RESUMEN
Inter-α-trypsin inhibitor heavy chain 4 (ITIH4) and C-reactive protein (CRP) have been isolated from acute phase dog sera by affinity chromatography with insolubilized polyclonal antibodies anti pig Major Acute phase Protein (Pig-MAP) and with p-Aminophenyl Phosphoryl Choline, respectively. Isolated proteins were used to prepare specific polyclonal rabbit antisera that have allowed quantifying their concentration in serum samples by single radial immunodifussion. Both proteins were quantified in sera from female dogs that had undergone ovariohysterectomy (OVH, n=9) or mastectomy (n=10). The observed increases in CRP concentrations showed that surgical traumas induced an acute phase response of a great magnitude in the dogs. In both surgeries a four-fold increase of ITIH4 concentrations was detected. It can be concluded that ITIH4 is a new positive acute phase protein in dogs, as reported in other species.
Asunto(s)
alfa-Globulinas/análisis , Anticuerpos/inmunología , Proteína C-Reactiva/análisis , alfa-Globulinas/inmunología , alfa-Globulinas/aislamiento & purificación , Animales , Proteína C-Reactiva/inmunología , Proteína C-Reactiva/aislamiento & purificación , Perros , Femenino , Sueros Inmunes/inmunología , ConejosRESUMEN
The acute-phase protein (APP) response to an infection caused by Haemophilus parasuis, the etiological agent of Glässer's disease in pigs, was characterized measuring serum concentrations of pig major acute-phase protein (pig MAP), haptoglobin (HPT), C-reactive protein (CRP) and apolipoprotein A-I (ApoA-I) in colostrum-deprived pigs. They were divided into six experimental groups: non-immunized control group (I); immunized with a non-commercial bacterin (II); with an OMP-vaccine (III); with a sublethal dose (IV); and with two commercial bacterins (V and VI). All groups were challenged intratracheally with 5 × 10(9)CFU of H. parasuis 37 days after immunisation. The highest levels of the positive APPs (pig MAP, HPT and CRP) and the lowest levels of the negative APPs (ApoA-I) were observed in the animals that died as a consequence of the infection, both those in the non-immunized and in the immunized groups. However, the surviving animals (all of them in groups II, V and VI, two pigs in group III, and three in group IV) showed a minor variation in APP response, mainly on day 1 post-challenge (p.c.), and then tended to recover the initial values. APP response was still less pronounced in the groups of pigs previously immunized with bacterins. In conclusion, APP response can reflect Glässer-disease ongoing, showing a correlation between the severity and duration of the clinical signs and lesions and the magnitude of changes in the APP levels.
Asunto(s)
Proteínas de Fase Aguda/análisis , Reacción de Fase Aguda , Infecciones por Haemophilus/veterinaria , Vacunas contra Haemophilus/inmunología , Haemophilus parasuis/inmunología , Enfermedades de los Porcinos/inmunología , Animales , Apolipoproteína A-I/sangre , Proteína C-Reactiva/análisis , Calostro , Infecciones por Haemophilus/inmunología , Infecciones por Haemophilus/metabolismo , Haptoglobinas/análisis , Inmunización/veterinaria , Masculino , Porcinos , Enfermedades de los Porcinos/metabolismoRESUMEN
A total of 240 pigs, 74 days old, half boars and half females, were included in a trial designed to assess the effect of the stress caused by changes in the pattern of food administration on the concentration of acute phase proteins (APP) and productive performance parameters. Half of the animals (pigs fed ad libitum, AL group) had free access to feed, while the rest were fed following a disorderly pattern (DIS group), in which animals had alternating periods of free access to feed and periods of no feeding, when food was removed from the feeder. The periods of free access to feed (two daily periods of 2-h duration) were randomly assigned, and varied from day to day. Total feed supplied per day was identical in both groups, and exceeded the minimal amount required for animals of these ages. Pen feed intake, individual body weights and the main positive pig APP pig major acute phase protein (Pig-MAP), haptoglobin, serum amyloid A (SAA), C-reactive protein (CRP), and the negative APP apolipoprotein A-I (ApoA-I) and transtherytin were determined every 2 weeks during the period 76 to 116 days of age. Animals fed ad libitum had better average daily gain (ADG) than DIS animals in the whole experimental period (P < 0.01) but the differences in ADG were only produced in the two first experimental sub-periods (60 to 74 and 74 to 116 days of age), suggesting that the stress diminished when the animals get used to the DIS feeding. Interestingly differences in ADG between DIS and AL pigs were due to males, whereas no differences were observed between females. The same differences observed for ADG were found for APP. DIS males had higher Pig-MAP concentration than AL males at 74 and 116 days of age, lower ApoA-I concentration at 74 days of age and higher haptoglobin and CRP concentration at 116 days of age (P < 0.05). The results obtained in this trial show an inverse relationship between weight gain and APP levels, and suggest that APP may be biomarkers for the evaluation of distress and welfare in pigs.
