Screening Serum Biomarkers for Rats Preconditioned with Hyperbaric Oxygen: Potential of Predicting Prognosis for Stroke.

BACKGROUND: Stroke is a major health concern and a leading cause of mortality and morbidity. We and other groups have documented that hyperbaric oxygen preconditioning could significantly alleviate neuronal damage in ischemia‒reperfusion models through various mechanisms. However, we found that some of the subjects did not benefit from preconditioning with hyperbaric oxygen. The preconditioning phenomenon is similar to vaccination, in which the endogenous survival system is activated to fight against further injuries. However, with vaccine inoculations, we could test for specific antibodies against the pathogens to determine if the vaccination was successful. Likewise, this experiment was carried out to explore a biomarker that can reveal the effectiveness of the preconditioning before neuronal injury occurs. METHODS: Middle cerebral artery occlusion (MCAO) was used to induce focal cerebral ischemia-reperfusion injury. 2D-DIGE-MALDI-TOF-MS/MS proteomic technique was employed to screen the differentially expressed proteins in the serum of rats among the control (Con) group (MCAO model without hyperbaric oxygen (HBO) preconditioning), hyperbaric oxygen protective (HBOP) group (in which the infarct volume decreased after HBO preconditioning vs. Con), and hyperbaric oxygen nonprotective (HBOU) group (in which the infarct volume remained the same or even larger after HBO preconditioning vs. Con). Candidate biomarkers were confirmed by western blot and enzyme linked immunosorbent assay (ELISA), and the relationship between the biomarkers and the prognosis of cerebral injury was further validated. RESULTS: Among the 15 differentially expressed protein spots detected in the HBOP group by Two-dimensional fluorescence difference gel electrophoresis (2D-DIGE), 3 spots corresponding to 3 different proteins (haptoglobin, serum albumin, and haemopexin) products were identified by MALDI-TOF-MS/MS. Serum albumin and haemopexin were upregulated, and haptoglobin was downregulated in the HBOP group (p < 0.05 vs. Con and HBOU groups). After the western blot study, only the changes in haemopexin were validated and exhibited similar changes in subjects from the HBOP group in accordance with MALDI-TOF-MS/MS proteomic analysis and enzyme linked immunosorbent assay (ELISA) analysis. The serum level of the hemopexin (HPX) at 2 h after HBO preconditioning was correlated with the infarct volume ratio after MCAO. CONCLUSIONS: Haemopexin may be developed as a predictive biomarker that indicated the effectiveness of a preconditioning strategy against cerebral ischaemic injury.

A magainin-2 like bacteriocin BpSl14 with anticancer action from fish gut Bacillus safensis SDG14.

Bacteriocins are gaining utmost importance in antimicrobial and chemotherapy due to their diverse structure and activity. This study centres on magainin-2 like bacteriocin with anticancer action, produced by Bacillus safensis strain SDG14 isolated from gut of marine fish Sardinella longiceps. The purified bacteriocin designated as BpSl14 was thermostable and pH tolerant. The molecular weight of BpS114 was estimated to be 6061.2 Da using MALDI-ToF MS. The partial primary sequence was elucidated by peptide mass fingerprinting using MALDI MS/MS. The tertiary structure of the partial sequence was similar to that of two magainin-2 α-helices joined together by extended indolicidin. The BpSl14 protein inhibited the cells of lung carcinoma, one of the deadliest cancers. Docking studies conducted with DR5 and TGF-ß, two of the most prominent apoptotic receptors in adenocarcinoma, also proved the anti-apoptotic action of BpSl14.

Expression regulation of chitin-binding protein and metal-binding peptide in new Bacillus velezensis: MALDI-TOF MS/MS analysis.

