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Biomedical subjects

M B Youdim

Publications and source records attributed to M B Youdim.

At least 73 records · Page 4Linked to original sources
PubMed1996

Regulation of cellular rabbit growth hormone (GH) receptor and GH-binding protein generation in vitro.

In rabbits and probably in man, GH-binding protein (GHBP) is generated from proteolysis of GH receptor (GHR). The present study describes the modulation of spontaneous release of GHBP into the culture medium in relation to cellular GH receptor (GHR) in Chinese hamster ovary cells transfected with rabbit GHR complementary DNA. Secretion of GHBP (approximately 50K protein) from these cells was dependent on time, percentage of FCS, temperature, and protein synthesis. GHBP was detected in the medium at 30 min, and a linear increase was observed over the next 4 h. GHBP release was reduced by low incubation temperature, suggesting that GHBP cleavage is an energy-requiring mechanism. N-Ethylmaleimide (500 microM for 30 min at 30 C) markedly increased GHBP secretion, matched by a corresponding decrease in GHR. However, the lack of effect of N-ethyl-maleimide observed at 4 C further confirms the temperature dependence of GHBP release. We have attempted to characterize the GHBP release protease with a number of recognized protease inhibitors. Benzamidine (10 mM) was the only protease inhibitor that reduced GHBP release; however, it also reduced the cellular GHR level. Cycloheximide (20 micrograms/ml) caused a parallel disappearance of cellular GHR and secreted GHBP with a half-life of about 50 min, but increased GHR messenger RNA expression (superinduction). Indeed, 4 h after removal of cycloheximide, GHR and GHBP were increased by 181% and 369%, respectively, compared to the control value. In summary, Chinese hamster ovary cells expressing rabbit GHR provide a useful cellular model system for studies on the mechanism of GHBP generation from GHR and its physiological importance.

Animals↗
PubMed1996

In search of the cellular site of growth hormone (GH)-binding protein cleavage from the rabbit GH receptor.

Transfection of Chinese hamster ovary cells with rabbit GH receptor (GHR) complementary DNA resulted in high expression of cellular GHR as well as markedly time- and temperature-dependent secretion of soluble GH-binding protein (GHBP) into the culture medium. In the present study, these cells were used as an in vitro model system to examine GHBP secretion in relation to GHR internalization, degradation, recycling, and biosynthesis. Incubation for 20 h with the lysosomotropic agents NH4Cl and monensin inhibited GH internalization and reduced cell surface GHR, whereas no significant effect on the level of secreted GHBP was observed. Cytochalasin B, a microfilament-disrupting agent, reduced the GHR level, but GHBP was not affected. Colchicine, a microfilament depolymerization agent, had no effect on the GHR level; however, it stimulated GHBP secretion approximately 2-fold. Brefeldin A (5 micrograms/ml), a transport blocker, incubated for 15-180 min resulted in a time-dependent decline in GHR, whereas no significant modulation effect on GHBP was apparent. The capacity of these cells to synthesize and incorporate GHR at the plasma membrane in relation to the generation of soluble GHBP was obtained by destruction of cell surface GHR by mild trypsinization and subsequently monitoring the rate of recovered GHR and GHBP. The rate of reappearance of GHR and GHBP was rapid, being observed within 1 h, whereas full recovery occurred within 2 and 3 h, respectively. The recovery was completely blocked by cycloheximide and brefeldin A. NH4Cl and monensin reduced GHR restoration by about 50%, but the recovery of GHBP was not affected. These data emphasize the importance of lysosomes and vesicular traffic in the regulation of secreted GHBP that might be derived from the internalized GHR and may provide insight into a better understanding of the cleavage process of GHBP from GHR.

Animals↗
PubMed1996

Pharmacology of selegiline.

