Biomedical subjects

J Uitto

Publications and source records attributed to J Uitto.

At least 379 records · Page 21Linked to original sources

PubMed1990

Evaluation of transforming growth factor beta and type I procollagen gene expression in fibrotic skin diseases by in situ hybridization.

Full thickness biopsies of affected skin and fascia from one patient with diffuse fasciitis and eosinophilia (DF), two patients with generalized morphea (GM), and five patients with progressive systemic sclerosis (PSS) of recent onset were examined for the expression of transforming growth factor beta 1 (TGF beta 1) and type I procollagen genes by in situ hybridization with human sequence-specific cDNA. An increased number of fibroblasts showing clearly detectable expression of pro alpha 1(I)collagen gene was found in all fibrotic lesions when compared with unaffected skin from the patient with DF and skin from two normal individuals examined in parallel. Expression of the TGF beta 1 gene was noted in a fibroblast subpopulation of the affected tissues from the patients with DF and GM. In contrast, the TGF beta 1 gene was not expressed at a detectable level in affected skin from the five patients with PSS. The results suggest that TGF beta 1 may play a role in the development of skin fibrosis in cases of DF and GM. However, from these studies, we cannot implicate TGF beta 1 in the pathogenesis of skin fibrosis in PSS.

DNA↗

PubMed1990

Regulation of elastin gene expression: evidence for functional promoter activity in the 5'-flanking region of the human gene.

Analysis of nucleotide sequences in the 5'-flanking region of the human elastin gene has revealed several unusual features, suggesting that regulation of elastin gene expression is complex. To identify any cis-acting regulatory promoter elements, a 35-kb fragment of DNA (CosE) was isolated from a human genomic cosmid library by hybridizations with a human elastin cDNA. Southern blots of EcoRI digests of CosE DNA, utilizing a 5'-end labeled 21-mer oligonucleotide corresponding to the signal sequence of elastin, revealed the presence of a single 7.8-kb genomic fragment. Partial dideoxynucleotide sequencing of this EcoRI genomic subclone revealed that it extended approximately 2.5 kb 3' of the translation initiation site (ATG), encompassing exon 1 and a portion of the first intron, while the remaining DNA encompassed the 5'-flanking region. Exonuclease III digestion (3'----5') was performed to remove sequences of the first intron and first exon, including the ATG site. One clone, approximately 5 kb in size, had the 3' end located 14 bp upstream of the ATG site. A 462-bp 3' portion of this 5-kb fragment was subcloned into a Bluescript/CAT chimeric plasmid (pBS0CAT) to generate an elastin gene promoter/CAT reporter gene construct (pEP6CAT). Transient transfection experiments with pEP6CAT using human skin fibroblasts, human HT-1080, mouse NIH-3T3, or freshly isolated neonatal rat aortic smooth muscle cells revealed significant CAT activity in each cell line. These results suggest that the 5'-flanking region of the elastin gene contains the cis-acting regulatory elements necessary for transcription. The chimeric plasmid pEP6CAT provides a means to study the transcriptional control of elastin gene expression by exogenous affector molecules, as well as in human dermatologic diseases.

Base Sequence↗

PubMed1990

Laser-induced photodynamic therapy with aluminum phthalocyanine tetrasulfonate as the photosensitizer: differential phototoxicity in normal and malignant human cells in vitro.

Photodynamic therapy (PDT) involves the use of laser or noncoherent light energy with photosensitizing dyes to induce a cytotoxic reaction in the target cells, resulting in cell injury and/or death. In this study, we have examined laser-induced phototoxicity in normal human skin fibroblasts and HT-1080 fibrosarcoma cells incubated with aluminum phthalocyanine tetrasulfonate (AlPcS) in vitro. The culture, laser, and photosensitizer parameters were varied in attempts to establish the conditions for differential cytotoxicity between normal and malignant human fibroblasts. Biochemical assays, as a measure of cytotoxicity, included [3H]thymidine incorporation (an index of DNA replication), [35S]methionine incorporation (a measure of protein synthetic activity), and the MTT assay (an indirect index of mitochondrial activity). In the absence of laser irradiation, AlPcS was non-toxic to both cell lines in concentrations up to 25 micrograms/ml. Laser light alone at 675 nm (the absorption maximum of AlPcS) had no effect on the cells at energy densities up to 16 J/cm2. In the presence of 3 or 10 micrograms/ml of AlPcS, both cell lines demonstrated marked energy-dependent toxicity. If an 8-h or a 24-h "efflux" period in AlPcS-free medium was allowed to take place prior to laser irradiation, normal fibroblasts were much less sensitive to PDT, whereas fibrosarcoma cells still exhibited a marked degree of toxicity. The results indicate that, under appropriate treatment conditions, AlPcS is capable of preferentially sensitizing a malignant mesenchymal cell line, while sparing its non-malignant normal cell counterpart.

