Giardan: structure, synthesis, regulation and inhibition.
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During encystment, Giardia trophozoites become encased in a filamentous extracellular matrix of their own making that consists of novel cyst wall proteins (Cwp) 1, 2 and 3, and a novel 2-acetamido-2-deoxy-d-galactan we are naming giardan. Giardan is synthesized from glucose via sugar phosphate intermediates to UDP-GalNAc by inducible, cytosolic enzymes. The UDP-GalNAc is fixed into giardan apparently by an inducible, particle-associated transferase. Regulation of this synthesis appears to centre around pyrophosphorylase, epimerase and cyst wall synthase (Cws) activities. Pyrophosphorylase seems to be involved in making sufficient UDP-N-acetylglucosamine (GlcNAc) to drive the epimerase kinetics toward UDP-GalNAc synthesis, while the Cws removes intracellular UDP-GalNAc, extruding it as giardan and thus preventing an increased intracellular concentration of UDP-GalNAc that could drive the reaction toward GlcNAc synthesis. Cyst wall proteins have been localized to encystment-specific vesicles (ESVs), but whether or not this is true for giardan is unknown. Also unknown is whether or not the cyst wall proteins and giardan are covalently linked. It remains unknown how or whether Giardia degrades giardan during excystation.Keywords:
Transferase
Abstract Collagens are abundant proteins in higher organisms, and are formed by a complex biosynthetic pathway involving intracellular and extracellular post‐translational modifications. Starting from simple soluble precursors, this interesting pathway produces insoluble functional fibrillar and non‐fibrillar elements of the extracellular matrix. The present review highlights recent progress and new insights into biological regulation of extracellular procollagen processing, and some novel functions of byproducts of these extracellular enzymatic transformations. These findings underscore the notion that released propeptides and other proteolytic products of extracellular matrix proteins have important biological functions, and that structural proteins are multifunctional. An emerging concept is that a dynamic interplay exists between extracellular products and byproducts with cells that helps to maintain normal cellular phenotypes and tissue integrity. J. Cell. Biochem. © 2005 Wiley‐Liss, Inc.
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The extracellular matrix is very well organized at the supramolecular and tissue levels and little is known on the potential role of intrinsic disorder in promoting its organization. We predicted the amount of disorder and identified disordered regions in the human extracellular proteome with established computational tools. The extracellular proteome is significantly enriched in proteins comprising more than 50% of disorder compared to the complete human proteome. The enrichment is mostly due to long disordered regions containing at least 100 consecutive disordered residues. The amount of intrinsic disorder is heterogeneous in the extracellular protein families, with the most disordered being collagens and the small integrin-binding ligand N-linked glycoproteins. Although most domains found in extracellular proteins are structured, the fibronectin III domains contain a variable amount of disordered residues (up to 92%). Binding sites for heparin and integrins are found in disordered sequences of extracellular proteins. Intrinsic disorder is evenly distributed in hubs and ends in the interaction network of extracellular proteins with their extracellular partners. In contrast, extracellular hubs are significantly enriched in disorder in the network of extracellular proteins with their extracellular, membrane and intracellular partners. Disorder could thus provide the structural plasticity required for the hubs to interact with membrane and intracellular proteins. Organization and assembly of the extracellular matrix, development of mineralized tissues and cell–matrix adhesion are the biological processes overrepresented in the most disordered extracellular proteins. Extracellular disorder is associated with binding to growth factors, glycosaminoglycans and integrins at the molecular level.
Proteome
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The aim of our study is to examine the effect of extracellular ATP whether it is correlated with intracellular Ca concentrations ([Ca2+]i) on human liver hepatocellular cells (HepG2). The extracellular ATP is responsible for regulating both cells signaling and cell functions. ATP maintains these effects through purinergic P2 receptors. The extracellular ATP promotes the release of Ca2+ from the Ca2+ stores to the cytoplasm in the cell and increases [Ca2+] I in the cell. In our study, various concentrations of ATP (10-3M-10-7M) were applied to HepG2 cells and incubated for 24 hours, 48 hours, and 72 hours. At these concentrations, the proliferation of cells and apoptosis of the cells was examined for 24 hours, 48 hours, and 72 hours. Similarly, cells with different ATP concentrations incubated for 24 hours and 48 hours were loaded with Indo 1FF AM calcium indicator to measure [Ca2+]i. Surprising results were obtained, 10-6M-10-7M extracellular ATP was found to be more toxic than 10-3 M-10-4 M extracellular ATP, (p<0.05). Low concentrations of ATP also reduced [Ca2+]i for 24 hours and 48 hours of incubations (p<0.01). As a result, low concentration extracellular ATP is more toxic in HepG2 cells. At the same time, the extracellular ATP correlates with [Ca2+]i.
