Rap1 promotes cell spreading by localizing Rac guanine nucleotide exchange factors

The Ras-related GTPase Rap1 stimulates integrin-mediated adhesion and spreading in various mammalian cell types. Here, we demonstrate that Rap1 regulates cell spreading by localizing guanine nucleotide exchange factors (GEFs) that act via the Rho family GTPase Rac1. Rap1a activates Rac1 and requires Rac1 to enhance spreading, whereas Rac1 induces spreading independently of Rap1. Active Rap1a binds to a subset of Rac GEFs, including VAV2 and Tiam1 but not others such as SWAP-70 or COOL-1. Overexpressed VAV2 and Tiam1 specifically require Rap1 to promote spreading, even though Rac1 is activated independently of Rap1. Rap1 is necessary for the accumulation of VAV2 in membrane protrusions at the cell periphery. In addition, if VAV2 is artificially localized to the cell edge with the subcellular targeting domain of Rap1a, it increases cell spreading independently of Rap1. These results lead us to propose that Rap1 promotes cell spreading by localizing a subset of Rac GEFs to sites of active lamellipodia extension

Spin Dynamics at Very Low Temperature in Spin Ice Dy2_2Ti2_2O7_7

We have performed AC susceptibility and DC magnetic relaxation measurements on the spin ice system Dy$_2$Ti$_2$O$_7$ down to 0.08 K. The relaxation time of the magnetization has been estimated below 2 K down to 0.08 K. The spin dynamics of Dy$_2$Ti$_2$O$_7$ is well described by using two relaxation times ($\tau_{\rm S}$ (short time) and $\tau_{\rm L}$ (long time)). Both $\tau_{\rm S}$ and $\tau_{\rm L}$ increase on cooling. Assuming the Arrhenius law in the temperature range 0.5-1 K, we obtained an energy barrier of 9 K. Below 0.5 K, both $\tau_{\rm S}$ and $\tau_{\rm L}$ show a clear deviation from the thermal activated dynamics toward temperature independent relaxation, suggesting a quantum dynamics.Comment: 4 page

Solid phase extraction for removal of matrix effects in lipophilic marine toxin analysis by liquid chromatography-tandem mass spectrometry

The potential of solid phase extraction (SPE) clean-up has been assessed to reduce matrix effects (signal suppression or enhancement) in the liquid chromatography-tandem mass spectrometry (LC¿MS/MS) analysis of lipophilic marine toxins. A large array of ion-exchange, silica-based, and mixed-function SPE sorbents was tested. Polymeric sorbents were found to retain most of the toxins. Optimization experiments were carried out to maximize recoveries and the effectiveness of the clean-up. In LC¿MS/MS analysis, the observed matrix effects can depend on the chromatographic conditions used, therefore, two different HPLC methods were tested, using either an acidic or an alkaline mobile phase. The recovery of the optimized SPE protocol was around 90% for all toxins studied and no break-through was observed. The matrix effects were determined by comparing signal response from toxins spiked in crude and SPE-cleaned extracts with those derived from toxins prepared in methanol. In crude extracts, all toxins suffered from matrix effects, although in varying amounts. The most serious effects were observed for okadaic acid (OA) and pectenotoxin-2 (PTX2) in the positive electrospray ionization mode (ESI+). SPE clean-up on polymeric sorbents in combination with the alkaline LC method resulted in a substantial reduction of matrix effects to less than 15% (apparent recovery between 85 and 115%) for OA, yessotoxin (YTX) in ESI¿ and azaspiracid-1 (AZA1), PTX2, 13-desmethyl spirolides C (SPX1), and gymnodimine (GYM) in ESI+. In combination with the acidic LC method, the matrix effects after SPE were also reduced but nevertheless approximately 30% of the matrix effects remained for PTX2, SPX1, and GYM in ESI+. It was concluded that SPE of methanolic shellfish extracts can be very useful for reduction of matrix effects. However, the type of LC and MS methods used is also of great importance. SPE on polymeric sorbents in combination with LC under alkaline conditions was found the most effective method