Fibroblasts↗

PubMed1990

Systemic isotretinoin: effects on dermal wound healing in a rabbit ear model in vivo.

Clinical observations have suggested that wound healing may be altered in patients treated with systemic isotretinoin. In this study, we examined the effects of systemic isotretinoin on dermal wound healing and connective tissue metabolism in a rabbit ear model. Forty 6-mm punch-biopsy wounds were created in the ears of two control rabbits as well as two experimental animals fed isotretinoin, 4 mg/kg per day. Clinical inspection and histologic examination revealed no difference between the control and isotretinoin-treated rabbits in terms of the time required for complete wound healing or the appearance of the final scar. The tissue removed from the wound site at days 0, 7, 14, and 21 after wounding was subjected to analysis of a collagen production and collagen gene expression. Collagen production, determined by the synthesis of [3H]hydroxyproline after incubation of tissue slices with [3H]proline in vitro or by the measurement of the steady-state levels of types I and III procollagen mRNAs, was not significantly different between the two groups. The results indicate that systemic administration of isotretinoin does not affect collagen synthesis in the rabbit ear model of wound healing.

Animals↗

PubMed1990

Tumor necrosis factor-alpha and interferon-gamma suppress the activation of human type I collagen gene expression by transforming growth factor-beta 1. Evidence for two distinct mechanisms of inhibition at the transcriptional and posttranscriptional levels.

Regulation of human type I procollagen gene expression was studied in cultured fibroblasts both at the transcriptional and posttranscriptional level. Transcriptional regulation was examined in cultures transfected with a human pro alpha 2(I) collagen promoter/reporter gene (chloramphenicol acetyltransferase) construct, while posttranscriptional regulation was assessed by parallel determinations of type I procollagen mRNA steady-state levels. Transforming growth factor-beta 1 (TGF-beta 1) elicited a marked, approximately 5-23-fold, enhancement of pro alpha 2(I) collagen promoter activity, which was accompanied by an elevation of type I procollagen mRNA levels. This enhancement of gene expression was suppressed by tumor necrosis factor-alpha (TNF-alpha) and interferon-gamma (IFN-gamma), as determined at mRNA steady-state level, but two distinct mechanisms were involved. TNF-alpha suppressed the pro alpha 2(I) collagen promoter activity, whereas IFN-gamma had only a minimal effect at transcriptional level. The effects of TNF-alpha and IFN-gamma were synergistic, suggesting that combination of these two factors may potentially provide pharmacologic means to counteract tissue deposition of collagen in diseases involving TGF-beta.

Animals↗

PubMed1990

Type VI collagen. In situ hybridizations and immunohistochemistry reveal abundant mRNA and protein levels in human neurofibroma, schwannoma and normal peripheral nerve tissues.

Cutaneous neurofibromas contain an extensive extracellular matrix composed of collagenous and non-collagenous macromolecules. In this study, the expression of type VI collagen genes in cutaneous neurofibromas was examined by a combination of in situ hybridizations and immunohistochemistry. In situ hybridizations with a 32P-labeled human type VI collagen-specific cDNA revealed that the majority of cells within neurofibromas expressed the gene for alpha 2(VI) collagen chain. The number of cells expressing clearly detectable levels of alpha 2(VI) collagen mRNA was considerably higher than that of cells actively expressing the pro alpha 1(I) or pro alpha 1(III) collagen genes. The presence of type VI collagen epitopes within the neurofibromas was also demonstrated by immunostaining with specific polyclonal antibodies. The expression of type VI collagen genes in neural tissues was further examined by immunostaining of a benign schwannoma tissue specimen consisting of Schwann cells. The results indicated close association of type VI collagen epitopes with the neoplastic Schwann cells. Immunolocalization of type VI collagen epitopes within normal human peripheral nerve revealed pericellular staining of perineurial cells and Schwann cells, suggesting synthesis of type VI collagen by these cell types. These results suggest that the expression of type VI collagen gene is active in nerve-derived tissues, and that type VI collagen may be a major component of the extracellular matrix in neural connective tissues.

Collagen↗

PubMed1990

Eosinophilic fasciitis. Increased collagen production and type I procollagen messenger RNA levels in fibroblasts cultured from involved skin.

