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.

Aggrecan structure in amphibian cartilage

The structure of the large proteoglycan present in the bullfrog epiphyseal cartilage was studied by immunochemical and biochemical methods. The isolated monomer showed a polydisperse behavior on Sepharose CL2B, with a peak at Kav = 0.14. Chondroitin sulfate chains were identified by HPLC analysis of the products formed by chondroitinase digestion and mercuric acetate treatment. These chains have approximately 38 disaccharides, a Di45:Di68 ratio of 1.6 and GalNAc4S + GalNAc4,6S are the main non-reducing terminals. Keratan sulfate was identified by the use of two monoclonal antibodies in Western blots after chondroitinase ABC treatment. A keratan sulfate-rich region (~110 kDa) was isolated by sequential treatment with chondroitinase ABC and proteases. We also employed antibodies in Western blotting experiments and showed that the full length deglycosylated core protein is about 300 kDa after SDS-PAGE. Domain-specific antibodies revealed the presence of immunoreactive sites corresponding to G1/G2 and G3 globular domains and the characterization of this large proteoglycan as aggrecan. The results indicate the high conservation of the aggrecan domain structure in this lower vertebrate.

Isolation of a ß-galactoside-binding lectin from cat liver

A lectin from cat liver has been identified and purified by affinity chromatography on asialofetuin-Sepharose. One hundred micrograms of lectin was obtained from one cat liver with a purification factor of 1561. The lectin agglutinates trypsin-treated rabbit and cow erythrocytes. Hemagglutination was inhibited only by saccharides containing ß-galactosyl residues, of which the 1-amine-1-deoxy-ß-D-galactose was the most potent one by inhibiting hemagglutination at a concentration of 12.5 mM, followed by melibiose, trehalose and galactose. The lectin has a subunit molecular mass of 14.4 kDa determined by SDS-PAGE under reducing conditions and a pI of 4.85. Compared with the composition of lectins from calf heart and porcine heart, cat liver lectin contains approximately the same amount of cysteine, half the amount of glycine, twice as much arginine and threonine, and three times the amounts of tyrosine and methionine. Cat liver lectin contains four cysteine residues per subunit, all of them in the reduced form. Their lack of reactivity towards thiol-reactive supports suggests they are not exposed on the lectin surface. The protein apparently has a blocked N-terminus. The purified lectin was stable for up to 20 months stored at +4ºC in buffer supplemented with 4 mM ß-mercaptoethanol. Results indicated that this lectin belongs to the family of soluble ß-galactoside-binding lectins, also known as galectins, which are expressed in a wide range of vertebrate tissues.

Restriction site heteroplasmy in the mitochondrial DNA of Brycon opalinus (Cuvier, 1819) (Characiformes, Characidae, Bryconiae)

Homoplasmy is a feature usually found in the mtDNA of higher animal taxa. On the other hand, the presence of two classes of mtDNA in the same cell or organism is rare and may appear in length or site variation. Data from mtDNA RFLP analysis of Brycon opalinus populations (Cuvier, 1819; Characiformes, Characidae, Bryconinae) revealed site heteroplasmy from endonuclease NheI digestion. Southern blotting hybridization was used to survey a total of 257 specimens with 24 restriction enzymes. Three different restriction fragment patterns of mtDNA were obtained from NheI digestion. Two individuals from hatchery broodstock were found to have two of them. NheI digests of heteroplasmic individuals yielded two fragments of approximately 1180 and 1260 bp. Despite the low frequency of this type of heteroplasmy in the whole B. opalinus population, the presence of site heteroplasmy in this species supports the evidence of this phenomenon in lower vertebrate groups.

