Targeting Lyn regulates Snail family shuttling and inhibits metastasis.

The acquisition of an invasive phenotype by epithelial cells occurs through a loss of cellular adhesion and polarity, heralding a multistep process that leads to metastatic dissemination. Since its characterization in 1995, epithelial-mesenchymal transition (EMT) has been closely linked to the metastatic process. As a defining aspect of EMT, loss of cell adhesion through downregulation of E-cadherin is carried out by several transcriptional repressors; key among them the SNAI family of transcription factors. Here we identify for the first time that Lyn kinase functions as a key modulator of SNAI family protein localization and stability through control of the Vav-Rac1-PAK1 (Vav-Rac1-p21-activated kinase) pathway. Accordingly, targeting Lyn in vitro reduces EMT and in vivo reduces metastasis of primary tumors. We also demonstrate the clinical relevance of targeting Lyn as a key player controlling EMT; patient samples across many cancers revealed a strong negative correlation between Lyn and E-cadherin, and high Lyn expression in metastatic tumors as well as metastasis-prone primary tumors. This work reveals a novel pancancer mechanism of Lyn-dependent control of EMT and further underscores the role of this kinase in tumor progression.

Lyn activity protects mice from DSS colitis and regulates the production of IL-22 from innate lymphoid cells.

Intestinal homeostasis requires a complex balance of interactions between diverse resident microbial communities, the intestinal epithelium, and the underlying immune system. We show that the Lyn tyrosine kinase, a critical regulator of immune cell function and pattern-recognition receptor (PRR) responses, has a key role in controlling gastrointestinal inflammation. Lyn⁻/⁻ mice were highly susceptible to dextran sulfate sodium (DSS)-induced colitis, whereas Lyn gain-of-function (Lyn(up)) mice exhibited attenuated colitis during acute and chronic models of disease. Lyn(up) mice were hypersensitive to lipopolysaccharide (LPS), driving enhanced production of cytokines and factors associated with intestinal barrier function, including interleukin (IL)-22. Oral administration of LPS was sufficient to protect antibiotic-treated Lyn(up) but not wild-type mice from DSS, highlighting how Lyn-dependent changes in the nature/magnitude of PRR responses can impact intestinal health. Furthermore, protection from DSS-induced colitis and increased IL-22 production in response to LPS did not depend on the adaptive immune system, with increased innate lymphoid cell-derived IL-22 correlating with Lyn activity in dendritic cells. These data reveal a key role for Lyn in the regulation of innate immune responses and control of intestinal inflammation.

Electronic real-time surveillance for influenza-like illness: experience from the 2009 influenza A(H1N1) pandemic in Denmark.

To enhance surveillance for influenza-like illness (ILI)in Denmark, a year-round electronic reporting system was established in collaboration with the Danish medical on-call service (DMOS). In order to achieve real-time surveillance of ILI, a checkbox for ILI was inserted in the electronic health record and a system for daily transfer of data to the national surveillance centre was implemented. The weekly number of all consultations in DMOS was around 60,000, and activity of ILI peaked in week 46 of 2009 when 9.5% of 73,723 consultations were classified as ILI. The incidence of ILI reached a maximum on 16 November 2009 for individuals between five and 24 years of age, followed by peaks in children under five years, adults aged between 25 and 64 years and on 27 November in senior citizens(65 years old or older). In addition to the established influenza surveillance system, this novel system was useful because it was timelier than the sentinel surveillance system and allowed for a detailed situational analysis including subgroup analysis on a daily basis.

[Hundred twentieth anniversary of the Berlin Surgical Society. Significance of military surgery]. / 120 Jahre Berliner Chirurgische Gesellschaft. Bedeutung der Militärchirurgie.

November 22, 2006 will mark the one hundred twentieth anniversary of the oldest regional surgical society in Germany, which was founded as the Free Association of Berlin Surgeons in 1886. For years, the chairmen were also chairmen of the German Surgical Society (established 1872). Thus they made important contributions to surgery in Germany as a whole. Professors such as Ernst von Bergmann, August Bier, and Ferdinand Sauerbruch furthered the reputation of the Berlin practitioners and German surgery throughout the world. In the states of Berlin and Brandenburg, development and promotion of surgery in the late eighteenth and nineteenth centuries owed much to the Prussian emperor Friedrich Wilhelm I and the necessities of Prussian battlefields (military surgical training). These battlefields also caused the sharp decline in worldwide importance of Berlin surgeons at the end of World War II. The special geopolitical situation of Berlin in post-war Germany constituted a negative turning point in this region, not only for surgery. As a result of the destruction of Berlin, most records and documents of the Berlin Surgical Society were lost. Research conducted in February 2006 revealed 20 membership lists from the founding years (1893-1914) which were presumed to be lost. These lists can now help us restore part of the Society's identity and roots. New insights have been made regarding the composition of the Society. For example, the large number of military surgeons in these lists reflects the spirit of the times around 1900 and emphasizes the importance of military medicine in imperial Germany.

