Facile crystallization of Escherichia coli ketol-acid reductoisomerase

Ketol-acid reductoisomerase (EC 1.1.1.86) catalyses the second reaction in the biosynthesis of branched-chain amino acids. The reaction involves an Mg2+-dependent alkyl migration followed by an NADPH-dependent reduction of the 2-keto group. Here, the crystallization of the Escherichia coli enzyme is reported. A form with a C-terminal hexahistidine tag could be crystallized under 18 different conditions in the absence of NADPH or Mg2+ and a further six crystallization conditions were identified with one or both ligands. With the hexahistidine tag on the N-terminus, 20 crystallization conditions were found, some of which required the presence of NADPH, NADP(+), Mg2+ or a combination of ligands. Finally, the selenomethionine-substituted enzyme with the N-terminal tag crystallized under 15 conditions. Thus, the enzyme is remarkably easy to crystallize. Most of the crystals diffract poorly but several data sets were collected at better than 3.2 Angstrom resolution; attempts to phase them are currently in progress

Analysis of Synaptic Proteins in the Cerebrospinal Fluid as a New Tool in the Study of Inborn Errors of Neurotransmission

Abstract In a few rare diseases, specialised studies in cerebrospinal fluid (CSF) are required to identify the underlying metabolic disorder. We aimed to explore the possibility of detecting key synaptic proteins in the CSF, in particular dopaminergic and gabaergic, as new procedures that could be useful for both pathophysiological and diagnostic purposes in investigation of inherited disorders of neurotransmission. Dopamine receptor type 2 (D2R), dopamine transporter (DAT) and vesicular monoamine transporter type 2 (VMAT2) were analysed in CSF samplesfrom 30 healthy controls (11 days to 17 years) by western blot analysis. Because VMAT2 was the only protein with intracellular localisation, and in order to compare results, GABA vesicular transporter, which is another intracellular protein, was also studied. Spearman’s correlation and Student’s t tests were applied to compare optical density signals between different proteins. All these synaptic proteins could be easily detected and quantified in the CSF. DAT, D2R and GABA VT expression decrease with age, particularly in the first months of life, reflecting the expected intense synaptic activity and neuronal circuitry formation. A statistically significant relationship was found between D2R and DAT expression, reinforcing the previous evidence of DAT regulation by D2R. To our knowledge, there are no previous studies on human CSF reporting a reliable analysis of these proteins. These kinds of studies could help elucidate new causes of disturbed dopaminergic and gabaergic transmission as well as understanding different responses to L-dopa in inherited disorders affecting dopamine metabolism. Moreover, this approach to synaptic activity in vivo can be extended to different groups of proteins and diseases

Advanced glycation end products (AGEs) in relation to atherosclerotic lipid profiles in middle-aged and elderly diabetic patients

<p>Abstract</p> <p>Objectives</p> <p>To evaluate the association between AGEs and atherosclerotic lipid profiles among aging diabetic patients in Taiwan.</p> <p>Design and Methods</p> <p>After age and gender matching, we selected 207 diabetic subjects and 174 diabetic subjects with proteinuria. Lipid profiles, including total cholesterol (TC), triglycerides (TG), high density cholesterol-lipoprotein (HDL-C) and low density lipoprotein-cholesterol (LDL-C) were measured using standard methods. AGEs were measured with the immunoassay method.</p> <p>Results</p> <p>In general, males were heavier; however, females had higher AGEs, fasting glucose (GLU), TC, HDL-C and LDL-C levels than males, and had higher TC/HDL-C, LDL-C/HDL-C, and TG/HDL-C ratios compared to males. AGEs were more strongly correlated with TG levels and TCL/LDL-C, LDL-C/HDL-C and TG/HDL-C ratios when compared to glucose or hemoglobin A1c. Subjects had higher AGEs levels (≧ 2.0 AU) with more adverse lipid profiles.</p> <p>Conclusion</p> <p>AGEs seem to be a good biomarker to evaluate the association between diabetes and atherosclerotic disorders in aging diabetes.</p

Meningitis and a Febrile Vomiting illness Caused by Echovirus Type 4, Northern Territory, Australia

A strain that emerged in July 2007 caused laboratory-confirmed meningitis

Finding the region of pseudo-periodic tandem repeats in biological sequences

SUMMARY: The genomes of many species are dominated by short sequences repeated consecutively. It is estimated that over 10% of the human genome consists of tandemly repeated sequences. Finding repeated regions in long sequences is important in sequence analysis. We develop a software, LocRepeat, that finds regions of pseudo-periodic repeats in a long sequence. We use the definition of Li et al. [1] for the pseudo-periodic partition of a region and extend the algorithm that can select the repeated region from a given long sequence and give the pseudo-periodic partition of the region. AVAILABILITY: LocRepeat is available a

