When an epithelium ceases to exist – an ultrastructural study on the fate of the embryonic coelom in Epiperipatus biolleyi (Onychophora, Peripatidae)

It is an accepted fact that fusion between the coelomic cavities and the primary body cavity occurs during development in the Arthropoda. However, such a fusion is much disputed in the Onychophora. In order to clarify this subject, the fate of embryonic coelomic cavities has been studied in an onychophoran. Ultrastructural investigations in this paper provide evidence that embryonic coelomic cavities fuse with spaces of the primary body cavity in Epiperipatus biolleyi. During embryogenesis, the somatic and splanchnic portions of the mesoderm separate and the former coelomic linings are transformed into mesenchymatic tissue. The resulting body cavity therefore represents a mixture of primary and secondary (coelomic) body cavities, i.e. the ‘mixocoel’. The nephridial anlage is already present, when the ‘mixocoel’ is formed, although there is no trace of a sacculus yet. The lumen of the nephridial anlage, thus, communicates with the newly formed ‘mixocoel’. Accordingly, the lumen of the nephridial sacculus cannot be regarded as a kind of ‘persisting coelomic cavity’ in E. biolleyi. Our findings support the hypothesis that the ‘mixocoel’ was already present in the common stem species of the Onychophora and Euarthropoda.     

Energy metabolism of autotrophically and heterotrophically grown cells of Nitrobacter winogradskyi

The adenine nucleotide pools and the NADH pool were compared in intact Nitrobacter winogradskyi cells grown under different conditions. The NADH pool was highest in nitrite-grown cells (22.0 nmol/mg N), less high in acetategrown cells (15.1 nmol/mg N),and lowest in pyruvate-grown cells (11.9 nmol/mg N).The adenine nucleotide pools and the NADH pool were determined after the transition from anaerobic to aerobic conditions.In both autotrophically and heterotrophically grown cells the ATP pool decreased within the first second after the addition of oxygen and then increased.In cells grown with nitrite or acetate the NADH pool increased the first second after the addition of oxygen then decreased below the initial value. In pyruvate-grown cells the changes in the NADH pool were less obvious.In the presence of rotenone autotrophic cells were able to generate ATP, but the reverse energy-dependent electron transport was inhibited. Consequently, NADH was not synthesized. N,N-dicyclohexylcarbodiimide an inhibitor of ATPase, prevented both ATP and NADH generation.Abbreviations DCCD N,N-dicyclohexylcarbodiimide     

Endobacterial morphotypes in nudibranch cerata tips: a SEM analysis

The SEM investigation of nudibranch cerata material exhibits endobacterial morphotypes found in 12 out of 13 species tested: Aeolidia papillosa, Berghia caerulescens, Coryphella brownii, Coryphella lineata, Coryphella verrucosa, Cuthona amoena, Facelina coronata, Flabellina pedata, Dendronotus frondosus, Doto coronata, Tritonia plebeia and Janolus cristatus. Endobacteria could not be detected inside Tritonia hombergi. Endobacterial morphology found inside nudibranch species was compared to bacterial morphotypes detected earlier in tentacles of cnidarian species. SEM micrographs show endobacterial analogy among nudibranch species, but also similarity to cnidarian endobacteria investigated earlier. Of course, morphological data of microbes do not allow their identification. However, since most of these nudibranch species prey on cnidaria, it cannot be excluded that many of the endobacteria detected inside nudibranch species may originate from their cnidarian prey. Our previous data describing genetic affiliation of endobacteria from nudibranchian and cnidarian species support this assumption. Dominant coccoid endobacteria mostly exhibit smooth surface and are tightly packed as aggregates and/or wrapped in envelopes. Such bacterial aggregate type has been described previously in tentacles of the cnidarian species Sagartia elegans. Similar coccoid bacteria, lacking envelopes were also found in other nudibranch species. A different type of coccoid bacteria, characterized by a rough surface, was detected inside cerata of the nudibranch species Berghia caerulescens, and surprisingly, inside tentacles of the cnidarian species Tubularia indivisa. In contrast to cnidarian endobacteria, rod-shaped microorganisms are largely absent in nudibranch cerata.     

Heterogeneity of CD4 and CD8+ memory T cells in localized and generalized Wegener's granulomatosis