[Procedures of temporary wall closure in abdominal trauma and sepsis]. / Verfahren zum temporären Bauchdeckenverschluss bei Trauma und Sepsis.

Temporary abdominal closure methods differ mainly between vacuum-assisted and conventional approaches. Each method has its indications. Vacuum-assisted methods seem to be superior especially for trauma indications--in terms of lethality, the possibility of secondary closure during primary hospital stay, and frequency of enterocutaneous fistulas. Skin-only closure might be used as a short-term application (e.g. when damage control closure is needed), and the Bogota bag silo gives space to protruding bowels in pending or manifest abdominal compartment syndrome. Temporary fascial mesh closure enables repetitive laparotomies through the mesh, thus sparing the fascia. For that reason it is to be preferred, especially for its good practicability in clinical situations and on mission abroad.

Gain- and loss-of-function Lyn mutant mice define a critical inhibitory role for Lyn in the myeloid lineage.

To investigate the role of the Lyn kinase in establishing signaling thresholds in hematopoietic cells, a gain-of-function mutation analogous to the Src Y527F-activating mutation was introduced into the Lyn gene. Intriguingly, although Lyn is widely expressed within the hematopoietic system, these mice displayed no propensity toward hematological malignancy. By contrast, analysis of aging cohorts of both loss- and gain-of-function Lyn mutant mice revealed that Lyn(-/-) mice develop splenomegaly, increased numbers of myeloid progenitors, and monocyte/macrophage (M phi) tumors. Biochemical analysis of cells from these mutants revealed that Lyn is essential in establishing ITIM-dependent inhibitory signaling and for activation of specific protein tyrosine phosphatases within myeloid cells. Loss of such inhibitory signaling may predispose mice lacking this putative protooncogene to tumorigenesis.

Severe obesity does not adversely affect perioperative mortality and morbidity in coronary artery bypass surgery.

OBJECTIVE: Obese patients are usually thought to have an increased risk for complications in coronary artery bypass surgery. METHODS: Therefore, the data of 500 consecutive patients undergoing coronary artery bypass grafting at our department in 1998 by use of cardiopulmonary bypass were analyzed. Severe obesity was defined as body mass index (BMI) > or = 30.0 kg/m(2). Obese patients (n=100; group O) were compared to the remaining 400 patients (group C). Both groups were comparable with respect to sex, history of prior myocardial infarction, chronic obstructive pulmonary disease, previous stroke, duration of cardiopulmonary bypass, aortic cross-clamp time and number of distal anastomoses performed. Obese patients were slightly younger and diabetes and hypertension were more common in these patients. RESULTS: Survival and potential complications including perioperative myocardial infarction, sternal wound infection, wound infection at the leg, renal failure, stroke, prolonged mechanical ventilation, pneumonia, reexploration for bleeding, and atrial arrhythmias were analyzed. No significant differences between obese and non-obese patients were detected. CONCLUSION: Severe obesity does not necessarily adversely affect perioperative mortality and morbidity in patients undergoing coronary artery bypass grafting in this study.

The psychophysics of imagery.

A series of experiments considers the extent to which the interrelations among subjective magnitudes aroused by images corresponds to those for subjective magnitudes aroused by physical stimuli. In Experiment 1, 68 undergraduates typed phrases in response to graded categories regarding the imagined magnitude of lights, sounds, and smells. In Experiment 2, 5 undergraduates and, in Experiment 3, 3 graduate students then magnitude estimated the image intensity aroused by each of these stimulus phrases. In Experiments 4 and 5, the same subjects performed cross-modality matches between phrases arousing images for different attributes (light, sound, and smell). Statistical analysis indicates that estimates based on images display many of the same patterns as those based on physical stimuli. The major exception involves sequence effects, present for actual stimuli but not for images.

Protein-tyrosine phosphatase alpha regulates Src family kinases and alters cell-substratum adhesion.

