A minimal TrpRS catalytic domain supports sense/antisense ancestry of class I and II aminoacyl-tRNA synthetases

The emergence of polypeptide catalysts for amino acid activation, the slowest step in protein synthesis, poses a significant puzzle associated with the origin of biology. This problem is compounded as the 20 contemporary aminoacyl-tRNA synthetases belong to two quite distinct families. We describe here the use of protein design to show experimentally that a minimal class I aminoacyl-tRNA synthetase active site might have functioned in the distant past. We deleted the anticodon binding domain from tryptophanyl-tRNA synthetase and fused the discontinuous segments comprising its active site. The resulting 130 residue minimal catalytic domain activates tryptophan. This residual catalytic activity constitutes the first experimental evidence that the conserved class I signature sequences, HIGH and KMSKS, might have arisen in-frame, opposite motifs 2 and 1 from class II, as complementary sense and antisense strands of the same ancestral gene.     

Depositional processes of ribbon carbonates in Middle Cambrian of Iran (Deh-Sufiyan Formation,Central Alborz)

A variety of ribbon carbonates of the Deh-Sufiyan Formation (Middle Cambrian) in Central Alborz Range of northern Iran are studied to provide facies characterization and paleoenvironmental interpretation of ribbon carbonates on shallow-marine carbonate platforms. Seven types of ribbon carbonates are divided based mainly on sedimentary structures, ichnofossils, and bed geometry, which represent deposition during different phases of storm-induced processes. The different features of the storm deposits in ribbon carbonates such as hummocky and swaley cross-stratification, planar lamination, and combined-flow-ripple cross-stratification were formed by combined flows. Identification and interpretation of ichnological signatures and the spatial arrangement of succession of sedimentary structures are used to further refine sedimentary interpretations of parameters such as wave energy, substrate properties, variability in sedimentation rates, and proximality-distality trends of a wave-dominated marine ramp sequence. Successions from individual storm events reflect deposition during increasing combined oscillatory and unidirectional flow succeeded by the waning stages. The study provides depositional processes and models of various ribbon carbonates that may be useful for facies interpretation of ribbon rocks elsewhere.     

Sequence-Level Analysis of the Major European Huntington Disease Haplotype

Huntington disease (HD) reflects the dominant consequences of a CAG-repeat expansion in HTT. Analysis of common SNP-based haplotypes has revealed that most European HD subjects have distinguishable HTT haplotypes on their normal and disease chromosomes and that ∼50% of the latter share the same major HD haplotype. We reasoned that sequence-level investigation of this founder haplotype could provide significant insights into the history of HD and valuable information for gene-targeting approaches. Consequently, we performed whole-genome sequencing of HD and control subjects from four independent families in whom the major European HD haplotype segregates with the disease. Analysis of the full-sequence-based HTT haplotype indicated that these four families share a common ancestor sufficiently distant to have permitted the accumulation of family-specific variants. Confirmation of new CAG-expansion mutations on this haplotype suggests that unlike most founders of human disease, the common ancestor of HD-affected families with the major haplotype most likely did not have HD. Further, availability of the full sequence data validated the use of SNP imputation to predict the optimal variants for capturing heterozygosity in personalized allele-specific gene-silencing approaches. As few as ten SNPs are capable of revealing heterozygosity in more than 97% of European HD subjects. Extension of allele-specific silencing strategies to the few remaining homozygous individuals is likely to be achievable through additional known SNPs and discovery of private variants by complete sequencing of HTT. These data suggest that the current development of gene-based targeting for HD could be extended to personalized allele-specific approaches in essentially all HD individuals of European ancestry.     

Transient Receptor Potential Canonical 1 (TRPC1) Channels as Regulators of Sphingolipid and VEGF Receptor Expression: IMPLICATIONS FOR THYROID CANCER CELL MIGRATION AND PROLIFERATION*

The identity of calcium channels in the thyroid is unclear. In human follicular thyroid ML-1 cancer cells, sphingolipid sphingosine 1-phosphate (S1P), through S1P receptors 1 and 3 (S1P₁/S1P₃), and VEGF receptor 2 (VEGFR2) stimulates migration. We show that human thyroid cells express several forms of transient receptor potential canonical (TRPC) channels, including TRPC1. In TRPC1 knockdown (TRPC1-KD) ML-1 cells, the basal and S1P-evoked invasion and migration was attenuated. Furthermore, the expression of S1P₃ and VEGFR2 was significantly down-regulated. Transfecting wild-type ML-1 cells with a nonconducting TRPC1 mutant decreased S1P₃ and VEGFR2 expression. In TRPC1-KD cells, receptor-operated calcium entry was decreased. To investigate whether the decreased receptor expression was due to attenuated calcium entry, cells were incubated with the calcium chelator BAPTA-AM (1,2-bis(o-aminophenoxy)ethane-N,N,N′,N′-tetraacetic acid). In these cells, and in cells where calmodulin and calmodulin-dependent kinase were blocked pharmacologically, S1P₃ and VEGFR2 expression was decreased. In TRPC1-KD cells, both hypoxia-inducible factor 1α expression and the secretion and activity of MMP2 and MMP9 were attenuated, and proliferation was decreased in TRPC1-KD cells. This was due to a prolonged G₁ phase of the cell cycle, a significant increase in the expression of the cyclin-dependent kinase inhibitors p21 and p27, and a decrease in the expression of cyclin D2, cyclin D3, and CDK6. Transfecting TRPC1 to TRPC1-KD cells rescued receptor expression, migration, and proliferation. Thus, the expression of S1P₃ and VEGFR2 is mediated by a calcium-dependent mechanism. TRPC1 has a crucial role in this process. This regulation is important for the invasion, migration, and proliferation of thyroid cancer cells.     

