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Biomedical subjects

J C White

Publications and source records attributed to J C White.

At least 91 records · Page 5Linked to original sources
PubMed1987

Membrane transport influences the rate of accumulation of cytosine arabinoside in human leukemia cells.

The role of membrane transport in the cellular accumulation of 1-beta-D-arabinofuranosylcytosine (ara-C) was studied in freshly isolated human acute leukemia cells. Patient cells had low rates for ara-C transport as compared with human and murine experimental cells and correspondingly low binding capacities for the nucleoside transport inhibitor, nitrobenzylmercaptopurine riboside (NBMPR). At 1 microM ara-C, the rate of net cellular accumulation was close to the membrane transport rate, and NBMPR inhibited transport and accumulation to the same extent. The rate of ara-C accumulation was half maximal at only 3-5 microM, a level much lower than that required for murine cells (67-85 microM). At concentrations below 1 microM the rate of ara-C accumulation was determined primarily by the transport rate, but at higher concentrations above 10 microM, phosphorylation capacity was the principal determinant of the net uptake rate. This difference in the role of transport at high and low ara-C concentrations may explain, in part, the efficacy of high-dose ara-C in patients refractory to standard dose protocols.

Acute Disease↗
PubMed1986

Membrane ordering effects of the anticancer agent VM-26.

The effect of the anticancer agent VM-26 on acyl chain order of cellular and model membranes was examined by electron spin resonance techniques. The order parameter for the paramagnetic probe 5-doxyl stearate was increased when VM-26 was incorporated into the bilayer of fluid-phase dimyristoylphosphatidylcholine (DMPC) or gel-phase dipalmitoylphosphatidylcholine (DPPC) liposomes at concentrations up to 4.8 mol%. The ordering effect of VM-26 in DMPC was greater than that of cholesterol on an equimolar basis. The less cytotoxic congener of VM-26, VP-16, was only one-third as active as VM-26 in its ordering effects on DMPC. Higher order parameters for 5-doxyl stearate were also noted in asolectin liposomes, Ehrlich ascites tumor cells, and CCRF-CEM cells treated with VM-26. We conclude that VM-26 has significant membrane associated activity in addition to its previously recognized nuclear effects.

1,2-Dipalmitoylphosphatidylcholine↗
PubMed1986

Teniposide-induced changes in the physical properties of phosphatidylcholine liposomes. A calorimetric study.

The anticancer agent teniposide has significant effects on plasma membrane components, in addition to its well known nuclear effects. Alterations in the properties and function of cellular membranes by various amphipathic compounds have been attributed previously to their relatively non-specific interactions with membrane components. We have examined the interaction of teniposide with defined model membranes by monitoring drug-induced changes in the melting profile of phospholipids by differential scanning calorimetry. The main phase transition temperature of dimyristoyl- or dipalmitoylphosphatidylcholine was lowered and broadened by the presence of teniposide in the liposomes. These effects were essentially linear over the concentration range of 1-5 mole %. The calorimetric enthalpy of the gel to liquid-crystalline transition of the phospholipids was not changed by the addition of the drug. The characteristic pretransition of these saturated phospholipids was decreased by teniposide concentrations as low as 0.1 mole % and was abolished at teniposide concentrations greater than 1 mole %. The data confirm the lipophilic nature of teniposide and indicate that the non-specific interactions with membrane lipids should be considered when evaluating the membrane-related effects of this agent.

Antineoplastic Agents↗
PubMed1986

Correlation of 1a afferent conduction with the ataxia of Fisher syndrome.

The pathophysiology of the ataxia in Fisher syndrome (the syndrome of acute ophthalmoplegia, ataxia, and areflexia) has been attributed to both peripheral nervous system pathology and cerebellar system dysfunction. Pathologic studies have demonstrated no consistent central nervous system abnormalities. We present a case in which abnormalities of 1a sensory conduction were found to correlate directly with the degree of ataxia, without impairment of motor or cutaneous sensory conduction. We propose that the ataxia seen in typical Fisher syndrome may be due to demyelination of 1a afferent fibers.

Adult↗
PubMed1986

Impairment of antibacterial defense mechanisms of the lung by extrapulmonary infection.

