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Biomedical subjects

E Conway

Publications and source records attributed to E Conway.

At least 19 recordsLinked to original sources
PubMed1992

Identification of transglutaminase substrates in HT29 colon cancer cells: use of 5-(biotinamido)pentylamine as a transglutaminase-specific probe.

A biotinamine probe, 5-(biotinamido)pentylamine, was used for biotin-labeling of proteins in HT29 colon cancer cell extracts by endogenous transglutaminase activity. The biotin-labeled protein substrates were isolated and recovered by avidin-affinity chromatography. The proteins were separated using SDS-polyacrylamide gel electrophoresis, electroblotted onto a polyvinylidene difluoride membrane, visualized using Coomassie blue, cut out, and sequenced. Amino acid sequence data identified human fructose-1,6-bisphosphate aldolase A, an intracellular protein, as a substrate for cellular transglutaminase.

Amines↗
PubMed1991

Localization of cellular transglutaminase on the extracellular matrix after wounding: characteristics of the matrix bound enzyme.

Extending our previous observation that tissue transglutaminase (TGase) binds to extracellular matrix (ECM) fibronectin, we report here that endogenous tissue TGase is localized on the adjacent ECM after puncture wounding embryonic human lung fibroblasts (WI-38). The bound TGase persisted at the wound site for many hours, demonstrated by immunofluorescence and by catalytic activity using an overlay assay. The binding characteristics of TGase with ECM were studied further by the addition of exogenous TGase to cell monolayers and monitoring by immunofluorescence or overlay catalytic activity assays. Binding occurred equally well at 4 degrees C or 37 degrees C. Prior incubation of exogenous TGase with guanosine 5'-triphosphate (GTP), guanosine 5'-diphosphate (GDP), or adenosine triphosphate (ATP) had little effect on the amount bound to matrix, but prior treatment with calcium, magnesium, strontium, or manganese ions enhanced binding 2- to 3-fold. The Ca(++)-dependent change was a concentration-dependent effect on soluble exogenous TGase, rather than an effect on ECM. Immunofluorescent techniques showed that binding of exogenous TGase to ECM was prevented by prior mixing with fibronectin or collagen, but not with several other ECM components, including laminin, elastin, chondroitin sulfate, heparan sulfate, and hyaluronic acid. ECM-bound TGase was released by 2 M potassium thiocyanate (KSCN) treatment but was not released by treatment with a variety of amino acids, salts, reducing agents, glycerol, or other chaotropic agents.

Adenosine Triphosphate↗
PubMed1990

Differential expression of transglutaminase in human erythroleukemia cells in response to retinoic acid.

Two human erythroleukemia cell lines, HEL and K562, express transglutaminase activity. The enzyme was identified as a tissue transglutaminase following chromatographic purification. All-trans-retinoic acid (10 microM) stimulated differentiation in HEL cells as judged by a 4-fold increase in hemoglobin content and a reduction in cell proliferation. The transglutaminase activity increased 9-fold. This increase in transglutaminase was the result of a pretranslational regulation of the gene as revealed by Northern blot analysis of mRNA. These changes were not a result of cell apoptosis, since parallel DNA degradation catalyzed by a Ca2(+)-dependent endonuclease could not be demonstrated. The K562 cells, in contrast, showed no transglutaminase induction following exposure to retinoic acid and displayed no changes in maturation markers or cell growth.

Cell Division↗
PubMed1990

Adrenal function in asthma.

A dose dependent suppression of daily cortisol excretion was shown in 25 children with asthma being treated with beclomethasone dipropionate. Cortisol metabolites tended to occur below the normal range when doses of beclomethasone of more than 400 micrograms/m2/day were given. Androgen excretion below the normal range was apparent in asthmatic children aged 8-13 years regardless of whether they were receiving inhaled steroids. This may be the reason for growth delay often seen in asthmatic children. These side effects of beclomethasone are not enough reason to discourage its prescription for the treatment of asthma, but endocrine assessment is desirable when the dose exceeds 400 micrograms/m2/day.

Adolescent↗
PubMed1989

Colorimetric assay for cellular transglutaminase.

