Estimation of the efficiency of 2D analysis and bioinformatics search in identification of protein markers for colon tumors

A modification of proteome differential analysis was developed; it comprises initial removal of the cell structural proteins by extraction with buffered saline and protein fractionation by gel filtration with subsequent separation by two-dimensional gel electrophoresis and identification by mass-spectrometry. This approach provided for detection of 12 proteins with significantly elevated expression levels in the majority of the analyzed malignant colorectal tumor specimens as compared with normal tissues. Increased contents of eight proteins were discovered for the first time. The efficiencies of the search for tumor markers by 2D analysis and serial analysis of gene expression (SAGE) were compared using the control panel of 19 potential colorectal tumor markers, identified earlier or independently found by other researchers. The 2D data for the control panel matched the earlier published data, whereas the search of SAGE database succeeded in detection only one-third of the markers.     

Postnatal exposure to chromium through mother’s milk accelerates follicular atresia in F1 offspring through increased oxidative stress and depletion of antioxidant enzymes

Hexavalent chromium, CrVI, is a heavy metal endocrine disruptor, known as a mutagen, teratogen, and a group A carcinogen. Environmental contamination with CrVI, including drinking water, has been increasing in more than 30 cities in the United States. CrVI is rapidly converted to CrIII intracellularly, and CrIII can cause DNA strand breaks and cancer or apoptosis through different mechanisms. Our previous study demonstrated that lactational exposure to chromium results in a delay or arrest in follicle development and a decrease in steroid hormone levels in F1 female rats, both of which are mitigated (partial inhibition) by vitamin C. The current study tested the hypothesis that lactational exposure to CrIII accelerates follicle atresia in F1 offspring by increasing reactive oxygen species (ROS) and decreasing cellular antioxidants. Results showed that lactational exposure to CrIII dose-dependently increased follicular atresia and decreased steroidogenesis in postnatal day 25, 45, and 65 rats. Vitamin C mitigated or inhibited the effects of CrIII at all doses. CrIII increased hydrogen peroxide and lipid hydroperoxide in plasma and ovary; decreased the antioxidant enzymes (AOXs) GPx1, GR, SOD, and catalase; and increased glutathione S-transferase in plasma and ovary. To understand the effects of CrVI on ROS and AOXs in granulosa (GC) and theca (TC) cell compartments in the ovary, ROS levels and mRNA expression of cytosolic and mitochondrial AOXs, such as SOD1, SOD2, catalase, GLRX1, GSTM1, GSTM2, GSTA4, GR, TXN1, TXN2, TXNRD2, and PRDX3, were studied in GCs and TCs and in a spontaneously immortalized granulosa cell line (SIGC). Overall, CrVI downregulated each of the AOXs; and vitamin C mitigated the effects of CrVI on these enzymes in GCs and SIGCs, but failed to mitigate CrVI effects on GSTM1, GSTM2, TXN1, and TXN2 in TCs. Thus, these data for the first time reveal that lactational exposure to CrIII accelerated follicular atresia and decreased steroidogenesis in F1 female offspring by altering the ratio of ROS and AOXs in the ovary. Vitamin C is able to protect the ovary from CrIII-induced oxidative stress and follicle atresia through protective effects on GCs rather than TCs.     

hSP, the domain-duplicated homolog of pS2 protein, is co-expressed with pS2 in stomach but not in breast carcinoma.

Approximately 50% of human breast tumors secrete a small cysteine-rich protein, pS2, of unknown function. pS2 protein was recently found to be homologous to a porcine protein with hormonogastric activity, pancreatic spasmolytic polypeptide (PSP), in which the 5-cysteine domain present in pS2 is tandemly duplicated. We have characterized cDNA species encoding PSP and its human and mouse counterparts, hSP and mSP. We show that hSP and pS2 are separately encoded in the genome, and that the two proteins are co-expressed in normal stomach epithelium. However, expression of hSP was not detected in breast tumors. Computer analysis revealed that the pattern of conserved cysteine residues in hSP and pS2, the P domain, is present at the N termini of two other mammalian proteins, intestinal sucrase-isomaltase and lysosomal alpha-glucosidase.     

