Role of Heme in Synthesis and Membrane Binding of Succinic Dehydrogenase in Bacillus subtilis

A 5-aminolevulinic acid-requiring mutant of Bacillus subtilis was isolated. When the mutant is shifted from medium containing 5-aminolevulinic acid to medium lacking this growth factor, the bacteria continued to grow at undiminished rate for about three generations. The membranes from these bacteria contained severely reduced amounts of cytochrome. The mutant was used to study the role of heme synthesis on synthesis and membrane binding of succinic dehydrogenase (SDH). The amount of SDH in whole-cell lysates in the soluble cytoplasmic fraction and in membranes was determined by one-dimensional (rocket) immunoelectrophoresis with an SDH-specific antiserum. After heme synthesis was blocked, the relative amount of SDH in the membrane decreased, whereas increasing amounts of SDH antigen were found in the cytoplasm. When heme synthesis was resumed on readdition of 5-aminolevulinic acid, the amount of membrane-bound SDH antigen increased at a much faster rate than net synthesis. During a 3-h growth period without 5-aminolevulinic acid, there was little change in the pattern of membrane proteins as determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis of radioactively labeled membranes, as compared to membranes from control cultures. However, both the 65,000-dalton and the 28,000-dalton polypeptides of the SDH complex (L. Hederstedt, E. Holmgren, and L. Rutberg, J. Bacteriol. 138:370-376, 1979) were present in decreasing amounts in membranes from 5-aminolevulinic acid-starved bacteria. From these results we suggest that SDH in B. subtilis is synthesized as a soluble protein and becomes membrane bound only when it attaches to a site in the membrane, (part of) which is a cytochrome of b type.     

Circadian rhythms of sensitivity to insecticides in Musca domestica (Diptera, Muscidae)

Circadian rhythms of LD₅₀ values to DDT, dieldrin and malathion, topically applied, were determined for houseflies reared under LD 14:10 with dawn at 06.00 hr. There was a marked increase in susceptibility at 05.00 hr in each case. With dawn at 18.00 hr., DDT LD₅₀ values were lowest at 17.00 hr indicating independence of the flies' biological clocks from clock time of day. Flies reared under LD 18:6 and 10:14 also had circadian rhythms of sensitivity to DDT. Mean daily LD₅₀ values were inversely related to photophase length. The ratios of mean daily LD₅₀ to pre-dawn values were greatest for the longer photophases. Flies reared under LD 14:10 until the pupal stage, then DD until testing showed a normal circadian rhythm. Flies reared in total darkness (DD) showed no diel variations in susceptibility. W.H.O. standard strain flies were used for all the experiments. A fully susceptible (Cooper) and a DDT resistant (DEH-DOV) strain also showed significant circadian rhythms of sensitivity to DDT.

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Zusammenfassung Circadianrhythmen der LD 50-Werte gegenüber DDT, Dieldrin und Malathion-topical angewandt wurden bei Stubenfliegen ermittelt, die bei 14:10 h-Tag mit Tagesanbruch um 6.00 Uhr gezüchtet wurden. In allen Fällen war die Empfindlichkeit um 5.00 Uhr wesentlich erhöht. Bei Tagesanbruch um 18.00 Uhr waren die niedrigsten LD 50-Werte um 17.00 Uhr. Dies weist auf die Unabhängigkeit der biologischen Uhr der Fliegen von der Tageszeit hin. Fliegen, die bei 18:6 oder bei 10:14 LD gezüchtet wurden, zeigten ebenfalls einen Circadianrhythmus hinsichtlich der Empfindlichkeit gegenüber DDT. Die mittleren LD 50-Werte waren umgekehrt proportional zur Länge der Photophase. Das Verhältnis der mittleren täglichen LD 50-Werte zu den Vortagesanbruchwerten war am grössten bei längerer Photophase. Fliegen, die bei 14:10 LD bis zum Puppenstadium und anschliessend bei DD bis zur Testung gehalten wurden, zeigten einen normalen circadianen Rhythmus. Bei Züchtung in völliger Dunkelheit zeigten sie keine Tagesschwankungen in der Empfindlichkeit. Für alle Versuche wurde ein WHO-Standardstamm benutzt. Zwei andere Stämme, einer voll empfindlich (Cooper), der andere resistent (DEH-DOV) zeigten ebenfalls signifikante Circadianrhythmen in der DDT-Empfindlichkeir.

     

Diel changes in DDT absorption and breakdown rates and respiratory rhythm in the housefly, Musca domestica

Absorption rates in three strains of housefly show little variation when DDT is applied at different times during the 24-hr cycle. Rates of breakdown of DDT to DDE, however, vary significantly at different times of the day with most rapid breakdown at 05.00–05.30 and 15.00 hr, corresponding to diel peaks in oxygen consumption. The pre-dawn peak breakdown rates occur at the times at which the flies were found, previously, to be most susceptible to DDT, thus this change in susceptibility must have other causes.

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Zusammenfassung Die Geschwindigkeit, mit der DDT von drei Stubenfliegenrassen absorbiert wurde, zeigte nur wenig Verschiedenheit, wenn es zu verschiedenen Zeiten im Verlauf des 24-h-Tages appliziert wurde. Die Rate des Abbaus von DDT zu DDE variierte dagegen im Lauf des Tages, sie war am grössten um 5.00 bis 5.30 Uhr und um 15.00 Uhr, entsprechend den Tagesspitzen des Sauerstoff-Verbrauchs. Das erste Abbaumaximum (vor Tagesanbruch) fiel zusammen mit der Zeit, in der die Fliegen höchste Empfindlichkeit gegenüber DDT zeigten (vgl. vorige Veröffentlichung). Diese tageszeitspezifische Empfindlichkeit liess sich demnach keineswegs mit einer (geringeren) Abbaugeschwindigkeit in Zusammenhang bringen.

