Verkenning sensorgestuurde dosering van loofdodingsmiddelen in pootaardappelen

In 2008 is een verkenning gedaan naar haalbaarheid van sensorgestuurde dosering van loofdodingsmiddelen in pootgoedteelt van aardappelen. Er werd een prototype gebouwd en getest waarmee Finale pleksgewijs gedoseerd werd d.m.v. Greenseeker-sensoren op een klap-spuit-combinatie en een rekenregel die plaatspecifieke meetwaarden doorvertaalde in een minimum effectieve dosering. Ook werden dosis-response proeven uitgevoerd. De eerste resultaten met het prototype en systeem waren positief. Met de sensoren konden variatie in gewasstand in kaart gebracht worden, en deze bleek groter dan verwacht. Bovendien kon de dosering effectief gestuurd worden met de sensormeetwaarden. Het onderzoek laat zien dat reductie in loofdodingsmiddel met sensorsturing mogelijk is. In het onderzoek bleken ras en dosering van Finale geen effect te hebben op verkleuring van navels van knollen. Nader onderzoek aan de beslisregels, validatie in de praktijk inclusief effecten op kwaliteit van de aardappelen is nodig voor implementatie in de praktij

Population Pharmacokinetic Modelling of FE 999049, a Recombinant Human Follicle-Stimulating Hormone, in Healthy Women After Single Ascending Doses

OBJECTIVE: The purpose of this analysis was to develop a population pharmacokinetic model for a novel recombinant human follicle-stimulating hormone (FSH) (FE 999049) expressed from a human cell line of foetal retinal origin (PER.C6(®)) developed for controlled ovarian stimulation prior to assisted reproductive technologies.METHODS: Serum FSH levels were measured following a single subcutaneous FE 999049 injection of 37.5, 75, 150, 225 or 450 IU in 27 pituitary-suppressed healthy female subjects participating in this first-in-human single ascending dose trial. Data was analysed by nonlinear mixed effects population pharmacokinetic modelling in NONMEM 7.2.0.RESULTS: A one-compartment model with first-order absorption and elimination rates was found to best describe the data. A transit model was introduced to describe a delay in the absorption process. The apparent clearance (CL/F) and apparent volume of distribution (V/F) estimates were found to increase with body weight. Body weight was included as an allometrically scaled covariate with a power exponent of 0.75 for CL/F and 1 for V/F.CONCLUSIONS: The single-dose pharmacokinetics of FE 999049 were adequately described by a population pharmacokinetic model. The average drug concentration at steady state is expected to be reduced with increasing body weight

Cell-type-specific modulation of hox gene expression by members of the TGF-beta superfamily: a comparison between human osteosarcoma and neuroblastoma cell lines

Contains fulltext : 25410___.PDF (publisher's version ) (Open Access

Genomic organization of the human bone morphogenetic protein-4 gene: molecular basis for multiple transcripts

Contains fulltext : 23948___.PDF (publisher's version ) (Open Access

Regulation of gluconate and ketogluconate production in <Emphasis Type="Italic">Gluconobacter oxydans</Emphasis> ATCC 621-H

