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Social relationships can have important fitness consequences. Although there is increasing evidence that social relationships carry over across contexts, few studies have investigated whether relationships formed early in life are carried over to adulthood. For example, juveniles of monogamous species go through a major life history stage transition, pair formation, during which the pair bond becomes a central unit of the social organization. At present, it remains unclear whether pair members retain their early life relationships after pair formation. We investigated whether same-sex associations formed early in life carry over into adulthood and whether carryover was dependent on season, in a monogamous species. We also investigated the role of familiarity, genetic relatedness and aggression on the perseverance of social associations. We studied the social structure before and after pair formation in captive barnacle geese, Branta leucopsis, a highly social, long-lived, monogamous species. We constructed association networks of groups of geese before pair formation, during the subsequent breeding season and in the following wintering season. Next, we studied how these associations carried over during seasonal changes. We found that early life associations in females were lost during the breeding season but resurfaced during the subsequent wintering season. In males, the early life associations persisted across both seasons. Association persistence was not mediated by genetic relatedness or familiarity. The high level of aggressiveness of males, but not females, in the breeding season suggests that males may have played a key role in shaping both their own social environment and that of their partners. We show that early life social relationships can be maintained well into later life. Such relationships can be sustained even if they are temporarily disrupted, for example due to reproductive behaviour. Our findings therefore highlight that the early life social environment can have lifelong consequences for individuals’ social environment.

// Tillmann Bedau 1 , Neele Schumacher 1 , Florian Peters 1 , Johannes Prox 1 , Philipp Arnold 2 , Tomas Koudelka 3 , Ole Helm 4 , Frederike Schmidt 1 , Bjorn Rabe 1 , Marlene Jentzsch 5 , Philip Rosenstiel 5 , Susanne Sebens 4 , Andreas Tholey 3 , Stefan Rose-John 1 and Christoph Becker-Pauly 1 1 Unit for Degradomics of the Protease Web, Institute of Biochemistry, University of Kiel, 24118 Kiel, Germany 2 Anatomical Institute, University of Kiel, 24118 Kiel, Germany 3 Institute of Experimental Medicine, University of Kiel, 24118 Kiel, Germany 4 Institute for Experimental Tumor Research, University of Kiel, 24118 Kiel, Germany 5 Institute of Clinical Molecular Biology, University of Kiel, 24118 Kiel, Germany Correspondence to: Christoph Becker-Pauly, email: cbeckerpauly@biochem.uni-kiel.de Keywords: CD99, meprin β, proteolytic shedding, lung cancer, inflammation Received: January 18, 2017 Accepted: June 17, 2017 Published: July 04, 2017 ABSTRACT Transendothelial cell migration (TEM) is crucial for inflammation and metastasis. The adhesion molecule CD99 was shown to be important for correct immune cell extravasation and is highly expressed on certain cancer cells. Recently, we demonstrated that ectodomain shedding of CD99 by the metalloprotease meprin β promotes TEM in vitro . In this study, we employed an acute inflammation model (air pouch/carrageenan) and found significantly less infiltrated cells in meprin β knock-out animals validating the previously observed pro-inflammatory activity. To further analyze the impact of meprin β on CD99 shedding with regard to cell adhesion and proliferation we characterized two lung cancer associated CD99 variants (D92H, D92Y), carrying point mutations at the main cleavage site. Interestingly, ectodomain shedding of these variants by meprin β was still detectable. However the cleavage site shifted to adjacent positions. Nevertheless, expression of CD99 variants D92H and D92Y revealed partial misfolding and proteasomal degradation. A previously observed influence of CD99 on Src activation and increased proliferation could not be confirmed in this study, independent of wild-type CD99 or the variants D92H and D92Y. However, we identified meprin β as a potent inducer of Src phosphorylation. Importantly, we found significantly increased cell migration when expressing the cancer-associated CD99 variant D92H compared to the wild-type protein.

