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Biomedical subjects

J Uitto

Publications and source records attributed to J Uitto.

At least 37 records · Page 2Linked to original sources
PubMed2001

Downregulation of human type VII collagen (COL7A1) promoter activity by dexamethasone. Identification of a glucocorticoid receptor binding region.

Type VII collagen is the major collagenous component of the anchoring fibrils, attachment structures that stabilize the association of the cutaneous basement membrane zone to the underlying dermis. It is expressed by both epidermal keratinocytes and dermal fibroblasts. In this study, we have examined the pharmacological control of COL7A1 gene expression by the glucocorticorticoid dexamethasone. We demonstrate that dexamethasone is a potent transcriptional inhibitor of COL7A1 promoter activity in dermal fibroblasts, and we identify a potential glucocorticoid response element in the region -318/-212 of the promoter. In addition, we have determined that dexamethasone antagonizes transforming growth factor-beta (TGF-beta) activation of the COL7A1 promoter. This effect occurred without dexamethasone interfering with TGF-beta-induced Smad-specific gene transcription. These results indicate potential deleterious effects of glucocorticosteroids on epidermal wound healing, as reduced COL7A1 expression likely leads to decreased anchoring fibril formation, which may translate into delayed or impaired reepithelialization.

Cells, Cultured↗
PubMed2001

The nitroxide Tempol affords protection against ultraviolet radiation in a transgenic murine fibroblast culture model of cutaneous photoaging.

The generation of reactive oxygen species is among the various mechanisms by which ultraviolet radiation damages skin. Tempol, a superoxide dismutase analogue which readily penetrates cell membranes when administered exogenously, has been shown to provide protection against some forms of oxygen-dependent damage. In this study, we measured the ability of Tempol to protect against ultraviolet A- and ultraviolet B-induced damage, using a previously described transgenic mouse model of cutaneous photoaging. The ability of Tempol to prevent ultraviolet radiation-induced elastin promoter activation was determined in vitro. Tempol provided over 50% protection against ultraviolet B and over 70% protection against ultraviolet A as measured in our in vitro system. These results demonstrate the ability of the superoxide dismutase mimic, Tempol, to protect against ultraviolet induced elastin promoter activation. This compound could be a useful pharmacological agent to prevent cutaneous photoaging.

Animals↗
PubMed2001

Molecular genetics of pseudoxanthoma elasticum.

Pseudoxanthoma elasticum (PXE), a systemic heritable connective tissue disorder, is characterized by progressive calcification of elastic structures in the skin, the eyes and the cardiovascular system, with considerable intra- and interfamilial phenotypic variability. Recently, underlying genetic defects have been identified in the ABCC6 gene, which resides on the chromosomal locus 16p13.1 and encodes the MRP6 protein, a member of the ATP-binding cassette (ABC) family of proteins. The affected individuals are homozygous or compound heterozygous for a spectrum of genetic lesions, including nonsense and missense mutations, or deletions and splice-site alterations, confirming the autosomal recessive nature of this condition. Analysis of the deduced primary sequence suggests that MRP6 is a transmembrane transporter, but its function has not been delineated yet. Surprisingly, however, MRP6 is expressed primarily, if not exclusively, in the liver and the kidneys, suggesting that PXE may be a primary metabolic disorder with secondary involvement of elastic fibers. Identification of mutations in the ABCC6 gene in PXE provides a means for prenatal and presymptomatic testing in families at risk for recurrence. DNA-based analyses will also identify heterozygous carriers who may be at risk for development of limited manifestations of the disease as a result of compounding genetic factors and/or environmental modifiers.

ATP-Binding Cassette Transporters↗
PubMed2001

Ultraviolet-filtering properties of commonly used tissue cell culture plasticware.

