Agarose Gel Assay Research Articles

Copper(II) and silver(I)-1,10-phenanthroline-5,6-dione complexes interact with double-stranded DNA: further evidence of their apparent multi-modal activity towards Pseudomonas aeruginosa

Tackling microbial resistance requires continuous efforts for the development of new molecules with novel mechanisms of action and potent antimicrobial activity. Our group has previously identified metal-based compounds, [Ag(1,10-phenanthroline-5,6-dione)2]ClO4 (Ag-phendione) and [Cu(1,10-phenanthroline-5,6-dione)3](ClO4)2.4H2O (Cu-phendione), with efficient antimicrobial action against multidrug-resistant species. Herein, we investigated the ability of Ag-phendione and Cu-phendione to bind with double-stranded DNA using a combination of in silico and in vitro approaches. Molecular docking revealed that both phendione derivatives can interact with the DNA by hydrogen bonding, hydrophobic and electrostatic interactions. Cu-phendione exhibited the highest binding affinity to either major (− 7.9 kcal/mol) or minor (− 7.2 kcal/mol) DNA grooves. In vitro competitive quenching assays involving duplex DNA with Hoechst 33258 or ethidium bromide demonstrated that Ag-phendione and Cu-phendione preferentially bind DNA in the minor grooves. The competitive ethidium bromide displacement technique revealed Cu-phendione has a higher binding affinity to DNA (Kapp = 2.55 × 106 M−1) than Ag-phendione (Kapp = 2.79 × 105 M−1) and phendione (Kapp = 1.33 × 105 M−1). Cu-phendione induced topoisomerase I-mediated DNA relaxation of supercoiled plasmid DNA. Moreover, Cu-phendione was able to induce oxidative DNA injuries with the addition of free radical scavengers inhibiting DNA damage. Ag-phendione and Cu-phendione avidly displaced propidium iodide bound to DNA in permeabilized Pseudomonas aeruginosa cells in a dose-dependent manner as judged by flow cytometry. The treatment of P. aeruginosa with bactericidal concentrations of Cu-phendione (15 µM) induced DNA fragmentation as visualized by either agarose gel or TUNEL assays. Altogether, these results highlight a possible novel DNA-targeted mechanism by which phendione-containing complexes, in part, elicit toxicity toward the multidrug-resistant pathogen P. aeruginosa.Graphical abstract

Polyamide Amine Dendrimer Nanoparticles Conjugated with Cis-Diamminedichloroplatinum (CDDP) and miRNA-200c Inhibitor Suppresses Lung Cancer Cell Proliferation

Our study elucidates the effect of folate polyamide amine dendrimer nanoparticles targeting delivery of miRNA-200c inhibitor and CDDP on lung cancer cells proliferation. We established polyamide amine dendrimer nanoparticles binding with CDDP and miRNA-200c inhibitor (Den-PEI-CDDP-siRNA-FA), TEM was employed to detect the morphology of nanoparticles. Agarose gel assay was selected for stabilization test. Cell proliferation were detected by trypanosoma blue exclusion method. The expression of miRNA-200c targeted APKPA12 and apoptosis-related proteins were detected by Western blot and PCR. Finally, apoptosis was analyzed by flow cytometry. Den-PEI-CDDP-siRNA-FA nanoparticles showed excellent stability and drug encapsulation ability. Nanoparticles targeting for FRA to co-deliver siRNA and CDDP could significantly promote cell apoptosis, increase apoptosis-related protein expression, and inhibit cell proliferation. Besides, nanoparticles exerted less venomous effect than untargeted nanoparticles in MRC9 lung fibroblast. Den nanoparticle targeting FRA might be used as the carrier for joint applications with siRNA and CDDP for treating lung cancer.

The absence of specific yeast heat-shock proteins leads to abnormal aggregation and compromised autophagic clearance of mutant Huntingtin proteins.

