Inhibition of colon cancer cell growth and antioxidant activity of bioactive compounds from Poncirus trifoliata (L.) Raf

Recently several plant derived natural compounds have been screened for their anticancer activity in order to identify putative compounds with novel structures or mechanism of action. In the present study, fruits of Poncirus trifoliata were extracted with acetone and loaded onto silica gel column chromatography. The column was eluted with different solvents to obtain two bioactive compounds. The purity of compounds was analyzed by HPLC and their structures were identified by 1H and 13C NMR experiments as beta-sitosterol and 2-hydroxy-1,2,3-propanetricarboxylic acid 2-methyl ester (HPCME). beta-Sitosterol, HPCME, and trolox were tested for their antioxidant capacity by oxygen radical absorbance capacity (ORAC) measurement. Further, these compounds were tested for their inhibition of cancer cell proliferation and apoptosis using human colon cancer cell line (HT-29). These results were compared with the corresponding activity on non-cancerous (COS-1 fibroblast) cells. Cell proliferation and induction of apoptosis were determined by MTT assay and nuclear staining. The MTT assay indicated that both the compounds exhibited differential inhibition at various concentrations. Significant arrest of cell growth was observed with beta-sitosterol even at low concentration such as 0.63 microM in 48 h and none of the compounds exerted any apparent cytostatic effects on the non-cancerous COS-1 fibroblast cells. Growth inhibition assay suggested the potential use of bioactive compounds as cancer chemopreventive and therapeutic agents. This is the first report on HPCME isolation and identification from Rutaceae family and beta-sitosterol from P. trifoliata.     

beta-sitosterol: esterification by intestinal acylcoenzyme A: cholesterol acyltransferase (ACAT) and its effect on cholesterol esterification

Rabbits were fed either 10% coconut oil, 10% coconut oil and 1% beta-sitosterol, 10% coconut oil and 1% cholesterol, or 10% coconut oil and 1% beta-sitosterol plus 1% cholesterol for 4 weeks. Microsomal membranes from intestines of animals fed the 1% beta-sitosterol diet had 48% less cholesterol and were enriched twofold in beta-sitosterol compared to membranes from animals fed the coconut oil diet alone. Acylcoenzyme A:cholesterol acyltransferase (ACAT) activity in jejunum and ileum was decreased significantly in animals fed the plant sterol alone. In membranes from animals fed 1% beta-sitosterol and 1% cholesterol, beta-sitosterol content increased 50% whereas cholesterol was modestly decreased compared to their controls fed only cholesterol. Intestinal ACAT was unchanged in the animals fed both sterols when compared to their controls. beta-Sitosterol esterification was determined by incubating intestinal microsomal membranes with either [(14)C]beta-sitosterol-albumin emulsion or [(14)C]beta-sitosterol:dipalmitoyl phosphatidylcholine (DPPC) liposomes to radiolabel the endogenous sterol pool. Oleoyl-CoA was then added. The CoA-dependent esterification rate of beta-sitosterol was very slow compared to that of cholesterol using both techniques. An increased amount of endogenous microsomal beta-sitosterol, which occurs in animals fed 1% beta-sitosterol, did not interfere with the stimulation of ACAT activity secondary to cholesterol enrichment of the membranes. Enriching microsomal membranes three- to five-fold with beta-sitosterol did not affect ACAT activity. Freshly isolated intestinal cells were incubated for 1 hour with [(3)H]oleic acid and beta-sitosterol:DPPC or 25-hydroxycholesterol:DPPC. Incorporation of oleic acid into cholesteryl esters did not change in the presence of beta-sitosterol but increased fourfold after the addition of 25-hydroxycholesterol. We conclude that the CoA-dependent esterification rate of cholesterol is at least 60 times greater than that of beta-sitosterol. Membrane beta-sitosterol does not interfere with nor compete with cholesterol esterification. Inadequate esterification of this plant sterol may play a role in the poor absorption of beta-sitosterol by the gut.-Field, F. J., and S. N. Mathur. beta-Sitosterol: esterification by intestinal acylcoenzyme A:cholesterol acyltransferase (ACAT) and its effect on cholesterol esterification.     

