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Entry Pathways (entry + pathway)
Kinds of Entry Pathways Selected AbstractsActivation of a calcium entry pathway by sodium pyrithione in the bag cell neurons of AplysiaDEVELOPMENTAL NEUROBIOLOGY, Issue 4 2004Ronald J. Knox Abstract The ability of sodium pyrithione (NaP), an agent that produces delayed neuropathy in some species, to alter neuronal physiology was accessed using ratiometric imaging of cytosolic free Ca2+ concentration ([Ca2+]i) in fura PE-filled cultured Aplysia bag cell neurons. Bath-application of NaP evoked a [Ca2+]i elevation in both somata and neurites with an EC50 of ,300 nM and a Hill coefficient of ,1. The response required the presence of external Ca2+, had an onset of 3,5 min, and generally reached a maximum within 30 min. 2-Methyl-sulfonylpyridine, a metabolite and close structural analog of NaP, did not elevate [Ca2+]i. Under whole-cell current-clamp recording, NaP produced a ,14 mV depolarization of resting membrane potential that was dependent on external Ca2+. These data suggested that NaP stimulates Ca2+ entry across the plasma membrane. To minimize the possibility that a change in cytosolic pH was the basis for NaP-induced Ca2+ entry, bag cell neuron intracellular pH was estimated with the dye 2,,7,-bis(carboxyethyl-5(6)-carboxy-fluorescein acetoxy methylester. Exposure of the neurons to NaP did not alter intracellular pH. The slow onset and sustained nature of the NaP response suggested that a cation exchange mechanism coupled either directly or indirectly to Ca2+ entry could underlie the phenomenon. However, neither ouabain, a Na+/K+ ATPase inhibitor, nor removal of extracellular Na+, which eliminates Na+/Ca2+ exchanger activity, altered the NaP-induced [Ca2+]i elevation. Finally, the possibility that NaP gates a Ca2+ -permeable ion channel in the plasma membrane was examined. NaP did not appear to activate two major forms of bag cell neuron Ca2+ -permeable ion channels, as Ca2+ entry was unaffected by inhibition of voltage-gated Ca2+ channels using nifedipine or by inhibition of a voltage-dependent, nonselective cation channel using a high concentration of tetrodotoxin. In contrast, two potential store-operated Ca2+ entry current inhibitors, SKF-96365 and Ni2+, attenuated NaP-induced Ca2+ entry. We conclude that NaP activates a slow, persistent Ca2+ influx in Aplysia bag cell neurons. © 2004 Wiley Periodicals, Inc. J Neurobiol 411,423, 2004 [source] Scavenger receptor class B type I is a key host factor for hepatitis C virus infection required for an entry step closely linked to CD81,HEPATOLOGY, Issue 6 2007Mirjam B. Zeisel Hepatitis C virus (HCV) is a major cause of chronic hepatitis worldwide. Scavenger receptor class B type I (SR-BI) has been shown to bind HCV envelope glycoprotein E2, participate in entry of HCV pseudotype particles, and modulate HCV infection. However, the functional role of SR-BI for productive HCV infection remains unclear. In this study, we investigated the role of SR-BI as an entry factor for infection of human hepatoma cells using cell culture,derived HCV (HCVcc). Anti,SR-BI antibodies directed against epitopes of the human SR-BI extracellular loop specifically inhibited HCVcc infection in a dose-dependent manner. Down-regulation of SR-BI expression by SR-BI,specific short interfering RNAs (siRNAs) markedly reduced the susceptibility of human hepatoma cells to HCVcc infection. Kinetic studies demonstrated that SR-BI acts predominately after binding of HCV at an entry step occurring at a similar time point as CD81,HCV interaction. Although the addition of high-density lipoprotein (HDL) enhanced the efficiency of HCVcc infection, anti,SR-BI antibodies and SR-BI,specific siRNA efficiently inhibited HCV infection independent of lipoprotein. Conclusion: Our data suggest that SR-BI (i) represents a key host factor for HCV entry, (ii) is implicated in the same HCV entry pathway as CD81, and (iii) targets an entry step closely linked to HCV,CD81 interaction. (HEPATOLOGY 2007.) [source] Calcium Channel TRPV6 Is Involved in Murine Maternal,Fetal Calcium Transport,,JOURNAL OF BONE AND MINERAL RESEARCH, Issue 8 2008Yoshiro Suzuki Abstract Maternal,fetal calcium (Ca2+) transport is crucial for fetal Ca2+ homeostasis and bone mineralization. In this study, the