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Cell-free System (cell-free + system)
Selected AbstractsGenotoxicity of inorganic lead salts and disturbance of microtubule functionENVIRONMENTAL AND MOLECULAR MUTAGENESIS, Issue 4 2005Daniela Bonacker Abstract Lead compounds are known genotoxicants, principally affecting the integrity of chromosomes. Lead chloride and lead acetate induced concentration-dependent increases in micronucleus frequency in V79 cells, starting at 1.1 ,M lead chloride and 0.05 ,M lead acetate. The difference between the lead salts, which was expected based on their relative abilities to form complex acetato-cations, was confirmed in an independent experiment. CREST analyses of the micronuclei verified that lead chloride and acetate were predominantly aneugenic (CREST-positive response), which was consistent with the morphology of the micronuclei (larger micronuclei, compared with micronuclei induced by a clastogenic mechanism). The effects of high concentrations of lead salts on the microtubule network of V79 cells were also examined using immunofluorescence staining. The dose effects of these responses were consistent with the cytotoxicity of lead(II), as visualized in the neutral-red uptake assay. In a cell-free system, 20,60 ,M lead salts inhibited tubulin assembly dose-dependently. The no-observed-effect concentration of lead(II) in this assay was 10 ,M. This inhibitory effect was interpreted as a shift of the assembly/disassembly steady-state toward disassembly, e.g., by reducing the concentration of assembly-competent tubulin dimers. The effects of lead salts on microtubule-associated motor-protein functions were studied using a kinesin-gliding assay that mimics intracellular transport processes in vitro by quantifying the movement of paclitaxel-stabilized microtubules across a kinesin-coated glass surface. There was a dose-dependent effect of lead nitrate on microtubule motility. Lead nitrate affected the gliding velocities of microtubules starting at concentrations above 10 ,M and reached half-maximal inhibition of motility at about 50 ,M. The processes reported here point to relevant interactions of lead with tubulin and kinesin at low dose levels. Environ. Mol. Mutagen., 2005. © 2005 Wiley-Liss, Inc. [source] Implication of allelic polymorphism of osteopontin in the development of lupus nephritis in MRL/lpr miceEUROPEAN JOURNAL OF IMMUNOLOGY, Issue 5 2005Tatsuhiko Miyazaki Abstract Potentially, autoimmune diseases develop from a combination of multiple genes with allelic polymorphisms. An MRL/Mp-Faslpr/lpr (MRL/lpr) strain of mice develops autoimmune diseases, including lupus nephritis, but another lpr strain, C3H/HeJ-Faslpr/lpr (C3H/lpr) does not. This indicates that MRL polymorphic genes are involved in the development of the diseases. By quantitative trait loci (QTL) analysis using 527 of the (MRL/lpr × C3H/lpr)F2 mice, we identified a novel locus for susceptibility to lupus nephritis at map position D5Mit115 on chromosome 5, the same alias of the osteopontin (Opn) gene (LOD score =4.0), susceptible in the MRL allele. In functional analyses of the MRL and C3H Opn alleles using synthetic osteopontin (OPN) made with a new method "cell-free system" with wheat germ ribosomes, the MRL-OPN induced higher expression and production of immunoglobulins as well as cytokines including TNF-,, IL-1, and IFN-, in splenocytes and/or macrophages than that of the C3H allele. These findings suggest that allelic polymorphism of OPN causes the functional differences in antibody production and macrophage activation between MRL and C3H strains, possibly involved in the development of lupus nephritis. [source] Evaluation of detergents for the soluble expression of ,-helical and ,-barrel-type integral membrane proteins by a preparative scale individual cell-free expression systemFEBS JOURNAL, Issue 23 2005Christian Klammt Cell-free expression has become a highly promising tool for the fast and efficient production of integral membrane proteins. The proteins can be produced as precipitates that solubilize in mild detergents usually without any prior denaturation sttif. Alternatively, membrane proteins can be synthesized in a soluble form by adding detergents to the cell-free system. However, the effects of a representative variety of detergents on the production, solubility and activity of a wider range of membrane proteins upon cell-free expression are currently unknown. We therefore analyzed the cell-free expression of three structurally very different membrane proteins, namely the bacterial ,-helical multidrug transporter, EmrE, the ,-barrel nucleoside transporter, Tsx, and the porcine vasopressin receptor of the eukaryotic superfamily of G-protein coupled receptors. All three membrane proteins could be produced in amounts of several mg per one ml of reaction mixture. In general, the detergent 1-myristoyl-2-hydroxy- sn -glycero-3-[phospho- rac -(1-glycerol)] was found to be most effective for the resolubilization