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Cell Extracts (cell + extract)
Kinds of Cell Extracts Selected AbstractsPhotoaffinity Isolation and Identification of Proteins in Cancer Cell Extracts that Bind to Platinum-Modified DNACHEMBIOCHEM, Issue 1 2009Evan R. Guggenheim Dr. Abstract The activity of the anticancer drug cisplatin is a consequence of its ability to bind DNA. Platinum adducts bend and unwind the DNA duplex, creating recognition sites for nuclear proteins. Following DNA damage recognition, the lesions will either be repaired, facilitating cell viability, or if repair is unsuccessful and the Pt adduct interrupts vital cellular functions, apoptosis will follow. With the use of the benzophenone-modified cisplatin analogue Pt-BP6, 25 bp DNA duplexes containing either a 1,2-d(G*pG*) intrastrand or a 1,3-d(G*pTpG*) intrastrand crosslink were synthesized, where the asterisks designate platinated nucleobases. Proteins having affinity for these platinated DNAs were photocrosslinked and identified in cervical, testicular, pancreatic and bone cancer-cell nuclear extracts. Proteins identified in this manner include the DNA repair factors RPA1, Ku70, Ku80, Msh2, DNA ligase III, PARP-1, and DNA,PKcs, as well as HMG-domain proteins HMGB1, HMGB2, HMGB3, and UBF1. The latter strongly associate with the 1,2-d(G*pG*) adduct and weakly or not at all with the 1,3-d(G*pTpG*) adduct. The nucleotide excision repair protein RPA1 was photocrosslinked only by the probe containing a 1,3-d(G*pTpG*) intrastrand crosslink. The affinity of PARP-1 for platinum-modified DNA was established using this type of probe for the first time. To ensure that the proteins were not photocrosslinked because of an affinity for DNA ends, a 90-base dumbbell probe modified with Pt-BP6 was investigated. Photocrosslinking experiments with this longer probe revealed the same proteins, as well as some additional proteins involved in chromatin remodeling, transcription, or repair. These findings reveal a more complete list of proteins involved in the early steps of the mechanism of action of the cisplatin and its close analogue carboplatin than previously was available. [source] Identification of human phosphoglucomutase 3 (PGM3) as N -acetylglucosamine-phosphate mutase (AGM1)ANNALS OF HUMAN GENETICS, Issue 2 2002H. PANG We performed phenotyping of human phosphoglucomutase 3 (PGM3) and screening for mutations in the human N -acetylglucosamine-phosphate mutase gene (AGM1) to identify PGM3 as AGM1. By sequencing the coding region of AGM1, two alleles containing a G or A base at nucleotide 1396, that can respectively encode aspartic acid or asparagine at codon 466, were identified. Cell extracts of COS7 cells after transfection with the pcDNA 3·1(+) plasmid containing an AGM1 allele with 1396G or 1396A showed similar electrophoretic patterns to the PGM3 1 or PGM3 2 protein, respectively, with the isozyme detection method used for PGM3 phenotyping. The genotypes determined by the two alleles of AGM1 coincided exactly with the PGM3 phenotypes in 20 individuals. We also investigated the allele frequency of the AGM1 nucleotide polymorphism in a Japanese population by DNA sequencing and found that the frequencies of alleles 1396G and 1396A were similar to previously reported PGM3*1 and PGM3*2 frequencies. Overall, the facts that the AGM1 gene product shows PGM activity, AGM1 is polymorphic, the electrophoretic mobility is similar between AGM1 allele-specific products and PGM3 1 and 2 proteins, PGM3 phenotypes and AGM1 genotypes completely coincide in 20 individuals, and AGM1 allele frequencies are similar to those of PGM3*1 and PGM3*2 in Japanese populations, suggest that PGM3 is identical to AGM1. [source] Role of myosin II activity and the regulation of myosin light chain phosphorylation in astrocytomasCYTOSKELETON, Issue 1 2008Bodour Salhia Abstract The generation of contractile force mediated by actin-myosin interactions is essential for cell motility. Myosin activity is promoted by phosphorylation of myosin light chain (MLC). MLC phosphorylation in large part is controlled by kinases that are effectors of Rho family GTPases. Accordingly, in this study we examined the effects of ROCK and Rac1 inhibition on MLC phosphorylation in astrocytoma cells. We found that low concentrations of the ROCK inhibitor Y27632 increased the phosphorylation state of the Triton X-100 soluble fraction of MLC, whereas higher concentrations of Y27632 decreased soluble phospho-MLC. These effects of Y27632 were dependent on Rac1. The soluble form of phospho-MLC comprises about 10% of total phospho-MLC in control cells. Interestingly, ROCK inhibition led to a decrease in the phosphorylation state of total MLC, whereas Rac1 inhibition had little effect. Thus, the soluble form of MLC is differentially regulated by ROCK and Rac1 compared with MLC examined in a total cell extract. We also observed that astrocytoma migration is stimulated by low concentrations of the myosin II inhibitor blebbistatin. However, higher concentrations of blebbistatin inhibit migration leading us to believe that migration has a biphasic dependence on myosin II activity. Taken together, our data show that modulation of myosin II activity is important in determining optimal astrocytoma migration. In addition, these findings suggest that there are at least two populations of MLC that are differentially regulated. Cell Motil. Cytoskeleton 2008. © 2007 Wiley-Liss, Inc. [source] Towards clarification of the biological role of microcystins, a family of cyanobacterial toxinsENVIRONMENTAL MICROBIOLOGY, Issue 4 2007Daniella Schatz Summary Microcystins constitute a serious threat to the quality of drinking water worldwide. These protein phosphatase inhibitors are formed by various cyanobacterial species, including Microcystis sp. Microcystins are produced by a complex microcystin synthetase, composed of peptide synthetases and polyketide synthases, encoded by the mcyA-J gene