The composition of cell wall skeleton and outermost lipids of Mycobacterium vaccae is modified by ethambutol treatment

Ethambutol (EMB) is a first line drug in tuberculosis treatment inhibiting the biosynthesis of arabinogalactan, which is a component of the mycobacterial cell wall. The growth of Mycobacterium vaccae cells in the presence of EMB increases cell wall permeability, which was monitored by beta-sitosterol biotransformation. GC/MS and GLC/MS (gas chromatography/mass spectrometry) analysis revealed dramatic changes in the content of covalently bound mycolic acids and in molar ratio galactose (Gal) to arabinose (Ara) in the cell envelopes of EMB-treated cells. The detected variations in the compositions of fatty acids indicate that both the cell wall skeleton and outer layer (free lipids) are decomposed due to EMB treatment.     

A facile and efficient synthesis of some (6E)-hydroximino-4-en-3-one steroids, steroidal oximes from Cinachyrella spp. sponges

Using beta-sitosterol as a starting material, (6E)-hydroximino-24-ethylcholest-4-en-3-one (1), a natural steroidal oxime from Cinachyrella alloclada and C. apion, was synthesized in four steps with a high overall yield. First, beta-sitosterol (5a) is transformed into the corresponding 24-ethylcholest-4-en-3,6-dione (6a) via oxidation with pyridinium chlorochromate (PCC). Selective reduction of 6a by NaBH(4) in the presence of CoCl(2) gives 24-ethylcholest- 4-en-3beta-ol-6-one (7a). The reaction of 7a with hydroxylamine hydrochloride offers the oxime 8a and the oxidation of 8a by Jones reagent gives the target steroid 1. (6E)-Hydroximinocholest-4-en-3-one (2) and (6E)-hydroximino-24-ethylcholest-4,22-dien-3-one (4) were synthesized by a similar method. The cytotoxicity of the synthesized compounds against sk-Hep-1 (human liver carcinoma cell line), H-292 (human lung carcinoma cell line), PC-3 (human prostate carcinoma cell line) and Hey-1B (human ovarian carcinoma cell line) cells were investigated. The presence of a cholesterol-type side chain appears to be necessary for the biological activity.     

Effect of phytosterols on cholesterol metabolism and MAP kinase in MDA-MB-231 human breast cancer cells

Epidemiological studies suggest that dietary phytosterols may offer protection form some types of cancer including breast cancer. In an attempt to investigate the mechanism by which phytosterols offer this protection, we investigated the effect of the two most common dietary phytosterols, beta-sitosterol and campesterol, on the mevalonate and MAP Kinase (MAPK) pathways in MDA-MB-231 cells. These pathways play a role in cell growth and apoptosis. MDA-MB-231 cell line was used in this study since it is a hormone-insensitive tumor cell line which represents the majority of advanced breast cancer cases. Cells grown in the presence of 16 microM beta-sitosterol or campesterol for 3 days exhibited a 70% and 6% reduction in cell growth, respectively, while cholesterol treatment had no effect on growth as compared to the control. Studies investigating the effect of sterol supplementation on the relative and total sterol composition of cells, showed that cells supplemented with cholesterol contained 23% more cholesterol than the control. Cells supplemented with campesterol had almost one-half the cholesterol of controls but accumulated campesterol to account for 40% of the total sterols. In the case of cells supplemented with beta-sitosterol, cells had only 25% of their sterols as cholesterol and the rest was in the form of beta-sitosterol. All sterols tested equally inhibited de novo cholesterol synthesis using 14C-acetate as substrate. beta-Sitosterol supplemented cells had reduced cholesterol synthesis when using 3H-mevalonolactone as substrate, which suggests that the inhibition in this pathway is downstream of mevalonate where processes such as isoprenylation of proteins may take place. Mevalonate supplementation to cells treated with beta-sitosterol did not completely correct the observed growth inhibition by beta-sitosterol. There was no effect of sterols on the concentrations of both low (21-26 kDa) or high (44-74 kDa) molecular weight isoprenylated proteins in these cells. On the other hand, both the quantity and activity of MAPK was elevated in the cells supplemented with beta-sitosterol. These data suggest that the down regulation of cholesterol synthesis from mevalonate and stimulation of the MAPK pathway may play roles in the inhibition of MDA-MB-231 cell growth by beta-sitosterol.     

