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Reindeer (Rangifer tarandus) are semi-domesticated animals adapted to the challenging conditions of northern Eurasia. Adipose tissues play a crucial role in northern animals by altering gene expression in their tissues to regulate energy homoeostasis and thermogenic activity. Here, we perform transcriptome profiling by RNA sequencing of adipose tissues from three different anatomical depots: metacarpal (bone marrow), perirenal, and prescapular fat in Finnish and Even reindeer (in Sakha) during spring and winter. A total of 16,212 genes are expressed in our data. Gene expression profiles in metacarpal tissue are distinct from perirenal and prescapular adipose tissues. Notably, metacarpal adipose tissue appears to have a significant role in the regulation of the energy metabolism of reindeer in spring when their nutritional condition is poor after winter. During spring, genes associated with the immune system are upregulated in the perirenal and prescapular adipose tissue. Blood and tissue parameters reflecting general physiological and metabolic status show less seasonal variation in Even reindeer than in Finnish reindeer. This study identifies candidate genes potentially involved in immune response, fat deposition, and energy metabolism and provides new information on the mechanisms by which reindeer adapt to harsh arctic conditions., Weldenegodguad et al. performed transcriptome profiling of adipose tissues from the bone marrow, perirenal, and prescapular fat in Finnish and Even reindeer (in Sakha) during spring and winter. They identified candidate genes potentially involved in immune responses, fat deposition, and energy metabolism that provide insight into the mechanisms by which reindeer adapt to harsh arctic conditions.

Summary • Expression of all known and newly found pathogenesis-related PR-10 proteins (PR-10a, b, c, d, e) was analysed from Cu-sensitive and –tolerant birch clones to find out whether they follow the same expression pattern. The relationship of PR-10 proteins, particularly PR-10c, to oxidative stress caused by metals or ozone was studied in tolerant and sensitive birch clones to find out possible linkages to tolerance. • Antibody developed to PR-10c was used in Western blot analysis. Other PR-10 proteins were studied with two-dimensional electrophoresis and mass spectrometry. Metal-sensitive yeasts were transformed with PR-10c. • Two new members of PR-10 family, PR-10d and PR-10e, were found. Various PR-10 proteins showed different expression patterns. The amount of PR-10c increased with increasing soil metal concentrations but was, in general, more prominent in Cu-sensitive than in Cu-tolerant clones. PR-10c did not alter metal tolerance in metal-sensitive yeasts. • The PR-10c protein appears not to confer metal- or ozone-tolerance in birch. However, this does not exclude the possibility that it is involved in the tolerance or sensitivity mechanism in an indirect manner.

Reindeer (Rangifer tarandus) are semi-domesticated animals adapted to the challenging arctic conditions of northern Eurasia. Adipose tissues play a crucial role in animals living in northern environments by altering gene expression in their tissues to regulate energy homeostasis and thermogenic activity. Here, we performed transcriptome profiling by RNA sequencing of adipose tissues from three different anatomical depots: metacarpal (bone marrow), perirenal, and prescapular fat in Finnish and Even reindeer (in Sakha) during two seasonal time points (spring and winter). On average 36.5 million pair-ended clean reads were obtained for each sample, and a total of 16,362 genes were expressed in our data. Gene expression profiles in metacarpal tissue were distinct and clustered separately from perirenal and prescapular adipose tissues. Notably, metacarpal adipose tissue appeared to have a significant role in the regulation of the energy metabolism of reindeer in spring when their nutritional condition is poor after winter. During spring, when the animals are in less optimal condition, genes associated with the immune system (e.g., CCL2, CCL11, CXCL14, IGSF3, IGHM, IGLC7, IGKC, JCHAIN, and IGSF10) were upregulated in the perirenal and prescapular adipose tissue, while genes involved in energy metabolism (e.g., ACOT2, APOA1, ANGPTL1, ANGPTL8, ELOVL7, MSMO1, PFKFB1, and ST3GAL6) were upregulated in metacarpal tissue. Even reindeer harboured relatively fewer significantly differentially expressed genes than Finnish reindeer, irrespective of the season, possibly owing to climatic and management differences. Moreover, blood and tissue parameters reflecting general physiological and metabolic status showed less seasonal variation in Even reindeer than in Finnish reindeer. This study identified adipose candidate genes potentially involved in immune response, fat deposition, energy metabolism, development, cell growth, and organogenesis. Taken together, this study provides new information on the mechanisms by which reindeer adapt to less optimal arctic conditions.

The Glanville fritillary (Melitaea cinxia) butterfly is a long-term model system for metapopulation dynamics research in fragmented landscapes. Here, we provide a chromosome level assembly of the butterfly’s genome produced from Pacific Biosciences sequencing of a pool of males, combined with a linkage map from population crosses. The final assembly size of 484 Mb is an increase of 94 Mb on the previously published genome. Estimation of the completeness of the genome with BUSCO, indicates that the genome contains 93 - 95% of the BUSCO genes in complete and single copies. We predicted 14,830 gene models using the MAKER pipeline and manually curated 1,232 of these gene models. The genome and its annotated gene models are a valuable resource for future comparative genomics, molecular biology, transcriptome and genetics studies on this species.

Silver birch (Betula pendula) is a pioneer boreal tree that can be induced to flower within 1 year. Its rapid life cycle, small (440-Mb) genome, and advanced germplasm resources make birch an attractive model for forest biotechnology. We assembled and chromosomally anchored the nuclear genome of an inbred B. pendula individual. Gene duplicates from the paleohexaploid event were enriched for transcriptional regulation, whereas tandem duplicates were overrepresented by environmental responses. Population resequencing of 80 individuals showed effective population size crashes at major points of climatic upheaval. Selective sweeps were enriched among polyploid duplicates encoding key developmental and physiological triggering functions, suggesting that local adaptation has tuned the timing of and cross-talk between fundamental plant processes. Variation around the tightly-linked light response genes PHYC and FRS10 correlated with latitude and longitude and temperature, and with precipitation for PHYC. Similar associations characterized the growth-promoting cytokinin response regulator ARR1, and the wood development genes KAK and MED5A.

Calamine accessions of the zinc/cadmium/nickel hyperaccumulator, Noccaea caerulescens, exhibit striking variation in foliar cadmium accumulation in nature. The Ganges accession (GA) from Southern France displays foliar cadmium hyperaccumulation (>1000 μg g −1 DW), whereas the accession La Calamine (LC) from Belgium, with similar local soil metal composition, does not (−1 DW). All calamine accessions are cadmium hypertolerant. To find out the differences between LC and GA in their basic adaptation mechanisms, we bypassed the cadmium excluding phenotype of LC by exposing the plants to 50 μm cadmium in hydroponics, achieving equal cadmium accumulation in the shoots. The iron content increased in the roots of both accessions. GA exhibited significant decreases in manganese and zinc contents in the roots and shoots, approaching those in LC. Altogether 702 genes responded differently to cadmium exposure between the accessions, 157 and 545 in the roots and shoots, respectively. Cadmium-exposed LC showed a stress response and had decreased levels of a wide range of photosynthesis-related transcripts. GA showed less changes, mainly exhibiting an iron deficiency-like response. This included increased expression of genes encoding five iron deficiency-regulated bHLH transcription factors, ferric reduction oxidase FRO2, iron transporters IRT1 and OPT3, and nicotianamine synthase NAS1, and decreased expression of genes encoding ferritins and NEET (a NEET family iron-sulfur protein), which is possibly involved in iron transfer, distribution and/or management. The function of the IRT1 gene in the accessions was compared. We conclude that the major difference between the two accessions is in the way they cope with iron under cadmium exposure.

