Two-dimensional Protein Analysis Research Articles

Molecular form identification of anterior pituitary gland-secreted prolactin in chicken

Endocrine changes during bird reproduction are well documented. Prolactin (PRL) exhibits a strong relationship between incubation and broody behavior. The molecular forms of PRL in the anterior pituitary gland during the reproductive cycle have already been previously identified but not those in the secreted form. To identify the molecular forms of secreted PRL during the reproductive cycle, we thus monitored the physiological status and incubation behavior of 10 Silkie hens by a video recording system over 1–2 years. Nine out of ten mature hens exhibited incubation behavior multiple times during the experiment. Ten hens demonstrated two interesting features. In a typical clutch, hens spent 10–15 min in the nest to lay an egg. Once they spent over 1 h in the nest, the nest occupancy increased incrementally. This shift in the nest occupancy occurred 7–10 days before the incubation onset and was highly repeatable. Based on the behavior of the hens, we cultured the anterior pituitary gland during four stages (premature non-laying, laying, trans, and incubation) with physiological PRL-releasing factor, vasoactive intestinal peptide (VIP). Based on our two-dimensional protein analysis, glycosylated PRL (G-PRL) displayed several isoforms with varying isoelectric points (pI), whereas we could detect one primary signal for non-glycosylated PRL (NG-PRL). However, 3–4 NG-PRL isoforms were detected in the anterior pituitary gland. These results suggested that secreted PRL, especially from the trans and incubation stages, contains various isoforms and it is post-translationally glycosylated and phosphorylated.

Change of Ras and its guanosine triphosphatases (GTPases) during development and regression in bovine corpus luteum

The aim of this study was to determine the change of Ras and its guanosine triphosphatases (GTPases) proteins in the bovine corpus luteum (CL) during estrous cycle and investigate protein-protein interaction between hormone receptors and Ras proteins via angiogenetic and apoptotic factors using bioinformatics database. The bovine CLs at proliferation phase (PP), secretion phase (SP), and regression phase (RP) were dissected from abattoir ovaries (n = 4/stage), whole of the tissue samples was used to analyze two-dimensional electrophoresis (2-DE), mRNA, and protein analysis. The protein-protein interaction between the Ras GTPases proteins and hormone receptors were analyzed using Search Tool for the Retrieval of Interacting Genes (STRING) database. The Ras protein activator like 3 (RASAL3), Ras GTPase activating protein 3 (RASA3), Ras guanine nucleotide exchange factors 1 beta (RasGEF1B) were discovered by the 2-DE and mass spectrometry in bovine CLs, and the protein spots of RASA3 and RASAL3 were significantly increased in the SPCL compared to the PPCL, whereas the RasGEF1B was reduced in the PPCL (P < 0.05). The mRNA and proteins expression of progesterone receptor, estrogen receptor alpha (ERα), vascular endothelial growth factor A (VEGFA), angiopoietin 1 (Ang1), VEGF receptor2 (VEGFR2), and Tie2 were significantly increased, but intrinsic and extrinsic apoptotic factors were decreased in PPCL and SPCL compared to RPCL (P < 0.05). Based on STRING database, we determined that RasGEF1B is activated by ERα via VEGFA and VEGFR2, then RasGEF1B activates H-Ras and R-Ras. In addition, the RasGAP protein was significantly increased, however, the RasGEF, H-Ras and R-Ras proteins were reduced in SPCL compared to PPCL and RPCL (P < 0.05). In summary, the RasGEF and Ras proteins were raised during the development, whereas the RasGAP was increased when development was completed, then the Ras and its GTPases dramatically decreased at the regression in bovine CL. In conclusion, these results suggest that Ras and Ras GTPases could be changed during development and regression, activated by the ERα via angiogenetic signaling during proliferation, and may be important to understanding of the Ras and its GTPases system for estrous cycle in bovine CL.