Metallothionein and metal-binding peptides are small cysteine-rich proteins produced by different organisms in stress conditions. In this study, the metal-binding peptide was detected in extracellular proteins of a new Bacillus velezensis strain, isolated from metal contaminated soil, and grown on the lead-enriched medium, for the first time. The presence of sulfide peptide was assayed by two simple tests (lead sulfide and Ellman's reagent test) for preliminary, and subsequently confirmed using polyacrylamide gel electrophoresis at media with different lead concentrations that the low-molecular-weight protein fragments (≈10 kDa) were observed while none were detected in the medium containing sodium chloride or calcium salt. The amino acids of the observed fragments were analyzed by matrix-assisted laser desorption ionization time-of-flight tandem mass spectrometry (MALDI-TOF MS/MS). Also, the metal adsorption was confirmed using sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) by staining with chromium solution. The results showed that the putative sulfide peptide is metallothionein, which is induced in stress conditions. It was interesting that in all SDS profiles, one protein fragment (≈18 kDa) was inhibited in lead-enriched media. The data from MALDI-TOF MS/MS analysis showed that this fraction was a chitin-binding protein whose production was regulated by metal contamination. It is anticipated that these two proteins regulate the toxicity of lead.

Antibacterial substances produced by pathogen inhibitory gut bacteria in Labeo rohita: Physico-chemical characterization, purification and identification through MALDI-TOF mass spectrometry.

Application of antibiotics to combat bacterial diseases in fish has been criticized due to likely emergence of drug resistance. Therefore, investigation of new bioactive compounds from natural sources has been taken into account. This study was designed to purify and characterize the bioactive compound in the cell free supernatant (CFSs) of autochthonous gut bacteria (Bacillus methylotrophicus KU556164, B. amyloliquefaciens KU556165, Pseudomonas fluorescens KU556166 and B. licheniformis KU556167) isolated from rohu, Labeo rohita. CFSs were antagonistic to fish pathogenic Aeromonas spp., moderately thermo-tolerant and active in wide range of pH (5-11). Antibacterial activity of the CFSs was reduced by the action of proteases (e.g., Proteinase K and Trypsin), indicating proteinaceous nature of the bioactive compound like the bacteriocins. Three-step purification procedure resulted in recovery of 16.97%, 18.04%, 33.33% and 6.38% activity of the antimicrobial protein produced by B. methylotrophicus, B. amyloliquefaciens, P. fluorescens and B. licheniformis, respectively. Purification at each step revealed decrease in protein content with gradual increase in the specific activity of the antimicrobial protein. The purified antibacterial compound ranged between 18.2 and 25.6 kDa. Identification through MALDI-TOF MS/MS and database search through Mascot search engine predicted that the bactericidal compound belonged to either alkaline proteases, or, transcriptional regulator and some hypothetical proteins. Apart from potential technological application of the antibacterial compound, the present study might show promise for application of gut-associated bacteriocinogenic bacteria to control diseases in fish caused by pathogenic bacteria.

Elevated levels of circulating ITIH4 are associated with hepatocellular carcinoma with nonalcoholic fatty liver disease: from pig model to human study.

BACKGROUND: Noninvasive biomarkers are urgently needed for optimal management of nonalcoholic fatty liver disease (NAFLD) for the prevention of disease progression into nonalcoholic steatohepatitis (NASH) and hepatocellular carcinoma (HCC). In order to identify the biomarkers, we generated the swine hepatocellular carcinoma (HCC) model associated with NAFLD and performed serum proteomics on the model. METHODS: Microminipigs were fed a high-fat diet to induce NAFLD and a normal diet as the control. To induce HCC, diethylnitrosamine was intraperitoneally administered. Biopsied liver samples were histopathologically analyzed every 12 weeks. Serum proteins were separated by blue native two-dimensional gel electrophoresis and proteins of interest were subsequently identified by MALDI-TOF MS/MS. Human serum samples were analyzed to validate the candidate protein using antibody-mediated characterization. RESULTS: In the NAFLD pigs, hepatic histology of nonalcoholic steatohepatitis (NASH) was observed at 36 weeks, and HCC developed at 60 weeks. Among serum proteins identified with MALDI-TOF MS/MS, serum inter-alpha-trypsin inhibitor heavy chain 4 (ITIH4), an acute response protein which is secreted primarily by liver, was identified as the most characteristic protein corresponding with NAFLD progression and HCC development in the NAFLD pigs. With immunoassay, serum ITIH4 levels in the NAFLD pigs were chronologically increased in comparison with those in control animal. Furthermore, immunohistochemistry showed ITIH4 expression in hepatocytes also increased in both the cancer lesions and parenchyma as NAFLD progressed. Human study is also consistent with this observation because serum ITIH4 levels were significantly higher in HCC-NAFLD patients than in the simple steatosis, NASH, and virus-related HCC patients. Of note, HCC-NAFLD patients who had higher serum ITIH4 levels exhibited poorer prognosis after hepatectomy. CONCLUSIONS: We established an HCC pig model associated with NAFLD. Serum proteomics on the swine HCC with NAFLD model implicated ITIH4 as a non-invasive biomarker reflecting NAFLD progression as well as subsequent HCC development. Most importantly, the results in the swine study have been validated in human cohort studies. Dissecting speciation of serum ITIH4 promises to have clinical utility in monitoring the disease.