Abstract. Gluconobacter spp. possess the enzymic potential for two pathways of direct glucose oxidation. It has been proposed that the major part of glucose is oxidized to gluconate via NADP-dependent glucose dehydrogenase and that reoxidation of NADPH under these conditions proceeds via recycling of gluconate through ketogluconates. This hypothesis was tested in experiments in which Gluconobacter oxydans ATCC 621-H was grown in glucoseyeast extract medium containing [~4C]2-ketogluconate. As expected, glucose was almost quantitatively oxidized to gluconate, without further accumulation of 2-and 5-ketogluconate. Interestingly, the total amount of neither [14C]2-ketogluconate nor [14C]gluconate did change significantly during this oxidation phase, indicating that recycling of gluconate through ketogluconates did not occur. An analysis of enzyme activities in cell-free extracts of glucose-grown cells of G. oxydans ATCC 621-H showed that the membranebound glucose dehydrogenase was far more active than the NADP-linked glucose dehydrogenase. The activity of the latter enzyme constituted only 10-15% of that of quinoprotein glucose dehydrogenase and was far too low to match the in vivo rates of gluconate production in batch cultures of G. oxydans. It is concluded that under these conditions glucose is mainly oxidized to gluconate via the membrane-bound glucose dehydrogenase. Implications of these results for the regulation of ketogluconate formation are discussed. Key words: Gluconobacter oxydans ATTC 621-H -Glucose oxidation -Gluconate formation -Ketogluconate formation -Regulation -[14C]ketogluconate -Enzymology The accumulation of gluconate and ketogluconates from glucose by Gluconobacter species has been subject of many studies. As a result, detailed information is currently available on cultivation conditions which favor their production. The underlying regulatory mechanisms that control the production of these compounds, however, are still poorly understood During growth of G. oxydans ATTC 621-H and other Gluconobacter species on glucose in pH controlled batch cultures, a sequential accumulation of gluconate and ketogluconates is generally observed 535 Inocula for experiments in fermenters were prepared from frozen stocks ( -7 0~ 2 ml) in 250 ml shake flasks containing 50 ml of mannitol (1% w/v)/yeast extract (0.2% w/v) medium. Incubation was overnight on a rotary shaker at 28~ and 200 rpm. Cultures to be used for analysis of enzymes were grown in Biostat E fermenters (B. Braun Melsungen AG, Melsungen, FRG) with a working volume of 5 1. The airflow rate was 60 1 h -1 and the dissolved-oxygen tension, as recorded polarographically with a Clark-type electrode, was maintained above 25% air saturation by automatic adjustment of the stirrer speed (500-1,000 rpm). The temperature was kept at 28~ and the pH was controlled at 5.5 by automatic titration with 4 M NaOH or 10% (v/v) H3PO4. Experiments with 14C-labelled compounds were carried out in a magnetically-stirred 1-1 fermenter (New Brunswick Scientific Co., Inc., Edison, NJ, USA) with a working volume of 0.5 1. The temperature of the cultures was 28 ~ C and the pH was maintained at 5.5 by automatic titration with 2 M NaOH or 1 M HC1. Sufficient aeration was obtained by vigorous stirring (750 rpm) and a high airflow rate (301 h-l). Growth of the cultures was monitored by measuring the absorbance at 650 nm using a Bausch and Lomb Spectronic 2000 spectrophotometer (Bausch and Lomb Inc. Rochester, NY, USA). For the assay of enzyme activities cells were harvested by centrifugation at 10,000 x g for 10 min at 4~ washed twice with 50 mM potassium phosphate buffer pH 6.0 containing 5 mM MgCI2 and resuspended in this buffer to a final concentration of 2 0 -4 0 mg dry weight ml-1. These suspensions were used immediately for enzyme assays or stored at -7 0~ until required. When used within 1 month, extracts of frozen cells exhibited enzyme activities of more that 90% of those obtained with freshly harvested cells. Materials and methods Microorganisms The Gluconobacter strains used in this study, G. oxydans subsp, suboxydans (ATCC 621-H) and G. oxydans subsp. albidus (IFO 3251 t2), and their maintenance have been described previously Media and cultivation The organisms were either grown in a complex medium containing glucose and yeast extract at concentrations as indicated in the Results section or in a mineral salts medium, supplemented with vitamins and L-glutamine. This medium contained per litre of deionized water: (NH4)2804, 2 g; MgSO4 " 7 H20, 0.2 g; KH2PO4, 2.2 g; NazHPO4 9 2 H20, 0.2 g; trace element solution according to Preparation of cell-free extracts and enzyme assays Suspensions were sonicated with a Branson Sonifier B-12 Cell Disruptor (Branson Sonic Power Co., Danbury, CT, USA) operating at 45 W output at 0~ for 15 x 15 s, with intermitting cooling periods of 30 s. Whole cells were removed by centrifugation at I0,000 x g for 5 rain at 4~ and the supernatant, which contained 7 -1 6 nag protein ml-1 was used as the cell-free extract for the determination of enzyme activities. All assays were performed at 28~ with a Bausch and Lomb Spectronic 2000 spectrophotometer (Bausch and Lomb Inc., Rochester, NY, USA). The observed rate was linear for at least 2 rain and was proportional to the amount of extract added. Enzyme activities are expressed as units rag-1 protein. One unit of activity is defined as that amount of enzyme catalyzing the transformation of 1 gmol of substrate in 1 rain. The molar extinction coefficients ( M -1 c m -1) of N A D P H at 340 nm and of 2,6-dichlorophenolindophenol at 600 nm and pH 5.5 were 6.22 x 103 and 7.0 x 103, respectively. In the assays of the NADP-dependent dehydrogenase and reductases Triton X-100 was added to inhibit the high spontaneous rate of N A D P H oxidation as was reported for rat liver microsomes (Boutin 1986). In this respect Triton X-100 turned out to be a more efficient inhibitor than potassium cyanide or sodium azide. 