Kristiina Vuori

Comparison of hypoxia-inducible factor-1 alpha in hypoxia-sensitive and hypoxia-tolerant fish species

Levels of oxygen can vary dramatically in aquatic environments. Aquatic organisms, including fishes, have adapted accordingly to survive. As there are both phylogenetically closely related fish species with differing oxygen requirements and distantly related species with similar oxygen requirements, fishes are good candidates for examining oxygen-related functions in vertebrates. We set out to investigate if sequence variation in the hypoxia-inducible factor-1 alpha (HIF-1alpha) gene is associated with variations in oxygen requirements. Since the teleost HIF-1alpha sequences available in databases represent a very limited dataset both phylogenetically and with regard to oxygen requirements, we have sequenced the protein coding sequence for HIF-1alpha from an additional 9 fish species. Our results indicate that the deduced HIF-1alpha proteins of teleost fishes are somewhat shorter than those of tetrapods. Additionally, the results suggest that tetrapod sequences more closely resemble the ancestral form of the protein than do teleost sequences. No clear signatures which could be associated with the oxygen requirements of the species were found. This study suggests that if species-specific differences in HIF-1alpha function with regards to oxygen dependence have evolved, they do not occur in the protein coding sequence but at other levels of the HIF-1alpha pathway.

M74 syndrome in Baltic salmon and the possible role of oxidative stresses in its development: present knowledge and perspectives for future studies.

Abstract Baltic salmon suffer from maternally transmitted yolk-sac fry mortality syndrome—M74. The incidence of M74 varies considerably on a year to year basis. In the 1990s the mortalities were 50–80% but in 2003–2005, below 10%. Before death, M74-affected fry have several typical symptoms. M74-eggs are characterized by low thiamine and carotenoid content, and affected fry show signs of oxidative stress. Although M74 is associated with thiamine deficiency and the symptoms of the fry can be alleviated with thiamine, the underlying causes of the syndrome have remained a mystery. We have studied the symptoms of M74 at the molecular level by investigating the global gene expression patterns using cDNA microarray and have quantified the changes in transcriptional regulation in M74-affected and healthy yolk-sac fry. Our and previous results suggest that M74 in Baltic salmon yolk-sac fry results from oxidative stresses disturbing several different developmental molecular pathways. Because the M74 syndrome is of maternal origin, factors in the Baltic Sea during salmon feeding and migration, i.e., the chemical composition of food, may be decisive in the development of M74. The possible mechanisms by which oxidative stresses may develop in adult salmon are discussed in the review.

Developmental disturbances in early life stage mortality (M74) of Baltic salmon fry as studied by changes in gene expression

BackgroundWe have studied alterations of gene expression associated with naturally-occurring early life stage mortality (M74) in Baltic salmon using a cDNA microarray and real time PCR. M74-affected fry have several typical neurological, cardiovascular and pathological symptoms. They are also characterized by low thiamine content and show signs of oxidative stress.ResultsAffected fry can be divided into three major groups with early, intermediate or late onset of mortality. If mortality starts during the first third of the yolk-sac stage, virtually all the responses are compatible with stress, which rapidly leads to the common terminal responses. If death occurs during the second third of the yolk sac stage, the terminal stage is preceded by a decrease in globin gene expression, which leads to internal hypoxia when the animals grow and shift from skin- to gill-breathing. Fry will eventually proceed to the terminal responses. The group developing M74 most slowly appears to compensate for reduced oxygen delivery by downregulation of metabolism, and hence some fry can escape death.ConclusionOur study is the first demonstration of diverse transcriptional responses to a naturally-occurring developmental disturbance. Since many of the genes differentially expressed in M74-fry are evolutionarily conserved, the M74 of Baltic salmon can serve as a model for developmental disturbances and environmental stress responses in vertebrates in general.

Abiotic Stress Modifies the Synthesis of Alpha-Tocopherol and Beta-Carotene in Phytoplankton Species

We performed laboratory experiments to investi‐gate whether the synthesis of the antioxidants α‐tocopherol (vitamin E) and β‐carotene in phytoplankton depends on changes in abiotic factors. Cultures of Nodularia spumigena, Phaeodactylum tricornutum, Skeletonema costatum, Dunaliella tertiolecta, Prorocentrum cordatum, and Rhodomonas salina were incubated at different tempe‐ratures, photon flux densities and salinities for 48 h. We found that abiotic stress, within natural ecological ranges, affects the synthesis of the two antioxidants in different ways in different species. In most cases antioxidant production was stimulated by increased abiotic stress. In P. tricornutum KAC 37 and D. tertiolecta SCCAP K‐0591, both good producers of this compound, α‐tocopherol accumulation was negatively affected by environmentally induced higher photosystem II efficiency (Fv/Fm). On the other hand, β‐carotene accumulation was positively affected by higher Fv/Fm in N. spumigena KAC 7, P. tricornutum KAC 37, D. tertiolecta SCCAP K‐0591 and R. salina SCCAP K‐0294. These different patterns in the synthesis of the two compounds may be explained by their different locations and functions in the cell. While α‐tocopherol is heavily involved in the protection of prevention of lipid peroxidation in membranes, β‐carotene performs immediate photo‐oxidative protection in the antennae complex of photosystem II. Overall, our results suggest a high variability in the antioxidant pool of natural aquatic ecosystems, which can be subject to short‐term temperature, photon flux density and salinity fluctuations. The antioxidant levels in natural phytoplankton communities depend on species composition, the physiological condition of the species, and their respective strategies to deal with reactive oxygen species. Since α‐tocopherol and β‐carotene, as well as many other nonenzymatic antioxidants, are exclusively produced by photo‐synthetic organisms, and are required by higher trophic levels through dietary intake, regime shifts in the phytoplankton as a result of large‐scale environmental changes, such as climate change, may have serious consequences for aquatic food webs.

