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Biomedical subjects

D R Edwards

Publications and source records attributed to D R Edwards.

At least 19 recordsLinked to original sources
PubMed1992

Involvement of AP1 and PEA3 binding sites in the regulation of murine tissue inhibitor of metalloproteinases-1 (TIMP-1) transcription.

Transcription of tissue inhibitor of metalloproteinases-1 (TIMP-1), a secreted protein that regulates the activities of the metalloproteinases, collagenase and stromelysin, is activated by serum growth factors. Transient transfection experiments have revealed several regions of cis-acting regulatory sequences involved in the response of the murine TIMP-1 gene to serum. One area is in the vicinity of the promoter, consisting of a non-consensus binding site (5'-TGAGTAA-3' at -59/-53) for transcription factor AP1 and an adjacent 24 bp region of dyad symmetry that contains a PEA3-binding site. A second is an upstream region (-1020 to -780) that acts as an enhancer when linked to a heterologous promoter, and contains a consensus AP1 binding site (at -803/ -797). Gel retardation assays revealed differences between nuclear factors in mouse C3H10T1/2 cells that bound to the TIMP(-59/ -53)AP1 site and a consensus collagenase TRE (TPA-response element). The TIMP(-59/ -53)AP1 site is a promiscuous motif that binds c-Fos/c-Jun AP1 translated in vitro and is an effective competitor for binding of nuclear AP1 factors to the consensus TRE, but in addition it binds factors that do not associate with the consensus TRE. The TIMP(-59/ -53)AP1 motif and the dyad symmetry region stimulated expression from a thymidine kinase promoter in an additive fashion, and competition experiments showed that excess copies of these factor binding sites reduced expression from a reporter plasmid driven by the TIMP-1 promoter. Our data show that binding sites for AP1 and PEA3 transcription factors are involved in the regulation of TIMP-1 transcription, which suggests that the coordinated induction of TIMP-1, collagenase and stromelysin may be achieved through the actions of a shared set of nuclear transcription factors. However, the properties of the TIMP-1(-59/ -53)AP1 motif likely reflect an additional type of transcriptional regulation restricted to TIMP-1.

Animals↗
PubMed1992

Differential regulation of TIMP-1 and TIMP-2 mRNA expression in normal and Ha-ras-transformed murine fibroblasts.

A cDNA containing the complete coding region of the murine tissue inhibitor of metalloproteinases-2 (TIMP-2) was isolated using reverse transcription-polymerase chain reaction amplification. The predicted murine TIMP-2 amino acid sequence shows 96% identity with human TIMP-2, but only 42% identity with murine TIMP-1. This high degree of evolutionary conservation between the human and mouse proteins suggests that TIMP-2 performs an essential biological function. The expression of the TIMP-1 and TIMP-2 mRNAs was examined in normal and ras-transformed murine fibroblasts. While TIMP-1 transcription was highly serum-inducible in normal murine C3H10T1/2 fibroblasts, TIMP-2 mRNA expression was largely constitutive. A series of ras-transformed derivatives of C3H10T1/2 fibroblasts showed great variability in TIMP-1 expression: some lines retained serum inducibility, others displayed constitutive expression at either high or low levels. In contrast, TIMP-2 expression was insensitive to transformation. Neither TIMP-1 nor TIMP-2 expression at the RNA level, or total TIMP activity in conditioned media could be correlated with the metastatic potential of the ras-transformed lines. Our data demonstrate that the mechanisms that regulate murine TIMP-1 and TIMP-2 expression are distinct arguing for different physiological roles for the two TIMPs.

Amino Acid Sequence↗
PubMed1992

Protein synthesis inhibitors differentially superinduce c-fos and c-jun by three distinct mechanisms: lack of evidence for labile repressors.

