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Biomedical subjects

D J Adams

Publications and source records attributed to D J Adams.

At least 145 records · Page 8Linked to original sources
PubMed1985

Identification of human cytochromes P-450 analogous to forms induced by phenobarbital and 3-methylcholanthrene in the rat.

Antibodies to four rat liver forms of cytochrome P-450, two phenobarbital-inducible (PB1 and PB2) and two 3-methylcholanthrene-inducible (MC1 and MC2) proteins, have been used to make a structural and functional comparison of rat and human cytochromes P-450. Proteins from both species were identified on Western blots by their reaction with these antibodies. In the human liver preparations, structurally related proteins to PB1 and to PB2 were identified in all the samples tested with apparent Mr values of 51 800 and 54 800 for PB1 and 53 600 and 57 200 for PB2. Considerable variation in the content of the lower-Mr proteins was measured between samples and, as with the rat enzymes, samples which reacted well with anti-PB1 also reacted with anti-PB2, indicating that these proteins are regulated at least to some degree, co-ordinately. The apparent Mr values of the major human proteins identified with anti-MC1 and anti-MC2 were 54 400 and 57 000 respectively. Only six (of 31) human samples contained significant amounts of these proteins. The same six samples which reacted with anti-MC1 also reacted with anti-MC2, again indicating co-ordinate regulation of these two proteins. Antibody inhibition of microsomal 7-ethoxycoumarin and 7-ethoxyresorufin metabolism demonstrated a degree of conservation of substrate specificity related to specific P-450 isoenzymes between the species. However, the contributions of the different P-450 isoenzymes to the human microsomal activity were not always related to the rat enzyme with the highest activity towards these substrates.

Animals↗
PubMed1985

Induction of drug metabolizing enzymes in human liver cell line Hep G2.

Human cytochrome P-450, UDP-glucuronosyltransferase and sulphotransferase activities have been measured in the cell line Hep G2 following treatment of cells with 3-methylcholanthrene or phenobarbital. 3-Methylcholanthrene treatment caused a 20-30-fold increase in the O-deethylation of 7-ethoxycoumarin. The glucuronidation and sulphation of the product 7-hydroxycoumarin were increased 36 and 7 fold, respectively. In comparison, phenobarbital treatment did not increase these activities significantly. However, phenobarbital-inducible proteins were identified on "Western blots' using antibodies to a rat liver phenobarbital inducible P-450 form. The molecular masses of the proteins did not coincide with those expected for cytochromes P-450. However, characteristic of P-450 forms, the synthesis of these proteins was suppressed by 3-methylcholanthrene treatment. The Hep G2 cell line represents a potentially useful model for studying the regulation of human P-450 genes.

Cell Line↗
PubMed1985

Some properties of acetylcholine receptors in human cultured myotubes.

The distribution and single channel properties of acetylcholine (ACh) receptors in human myotubes grown in tissue culture have been examined. Radioautography of myotubes labelled with [125I]alpha-bungarotoxin showed that ACh receptors are distributed uniformly over the myotube surface at a density of 3.9 +/- 0.5 receptors per square micrometre. Accumulations of ACh receptors (hot spots) were found rarely. The conductance and kinetics of ACh-activated channels were investigated with the patch-clamp technique. Cell-attached membrane patches were used in all experiments. A single channel conductance in the range 40-45 pS was calculated. No sublevels of conductance (substates) of the activated channel were observed. The distribution of channel open-times varied with ACh concentration. With 100 nM ACh, the distribution was best fitted by the sum of two exponentials, whereas with 1 microM ACh a single exponential could be fitted. The mean channel open-time at the myotube resting potential (ca. -70 mV, 22 degrees C) was 8.2 ms. The distribution of channel closed-times was complex at all concentrations of ACh studied (100 nM to 10 microM). With desensitizing doses of ACh (10 microM), channel openings occurred in obvious bursts; each burst usually appeared as part of a 'cluster' of bursts. Both burst duration and mean interval between bursts increased with membrane hyperpolarization. Individual channel open-times and burst durations showed similar voltage dependence (e-fold increase per 80 mV hyperpolarization), whereas both the channel closed-times within a burst and the number of openings per burst were independent of membrane potential.

Acetylcholine↗
PubMed1985

Inhibitors of calcium buffering depress evoked transmitter release at the squid giant synapse.

