A Modified phosphate-carrier protein theory is proposed as a non-target site mechanism For glyphosate resistance in weeds

Glyphosate is an herbicide that inhibits the enzyme 5-enolpyruvyl-shikimate-3-phosphate synthase (EPSPs) (EC 2.5.1.19). EPSPs is the sixth enzyme of the shikimate pathway, by which plants synthesize the aromatic amino acids phenylalanine, tyrosine, and tryptophan and many compounds used in secondary metabolism pathways. About fifteen years ago it was hypothesized that it was unlikely weeds would evolve resistance to this herbicide because of the limited degree of glyphosate metabolism observed in plants, the low resistance level attained to EPSPs gene overexpression, and because of the lower fitness in plants with an altered EPSPs enzyme. However, today 20 weed species have been described with glyphosate resistant biotypes that are found in all five continents of the world and exploit several different resistant mechanisms. The survival and adaptation of these glyphosate resistant weeds are related toresistance mechanisms that occur in plants selected through the intense selection pressure from repeated and exclusive use of glyphosate as the only control measure. In this paper the physiological, biochemical, and genetic basis of glyphosate resistance mechanisms in weed species are reviewed and a novel and innovative theory that integrates all the mechanisms of non-target site glyphosate resistance in plants is presented.

Surface characterization of chemically modified fiber, wood and paper

Inorganic-organic sol-gel hybrid coatings can be used for improving and modifying properties of wood-based materials. By selecting a proper precursor, wood can be made water repellent, decay-, moisture- or UV-resistant. However, to control the barrier properties of sol-gel coatings on wood substrates against moisture uptake and weathering, an understanding of the surface morphology and chemistry of the deposited sol-gel coatings on wood substrates is needed. Mechanical pulp is used in production of wood-containing printing papers. The physical and chemical fiber surface characteristics, as created in the chosen mechanical pulp manufacturing process, play a key role in controlling the properties of the end-use product. A detailed understanding of how process parameters influence fiber surfaces can help improving cost-effectiveness of pulp and paper production. The current work focuses on physico-chemical characterization of modified wood-based materials with surface sensitive analytical tools. The overall objectives were, through advanced microscopy and chemical analysis techniques, (i) to collect versatile information about the surface structures of Norway spruce thermomechanical pulp fiber walls and understand how they are influenced by the selected chemical treatments, and (ii) to clarify the effect of various sol-gel coatings on surface structural and chemical properties of wood-based substrates. A special emphasis was on understanding the effect of sol-gel coatings on the water repellency of modified wood and paper surfaces. In the first part of the work, effects of chemical treatment on micro- and nano-scale surface structure of 1st stage TMP latewood fibers from Norway spruce were investigated. The chemicals applied were buffered sodium oxalate and hydrochloric acid. The outer and the inner fiber wall layers of the untreated and chemically treated fibers were separately analyzed by light microscopy, atomic force microscopy and field-emission scanning electron microscopy. The selected characterization methods enabled the demonstration of the effect of different treatments on the fiber surface structure, both visually and quantitatively. The outer fiber wall areas appeared as intact bands surrounding the fiber and they were clearly rougher than areas of exposed inner fiber wall. The roughness of the outer fiber wall areas increased most in the sodium oxalate treatment. The results indicated formation of more surface pores on the exposed inner fiber wall areas than on the corresponding outer fiber wall areas as a result of the chemical treatments. The hydrochloric acid treatment seemed to increase the surface porosity of the inner wall areas. In the second part of the work, three silane-based sol-gel hybrid coatings were selected in order to improve moisture resistance of wood and paper substrates. The coatings differed from each other in terms of having different alkyl (CH3–, CH3-(CH2)7–) and fluorocarbon (CF3–) chains attached to the trialkoxysilane sol-gel precursor. The sol-gel coatings were deposited by a wet coating method, i.e. spraying or spreading by brush. The effect of solgel coatings on surface structural and chemical properties of wood-based substrates was studied by using advanced surface analyzing tools: atomic force microscopy, X-ray photoelectron spectroscopy and time-of-flight secondary ion spectroscopy. The results show that the applied sol-gel coatings, deposited as thin films or particulate coatings, have different effects on surface characteristics of wood and wood-based materials. The coating which has a long hydrocarbon chain (CH3-(CH2)7–) attached to the silane backbone (octyltriethoxysilane) produced the highest hydrophobicity for wood and wood-based materials.

