The Cytoplasmic Region of SARAF Reduces Triple-Negative Breast Cancer Metastasis through the Regulation of Store-Operated Calcium Entry.

Triple-negative breast cancer has a poor prognosis and is non-responsive to first-line therapies; hence, new therapeutic strategies are needed. Enhanced store-operated Ca2+ entry (SOCE) has been widely described as a contributing factor to tumorigenic behavior in several tumor types, particularly in breast cancer cells. SOCE-associated regulatory factor (SARAF) acts as an inhibitor of the SOCE response and, therefore, can be a potential antitumor factor. Herein, we generated a C-terminal SARAF fragment to evaluate the effect of overexpression of this peptide on the malignancy of triple-negative breast cancer cell lines. Using both in vitro and in vivo approaches, we showed that overexpression of the C-terminal SARAF fragment reduced proliferation, cell migration, and the invasion of murine and human breast cancer cells by decreasing the SOCE response. Our data suggest that regulating the activity of the SOCE response via SARAF activity might constitute the basis for further alternative therapeutic strategies for triple-negative breast cancer.

TMPRSS11a is a novel age-altered, tissue specific regulator of migration and wound healing.

Aging is a gradual biological process characterized by a decrease in cellular and organism functions. Aging-related processes involve changes in the expression and activity of several proteins. Here, we identified the transmembrane protease serine 11a (TMPRSS11a) as a new age-specific protein that plays an important role in skin wound healing. TMPRSS11a levels increased with age in rodent and human skin and gingival samples. Strikingly, overexpression of TMPRSS11a decreased cell migration and spreading, and inducing cellular senescence. Mass spectrometry, bioinformatics, and functional analyses revealed that TMPRSS11a interacts with integrin ß1 through an RGD sequence contained within the C-terminal domain and that this motif was relevant for cell migration. Moreover, TMPRSS11a was associated with cellular senescence, as shown by overexpression and downregulation experiments. In agreement with tissue-specific expression of TMPRSS11a, shRNA-mediated downregulation of this protein improved wound healing in the skin, but not in the skeletal muscle of old mice, where TMPRSS11a is undetectable. Collectively, these findings indicate that TMPRSS11a is a tissue-specific factor relevant for wound healing, which becomes elevated with aging, promoting cellular senescence and inhibiting cell migration and skin repair.

SPARC Induces E-Cadherin Repression and Enhances Cell Migration through Integrin αvß3 and the Transcription Factor ZEB1 in Prostate Cancer Cells.

Secreted protein acidic and rich in cysteine (SPARC), or osteonectin, is a matricellular protein that modulates interactions between cells and their microenvironment. SPARC is expressed during extracellular matrix remodeling and is abundant in bone marrow and high-grade prostate cancer (PCa). In PCa, SPARC induces changes associated with epithelial-mesenchymal transition (EMT), enhancing migration and invasion and increasing the expression of EMT transcriptional factor Zinc finger E-box-binding homeobox 1 (ZEB1), but not Zinc finger protein SNAI1 (Snail) or Zinc finger protein SNAI2 (Slug). It is unknown whether the SPARC-induced downregulation of E-cadherin in PCa cells depends on ZEB1. Several integrins are mediators of SPARC effects in cancer cells. Because integrin signaling can induce EMT programs, we hypothesize that SPARC induces E-cadherin repression through the activation of integrins and ZEB1. Through stable knockdown and the overexpression of SPARC in PCa cells, we demonstrate that SPARC downregulates E-cadherin and increases vimentin, ZEB1, and integrin ß3 expression. Knocking down SPARC in PCa cells decreases the tyrosine-925 phosphorylation of FAK and impairs focal adhesion formation. Blocking integrin αvß3 and silencing ZEB1 revert both the SPARC-induced downregulation of E-cadherin and cell migration enhancement. We conclude that SPARC induces E-cadherin repression and enhances PCa cell migration through the integrin αvß3/ZEB1 signaling pathway.

KCTD5, a novel TRPM4-regulatory protein required for cell migration as a new predictor for breast cancer prognosis.