BACKGROUND: Fluorescent sunlamps are a common source of ultraviolet radiation (UVR) for photobiology research. However, these lamps emit a significant amount of biologically "irrelevant" wavelengths that, if not removed, can drastically skew results and perhaps lead to mistaken conclusions regarding human photobiology. The use of a cellulose triacetate sheet (Kodacel) to filter the shorter ultraviolet wavelengths has become the accepted standard in photobiology. Over time, the transmission characteristics of this sheet may be altered due to photochemical changes. In addition, in vitro experiments utilizing filtered fluorescent sunlamps require the removal of plastic tissue cell culture lids, increasing the possibility of contamination. METHODS: We evaluated the transmission characteristics of various commercially available plastic lids used in tissue cell cultures. In addition, we used a biological system containing the human elastin promoter/chloramphenicol acetyltransferase reporter gene construct to compare the effects of filtering from these plastic lids. RESULTS: Here, we demonstrate that the transmission of UVR and the biological response through plastic culture dish lids is similar to that of Kodacel. CONCLUSION: Although this is an improvement for in vitro experiments, further improvements can be made using more realistic UVR sources, e.g. UVA-340 lamps, which mimic the short wavelengths of sunlight.

Animals↗
PubMed2001

Differential expression of tissue-specific promoters by gene gun.

BACKGROUND: Tissue-specific promoters are becoming increasingly important in light of their effects on gene expression in gene therapy experiments. The regulation of gene expression may be as important as the delivery of the gene itself. OBJECTIVES: To determine the effects of the involucrin (INV), keratin 14 (K14) and cytomegalovirus (CMV) promoters on the expression of the reporter gene beta-galactosidase. METHODS: In vivo, plasmid DNA was introduced to BALB/c mice by gene gun. Skin biopsies were taken after 24 h for histology and beta-galactosidase staining. In tissue culture cells, plasmid DNA was introduced by transient transfection to cell lines 293 (transformed primary human embryonal kidney cells), NIH 3T3 (immortalized mouse fibroblasts) and human keratinocytes. Reporter gene expression was assayed by histochemical staining and chemiluminescence. RESULTS: The K14 and INV promoter constructs showed beta-galactosidase gene expression only in the epidermis, while the CMV promoter showed gene expression in both the dermis and epidermis. In cell culture, the INV and K14 promoter constructs demonstrated significant beta-galactosidase expression in human keratinocytes, but minimal expression in 293 and NIH 3T3 cell types. CMV promoter constructs demonstrated significant expression in all cell types. CONCLUSIONS: Gene expression can be regulated by different promoters both in vivo and in cell culture. Based on the physiological expression of the different promoters, gene expression can be restricted to certain cell types, tissues and skin layers.

Animals↗
PubMed2001

Papillon-Lefèvre syndrome: mutations and polymorphisms in the cathepsin C gene.

The Papillon-Lefèvre syndrome, inherited in an autosomal recessive pattern, manifests with palmoplantar keratoderma and early, destructive periodontitis. Recently, mutations in the gene encoding cathepsin C have been disclosed in a limited number of families with Papillon-Lefèvre syndrome. We have examined two multiplex families with Papillon-Lefèvre syndrome, and evaluated the gene encoding cathepsin C for mutations. The mutation detection strategy consisted of polymerase chain reaction amplification of all seven exons and flanking intronic sequences, followed by direct nucleotide sequencing. This strategy identified two missense mutations, W39S and G301S, affecting highly conserved amino acid residues within the cathepsin C polypeptide. The affected individuals were homozygotes whereas heterozygous carriers of the mutations were clinically unaffected, confirming the recessive nature of the mutations. Addition of these cathepsin C gene mutations into the expanding Papillon-Lefèvre syndrome mutation database allows further development of genotype/phenotype correlations towards understanding this severe genodermatosis.

Cathepsin C↗
PubMed2001

The spectrum of pathogenic mutations in SPINK5 in 19 families with Netherton syndrome: implications for mutation detection and first case of prenatal diagnosis.

