An in vivo tethered toxin approach for the cell-autonomous inactivation of voltage-gated sodium channel currents in nociceptors

Understanding information flow in sensory pathways requires cell-selective approaches to manipulate the activity of defined neurones. Primary afferent nociceptors, which detect painful stimuli, are enriched in specific voltage-gated sodium channel (VGSC) subtypes. Toxins derived from venomous animals can be used to dissect the contributions of particular ion currents to cell physiology. Here we have used a transgenic approach to target a membrane-tethered isoform of the conotoxin MrVIa (t-MrVIa) only to nociceptive neurones in mice. T-MrVIa transgenic mice show a 44 ± 7% reduction of tetrodotoxin-resistant (TTX-R) VGSC current densities. This inhibition is permanent, reversible and does not result in functional upregulation of TTX-sensitive (TTX-S) VGSCs, voltage-gated calcium channels (VGCCs) or transient receptor potential (TRP) channels present in nociceptive neurones. As a consequence of the reduction of TTX-R VGSC currents, t-MrVIa transgenic mice display decreased inflammatory mechanical hypersensitivity, cold pain insensitivity and reduced firing of cutaneous C-fibres sensitive to noxious cold temperatures. These data validate the use of genetically encoded t-toxins as a powerful tool to manipulate VGSCs in specific cell types within the mammalian nervous system. This novel genetic methodology can be used for circuit mapping and has the key advantage that it enables the dissection of the contribution of specific ionic currents to neuronal function and to behaviour.This work was supported by grants from the DFG to I.I.-T. and G.R.L. within the collaborative research centre (SFB 665) and from the Alexander von Humboldt Foundation to E.St.J.S

Microarray analysis identifies a set of CXCR3 and CCR2 ligand chemokines as early IFNβ-responsive genes in peripheral blood lymphocytes in vitro: an implication for IFNβ-related adverse effects in multiple sclerosis

BACKGROUND: A substantial proportion of multiple sclerosis (MS) patients discontinue interferon-beta (IFNβ) treatment due to various adverse effects, most of which emerge at the early phase after initiation of the treatment and then diminish with time. At present, the molecular mechanism underlying IFNβ-related adverse effects remains largely unknown. The aim of this study is to identify a comprehensive list of early IFNβ-responsive genes (IRGs) in peripheral blood mononuclear cells (PBMC) that may play a key role in induction of adverse effects. METHODS: Total RNA of PBMC exposed to 50 ng/ml recombinant human IFNβ for 3 to 24 hours in vitro was processed for cDNA microarray analysis, followed by quantitative real-time RT-PCR analysis. RESULTS: Among 1,258 genes on the array, IFNβ elevated the expression of 107 and 87 genes, while it reduced the expression of 22 and 23 genes at 3 and 24 hours, respectively. Upregulated IRGs were categorized into conventional IFN-response markers, components of IFN-signaling pathways, chemokines, cytokines, growth factors, and their receptors, regulators of apoptosis, DNA damage, and cell cycle, heat shock proteins, and costimulatory and adhesion molecules. IFNβ markedly upregulated CXCR3 ligand chemokines (SCYB11, SCYB10 and SCYB9) chiefly active on effector T helper type 1 (Th1) T cells, and CCR2 ligand chemokines (SCYA8 and SCYA2) effective on monocytes, whereas it downregulated CXCR2 ligand chemokines (SCYB2, SCYB1 and IL8) primarily active on neutrophils. CONCLUSION: IFNβ immediately induces a burst of gene expression of proinflammatory chemokines in vitro that have potential relevance to IFNβ-related early adverse effects in MS patients in vivo

Isolation, Cloning and Structural Characterisation of Boophilin, a Multifunctional Kunitz-Type Proteinase Inhibitor from the Cattle Tick

