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C W Taylor

Publications and source records attributed to C W Taylor.

At least 163 records · Page 9Linked to original sources
PubMed1988

Characterization and reactivity of a monoclonal antibody that recognizes a 76 kilodalton human colon tumor antigen.

A monoclonal antibody (MAb 76) was produced by immunizing mice with a soluble cytoplasmic protein fraction from a human adenocarcinoma of the colon. MAb 76 showed specific immunoreactivity against a 76 kDa protein in immunoblot studies using total colon tumor cytosol proteins. Immunoprecipitation of phosphorylated cytosolic protein products with MAb 76 and subsequent analysis on SDS containing polyacrylamide gels revealed a single 38 kDa band, indicating that the 76 kDa antigen is associated with a 38 kDa phosphoprotein species. Indirect immunofluorescence analysis of primary tumor specimens and human colon tumor cell lines showed positive immunoreactivity with 6/7 human colon adenocarcinoma tissues and 15/18 human colon tumor cell lines. MAb 76 was unreactive with normal colon, liver and lung specimens from human, mouse and hamster. The epitope-bearing monomer detected by MAb 76 is immunologically conserved in a high percentage of colon tumor cells and tissues and may represent a cellular product that is characteristic of the transformed colon cell phenotype.

Animals↗
PubMed1988

Variation in cytosolic protein expression between human colon tumors that differ with regard to differentiation class.

Using a combination of two-dimensional gel electrophoresis, silver staining, and a 16-quadrant grid system, we established a set of composite patterns for the colon tumor cytosol proteins of well, moderately, and poorly differentiated tumors. These composite patterns were found to be characteristic of the three individual differentiation classes for colon tumors. The apparent relative molecular masses (Mr) of the resolved proteins ranged from 14,000 to 105,000 and their isoelectric points from pH 5.2 to 8.4. Although the vast majority of the proteins identified in the composite patterns were common, a comparison based upon these patterns revealed two qualitative and seven quantitative protein differences. The qualitative differences were identified by apparent Mr X 10(-3)/pI coordinates of 73/7.2 and 66/6.2. Quantitative differences were identified by Mr X 10(-3)/pI coordinates of 71/6.0, 59/6.7, 57/6.7, 56/5.4, 52/6.1, 30/5.8, and 18/6.2. These cytosolic differentiation marker proteins may facilitate the diagnosis, staging, and monitoring of human colon cancer.

Animals↗
PubMed1987

The B lymphocyte calcium response to anti-Ig is diminished by membrane immunoglobulin cross-linkage to the Fc gamma receptor.

We report the cytosolic free calcium, [Ca2+]i, responses of single murine B lymphocytes to whole and F(ab')2 fragments of anti-Ig measured in the flow cytometer with indo-1, a new fluorescent chelator of calcium. The principle advantages of this recording system are these: Indo-1 is highly fluorescent; hence, loading concentrations that introduce artifacts in the reported [Ca2+]i signal may be avoided. The measurement of [Ca2+]i by fluorescence ratio corrects for nonuniform dye uptake, making possible quantitative estimates of [Ca2+]i in single cells and an assessment of the variability of population responses. Baseline recordings of unstimulated lymphocytes indicated a narrow, stable range of [Ca2+]i (75 to 125 nM). The [Ca2+]i rise induced by various anti-Ig preparations exhibited considerable heterogeneity. The initial mean value for F(ab')2 anti-Ig-stimulated cells peaked above 1 microM and was due only to the release of Ca2+ from intracellular stores. A steady state elevation of [Ca2+]i was reached by 5 min and persisted for hours. Cells stimulated with intact anti-Ig reached similar initial peak [Ca2+]i values, but then declined toward baseline. This difference was due to membrane Ig-IgG Fc receptor (mIg-Fc gamma R) cross-linkage, because blocking the Fc gamma R with a monoclonal antibody made the [Ca2+]i responses to F(ab')2 and intact anti-Ig identical. The attenuation of the [Ca2+]i signal by mIg-Fc gamma R cross-linkage is proceeded by a corresponding Fc gamma-mediated reduction in anti-Ig-induced inositol trisphosphate elevation. These findings outline a biochemical basis for mIg- and Fc gamma R-mediated activation and regulation intrinsic to the B cell, and demonstrate the advantages of indo-1 over quin2 for fluorescent measurement of [Ca2+]i in small cells.

