Showing papers on "Gel electrophoresis published in 2016"

A Comparison of Membrane Proteins of Normal and Transformed Cells by Lactoperoxidase Labeling (iodination/fibroblasts/membranes/gel electrophoresis)

Abstract: The enzyme lactoperoxidase (iodide: hy- drogen-peroxide oxidoreductase, EC 1.11.1.8) was used to iodinate (1251) accessible proteins on membranes of intact virally transformed and untransformed cells. A number of labeled bands of proteins were detected by acrylamide gel electrophoresis. A heavily labeled band with a rmolecular weight of approximately 250,000 daltons was found in all untransformed cells but was absent from transformed cells. When Coomassie-blue-stained membrane preparations were compared, a band was seen in normal cells which co- migrated with the lactoperoxidase-labeled band. In trans- formed cell membranes, three discrete bands were present in the same position. Thus, the expression of tlhis protein may be altered when cells are in the transformed state. The identification of alterations in membrane proteins and glycoproteins after transformation by DNA or RNA onco- genic viruses has been confusing. In general, there is reported to be an overall decrease in numbers of membrane glyco- proteins (1, 2), but there are demonstrations of an increased glycosylation of a restricted number of membrane glyco- proteins in transformed cells (3, 4). Conversely, other studies have suggested an association with untransformed cell

Electrophoresis in Practice: A Guide to Methods and Applications of DNA and Protein Separations

Abstract: FUNDAMENTALS Electrophoresis Isotachophoresis Isoelectric Focusing Blotting Instrumentation Interpretation of Electropherograms METHODS Method 1: PAGE of Dyes Method 2: Agarose and Immunoelectrophoresis Method 3: Titration Curve Analysis Method 4: Native PAGE in Amphoteric Buffers Method 5: Agarose IEF Method 6: PAGIEF in Rehydrated Gels Method 7: SDS-Polyacrylamide Electrophoresis Method 8: Semi-Dry Blotting of Proteins Method 9: IEF in Immobilized pH Gradients Method 10: High Resolution 2D Electrophoresis Method 11: PAGE of Double Stranded DNA Method 12: Native PAGE of Single Stranded DNA Method 13: Denaturing Gradient Gel Electrophoresis Method 14: Denaturing PAGE of DNA Method 15: Vertical PAGE Appendix References Index.

Invariance and heterogeneity in the major structural and regulatory proteins of chick muscle cells revealed by two-dimensional gel electrophoresis (100-A filaments/muscle contractile proteins/two-dimensional isoelectric focusing/sodium dodecyl sulfate gel electrophoresis)

Abstract: A two-dimensional gel electrophoresis system is used to investigate some of the properties of desmin, the major subunit of the 100-A filaments from chick muscle cells, and to compare these properties to those of the other major contractile and regulatory proteins of muscle. Desmin from embryonic and adult smooth, skeletal, and cardiac muscle cells is resolved into two isoelectric variants, a and A, which possess slightly different electrophoretic mobilities in sodium dodecyl sulfate/polyac- rylamide gel electrophoresis. Both the a and the A variants from all six preparations appear to be identical in isoelectric point and apparent molecular weight. The a and A desmin are present in approximately equal amounts in all three types of muscle, suggesting that both isoelectric variants of desmin serve as the structural subunits of the 100-A filaments in chick muscle cells. Tropomyosin also can be resolved into two subunits, a and A, in all three types of muscle. However, in each type of muscle both subunits differ from their counterparts in the other types of muscle, either by molecular weight or by isoelectric point. These results indicate that, with regard to apparent isoelectric point and molecular weight, desmin, a major muscle structural protein, is invariant, while tropomyosin, a major muscle regu- latory protein, exhibits heterogeneity in the three types of muscle.

The NDH-1L-PSI Supercomplex Is Important for Efficient Cyclic Electron Transport in Cyanobacteria.

