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1.
Naunyn Schmiedebergs Arch Pharmacol ; 389(1): 17-32, 2016 Jan.
Article in English | MEDLINE | ID: mdl-26319048

ABSTRACT

Extrusion of chemotherapeutics by ATP-binding cassette (ABC) transporters like ABCB1 (P-glycoprotein) represents a crucial mechanism of multidrug resistance in cancer therapy. We have previously shown that the 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) reductase inhibitor simvastatin directly inhibits ABCB1, alters the glycosylation of the transporter, and enhances the intracellular accumulation of doxorubicin with subsequent anti-cancer action. Here, we show that simvastatin reduces endogenous dolichol levels and ABCB1 in human neuroblastoma SH-SY5Y cells. Coapplication with dolichol prevents the downregulation of the ABCB1 transporter. Importantly, dolichol also attenuated simvastatin-induced apoptosis, unmasking involvement of unfolded protein response. Direct monitoring of the fluorescent fusion protein YFP-ABCB1 further confirms concentration-dependent reduction of ABCB1 in HEK293 cells by simvastatin. In simvastatin-treated murine xenografts, ABCB1 was also reduced in the liver and rhabdomyosarcoma but did not reach significance in neuroblastoma. Nevertheless, the in vivo anti-cancer effects of simvastatin are corroborated by increased apoptosis in tumor tissues. These findings provide experimental evidence for usage of simvastatin in novel chemotherapeutic regimens and link dolichol depletion to simvastatin-induced anti-cancer activity.


Subject(s)
ATP Binding Cassette Transporter, Subfamily B, Member 1/metabolism , Antineoplastic Agents/pharmacology , Hydroxymethylglutaryl-CoA Reductase Inhibitors/pharmacology , Simvastatin/pharmacology , Animals , Antineoplastic Agents/therapeutic use , Apoptosis/drug effects , Caspase 3/metabolism , Cell Line, Tumor , Dolichols/pharmacology , Down-Regulation , Female , HEK293 Cells , Humans , Hydroxymethylglutaryl-CoA Reductase Inhibitors/therapeutic use , Liver/drug effects , Liver/metabolism , Mice , Neoplasms/drug therapy , Neoplasms/metabolism , Neoplasms/pathology , Simvastatin/therapeutic use , Tumor Burden/drug effects
2.
Fitoterapia ; 106: 184-93, 2015 Oct.
Article in English | MEDLINE | ID: mdl-26358482

ABSTRACT

The polyprenols and their derivatives are highlighted in this study. These lipid linear polymers of isoprenoid residues are widespread in nature from bacteria to human cells. This review primarily presents the synthesis and biological activities of polyprenyl derivatives. Attention is focused on the synthesis and biological activity of dolichols, polyprenyl ester derivatives and polyprenyl amines. Other polyprenyl derivatives, such as oxides of polyprenols, aromatic polyprenols, polyprenyl bromide and polyprenyl sulphates, are mentioned. It is noted that polyprenyl phosphates and polyprenyl-linked glycosylation have better antibacterial, gene therapy and immunomodulating performance, whereas polyprenyl amines have better for antibacterial and antithrombotic activity. Dolichols, polyprenyl acetic esters, polyprenyl phosphates and polyprenyl-linked glycosylation have pharmacological anti-tumour effects. Finally, the postulated prospect of polyprenols and their derivatives are discussed. Further in vivo studies on the above derivatives are needed. The compatibility of polyprenols and their derivatives with other drugs should be studied, and new preparations of polyprenyl derivatives, such as hydrogel glue and release-controlled drugs, are suggested for future research and development.


