Numerous Cellular Components Research Articles

Phosphate theft: a path to fungal pathogenic success

Inorganic phosphate/PO4 /Pi is an essential andmajor constituent of numerous cellular components in all eukaryotes, including fungi. These components include nucleic acids, phospholipids and ATP. Despite its abundance in organic compounds, Pi is relatively scarce in its free form.Tobecome successful pathogens, fungi must therefore acquire free Pi from the host environment via enzyme-mediated hydrolysis of Pi-containing molecules and/or via more efficient use of their own Pi. Fungal adaptation to a Pi-limited environment is governed by the phosphate (PHO) system, a cellular pathway consistingofPi transporters, Pimobilising enzymes and regulatory elements, such as kinases and transcription factors that respond to Pi levels. This system is well studied in the model non-pathogenic yeast, Saccharomyces cerevisiae, but not in fungal pathogens. In this review we present what is known about the PHO system in the model fungal pathogen, Cryptococcus neoformans, including our identification and characterisation of a secreted acid phosphatase, Aph1, which serves as a valuable reporter for identifying the less well-conserved PHO elements, including transcription factors.

CD47 does not mediate amyloid-β(1–42) protofibril-stimulated microglial cytokine release

Neuroinflammation triggered by accumulation of amyloid-β protein (Aβ) is a significant component of the Alzheimer’s disease (AD) brain. Senile plaques composed of Aβ attract and activate microglia cells resulting in cytokine secretion and a proinflammatory environment. The mechanism by which Aβ activates microglia is complex and involves numerous cellular components. One receptor potentially involved in Aβ recognition and the ensuing microglia proinflammatory response is CD47. Since there is significant interest in soluble aggregated Aβ species, we sought to determine if CD47 plays a key role in microglia cytokine release stimulated by soluble Aβ(1–42) protofibrils. Pretreatment of primary murine microglia with the CD47 antagonist peptide 4N1K significantly and potently inhibited both tumor necrosis factor-α (TNFα) and interleukin-1β (IL-1β) secretion stimulated by Aβ(1–42) protofibrils. 4N1K displayed toxicity to the microglia but only at concentrations much higher than the observed inhibition. Surprisingly, 4N1K also potently inhibited TNFα secretion triggered by lipopolysaccharide which is not known to signal through CD47. Treatment of the microglia with a neutralizing anti-CD47 antibody failed to block the Aβ protofibril response even though comparable samples were completely inhibited by 4N1K. Finally, Aβ(1–42) protofibrils stimulated similar levels of secreted TNFα production in both wild-type and CD47−/− microglia and 4N1K still potently inhibited the Aβ protofibril response even in the CD47−/− microglia. The overall findings demonstrated that the microglial proinflammatory response to Aβ(1–42) protofibril is not dependent on CD47 and that 4N1K exhibits CD47-independent inhibitory activity.

Signaling networks converge on TORC1-SREBP activity to promote endoplasmic reticulum homeostasis.

The function and capacity of the endoplasmic reticulum (ER) is determined by multiple processes ranging from the local regulation of peptide translation, translocation, and folding, to global changes in lipid composition. ER homeostasis thus requires complex interactions amongst numerous cellular components. However, describing the networks that maintain ER function during changes in cell behavior and environmental fluctuations has, to date, proven difficult. Here we perform a systems-level analysis of ER homeostasis, and find that although signaling networks that regulate ER function have a largely modular architecture, the TORC1-SREBP signaling axis is a central node that integrates signals emanating from different sub-networks. TORC1-SREBP promotes ER homeostasis by regulating phospholipid biosynthesis and driving changes in ER morphology. In particular, our network model shows TORC1-SREBP serves to integrate signals promoting growth and G1-S progression in order to maintain ER function during cell proliferation.

PWE-119 Decreased Frequency Of Peripheral And Intestinal Nkg2a-positive T Cells In Ulcerative Colitis Patients

IntroductionThe intestinal tract is home to a numerous immune cellular components that continuously encounter abundant exogenous stimuli, where remain in a state of controlled inflammation normally. Inflammatory bowel disease (IBD)...

