Exclusively Heteronuclear NMR Experiments to Obtain Structural and Dynamic Information on Proteins

Provided that ¹³C‐detected NMR experiments are either preferable or complementary to ¹H detection, we report here tools to determine C^α? C′, C′? N, and C^α? H^α residual dipolar couplings on the basis of the CON experiment. The coupling constants determined on ubiquitin are consistent with the subset measured with the ¹H‐detected HNCO sequences. Since the utilization of residual dipolar couplings may depend on the mobility of the involved nuclei, we also provide tools to measure longitudinal and transverse relaxation rates of N and C′. This new set of experiments is a further development of a whole strategy based on ¹³C direct‐detection NMR spectroscopy for the study of biological macromolecules.     

Norcholanic acids as substrates for recombinant 3β-hydroxysteroid dehydrogenase and progesterone 5β-reductase, enzymes of the 5β-cardenolide biosynthesis

The conversion of 23-nor-5,20(22)E-choladienic acid-3β-ol and other intermediates of the putative norcholanic acid pathway of cardenolide biosynthesis by recombinant 3β-hydroxysteroid dehydrogenase from Digitalis lanata in dehydrogenation and reduction reactions was investigated. 23-nor-4,20(22)E-choladienic acid-3-one was found to be a substrate of recombinant progesterone 5β-reductases from D. lanata and Arabidopsis thaliana. The role of various substrates in cardenolide biosynthesis is discussed.     

Genetic diversity among Mycoplasma species bovine group 7: clonal isolates from an outbreak of polyarthritis, mastitis, and abortion in dairy cattle.

A comprehensive genetic analysis of 60 Mycoplasma sp. bovine group 7 isolates from different geographic origins and epidemiological settings is presented. Twenty-four isolates were recovered from the joints of calves during sporadic episodes of polyarthritis in geographically distinct regions of Queensland and New South Wales, Australia, including two clones of the type strain PG5O. A further three Australian isolates were also recovered from the tympanic bulla, retropharyngeal lymph node and the lung and another three isolates had unconfirmed histories. Six isolates originated from Germany, Portugal, Nigeria, and France. Twenty-four epidemiologically related isolates of Mycoplasma sp. bovine group 7 were recovered from multiple tissue sites and body fluids of infected calves with polyarthritis, mastitic milk, and from the stomach contents, lung and liver from aborted foetuses in three large, centrally managed dairy herds in New South Wales, Australia. Restriction endonuclease analysis (REA) of genomic DNA differentiated 29 Cfol profiles among these 60 isolates and grouped all 24 epidemiologically related isolates in a defined pattern showing a clonal origin. Three isolates of this clonal cluster were recovered from mastitic milk and the synovial exudate of clinically-affected calves and appeared sporadically for periods up to 18 months after the initial outbreak of polyarthritis indicating a persistent, close association of the organism with cattle in these herds. The Cfol profile representative of the clonal cluster was distinguishable from profiles of isolates recovered from multiple, unrelated cases of polyarthritis in Queensland and New South Wales and from other countries. All 24 isolates from the clonal cluster possessed a plasmid (pBG7AU) with a molecular size of 1022 bp. DNA sequence analysis of pBG7AU identified two open reading frames sharing 81 and 99% DNA sequence similarity with hypothetical replication control proteins A and B respectively, previously described in plasmid pADB201 isolated from M. mycoides subspecies mycoides. Other isolates of bovine group 7, epidemiologically unrelated to the clonal cluster, including two clones of the type strain PG5O, possessed a similar-sized plasmid. These data confirm that Mycoplasma sp. bovine group 7 is capable of migrating to, and multiplying within, different tissue sites within a single animal and among different animals within a herd.     

Overhauser DNP and EPR in a Mobile Setup: Influence of Magnetic Field Inhomogeneity

Power-dependent Overhauser dynamic nuclear polarization (DNP) enhancements and continuous-wave electron paramagnetic resonance (EPR) spectra of nitroxide radicals were measured in the magnetic field of a mobile Halbach-array permanent magnet and compared with results from a commercially available electromagnet. DNP saturation factors for varying microwave power were obtained from both measurement series and used to investigate how the increased magnetic field inhomogeneity present in the Halbach magnet affects the saturation efficiency. An EPR detection system was designed to allow continuous-wave EPR measurements at microwave power up to 20?W. Our results show that despite the lower magnetic field homogeneity, a Halbach-array magnet can be used for EPR and DNP-enhanced nuclear magnetic resonance of high quality providing almost the same performance as a more homogeneous electromagnet.