Hybridization and genetic variation in Danish populations of European crab apple (Malus sylvestris)

The aim of the present study was to investigate the genetic variation in Danish populations of the endangered European crab apple (Malus sylvestris). Special emphasis was given to hybridization between the wild species and its cultivated relative Malus ×domestica. A total of 178 wild individuals from four Danish populations were studied along with a reference sample of 29 old cultivars. The genetic variation within and among samples was studied at ten microsatellite marker loci. Additionally, a morphological analysis was carried out to identify hybrids and test for correspondence between phenotypic and genotypic indices of hybridization. From application of ordination and a model-based cluster analysis to the molecular data, two clusters were identified consisting of wild and cultivated individuals, respectively. This indicates that pronounced admixture between the two species is not present. At the population level, a high correspondence was found between geographic isolation from M. ×domestica and genotypic and morphological indices of hybridization. As expected, isolated populations appeared less affected by hybridization than poorly isolated populations. Isolated “pure” M. sylvestris populations could thus be identified. However, morphological and molecular evidences of hybridization were found to be divergent at the individual level. This is suggestive of some historical introgression into the M. sylvestris gene pool and indicates that relying exclusively on either morphological or molecular characters as diagnostic markers in studies of hybridization between M. ×domestica and M. sylvestris might lead to fallible results. Combined application of genetic and morphological markers is therefore recommended.     

Identification of candidate substrates for ectodomain shedding by the metalloprotease-disintegrin ADAM8

ADAM proteases are type I transmembrane proteins with extracellular metalloprotease domains. As for most ADAM family members, ADAM8 (CD156a, MS2) is involved in ectodomain shedding of membrane proteins and is linked to inflammation and neurodegeneration. To identify potential substrates released under these pathologic conditions, we screened 10-mer peptides representing amino acid sequences from extracellular domains of various membrane proteins using the ProteaseSpot system. A soluble ADAM8 protease containing a pro- and metalloprotease domain was expressed in E. coli and purified as active protease owing to autocatalytic prodomain removal. From 34 peptides tested in the peptide cleavage assay, significant cleavage by soluble ADAM8 was observed for 14 peptides representing membrane proteins with functions in inflammation and neurodegeneration, among them the beta-amyloid precursor protein (APP). The in vivo relevance of the ProteaseSpot method was confirmed by cleavage of full-length APP with ADAM8 in human embryonic kidney 293 cells expressing tagged APP. ADAM8 cleaved APP with similar efficiency as ADAM10, whereas the inactive ADAM8 mutant did not. Exchanging amino acids at defined positions in the cleavage sequence of myelin basic protein (MBP) revealed sequence criteria for ADAM8 cleavage. Taken together, the results allowed us to identify novel candidate substrates that could be cleaved by ADAM8 in vivo under pathologic conditions.     

Phosphorus Translocation by Red Deer on a Subalpine Grassland in the Central European Alps

We examined the role of red deer (Cervus elaphus L.) in translocating phosphorus (P) from their preferred grazing sites (short-grass vegetation on subalpine grasslands) to their wider home range in a subalpine grassland ecosystem in the Central European Alps. Phosphorus was used because it is the limiting nutrient in these grasslands. When we compared P removal of aboveground biomass due to grazing with P input due to the deposit of feces on a grid of 268 cells (20 m × 20 m) covering the entire grassland, we detected distinct spatial patterns: the proportion of heavily grazed short-grass vegetation increased with increasing soil-P pool, suggesting that red deer preferably grazed on grid cells with a higher soil-P pool. Biomass consumption related to increased proportion of short-grass vegetation, and therefore P removal, increased with increasing soil-P pool. However, within the two vegetation types (short-grass and tall-grass), consumption was independent from soil-P pool. In addition, P input rates from defecation increased with increasing soil-P pool, resulting in a constant mean net P loss of 0.083 kg ha⁻¹ y⁻¹ (0.03%–0.07% of soil-P pool) independent of both soil-P pool and vegetation type. Thus, there was no P translocation between grid cells with different soil-P pools or between short-grass and tall-grass vegetation. Based on these results, it is likely that the net rate of P loss is too small to explain the observed changes in vegetation composition from tall-herb/meadow communities to short-grass and from tall-grass to short-grass on the grassland since 1917. Instead, we suggest that the grazing patterns of red deer directly induced succession from tall-herb/meadow communities to short-grass vegetation. Yet, it is also possible that long-term net soil-P losses indirectly drive plant succession from short-grass to tall-grass vegetation, because nutrient depletion could reduce grazing pressure in short-grass vegetation and enable the characteristic tall-grass species Carex sempervirens Vill. to establish.     

