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Extracellular Milieu (extracellular + milieu)
Selected AbstractsPosttranslational regulation of BCL2 levels in cerebellar granule cells: A mechanism of neuronal survivalDEVELOPMENTAL NEUROBIOLOGY, Issue 13 2009Laura Lossi Abstract Apoptosis can be modulated by K+ and Ca2+ inside the cell and/or in the extracellular milieu. In murine organotypic cultures, membrane potential-regulated Ca2+ signaling through calcineurin phosphatase has a pivotal role in development and maturation of cerebellar granule cells (CGCs). P8 cultures were used to analyze the levels of expression of B cell lymphoma 2 (BCL2) protein, and, after particle-mediated gene transfer in CGCs, to study the posttranslational modifications of BCL2 fused to a fluorescent tag in response to a perturbation of K+/Ca2+ homeostasis. There are no changes in Bcl2 mRNA after real time PCR, whereas the levels of the fusion protein (monitored by calculating the density of transfected CGCs under the fluorescence microscope) and of BCL2 (inWestern blotting) are increased. After using a series of agonists/antagonists for ion channels at the cell membrane or the endoplasmic reticulum (ER), and drugs affecting protein synthesis/degradation, accumulation of BCL2 was related to a reduction in posttranslational cleavage by macroautophagy. The ER functionally links the [K+]e and [Ca2+]i to the BCL2 content in CGCs along two different pathways. The first, triggered by elevated [K+]e under conditions of immaturity, is independent of extracellular Ca2+ and operates via IP3 channels. The second leads to influx of extracellular Ca2+ following activation of ryanodine channels in the presence of physiological [K+]e, when CGCs are maintained in mature status. This study identifies novel mechanisms of neuroprotection in immature and mature CGCs involving the posttranslational regulation of BCL2. © 2009 Wiley Periodicals, Inc. Develop Neurobiol, 2009 [source] Diverse regulatory roles for lysosomal proteases in the immune responseEUROPEAN JOURNAL OF IMMUNOLOGY, Issue 11 2009Jeff D. Colbert Abstract The innate and adaptive immune system utilise endocytic protease activity to promote functional immune responses. Cysteine and aspartic proteases (cathepsins) constitute a subset of endocytic proteases, the immune function of which has been described extensively. Although historically these studies have focused on their role in processes such as antigen presentation and zymogen processing within the endocytic compartment, recent discoveries have demonstrated a critical role for these proteases in other intracellular compartments, and within the extracellular milieu. It has also become clear that their pattern of expression and substrate specificities are more diverse than was first envisaged. Here, we discuss recent advances addressing the role of lysosomal proteases in various aspects of the immune response. We pay attention to reports demonstrating cathepsin activity outside of its canonical endosome/lysosome microenvironment. [source] Human proteoglycan testican-1 inhibits the lysosomal cysteine protease cathepsin LFEBS JOURNAL, Issue 19 2003Jeffrey P. Bocock Testican-1, a secreted proteoglycan enriched in brain, has a single thyropin domain that is highly homologous to domains previously shown to inhibit cysteine proteases. We demonstrate that purified recombinant human testican-1 is a strong competitive inhibitor of the lysosomal cysteine protease, cathepsin L, with a Ki of 0.7 nm, but it does not inhibit the structurally related lysosomal cysteine protease cathepsin B. Testican-1 inhibition of cathepsin L is independent of its chondroitin sulfate chains and is effective at both pH 5.5 and 7.2. At neutral pH, testican-1 also stabilizes cathepsin L, slowing pH-induced denaturation and allowing the protease to remain active longer, although the rate of proteolysis is reduced. These data indicate that testican-1 is capable of modulating cathepsin L activity both in intracellular vesicles and in the extracellular milieu. [source] The role of Sov protein in the secretion of gingipain protease virulence factors of Porphyromonas gingivalisFEMS MICROBIOLOGY LETTERS, Issue 2 2010Keitarou Saiki Abstract Porphyromonas gingivalis transports Arg-gingipains and Lys-gingipain across the outer membrane via an unknown pathway. Recently, we found that the sov gene of P. gingivalis W83 was required for this step. In the present study, we characterized the Sov protein. We constructed a P. gingivalis strain that expresses histidine-tagged Sov instead of Sov. Subcellular fractionations and a histidine-tag pulldown experiment showed that histidine-tagged Sov was present in an outer membrane fraction. Furthermore, antiserum raised against the terminal regions of Sov obstructed the secretion of Arg-gingipains from wild-type W83 cells. A deletion study showed that the region from Phe2495 to the C-terminus Gln2499 of Sov is essential for gingipain secretion. Anti-histidine-tag immunoglobulins interfered with the secretion of Arg-gingipains by P. gingivalis cells that expressed histidine-tagged Sov. In conclusion, we found that Sov is an outer membrane protein participating in the secretion of gingipains and that the C-terminal region of Sov is exposed to the extracellular milieu and involved in the modulation of Sov