Human DDX17 Unwinds Rift Valley Fever Virus Non-Coding RNAs

Rift Valley fever virus (RVFV) is a mosquito-transmitted virus from the Bunyaviridae family that causes high rates of mortality and morbidity in humans and ruminant animals. Previous studies indicated that DEAD-box helicase 17 (DDX17) restricts RVFV replication by recognizing two primary non-coding RNAs in the S-segment of the genome: the intergenic region (IGR) and 5′ non-coding region (NCR). However, we lack molecular insights into the direct binding of DDX17 with RVFV non-coding RNAs and information on the unwinding of both non-coding RNAs by DDX17. Therefore, we performed an extensive biophysical analysis of the DDX17 helicase domain (DDX17_135–555) and RVFV non-coding RNAs, IGR and 5’ NCR. The homogeneity studies using analytical ultracentrifugation indicated that DDX17_135–555, IGR, and 5’ NCR are pure. Next, we performed small-angle X-ray scattering (SAXS) experiments, which suggested that DDX17 and both RNAs are homogenous as well. SAXS analysis also demonstrated that DDX17 is globular to an extent, whereas the RNAs adopt an extended conformation in solution. Subsequently, microscale thermophoresis (MST) experiments were performed to investigate the direct binding of DDX17 to the non-coding RNAs. The MST experiments demonstrated that DDX17 binds with the IGR and 5’ NCR with a dissociation constant of 5.77 ± 0.15 µM and 9.85 ± 0.11 µM, respectively. As DDX17_135–555 is an RNA helicase, we next determined if it could unwind IGR and NCR. We developed a helicase assay using MST and fluorescently-labeled oligos, which suggested DDX17_135–555 can unwind both RNAs. Overall, our study provides direct evidence of DDX17_135–555 interacting with and unwinding RVFV non-coding regions.     

Die Bestimmung der Saccharose mit alkalischer Jodlösung

European Food Research and Technology - Das Verfahren zur titrimetrischen Bestimmung von Glykose mit alkalischer Jodlösung nach Willstätter und Schudel, in der Ausführungsform von...     

Hochleistungsoszillographen mit abgeschmolzener Braunscher Röhre

Zusammenfassung Es wird ein neuentwickelter Hochleistungsmeßoszillograph mit abgeschmolzener Braunscher Röhre beschrieben, der in seiner Meßleistung die bisherigen, an der Pumpe betriebenen Kathodenstrahloszillographen erreicht, im Gegensatz zu diesen aber als tragbares Gerät ausgebildet ist. Der Oszillograph arbeitet mit Außenaufnahme mittels Kamera und Linse. Weiterhin wird über einen ebenfalls neuentwickelten Demonstrations-Kathodenstrahloszillographen berichtet, der dazu bestimmt ist, auch in den größten Hörsälen elektrische Vorgänge bis zu Tonfrequenzen hinauf als einmalige Vorgänge zu projizieren.An der Entwicklung der Braunschen Röhrc haben die Herren Dr. H. Bode, Dr. J. Dosse, E. Hentschel und Dr. H. Hinderer, an der Entwicklung der Netzgeräte und des meßtechnischen Teils der Oszillographen hat Herr H. Diebold maßgebenden Anteil. — Frl. Dr. H. Riedl führte die experimentelle Bestimmung der Meßleistung durch. Ihnen allen möchten wir für ihre Mitarbeit unseren besten Dank aussprechen.     

Mechanism of Rab geranylgeranylation: formation of the catalytic ternary complex

Rab proteins are geranylgeranylated on one or two C-terminal cysteines by Rab geranylgeranyl transferase (RabGGTase). The reaction is dependent on a Rab-binding protein, termed Rab escort protein (REP). Here, we studied the role of REP in the geranylgeranylation reaction. We first characterized the interaction between REP and ungeranylgeranylated Rab using analytical ultracentrifugation and a fluorescence-based assay. We measured an equilibrium dissociation constant of 0.2 microM for the formation of a 1:1 REP-Rab complex and showed that this interaction relies mostly on ionic bonds and does not involve the two C-terminal cysteine residues. Second, we show that REP is required for recognition of Rab by RabGGTase and therefore that the REP-Rab complex is the true substrate for RabGGTase. Third, we show that free REP inhibits the geranylgeranylation reaction, suggesting that the complex is recognized by RabGGTase primarily via a REP-binding site. Our data suggest a model whereby REP behaves kinetically as an essential activator of the reaction.     

