Sociodemographic factors affecting the comprehension of clinical information by inpatients undergoing trauma surgery. / Determinantes sociodemográficos de la comprensión de la información clínica en pacientes hospitalizados intervenidos de cirugía traumatológica.

INTRODUCTION: The clinical information process is the basis of the doctor-patient relationship. It starts with the information provided before signing informed consent and ends on the termination of the doctor-patient relationship. The influence of demographic variables in the information process has not been thoroughly studied for inpatients undergoing surgery. In this study we aim to answer two questions: 1) Does gender have an influence on the information process for these patients? 2) Are there other factors that affect the process? METHOD: A prospective study carried out using an 'ad hoc' designed survey on a 200-inpatient sample after undergoing surgery in the trauma and orthopaedics department of our hospital. Sampling was simple random. RESULTS: We found differences in the consistency of the answers by gender in the question regarding surgical priority, with the women having a better understanding of it (p=.04). The rest of the questions show no differences by gender. However, in the population analyzed, age and educational level are the main modifiers of understanding, and they are both related to gender (p<.0001; p=.003, respectively). CONCLUSIONS: In clinical practice, it is fundamental to keep in mind the factors that affect the information process. According to our results, the factors that define greater vulnerability in relation to the information process are age and low educational level.

Chloroplasts regulate leaf senescence: delayed senescence in transgenic ndhF-defective tobacco.

Mitochondrial involvement has not been identified in the programmed cell death (PCD) of leaf senescence which suggests that processes such as those involving reactive oxygen species (ROS) are controlled by chloroplasts. We report that transgenic tobacco (DeltandhF), with the plastid ndhF gene knocked-out, shows low levels of the plastid Ndh complex, homologous to mitochondrial complex I, and more than a 30-day-delay in leaf senescence with respect to wt. The comparison of activities and protein levels and analyses of genetic and phenotypic traits of wtxDeltandhF crosses indicate that regulatory roles of mitochondria in animal PCD are assumed by chloroplasts in leaf senescence. The Ndh complex would increase the reduction level of electron transporters and the generation of ROS. Chloroplastic control of leaf senescence provides a nonclassical model of PCD and reveals an unexpected role of the plastid ndh genes that are present in most higher plants.

Arabidopsis thaliana ecotype Cvi shows an increased tolerance to photo-oxidative stress and contains a new chloroplastic copper/zinc superoxide dismutase isoenzyme.

A new chloroplastic Cu/Zn-superoxide dismutase (SOD) isoenzyme was identified in Arabidopsis thaliana ecotype Cvi. Genetic analyses indicated that the new isoenzyme was encoded by a Cvi-specific allele of Csd2 that was named Csd2-2. Paraquat treatments of A. thaliana ecotypes Ler and Cvi resulted in higher levels of chloroplastic Cu/Zn-SOD activity in Cvi, suggesting that the Cvi isoenzyme has a higher stability and/or turnover rate than the Ler variant under photo-oxidative conditions. In addition, Cvi showed a higher tolerance to paraquat treatments. Hybrid plant populations expressing Csd2-2 also exhibited an increased tolerance, suggesting that the Cvi isoenzyme is one of the factors that contribute to a better fitness in photo-oxidative stress conditions.

Hydrogen peroxide mediates the induction of chloroplastic Ndh complex under photooxidative stress in barley.

Chloroplast-encoded NDH polypeptides (components of the plastid Ndh complex) and the NADH dehydrogenase activity of the Ndh complex (NADH-DH) increased under photooxidative stress. The possible involvement of H2O2-mediated signaling in the photooxidative induction of chloroplastic ndh genes was thoroughly studied. We have analyzed the changes in the NADH-DH and steady-state levels of NDH-F polypeptide and ndhB and ndhF transcripts in barley (Hordeum vulgare cv Hassan) leaves. Subapical leaf segments were incubated in growing light (GL), photooxidative light (PhL), GL and H2O2 (GL + H2O2), or PhL and 50 nM paraquat in the incubation medium. Treatments with H2O2 under GL mimicked the photooxidative stimulus, causing a dose-dependent increase of NADH-DH and NDH-F polypeptide. The kinetic of Ndh complex induction was further studied in leaves pre-incubated with or without the H2O2-scavenger dimethyltiourea. NADH-DH and NDH-F polypeptide rapidly increased up to 16 h in PhL, GL+ H2O2, and, at higher rate, in PhL and paraquat. The observed increases of NADH-DH and NDH-F after 4 h in PhL and GL + H2O2 were not accompanied by significant changes in ndhB and ndhF transcripts. However, at 16-h incubations NADH-DH and NDH-F changes closely correlated with higher ndhB and ndhF transcript levels. All these effects were prevented by dimethylthiourea. It is proposed that the induction of chloroplastic ndh genes under photooxidative stress is mediated by H2O2 through mechanisms that involve a rapid translation of pre-existing transcripts and the increase of the ndh transcript levels.

