Processing of proenkephalin is tissue-specific.

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.

Analysis of a ubiquitous promoter element in a primitive eukaryote: early evolution of the initiator element.

Typical metazoan core promoter elements, such as TATA boxes and Inr motifs, have yet to be identified in early-evolving eukaryotes, underscoring the extensive divergence of these organisms. Towards the identification of core promoters in protists, we have studied transcription of protein-encoding genes in one of the earliest-diverging lineages of Eukaryota, that represented by the parasitic protist Trichomonas vaginalis. A highly conserved element, comprised of a motif similar to a metazoan initiator (Inr) element, surrounds the start site of transcription in all examined T. vaginalis genes. In contrast, a metazoan-like TATA element appears to be absent in trichomonad promoters. We demonstrate that the conserved motif found in T. vaginalis protein-encoding genes is an Inr promoter element. This trichomonad Inr is essential for transcription, responsible for accurate start site selection, and interchangeable between genes, demonstrating its role as a core promoter element. The sequence requirements of the trichomonad Inr are similar to metazoan Inrs and can be replaced by a mammalian Inr. These studies show that the Inr is a ubiquitous, core promoter element for protein-encoding genes in an early-evolving eukaryote. Functional and structural similarities between this protist Inr and the metazoan Inr strongly indicate that the Inr promoter element evolved early in eukaryotic evolution.

Initiator recognition in a primitive eukaryote: IBP39, an initiator-binding protein from Trichomonas vaginalis.

While considerable progress has been made in understanding the mechanisms of transcription in higher eukaryotes, transcription in single-celled, primitive eukaryotes remains poorly understood. Promoters of protein-encoding genes in the parasitic protist Trichomonas vaginalis, which represents one of the deepest-branching eukaryotic lineages, have a bipartite structure with gene-specific regulatory elements and a conserved core promoter encompassing the transcription start site. Core promoters in T. vaginalis appear to consist solely of a highly conserved initiator (Inr) element that is both a structural and a functional homologue of its metazoan counterpart. Using DNA affinity chromatography, we have isolated an Inr-binding protein from T. vaginalis. Cloning of the gene encoding the Inr binding protein identified a novel 39-kDa protein (IBP39). We show that IBP39 binds to both double and single Inr motifs found in T. vaginalis genes and that binding requires the conserved nucleotides necessary for Inr function in vivo. Analyses of the cloned IBP39 gene revealed no homology at the protein sequence level with identified proteins in other organisms or the presence of known DNA-binding domains. The relationship between IBP39 and Inr-binding proteins in metazoa presents interesting evolutionary questions.

Characterization of new enkephalin-containing peptides in the adrenal medulla by immunoblotting.

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.

Syntheses and anticholinesterase activity of tetrahydrobenzazepine carbamates.

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.

Syntheses, resolution, and structure-activity relationships of potent acetylcholinesterase inhibitors: 8-carbaphysostigmine analogues.

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).

Design and synthesis of 1-heteroaryl-3-(1-benzyl-4-piperidinyl)propan-1-one derivatives as potent, selective acetylcholinesterase inhibitors.

Herein is described the synthesis and structure--activity relationship of a novel series of aromatic and heteroaromatic 3-(1-benzyl-4-piperidinyl)propan-1-one derivatives that display potent and selective inhibition of the enzyme acetylcholinesterase (AChE). 1-(2-Methyl-6-benzothiazolyl)-3-(N-benzyl-4-piperidinyl)propan-1-one hydrochloride, 6d, is one of the most active compounds within this series exhibiting an IC50 for the inhibition of the AChE enzyme equal to 6.8 nM. Compound 6d has shown a dose-dependent elevation of total acetylcholine (ACh) levels in the mouse forebrain with an oral ED50 = 9.8 mg/kg. In addition, in vivo microdialysis experiments in the rat demonstrate that 6d increases extracellular ACh (100% over basal) 1-3 h postdose with an oral ED50 = 4.8 mg/kg.

Novel benzisoxazole derivatives as potent and selective inhibitors of acetylcholinesterase.