Memory T cells display phenotypic heterogeneity. Surface antigens previously regarded as exclusive markers of naive T cells, such as L-selectin (CD62L), can also be detected on some memory T cells. Moreover, a fraction of CD45RO+ (positive for the short human isoform of CD45) memory T cells reverts to the CD45RA+ (positive for the long human isoform of CD45) phenotype. We analyzed patients with biopsy-proven localized Wegener's granulomatosis (WG) (n = 5), generalized WG (n = 16) and age- and sex-matched healthy controls (n = 13) to further characterize memory T cells in WG. The cell-surface expression of CD45RO, CD45RA, CD62L, CCR3, CCR5 and CXCR3 was determined on blood-derived T cells by four-color flow cytometric analysis. The fractions of CCR5+ and CCR3+ cells within the CD4+CD45RO+ and CD8+CD45RO+ memory T cell populations were significantly expanded in localized and generalized WG. The mean percentage of Th1-type CCR5 expression was higher in localized WG. Upregulated CCR5 and CCR3 expression could also be detected on a fraction of CD45RA+ T cells. CD62L expression was seen on approximately half of the memory T cell populations expressing chemokine receptors. This study demonstrates for the first time that expression of the inducible inflammatory chemokine receptors CCR5 and CCR3 on CD45RO+ memory T cells, as well as on CD45RA+ T cells ('revertants'), contributes to phenotypic heterogeneity in an autoimmune disease, namely WG. Upregulated CCR5 and CCR3 expression suggests that the cells belong to the effector memory T cell population. CCR5 and CCR3 expression on CD4+ and CD8+ memory T cells indicates a potential to respond to chemotactic gradients and might be important in T cell migration contributing to granuloma formation and vasculitis in WG.     

EST sequencing of Onychophora and phylogenomic analysis of Metazoa

Onychophora (velvet worms) represent a small animal taxon considered to be related to Euarthropoda. We have obtained 1873 5' cDNA sequences (expressed sequence tags, ESTs) from the velvet worm Epiperipatus sp., which were assembled into 833 contigs. BLAST similarity searches revealed that 51.9% of the contigs had matches in the protein databases with expectation values lower than 10(-4). Most ESTs had the best hit with proteins from either Chordata or Arthropoda (approximately 40% respectively). The ESTs included sequences of 27 ribosomal proteins. The orthologous sequences from 28 other species of a broad range of phyla were obtained from the databases, including other EST projects. A concatenated amino acid alignment comprising 5021 positions was constructed, which covers 4259 positions when problematic regions were removed. Bayesian and maximum likelihood methods place Epiperipatus within the monophyletic Ecdysozoa (Onychophora, Arthropoda, Tardigrada and Nematoda), but its exact relation to the Euarthropoda remained unresolved. The "Articulata" concept was not supported. Tardigrada and Nematoda formed a well-supported monophylum, suggesting that Tardigrada are actually Cycloneuralia. In agreement with previous studies, we have demonstrated that random sequencing of cDNAs results in sequence information suitable for phylogenomic approaches to resolve metazoan relationships.     

Bacterial aggregates in the tentacles of the sea anemone <Emphasis Type="Italic">Metridium senile</Emphasis>

This paper provides first information on organ-like bacterial aggregates in the tentacles of the sea anemone Metridium senile. The specimens were collected from waters near Helgoland (German Bight, North Sea) and the Orkney Islands. Tentacles were prepared for morphological inspection by light and scanning electron microscopy as well as for the phylogenetic analysis of endocytic bacteria. Bacterial aggregates are located in caverns of the tentacles’ epidermis. The aggregates are enwrapped in thin envelopes, which contain coccoid and/or rod-shaped tightly packed bacteria of different division states. Most of the bacterial cells are connected by fine filamentous structures. The phylogenetic determination is based on the sequence data of the 16S rDNA derived from tentacle material. Sequence analysis revealed three different subgroups of intratentacular proteobacteria. The dominant band, detected in all of the samples tested, showed a close relationship (98%) to a gram-negative Endozoicimonas elysicola. Two bands, only detected in tentacles of M. senile from Helgoland were assigned to Pseudomonas saccherophilia (99%), a knallgas bacterium, and to Ralstonia pickettii (100%). The bacteria represent a specific bacterial community. Their DGGE profiles do not correspond to the profiles of the planktonic bacteria generated from seawater close to the habitats of the anemones. The allocation of DNA sequences to the different morphotypes, their isolation, culturing and the elucidation of the physiological functions of intratentacular bacteria are in progress.     

Competition versus facilitation: conspecific effects on pollinator visitation and seed set in the iris Lapeirousia oreogena

Ecological interactions between conspecific plants can range from facilitative to competitive depending on the spacing and abundance of individuals. Competitive interactions are expected when plants flower en‐masse and availability of pollinators is limited. We tested this prediction using Lapeirousia oreogena, a mass‐flowering South African iris that is pollinated by a single species of long‐proboscid fly. Controlled hand‐pollination experiments showed that L. oreogena is self‐compatible, but reliant on pollinator visits for seed set. Seed production per flower showed a significant negative relationship with patch size (and the correlated measure of number of individuals per patch), but was not affected by flower density or distance to neighbouring patches. There was a tendency for fly abundance to increase with patch size, but the rate of visits to individual flowers by flies was not affected by patch size. Seed set of hand‐pollinated flowers did not differ for plants in and out of dense patches, indicating that the large differences in seed set among patches were likely to reflect pollinator visits, rather than the genetic or physiological capacity of plants to produce seeds. The reduced fecundity of L. oreogena in large patches with a greater numbers of flowers is consistent with the idea that plants with highly specialized pollination systems can experience intra‐specific competition for pollination.     

