Proteomic and transcriptomic analysis of rice tranglutaminase and chloroplast-related proteins.

The recently cloned rice transglutaminase gene (tgo) is the second plant transglutaminase identified to date (Campos et al. Plant Sci. 205-206 (2013) 97-110). Similarly to its counterpart in maize (tgz), this rice TGase was localized in the chloroplast, although in this case not exclusively. To further characterise plastidial tgo functionality, proteomic and transcriptomic studies were carried out to identify possible TGO-related proteins. Some LHCII antenna proteins were identified as TGO related using an in vitro proteomic approach, as well as ATPase and some PSII core proteins by mass spectrometry. To study the relationship between TGO and other plastidial proteins, a transcriptomic in vivo Dynamic Array (Fluidigm™) was used to analyse the mRNA expression of 30 plastidial genes with respect to that of tgo, in rice plants subjected to different periods of continuous illumination. The results indicated a gene-dependent tendency in the expression pattern that was related to tgo expression and to the illumination cycle. For certain genes, including tgo, significant differences between treatments, principally at the initiation and/or at the end of the illumination period, connected with the day/night cycling of gene expression, were observed. The tgo expression was especially related to plastidial proteins involved in photoprotection and the thylakoid electrochemical gradient.

Rice transglutaminase gene: Identification, protein expression, functionality, light dependence and specific cell location.

Transglutaminases (TGases), that catalyze post-translational modification of proteins, are scarcely known in plants. As part of a project to characterize transglutaminase genes in new plant species, the identification and characterization of a TGase in rice is presented. Using differential primers, a cDNA (tgo) of 1767bp from genomic rice DNA amplification was obtained. The primers were designed from the rice DNA sequence relatively homologous to the gene encoding active maize chloroplast TGase. Amino acid sequence of the deduced rice TGase protein (TGO) indicated that it contains the enzyme catalytic triad (Cys-His-Asp), three repeats, myristoylation domains and a leucine zipper motif. The TGO recombinant protein was characterized, showing specific activity regulation, and indicating that tgo encoded for an authentic TGase. Substrate preference and Ca(2+) dependent activity were also detected. In the rice plant TGO protein was immunolocalized in the grana chloroplasts, in protein vesicles near them, and in the bulliform cells. Immunoblot analyses, tgo mRNA expression, and TGase activity indicated that TGO expression in rice was light dependent and regulated by the illumination period. This work increases significantly our plant TGase understanding. Its functional role in rice, which is a good model system for C3 plants, is discussed.

Characterisation of Zea mays L. plastidial transglutaminase: interactions with thylakoid membrane proteins.

Chloroplast transglutaminase (chlTGase) activity is considered to play a significant role in response to a light stimulus and photo-adaptation of plants, but its precise function in the chloroplast is unclear. The characterisation, at the proteomic level, of the chlTGase interaction with thylakoid proteins and demonstration of its association with photosystem II (PSII) protein complexes was accomplished with experiments using maize thylakoid protein extracts. By means of a specific antibody designed against the C-terminal sequence of the maize TGase gene product, different chlTGase forms were immunodetected in thylakoid membrane extracts from three different stages of maize chloroplast differentiation. These bands co-localised with those of lhcb 1, 2 and 3 antenna proteins. The most significant, a 58 kDa form present in mature chloroplasts, was characterised using biochemical and proteomic approaches. Sequential fractionation of thylakoid proteins from light-induced mature chloroplasts showed that the 58 kDa form was associated with the thylakoid membrane, behaving as a soluble or peripheral membrane protein. Two-dimensional gel electrophoresis discriminated, for the first time, the 58-kDa band in two different forms, probably corresponding to the two different TGase cDNAs previously cloned. Electrophoretic separation of thylakoid proteins in native gels, followed by LC-MS mass spectrometry identification of protein complexes indicated that maize chlTGase forms part of a specific PSII protein complex, which includes LHCII, ATPase and pSbS proteins. The results are discussed in relation to the interaction between these proteins and the suggested role of the enzyme in thylakoid membrane organisation and photoprotection.

Recent patents on transglutaminase production and applications: a brief review.

