Original article
An acidic sphingomyelinase Type C activity from Mycobacterium tuberculosis
Actividad de una esfingomielinasa ácida tipo C producida por Mycobacterium tuberculosis
Jorge Castro-Garzaa, Francisco González-Salazara,b, Frederick D. Quinnc, Russell K. Karlsc, Laura Hermila De La Garza-Salinasd, Francisco J. Guzmán-de la Garzae, Javier Vargas-Villarreala,
Corresponding author
a Centro de Investigación Biomédica del Noreste, Instituto Mexicano del Seguro Social, Monterrey, N.L., México
b Departamento de Ciencias Básicas, División de Ciencias de la Salud, Universidad de Monterrey, San Pedro Garza García, N.L., México
c Department of Infectious Diseases, University of Georgia, Athens, GA, USA
d Coordinación de Planeación y Enlace Institucional, Instituto Mexicano Del Seguro Social, Delegación Nuevo León, Monterrey, N.L., México
e Unidad de Investigación Epidemiológica y en Servicios de Salud, Centro de Investigación Biomédica del Noreste, Instituto Mexicano del Seguro Social, Monterrey, N.L., México
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"apellidos" => "Yantorno" ] 16 => array:2 [ "nombre" => "Alejandra" "apellidos" => "Bosch" ] ] ] ] ] "idiomaDefecto" => "es" "EPUB" => "https://multimedia.elsevier.es/PublicationsMultimediaV1/item/epub/S0325754115001613?idApp=UINPBA00004N" "url" => "/03257541/0000004800000001/v1_201603250025/S0325754115001613/v1_201603250025/es/main.assets" ] "itemAnterior" => array:19 [ "pii" => "S0325754115001601" "issn" => "03257541" "doi" => "10.1016/j.ram.2015.10.006" "estado" => "S300" "fechaPublicacion" => "2016-01-01" "aid" => "75" "copyright" => "Asociación Argentina de Microbiología" "documento" => "article" "crossmark" => 1 "licencia" => "http://creativecommons.org/licenses/by-nc-nd/4.0/" "subdocumento" => "fla" "cita" => "Rev Argent Microbiol. 2016;48:15-20" "abierto" => array:3 [ "ES" => true "ES2" => true "LATM" => true ] "gratuito" => true "lecturas" => array:2 [ "total" => 1599 "formatos" => array:3 [ "EPUB" => 43 "HTML" => 1065 "PDF" => 491 ] ] "en" => array:13 [ "idiomaDefecto" => true "cabecera" => "<span class="elsevierStyleTextfn">Original article</span>" "titulo" => "PCR-based method for the rapid identification of astaxanthin-accumulating yeasts (<span class="elsevierStyleItalic">Phaffia</span> spp.)" "tienePdf" => "en" "tieneTextoCompleto" => "en" "tieneResumen" => array:2 [ 0 => "en" 1 => "es" ] "paginas" => array:1 [ 0 => array:2 [ "paginaInicial" => "15" "paginaFinal" => "20" ] ] "titulosAlternativos" => array:1 [ "es" => array:1 [ "titulo" => "Un método basado en la PCR para la identificación rápida de levaduras acumuladoras de astaxantina (<span class="elsevierStyleItalic">Phaffia</span> spp.)" ] ] "contieneResumen" => array:2 [ "en" => true "es" => true ] "contieneTextoCompleto" => array:1 [ "en" => true ] "contienePdf" => array:1 [ "en" => true ] "resumenGrafico" => array:2 [ "original" => 0 "multimedia" => array:7 [ "identificador" => "fig0005" "etiqueta" => "Figure 1" "tipo" => "MULTIMEDIAFIGURA" "mostrarFloat" => true "mostrarDisplay" => false "figura" => array:1 [ 0 => array:4 [ "imagen" => "gr1.jpeg" "Alto" => 748 "Ancho" => 1314 "Tamanyo" => 56132 ] ] "descripcion" => array:1 [ "en" => "<p id="spar0015" class="elsevierStyleSimplePara elsevierViewall">Yeast strains tested with multiplex PhR, ITS3 and NL4. 1–7 <span class="elsevierStyleItalic">P. rhodozyma</span>: (1) CBS 5905 (Lineage C1/B); (2) CBS 7918<span class="elsevierStyleSup">T</span> (Lineage C2); (3) CRUB 1149 (Lineage A); (4) ATCC 24229 (Lineage D); (5) ZP 938 (Lineage E); (6) ZP 875 (Lineage F); (7) ZP 874 (Lineage B); (8) TSN-67; (9) <span class="elsevierStyleItalic">C. capitatum</span> CBS 7420; (10) <span class="elsevierStyleItalic">C. macerans</span> CBS 2206; N – negative control (no template).</p>" ] ] ] "autores" => array:1 [ 0 => array:2 [ "autoresLista" => "Fernando Colabella, Diego Libkind" "autores" => array:2 [ 0 => array:2 [ "nombre" => "Fernando" "apellidos" => "Colabella" ] 1 => array:2 [ "nombre" => "Diego" "apellidos" => "Libkind" ] ] ] ] ] "idiomaDefecto" => "en" "EPUB" => "https://multimedia.elsevier.es/PublicationsMultimediaV1/item/epub/S0325754115001601?idApp=UINPBA00004N" "url" => "/03257541/0000004800000001/v1_201603250025/S0325754115001601/v1_201603250025/en/main.assets" ] "en" => array:20 [ "idiomaDefecto" => true "cabecera" => "<span class="elsevierStyleTextfn">Original article</span>" "titulo" => "An acidic sphingomyelinase Type C activity from <span class="elsevierStyleItalic">Mycobacterium tuberculosis</span>" "tieneTextoCompleto" => true "paginas" => array:1 [ 0 => array:2 [ "paginaInicial" => "21" "paginaFinal" => "26" ] ] "autores" => array:1 [ 0 => array:4 [ "autoresLista" => "Jorge Castro-Garza, Francisco González-Salazar, Frederick D. 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Mycobacterial cell fractions (2<span class="elsevierStyleHsp" style=""></span>μg of total protein) were tested for SMase activity at incubation time points between 0 and 60<span class="elsevierStyleHsp" style=""></span>min. Symbols represent the mean<span class="elsevierStyleHsp" style=""></span>±<span class="elsevierStyleHsp" style=""></span>SE of [<span class="elsevierStyleSup">14</span>C]-phosphorylcholine released by <span class="elsevierStyleItalic">M. tuberculosis</span> whole cell extracts of strains H37Rv (○) or CDC1551 (●) in three independent experiments performed in triplicate.</p>" ] ] ] "textoCompleto" => "<span class="elsevierStyleSections"><span id="sec0005" class="elsevierStyleSection elsevierViewall"><span class="elsevierStyleSectionTitle" id="sect0025">Introduction</span><p id="par0005" class="elsevierStylePara elsevierViewall">Sphingomyelinases (EC 3.1.4.12) (SMases) are enzymes that catalyze the hydrolysis of sphingomyelin to ceramide and phosphorylcholine<a class="elsevierStyleCrossRef" href="#bib0155"><span class="elsevierStyleSup">5</span></a>. Sphingolipids are ubiquitous molecules of eukaryotic cell membranes and constitute a significant portion of membrane lipids<a class="elsevierStyleCrossRef" href="#bib0170"><span class="elsevierStyleSup">8</span></a>. Sphingolipids have been shown to be involved in a variety of physiological functions including regulation of cell growth, differentiation, cell migration, adhesion, inflammation and apoptosis<a class="elsevierStyleCrossRefs" href="#bib0140"><span class="elsevierStyleSup">2,22,24</span></a>.