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Biomedical subjects

J Uitto

Publications and source records attributed to J Uitto.

At least 73 records · Page 4Linked to original sources
PubMed2000

Mutation reports: epidermolysis bullosa simplex associated with severe mucous membrane involvement and novel mutations in the plectin gene.

We report a novel case of epidermolysis bullosa simplex with severe mucous membrane involvement and mutations in the plectin gene (PLEC1). The patient suffered from extensive blistering of the skin and oral and laryngeal mucous membranes. Electron microscopy of a lesional skin biopsy showed cleft formation within the basal cell layer of the epidermis. Antigen mapping displayed entirely negative staining for plectin, a large (>500 kDa) multifunctional adhesion protein present in hemidesmosomes of the basal keratinocytes. Mutation analysis revealed compound heterozygous, previously undisclosed nonsense mutations, Q1713X and R2351X, of paternal and maternal origin, respectively, within exon 32 of PLEC1. Based on earlier reports, plectin deficiency is associated with late onset muscular dystrophy in patients with epidermolysis bullosa. No signs of muscle weakness have been observed during the 4 y follow-up of our patient. This case illustrates the fact that molecular pathological analyses have prognostic implications in identification and evaluation of patients who appear to be at risk for development of muscular dystrophy later in life.

Biopsy↗
PubMed2000

Epidermolysis bullosa: novel and de novo premature termination codon and deletion mutations in the plectin gene predict late-onset muscular dystrophy.

Epidermolysis bullosa (EB) with late-onset muscular dystrophy (EB-MD) is a hemidesmosomal variant of EB due to mutations in the plectin gene (PLEC1). The age of onset of muscle involvement has been noted to vary from infancy to the fourth decade of life. Immunofluorescence of the patients' skin and muscle biopsies is usually negative for staining with antibodies recognizing plectin, a large cytoskeleton-associated anchorage protein. In this study we report novel plectin mutations in two families with EB. In both families, the proband was a newborn with neonatal blistering with no evidence for muscle weakness as yet. Peripheral blood DNA was isolated and examined by heteroduplex scanning strategy, protein truncation test (PTT), and/or direct sequencing of the plectin gene. One of the probands was compound heterozygote for nonsense mutations E2005X/K4460X, and the proband in the second family was compound heterozygote for deletion mutations 5083delG/2745-9del21, the latter mutation extending from -9 to +12 at the intron 22/exon 23 border. The mutations K4460X and 5083delG were not present in either one of the parents, thus being de novo events. In both cases, nonpaternity was excluded by microsatellite marker analysis. The stop codon mutations are predicted to result in the synthesis of a truncated protein lacking the carboxy-terminal globular domain of the protein and possibly causing nonsense-mediated decay of the corresponding mRNA. The 2745-9del21 deletion mutation abolishes the splice site at the intron 22/exon 23 junction, predicting abnormal splicing events. Because plectin deficiency is associated with muscular dystrophy, molecular diagnostics of the plectin gene provides prognostic value in evaluation of these patients who appear to be at risk to develop muscular dystrophy.

Adult↗
PubMed2000

Increased glycosaminoglycans production in sclerosing basal cell carcinoma-derived fibroblasts and stimulation of normal skin fibroblast glycosaminoglycans production by a cytokine-derived from sclerosing basal cell carcinoma.

