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In this report, we describe proteomic analysis of corpora amylacea collected by postmortem laser microdissection from multiple sclerosis (MS) brain lesions. Using low level protein loads (about 30 µg), a combination of two-dimensional electrophoresis with matrix-assisted laser desorption/ionization-time of flight mass spectrometry and database interrogations we identified 24 proteins of suspected neuronal origin. In addition to major cytoskeletal proteins like actin, tubulin, and vimentin, we identified a variety of proteins implicated specifically in cellular motility and plasticity (F-actin capping protein), regulation of apoptosis and senescence (tumor rejection antigen-1, heat shock proteins, valosin-containing protein, and ubiquitin-activating enzyme E1), and enzymatic pathways (glyceraldehyde-3-dehydrogenase, protein disulfide isomerase, protein disulfide isomerase related protein 5, lactate dehydrogenase). Samples taken from regions in the vicinity of corpora amylacea showed only traces of cellular proteins suggesting that these bodies may represent remnants of neuronal aggregates with highly polymerized cytoskeletal material. Our data provide evidence supporting the concept that biogenesis of corpora amylacea involves degeneration and aggregation of cells of neuronal origin.
Long chain base compositions of gangliosides containing mainly stearic acid could be determined without any chemical modification by matrix-assisted laser desorption ionization time-of-flight mass spectrometry with delayed ion extraction (DE MALDI-TOF MS). The analytical results for the long chain base compositions of various samples of GM1 from the brain tissues of patients with different diseases at different ages confirmed that the proportion of d20:1 (icosasphingosine) and d20 (icosa-sphinganine) of the total sphingosine bases increased quickly until adolescent or adult age and then remained constant slightly exceeding 50%; this value was evidently higher than the proportion of d20:1 and d20 of GM1 in various adult mammalian brains. A long chain base composition of GM1 from the brain tissue of a patient with infantile type of GM1-gangliosidosis at 4y2m was abnormal and so was in two sibling patients with Spielmeyer-Vogt type of juvenile amaurotic idiocy at 19y and 21y in spite of that in the latter there was no accumulation of GM1 in the brain tissue. On the other hand, a patient with adult type of GM1 gangliosidosis at 66y showed a local accumulation of GM1 in the putamen and caudate nucleus, but its long chain base composition was found to be normal. It was of interest that the white matter of Eker rat with hereditary renal carcinoma contained a large amount of plasmalocerebroside as compared with the amount of cerebroside and sphingomyelin. The individual molecular species of plasmalocerebroside were identified by DE MALDI-TOF MS.
We used complexes between a fourth generation polyamidoamine (PAMAM) dendrimer and one of two heterocyclic compounds — 1-(6-hydroxyhexyl)-3-(5-phenyl-isoxazole-3-yl)-urea or 5-phenyl-isoxazole-3-carboxylic acid — to reduce oxygen consumption in transverse slices of the hippocampus taken from 4-week old male rats. In vitro electrophysiological experiments revealed that the inhibitory effect of the hypoxic state on the evoked responses was enhanced in the presence of the complexes. The data were analyzed in terms of the potential antitumor effects of these complexes.
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Membrane association of N-oleoyl-dopamine in rat brain

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N-oleoyl-dopamine (OLDA) belongs to a novel class of bioactive amides of fatty acids. The compound, a lipid derivative of dopamine, holds promise as a potential prodrug or carrier of dopamine into the brain. In this context, a key issue concerning OLDA is the integrity of the compound once it enters the brain. We addressed this issue in the current study by assessing the propensity of OLDA for hydrolysis in rat brain tissue in vitro. The brains were dissected from surgically anesthetized rats after they had been sacrificed by perfusion with physiological saline through the heart. Membrane fractions of brain tissue were isolated and incubated with 1 mmol/l OLDA. Stability of the OLDA molecule was assessed from the spectrophotometric recordings of OLDA spectra in membrane fractions at hourly time points for up to 24 hours. The methodological assumption was that any major change in the shape of the OLDA spectrum would point to a structural, and thus also possibly functional, alteration of the molecule. We found that the OLDA spectrum remained unchanged in the assays for up to 17 h of incubation. We conclude that OLDA strongly resists hydrolysis in brain membrane fractions. The results suggest that dopamine-like biological effects of OLDA might have to do with the interaction of the integral OLDA compound, rather than a dissociated-off dopamine moiety, with the dopaminergic system.
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Stability of ascorbyl palmitate molecule in the rat brain

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Recent investigations have shown the ability of ascorbyl palmitate (AP), a bioactive, lipid-soluble ester of ascorbic acid (AA), to penetrate neural tissues. This study seeks to determine the occurrence of hydrolysis of AP molecule in brain tissue, which could rather point to the action of AA alone carried over the biological barrier and then released from the AP compound. The integrity of AP molecule was examined qualitatively in the rat brain by thin-layer-chromatography. AP was injected into an internal carotid artery in a dose of 75 mg per rat after tying off the common and external carotid arteries at the same side. The rats were sacrificed 15 min later, the brain tissue was extracted with chloroform/methanol and chromatographed. The AP bands plated from the samples ispilateral to the injection side strictly corresponded to the AP standard's location and were clearly separated from the AA standard with no overlap. The experiment showed that AP resists hydrolysis in the brain and thus the short-term biological effects of AP may be ascribed to the action of an intact ester molecule. The results may help elucidate the biological action of AP, a compound that increasingly attracts attention for biomedical use due to its antioxidant potential and ability to penetrate into the membrane signaling target sites.
The goal of regenerative medicine is to ameliorate irreversible destruction of brain tissue by harnessing the power of stem cells in the process of neurogenesis. Several types of stem cells, including mesenchymal stem cells, hematopoietic stem cells, as well as neural cells differentiated from embryonic stem cell lines, have been proposed as potential therapeutic vehicles. In this review paper we will discuss a perspective of stem cell therapies for neurological disorders with special emphasis on potential application of cells isolated from adult tissues. In support of this our group found that murine bone marrow contains a mobile population of Oct-4+CXCR4+SSEA-1+Sca-1+lin–CD45– very small embryonic-like stem cells (VSELs) that are mobilized into peripheral blood in a murine stroke model. The number of these cells in circulation increases also after pharmacological mobilization by administration of granulocyte colony stimulating factor (G-CSF). Recently we found that VSELs are present in various non-hematopoietic adult organs and, interestingly, our data indicate that the brain contains a high number of cells that display the VSEL phenotype. Based on our published data both in human and mice we postulate that VSELs are a mobile population of epiblast/germ line-derived stem cells and play an important role as an organ-residing reserve population of pluripotent stem cells that give rise to stem cells committed to particular organs and tissues - including neural tissue. In conclusion human VSELs could be potentially harnessed in regenerative medicine as a source of stem cells for neurogenesis.
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