Targeting cisplatin-resistant human tumor cells with metabolic inhibitors

Although cisplatin is the drug of choice in treating lung cancer patients, relapse and resistance is a common drawback to its clinical effectiveness. Based on cisplatin's reported ability to interfere with numerous cellular components, including mitochondria, we probed alterations in metabolism in cisplatin-resistant tumor cell lines to reveal targets for overcoming this important form of resistance. Cisplatin-resistant lung and ovarian cancer cell lines were used to evaluate the efficacy of metabolic inhibitors for selectively targeting cisplatin-resistant cells under varying oxygen conditions. Three cisplatin-resistant cancer cell lines expressed lower HKII protein when compared to the respective cisplatin-sensitive cancer cell lines from which they were derived. Under anaerobic and hypoxic conditions, treatment with the glycolytic inhibitors 2-deoxyglucose (2-DG) and 2-fluorodeoxyglucose (2-FDG) correlated with increased cytotoxicity and more pronounced decreases in lactate production in cisplatin-resistant cells, indicating a greater blockage of glycolysis. Knockdown of HKI or HKII with siRNA in the parental lung cancer cell lines led to increased 2-FDG-induced cell death under anaerobic conditions. Under normal oxygen conditions, blockage of either fatty acid oxidation or deprivation of glutamine resulted in cell death in cisplatin-resistant lung cancer cell lines. Altered hexokinase levels in cisplatin-resistant cancer cell lines leads to increased sensitivity to glycolytic inhibition under anaerobic conditions, whereas under normoxic conditions, blockage of either fatty acid oxidation or deprivation of glutamine leads to cell death. These findings may be clinically applicable when considering cisplatin resistance.

Two Wheat Glutathione Peroxidase Genes Whose Products Are Located in Chloroplasts Improve Salt and H2O2 Tolerances in Arabidopsis

Oxidative stress caused by accumulation of reactive oxygen species (ROS) is capable of damaging effects on numerous cellular components. Glutathione peroxidases (GPXs, EC 1.11.1.9) are key enzymes of the antioxidant network in plants. In this study, W69 and W106, two putative GPX genes, were obtained by de novo transcriptome sequencing of salt-treated wheat (Triticum aestivum) seedlings. The purified His-tag fusion proteins of W69 and W106 reduced H2O2 and t-butyl hydroperoxide (t-BHP) using glutathione (GSH) or thioredoxin (Trx) as an electron donor in vitro, showing their peroxidase activity toward H2O2 and toxic organic hydroperoxide. GFP fluorescence assays revealed that W69 and W106 are localized in chloroplasts. Quantitative real-time PCR (Q-RT-PCR) analysis showed that two GPXs were differentially responsive to salt, drought, H2O2, or ABA. Isolation of the W69 and W106 promoters revealed some cis-acting elements responding to abiotic stresses. Overexpression of W69 and W106 conferred strong tolerance to salt, H2O2, and ABA treatment in Arabidopsis. Moreover, the expression levels of key regulator genes (SOS1, RbohD and ABI1/ABI2) involved in salt, H2O2 and ABA signaling were altered in the transgenic plants. These findings suggest that W69 and W106 not only act as scavengers of H2O2 in controlling abiotic stress responses, but also play important roles in salt and ABA signaling.

Plastidal Pathways for Nitrogen Assimilation and the Inhibition of Amino Acid Biosynthesis by Herbicides

There are many cellular processes occurring in plastids. One of them concerns nitrogen assimilation (nitrate and ammonium). Photosynthesis taking place in the same organelle plays a key role in direct or indirect assimilation of nitrogen. This is involved in the building of the fundamental life molecules: nucleotides, proteins, chlorophyll and numerous other metabolites and cellular components. This review enlightens the mechanisms involved in the transport, reduction and assimilation of nitrate implying specific transporters in the plastid envelopes, specific enzymes as glutamine synthetase/ glutamate synthase pathway in the plastid stroma from plants and algae studied in the lab as models. EPSP synthase, AHA synthase and GS participating to amino acid biosynthesis have been used as targets for widely distributed herbicides. This paper will review the molecular basis of their inhibiting action and the various approaches to obtain herbicide- tolerant crops. Keywords: Algae, chloroplasts, herbicides, higher plants, herbicide tolerant crops, nitrate, nitrite, plastids, ransmembrane, lamina