The Comèl-Netherton syndrome is an autosomal recessive multisystemic disorder characterized by localized or generalized congenital ichthyosis, hair shaft abnormalities, immune deficiency, and markedly elevated IgE levels. Life-threatening complications during infancy include temperature and electrolyte imbalance, recurrent infections, and failure to thrive. To study the clinical presentations of the Comèl-Netherton syndrome and its molecular cause, we ascertained 19 unrelated families of various ethnic backgrounds. Results of initial linkage studies mapped the Comèl-Netherton syndrome in 12 multiplex families to a 12 cM interval on 5q32, thus confirming genetic homogeneity of Comèl-Netherton syndrome across families of different origins. The Comèl-Netherton syndrome region harbors the SPINK5 gene, which encodes a multidomain serine protease inhibitor (LEKTI) predominantly expressed in epithelial and lymphoid tissues. Recently, recessive mutations in SPINK5 were identified in several Comèl-Netherton syndrome patients from consanguineous families. We used heteroduplex analysis followed by direct DNA sequencing to screen all 33 exons and flanking intronic sequences of SPINK5 in the affected individuals of our cohort. Mutation analysis revealed 17 distinct mutations, 15 of which were novel, segregating in 14 Comèl-Netherton syndrome families. The nucleotide changes included four non-sense mutations, eight small deletions or insertions leading to frameshift, and five splice site defects, all of which are expected to result in premature terminated or altered translation of SPINK5. Almost half of the mutations clustered between exons 2 and 8, including two recurrent mutations. Genotype-phenotype correlations suggested that homozygous nucleotide changes resulting in early truncation of LEKT1 are associated with a severe phenotype. For the first time, we used molecular data to perform prenatal testing, thus demonstrating the feasibility of molecular diagnosis in the Comèl-Netherton syndrome.

Adolescent↗
PubMed2001

Tissue distribution and cell type-specific expression of p120ctn isoforms.

Cadherin-based molecular complexes play a major role in cell-cell adhesion. At the adherens junctions the intracellular domain of cadherins specifically interacts with beta-catenin and p120ctn, members of the Armadillo repeat protein family. Differential splicing and utilization of the alternative translation initiation codons lead to many p120ctn isoforms. Two major p120ctn isoforms are expressed in mouse tissues. In this study we used indirect immunofluorescence to demonstrate significant tissue specificity in expression of the p120ctn isoforms. The short isoform is abundant at cell-cell adhesion junctions in epidermis, palatal, and tongue epithelia, in the ducts of excretory glands, bronchiolar epithelium, and in mucosal epithelia of esophagus, forestomach, and small intestine. In contrast, the long isoform, containing an amino terminus highly conserved within the p120ctn subfamily, is expressed at vascular-endothelial cell junctions in blood vessels, at cell-cell junctions in the serosal epithelium lining the internal organs, in choroid plexus of brain, in the pigment epithelium of retina, and in structures such as the outer limiting membrane of retina and intercalated discs of cardiomyocytes. The tissue- and cell type-specific expression of p120ctn isoforms suggests a role for the long p120ctn isoform in cell structures responsible for stable tissue integrity, compared to the role of the short isoform in cell-cell adhesion in the external epithelia with rapid turnover.

Animals↗
PubMed2001

Epidermolysis bullosa with congenital pyloric atresia: novel mutations in the beta 4 integrin gene (ITGB4) and genotype/phenotype correlations.

Epidermolysis bullosa with pyloric atresia (EB-PA: OMIM 226730), also known as Carmi syndrome, is a rare autosomal recessive genodermatosis that manifests with neonatal mucocutaneous fragility associated with congenital pyloric atresia. The disease is frequently lethal within the first year, but nonlethal cases have been reported. Mutations in the genes encoding subunit polypeptides of the alpha 6 beta 4 integrin (ITGA6 and ITGB4) have been demonstrated in EB-PA patients. To extend the repertoire of mutations and to identify genotype-phenotype correlations, we examined seven new EB-PA families, four with lethal and three with nonlethal disease variants. DNA from patients was screened for mutations using heteroduplex analysis followed by nucleotide sequencing of PCR products spanning all beta 4 integrin-coding sequences. Mutation analysis disclosed 12 distinct mutations, 11 of them novel. Four mutations predicted a premature termination codon as a result of nonsense mutations or small out-of-frame insertions or deletions, whereas seven were missense mutations. This brings the total number of distinct ITGB4 mutations to 33. The mutation database indicates that premature termination codons are associated predominantly with the lethal EB-PA variants, whereas missense mutations are more prevalent in nonlethal forms. However, the consequences of the missense mutations are position dependent, and substitutions of highly conserved amino acids may have lethal consequences. In general, indirect immunofluorescence studies of affected skin revealed negative staining for beta 4 integrin in lethal cases and positive, but attenuated, staining in nonlethal cases and correlated with clinical phenotype. The data on specific mutations in EB-PA patients allows prenatal testing and preimplantation genetic diagnosis in families at risk.