Primary transcripts of ndhD of Liliaceae and Aloaceae require editing of the start and 20th codons.

About 350 nucleotide sequences around the start codon of the plastid ndhD gene were determined in six species to investigate the requirement of C to U editing of the cryptic start codons. The comparison with other sequences in databanks showed that, in contrast to grasses and similarly to dicots, Liliaceae and Aloaceae require editing of the primary transcript to generate the AUG start codon. Primary transcripts of Liliaceae and Aloaceae also show a new editing site at the 59th nucleotide, which has not been described in other plants. In some cases, transcripts showed partial editing which suggests that the synthesis of the protein may be controlled at the level of transcript processing.

Postsymbiotic plasmid acquisition and evolution of the repA1-replicon in Buchnera aphidicola.

Buchnera aphidicola is an obligate, strictly vertically transmitted, bacterial symbiont of aphids. It supplies its host with essential amino acids, nutrients required by aphids but deficient in their diet of plant phloem sap. Several lineages of Buchnera show adaptation to their nutritional role in the form of plasmid-mediated amplification of key-genes involved in the biosynthesis of tryptophan (trpEG) and leucine (leuABCD). Phylogenetic analyses of these plasmid-encoded functions have thus far suggested the absence of horizontal plasmid exchange among lineages of Buchnera. Here, we describe three new Buchnera plasmids, obtained from species of the aphid host families Lachnidae and Pemphigidae. All three plasmids belong to the repA1 family of Buchnera plasmids, which is characterized by the presence of a repA1-replicon responsible for replication initiation. A comprehensive analysis of this family of plasmids unexpectedly revealed significantly incongruent phylogenies for different plasmid and chromosomally encoded loci. We infer from these incongruencies a case of horizontal plasmid transfer in Buchnera. This process may have been mediated by secondary endosymbionts, which occasionally undergo horizontal transmission in aphids.

Identification of the Ndh (NAD(P)H-plastoquinone-oxidoreductase) complex in etioplast membranes of barley: changes during photomorphogenesis of chloroplasts.

In the last few years the presence in thylakoid membranes of chloroplasts of a NAD(P)H-plastoquinone oxidoreductase complex (Ndh complex) homologous to mitochondrial complex I has been well established. Herein, we report the identification of the Ndh complex in barley etioplast membranes. Two plastid DNA-encoded polypeptides of the Ndh complex (NDH-A and NDH-F) were relatively more abundant in etioplast membranes than in thylakoids from greening chloroplasts. Conversion of etioplast into chloroplast, after light exposure of barley seedlings grown in the dark, was accompanied by a decrease in the NADH dehydrogenase activity associated to plastid membranes. Using native-PAGE and immunolabelling techniques we have determined that a NADH specific dehydrogenase activity associated with plastid membranes, which was more active in etioplasts than in greening chloroplasts, contained the NDH-A and NDH-F polypeptides. These results complemented by those obtained through blue-native-PAGE indicated that NDH-A and NDH-F polypeptides are part of a 580 kDa NADH dependent dehydrogenase complex present in etioplast membranes. This finding proves that accumulation of the Ndh complex is independent of light. The decrease in the relative levels and specific activity of this complex during the transition from etioplast to chloroplasts was accompanied by a parallel decrease in the specific activity of peroxidase associated to plastid membranes. Based on the mentioned observations it is proposed that an electron transport chain from NADH to H2O2 could be active in barley etioplasts.

Transcripts of the ndhH-D operon of barley plastids: possible role of unedited site III in splicing of the ndhA intron.

