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Biomedical subjects

D Boyd

Publications and source records attributed to D Boyd.

At least 127 records · Page 7Linked to original sources
PubMed1989

Use of phoA fusions to study the topology of the Escherichia coli inner membrane protein leader peptidase.

A topology of the Escherichia coli leader peptidase has been previously proposed on the basis of proteolytic studies. Here, a collection of alkaline phosphatase fusions to leader peptidase is described. Fusions to the periplasmic domain of this protein exhibit high alkaline phosphatase activity, while fusions to the cytoplasmic domain exhibit low activity. Elements within the cytoplasmic domain are necessary to stably anchor alkaline phosphatase in the cytoplasm. The amino-terminal hydrophobic segment of leader peptidase acts as a weak export signal for alkaline phosphatase. However, when this segment is preceded by four lysines, it acts as a highly efficient export signal. The coherence of in vitro studies with alkaline phosphatase fusion analysis of the topology of leader peptidase further indicates the utility of this genetic approach to membrane protein structure and insertion.

Alkaline Phosphatase↗
PubMed1988

Insulin receptors with defective tyrosine kinase inhibit normal receptor function at the level of substrate phosphorylation.

Rat 1 fibroblasts have been transfected with the cDNA encoding a kinase-defective mutant human insulin receptor (A/K1018). Expression of this cDNA results in a receptor that is not only biologically inactive but also inhibits normal insulin action through the normal endogenous rat receptors in this fibroblast line (McClain, D. A., Maegawa, H., Lee, J., Dull, T. J., Ullrich, A., and Olefsky, J. M. (1987) J. Biol. Chem. 262, 14663-14671). We have investigated the mechanism of this inhibition and show that: 1) rat receptors are expressed at normal to increased levels in two cell lines which also express A/K1018 receptors at low (A/K1018-A, 5700 total receptors) or high (A/K1018-B, 2.2 x 10(5) total receptors) levels. 2) The rat receptors in the A/K1018 lines can be normally autophosphorylated under the control of insulin in vitro. 3) A/K1018 receptors do not inhibit the kinase activity of normal receptors when mixed together in vitro. 4) In intact A/K1018-B cells, the ability of insulin to stimulate autophosphorylation of the rat receptor is unimpaired; furthermore, the autophosphorylated rat receptor becomes normally activated as a tyrosine kinase. 5) The expression of receptors for insulin-like growth factor I and stimulation of hexose uptake mediated by this receptor are unaffected in cells expressing inhibitory A/K1018 receptors. 6) Expression of the A/K1018 receptor inhibits insulin-stimulated phosphorylation of two endogenous protein substrates (pp220 and pp170) by the native rat receptors. We conclude that the inhibition of insulin action seen in the A/K1018 cells is not mediated at the levels of native receptor expression or activation, nor is the effector (hexose uptake) mechanism affected by the A/K1018 receptors. The expression of this kinase-defective receptor does, however, inhibit the phosphorylation of substrate molecules by the normally activated endogenous rat receptors.

Animals↗
PubMed1988

Modulation of the urokinase receptor in human colon cell lines by N,N-dimethylformamide.

The present study documents the effect of the planar, polar differentiation promoter N,N-dimethylformamide (DMF) on urokinase binding to colon carcinoma cells. Exposure of the colon carcinoma cell lines to the agent resulted in enhanced specific binding of radioactive urokinase to all cells tested. Insulin binding to the cells was, however, unaffected by DMF. A DMF exposure period of 45 h was required to observe maximum urokinase binding to two representative cell lines FET and RKO. Optimal stimulation of both cell lines occurred with 0.8% DMF. Scatchard analysis revealed the dissociation constants to be unchanged by the agent with the increased binding of radioactive plasminogen activator reflecting an up-regulation of binding sites. In this regard, the cell line RKO upon exposure to DMF, displayed approx. 700,000 receptors/cell, the highest value published, to date, for any cell line.

Cell Line↗
PubMed1988

Determination of the levels of urokinase and its receptor in human colon carcinoma cell lines.

