RESUMEN
OBJECTIVES: The role of follow-up blood cultures (FUBCs) in the management of Gram-negative bacteraemia (GNB) is poorly understood. We aimed to determine the utility of FUBCs in identifying patients with increased mortality risk. METHODS: An observational study with a prospectively enrolled cohort of adult inpatients with GNB was conducted at Duke University Health System from 2002 to 2015. FUBCs were defined as blood cultures performed from 24 hours to 7 days from initial positive blood culture. RESULTS: Among 1702 patients with GNB, 1164 (68%) had FUBCs performed. When performed, FUBCs were positive in 20% (228/1113) of cases. FUBC acquisition was associated with lower all-cause in-hospital mortality (108/538, 20%, vs. 176/1164, 15%; p 0.01) and attributable in-hospital mortality (78/538, 15%, vs. 98/1164, 8%; p < 0.0001). Propensity score-weighted Cox proportional hazards models revealed that obtaining FUBCs was associated with reductions in all-cause (hazard ratio (HR) 0.629; 95% confidence interval (CI), 0.511-0.772; p < 0.0001) and attributable mortality (HR 0.628; 95% CI, 0.480-0.820; p 0.0007). Positive FUBCs were associated with increased all-cause mortality (49/228, 21%, vs. 110/885, 11%; p 0.0005) and attributable mortality (27/228, 12%, vs. 61/885, 7%; p 0.01) relative to negative FUBCs. Propensity score-weighted Cox proportional hazards models revealed that positive FUBCs were associated with increased all-cause (HR 2.099; 95% CI, 1.567-2.811; p < 0.0001) and attributable mortality (HR 1.800; 95% CI, 1.245-2.603; p 0.002). In a calibration analysis, a scoring system accurately identified patients at high risk of positive FUBCs. CONCLUSIONS: Rates of positive FUBCs were high and identified patients at increased risk for mortality. Clinical variables can identify patients at high risk for positive FUBCs. FUBCs should be considered in the management of GNB.
Asunto(s)
Bacteriemia/mortalidad , Cultivo de Sangre/métodos , Infecciones por Bacterias Gramnegativas/mortalidad , Anciano , Bacteriemia/microbiología , Femenino , Estudios de Seguimiento , Infecciones por Bacterias Gramnegativas/microbiología , Mortalidad Hospitalaria , Humanos , Pacientes Internos , Masculino , Persona de Mediana Edad , Estudios Prospectivos , Análisis de SupervivenciaRESUMEN
AIMS/HYPOTHESIS: Endothelial cells (ECs) and smooth muscle cells (SMCs) play key roles in the development of intimal hyperplasia in saphenous vein (SV) bypass grafts. In diabetic patients, insulin administration controls hyperglycaemia but cardiovascular complications remain. Insulin is synthesised as a pro-peptide, from which C-peptide is cleaved and released into the circulation with insulin; exogenous insulin lacks C-peptide. Here we investigate modulation of human SV neointima formation and SV-EC and SV-SMC function by insulin and C-peptide. METHODS: Effects of insulin and C-peptide on neointima formation (organ cultures), EC and SMC proliferation (cell counting), EC migration (scratch wound), SMC migration (Boyden chamber) and signalling (immunoblotting) were examined. A real-time RT-PCR array identified insulin-responsive genes, and results were confirmed by real-time RT-PCR. Targeted gene silencing (siRNA) was used to assess functional relevance. RESULTS: Insulin (100 nmol/l) augmented SV neointimal thickening (70% increase, 14 days), SMC proliferation (55% increase, 7 days) and migration (150% increase, 6 h); effects were abrogated by 10 nmol/l C-peptide. C-peptide did not affect insulin-induced Akt or extracellular signal-regulated kinase signalling (15 min), but array data and gene silencing implicated sterol regulatory element binding transcription factor 1 (SREBF1). Insulin (1-100 nmol/l) did not modify EC proliferation or migration, whereas 10 nmol/l C-peptide stimulated EC proliferation by 40% (5 days). CONCLUSIONS/INTERPRETATION: Our data support a causative role for insulin in human SV neointima formation with a novel counter-regulatory effect of proinsulin C-peptide. Thus, C-peptide can limit the detrimental effects of insulin on SMC function. Co-supplementing insulin therapy with C-peptide could improve therapy in insulin-treated patients.