A colorimetric assay for cellular transglutaminase using 5-(biotinamido)pentylamine and polyvinylidine difluoride membranes for crude cellular preparations and purified enzyme has been developed. The biotinpentylamine substrate was incorporated into N,N-dimethylcasein by transglutaminase, the biotinylated products were adsorbed onto the membrane disks and conjugated with streptavidin-beta-galactosidase, and the absorbance resulting from the formation of p-nitrophenol from hydrolysis of p-nitrophenyl-beta-D-galactopyranoside was measured at 405 nm. The validity of the assay was established by showing a good correlation, gamma = 0.922, between the colorimetric procedure and the commonly used radiometric filter paper method for the enzyme. The procedure offers a rapid, sensitive, and nonisotopic method for the estimation of cellular transglutaminase activity in as low as 20 ng of purified guinea pig liver transglutaminase and 10 micrograms of crude fibroblast cytosol protein.

Animals↗
PubMed1987

Induction of cellular transglutaminase biosynthesis by sodium butyrate.

Cellular transglutaminase activity was induced in simian virus-transformed human embryonic lung fibroblasts (WI-38 VA13A) by sodium butyrate. The level of enzyme activity approached a maximum by 6 days; 9-11-fold higher in the presence of sodium butyrate (1 mM) than in its absence. The observed increases in cellular transglutaminase activity could be entirely accounted for by equivalent increases in the levels of enzyme protein measured by inhibition enzyme-linked immunosorbent assay. Sodium butyrate also increased the rate of enzyme synthesis, but had no effect on the rate of cellular transglutaminase degradation. The increase in the rate of enzyme synthesis was matched by an increased level of translatable transglutaminase mRNA as measured in a cell-free translation system. Our results suggest that sodium butyrate regulates cellular transglutaminase at the pretranslational level.

Antibodies, Monoclonal↗
PubMed1987

Cellular transglutaminase has affinity for extracellular matrix.

Cellular transglutaminase (TGase) was demonstrated as an intracellular enzyme by immunofluorescence in WI-38 cells. Following cell membrane perturbation by Triton X-100 treatment, TGase was bound to the extracellular matrix and was found to coexist with fibronectin as visualized by immunofluorescence microscopy. The binding of TGase to the cell matrix was blocked by anti-fibronectin antibody. Exogenous sources of soluble TGase were transferred to the extracellular matrix of an untreated or methanol fixed cell. The experimental data indicated that "particulate bound" TGase is a consequence of soluble TGase binding to the extracellular matrix following cell rupture.

Extracellular Matrix↗
PubMed1985

Variable transglutaminase activity in human diploid fibroblasts during in vitro senescence.

The specific activity of transglutaminase (TGase) was followed in human diploid fibroblasts (HDF) as a function of in vitro age. It was determined that at least 90% of the TGase activity was found in a soluble fraction at all in vitro ages; but the activity was variable with age. It was high in cells that had completed less than 50% of their lifespan (%LSC), declined to a minimum between 60 and 85% LSC, and again became elevated at more than 90% LSC. These age related variations in TGase activity could not be attributed to cellular growth characteristics, enzyme amount, or clonal selection processes. It is postulated that the variable TGase activity observed during in vitro senescence of HDF may reflect a change in affinity of the enzyme for a particular molecule; possibly fibronectin.

Cell Division↗
PubMed1985

A monoclonal antibody to cellular transglutaminase.

A cellular enzyme-linked immunosorbent assay was developed for estimating cellular transglutaminase in situ using a monoclonal antibody produced to tissue transglutaminase. The minimum level of detection of TGase was 2-5 ng. The enzyme was present in greater amounts in WI-38 and IMR90 cells than in their simian virus-transformed counterparts. The levels of TGase in the virus-transformed cells increased significantly when the cells were grown in the presence of sodium butyrate to induce enzyme activity. Staining of confluent WI-38 cells by indirect immunofluorescence using the monoclonal antibody showed microscopic fibers suggesting that the enzyme may be associated with detergent-insoluble components.

Acyltransferases↗
PubMed1983

Enhanced transglutaminase activity in transformed human lung fibroblast cells after exposure to sodium butyrate.