Subject(s)
Lipids/chemistry , Terpenes/chemistry , Dolichols/chemistry , Dolichols/pharmacology , Humans , Lipids/pharmacology , Plant Extracts/chemistry , Plants/chemistry , Terpenes/pharmacology
3.
Mol Membr Biol ; 25(6-7): 547-56, 2008 Sep.
Article in English | MEDLINE | ID: mdl-18985511

ABSTRACT

The effect of dolichol C(95) on the structure and thermotropic phase behaviour of dipalmitoylphosphatidylcholine, dipalmitoylphosphatidylethanolamine and stearoyloleoylphosphatidylethanolamine has been examined by synchrotron X-ray diffraction and differential scanning calorimetry. The presence of dolichol C(95) had no detectable effects on the temperature of either the gel to ripple or the ripple to liquid-crystal phase transition of dipalmitoylphosphatidylcholine. A proportionate increase of a few degrees in the temperature of the gel to lamellar liquid-crystal phase transition is observed in dispersions of dipalmitoylphosphatidylethanolamine and significantly there is a decrease in the temperature of the lamellar to non-lamellar phase transition of stearoyloleoylphosphatidylethanolamine. There was no significant change in the bilayer repeat spacing of all three mixed dispersions in gel phase in the presence of up to 20 mol% dolichol C(95). Electron density calculations showed that there was no change of bilayer thickness of dipalmitoylphosphatidylcholine with incorporation of up to 7.5 mol% dolichol C(95). These data suggest that effect of dolichol on the phospholipid model membranes depend on both the head group and the hydrocarbon chains of the phospholipid molecules. The presence of dolichol in phosphatidylcholine bilayers conforms to a model in which the polyisoprene compound is phase separated into a central domain sandwiched between the two monolayers in gel phase. In bilayers of phosphatidylethanolamines dolichol tends to stabilize the bilayers in gel phase at low temperatures and destabilize the bilayers in lamellar disordered structure at high temperatures. Non-lamellar structures coexist with lamellar disordered phase over a wide temperature range suggesting that dolichol is enriched in domains of non-lamellar structure and depleted from lamellar phase. These findings are useful to understand the function of dolichol in cell membranes.


Subject(s)
Dolichols/pharmacology , Phase Transition/drug effects , Phospholipids , Lipid Bilayers , Models, Biological , Molecular Structure , Phosphatidylethanolamines , Transition Temperature/drug effects
4.
Biochemistry ; 47(42): 11134-43, 2008 Oct 21.
Article in English | MEDLINE | ID: mdl-18823126

ABSTRACT

Niemann-Pick disease type C (NPC) is caused by defects in either the NPC1 or NPC2 gene and is characterized by accumulation of cholesterol and glycolipids in the late endosome/lysosome compartment. NPC2 is an intralysosomal protein that binds cholesterol in vitro. Previous studies demonstrated rapid rates of cholesterol transfer from NPC2 to model membranes [Cheruku, S. R., et al. (2006) J. Biol. Chem. 281, 31594-31604]. To model the potential role of NPC2 as a lysosomal cholesterol export protein, in this study we used fluorescence spectroscopic approaches to examine cholesterol transfer from membranes to NPC2, assessing the rate, mechanism, and regulation of this transport step. In addition, we examined the effect of NPC2 on the rate and kinetic mechanism of intermembrane sterol transport, to model the movement of cholesterol from internal lysosomal membranes to the limiting lysosomal membrane. The results support the hypothesis that NPC2 plays an important role in endo/lysosomal cholesterol trafficking by markedly accelerating the rates of cholesterol transport. Rates of sterol transfer from and between membranes were increased by as much as 2 orders of magnitude by NPC2. The transfer studies indicate that the mechanism of NPC2 action involves direct interaction of the protein with membranes. Such interactions were observed directly using FTIR spectroscopy and protein tryptophan spectral shifts. Additionally, cholesterol transfer by NPC2 was found to be greatly enhanced by the unique lysosomal phospholipid lyso-bisphosphatidic acid (LBPA), suggesting an important role for LBPA in NPC2-mediated cholesterol trafficking.


Subject(s)
Carrier Proteins/metabolism , Glycoproteins/metabolism , Sterols/metabolism , Animals , Biological Transport, Active/drug effects , Carrier Proteins/chemistry , Carrier Proteins/genetics , Cattle , Cholesterol/metabolism , Dolichols/pharmacology , Endosomes/metabolism , Ergosterol/analogs & derivatives , Ergosterol/metabolism , Fluorescent Dyes , Gangliosides/pharmacology , Glycoproteins/chemistry , Glycoproteins/genetics , Humans , In Vitro Techniques , Intracellular Membranes/metabolism , Kinetics , Lysosomes/metabolism , Niemann-Pick Disease, Type C/genetics , Niemann-Pick Disease, Type C/metabolism , Recombinant Proteins/chemistry , Recombinant Proteins/genetics , Recombinant Proteins/metabolism , Spectrometry, Fluorescence , Spectroscopy, Fourier Transform Infrared , Tryptophan/chemistry , Vesicular Transport Proteins/chemistry , Vesicular Transport Proteins/genetics , Vesicular Transport Proteins/metabolism
5.
J Cell Biochem ; 100(2): 349-61, 2007 Feb 01.
Article in English | MEDLINE | ID: mdl-16924672

