Cloning and molecular analysis of the poly(3-hydroxybutyric acid) biosynthetic genes of Thiocystis violacea

From a genomic library of Thiocystis violaceae strain 2311 in L47, two adjacent EcoRI restriction fragments of 5361 base pairs (bp) and of 1978 bp were cloned. The 5361-bp EcoRI restriction fragment hybridized with a DNA fragment harbouring the Alcaligenes eutrophus poly(3-hydroxyalkanoate) (PHA) synthase operon (phbCAB) and restored the ability to synthesize and accumulate PHA in PHA-negative mutants derived from A. eutrophus. The nucleotide sequence analysis of both fragments revealed five open-reading frames (ORFs); at least three of them are probably relevant for PHA biosynthesis. The amino acid sequences of the putative proteins deduced from these genes indicate that they encode a -ketothiolase [phbA _Tv, relative molecular mass (M_r) 40850], which exhibited 87.3% amino acid identify with the -ketothiolase from Chromatium vinosum. The amino acid sequences of the putative proteins deduced from ORF2_Tv (M_r 41 450) and phbC _Tv (M_r 39 550), which were located upstream of and antilinear to phbA _Tv, exhibited 74.7% and 87.6% amino acid identify, respectively, with the corresponding gene products of C. vinosum. Downstream of and antilinear to phbC _Tv was located ORF5, which encodes for a protein of high relative molecular mass (M_r 76428) of unknown function. With respect to the divergent organisation of ORF2_Tv and phbC _Tv on one side and of phbA _Tv on the other side and from the homologies of the putative gene products, this region of the T. violaceae genome resembled very much the corresponding region of C. vinosum, which was identified recently. Correspondence to: A. Steinbüchel     

Structures of lysenin reveal a shared evolutionary origin for pore-forming proteins and its mode of sphingomyelin recognition

Pore-forming proteins insert from solution into membranes to create lesions, undergoing a structural rearrangement often accompanied by oligomerization. Lysenin, a pore-forming toxin from the earthworm Eisenia fetida, specifically interacts with sphingomyelin (SM) and may confer innate immunity against parasites by attacking their membranes to form pores. SM has important roles in cell membranes and lysenin is a popular SM-labeling reagent. The structure of lysenin suggests common ancestry with other pore-forming proteins from a diverse set of eukaryotes and prokaryotes. The complex with SM shows the mode of its recognition by a protein in which both the phosphocholine headgroup and one acyl tail are specifically bound. Lipid interaction studies and assays using viable target cells confirm the functional reliance of lysenin on this form of SM recognition.     

Global patterns in biomass models describing prey consumption by big cats

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A (1-->3)-beta-D-glucan recognition protein from the sponge Suberites domuncula. Mediated activation of fibrinogen-like protein and epidermal growth factor gene expression.

Sponges (phylum Porifera) live in a symbiotic relationship with microorganisms, primarily bacteria. Until now, molecular proof for the capacity of sponges to recognize fungi in the surrounding aqueous milieu has not been available. Here we demonstrate, for the demosponge Suberites domuncula (Porifera, Demospongiae, Hadromerida), a cell surface receptor that recognizes (1-->3)-beta-D-glucans, e.g. curdlan or laminarin. This receptor, the (1-->3)-beta-D-glucan-binding protein, was identified and its cDNA analysed. The gene coding for the 45 kDa protein was found to be upregulated in tissue after incubation with carbohydrate. Simultaneously with the increased expression of this gene, two further genes showed an elevated steady state level of expression; one codes for a fibrinogen-like protein and the other for the epidermal growth factor precursor. Expression of the (1-->3)-beta-D-glucan-binding protein and the fibrinogen-like protein occurred in cells on the sponge surface, in the pinacoderm. By Western blotting, the product of the fibrinogen-like protein gene was identified, the recombinant protein isolated, and antibodies raised to this protein. Their application revealed that a 5 kDa factor is produced, which is apparently processed from the 77 kDa epidermal growth factor precursor. Finally, we provided evidence that a tyrosine kinase pathway is initiated in response to exposure to D-glucan; its phosphorylation activity could be blocked by aeroplysinin. In turn, the increased expression of the downstream genes was suppressed. We conclude that sponges possess a molecular mechanism for recognizing fungi via the d-glucan carbohydrates on their surfaces.     

