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D P Thompson

Publications and source records attributed to D P Thompson.

At least 73 records · Page 4Linked to original sources
PubMed1990

Biophysical transport properties of the cuticle of Ascaris suum.

The transport properties of isolated cuticle from Ascaris suum were studied using standard two-chamber diffusion cells and a number of radiolabeled permeants which varied in molecular size, lipophilicity and electrical charge. The permeability coefficient of the collagen matrix (lipid-extracted cuticle) vs. molecular radius relationship showed the interdependence of molecular size and electrical charge of the permeants with respect to the aqueous pores of the negatively charged matrix. The permeability of neutral solutes decreased monotonically with size. Protonated amines permeated the aqueous pores faster than neutral solutes of comparable size, while the permeation of anions was slower. The average pore size was estimated to be 1.5 nm in radius. A biophysical model which accounted for diffusion of molecules within a fixed electrostatic field of force and for molecular sieving by the pore channels was used in the mechanistic interpretation of the data. The effective permeability coefficient of the non-lipid-extracted cuticle was delineated into the permeability coefficients of the water-filled collagen matrix and the lipoidal component of the cuticle to determine which layer was the rate-controlling barrier. While each solute was capable of penetrating the water-filled collagen matrix, the rate-determining step for the majority of compounds was passive diffusion across the lipid component, which controlled 75-99% of transport. The exception was water, for which transport kinetics was 75% matrix-controlled. In general, permeation across the lipid-filled tissue was more favorable for small lipophilic compounds because of molecular restriction not only in the aqueous pores, but also in the lipid-filled pores.

Animals↗
PubMed1989

The Madin Darby canine kidney (MDCK) epithelial cell monolayer as a model cellular transport barrier.

Two strains of Madin Darby canine kidney (MDCK) cells were grown on a polycarbonate membrane with 3-micron pores without any extracellular matrix treatment. The membrane, 2.45 cm in diameter, which is part of a commercially obtained presterilized culture insert, provides two chambers when placed in a regular six-well culture plate. This device was found to be convenient for investigating transport of a few selected fluid-phase markers across the MDCK cell monolayer. Both the strain from the American Type Culture Collection (ATCC) and the so-called highly resistant strain I, at a serial passage between 65 and 70, showed a seeding concentration-dependent lag phase followed by a growth phase with a 21-hr doubling time. When seeded at 5 x 10(4) cells/cm2, cell confluence was achieved in 5 days in a modified Eagle's minimum essential medium (MEM) containing 10% fetal bovine serum under a 5% CO2 atmosphere. Similarly, transepithelial electrical resistance (TEER) also reached a plateau value in 5 days. Both light and electron microscopic examinations revealed well-defined junctional structures. Transport of the fluid-phase markers, sucrose, lucifer yellow CH (LY), inulin, and dextran across the MDCK cell monolayers was studied primarily at 37 degrees C following the apical-to-basolateral as well as the basolateral-to-apical direction. Large variations in the steady-state transport rate were observed for a given marker between the cell layer preparations. Thus, the present study proposes an "internal standard" procedure for meaningful comparisons of the transport rate. When normalized to the rate of sucrose, the rate ratio was 1.00:0.80:0.67:0.15 for sucrose:LY:inulin:dextran.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1989

Neutrophil-mediated transport of liposomes across the Madin Darby canine kidney epithelial cell monolayer.

