2,3-Dihydroxybenzoate 2,3-oxygenase from the chloroplast fraction of Tecoma stans

2,3-Dihydroxybenzoate-2,3-oxygenase is mainly localized in the soluble and the chloroplast fractions of Tecoma leaves. It is associated with the lamellar structure of the chloroplast fraction. The chloroplast enzyme has properties similar to those of the soluble enzyme, but it has a longer half-life and is more stable to dialysis than the soluble enzyme. It is inhibited by sulfhydryl reagents and the inhibition is reversed by the addition of reduced glutathione. The chloroplast enzyme is insensitive to iron-chelating agents. The enzyme loses activity on dialysis against copper-chelating agents and the activity is completely recovered on the addition of copper; addition of iron does not restore the activity. Polyphenol oxidase is probably present only in the active form in the Tecoma chloroplast but it is not involved in the intradiol cleavage of 2,3-dihydroxybenzoic acid.

The averaging principle and the diagram technique

Rae and Davidson have found a striking connection between the averaging method generalised by Kruskal and the diagram technique used by the Brussels school in statistical mechanics. They have considered conservative systems whose evolution is governed by the Liouville equation. In this paper we have considered a class of dissipative systems whose evolution is governed not by the Liouville equation but by the last-multiplier equation of Jacobi whose Fourier transform has been shown to be the Hopf equation. The application of the diagram technique to the interaction representation of the Jacobi equation reveals the presence of two kinds of interactions, namely the transition from one mode to another and the persistence of a mode. The first kind occurs in the treatment of conservative systems while the latter type is unique to dissipative fields and is precisely the one that determines the asymptotic Jacobi equation. The dynamical equations of motion equivalent to this limiting Jacobi equation have been shown to be the same as averaged equations.

Lipids of some thermophilic fungi

Total lipid content in the thermophilic fungi—Thermoascus aurantiacus, Humicola lanuginosa, Malbranchea pulchella var.sulfurea, andAbsidia ramosa—varied from 5.3 to 19.1% of mycelial dry weight. The neutral and polar lipid fractions accounted for 56.4 to 80.2% and 19.8 to 43.6%, respectively. All the fungi contained monoglycerides, diglycerides, triglycerides, free fatty acids, and sterols in variable amounts. Sterol ester was detected only inA. ramosa. Phosphatide composition was: phosphatidyl choline (15.9–47%), phosphatidyl ethanolamine (23.4–67%), phosphatidyl serine (9.3–17.6%), and phosphatidyl inositol (1.9–11.9%). Diphosphatidyl glycerol occurred in considerable quantity only inH. lanuginosa andM. pulchella var.sulfurea. Phosphatidic acid, detected as a minor component only inM. pulchella var.sulfurea andA. ramosa, does not appear to be a characteristic phosphatide of thermophilic fungi as suggested earlier. The 16∶0, 16∶1, 18∶0, 18∶1, and 18∶2 acids were the main fatty acid components. In addition,A. ramosa contained 18∶3 acid. Total lipids contained an average of 0.93 double bonds per mole of fatty acids, and neutral lipids tend to be more unsaturated than phospholipids.

Mammalian brain plasma membranes: Isolation, enzymatic, and chemical characterization

Two variants of a simplified procedure for the isolation of plasma membrane fractions from monkey and rat brains, are described. The preparations show marked enrichments in the marker enzymes, (Na+-K+) adenosine triphosphatase, acetylcholinesterase, 5′-nucleotidase and adenylate cyclase. Lipid analysis and a protein electrophoretic pattern are presented. An enzymatic check has been made to assess for contamination by other cellular organelles. The amino acid composition of brain membrane proteins show a resemblance to the reported composition of erythrocyte ghost proteins but differ from myelin proteins.

Construction of stability multipliers for non-linear feedback systems

This paper is concerned with the analysis of the absolute stability of a non-linear autonomous system which consists of a single non-linearity belonging to a particular class, in an otherwise linear feedback loop. It is motivated from the earlier Popovlike frequency-domain criteria using the ' multiplier ' eoncept and involves the construction of ' stability multipliers' with prescribed phase characteristics. A few computer-based methods by which this problem can be solved are indicated and it is shown that this constitutes a stop-by-step procedure for testing the stability properties of a given system.