of membrane protein precipitates, while long chain polyoxyethylene-alkyl-ethers proved to be most suitable for the soluble expression of all three types of membrane proteins. The yield of soluble expressed membrane protein remained relatively stable above a certain threshold concentration of the detergents. We report, for the first time, the high-level cell-free expression of a ,-barrel type membrane protein in a functional form. Structural and functional variations of the analyzed membrane proteins are evident that correspond with the mode of expression and that depend on the supplied detergent. [source] ,-Fetoprotein positively regulates cytochrome c -mediated caspase activation and apoptosome complex formationFEBS JOURNAL, Issue 21 2003Lidia Semenkova Previous results have shown that the oncoembryonic marker ,-fetoprotein (AFP) is able to induce apoptosis in tumor cells through activation of caspase 3, bypassing Fas-dependent and tumor necrosis factor receptor-dependent signaling. In this study we further investigate the molecular interactions involved in the AFP-mediated signaling of apoptosis. We show that AFP treatment of tumor cells is accompanied by cytosolic translocation of mitochondrial cytochrome c. In a cell-free system, AFP mediates processing and activation of caspases 3 and 9 by synergistic enhancement of the low-dose cytochrome c -mediated signals. AFP was unable to regulate activity of caspase 3 in cell extracts depleted of cytochrome c or caspase 9. Using high-resolution chromatography, we show that AFP positively regulates cytochrome c/dATP-mediated apoptosome complex formation, enhances recruitment of caspases and Apaf-1 into the complex, and stimulates release of the active caspases 3 and 9 from the apoptosome. By using a direct protein,protein interaction assay, we show that pure human AFP almost completely disrupts the association between processed caspases 3 and 9 and the cellular inhibitor of apoptosis protein (cIAP-2), demonstrating its release from the complex. Our data suggest that AFP may regulate cell death by displacing cIAP-2 from the apoptosome, resulting in promotion of caspase 3 activation and its release from the complex. [source] Generation of catalytically active 6-phosphofructokinase from Saccharomyces cerevisiae in a cell-free systemFEBS JOURNAL, Issue 15 2000Anke Edelmann PFK1 and PFK2 coding for the subunits of 6-phosphofructokinase from Saccharomyces cerevisiae were cloned into plasmids suitable for runoff transcription. In vitro translation products of both kinds of subunit were obtained using rabbit reticulocyte lysate as the synthesis and folding system. They were monitored by chemiluminescent Western-blot analysis. Folding and assembly of the ,-subunit and ,-subunit of 6-phosphofructokinase were found to occur in the cell-free system resulting in an enzymatically active protein. The in vitro generated enzyme exhibits a folding state that is similar to that of the heterooctameric form of 6-phosphofructokinase in the presence of fructose 6-phosphate, ATP and ammonium sulfate, as demonstrated by size-exclusion HPLC followed by ELISA. [source] FLORIDOSIDE AS A CARBON PRECURSOR FOR THE SYNTHESIS OF CELL-WALL POLYSACCHARIDE IN THE RED MICROALGA PORPHYRIDIUM SP. (RHODOPHYTA),JOURNAL OF PHYCOLOGY, Issue 5 2002Shi-Yan Li Although red algae are known to be obligatory photoautotrophs, the red microalga Porphyridium sp. was shown to assimilate and metabolize floridoside. A pulse-chase experiment with [14C]floridoside showed that at the end of a 240-min pulse, 70% of total 14C-uptake by the cells remained in the floridoside fraction. To evaluate the assimilation of floridoside by Porphyridium sp. cells, we exposed Porphyridium sp. not only to [14C]floridoside but also to its constituents, [14C]glycerol and [14C]galactose, as compared with [14C]bicarbonate. The extent of incorporation of [14C] galactose by the Porphyridium sp. cells was insignificant (50,80 dpm·mL,1), whereas uptake of 14C from [14C]glycerol into the algal cells was evident (2.4 × 103 dpm·mL,1) after 60 min of the pulse. The pattern of 14C distribution among the major constituent sugars, xylose, glucose and galactose, of the labeled soluble polysaccharide was dependent on the 14C source. The relative content of [14C]galactose in the soluble polysaccharide was highest (28.8%) for [14C]floridoside-labeled culture and lowest (19.8%) for the [14C]glycerol-labeled culture. Upon incubation of [14C]floridoside with a crude extract of a cell-free system prepared from nonlabeled cells of Porphyridium sp., the label was indeed found to be incorporated into the sulfated polysaccharide. Our results suggested that the carbon metabolic pathway in Porphyridium sp. passes through the low molecular weight photoassimilatory product,floridoside,toward sulfated cell-wall polysaccharide production. [source] Ridogrel, a dual thromboxane synthase inhibitor and receptor antagonist: anti-inflammatory profile in inflammatory bowel diseaseALIMENTARY