Neurexins induce differentiation of GABA and glutamate postsynaptic specializations via neuroligins

Formation of synaptic connections requires alignment of neurotransmitter receptors on postsynaptic dendrites opposite matching transmitter release sites on presynaptic axons. beta-neurexins and neuroligins form a trans-synaptic link at glutamate synapses. We show here that neurexin alone is sufficient to induce glutamate postsynaptic differentiation in contacting dendrites. Surprisingly, neurexin also induces GABA postsynaptic differentiation. Conversely, neuroligins induce presynaptic differentiation in both glutamate and GABA axons. Whereas neuroligins-1, -3, and -4 localize to glutamate postsynaptic sites, neuroligin-2 localizes primarily to GABA synapses. Direct aggregation of neuroligins reveals a linkage of neuroligin-2 to GABA and glutamate postsynaptic proteins, but the other neuroligins only to glutamate postsynaptic proteins. Furthermore, mislocalized expression of neuroligin-2 disperses postsynaptic proteins and disrupts synaptic transmission. Our findings indicate that the neurexin-neuroligin link is a core component mediating both GABAergic and glutamatergic synaptogenesis, and differences in isoform localization and binding affinities may contribute to appropriate differentiation and specificity.     

The MHC class I-like IgG receptor controls perinatal IgG transport,IgG homeostasis,and fate of IgG-Fc-coupled drugs

Abs of the IgG isotype are efficiently transported from mother to neonate and have an extended serum t(1/2) compared with Abs of other isotypes. Circumstantial evidence suggests that the MHC class I-related protein, the neonatal FcR (FcRn), is the FcR responsible for both in vivo functions. To understand the phenotypes imposed by FcRn, we produced and analyzed mice with a defective FcRn gene. The results provide direct evidence that perinatal IgG transport and protection of IgG from catabolism are mediated by FcRn, and that the latter function is key to IgG homeostasis, essential for generating a potent IgG response to foreign Ags, and the basis of enhanced efficacy of Fc-IgG-based therapeutics. FcRn is therefore a promising therapeutic target for enhancing protective humoral immunity, treating autoimmune disease, and improving drug efficacy.     

Vascular endothelial cells express a functional fas-receptor due to lack of hemodynamic forces

The fas system is present in atherosclerotic lesions. However, its role in the initiation and progression is still unclear. Here we show that in endothelial cells (EC) the expression of the fas receptor is regulated by flow conditions. The EC of the vascular system are regularly exposed to a range of hemodynamic forces with great impact on cellular structures and functions. Recently it was reported that in endothelial cells the lack of hemodynamic forces as well as irregular flow conditions trigger apoptosis by induction of a mechanosensitive autocrine loop of thrombospondin-1 and the _V3 integrin/integrin-associated protein complex. Here we show that EC cultivated under regular laminar flow conditions are devoid of the fas-receptor whereas cultivation under static conditions as well as under turbulence leads to its expression. Stimulation of the fas-receptor by its ligand increases the amount of apoptotic cells by twofold; the increase can be prevented by blocking the fas-receptor. The availability of the expressed fas receptor for stimulation by its ligand hints at a role as a tool for progression of atherosclerosis.     

Development of an in vitro integration assay for the Bacteroides conjugative transposon CTnDOT

Integrated self-transmissible elements called conjugative transposons (CTns) are responsible for the transfer of antibiotic resistance genes in many different species of bacteria. One of the best characterized of these newly recognized elements is the Bacteroides CTn, CTnDOT. CTnDOT is thought to have a circular transfer intermediate that transfers to and integrates into the genome of the recipient cell. Previous investigations of the mechanism of CTnDOT integration have been hindered by the lack of an in vitro system for checking this model of integration and determining whether the CTnDOT integrase alone was sufficient to catalyze the integration reaction or whether host factors might be involved. We report here the development of an in vitro system in which a plasmid containing the joined ends of CTnDOT integrates into a plasmid carrying a CTnDOT target site. To develop this in vitro system, a His-tagged version of the integrase gene of CTnDOT was cloned and shown to be active in vivo. The protein produced by this construct was partially purified and then added to a reaction mixture that contained the joined ends of the circular form of CTnDOT and a plasmid carrying one of the CTnDOT target sites. Integration was demonstrated by using a fairly simple mixture of components, but integration was stimulated by a Bacteroides extract or by purified Escherichia coli integration host factor. The results of this study demonstrate both that the circular form of CTnDOT is the form that integrates into the target site and that host factors are involved in the integration process.     

Mechanosensitive induction of apoptosis in fibroblasts is regulated by thrombospondin-1 and integrin associated protein (CD47)

Fibroblasts are cultured in three-dimensional collagen matrices to investigate the effect of mechanical tension on the regulation of apoptosis. Under the influence of mechanical loading, the cells show little apoptosis whereas releasing of tension leads to an increase up to tenfold during the first 24 h and remains constant for further 48 h. An autocrine loop of the integrin _V3/CD47 receptor complex and thrombospondin-1 is identified as the molecular coupling device between mechanical loading and apoptosis: The integrin _V3 is expressed under mechanical loading as well as unloading whereas the CD47 could only be identified after the release of tension. The secreted thrombospondin binds to the active receptor and induces apoptosis. The presented mechanosensitive regulation of apoptosis in fibroblast cultures could be an essential mechanism for the regression of the granulation tissue by apoptosis in the process of wound healing.     

