Showing papers on "Gel electrophoresis published in 2019"
TL;DR: A mass-spectrometry-compatible surfactant called Azo effectively solubilizes proteins, is rapidly degraded by ultraviolet irradiation and enables top-down proteomic analysis of membrane proteins.
Abstract: We report the identification of a photocleavable anionic surfactant, 4-hexylphenylazosulfonate (Azo), which can be rapidly degraded by ultraviolet irradiation, for top-down proteomics. Azo can effectively solubilize proteins with performance comparable to that of sodium dodecyl sulfate (SDS) and is compatible with mass spectrometry. Azo-aided top-down proteomics enables the solubilization of membrane proteins for comprehensive characterization of post-translational modifications. Moreover, Azo is simple to synthesize and can be used as a general SDS replacement in SDS-polyacrylamide gel electrophoresis.
75 citations
TL;DR: A simple method to estimate MW by running SDS-PAGE of standard proteins is explained by an example in which proteins extracted from mouse retina were analyzed by two-dimensional isoelectric focusing (2-D IEF) SDS -PAGE followed by protein identification by peptide mass fingerprinting.
Abstract: An apparent molecular weight (MW) of a protein can be determined from the migration distance of a protein complexed with a strong cationic detergent sodium dodecyl sulfate (SDS) separated on sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). This method was established around 1969 and has been utilized substantially even today because of its simplicity. During the following half a century, although it has been reported that many proteins show some deviation in MW when determined on SDS-PAGE especially when their peptide chains are posttranslationally modified, this versatile method is still being used very often in current biochemical works. In this protocol, a simple method to estimate MW by running SDS-PAGE of standard proteins is explained by an example in which proteins extracted from mouse retina were analyzed by two-dimensional isoelectric focusing (2-D IEF) SDS-PAGE followed by protein identification by peptide mass fingerprinting.
36 citations
TL;DR: Gel electrophoresis had been the primary method in proteomics but turned downwards due to the progress of other separations including liquid chromatography and ionization techniques, resulting gel-free proteomics finally becoming dominant players at present.
29 citations
TL;DR: A novel simple method to detect protein phosphorylation, which is based on the construction of a variant protein displaying a electrophoretic mobility shift-related motif, which may be used as a general strategy to easily measure the ratio of proteinosphorylation in cells.
Abstract: Phosphorylation is the most important modification for protein regulation; it controls many signal transduction pathways in all organisms. While several tools to detect phosphorylated proteins have been developed to study a variety of basic cellular processes involving protein phosphorylation, these methods have several limitations. Many proteins exhibit a phosphorylation-dependent electrophoretic mobility shift (PDEMS) in sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), and the molecular mechanism responsible for this phenomenon has been elucidated recently. The method for detecting phosphorylated proteins can be simplified by the application of the PDEMS. Herein, we present a novel simple method to detect protein phosphorylation, which is based on the construction of a variant protein displaying a PDEMS. The PDEMS of proteins is caused by the distribution of negatively charged amino acids around the phosphorylation site, i.e. an electrophoretic mobility shift (EMS)-related motif (ΘX1-3ΘX1-3Θ, where Θ corresponds to an acidic or phosphorylated amino acid and X represents any amino acid). The EMS-related motif can be constructed by the introduction of a negative charge by phosphorylation; it results in the decreased binding of SDS to the proteins, consequently inducing the retardation of the mobility of the protein during SDS-PAGE. Based on these molecular analyses of the PDEMS, a protein with the EMSrelated motif is designed and used to determine the in vivo phosphorylation state of the protein. This method may be used as a general strategy to easily measure the ratio of protein phosphorylation in cells.
29 citations
TL;DR: It is unveiled that GAPDH, an essential enzyme in glycolysis, serves as a vital target of Ag+ in E. coli for the first time and is the first report on antimicrobial Ag+ targeting a key enzyme in the glyCOlytic pathway of E. bacteria.
Abstract: Silver has long been used as an antibacterial agent, yet its molecular targets remain largely unknown. Using a custom-designed coupling of gel electrophoresis with inductively coupled plasma mass spectrometry (GE-ICP-MS), we identified six silver-binding proteins in E. coli. The majority of the identified proteins are associated with the central carbon metabolism of E. coli. Among them, we unveil that GAPDH, an essential enzyme in glycolysis, serves as a vital target of Ag+ in E. coli for the first time. We demonstrate that silver inhibits the enzymatic function of GAPDH through targeting Cys149 in its catalytic site. The X-ray structure reveals that Ag+ coordinates to Cys149 and His176 with a quasi-linear geometry (S–Ag–N angle of 157°). And unexpectedly, two Ag+ ions coordinate to Cys288 in the non-catalytic site with weak argentophilic interaction (Ag⋯Ag distance of 2.9 A). This is the first report on antimicrobial Ag+ targeting a key enzyme in the glycolytic pathway of E. coli. The findings expand our knowledge on the mode of action and bio-coordination chemistry of silver, particularly silver-targeting residues in proteins at the atomic level.