RasGRP4 Is a Novel Ras Activator Isolated from Acute Myeloid Leukemia

Although a number of genetic defects are commonly associated with acute myeloid leukemia (AML), a large percentage of AML cases are cytogenetically normal. This suggests a functional screen for transforming genes is required to identify genetic mutations that are missed by cytogenetic analyses. We utilized a retrovirus-based cDNA expression system to identify transforming genes expressed in cytogenetically normal AML patients. We identified a new member of the Ras guanyl nucleotide-releasing protein (RasGRP) family of Ras guanine nucleotide exchange factors, designating it RasGRP4. Subsequently, cDNA sequences encoding rodent and human RasGRP4 proteins were deposited in GenBank. RasGRP4 contains the same protein domain structure as other members of the RasGRP family, including a Ras exchange motif, a CDC25 homology domain, a C1/diacyglycerol-binding domain, and putative calcium-binding EF hands. We show that expression of RasGRP4 induces anchorage-independent growth of Rat1 fibroblasts. RasGRP4 is a Ras-specific activator and, interestingly, is highly expressed in peripheral blood leukocytes and myeloid cell lines. Unlike other RasGRP proteins, RasGRP4 is not expressed in the brain or in lymphoid cells. We demonstrated that 32D myeloid cells expressing RasGRP4 have elevated levels of activated Ras compared with control cells, and phorbol 12-myristate 13-acetate (PMA) treatment greatly enhanced Ras activation. PMA induced membrane localization of RasGRP4 and 32D cells expressing RasGRP4 were capable of cytokine-independent proliferation in the presence of PMA. We conclude that RasGRP4 is a member of the RasGRP family of Ras guanine nucleotide exchange factors that may play a role in myeloid cell signaling growth regulation pathways that are responsive to diacylglycerol levels

A Molecular Redox Switch on p21 ras: STRUCTURAL BASIS FOR THE NITRIC OXIDE-p21rasINTERACTION

We have identified the site of molecular interaction between nitric oxide (NO) and p21(ras) responsible for initiation of signal transduction. We found that p21(ras) was singly S-nitrosylated and localized this modification to a fragment of p21(ras) containing Cys118. A mutant form of p21(ras), in which Cys118 was changed to a serine residue and termed p21(ras)C118S, was not S-nitrosylated. NO-related species stimulated guanine nucleotide exchange on wild-type p21(ras), resulting in an active form, but not on p21(ras)C118S. Furthermore, in contrast to parental Jurkat T cells, NO-related species did not stimulate mitogen-activated protein kinase activity in cells transfected with p21(ras)C118S. These data indicate that Cys118 is a critical site of redox regulation of p21(ras), and S-nitrosylation of this residue triggers guanine nucleotide exchange and downstream signaling

Analysis of physical pore space characteristics of two pyrolytic biochars and potential as microhabitat

