Biomedical subjects
Publications and source records attributed to B H Wainer.
Explore the source record for details and available documents.
Acetylcholinesterase-histochemistry has been widely used for localizing cholinergic neurons despite specificity problems. The distribution of cells stained with this method has never been directly compared on a histochemical level with the specific cholinergic marker, choline acetyltransferase. We recently reported the immunohistochemical localization of choline acetyltransferase using monoclonal antibodies [Levey A. I., Armstrong D., Atweh S. F., Terry R. D. & Wainer B. H. (1983) J. Neurosci 3, 1-9]. Here we report the development of a combined histochemical and immunohistochemical method for the co-localization of the 2 cholinergic markers, and their comparison in the rat cerebrum. Although the precise relationship between the markers was complex, the important results were: (1) all neurons which contained choline acetyltransferase also contained some acetylcholinesterase; (2) many acetylcholinesterase-containing neurons did not contain any demonstrable choline acetyltransferase; (3) all neurons which stained intensely for acetylcholinesterase in the neostriatum and basal forebrain also contained choline acetyltransferase; and (4) many choline acetyltransferase-containing neurons did not stain intensely for acetylcholinesterase. The results corroborate the assumption that choline acetyltransferase is a more specific marker for cholinergic neurons than acetylcholinesterase. Intense staining for acetylcholinesterase can be reliably used in some regions of the cerebrum for identifying cholinergic neurons, however, it should be recognized that this criterion s not essential for all cholinergic neurons.
Monoclonal antibodies to choline acetyltransferase and a histochemical method for the concurrent demonstration of acetylcholinesterase and horseradish peroxidase were used to investigate the organization of ascending cholinergic pathways in the central nervous system of the rat. The cortical mantle, the amygdaloid complex, the hippocampal formation, the olfactory bulb and the thalamic nuclei receive their cholinergic innervation principally, from cholinergic projection neurons of the basal forebrain and upper brainstem. On the basis of connectivity patterns, we subdivided these cholinergic neurons into six major sectors. The Ch1 and Ch2 sectors are contained within the medial septal nucleus and the vertical limb nucleus of the diagonal band, respectively. They provide the major cholinergic projections of the hippocampus. The Ch3 sector is contained mostly within the lateral portion of the horizontal limb nucleus of the diagonal band and provides the major cholinergic innervation to the olfactory bulb. The Ch4 sector includes cholinergic neurons in the nucleus basalis, and also within parts of the diagonal band nuclei. Neurons of the Ch4 sector provide the major cholinergic innervation of the cortical mantle and the amygdala. The Ch5-Ch6 sectors are contained mostly within the pedunculopontine nucleus of the pontomesencephalic reticular formation (Ch5) and within the laterodorsal tegmental gray of the periventricular area (Ch6). These sectors provide the major cholinergic innervation of the thalamus. The Ch5-Ch6 neurons also provide a minor component of the corticopetal cholinergic innervation. These central cholinergic pathways have been implicated in a variety of behaviors and especially in memory function. It appears that the age-related changes of memory function as well as some of the behavioral disturbances seen in the dementia of Alzheimer's Disease may be related to pathological alterations along central cholinergic pathways.
Immunohistochemical localization of choline acetyltransferase (ChAT) in cholinergic neurons has been difficult to achieve because of problems encountered in producing specific antisera. Here we describe the production and characterization of several distinct monoclonal antibodies to ChAT. Each of the monoclonal antibodies exhibits one of three general patterns of cross-species reactions; one pattern shows reactivity limited mainly to bovine ChAT, a second pattern shows reactivity only to ChAT from higher mammals including humans, and the third pattern shows reactivity to ChAT from all mammals tested. The antibodies bound specifically to two closely related bovine proteins of 68,000 and 70,000 daltons using the Western blotting technique. One of the antibodies was used to localize immunohistochemically known cholinergic structures in the rat brain, including motor neurons, basal forebrain neurons, and neostriatal neurons.
We have previously reported the first successful production of monoclonal antibodies against the cholinergic neuronal marker, choline acetyltransferase (ChAT). We now report some inter-and intraspecies cross-reactivity studies of the monoclonal antibody AB1. This antibody reacted most strongly with bovine ChAT and weakly with sheep and human enzyme. Similar amounts of binding were observed with the two forms of bovine ChAT separated by ion exchange chromatography. The antibody also reacted equally well with human ChAT derived from either brain or placenta.