Uranium (U), cobalt (Co), molybdenum (Mo), nickel (Ni), lead (Pb), thorium (Th) and zinc (Zn) occur naturally in soil but their radioactive isotopes can also be released into the environment during the nuclear fuel cycle. The transfer of these elements was studied in three different trophic levels in experimental mesocosms containing downy birch (Betula pubescens), narrow buckler fern (Dryopteris carthusiana) and Scandinavian small-reed (Calamagrostis purpurea ssp. Phragmitoides) as producers, snails (Arianta arbostorum) as herbivores, and earthworms (Lumbricus terrestris) as decomposers. To determine more precisely whether the element uptake of snails is mainly via their food (birch leaves) or both via soil and food, a separate microcosm experiment was also performed. The element uptake of snails did not generally depend on the presence of soil, indicating that the main uptake route was food, except for U, where soil contact was important for uptake when soil U concentration was high. Transfer of elements from soil to plants was not linear, i.e. it was not correctly described by constant concentration ratios (CR) commonly applied in radioecological modeling. Similar nonlinear transfer was found for the invertebrate animals included in this study: elements other than U were taken up more efficiently when element concentration in soil or food was low.

Silver birch (Betula pendula) is a pioneer boreal tree that can be induced to flower within 1 year. Its rapid life cycle, small (440-Mb) genome, and advanced germplasm resources make birch an attractive model for forest biotechnology. We assembled and chromosomally anchored the nuclear genome of an inbred B. pendula individual. Gene duplicates from the paleohexaploid event were enriched for transcriptional regulation, whereas tandem duplicates were overrepresented by environmental responses. Population resequencing of 80 individuals showed effective population size crashes at major points of climatic upheaval. Selective sweeps were enriched among polyploid duplicates encoding key developmental and physiological triggering functions, suggesting that local adaptation has tuned the timing of and cross-talk between fundamental plant processes. Variation around the tightly-linked light response genes PHYC and FRS10 correlated with latitude and longitude and temperature, and with precipitation for PHYC. Similar associations characterized the growth-promoting cytokinin response regulator ARR1, and the wood development genes KAK and MED5A., published version, peerReviewed

Tricholoma matsutake is an ectomycorrhizal fungus that forms commercially important mushrooms in coniferous forests. In this study, we explored the ability of T. matsutake to form mycorrhizae with Pinus sylvestris by inoculating emblings produced through somatic embryogenesis (SE) in an aseptic culture system. Two months after inoculation, clones with less phenolic compounds in the tissue culture phase formed mycorrhizae with T. matsutake, while clones containing more phenols did not. Effects of inoculation on embling growth varied among clones; two of the four tested showed a significant increase in biomass and two had a significant increase in root density. In addition, results suggest that clones forming well-developed mycorrhizae absorbed more Al, Fe, Na, P, and Zn after 8 weeks of inoculation. This study illustrates the value of SE materials in experimental work concerning T. matsutake as well as the role played by phenolic compounds in host plant response to infection by mycorrhizal fungi.

We studied phosphorous (P) export after clear-felling from eight Scots pine dominated, hydrologically isolated catchment areas located on a low-P sorptive peatland site in southern Finland, using the paired-catchments approach. Four of the catchment areas were clear-felled traditionally with stem-only harvesting, two areas were clear-felled using whole-tree harvesting, and the remaining two areas were treated with both whole-tree harvesting and stump harvesting. The effect of clear-felling on P loads varied considerably between the catchment areas from no increase to an increase of over 1.5 kg ha −1 of P during the three-year-study period. The most significant factor explaining the variation in P loads and the variation in P concentrations in outflow water from clear-felled peatland catchments was the post-harvest water level position. At water table levels

Volatile organic compounds (VOCs) emitted from vegetation to the atmosphere contribute to global climate change, but climate change factors also affect VOC emission from vegetation. Soil-grown Norway spruce seedlings were exposed to elevated ozone (1.4 × ambient ozone concentration) and elevated temperature (ambient + 1.3 °C) alone and in combination as well as to ambient ozone and temperature treatments under open-field conditions. VOC emissions (mainly terpenoids), genes involved in early steps of plastidial monoterpene and isoprene synthesis, photosynthetic parameters and growth were measured. In July, when daytime elevated ozone concentrations had been over 40 ppb, ozone doubled the total terpenoid emissions by increasing the emissions of many monoterpenes and sesquiterpenes. Elevated temperature changed the terpenoid profile by increasing the emissions of oxygenated monoterpenes, but did not influence total emissions. Terpenoid emission profiles also differed between elevated ozone alone and elevated ozone in combination with elevated temperature. In August, when daytime elevated ozone concentrations had been ca. 30 ppb, significant treatment effects were not found. Ozone and temperature reduced the expression of DXS2B (1-deoxy- d -xylulose 5-phosphate synthase type II), and ozone that of DXR (1-deoxy- d -xylulose 5-phosphate reductoisomerase) in August. Elevated temperature reduced the stem diameter growth, net photosynthesis and stomatal conductance, but elevated ozone did not have effects on these parameters. Results suggest that elevated temperature may not modify the ozone responses, or vice versa, in terms of gas exchange, growth or total terpenoid emission rates of young Norway spruces in a near-future climate. However, observed changes in terpenoid emission profiles may be important in the future climate, as reactivity in the troposphere differs between individual terpenoids.

Noccaea caerulescens of the Brassicaceae family has become the key model plant among the metal hyperaccumulator plants. Populations/accessions of N. caerulescens from geographic locations with different soil metal concentrations differ in their ability to hyperaccumulate and hypertolerate metals. Comparison of transcriptomes in several accessions provides candidates for detailed exploration of the mechanisms of metal accumulation and tolerance and local adaptation. This can have implications in the development of plants for phytoremediation and improved mineral nutrition. Transcriptomes from root and shoot tissues of four N. caerulescens accessions with contrasting Zn, Cd and Ni hyperaccumulation and tolerance traits were sequenced with Illumina Hiseq2000. Transcriptomes were assembled using the Trinity de novo assembler and were annotated and the protein sequences predicted. The comparison against the BUSCO plant early release dataset indicated high-quality assemblies. The predicted protein sequences have been clustered into ortholog groups with closely related species. The data serve as important reference sequences in whole transcriptome studies, in analyses of genetic differences between the accessions and other species, and for primer design., published version, peerReviewed

The intracytoplasmic aggregation of alpha-synuclein (alphaS) protein is a common denominator for a group of neurodegenerative disorders currently known as synucleinopathies. It is generally assumed that the incorporation of alphaS protein into compact inclusions compromises the function and viability of its host cell via mechanical disruption. Herein, we report a widespread and abundant alphaS pathology in an elderly subject, whose medical history gave no indication of any neurodegenerative disease. We compared neuronal and glial components in this neurologically unimpaired subject with a patient with a clinical syndrome of dementia with Lewy bodies (DLB) by using a range of antigenic determinants and an in situ end-labeling technique. We detected no differences in vascular pathologies, in gliosis, or in apoptosis that would have explained the incompatible clinical end-points. With respect to the Alzheimer's disease-related changes, the only differences noted were the beta-amyloid aggregates in the putamen found in the DLB patient alone. Our findings suggest that there must be some currently unidentified factors rather than alphaS-positive inclusions that are responsible for the neuronal dysfunction. The alphaS-positive inclusions may well represent detoxified reserves that cells can tolerate for years, and thus prevention of their development could actually accelerate the diseases process.