The essential roles of protein–protein interaction in sigma-1 receptor functions

The essential roles of protein–protein interaction in sigma-1 receptor functions

Proteolipidic Composition of Exosomes Changes during Reticulocyte Maturation

During the orchestrated process leading to mature erythrocytes, reticulocytes must synthesize large amounts of hemoglobin, while eliminating numerous cellular components. Exosomes are small secreted vesicles that play an important role in this process of specific elimination. To understand the mechanisms of proteolipidic sorting leading to their biogenesis, we have explored changes in the composition of exosomes released by reticulocytes during their differentiation, in parallel to their physical properties. By combining proteomic and lipidomic approaches, we found dramatic alterations in the composition of the exosomes retrieved over the course of a 7-day in vitro differentiation protocol. Our data support a previously proposed model, whereby in reticulocytes the biogenesis of exosomes involves several distinct mechanisms for the preferential recruitment of particular proteins and lipids and suggest that the respective prominence of those pathways changes over the course of the differentiation process.

Cardiopulmonary Arrest and Resuscitation Disrupts Cholinergic Anti-Inflammatory Processes: A Role for Cholinergic α7 Nicotinic Receptors

Cardiac arrest is a leading cause of death worldwide. While survival rates following sudden cardiac arrest remain relatively low, recent advancements in patient care have begun to increase the proportion of individuals who survive cardiac arrest. However, many of these individuals subsequently develop physiological and psychiatric conditions that likely result from ongoing neuroinflammation and neuronal death. The present study was conducted to better understand the pathophysiological effects of cardiac arrest on neuronal cell death and inflammation, and their modulation by the cholinergic system. Using a well validated model of cardiac arrest, here we show that global cerebral ischemia increases microglial activation, proinflammatory cytokine mRNA expression (interleukin-1β, interleukin-6, tumor necrosis factor-α), and neuronal damage. Cardiac arrest also induces alterations in numerous cellular components of central cholinergic signaling, including a reduction in choline acetyltransferase enzymatic activity and the number of choline acetyltransferase-positive neurons, as well as, reduced acetylcholinesterase and vesicular acetylcholine transporter mRNA. However, treatment with a selective agonist of the α7 nicotinic acetylcholine receptor, the primary receptor mediating the cholinergic anti-inflammatory pathway, significantly decreases the neuroinflammation and neuronal damage resulting from cardiac arrest. These data suggest that global cerebral ischemia results in significant declines in central cholinergic signaling, which may in turn diminish the capacity of the cholinergic anti-inflammatory pathway to control inflammation. Furthermore, we provide evidence that pharmacological activation of α7 nicotinic acetylcholine receptors provide significant protection against ischemia-related cell death and inflammation within a clinically relevant time frame.

Microenviromental factors controlling macrophage polarization in atherosclerosis

Microenviromental factors controlling macrophage polarization in atherosclerosis

ATP-Binding Membrane Cassette Transporter A1 (ABCA1): A Possible Link between Inflammation and Reverse Cholesterol Transport

Atherosclerosis is characterized by a chronic inflammatory condition that involves numerous cellular and molecular inflammatory components. A wide array of inflammatory mediators, such as cytokines and proteins produced by macrophages and other cells, play a critical role in the development and progression of the disease. ATP-binding membrane cassette transporter A1 (ABCA1) is crucial for cellular cholesterol efflux and reverse cholesterol transport (RCT) and is also identified as an important target in antiatherosclerosis treatment. Evidence from several recent studies indicates that inflammation, along with other atherogenic-related mediators, plays distinct regulating roles in ABCA1 expression. Proatherogenic cytokines such as interferon (IFN)-γ and interleukin (IL)-1β have been shown to inhibit the expression of ABCA1, while antiatherogenic cytokines, including IL-10 and transforming growth factor (TGF)-β1, have been shown to promote the expression of ABCA1. Moreover, some cytokines such as tumor necrosis factor (TNF)-α seem to regulate ABCA1 expression in species-specific and dose-dependent manners. Inflammatory proteins such as C-reactive protein (CRP) and cyclooxygenase (COX)-2 are likely to inhibit ABCA1 expression during inflammation, and inflammation induced by lipopolysaccharide (LPS) was also found to block the expression of ABCA1. Interestingly, recent experiments revealed ABCA1 can function as an antiinflammatory receptor to suppress the expression of inflammatory factors, suggesting that ABCA1 may be the molecular basis for the interaction between inflammation and RCT. This review aims to summarize recent findings on the role of inflammatory cytokines, inflammatory proteins, inflammatory lipids, and the endotoxin-mediated inflammatory process in expression of ABCA1. Also covered is the current understanding of the function of ABCA1 in modulating the immune response and inflammation through its direct and indirect antiinflammatory mechanisms including lipid transport, high-density lipoprotein (HDL) formation and apoptosis.

