Heterogeneous perfusion is a consequence of uniform shear stress in optimized arterial tree models

Using optimized computer models of arterial trees we demonstrate that flow heterogeneity is a necessary consequence of a uniform shear stress distribution. Model trees are generated and optimized under different modes of boundary conditions. In one mode flow is delivered to the tissue as homogeneously as possible. Although this primary goal can be achieved, resulting shear stresses between blood and the vessel walls show very large spread. In a second mode, models are optimized under the condition of uniform shear stress in all segments which in turn renders flow distribution heterogeneous. Both homogeneous perfusion and uniform shear stress are desirable goals in real arterial trees but each of these goals can only be approached at the expense of the other. While the present paper refers only to optimized models, we assume that this dual relation between the heterogeneities in flow and shear stress may represent a more general principle of vascular systems.     

Lipid Binding Defects and Perturbed Synaptogenic Activity of a Collybistin R290H Mutant That Causes Epilepsy and Intellectual Disability

Signaling at nerve cell synapses is a key determinant of proper brain function, and synaptic defects—or synaptopathies—are at the basis of many neurological and psychiatric disorders. In key areas of the mammalian brain, such as the hippocampus or the basolateral amygdala, the clustering of the scaffolding protein Gephyrin and of γ-aminobutyric acid type A receptors at inhibitory neuronal synapses is critically dependent upon the brain-specific guanine nucleotide exchange factor Collybistin (Cb). Accordingly, it was discovered recently that an R290H missense mutation in the diffuse B-cell lymphoma homology domain of Cb, which carries the guanine nucleotide exchange factor activity, leads to epilepsy and intellectual disability in human patients. In the present study, we determined the mechanism by which the Cb^R290H mutation perturbs inhibitory synapse formation and causes brain dysfunction. Based on a combination of biochemical, cell biological, and molecular dynamics simulation approaches, we demonstrate that the R290H mutation alters the strength of intramolecular interactions between the diffuse B-cell lymphoma homology domain and the pleckstrin homology domain of Cb. This defect reduces the phosphatidylinositol 3-phosphate binding affinity of Cb, which limits its normal synaptogenic activity. Our data indicate that impairment of the membrane lipid binding activity of Cb and a consequent defect in inhibitory synapse maturation represent a likely molecular pathomechanism of epilepsy and mental retardation in humans.     

ATGL and CGI-58 are lipid droplet proteins of the hepatic stellate cell line HSC-T6

Lipid droplets (LDs) of hepatic stellate cells (HSCs) contain large amounts of vitamin A [in the form of retinyl esters (REs)] as well as other neutral lipids such as TGs. During times of insufficient vitamin A availability, RE stores are mobilized to ensure a constant supply to the body. To date, little is known about the enzymes responsible for the hydrolysis of neutral lipid esters, in particular of REs, in HSCs. In this study, we aimed to identify LD-associated neutral lipid hydrolases by a proteomic approach using the rat stellate cell line HSC-T6. First, we loaded cells with retinol and FAs to promote lipid synthesis and deposition within LDs. Then, LDs were isolated and lipid composition and the LD proteome were analyzed. Among other proteins, we found perilipin 2, adipose TG lipase (ATGL), and comparative gene identification-58 (CGI-58), known and established LD proteins. Bioinformatic search of the LD proteome for α/β-hydrolase fold-containing proteins revealed no yet uncharacterized neutral lipid hydrolases. In in vitro activity assays, we show that rat (r)ATGL, coactivated by rat (r)CGI-58, efficiently hydrolyzes TGs and REs. These findings suggest that rATGL and rCGI-58 are LD-resident proteins in HSCs and participate in the mobilization of both REs and TGs.     

