ALIX binds a YPX(3)L motif of the GPCR PAR1 and mediates ubiquitin-independent ESCRT-III/MVB sorting.

The sorting of signaling receptors to lysosomes is an essential regulatory process in mammalian cells. During degradation, receptors are modified with ubiquitin and sorted by endosomal sorting complex required for transport (ESCRT)-0, -I, -II, and -III complexes into intraluminal vesicles (ILVs) of multivesicular bodies (MVBs). However, it remains unclear whether a single universal mechanism mediates MVB sorting of all receptors. We previously showed that protease-activated receptor 1 (PAR1), a G protein-coupled receptor (GPCR) for thrombin, is internalized after activation and sorted to lysosomes independent of ubiquitination and the ubiquitin-binding ESCRT components hepatocyte growth factor-regulated tyrosine kinase substrate and Tsg101. In this paper, we report that PAR1 sorted to ILVs of MVBs through an ESCRT-III-dependent pathway independent of ubiquitination. We further demonstrate that ALIX, a charged MVB protein 4-ESCRT-III interacting protein, bound to a YPX(3)L motif of PAR1 via its central V domain to mediate lysosomal degradation. This study reveals a novel MVB/lysosomal sorting pathway for signaling receptors that bypasses the requirement for ubiquitination and ubiquitin-binding ESCRTs and may be applicable to a subset of GPCRs containing YPX(n)L motifs

Overcoming bortezomib resistance in human B cells by anti-CD20/rituximab-mediated complement-dependent cytotoxicity and epoxyketone-based irreversible proteasome inhibitors

UNLABELLED: BACKGROUND: In clinical and experimental settings, antibody-based anti-CD20/rituximab and small molecule proteasome inhibitor (PI) bortezomib (BTZ) treatment proved effective modalities for B cell depletion in lymphoproliferative disorders as well as autoimmune diseases. However, the chronic nature of these diseases requires either prolonged or re-treatment, often with acquired resistance as a consequence. METHODS: Here we studied the molecular basis of acquired resistance to BTZ in JY human B lymphoblastic cells following prolonged exposure to this drug and examined possibilities to overcome resistance by next generation PIs and anti-CD20/rituximab-mediated complement-dependent cytotoxicity (CDC). RESULTS: Characterization of BTZ-resistant JY/BTZ cells compared to parental JY/WT cells revealed the following features: (a) 10-12 fold resistance to BTZ associated with the acquisition of a mutation in the PSMB5 gene (encoding the constitutive $β$5 proteasome subunit) introducing an amino acid substitution (Met45Ile) in the BTZ-binding pocket, (b) a significant 2-4 fold increase in the mRNA and protein levels of the constitutive $β$5 proteasome subunit along with unaltered immunoproteasome expression, (c) full sensitivity to the irreversible epoxyketone-based PIs carfilzomib and (to a lesser extent) the immunoproteasome inhibitor ONX 0914. Finally, in association with impaired ubiquitination and attenuated breakdown of CD20, JY/BTZ cells harbored a net 3-fold increase in CD20 cell surface expression, which was functionally implicated in conferring a significantly increased anti-CD20/rituximab-mediated CDC. CONCLUSIONS: These results demonstrate that acquired resistance to BTZ in B cells can be overcome by next generation PIs and by anti-CD20/rituximab-induced CDC, thereby paving the way for salvage therapy in BTZ-resistant disease

Evaluation of clay functionalized wafers and films for nicotine replacement therapy via buccal mucosa