Social relationships can have important fitness consequences. Although there is increasing evidence that social relationships carry over across contexts, few studies have investigated whether relationships formed early in life are carried over to adulthood. For example, juveniles of monogamous species go through a major life-history stage transition—pair formation—during which the pair bond becomes a central unit of the social organization. At present, it remains unclear if pair members retain their early-life relationships after pair formation. We investigated whether same-sex associations formed early in life carry over into adulthood and whether carry-over was dependent on season, in a monogamous species. Moreover, we investigated the role of familiarity, genetic relatedness and aggression on the perseverance of social associations. We studied the social structure before and after pair formation in captive barnacle geese (Branta leucopsis), a highly social, long-lived, monogamous species. We constructed association networks of groups of geese before pair formation, during the subsequent breeding season, and in the following wintering season. Next, we studied how these associations carried over during seasonal changes. We found that early-life associations in females were lost during the breeding season, but resurfaced during the subsequent wintering season. In males, the early-life associations persisted across both seasons. Association persistence was not mediated by genetic relatedness or familiarity. The high level of aggressiveness of males, but not females, in the breeding season suggests that males may have played a key role in shaping both their own social environment and that of their partners. We show that early-life social relationships can be maintained well into later life. Such relationships can be sustained even if they are temporarily disrupted, for example due to reproductive behaviour. Our findings therefore highlight that the early-life social environment can have life-long consequences on individuals’ social environment.

The meprin β metalloproteinase is an important enzyme in extracellular matrix turnover, inflammation, and neurodegeneration in humans and mice. Previous studies showed a diminished cleavage of certain meprin β substrates in the presence of calcium, although the mechanism was not clear. With the help of a specific fluorogenic peptide assay and the human amyloid precursor protein as substrate, we demonstrated that the influence of calcium is most likely a direct effect on human meprin β itself. Analyzing the crystal structures of pro- and mature meprin β helped to identify a cluster of negatively charged amino acids forming a potential calcium binding site. Mutation of 2 of these residues (D204A and D245A) led to severe differences in proteolytic activity and cellular localization of meprin β. D245A was almost completely inactive and largely stored into intracellular vesicles, indicating severe misfolding of the protein. Astonishingly, D204A was not transported to the cell surface, but exhibited strong β-secretase activity, resulting in massive accumulation of Aβ-peptides. This could be explained by constitutive maturation of this meprin β mutant already in the early secretory pathway. We hypothesize that lacking D204 abrogates the capability of binding calcium in the catalytic domain, an important step for proper folding of the propeptide and subsequent inhibition of the protease. This is supported by the inhibition constant of calcium for meprin β (inhibitory constant 50 = 11 mM), which resembles the physiologic concentrations found in the endoplasmic reticulum. For instance, it was shown for amyotrophic lateral sclerosis that a loss of calcium in the endoplasmic reticulum leads to the misfolding of calcium-dependent proteins, which might also be relevant for proper function of meprin β.

Proteolytic and amyloidogenic processing of amyloid precursor protein (APP) by β- and γ-secretases are pathological hallmarks of Alzheimer’s disease (AD). These proteolytic activities lead to release of the amyloid-β pep­tides believed to cause neurological pathol­ogy and be linked to pathological progres­sion in AD. Due to its capability to cleave APP within the toxic peptide sequence, the metalloproteinase ADAM10 (“a disintegrin and me­talloprotease”) is a known antagonist of the disease-causing pathway. ADAM10 also plays a major role in the ectodomain shedding of a number of important cell surface proteins. In addition, ADAM10 is involved in the proteolytic activation cascade of the Notch receptor, which is of crucial function in develop­mental processes. The study of ADAM10-deficient mice also revealed that ADAM10 regulates synaptic function and synaptogenesis. Pharmacological activation of ADAM10 is postulated to represent a valuable strategy for prevention of AD. However, due to the multiple roles of ADAM10 in the brain, it will be challenging to find a suitable therapeutic window.

The protease web is a synonym for highly regulated molecular networks comprising enzymes, substrates, inhibitors, and other regulatory proteins. Latest high-throughput methods provided huge data sets, revealing an amazing complexity of proteolytic systems important for health and disease. Based on our previous studies, we discuss major problems and questions that have to be solved to gain precise insight into the regulation of the protease web and its impact on pathophysiological conditions. The goal is a combination of different proteomic approaches that help to investigate specific protease function at a glance. Exemplarily, the characterization of the metalloproteases meprin α and meprin β by proteomic identification of cleavage sites and terminal amine isotopic labeling of substrates demonstrates the power of MS-based techniques. Meprins are rather orphan proteases and could not be assigned to precise biological functions until recently. Proteomics helped to identify meprin α and meprin β being important for collagen assembly and deposition in skin, which makes them potential therapeutic targets in fibrotic conditions. Additionally, identification of the cleavage site specificity provides the basis for the development of activity-based probes and small compound inhibitors, important for the regulation of meprin activity and subsequent treatment of associated diseases.