PHARMACOLOGY & THERAPEUTICS, Issue 6 2000Carty Background: Thromboxanes, prostaglandins, reactive oxygen metabolites and pro-inflammatory cytokines are produced in excess in inflammatory bowel disease. Preliminary reports suggest that ridogrel, a thromboxane synthesis inhibitor and receptor blocker, may have therapeutic benefits in ulcerative colitis. Aims: To investigate the anti-inflammatory profile of ridogrel. Methods: The effects of ridogrel on the production of eicosanoids, reactive oxygen metabolites and cytokines by cultured inflamed colorectal mucosal biopsies were made using ELISA and chemiluminescence, reactive oxygen metabolite generation in a cell-free system, and platelet activation using flow cytometry. The effects of oral ridogrel on mucosal release of eicosanoids in two patients with active ulcerative colitis were assessed using rectal dialysis. Results: Ridogrel significantly reduced the release of thromboxane B2, but not prostaglandin E2 or tumour necrosis factor-,, from biopsies (P < 0.01 for 10 ,M ridogrel). Ridogrel showed no direct antioxidant activity but significantly reduced reactive oxygen metabolite production from cultured biopsies (P < 0.01 for 10 ,M ridogrel). Platelet activation in vitro was inhibited by ridogrel (P , 0.05 for , 10 ,M ridogrel). Mean rectal mucosal thromboxane B2 release was reduced to 86% of pre-treatment levels in two patients treated with oral ridogrel. Conclusions: Its inhibition of mucosal production of thromboxane B2, reactive oxygen metabolites, and of platelet activation, suggests that ridogrel could have a therapeutic role in inflammatory bowel disease. [source] Apple polyphenols diminish the phosphorylation of the epidermal growth factor receptor in HT29 colon carcinoma cellsMOLECULAR NUTRITION & FOOD RESEARCH (FORMERLY NAHRUNG/FOOD), Issue 5 2007Diana Fridrich Abstract Previously, we showed that an apple juice extract (AE) potently inhibits the protein tyrosine kinase (PTK) activity of the epidermal growth factor receptor (EGFR). In the present study, an apple pomace extract (APE) was found to exceed the EGFR inhibitory properties of AE in a cell-free system. The impact of the extracts on the phosphorylation status of the EGFR in intact cells (HT29) was sensitive to catalase, added to suppress the accumulation of hydrogen peroxide. In the absence of catalase, the formation of hydrogen peroxide was observed, achieving 1.1 ± 0.1 ,M (AE) and 1.5 ± 0.1 ,M (APE) after 45 min of incubation. In the presence of catalase, suppressing the hydrogen peroxide level to the solvent control, APE effectively suppressed EGFR phosphorylation, even exceeding the effects of AE. Both extracts inhibited the growth of HT29 cells, albeit the enhanced EGFR inhibitory properties of APE compared to AE were not reflected by a higher growth inhibitory potential. The results clearly show that the effect of apple extracts on the EGFR and cell growth are not simply artefacts of hydrogen peroxide formation. However, the formation of hydrogen peroxide has to be considered to modulate and/or mask cellular responses to apple extracts. [source] OT-674 Suppresses Photooxidative Processes Initiated by an RPE Lipofuscin FluorophorePHOTOCHEMISTRY & PHOTOBIOLOGY, Issue 1 2008Jilin Zhou The pathological processes involved in age-related macular degeneration (AMD) include retinal pigment epithelial (RPE) cell degeneration; oxidative mechanisms likely contribute to the demise of these cells. Indeed, RPE cells may be particularly susceptible to photooxidative mechanisms since they accumulate retinoid-derived photoreactive compounds that constitute the lipofuscin of the cell. Thus we undertook to test the capacity of OT-674, the reduction product (Tempol-H) of the nitroxide Tempol, to suppress photooxidative processes initiated by the RPE lipofuscin fluorophore A2E. Accordingly, when ARPE-19 cells that had accumulated A2E were irradiated at 430 nm, pretreatment with OT-674 (0.01,10 mm) was found to confer a resistance to cell death. Monitoring by quantitative HPLC also showed that OT-674 reduced A2E photooxidation in a cell-free system. Moreover, when presented with a singlet oxygen generator, OT-674 served as a quencher of singlet oxygen that was more effective than Trolox and ,-tocopherol. We conclude that OT-674 is a potent antioxidant that suppresses photooxidative processes generated in cultured RPE cells by the lipofuscin fluorophore A2E. As oxidative damage to RPE cells is considered to be a risk factor for AMD, antioxidant therapy with OT-674 may serve a protective role. [source] Nitric oxide generation from hydroxylamine in the presence of neutrophils and in the cell-free systemAPMIS, Issue 7-8 2001Magdalena Klink Conversion of hydroxylamine (HA) to nitric oxide (NO) has been studied in the presence or absence of human neutrophils with or without myristate acetate phorbol (PMA), catalase (CAT), hydrogen peroxide (H2O2), and superoxide dismutase (SOD) and nitric oxide synthase (NOS) inhibitors. The generation of NO