Showing papers on "Gel electrophoresis published in 2014"

Single-cell western blotting

Abstract: To measure cell-to-cell variation in protein-mediated functions, we developed an approach to conduct ∼10(3) concurrent single-cell western blots (scWesterns) in ∼4 h. A microscope slide supporting a 30-μm-thick photoactive polyacrylamide gel enables western blotting: settling of single cells into microwells, lysis in situ, gel electrophoresis, photoinitiated blotting to immobilize proteins and antibody probing. We applied this scWestern method to monitor single-cell differentiation of rat neural stem cells and responses to mitogen stimulation. The scWestern quantified target proteins even with off-target antibody binding, multiplexed to 11 protein targets per single cell with detection thresholds of <30,000 molecules, and supported analyses of low starting cell numbers (∼200) when integrated with FACS. The scWestern overcomes limitations of antibody fidelity and sensitivity in other single-cell protein analysis methods and constitutes a versatile tool for the study of complex cell populations at single-cell resolution.

Experiments in Molecular Biology

Abstract: DNA Isolation and Transformation in Escherichia coli.- Isolation of Plasmid DNA.- Restriction Endonuclease Digestion and Agarose Gel Electrophoresis of DNA.- Restriction Site Mapping.- Southern Blotting.- Preparative Electrophoresis.- DNA Ligation and Escherichia coli Transformation.- Detection of Plasmid by Colony Hybridization.- Microisolation and Microanalysis of Plasmid DNA.- Isopycnic Centrifugation of DNA.- Purification of Plant Ribosomal DNA.- The Extraction and Affinity Chromatography of Messenger RNA.- Agarose Gel Electrophoresis of RNA.- In Vitro Protein Synthesis.- SDS Polyacrylamide Gel Electrophoresis of Proteins.- Isoelectric Focusing (IEF) of Proteins in Thin-Layer Polyacrylamide Gels.- Protein (Western) Blotting with Immunodetection.- Thin-Layer Gel Filtration.- The Dansyl-Edman Method for Peptide Sequencing.- The Double Immunodiffusion Technique.- Immunoelectrophoretic Techniques.- The Time Course of ?-Galactosidase Induction in Escherichia coli.- Experiments on Enzyme Induction in the Unicellular Green Alga Chlorella fusca.- Yeast Protoplast Fusion.- The Ames Test.

One-dimensional SDS-polyacrylamide gel electrophoresis (1D SDS-PAGE).

Abstract: This protocol describes a denaturing polyacrylamide gel system utilizing sodium dodecyl sulfate (SDS) to separate protein molecules based on size as first described by Laemmli (1970). SDS-PAGE can be used to monitor protein purifications, check the purity of samples, and to estimate molecular weights for unknown proteins.

The potato tuber mitochondrial proteome

Abstract: Mitochondria are called the powerhouses of the cell. To better understand the role of mitochondria in maintaining and regulating metabolism in storage tissues, highly purified mitochondria were isolated from dormant potato tubers (Solanum tuberosum ‘Folva’) and their proteome investigated. Proteins were resolved by one-dimensional gel electrophoresis, and tryptic peptides were extracted from gel slices and analyzed by liquid chromatography-tandem mass spectrometry using an Orbitrap XL. Using four different search programs, a total of 1,060 nonredundant proteins were identified in a quantitative manner using normalized spectral counts including as many as 5-fold more “extreme” proteins (low mass, high isoelectric point, hydrophobic) than previous mitochondrial proteome studies. We estimate that this compendium of proteins represents a high coverage of the potato tuber mitochondrial proteome (possibly as high as 85%). The dynamic range of protein expression spanned 1,800-fold and included nearly all components of the electron transport chain, tricarboxylic acid cycle, and protein import apparatus. Additionally, we identified 71 pentatricopeptide repeat proteins, 29 membrane carriers/transporters, a number of new proteins involved in coenzyme biosynthesis and iron metabolism, the pyruvate dehydrogenase kinase, and a type 2C protein phosphatase that may catalyze the dephosphorylation of the pyruvate dehydrogenase complex. Systematic analysis of prominent posttranslational modifications revealed that more than 50% of the identified proteins harbor at least one modification. The most prominently observed class of posttranslational modifications was oxidative modifications. This study reveals approximately 500 new or previously unconfirmed plant mitochondrial proteins and outlines a facile strategy for unbiased, near-comprehensive identification of mitochondrial proteins and their modified forms.