Protein synthesis inhibitors strongly augment and prolong the usually transient induction of c-fos and c-jun by growth factors, phorbol esters etc., a phenomenon termed superinduction which is conventionally regarded as a secondary consequence of translational arrest. Our recent demonstration that some inhibitors can act positively as nuclear signalling agonists compromises this view and necessitates a re-evaluation of superinduction. First, we show that labile repressors, widely postulated to act negatively on diverse superinducible genes, are not involved in regulating c-fos and c-jun. Secondly, two components of c-fos and c-jun superinduction, namely the delay in shutting off transcription and stabilization of their mRNAs, arise from translational arrest and are common to all protein synthesis inhibitors. Thirdly, the recently described capacity to act positively as nuclear signalling agonists to stimulate pp33/pp15 phosphorylation is restricted to compounds such as anisomycin and cycloheximide; these, but not emetine or puromycin, will induce c-fos/c-jun on their own. Fourthly, the translational arrest-related components of superinduction are dissociable from the signalling agonist effects at sub-inhibitory concentrations of anisomycin, under which conditions a new type of c-fos/c-jun superinduction with 'spike' kinetics is observed. Finally, we show that in response to EGF plus anisomycin, the nuclear signalling responses are themselves augmented and prolonged in a manner that corresponds to c-fos/c-jun superinduction under these conditions.

Animals↗
PubMed1992

Expression of metalloproteinases and their inhibitors in primary pulmonary carcinomas.

Nine primary pulmonary carcinomas, one metastatic carcinoma, and two malignant pleural mesotheliomas have been analysed for the expression at the mRNA level of metalloproteinases (MPs) and tissue inhibitors of MPs (TIMPs). In situ hybridisation showed TIMP-1 and TIMP-2 transcripts predominantly over tumour stroma and gelatinases evenly distributed over both stromal and tumour cells. While both TIMP-1 and TIMP-2 were expressed in non-neoplastic lungs (NNL) as well as in carcinomas, stromelysin 3 (ST3), 92 kDa gelatinase and interstitial collagenase were expressed only by carcinomas. Expression of these MPs by carcinomas was independent of histologic type and such tumour features as fibrosis or necrosis. The consistent expression of ST3 by all of the carcinomas examined and absence of its expression in NNL indicates that ST3 production is likely associated with the malignant phenotype. However, since 92 kDa gelatinase and interstitial collagenase transcripts were found in some but not all tumour samples, their expression is not a uniform feature of pulmonary carcinomas. The possible prognostic significance of the expression of the latter two enzymes by carcinomas remains to be established.

Aged↗
PubMed1992

Relationship between the clinical aggressiveness of large cell immunoblastic lymphomas and expression of 92 kDa gelatinase (type IV collagenase) and tissue inhibitor of metalloproteinases-1 (TIMP-1) RNAs.

Aberrant expression of secreted proteinases and their specific inhibitors is believed to represent an important factor in the pathogenesis of invasion and metastases of malignant neoplasms. Our previous data indicated a link between elevated expression of tissue inhibitor of metalloproteinases-1 (TIMP-1) and the clinical aggressiveness of malignant non-Hodgkin's lymphomas. Further studies are presented on eighteen cases of high grade, large cell immunoblastic lymphoma in which expression at the RNA level of TIMP-1 and the metalloproteinase, 92 kDa gelatinase, were analyzed. Factors that may influence production of 92 kDa gelatinase, such as necrosis, vascularity, proliferative activity, and extranodal extension, as well as clinical parameters, such as age and sex, stage, location, and survival were assessed. Statistical analysis showed that, although clinical stage was the most important predictor of survival, after controlling for age at diagnosis, levels of 92 kDa gelatinase transcripts added to the ability to predict survival.

Adolescent↗
PubMed1992

Expression of TIMP in fetal and adult mouse tissues studied by in situ hybridization.

We have studied the expression of TIMP mRNA during mouse embryogenesis and in adult tissues using ribonuclease protection assays and in situ hybridization. Low levels of transcripts were found in many tissues, including embryonic kidney, amnion, lung and maternal deciduum and in these cases expression was not restricted to a phenotypically distinct sub-population of cells. In situ hybridization revealed high levels of TIMP transcripts in the corpus luteum of the adult ovary. Also, we observed significant expression in areas of membrane and endochondral bone formation in the embryo, commencing at about 15.5 d p.c. and increasing until birth. The pattern of TIMP expression in developing bone overlaps significantly with the localization of transforming growth factor beta (TGF beta) implying a role for this factor in the control of TIMP production in vivo.

Animals↗
PubMed1991

Tissue inhibitor of metalloproteinases-1 (TIMP-1) RNA is expressed at elevated levels in malignant non-Hodgkin's lymphomas.