Abstract: Two mutants isolated from a tagging library of Synechocystis sp. strain PCC 6803 were sensitive to high light and had a tag in sll1471 encoding CpcG2, a linker protein for photosystem I (PSI)-specific antenna. Both mutants demonstrated strongly impaired NDH-1-dependent cyclic electron transport. Blue native-polyacrylamide gel electrophoresis followed by immunoblotting and mass spectrometry analyses of the wild type and a mutant containing CpcG2 fused with yellow fluorescent protein-histidine6 indicated the presence of a novel NDH-1L-CpcG2-PSI supercomplex, which was absent in the cpcG2 deletion mutant, the PSI-less mutant, and several other strains deficient in NDH-1L and/or NDH-1M. Coimmunoprecipitation and pull-down analyses on CpcG2-yellow fluorescent protein-histidine6, using antibody against green fluorescent protein and nickel column chromatography, confirmed the association of CpcG2 with the supercomplex. Conversely, the use of antibodies against NdhH or NdhK after blue native-polyacrylamide gel electrophoresis and in coimmunoprecipitation experiments verified the necessity of CpcG2 in stabilizing the supercomplex. Furthermore, deletion of CpcG2 destabilized NDH-1L as well as its degradation product NDH-1M and significantly decreased the number of functional PSI centers, consistent with the involvement of CpcG2 in NDH-1-dependent cyclic electron transport. The CpcG2 deletion, however, had no effect on respiration. Thus, we propose that the formation of an NDH-1L-CpcG2-PSI supercomplex in cyanobacteria facilitates PSI cyclic electron transport via NDH-1L.

A Universal Polymerase Chain Reaction Developer.

Abstract: The versatility of PCR, the gold standard for amplification of DNA targets, is hampered by the laborious, multi-step detection based on gel electrophoresis. We propose a one-step, one-tube method for the rapid (5 min) naked-eye detection of PCR products, based on controlled aggregation of gold nanoparticles. Our method is universal, instrument-free, and ultra-sensitive, as it could detect as low as 0.01 zeptomoles of HIV template DNA in an excess of interfering human genomic DNA.

Two ornithine decarboxylase mRNA species in mouse kidney arise from size heterogeneity at their 3' termini (androgen action/ornithine decarboxylase genes/polyadenylylation/blot hybridization/DNA sequencing)