Mixed ligand μ-phenoxo-bridged dinuclear copper(II) complexes with diimine co-ligands: efficient chemical nuclease and protease activities and cytotoxicity

Abstract: The water soluble mixed ligand copper(II) complexes of the type [Cu(sal)(diimine)(ClO4)]21–5, where sal is salicylaldehyde and diimine is 2,2′-bipyridine (bpy, 1), 1,10-phenanthroline (phen, 2), 5,6-dimethyl-1,10-phenanthroline (5,6-dmp, 3), 3,4,7,8-tetramethyl-1,10-phenanthroline (3,4,7,8-tmp, 4) or dipyrido[3,2-d:2′,3′-f]quinoxaline (dpq, 5), and [Cu(sal)(phen)(NO3)]2 (2a) have been successfully isolated and characterized by elemental analysis and other spectral techniques. The DNA binding and cleavage properties of 1–5 have been explored by using various physical and biochemical methods. The coordination geometry around copper(II) in the X-ray structures of 1, 2, 2a and 4 is described as an elongated octahedron. The UV-Vis and EPR spectral and ESI-MS studies reveal that in solution the dinuclear complexes dissociate into essentially mononuclear [Cu(sal)(diimine)]+ species with square-based geometry. The absorption spectral titrations and competitive DNA binding studies reveal that the intrinsic DNA binding affinity of the complexes depends upon the diimine co-ligand and is of the order of dpq (5) > 3,4,7,8-tmp (4) > 5,6-dmp (3) > phen (2) > bpy (1). The complexes 2 and 5 are involved in a partial intercalative interaction with DNA base pairs, while 3 and 4 are involved in a hydrophobic interaction with DNA and 1 is involved in an electrostatic interaction with DNA, which is supported by viscosity studies. Interestingly, only 3 and 4 are selective in exhibiting a positive induced CD band (ICD) upon binding to DNA suggesting that they induce a B to A conformational change in DNA. All the complexes exhibit an oxidative DNA cleavage ability, which varies as 5 > 4 > 3 > 2 > 1. While 4 and 5 are unique in displaying prominent double-strand DNA cleavage even in the absence of an activator, 2 and 3 display only single-strand DNA cleavage. Interestingly, all the complexes exhibit oxidative double-strand DNA cleavage in the presence of ascorbic acid, with 4 and 5 showing a DNA cleavage activity more prominent than 1 and 2. The ability of the complexes to bind and cleave the protein BSA varies in the order, 4 > 3 > 5 > 2 > 1. Interestingly, 3 and 4 cleave the protein in the presence of H2O2 as an activator in a non-specific manner suggesting that they can act as chemical proteases. It is remarkable that all the complexes exhibit cytotoxicity against human breast cancer cell lines (MCF-7) with a potency more than the widely used drug cisplatin indicating that they have the potential to act as effective anticancer drugs in a time dependent manner. The morphological assessment data obtained by using Hoechst 33258 staining reveal that 3 and 4 induce apoptosis much more effectively than the other complexes. Also, the alkaline single-cell gel electrophoresis study (comet assay) suggests that the same complexes induce DNA fragmentation more efficiently than others.

The Potato Tuber Mitochondrial Proteome 1(W)(OPEN)

Abstract: Mitochondria are called the powerhouses of the cell. To better understand the role of mitochondria in maintaining and regulating metabolism in storage tissues, highly purified mitochondria were isolated from dormant potato tubers (Solanum tuberosum ‘Folva’) and their proteome investigated. Proteins were resolved by one-dimensional gel electrophoresis, and tryptic peptides were extracted from gel slices and analyzed by liquid chromatography-tandem mass spectrometry using an Orbitrap XL. Using four different search programs, a total of 1,060 nonredundant proteins were identified in a quantitative manner using normalized spectral counts including as many as 5-fold more “extreme” proteins (low mass, high isoelectric point, hydrophobic) than previous mitochondrial proteome studies. We estimate that this compendium of proteins represents a high coverage of the potato tuber mitochondrial proteome (possibly as high as 85%). The dynamic range of protein expression spanned 1,800-fold and included nearly all components of the electron transport chain, tricarboxylic acid cycle, and protein import apparatus. Additionally, we identified 71 pentatricopeptide repeat proteins, 29 membrane carriers/transporters, a number of new proteins involved in coenzyme biosynthesis and iron metabolism, the pyruvate dehydrogenase kinase, and a type 2C protein phosphatase that may catalyze the dephosphorylation of the pyruvate dehydrogenase complex. Systematic analysis of prominent posttranslational modifications revealed that more than 50% of the identified proteins harbor at least one modification. The most prominently observed class of posttranslational modifications was oxidative modifications. This study reveals approximately 500 new or previously unconfirmed plant mitochondrial proteins and outlines a facile strategy for unbiased, near-comprehensive identification of mitochondrial proteins and their modified forms.

