Epidermal growth factor receptor levels are reduced in mice with targeted disruption of the protein kinase A catalytic subunit

<p>Abstract</p> <p>Background</p> <p>Epidermal Growth Factor Receptor (EGFR) is a key target molecule in current treatment of several neoplastic diseases. Hence, in order to develop and improve current drugs targeting EGFR signalling, an accurate understanding of how this signalling pathway is regulated is required. It has recently been demonstrated that inhibition of cAMP-dependent protein kinase (PKA) induces a ligand-independent internalization of EGFR. Cyclic-AMP-dependent protein kinase consists of a regulatory dimer bound to two catalytic subunits.</p> <p>Results</p> <p>We have investigated the effect on EGFR levels after ablating the two catalytic subunits, Cα and Cβ in two different models. The first model used targeted disruption of either Cα or Cβ in mice whereas the second model used Cα and Cβ RNA interference in HeLa cells. In both models we observed a significant reduction of EGFR expression at the protein but not mRNA level.</p> <p>Conclusion</p> <p>Our results suggest that PKA may represent a target that when manipulated can maintain EGFR protein levels at the single cell level as well as in intact animals.</p

The effect of preconditioning on liver regeneration after hepatic resection in cirrhotic rats

BACKGROUND/AIMS: Ischemic preconditioning (IP) decreases severity of liver necrosis and has anti-apoptotic effects in previous studies using liver regeneration in normal rats. This study assessed the effect of IP on liver regeneration after hepatic resection in cirrhotic rats. METHODS: To induce liver cirrhosis, thioacetamide (300 mg/kg) was injected intraperitoneally into Sprague-Dawley rats twice per week for 16 weeks. Animals were divided into four groups: non-clamping (NC), total clamping (TC), IP, and intermittent clamping (IC). Ischemic injury was induced by clamping the left portal pedicle including the portal vein and hepatic artery. Liver enzymes alanine transaminase (ALT) and aspartate aminotransferase (AST) were measured to assess liver damage. Terminal deoxynucleotidyl transferase-mediated deoxyuridine triphosphate nick-end labeling (TUNEL) staining for apoptosis and proliferating cell nuclear antigen (PCNA) staining for cell replication were also performed. RESULTS: Day-1 ALT and AST were highest in IP, however, levels in NC and IC were comparably low on days 1-7. There was no significant correlation of AST or ALT with experimental groups (P=0.615 and P=0.186). On TUNEL, numbers of apoptotic cells at 100x magnification (cells/field) were 31.8+/-24.2 in NC, 69.0+/-72.3 in TC, 80.2+/-63.1 in IP, and 21.2+/-20.8 in IC (P<0.05). When regeneration capacity was assessed by PCNA staining, PCNA-positive cells (cells/field) at 400x were 3.4+/-6.0 in NC, 16.9+/-69 in TC, 17.0+/-7.8 in IP and 7.4+/-7.6 in IC (P<0.05). CONCLUSIONS: Although regeneration capacity in IP is higher than IC, the liver is vulnerable to ischemic damage in cirrhotic rats. Careful consideration is needed in applying IP in the clinical setting.ope

Remodeling of secretory lysosomes during education tunes functional potential in NK cells

Inhibitory signaling during natural killer (NK) cell education translates into increased responsiveness to activation;however, the intracellular mechanism for functional tuning by inhibitory receptors remains unclear. Secretory lysosomes are part of the acidic lysosomal compartment that mediates intracellular signalling in several cell types. Here we show that educated NK cells expressing self-MHC specific inhibitory killer cell immunoglobulin-like receptors (KIR) accumulate granzyme B in dense-core secretory lysosomes that converge close to the centrosome. This discrete morphological phenotype is independent of transcriptional programs that regulate effector function, metabolism and lysosomal biogenesis. Meanwhile, interference of signaling from acidic Ca2+ stores in primary NK cells reduces target-specific Ca2+-flux, degranulation and cytokine production. Furthermore, inhibition of PI (3,5) P-2 synthesis, or genetic silencing of the PI(3,5) P-2-regulated lysosomal Ca2+-channel TRPML1, leads to increased granzyme B and enhanced functional potential, thereby mimicking the educated state. These results indicate an intrinsic role for lysosomal remodeling in NK cell education

Remodeling of secretory lysosomes during education tunes functional potential in NK cells

Inhibitory signaling during natural killer (NK) cell education translates into increased responsiveness to activation; however, the intracellular mechanism for functional tuning by inhibitory receptors remains unclear. Secretory lysosomes are part of the acidic lysosomal compartment that mediates intracellular signalling in several cell types. Here we show that educated NK cells expressing self-MHC specific inhibitory killer cell immunoglobulin-like receptors (KIR) accumulate granzyme B in dense-core secretory lysosomes that converge close to the centrosome. This discrete morphological phenotype is independent of transcriptional programs that regulate effector function, metabolism and lysosomal biogenesis. Meanwhile, interference of signaling from acidic Ca2+ stores in primary NK cells reduces target-specific Ca2+-flux, degranulation and cytokine production. Furthermore, inhibition of PI(3,5)P2 synthesis, or genetic silencing of the PI(3,5)P2-regulated lysosomal Ca2+-channel TRPML1, leads to increased granzyme B and enhanced functional potential, thereby mimicking the educated state. These results indicate an intrinsic role for lysosomal remodeling in NK cell education

