Development of the nervous system in the "head" of <Emphasis Type="Italic">Limulus polyphemus</Emphasis> (Chelicerata: Xiphosura): morphological evidence for a correspondence between the segments of the chelicerae and of the (first) antennae of Mandibulata

We investigated brain development in the horseshoe crab Limulus polyphemus and several other arthropods via immunocytochemical methods, i.e. antibody stainings against acetylated alpha-tubulin and synapsin. According to the traditional view, the first appendage-bearing segment in chelicerates (the chelicerae) is not homologous to the first appendage-bearing segment of mandibulates (first antenna, deutocerebrum) but to the segment of the second antenna (tritocerebrum) or the intercalary segment in hexapods and myriapods. Accordingly, the segment of the deutocerebrum in chelicerates would be completely reduced. The main arguments for this view are: (1) the postoral origin of the cheliceral ganglion, (2) a poststomodaeal commissure, and (3) a connection of the cheliceral ganglion to the stomatogastric system. Our data show that these arguments are not convincing. During the development of horseshoe crabs there is no evidence for a former additional segment in front of the chelicerae. Instead, comparison of the brain structure (neuropil ring) between chelicerates, crustaceans and insects shows remarkable similarities. Furthermore, the cheliceral commissure in horseshoe crabs runs mainly praestomodaeal, which would be unique for a tritocerebral commissure. An unbiased view of the developing nervous system in the "head" of chelicerates, crustaceans and insects leads to a homologisation of the cheliceral segment and that of the (first) antenna (= deutocerebrum) of mandibulates that is also congruous to the interpretation of the Hox gene expression patterns. Thus, our data provide morphological evidence for the existence of a chelicerate deutocerebrum.     

A developmentally regulated aconitase related to iron-regulatory protein-1 is localized in the cytoplasm and in the mitochondrion of Trypanosoma brucei

Mitochondrial energy metabolism and Krebs cycle activities are developmentally regulated in the life cycle of the protozoan parasite Trypanosoma brucei. Here we report cloning of a T. brucei aconitase gene that is closely related to mammalian iron-regulatory protein 1 (IRP-1) and plant aconitases. Kinetic analysis of purified recombinant TbACO expressed in Escherichia coli resulted in a K(m) (isocitrate) of 3 +/- 0.4 mM, similar to aconitases of other organisms. This was unexpected since an arginine conserved in the aconitase protein family and crucial for substrate positioning in the catalytic center and for activity of pig mitochondrial aconitase (Zheng, L., Kennedy, M. C., Beinert, H., and Zalkin, H. (1992) J. Biol. Chem. 267, 7895-7903) is substituted by leucine in the TbACO sequence. Expression of the 98-kDa TbACO was shown to be lowest in the slender bloodstream stage of the parasite, 8-fold elevated in the stumpy stage, and increased a further 4-fold in the procyclic stage. The differential expression of TbACO protein contrasted with only minor changes in TbACO mRNA, indicating translational or post-translational mechanisms of regulation. Whereas animal cells express two distinct compartmentalized aconitases, mitochondrial aconitase and cytoplasmic aconitase/IRP-1, TbACO accounts for total aconitase activity in trypanosomes. By cell fractionation and immunofluorescence microscopy, we show that native as well as a transfected epitope-tagged TbACO localizes in both the mitochondrion (30%) and in the cytoplasm (70%). Together with phylogenetic reconstructions of the aconitase family, this suggests that animal IRPs have evolved from a multicompartmentalized ancestral aconitase. The possible functions of a cytoplasmic aconitase in trypanosomes are discussed.     

Nonlinear changes of transmembrane potential caused by defibrillation shocks in strands of cultured myocytes

Organization of cardiac tissue into cell strands and layers has been implicated in changes of transmembrane potential (DeltaV(m)) during defibrillation. To determine the shock-induced DeltaV(m) in such structures, cell strands of variable width [strand width (SW) = 0.15-2 mm] were grown in culture. Uniform-field shocks with variable strength [shock strength (SS) = 2-50 V/cm] were applied across strands during the action potential (AP) plateau, and DeltaV(m) were measured optically. Three different types of DeltaV(m) were observed. Small DeltaV(m) [<40%AP amplitude (APA)] were linearly dependent on SS and SW and were symmetrically distributed about a strand centerline with maximal positive and negative DeltaV(m) on opposite strand sides being equal. Intermediate DeltaV(m) (<200%APA) were strongly asymmetric with negative DeltaV(m) > positive DeltaV(m) because of a negative time-dependent shift of V(m) at the depolarized side of the strands. For large DeltaV(m) (>200%APA), a second time-dependent shift of V(m) to more positive levels was observed in the hyperpolarized portions of strands, causing reduction of the DeltaV(m) asymmetry. We conclude that during application of shocks to cell strands during the AP plateau, passive changes of V(m) were followed by two voltage- and time-dependent shifts of V(m), possibly reflecting changes of ionic currents or membrane electroporation.     

