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Biomedical subjects

K D Brown

Publications and source records attributed to K D Brown.

At least 91 records · Page 5Linked to original sources
PubMed1989

A family of small inducible proteins secreted by leukocytes are members of a new superfamily that includes leukocyte and fibroblast-derived inflammatory agents, growth factors, and indicators of various activation processes.

We have isolated and characterized four cDNA clones that encode mRNA expressed more abundantly in Con A-activated mouse helper T cells than by resting T cells. One mRNA encoded a approximately 14-kDa protein with a hydrophobic N-terminal sequence and was abundantly expressed by the Th 2 subset of Th cells, but was not expressed by Th 1 cells. The remaining three mRNA encoded related approximately 8-kDa secreted proteins that are part of a family of small, secreted, and inducible mouse and human proteins. This family of proteins is itself distantly related to another family of growth and inflammatory factors that are associated with various lymphoid and fibroblast activation phenomena. One of the small, inducible, secreted proteins has a predicted mature N terminus identical to that of the previously described macrophage inflammatory protein.

Amino Acid Sequence↗
PubMed1989

Single-cell analysis of the mitogen-induced calcium responses of normal and protein kinase C-depleted Swiss 3T3 cells.

Single-cell fluorescence image analysis has been used to characterize the mitogen-induced increases in intracellular free [Ca2+] ([Ca2+]i) in control and protein kinase C-depleted Swiss 3T3 cells. More than 80% of the control cells exhibited fast, transient responses to bombesin, vasopressin, or prostaglandin F2 alpha (PGF2 alpha). In contrast, the [Ca2+]i responses induced by platelet-derived growth factor (PDGF) were markedly more heterogeneous, slower, and often biphasic, with fewer cells (60-70%) responding. The peak [Ca2+]i values obtained in response to each mitogen showed substantial variation between cells. Brief pretreatment of the cells with 12-O-tetradecanoyl phorbol 13-acetate (TPA) reduced the [Ca2+]i responses to bombesin, but did not affect the responses to PDGF. Long-term pretreatment of the cells with TPA to down-modulate protein kinase C resulted in substantially prolonged [Ca2+]i responses to bombesin, vasopressin, and PGF2 alpha, but had no such effect on the responses to PDGF. We conclude that differences between the [Ca2+]i responses to bombesin and PDGF, previously reported using cell populations, reflect differences occurring in individual cells, and that the [Ca2+]i responses to bombesin, vasopressin, and PGF2 alpha (but not PDGF) are subject to feedback inhibition via protein kinase C.

3T3 Cells↗
PubMed1989

Tissue-specific effects of castration and ovariectomy on murine epidermal growth factor and its mRNA.

The polypeptide mitogen, epidermal growth factor (EGF), was originally isolated from mouse submaxillary gland (SMG). In mice, these glands are sexually dimorphic; the SMG of male animals typically contains up to 400 pmol EGF/mg protein whereas EGF concentrations in the SMG of female mice are only 5-20 pmol/mg protein. When mice were castrated at 8 weeks of age, EGF mRNA levels in the SMG fell rapidly to the low levels observed in control female animals. Although the concentration of EGF in the SMG, measured by radioimmunoassay, also fell to very low levels (13.8 +/- 0.7 (S.E.M.) pmol/mg protein) in the castrated mice, the rate of decrease was considerably slower than that of the mRNA. In contrast to the loss of EGF and EGF mRNA from mice following castration, ovariectomy led to a rise in EGF mRNA levels in SMG, with a maximal increase (approximately 100-fold) 4-6 weeks after ovariectomy. Concentrations of EGF in the SMG also rose markedly in the ovariectomized animals, from a control value of 5.4 +/- 0.8 to 34.7 +/- 7.9 pmol/mg protein at 6 weeks. In mice, the kidney displays the second highest level of EGF gene expression. However, in contrast to the effects on SMG, kidney EGF mRNA was not affected by either castration or ovariectomy. These results provide further evidence for the tissue-specific control of EGF gene expression in response to steroid hormones.

Animals↗
PubMed1989

A hospital-wide blind control program for bedside glucose meters.