from HA in the presence of neutrophils was higher than in the cell-free system. We found that catalase did not influence the nitrite generation from HA in the cell-free system and in the presence of neutrophils. The H2O2 enhanced the NO generation from HA in the presence of neutrophils only. When catalase and H2O2 were added together, a high increase of NO generation from HA in both systems was observed. The addition of SOD decreased whereas addition of PMA enhanced the NO generation from HA in the presence of neutrophils. The presented data show the possible role of oxygen radicals in the decomposition of HA to NO. The addition of NOS inhibitors to the culture of neutrophils decreased the generation of nitrite from HA. Our results suggest that NO generation from HA, which is an intermediate in NO production from L-arginine, may be supported by an enzymatic pathway in which cellular NO synthase is involved. [source] Structure of a conserved CoA-binding protein synthesized by a cell-free systemACTA CRYSTALLOGRAPHICA SECTION D, Issue 7 2003Takashi Wada TT1466 is a hypothetical protein from the extremely thermophilic bacterium Thermus thermophilus HB8 and is highly conserved in bacteria and archaea. The selenomethionyl protein was synthesized by a cell-free system and the crystal structure was determined at 2.0,Å by MAD phasing. A native crystal was used for structure refinement to 1.7,Å. The structure is highly homologous to that of the CoA-binding domain of the succinyl-CoA synthetase from Escherichia coli, despite the protein having only 14% sequence identity to this domain. An isothermal titration calorimetry experiment was performed to investigate whether TT1466 binds CoA and revealed high-affinity CoA binding of TT1466. [source] High-level cell-free synthesis yields of proteins containing site-specific non-natural amino acidsBIOTECHNOLOGY & BIOENGINEERING, Issue 2 2009Aaron R. Goerke Abstract We describe an E. coli -based cell-free system for the production of proteins with a non-natural amino acid (nnAA) incorporated site-specifically (modified protein). The mutant Methanococcus jannaschii tyrosyl-tRNA synthetase (mTyrRS) and tRNATyr pair were used as orthogonal elements. The mTyrRS experienced proteolysis and modified protein yields improved with higher synthetase addition (200,300 µg/mL). Product yields were also improved by increasing levels of total protein to 20 mg protein/mL and available vesicle surface area to 0.5 m2/mL. This new E. coli -based cell-free procedure produced up to 400 µg/mL of eCAT109pAz, 660 µg/mL of eDHFR10pAz, and 210 µg/mL of mDHFR31pAz with p -azido- L -phenylalanine (pAz) incorporated site-specifically at the amber nonsense codon. O -methyl- L -tyrosine and p -acetyl- L -phenylalanine were incorporated by similar protocols. The desired specificity for incorporation of the nnAA by the cell-free system was confirmed. Additionally, the modified proteins were enzymatically active and reactive for copper(I)-catalyzed (3,+,2) cycloadditions (click chemistry). Biotechnol. Bioeng. 2009;102: 400,416. © 2008 Wiley Periodicals, Inc. [source] Simultaneous expression and maturation of the iron-sulfur protein ferredoxin in a cell-free systemBIOTECHNOLOGY & BIOENGINEERING, Issue 1 2006Marcus E. Boyer Abstract The model iron-sulfur (Fe-S) protein ferredoxin (Fd) from Synechocystis sp. PCC 6803 has been simultaneously produced and matured in a cell-free production system. After 6 h of incubation at 37°C, Fd accumulated to >450 µg/mL. Essentially all was soluble, and 85% was active. Production and maturation of the protein in the cell-free system were found to be dependent in a coupled manner on the concentration of the supplemented iron and sulfur sources, ferrous ammonium sulfate and cysteine, respectively. The recombinant expression of ISC helper proteins during cell extract preparation did not increase cell-free Fd accumulation or activity, although the efficiency of iron and cysteine utilization increased. Fd maturation was independent of protein production rate, and proceeded at a constant rate throughout the period of active translation. In addition, incubation of denatured apo Fd with cell-free reaction components resulted in recovery of Fd activity, supporting the interpretation that maturation mechanisms did not act co-translationally. Incubation at 28°C increased total and active protein accumulation, but decreased the ratio of active to total Fd produced. In summary, the high product yields and folding efficiency make the cell-free system described here an attractive platform for the study of Fe-S protein production and maturation. The system enables both small-volume, high throughput investigations as well as larger scale production. To our knowledge, this is the first demonstration of directed, high-yield production and maturation of an Fe-S protein in a cell-free system. © 2006 Wiley Periodicals, Inc. [source] A wheat embryo cell-free protein synthesis system not requiring an exogenous supply of