2-D Two Dimensional Gel Electrophoresis Research Publications

Plasmid dna replication and topology as visualized by two-dimensional agarose... Related Articles

Plasmid dna replication and topology as visualized by two-dimensional agarose gel electrophoresis.

Plasmid. 2009 Nov 16;

Authors: Schvartzman JB, Martínez-Robles ML, Hernández P, Krimer DB

During the last 20 years, two-dimensional agarose gel electrophoresis combined with other techniques such as Polymerase Chain Reaction, helicase assay and electron microscopy, helped to characterize plasmid DNA replication and topology. Here we describe some of the most important findings that were made using this method including the characterization of unidirectional replication, replication origin interference, DNA breakage at the forks, replication fork blockage, replication knotting, replication fork reversal, the interplay of supercoiling and catenation and other changes in DNA topology that take place as replication progresses.

PMID: 19925824 [PubMed - as supplied by publisher]

Characterization of Hepatitis C virus NS3 modifications in the context of replication.

J Gen Virol. 2009 Nov 18;

Authors: Liefhebber JM, Hensbergen PJ, Deelder AM, Spaan WJ, van Leeuwen H

Post-translational modifications (PTMs) of viral proteins regulate various stages of infection. With only 10 proteins Hepatitis C virus can orchestrate its complete viral lifecycle. The HCV non-structural protein 3 has many functions. It has protease and helicase activity, interacts with several host cell proteins and plays a role in translation, replication and virus particle formation. Organization of all these functions is necessary and could be regulated by PTMs. We therefore searched for modifications of the NS3 protein in the subgenomic HCV replicon. When performing a tag-capture approach coupled with two-dimensional gel electrophoresis analyses, we observed that isolated His6-NS3 yielded multiple spots. Individual protein spots were in-gel digested and analyzed by mass spectrometry. Differences observed between the individual peptide mass fingerprints suggested the presence of modified peptides and allowed us to identify N-terminal acetylation and an adaptive mutation of NS3 (Q1067R). Further analysis of other NS3 variants revealed phosphorylation of NS3.

PMID: 19923258 [PubMed - as supplied by publisher]

Protection in Glutamate-Induced Neurotoxicity by Imidazoline Receptor Agonist Moxonidine.

Int J Neurosci. 2009;119(10):1705-1717

Authors: Bakuridze K, Savli E, Gongadze N, Baş DB, Gepdi˙remen A

In the present study we investigated the effects of mixed imidazoline-1 and alpha(2)-adrenoceptor agonist, moxonidine, in glutamate-induced neurotoxicity in frontal cortical cell cultures of rat pups by dye exclusion test. Also, phosphorylated p38 mitogen activated protein kinases (p-p38 MAPK) levels were determined from rat frontal cortical tissue homogenates by two dimensional gel electrophoresis and semidry western blotting. Glutamate at a concentration of 10(-6) M was found neurotoxic when applied for 16 hr in cell cultures. Dead cell mean scores were 12.8 +/- 0.5 for control and 52.3 +/- 4.8 for glutamate (p < .001). On the other hand, p-p38 MAPK levels start to increase at a glutamate concentration of 10(-7) M for 20 min application. Moxonidine was found to have an U-shape neuroprotective effect in glutamate-induced neurotoxicity in neuronal cell culture experiments. Even though moxonidine did not induce neurotoxicity alone between the doses of 10(-8) to 10(-4) M concentrations in cell culture series, it caused the reduction of glutamate-induced dead cell population 23.07 +/- 3.6% in 10(-6) M and 26.7 +/- 2.1% in 10(-5) M concentrations (p <.001 for both, in respect to control values). The protective effect of moxonidine was confirmed in 10(-8) and 10(-7) M, but not in higher concentrations in glutamate neurotoxicity in gel electrophoresis and western blotting of p-p38 MAPK levels. In addition to other studies that revealed an antihypertensive feature of moxonidine, we demonstrated a possible partial neuroprotective role in lower doses for it in glutamate-mediated neurotoxicity model.

PMID: 19922382 [PubMed - as supplied by publisher]

Tuning the Helicity of Self-Assembled Structure of a Sugar-Based Organogelator by the Proper Choice of Cooling Rate.

Langmuir. 2009 Nov 18;

Authors: Cui J, Liu A, Guan Y, Zheng J, Shen Z, Wan X

A novel sugar-appended low-molecular-mass gelator, 4''-butoxy-4-hydroxy-p-terphenyl-beta-d-glucoside (BHTG), was synthesized. It formed thermally reversible gels in a variety of aqueous and organic solvents. Three-dimensional networks made up of helical ribbons were observed in the mixture of H(2)O/1,4-dioxane (40/60 v/v). The handedness of the ribbons depended on the rate of gel formation. Fast-cooling process led to right-handed ribbons, while slow-cooling process led to left-handed ones. A combinatory analyses of microscopic, spectroscopic, and diffraction techniques revealed that BHTG formed a twisted interdigitated bilayer structure with a d spacing of 3.1 nm in gels through a kinetically controlled nucleation-growth process. There were two kinds of molecular orientations of BHTG in the nuclei, clockwise and anticlockwise, which dictated the growth of ribbons. One was metastable and formed first during the cooling process of gel formation. It was able to gradually transform into the more stable latter one with further decreasing temperature. Fast-cooling process did not leave enough time for the nuclei to evolve from metastable to stable state and the ribbons grown from them exhibited right-handedness. However, the metastable nuclei transformed into the stable one when cooled slowly and directed the molecules of BHTG to grow into left-handed aggregates.