Proteomic Analysis of Salt-Responsive Proteins in the Leaves of Mangrove Kandelia candel during Short-Term Stress

Abstract: Salt stress is a major abiotic stress that limits crop productivity in many regions of the world. A comparative proteomic approach to identify salt stress-responsive proteins and to understand the molecular mechanisms was carried out in the woody halophyte Kandelia candel. Four-leaf-old K. candel seedlings were exposed to 150 (control), 300, 450, and 600 mM NaCl for 3 days. Proteins extracted from the leaves of K. candel seedlings were separated by two-dimensional gel electrophoresis (2-DE). More than 900 protein spots were detected on each gel, and 53 differentially expressed protein spots were located with at least two-fold differences in abundance on 2-DE maps, of which 48 were identified by matrix-assisted laser desorption ionization time-of-flight/time-of-flight mass spectrometry (MALDI-TOF-TOF/MS). The results showed that K. candel could withstand up to 450 mM NaCl stress by up-regulating proteins that are mainly involved in photosynthesis, respiration and energy metabolism, Na+ compartmentalization, protein folding and assembly, and signal transduction. Physiological data, including superoxide dismutase (SOD) and dehydroascorbate reductase (DHAR) activities, hydrogen peroxide (H2O2) and superoxide anion radicals (O2−) contents, as well as Na+ content and K+/Na+ ratios all correlated well with our proteomic results. This study provides new global insights into woody halophyte salt stress responses. Identification of differentially expressed proteins promotes better understanding of the molecular basis for salt stress reduction in K. candel.

Amino Acid Composition, Molecular Weight Distribution and Gel Electrophoresis of Walnut (Juglans regia L.) Proteins and Protein Fractionations

Abstract: As a by-product of oil production, walnut proteins are considered as an additional source of plant protein for human food. To make full use of the protein resource, a comprehensive understanding of composition and characteristics of walnut proteins are required. Walnut proteins have been fractionated and characterized in this study. Amino acid composition, molecular weight distribution and gel electrophoresis of walnut proteins and protein fractionations were analyzed. The proteins were sequentially separated into four fractions according to their solubility. Glutelin was the main component of the protein extract. The content of glutelin, albumin, globulin and prolamin was about 72.06%, 7.54%, 15.67% and 4.73% respectively. Glutelin, albumin and globulin have a balanced content of essential amino acids, except for methionine, with respect to the FAO pattern recommended for adults. SDS-PAGE patterns of albumin, globulin and glutelin showed several polypeptides with molecular weights 14.4 to 66.2 kDa. The pattern of walnut proteins in two-dimension electrophoresis (2-DE) showed that the isoelectric point was mainly in the range of 4.8–6.8. The results of size exclusion chromatogram indicated molecular weight of the major components of walnut proteins were between 3.54 and 81.76 kDa.

Synthesis, characterization and crystal structure of cobalt(III) complexes containing 2-acetylpyridine thiosemicarbazones: DNA/protein interaction, radical scavenging and cytotoxic activities.