Asunto(s)
Péptido C/metabolismo , Células Endoteliales/patología , Endotelio Vascular/patología , Insulina/metabolismo , Músculo Liso Vascular/patología , Vena Safena/patología , Túnica Íntima/patología , Análisis de Varianza , Western Blotting , Recuento de Células , Movimiento Celular/efectos de los fármacos , Proliferación Celular/efectos de los fármacos , Células Cultivadas , Células Endoteliales/efectos de los fármacos , Células Endoteliales/metabolismo , Endotelio Vascular/efectos de los fármacos , Endotelio Vascular/metabolismo , Humanos , Hiperplasia/tratamiento farmacológico , Hiperplasia/metabolismo , Hiperplasia/patología , Insulina/farmacología , Músculo Liso Vascular/efectos de los fármacos , Músculo Liso Vascular/metabolismo , Miocitos del Músculo Liso/efectos de los fármacos , Miocitos del Músculo Liso/metabolismo , Miocitos del Músculo Liso/patología , Fosforilación/efectos de los fármacos , Proteínas Proto-Oncogénicas c-akt/metabolismo , ARN Interferente Pequeño , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Vena Safena/efectos de los fármacos , Vena Safena/metabolismo , Transducción de Señal/efectos de los fármacos , Túnica Íntima/efectos de los fármacos , Túnica Íntima/metabolismoRESUMEN
BACKGROUND: Secretion of Weibel-Palade body (WPB) contents is regulated, in part, by the phosphorylation of proteins that constitute the endothelial exocytotic machinery. In comparison to protein kinases, a role for protein phosphatases in regulating endothelial exocytosis is undefined. OBJECTIVE AND METHOD: In this study, we investigated the role of protein phosphatase 2B (PP2B) in the process of endothelial exocytosis using pharmacological and gene knockdown approaches. RESULTS: We show that inhibition of protein phosphatase 2B (PP2B) activity by cyclosporine A (CsA), tacrolimus or a cell-permeable PP2B autoinhibitory peptide promotes the secretion of ultralarge von Willebrand factor (ULVWF) from human umbilical vein endothelial cells (HUVECs) in the absence of any other endothelial cell-stimulating agent. PP2B inhibitor-induced secretion and anchorage of ULVWF strings from HUVECs mediate platelet tethering. In support of a role for PP2B in von Willebrand factor (VWF) secretion, the catalytic subunit of PP2B interacts with the vesicle trafficking protein, Munc18c. Serine phosphorylation of Munc18c, which promotes granule exocytosis in other secretory cells, is increased in CsA-treated HUVECs, suggesting that this process may be involved in CsA-mediated WPB exocytosis. Furthermore, the plasma VWF antigen level is also enhanced in CsA-treated mice, and small interfering RNA-mediated knockdown of the alpha and beta isoforms of the PP2B-A subunit in HUVECs enhanced VWF secretion. CONCLUSIONS: These observations suggest that CsA promotes VWF release, in part by inhibition of PP2B activity, and are compatible with the clinically observed association of CsA treatment and increased plasma VWF levels in humans.
Asunto(s)
Inhibidores de la Calcineurina , Endotelio Vascular/metabolismo , Factor de von Willebrand/metabolismo , Animales , Secuencia de Bases , Calcineurina/genética , Células Cultivadas , Cartilla de ADN , Endotelio Vascular/citología , Inhibidores Enzimáticos/farmacología , Humanos , Ratones , Ratones Endogámicos BALB C , ARN Mensajero/genética , Reacción en Cadena de la Polimerasa de Transcriptasa InversaRESUMEN
During periods of stress, trophozoites of Entamoeba invadens (strain IP-1) undergo a process of differentiation (encystment) that results in a dormant cyst with a chitin-containing cyst wall. Encystment can be induced by resuspension of trophozoites from growth medium into a diluted glucose-free medium (47% LG) containing 5% adult bovine serum (ABS). ABS is thought to be a source of gal-terminated ligands that are required for high levels of encystment. After resuspension of trophozoites in 47% LG, encystment cultures were examined every 2h for responses to the (i) addition of 10mM free-galactose, (ii) resuspension of cells to serum-free medium, (iii) and dilution of encysting cultures to cell densities below that known to support full encystment (from 5 x 10(5) to 1 x 10(4)cells/ml). The role of serum components (and the gal-terminated ligand asialofetuin; ASF) adsorbed onto the surface upon which encystment proceeds, and their effect on the multi-cellular aggregation patterns formed during encystment, were also investigated. The addition of free-galactose reduced the levels of encystment (compared with the control) even when added at 10h after resuspension of trophozoites in 47% LG. The requirement for the presence of ABS during encystment was lost within 6h, with levels of encystment of cells washed free of serum reaching 80% of the control. The ability of cells to encyst when diluted to a cell density below that normally thought to support encystment reached over 50% by 8h. Efficient encystment could be obtained in 47% LG in the absence of ABS or ASF using pre-treated glass culture tubes. Encystment (47% LG; 5% ABS) using ultra low attachment plates was poor, suggesting attachment of cells to a surface via gal-terminated ligands was important for efficient encystment. The results suggest that ABS is probably not the only source of gal-terminated ligands necessary for high levels of encystment in 47% LG. While serum may provide a source of ligands which enhance the levels of encystment initially, other gal-terminated ligands possibly released by the encysting cells are still required for the completion of the encystment process and the formation of mature cysts. In addition, the gal-terminated ligands necessary for encystment efficiency may be adsorbed onto the glass surface of culture tubes and aid the initial aggregation process, as well as be involved in cell signaling during the encystment process.