cluster. Recent phylogenetic analysis suggested that the microcystin synthetase predated the metazoan lineage, thus dismissing the possibility that microcystins emerged as a means of defence against grazing, and their original biological role is not clear. We show that lysis of Microcystis cells, either mechanically or because of various stress conditions, induced massive accumulation of McyB and enhanced the production of microcystins in the remaining Microcystis cells. A rise in McyB content was also observed following exposure to microcystin or the protease inhibitors micropeptin and microginin, also produced by Microcystis. The extent of the stimulation by cell extract was strongly affected by the age of the treated Microcystis culture. Older cultures, or those recently diluted from stock cultures, hardly responded to the components in the cell extract. We propose that lysis of a fraction of the Microcystis population is sensed by the rest of the cells because of the release of non-ribosomal peptides. The remaining cells respond by raising their ability to produce microcystins thereby enhancing their fitness in their ecological niche, because of their toxicity. [source] Functional characterization of an orphan cupin protein from Burkholderia xenovorans reveals a mononuclear nonheme Fe2+ -dependent oxygenase that cleaves ,-diketonesFEBS JOURNAL, Issue 20 2009Stefan Leitgeb Cupins constitute a large and widespread superfamily of ,-barrel proteins in which a mononuclear metal site is both a conserved feature of the structure and a source of functional diversity. Metal-binding residues are contributed from two core motifs that provide the signature for the superfamily. On the basis of conservation of this two-motif structure, we have identified an ORF in the genome of Burkholderia xenovorans that encodes a novel cupin protein (Bxe_A2876) of unknown function. Recombinant Bxe_A2876, as isolated from Escherichia coli cell extract, was a homotetramer in solution, and showed mixed fractional occupancy of its 16.1 kDa subunit with metal ligands (0.06 copper; 0.11 iron; 0.17 zinc). Our quest for possible catalytic functions of Bxe_A2876 focused on Cu2+ and Fe2+ oxygenase activities known from related cupin enzymes. Fe2+ elicited enzymatic catalysis of O2 -dependent conversion of various ,-diketone substrates via a nucleophilic mechanism of carbon,carbon bond cleavage. Data from X-ray absorption spectroscopy (XAS) support a five-coordinate or six-coordinate Fe2+ center where the metal is bound by three imidazole nitrogen atoms at 1.98 Å. Results of structure modeling studies suggest that His60, His62 and His102 are the coordinating residues. In the ,best-fit' model, one or two oxygens from water and a carboxylate oxygen (presumably from Glu96) are further ligands of Fe2+ at estimated distances of 2.04 Å and 2.08 Å, respectively. The three-histidine Fe2+ site of Bxe_A2876 is compared to the mononuclear nonheme Fe2+ centers of the structurally related cysteine dioxygenase and acireductone dioxygenase, which also use a facial triad of histidines for binding of their metal cofactor but promote entirely different substrate transformations. [source] Cell-free synthesis of 15N-labeled proteins for NMR studiesIUBMB LIFE, Issue 9 2005Kiyoshi Ozawa Abstract Modern cell-free in vitro protein synthesis systems present powerful tools for the synthesis of isotope-labeled proteins in high yields. The production of selectively 15 N-labeled proteins from 15 N-labeled amino acids is particularly economic and yields are often sufficient to analyze the proteins very quickly by two-dimensional NMR spectra recorded of the crude reaction mixture without concentration or chromatographic purification of the protein. We review methodological aspects of cell-free in vitro protein synthesis based on an Escherichia coli cell extract, in particular with regard to the production of 15 N-labeled proteins for analysis by NMR spectroscopy. IUBMB Life, 57: 615-622, 2005 [source] Cytosolic NADP phosphatases I and II from Arthrobacter sp. strain KM: Implication in regulation of NAD+/NADP+ balanceJOURNAL OF BASIC MICROBIOLOGY, Issue 3 2004Shigeyuki Kawai NADP phosphatase (NADPase) is an enzyme that converts NADP+ into NAD+ through dephosphorylation of NADP+, and is considered to be one of the possible candidates for regulation of the NAD+/NADP+ balance in vivo. In order to obtain an intrinsic NADPase, the NADP+ -degrading activity in a membrane-free cell extract of a Gram-positive bacterium, Arthrobacter sp. strain KM, was first assessed and demonstrated to be mainly achieved through the NADPase reaction, indicating NADPase is essential for degradation of NADP+ and therefore for regulation of the NAD+/NADP+ balance in cytosol. Then, the isolation of cytosolic NADPase was attempted using NADP+ as a substrate. Two NADPase isozymes, designated as NADPases I and II, were purified from the cell extract of the bacterium, and were indicated to be the sole cytosolic NADPases regulating the balance of NAD+/NADP+. NADPases I and II are homodimers of 32 and 30 kDa subunits, respectively, and most active at pH 7,8. The N-terminal amino acid sequences of the two enzymes are similar to each other. Among the biological substrates tested, both enzymes showed the highest activity toward NADP+ and NADPH. AMP, ADP, and pyridoxal 5,-phosphate were also dephosphorylated, but to lower extents. Comparison of the features of NADPases I and II with those of other acid phosphatases possessing NADPase activity suggested that NADPases I and II are novel enzymes participating in regulation of the NAD+/NADP+ balance in the cytosol. (© 2004 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim) [source] Antigenicity of Streptococcus agalactiae extracellular products and vaccine efficacyJOURNAL OF FISH DISEASES, Issue 4 2005D J Pasnik Abstract Streptococcus agalactiae is a major bacterial pathogen that is the cause of serious economic losses in many species