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.     

Structure of the stridulation ofOrchelimum (Orthoptera: Tettigoniidae)

Summary The structure of the stridulation was investigated by re-playing tape-recordings at very slow speed. The findings were corrobrated by sonograms and mingograms.The central part of the song is the ripple, a fast succession of syllables around which isolated syllables (clicks) are distributed according to species and circumstances. The rate of syllables in the ripple is a linear function of temperature.A quantitative expression for the stridulatory activity is the actual number of syllables per time unit, including pauses. By changes in the combination of elements, at leastO. agile is able to increase the output of syllables four to six times.This work was made possible by a grant from the Carlsberg Foundation to whom my most sincere thanks are due. —As I do not have advanced equipment for sound analysis, I am very much indebted to Dr. Bondesen and cand. sci. Poul Hansen, Bioakustisk Laboratorium, Naturhistorisk Museum, Aarhus, Denmark, and Mr. W.B. Broughton and Dr. M. Samways, Animal Acoustic Unit, City of London Polytechnic, London, for analysing part of the material by sonograms and mingograms. The very valuable help of Dr. Th.J. Walker, University of Florida, Gainesville, Florida, in identifying the species is gratefully acknowledged. For friendly discussions and linguistic corrections my best thanks are due to H.T. Evans and F.D.S. Evans.