Abstract: Ornithine decarboxylase (OrnDCase; L-ornithine carboxy-lyase, EC 4.1.1.17) mRNA present In mouse kidney comprises two species with molecular sizes of =2.2 and -=2.7 kilobases (kb). cDNA clones prepared from murine kidney OrnDCase mRNA were used to determine the reason for the size heterogeneity of these mRNAs. Two of the cDNA clones (pODC16 and pODC74) that differed at the 3' termini were isolated and sequenced. DNA sequencing indicated that each cDNA had a poly(A) tail; however, pODC74 was 429 nucleotides longer than pODC16 at the 3' end and contained two AATAAA signals for poly(A) addition. That the longer cDNA corresponded to the larger mRNA was confirmed by hybridization of a unique Pst I/Pst I fragment from the 3' terminus of pODC74 only to the 2.7-kb OrnDCase mRNA. The two cDNAs did not represent fulllength copies of OrnDCase mRNAs and were 1199 (pODC16) and 1204 base pairs (bp) (pODC74) long. There were five mismatches in their 759-bplong overlapping nucleotide sequence, suggesting that the 2.2and 2.7-kb OrnDCase mRNAs may be products of two separate, yet very similar, OrnDCase genes. Androgen regulation of the accumulation of these two OrnDCase mRNAs appeared to occur coordinately, as testosterone administration brought about comparable increases in their concentrations in mouse kidney. Ornithine decarboxylase (OmDCase; L-ormithine carboxylyase, EC 4.1.1.17) catalyzes the conversion of ornithine to putrescine and is the first and apparently rate-controlling enzyme in polyamine biosynthesis (1-3). In the murine kidney, OrnDCase activity and the enzyme protein concentration are increased by androgen treatment with relatively rapid kinetics, with a maximal induction at 18-24 hr after steroid administration (4, 5). In addition to enhancing enzyme synthesis, androgen administration increases OrnDCase concentration through prolonging the biological half-life of the enzyme protein (5, 6). To better understand the mechanisms regulating OmDCase synthesis, workers in our own (7) and other laboratories (8-10) have prepared cDNA clones for the mRNA encoding OmDCase. When these cDNAs were used to identify OrnDCase mRNA by hybridization after agarose gel electrophoresis, two mRNA species of =2.2 and =2.7 kilobases (kb) were found (7, 10). The nucleotide sequence and deduced amino acid sequence of the shorter OrnDCase mRNA have been recently published (11, 12). We report here the nucleotide sequence of the 3' ends of the two OrnDCase mRNA species in mouse kidney. From the sequences and from RNA blot hybridization data using the unique 3'terminal probe from one of the clones, we conclude that the size heterogeneity of the two OrnDCase mRNAs is due to the dissimilar lengths of their 3' noncoding regions. In addition, The publication costs of this article were defrayed in part by page charge payment. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. ?1734 solely to indicate this fact. DNA sequence data suggest that they could be products of two similar but separate OrnDCase genes. MATERIALS AND METHODS Animnals. Mature male and female NCS [randomly bred strain Rku:NCS(s) SPF] and 129/J mice were from The Rockefeller University and The Jackson Laboratory, respectively. The animals were treated for 7 days with testosteronecontaining Silastic implants releasing either 40 or 200 .tg of testosterone per day. OrnDCase cDNAs. The preparation and cloning of the two OrnDCase eDNAs (pODC16 and pODC74) have been described (7, 13). The plasmids were propagated in Escherichia coli strain LE 392 and purified by a modification of the method of Ish-Horowicz and Burke (14) followed by CsCl density gradient centrifugation. End-Labeling and Sequencing of cDNAs. Plasmids carrying OrnDCase cDNAs were digested overnight with the appropriate restriction enzymes, ethanol-precipitated, and labeled at the 3' ends with [a-32P]dNTPs using the Klenow fragment of DNA polymerase I, or by the terminal deoxyribonucleotidyltransferase-catalyzed addition of cordycepin 5'-[a32P]triphosphate. Labeled fragments were isolated by electrophoresis on 6% polyacrylamide gels under non-denaturing conditions, recovered from the gel slices by electroelution, and ethanol-precipitated. Isolated fragments were recut, purified by electrophoresis, and sequenced by the chemical degradation method of Maxam and Gilbert (15), as modified by Catterall et al. (16). Each sample was subjected to electrophoresis on 20%, two 10% and 8% polyacrylamide sequencing gels with 7 M urea/90 mM Tris-HCl/90 mM borate/2 mM EDTA buffer, pH 8.3, and autoradiographed at -70?C using Kodak XAR-5 fim. Isolation of Renal RNA and Gel Blot Hybridizations. Total RNA was isolated from murine kidney by the lithium chloride/urea method (17) and enriched for poly(A)-containing RNA by oligo(dT)-cellulose chromatography (18). RNA samples (4-6 ug) were fractionated on 1% agarose gels containing 2.2 M formaldehyde (19), transferred to nitrocellulose filters, and hybridized with radioactive probes (20). The probes used for hybridization were isolated from pODC74 or pODC16 by preparative gel electrophoresis on 6% polyacrylamide gels after cleavage with appropriate restriction enzymes, and labeled with [a-32P]dCTP by nicktranslation. The terminal Pst I/Pst I fragment of pODC74 (from the cloning site to the first Pst I site at the 3' end) was the unique 3' probe, whereas the Hpa II/Hha I fragment of pODC74 or the 5' Pst I/Pst I fragment of pODC16 were the probes representing overlapping sequences (see Fig. 1). Quantitation of the signals was achieved by excision of the Abbreviations. OrnDCase, ornithine decarboxylase; kb, kilobase(s); bp, base pair(s). *To whom reprint requests should be addressed.

