Asunto(s)
Neoplasias de los Bronquios/diagnóstico , Linfoma de Células B de la Zona Marginal/diagnóstico , Anciano de 80 o más Años , Protocolos de Quimioterapia Combinada Antineoplásica/uso terapéutico , Clorhidrato de Bendamustina/administración & dosificación , Neoplasias de los Bronquios/tratamiento farmacológico , Broncoscopía , Femenino , Humanos , Linfoma de Células B de la Zona Marginal/tratamiento farmacológico , Membrana Mucosa , Rituximab/administración & dosificación , Tomografía Computarizada por Rayos XAsunto(s)
Antineoplásicos/uso terapéutico , Linfoma de Células B Grandes Difuso/terapia , Trasplante de Células Madre , Adulto , Anciano , Anticuerpos Monoclonales de Origen Murino/administración & dosificación , Femenino , Humanos , Linfoma de Células B Grandes Difuso/tratamiento farmacológico , Masculino , Persona de Mediana Edad , Pronóstico , Inducción de Remisión , Estudios Retrospectivos , Rituximab , Acondicionamiento Pretrasplante , Trasplante Autólogo , Resultado del TratamientoAsunto(s)
Aminoglicósidos/administración & dosificación , Anticuerpos Monoclonales/administración & dosificación , Antineoplásicos/administración & dosificación , Movilización de Célula Madre Hematopoyética , Leucemia Promielocítica Aguda/terapia , Trasplante de Células Madre de Sangre Periférica , Acondicionamiento Pretrasplante , Anticuerpos Monoclonales Humanizados , Protocolos de Quimioterapia Combinada Antineoplásica/administración & dosificación , Femenino , Gemtuzumab , Humanos , Persona de Mediana Edad , Inducción de Remisión , Trasplante AutólogoRESUMEN
Innate immunity responds to various pathogen-associated molecular patterns (PAMPs) to evaluate the biological nature of foreign materials by using limited numbers of receptors. Analyses of interactions between PAMPs and its receptors are essential to understand the molecular basis regarding how we discriminate self and non-self materials. Upon infection of horseshoe crabs, an arthropod species, rapid hemolymph coagulation is induced to engulf invading microorganisms by a cascade-type reaction. The reaction is very sensitive to lipopolysaccharide and (1-->3)-beta-D-glucans on Gram-negative bacteria and fungi, respectively, and hence is utilized as assay reagents that detect and quantitate these PAMPs with a name of "limulus test." In this mini-review, recognition of (1-->3)-beta-D-glucans by a unique serine protease zymogen factor G of horseshoe crab is described. Molecular dissection and detailed kinetic analyses have revealed that multivalent binding to polymers of a simple target structure is one of the principles that allows stable and specific recognition of PAMPs by pattern recognition receptors in innate immunity.
Asunto(s)
Cangrejos Herradura/inmunología , Inmunidad Innata/inmunología , beta-Glucanos/inmunología , Animales , Factores de Coagulación Sanguínea/inmunología , Cangrejos Herradura/metabolismo , Proteoglicanos , beta-Glucanos/químicaRESUMEN
Because invertebrates lack an adaptive immune system, they had to evolve effective intrinsic defense strategies against a variety of microbial pathogens. This ancient form of host defense, the innate immunity, is present in all multicellular organisms including humans. The innate immune system of the Japanese horseshoe crab Tachypleus tridentatus, serving as a model organism, includes a hemolymph coagulation system, which participates both in defense against microbes and in hemostasis. Early work on the evolution of vertebrate fibrinogen suggested a common origin of the arthropod hemolymph coagulation and the vertebrate blood coagulation systems. However, this conjecture could not be verified by comparing the structures of coagulogen, the clotting protein of the horseshoe crab, and of mammalian fibrinogen. Here we report the crystal structure of tachylectin 5A (TL5A), a nonself-recognizing lectin from the hemolymph plasma of T. tridentatus. TL5A shares not only a common fold but also related functional sites with the gamma fragment of mammalian fibrinogen. Our observations provide the first structural evidence of a common ancestor for the innate immunity and the blood coagulation systems.