Effect of resveratrol and beta-sitosterol in combination on reactive oxygen species and prostaglandin release by PC-3 cells

The objective of this project was to identify some possible mechanisms by which two common phytochemicals, resveratrol and beta-sitosterol, inhibit the growth of human prostate cancer PC-3 cells. These mechanisms include the effect of the phytochemicals on apoptosis, cell cycle progression, prostaglandin synthesis and the production of reactive oxygen species (ROS). Prostaglandins have been known to play a role in regulating cell growth and apoptosis. PC-3 cells were supplemented with 50 microM resveratrol or 16 microM beta-sitosterol alone or in combination for up to 5 days. Phytochemical supplementation resulted in inhibition in cell growth. beta-Sitosterol was more potent than resveratrol and the combination of the two resulted in greater inhibition than supplementation with either alone. Long-term supplementation with resveratrol or beta-sitosterol elevated basal prostaglandin release but beta-sitosterol was much more potent than resveratrol in this regard. beta-Sitosterol was more effective than resveratrol in inducing apoptosis and the combination had an intermediate effect after 1 day of supplementation. Cells supplemented with resveratrol were arrested at the G1 phase and at the G2/M phase in the case of beta-sitosterol while the combination resulted in cell arrest at the two phases of the cell cycle. beta-Sitosterol increased ROS production while resveratrol decreased ROS production. The combination of the two phytochemicals resulted in an intermediate level of ROS. The observed changes in prostaglandin levels and ROS production by these two phytochemicals may suggest their mediation in the growth inhibition. The reduction in ROS level and increase by resveratrol supplementation in PC-3 cells reflects the antioxidant properties of resveratrol. It was concluded that these phytochemicals may induce the inhibition of tumor growth by stimulating apoptosis and arresting cells at different locations in the cell cycle and the mechanism may involve alterations in ROS and prostaglandin production.     

Specific Cell Wall Proteins Confer Resistance to Nisin upon Yeast Cells

The cell wall of a yeast cell forms a barrier for various proteinaceous and nonproteinaceous molecules. Nisin, a small polypeptide and a well-known preservative active against gram-positive bacteria, was tested with wild-type Saccharomyces cerevisiae. This peptide had no effect on intact cells. However, removal of the cell wall facilitated access of nisin to the membrane and led to cell rupture. The roles of individual components of the cell wall in protection against nisin were studied by using synchronized cultures. Variation in nisin sensitivity was observed during the cell cycle. In the S phase, which is the phase in the cell cycle in which the permeability of the yeast wall to fluorescein isothiocyanate dextrans is highest, the cells were most sensitive to nisin. In contrast, the cells were most resistant to nisin after a peak in expression of the mRNA of cell wall protein 2 (Cwp2p), which coincided with the G2 phase of the cell cycle. A mutant lacking Cwp2p has been shown to be more sensitive to cell wall-interfering compounds and Zymolyase (J. M. Van der Vaart, L. H. Caro, J. W. Chapman, F. M. Klis, and C. T. Verrips, J. Bacteriol. 177:3104–3110, 1995). Here we show that of the single cell wall protein knockouts, a Cwp2p-deficient mutant is most sensitive to nisin. A mutant with a double knockout of Cwp1p and Cwp2p is hypersensitive to the peptide. Finally, in yeast mutants with impaired cell wall structure, expression of both CWP1 and CWP2 was modified. We concluded that Cwp2p plays a prominent role in protection of cells against antimicrobial peptides, such as nisin, and that Cwp1p and Cwp2p play a key role in the formation of a normal cell wall.     