</p><p id="par0010" class="elsevierStylePara elsevierViewall">SMases are classified into sub-type C and D based on the site of sphingomyelin hydrolysis and on the resulting products. Products from SMase C are ceramide and phosphorylcholine while SMase D degrades sphingomyelin to phosphoryl ceramide and choline<a class="elsevierStyleCrossRefs" href="#bib0185"><span class="elsevierStyleSup">11,19</span></a>. Furthermore, SMases are classified by their optimum pH activity and localization into (a) acid SMase (A-SMase), (b) secretory Zn<span class="elsevierStyleSup">2+</span>-dependent SMase (A-sSMase), (c) neutral Mg<span class="elsevierStyleSup">2+</span>-dependent SMase (N-SMase), (d) neutral Mg<span class="elsevierStyleSup">2+</span>-independent SMase (N-iSMase), (e) alkaline SMase, and (f) bacterial SMase-phospholipase C<a class="elsevierStyleCrossRef" href="#bib0155"><span class="elsevierStyleSup">5</span></a>. Several species of bacteria, viruses, and even parasites infect host cells by exploiting either the A-SMase or the N-SMase-ceramide system, or both<a class="elsevierStyleCrossRefs" href="#bib0145"><span class="elsevierStyleSup">3,6,14</span></a>. Alternatively, it has been shown that cellular stress produces plasma secretion of A-SMases resulting in the formation of membrane-embedded, ceramide-enriched lipid rafts and the reorganization of receptor complexes leading to signaling events and regulation of a cellular phenotype that protect mice in a sepsis model<a class="elsevierStyleCrossRef" href="#bib0175"><span class="elsevierStyleSup">9</span></a>.</p><p id="par0015" class="elsevierStylePara elsevierViewall">Moreover, several pathogenic bacteria produce SMases with direct activity on target host cells. These virulence factors include <span class="elsevierStyleItalic">Clostridium perfringens</span> alpha-toxin, possessing phospholipase C-SMase and biological activities causing hemolysis, dermonecrosis and potentially death of the host<a class="elsevierStyleCrossRef" href="#bib0230"><span class="elsevierStyleSup">20</span></a>. Inhibition of <span class="elsevierStyleItalic">Bacillus cereus</span> SMase prevented mortality in mice<a class="elsevierStyleCrossRef" href="#bib0205"><span class="elsevierStyleSup">15</span></a>. Phospholipase C/SMase from the opportunistic pathogen <span class="elsevierStyleItalic">Pseudomonas aeruginosa</span> has hemolytic activity and is selectively cytotoxic to mammalian endothelial cells<a class="elsevierStyleCrossRef" href="#bib0245"><span class="elsevierStyleSup">23</span></a>.</p><p id="par0020" class="elsevierStylePara elsevierViewall"><span class="elsevierStyleItalic">Staphylococcus aureus</span> invades non-professional phagocytes, eventually resides in acidified phago-endosomes and subsequently escapes from this compartment due to a synergistic activity of the cytolytic peptide, staphylococcal δ-toxin and the SMase β-toxin<a class="elsevierStyleCrossRef" href="#bib0150"><span class="elsevierStyleSup">4</span></a>. A similar effect has been reported for the <span class="elsevierStyleItalic">C. perfringens</span> SMase activity<a class="elsevierStyleCrossRef" href="#bib0255"><span class="elsevierStyleSup">25</span></a>.</p><p id="par0025" class="elsevierStylePara elsevierViewall">We previously reported the identification of alkaline, neutral, and acidic SMase activities from <span class="elsevierStyleItalic">Mycobacterium tuberculosis</span><a class="elsevierStyleCrossRef" href="#bib0260"><span class="elsevierStyleSup">26</span></a>. In the present study, we report a novel acidic mycobacterial Zn<span class="elsevierStyleSup">2+</span>-dependent SMase activity.</p></span><span id="sec0010" class="elsevierStyleSection elsevierViewall"><span class="elsevierStyleSectionTitle" id="sect0030">Materials and methods</span><span id="sec0015" class="elsevierStyleSection elsevierViewall"><span class="elsevierStyleSectionTitle" id="sect0035">Mycobacterial whole cell extracts</span><p id="par0030" class="elsevierStylePara elsevierViewall"><span class="elsevierStyleItalic">M. tuberculosis</span> strains H37Rv and CDC1551 were grown in Middlebroook 7H9 medium supplemented with OADC for 10 days at 37<span class="elsevierStyleHsp" style=""></span>°C. Cultures were harvested by centrifugation at 3000<span class="elsevierStyleHsp" style=""></span>×<span class="elsevierStyleHsp" style=""></span><span class="elsevierStyleItalic">g</span> for 15<span class="elsevierStyleHsp" style=""></span>min. An approximate mass of 800<span class="elsevierStyleHsp" style=""></span>mg (wet weight) in 1<span class="elsevierStyleHsp" style=""></span>ml of PBS plus 0.5<span class="elsevierStyleHsp" style=""></span>% Triton X100 was gamma-irradiated for 48<span class="elsevierStyleHsp" style=""></span>h in a Gammacell 3000 equipment (Elan, Nordion International Inc, Kanata, Ontario, Canada). The gamma-irradiated bacteria were washed twice with PBS and resuspended in 1<span class="elsevierStyleHsp" style=""></span>ml of sodium acetate-buffer solution 100<span class="elsevierStyleHsp" style=""></span>mM pH 5.5 (ABS) with 0.5<span class="elsevierStyleHsp" style=""></span>% Triton X-100 (Sigma Chemical Co., St. Louis, MO) and Boehringer Manheim complete EDTA-free protease inhibitor cocktail (cat. No. 1836-170). Bacterial suspensions were disrupted with the Fast Prep System (MP Biomedicals, Santa Ana, CA) by applying 5 cycles of 1<span class="elsevierStyleHsp" style=""></span>min at setting 6, with 2<span class="elsevierStyleHsp" style=""></span>min resting on ice in between runs. The whole cell extracts were centrifuged twice at 16<span class="elsevierStyleHsp" style=""></span>000<span class="elsevierStyleHsp" style=""></span>×<span class="elsevierStyleHsp" style=""></span><span class="elsevierStyleItalic">g</span> for 8<span class="elsevierStyleHsp" style=""></span>min at room temperature, to remove intact bacterial cells. The preparations were divided into 200<span class="elsevierStyleHsp" style=""></span>μl aliquots and stored at −70<span class="elsevierStyleHsp" style=""></span>°C until used. Protein quantification was done by the method proposed by Lowry et al.<a class="elsevierStyleCrossRef" href="#bib0195"><span class="elsevierStyleSup">13</span></a>.</p></span><span id="sec0020" class="elsevierStyleSection elsevierViewall"><span class="elsevierStyleSectionTitle" id="sect0040">Sphingomyelinase activity</span><p id="par0035" class="elsevierStylePara elsevierViewall">The measurement of SMase was based on the method described by Vargas-Villarreal <span class="elsevierStyleItalic">et al</span>.