Directed cortical information flow during human object recognition: analyzing induced EEG gamma-band responses in brain's source space

The increase of induced gamma-band responses (iGBRs; oscillations >30 Hz) elicited by familiar (meaningful) objects is well established in electroencephalogram (EEG) research. This frequency-specific change at distinct locations is thought to indicate the dynamic formation of local neuronal assemblies during the activation of cortical object representations. As analytically power increase is just a property of a single location, phase-synchrony was introduced to investigate the formation of large-scale networks between spatially distant brain sites. However, classical phase-synchrony reveals symmetric, pair-wise correlations and is not suited to uncover the directionality of interactions. Here, we investigated the neural mechanism of visual object processing by means of directional coupling analysis going beyond recording sites, but rather assessing the directionality of oscillatory interactions between brain areas directly. This study is the first to identify the directionality of oscillatory brain interactions in source space during human object recognition and suggests that familiar, but not unfamiliar, objects engage widespread reciprocal information flow. Directionality of cortical information-flow was calculated based upon an established Granger-Causality coupling-measure (partial-directed coherence; PDC) using autoregressive modeling. To enable comparison with previous coupling studies lacking directional information, phase-locking analysis was applied, using wavelet-based signal decompositions. Both, autoregressive modeling and wavelet analysis, revealed an augmentation of iGBRs during the presentation of familiar objects relative to unfamiliar controls, which was localized to inferior-temporal, superior-parietal and frontal brain areas by means of distributed source reconstruction. The multivariate analysis of PDC evaluated each possible direction of brain interaction and revealed widespread reciprocal information-transfer during familiar object processing. In contrast, unfamiliar objects entailed a sparse number of only unidirectional connections converging to parietal areas. Considering the directionality of brain interactions, the current results might indicate that successful activation of object representations is realized through reciprocal (feed-forward and feed-backward) information-transfer of oscillatory connections between distant, functionally specific brain areas.     

Expression of marker genes during early ear development in medaka

Induction of the otic placode involves a number of regulatory interactions. Early studies revealed that the induction of this program is initiated by instructive signals from the mesendoderm as well as from the adjacent hindbrain. Further investigations on the molecular level identified in zebrafish Fgf3, Fgf8, Foxi1, Pax8, Dlx3b and Dlx4b genes as key players during the induction phase. Thereafter an increasing number of genes participates in the regulatory interactions finally resulting in a highly structured sensory organ. Based on data from zebrafish we selected medaka genes with presumptive functions during early ear development for an expression analysis. In addition we isolated Foxi1 and Dlx3b gene fragments from embryonic cDNA. Altogether we screened the spatio-temporal distribution of more than 20 representative marker genes for otic development in medaka embryos, with special emphasis on the early phases. Whereas the spatial distribution of these genes is largely conserved between medaka and zebrafish, our comparative analysis revealed several differences, in particular for the timing of expression.     

Different capacity of monocyte subsets to phagocytose iron-oxide nanoparticles

Objective

To explore the capacity of human CD14⁺CD16⁺⁺ and CD14⁺⁺CD16^- monocytes to phagocyte iron-oxide nanoparticles in vitro.

Methods

Human monocytes were labeled with four different magnetic nanoparticle preparations (Ferumoxides, SHU 555C, CLIO-680, MION-48) exhibiting distinct properties and cellular uptake was quantitatively assessed by flow cytometry, fluorescence microscopy, atomic absorption spectrometry and Magnetic Resonance Imaging (MRI). Additionally we determined whether cellular uptake of the nanoparticles resulted in phenotypic changes of cell surface markers.

Results

Cellular uptake differed between the four nanoparticle preparations. However for each nanoparticle tested, CD14⁺⁺CD16^- monocytes displayed a significantly higher uptake compared to CD14⁺CD16⁺⁺ monocytes, this resulted in significantly lower T1 and T2 relaxation times of these cells. The uptake of iron-oxide nanoparticles further resulted in a remarkable shift of expression of cell surface proteins indicating that the labeling procedure affects the phenotype of CD14⁺CD16⁺⁺ and CD14⁺⁺CD16^- monocytes differently.

Conclusion

Human monocyte subsets internalize different magnetic nanoparticle preparations differently, resulting in variable loading capacities, imaging phenotypes and likely biological properties.     

Müller glial cell-provided cellular light guidance through the vital guinea-pig retina

In vertebrate eyes, images are projected onto an inverted retina where light passes all retinal layers on its way to the photoreceptor cells. Light scattering within this tissue should impair vision. We show that radial glial (Müller) cells in the living retina minimize intraretinal light scatter and conserve the diameter of a beam that hits a single Müller cell endfoot. Thus, light arrives at individual photoreceptors with high intensity. This leads to an optimized signal/noise ratio, which increases visual sensitivity and contrast. Moreover, we show that the ratio between Müller cells and cones-responsible for acute vision-is roughly 1. This suggests that high spatiotemporal resolution may be achieved by each cone receiving its part of the image via its individual Müller cell-light guide.