Nano-liquid chromatography-tandem mass spectrometry analysis of oxysterols in brain: monitoring of cholesterol autoxidation

Oxysterols are present in mammalian brain at ng/g–μg/g levels while cholesterol is present at the mg/g level. This makes oxysterol analysis of brain challenging. In an effort to meet this challenge we have developed, and validated, an isolation method based on solid phase extraction and an analytical protocol involving oxidation/derivatisation (i.e., charge-tagging) followed by nano-flow liquid chromatography (nano-LC) combined with tandem mass spectrometry utilising multi-stage fragmentation (MSⁿ). The oxidation/derivatisation method employed improves detection limits by two orders of magnitude, while nano-LC–MSⁿ provides separation of isomers and allows oxysterol quantification. Using this method 13 different oxysterols have been identified in rat brain including 24S-hydroxycholesterol, 24S,25-epoxycholesterol and 7α,26-dihydroxycholest-4-en-3-one. The level of 24S-hydroxycholesterol in rat brain was determined to be 20.3 ± 3.4 μg/g and quantitative estimates were made for the other oxysterols identified. The presence of a large excess of cholesterol over oxysterol in brain raises the problem of autoxidation during sterol isolation and sample preparation. Thus, in parallel to identification studies, the degree of cholesterol autoxidation occurring during sterol isolation and analysis has been evaluated with the aid of [²H₇]-labelled cholesterol and cholesterol autoxidation products identified.     

Simultaneous determination of isoniazid,rifampicin, levofloxacin in mouse tissues and plasma by high performance liquid chromatography–tandem mass spectrometry

A rapid and selective high performance liquid chromatography–tandem mass spectrometry (HPLC–MS/MS) method for simultaneous determination of isoniazid (INH), rifampicin (RFP) and levofloxacin (LVX) in mouse tissues and plasma has been developed and validated, using gatifloxacin as the internal standard (I.S.). The compounds and I.S. were extracted from tissue homogenate and plasma by a protein precipitation procedure with methanol. The HPLC separation of the analytes was performed on a Welch materials C4 column (250 mm × 4.6 mm, 5.0 μm, USA) at 25 °C, using a gradient elution program with the initial mobile phase constituting of 0.05% formic acid and methanol (93:7, v/v) at a flow-rate of 1.0 ml/min. For all the three analytes, the recoveries varied between 83.3% and 98.8% in tissues and between 75.5% and 90.8% in plasma, the accuracies ranged from 91.7% to 112.0% in tissues and from 94.6% to 108.8% in plasma, and the intra- and inter-day precisions were less than 13.3% in tissues and less than 8.2% in plsama. Calibration ranges for INH were 0.11–5.42 μg/g in tissues and 0.18–9.04 μg/ml in plasma, for RFP were 0.12–1200 μg/g in tissues and 4.0–200 μg/ml in plasma, and for LVX were 0.13–26.2 μg/g in tissues and 0.09–4.53 μg/ml in plasma. The lower limits of quantification (LLOQs) for INH, RFP and LVX in mouse tissues were 0.11, 0.12 and 0.13 μg/g and for those in mouse plasma were 18.1, 20.0 and 21.8 ng/ml, respectively. The limits of detection (LODs) for INH, RFP and LVX in mouse tissues were 0.04, 0.05 and 0.05 μg/g and for those in mouse plasma were 5.5, 6.0 and 6.6 ng/ml, respectively. The established method was successfully applied to simultaneous determination of isoniazid, rifampicin and levofloxacin in mouse plasma and different mouse tissues.     

Quantitative analysis of 3α,6α,24-trihydroxy-24,24-di(trifluoromethyl)-5β-cholane,a potent synthetic steroidal liver X receptor agonist in plasma using liquid chromatography–tandem mass spectrometry

The steroidal liver X receptor agonist, 3α,6α,24-trihydroxy-24,24-di(trifluoromethyl)-5β-cholane (ATI-829) is a potential therapeutic agent for the treatment of atherosclerosis. A sensitive and selective liquid chromatography–tandem mass spectrometry (LC–MS–MS) method for the quantification of ATI-829 in mouse plasma was developed and validated. Proteins in a 25 μL aliquot of mouse plasma were precipitated, and ATI-829 was extracted from the precipitate by the addition of 125 μL methanol. The overall extraction efficiency was greater than 99%. LC–MS–MS with negative ion electrospray and selected reaction monitoring was used for the quantitative analysis of ATI-829. The lower limit of quantitation of ATI-829 corresponded to 5.0 ng/mL (9.7 nM) plasma. Interference from matrix was negligible. The calibration curve was linear over the range 5–2000 ng/mL. The intra-day precision and inter-day precision of the analyses were <4.5% and <6%, respectively, and the accuracy ranged from 92% to 103%. ATI-829 in plasma was stable for at least 6 h at room temperature, 1 week at 4 °C, and 3 weeks at −20 °C. The validated method was then utilized for pharmacokinetic studies of ATI-829 administered to mice.     