physiological significance of the transient receptor potential, vanilloid 6 (TRPV6) Ca2+ channel in maternal,fetal Ca2+ transport was investigated using Trpv6 knockout mice. The Ca2+ concentration in fetal blood and amniotic fluid was significantly lower in Trpv6 knockout fetuses than in wildtypes. The transport activity of radioactive Ca2+ (45Ca) from mother to fetuses was 40% lower in Trpv6 knockout fetuses than in wildtypes. The ash weight was also lower in Trpv6 knockout fetuses compared with wildtype fetuses. TRPV6 mRNA and protein were mainly localized in intraplacental yolk sac and the visceral layer of extraplacental yolk sac, which are thought to be the places for maternal,fetal Ca2+ transport in mice. These expression sites were co-localized with calbindin D9K in the yolk sac. In wildtype mice, placental TRPV6 mRNA increased 14-fold during the last 4 days of gestation, which coincides with fetal bone mineralization. These results provide the first in vivo evidence that TRPV6 is involved in maternal,fetal Ca2+ transport. We propose that TRPV6 functions as a Ca2+ entry pathway, which is critical for fetal Ca2+ homeostasis. [source] Entry of pseudotyped hepatitis C virus into primary human hepatocytes depends on the scavenger class B type I receptorJOURNAL OF VIRAL HEPATITIS, Issue 12 2008M. Régeard Summary., Entry of the hepatitis C virus (HCV) into the cell seems to be a complex multi-step process involving several cellular factors such as the scavenger class B type I receptor (SRBI). Until now, all investigations conducted to assess the involvement of SRBI have been based on in vitro infection models which use human hepatoma-derived cell lines. However, the HCV entry pathway may be altered in these types of cells because of the impairment of some hepatic characteristics. In this study, we showed that SRBI also plays an essential role in HCV entry into primary human hepatocytes with two distinct approaches: gene extinction and antibodies neutralization assays. [source] Cytoplasmic calcium oscillations and store-operated calcium influxTHE JOURNAL OF PHYSIOLOGY, Issue 13 2008James W. Putney Intracellular calcium oscillations have fascinated scientists for decades. They provide an important cellular signal which, unlike most signalling mechanisms, is digitally encoded. While it is generally agreed that oscillations most frequently arise from cyclical release and re-uptake of intracellularly stored calcium, it is becoming increasingly clear that influx of calcium across the plasma membrane also plays a critical role in their maintenance and even in delivering their signal to the correct cellular locus. In this review we will discuss the role played by Ca2+ entry mechanisms in Ca2+ oscillations, and approaches to understanding the molecular nature of this Ca2+ entry pathway. [source] Calcium handling in afferent arteriolesACTA PHYSIOLOGICA, Issue 4 2004M. Salomonsson Abstract The cytosolic intracellular calcium concentration ([Ca2+]i) is a major determining factor in the vascular smooth muscle tone. In the afferent arteriole it has been shown that agonists utilizing G-protein coupled receptors recruit Ca2+ via release from intracellular stores and entry via pathways in the plasma membrane. The relative importances of entry vs. mobilization seem to differ between different agonists, species and preparations. The entry pathway might include different types of voltage sensitive Ca2+ channels located in the plasmalemma such as dihydropyridine sensitive L-type channels, T-type channels and P/Q channels. A role for non-voltage sensitive entry pathways has also been suggested. The importance of voltage sensitive Ca2+ channels in the control of the tone of the afferent arteriole (and thus in the control of renal function and whole body control of extracellular fluid volume and blood pressure) sheds light on the control of the membrane potential of afferent arteriolar smooth muscle cells. Thus, K+ and Cl, channels are of importance in their role as major determinants of membrane potential. Some studies suggest a role for calcium-activated chloride (ClCa) channels in the renal vasoconstriction elicited by agonists. Other investigators have found evidence for several types of K+ channels in the regulation of the afferent arteriolar tone. The available literature