GTPBIOTECHNOLOGY PROGRESS, Issue 5 2009Hirohisa Koga Abstract Most in vitro protein synthesis systems require a supply of GTP for the formation of translation initiation complexes, with two GTP molecules per amino acid needed as an energy source for a peptide elongation reaction. In order to optimize protein synthesis reactions in a continuous-flow wheat embryo cell-free system, we have examined the influence of adding GTP and found that the system does not require any supply of GTP. We report here the preparation of a wheat embryo extract from which endogenous GTP was removed by gel filtration, and the influence of adding GTP to the system on protein synthesis reactions. Using Green Fluorescent Protein (GFP) as a reporter, higher levels of production were observed at lower concentrations of GTP, with the optimal level of production obtained with no supply of GTP. A HPLC-based analysis of the extract and the translation mixture containing only ATP as an energy source revealed that GTP was not detectable in the extract, however, 35 ,M of GTP was found in the translation mixture. This result suggests that GTP could be generated from other compounds, such as GDP and GMP, using ATP. A similar experiment with a C-terminally truncated form of human protein tyrosine phosphatase 1B (hPTP1B1-320) gave almost the same result. The wheat embryo cell-free translation system worked most efficiently without exogenous GTP, producing 3.5 mg/mL of translation mixture over a 48-h period at 26°C. © 2009 American Institute of Chemical Engineers Biotechnol. Prog., 2009 [source] Design of a cytochrome P450BM3 reaction system linked by two-step cofactor regeneration catalyzed by a soluble transhydrogenase and glycerol dehydrogenaseBIOTECHNOLOGY PROGRESS, Issue 5 2009Tsuyoshi Mouri Abstract A cytochrome P450BM3-catalyzed reaction system linked by a two-step cofactor regeneration was investigated in a cell-free system. The two-step cofactor regeneration of redox cofactors, NADH and NADPH, was constructed by NAD+ -dependent bacterial glycerol dehydrogenase (GLD) and bacterial soluble transhydrogenase (STH) both from Escherichia coli. In the present system, the reduced cofactor (NADH) was regenerated by GLD from the oxidized cofactor (NAD+) using glycerol as a sacrificial cosubstrate. The reducing equivalents were subsequently transferred to NADP+ by STH as a cycling catalyst. The resultant regenerated NADPH was used for the substrate oxidation catalyzed by cytochrome P450BM3. The initial rate of the P450BM3-catalyzed reaction linked by the two-step cofactor regeneration showed a slight increase (approximately twice) when increasing the GLD units 10-fold under initial reaction conditions. In contrast, a 10-fold increase in STH units resulted in about a 9-fold increase in the initial reaction rate, implying that transhydrogenation catalyzed by STH was the rate-determining step. In the system lacking the two-step cofactor regeneration, 34% conversion of 50 ,M of a model substrate (p-nitrophenoxydecanoic acid) was attained using 50 ,M NADPH. In contrast, with the two-step cofactor regeneration, the same amount of substrate was completely converted using 5 ,M of oxidized cofactors (NAD+ and NADP+) within 1 h. Furthermore, a 10-fold dilution of the oxidized cofactors still led to approximately 20% conversion in 1 h. These results indicate the potential of the combination of GLD and STH for use in redox cofactor recycling with catalytic quantities of NAD+ and NADP+. © 2009 American Institute of Chemical Engineers Biotechnol. Prog., 2009 [source] Cell-Free Protein Synthesis System Prepared from Insect Cells by Freeze-ThawingBIOTECHNOLOGY PROGRESS, Issue 6 2006Toru Ezure We established a novel cell-free protein synthesis system derived from Trichoplusia ni (HighFive) insect cells by a simple extraction method. Luciferase and ,-galactosidase were synthesized in this system with active forms. We analyzed and optimized (1) the preparation method of the insect cell extract, (2) the concentration of the reaction components, and (3) the 5,-untranslated region (5,-UTR) of mRNA. The extract was prepared by freeze-thawing insect cells suspended in the extraction buffer. This preparation method was a simple and superior method compared with the conventional method using a Dounce homogenizer. Furthermore, protein synthesis efficiency was improved by the addition of 20% (v/v) glycerol to the extraction buffer. Concentrations of the reaction components were optimized to increase protein synthesis efficiency. Moreover, mRNAs containing 5,-UTRs derived from baculovirus polyhedrin genes showed high protein synthesis activity. Especially, the leader composition of the Ectropis obliqua nucleopolyhedrovirus polyhedrin gene showed the highest enhancement activity among the six 5,-UTRs tested. As a result, in a batch reaction approximately 71 ,g of luciferase was synthesized per milliliter of reaction volume at 25 °C for 6 h. Moreover, this method for the establishment of a cell-free system