We have instituted hospital-wide proficiency testing of nursing personnel who operate glucose-monitoring instruments. Whole blood samples with known glucose levels were distributed to all nursing units for analysis on a biweekly basis. Measurements obtained by nurses on glucose meters were compared with one another and with true glucose levels to identify discrepant performance and institute corrective action. After 10 months of operation, we found that the nursing units using glucose meters daily showed mean absolute variation from true glucose of 12.7% (range, 10.0% to 17.7%), whereas intermittent users showed mean absolute variation of 15.3% (range, 10.7% to 24.0%). Of 552 total paired determinations, 199 (36.9%) differed from true glucose by greater than 15%. This program was successful in establishing uniform quality control procedures among nonlaboratory personnel who had no previous experience in laboratory measurements.

Blood Glucose↗
PubMed1988

Guanine nucleotides stimulate hydrolysis of phosphatidylinositol and polyphosphoinositides in permeabilized Swiss 3T3 cells.

Hydrolysis-resistant analogues of GTP specifically stimulate the formation of [3H]inositol mono-, bis- and trisphosphates by saponin-permeabilized Swiss 3T3 cells prelabelled with [3H]inositol. Each inositol phosphate is formed largely by hydrolysis of its parent lipid and not by dephosphorylation of inositol 1,4,5-trisphosphate [(1,4,5)IP3]. Although hydrolysis of phosphatidylinositol 4,5-bisphosphate (PIP2) is most sensitive to guanine nucleotides, hydrolysis of phosphatidyl-inositol (PI) and phosphatidylinositol 4-phosphate (PIP) is quantitatively more important. These results suggest that a guanine nucleotide-dependent regulatory protein(s) (G-protein) is involved in regulating the hydrolysis of PI and PIP, as well as PIP2, and so may allow formation of diacylglycerol (DG) without simultaneous production of (1,4,5)IP3 and mobilization of intracellular Ca2+.

Animals↗
PubMed1988

Insulin-like growth factor 1 and insulin reduce epidermal growth factor binding to Swiss 3T3 cells by an indirect mechanism that is apparently independent of protein kinase C.

Insulin-like growth factor 1 and insulin reduced the binding of 125I-labelled epidermal growth factor (125I-EGF) to Swiss 3T3 cells by 15-20% at 37 degrees C, but not at 4 degrees C. Scatchard analysis indicated that IGF-1 and insulin affected the higher-affinity component of EGF binding, an effect previously associated with the activation of protein kinase C. However, the inhibition of 125I-EGF binding by IGF-1 and insulin was increased, not reduced, when the cells were treated with high concentrations of phorbol esters to down-modulate protein kinase C. We suggest that IGF-1 and insulin activate a protein kinase with similar or overlapping specificity to that of protein kinase C.

Animals↗
PubMed1988

Ligand-receptor interactions involved in the stimulation of Swiss 3T3 fibroblasts by insulin-like growth factors and insulin.

1. The binding of 125I-labelled insulin-like growth factor 1 (125I-IGF-1) to Swiss mouse 3T3 fibroblasts was time- and concentration-dependent. Unlabelled IGF-1 had a slightly higher potency than multiplication-stimulating activity (MSA) in inhibiting the binding of 125I-IGF-1, and insulin gave a parallel inhibition curve at 300-1000-fold lower potency. Chemical cross-linking of bound 125I-IGF-1 to its receptors, followed by polyacrylamide-gel electrophoresis under reducing conditions, revealed a major band of Mr 130,000, the labelling of which was inhibited by IGF-1 or high concentrations of insulin. 2. The binding of 125I-IGF-1 was not affected by either co-incubation or preincubation of the cells with a range of heterologous growth factors and mitogens. However, IGF-1 and MSA each induced down-regulation of 125I-IGF-1 binding sites. 3. The maximal stimulations of DNA synthesis induced by IGF-1, MSA and insulin, in the presence of a synergizing mitogen, were similar. The dose-response curve for insulin was not parallel to those for IGF-1 and MSA; in particular, low concentrations of insulin induced a greater stimulation than expected on the basis of its potency in the inhibition or down-regulation of 125I-IGF-1 binding. 4. The preincubation of 125I-IGF-1 with Swiss 3T3 cells at 37 degrees C decreased its ability to bind to a second batch of cells. This inactivation did not occur when the preincubation was performed at 4 degrees C or in the presence of cycloheximide. Chemical cross-linking revealed that the cells released an IGF-binding protein, giving a complex of Mr about 48,000. 5. It is concluded that type I IGF receptors mediate the stimulation of Swiss 3T3 cells by insulin-like mitogens, but that insulin probably stimulates the cells through insulin receptors. The cells can modulate the amount of ligand binding, both by down-regulation of the receptors and by the secretion of an IGF-binding protein.

