The effects of L-deprenyl treatment, alone and combined with GM1 ganglioside, on striatal dopamine content and substantia nigra pars compacta neurons

A novel amperometric HPLC detection method for the cyanobacterial (blue-green algal) peptide toxins microcystin-LR, -YR and -RR was developed. Purified microcystins and cyanobacterial extracts were chromatographed using an internal surface reversed-phase column with acetate- and phosphate-based mobile phase systems. Electrochemical oxidation reactions at 1.20 V vs. Ag/AgCl (glassy carbon working electrode) were show to originate in arginine and tyrosine residues of microcystins.     

Use of cyclodextrins for manipulating cellular cholesterol content

Previous studies from this laboratory have demonstrated that exposure of tissue culture cells to cyclodextrins results in rapid cholesterol depletion. In the present study, we have developed experimental systems for using solutions of cyclodextrins, either 2-hydroxypropyl beta-cyclodextrin or methylated beta-cyclodextrin, complexed with varying amounts of free cholesterol to manipulate cell cholesterol content. Cholesterol delivered via the cyclodextrin has been found to be metabolically active, as measured by the acyl-coenzyme A:cholesterol acyltransferase (ACAT)-mediated esterification of [3H]cholesterol in Fu5AH rat hepatoma cells and Chinese hamster ovary cells. The methylated beta-cyclodextrin was found to be a more efficient donor in all cell types studied, with an average cholesterol uptake of at least 100 microg cholesterol/mg protein within 6 h. By modifying the cyclodextrin:cholesterol molar ratio, it is possible to manipulate the cellular cholesterol content of cells, producing conditions ranging from net cholesterol enrichment to depletion. The use of cyclodextrins provides a convenient, precise and reproducible method for modulating the cholesterol content of tissue culture cells.     

Alterations in pallidal neuronal responses to peripheral sensory and striatal stimulation in symptomatic and recovered parkinsonian cats

The spontaneous activity, responses to peripheral sensory and ipsilateral caudate nucleus stimulation of globus pallidus (GP) and entopeduncular nucleus (ENTO) neurons were studied in cats while normal, symptomatic for 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) induced parkinsonism, and when spontaneously recovered from gross parkinsonian motor deficits. Administration of MPTP resulted in parkinsonian motor symptoms that spontaneously recovered approximately 4-6 weeks after the MPTP administration. Post-mortem dopamine levels in recovered animals was approximately 95% below levels previously measured in normal animals. In symptomatic animals, the mean spontaneous firing rate for GP units was decreased by 50% and increased by 55% for ENTO units recorded. Spontaneous firing rates for GP and ENTO units in recovered cats were not significantly different from those observed in normal cats. In normal cats, 31.4% of GP and 29% of ENTO units tested responded to tactile stimulation of the face. Only 12.2% of GP and 13% of ENTO units responded to such stimulation in parkinsonian animals while the responses were generally less specific (larger receptive fields, more bilateral receptive fields, and more responses to multiple stimulation types) than normal. In recovered cats GP and ENTO responses resembled those observed in normal cats. There was no difference in the overall percentage of pallidal units responding to striatal stimulation across the 3 experimental conditions. There was, however, an increase in the percentage of units responding with complex response sequences (i.e. decrease in activity followed by an increase in activity) in symptomatic animals as compared to normal and recovered animals. The results suggest that loss of striatal dopamine in parkinsonian animals has profound effects on the sensory responsiveness of GP and ENTO neurons and that these effects coincide with the appearance of and recovery from parkinsonian motor deficits. These data further support the notion that sensory information processing by the basal ganglia may play an important role in influencing motor output.     

Dietary modification of high density lipoprotein phospholipid and influence on cellular cholesterol efflux

African green monkeys fed fat-specific diets served as a model to investigate the effect of phospholipid acyl chain modification on high density lipoprotein (HDL)-mediated cellular cholesterol efflux. Diets enriched in saturated, monounsaturated, n-6 polyunsaturated, or n-3 polyunsaturated fats were provided during both low cholesterol and cholesterol-enriched stages; sera and HDL3 samples were obtained at specific points during the treatment period. Analysis of the HDL phospholipid composition revealed significant acyl chain modification, consistent with the respective fat-specific diet. Cholesterol efflux from mouse L-cell fibroblasts to HDL3 isolated from the specific diet groups was measured and revealed no differences in the abilities of the particles to accept cellular cholesterol; determination of the bidirectional flux of cholesterol between the cells and HDL3 species further demonstrated no effect of phospholipid acyl chain modification on this process. The effects of dietary modification of phospholipid acyl chains on cellular cholesterol efflux were directly examined by isolating the HDL phospholipid and combining it with human apolipoprotein A-I to form well-defined reconstituted HDL particles. These complexes did not display any differences with respect to their ability to stimulate cellular cholesterol efflux. Incubations with 5% sera further confirmed that the fat-specific diets do not influence cholesterol efflux. These results suggest that the established influences of specific dietary fats on the progression of atherosclerosis are due to effects on cholesterol metabolism other than the efflux of cellular cholesterol in the first step of reverse cholesterol transport.     

Landmark discrimination in the rat: A measure of allocentric spatial ability.