<a class="elsevierStyleCrossRef" href="#bib0260"><span class="elsevierStyleSup">26</span></a>, with minor modifications. The mycobacterial whole cell extracts were the sources of enzymes for all the described experiments. In brief, 2.5<span class="elsevierStyleHsp" style=""></span>μCi of [N-methyl-<span class="elsevierStyleSup">14</span>C]-sphingomyelin (47<span class="elsevierStyleHsp" style=""></span>mCi/mmol; PerkinElmer Life and Analytical Science, Boston, MA) was mixed with 200<span class="elsevierStyleHsp" style=""></span>mM sodium acetate (pH 5.5), 2<span class="elsevierStyleHsp" style=""></span>mM Zn<span class="elsevierStyleSup">2+</span>, 4<span class="elsevierStyleHsp" style=""></span>% Triton X-100 and 0.3<span class="elsevierStyleHsp" style=""></span>mM sphingomyelin. The mixtures were sonicated in an Ultratip Labsonic System (Lab-Line Instrument Inc., Melrose Park IL), applying one pulse of 40<span class="elsevierStyleHsp" style=""></span>W/min. The assays were performed by mixing 10<span class="elsevierStyleHsp" style=""></span>μl of the substrate preparation with 10<span class="elsevierStyleHsp" style=""></span>μl of whole cell extract suspension of <span class="elsevierStyleItalic">M. tuberculosis</span> strain H37Rv or CDC-1551 containing 0–2<span class="elsevierStyleHsp" style=""></span>μg of total protein. The mixture was vortexed for 10<span class="elsevierStyleHsp" style=""></span>sec and incubated at 37<span class="elsevierStyleHsp" style=""></span>°C for 60<span class="elsevierStyleHsp" style=""></span>min. The reaction was stopped by adding 25<span class="elsevierStyleHsp" style=""></span>μl of 1<span class="elsevierStyleHsp" style=""></span>mg/ml sphingomyelin, 1<span class="elsevierStyleHsp" style=""></span>mg/ml phosphorylcholine and 1<span class="elsevierStyleHsp" style=""></span>mg/ml choline (Sigma Chemical Co., St. Louis, MO) in 5<span class="elsevierStyleHsp" style=""></span>% trichloroacetic acid in n-butanol. Non-digested sphingomyelin was separated from the SMase hydrolysis products (phosphorylcholine and choline) by thin-layer chromatography (TLC). The assay mixtures were applied (45<span class="elsevierStyleHsp" style=""></span>μl) on 10<span class="elsevierStyleHsp" style=""></span>cm<span class="elsevierStyleHsp" style=""></span>×<span class="elsevierStyleHsp" style=""></span>10<span class="elsevierStyleHsp" style=""></span>cm silica-gel plates (0.25-mm thickness, 60-mesh; Merck, Germany). The plates were placed in a TLC tank containing chloroform:methanol:water (65:25:4, v/v) as a mobile phase. The tank was loaded 15<span class="elsevierStyleHsp" style=""></span>min before starting each chromatographic run with the mobile phase-vapors to saturate the internal atmosphere. The spots corresponding to choline and phosphorylcholine (origin), and sphingomyelin (<span class="elsevierStyleItalic">R</span><span class="elsevierStyleInf">f</span><span class="elsevierStyleHsp" style=""></span>=<span class="elsevierStyleHsp" style=""></span>0.29) were developed by exposing the TLC plates to iodine vapors<a class="elsevierStyleCrossRef" href="#bib0235"><span class="elsevierStyleSup">21</span></a>. To identify [<span class="elsevierStyleSup">14</span>C]-sphingomyelin, [<span class="elsevierStyleSup">14</span>C]-phosphorylcholine and [<span class="elsevierStyleSup">14</span>C]-choline spots, their respective relative migration coefficients (<span class="elsevierStyleItalic">R</span><span class="elsevierStyleInf">f</span>) were compared with those of their corresponding non-radioactive standards (Sigma Chemical Co., St. Louis, Mo.) obtained in the same run. The two spots corresponding to [<span class="elsevierStyleSup">14</span>C]-sphingomyelin, and [<span class="elsevierStyleSup">14</span>C]-phosphorylcholine and [<span class="elsevierStyleSup">14</span>C]-choline mixed were scraped from the TLC silica gel plates and placed into plastic vials containing 5<span class="elsevierStyleHsp" style=""></span>ml scintillation liquid (BCS, Biodegradable Counting Scintillation; Amersham International Corporation, Buckinghamshire, England). Radioactivity in each vial was determined with a 1600 Tri-Carb liquid scintillation spectrometer (Packard Instrument Company, Inc., Downers Grove, IL). The device was adjusted to work with unquenched samples at 96<span class="elsevierStyleHsp" style=""></span>% efficiency. One unit of SMase activity was defined as 1<span class="elsevierStyleHsp" style=""></span>pmole of [<span class="elsevierStyleSup">14</span>C]-sphingomyelin hydrolyzed (equivalent to picomoles of [<span class="elsevierStyleSup">14</span>C]-phosphorylcholine plus [<span class="elsevierStyleSup">14</span>C]-choline released) in 1<span class="elsevierStyleHsp" style=""></span>h of incubation. The specific activity was given as the number of units of SMase activity per mg of total mycobacterial proteins. The classification of the SMase type was determined as described previously<a class="elsevierStyleCrossRef" href="#bib0260"><span class="elsevierStyleSup">26</span></a>.</p></span><span id="sec0025" class="elsevierStyleSection elsevierViewall"><span class="elsevierStyleSectionTitle" id="sect0045">Effect of incubation time, dose, pH and cations on sphingomyelinase activity</span><p id="par0040" class="elsevierStylePara elsevierViewall"><span class="elsevierStyleItalic">Time</span>: The rate of hydrolysis of sphingomyelin was determined at various time points over 60<span class="elsevierStyleHsp" style=""></span>min by mixing 10<span class="elsevierStyleHsp" style=""></span>μl of whole mycobacterial extracts (containing 2<span class="elsevierStyleHsp" style=""></span>μg of total proteins) with 2.5<span class="elsevierStyleHsp" style=""></span>μCi [N-methyl-<span class="elsevierStyleSup">14</span>C]-sphingomyelin (final concentration 1<span class="elsevierStyleHsp" style=""></span>mg/ml) dissolved in 20<span class="elsevierStyleHsp" style=""></span>μl of 200<span class="elsevierStyleHsp" style=""></span>mM sodium acetate buffer (pH 5.5), 1<span class="elsevierStyleHsp" style=""></span>mM Zn<span class="elsevierStyleSup">2+</span>, and 0.2<span class="elsevierStyleHsp" style=""></span>% Triton X-100.</p><p id="par0045" class="elsevierStylePara elsevierViewall"><span class="elsevierStyleItalic">Dose</span>: Variable amounts of protein from whole cell extracts from strains H37Rv and CDC1551 were assayed by mixing 10<span class="elsevierStyleHsp" style=""></span>μl of whole mycobacterial extracts (containing various concentrations of protein between 0 and 2<span class="elsevierStyleHsp" style=""></span>μg) with 2.5<span class="elsevierStyleHsp" style=""></span>μCi [N-methyl-<span class="elsevierStyleSup">14</span>C]-sphingomyelin (final concentration 1<span class="elsevierStyleHsp" style=""></span>mg/ml) dissolved in 20<span class="elsevierStyleHsp" style=""></span>μl of 200<span class="elsevierStyleHsp" style=""></span>mM sodium acetate buffer (pH 5.5), 1<span class="elsevierStyleHsp" style=""></span>mM Zn<span class="elsevierStyleSup">2+</span>, and 0.2<span class="elsevierStyleHsp" style=""></span>% Triton X-100 at 37<span class="elsevierStyleHsp" style=""></span>°C, for 1<span class="elsevierStyleHsp" style=""></span>h at pH 5.5.</p><p id="par0125" class="elsevierStylePara elsevierViewall"><span class="elsevierStyleItalic">pH</span>: pH values were adjusted with appropriate proportions of sodium acetate (pH 3–6) or Trizma-Base (pH 7–10) buffers to mycobacterial extracts containing 2<span class="elsevierStyleHsp" style=""></span>μg of total protein and incubated for 1<span class="elsevierStyleHsp" style=""></span>h at 37<span class="elsevierStyleHsp" style=""></span>°C prior to running the standard assay as described above.