A review of screening mammography participation and utilization in Canada.

INTRODUCTION: Participation rate is an important indicator for a screening program's effectiveness; however, the current approach to measuring participation rate in Canada is not comparable with other countries. The objective of this study is to review the measurement of screening mammography participation in Canada, make international comparisons, and propose alternative methods. METHODS: Canadian breast cancer screening program data for women aged 50 to 69 years screened between 2004 and 2006 were extracted from the Canadian Breast Cancer Screening Database (CBCSD). The fee-for-services (FSS) mammography data (opportunistic screening mammography) were obtained from the provincial ministries of health. Both screening mammography program participation and utilization were examined over 24 and 30 months. RESULTS: Canada's screening participation rate increases from 39.4% for a 24-month cut-off to 43.6% for a 30-month cut-off. The 24-month mammography utilization rate is 63.1% in Canada, and the 30-month utilization rate is 70.4%. CONCLUSION: Due to the differences in health service delivery among Canadian provinces, both programmatic participation and overall utilization of mammography at 24 months and 30 months should be monitored.

Mucinous ovarian cancer.

Mucinous epithelial ovarian cancer (mEOC) accounts for approximately 10% of EOCs. Patients presenting with early-stage disease have an excellent prognosis, however, those with advanced disease have a poor outcome with relative resistance to standard ovarian cancer chemotherapy. Molecular and genetic studies demonstrate differences between mucinous and serous EOC supporting the concept that these tumors develop along separate pathways. Together with the observed differences in clinical behavior and outcome for mEOC, there is a need to develop specific therapeutic strategies for this histologic subtype. The relative rarity of advanced mEOC has resulted in few patients enrolled in major ovarian cancer trials. The results of such trials may not necessarily reflect those specific to mEOC. Separate trials testing alternative chemotherapeutics are required. Metastatic mucinous tumors from other sites such as the gastrointestinal tract may present with ovarian involvement. For all mucinous tumors of the ovary, establishing primary as opposed to metastatic cancers is important. Clinical presentation, tumor markers, histologic, and immunohistochemical features are helpful in distinguishing most cases.

Aerobic production of isoamyl acetate by overexpression of the yeast alcohol acetyl-transferases AFT1 and AFT2 in Escherichia coli and using low-cost fermentation ingredients.

Isoamyl acetate, produced via fermentation, is a natural flavor chemical with applications in the food industry. Two alcohol acetyltransferases from Saccharomyces cerevisiae (ATF1 and ATF2) can catalyze the esterification of isoamyl alcohol with acetyl coenzyme A. The respective genes were cloned and expressed in an appropriate ack-pta(-) strain of Escherichia coli. The engineered strains produce isoamyl acetate when isoamyl alcohol is added to the culture medium. Aerobic shake flask experiments examined isoamyl acetate production over various growth times, temperatures, and initial optical densities. The strain carrying the pBAD-ATF1 plasmid exhibited a high molar ester yield from glucose (1.13) after 48 h of aerobic growth at 25 degrees C. Low-cost media components, such as fusel oil, sorghum glucose and corn steep liquor, were found to give a high yield of isoamyl acetate. High-cell-density gave an increased isoamyl acetate yield of 0.18 g/g of glucose consumed.

Management of metastatic gastrointestinal stromal tumour in the Glivec era: a practical case-based approach.

Gastrointestinal stromal tumour is now recognized as a distinct pathological malignancy and has received much attention over the last few years. Despite almost universal resistance to chemotherapy, a novel therapy, Imatinib, which targets the KIT receptor, has changed the natural history of this disease. We have audited the first 26 consecutive patients with gastrointestinal stromal tumour treated over 4 years at a single institution. A practical guide to the management of common toxicities and drug resistance is reported with a review of the published reports. Many of the strategies used are likely to be widely applicable to the use of targeted therapies in other malignancies.

Small cell of the ovary, hypercalcemic type -- analysis of combined experience and recommendation for management. A GCIG study.