Detection of DNA Fragmentation in Apoptotic Cells by TUNEL

Abstract: Degradation of DNA into oligonucleosomal-sized fragments is a unique event in apoptosis that is orchestrated by caspase-activated DNase. Traditionally, this event is observed by resolving cellular DNA by gel electrophoresis, which results in a characteristic "ladder" pattern. However, this technique is time-consuming and cannot be used to quantitate the number of apoptotic cells in a sample. Terminal dUTP nick-end labeling (TUNEL) of fragmented DNA allows researchers to identify DNA fragmentation at the single-cell level. This method involves the specific addition of fluorescently labeled UTP to the 3'-end of the DNA fragments by terminal deoxynucleotidyl transferase. The TUNEL assay is both fast and sensitive. Here, we describe a protocol in which cells are treated with TUNEL reagent and counterstained with Hoechst 33342. In contrast to TUNEL, which only stains apoptotic cells, Hoechst 33342 stains the DNA of all cells.

Adapting capillary gel electrophoresis as a sensitive, high-throughput method to accelerate characterization of nucleic acid metabolic enzymes.

Abstract: Detailed biochemical characterization of nucleic acid enzymes is fundamental to understanding nucleic acid metabolism, genome replication and repair. We report the development of a rapid, high-throughput fluorescence capillary gel electrophoresis method as an alternative to traditional polyacrylamide gel electrophoresis to characterize nucleic acid metabolic enzymes. The principles of assay design described here can be applied to nearly any enzyme system that acts on a fluorescently labeled oligonucleotide substrate. Herein, we describe several assays using this core capillary gel electrophoresis methodology to accelerate study of nucleic acid enzymes. First, assays were designed to examine DNA polymerase activities including nucleotide incorporation kinetics, strand displacement synthesis and 3'-5' exonuclease activity. Next, DNA repair activities of DNA ligase, flap endonuclease and RNase H2 were monitored. In addition, a multicolor assay that uses four different fluorescently labeled substrates in a single reaction was implemented to characterize GAN nuclease specificity. Finally, a dual-color fluorescence assay to monitor coupled enzyme reactions during Okazaki fragment maturation is described. These assays serve as a template to guide further technical development for enzyme characterization or nucleoside and non-nucleoside inhibitor screening in a high-throughput manner.

Isolation of proteins associated with kinetoplast DNA networks in vivo (DNA-binding protein/DNA-protein crosslinking/mitochondrial DNA/presequence)

Abstract: Kinetoplast DNA (kDNA), the mitochondrial DNA of trypanosomes, is a highly condensed disc-shaped network of catenated DNA circles consisting of maxicircles, the equivalent of conventional mitochondrial DNA, and several thousand smaller circular DNAs termed minicircles. Upon cell lysis, kDNA expands, giving rise to a two-dimensional network of catenated circles with an overall diameter close to that of the whole cell. To identify proteins associated with the condensed form of kDNA in the cell, proteins were reversibly crosslinked to kDNA in whole cells of Crithidiafasciculata by formaldehyde treatment. Crosslinked networks were purified and found to retain a condensed structure which becomes fully expanded upon proteinase K treatment or reversal of the crosslinks by heating at 65?C. Five low molecular weight proteins released from the kDNA by heat treatment were purified by polyacryl- amide gel electrophoresis and their amino-terminal sequences were determined. PCR amplification and sequence analysis of cDNA sequences between these amino-terminal sequences and the miniexon (spliced leader) sequence present at the 5' end of all C. fasciculata mRNAs predicts the presence of 9-amino acid presequences with features characteristic of mitochondrial presequences on three of the proteins. Two of these proteins are lysine-rich basic proteins. These fmdings suggest that basic proteins may play a role in the condensation of kDNA in the kinetoplast and that these proteins are imported into the kinetoplast by a mechanism involving a cleavable presequence.