Asunto(s)
Coagulación Sanguínea , Proteínas Sanguíneas/química , Evolución Molecular , Lectinas/química , Secuencia de Aminoácidos , Animales , Proteínas Sanguíneas/inmunología , Proteínas Sanguíneas/fisiología , Cristalografía por Rayos X , Fibrinógeno/química , Cangrejos Herradura , Humanos , Inmunidad Innata , Lectinas/inmunología , Lectinas/fisiología , Ligandos , Modelos Moleculares , Datos de Secuencia Molecular , Unión Proteica , Pliegue de Proteína , Estructura Terciaria de Proteína , Homología de Secuencia de AminoácidoRESUMEN
Although genetic studies have revealed a critical role for the toll-like receptor (TLR) 4 in the biological response to lipopolysaccharide (LPS), the activities of ectopically expressed TLR4 and TLR2 are controversial. We have found that under appropriate transfection conditions, both TLR2 and TLR4 mediate LPS-induced NF-kappaB activation in human embryonic kidney 293 cells. The reconstitution systems we established here allow direct biochemical characterization and comparison of activation of each receptor. TLR4 is approximately 100-fold more sensitive to LPS than TLR2. In contrast to the response to commercial LPS preparations, TLR2 is unresponsive to repurified LPS or synthetic lipid A, indicating the requirement for an additional molecule(s). On the other hand, a lipid A-neutralizing reagent, polymyxin B, blocks the ability of the LPS preparation to stimulate both receptors, suggesting that lipid A is also involved in the activation of TLR2. Mutant TLRs harboring a point mutation in the cytoplasmic domain is inactive in transducing the signal upon stimulation, and act as dominant-negative mutants specifically inhibiting the activation of corresponding type of the receptor but not the other type. Thus, the two receptors are independently activated by distinguishable ligands. Nevertheless, the responses of both TLRs to the LPS preparation are strongly dependent on serum and CD14 and LPS-binding protein are essential for the activation of both of the two receptors. Supporting its functional significance, both receptors are found to associate with CD14.
Asunto(s)
Proteínas de Fase Aguda , Proteínas Portadoras/fisiología , Proteínas de Drosophila , Receptores de Lipopolisacáridos/fisiología , Glicoproteínas de Membrana/fisiología , Receptores de Superficie Celular/fisiología , Línea Celular , Humanos , Ligandos , Lípido A/farmacología , Lipopolisacáridos/farmacología , FN-kappa B/metabolismo , Polimixina B/farmacología , Receptor Toll-Like 2 , Receptor Toll-Like 4 , Receptores Toll-LikeRESUMEN
A 55-year-old Japanese man was hospitalized on October 5, 1999, because of high fever. Physical examination revealed neither lymphadenopathy nor hepato-splenomegaly. Laboratory data on admission showed a white blood cell count of 1,580/microliter, a hemoglobin level of 9.1 g/dl, and a platelet count of 113 x 10(3)/microliter. A small percentage of abnormal mononuclear cells were present in the peripheral blood. A bone marrow biopsy specimen demonstrated myelofibrosis and diffuse infiltration of abnormal monoculear cells with a mature B cell phenotype. A bone marrow aspirate showed 29% abnormal mononuclear cells, which had an indented or folded nucleus and reticular nuclear chromatin. Moderate to strong tartarate-resistant acid phosphatase activity was detected in these cells. Although the cytoplasmic projections were poorly preserved in specimens stained with May-Giemsa, fresh preparations showed numerous slender cytoplasmic projections by phase-contrast microscopy. The hairy cells had the phenotype CD5-, CD10-, CD11c+, CD19+, CD20+, CD25+, lambda. The patient was diagnosed as having European-American-type hairy cell leukemia (HCL) without splenomegaly, which is quite rare in Japan. The value of phase-contrast microscopy for recognition of the hairy cells was emphasized. The patient was treated successfully with deoxycoformycin (DCF).