The functionality of a protein depends on its correct folding, but newly synthesized proteins are susceptible to aberrant folding and aggregation. Heat shock proteins (HSPs) function as molecular chaperones that aid in protein folding and the degradation of misfolded proteins. Trinucleotide (CAG) repeat expansion in the Huntingtin gene (HTT) results in the expression of misfolded Huntingtin protein (Htt), which contributes to the development of Huntington’s disease. We previously found that the degradation of mutated Htt with polyQ expansion (Htt103QP) depends on both ubiquitin proteasome system and autophagy. However, the role of heat shock proteins in the clearance of mutated Htt remains poorly understood. Here, we report that cytosolic Hsp70 (Ssa family), its nucleotide exchange factors (Sse1 and Fes1), and a Hsp40 co-chaperone (Ydj1) are required for inclusion body formation of Htt103QP proteins and their clearance via autophagy. Extended induction of Htt103QP-GFP leads to the formation of a single inclusion body in wild-type yeast cells, but mutant cells lacking these HSPs exhibit increased number of Htt103QP aggregates. Most notably, we detected more aggregated forms of Htt103QP in sse1Δ mutant cells using an agarose gel assay. Increased protein aggregates are also observed in these HSP mutants even in the absence Htt103QP overexpression. Importantly, these HSPs are required for autophagy-mediated Htt103QP clearance, but are less critical for proteasome-dependent degradation. These findings suggest a chaperone network that facilitates inclusion body formation of misfolded proteins and the subsequent autophagic clearance.

DNA–EB in agarose gel assay: a simple methodology in the search for DNA-binders in crude extracts from actinomycetes

Here, a methodology was proposed to detect DNA-binders in a screening process with crude extracts produced by actinomycetes, positive extracts were subjected to replication studies by LC-MS/MS.

Photoinduced DNA damage and cytotoxicity by a triphenylamine-modified platinum-diimine complex

Many planar photosensitizers tend to self-aggregate via van der Waals interactions between π-conjugated systems. The self-aggregation of the photosensitizer may reduce the efficiency of the photosensitizer to generate singlet oxygen, thereby diminishing its photodynamic activity. Efforts have been made to improve the photodynamic activity of bis-(o-diiminobenzosemiquinonato)platinum(II) which has planar geometry by the introduction of the sterically hindered triphenylamine moiety into the ligand. Herein we report the photoinduced DNA damage and cytotoxicity by a triphenylamine-modified platinum-diimine complex in red light studied by fluorescence spectra, agarose gel assay and cell viability assay. The results suggest that the triphenylamine-modified platinum-diimine complex has better capability to generate singlet oxygen than bis-(o-diiminobenzosemiquinonato)platinum(II), and it can induce DNA damage in red light, causing high photocytotoxicity in HepG-2 cells in vitro.

Comparison of chitosan, alginate and chitosan/alginate nanoparticles with respect to their size, stability, toxicity and transfection

ObjectiveTo to compare the chitosan/alginate, chitosan and alginate nanoparticles as plasmid vectors, to determine the morphological characteristics, size and physicochemical properties of nanoparticle-pEGFP complexes and to evaluate the potential of these nanoparticles in transfection of pEGFP plasmid in to a cultured the human embryonic kidney cell line (HEK 293 cells). MethodsNanoparticles comprising chitosan, alginate and both chitosan-alginate polymers were formed through pregel preparation method. The ability of plasmid-complexes in preventing DNA migration were assessed by the agarose gel assay. The efficiency of nanoparticles in transfection of pEGFP plasmid in the cultured HEK 293 cells was measured by flow cytometry. The effect of the nanoparticle-plasmid complexes on the cell viability was determined using cytotoxicity assay. ResutlsChitosan, alginate and alginate/chitosan nanoparticles had a mean Z-average diameter of 620 nm, 235.8 nm and 161.8 nm and mean zeta potential of 45 mV, −18.6 mV and 29.3 mV, respectively. Chitosan and chitosan/alginate nanoparticles have greater capacity to maintain plasmid than alginate nanoparticles. Alginate nanoparticles had the greater transfection in comparison to the others. Cell viability assays indicated that nanoparticles had no toxic effect on HEK 293 cells after 4 h or 24 h. ConclusionsThe combination of particle surface, hydrophobicity size and zeta potential can influence on transfection efficiency and the cellular uptake of the nanoparticles. Our suitable candidate for gene delivery would be alginate/chitosan nanoparticles.