of freshwater, marine and estuarine fish worldwide. A highly efficacious S. agalactiae vaccine was developed using extracellular products (ECP) and formalin-killed whole cells of S. agalactiae. The vaccine efficacy following storage of S. agalactiae ECP and formalin-killed S. agalactiae cells at 4 °C for 1 year was determined. The stored ECP containing S. agalactiae formalin-killed cells failed to prevent morbidity and mortality among the vaccinated fish, and the relative percentage survival was 29. Serum antibody responses of the stored ECP and freshly prepared ECP against soluble whole cell extract of S. agalactiae indicated that significantly less antibody was produced in fish immunized with stored ECP and S. agalactiae cells than in those fish immunized with freshly prepared ECP and S. agalactiae cells at day 31 post-vaccination. Silver staining of sodium dodecyl sulphate-polyacrylamide gels and immunostaining of Western blots with tilapia antiserum to S. agalactiae revealed that predominant 54 and 55 kDa bands were present in the freshly prepared ECP fraction. The 55 kDa band was absent from the stored ECP and new bands below 54 kDa appeared on the Western blot. The results of this study on S. agalactiae ECP provide evidence for a correlation between protection and antibody production to ECP and for the importance of the 55 kDa ECP antigen for vaccine efficacy. [source] Protein purification using chromatography: selection of type, modelling and optimization of operating conditionsJOURNAL OF MOLECULAR RECOGNITION, Issue 2 2009J. A. Asenjo Abstract To achieve a high level of purity in the purification of recombinant proteins for therapeutic or analytical application, it is necessary to use several chromatographic steps. There is a range of techniques available including anion and cation exchange, which can be carried out at different pHs, hydrophobic interaction chromatography, gel filtration and affinity chromatography. In the case of a complex mixture of partially unknown proteins or a clarified cell extract, there are many different routes one can take in order to choose the minimum and most efficient number of purification steps to achieve a desired level of purity (e.g. 98%, 99.5% or 99.9%). This review shows how an initial 'proteomic' characterization of the complex mixture of target protein and protein contaminants can be used to select the most efficient chromatographic separation steps in order to achieve a specific level of purity with a minimum number of steps. The chosen methodology was implemented in a computer- based Expert System. Two algorithms were developed, the first algorithm was used to select the most efficient purification method to separate a protein from its contaminants based on the physicochemical properties of the protein product and the protein contaminants and the second algorithm was used to predict the number and concentration of contaminants after each separation as well as protein product purity. The application of the Expert System approach was experimentally tested and validated with a mixture of four proteins and the experimental validation was also carried out with a supernatant of Bacillus subtilis producing a recombinant , -1,3-glucanase. Once the type of chromatography is chosen, optimization of the operating conditions is essential. Chromatographic elution curves for a three-protein mixture (, -lactoalbumin, ovalbumin and , -lactoglobulin), carried out under different flow rates and ionic strength conditions, were simulated using two different mathematical models. These models were the Plate Model and the more fundamentally based Rate Model. Simulated elution curves were compared with experimental data not used for parameter identification. Deviation between experimental data and the simulated curves using the Plate Model was less than 0.0189 (absorbance units); a slightly higher deviation [0.0252 (absorbance units)] was obtained when the Rate Model was used. In order to optimize operating conditions, a cost function was built that included the effect of the different production stages, namely fermentation, purification and concentration. This cost function was also successfully used for the determination of the fraction of product to be collected (peak cutting) in chromatography. It can be used for protein products with different characteristics and qualities, such as purity and yield, by choosing the appropriate parameters. Copyright © 2008 John Wiley & Sons, Ltd. [source] Purification and partial characterization of a dipeptidyl peptidase from Prevotella intermediaMOLECULAR ORAL MICROBIOLOGY, Issue 3 2003Y. Shibata A peptidase hydrolyzed X-Pro- p -nitroanilide was purified from the cell extract of Prevotella intermedia ATCC 25611 by ion-exchange chromatography and hydrophobic interaction chromatography. The purified enzyme exhibited a molecular size of 74 kDa from sodium dodecyl sulfate-polyacrylamide gel electrophoresis and the maximum enzyme activity was found between pH 7.0 and pH 7.5. This peptidase was a serine enzyme and hydrolyzed Lys-Pro- p -nitroanilide, Arg-Pro- p -nitroanilide, and Ala-Pro- p -nitroanilide, but Lys-Ala- p -nitroanilide was not split. The enzyme may be classified as a dipeptidyl peptidase IV. [source] Identification of potential substrate proteins for the periplasmic Escherichia coli chaperone SkpPROTEINS: STRUCTURE, FUNCTION AND BIOINFORMATICS, Issue 23-24 2008Svenja Jarchow Abstract The "seventeen kilodalton protein" (Skp) is a predominant periplasmic chaperone of Escherichia coli, which is involved in the biogenesis of abundant outer membrane proteins (OMPs) such as OmpA, PhoE, and LamB. In this study the substrate profile of Skp was investigated in a proteomics approach. Skp was overexpressed in a deficient E. coli strain as a fusion protein with the Strep,tag and captured, together with any host proteins associated with it, from the periplasmic cell extract under mild conditions via one-step Strep,Tactin