</p><p id="par0050" class="elsevierStylePara elsevierViewall"><span class="elsevierStyleItalic">Cations</span>: Each metal cation was assayed separately. Assay mixtures with 1 or 10<span class="elsevierStyleHsp" style=""></span>mM MgCl<span class="elsevierStyleInf">2</span>, CaCl<span class="elsevierStyleInf">2</span>, ZnSO<span class="elsevierStyleInf">4</span>, HgCl<span class="elsevierStyleInf">2</span>, MnCl<span class="elsevierStyleInf">2</span>, or CoCl<span class="elsevierStyleInf">2</span>, or with 1 or 10<span class="elsevierStyleHsp" style=""></span>mM of chelating agent EDTA (Sigma Chemical Co., St. Louis, MO) were incubated for 1<span class="elsevierStyleHsp" style=""></span>h at 37<span class="elsevierStyleHsp" style=""></span>°C to run the standard assay as described above.</p></span><span id="sec0030" class="elsevierStyleSection elsevierViewall"><span class="elsevierStyleSectionTitle" id="sect0050">Statistics</span><p id="par0055" class="elsevierStylePara elsevierViewall">All the experiments were performed by triplicate (<span class="elsevierStyleItalic">n</span><span class="elsevierStyleHsp" style=""></span>=<span class="elsevierStyleHsp" style=""></span>9). Slopes of the effect of incubation time and dose of mycobacterial extracts were calculated by linear regression. Statistical significance was determined using ANOVA.</p></span></span><span id="sec0035" class="elsevierStyleSection elsevierViewall"><span class="elsevierStyleSectionTitle" id="sect0055">Results</span><span id="sec0040" class="elsevierStyleSection elsevierViewall"><span class="elsevierStyleSectionTitle" id="sect0060">Dependence of mycobacterial sphingomyelinase on incubation time and dose of extracts</span><p id="par0060" class="elsevierStylePara elsevierViewall">The SMase activity of both H37Rv and CDC1551 strains increased linearly between 0 and 60<span class="elsevierStyleHsp" style=""></span>min of incubation (<span class="elsevierStyleItalic">r</span><span class="elsevierStyleSup">2</span><span class="elsevierStyleHsp" style=""></span>=<span class="elsevierStyleHsp" style=""></span>0.99). After 60<span class="elsevierStyleHsp" style=""></span>min of incubation 37.89<span class="elsevierStyleHsp" style=""></span>pmol and 16.1<span class="elsevierStyleHsp" style=""></span>pmol of [<span class="elsevierStyleSup">14</span>C]-phosphorylcholine were recovered from chromatography plates of H37Rv and CDC1551, respectively (<a class="elsevierStyleCrossRef" href="#fig0005">Fig. 1</a>).</p><elsevierMultimedia ident="fig0005"></elsevierMultimedia><p id="par0065" class="elsevierStylePara elsevierViewall">The amount of [<span class="elsevierStyleSup">14</span>C]-phosphorylcholine released from [<span class="elsevierStyleSup">14</span>C]-sphingomyelin also increased linearly as a function of extract dose of both strains (<span class="elsevierStyleItalic">r</span><span class="elsevierStyleSup">2</span><span class="elsevierStyleHsp" style=""></span>=<span class="elsevierStyleHsp" style=""></span>0.99). After 60<span class="elsevierStyleHsp" style=""></span>min incubation with 2<span class="elsevierStyleHsp" style=""></span>μg of total protein extract 37.94<span class="elsevierStyleHsp" style=""></span>pmol and 17.65<span class="elsevierStyleHsp" style=""></span>pmol of [<span class="elsevierStyleSup">14</span>C]-phosphorylcholine were recovered from strains H37Rv and CDC1551, respectively (<a class="elsevierStyleCrossRef" href="#fig0010">Fig. 2</a>). Those values corresponded to a specific SMase activity of 37.9<span class="elsevierStyleHsp" style=""></span>×<span class="elsevierStyleHsp" style=""></span>10<span class="elsevierStyleSup">6</span><span class="elsevierStyleHsp" style=""></span>U/mg/h for H37Rv, which was 2.15 times higher (<span class="elsevierStyleItalic">p</span><span class="elsevierStyleHsp" style=""></span><<span class="elsevierStyleHsp" style=""></span>0.001) than that of CDC1551 (17.6<span class="elsevierStyleHsp" style=""></span>×<span class="elsevierStyleHsp" style=""></span>10<span class="elsevierStyleSup">6</span><span class="elsevierStyleHsp" style=""></span>U/mg/h).</p><elsevierMultimedia ident="fig0010"></elsevierMultimedia></span><span id="sec0045" class="elsevierStyleSection elsevierViewall"><span class="elsevierStyleSectionTitle" id="sect0065">Dependence on pH</span><p id="par0070" class="elsevierStylePara elsevierViewall">Assayed extracts from both strains produced one primary peak of SMase activity at pH 5.5, which corresponds to the maximum specific activity. The peak from strain H37Rv extracts was almost three times higher than that of strain CDC1551 (<a class="elsevierStyleCrossRef" href="#fig0015">Fig. 3</a>).</p><elsevierMultimedia ident="fig0015"></elsevierMultimedia></span><span id="sec0050" class="elsevierStyleSection elsevierViewall"><span class="elsevierStyleSectionTitle" id="sect0070">Effect of divalent cations</span><p id="par0075" class="elsevierStylePara elsevierViewall">The SMase activity detected in whole cell extracts with no extra reagents added was defined as the basal activity equivalent to 100<span class="elsevierStyleHsp" style=""></span>%. Subsequently, the activity was stimulated at different rates by 1 and 10<span class="elsevierStyleHsp" style=""></span>mM concentration of cations Co<span class="elsevierStyleSup">2+</span>, Mn<span class="elsevierStyleSup">2+</span>, Zn<span class="elsevierStyleSup">2+</span>, and Mg<span class="elsevierStyleSup">2+</span> or inhibited by Hg<span class="elsevierStyleSup">2+</span> and Ca<span class="elsevierStyleSup">2+</span>. As expected, chelating agent EDTA resulted in the greatest inhibition: 96–99<span class="elsevierStyleHsp" style=""></span>% for strain H37Rv and 75–77<span class="elsevierStyleHsp" style=""></span>% for CDC1551. Results are shown in <a class="elsevierStyleCrossRef" href="#tbl0005">Table 1</a>.</p><elsevierMultimedia ident="tbl0005"></elsevierMultimedia></span></span><span id="sec0055" class="elsevierStyleSection elsevierViewall"><span class="elsevierStyleSectionTitle" id="sect0075">Discussion</span><p id="par0080" class="elsevierStylePara elsevierViewall">Sphingomyelinases (SMases) are enzymes produced by mammalian cells; their main activity is considered to occur during digestive processes. However, these enzymes and the products derived from the hydrolysis of the substrate sphingomyelin are involved in many other cell regulating functions such as cell growth, differentiation, cell migration, adhesion, inflammation and apoptosis<a class="elsevierStyleCrossRefs" href="#bib0140"><span class="elsevierStyleSup">2,22,24</span></a>. A more recent interest in the enzymes has been in their protective roles against pathogens by inhibiting invasion, regulating cytokine responses and inducing apoptosis in infected host cells<a class="elsevierStyleCrossRef" href="#bib0165"><span class="elsevierStyleSup">7</span></a>.