Identification of possible cytotoxicity mechanism of polyethylenimine by proteomics analysis

Abstract: Polyethylenimine (PEI) is a polycation widely used for successful gene delivery both in vitro and in vivo experiments. However, different studies showed that PEI could be cytotoxic to transfected cells, and the mechanism of toxicity is poorly understood. Identification of PEI-interacting proteins may help in understanding the toxicity pathways. In this study, we investigated proteins that could interact with PEI in human colorectal adenocarcinoma cells (HT29). In order to identify the proteins interacting with PEI, PEI was immobilized to sepharose beads as solid matrix. The HT29 cell lysate were passed through the matrix. PEI-bound proteins were isolated, and further separation was performed by two-dimensional gel electrophoresis. After gel digestion, proteins were identified by matrix-assisted laser desorption/ionization-time-of-flight (TOF)/TOF mass spectrometry. Our data indicated that most of the identified PEI-interacting proteins such as shock proteins, glutathione-S-transferases, and protein disulfide isomerase are involved in apoptosis process in cells. Thus, although this is a preliminary experiment implicating the involvement of some proteins in PEI cytotoxicity, it could partly explain the mechanism of PEI cytotoxicity in cells.

Proteomic analysis reveals differentially secreted proteins in the urine from patients with clear cell renal cell carcinoma

Abstract: Objective The aim of this study was to evaluate the differentially secreted protein profile in the urine from patients with clear cell renal cell carcinoma (ccRCC) using mass spectrometry–based methods. Urine composition can reflect kidney physiology and can be used to detect markers for renal diseases. Moreover, characterization of the secretome is likely to assist in the investigation of new drugs for biological targets and diagnose the ccRCC at an early stage. Methods and materials Urine samples from patients were divided according to Fuhrman degree (FI-IV), which was associated with the cellular differentiation as good prognosis (GP) and poor prognosis (PP). Healthy individuals were used as the control group (CG). We used both qualitative and quantitative mass spectrometry–based analyses that involved the following approaches: 1-dimensional gel electrophoresis combined with liquid chromatography mass spectrometry in tandem (1DE LC-MS/MS), in-solution digestion combined with label-free 1-dimensional LC-MSE (1D LC-MSE), and bidimensional gel electrophoresis combined with matrix-assisted laser desorption/ionization time of flight in tandem (2DE MALDI-TOF/TOF) or combined with LC-MS/MS. Results All the strategies allowed the identification of 354 proteins from the CG, GP, and PP groups. Qualitative experiments using 1DE LC-MS/MS analysis detected different protein profiles, and 224 proteins were identified in all groups. The label-free MSE quantitative analysis identified 113 proteins and generated novel information on secreted protein profiles, including 49 up-secreted proteins in the urine from patients with ccRCC and 40 down-secreted proteins related to the CG. Proteins such as kininogen-1, uromodulin, apolipoprotein D, polyubiquitin, and CD59 glycoprotein were down secreted according to the groups CG>GP>PP. In contrast, apolipoprotein A, fibrinogen, and haptoglobin were up secreted in patient groups. The same expression profile observed for kininogen-1, apolipoprotein D, fibrinogen, and haptoglobin was corroborated by 2DE LC-MS/MS or 2DE MALDI-TOF/TOF analyses. These 2 strategies also showed 13 differentially secreted proteins among the 3 groups. Conclusions The proteins kininogen-1, apolipoprotein D, fibrinogen, and haptoglobin presented similar quantitative protein profiles according to MSE and 2DE approaches. The latter proteins were up secreted and the former ones were down-regulated. The strategies used proved to be valuable in identifying proteins that were differentially secreted in urine from patients with RCC.

Pulse Field Gel Electrophoresis

Abstract: Pulse Field Gel Electrophoresis (PFGE) is a powerful genotyping technique used for the separation of large DNA molecules (entire genomic DNA) after digesting it with unique restriction enzymes and applying to a gel matrix under the electric field that periodically changes direction. PFGE is a variation of agarose gel electrophoresis that permits analysis of bacterial DNA fragments over an order of magnitude larger than that with conventional restriction enzyme analysis. It provides a good representation of the entire bacterial chromosome in a single gel with a highly reproducible restriction profile, providing clearly distinct and well-resolved DNA fragments.

Mass spectrometry analysis of the excretory-secretory (E-S) products of the model cestode Hymenolepis diminut a reveals their immunogenic properties and the presence of new E-S proteins in cestodes.