Adenosine triphosphate
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Calcium in biology
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We studied the effects of varying extracellular concentrations of Ca++ and Mg++ on cytosolic Ca++ concentration and PTH release by incorporating the Ca++-sensitive, fluorescent dye "QUIN 2" into dispersed bovine parathyroid cells. Increasing extracellular Ca++ from 0.5 to 2.0 mM led to a dose-dependent increase in cytosolic Ca++ from 179 +/- 8 to 646 +/- 68 nM (mean +/- SE, N = 13; P less than 0.01), which correlated closely (r = -.987, P less than .001) with the suppression of PTH secretion by the same Ca++ concentrations. Raising extracellular Mg++ from 0.5 to 3 mM also caused a dose-related increase in cytosolic Ca++ from 176 +/- 10 to 277 +/- 15 nM (N = 12, P less than 0.01) which correlated (r = -.959, P less than .01) with inhibition of PTH release. These results show a close inverse relationship between cytosolic Ca++ and PTH release with alterations in extracellular Ca++ or Mg++. Cytosolic Ca++ may, therefore, act as a second messenger mediating the effects of these divalent cations on PTH release.
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Extracellular action potentials found close to the surface of motoneurons are related to the intracellular spikes. Evidence is cited to support the assumption that the extracellular spikes have the same time course as the membrane current at the site of recording. Simultaneously recorded intracellular and extracellular spikes are compared. Intracellular spikes are transformed, by means of a circuit which is equivalent to the extracellular recording situation, into transients that are like those appearing extracellularly. Evidence is given that the recordings are from the cell bodies of motoneurons. The results show that the membrane at the extracellular recording site does not produce a spike since the time course of the extracellular potentials is determined by the passive properties of the membrane.
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The cytotoxic action of adriamycin is influenced by its intracellular accumulation. Therefore, it is important to clarify the mechanisms of adriamycin influx and efflux. In the present study, the influence of the extracellular KCl and Ca2+ concentration, the extracellular pH and the presence of NaHC03 on the ADR accumulation was investigated in order to study the mechanisms in ADR accumulation influenced by ions. The extracellular KCl concentration did not affect the intracellular ADR accumulation. This suggests the cell membrane potential does not affect the ADR accumulation since it is influenced by the KCl concentration. The intracellular intensity of fluorescence of Fluo3, an indicator of Ca2+, increased between 0 and 20 mM of the extracellular concentration of Ca2+ ion. However, the ADR accumulation did not change between 0 and 20 mM of the extracellular concentration of Ca2+. This indicates that the extracellular concentration of Ca2+ does not affect the ADR accumulation under physiological conditions since 20 mM of Ca2+ is beyond normal physiological conditions. Further, the intracellular fluorescence of Fluo3 decreased as increasing the extracellular pH. In contrast, the ADR accumulation increased as increasing the extracellular pH. These suggest that the ADR accumulation increases as decreasing the intracellular Ca2+ as changing the extracellular pH. In wild type strain, the ADR accumulation did not change by the extracellular pH in the absence of NaHCO3, but increased as increasing the extracellular pH in the presence of NaHCO3. This suggests that the ADR accumulation in the wild type strain is influenced by NaHCO3 but the extracellular pH. In the ADR-resistant strain, the ADR accumulation decreased as increasing the extracellular pH regardless of NaHCO3. However, the accumulation of ADR increased as increasing the extracellular pH when the adjustment of pH was carried out with 1N HCl. This suggests that Cl- may play an important role in the ADR influx in the ADR-resistant strain.
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Entry of extracellular Ca2+ into the cytosol of exocrine cells plays an important role in the process of fluid transport, especially during periods of prolonged secretion. However, in parotid acinar cells, the process of Ca2+ entry and the identity of factors which regulate it remain obscure. In this report, we demonstrate that AlF-4, like carbachol, activates Ca2+ entry into dispersed rat parotid acini. In physiological Ca2(+)-containing (1.28 mM) medium, both agents elicit three phases of cytosolic Ca2+ change, an initial transient increase (intracellular Ca2+ dependent) followed sequentially by a decrease (intra- and extracellular Ca2+ dependent) and a small sustained increase (extracellular Ca2+ dependent). Cytosolic Ca2+ concentration ([Ca2+]i) during the last two phases is influenced by variations in extracellular [Ca2+]. Elevation of extracellular [Ca2+], at any time after the initial transient increase, results in a rise of cytosolic [Ca2+], thus demonstrating the existence of a Ca2+ entry pathway during the two later phases. These data suggest the likelihood that in parotid acini, G protein activation is involved in stimulating this Ca2+ entry pathway. Because in AlF-4-treated acini entry into the cytosol is detectable only after the initial intracellular Ca2+ release phase, we suggest that this Ca2+ entry process does not accompany initial intracellular Ca2+ mobilization. Furthermore, the sustained cytosolic [Ca2+] elevation which can be observed 15-30 min after initial stimulation of acini is likely determined by this Ca2+ entry process which, in physiological conditions, could support sustained fluid secretion.
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