A series of N-benzylpiperidine benzisoxazoles has been developed as potent and selective inhibitors of the enzyme acetylcholinesterase (AChE). The benzisoxazole heterocycle was found to be an appropriate bioisosteric replacement for the benzoyl functionality present in the N-benzylpiperidine class of inhibitors. The title compounds were synthesized by alkylating 3-methyl-1,2-benzisoxazoles with an iodo piperidine derivatives as the key step. Benzisoxazoles 1b-j,o displayed potent inhibition of AChE in vitro with IC50's = 0.8-14 nM. Particularly interesting were N-acetyl and morpholino derivatives 1g (IC50 = 3 nM) and 1j (IC50 = 0.8 nM), respectively, which displayed outstanding selectivity for acetyl-over butyrylcholinesterase, in excess of 3 orders of magnitude. N-Acetyl 1g also displayed a favorable profile in vivo. This analog showed a dose-dependent elevation of total acetylcholine in mouse forebrain after oral administration with an ED50 = 2.4 mg/kg. In addition, 1g was able to reverse amnesia in a mouse passive avoidance model at doses of 3.2 and 5.6 mg/kg with an average reversal of 89.7%. Molecular dynamics simulations were used to study the possible binding modes of N-benzylpiperidine benzisoxazoles to AChE from Torpedo californica. Key structural insights were obtained regarding the potency of this class of inhibitors. Specifically, Asp-72, Trp-84, Trp-279, Phe-288, and Phe-330 are implicated in the binding of these inhibitors. The N-benzylpiperidine benzisoxazoles may be suitable compounds for the palliative treatment of Alzheimer's Disease.

5,7-dihydro-3-[2-[1-(phenylmethyl)-4-piperidinyl]ethyl]-6H- pyrrolo[3,2-f]-1,2-benzisoxazol-6-one: a potent and centrally-selective inhibitor of acetylcholinesterase with an improved margin of safety.

A series of N-benzylpiperidines (2a-d, 10) with novel isoxazole-containing tricycles has been prepared. This series has shown potent in vitro inhibition of the enzyme acetylcholinesterase (AChE), with IC50S = 0.33 - 3.6 nM. Compound 2a was the most potent inhibitor with an IC50 = 0.33 +/- 0.09 nM. Derivatives 2a-d and 10 displayed weak in vitro inhibition of butyrylcholinesterase (BuChE) with IC50S = 600 - 23,000 nM. The most selective compound was 2a with a BuChE/AChE ratio in excess of 4 orders of magnitude (> 10,000). Pyrrolobenzisoxazole 2a also displayed a favorable profile in vivo. In microdialysis experiments, 2a produced a 200% increase in extracellular levels of acetylcholine (ACh) at a dose of 0.4 mg/kg in freely moving, conscious rats. Peripheral side effects (salivation ED50 = 26 +/- 1.5 mg/kg) and acute lethality (LD50[1 h] = 42 mg/kg) were observed at > 60-fold higher doses. These data indicate that 2a is an AChE inhibitor with good central selectivity and a favorable margin of safety. Compound 2a, designated as CP-118,954, is currently in clinical development for the treatment of cognitive disorders.

Distribution and characterization of synenkephalin immunoreactivity in the bovine brain and pituitary.

The distribution of synenkephalin, the N-terminal fragment of proenkephalin, was studied in various parts of the bovine brain (globus pallidus, caudate nucleus, hypothalamus) and in the posterior pituitary by the use of a radioimmunoassay. The distribution of synenkephalin-immunoreactivity (IR) was compared to the distribution of Met-enkephalin-IR. Gel exclusion chromatography was used to examine the molecular forms of the immunoreactivities present in the tissues. The distribution of synenkephalin-IR was similar to the distribution of Met-enkephalin-IR, with a molar ratio of Met-enkephalin/synenkephalin ranging between 2.7 and 5.9. In all regions tested except the hypothalamus the synenkephalin-IR was present as a single species. However, in the hypothalamus a small amount of IR material (3% of the total synenkephalin-IR) was detected in fractions where larger Met-enkephalin-containing peptides eluted. Based on the concordance between the molar ratio of Met-enkephalin to synenkephalin found in the tissues and the molar ratio present in the sequence of adrenal proenkephalin, it is concluded that the brain and adrenal glands utilize a similar precursor for enkephalin biosynthesis.