Absolute Determination of Single-Stranded and Self-Complementary Adeno-Associated Viral Vector Genome Titers by Droplet Digital PCR

Accurate titration of adeno-associated viral (AAV) vector genome copies is critical for ensuring correct and reproducible dosing in both preclinical and clinical settings. Quantitative PCR (qPCR) is the current method of choice for titrating AAV genomes because of the simplicity, accuracy, and robustness of the assay. However, issues with qPCR-based determination of self-complementary AAV vector genome titers, due to primer-probe exclusion through genome self-annealing or through packaging of prematurely terminated defective interfering (DI) genomes, have been reported. Alternative qPCR, gel-based, or Southern blotting titering methods have been designed to overcome these issues but may represent a backward step from standard qPCR methods in terms of simplicity, robustness, and precision. Droplet digital PCR (ddPCR) is a new PCR technique that directly quantifies DNA copies with an unparalleled degree of precision and without the need for a standard curve or for a high degree of amplification efficiency; all properties that lend themselves to the accurate quantification of both single-stranded and self-complementary AAV genomes. Here we compare a ddPCR-based AAV genome titer assay with a standard and an optimized qPCR assay for the titration of both single-stranded and self-complementary AAV genomes. We demonstrate absolute quantification of single-stranded AAV vector genomes by ddPCR with up to 4-fold increases in titer over a standard qPCR titration but with equivalent readout to an optimized qPCR assay. In the case of self-complementary vectors, ddPCR titers were on average 5-, 1.9-, and 2.3-fold higher than those determined by standard qPCR, optimized qPCR, and agarose gel assays, respectively. Droplet digital PCR-based genome titering was superior to qPCR in terms of both intra- and interassay precision and is more resistant to PCR inhibitors, a desirable feature for in-process monitoring of early-stage vector production and for vector genome biodistribution analysis in inhibitory tissues.

Chemotactic Response of Escherichia Coli to Repellents, CoCl2 and NiCl2

Negative chemotaxis refers to the motion of microorganisms away from harmful chemicals. Soft agarose gel assay has been traditionally used to characterize the response to various repellents. In this study, we use the “chemical-in-plug” method to quantify the motion of Escherichia coli in the presence of repellents, NiCl2 and CoCl2, over a broad range of concentrations. These experiments were complimented with drift velocity measurements of individual bacteria in controlled gradients using a capillary assay. The latter also revealed the tumbling frequency and steady state clockwise bias for varying concentrations of repellents thereby providing insight into adaptation to repellents. The experimental technique yielded the motion of the bacteria in space and time and further related the motion to the evolving concentration profile of the repellent. Results show that the bacteria exhibit logarithmic sensing to the repellents, i.e., the drift velocity of E.coli is proportional to the logarithmic concentration gradient suggesting Weber law. The predictions of a standard population based model agreed with the observed linear behavior when the binding of the repellent to the receptor was sub-sensitive. This was borne out by a low value of Hill coefficient (n<<1) used to describe the binding characteristics of the receptors. The analysis shows that the binding characteristics for the repellents was sub-sensitive in contrast to an ultra-sensitive response observed for attractants suggesting a negative cooperative behavior of receptors. The above experiments suggest that negative cooperativity allows the cells to respond to harmful chemicals without saturation even at high concentration.

New chitosan nanobubbles for ultrasound-mediated gene delivery: preparation and in vitro characterization.

BackgroundThe development of nonviral gene delivery systems is one of the most intriguing topics in nanomedicine. However, despite the advances made in recent years, several key issues remain unsettled. One of the main problems relates to the difficulty in designing nanodevices for targeted delivery of genes and other drugs to specific anatomic sites. In this study, we describe the development of a novel chitosan nanobubble-based gene delivery system for ultrasound-triggered release.Methods and resultsChitosan was selected for the nanobubble shell because of its low toxicity, low immunogenicity, and excellent biocompatibility, while the core consisted of perfluoropentane. DNA-loaded chitosan nanobubbles were formed with a mean diameter of less than 300 nm and a positive surface charge. Transmission electron microscopic analysis confirmed composition of the core-shell structure. The ability of the chitosan nanobubbles to complex with and protect DNA was confirmed by agarose gel assay. Chitosan nanobubbles were found to be stable following insonation (2.5 MHz) for up to 3 minutes at 37°C. DNA release was evaluated in vitro in both the presence and absence of ultrasound. The release of chitosan nanobubble-bound plasmid DNA occurred after just one minute of insonation. In vitro transfection experiments were performed by exposing adherent COS7 cells to ultrasound in the presence of different concentrations of plasmid DNA-loaded nanobubbles. In the absence of ultrasound, nanobubbles failed to trigger transfection at all concentrations tested. In contrast, 30 seconds of ultrasound promoted a moderate degree of transfection. Cell viability experiments demonstrated that neither ultrasound nor the nanobubbles affected cell viability under these experimental conditions.ConclusionBased on these results, chitosan nanobubbles have the potential to be promising tools for ultrasound-mediated DNA delivery.