ABSTRACT

The mechanism of induction of apoptosis by dolichyl phosphate (Dol-P) was investigated in U937 cells. Studies using isolated mitochondria revealed that the respiratory complex II activity was almost completely inhibited by 20 microg/ml of Dol-P but not by the same concentration of dolichol. Activities of complex I and III were also inhibited by Dol-P, but nearly 50% of activity still remained at 20 microg/ml. Dol-P induced release of cytochrome-c from the isolated mitochondria. Fluorometric microtiter plate assay revealed that generation of reactive oxygen species (ROS) increased in a time-dependent manner. Flow cytometric analysis also indicated that Dol-P caused loss of mitochondrial membrane potential (Deltapsi(m)) and increased ROS generation. The addition of the antioxidant pyrrolidine dithiocarbamate (PDTC) significantly inhibited Dol-P-induced ROS generation and activation of caspase-3. A specific inhibitor of respiratory complex II, thenoyltrifluoroacetone (TTFA), increased ROS generation, potentially mimicking the consequence of inhibition of electron flow at complex II by Dol-P in U937 cells. Electron microscopy revealed that mitochondria became swollen and spherical in shape by the treatment with Dol-P. Neither the tyrosine kinase inhibitor k252a nor mitogen activated protein kinase/extracellular signal-regulated kinase kinase (MEK) inhibitors PD98059 and U0126 inhibited the Dol-P-induced apoptosis. Together, these results suggest that the direct disruption of mitochondrial respiratory complexes and the consequent ROS generation play a critical role in the initiation of Dol-P-induced apoptosis.


Subject(s)
Apoptosis/drug effects , Dolichol Phosphates/pharmacology , Reactive Oxygen Species/metabolism , Benzoquinones/toxicity , Caspase 3/metabolism , Caspase Inhibitors , Cell Line, Tumor , Cell Respiration/drug effects , Cell Shape , Cytochromes c/metabolism , Dolichols/pharmacology , Enzyme Activation/drug effects , Enzyme Inhibitors/pharmacology , Humans , Lactams, Macrocyclic/toxicity , Microscopy, Electron, Transmission , Mitochondria/drug effects , Mitochondria/metabolism , Mitogen-Activated Protein Kinases/metabolism , Pyrrolidines/pharmacology , Rifabutin/analogs & derivatives , Thiocarbamates/pharmacology , Time Factors
7.
J Alzheimers Dis ; 6(2): 129-35, 2004 Apr.
Article in English | MEDLINE | ID: mdl-15096696

ABSTRACT

Dolichol is a polyprenol compound broadly distributed in membranes, biosynthetized by the general isoprenoid pathway from acetate via mevalonate and farnesyl pyrophosphate. Dolichol lays inside the membrane between the two leaflets of the lipid bilayer very close to the tail of phospholipid fatty acids. No definite catabolic pathways for this molecule have yet been identified. Evidence is produced that dolichol levels increase dramatically with increasing age; that anti-ageing caloric restriction retards this age-associated change; that dolichol may act as a radical scavenger of peroxidized lipids belonging to the cell membranes. In view of the polyunsaturated fatty acids (PUFA), dolichol and Vitamin E location and stechiometry, it is proposed that molecules might interact each-other to form a highly matched free-radical-transfer chain, whose malfunctioning might be involved in statin toxicity and neurodegenerative diseases.


