Seasonal variation in migratory body condition, corticosterone secretion, oxidative stress and biotransformation activity in barn swallows (Hirundo rustica L.)

I studied the associations between migration-related physiological regulation (corticosterone) and body condition of barn swallows (Hirundo rustica L.). An additional purpose was to determine whether oxidative stress and biotransformation activity vary seasonally. Since physiological regulation, biotransformation activity and the stress involved may be important factors for body condition during migration; they may have direct effects on migration success. This in turn may influence other important life history stages, such as breeding and moult. In the thesis I used barn swallow data of the Finnish Ringing Centre (1997–2009), consisting of all juveniles ringed in the nests and recaptured from night roosts later the same autumn. Before the autumn migration in Finland I also captured, ringed and sampled barn swallows from night roosts in 2003, 2006, 2007 and 2011. Samples preceding spring migration in South Africa were collected in 2007. Juvenile barn swallows started to migrate southward in mid-August (first broods). Second broods started their migration at a younger age and almost a month later than first broods (mid-September). Barn swallows increased body mass and accumulated fat for the autumn migration. In the course of the autumn they seemed to be able to prevent the loss of energy already accumulated, since the proportional overnight mass loss, fat loss and faecal production decreased. Surprisingly, corticosterone, the major energy-regulating hormone in birds, seemed not to be involved in the fuelling process. Previous studies with warblers, sparrows and shorebirds had shown that during migration, the baseline levels of corticosterone were elevated in order to facilitate fuelling. It is possible that for Finnish barn swallows the most important fuelling place is in southern Europe, since northern and eastern populations migrate via the Balkan Peninsula. However, the adrenocortical stress response of Finnish barn swallows in good body condition was lower than that of those in poor body condition. Birds clearly suppressed the response, probably to prevent the catabolic effects of excessive corticosterone levels; birds cannot afford to lose muscle mass before migration. South African barn swallows had high levels of baseline corticosterone, but this may have been associated with the high oxidative damage and biotransformation activity of those birds. Barn swallows in spring and summer had low biotransformation activity and intermediate oxidative stress, which was probably related to breeding. Autumn birds had low biotransformation activity and oxidative stress but high redox enzyme activities in some migration-related enzymes.

The Role of Oxidative Stress in Environmental Responses of Fennoscandian Animals

All aerobic organisms have to deal with the toxicity of oxygen. Oxygen enables more efficient energy production compared to anaerobic respiration or fermentation, but at the same time reactive oxygen species (ROS) are being formed. ROS can also be produced by external factors such as UV-radiation and contamination. ROS can cause damage to biomolecules such as DNA, lipids and proteins and organisms try to keep the damage as small as possible by repairing biomolecules and metabolizing ROS. All ROS are not harmful, because they are used as signaling molecules. To cope against ROS organism have an antioxidant (AOX) system which consists both enzymatic and non-enzymatic AOX defense. Some AOX are produced by the organism itself and some are gained via diet. In this thesis I studied environmentally caused changes in the redox regulation of different wild vertebrate animals to gain knowledge on the temporal, spatial and pollution-derived-effects on the AOX systems. As study species I used barn swallow, ringed seal and the Baltic salmon. For the barn swallow the main interest was the seasonal fluctuation in the redox regulation and its connection to migration and breeding. The more contaminated ringed seals of the Baltic Sea were compared to seals from cleaner Svalbard to investigate whether they suffered from contaminant induced oxidative stress. The regional and temporal variation in redox regulation and regional variation in mRNA and protein expressions of Baltic salmon were studied to gain knowledge if the salmon from different areas are equally stressed. As a comparative aspect the redox responses of these different species were investigated to see which parts of the AOX system are substantial in which species. Certain parts of AOX system were connected to breeding and others to migration in barn swallows, there was also differences in biotransformation between birds caught from Africa and Finland. The Baltic ringed seal did not differ much from the seals from Svalbard, despite the difference in contaminant load. A possible explanation to this could be the enhanced AOX mechanisms against dive-associated oxidative stress in diving air-breathing animals, which also helps to cope with ROS derived from other sourses. The Baltic salmon from Gulf of Finland (GoF) showed higher activities in their AOX defense enzymes and more oxidative damage than fish from other areas. Also on mRNA and proteomic level, stress related metabolic changes were most profound in in the fish from GoF. Mainly my findings on species related differences followed the pattern of mammals showing highest activities and least damage and birds showing lower activities and most damage, fish being intermediate. In general, the glutathione recycling-related enzymes and the ratio of oxidized and reduced glutathione seemed to be the most affected parameters in all of the species.

