The Levels of DAHP Synthase, the First Enzyme of the Shikimate Pathway, Are Related to Free Aromatic Amino Acids and Glutamine Content in Nicotiana plumbaginifolia Cell Cultures

Aromatic amino acid homeostasis was investigated in cell suspension cultures of Nicotiana plumbaginifolia and was related to the activity of the first enzyme in aromatic biosynthesis, 3-deoxy-D-arabino-heptulosonate-7-phosphate (DAHP) synthase. An inverse relationship was found between the intracellular content of free phenylalanine, tyrosine and tryptophan and enzyme specific activity levels, suggesting the occurrence of end-product control mechanisms. Two DAHP synthase isogenes are present in wild tobacco that showed a different expression pattern during the culture growth cycle. Intracellular levels of aromatic amino acids were increased or decreased by adding the culture medium with phenylalanine, tyrosine and tryptophan, or with sublethal doses of the shikimate pathway inhibitor glyphosate, respectively. As a consequence, enzyme levels varied in the opposite direction. The concomitant exogenous supply of glutamine further reduced enzyme activity in mid-log cells, suggesting induction by both aromatic amino acid depletion and nitrogen starvation

Desulfo-glucosinolate sulfotransferases isolated from several Arabidopsis thaliana ecotypes differ in their sequence and enzyme kinetics

The goal was to investigate whether the diverse glucosinolate (Gl) profiles described for different Arabidopsis thaliana (L.) Heynh. ecotypes are at least partially shaped by the kinetic properties of sulfotransferases (SOTs) (EC 2.8.2.-) catalyzing the final step in Gl core structure biosynthesis. This study focuses on only one of the three SOTs that contribute to Gl biosynthesis. Homologues of AtSOT18 proteins were characterized, which was inspired by earlier findings on SOTs from ecotypes Col-0 and C24 differing in two amino acids (aa) and specific enzyme activities. Could there be a correlation of AtSOT18 enzyme activities and differences in Gl profiles between the ecotypes? SOT18 sequences from eight Arabidopsis ecotypes with highly diverse Gl patterns differed in two aa at various positions in the protein sequence. The SOT18 sequence from Col-0 showed the highest similarity to the largest number of other sequences in the alignment. The small differences in the primary sequence lead to important structural changes in secondary and tertiary structure that might be the key of different kinetic activities towards a broad range of substrates. All recombinant AtSOT18 proteins showed low substrate specificity with an indolic Gl, while the specificity for aliphatic substrates varied. There is no correlation in the kinetic behavior with the major ds-Gl contents or with the ratio of C-3/C-4 ds-Gl in the respective ecotype. Therefore, is it unlikely that ds-Gl AtSOT18 proteins play a major role in shaping the Gl profile in Arabidopsis

Molecular and cellular correlates of the CIITA-mediated inhibition of HTLV-2 Tax-2 transactivator function resulting in loss of viral replication

<p>Abstract</p> <p>Background</p> <p>MHC class II transactivator CIITA inhibits the function of HTLV-2 Tax-2 viral transactivator and, consequently, the replication of the virus in infected cells. Moreover overexpression of the nuclear factor NF-YB, that cooperates with CIITA for the expression of MHC class II genes, results also in inhibition of Tax-2 transactivation. The purpose of this investigation was to assess the cellular and molecular basis of the CIITA-mediated inhibition on Tax-2, and the relative role of NF-YB in this phenomenon.</p> <p>Methods</p> <p>By co-immunoprecipitation of lysates from 293T cells cotransfected with CIITA or fragments of it, and Tax-2 it was assessed whether the two factors interact <it>in vivo</it>. A similar approach was used to assess Tax-2-NF-YB interaction. In parallel, deletion fragments of CIITA were tested for the inhibition of Tax-2-dependent HTLV-2 LTR-luciferase transactivation. Subcellular localization of CIITA and Tax-2 was investigated by immunofluorescence and confocal microscopy.</p> <p>Results</p> <p>CIITA and Tax-2 interact <it>in vivo </it>through at least two independent regions, at the 1-252 N-term and at the 410-1130 C-term, respectively. Interestingly only the 1-252 N-term region mediates Tax-2 functional inhibition. CIITA and Tax-2 are localized both in the cytoplasm and in the nucleus, when separately expressed. Instead, when coexpressed, most of Tax-2 colocalize with CIITA in cytoplasm and around the nuclear membrane. The Tax-2 minor remaining nuclear portion also co-localizes with CIITA. Interestingly, when CIITA nucleus-cytoplasm shuttling is blocked by leptomycin B treatment, most of the Tax-2 molecules are also blocked and co-localize with CIITA in the nucleus, suggesting that CIITA-Tax-2 binding does not preclude Tax-2 entry into the nucleus.</p> <p>Finally, the nuclear factor NF-YB, also strongly binds to Tax-2. Notably, although endogenous NF-YB does not inhibit Tax-2-dependent HTLV-2 LTR transactivation, it still binds to Tax-2, and in presence of CIITA, this binding seems to increase.</p> <p>Conclusions</p> <p>These results strongly suggest that CIITA inhibit Tax-2 by binding the viral transactivator both directly or through a tripartite interaction with NF-YB in. CIITA is therefore a viral restriction factor for HTLV-2 and this open the possibility to control HTLV-2 viral replication and spreading by the controlled induction of CIITA in infected cells</p

