RESUMEN

Dipeptidyl peptidases (DP) 8 and 9 are homologous, cytoplasmic N-terminal post-proline-cleaving enzymes that are anti-targets for the development of DP4 (DPPIV/CD26) inhibitors for treating type II diabetes. To date, DP8 and DP9 have been implicated in immune responses and cancer biology, but their pathophysiological functions and substrate repertoire remain unknown. This study utilizes terminal amine isotopic labeling of substrates (TAILS), an N-terminal positional proteomic approach, for the discovery of in vivo DP8 and DP9 substrates. In vivo roles for DP8 and DP9 in cellular metabolism and homeostasis were revealed via the identification of more than 29 candidate natural substrates and pathways affected by DP8/DP9 overexpression. Cleavage of 14 substrates was investigated in vitro; 9/14 substrates for both DP8 and DP9 were confirmed by MALDI-TOF MS, including two of high confidence, calreticulin and adenylate kinase 2. Adenylate kinase 2 plays key roles in cellular energy and nucleotide homeostasis. These results demonstrate remarkable in vivo substrate overlap between DP8/DP9, suggesting compensatory roles for these enzymes. This work provides the first global investigation into DP8 and DP9 substrates, providing a number of leads for future investigations into the biological roles and significance of DP8 and DP9 in human health and disease.

Asunto(s)

Adenilato Quinasa/metabolismo , Calreticulina/metabolismo , Dipeptidasas/metabolismo , Dipeptidil-Peptidasas y Tripeptidil-Peptidasas/metabolismo , Proteómica/métodos , Secuencia de Aminoácidos , Cationes , Línea Celular Tumoral , Separación Celular , Citoplasma/metabolismo , Metabolismo Energético , Citometría de Flujo , Homeostasis , Humanos , Marcaje Isotópico , Espectrometría de Masas , Datos de Secuencia Molecular , Estructura Terciaria de Proteína , Especificidad por Sustrato

RESUMEN

Resistance (R) proteins are key regulators of the plant innate immune system and are capable of pathogen detection and activation of the hypersensitive cell death immune response. To understand the molecular mechanism of R protein activation, we undertook a phenotypic and biochemical study of the flax nucleotide binding (NB)-ARC leucine-rich repeat protein, M. Using Agrobacterium-mediated transient expression in flax cotyledons, site-directed mutations of key residues within the P-loop, kinase 2, and MHD motifs within the NB-ARC domain of M were shown to affect R protein function. When purified using a yeast expression system and assayed for ATP and ADP, these mutated proteins exhibited marked differences in the quantity and identity of the bound nucleotide. ADP was bound to recombinant wild-type M protein, while the nonfunctional P-loop mutant did not have any nucleotides bound. In contrast, ATP was bound to an autoactive M protein mutated in the highly conserved MHD motif. These data provide direct evidence supporting a model of R protein function in which the "off" R protein binds ADP and activation of R protein defense signaling involves the exchange of ADP for ATP.

Asunto(s)

Adenosina Difosfato/metabolismo , Adenosina Trifosfato/metabolismo , Lino/metabolismo , Proteínas de Plantas/metabolismo , Lino/genética , Regulación de la Expresión Génica de las Plantas/fisiología , Mutagénesis Sitio-Dirigida , Mutación , Hojas de la Planta/metabolismo , Proteínas de Plantas/genética , Unión Proteica , Estructura Terciaria de Proteína , Proteínas Recombinantes , Rhizobium