Oxysterols in the brain of the cholesterol 24-hydroxylase knockout mouse

Oxysterols are oxidised forms of cholesterol or its precursors. In this study we utilised the cholesterol 24-hydroxylase knockout mouse (Cyp46a1−/−) to study the sterol and oxysterol content of brain. Despite a great reduction in the abundance of 24S-hydroxycholesterol, the dominant metabolite of cholesterol in wild type brain, no other cholesterol metabolite was found to quantitatively replace this oxysterol in the Cyp46a1−/− mouse. Only minor amounts of other side-chain oxysterols including 22R-, 24R-, 25- and (25R),26-hydroxycholesterols were detected. In line with earlier studies, levels of cholesterol were similar in Cyp46a1−/− and wild type animals. However, the level of the cholesterol precursor, desomsterol, and its parallel metabolite formed via a shut of the mevalonate pathway, 24S,25-epoxycholesterol, were reduced in the Cyp46a1−/− mouse. The reduction in abundance of 24S,25-epoxycholesterol is interesting in light of a recent report indicating that this oxysterol promotes dopaminergic neurogenesis.     

Modulation of advanced glycation endproduct synthesis by kynurenines in human lens proteins

Human lens proteins (HLP) become chemically modified by kynurenines and advanced glycation end products (AGEs) during aging and cataractogenesis. We investigated the effects of kynurenines on AGE synthesis in HLP. We found that incubation with 5 mM ribose or 5 mM ascorbate produced significant quantities of pentosidine, and this was further enhanced in the presence of two different kynurenines (200–500 µM): N-formylkynurenine (Nfk) and kynurenine (Kyn). Another related compound, 3-hydroxykynurenine (3OH-Kyn), had disparate effects; low concentrations (10–200 µM) promoted pentosidine synthesis, but high concentrations (200–500 µM) inhibited it. 3OH-Kyn showed similar effects on pentosidine synthesis from Amadori-enriched HLP or ribated lysine. Chelex-100 treatment of phosphate buffer reduced pentosidine synthesis from Amadori-enriched HLP by ∼ 90%, but it did not inhibit the stimulating effect of 3OH-Kyn and EDTA. 3OH-Kyn (100–500 μM) spontaneously produced copious amounts of H₂O₂ (10–25 μM), but externally added H₂O₂ had only a mild stimulating effect on pentosidine but had no effect on N^ε-carboxymethyl lysine (CML) synthesis in HLP from ribose and ascorbate. Further, human lens epithelial cells incubated with ribose and 3OH-Kyn showed higher intracellular pentosidine than cells incubated with ribose alone. CML synthesis from glycating agents was inhibited 30 to 50% by 3OH-Kyn at concentrations of 100–500 μM. Argpyrimidine synthesis from 5 mM methylglyoxal was slightly inhibited by all kynurenines at concentrations of 100–500 μM. These results suggest that AGE synthesis in HLP is modulated by kynurenines, and such effects indicate a mode of interplay between kynurenines and carbohydrates important for AGE formation during lens aging and cataract formation.     

Analysis of bioactive oxysterols in newborn mouse brain by LC/MS

Unesterified cholesterol is a major component of plasma membranes. In the brain of the adult, it is mostly found in myelin sheaths, where it plays a major architectural role. In the newborn mouse, little myelination of neurons has occurred, and much of this sterol comprises a metabolically active pool. In the current study, we have accessed this metabolically active pool and, using LC/MS, have identified cholesterol precursors and metabolites. Although desmosterol and 24S-hydroxycholesterol represent the major precursor and metabolite, respectively, other steroids, including the oxysterols 22-oxocholesterol, 22R-hydroxycholesterol, 20R,22R-dihydroxycholesterol, and the C₂₁-neurosteroid progesterone, were identified. 24S,25-epoxycholesterol formed in parallel to cholesterol was also found to be a major sterol in newborn brain. Like 24S- and 22R-hydroxycholesterols, and also desmosterol, 24S,25-epoxycholesterol is a ligand to the liver X receptors, which are expressed in brain. The desmosterol metabolites (24Z),26-, (24E),26-, and 7α-hydroxydesmosterol were identified in brain for the first time     

