Factor V Leiden increases the risk of thrombosis in patients with antiphospholipid antibodies.
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Antibodies, Antiphospholipid↗
Biomedical subjects
R Simantov
Publications and source records attributed to R Simantov.
Lack of effect of gamma-hydroxybutyrate on mu, delta and kappa opioid receptor binding.
gamma-Hydroxybutyrate (GHB) and morphine induce a number of similar effects. Moreover, the effects they elicit can be reversed by the opiate antagonist naloxone (NX), suggesting that GHB may produce at least some of its central effects by acting as an opiate agonist. The present study considered this possibility by examining the effect of GHB on mu, delta, and kappa-opioid receptor binding in concentrations of 1 nM-0.1 mM. GHB was inactive in each instance, at every dose examined. GHB is consequently not a direct opiate receptor agonist. It is also unlikely to be an indirect (enkephalin or dynorphin release-stimulating) agonist. The mechanism of action involved whereby NX can reverse the effects of GHB must therefore not involve opioid mechanisms; at least not directly.
Adjuvants, Anesthesia↗
The effect of human recombinant erythropoietin on the growth of a human neuroblastoma cell line.
Erythropoietin is a growth factor. Cancer can be described as disturbance of the fine balance of positive and negative growth control mechanisms. The effect of human recombinant erythropoietin (EPO) was studied on the cell growth and differentiation of a human neuroblastoma cell line (h-NMB). Cell growth curves, trypan blue staining and thymidine uptake were used to assess cell proliferation and death. To assess cell differentiation, neutral endopeptidase (cell membrane enzyme marker), creatine kinase (cytosolic enzyme marker), dopamine uptake (dopamine transporter marker) and cell morphology were determined. Specific EPO receptor mRNA, by RT-PCR technique, was demonstrated. The incubation of erythropoietin with the tumor cell line resulted in inhibition of cell proliferation as evidenced in a diminished cell growth. EPO was shown to have induced a differentiation process as seen from the two different enzymatic markers, membranal and cytosolic, and from the cells dopamine uptake studies. However, the morphological changes did not document a full differentiation effect. EPO specific antibodies blocked the effects of EPO on cell proliferation and creatine kinase activity. In this study, EPO did not produce any sign of proliferation in the nervous tumor cell line used.
Base Sequence↗
Selective role of glutathione in protecting human neuronal cells from dopamine-induced apoptosis.
The role of glutathione and other antioxidants in dopamine-induced apoptosis has been analyzed in cultures of the human neuronal cell line NMB. Apoptosis, induced by 0.1-0.3 mM dopamine, was blocked by glutathione in a dose- and time-dependent manner. This was observed by monitoring cell morphology, cell viability, and the release of the cytosolic enzyme lactate dehydrogenase into the culture medium. L-Cysteine and N-acetylcysteine had a similar effect in protecting against dopamine neurotoxicity, but at lower concentrations than glutathione. The dopamine-induced alteration in the cell cycle profile, detected by flow cytometry (FACS), and intranucleosomal DNA fragmentation, were both blocked by glutathione. Treatment of NMB cells with buthionine sulfoximine, an irreversible inhibitor of gamma-glutamylcysteine synthetase, increased the neurotoxic effect of, dopamine, suggesting that endogenous glutathione participates in reducing dopamine neurotoxicity. The relationship between glutathione and dopamine was further investigated by testing the effect of dopamine on the endogenous glutathione level. Dopamine decreased glutathione levels within 16-24 hr; however, this effect was preceded by a transient increase in the level of the tripeptide within the first 0.5-7 hr. Two other types of endogenous antioxidants, (+)-alpha-tocopherol (vitamin E) and ascorbic acid (vitamin C), were tested; vitamin E (at 1-100 microgram/ml) was inactive against dopamine toxicity, whereas vitamin C had no effect at 0.05-0.2 mM, but increased dopamine toxicity at 0.5-2 mM. The results indicate that glutathione has a selective role in protecting human neural cells from the toxic effect of dopamine. This study may contribute, therefore, to a better understanding of the mechanisms underling the excessive loss of dopaminergic neurons in neurodegenerative diseases, such as Parkinsonism, and in the aging process.
Antioxidants↗
Dopamine-induced apoptosis in human neuronal cells: inhibition by nucleic acids antisense to the dopamine transporter.