The acetylenic selective monoamine oxidase (MAO) type B suicide inhibitor selegiline (previously called L-deprenyl) has proved to be a useful adjuvant to levodopa therapy and monotherapy of Parkinson's disease (PD). Selegiline is readily absorbed from the gastrointestinal tract and rapidly enters the brain and spinal cord following oral administration. The drug binds to brain regions with a high MAO-B content, such as the thalamus, the striatum, the cortex, and the brainstem. It is extensively metabolized in humans, mainly in the liver, to form desmethylselegiline and methamphetamine, which are further metabolized to amphetamine. Eighty-six percent of the 10-mg dose was recovered in the urine within 24 hours. These data suggest that accumulation of metabolites does not occur. Although not all features of its anti-PD action are known, studies using brain obtained at autopsy from patients who had been treated with 10 mg of selegiline showed that selective inhibition of MAO-B, with the concomitant increase of phenylethylamine and dopamine (DA) but not of serotonin or noradrenaline, in the basal ganglia may be regarded as its mode of action. The protective effects afforded by selegiline in PD, resulting in a delayed need for levodopa therapy, have been variously interpreted in terms of the involvement of an endogenous neurotoxin or an oxygen free radical mechanism (oxidative stress) in the development of PD. However, although many different hypotheses have been advanced and recent findings have emphasized the significance of oxidative stress in the pathogenesis of the disease, the cause of chronic nigral cell death and the underlying mechanisms remain, as yet, elusive. Therefore, there is no clear knowledge regarding an understanding of the reported effects of selegiline on the progression of PD. Nevertheless, selegiline might be expected to have some protective effects in reducing the production of potentially neurotoxic compounds resulting in the MAO-catalyzed oxidation of DA. In addition, some evidence suggests both an indirect (via induction of radical-scavenging enzymes) and a direct antioxidant function for selegiline. On the other hand, the reported protective effect of selegiline might also receive a contribution from the diminished potentiation of the N-methyl-D-aspartate receptor by the polyamine binding site. Finally, the effects of selegiline might also involve preventing, or perhaps to some extent reversing, the decline in resistance normally associated with cellular aging because of its neurotrophine-like action. However, even in the early clinical stage of PD, the sequence of events leading to nigral cell death may be too far advanced for selegiline to exhibit its maximum potential.

Antiparkinson Agents↗
PubMed1995

Inhibition of mitochondrial complexes I and IV by 6-hydroxydopamine.

The enzymes of mitochondrial respiratory chain, NADH dehydrogenase (complex I) and cytochrome c oxidase (complex IV), were completely inhibited by 6-hydroxydopamine with IC50 = 10.5 microM and IC50 = 34 microM respectively. The enzyme inhibition was insensitive to the change of NADH or cytochrome c concentrations. The extent of complex I inhibition decreased as a consequence of both non-enzymatic and monoamine oxidase-catalyzed oxidation of 6-hydroxydopamine. Monoamine oxidase A and B inhibitors, tranylcypromine and clorgyline but not l-deprenyl increased the extent of 6-hydroxydopamine induced inhibition of complex I. Thus, 6-hydroxydopamine itself and not its oxidation products may be responsible for the neurotoxicity of this agent via inhibition of respiratory chain enzymes.

Animals↗
PubMed1995

Mössbauer spectroscopic studies of purified human neuromelanin isolated from the substantia nigra.

57Fe Mössbauer spectroscopy at different temperatures has been used to characterize the nature of purified human neuromelanin isolated from the substantia nigra. The quantitative determination of iron(III) by estimation of the overall area of the Mössbauer spectrum at room temperature reveals an iron content of 2.8 +/- 1.4%. No subspectra corresponding to divalent iron could be observed in these spectra. The derived Mössbauer parameters lead to the conclusion that the iron sites in the human neuromelanin are similar to those of human hemosiderin (or ferritin). However, owing to the water insolubility of the purified neuromelanin, it must be concluded that the neuromelanin hemosiderin (or ferritin) is bound in a protein matrix that makes it insoluble and difficult to stain histochemically. This protein attachment to neuromelanin is important in that it is what makes it different from synthetic dopamine melanin.

