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Biomedical subjects

K D Brown

Publications and source records attributed to K D Brown.

At least 109 records · Page 6Linked to original sources
PubMed1987

Synergistic stimulation of Swiss mouse 3T3 fibroblasts by epidermal growth factor and other factors in human mammary secretions.

The concentration of epidermal growth factor (EGF) in human mammary secretions was about 50 nmol/l for several weeks prepartum. It then fell to about 13 nmol/l within 4 days after parturition, in parallel with the decrease in protein concentration which is associated with the onset of lactation. In contrast, the concentration of EGF in urine samples from the same donors remained constant throughout this period. All the immunoreactive EGF in mammary secretions competed at the EGF receptors on Swiss mouse 3T3 fibroblasts. The stimulation of these cells by samples of mammary secretions was, however, much greater than that induced by EGF alone, indicating the presence of other factors which synergize with EGF. Gel filtration of mammary secretions revealed two major peaks of mitogenic activity, corresponding to EGF and a factor of higher molecular weight. The latter could synergize with added EGF, insulin or bombesin, and thus falls into a different functional class from any of these factors.

Animals↗
PubMed1987

Stimulation of intestinal epithelial cell proliferation in culture by growth factors in human and ruminant mammary secretions.

Samples of human and ruminant mammary secretions stimulated the proliferation of rat intestinal epithelial (RIE-1) cells in culture. The stimulation was dose-dependent, and samples taken prepartum had greater potency than those taken after parturition. When various hormones and growth factors known to be present in milk were tested, only epidermal growth factor (EGF), insulin and insulin-like growth factor I (IGF-I) stimulated the proliferation of RIE-1 cells. IGF-I was effective at lower concentrations than insulin, and the maximal stimulation induced by each of these two polypeptides was greater than that induced by EGF. The maximal stimulation induced by samples of mammary secretions was similar to that induced by insulin or IGF-I.

Animals↗
PubMed1986

Effects of bombesin and insulin on inositol (1,4,5)trisphosphate and inositol (1,3,4)trisphosphate formation in Swiss 3T3 cells.

The effects of bombesin and insulin, separately and in combination, have been studied in Swiss mouse 3T3 cells. Bombesin caused a rapid transfer of 3H from the lipid inositol pool of prelabeled cells into inositol phosphates. Label in inositol tetrakisphosphate (InsP4) and in Ins1,4,5P3 and Ins1,3,4P3 rose within 10 sec of stimulation and that in Ins1,4P2, another InsP2 and InsP1, more slowly. Insulin, which had little effect on its own, increased the turnover of inositol lipids due to acute bombesin stimulation and also enhanced the DNA synthesis evoked by prolonged bombesin treatment. The results suggest that bombesin acting as a growth factor, uses inositol lipids as part of its transduction mechanism and that insulin acts synergistically to enhance both inositol phosphate formation and DNA synthesis.

Animals↗
PubMed1986

Early phosphorylation events following the treatment of Swiss 3T3 cells with bombesin and the mammalian bombesin-related peptide, gastrin-releasing peptide.

Bombesin and the related mammalian peptides, such as gastrin-releasing peptide (GRP), are potent mitogens for some fibroblast cell lines. Here we have examined the bombesin- and GRP-mediated changes in the phosphorylation of proteins in Swiss 3T3 cells and compared these to the events observed after platelet-derived growth factor (PDGF), epidermal growth factor (EGF) and tumor promoter treatment. In agreement with previous reports, bombesin, GRP and PDGF, but not EGF, increased the activity of protein kinase C. This was assayed by an inhibition of [125I]EGF binding, stimulation in phosphorylation of pp60c-src on serine 12 and stimulation in phosphorylation of a group of 80 kd proteins. The different phosphorylated forms of the 80 kd proteins were examined by tryptic peptide mapping and shown to contain multiple phosphorylation sites. An investigation of the tyrosine phosphorylation events following mitogen treatment revealed a significant difference between PDGF and the bombesin peptides. PDGF treatment caused a marked increase in total cellular phosphotyrosine levels, and tyrosine phosphorylation both of known substrates and its own receptor. In contrast, bombesin and GRP treatments resulted in only a weak or undetectable increase in tyrosine phosphorylation of total cellular protein or known substrates. In this respect bombesin and GRP were more similar to EGF. The fact that the bombesin peptides do not induce a phosphorylation response identical with either PDGF or EGF suggests that there is not a single common signal pathway which is activated by all these mitogens.