Sclerosing basal cell carcinoma (S-BCC) is characterized by an abundant stroma. There is evidence that some tumor cells secrete cytokines that are mitogenic for stromal fibroblasts (FBs). From this study we report increased glycosaminoglycan (GAG) production by cultures of S-BCC FBs in comparison to cultures of nodular BCC (N-BCC) FBs and normal skin FBs. GAG production was measured by cetylpyridinium chloride precipitation of incorporated [3H]-glucosamine. The sclerosing BCC FBs demonstrated a significant increase in production of GAG over control FBs (P <.001) and over N-BCC FBs (P<.001). Values reported as a mean percentage +/- SEM for GAG production by S-BCC over control normal skin FBs are 359+/-28 and over N-BCC FBs are 266+/-27. In additional experiments, cell extract dilutions from S-BCC tumor, normal dermis, and normal epidermis were incubated with cultures of normal skin FBs. S-BCC-conditioned media was also incubated with normal FBs and GAG production was measured. For both S-BCC extracts and conditioned media, a dose response curve was established showing increased GAG production by normal FBs in relation to increasing the concentration of S-BCC extract or conditioned media. When S-BCC extract was added to normal FBs there was increased GAG production in comparison to normal FBs incubated with dermal or epidermal extracts (P<.001) for both. Two growth factors, transforming growth factor-beta (TGF-beta) and platelet-derived growth factor (PDGF), already known to be mitogenic for FBs, were incubated with N-BCC and normal FBs in an effort to elucidate the potential cytokine(s) released by S-BCC, causing increased GAG production by surrounding FBs. Neither of these cytokines proved to be effective in promoting a significant increase in GAG production. Our findings support the hypothesis that BCCs release factors that alter stromal FB production of GAG.

Basal Cell Carcinoma↗
PubMed2000

Congenital focal segmental glomerulosclerosis associated with beta4 integrin mutation and epidermolysis bullosa.

We report the occurrence of congenital nephrotic-range proteinuria secondary to focal segmental glomerulosclerosis in an infant with epidermolysis bullosa and pyloric atresia. A homozygous missense mutation, R1281W, in exon 31 of the beta4 integrin gene, ITGB4, was identified. By immunofluorescence, beta4 integrin expression was reduced in both dermal keratinocytes and glomerular podocytes. This is the first demonstration of beta4 integrin expression in human glomeruli. We postulate a role for altered beta4 integrin function in the mediation of the glomerular permeability defect.

Antigens, CD↗
PubMed2000

Common fluorescent sunlamps are an inappropriate substitute for sunlight.

Fluorescent sunlamps are commonly employed as convenient sources in photobiology experiments. The ability of Kodacel to filter photobiologically irrelevant UVC wavelengths has been described. Yet there still remains a major unaddressed issue--the over representation of UVB in the output. The shortest terrestrial solar wavelengths reaching the surface are approximately 295 nm with the 295-320 nm range comprising approximately 4% of the solar UV irradiance. In Kodacel-filtered sunlamps, 47% of the UV output falls in this range. Consequently, in studies designed to understand skin photobiology after solar exposure, the use of these unfiltered sunlamps may result in misleading, or even incorrect conclusions. To demonstrate the importance of using an accurate representation of the UV portion of sunlight, the ability of different ultraviolet radiation (UVR) sources to induce the expression of a reporter gene was assayed. Unfiltered fluorescent sunlamps (FS lamps) induce optimal chloramphenicol acetyltransferase (CAT) activity at apparently low doses (10-20 J/cm2). Filtering the FS lamps with Kodacel raised the delivered dose for optimal CAT activity to 50-60 mJ/cm2. With the more solar-like UVA-340 lamps somewhat lower levels of CAT activities were induced even though the apparent delivered doses were significantly greater than for either the FS or Kodacel-filtered sunlamp (KFS lamps). When DNA from parallel-treated cells was analyzed for photoproduct formation by a radioimmuneassay, it was shown that the induction of CAT activity correlated with the level of induced photoproduct formation regardless of the source employed.

Animals↗
PubMed1999

Human p120ctn catenin: tissue-specific expression of isoforms and molecular interactions with BP180/type XVII collagen.