Cyanobacterial bioactive compound EMTAHDCA recovers splenomegaly, affects protein profile of E. coli and spleen of lymphoma bearing mice.

The antibacterial and anticancerous properties of EMTAHDCA have already been reported in our previous study. However, mode of action of EMTAHDCA is still elusive. The present study was aimed to investigate the molecular targets in Escherichia coli and spleen of lymphoma-bearing mice in response to cyanocompound 9-ethyliminomethyl-12 (morpholin-4-ylmethoxy)-5, 8, 13, 16-tetraaza -hexacene-2, 3- dicarboxylic acid (EMTAHDCA) isolated from fresh water cyanobacterium Nostoc sp. MGL001. Differential expressions of proteins were observed in both E. coli and spleen of lymphoma-bearing mice after EMTAHDCA treatment. In continuation of our previous study, the present study revealed that the antibacterial agent, EMTAHDCA causes the drastic reduction in synthesis of proteins related to replication, transcription, translation and transportation in E. coli. Probably the direct or indirect interaction of this compound with these important metabolic processes led to the reduction in growth and cell death. Furthermore, the anticancerous property of the compound EMTAHDCA reflected as down regulation in proteins of cell cycle, cellular metabolism, signalling, transcription and transport together with up regulation of apoptosis, DNA damage and immunoprotection related proteins in spleen of lymphoma-bearing mice. In this study the EMTAHDCA induced modulations in expression of proteins of key metabolic pathways in E. coli and spleen cells of lymphoma bearing mice helped in understanding the mechanism underlying the antibacterial and anti-cancerous property.

Proteomic Characterization of Primary Mouse Hepatocytes in Collagen Monolayer and Sandwich Culture.

Dedifferentiation of primary hepatocytes in vitro makes their application in long-term studies difficult. Embedding hepatocytes in a sandwich of extracellular matrix is reported to delay the dedifferentiation process to some extent. In this study, we compared the intracellular proteome of primary mouse hepatocytes (PMH) in conventional monolayer cultures (ML) to collagen sandwich culture (SW) after 1 day and 5 days of cultivation. Quantitative proteome analysis of PMH showed no differences between collagen SW and ML cultures after 1 day. Glycolysis and gluconeogenesis were strongly affected by long-term cultivation in both ML and SW cultures. Interestingly, culture conditions had no effect on cellular lipid metabolism. After 5 days, PMH in collagen SW and ML cultures exhibit characteristic indications of oxidative stress. However, in the SW culture the defense system against oxidative stress is significantly up-regulated to deal with this, whereas in the ML culture a down-regulation of these important enzymes takes place. Regarding the multiple effects of ROS and oxidative stress in cells, we conclude that the down-regulation of these enzymes seem to play a role in the loss of hepatic function observed in the ML cultivation. In addition, enzymes of the urea cycle were clearly down-regulated in ML culture. Proteomics confirms lack in oxidative stress defense mechanisms as the major characteristic of hepatocytes in monolayer cultures compared to sandwich cultures. J. Cell. Biochem. 119: 447-454, 2018. © 2017 Wiley Periodicals, Inc.

Effects of novel brominated flame retardant TBBPA on human airway epithelial cell (A549) in vitro and proteome profiling.

