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Biomedical subjects

J Uitto

Publications and source records attributed to J Uitto.

At least 325 records · Page 18Linked to original sources
PubMed1992

Regulation of ornithine decarboxylase gene expression, polyamine levels, and DNA synthetic rates by all-trans-retinoic acid in cultured human keratinocytes.

Regulation of ornithine decarboxylase (ODC) gene expression and cell growth by all-trans-retinoic acid in the presence and absence of exogenous putrescine were examined in normal keratinocyte cultures maintained in serum-free medium containing 0.15 mM Ca++. Putrescine and the higher polyamines are negative feedback regulators of ODC synthesis and are essential for cell growth. Human keratinocytes were incubated with and without 1 microM putrescine and the effects of 5 x 10(-7) M retinoic acid on ODC mRNA levels, ODC activity, polyamine levels, and DNA synthetic rates were determined. Northern blot analysis of total RNA isolated from breast reduction keratinocytes treated with retinoic acid up to 24 h showed a time-dependent suppression of ODC mRNA levels that was unaffected by putrescine. ODC activity was suppressed more rapidly in keratinocytes grown in the absence of putrescine; however, at 24 h, ODC activity was suppressed to an equal extent under both culture conditions. The effect of retinoic acid on polyamine levels was determined in the absence of exogenous putrescine. Retinoic acid treatment markedly suppressed putrescine and N1-acetylspermidine levels, whereas spermidine and spermine levels were relatively unaffected. The effect of retinoic acid on DNA synthetic rates, as measured by 3H-thymidine incorporation, was variable. Retinoic acid either stimulated or had little effect on keratinocyte DNA synthetic rates in cells derived from breast reductions and cultured in the absence of putrescine; these effects were not opposed by the presence of exogenous putrescine. In contrast, DNA synthesis in keratinocytes derived from neonatal foreskins was consistently suppressed by retinoic acid, independent of the polyamine status. Our data, therefore, suggest that the effect of retinoic acid on cell growth, as indicated by DNA synthetic rates, does not necessarily parallel its effect on ODC activity and mRNA levels.

Biogenic Polyamines↗
PubMed1992

Transforming growth factor-beta up-regulates the expression of the genes for beta 4 integrin and bullous pemphigoid antigens (BPAG1 and BPAG2) in normal and transformed human keratinocytes.

Three distinct proteins, namely, beta 4 integrins, and the 230-kDa (BPAG1) and 180-kDa (BPAG2) bullous and pemphigoid antigens, have been shown to co-localize with hemidesmosomes at the dermal-epidermal basement membrane zone. In this study, we examined the expression of the corresponding genes in cultures of normal and transformed human epidermal keratinocytes. The expression of these genes was detected by Northern and in situ hybridizations, and the expression of beta 4 integrins was also demonstrated by indirect immunofluorescence. The results indicated clearly detectable expression of all three genes in normal keratinocytes, whereas extremely low or undetectable levels of expression were noted in two transformed cell lines. Addition of TGF-beta 1 or TGF-beta 2 (10 ng/ml) up-regulated mRNA levels for all three proteins (up to 4.6 times). The increase by TGF-beta 1 was particularly striking in keratinocyte cultures incubated in the presence of low (0.15 mM) Ca++, and somewhat less pronounced in the presence of high (1.2 mM) Ca++. The increase in beta 4 integrin synthesis was also documented by enhanced immunosignal of the corresponding epitopes. These results indicate that the three hemidesmosomal genes studied here are all responsive to TGF-beta. These observations, together with previous data on the effects of TGF-beta on other components of the skin, suggest that this cytokine may play a role in the development and repair of the cutaneous basement membrane zone.

Adult↗
PubMed1992

Cloning of partial cDNA for mouse 180-kDa bullous pemphigoid antigen (BPAG2), a highly conserved collagenous protein of the cutaneous basement membrane zone.