To determine whether extrapulmonary infection alters antibacterial defenses of the lung, we challenged mice with peritonitis due to Escherichia coli by aerosol inhalation with either Staphylococus aureus or Pseudomonas aeruginosa. In animals without peritonitis, 14% +/- 5% and 11% +/- 1% of the initially deposited viable S. aureus and P. aeruginosa, respectively, remained in the lungs at 4 hr. In contrast, in mice with peritonitis, at 4 hr 45% +/- 9% of the staphylococci were recoved, and the P. aeruginosa had increased to 948% +/- 354% of the initial inoculum. Proliferation of P. aeruginosa in mice with peritonitis was associated with impaired recruitment of polymorphonuclear neutrophils (PMNs) into the lungs. In contrast, a noninfectious stimulus induced more PMNs into the peritoneal cavity than did intraabdominal sepsis but only minimally impaired PMN recruitment into the lungs after aerosol challenge with P. aeruginosa. Sterile intraperitoneal stimulation did not significantly impair intrapulmonary killing of P. aeruginosa. Levels of antigenic C3 and functionally active C5 were significantly depleted in mice with peritonitis due to E. coli. We conclude that the systemic effects of sepsis, including complement depletion, contribute to the decreased pulmonary PMN recruitment and to impaired intrapulmonary bacterial killing of animals with peritonitis due to E. coli.

Animals↗
PubMed1985

Methylprednisolone impairs the bactericidal activity of alveolar macrophages.

Corticosteroid treatment of patients following acid aspiration has been reported to increase the incidence of bacterial pneumonia, with Staphylococcus aureus being a common isolate. We hypothesized that administration of methylprednisolone (MP) to mice with acid-injured lungs would impair pulmonary clearance of S. aureus by compromising the bactericidal oxidative metabolism of pulmonary phagocytes. Using an inhalational bacterial challenge, we established that MP decreased pulmonary clearance of S. aureus. In mice with normal lungs and without MP treatment 14 +/- 2% of all initially deposited Staphylococci remained at 4 hr compared to 28 +/- 2% remaining in the lungs of mice with MP treatment. In mice with acid-injured lungs, MP caused a greater impairment of S. aureus clearance at 4 hr with 44 +/- 10% of all initially deposited bacteria remaining in the lungs of control mice and 210 +/- 24% remaining in the lungs of MP-treated mice. After it was demonstrated that there was no difference in the numbers of phagocytic cells obtained by lung lavage from mice with or without MP treatment, the bactericidal oxidative metabolism of these cells was quantitated using luminol-amplified chemiluminescence. Phagocytic cells from mice not exposed to S. aureus displayed minimal chemiluminescence whether they were treated with saline (22 +/- 14 mV) or MP (14 +/- 8 mV). In contrast, phagocytes from saline-treated mice exposed to S. aureus showed a significant increase in chemiluminescence (159 +/- 22 mV). Pretreatment with MP, however, prevented this response to S. aureus (21 +/- 13 mV), indicating that bactericidal oxidative metabolism of these phagocytic cells had been suppressed.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1985

Immunochemistry, physical chemistry and biology of 2',5'-oligoadenylates.

Monoclonal antibodies directed against 2-5A were developed and characterized. 2',5'-Oligoadenylate-protein complexes possess at least three distinct antigenic surfaces defined primarily by the ribose-phosphate backbone. A schematic model for the three epitopes is presented. Antibodies directed against 2-5A, in conjunction with other techniques, were employed to quantify 2-5A in various tissues of pathogen-free mice. Levels of 2-5A were in the range of 400-800 fmole/gm. Mice injected with poly(I).poly(c) or encephalomyocarditis virus (EMCV) showed elevated levels of 2-5A. Administration of poly(I).poly(C) or EMCV increased the level of 2-5A in different tissues to different extents. Raman spectroscopy was employed to explore the conformation of 2-5A. Distinct differences were observed in bands arising from the backbone portion of 2-5A relative to those of 3-5A. The most striking finding was the appearance of a strong, sharp band at 1460 cm-1 in spectra of 5'-monophosphorylated 2-5A's. This band was barely detectable in core and triphosphorylated 2-5A. Its presence suggests that 5'-monophosphorylated 2-5A's possess a unique conformational feature that distinguish them from cores and 5'-triphosphorylated forms.

Adenine Nucleotides↗
PubMed1985

Inhibition of 1-beta-D-arabinofuranosylcytosine transport and net accumulation by teniposide and etoposide in Ehrlich ascites cells and human leukemic blasts.

