Identical Substitutions Research Articles

Mutagenesis and modeling to predict structural and functional characteristics of the Staphylococcus aureus MepA multidrug efflux pump.

MepA is a multidrug and toxin extrusion (MATE) family protein and the only MATE protein encoded within the Staphylococcus aureus genome. Structural data for MATE proteins are limited to a single high-resolution example, NorM of Vibrio cholerae. Substitution mutations were created in MepA using gradient plates containing both a substrate and reserpine as an efflux pump inhibitor. Site-directed mutagenesis of plasmid-based mepA was used to reproduce these mutations, as well as unique or low-frequency mutations identified in mepA-overexpressing clinical strains, and to mutagenize conserved acidic residues. The effect of these changes on protein function was quantitated in a norA-disrupted host strain by susceptibility testing with and without inhibitors and by determining the proficiency of ethidium efflux. Up-function substitutions clustered in the carboxy half of MepA, near the cytoplasmic face of the protein. Repeated application of the same gradient plate conditions frequently reproduced identical substitution mutations, suggesting that individual residues are required for interaction with specific substrates. Acidic residues critical to protein function were identified in helices 4 and 5. In silico modeling revealed an outward-facing molecule, with helices 1, 2, 4, 7, 8, and 10 having contact with a central cavity that may represent a substrate translocation pathway. Functionally important residues within this cavity included S81, A161, M291, and A302. These data provide a critical starting point for understanding how MATE multidrug efflux proteins function and will be useful in refining crystallographic data when they are available.

Hepatitis E virus from India exhibits significant amino acid mutations in fulminant hepatic failure patients

In India, hepatitis E virus (HEV) is the predominant cause of acute viral hepatitis (AVH) and fulminant hepatic failure (FHF) among pregnant women and adults. The present study evaluates association, if any, of the mutations in the viral genome with disease outcome. Ten genotype-1 complete genomes (five each from AVH and FHF patients) were sequenced. Phylogenetic analysis showed a distinct cluster including all five FHF-HEV sequences from western India (present study), one FHF isolate from northern India, and one AVH isolate detected in 2010 (present study). HEV genotype-1 sequences from fulminant cases exhibited 150 significantly different (p≤0.05) nucleotide substitutions when compared to all genotype-1-AVH sequences as well as isolates from the Indian subcontinent. At six positions, all FHF sequences showed identical substitutions (1 non-synonymous). Six amino acid changes in ORF1; F179S, A317T, T735I, L1110F, V1120I, and F1439Y were significantly associated with HEV-type-1 FHF. The data suggests that the nucleotide substitutions recorded and/or L1110F and V1120I amino acid substitutions in helicase domain may play important role in determining outcome of HEV infection.

A MYB Transcription Factor Controls Flower Color in Soybean

Purple-blue flower of soybean (Glycine max [L.] Merr.) is controlled by the W2 locus. Previous studies revealed that a MYB transcription factor gene GmMYB-G20-1 was located at a position similar to the W2 gene and that a base substitution generated a stop codon in the MYB domains of 2 soybean lines with purple-blue flowers. This study was conducted to confirm the relationship between GmMYB-G20-1 and the W2 gene. Cleaved amplified polymorphic sequence analysis to detect the base substitution suggested that a similar mutation occurred in 2 other soybean lines having purple-blue flowers, 037-E-8, and Yogetsu 1-blue. Thus, all genotypes having purple-blue flowers had identical base substitutions. To verify the function of GmMYB-G20-1, apple latent spherical virus (ALSV) vectors were constructed to perform virus-induced gene silencing of GmMYB-G20-1. A cultivar Harosoy with purple flowers (W2W2) was infected by the empty ALSV vector (wtALSV) or the GmMYB-G20-1-ALSV vector containing a fragment (nucleotide position 685-885) of GmMYB-G20-1. Plants infected by empty vectors had only purple flowers. In contrast, most flowers of plants infected with GmMYB-G20-1-ALSV had irregular gray/blue sectors in flower petals and some of the flowers had almost gray/blue petals. These results strongly suggest that silencing of GmMYB-G20-1 can alter flower color and that it may correspond to the W2 gene.

