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S A Jimenez

Publications and source records attributed to S A Jimenez.

At least 37 records · Page 2Linked to original sources
PubMed1998

Modulation of human alpha1(I) procollagen gene activity by interaction with Sp1 and Sp3 transcription factors in vitro.

In previous work, we delineated a proximal region of the human alpha1(I) collagen gene (COL1A1) promoter necessary to direct its basal transcription in fibroblasts. This region has potential recognition sites for a variety of DNA-binding proteins. Here, we show that the -129/-107-bp sequence in this region of the promoter, which harbors an inverted CCAAT motif closely linked to a GC-rich direct repeat and is perfectly conserved between mouse and human, specifically bound the transcription factors Sp1, Sp3, and CTF/NF-1 in nuclear extracts from human skin and lung fibroblasts. Drosophila Schneider L2 cells lacking endogenous Sp activity were used to investigate the effect of expression of Sp or CTF/NF-1 transcription factors on COLlAl promoter activity. Expression of Sp1 caused potent trans-activation of a COL1A1 promoter (-174 to +42bp). In contrast, expression of Sp3, which binds to the same recognition sites as Sp1, and CTF/NF-1 stimulated COL1A1 promoter activity only at higher concentrations, and Sp2 did not transactivate. Expression of a 10-fold excess of Sp3, but not CTF/NF-1 or Sp2, abrogated the stimulation of COL1A1 promoter activity induced by Sp1. TGF-beta at concentrations previously shown to increase COL1A1 transcription caused a decrease in the relative amount of Sp3 in fibroblast nuclear extracts. These results suggest that both Sp1 and Sp3 bind to the proximal COL1A1 promoter and stimulate its activity; however, their interaction with each other may result in repression of Sp1-induced COL1A1 transcription. Alterations in the relative amounts or DNA-binding activities of these transcription factors in a cell- or signal-specific manner may contribute to the control of transcription from the COL1A1 promoter. The present findings, and recent observations implicating Sp1 and its homologs in regulating the expression of several collagen genes, suggest that the family of Sp1 transcription factors play a role in physiological and pathological modulation of connective tissue accumulation.

Animals↗
PubMed1998

Differential regulation of cyclooxygenases 1 and 2 by interleukin-1 beta, tumor necrosis factor-alpha, and transforming growth factor-beta 1 in human lung fibroblasts.

In the present studies we found that incubation of human lung fibroblasts with transforming growth factor-beta 1 (TGF-beta 1) potentiated the interleukin-1 beta (IL-1 beta) and/or tumor necrosis factor-alpha (TNF-alpha)-stimulated production of prostaglandin E2 (PGE2). Analysis of fibroblast proteins showed the induction of cyclooxygenase-1 (Cox-1) by TGF-beta 1 and the induction of Cox-2 by IL-1 beta and TNF-alpha. The levels of transcripts for Cox-1 were minimally modified by IL-1 beta or TNF-alpha, however, they were increased by 12-fold by TGF-beta 1. Transcripts for Cox-2 were induced by IL-1 beta or TNF-alpha and their induction was potentiated by TGF-beta 1. TGF-beta 1 alone did not induce Cox-2 transcripts. In vitro transcription assays showed that IL-1 beta and TNF-alpha increased the transcription of the Cox-2 gene, whereas TGF-beta 1 had no effect. Addition of TGF-beta did not increase further the transcription of Cox-2 in IL-1 beta-treated cells, but increased the stability of the corresponding transcripts. The transcription rate of the Cox-1 gene was not increased by any of the cytokines studied. In summary, we demonstrate that the potentiation of PGE2 production by TGF-beta 1 in IL-1 beta and TNF-alpha-treated fibroblasts is the result of transcriptional stimulation of the Cox-2 gene by IL-1 beta and TNF-alpha and the stabilization of the resulting transcripts by TGF-beta 1.

Cells, Cultured↗
PubMed1998

Identification of fetal DNA and cells in skin lesions from women with systemic sclerosis.