The role of endothelial enzymes NOS, VAP-1 and CD73 in acute lung injury

Acute lung injury (ALI) is a syndrome of acute hypoxemic respiratory failure with bilateral pulmonary infiltrates that is not caused by left atrial hypertension. Since there is no effective treatment available, this frequent clinical syndrome significantly contributes to mortality of both medical and surgical patients. Great majority of the patients with the syndrome suffers from indirect ALI caused by systemic inflammatory response syndrome (SIRS). Sepsis, trauma, major surgery and severe burns, which represent the most common triggers of SIRS, often induce an overwhelming inflammatory reaction leading to dysfunction of several vital organs. Studies of indirect ALI due to SIRS revealed that respiratory dysfunction results from increased permeability of endothelium. Disruption of endothelial barrier allows extravasation of protein-rich liquid and neutrophils to pulmonary parenchyma. Both under normal conditions and in inflammation, endothelial barrier function is regulated by numerous mechanisms. Endothelial enzymes represent one of the critical control points of vascular permeability and leukocyte trafficking. Some endothelial enzymes prevent disruption of endothelial barrier by production of anti-inflammatory substances. For instance, nitric oxide synthase (NOS) down-regulates leukocyte extravasation in inflammation by generation of nitric oxide. CD73 decreases vascular leakage and neutrophil emigration to inflamed tissues by generation of adenosine. On the other hand, vascular adhesion protein-1 (VAP-1) mediates leukocyte trafficking to the sites of inflammation both by generation of pro-inflammatory substances and by physically acting as an adhesion molecule. The aims of this study were to define the role of endothelial enzymes NOS, CD73 and VAP-1 in acute lung injury. Our data suggest that increasing substrate availability for NOS reduces both lung edema and neutrophil infiltration and this effect is not enhanced by concomitant administration of antioxidants. CD73 protects from vascular leakage in ALI and its up-regulation by interferon-β represents a novel therapeutic strategy for treatment of this syndrome. Enzymatic activity of VAP-1 mediates neutrophil infiltration in ALI and its inhibition represents an attractive approach to treat ALI.

Type II aromatic polyketide biosynthetic tailoring enzymes: diversity and adaptation in Streptomyces secondary metabolism.

Members of the bacterial genus Streptomyces are well known for their ability to produce an exceptionally wide selection of diverse secondary metabolites. These include natural bioactive chemical compounds which have potential applications in medicine, agriculture and other fields of commerce. The outstanding biosynthetic capacity derives from the characteristic genetic flexibility of Streptomyces secondary metabolism pathways: i) Clustering of the biosynthetic genes in chromosome regions redundant for vital primary functions, and ii) the presence of numerous genetic elements within these regions which facilitate DNA rearrangement and transfer between non-progeny species. Decades of intensive genetic research on the organization and function of the biosynthetic routes has led to a variety of molecular biology applications, which can be used to expand the diversity of compounds synthesized. These include techniques which, for example, allow modification and artificial construction of novel pathways, and enable gene-level detection of silent secondary metabolite clusters. Over the years the research has expanded to cover molecular-level analysis of the enzymes responsible for the individual catalytic reactions. In vitro studies of the enzymes provide a detailed insight into their catalytic functions, mechanisms, substrate specificities, interactions and stereochemical determinants. These are factors that are essential for the thorough understanding and rational design of novel biosynthetic routes. The current study is a part of a more extensive research project (Antibiotic Biosynthetic Enzymes; www.sci.utu.fi/projects/biokemia/abe), which focuses on the post-PKS tailoring enzymes involved in various type II aromatic polyketide biosynthetic pathways in Streptomyces bacteria. The initiative here was to investigate specific catalytic steps in anthracycline and angucycline biosynthesis through in vitro biochemical enzyme characterization and structural enzymology. The objectives were to elucidate detailed mechanisms and enzyme-level interactions which cannot be resolved by in vivo genetic studies alone. The first part of the experimental work concerns the homologous polyketide cyclases SnoaL and AknH. These catalyze the closure of the last carbon ring of the tetracyclic carbon frame common to all anthracycline-type compounds. The second part of the study primarily deals with tailoring enzymes PgaE (and its homolog CabE) and PgaM, which are responsible for a cascade of sequential modification reactions in angucycline biosynthesis. The results complemented earlier in vivo findings and confirmed the enzyme functions in vitro. Importantly, we were able to identify the amino acid -level determinants that influence AknH and SnoaL stereoselectivity and to determine the complex biosynthetic steps of the angucycline oxygenation cascade of PgaE and PgaM. In addition, the findings revealed interesting cases of enzyme-level adaptation, as some of the catalytic mechanisms did not coincide with those described for characterised homologs or enzymes of known function. Specifically, SnoaL and AknH were shown to employ a novel acid-base mechanism for aldol condenzation, whereas the hydroxylation reaction catalysed by PgaM involved unexpected oxygen chemistry. Owing to a gene-level fusion of two ancestral reading frames, PgaM was also shown to adopt an unusual quaternary sturucture, a non-covalent fusion complex of two alternative forms of the protein. Furthermore, the work highlighted some common themes encountered in polyketide biosynthetic pathways such as enzyme substrate specificity and intermediate reactivity. These are discussed in the final chapters of the work.