The maternal JAK/STAT pathway of Drosophila regulates embryonic dorsal-ventral patterning

Activation of NFkappaB plays a pivotal role in many cellular processes such as inflammation, proliferation and apoptosis. In Drosophila, nuclear translocation of the NFkappaB-related transcription factor Dorsal is spatially regulated in order to subdivide the embryo into three primary dorsal-ventral (DV) domains: the ventral presumptive mesoderm, the lateral neuroectoderm and the dorsal ectoderm. Ventral activation of the Toll receptor induces degradation of the IkappaB-related inhibitor Cactus, liberating Dorsal for nuclear translocation. In addition, other pathways have been suggested to regulate Dorsal. Signaling through the maternal BMP member Decapentaplegic (Dpp) inhibits Dorsal translocation along a pathway parallel to and independent of Toll. In the present study, we show for the first time that the maternal JAK/STAT pathway also regulates embryonic DV patterning. Null alleles of loci coding for elements of the JAK/STAT pathway, hopscotch (hop), marelle (mrl) and zimp (zimp), modify zygotic expression along the DV axis. Genetic analysis suggests that the JAK kinase Hop, most similar to vertebrate JAK2, may modify signals downstream of Dpp. In addition, an activated form of Hop results in increased levels of Cactus and Dorsal proteins, modifying the Dorsal/Cactus ratio and consequently DV patterning. These results indicate that different maternal signals mediated by the Toll, BMP and JAK/STAT pathways may converge to regulate NFkappaB activity in Drosophila.

Apoptotic mimicry: an altruistic behavior in host/Leishmania interplay

Apoptosis is the most common phenotype observed when cells die through programmed cell death. The morphologic and biochemical changes that characterize apoptotic cells depend on the activation of a diverse set of genes. Apoptosis is essential for multicellular organisms since their development and homeostasis are dependent on extensive cell renewal. In fact, there is strong evidence for the correlation between the emergence of multicellular organisms and apoptosis during evolution. On the other hand, no obvious advantages can be envisaged for unicellular organisms to carry the complex machinery required for programmed cell death. However, accumulating evidence shows that free-living and parasitic protozoa as well as yeasts display apoptotic markers. This phenomenon has been related to altruistic behavior, when a subpopulation of protozoa or yeasts dies by apoptosis, with clear benefits for the entire population. Recently, phosphatidylserine (PS) exposure and its recognition by a specific receptor (PSR) were implicated in the infectivity of amastigote forms of Leishmania, an obligatory vertebrate intramacrophagic parasite, showing for the first time that unicellular organisms use apoptotic features for the establishment and/or maintenance of infection. Here we focus on PS exposure in the outer leaflet of the plasma membrane - an early hallmark of apoptosis - and how it modulates the inflammatory activity of phagocytic cells. We also discuss the possible mechanisms by which PS exposure can define Leishmania survival inside host cells and the evolutionary implications of apoptosis at the unicellular level.

Purification, partial characterization and role in lipid transport to developing oocytes of a novel lipophorin from the cowpea weevil, Callosobruchus maculatus

Lipid transport in arthropods is achieved by highly specialized lipoproteins, which resemble those described in vertebrate blood. Here we describe purification and characterization of the lipid-apolipoprotein complex, lipophorin (Lp), from adults and larvae of the cowpea weevil Callosobruchus maculatus. We also describe the Lp-mediated lipid transfer to developing oocytes. Lps were isolated from homogenates of C. maculatus larvae and adults by potassio bromide gradient and characterized with respect to physicochemical properties and lipid content. The weevil Lp (465 kDa) and larval Lp (585 kDa), with hydrated densities of 1.22 and 1.14 g/mL, contained 34 and 56% lipids and 9 and 7% carbohydrates, respectively. In both Lps, mannose was the predominant monosaccharide detected by paper chromatography. SDS-PAGE revealed two apolipoproteins in each Lp with molecular masses of 225 kDa (apolipoprotein-I) and 79 kDa (apolipoprotein-II). The lipids were extracted and analyzed by thin-layer chromatography. The major phospholipids found were phosphatidylserine, phosphatidylcholine and phosphatidylethanolamine in adult Lp, and phosphatidylcholine, phosphatidylethanolamine and sphingomyelin in larval Lp. Hydrocarbons, fatty acids and triacylglycerol were the major neutral lipids found in both Lps. Lps labeled in the protein moiety with radioactive iodine (125I-iodine) or in the lipid moiety with fluorescent lipids revealed direct evidence of endocytic uptake of Lps in live oocytes of C. maculatus.