Catenins, a family of structurally related proteins, are involved in epidermal keratinocyte cell-cell adhesion by interacting through their central Armadillo repeats with the intracellular domains of cadherins, transmembrane components of the adhesion junctions. p120ctn is a catenin expressed in different isoforms due to alternative splicing and multiple translation start sites. BP180 is a collagenous transmembrane protein (type XVII collagen) localized to hemidesmosomal attachment complexes in basal keratinocytes. In this study, we have delineated the molecular interaction between these two proteins utilizing the yeast two-hybrid system, which was confirmed by an in vitro protein-protein interaction assay. Specifically, it was shown that an amino-terminal segment of BP180 (aa. 13-25) contains the information necessary for binding to p120ctn isoforms 1-3, but not to the isoform 4, suggesting that the interacting domain is located immediately upstream from the Armadillo repeats and is encoded by exons 5 and 6, which are subject to alternative splicing only in a minority of transcripts. In addition to epidermal keratinocytes, p120ctn was shown to be expressed in a variety of adult and fetal tissues as well as in a number of human tumors. The expression pattern of various p120ctn transcripts, reflecting alternative splicing of the 5' exons, was strikingly similar between the corresponding adult and fetal tissues, while the expression patterns were discordant between certain tumors and their normal parental tissues, suggesting a functional role for the tissue-specific expression of the p120ctn isoforms. Finally, the tissue-specific expression of BP180 was shown to partially overlap with that of p120ctn, suggesting that the interaction of these two proteins may contribute to the modulation of cell-cell/matrix interactions in such tissues.

Alternative Splicing↗
PubMed1999

Nuclear factor-kappa B mediates TNF-alpha inhibitory effect on alpha 2(I) collagen (COL1A2) gene transcription in human dermal fibroblasts.

Among its plethora of activities as an inflammatory mediator, TNF-alpha has potent regulatory control on extracellular matrix production and degradation. Earlier studies have documented that TNF-alpha inhibits type I collagen gene (COL1A2) expression at the transcriptional level, but the characterization of the transcription factors involved has been elusive. In the present study, using transient cell transfection of human dermal fibroblasts with a battery of 5' end deletion/chloramphenicol acetyltransferase (CAT) reporter gene constructs, we have characterized the TNF-alpha response element of the COL1A2 promoter. The TNF-alpha response element was attributed to a specific region that comprises noncanonical activator protein-1 (AP-1) (CGAGTCA) and NF-kappa B (AGAGTTTCCC) binding sites. TNF-alpha effect was eliminated by a 2-bp substitution mutation in the NF-kappa B1 binding half site of the NF-kappa B cis element. Electrophoretic mobility shift assays (EMSA) showed that recombinant human NF-kappa B heterodimers as well as NF-kappa B1 and RelA homodimers, but not AP-1, were capable of binding this element. Further, EMSA with human fibroblast nuclear extracts demonstrated enhanced binding of a single, specific complex within 5 min of TNF-alpha stimulation, which reached a plateau by 1 h and was not affected by preincubation of cells with cycloheximide. Gel supershift assays identified the complex as the NF-kappa B (p50/p65) heterodimer, whereas Abs to nuclear factor of activated T cells (NF-AT) and Jun family members failed to recognize the complex. These data suggest that in fibroblasts TNF-alpha activates and initiates the nuclear translocation of NF-kappa B that binds a divergent NF-kappa B element and plays a critical role in the observed inhibition of alpha 2(I) collagen gene transcription.

Carrier Proteins↗
PubMed1999

Cooperation between SMAD and NF-kappaB in growth factor regulated type VII collagen gene expression.

We have previously demonstrated that transforming growth factor-beta (TGF-beta) and pro-inflammatory cytokines, such as tumor necrosis factor-alpha (TNF-alpha) or interleukin-1beta, synergistically enhance the expression of type VII collagen gene (COL7A1) in human dermal fibroblasts in culture (Mauviel et al., 1994). Recently, we identified a SMAD-containing complex, rapidly induced by TGF-beta and binding the region [-496/-444] of the COL7A1 promoter, responsible for COL7A1 gene transactivation (Vindevoghel et al., 1998a). In this report, we demonstrate that TGF-beta and TNF-alpha response elements are distinct entities within the COL7A1 promoter. In particular, we demonstrate that the TNF-alpha effect is mediated by NF-kappaB1/RelA (p50/p65) and RelA/RelA (p65/p65) NF-kappaB complexes binding the TNF-alpha response element (TaRE) located in the region [-252/-230], with RelA acting as the transcriptional activator. Finally, we provide definitive evidence for the role of both TGF-beta and TNF-alpha response elements as enhancer sequences, functioning in the context of a heterologous promoter in an additive manner in response to TGF-beta and TNF-alpha. This study provides the first identification of a functional interaction between the two immediate-early transcription factors, SMAD and NF-kappaB, to activate the expression of an extracellular matrix-related gene, COL7A1.