Evoked release of transmitter at the squid giant synapse was examined under conditions where the calcium ion concentration in the presynaptic terminal was manipulated by inhibitors of calcium sequestration. Simultaneous intracellular recordings of presynaptic and post-synaptic resting and action potentials were made during bath application of one of the following metabolic inhibitors: sodium cyanide (NaCN), carbonyl cyanide-p-trifluoromethoxyphenyl hydrazone (FCCP); ruthenium red (RuR) and sodium-free (lithium) sea water. Cyanide and lithium sea water reversibly depressed the post-synaptic potential (p.s.p.) whilst RuR and FCCP blocked the evoked post-synaptic response irreversibly. The progressive reduction of p.s.p. amplitude was accompanied by a reversible increase in synaptic delay. The time course of block of the p.s.p. was similar for different agents and dependent on the rate of presynaptic activity (30-40 min at 0.01 Hz). Recovery of the post-synaptic action potential following block by cyanide and lithium sea water was obtained within 40 min and 5 min respectively. Synaptic depression by the metabolic inhibitors does not result from changes in presynaptic resting or action potentials, nor from a change in post-synaptic receptor sensitivity. The post-synaptic response to the local ionophoresis of L-glutamate was unchanged following inhibition of evoked release of transmitter by cyanide. Injections of EGTA into presynaptic terminals poisoned by cyanide produced transient increases in p.s.p. amplitude, suggesting that cyanide is having its effect through raising intracellular calcium rather than lowering ATP. Control experiments injecting EGTA into unpoisoned nerve terminals showed no apparent effect on evoked transmitter release.

Action Potentials↗
PubMed1985

The presence of an estrogen-regulated protein detected by monoclonal antibody in abnormal human endometrium.

The cellular localization of a 24,000 (24K) mol wt protein was evaluated by monoclonal antibody immunocytochemistry in atrophic and persistent proliferative endometrium as well as in various forms of hyperplastic and neoplastic human endometria. 24K was chiefly located in the cytoplasm of ciliated cells and their precursor forms, clear cells. It was absent in atrophic, resting, and regressive forms of cystic glandular hyperplasia. Persistent proliferative endometria with a low degree of proliferation has occasional 24K immunostained cells, whereas those with active proliferation contained intense 24K immunostaining. A similar pattern characterized the active forms of cystic glandular hyperplasia. The immunostaining reaction decreased in adenomatous and atypical adenomatous hyperplasia as well as in well differentiated adenocarcinomas. In poorly differentiated carcinomas, 24K protein was virtually absent. These findings suggest that 24K protein is estrogen and ciliated cell related, and endometrial ciliogenesis and 24K protein are morphological and biochemical markers, respectively, of the estrogenic endometrial response. Their increase and decrease in hyperplastic and neoplastic endometria support the concept that early hyperplasia is highly sensitive to estrogenic stimulation, whereas with increasing architectural and cytologic atypia, the estrogenic response decreases as a reflection of growing cell populations that are independent of estrogenic influence. Immunostaining with 24K is not sensitive enough to discriminate estrogen-independent cells of atypical adenomatous hyperplasia from those of early, well differentiated adenocarcinoma.

Adult↗
PubMed1985

Quantitative enzyme-linked immunosorbent assay for the estrogen-regulated Mr 24,000 protein in human breast tumors: correlation with estrogen and progesterone receptors.

A competitive enzyme-linked immunoassay has been developed to quantitate the Mr 24,000 estrogen-regulated protein (p24) in human breast tumors and tumor cell lines. The concentration of monoclonal antibody used to coat polystyrene microtiter assay plates was critical in optimizing assay sensitivity versus precision and demonstrated marked differences between plate types. Analysis of 114 human breast tumor cytosols revealed a 4-log range in p24 concentration with a median value of 1 microgram/mg cytosol protein. The abundance of p24 in breast tumors in vivo is therefore similar to that observed previously in hormone-responsive breast tumor cells in vitro. Elevated expression of p24 in these specimens was found to correlate well with presence of estrogen and progesterone receptors (P = 0.0002 by Pearson chi 2 analysis) with the correlation coming primarily through estrogen receptor. This result was confirmed by immunoblot analysis of p24 in 76 additional tumor cytosols and suggests that p24 may be a valuable marker for differentiation in human breast cancer.

Biopsy↗
PubMed1985

Presence of an estrogen-regulated protein in endometrial cancer.

The presence of an estrogen-regulated protein of Mr 24,000 (24K) was studied in 43 patients diagnosed as having endometrial adenocarcinoma. using an anti-24K monoclonal antibody, a modified immunoperoxidase system (avidinbiotin complex) was used to detect the presence of 24K in formalin-fixed, paraffin-embedded tissue samples. The positivity for 24K was correlated with low tumor histologic grade, few mitotic figures, few nucleoli, and a low degree of nuclear pleomorphism. These data suggest that 24K may be a potential marker of tumor differentiation.

Adenocarcinoma↗
PubMed1984

Estrogen-induced 24K protein in MCF-7 breast cancer cells is localized in granules.