Adult hematopoiesis requires tightly regulated cell–cell interactions between hematopoietic cells and the bone marrow stromal microenvironment. We addressed the question if the ectodomain sheddase ADAM10 is essential to regulate adult hematopoiesis. Induced ADAM10 deletion in hematopoietic cells resulted in morphological and histological abnormalities that resemble an unclassified myeloproliferative disorder (MPD). The MPD was characterized by an expansion of granulocytic subpopulations and their infiltration of peripheral hematopoietic tissues, the development of hepatosplenomegaly with extramedullary erythropoiesis, lymphnodepathy and death of the mice around 20 weeks after induction. ADAM10 expression analysis during the different stages of the MPD revealed that non-targeted hematopoietic cells repopulated the immune system of the ADAM10-deficient mice. Examination of mice with a myeloid- or epidermis-specific deletion of ADAM10 and bone marrow transplantation (BMT) experiments indicated that the development of the MPD can be triggered by non-cell autonomous effects. We found that plasma levels of clinical markers for MPD such as G-CSF, TIMP-1 and IL-16 were significantly elevated in ADAM10-deficient mice. Our findings indicate that a tightly controlled ADAM10 expression is needed to balance hematopoietic cell-fate decisions in adult mice.

The cellular prion protein (PrP(C)) plays important roles in neurodegenerative diseases. First, it is the well-established substrate for the conformational conversion into its pathogenic isoform (PrP(Sc)) giving rise to progressive and fatal prion diseases. Moreover, several recent reports highlight important roles of PrP(C) in other neurodegenerative conditions such as Alzheimer's disease. Since PrP(C) is subject to proteolytic processing, here we discuss the two main cleavage events under physiological conditions, α-cleavage and shedding. We focus on how these cleavages and the resulting fragments may impact prion diseases as well as other neurodegenerative proteinopathies. Finally, we discuss the recently identified sheddase of PrP(C), namely the metalloprotease ADAM10, with regard to therapeutic potential against neurodegenerative diseases.

Meprin β is a dimeric type I transmembrane protein and acts as an ectodomain sheddase at the cell surface. It was shown that meprin β cleaves the amyloid precursor protein (APP), thereby releasing neurotoxic amyloid β peptides and implicating a role of meprin β in Alzheimer’s disease. In order to identify non-proteolytic regulators of meprin β, we performed a split ubiquitin yeast two-hybrid screen using a small intestinal cDNA library. In this screen we identified tetraspanin 8 (TSPAN8) as interaction partner for meprin β. Since several members of the tetraspanin family were described to interact with metalloproteases thereby affecting their localization and/or activity, we hypothesized similar functions of TSPAN8 in the regulation of meprin β. We employed cell biological methods to confirm direct binding of TSPAN8 to meprin β. Surprisingly, we did not observe an effect of TSPAN8 on the catalytic activity of meprin β nor on the specific cleavage of its substrate APP. However, both proteins were identified being present in tetraspanin-enriched microdomains. Therefore we hypothesize that TSPAN8 might be important for the orchestration of meprin β at the cell surface with impact on certain proteolytic processes that have to be further identified.

