ABA-dependent amine oxidases-derived H2O2 affects stomata conductance

Recently we showed that ABA is at least partly responsible for the induction of the polyamine exodus pathway in Vitis vinifera plants. Both sensitive and tolerant plants employ this pathway to orchestrate stress responses, differing between stress adaptation and programmed cell death. Herein we show that ABA is an upstream signal for the induction of the polyamine catabolic pathway in Vitis vinifera. Thus, amine oxidases are producing H₂O₂ which signals stomata closure. Moreover, the previously proposed model for the polyamine catabolic pathway is updated and discussed.Key words: plant growth, abscissic acid, polyamines, amine oxidases, signaling, oxidative stress, programmed cell deathWe have shown that tobacco salinity induces an exodus of the polyamine (PA) spermidine (Spd) into the apoplast where it is oxidized by polyamine oxidase (PAO) generating hydrogen peroxide (H₂O₂). Depending on the size of H₂O₂, it signals either tolerance-effector genes or the programmed cell death syndrome¹ (PCD). PAs are ubiquitous and biologically active molecules. In the recent years remarkable progress has been accomplished regarding the regulation of PAs biosynthesis and catalysis, not only under normal physiological but also under stress conditions.¹ The most studied PAs are the diamine Putrescine (Put) and its derivatives the triamine Spd and the tetramine spermine (Spm). They are present in the cells in soluble form (S), or conjugated either to low molecular weight compounds (soluble hydrolyzed form, SH) or to “macro” molecules or cell walls (pellet hydrolyzed form, PH). In higher plants, Put is synthesized either directly from ornithine via ornithine decarboxylase (ODC; EC 4.1.1.17) or indirectly from arginine via arginine decarboxylase (ADC; EC 4.1.1.19). Spd and Spm are synthesized via Spd synthase (EC 2.5.1.16, SPDS) and Spm synthase (EC 2.5.1.22, SPMS), respectively, by sequential addition of aminopropyl groups to Put, catalyzed by S-adenosyl-L-methionine decarboxylase (SAMDC; EC 4.1.1.50).^2,3 In plants, PAs are present in the cytoplasm, as well as in cellular organelles.⁴ Recently it was shown that during stress, they are secreted into the apoplast where they are oxidized by amine oxidases (AOs), such as diamine oxidase for Put (DAO, E.C. 1.4.3.6) and polyamine oxidase (PAO, E.C. 1.4.3.4) for Spd and Spm.^1,5,6 Oxidation of PAs generates, amongst other products, H₂O₂^1,7,8 which is involved in cell signaling processes coordinated by abscissic acid (ABA),⁹ but also acts as efficient oxidant and, at high concentration, orchestrates the PCD syndrome.^6,10 Two types of PA catabolism by PAO are known in plants: the terminal and the back-conversion pathways. The terminal one takes place in the apoplast, produces except H₂O₂, 1,3-diaminopropane and an aldehyde depending on the species. On the other hand, the back-conversion pathway is intracellular (cytoplasm and peroxisomes) resulting to the production of H₂O₂ and the sequential production of Put by Spm via Spd.^1,7 Now we have shown that PA exodus also occurs in Vitis vinifera and this phenomenon is at least partially induced by abscissic acid (ABA).¹¹ Thus, exogenous application of ABA results to PA exodus into the apoplast of grapevine. PA is oxidized by an AO resulting to production of H₂O₂. When the titer of H₂O₂ is below a threshold, expression of tolerance-effector genes is induced, while when it exceeds this threshold the programmed cell death (PCD) syndrome is induced.     

Transgenic tobacco plants overexpressing polyamine oxidase are not able to cope with oxidative burst generated by abiotic factors

The molecular and biochemical mechanism(s) of polyamine (PA) action remain largely unknown. Transgenic tobacco plants overexpressing polyamine oxidase (PAO) from Zea mays exhibited dramatically increased expression levels of Mpao and high 1,3-diaminopropane (Dap) content. All fractions of spermidine and spermine decreased significantly in the transgenic lines. Although Dap was concomitantly generated with H(2)O(2) by PAO, the latter was below the detection limits. To show the mode(s) of H(2)O(2) scavenging, the antioxidant machinery of the transgenics was examined. Specific isoforms of peroxidase, superoxide dismutase and catalase were induced in the transgenics but not in the wild-type (WT), along with increase in activities of additional enzymes contributing to redox homeostasis. One would expect that because the antioxidant machinery was activated, the transgenics would be able to cope with increased H(2)O(2) generated by abiotic stimuli. However, despite the enhanced antioxidant machinery, further increase in the intracellular reactive oxygen species (ROS) by exogenous H(2)O(2), or addition of methylviologen or menadione to transgenic leaf discs, resulted in oxidative stress as evidenced by the lower quantum yield of PSII, the higher ion leakage, lipid peroxidation and induction of programmed cell death (PCD). These detrimental effects of oxidative burst were as a result of the inability of transgenic cells to further respond as did the WT in which induction of antioxidant enzymes was evident soon following the treatments. Thus, although the higher levels of H(2)O(2) generated by overexpression of Mpao in the transgenics, with altered PA homeostasis, were successfully controlled by the concomitant activation of the antioxidant machinery, further increase in ROS was detrimental to cellular functions and induced the PCD syndrome.     

