Membrane Topology and Identification of Critical Amino Acid Residues in the Wzx O-Antigen Translocase from Escherichia coli O157:H4

Wzx belongs to a family of membrane proteins involved in the translocation of isoprenoid lipid-linked glycans, which is loosely related to members of the major facilitator superfamily. Despite Wzx homologs performing a conserved function, it has been difficult to pinpoint specific motifs of functional significance in their amino acid sequences. Here, we elucidate the topology of the Escherichia coli O157 Wzx (Wzx(EcO157)) by a combination of bioinformatics and substituted cysteine scanning mutagenesis, as well as targeted deletion-fusions to green fluorescent protein and alkaline phosphatase. We conclude that Wzx(EcO157) consists of 12 transmembrane (TM) helices and six periplasmic and five cytosolic loops, with N and C termini facing the cytoplasm. Four TM helices (II, IV, X, and XI) contain polar residues (aspartic acid or lysine), and they may form part of a relatively hydrophilic core. Thirty-five amino acid replacements to alanine or serine were targeted to five native cysteines and most of the aspartic acid, arginine, and lysine residues. From these, only replacements of aspartic acid-85, aspartic acid-326, arginine-298, and lysine-419 resulted in a protein unable to support O-antigen production. Aspartic acid-85 and lysine-419 are located in TM helices II and XI, while arginine-298 and aspartic acid-326 are located in periplasmic and cytosolic loops 4, respectively. Further analysis revealed that the charge at these positions is required for Wzx function since conservative substitutions maintaining the same charge polarity resulted in a functional protein, whereas those reversing or eliminating polarity abolished function. We propose that the functional requirement of charged residues at both sides of the membrane and in two TM helices could be important to allow the passage of the Und-PP-linked saccharide substrate across the membrane.

An amino acid substitution in Fasciola hepatica P-glycoprotein from triclabendazole-resistant and triclabendazole-susceptible populations

Control of fasciolosis is threatened by the development of anthelmintic resistance. Enhanced triclabendazole (TCBZ) efflux by ABC transporters such as P-glycoprotein (Pgp) has been implicated in this process. A putative full length cDNA coding for a Pgp expressed in adult Fasciola hepatica has been constructed and used to design a primer set capable of amplifying a region encoding part of the second nucleotide binding domain of Pgp when genomic DNA was used as a template. Application of this primer set to genomic DNA from TCBZ-resistant and -susceptible field populations has shown a significant difference in the alleles present. Analysis of an allele occurring at a three-fold higher frequency in the "resistant" population revealed that it was characterised by a serine to arginine substitution at residue 1144. Homology modelling studies have been used to locate this site in the Pgp structure and hence assess its potential to modify functional activity. © 2012 Elsevier B.V.

Sequence divergence of measles virus haemagglutinin during natural evolution and adaptation to cell culture

Phylogenetic analysis of the sequence of the H gene of 75 measles virus (MV) strains (32 published and 43 new sequences) was carried out. The lineage groups described from comparison of the nucleotide sequences encoding the C-terminal regions of the N protein of MV were the same as those derived from the H gene sequences in almost all cases. The databases document a number of distinct genotype switches that have occurred in Madrid (Spain). Well-documented is the complete replacement of lineage group C2, the common European genotype at that time, with that of group D3 around the autumn of 1993. No further isolations of group C2 took place in Madrid after this time. The rate of mutation of the H gene sequences of MV genotype D3 circulating in Madrid from 1993 to 1996 was very low (5 x 10(-4) per annum for a given nucleotide position). This is an order of magnitude lower than the rates of mutation observed in the HN genes of human influenza A viruses. The ratio of expressed over silent mutations indicated that the divergence was not driven by immune selection in this gene. Variations in amino acid 117 of the H protein (F or L) may be related to the ability of some strains to haemagglutinate only in the presence of salt. Adaptation of MV to different primate cell types was associated with very small numbers of mutations in the H gene. The changes could not be predicted when virus previously grown in human B cell lines was adapted to monkey Vero cells. In contrast, rodent brain-adapted viruses displayed a lot of amino acid sequence variation from normal MV strains. There was no convincing evidence for recombination between MV genotypes.