The plastid ndhH-D operon produces several transcripts containing ndhA sequence with and without its group II intron. After sequencing an 8125 bp fragment of barley plastid DNA including the ndhH-D operon, we investigated the editing-splicing status of transcripts in the range 1.0-7.8 kb. Reverse transcription and sequencing of RNA bands separated by electrophoresis were used to determine C-->U editing sites. Sites I, II and IV of ndhA and site V of ndhD were edited in all transcripts analysed and, probably, were edited before any splicing had taken place. In contrast, site III of ndhA (13 bp from the 5'-end base of the second exon) was not edited in transcripts containing the intron (including the 1.7 kb intermediary transcript consisting of the intron and the second exon) but was edited in all transcripts lacking the ndhA intron. Comparison of the secondary structures of the ndhA intron and intron-second exon intermediate suggests that G pairing prevents editing of site III in transcripts containing the intron and maintains the secondary structure required for splicing. Splicing of the ndhA intron releases the site III C from pairing and, probably, brings it close to cis-acting elements for editing upstream in the first exon.

Molecular characterization of the leucine plasmid from Buchnera aphidicola, primary endosymbiont of the aphid Acyrthosiphon pisum.

The complete sequence of the leucine plasmid of Buchnera aphidicola from the aphid Acyrthosiphon pisum (pLeu-BAp) is reported. Its gene organization was concordant with those of other leucine plasmids of Buchnera from aphids of the Aphidini and Macrosiphini tribes. Three inverted repeats are present in pLeu-BAp. Two of them are also present in pLeu from the family Aphididae: (i) SIR1, located downstream the leucine operon, resembles a rho-independent terminator of transcription, and (ii) LIR1, located upstream of the leucine operon, is suggested to be involved in transcription termination or messenger stability. The third, located near the putative ATGC repeats involved in the origin of replication, is specific in aphids of the Macrosiphini tribe. Phylogenetic analyses based on sequences of leuA, leuB, leuC, leuD, repA1 and ORF1 showed a closer relationship between Buchnera (A. pisum) and Buchnera (Diuraphis noxia). However, tree topologies indicate that the split between both aphid species took place soon after the formation of the Macrosiphini lineage.

Chlororespiration and poising of cyclic electron transport. Plastoquinone as electron transporter between thylakoid NADH dehydrogenase and peroxidase.

Polypeptides encoded by plastid ndh genes form a complex (Ndh) which could reduce plastoquinone with NADH. Through a terminal oxidase, reduced plastoquinone would be oxidized in chlororespiration. However, isolated Ndh complex has low activity with plastoquinone and no terminal oxidase has been found in chloroplasts, thus the function of Ndh complex is unknown. Alternatively, thylakoid hydroquinone peroxidase could oxidize reduced plastoquinone with H(2)O(2). By immunoaffinity chromatography, we have purified the plastid Ndh complex of barley (Hordeum vulgare L.) to investigate the electron donor and acceptor specificity. A detergent-containing system was reconstructed with thylakoid Ndh complex and peroxidase which oxidized NADH with H(2)O(2) in a plastoquinone-dependent process. This system and the increases of thylakoid Ndh complex and peroxidase activities under photooxidative stress suggest that the chlororespiratory process consists of the sequence of reactions catalyzed by Ndh complex, peroxidase (acting on reduced plastoquinone), superoxide dismutase, and the non-enzymic one-electron transfer from reduced iron-sulfur protein (FeSP) to O(2). When FeSP is a component of cytochrome b(6).f complex or of the same Ndh complex, O(2) may be reduced with NADH, without requirement of light. Chlororespiration consumes reactive species of oxygen and, eventually, may decrease their production by lowering O(2) concentration in chloroplasts. The common plastoquinone pool with photosynthetic electron transport suggests that chlororespiratory reactions may poise reduced and oxidized forms of the intermediates of cyclic electron transport under highly fluctuating light intensities.

Peroxidase activity in Aloe barbadensis commercial gel: probable role in skin protection.

A basic peroxidase (EC 1.11.1.7) (pl around 9.0) has been identified in commercial gel of Aloe barbadensis. In vivo, the activity is localised in the vascular system of inner aqueous leaf parenchyma. Some relevant properties of this basic peroxidase of Aloe have been investigated in leaf extract and in commercial gel where it is notably stable. The acid optimum pH (5.0) for activity and the low KM for H2O2 (0.14 mM) suggest that, when topically applied, Aloe peroxidase may scavenge H2O2 in skin surface.

Structure and evolution of the leucine plasmids carried by the endosymbiont (Buchnera aphidicola) from aphids of the family Aphididae.

