This study compared the whole cell proteome profiles of two isogenic colorectal cancer (CRC) cell lines (primary SW480 cell line and its lymph node metastatic variant SW620), as an in vitro metastatic model, to gain an insight into the molecular events of CRC metastasis. Using iTRAQ (isobaric tags for relative and absolute quantitation) based shotgun proteomics approach, we identified 1140 unique proteins, out of which 147 were found to be significantly altered in the metastatic cell. Ingenuity pathway analysis with those significantly altered proteins, revealed cellular organization and assembly as the top-ranked altered biological function. Differential expression pattern of 6 candidate proteins were validated by Western blot. Among these, the low expression level of β-catenin combined with the up-regulation of CacyBP (Calcyclin binding Protein), a β-catenin degrading protein, in the metastatic cell provided a rational guide for the downstream functional assays. The relative expression pattern of these two proteins was further validated in three other CRC cells by Western blot and quantitative immunofluorescence studies. Overexpression of CacyBP in three different primary CRC cell lines showed significant reduction in adhesion characteristics as well as cellular β-catenin level as confirmed by our experiments, indicating the possible involvement of CacyBP in CRC metastasis. In short, this study demonstrates successful application of a quantitative proteomics approach to identify novel key players for CRC metastasis, which may serve as biomarkers and/or drug targets to improve CRC therapy.

Current limitations in proteome analysis by high-throughput mass spectrometry (MS) approaches have sometimes led to incomplete (or inconclusive) data sets being published or unpublished. In this work, we used an iTRAQ reference data on hepatocellular carcinoma (HCC) to design a two-stage functional analysis pipeline to widen and improve the proteome coverage and, subsequently, to unveil the molecular changes that occur during HCC progression in human tumorous tissue. The first involved functional cluster analysis by incorporating an expansion step on a cleaned integrated network. The second used an in-house developed pathway database where recovery of shared neighbors was followed by pathway enrichment analysis. In the original MS data set, over 500 proteins were detected from the tumors of 12 male patients, but in this paper we reported an additional 1000 proteins after application of our bioinformatics pipeline. Through an integrative effort of network cleaning, community finding methods, and network analysis, we also uncovered several biologically interesting clusters implicated in HCC. We established that HCC transition from a moderate to poor stage involved densely connected clusters that comprised of PCNA, XRCC5, XRCC6, PARP1, PRKDC, and WRN. From our pathway enrichment analyses, it appeared that the HCC moderate stage, unlike the poor stage, is enriched in proteins involved in immune responses, thus suggesting the acquisition of immuno-evasion. Our strategy illustrates how an original oncoproteome could be expanded to one of a larger dynamic range where current technology limitations prevent/limit comprehensive proteome characterization.

Butyrate, a 4-carbon short chain fatty acid, is responsible for the protective effects of fiber in colorectal cancer prevention. To better understand the 'blueprint' of butyrate's chemopreventive role in this disease, we performed 2-dimensional difference gel electrophoresis (2-D DIGE) of butyrate-treated HCT-116 colorectal cancer cells after pre-fractionation using heparin affinity chromatography. A combination of this enrichment step with overlapping narrow range IPGs (pH 4-7 and pH 6-11) in 2-D DIGE resulted in the detection of 46 differentially expressed spots. Twenty-four of these were identified by MS analyses, and 5 spots were found to be heterogeneous nuclear ribonucleoprotein A1 (hnRNP A1). Three isoforms of 38 kDa were down-regulated while two with Mr approximately 26 kDa were up-regulated. These represent phosphorylated isoforms of hnRNP A1 as verified by immunoblotting with anti-phosphotyrosine and anti-phosphoserine antibodies. Using 2-DE, subcellular fractionation and western blot analysis, we further showed that full-length hnRNP A1 underwent down-regulation, cleavage and cytoplasmic retention upon butyrate treatment. These indicate that modulations of hnRNP A1 may play a significant role in the mediation of growth arrest and apoptosis by butyrate.

Hepatocellular carcinoma (HCC) is a major cause of cancer worldwide and is often characterized by aggressive tumour behaviour and poor prognosis. One of the major etiologies is hepatitis B or C virus (HBV or HCV) infections. In order to better comprehend the molecular mechanisms involved in HCC progression, we performed a systematic analysis on moderately and poorly differentiated human HCC tissues using 2-D DIGE coupled to MALDI-TOF/TOF MS. A total of 52 and 26 proteins were found to be dysregulated in moderately and poorly differentiated HCC tissues, respectively. For the first time, the over-expression of a novel protein family, far upstream binding proteins (FUBPs) was identified in both stages of HCC and confirmed by western blots. FUBPs are of particular interest due to their transcriptional activity on the oncogene, c-myc. It has generally been accepted that c-myc plays an important role in HCC progression but its exact activators remain poorly understood. Interestingly, we also observed elevated c-myc levels in the tissues used in this study by western blot analysis. We therefore propose that the FUBP family of proteins may be one of the possible upstream players that are involved in modulating the c-myc levels in HCC tumorigenesis.