Removal of naphthenic acids from crude oil using amino acid ionic liquids

A number of tetraalkylammonium and tetraalkylphosphonium amino acid based ionic liquids (AAILs) have been successfully used and recycled for the reactive extraction of naphthenic acids from crude oil and crude oil distillates. Spectral studies show that the mechanism by which this occurs is through the formation of a zwitterionic complex. Therein, the amino acid anion plays a key role in the formation of this complex. (C) 2013 Elsevier Ltd. All rights reserved.

Observation of Ultrafast Charge Migration in an Amino Acid

We present the first direct measurement of ultrafast charge migration in a biomolecular building block the amino acid phenylalanine. Using an extreme ultraviolet pulse of 1.5 fs duration to ionize molecules isolated in the gas phase, the location of the resulting hole was probed by a 6 fs visible/near-infrared pulse. By measuring the yield of a doubly charged ion as a function of the delay between the two pulses, the positive hole was observed to migrate to one end of the cation within 30 fs. This process is likely to originate from even faster coherent charge oscillations in the molecule being dephased by bond stretching which eventually localizes the final position of the charge. This demonstration offers a clear template for observing and controlling this phenomenon in the future.

Ultrafast charge dynamics in an amino acid induced by attosecond pulses

In the past few years, attosecond techniques have been implemented for the investigation of ultrafast dynamics in molecules. The generation of isolated attosecond pulses characterized by a relatively high photon flux has opened up new possibilities in the study of molecular dynamics. In this paper, we report on experimental and theoretical results of ultrafast charge dynamics in a biochemically relevant molecule, namely, the amino acid phenylalanine. The data represent the first experimental demonstration of the generation and observation of a charge migration process in a complexmolecule, where electron dynamics precede nuclear motion. The application of attosecond technology to the investigation of electron dynamics in biologically relevant molecules represents a multidisciplinary work, which can open new research frontiers: those in which few-femtosecond and even subfemtosecond electron processes determine the fate of biomolecules. It can also open new perspectives for the development of new technologies, for example, in molecular electronics, where electron processes on an ultrafast temporal scale are essential to trigger and control the electron current on the scale of the molecule.

Fluorescent Signalling of the Brain Neurotransmitter Gamma Amino Butyric Acid and Related Amino Acid Zwitterions

Fluorescent PET (photoinduced electron transfer) sensor 1 with monoaza-18-crown-6 ether and guanidinium receptor units shows a significant fluorescence enhancement with y-aminobutyric acid (GABA) in mixed aqueous solution.

Calluna vulgaris root cells show increased capacity for amino acid uptake when colonized with the mycorrhizal fungus Hymenoscyphus ericae

Ericoid mycorrhizas are believed to improve N nutrition of many ericaceous plant species that typically occur in habitats with impoverished nutrient status, by releasing amino acids from organic N forms. Despite the ubiquity of mycorrhizal formation the mechanisms and regulation of nutrient transport in mycorrhizal associations are poorly understood. We used an electrophysiological approach to study how amino acid transport characteristics of Calluna vulgaris were affected by colonization with the ericoid mycorrhiza fungus Hymenoscyphus ericae. Both the V_max and K_m parameters of amino acid uptake were affected by fungal colonization in a manner consistent with an increased availability of amino acid to the plant. The ecophysiological significance of altered amino acid transport in colonized root cells of C. vulgaris is discussed. © New Phytologist (2002).

Familial idiopathic methemoglobinemia revisited: original cases reveal 2 novel mutations in NADH cytochrome b5 reductase

In 1943, the first description of familial idiopathic methemoglobinemia in the United Kingdom was reported in 2 members of one family. Five years later, Quentin Gibson (then of Queen's University, Belfast, Ireland) correctly identified the pathway involved in the reduction of methemoglobin in the family, thereby describing the first hereditary trait involving a specific enzyme deficiency. Recessive congenital methemoglobinemia (RCM) is caused by a deficiency of reduced nicotinamide adenine dinucleotide (NADH)-cytochrome b5 reductase. One of the original propositi with the type 1 disorder has now been traced. He was found to be a compound heterozygote harboring 2 previously undescribed mutations in exon 9, a point mutation Gly873Ala predicting a Gly291Asp substitution, and a 3-bp in-frame deletion of codon 255 (GAG), predicting loss of glutamic acid. A brother and a surviving sister are heterozygous; each bears one of the mutations. Thirty-three different mutations have now been recorded for RCM. The original authors' optimism that RCM would provide material for future genetic studies has been amply justified.