Secreted metalloproteinases (MPs) and their specific inhibitors (TIMPs, tissue inhibitors of MPs) are important mediators of extracellular matrix metabolism. Previous studies have linked either excessive MP release or reduced TIMP-1 production to the invasive and metastatic phenotypes of cancer cells. In the present study we investigated the relationship between the expression of TIMP-1 and the clinical behavior of 28 non-Hodgkin's lymphomas. Northern blot analysis showed that levels of TIMP-1 mRNAs correlated directly with clinical aggressiveness: tumors in the high-grade category contained the highest levels of TIMP-1 transcripts approaching those found in maximally growth factor-stimulated fibroblasts in vitro. In situ hybridization localized the TIMP-1 expression to stromal cells of endothelial and fibroblastic origin. In contrast, transcripts hybridizing with metalloproteinase gene probes (interstitial collagenase and 72-Kd type IV collagenase) were expressed at very low levels in malignant lymphomas and their expression was not coordinately regulated with that of TIMP-1. The majority of tumors expressed either interstitial collagenase or 72-Kd type IV collagenase, and only a small number expressed both. Interstitial collagenase transcripts were only detected in high-grade tumors. The relative levels of TIMP-1 expression did not correlate with the degree of fibrosis of the tumors. Our data suggest the importance of tumor-stromal interactions in non-Hodgkin's lymphomas, and moreover, our results indicate a possible relationship between high-level, localized expression of TIMP-1 and the malignant phenotype of high-grade advanced-stage lymphomas.

Blotting, Northern↗
PubMed1991

Spontaneous immortalization of mouse embryo cells: strain differences and changes in gene expression with particular reference to retroviral gag-pol genes.

We have studied the kinetics with which cultures of primary mouse embryo cells pass through the crisis period, escape their terminal differentiation (cellular senescence), and give rise to an immortal cell line. The process is strain-dependent, with cells from the outbred Swiss CD-1 mouse being considerably more adept at forming an immortal 3T3 line than cells from the inbred SWR line; Balb/c cells appeared intermediate in their behavior. The continued presence of the tumor promoter 12-O-tetradecanoylphorbol-13-acetate or the poly(ADPribose)polymerase inhibitor 3-aminobenzamide affected the kinetics but did not seem to alter the outcome. Changes in expression of various genes, including those encoding mitogen-regulated protein (proliferin), endogenous gag-pol retrovirus sequences, insulin-like growth factor II, and a variety of protooncogenes, were monitored during the process of immortalization, and although certain changes were reproducibly characteristic of cells from a given mouse strain passed according to a specific regimen, none of the observed changes were reproducibly characteristic under all conditions of immortalization. In particular, our data indicate the absence of a strict correlation between cellular immortalization and the activation of endogenous gag-pol expression. We conclude from our observations that the establishment of permanent lines from primary mouse embryo cells in serum-containing medium reflects the selection of a variant subpopulation of cells that did not preexist but rather arose in response to the specific culture conditions by a process resembling differentiation. Multiple and complex changes in gene expression occur that are affected by the culture conditions and the strain (genotype) of the mouse.

Animals↗
PubMed1991

Hormonal regulation of matrix metalloproteinase inhibitors in rat granulosa cells and ovaries.

Metalloproteinases, such as collagenase or gelatinase, and their associated inhibitors appear to control connective tissue remodeling during follicular rupture. We examined the regulation of metalloproteinase inhibitor activity by various treatments in cultured rat granulosa cells. Granulosa cells were harvested from immature PMSG-primed rats and cultured with LH, the phorbol ester 12-O-tetradecanoylphorbol 13-acetate (TPA), cAMP, or forskolin. Inhibitor activity was measured in the medium. Increasing concentrations of either LH (0.1-1000 ng/ml) or TPA (2.5-100 nM) resulted in a dose-dependent increase in metalloproteinase inhibitor activity (2.9- and 2.4-fold increases above control, respectively). There was also a time-dependent induction of inhibitor activity in cells incubated in the presence of LH (100 ng/ml) for 6, 12, 18, or 24 h. Forskolin (0.1 mM) or cAMP (1 mM) treatment increased inhibitor activity 2.8- and 1.6-fold above that in control cultures. LH and TPA treatment in combination resulted in an additive increase in inhibitor activity compared to LH or TPA treatment alone. This finding suggested that the granulosa cell inhibitor activity might be induced through separate intracellular pathways. The inhibitor present in conditioned medium was isolated by chromatographic separation on a Sepharose 6B mol wt exclusion column. The inhibitor present was approximately 28,000 mol wt, which is consistent with the size of tissue inhibitor of metalloproteinase (TIMP). In addition to the granulosa cell experiments, changes in ovarian mRNA levels for TIMP were determined. There was a preovulatory increase in TIMP mRNA from whole rat ovaries, with the highest levels detected 12 h after hCG administration. The present study establishes that metalloproteinase inhibitor activity from rat granulosa cells is induced through separate pathways: a LH-cAMP-dependent protein kinase-A pathway and a cAMP-independent protein kinase-C pathway. Furthermore, a TIMP-like protein is observed in granulosa cell-conditioned medium, while TIMP mRNA is present in rat ovaries and increases before ovulation, suggesting that the granulosa cell metalloproteinase inhibitor is TIMP. We propose that TIMP acts in part to control the site and extent of follicular connective tissue remodeling associated with ovulation.