Tissue-specific expression of aryl hydrocarbon receptor and putative developmental regulatory modules in Baltic salmon yolk-sac fry

The aryl hydrocarbon receptor (AhR) is an ancient protein that is conserved in vertebrates and invertebrates, indicating its important function throughout evolution. AhR has been studied largely because of its role in toxicology-gene expression via AhR is induced by many aromatic hydrocarbons in mammals. Recently, however, it has become clear that AhR is involved in various aspects of development such as cell proliferation and differentiation, and cell motility and migration. The mechanisms by which AhR regulates these various functions remain poorly understood. Across-species comparative studies of AhR in invertebrates, non-mammalian vertebrates and mammals may help to reveal the multiple functions of AhR. Here, we have studied AhR during larval development of Baltic salmon (Salmon salar). Our results indicate that AhR protein is expressed in nervous system, liver and muscle tissues. We also present putative regulatory modules and module-matching genes, produced by chromatin immunoprecipitation (ChIP) cloning and in silico analysis, which may be associated with evolutionarily conserved functions of AhR during development. For example, the module NFKB-AHRR-CREB found from salmon ChIP sequences is present in human ULK3 (regulating formation of granule cell axons in mouse and axon outgrowth in Caernohabditis elegans) and SRGAP1 (GTPase-activating protein involved in the Slit/Robo pathway) promoters. We suggest that AhR may have an evolutionarily conserved role in neuronal development and nerve cell targeting, and in Wnt signaling pathway.

Stock‐specific variation of trophic position, diet and environmental stress markers in Atlantic salmon Salmo salar during feeding migrations in the Baltic Sea

This study investigated stock-specific variation in selected ecophysiological variables during the feeding migrations of Atlantic salmon Salmo salar in the Baltic Sea. Oxidative stress biomarkers and EROD (ethoxyresorufin-O-deethylase, Cyp1A enzyme) activity were used as indicators of possible environmental stress and stable isotopes as determinants of diet and trophic position. Latvian S. salar stocks Daugava and Gauja had distinct stable-isotope signatures compared to the other stocks, indicating differences in migration patterns, residency or arrival times, or dietary specialization among stocks. Salmo salar originating from Daugava and Gauja also had lower catalase enzyme activity than the other stocks. Post-smolts originating from rivers of the Gulf of Finland had elevated EROD activities compared to fish of the same age from Bothnian Bay rivers, which could indicate exposure to organochlorine pollutants. No other stock-specific differences in oxidative stress biomarkers were found. The study demonstrates how genetic, oxidative stress biomarker, EROD and stable-isotope data may be combined to study trophic position, prey prevalence and environmental stress of mixed S. salar stocks foraging in the sea.

Molecular Markers of Yolk Sac Fry Development in Nine Families of Lake Trout

Salmonids in certain areas of North America and northern Europe suffer from reproductive disturbances manifested through the death of yolk sac fry. These disturbances are referred to as early mortality syndrome (EMS) in the Great Lakes region and M74 in the Baltic Sea. Both of these syndromes have been associated with reduced concentrations of thiamine in affected females and their eggs. However, large variations in signs and mortality, both within and between the individual syndromes, have been reported. Yolk sac fry mortality (M74) in Atlantic salmon Salmo salar has been shown to be associated with reduced DNA binding of the hypoxia-inducible transcription factor 1 (HIF-1), reduced production of vascular endothelial growth factor (VEGF) protein, decreased capillary density, and down-regulation of adult-type globin gene transcription (which is responsible for the protein part of adult hemoglobin). One of the main effects of all of these changes is reduced oxygen transport to the tissues of affected fry. In this study, the developmental patterns of HIF-1 DNA binding, VEGF protein expression, and adult-type globin gene transcription were analyzed in nine family groups of Lake Michigan lake trout Salvelinus namaycush. The results indicate that HIF-1 DNA binding and globin gene transcription increase from hatch to the end of yolk sac stage. Interindividual and between-family biological variations were detected, especially in VEGF protein expression and globin gene transcription. Our results demonstrate the possibility of using these molecular markers in investigating the etiology of EMS and making comparisons between the mechanisms of different salmonid yolk sac fry mortalities.

TransLISA, a novel quantitative, nonradioactive assay for transcription factor DNA‐binding analyses

Transcription factors are DNA‐binding proteins that regulate key biological processes. Their interactions with DNA are commonly analyzed with gel‐based electrophoretic mobility shift assay (EMSA) using radioactively labeled probes. Within various fields of research, there exists an increasing demand to develop assays with faster sample throughput combined with improved sensitivity, increased analytical range, and precise quantification. Here, we describe the development and performance of a 384‐well plate immunoassay, termed TransLISA, which is a novel homogeneous assay for rapid and sensitive quantification of the DNA‐binding activity of transcription factors in cell and tissue lysates. TransLISA outperforms EMSAs, because it eliminates the need to use radioactive chemicals and allows fast and precise quantification of DNA‐binding activity of transcription factors from large number of samples simultaneously. We have used TransLISA to demonstrate the DNA‐binding activity of heat shock factor 1, representing a well‐known model of inductive transcriptional regulatory responses, but the method is easily adaptable for the study of any transcription factor. Thus, TransLISA can replace EMSAs and may be used in various applications and research fields where quantitative, cost‐effective and large‐scale measurements of the DNA‐binding activity of transcription factors are required, including screening of responses in multiple treatments in cellular and molecular biology, evolutionary research, environmental monitoring, and drug discovery.