28 citations
TL;DR: TPA-BI-C/DOPE is the first non-viral gene vector with the abilities of pH /lipase enzyme responsiveness, one/two photon fluorescent tracking of intracellular delivery of DNA, and successful transfection in vivo and in vitro, even better than Lipo2000.
Abstract: Three nonviral gene vectors, TPA-BI-A/B/C, have been designed and synthesized by the combination of one or two hydrophilic [12]aneN3 moieties and two-photon fluorescent triphenylamine-benzylideneimidazolone (TPA-BI) units through different ester linkage. Spectroscopic characterization demonstrated that TPA-BI-A/B/C had strong aggregation-induced emissions (AIE), large Stokes shifts (230, 284, and 263 nm), and large two-photon absorption cross sections (δ2PA) (67, 592, and 80 GM). Gel electrophoresis indicated that the three compounds completely condensed DNA at 15 μM in the presence of DOPE, and showed the lipase- and pH-triggered reversible release of DNA and the fluorescent recognition of the different lengths of ssDNA and dsDNA. The optimal TPA-BI-C/DOPE-mediated luciferase and GFP activity was 146% and 290% higher than those of Lipo2000. The transfection process of DNA could be traced clearly through one- and two-photon fluorescence spectra, and displayed in a 3D-video. TPA-BI-C/DOPE successfully transfected the GFP gene into zebrafish, which was superior to Lipo2000 (192%). In conclusion, TPA-BI-C/DOPE is the first nonviral gene vector with the abilities of pH/lipase enzyme responsiveness, one/two-photon fluorescent tracking of intracellular delivery of DNA, and successful transfection in vivo and in vitro, even better than Lipo2000.
28 citations
TL;DR: The ability of Fe1 to cleave DNA was greater than that of other complexes and these results are in conformity with their DNA-binding affinities, suggesting that the complex Fe1 could be a good candidate as a chemotherapeutic drug targeting DNA.
Abstract: One iron(III) and two manganese(III) complexes based on thiosemicarbazone were synthesized and characterized using analytical and spectroscopic data. The crystallographic analysis showed the square pyramid structures of the complexes. Electronic spectra analysis was performed to determine the nature of the interaction between the complexes and calf thymus DNA (CT-DNA). DNA cleavage activities of the complexes were examined by gel electrophoresis (pBR322 DNA). The cytotoxicity of the complexes was determined against human cervical carcinoma (HeLa) and human colorectal adenocarcinoma (HT-29) cell lines by MTT assay. The results indicated that complex Fe1 is bound to CT-DNA via the intercalation mode, while complexes Mn1 and Mn2 are bound to CT-DNA via groove binding and/or electrostatic interactions rather than the intercalation mode. In addition, they showed good binding activity, which followed the order of Fe1 > Mn2 > Mn1. Complexes were found to promote the cleavage of DNA from supercoiled form (SC, Form I) to nicked circular form (NC, Form II) without concurrent formation of Form III, revealing the single-strand DNA cleavage. No significant cleavage was found in the presence of Mn1 and Mn2; however, it was observed at 2000 and 3000 µM concentrations of Fe1. The ability of Fe1 to cleave DNA was greater than that of other complexes and these results are in conformity with their DNA-binding affinities. Cytotoxicity determination tests revealed that the complex Fe1 on HeLa and HT-29 cells exhibited a higher anti-proliferative effect than Mn1 and Mn2 (Fe1 > Mn2 > Mn1). These studies suggested that the complex Fe1 could be a good candidate as a chemotherapeutic drug targeting DNA.
25 citations
TL;DR: Genome sequencing of the three isolates revealed the presence of gene clusters associated with the production of non-ribosomally synthesized peptides and bacteriocins that suggest they can be considered as potent biocontrol strains that produce an arsenal of antimicrobial molecules active against Gram-positive and Gram-negative multi-resistant pathogens, fungi and insects.