Asunto(s)
Entamoeba/fisiología , Galactosa/metabolismo , Acetilglucosamina/metabolismo , Animales , Asialoglicoproteínas/metabolismo , Medios de Cultivo , Fetuínas , Ligandos , Factores de Tiempo , alfa-Fetoproteínas/metabolismoRESUMEN
AIMS: This study investigates the effects of biguanides during encystment of Acanthamoeba castellanii. METHODS AND RESULTS: A non-nutrient encystment system was used to investigate the changes in the levels of sorption (uptake) of three non-cysticidal concentrations (10, 20 and 50 microg ml(-1)) of chlorhexidine diacetate (CHA) and polyhexamethylene biguanide (PHMB) as well as their effects on viability and leakage of pentose sugars during the first 36 h of encystment. Trophozoites treated with CHA or PHMB were more sensitive and generally sorbed more of each biocide than cysts. During encystment, the largest increases in resistance developed between 18 and 36 h for both biguanides with the resistance emerging to biguanide concentrations of 10 or 20 microg ml(-1) between 18 and 24 h. At 50 microg ml(-1) resistance emerged between 24 and 36 h. There was a general decrease in biocide sorption during encystment between 0-24 and 0-21 h for CHA and PHMB, respectively, at a concentration of 50 microg ml(-1). The greatest decline in biguanide-induced pentose leakage was between 0 and 12 h. CONCLUSIONS: The results suggest that during encystment some of the changes in the susceptibility to CHA or PHMB may be related to decreases in the levels of biocide sorption, which is limited by the developing cyst wall. SIGNIFICANCE AND IMPACT OF THE STUDY: During encystation, changes occur in biguanide sensitivity. The physical barrier of the cyst wall may be an important factor in limiting biocide sorption.
Asunto(s)
Acanthamoeba/efectos de los fármacos , Amebicidas/farmacología , Biguanidas/farmacología , Desinfectantes/farmacología , Pentosas/metabolismo , Acanthamoeba/metabolismo , Amebicidas/farmacocinética , Animales , Biguanidas/farmacocinética , Clorhexidina/farmacocinética , Clorhexidina/farmacología , Desinfectantes/farmacocinética , Relación Dosis-Respuesta a Droga , Resistencia a MedicamentosRESUMEN
Under flow conditions, platelets adhere singly or in small aggregates on von Willebrand factor (VWF)-coated surfaces, but form large aggregates on immobilized fibrillar collagen. We developed a novel flow cytometric analysis to study the mechanisms underlying these distinct platelet deposition patterns. Flow cytometry was used to measure platelet activation after platelet adherence onto microspheres coated with either VWF or collagen fibrils. Two representative indices were calculated to quantify activated GpIIb-IIIa and P-selectin expression on adherent platelets. The signaling pathways responsible for platelet activation after interacting with fibrillar collagen were elucidated using various inhibitors. An in vitro endothelial cell wound model was also used to study the roles of VWF and fibrillar collagen in platelet deposition onto subendothelial matrixes. The adherent platelets on fibrillar collagen express more activated GpIIb-IIIa and P-selectin than those on VWF. Activation of GpIIb-IIIa and expression of P-selectin after platelet interaction with collagen occur via different intracellular signaling pathways; however, Ca2+ released from intracellular pools is common to both phenomena. Platelets were deposited singly or formed small aggregates on the endothelial cell wounded area, and this deposition pattern was dependent on VWF molecules secreted by endothelial cells and the absence of subendothelial collagen fibrils. As less activated GpIIb-IIIa and P-selectin are expressed after platelets interact with immobilized VWF alone, subsequent flowing platelet recruitment is minimal. Collagen fibrils, however, can activate adherent platelets sufficiently to promote the formation of large platelet aggregates.