Transient receptor potential melastatin 4 (TRPM4) is a Ca2+ -activated nonselective cationic channel that regulates cell migration and contractility. Increased TRPM4 expression has been related to pathologies, in which cytoskeletal rearrangement and cell migration are altered, such as metastatic cancer. Here, we identify the K+ channel tetramerization domain 5 (KCTD5) protein, a putative adaptor of cullin3 E3 ubiquitin ligase, as a novel TRPM4-interacting protein. We demonstrate that KCTD5 is a positive regulator of TRPM4 activity by enhancing its Ca2+ sensitivity. We show that through its effects on TRPM4 that KCTD5 promotes cell migration and contractility. Finally, we observed that both TRPM4 and KCTD5 expression are increased in distinct patterns in different classes of breast cancer tumor samples. Together, these data support that TRPM4 activity can be regulated through expression levels of either TRPM4 or KCTD5, not only contributing to increased understanding of the molecular mechanisms involved on the regulation of these important ion channels, but also providing information that could inform treatments based on targeting these distinct molecules that define TRPM4 activity.

Cellular response of human apical papilla cells to calcium hydroxide and tricalcium silicate-based cements.

BACKGROUND: This study aimed to evaluate the biological response of human apical papilla cells to different calcium hydroxide formulations and three tricalcium silicate-based materials. METHODS: Primary cells were obtained from explants of young immature premolars. 20,000 cells adhered for 24 h over discs of Biodentine™, ProRoot®MTA, BioRoot®RCS and calcium hydroxide mixed either with sodium chloride 0.9%w/v or polyethylene glycol and UltraCal® were used to evaluate cell adhesion by scanning electron microscopy and cell viability by MTT assay. RESULTS: Cells adhered to ProRoot®MTA showed an increase of F-actin like protrusions, suggesting bioactivity. Cells adhered to UltraCal® show protrusion such as filopodia. On the contrary, cells adhered to BioRoot®RCS showed no signs of any cellular protrusion. Regarding viability between the materials, we found a higher percentage of viability in cells cultured over discs of Biodentine™ and ProRoot®MTA. CONCLUSION: ProRoot®MTA and Biodentine™ exhibit a better cellular response of human apical papilla cells in vitro conditions compared to BioRoot® and calcium hydroxide diluted in sodium chloride.

EB1- and EB2-dependent anterograde trafficking of TRPM4 regulates focal adhesion turnover and cell invasion.

Transient receptor potential melastatin 4 (TRPM4) is a Ca2+-activated nonselective cationic channel involved in a wide variety of physiologic and pathophysiological processes. Bioinformatics analyses of the primary sequence of TRPM4 allowed us to identify a putative motif for interaction with end-binding (EB) proteins, which are microtubule plus-end tracking proteins. Here, we provide novel data suggesting that TRPM4 interacts with EB proteins. We show that mutations of the putative EB binding motif abolish the TRPM4-EB interaction, leading to a reduced expression of the mature population of the plasma membrane channel and instead display an endoplasmic reticulum-associated distribution. Furthermore, we demonstrate that EB1 and EB2 proteins are required for TRPM4 trafficking and functional activity. Finally, we demonstrated that the expression of a soluble fragment containing the EB binding motif of TRPM4 reduces the plasma membrane expression of the channel and affects TRPM4-dependent focal adhesion disassembly and cell invasion processes.-Blanco, C., Morales, D., Mogollones, I., Vergara-Jaque, A., Vargas, C., Álvarez, A., Riquelme, D., Leiva-Salcedo, E., González, W., Morales, D., Maureira, D., Aldunate, I., Cáceres, M., Varela, D., Cerda, O. EB1- and EB2-dependent anterograde trafficking of TRPM4 regulates focal adhesion turnover and cell invasion.

Effect of sevoflurane and propofol on tourniquet-induced endothelial damage: a pilot randomized controlled trial for knee-ligament surgery.

