Comparison of eyeblink conditioning in patients with superior and posterior inferior cerebellar lesions.

The aim of the present study was to compare eyeblink conditioning in cerebellar patients with lesions including the territory of the superior cerebellar artery (SCA) and in patients with lesions restricted to the territory of the posterior inferior cerebellar artery (PICA). The cerebellar areas known to be most critical in eyeblink conditioning based on animal data (i.e. Larsell lobule H VI and interposed nucleus) are commonly supplied by the SCA. Eyeblink conditioning was expected to be impaired in SCA, but not in PICA patients. A total of 27 cerebellar patients and 25 age-matched controls were tested. Cerebellar lesions were primarily unilateral (n = 20). Most patients suffered from ischaemic infarctions of the SCA (n = 11) or the PICA (n = 13). The other patients presented with cerebellar tumours (n = 2) and cerebellar agenesis (n = 1). The extent of the cortical lesion (i.e. which lobuli were affected) and possible involvement of the cerebellar nuclei was determined by 3D-MRI. As expected, the ability to acquire classically conditioned eyeblink responses was significantly reduced in the group of all cerebellar patients compared with the controls. In the patients with unilateral cerebellar lesions, conditioning deficits were present ipsilaterally. In SCA patients with lesions including hemispheral lobules VI and Crus I, eyeblink conditioning was significantly reduced on the affected side compared with the unaffected side. No significant difference between the affected and unaffected sides was present in patients with lesions restricted to the common PICA territory (i.e. Crus II and below). Conditioning deficits were neither significantly different in SCA patients with pure cortical lesions compared with SCA patients with additional nuclear impairment nor in SCA patients with unilateral lesions compared with SCA patients with bilateral lesions. To summarize, unilateral cortical lesions of the superior cerebellum appear to be sufficient to reduce eyeblink conditioning in humans significantly.

Human-bacteria nitric oxide cycles in HIV-1 infection.

The human intestinal tract harbors a complex microbiotic environment containing commensal bacteria and immunocompetent mucosal cells. There is considerable communication between the bacteria and host cells through dietary constituents and metabolic cycles. We propose that in the pathogenesis of acquired immunodeficiency syndrome (AIDS), the human immunodeficiency virus-1 (HIV-1) triggers a change in a coupled transorganism (human-bacteria) nitric oxide interchange cycle, that may influence the biosynthesis and recycling of nitric oxide (NO) in AIDS patients. Normally, nitric oxide (NO) is produced from arginine through nitrate NO(3)(-), which is ultimately eliminated in the urine and feces. In HIV infection, however, the NO(3)(-) is converted into NO and nitrite NO(2)(-) and recirculated in the body, perhaps as a result of concomitant opportunistic bacterial infections and cellular hypoxia. Due to the efficient coupling of the human-bacteria nitric oxide cycles, persistently high levels of nitrite and the free radicals peroxynitrite (ONOO(-)) may occur in AIDS patients, contributing to the etiology of AIDS-related dementia, persistent immunosuppression and Kaposi's sarcoma.

Motor deficits cannot explain impaired cognitive associative learning in cerebellar patients.

There is a strong evidence that the cerebellum is involved in associative motor learning. The exact role of the cerebellum in motor learning, and whether it is involved in cognitive learning processes too, are still controversially discussed topics. A common problem of assessing cognitive capabilities of cerebellar patients is the existence of additional motor demands in all cognitive tests. Even if the patients are able to cope well with the motor requirements of the task, their performance could still involve compensating strategies which cost them more attentional resources than the normal controls. To investigate such interaction effects of cognitive and motor demands in cerebellar patients, we conducted a cognitive associative learning paradigm and varied systematically the motor demands and the cognitive requirements of the task. Nine patients with isolated cerebellar disease and nine matched healthy controls had to learn the association between pairs of color squares, presented centrally on a computer monitor together with a left or right answer button. In the simple motor condition, the answer button had to be pressed once and in the difficult condition three times. We measured the decision times and evaluated the correctly named associations after the test was completed. The cerebellar subjects showed a learning deficit, compared to the normal controls. However, this deficit was independent of the motor difficulty of the task. The cerebellum seems to contribute to motor-independent processes, which are generally involved in associative learning.