Northern forests are currently experiencing increasing mean temperatures, especially during autumn and spring. Consequently, alterations in carbon sequestration, leaf biochemical quality and freezing tolerance (FT) are likely to occur. The interactive effects of elevated temperature and ozone (O(3)), the most harmful phytotoxic air pollutant, on Norway spruce (Picea abies (L.) Karst.) seedlings were studied by analysing phenology, metabolite concentrations in the needles, FT and gas exchange. Sampling was performed in September and May. The seedlings were exposed to a year-round elevated temperature (+1.3 °C), and to 1.4× ambient O(3) concentration during the growing season in the field. Elevated temperature increased the concentrations of amino acids, organic acids of the citric acid cycle and some carbohydrates, and reduced the concentrations of phenolic compounds, some organic acids of the shikimic acid pathway, sucrose, cyclitols and steroids, depending on the timing of the sampling. Although growth onset occurred earlier at elevated temperature, the temperature of 50% lethality (LT(50)) was similar in the treatments. Photosynthesis and the ratio of photosynthesis to dark respiration were reduced by elevated temperature. Elevated concentrations of O(3) reduced the total concentration of soluble sugars, and tended to reduce LT(50) of the needles in September. These results show that alterations in needle chemical quality can be expected at elevated temperatures, but the seedlings' sensitivity to autumn and spring frosts is not altered. Elevated O(3) has the potential to disturb cold hardening of Norway spruce seedlings in autumn, and to alter the water balance of the seedling through changes in stomatal conductance (g(s)), while elevated temperature is likely to reduce g(s) and consequently reduce the O(3)-flux inside the leaves.

Summary 1. Many plant species have two inducible indirect defences against herbivores, emission of volatile organic compounds (VOCs) and secretion of extrafloral nectar (EFN). Moreover, herbivore-induced VOCs can serve as airborne signals in plant–plant communication, inducing and/or priming defences in undamaged neighbouring plants. However, it remains largely unknown, particularly in woody plants, whether herbivore-induced VOCs induce and/or prime these two indirect defences simultaneously. 2. We investigated the VOC-mediated induction and priming of VOC emission and EFN secretion in the woody plant hybrid aspen (Populus tremula × Populus tremuloides) in the context of plant–plant communication. Hybrid aspen saplings were exposed to VOCs released either from herbivore-infested or uninfested conspecifics and subsequently challenged by herbivores. VOCs and EFN from exposed plants were collected throughout experiments. Additionally, local and systemic EFN induction was investigated. 3. Exposure to VOCs from infested plants directly induced secretion of EFN sugars but not emission of volatile terpenes. Upon subsequent herbivore attack, however, plants previously exposed to herbivore-induced VOCs released significantly more terpenes than unexposed plants, whereas EFN secretion was not increased, indicating that herbivore-induced VOCs prime VOC emission but not EFN secretion. This switching in the indirect defence priority from induced EFN secretion to primed VOC emission may reflect the potential fitness disadvantages of VOC emission and EFN secretion on their own. In addition, feeding by Epirrita autumnata larvae reduced EFN sugar production both locally and systemically, which disagrees with previous findings and suggests that EFN secretion varies depending on plant and herbivore species. 4. Thereby, our work has parallels and divergences from the most significant previous studies. Nevertheless, it suggests that plants can respond differently to early warning signals and ensuing herbivory and in doing so fine-tune their different indirect defences timely to achieve optimal use of resources.

Tricholoma matsutake is an economically important ectomycorrhizal fungus of coniferous woodlands. Mycologists suspect that this fungus is also capable of saprotrophic feeding. In order to evaluate this hypothesis, enzyme and chemical assays were performed in the field and laboratory. From a natural population of T. matsutake in southern Finland, samples of soil-mycelium aggregate (shiro) were taken from sites of sporocarp formation and nearby control (PCR-negative) spots. Soil organic carbon and activity rates of hemicellulolytic enzymes were measured. The productivity of T. matsutake was related to the amount of utilizable organic carbon in the shiro, where the activity of xylosidase was significantly higher than in the control sample. In the laboratory, sterile pieces of bark from the roots of Scots pine were inoculated with T. matsutake and the activity rates of two hemicellulolytic enzymes (xylosidase and glucuronidase) were assayed. Furthermore, a liquid culture system showed how T. matsutake can utilize hemicellulose as its sole carbon source. Results linked and quantified the general relationship between enzymes secreted by T. matsutake and the degradation of hemicellulose. Our findings suggest that T. matsutake lives mainly as an ectomycorrhizal symbiont but can also feed as a saprotroph. A flexible trophic ecology confers T. matsutake with a clear advantage in a heterogeneous environment and during sporocarp formation.

For northern Europe’s climate, models predict an increase in periods of drought and waterlogging. Knowledge of variation between genotypes of Betula pendula (birch) and Populus tremula (aspen) to drought and excess soil moisture are unavailable but relevant for future development of forest ecosystems. We studied variation between genotypes to soil moisture in birch and aspen with plant material representing naturally regenerated populations and showed differences in acclimation to soil moisture conditions. Genotypes showing most growth and biomass accumulation across treatments maintained most leaf area, high gas exchange and water-use efficiency and grew most root mass but had the lowest root length per unit root dry mass compared to other genotypes. This indicates that these genotypes are more efficient in harvesting water from the soil under adverse conditions. We also showed that birch and aspen employ different strategies to cope with soil moisture conditions, with aspen investing more in perennial parts, while birch efficiently maintains foliar processes. When the expression of some known drought responsive genes was measured, only ACC oxidase was induced by the drought treatment at the beginning of the experiment, while surprisingly LEA5, RD22 and ADH1 did not respond to drought, but were up-regulated in prolonged wet conditions indicating oxidative stress and hypoxia and that these genes are responding to multiple stress factors. We conclude that in plants micro-propagated from naturally regenerated birch and aspen populations, there is variation between genotypes in acclimation efficiency to soil moisture conditions.

Stress tolerance is currently one of the major research topics in plant biology because of the challenges posed by changing climate and increasing demand to grow crop plants in marginal soils. Increased Zn tolerance and accumulation has been reported in tobacco expressing the glyoxalase 1-encoding gene from Brassica juncea. Previous studies in our laboratory showed some Zn tolerance-correlated differences in the levels of glyoxalase 1-like protein among accessions of Zn hyperaccumulator Thlaspi caerulescens. We have now isolated the corresponding gene (named here TcGLX1), including ca. 570 bp of core and proximal promoter region. The predicted protein contains three glyoxalase 1 motifs and several putative sites for post-translational modification. In silico analysis predicted a number of cis-acting elements related to stress. The expression of TcGLX1 was not responsive to Zn. There was no correlation between the levels of TcGLX1 expression and the degrees of Zn tolerance or accumulation among T. caerulescens accessions nor was there co-segregation of TcGLX1 expression with Zn tolerance or Zn accumulation among F3 lines derived from crosses between plants from accessions with contrasting phenotypes for these properties. No phenotype was observed in an A. thaliana T-DNA insertion line for the closest A. thaliana homolog of TcGLX1, ATGLX1. These results suggest that glyoxalase 1 or at least the particular isoform studied here is not a major determinant of Zn tolerance in the Zn hyperaccumulator plant T. caerulescens. In addition, ATGLX1 is not essential for normal Zn tolerance in the non-tolerant, non-accumulator plant A. thaliana. Possible explanations for the apparent discrepancy between this and previous studies are discussed. © 2011 Springer-Verlag.

Metallothioneins (MTs) are ubiquitous cysteine-rich proteins present in plants, animals, fungi and cyanobacteria. In plants, MTs are suggested to be involved in metal tolerance or homeostasis, as they are able to bind metal ions through the thiol groups of their cysteine residues. Recent reports show that MTs are also involved in the scavenging of reactive oxygen species (ROS). The interplay between these roles is not entirely clear. Plants have many MT isoforms with overlapping expression patterns, and no specific role for any of them has been assigned. This review is focused on recent findings on plant MTs.