Influence of uncertainty inherent to heavy metal build-up and wash-off on stormwater quality

Uncertainty inherent to heavy metal build-up and wash-off stems from process variability. This results in inaccurate interpretation of stormwater quality model predictions. The research study has characterised the variability in heavy metal build-up and wash-off processes based on the temporal variations in particle-bound heavy metals commonly found on urban roads. The study outcomes found that the distribution of Al, Cr, Mn, Fe, Ni, Cu, Zn, Cd and Pb were consistent over particle size fractions <150µm and >150µm, with most metals concentrated in the particle size fraction <150µm. When build-up and wash-off are considered as independent processes, the temporal variations in these processes in relation to the heavy metals load are consistent with variations in the particulate load. However, the temporal variations in the load in build-up and wash-off of heavy metals and particulates are not consistent for consecutive build-up and wash-off events that occur on a continuous timeline. These inconsistencies are attributed to interactions between heavy metals and particulates <150µm and >150µm, which are influenced by particle characteristics such as organic matter content. The behavioural variability of particles determines the variations in the heavy metals load entrained in stormwater runoff. Accordingly, the variability in build-up and wash-off of particle-bound pollutants needs to be characterised in the description of pollutant attachment to particulates in stormwater quality modelling. This will ensure the accounting of process uncertainty, and thereby enhancing the interpretation of the outcomes derived from modelling studies.

Studies on the metabolism of β-carotene and apo-β-carotenoids in rats and chickens

1. (1) The relative abilities of the various cell fractions of rat and chicken liver to oxidize and reduce retinal and 8'- and 12'-apo-β-carotenal were investigated and it has been shown that, while retinal is exclusively oxidized by the soluble fraction, the apocarotenals are mostly oxidized by the particulate fractions of the homogenate. 2. (2) Addition of NAD+ or NADP+ markedly activated the oxidation of the apocarotenals, but not of retinal by the particulate fractions. 3. (3) Considerable amounts of retinal and 8'-, 10'- and 12'-apo-β-carotenal were isolated from the intestine of chickens fed β-carotene and these apocarotenoids were conclusively identified. 4. (4) Significant amounts of 8'-, 10'- and 12'-apo-β-carotenoic acids were isolated from the intestine of rats given 8'-apo-β-carotenal and these apocarotenoic acids were also conclusively identified. 5. (5) In the light of these observations it is suggested that during conversion to vitamin A, the β-carotene molecule is simultaneously attacked by the dioxygenase at several double bonds, the primary attack being at the central double bond and a tentative scheme for the mechanism of conversion is proposed.

Characterisation and source identification of pollution episodes caused by long-range transported aerosols

