Glucuronidase

We describe the identification and structural characterization of a novel family of Arabidopsis genes related to ATL2 which encode a variant of the RING zinc finger domain, known as RING-H2. Analysis of genes selected by us and of sequences from Arabidopsis stored in databases permitted the prediction of several RING-H2 proteins that contain highly homologous RING domains. The ATL gene family is represented by fifteen sequences that contain, in addition to the RING, a transmembrane domain which is located in most of them towards the N-terminal end. Transgenic Arabidopsis seedlings carrying the ATL2 promoter fused to the GUS reporter gene revealed that the expression of ATL2 is rapidly induced after exposure to chitin or inactivated crude cellulase preparations. Rapid induction of transcript accumulation of another member of the ATL family was also observed under the same conditions. These results suggest that some ATLs may be involved in the early stages of the defense response triggered in plants in response to pathogen attack.

Le septembre 2008 Titre : Caractérisation moléculaire et physiologique des Facteurs de Réponse à l'Ethylène (ERF) chez la tomate (Solanum lycopersicon) JURY Mr. Jean-Claude PECH Président (INP-ENSAT) Mr. Christophe ROTHAN Rapporteur (INRA Bordeaux) Mr. Guy ALBAGNAC Rapporteur (INRA Montpellier) Mme. Mathilde CAUSSE Examinatrice (INRA Avignon) Mr. Masaru OHME-TAKAGI Examinateur (AIST Tsukuba) Mr. Farid REGAD Examinateur (INP-ENSAT) Mr. Mondher BOUZAYEN Directeur de thèse (INP-ENSAT) Mr. Xavier BERTHET Membre invité (NINSAR Barcelone) Ecole doctorale : Biologie Santé et Biotechnologie Unité de recherche : UMR990 INP-ENSAT/INRA Génomique et Biotechnologie des Fruits Directeur de Thèse : Pr. Mondher BOUZAYEN Je remercie Mondher BOUZ AYEN et Masaru OHME-T AKAGI pour m'avoir acceuilli, encadré et fait confiance pendant ce travail de thèse. Je remercie Farid REGAD pour les discussions scientifiques ainsi que son soutien jusqu'à très tard dans la nuit. Je remercie Jean-Claude PECH et Alain LATCHE pour avoir échangé scientifiquement avec moi. Un merci tout spéciale à Corinne, Isabelle, Simone, Marie, Salma et Benoît pour tous les moments de sciences (ou pas) partagés. Merci à Prassad, Ravi, Wangshu et Lanlan d'avoir participé à cette aventure. Un grand merci à Hélène, Carole, Lydie, Olivie r et Dominique pour la transformation et le suivi des plantes transgéniques (et ce n'est pas peu dire). Un grand merci à l'ensemble des membres du laboratoire pour tous les moments partagés, de sciences, et de convivialité : Christian, Je tiens également à remercier tous les membres du Research Institute of Genome-based Biofactory Miho Ikeda, Akira Iwase, Yoshimi Umemura, Tomotsugu Koyama, Masahito Shikata, Kyoko Matsui, Nobut aka Mitsuda, Naoko Kaneta , sans qui cette thèse n'aurait pas eu le même parfum. ありがとう. Merci aussi à Toshi et Hiroshi Ez ura de m'avoir acceuilli. Merci à mes proches qui m'ont soutenu pendant ces années, mes parents et mon frère. Enfin, je tiens à remercier tout particulièrement Laëtitia qui m'a suivi au bout du monde (au sens propre et au sens figuré), pour son soutien permanent et sans qui ce travail aurait été un peu différent. (2007): Isolation and characterization of a Vitis vinifera transcription factor, VvWRKY1, and its effect on responses to fungal pathogens in transgenic tobacco plants J Exp Bot.;58 (8):

We found a tumor me&stasis-associa ted heparan sulfate (HS)degrading endoglycosidase in melanoma cells that is a unique endo-&ghzuronidase (hepamnam ) capable of speciftcally cleaving HS at intrachain sites (M. Nakajima, T. Irimura, N. DiFerrame, and G. L. Nicolson, 1984, J. Biof. Chem. 259.2283-2290. To perform rapid and microscale quantitative assays of heparanase we developed a solid-phase HS substrate by crosslinking radiolabeled HS onto agarose gel beads using one covalent linkage. The HS from bovine lung was partially Ndesulfated and labeled with ["C]acetic anhydride. Free HS amino groups were completely acetylated, and reducing terminal saccharides were reductively aminated. The HS derivatives with amino groups at their reducing termini were coupled to amino-reactive agarose beads Incubation of the solid-phase HS substrates with B 16 melanoma cell extracts in the presence of ~saccharic acid 1 +lactone (a potent exo-j3glucuronidase inhibitor) resulted in the time-and dosedependent release of [%]HS fragments.