UNLABELLED: Small cell carcinoma of the ovary, hypercalcemic type (SCCOHT) is a rare tumor typically affecting young women. It is an aggressive malignancy with a poor prognosis and few long-term survivors. OBJECTIVE: Investigate the outcome of patients with SCCOHT. METHOD: Data were collected for patients with SCCOHT treated in Australia, Canada and Europe. Information included stage, surgery, chemotherapy, radiotherapy, recurrence and survival. RESULTS: The median follow-up is 13 months for all patients and 35.5 months in surviving patients. Ten patients had FIGO stage I tumors, six stage III tumors and one stage unknown. All underwent surgical resection. Adjuvant platinum-based chemotherapy was given to all patients. Seven received adjuvant radiotherapy with either pelvic and para-aortic radiotherapy, average dose 46.5 Gy (40 Gy/25# - 50.4 Gy/23#), or pelvic and whole abdominal radiotherapy, average dose 45 Gy to pelvis and 25 Gy (22.5 Gy/22# - 30 Gy/25#) to abdomen. The median survival for stage I tumors was not reached and was 6 months for stage III tumors. For the ten patients with stage I tumors: six received adjuvant radiotherapy with five alive and disease-free; four received no adjuvant radiotherapy with one alive and disease-free, while three have relapsed with one alive and disease-free after resection. Of the seven patients with stage III or unknown stage tumors, all but one have died. Recurrences were most frequent in the pelvis and the abdomen. Patients receiving salvage treatment with chemotherapy and radiotherapy did poorly. CONCLUSION: We advocate a multi-modality treatment approach including surgery, chemotherapy with the addition of radiotherapy either sequentially or concurrently.

Visualization of Syk-antigen receptor interactions using green fluorescent protein: differential roles for Syk and Lyn in the regulation of receptor capping and internalization.

The cross-linking of the B cell Ag receptor (BCR) is coupled to the stimulation of multiple intracellular signal transduction cascades via receptor-associated, protein tyrosine kinases of both the Src and Syk families. To monitor changes in the subcellular distribution of Syk in B cells responding to BCR cross-linking, we expressed in Syk-deficient DT40 B cells a fusion protein consisting of Syk coupled to green fluorescent protein. Treatment of these cells with anti-IgM Abs leads to the recruitment of the kinase from cytoplasmic and nuclear compartments to the site of the cross-linked receptor at the plasma membrane. The Syk-receptor complexes aggregate into membrane patches that redistribute to form a cap at one pole of the cell. Syk is not demonstrably associated with the internalized receptor. Catalytically active Syk promotes and stabilizes the formation of tightly capped BCR complexes at the plasma membrane. Lyn is not required for the recruitment of Syk to the cross-linked receptor, but is required for the internalization of the clustered BCR complexes. In the absence of Lyn, receptor-Syk complexes at the plasma membrane are long lived, and the receptor-mediated activation of the NF-AT transcription factor is enhanced. Thus, Lyn appears to function to negatively regulate aspects of BCR-dependent signaling by stimulating receptor internalization and down-regulation.

Substrate recognition by the Lyn protein-tyrosine kinase. NMR structure of the immunoreceptor tyrosine-based activation motif signaling region of the B cell antigen receptor.

The immunoreceptor tyrosine-based activation motif (ITAM) plays a central role in transmembrane signal transduction in hematopoietic cells by mediating responses leading to proliferation and differentiation. An initial signaling event following activation of the B cell antigen receptor is phosphorylation of the CD79a (Ig-alpha) ITAM by Lyn, a Src family protein-tyrosine kinase. To elucidate the structural basis for recognition between the ITAM substrate and activated Lyn kinase, the structure of an ITAM-derived peptide bound to Lyn was determined using exchange-transferred nuclear Overhauser NMR spectroscopy. The bound substrate structure has an irregular helix-like character. Docking based on the NMR data into the active site of the closely related Lck kinase strongly favors ITAM binding in an orientation similar to binding of cyclic AMP-dependent protein kinase rather than that of insulin receptor tyrosine kinase. The model of the complex provides a rationale for conserved ITAM residues, substrate specificity, and suggests that substrate binds only the active conformation of the Src family tyrosine kinase, unlike the ATP cofactor, which can bind the inactive form.

