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Biomedical subjects

R F Murphy

Publications and source records attributed to R F Murphy.

At least 127 records · Page 7Linked to original sources
PubMed1988

A chloroquine-resistant Swiss 3T3 cell line with a defect in late endocytic acidification.

To investigate the role of acidification in cell proliferation, several cell lines resistant to chloroquine were isolated with the expectation that some would express altered endocytic acidification. The preliminary characterization of one of these lines, CHL60-64, is described. In contrast to endocytic mutants described previously, the initial phase of endocytic acidification, as measured by transferrin acidification, is normal in this cell line. However, a difference in subsequent endocytic acidification was observed in CHL60-64. In the parental cells, internalized dextran was fully acidified to approximately pH 5.5 within 1 h. In CHL60-64, the pH in the endocytic compartment was only 6.1 after 1 h and remained as high as 5.8 for at least 4 h. After an 8-h incubation, the pH decreased to 5.5, indicating that the second phase of acidification is only slowed in CHL60-64, and not blocked. Consistent with this retarded acidification, ATP-dependent acidification in vitro (as measured by acridine orange accumulation) was reduced in both the lysosomal fraction and the endosomal fraction isolated from CHL60-64. A decrease in the in vivo rate of acridine orange accumulation after perturbation with amine was also observed. In addition to amine resistance and defective acidification, CHL60-64 was found to be resistant to vacuolation in the presence of chloroquine and ammonium chloride, and was resistant to ouabain. Further studies on this new class of endocytosis mutant, in combination with existing mutants, should help to clarify the mechanisms responsible for the regulation of endocytic acidification.

Acid-Base Equilibrium↗
PubMed1988

The relationship between blood gastrin levels and gastric secretion in conscious dogs.

1. In conscious dogs with gastric fistulae and Heidenhain pouches, acid and pepsin secretion, immunoreactive plasma pancreatic polypeptide (PP) and gastrin were measured following intravenously administered pentagastrin, cholinomimetics, and intragastric administration of milk. 2. Pentagastrin did not raise endogenous plasma gastrin. There was a significant positive dose-response relationship between pentagastrin and acid and fistula pepsin secretions, but not between plasma gastrin of endogenous origin and gastric secretion. 3. Carbachol raised plasma gastrin immunoreactivity; but in no instance was there a significant relationship between gastric secretion and plasma gastrin immunoreactivity. Gastric acid secretion faded, but plasma gastrin concentrations did not. 4. PP plasma immunoreactivity was elevated by methacholine and carbachol. Its levels correlated significantly with gastric acid secretion. Pentagastrin did not raise PP and its levels did not, therefore, correlate with gastric acid secretion. 5. Intragastric milk raised plasma gastrin immunoreactivity, but the acid secretion per pg of plasma gastrin was much smaller than with the cholinomimetics. Ganglionic blocking agents depressed both plasma gastrin and acid and pepsin secretion. 6. The results suggest that cholinomimetics sensitize parietal cells to the stimulating action of gastrin.

Animals↗
PubMed1988

Bioavailability of oral trimetrexate in patients with acquired immunodeficiency syndrome.

The combination of the lipophilic antifolate trimetrexate and the rescue agent leucovorin has shown promise in the treatment of Pneumocystis carinii pneumonia in patients with acquired immunodeficiency syndrome. The pharmacokinetic behavior of trimetrexate administered either by intravenous bolus or orally was studied in six patients with acquired immunodeficiency syndrome with a reversed-phase high-pressure liquid chromatography assay. The mean clearance following bolus injection was 38 ml/min per m2, with a range of 15 to 55 ml/min per m2. The postdistributive half-life ranged from 6 to 16 h. With oral administration, the mean bioavailability was 44% (range, 19 to 67%). An oral dose of 60 mg/m2 (162 mumol/m2) resulted in concentrations in plasma that approximated those achieved with a 30-mg/m2 (81-mumol/m2) intravenous dose. The toxicity of this combination regimen was minimal. It appears that the oral route is a practical route of administration for trimetrexate in patients with acquired immunodeficiency syndrome requiring long-term outpatient treatment or prophylaxis for P. carinii pneumonia.