Hydrophobic thickness, lipid surface area and polar region hydration in monounsaturated diacylphosphatidylcholine bilayers: SANS study of effects of cholesterol and beta-sitosterol in unilamellar vesicles

The influence of a mammalian sterol cholesterol and a plant sterol beta-sitosterol on the structural parameters and hydration of bilayers in unilamellar vesicles made of monounsaturated diacylphosphatidylcholines (diCn:1PC, n=14-22 is the even number of acyl chain carbons) was studied at 30 degrees C using small-angle neutron scattering (SANS). Recently published advanced model of lipid bilayer as a three-strip structure was used with a triangular shape of polar head group probability distribution (Kucerka et al., Models to analyze small-angle neutron scattering from unilamellar lipid vesicles, Physical Review E 69 (2004) Art. No. 051903). It was found that 33 mol% of both sterols increased the thickness of diCn:1PC bilayers with n=18-22 similarly. beta-sitosterol increased the thickness of diC14:1PC and diC16:1PC bilayers a little more than cholesterol. Both sterols increased the surface area per unit cell by cca 12 A(2) and the number of water molecules located in the head group region by cca 4 molecules, irrespective to the acyl chain length of diCn:1PC. The structural difference in the side chain between cholesterol and beta-sitosterol plays a negligible role in influencing the structural parameters of bilayers studied.     

High temperature induced antibiotic sensitivity changes in Pseudomonas aeruginosa.

Pseudomonas aeruginosa, which was resistant to a wide variety of antibiotics, became sensitive to several of these antibiotics when grown and tested at 46 degrees C. Cell wall antibiotics such as penicillin G and ampicillin were only effective when added to cells growing at 46 degrees C prior to a temperature shift to 37 degrees C. Antibiotics which penetrate the cytoplasmic membrane to express their inhibiting action present a pattern different from those which are active against the outer cell wall. In order that these compounds be effective, the permeability of the cytoplasmic membrane must be further altered with agents such as EDTA which allow the penetration of actinomycin D. Inhibitors of protein synthesis, such as streptomycin and chloramphenicol, have increased access to their sites of action in cells grown at 46 degrees C. Cells grown at 46 degrees C have 40% less lipopolysaccharide (LPS) than cells grown at 37 degrees C and the LPS aggregates were of large molecular size in cells grown at 46 degrees C. Growth at 46 degrees C affects the permeability properties of the outer cell wall more than the permeability properties of the cytoplasmic membrane and this was due, in part, to the selective release of LPS of LPS-protein complexes at elevated growth temperatures.     

Surface Stress Induces a Conserved Cell Wall Stress Response in the Pathogenic Fungus Candida albicans

The human fungal pathogen Candida albicans can grow at temperatures of up to 45°C. Here, we show that at 42°C substantially less biomass was formed than at 37°C. The cells also became more sensitive to wall-perturbing compounds, and the wall chitin levels increased, changes that are indicative of wall stress. Quantitative mass spectrometry of the wall proteome using ¹⁵N metabolically labeled wall proteins as internal standards revealed that at 42°C the levels of the β-glucan transglycosylases Phr1 and Phr2, the predicted chitin transglycosylases Crh11 and Utr2, and the wall maintenance protein Ecm33 increased. Consistent with our previous results for fluconazole stress, this suggests that a wall-remodeling response is mounted to relieve wall stress. Thermal stress as well as different wall and membrane stressors led to an increased phosphorylation of the mitogen-activated protein (MAP) kinase Mkc1, suggesting activation of the cell wall integrity (CWI) pathway. Furthermore, all wall and membrane stresses tested resulted in diminished cell separation. This was accompanied by decreased secretion of the major chitinase Cht3 and the endoglucanase Eng1 into the medium. Consistent with this, cht3 cells showed a similar phenotype. When treated with exogenous chitinase, cell clusters both from stressed cells and mutant strains were dispersed, underlining the importance of Cht3 for cell separation. We propose that surface stresses lead to a conserved cell wall remodeling response that is mainly governed by Mkc1 and is characterized by chitin reinforcement of the wall and the expression of remedial wall remodeling enzymes.     