Growth of Erwinia chrysanthemi in media of elevated osmolarity can be achieved by the uptake and accumulation of various osmoprotectants. This study deals with the cloning and sequencing of the ousA gene-encoded osmoprotectant uptake system A from E. chrysanthemi 3937. OusA belongs to the superfamily of solute ion cotransporters. This osmotically inducible system allows the uptake of glycine betaine, proline, ectoine, and pipecolic acid and presents strong similarities in nucleotide sequence and protein function with the proline/betaine porter of Escherichia coli encoded by proP. The control of ousA expression is clearly different from that of proP. It is induced by osmotic strength and repressed by osmoprotectants. Its expression in E. coli is controlled by H-NS and is rpoS dependent in the exponential phase but unaffected by the stationary phase.

A continuous spectrophotometric assay has been developed for detecting beta-glucuronidase activity. In the assay, Para-nitrophenyl beta-D-glucuronide is cleaved to yield a chromophoric product. With the commercial E. coli enzyme, it is demonstrated that the reactions can be continuously monitored by the increase of absorbance at 405 nm. The method is highly sensitive and able to detect less than 1.4 x 10(-4) U/mL of the enzyme activity in solution. Such a new assay offers significant advantages over the existing discontinuous methods and should be useful for both routine enzyme assay and accurate kinetic studies.

Malignant pleural mesothelioma (MPM) is a fatal disease with a median survival of less than 14 months. For the first time, a genetically engineered vaccinia virus is shown to produce efficient infection, replication, and oncolytic effect against MPM. GLV-1h68 is a replication-competent engineered vaccinia virus carrying transgenes encoding Renilla luciferase, green fluorescent protein (both inserted at the F14.5L locus), ␤-galactosidase (inserted at the J2R locus, which encodes thymidine kinase), and ␤-glucuronidase (at the A56R locus, which encodes hemagglutinin). This virus was tested in six human MPM cell lines (MSTO-211H, VAMT, JMN, H-2373, H-2452, and H-2052. GLV-1h68 successfully infected all cell lines. For the most sensitive line, MSTO-211H, expression of green fluorescent protein (GFP) started within 4 hr with increasing intensity over time until nearly 100% of cells expressed GFP at 24 hr. All cell lines were sensitive to killing by GLV-1h68, with the degree of sensitivity predictable by infectivity assay. Even the most resistant cell line exhibited 44 Ϯ 3.8% cell survival by day 7 when infected at a multiplicity of infection of 1.0. Viral proliferation assays demonstrated 2to 4-fold logarithmic replication of GLV-1h68 in the cell lines tested. In an orthotopic model, GLV-1h68 effectively prevented development of cachexia and tumor-related morbidity, reduced tumor burden, and cured MPM in both early and late treatment groups. GLV-1h68 was successfully used to treat MPM in vitro and in an orthotopic model (in vivo). These promising results warrant clinical investigation of GLV-1h68 as a novel agent in the treatment of MPM.

The vesicles made of dipalmitoylphosphatidylcholine and stearylamine (9:1) were multilamellar and rather homogeneous in shape as seen by transmission electron microscopy. Upon addition of circular DNA plasmids of different lengths to the liposomes, the formation of vesicle clusters around the DNA filament was observed, with dimensions dictated by the ratio DNA/lipid. These liposomes were able to transfect lentil (Lens culinaris) protoplasts inside the cells two different reporter genes, chloramphenicol-acetyltransferase and I$-glucuronidase.

Plant non-speciWc lipid transfer proteins (nsLTPs) are encoded by a multigene family and support physiological functions, which remain unclear. We adapted an eYcient ligation-mediated polymerase chain reaction (LM-PCR) procedure that enabled isolation of 22 novel Triticum aestivum nsLtp (TaLtp) genes encoding types 1 and 2 nsLTPs. A phylogenetic tree clustered the wheat nsLTPs into ten subfamilies comprising 1-7 members. We also studied the activity of four type 1 and two type 2 TaLtp gene promoters in transgenic rice using the -Glucuronidase reporter gene. The activities of the six promoters displayed both overlapping and distinct features in rice. In vegetative organs, these promoters were active in leaves and root vascular tissues while no -Glucuronidase (GUS) activity was detected in stems. In Xowers, the GUS activity driven by the TaLtp7.2a, TaLtp9.1a, TaLtp9.2d, and TaLtp9.3e gene promoters was associated with vascular tissues in glumes and in the extremities of anther Wlaments whereas only the TaLtp9.4a gene promoter was active in anther epidermal cells. In developing grains, GUS activity and GUS immunolocalization data evidenced complex patterns of activity of the TaLtp7.1a, TaLtp9.2d, and TaLtp9.4a gene promoters in embryo scutellum and in the grain epicarp cell layer. In contrast, GUS activity driven by TaLtp7.2a, TaLtp9.1a, and TaLtp9.3e promoters was restricted to the vascular bundle of the embryo scutellum. This diversity of TaLtp gene promoter activity supports the hypothesis that the encoded TaLTPs possess distinct functions in planta.