PMID: 19921782 [PubMed - as supplied by publisher]

[Proteomic research of biomarker of colorectal cancer metastasis.]

Zhonghua Wei Chang Wai Ke Za Zhi. 2009 Nov;12(6):618-622

Authors: Zhang P, Huang L, Ma YL, Peng JY, Shen TY, Chen HQ, Zhou YK, Chu ZX, Zhang M, Qin HL

OBJECTIVE: To explore the potential markers of colorectal cancer metastasis and the influence of 5-FU on differentially expressed proteins by using proteomic technology, and to elucidate the mechanism of colorectal cancer metastasis. METHODS: Human colorectal carcinoma cell lines of different metastatic potential, Lovo and SW480 were conventionally cultured, and the protein was extracted. 50% inhibitory concentration(IC(50)) of 5-FU to these two cell lines was measured by MTT assay. Proteins of these two cell lines after intervention by 5-FU at IC(50) were extracted, then 2-dimensional gel electrophoresis was conducted for the proteins. The differential protein spots were examined by mass spectrometry and analyzed by bioinformatics. Difference of expressed proteins in two cell lines before and after the intervention of 5-FU was validated by Western blot and immunofluorescence. RESULTS: Eleven differentially expressed proteins were identified by 2-dimensional gel electrophoresis and mass spectrometry. The hnRNP K protein and PDI were selected to be examined by Western blot and immunofluorescence. Results revealed that the expression of hnRNP K in Lovo was higher than that in SW480, while the expression of PDI was lower in Lovo. After intervention by 5-FU at IC(50), the expression of hnRNP K in Lovo decreased more as compared to SW480, while the expression of PDI in SW480 increased more as compared to Lovo. CONCLUSION: There are significant differences in expression of hnRNP K and PDI proteins between Lovo and SW480 cell lines, and the proteins alter regularly after 5-FU intervention.

PMID: 19921578 [PubMed - as supplied by publisher]

[Proteomics study of intestinal mucosa after ischemic preconditioning against intestinal ischemic reperfusion injury in rats.]

Zhonghua Wei Chang Wai Ke Za Zhi. 2009 Nov;12(6):598-602

Authors: Liu KX, Li YS, Wang ZX, Li C, Liu JX, Huang WQ

OBJECTIVE: To identify associated proteins involved in the molecular response of ischemic preconditioning (IPC) against intestinal ischemia/reperfusion(II/R) in the intestinal mucosa of rats. METHODS: Sixteen SD rats were randomly divided into II/R and IPC groups. II/R injury in rats was produced by clamping superior mesenteric artery for 60 min followed by 60 min reperfusion. IPC was elicited by 20 min ischemia and 5 min reperfusion before index ischemia. The intestinal mucosa was scratched immediately after 60 min of reperfusion and total proteins were separated by immobilized pH gradient(IPG) based on two-dimensional gel electrophoresis (2-DE). The differentially expressed proteins were analyzed using Image Master 2D Elite 5.0 image analysis software and identified by MALDI-TOF-MS. The biological information of these proteins was searched in the database of these peptide mass finger-printing(PMF). Western blotting and RT-PCR were used to validate the differentially expressed proteins. RESULTS: Image analysis revealed that averages of 1404+/-20 and 1338+/-20 were detected in II/R and IPC groups. A total of 10 spots yielded good spectra, and 8 spots matched with known proteins after database searching. These proteins were mainly involved in anti-oxidation, inhibiting apoptosis and energy metabolism. Western blot confirmed up-regulation of aldehyde dehydrogenase and RT-PCR confirmed up-regulation of aldose reductase in IPC group. CONCLUSION: The clues provided by comparative proteome strategy will shed light on molecular mechanisms of IPC against II/R injury.

PMID: 19921573 [PubMed - as supplied by publisher]

Proteomic analysis of early phase of conidia germination in Aspergillus nidulans.

Fungal Genet Biol. 2009 Nov 14;

Authors: Oh YT, Ahn CS, Kim JG, Ro HS, Lee CW, Kim JW

In order to investigate proteins involved in early phase of conidia germination, proteomic analysis was performed using two-dimensional gel electrophoresis (2D-GE) in conjunction with MALDI-TOF mass spectrometry (MS). The expression levels of 241 proteins varied quantitatively with statistical significance (P<0.05) at the early phase of the germination stage. Out of these 57 were identified by MALDI-TOF MS. Through classification of physiological functions from Conserved Domain Database analysis, among the identified proteins, 21, 13, and 6 proteins were associated with energy metabolism, protein synthesis, and protein folding process, respectively. Interestingly, 8 proteins, which are involved in detoxification of reactive oxygen species (ROS) including catalase A, thioredoxin reductase, and mitochondrial peroxiredoxin, were also identified. The expression levels of the genes were further confirmed using Northern blot and reverse transcriptase (RT)-PCR analyses. This study represents the first proteomic analysis of early phase of conidia germination and will contribute to a better understanding of the molecular events involved in conidia germination process.

PMID: 19919853 [PubMed - as supplied by publisher]