Animals↗
PubMed1986

Effects of Ca2+ on phosphoinositide breakdown in exocrine pancreas.

Recent studies have established that inositol 1,4,5-trisphosphate [I(1,4,5)P3] provides the link between receptor-regulated polyphosphoinositide hydrolysis and mobilization of intracellular Ca2+. Here, we report the effects of Ca2+ on inositol trisphosphate (IP3) formation from phosphatidylinositol bisphosphate (PIP2) catalysed by phospholipase C in intact and electrically permeabilized rat pancreatic acinar cells. In permeabilized cells, the Ca2+-mobilizing agonist caerulein stimulated [3H]IP3 formation when the free [Ca2+] was buffered at 140 nM, the cytosolic free [Ca2+] of unstimulated pancreatic acinar cells. When the free [Ca2+] was reduced to less than 10 nM, caerulein did not stimulate [3H]IP3 formation. Ca2+ in the physiological range stimulated [3H]IP3 formation and reduced the amount of [3H]PIP2 in permeabilized cells. The effects of Ca2+ and the receptor agonist caerulein were additive, but we have not established whether this reflects independent effects on the same or different enzymes. The effect of Ca2+ on [3H]IP3 formation by permeabilized cells was unaffected by inhibitors of the cyclo-oxygenase and lipoxygenase pathways of arachidonic acid metabolism; nor were the effects of Ca2+ mimicked by addition of arachidonic acid. These results suggest that the effects of Ca2+ on phospholipase C activity are not a secondary consequence of Ca2+ activation of phospholipase A2. Changes in free [Ca2+] (less than 10 nM-1.2 mM) did not affect the metabolism of exogenous [3H]I(1,4,5)P3 by permeabilized cells. In permeabilized cells, breakdown of exogenous [3H]IP3 to [3H]IP2 (inositol bisphosphate), and formation of [3H]IP3 in response to receptor agonists were equally inhibited by 2,3-bisphosphoglyceric acid. This suggests that the [3H]IP2 formed in response to receptor agonists is entirely derived from [3H]IP3. In intact cells, [3H]IP3 formation was stimulated when ionomycin was used to increase the cytosolic free [Ca2+]. However, a maximal concentration of caerulein elicited ten times as much IP3 formation as did the highest physiologically relevant [Ca2+]. We conclude that the major effect of receptor agonists on IP3 formation does not require an elevation of cytosolic free [Ca2+], although the increase in free [Ca2+] that normally follows IP3 formation may itself have a small stimulatory effect on phospholipase C.

Animals↗
PubMed1986

Isomers of inositol trisphosphate in exocrine pancreas.

In rat pancreatic acinar cells, the Ca2+-mobilizing receptor-agonist, caerulein, at both maximal and submaximal concentrations, stimulated a rapid, transient, increase in [3H]inositol 1,4,5-trisphosphate [(1,4,5)IP3], followed by a slower, sustained, increase in [3H]inositol 1,3,4-trisphosphate [(1,3,4)IP3]. Neither activation of protein kinase C by phorbol dibutyrate nor prevention of the caerulein-stimulated elevation of cytosolic [Ca2+] significantly affected the pattern of formation of the two isomers of IP3. Although carbachol evoked an increase in cytosolic [Ca2+], it did not significantly stimulate [3H](1,4,5)IP3 accumulation, but did promote [3H](1,3,4)IP3 accumulation. Moreover, both carbachol and caerulein maintained hormone-sensitive intracellular Ca2+ pools in a Ca2+-depleted state after [3H](1,4,5)IP3 had returned to basal concentrations. One interpretation of these findings is that total cellular concentrations of [3H](1,4,5)IP3 may not accurately reflect the concentration of this putative mediator in biologically relevant compartments.

