Showing papers on "Gel electrophoresis published in 2015"
[...]
TL;DR: This chapter will outline the requisite steps including gel electrophoresis of a protein sample, transfer of protein from a gel to a membrane support, and immunodetection of a target antigen.
Abstract: Western blotting is a technique that involves the separation of proteins by gel electrophoresis, their blotting or transfer to a membrane, and selective immunodetection of an immobilized antigen. This is an important and routine method for protein analysis that depends on the specificity of antibody-antigen interaction and is useful for the qualitative or semiquantitative identification of specific proteins and their molecular weight from a complex mixture. This chapter will outline the requisite steps including gel electrophoresis of a protein sample, transfer of protein from a gel to a membrane support, and immunodetection of a target antigen.
121 citations
TL;DR: Gel electrophoresis indicated that the ALB has less disulfide bonds and hence a more open (flexible) structure; this was confirmed by the intrinsic fluorescence and circular dichroism data showing greater exposure of tyrosine residues when compared to GLB.
117 citations
TL;DR: Airborne MS2 bacteriophages exposed to atmospheric-pressure cold plasma produced using different power levels and gas carriers showed various levels of damage to both surface proteins and their related RNA genes after the APCP exposure, thus leading to the loss of their viability and infectivity.
Abstract: In this study, airborne MS2 bacteriophages were exposed for subsecond time intervals to atmospheric-pressure cold plasma (APCP) produced using different power levels (20, 24, and 28 W) and gas carriers (ambient air, Ar-O2 [2%, vol/vol], and He-O2 [2%, vol/vol]). In addition, waterborne MS2 viruses were directly subjected to the APCP treatment for up to 3 min. MS2 viruses with and without the APCP exposure were examined by scanning electron microscopy (SEM), reverse transcription-PCR (RT-PCR), and sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). Viral inactivation was shown to exhibit linear relationships with the APCP generation power and exposure time (R(2) > 0.95 for all energy levels tested) up to 95% inactivation (1.3-log reduction) after a subsecond airborne exposure at 28 W; about the same inactivation level was achieved for waterborne viruses with an exposure time of less than 1 min. A larger amount of reactive oxygen species (ROS), such as atomic oxygen, in APCP was detected for a higher generation power with Ar-O2 and He-O2 gas carriers. SEM images, SDS-PAGE, and agarose gel analysis of exposed waterborne viruses showed various levels of damage to both surface proteins and their related RNA genes after the APCP exposure, thus leading to the loss of their viability and infectivity.
100 citations
TL;DR: The results indicated the reliability of Nb-AP fusion protein-based dc-FEIA for monitoring OTA contamination in cereal.
Abstract: A rapid and sensitive direct competitive fluorescence enzyme immunoassay (dc-FEIA) for ochratoxin A (OTA) based on a nanobody (Nb)–alkaline phosphatase (AP) fusion protein was developed. The VHH (variable domain of heavy chain antibody) gene of Nb28 was subcloned into the expression vector pecan45 containing the AP double-mutant gene. The Nb28–AP construct was transformed into Escherichia coli BL21(DE3)plysS, and soluble expression in bacteria was confirmed by sodium dodecyl sulfate–polyacrylamide gel electrophoresis and Western blot. Both the Nb properties and AP enzymatic activity were validated by colorimetric and fluorometric analysis. The 50% inhibitory concentration and the detection limit of the dc-FEIA were 0.13 and 0.04 ng/mL, respectively, with a linear range of 0.06–0.43 ng/mL. This assay was compared with LC–MS/MS, and the results indicated the reliability of Nb–AP fusion protein-based dc-FEIA for monitoring OTA contamination in cereal.
78 citations
TL;DR: The INB method has high specificity, sensitivity, and quantitative capability, and it can be employed with conventional experimental apparatus, therefore, this method would be useful for research on RNA modifications and metabolism.
