Identification of differentially expressed proteins in colorectal cancer by proteomics: down-regulation of secretagogin

To identify proteins with colorectal cancer-specific regulation, comparative 2-DE of individual-matched normal and neoplastic colorectal tissue specimens was performed. We found 15 protein spots with concordantly increased and 20 protein spots with concordantly decreased intensity in tumor tissue (expression regulation more than fivefold). Nine of these proteins were identified by MS/MS. Interestingly, one of the proteins, which exhibited a marked down-regulation in colorectal cancer tissues, was the recently identified endocrine cell-expressed protein secretagogin. The reduction of the secretagogin content in colorectal cancer tissues was confirmed by comparative immunoblotting (n = 17) and RT-PCR (n = 22) as well as by immunohistochemistry (n = 45) of individual-matched neoplastic and normal colorectal tissue specimens. Immunohistochemistry revealed absence of secretagogin-expressing cells in most of the colorectal cancer tissue specimens. However, some colorectal cancers were characterized by secretagogin-expressing cells. In normal mucosa, positively stained cells exhibited a neuroendocrine cell-characteristic morphology and mucosal location. In colorectal cancer tissues, secretagogin-expressing cells were characterized by a malignant morphology. Our findings might represent the basis for the clinical application of secretagogin as a biomarker for a distinct subgroup of colorectal cancers.     

Quantitative proteomics of Arabidopsis shoot microsomal proteins reveals a cross‐talk between excess zinc and iron deficiency

Iron (Fe) deficiency significantly effects plant growth and development. Plant symptoms under excess zinc (Zn) resemble symptoms of Fe‐deficient plants. To understand cross‐talk between excess Zn and Fe deficiency, we investigated physiological parameters of Arabidopsis plants and applied iTRAQ‐OFFGEL quantitative proteomic approach to examine protein expression changes in microsomal fraction from Arabidopsis shoots under those physiological conditions. Arabidopsis plants manifested shoot inhibition and chlorosis symptoms when grown on Fe‐deficient media compared to basal MGRL solid medium. iTRAQ‐OFFGEL approach identified 909 differentially expressed proteins common to all three biological replicates; the majority were transporters or proteins involved in photosynthesis, and ribosomal proteins. Interestingly, protein expression changes between excess Zn and Fe deficiency showed similar pattern. Further, the changes due to excess Zn were dramatically restored by the addition of Fe. To obtain biological insight into Zn and Fe cross‐talk, we focused on transporters, where STP4 and STP13 sugar transporters were predominantly expressed and responsive to Fe‐deficient conditions. Plants grown on Fe‐deficient conditions showed significantly increased level of sugars. These results suggest that Fe deficiency might lead to the disruption of sugar synthesis and utilization.     

Identification of proteins differentially expressed between capillary endothelial cells of hepatocellular carcinoma and normal liver in an orthotopic rat tumor model using 2‐D DIGE

Hepatocellular carcinoma (HCC) is one of the deadliest cancers with few treatment options. It is a hypervascular tumor in which angiogenesis plays a critical role in its progression. Tumor capillary endothelial cells (TECs) in HCC are known to originate from liver sinusoid endothelial cells, which then go through a capillarization process to become morphologically as well as functionally different TECs. In this work, we investigated proteins differentially expressed between freshly isolated TECs and sinusoid endothelial cells from well‐formed rat HCC using 2‐D DIGE coupled with MALDI‐TOF/TOF MS. Thirty‐eight unique proteins were identified to be differentially expressed more than twofold between the two endothelial cell types. Amongst the differentially expressed proteins, two novel endothelial markers, EH domain‐containing protein 3 and galectin‐3, were confirmed by Western blot and immunohistochemistry in both rat and human HCC samples. We showed that EH domain‐containing protein 3 is significantly down‐regulated in TECs, but galectin‐3 is up‐regulated. We propose possible roles of these two proteins in tumor vessel development in HCC.     

Proteomic identification of differentially expressed plasma membrane proteins in renal cell carcinoma by stable isotope labelling of a von Hippel‐Lindau transfectant cell line model

The von Hippel‐Lindau (VHL) tumour suppressor gene plays a central role in development of clear cell renal cell carcinoma (RCC). Using a cell line pair generated from the VHL‐defective RCC cell line UMRC2 by transfection with vector control or VHL (?/+VHL) and stable isotope labelling with amino acids in cell culture (SILAC) followed by enrichment of plasma membrane proteins by cell surface biotinylation/avidin‐affinity chromatography and analysis by GeLC‐MS/MS, VHL‐associated changes in plasma membrane proteins were analysed. Comparative analysis of ‐/+VHL cells identified 19 differentially expressed proteins which were confirmed by reciprocal SILAC labelling. These included several proteins previously reported to be VHL targets, such as transferrin receptor 1 and the α3 and β1 integrin subunits and novel findings including upregulation of CD166 and CD147 in VHL‐defective cells. Western blotting confirmed these changes and also revealed VHL‐dependent alterations in protein form for CD147 and CD166, which in the case of CD166 was shown to be due to differential glycosylation. Analysis of patient‐matched normal and malignant renal tissues confirmed these differences were also present in vivo in a subset of clear cell RCCs. These results illustrate the potential of this approach for identifying VHL‐dependent proteins that may be important in tumorigenesis.     

Does the presence of a seawater gill morphology induced by dietary salt loading affect Cl(-) uptake and acid-base regulation in freshwater rainbow trout Oncorhynchus mykiss

The goal of this study was to determine the effect of the changes in gill morphology induced by dietary salt feeding on several aspects of gill function in rainbow trout Oncorhynchus mykiss maintained in fresh water with specific emphasis on Cl(-) uptake (J(IN)Cl(-)) and acid-base regulation. The addition of 11% NaCl to the diet caused J(IN)Cl(-) to be reduced by c. 45% from 214·4 ± 26·7 to 117·3 ± 17·4 μmol kg(-1) h(-1) (mean ± s.e.). Rates of Cl(-) efflux (J(OUT)Cl(-)), net Cl(-) flux (J(NET)Cl(-)), J(NET) Na(+) and plasma levels of Na(+) or Cl(-) were unaffected by salt feeding. On the basis of significant effect of the salt diet on decreasing the maximal uptake rate of Cl(-)(J(MAX)Cl(-)), it would appear that internal salt loading caused a decrease in the number of functional ion transport proteins involved in Cl(-) uptake (e.g. Cl(-) -HCO(3)(-) exchangers) and decreased the transporting capacity of existing proteins. The acid-base regulating capacity of control fish and salt-loaded fish was assessed by monitoring arterial blood acid-base status [partial pressure of CO(2) (PCO(2)), pH and HCO(3)(-)] during exposure to external hypercapnia (nominally 7·5 mm Hg). Both groups of fish exhibited typical compensatory responses to sustained hypercapnia consisting of the gradual accumulation of plasma HCO(3) (-) and thus metabolic restoration within 24 h of the initial respiratory acidosis elicited by hypercapnia. Overall, the results demonstrate that while Cl(-) uptake capacity is reduced in salt-fed fish, there is no associated alteration in acid-base regulating capability.