Cell-specific expression of the mercury-insensitive plasma-membrane aquaporin NtAQP1 from Nicotiana tabacum

 The aquaporin NtAQP1 from Nicotiana tabacum L. is insensitive to heavy-metal ions. In addition to water, the transport of urea or glycerol is facilitated by this plasma-membrane-located water channel. Northern hybridization and whole-mount in situ hybridization revealed a high steady-state level of NtAQP1-RNA in roots, a decreased content in shoots and a low content in leaves. By immunolocalization with an antibody targeted to the N-terminus of the aquaporin, the localization of NtAQP1-protein at sites of expected high water transport rates from and to the apoplast or symplast could be demonstrated. The specific pattern of NtAQP1 distribution in petioles strongly indicates a transcellular movement of water. Received: 12 August 1999 / Accepted: 27 December 1999     

Intracellular pathways and degradation of endosomal contents in basal epithelial cells of freshwater sponges (Porifera, Spongillidae)

 The digestive system expressed by basal epithelial cells of the freshwater sponges Spongilla lacustris and Ephydatia muelleri is mainly represented by a population of 30–50 preexisting lysosomes located in the close vicinity of the central nucleus. The strongly acidic vacuoles (pH 4–4.5) vary in size between 1 and 3 μm, and contain a set of different lysosomal enzymes. Immunocytochemical studies succeeded in the detection of β-hexosaminidase, cathepsin D, acid phosphatase, and α-glucosidase. Endosomes resulting from fluid-phase macropinocytosis, receptor-mediated endocytosis, or phagocytotic activity deliver their exogenous contents to the preexisting lysosomes for enzymatic degradation. Macropinosomes and phagosomes follow a rather reduced intracellular pathway by immediate fusion with the lysosomal compartment, whereas substances conveyed by coated vesicles pass through two additional vacuolar stages, namely early and late endosomes. Early endosomes serve as sorting organelles and segregate various constituents of complex ligands (BSA-AU_6, BSA-AU₁₂) by size into individual late endosomes, which then coalesce with preexisting lysosomes. As a whole, the intracellular pathways and hydrolytic processing of endosomal and phagosomal contents in freshwater sponge cells share certain similarities with the respective mechanisms in cells of higher eukaryotes. Accepted: 7 October 1997     

Gene Control in Somatic Embryos of Digitalis lanata: Expression of the β-Glucuronidase Gene Fused to a Plastocyanin Promoter

Digitalis lanata was transformed by agrobacteria-mediated gene transfer with a chimeric reporter gene encoding for β-glucuronidase (CUS) from Escherichia coll under the control of the plastocyanin 3 (Pc3) promoter from Spinada oleracea (Pc3::uidA fusion gene). Transformed cell lines were regenerated to plants via somatic embryos. CUS activity was determined fluorometrically and histochemically. The Pc3::uidA fusion gene was expressed in the late globular and bipolar stages of somatic embryos. Expression started in globular embryos (stage-1-globules) in that part of the parenchymatic tissue which later on formed the cotyledons. No GUS activity was detectable in the parenchymatic tissue forming the root pole, in cells of the developing procambium or in epidermal cells. These tissues were free of GUS activity also in bipolar embryos. The parenchymatic cells of the cotyledons and the primary cortex of the hypocotyl of germinating embryos showed GUS activity, in contrast to the epidermal cells and the cells of the central cylinder.     

Linking aphid honeydew, throughfall, and forest floor solution chemistry of Norway spruce

In a seminatural manipulation experiment with artificial irrigation we followed throughfall and forest floor solution chemistry collected underneath aphid infested and uninfested Norway spruce. Solutions underneath infested trees showed significantly higher concentrations of dissolved organic carbon (DOC) but lower concentrations of dissolved organic nitrogen (DON), NO₃-N, and NH₄-N in throughfall solutions and of NH₄-N in forest floor solutions. Average concentrations were 40.5% (DON), 27.5% (NO₃-N), and 46.2% lower (NH₄-N) underneath infested trees in throughfall solutions, and 19.5% (DON), 9.4% (NO₃-N), and 42.0% (NH₄-N) lower in forest floor solutions. Differences in throughfall were more pronounced than in forest floor leachates. It is likely that honeydew is fuelling the metabolism of micro-organisms and thus critically affects above and below ground nutrient cycles. We emphasize the importance of linking the biology of herbivores and micro-organisms with geochemical processes.     

