Falsocalcifolium nov. gen. and revision of the tribe Calcifolieae (Mississippian, incertae sedis algae)

A new genus, Falsocalcifolium is described. The type species is F. punctatum (Maslov) emend. The genus is placed in the tribe Calcifolieae Shuysky emend. of the family Calcifoliaceae Termier, Termier and Vachard nomen translat. Consequently, the generic make-up of the tribe is emended, and is now considered to be composed of Frustulata Saltovskaya emend., Falsocalcifolium nov. gen., and Calcifolium Shvetsov and Birina emend. Evolution within the tribe is shown by the progressive development of more petaloid morphologies and changes in the number and relationships of the internal structures called filaments (interpreted here as the emplacements containing endosymbiotic cyanobacteria). This evolution is also related to palaeoecological factors. The representatives of the tribe are considered excellent biostratigraphic markers in several western Tethyan regions (e.g. Great Britain, Ireland, southern France, southern Spain), where they are widely represented, but their geographical distribution in other basins seems to be incomplete, and thus their use in biostratigraphic correlation is at the moment limited.     

Contrasting spatial heterogeneity of sessile organisms within mussel (Perna perna L.) beds in relation to topographic variability

We examined the spatial heterogeneity in three sessile rocky shore organisms, the mussel Perna perna, the barnacle Octomeris angulosa (Sowerby) and the red alga Gelidium pristoides (Turn.) at a range of continuous local scales along horizontal transects within mid- and upper mussel beds of South African shores. We also examined the relationships between variability of organisms and topographic features (rock depressions, slope, aspect), and between mussel, barnacle and algal variability over the same scales. To estimate spatial heterogeneity, we analyzed scaling properties of semivariograms using a fractal approach. Relationships between different variables at the different scales were examined by cross-semivariograms. Spatial dependence of P. perna variability increased with spatial dependence of topographic variability, so that scaling regions of mussel and topographic distributions corresponded well. This relationship often improved with larger local scales (mussel cover increased with depressions, steeper slope and aspect towards waves), while at smaller spatial scales, variability in mussel cover was less well explained by variability in topography. The variability of the barnacle O. angulosa exhibited spatial dependence, even on topographically unstructured shores. In contrast, the distribution of the alga G. pristoides revealed high fractal dimensions, showing spatial independence on topographically unstructured shores. Algae also showed a very strong negative relationship with mussels at most local scales, and a negative relationship with barnacles in upper zones, especially at larger local scales. Barnacles may show clear spatial dependence because of hydrodynamics (at larger local scales) and the need to find a future mate in close proximity (at smaller local scales), while algae may show a strong negative relationship with mussels because of competition for space.     

Eukaryotic expression and secretion of EGFP-labeled annexin A5

The Ca²⁺-dependent binding of annexin A5 to phosphatidylserine on cell surfaces is a reliable marker for apoptosis that is widely used in flow cytometry based apoptosis assays. In this approach, annexin A5 must be coupled to a fluorescent dye, but standard dyes such as fluorescein are photolabile, and the heterogeneous chemical linkage partially inhibits binding to phosphatidylserine. Recombinant fusions comprising annexin A5 and fluorescent proteins are available for prokaryotic expression, but can be purified only at low concentrations due to their low solubility in the cytoplasm. Here we describe a eukaryotic expression system for the secretion of functional recombinant annexin A5, with and without fluorescent protein fusions, in different formats. Metal affinity purification yielded up to 18 μg of histidine-tagged annexin A5 fusions per ml processed cell culture supernatants. Furthermore the supernatant itself was sufficient for direct use in apoptosis assays. The availability of such fusion proteins offers new and more economical opportunities for the development and application of this widely utilized apoptosis assay.     