affinity chromatography. Copurified substrate proteins were then identified by high resolution 2-DE with immobilized pH-gradients, followed by MALDI-TOF MS. Apart from the known Skp substrates, including OmpA and LamB, more than 30 other interacting proteins were detected, especially from the outer membrane, among these FadL and BtuB, and from the periplasm such as MalE and OppA. Thus, Skp does not only serve as a specialized chaperone for a small set of OMPs, but it seems to exhibit a broader substrate spectrum, including soluble periplasmic proteins. These findings should prompt further investigation into the physiological role of Skp and may promote its use for the bacterial production of biochemically active heterologous proteins whose folding requires secretion into the oxidizing milieu of the periplasm. [source] Cell-free synthesis of functional proteins using transcription/translation machinery entrapped in silica sol,gel matrixBIOTECHNOLOGY & BIOENGINEERING, Issue 1 2009Kyeong-Ohn Kim Abstract Herewith we report the encapsulation of functional protein synthesis machinery in a silica sol,gel matrix. When the sol,gel reaction using alkoxysilane monomers was carried out in the presence of Escherichia coli cell extract, macromolecular protein synthesis machinery in the cell extract was successfully immobilized within a silica gel matrix, catalyzing the translation of co-immobilized DNA when supplied with small-molecular-weight substrates for protein synthesis. The efficiency of protein synthesis was affected by the pore size of the gel structure, which was controlled through the use of appropriate additives during the sol,gel reactions. To the best of our knowledge, this is the first report describing the reproduction of the entire set of complicated biological process within an inorganic gel matrix, and we expect that the developed technology will find many applications in numerous fields such as high-throughput gene expression and the development of multifunctional biosensors. Biotechnol. Bioeng. 2009;102: 303,307. © 2008 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] Optimization of Enzymatic Gas-Phase Reactions by Increasing the Long-Term Stability of the CatalystBIOTECHNOLOGY PROGRESS, Issue 3 2004Clara Ferloni Enzymatic gas-phase reactions are usually performed in continuous reactors, and thus very stable and active catalysts are required to perform such transformations on cost-effective levels. The present work is concerned with the reduction of gaseous acetophenone to enantiomerically pure ( R)-1-phenylethanol catalyzed by solid alcohol dehydrogenase from Lactobacillus brevis (LBADH), immobilized onto glass beads. Initially, the catalyst preparation displayed a half-life of 1 day under reaction conditions at 40 °C and at a water activity of 0.5. It was shown that the observed decrease in activity is due to a degradation of the enzyme itself (LBADH) and not of the co-immobilized cofactor NADP. By the addition of sucrose to the cell extract before immobilization of the enzyme, the half-life of the catalyst preparation (at 40 °C) was increased 40 times. The stabilized catalyst preparation was employed in a continuous gas-phase reactor at different temperatures (25,60 °C). At 50 °C, a space-time yield of 107 g/L/d was achieved within the first 80 h of continuous reaction. [source] Design of Affinity Tags for One-Step Protein Purification from Immobilized Zinc ColumnsBIOTECHNOLOGY PROGRESS, Issue 1 2000Richard S. Pasquinelli Affinity tags are often used to accomplish recombinant protein purification using immobilized metal affinity chromatography. Success of the tag depends on the chelated metal used and the elution profile of the host cell proteins. Zn(II)-iminodiacetic acid (Zn(II)-IDA) may prove to be superior to either immobilized copper or nickel as a result of its relatively low binding affinity for cellular proteins. For example, almost all Escherichiacoli proteins elute from Zn(II)-IDA columns between pH 7.5 and 7.0 with very little cellular protein emerging at pH values lower than 7.0. Thus, a large portion of the Zn(II)-IDA elution profile may be free of contaminant proteins, which can be exploited for one-step purification of a target protein from raw cell extract. In this paper we have identified several fusion tags that can direct the elution of the target protein to the low background region of the Zn(II)-IDA elution profile. These tags allow targeting of proteins to different regions of the elution profile, facilitating purification under mild conditions. [source] A FERM domain in a class XIV myosin interacts with actin and tubulin and localizes to the cytoskeleton, phagosomes, and nucleus in Tetrahymena thermophila,CYTOSKELETON, Issue 2 2010Michael Gotesman Abstract Previous studies have shown that Myo1(myosin class XIV) localizes to the cytoskeleton and is involved in amitosis of the macronucleus and trafficking of phagosomes. Myo1 contains a FERM domain that could be a site for interaction between Myo1 and the cytoskeleton. Here, we explore the function of FERM by investigating its cytoskeleton binding partners and involvement in localization of Myo1. Alignment of Myo1 FERM with a talin actin-binding sequence, a MAP-2 tubulin-binding sequence, the radixin FERM dimerization motif, and the SV40 nuclear localization sequence (NLS) revealed putative actin- and tubulin-binding sequences, a putative FERM dimerization motif, and NLS-like sequences in both the N-terminal and C-terminal regions of Myo1 FERM. Alignment of Myo1 with an ERM C-terminal motif revealed a similar sequence in the Myo1 motor domain. GFP-FERM and two truncated FERM domains were separately expressed in Tetrahymena. GFP-FERM contained the entire Myo1 FERM. Truncated Myo1 FERM domains contained either the N-terminal or the C-terminal region of FERM and one putative sequence for actin-binding, one for tubulin-binding, a putative dimerization motif, and a NLS-like sequence. Actin