RESUMEN
We have undertaken a study to characterize the lipolytic pathway responsible for the generation of free fatty acids (FFA) during Fas/CD95-induced apoptosis in Jurkat cells. It was initially shown that the cellular lipid fraction that suffered the major quantitative decrease during Fas-induced apoptosis was that of phosphatidylcholine (PC). In addition, the secretion of palmitic acid-derived FFA was largely prevented by D609, an inhibitor of PC-specific phospholipase C (PC-PLC) and also by the diacylglycerol lipase (DAGL) inhibitor RHC-80267, suggesting that the secretion of these FFA during Fas-induced apoptosis is mediated by the generation of DAG by a PC-PLC activity and, sequentially, by a 1-DAGL activity which generates the FFA from its sn-1 position. The endocannabinoid 2-arachidonoyl glycerol (2-AG) should be generated as a sub-product of this pathway, but it did not accumulate inside the cells nor was secreted into the supernatant. Interestingly, the complete inhibition of free AA secretion during Fas-induced apoptosis was only achieved by using the AA trifluoromethylketone, which not only inhibits all types of phospholipase-A(2) (PLA(2)) activities, but also the described lytic activities on 2-AG. Using a combination of RHC-80267 and the iPLA(2)-specific inhibitor bromoenol lactone, it was shown that the DAGL pathway also cooperates with iPLA(2) in the generation of free arachidonate.
Asunto(s)
Apoptosis , Ácidos Grasos no Esterificados/metabolismo , Lipólisis , Receptor fas/metabolismo , Apoptosis/efectos de los fármacos , Ácidos Araquidónicos/farmacología , Hidrocarburos Aromáticos con Puentes/farmacología , Ciclohexanonas/farmacología , Endocannabinoides , Inhibidores Enzimáticos/farmacología , Glicéridos/farmacología , Humanos , Células Jurkat , Cetonas/farmacología , Metabolismo de los Lípidos/efectos de los fármacos , Lipólisis/efectos de los fármacos , Lipoproteína Lipasa/antagonistas & inhibidores , Modelos Biológicos , Norbornanos , Ácido Palmítico/farmacología , Ácidos Fosfatidicos/metabolismo , Fosfatidilcolinas/metabolismo , Tiocarbamatos , Tionas/farmacología , Fosfolipasas de Tipo C/antagonistas & inhibidoresRESUMEN
In this work, apolipoprotein A-I (ApoA-I) was purified from pig sera. The responses of this protein after sterile inflammation and in animals infected with Actinobacillus pleuropneumoniae or Streptococcus suis were investigated. Decreases in the concentrations of ApoA-I, two to five times lower than the initial values, were observed at 2 to 4 days. It is concluded that ApoA-I is a negative acute-phase protein in pigs.
Asunto(s)
Infecciones por Actinobacillus/inmunología , Apolipoproteína A-I/sangre , Inflamación/inmunología , Infecciones Estreptocócicas/inmunología , Enfermedades de los Porcinos/inmunología , Enfermedades de los Porcinos/microbiología , Infecciones por Actinobacillus/microbiología , Infecciones por Actinobacillus/fisiopatología , Actinobacillus pleuropneumoniae/patogenicidad , Secuencia de Aminoácidos , Animales , Apolipoproteína A-I/química , Inflamación/etiología , Inflamación/fisiopatología , Datos de Secuencia Molecular , Infecciones Estreptocócicas/microbiología , Infecciones Estreptocócicas/fisiopatología , Streptococcus suis/patogenicidad , Porcinos , Enfermedades de los Porcinos/fisiopatología , TrementinaRESUMEN
We have isolated from calf serum a protein with an apparent M(r) of 120,000. The protein was detected by using antibodies against major acute-phase protein in pigs with acute inflammation. The amino acid sequence of an internal fragment revealed that this protein is the bovine counterpart of ITIH4, the heavy chain 4 of the inter-alpha-trypsin inhibitor family. The response of this protein in the sera was determined for animals during experimental bacterial and viral infections. In the bacterial model, animals were inoculated with a mixture of Actinomyces pyogenes, Fusobacterium necrophorum, and Peptostreptococcus indolicus to induce an acute-phase reaction. All animals developed moderate to severe clinical mastitis and exhibited remarkable increases in ITIH4 concentration in serum (from 3 to 12 times the initial values, peaking at 48 to 72 h after infection) that correlated with the severity of the disease. Animals with experimental infections with bovine respiratory syncytial virus (BRSV) also showed increases in ITIH4 concentration (from two- to fivefold), which peaked at around 7 to 8 days after inoculation. Generally, no response was seen after a second infection of the same animals with the virus. Because of the significant induction of the protein in the animals in the mastitis and BRSV infection models, we can conclude that ITIH4 is a new positive acute-phase protein in cattle.