in this field regarding afferent arterioles is, however, relatively sparse and not conclusive. This review is an attempt to summarize the results obtained by others and ourselves in the field of agonist induced afferent arteriolar Ca2+ recruitment, with special emphasis on the control of voltage sensitive Ca2+ entry. Outline of the Manuscript: This manuscript is structured as follows: it begins with an introduction where the general role for [Ca2+]i as a key factor in the regulation of the tone of vascular smooth muscles (VSMC) is detailed. In this section there is an emphasis is on observations that could be attributed to afferent arteriolar function. We then investigate the literature and describe our results regarding the relative roles for Ca2+ entry and intracellular release in afferent arterioles in response to vasoactive agents, with the focus on noradrenalin (NA) and angiotensin II (Ang II). Finally, we examine the role of ion channels (i.e. K+ and Cl, channels) for the membrane potential, and thus activation of voltage sensitive Ca2+ channels. [source] Characterization of host-range and cell entry properties of the major genotypes and subtypes of hepatitis C virus,HEPATOLOGY, Issue 2 2005Dimitri Lavillette Because of the lack of a robust cell culture system, relatively little is known about the molecular details of the cell entry mechanism for hepatitis C virus (HCV). Recently, we described infectious HCV pseudo-particles (HCVpp) that were generated by incorporating unmodified HCV E1E2 glycoproteins into the membrane of retroviral core particles. These initial studies, performed with E1E2 glycoproteins of genotype 1, noted that HCVpp closely mimic the cell entry and neutralization properties of parental HCV. Because sequence variations in E1 and E2 may account for differences in tropism, replication properties, neutralization, and response to treatment in patients infected with different genotypes, we investigated the functional properties of HCV envelope glycoproteins from different genotypes/subtypes. Our studies indicate that hepatocytes were preferential targets of infection in vitro, although HCV replication in extrahepatic sites has been reported in vivo. Receptor competition assays using antibodies against the CD81 ectodomain as well as ectopic expression of CD81 in CD81-deficient HepG2 cells indicated that CD81 is used by all the different genotypes/subtypes analyzed to enter the cells. However, by silencing RNA (siRNA) interference assays, our results show that the level of Scavenger Receptor Class-B Type-I (SR-BI) needed for efficient infection varies between genotypes and subtypes. Finally, sera from chronic HCV carriers were found to exhibit broadly reactive activities that inhibited HCVpp cell entry, but failed to neutralize all the different genotypes. In conclusion, we characterize common steps in the cell entry pathways of the major HCV genotypes that should provide clues for the development of cell entry inhibitors and vaccines. (HEPATOLOGY 2005;41:265,274.) [source] Characterization of Ca2+ signaling pathways in mouse adrenal medullary chromaffin cellsJOURNAL OF NEUROCHEMISTRY, Issue 5 2010Pei-Chun Wu J. Neurochem. (2010) 112, 1210,1222. Abstract In the present study, we characterized the Ca2+ responses and secretions induced by various secretagogues in mouse chromaffin cells. Activation of the acetylcholine receptor (AChR) by carbachol induced a transient intracellular Ca2+ concentration ([Ca2+]i) increase followed by two phases of [Ca2+]i decay and a burst of exocytic events. The contribution of the subtypes of AChRs to carbachol-induced responses was examined. Based on the results obtained by stimulating the cells with the nicotinic receptor (nAChR) agonist, 1,1-dimethyl-4-phenylpiperazinium iodide, high K+ and the effects of thapsigargin, it appears that activation of nAChRs induces an extracellular Ca2+ influx, which in turn activate Ca2+ -induced Ca2+ release via the ryanodine receptors. Muscarine, a muscarinic receptor (mAChRs) agonist, was found to induce [Ca2+]i oscillation and sustained catecholamine release, possibly by activation of both the receptor- and store-operated Ca2+ entry pathways. The RT-PCR results showed that mouse chromaffin cells are equipped with messages for multiple subtypes of AChRs, ryanodine