bluuebunny/crossref_metadata_2025 huggingface dataset:
A lipoprotein of Yersinia enterocolitica facilitates ferrioxamine uptake in Escherichia coli
Authors: Bäumler A J , Hantke K
Published: November 2016

A cloned fragment of Yersinia enterocolitica DNA complemented the defect in ferrioxamine B uptake of an Escherichia coli fhuE mutant lacking the outer membrane high-affinity transport protein FhuE. Subcloning revealed that a 13.7-kDa outer membrane protein was required for complementation. The amino acid sequence deduced from the nucleotide sequence showed extensive homology to PCPHi, an outer membrane lipoprotein of Haemophilus influenzae. We therefore termed this protein PCPYe. Plasmid-encoded pcpY mediated a low-affinity uptake of ferrioxamine B which may be caused by changes in the permeability of the outer membrane due to an overexpression of this outer membrane protein. A transposon insertion mutant in the plasmid-encoded pcpY gene was transferred into the chromosome of Y. enterocolitica. The resulting mutation had no effect on the high-affinity uptake of ferrioxamine B in Yersinia cells. Using the antibiotic ferrimycin we were able to isolate a Y. enterocolitica mutant lacking the high-affinity outer membrane receptor for ferrioxamine uptake, termed FoxA.
Involvement of rcsB in Klebsiella K2 capsule synthesis in Escherichia coli K-12
Authors: Wacharotayankun R , Arakawa Y , Ohta M , Hasegawa T , Mori M , Horii T , Kato N
Published: November 2016

Escherichia coli K-12 harboring a part of the structural genes for the Klebsiella K2 capsular polysaccharide (cpsK*) expresses a large amount of K2 capsular polysaccharide as a thick capsule in the presence of plasmids carrying rmpA and rcsB. We have previously shown that expression of the Klebsiella K2 capsule in E. coli HB101 harboring cpsK* depends on the presence of rmpA, a regulatory gene from a large plasmid of Klebsiella pneumoniae Chedid (O1:K2). E. coli K-12 JM109, however, produces only a small amount of K2 capsular polysaccharide, even in the presence of plasmids carrying rmpA as well as the cpsK* structural genes. Introduction of the rcsB gene, a positive regulator of colanic acid capsule synthesis in E. coli K-12 which was cloned from HB101 on a plasmid, into JM109 cells carrying cpsK* and rmpA, results in the expression of a thick K2 capsule. By Northern (RNA) hybridization analysis, rcsB has been found to enhance transcription of a long strand of mRNA (longer than 14 kb) from cpsK*. These E. coli transformants which produce a thick K2 capsule also express colanic acid production at high levels. Therefore, rcsB can act as a positive regulator of Klebsiella K2 capsule production and two capsular polysaccharides can be expressed in E. coli simultaneously. With a somewhat different strain background, we have found that both of the colanic acid regulators, rcsA and rcsB, contribute to the basal level of Klebsiella K2 capsule expression but that the presence of multicopy rcsB in either an rcsB or an rcsA mutant of E. coli is sufficient to increase the expression of K2 capsular polysaccharide. These results suggest further parallels between the regulation of colanic acid synthesis in E. coli and the regulation of Klebsiella K2 capsule synthesis.
Membrane properties of a plant-pathogenic mycoplasmalike organism
Authors: Lim P O , Sears B B , Klomparens K L
Published: November 2016

In terms of biosystematics, the plant-pathogenic mycoplasmalike organisms (MLOs) have been tentatively placed into the class Mollicutes. Certain physiological tests have been used to distinguish families within this class: the sterol-nonrequiring Acholeplasmataceae differ from the sterol-requiring Mycoplasmataceae in that the former are more resistant to lysis by digitonin and more sensitive to lysis in hypotonic salt solutions. To test MLOs for these membrane properties and thus assist in their definitive classification, a dot-blot microassay procedure was used to detect nucleic acids released from lysed cells. The results show that MLOs resemble acholeplasmas grown in the absence of sterols in that they are resistant to digitonin and sensitive to hypotonic salt solutions. The MLOs can be differentiated from acholeplasmas grown without sterols by their greater resistance to lysis in hypotonic sucrose solutions.
Characterization of a regulatory network that controls sigma B expression in Bacillus subtilis
Authors: Benson A K , Haldenwang W G
Published: November 2016

