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Biomedical subjects

E Gherardi

Publications and source records attributed to E Gherardi.

At least 55 records · Page 3Linked to original sources
PubMed1992

Transforming growth factor-beta 1 and interleukin-1 beta stimulate LDL receptor activity in Hep G2 cells.

The effect of transforming growth factor-beta 1 (TGF-beta 1) and interleukin-1 beta (IL-1 beta) on LDL receptor in Hep G2 cells was investigated. A greater than two-fold stimulation of the binding and internalisation of [125I]-labelled LDL at 37 degrees C was observed after an 18-h incubation of the cells with TGF-beta 1 at 50 ng/ml and IL-1 beta at 11,700 units/ml compared with control cells. Scatchard analysis of the binding of [125I]-labelled LDL at 4 degrees C after an 18-h incubation of the cells with 1170 units/ml IL-1 beta and 5 ng/ml TGF-beta 1 showed that they were both acting primarily by increasing LDL receptor number. The increase in LDL receptor activity could not be attributed to an increase in cell proliferation as TGF-beta 1 at concentrations from 0.05 ng/ml to 50 ng/ml had no significant effect on either cell number or [3H]thymidine incorporation into DNA whilst IL-1 beta inhibited DNA synthesis by more than 80% at a concentration of 11,700 units/ml but had significant effect on cell number. Cholesterol biosynthesis from [14C]acetate, in contrast to the stimulation of LDL receptor activity, was inhibited by approximately two-fold by incubation with TGF-beta 1 at 50 ng/ml and IL-1 beta at 11,700 units/ml.

Animals↗
PubMed1992

Down-regulation of scatter factor in MRC 5 fibroblasts by epithelial-derived cells. A model for scatter factor modulation.

Scatter factor/hepatocyte growth factor (SF/HGF) is a multifunctional cytokine produced by embryonic fibroblasts and other mesenchymal cells that affects the growth and/or the movement of certain epithelia. Here we report that expression of scatter factor activity by MRC 5 cells, a strain of normal human embryonic lung fibroblasts, is greatly reduced as a result of co-culture of these cells with SVK14, an SV40-transformed human keratinocyte cell line. Using a cDNA probe to the beta chain of human HGF, we have found that the fall in SF activity in MRC 5/SVK14 co-cultures is accompanied by the loss of SF/HGF transcripts. As the inhibition of SF activity coincides with the disappearance of SF/HGF transcript, we conclude that inhibition of the SF activity expressed by MRC 5 cells by co-culture with SVK14 involves transcriptional regulation.

Animals↗
PubMed1992

Growth requirements and expression of LDL receptor and HMG-CoA reductase in Hep G2 hepatoblastoma cells cultured in a chemically defined medium.

A serum-free chemically defined medium (CDM) has been developed which sustains the growth in culture of the highly differentiated human hepatoma cell line Hep G2. Unlike rodent hepatoma lines, Hep G2 cells in serum-free medium have an absolute requirement for lipoprotein lipids (either low density lipoprotein (LDL) or high density lipoprotein (HDL)) for growth. In the presence of LDL (or HDL) growth was further enhanced by insulin, triiodo-L-thyronine, 17 alpha-ethinylestradiol but not by epidermal growth factor (EGF). On type I collagen gels cells cultured in CDM were contact inhibited and formed monolayers. This contrasted with the pattern of growth of cells cultured in the presence of serum on type I collagen gels and cells cultured on tissue-culture plastic in either CDM or medium containing serum which formed foci of multilayered cells. Expression of the LDL receptor and HMG-CoA (3-hydroxy-3-methylglutaryl coenzyme A) reductase genes was comparable in Hep G2 cells cultured in CDM and serum-containing medium. Furthermore, the binding and internalisation of 125I-LDL at 37 degrees C was modulated by hormones that have previously been shown to affect LDL receptor levels in liver in vivo or in hepatocytes cultured in serum-containing medium in vitro. The culture system described provides a basis for studying the regulation of hepatocyte-specific functions by soluble factors (either plasma- or cell-derived) and cell-substratum interactions in a human liver cell line.

Carcinoma, Hepatocellular↗
PubMed1992

Porcine smooth muscle cell-conditioned medium stimulates LDL receptor activity in Hep G2 cells.

Paracrine factors may modulate low density lipoprotein (LDL) receptor activity in hepatocytes. To study this the effect of conditioned medium prepared from a range of cell types on the binding and internalisation of 125I-LDL in Hep G2 cells was studied. Seven of the fourteen conditioned media tested, including those from P388D1, U937, porcine smooth muscle (Pc SMC) Swiss 3T3, STO, = 48 and MDCK cells, were found to increase the binding and internalisation of 125I-LDL at 37 degrees C by Hep G2 cells (P < 0.01). The largest increase in LDL receptor activity was produced by conditioned medium from Pc SMC cells and was, therefore, selected for further analysis. The Pc SMC-conditioned medium increased LDL receptor number in Hep G2 cells by three-fold but had no effect on LDL receptor activity in human skin fibroblasts. DNA synthesis and cholesterol synthesis by Hep G2 cells were inhibited by Pc SMC-conditioned medium. Preliminary characterisation of the Pc SMC-derived factor(s) suggests that it is a protein(s) of low relative molecular mass.

