Biomedical subjects

D B McKay

Publications and source records attributed to D B McKay.

At least 91 records · Page 5Linked to original sources

PubMed1989

[3H]noradrenaline accumulation in cultured bovine adrenal medullary cells: modulation of accumulation by nicotine.

The mechanisms by which catecholamines are transported into adrenal medullary cells are not as well characterized as the high affinity catecholamine uptake system of sympathetic neurones. Therefore, we studied the accumulation of [3H]noradrenaline in 7-21 day old cultures of bovine adrenal cells. Cultured adrenal cells accumulate [3H]noradrenaline. The accumulation process has an apparent Km for noradrenaline of 0.47 mumol/l, is Na(+)-dependent, and is inhibited by desipramine, (-)-noradrenaline and (-)adrenaline (IC50's, 0.007, 0.9 and 3.8 mumol/l, respectively). Other aromatic amines also inhibit accumulation with an order of potency, (+)-amphetamine greater than or equal to dopamine greater than (-)-noradrenaline greater than (-)-adrenaline greater than or equal to 5-hydroxytryptamine. Nicotine and other stimulants of catecholamine release (acetylcholine and depolarizing K+ concentrations) were found to inhibit [3H]noradrenaline accumulation. Reserpine pretreatment (72 h) or omission of calcium did not prevent nicotine's inhibitory effect on accumulation, but did reduce or inhibit nicotine-induced catecholamine release, suggesting that adrenal [3H]noradrenaline accumulation may be regulated by processes involved with stimulus-secretion coupling (e.g., sodium gradient and/or depolarization) which are independent of release. These results suggest that the catecholamine transport mechanisms of cultured bovine adrenal cells and sympathetic neurones are similar and may be regulated by secretory processes.

Adrenal Medulla↗

PubMed1989

Pregnancy as a natural model of allograft tolerance. Interactions between adherent macrophages and trophoblast populations.

From an immunologic viewpoint, the fetus with its paternal antigens may be considered a successful allograft in the maternal host. Understanding the basis of this host-allograft relationship remains a fundamental unsolved problem in transplantation immunobiology. We have previously demonstrated that local immunoregulation in the murine placenta prevented macrophage activities necessary for an effective response against the intracellular bacterium Listeria monocytogenes. Given the central role of macrophages both as antigen-presenting and cytolytic effector cells, such local immuno-regulation may ordinarily help prevent rejection of the fetoplacental unit with its paternal alloantigens by the maternal immune system. We therefore examined two types of interaction between macrophages and the placental cells that populate the maternal-fetal interface. (1) Upon activation to kill listeria efficiently, macrophages also acquire cytolytic capacities against some tumor and embryonic cells. We tested the hypothesis that macrophage activation in the placenta was inhibited to prevent macrophages from lysing fetal trophoblasts. We found, however, that trophoblasts isolated by dispase dispersion, differential isopyknic centrifugation, and adherence were not lysed by three different populations of cytolytic macrophages: (a) those activated in vivo during listeriosis, (b) peptone-elicited macrophages activated in vitro by recombinant interferon gamma and other lymphokines, and (c) the macrophage cell line RAW 264.7 activated in vitro. (2) Previous studies had demonstrated that cells from the placental region and their conditioned media inhibited a variety of lymphocyte functions. However, we found that these did not inhibit activation of adherent macrophages as assessed by induction of cell-surface Ia and acquisition of tumoricidal activity. In addition, under conditions where placental cells inhibited the proliferative response of a cloned CD4+ anti-Listeria T cell line to fixed, antigen-pulsed macrophages, the secretion of macrophage-activating lymphokines was not affected. These studies are important because they indicate that previously described suppressor systems in the murine placental region do not account for the profound local deficits in macrophage function seen during listeriosis.

Animals↗

PubMed1989

Structure-activity study on the actions of taxol and related taxanes on primary cultures of adrenal medullary cells.