The sigB operon of Bacillus subtilis encodes sigma B and three additional open reading frames (orfV, orfW, and orfX). Having previously mapped several mutations that alter the induction pattern of a sigma B-dependent promoter (ctc) to regions of cloned B. subtilis DNA which contain these three open reading frames, we directly tested the regulatory potential of orfV, orfW, and orfX by creating null alleles of each of these genes and examining the effects of the mutations, either singly or in pairs, on transcription of ctc and the sigB operon. Using lacZ reporter gene fusions and Northern (RNA) blot analyses, we have determined that all three genes modulate the activation of the sigma B-dependent promoters at both the sigB operon and ctc. Our data are consistent with the three gene products participating in a single pathway of negative control. orfW and orfX single-mutant strains have high levels of sigB and ctc transcription. sigB and ctc transcription in an orfV strain is similar to that found in mutant strains which lack sigma B itself. The orfV mutation is dominant to orfX but recessive to orfW. These results suggest that OrfW is the primary inhibitor of sigma B-dependent transcription and that OrfV is capable of counteracting the negative control of OrfW but is prevented from doing this by the orfX gene product.
Molecular characterization of the 28- and 31-kilodalton subunits of the Legionella pneumophila major outer membrane protein
Authors: Hoffman P S , Seyer J H , Butler C A
Published: November 2016

The major outer membrane protein of Legionella pneumophila exhibits an apparent molecular mass of 100 kDa. Previous studies revealed the oligomer to be composed of 28- and 31-kDa subunits; the latter subunit is covalently bound to peptidoglycan. These proteins exhibit cross-reactivity with polyclonal anti-31-kDa protein serum. In this study, we present evidence to confirm that the 31-kDa subunit is a 28-kDa subunit containing a bound fragment of peptidoglycan. Peptide maps of purified proteins were generated following cyanogen bromide cleavage or proteolysis with staphylococcal V8 protease. A comparison of the banding patterns resulting from sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) revealed a common pattern. Selected peptide fragments were sequenced on a gas phase microsequencer, and the sequence was compared with the sequence obtained for the 28-kDa protein. While the amino terminus of the 31-kDa protein was blocked, peptide fragments generated by cyanogen bromide treatment exhibited a sequence identical to that of the amino terminus of the 28-kDa protein, but beginning at amino acid four (glycine), which is preceded by methionine at the third position. This sequence, (Gly-Thr-Met)-Gly-Pro-Val-Trp-Thr-Pro-Gly-Asn ... , confirms that these proteins have a common amino terminus. An oligonucleotide synthesized from the codons of the common N-terminal amino acid sequence was used to establish by Southern and Northern (RNA) blot analyses that a single gene coded for both proteins. With regard to the putative porin structure, we have identified two major bands at 70 kDa and at approximately 120 kDa by nonreducing SDS-PAGE. The former may represent the typical trimeric motif, while the latter may represent either a double trimer or an aggregate. Analysis of these two forms by two-dimensional SDS-PAGE (first dimensions, nonreducing; second dimensions, reducing) established that both were composed of 31- and 28-kDa subunits cross-linked via interchain disulfide bonds. These studies confirm that the novel L. pneumophila major outer protein is covalently bound to peptidoglycan via a modified 28-kDa subunit (31-kDa anchor protein) and cross-linked to other 28-kDa subunits via interchain disulfide bonds.
Cloning and sequencing of the genes involved in the conversion of 5-substituted hydantoins to the corresponding L-amino acids from the native plasmid of Pseudomonas sp. strain NS671
Authors: Watabe K , Ishikawa T , Mukohara Y , Nakamura H
Published: November 2016