The roles of protein-tyrosine phosphatases (PTPs) in processes such as cell growth and adhesion are poorly understood. To explore the ability of specific PTPs to regulate cell signaling pathways initiated by stimulation of growth factor receptors, we expressed the receptor-like PTP, PTPalpha, in A431 epidermoid carcinoma cells. These cells express high levels of the epidermal growth factor (EGF) receptor and proliferate in response to the autocrine production of transforming growth factor-alpha. Conversely, EGF stimulation of A431 cells in vitro leads to growth inhibition and triggers the rapid detachment of these cells from the substratum. Although PTPalpha expression did not alter the growth characteristics of either unstimulated or EGF-stimulated cells, this phosphatase was associated with increased cell-substratum adhesion. Furthermore, PTPalpha-expressing A431 cells were strikingly resistant to EGF-induced cell rounding. Overexpression of PTPalpha in A431 cells was associated with the dephosphorylation/activation of specific Src family kinases, suggesting a potential mechanism for the observed alteration in A431 cell-substratum adhesion. Src kinase activation was dependent on the D1 catalytic subunit of PTPalpha, and there was evidence of association between PTPalpha and Src kinase(s). PTPalpha expression also led to increased association of Src kinase with the integrin-associated focal adhesion kinase, pp125(FAK). In addition, paxillin, a Src and/or pp125(FAK) substrate, displayed increased levels of tyrosine phosphorylation in PTPalpha-expressing cells and was associated with elevated amounts of Csk. In view of these alterations in focal adhesion-associated molecules in PTPalpha-expressing A431 cells, as well as the changes in adhesion demonstrated by these cells, we propose that PTPalpha may have a role in regulating cell-substratum adhesion.

CD45 and RPTPalpha display different protein tyrosine phosphatase activities in T lymphocytes.

To examine the substrate specificity and function of two receptor protein tyrosine phosphatases, CD45 and RPTPalpha, RPTPalpha was expressed in a CD45(-), T-cell receptor (TCR)+, BW5147 T-lymphoma cell. High levels of expression of RPTPalpha did not fully restore either proximal or distal TCR-mediated signalling events. RPTPalpha was unable to reconstitute the phosphorylation of CD3zeta and did not increase the expression of the activation marker, CD69, on stimulation with TCR/CD3. RPTPalpha did not significantly alter the phosphorylation state or kinase activity of two CD45 substrates, p56(lck) or p59(fyn), suggesting that RPTPalpha does not have the same specificity or function as CD45 in T-cells. Further comparison of the two phosphatases indicated that immunoprecipitated RPTPalpha was approx. one-seventh to one-tenth as active as CD45 when tested against artificial substrates. This difference in activity was also observed in vitro with purified recombinant enzymes at physiological pH. Additional analysis with Src family phosphopeptides and recombinant p56(lck) as substrates indicated that CD45 was consistently more active than RPTPalpha, having both higher Vmax and lower Km values. Thus CD45 is intrinsically a much more active phosphatase than RPTPalpha, which provides one reason why RPTPalpha cannot effectively dephosphorylate p56(lck) and substitute for CD45 in T-cells. This work establishes that these two related protein tyrosine phosphatases are not interchangeable in T-cells and that this is due, at least in part, to quantitative differences in phosphatase activity.

Comparison of the specificity of bacterially expressed cytoplasmic protein-tyrosine phosphatases SHP and SH-PTP2 towards synthetic phosphopeptide substrates.

SHP and SH-PTP2 are related cytoplasmic protein-tyrosine phosphatases having two tandem amino-terminal src homology 2 domains linked to a single catalytic domain. There is growing evidence that these two molecules may exhibit opposing effects within specific signaling pathways. However, the relative contributions of the src homology 2 domains or the catalytic domains to these opposing effects are not well known. To evaluate the potential contribution of the catalytic domains, we compared the substrate specificity of the two phosphatases. As seen previously, the catalytic activities of bacterially expressed SHP and SH-PTP2 were regulated by the presence of the linked src homology 2 domains. In addition, we characterized a cryptic thrombin cleavage site within the carboxy-terminus of SHP that led to a striking increase in the activity of the catalytic domain. Employing a panel of phosphopeptide substrates whose sequences were modeled after intracellular phosphorylation sites, both SHP and SH-PTP2 demonstrated a similar specificity pattern. Similar to SH-PTP2, SHP failed to elicit detectable phosphate release from several phosphopeptide substrates, while displaying catalytic efficiencies that ranged over approximately 40-1.6 x 10(3) M-1 s-1 towards other substrates. In contrast, the PTP-1B phosphatase dephosphorylated all of the phosphopeptide substrates tested with approximately equal ease. The overall similarity demonstrated by the catalytic domains of SHP and SH-PTP2 suggested that differences in the in vivo behavior of these two molecules might not stem from differences in the substrate specificity of the catalytic domains, suggesting instead that the specificity of the src homology 2 domains is more important in this regard.