Targeted drug delivery to peripheral blood neutrophils (PMNs) should be of therapeutic potential in various disease states. In addition, substances taken up by PMNs in the circulation may be delivered to an extravascular site via the naturally occurring cell infiltration. The present study employs an in vitro chemotaxis model to test whether particulate drug carriers such as liposomes can be transported across a cellular barrier by migrating PMNs. The system contained 10(7) human PMNs/ml, 0.3-micron liposomes at a total lipid concentration of 2.5 mM, and 10% autologous human serum in the apical side of a confluent Madin Darby canine kidney (MDCK) epithelial cell monolayer of 4.71 cm2. The MDCK cells were grown on a polycarbonate membrane with 3-micron pores without any extracellular matrix, and 10(-7) M f-Met-Leu-Phe was added to the basolateral side as a trigger of chemotaxis. The aqueous phase of the reverse-phase evaporation vesicles (REVs) contained lucifer yellow CH (LY) and [14C]sucrose. The lipid bilayer of the REVs was spiked with [3H]dipalmitoylphosphatidylcholine (DPPC). Transmission electron micrographs showed that, in response to the formyl peptide, PMNs adhered to the apical surface of MDCK cells, emigrated across the MDCK cell layer, passed through the 3-micron pores in the polycarbonate membrane, and finally, appeared in the bottom well. Epifluorescence micrographs showed that most, if not all, of the migrated PMNs contained punctate fluorescence derived from LY. Transport data over a 3.5-hr period indicated that those markers that appeared in the basal side were indeed transported by phagocytosis of REVs by PMNs and that intact serum was an essential component in the process.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1989

Mechanisms of drug resistance to inhibitors directed at the individual subunits of ribonucleotide reductase.

Ribonucleotide reductase consists of two non-identical subunits, non-heme iron subunit (NHI) and effector-binding subunit (EB), that are encoded by different genes and that can be specifically and independently inhibited. L1210 cell lines were selected for resistance to hydroxyurea (HU-7), deoxyadenosine (Y-8), or the combination of pyrazoloimidazole and deoxyadenosine (ED2). The gene for the NHI was amplified in the HU-7, Y-8, and ED2 cell lines; there was no amplification of the gene for the EB. The mRNA for the NHI was increased in the HU-7, Y-8, and ED2 cells, but there was no change in the mRNA levels for the EB. Reductase activity was increased in the HU-7, ED2 cells, but not in the Y-8 cells. The reductase activities in the Y-8 and the ED2 cells were not subject to feedback inhibition by dATP. These data show that the mechanisms of resistance to inhibitors directed at this reductase are varied and do not require increased enzyme activity. Further, gene amplification or increased mRNA levels did not necessarily result in increased levels of cellular enzyme.

Animals↗
PubMed1989

Changes in messenger RNA levels for the subunits of ribonucleotide reductase during the cell cycle of leukemia L1210 cells.

Ribonucleotide reductase consists of two non-identical protein subunits that are required for enzyme activity. These subunits are encoded by different genes and are not expressed coordinately as the cells pass through the cell cycle. Using specific cDNAs for the non-heme iron (NHI) and the effector-binding (EB) subunits the levels of the mRNAs for these two subunits were determined in leukemia L1210 cells during the transition from the G0/G1 phase to the S and G2/M phases of the cell cycle. Synchronized populations of L1210 cells were obtained either by ethylene glycol-bis(beta-aminoethyl ether) N,N,N',N'-tetraacetic acid (EGTA) treatment or by enrichment by elutriation centrifugation. The changes in the levels of the mRNAs for NHI and EB subunits were compared with the changes in the levels of the mRNAs for actin, p53, c-myc, thymidine incorporation into DNA, and DNA content by flow cytometric measurements. Synchronization of the cells by the two methods resulted in quantitative differences in the responses. The EGTA synchronized L1210 cells showed maximal increases of 9.3- and 5.7-fold in the mRNAs for the NHI and EB subunits, respectively. The peak level of the NHI mRNA was observed at 12 hr after the addition of calcium ions. The peak increase in the level of the mRNA for the EB subunit was observed between 12 and 15 hr after the addition of calcium ions. The rate of increase for the mRNA for c-myc was greater than the increase in the mRNA for the NHI subunit.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1988

Identification of cDNA clones that code for protein-tyrosine kinases by screening expression libraries with antibodies against phosphotyrosine.

A phage lambda gt11 human fibroblast cDNA expression library was screened with antibodies against phosphotyrosine to determine the feasibility of this approach as a method for the identification of clones that code for protein-tyrosine kinases. Several antibody positive clones were isolated. One clone also scored positive with degenerate oligonucleotides designed to identify sequences coding for protein-tyrosine kinases. This cDNA clone was partially sequenced and proved to be identical to part of lyn, a recently reported putative protein-tyrosine kinase. A portion of the cDNA was cloned into an inducible plasmid expression vector. Phosphorylation of many bacterial proteins on tyrosine was observed upon addition of inducing agent. The results demonstrate that this method of screening can identify cDNAs that encode active protein-tyrosine kinases and could prove useful for the identification of novel ones.

