Biomedical subjects
Publications and source records attributed to D Liston.
The synthesis and SAR of a series of 6-(4-(substituted)phenyl)-2-aminopyridines as inhibitors of nitric oxide synthase are described. Compound 3a from this series shows potent and selective inhibition of the human nNOS isoform, with pharmacokinetics sufficient to provide in vivo inhibition of nNOS activity.
The synthesis of a series of alkylcarbamates of 1,5-methano-2,3,4,5-tetrahydro-1H-2-benzazepin-7-ol is reported. Many of these compounds are potent acetylcholinesterase (AChE) inhibitors. The in vitro AChE inhibition, cholinergic effects, acute toxicity, and elevation of brain acetylcholine levels in vivo of this series of compounds are described. A representative compound, 1d (5.6 mg/kg, po), was able to reverse hemicolinium-3-induced amnesia in the mouse passive avoidance assay.
Based on a national survey conducted in spring 1993 of 1,953 private and public employers, this DataWatch examines the design of employer-sponsored health benefits and how they have changed during the past five years. We contrast cost of coverage, employee cost sharing, and premium increases among small, mid-size, and large firms. Premiums increased 8.5 percent from 1992 to 1993, the lowest rate of increase since 1986-1987. Future premium increases should be modest by historical standards. Small firms and conventional plans experienced larger premium increases last year. Managed care plans now constitute 51 percent of enrollment, up from 29 percent in 1988. If current trends continue, even without health care reform legislation, the health care system of the future will contrast strikingly with the system most Americans remember from past decades.
To identify signals that direct the proteolytic processing and regulated secretion of human proenkephalin (hPE), we have transfected the hPE gene or minigene constructs into pituitary tumor cells, either rat GH4Cl cells or mouse AtT-20 cells. Cells transfected with either the hPE gene or minigene contained similar levels of methionine-enkephalin (ME)-containing peptides and hPE mRNA. In the GH4Cl clones, ME was present predominantly in high-molecular-mass forms (5-25 kDa). In contrast, the AtT-20 clones contained almost exclusively free ME and low-molecular-mass forms (< 5 kDa), with very little high-molecular-mass species present. Thus, among pituitary cells, corticotroph-derived cells appear better equipped to process hPE than lactotroph-derived cells. Despite limited proteolytic processing, GH4Cl clones secreted large amounts of unprocessed (> 20 kDa) hPE into the medium, making up to 10% of endogenous rat prolactin secretion. Both precursor and processed forms of ME were cosecreted acutely (< 1 h) with rat prolactin, and release of both polypeptides was stimulated up to 12-fold by secretagogues. Thus, complete proteolytic processing was not required for accurate targeting of hPE to the regulated secretory pathway. When transfected with constructs bearing deletions of amino-terminal amino acids 2-43 or 2-67, i.e., part or nearly all of the synenkephalin moiety, GH4Cl cells handled the modified protein much like cells expressing the complete protein. They did not process the modified hPE extensively, but the protein was correctly targeted to the regulated secretory pathway. AtT-20 cells transfected with truncated hPE cDNA constructs expressed and processed the protein as efficiently as cells expressing unmodified hPE and expressed predominantly low-molecular-mass forms of ME. Therefore, the structural features required for correct targeting and processing are not present in the cysteine-rich amino-terminal third of the prohormone. It is interesting that the deletions did not include the SHLL peptide motif in synenkephalin, a motif that has been proposed as a sorting signal.
The synthesis of a series of 1,2,3,3a,8,8a-hexahydroindeno[2,1-b]pyrrole 5-alkylcarbamates and their resolution are reported. These compounds are structurally related to physostigmine with substitution of a methylene group in place of the NMe group at position 8 of physostigmine. Many of these 8-carbaphysostigmine analogues are more potent acetylcholinesterase inhibitors in vitro and less toxic in vivo than physostigmine. The (-)-enantiomer (e.g., 1d and 1g) possessing the same absolute configuration at C3a and C8a as that of physostigmine, is about 6 to 12-fold more potent at inhibiting acetylcholinesterase than the corresponding (+)-enantiomer (e.g., 1e and 1h).
9-Amino-1,2,3,4-tetrahydroacridine (THA) has been reported to cause improvement in patients with senile dementia of the Alzheimer's type. We have examined some effects of THA in vitro and in vivo to define its mechanism of action. In vitro, THA inhibits acetylcholinesterase (AChE) (IC50 = 223 nM) and blocks [3H]AFDX-116 (M2) and [3H]telenzepine (M1) binding (IC50 s of 1.5 and 9.1 microM respectively). In vivo levels of THA were 10-fold higher in brain than plasma following 3.2 mg/kg i.p., a dose which was found to be active in reversing amnesia induced by scopolamine assessed in T-maze tests in rats and passive avoidance tests in mice. Additionally, these brain concentrations were above the IC50 of THA for AChE inhibition. THA (5.6-17.8 mg/kg i.p.) also elevated acetylcholine levels in the rat CNS. THA-induced side effects were blocked by the central muscarinic antagonist, scopolamine, but not by the peripheral antagonists methscopolamine and glycopyrrolate, nor by nicotinic antagonists. We conclude that brain AChE inhibition by THA is sufficient to explain its purported therapeutic activity in Alzheimer's disease and that its favorable brain/plasma distribution in vivo may account for its central cholinergic action without inducing the severe peripheral cholinergic effects typically seen with other AChE inhibitors.