Abstract 1590: Autophagic Processing Of Ubiquitinated Protein Aggregates During Unloading-induced Cardiac Atrophy

Background: Mechanical unloading of the diseased ventricle has emerged as an important treatment strategy in advanced heart failure. In rare instances, dramatic improvements in ventricular performance are seen, yet little is known regarding underlying mechanisms. Autophagy is an ancient mechanism of bulk protein degradation recently shown to be activated by a variety of forms of cardiac stress. Methods and Results: To test for a role of autophagy in atrophic remodeling of the unloaded left ventricle (LV), murine wild-type (WT) and “autophagy reporter” (αMHC-GFP-LC3 transgenic) hearts underwent heterotopic transplantation into the abdomen of same-strain recipient mice. The donor hearts were thereby mechanically unloaded while the native recipient hearts served as controls. In WT hearts, cardiomyocyte cell surface area declined 27±2% at 7 days. Processing of LC3 (microtubule-associated light chain 3) increased in atrophying donor hearts, indicative of increased autophagosome formation. Further, significant increases in the number of GFP-LC3 puncta in “autophagy reporter” hearts were observed. Levels of the pro-autophagic protein Beclin 1 increased in donor hearts, further evidence of increased autophagic flux during atrophic remodeling. In atrophic donor hearts, we detected significant increases in polyubiquitinated proteins as well as in the abundance of the ER chaperone proteins GRP78/BIP and GRP94, indicative of an unfolded protein response. As autophagy degrades numerous cellular components including polyubiquitinated protein aggregates, we transplanted hearts haploin-sufficient for beclin 1 , a gene required for early processing of autophagosomes (≈50% decrease in autophagy). In these studies, we detected robust increases in the abundance of polyubiquitin proteins in beclin 1 ± donor hearts relative to WT (62% vs 12%, p<0.05). Conclusions: Autophagy is activated during cardiac atrophy and participates in processing of protein aggregates. Together, these findings implicate autophagic activity in the remodeling of the unloaded LV. This research has received full or partial funding support from the American Heart Association, AHA South Central Affiliate (Arkansas, New Mexico, Oklahoma & Texas).

Cytokines and atherosclerosis: a comprehensive review of studies in mice

In the past few years, inflammation has emerged as a major driving force of atherosclerotic lesion development. It is now well-established that from early lesion to vulnerable plaque formation, numerous cellular and molecular inflammatory components participate in the disease process. The most prominent cells that invade in evolving lesions are monocyte-derived macrophages and T-lymphocytes. Both cell types produce a wide array of soluble inflammatory mediators (cytokines, chemokines) which are critically important in the initiation and perpetuation of the disease. This review summarizes the currently available information from mouse studies on the contribution of a specified group of cytokines expressed in atherosclerotic lesions, viz. interleukins (IL-1, IL-2, IL-3, IL-4, IL-5, IL-6, IL-10, IL-12, IL-18, IL-20) and macrophage-associated cytokines [tumour necrosis factor-α (TNF-α); macrophage migration inhibitory factor (MIF); interferon-γ (IFN-γ); colony stimulating factors G-CSF,-M-CSF,-GM-CSF) to atherogenesis. Emphasis is put on the consistency of the effects of these cytokines, i.e. inasmuch an effect depends on the experimental approach applied (overexpression/deletion, strain, gender, dietary conditions, and disease stage). An important outcome of this survey is (i) that only for a few cytokines there is sufficient consistent data allowing classifying them as typically proatherogenic (IL-1, IL-12, IL-18, MIF, IFN-γ, TNF-α, and M-CSF) or antiatherogenic (IL-10) and (ii) that some cytokines (IL-4, IL-6 and GM-CSF) can exert pro- or anti-atherogenic effects depending on the experimental conditions. This knowledge can be used for improved early detection, prevention and treatment of atherosclerosis.