BACKGROUND: The glycocalyx layer is a key structure in the endothelium. Tourniquet-induced ischemic periods are used during orthopedic surgery, and the reactive oxygen species generated after ischemia-reperfusion may mediate the shedding of the glycocalyx. Here, we describe the effects of tourniquet-induced ischemia-reperfusion and compare the effects of sevoflurane and propofol on the release of endothelial biomarkers after ischemia-reperfusion in knee-ligament surgery. METHODS: This pilot, single-center, blinded, randomized, controlled trial included 16 healthy patients. After spinal anesthesia, hypnosis was achieved with sevoflurane or propofol according to randomization. During the perioperative period, five venous blood samples were collected for quantification of syndecan-1, heparan sulfate, and thrombomodulin from blood serum by using ELISA assays kits. Sample size calculation was performed to detect a 25% change in the mean concentration of syndecan-1 with an alpha of 0.05 and power of 80%. RESULTS: For our primary outcome, a two-way ANOVA with post-hoc Bonferroni correction analysis showed no differences in syndecan-1 concentrations between the sevoflurane and propofol groups at any time point. In the sevoflurane group, we noted an increase in syndecan-1 concentrations 90 min after tourniquet release in the sevoflurane group from 34.6 ± 24.4 ng/mL to 47.9 ± 29.8 ng/mL (Wilcoxon test, p < 0.01) that was not observed in patients randomized to the propofol group. The two-way ANOVA showed no intergroup differences in heparan sulfate and thrombomodulin levels. CONCLUSIONS: Superficial endothelial damage without alterations in the cell layer integrity was observed after tourniquet knee-ligament surgery. There was no elevation in serum endothelial biomarkers in the propofol group patients. Sevoflurane did not show the protective effect observed in in vitro and in vivo studies. TRIAL REGISTRATION: The trial was registered in www.clinicaltrials.gov (ref: NCT03772054, Registered 11 December 2018).

Inhibition of the 3-hydroxy-3-methyl-glutaryl-CoA reductase induces orofacial defects in zebrafish.

BACKGROUND: Orofacial clefts (OFCs) are common birth defects, which include a range of disorders with a complex etiology affecting formation of craniofacial structures. Some forms of syndromic OFCs are produced by defects in the cholesterol pathway. The principal enzyme of the cholesterol pathway is the 3-hydroxy-3-methyl-glutaryl-CoA reductase (HMGCR). Our aim is to study whether defects of HMGCR function would produce orofacial malformation similar to those found in disorders of cholesterol synthesis. METHODS: We used zebrafish hmgcrb mutants and HMGCR inhibition assay using atorvastatin during early and late stages of orofacial morphogenesis in zebrafish. To describe craniofacial phenotypes, we stained cartilage and bone and performed in situ hybridization using known craniofacial markers. Also, we visualized neural crest cell migration in a transgenic fish. RESULTS: Our results showed that mutants displayed loss of cartilage and diminished orofacial outgrowth, and in some cases palatal cleft. Late treatments with statin show a similar phenotype. Affected-siblings displayed a moderate phenotype, whereas early-treated embryos had a minor cleft. We found reduced expression of the downstream component of Sonic Hedgehog-signaling gli1 in ventral brain, oral ectoderm, and pharyngeal endoderm in mutants and in late atorvastatin-treated embryos. CONCLUSION: Our results suggest that HMGCR loss-of-function primarily affects postmigratory cranial neural crest cells through abnormal Sonic Hedgehog signaling, probably induced by reduction in metabolites of the cholesterol pathway. Malformation severity correlates with the grade of HMGCR inhibition, developmental stage of its disruption, and probably with availability of maternal lipids. Together, our results might help to understand the spectrum of orofacial phenotypes found in cholesterol synthesis disorders. Birth Defects Research (Part A) 106:814-830, 2016. © 2016 Wiley Periodicals, Inc.

Casein kinase-mediated phosphorylation of serine 839 is necessary for basolateral localization of the Ca²âº-activated non-selective cation channel TRPM4.

