Biochemical markers in persons with preclinical familial Alzheimer disease.

BACKGROUND: Persons at risk for familial Alzheimer disease (FAD) provide a model in which biomarkers can be studied in presymptomatic disease. METHODS: Twenty-one subjects at risk for presenilin-1 (n = 17) or amyloid precursor protein (n = 4) mutations underwent evaluation with the Clinical Dementia Rating (CDR) scale. We obtained plasma from all subjects and CSF from 11. Plasma (Abeta(40), Abeta(42), F(2)-isoprostanes) and CSF (F(2)-isoprostanes, t-tau, p-tau(181), Abeta(40), Abeta(42), and Abeta(42)/Abeta(40) ratio) levels were compared between FAD mutation carriers (MCs) and noncarriers (NCs). RESULTS: Plasma Abeta(42) levels (25.1 pM vs 15.5 pM, p = 0.031) and the ratio of Abeta(42)/Abeta(40) (0.16 vs 0.11, p = 0.045) were higher in presymptomatic MCs. Among MCs, those with CDR scores of 0.5 had lower plasma Abeta(42) levels than those with CDR scores of 0 (14.1 pM vs 25.1, p = 0.02). The ratio of Abeta(42) to Abeta(40) was also reduced in the CSF (0.08 vs 0.15, p = 0.046) of nondemented MCs compared to NCs. Total CSF tau and p-tau(181) levels were elevated in presymptomatic FAD MCs. CSF levels of F(2)-isoprostanes were also elevated in MCs (n = 7, 48.6 pg/mL) compared to NCs (n = 4, 21.6 pg/mL, p = 0.031). CONCLUSIONS: Our data indicate that Abeta(42) is elevated in plasma in familial Alzheimer disease (FAD) mutation carriers (MCs) and suggests that this level may decrease with disease progression prior to the development of overt dementia. We also demonstrated that the ratio of Abeta(42) to Abeta(40) was reduced in the CSF of nondemented MCs and that elevations of t-tau and p-tau(181) are sensitive indicators of presymptomatic disease. Our finding of elevated F(2)-isoprostane levels in the CSF of preclinical FAD MCs suggests that oxidative stress occurs downstream to mismetabolism of amyloid precursor protein.

Pharmacology department.

Traditionally administered unfractionated heparin (UH) is a heterogeneous mixture of polysaccharide chains of varying length. Heparin is now available in new formulations, most notably the low-molecular-weight heparins (LMWHs), which possess pharmacology that is similar to but distinct from UH. Key advantages of LMWHs include improved bioavailability and longer half-life, more predictable anticoagulation that requires less laboratory monitoring, and fewer serious side effects. The article reviews the pharmacology of heparin and discusses the addition of LMWHs to the family of anticoagulant drugs.

Quantitative characterization of crude synaptosomal fraction (P-2) components by flow cytometry.

Flow cytometry, which definitively identifies each particle as positive or negative with respect to fluorescent markers, is used to characterize the P-2 fraction (crude synaptosomal fraction) with respect to primary components, size, and intactness. Particle size ranged from a few tenths of a microm to greater than 4.5 microm. The viable dye calcein AM labeled 90% of the preparation, indicating that the majority of particles were intact and esterase-positive. 66% of the P-2 fraction is neuronal in origin, as demonstrated by labeling with an antibody directed against SNAP-2. An antibody directed against glial fibrillary acidic protein (GFAP) labeled 35% of the particles in this preparation. The mitochondrial dye nonyl acridine orange (NAO) stained 74% of particles, indicating intra- and extrasynaptosomal mitochondria. Gating analysis reveals that SNAP-25 is enriched in the larger particles. These results suggest that flow cytometry may be used to take advantage of the increased viability, yield, and convenience of the P-2 fraction for studies of nerve terminal function.

Pharmacology department: pharmacologic approaches to abnormal blood lipids.

There are currently four classes of drugs available to treat dyslipidemia: niacin, bile acid-binding resins, 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) reductase inhibitors, and fibric acid derivatives. Each acts at a unique point in a complex set of interrelated lipid metabolic pathways. The mechanism of action and adverse effects of these four classes are reviewed briefly. The efficacy of antioxidants and the importance of compliance issues are described.

Pharmacology department. Antimicrobial resistance.

Patients with cardiovascular disease are often predisposed to multiple infections. The degree of resistance to antibiotics that has developed in the last decade makes a significant contribution to the severity of infections in this patient population. Mechanisms for development and spread of resistance between organisms are described and related to a clinical example.

Pharmacology.

Inadequate pain relief has been identified as a common problem after coronary artery bypass surgery, Understanding the pharmacology of analgesics commonly used after cardiovascular surgery is a fundamental part of effective pain management. The pharmacologic properties for two analgesic drug classes, opioid and nonopioid, are reviewed. Major drugs in each class are discussed in terms of their pharmacodynamic and pharmacokinetic properties.

cAMP decreases steady-state levels of delta-opioid receptor mRNA in NG108-15 cells.

We have compared several drug combinations for their ability to increase basal cAMP and to down-regulate delta-opioid receptor mRNA levels. Continuous treatment for up to 48 h with a phosphodiesterase inhibitor in combination with the adenylyl cyclase activator forskolin showed an early peak response, but cAMP levels returned to control after 8 and 24 h. Increases in cAMP level up to 150-fold were observed after treatment for 1 h with a series of drugs (rolipram, IBMX/forskolin, rolipram/forskolin, dibutyryl cAMP, and prostaglandin E2) that increase cAMP by different mechanisms. A significant decrease in DOR mRNA level, to 31% of control, followed the three treatments that produced the largest increases in cAMP level: IBMX/forskolin, rolipram/forskolin, and prostaglandin E2.