Occurrence of squalene and sterols in Cellulomonas dehydrogenans (Arnaudi 1942) comb. nov. Hester 1971.

The neutral lipid fraction of the photochromogenic, coryneform bacterium Cellulomonas dehydrogenans (Arnaudi 1942) comb. nov. contains the sterol precursor squalene and at least two sterols, cholesterol and beta-sitosterol. The compounds were characterized by mass spectrometry and combination gas-liquid chromatography--mass spectrometry. De novo sterol biosynthetic ability was shown from incorporation of 14C from D-[U-14C]glucose into squalene and the sterol fraction. The squalene concentration approximated 0.002 to 0.005% of the total dry cell weight, and the sterols approximated 0.03 to 0.05%.     

The moonlighting protein Tsa1p is implicated in oxidative stress response and in cell wall biogenesis in Candida albicans

Candida albicans is one of the most common fungal pathogens in humans. The cell wall is the first contact site between host and pathogen and thus is critical for colonization and infection of the host. We have identified Tsa1p, a protein that is differentially localized to the cell wall of C. albicans in hyphal cells but remains in the cytosol and nucleus in yeast-form cells. This is different from Saccharomyces cerevisiae, where the homologous protein solely has been found in the cytoplasm. We report here that TSA1 confers resistance towards oxidative stress as well as is involved in the correct composition of hyphal cell walls. However, no significant change of the cell wall composition was observed in a TSA1 deletion strain in yeast-form cells, which is in good agreement with the observation that Tsa1p is absent from the yeast-form cell wall. This indicates that Tsa1p of C. albicans might represent a moonlighting protein with specific functions correlating to its respective localization. Furthermore, the translocation of Tsa1p to the hyphal cell wall of C. albicans depends on Efg1p, suggesting a contribution of the cAMP/PKA pathway to the localization of this protein. In a strain deleted for TUP1 that filaments constitutively Tsa1p can be found in the cell wall under all conditions tested, confirming the result that Tsa1p localization to the cell wall is correlated to the morphology of C. albicans.     

Cell wall modifications during osmotic stress in Lactobacillus casei

AIMS: To study the modification of the cell wall of Lactobacillus casei ATCC 393 grown in high salt conditions. METHODS AND RESULTS: Differences in the overall structure of cell wall between growth in high salt (MRS + 1 mol l(-1) NaCl; N condition) and control (MRS; C condition) conditions were determined by transmission electronic microscopy and analytical procedures. Lactobacillus casei cells grown in N condition were significantly larger than cells grown under unstressed C condition. Increased sensitivity to mutanolysin and antibiotics with target in the cell wall was observed in N condition. Purified cell wall also showed the increased sensitivity to lysis by mutanolysin. Analysis of peptidoglycan (PG) from stressed cells showed that modification was at the structural level in accordance with a decreased PG cross-link involving penicillin-binding proteins (PBP). Nine PBP were first described in this species and these proteins were expressed in low percentages or presented a modified pattern of saturation with penicillin G (Pen G) during growth in high salt. Three of the essential PBP were fully saturated in N condition at lower Pen G concentrations than in C condition, suggesting differences in functionality in vivo. CONCLUSIONS: The results show that growth in high salt modified the structural properties of the cell wall. SIGNIFICANCE AND IMPACT OF STUDY: Advances in understanding the adaptation to high osmolarity, in particular those involving sensitivity to lysis of lactic acid bacteria.     