One-hundred-eighty kilodalton bullous pemphigoid antigen (BPAG2) is recognized by autoantibodies in the sera of patients with blistering skin diseases, bullous pemphigoid (BP), and herpes gestationis (HG). In this study, we have screened a mouse epidermal keratinocyte cDNA library with a 1.0-kb human BPAG2 cDNA, which has been shown to correspond to two collagenous domains (Giudice et al: J Clin Invest 87:734-738, 1991). Screening of the mouse library identified two cDNA clones, the larger one being 1.8 kb in size. Comparison of the mouse amino acid sequences, as deduced from cDNA, with the corresponding human sequences revealed 86% homology. Furthermore, Northern hybridizations of mouse epidermal RNA with these cDNA revealed the presence of an mRNA transcript of approximately 6 kb, the size of the human BPAG2 mRNA. Elucidation of the deduced amino-acid sequences revealed the presence of definitely one and possibly two putative membrane-associated segments, suggesting that the 180-kDa BP antigen is a transmembrane protein. The sequence analysis also identified a 7-amino-acid segment that was predicted by computer analysis to be antigenic. Elucidation of the divergence between the mouse and previously published human and chicken BPAG2 sequences indicated that this protein segment was relatively well conserved. These data suggest, therefore, that the 180-kDa bullous pemphigoid antigen associated with hemidesmosomes is a well-conserved transmembrane protein that may play a critical role in the attachment of epidermis to the underlying basement membrane.

Amino Acid Sequence↗
PubMed1992

Genetic linkage between the collagen VII (COL7A1) gene and the autosomal dominant form of dystrophic epidermolysis bullosa in two Dutch kindreds.

Epidermolysis bullosa is a heterogeneous group of heritable blistering skin diseases affecting epidermis and the dermal-epidermal junction zone. Recently, genetic linkage to the type VII collagen gene (Z = 8.77; theta = 0.00) localized on chromosome 3p21 was shown in three Finnish families with the autosomal dominant form of dystrophic epidermolysis bullosa. Two Dutch kindreds with intrafamilial characteristics of both the Cockayne-Touraine type and Bart's syndrome of autosomal dominant dystrophic epidermolysis bullosa have been studied. Two-point linkage analysis in these two families with the COL7A1 marker revealed a combined lod score of Z = 6.08 at theta = 0.00. These data strongly suggest that the type VII collagen gene is the candidate gene in these Dutch pedigrees. At least two (Cockayne-Touraine and Bart) of the three subtypes of dominant dystrophic epidermolysis bullosa seem to represent different forms of expression of the same gene defect.

Collagen↗
PubMed1992

Modulation of bullous pemphigoid antigen gene expression by gamma-interferon in cultured keratinocytes.

Bullous pemphigoid (BP) is characterized by the production of autoantibodies against BP antigens. gamma-interferon (gamma-IFN), a T-cell lymphokine, is known to enhance the expression of several cell-surface proteins. In this study, keratinocytes were cultured in the presence of gamma-IFN, the expression of BP antigen protein was examined by flow cytometry and BP antigen messenger RNA (mRNA) (encoding 230-kDa protein) was quantified by slot-blot hybridization. The results indicated that BP antigen gene expression by keratinocytes was upregulated by gamma-IFN. This enhancement of gene expression was detected at both the protein and mRNA level, suggesting pretranslational regulation. These results imply the involvement of not only humoral immunity but also cell-mediated immunity in the development of BP.

Animals↗
PubMed1992

Molecular biology and pathology of type VII collagen.

Type VII collagen is a genetically distinct member of the collagen family of proteins. Type VII collagen has been shown to be the major component of anchoring fibrils, attachment complexes which secure the cutaneous basement membrane of the skin to the underlying dermis. Understanding of the structure of type VII collagen has been advanced by recent cloning of the corresponding gene. Chromosomal mapping of the gene to the short arm of chromosome 3 and identification of intragenic polymorphic markers have allowed demonstration of strong genetic linkage between the type VII collagen locus and the dystrophic forms of EB (epidermolysis bullosa). This overview summarizes the progress made in the molecular genetics of type VII collagen.

Amino Acid Sequence↗
PubMed1992

Type VII collagen gene expression by cultured human cells and in fetal skin. Abundant mRNA and protein levels in epidermal keratinocytes.