The interactions of the epipodophyllotoxins, teniposide (VM-26) and etoposide (VP-16), with the nucleoside carrier were examined with emphasis on their effects on 1-beta-D-arabinofuranosylcytosine (ara-C) transport and net accumulation. VM-26 inhibited ara-C transport by Ehrlich ascites cells within 1 min of exposure, and inhibition was only partially reversed after 45 min in VM-26-free medium. ara-C transport was slowed by 50% by 7 microM VM-26 or by 35 microM VP-16. Since epipodophyllotoxins were noncompetitive inhibitors, fractional inhibition was independent of the ara-C concentration. Analysis of ara-C transport kinetics revealed only a single saturable transport route, and there was no indication of VM-26-insensitive transport. VM-26, VP-16, and ara-C were competitive inhibitors of the specific binding of nitrobenzylthioinosine to the nucleoside carrier with Ki values of 7.4 microM, 23 microM, and 2.2 microM, respectively. The rate of dissociation of nitrobenzylthioinosine (t 1/2 = 20.6 min) was accelerated by 5 microM ara-C (t 1/2 = 18.5 min) but slowed by 100 microM VM-26 (t 1/2 = 34.6). By these criteria, the interaction of VM-26 with the nucleoside carrier was qualitatively similar to that of dipyridamole. Although VM-26 inhibited ara-C transport, it did not significantly slow the rate of net intracellular accumulation of ara-C by Ehrlich cells, presumably because transport capacity far exceeds the capacity for phosphorylation in these cells. In freshly isolated human leukemic blasts, which have far less nucleoside transport activity, inhibition of ara-C accumulation by VM-26 was dependent on the ara-C concentration. At 1 microM ara-C, a concentration where transport was rate limiting for net uptake, VM-26 inhibited accumulation of ara-C over a 60-min time course. At 50 microM ara-C, transport was in excess, and VM-26 did not slow ara-C metabolism.

Animals↗
PubMed1984

Binding of radiolabeled N-(phosphonacetyl)-L-aspartate to aspartate transcarbamylase from Ehrlich ascites tumor cells.

Binding of N-(phosphonacetyl)-[3H]L-aspartate (PALA) to aspartate transcarbamylase (ATCase, EC 2.1.3.1) in crude extracts from Ehrlich ascites tumor cells was examined. At pH 7.4, the dissociation constant was 1.39 +/- 0.22 nM; the maximal binding capacity indicated an average intracellular ATCase concentration of 0.13 microM. The presence of phosphate, MgCl2, or CaCl2 increased the apparent dissociation constant for [3H]PALA without altering the maximal binding capacity. Phosphate, a product of the ATCase reaction, probably acts as a competitor for the PALA binding site; Mg2+ and Ca2+ may inhibit [3H]PALA binding by forming a chelate which reduces the effective concentration of the free [3H]PALA. Carbamyl phosphate was a relatively weak inhibitor of [3H]PALA binding in N-2-hydroxyethylpiperazine-N'-2-ethanesulfonic acid (HEPES) buffer alone. Addition of NaF, an inhibitor of nonspecific phosphatases, decreased the carbamyl phosphate "Ki" as an inhibitor of [3H]PALA binding to 7 microM, a value close to the Km. NaF appears to act as an inhibitor of carbamyl phosphatase activity present in the cell extract. A first-order dissociation rate constant of 0.050 +/- 0.004 min-1 (T1/2 = 14 min) was determined by following displacement of [3H]PALA with excess unlabeled PALA. The dissociation rate was strongly temperature dependent. A second-order rate constant of 3.6 X 10(7) liters mol-1 min-1 was calculated from this rate constant and the dissociation constant. Using these kinetic constants, a simple computer model predicted that PALA binding to ATCase is 95% complete within 14 min under the conditions of the assay; at intracellular ATCase concentrations, binding is slightly faster. These results are discussed in the context of both the kinetics of inhibition and the reversal of inhibition of pyrimidine synthesis within the intact cell.

Animals↗
PubMed1984

Use of a new generation ophthalmoscope.