Oxidation of 7-dehydrocholesterol and desmosterol by human cytochrome P450 46A1

Cytochrome P450 (P450 or CYP) 46A1 is expressed in brain and has been characterized by its ability to oxidize cholesterol to 24S-hydroxycholesterol. In addition, the same enzyme is known to further oxidize 24S-hydroxycholesterol to the 24,25- and 24,27-dihydroxy products, as well as to catalyze side-chain oxidations of 7α-hydroxycholesterol and cholestanol. As precursors in the biosynthesis of cholesterol, 7-dehydrocholesterol has not been found to be a substrate of P450 46A1 and desmosterol has not been previously tested. However, 24-hydroxy-7-dehydrocholesterol was recently identified in brain tissues, which prompted us to reexamine this enzyme and its potential substrates. Here we report that P450 46A1 oxidizes 7-dehydrocholesterol to 24-hydroxy-7-dehydrocholesterol and 25-hydroxy-7-dehydrocholesterol, as confirmed by LC-MS and GC-MS. Overall, the catalytic rates of formation increased in the order of 24-hydroxy-7-dehydrocholesterol < 24-hydroxycholesterol < 25-hydroxy-7-dehydrocholesterol from their respective precursors, with a ratio of 1:2.5:5. In the case of desmosterol, epoxidation to 24S,25-epoxycholesterol and 27-hydroxylation was observed, at roughly equal rates. The formation of these oxysterols in the brain may be of relevance in Smith-Lemli-Opitz syndrome, desmosterolosis, and other relevant diseases, as well as in signal transduction by lipids.     

Reduced de novo synthesis of 5-methyltetrahydrofolate and reduced taurine levels in ethanol-treated chick brains

In previous studies, exogenous ethanol (3 mmol EtOH/kg egg) caused a 1.6-fold increase in chick brain homocysteine (HoCys) levels at 11 days of development and the mixture of 3 mmol EtOH/kg egg and 34 μmol folic acid/kg egg attenuated EtOH-induced increases in chick brain HoCys levels. Because HoCys is converted to methionine utilizing the methyl donor, 5-methyltetrahydrofolate (5-methyl THF), we studied whether exogenous ethanol (3 mmol EtOH/kg egg) or the mixture of 3 mmol EtOH/kg egg and 34 μmol 5-methyl THF/kg egg inhibited chick brain 10-formyltetrahydrofolate dehydrogenase (10-FTHF DH; EC 1.5.1.6) activities and brain N⁵, N¹⁰-methylenetetrahydrofolate reductase (MTHFR; EC 1.5.1.20) activities at 11 days of development. Three daily dosages of 3 mmol EtOH/kg egg (E_0–2) caused approximately a 7-fold reduction in brain 10-FTHF DH activities and approximately a 1.9-fold reduction in brain MTHFR activities as compared to controls at 11 days of development (p ≤ 0.05). Because HoCys is also removed by the transsulfuration pathway, which synthesizes taurine, we studied whether exogenous ethanol (3 mmol EtOH/kg egg) or the mixture of 3 mmol EtOH/kg egg and 34 μmol 5-methyl THF/kg egg influenced chick brain taurine levels. In EtOH-treated and EtOH and 5-methyl THF-treated embryos, brain taurine levels decreased by approximately 5.5-fold and 6.2-fold as compared to controls, respectively (p ≤ 0.05). Exogenous 5-methyl THF failed to attenuate EtOH-induced decreased brain taurine levels at 11 days of development.     