Acid-base Balance and Oxidative Metabolism in Calcified Tissues

The calcified tissues, comprising bone and cartilage, are metabolically active tissues that bind and release calcium, bicarbonate and other substances according to systemic needs. Understanding the regulation of cellular metabolism in bone and cartilage is an important issue, since a link between the metabolism and diseases of these tissues is clear. An essential element in the function of bone-resorbing osteoclasts, namely regulation of bicarbonate transport, has not yet been thoroughly studied. Another example of an important but at the same time fairly unexplored subject of interest in this field is cartilage degeneration, an important determinant for development of osteoarthritis. The link between this and oxidative metabolism has rarely been studied. In this study, we have investigated the significance of bicarbonate transport in osteoclasts. We found that osteoclasts possess several potential proteins for bicarbonate transport, including carbonic anhydrase IV and XIV, and an electroneutral bicarbonate co-transporter NBCn1. We have also shown that inhibiting the function of these proteins has a significant impact on bone resorption and osteoclast morphology. Furthermore, we have explored oxidative metabolism in chondrocytes and found that carbonic anhydrase III (CA III), a protein linked to the prevention of protein oxidation in muscle cells, is also present in mouse chondrocytes, where its expression correlates with the presence of reactive oxygen species. Thus, our study provides novel information on the regulation of cellular metabolism in calcified tissues.

Structural studies on enzymes of biotechnological and biomedical interest

Structural studies of proteins aim at elucidating the atomic details of molecular interactions in biological processes of living organisms. These studies are particularly important in understanding structure, function and evolution of proteins and in defining their roles in complex biological settings. Furthermore, structural studies can be used for the development of novel properties in biomolecules of environmental, industrial and medical importance. X-ray crystallography is an invaluable tool to obtain accurate and precise information about the structure of proteins at the atomic level. Glutathione transferases (GSTs) are amongst the most versatile enzymes in nature. They are able to catalyze a wide variety of conjugation reactions between glutathione (GSH) and non-polar components containing an electrophilic carbon, nitrogen or sulphur atom. Plant GSTs from the Tau class (a poorly characterized class) play an important role in the detoxification of xenobiotics and stress tolerance. Structural studies were performed on a Tau class fluorodifen-inducible glutathione transferase from Glycine max (GmGSTU4-4) complexed with GSH (2.7 Å) and a product analogue Nb-GSH (1.7 Å). The three-dimensional structure of the GmGSTU4-4-GSH complex revealed that GSH binds in different conformations in the two subunits of the dimer: in an ionized form in one subunit and a non-ionized form in the second subunit. Only the ionized form of the substrate may lead to the formation of a catalytically competent complex. Structural comparison between the GSH and Nb-GSH bound complexes revealed significant differences with respect to the hydrogen-bonding, electrostatic interaction pattern, the upper part of -helix H4 and the C-terminus of the enzyme. These differences indicate an intrasubunit modulation between the G-and Hsites suggesting an induced-fit mechanism of xenobiotic substrate binding. A novel binding site on the surface of the enzyme was also revealed. Bacterial type-II L-asparaginases are used in the treatment of haematopoietic diseases such as acute lymphoblastic leukaemia (ALL) and lymphomas due to their ability to catalyze the conversion of L-asparagine to L-aspartate and ammonia. Escherichia coli and Erwinia chrysanthemi asparaginases are employed for the treatment of ALL for over 30 years. However, serious side-effects affecting the liver and pancreas have been observed due to the intrinsic glutaminase activity of the administered enzymes. Structural studies on Helicobacter pylori L-asparaginase (HpA) were carried out in an effort to discover novel L-asparaginases with potential chemotherapeutic utility in ALL treatment. Detailed analysis of the active site geometry revealed structurally significant differences between HpA and other Lasparaginases that may be important for the biological activities of the enzyme and could be further exploited in protein engineering efforts.

