Regulation of enzymes involved in C4 photosynthesis and the antioxidant metabolism by UV-B radiation in Egeria densa,a submersed aquatic species

Egeria densa, a submersed aquatic species, was exposed to different treatments under UV-B radiation, and the response of phosphoenolpyruvate carboxylase (PEPC) and NADP-malic enzyme (NADP-ME) was determined. Exposure to UV-B radiation for 4 h per day over 7–16 days caused an increase in both enzymes, together with an increase in the activity of some isoforms of several enzymes involved in the antioxidant metabolism, such as superoxide dismutase (SOD), ascorbate peroxidase (APX), catalase (CAT) and peroxidase (POD). The content of chlorophylls and carotenoids was considerably decreased, suggesting that degradation or repression of the synthesis of these molecules may be occurring after UV-B exposure. Reactive oxygen species (ROS) were also required for UV-B induction of PEPC and NADP-ME, as the addition of ascorbic acid before UV-B treatment prevented the induction of these enzymes, while salicylic acid was not effective in inducing NADP-ME but increased the expression of the lower molecular mass isoform of PEPC. On the other hand, damage to the photosynthetic machinery may be occurring after exposure to UV-B radiation for 8 per day over 1–2 days, as indicated by a decrease in the levels of Rubisco, PEPC and NADP-ME. Some of the enzymes involved in the antioxidant metabolism, such as CAT and APX, were also sensitive to continuous exposure, evidenced by a decrease in their activity. In this way, in E. densa, several enzymes involved in different metabolic pathways showed a distinct response, depending on the UV-B treatment. This revised version was published online in June 2006 with corrections to the Cover Date.     

The FDA approved PI3K inhibitor GDC‐0941 enhances in vitro the anti‐neoplastic efficacy of Axitinib against c‐myc‐amplified high‐risk medulloblastoma

Aberrant receptor kinase signalling and tumour neovascularization are hallmarks of medulloblastoma development and are both considered valuable therapeutic targets. In addition to VEGFR1/2, expression of PDGFR α/β in particular has been documented as characteristic of metastatic disease correlating with poor prognosis. Therefore, we have been suggested that the clinically approved multi‐kinase angiogenesis inhibitor Axitinib, which specifically targets these kinases, might constitute a promising option for medulloblastoma treatment. Indeed, our results delineate anti‐neoplastic activity of Axitinib in medulloblastoma cell lines modelling the most aggressive c‐myc‐amplified Non‐WNT/Non‐SHH and SHH‐TP53‐mutated tumours. Exposure of medulloblastoma cell lines to Axitinib results in marked inhibition of proliferation and profound induction of cell death. The differential efficacy of Axitinib is in line with target expression of medulloblastoma cells identifying VEGFR 1/2, PDGFR α/β and c‐kit as potential markers for drug application. The high specificity of Axitinib and the consequential low impact on the haematopoietic and immune system render this drug ideal multi‐modal treatment approaches. In this context, we demonstrate that the clinically available PI3K inhibitor GDC‐0941 enhances the anti‐neoplastic efficacy of Axitinib against c‐myc‐amplified medulloblastoma. Our findings provide a rational to further evaluate Axitinib alone and in combination with other therapeutic agents for the treatment of most aggressive medulloblastoma subtypes.     

UV‐B radiation delays flowering time through changes in the PRC2 complex activity and miR156 levels in Arabidopsis thaliana

UV‐B is a high‐energy component of the solar radiation perceived by the plant and induces a number of modifications in plant growth and development, including changes in flowering time. However, the molecular mechanisms underlying these changes are largely unknown. In the present work, we demonstrate that Arabidopsis plants grown under white light supplemented with UV‐B show a delay in flowering time, and this developmental reprogramming is mediated by the UVR8 photoreceptor. Using a combination of gene expression analyses and UV‐B irradiation of different flowering mutants, we gained insight into the pathways involved in the observed flowering time delay in UV‐B‐exposed Arabidopsis plants. We provide evidence that UV‐B light downregulates the expression of MSI1 and CLF, two of the components of the polycomb repressive complex 2, which in consequence drives a decrease in H3K27me3 histone methylation of MIR156 and FLC genes. Modification in the expression of several flowering time genes as a consequence of the decrease in the polycomb repressive complex 2 activity was also determined. UV‐B exposure of flowering mutants supports the involvement of this complex in the observed delay in flowering time, mostly through the age pathway.     

Condensation anomalies and exclusion in micronuclei of rearranged chromosomes in human fibroblasts cultured in vitro

Anomalies of chromatin condensation, such as fragmentation, uncoiling and pulverization, were observed in XP9UV25, a xeroderma pigmentosum fibroblast clone in which a high proportion of cells carried an end-to-end dicentric chromosome, dic (5;16) (p15.2;q24), that gives rise during propagation in culture to a variety of dicentric and monocentric derivatives. The coiling anomaly affected exclusively part of a rearranged chromosome, in particular the region previously involved in breakage events. The heterochromatic 16q region, which is a preferential breakpoint in the formation of dicentric and monocentric derivatives, was consistently the limit of the uncoiled or pulverized regions. This observation suggests that the anomalous chromatin behavior could derive from alteration of a region relevant for the correct condensation of the chromosome. In XP9UV25 the frequency of nuclei with associated micronuclei increased with time in culture, in parallel with that of mitoses with dicentric chromosomes. In situ hybridization with DNA probes specific for chromosomes 5 and 16 revealed hybridization signals in about 40% of micronuclei. Since the frequency of micronuclei is about ten times less than that of dicentrics, it is probable that only the rearranged chromosomes undergoing coiling anomalies are excluded in micronuclei.     