Release of synenkephalin from neuronal terminals in vitro.

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.

Distribution of enkephalin-containing peptides within bovine chromaffin granules.

The distribution of large enkephalin-containing peptides (ECP's) between soluble and membrane components of bovine chromaffin granules was examined by immunoblotting with synenkephalin antiserum which recognizes the NH2-terminus of proenkephalin. Immunoblots showed that the 23.3 and 18.2 kilodalton ECP's were present in both soluble and membrane granule compartments but the 12.6 kilodalton ECP was present only in the soluble fraction. These results suggest that the larger ECP's may be preferentially associated with the granule membrane and may be redistributed to the soluble granule compartment upon proteolytic processing.

Characterization of a new 23 kDalton enkephalin-containing protein in the bovine adrenal medulla.

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.

The health insurance picture in 1993: some rare good news.

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.

Correlation of brain levels of 9-amino-1,2,3,4-tetrahydroacridine (THA) with neurochemical and behavioral changes.

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.

Clozapine is a potent and selective muscarinic M4 receptor agonist.

Clozapine was studied in functional assays at human muscarinic M1-M5 receptors expressed in Chinese hamster ovary cells. Clozapine was a full agonist at the muscarinic M4 receptor (EC50 = 11 nM), producing inhibition of forskolin-stimulated cAMP accumulation. In contrast, clozapine potently antagonized agonist-induced responses at the other four muscarinic receptor subtypes. Selective stimulation of M4 receptors may, in part, explain the hypersalivation observed clinically with clozapine. Moreover, the unique overall muscarinic profile of clozapine may contribute to its atypical antipsychotic efficacy.

Methyl-beta-carboline-induced convulsions are antagonized by Ro 15-1788 and by propyl-beta-carboline.

Injected i.v. into baboons, Ro 15-1788 (a benzodiazepine antagonist) and propyl-beta-carboline-3-carboxylate did not modify either the behavior or the electroencephalogram at doses up to 2 mg/kg. Methyl-beta-carboline-3-carboxylate is a potent convulsant at doses of 20 micrograms/kg in photosensitive baboons and 100 micrograms/kg in non-photosensitive baboons. These convulsive doses of methyl-beta-carboline-3-carboxylate are effectively antagonized by 0.5 mg/kg of Ro 15-1788 and also by 2 mg/kg of propyl-beta-carboline-3-carboxylate.

Synenkephalin is coreleased with Met-enkephalin from neuronal terminals in vitro.

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.

Purification of the N-terminal fragment of proenkephalin from bovine adrenal medulla.

Two forms of the N-terminal fragment of proenkephalin have been purified from the bovine adrenal medulla and characterized. One of these proteins contains the sequence of Met-enkephalin and is composed of residues 1-77 of proenkephalin. The other protein does not contain Met-enkephalin and is composed of residues 1-72 of proenkephalin.

Calcium and vitamin D status of pregnant teenagers in Maiduguri, Nigeria.

This study investigates parameters related to calcium and bone metabolism by determining the concentrations of total calcium, 25-hydroxyvitamin D, 1,25-dihydroxyvitamin D, parathyroid hormone, and phosphorous in young pregnant women. The patient population was 30 pregnant Nigerian teenage women grouped by trimester (10 per group), 10 women immediately following delivery, and 21 healthy age-matched controls. On the basis of serum prealbumin levels, the general nutrition of the pregnant women was found to be significantly below that of the more privileged and better-educated nonpregnant controls. The mean total calcium concentration in sera of the third-trimester women was 8.83 mg/dL, which was significantly below that of the controls (9.77 mg/dL) and the first-trimester group (9.30 mg/dL). Despite the 10% to 15% decline in the serum level of total calcium during pregnancy, the parathyroid hormone level decreased markedly from 0.60 to 0.61 ng/mL in the first and second trimesters to 0.41 ng/mL in the third trimester. Serum vitamin D and 1,25-dihydroxyvitamin D levels in the second and third trimesters were within the normal range. These data indicate that toward the end of gestation, pregnant teenagers in northern Nigeria appear to become calcium deficient and do not exhibit the expected increase in serum parathyroid hormone levels normally seen in pregnant women.