Abstract: The synthesis, structure and biological studies of cobalt(III) complexes supported by NNS-tridentate ligands are reported. Reactions of 2-acetylpyridine N-substituted thiosemicarbazone (HL1–3) with [CoCl2(PPh3)2] resulted [Co(L1–3)2]Cl (1–3) which were characterized by elemental analysis and various spectral studies. The molecular structure of the complex 1 has been determined by single crystal X-ray diffraction studies. In vitro DNA binding studies of complexes 1–3 carried out by fluorescence studies and the results revealed the binding of complexes to DNA via intercalation. The binding constant (Kb) values of complexes 1–3 from fluorescence experiments showed that the complex 3 has greater binding propensity for DNA. The DNA cleavage activity of the complexes 1 and 3 were ascertained by gel electrophoresis assay which revealed that the complexes are good DNA cleavage agents. Further, the interactions of the complexes with bovine serum albumin (BSA) were also investigated using fluorescence spectroscopic method, which showed that the complexes 1–3 could bind strongly with BSA. The antioxidant property of the complexes was evaluated to test their free-radical scavenging ability. Furthermore, in vitro cytotoxicity of the complexes against MCF-7 and A431 cell lines was assayed which showed higher activity and efficiently vanished the cancer cells even at low concentrations.

Protein composition of wheat gluten polymer fractions determined by quantitative two-dimensional gel electrophoresis and tandem mass spectrometry

Abstract: Certain wheat gluten proteins form large protein polymers that are extractable in 0.5% SDS only after sonication. Although there is a strong relationship between the amounts of these polymers in the flour and bread-making quality, the protein components of these polymers have not been thoroughly investigated. Flour proteins from the US bread wheat Butte 86 were extracted in 0.5% SDS using a two-step procedure with and without sonication. Proteins were further separated by size exclusion chromatography (SEC) into monomeric and polymeric fractions and analyzed by quantitative two-dimensional gel electrophoresis (2-DE). When proteins in select 2-DE spots were identified by tandem mass spectrometry (MS/MS), overlapping spots from the different protein fractions often yielded different identifications. Most high-molecular-weight glutenin subunits (HMW-GS) and low-molecular-weight glutenin subunits (LMW-GS) partitioned into the polymer fractions, while most gliadins were found in the monomer fractions. The exceptions were alpha, gamma and omega gliadins containing odd numbers of cysteine residues. These proteins were detected in all fractions, but comprised the largest proportion of the SDS-extractable polymer fraction. Several types of non-gluten proteins also were found in the polymer fractions, including serpins, triticins and globulins. All three types were found in the largest proportions in the SDS-extractable polymer fraction. This is the first study to report the accumulation of gliadins containing odd numbers of cysteine residues in the SDS-extractable glutenin polymer fraction, supporting the hypothesis that these gliadins serve as chain terminators of the polymer chains. These data make it possible to formulate hypotheses about how protein composition influences polymer size and structure and provide a foundation for future experiments aimed at determining how environment affects glutenin polymer distribution. In addition, the analysis revealed additional layers of complexity to the wheat flour proteome that should be considered when evaluating quantitative 2-DE data.

Fibrinogen enhances the inflammatory response of alveolar macrophages to TiO2, SiO2 and carbon nanomaterials.

Abstract: Many studies have shown that the composition of the protein corona dramatically affects the response of cells to nanomaterials (NMs). However, the role of each single protein is still largely unknown. Fibrinogen (FG), one of the most abundant plasma proteins, is believed to mediate foreign-body reactions. Since this protein is absent in cell media used in in vitro toxicological tests the possible FG-mediated effects have not yet been assessed. Here, the effect of FG on the toxicity of three different kinds of inorganic NMs (carbon, SiO2 and TiO2) on alveolar macrophages has been investigated. A set of integrated techniques (UV–vis spectroscopy, dynamic light scattering and sodium dodecyl sulphate-polyacrylamide gel electrophoresis) have been used to study the strength and the kinetics of interaction of FG with the NMs. The inflammatory response of alveolar macrophages (MH-S) exposed to the three NMs associated with FG has also been investigated. We found that FG significantly enhances the cytotoxi...

Boron bridging of rhamnogalacturonan‐II, monitored by gel electrophoresis, occurs during polysaccharide synthesis and secretion but not post‐secretion