Transient receptor potential melastatin-like 4 (TRPM4) is a Ca(2+)-activated non-selective cation channel expressed in a wide range of human tissues. TRPM4 participates in a variety of physiological processes such as T cell activation, myogenic vasoconstriction, and allergic reactions. TRPM4 Ca(2+) sensitivity is enhanced by calmodulin (CaM) and phosphathydilinositol 4, 5-bisphosphate (PI(4,5)P2) binding, as well as, under certain conditions, PKC activation. However, information as to the mechanisms of modulation of this channel remains unknown, including direct identification of phosphorylation sites on TRPM4 and their role in channel features. Here, we use mass-spectrometric-based proteomic approaches (immunoprecipitation and tandem mass spectrometry) to unambiguously identify S839 as a phosphorylation site present on human TRPM4 expressed in a human cell line. Site-directed mutagenesis employing a serine to alanine mutation to eliminate phosphorylation, and a phospho-mimetic aspartate mutation, as well as biochemical and immunocytochemical experiments, revealed a role for S839 phosphorylation in the basolateral expression of TRPM4 channels in epithelial cells. Moreover, we demonstrated that casein kinase 1 (CK1) phosphorylates S839 and is responsible for the basolateral localization of TRPM4.

Increases in reactive oxygen species enhance vascular endothelial cell migration through a mechanism dependent on the transient receptor potential melastatin 4 ion channel.

A hallmark of severe inflammation is reactive oxygen species (ROS) overproduction induced by increased inflammatory mediators secretion. During systemic inflammation, inflammation mediators circulating in the bloodstream interact with endothelial cells (ECs) raising intracellular oxidative stress at the endothelial monolayer. Oxidative stress mediates several pathological functions, including an exacerbated EC migration. Because cell migration critically depends on calcium channel-mediated Ca(2+) influx, the molecular identification of the calcium channel involved in oxidative stress-modulated EC migration has been the subject of intense investigation. The transient receptor potential melastatin 4 (TRPM4) protein is a ROS-modulated non-selective cationic channel that performs several cell functions, including regulating intracellular Ca(2+) overload and Ca(2+) oscillation. This channel is expressed in multiple tissues, including ECs, and contributes to the migration of certain immune cells. However, whether the TRPM4 ion channel participates in oxidative stress-mediated EC migration is not known. Herein, we investigate whether oxidative stress initiates or enhances EC migration and study the role played by the ROS-modulated TRPM4 ion channel in oxidative stress-mediated EC migration. We demonstrate that oxidative stress enhances, but does not initiate, EC migration in a dose-dependent manner. Notably, we demonstrate that the TRPM4 ion channel is critical in promoting H2O2-enhanced EC migration. These results show that TRPM4 is a novel pharmacological target for the possible treatment of severe inflammation and other oxidative stress-mediated inflammatory diseases.

Characterization of the arginine decarboxylase gene (ORF HP0422, speA) involved in acid tolerance in Helicobacter pylori.

BACKGROUND: Helicobacter pylori is a motile microaerophilic bacterium that colonizes the human stomach. H. pylori infection triggers gastric diseases, such as gastritis, peptic ulcer and gastric cancer. Stomach represents a barrier for microorganism colonization, particularly because of its high hydrochloric acid concentration. The main mechanism developed by H. pylori to maintain intracellular pH homeostasis in this environment is the urease activity. However, urease negative strains can be also isolated from clinical samples, suggesting that H. pylori presents other components involved in acid resistance. OBJECTIVE: Here, we present some evidence that the arginine decarboxylase gene (speA) in H. pylori could be involved in an acid adaptation mechanism similar to the one in Enterobacteriaceae, which is dependent on the presence of arginine. METHODS: Indeed, speA mRNA and protein expression are acutely induced by acid stress. RESULTS: Moreover, we showed that H. pylori uses arginine in an acid response mechanism required for its growth in acid conditions. CONCLUSION: Altogether, these results provide novel information regarding the H. pylori physiology and acid response mechanism.

The power of computational proteomics platforms to decipher protein-protein interactions.