</p><p id="par0085" class="elsevierStylePara elsevierViewall">Pathogens also produce SMases and they may act as virulence factors as demonstrated with <span class="elsevierStyleItalic">C. perfringens</span><a class="elsevierStyleCrossRef" href="#bib0230"><span class="elsevierStyleSup">20</span></a>, <span class="elsevierStyleItalic">B. cereus</span><a class="elsevierStyleCrossRef" href="#bib0205"><span class="elsevierStyleSup">15</span></a>, <span class="elsevierStyleItalic">P. aeruginosa</span><a class="elsevierStyleCrossRef" href="#bib0245"><span class="elsevierStyleSup">23</span></a>, <span class="elsevierStyleItalic">Listeria spp.</span><a class="elsevierStyleCrossRef" href="#bib0215"><span class="elsevierStyleSup">17</span></a> and <span class="elsevierStyleItalic">S. aureus</span><a class="elsevierStyleCrossRef" href="#bib0150"><span class="elsevierStyleSup">4</span></a>. Previously, we reported the identification of acidic, alkaline and neutral SMases activities in <span class="elsevierStyleItalic">M. tuberculosis</span>. In that report, neutral SMase had the highest activity and was stimulated by Mg<span class="elsevierStyleSup">2+</span>, Mn<span class="elsevierStyleSup">2+</span> and Co<span class="elsevierStyleSup">2+</span> while acidic and alkaline SMase activities remained uncharacterized<a class="elsevierStyleCrossRef" href="#bib0260"><span class="elsevierStyleSup">26</span></a>. Therefore, in this study, <span class="elsevierStyleItalic">M. tuberculosis</span> acidic SMase activity is further characterized. Maximal SMase activity occurs in a pH range from 5 to 6, with optimal activity at pH at 5.5 (<a class="elsevierStyleCrossRef" href="#fig0015">Fig. 3</a>). The assay for cation-induced SMase activity at pH 5.5 resulted in elevated activities by Co<span class="elsevierStyleSup">2+</span>, Mn<span class="elsevierStyleSup">2+</span>, Zn<span class="elsevierStyleSup">2+</span>, and to a lesser extent Mg<span class="elsevierStyleSup">2+</span> (<a class="elsevierStyleCrossRef" href="#tbl0005">Table 1</a>). Zn<span class="elsevierStyleSup">2+</span>-inducible activity was not previously reported for <span class="elsevierStyleItalic">M. tuberculosis</span>. Thus, it can be classified as Zn<span class="elsevierStyleSup">2+</span>-dependent acidic SMase activity. By comparing its biochemical characteristics with other SMases, it may correspond to a secretory acidic SMase. In mammalian hosts, this type of SMase regulates immune response<a class="elsevierStyleCrossRef" href="#bib0180"><span class="elsevierStyleSup">10</span></a>. In this instance, the presence of exogenous SMase produced by <span class="elsevierStyleItalic">M. tuberculosis</span> during infection may interfere with the normal host inflammatory response thus allowing for the establishment of infection and the development of disease. Recently, Roca and Ramakrishnan<a class="elsevierStyleCrossRef" href="#bib0220"><span class="elsevierStyleSup">18</span></a> observed that the chemical blockade of host acid SMase activity prevents TNF-induced macrophage programmed necroptosis and mycobacterial release from the cell. Thus, <span class="elsevierStyleItalic">M. tuberculosis</span> acidic SMase may aid in macrophage escape through the induction of necroptosis by its enzymatic product ceramide.</p><p id="par0090" class="elsevierStylePara elsevierViewall">Presently, <span class="elsevierStyleItalic">M. tuberculosis</span> genome annotation in Tuberculist predicts that the protein products from genes <span class="elsevierStyleItalic">plcA</span> (<span class="elsevierStyleItalic">Rv2351c</span>), <span class="elsevierStyleItalic">plcB</span> (<span class="elsevierStyleItalic">Rv2350c</span>), <span class="elsevierStyleItalic">plcC</span> (<span class="elsevierStyleItalic">Rv2349c</span>), and <span class="elsevierStyleItalic">plcD</span> (<span class="elsevierStyleItalic">Rv1755c</span>, reported as a fragment) have phospholipase and SMase activities and they are noted as virulence factors implicated in the pathogenesis of <span class="elsevierStyleItalic">M. tuberculosis</span> at the level of intracellular survival by the alteration of cell signaling events or by direct cytotoxicity<a class="elsevierStyleCrossRefs" href="#bib0190"><span class="elsevierStyleSup">12,16</span></a>. Several bacterial phospholipases possess both phospholipase and SMase activities; an example is the alpha-toxin from the pathogen <span class="elsevierStyleItalic">C. perfringens</span><a class="elsevierStyleCrossRef" href="#bib0230"><span class="elsevierStyleSup">20</span></a>. The activity we have identified in this study may correspond to <span class="elsevierStyleItalic">M. tuberculosis</span> phospholipase C expressed from gene <span class="elsevierStyleItalic">plcC</span> (<span class="elsevierStyleItalic">Rv2349c</span>) because the proteins from the <span class="elsevierStyleItalic">plcA</span> and <span class="elsevierStyleItalic">plcB</span> genes are membrane-associated phospholipases C. Furthermore, another report where <span class="elsevierStyleItalic">M. smegmatis</span> was used as an expression system of four <span class="elsevierStyleItalic">M. tuberculosis plc</span> genes supports this idea; <span class="elsevierStyleItalic">plcC</span> product was found to be the most active of the four recombinant PLCs under acidic conditions<a class="elsevierStyleCrossRef" href="#bib0135"><span class="elsevierStyleSup">1</span></a>. However, a novel <span class="elsevierStyleItalic">M. tuberculosis</span> enzyme with SMase activity cannot be ruled out. More research work is needed to make this determination.</p><p id="par0095" class="elsevierStylePara elsevierViewall">Sphingomyelinases (SMases) and their hydrolysis products (ceramide and phosphorylcholine) are regulators of a multitude of cellular processes. Several pathogens produce SMases as virulence factors. In the present study, we report a novel acidic mycobacterial Zn<span class="elsevierStyleSup">2+</span>-dependent SMase activity. The relevance of this enzyme in tuberculosis pathogenesis remains to be elucidated.</p></span><span id="sec0060" class="elsevierStyleSection elsevierViewall"><span class="elsevierStyleSectionTitle" id="sect0080">Ethical disclosures</span><span id="sec0065" class="elsevierStyleSection elsevierViewall"><span class="elsevierStyleSectionTitle" id="sect0085">Protection of human and animal subjects</span><p id="par0100" class="elsevierStylePara elsevierViewall">The authors declare that no experiments were performed on humans or animals for this investigation.</p></span><span id="sec0070" class="elsevierStyleSection elsevierViewall"><span class="elsevierStyleSectionTitle" id="sect0090">Confidentiality of data</span><p id="par0105" class="elsevierStylePara elsevierViewall">The authors declare that no patient data appears in this article.