BACKGROUND: Systemic sclerosis is a disease of unknown origin which often occurs in women after their childbearing years. It has many clinical and histopathological similarities to chronic graft-versus-host disease. Recent studies indicate that fetal stem cells can survive in the maternal circulation for many years post partum. This finding suggests that fetal cells persisting in the maternal circulation or tissues could be involved in the pathogenesis of systemic sclerosis by initiating a graft-versus-host reaction. METHODS: We used the polymerase chain reaction (PCR) to identify Y-chromosome sequences in DNA extracted from peripheral-blood cells and skin lesions from women with systemic sclerosis of recent onset. To confirm the PCR findings, we used fluorescence in situ hybridization of peripheral-blood cells and cells within chronic inflammatory-cell infiltrates in biopsy specimens of affected skin. RESULTS: Y-chromosome sequences were found in DNA from peripheral-blood cells in 32 of 69 women with systemic sclerosis (46 percent), as compared with 1 of 25 normal women (4 percent, P<0.001), and in T lymphocytes from 3 women with systemic sclerosis who had male offspring. Furthermore, Y-chromosome sequences were identified in skin-biopsy specimens from 11 of 19 women with systemic sclerosis (58 percent); 9 of the 11 were known to have carried male fetuses. Nucleated cells containing Y chromosomes were detected by fluorescence in situ hybridization in paraffin-embedded sections of skin lesions from all seven women we tested whose skin-biopsy specimens contained Y-chromosome sequences. CONCLUSIONS: Fetal antimaternal graft-versus-host reactions may be involved in the pathogenesis of systemic sclerosis in some women.

Adolescent↗
PubMed1998

The Tight skin mouse: demonstration of mutant fibrillin-1 production and assembly into abnormal microfibrils.

Mice carrying the Tight skin (Tsk) mutation harbor a genomic duplication within the fibrillin-1 (Fbn 1) gene that results in a larger than normal in-frame Fbn 1 transcript. In this study, the consequences of the Tsk mutation for fibrillin-containing microfibrils have been examined. Dermal fibroblasts from Tsk/+ mice synthesized and secreted both normal fibrillin (approximately 330 kD) and the mutant oversized Tsk fibrillin-1 (approximately 450 kD) in comparable amounts, and Tsk fibrillin-1 was stably incorporated into cell layers. Immunohistochemical and ultrastructural analyses of normal and Tsk/+ mouse skin highlighted differences in the gross organization and distribution of microfibrillar arrays. Rotary shadowing of high Mr preparations from Tsk/+ skin demonstrated the presence of abundant beaded microfibrils. Some of these had normal morphology and periodicity, but others were distinguished by diffuse interbeads, longer periodicity, and tendency to aggregate. The presence of a structurally abnormal population of microfibrils in Tsk/+ skin was unequivocally demonstrated after calcium chelation and in denaturating conditions. Scanning transmission electron microscopy highlighted the presence of more mass in Tsk/+ skin microfibrils than in normal mice skin microfibrils. These data indicate that Tsk fibrillin-1 polymerizes and becomes incorporated into a discrete population of beaded microfibrils with altered molecular organization.

Animals↗
PubMed1998

Effects of interferon-gamma and tumor necrosis factor alpha on the expression of the genes encoding aggrecan, biglycan, and decorin core proteins in cultured human chondrocytes.

OBJECTIVE: To determine the effects of interferon-gamma (IFN gamma) and tumor necrosis factor alpha (TNF alpha), alone or in combination, on the expression of aggrecan, biglycan, and decorin core protein genes in human chondrocytes. METHODS: Isolated human chondrocytes were cultured on poly(2-hydroxyethyl methacrylate)-coated plastic dishes to prevent the loss of cartilage-specific phenotype, and the effects of IFN gamma and TNF alpha, alone or in combination, on aggrecan, biglycan, and decorin core protein gene transcription and steady-state messenger RNA (mRNA) levels were examined. RESULTS: The addition of IFN gamma (1.5 pM) or TNF alpha (0.3 pM) caused a decrease in the steady-state level of aggrecan mRNA (-25% and -15%, respectively), and the combination of these low-concentration cytokines caused a potent inhibition (-66%). These effects were the result of a decrease (-50%) in the transcription rate of the corresponding gene. At the concentrations used, IFN gamma did not alter the levels of biglycan mRNA or the transcription rates of the biglycan core protein gene. In contrast, TNF alpha decreased biglycan steady-state mRNA levels (-62%) and the biglycan core protein gene transcription rate (-18%). The combination of IFN gamma and TNF alpha resulted in a potentiation of the inhibitory effects of TNF alpha on biglycan mRNA levels (-79%) and transcription rate of the biglycan core protein gene (-46%). IFN gamma produced a modest decrease in decorin mRNA levels (-23%) and decorin core protein gene transcription rate (-17%). In contrast, TNF alpha resulted in a marked increase in decorin mRNA levels (+260%) that was not the result of transcriptional regulation. Notably, the combination of IFN gamma and TNF alpha potentiated the inhibitory effects of IFN gamma on decorin mRNA (-80%) and on the transcription of the corresponding gene (-43%). Similar results were obtained in fetal and adult articular chondrocytes. CONCLUSION: These data demonstrate that 1) the expression of the core protein genes encoding the cartilage proteoglycans aggrecan, biglycan, and decorin is differentially regulated by IFN gamma and TNF alpha; 2) these effects are mediated by transcriptional and posttranscriptional mechanisms; and 3) the combination of the 2 cytokines causes a potent inhibitory effect on the expression of the genes for the core proteins of these 3 proteoglycans, which occurs largely at the transcriptional level. The inhibition of aggrecan, decorin, and biglycan core protein gene expression by the combination of IFN gamma and TNF alpha may contribute to the cartilage destruction that is characteristic of inflammatory joint diseases.