This paper investigates differences in gene expression among the two Thlaspi caerulescens ecotypes La Calamine (LC) and Lellingen (LE) that have been shown to differ in metal tolerance and metal uptake. LC originates from a metalliferous soil and tolerates higher metal concentrations than LE which originates from a non-metalliferous soil. The two ecotypes were treated with different levels of zinc in solution culture, and differences in gene expression were assessed through application of a cDNA microarray consisting of 1,700 root and 2,700 shoot cDNAs. Hybridisation of root and shoot cDNA from the two ecotypes revealed a total of 257 differentially expressed genes. The regulation of selected genes was verified by quantitative reverse transcriptase polymerase chain reaction. Comparison of the expression profiles of the two ecotypes suggests that LC has a higher capacity to cope with reactive oxygen species and to avoid the formation of peroxynitrite. Furthermore, increased transcripts for the genes encoding for water channel proteins could explain the higher Zn tolerance of LC compared to LE. The higher Zn tolerance of LC was reflected by a lower expression of the genes involved in disease and defence mechanisms. The results of this study provide a valuable set of data that may help to improve our understanding of the mechanisms employed by plants to tolerate toxic concentrations of metal in the soil. © Springer-Verlag 2009.

Metal uptake and its effect on foliar metallothionein 2b (MT2b) mRNA levels were studied in hybrid aspen (Populus tremulaxtremuloides) in field conditions. The trees were planted on a site contaminated with several metals, including cadmium (mean 5.1 mgkg(-1)), chromium (80 mgkg(-1)), copper (180 mgkg(-1)), nickel (81 mgkg(-1)), vanadium (240 mgkg(-1)) and zinc (520 mgkg(-1)). Of the ten trace elements analyzed, only Cd and Zn accumulated in the leaves with maximal foliar concentrations of 35 and 2400 mgkg(-1) (dry weight), respectively. There was a strong correlation between Cd and Zn concentrations and bioaccumulation factors (concentration in plant/soil) in the leaves, branches and roots, suggesting similar transport mechanisms for these two metals. The levels of MT2b correlated with Cd and Zn concentrations in the leaves, demonstrating that increased MT2b expression is one of the responses of hybrid aspen to chronic metal exposure.

Two birch clones originating from metal-contaminated sites were exposed for 3 months to soils (sand-peat ratio 1:1 or 4:1) spiked with a mixture of polyaromatic hydrocarbons (PAHs; anthracene, fluoranthene, phenanthrene, pyrene). PAH degradation differed between the two birch clones and also by the soil type. The statistically most significant elimination (p < or = 0.01), i.e. 88% of total PAHs, was observed in the more sandy soil planted with birch, the clearest positive effect being found with Betula pubescens clone on phenanthrene. PAHs and soil composition had rather small effects on birch protein complement. Three proteins with clonal differences were identified: ferritin-like protein, auxin-induced protein and peroxidase. Differences in planted and non-planted soils were detected in bacterial communities by 16S rRNA T-RFLP, and the overall bacterial community structures were diverse. Even though both represent complex systems, trees and rhizoidal microbes in combination can provide interesting possibilities for bioremediation of PAH-polluted soils.

To study the role of metallothioneins (MTs) in Zn accumulation, the expression of TcMT2a, TcMT2b, and TcMT3 was analysed in three accessions and 15 F(3) families of two inter-accession crosses of the Cd/Zn hyperaccumulator Thlaspi caerulescens, with different degrees of Zn accumulation. The highest expression levels were found in the shoots of a superior metal-accumulating calamine accession from St Laurent le Minier, with >10-fold TcMT3 expression compared with another calamine accession and a non-metallicolous accession. Moreover, F(3) sibling lines from the inter-accession crosses that harboured the MT2a or MT3 allele from St Laurent le Minier had higher expression levels. However, there was no co-segregation of TcMT2a or TcMT3 expression and Zn accumulation. To examine the functions of TcMTs in plants, TcMT2a and TcMT3 were ectopically expressed in Arabidopsis. The transformant lines had reduced root length in control medium but not at high metal concentrations, suggesting that the ectopically expressed proteins interfered with the physiological availability of essential metals under limited supply. The Arabidopsis transformant lines did not show increased tolerance to Cd, Cu, or Zn, nor increased Cd or Zn accumulation. Immunohistochemical analysis indicated that in roots, MT2 protein is localized in the epidermis and root hairs of both T. caerulescens and Arabidopsis thaliana. The results suggest that TcMT2a, TcMT2b, and TcMT3 are not primarily involved in Zn accumulation as such. However, the elevated expression levels in the metallicolous accessions suggests that they do contribute to the metal-adapted phenotype, possibly through improving Cu homeostasis at high Zn and Cd body burdens. Alternatively, they might function as hypostatic enhancers of Zn or Cd tolerance.

Several populations with different metal tolerance, uptake and root-to-shoot transport are known for the metal hyperaccumulator plant Thlaspi caerulescens. In this study, genes differentially expressed under various Zn exposures were identified from the shoots of two T. caerulescens accessions (calaminous and non-calaminous) using fluorescent differential display RT-PCR. cDNA fragments from 16 Zn-responsive genes, including those encoding metallothionein (MT) type 2 and type 3, MRP-like transporter, pectin methylesterase (PME) and Ole e 1-like gene as well as several unknown genes, were eventually isolated. The full-length MT2 and MT3 sequences differ from those previously isolated from other Thlaspi accessions, possibly representing new alleles or isoforms. Besides the differential expression in Zn exposures, the gene expression was dependent on the accession. Thlaspi homologues of ClpP protease and MRP transporter were induced at high Zn concentrations. MT2 and PME were expressed at higher levels in the calaminous accession. The MTs and MRP transporter expressed in transgenic yeasts were capable of conferring Cu and Cd tolerance, whereas the Ole e 1-like gene enhanced toxicity to these metals. The MTs increased yeast intracellular Cd content. As no significant differences were found between Arabidopsis and Thlaspi MTs, they apparently do not differ in their capacity to bind metals. However, the higher levels of MT2 in the calaminous accession may contribute to the Zn-adapted phenotype. © 2006 Springer-Verlag.

PR-10c is a unique member of PR-10 proteins in birch, since it is the only one known to be post-translationally modified by glutathione and is not constitutively expressed in pollen. Both reduced and S-glutathiolated forms of PR-10c show low ribonuclease activity. However, the major function of the protein is apparently not yet resolved. Our protein-ligand interaction studies with saturation transfer difference (STD) NMR revealed that PR-10c interacts with several biologically important molecules, including cytokinin, flavonoid glycosides, sterols and emodin. Competition study with deoxycholate and kinetin revealed no statistically significant binding interference, indicating that these ligands have different binding sites in PR-10c. Ligand docking studies with a molecular model of PR-10c support the STD NMR results of ligand binding and binding epitopes, suggesting that there are three potential binding sites in PR-10c: two in the hydrophobic cavity and one in the glycine-rich loop. Our docking calculations suggested that only kinetin interacts with the glycine-rich loop, the binding occurring through its adenine moiety. Clear ligand specificity could be observed in the binding of nucleotide derivatives. S-glutathiolation of PR-10c did not affect kinetin binding. The present results suggest that birch PR-10c is a multifunctional protein, which has diverse roles in plant stress responses.