The cellular toxicity response of human airway epithelial cells (A549) to tetrabromobisphenol (TBBPA) was assessed in vitro. Cell viability, levels of intracellular reactive oxygen species (ROS), lipid peroxidation (MDA), and caspase-3 activity were determined after A549 treated with varying concentrations of TBBPA. A comparative proteomic analysis was performed in cells treated with different concentrations of TBBPA (0, 10, and 40 µg/mL). Two-way anova analysis showed that cell viability was significantly decreased after treatment by TBBPA with a concentration of 16 µg/mL for 48 hr, however, the caspase-3 activities, ROS generation, and MDA content increased. Ultrastructural observation revealed that the cell was morphological damaged after exposure to 64 µg/mL TBBPA, with mitochondria seriously injured and the smooth endoplasmic reticulum dilated. There was a good correlation between ROS generation and mitochondrial dysfunction. Seventeen differentially expressed proteins involved in various biological processes were identified. These findings provide a basis for understanding the mechanisms of cell dysfunction and perturbation of antioxidant status induced by additive flame retardant on airway epithelial cells.

Characterization of a novel allergenic protein from the octocoral Scleronephthya gracillima (Kuekenthal) that corresponds to a new GFP-like family named Akane.

Certain marine organisms have been known to cause allergic reactions among occupational fishermen. We have previously reported that bronchial asthma among the workers engaged in spiny lobster fishing in Japan was caused by octocorals such as Dendronephthya sp. and Scleronephthya gracillima (previously named Alcyonium gracillimum). Now we have found another octocoral, Scleronephthya gracillima (Kuekenthal), which causes the allergic disease in fishermen. The octocoral was characterized as a new green fluorescent protein (GFP)-like family. The new allergen has a molecular mass of 27 kDa in 1D and 2D SDS-PAGE under reduced conditions. The 27 kDa component was determined to be an allergen by western blotting, ECL immune staining method and absorption of patient sera with the antigen. Furthermore, the combination of analysis with LC-ESI-MS/MS and MASCOT search in the NCBInr database concluded the 27 kDa component had the sequence YPADI/LPDYFK, and that the 22 kDa component had the sequence QSFPEGFSWER, which both matched a GFP-like protein in Acropora aculeus and in Montastraea annularis. Further analysis by MALDI-TOF/MS/MS and MASCOT search in the NCBInr database of all 27 kDa eight spot components from 2D SDS-PAGE indicated that the sequence QSFPEGFSWER also matched as GFP-like protein in Lobophyllia hemprichii and Scleractinia sp. To our knowledge, this is the first report of the new allergenic protein that corresponds to a new GFP-like protein named Akane, and which has fluorescent emissions in the red and green part of the spectra at 628 nm and 508 nm, respectively.

Comparative proteome analysis of egg yolk plasma proteins during storage.

BACKGROUND: Physical changes such as chicken egg white thinning and egg yolk flattening occur during storage, implying a decline in egg quality. To reveal the deteriorative process related to chicken egg internal quality, a comparative proteomic method was used in this study to analyze the alterations in egg yolk plasma proteins at different storage times (0, 20 and 40 days) under an ambient temperature of 22 ± 2 °C. RESULTS: Using two-dimensional electrophoresis followed by matrix-assisted laser desorption/ionization time-of-flight tandem mass spectrometry, 33 protein spots representing 12 proteins were identified with significant (P < 0.05) alterations in abundance at different storage times. The proteins that showed significant changes in abundance included serum albumin, vitellogenin fragments, IgY chains, ovalbumin, ovoinhibitor, α2 -macroglobulin-like protein 1-like, hemopexin, transthyretin, apolipoprotein A-I and ß2 -glycoprotein I precursor. Accelerating degradation for most egg yolk plasma proteins was observed after prolonged storage (from day 20 to day 40). CONCLUSION: It is likely that the increased degradation of protease inhibitors such as ovoinhibitor and α2 -macroglobulin-like protein 1-like during prolonged storage lead to an imbalance of protease and antiprotease in egg yolk, which may play a key role in the degradation of egg yolk proteins. These findings will provide an insight into the effects of storage on egg yolk protein changes and give a deeper understanding of the deteriorative process of chicken egg yolk. © 2016 Society of Chemical Industry.

Identification and comparative proteomic study of quail and duck egg white protein using 2-dimensional gel electrophoresis and matrix-assisted laser desorption/ionization time-of-flight tandem mass spectrometry analysis.

