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Biomedical subjects

C W Taylor

Publications and source records attributed to C W Taylor.

At least 127 records · Page 7Linked to original sources
PubMed1992

Luminal Ca2+ promoting spontaneous Ca2+ release from inositol trisphosphate-sensitive stores in rat hepatocytes.

1. Spontaneous Ca2+ release from the inositol 1,4,5-trisphosphate (InsP3)-sensitive stores in permeabilized hepatocytes was monitored using Fluo-3 to measure the free [Ca2+] of the medium bathing the cells. 2. Permeabilized cells rapidly sequestered Ca2+, reducing the [Ca2+] to 103 +/- 5 nM. Under conditions that depended critically upon cell density and the amount of Ca2+ in the medium, this was followed by a slow increase in [Ca2+] culminating in a substantial Ca2+ spike representing synchronous discharge from the InsP3-sensitive stores. 3. During the latency preceding the Ca2+ spike, the stores increased their sensitivity to InsP3. This sensitization seemed to be an all-or-none phenomenon. 4. Oxidized glutathione and thimerosal promoted the spontaneous release by sensitizing the InsP3 receptor. 5. An increase in the [Ca2+] within the stores was required for both the increased sensitivity to InsP3 and the subsequent spike. 6. Caffeine (6 mM) antagonized the effect of very low InsP3 concentrations and abolished the Ca2+ spike, without itself releasing Ca2+. 7. Our results suggesting that luminal Ca2+ may sensitive InsP3-sensitive stores leading to spontaneous Ca2+ mobilization will be discussed in the light of a modified version of the two-pool model for explaining cytosolic Ca2+ oscillations.

Action Potentials↗
PubMed1992

Luminal Ca2+ increases the sensitivity of Ca2+ stores to inositol 1,4,5-trisphosphate.

Ca2+ within intracellular stores has been proposed to act with cytosolic inositol 1,4,5-trisphosphate (InsP3) to cause opening of the integral Ca2+ channel of the InsP3 receptor, leading to mobilization of intracellular Ca2+ stores [FEBS Lett. 263:5-9 (1990)]. We have tested that suggestion in saponin-permeabilized rat hepatocytes by manipulating the Ca2+ content of the stores and then determining their sensitivity to InsP3, while keeping the cytosolic Ca2+ concentration constant. Stores depleted of Ca2+ by incubation with ionomycin were significantly less sensitive to InsP3, an effect thought likely to result from the decrease in luminal free Ca2+ concentration rather than from direct effects of ionomycin on InsP3 binding or Ca2+ permeability. The luminal free Ca2+ concentration of stores loaded in the presence of pyrophosphate appeared to be substantially reduced, and again there was a significant inverse correlation between the estimated free Ca2+ concentration of the stores and their sensitivity to InsP3. By following the kinetics of 45Ca2+ uptake into empty stores in the presence of inositol trisphosphorothioate, a stable InsP3 analogue, we demonstrated that stores respond to inositol trisphosphorothioate only after their luminal free Ca2+ concentration exceeds a critical level. We conclude that InsP3 and luminal Ca2+ together regulate Ca2+ mobilization from intracellular stores, and we discuss some of the implications of this interaction for the complex Ca2+ signals evoked by extracellular stimuli.

Animals↗
PubMed1992

Different calcium-mobilizing receptors share the same guanine nucleotide-binding protein pool in hepatocytes.

High affinity binding of epinephrine to the alpha 1-adrenoceptor reflects the association of the ligand-receptor complex with a guanine nucleotide-binding protein (G protein) and thereby allows the receptor-G protein interaction to be assessed by radioligand binding methods. We have used [3H]prazosin/epinephrine competition binding to rat liver plasma membranes to examine the effects of other Ca(2+)-mobilizing hormones on the interaction between the alpha 1-adrenoceptor and its G protein. The aim of our experiments was to test whether the different Ca(2+)-mobilizing receptors in liver share the same limited pool of G proteins. [Arg8] Vasopressin (AVP) caused a concentration-dependent (EC50 = 0.49 +/- 0.03 nM) inhibition of the extent to which epinephrine formed a high affinity complex with the alpha 1-adrenoceptor; antagonist binding was unaffected by AVP. The effect of AVP was competitively antagonized (Kd = 0.27 +/- 0.10 nM) by a selective peptide antagonist of the V1 vasopressin receptor. We conclude that, in rat hepatocytes, alpha 1-adrenoceptors and V1 vasopressin receptors converge to interact with the same pool of G proteins.

Adrenergic beta-Agonists↗
PubMed1992

A phase I trial of fluorouracil, leucovorin, and recombinant interferon alpha-2b in patients with advanced malignancy.