Type VII collagen, a genetically distinct member of the collagen family, is present in the cutaneous basement membrane zone as an integral component of the anchoring fibrils. We have recently isolated several cDNAs that correspond to human type VII collagen sequences. One of these cDNAs (clone K-131) was utilized to examine type VII collagen gene expression in cultures of human cells by Northern analyses, in situ hybridizations and indirect immunofluorescence. Northern hybridizations revealed the presence of an approximately 9-kb mRNA transcript, and indicated a high level of expression in epidermal keratinocytes as well as in an oral epidermoid carcinoma cell line (KB), while the expression was considerably lower in skin fibroblasts and in several virally or spontaneously transformed epithelial cell lines. In situ hybridizations of cultured keratinocytes supported the notion of a high level of gene expression. Indirect immunofluorescence of skin from a 19-wk fetus revealed type VII collagen gene expression at the dermal-epidermal basement membrane zone. These results indicate that several different cell types including epidermal keratinocytes and dermal fibroblasts express the type VII collagen gene, but epidermal keratinocytes may be the primary cell source of type VII collagen in developing human skin.

Base Sequence↗
PubMed1992

Genetic linkage of type VII collagen (COL7A1) to dominant dystrophic epidermolysis bullosa in families with abnormal anchoring fibrils.

Epidermolysis bullosa (EB) in a group of genodermatoses characterized by the fragility of skin. Previous studies on the dystrophic (scarring) forms of EB have suggested abnormalities in anchoring fibrils, morphologically recognizable attachment structures that provide stability to the association of the cutaneous basement membrane to the underlying dermis. Since type VII collagen is the major component of the anchoring fibrils, we examined the genetic linkage of dominant dystrophic EB (EBDD) and the type VII collagen gene (COL7A1) locus, which we have recently mapped to chromosome 3p, in three large kindreds with abnormal anchoring fibrils. Strong genetic linkage of EBDD and COL7A1 loci was demonstrated with the maximum logarithm of odds (LOD) score of 8.77 at theta = 0. This linkage was further confirmed with two additional markers in this region of the short arm of chromosome 3, and these analyses allowed further refinement of the map locus of COL7A1. Since there were no recombinants between the COL7A1 and EBDD loci, our findings suggest that type VII collagen is the candidate gene that may harbor the mutations responsible for the EB phenotype in these three families.

Chromosome Mapping↗
PubMed1992

Genetic linkage of recessive dystrophic epidermolysis bullosa to the type VII collagen gene.

Generalized mutilating recessive dystrophic epidermolysis bullosa (RDEB) is characterized by extreme skin fragility owing to loss of dermal-epidermal adherence. Immunohistochemical studies have implicated type VII collagen, the major component of anchoring fibrils, in the etiology of RDEB. In this study, we demonstrate genetic linkage of the type VII collagen gene and the generalized mutilating RDEB phenotype. We first identified a Pvull polymorphic site by digestion of an amplified product of the type VII collagen gene, which was shown to reside within the coding region. Genetic linkage analysis between this marker and the RDEB phenotype in 19 affected families which were informative for this polymorphism showed no recombination events, and gave a maximum lod score of 3.97 at a recombination fraction (theta) of 0, demonstrating that this DNA region is involved in this form of RDEB. These data provide strong evidence that the type VII collagen gene, which has also been linked with the dominant form of the disease, harbors the mutation(s) causing the generalized mutilating form of RDEB in these families, thus underscoring the major functional importance of type VII collagen in basement membrane zone stability.

Base Sequence↗
PubMed1992

Glucose transporters of rat peripheral nerve. Differential expression of GLUT1 gene by Schwann cells and perineural cells in vivo and in vitro.

Expression of GLUTs in rat peripheral nerve was first studied at the mRNA level with Northern transfer analysis with cDNAs specific for GLUT1, GLUT2, GLUT3, and GLUT4. GLUT1 mRNA was the only GLUT mRNA detectable in rat sciatic nerve. In situ hybridization localized this mRNA to the perineurium and to some endo- and epineurial capillaries. Indirect immunofluorescence stainings demonstrated that GLUT1 protein epitopes were concentrated primarily in the perineurium and endoneurial capillaries. Also, some Schwann cells, a few epineurial capillaries, and medium-sized blood vessels showed a faintly positive immunoreaction. All cell types present in primary cultures initiated from rat sciatic nerve (perineurial cells, Schwann cells, and fibroblasts) expressed GLUT1 protein in vitro. Thus, Schwann cells, which expressed GLUT1 only occasionally at a low level in vivo, have the potential to express GLUT1 at a markedly higher level under cell culture conditions. Incubation of the cultures in 25 mM D-glucose for 7 days caused a 39% reduction in the amount of immunodetectable GLUT1 protein, and a marked (34%) decrease of GLUT1 mRNA compared with cultures incubated in 5.5 mM D-glucose. Interestingly, the reduction of [3H]-2-DG uptake in the same cultures exceeded 70%, suggesting that the reduced amount of GLUT1 protein alone did not explain the marked reduction in glucose uptake in these cultures. Immunostaining of the cell cultures suggested that perineurial cells were the main target for the glucose-induced decrease of GLUT1 protein.