Mitochondria-rich cells as experimental model in studies of epithelial chloride channels

The mitochondria-rich (mr) cell of amphibian skin epithelium is differentiated as a highly specialised pathway for passive transepithelial transport of chloride. The apical membrane of mr cells expresses several types of Cl(-) channels, of which the function of only two types has been studied in detail. (i) One type of channel is gated by voltage and external chloride concentration. This intriguing type of regulation leads to opening of channels only if [Cl(-)](o) is in the millimolar range and if the electrical potential is of a polarity that secures an inwardly directed net flux of this ion. Reversible voltage activations of the conductance proceed with long time constants, which depend on V in such a way that the rate of conductance activation increases when V is clamped at more negative values (serosal bath grounded). The gating seems to involve processes that are dependent on F-actin localised in the submembrane domain in the neck region of the flask-shaped mr cell. (ii) The other identified Cl(-) pathway of mr cells is mediated by small-conductance apical CFTR chloride channels as concluded from its activation via beta-adrenergic receptors, ion selectivity, genistein stimulation and inhibition by glibenclamide. bbCFTR has been cloned, and immunostaining has shown that the gene product is selectively expressed in mr cells. There is cross-talk between the two pathways in the sense that activation of the conductance of the mr cell by voltage clamping excludes activation via receptor occupation, and vice versa. The mechanism of this cross-talk is unknown.     

Effect of unilateral partial facial paralysis on periosteal growth at the muscle-bone interface of facial muscles and facial bones

In a previous study, the influence of the midfacial musculature upon growth and development of the maxilla and mandible was established macroscopically. Dry skull measurements revealed a reduced premaxillary, maxillary, mandibular, and anterior corpus length with a simultaneous increase in mandibular ramal height on the paralyzed side. It was demonstrated that these reduced premaxillary and maxillary lengths were among others the result of reduced nasofrontal growth, whereas the increased ramal height was accompanied by condylar growth alterations. This study investigated whether the growth alterations at the mandibular corpus region could be explained by altered periosteal growth at the muscle-bone interface of the zygomatico-auricular muscle and the mandibular corpus, caused by altered muscle activity acting upon the periosteal sleeve. Fifty-six 12-day-old New Zealand White rabbits were randomly assigned to either a control or an experimental group. In the experimental group, left-sided partial facial paralysis was induced surgically when the animals were 12 days old. To study the muscle-bone interface, seven follow-up time intervals were defined between 3.5 and 60 days following the surgery. At these time intervals, four randomly selected control animals and four randomly selected experimental animals were killed. The anterior mandibular corpus region with the muscle-bone interface of the left control hemimandible and the left and right experimental hemimandibles was processed for undecalcified tissue preparation. Quantitative analysis of the total bone area at the muscle-bone interface revealed no significant differences between the left control hemimandible and the left and right experimental hemimandibles. Also, qualitative study of the histologic sections showed no major changes in the appearance or development of the trabecular pattern between the groups. However, slight differences in the distribution pattern of osteoblasts and osteoclasts along the bony surface were found between the left control hemimandible and the left and right experimental hemimandibles, which seemed to explain the alterations in mandibular corpus shape between these groups. It was suggested that these changes in the distribution pattern of osteoblasts and osteoclasts were the result of changes in the loading distribution pattern acting upon the mandible, caused by an altered neuromuscular recruitment pattern of the remaining functionally intact, mandibularly attached muscles. The latter was probably the result of adaptive mandibular positioning in response to an altered occlusal relationship, which was induced by the abnormal maxillary growth as a result of the unilateral partial facial paralysis.     

Light-induced fluorescence changes in<Emphasis Type="Italic"> Phycomyces</Emphasis>: evidence for blue light-receptor associated flavo-semiquinones

Light-induced fluorescence changes (LIFCs) were detected in sporangiophores of the blue-light-sensitive fungus Phycomyces blakesleeanus (Burgeff). The LIFCs can be utilized as a spectrophotometric assay for blue-light photoreceptors and for the in vivo characterization of their photochemical primary reactions. Blue-light irradiation of sporangiophores elicited a transient decrease and subsequent regeneration of flavin-like fluorescence emission at 525 nm. The signals recovered in darkness in about 120 min. In contrast to blue light, near-UV (370 nm) caused an increase in the fluorescence emission at 525 nm. Because the LIFCs were altered in a light-insensitive madC mutant with a defective photoreceptor, the fluorescence changes must be associated with early photochemical events of the transduction chain. Action spectra for the fluorescence changes at 525 nm showed major peaks near 470 and 600 nm. Double-pulse experiments involving two consecutive pulses of either blue and near-UV, blue and red, or near-UV and red showed that the responses depended on the sequence in which the different wavelengths were applied. The results indicate a blue-light receptor with intermediates in the near-UV, blue and red spectral regions. We explain the results in the framework of a general model, in which the three redox states of the flavin photoreceptor, the oxidized flavin (Fl), the flavo-semiquinone (FlH^·), and the flavo-hydroquinone (FlH₂) are each acting as chromophores with their own characteristic photochemical primary reactions. These consist of the photoreduction of the oxidized flavin generating semiquinone, the photoreduction of the semiquinone generating hydroquinone, and the photooxidation of the flavo-hydroquinone regenerating the pool of oxidized flavins. The proposed mechanism represents a photocycle in which two antagonistic photoreceptor forms, Fl and FlH₂, determine the pool size of the biological effector molecule, the flavo-semiquinone. The redox changes that are associated with the photocycle are maintained by redox partners, pterins, that function in the near-UV as secondary chromophores.Abbreviations FAD flavin adenine dinucleotide - Fl oxidized flavin - FlH flavo-semiquinone radical - FlH₂ flavo-hydroquinone - LIAC light-induced absorbance change - LIFC light-induced fluorescence change - Pt oxidized pterin - PtH₂ dihydro-pterin - PtH₄ tetrahydro-pterin