We conducted a phase I trial of fluorouracil (5-FU), leucovorin, (LCV), and recombinant interferon-alpha-2b (rIFN-alpha-2b). The doses of each of the three agents were escalated sequentially. 5-FU and LCV were administered by IV bolus, weekly for 6 weeks and rIFN-alpha-2b was administered by subcutaneous injection, three times weekly for 6 weeks. Twenty-nine patients with advanced cancer (75% colon or pancreatic cancer) were treated. Partial remissions were observed in three patients (10%) with previously untreated colon cancer, colon cancer refractory to 5-FU plus LCV and previously untreated pancreatic cancer, respectively. An additional three patients with pancreatic, prostate, and rectal cancer had a 50% reduction in tumor markers but no change in objective tumor measurements. The toxicity of this regimen was tolerable. The most common toxicities were diarrhea, fatigue, flu-like symptoms, nausea/vomiting, and mucositis. However, no fatal or life-threatening toxicities were observed. We conclude that the combination of 5-FU, LCV, and rIFN-alpha-2b can be safely administered and recommend further evaluation of this regimen in patients with tumors of gastrointestinal origin using doses of 5-FU 600 mg/m2, LCV 500 mg/m2, and rIFN-alpha-2b 10 x 10(6) U.

Adult↗
PubMed1992

Growth and dissemination of human malignant melanoma cells in mice with severe combined immune deficiency.

BACKGROUND: The severe combined immune deficiency (SCID) mouse is lacking mature B and T lymphocytes and may be permissive for human tumor growth and metastasis. EXPERIMENTAL DESIGN: SCID mice received human melanoma cells of diverse origins including: 2 established cell lines, 4 early passage cell lines, and fresh or cryopreserved cells obtained directly from 9 patient biopsies. They were introduced into SCID mice via intraperitoneal, subcutaneous and intravenous injections. RESULTS: Tumor growth occurred with each of the 15 melanoma specimens for a take rate of 100% considering cell source. In addition, 60% of the 102 total mice injected displayed tumor growth in at least one site. The most consistent tumor growth (77%) occurred after intraperitoneal injection. Tumors developed in 41 and 48% of mice injected subcutaneously and intravenously, respectively. The mice developed both local tumor growth with palpable tumor nodules at injection sites and hematogenous and/or lymphatic dissemination to multiple sites in the abdominal and thoracic cavities. The number of metastases per animal averaged 16.3 and the number per organ ranged from 1 to 38. Melanotic and amelanotic tumor nodules obtained from a single patient retained their original characteristics with regard to melanin production after passage in the SCID mouse. The appearance of the human melanoma cells in SCID mouse tissues ranged from implants on the organ capsule to frank parenchymal organ involvement and vascular invasion. Some small foci of tumor were only detected using immunohistochemistry with monoclonal antibodies against the S-100 and HMB-45 to melanoma-related antigens. CONCLUSIONS: We conclude that the SCID mouse consistently supports growth, invasion, and metastatic spread of human melanoma cells, including specimens obtained from fresh patient biopsies. The SCID mouse will serve as a relevant in vivo model for studying the biology of human malignant melanoma and screening new therapeutic agents.

Animals↗
PubMed1991

Heparin and other polyanions uncouple alpha 1-adrenoceptors from G-proteins.

Several polyanionic compounds antagonize the interaction between receptors and the G-proteins that regulate adenylate cyclase or K+ channels, possibly by binding to a basic stretch of the receptor that is proposed to mediate its interaction with the G-proteins. We have studied the effects of polyanions on the interaction between the liver alpha 1-adrenoceptor and the G-protein through which it stimulates polyphosphoinositide turnover. Heparin [concn. causing 50% of maximal effect (EC50) = 0.5 microM], Trypan Blue (EC50 7.1 microM) or suramin (EC50 2.1 microM) prevented formation of the high-affinity adrenaline-receptor-G-protein complex without affecting antagonist binding. After alkaline treatment of the membranes, previously reported to cause G-protein removal, binding of agonists was insensitive to both guanine nucleotides and heparin. We conclude that these polyanions uncouple the alpha 1-adrenoceptor from its G-protein, suggesting that similar coupling mechanisms may underlie receptor activation of the G-proteins that activate polyphosphoinositide hydrolysis and those that regulate adenylate cyclase. This action of heparin severely limits its utility as a selective antagonist of the Ins(1,4,5)P3 receptor in intact cells.

Adenylate Cyclase Toxin↗
PubMed1991

Quantal Ca2+ mobilization stimulated by inositol 1,4,5-trisphosphate in permeabilized hepatocytes.