Animals↗
PubMed1988

Characterization of the high-affinity receptors on Swiss 3T3 cells which mediate the binding, internalization and degradation of the mitogenic peptide bombesin.

Bombesin and bombesin-related peptides such as gastrin-releasing peptide (GRP) stimulate DNA synthesis and proliferation of Swiss 3T3 cells in culture. We have used 125I-labelled [Tyr4]bombesin and 125I-labelled GRP to characterize and identify the receptors for these peptides on Swiss 3T3 cells. The binding of 125I-[Tyr4]bombesin, which retained full biological activity, was maximal between 20 and 30 min incubation at 37 degrees C, after which continued incubation led to a decline in cell-associated radioactivity. This decline was markedly slowed by the presence of lysosomal enzyme inhibitors. Specificity of the binding site was indicated by the competitive inhibition of binding by bombesin-related peptides, but not by unrelated peptides and growth factors. Scatchard analysis of binding data indicated a single class of high-affinity receptors. The calculated value for the dissociation constant (Kd) was 2.1 nM and each cell possesses approx. 240,000 receptors. Because [Tyr4]bombesin has no free amino group, 125I-GRP was used in chemical cross-linking studies. When disuccinimidyl suberate was used to covalently couple 125I-GRP to the cells, two major radiolabelled complexes were detected with molecular masses of approx. 80,000-85,000 and 140,000. The binding of 125I-[Tyr4]bombesin to the cells was pH-dependent with maximal binding at pH 6.5-7.5 and effectively no specific binding at pH values below 4.5. At 37 degrees C, cell-associated 125I-[Tyr4]bombesin quickly became resistant to removal by acidic buffers, suggesting its rapid transfer to an intracellular compartment. However, pre-incubation with unlabelled [Tyr4]bombesin did not induce down-regulation of bombesin receptors as measured by the subsequent binding of 125I-[Tyr4]bombesin. In contrast with the Swiss 3T3 cells, specific binding of 125I-[Tyr4]bombesin was not detectable in two cell lines which are biologically unresponsive to bombesin-related peptides.

Bombesin↗
PubMed1988

Effects of pertussis toxin on growth factor-stimulated inositol phosphate formation and DNA synthesis in Swiss 3T3 cells.

We have compared the effects of pretreatment of Swiss 3T3 cell with pertussis toxin on the stimulation of DNA synthesis and phosphoinositide hydrolysis in response to a wide variety of mitogens. The toxin substantially inhibited the stimulation of DNA synthesis in response to a phorbol ester or various peptide and polypeptide growth factors irrespective of their ability to activate phosphoinositidase C. Production of inositol phosphates in response to platelet-derived growth factor, fibroblast growth factor and prostaglandin F2 alpha were unaffected by the toxin while bombesin- and vasopressin-stimulated formation of inositol phosphates were inhibited by only 27 and 23% respectively. These results argue against a major role for a pertussis toxin-sensitive G protein in coupling any of these mitogen receptors to activation of a phosphoinositidase C. Furthermore, the results suggest that the widespread inhibitory effects of pertussis toxin on mitogen-stimulated DNA synthesis may be unrelated to the toxin's limited actions on phosphoinositide hydrolysis.

Animals↗
PubMed1988

DL-myo-inositol 1,4,5-trisphosphorothioate mobilizes intracellular calcium in Swiss 3T3 cells and Xenopus oocytes.

The initial water-soluble product of receptor-stimulate polyphosphoinositide hydrolysis, D-myo-inositol 1,4,5-trisphosphate is now accepted as the second messenger that stimulates release of Ca2+ from intracellular pools. We report here the first examples of Ca2+ release by a novel phosphatase-resistant inositol trisphosphate analogue, DL-myo-inositol 1,4,5-trisphosphorothioate, in Swiss 3T3 cells and Xenopus oocytes. L-myo-inositol 1,4,5-trisphosphate was inactive in the latter system.

Animals↗
PubMed1988

The insulin-like growth factor I content in human milk increases between early and full lactation.