Identification of tumor-associated plasma biomarkers using proteomic techniques: from mouse to human

In an effort to identify tumor-associated proteins from plasma of tumor-bearing mice that may be used as diagnostic biomarkers, we developed a strategy that combines a tumor xenotransplantation model in nude mice with comparative proteomic technology. Five human cancer cell lines (SC-M1, HONE-1, CC-M1, OECM1, GBM 8401) derived from stomach, nasopharyngeal, colon, oral and brain cancers were subcutaneously inoculated into nude mice and compared to control nude mice injected with phosphate-buffered saline. One month later, plasma from mice inoculated with cancer cells was collected for proteomic analysis using two-dimensional gel electrophoresis (2-DE) and mass spectrometry (MS). Comparison of plasma 2-DE maps from tumor-bearing mice with those produced from control mice revealed the overexpression of several mouse acute phase proteins (APPs) such as haptoglobin. Another APP, serum amyloid A (SAA), was found only in mice bearing tumors induced by the stomach cancer cell line SC-M1, which has not previously been demonstrated in xenotransplatation experiment. Furthermore, by using immunohistochemistry, SAA and haptoglobin were found to originate from the mouse hosts and not from the human cancer cell line donors. The protein alterations were further confirmed on patients with stomach cancers where up-regulated levels of SAA were also observed. These results indicate that APPs may be used as nonspecific tumor-associated serum markers. SAA in particular may serve as a potential marker for detecting stomach cancer. Taken together, the combination of the xenotransplatation model in nude mice and proteomics analysis provided a valuable impact for clinical applications in cancer diagnostics. In addition, our findings demonstrate that a panel of APPs might serve as screening biomarkers for early cancer detection.     

Sequence, heterologous expression and functional characterization of tryparedoxin1 from Crithidia fasciculata.

Tryparedoxin (TXN) has recently been discovered as a constituent of the complex peroxidase system in the trypanosomatid Crithidia fasciculata [Nogoceke et al. (1997) Biol. Chem. 378, 827-836] where it catalyzes the reduction of a peroxiredoxin-type peroxidase by trypanothione. Here we report on the full-length DNA sequence of the TXN previously isolated from C. fasciculata (TXN1). The deduced amino acid sequence comprises 147 residues and matches with all the peptide sequences of fragments obtained from TXN1. It shares a characteristic sequence motif YFSAxWCPPCR with some thioredoxin-related proteins of unknown function. This motif is homologous with the CXXC motif, which characterizes the thioredoxin superfamily of proteins and is known to catalyze disulfide reductions. Sequence conservations between TXNs and the typical thioredoxins are restricted to the intimate environment of the CXXC motif and three more remote residues presumed to contribute to the folding pattern of the thioredoxin-type proteins. The TXNs thus form a distinct molecular clade within the thioredoxin superfamily. TXN1 was expressed in Escherichia coli BL21 (DE3)pLysS as a C-terminally extended and His-tagged protein, isolated by chelate chromatography and characterized functionally. The recombinant product exhibited a kinetic pattern identical with, and kinetic parameters similar to those of the authentic enzyme in the trypanothione/peroxiredoxin oxidoreductase assay. The recombinant TXN1 can therefore be considered a valuable tool for the screening of specific inhibitors as potential trypanocidal agents.     

Redox Proteomics of the Inflammatory Secretome Identifies a Common Set of Redoxins and Other Glutathionylated Proteins Released in Inflammation,Influenza Virus Infection and Oxidative Stress