Taxol is a natural plant product which has antimitotic activity. It is currently being investigated for use both as a cancer chemotherapy agent and as a tool to investigate microtubule involvement with various cellular functions. In the studies reported here, the actions of taxol on cultured adrenal medullary cells are compared to those of two other taxanes, baccatin III and 19-hydroxybaccatin III, which are structurally similar but lacking a bulky ester linkage at the C13 position. Unlike taxol, baccatin III and 19-hydroxybaccatin III have little or no known actions on mammalian microtubules. These studies were designed 1) to establish structural requirements for taxol's effects on adrenal cells and 2) to investigate whether the effects of taxol on adrenal cells involve microtubules. The effects of taxol on basal catecholamine release, on nicotinic receptor-stimulated catecholamine release and on the adrenal microtubule network are quantitatively and qualitatively different from those of baccatin III and 19-hydroxybaccatin III. These results suggest that esterification of taxol at position C13 is important in taxol's inhibitory actions on nicotinic receptor-stimulated catecholamine release. Similar structure requirements have been reported for taxol's actions on microtubules. Catecholamine release stimulated through activation of nicotinic receptors is much more sensitive to the inhibitory actions of taxol than is release stimulated by other secretagogues. Finally, a direct effect of taxol on the adrenal microtubular network was demonstrated using treatment conditions which are similar to those used to interfere with nicotinic receptor function. The studies reported here offer further evidence that taxol's actions on nicotinic receptor function may involve microtubules.

Adrenal Medulla↗

PubMed1989

Renal parenchymal malakoplakia. Histologic spectrum and its relationship to megalocytic interstitial nephritis and xanthogranulomatous pyelonephritis.

We report five cases of renal parenchymal malakoplakia. They represent a histologic spectrum of this uncommon inflammatory process as it involves the renal parenchyma. Only one of these five cases presented the classical histologic picture of malakoplakia ("diagnostic stage"). Two cases--although with similar cellular infiltrate--had a marked paucity of Michaelis-Gutmann bodies and thus might have been classified as megalocytic interstitial nephritis were it not for the identification of such calcific intracellular inclusions under the electron microscope. The fourth case presented a pseudosarcomatous morphology and is similar to what has been described as a late or fibrous stage in malakoplakia of the urinary bladder. The last case had a destructive, frankly granulomatous histologic picture with a preponderance of foamy histiocytes reminiscent of xanthogranulomatous pyelonephritis, but with clusters of von Kossa-positive Michaelis-Gutmann bodies. The last two forms of the disease have not been previously reported to occur in the kidney. We believe that these cases represent various stages of development of the disease.

Adult↗

PubMed1988

Crystals of an ATPase fragment of bovine clathrin uncoating ATPase.

A 44,000 Mr amino-terminal, clathrin-independent ATPase fragment of the bovine clathrin uncoating ATPase has been crystallized in a form suitable for X-ray diffraction studies. The crystals are orthorhombic, space group P2(1)2(1)2(1), a = 145.3 A, b = 65.0 A, c = 46.9 A, with one protein molecule per asymmetric unit (1 A = 0.1 nm).

Adenosine Triphosphatases↗

PubMed1988

Mapping the enzymatic active site of Pseudomonas aeruginosa exotoxin A.

Pseudomonas aeruginosa exotoxin A is a representative of a class of enzymes, the mono-ADP-ribosyl transferases, which catalyze the covalent transfer of an ADP-ribose moiety of NAD+ to a target substrate. Availability of the three-dimensional structure of exotoxin A provides the opportunity for mapping substrate binding sites and suggesting which amino acid residues may be involved in catalysis. Data from several sources have been combined to develop a proposal for the NAD+ binding site of exotoxin A: the binding of NAD+ fragments adenosine, AMP, and ADP have been delineated crystallographically to 6.0, 6.0, and 2.7 A, respectively; significant sequence homology spanning 60 residues has been found between exotoxin A and diphtheria toxin, which has the identical enzymatic activity; iodination of exotoxin A, under conditions in which only tyrosine 481 is iodinated in the enzymatic domain, abolishes ADP-ribosyl transferase activity.

ADP Ribose Transferases↗

PubMed1987

Three-dimensional structure of interleukin-2.

Interleukin-2 is an effector protein that participates in modulating the immune response; it has become a focal point for the study of lymphokine structure and function. The three-dimensional structure of the interleukin molecule has been solved to 3.0 angstrom resolution. Interleukin-2 has a novel alpha-helical tertiary structure that suggests one portion of the molecule forms a structural scaffold, which underlies the receptor binding facets of the molecule.

Animals↗

PubMed1987

Crystals and a low resolution structure of interleukin-2.