New method for detection of p22-phox-deficient chronic granulomatous disease heterozygote carriers in Jeju

Chronic granulomatous disease (CGD) is a genetically heterogeneous disease caused by defects in the genes encoding any one of the NADPH oxidase components. The estimated prevalence of CGD is between 1 in 200,000 and 1 in 250,000 individuals, with variable rates in different countries. According to a compilation by the Korean College of Pediatric Clinical Immunology, the prevalence of CGD in Korea is 0.9 in 1,000,000 individuals. Surprisingly, the prevalence of CGD in Jeju Island is 20.7 in 1,000,000 individuals. We reported an identical homozygous single-base substitution of C to T in exon 1 (c.7C > T) of the CYBA gene from 12 CGD patients in Jeju Island. We hypothesized that the high prevalence of CGD in Jeju Island is associated with an identical mutation inherited from a common ancestor or proband. The aim of this study was to develop an assay to detect heterozygote carriers of the genotype specific to Jeju Island. We developed three specific primers, and nested polymerase chain reaction was employed using whole blood samples as a source of genomic DNA. Using the new detection method, 704 individuals were tested, 9 of which were detected as carriers, and the expected number of carriers is 1.3 in 100 individuals.

COULD VIBRATIONAL SPECTROSCOPY REVOLUTIONISE QUALITY CONTROL OF HERBAL MEDICINES?

s. PHYTOPHARM 2012 M 114 Obzory po kliniceskoj farmacologii i lekarstvennoj terapii [Reviews of clinical pharmacology and drug therapy] ТОМ 10/2012/2 COULD VIBRATIONAL SPECTROSCOPY REVOLUTIONISE QUALITY CONTROL OF HERBAL MEDICINES? © Viljoen Alvaro, Vermaak Ilze Department of Pharmaceutical Sciences, Faculty of Science, Tshwane University of Technology, Private Bag X680, Pretoria 0001, South Africa Vibrational spectroscopy includes near infrared (NiR) mid-infrared (MiR) and Raman spectroscopy. These methods in combination with multivariate data analysis have proved to be powerful analytical techniques and have been implemented in various industries such as the petrochemical, agricultural and food industries. The quality of herbal medicines is of great concern as many consumers now choose herbal medicines over Western medicines and cases of mistaken identity (substitutions) or adulteration with other species with sometimes dire health consequences or loss of consumer trust in the product due to ineffectiveness has been reported. The quality of herbal medicines is assessed both qualitatively (authentication) and quantitatively through the quantification of specific biomarkers. Traditional analytical methods used to determine quality such as high performance liquid chromatography and mass spectrometry are destructive, expensive and time-consuming. Vibrational spectroscopy methods have major advantages over these methods in that it is simple, rapid, relatively cheap and non-destructive. Various examples of quality control methods developed using vibrational spectroscopy techniques will be described for medicinally and commercially important plant species: to discriminate between closely related plant species (e. g. Agathosma betulina and A. crenulata; Pelargonium reniforme and P. sidoides), a case of mistaken identity (Eriocephalus punctulatus and E. tenuifolius) and as a quantification tool for biomarkers (e. g. pulegone, diosphenol, isomenthone etc. in A. betulina essential oils). hyperspectral imaging uses the platform of vibrational spectroscopy and multivariate data analysis but adds an extra data dimension opening up a range of novel applications in the quality control of herbal medicines such as authentication using whole dried fruits (e. g. Illicium anisatum and I. verum). from these examples it is evident, the revolution has already begun. THE METABOLISM OF LIGNANS FROM FRUCTUS SCHISANDRA © Yin Jun , Cao Yun-Feng , Yang Ling 2 School of Traditional Chinese Medicines, Shenyang Pharmaceutical University, Shenyang 110016, China Laboratory of Pharmaceutical Resource Discovery, Dalian Institute of Chemical Physics, Chinese Academy of Sciences, Dalian 116023, China After metabolites from dibenzocyclooctadiene lignans (Schizandrin, deoxyschizandrin and schisantherin A) of Schisandra chinensis incubated with human liver microsomes were isolated by hPlC and their structures were identified by lC–MS, h-NMR and C-NMR, the study combined correlation analysis, chemical inhibition studies, assays with recombinant CyPs and enzyme kinetics indicated that CyP3A4 was the main hepatic isoform. Three metabolites were isolated by hPlC and their structures were identified to be 8 (R)-hydroxylschizandrin, 2-demethyl-8 (R)-hydroxyl-schizandrin, 3-demethyl-8 (R)-hydroxyl-schizandrin, Rat and minipig liver microsomes were applied to evaluate species differences, and the results showed little difference among the mentioned species. Speculated that deoxyschizandrin might undergo hydroxylation in hlMs, and schizandrin could represent the corresponding metabolite. The data also show that deoxyschizandrin metabolism to form schizandrin specially mediated by CyP3A4, is the principal route of disposition in humans. The specificity, high affinity, and high turnover make deoxyschizandrin hydroxylation an excellent probe drug for CyP3A4 activity in vitro. The metabolic pathway of schisantherin A validated the inhibitory mechanism against CyP3A4 undergoes demethylenenation in the human liver microsomes. iC 50 drift and dynamics test indicate that schisantherin A is inhibitor which shows dependency to NAdP and time, which illustrates that the methylene of schisantherin A produced active intermediate during the catalyzed reaction, thereby presents forceful inhibitory effect against CyP3A4. The lignans containing methylenedioxy abundant in fructus Schisandra probably reduce clinical value thereof. in mice liver microsomes, schisantherin A, schizandrol b and schizandrin b showed strong inhibition against metablism schizandrin and deoxschizandrin. Our research results indicate that why deoxyschizandrin presented forceful anti-multidrug resistance in vitro but reduced or vanished in vivo, and the extract of fructus Schisandra containing deoxyschizandrin presents forceful anti-multidrug resistance in vivo.