Animals↗
PubMed1991

Autism: early identification and management in family practice.

Autism is a chronic, nonprogressive developmental disability with a unique triad of abnormalities in socialization, communication and behavior. Most, but not all, children with autism have some degree of mental retardation, and many develop epilepsy. No single biomedical etiology has been found, but a significant number of medical disorders occur in association with autism. The medical evaluation is directed toward finding underlying or associated neurologic, metabolic, genetic or infectious diseases. Psychoeducational and behavioral therapies are the most successful approaches to the treatment of autism. Community-based and structured programs that emphasize socialization, communication and family training are the most effective. Medical management focuses on treatment of underlying or associated diseases. Pharmacotherapy is sometimes beneficial, but no drug acts specifically on this complex of symptoms. Family physicians can provide early identification, continuing medical care and support to the child and the family.

Autistic Disorder↗
PubMed1990

Developmentally programmed induction of differentiation inhibiting activity and the control of stem cell populations.

Differentiation inhibiting activity/leukemia inhibitory factor (DIA/LIF) is a glycoprotein that controls differentiation of pluripotential stem cells. Alternative transcription generates both diffusible and matrix-associated forms of DIA/LIF. Transcriptional analysis using a sensitive ribonuclease protection assay revealed that the two messages are expressed independently, consistent with the proposition that the two forms of DIA/LIF have distinct biological roles. DIA/LIF expression was found to be activated early during differentiation of embryonic stem (ES) cells, providing a mechanism for feedback regulation of stem cell renewal. Expression of DIA/LIF by mesenchymal cells was shown to be controlled in a paracrine manner by polypeptide regulatory factors. Specific expression of the two forms of DIA/LIF was also demonstrated in the egg cylinder-stage mouse embryo. The combination of cell type-specific and signal-specific regulation enables very precise control over DIA/LIF expression and may represent an important component of the regulatory networks that govern stem cell proliferation and differentiation during mammalian development.

Animals↗
PubMed1990

Identification of a serum-inducible messenger RNA (5B10) as the mouse homologue of calcyclin: tissue distribution and expression in metastatic, ras-transformed NIH 3T3 cells.

A mouse mRNA, provisionally designated 5B10, has been cloned based on its inducibility by serum in quiescent murine fibroblasts. Here we report the full-length complementary DNA sequence and a partial characterization. There are about five copies of the gene in the mouse genome. Sequence analysis of the 5B10 coding region reveals 94 and 97% amino acid identity to human and rat calcyclin, respectively. Although the coding region has been highly conserved during evolution of the rodent and human genomes, the untranslated flanking sequences differ significantly. A protein of Mr about 8000 was produced by in vitro translation of the mRNA transcribed in vitro from 5B10 complementary DNA in a riboprobe vector. An antiserum raised against a portion of the predicted human calcyclin protein cross-reacted with this mouse protein. 5B10 mRNA was found in greatest amount in organs containing proliferating cells, e.g., epidermis, skin, stomach, uterus of pregnant mouse, placenta, and decidua. Brain, liver, mature thymus, and skeletal muscle had little or no detectable 5B10 mRNA. 5B10 mRNA levels were higher in cells treated with 7,12-dimethylbenzanthracene and 12-O-tetradecanoylphorbol-13-acetate than in their normal counterparts, suggesting a role in tumorigenesis. In addition, high 5B10 mRNA levels were associated with metastatic ability in a series of ras-transformed cells, in proportion to levels of ras p21 expressed by the cells, implicating 5B10 even more deeply in carcinogenesis.