The concentration of immunoreactive insulin-like growth factor I (IGF-I) in human mammary secretions, assayed after acid-ethanol extraction, was high [mean, 4.1 +/- 0.5 (+/- SE) nmol/L; n = 13] for several weeks prepartum. It then decreased during the first 3 days postpartum to 1.3 +/- 0.1 nmol/L (n = 28), in parallel with changes in epidermal growth factor (EGF) and protein concentrations. However, between the first and sixth weeks postpartum, the IGF-I concentration increased to 2.5 +/- 0.2 nmol/L (n = 18), while levels of EGF and protein decreased further. Given that the volume of milk produced increases during this period, the total IGF-I output rose by up to 4-fold, while EGF output remained constant. The increase in IGF-I and decrease in EGF in milk suggest that different regulatory mechanisms control the output of different growth factors by the mammary gland.

Epidermal Growth Factor↗
PubMed1988

Structural analysis of the 5'-flanking sequence of the mouse epidermal growth factor gene.

The 5'-flanking sequence of the mouse epidermal growth factor gene has been isolated from a mouse genomic DNA library. S1 nuclease mapping indicated that the transcription start sites used in the submaxillary gland and the kidney are identical. Computer-aided sequence comparisons have indicated regions of the gene which may be involved in hormonal regulation.

Animals↗
PubMed1988

From animal to molecule: aspects of the biology of insulin-like growth factors.

The synthesis of IGF-II mRNA in sheep foetal tissues is considerably higher than IGF-I. IGF-II probably has a paracrine role in the foetus; however it is likely that IGF-I originates mainly from the foetal liver and has an endocrine function. Although in the adult system IGF-I is tightly bound to serum carrier proteins it is potentially biologically active. Galactopoiesis in the goat mammary gland provides a useful model for demonstrating the importance of circulating IGF-I as a mediator of GH action. Ligand-receptor interactions involved in the stimulation of Swiss 3T3 fibroblasts by IGF-I, II and insulin were examined. It was found that the potency of binding to type I receptors was IGF-I greater than IGF-II much greater than insulin by competitive binding assays and chemical cross-linking studies, and that some cell lines secrete an IGF binding protein which is specific for IGF-I and II and which acts as an inhibitor in cellular binding assays. Maximal stimulation of DNA synthesis induced by IGF-I, II and insulin in the presence of synergising mitogens were similar. While the actions of the IGFs were consistent with type I receptor binding insulin appeared to act through its own receptor. The reduction of EGF receptor affinity following the addition of IGF-I and insulin to 3T3 cells may involve a protein kinase that is not sensitive to phorbol esters. 3T3 cell nuclei contain endogenous inositol phospholipids and their corresponding kinases and monoesterases.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1987

Two types of mouse helper T cell clone. III. Further differences in lymphokine synthesis between Th1 and Th2 clones revealed by RNA hybridization, functionally monospecific bioassays, and monoclonal antibodies.

Lymphokine synthesis patterns of a panel of 19 T cell clones have been evaluated, using mRNA hybridization methods to examine 11 different mRNAs induced by Con A. The two types of CD4+ Th cell clone described previously were clearly distinguished by this procedure, and the differences between the two types have now been extended to six induced products. With minor exceptions, only Th1 clones synthesized mRNA for IL-2, IFN-gamma, and lymphotoxin, and only Th2 clones synthesized mRNA for IL-4, IL-5, and another induced gene, P600. Four more induced products were expressed preferentially but not uniquely by one or another type of clone: mRNAs for GM-CSF, TNF, and another induced, secreted product (TY5) were produced in larger amounts by Th1 clones, whereas preproenkephalin was preferentially expressed by Th2 clones. IL-3 was produced in similar amounts by both types of clone. mAbs were used to establish three bioassays that were functionally monospecific for IL-2, IL-3, and IL-4, and a new anti-IFN gamma mAb, XMG1.2, was used to establish an ELISA for IFN-gamma. These four assays were used to show that secreted protein and mRNA levels correlated well for all cell lines. The implications of these findings for normal T cells are discussed.

Animals↗
PubMed1987

Protein kinase C-mediated negative-feedback inhibition of unstimulated and bombesin-stimulated polyphosphoinositide hydrolysis in Swiss-mouse 3T3 cells.