Abstract: The biological roles of RNA modifications are still largely not understood. Thus, developing a method for detecting RNA modifications is important for further clarification. We developed a method for detecting RNA modifications called immuno-northern blotting (INB) analysis and herein introduce its various capabilities. This method involves the separation of RNAs using either polyacrylamide or agarose gel electrophoresis, followed by transfer onto a nylon membrane and subsequent immunoblotting using antibodies against modified nucleosides for the detection of specific modifications. We confirmed that INB with the antibodies for 1-methyladenosine (m1A), N6-methyladenosine (m6A), pseudouridine, and 5-methylcytidine (m5C) showed different modifications in a variety of RNAs from various species and organelles. INB with the anti-m5C antibody revealed that the antibody cross-reacted with another modification on DNA, suggesting the application of this method for characterization of the antibody for modified nucleosides. Additionally, using INB with the antibody for m1A, which is a highly specific modification in eukaryotic tRNA, we detected tRNA-derived fragments known as tiRNAs under the cellular stress response, suggesting the application for tracking target RNA containing specific modifications. INB with the anti-m6A antibody confirmed the demethylation of m6A by the specific demethylases fat mass and obesity-associated protein (FTO) and ALKBH5, suggesting its application for quantifying target modifications in separated RNAs. Furthermore, INB demonstrated that the knockdown of FTO and ALKBH5 increased the m6A modification in small RNAs as well as in mRNA. The INB method has high specificity, sensitivity, and quantitative capability, and it can be employed with conventional experimental apparatus. Therefore, this method would be useful for research on RNA modifications and metabolism.
74 citations
TL;DR: The role of capillary electrophoresis in the discovery, design, and characterization of DNA aptamers for molecular recognition, and expanding and emerging techniques in the field are highlighted.
Abstract: This review of capillary electrophoresis methods for DNA analyses covers critical advances from 2009 to 2014, referencing 184 citations. Separation mechanisms based on free-zone capillary electrophoresis, Ogston sieving, and reptation are described. Two prevalent gel matrices for gel-facilitated sieving, which are linear polyacrylamide and polydimethylacrylamide, are compared in terms of performance, cost, viscosity, and passivation of electroosmotic flow. The role of capillary electrophoresis in the discovery, design, and characterization of DNA aptamers for molecular recognition is discussed. Expanding and emerging techniques in the field are also highlighted.
59 citations
TL;DR: Optimized conditions for coating CPs onto the surfaces of SPIONs, forming CPIOs, for magneto-gene delivery systems resulted in the formation of a stable particle as a good transgene carrier as well as chemically-conjugated chondroitin sulfate-PEI copolymers (CPs).
Abstract: MicroRNA-128 (miR-128) is an attractive therapeutic molecule with powerful glioblastoma regulation properties. However, miR-128 lacks biological stability and leads to poor delivery efficacy in clinical applications. In our previous study, we demonstrated two effective transgene carriers, including polyethylenimine (PEI)-decorated superparamagnetic iron oxide nanoparticles (SPIONs) as well as chemically-conjugated chondroitin sulfate-PEI copolymers (CPs). In this contribution, we report optimized conditions for coating CPs onto the surfaces of SPIONs, forming CPIOs, for magneto-gene delivery systems. The optimized weight ratio of the CPs and SPIONs is 2:1, which resulted in the formation of a stable particle as a good transgene carrier. The hydrodynamic diameter of the CPIOs is ∼136 nm. The gel electrophoresis results demonstrate that the weight ratio of CPIO/DNA required to completely encapsulate pDNA is ≥3. The in vitro tests of CPIO/DNA were done in 293 T, CRL5802, and U87-MG cells in the presence and absence of an external magnetic field. The magnetofection efficiency of CPIO/DNA was measured in the three cell lines with or without fetal bovine serum (FBS). CPIO/DNA exhibited remarkably improved gene expression in the presence of the magnetic field and 10% FBS as compared with a gold non-viral standard, PEI/DNA, and a commercial magnetofection reagent, PolyMag/DNA. In addition, CPIO/DNA showed less cytotoxicity than PEI/DNA and PolyMag/DNA against the three cell lines. The transfection efficiency of the magnetoplex improved significantly with an assisted magnetic field. In miR-128 delivery, a microRNA plate array and fluorescence in situ hybridization were used to demonstrate that CPIO/pMIRNA-128 indeed expresses more miR-128 with the assisted magnetic field than without. In a biodistribution test, CPIO/Cy5-DNA showed higher accumulation at the tumor site where an external magnet is placed nearby.
57 citations
TL;DR: It was found that GelRed™ was the most sensitive and safest dye to use with UV light excitation, and both GelGreen and Diamond Nucleic Acid Dye were sensitive and the safer dyes using blue lightexcitation.