Aerosol particles can cause detrimental environmental and health effects. The particles and their precursor gases are emitted from various anthropogenic and natural sources. It is important to know the origin and properties of aerosols to efficiently reduce their harmful effects. The diameter of aerosol particles (Dp) varies between ~0.001 and ~100 μm. Fine particles (PM2.5: Dp < 2.5 μm) are especially interesting because they are the most harmful and can be transported over long distances. The aim of this thesis is to study the impact on air quality by pollution episodes of long-range transported aerosols affecting the composition of the boundary-layer atmosphere in remote and relatively unpolluted regions of the world. The sources and physicochemical properties of aerosols were investigated in detail, based on various measurements (1) in southern Finland during selected long-range transport (LRT) pollution episodes and unpolluted periods and (2) over the Atlantic Ocean between Europe and Antarctica during a voyage. Furthermore, the frequency of LRT pollution episodes of fine particles in southern Finland was investigated over a period of 8 years, using long-term air quality monitoring data. In southern Finland, the annual mean PM2.5 mass concentrations were low but LRT caused high peaks of daily mean concentrations every year. At an urban background site in Helsinki, the updated WHO guideline value (24-h PM2.5 mean 25 μg/m3) was exceeded during 1-7 LRT episodes each year during 1999-2006. The daily mean concentrations varied between 25 and 49 μg/m3 during the episodes, which was 3-6 times higher than the mean concentration in the long term. The in-depth studies of selected LRT episodes in southern Finland revealed that biomass burning in agricultural fields and wildfires, occurring mainly in Eastern Europe, deteriorated air quality on a continental scale. The strongest LRT episodes of fine particles resulted from open biomass-burning fires but the emissions from other anthropogenic sources in Eastern Europe also caused significant LRT episodes. Particle mass and number concentrations increased strongly in the accumulation mode (Dp ~ 0.09-1 μm) during the LRT episodes. However, the concentrations of smaller particles (Dp < 0.09 μm) remained low or even decreased due to the uptake of vapours and molecular clusters by LRT particles. The chemical analysis of individual particles showed that the proportions of several anthropogenic particle types increased (e.g. tar balls, metal oxides/hydroxides, spherical silicate fly ash particles and various calcium-rich particles) in southern Finland during an LRT episode, when aerosols originated from the polluted regions of Eastern Europe and some open biomass-burning smoke was also brought in by LRT. During unpolluted periods when air masses arrived from the north, the proportions of marine aerosols increased. In unpolluted rural regions of southern Finland, both accumulation mode particles and small-sized (Dp ~ 1-3 μm) coarse mode particles originated mostly from LRT. However, the composition of particles was totally different in these size fractions. In both size fractions, strong internal mixing of chemical components was typical for LRT particles. Thus, the aging of particles has significant impacts on their chemical, hygroscopic and optical properties, which can largely alter the environmental and health effects of LRT aerosols. Over the Atlantic Ocean, the individual particle composition of small-sized (Dp ~ 1-3 μm) coarse mode particles was affected by continental aerosol plumes to distances of at least 100-1000 km from the coast (e.g. pollutants from industrialized Europe, desert dust from the Sahara and biomass-burning aerosols near the Gulf of Guinea). The rate of chloride depletion from sea-salt particles was high near the coasts of Europe and Africa when air masses arrived from polluted continental regions. Thus, the LRT of continental aerosols had significant impacts on the composition of the marine boundary-layer atmosphere and seawater. In conclusion, integration of the results obtained using different measurement techniques captured the large spatial and temporal variability of aerosols as observed at terrestrial and marine sites, and assisted in establishing the causal link between land-bound emissions, LRT and air quality.

Orientational ordering in sheared inelastic dumbbells

Using even driven simulations, we show that homogeneously sheared inelastic dumbbells in two dimensions are randomly orientated in the limit of low density. As the packing fraction is increased, particles first tend to orient along the extensional axis, and then as the packing fraction is further increased, the alignment shifts closer to the flow axis. The orientational order parameter displays a continuous increase with packing fraction and does not appear to exhibit a universal scaling with elongation. Except at the highest packing fractions, the orientational distribution function can be reconstructed with only the first coefficient of the Fourier expansion.

Microbial oil production from sugarcane bagasse hydrolysates by oleaginous yeast and filamentous fungi

This study investigated the potential use of sugarcane bagasse as a feedstock for oil production through microbial cultivation. Bagasse was subjected to dilute acid pretreatment with 0.4 wt% H2SO4 (in liquid) at a solid/liquid ratio of 1:6 (wt/wt) at 170 °C for 15 min, followed by enzymatic hydrolysis of solid residue. The liquid fractions of the pretreatment process and the enzymatic hydrolysis process were detoxified and used as liquid hydrolysate (SCBLH) and enzymatic hydrolysate (SCBEH) for the microbial oil production by oleaginous yeast (Rhodotorula mucilaginosa) and filamentous fungi (Aspergillus oryzae and Mucor plumbeus). The results showed that all strains were able to grow and produce oil from bagasse hydrolysates. The highest oil concentrations produced from bagasse hydrolysates were by M. plumbeus at 1.59 g/L (SCBLH) and 4.74 g/L (SCBEH). The microbial oils obtained have similar fatty acid compositions to vegetable oils, indicating that the oil can be used for the production of second generation biodiesel. On the basis of oil yields obtained by M. plumbeus, from 10 million t (wet weight) of bagasse generated annually from sugar mills in Australia, it is estimated that the total biodiesel that could be produced would be equivalent to about 9% of Queensland’s diesel consumption.

