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Biomedical subjects

J M Bidlack

Publications and source records attributed to J M Bidlack.

90 records · Page 5Linked to original sources
PubMed1985

A monoclonal antibody capable of modulating opioid binding to rat neural membranes.

A monoclonal antibody capable of inhibiting opioid binding to rat neural membranes has been produced. Spleen cells from a BALB/c mouse, immunized with a partially purified opioid receptor complex, were fused with P3-X63.Ag8.653.3 myeloma cells. The cell line OR-689.2.4 secreted an IgM that was capable of partially inhibiting opioid binding to rat neural membranes under equilibrium binding conditions, while not affecting the binding of nonopioid ligands. Control mouse immunoglobulins and heat-denatured OR-689.2.4 did not inhibit opioid binding to membranes. The purified immunoglobulin inhibited the binding of [3H]dihydromorphine in a titrable, saturable, and reversible manner, as well as the binding of the delta-ligand [3H][D-Ala2,D-Leu5]enkephalin, the kappa-ligand [3H] ethylketocyclazocine, and 3H-labeled antagonists. In addition to blocking the binding of opioids to membranes, the immunoglobulin could also displace bound [3H]dihydromorphine from neural membranes. The 125I-labeled immunoglobulin specifically bound to neural membranes with a Kd of 1.3 nM and a maximal number of binding sites of 41.8 fmol/0.25 mg of membrane protein. In a titrable manner, the immunoglobulin precipitated opioid binding sites from a solubilized preparation of neural membranes. When OR-689.2.4 conjugated to Sepharose was incubated with the partially purified opioid receptor complex, labeled with 125I, a 35,000-dalton protein was specifically bound by the immunoglobulin. This antibody provides a tool for probing the multiple opioid binding sites.

Animals↗
PubMed1985

Multiple sclerosis. Cerebrospinal fluid immune complexes that bind C1q.

We used a sensitive C1q-binding assay to measure levels of soluble immune complexes in 182 samples of cerebrospinal fluid (CSF) from control patients and patients with multiple sclerosis (MS). Soluble immune complexes in CSF were detected in 16% of patients with progressive MS, 38% of patients with exacerbating-remitting MS, 55% of patients with infectious or inflammatory diseases, 3% of patients with noninflammatory neurologic disorders, and in 0% of control patients with back pain. No correlations were found between the results of the C1q-binding assay and abnormalities of other CSF parameters. These included an elevated level of myelin basic protein, pleocytosis, oligoclonal bands, or an increased IgG level. Because of the lack of correlation to laboratory indexes of disease activity and the nonspecificity of a positive test, the C1q-binding assay seems to have little clinical usefulness in the diagnosis or management of patients with MS.

Antigen-Antibody Complex↗
PubMed1985

Multiple sclerosis: free light chains in cerebrospinal fluid.

We found free light chains in the CSF of 18 MS patients, but not in any of 14 patients with other neurologic disease. CSF from all MS patients contained kappa and lambda chain dimers, less frequently contained light chain monomers, and never contained free gamma heavy chains. Light chains were not detected in matched serum samples. CSF from MS patients did not release free light chains from whole IgG in CSF of controls. The findings suggest that these free light chains originate in plasma cells, not from degradation of whole IgG.

Adult↗
PubMed1984

A freezing method for cell fusions to distribute and reduce labor and permit more thorough early evaluation of hybridomas.

A method is described whereby cell fusions can be bulk-frozen shortly after the hybridization step. Recoveries are shown to be comparable to those obtained for control hybridomas cultured without freezing. Advantages are discussed in terms of labor distribution and antibody assay and evaluation strategies. It is further shown that peritoneal feeder cell preparations can be conveniently frozen as a means of workload reduction.

Animals↗
PubMed1983

Generating monoclonal antibodies to the opioid receptor.

BALB/c mice were immunized with an opioid receptor complex over the period of 1 year. Spleen cells from the mouse, whose serum inhibited opiate binding to rat neural membranes to the greatest extent, were fused with P3-X63-Ag8. 653.3 myeloma cells. By radioimmunoassay (RIA), 32 cell lines have been detected that secrete an antibody to a component of the isolated receptor complex. Antibodies from 2 of the cell lines have an effect on opiate binding to rat neural membranes. One antibody, OR-689.2.4 is an IgM cryoglobulin. This antibody partially inhibited the binding of 3H-dihydromorphine (3H-DHM), 3H-naloxone, 3H-ethylketocyclazocine (3H-EKC), and 3H-D-Ala2, D-Leu5 enkephalin (3H-DADLE) to rat neural membranes. The other antibody, OR-465.3, inhibited the binding of 3H-DHM and 3H-naloxone to rat neural membranes by a maximum of 70%. This antibody also inhibited the binding of 3H-DADLE to neural membranes but, did not affect the binding of this peptide to membranes from the neuroblastoma-glioma hybrid cell line, NG108-15. Work is ongoing to generate monoclonal antibodies specific for each subclass of opioid receptor.