</p></span><span id="sec0075" class="elsevierStyleSection elsevierViewall"><span class="elsevierStyleSectionTitle" id="sect0095">Right to privacy and informed consent</span><p id="par0110" class="elsevierStylePara elsevierViewall">The authors declare that no patient data appears in this article.</p></span></span><span id="sec0080" class="elsevierStyleSection elsevierViewall"><span class="elsevierStyleSectionTitle" id="sect0100">Conflict of interest</span><p id="par0115" class="elsevierStylePara elsevierViewall">The authors declare that they have no conflicts of interest.</p></span></span>" "textoCompletoSecciones" => array:1 [ "secciones" => array:12 [ 0 => array:3 [ "identificador" => "xres620791" "titulo" => "Abstract" "secciones" => array:1 [ 0 => array:1 [ "identificador" => "abst0005" ] ] ] 1 => array:2 [ "identificador" => "xpalclavsec635072" "titulo" => "Keywords" ] 2 => array:3 [ "identificador" => "xres620790" "titulo" => "Resumen" "secciones" => array:1 [ 0 => array:1 [ "identificador" => "abst0010" ] ] ] 3 => array:2 [ "identificador" => "xpalclavsec635071" "titulo" => "Palabras clave" ] 4 => array:2 [ "identificador" => "sec0005" "titulo" => "Introduction" ] 5 => array:3 [ "identificador" => "sec0010" "titulo" => "Materials and methods" "secciones" => array:4 [ 0 => array:2 [ "identificador" => "sec0015" "titulo" => "Mycobacterial whole cell extracts" ] 1 => array:2 [ "identificador" => "sec0020" "titulo" => "Sphingomyelinase activity" ] 2 => array:2 [ "identificador" => "sec0025" "titulo" => "Effect of incubation time, dose, pH and cations on sphingomyelinase activity" ] 3 => array:2 [ "identificador" => "sec0030" "titulo" => "Statistics" ] ] ] 6 => array:3 [ "identificador" => "sec0035" "titulo" => "Results" "secciones" => array:3 [ 0 => array:2 [ "identificador" => "sec0040" "titulo" => "Dependence of mycobacterial sphingomyelinase on incubation time and dose of extracts" ] 1 => array:2 [ "identificador" => "sec0045" "titulo" => "Dependence on pH" ] 2 => array:2 [ "identificador" => "sec0050" "titulo" => "Effect of divalent cations" ] ] ] 7 => array:2 [ "identificador" => "sec0055" "titulo" => "Discussion" ] 8 => array:3 [ "identificador" => "sec0060" "titulo" => "Ethical disclosures" "secciones" => array:3 [ 0 => array:2 [ "identificador" => "sec0065" "titulo" => "Protection of human and animal subjects" ] 1 => array:2 [ "identificador" => "sec0070" "titulo" => "Confidentiality of data" ] 2 => array:2 [ "identificador" => "sec0075" "titulo" => "Right to privacy and informed consent" ] ] ] 9 => array:2 [ "identificador" => "sec0080" "titulo" => "Conflict of interest" ] 10 => array:2 [ "identificador" => "xack209300" "titulo" => "Acknowledgements" ] 11 => array:1 [ "titulo" => "References" ] ] ] "pdfFichero" => "main.pdf" "tienePdf" => true "fechaRecibido" => "2015-08-07" "fechaAceptado" => "2016-01-15" "PalabrasClave" => array:2 [ "en" => array:1 [ 0 => array:4 [ "clase" => "keyword" "titulo" => "Keywords" "identificador" => "xpalclavsec635072" "palabras" => array:5 [ 0 => "<span class="elsevierStyleItalic">Mycobacterium tuberculosis</span>" 1 => "Tuberculosis" 2 => "Sphingomyelinase" 3 => "Virulence factors" 4 => "Mexico" ] ] ] "es" => array:1 [ 0 => array:4 [ "clase" => "keyword" "titulo" => "Palabras clave" "identificador" => "xpalclavsec635071" "palabras" => array:5 [ 0 => "<span class="elsevierStyleItalic">Mycobacterium tuberculosis</span>" 1 => "Tuberculosis" 2 => "Esfingomielinasa" 3 => "Factores de virulencia" 4 => "México" ] ] ] ] "tieneResumen" => true "resumen" => array:2 [ "en" => array:2 [ "titulo" => "Abstract" "resumen" => "<span id="abst0005" class="elsevierStyleSection elsevierViewall"><p id="spar0005" class="elsevierStyleSimplePara elsevierViewall">Sphingomyelinases (SMases) catalyze the hydrolysis of sphingomyelin to ceramide and phosphorylcholine. Sphingolipids are recognized as diverse and dynamic regulators of a multitude of cellular processes mediating cell cycle control, differentiation, stress response, cell migration, adhesion, and apoptosis. Bacterial SMases are virulence factors for several species of pathogens. Whole cell extracts of <span class="elsevierStyleItalic">Mycobacterium tuberculosis</span> strains H37Rv and CDC1551 were assayed using [N-methyl-<span class="elsevierStyleSup">14</span>C]-sphingomyelin as substrate. Acidic Zn<span class="elsevierStyleSup">2+</span>-dependent SMase activity was identified in both strains. Peak SMase activity was observed at pH 5.5. Interestingly, overall SMase activity levels from CDC1551 extracts are approximately 1/3 of those of H37Rv. The presence of exogenous SMase produced by <span class="elsevierStyleItalic">M. tuberculosis</span> during infection may interfere with the normal host inflammatory response thus allowing the establishment of infection and disease development. This Type C activity is different from previously identified <span class="elsevierStyleItalic">M. tuberculosis</span> SMases. Defining the biochemical characteristics of <span class="elsevierStyleItalic">M. tuberculosis</span> SMases helps to elucidate the roles that these enzymes play during infection and disease.</p></span>" ] "es" => array:2 [ "titulo" => "Resumen" "resumen" => "<span id="abst0010" class="elsevierStyleSection elsevierViewall"><p id="spar0010" class="elsevierStyleSimplePara elsevierViewall">Las esfingomielinasas (SMasas) catalizan la hidrólisis de esfingomielina a ceramida y fosforilcolina. Los esfingolípidos son reconocidos como reguladores diversos y dinámicos de una multitud de procesos celulares que median en el control del ciclo celular, la diferenciación, la respuesta al estrés, la migración celular, la adhesión y la apoptosis. Las esfingomielinasas bacterianas son factores de virulencia reconocidos en varias especies de patógenos. En este trabajo se analizaron los extractos de células enteras de las cepas de <span class="elsevierStyleItalic">Mycobacterium tuberculosis</span> H37Rv y CDC1551 utilizando [N-metil-<span class="elsevierStyleSup">14</span>C]-esfingomielina como sustrato. Se identificó actividad de SMasa-ácida dependiente de zinc en ambas cepas. La actividad máxima se observó a pH 5.5. Curiosamente, los niveles de actividad de SMasa generados a partir de extractos de la cepa CDC1551 son aproximadamente un tercio de los de la cepa H37Rv. La presencia de una SMasa exógena producida por <span class="elsevierStyleItalic">M. tuberculosis</span> durante la infección puede interferir con la respuesta inflamatoria del huésped, permitiendo así el establecimiento de la infección y el desarrollo de la enfermedad. Esta actividad tipo C es distinta de las actividades previamente reportadas para <span class="elsevierStyleItalic">M. tuberculosis</span>. Definir las características bioquímicas de las esfingomielinasas de <span class="elsevierStyleItalic">M. tuberculosis</span> ayudará a dilucidar el papel que desempeñan estas enzimas durante la infección y la enfermedad.