The cell wall is a multifunctional structure which is implicated in plant growth and development as well as responding to any environmental changes including biotic and abiotic stresses. One of the practical approaches in cell wall integrity studies is the modification of the quality and quantity of particular cell wall components or destroying the specific step in cell wall synthesis pathway using Cellulose Biosynthesis Inhibitors (CBIs). In this case, chemical screen for swollen organ phenotype has proved to be an important technique to identify the genes that are directly or indirectly involved in cellulose biosynthesis. In the present research, a number of synthetic CBIs were obtained through a chemical library screen from Chembridge Company for the root swollen phenotype which is believed to be the response to a defect in cellulose biosynthesis. Therefore, a genome-wide expression profiling based on Affymetrix ATH1 GeneChip arrays (contains 22810 probe sets) were applied to investigate the altered transcriptome of four different CBIs including CBI-15, 18, 22, and 27 and isoxaben in 5 day-old Arabidopsis thaliana seedlings. The results of this project revealed overlapped up and down-regulated genes as well as discriminate responses to each CBI. The most striking modification were found in genes involve in response to the stress as well as cell wall integrity and restructuring. Thus, the identification of regulated genes under CBIs treatment suggests a robust candidate group of genes that likely to be correlated to cell wall biosynthesis. / UOIT

No description available.

Intensive swine operations produce large amounts of manure that must be dealt with responsibly. Liquid swine manure (LSM) collected in storage units is applied to cropland as a nutrient source. Maximizing crop utilization of the nitrogen (N) added in manure is important to achieve economic and environmental benefits. The objectives of this research were to evaluate the effect of 1) adding a nitrification inhibitor and 2) using supplemental phosphorus (P) and sulfur (S) fertilizers as means of enhancing crop recovery of LSM-N.<p>Field experiments were conducted at two long-term manure management sites in Saskatchewan; 1) Dixon (Black Chernozem) and 2) Melfort (Dark Grey Luvisol). At the Dixon site, plant and soil samples were collected throughout the 2005 and 2006 growing season, and ammonium-N (NH4+-N) and nitrate-N (NO3--N) concentration in soil, and total N content in plant were measured. Plant root simulator (PRSTM) probes were used to measure NH4+-N and NO3--N supply rates at the Dixon site to determine the effectiveness of a nitrification inhibitor dicyandiamide (DCD) added to LSM. Crop recovery of N applied through LSM application was assessed by measuring seed and straw yield and total N content. The effect of adding supplemental P fertilizer at 6.5 kg P ha-1 to swine manure amended soil on N recovery was also evaluated at the Dixon site. At the S deficient Melfort site, the effect of supplemental S fertilizer added at 40 kg S ha-1 as ammonium sulfate and elemental S was evaluated.<p>The addition of DCD (0.275 mL kg-1) to LSM in 2005 and 2006 at Dixon did not significantly affect the proportion of LSM-N recovered or the seed yield. However, measurements of available NH4+-N and NO3--N concentrations and supply rates at the beginning of the growing season in 2005 indicated that the nitrification inhibitor was effective in keeping more of the LSM-N in the NH4+ form for approximately 14 days after LSM application. <p> The addition of supplemental P fertilizer to plots fertilized with LSM at the Dixon site, generally did not produce any significant increase in crop N recovery or seed yield. However, increase in crop N recovery and seed yield in 100 kg N ha-1 urea treatments indicates that there was insufficient P available in the soils to maximize crop N recovery and seed yield. It appears that LSM is able to provide sufficient amounts of available P when applied annually at rates of 37,000 L ha-1 or higher. <p>At the Melfort site, the addition of supplemental S fertilizer did not significantly affect crop N recovery or seed yield in LSM treatments. Annual applications of the low rate of LSM of 37,000 L ha-1 supplied sufficient amounts of N and S to maximize seed yield and crop N recovery. However, large significant increases in seed yield and crop N recovery with supplemental S fertilizers were observed in the 80 kg N ha-1 urea treatment.<p>The use of a nitrification inhibitor added to LSM was effective at maintaining N in NH4+ form longer; however there was no significant effect on final yield, grain N or %N recovery. This may be due to the low N loss potential on prairies. Supplemental S and P fertilizer may be required with liquid swine manure. Supplemental commercial fertilizers with LSM are dependant on: the crop nutrient requirements, soil nutrient status and manure nutrient composition.

