S100A8/A9 (Calprotectin) Negatively Regulates G2/M Cell Cycle Progression and Growth of Squamous Cell Carcinoma

Malignant transformation results in abnormal cell cycle regulation and uncontrolled growth in head and neck squamous cell carcinoma (HNSCC) and other cancers. S100A8/A9 (calprotectin) is a calcium-binding heterodimeric protein complex implicated in cell cycle regulation, but the specific mechanism and role in cell cycle control and carcinoma growth are not well understood. In HNSCC, S100A8/A9 is downregulated at both mRNA and protein levels. We now report that downregulation of S100A8/A9 correlates strongly with a loss of cell cycle control and increased growth of carcinoma cells. To show its role in carcinogenesis in an in vitro model, S100A8/A9 was stably expressed in an S100A8/A9-negative human carcinoma cell line (KB cells, HeLa-like). S100A8/A9 expression increases PP2A phosphatase activity and p-Chk1 (Ser345) phosphorylation, which appears to signal inhibitory phosphorylation of mitotic p-Cdc25C (Ser216) and p-Cdc2 (Thr14/Tyr15) to inactivate the G2/M Cdc2/cyclin B1 complex. Cyclin B1 expression then downregulates and the cell cycle arrests at the G2/M checkpoint, reducing cell division. As expected, S100A8/A9-expressing cells show both decreased anchorage-dependent and -independent growth and mitotic progression. Using shRNA, silencing of S100A8/A9 expression in the TR146 human HNSCC cell line increases growth and survival and reduces Cdc2 inhibitory phosphorylation at Thr14/Tyr15. The level of S100A8/A9 endogenous expression correlates strongly with the reduced p-Cdc2 (Thr14/Tyr14) level in HNSCC cell lines, SCC-58, OSCC-3 and UMSCC-17B. S100A8/A9-mediated control of the G2/M cell cycle checkpoint is, therefore, a likely suppressive mechanism in human squamous cell carcinomas and may suggest new therapeutic approaches.     

Fe(III), Cr(VI), and Fe(III) mediated Cr(VI) reduction in alkaline media using a <Emphasis Type="Italic">Halomonas</Emphasis> isolate from Soap Lake,Washington

Hexavalent chromium is one of the most widely distributed environmental contaminants. Given the carcinogenic and mutagenic consequences of Cr(VI) exposure, the release of Cr(VI) into the environment has long been a major concern. While many reports of microbial Cr(VI) reduction are in circulation, very few have demonstrated Cr(VI) reduction under alkaline conditions. Since Cr(VI) exhibits higher mobility in alkaline soils relative to pH neutral soils, and since Cr contamination of alkaline soils is associated with a number of industrial activities, microbial Cr(VI) reduction under alkaline conditions requires attention. Soda lakes are the most stable alkaline environments on earth, and contain a wide diversity of alkaliphilic organisms. In this study, a bacterial isolate belonging to the Halomonas genus was obtained from Soap Lake, a chemically stratified alkaline lake located in central Washington State. The ability of this isolate to reduce Cr(VI) and Fe(III) was assessed under alkaline (pH = 9), anoxic, non-growth conditions with acetate as an electron donor. Metal reduction rates were quantified using Monod kinetics. In addition, Cr(VI) reduction experiments were carried out in the presence of Fe(III) to evaluate the possible enhancement of Cr(VI) reduction rates through electron shuttling mechanisms. While Fe(III) reduction rates were slow compared to previously reported rates, Cr(VI) reduction rates fell within range of previously reported rates.     

Structure of the Essential Plasmodium Host Cell Traversal Protein SPECT1

Host cell traversal by Plasmodium, the protozoan cause of malaria, is an essential part of this parasite''s virulence. In this process, the parasite enters a host cell through a parasite-induced pore, traverses the host cell, and then exits the host cell. Two P. berghei proteins, SPECT1 and SPECT2, are required for host cell traversal by the sporozoite form of the parasite. In the absence of either, no pore formation is observed. While SPECT2 has sequence homology to pore-forming proteins, SPECT1 has no homology to proteins of known structure or function. Here we present the 2.75 Å resolution structure of a slightly truncated version of P. berghei SPECT1. The structure reveals that the protein forms a four-helix bundle, with the rare feature of having all of these helices in parallel or antiparallel alignment. Also notable is the presence of a large, conserved, hydrophobic internal cavity in the protein, which may constitute a ligand-binding site or be indicative of partial instability in SPECT1, or both. The structure of SPECT1 will make possible targeted mutagenesis experiments aimed at understanding its mechanism of action in host cell traversal.     

Development of Ultra-High-Density Screening Tools for Microbial “Omics”

High-throughput genetic screens in model microbial organisms are a primary means of interrogating biological systems. In numerous cases, such screens have identified the genes that underlie a particular phenotype or a set of gene-gene, gene-environment or protein-protein interactions, which are then used to construct highly informative network maps for biological research. However, the potential test space of genes, proteins, or interactions is typically much larger than current screening systems can address. To push the limits of screening technology, we developed an ultra-high-density, 6144-colony arraying system and analysis toolbox. Using budding yeast as a benchmark, we find that these tools boost genetic screening throughput 4-fold and yield significant cost and time reductions at quality levels equal to or better than current methods. Thus, the new ultra-high-density screening tools enable researchers to significantly increase the size and scope of their genetic screens.