antibody coprecipitated GFP-fusion polypeptides and tubulin from lysate of cells expressing GFP-fusions. Cosedimentation assays performed with either whole cell extracts or anti-actin immunoprecipitation pellets revealed that F-actin (independent of ATP) and microtubules cosedimented with GFP-fusion polypeptides. GFP-FERM localized to the cytoskeleton, phagosomes, and nucleus. Truncated GFP-FERM domains localized to phagosomes but not to the cytoskeleton or nucleus. © 2009 Wiley-Liss, Inc. [source] Quantitative evaluation of sample application methods for semipreparative separations of basic proteins by two-dimensional gel electrophoresisELECTROPHORESIS, Issue 19-20 2003Richard C. Barry Abstract The use of cup-loading for sample application has become widely used in two-dimensional electrophoresis (2-DE) for resolution of basic proteins, but no side-by-side quantitative study has been published which compares cup-loading with the alternative passive and active rehydration methods to fully promote one type of loading method over another. Replicate 2-D gels from each loading method were quantitatively evaluated for gel-to-gel reproducibility using IPG 6,11 strips and semipreparative protein loads (300 ,g). Gels were stained with SYPRO Ruby and analyzed with PDQuest. An inexpensive home-made assembly for cup-loading was used with the Protean IEF Cell for separation of whole cell extracts from the archaeon, Sulfolobus solfataricus. Cup-loading was determined to be far superior for IPG 6,11 separations than active or passive rehydration methods. Cup-loading consistently produced the greatest number of detectable spots, the best spot matching efficiency (56%), lowest spot quantity variations (28% coefficient of variation, CV), and the best-looking gels qualitatively. The least satisfactory results were obtained with active rehydration, followed closely by passive rehydration in off-line tubes. Passive rehydration experiments, performed using an on-line isoelectric focusing (IEF) tray, produced comparable spot numbers to cup-loading (84%), with 55% of the spots having higher intensity but 10% more spot quantity variance than cup-loading. [source] High-throughput screening of kinase inhibitors by multiplex capillary electrophoresis with UV absorption detectionELECTROPHORESIS, Issue 1-2 2003Yan He Abstract Protein kinases play a major role in the transformation of cells and are often used as molecular targets for the new generation of anticancer drugs. We present a novel technique for high-throughput screening of inhibitors of protein kinases. The technique involves the use of multiplexed capillary electrophoresis (CE) for the rapid separation of the peptides, phosphopeptides, and various inhibitors. By means of UV detection, diversified peptides with native amino acid sequences and their phosphorylated counterparts can be directly analyzed without the need for radioactive or fluorescence labeling. The effects of different inhibitors and their IC50 value were determined using three different situations involving the use of a single purified kinase, two purified kinases, and crude cell extracts, respectively. The results suggest that multiplexed CE/UV may prove to be a straightforward and general approach for high-throughput screening of compound libraries to find potent and selective inhibitors of the various protein kinases. [source] Mutagenicity of nitroaromatic degradation compoundsENVIRONMENTAL TOXICOLOGY & CHEMISTRY, Issue 10 2003Ranjit S. Padda Abstract The mutagenicity of 2,4-dinitrotoluene (24DNT), and 2,6-dinitrotoluene (26DNT), and their related transformation products such as hydroxylamine and amine derivatives, which are formed by Clostridium acetobutylicum, were tested in crude cell extracts using Salmonella typhimurium TA100. A previous publication already reported the mutagenic activities of 2,4,6-trinitrotoluene (TNT) and its related hydroxylamine derivatives in this test system. A time course of the mutagenicity during the anaerobic transformation of TNT, 24DNT, and 26DNT was also investigated under the same conditions to compare with the results from the pure compounds. The monohydroxylamino intermediates 2-hydroxylamino-4-nitrotoluene (2HA4NT), 4-hydroxylamino-2-nitrotoluene (4HA2NT) and 2-hydroxylamino-6-nitrotoluene (2HA6NT) formed during anaerobic transformation of dinitrotoluenes were proven to be mutagenic in the Ames test using Salmonella typhimurium TA100. This study reports that 4HA2NT is the most stable derivative, whereas 2HA4NT and 2HA6NT are less stable and these intermediates are mutagenic in the Ames test. Both 24DNT and 26DNT and their final metabolites 2,4-diaminotoluene (24DAT) and 2,6-aminotoluene (26DAT) appeared nonmutagenic. In a time-course study of TNT degradation, the temporal sample containing 85% of 2,4-dihydroxylamino-6-nitrotoluene (24HA6NT) is most mutagenic. These observations suggest that the bioremediation approach for treatment of 24DNT and 26DNT should be carried past the hydroxylamino intermediate. [source] Molecular genetics of Xeroderma pigmentosum variantEXPERIMENTAL DERMATOLOGY, Issue 5 2003Alexei Gratchev Skin abnormalities result from an inability to repair UV-damaged DNA because of defects in the nucleotide excision repair (NER) machinery. Xeroderma pigmentosum is genetically heterogeneous and is classified into seven complementation groups (XPA-XPG) that correspond to genetic alterations in one of seven genes involved in NER. The variant type of XP (XPV), first described in 1970 by Ernst G. Jung as ,pigmented xerodermoid', is caused by defects in the post replication repair machinery while NER is not impaired. Identification of the XPV gene was only achieved in 1999 by biochemical purification and sequencing of a protein from HeLa cell extracts complementing the PRR defect in XPV cells. The XPV protein, polymerase (pol),, represents a novel member of the Y family of bypass DNA polymerases that facilitate DNA translesion synthesis. The