Aged↗
PubMed1995

The therapeutic potential of moclobemide, a reversible selective monoamine oxidase A inhibitor in Parkinson's disease.

Dopamine is equally well deaminated oxidatively by monoamine oxidase (MAO) A and B types. Selegiline (L-deprenyl), a selective inhibitor of MAO-B, ameliorates the "wearing off" akinesia and delays the need for levodopa in mild, previously untreated Parkinson's disease. The therapeutic potential of selective inhibition of MAO-A in Parkinson's disease has not been examined in detail. MAO-A accounts for only about 20% of total MAO activity in the human basal ganglia, and it differs from MAO-B in distribution. In contrast to MAO-B, which is confined to the extraneuronal compartment, MAO-A is found both extraneuronally and within the presynaptic dopaminergic terminals. The inhibition of MAO-A might alter the intraneuronal handling of dopamine reuptaken from synaptic clefts and thereby prolong oral levodopa benefit. We have given moclobemide, a selective, reversible inhibitor of MAO-A, to nondepressed patients with Parkinson's disease receiving standard levodopa/peripheral decarboxylase inhibitor or levodopa with dopaminergic agonist (bromocriptine, pergolide). Selegiline was discontinued at least 8 weeks earlier. A standard oral levodopa challenge was performed at the patient's entry to the study and repeated on the 22nd day of moclobemide treatment (150 mg thrice daily). The overall time spent "on" and "off" before the onset of treatment and during the last week on the drug was estimated from the patients' diaries. Neuropsychological assessments were also made before and after 3 weeks of moclobemide to measure possible effects on cognitive performance and mood. In acute levodopa challenge, the latency of motor response was significantly shortened and its duration was prolonged during moclobemide treatment.(ABSTRACT TRUNCATED AT 250 WORDS)

Adult↗
PubMed1995

Ultrastructural changes in mitochondria of the adrenal cortex of iron-deficient rats.

Male Sprague-Dawley rats aged 3 weeks that were maintained on an iron-deficient diet for 4-5 weeks developed severe anemia with markedly reduced hemoglobin levels (4.11 +/- 0.20 Hb g% versus controls 12.74 +/- 0.15 Hb g%). On sacrifice, the adrenal glands were removed and processed for light and transmission electron microscopy and enzyme cytochemistry. The major histological and ultrastructural changes in the adrenal cortex in response to the iron deficiency were seen in cells of the zona fasciculata, especially in its outer region, and to a lesser degree in cells of the zona reticularis. Structural changes were seen in the mitochondria of these cells, which often became grossly enlarged and developed unusual electron-dense inclusions. In addition, the lipid droplets in the iron-deficient cells of these regions were much less developed and less prominent compared with controls. Quantitative cytochemical localization of succinic dehydrogenase (SDH) activity in the adrenal glands showed that in iron-deficient rats there was an increase in SDH activity in the zona fasciculata (46%) and in the zona reticularis (74%), whereas there was a reduction of approximately 41% in SDH activity in the zona glomerulosa. Serum corticosterone levels were significantly raised in the iron-deficient rats compared with the control rats. Our results indicate that severe nutritional iron deficiency in rats causes ultrastructural and cytochemical changes in the mitochondria of the adrenal cortex accompanied by increased secretion of corticosterone.

Adrenal Cortex↗
PubMed1995

Interaction of neuroprotective substances with human brain superoxide dismutase. An in vitro study.