The functional physicochemical properties of nicotine (NIC)-loaded composite freeze-dried wafers and solvent-evaporated films comprising hydroxypropylmethylcellulose (HPMC) and sodium alginate (SA), stabilized with magnesium aluminium silicate (MAS), have been reported. The formulations were characterized for swelling capacity, mucoadhesion, in vitro drug dissolution properties in simulated saliva (SS) and PBS at pH 6.8, and ex vivo and in vitro permeation using pig buccal mucosa membrane and EpiOralTM buccal tissue culture, respectively; finally, the cell viability of the EpiOralTM tissues after contact with the NIC-loaded formulations was investigated using 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay and the functional characteristics compared with those of commercially available NIC strips. Swelling and NIC release from the HPMC–SA wafers were more prolonged (30 min) compared to the commercially available NIC strips which disintegrated rapidly and released the drug within 5 min. Generally, swelling, mucoadhesion, and drug release was faster in PBS than in SS, and the presence of MAS was essential for maintaining a high dose recovery compared to non-MAS formulations and commercial NIC strips, which showed lower percentage of NIC content, possibly due to evaporation during analysis. Permeation studies showed that the NIC released was able to cross both porcine buccal membrane and the EpiOralTM buccal tissue, with the latter showing higher permeation flux for all the formulations tested. All the NIC-loaded, MAS-stabilized formulations showed high tissue viability, with values above 80%, showing their great potential for use as buccal delivery platforms for NIC replacement therapy to aid smoking cessation

Monitoring van Natura 2000 habitattypen - advies voor een landelijk meetprogramma

Contains fulltext : 72744.pdf (publisher's version ) (Open Access)73 p

Effects of insulin on intracellular GLUT4 vesicles in adipocytes: Evidence for a secretory mode of regulation

The facilitative glucose transporter, GLUT4 undergoes insulin-dependent movement to the cell surface in adipocytes, The magnitude of the insulin effect is much greater for CLUT4 than other recycling proteins such as the CD-MPR, In the present study we have studied the colocalisation of these proteins in adipocytes in an effort to explain this selective insulin-dependent recruitment of CLUT4, Using immunofluorescence microscopy or immuno-EM on 3T3-L1 adipocytes we find that there is considerable colocalisation between these proteins particularly within the area of the TGN, However, the distribution of CD-MPR was not significantly effected by insulin. The insulin-dependent recruitment of GLUT4 was concomitant with a selective decrease in GLUT4 labelling of cytoplasmic vesicles whereas the amount of CLUT4 in the TGN region (approx. 50% of total GLUT4) was relatively unaffected. To explore the possibility that the cytoplasmic GLUT4(+) vesicles represent an intracellular insulin-responsive storage compartment we performed quantitative immuno-EM on whole mounts of intracellular vesicles isolated from basal and insulin-stimulated adipocytes. These studies revealed that: (1) GLUT4 and CD-MPR were concentrated in small (30-200 nm) vesicles at a labelling density of 1-20+ gold particles/vesicle; (2) there was significant overlap between both proteins in that 70% of the total GLUT4 pool colocalised with CD-MPR; (3) a significant amount of GLUT4 (approx, 50% of total) was found in a subpopulation of vesicles that contained as little as 5% of the total CD-MPR pool; (4) the GLUT4(+)/CD-MPR(-) vesicles were highly insulin-responsive, and (5) the total number of GLUT4(+) vesicles, but not CD-MPR(+) vesicles, decreased by approx. 30% in response to insulin treatment. These data are consistent with a model in which GLUT4 is selectively sorted into a vesicular compartment in adipocytes that is recruited to the plasma membrane by insulin stimulation

Reversal of Tetracycline Resistance by Cepharanthine, Cinchonidine, Ellagic Acid and Propyl Gallate in a Multi-drug Resistant Escherichia coli

Bacterial resistance to antibiotics is an increasing threat to global healthcare systems. We therefore sought compounds with potential to reverse antibiotic resistance in a clinically relevant multi-drug resistant isolate of Escherichia coli (NCTC 13400). 200 natural compounds with a history of either safe oral use in man, or as a component of a traditional herb or medicine, were screened. Four compounds; ellagic acid, propyl gallate, cinchonidine and cepharanthine, lowered the minimum inhibitory concentrations (MICs) of tetracycline, chloramphenicol and tobramycin by up to fourfold, and when combined up to eightfold. These compounds had no impact on the MICs of ampicillin, erythromycin or trimethoprim. Mechanistic studies revealed that while cepharanthine potently suppressed efflux of the marker Nile red from bacterial cells, the other hit compounds slowed cellular accumulation of this marker, and/or slowed bacterial growth in the absence of antibiotic. Although cepharanthine showed some toxicity in a cultured HEK-293 mammalian cell-line model, the other hit compounds exhibited no toxicity at concentrations where they are active against E. coli NCTC 13400. The results suggest that phytochemicals with capacity to reverse antibiotic resistance may be more common in traditional medicines than previously appreciated, and may offer useful scaffolds for the development of antibiotic-sensitising drugs