Abstract: Bacteria active against multi-drug resistant pathogens, isolated by direct selection of colonies from clover silage samples, produce zones of inhibition against two Gram-negative (Klebsiella pneumoniae Ni9 and Pseudomonas aeruginosa MMA83) and two Gram-positive (Staphylococcus aureus ATCC25923 and Listeria monocytogenes ATCC19111) pathogens. Isolates BGSP7, BGSP9, BGSP11 and BGSP12 produced the largest zones of inhibition against all four pathogens when grown in LB broth with aeration at 37°C. Isolates BGSP7, BGSP9, BGSP11 and BGSP12 were identified as Brevibacillus laterosporus and pulsed field gel electrophoresis and extracellular protein profiles showed that three different strains (BGSP7, BGSP9 and BGSP11) were isolated. A semi-native SDS-PAGE (sodium dodecyl sulphate-polyacrylamide gel electrophoresis) gel overlay assay showed that BGSP7 and BGSP9 produce small antimicrobial molecules of about 1.5 kDa, while BGSP11 produces antimicrobial molecules of 1.5 and 6 kDa active against S. aureus ATCC25923. Amino acid analysis of two antimicrobial molecules (1583.73 Da; from BGSP7 and 1556.31 Da; from BGSP11) revealed that they have a similar composition and differ only by virtue of the presence of a methionine which is present only in BGSP11 molecule. Genome sequencing of the three isolates revealed the presence of gene clusters associated with the production of non-ribosomally synthesized peptides (brevibacillin, bogorol, gramicidin S, plipastatin and tyrocin) and bacteriocins (laterosporulin, a lactococcin 972-like bacteriocin, as well as putative linocin M18, sactipeptide, UviB and lantipeptide-like molecules). Ultimately, the purification of a number of antimicrobial molecules from each isolate suggests that they can be considered as potent biocontrol strains that produce an arsenal of antimicrobial molecules active against Gram-positive and Gram-negative multi-resistant pathogens, fungi and insects.
21 citations
TL;DR: Five imidazo [1,2-a]pyridine derivatives and their Au(III) complexes were synthesized and the results suggest intercalation mode of binding in compound-DNA interaction.
21 citations
TL;DR: Results indicate that pELF1 has the ability to confer multidrug resistance to Enterococcus spp.
Abstract: Vancomycin-resistant enterococci are troublesome pathogens in clinical settings because of few treatment options. A VanA/VanM-type vancomycin-resistant Enterococcus faecium clinical isolate was identified in Japan. This strain, named AA708, harbored five plasmids, one of which migrated during agarose gel electrophoresis without S1 nuclease treatment, which is indicative of a linear topology. We named this plasmid pELF1. Whole genome sequencing (WGS) analysis of the AA708 strain revealed that the complete sequence of pELF1 was 143,316 bp long and harbored both the vanA and vanM gene clusters. Furthermore, mfold analysis and WGS data show that the left end of pELF1 presumably forms a hairpin structure, unlike its right end. The pELF1 plasmid was not digested by lambda exonuclease, indicating that terminal proteins were bound to the 5' end of the plasmid, similar to the Streptomyces linear plasmids. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis results were also consistent with the exonuclease assay results. In retardation assays, DNAs containing the right end of proteinase K-untreated pELF1 did not appear to move as well as the proteinase K-treated pELF1, suggesting that terminal proteins might be attached to the right end of pELF1. Palindromic sequences formed hairpin structures at the right terminal sequence of pELF1; however, sequence similarity with the well-known linear plasmids of Streptomyces spp. was not high. pELF1 was unique as it possessed two different terminal structures. Conjugation experiments revealed that pELF1 could be transferred to E. faecalis, E. faecium, E. casseliflavus, and E. hirae. These transconjugants exhibited not only high resistance levels to vancomycin, but also resistance to streptomycin, kanamycin, and erythromycin. These results indicate that pELF1 has the ability to confer multidrug resistance to Enterococcus spp. simultaneously, which might lead to clinical hazards.
19 citations
TL;DR: The addition of the dangling amine groups resulting in structures of SI-PPCs, which differ so markedly from the derivatives of cisplatin used in the clinic, appears to contribute to the overall biological activity of these molecules.