Asunto(s)
Citometría de Flujo/métodos , Ácido Gálico/análogos & derivados , Activación Plaquetaria/fisiología , Bloqueadores de los Canales de Calcio/farmacología , Señalización del Calcio , Células Cultivadas , Colágeno , Endotelio Vascular/lesiones , Endotelio Vascular/fisiopatología , Ácido Gálico/farmacología , Humanos , Técnicas In Vitro , Selectina-P/sangre , Activación Plaquetaria/efectos de los fármacos , Adhesividad Plaquetaria/fisiología , Agregación Plaquetaria/fisiología , Complejo GPIIb-IIIa de Glicoproteína Plaquetaria/metabolismo , Propiedades de Superficie , Factor de von WillebrandRESUMEN
Saphenous vein (SV) grafts are commonly used to bypass coronary arteries that are diseased due to atherosclerosis. However, the development of intimal hyperplasia in such grafts can lead to patency-threatening stenosis and re-occlusion of the vessel. The proliferation and migration of smooth muscle cells (SMC) play key roles in the development of intimal hyperplasia, and an agent that inhibits both of these processes therefore has therapeutic potential. A prerequisite for SMC proliferation and migration in vivo is degradation of the basement membrane, achieved by secretion of the matrix-degrading gelatinases matrix metalloproteinase-2 (MMP-2) and MMP-9. Statins are cholesterol-lowering drugs that also have direct effects on SMC function. Here we report that neointima formation in organ-cultured human SV segments is inhibited by simvastatin, an effect that is associated with reduced MMP-9 activity. Additionally, our work shows that simvastatin not only inhibits proliferation, but importantly also inhibits invasion (migration through a matrix barrier), of cultured human SV SMC. Thus simvastatin treatment appears to inhibit neointima formation as a result of combined inhibition of SMC proliferation and invasion. The potential intracellular mechanisms by which statins affect SMC proliferation and migration, and thus attenuate intimal hyperplasia, are discussed, with particular emphasis on the role of MMP-9.
Asunto(s)
Puente de Arteria Coronaria/efectos adversos , Inhibidores de Hidroximetilglutaril-CoA Reductasas/farmacología , Inhibidores de la Metaloproteinasa de la Matriz , Vena Safena/efectos de los fármacos , Vena Safena/trasplante , Células Cultivadas , Constricción Patológica , Humanos , Hiperplasia , Músculo Liso Vascular/efectos de los fármacos , Músculo Liso Vascular/patología , Técnicas de Cultivo de Órganos , Vena Safena/enzimología , Vena Safena/patologíaRESUMEN
Using heparinized whole blood and flow conditions, it was shown that adenosine 5'-diphosphate (ADP) receptors P2Y(12) and P2Y(1) are both important in direct shear-induced platelet aggregation and platelet aggregation subsequent to initial adhesion onto von Willebrand factor (vWf)-collagen. In the viscometer, whole blood was subjected to shear rates of 750, 1500, and 3000 s(-1) for 30 seconds at room temperature. The extent of aggregation was determined by flow cytometry. The P2Y(12) antagonist AR-C69 931MX (ARMX) reduced shear-induced aggregation at these rates by 56%, 54%, and 16%, respectively, compared to control samples. Adenosine 3',5'-diphosphate (A3P5P; P2Y(1) antagonist) inhibited shear-induced aggregation by 40%, 30% and 29%, respectively, compared to control samples. Blockade of both ADP receptors at 3000 s(-1) with ARMX plus A3P5P further reduced the platelet aggregation by 41% compared to the addition of ARMX alone (57% compared to control samples). Using a parallel-plate flow chamber, whole blood was perfused over bovine collagen type 1 at a wall shear rate of 3000 s(-1) for 60 seconds. Platelet deposition was quantified with epifluorescence video microscopy and digital image processing. Blockade of P2Y(12) alone or blockade of P2Y(1) alone did not reduce thrombus formation on vWf-collagen. In contrast, blockade of both P2Y(12) and P2Y(1) reduced platelet deposition by 72%. These results indicate that combinations of antagonists of the ADP receptors P2Y(12) and P2Y(1) are effective inhibitors of direct shear-induced platelet aggregation and of platelet aggregation subsequent to initial adhesion under flow conditions. Inhibitors of these pathways are potentially useful as antiarterial thrombotic agents.