Animals↗
PubMed1986

Evidence suggesting that a novel guanine nucleotide regulatory protein couples receptors to phospholipase C in exocrine pancreas.

The initial response of many cells to 'Ca2+-mobilizing' agonists is phospholipase C-mediated hydrolysis of phosphatidylinositol bisphosphate to inositol trisphosphate (IP3) and diacylglycerol. It has been suggested, by analogy with receptor regulation of adenylate cyclase, that 'Ca2+-mobilizing' receptors may interact with a guanine nucleotide-binding protein (G protein) to regulate phospholipase C activity. Here we report increased accumulation of IP3 in response to caerulein or carbachol in electrically permeabilized rat pancreatic acinar cells. The stable analogues of GTP (guanosine 5'-[gamma-thio]trisphosphate and guanosine 5'-[beta, gamma-imido]triphosphate) stimulate IP3 accumulation and potentiate the effects of caerulein and carbachol. This synergism demonstrates an interaction between receptors, a G protein and phospholipase C. These responses are unaffected by pretreatment of the cells with pertussis or cholera toxins under conditions that produce substantial covalent modification of Gi and Gs, the proteins that couple receptors to adenylate cyclase. We therefore conclude that the G protein that couples receptors to phospholipase C in exocrine pancreas is probably neither Gi nor Gs; instead, we propose that a different G protein mediates this effect.

Adenylate Cyclase Toxin↗
PubMed1986

A guanine nucleotide-dependent regulatory protein couples substance P receptors to phospholipase C in rat parotid gland.

Electrically permeabilized cells of rat parotid gland, prelabelled with [3H]-inositol, synthesized [3H]-inositol phosphates (IP3 and IP2) when stimulated with alpha 1-adrenergic, muscarinic-cholinergic, and substance P receptor-agonists. Non-hydrolyzable analogues of GTP (GTP gamma S and GppNHp) also stimulated [3H]-IP3 formation by permeabilized cells and they potentiated the stimulation by receptor-agonists. These effects of guanine nucleotides occurred only with GTP analogues and only in permeabilized cells indicating an intracellular site of action. NaF stimulated [3H]-IP3 accumulation, an effect that was not entirely attributable to the ability of F- to inhibit (1,4,5)IP3 degradation. These results suggest that a guanine nucleotide-dependent regulatory protein couples Ca2+-mobilizing receptors to phospholipase C in parotid gland.

Animals↗
PubMed1986

Formation and biological action of inositol 1,4,5-trisphosphate.

A wide variety of receptors appear to be coupled to a phospholipase C (EC 3.1.4.3) that hydrolyzes inositol lipids. This reaction is believed to provide a link between receptor activation and cellular Ca2+ mobilization. The mechanisms by which this occurs are believed to involve inositol 1,4,5-trisphosphate (1,4,5-IP3), which signals release of Ca2+ from the endoplasmic reticulum. In rat parotid acinar cells made permeable with saponin, 1,4,5-IP3 induced rapid release of sequestered Ca2+. In intact parotid cells, the concentration-response relationship for methacholine-induced IP3 formation was similar to the relationship for muscarinic receptor occupancy by methacholine. About 10-fold lower concentrations of methacholine were sufficient to increase cytosolic [Ca2+] and to activate secretion, indicating an excess IP3 forming capacity for the muscarinic receptor. The mechanisms for the coupling of receptors to IP3 formation were studied in pancreatic acinar cells made permeable electrically. In this preparation, nonhydrolyzable derivatives of GTP potentiated agonist-induced IP3 production, which suggests the involvement of a guanine nucleotide-dependent regulatory protein. The effects of agonists and guanine nucleotides were not altered by pretreating the acinar cells with cholera or pertussis toxins, which indicated that the regulatory protein linking receptors to IP3 formation is distinct from the ones involved in the regulation of adenylate cyclase.

Animals↗
PubMed1985

Size of the inositol 1,4,5-trisphosphate-sensitive calcium pool in guinea-pig hepatocytes.

