RESUMEN
Rapid isolation and identification of pathogens is a major goal of diagnostic microbiology. In order to isolate and identify Staphylococcus aureus, a number of authors have used a variety of selective and/or differential culture media. However, to date, there are no reports comparing the efficacy of selective and differential culture media for S. aureus isolation from bovine mastitis cases using the 16S rRNA (rrs) gene sequence as a gold standard test. In the present study, we evaluated the efficacy of four selective and/or differential culture media for the isolation of S. aureus from milk samples collected from cows suffering from bovine mastitis. Four hundred and forty isolates were obtained using salt-mannitol agar (SMA, Bioxon), Staphylococcus-110 agar (S110, Bioxon), CHROMAgar Staph aureus (CSA, BD-BBL) and sheep's blood agar (SBA, BD-BBL). All bacterial isolates were identified by their typical colony morphology in the respective media, by secondary tests (for coagulase and ß-haemolysis) and by partial 16S rRNA (rrs) gene sequencing as a gold standard test. Sensitivity, positive predictive and negative predictive values were higher for SMA (86.96, 52.63 and 95.95%, respectively) compared with S110 (70.00, 23.73 and 90.91%, respectively), CSA (69.23, 28.13 and 95.74%, respectively) and SBA (68.75, 37.93 and 89.58%, respectively) while specificity values were similar for all media. Data indicated that the use of culture media for S. aureus isolation combined with determination of coagulase activity and haemolysis as secondary tests improved accuracy of the identification and was in accordance with rrs gene sequence-analysis compared with the use of the culture media alone.
Asunto(s)
Medios de Cultivo/química , Mastitis Bovina/microbiología , Infecciones Estafilocócicas/veterinaria , Staphylococcus aureus/clasificación , Staphylococcus aureus/aislamiento & purificación , Animales , Técnicas Bacteriológicas/veterinaria , Bovinos , Femenino , Datos de Secuencia Molecular , Valor Predictivo de las Pruebas , Sensibilidad y Especificidad , Infecciones Estafilocócicas/microbiologíaRESUMEN
Microbial fructosyltransferases are polymerases that are involved in microbial fructan (levan, inulin and fructo-oligosaccharide) biosynthesis. Structurally, microbial fructosyltransferase proteins share the catalytic domain of glycoside hydrolases 68 family and are grouped in seven phylogenetically related clusters. Fructosyltransferase-encoding genes are organized in operons or in clusters associated with other genes related to carbohydrate metabolism or fructosyltransferase secretion. Fructosyltransferase gene expression is mainly regulated by two-component systems or phosphorelay mechanisms that respond to sucrose availability or other environmental signals. Microbial fructans are involved in conferring resistance to environmental stress such as water deprivation, nutrient assimilation, biofilm formation, and as virulence factors in colonization. As a result of the biological and industrial importance of fructans, fructosyltransferases have been the subject of extensive research, conducted to improve their enzymatic activity or to elucidate their biological role in nature.
Asunto(s)
Bacterias/enzimología , Fructanos/biosíntesis , Hexosiltransferasas/química , Hexosiltransferasas/genética , Bacterias/genética , Bacterias/metabolismo , Metabolismo de los Hidratos de Carbono , Regulación de la Expresión Génica , OperónRESUMEN
Bacterial internalization is an important process in the pathogenesis of infectious diseases in which nuclear factor kappaB (NF-kappaB) plays a prominent role. We present pharmacological evidence indicating that in bovine endothelial cells (BEC) the internalization of Staphylococcus aureus, a pathogenic bacterium that causes mastitis in bovine cattle, was associated with the activation of NF-kappaB. The internalization of S. aureus increased when BEC were stimulated with alpha-tumour necrosis factor (TNF-alpha) or beta-interleukin 1 (IL-1beta) which are known activators of NF-kappaB. SN50 (an inhibitor peptide of NF-kappaB nuclear translocation) and BAY 11-7083 (a chemical that inhibits the IkappaBalpha phosphorylation) caused significant reduction in S. aureus intracellular number, indicating that its internalization was associated with the NF-kappaB activity. Furthermore, specific inhibition of c-Jun N-terminal kinase with SP600125 (SP) or p-38 with SB203580 (SB) did not cause any change in the S. aureus intracellular number compared with the untreated control. Finally, TNF-alpha treatment of BEC after the addition of both SP and SB, induced a significant increase in S. aureus internalization above the control value. These data indicate that NF-kappaB activity is associated with S. aureus internalization and suggest that this transcription factor may play a role in the pathophysiology of bovine mastitis caused by this bacterium.