Structural requirements for arachidonylethanolamide interaction with CB1 and CB2 cannabinoid receptors: pharmacology of the carbonyl and ethanolamide groups.

Analogs of arachidonylethanolamide (anandamide) were prepared to investigate the structural requirements for ligand binding to and activation of the CB1 and CB2 cannabinoid receptors. The importance of the presence and the placement of the carbonyl was examined with analogs lacking the carbonyl or with the carbonyl amide order reversed. The presence and location of the carbonyl is essential for high-affinity binding to both cannabinoid receptor subtypes, and for determination of signal transduction via G-proteins. Methyl groups were substituted on the 1'- and 2'-positions of arachidonylethanolamide and the significance of chirality was examined. Stereochemical differences in the ethanolamide group influence the affinity for both cannabinoid receptor subtypes and the signal transduction capabilities of the methanandamide derivatives.

Cerebrodiene, arachidonyl-ethanolamide, and hybrid structures: potential for interaction with brain cannabinoid receptors.

Cerebrodiene (cis-9, 10-octadecenoamide) was recently isolated from cerebral fluid of sleep-deprived cats and shown to induce sleep in rats. Because this lipid amide is related to arachidonylethanolamide (AEA), and because AEA binds to the cannabinoid receptor with high affinity, we investigated the binding affinity of cerebrodiene and some analogs to the cannabinoid receptor. In addition, we tested the ability of these compounds to act as cannabinoid receptor agonists by determining GTP gamma S binding. Each of the analogs competed for [3H] CP55940 binding, but with relatively low affinity (Ki = 26-44 microM). These analogs were not able to stimulate binding of GTP gamma S at concentrations of 100 microM or 1 mM. We conclude that the sleep-inducing actions of cerebrodiene are not mediated via activation of the cannabinoid receptor.

Forskolin carbamates: binding and activation studies with type I adenylyl cyclase.

Three series of analogs were regioselectively prepared from a protected forskolin precursor to afford 7-carbamoyl-7-desacetylforskolins (series 1), 6-carbamoyl-7-desacetylforskolins (series 2), and 6-carbamoylforskolins (series 3). The analogs were pharmacologically evaluated for binding (IC50) to and activation (EC50) of type I adenylyl cyclase in membranes from stably transfected Sf9 cell lines expressing a single adenylate cyclase subtype. The following ranges were determined for the IC50's and EC50's of each individual series: series 1, IC50 = 43-1600 nM, EC50 = 0.5-9.6 microM; series 2, IC50 = 65-680 nM, EC50 = 0.63-6.5 microM; series 3, IC50 = 21-271 nM, EC50 = 0.5-8.1 microM (forskolin IC50 = 41 nM and EC50 = 0.5 microM). Activation paralleled binding; however, some analogs exhibited poor binding and good activation whereas others demonstrated good binding but poor activation. Steric bulk tended to diminish binding and activation when at the 6- or 7-position, although bulk was accommodated at the 6-position if the 7-site was reacetylated. Acylation of the 7-position by the carbamoyl linker or acetyl was important for obtaining good binding and activation; however, the effect was more pronounced with binding. For both binding and activation, small, linear, lipophilic substituents (propyl, allyl, isopropyl) are well tolerated at the 7-position but less so in the 6-position, even when the 7-site is reacetylated. Planar aromatic moieties (phenyl and 2-pyridinyl) demonstrated moderate to good potency for binding and activation when located at either the 6- or 7-positions. There is an overall trend toward increasing potency for both binding and activation with polar substituents.

Cannabinoid receptor binding and agonist activity of amides and esters of arachidonic acid.