Anticancer potential of Thevetia peruviana fruit methanolic extract

Abstract Background: Thevetia peruviana (Pers.) K. Schum or Cascabela peruviana (L.) Lippold (commonly known as ayoyote, codo de fraile, lucky nut, or yellow oleander), native to Mexico and Central America, is a medicinal plant used traditionally to cure diseases like ulcers, scabies, hemorrhoids and dissolve tumors. The purpose of this study was to evaluate the cytotoxic, antiproliferative and apoptotic activity of methanolic extract of T. peruviana fruits on human cancer cell lines. Methods: The cytotoxic activity of T. peruviana methanolic extract was carried out on human breast, colorectal, prostate and lung cancer cell lines and non-tumorigenic control cells (fibroblast and Vero), using the MTT assay. For proliferation and motility, clonogenic and wound-healing assays were performed. Morphological alterations were monitored by trypan blue exclusion, as well as DNA fragmentation and AO/EB double staining was performed to evaluate apoptosis. The extract was separated using flash chromatography, and the resulting fractions were evaluated on colorectal cancer cells for their cytotoxic activity. The active fractions were further analyzed through mass spectrometry. Results: The T. peruviana methanolic extract exhibited cytotoxic activity on four human cancer cell lines: prostate, breast, colorectal and lung, with values of IC50 1.91 ± 0.76, 5.78 ± 2.12, 6.30 ± 4.45 and 12.04 ± 3.43 μg/mL, respectively. The extract caused a significant reduction of cell motility and colony formation on all evaluated cancer cell lines. In addition, morphological examination displayed cell size reduction, membrane blebbing and detachment of cells, compared to non-treated cancer cell lines. The T. peruviana extract induced apoptotic cell death, which was confirmed by DNA fragmentation and AO/EB double staining. Fractions 4 and 5 showed the most effective cytotoxic activity and their MS analysis revealed the presence of the secondary metabolites: thevetiaflavone and cardiac glycosides. Conclusion: T. peruviana extract has potential as natural anti-cancer product with critical effects in the proliferation, motility, and adhesion of human breast and colorectal cancer cells, and apoptosis induction in human prostate and lung cancer cell lines, with minimal effects on non-tumorigenic cell lines. Keywords: Cytotoxic activity, Anti-proliferative activity, Motility, Apoptosis, Human cancer cells, Flavonoid, Cardiac glycoside

Laboratory-based ergometry for swimmers: a narrative review

INTRODUCTION: First widely available dry-land training machines for swimmers were introduced about 40 years ago. They were designed so that swimmers could perform resistance exercise whilst more-closely replicating the movements of swimming, than when using other gymnasium-based resistance training machines. This narrative review categorises and summarises what has been shown by the studies that have utilised laboratory-based ergometry for swimmers. EVIDENCE ACQUISITION: A systematic search was conducted in PubMed, Web of Science, ScienceDirect and Scopus (1970-2018) and relevant publications were included. Publications were grouped into 4 main areas of research: (i) physiological responses to exercise, (ii) functional evaluation of swimmers, (iii) monitoring of training, and (iv) muscular work output of swimmers. EVIDENCE SYNTHESIS: Significant differences were showed between swim bench exercise and real swimming, especially in regard to the muscles involved. The difficulties of accurate reproduction of the movements and coordinated dynamic actions of swimming have not been overcome. Nevertheless, the literature shows that the use of these devices has provided a valuablecontribution to swimming physiology, while overcoming difficulties presented by attempting to make physiological measurements in the water. CONCLUSIONS: In spite of its limitations, laboratory-based ergometry has allowed a valuable contribution to the understanding of the physiology, effects of training and efficiency of swimming