At present, there is a lack of availability of differentiation markers for colon carcinoma. This may, in part, be a consequence of the diversified function of the normal human colon. This study addresses the possibility that the expression of urokinase and its receptor is inversely related to differentiation in colon carcinoma. Six colon carcinoma cell lines including three well-differentiated (CBS, GEO, FET) and three poorly differentiated ones (HCT116, HCT116b, RKO) were screened for urokinase receptor display and secretion of the plasminogen activator. A radioreceptor assay was used to determine receptor levels. Binding of radioactive urokinase to colon cells was saturable, specific, and time dependent. Cell-bound 125I-labeled protease was unaffected by the presence of epidermal growth factor, low-molecular-weight urokinase, plasminogen, or transferrin. Time course studies revealed that maximum amounts of radioactive tracer were bound in a 30-min period with no change occurring over the course of a 90-min incubation. Scatchard analysis of ligand binding indicated that the well-and poorly differentiated cells could be separated on the basis of receptor display; the aggressive RKO, HCT116, and HCT116b expressed in excess of 10(5) sites per cell, while the more indolent CBS, GEO, and FET possessed less than 1.5 X 10(4) receptors per cell. The colon carcinoma cells were also analyzed for urokinase in the conditioned medium. Low levels of the plasminogen activator (0.8 to 1.3 ng/ml/10(6) cells/72 h) were associated with the more "mature" cells. This was in contrast to the elevated levels of the protease (3.9 to 11.4 ng/ml/10(6) cells/72 h) present in the medium derived from the more aggressive cells (HCT 116, HCT116b, RKO). Thus, secreted urokinase and/or the expression of cellular receptor for the plasminogen activator may provide useful measurements of the degree of undifferentiation of in vitro colon carcinoma.

Binding, Competitive↗
PubMed1988

Alterations of the biological characteristics of a colon carcinoma cell line by colon-derived substrata material.

This study documents the ability of substrata material derived from well but not poorly differentiated colon carcinoma cells to alter the biological characteristics of a separate colon carcinoma cell line (MOSERSF). To assess changes induced by the presence of these substrata, MOSERSF cells were screened for (a) morphological features, (b) secretion of carcinoembryonic antigen (CEA), (c) alteration of urokinase levels, and (d) sensitivity to the growth-inhibitory peptide transforming growth factor beta. Morphologically, MOSERSF cells grown on plastic displayed a rounded shape and could be detached by agitation. Subculturing of these cells onto substrata laid down by well differentiated (mature) colon carcinoma cells resulted in cell attachment and spreading. These changes did not manifest themselves when cells were plated on material derived from poorly differentiated (primitive) colon cells. Conditioned medium from MOSERSF cells grown on plastic or on colon-derived material from the well and poorly differentiated colon cells were compared for CEA levels. Substrata derived from undifferentiated cells were without effect on assayable CEA (substrata absent, 1.4 ng/ml/10(6) cells/72 h; substrata present, 1.4-1.7 ng/ml/10(6) cells/72 h). However, growth of MOSERSF cells on material deposited by well differentiated colon cells resulted in a 3-fold increase in the level of CEA. Spent medium was also analyzed for urokinase. A high level of the protease (20.3 ng/ml/10(6) cells/72 h) was expressed by MOSERSF cells. The concentration of the enzyme was reduced by over 50% when MOSERSF cells were propagated on substrata laid down by well differentiated cells. An enhanced sensitivity to the growth-retarding effects of transforming growth factor beta was seen with certain substrata. On plastic, transforming growth factor beta inhibited proliferation of MOSERSF cells with a median effective concentration of 0.65 ng/ml. However, on substrata from mature but not primitive cells, MOSERSF cells exhibited an increased sensitivity to the peptide (median effective concentration, 0.16 ng/ml). Colon-derived material obtained from both well differentiated and poorly differentiated colon carcinoma cells was compared after [35S]-methionine metabolic labeling. More [35S]methionine was incorporated into the material from the "mature" colon cells. The substrata could also be distinguished by quantitative differences in a number of high molecular weight proteins. Immunofluorescence of colon-deposited material revealed the presence of laminin and fibronectin.