Protein cysteines can form transient disulfides with glutathione (GSH), resulting in the production of glutathionylated proteins, and this process is regarded as a mechanism by which the redox state of the cell can regulate protein function. Most studies on redox regulation of immunity have focused on intracellular proteins. In this study we have used redox proteomics to identify those proteins released in glutathionylated form by macrophages stimulated with lipopolysaccharide (LPS) after pre-loading the cells with biotinylated GSH. Of the several proteins identified in the redox secretome, we have selected a number for validation. Proteomic analysis indicated that LPS stimulated the release of peroxiredoxin (PRDX) 1, PRDX2, vimentin (VIM), profilin1 (PFN1) and thioredoxin 1 (TXN1). For PRDX1 and TXN1, we were able to confirm that the released protein is glutathionylated. PRDX1, PRDX2 and TXN1 were also released by the human pulmonary epithelial cell line, A549, infected with influenza virus. The release of the proteins identified was inhibited by the anti-inflammatory glucocorticoid, dexamethasone (DEX), which also inhibited tumor necrosis factor (TNF)-α release, and by thiol antioxidants (N-butanoyl GSH derivative, GSH-C4, and N-acetylcysteine (NAC), which did not affect TNF-α production. The proteins identified could be useful as biomarkers of oxidative stress associated with inflammation, and further studies will be required to investigate if the extracellular forms of these proteins has immunoregulatory functions.     

Permutation of the active site motif of tryparedoxin 2

Tryparedoxins (TXN) are thioredoxin-related proteins which, as trypanothione:peroxiredoxin oxidoreductases, constitute the trypanothione-dependent antioxidant defense and may also serve as substrates for ribonucleotide reductase in trypanosomatids. The active site motif of TXN2, 40WCPPCR45, of Crithidia fasciculata was mutated by site-directed mutagenesis and eight corresponding muteins were expressed in E. coli as terminally His-tagged proteins, purified to homogeneity by nickel chelate chromatography, and characterized in terms of specific activity, specificity and, if possible, kinetics. Exchange of Cys41 and Cys44 by serine yielded inactive products confirming their presumed involvement in catalysis. Exchange of Arg45 by aspartate resulted in loss of activity, suggesting an activation of active site cysteines by the positive charge of Arg45. Substitution of Trp40 by phenylalanine or tyrosine resulted in moderate decrease of specific activity, as did exchange of Pro42 by glycine. Kinetic analysis of these three muteins revealed that primarilythe reaction with trypanothione is affected by the mutations. Simulation of thioredoxin or glutaredoxin-like active sites in TXN2 (P42G and W40T/P43Y, respectively) did not result in thioredoxin or glutaredoxin-like activities. These data underscore that TXNs, although belonging to the thioredoxin superfamily, represent a group of enzymes distinct from thioredoxins and glutaredoxins in terms of specificity, and appear attractive as molecular targets for the design of trypanocidal compounds.     

Capacities of ultrasound study in the diagnosis of diffuse gastric carcinoma

By using 237 verified cases of diffuse carcinoma and benign diseases of the stomach as an example, the authors assess the capacities of routine transabdominal ultrasound study (USS) of the stomach in the diagnosis of tumors and present their modification of the procedure facilitating the imaging of problem areas. In the authors' opinion, the symptoms of thickening of the wall of the stomach and abnormalities in its normal five-layered structure indicate diffuse gastric carcinoma. The authors consider USS to be a supplement to the basic diagnostic techniques--X-ray study and gastroscopy.     

Aberrant expression of proteins involved in signal transduction and DNA repair pathways in lung cancer and their association with clinical parameters

BACKGROUND: Because cell signaling and cell metabolic pathways are executed through proteins, protein signatures in primary tumors are useful for identifying key nodes in signaling networks whose alteration is associated with malignancy and/or clinical outcomes. This study aimed to determine protein signatures in primary lung cancer tissues. METHODOLOGY/ PRINCIPAL FINDINGS: We analyzed 126 proteins and/or protein phosphorylation sites in case-matched normal and tumor samples from 101 lung cancer patients with reverse-phase protein array (RPPA) assay. The results showed that 18 molecules were significantly different (p<0.05) by at least 30% between normal and tumor tissues. Most of those molecules play roles in cell proliferation, DNA repair, signal transduction and lipid metabolism, or function as cell surface/matrix proteins. We also validated RPPA results by Western blot and/or immunohistochemical analyses for some of those molecules. Statistical analyses showed that Ku80 levels were significantly higher in tumors of nonsmokers than in those of smokers. Cyclin B1 levels were significantly overexpressed in poorly differentiated tumors while Cox2 levels were significantly overexpressed in neuroendocrinal tumors. A high level of Stat5 is associated with favorable survival outcome for patients treated with surgery. CONCLUSIONS/ SIGNIFICANCE: Our results revealed that some molecules involved in DNA damage/repair, signal transductions, lipid metabolism, and cell proliferation were drastically aberrant in lung cancer tissues, and Stat5 may serve a molecular marker for prognosis of lung cancers.     