In several cell types, including hepatocytes, submaximal concentrations of Ins(1,4,5)P3 stimulate an initial rapid mobilization of intracellular Ca2+ stores that is followed by either no further Ca2+ release or very much slower release. Further additions of Ins(1,4,5)P3 then evoke further Ca2+ mobilization. Such 'incremental' responses [Meyer & Stryer (1990) Proc. Natl. Acad. Sci. U.S.A. 87, 3841-3845] could result from all-or-nothing emptying of stores that differ in their sensitivities to Ins(1,4,5)P3 or from partial emptying of stores that are more uniformly sensitive, but unable to release all of their Ca2+ because the response to Ins(1,4,5)P3 rapidly attenuates. By measuring unidirectional 45Ca2+ efflux from intracellular stores stimulated with Ins(1,4,5)P3 under conditions where they continue to sequester 40Ca2+, we provide evidence suggesting that Ins(1,4,5)P3 stimulates all-or-nothing emptying of stores that differ in their sensitivities to Ins(1,4,5)P3, a quantal response pattern.

Animals↗
PubMed1991

Spontaneous calcium release from inositol trisphosphate-sensitive calcium stores.

Inositol 1,4,5-trisphosphate (InsP3) functions as a second messenger to mobilize Ca2+ from intracellular reservoirs. The release mechanism displays all-or-none characteristics, that may account for other observations that the InsP3-induced mobilization of Ca2+ is quantal. Quantal release may depend on the sensitivity of the InsP3 receptor being regulated by the Ca2+ concentration in the lumen of the endoplasmic reticulum. We report here that the InsP3-sensitive store in hepatocytes discharges spontaneously when overloaded with Ca2+. The release, which is blocked by heparin, is preceded by an increasing sensitivity of the InsP3 receptor to endogenous InsP3, and is promoted by those sulphydryl reagents (oxidized glutathione and thimerosal) that induce Ca2+ oscillations in intact cells (ref. 8, and T. A. Rooney, D. C. Renard, E. J. Sass and A. P. Thomas, manuscript in preparation). This novel process could have a role in generating both Ca2+ oscillations and Ca2+ waves.

Animals↗
PubMed1991

Altered expression of P-glycoprotein and cellular adhesion molecules on human multi-drug-resistant tumor cells does not affect their susceptibility to NK- and LAK-mediated cytotoxicity.

Drug resistance has been associated with resistance to NK- and LAK-cell-mediated cytotoxicity. We evaluated this issue in human cell lines, using multiple myeloma cells (8226) and 2 multi-drug-resistant (MDR) sublines selected using doxorubicin (8226/Dox40) and mitoxantrone (8226/MR40). In parallel, we studied the human breast carcinoma cell line series MCF7, MCF7/D40 and MCF7/Mitox. Unlike the sensitive parental cell lines, all 4 sublines display MDR-patterns of resistance, with the P-glycoprotein pump (P-170) detected only in the doxorubicin-selected sublines. Flow cytometric and immunocytochemical analyses showed expression of cellular adhesion molecules ICAM-I and LFA-3, and MHC-Class-I (MCF7/D40 only), to be decreased in the doxorubicin-selected MDR-sublines, whereas expression of CD56 (Leu 19) was strongly up-regulated in 8226/Dox40. Lysis of P-170-positive MDR tumor cells by NK or LAK cells was, however, unaffected by these alterations, suggesting redundancy in effector:target-cell adhesion pathways. Mitoxantrone-selected tumor cells did not display P-170, nor did they show altered expression of cellular adhesion molecules. Their susceptibility to NK or LAK cytolysis was also unimpaired as compared to the parental cell lines. Clinically, these results imply that immunotherapeutic modalities aiming at increased natural killer functions deserve full consideration even in patients who have become refractory to further cytostatic drug treatment.

ATP Binding Cassette Transporter, Subfamily B, Mem↗
PubMed1991

Squamous-cell carcinoma of the anal canal: management with combined chemo-radiation therapy.

Twenty patients with squamous-cell carcinoma of the anal canal received combined chemo-radiation therapy as their primary treatment. There were 18 women and two men with a mean age of 63 years (range, 34-91 years). The mean follow-up was 34 months (range, 6-62 months). Anal margin cancers and adenocarcinomas were excluded. Fourteen of 20 patients treated had a complete response. There were six local failures: three with residual disease at the end of treatment and three with recurrent disease at a later date. Of the three with residual disease, one underwent abdominoperineal resection and two received salvage therapy (one with chemo-radiation and one with radiation alone). All three patients with recurrent disease were treated with abdominoperineal resection. All six were disease free at the end of the study. Of the 14 patients with complete local response, one presented with liver metastases 19 months later. Sixteen patients (80 percent) were alive at the end of the study, and 19 patients (95 percent) had no evidence of disease. These data add support for salvage therapy in the treatment of patients with residual disease following initial chemo-radiation therapy. Salvage options for patients with squamous-cell carcinoma of the anus who fail the Nigro protocol will be discussed.