was applied also to Spodoptera frugiperda 21 (Sf21) insect cells. After optimizing the concentrations of the reaction components and the 5,-UTR of mRNA, approximately 45 ,g/mL of luciferase was synthesized in an Sf21 cell-free system at 25 °C for 3 h. These productivities were sufficient to perform gene expression analyses. Thus, these cell-free systems may be a useful tool for simple synthesis in post-genomic studies as a novel protein production method. [source] Prolonging Cell-Free Protein Synthesis by Selective Reagent AdditionsBIOTECHNOLOGY PROGRESS, Issue 3 2000Dong-Myung Kim Factors causing the early cessation of protein synthesis have been studied in a cell-free system from Escherichia coli. We discovered that phosphoenol pyruvate (PEP), the secondary energy source for ATP regeneration, and several amino acids are rapidly degraded during the cell-free protein synthesis reaction. The degradation of such compounds takes place even in the absence of protein synthesis. This degradation severely reduces the capacity for protein synthesis. The lost potency was completely recovered when the reaction mixture was supplied with additional PEP and amino acids. Of the 20 amino acids, only arginine, cysteine, and tryptophan were required to restore system activity. Through repeated additions of PEP, arginine, cysteine,and tryptophan, the duration of protein synthesis was greatly extended. In this fed-batch reaction, after a 2 h incubation, the level of cell-free synthesized chloramphenicol acetyl transferase (CAT) reached 350 ,g/mL, which is 3.5 times the yield of the batch reaction. Addition of fresh magnesium further extended the protein synthesis. As a result, through coordinated additions of PEP, arginine, cysteine, tryptophan, and magnesium, the final concentration of cell-free synthesized CAT increased more than 4-fold compared to a batch reaction. SDS-PAGE analysis of such a fed-batch reaction produced an obvious band of CAT upon Coomassie Blue staining. [source] Effects of policosanol treatment on the susceptibility of low density lipoprotein (LDL) isolated from healthy volunteers to oxidative modification in vitroBRITISH JOURNAL OF CLINICAL PHARMACOLOGY, Issue 3 2000Roberto Menéndez Aims The aim of this study was to investigate the effect of policosanol on the susceptibility of LDL-C to in vitro lipid peroxidation in human healthy volunteers. Methods The effect of policosanol (5 and 10 mg day,1) on LDL-C oxidation was studied in a double-blind, randomized, placebo-controlled trial conducted in 69 subjects. LDL-C samples isolated at baseline and after 8 weeks were subjected to in vitro tests of LDL-C oxidation. We tested the susceptibility of LDL-C to lipid peroxidation in a cell-free system by the addition of copper ions as well as in a more physiological system, macrophage-mediated oxidation. Results At baseline all groups were well matched regarding all variables. After 8 weeks of therapy policosanol administered at 5 and 10 mg, significantly and in a dose-dependent manner increased the lag phase of conjugated diene generation (mean ± s.d.) from 83.79 ± 29.16 min to 94.90 ± 25.50 min (5 mg day,1) and from 82.74 ± 17.16 min to 129.89 ± 35.71 min (10 mg day,1), while in the placebo group LDL-C oxidation did not change significantly. Policosanol (10 mg day,1), but not placebo, significantly decreased the rate of conjugated diene generation. Comparison with placebo after therapy also showed significant differences. Macrophage mediated-oxidation was also inhibited by policosanol as evident by measuring thiobarbituric acid reactive substances (TBARS). Policosanol (10 mg day,1) significantly lowered malondialdehyde (MDA) generation from 8.50 ± 0.91 to 5.76 ± 1.01 nmol mg,1 protein. Comparison with placebo after 5 and 10 mg day,1 showed significant differences. Policosanol significantly lowered total cholesterol by 10.5% (5 mg day,1) and 12.4% (10 mg day,1) and LDL-C by 16.7% and 20.2%, respectively. Also, policosanol (10 mg day,1) increased HDL-C by 15.2%. Five subjects withdrew from the study, none because of adverse experiences. No clinical or blood biochemical drug-related disturbances were found. Conclusions The present study demonstrated that policosanol administered within its therapeutic dosage for lowering cholesterol (5 and 10 mg day,1), decreased the susceptibility of LDL-C to lipid peroxidation in vitro. [source] Romidepsin (FK228), a potent histone deacetylase inhibitor, induces apoptosis through the generation of hydrogen peroxideCANCER SCIENCE, Issue 10 2010Hideki Mizutani Romidepsin (FK228) is a potent histone deacetylase (HDAC) inhibitor, which has a potent anticancer activity, but its molecular mechanism is unknown. We investigated the mechanism of FK228-induced apoptosis in the human leukemia cell line HL-60 and its hydrogen peroxide (H2O2)-resistant sub-clone, HP100, and the human colon cancer cell line Caco-2. Cytotoxicity and DNA ladder formation induced by FK228 could be detected in HL-60 cells after a 24-h