Human neuroblastoma NMB cells take up [3H]dopamine in a selective manner indicating that dopamine transporters are responsible for this uptake. These cells were therefore used as a model to study dopamine neurotoxicity, and to elucidate the role of dopamine transporters in controlling cell death. Treatment with 0.05 0.4 mM dopamine changed cells' morphology within 4 h, accompanied by retraction of processes, shrinkage, apoptosis-like atrophy, accumulation of apoptotic particles, DNA fragmentation and cell death. Cycloheximide inhibited dopamine's effect suggesting that induction of apoptosis by dopamine was dependent upon protein synthesis. Dopamine cytotoxicity, monitored morphologically by flow cytometric analysis, and by lactate dehydrogenase released, was blocked by cocaine but not by the noradrenaline and serotonin uptake blockers desimipramine and imipramine, respectively. Attempting to inhibit dopamine transport and toxicity in a drug-free and highly selective way, three 18-mer dopamine transporter antisense phosphorothioate oligonucleotides (numbers 1, 2 and 3) and a new plasmid vector expressing the entire rat dopamine transporter complementary DNA in the antisense orientation were prepared and tested. Antisense phosphorothioate oligonucleotide 3 inhibited [3H]dopamine uptake in a time- and dose-dependent manner. Likewise, transient transfection of NMB cells with the plasmid expressing dopamine transporter complementary DNA in the antisense orientation partially blocked [3H]dopamine uptake. Antisense phosphorothioate oligonucleotide 3 also decreased, dose-dependently, the toxic effect of dopamine and 6-hydroxydopamine. Western blot analysis with newly prepared anti-human dopamine transporter antibodies showed that antisense phosphorothioate oligonucleotide 3 decreased the transporter protein level. These studies contribute to better understand the mechanism of dopamine-induced apoptosis and neurotoxicity.
Apoptosis↗
Low power laser irradiation enhances migration and neurite sprouting of cultured rat embryonal brain cells.
We used fetal brain cells grown in tissue culture to study some basic features of the interaction between low-power laser irradiation and biological systems. Seven- to nine-day-old rat fetal brain cell aggregates in culture were subjected to direct focused irradiation of low-power helium-neon laser (0.3 mW, 632.8 nm). An 8-minute dose of laser irradiation enhanced the appearance of brain cells around the treated aggregates, as monitored under the microscope of the stained cultures. Two and three doses of laser irradiations were correlated with 97% and 142% respective increases of the numbers of cells surrounding the aggregates. To identify the type of cells grown in the outgrowth of the treated aggregate, specific tetanus-anti-tetanus antibodies were used. Rhodamine-labeled antibodies bound to receptors on cells indicated massive neurite sprouting and outgrowth of migrating brain cells in culture.
Animals↗
Antiphospholipid antibodies activate vascular endothelial cells.
Antiphospholipid antibodies (aPL) are associated with a syndrome of arterial and venous thrombosis and recurrent fetal loss. We have shown that IgG purified from patients with aPL activate vascular endothelial cells (EC), converting the steady-state, non-thrombotic endothelial surface to a pro-thrombotic state. The aPL-activated EC are characterized by the expression of leukocyte adhesion molecules, including ICAM-1, VCAM, and E-selectin. EC activation is dependent upon the presence of beta 2-GP-I, a cofactor necessary for anticardiolipin reactivity. In addition, EC activation is not attributable to endotoxin contamination, Fc receptor interactions, or immune complexes, but rather is the result of the specific anticardiolipin reactivity of the IgG. Endothelial activation by aPL may be an important mechanism by which these antibodies cause a hypercoagulable state.
Antibodies, Antiphospholipid↗
Antisense knockout of a neuropeptide gene reduces fibroblast proliferation.
The opioid peptides enkephalins are widely expressed throughout the body. While their role in the nervous system is well characterized, their function in other tissues is unclear. The antisense knockout approach was used to investigate the involvement of proenkephalin A gene in proliferation of cultured embryonic fibroblasts. Transfection of fibroblasts with enkephalin antisense vectors reduced [3H] thymidine incorporation and fibroblast colony growth. Moreover, FACS analysis indicated that transient or stable transfection with the enkephalin antisense vectors shifted fibroblasts from their normal G1 restriction point to one in the S-phase.
Animals↗
Neurotransporters: regulation, involvement in neurotoxicity, and the usefulness of antisense nucleic acids.
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Animals↗
Activation of cultured vascular endothelial cells by antiphospholipid antibodies.