Carcinoembryonic Antigen↗
PubMed1988

Genetic analysis of the membrane insertion and topology of MalF, a cytoplasmic membrane protein of Escherichia coli.

MalF is an essential cytoplasmic membrane protein of the maltose transport system of Escherichia coli. We have developed a general approach for analysis of the mechanism of integration of membrane proteins and their membrane topology by characterizing a series of fusions of beta-galactosidase to MalF. The properties of the fusion proteins indicate the following. (1) The first two presumed transmembrane segments of MalF are sufficient to anchor beta-galactosidase firmly to the inner membrane. (2) Hybrid proteins with beta-galactosidase fused to a presumed cytoplasmic domain of MalF have high beta-galactosidase specific activity; fusions to periplasmic domains have low activity. We propose therefore, that periplasmic and cytoplasmic domains of integral membrane proteins can be distinguished by the enzymatic properties of such hybrid proteins. In general, it appears that cleaved or non-cleaved signal sequences when attached to beta-galactosidase cause it to become embedded in the membrane, and this results in the inability of the hybrid proteins to assemble into active enzyme. Additional properties of these fusion proteins contribute to our understanding of the regulation of MalF synthesis. The MalF protein, synthesized as part of the malEFG operon of E. coli, is approximately 30-fold less abundant in the cell than MalE protein (the maltose-binding protein). Differential amounts of the fusion proteins indicate that a regulatory signal occurs within the malF gene that is responsible for the step-down in expression from the malE gene to the malF gene.

ATP-Binding Cassette Transporters↗
PubMed1988

Alteration in the behavior of a colon carcinoma cell line by extracellular matrix components.

It was previously demonstrated that substrata derived from well differentiated colon carcinoma cell lines induced a more benign program in a separate malignant colon cell line, MOSERsf. This study attempts to define a role for extracellular matrix components in the biological events of MOSERsf cells. Alterations in morphology, secreted carcinoembryonic antigen (CEA) and urokinase brought about by individual components were determined. Laminin induced similar changes to colon-derived substrata in that there was increased cell attachment and spreading, a 4-fold elevation in CEA and a 45% reduction in urokinase. Fibronectin stimulated cell attachment without altered morphology and reduced the amount of plasminogen activator. CEA values, however, remained unchanged. Growth of MOSERsf cells on all types of collagen failed to elicit any change in cell shape or CEA. However, type I/III collagen raised urokinase levels by 40%. Transforming growth factor beta (TGF-beta) induces cellular laminin and fibronectin, promotes cell attachment, and spreading, elevates CEA and diminishes urokinase. These data argue for a role for laminin and possibly fibronectin in the governing of biological events culminating in a more mature colon cell.

Carcinoembryonic Antigen↗
PubMed1987

Effects of difluoromethylornithine and dicyclohexylammonium sulfate on the transformed state of AKR-MCA cells.

The effect of two inhibitors of polyamine biosynthesis, difluoromethylornithine and dicyclohexylammonium sulfate, on the transformed fibroblastic cell line AKR-MCA and its parental counterpart AKR-2B was investigated. Treatment of monolayer AKR-MCA cells with either agent results in morphological changes akin to AKR-2B; the cells appear to be flattened with a polygonal shape. The ability of the inhibitors to alter the phenotype is lost when the cells are cocultured with polyamines. More specifically, putrescine and spermidine abrogate the effects of difluoromethylornithine while only spermidine is effective in reversing the dicyclohexylammonium sulfate induced phenomenon. Further evidence that these enzyme inhibitors are reversing the transformed state of AKR-MCA is obtained from soft agarose experiments. AKR-MCA cells will generate colonies only in the absence of either difluoromethylornithine or dicyclohexylammonium sulfate. Polyamine levels were determined in parental AKR-2B and AKR-MCA cells. The levels of putrescine and spermine were similar in both cell types. In contrast, significantly more (P less than or equal to 0.01) spermidine was expressed by the malignant line [7.3 +/- 0.8 (SD) nmol/10(6) cells] when compared with the untransformed AKR-2B (5.4 +/- 0.8 nmol/10(6)) cells. Intracellular putrescine and spermidine were sensitive to difluoromethylornithine, dicyclohexylammonium sulfate, and dimethylformamide, a planar, polar solvent which has been reported to "normalize" the transformed phenotype. AKR-MCA treated with difluoromethylornithine or dimethylformamide manifested time dependent reductions in both polyamines which preceded morphological changes. Dicyclohexylammonium sulfate similarly caused a 70% reduction in spermidine, but in contrast to the other agents there was a marked accumulation of putrescine. These data concur with the established molecular actions of the two enzyme inhibitors as blockers of ornithine decarboxylase (difluoromethylornithine) and spermidine synthase (dicyclohexylammonium sulfate). The normalizing capacity of dimethylformamide was not compromised by cotreatment with putrescine or spermidine. Both difluoromethylornithine and dicyclohexylammonium sulfate inhibited the growth of monolayer AKR-2B and AKR-MCA. In view of the well documented cytostatic effects of polyamine inhibitors, it is suggested that a decrease in growth by these agents triggers a more normal phenotype in AKR-MCA cells.