Asunto(s)
Interleucina-1beta/inmunología , Mastitis Bovina/microbiología , FN-kappa B/inmunología , Infecciones Estafilocócicas/veterinaria , Staphylococcus aureus/inmunología , Factor de Necrosis Tumoral alfa/inmunología , Animales , Antracenos/farmacología , Bovinos , Recuento de Colonia Microbiana , Células Endoteliales/efectos de los fármacos , Células Endoteliales/enzimología , Células Endoteliales/inmunología , Células Endoteliales/microbiología , Inhibidores Enzimáticos/farmacología , Femenino , Imidazoles , Interleucina-1beta/farmacología , Proteínas Quinasas JNK Activadas por Mitógenos/antagonistas & inhibidores , Proteínas Quinasas JNK Activadas por Mitógenos/inmunología , Mastitis Bovina/inmunología , Microscopía Electrónica/veterinaria , FN-kappa B/antagonistas & inhibidores , Nitrilos/farmacología , Péptidos/farmacología , Piridinas , Infecciones Estafilocócicas/inmunología , Infecciones Estafilocócicas/microbiología , Sulfonas/farmacología , Factor de Necrosis Tumoral alfa/farmacologíaRESUMEN
Inward-rectifying potassium (K+(in)) channels in guard cells have been suggested to provide a pathway for K+ uptake into guard cells during stomatal opening. To test the proposed role of guard cell K+(in) channels in light-induced stomatal opening, transgenic Arabidopsis plants were generated that expressed dominant negative point mutations in the K+(in) channel subunit KAT1. Patch-clamp analyses with transgenic guard cells from independent lines showed that K+(in) current magnitudes were reduced by approximately 75% compared with vector-transformed controls at -180 mV, which resulted in reduction in light-induced stomatal opening by 38% to 45% compared with vector-transformed controls. Analyses of intracellular K+ content using both sodium hexanitrocobaltate (III) and elemental x-ray microanalyses showed that light-induced K+ uptake was also significantly reduced in guard cells of K+(in) channel depressor lines. These findings support the model that K+(in) channels contribute to K+ uptake during light-induced stomatal opening. Furthermore, transpirational water loss from leaves was reduced in the K+(in) channel depressor lines. Comparisons of guard cell K+(in) current magnitudes among four different transgenic lines with different K+(in) current magnitudes show the range of activities of K+(in) channels required for guard cell K+ uptake during light-induced stomatal opening.
Asunto(s)
Arabidopsis/fisiología , Epidermis de la Planta/fisiología , Canales de Potasio de Rectificación Interna , Canales de Potasio/fisiología , Potasio/metabolismo , Arabidopsis/genética , Arabidopsis/efectos de la radiación , Proteínas de Arabidopsis , Transporte Biológico Activo , Regulación de la Expresión Génica de las Plantas , Activación del Canal Iónico/genética , Luz , Potenciales de la Membrana/efectos de los fármacos , Técnicas de Placa-Clamp , Epidermis de la Planta/genética , Proteínas de Plantas , Transpiración de Plantas/fisiología , Plantas Modificadas Genéticamente/fisiología , Plantas Modificadas Genéticamente/efectos de la radiación , Mutación Puntual , Canales de Potasio/genéticaRESUMEN
The Arabidopsis thaliana cDNA, KAT1 encodes a hyperpolarization-activated K+ (K+in) channel. In the present study, we identify and characterize dominant negative point mutations that suppress K+in channel function. Effects of two mutations located in the H5 region of KAT1, at positions 256 (T256R) and 262 (G262K), were studied. The co-expression of either T256R or G262K mutants with KAT1 produced an inhibition of K+ currents upon membrane hyperpolarization. The magnitude of this inhibition was dependent upon the molar ratio of cRNA for wild-type to mutant channel subunits injected. Inhibition of KAT1 currents by the co-expression of T256R or G262K did not greatly affect the ion selectivity of residual currents for Rb+, Na+, Li+, or Cs+. When T256R or G262K were co-expressed with a different K+ channel, AKT2, an inhibition of the channel currents was also observed. Voltage-dependent Cs+ block experiments with co-expressed wild type, KAT1 and AKT2, channels further indicated that KAT1 and AKT2 formed heteromultimers. These data show that AKT2 and KAT1 are able to co-assemble and suggest that suppression of channel function can be pursued in vivo by the expression of the dominant negative K+in channel mutants described here.
