Protective activity of plicatin B against human LDL oxidation induced in metal ion-dependent and -independent processes. Experimental and theoretical studies.

Oxidation of low-density lipoproteins (LDL) is thought to be a major factor in the pathophysiology of atherosclerosis. Natural antioxidants have been shown to protect LDL from oxidation and to inhibit atherogenic developments in animals. Structurally related prenylated pterocarpans, erybraedin C and bitucarpin A, and the prenylchalcone plicatin B were examined for their ability to inhibit LDL oxidation in vitro. The kinetic profile of peroxidation is characterized by the lag time of oxidation (t(lag)), the maximal rate of oxidation (V(max)) and the maximal accumulation of oxidation products (OD(max)). Specific variation of the set of kinetic parameters by antioxidants may provide important information about the mechanism of inhibitory action of a given compound. At equimolar concentrations (1 microM) the prenylated derivatives tested were found to inhibit 1 microM copper sulphate-induced oxidation of LDL (50 microg protein/ml) in accordance with the following order of activity: plicatin B>erybraedin Cbitucarpin A. Structural aspects, such as hydrogen-donating substituents, their number and arrangement in the aromatic ring moieties, and the prenyl and methoxy substituents, were investigated in order to explain the findings obtained. It is well known that the antioxidant activity of flavonoids is believed to be caused by a combination of transition metal chelation and free-radical-scavenging activities. To investigate these differences we comparatively studied the protective mechanism of plicatin B in copper-dependent or -independent LDL oxidation. The latter was mediated by 2,2'-azo-bis-(2-amidinopropane) dihydrochloride (ABAP). We measured the formation of conjugated dienes (OD(234 nm)). Plicatin B (0.2-1.5 microM) delayed the Cu(2+) (1 microM) promoted oxidation as conjugate diene formation (t(lag)) of the LDL by 45.2-123.5 min and reduced V(max) by 0.46-0.29 microM/min. In the ABAP (0.2mM) promoted LDL oxidation t(lag) increased by 67.2-110.2 min through plicatin B (0.5-2.5 microM). In experiments in which Cu(2+) concentrations increased (0.5 - 3 microM) and the amount of plicatin B (1 microM) was maintained constant, a significant decrease in t(lag) and an increase in V(max) was observed. In this study plicatin B appeared to exhibit a mixed mechanism, interfering with the formation of the radicals by chelating copper involved in the initiation/propagation reaction, but also by scavenging free hydroperoxyl radicals resulting from ABAP thermolysis. In addition, theoretical analysis indicated that plicatin B preferentially established the chelating complex with Cu(2+), because its affinity value is notably higher (by a factor of 5) than that for Cu(+).

Acute changes in clinical parameters and thyroid function peripheral markers following L-T4 withdrawal in patients totally thyroidectomized for thyroid cancer.

After total thyroidectomy, differentiated thyroid cancer (DTC) patients have to undergo L-T4 withdrawal for measuring serum thyroglobulin and 131I whole-body scan (131I WBS) to evaluate residual/recurrent malignant disease. The aim of the present work was to study in these patients the effects of acute thyroid hormone deficiency on various target organs and tissues. Clinical parameters and thyroid function peripheral markers were evaluated in 20 DTC patients, both before and after L-T4 withdrawal. A 24-h urine collection, a fasting blood sample for laboratory examinations, a clinical score for hypothyroidism and cardiovascular, neurological and neuropsychological evaluations were carried out. After L-T4 withdrawal, the clinical score significantly increased, as well as total cholesterol, triglycerides, creatine kinase, lactate dehydrogenase, aspartate aminotransferase and alanine aminotransferase, whereas SHBG, osteocalcin and urine hydroxyproline levels significantly decreased. The acute thyroid hormone deficiency caused a systolic dysfunction of the left ventricle associated with an increase in systemic vascular resistance without cardiac contractility alterations. A significant increase in the left ventricular mass and thickness was also observed. Carpal tunnel syndrome appeared in 30% of patients and a significant reduction in the immediate auditive memorization and in attentive performance was also detected. These observations indicate that acute hypothyroidism causes significant clinical alterations of peripheral tissue function. In the follow-up of DTC patients, therefore, L-T4 withdrawal procedure should be restricted to cases where the cost/benefit ratio is favorable. Alternative procedures, such as the use of recombinant human TSH, should be used whenever possible.