Human brain total superoxide dismutase activity (SOD) was assayed in the presence of increasing concentrations of neuroprotectives. Superoxide-dependent nitrobluetetrazolium (NBT) reduction served as control for direct radical interaction of these substances. High concentrations of the dopamimetic substances L-DOPA slightly and the monoamine oxidase B inhibitor selegiline more effectively inhibit SOD activity. The MAO-B inhibitor RO 16-6491 (N-(2-aminoethyl)-4-chlorobenzamide hydrochloride) has no effect on SOD enzyme activity. Reduced glutathione stimulates SOD activity. Moreover it exhibits slight activity in scavenging radicals in vitro. Oxidized glutathione and vitamin E are unable to do so. Ascorbic acid mimics the activity of reduced glutathione, but directly interacts with NBT reduction. Thioctic acid shows no effect on SOD activity but stimulates superoxide-dependent NBT reduction. The Ginkgo biloba extract EGb 761 is highly active in inhibiting superoxide-dependent NBT reduction as well as SOD activity.

Adult↗
PubMed1995

Chemical evidence for 6-hydroxydopamine to be an endogenous toxic factor in the pathogenesis of Parkinson's disease.

The presence of 5-Hydroxydopamine (5-OHDA) and 6-Hydroxydopamine (6-OHDA) in the urine of parkinsonian patients on levodopa medication was reported by Andrew et al. (1993). To answer the question about the putative relevance of 6-OHDA endogenously formed in the brain for the pathogenesis of Parkinson's disease (PD), the chemical mechanisms leading to dopamine-coordinative complexes were investigated in vitro. Kinetic studies of the reaction of dopamine (DA) with dioxygen over the pH range 7.0-9.0, where it reacts spontaneously without the necessity of metal-ion analysis, show that stoichiometric amounts of H2O2 are produced. Pink dopaminochrome, another oxidation product, is not stable and further reacts--without the consumption of dioxygen--to form the insoluble polymeric material known as melanin. Based on these results, the in vitro chemistry of the reactions of DA, 5-OHDA, and 6-OHDA in the presence of Fe3+ and dioxygen are studied. A mechanism for the initiation of a chain reaction is suggested by which excess Fe3+ could arise, and its relevance for the degeneration of dopaminergic neurons in PD is discussed. Detailed studies on the release of ferritin bound iron (0.2-1.4 microM Fe3+) by synthetic DA (200 microM) may provide further insight into the pathogenesis of PD, but further studies are warranted to elucidate the molecular basis of this neurodegenerative disorder of the extrapyramidal system.

Dopamine↗
PubMed1994

Ultrastructural and cytochemical changes in the heart of iron-deficient rats.

Male Sprague-Dawley rats aged 3 weeks that were maintained on an iron-deficient diet for 4-5 weeks developed severe anemia with markedly reduced hemoglobin levels (3.94 +/- 0.14 Hb g% versus controls 12.9 +/- 0.11 Hb g%). Iron-deficiency resulted in marked cardiac hypertrophy (cardiomegaly). On sacrifice, the hearts were processed for light and transmission electron microscopy. The major ultrastructural changes were found in the hypertrophied left ventricle and left papillary muscles. Iron-deficiency caused marked edema in myocytes, sarcomeres were out of register, and degeneration and discontinuities in myofilaments were common. Iron-deficiency resulted in the enlargement of the interfibrillar mitochondria, changes in the matrix and the formation of electron-dense amorphous bodies. The ultrastructural changes in myocytes in response to experimental iron-deficiency were similar to those described by others in cases of experimental ischemia or hypoxia. Mitochondrial changes were also found in the atria of iron-deficient rats. Quantitative cytochemical measurement of succinate dehydrogenase activity was determined and was shown to be substantially reduced in the iron-deficient heart. In severely iron-deficient rats restored to a normal iron-sufficient diet for two weeks, hemoglobin levels recovered, however the myocytes of the hypertrophied left ventricles and papillary muscles continued to show severe degenerative changes.

Animals↗
PubMed1994

Unaltered aconitase activity, but decreased complex I activity in substantia nigra pars compacta of patients with Parkinson's disease.