An Acvr1 R206H knock-in mouse has fibrodysplasia ossificans progressiva

Fibrodysplasia ossificans progressiva (FOP; MIM #135100) is a debilitating genetic disorder of dysregulated cellular differentiation characterized by malformation of the great toes during embryonic skeletal development and by progressive heterotopic endochondral ossification postnatally. Patients with these classic clinical features of FOP have the identical heterozygous single nucleotide substitution (c.617G > A; R206H) in the gene encoding ACVR1/ALK2, a bone morphogenetic protein (BMP) type I receptor. Gene targeting was used to develop an Acvr1 knock-in model for FOP (Acvr1(R206H/+)). Radiographic analysis of Acvr1(R206H/+) chimeric mice revealed that this mutation induced malformed first digits in the hind limbs and postnatal extraskeletal bone formation, recapitulating the human disease. Histological analysis of murine lesions showed inflammatory infiltration and apoptosis of skeletal muscle followed by robust formation of heterotopic bone through an endochondral pathway, identical to that seen in patients. Progenitor cells of a Tie2(+) lineage participated in each stage of endochondral osteogenesis. We further determined that both wild-type (WT) and mutant cells are present within the ectopic bone tissue, an unexpected finding that indicates that although the mutation is necessary to induce the bone formation process, the mutation is not required for progenitor cell contribution to bone and cartilage. This unique knock-in mouse model provides novel insight into the genetic regulation of heterotopic ossification and establishes the first direct in vivo evidence that the R206H mutation in ACVR1 causes FOP.

Analogues of doxanthrine reveal differences between the dopamine D 1 receptor binding properties of chromanoisoquinolines and hexahydrobenzo[ a]phenanthridines

Efforts to develop selective agonists for dopamine D 1-like receptors led to the discovery of dihydrexidine and doxanthrine, two bioisosteric β-phenyldopamine-type full agonist ligands that display selectivity and potency at D 1-like receptors. We report herein an improved methodology for the synthesis of substituted chromanoisoquinolines (doxanthrine derivatives) and the evaluation of several new compounds for their ability to bind to D 1- and D 2-like receptors. Identical pendant phenyl ring substitutions on the dihydrexidine and doxanthrine templates surprisingly led to different effects on D 1-like receptor binding, suggesting important differences between the interactions of these ligands with the D 1 receptor. We propose, based on the biological results and molecular modeling studies, that slight conformational differences between the tetralin and chroman-based compounds lead to a shift in the location of the pendant ring substituents within the receptor.