Adopting computational tools for analyzing extensive biological datasets has profoundly transformed our understanding and interpretation of biological phenomena. Innovative platforms have emerged, providing automated analysis to unravel essential insights about proteins and the complexities of their interactions. These computational advancements align with traditional studies, which employ experimental techniques to discern and quantify physical and functional protein-protein interactions (PPIs). Among these techniques, tandem mass spectrometry is notably recognized for its precision and sensitivity in identifying PPIs. These approaches might serve as important information enabling the identification of PPIs with potential pharmacological significance. This review aims to convey our experience using computational tools for detecting PPI networks and offer an analysis of platforms that facilitate predictions derived from experimental data.

The odyssey of the TR(i)P journey to the cellular membrane.

Ion channels are integral membrane proteins mediating ion flow in response to changes in their environment. Among the different types of ion channels reported to date, the super-family of TRP channels stands out since its members have been linked to many pathophysiological processes. The family comprises 6 subfamilies and 28 members in mammals, which are widely distributed throughout most tissues and organs and have an important role in several aspects of cellular physiology. It has been evidenced that abnormal expression, post-translational modifications, and channel trafficking are associated with several pathologies, such as cancer, cardiovascular disease, diabetes, and brain disorders, among others. In this review, we present an updated summary of the mechanisms involved in the subcellular trafficking of TRP channels, with a special emphasis on whether different post-translational modifications and naturally occurring mutagenesis affect both expression and trafficking. Additionally, we describe how such changes have been associated with the development and progress of diverse pathologies associated with the gain or loss of functional phenotypes. The study of these processes will not only contribute to a better understanding the role of TRP channels in the different tissues but will also present novel possible therapeutic targets in diseases where their activity is dysregulated.

Mass spectrometry-based phosphoproteomics reveals multisite phosphorylation on mammalian brain voltage-gated sodium and potassium channels.

Voltage-gated sodium and potassium channels underlie electrical activity of neurons, and are dynamically regulated by diverse cell signaling pathways that ultimately exert their effects by altering the phosphorylation state of channel subunits. Recent mass spectrometric-based studies have led to a new appreciation of the extent and nature of phosphorylation of these ion channels in mammalian brain. This has allowed for new insights into how neurons dynamically regulate the localization, activity and expression through multisite ion channel phosphorylation.

Activity-dependent phosphorylation of neuronal Kv2.1 potassium channels by CDK5.

Dynamic modulation of ion channel expression, localization, and/or function drives plasticity in intrinsic neuronal excitability. Voltage-gated Kv2.1 potassium channels are constitutively maintained in a highly phosphorylated state in neurons. Increased neuronal activity triggers rapid calcineurin-dependent dephosphorylation, loss of channel clustering, and hyperpolarizing shifts in voltage-dependent activation that homeostatically suppress neuronal excitability. These changes are reversible, such that rephosphorylation occurs after removal of excitatory stimuli. Here, we show that cyclin-dependent kinase 5 (CDK5), a Pro-directed Ser/Thr protein kinase, directly phosphorylates Kv2.1, and determines the constitutive level of Kv2.1 phosphorylation, the rapid increase in Kv2.1 phosphorylation upon acute blockade of neuronal activity, and the recovery of Kv2.1 phosphorylation after stimulus-induced dephosphorylation. We also demonstrate that although the phosphorylation state of Kv2.1 is also shaped by the activity of the PP1 protein phosphatase, the regulation of Kv2.1 phosphorylation by CDK5 is not mediated through the previously described regulation of PP1 activity by CDK5. Together, these studies support a novel role for CDK5 in regulating Kv2.1 channels through direct phosphorylation.

Relationship Between Endothelial and Angiogenesis Biomarkers Envisage Mortality in a Prospective Cohort of COVID-19 Patients Requiring Respiratory Support.