Acquired Immunodeficiency Syndrome↗
PubMed1988

Pharmacokinetics of subcutaneous methotrexate.

The pharmacokinetics of subcutaneously administered methotrexate was studied as a parenteral alternative to oral administration. An initial feasibility study was performed in Rhesus monkeys comparing the subcutaneous route to intravenous (IV) injection and oral administration. The subcutaneous dose was completely absorbed and a sustained-release effect was observed when compared with the IV dose. No local or systemic toxicities resulted from subcutaneous methotrexate in the animals. Twelve children with acute lymphoblastic leukemia on maintenance therapy protocols prescribing either 7.5 mg/m2 biweekly or 40 mg/m2 weekly were also monitored after both a subcutaneous and an oral dose of methotrexate. Four children at the higher dosage level were also studied after an equal IV dose. The subcutaneous dose was again completely absorbed in these children at both dose levels, whereas the oral dose, which produced comparable plasma drug concentrations at the lower dosage level, resulted in a total drug exposure (area under the plasma concentration-time curve) that was one third that of the equal subcutaneous dose at the higher dosage level. No local or systemic toxicity was attributed to the subcutaneous methotrexate. Subcutaneous administration of methotrexate is well tolerated and well absorbed and appears to overcome the problems associated with oral administration, including variable absorption and saturation of the absorption mechanism with increasing doses.

Absorption↗
PubMed1987

A modified spectrofluorometric assay for DNA using adriamycin.

A spectrofluorometric assay for DNA is described. The changes in the fluorescence of adriamycin when it binds to DNA may be quantitated in terms of the amount of DNA present. The assay is specific and reproducible, being free from interference from RNA, protein, and sample turbidity.

Breast Neoplasms↗
PubMed1987

Flow cytometric DNA analysis of adrenocortical tumors in children.

Flow cytometric DNA analysis of isolated nuclei was performed on 14 lesions occurring in ten children with adrenocortical tumors. Unimodal DNA content distributions were obtained from seven tumors occurring in patients without metastases 2 to 18 years after diagnosis. Abnormal DNA contents were detected in all four primary lesions, which subsequently metastasized, and in the tumor of one patient who was followed for less than 2 years. Paraffin and frozen preparations were virtually identical, as were the analyses of the primary, recurrent, and metastatic disease occurring in one patient. These observations suggest that DNA content abnormalities detected by flow cytometry correlate with metastases, and may provide an objective measure of the biologic potential of these tumors.

Adrenal Gland Neoplasms↗
PubMed1987

Kinetics and temperature dependence of exposure of endocytosed material to proteolytic enzymes and low pH: evidence for a maturation model for the formation of lysosomes.

The temperature dependence of acidification of internalized dextran by Swiss 3T3 cells was determined using dual fluorescence flow cytometry. Essentially no acidification was observed at 11 degrees C; acidification was limited to pH 6-6.5 at temperatures between 13 degrees C and 17 degrees C. In contrast, a rapid drop to pH 6-6.5 followed by acidification to pH 5-5.5 was observed at temperatures above 19 degrees C. These results confirm the biphasic nature of the acidification process (J. Cell Biol. (1984) 98: 1757-1762). The timing of exposure of material internalized by fluid-phase endocytosis to lysosomal enzymes was determined for Swiss 3T3 cells by using a fluorogenic substrate specific for Cathepsin B. Hydrolysis of the substrate, as measured by both fluorometry and flow cytometry, began within minutes of its addition to cells at 37 degrees C, and was inhibited by coincubation with leupeptin, a competitive inhibitor of the enzyme, or by weak bases, which raise the pH of acidic compartments. At temperatures between 13 degrees (and 21 degrees C, the rate of hydrolysis was reduced to 31-44% of that at 37 degrees C. Thus, in contrast to previous reports, exposure of endocytosed material to at least one lysosomal enzyme is not inhibited below 20 degrees C; the reduction in hydrolysis rate may be explained by the temperature effects on the efficiency of the enzyme. The results for acidification and proteolysis are consistent with, but do not prove, a maturation model for the formation of lysosomes. We suggest that at lower temperatures, part of the maturation involving recycling and/or concentration of the contents of the endosome is inhibited. This causes the endosome to remain as a mildly acidic, low-density organelle containing lysosomal enzymes.