Animals↗
PubMed1991

Probing of the expression of the low-density lipoprotein receptor in vivo using an anti-receptor monoclonal antibody.

MAC188 is a rat anti-[low-density lipoprotein (LDL) receptor] monoclonal antibody (McAb) which binds to the cell surface receptor with high affinity at physiological temperatures, even in the presence of high concentrations of the natural ligand, LDL. Binding of McAb MAC188 at 37 degrees C is followed by internalization and intracellular sequestering of the receptor, which results in the transient disappearance of the receptor from the cell surface. The high binding affinity and epitope specificity of McAb MAC188 suggested that this antibody could be used to quantify receptor expression in vivo. Mixtures of radiolabelled anti-receptor antibody and a control McAb (MAC221) were injected intravenously into rabbits, and the clearance from serum and uptake into tissues was determined. A fraction of the anti-receptor McAb was cleared rapidly from the circulation by a high-affinity and saturable (receptor-dependent) process. Receptor-dependent uptake of the anti-receptor McAb was measurable in liver, adrenal glands, kidneys, spleen, kidney, thoracic aorta and heart. It was highest in liver and adrenal glands and correlated well with the level of receptor protein and the rate of LDL transport in individual tissues. Anti-receptor McAbs such as MAC188, with suitable domain specificity and binding affinity at physiological temperatures, have important advantages over the natural ligand as tracers for the receptor in vivo, and may find widespread applications in studies of the receptor status (activity) in animals and man.

Animals↗
PubMed1991

Mutation and selection during the secondary response to 2-phenyloxazolone.

The most characteristic feature of the mouse antibody response to the hapten 2-phenyloxazolone is the recurrent expression of the light-chain variable region Igk-VO chi 1 gene in its germ-line or mutated configuration. The analysis of somatic mutants of the Igk-VO chi 1 gene reported here indicates that, as found during the primary response, hypermutation is also activated during the secondary response. Somatic mutations in the Igk-VO chi 1 gene increased in sequences obtained at day 14 and day 21 in the primary response and again in the secondary response at days 3, 5, and 7. The ratio of replacement to silent mutations also increased, particularly between days 5 and 7, suggesting that a stage of negative selection operates on new somatic mutants generated in the secondary response. Most Igk-VO chi 1 mutants isolated in the secondary response had the features of selected memory clones (i.e., they carried mutations known to increase binding affinity for the hapten). However, some clones had chain-termination codons, and others had mutations predicting a nonfunctional light chain. At least three and possibly five of these clones also expressed the mutation characteristic of the memory response to 2-phenyloxazolone (His-34----Asn-34/Gln-34). We conclude that after a second antigenic challenge, new somatic variants, including some leading to the loss of antigen binding, are generated by hypermutation of cells derived from the memory pool.

Amino Acid Sequence↗
PubMed1991

Colony assays for antibody fragments expressed in bacteria.

This paper describes procedures for the detection and selection of bacterial colonies expressing antibody fragments of desired antigen specificity. Fab and Fv fragments are detected in a filter assay in which bacterial colonies are grown on a master filter in contract with a second, antigen-coated filter. Ab fragments diffusing onto the second filter bind antigen directly and specifically and are detected with a monoclonal antibody directed against a myc-tag sequence fused to the carboxy-terminal end of the light chain or heavy chain (direct assay). Single-chain Fv (scFv) in which the VH and V1 sequences are joined by a short linker peptide are detected by a modified procedure in which scFv are immobilized on filters coated with the anti-myc-tag sequence and subsequently detected by specific binding to radiolabeled antigen (indirect assay). A single positive bacterial colony expressing antigen-specific Fv (or scFv) can be recovered among at least 10,000 negative colonies using the procedures described. The direct assay has been successfully used to discriminate Fv fragments which express point mutations known to increase the binding affinity of antibodies to the hapten 2-phenyl-oxazolone. The procedures described may thus prove generally useful for the selection of antigen-specific clones expressed in bacteria and/or higher-affinity variants of such antibodies.

Antibodies↗
PubMed1991

Purification and characterization of scatter factor.