A widely distributed putative mammalian transcriptional regulator containing multiple paired amphipathic helices, with similarity to yeast SIN3.

The mammalian Sin3 gene (mSin3) encodes four paired amphipathic helix (PAH) motifs, three of which and an extended region beyond PAH3 share between 59 and 70% sequence similarity with the yeast transcriptional regulator, SIN3. However, mSin3/SIN3 fusion proteins were not able to substitute for the yeast molecule in complementation assays. Transcripts encoding this putative transcriptional regulator, which maps to human chromosome 15q24, were detected in multiple mouse tissues, with highest levels seen in testis, lung, and thymus. Its wide tissue distribution suggests that mSin3, like yeast SIN3, may regulate the transcription of multiple genes.

Non-radioactive method to measure CD45 protein tyrosine phosphatase activity isolated directly from cells.

Preparation of radioactive phosphorylated substrates is laborious, yields a limited amount of substrate with a short half-life and generates a low percentage of phosphorylated product which then has to be separated from non-phosphorylated material. These factors limit the usefulness of radioactive phosphorylated substrates in phosphatase assays and prohibit their use for kinetic analysis, which often requires large amounts of substrate. An alternative method for the kinetic analysis of purified or recombinant soluble phosphatases uses the malachite green reagent which can detect nanomoles of phosphate released from chemically synthesized phosphorylated peptides. In this report we describe a rapid and sensitive non-radioactive method that can be used to measure protein tyrosine phosphatase (PTP) activities of both transmembrane and soluble phosphatases immunoprecipitated directly from cells. This colorimetric microassay is performed in 96 well microtitre plates and can reliably detect 100 pmol of free phosphate released, using a standard microplate reader. The phosphatase activity of CD45, a transmembrane PTP, was determined from as few as 1 x 10(4) lymphoid cells. The development of this colorimetric assay to measure immunoprecipitated CD45 PTP activity isolated from very small numbers of cells has general applicability for other PTPs and will help identify the cellular situations and conditions that result in changes in PTP activity.

High-sensitivity determination of tyrosine-phosphorylated peptides by on-line enzyme reactor and electrospray ionization mass spectrometry.

We describe a simple, fast, sensitive, and nonisotopic bioanalytical technique for the detection of tyrosine-phosphorylated peptides and the determination of sites of protein tyrosine phosphorylation. The technique employs a protein tyrosine phosphatase micro enzyme reactor coupled on-line to either capillary electrophoresis or liquid chromatography and electrospray ionization mass spectrometry instruments. The micro enzyme reactor was constructed by immobilizing genetically engineered, metabolically biotinylated human protein tyrosine phosphatase beta onto the inner surface of a small piece of a 50-microns inner diameter, 360-microns outer diameter fused silica capillary or by immobilization of the phosphatase onto 40-90-microns avidin-activated resins. By coupling these reactors directly to either a capillary electrophoresis column or a liquid chromatography column, we were able to rapidly perform enzymatic dephosphorylation and separation of the reaction products. Detection and identification of the components of the reaction mixture exiting these reactors were done by mass analysis with an on-line electrospray ionization mass spectrometer. Tyrosine-phosphorylated peptides, even if present in a complex peptide mixture, were identified by subtractive analysis of peptide patterns generated with or without phosphatase treatment. Two criteria, namely a phosphatase-induced change in hydropathy and charge, respectively, and a change in molecular mass by 80 Da, were used jointly to identify phosphopeptides. We demonstrate that, with this technique, low picomole amounts of a tyrosine-phosphorylated peptide can be detected in a complex peptide mixture generated by proteolysis of a protein and that even higher sensitivities can be realized if more sensitive detection systems are applied.

Antitumor drug fostriecin inhibits the mitotic entry checkpoint and protein phosphatases 1 and 2A.

In most eukaryotic cells, entry into mitosis is tightly controlled and requires completely replicated and undamaged DNA. We show that the antitumor drug, fostricin, interferes with this control; it induces cycling cells to enter mitosis prematurely, and it can overcome the mitotic entry checkpoint, forcing into mitosis cells that were arrested in the division cycle by treatment with the DNA replication inhibitor aphidicolin or with the DNA-damaging agents camptothecin and teniposide. This effect was observed in all rodent, simian, and human cell lines tested. Fostriecin also hampers progression through the later stages of mitosis as determined by the absence of normal half-spindles, anaphase figures, and telophase figures. The only previously known target for fostriecin is topoisomerase II, which is inhibited in vitro with a 50% inhibitory concentration of 40 microM (T. J. Boritzki, T. S. Wolfard, J. A. Besserer, R. C. Jackson, and D. W. Fry. Inhibition of type II topoisomerase by fostriecin. Biochem. Pharmacol., 37: 4063-4068, 1988). We show that fostriecin is a more potent inhibitor of protein phosphatase 1, with a 50% inhibitory concentration of 4 microM and protein phosphatase 2A, with a 50% inhibitory concentration of 40 nM. Inhibition of the mitotic entry checkpoint and inhibition of protein phosphatases are novel properties for antitumor drugs with potential or proven therapeutic value.