Animals↗
PubMed1986

Contradistinctive growth responses of cultured rat intestinal epithelial cells to epidermal growth factor depending on cell population density.

The growth response of cultured rat intestinal epithelial (RIE-1) cells to epidermal growth factor (EGF) depends on the cell population density. EGF stimulated the proliferation of RIE-1 cells in dense cultures, but inhibited the proliferation of cells growing at low population densities. In contrast, insulin enhanced RIE-1 cell growth irrespective of the population density. The tumour promoter 12-0-tetradecanoylphorbol 13-acetate (TPA), like EGF, inhibited the proliferation of low-density RIE-1 cells, but differed from EGF in that it did not stimulate the growth of dense cultures.

Animals↗
PubMed1986

Kinetics of transfer of 125I-labelled epidermal growth factor from blood into mammary secretions of goats.

125I-Labelled mouse epidermal growth factor (125I-EGF) was transferred intact and undegraded from circulating blood into milk in conscious lactating goats. Greater than 90% of the total radioactivity present in milk from the infused gland was in the aqueous phase and more than 72% was acid-precipitable. This radiolabelled material co-eluted with authentic EGF through gel filtration and was immunoprecipitable by a specific rabbit anti-mouse EGF immunoglobulin. Mammary uptake of 125I-EGF infused into mammary arterial blood (close-arterial infusion) for 1 h varied from 20 to 83% at different stages of the reproductive cycle. Only 0.5-2.9% of the infused 125I-EGF was transferred into milk during the first 3 h after the start of the infusion, which represents 0.7-6.3% of mammary uptake of EGF. The kinetics of transfer of 125I-EGF were followed in two lactating goats. Radioactivity reached peak levels in milk about 120 min after the start of a 1 h close-arterial infusion into the mammary gland, with an initial lag of about 30 min when little transfer occurred. Transfer was slower in two non-lactating goats with maximal levels of activity in milk being reached after about 180 min. The results are consistent with a transcellular transfer, whereby the factor is bound to receptors on the baso-lateral membrane, internalized by epithelial cells and subsequently secreted across the apical membrane into the alveolar lumen. The low level of degraded labelled EGF in milk (and mammary vein blood) suggests a modification of the normal pathway of EGF degradation such that the delivery of internalized factor to lysosomes is avoided.

Animals↗
PubMed1986

Mechanisms of transfer of steroid hormones and growth factors into milk.

In this paper we examine the ability of the mammary gland to remove from circulating blood three compounds which differ in their physico-chemical and structural properties. Mammary extraction of progesterone, oestrone sulphate and epidermal growth factor (EGF) is similar at peak lactation in goats, but the proportion of labelled infusate that is transferred into milk is greater for oestrone sulphate and EGF than progesterone which is rapidly metabolised by mammary tissue. The kinetics of transfer of progesterone, oestrone sulphate and EGF from blood into milk show that transcellular processes are involved, and on the basis of earlier hypotheses and new information reported here the results indicate the probable importance of simple and facilitated diffusion pathways for progesterone and oestrone sulphate, and secretory mechanisms for oestrone sulphate and EGF. Although evidence is lacking for a direct effect of hormones in milk on mammary function, their concentration in milk may reflect changes in local regulation of mammary secretion. Considerable practical value is attached to the immunodiagnostic use of milk hormone concentrations to determine ovarian and placental endocrine activity during pregnancy in domestic ruminants.

Animals↗
PubMed1985

A peptide that inhibits the mitogenic stimulation of Swiss 3T3 cells by bombesin or vasopressin.