Asunto(s)
Adenosina Monofosfato/análogos & derivados , Hemorreología , Proteínas de la Membrana , Inhibidores de Agregación Plaquetaria/farmacología , Agregación Plaquetaria/fisiología , Antagonistas del Receptor Purinérgico P2 , Adenosina Difosfato/farmacología , Adenosina Monofosfato/farmacología , Viscosidad Sanguínea , Colágeno Tipo I/farmacología , Citometría de Flujo , Humanos , Procesamiento de Imagen Asistido por Computador , Microscopía Fluorescente , Receptores Purinérgicos P2Y1 , Receptores Purinérgicos P2Y12RESUMEN
Enantioselectivity of enzyme catalysis is often rationalized via active site models. These models are constructed on the basis of comparing the enantiomeric excess of product observed in a series of reactions which are conducted with a range of homologous substrates, typically carrying various side chain substitutions. Surprisingly the practical application of these simple but informative 'pocket size' models has been rarely tested in genetic engineering experiments. In this paper we report the construction, purification and enantioselectivity of two recombinant Rhizomucor miehei lipases which were designed to check the validity of such a model in reactions of ring opening of oxazolin-5(4H)-ones.
Asunto(s)
Lipasa/metabolismo , Oxazoles/metabolismo , Rhizomucor/enzimología , Dominio Catalítico , Clonación Molecular , Simulación por Computador , Proteínas Fúngicas , Enlace de Hidrógeno , Cinética , Lipasa/genética , Modelos Moleculares , Mutagénesis Sitio-Dirigida , Oxazoles/química , Oxazolona , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , EstereoisomerismoRESUMEN
A library of heptapeptides displayed on the surface of filamentous phage M13 was evaluated as a potential source of affinity ligands for the purification of Rhizomucor miehei lipase. Two independent selection (biopanning) protocols were employed: the enzyme was either physically adsorbed on polystyrene or chemically immobilized on small magnetic beads. From screening with the polystyrene-adsorbed lipase it was found that there was a rapid enrichment of the library with "doublet" clones i.e. the phage species which carried two consecutive sequences of heptapeptides, whilst no such clones were observed from the screening using lipase attached to magnetic beads. The binding of the best clones to the enzyme was unambiguously confirmed by ELISA. However the synthetic heptapeptide of identical sequence to the best "monomeric" clone did not act as a satisfactory affinity ligand after immobilization on Sepharose. This indicated that the interaction with lipase was due to both the heptapeptide and the presence of a part of the phage coat protein. This conclusion was further verified by immobilizing the whole phage on the surface of magnetic beads and using the resulting conjugate as an affinity adsorbent. The scope of application of this methodology and the possibility of preparing phage-based affinity materials are briefly discussed.
RESUMEN
The feasibility of altering the chain length specificity of industrially important Rhizomucor miehei lipase was investigated by randomly mutating Phe94 in the protein groove which is responsible for accommodating the acyl chain of the substrate. The recombinant lipase was initially expressed in E. coli. Individual colonies were selected, grown, and the DNA sequence of the lipase gene determined. Fourteen of the 19 possible mutants were identified and each of these was transformed into Pichia pastoris which expresses the enzyme extracellularly. The yeast was grown and the supernatants assessed in several assays with long and short chain substrates. Based on this preliminary screen, one mutant, Phe94Gly, was selected and purified to homogeneity for further analysis. It was found that the substitution of phenylalanine 94 with glycine led to an enzyme which was about six times less active against resorufin ester but displayed 3-4 times higher activity with short chain substrates such as butyric acid esters. The observed alteration to the enzyme specificity was rationalised using the available 3D structure of the lipase.
Asunto(s)
Ésteres/metabolismo , Lipasa/genética , Lipasa/metabolismo , Mutagénesis , Ingeniería de Proteínas/métodos , Sustitución de Aminoácidos , Sitios de Unión , Ácido Butírico/química , Ácido Butírico/metabolismo , Cromatografía en Agarosa/métodos , Escherichia coli/genética , Ésteres/química , Hidrólisis , Lipasa/química , Fenilalanina , Pichia/genética , Conformación Proteica , Proteínas Recombinantes/genética , Proteínas Recombinantes/aislamiento & purificación , Proteínas Recombinantes/metabolismo , Rhizomucor/enzimología , Especificidad por SustratoRESUMEN
Liver microsomes contain two carnitine acyltransferase activities. One of these has properties closely corresponding to those of 88 kDa mitochondrial carnitine palmitoyltransferase-1 (CPT-1). Antisera against CPT-1 cross-react with an 88 kDa microsomal protein, suggesting that CPT-1 may be targeted to both microsomal and mitochondrial membranes. However, no experiments using cDNAs corresponding to CPT-1 involving in vitro translation with microsomes or involving in vivo COS-1 cell transfection provided any evidence to support this hypothesis.