Structuring time and questioning to achieve tactical awareness in games lessons

Background: A paradigm shift in educational policy to create problem solvers and critical thinkers produced the games concept approach (GCA) in Singapore's Revised Syllabus for Physical Education (1999). A pilot study (2001) conducted on 11 primary school student teachers (STs) using this approach identified time management and questioning as two of the major challenges faced by novice teachers. Purpose: To examine the GCA from three perspectives: structure—lesson form in terms of teacher-time and pupil-time; product—how STs used those time fractions; and process—the nature of their questioning (type, timing, and target). Participants and setting: Forty-nine STs from three different PETE cohorts (two-year diploma, four-year degree, two-year post-graduate diploma) volunteered to participate in the study conducted during the penultimate week of their final practicum in public primary and secondary schools. Intervention: Based on the findings of the pilot study, PETE increased the emphasis on GCA content specific knowledge and pedagogical procedures. To further support STs learning to actualise the GCA, authentic micro-teaching experiences that were closely monitored by faculty were provided in schools nearby. Research design: This is a descriptive study of time-management and questioning strategies implemented by STs on practicum. Each lesson was segmented into a number of sub-categories of teacher-time (organisation, demonstration and closure) and pupil-time (practice time and game time). Questions were categorised as knowledge, technical, tactical or affective. Data collection: Each ST was video-taped teaching a GCA lesson towards the end of their final practicum. The STs individually determined the timing of the data collection and the lesson to be observed. Data analysis: Each lesson was segmented into a number of sub-categories of both teacher- and pupil-time. Duration recording using Noldus software (Observer 4.0) segmented the time management of different lesson components. Questioning was coded in terms of type, timing and target. Separate MANOVAs were used to measure the difference between programmes and levels (primary and secondary) in relation to time-management procedures and questioning strategies. Findings: No differences emerged between the programmes or levels in their time-management or questioning strategies. Using the GCA, STs generated more pupil time (53%) than teacher time (47%). STs at the primary level provided more technical practice, and those in secondary schools more small-sided game play. Most questions (58%) were asked during play or practice but were substantially low-order involving knowledge or recall (76%) and only 6.7% were open-ended or divergent and capable of developing tactical awareness. Conclusions: Although STs are delivering more pupil time (practice and game) than teacher-time, the lesson structure requires further fine-tuning to extend the practice task beyond technical drills. Many questions are being asked to generate knowledge about games but lack sufficient quality to enhance critical thinking and tactical awareness, as the GCA intends.