Background and Aims Biochar amendment to soil is a promising practice of enhancing productivity of agricultural systems. The positive effects on crop are often attributed to a promotion of beneficial soil microorganisms while suppressing pathogens e.g. This study aims to determine the influence of biochar feedstock on (i) spontaneous and fungi inoculated microbial colonisation of biochar particles and (ii) physical pore space characteristics of native and fungi colonised biochar particles which impact microbial habitat quality. Methods Pyrolytic biochars from mixed woods and Miscanthus were investigated towards spontaneous colonisation by classical microbiological isolation, phylogenetic identification of bacterial and fungal strains, and microbial respiration analysis. Physical pore space characteristics of biochar particles were determined by X-ray μ-CT. Subsequent 3D image analysis included porosity, surface area, connectivities, and pore size distribution. Results Microorganisms isolated from Wood biochar were more abundant and proliferated faster than those from the Miscanthus biochar. All isolated bacteria belonged to gram-positive bacteria and were feedstock specific. Respiration analysis revealed higher microbial activity for Wood biochar after water and substrate amendment while basal respiration was on the same low level for both biochars. Differences in porosity and physical surface area were detected only in interaction with biochar-specific colonisation. Miscanthus biochar was shown to have higher connectivity values in surface, volume and transmission than Wood biochars as well as larger pores as observed by pore size distribution. Differences in physical properties between colonised and non-colonised particles were larger in Miscanthus biochar than in Wood biochar. Conclusions Vigorous colonisation was found on Wood biochar compared to Miscanthus biochar. This is contrasted by our findings from physical pore space analysis which suggests better habitat quality in Miscanthus biochar than in Wood biochar. We conclude that (i) the selected feedstocks display large differences in microbial habitat quality as well as physical pore space characteristics and (ii) physical description of biochars alone does not suffice for the reliable prediction of microbial habitat quality and recommend that physical and surface chemical data should be linked for this purpose

Isolation of a NCK-associated Kinase, PRK2, an SH3-binding Protein and Potential Effector of Rho Protein Signaling

The NCK adapter protein is comprised of three consecutive Src homology 3 (SH3) protein-protein interaction domains and a C-terminal SH2 domain. Although the association of NCK with activated receptor protein-tyrosine kinases, via its SH2 domain, implicates NCK as a mediator of growth factor-induced signal transduction, little is known about the pathway(s) downstream of NCK recruitment. To identify potential downstream effectors of NCK we screened a bacterial expression library to isolate proteins that bind its SH3 domains. Two molecules were isolated, the Wiskott-Aldrich syndrome protein (WASP, a putative CDC42 effector) and a serine/threonine protein kinase (PRK2, closely related to the putative Rho effector PKN). Using interspecific backcross analysis the Prk2 gene was mapped to mouse chromosome 3. Unlike WASP, which bound the SH3 domains of several signaling proteins, PRK2 specifically bound to the middle SH3 domain of NCK and (weakly) that of phospholipase Cgamma. PRK2 also specifically bound to Rho in a GTP-dependent manner and cooperated with Rho family proteins to induce transcriptional activation via the serum response factor. These data suggest that PRK2 may coordinately mediate signal transduction from activated receptor protein-tyrosine kinases and Rho and that NCK may function as an adapter to connect receptor-mediated events to Rho protein signaling

Adverse outcomes after colposcopy

Abstract Background Colposcopy is an essential part of the National Health Service Cervical Screening Programme (NHSCSP). It is used for both diagnosis and treatment of pre-cancerous cells of the cervix. Despite colposcopy being a commonly performed and relatively invasive procedure, very little research has explored the potential long-term impacts of colposcopic examination upon patient quality of life. The aim of this study is to investigate and quantify any potential reduction in women's quality of life following a colposcopy procedure. More specifically, the degree of female sexual dysfunction and the excess risk of adverse events in those undergoing colposcopy will be explored. If such risks are identified, these can be communicated to women before undergoing colposcopy. It will also assist in identifying whether there are particular sub-groups at greater risk and if so, this may lead to a re-evaluation of current recommendations concerning colposcopically directed treatments. Methods/design Cohort study using postal surveys to assess sexual function and quality of life in women who have attended for colposcopy (cases), compared with those who have not attended colposcopy (controls). The prevalence and excess risk of female sexual dysfunction will be determined. Logistic regression will identify the predictors of adverse outcomes. Discussion There are more than 400,000 colposcopy appointments each year in England, of which 134,000 are new referrals. There is some evidence that there may be long-term implications for women treated under colposcopy with respect to adverse obstetric outcomes, persisting anxiety, increased rates of sexual dysfunction and reduced quality of life. Reliably establishing whether such adverse outcomes exist and the excess risk of adverse events will facilitate informed decision-making and patient choice.</p