function. [source] Analysis of the mechanism for extracellular processing in the presentation of human immunodeficiency virus-1 envelope protein-derived peptide to epitope-specific cytotoxic T lymphocytesIMMUNOLOGY, Issue 1 2000Y. Nakagawa Summary An immunodominant epitope of human immunodeficiency virus-1 (HIV-1) gp160 recognized by Dd class I major histocompatibility complex (MHC) molecule-restricted, CD8+ cytotoxic T lymphocytes (CTL) was originally identified as a peptide composed of 15 amino acids (P18IIIB: RIQRGPGRAFVTIGK). However, further study has indicated that a 10-mer peptide, I-10 (RGPGRAFVTI), within P18IIIB is the minimal-sized epitope and the trimming step(s) of two carboxyl terminal amino acids (GK) is essential to produce I-10 from P18IIIB. In the processing, angiotensin-1-converting enzyme (ACE), found in sera, plays a central role in generating I-10. Target cells could be sensitized with I-10 under conditions where ACE activity in the sera was abrogated. In contrast, in the case of P18IIIB, requiring further processing to delete the C-terminus of two amino acids in order to act, sensitization of target cells was completely abrogated under the conditions. Pretreatment of target cells with brefeldin A (BFA), preventing the presentation of endogenous antigens from the class I MHC molecule pathway, did not inhibit the presentation of P18IIIB. Moreover, glutaraldehyde-fixed cells, which can not process native protein, though they could present the exogenously added peptides, were also sensitized by P18IIIB. These results clearly demonstrate that the fine processing to produce I-10 occurred in the extracellular milieu. Furthermore, our result suggests that the longer P18IIIB can bind to the class I molecules on the cell surface, and then be trimmed by ACE while it is bound. The mechanisms behind the extracellular processing outlined in this paper will offer important information for designing peptide-based vaccines to elicit MHC molecule-restricted effectors. [source] From collagen chemistry towards cell therapy , a personal journeyINTERNATIONAL JOURNAL OF EXPERIMENTAL PATHOLOGY, Issue 4 2007Michael E. Grant Summary The Fell,Muir Award requires the recipient to deliver a lecture and a review manuscript which provides a personal overview of significant scientific developments in the field of matrix biology over the period of the recipient's career. In this context, this review considers the collagen family of structural proteins and the advances in biochemical, molecular biological and genetic techniques which led to the elucidation of the structure, synthesis and function of this important group of extracellular matrix constituents. Particular attention is focussed on early research on the identification and assembly of the soluble precursors of collagen types I and II, and the identification of the precursor of basement membrane collagen type IV. In subsequent studies investigating the maintenance of the chick chondrocyte phenotype in culture, the influence of the extracellular milieu was found to influence markedly both cell morphology and collagen gene expression. These studies led to the discovery of collagen type X whose expression is restricted to hypertrophic chondrocytes at sites of endochondral ossification. Such research provided a prelude to investigations of mammalian endochondral ossification which is known to be aberrant in a variety of human chondrodysplasias and is reactivated in bone fracture repair and in osteoarthritis. The cloning of bovine and then human collagen type X genes facilitated studies in relevant human diseases and contributed to the discovery of mutations in the COL10A1 gene in families with metaphyseal chondrodysplasia type Schmid. Clustering of mutations in the C-terminal domain of the type X collagen molecule has now been widely documented and investigations of the pathogenic mechanisms in animal models are beginning to suggest the prospect of novel treatment strategies. [source] Membrane vesicles containing matrix metalloproteinase-9 and fibroblast growth factor-2 are released into the extracellular space from mouse mesoangioblast stem cells,JOURNAL OF CELLULAR PHYSIOLOGY, Issue 1 2010Maria Elena Candela Certain proteins, including fibroblast growth factor-2 (FGF-2) and matrix metalloproteinase-9 (MMP-9), have proved very effective in increasing the efficacy of mesoangioblast stem cell therapy in repairing damaged tissue. We provide the first evidence that mouse mesoangioblast stem cells release FGF-2 and MMP-9 in their active form through the production of membrane vesicles. These vesicles are produced and turned over continuously, but are stable for some time in the extracellular milieu. Mesoangioblasts shed membrane vesicles even under oxygen tensions that are lower than those typically used for cell culture and more like those of mouse tissues. These findings suggest that mesoangioblasts may themselves secrete paracrine signals and factors that make damaged tissues more amenable to cell therapy through the release of membrane vesicles. J. Cell. Physiol. 