Characterization,expression and localization of valosin-containing protein in ovaries of the giant tiger shrimp Penaeus monodon

Valosin-containing protein (VCP), a member of the ATPase-associated with diverse cellular activity (AAA) family, was identified from the giant tiger shrimp (Penaeus monodon). The full-length cDNA of the PmVCP mRNA consisted of 2724 bp containing an ORF of 2367 bp corresponding to a deduced polypeptide of 788 amino acids. The deduced PmVCP protein contained two putative Cdc48 domains (positions 17–103, E-value = 2.00e− 36 and 120–186, E-value = 3.60e− 11) and two putative AAA domains (positions 232–368, E-value = 3.67e− 24 and 505–644, E-value = 3.73e− 25). PmVCP mRNA expression in ovaries was greater than that in testes in both juveniles and broodstock. PmVCP was significantly up-regulated in stages II and IV ovaries in intact wild broodstock (P < 0.05) but it was not differentially expressed during ovarian development in eyestalk-ablated broodstock (P > 0.05). The expression level of PmVCP mRNA in ovaries of 14-month-old shrimp was not affected by progesterone injection (0.1 μg/g body weight, P > 0.05). In contrast, exogenous 5-HT administration (50 μg/g body weight) resulted in an increase of PmVCP mRNA in ovaries of 18-month-old shrimp at 6 and 24 h post-injection (hpi) (P < 0.05). The rPmCdc48-VCP protein and its polyclonal antibody were successfully produced. Cellular localization revealed that PmVCP was localized in the ooplasm of previtellogenic oocytes. Subsequently, it was translocated into the germinal vesicle of vitellogenic oocytes. Interestingly, PmVCP was found in nucleo-cytoplasmic compartments, in the cytoskeletal architecture and in the plasma membrane of mature oocytes in both intact and eyestalk-ablated broodstock.     

Inhibition of carboxylesterase activity of THP1 monocytes/macrophages and recombinant human carboxylesterase 1 by oxysterols and fatty acids

Two major isoforms of human carboxylesterases (CEs) are found in metabolically active tissues, CES1 and CES2. These hydrolytic enzymes are involved in xenobiotic and endobiotic metabolism. CES1 is abundantly expressed in human liver and monocytes/macrophages, including the THP1 cell line; CES2 is expressed in liver but not in monocytes/macrophages. The cholesteryl ester hydrolysis activity in human macrophages has been attributed to CES1. Here, we report the direct inhibitory effects of several endogenous oxysterols and fatty acids on the CE activity of THP1 monocytes/macrophages and recombinant human CES1 and CES2. Using THP1 whole-cell lysates we found: (1) 27-hydroxycholesterol (27-HC) is a potent inhibitor of carboxylesterase activity (IC₅₀ = 33 nM); (2) 24(S),25-epoxycholesterol had moderate inhibitory activity (IC₅₀ = 8.1 μM); and (3) cholesterol, 7-ketocholesterol, 22(R)-hydroxycholesterol, 24(S)-hydroxycholesterol, and 25-hydroxycholesterol each had little inhibitory activity. 27-HC was a partially noncompetitive inhibitor of recombinant CES1 (K_iapp = 10 nM) and impaired intracellular CES1 activity following treatment of intact THP1 cells. In contrast, recombinant CES2 activity was not inhibited by 27-HC, suggesting isoform-selective inhibition by 27-HC. Furthermore, unsaturated fatty acids were better inhibitors of CES1 activity than saturated fatty acids, while CES2 activity was unaffected by any fatty acid. Arachidonic acid (AA) was the most potent fatty acid inhibitor of recombinant CES1 and acted by a noncompetitive mechanism (K_iapp = 1.7 μM); when not complexed to albumin, exogenous AA penetrated intact THP1 cells and inhibited CES1. Inhibition results are discussed in light of recent structural models for CES1 that describe ligand binding sites separate from the active site. In addition, oxysterol-mediated inhibition of CES1 activity was demonstrated by pretreatment of human liver homogenates or intact THP1 cells with exogenous 27-HC, which resulted in significantly reduced hydrolysis of the pyrethroid insecticide bioresmethrin, a CES1-specific xenobiotic substrate. Collectively, these findings suggest that CE activity of recombinant CES1, cell lysates, and intact cells can be impaired by naturally occurring lipids, which may compromise the ability of CES1 to both detoxify environmental pollutants and metabolize endogenous compounds in vivo.     