Asunto(s)
Antibióticos Antineoplásicos/uso terapéutico , Leucemia de Células Pilosas/tratamiento farmacológico , Pentostatina/uso terapéutico , Humanos , Leucemia de Células Pilosas/clasificación , Leucemia de Células Pilosas/patología , Masculino , Microscopía de Contraste de Fase , Persona de Mediana Edad , EsplenomegaliaRESUMEN
The transcription factor nuclear factor-kappaB (NF-kappaB) plays crucial roles in a wide variety of cellular functions and its activity is strictly regulated by cytosolic inhibitors known as IkappaBs. We here report a new member of the IkappaB protein family, IkappaB-zeta, harboring six ankyrin repeats at its carboxyl terminus. IkappaB-zeta mRNA is strongly induced after stimulation by lipopolysaccharide. The induction of IkappaB-zeta is also observed by stimulation with interleukin-1beta but not by tumor necrosis factor-alpha. In contrast to cytosolic IkappaB-alpha, -beta, and -epsilon, the induced IkappaB-zeta localizes in the nucleus via its amino-terminal region, which shows no homology with other proteins. Transiently expressed IkappaB-zeta inhibits the NF-kappaB activity without affecting the nuclear translocation of NF-kappaB upon stimulation. The expressed IkappaB-zeta preferentially associates with the NF-kappaB subunit p50 rather than p65 and recombinant IkappaB-zeta proteins inhibit the DNA binding of the p65/p50 heterodimer and the p50/p50 homodimer. Thus, IkappaB-zeta negatively regulates NF-kappaB activity in the nucleus, possibly in order to prevent excessive inflammation. Moreover, transfection of IkappaB-zeta renders cells more susceptible to apoptosis induced by tumor necrosis factor-alpha. The proapoptotic activity of IkappaB-zeta further suggests that it might be one of key regulators for inflammation and other biologically relevant processes.
Asunto(s)
Núcleo Celular/metabolismo , Proteínas I-kappa B/fisiología , Lipopolisacáridos/farmacología , FN-kappa B/metabolismo , Isoformas de Proteínas/fisiología , Secuencia de Aminoácidos , Animales , Secuencia de Bases , Cartilla de ADN , Proteínas I-kappa B/biosíntesis , Macrófagos Peritoneales/efectos de los fármacos , Macrófagos Peritoneales/metabolismo , Ratones , Datos de Secuencia Molecular , Isoformas de Proteínas/biosíntesis , ARN Mensajero/genética , Homología de Secuencia de AminoácidoRESUMEN
We present three cases of cervical spinal fracture, involving two columns without an obvious neurological deficit. Usually if two of three columns are fractured, the injury is considered unstable structurally and clinically. Fortunately our cases did not involve sensory or motor impairment because of an enlargement of the spinal canal.