Subject(s)
Aging/drug effects , Aging/physiology , Antioxidants/physiology , Dolichols/physiology , Oxidative Stress/drug effects , Animals , Cell Membrane/metabolism , Dolichols/metabolism , Dolichols/pharmacology , Energy Intake , Free Radical Scavengers/pharmacology , Lipid Peroxidation/drug effects , Rats , Time Factors
8.
Glycoconj J ; 13(4): 675-80, 1996 Aug.
Article in English | MEDLINE | ID: mdl-8872125

ABSTRACT

Mannosyl-P-dolichol (man-P-dol) has been shown to stimulate the early reactions of the dolichol pathway, specifically, the biosynthesis of GlcNAc-P-P-dol and GlcNAc-GlcNAc-P-P-dol, and thus may play a regulatory role in glycoprotein biosynthesis. The site of action of man-P-dol has previously been suggested to be the GlcNAc-transferase concerned with the formation of the monoglucosaminyl derivative. Since the concentration of the chitobiosyl compound also increases as a result of the presence of man-P-dol, the immediate site of the activation was reexamined. The effect of man-P-dol on the formation of GlcNAc-GlcNAc-P-P-dol using GlcNAc-P-P-dol synthesized in situ or added exogenously as the substrate was investigated. In addition, the distribution of radioactivity in the glucosaminyl constituents of the products under the stimulatory conditions was determined. The results of these studies supported the conclusion that the stimulation of GlcNAc-lipid synthesis by man-P-dol is due to the enhanced synthesis of GlcNAc-P-P-dol. It is not a result of the activation of the GlcNAc-transferase catalyzing the attachment of the second GlcNAc residue for the biosynthesis of the chitobiosyl derivative.


Subject(s)
Acetylglucosamine/biosynthesis , Dolichols/pharmacology , Lipids/biosynthesis , Microsomes/drug effects , Retina/drug effects , Animals , Chick Embryo , Microsomes/metabolism , Retina/embryology , Retina/metabolism
9.
Biochem Biophys Res Commun ; 216(3): 848-53, 1995 Nov 22.
Article in English | MEDLINE | ID: mdl-7488203

ABSTRACT

Exposure of rat glioma C6 cells to dolichyl phosphate resulted in cell shrinkage followed by nuclear fragmentation and internucleosomal cleavage of genomic DNA, yielding ladder patterns of oligonucleosomal fragments, all characteristics of apoptosis. This phenomenon occurred in a dose and time dependent manner. Dolichol and prenol failed to induce apoptosis. The inhibitors of N-glycosylation, tunicamycin and swainsonine had no apparent effect on dolichyl phosphate-induced apoptosis. Apoptotic changes were also observed in HL-60 cells, SIRC cells and HeLa cells. Thus, dolichyl phosphate functions as a potential apoptosis inducer as well as an essential carrier lipid in the biosynthesis of N-linked glycoprotein.


Subject(s)
Apoptosis/drug effects , Dolichol Phosphates/pharmacology , Glioma/pathology , Animals , DNA/metabolism , Dolichols/pharmacology , Electrophoresis, Agar Gel , Glioma/metabolism , Glycoproteins/antagonists & inhibitors , Glycosylation/drug effects , HeLa Cells , Hemiterpenes , Humans , Kinetics , Pentanols/pharmacology , Rats , Swainsonine/pharmacology , Tumor Cells, Cultured , Tunicamycin/pharmacology
10.
Mol Biochem Parasitol ; 72(1-2): 121-7, 1995 Jun.
Article in English | MEDLINE | ID: mdl-8538683

ABSTRACT

We report the ability of Giardia lamblia to modify several of its cellular proteins by isoprenylation. Trophozoites cultured in the presence of [3H]mevalonate synthesized radiolabeled proteins of approx. 50 and 21-26 kDa. Chemical analysis indicated that farnesyl and geranylgeranyl isoprenoids comprised the majority of the radiolabel covalently associated with trophozoite proteins. In addition, antibodies to human p21ras immunoprecipitated mevalonate-labelled species of approx. 21 kDa. Inhibitors of several enzymatic steps of the mevalonate pathway dramatically affected Giardia metabolism. Protein isoprenylation and cell growth were blocked by compactin and mevinolin, competitive inhibitors of HMG-CoA reductase, the rate-limiting enzyme in isoprenoid biosynthesis. In the presence of these inhibitors, Giardia growth was restored by the addition of mevalonate to the culture medium. In contrast, cell growth was blocked irreversibly by inhibitors of subsequent steps in the protein isoprenylation pathway. Trophozoite growth inhibition by limonene, perillic acid, perillyl alcohol and N-acetyl-S-farnesyl-L-cysteine was not reversed after the addition of mevalonate, dolichol, ubiquinone or cholesterol to the medium. These observations constitute the first description of protein isoprenylation in any protozoan and indicate that this post-translational modification is an important step in the regulation of the growth of this primitive eukaryote.