Abstract: The substitution-inert polynuclear platinum complexes (SI-PPCs) are now recognized as a distinct subclass of platinum anticancer drugs with high DNA binding affinity. Here, we investigate the effects of SI-PPCs containing dangling amine groups in place of NH3 as ligands to increase the length of the molecule and therefore overall charge and its distribution. The results obtained with the aid of biophysical techniques, such as total intensity light scattering, gel electrophoresis, and atomic force microscopy, show that addition of dangling amine groups considerably augments the ability of SI-PPCs to condense/aggregate nucleic acids. Moreover, this enhanced capability of SI-PPCs correlates with their heightened efficiency to inhibit DNA-related enzymatic activities, such as those connected with DNA transcription, catalysis of DNA relaxation by DNA topoisomerase I, and DNA synthesis catalyzed by Taq DNA polymerase. Thus, the addition of the dangling amine groups resulting in structures of SI-PPCs, which differ so markedly from the derivatives of cisplatin used in the clinic, appears to contribute to the overall biological activity of these molecules.
TL;DR: In this article, the authors reported the molecular design and synthesis of [Ru(η6-p-cymene)-(chromone)Cl] complex (1) as a potential topoisomerase I inhibitor.
Abstract: In the present study, we report the molecular design and synthesis of [Ru(η6-p-cymene)-(chromone)Cl] complex (1) as a potential topoisomerase I inhibitor. The structural elucidation of complex 1 was carried out by analytical, spectral and single crystal X-ray crystallographic techniques. Complex 1 crystallized in the monoclinic Pc space group with a Ru(II) ion coordinated to a p-cymene arene unit in a usual “piano-stool” geometry. In vitro binding interaction studies of complex 1 with ct-DNA and HSA were carried out and suggested a strong binding affinity towards DNA via an intercalative mode. DNA photocleavage activity was carried out with a pBR322 plasmid DNA substrate to ascertain the cleaving ability and the mechanistic pathway of the cleavage process. Topoisomerase I inhibition assay of complex 1 was performed via gel electrophoresis which revealed a significant inhibitory effect on the enzyme catalytic activity at a minimum concentration of 20 μM. Molecular docking studies of the complex were carried out with DNA, HSA and topoisomerase I to determine the specific binding preferences at the target site and complement the spectroscopic studies. Cytotoxic studies of complex 1 on a series of five human cancer cell lines, viz. A-498, HepG2, HeLa, MCF-7 and MIA-PA-CA-2, by the SRB assay revealed a moderate but selective activity towards pancreatic and cervical cancer cell lines.
TL;DR: Enzymatic treatment mitigated the allergenicity of fish PV, which was closely related to the alterations in the conformational structure, and showed potential for developing hypoallergenic fish products under mild reaction conditions.
Abstract: Enzymatic treatment of allergenic protein can alter their functional properties under a mild reaction condition due to specificity of enzymes. Phenolic compounds act as mediators and enhance the crosslinking reactions. The study aimed to assess the changes in the structure and immunoglobulin G (IgG) binding capacity of turbot parvalbumin (PV) upon crosslinking with tyrosinase (Tyr) in the absence and presence of caffeic acid. RESULTS Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) analysis revealed the appearance of higher molecular weight bands (24, 36 kDa) in the crosslinked PV. The secondary structure of crosslinked PV became loosened and disordered. The results of intrinsic fluorescence and ultraviolet absorption spectral analyses, as well as surface hydrophobicity and free amino group analyses also revealed structural changes. As observed by western blot analysis, the intensity of the PV bands reduced upon Tyr treatment, indicating reduced binding of specific IgG to PV. Moreover, the indirect ELISA (enzyme-linked immunosorbent assay) analysis confirmed that the IgG binding ability of crosslinked PV was reduced 34.94%. CONCLUSION Enzymatic treatment mitigated the allergenicity of fish PV, which was closely related to the alterations in the conformational structure. This treatment showed potential for developing hypoallergenic fish products under mild reaction conditions. ? 2019 Society of Chemical Industry
TL;DR: The grass carp gill is involved in response processes, including energy generation, metabolic processes, cellular structure, antioxidation, immunity, and signal transduction, to hypoxic stress, and this study will help increase the understanding of the molecular mechanisms involved in Hypoxic stress responses in fish at the protein level.