Novel technology for preparation of optically active chemicals

Asymmetric synthesis using modified heterogeneous catalysts has gained lots of interest in the production of optically pure chemicals, such as pharmaceuticals, nutraceuticals, fragrances and agrochemicals. Heterogeneous modified catalysts capable of inducing high enantioselectivities are preferred in industrial scale due to their superior separation and handling properties. The topic has been intensively investigated both in industry and academia. The enantioselective hydrogenation of ethyl benzoylformate (EBF) to (R)-ethyl mandelate over (-)-cinchonidine (CD)-modified Pt/Al2O3 catalyst in a laboratory-scale semi-batch reactor was studied as a function of modifier concentration, reaction temperature, stirring rate and catalyst particle size. The main product was always (R)-ethyl mandelate while small amounts of (S)-ethyl mandelate were obtained as by product. The kinetic results showed higher enantioselectivity and lower initial rates approaching asymptotically to a constant value as the amount of modifier was increased. Additionally, catalyst deactivation due to presence of impurities in the feed was prominent in some cases; therefore activated carbon was used as a cleaning agent of the raw material to remove impurities prior to catalyst addition. Detailed characterizations methods (SEM, EDX, TPR, BET, chemisorption, particle size distribution) of the catalysts were carried out. Solvent effects were also studied in the semi-batch reactor. Solvents with dielectric constant (e) between 2 and 25 were applied. The enantiomeric excess (ee) increased with an increase of the dielectric coefficient up to a maximum followed by a nonlinear decrease. A kinetic model was proposed for the enantioselectivity dependence on the dielectric constant based on the Kirkwood treatment. The non-linear dependence of ee on (e) successfully described the variation of ee in different solvents. Systematic kinetic experiments were carried out in the semi-batch reactor. Toluene was used as a solvent. Based on these results, a kinetic model based on the assumption of different number of sites was developed. Density functional theory calculations were applied to study the energetics of the EBF adsorption on pure Pt(1 1 1). The hydrogenation rate constants were determined along with the adsorption parameters by non-linear regression analysis. A comparison between the model and the experimental data revealed a very good correspondence. Transient experiments in a fixed-bed reactor were also carried out in this work. The results demonstrated that continuous enantioselective hydrogenation of EBF in hexane/2-propanol 90/10 (v/v) is possible and that continuous feeding of (-)-cinchonidine is needed to maintain a high steady-state enantioselectivity. The catalyst showed a good stability and high enantioselectivity was achieved in the fixed-bed reactor. Chromatographic separation of (R)- and (S)-ethyl mandelate originating from the continuous reactor was investigated. A commercial column filled with a chiral resin was chosen as a perspective preparative-scale adsorbent. Since the adsorption equilibrium isotherms were linear within the entire investigated range of concentrations, they were determined by pulse experiments for the isomers present in a post-reaction mixture. Breakthrough curves were measured and described successfully by the dispersive plug flow model with a linear driving force approximation. The focus of this research project was the development of a new integrated production concept of optically active chemicals by combining heterogeneous catalysis and chromatographic separation technology. The proposed work is fundamental research in advanced process technology aiming to improve efficiency and enable clean and environmentally benign production of enantiomeric pure chemicals.

Bio-ethanol valorization towards C4s including 1-butanol over metal modified alumina and zeolite catalysts