Targeted On‐line SPE‐LC‐MS/MS Assay for the Quantitation of 12 Apolipoproteins from Human Blood

Laborious sample pretreatment of biological samples represents the most limiting factor for the translation of targeted proteomics assays from research to clinical routine. An optimized method for the simultaneous quantitation of 12 major apolipoproteins (apos) combining on‐line SPE and fast LC‐MS/MS analysis in 6.5 min total run time was developed, reducing the manual sample pretreatment time of 3 μL serum or plasma by 60%. Within‐run and between‐day imprecisions below 10 and 15% (n = 10) and high recovery rates (94–131%) were obtained applying the high‐throughput setup. High‐quality porcine trypsin was used, which outperformed cost‐effective bovine trypsin regarding digestion efficiency. Comparisons with immunoassays and another LC‐MS/MS assay demonstrated good correlation (Pearson's R: 0.81–0.98). Further, requirements on sample quality concerning sampling, processing, and long‐term storage up to 1 year were investigated revealing significant influences of the applied sampling material and coagulant on quantitation results. Apo profiles of 1339 subjects of the LIFE‐Adult‐Study were associated with lifestyle and physiological parameters as well as establish parameters of lipid metabolism (e.g., triglycerides, cholesterol). Besides gender effects, most significant impact was seen regarding lipid‐lowering medication. In conclusion, this novel highly standardized, high‐throughput targeted proteomics assay utilizes a fast, simultaneous analysis of 12 apos from least sample amounts.     

Plants,microorganisms, and soil temperatures contribute to a decrease in methane fluxes on a drained Arctic floodplain

As surface temperatures are expected to rise in the future, ice‐rich permafrost may thaw, altering soil topography and hydrology and creating a mosaic of wet and dry soil surfaces in the Arctic. Arctic wetlands are large sources of CH₄, and investigating effects of soil hydrology on CH₄ fluxes is of great importance for predicting ecosystem feedback in response to climate change. In this study, we investigate how a decade‐long drying manipulation on an Arctic floodplain influences CH₄‐associated microorganisms, soil thermal regimes, and plant communities. Moreover, we examine how these drainage‐induced changes may then modify CH₄ fluxes in the growing and nongrowing seasons. This study shows that drainage substantially lowered the abundance of methanogens along with methanotrophic bacteria, which may have reduced CH₄ cycling. Soil temperatures of the drained areas were lower in deep, anoxic soil layers (below 30 cm), but higher in oxic topsoil layers (0–15 cm) compared to the control wet areas. This pattern of soil temperatures may have reduced the rates of methanogenesis while elevating those of CH₄ oxidation, thereby decreasing net CH₄ fluxes. The abundance of Eriophorum angustifolium, an aerenchymatous plant species, diminished significantly in the drained areas. Due to this decrease, a higher fraction of CH₄ was alternatively emitted to the atmosphere by diffusion, possibly increasing the potential for CH₄ oxidation and leading to a decrease in net CH₄ fluxes compared to a control site. Drainage lowered CH₄ fluxes by a factor of 20 during the growing season, with postdrainage changes in microbial communities, soil temperatures, and plant communities also contributing to this reduction. In contrast, we observed CH₄ emissions increased by 10% in the drained areas during the nongrowing season, although this difference was insignificant given the small magnitudes of fluxes. This study showed that long‐term drainage considerably reduced CH₄ fluxes through modified ecosystem properties.     

Step by step removal of hyperforin and hypericin: activity profile of different Hypericum preparations in behavioral models

Herbal extracts of Hypericum perforatum L. (St. John's wort, SJW) are now successfully competing for status as a standard antidepressant therapy. Because of this, great effort has been devoted to identifying the antidepressive active compounds. In the present study we used the following strategy to evaluate the relative pharmacological importance of various extract components: 1. preparation of an hydroalcoholic SJW extract containing both hyperforin (3.2%) and hypericin (0.15%) (extract A); 2. step by step removal of hyperforin and hypericin led to the following extracts: Extract B, devoid of hyperforin but still containing hypericin (0.14%) and Extract C, free of hypericin and hyperforin but enriched in flavonoids ( approximately 12%). We characterized the in vivo activity profile of all three preparations using the tail suspension test (TST) in mice and the forced swimming test (FST) in rats as screening models. We further investigated the activity of pure hyperforin. Extract B and C (500 mg/kg each) as well as pure hyperforin (8 mg/kg) significantly shortened immobility time in the TST after acute pre-treatment whereas extract A was inactive. In the FST all three extracts decreased immobility time in a dosage of 500 mg/kg after acute as well as after repeated treatment. The present results clearly show that an SJW extract free of hyperforin and hypericin exerts antidepressant activity in behavioral models, supporting our working hypothesis that flavonoids are part of the constituents responsible for the therapeutic efficacy of SJW extracts. We also could show that hyperforin contributes to the beneficial properties of SJW extract, confirming the hypothesis that the crude SJW extract contains several constituents with antidepressant activity.