Biosynthesis of pyranonaphthoquinone polyketides:characterization of the alnumycin pathway

Alnumycin A is an aromatic pyranonaphthoquinone (PNQ) polyketide closely related to the model compound actinorhodin. While some PNQ polyketides are glycosylated, alnumycin A contains a unique sugar-like dioxane moiety. This unusual structural feature made alnumycin A an interesting research target, since no information was available about its biosynthesis. Thus, the main objective of the thesis work became to identify the steps and the enzymes responsible for the biosynthesis of the dioxane moiety. Cloning, sequencing and heterologous expression of the complete alnumycin gene cluster from Streptomyces sp. CM020 enabled the inactivation of several alnumycin biosynthetic genes and preliminary identification of the gene products responsible for pyran ring formation, quinone formation and dioxane biosynthesis. The individual deletions of the genes resulted in the production of several novel metabolites, which in many cases turned out to be pathway intermediates and could be used for stepwise enzymatic reconstruction of the complete dioxane biosynthetic pathway in vitro. Furthermore, the in vitro reactions with purified alnumycin biosynthetic enzymes resulted in the production of other novel compounds, both pathway intermediates and side products. Identification and molecular level studies of the enzymes AlnA and AlnB catalyzing the first step of dioxane biosynthesis – an unusual C-ribosylation step – led to a mechanistic proposal for the C-ribosylation of the polyketide aglycone. The next step on the dioxane biosynthetic pathway was found to be the oxidative conversion of the attached ribose into a highly unusual dioxolane unit by Aln6 belonging to an uncharacterized protein family, which unexpectedly occurred without any apparent cofactors. Finally, the last step of the pathway was found to be catalyzed by the NADPH-dependent reductase Aln4, which is able to catalyze the conversion of the formed dioxolane into a dioxane moiety. The work presented here and the knowledge gained of the enzymes involved in dioxane biosynthesis enables their use in the rational design of novel compounds containing C–C bound ribose, dioxolane and dioxane moieties.

Molecular Mechanisms and Interactions in Regulation of Photosynthetic Reactions

In photosynthesis, light energy is converted to chemical energy, which is consumed for carbon assimilation in the Calvin-Benson-Bassham (CBB) cycle. Intensive research has significantly advanced the understanding of how photosynthesis can survive in the ever-changing light conditions. However, precise details concerning the dynamic regulation of photosynthetic processes have remained elusive. The aim of my thesis was to specify some molecular mechanisms and interactions behind the regulation of photosynthetic reactions under environmental fluctuations. A genetic approach was employed, whereby Arabidopsis thaliana mutants deficient in specific photosynthetic protein components were subjected to adverse light conditions and assessed for functional deficiencies in the photosynthetic machinery. I examined three interconnected mechanisms: (i) auxiliary functions of PsbO1 and PsbO2 isoforms in the oxygen evolving complex of photosystem II (PSII), (ii) the regulatory function of PGR5 in photosynthetic electron transfer and (iii) the involvement of the Calcium Sensing Receptor CaS in photosynthetic performance. Analysis of photosynthetic properties in psbo1 and psbo2 mutants demonstrated that PSII is sensitive to light induced damage when PsbO2, rather than PsbO1, is present in the oxygen evolving complex. PsbO1 stabilizes PSII more efficiently compared to PsbO2 under light stress. However, PsbO2 shows a higher GTPase activity compared to PsbO1, and plants may partially compensate the lack of PsbO1 by increasing the rate of the PSII repair cycle. PGR5 proved vital in the protection of photosystem I (PSI) under fluctuating light conditions. Biophysical characterization of photosynthetic electron transfer reactions revealed that PGR5 regulates linear electron transfer by controlling proton motive force, which is crucial for the induction of the photoprotective non-photochemical quenching and the control of electron flow from PSII to PSI. I conclude that PGR5 controls linear electron transfer to protect PSI against light induced oxidative damage. I also found that PGR5 physically interacts with CaS, which is not needed for photoprotection of PSII or PSI in higher plants. Rather, transcript profiling and quantitative proteomic analysis suggested that CaS is functionally connected with the CBB cycle. This conclusion was supported by lowered amounts of specific calciumregulated CBB enzymes in cas mutant chloroplasts and by slow electron flow to PSI electron acceptors when leaves were reilluminated after an extended dark period. I propose that CaS is required for calcium regulation of the CBB cycle during periods of darkness. Moreover, CaS may also have a regulatory role in the activation of chloroplast ATPase. Through their diverse interactions, components of the photosynthetic machinery ensure optimization of light-driven electron transport and efficient basic production, while minimizing the harm caused by light induced photodamage.