Subject(s)
Giardia lamblia/metabolism , Monoterpenes , Protein Prenylation , Protozoan Proteins/metabolism , Acetylcysteine/analogs & derivatives , Acetylcysteine/pharmacology , Animals , Cholesterol/pharmacology , Cyclohexenes , Dolichols/pharmacology , Giardia lamblia/drug effects , Giardia lamblia/growth & development , Humans , Hydroxymethylglutaryl-CoA Reductase Inhibitors , Limonene , Lipid Metabolism , Lovastatin/analogs & derivatives , Lovastatin/pharmacology , Mevalonic Acid/metabolism , Mevalonic Acid/pharmacology , Protein Prenylation/drug effects , Proto-Oncogene Proteins p21(ras)/immunology , Terpenes/pharmacology , Ubiquinone/pharmacology
11.
Anticancer Res ; 14(1A): 123-8, 1994.
Article in English | MEDLINE | ID: mdl-8166438

ABSTRACT

The possible growth regulatory role of isoprenoids (mevalonate-derived products) in secondary cultures of normal human mammary epithelial cells (HMEC), as compared to the two human breast cancer cell lines Hs578T and MDA231, was investigated. All three cell types responded promptly to inhibitors of HMG CoA reductase and thereby became arrested. Whereas the growth of MDA231 cells was totally independent of exogenous growth factors, the proliferation of HMEC and Hs578T was blocked or partially blocked, respectively, following growth factor-depletion. Closer analysis showed that the depressive effects on cell growth, induced by HMG CoA reductase inhibition and growth factor depletion, were from a kinetic point of view identical. These data suggest that the biosynthesis of isoprenoids may comprise one event involved in the intracellular mechanisms lying behind the growth factor-mediated growth of mammary epithelial cells. The effects of addition of different known isoprenoids on growth of cells subjected to HMG CoA reductase inhibition or growth factor depletion were also investigated. It was found that coenzyme Q and dolichol significantly delayed growth arrest in all three cell types. In contrast, cholesterol and isopentenyladenine were ineffective.


Subject(s)
Breast Neoplasms/drug therapy , Breast Neoplasms/pathology , Breast/cytology , Breast/drug effects , Butadienes/pharmacology , Mevalonic Acid/analogs & derivatives , Cell Division/drug effects , DNA/biosynthesis , DNA, Neoplasm/biosynthesis , Dolichols/pharmacology , Epithelial Cells , Epithelium/drug effects , Humans , Hydroxymethylglutaryl-CoA Reductase Inhibitors , Mevalonic Acid/pharmacology , Stimulation, Chemical , Ubiquinone/pharmacology
12.
J Cell Physiol ; 155(3): 539-48, 1993 Jun.
Article in English | MEDLINE | ID: mdl-8491791

ABSTRACT

Growth arrest induced by serum depletion and/or treatment with mevinolin (an inhibitor of mevalonate synthesis) in the human breast cancer cell line Hs578T was overcome by exogenous mevalonate, indicating that some product or metabolite of mevalonate may be involved in the mediation of serum-regulated growth of these cells. In the search for such compounds we first tested a variety of known end products of mevalonate with respect to their ability to counteract the inhibition of DNA synthesis caused by serum-free medium and mevinolin. Thereby high doses (10 micrograms/ml) of dolichol-20 were found to cause a partial counteraction. After straight-phase HPLC purification of endogenous lipids, isolated from 3H- or 14C-mevalonate-labelled Hs578T cultures, we found that non-sterol lipids co-eluting with dolichols efficiently induced DNA synthesis. After further purification with reverse-phase HPLC it was confirmed that virtually all of this effect was achieved by compounds(s) (seen as a single UV and radioactive peak) co-eluting with dolichol-20. Nanogram doses, at most, of this (these) compound(s) elicited a substantial stimulation of DNA synthesis. The lipid(s) also counteracted the inhibition by mevinolin of N-linked glycosylation, indicating that it (they) also interfere(s) with this processing. Since treatment with tunicamycin (an inhibitor of N-linked glycosylation) abolished this growth-stimulative effect, N-linked glycosylation seems to be a necessary event in the processes leading to lipid-induced initiation of DNA synthesis.