Abstract: †‡ SUMMARY The cell-wall pectic domain rhamnogalacturonan-II (RG-II) is cross-linked via borate diester bridges, which influence the expansion, thickness and porosity of the wall. Previously, little was known about the mechanism or subcellular site of this cross-linking. Using polyacrylamide gel electrophoresis (PAGE) to separate monomeric from dimeric (boron-bridged) RG-II, we confirmed that Pb 2+ promotes H3BO3-dependent dimerisation in vitro .H 3BO3 concentrations as high as 50 mM did not prevent cross-linking. For in-vivo experiments, we successfully cultured ‘Paul’s Scarlet’ rose (Rosa sp.) cells in boron-free medium: their wall-bound pectin contained monomeric RG-II domains but no detectable dimers. Thus pectins containing RG-II domains can be held in the wall other than via boron bridges. Re-addition of H3BO3 to 3.3 lM triggered a gradual appearance of RG-II dimer over 24 h but without detectable loss of existing monomers, suggesting that only newly synthesised RG-II was amenable to boron bridging. In agreement with this, Rosa cultures whose polysaccharide biosynthetic machinery had been compromised (by carbon starvation, respiratory inhibitors, anaerobiosis, freezing or boiling) lost the ability to generate RG-II dimers. We conclude that RG-II normally becomes boron-bridged during synthesis or secretion but not post-secretion. Supporting this conclusion, exogenous [ 3 H]RG-II was neither dimerised in the medium nor cross-linked to existing wall-associated RG-II domains when added to Rosa cultures. In conclusion, in cultured Rosa cells RG-II domains have a brief window of opportunity for boron-bridging intraprotoplasmically or during secretion, but secretion into the apoplast is a point of no return beyond which additional boron-bridging does not readily occur.

Synthesis, characterization, DNA binding, cleavage activity and cytotoxicity of copper(II) complexes

Abstract: Three new mononuclear copper(II) complexes, [Cu(L2)]2+ (1), [Cu(acac)(L)]+ (2), and [Cu(acac-Cl)(L)]+ (3) (L = 2-(4-pyridine)oxazo[4,5-f]1,10-phenanthroline (4-PDOP); acac = acetylacetone; acac-Cl = 3-chloroacetylacetone), have been synthesized and characterized by elemental analysis, high resolution mass spectrometry (Q-TOF), and IR spectroscopy Two of the complexes were structurally characterized by single-crystal X-ray diffraction techniques Their interactions with DNA were studied by UV-vis absorption and emission spectra, viscosity, thermal melting, DNA unwinding assay and CD spectroscopy The nucleolytic cleavage activity of the compounds was carried out on double stranded pBR322 circular plasmid DNA by using a gel electrophoresis experiment in the presence and absence of an oxidant (H2O2) Active oxygen intermediates such as hydroxyl radicals and hydrogen peroxide generated in the presence of L and complexes 1–3 may act as active species for the DNA scission The cytotoxicity of the complexes against HepG2 cancer cells was also studied

Northern blots for small RNAs and microRNAs.

Abstract: This protocol describes the detection of small RNAs (10–200 nucleotides) by blot hybridization. The RNA samples, denatured in formamide, are separated by denaturing polyacrylamide gel electrophoresis. Becausehigh-percentage polyacrylamide gels arerequired toseparateRNAs in this sizerange, it is necessary to perform electrophoretic transfer to positively charged nylon membranes. After transfer, the immobilized RNAs are subjected to hybridization with a 32 P-radiolabeled DNA or RNA probe and detected by phosphorimaging or autoradiography. This procedure is commonly used to detect small, U-rich spliceosomal small nuclear RNAs (snRNAs) and miRNAs. It should be possible also to detect most miRNAs using high-percentage (e.g., 15%) urea–polyacrylamide gel electrophoresis.

Synthesis, crystal structures, in vitro DNA binding, antibacterial and cytotoxic activities of new di- and polynuclear silver(I) saccharinate complexes with tertiary monophosphanes