incubation, whereas they could not be detected in HP100 cells. Trichostatin A (TSA), an HDAC inhibitor, induced DNA ladder formation in both HL-60 and HP100 cells. In contrast, FK228 inhibited HDAC activity in both HL-60 and HP100 cells to a similar extent. These findings suggest that FK228-induced apoptosis involves H2O2 -mediated pathways and that TSA-induced apoptosis does not. Flow cytometry revealed H2O2 formation and a change in mitochondrial membrane potential (,,m) in FK228-treated cells. FK228 also induced apoptosis in Caco-2 cells, which was prevented by N -acetyl-cysteine, suggesting that reactive oxygen species participate in apoptosis in various types of tumor cells. Interestingly, in a cell-free system, FK228 generated superoxide (O2,) in the presence of glutathione, suggesting that H2O2 is derived from dismutation of O2, produced through redox-cycle of FK228. Therefore, in addition to HDAC inhibition, H2O2 generated from FK228 may participate in its apoptotic effect. (Cancer Sci 2010;) [source] A New Protein Engineering Approach Combining Chemistry and Biology, Part I; Site-Specific Incorporation of 4-Iodo- L -phenylalanine in vitro by Using Misacylated Suppressor tRNAPheCHEMBIOCHEM, Issue 10 2006Koichiro Kodama Abstract An Escherichia coli suppressor tRNAPhe (tRNAPheCUA) was misacylated with 4-iodo- L -phenylalanine by using the A294G phenylalanyl,tRNA synthetase mutant (G294-PheRS) from E. coli at a high magnesium-ion concentration. The preacylated tRNA was added to an E. coli cell-free system and a Ras protein that contained the 4-iodo- L -phenylalanine residue at a specific target position was synthesized. Site-specific incorporation of 4-iodo- L -phenylalanine was confirmed by using LC,MS/MS. Free tRNAPheCUA was not aminoacylated by aminoacyl,tRNA synthetases (aaRSs) present in the E. coli cell-free system. Our approach will find wide application in protein engineering since an aryl iodide tag on proteins can be used for site-specific functionalization of proteins. [source] Stabilization of G-Quadruplex DNA with Platinum(II) Schiff Base Complexes: Luminescent Probe and Down-Regulation of c- myc Oncogene ExpressionCHEMISTRY - A EUROPEAN JOURNAL, Issue 47 2009Peng Wu Dr. Abstract The interactions of a series of platinum(II) Schiff base complexes with c- myc G-quadruplex DNA were studied. Complex [PtL1a] (1,a; H2L1a=N,N,-bis(salicylidene)-4,5-methoxy-1,2-phenylenediamine) can moderately inhibit c- myc gene promoter activity in a cell-free system through stabilizing the G-quadruplex structure and can inhibit c- myc oncogene expression in cultured cells. The interaction between 1,a and G-quadruplex DNA has been examined by 1H NMR spectroscopy. By using computer-aided structure-based drug design for hit-to-lead optimization, an in silico G-quadruplex DNA model has been constructed for docking-based virtual screening to develop new platinum(II) Schiff base complexes with improved inhibitory activities. Complex [PtL3] (3; H2L3=N,N,-bis{4-[1-(2-propylpiperidine)oxy]salicylidene}-4,5-methoxy-1,2-phenylenediamine) has been identified with a top score in the virtual screening. This complex was subsequently prepared and experimentally tested in vitro for its ability to stabilize or induce the formation of the c -myc G-quadruplex. The inhibitory activity of 3 (IC50=4.4,,M) is tenfold more than that of 1,a. The interaction between 1,a or 3 with c- myc G-quadruplex DNA has been examined by absorption titration, emission titration, molecular modeling, and NMR titration experiments, thus revealing that both 1,a and 3 bind c- myc G-quadruplex DNA through an external end-stacking mode at the 3' terminal face of the G-quadruplex. Such binding of G-quadruplex DNA with 3 is accompanied by up to an eightfold increase in the intensity of photoluminescence at ,max=652,nm. Complex 3 also effectively down-regulated the expression of c- myc in human hepatocarcinoma cells. [source] Insights into electromagnetic interaction mechanismsJOURNAL OF CELLULAR PHYSIOLOGY, Issue 1 2002Reba Goodman Low frequency (<,300 Hz) electromagnetic (EM) fields induce biological changes that include effects ranging from increased enzyme reaction rates to increased transcript levels for specific genes. The induction of stress gene HSP70 expression by exposure to EM fields provides insight into how EM fields interact with cells and tissues. Insights into the mechanism(s) are also provided by examination of the interaction of EM fields with moving charges and their influence on enzyme reaction rates in cell-free systems. Biological studies with in vitro model systems have focused, in general, on the nature of the signal transduction pathways involved in response to EM fields. It is likely, however, that EM fields also interact directly with electrons in DNA to stimulate biosynthesis. Identification of an EM field-sensitive DNA sequence in the heat shock 70 (HSP70) promoter, points to the application of EM fields in two biomedical applications: cytoprotection and gene