Towards reducing the immunogenic potential of wheat flour: omega gliadins encoded by the D genome of hexaploid wheat may also harbor epitopes for the serious food allergy WDEIA

BackgroundOmega-5 gliadins are a group of highly repetitive gluten proteins in wheat flour encoded on the 1B chromosome of hexaploid wheat. These proteins are the major sensitizing allergens in a severe form of food allergy called wheat-dependent exercise-induced anaphylaxis (WDEIA). The elimination of omega-5 gliadins from wheat flour through biotechnology or breeding approaches could reduce the immunogenic potential and adverse health effects of the flour.ResultsA mutant line missing low-molecular weight glutenin subunits encoded at the Glu-B3 locus was selected previously from a doubled haploid population generated from two Korean wheat cultivars. Analysis of flour from the mutant line by 2-dimensional gel electrophoresis coupled with tandem mass spectrometry revealed that the omega-5 gliadins and several gamma gliadins encoded by the closely linked Gli-B1 locus were also missing as a result of a deletion of at least 5.8 Mb of chromosome 1B. Two-dimensional immunoblot analysis of flour proteins using sera from WDEIA patients showed reduced IgE reactivity in the mutant relative to the parental lines due to the absence of the major omega-5 gliadins. However, two minor proteins showed strong reactivity to patient sera in both the parental and the mutant lines and also reacted with a monoclonal antibody against omega-5 gliadin. Analysis of the two minor reactive proteins by mass spectrometry revealed that both proteins correspond to omega-5 gliadin genes encoded on chromosome 1D that were thought previously to be pseudogenes.ConclusionsWhile breeding approaches can be used to reduce the levels of the highly immunogenic omega-5 gliadins in wheat flour, these approaches are complicated by the genetic linkage of different classes of gluten protein genes and the finding that omega-5 gliadins may be encoded on more than one chromosome. The work illustrates the importance of detailed knowledge about the genomic regions harboring the major gluten protein genes in individual wheat cultivars for future efforts aimed at reducing the immunogenic potential of wheat flour.

Proteome analysis reveals global response to deletion of mrflbA in Monascus ruber.

Monascus spp. are commonly used for a wide variety of applications in the food and pharmaceutical industries. In previous studies, the knock-out of mrflbA (a putative regulator of the G protein α subunit) in M. ruber led to autolysis of the mycelia, decreased pigmentation and lowered mycotoxin production. Therefore, we aimed to obtain a comprehensive overview of the underlying mechanism of mrflbA deletion at the proteome level. A two-dimensional gel electrophoresis analysis of mycelial proteins indicated that the abundance of 178 proteins was altered in the ΔmrflbA strain, 33 of which were identified with high confidence. The identified proteins are involved in a range of activities, including carbohydrate and amino acid metabolism, hyphal development and the oxidative stress response, protein modification, and the regulation of cell signaling. Consistent with these findings, the activity of antioxidative enzymes and chitinase was elevated in the supernatant of the ΔmrflbA strain. Furthermore, deletion of mrflbA resulted in the transcriptional reduction of secondary metabolites (pigment and mycotoxin). In short, the mutant phenotypes induced by the deletion of mrflbA were consistent with changes in the expression levels of associated proteins, providing direct evidence of the regulatory functions mediated by mrflbA in M. ruber.

Rhodococcus wratislaviensis strain 9: An efficient p-nitrophenol degrader with a great potential for bioremediation

A Gram-positive bacterium, Rhodococcus wratislaviensis strain 9, was isolated from groundwater contaminated with nitrophenolics and trichloroethene following enrichment culture technique. The cells of strain 9 grown on LB broth (uninduced) degraded 720 μM p-nitrophenol (PNP) within 12 h, and utilized as a source of carbon and energy. Orthogonal experimental design analysis to determine optimal conditions for biodegradation of PNP showed that pH had a significant positive effect (P ≤ .05) on bacterial degradation of PNP, while glucose, di- and tri-nitrophenols exhibited significant negative effect. Cell-free extracts obtained from PNP-grown culture that contained 20 μg mL−1 protein degraded 90% of 720 μM PNP within 5 h of incubation. Two-dimensional protein analysis revealed differential expression of the oxygenase component of PNP monooxygenase and an elongation factor Tu in PNP-grown cells, but not in those grown on glucose. The strain 9 remediated laboratory wastewater containing 900 μM PNP efficiently within 14 h, indicating its great potential in bioremediation of PNP-contaminated waters.