The synthetic peptide [D-Arg1,D-Pro2,D-Trp7,9,Leu1]substance P inhibits the stimulation of DNA synthesis induced in Swiss 3T3 cells by bombesin or vasopressin, but not that induced by a wide range of other growth factors and mitogens. The stimulation induced by 10 pM-3 nM-bombesin is inhibited by 1-30 microM-antagonist in a manner consistent with competition at the bombesin receptor. The inhibition by the antagonist of the stimulation induced by vasopressin suggests a previously unrecognized interaction of the antagonist with vasopressin receptors. The antagonist should be useful in studies of cell proliferation both in vivo and in vitro.

Animals↗
PubMed1985

Loss of epidermal growth factor receptors and release of transforming growth factors do not correlate with sarcoma virus-transformation in clonally-related NIH/3T3-derived cell lines.

Transformation of NIH/3T3 cells by Kirsten murine sarcoma virus (MSV) caused a dramatic reduction in the number of cell-surface receptors for epidermal growth factor (EGF). However, the number of EGF receptors remained at a very low level in a non-tumourigenic revertant cell line isolated from the virus-transformed cells, indicating that an increase in EGF receptors is not a requirement for the phenotypic reversion of Kirsten MSV-transformed 3T3 cells. Serum-free conditioned medium from normal and virus-transformed cell lines contained similar amounts of cell growth-promoting activity as assayed by the ability to stimulate DNA synthesis in quiescent Swiss 3T3 cell cultures. However, the concentrated conditioned medium from these cell lines showed no evidence of beta-transforming growth factor (TGF) activity as assayed by promotion of anchorage-independent growth of untransformed normal rat kidney (NRK) fibroblasts in agarose. The cellular release of alpha-TGF activity was assayed by measuring the ability of concentrated conditioned medium to inhibit the binding of 125I-EGF to Swiss 3T3 cells. Conditioned medium protein from the virus-transformed cell line inhibited 125I-EGF binding but only to the same extent as conditioned medium protein prepared from the untransformed cell line. The alpha-TGF secretion by these cell lines was estimated to be 30-45-fold lower than the level of alpha-TGF released by a well-characterized alpha-TGF-producing cell line (3B11). These results suggest that the induction of TGF release is not a necessary event in the transformation of NIH/3T3 cells by Kirsten MSV.

Animals↗
PubMed1985

Functional receptors for epidermal growth factor in an epithelial-cell line derived from the rat small intestine.

Epidermal growth factor (EGF) regulates the proliferation of cells of a rat intestinal epithelial-cell line (RIE-1) in culture. Confluent RIE-1 cells were stimulated to proliferate by EGF with a half-maximal effect at 1-2 ng/ml. In contrast, the growth of sparse RIE-1 cells was inhibited by the growth factor. Binding studies at 4 degrees C with 125I-EGF identified two classes of binding sites for EGF on RIE-1 cells, one of high affinity (KD = 1.8 X 10(-10)M; 1.8 X 10(4) receptors/cell) and one of lower affinity (KD = 5.2 X 10(-9)M; 6.3 X 10(4) receptors/cell). After binding to the cells at 37 degrees C, 125I-EGF was rapidly internalized and subsequently degraded. Degradation products were released into the medium after a lag of 15-30 min. The degradation of 125I-EGF did not occur at 4 degrees C and was inhibited at 37 degrees C by chloroquine, methylamine or NH4Cl, but not by colchicine. Exposure of RIE-1 cells to EGF caused a time- and dose-dependent loss of EGF receptors from the cell surface. The recovery of receptors after the removal of EGF was retarded in the absence of serum and prevented by the presence of cycloheximide or actinomycin D. Sodium dodecyl sulphate/polyacrylamide-gel electrophoresis separation of the 125I-EGF-receptor complex from RIE-1 cells after covalently cross-linking with disuccinimidyl suberate indicated a receptor of Mr congruent to 160 000. The demonstration of functional EGF receptors in this cell line provides further evidence that EGF may regulate intestinal-epithelial-cell physiology.

Animals↗
PubMed1985

Epidermal growth factor promotes the chemotactic migration of cultured rat intestinal epithelial cells.