A Novel Disease Affecting the Predatory Mite Phytoseiulus persimilis (Acari, Phytoseiidae): 2. Disease Transmission by Adult Females

Adult female Phytoseiulus persimilis Athias-Henriot (Acari, Phytoseiidae) of one of our laboratory populations (=NR-population), show the following set of symptoms: predators shrink several days after mating, cease egg production and die several days after shrinking, show a lower degree of attraction to herbivore-induced plant volatiles and a shorter choice time in olfactometer tests, have the tendency to leave a prey patch with ample food, may carry excretory crystals in the legs, may cease prey consumption, and have a lower excretion rate. We hypothesized earlier that this characteristic syndrome, called non-responding (=NR-) syndrome, is caused by a pathogen infecting P. persimilis. To further support this hypothesis we here study several transmission modes of the factor causing the NR-syndrome. In all tests we measured size, short-term fecundity, mortality, predator position, response to plant odors and crystal location, thus including 6 of the 9 symptoms known yet. No evidence was found for vertical transmission from parent to offspring. Eggs from symptomatic females of the NR-population mated by males of the NR-population gave rise to normal-sized, well performing predators, when they had been surface sterilized or transferred to a new leaf. However, such eggs gave rise to shrunken females (17%) when left on the leaf where they had been laid. In the latter case transmission via products deposited on the leaf by the mothers was possible. We therefore tested several modes of horizontal transmission by exposing females of a commercial population that never showed the NR-syndrome (=R1-population) to products related to the symptomatic NR-population. No evidence was found for transmission via food or via squashed adult females. However, symptoms were induced in adult females of the R1-population after a 3-day exposure to a live adult female of the NR-population (incubation period=3–7 days, fraction shrunken females=53%) and after a 1-day exposure to feces and debris collected from such females (incubation period=2–4 days, fraction shrunken females=65%). Contact with live females and feces of the R1-population did not induce the syndrome. These results clearly indicate that the NR-syndrome is a contagious phenomenon and that the factor inducing the syndrome is transmitted horizontally among and between generations via feces and debris deposited by symptomatic females. The results are discussed in the context of mite pathology and biological control.     

Comparison of growth inhibition profiles and mechanisms of apoptosis induction in human colon cancer cell lines by isothiocyanates and indoles from Brassicaceae

The isothiocyanates sulforaphane and PEITC (beta-phenethyl isothiocyanate) as well as the indoles indole-3-carbinol and its condensation product 3,3'-diindolylmethane are known to inhibit cancer cell proliferation and induce apoptosis. In this study, we compared the cell growth inhibitory potential of the four compounds on the p53 wild type human colon cancer cell line 40-16 (p53(+/+)) and its p53 knockout derivative 379.2 (p53(-/-)) (both derived from HCT116). Using sulforhodamin B staining to assess cell proliferation, we found that the isothiocyanates were strongly cytotoxic, whereas the indoles inhibited cell growth in a cytostatic manner. Half-maximal inhibitory concentrations of all four compounds in both cell lines ranged from 5-15 microM after 24, 48 and 72 h of treatment. Apoptosis induction was analyzed by immunoblotting of poly(ADP-ribose)polymerase (PARP). Treatment with sulforaphane (15 microM), PEITC (10 microM), indole-3-carbinol (10 microM) and 3,3'-diindolylmethane (10 microM) induced PARP cleavage after 24 and 48 h in both 40-16 and the 379.2 cell lines, suggestive of a p53-independent mechanism of apoptosis induction. In cultured 40-16 cells, activation of caspase-9 and -7 detected by Western blotting indicated involvement of the mitochondrial pathway. We detected time- and concentration-dependent changes in protein expression of anti-apoptotic Bcl-x(L) as well as pro-apoptotic Bax and Bak proteins. Of note is that for sulforaphane only, ratios of pro- to anti-apoptotic Bcl-2 family protein levels directly correlated with apoptosis induction measured by PARP cleavage. Taken together, we demonstrated that the glucosinolate breakdown products investigated in this study have distinct profiles of cell growth inhibition, potential to induce p53-independent apoptosis and to modulate Bcl-2 family protein expression in human colon cancer cell lines.     

Transient expression of hemagglutinin antigen from low pathogenic avian influenza A (H7N7) in Nicotiana benthamiana

The influenza A virus is of global concern for the poultry industry, especially the H5 and H7 subtypes as they have the potential to become highly pathogenic for poultry. In this study, the hemagglutinin (HA) of a low pathogenic avian influenza virus of the H7N7 subtype isolated from a Swedish mallard Anas platyrhynchos was sequenced, characterized and transiently expressed in Nicotiana benthamiana. Recently, plant expression systems have gained interest as an alternative for the production of vaccine antigens. To examine the possibility of expressing the HA protein in N. benthamiana, a cDNA fragment encoding the HA gene was synthesized de novo, modified with a Kozak sequence, a PR1a signal peptide, a C-terminal hexahistidine (6×His) tag, and an endoplasmic retention signal (SEKDEL). The construct was cloned into a Cowpea mosaic virus (CPMV)-based vector (pEAQ-HT) and the resulting pEAQ-HT-HA plasmid, along with a vector (pJL3:p19) containing the viral gene-silencing suppressor p19 from Tomato bushy stunt virus, was agro-infiltrated into N. benthamiana. The highest gene expression of recombinant plant-produced, uncleaved HA (rHA0), as measured by quantitative real-time PCR was detected at 6 days post infiltration (dpi). Guided by the gene expression profile, rHA0 protein was extracted at 6 dpi and subsequently purified utilizing the 6×His tag and immobilized metal ion adsorption chromatography. The yield was 0.2 g purified protein per kg fresh weight of leaves. Further molecular characterizations showed that the purified rHA0 protein was N-glycosylated and its identity confirmed by liquid chromatography-tandem mass spectrometry. In addition, the purified rHA0 exhibited hemagglutination and hemagglutination inhibition activity indicating that the rHA0 shares structural and functional properties with native HA protein of H7 influenza virus. Our results indicate that rHA0 maintained its native antigenicity and specificity, providing a good source of vaccine antigen to induce immune response in poultry species.     