Two patients with eosinophilic fasciitis were studied to elucidate the activation of collagen production in this disorder. Histologic examination of biopsy specimens from the involved area of skin revealed the presence of inflammatory cell infiltrates and various degrees of collagen accumulation in the dermis, subcutis, fascia, and underlying muscle. Fibroblast cultures initiated from the involved skin exhibited 2.0- to 3.7-fold increased collagen production when compared with control fibroblast cultures established from the uninvolved skin of the same patients. Eosinophilic fasciitis fibroblasts also displayed 2.4- to 6.2-fold higher steady-state levels of type I procollagen messenger RNA than did the control cells, indicating pretranslational activation of type I procollagen gene expression. In addition, cellular fibronectin messenger RNA steady-state levels were elevated 1.9- to 3.3-fold in eosinophilic fasciitis fibroblasts. These results suggest that fibroblasts in the involved skin of patients with eosinophilic fasciitis exhibit an activated phenotype, similar to that of scleroderma fibroblasts, leading to accumulation of collagen in the skin and the underlying structures.

Adult↗

PubMed1989

Characterization of the complete human elastin gene. Delineation of unusual features in the 5'-flanking region.

Genomic clones encompassing the entire human elastin gene, including 11 kilobases flanking the ATG translation initiation codon, have been obtained and characterized by restriction enzyme analysis and extensive DNA sequencing. These analyses demonstrated that functionally distinct hydrophobic and cross-linking domains of the protein are segregated into separate exons throughout the gene. All exons are multiples of three nucleotides, and exon:intron borders always split codons in the same way which permits cassette-like alternative splicing. The 5'-flanking region lacks a canonical TATA sequence, is G + C-rich, and contains several SP1 binding sites and an AP2 binding site. Primer extension and S1 nuclease protection experiments indicate that transcription is initiated at multiple sites in the gene.

Amino Acid Sequence↗

PubMed1989

Expression of extracellular matrix genes by cultured human cells: localization of messenger RNAs and antigenic epitopes.

Simplified and expedient methodologies for examination of cellular gene expression at the mRNA and protein levels, utilizing in situ hybridization and peroxidase-anti-peroxidase immunodetection, were developed. These techniques were first optimized for the detection of extracellular matrix genes expressed by cultured human skin fibroblasts and keratinocytes, the two principal cell types of human skin. In situ hybridizations and Northern transfer analyses with human-sequence-specific cDNAs encoding collagenous and noncollagenous protein sequences demonstrated selective expression of different matrix genes by these two cell types, indicating different biosynthetic capacities of these cells and attesting to the specificity of the hybridizations. The utility of in situ hybridization was also demonstrated in mixed primary cell cultures established from cutaneous neurofibromas consisting of Schwann cells, perineurial cells, and fibroblasts. The methodologies developed here were further utilized for simultaneous detection of fibronectin mRNA and immunoreactive protein in fibroblast cultures. This procedure allowed detection of grains representative of radioactively labeled cDNA-mRNA hybrids and protein epitopes, as visualized by peroxidase-anti-peroxidase immunodetection on the same cells. This methodology, with appropriate modifications, may be applicable to other cell types as well as tissue specimens.

Cells, Cultured↗

PubMed1989

Human laminin A chain (LAMA) gene: chromosomal mapping to locus 18p11.3.

Laminin, an integral component of basement membranes, consists of three subunit polypeptides, A, B1, and B2 chains. We have recently isolated cDNAs corresponding to human laminin A chain. These cDNAs were utilized for chromosomal in situ hybridizations to establish the genomic location of the laminin A chain gene. Metaphase chromosomes of PHA-stimulated human peripheral blood leukocytes were examined by in situ hybridization with 3H-labeled cDNAs, and the chromosomes were identified by R-banding (fluorochrome-photolysis-Giemsa method). The results indicated that the human laminin A chain is at locus 18p11.3. Since human laminin B1 and B2 chain genes have been previously mapped to chromosomes 7 and 1, respectively, the results indicate that genes encoding human laminin chains reside in separate chromosomes.

Blotting, Southern↗

PubMed1989

Differential expression of type IV procollagen and laminin genes by fetal vs adult skin fibroblasts in culture: determination of subunit mRNA steady-state levels.