Adult↗
PubMed1998

Oral iloprost treatment in patients with Raynaud's phenomenon secondary to systemic sclerosis: a multicenter, placebo-controlled, double-blind study.

OBJECTIVE: To evaluate the efficacy and tolerability of an oral preparation of iloprost, a prostacyclin analog, in patients with Raynaud's phenomenon (RP) secondary to systemic sclerosis (scleroderma). METHODS: A multicenter, randomized, parallel-group, placebo-controlled double-blind study was performed at university and community-based medical centers. Patients were randomly assigned to receive either 50 microg of iloprost orally twice daily or an identical gelatin-coated capsule containing placebo for 6 weeks. Outcome measures included average total daily duration of RP attacks, average number of RP attacks, and RP condition scored via a standardized daily diary. RESULTS: Three hundred eight patients with scleroderma (272 women, 36 men, mean age 49 years [range 18-80]) were enrolled. One hundred fifty seven were assigned to receive iloprost and 151 to receive placebo. One hundred forty-three patients in the iloprost group (91.1%) and 144 in the placebo group (95.4%) completed the 6-week treatment phase. Fifteen of these treated patients (8 iloprost, 7 placebo) failed to complete all of the followup visits. The mean reduction in the average duration of attacks from baseline to week 5-6 was 24.32 minutes in the iloprost group and 34.34 minutes in the placebo group (P = 0.569). Likewise, the mean reduction from baseline to week 5-6 in the daily frequency of attacks was 1.02 in the iloprost group and 0.83 in the placebo group (P = 0.459). The Raynaud's condition score, a patient-completed assessment of the severity of RP attacks, was reduced by 1.32 in the iloprost group and 1.00 in the placebo group (P = 0.323). The lack of significant difference between treatment groups did not change when a variety of factors, including use of other vasodilators, duration of disease, classification of scleroderma (limited versus diffuse), or number of baseline digital ulcers were taken into account. Premature withdrawal from the study due to adverse events occurred in 10 patients (6.4%) in the iloprost group and 3 (2.0%) in the placebo group (P = 0.058). CONCLUSION: Oral iloprost at a dosage of 50 microg twice daily is no better than placebo for management of RP secondary to scleroderma, either during 6 weeks of treatment or during 6 weeks of posttreatment followup.

Administration, Oral↗
PubMed1998

Modulation of basal expression of the human alpha1(I) procollagen gene (COL1A1) by tandem NF-1/Sp1 promoter elements in normal human dermal fibroblasts.

We previously demonstrated that a segment of the human alpha1 type I procollagen gene (COL1A1) promoter encompassing nt -174 to -84 is responsible for the highest transcriptional activity in collagen producing cells in vitro. Here, we identified two almost identical tandem NF-1/Sp1 binding sites located between nt -129 to -107 (distal element) and nt -104 to -77 (proximal element) that are responsible for the basal regulation of COL1A1 transcription in normal human dermal fibroblasts. Transient transfection studies revealed that 85% of the basal COL1A1 promoter activity resides within the distal element; however, site-directed mutagenesis within the CCAAT motif in the proximal element resulted in a 98% decrease of the COL1A1 promoter activity. We conclude that each of the NF-1/Sp1 tandem binding sites has a different function. The distal element drives the transcriptional activity of the COL1A1 promoter but is not sufficient for its basal expression, whereas the NF-1 binding site in the proximal element is essential for in vitro COL1A1 gene transcription.