Asunto(s)
Carnitina Aciltransferasas/metabolismo , Malonil Coenzima A/metabolismo , Microsomas Hepáticos/enzimología , Animales , Carnitina O-Palmitoiltransferasa/metabolismo , Retículo Endoplásmico/enzimología , Retículo Endoplásmico/metabolismo , Hígado/enzimología , Hígado/metabolismo , Microsomas Hepáticos/metabolismo , Mitocondrias/enzimología , Mitocondrias/metabolismo , Isoformas de Proteínas/metabolismo , Transporte de ProteínasRESUMEN
The aim of this study was to determine whether internalisation of the angiotensin II (Ang II) AT(1A) receptor (AT(1A)R) was a prerequisite for Ang II-induced activation of the extracellular signal-regulated kinases, ERK-1/2. The human embryonic kidney (HEK293) cell line stably transfected with either the wild-type rat AT(1A)R or an internalisation-deficient C-terminal truncated mutant of the AT(1A)R (AT(1A)T318R) was used as a model for these studies. Inhibition of AT(1A)R internalisation by treatment with an inhibitor of clathrin-mediated endocytosis, Concanavalin A (Con A), did not inhibit Ang II-induced ERK-1/2 activation. Furthermore, cells transfected with the internalisation-deficient AT(1A)T318R mutant readily activated ERK-1/2 in response to Ang II. Ang II activated ERK-1/2 via two distinct signalling pathways in HEK-AT(1A)R cells. Approximately half of Ang II-induced ERK-1/2 activation was protein kinase C (PKC)-dependent, and the remainder was calcium- and c-Src-dependent and involved transactivation of the epidermal growth factor receptor (EGFR). In summary, Ang II-induced activation of ERK-1/2 occurs via two distinct pathways in HEK293 cells, neither of which requires AT(1A)R internalisation.
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Angiotensina II/metabolismo , Proteína Quinasa 1 Activada por Mitógenos/metabolismo , Proteínas Quinasas Activadas por Mitógenos/metabolismo , Receptores de Angiotensina/metabolismo , Angiotensina II/genética , Animales , Calcio/fisiología , Línea Celular , Concanavalina A/farmacología , Relación Dosis-Respuesta a Droga , Activación Enzimática , Receptores ErbB/metabolismo , Humanos , Immunoblotting , Proteína Quinasa 3 Activada por Mitógenos , Modelos Biológicos , Mutación , Proteína Quinasa C/metabolismo , Proteína Quinasa C/fisiología , Proteínas Proto-Oncogénicas pp60(c-src)/metabolismo , Ratas , Receptor de Angiotensina Tipo 1 , Receptores de Angiotensina/química , Receptores de Angiotensina/genética , Transducción de Señal , Factores de Tiempo , Activación Transcripcional , TransfecciónRESUMEN
Since the early 1960s, axenic culture and the development of procedures for the induction of encystation have made Acanthamoeba spp. superb experimental systems for studies of cell biology and differentiation. More recently, since their roles as human pathogens causing keratitis and encephalitis have become widely recognized, it has become urgent to understand the parameters that determine differentiation, as cysts are much more resistant to biocides than are the trophozoites. Viability of trophozoites of the soil amoeba Acanthamoeba castellanii (Neff), is conveniently measured by its ability to form plaques on a lawn of Escherichia coli. Use of confocal laser scanning microscopy with Calcofluor white, Congo Red or the anionic oxonol dye, DiBAC4(3) or flow cytometry with propidium iodide diacetate and fluorescein or oxonol provides more rapid assessment. For cysts, the plaque method is still the best, because dye exclusion does not necessarily indicate viability and therefore the plate count method has been used to study the sequence of development of biocide resistance during the differentiation process. After two hours, resistance to HCl was apparent. Polyhexamethylene biguanide, benzalkonium chloride, propamidine isethionate, pentamidine isethionate, dibromopropamine isethionate, and H2O2 and moist heat, all lost effectiveness at between 14 and 24 h after trophozoites were inoculated into encystation media. Chlorhexidine diacetate resistance was observed at between 24 and 36 h. The molecular biology and biochemistry of the modifications that underlie these changes are now being investigated.