Abstract: Two-dimensional gel electrophoresis (2-DE) was combined with liquid chromatography-mass spectrometry (LC-MS/MS) to identify the differential proteomics of grass carp gills after hypoxic stress to better understand the roles of proteins in the hypoxic response and to explore the possible molecular mechanisms. Protein spots were obtained from a hypoxia-stressed group (372 ± 11 individuals) and a control group (406 ± 14 individuals) using the lmage Master 2D Platinum 7.0 analysis software. Fifteen protein spots were expressed differentially in the hypoxia-stressed group and varied significantly after exposure to the hypoxic conditions. In addition, these differential proteins were identified by mass spectrometry and then searched in a database. We found the expression and upregulation of the toll-like receptor 4, ephx1 protein, isocitrate dehydrogenase, L-lactate dehydrogenase, GTP-binding nuclear protein Ran, and glyceraldehyde-3-phosphate dehydrogenase; however, the expression of the keratin type II cytoskeletal 8, type I cytokeratin, ARP3 actin-related protein 3 homolog, thyroid hormone receptor alpha-A, ATP synthase subunit beta, citrate synthase, tropomyosin 2, and tropomyosin 3 were downregulated. Six proteins were found in the hypoxia-inducible factor-1 (HIF-1) signaling pathway. We concluded that the grass carp gill is involved in response processes, including energy generation, metabolic processes, cellular structure, antioxidation, immunity, and signal transduction, to hypoxic stress. To our knowledge, this is the first study to conduct a proteomics analysis of expressed proteins in the gills of grass carp, and this study will help increase the understanding of the molecular mechanisms involved in hypoxic stress responses in fish at the protein level.
TL;DR: Both GSC and PSC showed distinct differences from each other and the type I collagen from grass carp muscle on sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and had imino acid of 150 and 155 residues/1000 residues, respectively.
Abstract: The aim of this study was to characterize the collagens from the body of surf clam shell (Coelomactra antiquata). Guanidine hydrochloride and pepsin were used to extract collagens. Guanidine hydrochloride soluble collagen (GSC) and pepsin soluble collagen (PSC) were separately isolated from the body of surf clam shell. Results showed that the moisture, protein, carbohydrate, and ash contents of the body of surf clam shell were 82.46%, 11.56%, 3.05%, and 2.38%, respectively, but the fat content was only 0.55%. The yields were 0.59% for GSC and 3.78% for PSC. Both GSC and PSC were composed of α₁ and α₂ chains and a β chain, however, GSC and PSC showed distinct differences from each other and the type I collagen from grass carp muscle on sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). GSC and PSC contained glycine as the major amino acid and had imino acid of 150 and 155 residues/1000 residues, respectively. Fourier transform infrared spectroscopy (FTIR) spectra of GSC and PSC revealed the presence of a triple helix. The GSC appeared to have a dense sheet-like film linked by random-coiled filaments and PSC had fine globular filaments under scanning electron microscopy (SEM). The maximum transition temperature (Tmax) of GSC and PSC was 33.05 °C and 31.33 °C, respectively. These results provide valuable scientific information for the texture study and development of surf clam shell or other bivalve mollusks.
TL;DR: A device that is compatible with coupling droplets to gel and free-solution electrophoresis, and based on a novel mechanism of carrier phase separation using the difference in density of the carrier phase and the running buffer is presented.
Abstract: Electrophoresis has demonstrated utility as tool for screening of small molecule modulators of protein-protein interactions and enzyme targets. Screening of large chemical libraries requires high-throughput separations. Such fast separation can be accessed by microchip electrophoresis. Here, microchip gel electrophoresis separations of proteins are achieved in 2.6 s with 1200 V/cm and 3-mm separation lengths. However, such fast separations can still suffer from limited overall throughput from sample introduction constraints. Automated introduction of microfluidic droplets has been demonstrated to overcome this limitation. Most devices for coupling microfluidic droplets to microchip electrophoresis are only compatible with free-solution separations. Here, we present a device that is compatible with coupling droplets to gel and free-solution electrophoresis. In this device, automated sample introduction is based on a novel mechanism of carrier phase separation using the difference in density of the carrier phase and the running buffer. This device is demonstrated for microchip gel electrophoresis and free-solution electrophoresis separations of protein-protein interaction and enzyme samples, respectively. Throughputs of about 10 s per sample are achieved and over 1000 separations are demonstrated without reconditioning of the device.
TL;DR: It is demonstrated that PflLys49-PLA2 BPII spontaneously oligomerized in the presence of SDS, which is one of the strongest protein denaturants.