Asunto(s)
Proteínas de Unión al Calcio/aislamiento & purificación , Bovinos/metabolismo , Glicoproteínas/aislamiento & purificación , Infecciones/metabolismo , Secuencia de Aminoácidos , Animales , Proteínas de Unión al Calcio/metabolismo , Electroforesis en Gel de Poliacrilamida , Femenino , Glicoproteínas/metabolismo , Mastitis Bovina/metabolismo , Proteínas Inhibidoras de Proteinasas SecretorasRESUMEN
Preformed Fas ligand (FasL) and APO2 ligand (APO2L)/TNF-related apoptosis-inducing ligand (TRAIL) are stored in the cytoplasm of the human Jurkat T cell line and of normal human T cell blasts. The rapid release of these molecules in their bioactive form is involved in activation-induced cell death. In this study, we show by confocal microscopy that FasL and APO2L/TRAIL are mainly localized in lysosomal-like compartments in these cells. We show also by immunoelectron microscopy that FasL and APO2L/TRAIL are stored inside cytoplasmic compartments approximately 500 nm in diameter, with characteristics of multivesicular bodies. Most of these compartments share FasL and APO2L/TRAIL, although exclusive APO2L/TRAIL labeling can be also observed in separate compartments. Upon PHA activation, the mobilization of these compartments toward the plasma membrane is evident, resulting in the secretion of the internal microvesicles loaded with FasL and APO2L/TRAIL. In the case of activation with anti-CD59 mAb, the secretion of microvesicles labeled preferentially with APO2L/TRAIL predominates. These data provide the basis of a new and efficient mechanism for the rapid induction of autocrine or paracrine cell death during immune regulation and could modify the interpretation of the role of FasL and APO2L/TRAIL as effector mechanisms in physiological and pathological situations.
Asunto(s)
Muerte Celular , Activación de Linfocitos , Glicoproteínas de Membrana/metabolismo , Vesículas Secretoras/química , Linfocitos T/inmunología , Factor de Necrosis Tumoral alfa/metabolismo , Anticuerpos Monoclonales/farmacología , Proteínas Reguladoras de la Apoptosis , Biomarcadores/análisis , Antígenos CD59/inmunología , Células Cultivadas , Proteína Ligando Fas , Citometría de Flujo , Humanos , Células Jurkat , Lisosomas/química , Microscopía Confocal , Microscopía Inmunoelectrónica , Fitohemaglutininas/farmacología , Vesículas Secretoras/ultraestructura , Linfocitos T/ultraestructura , Ligando Inductor de Apoptosis Relacionado con TNFRESUMEN
The objective of this study was to investigate, using a pulse-chase technique, the different incorporation of (1-(14)C) n -9, n -6 and n 3 fatty acids into hepatoma lipids and their secretion to the culture medium. Docosahexaenoic acid (DHA) accumulated preferentially into the triacylglycerol while arachidonic acid (AA) did into the phospholipid fraction. DHA was poorly secreted to the culture medium whereas AA was secreted to a large extent. The fatty acids were initially esterified mainly into phosphatidylcholine and phosphatidylethanolamine. During the 24 h chase, a general shift from phosphatidylcholine to phosphatidylethanolamine was observed. Linoleic acid was esterified in cardiolipin to a much greater extent than any other fatty acid and it was not converted to more polyunsaturated fatty acids. The supplementation of the culture medium with polyunsaturated fatty acids had no inhibitory effect on the growth of the hepatoma cells, in marked contrast to observations made in other tumoral cells. The reasons for the resistance of the hepatoma cells to polyunsaturated fatty acid toxicity, including the possible antioxidant effect of linoleic acid accumulation in cardiolipin, are also discussed.
Asunto(s)
Ácidos Grasos/metabolismo , Neoplasias Hepáticas Experimentales/metabolismo , Albúminas/metabolismo , Animales , Radioisótopos de Carbono , Cardiolipinas/química , División Celular/efectos de los fármacos , Ácidos Grasos/farmacología , Ácidos Grasos Insaturados/metabolismo , Ácidos Grasos Insaturados/farmacología , Ácido Linoleico/metabolismo , Fosfolípidos/química , Fosfolípidos/metabolismo , Ratas , Factores de Tiempo , Triglicéridos/química , Triglicéridos/metabolismo , Células Tumorales Cultivadas/química , Células Tumorales Cultivadas/metabolismoRESUMEN
A prominent tyrosine phosphorylated protein of 85 kDa (p85) was detected in highly proliferative sublines derived from the Jurkat T cell leukemia. We undertook a study to characterize the identity of this protein and its possible role in the hyperproliferative phenotypes observed. Using immunoblot and immunoprecipitation techniques, this protein was characterized as the p85 regulatory subunit of phosphatidylinositol 3-kinase. Cell proliferation and p85 tyrosine phosphorylation was not affected by tyrphostin AG-490, an inhibitor of Jak kinases, wortmannin or LY294002, inhibitors of the activity of the catalytic phosphatidylinositol 3-kinase subunit. Herbimycin-A and PPI, inhibitors of src-like protein tyrosine kinases, and genistein, a general tyrosine kinase inhibitor, inhibited p85 tyrosine phosphorylation and induced cell death in the sublines. PD98059, an inhibitor of Mek, inhibited cell growth of the sublines, but not that of the parental cells. It was concluded that tyrosine phosphorylation of p85 is associated with highly proliferative tumoral phenotypes, at least in T cell leukemias, independent of the phosphatidylinositol 3-kinase activity of the catalytic subunit.