Circulating antiphospholipid antibodies (aPL) are associated with a syndrome of thrombosis, recurrent fetal loss, and thrombocytopenia. We have demonstrated the activation of cultured human umbilical vein endothelial cells (HUVEC) by IgG from patients with anticardiolipin antibodies (aCL). Incubation of HUVEC for 4 h with purified IgG (100 micrograms/ml) from patients with high-titer aCL induced a 2.3-fold increase in monocyte adhesion over that seen in HUVEC incubated with IgG's from normal subjects. The effect of aCL was not attributable to LPS contamination, Fc receptors, or immune complexes. Monocyte adhesion was not induced when the aCL were added in serum-free media but was restored by the addition of purified beta 2GP1, previously described as a necessary cofactor for aCL reactivity. Purified rabbit polyclonal IgG raised against beta 2GP1 also induced monocyte adhesion when incubated with HUVEC. Preadsorption of patient serum with cardiolipin reduced monocyte adhesion by 60%. Immunofluorescent microscopy demonstrated that endothelial cells incubated with patient IgG expressed cell adhesion molecules, including E-selectin, vascular cell adhesion molecule-1, and intracellular adhesion molecule-1. These data support the hypothesis that aPL activate vascular endothelial cells, thereby leading to a pro-thrombotic state.
Antibodies, Antiphospholipid↗
Chronic morphine alters dopamine transporter density in the rat brain: possible role in the mechanism of drug addiction.
The effect of acute or chronic morphine treatment on dopamine transporters was studied with the selective transporter blocker [3H]GBR12935. Chronic, but not acute treatment of rats with morphine significantly decreased the Bmax of [3H]GBR12935 binding to membranes from the anterior basal forebrain, that includes the nucleus accumbens, but had no such effect on binding to striatum membranes. No effect on the affinity (Kd) of the radioligand to either one of the two brain regions was observed. The selectivity of morphine interaction with the dopamine system was tested with a ligand that binds selectively to serotonin transporters, [3H]citalopram. Neither acute nor chronic morphine altered [3H]citalopram binding to the anterior basal forebrain, or the striatum membranes. It is suggested, therefore, that chronic morphine treatment has a long-lasting and selective effect on the activity of dopamine transporters in the dopaminergic reward pathway, but not in the striatum.
Animals↗
A cocaine analog and a GBR analog label the same protein in rat striatal membranes.
Because some evidence suggests that cocaine and GBR12935 bind to different sites, we utilized photoaffinity probes from both classes of compounds to see if they label the same protein. [125I]RTI-82 a cocaine analog, and [125I]DEEP, a GBR analog, labeled protein(s) showing the same molecular weight, a similar pharmacological profile and a similar sensitivity to neuraminidase.
Animals↗
Dopamine transporter: deglycosylation with exo- and endoglycosidases.
The dopamine transporter from rat caudate-putamen was photolabeled with [125I]DEEP as previously described. Treatment of photolabeled membranes with neuraminidase and N-glycanase reduced the molecular weight of the [125I]DEEP photolabeled dopamine transporter complex, whereas treatment with alpha-mannosidase had no effect. The solubilized [125I]DEEP photolabeled dopamine transporter complex readily bound to wheat-germ agglutinin but not to concanavalin-A sepharose columns. These results suggest that the carbohydrate moiety of the dopamine transporter is N-linked and contains significant quantities of sialic acid but not high mannose residues. A DEEP binding protein was readily detectable in other brain regions including the nucleus accumbens and olfactory tubercle, but not in the prefrontal cortex, olfactory bulb or hypothalamus under similar conditions. The DEEP binding protein in the other brain regions was similar to that in the striatum.
Animals↗
Dopamine transporters in the nucleus accumbens and the striatum have different apparent molecular weights.
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Animals↗
Regulation of proenkephalin A gene expression in aggregating fetal rat brain cells.
1. Aggregating fetal rat brain cells express a significant amount of proenkephalin A (PENK) mRNA, a selective radioimmunoassay shows that this mRNA is also translated into enkephalins. 2. Depolarization with potassium chloride (KCl) or veratridine increases the expression of PENK mRNA in a time-dependent fashion, with a maximal increase of sixfold. It is interesting, however, that depolarization of the same cultures with KCl has no effect on the expression of prodynorphin mRNA. 3. An increase in PENK mRNA levels has been also observed in cultures treated with 8-Br-cAMP, phorbol 12-myristate-13-acetate (TPA), or dexamethasone. 4. However, incubation of the cultures with the opioid agonist etorphine or the antagonist naltrexone did not alter PENK gene expression, suggesting that there is not feedback control of opioids on PENK biosynthesis in these cells. 5. The increase in PENK mRNA in depolarized and in TPA-dexamethasone-, or 8-Br-cAMP-treated cultures was not accompanied by a significant increase in the amount of free immunoreactive met-enkephalin. Fetal brain cell cultures are therefore a useful neuronal model system for studying the mechanism that regulated the expression of PENK mRNA.
