Proteomic analysis of advanced colorectal cancer by laser capture microdissection and two-dimensional difference gel electrophoresis.

The emergence of laser capture microdissection (LCM) and two-dimensional difference gel electrophoresis (2D-DIGE) has been shown to greatly improve the accuracy and sensitivity of global protein expression analysis. However, their combined use in profiling tumour proteome has rarely been reported. In this study, we applied these techniques to profile the protein expression changes of the late stage colorectal cancer (CRC) and its liver metastases. The study revealed that both the primary and secondary tumours showed a distinct protein expression profile compared to normal tissues, but were indistinguishable from each other. Differential analysis between the primary tumour and patient-matched normal colon mucosa identified a total of 71 proteins to be altered in CRC. Over 40% of these proteins have been previously reported as CRC-related proteins, validating the accuracy of the current analysis. We have also identified many previously unknown changes including overexpression of ACY1, HSC70, HnRNP I, HnRNP A3, SET, ANP32A and TUFM in CRC, which have been further verified by western blotting and immunohistochemistry. This study demonstrated that LCM in combination with 2D-DIGE is a powerful tool to analyse the proteome of tumour tissues and may lead to the identification of potential novel protein markers and therapeutic targets for cancer.

The BPI-like/PLUNC family proteins in cattle.

Members of the protein family having similarity to BPI (bactericidal/permeability increasing protein) (the BPI-like proteins), also known as the PLUNC (palate, lung and nasal epithelium clone) family, have been found in a range of mammals; however, those in species other than human or mouse have been relatively little characterized. Analysis of the BPI-like proteins in cattle presents unique opportunities to investigate the function of these proteins, as well as address their evolution and contribution to the distinct physiology of ruminants. The present review summarizes the current understanding of the nature of the BPI-like locus in cattle, including the duplications giving rise to the multiple BSP30 (bovine salivary protein 30 kDa) genes from an ancestral gene in common with the single PSP (parotid secretory protein) gene found in monogastric species. Current knowledge of the expression of the BPI-like proteins in cattle is also presented, including their pattern of expression among tissues, which illustrate their independent regulation at sites of high pathogen exposure, and the abundance of the BSP30 proteins in saliva and salivary tissues. Finally, investigations of the function of the BSP30 proteins are presented, including their antimicrobial, lipopolysaccharide-binding and bacterial aggregation activities. These results are discussed in relation to hypotheses regarding the physiological role of the BPI-like proteins in cattle, including the role they may play in host defence and the unique aspects of digestion in ruminants.

Metastatic susceptibility locus, an 8p hot-spot for tumour progression disrupted in colorectal liver metastases: 13 candidate genes examined at the DNA, mRNA and protein level.

BACKGROUND: Mortality from colorectal cancer is mainly due to metastatic liver disease. Improved understanding of the molecular events underlying metastasis is crucial for the development of new methods for early detection and treatment of colorectal cancer. Loss of chromosome 8p is frequently seen in colorectal cancer and implicated in later stage disease and metastasis, although a single metastasis suppressor gene has yet to be identified. We therefore examined 8p for genes involved in colorectal cancer progression. METHODS: Loss of heterozygosity analyses were used to map genetic loss in colorectal liver metastases. Candidate genes in the region of loss were investigated in clinical samples from 44 patients, including 6 with matched colon normal, colon tumour and liver metastasis. We investigated gene disruption at the level of DNA, mRNA and protein using a combination of mutation, semi-quantitative real-time PCR, western blotting and immunohistochemical analyses. RESULTS: We mapped a 2 Mb region of 8p21-22 with loss of heterozygosity in 73% of samples; 8/11 liver metastasis samples had loss which was not present in the corresponding matched primary colon tumour. 13 candidate genes were identified for further analysis. Both up and down-regulation of 8p21-22 gene expression was associated with metastasis. ADAMDEC1 mRNA and protein expression decreased during both tumourigenesis and tumour progression. Increased STC1 and LOXL2 mRNA expression occurred during tumourigenesis. Liver metastases with low DcR1/TNFRSF10C mRNA expression were more likely to present with extrahepatic metastases (p = 0.005). A novel germline truncating mutation of DR5/TNFRSF10B was identified, and DR4/TNFRSF10A SNP rs4872077 was associated with the development of liver metastases (p = 0.02). CONCLUSION: Our data confirm that genes on 8p21-22 are dysregulated during colorectal cancer progression. Interestingly, however, instead of harbouring a single candidate colorectal metastasis suppressor 8p21-22 appears to be a hot-spot for tumour progression, encoding at least 13 genes with a putative role in carcinoma development. Thus, we propose that this region of 8p comprises a metastatic susceptibility locus involved in tumour progression whose disruption increases metastatic potential.