Ellagitannins in Finnish plant species – Characterization, distribution and oxidative activity

Ellagitannins are secondary metabolites that are produced by plants. Among other features, they are assumed to function as plants’ defensive compounds against plant-eating herbivores. This thesis focuses on a theory, which suggests that the biological activity of ellagitannins is based on their tendency to oxidize at the highly alkaline gut conditions of insect herbivores (oxidative activity). To study the biological activities of ellagitannins, a wide variety of structurally different ellagitannins were purified from different plant species by using liquid chromatographic techniques. The structures were characterized with the aid of spectrometric methods. Based on the acquired data, it was also possible to create a scheme, which enables the classification and even identification of ellagitannins from plant extracts without the need to isolate each compound for individual characterization. The biological activities of ellagitannins were determined with methods that are based on the abilities of the compounds to scavenge radicals, chelate iron ions, and on their rate of oxidation at high pH. The results showed that ellagitannins possess oxidative activities both at high and neutral pH, and that their activities depend on structure. The occurrence, distribution and content of ellagitannins in Finnish plant species were also studied. The specific ellagitannin profiles of the studied plant species were found to correlate well with their taxonomic classification.

Structural and functional Studies of angucycline tailoring enzymes

Polyketides are a diverse group of natural products produced in many bacteria, fungi and plants. These metabolites have diverse biological activities and several members of this group are in clinical use as antibiotics, anticancer agents, antifungals and immunosuppressants. The different polyketides are produced by polyketide synthases, which catalyze the condensation of extender units into various polyketide scaffolds. After the biosynthesis of the polyketide backbone, more versatility is created to the molecule by tailoring enzymes catalyzing for instance hydroxylations, methylations and glycosylations. Flavoprotein monooxygenases (FPMO) and short-chain alcohol dehydrogenases/reductases (SDR) are two enzyme families that catalyze unusual tailoring reactions in the biosynthesis of natural products. In the experimental section, functions of homologous FPMO and SDR tailoring enzymes from five different angucycline pathways were studied in vitro. The results revealed how different angucyclinones are produced from a common intermediate and that FPMO JadH and SDR LanV are responsible for the divergence of jadomycins and landomycins, respectively, from other angucyclines. Structural studies of these tailoring enzymes revealed differences between homologous enzymes and enabled the use of structure-based protein engineering. Mutagenesis experiments gave important information about the enzymes behind the evolution of distinct angucycline metabolites. These experiments revealed a correlation between the substrate inhibition and bi-functionality in JadH homologue PgaE. In the case of LanV, analysis of mutagenesis results revealed that the difference between the stereospecificities of LanV and its homologues CabV and UrdMred is unexpectedly related to the conformation of the substrate rather than to the structure of the enzyme. Altogether, the results presented here have improved our knowledge about different steps of angucycline biosynthesis and the reaction mechanisms used by the tailoring enzymes behind these steps. This information can hopefully be used to modify these enzymes to produce novel metabolites, which have new biological targets or possess novel modes-of-action. The understanding of these unusual enzyme mechanisms is also interesting to enzymologists outside the field of natural product research.