Amino Acid Sequence↗
PubMed1986

Calmodulin: biochemical, physiological, and morphological effects on Schistosoma mansoni.

Results of radioimmunoassays for the Ca2+-binding protein, calmodulin, revealed that this receptor constitutes 0.53 +/- 0.12% of the total protein in adult male Schistosoma mansoni. Schistosome calmodulin purified by Ca2+-dependent hydrophobic interaction chromatography showed an apparent molecular weight of 19 kDa, and its mobility on sodium dodecyl sulfate polyacrylamide gels was influenced by the presence of Ca2+ but not the antischistosomal drug praziquantel. Calmodulin from the parasite effected a four-fold stimulation of bovine heart adenosine 3',5'-cyclic monophosphate phosphodiesterase; this process was inhibited by removal of Ca2+ with ethyleneglycol-bis(B-aminoethylether)-N,N'-tetraacetic acid but not by praziquantel. Inhibition of calmodulin-activated processes with antipsychotic compounds in vitro resulted in a number of time- and concentration-dependent changes, including inhibition of schistosome calmodulin stimulation of bovine heart phosphodiesterase, disruption and depolarization of the parasite's tegument, and positive inotropic effects on longitudinal musculature. Results of this study indicate that calmodulin is a functional component of schistosomes and suggest that the role it serves is analogous to that obtained in other eukaryotes; i.e., it is an important component of numerous processes regulated, in part, by Ca2+.

3',5'-Cyclic-AMP Phosphodiesterases↗
PubMed1984

Changes in glucose metabolism and cyanide sensitivity in Schistosoma mansoni during development.

Schistosoma mansoni was studied by biochemical and electrophysiological techniques to follow the physiological changes occurring during transformation in the mammalian host. Volume conducted electrical potentials and measurement of CO2 evolution indicate that 3 h post-transformational schistosomula are highly sensitive to cyanide. By 24 h after transformation, evolution of CO2 under control conditions is reduced by 77% from 3 h levels, while lactate excretion rises by 84%. Cyanide does not affect the frequency or magnitude of endogenous electrical transients, but does eliminate 83% of the already reduced levels of CO2 evolved in 24 schistosomula. Electrophysiological analyses indicate that the timecourse of metabolic changes in skin- and mechanically transformed schistosomula are similar, and incubation of schistosomula in 200 micrograms ml-1 puromycin does not alter the onset of cyanide insensitivity. The adult parasite evolves a low level of CO2 which is reduced by 88% in the presence of 1 mM cyanide. No significant Pasteur effect is detected, however, and endogenous electrical activity as well as mechanical responses of the adult musculature are unaffected by cyanide exposure. Our results indicate that schistosomula continue to rely on cyanide-sensitive respiratory components for at least 3 h after transformation; by 24 h, however, the parasites are metabolically similar to the adult stage, i.e., they depend on lactate fermentation for most of their energy requirements.

Aerobiosis↗
PubMed1984

Schistosoma mansoni: a comparative study of schistosomula transformed mechanically and by skin penetration. Electrophysiological responses to a wide range of substances.

Volume conducted potentials were recorded from schistosomula of Schistosoma mansoni transformed mechanically (MS) and by skin penetration (SS). The spontaneous electrical activity recorded consisted of bi- and triphasic transients ranging from 20 to 200 microV in amplitude and 10 to 300 msec in duration. Low amplitude potentials occurred at a much greater frequency than large amplitude potentials, which appeared to correlate with peristaltic-like contractions of the schistosomulum's musculature. Electrical activity in the schistosomulum was highly sensitive to a number of agents believed to affect metabolic pathways, the neuromuscular system or the host/parasite interface of adult schistosomes. Among the most reactive substances were potassium antimony tartrate, eserine, poly-L-arginine and potassium cyanide. Over a wide range of experimental treatments, electrophysiological responses in schistosomula transformed from cercariae by mechanical decaudation and skin penetration were remarkably similar, supporting the notion that MS are suitable material for in vitro immunochemical, biochemical and physiological study. Some treatments, however, were more or less effective in altering electrophysiological activity and motility in the schistosomulum than in adult S. mansoni. This suggests that significant physiological alterations may occur during development from skin stage to adult parasites concomitant with immunochemical and morphological changes already known to occur.