A proteomic study of egg white proteins from 2 major poultry species, namely quail (Coturnix coturnix) and duck (Anas platyrhynchos), was performed with comparison to those of chicken (Gallus gallus) through 2-dimensional polyacrylamide gel electrophoresis (2-DE) analysis. By using matrix-assisted laser desorption/ionization time-of-flight tandem mass spectrometry (MALDI-TOF MS/MS), 29 protein spots representing 10 different kinds of proteins as well as 17 protein spots designating 9 proteins were successfully identified in quail and duck egg white, respectively. This report suggested a closer relationship between quail and chicken egg white proteome patterns, whereas the duck egg white protein distribution on the 2-DE map was more distinct. In duck egg white, some well-known major proteins, such as ovomucoid, clusterin, extracellular fatty acid-binding protein precursor (ex-FABP), and prostaglandin D2 synthase (PG D2 synthase), were not detected, while two major protein spots identified as "deleted in malignant brain tumors 1" protein (DMBT1) and vitellogenin-2 were found specific to duck in the corresponding range on the 2-DE gel map. These interspecies diversities may be associated with the egg white protein functions in cell defense or regulating/supporting the embryonic development to adapt to the inhabiting environment or reproduction demand during long-term evolution. The findings of this work will give insight into the advantages involved in the application on egg white proteins from various egg sources, which may present novel beneficial properties in the food industry or related to human health.

Thermostable lipase from Geobacillus sp. Iso5: bioseparation, characterization and native structural studies.

The extracellular thermoalkaline lipase from Geobacillus sp. Iso5 was purified to homogeneity by ultrafiltration, 6% cross-linked agarose and Phenyl spehrose HIC column chromatography. The final purified lipase resulted in 8.7-fold with 6.2% yield. The relative molecular weight of the enzyme was determined to be a monomer of 47 kDa by SDS-PAGE and MALDI-TOF MS/MS spectroscopy. The purified enzyme exhibit optimum activity at 70 °C and pH 8.0. The enzyme retained above 90% activity at temperatures of 70 °C and about 35% activity at 85 °C for 2 h. However, the stability of the enzyme decreased at the temperature over 90 °C. The enzyme activity was promoted in the presence of Ca(2+) and Mg(2+) and strongly inhibited by HgCl2 , PMSF, DTT, K(+) , Co(2+) , and Zn (2+) . EDTA did not affect the enzyme activity. The secondary structure of purified lipase contains 36% α-helix and 64% ß-sheet which was determined by Circular dichromism, FTIR, and Raman Spectroscopy.

Serine protease identification (in vitro) and molecular structure predictions (in silico) from a phytopathogenic fungus, Alternaria solani.

Serine proteases are involved in an enormous number of biological processes. The present study aims at characterizing three-dimensional (3D) molecular architecture of serine proteases from early blight pathogen, Alternaria solani that are hypothesized to be markers of phytopathogenicity. A serine protease was purified to homogeneity and MALDI-TOF-MS/MS analysis revealed that protease produced by A. solani belongs to alkaline serine proteases (AsP). AsP is made up of 403 amino acid residues with molecular weight of 42.1 kDa (Isoelectric point - 6.51) and its molecular formula was C1859 H2930 N516 O595 S4 . AsP structure model was built based on its comparative homology with serine protease using the program, MODELER. AsP had 16 ß-sheets and 10 α-helices, with Ser(350) (G347-G357), Asp(158) (D158-H169), and His(193) (H193-G203) in separate turn/coil structures. Biological metal binding region situated near 6th-helix and His(193) residue is responsible for metal binding site. Also, calcium ion (Ca(2+)) is coordinated by the carboxyl groups of Lys(84), Ile(85), Lys(86), Asp(87), Phe(88), Ala(89), Ala(90) (K84-A90) for first Ca(2+) binding site and carbonyl oxygen atom of Lys(244), Gly(245), Arg(246), Thr(247), Lys(248), Lys(249), and Ala(250) (K244-A250), for second Ca(2+) binding site. Moreover, Ramachandran plot analysis of protein residues falling into most favored secondary structures were determined (83.3%). The predicted molecular 3D structural model was further verified using PROCHECK, ERRAT, and VADAR servers to confirm the geometry and stereo-chemical parameters of the molecular structural design. The functional analysis of AsP 3D molecular structure predictions familiar in the current study may provide a new perspective in the understanding and identification of antifungal protease inhibitor designing.

Analysis of Caenorhabditis Protein Glycosylation.