The Structural and Functional Basis of the p97/Valosin-containing Protein (VCP)-interacting Motif (VIM): MUTUALLY EXCLUSIVE BINDING OF COFACTORS TO THE N-TERMINAL DOMAIN OF p97

The AAA (ATPase associated with various cellular activities) ATPase p97, also referred to as valosin-containing protein (VCP), mediates essential cellular processes, including ubiquitin-dependent protein degradation, and has been linked to several human proteinopathies. p97 interacts with multiple cofactors via its N-terminal (p97N) domain, a subset of which contain the VCP-interacting motif (VIM). We have determined the crystal structure of the p97N domain in complex with the VIM of the ubiquitin E3 ligase gp78 at 1.8 Å resolution. The α-helical VIM peptide binds into a groove located in between the two subdomains of the p97N domain. Interaction studies of several VIM proteins reveal that these cofactors display dramatically different affinities, ranging from high affinity interactions characterized by dissociation constants of ∼20 nm for gp78 and ANKZF1 to only weak binding in our assays. The contribution of individual p97 residues to VIM binding was analyzed, revealing that identical substitutions do not affect all cofactors in the same way. Taken together, the biochemical and structural studies define the framework for recognition of VIM-containing cofactors by p97. Of particular interest to the regulation of p97 by its cofactors, our structure reveals that the bound α-helical peptides of VIM-containing cofactors overlap with the binding site for cofactors containing the ubiquitin regulatory X (UBX) domain present in the UBX protein family or the ubiquitin-like domain of NPL4 as further corroborated by biochemical data. These results extend the concept that competitive binding is a crucial determinant in p97-cofactor interactions.

Referential Opacity and Epistemic Logic

Referential opacity is the failure of substitutivity of identity (SI, for short) and in Quine’s view of existential generalization (EG, for short) as well. Quine thinks that its “solution” in epistemic and doxastic contexts, which relies on the notion of exportation, leads to undesirable results. But epistemic logicians such as Jaakko Hintikka and Wolfgang Lenzen provide another solution based on a different diagnosis: opacity is not, as in Quine’s view, due to the absence of reference, it is rather due to the plurality of references; therefore, one must stabilize the reference to restore SI and EG. However, Hintikka’s semantics remains classical in its treatment of existence, which makes EG non-applicable to possible objects, while in Lenzen’s system, EG is applicable by means of a particular quantifier. But this requires adding the predicate of existence to account for real objects. In this paper, I will show the advantages and disadvantages of both solutions and will end by providing an alternative approach to the problem of non-existent objects, which stays into the frame of possible worlds semantics, but introduces some more restrictions, eliminates the problematic predicate of existence, and applies a neutral quantifier to possible non-existent objects.

Chromone, a Privileged Scaffold for the Development of Monoamine Oxidase Inhibitors

Two series of novel chromone derivatives were synthesized and investigated for their ability to inhibit the activity of monoamine oxidase. The SAR data indicate that chromone derivatives with substituents in position 3 of γ-pyrone nucleus act preferably as MAO-B inhibitors, with IC(50) values in the nanomolar to micromolar range. Almost all chromone 3-carboxamides display selectivity toward MAO-B. Identical substitutions on position 2 of γ-pyrone nucleus result in complete loss of activity in both isoforms (chromones 2-12 except 3 and 5). Notably, chromone (19) exhibits an MAO-B IC(50) of 63 nM, greater than 1000-fold selectivity over MAO-A, and behaves as a quasi-reversible inhibitor. Docking experiments onto the MAO binding of the most active compound highlight different interaction patterns among the isoforms A and B. The differential analysis of the solvation effects among the chromone isomers gave additional insight about the superior outline of the 3-substituted chromone derivatives.

Do some IAA proteins have two repression domains?

The plant hormone auxin regulates the transcription of specific genes through the interplay of Auxin Response Factors (ARFs) and Aux/IAA (IAA) repressors. We have recently shown that stabilized IAA repressors with identical amino acid substitutions in their conserved repression domains (i.e., domain I) confer either “low auxin” or “high auxin” phenotypes when the IAA proteins are constitutively expressed in transformed Arabidopsis plants. We have suggested that when domain I loses its capacity to repress, “high auxin” phenotypes generally result, but a subset of IAA proteins (e.g., IAA17) appear to contain a second repression domain resulting in the maintenance of “low auxin” phenotypes. Here we provide evidence for a second repression domain that lies between domains I and II in IAA7, an IAA repressor within the same clade as IAA17.