</p></span>" ] ] "multimedia" => array:4 [ 0 => array:7 [ "identificador" => "fig0005" "etiqueta" => "Figure 1" "tipo" => "MULTIMEDIAFIGURA" "mostrarFloat" => true "mostrarDisplay" => false "figura" => array:1 [ 0 => array:4 [ "imagen" => "gr1.jpeg" "Alto" => 1215 "Ancho" => 1229 "Tamanyo" => 73344 ] ] "descripcion" => array:1 [ "en" => "<p id="spar0015" class="elsevierStyleSimplePara elsevierViewall">Time-course of mycobacterial SMase activity. Mycobacterial cell fractions (2<span class="elsevierStyleHsp" style=""></span>μg of total protein) were tested for SMase activity at incubation time points between 0 and 60<span class="elsevierStyleHsp" style=""></span>min. Symbols represent the mean<span class="elsevierStyleHsp" style=""></span>±<span class="elsevierStyleHsp" style=""></span>SE of [<span class="elsevierStyleSup">14</span>C]-phosphorylcholine released by <span class="elsevierStyleItalic">M. tuberculosis</span> whole cell extracts of strains H37Rv (○) or CDC1551 (●) in three independent experiments performed in triplicate.</p>" ] ] 1 => array:7 [ "identificador" => "fig0010" "etiqueta" => "Figure 2" "tipo" => "MULTIMEDIAFIGURA" "mostrarFloat" => true "mostrarDisplay" => false "figura" => array:1 [ 0 => array:4 [ "imagen" => "gr2.jpeg" "Alto" => 1300 "Ancho" => 1246 "Tamanyo" => 76555 ] ] "descripcion" => array:1 [ "en" => "<p id="spar0020" class="elsevierStyleSimplePara elsevierViewall">Dose-dependence of <span class="elsevierStyleItalic">M. tuberculosis</span> SMase activity. Mycobacterial cell fractions containing 0–2<span class="elsevierStyleHsp" style=""></span>μg of protein per assay were tested. Symbols represent the mean<span class="elsevierStyleHsp" style=""></span>±<span class="elsevierStyleHsp" style=""></span>SE of [<span class="elsevierStyleSup">14</span>C]-phosphorylcholine released [in pmol] by <span class="elsevierStyleItalic">M. tuberculosis</span> whole cell extracts of strains H37Rv (○) or CDC1551 (●). All assays were performed at pH 5.5 with 1<span class="elsevierStyleHsp" style=""></span>h incubation.</p>" ] ] 2 => array:7 [ "identificador" => "fig0015" "etiqueta" => "Figure 3" "tipo" => "MULTIMEDIAFIGURA" "mostrarFloat" => true "mostrarDisplay" => false "figura" => array:1 [ 0 => array:4 [ "imagen" => "gr3.jpeg" "Alto" => 1203 "Ancho" => 1222 "Tamanyo" => 71619 ] ] "descripcion" => array:1 [ "en" => "<p id="spar0025" class="elsevierStyleSimplePara elsevierViewall">Effect of pH on mycobacterial SMase activity. Mycobacterial cell fractions containing 2<span class="elsevierStyleHsp" style=""></span>μg of proteins were tested at different pH levels (range 3–9). Symbols represent the means<span class="elsevierStyleHsp" style=""></span>±<span class="elsevierStyleHsp" style=""></span>SE of SMase specific activity of <span class="elsevierStyleItalic">M. tuberculosis</span> whole cell extracts of strains H37Rv (○) or CDC1551 (●).</p>" ] ] 3 => array:7 [ "identificador" => "tbl0005" "etiqueta" => "Table 1" "tipo" => "MULTIMEDIATABLA" "mostrarFloat" => true "mostrarDisplay" => false "tabla" => array:1 [ "tablatextoimagen" => array:1 [ 0 => array:2 [ "tabla" => array:1 [ 0 => """ <table border="0" frame="\n \t\t\t\t\tvoid\n \t\t\t\t" class=""><thead title="thead"><tr title="table-row"><th class="td" title="table-head " align="" valign="top" scope="col" style="border-bottom: 2px solid black"> \t\t\t\t\t\t\n \t\t\t\t</th><th class="td" title="table-head " colspan="4" align="center" valign="top" scope="col" style="border-bottom: 2px solid black"><span class="elsevierStyleItalic">Mycobacterium tuberculosis</span> strain</th></tr></thead><tbody title="tbody"><tr title="table-row"><td class="td" title="table-entry " align="" valign="top"> \t\t\t\t\t\t\n \t\t\t\t</td><td class="td" title="table-entry " colspan="2" align="center" valign="top" style="border-bottom: 2px solid black">H37Rv</td><td class="td" title="table-entry " colspan="2" align="center" valign="top" style="border-bottom: 2px solid black">CDC-1551</td></tr><tr title="table-row"><td class="td" title="table-entry " align="" valign="top"> \t\t\t\t\t\t\n \t\t\t\t</td><td class="td" title="table-entry " colspan="4" align="center" valign="top" style="border-bottom: 2px solid black">Concentration (mM)</td></tr><tr title="table-row"><td class="td" title="table-entry " align="" valign="top"> \t\t\t\t\t\t\n \t\t\t\t</td><td class="td" title="table-entry " align="left" valign="top">1.0 \t\t\t\t\t\t\n \t\t\t\t</td><td class="td" title="table-entry " align="left" valign="top">10.0 \t\t\t\t\t\t\n \t\t\t\t</td><td class="td" title="table-entry " align="left" valign="top">1.0 \t\t\t\t\t\t\n \t\t\t\t</td><td class="td" title="table-entry " align="left" valign="top">10.0 \t\t\t\t\t\t\n \t\t\t\t</td></tr><tr title="table-row"><td class="td" title="table-entry " align="" valign="top"> \t\t\t\t\t\t\n \t\t\t\t</td><td class="td" title="table-entry " colspan="4" align="center" valign="top" style="border-bottom: 2px solid black">Normalized specific SMase activity</td></tr><tr title="table-row"><td class="td-with-role" title="table-entry ; entry_with_role_rowhead " align="left" valign="top">None \t\t\t\t\t\t\n \t\t\t\t</td><td class="td" title="table-entry " align="char" valign="top">1.00 \t\t\t\t\t\t\n \t\t\t\t</td><td class="td" title="table-entry " align="char" valign="top">1.00 \t\t\t\t\t\t\n \t\t\t\t</td><td class="td" title="table-entry " align="char" valign="top">1.00 \t\t\t\t\t\t\n \t\t\t\t</td><td class="td" title="table-entry " align="char" valign="top">1.00 \t\t\t\t\t\t\n \t\t\t\t</td></tr><tr title="table-row"><td class="td-with-role" title="table-entry ; entry_with_role_rowhead " align="left" valign="top">EDTA \t\t\t\t\t\t\n \t\t\t\t</td><td class="td" title="table-entry " align="char" valign="top">0.04 \t\t\t\t\t\t\n \t\t\t\t</td><td class="td" title="table-entry " align="char" valign="top">0.01 \t\t\t\t\t\t\n \t\t\t\t</td><td class="td" title="table-entry " align="char" valign="top">0.23 \t\t\t\t\t\t\n \t\t\t\t</td><td class="td" title="table-entry " align="char" valign="top">0.25 \t\t\t\t\t\t\n \t\t\t\t</td></tr><tr title="table-row"><td class="td-with-role" title="table-entry ; entry_with_role_rowhead " align="left" valign="top">MgCl<span class="elsevierStyleInf">2</span> \t\t\t\t\t\t\n \t\t\t\t</td><td class="td" title="table-entry " align="char" valign="top">1.02 \t\t\t\t\t\t\n \t\t\t\t</td><td class="td" title="table-entry " align="char" valign="top">1.57 \t\t\t\t\t\t\n \t\t\t\t</td><td class="td" title="table-entry " align="char" valign="top">1.29 \t\t\t\t\t\t\n \t\t\t\t</td><td class="td" title="table-entry " align="char" valign="top">1.74 \t\t\t\t\t\t\n \t\t\t\t</td></tr><tr title="table-row"><td class="td-with-role" title="table-entry ; entry_with_role_rowhead " align="left" valign="top">MnCl<span