Asunto(s)
Fosfatidilinositol 3-Quinasas/metabolismo , Tirosina/metabolismo , División Celular/efectos de los fármacos , Humanos , Células Jurkat , Proteína Tirosina Quinasa p56(lck) Específica de Linfocito/metabolismo , Inhibidores de las Quinasa Fosfoinosítidos-3 , Fosforilación , Proteínas Proto-Oncogénicas/metabolismo , Proteínas Proto-Oncogénicas c-fynRESUMEN
Jurkat cells and the derived TCR / CD3-defective subline, J.RT3.T3.5 undergo activation induced cell death (AICD) when stimulated with phytohemagglutinin (PHA). Since J.RT3.T3.5 cells do not express antigen receptor, we searched for the molecules that could be ligated by PHA and induce AICD in this cell line. We show here that the glycosylphosphatidylinositol linked CD59 molecule is expressed at the surface of Jurkat and J.RT3.T3.5 cells, and when cross-linked by specific antibodies can induce cell death. The toxicity of supernatants from PHA-stimulated Jurkat or J.RT3.T3.5 cells was prevented by a combination of the blocking anti-Fas mAb SM1 / 23 and anti-APO2L / TRAIL mAb 5C2. However, toxicity of supernatants from anti-CD59 stimulated cells was specifically prevented by the anti-APO2L blocking antibody. Anti-CD59 cross-linking induced AICD also in normal human T cell blasts, which secreted toxic molecules into the supernatant. The toxicity of these supernatants on Jurkat cells was fully prevented by the anti-APO2L blocking antibody, showing that CD59 crosslinking induces the preferential release of APO2L also in normal T cells. The possible physiological and / or pathological consequences of this observation are discussed.
Asunto(s)
Antígenos CD59/metabolismo , Activación de Linfocitos/inmunología , Glicoproteínas de Membrana/metabolismo , Agregación de Receptores , Linfocitos T/inmunología , Linfocitos T/metabolismo , Factor de Necrosis Tumoral alfa/metabolismo , Secuencia de Aminoácidos , Anticuerpos Bloqueadores/inmunología , Anticuerpos Monoclonales/inmunología , Apoptosis/efectos de los fármacos , Proteínas Reguladoras de la Apoptosis , Biomarcadores/análisis , Antígenos CD59/inmunología , Caspasa 3 , Caspasas/metabolismo , Membrana Celular/metabolismo , Células Cultivadas , Medios de Cultivo Condicionados , Proteína Ligando Fas , Humanos , Células Jurkat , Activación de Linfocitos/efectos de los fármacos , Proteína Tirosina Quinasa p56(lck) Específica de Linfocito/metabolismo , Glicoproteínas de Membrana/inmunología , Datos de Secuencia Molecular , Fitohemaglutininas/farmacología , Proteínas Proto-Oncogénicas/metabolismo , Proteínas Proto-Oncogénicas c-fyn , Receptores de Antígenos de Linfocitos T/genética , Receptores de Antígenos de Linfocitos T/fisiología , Linfocitos T/citología , Linfocitos T/efectos de los fármacos , Ligando Inductor de Apoptosis Relacionado con TNF , Factor de Necrosis Tumoral alfa/inmunología , Receptor fas/inmunología , Receptor fas/metabolismoRESUMEN
The acute-phase expression of pig MAP (major acute-phase protein)/ITIH4 (inter-alpha-trypsin inhibitor heavy chain 4) and haptoglobin were analysed in primary cultures of isolated pig hepatocytes in response to recombinant human (rh) cytokines: tumor necrosis factor-alpha (TNF-alpha), interleukin-1 (IL-1), interleukin-6 (IL-6), as well as to bacterial lipopolysaccharide (LPS). Analysis of pig MAP/ITIH4 and haptoglobin mRNAs was carried out by RT-PCR amplification. Secreted proteins from the cytokine-treated hepatocytes were quantified by immunochemical techniques. Time-course and dose-response experiments show that pig MAP/ITIH4 and haptoglobin belong to the type II acute-phase proteins, as they are specifically induced by rhIL-6 and not by rhTNF-alpha or rhIL-1. Stimulation of cultured pig hepatocytes with rhIL-6 for 48 h at doses of 1000 U.mL-1 showed a fourfold to fivefold increase in pig MAP/ITIH4 concentration in the medium, while the concentration of haptoglobin only increased twofold. A similar increase in the concentration of pig MAP/ITIH4 was also observed in media of LPS-treated hepatocytes with the simultaneous generation of IL-6 by the Kupffer cells present in the cultures. Albumin secretion decreased after stimulation with doses of 100 or 1000 U.mL-1 rhTNF-alpha, rhIL-1 or rhIL-6. Therefore, it can be concluded that pig MAP/ITIH4 behaves as a major acute-phase protein produced by porcine hepatocytes under the effect of inflammatory cytokines.