Tetracycline inducible gene manipulation in serotonergic neurons

The serotonergic (5-HT) neuronal system has important and diverse physiological functions throughout development and adulthood. Its dysregulation during development or later in adulthood has been implicated in many neuropsychiatric disorders. Transgenic animal models designed to study the contribution of serotonergic susceptibility genes to a pathological phenotype should ideally allow to study candidate gene overexpression or gene knockout selectively in serotonergic neurons at any desired time during life. For this purpose, conditional expression systems such as the tet-system are preferable. Here, we generated a transactivator (tTA) mouse line (TPH2-tTA) that allows temporal and spatial control of tetracycline (Ptet) controlled transgene expression as well as gene deletion in 5-HT neurons. The tTA cDNA was inserted into a 196 kb PAC containing a genomic mouse Tph2 fragment (177 kb) by homologous recombination in E. coli. For functional analysis of Ptet-controlled transgene expression, TPH2-tTA mice were crossed to a Ptet-regulated lacZ reporter line (Ptet-nLacZ). In adult double-transgenic TPH2-tTA/Ptet-nLacZ mice, TPH2-tTA founder line L62-20 showed strong serotonergic β-galactosidase expression which could be completely suppressed with doxycycline (Dox). Furthermore, Ptet-regulated gene expression could be reversibly activated or inactivated when Dox was either withdrawn or added to the system. For functional analysis of Ptet-controlled, Cre-mediated gene deletion, TPH2-tTA mice (L62-20) were crossed to double transgenic Ptet-Cre/R26R reporter mice to generate TPH2-tTA/Ptet-Cre/R26R mice. Without Dox, 5-HT specific recombination started at E12.5. With permanent Dox administration, Ptet-controlled Cre-mediated recombination was absent. Dox withdrawal either postnatally or during adulthood induced efficient recombination in serotonergic neurons of all raphe nuclei, respectively. In the enteric nervous system, recombination could not be detected. We generated a transgenic mouse tTA line (TPH2-tTA) which allows both inducible and reversible transgene expression and inducible Cre-mediated gene deletion selectively in 5-HT neurons throughout life. This will allow precise delineation of serotonergic gene functions during development and adulthood.     

The Time Required for Dormancy Release in Arabidopsis Is Determined by DELAY OF GERMINATION1 Protein Levels in Freshly Harvested Seeds

Seed dormancy controls the start of a plant's life cycle by preventing germination of a viable seed in an unfavorable season. Freshly harvested seeds usually show a high level of dormancy, which is gradually released during dry storage (after-ripening). Abscisic acid (ABA) has been identified as an essential factor for the induction of dormancy, whereas gibberellins (GAs) are required for germination. The molecular mechanisms controlling seed dormancy are not well understood. DELAY OF GERMINATION1 (DOG1) was recently identified as a major regulator of dormancy in Arabidopsis thaliana. Here, we show that the DOG1 protein accumulates during seed maturation and remains stable throughout seed storage and imbibition. The levels of DOG1 protein in freshly harvested seeds highly correlate with dormancy. The DOG1 protein becomes modified during after-ripening, and its levels in stored seeds do not correlate with germination potential. Although ABA levels in dog1 mutants are reduced and GA levels enhanced, we show that DOG1 does not regulate dormancy primarily via changes in hormone levels. We propose that DOG1 protein abundance in freshly harvested seeds acts as a timer for seed dormancy release, which functions largely independent from ABA.     

Only plant-type (GLYK) glycerate kinases produce d-glycerate 3-phosphate

d-Glycerate kinases (GK) occur in three phylogenetically distinct classes. Class II GKs produce glycerate 2-phosphate, while both class I GK and class III GK (GLYK) are thought to produce glycerate 3-phosphate. We report on the identification of a bacterial-type class I GK in the unicellular cyanobacterium Synechocystis sp. strain PCC 6803 and of a plant-type GLYK in the filamentous cyanobacterium Nostoc sp. strain PCC 7120. The comparison with other prokaryotic and eukaryotic GKs of both classes shows that glycerate 3-phosphate is produced only by the GLYKs, but, in contrast to current thinking, not by any of the examined class I enzymes.