A model investigation of ab initio geometries for identity and nonidentity substitution with three‐center four‐ and three‐electron transition states

AbstractWith the van't Hoff model derived from the dynamics of a regular tetrahedron in which the interstitial carbon changes its position from tetrahedral into a trigonal (bi)pyramidal configuration, the introduction of dimensionless ratio numbers (R) can serve to localize significant points on the principal reaction coordinate. These numbers are expressed as R(d) and R(n) based on transition state geometries and the number of electrons involved in the three‐center bonding, respectively. Using this concept, we obtain a model for the evaluation of different ab initio calculations based on identity and nonidentity substitution reactions for three‐center four‐ and three‐electron bonding transition states. Similar ratio numbers have been derived for proton exchange reactions. The reactions under investigation show clearly that in spite of the differences in chemical outcome the transition steps reduced to the first principles of chemical bonding are similar. © 2010 Wiley Periodicals, Inc. Int J Quantum Chem, 2011

Rapid Evolution of Lifespan in a Novel Environment: Sex-Specific Responses and Underlying Genetic Architecture

Animal lifespans can vary substantially among closely related species and even among conspecific populations, but it is often difficult to identify environmental and genetic factors producing such variation. We used experimental evolution to examine how transfer to a novel environment affects adult lifespan and rates of senescence in a seed-feeding beetle. Three replicate lines of Callosobruchus maculatus (F.) were switched to a new host plant (cowpea), and each evolved shorter adult lifespans compared to a line maintained on the ancestral host (mung bean). However, the evolution of lifespan differed between the sexes; female lifespan was reduced by ~11% in all cowpea replicates, whereas male lifespan decreased by an average of only 5.6% and the magnitude of the reduction varied among replicates. Reduced lifespan in lines switched to cowpea mirrored the shorter lifespan observed in a separate population chronically associated with cowpea. We then performed crosses between the mung bean and cowpea lines to estimate the genetic architecture underlying the rapid evolution of a shorter lifespan on cowpea. Dominance (overdominance) contributed substantially to the difference between the cowpea and mung bean lines for female lifespan but not for male lifespan. However, details of the genetic architecture varied among the three replicate crosses, so that the convergent evolution of shorter female lifespan in the different cowpea lines did not arise from identical allelic substitutions. Our study demonstrates that insect lifespan can be predictably modified by a switch to a novel host plant, that both the magnitude of this response and its underlying genetic architecture can be sex-specific, and that convergent evolution of a complex trait such as lifespan can arise from different genetic mechanisms.

Potential role of molecular mimicry between human U1-70kDa and fungal proteins in the development of T-cell mediated anti-U1-70kDa autoimmunity

Context: Molecular mimicry between human and microbial antigens is a possible trigger of autoimmunity. The possible role of this mechanism in the onset of autoimmunity against the human autoantigen U1-70kDa, typical of mixed connective tissue disease, is not fully elucidated.Objective: We aimed to identify microbial proteins highly similar to U1-70kDa and potentially triggering anti-U1-70kDa autoimmunity.Materials and methods: We compared in silico the amino acid sequence of human U1-70kDa and those of all the sequenced fungal, viral and bacterial proteins.Results: Human U1-70kDa shares highly significant (E<10−20) amino acid sequence homology, spanning a segment containing T-cell epitopes, with 13 fungal (but no viral or bacterial) proteins, belonging to human pathogens. Nine of these proteins include the amino acid sequence VLVDVERGRTV, identical to the most frequent U1-70kDa T-cell epitope in anti-U1-70kDa positive patients, and sequences highly similar to the epitope DAFKTLFVARVN (identical residues or conservative residue substitutions in positions crucial for epitope binding).Discussion and conclusion: Cross-reactivity between human U1-70kDa and microbial proteins was demonstrated for B-cell epitopes, but never investigated before for T-cell epitopes. Our data identify some fungal proteins as possible triggers of anti-U1-70kDa autoimmunity via molecular mimicry. Research in this field could improve the understanding of the mechanisms leading to anti-U1-70kDa autoimmunity, with potential consequences on prevention.

Identical Amino Acid Substitutions in the Repression Domain of Auxin/Indole-3-Acetic Acid Proteins Have Contrasting Effects on Auxin Signaling