A compact unc45b‐promoter drives muscle‐specific expression in zebrafish and mouse

Summary: Gene therapeutic approaches to cure genetic diseases require tools to express the rescuing gene exclusively within the affected tissues. Viruses are often chosen as gene transfer vehicles but they have limited capacity for genetic information to be carried and transduced. In addition, to avoid off‐target effects the therapeutic gene should be driven by a tissue‐specific promoter in order to ensure expression in the target organs, tissues, or cell populations. The larger the promoter, the less space will be left for the respective gene. Thus, there is a need for small but tissue‐specific promoters. Here, we describe a compact unc45b promoter fragment of 195 bp that retains the ability to drive gene expression exclusively in skeletal and cardiac muscle in zebrafish and mouse. Remarkably, the described unc45b promoter fragment not only drives muscle‐specific expression but presents heat‐shock inducibility, allowing a temporal and spatial quantity control of (trans)gene expression. Here, we demonstrate that the transgenic expression of the smyd1b gene driven by the unc45b promoter fragment is able to rescue the embryonically lethal heart and skeletal muscle defects in smyd1b‐deficient flatline mutant zebrafish. Our findings demonstrate that the described muscle‐specific unc45b promoter fragment might be a valuable tool for the development of genetic therapies in patients suffering from myopathies. genesis 54:431–438, 2016. © 2016 The Authors. Genesis Published by Wiley Periodicals, Inc.     

Feinstruktur und Histochemie der Sexualduftdrüse des Seidenspinners Bombyx mori L.

Zusammenfassung Das Weibchen des Seidenspinners, Bombyx mori L., erzeugt zur Anlockung der männlichen Artgenossen in paarigen, ausstülpbaren Drüsen, den am Abdomenende gelegenen Sacculi laterales, einen spezifischen Sexuallockstoff. Dieser Lockstoff, das Bombykol, ist in seiner chemischen Konstitution bekannt und auch in synthetischer Form verfügbar.Das Drüsenepithel stellt eine differenzierte Form der normalen Insekten-epidermis dar. Wie diese besteht es aus einer einschichtigen Zellage, die an ihrer Außenfläche eine chitinhaltige Cuticula und innen, an der Grenze zum Hämolymphraum, eine Basalmembran trägt. Laterale Verzahnungen (Interdigitationen) und Desmosomen sichern den Zusammenhalt der Zellen, die beim Aus- und Einstülpen der Drüse starken Formveränderungen ausgesetzt sind.Die Zellen enthalten große, gelappte Zellkerne mit sehr locker strukturiertem Chromatin; im Cytoplasma ist ein agranuläres endoplasmatisches Reticulum stark ausgeprägt, das mit dem Ansteigen der Lockaktivität an die Stelle eines granulären endoplasmatischen Reticulums tritt. Der Golgi-Apparat ist nur unscheinbar; Mitochondrien sind in großer Zahl vorhanden.Im Gegensatz zur undifferenzierten Epidermis treten im Drüsenepithel mit Beginn der Lockaktivität in zunehmendem Maße Lipidtröpfchen auf. In diesen wird auf Grund histologischer und histochemischer Befunde eine Vorstufe des Lockstoffes vermutet.Die Grenzfläche der Zelle zur Cuticula ist durch Ausbildung eines Falten-saums 30–60fach vergrößert. Dieser wird von lamellenartigen Zellvorsprüngen gebildet, die sehr dicht stehen und weitgehend parallel zueinander verlaufen.Die Ausbildung des Faltensaums kann mit dem Anstieg der Lockwirkung der Drüse korreliert werden. Es wird ein Zusammenhang zwischen der Vergrößerung der apikalen Zelloberfläche und der Lockstoffsekretion vermutet.Das Drüsenepithel unterscheidet sich von der Intersegmentalmembran durch eine bedeutend stärkere Aktivität der NADP-Tetrazolium-Reduktase (früher als TPN-Diaphorase bezeichnet), was mit der stärkeren Synthesetätigkeit der Drüsenzellen in Zusammenhang gebracht wird.Der Weg des Lockstoffs durch die Zellmembran und die Cuticula konnte nicht verfolgt werden. Die Cytoplasmamembran bleibt stets intakt; die Cuticula läßt keine Kanalbildungen erkennen. Es wird vermutet, daß sich die Absonderung des Lockstoffs auf molekularer Ebene abspielt.Herrn Priv.-Doz. Dr. D. Schneider danke ich für die Anregung und stete Förderung der Arbeit, Herrn Prof. Dr. G. Peters für die Überlassung eines Arbeitsplatzes, den Herren Priv.-Doz. Dr. Dr. H. Hager und Dr. K. Blinzinger (Abteilung für Neurozytologie) und Dr. G. Kreutzberg (Hirnpathologisches Institut) für fördernde Kritik und technische Unterstützung.Dissertation der Naturwissenschaftlichen Fakultät der Universität München.     