Pseudomonas sp. strain NS671, which produces L-amino acids asymmetrically from the corresponding racemic 5-substituted hydantoins, harbored a plasmid of 172 kb. Curing experiments suggest that this plasmid, designated pHN671, is responsible for the conversion of 5-substituted hydantoins to their corresponding L-amino acids by strain NS671. DNA fragments containing the genes involved in this conversion were cloned from pHN671 in Escherichia coli by using pUC18 as a cloning vector. The smallest recombinant plasmid, designated pHPB12, contained a 7.5-kb insert DNA. The nucleotide sequence of the insert DNA was determined, and three closely spaced open reading frames predicted to encode peptides with molecular masses of 75.6, 64.9, and 45.7 kDa were found. These open reading frames were designated hyuA, hyuB, and hyuC, respectively. Cell extracts from E. coli carrying deletion derivatives of pHPB12 were analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, and the gene products of hyuA, hyuB, and hyuC were identified. The functions of these gene products were also examined with the deletion derivatives. The results indicate that both hyuA and hyuB are involved in the conversions of D- and L-5-substituted hydantoins to corresponding N-carbamyl-D- and N-carbamyl-L-amino acids, respectively, and that hyuC is involved in the conversion of N-carbamyl-L-amino acids to L-amino acids.
Characterization of the Agrobacterium tumefaciens heat shock response: evidence for a sigma 32-like sigma factor
Authors: Mantis N J , Winans S C
Published: November 2016

We have characterized the heat shock response of Agrobacterium tumefaciens and compared it with the well-characterized Escherichia coli heat shock response. Four major heat shock proteins with apparent molecular masses of 98, 75, 65, and 20 kDa were identified by pulse-labelling cultures after temperature upshift. The three largest proteins comigrated with proteins that were antigenically related to the E. coli heat shock proteins sigma 70, DnaK, and GroEL, respectively. The heat shock proteins were also strongly induced by ethanol and cadmium chloride and were mildly induced by mitomycin C. To determine whether the A. tumefaciens heat shock regulatory system was similar to that of E. coli, we introduced the E. coli dnaK gene into A. tumefaciens. The E. coli DnK protein was expressed in A. tumefaciens, and its synthesis was induced after heat shock. Primer extension analysis of the E. coli dnaK gene in A. tumefaciens indicated that transcription initiated from one or possibly both of the E. coli heat shock promoters. We conclude that A. tumefaciens has a heat shock response similar to that of E. coli, in that (i) similar proteins are induced by heat shock, (ii) synthesis of these proteins is induced in response to similar stimuli, and (iii) A. tumefaciens can recognize an E. coli heat shock promoter, suggesting that A. tumefaciens has a sigma factor similar to sigma 32.
Role of alternative promoter elements in transcription from the nar promoter of Escherichia coli
Authors: Walker M S , DeMoss J A
Published: November 2016

The effects of mutations in the -10, -35, and Fnr box regions of the narGHJI promoter of Escherichia coli were determined by assaying the expression of beta-galactosidase from narG::lacZ fusion plasmids under aerobic and anaerobic conditions. A 1-base change in the -10 hexamer completely abolished expression, whereas a 3-base change to create the consensus TATAAT resulted in significant aerobic as well as anaerobic expression. A mutation in the putative -35 hexamer did not affect anaerobic expression but reduced aerobic expression from the construction with the -10 consensus sequence. A mutation in the Fnr box severely reduced anaerobic expression but did not affect aerobic expression. When the complete 5' region of the nar operon including the NarL box was present, nitrate stimulated both aerobic and anaerobic expression. Stimulation of expression by nitrate occurred in an fnr mutant but not in a narL mutant. We conclude that the rate of transcription of the nar operon is dependent on two distinct modes of transcription. One mode, which occurs at low levels, depends on the -10 and -35 hexamer sequences and is dramatically enhanced by changing the -10 sequence to the consensus TATAAT. The second depends on the -10 and Fnr box sequences but is independent of the -35 sequence. This second mode occurs at a very high level under anaerobic conditions when Fnr is activated and is also enhanced by changing the -10 sequence to the consensus TATAAT. NarL, activated by nitrate, stimulated both modes of transcription, indicating that it does not act through Fnr but that it directly affects the interaction of RNA polymerase with the promoter.
Mutagenesis of ribosomal protein S8 from Escherichia coli: defects in regulation of the spc operon
Authors: Wower I , Kowaleski M P , Sears L E , Zimmermann R A
Published: November 2016