Abstract: Phospholipase A2 (PLA2) is one of the representative toxic components of snake venom. PLA2s are categorized into several subgroups according to the amino acid at position 49, which comprises either Asp49, Lys49, Arg49 or Ser49. Previous studies suggested that the Lys49-PLA2 assembles into an extremely stable dimer. Although the behavior on Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) under reducing or non-reducing conditions suggested the presence of intermolecular disulfide bonds, these bonds were not observed in the crystal structure of Lys49-PLA2. The reason for this discrepancy between the crystal structure and SDS-PAGE of Lys49-PLA2 remains unknown. In this study, we analyzed a Lys49-PLA2 homologue from Protobothrops flavoviridis (PflLys49-PLA2 BPII), by biophysical analyses including X-ray crystallography, SDS-PAGE, native-mass spectrometry, and analytical ultracentrifugation. The results demonstrated that PflLys49-PLA2 BPII spontaneously oligomerized in the presence of SDS, which is one of the strongest protein denaturants.
TL;DR: A combination between gel electrophoresis and smartphone technology is applied for quantifying proteins in a serum sample and SDS-PAGE provides multiple information regarding the quality of the protein, such as the presence of degradation products, as well as if there was interaction reaction between proteins.
TL;DR: An on-chip method for the extraction of RNA within a specific size range from low-abundance samples using isotachophoresis with an ionic spacer and a sieving matrix to enable size-selection with a high yield of RNA in the target size range is presented.
Abstract: We present an on-chip method for the extraction of RNA within a specific size range from low-abundance samples. We use isotachophoresis (ITP) with an ionic spacer and a sieving matrix to enable size-selection with a high yield of RNA in the target size range. The spacer zone separates two concentrated ITP peaks, the first containing unwanted single nucleotides and the second focusing RNA of the target size range (2–35 nt). Our ITP method excludes >90% of single nucleotides and >65% of longer RNAs (>35 nt). Compared to size selection using gel electrophoresis, ITP-based size-selection yields a 2.2-fold increase in the amount of extracted RNAs within the target size range. We also demonstrate compatibility of the ITP-based size-selection with downstream next generation sequencing. On-chip ITP-prepared samples reveal higher reproducibility of transcript-specific measurements compared to samples size-selected by gel electrophoresis. Our method offers an attractive alternative to conventional sample preparation for sequencing with shorter assay time, higher extraction efficiency and reproducibility. Potential applications of ITP-based size-selection include sequencing-based analyses of small RNAs from low-abundance samples such as rare cell types, samples from fluorescence activated cell sorting (FACS), or limited clinical samples.
TL;DR: A review is given on the theory of the gel electrophoresis of a spherical soft particle covered with an ion-penetrable surface layer of polyelectrolytes moving in a polymer gel medium, which may either be charged or uncharged.
26 Jan 2019
TL;DR: These new complexes showed moderate cytotoxicity against hepatocellular carcinoma (HepG2) and neuroblastoma (SHSY5Y) cells, similarly to previous compounds in this series.
Abstract: A new oxindolimine ligand derived from isatin (1H-indole-2,3-dione) and 2-aminomethylbenzimidazole was synthesized, leading to two novel complexes after metalation with copper(II) perchlorate or zinc(II) chloride, [Cu(isambz)2](ClO4)2 (complex 1) and [Zn(isambz)Cl2] (complex 2). This new ligand was designed as a more lipophilic compound, in a series of oxindolimine–metal complexes with antitumor properties, having DNA, mitochondria, and some proteins, such as CDK1 kinase and topoisomerase IB, as key targets. The new complexes had their reactivity to human serum albumin (HSA) and DNA, and their cytotoxicity toward tumor cells investigated. The binding to CT-DNA was monitored by circular dichroism (CD) spectroscopy and fluorescence measurements using ethidium bromide in a competitive assay. Consequent DNA cleavage was verified by gel electrophoresis with complex 1, in nmolar concentrations, with formation of linear DNA (form III) after 60 min incubation at 37 °C, in the presence of hydrogen peroxide, which acts as a reducing agent. Formation of reactive oxygen species (ROS) was observed, monitored by spin trapping EPR. Interaction with HSA lead to α-helix structure disturbance, and formation of a stable radical species (HSA–Tyr·) and carbonyl groups in the protein. Despite showing oxidative ability to damage vital biomolecules such as HSA and DNA, these new complexes showed moderate cytotoxicity against hepatocellular carcinoma (HepG2) and neuroblastoma (SHSY5Y) cells, similarly to previous compounds in this series. These results confirm DNA as an important target for these compounds, and additionally indicate that oxidative damage is not the leading mechanism responsible for their cytotoxicity. Additionally, this work emphasizes the importance of ligand characteristics and of speciation in activity of metal complexes.