Bio-ethanol has been used as a fuel additive in modern society aimed at reducing CO2-emissions and dependence on oil. However, ethanol is unsuitable as fuel supplement in higher proportions due to its physico-chemical properties. One option to counteract the negative effects is to upgrade ethanol in a continuous fixed bed reactor to more valuable C4 products such as 1-butanol providing chemical similarity with traditional gasoline components. Bio-ethanol based valorization products also have other end-uses than just fuel additives. E.g. 1-butanol and ethyl acetate are well characterised industrial solvents and platform chemicals providing greener alternatives. The modern approach is to apply heterogeneous catalysts in the investigated reactions. The research was concentrated on aluminium oxide (Al2O3) and zeolites that were used as catalysts and catalyst supports. The metals supported (Cu, Ni, Co) gave very different product profiles and, thus, a profound view of different catalyst preparation methods and characterisation techniques was necessary. Additionally, acidity and basicity of the catalyst surface have an important role in determining the product profile. It was observed that ordinary determination of acid strength was not enough to explain all the phenomena e.g. the reaction mechanism. One of the main findings of the thesis is based on the catalytically active site which originates from crystallite structure. As a consequence, the overall evaluation of different by-products and intermediates was carried out by combining the information. Further kinetic analysis was carried out on metal (Cu, Ni, Co) supported self-prepared alumina catalysts. The thesis gives information for further catalyst developments aimed to scale-up towards industrially feasible operations.

Synecological comparisons sustained by ecophysiological fingerprinting of intrinsic photosynthetic capacity of plants as assessed by measurements of light response curves

In some literature variations in photosynthetic rates are considered to be of little relevance for individual fitness. This depends among other things on how one defines fitness, i.e. if one takes strictly Darwinian fitness as seed production or if one needs to evaluate particular traits and consider plant establishment. It also matters if one takes the Darwinian "organism individual" as the central entity in evolution ("individual fitness") or the "species individual" in a modified "Structure of Evolutionary Theory" sensu Stephen Jay Gould. A phenotypically expressed trait like photosynthetic rate, even if intra- and interspecific differences may be small, can matter in habitat performance and niche acquisition. Light dependence curves (LCs) of photosynthetic rates are now readily measured under field conditions using miniaturized equipment of pulse amplitude modulated fluorometers. In contrast to actual momentary measurements of quantum yield of photosynthesis under actually prevailing ambient conditions, LC measurements reflect the expressed intrinsic capacity of photosynthesis. In this review we explore the power of LC measurements yielding cardinal points such as maximum apparent electron transport rate of photosystem II (ETRmax) and saturating photosynthetically active radiation (PARsat) in making intra- and interspecific comparisons of plant performance and synecological fingerprinting in ecophysiological studies across species, sites, habitats and ecosystems.

Plasma clearance and biodistribution of oxidatively modified 99mTc-ß-VLDL in rabbits

The biodistribution and removal from plasma (measured as fractional clearance rate, FCR, per hour) of native and oxidatively modified 99mtechnetium-labeled ß-very low density lipoprotein (99mTc-ß-VLDL) were investigated in hypercholesterolemic (HC) and control (C) three-month old New Zealand rabbits. The intracellular accumulation of ß-VLDL labeled with 99mTc was studied in vitro in THP-1 cells and monocyte-derived macrophages isolated from rabbits. After intravenous injection into C rabbits, copper-oxidized ß-VLDL (99mTc-ox-ß-VLDL) was cleared from the circulation faster (0.362 ± 0.070/h) than native ß-VLDL (99mTc-nat-ß-VLDL, 0.241 ± 0.070/h). In contrast, the FCR of 99mTc-ox-ß-VLDL in HC rabbits was lower (0.100 ± 0.048/h) than that of 99mTc-nat-ß-VLDL (0.163 ± 0.043/h). The hepatic uptake of radiolabeled lipoproteins was lower in HC rabbits (0.114 ± 0.071% injected dose/g tissue for 99mTc-nat-ß-VLDL and 0.116 ± 0.057% injected dose/g tissue for 99mTc-ox-ß-VLDL) than in C rabbits (0.301 ± 0.113% injected dose/g tissue for 99mTc-nat-ß-VLDL and 0.305 ± 0.149% injected dose/g tissue for 99mTc-ox-ß-VLDL). The uptake of 99mTc-nat-ß-VLDL and 99mTc-ox-ß-VLDL by atherosclerotic aorta lesions isolated from HC rabbits (99mTc-nat-ß-VLDL: 0.033 ± 0.012% injected dose/g tissue and 99mTc-ox-ß-VLDL: 0.039 ± 0.017% injected dose/g tissue) was higher in comparison to that of non-atherosclerotic aortas from C rabbits (99mTc-nat-ß-VLDL: 0.023 ± 0.010% injected dose/g tissue and 99mTc-ox-ß-VLDL: 0.019 ± 0.010% injected dose/g tissue). However, 99mTc-nat-ß-VLDL and 99mTc-ox-ß-VLDL were taken up by atherosclerotic lesions at similar rates. In vitro studies showed that both monocyte-derived macrophages isolated from rabbits and THP-1 macrophages significantly internalized more 99mTc-ox-ß-VLDL than 99mTc-nat-ß-VLDL. These results indicate that in cholesterol-fed rabbits 99mTc-ox-ß-VLDL is slowly cleared from plasma and accumulates in atherosclerotic lesions. However, although the extent of in vitro uptake of 99mTc-ox-ß-VLDL by macrophages was high, the in vivo accumulation of this radiolabeled lipoprotein by atherosclerotic lesions did not differ from that of 99mTc-nat-ß-VLDL.