Eccentric muscle contraction causes fibre injury associated with disruption of the myofibrillar cytoskeleton. The medicinal plant Panax ginseng C.A. Meyer, known for its therapeutic properties, was studied to explore its protective Ž . effects after eccentric contraction. A crude extract and a standardised extract G115 of different saponin compositions were tested as to their efficacy in reducing lipid peroxidation, inflammation and release of myocellular proteins after the Ž . realisation of an eccentric contraction protocol on a rat treadmill. Plasma creatine kinase CK levels were significantly reduced by approximately 25% after ingestion of both extracts of ginseng. Both extracts reduced lipid peroxidation by approximately 15% as measured by malondialdehyde levels. ␤-Glucuronidase concentrations and glucose-6-phosphate Ž . dehydrogenase G6PDH levels, which can be considered markers of inflammation, were also significantly reduced. The values of ␤-glucuronidase were increased from 35.9" 1.5 to 128.4" 8.1 in vastus and to 131.1" 12.1 U g y1 in rectus, the protection due to ginseng administration being approximately 40% in both muscles. Both extracts appeared to be equally effective in reducing injuries and inflammation caused by eccentric muscle contractions. ᮊ

Three different regeneration systems, viz. direct regeneration of adventitious shoot buds from explant, regeneration through callus cultures and somatic embryos were compared to see their effect on transfer of neomycin phosphotransferase (nptII) and β-glucuronidase (GUS) reporter gene (gus) to Morus alba clone M5, through Agrobacterium tumefaciens mediated transformation. Pre-conditioning and co-cultivation durations had a marked effect on transformation frequency. The highest transformation frequency of 18.6% was obtained using direct induction of adventitious shoot buds. Expression and presence of transgene were assayed histochemically and through polymerase chain reaction. Southern analysis of GUS and PCR positive transformants confirmed stable integration of transgenes with two to four copy numbers. The selected transformants showed normal phenotype under in vitro and field conditions.

The budding yeast Pichia pastoris is an attractive system for exploring certain questions in cell biology, but experimental use of this organism has been limited by a lack of convenient expression vectors. Here we describe a set of compact vectors that should allow for the expression of a wide range of endogenous or foreign genes in P. pastoris. A gene of interest is inserted into a modified pUC19 polylinker; targeted integration into the genome then results in stable and uniform expression of this gene. The utility of these vectors was illustrated by expressing the bacterial -glucuronidase (GUS) gene. Constitutive GUS expression was obtained with the strong GAP promoter or the moderate YPT1 promoter. The regulatable AOX1 promoter yielded very strong GUS expression in methanol-grown cells, negligible expression in glucose-grown cells, and intermediate expression in mannitol-grown cells.

A high copy number, selectable marker gene (SMG)-free Agrobacterium binary vector pBin19 nptII was constructed by deleting the nptII gene from pBin19. The binary vectors with the RK2 and pVS replication origins exist in 12 and 3 copies, respectively, in Agrobacterium. The tobacco osmotin gene (ap24) was cloned in pBin19 nptII and the resultant plasmid pBin19 nptII-ap24 was mobilized into the Agrobacterium tumefaciens strain C58C1 Rif r harbouring the single-copy cointegrate vector pGV2260::pSSJ1. The T-DNA of the cointegrate vector harboured the hph (SMG) and gus genes. Transformation of Oryza sativa L. var. Pusa Basmati1 with Agrobacterium tumefaciens (pGV2260::pSSJ1, pBin19 nptII-ap24) yielded 14 independent hyg + /GUS + transgenic plants. Southern blot analysis with hph and ap24 probes revealed that 12 out of the 14 transgenic plants were co-transformed and harboured hph, gus and ap24 genes. The new multi-copy binary vector yielded 86% co-transformation efficiency. SMG elimination by genetic separation of the cointegrate T-DNA with the hph/gus genes and binary vector T-DNA with the ap24 gene was accomplished in four out of ten primary co-transformants that were forwarded to the T 1 generation.

Objective There are no specific biomarkers available for the definitive diagnosis of pancreatic cancer. Analysis of d-glucuronic acid (GlcUA) in bile could be valuable in this regard. Materials and methods Bile samples obtained from patients with pancreatic cancer (n = 4), chronic pancreatitis (n = 3) and control patients with biliary obstruction (n = 10) were analyzed by 1 H NMR spectroscopy. GlcUA was quantified from the peak area of the α-1 CH signal (at 5.24 ppm) obtained by deconvolution. Results GlcUA was detected in human bile by one-dimensional 1 H NMR and two-dimensional 1 H-1 H COSY and TOCSY experiments. Quantification of GlcUA was achieved by measuring the peak area of the α-1 CH signal using CPMG experiment, and the quantities of GlcUA were calibrated to account for the attenuation due to T 2 relaxation. GlcUA was observed at elevated levels in bile samples obtained from pancreatic cancer patients, whereas it was either absent or found in negligible amounts in control and chronic pancreatitis patients. The reason for the presence of elevated levels of GlcUA could be the hydrolysis of biliary bilirubin diglucuronide by β-glucuronidase, released excessively from pancreatic tissue during the course of malignancy.