Transcranial magnetic stimulation after pure motor stroke.

OBJECTIVES: The objective of this study was to assess the sensitivity of motor-evoked potentials (MEPs) elicited by transcranial magnetic stimulation (TMS) in demonstrating the possible subclinical impairment of the corticospinal pathway after recovery, in patients with a clinical history of pure motor stroke (PMS) due to a single lacunar infarct detectable by magnetic resonance imaging (MRI). METHODS: MEPs were recorded from the first dorsal interosseous muscle of 20 healthy subjects and 40 patients, 6 months or more after PMS onset. Patients were evaluated clinically by means of the NIH stroke scale, the Medical Research Council (MRC) scale and the Barthel Index. The patients with full hand strength recovery and the normal controls were also tested by means of the 9-hole peg test. RESULTS: Motor threshold (MT), MEP amplitude and central motor conduction time (CMCT) of the affected side were significantly different from those of the normal side and of the control subjects. MT, MEP amplitude and CMCT obtained after stimulation of the affected hemisphere were significantly correlated with the MRC scale values of the affected hand. Eighty-six percent of patients with persistent hand strength deficit showed MEP abnormalities. In 21 patients with complete clinical recovery, a significant increase in MT and decrease in MEP amplitude on the affected side were observed. CONCLUSIONS: After PMS, neurophysiological changes may persist despite complete clinical recovery. TMS represents a sensitive tool that enables to demonstrate objectively the clinical and subclinical impairment of the corticospinal pathway.

Reduced excitability of the motor cortex in untreated patients with de novo idiopathic "grand mal" seizures.

OBJECTIVES: Transcranial magnetic stimulation (TMS) was used to investigate motor cortex excitability, intracortical excitatory, and inhibitory pathways in 18 patients having experienced a first "grand mal" seizure within 48 hours of the electrophysiological test. All had normal brain MRI, and were free of any treatment, drug, or alcohol misuse. Results were compared with those of 35 age matched normal volunteers. METHODS: The following parameters of responses to TMS were measured: motor thresholds at rest and with voluntary contraction, amplitudes of responses, cortical silent periods, and responses to paired pulse stimulation with interstimulus intervals of 1 to 20 ms. RESULTS: In patients, there were significantly increased motor thresholds with normal amplitudes of motor evoked potentials (MEPs), suggesting decreased cortical excitability. Cortical silent periods were not significantly different from those of normal subjects. Paired TMS with short interstimulus intervals (1-5 ms) induced normal inhibition of test MEPs, suggesting preserved function of GABAergic intracortical inhibitory interneurons. On the contrary, the subsequent period of MEP facilitation found in normal subjects (ISIs of 6-20 ms) was markedly reduced in patients. This suggests the existence of abnormally prolonged intracortical inhibition or deficient intracortical excitation. In nine patients retested 2 to 4 weeks after the initial seizure, these abnormalities persisted, although to a lesser extent. CONCLUSION: The present findings together with abnormally high motor thresholds could represent protective mechanisms against the spread or recurrence of seizures.

Transcranial magnetic stimulation in Alzheimer disease: motor cortex excitability and cognitive severity.

To study the possible changes of cortical excitability in the Alzheimer disease (AD) by transcranial magnetic stimulation (TMS) and to evaluate their eventual correlation with its stage twenty-one AD patients and 18 normal controls underwent TMS. Motor threshold, amplitudes of motor evoked potentials (MEPs), central motor conduction time (CMCT) and silent period (SP) were considered. The motor threshold in AD patients was lower than in normal subjects with a significant correlation between the stage of cognitive severity. The amplitude of MEPs was increased and the SP duration was reduced in AD patients. No significant differences were obtained for CMCT. These findings could suggest a correlation between increased motor cortical excitability and cognitive severity. Moreover, the increased cortical excitability could represent a key to understand the mechanism of AD and may have implication for novel treatment strategies.