Intensive swine operations produce large amounts of manure that must be dealt with responsibly. Liquid swine manure (LSM) collected in storage units is applied to cropland as a nutrient source. Maximizing crop utilization of the nitrogen (N) added in manure is important to achieve economic and environmental benefits. The objectives of this research were to evaluate the effect of 1) adding a nitrification inhibitor and 2) using supplemental phosphorus (P) and sulfur (S) fertilizers as means of enhancing crop recovery of LSM-N.<p>Field experiments were conducted at two long-term manure management sites in Saskatchewan; 1) Dixon (Black Chernozem) and 2) Melfort (Dark Grey Luvisol). At the Dixon site, plant and soil samples were collected throughout the 2005 and 2006 growing season, and ammonium-N (NH4+-N) and nitrate-N (NO3--N) concentration in soil, and total N content in plant were measured. Plant root simulator (PRSTM) probes were used to measure NH4+-N and NO3--N supply rates at the Dixon site to determine the effectiveness of a nitrification inhibitor dicyandiamide (DCD) added to LSM. Crop recovery of N applied through LSM application was assessed by measuring seed and straw yield and total N content. The effect of adding supplemental P fertilizer at 6.5 kg P ha-1 to swine manure amended soil on N recovery was also evaluated at the Dixon site. At the S deficient Melfort site, the effect of supplemental S fertilizer added at 40 kg S ha-1 as ammonium sulfate and elemental S was evaluated.<p>The addition of DCD (0.275 mL kg-1) to LSM in 2005 and 2006 at Dixon did not significantly affect the proportion of LSM-N recovered or the seed yield. However, measurements of available NH4+-N and NO3--N concentrations and supply rates at the beginning of the growing season in 2005 indicated that the nitrification inhibitor was effective in keeping more of the LSM-N in the NH4+ form for approximately 14 days after LSM application. <p> The addition of supplemental P fertilizer to plots fertilized with LSM at the Dixon site, generally did not produce any significant increase in crop N recovery or seed yield. However, increase in crop N recovery and seed yield in 100 kg N ha-1 urea treatments indicates that there was insufficient P available in the soils to maximize crop N recovery and seed yield. It appears that LSM is able to provide sufficient amounts of available P when applied annually at rates of 37,000 L ha-1 or higher. <p>At the Melfort site, the addition of supplemental S fertilizer did not significantly affect crop N recovery or seed yield in LSM treatments. Annual applications of the low rate of LSM of 37,000 L ha-1 supplied sufficient amounts of N and S to maximize seed yield and crop N recovery. However, large significant increases in seed yield and crop N recovery with supplemental S fertilizers were observed in the 80 kg N ha-1 urea treatment.<p>The use of a nitrification inhibitor added to LSM was effective at maintaining N in NH4+ form longer; however there was no significant effect on final yield, grain N or %N recovery. This may be due to the low N loss potential on prairies. Supplemental S and P fertilizer may be required with liquid swine manure. Supplemental commercial fertilizers with LSM are dependant on: the crop nutrient requirements, soil nutrient status and manure nutrient composition.

C1 inhibitor (C1 INH) is a member of the serine protease inhibitor (serpin) superfamily. It is the only physiological inhibitor of protease C1r and C1s in the complement system. C1 INH is a single chain glycoprotein with apparent molecular weight of 105 KDa, consisting of 478 amino acids. C1 INH N-terminal domain includes first 98 amino acids with 10 definite and 7 potential glycosylation site. Various of carbohydrates are present on the cell surface and component of ECM (extracellular matrix) in every eukaryotic cell, including both cancer cells and cells that are important for tumur survival. Carbohydrates on the cancer cell surface have been shown to be important in many aspects of cancer cell physiological processes, involved in cell growth and cell adhesion. Carbohydrates are also able to bind and interact with growth factors and other proteins that trigger signal transduction. Interfere carbohydrates maybe offer a useful therapeutic approach for treating cancers. In order to understand whether the C1 INH NT98 polypeptides can influences cancer or not, we amplified a DNA fragment encoding C1 INH N-terminal domain 98 residues (C1 INH NT98) by PCR, and transfer to the plasmid pGEX-2T, than use E.coli (BL21 strain) to express the non-glycosylated polypeptides, and further analyze the influence of the effective roles exhibited by the polypeptides non-glycosylated on breast cancer cell MDA-MB-435s. Proliferation and migration assays in our experiment showed that non-glycosylated C1 INH NT98 can inhibited breast cancer cell growth and migration, and the mechanism needed to be clarified clearly through extensive research.