Adaptation of the fungal parasite Zygorhizidium planktonicum during 200 generations of growth on homogeneous and heterogeneous populations of its host, the diatom Asterionella formosa

We followed adaptation of the chytrid parasite Zygorhizidium planktonicum during 200 generations of growth on its host, the freshwater diatom Asterionella formosa, in a serial passage experiment. Evolution of parasite fitness was assessed both on a homogenous and heterogeneous host population, consisting of respectively a single new and ten different new host strains. These 10 host strains were genetically different and also varied in their initial susceptibility to the parasite. Parasite fitness increased significantly and rapidly on the new, genetically homogenous host population, but remained unaltered during 200 generations of growth on the heterogeneous host population. Enhanced parasite fitness was the result of faster and more efficient transmission, resulting in higher values of R0 (number of secondary infections). Consequently, parasites that evolved within the uniclonal host population infected significantly more of these hosts than did their ancestors. We thus provide experimental evidence for the widely held view that host genetic diversity restricts evolution of parasites and moderates their harmful effects. Genetically uniform host populations are not only at increased risk from fungal epidemics because they all share the same susceptibility, but also because new parasite strains are able to adapt quickly to new host environments and to improve their fitness.     

High rates of nitrogen fixation (acetylene reduction) on coral skeletons following bleaching mortality

Nubbins of the coral Acropora aspera were artificially bleached and nitrogen fixation (acetylene reduction) rates were measured on the developing epilithic communities. Seasonal comparisons were made between corals that died in summer of heat stress and corals that died in winter from natural cold stress. Rates of acetylene reduction from artificially bleached corals peaked at 26.66 nmol cm⁻² h⁻¹ 2 weeks after summer mortality, while rates from natural winter mortality peaked at 18.07 nmol cm⁻² h⁻¹ 12 days after coral death. Comparative rates of acetylene reduction taken from live corals and coral rubble ranged between 0.56 and 1.16 nmol cm⁻² h⁻¹, and 0.15 and 12.77 nmol cm⁻² h⁻¹, respectively. N₂-fixation rates from dead corals were up to 30 times greater than those measured on live corals. The observed increase in N₂-fixation from dead corals may increase the availability of nitrogen for use in trophic processes within the reef for an extended period following the initial mortality event. If the spatial scale over which coral mortality has occurred in past thermal bleaching events is considered the ramifications of such an increase may be substantial.     

Benthic algal response to hyporheic-surface water exchange in an alluvial river

We studied the response of benthic algae to points of hyporheic-surface water exchange in the main channel of the Middle Fork Flathead River within the Nyack Flood Plain, Montana. We examined hyporheic exchange at 120 sites using piezometers and measuring vertical hydraulic gradient (VHG), hydraulic conductivity, and vertical discharge. We removed benthic algae from a single cobble at each site, and we used VHG to group sampling sites for statistical analysis. Algal cell density and chlorophyll a concentration were significantly higher at sites with hyporheic discharge (+VHG, upwelling) compared to both sites with hyporheic recharge (−VHG, downwelling) and sites with no hyporheic-surface water exchange (=VHG, neutral) (ANOVA, P < 0.05). The assemblages of algae at upwelling sites were also significantly different from downwelling and neutral exchange sites (ANOSIM, P < 0.05). Filamentous green algae Stigeoclonium sp. and Zygnema sp. and a chrysophyte, Hydrurus foetidus (Villars) Trevisan were abundant at upwelling sites, whereas an assemblage of diatoms Achnanthidium minutissimum (Kützing) Czarnecki, Cymbella excisa Kützing, Diatoma moniliformis Kützing, and Gomphonema olivaceoides Hustedt, were the most abundant taxa at downwelling and neutral exchange sites, occurring attached to, or in close association with the stalks of Didymosphenia geminata (Lyngbye) Schmidt. These data show that benthic algal communities are structured differently depending on the direction of hyporheic flux in the main channel of a large alluvial river, suggesting that hyporheic-surface exchange may influence the spatial distribution of main-channel benthic algae in rivers with hyporheic-surface water connectivity. Handling editor: J. Padisak     

Seasonal changes of fatty acid composition and thermotropic behavior of polar lipids from marine macrophytes