TL;DR: This protocol describes the analysis of mitochondrial respiratory chain complexes by blue native polyacrylamide gel electrophoresis (BN-PAGE) in application to cultured human cells, a convenient and inexpensive technique that can be used to evaluate the assembly of entire OXPHOS complexes, in contrast to the basic SDS-PAGES allowing the study of only individual OX PHOS complex subunits.
Abstract: Mitochondrial respiration is performed by oxidative phosphorylation (OXPHOS) complexes within mitochondria. Internal and environmental factors can perturb the assembly and stability of OXPHOS complexes. This protocol describes the analysis of mitochondrial respiratory chain complexes by blue native polyacrylamide gel electrophoresis (BN-PAGE) in application to cultured human cells. First, mitochondria are extracted from the cells using digitonin, then using lauryl maltoside, the intact OXPHOS complexes are isolated from the mitochondrial membranes. The OXPHOS complexes are then resolved by gradient gel electrophoresis in the presence of the negatively charged dye, Coomassie blue, which prevents protein aggregation and ensures electrophoretic mobility of protein complexes towards the cathode. Finally, the OXPHOS complexes are detected by standard immunoblotting. Thus, BN-PAGE is a convenient and inexpensive technique that can be used to evaluate the assembly of entire OXPHOS complexes, in contrast to the basic SDS-PAGE allowing the study of only individual OXPHOS complex subunits.
TL;DR: Study of the effect of the sequence Gly1127-Cys1140 on the structural characteristics of sphingomyelinase- (SMase) and phospholipase 2 (PLA2)-modified LDL particles suggests that these LRP1(CR9) derived peptides could be promising tools to prevent LDL aggregation induced by the main proteolytic enzymes acting in the arterial intima.
TL;DR: In the present contribution, new binuclear ternary complexes were synthesized, characterized and screened for their antimicrobial activity and cytotoxicity against human liver carcinoma cells as well as non-malignant human embryonic kidney cells (HEK-293).
Abstract: In the present contribution, new binuclear ternary complexes; [M2(bpy)4L](ClO4)4 (M = Co(II) (1) and Ni(II) (2); bpy = 2,2′-bipyridine; L = 1,1′-(hexane-1,6-diyl)bis[2-(pyridin-2-yl)1H-benzimidazole] and [Cu2(bpy)2(OH2)2L](BF4)4 (3) were synthesized, characterized and screened for their antimicrobial activity and cytotoxicity against human liver carcinoma cells (HepG-2) as well as non-malignant human embryonic kidney cells (HEK-293). The structural studies were complemented by density functional theory (DFT) calculations. DNA binding of 1–3 was spectrophotometrically studied. The DNA cleavage ability of 1–3 towards the supercoiled plasmid DNA (pBR322 DNA) was examined through gel electrophoresis. Compound 3 has the highest cytotoxic activity (IC50 = 3.5 μg mL−1) against HepG-2 among the investigated complexes and is non cytotoxic to noncancerous HEK-293. Complexes (1 and 2) exhibited toxicity to HEK-293 with IC50 values of 30.3 and 23.5 μg mL−1 in that order. While compound 1 showed antifungal activity against Cryptococcus neoformans, complex 2 exhibited its toxicity against Candida albicans.
TL;DR: Some proteins associated with carbon metabolism, proteolytic enzymes, F-box containing transcription factors, RNA helicases, DNA replication/transcription/repair enzymes and ankyrins were detected in Cd-tolerant line D28, which might alleviate the abiotic stress caused by Cd treatment.
TL;DR: In vivo data revealed that Gm-SGPP increased the calcium and phosphorus content of tibias and promoted longitudinal bone growth in adolescent rats, and high-performance size exclusion chromatography and sodium dodecyl sulfate-polyacrylamide gel electrophoresis demonstrated that it is a 7000-Da pure sialoglycopeptide.