RESUMEN

Dipeptidyl peptidase (DP) 8 belongs to the dipeptidyl peptidase IV gene family. DP8 has been implicated in immune function and asthma, although its biological function is yet unknown. Structures of the homologs, fibroblast activation protein (FAP) and DPIV, are known but the DP8 structure is yet to be resolved. To help characterise the DP8 substrate pocket, mutants of residues lining the pocket were produced at DP8(D772), DP8(Y315), DP8(H434) and DP8(D435) and assessed by substrate kinetics and size-exclusion chromatography. Mutations of DP8(D772A/E/S/V) affected catalysis but did not confer endopeptidase activity. Mutations of DP8(H434F), DP8(D435F) and DP8(Y315F) reduced catalytic activity. Furthermore, mutations to DP8(D772A/E/S/V), DP8(H434F), DP8(D435F) and DP8(Y315F) affected dimer stabilisation. Homology modelling of DP8 using DPIV and FAP crystal structures suggested that DP8(D772), DP8(H434) and DP8(D435) were located at the edge of the S2 catalytic pocket, contributing to the junction between the alpha-beta hydrolase and beta-propeller domains. This study provides insights into how the DP8 substrate pocket and dimer interface differ from DPIV and FAP which could be utilised for designing more selective DP8 inhibitors.

Asunto(s)

Biocatálisis , Dominio Catalítico , Dimerización , Dipeptidasas/química , Dipeptidasas/metabolismo , Dominios y Motivos de Interacción de Proteínas , Bases de Datos de Proteínas , Dipeptidasas/genética , Dipeptidasas/aislamiento & purificación , Dipeptidil Peptidasa 4/química , Dipeptidil-Peptidasas y Tripeptidil-Peptidasas/química , Endopeptidasas , Gelatinasas/química , Humanos , Concentración de Iones de Hidrógeno , Interacciones Hidrofóbicas e Hidrofílicas , Cinética , Proteínas de la Membrana/química , Mutagénesis Sitio-Dirigida , Proteínas Mutantes/química , Proteínas Mutantes/aislamiento & purificación , Proteínas Mutantes/metabolismo , Estabilidad Proteica , Estructura Cuaternaria de Proteína , Proteínas Recombinantes/química , Proteínas Recombinantes/aislamiento & purificación , Proteínas Recombinantes/metabolismo , Alineación de Secuencia , Homología de Secuencia de Aminoácido , Serina Endopeptidasas/química , Especificidad por Sustrato

RESUMEN

The developmental stages of Hepatozoon tuatarae were elucidated in both the tuatara host, Sphenodon punctatus and the tick, Amblyomma sphenodonti. PCR amplicons from A. sphenodonti samples identified DNA matching H. tuatarae. Dissection of tick samples showed oogenesis and sporogony occurring in the haemocoel of A. sphenodonti with the average mature oocyst size being 236 x 228 microm. Partial sequence data of the parasite's small subunit ribosomal gene, obtained by PCR, was used for phylogenetic comparison. Characterisation of the H. tuatarae lifecycle will help in conservation management of the tuatara.

Asunto(s)

Vectores Arácnidos/parasitología , Coccidiosis/veterinaria , Eucoccidiida/clasificación , Eucoccidiida/crecimiento & desarrollo , Ixodidae/parasitología , Lagartos/parasitología , Filogenia , Animales , Coccidiosis/parasitología , Eucoccidiida/genética , Eucoccidiida/aislamiento & purificación , Datos de Secuencia Molecular , Proteínas Protozoarias/genética

RESUMEN

Peroxiredoxins (Prxs, EC: 1.11.1.15) are cysteine-dependent peroxidases proposed to function as antioxidant enzymes and also in H2O2-mediated cell signaling. They have been well characterized in yeast, mammals, protists and bacteria but not yet in fish. Here we describe the cloning and functional characterization of a Prx 2 cDNA from southern bluefin tuna (SBT, Thunnus maccoyii), an important aquaculture species in South Australia. The SBT Prx sequence was closely related (76-92% identical) to Prx 1 and 2 sequences from other fish and mammals and phylogenetic analyses showed that it was most likely a Prx 2. The deduced amino acid sequence contained the peroxidatic and resolving Cys residues characteristic of typical 2-Cys Prx proteins from all kingdoms of life. It also contained the GGLG motif associated with the sensitivity of eukaryotic typical 2-Cys Prx proteins to overoxidation and consequent inactivation by H2O2. When the SBT Prx 2 was expressed in E. coli, it showed thioredoxin (Trx)-dependent peroxidase activity with H2O2, cumene hydroperoxide (CuOOH) and t-butyl hydroperoxide (t-bOOH). The SBT Prx displayed Michaelis-Menten kinetics with Trx but sigmoidal kinetics with H2O2 and CuOOH. The K(m)(Trx) was 12 microM and the S(0.5) values for H2O2 and CuOOH were 29 and 25 microM, respectively. At mM concentrations of H2O2, SBT Prx progressively lost its peroxidase activity as has been observed for other eukaryotic typical 2-Cys Prx proteins. The native SBT Prx enzyme existed as a mixture of dimers, tetramers, decamers and a higher order aggregate.