Cardiodepressive effect elicited by the essential oil of Alpinia speciosa is related to L-type Ca current blockade

This study was undertaken to elucidate the effect of the essential oil from Alpinia speciosa (EOAs) on cardiac contractility and the underlying mechanisms. The essential oil was obtained from Alpinia speciosa leaves and flowers and the oil was analyzed by GC-MS method. Chemical analysis revealed the presence of at least 18 components. Terpinen-4-ol and 1,8-cineole corresponded to 38% and 18% of the crude oil, respectively. The experiments were conducted on spontaneously-beating right atria and on electrically stimulated left atria isolated from adult rats. The effect of EOAs on the isometric contractions and cardiac frequency in vitro was examined. EOAs decreased rat left atrial force of contraction with an EC₅₀ of 292.2 ± 75.7 μg/ml. Nifedipine, a well known L-type Ca²⁺ blocker, inhibited in a concentration-dependent manner left atrial force of contraction with an EC₅₀ of 12.1 ± 3.5 μg/ml. Sinus rhythm was diminished by EOAs with an EC₅₀ of 595.4 ± 56.2 μg/ml. Whole-cell L-type Ca²⁺ currents were recorded by using the patch-clamp technique. EOAs at 25 μg/ml decreased I_Ca,L by 32.6 ± 9.2% and at 250 μg/ml it decreased by 89.3 ± 7.4%. Thus, inhibition of L-type Ca²⁺ channels is involved in the cardiodepressive effect elicited by the essential oil of Alpinia speciosa in rat heart.     

Regulation of environmental factors on the expression of a solid-state specific lipase (Lip1) with Rhizopus chinensis by western blot and indirect Elisa

To identify which solid-state typical environmental factors are involved in the induction of a solid-state special lipase (Lip1), western blot and Elisa based on Lip1 antibody were used. A low water activity played a significant role in the induction of Lip1, as evidenced by the increased expression level (20–46 μg/g dry cell) along with the decrease of water activity (0.927–0.969). Physical barrier against hyphal extension was found to be another required factor, since the expression of Lip1 was significantly enhanced by 3-fold using a membrane with smaller pore size (0.45 and 0.22 μm) covered on top of surface culture.     

The sedimentation properties of ferritins. New insights and analysis of methods of nanoparticle preparation

Background

Ferritin exhibits complex behavior in the ultracentrifuge due to variability in iron core size among molecules. A comprehensive study was undertaken to develop procedures for obtaining more uniform cores and assessing their homogeneity.

Methods

Analytical ultracentrifugation was used to measure the mineral core size distributions obtained by adding iron under high- and low-flux conditions to horse spleen (apoHoSF) and human H-chain (apoHuHF) apoferritins.

Results

More uniform core sizes are obtained with the homopolymer human H-chain ferritin than with the heteropolymer horse spleen HoSF protein in which subpopulations of HoSF molecules with varying iron content are observed. A binomial probability distribution of H- and L-subunits among protein shells qualitatively accounts for the observed subpopulations. The addition of Fe²⁺ to apoHuHF produces iron core particle size diameters from 3.8 ± 0.3 to 6.2 ± 0.3 nm. Diameters from 3.4 ± 0.6 to 6.5 ± 0.6 nm are obtained with natural HoSF after sucrose gradient fractionation. The change in the sedimentation coefficient as iron accumulates in ferritin suggests that the protein shell contracts ∼ 10% to a more compact structure, a finding consistent with published electron micrographs. The physicochemical parameters for apoHoSF (15%/85% H/L subunits) are M = 484,120 g/mol, ν? = 0.735 mL/g, s_20,w = 17.0 S and D_20,w = 3.21 × 10⁻⁷ cm²/s; and for apoHuHF M = 506,266 g/mol, ν? = 0.724 mL/g, s_20,w = 18.3 S and D_20,w = 3.18 × 10⁻⁷ cm²/s.

Significance

The methods presented here should prove useful in the synthesis of size controlled nanoparticles of other minerals.     

One-pot microwave derivatization of target compounds relevant to metabolomics with comprehensive two-dimensional gas chromatography

Metabolomics has been defined as the quantitative measurement of all low molecular weight metabolites (sugars, amino acids, organic acids, fatty acids and others) in an organism's cells at a specified time under specific environmental/biological conditions. Currently, there is considerable interest in developing a single method of derivatization and separation that satisfies the needs for metabolite analysis while recognizing the many chemical classes that constitute the metabolome. Chemical derivatization considerably increases the sensitivity and specificity of gas chromatography–mass spectrometry for compounds that are polar and have derivatizable groups. Microwave-assisted derivatization (MAD) of a set of standards spanning a wide range of metabolites of interest demonstrates the potential of MAD for metabolic profiling. A final protocol of 150 W power for 90 s was selected as the derivatization condition, based upon the study of each chemical class. A study of the generation of partially derivatized components established the conditions where this could potentially be a problem; the use of greater volumes of reagent ensured this would not arise. All compounds analyzed by comprehensive two-dimensional gas chromatography–time-of-flight mass spectrometry in a standard mixture showed good area ratio reproducibility against a naphthalene internal standard (RSD < 10% in all but one case). Concentrations tested ranged from 1 μg/mL to 1000 μg/mL, and the calibration curves for the standard mixtures were satisfactory with regression coefficients generally better than 0.998. The application to gas chromatography–quadrupole mass spectrometry and comprehensive two-dimensional gas chromatography–time-of-flight mass spectrometry for a typical reference standard of relevance to metabolomics is demonstrated.     