We have previously reported the production of monoclonal antibodies which detect, by immunohistochemistry, an estrogen-induced protein of molecular weight 24,000 daltons (24K). This protein, of unknown function, has been detected in: a) estrogen receptor-positive breast cancer cell lines but not in receptor-negative lines; b) several human normal estrogen target organs; and c) certain human carcinomas, including breast tumors. To examine the subcellular localization of this 24K estrogen-induced protein, we have done immunohistochemical studies at light and electron microscopic levels using a human breast tumor cell line (MCF-7) grown in vitro and also in nude mice in vivo. MCF-7 cells grown in the ascites fluid of nude mice and processed for paraffin sections showed a defined polarity, and the 24K protein was localized in the apical cytoplasm of the cells. After cytocentrifugation, MCF-7 cells grown in vitro displayed 24K protein mainly confined to large cytoplasmic granules. The presence of 24K protein in cytoplasmic granules was also seen by immunoelectronmicroscopy in MCF-7 cells grown both in vitro and in vivo. The granules had different sizes, shapes, and 24K immunostaining intensity. The morphological evidence suggests that the 24K estrogen-induced protein is secreted from the cells.

Animals↗
PubMed1984

Estrogen-regulated protein in human breast cancer.

An estrogen-regulated cytoplasmic protein has been purified from MCF-7 human breast cancer cells with anion exchange and monoclonal antibody affinity chromatographies. The purified protein has a monomeric mol. wt of 28,000 and isoelectric species with pI's between 5.9 and 6.0. Amino acid analysis indicates the protein is acidic, is probably hydrophilic, and contains unusually low amounts of methionine and half cystine. The monoclonal antibodies are of the IgG1 subclass, exhibit a high affinity for the 28K protein, and recognize an antigenic site that is stable to sodium dodecyl sulfate (SDS) gel electrophoresis and electrophoretic transfer techniques. These monoclonal antibodies have been used to detect 28K in certain human breast tumors.

Amino Acids↗
PubMed1983

Interaction of internal anions with potassium channels of the squid giant axon.

The interaction of internal anions with the delayed rectifier potassium channel was studied in perfused squid axons. Changing the internal potassium salt from K+ glutamate- to KF produced a reversible decline of outward K currents and a marked slowing of the activation of K channels at all voltages. Fluoride ions exert a differential effect upon K channel gating kinetics whereby activation of IK during depolarizing steps is slowed dramatically, but the rate of closing after the step is not much altered. These effects develop with a slow time course (30-60 min) and are specific for K channels over Na channels. Both the amplitude and activation rate of IK were restored within seconds upon return to internal glutamate solutions. The fluoride effect is independent of the external K+ concentration and test membrane potential, and does not recover with repetitive application of depolarizing voltage steps. Of 11 different anions tested, all inorganic species induced similar decreases and slowing of IK, while K currents were maintained during extended perfusion with several organic anions. Anions do not alter the reversal potential or shape of the instantaneous current-voltage relation of open K channels. The effect of prolonged exposure to internal fluoride could be partially reversed by the addition of cationic K channel blocking agents such as TEA+, 4-AP+, and Cs+. The competitive antagonism between inorganic anions and internal cationic K channel blockers suggests that they may interact at a related site(s). These results indicate that inorganic anions modify part of the K channel gating mechanism (activation) at a locus near the inner channel surface.

Action Potentials↗
PubMed1983

Monoclonal antibody storage conditions, and concentration effects on immunohistochemical specificity.

Monoclonal antibodies against a 24,000 dalton intracellular estrogen-regulated protein in human breast cancer cells were used to study storage conditions and the effects of monoclonal antibody concentrations on immunohistochemical antigen localization. Both hybridoma supernatants and ascites fluid obtained from mice injected with hybridoma cells were used as sources of monoclonal antibodies; the monoclonal antibodies in the ascites fluid were concentrated and purified. Both antibody preparations were stored at 4, -20, or -70 degrees C and periodically tested for activity at these storage conditions. There was no difference in activity for the antibodies between storage at -20 and -70 degrees C. However, when highly diluted antibody was stored at 4 degrees C, the activity was lost within 2 weeks if carrier proteins were not added. These monoclonal antibodies were applied to immunohistochemical staining of different mouse and human tissues processed for routine paraffin sections, using the avidin-biotin-peroxidase procedure. A monoclonal antibody of unrelated specificity was used as control. When these antibodies were used at high concentrations, all the different tissues examined were immunostained. With reduction of the antibody concentration, an immunohistochemical dissection of the tissues was seen until specific immunostaining was reached. When even more highly diluted monoclonal antibody was used, heterogeneity in the staining pattern became very high. On the basis of these results, certain immunohistochemical criteria are proposed for the selection of the optimum concentration of monoclonal antibodies for specific antigen detection.