Behavioral profiles displayed by rats in an elevated asymmetric plus-maze: effects of diazepam

When rats are exposed to unknown environments where novelty and fear-inducing characteristics are present (conflictive environments), some specific behaviors are induced and exploration is apparently modulated by fear. In our laboratory, a new type of plus-maze was designed as a model of conflictive exploration. The maze is composed of four arms with different geometrical characteristics, differing from each other by the presence or absence of walls. The degree of asymmetry was as follows: NW, no wall arm; SW, a single high wall present; HL, a low and a high wall present, and HH, two high walls present. The four arms were arranged at 90o angles and the apparatus was called the elevated asymmetric plus-maze (APM). The purpose of the present study was to assess the behavioral profile of rats exposed for a single time to the APM with or without treatment with benzodiazepine. Increasing doses of diazepam were injected intraperitoneally in several groups of male, 90-day-old Holtzman rats. Distilled water was injected in control animals. Thirty minutes after treatment all rats were exposed singly to a 5-min test in the APM. Diazepam induced a biphasic modification of exploration in the NW and SW arms. The increase in the exploration score was evident at low doses of diazepam (0.25-1.0 mg/kg body weight) and the decrease in exploration was found with the higher doses of diazepam (2.0-3.0 mg/kg body weight). Non-exploratory behaviors (permanency) were not affected by benzodiazepine treatment. In the HL arm, exploration was not modified but permanency was increased in a dose-dependent manner. In the HH arm, exploration and permanency were not affected. Results are compatible with the idea that exploration-processing mechanisms in conflictive environments are modulated by fear-processing mechanisms of the brain.

Zymosan phagocytosis by mouse peritoneal macrophages is increased by apoHDL- and not by intact HDL-covered particles

The uptake of lipids and lipoprotein particles by macrophages undergoes phagocytic activation and the formation of foam cells are key events in atherosclerosis. In this study we determined how intact high density lipoproteins (HDL) and apolipoproteins-HDL (removal of the lipid component from HDL, i.e., apoHDL) influence the phagocytosis of zymosan by mouse peritoneal macrophages. Zymosan particles preincubated together with lipoproteins or alone (control) were incubated with the macrophages. Phagocytosis activity was reported as the percent of macrophages that internalized three or more zymosan particles. HDL co-incubated with zymosan did not influence the over-all uptake of zymosan particles compared to apoHDL, which greatly enhanced the ability of the particle to be phagocytized (P<0.001). Part of this effect might be related to a greater binding of apoHDL to the particles compared to that of HDL (P<0.05). We conclude that this can be a useful method to study the ability of lipoproteins, including modified lipoproteins obtained from subjects with genetic forms of hyperlipidemia, to opsonize particles such as red blood cells and thus to investigate the processes that control the formation of foam cells and the mechanisms of atherogenesis.

Nonconventional amide bond formation catalysis: programming enzyme specificity with substrate mimetics

This article reports on the design and characteristics of substrate mimetics in protease-catalyzed reactions. Firstly, the basis of protease-catalyzed peptide synthesis and the general advantages of substrate mimetics over common acyl donor components are described. The binding behavior of these artificial substrates and the mechanism of catalysis are further discussed on the basis of hydrolysis, acyl transfer, protein-ligand docking, and molecular dynamics studies on the trypsin model. The general validity of the substrate mimetic concept is illustrated by the expansion of this strategy to trypsin-like, glutamic acid-specific, and hydrophobic amino acid-specific proteases. Finally, opportunities for the combination of the substrate mimetic strategy with the chemical solid-phase peptide synthesis and the use of substrate mimetics for non-peptide organic amide synthesis are presented.