Comparison of fractionation strategies for offline two-dimensional liquid chromatography tandem mass spectrometry analysis of proteins from mouse adipose tissue

In the frame of protein identification from mouse adipose tissue, two strategies were compared for the offline elution of peptides from a strong cation exchange (SCX) column in two-dimensional liquid chromatography tandem mass spectrometry (2D–LC–MS/MS) analyses. First, the salt gradient (using K+ as displacing agent) was evaluated from 25 to 500mM KCl. Then, a less investigated elution mode using a pH gradient (using citric acid and ammonium hydroxide) was carried out from pH 2.5 to 9.0. Equal amounts of peptide digest derived from mouse adipose tissue were loaded onto the SCX column and fractionated according to the two approaches. A total of 15 fractions were collected in two independent experiments for each SCX elution strategy. Then, each fraction was analyzed on a nanoLC–MS/MS platform equipped with a column-switching unit for desalting and enrichment. No substantial differences in peptide quality characteristics (molecular weight, isoelectric point, or GRAVY [grand average of hydropathicity] index distributions) were observed between the two datasets. The pH gradient approach was found to be superior, with 27.5% more unique peptide identifications and 10% more distinct protein identifications compared with the salt-based elution method. In conclusion, our data imply that the pH gradient SCX fractionation is more desirable for proteomics analysis of entire adipose tissue.

Proteomic identification, characterization and expression analysis of Ctenopharyngodon idella VDAC1 upregulated by grass carp reovirus infection

Voltage-dependent anion channels (VDACs) located in the mitochondrial outer membrane are mitochondrial porins that play central roles in regulating cell life and death. In this present report, the VDAC protein 1 from grass carp Ctenopharyngodon idella (designated as CiVDAC1) was found to be upregulated by grass carp reovirus (GCRV) infection through two-dimensional gel electrophoresis and protein analysis of infected C. idella kidney (CIK) cells. The full-length cDNA of CiVDAC1 was 995 bp with an open reading frame (ORF) of 852 bp that encodes a putative 283-amino acid protein. Phylogenic analysis revealed that the complete ORF of CiVDAC1 demonstrated high identity with well characterized mammalian homologs. The deduced CiVDAC1 protein contains an α-helix at the amino terminal, 19 membrane-spanning β-strands, and one eukaryotic mitochondrial porin signature motif. Tissue tropism analysis indicated that CiVDAC1 is abundant in muscle, heart, skin, swim bladder, trunk kidney and spleen. Transcriptional expression profiles indicated that the CiVDAC1 gene was upregulated upon viral challenge in a manner similar to the Mx2 gene, which is a marker gene used to indicate activation of innate antiviral immunity. Similar expression patterns of the CiVDAC1 gene were observed in CIK cells stimulated with poly (I:C), as well as grass carp kidney tissue challenged with GCRV in vivo. CiVDAC1 silencing in CIK cells had no impact on progeny virus production, but over-expression of CiVDAC1 in vivo showed strongly protect against challenge with live virus. To interpret the role of other VDAC proteins in viral pathogenesis, CiVDAC2 was characterized and showed to respond positively to GCRV challenge, which suggested that CiVDAC2 might functionally complement CiVDAC1 in C. idella. The present data did demonstrate that CiVDAC1 might be mediated grass carp antiviral immune response.

Whole-Exome Capture and Sequencing Identifies HEATR2 Mutation as a Cause of Primary Ciliary Dyskinesia