Animals↗
PubMed1983

Purification of an estrogen-regulated breast cancer protein by monoclonal antibody affinity chromatography.

An estrogen-regulated cytoplasmic protein has been purified from MCF-7 human breast cancer cells with anion exchange and monoclonal antibody affinity chromatographies. The purified protein has a monomeric mol wt of 28,000 and isoelectric species with pI's between 5.9 and 6.0. Amino acid analysis indicates the protein is acidic, is probably hydrophilic, and contains unusually low amounts of methionine and half cystine. This rapid, two-step procedure produces purified protein in high yield and demonstrates the power of monoclonal antibodies in protein purification.

Amino Acids↗
PubMed1983

Evidence for modulation of a 24K protein in human endometrium during the menstrual cycle.

The presence and distribution of a protein with a molecular weight of 24,000 (24K) was determined in endometrial biopsies from regularly cycling women. This protein, of as yet unknown function and originally found in a breast cancer cell line, was detected by immunohistochemistry using a monoclonal antibody. In the glandular epithelium, the 24K protein began to appear during the late proliferative phase and decreased after ovulation. In contrast, the strongest immunostaining was observed in the superficial epithelium during the secretory phase. In these cells, the 24K immunoactivity reached the maximum around day 21 of the cycle and was clearly seen in the bulbous projections of the apical cytoplasm. These results suggest that the 24K protein may be a marker for hormonal events during the menstrual cycle.

Cytoplasm↗
PubMed1983

Detection of a Mr 24,000 estrogen-regulated protein in human breast cancer by monoclonal antibodies.

Monoclonal antibodies have been prepared that can specifically detect an estrogen-regulated protein with a molecular weight of 24,000 in the MCF-7 human breast tumor cell line and in human breast tumor biopsies. These antibodies are of the immunoglobulin G1 subclass, exhibit a high affinity for the Mr 24,000 protein, and recognize an antigenic site that is stable to sodium dodecyl sulfate gel electrophoresis and electrophoretic transfer techniques. These monoclonal antibodies have been used to confirm estrogen regulation of the Mr 24,000 protein in MCF-7 cells and to detect Mr 24,000 protein in certain human breast tumors.

Animals↗
PubMed1982

Inhibitory postsynaptic currents at Aplysia cholinergic synapses: effects of permeant anions and depressant drugs.

Inhibitory postsynaptic potentials (i.p.s.ps) and, under voltage-clamp conditions, inhibitory postsynaptic currents (i.p.s.cs) were recorded in neurons in buccal ganglia of Aplysia juliana. The decay of i.p.s.cs was exponential with a single time constant, tau, which decreased with membrane depolarization. In external solutions containing iodide or bromide ions instead of chloride ions, tau varied according to the sequence tau (I) greater than tau (Br) greater than tau (Cl), and the voltage sensitivity of tau was altered. In iodide solution, the voltage sensitivity of tau was reversed. Furthermore, the foreign halides depressed the peak current amplitude and shifted the reversal (zero-current) potential to more positive membrane potentials. In low concentrations of sodium pentobarbitone (100-200 microns), the decay of i.p.s.cs became biphasic. Increasing drug concentration and membrane hyperpolarization had differential effects on the rates and relative amplitudes of the two components of i.p.s.c. decay. Octanol (0.5-1 mM) reduced the amplitude of i.p.s.ps and increased the rate of decay of i.p.s.cs without changing the voltage sensitivity of tau. The effect of foreign halides and barbiturates on i.p.s.c. decay were interpreted in terms of a reaction between the anion and an ion-binding site(s) associated with the anion-selective channel, which affects the probability of anions entering the channel and normal channel closure.

Acetylcholine↗
PubMed1982

Protamine precipitation of two reovirus particle types from polluted waters.

Two forms of virus particle are released from reovirus-infected cell cultures, infectious reovirus and potentially infectious reovirus (PIV). PIV particle forms have a complete outer coat and are not infectious until the outer coat is altered or removed. The PIV concentration in polluted waters, however, has not been determined. Protamine sulfate precipitation, using 0.25% fetal bovine serum and 0.005% protamine sulfate for the first precipitation of the sample and 0.0025% for the second, was employed to concentrate infectious reovirus and PIV from water and sewage. Infectious reovirus and PIV particles were concentrated over 500-fold from river water inoculated with virus, and virus recoveries of between 80 and 100% were achieved. Virus precipitates stored at -20 degrees C as a protamine-virus concentrate showed a 5% loss of PIV after 14 days. Virus preparations were assayed, before and after treatment, with 200 micrograms of chymotrypsin per ml, using a fluorescent-antibody procedure. Protamine sulfate precipitation and fluorescent-antibody detection are effective ways to recover and assay reoviruses present in raw sewage.

Chemical Precipitation↗