Abstract: Nucleic acid staining dyes are used for detecting nucleic acids in electrophoresis gels. Historically, the most common dye used for gel staining is ethidium bromide, however due to its toxicity and mutagenicity other dyes that are safer to the user and the environment are preferred. This Short Communication details the properties of dyes now available and their sensitivity for detection of DNA and their ability to permeate the cell membrane. It was found that GelRed™ was the most sensitive and safest dye to use with UV light excitation, and both GelGreen™ and Diamond™ Nucleic Acid Dye were sensitive and the safer dyes using blue light excitation.
57 citations
TL;DR: Metal complexes (1-3) exhibit a good binding propensity to bovine serum albumin (BSA) proteins having relatively high binding constant values and the cytotoxicity studies of complexes 1-3 were tested in vitro on breast cancer cell line (MCF-7) and they found to be active.
56 citations
TL;DR: The results demonstrate that, in addition to the well-recognized immune reaction to gluten, celiac disease is associated with a robust humoral response directed at a specific subset of the nongluten proteins of wheat.
Abstract: While the antigenic specificity and pathogenic relevance of immunologic reactivity to gluten in celiac disease have been extensively researched, the immune response to nongluten proteins of wheat has not been characterized. We aimed to investigate the level and molecular specificity of antibody response to wheat nongluten proteins in celiac disease. Serum samples from patients and controls were screened for IgG and IgA antibody reactivity to a nongluten protein extract from the wheat cultivar Triticum aestivum Butte 86. Antibodies were further analyzed for reactivity to specific nongluten proteins by two-dimensional gel electrophoresis and immunoblotting. Immunoreactive molecules were identified by tandem mass spectrometry. Compared with healthy controls, patients exhibited significantly higher levels of antibody reactivity to nongluten proteins. The main immunoreactive nongluten antibody target proteins were identified as serpins, purinins, α-amylase/protease inhibitors, globulins, and farinins. Assessme...
55 citations
TL;DR: It is remarkable that copper(bimda) complexes exhibit cytotoxicity against human breast cancer cell lines (MCF-7) with potency higher than the widely used drug cisplatin indicating that they have the potential to act as effective anticancer drugs in a time dependent manner.
Abstract: A few water soluble mixed ligand copper(II) complexes of the type [Cu(bimda)(diimine)] 1–5, where bimda is N-benzyliminodiacetic acid and diimine is 2,2′-bipyridine (bpy, 1) or 1,10-phenanthroline (phen, 2) or 5,6-dimethyl-1,10-phenanthroline (5,6-dmp, 3) or 3,4,7,8-tetramethyl-1,10-phenanthroline (3,4,7,8-tmp, 4) and dipyrido[3,2-d: 2′,3′-f]quinoxaline (dpq, 5), have been successfully isolated and characterized by elemental analysis and other spectral techniques. The coordination geometry around copper(II) in 2 is described as distorted square based pyramidal while that in 3 is described as square pyramidal. Absorption spectral titrations and competitive DNA binding studies reveal that the intrinsic DNA binding affinity of the complexes depends upon the diimine co-ligand, dpq (5) > 3,4,7,8-tmp (4) > 5,6-dmp (3) > phen (2) > bpy (1). The phen and dpq co-ligands are involved in the π-stacking interaction with DNA base pairs while the 3,4,7,8-tmp/5,6-dmp and bpy co-ligands are involved in respectively hydrophobic and surface mode of binding with DNA. The small enhancement in the relative viscosity of DNA upon binding to 1–5 supports the DNA binding modes proposed. Interestingly, 3 and 4 are selective in exhibiting a positive induced CD band (ICD) upon binding to DNA suggesting that they induce B to A conformational change. In contrast, 2 and 5 show CD responses which reveal their involvement in strong DNA binding. The complexes 2–4 are unique in displaying prominent double-strand DNA cleavage while 1 effects only single-strand DNA cleavage, and their ability to cleave DNA in the absence of an activator varies as 5 > 4 > 3 > 2 > 1. Also, all the complexes exhibit oxidative double-strand DNA cleavage activity in the presence of ascorbic acid, which varies as 5 > 4 > 3 > 2 > 1. 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 non-specifically in the presence of H2O2 as an activator suggesting that they can act also as chemical proteases. It is remarkable that 2–5 exhibit cytotoxicity against human breast cancer cell lines (MCF-7) with potency higher 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 other complexes. Also, the alkaline single-cell gel electrophoresis study (comet assay) suggests that the same complexes induce DNA fragmentation more efficiently than others.