8-Bromo Cyclic Adenosine Monophosphate↗
A rapid binding assay for solubilized dopamine transporters using [3H]WIN 35,428.
The cocaine analog [3H]WIN 35,428 was used to label digitonin-solubilized dopamine transporters from dog caudate nucleus. The assay consists of incubation of extracts with the ligand followed by separation of free from bound ligand by centrifugation after adding activated charcoal. Specific binding was observed in dog caudate but was absent in dog cerebellar extracts. Binding was linear with tissue, saturable, and of high affinity (Kd = 16 nM). In competition studies, soluble [3H]WIN 35,428 binding was inhibited strongly by mazindol, GBR 12909, and (-)-cocaine but only weakly by citalopram, desipramine, and (+)-cocaine; this is typical of binding to the dopamine transporter. Compared to assays using [3H]GBR 12935, (-)-cocaine was relatively more potent, suggesting that the cocaine and GBR 12935 binding sites are somewhat different. When soluble extract was chromatographed on a wheat germ agglutinin-Sepharose column, [3H]WIN 35,428 binding activity was eluted with N-acetylglucosamine in a manner similar to photoaffinity-labeled dopamine transporters.
Adsorption↗
Transient expression of opioid receptors in defined regions of developing brain: are embryonic receptors selective?
The developmental profile of opioid receptors was studied in rat and guinea pig striatum and hippocampus. The two brain regions show different receptor profiles during development, which are characteristic for each animal. Yet, both tissues and animal species share one common feature; the binding of the universal opioid ligand [3H]diprenorphine per milligram of protein is high at the early embryonic period, it decreases toward birth, and then gradually increases to the adult levels. This apparent transient expression of the receptors during the early developmental stage was manifested in the guinea pig as an actual decrease in the total receptor number. As an attempt to characterize the receptors involved in this process, the binding of the selective mu-opioid ligand [3H]Tyr-D-Ala-Gly-MePhe-NH(CH2)OH [( 3H]DAGO) was studied in striatal membranes of young (P1) and adult (P60) rats. Competition between [3H]DAGO and the delta-selective peptide Tyr-D-Pen-Gly-Phe-D-Pen (DPDPE) shows higher affinity of the delta opioid to P1 membranes than to P60 membranes, though the number of delta receptors in P1 membranes is very small. This observation is in line with a previous study suggesting that opioid receptors in embryonic striatum and hippocampus are less selective to various opioids than those of adult brain. An additional difference between adult and embryonic tissue was observed on Scatchard analysis of [3H]DAGO binding; striatum P60 membranes exhibit one binding site with a KD of 0.8 +/- 0.1 nM and Hill coefficient of 0.96, whereas striatum P1 membranes bind the peptide in an apparent cooperative fashion with an overall Hill coefficient of 1.30.(ABSTRACT TRUNCATED AT 250 WORDS)
Aging↗
Factors regulating the expression of acetylcholinesterase-containing neurons in striatal cultures: effects of chemical depolarization.
The influence of chemical depolarization on the survival and differentiation of acetylcholinesterase (AChE)-containing neurons was examined in primary rat striatal cultures, maintained in different types of media (serum-free and serum-supplemented) and substrate (poly-ornithine and astrocyte monolayer). Chronic application of 5 microM veratridine resulted in a significant loss of neurites by AChE-positive cells, while a higher concentration (20 microM) reduced the number of stained cell bodies. These effects appeared to be selective with regard to AChE-positive cells, as indicated by morphological observations of the cells in the treated cultures and receptor binding measurements. Similarly, elevation of extracellular KCl levels (20-60 mM) produced a dose-dependent neurite loss by AChE-containing cells. Blockers of voltage-sensitive Ca2+ channels--verapamil (1 microM) and nifedipine (1 microM)--did not affect the veratridine-induced neurite loss, while tetrodotoxin (0.1 microM) had a partial effect. When cultures treated with 5 microM veratridine were allowed to recuperate for several days, the number of AChE-positive cells possessing neurites returned close to control values, thus indicating the reversibility of the effect of chemical depolarization. The possibility that chronic neuronal depolarization in the striatum might play a role in regulation of the neuronal processes outgrowth by AChE-containing cells is discussed.
Acetylcholinesterase↗