Asunto(s)
Vértebras Cervicales/lesiones , Imagen por Resonancia Magnética , Fracturas de la Columna Vertebral/diagnóstico , Fracturas de la Columna Vertebral/cirugía , Moldes Quirúrgicos , Femenino , Estudios de Seguimiento , Humanos , Puntaje de Gravedad del Traumatismo , Masculino , Persona de Mediana Edad , Enfermedades del Sistema Nervioso/diagnóstico , Enfermedades del Sistema Nervioso/etiología , Procedimientos Ortopédicos/métodos , Fracturas de la Columna Vertebral/complicaciones , Fracturas de la Columna Vertebral/rehabilitación , Resultado del TratamientoRESUMEN
Activators of peroxisome proliferator-activated receptor gamma (PPAR gamma), which forms a heterodimer with retinoic X receptor (RXR), inhibit the production of certain inflammatory mediators. To clarify the role of the PPAR gamma:RXR signaling pathway in Kupffer cells, we studied the effect of an RXR agonist and PPARgamma agonist on LPS-induced nitric oxide (NO) and tumor necrosis factor-alpha (TNF-alpha) production. An RXR-specific agonist, Ro47-5944, and a PPAR gamma-specific agonist, AD4833 (pioglitazone hydrochloride), each inhibited LPS-induced NO and TNF-alpha production. The combined treatment of Ro47-5944 and AD4833 resulted in enhanced inhibition, and suppressed the mRNA levels of NO and TNF-alpha. PPAR gamma:RXR activation did not affect the level of LPS-induced phosphorylation of c-jun N-terminal kinase and p38 mitogen-activated protein kinase. PPAR gamma:RXR activation also did not affect nuclear factor kappa B (NF-kappa B) nuclear translocation nor NF-kappa B and activator protein 1 (AP-1) activation in the electrophoretic mobility-shift assay. Finally, PPAR gamma:RXR activation suppressed the LPS-induced promoter activity of the NF-kappa B-luciferase reporter gene in RAW 264.7 cells. These data imply that PPARgamma:RXR activation suppresses LPS-induced NO and TNF-alpha production in Kupffer cells, and that this inhibition occurred at the transcriptional level. Although no consensus PPAR gamma:RXR-responsive element in the promoter regions of the inducible isoform of nitric oxide synthase (iNOS) and TNF-alpha genes was found, PPAR gamma:RXR may interfere with NF-kappa B and AP-1 transcriptional activity. Our data also suggest a potential therapeutic approach for moderating hepatic injury such as endotoxin shock in which Kupffer cell activation has been implicated.
Asunto(s)
Macrófagos del Hígado/metabolismo , Óxido Nítrico/antagonistas & inhibidores , Receptores Citoplasmáticos y Nucleares/fisiología , Receptores de Ácido Retinoico/fisiología , Factores de Transcripción/fisiología , Factor de Necrosis Tumoral alfa/antagonistas & inhibidores , Animales , Transporte Biológico/fisiología , Células Cultivadas , Combinación de Medicamentos , Sinergismo Farmacológico , Electroforesis , Genes Reporteros/genética , Lipopolisacáridos/farmacología , Luciferasas/genética , Masculino , FN-kappa B/genética , FN-kappa B/metabolismo , Óxido Nítrico/biosíntesis , Óxido Nítrico Sintasa/genética , Óxido Nítrico Sintasa de Tipo II , Regiones Promotoras Genéticas/fisiología , ARN Mensajero/antagonistas & inhibidores , Ratas , Ratas Wistar , Receptores Citoplasmáticos y Nucleares/agonistas , Receptores de Ácido Retinoico/agonistas , Receptores X Retinoide , Transducción de Señal/fisiología , Factor de Transcripción AP-1/metabolismo , Factores de Transcripción/agonistas , Factor de Necrosis Tumoral alfa/biosíntesis , Factor de Necrosis Tumoral alfa/genéticaRESUMEN
A clottable protein coagulogen of the horseshoe crab Tachypleus tridentatus is proteolytically converted into an insoluble coagulin gel through non-covalent self-polymerization. Here we identified binding sites for the polymerization. A tryptic fragment, derived from the coagulin polymer chemically cross-linked by a bifunctional cross-linker, was isolated. Amino acid sequence analysis indicated that the fragment consists of two peptides cross-linked between Lys(85) and Lys(156). The two lysine residues are oppositely located at the head and tail regions of the elongated molecule separated by a much greater distance than the length of the cross-linker, which suggests that the cross-linking occurs intermolecularly. Based on the x-ray structural analysis, exposure of a hydrophobic cove on the head in response to the release of peptide C has been postulated (Bergner, A., Oganessyan, V., Muta, T., Iwanaga, S., Typke, D., Huber, R., and Bode, W. (1996) EMBO J. 15, 6789-6797). An octapeptide containing Tyr(136), which occupies the tail end of coagulin, was found to inhibit the polymerization. Replacement of Tyr(136) of the peptide with Ala resulted in loss of the inhibitory activity. These results indicated that the polymerization of coagulin proceeds through the interaction between the newly exposed hydrophobic cove on the head and the wedge-shaped hydrophobic tail.