Auxin/indole-3-acetic acid (Aux/IAA) proteins function as repressors of auxin response gene expression when auxin concentrations in a cell are low. At elevated auxin concentrations, these repressors are destroyed via the ubiquitin-proteasome pathway, resulting in derepression/activation of auxin response genes. Most Aux/IAA repressors contain four conserved domains, with one of these being an active, portable repression domain (domain I) and a second being an auxin-dependent instability domain (domain II). Here, we have analyzed the effects of amino acid substitutions in the repression domain of selected Aux/IAA proteins. We show that stabilized versions of Aux/IAA proteins with amino acid substitutions in domain I display contrasting phenotypes when expressed in transformed Arabidopsis (Arabidopsis thaliana) plants. An alanine-for-leucine substitution in the LxLxL (where L is leucine and x is another amino acid) repression domain of IAA3, IAA6, or IAA19 confers enhanced auxin response gene expression and "high-auxin" phenotypes when expressed from the 35S or IAA19 promoter (as tested with IAA19) in transformed Arabidopsis plants. In marked contrast, a single alanine-for-leucine substitution in domain I of IAA12 or IAA17 confers repression of auxin response genes and "low-auxin" phenotypes. These results point to intrinsic differences in the repression domain(s) of IAA proteins and suggest that some IAA proteins have stronger or more complex repression domains than others.

Identical Glycine Substitution Mutations in Type VII Collagen May Underlie Both Dominant and Recessive Forms of Dystrophic Epidermolysis Bullosa

Autosomal dominant and recessive forms of dystrophic epidermolysis bullosa (DEB) result from mutations in the type VII collagen gene (COL7A1). Although paradigms have emerged for genotype/phenotype correlation in DEB, some pathogenic mutations in COL7A1, notably glycine substitutions within the type VII collagen triple helix, may lead to diagnostic difficulties, since certain glycine substitutions can result in either dominant or recessive mutant alleles. Delineation of glycine substitution mutations into two discrete groups, however, is made difficult by observations that, for some particular glycine substitutions in type VII collagen, the same mutation can result in both dominant and recessive disease. In this report we describe four further glycine missense mutations: p.Gly1483Asp, p.Gly1770Ser, p.Gly2213Arg and p.Gly2369Ser, which can lead to either dominant or recessive DEB, and which result in a spectrum of clinical abnormalities. We also identify a further 30 new glycine substitution mutations that cause either dominant or recessive DEB, but not both. In screening the COL7A1 gene for mutations in individuals with DEB our data highlight that delineation of glycine substitutions in type VII collagen has important implications for genetic counselling.

A Presenilin-1 Mutation Identified in Familial Alzheimer Disease with Cotton Wool Plaques Causes a Nearly Complete Loss of γ-Secretase Activity

Mutations in presenilin-1 and presenilin-2 (PS1 and PS2) are the most common cause of familial Alzheimer disease. PS1 and PS2 are the presumptive catalytic components of the multisubunit gamma-secretase complex, which proteolyzes a number of type I transmembrane proteins, including the amyloid precursor protein (APP) and Notch. APP processing by gamma-secretase produces beta-amyloid peptides (Abeta40 and Abeta42) that accumulate in the Alzheimer disease brain. Here we identify a pathogenic L435F mutation in PS1 in two affected siblings with early-onset familial Alzheimer disease characterized by deposition of cerebral cotton wool plaques. The L435F mutation resides in a conserved C-terminal PAL sequence implicated in active site conformation and catalytic activity. The impact of PS1 mutations in and around the PAL motif on gamma-secretase activity was assessed by expression of mutant PS1 in mouse embryo fibroblasts lacking endogenous PS1 and PS2. Surprisingly, the L435F mutation caused a nearly complete loss of gamma-secretase activity, including >90% reductions in the generation of Abeta40, Abeta42, and the APP and Notch intracellular domains. Two nonpathogenic PS1 mutations, P433L and L435R, caused essentially complete loss of gamma-secretase activity, whereas two previously identified pathogenic PS1 mutations, P436Q and P436S, caused partial loss of function with substantial reductions in production of Abeta40, Abeta42, and the APP and Notch intracellular domains. These results argue against overproduction of Abeta42 as an essential property of presenilin proteins bearing pathogenic mutations. Rather, our findings provide support for the hypothesis that pathogenic mutations cause a general loss of presenilin function.

