Hits and misses in research trends to monitor contaminants in foods.

Monitoring of chemicals of toxicological concern in food is commonly needed for many purposes, which include (in part) food safety, regulatory enforcement, risk assessment, international food trade, label claims, environmental protection, industry needs, academic research, and consumer confidence. Chemicals of current concern include a variety of toxins, pesticides, veterinary drugs, growth promoters, environmental contaminants, toxic metals, allergens, endocrine disruptors, genetically modified organisms, melamine, acrylamide, furans, nitrosamines, food additives, packaging components, and miscellaneous other chemicals. In light of past crises, the potential harm from known or unknown chemicals not currently monitored are a source of additional concern by the food industry, regulators, scientists, and consumers. As global food trade has expanded and detection techniques have improved, chemical contaminant analysis of foods has also increased in importance and activity. This critical review article is aimed to highlight current trends in the literature, including neglected research needs, on the analysis of chemicals of toxicological concern in foods. Graphical abstract.

Recombinant nucleases CEL I from celery and SP I from spinach for mutation detection.

BACKGROUND: The detection of unknown mutations is important in research and medicine. For this purpose, a mismatch-specific endonuclease CEL I from celery has been established as a useful tool in high throughput projects. Previously, CEL I-like activities were described only in a variety of plants and could not be expressed in an active form in bacteria. RESULTS: We describe expression of active recombinant plant mismatch endonucleases and modification of their activities. We also report the cloning of a CEL I ortholog from Spinacia oleracea (spinach) which we termed SP I nuclease. Active CEL I and SP I nucleases were expressed as C-terminal hexahistidine fusions and affinity purified from the cell culture media. Both recombinant enzymes were active in mutation detection in BRCA1 gene of patient-derived DNA. Native SP nuclease purified from spinach is unable to incise at single-nucleotide substitutions and loops containing a guanine nucleotide, but the recombinant SP I nuclease can cut at these sites. CONCLUSION: The insect cell-expressed CEL I orthologs may not be identical to their native counterparts purified from plant tissues. The present expression system should facilitate further development of CEL I-based mutation detection technologies.

Expression of MTAP inhibits tumor-related phenotypes in HT1080 cells via a mechanism unrelated to its enzymatic function.

Methylthioadenosine Phosphorylase (MTAP) is a tumor suppressor gene that is frequently deleted in human cancers and encodes an enzyme responsible for the catabolism of the polyamine byproduct 5'deoxy-5'-methylthioadenosine (MTA). To elucidate the mechanism by which MTAP inhibits tumor formation, we have reintroduced MTAP into MTAP-deleted HT1080 fibrosarcoma cells. Expression of MTAP resulted in a variety of phenotypes, including decreased colony formation in soft-agar, decreased migration, decreased in vitro invasion, increased matrix metalloproteinase production, and reduced ability to form tumors in severe combined immunodeficiency mice. Microarray analysis showed that MTAP affected the expression of genes involved in a variety of processes, including cell adhesion, extracellular matrix interaction, and cell signaling. Treatment of MTAP-expressing cells with a potent inhibitor of MTAP's enzymatic activity (MT-DADMe-ImmA) did not result in a MTAP- phenotype. This finding suggests that MTAP's tumor suppressor function is not the same as its known enzymatic function. To confirm this, we introduced a catalytically inactive version of MTAP, D220A, into HT1080 cells and found that this mutant was fully capable of reversing the soft agar colony formation, migration, and matrix metalloproteinase phenotypes. Our results show that MTAP affects cellular phenotypes in HT1080 cells in a manner that is independent of its known enzymatic activity.

A Phosphotyrosine Proteomic Screen Identifies Multiple Tyrosine Kinase Signaling Pathways Aberrantly Activated in Malignant Mesothelioma.

Malignant mesothelioma (MM) is a highly aggressive cancer that is refractory to all current chemotherapeutic regimens. Therefore, uncovering new rational therapeutic targets is imperative in the field. Tyrosine kinase signaling pathways are aberrantly activated in many human cancers and are currently being targeted for chemotherapeutic intervention. Thus, we sought to identify tyrosine kinases hyperactivated in MM. An unbiased phosphotyrosine proteomic screen was employed to identify tyrosine kinases activated in human MM cell lines. From this screen, we have identified novel signaling molecules, such as JAK1, STAT1, cortactin (CTTN), FER, p130Cas (BCAR1), SRC and FYN as tyrosine phosphorylated in human MM cell lines. Additionally, STAT1 and SRC family kinases (SFK) were confirmed to be active in primary MM specimens. We also confirmed that known signal transduction pathways previously implicated in MM, such as EGFR and MET signaling axes, are co-activated in the majority of human MM specimens and cell lines tested. EGFR, MET, and SFK appear to be co-activated in a significant proportion of MM cell lines, and dual inhibition of these kinases was demonstrated to be more efficacious for inhibiting MM cell viability and downstream effector signaling than inhibition of a single tyrosine kinase. Consequently, these data suggest that TKI mono-therapy may not represent an efficacious strategy for the treatment of MM, due to multiple tyrosine kinases potentially signaling redundantly to cellular pathways involved in tumor cell survival and proliferation.

Searchable high-resolution 2D gel proteome of the human colon crypt.

We seek alterations in protein patterns at the earliest possible step on the path to cancer, namely, in cells of the target tissue from normal persons versus the corresponding normally appearing cells from persons who are heterozygous for mutation in a tumor suppressor gene that predisposes strongly to carcinoma in that tissue. To begin a systematic comparison of the proteomes of cells from normal and from neoplastic colons, we have undertaken the isolation of human colon crypts that are derived from the normal-appearing mucosa of left (descending) colon of patients with sporadic colorectal cancer. Two-dimensional (2D) gel electrophoresis is a proteomic approach that excels in the resolution of protein isoforms. Here, we document the practicality of this approach with human samples using gels of three overlapping pH ranges. For the first time, about 800 nonredundant proteins and 900 isoforms from purified human colonic crypts were identified, permitting an assessment of the contributions of protein isoforms. These interactive, searchable, hyperlink-enabled proteome maps and gene ontology analyses will facilitate future studies to discover the earliest markers and intervention targets during progression to colon cancer.