Motile cilia are essential components of the mucociliary escalator and are central to respiratory-tract host defenses. Abnormalities in these evolutionarily conserved organelles cause primary ciliary dyskinesia (PCD). Despite recent strides characterizing the ciliome and sensory ciliopathies through exploration of the phenotype-genotype associations in model organisms, the genetic bases of most cases of PCD remain elusive. We identified nine related subjects with PCD from geographically dispersed Amish communities and performed exome sequencing of two affected individuals and their unaffected parents. A single autosomal-recessive nonsynonymous missense mutation was identified in HEATR2, an uncharacterized gene that belongs to a family not previously associated with ciliary assembly or function. Airway epithelial cells isolated from PCD-affected individuals had markedly reduced HEATR2 levels, absent dynein arms, and loss of ciliary beating. MicroRNA-mediated silencing of the orthologous gene in Chlamydomonas reinhardtii resulted in absent outer dynein arms, reduced flagellar beat frequency, and decreased cell velocity. These findings were recapitulated by small hairpin RNA-mediated knockdown of HEATR2 in airway epithelial cells from unaffected donors. Moreover, immunohistochemistry studies in human airway epithelial cells showed that HEATR2 was localized to the cytoplasm and not in cilia, which suggests a role in either dynein arm transport or assembly. The identification of HEATR2 contributes to the growing number of genes associated with PCD identified in both individuals and model organisms and shows that exome sequencing in family studies facilitates the discovery of novel disease-causing gene mutations.

Two-dimensional Electrophoresis Analysis of Proteins in Response to Cold Stress in Extremely Cold-resistant Winter Wheat Dongnongdongmai 1 Tillering Nodes

The overwintering survival ratio of the cultivar Dongnongdongmai 1 with strong cold-resistance in paramos of Heilongjiang Province in China are over 85%. The tillering nodes are the most important organs for overwintering survival of winter wheat, because there are more substances associated with cold resistance in tillering nodes than those in leaves and roots. Proteins in the tillering nodes of the cold-resistant cultivar Dongnongdongmai 1 grown under field conditions with or without any low-temperature stress were analyzed by 2–dimensional electrophoresis and identified by mass spectrometry. In the range of pH 4–7, the expression of 37 proteins showed obvious difference (±more than two fold) in the proteomic maps of cold-stressed and non-stressed tillering nodes, including a new protein spot. All proteins exhibiting the difference in expression were identified using matrix-assisted laser desorption/ionization time-of-flight mass spectrometry, followed by a database search for protein identification and function prediction. Five groups of proteins were confirmed, namely stress-related proteins (22%), metabolism-associated proteins (35%), and signaling molecules (24%), cell wall-binding proteins (5%), unclear proteins (14%). This indicated that tillering node cells supported the energy requirements of plant growth and stress resistance by signal transduction adapting to metabolism and structure.

Analysis of the Aspergillus fumigatus Proteome Reveals Metabolic Changes and the Activation of the Pseurotin A Biosynthesis Gene Cluster in Response to Hypoxia

The mold Aspergillus fumigatus is the most important airborne fungal pathogen. Adaptation to hypoxia represents an important virulence attribute for A. fumigatus. Therefore, we aimed at obtaining a comprehensive overview about this process on the proteome level. To ensure highly reproducible growth conditions, an oxygen-controlled, glucose-limited chemostat cultivation was established. Two-dimensional gel electrophoresis analysis of mycelial and mitochondrial proteins as well as two-dimensional Blue Native/SDS-gel separation of mitochondrial membrane proteins led to the identification of 117 proteins with an altered abundance under hypoxic in comparison to normoxic conditions. Hypoxia induced an increased activity of glycolysis, the TCA-cycle, respiration, and amino acid metabolism. Consistently, the cellular contents in heme, iron, copper, and zinc increased. Furthermore, hypoxia induced biosynthesis of the secondary metabolite pseurotin A as demonstrated at proteomic, transcriptional, and metabolite levels. The observed and so far not reported stimulation of the biosynthesis of a secondary metabolite by oxygen depletion may also affect the survival of A. fumigatus in hypoxic niches of the human host. Among the proteins so far not implicated in hypoxia adaptation, an NO-detoxifying flavohemoprotein was one of the most highly up-regulated proteins which indicates a link between hypoxia and the generation of nitrosative stress in A. fumigatus.