     

Glutamine-dependent asparagine synthetase from Lupinus luteus

Asparagine synthetase (glutamine-hydrolyzing [l-aspartate: l-glutamine amido-ligase (AMP-forming), E.C. 6.3.5.4] was purified over 500-fold from cotyledon extracts of 1-week-old yellow lupin seedlings. The enzyme was labile and required protection by high levels of thiols; glycerol and the substrates also stabilized it. The reaction products were shown to be asparagine, AMP, PPi and glutamate. The limiting K_m values were for aspartate 1·3 mM, for MgATP 0·14 mM and for glutamine 0·16 mM. Positive homotropic cooperativity was observed for MgATP only, and gel filtration studies indicated that the substrate-free enzyme (MW 160 000) associated to a dimer (MW 320 000 in the presence of MgCl₂ and ATP. The purified enzyme, which had some glutaminase activity, catalyzed an aspartate- and glutamine-independent ATP-PPi exchange reaction at a rate 5–7-fold higher than the rate of asparagine synthesis. Initial velocity studies and exchange data indicated an overall ping-pong mechanism. Compared to similar enzymes isolated from mammalian tumor cells, the lupin enzyme appears to be unique with respect to MW, reaction mechanism and regulatory properties. The allosteric properties observed suggest an important role for this enzyme in the regulation of asparagine biosynthesis.     

Spectral imaging for precise surgical intervention in Hirschsprung's Disease

We used advanced spectral imaging for intrasurgical decision making in a preclinical study, on a mouse model of Hirschsprung's Disease. Our imaging device sampled areas from normal and abnormal (aganglionic) colon in these animals. Spectral segmentation and classification of the resulting images showed a clear distinction between the normal and aganglionic regions, as confirmed by pathological analysis and use of mutant mice. We developed a simple algorithm that could distinguish normal from aganglionic colon with high spatial resolution and reproducibility, and the following statistics: sensitivity = 97%, specificity = 94%, positive predictive value = 92%, negative predictive value = 98%. These studies showed translational proof of concept that spectral imaging could be used during operations, in real time, to help surgeons precisely distinguish normal from abnormal tissue without requiring traditional biopsy. (© 2008 WILEY‐VCH Verlag GmbH & Co. KGaA, Weinheim)     

Caffeine Junkie: an Unprecedented Glutathione S-Transferase-Dependent Oxygenase Required for Caffeine Degradation by Pseudomonas putida CBB5

Caffeine and other N-methylated xanthines are natural products found in many foods, beverages, and pharmaceuticals. Therefore, it is not surprising that bacteria have evolved to live on caffeine as a sole carbon and nitrogen source. The caffeine degradation pathway of Pseudomonas putida CBB5 utilizes an unprecedented glutathione-S-transferase-dependent Rieske oxygenase for demethylation of 7-methylxanthine to xanthine, the final step in caffeine N-demethylation. The gene coding this function is unusual, in that the iron-sulfur and non-heme iron domains that compose the normally functional Rieske oxygenase (RO) are encoded by separate proteins. The non-heme iron domain is located in the monooxygenase, ndmC, while the Rieske [2Fe-2S] domain is fused to the RO reductase gene, ndmD. This fusion, however, does not interfere with the interaction of the reductase with N₁- and N₃-demethylase RO oxygenases, which are involved in the initial reactions of caffeine degradation. We demonstrate that the N₇-demethylation reaction absolutely requires a unique, tightly bound protein complex composed of NdmC, NdmD, and NdmE, a novel glutathione-S-transferase (GST). NdmE is proposed to function as a noncatalytic subunit that serves a structural role in the complexation of the oxygenase (NdmC) and Rieske domains (NdmD). Genome analyses found this gene organization of a split RO and GST gene cluster to occur more broadly, implying a larger function for RO-GST protein partners.     

Mutation K42R in Ribosomal Protein S12 Does Not Affect Susceptibility of Mycobacterium smegmatis 16S rRNA A-Site Mutants to 2-Deoxystreptamines

Recent studies have suggested that ribosomal protein S12 modulates 16S rRNA function and susceptibility to 2-deoxystreptamine aminoglycosides. To study whether the non-restrictive K42R mutation in RpsL affects 2-deoxystreptamine susceptibility in Mycobacterium smegmatis, we studied the drug susceptibility pattern of various mutants with genetic alterations in the 16S rRNA decoding A-site in the context of wild-type and mutant protein S12. RpsL K42R substitution was found not to affect the drug resistance pattern associated with mutational alterations in 16S rRNA H44.     

Failure of duplexes based on polylaurusin [poly(L), “polyformycin B”] to induce interferon

Polylaurusin[poly(L) or “polyformycin B”] forms double-stranded complexes with polycytidylic acid (poly(C)) and with poly(5-bromocytidylic acid) [poly(br⁵C)] with T_m's of 46.5° (0.2 $\text{M}$ NaCl, pH 7) and 72.5° (0.15 M NaCl, pH 7), respectively. Both complexes fail to provide antiviral resistance (against vesicular stomatitis virus in primary rabbit kidney cells) or to induce interferon in “superinduced” primary rabbit kidney cells, even though they fulfill all previously recognized requirements for effective interferon inducers.