Lignin biosynthesis in Norway spruce: from a model system to the tree

Lignin is a hydrophobic polymer that is synthesised in the secondary cell walls of all vascular plants. It enables water conduction through the stem, supports the upright growth habit and protects against invading pathogens. In addition, lignin hinders the utilisation of the cellulosic cell walls of plants in pulp and paper industry and as forage. Lignin precursors are synthesised in the cytoplasm through the phenylpropanoid pathway, transported into the cell wall and oxidised by peroxidases or laccases to phenoxy radicals that couple to form the lignin polymer. This study was conducted to characterise the lignin biosynthetic pathway in Norway spruce (Picea abies (L.) Karst.). We focused on the less well-known polymerisation stage, to identify the enzymes and the regulatory mechanisms that are involved. Available data for lignin biosynthesis in gymnosperms is scarce and, for example, the latest improvements in precursor biosynthesis have only been verified in herbaceous plants. Therefore, we also wanted to study in detail the roles of individual gene family members during developmental and stress-induced lignification, using EST sequencing and real-time RT-PCR. We used, as a model, a Norway spruce tissue culture line that produces extracellular lignin into the culture medium, and showed that lignin polymerisation in the tissue culture depends on peroxidase activity. We identified in the culture medium a significant NADH oxidase activity that could generate H2O2 for peroxidases. Two basic culture medium peroxidases were shown to have high affinity to coniferyl alcohol. Conservation of the putative substrate-binding amino acids was observed when the spruce peroxidase sequences were compared with other peroxidases with high affinity to coniferyl alcohol. We also used different peroxidase fractions to produce synthetic in vitro lignins from coniferyl alcohol; however, the linkage pattern of the suspension culture lignin could not be reproduced in vitro with the purified peroxidases, nor with the full complement of culture medium proteins. This emphasised the importance of the precursor radical concentration in the reaction zone, which is controlled by the cells through the secretion of both the lignin precursors and the oxidative enzymes to the apoplast. In addition, we identified basic peroxidases that were reversibly bound to the lignin precipitate. They could be involved, for example, in the oxidation of polymeric lignin, which is required for polymer growth. The dibenzodioxocin substructure was used as a marker for polymer oxidation in the in vitro polymerisation studies, as it is a typical substructure in wood lignin and in the suspension culture lignin. Using immunolocalisation, we found the structure mainly in the S2+S3 layers of the secondary cell walls of Norway spruce tracheids. The structure was primarily formed during the late phases of lignification. Contrary to the earlier assumptions, it appears to be a terminal structure in the lignin macromolecule. Most lignin biosynthetic enzymes are encoded for by several genes, all of which may not participate in lignin biosynthesis. In order to identify the gene family members that are responsible for developmental lignification, ESTs were sequenced from the lignin-forming tissue culture and developing xylem of spruce. Expression of the identified lignin biosynthetic genes was studied using real-time RT-PCR. Candidate genes for developmental lignification were identified by a coordinated, high expression of certain genes within the gene families in all lignin-forming tissues. However, such coordinated expression was not found for peroxidase genes. We also studied stress-induced lignification either during compression wood formation by bending the stems or after Heterobasidion annosum infection. Based on gene expression profiles, stress-induced monolignol biosynthesis appeared similar to the developmental process, and only single PAL and C3H genes were specifically up-regulated by stress. On the contrary, the up-regulated peroxidase genes differed between developmental and stress-induced lignification, indicating specific responses.

Substance flow analysis in Finland : Four case studies on N and P flows

Nitrogen (N) and phosphorus (P) are essential elements for all living organisms. However, in excess, they contribute to several environmental problems such as aquatic and terrestrial eutrophication. Globally, human action has multiplied the volume of N and P cycling since the onset of industrialization. The multiplication is a result of intensified agriculture, increased energy consumption and population growth. Industrial ecology (IE) is a discipline, in which human interaction with the ecosystems is investigated using a systems analytical approach. The main idea behind IE is that industrial systems resemble ecosystems, and, like them, industrial systems can then be described using material, energy and information flows and stocks. Industrial systems are dependent on the resources provided by the biosphere, and these two cannot be separated from each other. When studying substance flows, the aims of the research from the viewpoint of IE can be, for instance, to elucidate the ways how the cycles of a certain substance could be more closed and how the flows of a certain substance could be decreased per unit of production (= dematerialization). In Finland, N and P are studied widely in different ecosystems and environmental emissions. A holistic picture comparing different societal systems is, however, lacking. In this thesis, flows of N and P were examined in Finland using substance flow analysis (SFA) in the following four subsystems: I) forest industry and use of wood fuels, II) food production and consumption, III) energy, and IV) municipal waste. A detailed analysis at the end of the 1990s was performed. Furthermore, historical development of the N and P flows was investigated in the energy system (III) and the municipal waste system (IV). The main research sources were official statistics, literature, monitoring data, and expert knowledge. The aim was to identify and quantify the main flows of N and P in Finland in the four subsystems studied. Furthermore, the aim was to elucidate whether the nutrient systems are cyclic or linear, and to identify how these systems could be more efficient in the use and cycling of N and P. A final aim was to discuss how this type of an analysis can be used to support decision-making on environmental problems and solutions. Of the four subsystems, the food production and consumption system and the energy system created the largest N flows in Finland. For the creation of P flows, the food production and consumption system (Paper II) was clearly the largest, followed by the forest industry and use of wood fuels and the energy system. The contribution of Finland to N and P flows on a global scale is low, but when compared on a per capita basis, we are one of the largest producers of these flows, with relatively high energy and meat consumption being the main reasons. Analysis revealed the openness of all four systems. The openness is due to the high degree of internationality of the Finnish markets, the large-scale use of synthetic fertilizers and energy resources and the low recycling rate of many waste fractions. Reduction in the use of fuels and synthetic fertilizers, reorganization of the structure of energy production, reduced human intake of nutrients and technological development are crucial in diminishing the N and P flows. To enhance nutrient recycling and replace inorganic fertilizers, recycling of such wastes as wood ash and sludge could be promoted. SFA is not usually sufficiently detailed to allow specific recommendations for decision-making to be made, but it does yield useful information about the relative magnitude of the flows and may reveal unexpected losses. Sustainable development is a widely accepted target for all human action. SFA is one method that can help to analyse how effective different efforts are in leading to a more sustainable society. SFA's strength is that it allows a holistic picture of different natural and societal systems to be drawn. Furthermore, when the environmental impact of a certain flow is known, the method can be used to prioritize environmental policy efforts.