Immunochemical properties of bovine and human choline acetyltransferase (ChAT, EC 2.3.1.6, acetyl-CoA:choline-O-acetyltransferase) were studied using six monoclonal antibodies (AB1, AB5, AB6, AB7, AB8, and AB9) reactive with the enzyme. All antibodies except AB1 bound specifically to two proteins of 68,000 and 70,000 MW on "Western" blots of sodium dodecyl sulfate-polyacrylamide gels containing human or bovine ChAT. The enzyme was specifically absorbed to immobilized antibody and could not be eluted by low pH and/or high salt concentrations although the enzyme retained activity on the immunoabsorbent. Pure bovine enzyme consisting of the same two proteins as seen in the Western blotting studies was eluted from immobilized AB1 in the presence of sodium dodecyl sulfate. Although active enzyme could not be eluted from immobilized antibodies by standard conditions, various combinations of free and immobilized antibodies were effective in competing off bound enzyme. Free antibody AB1 quantitatively eluted the active enzyme from immobilized AB1. The different capacities of the antibodies to elute enzyme from various immunoabsorbents reflect interesting properties of both the enzyme and the antibodies.
Explore the source record for details and available documents.
Explore the source record for details and available documents.
Explore the source record for details and available documents.
Antimorphine globulin was used to study the mechanism by which naloxone produces a contracture in ileal strips removed from morphine pellet-implanted guinea pigs. Although the antibodies effectively reversed the depressant actions of morphine on electrically stimulated contractions in both naive and tolerant tissues, neither monkey antimorphine (Kd = 1.43 x 10(-7)M) nor goat antimorphine (Kd = 1.36 x 10(-9)M) were able to elicit a naloxone-like contracture. The rate of attenuation of tissue sensitivity to naloxone was measured and no changes were observed after incubation in the presence of monkey antimorphine globulin. High-affinity goat antimorphine globulin, however, was able to accelerate the rate of attenuation of tissue sensitivity to naloxone. The naloxone contracture was accompanied by release of material reactive with morphine antiserum in a radioimmunoassay. The quantities of material were in excess of what could be bound by opiate receptors present in the tissue. Possible mechanisms of naloxone sensitivity observed in the isolated ileum are discussed.
We have encountered two cases of late calcification of the porcine heterograft. A patient in chronic renal failure died of sepsis and endocarditis fifteen months after replacement of the mitral and tricuspid valves. At postmortem examination, both heterograft valves exhibited severe calcification and thrombosis. A second patient with rheumatic heart disease and sickle cell disease underwent mitral valve replacement for severe regurgitation. Thirty months later, cardiac catheterization revealed prosthetic valve stenosis. The valve was replaced successfully, and the excised heterograft exhibited severe calcification with restriction of leaflet motion. Although calcification of the porcine heterograft is known to occur in patients with infection or disorders of calcium metabolism, dysfunction of the heterograft is rare in our experience.
The specificity of antimorphine and antimeperidine antisera was measured by competitive displacement of immunizing radiolabeled haptens. Antimorphine antisera demonstrated a high degree of specificity for a conformation of the phenylpiperidine moiety contained within the structures of morphine and its congeners of the morphinan and benzomorphan series. Antimeperidine antisera demonstrated a high degree of specificity for a different conformation of the phenylpiperidine moiety represented within the structures of meperidine and its semisynthetic derivatives. The reactivity of methionine- and leucine-enkephalin, several synthetic enkephalin analogs, and alpha- and beta-endorphin with the antibodies was tested using purified immunoglobulin G in order to avoid serum-induced proteolysis. No significant cross-reactivity of antimorphine antibodies with any of the opioid peptides was detected. All of the opioid peptides tested exhibited weak but immunologically specific cross-reactivity with antimeperidine antibodies. These findings suggest that conformations analogous to the phenylpiperidine moiety in morphine as have been proposed for [Tyr1] in opioid peptides do not appear to be present as measured by immunochemical methods. A conformation with weak stereochemical similarity to the phenylpeperidine moiety in meperidine does appear to be present. The possible homologies between [Phe4] of opioid peptides, meperidine and hydrophobic side chains of certain oripavine derivatives are discussed.