Glycoproteins result from post-translational modification of proteins by glycans attached to certain side chains, with possible heterogeneity due to different structures being possible at the same glycosylation site.In contrast to the mammalian systems, analysis of invertebrate glycans presents a challenge in analysis as there exist unfamiliar epitopes and a high degree of structural and isomeric variation between different species-Caenorhabditis elegans is no exception. Simple screening using lectins and antibodies can yield hints regarding which glycan epitopes are present in wild-type and mutant strains, but detailed analysis is necessary for determining more exact glycomic information. Here, our analytical approach is to analyze N- and O-glycans involving "off-line" RP-HPLC MALDI-TOF MS/MS. Enrichment and labeling steps facilitate the analysis of single structures and provide isomeric separation. Thereby, the "simple" worm expresses over 200 N-glycan structures varying depending on culture conditions or the genetic background.

A high-throughput sequence determination for one cyclic peptide immobilized on a single bead by the one-pot cyanidation followed by MALDI-TOF-MS/MS for discovery of interacting peptides.

One cyclic peptide immobilized on one gel-type bead has been employed for the discovery of both interacting peptides and/or medicinal medium-sized molecules. Although high-throughput characterization of recognized peptides has been a bottleneck, here, we describe direct liberation from beads by a one-pot reaction using 2-nitro-5-thiocyanatobenzoic acid followed by mass spectrometry to realize faster and routine sequencing of the peptide on the beads. This is useful for the investigation of protein-protein interactions as well as discovery of drug candidates.

Differences in protein changes between stress-induced premature senescence and replicative senescence states.

Replicative senescence and stress-induced premature senescence (SIPS) cells are known to share certain traits. However, whether these cells are different at the protein level is unclear. Thus, this study has utilized proteomics to identify differences in the proteomes of replicative senescence and SIPS cells compared to normal cells. Replicative senescence was induced by serial passage of normal cells in culture. SIPS was established by exposure to H2 O2 at a subcytotoxic concentration of 20 µM for two weeks. Following 2DE, protein profiles were compared and protein spots that changed in abundance were identified by MALDI-TOF MS. Quantitative real-time RT-PCR was then performed to evaluate the transcript expression of selected altered proteins. A total of 24 and 10 proteins were found to have changed in abundance in replicative senescence and SIPS cells, respectively, when compared to young cells. Quantitative RT-PCR revealed that nine genes showed the same direction of change as observed in the proteomics analysis. Very little overlap was observed between proteins that changed in replicative senescence and SIPS cells, suggesting that although both SIPS and replicative senescence cells share hallmarks of cellular senescence, they were different in terms of proteins that changed in abundance.

Angiotensin I-converting enzyme inhibitory peptides derived from bovine casein and identified by MALDI-TOF-MS/MS.

BACKGROUND: Hypertension is a major and common threat to the health of individuals around the world. Although agents such as captopril have been shown to regulate high blood pressure effectively, they bring unfavourable side effects such as dry cough and angioedema. If angiotensin I-converting enzyme (ACE) inhibitors derived from natural substances such as milk proteins can be shown to be safe and efficient at managing hypertension, such inhibitors will be a valuable auxiliary to agents such as captopril. RESULTS: Low-molecular-weight casein-derived peptides hydrolysed by cell envelope proteinase of Lactobacillus casei subsp. casei (ATCC 15008) showed quite high ACE-inhibitory activity. The peptide fraction from α-casein with molecular weight between 5 and 10 kDa showed the highest ACE-inhibitory activity of 82.35%, with a 50% inhibition concentration (IC50 ) of 2.36 mg mL(-1). Peptides from ß-casein exhibited lower ACE-inhibitory activity (56.67%, IC50 4.00 mg mL(-1)). Three distinct peptide sequences derived from α-casein (α(s1) -cn f95-105, f106-115 and f148-166) were identified using two-dimensional gel electrophoresis coupled with matrix-assisted laser desorption/ionisation time-of-flight tandem mass spectrometry. CONCLUSION: This work investigated the ACE-inhibitory properties of casein-derived peptides. Three distinct peptide sequences derived from α-casein were identified. Characterisation of such peptides furthers the investigation of casein-derived ACE-inhibitory peptides from fermented dairy products.