Inhibition of signaling through the B cell antigen receptor by the protooncogene product, c-Cbl, requires Syk tyrosine 317 and the c-Cbl phosphotyrosine-binding domain.

The Syk protein-tyrosine kinase couples the B cell Ag receptor (BCR) to intracellular biochemical pathways. Syk becomes phosphorylated on multiple tyrosine residues upon receptor cross-linking. Tyrosine 317 is a site of phosphorylation located within the linker region of Syk that separates the amino-terminal, tandem pair of SH2 domains from the carboxyl-terminal catalytic domain. The amino acid sequence surrounding phosphotyrosine 317 matches the consensus sequence for recognition by the phosphotyrosine-binding (PTB) domain of the protooncogene product, c-Cbl. The overexpression of c-Cbl in DT40 B cells inhibits Ag receptor-mediated activation of the NF-AT transcription factor. The ability of overexpressed c-Cbl to inhibit signaling requires both Syk tyrosine 317 and a functional c-Cbl PTB domain. Mutant forms of Syk lacking tyrosine 317 exhibit an enhanced ability to couple the BCR to pathways leading to the activation of both NF-AT and Elk-1. Coimmunoprecipitation experiments indicate that Syk phosphotyrosine 317 and the c-Cbl PTB domain enhance, but are not required for, all interactions between these two proteins. In unstimulated cells, c-Cbl and Syk can be isolated in a complex that also contains tubulin. A mutant form of Syk lacking tyrosine at position 317 exhibits an enhanced ability to interact with a diphosphopeptide modeled on the immunoreceptor tyrosine-based activation motif of the CD79a component of the Ag receptor. These studies indicate that c-Cbl may contribute to the regulation of BCR signaling by modulating the ability of Syk to associate with the BCR and couple the receptor to intracellular signaling pathways.

The engagement of beta1 integrins on promonocytic cells promotes phosphorylation of Syk and formation of a protein complex containing Lyn and beta1 integrin.

The protein-tyrosine kinase Syk participates in signal transduction pathways downstream from multiple immune recognition receptors. Recent evidence indicates that Syk is also functionally coupled to cell surface integrins, which mediate interactions between the actin cytoskeleton and extracellular matrix proteins. The interactions of undifferentiated, promonocytic HL60 or U937 cells with fibronectin or anti-beta1 integrin antibodies leads to an apparent activation and tyrosine phosphorylation of Syk that is independent of tight cellular adhesion and spreading. In response to fibronectin or anti-beta1 integrin antibodies, beta1 integrins become associated with a complex of proteins that include the Lyn protein tyrosine kinase and endogenous kinase substrates of 29 and 75-80 kDa. Lyn becomes transiently activated following integrin engagement and co-localizes with the actin cytoskeleton. These studies suggest a major role for Lyn in coupling beta1 integrins to the activation of Syk.

Phosphorylation- and activation-independent association of the tyrosine kinase Syk and the tyrosine kinase substrates Cbl and Vav with tubulin in B-cells.

Aggregation of the B-cell antigen receptor leads to the activation of the 72-kDa Syk protein-tyrosine kinase and the phosphorylation of tubulin on tyrosine. To explore the requirement of Syk catalytic activity for tubulin phosphorylation, tubulin was isolated from cytosolic fractions from anti-IgM-activated B-cells (DT40) that lacked endogenous Syk and immunoblotted with anti-phosphotyrosine antibodies. Tubulin was not tyrosine-phosphorylated in Syk- B-cells. Phosphorylation could be restored by the expression of wild-type, but not catalytically inactive, Syk. However, both catalytically inactive and wild-type Syk were capable of constitutive association with tubulin, indicating that tubulin phosphorylation is not required for this interaction. Anti-phosphotyrosine antibody immunoblotting of proteins adsorbed to colchicine-agarose revealed the presence of three major tubulin-associated phosphoproteins of 110, 90, and 74 kDa, the phosphorylation of which was dependent on Syk expression. The proteins of 110 and 90 kDa were identified as Cbl and Vav, two proto-oncogene products known to become prominently phosphorylated following receptor engagement. Both proteins were shown to be constitutively associated with tubulin.