TL;DR: The interaction of 1-4 with calf thymus DNA and bovine serum albumin was investigated using UV-visible and fluorescence spectrophotometric methods and a gel electrophoresis assay demonstrated that the complexes cleave pUC19 plasmid DNA.
TL;DR: This study showed the coproduction of two important carbapenemases (KPC-2 and NDM-1) associated with mobile genetic elements of worldwide epidemiological importance (Tn4401 and ISAba125, respectively), reinforcing the idea that urgent measures are necessary to reduce and prevent the spreading of these carbapanemases primarily in the hospital settings.
Abstract: The most important resistance mechanism against β-lactam drugs is the production of carbapenemases. In this study, we report the first identification of Klebsiella pneumoniae carbapenemase (KPC)-2 and New Delhi metallo-β-lactamase (NDM)-1 in Enterobacter hormaechei subps. oharae from Brazil. The detection of carbapenemases was done by phenotypic assays, PCR, and DNA sequencing, whereas the identification was performed by conventional techniques, sequencing of the 16S rDNA gene, and hsp60-genotyping. Molecular typing was performed using pulsed-field gel electrophoresis, and antimicrobial susceptibility was surrogated by the Etest methodology. Using the whole genome sequencing approach, we searched for resistance genes, plasmid incompatibility group genes, and the genetic environment of blaNDM and blaKPC. The plasmid identification was done by restriction digests with the S1 nuclease followed by hybridization using digoxigenin labeled specific probes. The isolate was considered multiresistant, being suscept...
TL;DR: In silico molecular docking studies not only validated the observed binding trend but also provided insight into the binding mode of the polyelectrolyte-DNA complex, which is dominated by electrostatic interactions.
TL;DR: The method was successfully applied for the identification of two complex I subunits that showed enhanced HNE-modifications in diabetic kidney mitochondria.
Abstract: The lipid peroxidation product 4-hydroxynonenal (HNE) can form protein-linked HNE adducts, thereby impacting protein structure and function. Mitochondrial complex I (NADH-ubiquinone oxidoreductase), containing at least 45 subunits in mammalian cells, sits in a lipid-rich environment and is thus very susceptible to HNE modifications. In this paper, a procedure for the identification of HNE-modified complex I subunits is described. Complex I was isolated by first dimensional non-gradient blue native polyacrylamide gel electrophoresis (BN-PAGE). The isolated complex I band, visualized by either Coomassie blue staining or silver staining, was further analyzed by second dimensional sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). HNE-modified proteins were visualized by Western blotting probed with anti-HNE antibodies. HNE-positive bands were then excised and the proteins contained in them were identified by mass spectrometric peptide sequencing. The method was successfully applied for the identification of two complex I subunits that showed enhanced HNE-modifications in diabetic kidney mitochondria.
TL;DR: The cow urinary proteome is profiled and functional significance of few selected proteins seems to play important role in their physiology, and for the first time evidence of more than 1550 proteins in urine of healthy cow donors is reported.
TL;DR: The findings demonstrated that 'particle- specific' effect, not 'ion-specific' one, was responsible for the NP action, and may be used as a sterilizing agent in various bioprocesses and as substituent of antibiotics, after thorough toxicological study.
TL;DR: Data highlight that aging affects the meat tenderness and proteolysis with different intensities in each muscle and could be useful for the development of muscle specific strategies for improving the quality and value in different commercial cuts.