Asunto(s)
Acanthamoeba/efectos de los fármacos , Acanthamoeba/crecimiento & desarrollo , Amebiasis/parasitología , Amebicidas/farmacología , Acanthamoeba/ultraestructura , Animales , Resistencia a Medicamentos , HumanosRESUMEN
In this study, an analysis of the transition between the inactive ("closed") and active ("open") conformations of Candida rugosa lipase in solution is performed using irreversible enzyme inhibitors, cyclic saligenin phosphates. It is shown that >90% inhibition of the enzyme activity toward water-soluble substrates (esterolytic activity) can be achieved with as little as 0.3 mol of the inhibitor per mole of enzyme, whereas activity toward emulsified substrates decreases by approximately 20% under the same conditions. It is also shown that short-term exposure of this inhibited enzyme preparation to an interface leads to a significant increase in esterolytic activity, which even exceeds that of the untreated control. These experimental observations suggest that the inhibitors interact predominantly, if not exclusively, with the open form of the enzyme and that any transitions occurring between the two conformers of the enzyme in solution, in the absence of an interface, are extremely slow. This conclusion is verified by separating the open and closed forms of the enzyme by hydrophobic interaction column chromatography on phenyl-sepharose. Fractions enriched with the respective conformations of the enzyme are further purified using gel-permeation chromatography. On the basis of the elution pattern from this step, and sodium dodecylsulfate-polyacrylamide gel electrophoresis (SDS-PAGE), the open (active in the absence of interface) form of the lipase is found to be present in solution as a dimer, whereas the closed form appears to be a monomer. The latter form of the enzyme may be activated by up to 60-fold when exposed to triolein.
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Candida/enzimología , Lipasa/química , Lipasa/metabolismo , Alcoholes Bencílicos/síntesis química , Alcoholes Bencílicos/farmacología , Cromatografía en Gel , Cromatografía por Intercambio Iónico , Activación Enzimática , Inhibidores Enzimáticos/síntesis química , Inhibidores Enzimáticos/farmacología , Intercambio Iónico , Cinética , Lipasa/aislamiento & purificación , Conformación Proteica/efectos de los fármacosAsunto(s)
Antibacterianos/farmacología , Bacterias/efectos de los fármacos , Acanthamoeba/efectos de los fármacos , Acanthamoeba/fisiología , Animales , Bacillus/efectos de los fármacos , Bacillus/fisiología , Bacterias/citología , Bacterias/crecimiento & desarrollo , Bacterias/metabolismo , Fenómenos Fisiológicos Bacterianos , Ciclo Celular/efectos de los fármacos , Clostridium/efectos de los fármacos , Clostridium/fisiología , Eucariontes/citología , Eucariontes/efectos de los fármacos , Giardia/efectos de los fármacos , Giardia/fisiología , Esporas Bacterianas/efectos de los fármacos , Esporas Bacterianas/fisiologíaRESUMEN
A synchronous encystment method was used to study the order of development of resistance of Acanthamoeba castellanii to a range of biocides. The emerging resistance during encystation to short-term exposure to the minimum amoebicidal concentrations of each biocide tested was recorded during the first 36 h of the differentiation process. Hydrochloric acid and moist heat were tested as possible resistance markers. Development of the acid-insoluble, proteincontaining, ectocyst wall and the cellulose endocyst wall was followed by quantification of the acid- and alkali-insoluble residues of cell samples removed from synchronous encystment cultures up to 36 h. Resistance to chemical agents (polyhexamethylene biguanide, benzalkonium chloride, propamidine isethionate, pentamidine isethionate, dibromopropamidine isethionate, hydrogen peroxide) and to moist heat was seen to develop between 14 and 24 h after trophozoites were inoculated into the encystment media. Resistance to hydrochloric acid developed between 0 and 2 h and to chlorhexidine diacetate between 24 and 36 h. Levels of acid-insoluble residues began to increase after 8 h and alkali-insoluble residues (cellulose) were detected after 16 h and coincided with the emergence of resistance to all the agents tested except hydrochloric acid. The results suggest that resistance to the biocides tested probably results largely from the physical barrier of the cyst walls rather than as a consequence of a metabolically dormant cyst.