Subject(s)
Blood , Breast Neoplasms/metabolism , DNA/biosynthesis , Mevalonic Acid/pharmacology , Cell Division/drug effects , Chromatography, High Pressure Liquid , Culture Media , Dolichols/pharmacology , Glycosylation , Humans , Lovastatin/pharmacology , Mevalonic Acid/administration & dosage , Mevalonic Acid/metabolism , Tumor Cells, Cultured , Tunicamycin/pharmacology
13.
J Cell Sci ; 103 ( Pt 4): 1065-72, 1992 Dec.
Article in English | MEDLINE | ID: mdl-1487489

ABSTRACT

It is well-established that some product(s) or metabolite(s) of mevalonate is (are) critical for growth of mammalian cells. In the search for this (these) compound(s) it seems meaningful to distinguish between compounds needed for cell cycle progression in proliferating cells and compounds needed for growth activation of arrested cells. By using time-lapse video recording we have studied the possible regulatory role of cholesterol, dolichol and mevalonate in the cell cycle of human diploid fibroblasts (HDF). HDF, which are serum-dependent, were rapidly growth-arrested in the first part of G1 upon removal of serum factors. They also responded to mevinolin (an HMG CoA reductase inhibitor) by a similar G1-block, indicating that a mevalonate-derived product is involved in the G1-located cell cycle control of HDF. Interestingly, dolichol counteracted the G1-block caused by both types of treatment. Hence, the early G1-cells could traverse the remainder of the cell cycle and divide despite depletion of serum or mevalonate. We also demonstrated that addition of dolichol resulted in a significant decrease in the rate of protein degradation. This protein stabilizing effect may constitute the mechanism by which dolichol delays the G1-arrest of HDF.


Subject(s)
Culture Media, Serum-Free/pharmacology , Dolichols/pharmacology , Fibroblasts/drug effects , Interphase/drug effects , Mevalonic Acid/pharmacology , Cell Cycle/drug effects , Cells, Cultured , Fibroblasts/cytology , Humans , Hydroxymethylglutaryl-CoA Reductase Inhibitors , Lovastatin/pharmacology , Proteins/metabolism
14.
Biochem Biophys Res Commun ; 186(1): 543-8, 1992 Jul 15.
Article in English | MEDLINE | ID: mdl-1632790

ABSTRACT

Acute leukemia cells of the established line CEM-C1 were treated during growth in serum-free medium with various concentrations of trans-trans farnesol. At concentrations ranging from 9.0 to 31.5 microM, farnesol inhibited growth of these cells without causing cell lysis. This effect was preceded by very rapid inhibition of choline incorporation in cellular lipid fraction. The growth inhibitory effect was prevented to a large extent by incubation with phosphatidylcholine or diacylglycerol.


Subject(s)
Cell Division/drug effects , Diglycerides/pharmacology , Farnesol/pharmacology , Phosphatidylcholines/pharmacology , Acyclic Monoterpenes , Cell Line , Dolichols/pharmacology , Dose-Response Relationship, Drug , Humans , Isomerism , Kinetics , Leukemia-Lymphoma, Adult T-Cell , Squalene/pharmacology , Terpenes/pharmacology
15.
Chem Phys Lipids ; 51(3-4): 219-26, 1989 Nov.
Article in English | MEDLINE | ID: mdl-2611963

ABSTRACT

Dolichol content and the effects of dolichol in vitro on synaptic plasma membrane (SPM) structure of different age groups of C57BL/6NNIA mice were examined. SPM of aged mice had higher amounts of dolichol as compared to younger animals. Age differences also were observed in dolichol isoprenologue distribution. In older animals, dolichol isoprenologue chain length was shifted towards shorter chain length values. The structure of SPM was determined by differential polarized phase fluorometry of 1,6-diphenyl-1,3,5-hexatriene (DPH). The SPM of old mice were more rigid, as indicated by higher limiting anisotropy, than those of young mice. Dolichol administered in vitro to SPM fluidized the membranes. However, dolichol administered in vitro fluidized SPM of younger mice significantly more than SPM of aged mice. The functional role of increased dolichol content in aged individuals is not known. Dolichol may act to regulate membrane fluidity of aged animals.