Asunto(s)
Proteínas de Arabidopsis , Arabidopsis/fisiología , Proteínas de Plantas/fisiología , Canales de Potasio de Rectificación Interna , Canales de Potasio/fisiología , Animales , Activación del Canal Iónico/genética , Técnicas de Placa-Clamp , Mutación PuntualRESUMEN
Sustained (noninactivating) outward-rectifying K+ channel currents have been identified in a variety of plant cell types and species. Here, in Arabidopsis thaliana guard cells, in addition to these sustained K+ currents, an inactivating outward-rectifying K+ current was characterized (plant A-type current: IAP). IAP activated rapidly with a time constant of 165 ms and inactivated slowly with a time constant of 7.2 sec at +40 mV. IAP was enhanced by increasing the duration (from 0 to 20 sec) and degree (from +20 to -100 mV) of prepulse hyperpolarization. Ionic substitution and relaxation (tail) current recordings showed that outward IAP was mainly carried by K+ ions. In contrast to the sustained outward-rectifying K+ currents, cytosolic alkaline pH was found to inhibit IAP and extracellular K+ was required for IAP activity. Furthermore, increasing cytosolic free Ca2+ in the physiological range strongly inhibited IAP activity with a half inhibitory concentration of approximately 94 nM. We present a detailed characterization of an inactivating K+ current in a higher plant cell. Regulation of IAP by diverse factors including membrane potential, cytosolic Ca2+ and pH, and extracellular K+ and Ca2+ implies that the inactivating IAP described here may have important functions during transient depolarizations found in guard cells, and in integrated signal transduction processes during stomatal movements.
RESUMEN
Several plasma-membrane proteins from beet root (Beta vulgaris L.) have been functionally incorporated into reconstituted proteoliposomes. These showed H(+)-ATPase activity, measured both as ATP hydrolysis and H+ transport. The proton-transport specific activity was 10 times higher than in plasma membranes, and was greatly stimulated by potassium and valinomycin. These proteoliposomes also showed calcium-regulated protein kinase activity. This kinase activity is probably due to a calmodulin-like domain protein kinase (CDPK), since two protein bands were recognized by antibodies against soybean and Arabidopsis CDPK. This kinase phosphorylated histone and syntide-2 in a Ca(2+)-dependent manner. Among the plasma-membrane proteins phosphorylated by this kinase, was the H(+)-ATPase. When the H(+)-ATPase was either prephosphorylated or assayed in the presence of Ca2+, both the ATP-hydrolysis and the proton-transport activities were slower. This inhibition was reversed by an alkaline-phosphatase treatment. A trypsin treatment (that has been reported to remove the C-terminal autoinhibitory domain from the H(+)-ATPase) also reversed the inhibition caused by phosphorylation. These results indicate that a Ca(2+)-dependent phosphorylation, probably caused by a CDPK, inhibits the H(+)-ATPase activities. The substrate of this regulatory phosphorylation could be the H(+)-ATPase itself, or a different protein influencing the ATPase activities.
Asunto(s)
Calcio/metabolismo , Chenopodiaceae/enzimología , Raíces de Plantas/enzimología , ATPasas de Translocación de Protón/metabolismo , Hidrólisis , Transporte Iónico , Fosforilación , Proteínas Quinasas/metabolismo , Proteolípidos , ATPasas de Translocación de Protón/antagonistas & inhibidores , Tripsina/farmacologíaRESUMEN
Inward-rectifying K+ (K+in) channels in the guard cell plasma membrane have been suggested to function as a major pathway for K+ influx into guard cells during stomatal opening. When K+in channels were blocked with external Cs+ in wild-type Arabidopsis guard cells, light-induced stomatal opening was reduced. Transgenic Arabidopsis plants were generated that expressed a mutant of the guard cell K+in channel, KAT1, which shows enhanced resistance to the Cs+ block. Stomata in these transgenic lines opened in the presence of external Cs+. Patch-clamp experiments with transgenic guard cells showed that inward K+(in) currents were blocked less by Cs+ than were K+ currents in controls. These data provide direct evidence that KAT1 functions as a plasma membrane K+ channel in vivo and that K+in channels constitute an important mechanism for light-induced stomatal opening. In addition, biophysical properties of K+in channels in guard cells indicate that components in addition to KAT1 may contribute to the formation of K+in channels in vivo.
Asunto(s)
Arabidopsis/efectos de la radiación , Cesio/farmacología , Plantas Modificadas Genéticamente/efectos de la radiación , Canales de Potasio/efectos de los fármacos , Arabidopsis/fisiología , Expresión Génica , Activación del Canal Iónico , Técnicas de Placa-Clamp , Plantas Modificadas Genéticamente/fisiología , Canales de Potasio/genéticaRESUMEN
The effect of NADH, NAD+, NADPH, and NADP+ on succinate driven ATP synthesis of Mg-ATP submitochondrial particles and on ATP hydrolysis in soluble and particulate F1 from bovine heart was studied. Hydrolysis was inhibited competitively by all pyridine nucleotides when the concentration of Mg [gamma 32P]-ATP was varied between 0.1 mM to 3 mM, but NAD+ was less effective. Succinate driven ATP synthesis at various concentrations of ADP was also inhibited by the four pyridine nucleotides, but NADH was much more effective. Increasing concentrations of phosphate decreased the extent of the NADH induced inhibition of ATP synthesis. The effect of various parts of the NADH molecule on ATP synthesis was tested; the order of inhibition by these compounds was: NADH greater than ADP-ribose greater than NAD+ greater than NADPH greater than NADP+ greater than nicotinamide mononucleotide greater than nicotinamide.