The interaction of mammalian mitochondrial translational initiation factor 3 with ribosomes: evolution of terminal extensions in IF3mt

Mammalian mitochondrial initiation factor 3 (IF3mt) has a central region with homology to bacterial IF3. This homology region is preceded by an N-terminal extension and followed by a C-terminal extension. The role of these extensions on the binding of IF3mt to mitochondrial small ribosomal subunits (28S) was studied using derivatives in which the extensions had been deleted. The Kd for the binding of IF3mt to 28S subunits is ∼30 nM. Removal of either the N- or C-terminal extension has almost no effect on this value. IF3mt has very weak interactions with the large subunit of the mitochondrial ribosome (39S) (Kd = 1.5 μM). However, deletion of the extensions results in derivatives with significant affinity for 39S subunits (Kd = 0.12−0.25 μM). IF3mt does not bind 55S monosomes, while the deletion derivative binds slightly to these particles. IF3mt is very effective in dissociating 55S ribosomes. Removal of the N-terminal extension has little effect on this activity. However, removal of the C-terminal extension leads to a complex dissociation pattern due to the high affinity of this derivative for 39S subunits. These data suggest that the extensions have evolved to ensure the proper dissociation of IF3mt from the 28S subunits upon 39S subunit joining

Amino acid "little Big Bang": Representing amino acid substitution matrices as dot products of Euclidian vectors

<p>Abstract</p> <p>Background</p> <p>Sequence comparisons make use of a one-letter representation for amino acids, the necessary quantitative information being supplied by the substitution matrices. This paper deals with the problem of finding a representation that provides a comprehensive description of amino acid intrinsic properties consistent with the substitution matrices.</p> <p>Results</p> <p>We present a Euclidian vector representation of the amino acids, obtained by the singular value decomposition of the substitution matrices. The substitution matrix entries correspond to the dot product of amino acid vectors. We apply this vector encoding to the study of the relative importance of various amino acid physicochemical properties upon the substitution matrices. We also characterize and compare the PAM and BLOSUM series substitution matrices.</p> <p>Conclusions</p> <p>This vector encoding introduces a Euclidian metric in the amino acid space, consistent with substitution matrices. Such a numerical description of the amino acid is useful when intrinsic properties of amino acids are necessary, for instance, building sequence profiles or finding consensus sequences, using machine learning algorithms such as Support Vector Machine and Neural Networks algorithms.</p

Crystal structure of human selenocysteine tRNA

Selenocysteine (Sec) is the 21st amino acid in translation. Sec tRNA (tRNASec) has an anticodon complementary to the UGA codon. We solved the crystal structure of human tRNASec. tRNASec has a 9-bp acceptor stem and a 4-bp T stem, in contrast with the 7-bp acceptor stem and the 5-bp T stem in the canonical tRNAs. The acceptor stem is kinked between the U6:U67 and G7:C66 base pairs, leading to a bent acceptor-T stem helix. tRNASec has a 6-bp D stem and a 4-nt D loop. The long D stem includes unique A14:U21 and G15:C20a pairs. The D-loop:T-loop interactions include the base pairs G18:U55 and U16:U59, and a unique base triple, U20:G19:C56. The extra arm comprises of a 6-bp stem and a 4-nt loop. Remarkably, the D stem and the extra arm do not form tertiary interactions in tRNASec. Instead, tRNASec has an open cavity, in place of the tertiary core of a canonical tRNA. The linker residues, A8 and U9, connecting the acceptor and D stems, are not involved in tertiary base pairing. Instead, U9 is stacked on the first base pair of the extra arm. These features might allow tRNASec to be the target of the Sec synthesis/incorporation machineries

Nematode-specific tRNAs that decode an alternative genetic code for leucine

Class II transfer RNAs (tRNAs), including tRNALeu and tRNASer, have an additional stem and loop structure, the long variable arm (V-arm). Here, we describe Class II tRNAs with a unique anticodon corresponding to neither leucine nor serine. Because these tRNAs are specifically conserved among the nematodes, we have called them ‘nematode-specific V-arm-containing tRNAs’ (nev-tRNAs). The expression of nev-tRNA genes in Caenorhabditis elegans was confirmed experimentally. A comparative sequence analysis suggested that the nev-tRNAs derived phylogenetically from tRNALeu. In vitro aminoacylation assays showed that nev-tRNAGly and nev-tRNAIle are only charged with leucine, which is inconsistent with their anticodons. Furthermore, the deletion and mutation of crucial determinants for leucylation in nev-tRNA led to a marked loss of activity. An in vitro translation analysis showed that nev-tRNAGly decodes GGG as leucine instead of the universal glycine code, indicating that nev-tRNAs can be incorporated into ribosomes and participate in protein biosynthesis. Our findings provide the first example of unexpected tRNAs that do not consistently obey the general translation rules for higher eukaryotes