Abstract: Four new silver(I) saccharinate (sac) complexes, [Ag(μ-sac)(PPh3)]2 (1), [Ag(μ-sac)(PPh2Cy)]2 (2), [Ag(μ-sac)(PPhCy2)]2 (3) and [Ag(μ-sac)(PCy3)]n (4), where PPh3 = triphenylphosphane, PPh2Cy = cyclohexyldiphenylphosphane, PPhCy2 = dicyclohexylphenylphosphane and PCy3 = tricyclohexylphosphane, have been synthesized and fully characterized by elemental analysis, IR, NMR, ESI-MS and single crystal X-ray diffraction. Fluorescence ethidium bromide displacement indicate that all complexes bind to fish sperm (FS) DNA by intercalation with binding constants (KA) of 29.1 ± 0.26 × 105 M−1 for 1, 2.54 ± 0.12 × 105 M−1 for 2, 2.42 ± 0.08 × 105 M−1 for 3, 0.19 ± 0.03 × 105 M−1 for 4. The relative viscosities of the FS-DNA solutions increase with increasing of the complex concentration, providing strong evidence for the intercalation mode. The gel electrophoresis assay further confirms their binding with the pBR322 plasmid DNA. The MIC values of the silver(I) complexes are generally higher than those of AgNO3 and silver sulfadiazine, but 1 presents a promising activity against Salmonella typhimurium and Staphylococcus aureus. All complexes are highly cytotoxic on human lung carcinoma (A549) and human breast adenocarcinoma (MCF-7) cell lines with IC50 values ranging from 0.82 to 3.13 μM.

Rapid and sensitive PCR-dipstick DNA chromatography for multiplex analysis of the oral microbiota.

Abstract: A complex of species has been associated with dental caries under the ecological hypothesis. This study aimed to develop a rapid, sensitive PCR-dipstick DNA chromatography assay that could be read by eye for multiplex and semiquantitative analysis of plaque bacteria. Parallel oligonucleotides were immobilized on a dipstick strip for multiplex analysis of target DNA sequences of the caries-associated bacteria, Streptococcus mutans, Streptococcus sobrinus, Scardovia wiggsiae, Actinomyces species, and Veillonella parvula. Streptavidin-coated blue-colored latex microspheres were to generate signal. Target DNA amplicons with an oligonucleotide-tagged terminus and a biotinylated terminus were coupled with latex beads through a streptavidin-biotin interaction and then hybridized with complementary oligonucleotides on the strip. The accumulation of captured latex beads on the test and control lines produced blue bands, enabling visual detection with the naked eye. The PCR-dipstick DNA chromatography detected quantities as low as 100 pg of DNA amplicons and demonstrated 10- to 1000-fold higher sensitivity than PCR-agarose gel electrophoresis, depending on the target bacterial species. Semiquantification of bacteria was performed by obtaining a series of chromatograms using serial 10-fold dilution of PCR-amplified DNA extracted from dental plaque samples. The assay time was less than 3 h. The semiquantification procedure revealed the relative amounts of each test species in dental plaque samples, indicating that this disposable device has great potential in analysis of microbial composition in the oral cavity and intestinal tract, as well as in point-of-care diagnosis of microbiota-associated diseases.

Rheological and mechanical behavior of polyacrylamide hydrogels chemically crosslinked with allyl agarose for two-dimensional gel electrophoresis.

Abstract: Two-dimensional (2-D) gel electrophoresis currently represents one of the most standard techniques for protein separation. In addition to the most commonly employed polyacrylamide crosslinked hydrogels, acrylamide-agarose copolymers have been proposed as promising systems for separation matrices in 2-D electrophoresis, because of the good resolution of both high and low molecular mass proteins made possible by careful control and optimization of the hydrogel pore structure. As a matter of fact, a thorough understanding of the nature of the hydrogel pore structure as well as of the parameters by which it is influenced is crucial for the design of hydrogel systems with optimal sieving properties. In this work, a series of acrylamide-based hydrogels covalently crosslinked with different concentrations of allyl agarose (0.2-1%) is prepared and characterized by creep-recovery measurements, dynamic rheology and tensile tests, in the attempt to gain a clearer understanding of structure-property relationships in crosslinked polyacrylamide-based hydrogels. The rheological and mechanical properties of crosslinked acrylamide-agarose hydrogels are found to be greatly affected by crosslinker concentration. Dynamic rheological tests show that hydrogels with a percentage of allyl agarose between 0.2% and 0.6% have a low density of elastically effective crosslinks, explaining the good separation of high molecular mass proteins in 2-D gel electrophoresis. Over the same range of crosslinker concentration, creep-recovery measurements reveal the presence of non-permanent crosslinks in the hydrogel network that justifies the good resolution of low molecular mass proteins as well. In tensile tests, the hydrogel crosslinked with 0.4% of allyl agarose exhibits the best results in terms of mechanical strength and toughness. Our results show how the control of the viscoelastic and the mechanical properties of these materials allow the design of mechanically stable hydrogels with improved sieving ability in protein electrophoresis over a wide range of molecular masses.