Stress tolerance is a multigenic character and there are many stress responsive genes, which are stress speciWc. Although many of these have been cloned, their functional signiWcance remains fragmentary. Hence it is important to identify the relevant stress genes involved in altering the metabolism for adaptation. Overexpression is one of the several approaches and Chlamydomonas is a suitable system to study the functional relevance of stress genes. Stress responses can only be assessed on prior exposure to sublethal induction stress. In this study the acclimation response of Chlamydomonas was assessed for diVerent abiotic stresses using physiological screens like chlorophyll stability, membrane damage, cell viability, accumulation of free radicals, survival and recovery growth. We demonstrate that Chlamydomonas responds to diverse stresses and is a potential system to study the relevance of stress genes. The relevance of choline oxidase A (codA), a key enzyme in glycinebetaine biosynthesis, was examined by developing transformants expressing codA gene from Arthrobacter globiformis. Southern positive transformants showed enhanced accumulation of glycinebetaine. The transformants also showed enhanced growth under salinity, high light coupled with methylviologen-induced oxidative stress, high temperature and cold stress. However the transgenics were not tolerant to PEG-mediated simulated osmotic stress, LiCl, menadione and UV stress. Increased cell survival and decreased chlorophyll degradation in transformants under acclimated conditions further con-Wrmed the relevance of codA in imparting stress tolerance. Our results indicated that the relevance of stress responsive genes can be eYciently validated for diverse abiotic stresses using Chlamydomonas system.

To monitor the colonization of wheat roots by Azospirillum brasilense, we constructed several plasmids based on the pBBR1 replicon expressing the gfp and gusA genes constitutively. Both genes were placed under control of the gentamycin resistance gene promoter resulting in high levels of expression in Escherichia coli and A. brasilense. The constructed plasmids were stably maintained in A. brasilense strains even in the absence of selective pressure. The colonization of wheat plants grown under controlled conditions in sterilized vermiculite by A. brasilense strain FP2 (a Sp7-derivative) transconjugants containing these plasmids was monitored. Bacteria expressing GFP were easily observed in fresh plant material by fluorescence microscopy. Cell aggregates and single bacteria were visualized on the surfaces of young root zones, such as root hairs and lateral roots. Large cellular clumps were observed at the points of lateral root emergence or at intercellular spaces of root epidermal cells 30 days after inoculation. Although we failed to detected bacteria in internal cortical and xylem tissues of wheat roots, the initial stage of endophytic colonization by A. brasilense may involve the sites detected in this work.

Vectors have been developed for inducible gene expression in Lactobacillus sakei and Lactobacillus plantarum in which expression of the gene of interest is driven by strong, regulated promoters from bacteriocin operons found in L. sakei strains. The activity of these promoters is controlled via a two-component signal transduction system, which responds to an externally added peptide pheromone. The vectors have a modular design, permitting easy exchange of all essential elements: the inducible promoter, the cognate regulatory system, the gene of interest, the antibiotic resistance marker and the replicon. Various variants of these so-called 'pSIP' vectors were constructed and tested, differing in terms of the bacteriocin regulon from which the regulatory elements were derived (sakacin A or sakacin P), the regulated promoter selected from these regulons, and the replicon (derived from p256 or pSH71). Using b-glucuronidase (GusA) and aminopeptidase N (PepN) as reporters, it was shown that the best vectors permitted inducible, pheromone-dose-dependent gene expression at very high levels, while displaying moderate basal activities when not induced. The most effective set-up was obtained using a vector containing the pSH71 replicon, the orfX promoter from the sakacin P regulon, and the cognate regulatory genes, in a L. sakei host. GusA levels obtained with this set-up were approximately ten times higher than the levels obtained with prototype pSIP versions, whereas PepN levels amounted to almost 50 % of total cellular protein.

Agrobacterium tumefaciens is a soil bacterium that for more than a century has been known as a pathogen causing the plant crown gall disease. Unlike many other pathogens, Agrobacterium has the ability to deliver DNA to plant cells and permanently alter the plant genome. The discovery of this unique feature 30 years ago has provided plant scientists with a powerful tool to genetically transform plants for both basic research purposes and for agricultural development.

Rubber tree (Hevea brasiliensis Muell. Arg.) is an important industrial crop for natural rubber production. At present, more than 9.5 million hectares in about 40 countries are devoted to rubber tree cultivation with a production about 6.5 million tons of dry rubber each year. The world supply of natural rubber is barely keeping up with a global demand for 12 million tons of natural rubber in 2020. Tapping panel dryness (TPD) is a complex physiological syndrome widely found in rubber tree plantations, which causes severe yield and crop losses in natural rubber producing countries. Currently, there is no effective prevention or treatment for this serious malady. As it is a perennial tree crop, the integration of specific desired traits through conventional breeding is both time-consuming and labour-intensive. Genetic transformation with conventional breeding is certainly a more promising tool for incorporation of agronomically important genes that could improve existing Hevea genotyp...