Asunto(s)

Proteínas de Peces/metabolismo , Peroxirredoxinas/metabolismo , Atún/metabolismo , Secuencia de Aminoácidos , Animales , Cromatografía de Afinidad , Clonación Molecular , Cisteína/análisis , Escherichia coli/genética , Proteínas de Peces/química , Proteínas de Peces/genética , Datos de Secuencia Molecular , Peroxirredoxinas/química , Peroxirredoxinas/genética , Filogenia , Alineación de Secuencia , Atún/genética

RESUMEN

Grape thaumatin-like (TL) proteins and chitinases play roles in plant-pathogen interactions and can cause protein haze in white wine unless removed prior to bottling. A two-step method is described that highly purified hundreds of milligrams of TL proteins and chitinases from two juices by strong cation exchange (SCX) and hydrophobic interaction chromatography (HIC). The method was fast and separated isoforms of TL proteins and chitinases from within the same juice, in most cases to >97% purity. The isolated proteins were identified by peptide nanoLC-MS/MS and crystallized using a high-throughput screening method. Crystals from three protein fractions produced high-resolution X-ray crystallography data.

Asunto(s)

Quitinasas/química , Quitinasas/aislamiento & purificación , Cromatografía/métodos , Proteínas de Plantas/química , Proteínas de Plantas/aislamiento & purificación , Vitis/química , Cristalización , Interacciones Huésped-Patógeno , Vitis/microbiología

RESUMEN

N-terminal truncation of chemokines by proteases including dipeptidyl peptidase (DP) IV significantly alters their biological activity; generally ablating cognate G-protein coupled receptor engagement and often generating potent receptor antagonists. DP8 is a recently recognised member of the prolyl oligopeptidase gene family that includes DPIV. Since DPIV is known to process chemokines we surveyed 27 chemokines for cleavage by DP8. We report DP8 cleavage of the N-terminal two residues of IP10 (CXCL10), ITAC (CXCL11) and SDF-1 (CXCL12). This has implications for DP8 substrate specificity. Chemokine cleavage and inactivation may occur in vivo upon cell lysis and release of DP8 or in the inactivation of internalized chemokine/receptor complexes.

Asunto(s)

Quimiocina CXCL10/metabolismo , Quimiocina CXCL11/metabolismo , Quimiocina CXCL12/metabolismo , Dipeptidasas/metabolismo , Quimiocina CXCL10/química , Quimiocina CXCL11/química , Quimiocina CXCL12/química , Dipeptidasas/aislamiento & purificación , Dipeptidil Peptidasa 4/aislamiento & purificación , Dipeptidil Peptidasa 4/metabolismo , Semivida , Humanos , Cinética , Peso Molecular , Procesamiento Proteico-Postraduccional , Proteínas Recombinantes/aislamiento & purificación , Proteínas Recombinantes/metabolismo , Solubilidad , Espectrometría de Masa por Láser de Matriz Asistida de Ionización Desorción , Especificidad por Sustrato