Anti-ulcer effect of Ranunculus millefoliatus on absolute alcohol-induced stomach ulceration

Ranunculus millefoliatus (RM) has been reported to have a numeral of biological properties. Though, the influence of this plant extract on stomach ulceration is yet stated. Thirty rats arbitrarily alienated 5 groups: the normal group, the ulcerated control group, the omeprazole group, and 2 investigational groups. Normal and ulcerated control groups were gavage by mouth 10% Tween 20. Omeprazole group fed orally 20 mg/kg omeprazole. Investigational group’s gavage of 250 mg/kg and 500 mg/kg ethanol extracted RM 10% Tween 20, correspondingly. Later another hour, the normal group gavage 10% Tween 20, and groups 2–5 gavage absolute ethanol. Afterward additional hours altogether rats were sacrificed. The ulcerated control group displayed extensive apparent stomach epithelial damage escorted by reduced stomachs mucus excretion and pH stomach contented. RM extract meaningfully condensed ethanol-induced gastric lacerations, for example, demonstrated via augmented gastric mucus and pH stomach contents, condensed ulceration expanse, decreased or lack of edema, and leucocyte penetration hypodermic coat. In stomach epithelial homogenate, RM extract revealed important upsurge superoxide dismutase (SOD), catalase (CAT) actions, expressively diminished malondialdehyde (MDA) level. Furthermore, RM extract augmented strength periodic acid–Schiff (PAS) stain stomach mucosa, besides formed up-regulation heat shock protein 70 (HSP 70) proteins down-regulation the Bcl-2-associated X protein (Bax) protein gastric mucosal. RM extract lessened the level of tumor necrosis factor-α (TNF-α), interleukin-6 (IL-6), and improved the quantity of interleukin-10 (IL-10). Acute toxicity greater dosage of 500 mg/kg RM extract organized not obvious at all toxicology symbols might improve self-protective tools against stomach epithelial abrasions. RM extract presented gastroprotective effects that could be due to capability upsurge pH then mucus discharge, rise SOD and CAT, decrease MDA quantity, up-regulating HSP 70 proteins, down-regulating Bax protein, and moderate provocative cytokines.     

Cellular expression patterns of genes upregulated in murine and human colonic neoplasms.

Markers overexpressed in colonic tumors of the multiple intestinal neoplasia (Min) mouse have been recently identified by cDNA subtractive hybridization and by microarray analysis. The significance of such a marker depends on its expression in tumor vs stromal lineages and on its expression pattern in normal tissue. From 34 differentially expressed markers, 14 were found to be expressed from supporting lineages. The markers expressed in the tumor lineage were grouped into three classes on the basis of ISH in mouse models and IHC in human adenomas. The first class includes markers expressed both in neoplastic cells and in the proliferating cells residing at the bottom of normal colonic crypts. The second class of markers shows elevated expression in neoplastic cells and also in the postmitotic Paneth cells of the small intestine. Finally, the third class of marker shows detectable intestinal expression only within tumors but not in the normal intestinal epithelium. Is such a tumor-associated marker uniquely essential for tumor growth? Deficiency for the tumor-associated glycoprotein clusterin does not affect the multiplicity or growth rate of intestinal tumors in Min mice. Thus, clusterin is a candidate secreted colon cancer marker but not a single target for chemoprevention or therapy. (J Histochem Cytochem 56:433–441, 2008)