Amino Acid Sequence↗
PubMed2001

trans-dominant inhibition of connexin-43 by mutant connexin-26: implications for dominant connexin disorders affecting epidermal differentiation.

Dominant mutations of GJB2-encoding connexin-26 (Cx26) have pleiotropic effects, causing either hearing impairment (HI) alone or in association with palmoplantar keratoderma (PPK/HI). We examined a British family with the latter phenotype and identified a new dominant GJB2 mutation predicted to eliminate the amino acid residue E42 (DeltaE42) in Cx26. To dissect the pathomechanisms that result in diverse phenotypes of dominant GJB2 mutations, we studied the effect of three Cx26 mutants (DeltaE42, D66H and R75W) identified in individuals with PPK/HI, and another (W44C) present in individuals with non-syndromic HI on gap junctional intercellular communication. We expressed mutant Cx26 alone and together with the epidermal connexins Cx26, Cx37 and Cx43 in paired Xenopus oocytes, and measured the intercellular coupling by dual voltage clamping. Homotypic expression of each connexin as well as co-expression of wild-type (wt) Cx26/wtCx43 and wtCx26/wtCx37 yielded variable, yet robust, levels of channel activity. However, all four Cx26 mutants were functionally impaired and failed to induce intercellular coupling. When co-expressed with wtCx26, all four mutants suppressed the wtCx26 channel activity consistent with a dominant inhibitory effect. However, only those Cx26 mutants associated with a skin phenotype also significantly (P<0.05) inhibited intercellular conductance of co-expressed wtCx43, indicating a direct interaction of mutant Cx26 units with wtCx43. These results demonstrate, for the first time, a trans-dominant negative effect of Cx26 mutants in vitro. Furthermore, they support a novel concept suggesting that the principal mechanism for manifestation of dominant GJB2 mutations in the skin is their dominant interference with the function of wtCx43. This assumption is further corroborated by our finding that Cx26 and Cx43 focally colocalize at gap junctional plaques in affected skin tissue of two carriers of DeltaE42.

Adolescent↗
PubMed2001

UVA-340 as energy source, mimicking natural sunlight, activates the transcription factor AP-1 in cultured fibroblasts: evidence for involvement of protein kinase-C.

Ultraviolet radiation (UVR) is known to affect a variety of cellular functions, including gene expression. A number of signaling pathways have been suggested to mediate these effects, including the participation of activator protein-1 (AP-1), activator protein-2 (AP-2) and nuclear factor-kappa B (NF-kappa B). The divergent results from previous studies could be explained, at least in part, by the source of UVR with different spectral characteristics as well as the type of cells employed as targets. In this study we have utilized UVA-340 as an energy source with output which closely matches the spectrum of natural sunlight over the range of 295-350 nm for irradiation of cultured fibroblasts. Combination of electrophoretic mobility shift assays and Northern analyses revealed activation of AP-1 but not NF-kappa B or AP-2. Inhibition studies further suggested the participation of protein kinase-C, but not protein kinase-A, and that an inhibitor of mitogen-activated protein kinase (MEK-1/2) did not alter the AP-1 activation. Free radical quenchers, sodium azide and N-acetylcysteine, did not affect the AP-1 binding activity. Finally, UVA-340 was shown to enhance transcriptional expression of the type-VII collagen gene (COL7A1), which is endogenously expressed in dermal fibroblast in an AP-1 dependent manner. Introduction of a mutation into the AP-1 site of the COL7A1 promoter abolished this activation. Thus, our results obtained by utilizing a novel energy source, UVA-340, mimicking natural sunlight at UVB and lower UVA range indicate a role for AP-1 in mediating the enhanced gene expression by UVR. Collectively, these results suggest that AP-1 is an important mediator of UVR action in fibroblasts.