G-NNACI, a Naja naja atra chymotrypsin inhibitor consists of 57 amino acid residues cross-linked by three disulfide bridges and belongs to the Kunitz/BPTI superfamily, has been successfully cloned and expressed in our laboratory. Since snake venom non-neurotoxic Kunitz/BPTI inhibitors are most conserved in the core and in the N-terminal surface area, Ala-screening mutagenesis, deletion and Domain swapping on the N-terminus were carried out in this study to assess the role of N-terminus in G-NNACI. G-NNACI mutants with single amino acid substitution and deleted mutants were prepared. The secondary structure of all mutated proteins did not significantly alter as evidenced by CD spectra. Although all mutants are found to be functionally active as an inhibitor, their inhibitory potency against chymotrypsin differed. In contrast to G-NNACI and other mutants, R1A¡BP2A and ¡µN3 mutants had a propensity to alter their disulfide linkages under basic conditions. The results of thermal and urea denaturation suggested that amino acid substitution and deletion at the N-terminus lead to a change in the structural stability of G-NNACI. Consequently, the inhibitory potency of G-NNACI mutants along with time was affected. B chain of

Endostatin, an angiogensis inhibitor, was discovered by Dr. Judah Folkman¡¦s group in 1997. From their series studies, they demonstrated that the angiogenesis inhibition approach, which abolished the formation of new blood vessels and led to starvation of cancer cells, is a safe, effective anticancer method without side effect and drug resistance. Phase clinical trial on endostatin was carried out in 1999 and completed in 2001, heralding the approaching of a new arsenal of cancer therapy drugs. Endostatin is also a proteolytic fragment (~20 kDa) from an extracellular protein, collagen XVIII. It potently inhibits endothelial cell proliferation and angiogenesis, but has no cytotoxic effects on other cells. Above all, cycled therapy of experimental cancer in rodents with endostatin led to tumor dormancy without drug resistance. However, the exact mechanism on how endostatin inhibited endothelial cells proliferation remains largely unknown. We have cloned mouse endostatin cDNA from mice liver by RT-PCR. After verification by DNA sequencing, endostatin cDNA was subcloned in to E. coli expression vector to express and generate large quantities of recombinant GST-fused endostatin. Unlike His-tagged endostatin, GST-endostatin is soluble and capable of inhibiting endothelial cell lines EA.hy926 with a half-maximal inhibition concentration (IC50) of 20 nM. In present study, we investigated whether GST-endostatin caused alterations in cytoskeleton in endothelial cells. By using a fluorescence dye to visualize the actin filament under confocal microscope, it was found that endostatin induced the corruption of actin network in endothelial cells. Western blot analysis revealed that GST-endostatin treatment caused downregulation of cytoskeleton proteins such as tubulin, vimentin and ECM-related signaling molecules such as focal adhesion kinase (FAK), mitogen activated protein kinse (MAPK), Erk in a dose-dependent manner. Moreover, GST-endostatin decreased the levels of cell survival factor such as AKT and NF-£eB. Since GST-endostatin induced sustained calcium rise, the effect of endostatin on protein kinase Cs (PKCs) were studied and revealed that endostatin reduced the levels of PKCK1¡BPKC eta¡BPKC iota and PKC lamda. Other than endothelial cell, the cytotoxicity of GST-endostatin in hepatoma cells were investigated since liver the primary expression site of collagen XVIII, precursor of endostatin. Unexpectedly, endostatin also inhibited the proliferation of hepatoma cells. Flow cytometry and nucleus staining indicated that GST-endostatin also induced apoptosis in hepatoma cells. Moreover, GST-endostatin exhibited differential cytotoxic effect against well-differentiated (such as HepG2, Hep3B) and poor differentiated (such as Mahlavu, Sk-hep-1) hepatoma cells that the IC50 for well differentiated hepatoma cells were 8-10 folds lower than for poor-differentiated cells. Above all, GST-endostatin inhibited the migration of SK-hep-1 and modulated the secretion of matrix-metalloproteinases (MMPs) by Mahlavu and SK-hep-1 cells. In summary, present study explored the role of alterations in cytoskeleon network in the cytotoxic mechanism of GST-endostatin. Moreover, the inhibitory effects of GST-endostatin on proliferation of hepatoma cells were reported for the first time.