Antimorphine antibodies produced in Rhesus monkeys immunized with morphine-6-hemisuccinate-BSA were passively administered to recipient monkeys trained to self-administer heroin and cocaine. Following antibody administration, changes in heroin self-administration behavior were observed which were similar to those achieved with low doses of naloxone. Both manipulations increased heroin self-administration without affecting cocaine responding.
Meperidinic acid was converted to O-meperidinyl-glycollic acid and covalently attached to bovine serum albumin. Rabbits injected with this conjugate produced antibodies reactive with meperidine which were measured by the ammonium sulfate method. The specificities of these antisera were studied by competitive inhibition of the binding of 100 pmol/ml of 3H-meperidine to antibody by the prior addition of increasing concentrations of various unlabeled compounds. The concentrations in nanomoles per milliliter of various unlabeled opiods required to inhibit 3H-meperidine binding by 50% (I50) were: meperidine, 0.08; O-meperidinyl-glycollic acid, 1.7; methadone, 580; heroin, 1750; codeine, 2600; and morphine, 4200. Several psychopharmacologically active compounds were found to have I50 values comparable to the nonmeperidine opioids: hydroxyzine. HCl, 460; propoxyphene, 4,500; diazepam, 6,500; and cocaine, 10,800. The metabolites of meperidine exhibited the following I50 values: normeperidine, 0.7; meperidinic acid and normeperidinic acid, 210. A radioimmunoassay for meperidine which employs this antiserum was shown to be approximately 100 times more sensitive than the spectrophotometric method of Burns et al. (J. Pharmacol. Exp Ther. 114: 289-293, 1955). In this assay only normeperidine and some of the meperidine congeners might be expected to interfere with the measurement of meperidine. The degree of normeperidine interference was shown to be comparable to that present in the existing assay method.
Meperidinic acid was converted to O-meperidinyl-glycollic acid and covalently attached to bovine serum albumin. Rabbits injected with this conjugate produced antibodies reactive with meperidine which were measured by the ammonium sulfate method. The specificities of these antisera were studied by competitive inhibition of the binding of 100 pmol/ml of 3H-meperidine to antibody by the prior addition of increasing concentrations of various unlabeled compounds. The concentrations in nanomoles per milliliter of various unlabeled opiods required to inhibit 3H-meperidine binding by 50% (I50) were: meperidine, 0.08; O-meperidinyl-glycollic acid, 1.7; methadone, 580; heroin, 1750; codeine, 2600; and morphine, 4200. Several psychopharmacologically active compounds were found to have I50 values comparable to the nonmeperidine opioids: hydroxyzine-HC1, 460; propoxyphene, 4,500; diazepam, 6,500; and cocaine, 10,800. The metabolites of meperidine exhibited the following I50 values: normeperidine, 0.7; meperidinic acid and normeperidinic acid, 210. A radioimmunoassay for meperidine which employs this antiserum was shown to be approximately 100 times more sensitive than the spectrophotometric method of Burns et al. (J. Pharmacol. Exp. Ther. 114:289-293, 1955). In this assay only normeperidine and some of the meperidine congeners might be expected to interfere with the measurement of meperidine. The degree of normeperidine interference was shown to be comparable to that present in the existing assay method.
Explore the source record for details and available documents.
Morphine clearance from the circulation of normal rabbits and rabbits with circulating anti-morphine antibody was studied. Individual animals were injected with 14C trace-labeled morphine in saline (6 mg morphine/kg body weight). The rabbits were bled at various times after morphine injection and the amount of morphine present in the serum at each time interval was determined. Morphine could be detected in the serum of normal animals for 1 week following injection and up to 12 weeks following injection in at least one animal with antibodies to morphine. The rate of morphine clearance in animals immunized to morphine-6-hemisuccinate-bovine serum albumin was not significantly different from normal during the first 4 hr after morphine injection. However, by 24 hr after injection, the rate of morphine clearance in morphine-6-hemisuccinate-bovine serum albumin-immunized animals was significantly slower than in normal animals. The amount of morphine present in the sera of the rabbits 24 hr after injection was related to the antigen-binding capacity and independent of antibody avidity. However, the rate of morphine clearance at times greater than 24 hr after injection was related to the average antibody avidity and not related to the antigen-binding capacity.
Explore the source record for details and available documents.