Abstract: The aim of this study was to understand the relationship between changes in postmortem degradation of myofibrillar proteins and tenderness development in 3 different muscles from 10 Podolian young bulls aged 1, 7, 14, and 21 d. Psoas major (PM), longissimus dorsi (LD), and semitendinosus muscle () were removed from each half carcass 24 h postmortem. Meat chemical composition, Warner-Bratzler shear force (WBSF), myofibril fragmentation index (MFI), total collagen content, and changes in myofibrillar proteins were estimated in triplicate at each aging time. A significant muscle effect was found on meat chemical composition. Semitendinosus muscle showed the lowest intramuscular fat percentage ( < 0.01) and the highest total collagen content ( < 0.01) with respect to LD and PM. Warner-Bratzler shear force decreased during aging in all muscles ( < 0.001), and semitendinosus was the toughest muscle whereas PM was the most tender ( < 0.001). Myofibril fragmentation index significantly increased ( < 0.001) in LD and PM meat throughout aging time whereas in semitendinosus it increased from 14 d of aging. Proteolysis was investigated by SDS-PAGE, western blotting, and 2-dimensional electrophoresis. Throughout postmortem aging, some structural proteins changed in intensity in all muscles analyzed. The blotting profile highlighted that desmin and troponin-T (TnT) bands were affected by both muscle and aging effects. Desmin degradation was more intense and faster in LD muscle than in semitendinosus and PM muscles. A progressive increase of the degraded isoforms of TnT (33 and 30 kDa polypeptides) was found during aging in LD, while, in PM these bands appeared earlier showing a greater intensity from 1 d. During aging, 2-dimensional gel electrophoresis (2DE) image analyses results showed a significant increase of the total number of spots reaching the highest value in the LD muscle at 21 d of aging ( < 0.01). Proteins separation also revealed differences in the spot number and expression patterns of myosin light chain (MLC) isoforms among muscles. A principal components analysis applied to meat chemical composition, tenderness, and myofibrillar proteins accounted for approximately 96.09% of total variance. Data highlight that aging affects the meat tenderness and proteolysis with different intensities in each muscle. These results provide knowledge about the tenderness mechanism in different muscles from a rustic breed and could be useful for the development of muscle specific strategies for improving the quality and value in different commercial cuts.
TL;DR: This work screened seed from 520 soybean accessions using a combination of sodium dodecyl sulfate-polyacrylamide gel electrophoresis and immunoblots with anti-Kunitz trypsin inhibitor antibodies and observed significant proteome rebalancing in all KTi mutant lines, resulting in dramatically increased BBI protein levels.
Abstract: Soybean seed contains antinutritional compounds that inactivate digestive proteases, principally corresponding to two families: Kunitz trypsin inhibitors (KTi) and Bowman-Birk inhibitors (BBI). High levels of raw soybean/soybean meal in feed mixtures can cause poor weight gain and pancreatic abnormalities via inactivation of trypsin/chymotrypsin enzymes. Soybean protein meal is routinely heat-treated to inactivate inhibitors, a practice that is energy-intensive and costly and can degrade certain essential amino acids. In this work, we screened seed from 520 soybean accessions, using a combination of sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and immunoblots with anti-Kunitz trypsin inhibitor antibodies. A soybean germplasm accession was identified with a mutation affecting an isoform annotated as nonfunctional (KTi1), which was determined to be synergistic with a previously identified mutation (KTi3-). We observed significant proteome rebalancing in all KTi mutant lines, resulting in dramatically increased BBI protein levels.
TL;DR: The glycoprotein and sulphated proteoglycan-like substance are assumed to contribute to anaerobic granule strength, thanks to their ability to perform interactions of various nature (ionic, hydrophobic, Ca2+ as divalent cation bridging, etc.).
Abstract: The protein fraction of extracellular polymeric substance (EPS) from two anaerobic granular sludge samples was characterized with sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE) and a far western blotting method SDS-PAGE was used with various staining applications to obtain a protein (silver), glycoprotein [periodic acid–Shiff's (PAS)] or proteoglycan-like (Alcian blue at pH 25 (carboxylic group) or 1 (sulphated group)) fingerprint The fingerprints of the EPS denatured protein from the two sludge samples differed Some proteins are specific to Soluble (S) or Bound (B)-EPS (20–100 kDa) Denatured proteins with a polysaccharide moieties characterization are more present in B-EPS Glycoproteins with α-d-mannosyl and/or α-d-glucosyl (90, 50, 40 kDa) were detected Proteoglycan-like and sulphated proteoglycan-like substances are also detected, mainly in B-EPS A 68 kDa sulphated proteoglycan-like substance contains two glucidic residue types: α-d-mannosyl and/or α-d-glucosyl and N-acet
TL;DR: This protocol describes the preparation of an agarose gel with formaldehyde and its setup in a horizontal electrophoresis apparatus, and the stained gel is stained to visualize RNA markers or rRNA using one of several different types of stains.
Abstract: Electrophoretic size fractionation can be used to denature and separate large mRNA molecules (0.5-10 kb) on formaldehyde-containing agarose gels. Formaldehyde contains a carbonyl group that reacts to form Schiff bases with the imino or amino groups of guanine, adenine, and cytosine. These covalent adducts prevent normal base pairing and maintain the RNA in a denatured state. Because these adducts are unstable, formaldehyde must be present in the gel to maintain the RNA in the denatured state. This protocol describes the preparation of an agarose gel with formaldehyde and its setup in a horizontal electrophoresis apparatus. RNA samples are prepared and denatured in a solution of formamide and formaldehyde and, with 0.5- to 10-kb size markers, subjected to electrophoresis through the gel. Following electrophoresis, the gel is stained to visualize RNA markers or rRNA using one of several different types of stains.