Two-dimensional immunoblot analysis of antigenic proteins of Spirometra plerocercoid recognized by human patient sera

Sparganosis is caused by invasion of Spirometra plerocercoid into various tissues/organs. Subcutaneous sparganosis can be diagnosed and treated by worm removal, while visceral/cerebral sparganosis is not easy to diagnose. The diagnosis depends largely on the detection of specific antibodies circulating in the patients' sera. Previous studies demonstrated that 31 and 36 kDa proteins of the sparganum invoked specific and sensitive antibody responses, but also showed cross reactions with cysticercosis sera. We enriched protein fractions containing 31–36 kDa through gel filtration and examined immune recognition pattern against the patient sera by 2-dimensional electrophoresis (2-DE) followed by immunoblotting. Serum samples from sparganosis patients recognized 8–10 protein spots of 31 and 36 kDa with different isoelectric point (p I) values with variable combinations, in which four spots of 31 kDa with p Is 3.4, 3.9, 4.0 and 4.1, and one 36 kDa spot (p I 3.5) appeared to be specifically reactive. One 31 kDa protein spot with p I 3.3 and two spots of 36 kDa with p Is 3.3 and 3.5 reacted crossly with neurocysticercosis sera. Neither sera from patients with other parasitic infections nor those from healthy controls showed positive reaction. Two-DE/immunoblot analysis might be highly available in differential serodiagnosis of human sparganosis.

Clinical reactivity to raw peanut correlates with IgE binding to conformational epitopes of Ara h 1: a Case Report

Most peanut allergic individuals have reactivity to the dominant allergens, Ara h 1, 2 or 3, and a limited number of that promote IgE of responses (1). While IgE recognition of aeroallergens is frequently against conformational epitopes, the responses to peanuts, and foods in general, is toward linear epitopes (2). Conformational epitopes to foods, mainly egg or milk, are actually associated with development of tolerance (3). Conversely, reactivity to linear epitopes is associated with food allergy persistence (4). Here, we report data on an unusual case of peanut allergy that supports IgE-mediated clinical reactivity to conformational epitopes in Ara h1. We show specific IgE reactivity to Ara h1 in peanut that is because of conformational epitopes. A 35-year-old male patient with a clinical history of chronic nasal congestion, intermittent postnasal drip, ocular pruritus increased in Spring and Fall or with cat exposure, and oral pruritus to several foods presented for allergic evaluation. The oral pruritus, similar in presentation to oral allergy syndrome (OAS), was often accompanied by tongue swelling but not with difficulty breathing and occurred with uncooked tree nuts and uncooked peanuts. He had similar symptoms in the past with banana. Surprizingly, he reported not having these symptoms when eating food products in which the peanuts had been cooked. He was found to be highly reactive upon skin testing to the following allergens: peanut, brazil nut, almond, hazelnut, pecan, pistachio, and pine nut, as well as trees, grasses, ragweed, mold, house dust mite and cat, but negative to cashew and walnut. He was found to have a peanut-specific IgE titer of 5.5 kU/l. To test whether reactivity was being lost upon heating, whole peanut extract was heated to boiling and immunoblotted using the patient's serum. IgE reactivity was detected using HRP-conjugated anti-human IgE. Oral allergy syndrome has been reported to associate with reactivity toward the relatively unstable 16- to 17-kD-sized Ara h 8 protein (5). While we did observe a heat-affected protein at this size, as well as an additional band at approximately 20 kD, Fig. 1A demonstrates that the reactivity to another specific band at approximately 60 kDa was rapidly lost upon boiling, while most others were unaffected. To identify this protein, we performed two-dimensional protein analysis on raw, whole peanut extracts and resolved this into a number of potential protein spots (Fig. 1B). Six potential proteins were selected for mass spectrometry-based identification based on immunoblotting of the protein map using the patients serum and included one protein of a size that was unaffected by boiling (Fig. 1B). The protein identities showed that spots 2–6 all contained the major peanut allergen Ara h 1 while protein spot 1 was Arachin 6. We utilized a peptide array-based immunoassay of sequential linear epitopes for Ara h1, 2 and 3, as we have previously described and shown to correlate with symptom severity (1) and found no IgE recognition of linear epitopes of Ara h1, 2 or 3. Figure 1 (A) IgE reactivity to peanut proteins is rapidly lost upon boiling. Whole peanut protein was boiled for the indicated times and immunoblotted using the patient's sera. Anti-human IgE was used to detect IgE reactivity. (B) Two-dimensional analysis of peanut ... Thermal processing has been the subject of a number of studies, in large part because of the dietary differences in peanut consumption between westernized and other societies. Some report reductions in the IgE-binding capacity of several peanut proteins, including Ara h 1 (6, 7). However, these studies show relatively small reductions and only after long periods of boiling (>20 min). This response is highly suggestive of a conformational epitope or reactivity toward a secondary modification, such as glycosylation or phosphorylation. However, two-dimensional resolution discriminates proteins with different modifications and because the patient's IgE showed reactivity toward six different modifications of Ara h 1, it is most likely that a conformational epitope is involved. In conclusion, we report a patient with novel form of peanut allergy, wherein IgE reactivity is rapidly lost upon cooking and symptoms are dominated by oral pruritus. We demonstrate that this reactivity is to Ara h 1 but believe it is against a conformational epitope, which has not been reported before for peanut allergens.