Purpose: Endothelial damage and angiogenesis are fundamental elements of neovascularisation and fibrosis observed in patients with coronavirus disease 2019 (COVID-19). Here, we aimed to evaluate whether early endothelial and angiogenic biomarkers detection predicts mortality and major cardiovascular events in patients with COVID-19 requiring respiratory support. Methods: Changes in serum syndecan-1, thrombomodulin, and angiogenic factor concentrations were analysed during the first 24 h and 10 days after COVID-19 hospitalisation in patients with high-flow nasal oxygen or mechanical ventilation. Also, we performed an exploratory evaluation of the endothelial migration process induced by COVID-19 in the patients' serum using an endothelial cell culture model. Results: In 43 patients, mean syndecan-1 concentration was 40.96 ± 106.9 ng/mL with a 33.9% increase (49.96 ± 58.1 ng/mL) at day 10. Both increases were significant compared to healthy controls (Kruskal-Wallis p < 0.0001). We observed an increase in thrombomodulin, Angiopoietin-2, human vascular endothelial growth factor (VEGF), and human hepatocyte growth factor (HGF) concentrations during the first 24 h, with a decrease in human tissue inhibitor of metalloproteinases-2 (TIMP-2) that remained after 10 days. An increase in human Interleukin-8 (IL-8) on the 10th day accompanied by high HGF was also noted. The incidence of myocardial injury and pulmonary thromboembolism was 55.8 and 20%, respectively. The incidence of in-hospital deaths was 16.3%. Biomarkers showed differences in severity of COVID-19. Syndecan-1, human platelet-derived growth factor (PDGF), VEGF, and Ang-2 predicted mortality. A multiple logistic regression model with TIMP-2 and PDGF had positive and negative predictive powers of 80.9 and 70%, respectively, for mortality. None of the biomarkers predicted myocardial injury or pulmonary thromboembolism. A proteome profiler array found changes in concentration in a large number of biomarkers of angiogenesis and chemoattractants. Finally, the serum samples from COVID-19 patients increased cell migration compared to that from healthy individuals. Conclusion: We observed that early endothelial and angiogenic biomarkers predicted mortality in patients with COVID-19. Chemoattractants from patients with COVID-19 increase the migration of endothelial cells. Trials are needed for confirmation, as this poses a therapeutic target for SARS-CoV-2.

Selective TASK-1 Inhibitor with a Defined Structure-Activity Relationship Reduces Cancer Cell Proliferation and Viability.

Chemical structures of selective blockers of TASK channels contain aromatic groups and amide bonds. Using this rationale, we designed and synthesized a series of compounds based on 3-benzamidobenzoic acid. These compounds block TASK-1 channels by binding to the central cavity. The most active compound is 3-benzoylamino-N-(2-ethyl-phenyl)-benzamide or F3, blocking TASK-1 with an IC50 of 148 nM, showing a reduced inhibition of TASK-3 channels and not a significant effect on different K+ channels. We identified putative F3-binding sites in the TASK-1 channel by molecular modeling studies. Mutation of seven residues to A (I118A, L122A, F125A, Q126A, L232A, I235A, and L239A) markedly decreased the F3-induced inhibition of TASK-1 channels, consistent with the molecular modeling predictions. F3 blocks cell proliferation and viability in the MCF-7 cancer cell line but not in TASK-1 knockdown MCF-7 cells, indicating that it is acting in TASK-1 channels. These results indicated that TASK-1 is necessary to drive proliferation in the MCF-7 cancer cell line.

P2X4 activation modulates volume-sensitive outwardly rectifying chloride channels in rat hepatoma cells.