Proteomics studies of childhood pilocytic astrocytoma

Childhood pilocytic astrocytoma is the most frequent brain tumor affecting children. Proteomics analysis is currently considered a powerful tool for global evaluation of protein expression and has been widely applied in the field of cancer research. In the present study, a series of proteomics, genomics, and bioinformatics approaches were employed to identify, classify and characterize the proteome content of low-grade brain tumors as it appears in early childhood. Through bioinformatics database construction, protein profiles generated from pathological tissue samples were compared against profiles of normal brain tissues. Additionally, experiments of comparative genomic hybridization arrays were employed to monitor for genetic aberrations and sustain the interpretation and evaluation of the proteomic data. The current study confirms the dominance of MAPK pathway for the childhood pilocytic astrocytoma occurrence and novel findings regarding the ERK-2 expression are reported.     

Most IL-4-producing gamma delta thymocytes of adult mice originate from fetal precursors

Thy-1(dull) gammadelta T cells constitute a distinct adult gammadelta T cell subset characterized by the expression of a TCR composed of Vgamma1Cgamma4 and Vdelta6Cdelta chains with limited junctional sequence diversity. However, several features of the expressed Thy-1(dull) TCR-gammadelta genes, in particular the absence or minimal presence of N region diversity and the almost invariable Ddelta2-Jdelta1 junction, are typical of rearrangements often found in the fetal thymus. In this study, we have investigated the origin of these cells. Few Thy-1(dull) gammadelta thymocytes developed in syngeneic radiation adult chimeras, regardless of whether the recipient mice were given adult bone marrow or fetal liver cells as a source of hemopoietic precursors. In contrast, normal numbers of Thy-1(dull) gammadelta T cells developed in fetal thymi grafted into adult syngeneic recipients. Interestingly, the majority of Thy-1(dull) gammadelta thymocytes present in the grafts were of graft origin, even when most conventional gammadelta and alphabeta thymocytes in the grafted thymi originated from T cell precursors of recipient origin. Single-cell PCR analyses of the nonselected TCR-gamma rearrangements present in adult Thy-1(dull) gammadelta thymocytes revealed that more than one-half of these cells represent the progenies of a limited number of clones that greatly expanded possibly during the first weeks of life. Finally, the second TCR-delta allele of a large number of Thy-1(dull) gammadelta T cells contained incomplete TCR-delta rearrangements, thus providing an explanation for the adult-type rearrangements previously found among nonfunctional V(D)J rearrangements present in Thy-1(dull) gammadelta thymocytes.     

Functional expression in Escherichia coli of the Haemophilus influenzae gene coding for selenocysteine-containing selenophosphate synthetase

The selenophosphate synthetases from several organisms contain a selenocysteine residue in their active site where the Escherichia coli enzyme contains a cysteine. The synthesis of these enzymes, therefore, depends on their own reaction product. To analyse how this self-dependence is correlated with the selenium status, e.g. after recovery from severe selenium starvation, we expressed the gene for the selenocysteine-containing selenophosphate synthetase from Haemophilus influenzae (selD _HI) in an E. coliΔselD strain. Gene selD _HI gave rise to a selenium-containing gene product and also supported – via its activity – the formation of E. coli selenoproteins. The results provide evidence either for the suppression of the UGA_Sec codon with the insertion of an amino acid allowing the formation of a functional product or for a bypass of the selenophosphate requirement. We also show that the selenocysteine synthesis and the insertion systems of the two organisms are fully compatible despite conspicuous differences in the mRNA recognition motif. Received: 8 July 1997 / Accepted: 3 September 1997     

Proteomics approaches in cervical cancer: focus on the discovery of biomarkers for diagnosis and drug treatment monitoring

Introduction: The HPV virus accounts for the majority of cervical cancer cases. Although a diagnostic tool (Pap Test) is widely available, cervical cancer incidence still remains high worldwide, and especially in developing countries, attributed to a large extent to suboptimal sensitivities of the Pap test and unavailability of the test in developing countries.

Areas covered: Proteomics approaches have been used in order to understand the HPV virus correlation to cervical cancer pathology, as well as to discover putative biomarkers for early cervical cancer diagnosis and drug mode of action.

Expert commentary: The present review summarizes the latest in vitro and in vivo proteomic studies for the discovery of putative cervical cancer biomarkers and the evaluation of available drugs and treatments.