The PR-10c (previously termed as Bet v 1-Sc3) protein of birch belongs to the family of intracellular pathogenesis-related proteins. The high-performance liquid chromatography electrospray ionization ion trap mass spectrometry (HPLC-ESI-MS) analysis of PR-10c-His fusion protein, produced in Escherichia coli, revealed three major peaks and masses. Enzymatic digestions and HPLC-ESI-MS and matrix assisted laser desorption/ionization - time of flight mass spectrometry (MALDI-TOF-MS) analyses of each fraction indicated that PR-10c-His protein is post-translationally modified by carbamylation and S-glutathiolation. Carbamylation was localized into the N-terminal end of PR-10c-His and does not represent a biologically significant modification. The possible nuclease activity of PR-10c was analysed with S-glutathiolated and reduced fractions of PR-10c-His fusion protein. Both forms of PR-10c-His as well as the dimeric form of the protein possess RNase activity which is capable of digesting different RNA substrates. None of the fractions showed activity against single- or double-stranded DNA. The MALDI-TOF-MS analysis of PR-10c polypeptide extracted from zinc-exposed birch roots showed that the protein is post-translationally modified by glutathione (gamma-Glu-Cys-Gly) also in vivo. The S-glutathiolated cysteine residue of PR-10c is not conserved among Bet v 1 homologous proteins and is also unique in the PR-10 family. As far as we know this is the first observation of S-glutathiolation in plants, or any post-translational modification in the PR-10 family of proteins.

The metal hyperaccumulator Noccaea caerulescens is an established model to study the adaptation of plants to metalliferous soils. Various comparators have been used in these studies. The choice of suitable comparators is important and depends on the hypothesis to be tested and methods to be used. In high-throughput analyses such as microarray, N. caerulescens has been compared to non-tolerant, non-accumulator plants like Arabidopsis thaliana or Thlaspi arvense rather than to the related hypertolerant or hyperaccumulator plants. An underutilized source is N. caerulescens populations with considerable variation in their capacity to accumulate and tolerate metals. Whole transcriptome sequencing (RNA-Seq) is revealing interesting variation in their gene expression profiles. Combining physiological characteristics of N. caerulescens accessions with their RNA-Seq has a great potential to provide detailed insight into the underlying molecular mechanisms, including entirely new gene products. In this review we will critically consider comparative transcriptome analyses carried out to explore metal hyperaccumulation and hypertolerance of N. caerulescens, and demonstrate the potential of RNA-Seq analysis as a tool in evolutionary genomics.

Commercial inactivated parenteral influenza vaccines reduced febrile (or = 38 degrees C) respiratory illness by 53% (95% CL: 41-63%) during a 3 week outbreak in 1998 when A/Sydney/5/97(H3N2)-like influenza viruses were shown to be the predominant etiological agents and an older antigenic variant, A/Nanchang/933/95, served as the vaccine virus. The calculatory efficacy for preventing virologically diagnosed influenza infections was 57% (95% CL: 40-68%). The study population consisted of 1374 young male military conscripts. Vaccination coverage on a voluntary basis was 67%. Vaccination was ineffective in preventing febrile illness during a second epidemic wave lasting 2 weeks when mainly adenoviruses were shown to have been circulating in the garrison. Out of the 36 nasopharyngeal aspirates positive for influenza A by antigen detection, 18 A/Sydney/5/97-like strains (10 from non-vaccinated and eight from vaccinated subjects) and two A/Nanchang/933/95-like strains (both from non-vaccinated subjects) were isolated in MDCK cell cultures. Intraepidemic variation was detected among the A/Sydney/5/97-like field strains in their HA1 sequences and reactivity in HI tests, but no evidence was obtained that this variation would have been of significance to the virus in breaking through the vaccination-induced immunity.

Silene vulgaris (Moench) Garcke has evolved populations with extremely high levels of copper tolerance. To evaluate the role of metallothioneins (MTs) in copper tolerance in S. vulgaris, we screened a cDNA library derived from a highly copper-tolerant population using Arabidopsis-based MT probes and identified an MT2b-like gene. When expressed in yeast, this gene,SvMT2b, restored cadmium and copper tolerance in different hypersensitive strains. Northern-blot analysis and quantitative reverse transcriptase-PCR showed that plants from the copper-tolerant S. vulgaris populations had significantly higher transcript levels of SvMT2b than plants from the copper-sensitive populations, both in roots and shoots and with and without copper exposure. Southern-blot analysis suggested that the higher expression of the latter allele was caused by gene amplification. Segregating families of crosses between copper-sensitive and copper-tolerant plants exhibited a 1 to 3 segregation forSvMT2b expression. Allele-specific PCR showed that low-expression F3 plants were homozygous for the allele inherited from the copper-sensitive parent, whereas high-expression plants possessed at least one allele from the tolerant parent.SvMT2b expression did not cosegregate with copper tolerance in crosses between sensitive and tolerant plants. However, a significant cosegregation with copper tolerance did occur in families derived from crosses between moderately tolerant F3 plants with different SvMT2b genotypes. Thus, overexpression ofSvMT2b conferred copper tolerance although only within the genetic background of a copper tolerant plant.

Metal concentrations in soils are locally quite high, and are still increasing due to many human activities, leading to elevated risk for health and the environment. Phytoremediation may offer a viable solution to this problem, and the approach is gaining increasing interest. Improvement of plants by genetic engineering, i.e. by modifying characteristics like metal uptake, transport and accumulation as well as metal tolerance, opens up new possibilities for phytoremediation. So far, only a few cases have been reported where one or more of these characteristics have been successfully altered; e.g. mercuric ion reduction causing improved resistance and phytoextraction, and metallothionein causing enhanced cadmium tolerance. These, together with other approaches and potentially promising genes for transformation of target plants are discussed. Copyright (C) 2000 Elsevier Science Ltd.

Populations of Noccaea caerulescens show tremendous differences in their capacity to hyperaccumulate and hypertolerate metals. To explore the differences that could contribute to these traits, we undertook SOLiD high-throughput sequencing of the root transcriptomes of three phenotypically well-characterized N. caerulescens accessions, i.e., Ganges, La Calamine, and Monte Prinzera. Genes with possible contribution to zinc, cadmium, and nickel hyperaccumulation and hypertolerance were predicted. The most significant differences between the accessions were related to metal ion (di-, trivalent inorganic cation) transmembrane transporter activity, iron and calcium ion binding, (inorganic) anion transmembrane transporter activity, and antioxidant activity. Analysis of correlation between the expression profile of each gene and the metal-related characteristics of the accessions disclosed both previously characterized (HMA4, HMA3) and new candidate genes (e.g., for nickel IRT1, ZIP10, and PDF2.3) as possible contributors to the hyperaccumulation/tolerance phenotype. A number of unknown Noccaea-specific transcripts also showed correlation with Zn(2+), Cd(2+), or Ni(2+) hyperaccumulation/tolerance. This study shows that N. caerulescens populations have evolved great diversity in the expression of metal-related genes, facilitating adaptation to various metalliferous soils. The information will be helpful in the development of improved plants for metal phytoremediation.

The effects of slightly elevated temperature (+0.8 ° C), ozone (O3) concentration (1.3 × ambient O3 concentration) and their combination on over-wintering buds of Betula pendula Roth were studied after two growing seasons of exposure in the field. Carbohydrate concentrations, freezing stress resistance (FSR), bud dry weight to fresh weight ratio, and transcript levels of cytochrome oxidase (COX), alternative oxidase (AOX) and dehydrin (LTI36) genes were studied in two clones (clones 12 and 25) in December. Elevated temperature increased the bud dry weight to fresh weight ratio and the ratio of raffinose family oligosaccharides to sucrose and the transcript levels of the dehydrin (LTI36) gene (in clone 12 only), but did not alter the FSR of the buds. Genotype-specific alterations in carbohydrate metabolism were found in the buds grown under elevated O 3. The treatments did not significantly affect the transcript level of the COX or AOX genes. No clear pattern of an interactive effect between elevated temperature and O3 concentration was found. According to these data, the increase in autumnal temperatures and slightly increasing O3 concentrations do not increase the risk for freeze-induced damage in winter in silver birch buds, although some alterations in bud physiology occur.