Recent studies have indicated a normal gene dose for the amyloid precursor protein (APP) in Alzheimer's disease (AD). These findings leave open the possibility that elevated levels of messenger RNA (mRNA) for this protein may contribute to the pathogenesis of AD. Using Northern analysis, we compared the levels of mRNA for the APP and 3 cytoskeletal proteins in parietal cortex of 6 brains having marked AD-type degeneration with the levels of these mRNAs in 6 control samples. The cytoskeletal mRNAs studied were those for the human neurofilament 68-kDa subunit (HNFL), for alpha-tubulin, and for glial fibrillary acidic protein (GFAP). A ribonuclease (RNase) protection assay was also used to compare AD and control HNFL mRNA levels. The mRNAs for APP, HNFL, and alpha-tubulin were diminished in AD cortex. The decrement for APP mRNA was less than that for HNFL or alpha-tubulin. The message for GFAP in AD cortex showed no loss. The findings support a general deficit in neuronal mRNAs, including that for APP. They do not exclude the possibility of elevated levels of the message for the APP in small neuronal subsets, in subcortical neurons projecting to cortex, or as a generalized phenomenon in earlier stages of the disease.
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DNA probes derived from rat and human proenkephalin and prodynorphin genes have been used to localize these two opiate neuropeptide genes on human chromosomes. Hybridization of probes to Southern blots made with DNAs from a rodent-human somatic-cell hybrid panel indicates localization of proenkephalin to human chromosome 8 and of prodynorphin to human chromosome 20. In situ hybridization to metaphase chromosomes confirms these assignments and indicates regional localizations of proenkephalin to 8q23-q24 and of prodynorphin to 20p12-pter. A human genomic prodynorphin clone reveals a frequent two-allele TaqI polymorphism.
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Synenkephalin, which comprises 70 residues at the aminoterminal of proenkephalin, was studied with immunocytochemical methods in the human and bovine spinal cord. Immunoreactive fibers had the same general distribution as methionine-enkephalin, but not as leucine-enkephalin fibers. They were found in all spinal layers and were most numerous in lamina II (outer zone) and V-VI (lateral portion). Synenkephalin immunoreactivity was overall less dense than that of the enkephalins. These results suggest that proenkephalin is the precursor protein also in enkephalinergic neurons of the human spinal cord.
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A recombinant plasmid containing the human proenkephalin gene ligated to pBR322 was introduced into a mouse pituitary cell line (AtT-20D16v) that normally expresses pro-opiomelanocortin but not proenkephalin. The plasmid was introduced by co-transformation with the G418-selectable plasmid, pRSVneo. Stable transformants were isolated and analyzed for the presence of the human proenkephalin gene. AtT-20 transformants which had one or more copies of the human proenkephalin gene integrated stably into the mouse chromosomal DNA expressed a 1.45 kb mRNA identical in size to human proenkephalin mRNA. Primer extension analysis indicated that the human proenkephalin gene was accurately and efficiently transcribed from its own promoter. AtT-20 transformants that expressed the 1.45 kb human proenkephalin mRNA also expressed proenkephalin protein and cleaved the protein to form free Met-enkephalin. This is of particular interest because these cells do not cleave all of the available pairs of basic amino acids in the endogenous protein, pro-opiomelanocortin, the precursor to ACTH, beta-endorphin and melanocyte stimulating hormones. The release of both ACTH and Met-enkephalin from these cells is stimulated by corticotropin releasing factor, a natural secretagogue for ACTH, indicating that the two classes of peptide share a related secretory pathway.
Immunoblots combined with specific radioimmunoassays (RIAs) have been used to visualize simultaneously all the enkephalin-containing peptides (ECPs) present in a crude extract of bovine adrenal medulla. They have allowed the characterization of a new high molecular weight ECP which has a molecular weight of 23.3 kDalton, contains the amino-terminal part of proenkephalin and ends with the sequence of Leu-enkephalin at its carboxy-terminus.
Synenkephalin, the amino-terminal 1-70 residues of proenkephalin is released intact from bovine globus pallidus following potassium-induced depolarization in vitro via a Ca++ dependent mechanism. The release of synenkephalin accompanies that of Met-enkephalin in a molar ratio of 1/4. In contrast to Met-enkephalin which is readily destroyed when released, synenkephalin is not destroyed.
Most neuropeptides are synthesized as large precursor proteins. These precursors undergo a maturation process involving several proteolytic events that generate the biologically active peptides. The enzymatic mechanisms underlying this processing are still largely unknown. The processing of the precursor protein proenkephalin was studied in two different bovine tissues, the hypothalamus and adrenal medulla. The high molecular weight enkephalin-containing peptides that accumulate in these two tissues were found to be different, indicating the existence of two processing pathways for this neuropeptide precursor.
Synenkephalin, the amino-terminal 1-70 residues of proenkephalin, is released intact from bovine globus pallidus and neurohypophysis following potassium-induced depolarization in vitro via a Ca2+-dependent mechanism. The release of synenkephalin accompanies that of Met-enkephalin in a molar ratio of 1/4. In contrast to Met-enkephalin, which is readily destroyed when released, synenkephalin is not destroyed.
Immunoblotting combined with radioimmunoassays (RIAs) directed specifically towards certain sequences of the proenkephalin molecule has been used to characterize the enkephalin-containing peptides (ECPs) present in the bovine adrenal medulla. Immunoblotting allowed the simultaneous visualization of all ECPs present in a crude extract of this gland. Combining this technique with RIAs we have been able to characterize a new high molecular mass ECP, a 23.3-kDa protein which contains the amino-terminal part of proenkephalin and ends with the sequence of Leu-enkephalin at its carboxy-terminus.