RESUMEN

Dihydroorotase (DHOase, EC 3.5.2.3) from the extreme thermophile Bacillus caldolyticus has been subcloned, sequenced, expressed, and purified as a monomer. The catalytic properties of this thermophilic DHOase have been compared with another type I enzyme, the DHOase domain from hamster, to investigate how the thermophilic enzyme is adapted to higher temperatures. B. caldolyticus DHOase has higher Vmax and Ks values than hamster DHOase at the same temperature. The thermodynamic parameters for the binding of L-dihydroorotate were determined at 25 degrees C for hamster DHOase (deltaG = -6.9 kcal/mol, deltaH = -11.5 kcal/mol, TdeltaS = -4.6 kcal/mol) and B. caldolyticus DHOase (deltaG = -5.6 kcal/mol, deltaH = -4.2 kcal/mol, TdeltaS = +1.4 kcal/mol). The smaller enthalpy release and positive entropy for thermophilic DHOase are indicative of a weakly interacting Michaelis complex. Hamster DHOase has an enthalpy of activation of 12.3 kcal/mol, similar to the release of enthalpy upon substrate binding, rendering the kcat/Ks value almost temperature independent. B. caldolyticus DHOase shows a decrease in the enthalpy of activation from 12.2 kcal/mol at temperatures from 30 to 50 degrees C to 5.3 kcal/mol for temperatures of 50-70 degrees C. Vibrational energy at higher temperatures may facilitate the transition ES --> ES(double dagger), making kcat/Ks almost temperature independent. The pseudo-first-order rate constant for water attack on L-dihydroorotate, based on experiments at elevated temperature, is 3.2 x 10(-11) s(-1) at 25 degrees C, with deltaH(double dagger) = 24.7 kcal/mol and TdeltaS(double dagger) = -6.9 kcal/mol. Thus, hamster DHOase enhances the rate of substrate hydrolysis by a factor of 1.6 x 10(14), achieving this rate enhancement almost entirely by lowering the enthalpy of activation (delta deltaH(double dagger) = -19.5 kcal/mol). Both the rate enhancement and transition state affinity of hamster DHOase increase steeply with decreasing temperature, consistent with the development of H-bonds and electrostatic interactions in the transition state that were not present in the enzyme-substrate complex in the ground state.

Asunto(s)

Bacillus/enzimología , Dihidroorotasa/química , Termodinámica , Secuencia de Aminoácidos , Animales , Catálisis , Cricetinae , Dihidroorotasa/antagonistas & inhibidores , Dihidroorotasa/genética , Activación Enzimática , Hidrólisis , Datos de Secuencia Molecular , Ácido Orótico/análogos & derivados , Ácido Orótico/química , Desnaturalización Proteica , Alineación de Secuencia , Temperatura

Asunto(s)

Dipeptidasas/química , Modelos Moleculares , Estructura Terciaria de Proteína , Sitios de Unión , Dipeptidasas/genética , Dipeptidil Peptidasa 4/química , Dipeptidil Peptidasa 4/genética , Dipeptidil-Peptidasas y Tripeptidil-Peptidasas , Humanos , Datos de Secuencia Molecular , Proteínas del Tejido Nervioso/química , Proteínas del Tejido Nervioso/genética , Péptido Hidrolasas/química , Péptido Hidrolasas/genética , Canales de Potasio/química , Canales de Potasio/genética , Prolil Oligopeptidasas , Homología de Secuencia de Aminoácido , Serina Endopeptidasas/química , Serina Endopeptidasas/genética

RESUMEN

We have cloned genes encoding three enzymes of the de novo pyrimidine pathway using genomic DNA from Plasmodium falciparum and sequence information from the Malarial Genome Project. Genes encoding dihydroorotase (reaction 3), orotate phosphoribosyltransferase (reaction 5), and OMP decarboxylase (reaction 6) have been cloned into the plasmid pET 3a or 3d with a thrombin cleavable 9xHis tag at the C-terminus and the enzymes were expressed in Escherichia coli. To overcome the toxicity of malarial OMP decarboxylase when expressed in E. coli, and the unusual codon usage of the malarial gene, a hybrid plasmid, pMICO, was constructed which expresses low levels of T7 lysozyme to inhibit T7 RNA polymerase used for recombinant expression, and extra copies of rare tRNAs. Catalytically-active OMP decarboxylase has been purified in tens of milligrams by chromatography on Ni-NTA. The gene encoding orotate phosphoribosyltransferase includes an extension of 66 amino acids from the N-terminus when compared with sequences for this enzyme from other organisms. We have found that other pyrimidine enzymes also contain unusual protein inserts. Milligram quantities of pure recombinant malarial enzymes from the pyrimidine pathway will provide targets for development of novel antimalarial drugs.