Improving the usability of extruded wood-plastic composites by using modification technology

Using cellulosic reinforcement to produce plastic composites is a globally growing trend. One of such materials are wood-plastic composites, which are an extensively studied group of materials for which the global industry is looking for new applications. Issues such as bondability, durability and fire resistance still require development to improve the usability of the wood-plastic composite material. Improvement of the usability of wood-plastic composites is studied in this thesis through the effects of using selected modification technology in wood and plastic industry. The applied modification methods are surface by mechanical abrasion and plasma, chemical impregnation of wood flour, and structural modification by the co-extrusion process. The study shows that the properties of WPC can be influenced by the selected modification methods. The selected methods are also found to be able to result as improvement in the properties of the material. The may also affect other than just the targeted properties of the end-product, either in a positive or a negative manner. Therefore modification as performance improvement should be considered as a caseby- case study. Introducing WPC materials for new applications can be done by using modification technology. Structuralmodification can possibly be used to reduce material costs of the modified WPC material.

Inhibition of gastric emptying and intestinal transit in anesthetized rats by a Tityus serrulatus scorpion toxin

The effects of a fraction (T1) of Tityus serrulatus scorpion venom prepared by gel filtration on gastric emptying and small intestinal transit were investigated in male Wistar rats. Fasted animals were anesthetized with urethane, submitted to tracheal intubation and right jugular vein cannulation. Scorpion toxin (250 µg/kg) or saline was injected iv and 1 h later a bolus of saline (1.0 ml/100 g) labeled with 99m technetium-phytate (10 MBq) was administered by gavage. After 15 min, animals were sacrificed and the radioactivity remaining in the stomach was determined. Intestinal transit was evaluated by instillation of a technetium-labeled saline bolus (1.0 ml) through a cannula previously implanted in the duodenum. After 60 min, the progression of the marker throughout 7 consecutive gut segments was estimated by the geometric center method. Gastric retention of the liquid test meal in rats injected with scorpion toxin (median: 88%; range: 52-95%) was significantly higher (P<0.02) than in controls (54%; 21-76%), an effect which was not modified by gastric secretion blockade with ranitidine. The progression of the isotope marker throughout the small intestine was significantly slower (P<0.05) in rats treated with toxin (1.2; 1.0-2.5) than in control animals (2.3; 1.0-3.2). Inhibition of both gastric emptying and intestinal transit in rats injected with scorpion toxin suggests an increased resistance to aboral flow, which might be caused by abnormal neurotransmitter release or by the local effects of venom on smooth muscle cells.

Design and applications of modified oligonucleotides

Oligonucleotides have a wide range of applications in fields such as biotechnology, molecular biology, diagnosis and therapy. However, the spectrum of uses can be broadened by introducing chemical modifications into their structures. The most prolific field in the search for new oligonucleotide analogs is the antisense strategy, where chemical modifications confer appropriate characteristics such as hybridization, resistance to nucleases, cellular uptake, selectivity and, basically, good pharmacokinetic and pharmacodynamic properties. Combinatorial technology is another research area where oligonucleotides and their analogs are extensively employed. Aptamers, new catalytic ribozymes and deoxyribozymes are RNA or DNA molecules individualized from a randomly synthesized library on the basis of a particular property. They are identified by repeated cycles of selection and amplification, using PCR technologies. Modified nucleotides can be introduced either during the amplification procedure or after selection.