TL;DR: This work was focused on a study of the DNA binding and cleavage properties of lomefloxacin and its ternary transition metal complexes with glycine and revealed that LMF and its complexes could interact with CT-DNA via partial/moderate intercalative mode.
TL;DR: The interaction of two copper(II) complexes with DNA has been thoroughly investigated using different characterization techniques, including electronic absorption spectroscopy, viscosity measurements, fluorescence spectrograms, circular dichroism spectrograph, thermal denaturation, stoichiometric determination, gel electrophoresis and atomic-force microscopy as discussed by the authors.
Abstract: The interaction of two copper(II) complexes, namely [Cu(L1)Cl2]2 (1) and [Cu(L2)Cl2]2 (2), where L1 = 1-amidino-O-methylurea and L2 = N-(benzyl)-amidino-O-methylurea, with DNA has been thoroughly investigated using different characterization techniques, including electronic absorption spectroscopy, viscosity measurements, fluorescence spectroscopy, circular dichroism spectroscopy, thermal denaturation, stoichiometric determination, gel electrophoresis and atomic-force microscopy. The coordination compounds exhibit DNA binding potential by non-intercalation and DNA-cleaving ability through the oxidative pathway. Indeed, both complexes display antibacterial properties (against three bacteria involved in human-food poisoning, i.e. Salmonella, E. coli and Campylobacter). Furthermore, their cytotoxicity has been tested against three cancer cell lines, which are the small cell lung carcinoma (NCI-H187), the oral cavity carcinoma (KB) and the breast adenocarcinoma (MCF-7) and it was revealed that they are more cytotoxic than cisplatin against the NCI-H187 cancer cell line.
TL;DR: LPS and A-LPS-modified proteins in OMVs carry immunodominant determinants and eventually elicit P. gingivalis-specific antibodies in mice, leading to strong immunogenicity of OMVs using an intranasal vaccine mouse model.
Abstract: Outer membrane vesicles (OMVs) of the periodontopathic bacterium Porphyromonas gingivalis contain a wide range of virulence factors including lipopolysaccharide (LPS), fimbriae and gingipains. We have recently reported strong immunogenicity of OMVs using an intranasal vaccine mouse model. In the present study, we performed sub-immunoproteome analysis of OMV-immunized mouse serum samples from six different mice in order to identify immunodominant antigens. The combination of two-dimensional (2D) gel electrophoresis and mass spectrometry analysis identified OMV proteins of 53 spots on a 2D map, and it was notable that OMV proteins were largely distributed within a low pH range, in marked contrast to the ubiquitous distribution of outer membrane proteins. Western blot using the six serum samples after 2D electrophoresis revealed that all showed immunoreactivity to some diffuse signals at extremely low pH, which was similar to the distribution of immunoreactive signals when the A-LPS antibody was used. Mass spectrometry analysis also demonstrated that the signals corresponded to a wide range of virulence factors including A-LPS-modified proteins such as gingipains. Absorption of serum with LPS resulted in a dramatic reduction of immmunoreactivity. We conclude that LPS and A-LPS-modified proteins in OMVs carry immunodominant determinants and eventually elicit P. gingivalis-specific antibodies in mice.
TL;DR: The application of different scavengers of reactive oxygen species provided a conclusion that DNA cleavage caused by copper complexes might occur via hydrolytic pathway.
Abstract: This work is focused on the study of DNA binding and cleavage properties of 2′-deoxyadenosines modified with ester/amide of histidine (his6dA ester, his6dA amide) and their copper(II) complexes. To determine the coordination mode of the complex species potentiometric and spectroscopic (UV–visible, CD, EPR) studies have been performed. The analysis of electronic absorption and fluorescence spectra has been used to find the nature of the interactions between the compounds and calf thymus DNA (CT-DNA). There is significant influence of the –NH2 and –OCH3 groups on binding of the ligands or the complexes to DNA. Only amide derivative and its complex reveal intercalative ability. In the case of his6dA ester and Cu(II)–his6dA ester the main interactions can be groove binding. DNA cleavage activities of the compounds have been examined by gel electrophoresis. The copper complexes have promoted the cleavage of plasmid DNA, but none of the ligands exhibited any chemical nuclease activity. The application of different scavengers of reactive oxygen species provided a conclusion that DNA cleavage caused by copper complexes might occur via hydrolytic pathway.