A disintegrin and metalloproteinase10 (ADAM10) has been implicated as a major sheddase responsible for the ectodomain shedding of a number of important surface molecules including the amyloid precursor protein and cadherins. Despite a well-documented role of ADAM10 in health and disease, little is known about the regulation of this protease. To address this issue we conducted a split-ubiquitin yeast two-hybrid screen to identify membrane proteins that interact with ADAM10. The yeast experiments and co-immunoprecipitation studies in mammalian cell lines revealed tetraspanin15 (TSPAN15) to specifically associate with ADAM10. Overexpression of TSPAN15 or RNAi-mediated knockdown of TSPAN15 led to significant changes in the maturation process and surface expression of ADAM10. Expression of an endoplasmic reticulum (ER) retention mutant of TSPAN15 demonstrated an interaction with ADAM10 already in the ER. Pulse-chase experiments confirmed that TSPAN15 accelerates the ER-exit of the ADAM10-TSPAN15 complex and stabilizes the active form of ADAM10 at the cell surface. Importantly, TSPAN15 also showed the ability to mediate the regulation of ADAM10 protease activity exemplified by an increased shedding of N-cadherin and the amyloid precursor protein. In conclusion, our data show that TSPAN15 is a central modulator of ADAM10-mediated ectodomain shedding. Therapeutic manipulation of its expression levels may be an additional approach to specifically regulate the activity of the amyloid precursor protein alpha-secretase ADAM10.

Nectin-1 is known to undergo ectodomain shedding by alpha-secretase and subsequent proteolytic processing by gamma-secretase. How secretase-mediated cleavage of nectin-1 is regulated in neuronal cells and how nectin-1 cleavage affects synaptic adhesion is poorly understood. We have investigated alpha-and gamma-secretase-mediated processing of nectin-1 in primary cortical neurons and identified which protease acts as a alpha-secretase. We report here that NMDA receptor activation, but not stimulation of AMPA or metabotropic glutamate receptors, resulted in robust alpha- and gamma-secretase cleavage of nectin-1 in mature cortical neurons. Cleavage of nectin-1 required influx of Ca(2+) through the NMDA receptor, and activation of calmodulin, but was not dependent on calcium/calmodulin-dependent protein kinase II (CaMKII) activation. We found that ADAM10 is the major secretase responsible for nectin-1 ectodomain cleavage in neurons and the brain. These observations suggest that alpha- and gamma-secretase processing of nectin-1 is a Ca(2+)/calmodulin-regulated event that occurs under conditions of activity-dependent synaptic plasticity and ADAM10 and gamma-secretase are responsible for these cleavage events.

The metalloproteinase and major amyloid precursor protein (APP) α-secretase candidate ADAM10 is responsible for the shedding of proteins important for brain development, such as cadherins, ephrins, and Notch receptors.Adam10−/−mice die at embryonic day 9.5, due to major defects in development of somites and vasculogenesis. To investigate the function of ADAM10 in brain, we generatedAdam10conditional knock-out (cKO) mice using a Nestin-Cre promotor, limiting ADAM10 inactivation to neural progenitor cells (NPCs) and NPC-derived neurons and glial cells. The cKO mice die perinatally with a disrupted neocortex and a severely reduced ganglionic eminence, due to precocious neuronal differentiation resulting in an early depletion of progenitor cells. Premature neuronal differentiation is associated with aberrant neuronal migration and a disorganized laminar architecture in the neocortex. Neurospheres derived fromAdam10cKO mice have a disrupted sphere organization and segregated more neurons at the expense of astrocytes. We found that Notch-1 processing was affected, leading to downregulation of several Notch-regulated genes inAdam10cKO brains, in accordance with the central role of ADAM10 in this signaling pathway and explaining the neurogenic phenotype. Finally, we found that α-secretase-mediated processing of APP was largely reduced in these neurons, demonstrating that ADAM10 represents the most important APP α-secretase in brain. Our study reveals that ADAM10 plays a central role in the developing brain by controlling mainly Notch-dependent pathways but likely also by reducing surface shedding of other neuronal membrane proteins including APP.