receptors and all known components of the receptor- and store-operated Ca2+ entry. Furthermore, results obtained by directly monitoring endoplasmic reticulum (ER) and mitochondrial Ca2+ concentration and by disabling mitochondrial Ca2+ uptake suggest that the ER acts as a Ca2+ source, while the mitochondria acts as a Ca2+ sink. Our results show that both nAChRs and mAChRs contribute to the initial carbachol-induced [Ca2+]i increase which is further enhanced by the Ca2+ released from the ER mediated by Ca2+ -induced Ca2+ release and mAChR activation. This information on the Ca2+ signaling pathways should lay a good foundation for future studies using mouse chromaffin cells as a model system. [source] ,-Synuclein modulation of Ca2+ signaling in human neuroblastoma (SH-SY5Y) cellsJOURNAL OF NEUROCHEMISTRY, Issue 5 2009Nishani T. Hettiarachchi Abstract Parkinson's disease (PD) is characterized in part by the presence of ,-synuclein (,-syn) rich intracellular inclusions (Lewy bodies). Mutations and multiplication of the ,-synuclein gene (SNCA) are associated with familial PD. Since Ca2+ dyshomeostasis may play an important role in the pathogenesis of PD, we used fluorimetry in fura-2 loaded SH-SY5Y cells to monitor Ca2+ homeostasis in cells stably transfected with either wild-type ,-syn, the A53T mutant form, the S129D phosphomimetic mutant or with empty vector (which served as control). Voltage-gated Ca2+ influx evoked by exposure of cells to 50 mM K+ was enhanced in cells expressing all three forms of ,-syn, an effect which was due specifically to increased Ca2+ entry via L-type Ca2+ channels. Mobilization of Ca2+ by muscarine was not strikingly modified by any of the ,-syn forms, but they all reduced capacitative Ca2+ entry following store depletion caused either by muscarine or thapsigargin. Emptying of stores with cyclopiazonic acid caused similar rises of [Ca2+]i in all cells tested (with the exception of the S129D mutant), and mitochondrial Ca2+ content was unaffected by any form of ,-synuclein. However, only WT ,-syn transfected cells displayed significantly impaired viability. Our findings suggest that ,-syn regulates Ca2+ entry pathways and, consequently, that abnormal ,-syn levels may promote neuronal damage through dysregulation of Ca2+ homeostasis. [source] Prevention of a hypoxic Ca2+i response by SERCA inhibitors in cerebral arteriolesBRITISH JOURNAL OF PHARMACOLOGY, Issue 4 2002C Guibert The aim of the study was to investigate the mechanism of a novel effect of hypoxia on intracellular Ca2+ signalling in rabbit cerebral arteriolar smooth muscle cells, an effect that was resistant to the L-type Ca2+ channel antagonist methoxyverapamil (D600). [Ca2+]i of smooth muscle cells in intact arteriolar fragments was measured using the Ca2+ -indicator dye fura-PE3. Hypoxia (PO2 10 , 20 mmHg) lowered basal [Ca2+]i but did not inhibit Ca2+ entry pathways measured by Mn2+ -quenching of fura-PE3. The effect of hypoxia was completely prevented by thapsigargin or cyclopiazonic acid, selective inhibitors of sarcoplasmic reticulum Ca2+ ATPase (SERCA). Since these inhibitors do not block Ca2+ extrusion or uptake via the plasma membrane, the data indicate that the effect of hypoxia depends on a functional sarcoplasmic reticulum. Because actions of nitric oxide (NO) on vascular smooth muscle are also prevented by SERCA inhibitors it was explored whether the effect of hypoxia occurred via modulation of endogenous NO release. Residual NOS-I and NOS-III were detected by immunostaining, and there were NO-dependent effects of NOS inhibitors on Ca2+i -signalling. Nevertheless, inhibition of endogenous NO production did not prevent the effect of hypoxia on [Ca2+]i. The experiments reveal a novel nitric oxide-independent effect of hypoxia that is prevented by SERCA inhibitors. British Journal of Pharmacology (2002) 135, 927,934; doi:10.1038/sj.bjp.0704547 [source] Differences in human macrophage receptor usage, lysosomal fusion kinetics and survival between logarithmic and metacyclic Leishmania infantum chagasi promastigotesCELLULAR MICROBIOLOGY, Issue 12 2009Norikiyo Ueno Summary The obligate intracellular protozoan, Leishmania infantum chagasi (Lic) undergoes receptor-mediated phagocytosis by macrophages followed by a transient delay in phagolysosome maturation. We found