Amides derived from heteroaromatic amines and selected steryl hemiesters

The current interest of the team has been focused on investigation of novel amides with potential cytotoxicity. The presented series of compounds was synthesized from selected steryl hemiesters and heteroaromatic amines. The synthetic protocol was designed in a simple and economic way, and divided into several general methodologies applicable to the compounds synthesized. The cytotoxicity was tested on cells derived from human T-lymphoblastic leukemia, breast adenocarcinoma and cervical cancer, and compared with tests on normal human fibroblasts. Most of the lanosterol-based compounds (3–5 and 7–10) showed medium to good cytotoxicity, while only two derivatives of cholesterol (18 and 19) showed medium cytotoxicity on human T-lymphoblastic leukemia cell line. The compounds 8 and 9 displayed the reasonable cytotoxicity among this series of amides, tested on the cell lines of T-lymphoblastic leukemia [14.5 ± 0.4 μM (8) and 18.5 ± 3.9 μM (9)], breast adenocarcinoma [19.5 ± 2.1 μM (8) and 23.1 ± 4.0 μM (9)] and cervical cancer [24.8 ± 5.3 μM (8) and 29.1 ± 4.7 μM (9)]. Only the compound 8 was adequately less active on normal human fibroblasts (40.4 ± 11.1 μM).     

Long-term peripheral infusion of nociceptin/orphanin FQ promotes hyperplasia, activation and migration of mucosal mast cells in the rat gastric fundus

The endogenous neuropeptide nociceptin/orphanin FQ (N/OFQ) modulates behavioral and gastrointestinal responses to stress. Mucosal mast cells (MMCs) are primary mediators of stress-related responses in the gastrointestinal tract. We investigated the influence of N/OFQ and of the N/OFQ peptide (NOP) receptor antagonist, UFP-101, on MMCs in the rat gastric fundus. N/OFQ was infused subcutaneously for 52 h at 0.1, 1 and 10 μg/kg/h and at 1 μg/kg/h for 4 h, 52 h, 7 days and 14 days via Alzet osmotic minipumps. Density of MMCs and connective tissue mast cells (CTMCs) was assessed histochemically and immunohistochemically. Activation and location of MMCs were assessed by transmission electron microscopy. Contacts between MMCs and nerve elements were assessed by double immunofluorescence. N/OFQ (1 μg/kg/h) and UFP-101 (10 and 30 μg/kg/h) were infused subcutaneously in the absence and presence of acute cold-restraint stress and density of MMCs was assessed. Peripheral N/OFQ dose-dependently increased the density of MMCs, while not influencing CTMCs. The increasing effect was maintained up to 14 days following continuous infusion, while after termination of the 4-h infusion, the effect declined rapidly. The peptide promoted the activation of MMCs and their migration from the lamina propria toward the epithelial layer. The association between MMCs and nerve fibers was time-dependently down-regulated following N/OFQ infusion. The stress-induced hyperplasia of MMCs was not influenced by N/OFQ and abolished by UFP-101. UFP-101 alone was ineffective. The present results suggest that endogenous N/OFQ could be considered a potential component of the circuit neuropeptides-mast cells-stress.     

Regulation of post-translational protein arginine methylation during HeLa cell cycle

Background

Post-translational arginine methylation which modifies protein-arginyl residues by protein arginine methyltransferase (PRMT) was investigated during synchronized HeLa cell cycle.

Methods

The lysates of cells synchronized at each stage were subjected to one and/or two dimensional electrophoresis followed by Western immunoblot using against anti-asymmetric-dimethyl-arginine (ASYM24), anti-symmetric-dimethyl-arginine (SYM10), and subclasses of PRMTs, including PRMT1, PRMT3, PRMT4 (CARM1), PRMT5, PRMT6, and PRMT7 antibodies.