Purification of a novel chitin-binding lectin with antimicrobial and antibiofilm activities from a bangladeshi cultivar of potato (Solanum tuberosum).

Abstract: A new chitin-binding lectin was purified from a Bangladeshi cultivar 'Deshi' of potato (Solanum tuberosum L.) through anion-exchange and affinity chromatographies using a chitin column. Sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE) showed the molecular mass of the lectin as 20,000 Daltons. This molecular mass was almost half of the molecular masses of chitin-binding lectins derived from other potatoes. The lectin showed both bactericidal and growth-inhibiting activities against Gram-positive (Listeria monocytogenes) and Gram-negative (Escherichia coli, Salmonella enteritidis and Shigella boydii) pathogenic bacteria. It also showed antifungal activity against Rhizopus spp., Penicillium spp. and Aspergillus niger. Biofilm produced by the bacterium Pseudomonas aeruginosa was dose-dependently reduced by 5-20% in 24 h after administration of the lectin, which was attributed to the glycan-binding property of the lectin having affinity to GlcNAc polymers. It was the first observation that any potato lectin prevented biofilm formation by P. aeruginosa and, therefore, could have possible applications in clinical microbiology and biomedical science.

Proteomic analysis of albumin and globulin fractions of pea (Pisum sativum L.) seeds.

Abstract: Background. Proteomic analysis is emerging as a highly useful tool in food research, including studies of food allergies. Two-dimensional gel electrophoresis involving isoelectric focusing and sodium dodecyl sulfate polyacrylamide gel electrophoresis is the most effective method of separating hundreds or even thou- sands of proteins. In this study, albumin and globulin fractions of pea seeds cv. Ramrod were subjected to proteomic analysis. Selected potentially alergenic proteins were identifi ed based on their molecular weights and isoelectric points. Material and methods. Pea seeds (Pisum sativum L.) cv. Ramrod harvested over a period of two years (Plant Breeding Station in Piaski-Szelejewo) were used in the experiment. The isolated albumins, globu- lins and legumin and vicilin fractions of globulins were separated by two-dimensional gel electrophoresis. Proteomic images were analysed in the ImageMaster 2D Platinum program with the use of algorithms from the Melanie application. The relative content, isoelectric points and molecular weights were computed for all identifi ed proteins. Electrophoregrams were analysed by matching spot positions from three independent replications. Results. The proteomes of albumins, globulins and legumin and vicilin fractions of globulins produced up to several hundred spots (proteins). Spots most characteristic of a given fraction were identifi ed by com- puter analysis and spot matching. The albumin proteome accumulated spots of relatively high intensity over a broad range of pI values of ~4.2-8.1 in 3 molecular weight (MW) ranges: I - high molecular-weight albu- mins with MW of ~50-110 kDa, II - average molecular-weight albumins with MW of ~20-35 kDa, and III - low molecular-weight albumins with MW of ~13-17 kDa. 2D gel electrophoregrams revealed the presence of 81 characteristic spots, including 24 characteristic of legumin and 14 - of vicilin. Conclusions. Two-dimensional gel electrophoresis proved to be a useful tool for identifying pea proteins. Patterns of spots with similar isoelectric points and different molecular weights or spots with different iso- electric points and similar molecular weights play an important role in proteome analysis. The regions char- acteristic of albumin, globulin and legumin and vicilin fractions of globulin with typical MW and pI values were identifi ed as the results of performed 2D electrophoretic separations of pea proteins. 2D gel electropho- resis of albumins and the vicilin fraction of globulins revealed the presence of 4 and 2 spots, respectively, representing potentially allergenic proteins. They probably corresponded to vicilin fragments synthesized during post-translational modifi cation of the analysed protein.