Base Sequence↗
PubMed1998

Type X collagen biosynthesis and expression in avian tibial dyschondroplasia.

OBJECTIVE: Tibial dyschondroplasia (TD) is an abnormality of growth plate cartilage characterized by the presence of non-vascularized, non-mineralized tissue. The objective of this study was to examine structural and functional alterations of the growth plate-specific type X collagen in TD cartilage. DESIGN: Collagen biosynthesis was examined in organ cultures and in cultured chondrocytes from normal growth plate and TD cartilage. Thermal stability of type X collagen extracted from normal and TD cartilage organ cultures to protease digestions by trypsin plus chymotrypsin or bacterial collagenase was determined. The expression of collagen genes was examined in cultured normal and TD chondrocytes. RESULTS: Synthesis of total collagen and of type X collagen was greater than threefold higher in organ cultures from the TD lesion compared with normal growth plate. The increase in type X collagen synthesis in the lesion was compensated by a reduction in the relative proportions of types II and XI collagens. The thermal denaturation and collagenase cleavage properties of purified types II and X collagens from TD cartilage were normal. The expression of type X collagen gene was threefold higher in cultured TD chondrocytes compared to chondrocytes from normal growth plate. Normal growth plate chondrocytes in primary cultures synthesized predominantly type X collagen (80% of total collagen). In contrast, TD chondrocytes synthesized mainly types I and II collagens and type X collagen represented only 22% of total collagen. TD cells initiated the synthesis of type I collagen within 5 days of primary culture, whereas normal chondrocytes did not synthesize this collagen during the same culture period. Although type X collagen synthesis was reduced in TD chondrocytes, the mRNA levels for type X collagen were substantially higher than in normal chondrocytes. CONCLUSION: Accumulation of type X collagen in TD cartilage results from its increased biosynthesis which is due largely to increased expression of the gene for this collagen, although, the chondrocyte culture studies suggest the possibility of postranscriptional defect in type X collagen synthesis or processing in TD lesion. Moreover, the TD chondrocytes in contrast with normal chondrocytes display evidence of prompt loss of their specific phenotype during short-term primary cultures.

Animals↗
PubMed1998

Production of cartilage oligomeric matrix protein (COMP) by cultured human dermal and synovial fibroblasts.

OBJECTIVE: Cartilage oligomeric matrix protein (COMP) is a large disulfide-linked pentameric protein. Each of its five subunits is approximately 100,000 Da in molecular weight. COMP was originally identified and characterized in cartilage and it has been considered a marker of cartilage metabolism because it is currently thought not to be present in other joint tissues, except for tendon. To confirm the tissue specificity of COMP expression we examined cultured human dermal fibroblasts, human foreskin fibroblasts, and normal human synovial cells for the synthesis of COMP in culture. METHOD: Normal synovial cells and normal human dermal foreskin fibroblasts were isolated from the corresponding tissues by sequential enzymatic digestions and cultured in media containing 10% fetal bovine serum until confluent. During the final 24 h of culture, the cells were labeled with 35S-methionine and 35S-cysteine in serum- and cysteine/methionine-free medium. The newly synthesized COMP molecules were immunoprecipitated from the culture media with a COMP-specific polyclonal antiserum, or with monoclonal antibodies or affinity-purified COMP antibodies. The immunoprecipitated COMP was analyzed by electrophoresis in 5.5% polyacrylamide gels. For other experiments, synovial cells cultured from the synovium of patients with rheumatoid arthritis (RA) and osteoarthritis (OA) were similarly examined. RESULTS: A comparison of the amounts of COMP produced by each cell type (corrected for the DNA content) revealed that synovial cells produced > or = 9 times more COMP than chondrocytes or dermal fibroblasts. COMP could be easily detected by immunoprecipitation in all cell types. Electrophoretic analysis revealed a distinct band with an apparent MW of 115-120 kDa in samples from each of the three cell types, regardless of the antibody used. COMP expression in cultures of synoviocytes derived from OA and RA patients showed that OA and RA synovial cells produced similar amounts of monomeric COMP of identical size to those COMP monomers produced by normal synovial cells. The addition of TGF-beta to these cultures resulted in an increase in COMP production in normal, OA and RA synovial cells (45, 116 and 115% respectively). CONCLUSION: These studies demonstrate that substantial amounts of COMP are produced by several mesenchymal cells including synoviocytes and dermal fibroblasts. These findings raise important concerns regarding the utility of measurements of COMP levels in serum or in synovial fluid as markers of articular cartilage degradation because of the likelihood that a substantial proportion of COMP or COMP fragments present in serum or synovial fluid may be produced by cells other than articular chondrocytes.