Abstract: Hymenolepis diminuta is an important model species in studies of therapeutics, biochemical processes, immune responses and other aspects of cestodiasis. The parasite produces numerous excretory-secretory (E-S) proteins and a glycocalyx covering its body. Our study focused on the mass spectrometry analysis of the E-S material with an objective to determine if E-S contains any new proteins, in particular those that can be identified as: antigens, vaccine candidates and drug targets. These proteins might engage directly in host-parasite interactions. Adult parasites collected from experimentally infected rats were cultured in vitro for 5 and 18h. Immunoblotting was used to verify which E-S protein bands separated in SDS-PAGE (sodium dodecyl sulfate-polyacrylamide gel electrophoresis) react with specific antibodies from sera of infected rats. We identified thirty-nine proteins by LC-MS/MS (liquid chromatography mass spectrometry). Results indicated the presence of proteins that have never been identified in cestode E-S material. Immunoblotting showed the immunogenicity of E-S products of H. diminuta, most probably associated with the presence of proteins known as antigens in other flatworm species. Among identified proteins are those engaged in immunomodulatory processes (eg. HSP), in response to oxidative stress (peroxidasin) or metabolism (eg. GAPDH). The predominant functions are associated with metabolism and catalytic activity. This is the first study identifying E-S-proteins in adult tapeworms, thus providing information for better understanding host-parasite interrelationships, and may point out potential targets for vaccines or drug discovery studies, as among the proteins observed in our study are those known to be antigens.

[12]aneN3 Modified Tetraphenylethene Molecules as High-Performance Sensing, Condensing, and Delivering Agents toward DNAs

Abstract: Four [12]aneN3 modified tetraphenylethene (TPE) compounds with different numbers of polyamine units and structure configurations, namely 1, 2, 3, and 4, were designed and synthesized. All compounds showed strong aggregation-induced emission (AIE) features. Compounds 2 and 4 showed significant emission enhancement after the addition of ssDNAs and dsDNAs of different lengths as well as calf thymus DNA (ctDNA). Compounds 1 and 3 showed very poor fluorescent responses toward DNAs. Gel electrophoresis demonstrated the abilities of 1–4 to condense DNA effectively. Complete retardation of plasmid DNA can be achieved at a concentration of 25 μM (1), 8 μM (for 2 and 3) and 4 μM (4). Experiments including fluorescent contrastive titrations, scanning electron microscopy, dynamic laser scattering, EB displacement, and gel electrophoresis demonstrated that the four compounds were able to integrate with DNA through electrostatic interactions and supramolecular stacking. A vicinal configuration around TPE (2) and more t...

Comparative Skeletal Muscle Proteomics Using Two-Dimensional Gel Electrophoresis

Abstract: The pioneering work by Patrick H. O’Farrell established two-dimensional gel electrophoresis as one of the most important high-resolution protein separation techniques of modern biochemistry (Journal of Biological Chemistry 1975, 250, 4007–4021). The application of two-dimensional gel electrophoresis has played a key role in the systematic identification and detailed characterization of the protein constituents of skeletal muscles. Protein changes during myogenesis, muscle maturation, fibre type specification, physiological muscle adaptations and natural muscle aging were studied in depth by the original O’Farrell method or slightly modified gel electrophoretic techniques. Over the last 40 years, the combined usage of isoelectric focusing in the first dimension and sodium dodecyl sulfate polyacrylamide slab gel electrophoresis in the second dimension has been successfully employed in several hundred published studies on gel-based skeletal muscle biochemistry. This review focuses on normal and physiologically challenged skeletal muscle tissues and outlines key findings from mass spectrometry-based muscle proteomics, which was instrumental in the identification of several thousand individual protein isoforms following gel electrophoretic separation. These muscle-associated protein species belong to the diverse group of regulatory and contractile proteins of the acto-myosin apparatus that forms the sarcomere, cytoskeletal proteins, metabolic enzymes and transporters, signaling proteins, ion-handling proteins, molecular chaperones and extracellular matrix proteins.

Recombinant human oviductin regulates protein tyrosine phosphorylation and acrosome reaction.

