Arthur J. Blume

Interleukin-1β decreases acetylcholine measured by microdialysis in the hippocampus of freely moving rats

Interleukin (IL-1) is a cytokine which plays an important role in the modulation of the acute response in host defense. This cytokine is also increased in patients with Alzheimers disease. In the present experiment systemic injection of IL-1 beta (7.5-50 micrograms/kg) decreased extracellular acetylcholine in the hippocampus. This effect could not be attributed entirely to general malaise since lithium chloride (130 mg/kg) had the opposite effect. Heat-inactivation of the cytokine eliminated the reduction of extracellular ACh. The results give further evidence of a relationship between the immune system and the central nervous system and suggest a possible relationship between IL-1 and cholinergic function or dysfunction in the hippocampus.

INTERLEUKIN-1 STIMULATES THE BETA-AMYLOID PRECURSOR PROTEIN PROMOTER

Summary1.Amyloid plaques found in the brains of Alzheimers diseased patients are composed of the 42 amino acid beta-amyloid peptide (BAP) which is processed out of the larger amyloid precursor protein (APP).2.To study the regulation of the APP gene expression, we have isolated the promoter region of this angle of this single-copy gene and produced a reporter gene system to determine if the promoter is responsive to agents that may cause the overproduction of APP leading to the abnormal accumulation of plaques in AD.3.The promoter contains sequences homologous to heat shock elements, AP-1 binding sites, and phorbol ester-inducible sequences as well as GG-rich regions found in other constitutively expressed genes.4.We show here that this promoter is inducible in cultured cells by interleukin-1 (IL-1) in a transient assay system and that the HSE and AP-1 binding site are required for this inducibility.5.This induction of transcription from the APP promoter implies that this gene is responsive to tropic and/or trophic agents which may be present in the diseased brain.

Quantitative measurement of alternatively spliced amyloid precursor protein mRNA expression in Alzheimer's disease and normal brain by S1 nuclease protection analysis

We have used an S1 nuclease protection strategy to measure alternatively spliced amyloid precursor protein (APP) mRNAs associated with Alzheimers disease (AD) to determine whether the expression of either one or more of the transcripts correlate with observed amyloid plaque pathology. Comparison of AD with normal cortex reveals that increasing plaque density parallels an increase in the fraction of APP-695 and a corresponding decrease in APP-770 and 751 mRNA fractions. A specific increase of APP-695, the protease inhibitor-lacking APP RNA form, in those brain regions most involved with amyloid plaque formation, suggests that an imbalance in the protease inhibitor is potentially significant in the disease. These data are consistent with cellular/tissue region-specific regulation of alternative splicing accounting for AD-related changes in the expression of APP mRNA forms.

Screening for potassium channel modulators by a high through-put 86-rubidium efflux assay in a 96-well microtiter plate

A rapid and sensitive 86Rb efflux assay to detect chemical compounds capable of modulating the ATP-dependent potassium (KATP) channel is described. This assay, which is performed in a 96-well microtiter plate, utilizes a substrate adherent cell line as the target, requires a small amount of 86Rb as the tracer, and is a suitable system for performing the biochemical and pharmacological characterization of the KATP-channel and its activators. Because this assay is amenable to automation, it presents a useful means for high-volume screening of chemical compounds on a routine basis.

Focusing on IL1-promotion of β-amyloid precursor protein synthesis as an early event in Alzheimer's disease

Abstract A rationale for increased synthesis of beta-amyloid peptide precursor (APP) protein in Alzheimers disease (AD) is developed in which Interleukin-1 (IL-1) plays a key role. This cytokine is elevated in AD, its receptors are on APP mRNA positive cells and it promotes APP gene expression. Potential involvement of the protease inhibitor (PI) activity of certain APP proteins in the activation process for IL-1 and Nerve Growth Factor (NGF) are proposed. The possibility of feedback loops among IL-1, APP and NGF and the implications for neuronal survival and function are discussed.

Mast cell degranulating peptide: a multi-functional neurotoxin.

Abstract— This review discusses our present knowledge of the structure and activities of the mast cell degranulating peptide (MCDP). This peptide is a basic, 22 amino acid residue component of honey bee venom with striking immunological and pharmacological activities. MCDP is a potent anti‐inflammatory agent, but at low concentrations it is a strong mediator of mast cell degranulation and histamine release. MCDP is also an epileptogenic neurotoxin, an avid blocker of the potassium channels and can cause a significant lowering of the blood pressure in rats. Some of the biological activities of MCDP appear to have distinct mechanisms and may represent a good illustration of the structure‐function relationship.