The African sharptooth catfish Clarias gariepinus lives in freshwater, is an obligatory air breather, and exhibits high tolerance of environmental ammonia. This study aimed at elucidating the strategies adopted by C. gariepinus to defend against ammonia toxicity during ammonia exposure. No carbamoyl phosphate synthetase (CPS) I or III activities were detected in the liver or muscle of the adult C. gariepinus. In addition, activities of other ornithine-urea cycle (OUC) enzymes, especially ornithine transcarbamylase, were low in the liver, indicating that adult C. gariepinus does not have a "functional" hepatic OUC. After being exposed to 50 or 100 mM NH4Cl for 5 d, there was no induction of hepatic OUC enzymes and no accumulation of urea in tissues of the experimental animals. In addition, the rate of urea excretion remained low and unchanged. Hence, ammonia exposure did not induce ureogenesis or ureotely in C. gariepinus as suggested elsewhere for another obligatory air-breathing catfish of the same genus, Clarias batrachus, from India. Surprisingly, the local C. batrachus did not possess any detectable CPS I or III activities in the liver or muscle as had been reported for the Indian counterpart. There were no changes in levels of alanine in the muscle, liver, and plasma of C. gariepinus exposed to 50 or 100 mM NH4Cl for 5 d; neither were there any changes in the glutamine levels in these tissues. Yet even after being exposed to 100 mM NH4Cl for 5 d, there was no significant increase in the level of ammonia in the muscle, which constitutes the bulk of the specimen. In addition, the level of ammonia accumulated in the plasma was relatively low compared to other tropical air-breathing fishes. More importantly, for all NH4Cl concentrations tested (10, 50, or 100 mM), the plasma ammonia level was maintained relatively constant (2.2-2.4 mM). These results suggest that C. gariepinus was able to excrete endogenous ammonia and infiltrated exogenous ammonia against a very steep ammonia gradient. When exposed to freshwater (pH 7.0) with or without 10 mM NH4Cl, C. gariepinus was able to excrete ammonia continuously to the external medium for at least 72 h. This was achieved while the plasma NH4+ and NH3 concentrations were significantly lower than those of the external medium. Diffusion trapping of NH3 through boundary layer acidification can be eliminated as the pH of the external medium became more alkaline instead. These results represent the first report on a freshwater fish (C. gariepinus) adopting active excretion of ammonia (probably NH4+) as a major strategy to defend against ammonia toxicity when exposed to environmental ammonia.

Lipase class 3 is part of the triacylglycerol lipase family involved in lipid degradation, esterification, and transesterification processes in plants. In this study, a lipase class 3 gene and promoter from oil palm (Elaeis guineensis Jacq.) were isolated and characterized by Northern blot, Southern blot, oil palm genome sequence, and transient expression GUS assay. The full-length lipase class 3 (FLL1) deduced polypeptide encoded 483 amino acids and was identical to that deduced from lipase (EgLip1) cDNA (GI: 409994625). It contained the lipase consensus sequence, GxSxG motif, and a putative catalytic triad and had a 3-dimensional protein model similar to that of a lipase from Giberella zeae with a 50 % identity. The Northern blot and reverse transcription polymerase chain reaction (RT-PCR) show that FLL1 was predominantly expressed in the mesocarp and the expression increased as fruits reached maturity. A lower expression was detected in germinated seedlings and especially in roots. The expression of FLL1 was also enhanced in the mesocarp of cold treated fruits. A high oil accumulation in the mesocarp during fruit development makes this tissue a suitable target for a genetic modification, hence the isolation of the FLL1 promoter. The transient expression of the β-glucuronidase (GUS) gene driven by the FLL1 promoter detected the GUS expression in mesocarp slices, especially in vascular bundles. This suggests the potential role of using the promoter as tool to direct the expression of a transgene to the mesocarp of transgenic oil palm.

No abstract

A previous study on transient expression of oil palm tissues has shown that the oil palm metallothioneinlike type 3 (MT3-A) gene promoter is specifically expressed in the mesocarp and not in other tissues.This study was conducted to determine whether or not Arabidopsis can be used as a model system to study oil palm promoter. Functional characterisation of the oil palm MT3-A promoter was performed using promoter::GUS fusion analysis in transgenic Arabidopsis. The localisation of β-glucuronidase (GUS) expression in several different tissues of transgenic Arabidopsis homozygous lines driven by the oil palm MT3-A promoter was determined. Histochemical GUS analysis in transgenic Arabidopsis revealed the highest expression in the cotyledon and hypocotyls as well as at the early stage of plant development and gradually decreased as the plant grew. Lower expression was detected in organs of mature plant and no expression was observed in reproductive tissues. This observation may suggest that MT3-A promoter might be involved during early stage of plant development. Since we use Arabidopsis which is neither a fruit nor mesocarp plant to check mesocarp-specific promoter it might not give a faithful pattern of expression but it may be suited to study oil palm specific promoter derived from other tissues. ABSTRACT A previous study on transient expression of oil palm tissues has shown that the oil palm metallothioneinlike type 3 (MT3-A) gene promoter is specifically expressed in the mesocarp and not in other tissues. This study was conducted to determine whether or not Arabidopsis can be used as a model system to study oil palm promoter. Functional characterisation of the oil palm MT3-A promoter was performed using promoter::GUS fusion analysis in transgenic Arabidopsis. The localisation of β-glucuronidase (GUS) expression in several different tissues of transgenic Arabidopsis homozygous lines driven by the oil palm MT3-A promoter was determined. Histochemical GUS analysis in transgenic Arabidopsis revealed the highest expression in the cotyledon and hypocotyls as well as at the early stage of plant development and gradually decreased as the plant grew. Lower expression was detected in organs of mature plant and no expression was observed in reproductive tissues. This observation may suggest that MT3-A promoter might be involved during early stage of plant development. Since we use Arabidopsis which is neither a fruit nor mesocarp plant to check mesocarp-specific promoter it might not give a faithful pattern of expression but it may be suited to study oil palm specific promoter derived from other tissues.