/ 5 gene in an indoleacetic acid (IAA)-dependent fashion. GUS expression was preferentially enhanced in the root elongation zone after treatment of young seedlings with 10 ؊ 7 M IAA. Expression of the BA-GUS gene in the axr1 , axr4 , and aux1 mutants required 10-to 100-fold higher auxin concentration than that in the wild-type background. GUS expression was nil in the axr2 and axr3 mutants. The transgene was used to isolate mutants exhibiting altered auxin-responsive gene expression ( age ). Two mutants, age1 and age2 , were isolated and characterized. age1 showed enhanced sensitivity to IAA, with strong GUS expression localized in the root elongation zone in the presence of 10 ؊ 8 M IAA. In contrast, age2 exhibited ectopic GUS expression associated with the root vascular tissue, even in the absence of exogenous IAA. Morphological and molecular analyses indicated that the age1 and age2 alleles are involved in the regulation of gene expression in response to IAA. Figure 1. Promoter-GUS Constructs and GUS Activity in Transgenic Arabidopsis Plants. (A) Schematic representation of the AuxRD-GUS fusions. The transcription start site (ϩ1) is shown, and the lengths of the promoter elements are indicated by the numbers over the constructs. CORE, nonauxin responsive segment spanning positions Ϫ92 to ϩ96 of PS-IAA4/5 gene; UBQ, UBQ3 promoter. (B) The mean GUS activity observed in seedlings analyzed without auxin treatment. The degree of auxin inducibility mediated by each promoter-GUS construct is shown as the ratio of GUS activity in seedlings treated with 2 ϫ 10 Ϫ5 M IAA relative to GUS activity observed in untreated seedlings. Each point is representative of seedlings from a single line. (C) Typical GUS staining patterns seen in No-0 plants harboring the indicated promoter-GUS fusions. The specific lines shown contain Ϫ318-GUS (line 37), CORE-GUS (line 53), A-GUS (line 5.1), B-GUS (line 20), BA-GUS (line 1.3), 4ϫA-GUS (line 30.4), and 4ϫB-GUS (line 13.2).

Background The future of genetic transformation as a tool for the improvement of fruit trees depends on the development of proper systems for the assessment of unintended effects in field-grown GM lines. In this study, we used eight transgenic lines of two different citrus types (sweet orange and citrange) transformed with the marker genes β-glucuronidase (uidA) and neomycin phosphotransferase II (nptII) as model systems to study for the first time in citrus the long-term stability of transgene expression and whether transgene-derived pleiotropic effects occur with regard to the morphology, development and fruit quality of orchard-grown GM citrus trees. Results The stability of the integration and expression of the transgenes was confirmed in 7-year-old, orchard-grown transgenic lines by Southern blot analysis and enzymatic assays (GUS and ELISA NPTII), respectively. Little seasonal variation was detected in the expression levels between plants of the same transgenic line in differe...

Twelve wastewater treatment plants (WWTPs) were sampled in France and Belgium in 1999 and 2000 in order to estimate the fecal coliform (FC) removal efficiency of various types of treatment. Only one of these WWTPs was equipped with a specific step to eliminate microorganisms (UV disinfection preceded by sand filtration). FC abundance was measured in raw and treated sewage by plate counts on selective medium and rapid b-d-glucuronidase (GLUase)based assays. Removal of culturable FC was the most efficient in treatments with high retention time (activated sludge process with nitrification and denitrification, lagooning), in biofiltration and in the treatment with a tertiary disinfection step. GLUase activity measurements showed the same removal pattern as plate counts except for UV disinfection, where no reduction of GLUase activity was measured. Specific loads of culturable FC and GLUase activity, i.e. daily amounts of culturable FC or GLUase activity in sewage per inhabitant-equivalent, were calculated in raw and treated wastewater for the different WWTPs. r

A series of 15 novel compounds incorporating the thieno[2,3-b]thiophene moiety were synthesized. The chemical structures of these compounds were deduced from elemental analyses, (1)H NMR, (13)C NMR, and ESI-mass spectral data. The enzyme inhibition potential of these compounds was evaluated, in vitro, against β-glucuronidase, xanthine oxidase, and α-chymotrypsin enzymes. The cytotoxicity was evaluated by a cell viability assay utilizing the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) dye. Among the compounds tested, compound 3 was the most potent β-glucuronidase inhibitor with an IC50 value of 0.9±0.0138μM; it was much more active than the standard, d-saccharic acid 1,4-lactone (IC50=45.75±2.16μM). Compound 12, on the other hand, was the most potent as a xanthine oxidase inhibitor with an IC50 of 14.4±1.2μM. With the characterization of their mechanism of action and with further testing, these compounds could be useful candidates as anticancer drugs. In additi...