Boron deficiency results in induction of pathogenesis-related proteins from the PR-10 family during the legume–rhizobia interaction

Boron (B) deficiency has a strong effect on molecular and cellular plant–bacteria interactions during the development of the legume–rhizobia symbiosis, leading to reduced infection and early necrosis of nodules, resembling a pathogenic-like rather than a symbiotic interaction. Therefore, induction of pathogenesis-related (PRs) proteins was investigated here in legume root nodules. Following two-dimensional electrophoresis and MALDI-TOF spectrometry analysis of proteins extracted from Pisum sativum B-sufficient (+B) or B-deficient (–B) root nodules, two proteins from the family PR10, ABR17 and PR10.1, were identified as highly induced in –B nodules. Analysis of gene expression and the use of anti-ABR17 confirmed that induction occurred in B-deficient young nodules and increased during nodule development. ABR17 was also induced in –B nodules of Phaseolus vulgaris. Boron deficiency did not significantly increase the expression of these PR10 in uninfected plant tissues. Moreover, independent of B, induction was detected in senescent tissues, although at a level weaker than in −B nodules. The immunochemical study of ABR17 antigen distribution showed that it was localized in all tissues of poorly invaded B-deficient nodules and accumulated around bacteria, which showed advanced degradation. These results suggest that, under B deficiency, the rhizobia–legume dialogue fails and the bacterium is recognized as a pathogen by the plant, which reacts to prevent infection by inducing at least these two identified PR10 proteins.

Proteomic profiling of medial degeneration in human ascending aorta

Objective: The objective of this study was the construction of a reference map for aortic medial degeneration by a proteomic approach. Design and methods: A proteomic profiling of the media of human ascending aorta was performed by two-dimensional electrophoresis and MALDI-TOF mass spectrometry. Results: A reliable protocol for two-dimensional electrophoresis analysis of human aortic media proteins was developed allowing the selection and identification of 52 spots. Protein identifications revealed that the predominant vascular smooth muscle cell proteins isolated from grade 1 aortic medial degeneration (MD) included proteins involved in muscle contraction, protein folding, cytoskeletal structure and metabolic processes, and those with antioxidant or transport functions. The most populated functional classes were those related to muscle contraction and cytoskeletal proteins, including actin, calmodulin, calponin, myosin light chain, tropomyosin, vimentin, profilin and transgelin. Conclusions: The obtained aortic MD proteomic profile provides a relevant background for future studies aimed to find further specific molecular changes potentially related to the aortic MD process.

NOVEL PROTEINS COMPRISING THE STRAMENOPILE TRIPARTITE MASTIGONEME IN OCHROMONAS DANICA (CHRYSOPHYCEAE)(1).