Volume-sensitive outwardly rectifying (VSOR) Cl(-) channels are critical for the regulatory volume decrease (RVD) response triggered upon cell swelling. Recent evidence indicates that H(2)O(2) plays an essential role in the activation of these channels and that H(2)O(2) per se activates the channels under isotonic isovolumic conditions. However, a significant difference in the time course for current onset between H(2)O(2)-induced and hypotonicity-mediated VSOR Cl(-) activation is observed. In several cell types, cell swelling induced by hypotonic challenges triggers the release of ATP to the extracellular medium, which in turn, activates purinergic receptors and modulates cell volume regulation. In this study, we have addressed the effect of purinergic receptor activation on H(2)O(2)-induced and hypotonicity-mediated VSOR Cl(-) current activation. Here we show that rat hepatoma cells (HTC) exposed to a 33% hypotonic solution responded by rapidly activating VSOR Cl(-) current and releasing ATP to the extracellular medium. In contrast, cells exposed to 200 microm H(2)O(2) VSOR Cl(-) current onset was significantly slower, and ATP release was not detected. In cells exposed to either 11% hypotonicity or 200 microm H(2)O(2), exogenous addition of ATP in the presence of extracellular Ca(2+) resulted in a decrease in the half-time for VSOR Cl(-) current onset. Conversely, in cells that overexpress a dominant-negative mutant of the ionotropic receptor P2X4 challenged with a 33% hypotonic solution, the half-time for VSOR Cl(-) current onset was significantly slowed down. Our results indicate that, at high hypotonic imbalances, swelling-induced ATP release activates the purinergic receptor P2X4, which in turn modulates the time course of VSOR Cl(-) current onset in a extracellular Ca(2+)-dependent manner.

Hydrogen peroxide removes TRPM4 current desensitization conferring increased vulnerability to necrotic cell death.

Necrosis is associated with an increase in plasma membrane permeability, cell swelling, and loss of membrane integrity with subsequent release of cytoplasmic constituents. Severe redox imbalance by overproduction of reactive oxygen species is one of the main causes of necrosis. Here we demonstrate that H(2)O(2) induces a sustained activity of TRPM4, a Ca(2+)-activated, Ca(2+)-impermeant nonselective cation channel resulting in an increased vulnerability to cell death. In HEK 293 cells overexpressing TRPM4, H(2)O(2) was found to eliminate in a dose-dependent manner TRPM4 desensitization. Site-directed mutagenesis experiments revealed that the Cys(1093) residue is crucial for the H(2)O(2)-mediated loss of desensitization. In HeLa cells, which endogenously express TRPM4, H(2)O(2) elicited necrosis as well as apoptosis. H(2)O(2)-mediated necrosis but not apoptosis was abolished by replacement of external Na(+) ions with sucrose or the non-permeant cation N-methyl-d-glucamine and by knocking down TRPM4 with a shRNA directed against TRPM4. Conversely, transient overexpression of TRPM4 in HeLa cells in which TRPM4 was previously silenced re-established vulnerability to H(2)O(2)-induced necrotic cell death. In addition, HeLa cells exposed to H(2)O(2) displayed an irreversible loss of membrane potential, which was prevented by TRPM4 knockdown.

tlpA gene expression is required for arginine and bicarbonate chemotaxis in Helicobacter pylori.

About half of the human population is infected with Helicobacter pylori, a bacterium causing gastritis, peptic ulcer and progression to gastric cancer. Chemotaxis and flagellar motility are required for colonization and persistence of H. pylori in the gastric mucus layer. It is not completely clear which chemical gradients are used by H. pylori to maintain its position. TlpA, a chemotaxis receptor for arginine/ bicarbonate, has been identified. This study aimed to find out whether tlpA gene expression is required for the chemotactic response to arginine/bicarbonate. Wild-type motile H. pylori ATCC 700392 and H. pylori ATCC 43504, a strain having an interrupted tlpA gene, were used. Also, a tlpA-knockout mutant of H. pylori 700392 (H. pylori 700-tlpA::cat) was produced by homologous recombination. Expression of tlpA was assessed by a Reverse Transcriptase-Polymerase Chain Reaction (RT-PCR) assay. Chemotaxis was measured as a Relative Chemotaxis Response (RCR) by a modified capillary assay. H. pylori 700392 presented chemotaxis to arginine and sodium bicarbonate. H. pylori 700-tlpA::cat showed neither tlpA gene expression nor chemotaxis towards arginine and bicarbonate. Besides confirming that TlpA is a chemotactic receptor for arginine/bicarbonate in H. pylori, this study showed that tlpA gene expression is required for arginine/bicarbonate chemotaxis.