人类致病菌新型隐球酵母Snf1/AMPK 蛋白激酶调节细胞壁的完整性

摘要：【目的】Snf1/AMPK在真核生物中是重要的且高度保守的一类蛋白激酶。在新型隐球酵母中,SNF1 基因在调节致病因子的生物合成和细胞毒力方面具有重要作用。本文进一步报道了该基因在维持细胞壁完整方面的新功能,这一功能在其他微生物中未见报道。【方法】利用荧光增白剂染料（Calcofluor white dye）染色,荧光显微观察细胞分离、胞壁完整性;利用恒定流速和压力水流冲击菌落,测定细胞黏附琼脂糖表面能力;在含有十二烷基硫酸钠（Sodium dodecyl sulfate,SDS）,刚果红（Congo red）染料和增白剂（Fluorescent Brightener 28）的培养基上观察突变株的生长情况,以验证细胞壁完整性。【结果】SNF1 基因突变菌株对细胞壁抑制剂SDS等敏感,表明细胞壁完整性的损坏;在葡萄糖固体培养基上表现为细胞与琼脂间的黏附力丧失;在热击压力下,该菌株不能正常生长,而这种生长缺陷能够被渗透平衡抑制。【结论】新型隐球酵母SNF1 基因对于维持细胞壁完整性是非常重要的,并且影响细胞与琼脂间黏附作用以及细胞对抗热的能力。     

Newbouldiaquinone A: A naphthoquinone-anthraquinone ether coupled pigment, as a potential antimicrobial and antimalarial agent from Newbouldia laevis

The study of the chemical constituents of the roots of Newbouldia laevis (Bignoniaceae) has resulted in the isolation and characterization of a naphthoquinone-anthraquinone coupled pigment named newbouldiaquinone A (1) together with 14 known compounds: apigenin, chrysoeriol, newbouldiaquinone, lapachol, 2-methylanthraquinone, 2-acetylfuro-1,4-naphthoquinone, 2,3-dimethoxy-1,4-benzoquinone, oleanolic acid, canthic acid, 2-(4-hydroxyphenyl)ethyl triacontanoate, newbouldiamide, 5,7-dihydroxydehydroiso-alpha-lapachone, beta-sitosterol, and beta-sitosterol glucopyranoside. The structure elucidation of the isolated compounds was established based on spectroscopic studies, notably of the 2D NMR spectra. The antimalarial activity of compound (1) against Plasmodium falciparum in vitro shows moderate chemo suppression of parasitic growth. Its antimicrobial activity against a wide range of microorganisms was 13- and 24-fold more active against Candida gabrata and Enterobacter aerogens than the reference antibiotics nystatin and gentamycin.     

Studies on the chemical constituents and anticancer activity of Saxifraga stolonifera (L) Meeb

Saxifraga stolonifera, an evergreen dicotyledon, has been identified as an important resource in Chinese medicine due to its anticancer activity. In the present report, chemical components of S. stolonifera (L) Meeb which is found in Guizhou province were investigated. Ten compounds were isolated from ethanol extracts of S. stolonifera plant and were identified as n-C(31)H(64) (1), (n-C(17)H(35))(2)CO (2), beta-sitosterol (3), n-C(29)H(60) (4), Bergenin (5), Protocatechuic acid (6), Gallic acid (7), Quercitrin 3-O-alpha-l-rhamnoside (8), Quercetin (9), and Quercetin 3-O-beta-d-glucopyranoside (10). Among them, n-C(31)H(64) (1), (n-C(17)H(35))(2)CO (2), beta-sitosterol (3), and n-C(29)H(60) (4) were isolated from this plant for the first time. The anticancer activities of S. stolonifera constituents on human gastric carcinoma cell line BGC-823 were evaluated by MTT assay and microscopic observation, DNA fragmentation, and flow cytometry analysis assay. It was found that quercetin could inhibit cell viability after 72 h of exposure. Furthermore, DNA ladder assay revealed that quercetin could induce DNA strand break in a concentration- and time-dependent fashion. Flow cytometric analysis shows that quercetin can induce 11.82% BGC-823 cell apoptosis in 48 h. These data reveal that quercetin is a potential agent capable of inducing apoptosis in BGC-823 cells.     