Developing New Anti-Arrhythmics: Clues from the Molecular Basis of Cardiac Ryanodine Receptor (RyR2) Ca2+-Release Channel Dysfunction

Sudden cardiac death (SCD) remains a major cause of mortality, and despite our knowledge of the causative genetic, molecular and biochemical cellular mechanisms involved, effective therapeutic strategies are lacking. Perturbations in cardiac Ca2+ handling promote arrhythmias and there is enormous interest in developing new anti-arrhythmics aimed at correcting Ca2+ release dysfunction. In particular, abnormal Ca2+ release arising as a result of acquired or genetic defects in cardiac ryanodine receptors (RyR2) has emerged as an important arrhythmogenic trigger in heart failure, and in a devastating genetic arrhythmia syndrome termed catecholaminergic polymorphic ventricular tachycardia (CPVT). Here, we evaluate how experimental insights into RyR2 structure-function are unravelling the precise molecular basis of channel dysfunction and are advancing the development of new therapeutic strategies. We also discuss the functional role of RyR2 in the context of the exquisite synergism existing between numerous cellular components involved in cardiac Ca2+ signalling, and how these complex interactions may be used to design new anti-arrhythmic approaches that target multiple facets of RyR2 regulation.

Repression of ergosterol level during oxidative stress by fission yeast F-box protein Pof14 independently of SCF

We describe a new member of the F-box family, Pof14, which forms a canonical, F-box dependent SCF (Skp1, Cullin, F-box protein) ubiquitin ligase complex. The Pof14 protein has intrinsic instability that is abolished by inactivation of its Skp1 interaction motif (the F-box), Skp1 or the proteasome, indicating that Pof14 stability is controlled by an autocatalytic mechanism. Pof14 interacts with the squalene synthase Erg9, a key enzyme in ergosterol metabolism, in a membrane-bound complex that does not contain the core SCF components. pof14 transcription is induced by hydrogen peroxide and requires the Pap1 transcription factor and the Sty1 MAP kinase. Pof14 binds to and decreases Erg9 activity in vitro and a pof14 deletion strain quickly loses viability in the presence of hydrogen peroxide due to its inability to repress ergosterol synthesis. A pof14 mutant lacking the F-box and an skp1-3 ts mutant behave as wild type in the presence of oxidant showing that Pof14 function is independent of SCF. This indicates that modulation of ergosterol level plays a key role in adaptation to oxidative stress.

Cellular, molecular and immunological mechanisms in the pathophysiology of vein graft intimal hyperplasia

Coronary artery disease, leading to myocardial infarction and ischaemia, affects millions of persons and is one of the leading causes of morbidity and mortality worldwide. Invasive techniques such as coronary artery bypass grafting are used to alleviate the sequelae of arterial occlusion. Unfortunately, restenosis or occlusion of the grafted conduit occurs over a time frame of months to years with a gradual reduction in patency, especially in vein grafts. The events leading to intimal hyperplasia (IH) formation involve numerous cellular and molecular components. Various cellular elements of the vessel wall are involved as are leucocyte-endothelial interactions that trigger the coagulation cascade leading to localized thrombus formation. Subsequent phenotypic modification of the medial smooth muscle cells and their intimal migration is the basis of the lesion formation that is thought to be propagated by an immune-mediated reaction. Despite intense scrutiny, the pathophysiology of IH remains an enigma. Although several growth factors, cytokines and numerous other biomolecules have been implicated and their relationship to prohyperplasia pathways such as the phosphatidyl-inositol 3-kinase (PI3K)-Akt pathway has been established, many pieces of the puzzle are still missing. An in-depth understanding of early vein graft adaptation and progression is necessary to improve the long-term prognosis and develop more effective therapeutic measures. In this review, we have critically evaluated and summarized the literature to elucidate and interlink the numerous established and emerging factors that play a key role in the development of IH leading to vein graft restenosis.