Recombinant derived human interleukin-2 and an analog in which cysteine 125 has been replaced with alanine have been crystallized in a form suitable for x-ray diffraction. The crystals are triclinic, space group P1, with two protein molecules in the unit cell; unit cell parameters are a = 55.8 A, b = 40.1 A, c = 33.7 A, alpha = 90.0 degrees, beta = 109.3 degrees, gamma = 93.2 degrees. The interleukin-2 structure has been solved to 5.5 A resolution using heavy atom isomorphous replacement methods. The resultant low resolution model reveals a significant fraction of alpha helical secondary structure and outlines the overall tertiary structure of the molecule.

Crystallization↗

PubMed1987

Effects of a cytosolic protein on the interaction of rat pancreatic zymogen granules in vitro.

Photon correlation spectroscopy has been used to study the kinetics of aggregation of isolated rat pancreatic zymogen granules in vitro by monitoring time-dependent changes in mean particle size derived from the photon count autocorrelation function, g2(tau). Isolated granules were stable in isotonic sucrose (pH 5.4-7.0). At pH 6.0 they maintained a mean diameter of 1225 +/- 18 nm with a polydispersity index of 0.199 +/- 0.007. The mean granule diameter showed a limited decrease (approx. 20%) with increasing pH within the range 5.4-7.0, but the polydispersity index was unaltered. At pH greater than 7.0 granule instability was indicated by a rapid reduction in total photon counts. In solutions of monovalent cations ([M+] greater than 10 mM) and divalent cations ([M2+] greater than 0.5 mM) zymogen granules aggregated at a rate dependent upon both ion and granule concentration. These effects were consistent with the bimolecular nature of the interaction mechanism and were clearly distinguishable from the limited size changes associated with osmolarity. At concentrations of Na+ or K+ salts greater than 50 mM granule aggregation was accompanied by anion-dependent solubilisation. A soluble protein fraction separated from the pancreatic acinar cell cytosol by gel filtration reduced the mean diameter and polydispersity index of zymogen granules suspended in isotonic sucrose, inhibited cation-induced aggregation and stabilised granules to solubilisation induced by raising pH greater than 7.0 or exposure to high ionic strength media. The inhibitory effects of this protein were apparent at concentrations less than or equal to 10 micrograms X ml-1 (i.e. at inhibitor: granule protein ratios less than 1:20) and could not be mimicked by bovine serum albumin, the Ca2+-binding proteins calmodulin and troponin C (less than or equal to 100 micrograms X ml-1), nor the highly negatively charged polymer polyglutamate (less than or equal to 10 micrograms X ml-1). Inhibitory activity was also absent from fractions of rat liver cytosol prepared identically to pancreatic acinar cytosol. These observations are consistent with the presence in pancreatic acinar cells of a specific cytosolic granule stabilisation factor (or factors) that normally restricts zymogen granule interaction and may therefore play an important role in the regulation of granule mobility and exocytosis.

Animals↗

PubMed1987

Control of secretory granule interaction and exocytosis in pancreatic cells.

Application of the laser-based technique of photon correlation spectroscopy to an in vitro study of the ionic stability and interaction kinetics of zymogen granules isolated from rat exocrine pancreas is described here. In addition the separation from pancreatic acinar cell cytosol of a factor which stabilizes isolated zymogen granules and inhibits cation-induced granule aggregation is outlined. The basis of this action and the significance of the cytosolic inhibitory factor in the regulation of granule mobility and exocytosis in vivo is discussed.

Animals↗

PubMed1987

Detection of polyols and sugars by cuprammonium ion in the presence of strong base.

A new and more sensitive cuprammonium reagent containing strong base has been developed for the colorimetric detection of polyols and sugars. A reagent blank with an absorbance at 280 nm, less than half that of previous methods, was achieved by reducing the cupric ion and ammonia concentrations to 1 mM (from 5 mM) and 0.4 M (from 2.25 M), respectively, and adding 0.2 M sodium hydroxide. Polyols are particularly well suited to the new method, showing up to an eightfold increase in sensitivity. The new reagent is readily applicable to the postcolumn detection of polyols and sugars after high-performance liquid chromatography and is linear over the range 40-180 nmol for D-glucitol (sorbitol) and 10-300 nmol for glucose, using standard HPLC equipment.