Fish hemoglobins

Vertebrate hemoglobin, contained in erythrocytes, is a globular protein with a quaternary structure composed of 4 globin chains (2 alpha and 2 beta) and a prosthetic group named heme bound to each one. Having myoglobin as an ancestor, hemoglobin acquired the capacity to respond to chemical stimuli that modulate its function according to tissue requirements for oxygen. Fish are generally submitted to spatial and temporal O2 variations and have developed anatomical, physiological and biochemical strategies to adapt to the changing environmental gas availability. Structurally, most fish hemoglobins are tetrameric; however, those from some species such as lamprey and hagfish dissociate, being monomeric when oxygenated and oligomeric when deoxygenated. Fish blood frequently possesses several hemoglobins; the primary origin of this finding lies in the polymorphism that occurs in the globin loci, an aspect that may occasionally confer advantages to its carriers or even be a harmless evolutionary remnant. On the other hand, the functional properties exhibit different behaviors, ranging from a total absence of responses to allosteric regulation to drastic ones, such as the Root effect.

Autonomic control of cardiorespiratory interactions in fish, amphibians and reptiles

Control of the heart rate and cardiorespiratory interactions (CRI) is predominantly parasympathetic in all jawed vertebrates, with the sympathetic nervous system having some influence in tetrapods. Respiratory sinus arrhythmia (RSA) has been described as a solely mammalian phenomenon but respiration-related beat-to-beat control of the heart has been described in fish and reptiles. Though they are both important, the relative roles of feed-forward central control and peripheral reflexes in generating CRI vary between groups of fishes and probably between other vertebrates. CRI may relate to two locations for the vagal preganglionic neurons (VPN) and in particular cardiac VPN in the brainstem. This has been described in representatives from all vertebrate groups, though the proportion in each location is variable. Air-breathing fishes, amphibians and reptiles breathe discontinuously and the onset of a bout of breathing is characteristically accompanied by an immediate increase in heart rate plus, in the latter two groups, a left-right shunting of blood through the pulmonary circuit. Both the increase in heart rate and opening of a sphincter on the pulmonary artery are due to withdrawal of vagal tone. An increase in heart rate following a meal in snakes is related to withdrawal of vagal tone plus a non-adrenergic-non-cholinergic effect that may be due to humoral factors released by the gut. Histamine is one candidate for this role.

Modulation of rhodopsin gene expression and signaling mechanisms evoked by endothelins in goldfish and murine pigment cell lines

Endothelins (ETs) and sarafotoxins (SRTXs) belong to a family of vasoconstrictor peptides, which regulate pigment migration and/or production in vertebrate pigment cells. The teleost Carassius auratus erythrophoroma cell line, GEM-81, and Mus musculus B16 melanocytes express rhodopsin, as well as the ET receptors, ETB and ETA, respectively. Both cell lines are photoresponsive, and respond to light with a decreased proliferation rate. For B16, the doubling time of cells kept in 14-h light (14L):10-h darkness (10D) was higher compared to 10L:14D, or to DD. The doubling time of cells kept in 10L:14D was also higher compared to DD. Using real-time PCR, we demonstrated that SRTX S6c (12-h treatment, 100 pM and 1 nM; 24-h treatment, 1 nM) and ET-1 (12-h treatment, 10 and 100 pM; 24- and 48-h treatments, 100 pM) increased rhodopsin mRNA levels in GEM-81 and B16 cells, respectively. This modulation involves protein kinase C (PKC) and the mitogen-activated protein kinase cascade in GEM-81 cells, and phospholipase C, Ca2+, calmodulin, a Ca2+/calmodulin-dependent kinase, and PKC in B16 cells. Cells were kept under constant darkness throughout the gene expression experiments. These results show that rhodopsin mRNA levels can be modulated by SRTXs/ETs in vertebrate pigment cells. It is possible that SRTX S6c binding to the ETB receptors in GEM-81 cells, and ET-1 binding to ETA receptors in B16 melanocytes, although activating diverse intracellular signaling mechanisms, mobilize transcription factors such as c-Fos, c-Jun, c-Myc, and neural retina leucine zipper protein. These activated transcription factors may be involved in the positive regulation of rhodopsin mRNA levels in these cell lines.