Asunto(s)

Dihidroorotasa/genética , Orotato Fosforribosiltransferasa/genética , Orotidina-5'-Fosfato Descarboxilasa/genética , Plasmodium falciparum/genética , Animales , Catálisis , Clonación Molecular , Codón , Cartilla de ADN/química , ARN Polimerasas Dirigidas por ADN/metabolismo , Escherichia coli/genética , Escherichia coli/metabolismo , Vectores Genéticos , Malaria/genética , Modelos Químicos , N-Acetil Muramoil-L-Alanina Amidasa/química , Sistemas de Lectura Abierta , Plásmidos/metabolismo , Plasmodium falciparum/enzimología , Estructura Terciaria de Proteína , Proteínas Recombinantes/química , Proteínas Virales

RESUMEN

The coding region of a putative orotidine 5'-monophosphate decarboxylase gene from Plasmodium falciparum was identified in genomic data from the Malarial Genome Sequencing Project. The gene encodes a protein of 323 amino acids with a predicted molecular weight of 37.8 kDa. The gene was cloned into a bacterial expression vector and over-expressed in Escherichia coli. The recombinant protein was purified and shown to have orotidine 5'-monophosphate decarboxylase activity, confirming the identity of the gene.

Asunto(s)

Genes Protozoarios/genética , Orotidina-5'-Fosfato Descarboxilasa/genética , Plasmodium falciparum/genética , Proteínas Protozoarias/genética , Secuencia de Aminoácidos , Animales , Clonación Molecular , ADN Protozoario/genética , Expresión Génica , Datos de Secuencia Molecular , Plasmodium falciparum/enzimología , Alineación de Secuencia , Especificidad de la Especie

Asunto(s)

Escherichia coli/enzimología , Orotidina-5'-Fosfato Descarboxilasa/genética , Orotidina-5'-Fosfato Descarboxilasa/metabolismo , Plásmidos/genética , Plasmodium falciparum/enzimología , Animales , Codón/genética , Escherichia coli/efectos de los fármacos , Escherichia coli/genética , Dosificación de Gen , Humanos , Orotidina-5'-Fosfato Descarboxilasa/toxicidad , Plasmodium falciparum/genética , Proteínas Protozoarias/genética , Proteínas Protozoarias/metabolismo , Proteínas Protozoarias/toxicidad , ARN de Transferencia/genética

RESUMEN

For routine QA measurements of the radiation output of a linear accelerator, an ionization chamber is usually used, the type of which may depend on the radiation quality. In this study, a comparison of the standard ionization chamber and the central diodes of an array, was performed both used simultaneously for the constancy check of the radiation output. It was investigated to which degree the two detectors deviate from each other over a long period of time for different radiation qualities. The aim of the study was to show whether the radiation output can be checked using the diode array, which is usually used for measuring beam profiles, by averaging the values of the central diodes within a reasonable accuracy. The results showed a maximum deviation of the two detectors of 0.9-2.2%, whereas the mean deviation is 0.4-0.8%, both depending on the radiation quality.

Asunto(s)

Física Nuclear/normas , Aceleradores de Partículas/normas , Humanos , Control de Calidad , Radioterapia/normas , Reproducibilidad de los Resultados

RESUMEN

The crystal structure of a novel aluminium fluoride inhibited form of bovine mitochondrial F(1)-ATPase has been determined at 2 A resolution. In contrast to all previously determined structures of the bovine enzyme, all three catalytic sites are occupied by nucleotide. The subunit that did not bind nucleotide in previous structures binds ADP and sulfate (mimicking phosphate), and adopts a "half-closed" conformation. This structure probably represents the posthydrolysis, pre-product release step on the catalytic pathway. A catalytic scheme for hydrolysis (and synthesis) at physiological rates and a mechanism for the ATP-driven rotation of the gamma subunit are proposed based on the crystal structures of the bovine enzyme.