Animals↗
PubMed1983

14 beta-(2-bromoacetamido)morphine and 14 beta-(2-bromoacetamido)morphinone.

14 beta-(2-Bromoacetamido)morphine (6) and 14 beta-(2-bromoacetamido)morphinone (9) were prepared preferably from the adduct of thebaine and 1-chloro-1-nitrosocyclohexane, which on reduction in methanol solution gave 14-aminocodeinone (2) and the corresponding ketal (3). When tested in a receptor-binding assay, the IC50 values of 6 and 9 were 15 and 10 nM, respectively. If the incubation time during the assay was increased from 15 to 30 min, irreversible binding of both ligands was observed.

Animals↗
PubMed1983

Molecular characteristics of brain opiate and nicotine receptors.

The present review has dealt with some of the molecular characteristics of the receptors associated with the action of opiates and nicotine. The binding characteristics of the opiate receptor in brain membrane preparations has been compared with those of a Triton X-100-solubilized extract of membranes as well as a preparation purified from rat brain by affinity chromatography. The purified opiate receptor appears to consist of three proteins; and the major component, which has a molecular weight of 35,000, may be the site of attachment of the opiate. Although phosphatidylserine appears to be associated with the opiate receptor within the membrane, its exact role in receptor binding is not known. The brain receptor for nicotine, which has a Kd in the nanomolar range, appears to have binding characteristics which differ from that of the classical nicotinic cholinergic receptor. This conclusion is corroborated by a characteristic behavioral response elicited by nicotine and not by cholinergic agonists. Finally, a discussion is presented on the presence of opiate receptors in human erythrocytes and leukocytes and the changes occurring in opiate addicts.

Animals↗
PubMed1982

Affinity labeling and purification of the opiate receptor from rat brain.

Affinity labeling of the opiate receptor has been performed on neural membranes from rat brain utilizing[125I]-14-bromoacetamidomorphine, an opiate agonist, and [125I]-14-chloracetylmorphine, an antagonist. With the use of SDS gel electrophoresis it could be shown that the agonist labeled three proteins with molecular weights of 43,000, 35,000 and 23,000, whereas the antagonist only labeled the 23,000 component. The preferential labeling of the 23,000 protein by the antagonist suggests that this component may be a primary recognition site for opiate antagonists. Calcium was stimulatory to the affinity labeling of all three proteins while sodium was inhibitory. With the use of affinity columns prepared by conjugating either ligand to omega-aminohexyl Sepharose, a receptor complex was obtained consisting all three proteins. Stereospecific opiate binding was demonstrable in the complex prepared from either column.

Affinity Labels↗
PubMed1981

Sites and mechanisms for nicotine's action in the brain.

A variety of pharmacologic, behavioral, and receptor-binding studies were performed in an effort to determine the mechanism and site of action of nicotine on the rat brain. When nicotine was given into the lateral or fourth ventricles or directly into the lateral vestibular nuclei of rats, it produced a characteristic prostration often accompanied by tonic seizures and body rotation along a longitudinal axis. Of a variety of brain areas studied, the prostration response could only be elicited from the lateral and, to a lesser extent, medial vestibular nuclei. The response could not be produced by a variety of cholinergic agonists or antagonized with nicotinic cholinergic antagonists, with the possible exception of mecamylamine. A good correlation was observed between the ability of nicotine analogues to antagonize the nicotine-induced prostration and their ability to compete with 3H-nicotine binding to rat brain membranes. 3H-nicotine binding had a high affinity, was stereoselective and concentrated in nerve endings and such brain regions as the thalamus, cerebrum, and hippocampus. When nicotine was administered intraventricularly to rats, it significantly elevated the threshold to an aversive shock. It was concluded that many of the central actions of nicotine could not be explained on the basis of traditional nicotinic cholinergic mechanisms.

Animals↗
PubMed1981

Purification of the opiate receptor from rat brain.

The opiate receptor was purified from a Triton-solubilized preparation of rat neural membranes by the use of affinity chromatography. The affinity gel was prepared by coupling 14-beta-bromoacetamidomorphine, a newly synthesized ligand, to omega-aminohexyl-Sepharose. After elution of the nonspecific proteins with 50 mM Tris (pH 7.5), the receptor proteins were eluted with 1 microM levorphanol or etorphine. NaDodSO4/polyacrylamide gel electrophoresis revealed three major proteins associated with the opiate receptor, having molecular weights of 43,000, 35,000, and 23,000. The purified receptor binds 10(-11) mol of dihydromorphine/per mg of protein, with a Kd of 3.8 X 10(-9) M. Other opiates, naloxone, and methionine-enkephalin, inhibit [3H]dihydromorphine binding in a manner similar to that observed with intact and solubilized neural membranes.

Animals↗
PubMed1980

Adenosine 3',5'-monophosphate-dependent phosphorylation of a 6000 and a 22,000 dalton protein from cardiac sarcoplasmic reticulum.