Malignom und Thrombose: eine wechselseitige klinische Beziehung

Venous thromboembolism (VTE) is the second most common cause of mortality in cancer patients and also points towards unfavourable prognosis. In about 10% of patients with idiopathic VTE there is underlying malignancy. However, the efficacy of extensive tumour screening in those patients is not yet established. Numerous plasmatic and cellular components contribute to the phenomenon of hypercoagulability in cancer patients, including Cancer Procoagulant and activation of coagulation with high levels of coagulation factors. Cancer surgery carries an increased risk compared to non-cancer patients necessating more intensive and longer thromboprophylaxis in those patients. In medical patients active cancer is associated with increased risk for VTE. In the treatment of VTE, cancer patients have a significantly higher recurrence rate for VTE when treated with vitamin K-antagonists (VKA) compared to non-cancer patients. Compared to VKA the use of low molecular weight heparin for long-term secondary prevention is more effective than vitamin K-antagonists. Thus, there is a grade 1A recommendation to use low molecular weight heparin for cancer patients during the first 3-6 months of secondary prevention of VTE. Several studies indicate that low molecular weight heparin may also improve the prognosis of cancer patients quoad vitam, particularly for cancer patients at an earlier stage of disease; this needs to be confirmed in further studies.

Changes in P-glycoprotein activity are mediated by the growth of a tumour cell line as multicellular spheroids.

BackgroundExpression of P-glycoprotein (P-gp), the multidrug resistance (MDR) 1 gene product, can lead to multidrug resistance in tumours. However, the physiological role of P-gp in tumours growing as multicellular spheroids is not well understood. Recent evidence suggests that P-gp activity may be modulated by cellular components such as membrane proteins, membrane-anchoring proteins or membrane-lipid composition. Since, multicellular spheroids studies have evidenced alterations in numerous cellular components, including those related to the plasma membrane function, result plausible that some of these changes might modulate P-gp function and be responsible for the acquisition of multicellular drug resistance. In the present study, we asked if a human lung cancer cell line (INER-51) grown as multicellular spheroids can modify the P-gp activity to decrease the levels of doxorubicin (DXR) retained and increase their drug resistance.ResultsOur results showed that INER-51 spheroids retain 3-folds lower doxorubicin than the same cells as monolayers however; differences in retention were not observed when the P-gp substrate Rho-123 was used. Interestingly, neither the use of the P-gp-modulating agent cyclosporin-A (Cs-A) nor a decrease in ATP-pools were able to increase DXR retention in the multicellular spheroids. Only the lack of P-gp expression throughout the pharmacological selection of a P-gp negative (P-gpneg) mutant clone (PSC-1) derived from INER-51 cells, allow increase of DXR retention in spheroids.ConclusionThus, multicellular arrangement appears to alter the P-gp activity to maintain lower levels of DXR. However, the non expression of P-gp by cells forming multicellular spheroids has only a minor impact in the resistance to chemotherapeutic agents.

Microarray Analysis of Host Gene‐Expression during Intracellular Nests Formation of Trypanosoma cruzi Amastigotes

The intracellular pathogens utilize numerous cellular components of host cells to advance the infection as well as to enter the host cell. Analyzing the host cellular response enables us to get a better understanding of the pathogenesis, and subsequently indicate possible therapeutic targets. We therefore analyzed gene-expression profile of NIH3T3 fibroblast cells infected by Trypanosoma, a representative intracellular pathogen similar to Leishmania, using custom-designed cDNA microarray consisting of 1,701 mKIAA cDNAs. Focusing on intracellular nest formation of Trypanosoma cruzi amastigotes, we profiled the host gene-expression at 8 days post-infection and found several degrees of change in 16 mKIAA genes. Among these genes, 10 were up-regulated and 6 were down-regulated. Assuming that these genes had important roles in the infection's progression, we performed semi-quantitative RT-PCR analysis and con-firmed the gene expression change of 4 genes. Furthermore, 5 genes were mapped on cadherin signaling pathway using pathway analysis software. These results indicate significance of the host cellular pathway in the proliferative stage of Trypanosoma cruzi amastigotes.