HEBE, a novel positive regulator of senescence in Solanum lycopersicum

Leaf senescence and plant aging are traits of great interest for breeders. Senescing cells undergo important physiological and biochemical changes, while cellular structures such as chloroplasts are degraded with dramatic metabolic consequences for the whole plant. The possibility of prolonging the photosynthetic ability of leaves could positively impact the plant\u2019s life span with benefits for biomass production and metabolite accumulation; plants with these characteristics display a stay-green phenotype. A group of plant transcription factors known as NAC play a pivotal role in controlling senescence: here we describe the involvement of the tomato NAC transcription factor Solyc12g036480, which transcript is present in leaves and floral buds. Since its silencing delays leaf senescence and prevents plants from ageing, we renamed Solyc12g0364 H\u1e16B\u112, for the Greek goddess of youth. In this manuscript we describe how HEB downregulation negatively affects the progression of senescence, resulting in changes in transcription of senescence-promoting genes, as well as the activity of enzymes involved in chlorophyll degradation, thereby explaining the stay-green phenotype

Genetic relationships among local Vitis vinifera cultivars from Campania (Italy)

A total of 114 accessions putatively corresponding to 69 local grape cultivars from Campania (Southern Italy) were analysed with 8 microsatellite markers (VVS2, VVMD5, VVMD7, VVMD25, VVMD27, VVMD31, VrZAG62 and VrZAG79) in order to evaluate their genetic diversity and relationships. According to their unique genotype at SSR loci finally 56 varieties were found. Interesting cases of synonymy, i.e. Greco di Tufo and Asprinio, Palummina and Piedirosso, and homonymy were disclosed. Pairwise genetic distances were calculated between all cultivars. Clustering of cultivars did not reflect their current distribution and this suggests that grape cultivars of Campania might have been introduced from various and distinct geographic areas

Boosting the MHC class II-restricted tumor antigen presentation to CD4+ T helper cells: A critical issue for triggering protective immunity and re-orienting the tumor microenvironment toward an anti-tumor state

6noopenAlthough the existence of an immune response against tumor cells is well documented, the fact that tumors take off in cancer patients indicates that neoplastic cells can circumvent this response. Over the years many investigators have described strategies to rescue the anti-tumor immune response with the aim of creating specific and long-lasting protection against the disease. When exported to human clinical settings, these strategies have revealed in most cases a very limited, if any, positive outcome. We believe that the failure is mostly due to the inadequate triggering of the CD4+ T helper (TH) cell arm of the adaptive immunity, as TH cells are necessary to trigger all the immune effector mechanisms required to eliminate tumor cells. In this review, we focus on novel strategies that by stimulating MHC class II-restricted activation of TH cells generate a specific and persistent adaptive immunity against the tumor. This point is of critical importance for both preventive and therapeutic anti-tumor vaccination protocols, because adaptive immunity with its capacity to produce specific, long-lasting protection and memory responses is indeed the final goal of vaccination. We will discuss data from our as well as other laboratories which strongly suggest that triggering a specific and persistent anti-tumor CD4+ TH cell response stably modify not only the tumor microenvironment but also tumor-dependent extratumor microenvironments by eliminating and/or reducing the blood-derived tumor infiltrating cells that may have a pro-tumor growth function such as regulatory CD4+/CD25+ T cells and myeloid-derived-suppressor cells. Within this frame, therefore, we believe that the establishment of a pro-tumor environment is not the cause but simply the consequence of the tumor strategy to primarily counteract components of the adaptive cellular immunity, particularly TH lymphocytes.openAccolla, R.S.; Lombardo, L.; Abdallah, R.; Raval, G.; Forlani, G.; Tosi, G.Accolla, Roberto; Lombardo, L.; Abdallah, R.; Raval, G.; Forlani, Greta; Tosi, Giovann

New insights into structural and functional roles of indole-3-acetic acid (IAA) : Changes in DNA topology and gene expression in bacteria

Indole-3-acetic acid (IAA) is a major plant hormone that affects many cellular processes in plants, bacteria, yeast, and human cells through still unknown mechanisms. In this study, we demonstrated that the IAA-treatment of two unrelated bacteria, the Ensifer meliloti 1021 and Escherichia coli, harboring two different host range plasmids, influences the supercoiled state of the two plasmid DNAs in vivo. Results obtained from in vitro assays show that IAA interacts with DNA, leading to DNA conformational changes commonly induced by intercalating agents. We provide evidence that IAA inhibits the activity of the type IA topoisomerase, which regulates the DNA topological state in bacteria, through the relaxation of the negative supercoiled DNA. In addition, we demonstrate that the treatment of E. meliloti cells with IAA induces the expression of some genes, including the ones related to nitrogen fixation. In contrast, these genes were significantly repressed by the treatment with novobiocin, which reduces the DNA supercoiling in bacterial cells. Taking into account the overall results reported, we hypothesize that the IAA action and the DNA structure/function might be correlated and involved in the regulation of gene expression. This work points out that checking whether IAA influences the DNA topology under physiological conditions could be a useful strategy to clarify the mechanism of action of this hormone, not only in plants but also in other unrelated organisms