A landmark discrimination task similar to that previously used with monkeys (W. Pohl, 1973) was adapted to measure allocentric spatial ability in rats. Rats were trained to approach 1 of 2 food wells, placed 36 cm apart, based on the proximity of the landmark. During initial training, the landmark was adjacent to the baited food well (0 cm). As training progressed, the distance of the landmark from the baited food well was increased in 1.25-cm increments. Results show that rats were able to successfully use a landmark as an external referent up to a distance of 11 cm (when the midpoint between food wells was 18 cm). Following the stepwise training phase, a 64-trial test of mixed distances was administered, and performance was above 80% up to and including 12.5 cm from the near landmark. Results suggest that this landmark discrimination task provides a means to assess allocentric spatial ability in rats and to explore underlying neural mechanisms across species. (PsycINFO Database Record (c) 2010 APA, all rights reserved)     

Origin of fatty acids of cholesteryl ester accumulated by Fu5AH cells in culture

The Fu5AH rat hepatoma cell line accumulates cholesteryl ester (CE) upon incubation in medium supplemented with hyperlipemic serum or hyperlipemic serum lipoproteins. This cell line was used to investigate the origin of the fatty acids esterified to cholesterol in intracellular accumulations of CE. The intracellular CE-fatty acid distribution was found to be markedly different from that of the lipoprotein which stimulated the accumulation. Free fatty acids added to the culture medium were found esterified to cholesterol in the cells, demonstrating that cellular esterification contributes to the accumulation of CE. Using a subline of Fu5AH cells containing radioactively labeled intracellular fatty acids, it was found that about one-third of the fatty acid moiety of CE accumulated by the cells during a 24 hr incubation with hyperlipemic serum was derived from endogenous fatty acids. The drug chloroquine was found to inhibit cellular cholesterol esterification, so that only 4% of CE-fatty acids were derived from endogenous fatty acids. Evidence is presented suggesting a major role for cellular esterification in CE accumulation by Fu5AH cells.     

Regional differences in striatal dopamine uptake and release associated with recovery from MPTP-induced parkinsonism: an in vivo electrochemical study

This study directly assessed striatal dopamine (DA) uptake rates and peak release in response to KCl in normal, symptomatic, and recovered 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP)-treated cats using in vivo electrochemistry. DA uptake rates measured after direct application of known concentrations of DA to the striatum were slowed significantly in both dorsal and ventral striatum in symptomatic cats compared with rates recorded in normal animals. DA uptake rates remained significantly slowed in recovered cats and were not significantly different from the rates recorded in symptomatic animals. In symptomatic cats, both DA uptake rates and the signal recorded in response to KCl stimulation were significantly decreased from normal in all dorsal and ventral striatal regions sampled. Reduction/oxidation (redox) ratios recorded in response to KCl stimulation suggested DA to be the predominant electroactive species. In spontaneously recovered MPTP-treated cats, recordings in the ventral striatum subsequent to KCl stimulation again suggested DA to be the predominant electroactive species released, and peak levels were significantly higher than those recorded in symptomatic animals. In the dorsal striatum of recovered cats, redox ratios recorded subsequent to KCl stimulation suggested serotonin rather than DA to be the predominant electroactive species released. Peak levels of release in the dorsal striatum were not significantly greater than those recorded in symptomatic animals. These results suggest that in spontaneously recovered MPTP-treated cats, there is partial recovery of ventral striatal DAergic terminals, persistent loss of dorsal striatal DAergic terminals, and a down-regulation of DA transporter number/function throughout the striatum. These processes may contribute to volume transmission of DA in the striatum and promote functional recovery.     

Parkinson's disease: improved function with GM1 ganglioside treatment in a randomized placebo-controlled study

BACKGROUND/OBJECTIVE: Studies in animal models of Parkinson's disease (PD) suggest that GM1 ganglioside treatment can restore neurologic and dopaminergic function. In view of positive preclinical findings and the results of a previous open-label study demonstrating efficacy of GM1 in PD patients, this study compared effects of GM1 ganglioside and placebo on motor functions in PD patients. METHODS: Forty-five patients with mild to moderate PD were studied. The primary efficacy measure was change in the Unified Parkinson's Disease Rating Scale (UPDRS) motor score. After three independent baseline assessments, patients received IV infusion of the test drug (1,000 mg GM1 or placebo) and then self-administered either GM1 or placebo twice daily (200 mg/day, subcutaneously) for 16 weeks. Patients were examined during monthly follow-up visits. RESULTS: There was a significant difference between groups in UPDRS motor scores at 16 weeks (p=0.0001). The activities of daily living portion of the UPDRS (off-period assessment) also showed a significant effect in favor of the GM1-treated patients (p=0.04). GM1-treated patients also had significantly greater mean improvements than placebo-treated patients in performance of timed motor tests including tests of arm, hand, and foot movements, and walking. GM1 was well tolerated and no serious adverse events were reported. CONCLUSIONS: This study demonstrates that GM1 ganglioside treatment enhances neurologic function significantly in PD patients. Further study is warranted to evaluate long-term effects of GM1 in PD patients and to elucidate further the mechanisms underlying patient improvements.     

Cholesteryl ester metabolism in tissue culture cells: II. Source of accumulated esterified cholesterol in Fu5AH rat hepatoma cells

Previous investigations had demonstrated that Fu5AH rat hepatoma cells accumulated large quantities of esterified cholesterol when grown in hyperlipemic rabbit serum. The present investigation has determined the sources of the cellular esterified cholesterol when the cells were grown in hyperlipemic serum. Cellular esterification of endogenous and exogenous free cholesterol contributed 10% and 30%, respectively. The remaining 60% of the accumulated cellular esterified cholesterol was derived from exogenous (serum) cholesteryl esters. Evidence for the hydrolysis of a portion of the incorporated esterified cholesterol is presented. A stimulation of free cholesterol incorporation and cellular esterification is elicited by hyperlipemic serum and serum lipoproteins when compared to normolipemic serum present at equivalent exogenous cholesterol concentrations. The effect of hyperlipemic serum is reduced by Tween-80 and Triton WR-1339. Comparative data on esterified cholesterol accumulation, free cholesterol incorporation, and cellular cholesterol esterification in Fu5-5 rat hepatoma cells, L-cells, and rabbit aortic medial cells are presented. This work was done during the tenure as Established Investigator of the American Heart Association.