Basement membrane zone gene expression by fibroblast cultures, established from individuals varying in age from 14 fetal weeks to 61 years, was examined by molecular hybridizations with human sequence specific cDNAs corresponding to type IV procollagen and laminin subunit polypeptides. Northern transfer analysis with poly(A)+RNA revealed the presence of specific mRNA transcripts for alpha 1(IV) and alpha 2(IV) chains of type IV procollagen as well as B1 and B2 chains of laminin. Laminin A chain mRNAs were not detected using the same RNA preparations. Quantitative estimates of the steady-state levels of type IV procollagen and laminin mRNAs indicated that they were of relatively low abundance, as compared with mRNA to type I procollagen. The expression of alpha 1(IV) and alpha 2(IV) collagen genes was high in fetal fibroblasts but was reduced to low, yet detectable, levels in cultures established from 3-d to 61-year-old individuals. In contrast, the laminin B1 and B2 chain mRNA levels showed little age-associated variation within the cultures examined. These results provide evidence for differential regulation of the expression of different basement membrane zone macromolecules during chronologic aging.

Aging↗

PubMed1989

Extracellular matrix of the skin: 50 years of progress.

The extracellular connective tissue matrix of the skin is a complex aggregate of distinct collagenous and non-collagenous components. Optimal quantities and delicate interactions of these components are necessary to maintain normal physiologic properties of skin. This overview summarizes the progress made in understanding the normal biology and biochemistry of the extracellular matrix, and will highlight cutaneous diseases with underlying molecular defects in the structure and expression of extracellular matrix components.

Animals↗

PubMed1989

Collagen gene expression by cultured human skin fibroblasts. Abundant steady-state levels of type VI procollagen messenger RNAs.

Previous studies have suggested that procollagen types I and III are the major collagenous gene products of cultured human skin fibroblasts. In this study the expression of 10 different genes, encoding the subunit polypeptides for collagen types I-VI, by human skin fibroblasts in culture was analyzed by molecular hybridizations. Northern transfer analysis demonstrated the presence of specific mRNA transcripts for collagen types I, III, IV, V, and VI, but not for type II collagen. Quantitation of the abundance of these mRNAs by slot blot hybridizations revealed that type I, III, and VI procollagens were the major collagenous gene products of skin fibroblasts in culture. The mRNAs for type IV and V collagens represented only a small percentage of the total collagenous mRNA transcripts. Further analysis by in situ hybridization demonstrated that the majority of the cultured cells coexpressed the genes for type I, III, and VI procollagen pro-alpha chains. Further in situ hybridization analyses revealed the expression of type VI collagen genes in normal human skin. These data demonstrate that human skin fibroblast cultures can be used to study the transcriptional regulation of at least nine genetically distinct procollagen genes. The data further suggest that type VI collagen, in addition to types I and III, may be a major collagenous component of human skin.

Cells, Cultured↗

PubMed1989

Type 1 neurofibromatosis: selective expression of extracellular matrix genes by Schwann cells, perineurial cells, and fibroblasts in mixed cultures.

Cutaneous neurofibromas, characteristic lesions of neurofibromatosis 1, are composed of an abundant extracellular matrix and nerve connective tissue-derived cell types: Schwann cells, perineurial cells, and fibroblasts. In this study, the extracellular matrix gene expression by these cells was examined under culture conditions that allowed them to be metabolically active and readily identifiable by morphologic and immunocytochemical criteria. Northern hybridizations demonstrated expression of genes for type I, III, IV, and VI collagens, as well as for fibronectin, laminin, and elastin. In situ hybridizations revealed that all three cell types expressed pro alpha 1 (I), pro alpha 2 (VI), and laminin B1 chain genes. However, fibroblasts did not contain [35S]cDNA-mRNA hybrids specific for type IV collagen, whereas both Schwann cells and perineurial cells expressed these genes. Perineurial cells and fibroblasts readily expressed the fibronectin gene whereas Schwann cells were essentially devoid of the corresponding mRNA. Perineurial cells also expressed the gene for laminin A chain. The results indicate that the extracellular matrix gene expression profiles of Schwann cells, perineurial cells, and fibroblasts are distinct: all three cell types are capable of expressing some of the genes for extracellular matrix components, such as type I and VI collagens, whereas Schwann cells and perineurial cells may have the primary role in synthesizing basement membrane zone components, type IV collagen and laminin. These observations potentially relate to the mechanisms of growth and development of human neurofibromas. The results attest to the applicability of the methodology utilized here to study other human tumors with mixed cell populations.

Blotting, Northern↗

PubMed1989

Localization of integrin receptors for fibronectin, collagen, and laminin in human skin. Variable expression in basal and squamous cell carcinomas.