Adult↗
PubMed1998

T cells infiltrating the skin of Tsk2 scleroderma-like mice exhibit T cell receptor bias.

The T cell repertoire expressed by Tsk2 mice, a novel experimental model of systemic sclerosis, was examined to determine whether cells infiltrating the areas of involved skin exhibit a T cell receptor (TCR) bias. Reverse transcription-polymerase chain reactions (RT-PCR) were conducted using RNA extracted from lymph nodes and skin from TSk2 mice and from normal mice, with an oligonucleotide primer library specific for the variable region of the TCR (beta) chain. RT-PCR signals were observed in all lymph node cell (LNC) samples from both Tsk2 mice and control mice, with eighteen of the twenty-one Vbeta types present. In contrast, cDNA extracted from areas of involved skin from Tsk2 mice exhibited a restricted pattern, with positive Vbeta signals corresponding to eight T cell subtypes (Vbeta1, 6, 8.1, 8.2, 10, 11, 16, and 18). Band strength analysis revealed that three Vbeta subtypes dominated within this restricted pattern (Vbeta8.1, 11, and 18). Moreover, this pattern of Vbeta bias was consistent among the four skin samples from different Tsk2 mice. These data suggest that a restricted T cell population participates in the inflammatory cell infiltrate of Tsk2 skin.

Animals↗
PubMed1998

A pilot study of intermittent intravenous cyclophosphamide for the treatment of systemic sclerosis associated lung disease.

OBJECTIVE: Pulmonary fibrosis, a frequent manifestation of systemic sclerosis (SSc), is considered incurable. Our aim was to assess the effect of therapy with intravenous (i.v.) cyclophosphamide on the course of pulmonary fibrosis in patients with SSc. METHODS: Five patients with SSc and clinical, laboratory, or radiographic findings of interstitial lung disease were treated with cyclophosphamide (1 g) administered i.v. monthly for 48 weeks. The dyspnea score, pulmonary function tests, arterial blood oxygen content, radiologic abnormalities, and bronchoalveolar lavage (BAL) fluid cellularity were determined before and after therapy. RESULTS: The dyspnea score decreased by 42% after 48 weeks of therapy. Forced vital capacity (FVC, percentage of predicted) increased by 7%, and carbon monoxide diffusing capacity (DLCO) decreased by 12%, but these changes were not statistically significant. Arterial blood oxygenation remained unchanged. At baseline, high resolution computed tomography of the lungs showed honeycombing, reticulonodular, or ground glass patterns in each patient examined. These radiologic abnormalities did not improve during treatment. A marked decrease in BAL fluid cell number, but not in the percentage of neutrophils, was observed after therapy. Nausea and leukopenia were frequent but mild side effects. One patient developed hemorrhagic cystitis. CONCLUSION: The results suggest that intermittent treatment with i.v. cyclophosphamide reduces the severity of dyspnea, but fails to improve FVC or DLCO, or cause resolution of radiologic abnormalities, in patients with SSc.

Adult↗
PubMed1997

Detection and characterization of Sp1 binding activity in human chondrocytes and its alterations during chondrocyte dedifferentiation.