The cannabinoid receptor in brain (CB1) specifically binds delta 9-tetrahydrocannabinol, the predominant central nervous system-active component of marijuana. An eicosanoid found in brain, N-(2-hydroxyethyl)arachidonylamide (anandamide), binds to CB1 with similar affinity. This report considers structure-activity requirements for a series of novel amides and rigid hairpin conformations typified by N-(2-hydroxyethyl)prostaglandin amides, assayed with phenylmethylsulfonyl fluoride inactivation of esterases/amidases. Arachidonyl esters were 30-fold less potent than N-(2-hydroxyethyl)arachidonylamide, showing a rank order of potency of methyl = ethyl > propyl = isopropyl. Within the N-(hydroxyalkyl)arachidonylamide series, a one-carbon increase in chain length increased the potency 2-fold, but continued extension decreased affinity. Substituting the amide for the N-(2-hydroxyethyl)amide function produced a 4-fold loss of affinity. The N-(propyl)-, N-(butyl)-, and N-(benzyl)arachidonylamide derivatives exhibited a 3-fold increase, no change, and a 5-fold decrease, respectively, in affinity, compared with N-(2-hydroxyethyl)arachidonylamide. Both the methoxy ether and the formamide derivatives suffered > 20-fold loss of potency, compared with N-(2-hydroxyethyl)arachidonylamide. N-(2-Aminoethyl)arachidonylamide interacted poorly with CB1. At 100 microM, N-(2-hydroxyethyl)amide analogs of prostaglandin E2, A2, B2, and B1 failed to alter [3H]CP55940 binding to CB1. N-(2-Hydroxyethyl)arachidonylamide inhibited adenylate cyclase with lesser potency but with similar efficacy, compared with desacetyllevonantradol. Extending the length of the hydroxyalkyl moiety by one carbon increased the apparent potency by 1 order of magnitude. The N-(propyl) derivative exhibited a 5-fold greater potency than did the N-(2-hydroxyethyl) analog. It appears that the bulk and length of the moiety appended to arachidonic acid are more important determinants of affinity for CB1 than is hydrogen-bonding capability.

Trifunctional agents as a design strategy for tailoring ligand properties: irreversible inhibitors of A1 adenosine receptors.

The 1,3-phenylene diisothiocyanate conjugate of XAC (8-[4-[[[[(2-aminoethyl)amino]carbonyl]methyl]- oxy]phenyl]-1,3-dipropylxanthine, a potent A1 selective adenosine antagonist) has been characterized as an irreversible inhibitor of A1 adenosine receptors. To further extend this work, a series of analogues were prepared containing a third substituent in the phenyl isothiocyanate ring, incorporated to modify the physiochemical or spectroscopic properties of the conjugate. Symmetrical trifunctional cross-linking reagents bearing two isothiocyanate groups were prepared as general intermediates for cross-linking functionalized congeners and receptors. Xanthine isothiocyanate derivatives containing hydrophilic, fluorescent, or reactive substituents, linked via an amide, thiourea, or methylene group in the 5-position, were synthesized and found to be irreversible inhibitors of A1 adenosine receptors. The effects of the 5-substituent on water solubility and on the A1/A2 selectivity ratio derived from binding assays in rat brain membranes were examined. Inhibition of binding of [3H]-N6-(2-phenylisopropyl)-adenosine and [3H] CGS21680 (2-[2-[4-carboxyethyl)phenyl]ethyl]amino] adenosine-5'-N-ethylcarboxamide) at central A1 and A2 adenosine receptors, respectively, was measured. A conjugate of XAC and 1,3,5-triisothiocyanatobenzene was 894-fold selective for A1 receptors. Reporter groups, such as fluorescent dyes and a spin-label, were included as chain substituents in the irreversible binding analogues, which were designed for spectroscopic assays, histochemical characterization, and biochemical characterization of the receptor protein.