3T3 Cells↗
PubMed2001

Mutation analysis in the family of a Taiwanese boy with with epidermolysis bullosa simplex dowling-meara.

Epidermolysis bullosa simplex (EBS) is a group of hereditary bullous diseases characterized by intraepidermal blistering due to mechanical stress-induced degeneration of basal keratinocytes. The major subtypes of EBS, including EBS Dowling-Meara (EBS-DM), are caused by mutations of the basal keratin genes, keratin 5 (KRT5) or keratin 14 (KRT14). Here, we describe the first reported pedigree of EBS-DM in Taiwan. The proband was a 5-day-old newborn, who presented with numerous blisters of various sizes, some of which were hemorrhagic, as well as erosions on the extremities and hard palate since birth. Biopsy of a new vesicle showed subepidermal and basal cleavage with infiltration of eosinophils and neutrophils. Electron microscopy revealed cytolysis of basal cells and clumping of tonofilaments forming thick bundles and peculiar electron-dense round or oval basket-weave bodies. These features are characteristic of EBS-DM. The proband's mother had also suffered from a similar blistering disorder since birth, with gradual appearance of mottled pigmentation on the trunk, diffuse irregular or linear palmoplantar hyperkeratosis, and nail dystrophy. Mutation analysis revealed a heterozygous point mutation (R125C) in helix 1A of keratin 14 in the proband and his mother. The detection of this pathogenic point mutation enables future prenatal diagnosis in this family.

DNA Mutational Analysis↗
PubMed2000

Tumor necrosis factor-alpha induces distinctive NF-kappa B signaling within human dermal fibroblasts.

The TNF-alpha receptor-associated factor 2 (TRAF2) and its downstream mediator, the NF-kappa B-inducing kinase (NIK), have been shown to induce NF-kappa B activation in 293 cells. Investigating the role these mediators play in human skin, we found that both NIK and TRAF2 failed to evoke transcription from NF-kappa B-dependent promoters linked to the CAT reporter in human dermal fibroblast cultures, while epidermal keratinocyte cultures demonstrated NIK-dependent signaling. Further, NF-kappa B activation by TNF-alpha was unaffected by overexpression of a dominant negative mutant NIK in fibroblasts, despite detection of endogenous TRAF2 and NIK by Western analysis. To explore alternative signaling mechanisms in dermal fibroblasts, we found that the intracellular calcium chelator, 3,4,5-trimethoxybenzoic acid, and the calpain inhibitor, N-acetyl-Leu-Leu-norleucinal, both blocked NF-kappa B activation; however, the specific proteosome inhibitor, lactacystin, failed to do so. Furthermore, TNF-alpha receptor mutants lacking a functional death domain failed to stimulate NF-kappa B, while phosphatidylcholine-phospholipase C inhibition and alkalization of endolysosomal compartments blocked its activation by TNF-alpha. These data indicate that, while epidermal keratinocytes utilize previously defined, NIK-dependent NF-kappa B pathways, dermal fibroblasts demonstrate unique NIK/TRAF2-independent signal transduction, where both acidic sphingomyelinase and calpain activity act as surrogate mediators for NF-kappa B activation.

Arabidopsis Proteins↗
PubMed2000

The genodermatoses: candidate diseases for gene therapy.

Tremendous progress has been made in understanding the genetic basis of different forms of genodermatoses, a group of heritable diseases displaying a spectrum of phenotypic manifestations and clinical severity. The information about the underlying mutations in the candidate gene/protein systems has provided the basis for initial development of cutaneous gene therapy, and these heritable conditions appear to serve as appropriate candidate diseases for such efforts. Because of its accessibility and the fact that resident skin cells, such as epidermal keratinocytes and dermal fibroblasts, can be readily propagated in culture, skin serves as an appropriate target tissue for gene therapy. Various strategic considerations, including the use of in vivo or ex vivo approaches, gene replacement versus gene repair, utilization of different delivery systems, etc., require careful prioritization depending on the type of mutations and their pathogenetic consequences at the mRNA and protein levels.

DNA Repair↗