Di- and polynuclear silver(I) saccharinate complexes of tertiary diphosphane ligands: synthesis, structures, in vitro DNA binding, and antibacterial and anticancer properties

Abstract: A series of new silver(I) saccharinate (sac) complexes, [Ag2(sac)2(μ-dppm)H2O]·H2O (1), {[Ag2(μ-sac)2(μ-dppe)]·3H2O·CH2Cl2} n (2), [Ag2(μ-sac)2(μ-dppp)] n (3), and [Ag(sac)(μ-dppb)] n (4) [dppm is 1,1-bis(diphenylphosphino)methane, dppe is 1,2-bis(diphenylphosphino)ethane, dppp is 1,3-bis(diphenylphosphino)propane, and dppb is 1,4-bis(diphenylphosphino)butane], have been synthesized and characterized by C, H, N elemental analysis, IR spectroscopy, 1H NMR, 13C NMR, and 31P NMR spectroscopy, electrospray ionization mass spectrometry, and thermogravimetry–differential thermal analysis. Single-crystal X-ray studies show that the diphosphanes act as bridging ligands to yield a dinuclear complex (1) and one-dimensional coordination polymers (2 and 4), whereas the sac ligand adopts a μ2-N/O bridging mode in 2, and is N-coordinated in 1 and 4. The interaction of the silver(I) complexes with fish sperm DNA was investigated using UV–vis spectroscopy, fluorescence spectroscopy, and agarose gel electrophoresis. The binding studies indicate that the silver(I) complexes can interact with fish sperm DNA through intercalation, and complexes 1 and 3 have the highest binding affinity. The gel electrophoresis assay further confirms the binding of the complexes with the pBR322 plasmid DNA. The minimum inhibitory concentrations of the complexes indicate that complex 1 exhibits very high antibacterial activity against standard bacterial strains of Escherichia coli, Salmonella typhimurium, and Staphylococcus aureus, being much higher than those of AgNO3, silver sulfadiazine, ciprofloxacin, and gentamicin. Moreover, complexes 1–3 exhibit very high cytotoxic activity against A549 and MCF-7 cancer cell lines, compared with AgNO3 and cisplatin. The bacterial and cell growth inhibitions of the silver(I) complexes are closely related to their DNA binding affinities.

Cloning, expression, and characterization of thermophilic L-asparaginase from Thermococcus kodakarensis KOD1.

Abstract: The present study demonstrates cloning, expression, and characterization of hyperthermostable L-asparaginase from Thermococcus kodakarensis KOD1 in Escherichia coli BLR(DE3). The recombinant 6× His-tagged protein L-asparaginase from T. kodakarensis (TkAsn), was purified to homogeneity by heat treatment followed by affinity chromatography using a nickel-nitrilotriacetic acid (Ni-NTA) column. The molecular mass of the purified enzyme was found to be approximately 37 kDa by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). The enzymatic properties, such as optimum temperature and pH, were 90 °C and 8.0, respectively. Its appearent Km , Vmax , and Kcat values were 2.6 mM, 1121 µmol min(-1) mg(-1) , and 694 S(-1) , respectively. The enzyme displayed high thermal stability at optimum temperature with an insignificant loss in enzymatic activity, retaining almost 90% of its activity over a time period of 32 h. The relative activity of the enzyme was significantly inhibited by the supplementation of Cu(2+) and Ni(2+) ions, while moderately inhibited by other ions. In contrast, Mg(2+) ions enhanced the relative activity compared to the control. The acrylamide contents in baked dough were reduced to sixty percent after treatment with recombinant TkAsn as compared to the untreated control. Results of the present study revealed that the enzyme was highly active at broader range of temperatures and pH, which reflect the potential of recombinant TkAsn in the food processing industry. In addition, the high thermal stability of the enzyme may facilitates its handling, storage, and transportation.

A simple method for RNA isolation from various tissues of the tree Neolamarckia cadamba

Abstract: Plant tissues contain abundant polysaccharides, phenolic compounds and other metabolites, which makes it difficult to isolate high-quality RNA from them. In addition, Neolamarckia cadamba contains large quantities of other components, particularly RNA-binding alkaloids, which makes the isolation even more challenging. Here, we describe a concise and efficient RNA isolation method that combines the cetyltrimethyl ammonium bromide (CTAB) and Plant RNA Kit (Omega) protocols. Gel electrophoresis showed that RNA extracted from all tissues, using this protocol, was of good integrity and without DNA contamination. Furthermore, the isolated RNA was of high purity, with an A260/A280 ratio of 2.1 and an A260/A230 ratio of >2.0. The isolated RNA was also suitable for downstream applications, such as reverse transcription-polymerase chain reaction (RT-PCR) and quantitative RT-PCR (RT-qPCR). The RNA isolation method was also efficient for recalcitrant plant tissues.