Asunto(s)
Proteínas de Fase Aguda/biosíntesis , Reacción de Fase Aguda/genética , Regulación de la Expresión Génica/efectos de los fármacos , Haptoglobinas/biosíntesis , Hígado/metabolismo , Proteínas de Fase Aguda/genética , Animales , Secuencia de Bases , Células Cultivadas/efectos de los fármacos , ADN Complementario/genética , Relación Dosis-Respuesta a Droga , Haptoglobinas/genética , Humanos , Interleucina-1/farmacología , Interleucina-6/farmacología , Macrófagos del Hígado/metabolismo , Lipopolisacáridos/farmacología , Hígado/citología , Hígado/efectos de los fármacos , Masculino , Datos de Secuencia Molecular , ARN Mensajero/biosíntesis , Proteínas Recombinantes/farmacología , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Porcinos , Factor de Necrosis Tumoral alfa/farmacologíaRESUMEN
The serum concentration of the inter-alpha trypsin inhibitor heavy chain 4 protein (ITIH4) increases (from 1.4-3 times) in male patients suffering of different acute-phase processes (myocardial infarction, unstable angina or programmed surgery). The concentration of C-reactive protein (CRP) in these samples ranged from 15 microg/ml to 133 microg/ml. Using the hepatocarcinoma HepG2 cell line we have observed up-regulation of ITIH4 mRNA expression upon dose-response treatments with interleukin-6 (IL-6). This effect correlates with the increase of radiolabeled ITIH4 in the cellular media of (35)S-labeled HepG2 cells treated with the cytokine. A similar effect was observed for haptoglobin mRNA, used as a control for acute-phase protein expression. IL-1beta, although up-regulating the expression of alpha(1)-acid glycoprotein in these cells, did not induce any effect in the expression of ITIH4. No changes were observed after TNF-alpha treatments. The results presented here indicate that ITIH4 is a type II acute-phase protein in humans.
Asunto(s)
Reacción de Fase Aguda/sangre , Proteínas de Unión al Calcio/sangre , Proteínas de Unión al Calcio/genética , Carcinoma Hepatocelular/genética , Glicoproteínas/sangre , Glicoproteínas/genética , Interleucina-6/farmacología , Neoplasias Hepáticas/genética , Adulto , Anciano , Angina Inestable/sangre , Secuencia de Bases , Proteínas de Unión al Calcio/biosíntesis , Cartilla de ADN/genética , Glicoproteínas/biosíntesis , Haptoglobinas/biosíntesis , Haptoglobinas/genética , Humanos , Masculino , Persona de Mediana Edad , Infarto del Miocardio/sangre , Orosomucoide/biosíntesis , Orosomucoide/genética , Proteínas Inhibidoras de Proteinasas Secretoras , ARN Mensajero/genética , ARN Mensajero/metabolismo , ARN Neoplásico/genética , ARN Neoplásico/metabolismo , Células Tumorales Cultivadas , Regulación hacia Arriba/efectos de los fármacosRESUMEN
Activation-induced cell death is a process by which overactivated T cells are eliminated, thus preventing potential autoimmune attacks. Two known mediators of activation-induced cell death are Fas(CD95) ligand (FasL) and APO2 ligand (APO2L)/TNF-related apoptosis-inducing ligand (TRAIL). We show here that upon mitogenic stimulation, bioactive FasL and APO2L are released from the T cell leukemia Jurkat and from normal human T cell blasts as intact, nonproteolyzed proteins associated with a particulate, ultracentrifugable fraction. We have characterized this fraction as microvesicles of 100-200 nm in diameter. These microvesicles are released from Jurkat and T cell blasts shortly (=1 h) after PHA stimulation, well before the cell enters apoptosis. FasL- and APO2L-containing vesicles are also present in supernatants from PHA-activated fresh human PBMC. These observations provide the basis for a new and efficient mechanism for the rapid induction of autocrine or paracrine cell death during immune regulation.