Mature tissue protocol for production of transgenic Citrus plants via Agrobacterium -mediated transformation uses explants derived from branches of mature, fruit-bearing trees. Through the multiple cleaning steps consisting of grafting of apical tip meristems on rootstock plants grown under sanitary conditions, "mother" plants are produced that will serve as a source of budding material. These buds are grafted onto rootstock plants grown under the same, highly sanitary conditions. Newly obtained, one meter tall, young grafted plants serve as a source of explants for co-incubation experiments with Agrobacterium . Following successful transformation with Agrobacterium , selected transgenic shoots are micrografted onto rootstock plants in vitro where they are allowed to grow for a couple of months. Grafted transgenic plantlet together with the associated rootstock plant is taken out of culture tubes, severed from the root, and regrafted in terra on a 1-year-old rootstock plant. With the application of proper horticultural techniques, such a plant will yield fi rst fruit about 12-15 months later.

Aims: Investigate the interaction of bioluminescent Escherichia coli and Salmonella Montevideo with germinating mung bean sprouts. Methods and Results: E. coli or Salm. Montevideo introduced on mung beans became established both internally and externally on sprouts after the initial 24 h germinating period. In both cases the inoculated bacterium formed the predominant microflora on the sprouted beans throughout. From the bioluminescent profile of inoculated sprouting beans, bacterial growth was found to be in close proximity to the roots but not on the hypocotyls. Clumps (biofilms) of cells with low viability were observed within the grooves between epidermal cells on hypocotyls. Treatment with 20 000 ppm sodium hypochlorite removed the majority of bacteria from the surface of hypocotyls although nonviable single cells were occasionally observed. However, viable bacteria were recovered from the apoplastic fluid, and extracts of surface-sterilized sprouts indicating that the internal bacterial populations had been protected. This was confirmed using in situ b-glucuronidase staining of surface-sterilized sprouts where cleaved enzyme substrate (by the action of internalized E. coli) was visualized within the plant vascular system. Conclusions: E. coli or Salmonella present on seeds become internalized within the subsequent sprouts and cannot be removed by postharvest biocidal washing. Significance and Impact of the Study: Mung bean production should be carefully controlled to prevent contamination occurring in order to minimize the health risk associated with raw bean sprouts.

Heparin and heparan sulfate (HS) are glycosaminoglycans (GAGs) that are synthesized in the tissues and organs of mammals. They are synthesized and attached to a core protein as proteoglycans through serine-glycine concensus motifs along the core protein. These GAGs are linear polysaccharides composed of repeating disaccharide saccharide units that are variously modified along their length. As a consequence of these modifications naturally occurring heparin and HS are extremely heterogeneous in their structures. A diverse range of proteins bind heparin and HS. The types of proteins that bind are dictated by the structure of the HS or heparin chains with which they are interacting. Heparan sulfates play major roles in tissue development and in maintaining homeostasis within healthy individuals. Recent genetic studies illustrate that alterations in their structural organization can have important consequences often giving rise to, or directly causing, a disease situation. A greater und...

Variations in transgene expression due to position effect and copy number are normalized when analysing and comparing the strengths of different promoters. In such experiments, the promoter to be tested is placed upstream to a reporter gene and a second expression cassette is introduced in a linked fashion in the same transfer DNA (T-DNA). Normalization in the activity of the test promoter is carried out by calculating the ratio of activities of the test and reference promoters. When an appropriate number of independent transgenic events are analysed, normalization facilitates assessment of the relative strengths of the test promoters being compared. In this study, using different modifi ed versions of the Caulifl ower Mosaic Virus (CaMV) 35S promoter expressing the reporter gene β-glucuronidase (gus) (test cassette) linked to a chloramphenicol acetyl transferase (cat) gene under the wild-type 35S promoter (reference cassette) in transgenic tobacco lines, we observed that cat gene expression varied depending upon the strength of the modifi ed 35S promoter expressing the gus gene. The 35S promoter in the reference cassette was found to have been upregulated in cases where the modifi ed 35S promoter was weaker than the wild-type 35S promoter. Many studies have been carried out in different organisms to study the phenomenon of transcriptional interference, which refers to the reduced expression of the downstream promoter by a closely linked upstream promoter. However, we observed a positive interaction wherein the weakened activity of a promoter led to upregulation of a contiguous promoter. These observations suggest that, in situations where the promoters of the test and reference gene share the same transcription factors, the activity of the test promoter can infl uence the activity of the reference promoter in a way that the test promoter's strength is underestimated when normalized by the reference promoter.