Among children with relapsed or refractory neuroblastoma, the prognosis is poor and novel therapeutic strategies are needed to improve long-term survival. As with other solid tumors, high vascular density within neuroblastoma is associated with advanced disease, and therapeutic regimens directed against the tumor vasculature may provide clinical benefit. The receptor tyrosine kinase RET is widely expressed in neuroblastoma and is known to activate key signal transduction pathways involved in tumor cell survival and progression including Ras/mitogen-activated protein kinase and phosphatidylinositol 3-kinase/Akt. We investigated the effect of dual targeting of tumor cells and tumor endothelium with ZD6474, a small-molecule tyrosine kinase inhibitor of vascular endothelial growth factor (VEGF) receptor 2, epidermal growth factor receptor, and RET. ZD6474 inhibited the phosphorylation of RET in neuroblastoma cells and had a direct effect on tumor cell viability in seven neuroblastoma cell lines. In a human neuroblastoma xenograft model, ZD6474 inhibited tumor growth by 85% compared with treatment with vehicle alone. In contrast, no significant inhibition of tumor growth was observed after treatment with bevacizumab, an antihuman VEGF monoclonal antibody, or the epidermal growth factor receptor inhibitor erlotinib, either alone or in combination. Immunohistochemical analysis showed that ZD6474 treatment led to an increase in endothelial cell apoptosis along with inhibition of VEGF receptor-2 activation on tumor endothelium. In conclusion, dual targeting of tumor cells, potentially through RET inhibition, and tumor vasculature with ZD6474 leads to potent antitumor effects. This approach merits further investigation for patients with neuroblastoma. [Mol Cancer Ther 2008;7(2):418–24]

The prion protein (PrPC) is highly expressed in the nervous system and critically involved in prion diseases where it misfolds into pathogenic PrPSc. Moreover, it has been suggested as a receptor mediating neurotoxicity in common neurodegenerative proteinopathies such as Alzheimer's disease. PrPC is shed at the plasma membrane by the metalloprotease ADAM10, yet the impact of this on prion disease remains enigmatic. Employing conditional knockout mice, we show that depletion of ADAM10 in forebrain neurons leads to posttranslational increase of PrPC levels. Upon prion infection of these mice, clinical, biochemical, and morphological data reveal that lack of ADAM10 significantly reduces incubation times and increases PrPSc formation. In contrast, spatiotemporal analysis indicates that absence of shedding impairs spread of prion pathology. Our data support a dual role for ADAM10-mediated shedding and highlight the role of proteolytic processing in prion disease. DOI: http://dx.doi.org/10.7554/eLife.04260.001, eLife digest Prion proteins are anchored to the surface of brain cells called neurons. Normally, prion proteins are folded into a specific three-dimensional shape that enables them to carry out their normal roles in the brain. However, they can be misfolded into a different shape known as PrPSc, which can cause Creutzfeldt-Jakob disease and other serious conditions that affect brain function and ultimately lead to death. The PrPSc proteins can force normal prion proteins to change into the PrPSc form, so that over time this form accumulates in the brain. They are essential components of infectious particles termed ‘prions’ and this is why prion diseases are infectious: if prions from one individual enter the brain of another individual they can cause disease in the recipient. The UK outbreak of variant Creutzfeldt-Jakob disease in humans in the 1990s is thought to be due to the consumption of meat from cattle with a prion disease known as mad cow disease. An enzyme called ADAM10 can cut normal prion proteins from the surface of neurons. However, it is not clear whether ADAM10 can also target the PrPSc proteins and what impact this may have on the development of prion diseases. Here, Altmeppen et al. studied mutant mice that were missing ADAM10 in neurons in the front portion of their brain. These mice had a higher number of normal prion proteins on the surface of their neurons than normal mice did. When mice missing ADAM10 were infected with prions, more PrPSc accumulated in their brain and disease symptoms developed sooner than when normal mice were infected. This supports the view that mice with higher numbers of prion proteins are more vulnerable to prion disease. However, disease symptoms did not spread as quickly to other parts of the brain in the mice missing ADAM10. This suggests that by releasing prion proteins from the surface of neurons, ADAM10 helps PrPSc proteins to spread around the brain. Recently, it has been suggested that prion proteins may also play a role in Alzheimer's disease and other neurodegenerative conditions. Therefore, Altmeppen et al.'s findings may help to develop new therapies for other forms of dementia. The next challenge is to understand the precise details of how ADAM10 works. DOI: http://dx.doi.org/10.7554/eLife.04260.002