The RIE-1 cell line is an untransformed, epithelial cell line derived from the rat small intestine. We report that epidermal growth factor (EGF), which regulates the proliferation of RIE-1 cells, also directs their movement. We measured cell migration through gelatin-coated filters in blind-well Boyden chambers. The migration of RIE-1 cells was stimulated up to approximately 100-fold by EGF, with a half-maximal response at 1-2 ng/ml and a maximal effect at 10 ng/ml. Further analysis showed that the RIE-1 cells responded directionally to a gradient of EGF in solution. Other growth factors tested did not stimulate RIE-1 cell migration, and EGF did not stimulate the migration of fibroblasts in this assay. We conclude that EGF is a potent and specific chemo-attractant for RIE-1 intestinal epithelial cells and suggest that EGF might influence epithelial cell migration in vivo.

Animals↗
PubMed1985

Phosphoinositides and cell proliferation.

Certain growth factors act by stimulating the hydrolysis of inositol lipids to yield putative second messengers such as diacylglycerol (DG) and inositol trisphosphate (IP3). One function of the former is to stimulate C-kinase, which may act by switching on a sodium/hydrogen exchanger to induce the increase in pH that appears to have a permissive effect on DNA synthesis. Studies on Swiss 3T3 cells have revealed that growth factors stimulate an increase in two separate isomers of IP3. In addition to inositol 1,4,5-trisphosphate there was a large increase in inositol 1,3,4-trisphosphate. While the former functions to elevate intracellular calcium, which has been implicated in the control of growth of many different cell types, the function of the latter is unknown. Since the 1,3,4 isomer turns over very slowly, it may control long-term events and thus could play a role in cell growth. There are other growth factors such as insulin and epidermal growth factor (EGF), which apparently do not work through the inositol lipids but they may initiate ionic events similar to those just described for calcium-mobilizing receptors. The bifurcating signal pathway based on IP3/Ca2+ and DG/C-kinase provides an interesting framework within which to consider the mode of action of oncogenes.

Animals↗
PubMed1985

Hydroxyurea interferes negatively with triglyceride measurement by a glycerol oxidase method.

Measured triglyceride concentrations were extremely low (less than 100 mg/L) in the serum of some patients who were receiving hydroxyurea for myeloproliferative diseases. The assay being used to quantify triglycerides was a "cascaded" enzymatic method involving (a) lipase, to generate glycerol from triglycerides; (b) glycerol oxidase, to convert glycerol to glyceraldehyde, with generation of hydrogen peroxide; and (c) peroxidase, which acts on the hydrogen peroxide with subsequent coupled generation of a red-violet quinone (reagent system used in the Technicon RA-1000). Hydroxyurea added to serum samples appeared to inhibit the action of glycerol oxidase, with a stoichiometric relation to the concentration of substrate (a decrease of roughly 2.4 mmol/L in measured triglyceride per 1 mmol of hydroxyurea per liter). A different enzymatic assay for triglycerides, which involves glycerol kinase (Beckman Instruments) did not show this effect of hydroxyurea.

Autoanalysis↗
PubMed1984

Reduction of epidermal growth factor receptor affinity by heterologous ligands: evidence for a mechanism involving the breakdown of phosphoinositides and the activation of protein kinase C.

The tetradecapeptide bombesin converts epidermal growth factor (EGF) receptors on Swiss 3T3 cells from a high affinity state (KD = 9.8 X 10(-11)M) to a lower affinity state (KD = 1.8 X 10(-9)M). This conversion occurs when the cells are incubated with bombesin at 37 degrees C but not when incubated at 4 degrees C. Previously, a number of other (chemically unrelated) cell growth-promoting peptides and polypeptides have been shown to induce a similar indirect, temperature-dependent reduction of EGF receptor affinity. We have now demonstrated that hormones and growth factors which cross-regulate EGF receptor affinity in Swiss 3T3 cells have a common ability to stimulate the breakdown of phosphoinositides in these cells. We propose that the reduction of EGF receptor affinity is a consequence of the activation of protein kinase C by the diacylglycerol generated by this breakdown. In support of this proposal we have found that exogenously added diacylglycerol reduces the affinity of the Swiss 3T3 cell EGF receptor.