Carbohydrates↗

PubMed1987

Comparison of the effects of colchicine and beta-lumicolchicine on cultured adrenal chromaffin cells: lack of evidence for an action of colchicine on receptor-associated microtubules.

The involvement of microtubules in adrenomedullary secretion is presently unclear. Evidence exists for a possible role of microtubules in cholinergic nicotinic receptor-related events. We now describe the actions of the microtubule disrupter, colchicine, on primary cultures of bovine adrenal chromaffin cells and compare these with corresponding actions of beta-lumicolchicine. beta-Lumicolchicine is a structural isomer of colchicine which neither binds microtubular protein (tubulin) nor interferes with microtubule assembly. Both colchicine and beta-lumicolchicine were found to inhibit acetylcholine-induced secretion with similar potencies (half maximal inhibitory concentration 0.2-0.5 mM). The inhibitory actions of both drugs are time-dependent and reversible. However, unlike colchicine which has no inhibitory effects on secretion evoked by depolarization with excess K+, beta-lumicolchicine also inhibits K+-induced secretion. Because colchicine and beta-lumicolchicine have similar effects, the selective inhibitory actions of colchicine on nicotinic receptor-mediated secretion cannot in itself be used as evidence in support of a role of microtubules in receptor-mediated events. However, our data do not preclude such a role. Differences in the effect of colchicine and beta-lumicolchicine on K+-evoked secretion suggests different modes of action of these structural isomers on chromaffin cell function.

Acetylcholine↗

PubMed1986

Structure of exotoxin A of Pseudomonas aeruginosa at 3.0-Angstrom resolution.

Exotoxin A of Pseudomonas aeruginosa is a secreted bacterial toxin capable of translocating a catalytic domain into mammalian cells and inhibiting protein synthesis by the ADP-ribosylation of cellular elongation factor 2. The protein is a single polypeptide chain of 613 amino acids. The x-ray crystallographic structure of exotoxin A, determined to 3.0-A resolution, shows the following: an amino-terminal domain, composed primarily of antiparallel beta-structure and comprising approximately half of the molecule; a middle domain composed of alpha-helices; and a carboxyl-terminal domain comprising approximately one-third of the molecule. The carboxyl-terminal domain is the ADP-ribosyltransferase of the toxin. The other two domains are presumably involved in cell receptor binding and membrane translocation.

ADP Ribose Transferases↗

PubMed1985

Crystallization of the protective antigen protein of Bacillus anthracis.

The protective antigen protein, one of the three separate proteins constituting the exotoxin system of Bacillus anthracis, has been crystallized in a form suitable for structural studies. The crystal form which is most amenable to x-ray analysis is orthorhombic, space group P2(1)2(1)2(1), a = 101.1 A, b = 95.4 A, c = 87.3 A, with one protective antigen monomer/asymmetric unit. The crystals diffract to approximately 3.0-A resolution.

Antigens, Bacterial↗

PubMed1985

Interactions of microtubule-active agents with nicotinic acetylcholine receptors. Relationship to their inhibition of catecholamine secretion by adrenal chromaffin cells.

Several microtubule-active drugs block cholinergically mediated catecholamine secretion from adrenal chromaffin cells without affecting secretion induced by other secretagogues. Interactions of these agents with nicotinic acetylcholine receptor-ion channel complexes from Torpedo californica electric organs were studied using radiolabeled probes for receptor and associated ion channel-binding sites. Colchicine, taxol, and the Vinca alkaloids had minimal affinity for cholinergic receptor-binding sites (nicotinic or muscarinic). The Vinca alkaloids (vinblastine, vincristine, vindesine) and colchicine inhibited [3H]perhydrohistrionicotoxin ([3H]H12-HTX) binding to the receptor-gated ion channel with IC50 values of 2-32 microM and 6 mM, respectively. The ability of the microtubule-active drugs to inhibit [3H]H12-HTX binding was increased by up to 5-fold in the presence of 1 microM carbamylcholine. The IC50 values for inhibition of [3H]H12-HTX binding by colchicine and three Vinca alkaloids were closely correlated with their abilities to inhibit acetylcholine-induced catecholamine secretion from cultured bovine adrenal chromaffin cells. As a consequence of its interaction (direct or indirect) with the ion channel, at least one Vinca alkaloid (vinblastine) stabilized a high agonist affinity conformation of the nicotinic receptor complex. beta-Lumicolchicine, an analog of colchicine devoid of microtubule activity, also blocked ion channel binding. On the other hand, taxol, a microtubule-stabilizing agent which also selectively blocks cholinergically mediated secretion, did not affect receptor or ion channel binding. The present results indicate that interactions with the nicotinic receptor-ion channel complex may underlie the actions of certain microtubule-active agents on catecholamine secretion by adrenal chromaffin cells.