In canine cardiac sarcoplasmic reticulum, adenosine 3',5'-monophosphate (cyclic AMP)-dependent protein kinase specifically phosphorylates two proteins, as seen by sodium dodecyl sulfate-slab gel electrophoresis and autoradiography. One protein has a molecular weight ranging between 22,000 and 24,000 daltons and has previously been identified and named phospholamban (Tada, M., Kirchberger, M.A. and Katz, A.M. (1975) J. Biol. Chem. 250, 2640-2647). The other protein that the 32P label incorporates into has a molecular weight of approximately 6000. Like the 22,000 dalton protein, the 6000 dalton protein has characteristics of phosphoester bonding. The time-dependent course of phosphorylation shows that initially the 32P label is incorporated more rapidly into the 22,000 dalton protein than the 6000 dalton protein, with both proteins reaching a steady-state level of phosphorylation after 10 min of incubation. When both protein kinase and cyclic AMP are eliminated from the incubation medium, both the 22,000 and the 6000 dalton protein are still phosphorylated, but only to about a quarter of the activity found when cyclic AMP and protein kinases are included in the incubation mixture. The addition of phosphodiesterase completely eliminates the phosphorylation of both proteins. Treating the microsomes with trypsin prevents subsequent phosphorylation of either protein. Phosphorylating the microsomes first, then treating with trypsin, renders both the 22,000 and the 6000 dalton proteins resistant to even prolonged trypsin attack. Unphosphorylated, both proteins are solubilized by a very low concentration of deoxycholate. After phosphorylation the proteins cannot be solubilized by deoxycholate. Phosphorylation appears to alter greatly the physical properties of these proteins. Control experiments exclude the possibility that a lipid is being phosphorylated. After phosphorylation the phosphorylated 22,000 dalton protein is separated from the 6000 dalton protein by proteolipid extraction. After first treating the microsomes with methanol, the 22,000 dalton protein is then soluble in acidified chloroform/methanol, while the 6000 dalton protein remains insoluble. The finding that both proteins have much different biochemical properties when phosphorylated than when not, may be relevant in how they regulate calcium transport in the sarcoplasmic reticulum.

Animals↗
PubMedDate not supplied

Beta-endorphin stimulates rat T lymphocyte proliferation.

beta-Endorphin 1-31 and several structurally related peptides were tested for their ability to alter mitogen-induced T cell proliferation. Rat beta-endorphin 1-31 and human beta-endorphin 1-27 increased phytohemagglutinin (PHA)-induced [3H]thymidine incorporation into rat lymph node cells. However, when PHA-induced proliferation was suppressed by the inclusion of prostaglandin E1 (PGE1), human beta-endorphin 1-31 and a number of structurally similar peptides, including some peptides that did not alter mitogen-induced proliferation, significantly reduced the PGE1 inhibition of PHA-stimulated T cell proliferation. Although the N-terminus of beta-endorphin was necessary for potency, inclusion of the opioid antagonist naloxone together with beta-endorphin 1-31 did not alter the blockage of PGE1 inhibition of PHA-induced proliferation caused by beta-endorphin. The inhibition of mitogen-stimulated proliferation by either cholera toxin or forskolin, two additional compounds that like PGE1 also elevate cyclic AMP levels, was not blocked by beta-endorphin. Verapamil suppression of proliferation was not modified by beta-endorphin, indicating that the beta-endorphin stimulatory effect was probably not due to Ca2+ influx through verapamil-sensitive Ca2+ channels. These data suggest that beta-endorphin, acting through a nonopioid beta-endorphin receptor, may modulate immunocompetence by stimulating T cell proliferation and by counteracting the inhibitory effects of PGE1.

Alprostadil↗
PubMedDate not supplied

Six highly active mu-selective opioid peptides identified from two synthetic combinatorial libraries.

Two synthetic combinatorial libraries (SCLs) were prepared, each composed of 52,128,400 L-amino acid hexapeptides, one with and the other without an N-terminal acetyl moiety. The two libraries were used in conjunction with an iterative selection process to identify individual peptides capable of inhibiting the binding of the mu-selective opioid peptide [3H]-[D-Ala2,MePhe4,Gly-ol5]enkephalin to rat brain homogenates. As reported previously, when using the nonacetylated SCL the first five residues identified corresponded exactly to methionine- and leucine-enkephalin, both of which are endogenous opioid peptides. The iterative identification process has now been completed for two additional mixtures found to have activity in the initial screening of this SCL. Two new series unrelated to the enkephalins have been identified: YPFGFO-NH2 and WWPKHO-NH2 (where O = one of the 20 L-amino acids). Individual peptides from each of these were found to be agonists at the mu receptor and have high affinity (IC50 values of the most active peptides were 10-15 nM) and selectivity for the mu receptor. In addition to the acetalins (described previously), two new series have now been identified from the acetylated library: Ac-FRWWYO-NH2 and Ac-RWIG-WO-NH2 (IC50 values of the most active peptides were 5-30 nM). Ac-FRWWYM-NH2 was determined to be an agonist at the mu receptor, whereas Ac-RWIGWR-NH2 was found to be an antagonist at this receptor.

Acetylation↗