224:144,151, 2010 © 2010 Wiley-Liss, Inc. [source] Chromogranins as regulators of exocytosisJOURNAL OF NEUROCHEMISTRY, Issue 2 2010Ricardo Borges J. Neurochem. (2010) 114, 335,343. Abstract Chromogranins (Cgs) constitute the main protein component in the vesicular matrix of large dense core vesicles (LDCV). These acidic proteins have been implicated in several physiological processes such as vesicle sorting, the generation of bioactive peptides and the accumulation of soluble species inside LDCV. This latter feature of Cgs accounts for the ability of vesicles to concentrate catecholamines and Ca2+. Indeed, the low affinity and high capacity of Cgs to bind solutes at the low pH of the LDCV lumen seems to be behind the delay in the neurotransmitter exit towards the extracellular milieu after vesicle fusion. The availability of new mouse strains lacking Cgs in combination with the arrival of several techniques for the direct monitoring of exocytosis (like amperometry, patch-amperometry and intracellular electrochemistry), have helped advance our understanding of how these granins concentrate catecholamines and Ca2+ in LDCV, and how they influence the kinetics of exocytosis. In this review, we will discuss the roles of Cgs A and B in maintaining the intravesicular environment of secretory vesicles and in exocytosis, bringing together the most recent findings from adrenal chromaffin cells. [source] Mechanism of association of adenylate cyclase toxin with the surface of Bordetella pertussis: a role for toxin,filamentous haemagglutinin interactionMOLECULAR MICROBIOLOGY, Issue 6 2002Franca R. Zaretzky Summary Adenylate cyclase (AC) toxin from Bordetella per-tussis is unusual in that, unlike most other members of the repeats-in-toxin family that are released into the extracellular milieu, it remains associated with the bacterial surface. In this study, we investigated the nature of the association of this toxin with the surface of B. pertussis. AC toxin was extracted from crude outer membrane preparations of B. pertussis with 8 M urea, but only partially with alkaline sodium carbonate and not at all with octylglucoside, suggesting that denaturation of the toxin is necessary for its removal from the membrane. B. pertussis mutants lacking filamentous haemagglutinin (FHA) released significantly more AC toxin into the medium, and AC toxin association with the bacterial surface was partially restored by expression of FHA from a plasmid, suggesting a role for FHA in surface retention of AC toxin. AC toxin distribution was unaffected by the absence of pertactin, or full-length lipopolysaccharide, or a defect in secretion of pertussis toxin. Using overlay and immunoprecipitation, we found that a direct physical association can occur between AC toxin and FHA. Combined, these findings suggest that FHA may play a role in AC toxin retention on the surface of B. pertussis and raise the possibility of an involvement of adherence mediated by FHA in delivery of AC toxin from the bacterium to the target cell. [source] P2 receptors: new potential players in atherosclerosisBRITISH JOURNAL OF PHARMACOLOGY, Issue 4 2002Francesco Di Virgilio Atherosclerosis is a focal inflammatory disease of the arterial wall. It starts with the formation of fatty streaks on the arterial wall that evolve to form a raised plaque made of smooth muscle cells (SMCs), and infiltrating leukocytes surrounding a necrotic core. The pathogenesis of the atherosclerotic lesion is incompletely understood, but it is clear that a dysfunction of the endothelium, recruitment and activation of inflammatory cells and SMC proliferation have a pivotal role. Over recent years receptors for extracellular nucleotides, the P2 receptors, have been recognized as fundamental modulators of leukocytes, platelets, SMCs and endothelial cells. P2 receptors mediate chemotaxis, cytokine secretion, NO generation, platelet aggregation and cell proliferation in response to accumulation of nucleotides into the extracellular milieu. Clinical trials have shown the benefit of antagonists of the ADP platelet receptor(s) in the prevention of vascular accidents in patients with atherosclerosis. Therefore, we anticipate that a deeper understanding of the involvement of P2 receptors in atheroma formation will open new avenues for drug design and therapeutic intervention. British Journal of Pharmacology (2002) 135, 831,842; doi:10.1038/sj.bjp.0704524 [source] Roles of pericellular proteolysis by membrane type-1 matrix metalloproteinase in cancer invasion and angiogenesisCANCER SCIENCE, Issue 7 2003Motoharu Seiki Behavior of cancer cells is profoundly affected by their microenvironment, which is often controlled by pericellular proteolysis or the processing of protein components, including extracellular matrices, growth factors, cytokines, receptors, cell adhesion molecules, and so on. Matrix metalloproteinases (MMPs) are a family of zinc-dependent proteases responsible for the proteolytic events in the extracellular milieu. Among the multiple MMPs expressed in a wide range of tumors, membrane type-1 MMP (MT1-MMP), which is expressed especially in tumor cells with significant invasive properties, is thought to be particularly important for pericellular proteolysis. Recent studies have elucidated in part how MT1-MMP is regulated biologically for the promotion of invasion by tumors or for angiogenesis by endothelial cells. Understanding of the proteolysis by, and the regulation of MT1-MMP, which probably promotes cell invasion, could provide a therapeutic hint as to how to block or delay the progression of cancer. [source] | |||||||||||||