VLA integrins in human skin were examined by indirect immunofluorescence utilizing antibodies recognizing the beta 1, alpha 2, alpha 3, or alpha 5 subunits. Staining of fetal, newborn, or adult skin with antibodies to beta 1, alpha 2, or alpha 3 subunits gave essentially similar staining patterns: intense staining was associated with the basal layer of the epidermis, hair follicles, and blood vessel walls. The alpha 5 subunit could be detected only in epidermis and the inner root sheath of hair follicles in fetal skin. In epidermis, the staining reaction for the beta 1 subunit was not only found in sites interfacing with the basement membrane zone, but also around the entire periphery of these cells. We speculate that these receptors might have previously unrecognized functions in cell-cell interactions or that these findings may suggest the presence of previously unrecognized ligands in the intercellular spaces of keratinocytes. Examination of nine nodular basal cell carcinomas revealed a prominent staining reaction with anti-beta 1 and anti-alpha 3 antibodies at the periphery of the tumor islands. In contrast, staining of five squamous cell carcinomas revealed either the absence of integrins or altered and variable expression. Thus, matrix components and their receptors may participate in modulation of growth, development, and organization of human skin.

Adult↗

PubMed1989

Human nidogen: cDNA cloning, cellular expression, and mapping of the gene to chromosome Iq43.

A human placental lambda gt11 expression cDNA library was screened for nidogen cDNAs by hybridizations with a heterologous mouse nidogen cDNA. A total of four positive overlapping clones were identified, and the sizes of the inserts were shown to vary from 0.8 to 2.8 kb. Nucleotide sequencing of the human cDNAs revealed that the largest clone, cHPN-16, contained both a 5' open reading frame encoding 582 amino acids and a 3' untranslated region of 1,063 nucleotides. Comparison of human cDNA sequences with mouse nidogen sequences revealed 84% identity on the nucleotide level and 88% identity with the deduced amino acid sequence. The deduced amino acid sequence of the human cDNAs revealed the presence of cysteine-rich epidermal growth factor-like repeats and the sequence Arg-Gly-Asp (RGD), a potential cell binding site, two features previously identified in mouse nidogen. The sequence Asn-Pro-Ser, a consensus sequence for N-linked glycosylation, was also noted. The newly isolated human cDNAs were utilized to analyze the expression of the nidogen gene by cultured human cells. Northern hybridizations revealed a single mRNA transcript of approximately 6.0 kb in human skin fibroblast and in HT 1080 fibrosarcoma cell cultures. However, the human choriocarcinoma cell line JEG-3, which expressed laminin genes, did not contain detectable levels of nidogen mRNAs. Quantitation of the relative nidogen mRNA abundance in HT 1080 fibrosarcoma cells indicated that nidogen mRNA levels were approximately the same as those for the laminin B2 chain. Finally, one of the nidogen cDNAs was used to map the nidogen gene onto locus q43 of chromosome 1.

Amino Acid Sequence↗

PubMed1989

Genetic linkage analysis of hereditary arthro-ophthalmopathy (Stickler syndrome) and the type II procollagen gene.

Hereditary arthro-ophthalmopathy (AO), or Stickler syndrome, is a dominantly inherited disorder characterized by vitreo-retinal degeneration and frequently accompanied by epiphyseal dysplasia and premature degenerative joint disease. Three large families with AO were analyzed for clinical manifestations of the disease and for coinheritance of the genetic defect with RFLPs in the type II procollagen gene (COL2A1). Genetic linkage between AO and COL2A1 was demonstrated in the largest family, with a maximum LOD score of 3.52 at a recombination distance of zero. Data from a second family also supported linkage of AO and COL2A1, with a LOD score of 1.20 at a recombination distance of zero. These results are consistent with the conclusion that mutations in the COL2A1 gene are responsible for AO in these two families. In a third AO family, however, recombination between AO and COL2A1 occurred in at least one meiosis, and the data were inconclusive with respect to linkage.

Chromosomes, Human, Pair 12↗

PubMed1989

Connective tissue biochemistry of the aging dermis. Age-associated alterations in collagen and elastin.

Cutaneous aging represents a complex situation in which at least two independent factors--innate aging and solar exposure--contribute to the development of degenerative changes in the dermis. The biochemical and ultrastructural evidence reviewed in this article indicates that reduced collagen deposition, as a result of diminished collagen biosynthesis and reduced proliferative capacity of the fibroblasts, could explain the development of dermal atrophy and would relate to poor wound healing in the elderly. At the same time, perturbations in the supramolecular organization of the elastic fiber network lead to alterations in the mechanical properties of the skin, as manifested by loose and sagging skin with reduced resilience and elasticity.

Aging↗