Animals↗
PubMed1992

Transforming growth factor-beta up-regulates elastin gene expression in human skin fibroblasts. Evidence for post-transcriptional modulation.

Transforming growth factor-beta s (TGF-beta) are potent enhancers of the expression of several connective tissue genes. In this study we examined the effects of TGF-beta 1 and TGF-beta 2 on human elastin mRNA abundance, promoter activity, and mRNA stability in cultured human skin fibroblasts. Treatment of cell cultures with varying concentrations of TGF-beta 1 or TGF-beta 2 for 24 hours resulted in a dose-dependent increase in the elastin mRNA steady-state levels, with a maximum enhancement of approximately 30-fold being noted with 1 ng/ml. Addition of cycloheximide (10 micrograms/ml) failed to block up-regulation of elastin gene expression by TGF-beta, indicating that this effect can occur in the absence of active protein synthesis. Furthermore, TGF-beta elicited enhancement of elastin mRNA levels could be abrogated by tumor necrosis factor-alpha and partially counteracted by interferon-gamma. Transient transfections of human skin fibroblasts with elastin promoter/chloramphenicol acetyl-transferase reporter gene constructs, which contained up to approximately 5 kb of the 5' flanking DNA, revealed no change in the promoter activity in the presence of TGF-beta. However, TGF-beta appeared to stabilize the elastin mRNA transcripts as determined by Northern hybridizations after inhibition of initiation of the transcription. As a result of this stabilization, the elastin mRNA levels were clearly detectable in TGF-beta 1-treated cultures even up to 48 hours after inhibition of transcription while they were undetectable in the control cells after 24 hours of incubation. These results demonstrate that TGF-beta 1 and TGF-beta 2 are potent enhancers of elastin gene expression and that this effect is mediated, at least in part, post-transcriptionally. These results suggest that TGF-beta s are involved in regulation of elastin deposition during fetal development and tissue repair, as well as in pathological conditions.

Dinoprostone↗
PubMed1991

Genomic organization of collagenous domains and chromosomal assignment of human 180-kDa bullous pemphigoid antigen-2, a novel collagen of stratified squamous epithelium.

We have recently isolated a 1.0-kilobase (kb) cDNA encoding 180-kDa bullous pemphigoid antigen (BPAG2), an autoantigen in blistering skin disease, bullous pemphigoid (Giudice, G. J., Squiquera, H. L., Elias, P. M., and Diaz, L. (1991) J. Clin. Invest. 87, 734-738). The deduced amino acid sequence identified two collagenous domains characterized by Gly-X-Y repeats. In this study we have elucidated the genomic organization of the corresponding segment in the human BPAG2 gene. Screening of a genomic lambda-phage DNA library yielded six overlapping clones, and the sequences corresponding to the 1.0-kb cDNA spanned approximately 12 kb of genomic DNA. The coding segment consisted of 19 separate exons varying in size from 27 to 222 base pairs. The organization of these exons and the splice sites at the intron-exon junctions were clearly different from other fibrillar and nonfibrillar collagen genes described to date. The BPAG2 gene was mapped by chromosomal in situ hybridization to the long arm of human chromosome 10, at locus 10q24.3; this location is distinct from any previously mapped collagens, and it also distinguishes BPAG2 from BPAG1, a 230-kDa bullous pemphigoid antigen, which has been mapped to chromosome 6p (Sawamura, D., Nomura, K., Sugita, Y., Mattei, M.-G., Chu, M.-L., Knowlton, R.G., and Uitto, J. (1990) Genomics 8, 722-726). These data suggest that BPAG2 is a novel collagen present in stratified squamous epithelia.

Amino Acid Sequence↗