Two-dimensional (2-D) protein analysis of the mastigoneme fraction of the chromophyte alga Ochromonas danica E. G. Pringsh. showed the presence of several component proteins of the tubular mastigoneme. Adding to the reported gene Ocm1, three new genes (Ocm2, Ocm3, and Ocm4) belonging to the Ocm gene family were isolated using degenerate primers designed from predicted Ocm1 amino acid sequences. The predicted polypeptides encoded by Ocm2, Ocm3, and Ocm4 were smaller in size than Ocm1. However, they shared four highly conserved, cysteine-rich, epithelial growth factor (EGF)-like motifs, potentially involved in protein-protein interaction. In addition, Ocm2, Ocm3, and Ocm4 showed homology to the SIG protein family in the centric diatom Thalassiosira weissflogii (Grunow) Fryxell et Hasle, which is up-regulated during early stages of sexual reproduction. Immunofluorescence analysis with a polyclonal antibody against the partial amino acid sequences of Ocm2, Ocm3, and Ocm4 showed that Ocm2 and Ocm3 were located in the basal segment region of mastigonemes attached on the surface of the anterior flagellum, and that Ocm4 was located within the tubular shaft portion similar to Ocm1.

Two-dimensional gel electrophoresis of dog plasma proteins: genetic polymorphism of an alpha 1-protease inhibitor and another postalbumin.

Two-dimensional electrophoretic analysis of dog plasma proteins was done by a first-dimension separation in agarose gel (pH 5.4) followed by a second-dimension separation in horizontal poly-acrylamide gel (pH 9.0). Two groups of α1-globulins, designated Pi-1 and Pi-2, were found to be protease inhibitors. Extensive genetic polymorphism of Pi-1 and of another unidentified α-globulin, tentatively designated postalbumin (Pa), was observed. Family data supported the hypothesis that Pi-1 and Pa polymorphisms were controlled by codominant, autosomal alleles. Three Pi-1 alleles and two Pa alleles were observed in 60 breeds (1119 dogs) studied. Each of the Pi-1 alleles gave rise to a single strong fraction while each of the Pa alleles showed a major and a minor anodic fraction. The present study, along with earlier reports, showed that some of the proteins and enzymes (albumin, transferrin, eserine resistant esterase, α1-protease inhibitor and postalbumin) in plasma exhibit considerably more polymorphism than that reported for hemoglobin and some of the red cell and tissue enzymes of the domestic dog.

An Effective Method for the Analysis of Human Plasma Proteome using Two-dimensional Gel Electrophoresis

Proteome analysis of plasma is increasingly leading to biomarker discovery of human diseases. However the highabundant proteins, excess of salt and lipid in plasma makes the analysis very challenging. Therefore it is necessary to improve the sample preparation procedures before/after the two-dimensional gel electrophoresis analysis of plasma proteins. The objective of this study was to develop a reproducible method by examining the following parameters: (1) depletion of the high-abundant proteins (2) effect of different precipitation methods (3) comparing optimised rehydration buffer using modified Taguchi method with the standard rehydration buffer and (4) comparing the effects of different staining methods. Our results showed that the depletion of two high-abundant proteins improved the visualization of less abundant proteins present in human plasma and precipitation with TCA/acetone resulted in an efficient sample concentration and desalting. We found that using optimized rehydration buffer as compared to standard rehydration buffer increased protein solubility, improved resolution and reproducibility of 2D gels. We also found that visualization of 2D gel profiles by silver staining and fluorescent staining enhanced the detection of low abundant plasma proteins as compared to Coomassie staining. In conclusion, the optimized conditions in our study can be applied to produce a better reference 2-DE gel of plasma samples for the identification of novel disease markers.

Genomic and Proteomic Analysis Reveals a Threshold Level of MYC Required for Tumor Maintenance

MYC overexpression has been implicated in the pathogenesis of most types of human cancers. MYC is likely to contribute to tumorigenesis by its effects on global gene expression. Previously, we have shown that the loss of MYC overexpression is sufficient to reverse tumorigenesis. Here, we show that there is a precise threshold level of MYC expression required for maintaining the tumor phenotype, whereupon there is a switch from a gene expression program of proliferation to a state of proliferative arrest and apoptosis. Oligonucleotide microarray analysis and quantitative PCR were used to identify changes in expression in 3,921 genes, of which 2,348 were down-regulated and 1,573 were up-regulated. Critical changes in gene expression occurred at or near the MYC threshold, including genes implicated in the regulation of the G(1)-S and G(2)-M cell cycle checkpoints and death receptor/apoptosis signaling. Using two-dimensional protein analysis followed by mass spectrometry, phospho-flow fluorescence-activated cell sorting, and antibody arrays, we also identified changes at the protein level that contributed to MYC-dependent tumor regression. Proteins involved in mRNA translation decreased below threshold levels of MYC. Thus, at the MYC threshold, there is a loss of its ability to maintain tumorigenesis, with associated shifts in gene and protein expression that reestablish cell cycle checkpoints, halt protein translation, and promote apoptosis.