Asunto(s)
Apoptosis/inmunología , Activación de Linfocitos , Glicoproteínas de Membrana/metabolismo , Linfocitos T/inmunología , Linfocitos T/metabolismo , Factor de Necrosis Tumoral alfa/metabolismo , Receptor fas/metabolismo , Secuencia de Aminoácidos , Apoptosis/efectos de los fármacos , Proteínas Reguladoras de la Apoptosis , Sistema Libre de Células/química , Sistema Libre de Células/inmunología , Sistema Libre de Células/metabolismo , Sistema Libre de Células/ultraestructura , Citocalasina B/farmacología , Endopeptidasas , Proteína Ligando Fas , Citometría de Flujo , Humanos , Hidrólisis , Células Jurkat , Ligandos , Glicoproteínas de Membrana/antagonistas & inhibidores , Glicoproteínas de Membrana/toxicidad , Microscopía Electrónica de Transmisión de Rastreo , Datos de Secuencia Molecular , Fitohemaglutininas/farmacología , Linfocitos T/química , Linfocitos T/ultraestructura , Ligando Inductor de Apoptosis Relacionado con TNF , Factor de Necrosis Tumoral alfa/antagonistas & inhibidores , Factor de Necrosis Tumoral alfa/toxicidad , Ultracentrifugación , Vacuolas/química , Vacuolas/inmunología , Vacuolas/metabolismo , Vacuolas/ultraestructura , Receptor fas/toxicidadRESUMEN
Freshly isolated fetal hepatocytes transformed 4.3, 8.5 and 19.2 pmol/min/10(6) cells of stearic, linoleic and alpha-linolenic acids, respectively, complexed to albumin or alpha-fetoprotein (AFP), to more unsaturated derivatives. Thus, fetal hepatocytes displayed high elongase and delta9, delta6, delta5-desaturase activities, as well as an ability to synthesize hexaene derivatives. Desaturase activities decreased when the time of culture of fetal hepatocytes (previous to incubation with the substrate) was prolonged, being practically undetectable after 24 h of culture. However, the rate of fatty acid uptake remained nearly constant. When AFP was used as the carrier the amount of hexaene fatty acid derivatives of alpha-linolenic acid recovered in cells was reduced up to 50% by albumin. This effect was associated with an increase of radioactivity found in the culture medium of hepatocytes incubated with AFP compared to albumin. Both observations taken together could be explained by an efflux of hexaene derivatives from cells caused by AFP.
Asunto(s)
Ácido Graso Desaturasas/metabolismo , Hígado/embriología , Hígado/metabolismo , alfa-Fetoproteínas/farmacología , Ácido alfa-Linolénico/metabolismo , Animales , Transporte Biológico , Ácido Graso Desaturasas/efectos de los fármacos , Ácidos Grasos/análisis , Ácidos Grasos/metabolismo , Ácido Linoleico/metabolismo , Hígado/citología , Hígado/efectos de los fármacos , Ratas , Ratas Wistar , Ácidos Esteáricos/metabolismoRESUMEN
Alpha-fetoprotein (AFP) is a major globulin of embryonic plasma and a physiological carrier of unesterified fatty acids. In the present work, we have characterized the interaction of AFP and albumin, a major serum protein of adult mammals which presents numerous biochemical analogies with AFP, with the plasma membrane of the rat Morris 7777 hepatoma cells. Time course analysis of the uptake of AFP and albumin by these cells showed a saturable profile at 4 degrees C and 37 degrees C. Saturable binding of 125I-AFP or 125I-albumin were observed when the concentration of these proteins increased (ranging from 0.3 to 4.5 microM). The Hill and Scatchard analysis revealed the existence of binding sites in the surface of hepatoma cells, with a k'd = 2.2 x 10(-6) M (2.9 x 10(6) sites/cell) in the case of AFP and a k'd = 4.5 x 10(-6) M (3.9 x 10(6) sites/cell) in the case of albumin. 125I-AFP and 125I-albumin bound to the cells were completely displaced in the presence of a 200-fold excess of unlabeled AFP or albumin, respectively, suggesting that these interactions were specific. We have observed crossed competition between AFP and albumin for their respective binding sites; no such crossed competition was observed when an excess of unlabeled transferrin was added. Pulse-chase experiments showed that about 50% of the AFP and 75% of the albumin taken up by the cells were released undegraded into the medium after 1 h. Cytochemical studies performed with covalent conjugates of AFP, albumin and transferrin with horseradish peroxidase have shown that AFP and albumin entered the cells via a vesicular system. This intracellular pathway is different from that of transferrin, a plasma protein whose internalization mediated by specific receptors via coated pits has been reported in other cells. The results presented here suggest that AFP and albumin interact with sites in the membrane of hepatoma cells, probably physically related, and then they are transported inside the cells by a mechanism different from that described for transferrin.