Substantia nigra pars compacta of seven patients who had died of Parkinson's disease, has been investigated for the iron-depending aconitase (reactions I and II). In addition we analysed respiratory chain enzymes. While complex I activity of the respiratory chain was significantly reduced, other enzymes of this pathway were unaltered. The citric acid cycle enzyme aconitase (reactions I and II) showed no difference between patients and controls. Thus this ferrous iron dependent and oxidatively sensitive enzyme is not affected by the unphysiological high amount of ferric iron and the 'oxidative stress' present in substantia nigra of parkinsonian patients.

Aconitate Hydratase↗
PubMed1994

The interactions of milacemide with monoamine oxidase.

The interactions of the anticonvulsant drug milacemide (2-n-pentylaminoacetamide) with rat liver mitochondrial monoamine oxidases-A and -B have been studied. The compound acts as a substrate for the B-form of the enzyme, with an apparent Km value of 49 +/- 4.7 microM and a Vmax value of 1.1 +/- 0.2 nmol/min/mg. It is also a time-dependent irreversible inhibitor of that enzyme. Any activity of monoamine oxidase-A towards this substrate was too low to allow accurate determinations to be made by either luminometric determination of H2O2 formation or spectrophotometric coupling of aldehyde formation to NAD+ reduction in the presence of aldehyde dehydrogenase. Milacemide was a reversible competitive inhibitor towards monoamine oxidase-A. The inhibitor constant (Ki) was 115 +/- 35 microM indicating a higher affinity than that towards monoamine oxidase-B, which was also competitively inhibited in the absence of enzyme-inhibitor preincubation (Ki = 331 +/- 185 microM). Determination of the formation of H2O2 and the aldehyde product of the oxidative cleavage of milacemide by purified monoamine oxidase-B from ox liver indicated that cleavage resulted solely in the formation of pentanal and glycinamide. There was no evidence for alternative cleavage to pentylamine and oxamaldehyde.

Acetamides↗
PubMed1994

Comparison of the effect of reversible and irreversible MAO inhibitors on renal nerve activity in the anesthetized rat.

Cardiovascular effects of the irreversible MAO-A inhibitor clorgyline and reversible MAO-inhibitors, moclobemide and brofaromine, were compared in the anesthetized rat. Electrical activity of the sympathetic renal nerve was monitored as an index of central sympathetic output. A long lasting decrease in the recorded parameters: blood pressure (BP), renal nerve activity (RNA) and heart rate (HR) was produced by acute administration of clorgyline (2 mg/Kg, IP). Acute treatment with moclobemide (10 mg/Kg, IP) or brofaromine (10 mg/Kg, IP) caused only a transient decrease in RNA. Pretreatment with the alpha 2 antagonist yohimbine, decreased significantly the inhibitory effect of clorgyline on all three parameters. The selective alpha 2 antagonist CH-38083 blocked the sympathoinhibitory effect of brofaromine. These results indicate an alpha 2 adrenoceptor involvement in the central sympathoinhibitory effect of MAO inhibitors, which may be manifested as a hypotensive effect, including orthostatic hypotension, in patients treated with irreversible selective MAO inhibitors.

Animals↗
PubMed1994

Is selegiline neuroprotective in Parkinson's disease?

Recent findings emphasize the significance of oxidative mechanisms, involving the activity of monoamine oxidase (MAO) and the formation of free radicals, in the pathogenesis of Parkinson's disease. The possible role of such mechanisms in the degeneration of neurones in the substantia nigra has led to clinical trials aimed at preventing or slowing the progressively disabling course of the disease. However, conclusive clinical evidence of a neuroprotective effect in PD is still lacking. In this paper, we discuss possible mechanisms by which selegiline manifests neuroprotective effects in experimental and clinical situations. Besides MAO-B inhibition, which above all explains the prevention of protoxin activation and substrate oxidation by MAO-B, selegiline appears to exhibit other mechanisms of action (induction of superoxide dismutase, stimulation of neurotrophic factor synthesis, antagonistic modulation of the polyamine binding site of the NMDA-receptor) which are independent of its action on MAO-B.

Animals↗