Ipsilateral motor responses to focal transcranial magnetic stimulation in healthy subjects and acute-stroke patients.

BACKGROUND AND PURPOSE: Prevalence and characteristics of ipsilateral upper limb motor-evoked potentials (MEPs) elicited by focal transcranial magnetic stimulation (TMS) were compared in healthy subjects and patients with acute stroke. METHODS: Sixteen healthy subjects and 25 patients with acute stroke underwent focal TMS at maximum stimulator output over motor and premotor cortices. If present, MEPs evoked in muscles ipsilateral to TMS were analyzed for latency, amplitude, shape, and center of gravity (ie, preferential coil location to elicit them). In stroke patients, possible relationships between early ipsilateral responses and functional outcome at 6 months were sought. RESULTS: With relaxed or slightly contracting target muscle, maximal TMS over the motor cortex failed to elicit ipsilateral MEPs in the first dorsal interosseous (FDI) or biceps of any of 16 normal subjects. In 5 of 8 healthy subjects tested, ipsilateral MEPs with latencies longer than contralateral MEPs were evoked in FDI muscle (in biceps, 6 of 8 subjects) during strong (>50% maximum) contraction of the target muscle. In 15 of 25 stroke patients, ipsilateral MEPs in the unaffected relaxed FDI (in biceps, 6 of 25 stroke patients) were evoked by stimulation of premotor areas of the affected hemisphere. Their latencies were shorter than those that MEPs evoked in the same muscle by stimulation of the motor cortex of the contralateral unaffected hemisphere. Such responses were never obtained in normal subjects and were mostly observed in patients with subcortical infarcts. Patients harboring these responses had slightly better bimanual dexterity after 6 months. CONCLUSIONS: Ipsilateral MEPs obtained in healthy individuals and stroke patients have different characteristics and probably different origins. In the former, they are probably conveyed via corticoreticulospinal or corticopropriospinal pathways, whereas in the latter, early ipsilateral MEPs could originate in hyperexcitable premotor areas.

Ab initio and density functional evaluations of the molecular conformations of beta-caryophyllene and 6-hydroxycaryophyllene.

The four conformations of beta-caryophyllene (alphaalpha, alphabeta, betaalpha, and betabeta) were investigated ab initio at the 6-31G/HF and MP2 levels and additionally with density functional methods (B3LYP/6-31G), as it concerns their relative thermodynamic stabilities. The alphaalpha is predicted to be the most stable geometry, in agreement with low-temperature NMR measurements. In the case of 6-hydroxycaryophyllene, the alphaalpha is still the most stable conformation when the configuration at C-6 is S, but when the configuration is reversed to R the betabeta geometry becomes the most stable one. This is again in agreement with NMR data. On the other hand, for both molecules the AM1 semiempirical model Hamiltonian fails to predict the alphaalpha as a low-energy geometry, mainly due to an incorrect description of the cyclobutane ring puckering. The interconversion paths among the different minima are also analyzed and discussed. The solvent effect (either chloroform or water) on the stability of the different conformers of beta-caryophyllene and 6-hydroxycaryophyllene was studied in the polarizable continuum model framework.

A theoretical study on reaction pathways to carbanions.

Different substituents (NO2, C6H5, NH2, NH-CH=CH-CHO) to a methylene group were taken into account to investigate under which conditions the mechanism of formation of carbanions by proton transfer to a base (methylamine) can be favorable, as a preliminary study of the reaction catalyzed by semicarbazide-sensitive amine oxidases. Three different approaching paths of methylamine to C(alpha) in NO2-C(alpha)H2-NO2, and the relevant potential energy surfaces, were examined at the SCF/3-21G and 6-31G* levels. The proton transfer along the first two paths occurred with a similar barrier, which became fairly consistent after including the MP2 correlation correction, with either basis set, while the last approaching path was abandoned. For the other model systems the minimum was searched only at the 3-21G level in the vicinity of the first reaction path. The substitution of a nitro group with a phenyl group sharply raised the barrier for the proton transfer to methylamine. Also by substituting the second nitro group with either -NH2 or -NH-CH=CH-CHO, a steep uphill pathway was found. A more realistic model of the substrate-cofactor complex, namely the Schiff base between benzylamine and pyridoxal, again produced a barrier, almost matching that obtained for C6H5-C(alpha)H2-NO2. In both cases, the energy profiles for the rotation about the CC(alpha)NC dihedral and the proton shift tautomers were also considered at the 3-21G and 6-31G* levels. A preliminary scan of the effect of methyl (or methylphosphate) substitutions to the pyridoxal ring was performed and the stability of the Schiff bases involving other cofactors was also considered.