Animals↗
PubMed1987

Evaluation of platelet function in pregnancy. Comparative studies in non-smokers and smokers.

We have evaluated the possible role of platelet functional abnormalities as a contributory cause of thrombosis during pregnancy and to the increased fetal mortality and morbidity among women who smoke. Fifty-three pregnant women were enrolled and evaluated on two separate prenatal visits held between the 20th and 36th week of pregnancy and, when possible, post partum. Smoking status was evaluated by personal statement and alveolar carbon monoxide levels. Women in the smoking group deliberately avoided cigarettes for at least 20 minutes before sampling. Plasma levels of beta-thromboglobulin, thromboxane B2, and 6-Keto PGF1 alpha were evaluated. A significant increase in 6-Keto PGF1 alpha was noted among smoking women as pregnancy advanced. 6-Keto PGF1 alpha levels decreased among non-smoking women while beta-thromboglobulin increased significantly between the 20th and 33rd week of pregnancy in non-smokers. Platelet aggregation, both in platelet rich plasma and in whole blood (by impedance aggregometry), was evaluated by five different parameters and four different aggregating agents. Significant differences between the non-smoking and smoking pregnant women were noted for selected age cohorts and aggregating agents. An increase in platelet reactivity among smokers was observed in whole blood by impedance aggregometry with adenosine diphosphate and in two age cohorts using platelet rich plasma. In two groups in which aggregation was significantly accelerated among non-smokers, epinephrine was used as the aggregating agent.

6-Ketoprostaglandin F1 alpha↗
PubMed1987

Human ferritin H and L sequences lie on ten different chromosomes.

In humans, the H (heavy) and L (light) chains of the iron-storage protein ferritin, are derived from multigene families. We have examined the chromosomal distribution of these H and L sequences by Southern analysis of hybrid cell DNA and by chromosomal in situ hybridization. Our results show that human ferritin H genes and related sequences are found on at least seven different chromosomes while L genes and related sequences are on at least three different chromosomes. Further, we have mapped the chromosomal location of expressed genes for human H and L ferritin chains and have found an H sequence which may be a useful marker for idiopathic hemochromatosis.

Animals↗
PubMed1987

The specific binding of transferrin to the murine fibroblast cell lines AKR-2B and its malignant counterpart AKR-MCA may be related to the transformed state.

The specific binding of radiolabelled transferrin to AKR-2B and AKR-MCA murine fibroblasts was studied. The binding of radioligand to both cell lines was specific, being displaced by excess of unlabelled transferrin but not myoglobin, or lactoperoxidase. Under equilibrium conditions the transformed line AKR-MCA bound significantly more radioactive transferrin (22.5 +/- 3.5 fmol/micrograms DNA) than the parental line AKR-2B (14.5 +/- 1.5 fmol/micrograms DNA). The differences in the amount of ligand bound was due to altered receptor numbers. Treatment of AKR-MCA and AKR-2B cells with DMF eliminated the difference in transferrin binding capacities. The maximum decrease in specific ligand binding to AKR-MCA cells brought about by the polar solvent was observed after 48 h. These data suggest an association between transferrin binding and the transformed state of AKR-2B fibroblasts.

