genodermatosis characterized by hyperkeratosis and blistering of the skin. Histopathology demonstrates suprabasilar blister formation with aggregation of tonofilaments. In this study, we tested the hypothesis that the EHK phenotype is linked to one of the suprabasilar keratins (KRT10 or KRT1) present in the types I and II keratin gene clusters in chromosomes 17q and 12q, respectively. For this purpose, Southern hybridizations were performed with DNA from a large kindred with EHK, consisting of 11 affected individuals in three generations. Segregation analysis with markers flanking the keratin gene clusters demonstrated linkage (Z = 3.61 at theta = 0) to a locus on 12q, while markers on 17q were excluded. These data implicate KRT1, the type II keratin expressed in suprabasilar keratinocytes, as a candidate gene in this family with EHK.

Blotting, Southern↗
PubMed1993

The human 230-kD bullous pemphigoid antigen gene (BPAG1). Exon-intron organization and identification of regulatory tissue specific elements in the promoter region.

The 230-kD bullous pemphigoid antigen (BPAG1), a hemidesmosomal protein, is encoded by a gene at the human chromosomal locus 6p11-12. We have elucidated the exon-intron organization of the entire human BPAG1 gene, including approximately 2.6 kb of 5'-flanking DNA. Seven overlapping genomic clones, spanning approximately 20 kb, contained the entire approximately 9 kb coding sequence of BPAG1 and consisted of 22 separate exons, which varied from 78 to 2,810 bp in size. The 5' flanking region of DNA, upstream from the ATG initiation codon for translation, was found to contain several putative transcriptional response elements. Most interestingly, two motifs potentially conferring keratinocyte specific expression to the gene were detected. The presence of such elements was suggested by approximately 20-fold higher expression of a promoter/chloramphenicol acetyl transferase (CAT) construct in normal human epidermal keratinocytes that express the endogenous gene, as compared to several non-expressing cell types. Transient transfections with 5'-deletion clones of the promoter/reporter gene (CAT) constructs identified a region containing a putative tissue specific element, KRE2, which also conferred tissue specificity to the expression of the truncated promoter downstream from this element, however, a mutated derivative of KRE2 was not functional. Detailed knowledge of the structure and regulation of the BPAG1 gene will aid in further elucidation of diseases affecting the cutaneous basement membrane zone.

3T3 Cells↗
PubMed1993

Epitope mapping of type VII collagen. Identification of discrete peptide sequences recognized by sera from patients with acquired epidermolysis bullosa.

Epidermolysis bullosa acquisita (EBA) is an acquired blistering skin disease characterized by the presence of IgG autoantibodies that recognize type VII (anchoring fibril) collagen. In this study, we have mapped the antigenic epitopes within the type VII collagen alpha chain by Western immunoblotting analysis with sera from 19 patients with EBA, using bacterial collagenase- or pepsin-resistant portions of type VII collagen and a panel of 12 recombinant fusion proteins corresponding to approximately 80% of the primary sequence of the alpha 1 (VII) collagen polypeptide. These studies identified four major immunodominant epitopes localized within the amino-terminal, noncollagenous (NC-1) domain. In addition to EBA, sera from three patients with bullous systemic lupus erythematosus (BSLE) were tested. The pattern of epitopes recognized by these sera were similar to those noted with EBA, suggesting that the same epitopes could serve as autoantigens in both blistering conditions. In contrast, sera from healthy controls or from patients with unrelated blistering skin diseases did not react with type VII collagen epitopes. Collectively, the results indicate that the immunodominant epitopes in EBA and BSLE lie within the noncollagenous regions of type VII collagen. The precise role of the circulating autoantibodies in the pathogenesis of these blistering diseases remains to be elucidated. Conceivably, however, such antibodies could disrupt the assembly of type VII collagen into anchoring fibrils and/or interfere with their interactions with other extracellular matrix molecules within the cutaneous basement membrane zone.

Amino Acid Sequence↗
PubMed1993

Expression of laminin isoforms by peripheral nerve-derived connective tissue cells in culture. Comparison with epitope distribution in normal human nerve and neural tumors in vivo.

BACKGROUND: Laminins are a family of multifunctional glycoproteins that play a role in various aspects of cell biology. Three different isoforms of laminin have been described, and each comprises a molecule consisting of three subunit polypeptides, the A, B1, B2, M or S chain. EXPERIMENTAL DESIGN: The expression pattern of laminin isoforms was studied by indirect immunofluorescence staining of human peripheral nerve in situ or cell cultures derived from such nerve by using monoclonal antibodies recognizing the subunit epitopes. RESULTS: Selective expression of the subunit polypeptides of laminin isoforms in endoneurium and perineurium was demonstrated. Specifically, an intense immunoreaction for A, B2 and S chain epitopes could be detected in perineurium, whereas endoneurium revealed the presence of B1, B2, M and S chains. Examination of the laminin isoform expression in perineurial cells, Schwann cells, and fibroblasts in cultures derived from normal human nerve indicated, however, that these cells under in vitro conditions were capable of expressing all five laminin chains. Cutaneous neurofibromas, tumors characterized by the presence of mixed cell populations consisting of Schwann cells, perineurial cells, and fibroblasts, demonstrated the expression of B1, B2 and M chain epitopes, whereas only a weak immunostaining could be detected with antibodies recognizing the A and S chains. Similar observations were made on schwannomas, a Schwann cell tumor. CONCLUSIONS: Collectively, the observations of this study attest to the plasticity of neural-derived connective tissue cells with respect to laminin isoform expression. Such plasticity may relate to the cell-cell and cell-matrix interactions during development of peripheral nerves and the potential for neural regeneration.

Adult↗
PubMed1993

DNA-based prenatal diagnosis of heritable skin diseases.

BACKGROUND: Recent success in identifying candidate genes and demonstrating mutations in such genes has set the stage for DNA-based prenatal diagnosis of genodermatoses. An example of such conditions is epidermolysis bullosa in which discrete mutations have been identified in different forms of the disease. The severity of the clinical phenotype in some forms of epidermolysis bullosa appears to justify prenatal diagnosis in families at risk for recurrence of the disease. OBSERVATIONS: DNA-based prenatal diagnosis can be performed on chorionic villi, which can be obtained as early as the eighth week of gestation. Thus, the approaches that use sensitive and specific molecular probes will allow identification of a fetus at risk relatively early during the pregnancy. Such prenatal diagnosis has successfully been performed in families with recessive dystrophic epidermolysis bullosa using type VII collagen-specific markers. CONCLUSIONS: Combination of informative intragenic and flanking DNA markers, and eventual identification of specific mutations in additional families with recessive dystrophic epidermolysis bullosa, is expected to allow early prenatal diagnosis in most families at risk for this devastating skin disease. An emerging technology in the field of prenatal genetics involves blastomere analysis prior to implantation. These DNA-based technologies will undoubtedly replace invasive skin biopsy in cases where candidate genes or specific mutations have been identified. Finally, identification of specific mutations will provide the foundation for potential gene replacement therapy in individuals affected with severe skin diseases.

Alleles↗