We have detected DNA binding activity for a synthetic oligonucleotide containing an Sp1 consensus sequence in nuclear extracts from human chondrocytes. Changes in the levels of Sp1 oligonucleotide binding activity were examined in nuclear extracts from freshly isolated human chondrocytes, from chondrocytes that had been cultured under conditions that allowed the maintenance of a chondrocyte-specific phenotype on plastic dishes coated with the hydrogel poly(2-hydroxyethyl methacrylate), and from chondrocytes induced to dedifferentiate into fibroblast-like cells by passage in monolayer culture on plastic substrata. It was observed that Sp1 binding was 2-3-fold greater in nuclear extracts from dedifferentiated chondrocytes than in nuclear extracts from either freshly isolated chondrocytes or from cells cultured in suspension. The Sp1 binding activity was specific, since it was competed by unlabeled Sp1 but not by AP1 or AP2. The addition of a polyclonal antibody against Sp1 to nuclear extracts from freshly isolated chondrocytes or to extracts isolated from chondrocytes cultured in monolayer decreased the binding of Sp1 by approximately 85%. However, when the same experiment was carried out with nuclear extracts prepared from cells cultured on poly(2-hydroxyethyl methacrylate)-coated plates, only a very slight inhibition of Sp1 binding was observed. When fragments of the COL2A1 promoter containing putative Sp1 binding sites amplified by polymerase chain reaction were examined, it was found that the amounts of DNA-protein complex formed with nuclear extracts from dedifferentiated chondrocytes were 2-3-fold greater than the amounts formed with nuclear extracts from freshly isolated chondrocytes or from cells cultured in suspension. Quantitation of DNA binding activity by titration experiments demonstrated that nuclear extracts from fibroblast-like cells contained approximately 2-fold greater Sp-1 specific binding activity than nuclear extracts from chondrocytes. The direct role of Sp1 in type II collagen gene transcription was demonstrated by co-transfection experiments of COL2A1 promoter-CAT constructs in Drosophila Schneider line L2 cells that lack Sp1 homologs. This is the first demonstration of Sp1 binding activity in human chondrocytes and of differences in Sp1 DNA binding activity between differentiated and dedifferentiated chondrocytes.

Animals↗
PubMed1997

Fetal-maternal HLA compatibility confers susceptibility to systemic sclerosis.

Systemic sclerosis (SSc) is a disease of unknown origin, which occurs predominantly in women after childbearing years. There are prominent clinical and histopathologic similarities between SSc and chronic graft-versus-host disease (GVHD). GVHD can occur after blood transfusions or after transplantation with HLA-compatible bone marrow. Here we examined the hypothesis that SSc may be caused by fetal cells crossing the placenta into the maternal circulation and providing donor lymphocytes which recognize disparate HLA antigens, resulting in a reaction similar to chronic GVHD. To test the hypothesis we analyzed the inheritance of HLA class I and class II haplotypes in the families of 37 SSc patients and 42 control individuals. Twenty-six (70.2%) SSc patients had HLA class II alleles compatible either for their offspring or mother, compared with only nine (21%) control individuals. The four patients with juvenile onset SSc we analyzed had alleles compatible with their mothers. These results suggest that in some patients, SSc may, indeed, be a form of chronic GVHD caused by fetal or maternal cells which have crossed the placenta during pregnancy and have remained unrecognized by the host due to class II HLA compatibility, and that subsequent activation of these cells by as yet unknown stimuli result in the development of the disease.

Adolescent↗
PubMed1997

Characterization of human type II procollagen and collagen-specific antibodies and their application to the study of human type II collagen processing and ultrastructure.

Type II collagen is the most abundant collagen in articular cartilage and, together with other tissue-specific collagens and proteoglycans, provides the tissue with its shock-absorbing properties and its resiliency to stress. Specific antibodies which recognize various collagen types have been very useful in the study of collagen biosynthesis, structure and metabolism in normal and pathological conditions. Antibodies which recognize epitopes of type II collagen have been described previously; however, many of these antibodies display cross-reactivity with other collagens or with type II collagen from other species, reflecting the high degree of homology of the helical domains of fibrillar collagens. In this study, we prepared antibodies to sequential determinants of human type II procollagen employing synthetic peptides with sequences deduced from the nucleotide sequence of the human alpha 1 (II) procollagen cDNA. The antibodies were highly specific for epitopes in either the C-terminal propeptide or the telopeptide of the human type II collagen and did not cross-react with other human interstitial collagens or with murine type II collagen. These antibodies were used in conjunction with biosynthetic labeling to study the secretion and processing of human type II procollagen and collagen in human chondrocytes in vitro. The results indicated that a lag period of about 90 min was required for the secretion of newly synthesized type II procollagen. Conversion of the secreted procollagen into fully processed alpha-chains and their deposition in the cell layer were first apparent 240 min following the initiation of biosynthetic labeling. The antibodies were also used to examine, by immunoelectron microscopy, the structure of the extracellular matrix produced by human chondrocytes maintained in long-term cultures under conditions which permit the preservation of the cartilage-specific phenotype. These highly specific antibodies provide valuable tools to study the metabolism and structure of human type II procollagen and collagen in normal and pathologic conditions.

Amino Acid Sequence↗
PubMed1997

Increased activity of the alpha 1(I) procollagen promoter in skin fibroblasts from patients with chronic eosinophilia-myalgia syndrome.