A detailed description of an economical setup for electroporation of chick embryos in ovo

One of the challenges of the postgenomic era is characterizing the function and regulation of specific genes. For various reasons, the early chick embryo can easily be adopted as an in vivo assay of gene function and regulation. The embryos are robust, accessible, easily manipulated, and maintained in the laboratory. Genomic resources centered on vertebrate organisms increase daily. As a consequence of optimization of gene transfer protocols by electroporation, the chick embryo will probably become increasingly popular for reverse genetic analysis. The challenge of establishing chick embryonic electroporation might seem insurmountable to those who are unfamiliar with experimental embryological methods. To minimize the cost, time, and effort required to establish a chick electroporation assay method, we describe and illustrate in great detail the procedures involved in building a low-cost electroporation setup and the basic steps of electroporation.

Analysis of the feeding behaviour of the mosquito Culex pipiens L. (Diptera: Culicidae) in relation to West Nile virus

The goal ofthis literature review is to inform the reader on several aspects of West Nile Virus (WNV) transmission by its mosquito vector, Culex pipiens and to elucidate how Cx. pipiens and WNV are intertwined. The first few sections of the literature review describe the life cycle and blood feeding behaviours ofmosquitoes so that baseline data ofmosquito biology are established. In addition to explaining how and why a mosquito blood feeds, the section on "Blood Meal Analysis" describes the different methods for determining the vertebrate source of mosquito blood meals and a brief history of these testing methods. Since this thesis looks at the feeding behaviour of Cx. pipiens, it is important to know how to determine what they are feeding upon. Discussion on other mosquito-borne diseases related to WNV gives a broader perspective to the thesis, and examines other diseases that have occurred in Ontario in the past. This is followed by background information on WNV and theories on how this virus came to North America and how it relates to Cx. pipiens. The final sections discuss Cx. pipiens and give background information to how this species of mosquito exists and behaves within North America.

Intracellular antioxidant and DNA repair enzymes as correlates of stress resistance and longevity in vertebrates

In animals, both stress resistance and longevity appear to be influenced by the insulin/insulin-like growth factor-l signaling (lIS) pathway, the basic organization of which is highly conserved from invertebrates to vertebrates. Reduced lIS or genetic disruption of the lIS pathway leads to the activation of forkhead box transcription factors, which is thought to upregulate the expression of genes involved in enhancing stress resistance, including perhaps key antioxidant enzymes as well as DNA repair enzymes. Enhanced antioxidant and DNA repair capacities may underlie the enhanced cellular stress resistance observed in long-lived animals, however little data is available that directly supports this idea. I used three. experimental approaches to test the association of intracellular antioxidant and DNA base excision repair (BER) capacities with stress resistance and longevity: (1) a comparison of multiple vertebrate endotherm species of varying body masses and longevities; (2) a comparison of long-lived Snell dwarf mice and their normallittermates; and (3) a comparison of hypometabolic animals undergoing hibernation or estivation with their active counterparts. The activities of the five major intracellular antioxidant enzymes as well as the two rate-limiting enzymes in the BER pathway, apurininc/apyrimidinic (AP) endonuclease and polymerase ~, were measured. These measurements were performed in one or more of the following: (1) cultured dermal fibroblasts; (2) brain tissue; (3) heart tissue; (4) liver tissue. My results indicate that antioxidant enzymes are not universally upregulated in association with enhanced stress resistance and longevity. I also did not find that BER enzyme activity was positively correlated with longevity, in an inter-species context, though there was evidence for enhanced BER in long-lived Snell dwarf mice. Thus, while there were instances in which enhanced antioxidant and BER enzyme activities were associated with increased stress resistance and/or longevity, this was not universally the case, indicating that other mechanisms must be involved. These results suggest the need to re-examine existing 'oxidative stress' hypotheses of longevity and probe further into the molecular physiology of longevity to discover its mechanistic basis.