Syk- and Lyn-dependent phosphorylation of Syk on multiple tyrosines following B cell activation includes a site that negatively regulates signaling.

The Syk protein tyrosine kinase is an essential component of the B cell Ag receptor signaling pathway. Syk is phosphorylated on tyrosine following B cell activation. However, the sites that are modified and the kinases responsible for these modifications have yet to be determined. To approach this problem, we used a mapping strategy based on the electrophoretic separation of peptides on alkaline polyacrylamide gels to identify the tryptic phosphopeptides derived from metabolically labeled Syk. In this work, we report that Syk from activated B cells is phosphorylated principally on six tyrosines: one located between the tandem SH2 domains (Tyr130); three in the linker region (Tyr317, Tyr342, and Tyr346); and two in the catalytic domain (Tyr519 and Tyr520). The linker region sites are the primary targets of the Src family protein tyrosine kinase, Lyn, and include a site that negatively (Tyr317) regulates receptor signaling. Efficient phosphorylation of the catalytic domain and inter-SH2 domain tyrosines is catalyzed primarily by Syk itself, but only occurs to an appreciable extent in cells that express Lyn. We propose that these sites are phosphorylated following the binding of Syk to immunoreceptor tyrosine-based activation motif.

Oxidative stress and thiol depletion in plasma and peripheral blood lymphocytes from HIV-infected patients: toxicological and pathological implications.

OBJECTIVES: To determine, first, whether the plasma and lymphocytes of HIV-positive individuals and AIDS patients have alterations in the major thiols glutathione and cysteine, and/or their oxidative disulphide and mixed disulphide products; and, secondly, whether thiol/disulphide status differs in patients with sulphonamide drug hypersensitivity reactions. DESIGN: Thiols provide critical cellular defence against toxic drug reactive intermediates and endogenous oxidative stress, and may modulate HIV replication. Glutathione is reported to be low in HIV-positive individuals and AIDS patients, but this is controversial and the mechanism responsible is unknown. Also unknown is whether altered thiol/disulphide status determines the predisposition of HIV-positive and AIDS patients to drug reactions. METHODS: Thiols and disulphides were measured by high-performance liquid chromatography. RESULTS: Both plasma thiols were decreased by approximately 58% in HIV-positive individuals and AIDS patients compared with uninfected controls (P < 0.05), with increases of up to threefold in oxidized products (P < 0.05). Similarly, in lymphocytes, thiols were decreased by 30-35% (P < 0.05), with apparent increases in oxidized products. For both glutathione and cysteine, the thiol/disulphide ratios also were decreased (P < 0.05). The plasma and lymphocyte glutathione thiol/disulphide ratios were highly correlated (r = 0.7661; P = 0.0001) among all subjects. No parameters differed in patients with drug reactions, or with antiretroviral therapy. CONCLUSIONS: The enhanced thiol oxidation in HIV-positive individuals and AIDS patients indicates oxidative stress, which also contributes to thiol depletion, and may enhance damage to macromolecular targets. These mechanisms may contribute to enhanced viral replication and other pathological outcomes. HIV-positive individuals' and AIDS patients' predisposition to drug hypersensitivity reactions appears to be unrelated to thiol/disulphide status.

The Annonaceous acetogenin bullatacin is cytotoxic against multidrug-resistant human mammary adenocarcinoma cells.

Cytotoxic effects of the Annonaceous acetogenin, bullatacin, were studied in multidrug-resistant (MDR) human mammary adenocarcinoma (MCF-7/Adr) cells vs. the parental non-resistant wild type (MCF-7/wt) cells. Bullatacin was effectively cytotoxic to the MCF-7/Adr cells while it was more cytostatic to the MCF-7/wt cells. ATP depletion is the mode of action of the Annonaceous acetogenins, and these agents offer a special advantage in the chemotherapeutic treatment of MDR tumors that have ATP-dependent mechanisms.

Syk activation and dissociation from the B-cell antigen receptor is mediated by phosphorylation of tyrosine 130.

