Association of fucoxanthin chlorophyll a/c-binding polypeptides with photosystems and phosphorylation in the centric diatom Cyclotella cryptica

Solubilization of thylakoid membranes of Cyclotella cryptica with dodecyl-beta maltoside followed by sucrose density gradient centrifugation or deriphate polyacrylamide gel electrophoresis resulted in the isolation of pigment protein complexes. These complexes were characterized by absorption and fluorescence spectroscopy, sodium dodecyl sulfate polyacrylamide gel electrophoresis and Western immunoblotting using antisera against fucoxanthin chlorophyll a/c-binding proteins and the reaction center protein D2 of photosystem II. Sucrose density gradient centrifugation yielded four bands. Band 1 consisted of free pigments with minor amounts of fucoxanthin chlorophyll a/c-binding proteins. Bands 2, 3, and 4 represented a major fucoxanthin chlorophyll a/c-binding protein fraction, photosystem II, and photosystem I, respectively. Deriphate polyacrylamide gel electrophoresis gave rise to five bands, representing photosystem I, photosystem II, two fucoxanthin chlorophyll a/c-binding protein complexes, and a band mostly consisting of free pigments. In the Western immunoblotting experiments, the specific association of two fucoxanthin chlorophyll a/c-binding proteins, Fcp2 and Fcp4, to the photosystems could be demonstrated. In vivo experiments using antibodies against phosphothreonine residues and in vitro studies using [gamma-32P]ATP showed that fucoxanthin chlorophyll a/c binding-proteins of 22 kDa became phosphorylated.     

Myelin basic protein-dependent plasma membrane reorganization in the formation of myelin

During vertebrate development, oligodendrocytes wrap their plasma membrane around axons to produce myelin, a specialized membrane highly enriched in galactosylceramide (GalC) and cholesterol. Here, we studied the formation of myelin membrane sheets in a neuron-glia co-culture system. We applied different microscopy techniques to visualize lipid packing and dynamics in the oligodendroglial plasma membrane. We used the fluorescent dye Laurdan to examine the lipid order with two-photon microscopy and observed that neurons induce a dramatic lipid condensation of the oligodendroglial membrane. On a nanoscale resolution, using stimulated emission depletion and fluorescence resonance energy transfer microscopy, we demonstrated a neuronal-dependent clustering of GalC in oligodendrocytes. Most importantly these changes in lipid organization of the oligodendroglial plasma membrane were not observed in shiverer mice that do not express the myelin basic protein. Our data demonstrate that neurons induce the condensation of the myelin-forming bilayer in oligodendrocytes and that MBP is involved in this process of plasma membrane rearrangement. We propose that this mechanism is essential for myelin to perform its insulating function during nerve conduction.     

Structure-functional analysis of the Dictyoglomus cell envelope

Several closely related strains of the thermophilic bacterium Dictyoglomus have been isolated from various hot springs on the Philippine archipelago. These strains as well as Dictyoglomus thermophilum H-6-12 were analyzed in view of the structure-functional relationships of the cell envelopes. All envelopes of Dictyoglomus strains show several peculiar features that are apparently either unique for the genus or common for other phylogenetically related Thermotogales. The filamentous cells develop pili at the cell poles, guided by large columnar protein assemblies that traverse the periplasm. Filamentous protein complexes span the periplasmic space at the longitudinal sides of the cell. By the end of the exponential growth phase, Dictyoglomus strains form multicellular aggregates ("rotund bodies") inside a compartment surrounded by a single, continuous outer envelope. The formation of these rotund bodies which are also found in some other deeply branching thermophilic phyla (Thermotoga, Thermus) was studied in detail. The transition between unicellular and multicellular lifestyle can be explained by the partial detachment of a protoplast from the outer envelope during cell division. When the outer envelope is partially detached from the protoplast, mechanical forces generated by protoplast elongation may drive cell rearrangement of daughter cells inside the compartment. During the following rounds of cell division, the overall shape of the compartment changes from spindle-like to globular geometry. Analysis of subcellular fractions of Dictyoglomus cells shows that glucan hydrolases are associated with the compartment. This feature is discussed in view of the multicellular life style of Dictyoglomus.     