Major glyco- and phospholipids as well as betaine lipid 1,2-diacylglycero-O-4'-(N,N,N-tri-methyl)-homoserine (DGTS) were isolated from five species of marine macrophytes harvested in the Sea of Japan in summer and winter at seawater temperatures of 20-23 and 3 degrees C, respectively. GC and DSC analysis of lipids revealed a common increase of ratio between n-3 and n-6 polyunsaturated fatty acids (PUFAs) of polar lipids from summer to winter despite their chemotaxonomically different fatty acid (FA) composition. Especially, high level of different n-3 PUFAs was observed in galactolipids in winter. However, the rise in FA unsaturation did not result in the lowering of peak maximum temperature of phase transition of photosynthetic lipids (glycolipids and phosphatidylglycerol (PG)) in contrast to non-photosynthetic ones [phosphatidylcholine (PC) and phosphatidylethanolamine (PE)]. Different thermotropic behavior of these lipid groups was accompanied by higher content of n-6 PUFAs from the sum of n-6 and n-3 PUFAs in PC and PE compared with glycolipids and PG in both seasons. Seasonal changes of DSC transitions and FA composition of DGTS studied for the first time were similar to PC and PE. Thermograms of all polar lipids were characterized by complex profiles and located in a wide temperature range between -130 and 80 degrees C, while the most evident phase separation occurred in PGs in both seasons. Polarizing microscopy combined with DSC has shown that the liquid crystal - isotropic melt transitions of polar lipids from marine macrophytes began from 10 to 30 degrees C mostly, which can cause the thermal sensitivity of plants to superoptimal temperatures in their environment.     

HPV16l1基因的克隆及其在真核细胞中的表达

克隆并表达人乳头瘤病毒16型(HPV16)晚期基因l1,以期为研制防治宫颈癌的DNA疫苗奠定基础。本实验采用PCR方法从质粒p16L1BN1中获得HPV16l1基因片段,利用基因重组技术,将其克隆至含巨细胞病毒(CMV)启动子的真核表达载体中,核酸序列鉴定HPV16l1基因真核表达质粒构建成功,再用脂质体介导基因转染7721人肝癌细胞。转化阳性细胞经SDS-PAGE显示在分子量大约为55kDa的位置出现一条特异性条带,与HPV16L1分子量大小相符。表达产物经Western blotting分析:能与HPV16L1单克隆抗体特异结合。真核表达质粒pcDNA3-HPV16L1构建成功并能在真核细胞7721中有效表达,为下一步进行动物DNA免疫实验奠定了基础。     

Morphological and molecular characterization of cyanobacteria from a Brazilian facultative wastewater stabilization pond and evaluation of microcystin production

The cyanobacterial population in the Cajati waste stabilization pond system (WSP) from São Paulo State, Brazil was assessed by cell isolation and direct microscope counting techniques. Ten strains, belonging to five genera (Synechococcus, Merismopedia, Leptolyngbya, Limnothrix, and Nostoc), were isolated and identified by morphological and molecular analyses. Morphological identification of the isolated strains was congruent with their phylogenetic analyses based on 16S rDNA gene sequences. Six cyanobacterial genera (Synechocystis, Aphanocapsa, Merismopedia, Lyngbya, Phormidium, and Pseudanabaena) were identified by direct microscope inspection. Both techniques were complementary, since, of the six genera identified by direct microscopic inspection, only Merismopedia was isolated, and the four other isolated genera were not detected by direct inspection. Direct microscope counting of preserved cells showed that cyanobacteria were the dominant members (>90%) of the phytoplankton community during both periods evaluated (summer and autumn). ELISA tests specific for hepatotoxic microcystins gave positive results for six strains (Synechococcus CENA108, Merismopedia CENA106, Leptolyngbya CENA103, Leptolyngbya CENA112, Limnothrix CENA109, and Limnothrix CENA110), and for wastewater samples collected from raw influent (3.70 μg microcystins/l) and treated effluent (3.74 μg microcystins/l) in summer. Our findings indicate that toxic cyanobacteria in WSP systems are of concern, since the treated effluent containing cyanotoxins will be discharged into rivers, irrigation channels, estuaries, or reservoirs, and can affect human and animal health.