9,10-Dimethyl-1,2-benzanthracene↗
PubMed1989

Differential screening of a cDNA library with cDNA probes amplified in a heterologous host: isolation of murine GRP78 (BiP) and other serum-regulated low-abundance mRNAs.

A novel method was used to screen differentially a cDNA library for clones representing serum-regulated mRNA species of low abundance. To increase the amount of probe available for screening, the cDNA probe was cloned and amplified. Two separate cDNA 'probe' libraries were constructed in the Escherichia coli plasmid vector pDE613, using poly(A)+mRNA from murine cells at 0 and 16 h after stimulation of a G0 population. Radiolabelled plasmid DNA from each library was hybridized sequentially to colony blots of the third 'target' library, constructed with mRNA from serum-stimulated cells in the Bacillus subtilis vector pBD214. Differential screening of the target cDNA library with the two probe libraries identified novel murine cDNA clones, some representing cytoplasmic poly(A)+RNA species of low (0.01%) abundance, accumulating after serum stimulation of a quiescent mouse embryo fibroblast population. One cDNA clone was found to correspond to mitochondrial 16S rRNA and a second was identified as the murine equivalent of previously described cDNA clones for the hamster 78-kDa glucose-regulated protein (GRP78) and the rat immunoglobulin heavy-chain-binding protein. GRP78 mRNA has not previously been recognized as a serum-inducible message.

Amino Acid Sequence↗
PubMed1989

Developmental expression of tissue inhibitor of metalloproteinase (TIMP) RNA.

Single-stranded antisense RNA probes have been used to study the expression of the metalloproteinase inhibitor TIMP (tissue inhibitor of metalloproteinases), during mouse embryogenesis and in adult tissues. Using a sensitive RNase protection assay, low levels of transcript can be detected in a variety of tissues, including maternal deciduum, embryonic kidney, lung and amnion. Higher levels are seen in osteogenic tissues such as calvaria, while the highest level in any tissue is found in the ovary, though even here expression is an order of magnitude below that observed in growth factor-treated fibroblasts in vitro. Using the technique of in situ hybridization, TIMP transcripts can first be detected in osteogenic tissues in the head and limb at about 15.5 days post coitum, and increase in amount until birth. The high levels of TIMP RNA in the ovary are localized to cells of the corpora lutea.

Animals↗
PubMed1989

Expression of multiple heparin-binding growth factor species by murine embryonal carcinoma and embryonic stem cells.

Culture medium conditioned by P19 embryonal carcinoma (EC) cells contains potent mitogenic activity which is markedly potentiated when the medium is conditioned in the presence of heparin. Fractionation of P19 medium conditioned in the presence of heparin reveals the existence of two biochemically distinct growth factor species both of which exhibit high affinity for immobilised heparin and significant activity as amphibian mesoderm-inducing agents. One of the species is recovered as a single polypeptide of apparent Mr = 15,000. This molecule is immunologically related to the protein product of the human K-FGF proto-oncogene. Transcripts derived from the murine K-FGF gene are also expressed by both differentiated and undifferentiated EC cells and embryonic stem cells. The second heparin-binding growth factor is recovered as a complex of four polypeptides, the largest of which has an apparent Mr = 17,000. This agent is immunologically and biochemically distinct from both acidic and basic fibroblast growth factor as well as K-FGF, and represents the predominant mitogenic activity in EC-cell-conditioned medium.

Animals↗
PubMed1989

Expression of genes for non-collagenous proteins during embryonic bone formation.

In situ hybridization has been used to localize RNAs for a variety of non-collagenous proteins during embryogenesis of the mouse, with particular reference to bone formation. Transcripts of the Sparc (osteonectin) gene are first detected around 14.5d post coitum (p.c.) in the periosteal layer around the cartilage model of long bones and in membrane bone in the head. By 15.5d p.c. transcripts are seen in hypertrophic cartilage and later in cells within the bone closely associated with new matrix. By contrast, RNA for 2ar (osteopontin) is not seen in the periosteal layer but is confined to cells in the marrow region. TIMP (tissue inhibitor of metalloproteinase) transcripts show a very similar distribution to those for Sparc.

Animals↗