Identification of rounded atelectasis in workers exposed to asbestos by contrast helical computed tomography

Rounded atelectasis (RA) is a benign and unusual form of subpleural lung collapse that has been described mostly in asbestos-exposed workers. This form of atelectasis manifests as a lung nodule and can be confused with bronchogenic carcinoma upon conventional radiologic examination. The objective of the present study was to evaluate the variation in contrast uptake in computed tomography for the identification of asbestos-related RA in Brazil. Between January 1998 and December 2000, high-resolution computed tomography (HRCT) was performed in 1658 asbestos-exposed workers. The diagnosis was made in nine patients based on a history of prior asbestos exposure, the presence of characteristic (HRCT) findings and lesions unchanged in size over 2 years or more. In three of them the diagnosis was confirmed during surgery. The dynamic contrast enhancement study was modified to evaluate nodules and pulmonary masses. All nine patients with RA received iodide contrast according to weight. The average enhancement after iodide contrast was infused, reported as Hounsfield units (HU), increased from 62.5 ± 9.7 to 125.4 ± 20.7 (P < 0.05), with a mean enhancement of 62.5 ± 19.7 (range 40 to 89) and with a uniform dense opacification. In conclusion, in this study all patients with RA showed contrast enhancement with uniform dense opacification. The main clinical implication of this finding is that this procedure does not permit differentiation between RA and malignant pulmonary neoplasm.

Screening for mutations in human alpha-globin genes by nonradioactive single-strand conformation polymorphism

Point mutations and small insertions or deletions in the human alpha-globin genes may produce alpha-chain structural variants and alpha-thalassemia. Mutations can be detected either by direct DNA sequencing or by screening methods, which select the mutated exon for sequencing. Although small (about 1 kb, 3 exons and 2 introns), the alpha-globin genes are duplicate (alpha2 and alpha1) and highy G-C rich, which makes them difficult to denature, reducing sequencing efficiency and causing frequent artifacts. We modified some conditions for PCR and electrophoresis in order to detect mutations in these genes employing nonradioactive single-strand conformation polymorphism (SSCP). Primers previously described by other authors for radioactive SSCP and phast-SSCP plus denaturing gradient gel electrophoresis were here combined and the resultant fragments (6 new besides 6 original per alpha-gene) submitted to silver staining SSCP. Nine structural and one thalassemic mutations were tested, under different conditions including two electrophoretic apparatus (PhastSystem™ and GenePhor™, Amersham Biosciences), different polyacrylamide gel concentrations, run temperatures and denaturing agents, and entire and restriction enzyme cut fragments. One hundred percent of sensitivity was achieved with four of the new fragments formed, using the PhastSystem™ and 20% gels at 15ºC, without the need of restriction enzymes. This nonradioactive PCR-SSCP approach showed to be simple, rapid and sensitive, reducing the costs involved in frequent sequencing repetitions and increasing the reliability of the results. It can be especially useful for laboratories which do not have an automated sequencer.

Protection of plasmid DNA by a Ginkgo biloba extract from the effects of stannous chloride and the action on the labeling of blood elements with technetium-99m

Ginkgo biloba extract (EGb) is a phytotherapeutic agent used for the treatment of ischemic and neurological disorders. Because the action of this important extract is not fully known, assays using different biological systems need to be performed. Red blood cells (RBC) are labeled with technetium-99m (Tc-99m) and used in nuclear medicine. The labeling depends on a reducing agent, usually stannous chloride (SnCl2). We assessed the effect of different concentrations of EGb on the labeling of blood constituents with Tc-99m, as sodium pertechnetate (3.7 MBq), and on the mobility of a plasmid DNA treated with SnCl2 (1.2 µg/ml) at room temperature. Blood was incubated with EGb before the addition of SnCl2 and Tc-99m. Plasma (P) and RBC were separated and precipitated with trichloroacetic acid, and soluble (SF-P and SF-RBC) and insoluble (IF-P and IF-RBC) fractions were isolated. The plasmid was incubated with Egb, SnCl2 or EGb plus SnCl2 and agarose gel electrophoresis was performed. The gel was stained with ethidium bromide and the DNA bands were visualized by fluorescence in an ultraviolet transilluminator system. EGb decreased the labeling of RBC, IF-P and IF-RBC. The supercoiled form of the plasmid was modified by treatment with SnCl2 and protected by 40 mg/ml EGb. The effect of EGb on the tested systems may be due to its chelating action with the stannous ions and/or pertechnetate or to the capability to generate reactive oxygen species that could oxidize the stannous ion.