Asunto(s)
Tromboplastina/química , Tromboplastina/metabolismo , Alanina/química , Secuencia de Aminoácidos , Animales , Sitios de Unión , Unión Competitiva , Cromatografía Líquida de Alta Presión , Reactivos de Enlaces Cruzados/farmacología , Electroforesis en Gel de Poliacrilamida , Cangrejos Herradura , Cinética , Lisina/química , Modelos Moleculares , Datos de Secuencia Molecular , Biosíntesis de Péptidos , Péptidos/química , Unión Proteica , Estructura Terciaria de Proteína , Resonancia por Plasmón de Superficie , Tromboplastina/antagonistas & inhibidores , Factores de Tiempo , Tirosina/química , Rayos XRESUMEN
Stimulation of monocytes/macrophages with lipopolysaccharide (LPS) results in activation of nuclear factor-kappaB (NF-kappaB), which plays crucial roles in regulating expression of many genes involved in the subsequent inflammatory responses. Here, we investigated roles of transforming growth factor-beta activated kinase 1 (TGF-TAK1), a mitogen-activated protein kinase kinase kinase (MAPKKK), in the LPS-induced signaling cascade. A kinase-negative mutant of TAK1 inhibited the LPS-induced NF-kappaB activation both in a macrophage-like cell line, RAW 264.7, and in human embryonic kidney 293 cells expressing toll-like receptor 2 or 4. Furthermore, we demonstrated that endogenous TAK1 is phosphorylated upon simulation of RAW 264.7 cells with LPS. These results indicate that TAK1 functions as a critical mediator in the LPS-induced signaling pathway.
Asunto(s)
Proteínas de Drosophila , Quinasas Quinasa Quinasa PAM/metabolismo , Macrófagos/fisiología , Glicoproteínas de Membrana/metabolismo , FN-kappa B/metabolismo , Receptores de Superficie Celular/metabolismo , Animales , Línea Celular , Humanos , Lipopolisacáridos , Quinasas Quinasa Quinasa PAM/genética , Activación de Macrófagos , Macrófagos/efectos de los fármacos , Glicoproteínas de Membrana/genética , Ratones , Fosforilación/efectos de los fármacos , Receptores de Superficie Celular/genética , Transducción de Señal , Receptor Toll-Like 2 , Receptores Toll-Like , TransfecciónRESUMEN
1-Methyl-4-phenyl-1,2,3,6-tetrahydropyridine has been reported to cause parkinsonism via its neurotoxic form, 1-methyl-4-phenylpyridinium ion (MPP+), which inhibits complex I of the mitochondrial respiratory chain. Its parkinsonism-causing mechanisms attract a great deal of interest as a model of the disease. Recently, we reported that MPP+ strongly decreases the amount of mtDNA independent of the inhibition of complex I. Maintenance of a proper amount of mtDNA is essential for the normal function of mitochondria as exemplified in many mitochondrial diseases. The most characteristic feature in vertebral mtDNA replication is that H-strand synthesis proceeds displacing the parental H-strand as a long single strand. It forms the D-loop, a triplex replication intermediate composed of the parental L-strand, nascent H-strand and displaced H-strand. Here we show that MPP+ does not inhibit DNA synthesis by DNA polymerase gamma, but rather releases the nascent H-strands from mtDNA both in organello and in vitro. This indicates that MPP+ directly destabilizes the D-loop structure, thereby inhibiting replication. This study raises a new mechanism, i.e. destabilization of replication intermediates, for depletion of mtDNA.