Abstract Vasostatin, the N-terminal 180 amino acids domain of calreticulin, induces apoptosis in endothelial cells and inhibits angiogenesis. However, the mechanism underlying the apoptosis induce by vasostatin remains elusive. In the present study, we investigated the role of (1) Fas /FasL pathway, (2) oxidative stress, and (3) nitric oxide (NO) in the apoptotic mechanism of vasostatin in endothelial cells. Recombinant vasostatin was generated and shown to induce apoptosis of bovine aortic endothelial cells (BAEC) as demonstrated by flow cytometry analysis, nucleus staining, and DNA fragmentation assay. Vasostatin elevated the levels of Fas and its adaptor, FADD, in BAEC. Furthermore, vasostatin treatment increased the activities as well as the expression of active form of caspase-8 and caspase-3 in BAEC. However, pretreatment with either caspase-3 inhibitor or caspase-8 inhibitor alone was not sufficient to blockade the vasostatin-mediated apoptosis, suggesting the involvement of other pathways. Extensive screening using an array of caspase inhibitors further supported such notion. Oxidative stress is frequently involved in the apoptosis of endothelial cells. Previous studies indicated that vasostatin enhanced WST-1-derived formazan formation despite its cytotoxic effect, suggesting vasostatin treatment might enhance the production of superoxide. By measuring the level of superoxide anion in cultured media by cytochrome c reducing test, it was found that vasostatin treatment increased the production of superoxide anion in endothelial cells. Antioxidants such as NAC, GSH, BHA partially attenuated the vasostatin-mediated cytotoxicity and cell death in endothelial cells. Noteworthingly, adding allopurinol, inhibitor of xanthine oxidase, but not other oxidase inhibitors abrogated the cytotoxicity of vasostatin, indicating that xanthine oxidase could be the source of ROS produced by vasostatin relate with apoptosis. The elecctrophoretic mobility shift assays (EMSA) suggested that vasostatin treatment increased the NF£eB DNA binding activity. Western blot analysis indicated vasostatin increased the levels of NF£eB but decreased I£eB level, which seemed to coincide with the EMSA findings. NO plays an important role in endothelial function. To investigate the role of NO in the cytotoxicity by vasostatin, analyzed the levels of NO metabolites in cultured media of endothelial cells and found that vasostatin treatment increased NO release in time- dependent manners. The expression of eNOS, but not iNOS, in endothelial cells was upregulated by vasostatin. Besides, vasostatin treatment also increased the AP-1 binding activities. Moreover, NOS inhibitor, L-NAME, or NO scavenger, carboxy-PTIO, slightly attenuated the cytotoxic effects of vasostatin in endothelial cells. In addition to direct cytotoxicity, NO may react with superoxide (O2-) to form peroxynitrite (ONOO-), which attacked the intracellular protein and caused the cell damage. Indeed, we also detected a dose-dependent increment in the nitrotyrosination of cellular protein by vasostatin treatment. Taking together, these results indicate that vasostatin induces apoptosis in endothelial cells via multiple pathways. The interactions between these distinct pathways remain to be elucidated in the future.

Zugl.: Giessen, Universiẗat, Diss., 2009.