Animals↗
PubMed1987

The regulation of basal pancreatic polypeptide levels in dogs.

In response to various stimuli, pancreatic polypeptide (PP) release is predominantly mediated by cholinergic mechanisms, and may be modulated by sympathetic and opiate (inhibitory) effects. However, the mechanisms regulating basal PP levels remain unclear. We examined the possible role of the sympathetic nervous system and endogenous opiates in the regulation of basal levels of pancreatic polypeptide in trained conscious dogs. During prolonged (150 min) alpha- or beta-adrenergic blockade with phentolamine and propranolol, separately or in combination, there was no change in the basal PP levels of 154 +/- 20 pg/ml. Effective adrenergic modulation of pancreatic hormones was evident since alpha blockade led to a rise in insulin and glucagon, beta blockade led to a fall in insulin and glucagon, while combined alpha- and beta-adrenergic blockade did not affect insulin or glucagon. Opiate blockade with naloxone (1.25 mg followed by 1 microgram/kg/min) led to a delayed fall in PP from 153 +/- 22 to 89 +/- 15 pg/ml at 90 min (no change by 30 min), without a change of insulin or glucagon. Infusion of a potent morphine analogue D-Met2-Pro5-enkephalinamide (0.5 microgram/kg/min) led to a sustained fall in PP to 91 +/- 8 pg/ml by 30 min without a change in insulin or glucagon. Somatostatin infusion (0.2 microgram/kg/min) with insulin and glucagon replacement, led to a similar sustained fall in PP. It is concluded that in dogs: in contrast to insulin and glucagon, at basal conditions the plasma level of PP is not modulated by endogenous alpha- or beta-adrenergic influences.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1987

The effect of methotrexate on the bioavailability of oral 6-mercaptopurine.

Fourteen children (aged 3 to 14 years) with average-risk acute lymphoblastic leukemia were studied after an oral dose of 6-mercaptopurine (6-MP) (75 mg/m2) administered alone and, on the next day, concurrently with oral methotrexate (20 mg/m2). When 6-MP was administered alone, both the peak plasma concentration (15 to 150 ng X ml-1) and the AUC (36 to 340 ng X ml-1 X hr) were highly variable. Concurrent methotrexate resulted in a 31% increase in the AUC (P less than 0.01) and a 26% increase in peak plasma levels (P less than 0.05) of 6-MP. The AUC of methotrexate correlated with the degree of increase in 6-MP plasma concentrations. These findings are consistent with previous in vitro studies demonstrating that methotrexate is an inhibitor of xanthine oxidase, the enzyme that catabolizes 6-MP to the inactive metabolite thiouric acid. Although the increases in 6-MP AUC and peak plasma concentrations resulting from concurrent methotrexate administration were statistically significant, this interaction is probably not clinically significant at standard low oral doses of methotrexate in light of the wide interpatient variability in these pharmacokinetic parameters of 6-MP.

Administration, Oral↗
PubMed1987

High-resolution kinetics of transferrin acidification in BALB/c 3T3 cells: exposure to pH 6 followed by temperature-sensitive alkalinization during recycling.

The kinetics of acidification of diferric human transferrin in BALB/c mouse 3T3 cells were determined by flow cytometry using a modification of the fluorescein-rhodamine fluorescence ratio technique. For cells labeled at 0 degrees C and warmed to 37 degrees C, the minimum pH observed was 6.1 +/- 0.2, occurring 5 min after warmup. This step was followed by a slower alkalinization to the pH of the external medium, occurring with a half-time of 5 min. Warmup to 24 degrees C or 17 degrees C resulted in slowing of the time of onset of acidification such that the minimum pH was 6.3 +/- 0.2, attained 15 and 25 min after warmup, respectively; the alkalinization step was completely blocked. The limited acidification observed for transferrin corresponds to the initial phase of acidification normally observed for other (nonrecycled) ligands. Since transferrin is not further acidified, the results confirm the existence of two phases of acidification during endocytosis. Measurements of transferrin dissociation at neutral pH after exposure to mildly acidic pH support the conclusion that the transferrin cycle may be completed without exposure of transferrin to a pH below 6. The mildly acidic pH of the endocytic compartments involved in recycling may play a role in regulating enzymatic processing of endocytosed material.