Raman spectroscopy was used to discriminate between Legionella strains and between E. coli and coliform strains. The relationship between triplicate Raman spectra derived from Legionella bacteria was compared with that derived from a blind set of samples and amplified fragment length polymorphism (AFLP) data from the same strains. Triplicate Raman spectra of E. coli and coliform bacteria were compared with their 16S phylogeny. In all cases Raman spectra were reproducible and could be distinguished from spectra of other organisms down to the strain level. All samples in a blind fourth set were identified correctly. Raman spectra of organisms of the same coliform species clustered according to 16S rRNA gene phylogeny, except for Enterobacter spp. At higher taxonomic levels the relationship between species was less comparable. For Legionella strains the Raman spectra grouped according to AFLP groups, based on the dataset used in this study. Raman spectroscopy could correctly distinguish E. coli from other coliform bacteria and L. pneumophila from non-pneumophila strains. Incubation of Legionella strains in different types of drinking water at different temperatures over a period of one week introduced so little variation in the Raman spectra that only very closely related L. pneumophila strains could not be distinguished from each other. Temperature, ageing and water type did not influence the identification potency of Raman spectroscopy in all cases. Given the accuracy, speed and simplicity of the Raman spectroscopy technique this method seems a welcome addition to the current tools for identification of waterborne bacteria.

We addressed how restoration of forestry-drained peatlands affects CH 4 -cycling microbes. Despite similar community compositions, the abundance of methanogens and methanotrophs was lower in restored than in natural sites and correlated with CH 4 emission. Poor establishment of methanogens may thus explain low CH 4 emissions on restored peatlands even 10 to 12 years after restoration.

Human papillomaviruses have been implicated in the pathogenesis of a variety of malignancies, particularly those of the anogenital tract. Some recent reports on the presence of human papillomavirus in bladder cancer have raised the possibility that it might be involved in the development of this malignancy as well. To study this concept, a series of 108 transitional cell carcinomas of the bladder were screened for the presence of human papillomavirus deoxyribonucleic acid (DNA) by in situ hybridization with biotin-labeled human papillomavirus cocktail probe and polymerase chain reaction with human papillomavirus L1 consensus primers. Although the positive controls showed strong hybridization signals, no evidence for human papillomavirus DNA was found in any of the bladder carcinomas by in situ hybridization. Similarly, despite the amplification of a 450 bp product in cervical human papillomavirus lesions (used as positive controls), no signals were obtained in any of the bladder tumors studied. beta-globin gene sequences (110 bp), serving as internal controls, were consistently amplified from all tumor samples, suggesting that cellular DNAs from the carcinoma specimens were sufficient for the amplification reaction. These data indicate that human papillomavirus infection is rare in transitional cell carcinoma of the bladder. The significance of these findings is discussed in relation to previous reports on human papillomavirus involvement in bladder carcinomas.

Epidemiological evidence suggests that alcohol intake, use of tobacco, ingestion of mycotoxins and nitrosamines and nutritional deficiencies are high-risk factors for the development of oesophageal cancer. Similarly, viral infections have been postulated to play a role in some tumours. However, the molecular events underlying the development of oesophageal carcinoma are poorly understood as yet. Loss of p53 tumour-suppressor gene function has been found in different human malignancies, and it can occur in a variety of ways, including gene mutation and interaction with the E6 protein of oncogenic human papillomaviruses (HPVs). Because the oesophageal mucosa is potentially exposed to mutagens and HPVs, we studied DNA samples derived from nine HPV-positive squamous cell carcinomas and 12 HPV-negative tumours. Exons 5-9 of the p53 gene containing phylogenetically conserved domains were examined using the polymerase chain reaction-single-strand conformation polymorphism (PCR-SSCP) technique. HPV detection was done using DNA in situ hybridisation with biotin-labelled HPV DNA probes. Mutations were detected in eight (38%) out of the 21 cases. Three mutations were found in exons 5/6, three in exon 7 and two in exon 8/9. Six (50%) of the 12 HPV-negative carcinomas showed p53 mutations. Two (22.2%) of the nine HPV-positive carcinomas were found to contain p53 mutations as well; one contained HPV 16 DNA sequences and showed p53 mutation in exon 8/9, and the other was HPV 6/11 positive with the mutation in exon 5/6. Although mutations were more common in HPV-negative tumours (50.0% vs 22.2%), the difference in p53 mutations in HPV-positive and -negative tumours did not reach statistical significance (P = 0.1946). These data indicate that inactivation of the p53 gene is a frequent event in oesophageal squamous cell carcinomas and such an inactivation might be an important molecular pathway for the development of oesophageal cancer. The findings of p53 mutations in HPV-positive oesophageal carcinomas suggest that HPV and p53 mutation were not mutually exclusive events. The presence of frequent mutations of p53 gene in both HPV-positive and -negative oesophageal carcinomas suggests a dominant role of environmental carcinogens in oesophageal carcinogenesis. Images Figure 1

Volatile organic compounds (VOCs) are expected to have an important role in plant adaptation to high temperatures. The impacts of increasing night-time temperature on daytime terpenoid emissions and related gene expression in silver birch (Betula pendula) and European aspen (Populus tremula) clones were studied. The plants were grown under five different night-time temperatures (6, 10, 14, 18, and 22 degrees C) while daytime temperature was kept at a constant 22 degrees C. VOC emissions were collected during the daytime and analysed by gas chromatography-mass spectrometry (GC-MS). In birch, emissions per leaf area of the C11 homoterpene 4,8-dimethy1-nona-1,3,7-triene (DMNT) and several sesquiterpenes were consistently increased with increasing night-time temperature. Total sesquiterpene (SQT) emissions showed an increase at higher temperatures. In aspen, emissions of DMNT and beta-ocimene increased from 6 degrees C to 14 degrees C, while several other monoterpenes and the SQTs (Z,E)-alpha-farnesene and (E,E)-alpha-farnesene increased up to 18 degrees C. Total monoterpene and sesquiterpene emission peaked at 18 degrees C, whereas isoprene emissions decreased at 22 degrees C. Leaf area increased across the temperature range of 6-22 degrees C by 32% in birch and by 59% in aspen. Specific leaf area (SLA) was also increased in both species. The genetic regulation of VOC emissions seems to be very complex, as indicated by several inverse relationships between emission profiles and expression of several regulatory genes (DXR, DXS, and IPP). The study indicates that increasing night temperature may strongly affect the quantity and quality of daytime VOC emissions of northern deciduous trees.