Increased expression of metalloprotease meprin β is associated with fibrotic syndromes and Alzheimer's disease (AD). Hence, regulation of meprin activity might be a suitable strategy for the treatment of these conditions. Meprin β is a type 1 transmembrane protein, but can be released from the cell surface by ectodomain shedding. The protease is expressed as an inactive zymogen and requires proteolytic maturation by tryptic serine proteases. In the present study, we demonstrate, for the first time, the differences in the activation of soluble and membrane bound meprin β and suggest transmembrane serine protease 6 [TMPRSS6 or matriptase-2 (MT2)] as a new potent activator, cleaving off the propeptide of meprin β between Arg61 and Asn62 as determined by MS. We show that MT2, but not TMPRSS4 or pancreatic trypsin, is capable of activating full-length meprin β at the cell surface, analysed by specific fluorogenic peptide cleavage assay, Western blotting and confocal laser scanning microscopy (CLSM). Maturation of full-length meprin β is required for its activity as a cell surface sheddase, releasing the ectodomains of transmembrane proteins, as previously shown for the amyloid precursor protein (APP).

The metalloproteinase ADAM10 is of importance for Notch-dependent cortical brain development. The protease is tightly linked with α-secretase activity toward the amyloid precursor protein (APP) substrate. Increasing ADAM10 activity is suggested as a therapy to prevent the production of the neurotoxic amyloid β (Aβ) peptide in Alzheimer's disease. To investigate the function of ADAM10 in postnatal brain, we generated Adam10 conditional knock-out (A10cKO) mice using a CaMKIIα-Cre deleter strain. The lack of ADAM10 protein expression was evident in the brain cortex leading to a reduced generation of sAPPα and increased levels of sAPPβ and endogenous Aβ peptides. The A10cKO mice are characterized by weight loss and increased mortality after weaning associated with seizures. Behavioral comparison of adult mice revealed that the loss of ADAM10 in the A10cKO mice resulted in decreased neuromotor abilities and reduced learning performance, which were associated with altered in vivo network activities in the hippocampal CA1 region and impaired synaptic function. Histological and ultrastructural analysis of ADAM10-depleted brain revealed astrogliosis, microglia activation, and impaired number and altered morphology of postsynaptic spine structures. A defect in spine morphology was further supported by a reduction of the expression of NMDA receptors subunit 2A and 2B. The reduced shedding of essential postsynaptic cell adhesion proteins such as N-Cadherin, Nectin-1, and APP may explain the postsynaptic defects and the impaired learning, altered network activity, and synaptic plasticity of the A10cKO mice. Our study reveals that ADAM10 is instrumental for synaptic and neuronal network function in the adult murine brain.

Alzheimer's disease causing mutations in the amyloid precursor protein (APP) or in the Presenilin 1 (PS1) or Presenilin 2 (PS2) genes increase the production of amyloid peptides (Aβ) that precipitate in amyloid plaques. Since amyloid plaques are also a prominent feature of sporadic Alzheimer's disease (AD), abnormal proteolysis of APP and the generation of amyloid beta (Aβ) are key events in the pathogenesis of AD. The proteases (secretases) that cleave APP are therefore important therapeutic targets, both for the rare familial forms but likely also for the sporadic forms of AD. The identification and understanding of the (neuro)biological functions of the α-, β-, and presenilin/γ-secretase (complexes) is important for the development of drugs and the delineation of their associated side effects. The potential impact of this type of research exceeds the AD field since the function of these secretases are also linked to cellular pathways like ectodomain shedding of growth factors and regulated intramembrane proteolysis of receptors in developmental biology, tissue homeostasis, and tumorigenesis. The generation of mice deficient in presenilin 1, presenilin 2, the α-secretase ADAM10, and the β-secretases BACE1 and BACE2 were instrumental for the elucidation of the physiological functions of these proteases. Using these mouse models understanding how these secretases regulate amyloid peptide formation and how they exert their diverse biological functions could be significantly increased. This review attempts to summarize selected aspects of the current view of the multiple roles such proteases play in health and disease.