Glycosyltransferase structural biology and its role in the design of catalysts for glycosylation

Glycosyltransferases (GTs) are ubiquitous in nature and are required for the transfer of sugars to a variety of important biomolecules. This essential enzyme family has been a focus of attention from both the perspective of a potential drug target and a catalyst for the development of vaccines, biopharmaceuticals and small molecule therapeutics. This review attempts to consolidate the emerging lessons from Leloir (nucleotide-dependent) GT structural biology studies and recent applications of these fundamentals toward rational engineering of glycosylation catalysts.     

Structural characterization of the mitomycin 7-O-methyltransferase

Mitomycins are quinone‐containing antibiotics, widely used as antitumor drugs in chemotherapy. Mitomycin‐7‐O‐methyltransferase (MmcR), a key tailoring enzyme involved in the biosynthesis of mitomycin in Streptomyces lavendulae, catalyzes the 7‐O‐methylation of both C9β‐ and C9α‐configured 7‐hydroxymitomycins. We have determined the crystal structures of the MmcR–S‐adenosylhomocysteine (SAH) binary complex and MmcR–SAH–mitomycin A (MMA) ternary complex at resolutions of 1.9and 2.3 Å, respectively. The study revealed MmcR to adopt a common S‐adenosyl‐L ‐methionine‐dependent O‐methyltransferase fold and the presence of a structurally conserved active site general acid–base pair is consistent with a proton‐assisted methyltransfer common to most methyltransferases. Given the importance of C7 alkylation to modulate mitomycin redox potential, this study may also present a template toward the future engineering of catalysts to generate uniquely bioactive mitomycins. Proteins 2011. © 2011 Wiley‐Liss, Inc.     

Semi-targeted plasma proteomics discovery workflow utilizing two-stage protein depletion and off-line LC-MALDI MS/MS

A quantitative proteomics workflow was implemented that provides extended plasma protein coverage by extensive protein depletion in combination with the sensitivity and breadth of analysis of two-dimensional LC-MS/MS shotgun analysis. Abundant proteins were depleted by a two-stage process using IgY and Supermix depletion columns in series. Samples are then extensively fractionated by two-dimensional chromatography with fractions directly deposited onto MALDI plates. Decoupling sample fractionation from mass spectrometry facilitates a targeted MS/MS precursor selection strategy that maximizes measurement of a consistent set of peptides across experiments. Multiplexed stable isotope labeling provides quantification relative to a common reference sample and ensures an identical set of peptides measured in the set of samples (set of eight) combined in a single experiment. The more extensive protein depletion provided by the addition of the Supermix column did not compromise overall reproducibility of the measurements or the ability to reliably detect changes in protein levels between samples. The implementation of this workflow is presented for a case study aimed at generating molecular signatures for prediction of first heart attack.     

Conserving pool-breeding amphibians in human-dominated landscapes through local implementation of Best Development Practices

Seasonal forest pools in the northeastern USA are unique ecosystems whose functions are intimately associated with adjacent upland habitats. This connection, coupled with their small size and ephemeral surface water, has made conservation of pool resources challenging. Seasonal pools provide optimal breeding habitat for animals adapted to temporary waters including ambystomatid salamanders (Ambystoma spp.), wood frogs (Rana sylvatica LeConte), and some invertebrates and plants. To date, wetland conservation efforts have been primarily limited to 2 pathways: land use regulation and preservation. Although both of these pathways have the potential to conserve some pool resources, they are often insufficient to maintain an array of pools in the landscape that support local population dynamics of amphibians. We propose a third pathway – local land-use planning – that can complement regulatory and preservation efforts. This suite of strategies, embodied in our Best Development Practices (BDPs), recognizes that not all pools will be conserved; local governances will need to develop priorities for conservation. The BDPs encourage local governances to (1) proactively identify their pool resources, (2) rank those pools according to their relative ecological value, and (3) establish management procedures and apply recommended guidelines in accordance with the relative rankings. We recommend that pools be ranked using biological criteria (e.g., presence of listed species, presence of breeding species, and egg mass abundance) and on the availability and quality of adjacent terrestrial habitat. We recommend 3 management zones: the pool depression, the pool envelope (i.e., land within 30 m of the pool), and the critical terrestrial habitat (i.e., 30–230 m from the pool). Residential, industrial, and commercial development, which may compromise pool habitat (e.g., through building and road construction, site clearing, stormwater management, and lighting), should follow the recommended guidelines presented in Appendix 1 of this paper. Planning at the watershed level, using such tools as overlay zones, wetland ordinances, and easements, should lead to more effective, long-term management of, at a minimum, the most ecologically important seasonal forest pool resources and will provide developers with clear development guidelines. This process is already being successfully implemented in a number of New England towns.     

Genome sequence of Leuconostoc argentinum KCTC 3773

Leuconostoc argentinum is one of the most prevalent lactic acid bacteria present during the manufacturing process of kimchi, the best-known traditional Korean dish. Here, we present the draft genome sequence of type strain KCTC 3773 of Leuconostoc argentinum (1,720,683 bp, with a G+C content of 42.9%), which consists of 98 large contigs (>100 bp in size).