Animals↗
PubMed1987

Determinants of membrane protein topology.

The topology of the integral membrane protein MalF, which is required for maltose transport in Escherichia coli, has been analyzed using fusions of alkaline phosphatase (EC 3.1.3.1). The properties of such fusion strains support a MalF structure previously proposed on theoretical grounds. Several transmembrane segments within MalF can act as signal sequences in exporting alkaline phosphatase. Other transmembrane sequences, in conjunction with cytoplasmic domains, can stably anchor alkaline phosphatase in the cytoplasm. Our results suggest that features of the amino acid sequence (possibly the positively charged amino acids) of the cytoplasmic domains of membrane proteins are important in anchoring these domains in the cytoplasm. These studies in conjunction with our earlier results show that alkaline phosphatase fusions to membrane proteins can be an important aid in analyzing membrane topology and its determinants.

ATP-Binding Cassette Transporters↗
PubMed1986

Comparative evaluation of antepartum and postpartum platelet function in smokers and nonsmokers.

Platelet aggregation was analyzed during normal pregnancy by evaluation of the same patient cohort (n = 19) on two separate prenatal visits and at 4 weeks post partum. To analyze platelet aggregation five different parameters were evaluated in platelet-rich plasma and whole blood (by impedance aggregometry) with the use of four different aggregating agents: adenosine diphosphate, collagen, sodium arachidonate, and epinephrine. Plasma levels of beta-thromboglobulin, thromboxane B2, and 1,6-keto-prostaglandin F1 alpha were also analyzed. Platelet counts rose 19% after delivery. Plasma levels of beta-thromboglobulin and thromboxane B2 were not significantly changed during the two antepartum visits, but B-thromboglobulin levels were modestly elevated post partum. Levels of 1,6-keto-prostaglandin F1 alpha decreased significantly from a mean of 125 pg/ml before delivery to 50 pg/ml post partum. All antepartum parameters on whole blood aggregation that were significantly different from postpartum values showed enhanced antepartum reactivity. In platelet-rich plasma, pregnancy was associated with greater platelet reactivity except for two variables, time to half-maximal aggregation for epinephrine and the extent of aggregation by collagen, both noted during the first antepartum evaluation. When the patient cohort of 19 was subdivided according to smoking status, all aggregation parameters of significance (p less than 0.05) demonstrated increased platelet reactivity during pregnancy compared with postpartum values. Smoking women accounted for a disproportionate number of significantly enhanced reactivity (seven variables versus three).

6-Ketoprostaglandin F1 alpha↗
PubMed1985

Structural and functional relationships of human ferritin H and L chains deduced from cDNA clones.

We have isolated essentially full-length cDNA clones for human ferritin H and L chains from a human liver cDNA library. This allows the first comparison of H and L nucleotide and amino acid sequences from the same species as well as ferritin L cDNA sequences from different species. We conclude that human H and L ferritins are related proteins which diverged about the time of evolution of birds and mammals. We also deduce the secondary structure of the H and L subunits and compare this with the known structure of horse spleen ferritin. We find that residues involved in subunit interaction in shell assembly are highly conserved in H and L sequences. However, we find several interesting differences in H subunits at the amino acid residues involved in iron transport and deposition. These substitutions could account for known differences in the uptake, storage, and release of iron from isoferritins of different subunit composition.

Amino Acid Sequence↗
PubMed1985

Ferritin H and L chains are derived from different multigene families.

This paper explores the complexity of human ferritin H and L genes. We show that essentially full-length cDNA clones for human ferritin H and L chains do not cross-hybridize under moderate stringency conditions and present the first comparisons of H and L mRNAs and genes from the same species. Northern blot analyses indicate that the H and L mRNAs each contain about 1100 nucleotides. Subprobe analyses of Southern blots show that both H and L genes exist as multiple gene families. Both the 5' and 3' sequences of the H genes are heterogeneous, whereas the 3' end of the L gene is relatively conserved.

Base Sequence↗