Comparative inhibitory activity of 3′- and 5′-functionalized nucleosides on ribonuclease A

Modified nucleosides, molecules, functionalized with various polar groups at different positions have been synthesized to rationalize the impact of structural modification on their inhibitory activity. Agarose gel and precipitation assays indicate their improved inhibitory activity on ribonuclease A (RNase A). Kinetic experiments clearly categorize them as competitive inhibitors of RNase A with improved inhibition constant ( K i) values (37 ± 9, 67 ± 6, and 193±7 μM for compounds 10, 3, and 7, respectively). The preferential hydrogen bonding network formation between His-12 and His-119 of RNase A with the polar carboxylic and amino groups of these compounds has been evidenced from the docking studies. The relationship between structural modifications and inhibitory activity of these compounds is further justified in terms of energetics using PEARLS.

Establishment and Application of Multiplex-PCR for Wheat Glutenin Subunits Relative to Superior End-Use Quality

Wheat processing qualities are largely dependent on the number and composition of high-molecular-weight glutenin subunits (HMW-GS). It has been demonstrated that Ax1/Ax2*, Dx5, and overexpressed Bx7 (Bx7OE) are normally associated with superior end-use quality, especially dough strength. To date, Bx7OE allele has not been exploited widely in China wheat breeding program. With the optimization of reaction components and amplification condition, three pairs of PCR primers targeting for Ax1/Ax2*, Bx7OE, and Dx5 genes were used to establish a multiplex PCR for the purpose of enhanced molecular marker-assisted breeding with good reliability and low cost. The validation with wheat cultivars (lines) carrying known genes displayed that the developed multiplex PCR permitted the discrimination of these major HMW-GS in a single PCR reaction and agarose gel assay. A total of 89 wheat cultivars and advanced lines from Tibet, China were tested by the multiplex PCR-based assay. The results showed that the frequencies of Ax1/Ax2* and Dx5 were both 12.4%, and only 10.1% of the accessions possessed both Ax1/Ax2* and Dx5, whereas no accessions carried Bx7OE. Introduction of HMW-GS relative to good gluten quality should enhance the improvement of wheat quality in this region.

A novel nonviral gene delivery vector: Low‐molecular‐weight polyethylenimine‐graft‐ovalbumin

AbstractA novel vector for gene delivery was synthesized. Here the ovalbumin (OVA) acts as a core and low‐molecular‐weight PEI600 was grafted to its surface. The finally product was characterized (1H‐NMR, UV, and TGA) and its biophysical properties such as DNA condensing, particle size, and zeta potential were determined. The agarose gel assay indicated that OVA‐PEI600 could efficiently condense plasmid DNA. Its particle size was about 150 nm and zeta potential was around +20 mV. The MTT assay showed that the cytotoxicity of OVA‐PEI600 was less than PEI25 kDa. Its transfection efficiency in SKOV‐3 and HepG2 cell lines was higher than that of PEI600 and comparable to PEI25 kDa. In vivo, luciferase activity could be tested in liver, spleen, kidney, lung, and blood serum, respectively, in mice. The core‐shell structure of OVA‐PEI600 provided a novel strategy for nonviral gene delivery. © 2009 Wiley Periodicals, Inc. J Appl Polym Sci, 2009

Inhibition of ribonuclease A by nucleoside–dibasic acid conjugates

We report the inhibition of the ribonucleolytic activity of ribonuclease A (RNase A) by nucleoside–dibasic acid conjugates for the first time. Agarose gel and precipitation assays show that the spacer length and the pK a of the carboxylic group have an important role in the inhibitory capacity. Kinetic experiments indicate a competitive mode of inhibition with inhibition constant ( K i) value of 132 ± 3 μM for Oxa- aT. Docking studies revealed that the carboxylic group of the most active compounds is within hydrogen bonding distance of His-12, Lys-41 and His-119.