Asunto(s)

Compuestos de Aluminio/metabolismo , Inhibidores Enzimáticos/metabolismo , Fluoruros/metabolismo , Mitocondrias/enzimología , Nucleótidos/metabolismo , ATPasas de Translocación de Protón/química , ATPasas de Translocación de Protón/metabolismo , Adenosina Difosfato/metabolismo , Adenosina Trifosfato/metabolismo , Regulación Alostérica , Animales , Sitios de Unión , Catálisis , Bovinos , Cristalografía por Rayos X , Hidrólisis , Modelos Biológicos , Modelos Moleculares , Estructura Cuaternaria de Proteína , Estructura Terciaria de Proteína , Subunidades de Proteína , ATPasas de Translocación de Protón/antagonistas & inhibidores , Rotación , Relación Estructura-Actividad , Sulfatos/metabolismo

RESUMEN

Analysis of tryptophan mutants of F(1)-ATPase from Escherichia coli [Löbau et al. (1997) FEBS Lett. 404, 15-18] suggested that nucleotide concentrations used to grow crystals for the determination of the structure of bovine F(1)-ATPase [Abrahams et al. (1994) Nature 370, 621-628] would be sufficient to occupy only two catalytic sites, and that higher concentrations of nucleotide would result in all three sites being occupied. We have determined the structure of bovine F(1)-ATPase at 2.9 A resolution with crystals grown in the presence of 5 mM AMPPNP and 5 microM ADP. Similar to previous structures of bovine F(1)-ATPase determined with crystals grown in the presence of lower nucleotide concentrations, only two beta-subunits have bound nucleotide and the third subunit remains empty.

Asunto(s)

Adenosina Difosfato/química , Adenilil Imidodifosfato/química , Mitocondrias/enzimología , ATPasas de Translocación de Protón/química , Animales , Dominio Catalítico , Bovinos , Cristalización , Modelos Moleculares , Nucleótidos , Estructura Secundaria de Proteína

RESUMEN

BACKGROUND: The globular domain of the membrane-associated F(1)F(o)-ATP synthase complex can be detached intact as a water-soluble fragment known as F(1)-ATPase. It consists of five different subunits, alpha, beta, gamma, delta and epsilon, assembled with the stoichiometry 3:3:1:1:1. In the crystal structure of bovine F(1)-ATPase determined previously at 2.8 A resolution, the three catalytic beta subunits and the three noncatalytic alpha subunits are arranged alternately around a central alpha-helical coiled coil in the gamma subunit. In the crystals, the catalytic sites have different nucleotide occupancies. One contains the triphosphate form of the nucleotide, the second contains the diphosphate, and the third is unoccupied. Fluoroaluminate complexes have been shown to mimic the transition state in several ATP and GTP hydrolases. In order to understand more about its catalytic mechanism, F(1)-ATPase was inhibited with Mg(2+)ADP and aluminium fluoride and the structure of the inhibited complex was determined by X-ray crystallography. RESULTS: The structure of bovine F(1)-ATPase inhibited with Mg(2+)ADP and aluminium fluoride determined at 2.5 A resolution differs little from the original structure with bound AMP-PNP and ADP. The nucleotide occupancies of the alpha and beta subunits are unchanged except that both aluminium trifluoride and Mg(2+)ADP are bound in the nucleotide-binding site of the beta(DP) subunit. The presence of aluminium fluoride is accompanied by only minor adjustments in the surrounding protein. CONCLUSIONS: The structure appears to mimic a possible transition state. The coordination of the aluminofluoride group has many features in common with other aluminofluoride-NTP hydrolase complexes. Apparently, once nucleotide is bound to the catalytic beta subunit, no additional major structural changes are required for catalysis to occur.

