GENETIC VARIABILITY AND MOLECULAR PHYLOGENY OF DINOPHYSIS SPECIES (DINOPHYCEAE) FROM NORWEGIAN WATERS INFERRED FROM SINGLE CELL ANALYSES OF rDNA1

The objectives of this study were to determine rDNA sequences of the most common Dinophysis species in Scandinavian waters and to resolve their phylogenetic relationships within the genus and to other dinoflagellates. A third aim was to examine the intraspecific variation in D. acuminata and D. norvegica, because these two species are highly variable in both morphology and toxicity. We obtained nucleotide sequences of coding (small subunit [SSU], partial large subunit [LSU], 5.8S) and noncoding (internal transcribed spacer [ITS]1, ITS2) parts of the rRNA operon by PCR amplification of one or two Dinophysis cells isolated from natural water samples. The three photosynthetic species D. acuminata, D. acuta, and D. norvegica differed in only 5 to 8 of 1802 base pairs (bp) within the SSU rRNA gene. The nonphotosynthetic D. rotundata (synonym Phalacroma rotundatum[Claparède et Lachmann] Kofoid et Michener), however, differed in approximately 55 bp compared with the three photosynthetic species. In the D1 and D2 domains of LSU rDNA, the phototrophic species differed among themselves by 3 to 12 of 733 bp, whereas they differed from D. rotundata by more than 100 bp. This supports the distinction between Dinophysis and Phalacroma. In the phylogenetic analyses based on SSU rDNA, all Dinophysis species were grouped into a common clade in which D. rotundata diverged first. The results indicate an early divergence of Dinophysis within the Dinophyta. The LSU phylogenetic analyses, including 4 new and 11 Dinophysis sequences from EMBL, identified two major clades within the phototrophic species. Little or no intraspecific genetic variation was found in the ITS1–ITS2 region of single cells of D. norvegica and D. acuminata from Norway, but the delineation between these two species was not always clear.     

ZNF385B and VEGFA Are Strongly Differentially Expressed in Serous Ovarian Carcinomas and Correlate with Survival

Background

The oncogenesis of ovarian cancer is poorly understood. The aim of this study was to identify mRNAs differentially expressed between moderately and poorly differentiated (MD/PD) serous ovarian carcinomas (SC), serous ovarian borderline tumours (SBOT) and superficial scrapings from normal ovaries (SNO), and to correlate these mRNAs with clinical parameters including survival.

Methods

Differences in mRNA expression between MD/PD SC, SBOT and SNO were analyzed by global gene expression profiling (n = 23), validated by RT-qPCR (n = 41) and correlated with clinical parameters.

Results

Thirty mRNAs differentially expressed between MD/PD SC, SBOT and SNO were selected from the global gene expression analyses, and 21 were verified (p<0.01) by RT-qPCR. Of these, 13 mRNAs were differentially expressed in MD/PD SC compared with SNO (p<0.01) and were correlated with clinical parameters. ZNF385B was downregulated (FC = −130.5, p = 1.2×10⁻⁷) and correlated with overall survival (p = 0.03). VEGFA was upregulated (FC = 6.1, p = 6.0×10⁻⁶) and correlated with progression-free survival (p = 0.037). Increased levels of TPX2 and FOXM1 mRNAs (FC = 28.5, p = 2.7×10⁻¹⁰ and FC = 46.2, p = 5.6×10⁻⁴, respectively) correlated with normalization of CA125 (p = 0.03 and p = 0.044, respectively). Furthermore, we present a molecular pathway for MD/PD SC, including VEGFA, FOXM1, TPX2, BIRC5 and TOP2A, all significantly upregulated and directly interacting with TP53.

Conclusions

We have identified 21 mRNAs differentially expressed (p<0.01) between MD/PD SC, SBOT and SNO. Thirteen were differentially expressed in MD/PD SC, including ZNF385B and VEGFA correlating with survival, and FOXM1 and TPX2 with normalization of CA125. We also present a molecular pathway for MD/PD SC.     

Phosphorus availability and microbial respiration across different tundra vegetation types

Phosphorus (P) is an important nutrient in tundra ecosystems that co-limits or in some cases limits primary production. The availability of P is largely driven by soil characteristics, e.g., pH, organic carbon, and abundance of P-sorbing elements such as aluminium (Al) or iron (Fe). We tested how vegetation and soil properties relate to P availability across different tundra vegetation types. The different soil P fractions in the organic horizon were measured and plant foliar nitrogen (N) to P ratio and a plant bioassay was used as indicators of plant nutrient status. Microbial bioassays were used to study microbial respiration kinetics and in response to carbon, N, and P amendments. The distribution of P fractions differed significantly across vegetation types; labile fractions of P were less abundant in meadow sites compared to heath sites. Calcium-phosphates seemed to be an important P-fraction in meadows, but were only found in lower concentrations in the heath. There were only small differences in NaOH–extractable P between the vegetation types and this correlated with the distribution of oxalate-extractable Al. Plant N:P ratios and the plant bioassay indicated decreasing P availability from dry heath to mesic heath to mesic meadow. The microbial bioassay suggests that the heterotrophic microbial community is C-limited with N as a secondary limiting nutrient although there were indications that microbial P availability was lower in the meadow sites. Overall, we suggest that the observed variations in soil P across vegetation types are affecting both plant and microbial function although the differences seem to be relatively small.     