Nucleoside–amino acid conjugates: An alternative route to the design of ribonuclease A inhibitors

Nucleoside–amino acid conjugates have been employed to inhibit the ribonucleolytic activity of ribonuclease A (RNase A) and affect the protonation/deprotonation equilibrium of its active site histidine residues. Agarose gel and precipitation assays indicate inhibition of RNase A activity by these molecules with a possible role of the polar side chains of the amino acids in RNase A inhibition. Kinetic experiments demonstrated that the mode of inhibition is competitive in nature with inhibition constants ( K i) in the micromolar range. The nucleoside–serine conjugate occupies the active site of RNase A and preferential perturbs the p K a value of His-119 by its ‘free amino group’ as found from 1H NMR studies. Docking studies revealed that the free amino groups of the most active compounds are within hydrogen bonding distance of His-119 in inhibitor–RNase A complexes.

Plasmid Loss in Plasmid-carrying Strains of Escherichia coli Treated with Phenoxazines and an Approach to Study Their DNA Binding Properties

The effect of subinhibitory concentrations of 2-trifluoromethyl-N10-substituted phenoxazines on plasmid-coded antibiotic resistance in Escherichia coli was investigated. Phenoxazine treatment resulted in the loss of resistance markers to an extent of 8-63% in all the strains tested, and the disappearance of plasmid DNA in phenoxazine sensitive colonies was evidenced by agarose gel electrophoresis. The resistant strains were sensitized in the presence of phenoxazines with a concomitant reduction in the MIC (minimum inhibitory concentration) values. The UV, fluorescence spectral, and ethidium bromide displacement agarose gel assay methods revealed that phenoxazines are intercalated with plasmid DNA. Progressive addition of DNA led to a significant reduction in the peak intensity of the absorption maximum of phenoxazine derivative. Further, destabilization of ethidium bromide-DNA complex as seen from fluorescence microscopy in the presence of phenoxazines was observed. The potency of phenoxazines to sensitize the resistant organisms follows the order butyl > propyl > acetyl derivatives.

The rice pathogen-related protein 10 (JIOsPR10) is induced by abiotic and biotic stresses and exhibits ribonuclease activity

We previously reported that rice blast fungus or jasmonic acid induced the expression of rice pathogenesis-related class 10 (JIOsPR10) proteins (Kim et al. 2003, 2004). However, no further studies have been carried out to examine the expression, localization, and enzymatic activity of this protein in either developmental tissues or in tissues under abiotic stress conditions. In this study, rice JIOsPR10 was examined by Western blot analysis, immunolocalization, and biochemical assays. Western blots revealed that the JIOsPR10 protein was expressed in developmental tissues, including in flower and root. The protein was also expressed under abiotic stresses, such as occurs during senescence and wounding. Using immunohistochemical techniques, we determined that expression of JIOsPR10 was localized to the palea of flower, in the exodermis, and inner part of the endodermis of the root. In senescencing tissues of leaf and coleoptiles, its expression was localized in vascular bundles. The RNase activity using JIOsPR10 recombinant protein was determined and abolished after treatment with DTT in a native in-gel assay. To test this, we created JIOsPR10 mutant proteins containing serine substitutions of amino acids C81S, C83S, or both and examined their RNase activities. The activity of the C83S mutant was decreased in the agarose gel assay compared to the wild type. Taken together, we hypothesize that the JIOsPR10 protein possesses RNase activity that is sensitive to DTT, suggesting the importance of the disulfide bonding between cysteine residues and that it might play a role in constitutive self-defense mechanisms in plants against biotic and abiotic stresses.

Improved Agarose Gel Assay for Quantification of Growth Factor-Induced Cell Motility

Cell chemotaxis is frequently required in normal or pathological situations such as invasion, metastasis, and tumor angiogenesis and may involve many different cell types. At present, no device can simultaneously (i) make morphological observations, (ii) quantify cell migration, (iii) test multiple chemoattracting gradients, and (iv) analyze cell-cell interactions. We developed an agarose-based assay to address these questions. Two glass molds were designed, around which agarose gel could be poured to form specific well shapes. Using a vital nuclear stain (Hoechst 33258), we characterized the migration profile of adherent or suspension cells. Cells could be observed during the entire migration process. We were able to follow cells moving toward chemoattractants or being repulsed by other molecules, and we could estimate average migration speed. Using this inexpensive assay, we were able to obtain precise, reproducible results concerning the chemotactic behavior of different cell types. The resulting data differentiated between chemokinetic and chemotactic movement. Chemotactic potencies could be compared using different criteria, such as the number of attracted cells, induced speed, and morphological aspect. This improved agarose assay appears to be a reliable and inexpensive alternative to other available chemotaxis study tools.