Asunto(s)

Adenosina Difosfato/farmacología , Compuestos de Aluminio/farmacología , Inhibidores Enzimáticos/farmacología , Fluoruros/farmacología , ATPasas de Translocación de Protón/antagonistas & inhibidores , ATPasas de Translocación de Protón/química , Animales , Dominio Catalítico , Bovinos , Cristalografía por Rayos X , Técnicas In Vitro , Mitocondrias/enzimología , Modelos Moleculares , Conformación Proteica , ATPasas de Translocación de Protón/metabolismo

RESUMEN

There is now compelling evidence in support of a rotary catalytic mechanism in F1-ATPase, and, by extension, in the intact ATP synthase. Although models have been proposed to explain how protein translocation in F0 results in rotation of the gamma-subunit relative to the alpha 3/beta 3 assembly in F1 [22], these are still speculative. It seems likely that a satisfactory explanation of this mechanism will ultimately depend on structural information on the intact ATP synthase.

Asunto(s)

Mitocondrias/enzimología , ATPasas de Translocación de Protón/química , ATPasas de Translocación de Protón/metabolismo , Animales , Catálisis , Dominio Catalítico , Bovinos , Inhibidores Enzimáticos/química , Técnicas In Vitro , Modelos Moleculares , Conformación Proteica , ATPasas de Translocación de Protón/antagonistas & inhibidores , Rotación

Asunto(s)

Vendajes , Medicina Familiar y Comunitaria , Humanos

RESUMEN

Changes in the respiratory rate and the contribution of the cytochrome (Cyt) c oxidase and alternative oxidase (COX and AOX, respectively) were investigated in soybean (Glycine max L. cv Stevens) root seedlings using the 18O-discrimination method. In 4-d-old roots respiration proceeded almost entirely via COX, but by d 17 more than 50% of the flux occurred via AOX. During this period the capacity of COX, the theoretical yield of ATP synthesis, and the root relative growth rate all decreased substantially. In extracts from whole roots of different ages, the ubiquinone pool was maintained at 50% to 60% reduction, whereas pyruvate content fluctuated without a consistent trend. In whole-root immunoblots, AOX protein was largely in the reduced, active form at 7 and 17 d but was partially oxidized at 4 d. In isolated mitochondria, Cyt pathway and succinate dehydrogenase capacities and COX I protein abundance decreased with root age, whereas both AOX capacity and protein abundance remained unchanged. The amount of mitochondrial protein on a dry-mass basis did not vary significantly with root age. It is concluded that decreases in whole-root respiration during growth of soybean seedlings can be largely explained by decreases in maximal rates of electron transport via COX. Flux via AOX is increased so that the ubiquinone pool is maintained in a moderately reduced state.

RESUMEN

An assay for biological dosimetry based on the induction of apoptosis in human T-lymphocytes is described. Radiation-induced apoptosis was assessed by flow cytometric identification of cells displaying apoptosis-associated DNA condensation. CD4 and CD8 T-lymphocytes were analysed. They were recognized on the basis of their cell-surface antigens. Four parameters were measured for both cell types: cell size, granularity, antigen immunofluorescence and DNA content. Apoptosis was quantified as the fraction of CD4-, or CD8-positive cells with a characteristic reduction of cell size and DNA content. At doses below 1 Gy, levels of radiation-induced apoptosis increased for up to 5 days after irradiation. Optimal dose discrimination was observed 4 days after irradiation, at which time the dose-response curves were linear, with a slope of 8% +/- 0.5% per 0.1 Gy. In controlled, dose-response experiments the lowest dose level at which the radiation-induced apoptosis frequency was still significantly above control was 0.05 Gy. After 5 days post-irradiation incubation, intra- and interdonor variations were measured and found to be similar; thus, apoptotic levels depend more on the dose than on the donor. The results demonstrate the potential of this assay as a biological dosimeter.

Asunto(s)

Apoptosis/efectos de la radiación , Linfocitos T CD4-Positivos/efectos de la radiación , Linfocitos T CD8-positivos/efectos de la radiación , Radiometría , Linfocitos T CD4-Positivos/patología , Linfocitos T CD8-positivos/patología , Humanos , Reproducibilidad de los Resultados