major function of pol, is to allow DNA translesion synthesis of UV-induced TT-dimers in an error-free manner; it also possesses the capability to bypass other DNA lesions in an error-prone manner. Xeroderma pigmentosum V is caused by molecular alterations in the POLH gene, located on chromosome 6p21.1,6p12. Affected individuals are homozygous or compound heterozygous for a spectrum of genetic lesions, including nonsense mutations, deletions or insertions, confirming the autosomal recessive nature of the condition. Identification of POLH as the XPV gene provides an important instrument for improving molecular diagnostics in XPV families. [source] Functional association of human Ki-1/57 with pre-mRNA splicing eventsFEBS JOURNAL, Issue 14 2009Gustavo C. Bressan The cytoplasmic and nuclear protein Ki-1/57 was first identified in malignant cells from Hodgkin's lymphoma. Despite studies showing its phosphorylation, arginine methylation, and interaction with several regulatory proteins, the functional role of Ki-1/57 in human cells remains to be determined. Here, we investigated the relationship of Ki-1/57 with RNA functions. Through immunoprecipitation assays, we verified the association of Ki-1/57 with the endogenous splicing proteins hnRNPQ and SFRS9 in HeLa cell extracts. We also found that recombinant Ki-1/57 was able to bind to a poly-U RNA probe in electrophoretic mobility shift assays. In a classic splicing test, we showed that Ki-1/57 can modify the splicing site selection of the adenoviral E1A minigene in a dose-dependent manner. Further confocal and fluorescence microscopy analysis revealed the localization of enhanced green fluorescent protein,Ki-1/57 to nuclear bodies involved in RNA processing and or small nuclear ribonucleoprotein assembly, depending on the cellular methylation status and its N-terminal region. In summary, our findings suggest that Ki-1/57 is probably involved in cellular events related to RNA functions, such as pre-mRNA splicing. Structured digital abstract ,,MINT-7041074: Ki-1/57 (uniprotkb:Q5JVS0) physically interacts (MI:0915) with SF2P32 (uniprotkb:Q07021) by two hybrid (MI:0018) ,,MINT-7041232: Ki-1/57 (uniprotkb:Q5JVS0) physically interacts (MI:0915) with SFRS9 (uniprotkb:Q13242) by pull down (MI:0096) ,,MINT-7041203: P80-Coilin (uniprotkb:P38432) and Ki-1/57 (uniprotkb:Q5JVS0) colocalize (MI:0403) by fluorescence microscopy (MI:0416) ,,MINT-7041217: SMN (uniprotkb:Q16637) and Ki-1/57 (uniprotkb:Q5JVS0) colocalize (MI:0403) by fluorescence microscopy (MI:0416) ,,MINT-7041189: SC-35 (uniprotkb:Q01130) and Ki-1/57 (uniprotkb:Q5JVS0) colocalize (MI:0403) by fluorescence microscopy (MI:0416) ,,MINT-7041169: NPM (uniprotkb:P06748) and Ki-1/57 (uniprotkb:Q5JVS0) colocalize (MI:0403) by fluorescence microscopy (MI:0416) ,,MINT-7041249: Ki-1/57 (uniprotkb:Q5JVS0) physically interacts (MI:0915) with SFRS9 (uniprotkb:O60506) by pull down (MI:0096) ,,MINT-7041065: Ki-1/57 (uniprotkb:Q5JVS0) physically interacts (MI:0915) with SFRS9 (uniprotkb:Q13242) by two hybrid (MI:0018) ,,MINT-7041069: Ki-1/57 (uniprotkb:Q5JVS0) physically interacts (MI:0915) with YB1 (uniprotkb:P67809) by two hybrid (MI:0018) ,,MINT-7041079: Ki-1/57 (uniprotkb:Q5JVS0) physically interacts (MI:0915) with HNRPQ (uniprotkb:O60506) by two hybrid (MI:0018) ,,MINT-7041087: Ki-1/57 (uniprotkb:Q5JVS0) physically interacts (MI:0218) with HNRPQ3 (uniprotkb:O60506-1), HNRPQ2 (uniprotkb:O60506-2) and HNRPQ-1 (uniprotkb:O60506-3) by anti bait coimmunoprecipitation (MI:0006) [source] Acetylcholinesterase from the invertebrate Ciona intestinalis is capable of assembling into asymmetric forms when co-expressed with vertebrate collagenic tail peptideFEBS JOURNAL, Issue 6 2008Adam Frederick To learn more about the evolution of the cholinesterases (ChEs), acetylcholinesterase (AChE) and butyrylcholinesterase in the vertebrates, we investigated the AChE activity of a deuterostome invertebrate, the urochordate Ciona intestinalis, by expressing in vitro a synthetic recombinant cDNA for the enzyme in COS-7 cells. Evidence from kinetics, pharmacology, molecular biology, and molecular modeling confirms that the enzyme is AChE. Sequence analysis and molecular modeling also indicate that the cDNA codes for the AChET subunit, which should be able to produce all three globular forms of AChE: monomers (G1), dimers (G2), and tetramers (G4), and assemble into asymmetric forms in association with the collagenic subunit collagen Q. Using velocity sedimentation on sucrose gradients, we found that all three of the globular forms are either expressed in cells or secreted into the medium. In cell extracts, amphiphilic monomers (G1a) and non-amphiphilic tetramers (G4na) are found. Amphiphilic dimers (G2a) and non-amphiphilic tetramers (G4na) are secreted into the medium. Co-expression of the catalytic subunit with Rattus norvegicus collagen Q produces the asymmetric A12 form of the enzyme. Collagenase digestion of the A12 AChE produces a lytic G4 form. Notably, only globular forms are present in vivo. This is the first demonstration that an invertebrate AChE is capable of assembling into asymmetric forms. We also performed a phylogenetic analysis of the sequence. We discuss the relevance of our results with respect to the evolution of the ChEs in general, in deuterostome invertebrates, and in chordates including vertebrates. [source] Surface nucleolin participates in both the binding and endocytosis of lactoferrin in target cellsFEBS JOURNAL, Issue 2 2004Dominique Legrand Lactoferrin (Lf), a multifunctional molecule present in mammalian secretions and blood, plays important roles in host defense and cancer. Indeed, Lf has been reported to inhibit the proliferation of cancerous mammary gland epithelial cells and manifest a potent antiviral activity against human immunodeficiency virus and human cytomegalovirus. The Lf-binding sites on the cell surface appear to be proteoglycans and other as yet undefined protein(s). Here, we