Animals↗
PubMed1984

Inositol trisphosphate formation and calcium mobilization in Swiss 3T3 cells in response to platelet-derived growth factor.

Swiss 3T3 cells incubated for 60 h with [3H]inositol incorporated radioactivity into phosphatidylinositol (PI) and the two polyphosphoinositides phosphatidylinositol 4-phosphate (PIP) and phosphatidylinositol 4,5-bisphosphate (PIP2). On stimulation with platelet-derived growth factor (PDGF) there were significant increases in the levels of inositol 1-phosphate (IP1), inositol 1,4-bisphosphate (IP2) and inositol 1,4,5-trisphosphate (IP3). The effect of PDGF and IP3 on Ca2+ mobilization was studied in both intact cells and in 'leaky' cells that had been permeabilized with saponin. In intact cells, PDGF stimulated the efflux of 45Ca2+, whereas IP3 had no effect. Conversely, IP3 stimulated 45Ca2+ efflux from 'leaky' cells, which were insensitive to PDGF. 'Leaky' cells, which accumulated 45Ca2+ to a steady state within 20 min, were found to release approx. 40% of the label within 1 min after addition of 10 microM-IP3. This stimulation of 45Ca2+ release by IP3 was reversible and was also dose-dependent, with a half-maximal effect at approx. 0.3 microM. It seems likely that an important action of PDGF on Swiss 3T3 cells is to stimulate the hydrolysis of PIP2 to form IP3 and diacylglycerol, both of which may function as second messengers. Our results indicate that IP3 mobilizes intracellular Ca2+, and we propose that diacylglycerol may act through C-kinase to activate the Na+/H+ antiport. By generating two second messengers, PDGF can simultaneously elevate the intracellular level of Ca2+ and alkalinize the cytoplasm by lowering the level of H+.

Animals↗
PubMed1984

Specificity of inositol trisphosphate-induced calcium release from permeabilized Swiss-mouse 3T3 cells.

Swiss-mouse 3T3 cells permeabilized with saponin were used to study the specificity of the inositol trisphosphate-induced release of 45Ca2+ from their intracellular stores. Inositol 1,4,5-trisphosphate was the most potent compound studied (dose giving half-maximal effect 0.3 microM). 45Ca2+ was also released by inositol 2,4,5-trisphosphate, glycerophosphoinositol 4,5-bisphosphate and inositol 4,5-bisphosphate, with doses giving half-maximal effect of respectively 1.6 microM, 1.6 microM and 20 microM, but not by inositol 1,4-bisphosphate (50 microM). These data suggest that the trans-vicinal phosphates on the 4- and 5-positions are essential for the Ca2+-mobilizing effect of inositol trisphosphate, and that in addition there is a requirement for a phosphate group on the opposite side of the molecule, with a preference for the 1-position.

Animals↗
PubMed1984

Partial purification and characterization of a growth factor present in goat's colostrum. Similarities with platelet-derived growth factor.

A factor in goat's colostrum which stimulates DNA synthesis and cell proliferation in Swiss 3T3 fibroblasts has been purified approx. 350-fold by a sequence of acid precipitation, cation-exchange chromatography and gel filtration. The growth factor is a highly basic, heat stable (100 degrees C for 5 min) polypeptide with Mr approx. 35000. The polypeptide resists denaturation by guanidinium chloride or urea but is totally inactivated by treatment with reducing agents. The factor, which we have termed colostric basic growth factor ( CBGF ), inhibits the binding of 125I-labelled epidermal growth factor (125I-EGF) to Swiss 3T3 fibroblasts but does not inhibit 125I-EGF binding to epidermoid A431 cells. CBGF interacts synergistically with plasma in stimulating DNA synthesis in quiescent Swiss 3T3 cells. The chemical and biological properties of CBGF are thus very similar to the properties reported for the human platelet-derived growth factor. Although high concentrations of CBGF are present in the colostrum of goats, cows, and sheep, the milk of these species contains little or no factor. The origin and possible functions of CBGF are unknown.

Animals↗