Scatter factor is a fibroblast-derived protein which disrupts and scatters epithelial colonies and enhances the local movement of individual epithelial and endothelial cells. The factor purified from mouse fibroblasts by cation-exchange and reverse phase chromatography is a dimer of 57 kD and 30 kD protein subunits (A and B subunits), is active at picomolar concentrations and requires intact intra- and/or inter-chain disulphide bonds for activity. In serum-free conditioned medium the factor is highly aggregated but in the presence of high-salt buffers or protein denaturants elutes from gel filtration columns with an apparent Mr of approximately 50 kD. From a combination of molecular sieving and ultracentrifugation studies, a calculated Mr of 61.4 kD is obtained for native mouse scatter factor, a value which agrees well with the Mr estimates obtained by SDS-PAGE (62-67 kD). Mouse fibroblast scatter factor is a heparin-binding, basic protein (pI 8.5-9.5) which contains N-linked carbohydrates which are not, however, essential for activity. The factor has no metallo- or serine protease activity and there is no evidence so far that its junctional-breaking activity involves proteolytic cleavage of surface molecules on target cells. Scatter factor is either identical or closely related to hepatocyte growth factor/hepatopoietin A (a potent mitogen for rat hepatocytes recently purified from human and rabbit serum and rat platelets). The factor is thus an effector of mesenchymal-epithelial interactions which affects the movement or the growth of different epithelia.

Animals↗
PubMed1991

A sensitive RNase protection assay for the quantitation of the mRNAs for the LDL receptor and HMG-CoA reductase in human total RNA. Effects of treatments on cells in culture designed to up- and down-regulate expression of the LDL receptor.

Regulation of expression of the genes for the low density lipoprotein receptor (LDLR) and 3-hydroxy-3-methylglutaryl-coenzyme A reductase (HMGR) is of central importance in the control of cholesterol metabolism and thus in influencing the concentration of low density lipoprotein in the plasma. This can be studied by investigating the effects of factors (hormones, drugs, etc.) on the levels of mRNA for these genes. An RNase protection assay is reported for measurement of the levels of mRNA for the LDLR and HMGR. Several probes have been developed for these genes, together with probes for the "housekeeping" genes, beta-actin and glyceraldehyde-3-phosphate dehydrogenase. Various conditions in the assay have been examined and optimised, e.g. conditions for solution hybridization and RNase digestion and the use of "sense" RNA standards. The assay allows accurate measurement of approximately 2 x 10(7) copies of LDLR and HMGR mRNAs, which is equivalent to the number of copies present in approximately 1 x 10(6) human dermal fibroblasts and approximately 5 x 10(5) Hep G2 liver hepatoma cells cultured in 10% fetal calf serum. The average number of copies of mRNA per cell was estimated in fibroblasts and Hep G2 cells under various conditions of regulation of the LDLR and revealed the following: [table: see text] Under the chosen conditions 10 copies per cell was the detection limit for the assay. The effect of these treatments on the number of copies of mRNA per cell for beta-actin and glyceraldehyde-3-phosphate dehydrogenase was also determined.

Actins↗
PubMed1990

A single-step procedure for cloning and selection of antibody-secreting hybridomas.

A procedure is described which permits high-yield direct cloning of newly established hybridomas on STO fibroblast feeders in soft agarose. Several thousand independent clones are typically obtained from each fusion (1 X 10(8) spleen cells). These are screened using a colony replica assay in which secreted antibodies diffuse through an agar overlay and bind to antigen immobilised on nitrocellulose. Bound antibodies are then detected with enzyme-labelled second antibody. The procedure is fast and efficient and permits the isolation and selection of antigen-specific clones in less than 2 weeks from fusion. It has been successfully employed for the derivation and selection of high-affinity anti-hapten antibodies. Other potential applications of the assay are in the detection of non-immobilised antigens by an indirect method using anti-globulin on nitrocellulose, in the generation of bispecific antibodies and the selection and characterisation of antibody specificities generated by the expression of antibody fragments in bacteria or yeasts.

Animals↗
PubMed1990

Microcirculation and hemorheology in NIDDM patients.

The authors studied 10 patients with non-insulin-dependent diabetes mellitus and 5 controls matched for age, sex, blood lipids, and smoking habit. The two groups were also comparable for hemorheologic characteristics as evaluated by viscosimetry on whole blood, plasma and serum, erythrocyte filtration and aggregation. The microcirculation was studied in the subjects of both groups by microalbuminuria determination, retinal fluorangiography, and capillaroscopic examination of the bulbar conjunctive and nail folds. None of the patients presented microalbuminuria values higher than the upper limit of normal (20mg/24h). Fluoroangiographic alterations were observed in 4 patients, and all 10 presented capillaroscopic alterations at the bulbar conjunctiva (microaneurysms, erythrocyte aggregates) and nail folds (more frequently of the fingers than toes). Similar alterations were detected in controls. Thus these abnormalities seem independent of hemorheologic values.

Blood Viscosity↗
PubMed1990

Epithelial scatter factor and development of the chick embryonic axis.

Scatter factor, a recently characterised protein secreted by certain embryonic fibroblasts, affects cultured epithelial by increasing cell motility, the breakdown of cell junctions and cell scattering. The process of gastrulation in higher vertebrate embryos, during which the primitive streak forms, involves an epithelial-to-mesenchymal transformation resembling the effects of the factor on cultured cells. The factor was applied locally to chick embryos, using both scatter-factor-secreting cell lines and inert carriers. We found that scatter factor can generate local supernumerary axial structures resembling primitive streak and/or neural plate and conclude that it may have primitive-streak and/or neural-inducing activity in chick embryos.

Animals↗