Expansion of the Bactericidal/Permeability Increasing-like (BPI-like) protein locus in cattle.

BACKGROUND: Cattle and other ruminants have evolved the ability to derive most of their metabolic energy requirement from otherwise indigestible plant matter through a symbiotic relationship with plant fibre degrading microbes within a specialised fermentation chamber, the rumen. The genetic changes underlying the evolution of the ruminant lifestyle are poorly understood. The BPI-like locus encodes several putative innate immune proteins, expressed predominantly in the oral cavity and airways, which are structurally related to Bactericidal/Permeability Increasing protein (BPI). We have previously reported the expression of variant BPI-like proteins in cattle (Biochim Biophys Acta 2002, 1579, 92-100). Characterisation of the BPI-like locus in cattle would lead to a better understanding of the role of the BPI-like proteins in cattle physiology RESULTS: We have sequenced and characterised a 722 kbp segment of BTA13 containing the bovine BPI-like protein locus. Nine of the 13 contiguous BPI-like genes in the locus in cattle are orthologous to genes in the human and mouse locus, and are thought to play a role in host defence. Phylogenetic analysis indicates the remaining four genes, which we have named BSP30A, BSP30B, BSP30C and BSP30D, appear to have arisen in cattle through a series of duplications. The transcripts of the four BSP30 genes are most abundant in tissues associated with the oral cavity and airways. BSP30C transcripts are also found in the abomasum. This, as well as the ratios of non-synonymous to synonymous differences between pairs of the BSP30 genes, is consistent with at least BSP30C having acquired a distinct function from the other BSP30 proteins and from its paralog in human and mouse, parotid secretory protein (PSP). CONCLUSION: The BPI-like locus in mammals appears to have evolved rapidly through multiple gene duplication events, and is thus a hot spot for genome evolution. It is possible that BSP30 gene duplication is a characteristic feature of ruminants and that the BSP30 proteins contribute to an aspect of ruminant-specific physiology.

The mammalian innate immune system: potential targets for drug development.

The innate immune system is the oldest mammalian defence against invading micro-organisms and provides the first line of defence against them, however until recently a detailed understanding of its complexity has been lacking. This review describes recent advances that have been made in understanding the components of the innate immune system, including the pathogen sensing mechanisms, receptor and intracellular signalling pathways, linkage to the acquired immune system, and effectors of the innate immune response. These discoveries have created an opportunity for the development of novel drugs through the identification of targets for rational drug design. The opportunity for the development of novel anti-inflammatory and antimicrobial drugs through modulation of pro-inflammatory or antimicrobial signals within the innate immune system, are discussed. A more detailed understanding of the effectors of the innate immune system is providing an opportunity for the design of effector mimetics as novel antimicrobial drugs. The innate immune system is providing the basis for much-needed alternative approaches to controlling infection and inflammation in human medicine.

Peloruside A, a novel antimitotic agent with paclitaxel-like microtubule- stabilizing activity.

Peloruside A is a novel secondary metabolite isolated from a New Zealand marine sponge, Mycale hentscheli, that has potent paclitaxel-like microtubule-stabilizing activity and is cytotoxic at nanomolar concentrations. Its 16-membered macrolide ring is similar to that of epothilone, a drug currently under clinical investigation as an anticancer agent. Like paclitaxel, peloruside A arrests cells in the G(2)-M phase of the cell cycle and induces apoptosis. The relatively simple structure of peloruside makes it suitable for the design and synthesis of analogues with improved tumor targeting and reduced tumor cross-resistance.