Alkaloids↗

PubMed1984

Selective inhibition of cholinergic receptor-mediated 45Ca++ uptake and catecholamine secretion from adrenal chromaffin cells by taxol and vinblastine.

We have characterized the actions of taxol, a novel drug promoting microtubule formation, on 45Ca++ uptake and catecholamine release by isolated and cultured bovine adrenal chromaffin cells. The effects of taxol are compared with corresponding actions of vinblastine. We also have measured the effects of microtubule-active drugs on the dynamic pattern of release by means of column perifusion of isolated chromaffin cells. Taxol inhibits acetylcholine-stimulated catecholamine secretion (IC50: approximately 1 microM) and 45Ca++ uptake. The inhibitory effects of both taxol and vinblastine on secretion are rapid in onset (approximately 1 min) and reversible. Taxol and vinblastine (5 microM) exert little or no inhibitory effect on catecholamine secretion induced by 1) the nonreceptor mediated secretagogues K+, Ba++ or veratridine or by 2) the receptor-mediated secretagogues histamine or bradykinin. Similarly, taxol and vinblastine had no effect on K+-induced 45Ca++ uptake into chromaffin cells. The inhibitory effects of taxol and vinblastine during a secretory challenge are specific for cholinergic receptor-mediated 45Ca++ uptake and catecholamine release and prevent receptor-mediated membrane depolarization. These results do not support a role for microtubules either in the exocytosis event or in granule transport during an initial secretory challenge. The results would be consistent with either an interaction of microtubule protein with the acetylcholine receptor or a direct action of the drugs on the acetylcholine receptor.

Acetylcholine↗

PubMed1983

Factors to consider in the assessment of viability of cryopreserved islets of Langerhans.

With the development of techniques for the isolation and transplantation of pancreatic islets of Langerhans, research has been directed toward low-temperature storage of islets as a means of preservation. For successful islet cryopreservation several factors must be considered. In these studies we have investigated the effects of the cryoprotectant dimethyl sulfoxide (Me2SO) on islet function in the absence of freezing. We have found that Me2SO pretreatment can inhibit subsequent glucose-induced insulin release, but this effect can be minimized by hypothermic exposure to the cryoprotectant using a stepwise addition and dilution protocol for treatment. By studying islet function after freezing and thawing, we have found also that a slow cooling rate (0.3 degrees C/min) results in optimal survival and that islet function can be significantly improved by increasing the duration of post-thaw culture. The results of these studies address only a few of the many questions that need to be answered before clinical application of cryopreserved islet transplantation occurs.

Animals↗

PubMed1983

A functional analysis on isolated rat islets of Langerhans: effects of dimethylsulfoxide and low-temperature preservation.

Cryopreservation of pancreatic islets of Langerhans offers the possibility of storage of sufficient quantities of this tissue for transplantation in the treatment of certain forms of diabetes, as well as providing a means of precise histocompatibility matching. In these studies, the effects of dimethylsulfoxide and various cooling rates on islet function are examined. These studies demonstrate that islets treated with 1.4 M dimethylsulfoxide and slowly cooled at a rate of 0.3 degrees C/min release insulin biphasically upon glucose challenge. In addition, this stimulated release is significantly improved (P less than 0.05) by increasing the duration of post-thaw culture. After thawing, these cryopreserved islets also retain the capacity to synthesize insulin. Islets frozen at faster cooling rates (3, 14, and 48 degrees C/min) exhibit varying degrees of glucose-induced insulin release, indicative of freeze-induced damage. These manifestations of freeze-induced damage include high basal (nonstimulatory) insulin release rates, little or no increase in the stimulated rate versus the nonstimulated rate, and failure of the stimulated release to return to basal levels when the glucose concentration is reduced.

Animals↗