Cybr, a cytokine-inducible protein that binds cytohesin-1 and regulates its activity

Cytokines regulate lymphocyte development and differentiation, but precisely how they control these processes is still poorly understood. By using microarray technology to detect cytokine-induced genes, we identified a cDNA encoding Cybr, which was increased markedly in cells incubated with IL-2 and IL-12. The mRNA was most abundant in hematopoietic cells and tissues. The predicted amino acid sequence is similar to that of GRP-1-associated protein (GRASP), a recently identified retinoic acid-induced cytohesin-binding protein. Physical interaction, dependent on the coiled-coil domains of Cybr and cytohesin-1, was demonstrated by coimmunoprecipitation of the overexpressed proteins from 293T cells. Cytohesin-1, in addition to its role in cell adhesion, is a guanine nucleotide-exchange protein activator of ARF GTPases. Acceleration of guanosine 5'-O-(thiotriphosphate) binding to ARF by cytohesin-1 in vitro was enhanced by Cybr. Because the binding protein modified activation of ADP ribosylation factor by cytohesin-1, we designate this cytokine-inducible protein Cybr (cytohesin binder and regulator).

Elements of the granular gland peptidome and transcriptome persist in air-dried skin of the South American orange-legged leaf frog, Phyllomedusa hypocondrialis.

The defensive strategy of amphibians against predator attack relies heavily on the secretion of noxious/toxic chemical cocktails from specialized skin granular glands. Bioactive peptides constitute a major component of secretions in many species and the most complex are produced by neotropical leaf frogs of the sub-family Phyllomedusinae. We recently reported that these skin secretions contain elements of both the granular gland peptidome and transcriptome and that polyadenylated mRNAs constituting the latter are protected from degradation by interactions with endogenous amphipathic peptides. This thus permits parallel amino acid sequencing of peptides and nucleic acid sequencing of cloned precursor transcripts from single lyophilized samples of secretion. Here we report that the protection afforded is sufficiently robust to permit transcriptome studies by cloning of full-length polyadenylated peptide precursor encoding mRNAs from libraries constructed using ambient temperature air-dried skin from recently deceased specimens as source material. The technique was sufficiently sensitive to permit the identification of cDNAs encoding antimicrobial peptides constituted by six different isoforms of phylloseptin and two dermaseptins. Also, for the first time, establishment of the nucleic acid and amino acid sequence of the precursor encoding the phyllomedusine frog skin bradykinin-related peptide, phyllokinin, from cloned cDNA, was achieved. These data unequivocally demonstrate that the granular gland transcriptome persists in air-dried amphibian skin—a finding that may have fundamental implications in the study of archived materials but also in the wider field of molecular biology.

A potent novel antimicrobial peptide related to caerin 1.12 from the skin secretion of the Australian frog, Litoria aurea

Skin secretions from Australian frogs of the genus Litoria have been extensively studied for many years and are known to contain a large array of antimicrobial peptides that often bear their specific names — caerins (L. caerulea), aureins (L. aurea), citropins (L. citropa) and maculatins (L. genimaculata) — and each group displays distinct primary structural attributes. During a systematic transcriptome cloning study using a cDNA library derived from skin secretion of L. aurea, a series of identical clones were identified that encoded a novel 25-mer antimicrobial peptide that displayed 92% structural identity with caerin 1.12 from L. caerulea, differing in amino acid sequence at only two positions — Arg for Gly at position 7 and Leu amide for Ser amide at the C-terminus. The novel peptide had conserved Pro residues at positions 15 and 19 that flank a flexible hinge region which previous studies have suggested are important for effective orientation of the two alpha-helices within the bacterial membrane resulting in lysis of cells. As the two substitutions in the novel peptide serve to increase both positive charge and hydrophobicity, we synthesised a replicate and determined its minimal inhibitory concentration (MIC) against Gram positive Staphylococcus aureus and Gram negative Escherichia coli. The MICs for these organisms were 3 µM and 4 µM, respectively, indicating a high potency and haemolysis was