Phenolic glycosides from Symplocos racemosa: natural inhibitors of phosphodiesterase I

One new phenolic glycoside named benzoylsalireposide (1) along with one known phenolic glycoside named salireposide (2) have been isolated from Symplocos racemosa. Four other known compounds i.e. beta-amyrin (3), oleonolic acid (4), beta-sitosterol (5) and beta-sitosterol glycoside (6) were also isolated from this plant. The structure elucidation of the isolated compounds was based primarily on 1D- and 2D-NMR analysis, including COSY, HMQC, and HMBC correlations. The compound 1 and 2 showed inhibitory activity against snake venom phosphodiesterase I.     

Distribution and movement of sterols with different side chain structures between the two leaflets of the membrane bilayer of mycoplasma cells

Mycoplasma gallisepticum was adapted to grow with delta 5-sterols modified in the aliphatic side chain, and stopped-flow kinetic measurements of filipin association were made to estimate the sterol distribution between the two leaflets of the membrane. Cholesterol derivatives with unsaturated side chains (desmosterol, cis- and trans-22-dehydrocholesterol, and cholesta-5,22E,24-trien-3 beta-ol) or an alkyl substituent (beta-sitosterol) were predominantly (86-94%) localized in the outer leaflet of the bilayer. However, cholesterol, 20-isocholesterol, and sterols with side chains of varying lengths (in the 20(R)-n-alkylpregn-5-en-3 beta-ol series where the alkyl group ranged from ethyl to undecyl) were distributed nearly symmetrically between the two halves of the bilayer. Kinetic measurements of beta-[14C]sitosterol and [14C]desmosterol exchange between M. gallisepticum cells and an excess of sonicated sterol/phosphatidylcholine vesicles confirmed the filipin-binding studies. More than 90% of these radiolabeled sterols underwent exchange at 37 degrees C with unlabeled sterols in vesicles over a period of 12-14 h in the presence of 2% (w/v) albumin. beta-[14C]Sitosterol exchange was characterized by biphasic exchange kinetics, indicative of two pools of sitosterol molecules in the cell membrane. Only a single kinetic pool was detected for [14C]desmosterol exchange. Stopped flow measurements of filipin binding to beta-sitosterol and stigmasterol also revealed an asymmetrical localization of these sterols in membranes of growing Mycoplasma. capricolum cells. When an early exponential culture of beta-sitosterol- or stigmasterol-adapted M. capricolum was transferred to a sterol-rich medium at 37 degrees C, approximately three-quarters of the beta-sitosterol or stigmasterol was localized in the outer leaflet after growth was continued for 6 h; in contrast, cholesterol was distributed symmetrically after about 1 h. The asymmetric localization of sterols with alkylated or unsaturated side chains suggests that growth-supporting sterols need not be translocated extensively into the inner leaflet of the bilayers of M. gallisepticum and M. capricolum.     

Neuroprotective properties of cannabinoids against oxidative stress: role of the cannabinoid receptor CB1

Neuroprotective effects have been described for many cannabinoids in several neurotoxicity models. However, the exact mechanisms have not been clearly understood yet. In the present study, antioxidant neuroprotective effects of cannabinoids and the involvement of the cannabinoid receptor 1 (CB1) were analysed in detail employing cell-free biochemical assays and cultured cells. As it was reported for oestrogens that the phenolic group is a lead structure for antioxidant neuroprotective effects, eight compounds were classified into three groups. Group A: phenolic compounds that do not bind to CB1. Group B: non-phenolic compounds that bind to CB1. Group C: phenolic compounds that bind to CB1. In the biochemical assays employed, a requirement of the phenolic lead structure for antioxidant activity was shown. The effects paralleled the protective potential of group A and C compounds against oxidative neuronal cell death using the mouse hippocampal HT22 cell line and rat primary cerebellar cell cultures. To elucidate the role of CB1 in neuroprotection, we established stably transfected HT22 cells containing CB1 and compared the protective potential of cannabinoids with that observed in the control transfected HT22 cell line. Furthermore, oxidative stress experiments were performed in cultured cerebellar granule cells, which were derived either from CB1 knock-out mice or from control wild-type littermates. The results strongly suggest that CB1 is not involved in the cellular antioxidant neuroprotective effects of cannabinoids.