Animals↗
PubMed1984

Extracellular enzyme production by Rhizopus and Mucor species on solid media.

Solid media were employed to determine the presence and absence of extracellular enzyme production by two genera of fruit-rot fungi, Rhizopus and Mucor. The results of this investigation revealed that phosphatase was released into the cultural medium by all the fungi examined; however, only R. oryzae, R. tritici, M. mucedo, and M. piriformis showed the possibility of being high producers of the enzyme. Protease, urease, ribonuclease, pectate lyase, and polygalacturonase, at varying levels of activity, were detected, in the majority of the fungi, in the cultural medium.

Culture Media↗
PubMed1983

The relationship between tegumental disruption and muscle contraction in Schistosoma mansoni exposed to various compounds.

The antischistosomal drugs praziquantel and Ro 11-3128, which cause a tetanic contraction of Schistosoma musculature, concurrently produce a disruption of the parasite's tegument. Both phenomena are attenuated by preincubation of the parasites in high Mg2+ containing media before addition of the drugs, and the therapeutically inactive stereoisomers of these drugs cause neither effect. Other antischistosomal drugs tested cause neither muscle contraction nor tegumental disruption. Some unrelated agents which cause muscle contraction (but have no antischistosomal effect) alter the parasite's tegument, while others do not. Cytochalasin B, but not colchicine, causes tegumental disruption without affecting muscle tension. Thus, there is no simple correlation between a drug's antischistosomal activity or ability to contract the parasite's muscle and its ability to damage the parasite's tegument.

Animals↗
PubMed1983

Neurophysiology of adult male Schistosoma mansoni.

From the studies on the neurophysiology of schistosomes it appears that in spite of the unique hexilaminar arrangement of the tegument's outer membrane, it has biophysical properties not markedly different from those of a variety of other multi-dimensional syncytia. The close electrical coupling of the muscle and tegument must be taken into account when one attempts to define sites and modes of action of drugs which affect motor activity. Agents which disrupt muscle function in the schistosome may exert their action indirectly by way of an effect on the tegumental membrane. The syncytial nature of the musculature and the possibility that longitudinal contraction waves are myogenic suggests that neurotransmitters may simply function as modulators of muscle activity as is the case for many vertebrate visceral muscles. External recordings indicate the presence of a variety of electrically active tissues within the schistosomes. There is no clear correlation of this activity with longitudinal muscle activity or with active membrane responses in this muscle. From this it would appear the bulk of this activity may have its origins in tissues other than the longitudinal muscle such as other muscle groups, nerve trunks, or the peripheral nerve net.

Animals↗
PubMed1983

Tegumental disruption with Triton X-100 and its effects on longitudinal muscle function in male Schistosoma mansoni.

Longitudinal muscles in adult male Schistosoma mansoni appear to remain in almost all respects functional after tegumental disruption by Triton X-100. Responses to high K+, ouabain, low temperature, praziquantel and neurotransmitters are still present. Muscle membrane potentials remain near those of control animals and lanthanum nitrate is still excluded from the muscle after the Triton treatment. Although muscle contractility remains after tegumental disruption, sodium and calcium levels, as measured by plasma emission spectroscopy, increase by 20-30% while potassium and magnesium levels decrease by 15%. 45Ca2+ accumulation is double that of control animals. Upon exposure to a bathing medium lacking Ca2+, Triton-treated animals lose responsiveness to high K+, ouabain, low temperature and praziquantel more rapidly than do control animals. D-600, which effectively blocks the high K+ response in control animals, has no measurable effect on the high K+ response in Triton-treated animals, suggesting that voltage-dependent Ca2+ channels are present in the tegument.

Animals↗