Animals↗
PubMed1987

Apparent endocytosis of fluorescein isothiocyanate-conjugated dextran by Saccharomyces cerevisiae reflects uptake of low molecular weight impurities, not dextran.

Concurrent with Riezman's report (Riezman, H. 1985, Cell. 40:1001-1009) that fluid-phase endocytosis of the small molecule Lucifer yellow occurs in the yeast Saccharomyces cerevisiae, Makarow (Makarow, M. 1985. EMBO [Eur. Mol. Biol. Organ.] J. 4:1861-1866) reported the endocytotic uptake of 70-kD FITC-dextran (FD) and its subsequent compartmentation into the yeast vacuole. Samples of FD synthesized and purified here failed to label yeast vacuoles under conditions that allowed labeling using commercial FD. Chromatography revealed that the commercial FD was heavily contaminated with at least three low molecular weight fluorescent compounds. Dialysis was ineffective for removing the contaminants. After purification (Sephadex G25, ethanol extraction), commercial FD was incapable of labeling vacuoles. Extracts of cells labeled with partially purified FD contained FITC, not FD, based on Sephadex and thin layer chromatography. In either the presence or absence of unlabeled 70-kD dextran, authentic FITC (10 micrograms/ml) was an effective labeling agent for vacuoles. The rapid kinetics (0.28 pmol/min per 10(6) cells at pH 5.5) and the pH dependence of FITC uptake suggest that the mechanism of FITC uptake involves diffusion rather than endocytosis. In view of these results, labeling experiments that use unpurified commercial FD should be interpreted with caution.

Antigens↗
PubMed1987

Stability of 5-fluorouracil in whole blood and plasma.

We studied the stability of 5-fluorouracil (5-FU) in plasma and whole blood kept at room temperature and on ice for 1 to 24 h. At room temperature, there was a steady loss of 94% of the parent drug over 24 h in whole blood and 52% in plasma. In the presence of an excess of uracil, 5-FU was stable for 24 h, suggesting that the loss of 5-FU is the result of enzymatic degradation. 5-FU is more stable in whole blood and plasma when samples are kept cold. For blood and plasma samples maintained on ice, the loss was only 30% and 10% of the parent drug in the respective samples over 24 h. Frozen plasma samples (-20 degrees C) were stable for five weeks. Blood specimens collected for quantifying 5-FU should be immediately placed on ice, and the plasma should be separated and frozen as promptly as possible.

Drug Stability↗
PubMed1987

Synthesis of neuropeptide Y.

Neuropeptide Y (NPY), a hexatriacontapeptide amide, was synthesized on benzhydrylamine resin. The peptide product obtained by HF treatment contained 63% of the target peptide, NPY. A comparison of the chemical, immunochemical and biological properties of the synthetic peptide with natural NPY indicated that they were identical.

Amino Acid Sequence↗
PubMed1986

Enhancement of methotrexate cytotoxicity towards the MDA.MB.436 human breast cancer cell line by dipyridamole. The role of methotrexate polyglutamates.

MTX and dipyridamole are synergistic in their toxicity towards the MDA.MB.436 human breast cancer cell line. Dipyridamole increases net MTX uptake into the cells and increases the intracellular levels of MTXG7 to G10, the highest molecular weight polyglutamyl derivatives of MTX detected. During a recovery period, after completion of exposure to MTX with and without dipyridamole, levels of MTXG7 to G10 remained elevated in dipyridamole treated cells by comparison with controls. Dipyridamole, which has no intrinsic effect on cell growth, transforms a cytostatic response of MDA.MB.436 cells towards MTX into a cytotoxic response. The effect of dipyridamole is not mediated through an increase in prostacyclin biosynthesis.