The disintegrin and metalloproteinase Adam10 has been implicated in the regulation of key signaling pathways that determine skin morphogenesis and homeostasis. To address the in vivo relevance of Adam10 in the epidermis, we have selectively disrupted Adam10 during skin morphogenesis and in adult skin. K14-Cre driven epidermal Adam10 deletion leads to perinatal lethality, barrier impairment and absence of sebaceous glands. A reduction of spinous layers, not associated with differences in either proliferation or apoptosis, indicates that loss of Adam10 triggers a premature differentiation of spinous keratinocytes. The few surviving K14-Adam10-deleted mice and mice in which Adam10 was deleted postnatally showed loss of hair, malformed vibrissae, epidermal hyperproliferation, cyst formation, thymic atrophy and upregulation of the cytokine thymic stromal lymphopoetin (TSLP), thus indicating non cell-autonomous multi-organ disease resulting from a compromised barrier. Together, these phenotypes closely resemble skin specific Notch pathway loss-of-function phenotypes. Notch processing is indeed strongly reduced resulting in decreased levels of Notch intracellular domain fragment and functional Notch signaling. The data identify Adam10 as the major Site-2 processing enzyme for Notch in the epidermis in vivo, and thus as a central regulator of skin development and maintenance.

Reelin is a secreted glycoprotein whose function is regulated by proteolysis. One of the specific cleavage sites of Reelin, called C-t, is located approximately between the sixth and seventh Reelin repeat but its exact site was unknown. We here show that a metalloprotease present in the culture supernatant of cerebellar granular neurons (CGN) cleaves Reelin between Ala2688 and Asp2689. A Reelin mutant in which Asp2689 is replaced by Lys (Reelin-DK) is resistant to C-t cleavage by culture supernatant of CGN. From biochemical characteristics and the cleavage site preference, meprin α and meprin β were suggested candidate proteases and both were confirmed to cleave Reelin at the C-t site. Meprin α cleaved Reelin-DK but meprin β did not. Actinonin, a meprin α and meprin β inhibitor, did not inhibit the Reelin-cleaving activity of CGN and the amount of Reelin fragments in brains of meprin β knock-out mice was not significantly different from that of the wild-type, indicating that meprin β does not play a major role in Reelin cleavage under basal conditions. We propose that meprin α and meprin β join the modulators of Reelin signalling as they cleave Reelin at a specific site and are upregulated under specific pathological conditions.

The effect of in situ freezing of the monkey (macaque) prostate on the development of antibodies reactive with allogeneic and autologous extract preparations of the cranial and caudal lobe of prostatic tissue and their tissue specificity were evaluated by the method of tanned cell hemagglutination. A representative percentage of the animals receiving cryosurgery developed antibodies to prostatic tissue components; however the intensity of this response was considerably modest when contrasted to that obtained following similar treatment of the rabbit prostate (coagulating gland) and generally did not appear, as in the latter, to increase to any significant degree following multiple freezing. The possible relationship of this modest humoral response to the “cryosensitivity” of the target organ and of the animals evaluated in the present study, i.e., the concentration of glandular secretions (autoantigens), physiologic state (elaboration of androgen) and immunocompetence, are considered.

Das antitoxische Cyclodecapeptid Antamanid aus dem grunen Knollenblatterpilz (Amanita phalloides) enthalt die Aminosauren Phe4Ala1Val1Pro4. Die kombinierte Anwendung von Gaschromatographie und Massenspektrometrie auf die N-trifluoracetylierten Peptidester aus der sauren Partial-Spaltung und die Interpretation seines hochaufgelosten Massenspektrums ergibt die Struktur 3. Bei seiner sauren oder alkalischen Partial-Spaltung entstehen infolge intramolekularer Umpeptidierungen auch falsche Sequenz-Bruchstucke. Sequence Analysis by Combined Gas Chromatography and Mass Spectrometry, II1) ). Structure of Antamanid The antitoxic cyclodecapeptide antamanid from Amanita phalloides contains the amino acids Phe4Ala1Val1Pro4. Partial hydrolysis and interpretation of the high resolution mass spectrum leads to structure 3. The peptide mixture from partial hydrolysis is separated by gas chromatography using N-trifluoracetylated peptide methylesters. As a consequence of intramolecular transpeptidation, wrong sequences from acidic or alkaline partial hydrolysis of antamanid are found.