Binding Sites↗
PubMed1999

180-kD bullous pemphigoid antigen/type XVII collagen: tissue-specific expression and molecular interactions with keratin 18.

The 180-kD bullous pemphigoid antigen (BPAG2) is a hemidesmosomal transmembrane protein, also known as type XVII collagen. In this study, potential interactions of BPAG2 with other proteins expressed in epidermal keratinocytes were explored by yeast two-hybrid system using the amino-terminal intracellular domain of BPAG2 as a bait. Several independent interacting clones encoding keratin 18 (K18) were identified when the keratinocyte cDNA library, cloned into the yeast two-hybrid activation domain vector, was screened. The peptide sequence responsible for the interaction of BPAG2 was restricted to amino acids 15-25, and substitution of a valine residue in the middle of this sequence by a proline (V23P) by site-directed mutagenesis abolished the interaction. Further examination of the K18 sequences by restricted cDNA constructs in yeast two-hybrid system identified a carboxyl-terminal segment corresponding to helix 2B domain as critical for BPAG2 binding. The interaction of BPAG2/K18 was confirmed by an in vitro protein-protein interaction assay, which also confirmed that normal human keratinocytes express K18 in culture. The tissue specific expression of BPAG2 was first examined using a multi-tissue RNA blot. Human multiple tissue cDNA panels representing a variety of adult and fetal tissues as well as tumor cells were used as PCR-templates to study the expression patterns of both BPAG2 and K18. The results demonstrated significant level of expression of BPAG2, besides in epidermal keratinocytes, also in a variety of tissues with predominant epithelial component, such as mammary, salivary and thyroid glands, colon, prostate, testis, placenta, and adult and fetal thymus, as well as in colon, pancreatic and prostatic adenocarcinoma cell lines, and an ovarian carcinoma. As expected, K18 transcript is present in liver, pancreas, colon, placenta, and in fetal kidney. Collectively, the results suggest that BPAG2 has a relatively broad tissue distribution including specialized and simple epithelia, and that within the tissues such as colon and placenta, BPAG2 may have direct interactions with K18, a keratin characteristically expressed in a simple epithelia.

Adult↗
PubMed1999

Human periplakin: genomic organization in a clonally unstable region of chromosome 16p with an abundance of repetitive sequence elements.

Periplakin, a member of the plakin family of proteins, has been recently characterized by cDNA cloning, and the corresponding gene, PPL, has been mapped to human chromosome 16p13.3 (Aho et al., 1998, Genomics 48: 242-247). Periplakin has also been shown to serve as an autoantigen in a malignancy-associated autoimmune blistering disease, paraneoplastic pemphigus (Mahoney et al., 1998, J. Invest. Dermatol. 111: 308-313). In this study, we have elucidated the intron-exon organization of human PPL and characterized its promoter region. The flanking 5' sequences were rich in G and C ( approximately 80%) and included multiple AP2 sites and a SP1 site, while no canonical TATA or CCAAT sequences were found. The functionality of the upstream sequences (-709 to +135) as a promoter in cultured epidermal keratinocytes was detected by a CAT reporter gene, and a limited region (-382 to +135) showed activity in cultured dermal fibroblasts, attesting to cell-type specificity of the promoter. The genomic organization, including the intron-exon borders, was determined by direct nucleotide sequencing of human genomic P1 clones. Comparative analysis of cDNA and genomic sequences revealed that PPL consists of 22 exons, with the distribution of exons in PPL being consistent with that of other plakin genes: 21 small exons, separated by large introns, encode the amino-terminal globular domain, and 1 large exon encodes the entire rod and the tail domains. Characterization of four P1 clones spanning the PPL locus revealed multiple Alu repeats, 20 of them within 33 kb of the entirely sequenced segments (0.60/kb), in addition to numerous MIR and L1 elements. These repetitive elements could lead to the clonal instability detected throughout the genomic P1 clones and may give rise to the genomic rearrangements possibly underlying the paraneoplastic pemphigus.

Base Sequence↗