2D-DIGE Analysis of Liver Disease in Mice.

Hepatocellular carcinoma (HCC) is the major type of primary liver cancer. In this chapter, we describe our routine two-dimensional difference gel electrophoresis (2D-DIGE) workflow for analysis of mouse liver tissue in physiological conditions, as well as of mouse HCC. 2D-DIGE still constitutes a valuable comparative proteomics technique, not only providing information on global protein expression in a sample but also on potential posttranslational protein modifications, occurrence of protein degradation fragments, and the existence of protein isoforms. Thus, 2D-DIGE analysis provides highly complementary data to non-gel-based shotgun mass spectrometry (MS) methods (e.g., liquid chromatography (LC)-MS/MS)-allowing, for example, identification of novel protein biomarkers for HCC or increasing insights into the molecular mechanisms underlying hepatocarcinogenesis.

Analysis of Modification of Liver Proteome in Diabetic Rats by 2D Electrophoresis and MALDI-TOF-MS.

The uncontrolled hyperglycemia can lead to disturbances in the cell structure and functions of organs. This study was performed to analyze the "differential proteome" change in rat liver associated with diabetes mellitus in relation to effects of an anti-diabetic herb, Cynodon dactylon leaf extracts. Rats were intraperitoneally injected with alloxan (150 mg/kg/bw) and treated with C. dactylon leaf extracts (450 mg/kg/bw/day/orally). The liver proteins were subjected to proteome analysis using the advanced technologies i.e., 2D electrophoresis (2-DE) and mass spectrometry. Comparison of 2-DE protein distribution profiles among the livers from normal, alloxan-induced diabetic rats and alloxan-induced diabetic rats treated with C. dactylon leaves identified three proteins that were up-regulated in alloxan-induced diabetic rats i.e., nucleophosmin, l-xylulose reductase and carbonic anhydrase III which are known to be mainly involved in ribosome biogenesis, centrosome duplication, cell proliferation, tumor suppression, glucose metabolism, osmo-regulation, water-CO2 balance and acid-base balance. These results help us to understand the elucidation of molecular mechanism connected to liver function and insulin associated with diabetes mellitus. These identified proteins were primarily involved in cell proliferation and homoeostasis of liver tissues upon the treatment with C. dactylon leaf extracts.

Proteomics analysis reveals several metabolic alterations in cyanobacterium Anabaena sp. NC-K1 in response to alpha-cypermethrin exposure.

In the current study, the effect of the EC50 and LC90 concentrations of pyrethroid insecticide alpha-cypermethrin to cyanobacteria Anabaena sp. NC-K1 was investigated at different time exposures (1st day, 4th day and 7th day) with reference to growth, photosynthetic pigments, oxidative damage and antioxidant defence system. Superoxide dismutase (1.38-fold), peroxidase (5.04) and proline content (2.27-fold) were enhanced compared to the control. After performing 2D gel electrophoresis at 1st day EC50 exposure, where appropriate differences in the biochemical and physiological parameters were observed, 22 differentially accumulated proteins (20 upregulated and 2 downregulated) were selected for mass spectrometry. Out of 42 proteins identified, 20 upregulated protein spots were classified into twelve categories according to their metabolic functions. Proteins related to photosynthesis (phycobilisome rod-core linker polypeptide, rubisco), stress responses (Hsp70, Hsp40, catalase family peroxidase), translation (elongation factor Tu) and amino acid biosynthesis and metabolism (3-phosphoshikimate 1-carboxyvinyl transferase) were significantly upregulated. Additionally, proteins involved in transcription and DNA repair (Snf-2 histone linker phd ring helicase, RNA polymerase sigma factor RpoD and Holliday junction ATP-dependent DNA helicase RuvA) were considerably upregulated. Upregulation of these proteins against pesticide stress presumably maintained the photosynthesis, energy metabolism, carbohydrate metabolism, transport and signalling proteins, transcription, translation and DNA repair. Additionally, these proteins might involve in sufficient detoxification of ROS and play a crucial role in damage removal and repair of oxidized proteins, lipids and nucleic acids. Taken together, Anabaena sp. NC-K1 responded towards alpha-cypermethrin stress via modulating its proteome to maintain its cellular metabolism and homeostasis.