O-␤-glucuronyl carbamate (DOX-GA3) has been developed to improve the antitumor effects of doxorubicin (DOX). The prodrug was originally designed to be activated into drug by human ␤-glucuronidase (GUS) released from tumor cells in necrotic areas of tumor lesions. The aim of this study was to further improve the antitumor effects of DOX-GA3 by means of antibody-directed enzyme prodrug therapy (ADEPT). We thus investigated if the administration of an enzyme-immunoconjugate prepared from the pancarcinoma Ep-CAM specific monoclonal antibody (MAb) 323/A3 and ␤-glucuronidase would result in improved antitumor effects because of additional enzyme localization in tumor tissue. In vitro, the prodrug DOX-GA3 was found to be 12-times less toxic than the parent drug DOX in a human ovarian cancer cell line. Immunospecific and complete activation of the prodrug took place when the cells were pretreated with 323/A3-␤-glucuronidase conjugate. In nude mice bearing s.c. human ovarian cancer xenografts (FMa) the maximum tolerated dose (MTD) of DOX-GA3 (500 mg/kg weekly ؋ 2) was much higher when compared with that of DOX (8 mg/kg weekly ؋ 2). In mice bearing well-established FMa xenografts, the standard treatment of DOX at the MTD (8 mg/kg weekly ؋ 2) resulted in a tumor growth inhibition of 67%. Treatment with DOX-GA3 at a single dose of 500 mg/kg resulted in a better tumor growth inhibition of 87%. The combination of DOX-GA3 (500 mg/kg) with 323/A3-mGUS conjugate and anti-GUS MAb 105, to clear circulating conjugate, improved the antitumor effect even further to 98%. At the lower dose of 250 mg/kg DOX-GA3 tumor growth inhibition (34%) was not better than that of DOX. The combination, however, of DOX-GA3 at 250 mg/kg and 323/A3-mGUS conjugate plus MAb 105 again greatly improved the antitumor effect (growth inhibition of 93%). DOX given at 8 mg/kg weekly x 2 did not result in tumor regressions. As a result of ADEPT, the number of regressions of tumors improved from 0 out of 12 to 9 out of 11 at a dose of 250 mg/kg DOX-GA3. At the higher prodrug dose (500 mg/kg) the number of regressions improved from 2 out of 12 to 9 out of 10 as a result from the addition of enzyme-immunoconjugate. Our studies show that the efficacy of the widely used anti-cancer agent DOX may be improved by using the prodrug DOX-GA3, in combination with the tumor-specific enzyme-immunoconjugate 323/A3-mGUS and a conjugate clearing antibody.

Jasmonate and related signalling compounds have a crucial role in both host immunity and development in plants, but the molecular details of the signalling mechanism are poorly understood. Here we identify members of the jasmonate ZIM-domain (JAZ) protein family as key regulators of jasmonate signalling. JAZ1 protein acts to repress transcription of jasmonate-responsive genes. Jasmonate treatment causes JAZ1 degradation and this degradation is dependent on activities of the SCF COI1 ubiquitin ligase and the 26S proteasome. Furthermore, the jasmonoyl-isoleucine (JA-Ile) conjugate, but not other jasmonate-derivatives such as jasmonate, 12-oxo-phytodienoic acid, or methyl-jasmonate, promotes physical interaction between COI1 and JAZ1 proteins in the absence of other plant proteins. Our results suggest a model in which jasmonate ligands promote the binding of the SCF COI1 ubiquitin ligase to and subsequent degradation of the JAZ1 repressor protein, and implicate the SCF COI1 -JAZ1 protein complex as a site of perception of the plant hormone JA-Ile.

pMADS3, a petunia class C gene, is the candidate homologue of Arabidopsis AGAMOUS (AG), which is involved in the specification of stamens and carpels. We report the characterization of loss-of-function phenotype of pMADS3 that resulted from silencing of this gene. Silencing of pMADS3 resulted in homeotic conversion of stamens into petaloid structures, whereas the carpels were only weakly affected. Ectopic secondary inflorescences emerged from the interstamenal region in the third whorl, which is unique and has not been reported for any class C gene of other plant species. Third-order inflorescences emerged at corresponding positions in the third whorl of inner flowers of secondary inflorescences, indicating reiterative conversion of parts of the floral meristem into inflorescence meristem. On the basis of phenotypic analysis of the pMADS3-silenced plants, we propose that pMADS3 is involved in determination of floral organ and floral meristem identity in petunia. Two hybrid studies in yeast showed that PMADS3 protein interacted specifically with FBP2, a candidate homologue of Arabidopsis SEPALLATA3 (SEP3). The evidence presented here suggest that a complex involving PMADS3 and FBP2 is responsible for specification of organ identity in the third whorl.