Rate-Determining Factors in Nucleophilic Aromatic Substitution Reactions

Quantum chemical calculations (OPBE/6-311++G(d,p)) have been performed to uncover the electronic factors that govern reactivity in the prototypical S(N)Ar reaction. It was found that intrinsic nucleophilicity--expressed as the critical energy (the energy required for forming the Meisenheimer structure Ph(X)(2)(-)) in the identity substitution reaction X(-) + PhX --> X(-) + PhX (Ph = phenyl)--shows the following approximate trend: NH(2)(-) approximately OH(-) approximately F(-) >> PH(2)(-) approximately SH(-) approximately Cl(-) > AsH(2)(-) approximately SeH(-) approximately Br(-). The periodic trends are discussed in terms of molecular properties (proton affinity of X(-) expressing Lewis basicity of the nucleophile and C(1s) orbital energy expressing Lewis acidity of the substrate) based on a dative bonding model. Furthermore, the stepwise progress of the reactions and the critical structures are analyzed applying energy decomposition analysis. Increased stability, and thereby increased intrinsic nucleophilicity, correlates with decreasing aromatic character of the Meisenheimer structure. This apparent contradiction is explained in consistency with the other observations using the same model.

Molecular characterization of moxifloxacin resistance from Canadian Clostridium difficile clinical isolates

Fluoroquinolone resistance in Clostridium difficile has been implicated in recent outbreaks of C. difficile infection. The purpose of this report was to characterize the molecular mechanism conferring resistance to moxifloxacin among C. difficile clinical isolates. Eighty-four C. difficile clinical isolates (collected as part of the Canadian Nosocomial Infection Surveillance Program) were evaluated in the current study. Pulsed-field gel electrophoresis was used to type the isolates. Susceptibility testing was performed using Clinical and Laboratory Standards Institute agar dilution methods. The quinolone resistance-determining region of both gyrA and gyrB was amplified using polymerase chain reaction and sequenced for each isolate. The proportion of isolates studied by the North American pulsed-field (NAP) type was as follows: NAP1 (47.6%), NAP2 (20.2%), NAP3 (5.9%), NAP4 (4.8%), NAP5 (2.4%), NAP6 (3.6%), and other patterns (15.5%). All isolates were resistant to ciprofloxacin. Among moxifloxacin-susceptible isolates (MIC ≤2 μg/mL), no amino acid substitutions were detected in either GyrA or GyrB. Three distinct amino acid substitutions were observed among the 3 isolates that had a moxifloxacin MIC of 8 μg/mL (GyrA Asp71 to Val, GyrB Asp426 to Asn, or Glu466 to Val). Isolates with a moxifloxacin MIC of 16 or 32 μg/mL (moderate-level resistance) all had a single identical amino acid substitution in GyrA (Thr82 to Ile). For isolates with a moxifloxacin MIC of ≥64 μg/mL (high-level resistance), this Thr82 to Ile substitution in GyrA was accompanied by at least 1 other amino acid substitution in either GyrA (Asp71 to Glu, Pro116 to Ala, or Ala118 to Ser) or GyrB (Ser366 to Ala, Asp426 to Asn, Asp426 to Val, or Leu444 to Phe) in all but 1 case. Moderate-level moxifloxacin resistance was associated with a single substitution in GyrA. High-level moxifloxacin resistance was associated with this GyrA substitution plus at least 1 other substitution in GyrA or GyrB.

Docking Simulation and Competitive Experiments Validate the Interaction Between the 2,5-Xylidine Inhibitor and Rigidoporus lignosus Laccase

Laccases are polyphenol oxidases which oxidize a broad range of reducing substrates, preferably phenolic compounds, and their use in biotechnological applications is increasing. Recently, the first X-ray structure of active laccase from white rot fungus Rigidoporus lignosus has been reported containing a full complement of copper ions. Comparison among selected fungal laccases of known 3D structure has shown that the Rigidoporus lignosus laccase has a very high similarity with the Trametes versicolor laccase that, being co-crystallized with 2,5-xylidine, shows a well defined binding pocket for the substrate. Global sequence alignment between Rigidoporus lignosus and Trametes versicolor laccases shows 73% of identity but, surprisingly, there is no identity and neither conservative substitutions between the residues composing the loops directly contacting the 2,5-xylidine. Moreover the structural alignment of these two enzymes identifies in these loops a striking structural similarity proposing the question if 2,5-xylidine may bind in same enzyme pocket. Here we report the protein-ligand docking simulation of 3D structure of Rigidoporus lignosus laccase and 2,5-xylidine. Docking simulation analyses show that spatial conformation of the two 2,5-xylidine binding pockets, despite differences in the residues directly contacting the ligand, may arrange a similar pocket that allows a comparable accommodation of the inhibitor. To validate these results the binding of 2,5-xylidine in the substrate cavity has been confirmed by kinetic competitive experiments.