Eosinophilia-myalgia syndrome (EMS), a novel L-tryptophan-associated disease, which occurred as an epidemic in 1989, is characterized by diffuse fibrosis of the skin. The objective of these studies was to compare the in vitro expression of the gene encoding the human alpha 1(I) procollagen (COL1A1) in dermal fibroblasts derived from patients with long-standing EMS and from healthy controls. Early passage fibroblasts in culture were transiently transfected with a series of progressively 5' deleted human COL1A1 promoter-CAT reporter gene DNA constructs. Resultant CAT activity was assayed in the cytoplasmic extracts. Following transient transfection, COL1A1 promoter activity in 4/5 fibroblast cell lines derived from patients with EMS was two-to threefold greater than in matched normal fibroblasts. Deletion analysis indicated that CAT activity was highest, and displayed the greatest increase in EMS vs normal fibroblasts, with promoter constructs spanning 174 bp upstream from the COL1A1 transcription start site. The study shows increased CAT activity driven by a 174 bp fragment of COL1A1 in transiently transfected skin fibroblasts from patients with EMS even in the chronic stage of disease. These findings expand on previous observations indicating increased type I collagen gene expression in EMS fibroblasts, and suggest that fibrosis in L-tryptophan-induced EMS is associated with transcriptional activation of type I collagen gene expression, which persists even following cessation of L-tryptophan use.

Adult↗
PubMed1996

The transcription of human alpha 1(I) procollagen gene (COL1A1) is suppressed by tumour necrosis factor-alpha through proximal short promoter elements: evidence for suppression mechanisms mediated by two nuclear-factorbinding sites.

Recent studies have demonstrated that tumour necrosis factor-alpha (TNF-alpha) decreases alpha 1(I) procollagen gene (COL1A1) expression in cultured human dermal fibroblasts. The purpose of this study was to analyse the transcriptional control of COL1A1 by TNF-alpha. Cultured human dermal fibroblasts were transiently transfected with plasmids containing 5' flanking sequences of COL1A1 fused to the chloramphenicol acetyltransferase (CAT) gene, and were incubated for 48 h in medium with or without TNF-alpha. TNF-alpha inhibited the CAT activity of fibroblasts transfected with plasmids containing 2.3 kb of 5' flanking sequences of COL1A1, whereas the activity of control plasmids containing the herpes simplex thymidine kinase promoter gene (pBLCAT) was unaltered. A series of deletion constructs of various small substitution mutations of the COL1A1 5' flanking region fused to the CAT gene were also transfected, and CAT activity was measured after incubation with TNF-alpha. TNF-alpha suppressed COL1A1 promoter activity through proximal short promoter elements containing only 107 bp. Short substitution mutations between -101 and -97 bp or between -46 and -38 bp abolished TNF-alpha suppression of COL1A1 promoter activity. DNA-protein complex formation was observed involving both sites in gel retardation assays. These results suggest that TNF-alpha suppressed COL1A1 promoter activity through elements located between -101 and -97 bp and between -46 and -38 bp of the COL1A1 promoter, and that the suppression involved DNA-protein interactions.

Base Sequence↗
PubMed1996

Alteration in the conformational stability of collagen caused by the incorporation of the lysine analogue S-2-aminoethylcysteine.

We studied the effects of the lysine analogue S-2-aminoethylcysteine on the activation of lysyl tRNA and on the secretion and conformational stability of newly synthesized type I collagen in embryonic chick tendon fibroblasts. The analogue competed efficiently with lysine for activation onto tRNA without affecting significantly the activation of other amino acids (Km for lysine: 1.6 microM; Ki for S-2-aminoethylcysteine: 1.4 microM). The analogue also profoundly inhibited the synthesis and secretion of [14C]procollagen but did not affect the synthesis or secretion of non-collagenous proteins. Although the [14C]proline-labeled procollagen synthesized in the presence of S-2-aminoethylcysteine contained normal levels of hydroxyproline, it was susceptible to digestion with pepsin at 25 degrees C, indicating that incorporation of the analogue altered the conformational stability of the collagen triple helix. This analogue should be a powerful tool to further study the role of lysine on collagen structure and to determine how altered collagen structure affects its synthesis and secretion. Furthermore, this analogue may be a potent and selective inhibitor of collagen accumulation in pathologic conditions accompanied by tissue fibrosis.

Amino Acids↗