In all examined species of the family Aphididae, the bacterial endosymbiont Buchnera aphidicola carries a plasmid encoding the genes leuABCD (involved in leucine biosynthesis) along with repA1, repA2 and ORF1. The gene organisation of the leucine plasmids was conserved, except in Buchnera isolated from Pterocomma populeum, where ORF1 was located in a different position. An inverted repeat (LIR1) located between repA2 and leuA is found in all of the Buchnera leucine plasmids examined. The predicted secondary structure of the LIR1 transcript conforms to a long hairpin loop, suggesting an involvement in transcription termination or messenger stability. Phylogenetic reconstruction based on repA2 sequences suggests that horizontal transfer of Buchnera leucine plasmids has not occurred.

Expression of the plastid ndhF gene product in photosynthetic and non-photosynthetic tissues of developing barley seedlings.

A fragment of the NDH-F subunit of the plastid NAD(P)H dehydrogenase complex (NAD(P)H-plastoquinone-oxidoreductase) from barley was expressed as a fusion protein in Escherichia coli and an antibody to the fusion protein was prepared. Western blot analysis using the anti-NDH-F antibody showed specificity towards a plastid polypeptide of approximately 70 kDa present in both photosynthetic and non-photosynthetic barley tissue. The polypeptide was found in thylakoid membranes of green leaves whereas in etiolated leaves it was shown to be associated with the membrane fraction of etioplasts. NDH-F levels were higher in roots and etiolated tissue than in greening or young leaves. During leaf ontogeny, NDH-F levels decreased from young to mature tissue but increased during senescence. The accumulation of NDH-F in thylakoids of young leaves was stimulated by photooxidative treatment. The results indicate a high degree of expression of plastid ndh genes (which encode NAD(P)H dehydrogenase subunits) in non-photosynthetic plastids and under conditions which impair the photosynthetic activity of chloroplasts. In addition to its putative implication in photosynthetic electron transport, a non-photosynthetic role, such as chlororespiration, is proposed for the plastid NAD(P)H dehydrogenase complex.

Identification of the product of ndhA gene as a thylakoid protein synthesized in response to photooxidative treatment.

A 76 amino acid sequence of NDH-A (the protein encoded by plastid ndhA gene) from barley (Hordeum vulgare L.) was expressed as a fusion protein with beta-galactosidase in E. coli. The corresponding antibody generated in rabbits was used to investigate localization, expression and synthesis in vitro of NDH-A. NDH-A was identified as a 35 kDa polypeptide localized in thylakoid membrane. Western blots shows a large increase in NDH-A levels when barley leaves were incubated under photooxidative conditions, which was more pronounced in mature-senescent leaves than in young leaves. Immunoprecipitation of the [35S]methionine labelled proteins, synthesized in vitro by isolated chloroplasts, demonstrated the synthesis in chloroplasts of the NDH-A 35 kDa polypeptide when barley leaves had been incubated under photooxidative conditions. The results indicate that ndh genes may be involved in the protection of chloroplasts against photooxidative stress, particularly in mature-senescent leaves.

Editing of the chloroplast ndhB encoded transcript shows divergence between closely related members of the grass family (Poaceae).

The ndhB-encoded transcript from barley chloroplasts deviates from the genomic ndhB sequence by nine C-to-U transitions, which is the maximum number of editing events for a chloroplast mRNA reported so far. Comparison with ndhB transcripts from other chloroplast species shows that six of the nine editing sites observed in barley are structurally and functionally conserved in maize, rice and tobacco. The remaining three sites, however, show divergent patterns of conservation even within the three members of the grass family. The conservation of two of these sites in tobacco but not in the closely related graminean species suggests that divergence of the ndhB editing sites is caused by the loss of preexisting editing sites rather than by gain of new sites.

Sensitivity of Superoxide Dismutase Transcript Levels and Activities to Oxidative Stress Is Lower in Mature-Senescent Than in Young Barley Leaves.

Antioxidant enzyme activities are inducible by oxidative stress and decrease during senescence. To determine if the age-dependent decrease of superoxide dismutase (SOD) activities is due to decreased sensitivity to oxidative stress, we have investigated the changes in steady-state levels of transcripts and activities of mitochondrial Mn-SOD (SOD1), chloroplastic Fe-SOD (SOD2), and cytoplasmic Cu-Zn-SOD (SOD3) in young and mature-senescent detached barley (Hordeum vulgare L.) leaves in response to incubation in darkness, growth light (20 W m-2), and photooxidative stress conditions (100 W m-2 with 21 or 100% O2). For a comparison, changes in the mRNA for ribulose bisphosphate carboxylase were also measured. After leaf detachment, the abundance of all three SOD mRNAs increased, then decreased and eventually stabilized after 6 h of incubation. After 20 h of incubation under darkness SOD transcripts decreased in both young and mature-senescent leaves. While under strong photooxidative stress the levels of the three SOD transcripts significantly increased in young leaves; in mature-senescent leaves SOD2 and, to lesser extent, SOD1 and SOD3 transcripts decreased. Generally, SOD activity changes were similar to those of mRNAs. It is proposed that oxidative damage during senescence could be favored by the inability of senescing leaves to modulate the steady-state level of SOD mRNA, and probably those of other antioxidant enzymes, concomitant with the rate of oxyradical formation.

