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Nonalcoholic fatty liver disease (NAFLD) and nonalcoholic steatohepatitis (NASH) are emerging as leading causes of liver disease worldwide and have been recognized as one of the major unmet medical needs of the 21st century. Our recent translational studies in mouse models, human cell lines, and well-characterized patient cohorts have identified serine/threonine kinase (STK)25 as a protein that coats intrahepatocellular lipid droplets (LDs) and critically regulates liver lipid homeostasis and progression of NAFLD/NASH. Here, we studied the mechanism-of-action of STK25 in steatotic liver by relative quantification of the hepatic LD-associated phosphoproteome from high-fat diet-fed Stk25knockout mice compared with their wild-type littermates. We observed a total of 131 proteins and 60 phosphoproteins that were differentially represented in STK25-deficient livers. Most notably, a number of proteins involved in peroxisomal function, ubiquitination-mediated proteolysis, and antioxidant defense were coordinately regulated in Stk25−/−versus wild-type livers. We confirmed attenuated peroxisomal biogenesis and protection against oxidative and ER stress in STK25-deficient human liver cells, demonstrating the hepatocyte-autonomous manner of STK25's action. In summary, our results suggest that regulation of peroxisomal function and metabolic stress response may be important molecular mechanisms by which STK25 controls the development and progression of NAFLD/NASH.

We show that UBR5 is necessary for ciliogenesis and identify CSPP1 as a UBR5-interacting protein. UBR5 ubiquitylates CSPP1, and UBR5 is required for cytoplasmic organization of CSPP1-comprising centriolar satellites. This key role for UBR5 in ciliogenesis may have important implications in understanding cancer pathophysiology.

Highcontent microscopic screening systems are powerful tools for extracting quantitative multiparameter measures from large number of cells under numerous conditions. These systems perform well in applications that monitor the presence of objects, but lack in their ability to accurately estimate object intensities and summarize these findings due to variations in background, aberrations in illumination, and variability in staining over the image andor sample wells. We present effective and automated methods that are applicable to analyzing intensitybased cell cycle assays under highthroughput screening conditions. We characterize the system aberration response from images of calibration beads and then enhance the detection and segmentation accuracy of traditional algorithms by preprocessing images for local background variations. We also provide a rapid, adaptive, cellcycle partitioning algorithm to characterize each sample well based on the estimated locally and globally corrected cell intensity measures of BrdU and DAPI incorporation. We demonstrated the utility and range of our cell ploidy and probe density measurement methods in a pilot screen using a siRNA library against 779 human protein kinases. With our method, multiple imagebased quantitative phenotypes can be realized from a single highthroughput imagebased microtiterplate screen. © 2008 International Society for Advancement of Cytometry

The cellular response to DNA damage is critical for maintenance of genomic integrity and inhibition of tumorigenesis. Mutations or aberrant expression of the E3 ubiquitin ligase EDD have been observed in a number of carcinomas and we recently reported that EDD modulates activity of the DNA damage checkpoint kinase, CHK2. Here, we demonstrate that EDD is necessary for G1/S and intra S phase DNA damage checkpoint activation and for the maintenance of G2/M arrest after double strand DNA breaks. Defective checkpoint activation in EDD-depleted cells led to radio-resistant DNA synthesis, premature entry into mitosis, accumulation of polyploid cells, and cell death via mitotic catastrophe. In addition to decreased CHK2 activation in EDD-depleted cells, the expression of several key cell cycle mediators including Cdc25A/C and E2F1 was altered, suggesting that these checkpoint defects may be both CHK2-dependent and -independent. These data support a role for EDD in the maintenance of genomic stability, emphasising the potential importance of dysregulated EDD expression and/or function in the evolution of cancer.