Subject(s)
Dolichols/metabolism , Synaptic Membranes/metabolism , Aging/metabolism , Animals , Dolichols/pharmacology , In Vitro Techniques , Male , Membrane Fluidity/drug effects , Mice , Mice, Inbred C57BL , Synaptic Membranes/drug effects
16.
Mol Chem Neuropathol ; 11(2): 77-86, 1989 Oct.
Article in English | MEDLINE | ID: mdl-2635593

ABSTRACT

The effects of dolichol on high affinity [3H]muscimol binding to synaptic plasma membrane (SPM) and [3H]GABA uptake into synaptosomes from rat brain were analyzed. Membranes were enriched with dolichol, by preincubation, in the presence of bovine serum albumin (BSA) as a vehicle (40-100 micrograms of dolichol/mg protein + 5% BSA). The rate of dolichol incorporation into the membrane was determined using [1-3H]dolichol C95, and it was in the range of 5-7 nmol/mg protein/h for synaptosomes and SPM, respectively. The uptake of [3H]GABA into synaptosomes enriched with dolichol decreased significantly by about 30%. Dolichol alone added into the incubation medium produced only a negligible effect. Specific binding of [3H]muscimol, which was higher than 90% of total binding, was significantly reduced to SPM enriched with dolichol as compared to controls. The Kd of the high affinity sites was significantly elevated by about 30% in SPM enriched with dolichol (10.8 +/- 0.3 nM vs 7.3 +/- 0.2 nM in control). This difference was more pronounced for SPM isolated from cerebellum (Kd increased by about 50%). The Bmax value was not changed. Dolichol alone did not alter the agonist binding. These results indicate for the first time that the higher level of dolichol in SPM might influence the GABAergic transmission system. An increase in dolichols in membranes may be an important factor in the decline of brain function during aging.


Subject(s)
Brain/metabolism , Dolichols/pharmacology , Muscimol/metabolism , Receptors, Muscarinic/metabolism , Synaptosomes/metabolism , gamma-Aminobutyric Acid/pharmacokinetics , Animals , Brain/drug effects , Male , Rats , Rats, Inbred Strains , Receptors, Muscarinic/drug effects , Synaptosomes/drug effects
17.
Biochim Biophys Acta ; 905(1): 133-42, 1987 Nov 27.
Article in English | MEDLINE | ID: mdl-3676305

ABSTRACT

Small vesicles containing the tetra-anionic fluorescent probe calcein were prepared by sonication of mixtures of plant phosphatidylethanolamine, plant phosphatidylcholine, and dolichol. Following chromatography, the isolated vesicles were found to retain entrapped calcein over the temperature range of 15 to 40 degrees C. Utilizing an assay measuring the fluorescence quenching of entrapped calcein by cobalt ions, the presence of dolichol in the membranes was found to promote the permeability of the phospholipid bilayers to the divalent cation. The permeability was shown to be dependent on temperature with an increase in rate of 17-fold between 15 and 35 degrees C although the plant phospholipids used in these experiments have no known phase transition within this temperature range. The incorporated dolichol was distributed uniformly throughout the vesicle population. Similar vesicles prepared from phosphatidylethanolamine and phosphatidylcholine without added dolichol, from phosphatidylcholine alone, or with phosphatidylcholine and dolichol were far less permeable to the divalent cation under the same assay conditions. These results demonstrate that dolichols have significant effects on the permeability properties of phospholipid bilayers that contain phosphatidylethanolamine.