therapy. EM field induction of the stress protein hsp70 may also provide a useful biomarker for establishing a science-based safety standard for the design of cell phones and their transmission towers. © 2002 Wiley-Liss, Inc. [source] Interactions between melatonin and nicotinamide nucleotide: NADH preservation in cells and in cell-free systems by melatoninJOURNAL OF PINEAL RESEARCH, Issue 2 2005Dun-Xian Tan Abstract:, Interactions of melatonin and nicotinamide adenine dinucleotide (NADH) have been studied in different experimental models including NADH-promoted oxyhemoglobin oxidation, vanadate-induced NADH oxidation and paraquat-induced NADH depletion in cultured PC12 cells. Our findings indicate that melatonin preserves NADH levels under oxidative stress both in cell-free systems and in cultured PC12 cells. These interactions likely involve electron donation by melatonin and reduction of the NAD radical. As a result, the NAD radical is recycled to NADH and melatonin is oxidized to N1 -acetyl- N2 -formyl-5-methoxykynuramine (AFMK). NADH is a central molecule at the crossroads between energy metabolism and the antioxidant defense system in organisms. Recycling of NADH by melatonin might improve the efficiency of NADH as an energy carrier and as an antioxidant. Interactions between melatonin and NADH may be implicated in mitochondrial metabolism. [source] Efficient and scalable method for scaling up cell free protein synthesis in batch modeBIOTECHNOLOGY & BIOENGINEERING, Issue 4 2005Alexei M. Voloshin A novel method for general cell free system scale-up in batch mode was applied to expression of E. coli chloramphenicol acetyl transferase (CAT) and a GMCSF-scFv fusion protein being developed as a B-cell lymphoma vaccine candidate (GLH). Performance of two different E. coli based cell-free systems was evaluated using the new scale-up approach. Reaction volumes from 15 to 500 µL were tested for both products and both reaction systems. In each case, the new scale-up method preserved total, soluble, and active volumetric yields of GLH and CAT at every reaction volume. At the 500 µL reaction volume, the PANOx SP system produced 560,±,36 µg/mL of active CAT and 99,±,10 µg/mL of active GLH protein using the new thin film approach whereas 500 µL test tube reactions produced 250,±,42 µg/mL and 72,±,7 µg/mL of active CAT and GLH respectively. Similarly, 500 µL cell-free synthesis reactions with the Cytomim system produced 481,±,38 µg/mL of active CAT and 109,±,15 µg/mL active GLH respectively in thin films compared to 29,±,7 µg/mL of active CAT and 5,±,2 µg/mL of active GLH protein in 500 µL test tube reactions. The new thin film approach improves oxygen supply for the Cytomim system, and increases the availability of hydrophobic surfaces. Analysis suggests that these surfaces provide significant benefit for protein expression and folding. We believe that this approach provides a general reaction scale-up technology that will be suitable for any protein target, cell free system, and reaction volume. © 2005 Wiley Periodicals, Inc. [source] Cell-Free Protein Synthesis System Prepared from Insect Cells by Freeze-ThawingBIOTECHNOLOGY PROGRESS, Issue 6 2006Toru Ezure We established a novel cell-free protein synthesis system derived from Trichoplusia ni (HighFive) insect cells by a simple extraction method. Luciferase and ,-galactosidase were synthesized in this system with active forms. We analyzed and optimized (1) the preparation method of the insect cell extract, (2) the concentration of the reaction components, and (3) the 5,-untranslated region (5,-UTR) of mRNA. The extract was prepared by freeze-thawing insect cells suspended in the extraction buffer. This preparation method was a simple and superior method compared with the conventional method using a Dounce homogenizer. Furthermore, protein synthesis efficiency was improved by the addition of 20% (v/v) glycerol to the extraction buffer. Concentrations of the reaction components were optimized to increase protein synthesis efficiency. Moreover, mRNAs containing 5,-UTRs derived from baculovirus polyhedrin genes showed high protein synthesis activity. Especially, the leader composition of the Ectropis obliqua nucleopolyhedrovirus polyhedrin gene showed the highest enhancement activity among the six 5,-UTRs tested. As a result, in a batch reaction approximately 71 ,g of luciferase was synthesized per milliliter of reaction volume at 25 °C for 6 h. Moreover, this method for the establishment of a cell-free system was applied also to Spodoptera frugiperda 21 (Sf21) insect cells. After optimizing the concentrations of the reaction components and the 5,-UTR of mRNA, approximately 45 ,g/mL of luciferase was synthesized in an Sf21 cell-free system at 25 °C for 3 h. These productivities were sufficient to perform gene expression analyses. Thus, these cell-free systems may be a useful tool for simple synthesis in post-genomic studies as a novel protein production method. [source] | ||||||||||||||||