Characterization and kinetic analysis of the intracellular domain of human protein tyrosine phosphatase beta (HPTP beta) using synthetic phosphopeptides.

The intracellular domain of human protein tyrosine phosphatase beta (HPTP beta) (44 kDa) was expressed in bacteria, purified using epitope 'tagging' immunoaffinity chromatography, and characterized with respect to kinetic profile, substrate specificity and potential modulators of enzyme activity. A chromogenic assay based on the Malachite Green method was employed for the detection of inorganic phosphate (Pi) released from phosphopeptides by HPTP beta. This assay, modified so as to improve its sensitivity, was adapted to a 96-well microtitre plate format, and provided linear detection between 50 and 1000 pmol of Pi. The cytoplasmic domain of HPTP beta was strongly inhibited by vanadate, molybdate, heparin, poly(Glu, Tyr) (4:1) and zinc ions. In order to explore the substrate preferences of this PTPase, we generated 13-residue synthetic phosphotyrosine-containing peptides that corresponded to sites of physiological tyrosine phosphorylation. HPTP beta demonstrated kcat. values between 76 and 258 s-1 using four different phosphopeptides. The substrate preference of HPTP beta was in the order srcTyr-527 > PDGF-RTyr-740 > ERK1Tyr-204 >> CSF-1RTyr-708 with Km values ranging from 140 microM to greater than 10 mM. The variations in affinity were probably due to differences among the four phosphopeptides compared, particularly with respect to the character of the charged amino acids flanking the phosphotyrosine residue.

Annoyance perception of sound and information extraction.

The judgment of annoyance of distorted speech differs radically for different language groups. The results show that those who do comprehend a spoken language, base their annoyance-judgments on the informational content extracted while those who do not base it on the perceptual characteristics of meaningless sound (particularly loudness). A series of distorted German speech sounds were presented to two subject groups consisting of native Swedish and English speakers, and the results were compared with earlier results from groups of native German and Polish subjects. The 50 stimuli were generated from the very same speech signal distorted in two principle ways, either with repeated silent gaps or superimposed noise impulses. The perceived annoyance of the distorted speech was judged both by category scaling for all subject groups, and as a control for "ceiling" effects, also by magnitude estimation for the Swedish and the English subjects. There is a pronounced tendency for German subjects to judge the German speech distorted with silent gaps as more annoying than that distorted with superimposed noise impulses. In contrast, the Swedish, English, and Polish subjects judged the two German-speech distortions in reversed order with regard to annoyance. Thus for noncomprehending listeners, noise-distorted speech is more annoying but for comprehending listeners it is speech distorted by gaps. This means that impaired communication intrusiveness rather than loudness predominates in annoyance judgments from comprehending listeners.

Characterization of protein tyrosine phosphatase SH-PTP2. Study of phosphopeptide substrates and possible regulatory role of SH2 domains.

The src homology 2 (SH2) domain containing protein-tyrosine-phosphatase SH-PTP2, was over-expressed in Escherichia coli for a kinetic study employing a set of synthetic 13- to 14-mer phosphopeptide substrates. The full-length SH-PTP2 protein, as well as a truncated form, lacking the two amino terminus SH2 domains (SH-PTP2(delta SH2)), exhibited Michaelis-Menten kinetics, and demonstrated striking substrate preferences on phosphopeptides having sequences based on sites of intracellular protein tyrosine phosphorylation. For example, while a KM of 59 microM and kcat/KM of 1.1 x 10(5) were obtained using SH-PTP2(delta SH2) and PDGFRY1021, a phosphorylation site within the platelet-derived growth factor receptor, other peptides revealed no detectable phosphate release. PDGFRY1009, modeled after a sequence identified as an in vivo binding site for SH-PTP2, was also a good substrate for this enzyme. The truncated form, lacking the SH2 domains demonstrated higher catalytic efficiency than the full-length enzyme. Interestingly, soluble SH2 domains were found to inhibit the catalytic activity of SH-PTP2 in a concentration-dependent manner. There was also evidence of a non-phosphotyrosine-mediated association between the two domains. These observations suggested that the SH2 domains have a direct role in regulating the catalytic activity of SH-PTP2.