Local nitric oxide levels reflect the degree of allergic airway inflammation after segmental allergen challenge in asthmatics.

Nitric oxide (NO) levels are increased in the exhaled air of asthmatics. As NO levels correlate with allergic airway inflammation, NO measurement has been suggested for disease monitoring. In patients with asthma, we previously demonstrated that intrabronchial treatment with a natural porcine surfactant enhanced airway inflammation after segmental allergen provocation. We studied whether local levels of NO reflect the degree of allergic airway inflammation following segmental allergen challenge with or without surfactant pretreatment. Segmental NO, as well as nitrite and nitrate in bronchoalveolar lavage (BAL) fluid, was measured before and after segmental challenge with either saline, saline plus allergen, or surfactant plus allergen in 16 patients with asthma and five healthy subjects. The data were compared with inflammatory BAL cells. Segmental NO levels were increased after instillation of saline (p < 0.05), or surfactant plus allergen in asthmatics (p < 0.05), and values were higher after surfactant plus allergen compared to saline challenge. Nitrate BAL levels were not altered after saline challenge but increased after allergen challenge (p < 0.05) and further raised by surfactant (p < 0.05), whereas nitrite levels were not altered by any treatment. Segmental NO and nitrate levels correlated with the degree of eosinophilic airway inflammation, and nitrate levels also correlated with neutrophil and lymphocyte numbers in BAL. In healthy subjects, NO, nitrite, and nitrate were unaffected. Thus, segmental NO and nitrate levels reflect the degree of allergic airway inflammation in patients with asthma. Measurement of both markers can be useful in studies using segmental allergen provocation, to assess local effects of potential immunomodulators.     

Crystal structure of NblA from Anabaena sp. PCC 7120, a small protein playing a key role in phycobilisome degradation

Cyanobacterial light-harvesting complexes, the phycobilisomes, are proteolytically degraded when the organisms are starved for combined nitrogen, a process referred to as chlorosis or bleaching. Gene nblA, present in all phycobilisome-containing organisms, encodes a protein of about 7 kDa that plays a key role in phycobilisome degradation. The mode of action of NblA in this degradation process is poorly understood. Here we presented the 1.8-A crystal structure of NblA from Anabaena sp. PCC 7120. In the crystal, NblA is present as a four-helix bundle formed by dimers, the basic structural units. By using pull-down assays with immobilized NblA and peptide scanning, we showed that NblA specifically binds to the alpha-subunits of phycocyanin and phycoerythrocyanin, the main building blocks of the phycobilisome rod structure. By site-directed mutagenesis, we identified amino acid residues in NblA that are involved in phycobilisome binding. The results provided evidence that NblA is directly involved in phycobilisome degradation, and the results allowed us to present a model that gives insight into the interaction of this small protein with the phycobilisomes.     

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.     

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     

Risk and protective factors for mental disorders beyond genetics: an evidence‐based atlas