Syk (p72(syk)) is a 72-kDa cytoplasmic protein-tyrosine kinase that serves as an essential component of the signal transduction machinery coupled to the B-cell antigen receptor. Syk is recruited to the receptor when it is cross-linked and, in response, becomes tyrosine-phosphorylated and activated before it dissociates from the receptor and appears in the cytoplasm. To begin to explore how tyrosine phosphorylation affects Syk activation and receptor binding, Tyr-130, which is localized within the Syk inter-Src homology 2 domain region, was substituted with Phe or Glu. Substitution of Tyr-130 with Phe enhanced the binding of Syk to the receptor and increased receptor-mediated protein tyrosine phosphorylation, while substitution with Glu greatly reduced this interaction. Replacement of Tyr-130 with Glu also increased the basal activity of the kinase, while replacement with Phe decreased its activity and uncoupled kinase activation from receptor engagement. These data suggest that the phosphorylation of Tyr-130 normally plays an important role in mediating both the activation of Syk and its release from the antigen receptor.

Identification of the major sites of autophosphorylation of the murine protein-tyrosine kinase Syk.

The protein tyrosine kinase p72syk (Syk) is expressed in a variety of hematopoietic cell types, including B cells, thymocytes, mast cells and others. Both the activity and phosphotyrosine content of this enzyme increase in these cells in response to engagement of the appropriate cell surface receptors. Herein, we describe the cloning of murine Syk and its expression in Sf9 cells as a catalytically active protein. Full-length Syk and a catalytically active 42.5 kDa carboxyl terminal fragment were also expressed as glutathione S-transferase fusion proteins. Comparative reverse phase HPLC and 40% alkaline gel analysis of tryptic digests of phosphorylated Syk demonstrated that all of the major sites of autophosphorylation were also present in GST-Syk and all but one were contained in the 42.5 kDa fragment. The sites of autophosphorylation were identified using a combination of Edman sequencing and mass spectrometric analysis. Ten sites were identified. One site is located in the amino terminal half of the molecule between the two tandem Src homology 2 (SH2) domains. Five sites are located in the hinge region located between the carboxyl terminal SH2 domain and the kinase domain. Two sites lie in the kinase domain within the catalytic loop and two near the extreme carboxyl terminus. Sequences of phosphorylation sites located within the hinge region predict that Syk serves as a docking site for other SH2 domain-containing proteins. Consistent with this prediction, autophosphorylated Syk efficiently binds the carboxyl terminal SH2 domain of phospholipase C-gamma 1.

Activation of T cell Raf-1 at mitosis requires the protein-tyrosine kinase Lck.

The serine/threonine protein kinase Raf-1 is activated in response to a variety of growth factors in fibroblasts and hematopoietic cells. In T cells, Raf-1 is activated in response to stimulation through the T cell antigen receptor, the interleukin-2 receptor, and by stimulation of protein kinase C. We demonstrate here that in T cells, Raf-1 is also activated during mitosis. The mitotic activation of Raf-1 was not observed in the Lck-deficient cell line, J.CaM.1. During mitosis, Raf-1 was found to interact selectively with a mitotic form of Lck that migrated with a reduced electrophoretic mobility on SDS-polyacrylamide gels. We conclude that Raf-1 is activated during mitosis in T cells and that this mitotic activation of Raf-1 is dependent on the presence of Lck.

The protein-tyrosine kinase Lck associates with and is phosphorylated by Cdc2.

The protein-tyrosine kinase Lck is essential for signaling through the T-cell antigen receptor. Treatment of T-cells with a variety of extracellular stimuli increases the phosphorylation of Lck on serine residues. This results in shifts in the apparent molecular weight of Lck to forms that exhibit reduced electrophoretic mobility on SDS-polyacrylamide gels. We found that as a result of arresting cells in mitosis, forms of Lck were generated that migrated with slower mobilities on SDS-polyacrylamide gels. This suggested that a serine/threonine kinase, active at mitosis, was phosphorylating Lck. Using antibodies to Lck and to the cyclin-dependent serine kinase, Cdc2, as well as the cyclin-dependent kinase affinity resin, Suc1-agarose, we detected a stable interaction between Lck and Cdc2. The interaction was mediated through the Src homology 3 domain of Lck and was selective, as only the active form of Cdc2 was found to associate with Lck. Moreover, Cdc2 was able to phosphorylate Lck in vitro and shift its electrophoretic mobility to a more slowly migrating form. An association between active Cdc2 and the Src-related kinases Lyn and Fyn was also demonstrated, although Cdc2 was not found associated with the tyrosine kinases, Csk and Syk. These results demonstrate that at mitosis, Cdc2 associates with and phosphorylates Lck.