The transglutaminases (TGase; EC2.3.2.13) (R-glutaminyl-peptide-aminase-gamma-glutamyltransferase) are a family of enzymes that catalyse post-translational modifications in proteins, producing covalent amide bonds between a primary amine group in a polyamine or lysine (amine donor), and a gamma-carboxamide group of the glutamyl residue of some proteins(amine receptor). These enzymes were detected for the first time in animals, where they modify structural proteins, and are widely distributed in bacteria, animals, and plants. Of all the reactions that are catalysed by TGases, protein crosslinking has probably attracted the greatest interest owing the industrial applications of these catalytic reactions. A review of the patents related to the different cloned TGases and its putative applications in medicine, food processing, and other applications as polymer obtaining, etc., is presented here. Due to the widespread number of recently published patents about this enzyme, a classification in relation to a) species origin and b) application field, is presented.

Immunogold localization of trehalose-6-phosphate synthase in leaf segments of wild-type and transgenic tobacco plants expressing the AtTPS1 gene from Arabidopsis thaliana.

Following the establishment of a transgenic line of tobacco (B5H) expressing the trehalose-6-phosphate synthase (TPS) gene from Arabidopsis thaliana, a preliminary immunolocalization study was conducted using leaves of adequately watered B5H and wild-type plants. Immunocytochemical staining, followed by electron microscopy showed that the enzyme could be detected in both B5H and wild-type plants at two different levels. Quantification showed the signal to be two to three times higher in transgenic plants than in the wild type. This enzyme was markedly present in the vacuoles and the cell wall, and to a lesser extent in the cytosol. Moreover, a high profusion of gold particles was detected in adjacent cells and in the sieve elements. Occasional spots were also detected in chloroplasts and the nucleus, especially in the transgenic B5H line. No labeling signal was detected in mitochondria. Protein localization seems to confirm the important role of TPS in sugar metabolism and transport through the plant, which could explain its role in plant stress tolerance. Finally, it can be expected that TPS from tobacco has a relatively high similarity to the TPS of Arabidopsis thaliana.

Linfadenectomía axilar completa frente observación en pacientes con micrometástasis en el ganglio centinela por neoplasia de mama. Resultados preliminares del ensayo clínico multicéntrico AATM 048/13/2000 / Complete axillary lymph node dissection versus clinical follow-up in breast cancer patients with sentinel node micrometastases. Interim analysis of the Spanish multicenter clinical trial. AATM 048/13/2000

Objetivo: Conocer la supervivencia, control o recidiva dela enfermedad, y calidad de vida de las pacientes con micrometástasisen el ganglio centinela no sometidas a vaciamientoaxilar.Pacientes y método: Un ensayo clínico prospectivo aleatorizadoen dos brazos, uno con pacientes con micrometástasis enel ganglio centinela que serían sometidas a observación clínica yotro brazo similar pero con linfadenectomía en un segundotiempo quirúrgico. Las pacientes serían seguidas detalladamentedurante cinco años. La muestra constaría de 176 pacientes porbrazo (total 352) para conseguir solidez estadística.Resultados: Hasta el momento se han incluido 120 pacientes(cabe decir que el estudio se mantiene activo). Descontandolos abandonos (7 en el grupo control y 2 en el experimental)permanecen en el estudio 54 en la rama control y 57en la experimental. En el grupo control se encontraron 8 vaciadospositivos (7 de ellos presentaban únicamente un segundoganglio afectado), mientras que en el grupo experimentalapareció una recidiva axilar local (por palpación, siendo el únicoganglio afectado en la linfadenectomía). La paciente se encuentraen la actualidad libre de enfermedad.El estudio no ha terminado. El tiempo máximo de seguimientoes de 47 meses, insuficiente para concluir. Queda sinembargo, abierto, pues consideramos que la pregunta planteadasigue siendo todavía de actualidad