Usefulness of the Serum Galactomannan Assay for Early Response Assessment and Treatment Stratifications of Invasive Aspergillosis

The galactomannan (GM) Enzyme Immunoassay (EIA) is an upcoming tool not only for diagnosis but also monitoring of invasive Aspergillosis (IA). Various studies were performed over the last years to apply such a promising instrument correctly. New findings show the potential of this test to segregate affected patients into treatment responders and non-responders at a time point as early as 7–14 days after initiation of antifungal therapy. Current data suggest that serial GM testing in patients receiving antifungal therapy for IA is essential as GMI kinetics may offer the clinician a substantial support in decision making concerning early therapeutic stratifications. The correct interpretation of GM EIA results with respect to the individual context of the patient is, however, absolutely necessary. The following review shall give an overview about the GM-EIA as a tool for IA monitoring and therapy stratification.     

Fbxw7 controls angiogenesis by regulating endothelial notch activity

Notch signaling controls fundamental aspects of angiogenic blood vessel growth including the selection of sprouting tip cells, endothelial proliferation and arterial differentiation. The E3 ubiquitin ligase Fbxw7 is part of the SCF protein complex responsible for the polyubiquitination and thereby proteasomal degradation of substrates such as Notch, c-Myc and c-Jun. Here, we show that Fbxw7 is a critical regulator of angiogenesis in the mouse retina and the zebrafish embryonic trunk, which we attribute to its role in the degradation of active Notch. Growth of retinal blood vessel was impaired and the Notch ligand Dll4, which is also a Notch target, upregulated in inducible and endothelial cell-specific Fbxw7(iECKO) mutant mice. The stability of the cleaved and active Notch intracellular domain was increased after siRNA knockdown of the E3 ligase in cultured human endothelial cells. Injection of fbxw7 morpholinos interfered with the sprouting of zebrafish intersegmental vessels (ISVs). Arguing strongly that Notch and not other Fbxw7 substrates are primarily responsible for these phenotypes, the genetic inactivation of Notch pathway components reversed the impaired ISV growth in the zebrafish embryo as well as sprouting and proliferation in the mouse retina. Our findings establish that Fbxw7 is a potent positive regulator of angiogenesis that limits the activity of Notch in the endothelium of the growing vasculature.     

Genomic Variation and Its Impact on Gene Expression in Drosophila melanogaster

Understanding the relationship between genetic and phenotypic variation is one of the great outstanding challenges in biology. To meet this challenge, comprehensive genomic variation maps of human as well as of model organism populations are required. Here, we present a nucleotide resolution catalog of single-nucleotide, multi-nucleotide, and structural variants in 39 Drosophila melanogaster Genetic Reference Panel inbred lines. Using an integrative, local assembly-based approach for variant discovery, we identify more than 3.6 million distinct variants, among which were more than 800,000 unique insertions, deletions (indels), and complex variants (1 to 6,000 bp). While the SNP density is higher near other variants, we find that variants themselves are not mutagenic, nor are regions with high variant density particularly mutation-prone. Rather, our data suggest that the elevated SNP density around variants is mainly due to population-level processes. We also provide insights into the regulatory architecture of gene expression variation in adult flies by mapping cis-expression quantitative trait loci (cis-eQTLs) for more than 2,000 genes. Indels comprise around 10% of all cis-eQTLs and show larger effects than SNP cis-eQTLs. In addition, we identified two-fold more gene associations in males as compared to females and found that most cis-eQTLs are sex-specific, revealing a partial decoupling of the genomic architecture between the sexes as well as the importance of genetic factors in mediating sex-biased gene expression. Finally, we performed RNA-seq-based allelic expression imbalance analyses in the offspring of crosses between sequenced lines, which revealed that the majority of strong cis-eQTLs can be validated in heterozygous individuals.     