Results

Proteins with approximate molecular masses of 80 kDa, 68 kDa, and 64 kDa, containing asymmetric-dimethyl-arginine (aDMA) were increased at G0/G1 to G1, which lasted until S phase. In addition, 25 kDa protein of symmetric-dimethyl-arginine (sDMA) was also markedly up-regulated from G0/G1 to G1. The levels of PRMT3, PRMT6 and PRMT7 were concurrently increased during the cell cycle. Two-dimensional gel electrophoresis followed by MALDI-TOF-MS was identified as aDMA-80 kDa and aDMA-68 kDa proteins as heterogeneous nuclear ribonucleoprotein R (hnRNPR), aDMA-64 kDa proteins as cleavage stimulation factor 64 kDa subunit (CstF-64), and sDMA-25 kDa protein as triosephosphate isomerase (TPI). The levels of increased aDMA of hnRNPR were reduced, when HeLa cells were transfected with siRNA for PRMT1, and the aDMA of CstF-64 with siRNA for PRMT3, while depletion of PRMT5 down-regulated sDMA of TPI.

Conclusion

Protein arginine dimethylations of hnRNPR, CstF-64, and TPI were regulated during HeLa cell cycle by respective PRMTs.

General significance

These results suggest that regulation of arginine dimethylation of hnRNPR, CstF-64, and TPI at G0/G1 to G1 are most likely to modulate the cellular growth and proliferation in HeLa cell cycle.     

Effects of various squalene epoxides on coenzyme Q and cholesterol synthesis

2,3-Oxidosqualene is an intermediate in cholesterol biosynthesis and 2,3:22,23-dioxidosqualene act as the substrate for an alternative pathway that produces 24(S),25-epoxycholesterol which effects cholesterol homeostasis. In light of our previous findings concerning the biological effects of certain epoxidated all-trans-polyisoprenes, the effects of squalene carrying epoxy moieties on the second and third isoprene residues were investigated here. In cultures of HepG2 cells both monoepoxides of squalene and one of their hydrolytic products inhibited cholesterol synthesis and stimulated the synthesis of coenzyme Q (CoQ). Upon prolonged treatment the cholesterol content of these cells and its labeling with [³H]mevalonate were reduced, while the amount and labeling of CoQ increased. Injection of the squalene monoepoxides into mice once daily for 6 days elevated the level of CoQ in their blood, but did not change the cholesterol level. The same effects were observed upon treatment of apoE-deficient mice and diabetic GK-rats. This treatment increased the hepatic level of CoQ10 in mice, but the amount of CoQ9, which is the major form, was unaffected. The presence of the active compounds in the blood was supported by the finding that cholesterol synthesis in the white blood cells was inhibited. Since the ratio of CoQ9/CoQ10 varies depending on the experimental conditions, the cells were titrated with substrate and inhibitors, leading to the conclusion that the intracellular isopentenyl-PP pool is a regulator of this ratio. Our present findings indicate that oxidosqualenes may be useful for stimulating both the synthesis and level of CoQ both in vitro and in vivo.     

Determination of the γ-secretase inhibitor MK-0752 in human plasma by online extraction and electrospray tandem mass spectrometry (HTLC–ESI-MS/MS)

A sensitive and rapid HTLC–ESI-MS/MS method with an advanced online sample preparation was developed for determination of the γ-secretase inhibitor MK-0752 in human plasma using an internal standard. Plasma samples (100 μL) were diluted and injected directly onto an online extraction column (Cohesive Cyclone MAX 0.5 mm × 50 mm, >30 μm), the sample matrix was washed out with an aqueous solution, and retained analytes were eluted out and transferred directly to the analytical column (Phenomenex Gemini 3μ C18 110A, 50 mm × 2.0 mm at 50 °C) for separation using a gradient mobile phase. The eluted analytes were then detected on an API-3000 LC–MS/MS System with ESI and a negative multiple reaction monitoring mode. The monitored ion transitions were m/z 441 → 175 for MK-0752 and 496 → 175 for the internal standard. Online extraction recoveries were 81%. The method was validated and was linear in the range of 0.05–50 μg/mL. Within-day and between-day precisions were < 8.6%, and accuracies were 0.7 and 7.1%. This method was applied to the measurement of plasma MK-0752 levels in a Phase I study of pediatric patients with recurrent or refractory brain tumors.