Selectivity of hemicholinium mustard, an affinity ligand, for the high-affinity choline transport system.

The selectivity of the irreversible inhibition of high-affinity choline uptake (HACU) by hemicholinium mustard (HCM; 2,2'-(4,4'-biphenylene)bis[2-hydroxy-4-(2-bromoethyl)-morpholine] hydrochloride) with respect to other cholinergic proteins and other sodium-dependent transport systems was examined. Preincubation of rat forebrain membranes with HCM, followed by washing and measurement of [3H]-hemicholinium-3 binding to the high-affinity choline transporter, was shown to decrease binding capacity (Bmax) by 70% without affecting the apparent affinity of the ligand. However, a similar preincubation, wash and binding experiment using [3H]-NMS as a ligand for muscarinic receptors showed no HCM effect on binding parameters. To measure the effects of HCM on choline acetyltransferase (ChAT), synaptosomes were incubated in HCM, then washed. The synaptosomes were lysed and ChAT activity was measured. Treatment with 50 microM HCM, a concentration that inhibits 100% of synaptosomal HACU, results in a 24% decrease in ChAT activity. HCM demonstrates little residual inhibition of other sodium-dependent neurotransmitter transporter transporters: preincubation with 50 microM HCM results in a decrease of 12% in transport of [3H]-dopamine and a decrease of 6% in the transport of [3H]-GABA. The binding of HCM, like that of hemicholinium-3 is sodium-dependent. HCM preincubation in the presence of sodium results in inhibition of HACU to 32% of control; in the absence of sodium HACU is 65% of control. This represents a loss of 51% of the observed irreversible inhibition produced by HCM. Irreversible inhibition by HCM can also be prevented by co-incubation with hemicholinium-3. Co-incubation with hemicholinium-3 results in residual HACU inhibition that decreases from 51% (HCM alone) to 28% (HCM + hemicholinium-3). When atropine instead of hemicholinium-3 is co-incubated with HCM, HCM still inhibits 40% of transport, demonstrating the pharmacological specificity of the protective effect of hemicholinium-3. Experiments in the guinea-pig myenteric plexus preparation demonstrate a gradual recovery from the residual effects of HCM. Evoked ACh release decreases to 24% immediately following treatment with 1 microM HCM. After 2 hr of recovery, tissues have recovered to about 50% of control levels, after which recovery continues at a slower rate.

Regulation of acetylcholine synthesis in the presence of hemicholinium mustard.

High affinity choline uptake (HACU) is a critical element in the synthetic pathway for acetylcholine (ACh), and is known to demonstrate activity-dependent regulation in vivo and in vitro. However, little is known about this important sodium-dependent transport protein at the biochemical level, and about the nature of its interaction with the ACh synthetic enzyme ChAT. Hemicholinium mustard (HCM), an irreversibly binding analog of hemicholinium-3 (HC3), was used to create a preparation with HACU that is completely inhibited in order to investigate the immediate source of Ch for ACh synthesis. Rat brain synaptosomes were pre-incubated with HCM and washed before transport incubations of increasing length (0-6 min) were carried out. The contribution of endogenous and extracellular (tracer) Ch to the ACh level was measured at each time point using a gas chromatography mass spectrometry (GCMS) system that allows quantitative measurement of endogenous (unlabelled; [2Ho]) Ch as well as tracer (deuterium-labelled; [2H4]) Ch. The hypothesis was that if an endogenous intraterminal Ch pool can be used for ACh synthesis, an increase in unlabelled ACh across time would be observed. In neither HCM-treated nor control synaptosomes was an increase observed in intraterminal (pellet) unlabelled ACh. To test the effects of high tissue demand, in other experiments synaptosomes were depolarized with addition of 40 mM KCl to the buffer after HCM treatment; again, no significant increase in intraterminal unlabelled ACh was observed across time. These experiments demonstrate that endogenous unlabelled Ch does not contribute to ACh synthesis, even when HACU is inactivated, and under conditions of high demand.

Characterization of the irreversible inhibition of high-affinity choline transport produced by hemicholinium mustard.

The inhibition of high-affinity choline transport by hemicholinium mustard (HCM), an alkylating analogue of hemicholinium-3, was examined in rat brain synaptosomes and guinea pig myenteric plexus. In synaptosomes, 50% high-affinity choline transport inhibition occurs with an HCM concentration of 104 nM (4-min incubation). A 10-min preincubation with 10 microM HCM results in essentially complete (greater than 95%) inactivation that persists after washing. Low-affinity choline transport in synaptosomes is unaffected by HCM inhibition at all concentrations examined (1-50 microM). Time course experiments indicate that the maximum irreversible inhibition (58%) seen after a 1-min preincubation with 500 nM HCM decreases to 46% inhibition after a 15-min preincubation; however, analysis of variance reveals that this difference is not significant. HCM inhibition of acetylcholine release from myenteric plexus-longitudinal muscle preparations persists for at least 2 h after removal of drug from the incubation bath; this inactivation can be prevented by coincubation with a high choline concentration during treatment with the mustard. In contrast, inhibition produced by the parent compound hemicholinium-3 is largely reversed by washing in both preparations examined. The observed potency and selectivity of HCM suggest its usefulness as a covalent probe for high-affinity choline transport.