536 Enzyme assays were performed as described previously Glucose dehydrogenase (EC 1.1.99.17). The reaction mixture (1 ml) contained: potassium phosphate buffer pH 5.5, 75 ~mol; 2,6-dichlorophenolindophenol, 0.15 ~mol; phenazinc methosulphate, 2 ~tmol; and extract. The reaction was started by the addition of 20 ~mol glucose. Glucose dehydrogenase (NADP-dependent) (EC 1.1.1.47). The reaction mixture (1 ml) contained: potassium phosphate buffer pH 7.4, 75 ~mol; NADP, 0.5 ~tmol; Triton X-100, 0.2% (v/v); and extract. The reaction was started by the addition of 60 ~tmol glucose. Gluconate dehydrogenase . The reaction mixture (1 ml) was the same as for the particulate glucose dehydrogenase (EC 1.1,99.17), except that 201xmol potassium gluconate was used as the substrate. 5-Ketogluconate reductase (NADP-dependent) (EC 1.1.1.69). The reaction mixture (1 ml) contained: potassium phosphate buffer pH 7.4, 75 l~mol; NADPH, 0.15 ~tmol; Triton X-100, 0.2% (v/v); and extract. The reaction was started by the addition of 2.51xmol potassium 5-ketogluconate. 2-Ketogluconate reductase The reaction mixture (1 ml) was the same as for 5-ketogluconate reductase, except that 10 ~tmol sodium 2-ketogluconate was used as the substrate. Preparation of [14 C] 2-ketogluconate Since 14C-labelled ketogluconates are not commercially available, it was decided to prepare [x4C]2-ketogluconate fermentatively from [U-lgC]glucose by a culture of G. oxydans subsp, albidus (IFO 3251 t2), as described above. The total amount of radioactivity of the 2-ketogluconate preparation obtained in this way was determined by liquid scintillation counting. Samples (10 or 20 Ixl) were diluted in 2 ml Pico-fluor 30 scintillation fluid and measured in a Packard Tri-Carb model 2000 CA analyzer (Packard Instrument Co., Downers Grove, IL, USA). Counting efficiences were calculated from internal quenching calibration curves. Protein determinations Protein was determined by the method of Substrate/product analyses Samples from cultures for the determination of substrates and products were obtained by centrifugation and subsequent filtration through a 0.2 ~tm filter (Sartorius GmbH, Gtttingen, FRG). Glucose was assayed enzymatically (GOD-Perid method of Boehringer Mannheim GmbH, FRG). Gluconate was either determined with gluconate kinase/ 6-phosphogluconate dehydrogenase (Boehringer Mannheim GmbH, FRG) or by a HPLC method, which was also used for the analysis of 2-and 5-ketogluconate (see below). Prior to the enzymatic analysis the samples were diluted 10-fold in 0.1 M K2HPO4 and incubated overnight, in order to ensure complete hydrolysis of glucono-5-1actone (the primary product of glucose oxidation) to gluconate. Gluconate and 2-and 5-ketogluconate were detected by High-Performance Liquid Chromatography using a Bio-Sil TSK DEAE-2 SW column (250 x 4.5 ram) (Bio-Rad Laboratories, Richmond, CA, USA). Sample volumes of 10 ~tl were injected. The temperature during the chromatography was kept constant at 40~ Each run was carried out isocratically using 60 mM KHzPO4 elution buffer, adjusted to pH 3.75 with phosphoric acid. Complete hydrolysis of glucono-6-1actone was achieved by adding 0.05 ml 1 M K/HPO4 to I ml of sample. After overnight incubation these solutions were diluted with a minimum of 0.95 ml 50 mM KH2PO4 (adjusted to pH 3.9 with phosphoric acid). Chromatography was performed with a flow rate of 0.5 ml rain-~ and gluconate, 5-ketogluconate and 2-ketogluconate peaks were detected by UV absorbance at 210 nm after approx. 13 rain, 15.5 rain and 17 rain, respectively. The detection limit was 1 raM. Radioactivity 14C present in these peaks was measured continuously by using a Ramona-D detector equipped with an internal CaF scintillator (400 ~tl volume) (Isomess GmbH, Straubenhardt, FRG), in series with the UV-detector. Chemicals [U-14C]glucose was obtained from NEN Chemicals (Boston, MA, USA) as dry powder. All biochemicals used in enzyme assays were from Sigma Chemical Co. (St. Louis, MO, USA), except for NADP and NADPH, which were purchased from Boehringer Mannheim GmbH (FRG). Pico-fiuor 30 was obtained from Packard Instrument Co. (Downers Grove, IL, USA). Results Production of [ l ~ C]2-ketogluconate from [14 C]glucose by Gluconobacter oxydans subsp, albidus (IFO 3251 t2) Radioactive ketogluconate was prepared by using G. oxydans subsp, albidus (IFO 3251 t2), which was selected for its high selectivity and rate of glucose conversion into 2-ketogluconate Fate of [14 C]2-ketogluconate during the oxidation of excess glucose in batch cultures of Gluconobacter oxydans ATCC 621-H The availability of radioactive ketogluconate allowed us to test the hypothesis that gluconate is being recycled during Enzymology of glucose utilization by Gluconobacter oxydans ATCC 621-H The gluconate-recycling model was originally put forward following the observation that NADP-dependent glucose dehydrogenase is the more active enzyme in the first phase of glucose utilization in batch culture. The results obtained in the experiment described above prompted us to critically reexamine the role of the various enzymes involved in the direct pathway of glucose oxidation Discussion The ability of Gluconobacter to produce gluconate from glucose has been recognized for a long time (Boutroux 1886). Over the years it has become clear that this is not a property 538 peculiar for this genus, but that several strains of Pseudomonas (Lessie and Phibbs 1984), Escherichia eoli (Hommes et al. 1984), Klebsiella aerogenes The results of the experiment with [~4C]2-ketogluconat