Distribution of neuropeptides in the primary olfactory center of the heliothine moth <Emphasis Type="Italic">Heliothis virescens</Emphasis>

Neuropeptides are a diverse widespread class of signaling substances in the nervous system. As a basis for the analysis of peptidergic neurotransmission in the insect olfactory system, we have studied the distribution of neuropeptides in the antennal lobe of the moth Heliothis virescens. Immunocytochemical experiments with antisera recognizing A-type allatostatins (AST-As), Manduca sexta allatotropin (Mas-AT), FMRFamide-related peptides (FaRPs), and tachykinin-related peptides (TKRPs) have shown that members of all four peptide families are present in local interneurons of the antennal lobe. Whereas antisera against AST-As, Mas-AT, and FaRPs give similar staining patterns characterized by dense meshworks of processes confined to the core of all antennal-lobe glomeruli, TKRPs are present only in neurons with blebby processes distributed throughout each glomerulus. In addition to local neurons, a pair of centrifugal neurons with cell bodies in the lateral subesophageal ganglion, arborizations in the antennal lobe, and projections in the inner antenno-cerebral tracts exhibits tachykinin immunostaining. Double-label immunofluorescence has detected the co-localization of AST-As, Mas-AT, and FaRPs in certain local interneurons, whereas TKRPs occurs in a distinct population. MALDI-TOF mass spectrometry has revealed nearly 50 mass peaks in the antennal lobe. Seven of these masses (four AST-As, two N-terminally extended FLRFamides, and Mas-AT) match known moth neuropeptides. The data thus show that local interneurons of the moth antennal lobe are highly differentiated with respect to their neuropeptide content. The antennal lobe therefore represents an ideal preparation for the future analysis of peptide signaling in insect brain.     

The major outer membrane protein of Fusobacterium nucleatum (FomA) folds and inserts into lipid bilayers via parallel folding pathways

Membrane protein insertion and folding was studied for the major outer membrane protein of Fusobacterium nucleatum (FomA), which is a voltage-dependent general diffusion porin. The transmembrane domain of FomA forms a beta-barrel that is predicted to consist of 14 beta-strands. Here, unfolded FomA is shown to insert and fold spontaneously and quantitatively into phospholipid bilayers upon dilution of the denaturant urea, which was shown previously only for outer membrane protein A (OmpA) of Escherichia coli. Folding of FomA is demonstrated by circular dichroism and fluorescence spectroscopy, by SDS-polyacrylamide gel electrophoresis, and by single-channel recordings. Refolded FomA had a single-channel conductance of 1.1 nS at 1 M KCl, in agreement with the conductance of FomA isolated from membranes in native form. In contrast to OmpA, which forms a smaller eight-stranded beta-barrel domain, folding kinetics of the larger FomA were slower and provided evidence for parallel folding pathways of FomA into lipid bilayers. Two pathways were observed independent of membrane thickness with two different lipid bilayers, which were either composed of dicapryl phosphatidylcholine or dioleoyl phosphatidylcholine. This is the first observation of parallel membrane insertion and folding pathways of a beta-barrel membrane protein from an unfolded state in urea into lipid bilayers. The kinetics of both folding pathways depended on the chain length of the lipid and on temperature with estimated activation energies of 19 kJ/mol (dicapryl phosphatidylcholine) and 70 kJ/mol (dioleoyl phosphatidylcholine) for the faster pathways.     

Molecular phylogeography and hybridization in members of the circumpolar Potentilla sect. Niveae (Rosaceae)

Glacial events and the formation of ice-free areas serving as refugia for plants and animals are important in shaping present patterns of genetic diversity in arctic areas. Beringia, situated in northeastern Russia and Alaska, has been pointed out as a major refugium. This study focuses on the historical biogeography of the circumpolar taxon Potentilla sect. Niveae. The taxonomy of the group is complex, most likely highly influenced by hybridization and apomixis. cpDNA microsatellites together with AFLP fragments were used to map the genetic variability in the section, from Beringia across the Canadian Arctic to Greenland. The data support the hypothesis that Beringia, as well as parts of adjacent arctic Canada, served as refugia during the Wisconsinan glaciation, and there is some evidence for a northern and a southern migration route out of Beringia. The hair type groups within sect. Niveae are more or less genetically distinct, and hybridization, especially with sect. Multifida, takes place. Haplotype diversity as well as frequency is at its maximum close to the Last Glacial Maximum ice cap edge. This pattern can be explained by merging of previously isolated refugia, by repeated extinction/colonization events close to the ice edge, and by hybridization among sympatric taxonomical lineages.     