Arachidonic Acid↗
PubMed1986

Interaction of synthetic peptides of apolipoprotein C-II and lipoprotein lipase at monomolecular lipid films.

The triacylglycerol hydrolyase and phospholipase A1 activities of bovine milk lipoprotein lipase toward long-chain fatty acyl ester substrates were investigated with monomolecular lipid films containing trioleoylglycerol and phosphatidylcholine. In a monolayer of egg phosphatidylcholine containing 3 mol% [14C]trioleoylglycerol, and in the presence of apolipoprotein C-II, a 79 amino acid activator protein for lipoprotein lipase, enzyme activity was maximal at a surface pressure of 21-22 mN X m-1 (37 mumol oleic acid released/h per mg enzyme); enzyme activity was enhanced 9-fold by apolipoprotein C-II. At surface pressures between 22 and 30 mN X m-1, lipoprotein lipase activity decreased over a broad range and was nearly zero at 30 mN X m-1. Apolipoprotein C-II and the synthetic fragments of the activator protein containing residues 56-79, 51-79 and 44-79 were equally effective at 20 mN X m-1 in enhancing lipoprotein lipase catalysis. However, at surface pressures between 25 and 29 mN X m-1, only apolipoprotein C-II and the phospholipid-associating fragment containing residues 44-79 enhanced enzyme catalysis. The effect of apolipoprotein C-II and synthetic peptides on the phospholipase A1 activity of lipoprotein lipase was examined in sphingomyelin:cholesterol (2:1) monolayers containing 5 mol% di[14C]myristoylphosphatidylcholine. At 22 mN X m-1, apolipoprotein C-II and the synthetic fragments containing residues 44-79 or 56-79 enhanced lipoprotein lipase activity (70-80 nmol/h per mg enzyme). In contrast to trioleoylglycerol hydrolysis, the synthetic fragments were not as effective as apolipoprotein C-II enhancing enzyme activity towards di[14C]myristoylphosphatidylcholine at higher surface pressures. We conclude that the minimal amino acid sequence of apolipoprotein C-II required for activation of lipoprotein lipase is dependent both on the lipid substrate and the packing density of the monolayer.

Amino Acids↗
PubMed1986

Cell-by-cell autofluorescence correction for low signal-to-noise systems: application to epidermal growth factor endocytosis by 3T3 fibroblasts.

Autofluorescence of cells can be a major portion of the fluorescence signal in many systems, especially when fluorescent conjugates are used to study receptor-ligand systems for which there are less than 70,000 receptors per cell. We have devised a method for the cell-by-cell correction of autofluorescence for flow cytometric data by using an additional parameter to measure and correct for autofluorescence in the fluorescence channel. The principle has been extended to allow simultaneous correction for autofluorescence and dual-fluorescence spillover compensation in samples labeled with two different fluorochromes; all corrections were done in software, making them applicable to any flow cytometer. The autofluorescence correction method was used to analyze the acidification of epidermal growth factor (EGF) by Swiss 3T3 cells. EGF is acidified to pH 6.2 starting two min after labeling, with a half-time for acidification of 45 s.

Acids↗
PubMed1986

Growth inhibition of 3T3 fibroblasts by lysosomotropic amines: correlation with effects on intravesicular pH but not vacuolation.

The effects of five lysosomotropic amines on the growth of Swiss 3T3 fibroblasts were measured and compared with effects on intravesicular pH. Tributylamine and benzylamine, amines that affect intravesicular pH without causing vacuolation, were found to inhibit cell growth to a similar extent as vacuologenic amines previously tested. Excellent correlation between the half-maximal concentrations for the growth and pH effects were found for tributylamine, benzylamine, chloroquine, and ammonium chloride. The results suggest that growth inhibition by these amines is a direct result of their effects on pH and not due to other effects (such as vacuolation). In contrast, a 100-fold difference in the half-maximal concentrations was found for methylamine, suggesting that methylamine inhibits growth by a mechanism unrelated to pH.

Amines↗