The low level of transglutaminase activity in virus-transformed human embryonic lung fibroblasts (WI-38 VA13A) increased markedly when cells were exposed to sodium butyrate. The effect of sodium butyrate was time- and concentration-dependent and fully reversible. Transformed cells exposed for 5 days to 1 mM sodium butyrate had fewer cells, showed an 8-10 fold higher transglutaminase activity, and stained more abundantly for transglutaminase and pericellular fibronectin than control cells when examined by indirect immunofluorescence. Non-transformed cells (WI-38) showed only a 2-4-fold increase in transglutaminase activity when treated in a similar manner. Studies with metabolic inhibitors revealed the increase in activity was the result of synthesis of new protein. Kinetic studies showed the affinity of putrescine for the enzyme was essentially unchanged but the number of active sites increased 9-fold following exposure to sodium butyrate. Enhanced transglutaminase activity returned to control levels within 7 days after subculture and sodium butyrate removal. These findings suggest that sodium butyrate offers a potential model system to understand better the role of transglutaminase in cells in culture; particularly growth control in transformed cells.

Acyltransferases↗
PubMed1981

The definitive identification of the lignans trans-2,3-bis(3-hydroxybenzyl)-gamma-butyrolactone and 2,3-bis(3-hydroxybenzyl)butane-1,4-diol in human and animal urine.

The definitive identification of the first lignans to be found in humans and animals is described. Gas chromatography--mass spectrometry, n.m.r. spectroscopy, i.r. spectroscopy and chemical techniques were employed to establish the structures of two lignans as trans-2,3-bis(3-hydroxybenzyl)-gamma-butyrolactone and 2,3-bis(3-hydroxybenzyl)butane-1,4-diol. Both compounds are essetially racemic. Evidence was also found for several methoxy analogues of these lignans in the vervet monkey.

4-Butyrolactone↗
PubMed1981

Increase in proliferative markers after inhibition of transglutaminase.

Cystamine inhibited transglutaminase activity (R-glutaminyl-peptide:amine gamma-glutamyltransferase, EC 2.3.2.13) of proliferating WI-38 cells in a dose-dependent manner over the concentration range 0.005-0.25 mM when added to the culture medium. The epsilon-(gamma-glutamyl)lysine content in the cells was decreased and several proliferation markers were enhanced. "Non-mitotic" cells were stimulated by cystamine (about 25% of that observed with 10% fetal bovine serum) to undergo DNA synthesis with subsequent increases in nuclei number. Numerous other disulfides, thiols, and amines were ineffective when added to culture medium. The findings are supportive of the concept that growth control involves a relationship between isopeptide crosslinks and proliferation.

Cell Division↗
PubMed1977

Transglutaminase activity in normal and transformed cells.

Transglutaminase activity was determined in normal and transformed paired cell systems. Reduced enzyme activity was found in virus-transformed human and hamster cells and in chemically transformed mouse cells relative to normal counterparts. Most of the enzyme activity was localized in the particulate fraction sedimenting at 105,000 X g. Enzyme activity was highest when normal cell populations were in an essentially nonmitotic state. Protein capable of incorporating putrescine was present in normal and transformed human cells, although the rate of incorporation was lower in the latter. The putrescine acceptor in the normal cell paralleled enzyme activity and enzyme distribution. Trypsin (5 microng/ml) treatment of the normal cell resulted in a 3-fold increase in enzyme activity, which occurred independently of protein synthesis.

Cell Division↗
PubMed1976

Amino acid and hexose transport of normal and simian virus 40-transformed human cells.

Studies on amino acid and hexose transport were performed on human WI-38 cells and WI-38 SV40-transformed cells (VA13A and VA13-2RA). Depending upon cell line or conditions, either no difference or a relative decrease in initial uptake by transformed cells was found. Under similar growth conditions, transformed hamster cells (PyBHK-21/C13) had increased uptake, compared with the normal hamster cells (BHK-21/C13). The normal and transformed human cells were also similar in sialic acid content and agglutinated when treated with concanavalin A.

Agglutination Tests↗