Impairment of neuromuscular transmission in a subgroup of migraine patients.

Neuronal voltage-dependent P/Q Ca2+ channels are genetically abnormal in many cases of familial hemiplegic migraine and possibly associated with the more common forms of migraine with and without aura. Besides the brain, these channels are found in motor nerve endings where they control stimulation-induced acetylcholine release. Using single fiber EMG recordings we were able to demonstrate subclinical abnormalities of neuromuscular transmission in a subgroup of patients suffering from migraine with aura. This could be related to genetic abnormalities of P/Q Ca2+ channels in certain patients suffering from migraine with aura, which needs to be explored by proper genetic analyses.

4-Diazinyl- and 4-pyridinylimidazoles: potent angiotensin II antagonists. A study of their activity and computational characterization.

A series of N-[biphenylyl(tetrazolyl)methyl]-2-butylimidazoles containing variously substituted diazine or pyridine moieties either as their free bases or N-oxide derivatives attached to the 4-position of the imidazole ring was synthesized and tested for interaction with the AT1 receptors of rat adrenal cortex membranes (receptor binding assay). Some compounds were then chosen for further evaluation in vivo in the A II-induced pressor response in conscious normotensive rats. The most potent in the AT1 binding assay were found to be compounds in which the diazine or pyridine ring nitrogen is adjacent to the point of attachment between the two heteroaromatic rings such as 2-butyl-4-(3,6-dimethylpyrazin-2-yl)-1-[[2'-(1H-tetrazol-5-y l)-biphenyl-4- yl]methyl]-1H-imidazole (3b) or 2-butyl-4-[5-(methoxycarbonyl)pyrid-2-yl]-1-[[2'-(1H-tetrazol++ +-5- yl)biphenyl-4-yl]methyl]-1H-imidazole (6c). The binding affinities and oral activities of the pyridine N-oxide imidazoles in which a stabilizing group ortho to the pyridine ring nitrogen is present were markedly improved as in 2-butyl-4-[(3-methoxycarbonyl)-6-methyl-N-oxopyridin-2-yl]-1-[[2'- (1H- tetrazol-5-yl)biphenyl-4-yl]methyl]-1H-imidazole 31b. Molecular modeling studies were carried out to determine the molecular electrostatic potential values of related model systems and to correlate their receptor interaction energies with the observed activities of our compounds.

Simple model for the effect of Glu165----Asp165 mutation on the rate of catalysis in triose phosphate isomerase.

We present an ab-initio self-consistent field calculation with a 4-31G basis set on a simple model for proton abstraction from hydroxyacetone (a model for dihydroxyacetone phosphate; DHAP) by formate, which is a model for Glu165 in triose phosphate isomerase. Earlier, we showed that the electrophilic groups on the enzyme (the NH3+ of Lys13 and the NH of His95) were essential to efficient catalysis by triose phosphate isomerase. These groups stabilized the enediolate formed by proton abstraction from the DHAP model so that proton transfer from this molecule to Glu165 became likely. In this study, we carry this analysis one step further. First, we re-examine the energy profile for proton transfer, using the fact that our earlier calculations showed that the combined effect of His95 and Lys13 on the reactant DHAP and intermediate enediolate was to make them equal in energy. Then, we analyze the likely effect of changing Glu165 to Asp165 and relate this to experiments on the kinetics of enzyme catalysis by the Glu165----Asp165 mutant.