Syk, activated by cross-linking the B-cell antigen receptor, localizes to the cytosol where it interacts with and phosphorylates alpha-tubulin on tyrosine.

Syk (p72syk) is a 72-kDa, nonreceptor, protein-tyrosine kinase that becomes tyrosine-phosphorylated and activated in B lymphocytes following aggregation of the B-cell antigen receptor. To explore the subcellular location of activated Syk, anti-IgM-activated B-cells were fractionated into soluble and particulate fractions by ultracentrifugation. Activated and tyrosine-phosphorylated Syk was found predominantly in the soluble fraction and was not associated with components of the antigen receptor. Similarly, the activated forms of Syk and its homolog, ZAP-70, were found in soluble fractions prepared from pervanadate-treated Jurkat T-cells. A 54-kDa protein that co-immunoprecipitated with Syk from the soluble fraction of activated B-cells was identified by peptide mapping as alpha-tubulin. alpha-Tubulin was an excellent in vitro substrate for Syk and was phosphorylated on a single tyrosine present within an acidic stretch of amino acids located near the carboxyl terminus. alpha-Tubulin was phosphorylated on tyrosine in intact cells following aggregation of the B-cell antigen receptor in a reaction that was inhibited by the Syk-selective inhibitor, piceatannol. Thus, once activated, Syk releases from the aggregated antigen receptor complex and is free to associate with and phosphorylate soluble proteins including alpha-tubulin.

Discharging patients earlier from Winnipeg hospitals: does it adversely affect quality of care?

OBJECTIVE: To determine whether decreasing lengths of stay over time for selected diagnostic categories were associated with increased hospital readmission rates and mean number of physician visits after discharge. DESIGN: Retrospective descriptive study. SETTING: The seven large (125 beds or more) acute care hospitals in Winnipeg. PATIENTS: Manitoba residents admitted to any one of the seven hospitals because acute myocardial infarction (AMI), bronchitis or asthma, transurethral prostatectomy (TURP) and uterine or adnexal procedures for nonmalignant disease during the fiscal years 1989-90 to 1992-93. Patients from out of province, those who died in hospital, those with excessively long stays (more than 60 days) and those who were transferred to or from another institution were excluded. OUTCOME MEASURES: Length of hospital stay, and rate of readmission within 30 days after discharge for all four categories and mean number of physician visits within 30 days after discharge for two categories (AMI and bronchitis or asthma. RESULTS: The length of stay decreased significantly over the 4 years for all of the four categories, the smallest change being observed for patients with AMI (11.1%) and the largest for those with bronchitis or asthma (22.0%). The readmission rates for AMI, bronchitis or asthma, and TURP showed no consistent change over the 4 years. The readmission rate for uterine or adnexal procedures increased significantly between the first and second year (chi 2 = 4.28, p = 0.04) but then remained constant over the next 3 years. The mean number of physician visits increased slightly for AMI in the first year (1.92 to 2.01) and then remained virtually the same. It decreased slightly for bronchitis or asthma over the 4 years. There was no significant correlation between length of stay and readmission rates for individual hospitals in 1992-93 in any of the four categories. Also, no correlation was observed between length of stay and mean number of physician visits for individual hospitals in 1992-93 in the categories AMI and bronchitis or asthma. CONCLUSIONS: Improving hospital efficiency by shortening length of stay does not appear to result in increased rates of readmission or numbers of physician visits within 30 days after discharge from hospital. Research is needed to identify optimal lengths of stay and expected readmission rates.