Dialogue between centrosomal entrance and exit scaffold pathways regulates mitotic commitment.

The fission yeast scaffold molecule Sid4 anchors the septum initiation network to the spindle pole body (SPB, centrosome equivalent) to control mitotic exit events. A second SPB-associated scaffold, Cut12, promotes SPB-associated Cdk1-cyclin B to drive mitotic commitment. Signals emanating from each scaffold have been assumed to operate independently to promote two distinct outcomes.

A PP1-PP2A phosphatase relay controls mitotic progression.

The widespread reorganization of cellular architecture in mitosis is achieved through extensive protein phosphorylation, driven by the coordinated activation of a mitotic kinase network and repression of counteracting phosphatases. Phosphatase activity must subsequently be restored to promote mitotic exit. Although Cdc14 phosphatase drives this reversal in budding yeast, protein phosphatase 1 (PP1) and protein phosphatase 2A (PP2A) activities have each been independently linked to mitotic exit control in other eukaryotes.

A global non-coding RNA system modulates fission yeast protein levels in response to stress.

Non-coding RNAs (ncRNAs) are frequent and prevalent across the taxa. Although individual non-coding loci have been assigned a function, most are uncharacterized. Their global biological significance is unproven and remains controversial.

Mass spectral enhanced detection of Ubls using SWATH acquisition: MEDUSA--simultaneous quantification of SUMO and ubiquitin-derived isopeptides.

Protein modification by ubiquitination and SUMOylation occur throughout the cell and are responsible for numerous cellular functions such as apoptosis, DNA replication and repair, and gene transcription. Current methods for the identification of such modifications using mass spectrometry predominantly rely upon tryptic isopeptide tag generation followed by database searching with in vitro genetic mutation of SUMO routinely required. We have recently described a novel approach to ubiquitin and SUMO modification detection based upon the diagnostic a' and b' ions released from the isopeptide tags upon collision-induced dissociation of reductively methylated Ubl isopeptides (RUbI) using formaldehyde.

Chemically facilitating the generation of diagnostic ions from SUMO(2/3) remnant isopeptides.

Rationale: Mapping sites of wild-type SUMO modification is a challenging endeavour. Here we postulate that a combination of chemical derivatistation and collision-induced dissociation (CID) could be used to generate SUMO remnant diagnostic ions to aid both detection of these isopeptides and increase the analytical value of the product ion spectra required to characterize the nature and position of modification.

Methods: SUMO(2/3)ylated proteins were digested with trypsin to generate isopeptides bearing TGG and QTGG isotags.

Enhanced detection of ubiquitin isopeptides using reductive methylation.

Identification of ubiquitination (Ub) sites is of great interest due to the critical roles that the modification plays in cellular regulation. Current methods using mass spectrometry rely upon tryptic isopeptide diglycine tag generation followed by database searching. We present a novel approach to ubiquitin detection based upon the dimethyl labeling of isopeptide N-termini glycines.

Removal of centrosomal PP1 by NIMA kinase unlocks the MPF feedback loop to promote mitotic commitment in S. pombe.

Background: Activation of the Cdk1/cyclin B complex, also known as mitosis-promoting factor (MPF), drives commitment to mitosis. Interphase MPF is inhibited through phosphorylation of Cdk1 by Wee1-related kinases. Because Cdc25 phosphatases remove this phosphate, Cdc25 activity is an essential part of the switch that drives cells into mitosis.

A novel approach to the analysis of SUMOylation with the independent use of trypsin and elastase digestion followed by database searching utilising consecutive residue addition to lysine.

Rationale: Identification of sites of protein SUMOylation is of great importance due its functional diversity within the cell. To date, most approaches to this problem rely on site-directed mutagenesis and/or highly specialised mass spectrometry approaches. We present a novel alternative approach to the site mapping of SUMOylation using trypsin and elastase digestion, routine mass spectrometry and an unbiased isotag database searching strategy.

The S. pombe cytokinesis NDR kinase Sid2 activates Fin1 NIMA kinase to control mitotic commitment through Pom1/Wee1.

Mitotic exit integrates the reversal of the phosphorylation events initiated by mitotic kinases with a controlled cytokinesis event that cleaves the cell in two. The mitotic exit network (MEN) of budding yeast regulates both processes, whereas the fission yeast equivalent, the septum initiation network (SIN), controls only the execution of cytokinesis. The components and architecture of the SIN and MEN are highly conserved.

Transient structure associated with the spindle pole body directs meiotic microtubule reorganization in S. pombe.

Background: Vigorous chromosome movements driven by cytoskeletal assemblies are a widely conserved feature of sexual differentiation to facilitate meiotic recombination. In fission yeast, this process involves the dramatic conversion of arrays of cytoplasmic microtubules (MTs), generated from multiple MT organizing centers (MTOCs), into a single radial MT (rMT) array associated with the spindle pole body (SPB), the major MTOC during meiotic prophase. The rMT is then dissolved upon the onset of meiosis I when a bipolar spindle emerges to conduct chromosome segregation.

The utility of porous graphitic carbon as a stationary phase in proteomics workflows: two-dimensional chromatography of complex peptide samples.

We present the first investigation into the utility of porous graphitic carbon (PGC) as a stationary phase in proteomic workflows involving complex samples. PGC offers chemical and physical robustness and is capable of withstanding extremes of pH and higher temperatures than traditional stationary phases, without the likelihood of catastrophic failure. In addition, unlike separations driven by ion exchange mechanisms, there is no requirement for high levels of non-volatile salts such as potassium chloride in the elution buffers, which must be removed prior to LC-MS analysis.