Asunto(s)
Neoplasias Hepáticas Experimentales/metabolismo , Albúmina Sérica/farmacocinética , alfa-Fetoproteínas/farmacocinética , Animales , Membrana Celular/metabolismo , Endocitosis , Peroxidasa de Rábano Silvestre/farmacocinética , Radioisótopos de Yodo , Ratas , Transferrina/farmacocinética , Células Tumorales CultivadasRESUMEN
The interaction of Fas with Fas ligand (FasL) mediates activation-induced cell death (AICD) of T hybridomas and of mature T lymphocytes. The TNF/TNF receptor system also plays a significant role in AICD of mature T cells and in the maintenance of peripheral tolerance. We previously demonstrated that in human Jurkat leukemia cells, AICD is triggered mainly by the rapid release of preformed FasL upon TCR stimulation. In the present work, we show that the cytotoxic cytokine APO2 ligand (APO2L; also known as TRAIL) is constitutively expressed as an intracytoplasmic protein in Jurkat T cells and derived sublines. APO2L is also detected in fresh human peripheral blood mononuclear cells (PBMC) from a significant number of donors, and the amount of both FasL and APO2L substantially increases upon blast generation. A neutralizing anti-APO2L monoclonal antibody (mAb) partially suppresses the cytotoxicity induced by supernatants of phytohemagglutinin (PHA)-prestimulated Jurkat or human PBMC on non-activated Jurkat cells, indicating that APO2L is released by these cells and contributes to AICD. A combination of neutralizing anti-APO2L and anti-Fas mAb blocks around 60 % of the toxicity associated with supernatants from PHA-activated human PBMC. These results show that FasL and APO2L account for the majority of cytotoxic activity released during AICD, and suggest that additional uncharacterized factors may also contribute to this process.
Asunto(s)
Apoptosis/inmunología , Células Jurkat/inmunología , Activación de Linfocitos , Glicoproteínas de Membrana/inmunología , Linfocitos T/inmunología , Factor de Necrosis Tumoral alfa/inmunología , Receptor fas/inmunología , Proteínas Reguladoras de la Apoptosis , Proteína Ligando Fas , Humanos , Células Jurkat/patología , Transducción de Señal/inmunología , Linfocitos T/patología , Ligando Inductor de Apoptosis Relacionado con TNFRESUMEN
In an experimental infection model mimicking acute Actinobacillus pleuropneumoniae (Ap) infection in swine (Sus scrofa) by aerosol inoculation, the development of a number of typical clinical signs was accompanied by a prototypic acute phase reaction encompassing fever and an acute phase protein response peaking at around 2 days after infection. Haptoglobin, C-reactive protein (CRP), and major acute phase protein (MAP) responded with large increases in serum levels, preceding the development of specific antibodies by 4-5 days. Serum amyloid A protein (SAA) was also strongly induced. The increase, kinetics of induction and normalization were different between these proteins. It is concluded that experimental Ap-infection by the aerosol route induces a typical acute phase reaction in the pig, and that pig Hp, CRP, MAP, and SAA are major acute phase reactants. These findings indicate the possibility of using one or more of these reactants for the nonspecific surveillance of pig health status.
Asunto(s)
Infecciones por Actinobacillus/sangre , Actinobacillus pleuropneumoniae , Proteínas de Fase Aguda/metabolismo , Reacción de Fase Aguda , Animales , Proteína C-Reactiva/metabolismo , Haptoglobinas/metabolismo , Masculino , Sensibilidad y Especificidad , Proteína Amiloide A Sérica/metabolismo , PorcinosRESUMEN
TCR engagement activates phospholipase C gamma 1 (PLC gamma 1) via a tyrosine phosphorylation-dependent mechanism. PLC gamma 1 contains a pair of Src homology 2 (SH2) domains whose function is that of promoting protein interactions by binding phosphorylated tyrosine and adjacent amino acids. The role of the PLC gamma 1 SH2 domains in PLC gamma 1 phosphorylation was explored by mutational analysis of an epitope-tagged protein transiently expressed in Jurkat T cells. Mutation of the amino-terminal SH2 domain (SH2(N) domain) resulted in defective tyrosine phosphorylation of PLC gamma 1 in response to TCR/CD3 perturbation. In addition, the PLC gamma 1 SH2(N) domain mutant failed to associate with Grb2 and a 36- to 38-kDa phosphoprotein (p36-38), which has previously been recognized to interact with PLC gamma 1, Grb2, and other molecules involved in TCR signal transduction. Conversely, mutation of the carboxyl-terminal SH2 domain (SH2(C) domain) did not affect TCR-induced tyrosine phosphorylation of PLC gamma 1. Furthermore, binding of p36-38 to PLC gamma 1 was not abrogated by mutations of the SH2(C) domain. In contrast to TCR/CD3 ligation, treatment of cells with pervanadate induced tyrosine phosphorylation of either PLC gamma 1 SH2(N) or SH2(C) domain mutants to a level comparable with that of the wild-type protein, indicating that pervanadate treatment induces an alternate mechanism of PLC gamma 1 phosphorylation. These data indicate that the SH2(N) domain is required for TCR-induced PLC gamma 1 phosphorylation, presumably by participating in the formation of a complex that promotes the association of PLC gamma 1 with a tyrosine kinase.