The regulation of extracellular proteolytic activity via the plasminogen activation system is complex, involving numerous activators, inhibitors, and receptors. Previous studies on monocytic and colon cell lines suggest that plasmin pre-treatment can increase plasminogen binding, allowing the active enzyme to generate binding sites for its precursor. Other studies have shown the importance of pre-formed receptors such as annexin II heterotetramer. However, few studies have used techniques that exclusively characterise cell-surface events and these mechanisms have not been investigated at the breast cancer cell surface. We have studied plasminogen binding to MCF-7 in which urokinase plasminogen activator receptor (uPAR) levels were upregulated by PMA (12-O-tetradecanoylphorbol-13-acetate) stimulation, allowing flexible and transient modulation of cell-surface uPA. Similar experiments were also performed using MDA-MB-231 cells, which overexpress uPAR/uPA endogenously. Using techniques that preserve cell integrity, we characterise the role of uPA as both a plasminogen receptor and activator and quantify the relative contribution of pre-formed and cryptic plasminogen receptors to plasminogen binding. Cell-surface plasminogen binding was significantly enhanced in the presence of elevated levels of uPA in an activity-dependent manner and was greatly attenuated in the presence of the plasmin inhibitor aprotinin. Pre-formed receptors were also found to contribute to increased plasminogen binding after PMA stimulation and to co-localise with uPA/uPAR and plasminogen. Nevertheless, a relatively modest increase in plasminogen-binding capacity coupled with an increase in uPA led to a dramatic increase in the proteolytic capacity of these cells. We show that the majority of lysine-dependent plasminogen binding to breast cancer cells is ultimately regulated by plasmin activity and is dependent on the presence of significant levels of active uPA. The existence of a proteolytic positive feedback loop in plasminogen activation has profound implications for the ability of breast cancer cells expressing high amounts of uPA to accumulate a large proteolytic capacity at the cell surface, thereby conferring invasive potential.

Both human and non-human faecal contamination of environmental waters pose a risk to human health that can be reduced if the source can be identified and responsibility for mitigation accepted. Traditional microbiological indicators are rarely adequate for source identification but some chemical indicators offer promise. In this work, the similarities and differences of selected chemical profiles of different faecal effluent types have been modelled using classification and regression tree (CART) analysis. Human effluents can be distinguished from non-human effluents by the presence of fluorescent whitening agents, and the concentrations or ratios of concentrations of the faecal stanols coprostanol and 24-ethylcoprostanol can distinguish most of the non-human effluent types considered. To allow for the effect of variable dilution when effluents enter environmental waters, ratios of concentrations will be required to establish a model, and a model more complex than CART will be required to apportion multiple effluent-type contamination.

The physiological roles of plasminogen activator inhibitor-2 (PAI-2) are not yet well understood. Kinetic studies suggest a role in the regulation of plasminogen activator-driven proteolysis in many cell types. This study describes a monoclonal antibody (2H5), which uniquely recognizes neoepitope determinants on PAI-2 appearing after thermodynamic relaxation of the molecule. Enzyme-linked immunosorbent assays and native polyacrylamide gel electrophoresis immunoblotting confirmed the specificity of 2H5 for urokinase type plasminogen activator·PAI-2 complexes. Examination of the affinity of 2H5 for complexes formed between PAI-2 and a synthetic 14-mer reactive site loop peptide, PAI-2 treated with tissue plasminogen activator, or thrombin suggests that the 2H5 epitope is determined exclusively by sequences found only on PAI-2 following proteolytic cleavage of the Arg380–Thr381bond and insertion of the reactive site loop into β-sheet A. Peptides lacking both the P13 (Glu368) and P14 (Thr367) residues did not induce a conformational change or affect the inhibitory activity of PAI-2, indicating that one or both of these residues are critical for PAI-2 function. To our knowledge, this is the first description of a monoclonal antibody that can distinguish conformational changes in PAI-2 related specifically to its potential biological function(s).

AimsThe excretion of phospholipids in urine may be a marker of the early renal toxicity of the aminoglycoside antibiotics. Urinary phospholipids are formed in myeloid bodies which develop in the lysosomes of proximal tubules during treatment with the aminoglycosides, and overflow into the urine.

Aims The excretion of phospholipids in urine may be a marker of the early renal toxicity of the aminoglycoside antibiotics. Urinary phospholipids are formed in myeloid bodies which develop in the lysosomes of proximal tubules during treatment with the aminoglycosides, and overflow into the urine.

Is most cancer just bad luck or does lifestyle still matter, asks Darren Saunders

The BiCAP method reveals protein partner–specific signaling with implications for breast cancer.

Presents a letter to the editor commenting about the letters to the press after the removal of anti-Bush sign from the Summer causeway published in the December 2004 issue of the journal "New Zealand Listener."

The Raman spectra of 29 alkenyl methyl ethers, CnH2n–1OMe, of diverse structure containing up to seven carbon atoms reveal reliable correlations between the wavenumber of the ν(C&z.dbd;C) and ν(sp2-CH) stretching bands and the position, stereochemistry and substitution pattern of the double bond.