Asunto(s)
1-Metil-4-fenilpiridinio/farmacología , Replicación del ADN/efectos de los fármacos , ADN Mitocondrial/química , Conformación de Ácido Nucleico/efectos de los fármacos , Enfermedad de Parkinson Secundaria/inducido químicamente , ADN Polimerasa gamma , Replicación del ADN/genética , ADN Mitocondrial/biosíntesis , ADN Mitocondrial/genética , ADN Mitocondrial/metabolismo , ADN de Cadena Simple/biosíntesis , ADN de Cadena Simple/química , ADN de Cadena Simple/genética , ADN de Cadena Simple/metabolismo , ADN Superhelicoidal/química , ADN Superhelicoidal/genética , ADN Superhelicoidal/metabolismo , ADN Polimerasa Dirigida por ADN/metabolismo , Células HeLa , Calor , Humanos , Concentración de Iones de Hidrógeno , Cinética , Mitocondrias/efectos de los fármacos , Mitocondrias/enzimología , Mitocondrias/genética , Desnaturalización de Ácido Nucleico/efectos de los fármacos , Enfermedad de Parkinson Secundaria/genética , Yoduro de Potasio/farmacología , Proteínas Recombinantes/metabolismoRESUMEN
Innate immunity against microbial pathogens relies on the pattern recognition of cell wall components on invading microbes. Recent evidence has shown that a mammalian Toll-like receptor (TLR) is activated by bacterial lipopolysaccharides (LPS). The innate immunity in invertebrates is also triggered by LPS, as seen in the hemolymph coagulation in horseshoe crab. We report the cloning of a TLR from the Japanese horseshoe crab Tachypleus tridentatus. A cDNA coding for Tachypleus Toll was isolated from a hemocyte cDNA library and the open reading frame codes for a proprotein including a signal sequence. Like Drosophila Toll, Tachypleus Toll is a type I transmembrane protein with an extracellular domain consisting of two leucine-rich repeats flanked by two cystein-rich clusters and a cytoplasmic domain exhibiting striking similarity with the cytoplasmic domain of interleukin-1 receptor. Tachypleus Toll is most similar to Drosophila Toll in the domain architecture and the overall length.
Asunto(s)
Proteínas de Drosophila , Drosophila/inmunología , Cangrejos Herradura/inmunología , Proteínas de Insectos/química , Proteínas de Insectos/inmunología , Glicoproteínas de Membrana/química , Glicoproteínas de Membrana/inmunología , Receptores de Superficie Celular , Animales , Clonación Molecular , ADN Complementario/genética , Drosophila/genética , Cangrejos Herradura/genética , Proteínas de Insectos/genética , Lipopolisacáridos/farmacología , Glicoproteínas de Membrana/genética , Estructura Terciaria de Proteína , Especificidad de la Especie , Receptores Toll-LikeRESUMEN
A 57-year-old woman was hospitalized because of gait disturbance and dysuria. Close examination revealed a cauda equina tumor at the level of L2 and L3. Tumor resection was performed, with posterolateral fusion and spinal instrumentation. On the eleventh day after the surgery, she experienced dyspnea and chest pain during standing and walking exercise. Pulmonary thromboembolism was diagnosed, based on: (1) blood gas analysis findings of hypoxemia and (2) defective images in both of the upper lobes on urgent pulmonary blood flow scintigram. Her clinical status improved with urgent thrombolytic therapy (with tisokinase and urokinase) and anticoagulation therapy (with heparin and warfarin), and her life was saved. When pulmonary thromboembolism occurs, early diagnosis by pulmonary blood flow scintigram and early thrombolytic and anticoagulative therapies are necessary. Special attention should be paid to symptoms of pulmonary thromboembolism in patients after spinal surgery.