Asunto(s)
Proteínas Adaptadoras Transductoras de Señales , Isoenzimas/metabolismo , Isoenzimas/fisiología , Complejo Receptor-CD3 del Antígeno de Linfocito T/fisiología , Fosfolipasas de Tipo C/metabolismo , Fosfolipasas de Tipo C/fisiología , Tirosina/metabolismo , Dominios Homologos src/inmunología , Sustitución de Aminoácidos/genética , Animales , Arginina/genética , Bovinos , Receptores ErbB/metabolismo , Proteína Adaptadora GRB2 , Glutatión Transferasa/genética , Humanos , Isoenzimas/genética , Células Jurkat , Mutagénesis Sitio-Dirigida , Fosfolipasa C gamma , Fosfoproteínas/genética , Fosforilación/efectos de los fármacos , Unión Proteica/genética , Estructura Terciaria de Proteína , Proteínas/metabolismo , Proteínas Recombinantes de Fusión/metabolismo , Fosfolipasas de Tipo C/genética , Vanadatos/farmacología , Dominios Homologos src/efectos de los fármacos , Dominios Homologos src/genéticaRESUMEN
Apoptosis induced by effector cells of the immune system or by cytotoxic drugs is a main mechanism mediating the prevention or elimination of tumoral cells. For instance, the human T-cell leukemia Jurkat is sensitive to Fas-induced apoptosis and to activation-induced cell death (AICD), and the promonocytic leukemia U937 is sensitive to Fas- and TNF-induced apoptosis. In this work, we have analyzed the mechanisms of resistance to physiological or pharmacological apoptosis in human leukemia by generating highly proliferative (hp) sub-lines derived from Jurkat and U937 cells. These hp sub-lines were resistant to Fas- and TNF-induced apoptosis, as well as to AICD. This was due to the complete loss of Fas and TNFR surface expression and, in the case of Jurkat-derived sub-lines, also of CD3, CD2 and CD59 molecules. The sub-lines also completely lacked the expression of the apoptotic protease CPP32, present in parental cells. Moreover, these sub-lines were no longer sensitive to doxorubicin-induced apoptosis, which was efficiently blocked by the general caspase inhibitor Z-VAD-fmk in the parental cell lines. These data suggest a molecular mechanism for the development of resistance of leukemic cells to physiological and pharmacological apoptosis inducers, giving rise to highly proliferative tumoral phenotypes. These results also indicate that Fas and CPP32 could be useful prognostic markers for the progression and/or therapy outcome of human leukemias.
Asunto(s)
Apoptosis/fisiología , Cisteína Endopeptidasas/biosíntesis , Células Jurkat/metabolismo , Células Jurkat/patología , Leucemia Promielocítica Aguda/metabolismo , Leucemia Promielocítica Aguda/patología , Receptor fas/biosíntesis , Antibióticos Antineoplásicos/farmacología , Apoptosis/efectos de los fármacos , División Celular/fisiología , Doxorrubicina/farmacología , Ensayos de Selección de Medicamentos Antitumorales , Citometría de Flujo , Humanos , Células Jurkat/enzimología , Leucemia Promielocítica Aguda/enzimología , Fenotipo , Receptores del Factor de Necrosis Tumoral/biosíntesis , Receptores del Factor de Necrosis Tumoral/metabolismo , Células Tumorales Cultivadas , Receptor fas/metabolismoRESUMEN
It has recently been proposed that doxorubicin (DOX) can induce apoptosis in human T-leukemia cells via the Fas/FasL system in an autocrine/paracrine way. We show here that treatment of Jurkat cells with either anti-Fas antibodies, anthracyclin drugs or actinomycin D induces the activation of CPP32 (caspase-3) and apoptosis. However, DOX treatment did not induce the expression of membrane FasL or the release of soluble FasL and co-incubation with blocking anti-Fas antibodies prevented Fas-induced but not DOX-induced apoptosis. All the morphological and biochemical signs of apoptosis induced by anti-Fas or DOX can be prevented by Z-VAD-fmk, a general caspase inhibitor. DEVD-cho, a specific inhibitor of CPP32-like caspases which completely blocks Fas-mediated apoptosis, prevented drug-induced nuclear apoptosis but not cell death. We conclude that: (i) DOX-induced apoptosis in human T-leukemia/lymphoma is Fas-independent and (ii) caspase-3 is responsible of DOX-induced nuclear apoptosis but other Z-VAD-sensitive caspases are implicated in cell death.