The beta-glucuronidase-deficient mucopolysaccharidosis type VII (MPS VII) mouse accumulates partially degraded glycosaminoglycans in many cell types, including retinal pigmented epithelial (RPE) cells in the eye. This lysosomal storage in RPE cells leads to progressive retinal degeneration and reduced function as measured by flash electroretinography (ERG). The impact of AAV-mediated intraocular gene therapy on pathology and retinal function was examined in normal and MPS VII mice treated at 4 weeks of age, when lysosomal storage is evident but functional impairment is minimal in affected animals. At 16 weeks, an age at which untreated MPS VII mice have advanced histologic lesions and significantly reduced ERG amplitudes, treated eyes had nearly normal levels of beta-glucuronidase activity, preservation of cells in the outer nuclear layer of the retina, and decreased lysosomal storage within the RPE. The AAV-treated MPS VII mice also had significantly increased dark-adapted ERG ampl...

Protein and lipid oxidation-mainly by mitochondrial reactive oxygen species (mtROS)-was proposed as a crucial determinant of health and lifespan. Angiotensin II (Ang II) enhances ROS production by activating NAD(P)H oxidase and uncoupling endothelial nitric oxide synthase (NOS). Ang II also stimulates mtROS production, which depresses mitochondrial energy metabolism. In rodents, renin-angiotensin system blockade (RAS blockade) increases survival and prevents age-associated changes. RAS blockade reduces mtROS and enhances mitochondrial content and function. This suggests that Ang II contributes to the ageing process by prompting mitochondrial dysfunction. Since Ang II is a pleiotropic peptide, the age-protecting effects of RAS blockade are expected to involve a variety of other mechanisms. Caloric restriction (CR)-an age-retarding intervention in humans and animals-and RAS blockade display a number of converging effects, i.e. they delay the manifestations of hypertension, diabetes, nephropathy, cardiovascular disease, and cancer; increase body temperature; reduce body weight, plasma glucose, insulin, and insulin-like growth factor-1; ameliorate insulin sensitivity; lower protein, lipid, and DNA oxidation, and mitochondrial H 2 O 2 production; and increase uncoupling protein-2 and sirtuin expression. A number of these overlapping effects involve changes in mitochondrial function. In CR, peroxisome proliferator-activated receptors (PPARs) seem to contribute to age-retardation partly by regulating mitochondrial function. RAS inhibition up-regulates PPARs; therefore, it is feasible that PPAR modulation is pivotal for mitochondrial protection by RAS blockade during rodent ageing. Other potential mechanisms that may underlie RAS blockade's mitochondrial benefits are TGF-b down-regulation and up-regulation of Klotho and sirtuins. In conclusion, the available data suggest that RAS blockade deserves further research efforts to establish its role as a potential tool to mitigate the growing problem of age-associated chronic disease.

We have previously reported the isolation and characterization of a gene (Zm 13) from Zea mays which shows a pollen-specific pattern of expression. Stably transformed tobacco plants containing a reporter gene linked to portions of the Zm 13 5′ flanking region show correct temporal and spatial expression of the gene. Here we present a more detailed analysis of the 5′ regions responsible for expression in pollen by utilizing a transient expression system. Constructs containing the β-glucuronidase (GUS) gene under the control of various sized fragments of the Zm13 5′ flanking region were introduced into Tradescantia and Zea mays pollen via high-velocity microprojectile bombardment, and monitored both visually and with a fluorescence assay. The results suggest that sequences necessary for expression in pollen are present in a region from −100 to −54, while other sequences which amplify that expression reside between −260 and −100. The replacement of the normal terminator with a portion of the Zm13 3′ region containing the putative polyadenylation signal and site also increased GUS expression. While the −260 to −100 region contains sequences similar to other protein-binding domains reported for plants, the −100 to −54 region appears to contain no significant homology to other known promoter fragments which direct pollen-specific expression. The microprojectile bombardment of Tradescantia pollen appears to be a good test system for assaying maize and possibly other monocot promoter constructs for pollen expression.

In this study, a versatile method was developed to assess biocide efficacy against Escherichia coli biofilm growth on carriers made of five different materials. The glucuronidase activity of live E. coli on a fluorogenic substrate (4-methylumbellyferyl-β-D-glucuronide, MUG) was used as a viability test. Fluorescence emissions from cellular suspensions of E. coli in the test range displayed a linear response with a MUG concentration of 10 μg ml −1 . A glucuronidase activity curve with cellular suspensions of E. coli calculated as colony-forming units per milliliter showed a good correlation (0.9487 and 0.917 for 1 and 18 h of incubation, respectively), with counts obtained from biofilm containing this organism; E. coli cultures in suspension were used as standard. Three agents commonly used as disinfectants, sodium hypochlorite, hydrogen peroxide, and ethanol, were tested at use concentrations and at one-half and decimal dilutions. At decimal dilutions, ethanol at 70% proved to be the least active disinfectant on E. coli biofilm. Unlike other methods, our method permits the testing of disinfectant efficacy against biofilm growth on different materials. In preliminary assays, glass, polyvinyl chloride, polypropylene, polycarbonate, and silicon were tested. Because they gave the lowest E. coli counts after 24 and 48 h, glass and polypropylene were the two materials to which biofilm adhered least strongly.