Chloroplast polypeptides synthesized by leaf segments and isolated chloroplasts during senescence in barley.

Starting from senescent barley (Hordeum vulgare L. cv Hassan) leaf segments receiving light and hormone treatments affecting senescence, the plastid polypeptides synthesized by isolated chloroplasts and by leaf segments were analyzed by radiolabelling followed SDS-PAGE and fluorography. Among 20 to 30 polypeptides detected, a few were specifically synthesized (by chloroplasts and/or leaf segments) after each senescence treatment. Apparently, the polypeptides labelled in assays with isolated chloroplasts are truly synthesized in vivo, because most of them were also labelled in assays with leaf segments. The comparison of polypeptide profiles, for every senescence treatment, after labelling with isolated chloroplasts or leaf segments, suggests that most plastid polypeptides synthesized during senescence are coded in plastid DNA.

Phytochrome and hormone control of polypeptides synthesized by chloroplasts of senescent barley leaves.

To identify the polypeptides involved in the mechanism of leaf senescence, light-driven protein synthesis was assayed with chloroplasts isolated from barley leaf segments incubated during 20 h under different light and hormone treatments affecting senescence. The radioactive products were analyzed by SDS-PAGE and fluorography. The synthesis of some polypeptides was stimulated by ABA (66, 44, 30, 22, 20, kDa) and ethylene (66, 50, 48, 44, kDa) which accelerate senescence. Kinetin and red light (in an effect mediated by phytochrome), which retard senescence, inhibited the synthesis of some polypeptides (50, 48, 37, kDa) and stimulated the synthesis of others (54, 32, kDa). Probably phytochrome and hormones control senescence by affecting the synthesis of specific polypeptides.

[L-alanine: 2-ketoglutarate aminotransferase from maize embryo with complex kinetic properties for l-alanine (author's transl)]. / L-alanina:2-cetoglutarato aminotransferasa de embrión de maíz con propiedades cinéticas complejas para L-alanina

The activity of L-Alanine: 2-oxoglutarate aminotransferase (E.C. 2.6.1.2) has been studied in maize (Zea mays) embryo. Crude extracts were fractioned with ammoniun sulfate to obtain low activity preparations of other aminotransferases present in crude extracts. The enzyme shows normal hyperbolic saturation curves for the substrasts: Pyruvate (Km 1 mM), 2-oxoglutarate (Km 0.4 mM) and L-Glutamate (Km 0.07 mM). However, it shows complex kinetics properties for the substrate L-Alanine, giving sigmoid saturation curves for L-Alanine at low but not at high fixed 2-oxoglutarate concentrations. These last results point to a regulation of the of L-Alanine degradation, which takes place during the germination of maize. Together with L-Glutamate and L-Alanine, the enzyme only seems to use L-Serine and L-Cysteine and their cetoacids.

Kinetic properties and related changes of molecular weight in a fructokinase from Streptomyces violaceoruber.

1. A study of the initial reaction rates at variable substrate concentrations and of the molecular weight of the enzyme in the presence of different effectors, has been carried out using fructokinase (ATP: fructose 6-phosphotransferase, EC 2.7.1.4) from Streptomyces violaceoruber. 2. Saturation curves for MgATP or CoATP are sigmoidal and they change to hyperbolic in the presence of 10 mM Mg2+ or Co2+ in excess over the nucleoside triphosphate. 3. Saturation cuvves for fructose show intermediary plateaux at high (but not at low) concentrations of ATP or Mg2+. 4. The molecular weight of the enzyme in the presence of high concentrations of MgATP is 80 000. In the presence of fructose, and/or Mg2+, the molecular weight is 20 000. 5. The effects of MgADP, uncomplexed ADP or ATP, and low concentrations of detergent on the kinetics have been studied. The results are interpreted as showing the existence of cooperative effects.