Abstract: The mammalian oviduct synthesizes and secretes a major glycoprotein known as oviductin (OVGP1), which has been shown to interact with gametes and early embryos. Here we report the use of recombinant DNA technology to produce, for the first time, the secretory form of human OVGP1 in HEK293 cells. HEK293 colonies stably expressing recombinant human OVGP1 (rHuOVGP1) were established by transfecting cells with an expression vector pCMV6-Entry constructed with OVGP1 cDNA. Large quantities of rHuOVGP1 were obtained from the stably transfected cells using the CELLSPIN cell cultivation system. A two-step purification system was carried out to yield rHuOVGP1 with a purity of >95%. Upon gel electrophoresis, purified rHuOVGP1 showed a single band corresponding to the 120-150 kDa size range of human OVGP1. Mass spectrometric analysis of the purified rHuOVGP1 revealed its identity as human oviductin. Immunofluorescence showed the binding of rHuOVGP1 to different regions of human sperm cell surfaces in various degrees of intensity. Prior treatment of sperm with 1% Triton X-100 altered the immunostaining pattern of rHuOVGP1 with an intense immunostaining over the equatorial segment and post-acrosomal region as well as along the length of the tail. Addition of rHuOVGP1 in the capacitating medium further enhanced tyrosine phosphorylation of sperm proteins in a time-dependent manner. After 4-h incubation in the presence of rHuOVGP1, the number of acrosome-reacted sperm induced by calcium ionophore significantly increased. The successful production of rHuOVGP1 can now facilitate the study of the role of human OVGP1 in fertilization and early embryo development.

Direct and Auger Electron-Induced, Single- and Double-Strand Breaks on Plasmid DNA Caused by 99mTc-Labeled Pyrene Derivatives and the Effect of Bonding Distance

Abstract: It is evident that 99mTc causes radical-mediated DNA damage due to Auger electrons, which were emitted simultaneously with the known γ-emission of 99mTc. We have synthesized a series of new 99mTc-labeled pyrene derivatives with varied distances between the pyrene moiety and the radionuclide. The pyrene motif is a common DNA intercalator and allowed us to test the influence of the radionuclide distance on damages of the DNA helix. In general, pUC 19 plasmid DNA enables the investigation of the unprotected interactions between the radiotracers and DNA that results in single-strand breaks (SSB) or double-strand breaks (DSB). The resulting DNA fragments were separated by gel electrophoresis and quantified by fluorescent staining. Direct DNA damage and radical-induced indirect DNA damage by radiolysis products of water were evaluated in the presence or absence of the radical scavenger DMSO. We demonstrated that Auger electrons directly induced both SSB and DSB in high efficiency when 99mTc was tightly bound to the plasmid DNA and this damage could not be completely prevented by DMSO, a free radical scavenger. For the first time, we were able to minimize this effect by increasing the carbon chain lengths between the pyrene moiety and the 99mTc nuclide. However, a critical distance between the 99mTc atom and the DNA helix could not be determined due to the significantly lowered DSB generation resulting from the interaction which is dependent on the type of the 99mTc binding motif. The effect of variable DNA damage caused by the different chain length between the pyrene residue and the Tc-core as well as the possible conformations of the applied Tc-complexes was supplemented with molecular dynamics (MD) calculations. The effectiveness of the DNA-binding 99mTc-labeled pyrene derivatives was demonstrated by comparison to non-DNA-binding 99mTcO4–, since nearly all DNA damage caused by 99mTcO4– was prevented by incubating with DMSO.