N‐Methyl‐d‐aspartate receptor antagonist MK‐801 attenuates morphine tolerance and associated glial fibrillary acid protein up‐regulation: a proteomic approach

It is well known that long-term morphine administration results in tolerance, which limits the clinical use of this drug in pain management. Male Wistar rats were randomly assigned to receive one of four different infusions: morphine [15 microg/h, intrathecal (i.t.)], saline, MK-801 (5 microg/h, i.t.) plus morphine (15 microg/h, i.t.), or MK-801 (5 microg/h, i.t.) alone. Morphine infusion induced a maximal antinociceptive effect on day 1 and tolerance on day 3, and the maximal anti-receptive tolerance was observed on day 5. Co-infusing MK-801 with morphine attenuated morphine's anti-receptive tolerance. Two-dimensional gel electrophoretic analysis of spinal proteins revealed that eight protein spots were up-regulated in morphine-tolerant rats, and that they were significantly inhibited by MK-801 co-infusion. Among the up-regulated proteins, glial fibrillary acid protein (GFAP), a glial-specific maker, was identified by mass spectrometry. This finding was also confirmed by Western blot analysis. Using proteomic analysis, we identified eight GFAP protein spots that were up-regulated in the dorsal horn of morphine-tolerant rat spinal cords. This up-regulation was partly inhibited by N-methyl-D-aspartate receptor antagonist MK-801 co-infusion, which suggests that GFAP protein can be considered to be a pathogenesis marker of morphine tolerance.

Excess copper induces anoxygenic photosynthesis in Anabaena doliolum: a homology based proteomic assessment of its survival strategy

This study is the first to demonstrate operation of anoxygenic photosynthesis in copper acclimated Anabaena doliolum and to offer proteomic comparison with the control cells. The Cu-treated control strain showed a negative correlation in growth and intracellular Cu, partial inhibition of O(2)-evolution, PS II, PS I, whole chain, chlorophyll absorption, and nitrogenase activity. However, the acclimated strain growing in 250-fold excess Cu exhibited near normal growth, ATP content, PS I activity, carbon fixation, and almost complete inhibition of O(2)-evolution, PS II and chlorophyll absorption, but increased nitrogenase activity as compared to control. Proteomic decoding of the survival strategy of Cu-treated control and the acclimated strain using two-dimensional gel electrophoresis and MALDI-TOF MS analysis of proteins displaying significant and reproducible changes demonstrated involvement of transketolase, phycoerythrocyanin alpha-chain, iron superoxide dismutase (Fe-SOD), hypothetical protein alr 0803, manganese superoxide dismutase (Mn-SOD), phosphoribulokinase, and plastocyanin (PLC). Expression pattern of these proteins was attested at the transcriptional level using RT-PCR. Time course analysis of proteins of Cu-treated control strain revealed almost no change in PLC level, and a minor accumulation of transketolase, phycoerythrocyanin alpha-chain and both isoforms of SOD after 7 and recovery after 10 days. Acclimated strain under excess Cu, however, exhibited significant accumulation of both isoforms of SOD, plastocyanin, phosphoribulokinase and transketolase, which seem to counteract oxidative damage, serve as an alternate electron carrier from cytochrome b6/f complex to photosystem I and meet the NADPH and ATP requirements, respectively, under anoxygenic photosynthesis. In view of the kinetics of the hypothetical protein alr0803 (no change in expression level for 7, maximum after 10 and decline after 15 days) its involvement in metal homeostasis is suggested.