Elucidation of the Retinoid Signalling Pathway Involved in Axon Guidance in Lymnaea stagnalis and Xenopus laevis

The vitamin A metabolite, retinoic acid (RA), is known to play a crucial role in several developmental processes including axial patterning and differentiation. More recently, RA has been implicated in the regenerative process acting through its classical signaling pathway, the nuclear receptors, retinoic acid receptor (RAR) and retinoid X receptor (RXR), to mediate gene transcription. Moreover, RA has been shown to act as a guidance molecule for growth cones of regenerating motorneurons of the pond snail, Lymnaea stagnalis. Our lab has recently shown that RA can induce this morphological response independent of nuclear transcription, however, the role of the retinoid receptors in RA-induced chemoattraction is still unknown. Here, I show that the retinoid receptors, RXR and RAR, may mediate the growth cones response to the metabolically active retinoic acid isomers, all-trans and 9-cis RA, in Lymnaea stagnalis. Data presented here show that both an RXR and RAR antagonist can block growth cone turning in response to application of both isomers. Because no prior investigations have shown growth cone turning of individual vertebrate neurons, I aimed to show that both retinoic acid isomers were capable of inducing growth cone turning of embryonic spinal cord neurons in the frog, Xenopus laevis. For the first time in Xenopus, I showed that both all-trans and 9-cis RA were able to induce significantly more neurite outgrowth from cultured embryonic spinal cord neurons and induce positive growth cone turning of individual growth cones. In addition, I showed that the presence of the RXR antagonist, HX531, blocked 9-cis RA-induced growth cone turning and the RARβ antagonist, LE135, blocked all-trans RA-induced growth cone turning in this species. Evidence provided here shows for the first time, conservation of retinoic acid-induced growth cone turning in a vertebrate model system. In addition, these data show that the receptors involved in this morphological response may be the same in vertebrates and invertebrates.

Alternative splicing of DNA polymerase beta in vertebrates

Alternative splicing (AS) is the predominant mechanism responsible for increasing eukaryotic transcriptome and proteome complexity. In this phenomenon, numerous mRNA transcripts are produced from a single pre-mRNA sequence. AS is reported to occur in 95% of human multi-exon genes; one specific gene that undergoes AS is DNA polymerase beta (POLB). POLB is the main DNA repair gene which performs short patch base excision repair (BER). In primate untransformed primary fibroblast cell lines, it was determined that the splice variant (SV) frequency of POLB correlates positively with species lifespan. To date, AS patterns of POLB have only been examined in mammals primarily through the use of cell lines. However, little attention has been devoted to investigating if such a relationship exists in non-mammals and whether cell lines reflect what is observed in vertebrate tissues. This idea was explored through cloning and characterization of 1,214 POLB transcripts from four non-mammalian species (Gallus gallus domesticus, Larus glaucescens, Xenopus laevis, and Pogona vitticeps) and two mammalian species (Sylvilagus floridanus and Homo sapiens) in two tissue types, liver and brain. POLB SV frequency occurred at low frequencies, < 3.2%, in non-mammalian tissues relative to mammalian (>20%). The highest POLB SV frequency was found in H. sapiens liver and brain tissues, occurring at 65.4% and 91.7%, respectively. Tissue specific AS of POLB was observed in L. glaucescens, P. vitticeps, and H. sapiens, but not G. gallus domesticus, X. laevis and S. floridanus.The AS patterns of a second gene, transient receptor potential cation channel subfamily V member 1 (TRPV1), were compared to those of POLB in liver and brain tissues of G. gallus domesticus, X. laevis and H. sapiens. This comparison was performed to investigate if any changes (either increase or decrease) observed in the AS of POLB were gene specific or if they were tissue specific, in which case similar changes in AS would be seen in POLB and TRPV1. Analysis did not reveal an increase or decrease in both the AS of POLB and TRPV1 in either the liver or brain tissues of G. gallus domesticus and H. sapiens. This result suggested that the AS patterns of POLB were not influenced by tissue specific rates of AS. Interestingly, an increase in the AS of both genes was only observed in X. laevis brain tissue. This result suggests that AS in general may be increased in the X. laevis brain as compared to liver tissue. No positive correlation between POLB SV frequency and species lifespan was found in non-mammalian tissues. The AS patterns of POLB in human primary untransformed fibroblast cell lines were representative of those seen in human liver tissue but not in brain tissue. Altogether, the AS patterns of POLB from vertebrate tissues and primate cell lines revealed a positive correlation between POLB SV frequency and lifespan in mammals, but not in non-mammals. It appears that this positive correlation does not exist in vertebrate species as a whole.