Exogenous glutathione induces sister chromatid exchanges,clastogenicity and endoreduplication in V79-E Chinese hamster cells

Glutathione (GSH) dissolved in Eagle's MEM and added to cultures o of V79-E cells in concentrations between 2.5 × 10^–4 and 10^–3 moles/l for 1 h induces a dose-dependent cell cycle delay, sister chromatid exchanges and clastogenic damage. 7–8% of the metaphases showed endoreduplication at a recovery phase of 25 and 30 h after treatment with 10^–3 molesll GSH. Higher concentrations were lethal. The highest tolerated dose corresponds to the intracellular GSH level in V79-E cells. In the same range of concentrations, glutathione disulfide was inactive. Endoreduplication induction by GSH is G2-phase specific and endoreduplication metaphases show a reduced occurrence of single SCEs when extrapolated to the diploid complement. The adverse effects of GSH are independent of the presence of serum in the culture fluid but completely abolished when the treatment is performed in Hank's solution instead of MEM. The mechanism of genotoxicity of exogenous GSH is discussed but, at present, no pertinent explanation can be given.Abbreviations BUdR 5-bromodeoxyuridine - GSH glutathione - GSSG glutathione disulfide - SCE sister chromatid exchange     

Die Feinstruktur von Milz und Leber bei experimenteller Amyloidose

Zusammenfassung Experimentell erzeugtes Amyloid besteht aus einer faserigen Komponente und einer homogenen Kittsubstanz. Darin eingelagert sieht man regelmäßig Thrombocyten. Die faserige Komponente des Amyloids besitzt eine andere Struktur als Reticulum- und Kollagenfasern und verhält sich nach Kontrastierung mit Schwermetallsalzen anders als diese. Mit großer Wahrscheinlichkeit handelt es sich bei den Fasern um die Eiweißkomponente der amyloiden Substanz. Das Amyloid stellt ein rein zwischenzelliges Differenzierungsprodukt dar, es tritt niemals intrazellulär auf.Während der amyloiderzeugenden Behandlung kommt es zu einer plasmazellulären Transformation von Zellen in der roten Pulpa der Milz. Vergleichbare Erscheinungen zeigen sich auch in der Leber. Die Amyloiddepots liegen häufig in unmittelbarer Nachbarschaft von Plasmazellen. Das Cytoplasma, insbesondere Ergastoplasmalamellen von Plasmazellen stehen oft mit dem Amyloid ohne Zwischenschaltung von Zellmembranen in unmittelbarem Kontakt. Diese Befunde sprechen für eine Beteiligung von Plasmazellen bei der Amyloidentstehung. Insbesondere können sie das regelmäßige Auftreten von Antigen-Antikörper-komplexen im Amyloid verständlich machen.Die Untersuchung wurde mit Hilfe der Deutschen Forschungsgemeinschaft durchgeführt.Herrn Prof. Dr. E. Letterer zum 30. 6. 60 gewidmet.     

Characterizing associations and SNP-environment interactions for GWAS-identified prostate cancer risk markers--results from BPC3

Genome-wide association studies (GWAS) have identified multiple single nucleotide polymorphisms (SNPs) associated with prostate cancer risk. However, whether these associations can be consistently replicated, vary with disease aggressiveness (tumor stage and grade) and/or interact with non-genetic potential risk factors or other SNPs is unknown. We therefore genotyped 39 SNPs from regions identified by several prostate cancer GWAS in 10,501 prostate cancer cases and 10,831 controls from the NCI Breast and Prostate Cancer Cohort Consortium (BPC3). We replicated 36 out of 39 SNPs (P-values ranging from 0.01 to 10⁻²⁸). Two SNPs located near KLK3 associated with PSA levels showed differential association with Gleason grade (rs2735839, P = 0.0001 and rs266849, P = 0.0004; case-only test), where the alleles associated with decreasing PSA levels were inversely associated with low-grade (as defined by Gleason grade <8) tumors but positively associated with high-grade tumors. No other SNP showed differential associations according to disease stage or grade. We observed no effect modification by SNP for association with age at diagnosis, family history of prostate cancer, diabetes, BMI, height, smoking or alcohol intake. Moreover, we found no evidence of pair-wise SNP-SNP interactions. While these SNPs represent new independent risk factors for prostate cancer, we saw little evidence for effect modification by other SNPs or by the environmental factors examined.