class="elsevierStyleInf">2</span> \t\t\t\t\t\t\n \t\t\t\t</td><td class="td" title="table-entry " align="char" valign="top">1.86 \t\t\t\t\t\t\n \t\t\t\t</td><td class="td" title="table-entry " align="char" valign="top">1.88 \t\t\t\t\t\t\n \t\t\t\t</td><td class="td" title="table-entry " align="char" valign="top">1.75 \t\t\t\t\t\t\n \t\t\t\t</td><td class="td" title="table-entry " align="char" valign="top">2.01 \t\t\t\t\t\t\n \t\t\t\t</td></tr><tr title="table-row"><td class="td-with-role" title="table-entry ; entry_with_role_rowhead " align="left" valign="top">CoCl<span class="elsevierStyleInf">2</span> \t\t\t\t\t\t\n \t\t\t\t</td><td class="td" title="table-entry " align="char" valign="top">2.05 \t\t\t\t\t\t\n \t\t\t\t</td><td class="td" title="table-entry " align="char" valign="top">2.23 \t\t\t\t\t\t\n \t\t\t\t</td><td class="td" title="table-entry " align="char" valign="top">2.10 \t\t\t\t\t\t\n \t\t\t\t</td><td class="td" title="table-entry " align="char" valign="top">2.20 \t\t\t\t\t\t\n \t\t\t\t</td></tr><tr title="table-row"><td class="td-with-role" title="table-entry ; entry_with_role_rowhead " align="left" valign="top">CaCl<span class="elsevierStyleInf">2</span> \t\t\t\t\t\t\n \t\t\t\t</td><td class="td" title="table-entry " align="char" valign="top">1.02 \t\t\t\t\t\t\n \t\t\t\t</td><td class="td" title="table-entry " align="char" valign="top">0.67 \t\t\t\t\t\t\n \t\t\t\t</td><td class="td" title="table-entry " align="char" valign="top">1.08 \t\t\t\t\t\t\n \t\t\t\t</td><td class="td" title="table-entry " align="char" valign="top">0.77 \t\t\t\t\t\t\n \t\t\t\t</td></tr><tr title="table-row"><td class="td-with-role" title="table-entry ; entry_with_role_rowhead " align="left" valign="top">HgCl<span class="elsevierStyleInf">2</span> \t\t\t\t\t\t\n \t\t\t\t</td><td class="td" title="table-entry " align="char" valign="top">0.56 \t\t\t\t\t\t\n \t\t\t\t</td><td class="td" title="table-entry " align="char" valign="top">0.36 \t\t\t\t\t\t\n \t\t\t\t</td><td class="td" title="table-entry " align="char" valign="top">0.82 \t\t\t\t\t\t\n \t\t\t\t</td><td class="td" title="table-entry " align="char" valign="top">0.41 \t\t\t\t\t\t\n \t\t\t\t</td></tr><tr title="table-row"><td class="td-with-role" title="table-entry ; entry_with_role_rowhead " align="left" valign="top">ZnSO<span class="elsevierStyleInf">4</span> \t\t\t\t\t\t\n \t\t\t\t</td><td class="td" title="table-entry " align="char" valign="top">1.32 \t\t\t\t\t\t\n \t\t\t\t</td><td class="td" title="table-entry " align="char" valign="top">1.50 \t\t\t\t\t\t\n \t\t\t\t</td><td class="td" title="table-entry " align="char" valign="top">1.39 \t\t\t\t\t\t\n \t\t\t\t</td><td class="td" title="table-entry " align="char" valign="top">1.87 \t\t\t\t\t\t\n \t\t\t\t</td></tr></tbody></table> """ ] "imagenFichero" => array:1 [ 0 => "xTab1017862.png" ] ] ] ] "descripcion" => array:1 [ "en" => "<p id="spar0030" class="elsevierStyleSimplePara elsevierViewall">Normalized sphingomyelinase (SMase) activity modulated by cations or EDTA addition</p>" ] ] ] "bibliografia" => array:2 [ "titulo" => "References" "seccion" => array:1 [ 0 => array:2 [ "identificador" => "bibs0005" "bibliografiaReferencia" => array:26 [ 0 => array:3 [ "identificador" => "bib0135" "etiqueta" => "1" "referencia" => array:1 [ 0 => array:2 [ "contribucion" => array:1 [ 0 => array:2 [ "titulo" => "Evidence for the cytotoxic effects of <span class="elsevierStyleItalic">Mycobacterium tuberculosis</span> phospholipase C towards macrophages" "autores" => array:1 [ 0 => array:2 [ "etal" => false "autores" => array:5 [ 0 => "J.C. 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| Year/Month | Html | Total | |
|---|---|---|---|
| 2024 November | 1 | 0 | 1 |
| 2024 October | 15 | 12 | 27 |
| 2024 September | 22 | 15 | 37 |
| 2024 August | 17 | 18 | 35 |
| 2024 July | 9 | 4 | 13 |
| 2024 June | 19 | 11 | 30 |
| 2024 May | 14 | 4 | 18 |
| 2024 April | 23 | 10 | 33 |
| 2024 March | 33 | 12 | 45 |
| 2024 February | 24 | 1 | 25 |
| 2024 January | 12 | 5 | 17 |
| 2023 December | 18 | 3 | 21 |
| 2023 November | 13 | 7 | 20 |
| 2023 October | 19 | 12 | 31 |
| 2023 September | 8 | 1 | 9 |
| 2023 August | 14 | 6 | 20 |
| 2023 July | 13 | 7 | 20 |
| 2023 June | 18 | 3 | 21 |
| 2023 May | 38 | 6 | 44 |
| 2023 April | 21 | 5 | 26 |
| 2023 March | 24 | 0 | 24 |
| 2023 February | 26 | 10 | 36 |
| 2023 January | 26 | 10 | 36 |
| 2022 December | 22 | 7 | 29 |
| 2022 November | 30 | 8 | 38 |
| 2022 October | 19 | 13 | 32 |
| 2022 September | 19 | 6 | 25 |
| 2022 August | 14 | 8 | 22 |
| 2022 July | 13 | 14 | 27 |
| 2022 June | 13 | 13 | 26 |
| 2022 May | 9 | 5 | 14 |
| 2022 April | 13 | 14 | 27 |
| 2022 March | 19 | 16 | 35 |
| 2022 February | 13 | 4 | 17 |
| 2022 January | 8 | 9 | 17 |
| 2021 December | 27 | 15 | 42 |
| 2021 November | 17 | 9 | 26 |
| 2021 October | 18 | 11 | 29 |
| 2021 September | 14 | 11 | 25 |
| 2021 August | 19 | 4 | 23 |
| 2021 July | 14 | 13 | 27 |
| 2021 June | 16 | 10 | 26 |
| 2021 May | 18 | 9 | 27 |
| 2021 April | 63 | 24 | 87 |
| 2021 March | 55 | 21 | 76 |
| 2021 February | 18 | 16 | 34 |
| 2021 January | 15 | 14 | 29 |
| 2020 December | 18 | 11 | 29 |
| 2020 November | 22 | 11 | 33 |
| 2020 October | 23 | 6 | 29 |
| 2020 September | 25 | 12 | 37 |
| 2020 August | 21 | 9 | 30 |
| 2020 July | 22 | 11 | 33 |
| 2020 June | 18 | 9 | 27 |
| 2020 May | 19 | 9 | 28 |
| 2020 April | 27 | 6 | 33 |
| 2020 March | 17 | 11 | 28 |
| 2020 February | 23 | 11 | 34 |
| 2020 January | 17 | 5 | 22 |
| 2019 December | 36 | 7 | 43 |
| 2019 November | 15 | 1 | 16 |
| 2019 October | 20 | 8 | 28 |
| 2019 September | 29 | 3 | 32 |
| 2019 August | 12 | 6 | 18 |
| 2019 July | 15 | 12 | 27 |
| 2019 June | 27 | 24 | 51 |
| 2019 May | 58 | 37 | 95 |
| 2019 April | 36 | 6 | 42 |
| 2019 March | 8 | 2 | 10 |
| 2019 February | 10 | 2 | 12 |
| 2019 January | 11 | 2 | 13 |
| 2018 December | 16 | 7 | 23 |
| 2018 November | 15 | 10 | 25 |
| 2018 October | 28 | 27 | 55 |
| 2018 September | 10 | 6 | 16 |
| 2018 August | 10 | 7 | 17 |
| 2018 July | 7 | 3 | 10 |
| 2018 June | 10 | 3 | 13 |
| 2018 May | 4 | 11 | 15 |
| 2018 April | 4 | 8 | 12 |
| 2018 March | 5 | 1 | 6 |
| 2018 February | 3 | 0 | 3 |
| 2018 January | 9 | 1 | 10 |
| 2017 December | 3 | 3 | 6 |
| 2017 November | 17 | 1 | 18 |
| 2017 October | 3 | 4 | 7 |
| 2017 September | 7 | 3 | 10 |
| 2017 August | 9 | 2 | 11 |
| 2017 July | 14 | 2 | 16 |
| 2017 June | 18 | 4 | 22 |
| 2017 May | 21 | 11 | 32 |
| 2017 April | 17 | 14 | 31 |
| 2017 March | 18 | 56 | 74 |
| 2017 February | 18 | 6 | 24 |
| 2017 January | 23 | 8 | 31 |
| 2016 December | 35 | 7 | 42 |
| 2016 November | 29 | 9 | 38 |
| 2016 October | 54 | 10 | 64 |
| 2016 September | 45 | 7 | 52 |
| 2016 August | 29 | 7 | 36 |
| 2016 July | 41 | 8 | 49 |
| 2016 June | 63 | 30 | 93 |
| 2016 May | 52 | 36 | 88 |
| 2016 April | 36 | 29 | 65 |
| 2016 March | 2 | 2 | 4 |
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