Extraction and long‐term storage of S‐layer proteins and flagella from Lysinibacillus sphaericus NCTC 9602: Building blocks for nanotechnology

Self‐assembling surface layer (SL) proteins of bacteria have been widely studied, in particular their use as molecularly defined, 2D coatings of technical surfaces. An important prerequisite is the availability of a sufficient amount of protein. However, a detailed and optimized protocol for the complete SL extraction is so far not available. Here, we describe the complete purification and reassembly procedure of an SL protein of Lysinibacillus sphaericus NCTC 9602, starting from the cultivation of cells, the preparation and purification of SL proteins up to the long‐term storage and in vitro self‐assembly of the proteins. All crucial steps of the procedure are assessed by different microscopic techniques, such as light microscopy, atomic force microscopy, and scanning electron microscopy as well as by SDS‐PAGE as a biochemical method. We demonstrate that storage of the protein in the presence of sodium azide or upon lyophilization allows the preservation of the self‐assembly properties for at least 9 years. Additionally, we describe a method allowing the extraction of intact flagella with lengths in the range up to 4 μm. Flagella may have applications in bio‐nanotechnology, for example as templates for metallic nanowires.     

A Phase I Double Blind,Placebo-Controlled,Randomized Study of the Safety and Immunogenicity of an Adjuvanted HIV-1 Gag-Pol-Nef Fusion Protein and Adenovirus 35 Gag-RT-Int-Nef Vaccine in Healthy HIV-Uninfected African Adults

Background

Sequential prime-boost or co-administration of HIV vaccine candidates based on an adjuvanted clade B p24, RT, Nef, p17 fusion protein (F4/AS01) plus a non-replicating adenovirus 35 expressing clade A Gag, RT, Int and Nef (Ad35-GRIN) may lead to a unique immune profile, inducing both strong T-cell and antibody responses.

Methods

In a phase 1, double-blind, placebo-controlled trial, 146 healthy adult volunteers were randomized to one of four regimens: heterologous prime-boost with two doses of F4/AS01_E or F4/AS01_B followed by Ad35-GRIN; Ad35-GRIN followed by two doses of F4/AS01_B; or three co-administrations of Ad35-GRIN and F4/AS01_B. T cell and antibody responses were measured.

Results

The vaccines were generally well-tolerated, and did not cause serious adverse events. The response rate, by IFN-γ ELISPOT, was greater when Ad35-GRIN was the priming vaccine and in the co-administration groups. F4/AS01 induced CD4+ T-cells expressing primarily CD40L and IL2 +/- TNF-α, while Ad35-GRIN induced predominantly CD8+ T-cells expressing IFN-γ +/- IL2 or TNF-α. Viral inhibition was induced after Ad35-GRIN vaccination, regardless of the regimen. Strong F4-specific antibody responses were induced. Immune responses persisted at least a year after the last vaccination. The complementary response profiles, characteristic of each vaccine, were both expressed after co-administration.

Conclusion

Co-administration of an adjuvanted protein and an adenovirus vector showed an acceptable safety and reactogenicity profile and resulted in strong, multifunctional and complementary HIV-specific immune responses.

Trial Registration

ClinicalTrials.gov NCT01264445     

The Temporal Expression Pattern of Alpha-Synuclein Modulates Olfactory Neurogenesis in Transgenic Mice

Background

Adult neurogenesis mirrors the brain´s endogenous capacity to generate new neurons throughout life. In the subventricular zone/ olfactory bulb system adult neurogenesis is linked to physiological olfactory function and has been shown to be impaired in murine models of neuronal alpha-Synuclein overexpression. We analyzed the degree and temporo-spatial dynamics of adult olfactory bulb neurogenesis in transgenic mice expressing human wild-type alpha-Synuclein (WTS) under the murine Thy1 (mThy1) promoter, a model known to have a particularly high tg expression associated with impaired olfaction.

Results

Survival of newly generated neurons (NeuN-positive) in the olfactory bulb was unchanged in mThy1 transgenic animals. Due to decreased dopaminergic differentiation a reduction in new dopaminergic neurons within the olfactory bulb glomerular layer was present. This is in contrast to our previously published data on transgenic animals that express WTS under the control of the human platelet-derived growth factor β (PDGF) promoter, that display a widespread decrease in survival of newly generated neurons in regions of adult neurogenesis, resulting in a much more pronounced neurogenesis deficit. Temporal and quantitative expression analysis using immunofluorescence co-localization analysis and Western blots revealed that in comparison to PDGF transgenic animals, in mThy1 transgenic animals WTS is expressed from later stages of neuronal maturation only but at significantly higher levels both in the olfactory bulb and cortex.

Conclusions

The dissociation between higher absolute expression levels of alpha-Synuclein but less severe impact on adult olfactory neurogenesis in mThy1 transgenic mice highlights the importance of temporal expression characteristics of alpha-Synuclein on the maturation of newborn neurons.