RAC2, AEP, and ICAM1 expression are associated with CNS disease in a mouse model of pre-B childhood acute lymphoblastic leukemia.

We developed a murine model of CNS disease to obtain a better understanding of the pathogenesis of CNS involvement in pre-B-cell acute lymphoblastic leukemia (ALL). Semiquantitative proteomic discovery-based approaches identified unique expression of asparaginyl endopeptidase (AEP), intercellular adhesion molecule 1 (ICAM1), and ras-related C3 botulinum toxin substrate 2 (RAC2), among others, in an invasive pre-B-cell line that produced CNS leukemia in NOD-SCID mice. Targeting RAC2 significantly inhibited in vitro invasion and delayed disease onset in mice.

Image analysis as an adjunct to manual HER-2 immunohistochemical review: a diagnostic tool to standardize interpretation.

Aims: Accurate determination of HER-2 status is critical to identify patients for whom trastuzumab treatment will be of benefit. Although the recommended primary method of evaluation is immunohistochemistry, numerous reports of variability in interpretation have raised uncertainty about the reliability of results. Recent guidelines have suggested that image analysis could be an effective tool for achieving consistent interpretation, and this study aimed to assess whether this technology has potential as a diagnostic support tool.

Gender differences in adolescents attending a drug and alcohol withdrawal service.

Introduction And Aims: Gender differences have been reported in adult substance users, but little research has examined gender differences in adolescents presenting to treatment services. This study aimed to explore gender differences in adolescents presenting to a withdrawal service.

Design And Methods: All presentations to a withdrawal service between March 2000 and September 2004 were identified.

A UK NEQAS ICC and ISH multicentre study using the Kreatech Poseidon HER2 FISH probe: intersite variation can be rigorously controlled using FISH.

Aim: To assess a new HER2 fluorescence in situ hybridization (FISH) test and report on multicentre intrasite and intersite variation.

Methods And Results: HER2 results were scored from 45 breast cancers in eight laboratories using the Kreatech Poseidon HER2 FISH probe (Kreatech Diagnostics, Amsterdam, the Netherlands). Overall, 80.

An integrated mass-spectrometry pipeline identifies novel protein coding-regions in the human genome.

Background: Most protein mass spectrometry (MS) experiments rely on searches against a database of known or predicted proteins, limiting their ability as a gene discovery tool.

Results: Using a search against an in silico translation of the entire human genome, combined with a series of annotation filters, we identified 346 putative novel peptides [False Discovery Rate (FDR)<5%] in a MS dataset derived from two human breast epithelial cell lines. A subset of these were then successfully validated by a different MS technique.

Chromogenic in situ hybridization: a multicenter study comparing silver in situ hybridization with FISH.

Our purposes were to perform a robust assessment of a new HER2 chromogenic in situ hybridization test and report on concordance of silver in situ hybridization (SISH) data with fluorescence in situ hybridization (FISH) data and on intraobserver and interlaboratory scoring consistency. HER2 results were scored from 45 breast cancers in 7 laboratories using the Ventana (Tucson, AZ) INFORM HER-2 SISH assay and in 1 central laboratory using a standard FISH assay. Overall, 94.

Mutation of a phosphorylatable residue in Put3p affects the magnitude of rapamycin-induced PUT1 activation in a Gat1p-dependent manner.

Saccharomyces cerevisiae can utilize high quality (e.g. glutamine and ammonia) as well as low quality (e.

SRC-induced disassembly of adherens junctions requires localized phosphorylation and degradation of the rac activator tiam1.

The Rac activator Tiam1 is required for adherens junction (AJ) maintenance, and its depletion results in AJ disassembly. Conversely, the oncoprotein Src potently induces AJ disassembly and epithelial-mesenchymal transition (EMT). Here, we show that Tiam1 is phosphorylated on Y384 by Src.

External quality assurance of HER2 FISH and ISH testing: three years of the UK national external quality assurance scheme.

The American Society of Clinical Oncology/College of American Pathologists guidelines highlighted the critical importance of quality assurance in diagnostic testing for HER2. Unstained formalin-fixed, paraffin-embedded human breast carcinoma cell line sections were circulated to scheme participants on 9 occasions. "Reference laboratories" reported results for the HER2/chromosome 17 ratio and HER2 copy number for 3 years for each cell line, including 418 sets of results (1,671 results total).

Exon level integration of proteomics and microarray data.

Background: Previous studies comparing quantitative proteomics and microarray data have generally found poor correspondence between the two. We hypothesised that this might in part be because the different assays were targeting different parts of the expressed genome and might therefore be subjected to confounding effects from processes such as alternative splicing.

Results: Using a genome database as a platform for integration, we combined quantitative protein mass spectrometry with Affymetrix Exon array data at the level of individual exons.

An evaluation of the prognostic significance of vascular endothelial growth factor in node positive primary breast carcinoma.

Angiogenesis is intimately related to the growth and progression of tumours and must be induced to facilitate growth beyond a minimum size. It has been implicated in the development of metastases and survival in breast carcinoma. VEGF is a cytokine that plays an important role in angiogenesis.

An immunohistochemical study of p21 and p53 expression in primary node-positive breast carcinoma.

Aims: p21, an inhibitor of cyclin-dependent kinase, is involved in the p53 pathway of growth control. Its expression has been linked to cellular differentiation. It has been implicated in p53-mediated growth arrest following DNA damage and in terminally differentiated cells.