Adult↗
PubMed1991

Structure and function of inositol trisphosphate receptors.

Inositol 1,4,5-trisphosphate (Ins(1,4,5)P3) is a soluble intracellular messenger formed rapidly after activation of a variety of cell-surface receptors that stimulate phosphoinositidase C activity. The initial response to Ins(1,4,5)P3 is a rapid Ca2+ efflux from nonmitochondrial intracellular stores which are probably specialized subcompartments of the endoplasmic reticulum, although their exact identities remain unknown. This initial response is followed by more complex Ca2+ signals: regenerative Ca2+ waves propagate across the cell, repetitive Ca2+ spikes occur, and stimulated Ca2+ entry across the plasma membrane contributes to the sustained Ca2+ signal. The mechanisms underlying these complex Ca2+ signals are unknown, although Ins(1,4,5)P3 is clearly involved. The intracellular receptor that mediates Ins(1,4,5)P3-stimulated Ca2+ mobilization has been purified and functionally reconstituted, and its amino acid sequence deduced from its cDNA sequence. These studies demonstrate that the Ins(1,4,5)P3 receptor has an integral Ca2+ channel separated from the Ins(1,4,5)P3 binding site by a long stretch of residues some of which form binding sites for allosteric regulators, and some of which are substrates for phosphorylation. In this review, we discuss the ligand recognition characteristics of Ins(1,4,5)P3 receptors, and their functional properties in their native environment and after purification, and we relate these properties to what is known of the structure of the receptor. In addition to regulation by Ins(1,4,5)P3, the Ins(1,4,5)P3 receptor is subject to many additional regulatory influences which include Ca2+, adenine nucleotides, pH and phosphorylation by protein kinases. Many of the functional and structural characteristics of the Ins(1,4,5)P3 receptor show striking similarities to another intracellular Ca2+ channel, the ryanodine receptor. These properties of the Ins(1,4,5)P3 are discussed, and their possible roles in contributing to the complex Ca2+ signals evoked by extracellular stimuli are considered.

Animals↗
PubMed1991

Solubilization of rat liver inositol 1,4,5-trisphosphate receptor.

High affinity Ins(1,4,5)P3-binding sites of permeabilized hepatocytes are probably the ligand recognition sites of the receptors that mediate the effects of Ins(1,4,5)P3 on intracellular Ca2+ mobilization. We have now solubilized these sites from rat liver membranes in the zwitterionic detergent, CHAPS, and shown that the solubilized sites bind Ins(1,4,5)P3 with an affinity (Kd = 7.26 +/- 0.52 nM, Hill coefficient h = 1.05 +/- 0.06) similar to that of the sites in native membranes (Kd = 6.02 +/- 1.57 nM, h = 0.99 +/- 0.02). ATP and a range of inositol phosphates (Ins(2,4,5)P3 Ins(4,5)P2, and inositol 1,4,5-trisphosphorothioate) also bound with similar affinities to the native and solubilized sites. Solubilization of the liver InsP3 receptor will allow its further characterization, purification, and comparison of its properties with those of InsP3 receptors already purified from cerebellum and smooth muscle.

Animals↗
PubMed1991

Different mechanisms of decreased drug accumulation in doxorubicin and mitoxantrone resistant variants of the MCF7 human breast cancer cell line.

We selected two drug resistant variants of the MCF7 human breast cancer cell line by chronic in vitro exposure to doxorubicin (MCF7/D40 cell line) and mitoxantrone (MCF7/Mitox cell line), respectively. The cell lines are similar in growth characteristics including doubling time, DNA synthetic phase and cell size. Resistance to mitoxantrone conferred only partial resistance to doxorubicin; whereas resistance selected for doxorubicin appeared to confer complete resistance to mitoxantrone. Both agents selected for cross resistance to the Vinca alkaloids. MCF7/D40 cells display a classic-multi-drug resistance phenotype with expression of P-glycoprotein, decreased drug accumulation relative to the parental line and reversal of drug accumulation and drug resistance by verapamil. MCF7/Mitox cells likewise display resistance to multiple drugs, but in contrast to MCF7/D40 cells do not express P-glycoprotein by immunoblot or RNA blot analysis. Net drug accumulation in MCF7/Mitox cells was decreased relative to the parental cells but there was no selective modulation of drug accumulation or in vitro drug resistance by the addition of verapamil. Efflux of mitoxantrone was enhanced in both the MCF7/D40 and MCF7/Mitox cell lines relative to the MCF7/S cell line. We conclude that the two drug resistant cell lines have different mechanisms of decreased drug accumulation.

ATP Binding Cassette Transporter, Subfamily B, Mem↗