Metal hyperaccumulator plants have previously been characterized by transcriptomics, but reports on other profiling techniques are scarce. Protein profiles of Thlaspi caerulescens accessions La Calamine (LC) and Lellingen (LE) and lines derived from an LCxLE cross were examined here to determine the co-segregation of protein expression with the level of zinc (Zn) hyperaccumulation. Although hydrophobic proteins such as membrane transporters are not disclosed, this approach has the potential to reveal other proteins important for the Zn hyperaccumulation trait. Plants were exposed to metals. Proteins were separated using two-dimensional electrophoresis and those showing differences among accessions, lines or metal exposures were subjected to mass-spectrometric analysis for identification. Crossing decreased the number of different proteins in the lines compared with the parents, more so in the shoots than in the roots, but the frequencies of Zn-responsive proteins were about the same in the accessions and the selection lines. This supports the finding that the Zn accumulation traits are mainly determined by the root and that Zn accumulation itself is not the reason for the co-segregation. This study demonstrates that crossing accessions with contrasting Zn accumulation traits is a potent tool to investigate the mechanisms behind metal hyperaccumulation. Four tentatively identified root proteins showed co-segregation with high or low Zn accumulation: manganese superoxide dismutase, glutathione S-transferase, S-formyl glutathione hydrolase, and translation elongation factor 5A-2. However, these proteins may not be the direct determinants of Zn accumulation. The role of these and other tentatively identified proteins in Zn accumulation and tolerance is discussed.

The leaf proteome of 3-week-old Arabidopsis thaliana seedlings exposed for 1 week to low, environmentally realistic Cd concentrations was investigated. The data indicated that at 1 μM Cd, A. thaliana plants adapted their metabolism to cope with the Cd exposure. As a result, only moderate protein changes were observed. However, at 10 μM Cd, severe stress was indicated by growth reduction and chlorosis of rosette leaves at the macroscopic level and by lipid peroxidation and enhanced peroxidase activity at the cellular level. Of the 730 reproducible proteins among all gels, 21 were statistically upregulated in response to Cd. These proteins can be functionally grouped into 5 classes: proteins involved in (1) oxidative stress response, (2) photosynthesis and energy production, (3) protein metabolism, (4) gene expression and finally, (5) proteins with various or unknown function. In order to provide greater insight into the mechanisms induced on Cd exposure, a working model is proposed.

A simple method of processing formalin-fixed, paraffin-embedded tissue sections for DNA amplification by polymerase chain reaction (PCR) is described. In this procedure, deparaffinized sections are readily subjected to DNA isolation simply by boiling and the released DNA can be directly employed for PCR. The method allows analysis of single-copy genes or viral sequences at least up to 300 base pairs long in one working day. This method is particularly useful in analysing retrospective materials when the simplicity and low cost of the assay are preferable. Furthermore, the simplicity of the procedure reduces the risk of contamination.

Suppression subtractive hybridization (SSH) was used to isolate genes differentially expressed following exposure to copper (Cu) in a naturally selected Cu-tolerant birch (Betula pendula Roth.) clone originating from a disused lead/zinc smelter. Of the 352 cDNA fragments initially isolated, 108 were up-regulated by Cu, of which 55 showed over twofold induction by macroarray analysis. Searches against protein databases (Blastx) and sequence analysis provided the tentative identity of 21 genes. Three fragments lacked homology to any sequences in the databases. Most of the identified genes are involved in cellular transport, regulation or cell rescue and defense. Several genes have not previously been reported to be up-regulated by Cu, e.g., plasma intrinsic protein 2, glutamine synthetase and multi-drug resistance-associated protein (MRP4). The expression of MRP4, a vacuolar sorting receptor-like protein and an unidentified gene was studied in more detail by quantitative real-time PCR. These genes showed stronger up-regulation by Cu in the roots and shoots of the Cu-tolerant birch clone compared with a less tolerant clone. Clear clonal differences in gene expression were observed, e.g., for the regulator of chromosome condensation family protein, DnaJ protein homolog, vacuolar sorting receptor-like protein and MRP4. These findings contribute to our understanding of Cu tolerance in birch, a pioneer plant in metal-contaminated soils.

Thlaspi caerulescens is increasingly acknowledged as one of the best models for studying metal hyperaccumulation in plants. In order to study the mechanisms underlying metal hyper-accumulation, we used proteomic profiling to identify differences in protein intensities among three T caerulescens accessions with pronounced differences in tolerance, uptake and root to shoot translocation of Zn and Cd. Proteins were separated using two-dimensional electrophoresis and stained with SYPRO Orange. Intensity values and quality scores were obtained for each spot by using PDQuest software. Principal component analysis was used to test the separation of the protein profiles of the three plant accessions at various metal exposures, and to detect groups of proteins responsible for the differences. Spot sets representing individual proteins were analysed with the analysis of variance and non-parametric Kruskal-Wallis test. Clearest differences were seen among the Thlaspi accessions, while the effects of metal exposures were less pronounced. The 48 tentatively identified spots represent core metabolic functions (e.g. photosynthesis, nitrogen assimilation, carbohydrate metabolism) as well as putative signalling and regulatory functions. The possible roles of some of the proteins in heavy metal accumulation and tolerance are discussed. © 2006 Wiley-VCH Verlag GmbH & Co. KGaA.

Roughly half (54%) of the 910 young conscripts at a garrison in Finland were vaccinated with commercial influenza vaccines in autumn 1998. During the influenza outbreak in February 1999, 12 H3N2-subtype virus strains were isolated from vaccinated patients, and 11 such strains were isolated from unvaccinated patients. The isolates were related to the vaccine strain A/Sydney/5/97 and could be classified into three subgroups based on sequence variation in the HA1 gene coding for the variable domain of viral haemagglutinin (HA). A total of 6–10 amino-acid substitutions in HA1, three of these in the receptor-binding site, differentiated the field strains from the vaccine virus. In haemagglutination inhibition (HI) tests, eight strains from the study population exhibited reduced reactivity with a variety of antisera including human post-vaccination sera. Six of these strains were isolated from vaccinated and two from unvaccinated patients. The reduced reactivity did not correlate with particular amino-acid changes in HA1. We suggest that low-reactivity viruses may have an advantage over other co-circulating variants under some circumstances characterized by enhanced immunity-mediated selection and high infection pressure. Whether the frequency of these viruses increased in our vaccinated study population cannot be determined, nor can their effect on vaccine efficacy.

Terrestrial metal-toxic soils are found all over the world. They originate either from geological anomalies (e.g., ore outcrops, serpentines), or from human activities (e.g., mining, smelting, agricultural use of metal containing fertilizers, pesticides, sludges, etc.). Even extremely metal-toxic soils eventually become vegetated, though often sparsely and without trees. These vegetations are usually poor in species, particularly in parts of the world that have been glaciated during the Pleistocene (Brooks et al., 1985), most probably mainly due to a strong selective effect of metal-toxicity as such (Schat and Verkleij, 1998).