A new optical configuration for an ophthalmoscope is a hybrid of the present direct and indirect instruments, combining the strengths of both to increase overall efficiency. Cross polarization is used as a means of reducing glare and increasing clarity of detail. A fiberoptic light source increases light intensity, but reduces heat, weight, and inconvenience. These characteristics result in a self-contained, handheld indirect instrument. This ophthalmoscope is mechanically aligned and therefore easy to use. Both ophthalmologist and nonophthalmologist may find this instrument of benefit in ocular examinations.

Eye Burns↗
PubMed1984

Role of endocytosis and lysosomal pH in uptake of N-(phosphonacetyl)-L-aspartate and its inhibition of pyrimidine synthesis.

The mechanism of uptake and retention of N-(phosphonacetyl)-L-aspartate (PALA) was examined. Uptake of [3H]PALA by Ehrlich ascites tumor cells appeared to be biphasic. A small, variable quantity of PALA associated with cells within 5 min; the significance of this rapid uptake component is unclear. Between 15 min and 5 h, uptake was linear and consistent from experiment to experiment. The properties of the slow phase of PALA uptake are consistent with fluid-phase endocytosis. The intracellular PALA concentration approached the extracellular level very slowly, at a rate of approximately 1%/hr. The velocity of PALA uptake in these cells was proportional to the concentration in the media from 10(-6) to 10(-2) M. Uptake of PALA was identical to that of the extracellular marker inulin. Uptake of both PALA and inulin was inhibited by colchicine and stimulated by phorbol myristate acetate. The microtubule antagonist and the phorbol ester are known to, respectively, inhibit and stimulate endocytosis in other cell types. Phorbol myristate acetate enhanced the ability of PALA to inhibit incorporation of [14C]bicarbonate into pyrimidine nucleotides, presumably through an increase in PALA uptake. This inhibitory action of PALA was almost completely blocked by two agents known to neutralize lysosomal pH, NH4Cl and methylamine. These results suggest that intracellular PALA is initially compartmentalized in a pinosomal vesicle which may later fuse with cellular lysosomes. Neutralization of lysosomal pH prevents the protonation of some or all of the four negatively charged groups found in the structure of PALA which may be necessary for its diffusion across the lysosomal membrane and eventual inhibition of aspartate transcarbamylase in the cytoplasm. Since partitioning of the fully charged molecule into the lipid phase of the plasma membrane for diffusion out of the cell should be minimal, the effects of PALA on cellular metabolism are expected to be prolonged.

Animals↗
PubMed1983

Predictions of a network thermodynamics computer model relating to the mechanism of methotrexate rescue by 5-formyltetrahydrofolate and to the importance of inhibition of thymidylate synthase by methotrexate-polyglutamates.

Computer modeling has been a valuable tool for clarifying the mechanism of action of antifolates. Some consequences of folyl and antifolyl polyglutamate synthesis can be addressed by adaptation of a network thermodynamic computer model of methotrexate action. Reversal or prevention of methotrexate cytotoxicity by 5-formyltetrahydrofolate has widely been assumed to occur through the delivery of reduced folate in substrate amounts for thymidylate synthesis, by-passing the effects of methotrexate at dihydrofolate reductase. This mechanism is inconsistent with experimental data which shows that "rescue" is a competitive phenomenon and that the transport process is incapable of delivering reduced folate at an adequate rate. Computer modeling studies are presented which predict that expansion of the total folate pool as folylpolyglutamates with "rescue" would reduce the inhibitory effect of MTX on thymidylate synthesis. Dihydrofolate polyglutamates could then accumulate to the high level needed to displace methotrexate from the small fraction of sites on dihydrofolate reductase that are sufficient to sustain tetrahydrofolate synthesis. Experimental studies with Ehrlich ascites tumor cells support this prediction. It is likely that a critical step in the protection of normal host tissues in high dose-rescue treatment regimens is the conversion of exogenously supplied 5-formyltetrahydrofolate to polyglutamyl derivatives and accumulation of total intracellular folate to higher than normal levels. Other computer simulations are presented which examine the potential significance of direct inhibition of thymidylate synthase by polyglutamyl forms of methotrexate. The model predicts that in cells with biochemical properties similar to methotrexate sensitive L1210 cells, inhibition of dihydrofolate reductase would still be the predominant site of action unless the thymidylate synthase Ki for a methotrexate polyglutamate is below about 0.1 microM. However, in methotrexate-resistant cells with elevated dihydrofolate reductase but normal membrane transport and polyglutamylation, thymidylate synthase may be the more important target enzyme.

Computers↗