Interactions between harmful algae and calanoid copepods in the Baltic Sea

The aim of the studies reported in this thesis was to examine the feeding interactions between calanoid copepods and toxic algae in the Baltic Sea. The central questions in this research concerned the feeding, survival and egg production of copepods exposed to toxic algae. Furthermore, the importance of copepods as vectors in toxin transfer was examined. The haptophyte Prymnesium parvum, which produces extracellular toxins, was the only studied species that directly harmed copepods. Beside this, it had allelopathic effects (cell lysis) on non-toxic Rhodomonas salina. Copepods that were exposed to P. parvum filtrates died or became severely impaired, although filtrates were not haemolytic (indicative of toxicity in this study). Monospecific Prymnesium cell suspensions, in turn, were haemolytic and copepods in these treatments became inactive, although no clear effect on mortality was detected. These results suggest that haemolytic activity may not be a good proxy of the harmful effects of P. parvum. In addition, P. parvum deterred feeding, and low egestion and suppressed egg production were consequently observed in monospecific suspensions of Prymnesium. Similarly, ingestion and faecal pellet production rates were suppressed in high concentration P. parvum filtrates and in mixtures of P. parvum and R. salina. These results indicate that the allelopathic effects of P. parvum on other algal species together with lowered viability as well as suppressed production of copepods may contribute to bloom formation and persistence. Furthermore, the availability of food for planktivorous animals may be affected due to reduced copepod productivity. Nodularin produced by Nodularia spumigena was transferred to Eurytemora affinis via grazing on filaments of small N. spumigena and by direct uptake from the dissolved pool. Copepods also acquired nodularin in fractions where N. spumigena filaments were absent. Thus, the importance of microbial food webs in nodularin transfer should be considered. Copepods were able to remove particulate nodularin from the system, but at the same time a large proportion of the nodularin disappeared. This indicates that copepods may possess effective mechanisms to remove toxins from their tissues. The importance of microorganisms, such as bacteria, in the degradation of cyanobacterial toxins could also be substantial. Our results were the first reports of the accumulation of diarrhetic shellfish toxins (DSTs) produced by Dinophysis spp. in copepods. The PTX2 content in copepods after feeding experiments corresponded to the ingestion of <100 Dinophysis spp. cells. However, no DSTs were recorded from field-collected copepods. Dinophysis spp. was not selected by the copepods and consumption remained low. It seems thus likely that copepods are an unimportant link in the transfer of DSTs in the northern Baltic Sea.

Elastic modulus of Ti-6Al-4V-xB alloys with B up to 0.55 wt.%

An anomalous variation in the experimental elastic modulus, E, of Ti-6Al-4V-xB (with x up to 0.55 wt.%) is reported. Volume fractions and moduli of the constituent phases were measured using microscopy and nanoindentation,respectively. These were used in simple micromechanical models to examine if the E values could be rationalized. Experimental E values higher than the upper bound estimates suggest complex interplay between microstructural modifications, induced by the addition of B, and properties.