Pyruvate dehydrogenase multienzyme complex. Characterization of assembly intermediates by sedimentation velocity analysis

The pyruvate dehydrogenase complex is a large, highly organized assembly of several different catalytic and regulatory component enzymes. The structural core of the complex is the E2-X subcomplex, consisting of 60 dihydrolipoamide transacetylase (E2) subunits arranged in a pentagonal dodecahedron; 6 protein X and 2 pyruvate dehydrogenase kinase molecules are tightly associated with this E2 60-mer. The native E2-X subcomplex exhibits a sedimentation coefficient of 32 S. The effects of several chaotropes (guanidinium chloride, potassium thiocyanide, and urea) on the E2-X subcomplex were assessed. Treatment of the E2-X subcomplex with 4 M guanidinium chloride caused a complete loss of enzymatic activity and the dissociation of the subcomplex into monomeric 1.5-3 S species. Removal of the chaotrope by dialysis for 18 h resulted in complete restoration of E2 enzymatic activity and reassembly of a 32 S subcomplex; this reassembled subcomplex contained less protein X than the native subcomplex. Sedimentation velocity analysis of reassembled E2-X subcomplex demonstrated the presence of an 8 S assembly intermediate; this sedimentation coefficient is characteristic of globular proteins of molecular weights similar to that expected for a trimer of E2. Shorter periods of dialysis also gave rise to the 8 S species; the amount of this intermediate decreased with increasing times of dialysis. The 8 S species associated non-cooperatively to yield additional assembly intermediates exhibiting sedimentation coefficients of 10-32 S.     

Zerstreuung von Elektronenstrahlen durch eigene Raumladung

Zusammenfassung Die durch die eigene Raumladung verursachte Durchmesserzunahme eines Elektronenstrahles wird unter der Voraussetzung unveränderlicher Stromdichte über den jeweiligen Strahlquerschnitt und einheitlicher Elektronengeschwindigkeit berechnet. Im Gegensatz zu früheren Veröffentlichungen, in denen nur die Durchmesserzunahme des anfangs parallelen Strahles angegeben wurde, wird für den meist vorliegenden Fall bestimmter (positiver oder negativer) Apertur des Strahles die zahlenmäßige Berechnung der gesamten Durchmesserveränderung, also der geometrischen Durchmesserzunahme oder -abnähmezusammen mit der durch Raumladung verursachten Durchmesserzunahme, auf die Benutzung einer Kurventafel mit Hilfe zweier GrößenA undB zurückgeführt, die aus den gegebenen Werten (Strahlspannung und -strom, Strahllänge, Anfangshalbmesser und -richtung) berechnet oder aus Nomogrammen abgelesen werden können. Die Kurvendarstellungen werden durch Angabe von Näherungsformeln und deren Gültigkeitsbereichen ergänzt. An Hand einiger Beispiele und weiterer Anwendungen wird die Benutzung der Ergebnisse und der Tafeln erläutert.     

A Regional Comparison of Calibration Results for Type K Thermocouple Wire from (100 to 1,100) °C

Under the auspices of the Inter-American Metrology System (SIM), the National Institute of Standards and Technology (NIST) initiated a regional comparison for type K thermocouples from (100 to 1,100) °C with 11 participating countries. The use of type K material above approximately 200 °C is considered destructive. Therefore, each participating laboratory was sent new, unused wire from a lot of material characterized by NIST. The uniformity of the lot was remarkable, especially at temperatures above 500 °C; the standard deviation of the thermocouple emf values of multiple cuts tested at NIST was 2.7 μV or less over the full temperature range. The high uniformity eliminated any need to correct for variations of the transfer standard among the laboratories, greatly simplifying the analysis. The level of agreement among the laboratories’ results was quite good. Even though test procedures and equipment varied significantly among the participants, the standard deviation of all emf values at each test temperature was less than the equivalent of 0.20 °C at 200 °C and below, and less than 0.60 °C from (400 to 1,100) °C. Of the 380 total bilateral combinations of the data at the eight test temperatures, only 13 (i.e., 3.4% of all combinations) are outside the k = 2 limits, and of these 13, only 3 are outside k = 3 limits. All the outliers occur at temperatures of 800 °C and below, which suggests that drift of the type K wire due to high-temperature oxidation did not cause changes in the thermocouple emf comparable to or larger than the claimed uncertainties.     

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Ohne Zusammenfassung