Consequences of altered eicosanoid patterns for nociceptive processing in mPGES-1-deficient mice

Cyclooxygenase-2 (COX-2)-dependent prostaglandin (PG) E(2) synthesis in the spinal cord plays a major role in the development of inflammatory hyperalgesia and allodynia. Microsomal PGE(2) synthase-1 (mPGES-1) isomerizes COX-2-derived PGH(2) to PGE(2). Here, we evaluated the effect of mPGES-1-deficiency on the nociceptive behavior in various models of nociception that depend on PGE(2) synthesis. Surprisingly, in the COX-2-dependent zymosan-evoked hyperalgesia model, the nociceptive behavior was not reduced in mPGES-1-deficient mice despite a marked decrease of the spinal PGE(2) synthesis. Similarly, the nociceptive behavior was unaltered in mPGES-1-deficient mice in the formalin test. Importantly, spinal cords and primary spinal cord cells derived from mPGES-1-deficient mice showed a redirection of the PGE(2) synthesis to PGD(2), PGF(2alpha) and 6-keto-PGF(1alpha) (stable metabolite of PGI(2)). Since the latter prostaglandins serve also as mediators of nociception they may compensate the loss of PGE(2) synthesis in mPGES-1-deficient mice.     

Sphingosine 1-phosphate modulates spinal nociceptive processing

Sphingosine 1-Phosphate (S1P) modulates various cellular functions such as apoptosis, cell differentiation, and migration. Although S1P is an abundant signaling molecule in the central nervous system, very little is known about its influence on neuronal functions. We found that S1P concentrations were selectively decreased in the cerebrospinal fluid of adult rats in an acute and an inflammatory pain model. Pharmacological inhibition of sphingosine kinases (SPHK) decreased basal pain thresholds and SphK2 knock-out mice, but not SphK1 knock-out mice, had a significant decrease in withdrawal latency. Intrathecal application of S1P or sphinganine 1-phosphate (dihydro-S1P) reduced the pain-related (nociceptive) behavior in the formalin assay. S1P and dihydro-S1P inhibited cyclic AMP (cAMP) synthesis, a key second messenger of spinal nociceptive processing, in spinal cord neurons. By combining fluorescence resonance energy transfer (FRET)-based cAMP measurements with Multi Epitope Ligand Cartography (MELC), we showed that S1P decreased cAMP synthesis in excitatory dorsal horn neurons. Accordingly, intrathecal application of dihydro-S1P abolished the cAMP-dependent phosphorylation of NMDA receptors in the outer laminae of the spinal cord. Taken together, the data show that S1P modulates spinal nociceptive processing through inhibition of neuronal cAMP synthesis.     

Subtypes of melanocytes and melanoma cells distinguished by their intercellular contacts: heterotypic adherens junctions,adhesive associations,and dispersed desmoglein 2 glycoproteins

In the tissue integration of melanocytes and melanoma cells, an important role is attributed to cell adhesion molecules, notably the cadherins. In cultured melanoma cells, we have previously described a more heterogeneous repertoire of cadherins than normal, including some melanoma subtypes synthesizing the desmosomal cadherin, desmoglein 2, out of the desmosomal context. Using biochemical and immunological characterization of junctional molecules, confocal laser scanning, and electron and immunoelectron microscopy, we now demonstrate homo- and heterotypic cell-cell adhesions of normal epidermal melanocytes. In human epidermis, both in situ and in cell culture, melanocytes and keratinocytes are connected by closely aligned membranes that are interspersed by small puncta adhaerentia containing heterotypic complexes of E- and P-cadherin. Moreover, melanocytes growing in culture often begin to synthesize desmoglein 2, which is dispersed over extended areas of intimate adhesive cell-cell associations. As desmoglein 2 is not found in melanocytes in situ, we hypothesize that its synthesis is correlated with cell proliferation. Indeed, in tissue microarrays, desmoglein 2 has been demonstrated in a sizable subset of nevi and primary melanomas. The biological meanings of these cell-cell adhesion molecule arrangements, the possible diagnostic and prognostic significance of these findings, and the implications of the heterogeneity types of melanomas are discussed. Electronic supplementary material  The online version of this article (doi:) contains supplementary material, which is available to authorized users. This work was supported in parts by grants from the Deutsche Forschungsgemeinschaft to W. K. Peitsch (project PE 896/1) and the Deutsche Krebshilfe to W. W. Franke (project 10-2049).