differences in the pathway through which virulent Lic metacyclic promastigotes or avirulent logarithmic promastigotes are phagocytosed by human monocyte-derived macrophages (MDMs). Both logarithmic and metacyclic promastigotes entered MDMs through a compartment lined by the third complement receptor (CR3). In contrast, many logarithmic promastigotes entered through vacuoles lined by mannose receptors (MR) whereas most metacyclic promastigotes did not (P < 0.005). CR3-positive vacuoles containing metacyclic promastigotes stained for caveolin-1 protein, suggesting CR3 localizes in caveolae during phagocytosis. Following entry, the kinetics of phagolysosomal maturation and intracellular survival also differed. Vacuoles containing metacyclic parasites did not accumulate lysosome-associated membrane protein-1 (LAMP-1) at early times after phagocytosis, whereas vacuoles with logarithmic promastigotes did. MDMs phagocytosed greater numbers of logarithmic than metacyclic promastigotes, yet metacyclics ultimately replicated intracellularly with greater efficiency. These data suggest that virulent metacyclic Leishmania promastigotes fail to ligate macrophage MR, and enter through a path that ultimately enhances intracellular survival. The relatively quiescent entry of virulent Leishmania spp. into macrophages may be accounted for by the ability of metacyclic promastigotes to selectively bypass deleterious entry pathways. [source] Alternative infectious entry pathways for dengue virus serotypes into mammalian cellsCELLULAR MICROBIOLOGY, Issue 10 2009Eliana G. Acosta Summary The entry of two dengue virus (DENV) serotypes into Vero cells was analysed using biochemical inhibitors, dominant negative mutants of cellular proteins involved in endocytic pathways, fluorescence microscopy and infectivity determinations. By treatment with dansylcadaverine and chlorpromazine and overexpression of a dominant negative form of the Eps15 protein, a clathrin-mediated endocytosis for productive DENV-1 internalization into Vero cells was demonstrated whereas the infectious entry of DENV-2 in the same cell system was independent of clathrin. Treatment with the inhibitors nystatin and methyl-,-cyclodextrin, as well as transfection of Vero cells with dominant negative caveolin-1, had no effect on DENV-2 virus infection. It was also shown, by using the K44A mutant and the inhibitor dynasore, that dynamin was required for DENV-2 entry. Consequently, the infectious entry of DENV-2 into Vero cells occurs by a non-classical endocytic pathway independent of clathrin, caveolae and lipid rafts, but dependent on dynamin. By contrast, DENV-2 entry into A549 cells was clathrin-dependent, as previously reported in HeLa, C6/36 and BS-C-1 cells. Our results conclusively show, for the first time, a differential mode of infective entry for DENV-1 and DENV-2 into a common host cell, Vero cells, as well as alternative entry pathways for a given serotype, DENV-2, into different types of cells. [source] HeLa Cell Entry by Guanidinium-Rich ,-Peptides: Importance of Specific Cation,Cell Surface InteractionsCHEMBIOCHEM, Issue 8 2007Terra B. Potocky Abstract Short cationic oligomers, including arginine-rich peptides and analogous ,-amino acid oligomers (", -peptides"), can enter the cytoplasm and nucleus of a living cell from the extracellular medium. It seems increasingly clear that multiple entry pathways are possible, depending upon the structure of the guanidinium-rich molecule, the type of cell, and other factors. We have previously shown that conformational stability and spatial clustering of guanidinium groups increase the HeLa cell entry efficiency of short helical , -peptides bearing six guanidinium groups, results that suggest that these , -peptides could be useful tools for studying the entry process. Here we describe studies intended to identify the point in the entry process at which helix stability and spatial arrangement of guanidinium groups exert their effect. Our results suggest that key distinctions involve the mode of interaction between different guanidinium-rich ,-peptides and the HeLa cell surface. A specific guanidinium display appears to be required for proper engagement of cell-surface heparan sulfate proteoglycans and concomitant induction of endocytic uptake. [source] | |||||||||||||