Die sich an der Luft in Gegenwart von Oxydasen blau verfärbenden Pigmente von Boletus erythropus, B. calopus und Xerocomus chrysenteron gehören nicht dem Anthrachinon- 1, sondern dem Pulvinsäuretyp an. In allen Fällen ist Variegatsäure 2 4 die Hauptkomponente, daneben kommen ein neues blauendes Pigment, Xerocomsäure, und in X. chrysenteron noch Spuren von Atromentinsäure und einer Chlor-xerocomsäure vor. Die für Xerocomsäure massenspektrometrisch abgeleitete Struktur einer 3.4.4′-Trihydroxy-pulvinsäure (3 a bzw. 3 b) wird synthetisch bewiesen. 1.2.4-Trihydroxy-anthrachinon-5-(oder 8) -carbonsäure (1 a oder 1 b) zeigt im Gegensatz zu den Angaben von KÖGL und DEIJS 1 mit Oxydasen keine Blaufärbung, so daß sie als blauendes Prinzip der Boletaceen aus der Literatur zu streichen ist. Die Struktur der blauen Oxydationsprodukte wird diskutiert.

The glucose oxidase—peroxidase procedure for determining glucose in serum or plasma has been modified by changing the assay pH to 5.5, altering the ratio of glucose oxidase to peroxidase, and using a soluble chromogen, p-diphenylamine sulfonate, to prepare a single complete reagent that develops stable color within 2 min without deproteinization. Most interferences were inconsequential. Serum that clearly is icteric or hemolytic is Somogyi-precipitated. Recovery was 99% and the CV was 3.3%. Correlation with results obtained by the neocuproine procedure, with use of the SMA 12/60, averaged 12 mg/dl lower values (r2 = 0.98).

SummaryNeuroligin (NLG), a postsynaptic adhesion molecule, is involved in the formation of synapses by binding to a cognate presynaptic ligand, neurexin. Here we report that neuroligin-1 (NLG1) undergoes ectodomain shedding at the juxtamembrane stalk region to generate a secreted form of NLG1 and a membrane-tethered C-terminal fragment (CTF) in adult rat brains in vivo as well as in neuronal cultures. Pharmacological and genetic studies identified ADAM10 as the major protease responsible for NLG1 shedding, the latter being augmented by synaptic NMDA receptor activation or interaction with soluble neurexin ligands. NLG1-CTF was subsequently cleaved by presenilin/γ-secretase. Secretion of soluble NLG1 was significantly upregulated under a prolonged epileptic seizure condition, and inhibition of NLG1 shedding led to an increase in numbers of dendritic spines in neuronal cultures. Collectively, neuronal activity-dependent proteolytic processing of NLG1 may negatively regulate the remodeling of spines at excitatory synapses.

An improved diazonium coupling procedure is described for measurement of L-aspartate: 2-oxoglutarate aminotransferase (EC 2.6.1.1, formerly termed glutamic oxalacetic transaminase) activity in serum. A relatively pH-stable dye, 5-chloro-2-methoxyaniline-1-diazonium chloride (Fast Red RC) is used, with dimethylformamide and methanol to enhance and stabilize color development. A 15-min incubation is part of the procedure, and color development stabilizes within 5 min.

Background The cellular prion protein (PrPC) fulfils several yet not completely understood physiological functions. Apart from these functions, it has the ability to misfold into a pathogenic scrapie form (PrPSc) leading to fatal transmissible spongiform encephalopathies. Proteolytic processing of PrPC generates N- and C-terminal fragments which play crucial roles both in the pathophysiology of prion diseases and in transducing physiological functions of PrPC. A-disintegrin-and-metalloproteinase 10 (ADAM10) has been proposed by cell culture experiments to be responsible for both shedding of PrPC and its α-cleavage. Here, we analyzed the role of ADAM10 in the proteolytic processing of PrPC in vivo. Results Using neuron-specific Adam10 knockout mice, we show that ADAM10 is the sheddase of PrPC and that its absence in vivo leads to increased amounts and accumulation of PrPC in the early secretory pathway by affecting its posttranslational processing. Elevated PrPC levels do not induce apoptotic signalling via p53. Furthermore, we show that ADAM10 is not responsible for the α-cleavage of PrPC. Conclusion Our study elucidates the proteolytic processing of PrPC and proves a role of ADAM10 in shedding of PrPC in vivo. We suggest that ADAM10 is a mediator of PrPC homeostasis at the plasma membrane and, thus, might be a regulator of the multiple functions discussed for PrPC. Furthermore, identification of ADAM10 as the sheddase of PrPC opens the avenue to devising novel approaches for therapeutic interventions against prion diseases.