Asunto(s)
Acanthamoeba/efectos de los fármacos , Amebicidas/farmacología , Desinfectantes/farmacología , Animales , Compuestos de Benzalconio/farmacología , Benzamidinas/farmacología , Clorhexidina/farmacología , Resistencia a Medicamentos , Peróxido de Hidrógeno/farmacología , Pentamidina/farmacologíaRESUMEN
BACKGROUND: Abciximab and ticlopidine reduce adverse cardiovascular events after percutaneous transluminal coronary angioplasty (PTCA). The goal of the current study was to determine if combined abciximab/ticlopidine therapy inhibits arterial thrombosis more effectively than either treatment alone. The effect of each therapy on platelet-leukocyte interactions was also investigated. METHODS AND RESULTS: Whole blood samples from 14 patients undergoing PTCA who received abciximab therapy, ticlopidine therapy, or both treatments were evaluated using dynamic experimental systems. Mural thrombus formation under arterial shear conditions (1500 s(-1)) was determined in a parallel plate flow chamber. Shear-induced platelet aggregation was evaluated using a cone-and-plate viscometer at a shear rate of 3000 s(-1). Of the 3 treatments, combined abciximab/ticlopidine therapy produced the most consistent reduction in shear-induced platelet aggregation and the most prolonged inhibition of mural thrombosis. Three days after PTCA, abciximab/ticlopidine treatment decreased mural thrombus formation to approximately 50% of baseline values. Abciximab treatment alone inhibited mural thrombosis for only 1 day after PTCA, whereas ticlopidine treatment alone had no significant effect. Two hours after PTCA, abciximab therapy significantly decreased the number of circulating platelet-neutrophil aggregates but significantly enhanced P-selectin-mediated leukocyte adhesion on the collagen/von Willebrand factor-platelet surface. CONCLUSIONS: Combined therapy with abciximab and ticlopidine has a prolonged inhibitory effect on mural thrombosis formation relative to either treatment alone. Further, we demonstrated an unexpected effect of abciximab in enhancing P-selectin-mediated leukocyte adhesion.
Asunto(s)
Angioplastia Coronaria con Balón , Anticuerpos Monoclonales/uso terapéutico , Anticoagulantes/uso terapéutico , Plaquetas/efectos de los fármacos , Fragmentos Fab de Inmunoglobulinas/uso terapéutico , Leucocitos/efectos de los fármacos , Trombosis/prevención & control , Ticlopidina/uso terapéutico , Abciximab , Anticuerpos Monoclonales/farmacología , Anticoagulantes/farmacología , Plaquetas/fisiología , Quimioterapia Combinada , Humanos , Fragmentos Fab de Inmunoglobulinas/farmacología , Leucocitos/fisiología , Glicoproteínas de Membrana/fisiología , Selectina-P/fisiología , Activación Plaquetaria/efectos de los fármacos , Agregación Plaquetaria/efectos de los fármacos , Trombosis/etiología , Ticlopidina/farmacologíaRESUMEN
The aim of this study was to test the hypothesis that oxidative stress induces apoptosis in the H9c2 cardiac muscle cell line, and that signaling via mitogen-activated protein kinase (MAPK) pathways is involved. Three forms of oxidative stress were utilized: the superoxide generator menadione; hydrogen peroxide; or simulated ischemia followed by reperfusion. Relatively low concentrations of menadione (10 micrometer) or H2O2 (250 micrometer) caused maximal DNA fragmentation and caspase activation, both markers for apoptotic cell death, and preferential activation of the c-Jun NH 2-terminal kinase (JNK) and p38 MAPK pathways. In contrast, higher concentrations of menadione or H 2O2 caused less DNA fragmentation, more necrotic cell death and preferential activation of the extracellular signal-regulated kinase (ERK) pathway. Simulated ischemia alone did not induce DNA fragmentation or caspase activation and activated only the p38 MAPK pathway. However, ischemia plus reperfusion resulted in DNA fragmentation, caspase activation, necrotic cell death and activation of all three MAPK pathways. Selective inhibition of the ERK or p38 MAPK pathways (by PD98059 or SB-203580, respectively) had no effect on the extent of oxidative stress-induced DNA fragmentation or caspase activation. In contrast, inhibition of the JNK pathway by transfection of a dominant negative mutant of JNK markedly reduced the extent of DNA fragmentation and caspase activation induced by oxidative stress. In conclusion, these data suggest that the JNK pathway plays an important role in signaling oxidative stress-induced apoptosis of H9c2 cardiac muscle cells.