Bombesin-related peptides stimulate a rapid increase in polyphosphoinositide hydrolysis in Swiss-mouse 3T3 cells. These peptides generate an increase in the efflux of 45Ca2+ from pre-labelled cells, a response consistent with an inositol trisphosphate-mediated mobilization of intracellular Ca2+. The bombesin-stimulated release of cellular 45Ca2+ is inhibited by tumour-promoting phorbol esters (e.g. 12-O-tetradecanoylphorbol 13-acetate, TPA). Although there are several possible sites of action at which this effect might occur, our results indicate that TPA induces an uncoupling of bombesin-stimulated hydrolysis of phosphatidylinositol 4,5-bisphosphate (PIP2) without decreasing cellular binding of bombesin. In cultured cells, protein kinase C can be down-modulated by a prolonged incubation of the cells with phorbol esters. Such pretreatment greatly decreased the inhibitory effect of TPA on bombesin-stimulated PIP2 hydrolysis, suggesting that this action of the phorbol ester is mediated via protein kinase C. Since diacylglycerol is an endogenous activator of protein kinase C and a direct product of PIP2 hydrolysis, these results suggest that protein kinase C inhibition of polyphosphoinositide hydrolysis may function as a negative-feedback pathway. Cells in which protein kinase C has been down-modulated show elevated basal and bombesin-stimulated production of inositol phosphates, providing evidence that such a feedback loop limits polyphosphoinositide turnover in both unstimulated and mitogen-stimulated cells.

Animals↗
PubMed1987

Insulin-like synergistic stimulation of DNA synthesis in Swiss 3T3 cells by the BSC-1 cell-derived growth inhibitor related to transforming growth factor type beta.

A cell growth inhibitor (GI), purified from BSC-1 cell-conditioned medium, has little if any effect on DNA synthesis when added alone to monolayer cultures of quiescent Swiss mouse 3T3 cells in serum-free medium. However, the inhibitor, which is closely related to transforming growth factor type beta (TGF-beta), exhibits a pronounced synergistic stimulation of DNA synthesis in combination with certain peptide (bombesin, vasopressin) or polypeptide (platelet-derived growth factor) mitogens. A similar synergistic response has been demonstrated for TGF-beta purified from human platelets. In the presence of 3 nM bombesin, a half-maximal stimulation of DNA synthesis was obtained at a GI concentration of approximately 60 pg/ml, with a maximal response at approximately 600 pg/ml. The synergistic interactions demonstrated by GI or TGF-beta in stimulating Swiss 3T3 cells closely resemble those previously shown for insulin, and we have observed that GI does not synergize with insulin to stimulate DNA synthesis in these cells. Like insulin, and in contrast to bombesin, vasopressin, and platelet-derived growth factor, GI does not activate cellular inositolphospholipid hydrolysis, calcium mobilization, or cross-regulation of epidermal growth factor receptor affinity. These results raise the possibility that the biochemical pathways activated by GI/TGF-beta and insulin converge at a post-receptor stage.

Animals↗
PubMed1987

Defective regulation of the phenylalanine biosynthetic operon in mutants of the phenylalanyl-tRNA synthetase operon.

Among mutants of Escherichia coli resistant to p-fluorophenylalanine (PFP) were some with constitutive expression of the phenylalanine biosynthetic operon (the pheA operon). This operon is repressed in the wild type by phenylalanine. The mutation in three of these mutants mapped in the aroH-aroD region of the E. coli chromosome at 37 min. A plasmid bearing wild-type DNA from this region restored p-fluorophenylalanine sensitivity and wild-type repression of the pheA operon. Analysis of subclones of this plasmid and comparison of its restriction map with published maps indicated that the mutations affecting regulation of the pheA operon lie in the structural genes for phenylalanyl-tRNA synthetase, pheST, probably in pheS. Thus, the pheST operon has a role in the regulation of phenylalanine biosynthesis, the most likely being that wild-type phenylalanyl-tRNA synthetase maintains a sufficient intracellular concentration of Phe-tRNA(Phe) for attenuation of the pheA operon in the presence of phenylalanine. A revised gene order for the 37-min region of the chromosome is reported. Read clockwise, the order is aroD, aroH, pheT, and pheS.

Amino Acyl-tRNA Synthetases↗