In HPV-associated genital lesions, low or absent expression of p53 has been attributed to the rapid degradation of p53 through its binding with HPV E6 protein. In this study, we examined p53 protein expression with two antibodies (CM1 polyclonal and PAb 1801 monoclonal antibodies), and Ki-67 proliferation antigen (monoclonal antibody) using an immunohistochemical (IHC) double-staining technique in 77 HPV-positive cervical lesions (HPV6, HPV11, HPV16, HPV18, HPV31, and HPV33) and in 15 HPV-negative cases. p53 protein expression was detected in 36/92 (39.1%) of the specimens. of the p53-positive cases, 80.6% (29/36) were HPV-positive samples, including 10/23 (43.5%) of HPV16- and 3/10 (30%) of HPV18-positive biopsies. In 52.8% of the p53-positive samples, the expression was found in less than 5% of the basal cells which were also positive for Ki-67. Ki-67 proliferation marker was found in 91/92 specimens, most intensely in those infected by HPV16. p53 was more abundant in progressive or persistent lesions, but no differences were found between HPV-positive and HPV-negative samples. the positive IHC double-staining of both p53 and Ki-67 proliferation antigen in the same basal (and parabasal) cells indicates that these two normal cell-cycle proteins are being expressed while the cells are entering from the G1 to the S phase of the cell cycle. Since the latter property is only attributed to the wild-type p53 (but not to mutated p53), the p53 protein detected in HPV lesions by IHC is likely to be the wild-type p53 rather than mutated p53, and the result was also confirmed by using p53 mutant specific antibody PAb 240. Accordingly, the concept of HPV inactivating the wild-type p53 protein should be re-examined, and other mechanisms for HPV-mediated carcinogenesis should be considered. L'expression faible ou nulle de p53 dans les lesions genitales associees au virus HPV a ete attribuee a la degradation rapide de p53 due a la liaison avec la proteine E6 du virus HPV. Dans cette etude, nous avons examine l'expression de la proteine p53 avec 2 anticorps (CM1 polyclonal-et PAb 1801 monoclonal), ainsi que la reactivite vis-a-vis de l'antig`ne de proliferation Ki-67 (anticorps monoclonal) grace a une technique immunohistochimique de double marquage dans 77 lesions cervicales positives pour HPV (HPV6, HPVII, HPV16, HPV18, HPV31, et HPV33) et dans 15 cas HPV negatifs. L'expression de la proteine p53 a ete decelee dans 36 cas sur 92 (39.1%). Parmi les cas p53-positifs, 80.6% (29/36) etaient HPV-positifs. incluant 10/23 (43.5%) de biopsies positives pour HPV16 et 3/10 (30%) pour HPV18. Dans 52.9% des cas p53-positifis, l'expression de la proteine est visible dans moins de 5% des cellules basales qui etaient aussi positives pour Ki-67. L'antigene de proliferation Ki-67 a ete detect6 dans 91 cas sur 92, avec une intensite plus forte dans les cas infectes par HPV16. La proteine p53 est plus fortement detectee dans les lesions persistantes ou progressives, mais aucune difference n'est observee entre les cas HPV-positifs et HPV-negatifs. Le double marquage positif pour p53 et pour I'antigene de proliferation Ki-67 dans les mZmes cellules basales (et parabasales) indiquent que ces deux proteines normales du cycle cellulaire sont exprimees lorsque les cellules passent de la phase G1, a la phase S du cycle cellulaire. Comme cette propriete n'est l'apanage que de la forme sauvage de p53 (mais pas de p53 mutee), la proteine p53 detectee dans les lesions a HPV par immunohistochimie est plus probablement de type sauvage que de type mute, ce resultat etant egalement confirme en utilisant I'anticorps PAb 240 specifique de p53 mutee. En consequence, la notion que la prothe p53 sauvage est inactivee par HPV doit etre reexaminee, et d'autres mecanismes de la carcinogenese like a HPV doivent etre reexaminee, et d'autres mecanismes de la carcinogenese liee a HPV doivent etre envisages. Der schwache oder fehlende Nachweis von p53 in HPV bedingten Lasionen wird durch einen raschen Abbau von p53 mittels Bindung an HPV E6-Protein erkart. In der vorliegenden Untersuchung wurde das p53-Protein mit 2 Antikorpern (CMI Polyklonal und PAb 1801 monoklonal) und Ki-67 (monoklonal) mittels einer immunhistochemischen Doppelfarbung untersucht. Das Material bestand aus 77 HPV-positiven Veranderungen (HPV6, HPVl1, HPV16, HPV18, HPV31 und HPV331 und aus 15 negativen Fallen. Eine p53-Expression wurde in 36/92 (39.1%) der Falle nachgewiesen, 80.6% (29/36) davon waren HPV-positiv, wobei es sich in 10/23 (43.5%) um HPV16 und in 3/10 (30%) um HPV18-positive Proben handelte. In 52.8% der positiven Proben waren weniger als 5% der Basalzellen positiv fur Ki-67. Dieses Proliferationsasntigen war in 91/92 Proben nachweisbar, am starksten jedoch in HPVl6 infizierten Fallen. p53 war besonders reichlich bei progressiven oder persistierenden Lasionen. Unterschiede zwischen HPV-positiv und HPV-negativ konnten jedoch nicht festgestellt werden. Eine doppelte Positivitat in denselben Basal-oder Parabasalzellen belegt, das diese Proteine des normalen Zellzyklus beim Ubergang von G1 zur S-Phase exprimiert werden. Da dies jedoch nur fur den Wildtyp von p53 nicht jedoch fur die Mutationen gilt, durfte es sich in der Regel um diesen handeln, was durch den Einsatz des p53-mutanten Antikorpers PAb 240 bestatigt wird. Demzulfolge sollte die Vorstellung uber die HPV-Inaktivierung von p53 und die carcinogene Wirkung uberdacht werden.

Summary: The expression of epidermal growth factor receptor (EGF-R), c-erbB-2 proto-oncogene, and estrogen receptor (ER) was studied immunohistochemically in a series of 97 human papillomavirus (HPV) lesions of the uterine cervix, with special emphasis on their association with the HPV type, grade of intraepithelial neoplasia (CIN), and the natural history of the disease. EGF-R expression was found in 95 of 97 (98%) specimens, mainly in basal and parabasal cells. Diffuse nuclear and cytoplasmic staining was detected in 36 of 97 (37%) samples, of which 29 were HPV positive. This staining pattern was most prominent in HPV 18–positive and in CIN lesions. Weak or moderate c-erbB-2 expression was found in 26 of 97 (27%) specimens. Estrogen receptor expression was observed in 28 of 77 (36%) samples, epithelial staining was seen in 11 of 77 (14%), and stromal staining occurred in 24 of 77 (31%) specimens. No clear-cut associations were established between the EGF-R, c-erbB-2, or ER expression and HPV type, nor in CIN or the clinical course of HPV infections. This failure for EGF-R, c-erbB-2, and ER to be associated with the specific HPV types, grade of CIN, or the clinical course of cervical HPV lesions suggests that the assessment of these factors is of limited value in explaining the development of HPV-associated CIN and in predicting the prognosis of this disease.

The p53 gene is contained within 16-20 kb of cellular DNA located on the short arm of human chromosome 17 at position 17p13.1. This gene encodes a 393-amino-acid nuclear phosphoprotein involved in the regulation of cell proliferation. Current evidence suggests that loss of normal p53 function is associated with cell transformation in vitro and development of neoplasms in vivo. More than 50% of human malignancies of epithelial, mesenchymal, haematopoietic, lymphoid, and central nervous system origin analysed thus far, were shown to contain an altered p53 gene. The oncoproteins derived from several tumour viruses, including the SV40 large T antigen, the adenovirus E1B protein and papillomavirus E6 protein, as well as specific cellular gene products, e.g. murine double minute-2 (MDM2), were found to bind to the wild-type p53 protein and presumably lead to inactivation of this gene product. Therefore, the inactivation of p53 tumour suppressor gene is currently regarded as an almost universal step in the development of human cancers. The current data on p53-associated tumourigenesis are briefly discussed in this minireview.

In a 30-year follow-up survey of the Finnish cohorts of the Seven Countries Study in 1989, 470 men aged 70-89 years were examined in two rural areas of Finland, in the East and in the West. Life-style-related coronary heart disease risk factors were at high levels in both groups, but the difference between areas found in the same cohort in middle age had mostly disappeared or partially reversed. Mean levels of systolic/diastolic blood pressure, body mass index, fasting blood glucose, blood glucose after 2-hour oral glucose tolerance test, and plasma fibrinogen were higher in the West than in the East (p < or = 0.05), but serum thiocyanate which reflects smoking habits was higher in the East (p < 0.001). No significant differences between areas were found in mean levels of serum total cholesterol, LDL-cholesterol, HDL-cholesterol, triglycerides, apolipoprotein A-I, apolipoprotein B, lipoprotein (a), plasma coagulation factor VIIc, and prevalence of current smoking.