Abstract: Gadus morhua eggs contain several nutrients, including polyunsaturated fatty acids, lecithin and glycoproteins. A novel sialoglycopeptide from the eggs of G. morhua (Gm-SGPP) was extracted with 90% phenol and purified by Q Sepharose Fast Flow (QFF) ion exchange chromatography, followed by S-300 gel filtration chromatography. Gm-SGPP contained 63.7% carbohydrate, 16.2% protein and 18.6% N-acetylneuraminic acid. High-performance size exclusion chromatography and sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) demonstrated that Gm-SGPP is a 7000-Da pure sialoglycopeptide. β-elimination reaction suggested that Gm-SGPP contained N-glycan units. Amino acid N-terminal sequence analysis indicated the presence of Ala-Ser-Asn-Gly-Thr-Gln-Ala-Pro amino acid sequence. Moreover, N-glycan was connected at the third Asn location of the peptide chain through GlcNAc. Gm-SGPP was composed of D-mannose, D-glucuronic acid and D-galactose. Fourier transform-infrared spectroscopy (FT-IR), 1H-nuclear magnetic resonance spectroscopy (1H-NMR) and methylation analysis were performed to reveal the structure profile of Gm-SGPP. In vitro results showed that the proliferation activity of MC3T3-E1 cells was significantly promoted by Gm-SGPP. In vivo data revealed that Gm-SGPP increased the calcium and phosphorus content of tibias and promoted longitudinal bone growth in adolescent rats.
TL;DR: The protein profile of black cumin seed protein concentrates prepared by the alkali extraction-acid precipitation technique (AE-IP) is analyzed using gel-based proteome mapping which included two-dimensional gel electrophoresis followed by matrix-assisted laser desorption/ionization-time of flight (MALDI-TOF/TOF).
Abstract: The amount of cold press oil manufacture is globally rising, which in turn leads to the accumulation of deoiled plant seeds at significant quantities and consequent manufacture of plant protein products. In this study, we made an attempt to analyze the protein profile of black cumin seed protein concentrates prepared by the alkali extraction-acid precipitation technique (AE-IP). The analytical strategy relied on gel-based proteome mapping which included two-dimensional gel electrophoresis followed by matrix-assisted laser desorption/ionization-time of flight (MALDI-TOF/TOF). 14 different protein bands were identified, and in gel-trypsinolysis was carried out for the corresponding gel spots. Using the MASCOT database, current findings on 10 proteins were compared with the existing data. The highest similarity was 46 which was obtained between the highest pI black cumin protein observed here and the cyclin dependent kinase inhibitor of Arabidopsis thaliana. The molecular mass of the intact protein was determined by linear MALDI-TOF/TOF-MS as 23,711.2186 Da. The peptide constructs of this protein have been further studied in order to identify potential biological activity. Matching sequences generated bioactive peptides in silico such as IR, AL, and SL dipeptides during sequential enzymatic digestion with pepsin and trypsin. Since the majority of bioactivity investigations on black cumin seeds have been related to black cumin oil and its oil soluble components, the structure and bioactivities of black cumin proteins deserve further research.
TL;DR: A simple enrichment of mucins without trypsinization is described that revealed that the concentrations of these molecules were lower in aged mice than in young mice, and that an unknown mucin-like molecule was detected only from the aged mouse submandibular gland.
TL;DR: It is suggested that binding of the charged nitrogen of the matrix to the phosphate backbone produces an asymmetry in charge neutralization that induces bending and base exposure in oc DNA, sc DNA and RNA.
TL;DR: It was shown that the green fruits are more responsive to infection, showing the production of antimicrobial peptides in response to injury and inoculation of the fungus, what did not occur in ripe fruits under any treatment.
Abstract: There are several phytosanitary problems that have been causing serious damage to the Capsicum crops, including anthracnose. Upon attack by certain pathogens, various protein molecules are produced, which are known as proteins related to pathogenesis (PR proteins), including antimicrobial peptides such as protease inhibitors, defensins and lipid transfer proteins (LTPs). The objective of this work is to identify antimicrobial proteins and/or peptides of two genotypes from Capsicum annuum fruits infected with Colletotrichum gloeosporioides The fungus was inoculated into Capsicum fruits by the deposition of a spore suspension (106 conidia ml-1), and after 24 and 48 h intervals, the fruits were removed from the humid chamber and subjected to a protein extraction process. Protein analysis of the extracts was performed by tricine gel electrophoresis and Western blotting. The distinctive bands between genotypes in the electrophoresis profiles were subjected to mass spectrometry sequencing. Trypsin inhibition assays, reverse zymographic detection of protease inhibition and β-1,3-glucanase activity assays were also performed and extracts were also tested for their ability to inhibit the growth of C. gloeosporioides fungi 'in vitro' There were several low molecular weight proteins in all treated samples, and some treatments in which antimicrobial peptides such as defensin, lipid transfer protein (LTP) and protease inhibitor have been identified. It was shown that the green fruits are more responsive to infection, showing the production of antimicrobial peptides in response to injury and inoculation of the fungus, what did not occur in ripe fruits under any treatment.