isolated a Lf-binding 105 kDa molecular mass protein from cell extracts and identified it as human nucleolin. Medium,affinity interactions (, 240 nm) between Lf and purified nucleolin were further illustrated by surface plasmon resonance assays. The interaction of Lf with the cell surface-expressed nucleolin was then demonstrated through competitive binding studies between Lf and the anti-human immunodeficiency virus pseudopeptide, HB-19, which binds specifically surface-expressed nucleolin independently of proteoglycans. Interestingly, binding competition studies between HB-19 and various Lf derivatives in proteoglycan-deficient hamster cells suggested that the nucleolin-binding site is located in both the N- and C-terminal lobes of Lf, whereas the basic N-terminal region is dispensable. On intact cells, Lf co-localizes with surface nucleolin and together they become internalized through vesicles of the recycling/degradation pathway by an active process. Morever, a small proportion of Lf appears to translocate in the nucleus of cells. Finally, the observations that endocytosis of Lf is inhibited by the HB-19 pseudopeptide, and the lack of Lf endocytosis in proteoglycan-deficient cells despite Lf binding, point out that both nucleolin and proteoglycans are implicated in the mechanism of Lf endocytosis. [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] Purification, characterization and subunits identification of the diol dehydratase of Lactobacillus collinoidesFEBS JOURNAL, Issue 22 2002Nicolas Sauvageot The three genes pduCDE encoding the diol dehydratase of Lactobacillus collinoides, have been cloned for overexpression in the pQE30 vector. Although the three subunits of the protein were highly induced, no activity was detected in cell extracts. The enzyme was therefore purified to near homogeneity by ammonium sulfate precipitation and gel filtration chromatography. In fractions showing diol dehydratase activity, three main bands were present after SDS/PAGE with molecular masses of 63, 28 and 22 kDa, respectively. They were identified by mass spectrometry to correspond to the large, medium and small subunits of the dehydratase encoded by the pduC, pduD and pduE genes, respectively. The molecular mass of the native complex was estimated to 207 kDa in accordance with the calculated molecular masses deduced from the pduC, D, E genes (61, 24.7 and 19,1 kDa, respectively) and a ,2,2,2 composition. The Km for the three main substrates were 1.6 mm for 1,2-propanediol, 5.5 mm for 1,2-ethanediol and 8.3 mm for glycerol. The enzyme required the adenosylcobalamin coenzyme for catalytic activity and the Km for the cofactor was 8 µm. Inactivation of the enzyme was observed by both glycerol and cyanocobalamin. The optimal reaction conditions of the enzyme were pH 8.75 and 37 °C. Activity was inhibited by sodium and calcium ions and to a lesser extent by magnesium. A fourth band at 59 kDa copurified with the diol dehydratase and was identified as the propionaldehyde dehydrogenase enzyme, another protein involved in the 1,2-propanediol metabolism pathway. [source] Proteasome-driven turnover of tryptophan hydroxylase is triggered by phosphorylation in RBL2H3 cells, a serotonin producing mast cell lineFEBS JOURNAL, Issue 19 2002Yoshiko Iida We previously demonstrated in mast cell lines RBL2H3 and FMA3 that tryptophan hydroxylase (TPH) undergoes very fast turnover driven by 26S-proteasomes [Kojima, M., Oguro, K., Sawabe, K., Iida, Y., Ikeda, R., Yamashita, A., Nakanishi, N. & Hasegawa, H. (2000) J. Biochem (Tokyo) 2000, 127, 121,127]. In the present study, we have examined an involvement of TPH phosphorylation in the rapid turnover, using non-neural TPH. The proteasome-driven degradation of TPH in living cells was accelerated by okadaic acid, a protein phosphatase inhibitor. Incorporation of 32P into a 53-kDa protein, which was judged to be TPH based on autoradiography and Western blot analysis using anti-TPH serum and purified TPH as the size marker, was observed in FMA3 cells only in the presence of both okadaic acid and MG132, inhibitors of protein phosphatase and proteasome, respectively. In a cell-free proteasome system constituted mainly of RBL2H3 cell extracts, degradation of exogenous TPH isolated from mastocytoma P-815 cells was inhibited by protein kinase inhibitors KN-62 and K252a but not by H89. Consistent with the inhibitor specificity, the same TPH was phosphorylated by exogenous Ca2+/calmodulin-dependent protein kinase II in the presence of Ca2+ and calmodulin but not by protein kinase A (catalytic subunit). TPH protein thus phosphorylated by Ca2+/calmodulin-dependent protein kinase II was digested more rapidly in the cell-free proteasome system than was the nonphosphorylated enzyme. These results indicated that the phosphorylation of TPH was a prerequisite for proteasome-driven TPH degradation. [source] Identification and characterization of a new gene from Variovorax paradoxus Iso1 encoding N -acyl- d -amino acid amidohydrolase responsible for d -amino acid productionFEBS JOURNAL, Issue 19 2002Pei-Hsun Lin An N -acyl- d -amino acid amidohydrolase (N -D-AAase) was identified in cell extracts of a strain, Iso1, isolated from an environment containing N -acetyl- d -methionine. The bacterium was classified as Variovorax paradoxus by phylogenetic analysis. The gene was cloned and sequenced. The gene consisted of a 1467-bp ORF encoding a polypeptide of 488 amino acids. The V. paradoxusN -D-AAase showed significant amino acid similarity to the N -acyl- d -amino acid amidohydrolases of the two eubacteria Alcaligenes xylosoxydans A-6 (44,56% identity), Alcaligenes facelis DA1 (54% identity) and the hyperthermophilic archaeon Pyrococcus abyssi (42% identity). After over-expression of the N -D-AAase protein in Escherichia coli, the enzyme was purified by multistep chromatography. The native molecular mass was 52.8 kDa, which agreed with the predicted molecular mass of 52 798 Da