The use of urea fertiliser has been associated with relatively poor nitrogen (N) use efficiency (NUE) due to heavy N losses such as gaseous emissions of ammonia (NH₃) and nitrous oxide (N₂O) and nitrate (NO₃⁻) leaching into surface and ground waters. Improving N use-efficiency of applied urea is therefore critical to maximise its uptake and to minimise its footprint on the environment. The study was conducted under laboratory-glasshouse conditions (Chapter 2-4)and lysimiter-field plot studies (Chapter 5). In chapter 2, Two glasshouse-based experimentswere conducted to investigate the potential of incorporating urea fertiliser with ureaseinhibitor, (N-(n-butyl) thiophosphoric triamide (nBTPT) or ‘Agrotain’) to enhance fertiliser N uptake efficiency. Urea, with or without Agrotain, was applied to Ryegrass (Lolium perenne L.) grown in standard plant trays maintained at soil moisture contents of 75–80% field capacity, at rates equivalent to 25 or 50 kg Nha⁻¹. These treatments were compared with other common forms of N fertilisers (ammonium nitrate, ammonium sulphate and sodium nitrate). In a separate pot experiment, granular ¹⁵N urea (10 atom %) with or without Agrotain, was applied at 25 kg Nh⁻¹ to track N use-efficiency and the fate of ¹⁵N-labelled fertiliser. In both experiments, Agrotain-treated urea improved bioavailability (defined as the fraction of total soil N that can interact with a biological target in the plant or that can be taken up by plant) of added N and resulted in significantly higher herbage DM yield and N uptake than urea alone or other forms of N fertilisers. Results from the ¹⁵N experiment support the suggestion that a delay in urea hydrolysis by Agrotain provided an opportunity for direct plant uptake of an increased proportion of the applied urea-N than in the case of urea alone. In chapter 3, two more glasshouse-based experiments were conducted to investigate if urea applied in fine particle application (FPA), with or without Agrotain, had any effect on fertiliser-N uptake efficiency (defined as the difference in N uptake between the fertiliser treatment and the control as a percentage of the amount of N applied) under optimum soil moisture (75-80% field capacity) and temperature (25 °C) conditions, in comparison with other common forms of N fertilisers applied, either in FPA or in granular form. In a separate pot experiment, ¹⁵N urea (10 atom %), with or without Agrotain, was applied to either shoots or leaves only or to the soil surface (avoiding the shoots and leaves) to determine urea hydrolysis, herbage DM and ¹⁵N uptake. In both experiments, herbage DM yield and N uptake were significantly greater in the FPA treatments than in those receiving granular application. Agrotain-treated urea FPA resulted in significantly higher N response efficiency (difference between the dry matter produced by the various fertiliser treatments and the control, divided by the amount of N applied) than urea FPA alone or other forms of N fertilisers. Results from the ¹⁵N experiment support the idea that Agrotain treatment improves the N response of urea applied in FPA form due to a delay in hydrolysis of urea, thus providing herbage an extended opportunity to absorb added urea directly through leaves, cuticles and roots. A further glasshouse-based study was conducted to investigate the effect of Agrotain and irrigation on urea hydrolysis and its movement in a Typic Haplustepts silt loam soil (Chapter 4). A total of 72 repacked soil cores (140 mm inner diameter and 100 mm deep) were used - half (36) of these cores were adjusted to soil moisture contents of 80% field capacity (FC) and the remaining 36 cores to 50% FC. Granular urea, with or without Agrotain, was applied at a rate equivalent to 100 kg N ha⁻¹. Twelve pots were destructively sampled at each day after 1, 2, 3, 4, 7, and 10 days of treatment application to determine urea hydrolysis and its lateral and vertical movement in different soil layers. Agrotain-treated urea delayed urea hydrolysis compared with urea alone during the first 7 days of its application. This delay in urea hydrolysis by Agrotain enabled added urea to disperse and move away from the surface soil layer to the sub-surface soil layer both vertically and laterally. In contrast, most urea in the absence of Agrotain hydrolysed within 2 days of its application. Irrigation after 1 day resulted in further urea movement from the surface soil layer (0-10 mm) to the sub-soil layer (30-50 mm) in Agrotain-treated urea. These results suggest that Agrotain delayed urea hydrolysis and allowed more time for rainfall or irrigation to move the added urea from the surface layer to sub-soil layers where it is likely to make good contact with plant roots. This distribution of urea in the rooting zone (0-200 mm) has the potential to enhance N use efficiency and minimise N losses via ammonia (NH₃) volatilisation from surface-applied urea. Finally, a field study using lysimeters (300 mm inner diameter and 400 mm deep), and small field plots (1 m² in area) was established using a silt loam Typic Haplustepts soil (Soil Survey Staff 1998) to investigate the effect of FPA and granular applications of urea, with or without Agrotain, on N losses and N use efficiency (Chapter 5). The five treatments were: control (no N) and ¹⁵N-labelled urea (10 atom %), with or without Agrotain, applied to lysimeters or mini plots (un-labelled urea), either in granular form to the soil surface or in FPA form (through a spray) at a rate equivalent to 100 kg N ha⁻¹. Gaseous emissions of NH₃ and N₂O, NO₃⁻ leaching, herbage production, N response efficiency, total N uptake and total recovery of applied ¹⁵N in the plant and soil were determined up to 63 days. Urea-alone and urea with Agrotain, applied in FPA form, was more effective than its granular form and reduced N2O emissions by 5-12% and NO3- leaching losses by 31-55%. Urea-alone applied in FPA form had no significant effect in reducing NH₃ losses compared with granular form. However, urea with Agrotain applied in FPA form reduced NH₃ emissions by 69% compared with the equivalent granular treatment. Urea-alone and with Agrotain applied in FPA form increased herbage dry matter production by 27% and 38%, and N response efficiency compared with the equivalent granular urea application, respectively. Urea applied in FPA form resulted in significantly higher ¹⁵N recovery in the shoots compared with granular treatments – this was improved further when urea in FPA form was applied with Agrotain. Thus, treating urea with Agrotain in FPA under field conditions has the potential to delay its hydrolysis, minimise N losses and improve N use efficiency and herbage production. The lower dry matter production and N-response efficiency to urea applied in FPA form in Chapter 3 are probably because of additional factors such as lower application rates (25 kg N ha⁻¹ ) or lack of interception of urea by the leaves. Applying urea in FPA form is a good management strategy and I conclude that combining FPA urea with Agrotain has the potential to increase N use efficiency and herbage production further.