TL;DR: An endo-actingfucoidanase that degrades Miyeokgui fucoidan into smaller-sized galactofuco-oligosaccharides is purified from a marine bacterium by ammonium sulfate precipitation, diethylaminoethyl-Sepharose column chromatography, and chromatofocusing.
Abstract: The Search for enzyme activities that efficiently degrade marine polysaccharides is becoming an increasingly important area for both structural analysis and production of lower-molecular weight oligosaccharides. In this study, an endo-acting fucoidanase that degrades Miyeokgui fucoidan (MF), a sulfated galactofucan isolated from the sporophyll (called Miyeokgui in Korean) of Undaria pinnatifida, into smaller-sized galactofuco-oligosaccharides (1000–4000 Da) was purified from a marine bacterium, Sphingomonas paucimobilis PF-1, by ammonium sulfate precipitation, diethylaminoethyl (DEAE)-Sepharose column chromatography, and chromatofocusing. The specific activity of this enzyme was approximately 112-fold higher than that of the crude enzyme, and its molecular weight was approximately 130 kDa (FNase S), as determined by native gel electrophoresis and 130 (S1), 70 (S2) and 60 (S3) kDa by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). The optimum pH and temperature of FNase S were pH 6.0–7.0 and 40–45 °C, respectively. FNase S activity was enhanced by Mn2+ and Na+ (115.7% and 131.2%), but it was inhibited by Ca2+, K+, Ba2+, Cu2+ (96%, 83.7%, 84.3%, and 89.3%, respectively), each at 1 mM. The Km, Vmax and Kcat values of FNase S on MF were 1.7 mM, 0.62 mg·min−1, and 0.38·S−1, respectively. This enzyme could be a valuable tool for the structural analysis of fucoidans and production of bioactive fuco-oligosaccharides.
TL;DR: The homologous sandwich ELISA based on the anti-goldfish Vtg antibody could provide a valuable tool for the study of estrogenic effects of exogenous chemicals on goldfish.
TL;DR: The mononuclear copper(II) complexes with P, O-donor ligand and different fluoroquinolones have been synthesized and characterized and suggest the classical intercalative mode of DNA binding.
TL;DR: In this article, new copper(I) and copper(II) complexes with phosphine derivatives of two fluoroquinolones (ciprofloxacin and norfloxacsin) are presented.
Abstract: In this paper, new copper(I) and copper(II) complexes with phosphine derivatives of two fluoroquinolones (ciprofloxacin and norfloxacin) are presented. The synthesized compounds ([CuI-PCp], [CuI-PNr], [OPCp-CuII]+ and [OPNr-CuII]+) were characterized by elemental analysis and MS as well as by the NMR, EPR and IR spectroscopies. X-ray techniques were used to determine the crystal and molecular structures of [CuI-PCp]·CH2Cl2·CH3CN and OPCp-CuII]NO3·3H2O. For all the studied compounds, the ability to interact with DNA was determined using three different methods. The results of gel electrophoresis revealed that in the presence or absence of H2O2, the copper(I) complexes caused only single-stranded cleavage of the sugar–phosphate backbone of DNA. In turn, the copper(II) complexes damaged the plasmid exclusively in the presence of the oxidant. The addition of H2O2 caused distinct changes in the plasmid structure, resulting in a complete disappearance of its native form. Forms II and III arising from single- and double-strand cleavage were detected. Studies of the interactions with calf thymus DNA in the presence of ethidium bromide (EB) showed that the tested complexes and phosphines interact with DNA in a partial intercalation mode, contrary to unmodified antibiotic and oxide derivatives, which do not displace EB from the system. Molecular docking (AutoDock Vina program) was performed using the synthetic double-stranded hexadecanucleotide (sequence: ATATCGCGATATCGCG). Data analysis showed that a majority of the compounds preferably bound to the minor or major grooves, however most of them were also able to intercalate with the DNA double helix.
TL;DR: This represents the first time these bacterial proteins are being reported, following treatments with the antibacterial plant extracts, and could lead to the detailed understanding of their inhibition mechanism and discovery of target-specific antibacterial agents.