Asunto(s)
Cauda Equina/cirugía , Neoplasias del Sistema Nervioso Periférico/cirugía , Complicaciones Posoperatorias , Embolia Pulmonar/etiología , Anticoagulantes/uso terapéutico , Dolor en el Pecho/etiología , Disnea/etiología , Femenino , Heparina/uso terapéutico , Humanos , Hipoxia/etiología , Vértebras Lumbares/cirugía , Persona de Mediana Edad , Activadores Plasminogénicos/uso terapéutico , Complicaciones Posoperatorias/tratamiento farmacológico , Embolia Pulmonar/tratamiento farmacológico , Fusión Vertebral/efectos adversos , Fusión Vertebral/instrumentación , Terapia Trombolítica , Activador de Tejido Plasminógeno/uso terapéutico , Activador de Plasminógeno de Tipo Uroquinasa/uso terapéutico , Warfarina/uso terapéuticoRESUMEN
We have characterized and cloned newly isolated lectins from hemolymph plasma of the horseshoe crab Tachypleus tridentatus, which we named tachylectins 5A and 5B (TLs-5). TLs-5 agglutinated all types of human erythrocytes and Gram-positive and Gram-negative bacteria. TLs-5 specifically recognize acetyl group-containing substances including noncarbohydrates; the acetyl group is required and is sufficient for recognition. TLs-5 enhanced the antimicrobial activity of a horseshoe crab-derived big defensin. cDNA sequences of TLs-5 indicated that they consist of a short N-terminal Cys-containing segment and a C-terminal fibrinogen-like domain with the highest sequence identity (51%) to that of mammalian ficolins. TLs-5, however, lack the collagenous domain found in a kind of "bouquet arrangement" of ficolins and collectins. Electron microscopy revealed that TLs-5 form two- to four-bladed propeller structures. The horseshoe crab is equipped with a unique functional homologue of vertebrate fibrinogen, coagulogen, as the target protein of the clotting cascade. Our observations clearly show that the horseshoe crab has fibrinogen-related molecules in hemolymph plasma and that they function as nonself-recognizing lectins. An ancestor of fibrinogen may have functioned as a nonself-recognizing protein.
Asunto(s)
Proteínas Sanguíneas/química , Proteínas Sanguíneas/inmunología , Eritrocitos/inmunología , Fibrinógeno/química , Cangrejos Herradura/inmunología , Lectinas/química , Lectinas/inmunología , Sistema del Grupo Sanguíneo ABO , Secuencia de Aminoácidos , Animales , Proteínas Sanguíneas/farmacología , Carbohidratos , ADN Complementario , Escherichia coli/efectos de los fármacos , Pruebas de Hemaglutinación , Humanos , Cinética , Lectinas/farmacología , Mamíferos , Pruebas de Sensibilidad Microbiana , Datos de Secuencia Molecular , Fragmentos de Péptidos/química , Salmonella/efectos de los fármacos , Alineación de Secuencia , Homología de Secuencia de AminoácidoRESUMEN
The most unique feature in the replication of mitochondrial DNA (mtDNA) is that most of the newly synthesized heavy strands (H-strands) terminate prematurely, resulting in the formation of displacement loop (D-loop) strands. Only the H-strand which proceeds past the termination site is a true nascent H-strand leading to the overall replication on a circular mtDNA molecule. The physiological significance of the D-loop formation has long been unclear. To examine the role of premature termination in mtDNA replication, we therefore developed a method for selectively measuring both the total amount of nascent H-strands and the amount of true nascent H-strands using ligation-mediated polymerase chain reaction, which, for the first time, enabled us to estimate the frequency of premature termination. The stimulation of cell proliferation with interleukin 2 and phytohemagglutinin in human peripheral T lymphocytes caused an increase in the net replication rate of mtDNA. In stimulated cells, in comparison to resting ones, the amount of true nascent H-strands increased approx. 2.6-fold while the total amount of nascent H-strands remained unchanged, indicating that premature termination decreased while the initiation of replication remained the same. Our findings thus demonstrate the first clear example that premature termination plays a primary role in the up-regulation of the net rate of mtDNA replication in human cells.