Objective: To asses the impact of SN technique on thewellbeing and performance status of breast cancer patients,specifically targeting the finding of axillary micrometastatic disease.Patients and method: To achieve such objective, a randomizedprospective clinical trial was devised with two arms. Inone arm, patients with SN micrometastases are the subject ofplain clinical follow-up (experimental arm). In the other arm, patientsare submited for second-sugery completion ALND (controlarm). All patients are intended for a five-year follow-up period,and the accrual goal is 176 patients per arm (352 overall).Results: The study is still in the accrual phase. Up untilnow, 120 patients have been entered. There have been 7withdrawals in the control group and 2 in the experimentalgroup. 54 patients are being followed in the control arm, and57 in the study arm. In the control arm, 8 completion ALNDturned out positive, in seven only with one additional (non-sentinel)lymph-node metastasis. In the study arm, one axillary recurrencehas been observed in a single lymph-node, one yearafter primary surgery. Two years after completion ALND, thepatient is free of disease. This is a still ongoing study. A maximum follow-up periodof 47 months is obviously not enough to draw solid conclusions.It will be open for further accrual as the proposed questionis still in need of a definite answer

Molecular cloning and characterization of a maize transglutaminase complementary DNA.

Two related complementary DNA clones, TGZ15 and TGZ21, encoding active maize transglutaminase (TGase) have been isolated for the first time in plants by molecular cloning (Patent Pending PCT/ES03/00247). Southern and northern blot analyses indicate that the two cDNAs probably corresponded to two different single-copy genes in the maize genome. Northern blot analyses revealed that the transcript is expressed preferentially in young leaves and differentiated embryogenic maize callus. This expression is dependent on light exposure time. TGase activity of the proteins encoded by clones TGZ15 and TGZ21 was detected in bacterial extracts overexpressing them, using two enzymatic assays. TGase activity was significantly higher than that of the empty-phagemid bacterial extracts. As in other TGases, this activity was inhibited by monodansyl cadaverine (MDC), GTP and the absence of exogenous Ca(2+). Likewise, light-stimulated Ca(2+)-dependent TGase activity was detected in thylakoids and grana of maize chloroplast, which was inhibited by MDC, GTP, DIECA and Diuron.

Immunogold localization of a transglutaminase related to grana development in different maize cell types.

A comparative study of the subcellular localization of a plant transglutaminase (TGase; EC 2.3.2.13) in various in vivo and in vitro maize cell types was carried out with a polyclonal antibody raised against a 58 kDa TGase purified from Helianthus tuberosus leaves. Immunocytochemical staining, followed by electron microscopy, showed that this enzyme was markedly present in the grana-appressed thylakoids of mature chloroplasts of the light-exposed cells. Moreover, during embryogenic callus chloroplast differentiation, the abundance of TGase in the grana-appressed thylakoids depended on the degree of grana development and was greater than in mature leaf chloroplasts. In addition to the 58 kDa form, two other forms of the protein (of 77 and 34 kDa) were obtained by Western blot. The 77 kDa form might correspond to the inactive form and was immunodetected in dense vesicles observed in dark-grown embryogenic callus cells. In adult leaves, the enzyme was also markedly present in the grana-appressed thylakoids of the mesophyll cell chloroplasts, though very scarce and dispersed in the bundle-sheath cell chloroplasts (which do not contain grana). The concordance of these localizations with those described for the light-harvesting antenna proteins of the photosystem II suggests that it is possible that this TGase has a functional role in photosynthesis, perhaps modulating the photosynthetic efficiency and the absorption of excess light by means of polyamine conjugation to the antenna proteins.

[Tachycardia-induced tachycardia: definitive treatment with radiofrequency ablation. Presentation of three cases]. / Taquicardia que induce taquicardia: tratamiento definitivo con ablación con radiofrecuencia. Presentación de tres casos.

Tachycardia-induced tachycardia is the phenomenon in which one tachycardia degenerates into another. Few data are available in patients suffering from AV nodal reentrant tachycardia an atrial fibrillation. For related to AV nodal reentrant triggered by tachycardia; there is a possible effective treatment by eliminating the slow nodal pathway, with radiofrequency ablation, as shown by other authors. In this study we present data on three patients with repeated episodes of documented atrial fibrillation and at least one episode of AV nodal reentrant tachycardia or regular palpitations. Radiofrequency ablation of the slow AV nodal pathway was successfully performed in both, and at a follow up of 6, 9 and 10 months, respectively, no new episode of AV nodal reentrant tachycardia or atrial fibrillation was documented.