Subject(s)
Diterpenes/pharmacology , Dolichols/pharmacology , Lipid Bilayers/metabolism , Cobalt , Fluoresceins , Permeability , Phosphatidylcholines , Phosphatidylethanolamines , Temperature
18.
Biochim Biophys Acta ; 902(3): 385-93, 1987 Sep 03.
Article in English | MEDLINE | ID: mdl-3040098

ABSTRACT

Dolichols are isoprenologues which constitute an important component of biological membranes. However, an understanding of the effects of dolichols on the organization and dynamics of biological membranes has not been forthcoming. The experiments reported here are aimed at understanding the effects of dolichols on the physical properties of mouse brain synaptic plasma membranes. The effect of dolichols incorporated into mouse brain synaptic plasma membranes on fluorescent and electron spin resonance probes sensing the hydrophobic core differed from that of probes reporting closer to the surface of membrane bilayers. Dolichols significantly (P less than 0.01) lowered the polarization, limiting anisotropy, and order parameter of diphenylhexatriene in synaptic plasma membranes and liposomes extracted from synaptic plasma membranes, without changing the rotational relaxation time. Similarly, dolichol increased the fluidity reported by 16-doxylstearic acid in synaptic plasma membranes or liposomes extracted from synaptic plasma membranes. In contrast, dolichols exerted no effect on those properties for trans-parinaric acid or 5-doxylstearic acid in synaptic plasma membranes or liposomes derived therefrom. Dolichols can dramatically alter the structure and dynamics of lipid motion in synaptic plasma membranes and these effects are dependent on the location of the probe in the membrane.


Subject(s)
Diterpenes/pharmacology , Dolichols/pharmacology , Membrane Fluidity/drug effects , Synaptic Membranes/physiology , Animals , Brain/physiology , Brain/ultrastructure , Diphenylhexatriene/pharmacology , Electron Spin Resonance Spectroscopy , Fluorescence Polarization , Mice , Stearic Acids , Synaptosomes , Temperature
19.
Biochem Cell Biol ; 65(7): 635-41, 1987 Jul.
Article in English | MEDLINE | ID: mdl-3435661

ABSTRACT

The concanavalin A resistant, glycosylation-deficient, Chinese hamster ovary cell variant CR-7 is auxotrophic for cholesterol owing to an inability to adequately convert lanosterol to cholesterol. It is also temperature sensitive for growth, being unable to proliferate at 39 degrees C. Temperature sensitivity was relieved by addition of mevalonolactone, dolichol, or dolichyl-P to the growth medium, provided that cholesterol was also present in amounts sufficient to overcome cholesterol auxotrophy at 34 degrees C. Other metabolites of mevalonolactone (squalene, ubiquinone, lanosterol, and isopentenyladenine) were inactive in this regard. Measurement of dolichol levels in CR-7 and wild-type cells at 34 degrees C and after exposure to 39 degrees C showed that dolichol increased at 39 degrees C to an approximately equal extent in both cell types. Dolichol, dolichyl-P, ubiquinone, and isopentenyladenine had no effect on the sensitivity of either wild-type or CR-7 cells to the cytotoxic effects of concanavalin A. Mevalonolactone or lanosterol markedly increased the resistance of CR-7 to the lectin, but had no effect on wild-type cells. This raises the possibility that the presence of unusually large amounts of lanosterol, coupled with low amounts of cholesterol, in the membranes of CR-7 may be related to its concanavalin A resistance and other characteristic phenotypic abnormalities.


Subject(s)
Cell Division , Cholesterol/metabolism , Concanavalin A/pharmacology , Temperature , Animals , Cell Division/drug effects , Cell Line , Dolichols/metabolism , Dolichols/pharmacology , Drug Resistance , Kinetics , Mevalonic Acid/analogs & derivatives , Mevalonic Acid/pharmacology
20.
Article in Russian | MEDLINE | ID: mdl-3663769

ABSTRACT

The effect of C35-dolichol on black planar bilayers from mixture of azolectin and cholesterol has been studied. C35-dolichol in concentration of about 10(-6) g/ml has been found to raise the bilayer conductivity, inducing current jumps with unclearly expressed conductivity level of 0.4-0.5 nSm. According to the data of the first and third harmonic during the several minutes after C35-dolichol addition along one side of the bilayer the capacity of the membranes and transversal module Young increase. It is supposed that dolichols induce the nonbilayer structure in membranes that is the basis for their transfer of oligosaccharides blocks across the membranes of Golgi apparatus.


Subject(s)
Diterpenes/pharmacology , Dolichols/pharmacology , Electric Conductivity , Lipid Bilayers , Dolichols/analogs & derivatives , Permeability
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