Decades of research have revealed numerous risk factors for mental disorders beyond genetics, but their consistency and magnitude remain uncer­tain. We conducted a “meta‐umbrella” systematic synthesis of umbrella reviews, which are systematic reviews of meta‐analyses of individual studies, by searching international databases from inception to January 1, 2021. We included umbrella reviews on non‐purely genetic risk or protective factors for any ICD/DSM mental disorders, applying an established classification of the credibility of the evidence: class I (convincing), class II (highly suggestive), class III (suggestive), class IV (weak). Sensitivity analyses were conducted on prospective studies to test for temporality (reverse causation), TRANSD criteria were applied to test transdiagnosticity of factors, and A Measurement Tool to Assess Systematic Reviews (AMSTAR) was employed to address the quality of meta‐analyses. Fourteen eligible umbrella reviews were retrieved, summarizing 390 meta‐analyses and 1,180 associations between putative risk or protective factors and mental disorders. We included 176 class I to III evidence associations, relating to 142 risk/protective factors. The most robust risk factors (class I or II, from prospective designs) were 21. For dementia, they included type 2 diabetes mellitus (risk ratio, RR from 1.54 to 2.28), depression (RR from 1.65 to 1.99) and low frequency of social contacts (RR=1.57). For opioid use disorders, the most robust risk factor was tobacco smoking (odds ratio, OR=3.07). For non‐organic psychotic disorders, the most robust risk factors were clinical high risk state for psychosis (OR=9.32), cannabis use (OR=3.90), and childhood adversities (OR=2.80). For depressive disorders, they were widowhood (RR=5.59), sexual dysfunction (OR=2.71), three (OR=1.99) or four‐five (OR=2.06) metabolic factors, childhood physical (OR=1.98) and sexual (OR=2.42) abuse, job strain (OR=1.77), obesity (OR=1.35), and sleep disturbances (RR=1.92). For autism spectrum disorder, the most robust risk factor was maternal overweight pre/during pregnancy (RR=1.28). For attention‐deficit/hyperactivity disorder (ADHD), they were maternal pre‐pregnancy obesity (OR=1.63), maternal smoking during pregnancy (OR=1.60), and maternal overweight pre/during pregnancy (OR=1.28). Only one robust protective factor was detected: high physical activity (hazard ratio, HR=0.62) for Alzheimer’s disease. In all, 32.9% of the associations were of high quality, 48.9% of medium quality, and 18.2% of low quality. Transdiagnostic class I‐III risk/protective factors were mostly involved in the early neurodevelopmental period. The evidence‐based atlas of key risk and protective factors identified in this study represents a benchmark for advancing clinical characterization and research, and for expanding early intervention and preventive strategies for mental disorders.     

The serine protease domain of hepatitis C viral NS3 activates RNA helicase activity by promoting the binding of RNA substrate

Nonstructural (NS) protein 3 is a DEXH/D-box motor protein that is an essential component of the hepatitis C viral (HCV) replicative complex. The full-length NS3 protein contains two functional modules, both of which are essential in the life cycle of HCV: a serine protease domain at the N terminus and an ATPase/helicase domain (NS3hel) at the C terminus. Truncated NS3hel constructs have been studied extensively; the ATPase, nucleic acid binding, and helicase activities have been examined and NS3hel has been used as a target in the development of antivirals. However, a comprehensive comparison of NS3 and NS3hel activities has not been performed, so it remains unclear whether the protease domain plays a vital role in NS3 helicase function. Given that many DEXH/D-box proteins are activated upon interaction with cofactor proteins, it is important to establish if the protease domain acts as the cofactor for stimulating NS3 helicase function. Here we show that the protease domain greatly enhances both the direct and functional binding of RNA to NS3. Whereas electrostatics plays an important role in this process, there is a specific allosteric contribution from the interaction interface between NS3hel and the protease domain. Most importantly, we establish that the protease domain is required for RNA unwinding by NS3. Our results suggest that, in addition to its role in cleavage of host and viral proteins, the NS3 protease domain is essential for the process of viral RNA replication and, given its electrostatic contribution to RNA binding, it may also assist in packaging of the viral RNA.     

Lipidomic analysis of lipid droplets from murine hepatocytes reveals distinct signatures for nutritional stress

Liver steatosis can be induced by fasting or high-fat diet. We investigated by lipidomic analysis whether such metabolic states are reflected in the lipidome of hepatocyte lipid droplets (LDs) from mice fed normal chow diet (FED), fasted (FAS), or fed a high-fat diet (HFD). LC-MS/MS at levels of lipid species profiles and of lipid molecular species uncovered a FAS phenotype of LD enriched in triacylglycerol (TG) molecular species with very long-chain (VLC)-PUFA residues and an HFD phenotype with less unsaturated TG species in addition to characteristic lipid marker species. Nutritional stress did not result in dramatic structural alterations in diacylglycerol (DG) and phospholipid (PL) classes. Moreover, molecular species of bulk TG and of DG indicated concomitant de novo TG synthesis and lipase-catalyzed degradation to be active in LDs. DG species with VLC-PUFA residues would be preferred precursors for phosphatidylcholine (PC) species, the others for TG molecular species. In addition, molecular species of PL classes fitted the hepatocyte Kennedy and phosphatidylethanolamine methyltransferase pathways. We demonstrate that lipidomic analysis of LDs enables phenotyping of nutritional stress. TG species are best suited for such phenotyping, whereas structural analysis of TG, DG, and PL molecular species provides metabolic insights.     

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.