Inhibitors of coagulation factors from blood-feeding animals display a wide variety of structural motifs and inhibition mechanisms. We have isolated a novel inhibitor from the cattle tick Boophilus microplus, one of the most widespread parasites of farm animals. The inhibitor, which we have termed boophilin, has been cloned and overexpressed in Escherichia coli. Mature boophilin is composed of two canonical Kunitz-type domains, and inhibits not only the major procoagulant enzyme, thrombin, but in addition, and by contrast to all other previously characterised natural thrombin inhibitors, significantly interferes with the proteolytic activity of other serine proteinases such as trypsin and plasmin. The crystal structure of the bovine α-thrombin·boophilin complex, refined at 2.35 Å resolution reveals a non-canonical binding mode to the proteinase. The N-terminal region of the mature inhibitor, Q16-R17-N18, binds in a parallel manner across the active site of the proteinase, with the guanidinium group of R17 anchored in the S1 pocket, while the C-terminal Kunitz domain is negatively charged and docks into the basic exosite I of thrombin. This binding mode resembles the previously characterised thrombin inhibitor, ornithodorin which, unlike boophilin, is composed of two distorted Kunitz modules. Unexpectedly, both boophilin domains adopt markedly different orientations when compared to those of ornithodorin, in its complex with thrombin. The N-terminal boophilin domain rotates 9° and is displaced by 6 Å, while the C-terminal domain rotates almost 6° accompanied by a 3 Å displacement. The reactive-site loop of the N-terminal Kunitz domain of boophilin with its P1 residue, K31, is fully solvent exposed and could thus bind a second trypsin-like proteinase without sterical restraints. This finding explains the formation of a ternary thrombin·boophilin·trypsin complex, and suggests a mechanism for prothrombinase inhibition in vivo

Einsatz des objektiven Bera-Verfahrens (MB 11) beim Neugeborenen Hörscreening in Marburg

In Marburg werteten wir im Untersuchungszeitraum insgesamt 605 Messergebnisse von 304 Neugeborenen aus. Bei insgesamt 287 Neugeborenen konnte in Stufe 1 ein Pass ermittelt werden. Die Spezifität nach dieser 1. Messung lag bei 97,1 %, nach Re-Testung (Stufe 2) bei 99.3 %. Die Messungen wurden von unterschiedlichen Untersuchern durchgeführt. Die ermittelten Ergebnisse hinsichtlich der Messzeit sind dennoch nahezu gleich. Wir sehen dies als wichtigen Hinweis für ein technisch ausgereiftes Untersuchungsverfahren an. Der große Vorteil hingegen liegt in der Tatsache, dass in dieser kurzen Untersuchungszeit Aussagen zur Funktionsfähigkeit der akustischen Bahn bis zum Hirnstamm vorgenommen werden können. Damit lassen sich auch Kinder mit einer auditorischen Neuropathie ausreichend gut diagnostizieren. Die schnelle Auswertung rechtfertigt erneut die von uns bereits durchgeführte beidseitige Messung. Wir sind der Ansicht, dass ein beidohriges Hörscreening erfolgen sollte, da eine einseitige unerkannte Hörstörung Einschränkungen in der zentralen Hörverarbeitung zur Folge hat. In Marburg wurde eine bereits gut funktionierende Datenbank entwickelt, die einerseits die Nachkontrolle der von uns selbst ermittelten auffälligen Kinder erleichtert, die aber auch für die Nachkontrolle von gemeldeten Kindern anderer screenender Einrichtungen Nordhessens genutzt wird

IPA free texturization of n type Si wafers Correlation of optical, electronic and morphological surface properties

The application of an Isopropanol IPA free potassium hydroxide KOH solution was evaluated in order to prepare random pyramids on as cut crystalline n type Si wafers to reduce reflection losses of substrates for high efficiency hetero junction solar cells. The influence of saw damage removal and texturization processes on the resulting pyramid morphology and the corresponding interplay between optical and electronic properties are revealed. It is shown that both the depth of the saw damage etching SDE and the duration of the texturization etching have crucial influence on the resulting pyramid size distribution. Reflection losses can be reduced with decreasing fraction of small pyramids. By intermediate saw damage removal and texture etching times in IPA free KOH solution the densities of electronic interface states were found to be strongly decreased Dit,min lt; 5 E 11 cm 2eV 1 , in comparison to pyramids prepared in IPA containing solutions. For the purpose of fabricating amorphous crystalline a Si H c Si heterojunction solar cells the Si substrate surfaces were passivated with an intrinsic layer of amorphous silicon a Si H i leading to minority charge carrier lifetimes taueff of 2 to 4 ms, depending on the preceding texturization process