Reconstitution of affinity-purified dopamine D2 receptor binding activities by specific lipids

The role of lipids in maintaining ligand binding properties of affinity-purified bovine striatal dopamine D2 receptor was investigated in detail. The receptor, purified on a haloperidol-linked Sepharose CL6B affinity column, exhibited low [3H]spiroperidol binding unless reconstituted with soybean phospholipids. In order to understand the role of individual phospholipids in maintaining the receptor binding activity, the purified preparation was reconstituted separately with individual phospholipids and assayed for [3H]spiroperidol binding. Except for phosphatidylcholine and phosphatidylethanolamine, that respectively restored 30 and 20% binding as compared to that obtained with soybean lipids, reconstitution with other lipids had very little effect. When various combinations of phospholipids were used for reconstitution, a phosphatidylcholine and phosphatidylserine mixture seemed to almost fully restore the receptor binding. A mixture of phosphatidylcholine and phosphatidylethanolamine was as effective as phosphatidylcholine alone in reconstituting ligand binding; however, when phosphatidylserine was also included in the mixture, there was a pronounced increase in binding (about 2-fold compared to the soybean lipids and about 6-fold compared to the phosphatidylcholine-phosphatidylethanolamine mixture). Substitution of other phospholipids or cholesterol for phosphatidylserine in phosphatidylcholine and phosphatidylethanolamine mixture had little effect. Maximal reconstitution of [3H]spiroperidol binding was obtained with phosphatidylcholine, phosphatidylethanolamine and phosphatidylserine mixture (2:2:1, w/w) at a concentration of 0.5 mg/ml. The reconstituted receptor exhibited high affinity binding for [3H]spiroperidol which was comparable to that obtained with membrane or solubilized preparations. Various dopaminergic antagonists and agonists showed appropriate order of potency for the reconstituted receptor. The presently described reconstitution data suggest a role of specific phospholipids in preserving the binding properties of dopamine D2 receptor and should prove useful in studies on functional reconstitution of the receptor.

Gene expression in Xenopus oocytes

1. Gene expression in Xenopus oocytes is now an integral part of many molecular cloning strategies. 2. For some genes, such as those encoding the ion channels, this system has emerged as the only available means to authenticate and examine the biological activities of the cloned DNA. 3. This review discusses some of the current applications of Xenopus oocytes in modern molecular biology.

Effect of Halide Ions on t-[35S]Butylbicyclophosphorothionate Binding

The binding of t‐[35S]butylbicyclophosphoro‐thionate ([35S]TBPS) to a site on the GABAA receptor complex is ion dependent. This study was conducted to determine the effects of ion species and concentration on the time course, affinity, and number of sites of [35S]TBPS binding. At a concentration of 200 mM ion, the time to equilibrium for [35S]TBPS binding was shortest for I‐, followed by Br‐< CI‐< F‐. A similar rank order was observed for the concentration of ion required to produce half‐maximal [35S]TBPS binding. Saturation binding experiments were conducted to evaluate the effect of increasing ion concentration on the KD and Bmax of [35S]TBPS binding. The Bmax was independent of both ion species and concentration. The receptor affinity, however, increased with increasing concentration for each ion. Calculated maximal affinity values were not different between ions; however, the EC50 to produce those values was different among ions and ranked in the same order as that for time course and maximal binding data. Association and dissociation rates for [35S]TBPS binding were greater in I‐ than in Cl‐. These data emphasize the importance of ion selection and incubation times on [35S]TBPS binding.

Differential effects of iodide and chloride on allosteric interactions of the GABAA receptor

t‐[35S]Butylbicyclophosphorothionate ([35S]TBPS) has been shown to bind to the GABAA receptor complex. The binding is modulated allosterically by drugs that interact at components of the receptor complex. The present studies were designed to evaluate the influence of ionic environment and state of equilibrium on the allosteric modification of [35S]TBPS binding. In both I‐ and Cl‐ under nonequilibrium conditions, diazepam, γ‐aminobutyric acid (GABA), and pentobarbital (PB) stimulate and methyl 6,7‐dimethoxy‐4‐ethyl‐β‐carboline‐3‐carboxylate (DMCM) inhibits [35S]TBPS binding. In addition, there is an inhibitory component to the effect of GABA and PB at higher drug concentrations. These effects are blocked by the appropriate antagonists for each drug. In Cl‐, the stimulation of [35S]TBPS binding by drugs disappears at equilibrium, whereas the inhibition by GABA and PB persists. The inhibitory effect of DMCM in Cl‐ also disappears at equilibrium. When assayed in I‐ at equilibrium. however, DMCM stimulates [35S]TBPS binding. In addition, bicuculline, which is without effect under nonequilibrium conditions in either Cl‐ or I‐, stimulates [35S]TBPS binding in I‐ at equilibrium. The persistent effects of DMCM, bicuculline, and GABA in I‐ are accompanied by alterations in the affinity of [35S]TBPS for its receptor. In addition, the stimulation of [35S]TBPS binding by GABA is associated with a decreased number of [35S]TBPS binding sites. These data demonstrate that receptor complex interactions with anions influence the responsiveness to drug binding.