Permeabilized hepatocytes accumulated 45Ca2+ into a non-mitochondrial pool when provided with ATP. 45Ca2+ efflux from this pool was revealed by removal of ATP with glucose and hexokinase or by inhibiting uptake with NaVO3. The effect of inositol 1,4,5-trisphosphate (IP3) on 45Ca2+ efflux from the pool was investigated. IP3 (5 microM) evoked a rapid increase in the rate of 45Ca2+ efflux. Kinetic analysis of the effect of IP3 indicated the existence of two distinct Ca2+ fractions within the pool; only one, accounting for about one-third of the ATP-dependent Ca2+ content of the pool, was responsive to IP3. The effect of IP3 on 45Ca2+ efflux from the non-mitochondrial pool does not require ATP, a finding that is inconsistent with a previous suggestion that this effect may be mediated by protein phosphorylation.

Adenosine Triphosphate↗
PubMed1985

Calcium regulation in blowflies: absence of a role for midgut.

Adult blowflies (Calliphora vicina) survive many days without increased mortality if fed 12.5 mM CaCl2 solution in place of water. Initially the calcium content of these flies rapidly increases to more than double the original content but then remains stable for many days afterward. Calcium absorption continues at a similar rate throughout this time. Midguts isolated from flies fed either 12.5 mM CaCl2 solution or distilled water have the same capacity to absorb calcium. Dopamine, octopamine, proctolin, and 5-hydroxytryptamine, at pharmacological doses, do not affect calcium absorption by the isolated midgut. Malpighian tubules are the site of most rapid calcium turnover when flies are fed excess calcium. It is concluded that excretion and not absorption is the major site of hemolymph calcium regulation in C. vicina. This contrasts to the situation in mammals where transcellular calcium absorption by the duodenum is precisely controlled. It is suggested that to avoid high luminal calcium concentrations, fast calcium absorption from the blowfly midgut is adapted to remove calcium from the lumen rather than to regulate hemolymph calcium concentration.

Animals↗
PubMed1985

Occurrence of cytosolic protein and phosphoprotein changes in human colon tumor cells with the development of resistance to mitomycin C.

The cytosolic proteins and phosphoproteins of a mitomycin C-sensitive cell line were examined as progressively greater doses of mitomycin C were administered over a period of 44 weeks. Resistance of the human colon carcinoma cell line increased from a 50% inhibitory concentration of 1 to 6 microM over this time period. Changes in cytosolic protein patterns included increases in the amounts of three proteins with molecular weight X 10(-3)/apparent isoelectric point (Mr/pl) values of 56/6.2, 37/7.3, and 27/6.1. Analysis of in vitro 32P-labeled phosphoprotein patterns revealed reductions in the amounts of four proteins with Mr/pl values of 42/6.3, 40/6.7, 31/6.3, and 25/6.1. One increase was detected in a phosphoprotein with a Mr/pl value of 33/6.1. These changes in cytosolic components paralleled the development of resistance to mitomycin C and may reflect changes in the clonal composition of the cell line as it becomes progressively more resistant to mitomycin C or changes in critical proteins or enzymes involved in the activation or biotransformation of the drug.

Autoradiography↗
PubMed1984

Transcellular calcium transport by the midgut of the blowfly, Calliphora vicina.

Transcellular calcium transport by the internally perfused Calliphora midgut has been measured by simultaneously monitoring 45Ca removal from the perfusing saline (entry to the cells) and its appearance in the bathing saline (exit from the cells). Reduction of the Na+ gradient across the basolateral membranes of midgut epithelial cells by removal of bathing Na+ or by addition of monensin or ouabain inhibits calcium transport across the basolateral membranes. Calcium entry at the apical membranes is inhibited in parallel. The calmodulin inhibitors, trifluoperazine or calmidazolium, do not directly affect calcium transport nor do they dissociate the parallel changes in calcium entry and exit when calcium exit is inhibited. Experiments with A23187 are consistent with a role for intracellular calcium in regulating calcium entry at the apical membranes. It is suggested that calcium transport out of midgut epithelial cells is largely by Na+-Ca2+ countertransport, and that entry may be regulated by cytoplasmic calcium so that the calcium influx never exceeds the capacity of the transport mechanisms to pump it out of the cells.