and the enzyme appeared to be a monomer protein by gel-filtration chromatography. A homogenous protein with a specific activity of 516 U·mg,1 was finally obtained. After peptide sequencing by LC/MS/MS, the results were in agreement with the deduced amino acid sequence of the N -D-AAase. The pI of the enzyme was 5.12 and it had an optimal pH and temperature of 7.5 and 50 °C, respectively. After 30 min heat treatment at 45 °C, between pH 6 and pH 8, 80% activity remained. The N -D-AAase had higher hydrolysing activity against N -acetyl- d -amino acid derivates containing d -methionine, d -leucine and d -alanine and against N -chloroacetyl- d -phenylalanine. Importantly, the enzyme does not act on the N -acetyl- l -amino acid derivatives. The enzyme was inhibited by chelating agents and certain metal ions, but was activated by 1 mm of Co2+ and Mg2+. Thus, the N -D-AAase from V. paradoxus can be considered a chiral specific and metal-dependent enzyme. [source] Energy metabolism and lipid peroxidation of human erythrocytes as a function of increased oxidative stressFEBS JOURNAL, Issue 3 2000Barbara Tavazzi To study the influence of oxidative stress on energy metabolism and lipid peroxidation in erythrocytes, cells were incubated with increasing concentrations (0.5,10 mm) of hydrogen peroxide for 1 h at 37 °C and the main substances of energy metabolism (ATP, AMP, GTP and IMP) and one index of lipid peroxidation (malondialdehyde) were determined by HPLC on cell extracts. Using the same incubation conditions, the activity of AMP-deaminase was also determined. Under nonhaemolysing conditions (at up to 4 mm H2O2), oxidative stress produced, starting from 1 mm H2O2, progressive ATP depletion and a net decrease in the intracellular sum of adenine nucleotides (ATP + ADP + AMP), which were not paralleled by AMP formation. Concomitantly, the IMP level increased by up to 20-fold with respect to the value determined in control erythrocytes, when cells were challenged with the highest nonhaemolysing H2O2 concentration (4 mm). Efflux of inosine, hypoxanthine, xanthine and uric acid towards the extracellular medium was observed. The metabolic imbalance of erythrocytes following oxidative stress was due to a dramatic and unexpected activation of AMP-deaminase (a twofold increase of activity with respect to controls) that was already evident at the lowest dose of H2O2 used; this enzymatic activity increased with increasing H2O2 in the medium, and reached its maximum at 4 mm H2O2 -treated erythrocytes (10-fold higher activity than controls). Generation of malondialdehyde was strictly related to the dose of H2O2, being detectable at the lowest H2O2 concentration and increasing without appreciable haemolysis up to 4 mm H2O2. Besides demonstrating a close relationship between lipid peroxidation and haemolysis, these data suggest that glycolytic enzymes are moderately affected by oxygen radical action and strongly indicate, in the change of AMP-deaminase activity, a highly sensitive enzymatic site responsible for a profound modification of erythrocyte energy metabolism during oxidative stress. [source] Functional characterization of human nucleosome assembly protein 1-like proteins as histone chaperonesGENES TO CELLS, Issue 1 2010Mitsuru Okuwaki Nucleosome Assembly Protein 1 (NAP1) is a highly conserved histone chaperone protein suspected to be involved in the dynamical regulation of the histone H2A-H2B hetero-dimer. However, the exact mechanism by which NAP1-like proteins act is currently unknown. In this work, we characterized the biochemical properties of two human NAP1-like proteins, hNAP1L1 and hNAP1L4, including a previously uncharacterized subtype, with the aim of determining their exact mechanistic role. Both hNAP1L1 and hNAP1L4 were found to be localized mainly to the cytoplasm and a minor population of them was suggested to be in the nucleus. Biochemical analyses demonstrated that both hNAP1L1 and hNAP1L4 mediated nucleosome formation. In addition, hNAP1L1 was shown to possess a significantly greater nucleosome disassembly activity than hNAP1L4, suggesting that hNAP1L1 and hNAP1L4 may play distinct roles in the regulation of histone dynamics. Building upon this initial discovery we also found that histone H2A-H2B and various histone H2A variants-H2B dimers were found to associate with both hNAP1L1 and hNAP1L4 in cell extracts. These results suggest that human NAP1-like proteins play overlapping roles in transport and deposition of histone H2A-H2B or H2A variants-H2B dimers on chromatin and nonoverlapping roles in nucleosome disassembly. [source] Mechanism of H-8 inhibition of Cyclin-dependent kinase 9: study using inhibitor-immobilized matricesGENES TO CELLS, Issue 3 2003Daisuke Shima Background: Positive transcription elongation factor b (P-TEFb), which phosphorylates the carboxyl-terminal domain (CTD) of RNA polymerase II (RNAPII), is comprised of the catalytic subunit cyclin-dependent kinase 9 (CDK9) and the regulatory subunit cyclin T. The kinase activity and transcriptional activation potential of P-TEFb is sensitive to various compounds, including H-8, 5,6-dichloro-1-,-d-ribofuranosylbenzimidazole (DRB), and flavopiridol. Results: We investigated the molecular mechanism of the H-8 inhibition of CDK9 using matrices to which H-9, an amino derivative of H-8, was immobilized. CDK9 bound specifically to H-9, and this interaction was competitively inhibited by ATP and DRB, but not by flavopiridol. Mutational analyses demonstrated that the central region of CDK9, which encompasses the T-loop region, was important for its binding to H-9. Conclusions: H-9-immobilized latex beads are useful for trapping CDK9 and a subset of kinases from crude cell extracts. The flavopiridol-binding region of CDK9 is most likely different from its H-9-binding region. These biochemical data support previously reported observations which were based on crystallographic data. [source] | ||||||||||||||||||||||||||||||||||