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[en] Full text: MiAMP1 is a recently discovered 76 amino acid, highly basic protein from the nut kernel of Macadamia integrifolia which possesses no sequence homology to any known protein. A study of its antimicrobial activity revealed that it inhibited the growth of several microbial plant pathogens in vitro but had no effect on mammalian or plant cells. For these reasons, MiAMP1 is considered to be a potentially useful tool for the genetic engineering of disease resistance in transgenic crop plants and for the design of new fungicides. The three-dimensional structure of MiAMP1 was determined through homonuclear and heteronuclear (¹⁵N) 2D NMR spectroscopy and subsequent simulated annealing calculations. MiAMP1 is made up of eight β-strands which are arranged in two Greek key motifs. These Greek key motifs associate to form a Greek key β-barrel. This structure is unique amongst plant antimicrobial proteins and forms a new class which we term the β-Barrelins. Interestingly, the structure of MiAMP1 bears remarkable similarity to a yeast killer toxin from Williopsis mrakii. The structural similarity of MiAMP1 and WmKT, which originate from plant and fungal phyla respectively, may reflect a similar mode of action

[en] Full text: The arrest of replication forks during the termination of DNA replication in Bacillus subtilis is dependent upon the binding of the 30 kDa replication terminator protein (RTP) to its cognate Ter binding site. Two adjacently bound dimers of RTP form a termination complex that can prevent the progression of a replication fork approaching from one direction, but not the other. The crystal structure of free RTP has previously been solved, but the precise orientation with which it binds to Ter sites remains unknown. This information is important for understanding the molecular mechanism of replication fork arrest. We have used NMR spectroscopy to observe ¹H-¹⁵N correlations arising from ¹⁵N-labelled RTP mutant, and to track their perturbations upon the addition of DNA. This showed that 60% of the amino acid residues are affected by the DNA interaction, and also that the complex is symmetrical. Assignment of the ¹H-¹⁵N correlations was achieved using a suite of triple resonance NMR experiments with ¹⁵N,¹³C,²H enriched protein recorded at 800 MHz and using TROSY pulse sequences. This revealed that α3-helices are involved in the binding interaction, and that the 'wings' of RTP may not be contributing to binding. Crystals of the complex have been grown from the NMR sample, and data collected to 3.1 Angstroms is anticipated to provide further molecular detail

[en] Full text: The techniques of Magnetic Resonance Spectroscopy (MRS) and Imaging (MRI) are outlined, and compared with Positron Emission Tomography (PET). Invasive PET techniques using ¹⁹F-fluorodeoxyglucose (FDG) and ¹⁸O₂ form the main basis of brain activation studies, and with ¹⁹F-fluoroDOPA, make major contributions to studies on neurological disorders such as stroke, Alzheimer's disease and Parkinson's disease. However the technique has no chemical specificity so can provide no knowledge of intermediary metabolism. Non-invasive MRI is also being applied to brain activation studies but also has no chemical specificity. On the other hand MRS has superb chemical specificity, although it suffers from low sensitivity. A most interesting example of this is the use of ¹³C-MRS. If glucose is labelled on the no. 1 or no. 2 positions with ¹³C, the passage of the label through different neuronal and glial metabolic pathways can be followed. If acetate is similarly labelled, metabolic routes through specifically glial pathways can be monitored, since acetate is taken up only by glia. These studies contributed to knowledge on metabolic trafficking, in that glia produce alanine, citrate and lactate in addition to the previously characterised production of glutamine. Studies on the hypoxic brain revealed increased production of alanine, lactate and glycerol 3-phosphate, providing further understanding of the role of the NADH redox state. 'Isotopomer analysis' of ¹³C resonances provides more information on metabolic pathways, because the chemical shift of a ¹³C atom is specifically affected by a neighbouring ¹³C within the same molecule. This approach was used to demonstrate that neurotransmitter γ-aminobutyrate (GABA) is partly derived from glial glutamine. Analogous ¹³C MRS studies are now providing novel information on metabolic flux rates within the human brain, and the most exciting developments are to follow changes in these rates on brain activation which can be compared with results from PET activation studies. Thus we have new windows on human brain function