Animals↗
PubMed1984

Rate of growth and extent of differentiation reflected by cytoplasmic proteins and antigens of human colon tumor cell lines.

The cytosolic proteins and antigens of 11 human colon tumor cell lines were examined with respect to their rate of growth and state of differentiation. Coomassie blue-stained protein analysis of sodium dodecyl sulfate-containing polyacrylamide gels revealed protein bands at Mr 30,000, 31,000, and 58,000, which were characteristic of slower growing and more differentiated cell lines. More rapidly dividing and disdifferentiated colon cell lines lacked the Mr 30,000 and Mr 58,000 bands; instead, they produced a single protein band that ran between the Mr 30,000 and Mr 31,000 positions on the gel. Western transfer analysis of cytoplasmic antigens further subdivided the 11 cell lines into 3 separate categories. Slowly growing and more differentiated lines produced a Mr 52,000 antigen. Intermediate lines, with respect to growth rate and state of differentiation, produced a Mr 38,000 antigen. The rapidly growing and highly disdifferentiated cell lines contained three cytosolic antigens with molecular weights of 37,000, 39,000, and 48,000. These criteria made it possible to classify these 11 human colon tumor tissue culture cell lines into 3 groups which reflect their state of growth activity and degree of differentiation.

Antibodies↗
PubMed1983

Identification of cytosolic antigens from GW-39 adenocarcinoma cells by crossed immunoelectrophoresis and immunofluorescence.

Rabbits were immunized with a cytosolic fraction prepared from human adenocarcinoma cells of the colon (GW-39). The antibodies obtained were analyzed using crossed immunoelectrophoresis and were found to precipitate 23 distinct antigens in the cytosolic fraction from colon tumor cells. After preabsorption with human serum and plasma as well as acetone powders prepared from 2 normal human tissues and 4 normal hamster tissues, 1 major immunodominant cytosol antigen (CA-3) and two less intense immunoprecipitin peaks (CA-1 and CA-5) remained detectable by the crossed immunoelectrophoretic method. The preabsorptions with normal tissues were sufficiently complete to remove anti-carcinoembryonic antigen antibodies and showed that antigens CA-1, CA-3 and CA-5 are immunologically distinct from carcinoembryonic antigen. Indirect immunofluorescence localization studies with preabsorbed anti-cytosol antibodies and FITC-conjugated second antibody showed that these antigens were expressed in the cytoplasm and at the cell surface in several human colon tumor cell lines, their subclones and in primary colon tumor specimens.

Adenocarcinoma↗
PubMed1983

Isolation and partial characterization of a 700 kilodalton human colon carcinoma associated antigen.

An antigen of high molecular weight (CA-3) was isolated from the cytosol fraction of GW-39 human colon tumor cells by antibody affinity chromatography. CA-3 was characterized by an acidic pI value of 4.5-4.9, a molecular weight of 700 kilodaltons and a sedimentation coefficient of 13S. It contained all of the commonly occurring amino acids and had an acidic to basic amino acid ratio of 1.4. CA-3 was resistant to dissociation by reducing agents as well as by sodium dodecylsulfate. Quantitation of CA-3 by a radioimmunoassay employing rabbit anti-CA-3 antiserum revealed a marked elevation of CA-3 in the cytosol extracts of human primary colon carcinoma in comparison to normal colon. The molecular properties of CA-3 are compared to those of carcinoembryonic antigen, high molecular weight colon specific antigen CSAp and two other high molecular weight proteins, fibronectin and conglutinin. Colon antigen CA-3 appears to be different from these other molecules in terms of its molecular weight, sedimentation value, isoelectric point and amino acid composition.

Animals↗
PubMed1982

Salivary gland heterotopia: a unique cerebellopontine angle tumor.

An unusual maldevelopmental lesion, a salivary gland heterotopia of the right cerebellopontine angle, was studied. There was an associated primary adenoid cystic carcinoma. Possible modes of development and the relationship to salivary heterotopia of the middle ear are discussed.

Adult↗