Novel Bioreducible Poly(amido amine)s for Highly Efficient Gene Delivery

A series of novel bioreducible poly(amido amine)s containing multiple disulfide linkages (SS-PAAs) were synthesized and evaluated as nonviral gene vectors. These linear SS-PAAs could be easily obtained by Michael-type polyaddition of various primary amines to the disulfide-containing cystamine bisacrylamide. The SS-PAA polymers are relatively stable in medium mimicking physiological conditions (pH 7.4, 150 mM PBS, 37 degrees C), but are rapidly degraded in the presence of 2.5 mM DTT, mimicking the intracellular reductive environment (pH 7.4, [R-SH] = 5 mM, 37 degrees C). The polymers efficiently condense DNA into nanoscaled (<200 nm) and positively charged (>+20 mV) polyplexes that are stable under neutral conditions but are rapidly destabilized in a reductive environment, as was revealed by both dynamic light scatting measurement and agarose gel assays. Moreover, most of the poly(amido amine)s possess buffer capacities in the pH range pH 7.4-5.1 that are even higher than polyethylenimine (pEI), a property that may favorably contribute to the endosomal escape of the polyplexes. Polyplexes of four of the seven SS-PAAs studied were able to transfect COS-7 cells in vitro with transfection efficiencies significantly higher than those of branched pEI, being one of the most effective polymeric gene carriers reported to date. Importantly, also in the presence of serum, a high level of gene expression could be observed when the incubation time was elongated from 1 h to 4 h. XTT assays showed that SS-PAAs and their polyplexes possess essentially no or only very low cytotoxicity at concentrations where the highest transfection activity is observed. The results indicate that bioreducible poly(amido amine)s have excellent properties for the development of highly potent and nontoxic polymeric gene carriers.

Cell death during preoviposition period in Boophilus microplus tick

Programmed cell death (PCD) is present during the development of multicellular organisms and occurs from embryogenesis to death. In females of Boophilus microplus, the mass of several organs is reduced after the detachment from the host. In order to better characterize the cell death process that eliminates unnecessary tissues, the degeneration of salivary glands, ovaries and synganglia was investigated using DNA fragmentation in agarose gel, comet and TUNEL assays, and apoptosis activation pathway by the caspase assay. DNA fragmentation and enzymatic activity of caspase-3 were observed in salivary glands and ovaries at 48 and 72 h after tick removal from the host; in synganglia these parameters were maintained at low levels upon 48 h. The results obtained suggest that there is a refined control of tissue maintenance through apoptosis.

Quantitative real-time polymerase chain reaction for determination of plasmid copy number in bacteria

A method for determination of plasmid copy number (PCN) in bacteria by real-time quantitative polymerase chain reaction (QPCR) was developed as an alternative to current PCN assays. Conventional methods for PCN estimation are generally not of high throughput, laborious, have low reproducibility, require large amounts of biological samples and are applicable only for a narrow dynamic range. Real-time QPCR, using the ABI Prism® 7000, was able to sensitively detect the quantity of the pUC ori based plasmid, NS3, transformed into Escherichia coli host, DH5α, to be 411 ± 6.1. The PCN of pBR322 plasmid DNA in DH5α was estimated to be 40 ± 0.6 which is within its previously reported PCN range of approximately 30 to 70. QPCR was found to show good reproducibility and high sensitivity in detecting a two fold difference in template concentration, and a wide linear dynamic range covering 0.5 pg to 50 ng of DNA. PCNs of DH5α bearing plasmids pBR322 and NS3 computed from real-time QPCR assay were validated by that of agarose gel assay, and a marginal difference of only 13.0% and 10.7% was found for the two plasmids respectively. The QPCR assay was able to detect changes in PCN of plasmid producing DH5α during the course of a 2 l batch fermentation.