The chemical chaperone sodium 4-phenylbutyrate improves the secretion of the protein CA267T mutant in CHO-K1 cells trough the GRASP55 pathway

Some inherited coagulation factor deficiencies are caused by intracellular retention or degradation of misfolded proteins, and chemical chaperones have been shown to reverse protein misfolding. The purpose of the present study was to investigate whether chemical chaperones may improve secretion of the protein CA267T (PCA267T) mutant in a cellular model. Using stably transfected Chinese hamster ovary cells (CHO-K1) expressing PCA267T we demonstrate that sodium 4-phenylbutyrate (PBA) increased the secretion of PCA267T by approximately 4-fold in comparison with untreated cells, and that this secretion seemed to follow an unconventional pathway via the Golgi reassembly stacking protein (GRASP55)

Hvordan kan en sykepleier bruke kommunikasjon til å oppnå en god relasjon til innlagte pasienter med diagnosen schizofreni?

Bakgrunn: Schizofreni rammer cirka en prosent av befolkningen og er den alvorligste diagnosen innenfor psykoselidelsene. En utfordring som ses ved behandling av lidelsen er svikt i å mestre relasjoner. Pasientens kognitive funksjon og kommunikasjonsevne er noe sykepleier er nødt til å ta hensyn til, da mange pasienter med schizofreni lar seg lettere distrahere. Hensikt: Å belyse hva videre forskning og annen litteratur sier om hvordan sykepleiere effektivt kan bruke god kommunikasjon til å oppnå en god relasjon til pasienter med schizofreni. Metode: En litteraturstudie som benytter kvalitative og kvantitative forskningsartikler. Litteraturstudien inneholder syv forskningsartikler, der en er pilotstudie. Databaser som ble benyttet til søket var Ovid Medline og Cinahl Complete. Artiklene ble funnet ved hjelp av søkeordene: schizophrenia, psychiatric patients, communication, nursing/nursing care og nurse-patient relations. Resultat: Analyseprosessen avdekket tre temaer: “Å bli sett som et menneske”, “kommunikasjon og kommunikasjonsferdigheter”, relasjonens betydning” Konklusjon: Litteraturstudien belyser hvor viktig gode kommunikasjonsferdigheter er i møte med pasienter med schizofreni. Studien kommer fram til flere forhold som er relevante for kommunikasjonen med pasientgruppen: behov for økt kompetanse og opplæring av kommunikasjonsferdigheter, det å skape en relasjon og anerkjenne pasienten med schizofreni, og til slutt betydningen av sykepleierens holdninger og tidsforbruk. Det er et økt behov for kunnskap om kommunikasjonsferdigheter og forståelse av pasientens erfaringer. Faktorer som oppmerksomhet, lytting, vise respekt, skape tillit og empati er vesentlig. Bruk av blikkontakt, ansiktsmimikk, overdreven bruk av verbal og nonverbal språk og humor viste seg å være effektive tiltak for å etablere kontakt

Isolation and mass spectrometry analysis of urinary extraexosomal proteins

The aim of the present study was to develop a LC-MS/MS-based proteomic analysis method of urinary exosomal proteins that has the potential to discover disease biomarkers. In short, urinary exosomes from healthy subjects were isolated by immunocapture on magnetic beads, detected by immunofluorescence and TEM, trypsin digested directly on the beads for an accelerated time with no addition of detergents before performing an LC-MS analysis of the trypsinate. To our knowledge, this is the first proteomic analysis of proteins displayed on the outer surface of exosomes. The outer exosome proteome may contain proteins that are of higher biomarker value compared to soluble cargo protein as the proteins projecting into the extracellular milieu might be more directly involved in physiological functions of exosomes. The proteomic analysis identified 49 proteins that were considered significant; the majority is involved in carbohydrate and lipid metabolism or in immune responses. Thirty of the proteins are linked to diseases. The developed proteomic method exploiting urinary exosomes might be of great value in search for diagnostic or prognostic biomarkers of especially metabolic and immune-related diseases

Photochemical activation of MH3-B1/rGel: a HER2-targeted treatment approach for ovarian cancer

HER2-targeted therapy has been shown to have limited efficacy in ovarian cancer despite frequent overexpression of this receptor. Photochemical internalization (PCI) is a modality for cytosolic drug delivery, currently undergoing clinical evaluation. In the present project we studied the application of PCI in combination with the HER2-targeted recombinant fusion toxin, MH3-B1/rGel, for the treatment of ovarian cancer. The SKOV-3 cell line, resistant to trastuzumab- and MH3-B1/rGel- monotherapy, was shown to respond strongly to PCI of MH3-B1/rGel to a similar extent as observed for the treatment-sensitive SK-BR-3 breast cancer cells. Extensive hydrolytic degradation of MH3-B1/rGel in acidic endocytic vesicles was indicated as the mechanism of MH3-B1/rGel resistance in SKOV-3 cells. This was shown by the positive Pearson’s correlation coefficient between Alexa488-labeled MH3-B1/rGel and Lysotracker in SKOV-3 cells in contrast to the negative Pearson’s correlation coefficient in SK-BR-3 cells. The application of PCI to induce the release of MH3-B1/rGel was also demonstrated to be effective on SKOV-3 xenografts. Application of PCI with MH3-B1/rGel was further found highly effective in the HER2 expressing HOC-7 and NuTu-19 ovarian cancer cell lines. The presented results warrant future development of PCI in combination with MH3-B1/rGel as a novel therapeutic approach in preclinical models of ovarian cancer