A new method to study changes in microvascular blood volume in muscle and adipose tissue: real-time imaging in humans and rat

We employed and evaluated a new application of contrast-enhanced ultrasound for real-time imaging of changes in microvascular blood volume (MBV) in tissues in females, males, and rat. Continuous real-time imaging was performed using contrast-enhanced ultrasound to quantify infused gas-filled microbubbles in the microcirculation. It was necessary to infuse microbubbles for a minimum of 5-7 min to obtain steady-state bubble concentration, a prerequisite for making comparisons between different physiological states. Insulin clamped at a submaximal concentration (～75 μU/ml) increased MBV by 27 and 39% in females and males, respectively, and by 30% in female subcutaneous adipose tissue. There was no difference in the ability of insulin to increase muscle MBV in females and males, and microvascular perfusion rate was not increased significantly by insulin. However, perfusion rate of the microvascular space was higher in females compared with males. In rats, insulin clamped at a maximal concentration increased muscle MBV by 60%. Large increases in microvascular volume and perfusion rate were detected during electrical stimulation of muscle in rats and immediately after exercise in humans. We have demonstrated that real-time imaging of changes in MBV is possible in human and rat muscle and in subcutaneous adipose tissue and that the method is sensitive enough to pick up relatively small changes in MBV when performed with due consideration of steady-state microbubble concentration. Because of real-time imaging, the method has wide applications for determining MBV in different organs during various physiological or pathophysiological conditions.     

Floral ontogeny in tribe Dalbergieae (Leguminosae: Papilionoideae):Dalbergia brasiliensis, Machaerium villosum s. l.Platymiscium floribundum, andPterocarpus rotundifolius

Floral organogenesis and development of the tropical legume treesDalbergia brasiliensis, Machaerium villosum, Platymiscium floribundum, andPterocarpus rotundifolius were studied using scanning electron microscopy. The aims were to compare ontogenies and to elucidate if floral ontogenetic data will provide new character states diagnostic of the tribe Dalbergieae, which is considered a basal papilionoid tribe and primarily defined on fruit characters. Organ inception is principally acropetal in all taxa studied. Carpel inception is, however, consistently precocious. InD. brasiliensis sepals are initiated in an order not previously reported in Papilionoideae. It may be considered modified helical. InP. rotundifolius the inner whorl of stamens initiate in an unusual way, this is lateral two stamens first, then the two abaxial ones, and last the adaxial one, opposed to the unidirectional order usually seen in Papilionoideae. Generally the differences in flower development among the studied genera appear at initiation and late stage in ontogenesis, rather than at mid-stage.     

Is fertility reduced among men exposed to radiofrequency fields in the Norwegian Navy?

The effects of radiofrequency fields on human health are not well understood, and public concern about negative health effects has been rising. The aim of this study was to examine the relationship between workers exposed to electromagnetic fields and their reproductive health. We obtained data using a questionnaire in a cross-sectional study of naval military men, response rate 63% (n = 1487). We asked the respondents about exposure, lifestyle, reproductive health, previous diseases, work and education. An expert group categorized the work categories related to electromagnetic field exposure. We categorized the work categories "tele/communication," "electronics" and "radar/sonar" as being exposed to electromagnetic fields. Logistic regression adjusted for age, ever smoked, military education, and physical exercise at work showed increased risk of infertility among tele/communication odds ratio (OR = 1.72, 95% confidence interval 1.04-2.85), and radar/sonar odds ratio (OR = 2.28, 95% confidence interval 1.27-4.09). The electronics group had no increased risk. This study shows a possible relationship between exposure to radiofrequency fields during work with radiofrequency equipment and radar and reduced fertility. However, the results must be interpreted with caution.     

Small angle X-ray studies reveal that Aspergillus niger glucoamylase has a defined extended conformation and can form dimers in solution

The industrially important glucoamylase 1 is an exo-acting glycosidase with substrate preference for alpha-1,4 and alpha-1,6 linkages at non-reducing ends of starch. It consists of a starch binding and a catalytic domain interspersed by a highly glycosylated polypeptide linker. The linker function is poorly understood and structurally undescribed, and data regarding domain organization and intramolecular functional cooperativity are conflicting or non-comprehensive. Here, we report a combined small angle x-ray scattering and calorimetry study of Aspergillus niger glucoamylase 1, glucoamylase 2, which lacks a starch binding domain, and an engineered low-glycosylated variant of glucoamylase 1 with a short linker. Low resolution solution structures show that the linker adopts a compact structure rendering a well defined extended overall conformation to glucoamylase. We demonstrate that binding of a short heterobidentate inhibitor simultaneously directed toward the catalytic and starch binding domains causes dimerization of glucoamylase and not, as suggested previously, an intramolecular conformational rearrangement mediated by linker flexibility. Our results suggest that glucoamylase functions via transient dimer formation during hydrolysis of insoluble substrates and address the question of the cooperative effect of starch binding and hydrolysis.