[Clinical effectiveness of naloxone in acute ethanol intoxication]. / Eficacia clínica de la naloxona en la intoxicación etílica aguda.

The first articles trying to link the effects of ethyl alcohol with those of opiates appeared in 1970. The first case of reversion from ethylic coma due to naloxone was described in 1978. Since then, many authors have reported different results related to the effectiveness of opiate antagonists in this clinical situation, without any of the studies showing a beneficial effect from its use. The purpose of the present study is to perform a double-blinded clinical trial with placebo to evaluate naloxone's clinical effectiveness in severe ethylic intoxication, using the Glasgow test (GT) to measure the level of consciousness. Treatment was randomly assigned to 38 patients of whom 18 received 2 mg of naloxone and 20 a placebo. Among the results, it was found that the best approximation of the level of alcoholemia responsible for ethylic coma, without the involvement of other toxic psychotropes, was made using the Glasgow test, especially in patients who are occasional drinkers. It was shown that naloxone indeed lacks effectiveness in ethylic coma, raising the level of consciousness only one or two points in the Glasgow test for the 15 to 45 minutes after its administration and primarily in the group of patients with the lowest levels of consciousness.

[Acquired immunodeficiency syndrome: a descriptive study and analysis of survival in 73 cases]. / Síndrome de inmunodeficiencia adquirida: estudio descriptivo y análisis de la supervivencia en 73 casos.

The clinical features and prognostic factors influencing survival in 73 patients diagnosed of acquired immunodeficiency syndrome (AIDS) from June 1984 to November 1988 were evaluated. Mean age was 32 years. The predominant risk group were drug abusers (67%). The most common opportunistic infections were extrapulmonary tuberculosis and esophageal candidiasis. After 6 months, with 42 patients followed up, the probability of survival was 69% +/- 11 (95% confidence interval); after 12 months, with 28 patients, it was 65% +/- 12 (95% confidence interval); and after 18 months, with 11 patients, it was 54% +/- 15 (95% confidence interval). Patients younger than 30 years and those with extrapulmonary tuberculosis had a longer survival than the rest (p = 0.046 and p = 0.014, respectively). The remaining evaluated variables did not have any influence on survival.

Gene sequence, developmental expression, and protein phosphorylation of RAB-17 in maize.

The ABA-induced MA12 cDNA from maize, which encodes a set of highly phosphorylated embryo proteins, was used to isolate the corresponding genomic clone. This gene, called RAB-17 (responsive to ABA), encodes a basic, glycine-rich protein (mol. wt. 17,164) containing a cluster of 8 serine residues, seven of them contiguous. It is a homologue of the rice RAB-21 gene (Mundy J, Chua NH, EMBO J 7; 2279-2286, 1988). Phosphoamino acid analysis of the isolated protein indicates that only the serine residues are phosphorylated and a putative casein-type kinase phosphorylatable sequence was identified in the protein. The pattern of expression and in vivo phosphorylation of the RAB-17 protein was studied during maize embryo germination and in calli of both meristematic or embryonic origin. ABA treatment induced the synthesis of RAB-17 mRNA and protein in calli, however, the RAB-17 proteins were found to be highly phosphorylated only in embryos.

Expression of genes for cell-wall proteins in dividing and wounded tissues ofZea mays L.

Hydroxyproline-rich glycoproteins (HRGPs) fromZea mays have been immunolocalized in the cell wall of root tip cells using ultrathin sections and antibodies ellicited against the purified protein. The accumulation of mRNA corresponding to this protein was studied using the cDNA probe. Maximum accumulation of the mRNA was found in tissues with a high proportion of dividing cells such as those in the root tip of young maize seedlings and a close relationship with cellular division was also observed in in-vitro cultures. However, the level of the mRNA in elongating tissues was minimal, as shown by studies carried out on the elongation zones of root tips and coleoptiles. The mRNA was induced by stress conditions, particularly by wounding young leaves and coleoptiles. It is concluded that in maize this group of proline-rich cell-wall proteins accumulates during cell division and not during cell elongation or differentiation, and participates in the stress-response mechanisms of the plant.