The Application of Pulsed Field Gel Electrophoresis in Clinical Studies

Abstract: Pulsed-field gel electrophoresis is a method applied in separating large segments of deoxyribonucleotide using an alternating and cross field. In a uniform magnetic field, components larger than 50kb pass a route through the gel and since the movement of DNA (Deoxyribonucleic acid) molecules are in a Zigzag form, separation of DNAs as bands carried out better via gel. PFGE in microbiology is a standard method which is used for typing of bacteria. It is also a very useful tool in epidemiological studies and gene mapping in microbes and mammalian cell, also motivated development of large-insert cloning system such as bacterial and yeast artifical chromosomes. In this method, close and similar species in terms of genetic patterns show alike profiles regarding DNA separation, and those ones which don't have similarity or are less similar, reveal different separation profiles. So this feature can be used to determine the common species as the prevalence agent of a disease. PFGE can be utilized for monitoring and evaluating different micro-organisms in clinical samples and existing ones in soil and water. This method can also be a reliable and standard method in vaccine preparation. In recent decades, PFGE is highly regarded as a powerful tool in control, prevention and monitoring diseases in different populations.

Cr3+ Binding to DNA Backbone Phosphate and Bases: Slow Ligand Exchange Rates and Metal Hydrolysis

Abstract: The interaction between chromium ions and DNA is of great interest in inorganic chemistry, toxicology, and analytical chemistry. Most previous studies focused on in situ reduction of Cr(VI), producing Cr3+ for DNA binding. Recently, Cr3+ was reported to activate the Ce13d DNAzyme for RNA cleavage. Herein, the Ce13d is used to study two types of Cr3+ and DNA interactions. First, Cr3+ binds to the DNA phosphate backbone weakly through reversible electrostatic interactions, which is weakened by adding competing inorganic phosphate. However, Cr3+ coordinates with DNA nucleobases forming stable cross-links that can survive denaturing gel electrophoresis condition. The binding of Cr3+ to different nucleobases was further studied in terms of binding kinetics and affinity by exploiting carboxyfluorescein-labeled DNA homopolymers. Once binding takes place, the stable Cr3+/DNA complex cannot be dissociated by EDTA, attributable to the ultraslow ligand exchange rate of Cr3+. The binding rate follows the order of G >...

Antiproliferative activity of ruthenium(II) arene complexes with mono- and bidentate pyridine-based ligands

Abstract: A series of RuII arene complexes of mono- and bidentate N-donor ligands with carboxyl or ester groups and chlorido ancillary ligands were synthesised and structurally characterised. The complexes have a distorted tetrahedral piano-stool geometry. The binding interaction was studied with calf thymus DNA (CT-DNA) by absorption titration, viscosity measurement, thermal melting, circular dichroism, ethidium bromide displacement assay and DNA cleavage of plasmid DNA (pBR322), investigated by gel electrophoresis. The dichlorido complexes bind covalently to DNA in the dark, similar to cisplatin, while the monochlorido complexes bind covalently on irradiation, similar to cisplatin analogues. The compounds are selectively cytotoxic against several tumour cell lines and show specific nonlinear correlation between dose and activity. This phenomenon is closely related to their potential to act preferentially as inhibitors of cell division.

Scrutinizing the DNA damaging and antimicrobial abilities of triazole appended metal complexes.

Abstract: New mononuclear transition metal complexes 1-12 bearing the bioactive triazole analogues were synthesized and characterized by elemental analysis and spectroscopic techniques. The interaction of calf thymus DNA (CT-DNA) with the synthesized compounds was studied at physiological pH by spectrophotometric, spectrofluorometric, cyclic voltammetry, and viscometric techniques. The entire DNA binding results suggested the intercalative mode of binding for the synthesized compounds. Interestingly, the binding strength of the complexes is found to be greater than that of the free ligands. Among the complexes explored, complex 5 reveals strong hypochromism and a slight red shift as compared to the other complexes highlighting its higher DNA binding propensity. The intrinsic binding constant values of the complexes compared to cisplatin reveal that all the complexes are greater in magnitude than that of cisplatin. Fluorescence titrations show that the Cu(II) complexes have the ability to displace DNA-bound ethidium bromide. Also, these compounds induce cleavage in pBR322 plasmid DNA as indicated in gel electrophoresis and exhibit excellent nuclease activity in the presence of H2O2. Moreover, the complexes were screened for in vitro antimicrobial activity along with free ligands and solvent control. The outcome is that the complexes possess good activity than the free ligands. These complexes may have further scope in developing them into antimicrobial drugs and DNA probes.