NADP-malic enzyme: immunolocalization in different tissues34 plant maize and the C3 plant wheat

In situimmunolocalization and Western blot analysis of separatedcellular and subcellular fractions, were used to determine thelocalization of different isoforms of NADP-malic enzyme in bothwheat (C₃) and maize (C₄) plants. In both techniques, an affinitypurified anti-(maize 62 kDa NADP-ME) lgG from the maize greenleaf isoform also reacted with a 72 kDa protein in tissues ofC4 plants as well as C3 plants. The light- inducible 62 kDaisofomi is located in bundle sheath chioroplasts of maize leaves.In etiolated leaves and in roots of maize there is evidencefor the occurrence of a 72 kDa isoform which co-migrates on2-D (SDS and isoelectric focusing) PAGE. The 72 kDa isoformis also present in low levels in green leaves. This form mayoccur in multiple intracellular compartments; but in situ immunolocalizationexperiments and Western blot and activity assays on fractionatedprotoplasts indicate that a significant amount of this isoformoccurs in plastids. With regards to C³ plants such as wheat,a 72 kDa isoform in leaves is largely confined to the chloroplastsbased on in situ immunolocalization and Western blots and enzymeactivity assays with fractionated protoplasts. In maize, itappears that the constitutive expression pattern of a possibleC₃ ancestral gene for NADP-malic enzyme has been maintained,and a high level expression of a light-inducible isoform locatedin bundle sheath chloroplasts (62 kDa) has been acquired duringits evolution. Key words: NADP-malic enzyme, Triticum aestivum, Zea mays     

Spectrophotometric Assay of Lipase Activity: A New 4-nitrophenyl Ester of a Dialkylglycerol Suitable as a Chromogenic Substrate of Pseudomonas cepacia Lipase

Evaluation of Pseudomonas cepacia lipase (PCL) activity by a titrimetric method with triacylglycerols (TAG) and synthetic dialkylglycerol esters (DAGE) established the chain length selectivity of the enzyme and this information has been used to design a new chromogenic substrate [1,2-di-O-octyl-sn-glycerol-3-O-(4-nitrophenyl) glutarate] for the determination of the lipolytic activity of PCL.     

The FRAME alternatives laboratory database. 1. In vitro basal cytotoxicity determined by the Kenacid blue total protein assay

A database of over 280 chemicals has been compiled by using a mouse 3T3-L1 fibroblast-like cell line in exponential growth, exposed to chemicals for 72 hours in a 96-well tissue culture plate format, and determining cell number via the Kenacid blue (KB) assay for total protein. Ranking the chemicals according to their basal cytotoxicity, expressed as the concentration (mM) that inhibits increase in total cellular protein over 72 hours by 50% (the ID50 value) shows a wide range of ID50 values, from 0.00003 mM to 10,096 mM. This information includes the results for MEIC chemicals 1-50, and we have now added basal cytotoxicity data for 23 of the next 25 MEIC chemicals. When the neutral red uptake (NRU) assay was performed with the same cell cultures, before the KB assay, very similar indications of basal cytotoxicity were obtained. Comparisons between the results with 3T3-L1 cells and with a human fibroblast-like cell line, BCL-D1 showed a significant difference in order of magnitude of the ID50 value for only 5 of 52 chemicals. However, there was a difference in ID50 value of more than one order of magnitude for 8 of 24 chemicals tested with an undifferentiated teratocarcinoma cell line, F9.     

New 16Sr subgroups and distinct single nucleotide polymorphism lineages among grapevine Bois noir phytoplasma populations

Bois noir (BN) is an insect-transmitted grapevine yellows disease caused by phytoplasmas belonging to the stolbur subgroup 16SrXII-A. In Italy, increasing prevalence of stolbur phytoplasma strains in vineyards suggests progressive spread of the disease and potential for heavy impacts on the wine industry. In this study, we investigated the genetic diversity of stolbur phytoplasma strains in BN phytoplasma populations. Nucleotide sequences of 16S rRNA genes from stolbur phytoplasma strains affecting vineyards in the Lombardy region of Italy and stolbur phytoplasma 16S rDNA sequences retrieved from GenBank were subjected to virtual restriction fragment length polymorphism analysis. Calculation of virtual restriction similarity coefficients revealed the presence of new subgroups in group 16SrXII (stolbur phytoplasma group). Representative strains of confirmed new subgroups 16SrXII-F (XII-F) and XII-G and tentative new subgroups XII-A1 through XII-A19, XII-H, XII-I, and XII-J as well as known subgroup XII-A were from grapevines; strains representing three additional tentative new subgroups (XII-K, XII-L and XII-M) were from other plant hosts. Nucleotide sequence alignments identified no less than nine genetically distinct 16S rDNA single nucleotide polymorphism lineages from grapevine, indicating a high degree of genetic heterogeneity within BN phytoplasma populations. The findings open new opportunities for in-depth studies of the distribution of grapevine-associated 16SrXII phytoplasma strains in weeds, insect vector populations and grapevines from vineyards located in different geographic areas.     

Ultrasensitive glycogen synthesis in Cyanobacteria

Cyanobacter ADPglucose pyrophosphorylase exhibits a ultrasensitive response in activity towards its allosteric effector 3-phosphoglycerate, elicited by orthophosphate and polyethyleneglycol-induced molecular crowding. The ultrasensitive response was observed either when the enzyme operates in the zero or first order region for its physiological substrates. The ultrasensitivity exhibited maximal amplification factors of 15-19-fold with respect to 1% of the maximal system velocity. Only a 2.4-3.8-fold increase in 3PGA concentration was necessary to augment the flux from 10% to 90% through AGPase as compared with 200-fold required for the control. The results are discussed in terms of finely tuned regulatory mechanisms of polysaccharide synthesis in oxygenic photosynthetic organisms.