The structural features of Escherichia coli ribosomal protein S8 that are involved in translational regulation of spc operon expression and, therefore, in its interaction with RNA have been investigated by use of a genetic approach. The rpsH gene, which encodes protein S8, was first inserted into an expression vector under the control of the lac promoter and subsequently mutagenized with methoxylamine or nitrous acid. A screening procedure based on the regulatory role of S8 was used to identify mutants that were potentially defective in their ability to associate with spc operon mRNA and, by inference, 16S mRNA. In this way, we isolated 39 variants of the S8 gene containing alterations at 34 different sites, including 37 that led to single amino acid substitutions and 2 that generated premature termination codons. As the mutations were distributed throughout the polypeptide chain, our results indicate that amino acid residues important for the structural integrity of the RNA-binding domain are not localized to a single segment. Nonetheless, the majority were located within three short sequences at the N terminus, middle, and C terminus that are phylogenetically conserved among all known eubacterial and chloroplast versions of this protein. We conclude that these sites encompass the main structural determinants required for the interaction of protein S8 with RNA.
Effect of Escherichia coli nusG function on lambda N-mediated transcription antitermination
Authors: Sullivan S L , Ward D F , Gottesman M E
Published: November 2016

The Escherichia coli Nus factors act in conjunction with the bacteriophage lambda N protein to suppress transcription termination on the lambda chromosome. NusA binds both N and RNA polymerase and may also interact with other Nus factors. To search for additional components of the N antitermination system, we isolated host revertants that restored N activity in nusA1 mutants. One revertant, nusG4, was mapped to the rif region of the E. coli chromosome and shown to represent a point mutation near the 3' end of the nusG gene. The nusG4 mutation also suppressed nusE71 but not nusASal, nusB5, nusC60 (rpoB60), or nusD026 (rho026). However, nusG+ expressed from a multicopy plasmid suppressed nusD026 and related rho mutants for both lambda and phage T4 growth. These results suggest that NusG may act as a component of the N antitermination complex. In addition, the data imply a role for NusG in Rho-dependent termination.
Aspartate and maltose-binding protein interact with adjacent sites in the Tar chemotactic signal transducer of Escherichia coli
Authors: Gardina P , Conway C , Kossman M , Manson M
Published: November 2016

The Tar protein of Escherichia coli is a chemotactic signal transducer that spans the cytoplasmic membrane and mediates responses to the attractants aspartate and maltose. Aspartate binds directly to Tar, whereas maltose binds to the periplasmic maltose-binding protein, which then interacts with Tar. The Arg-64, Arg-69, and Arg-73 residues of Tar have previously been shown to be involved in aspartate sensing. When lysine residues are introduced at these positions by site-directed mutagenesis, aspartate taxis is disrupted most by substitution at position 64, and maltose taxis is disrupted most by substitution at position 73. To explore the spatial distribution of ligand recognition sites on Tar further, we performed doped-primer mutagenesis in selected regions of the tar gene. A number of mutations that interfere specifically with aspartate taxis (Asp-), maltose taxis (Mal-), or both were identified. Mutations affecting residues 64 to 73 or 149 to 154 in the periplasmic domain of Tar are associated with an Asp- phenotype, whereas mutations affecting residues 73 to 83 or 141 to 150 are associated with a Mal- phenotype. We conclude that aspartate and maltose-binding protein interact with adjacent and partially overlapping regions in the periplasmic domain of Tar to initiate attractant signalling.
Suppression of insertions in the complex pdxJ operon of Escherichia coli K-12 by lon and other mutations
Authors: Lam H M , Tancula E , Dempsey W B , Winkler M E
Published: November 2016

Complementation analyses using minimal recombinant clones showed that all known pdx point mutations, which cause pyridoxine (vitamin B6) or pyridoxal auxotrophy, are located in the pdxA, pdxB, serC, pdxJ, and pdxH genes. Antibiotic enrichments for chromosomal transposon mutants that require pyridoxine (vitamin B6) or pyridoxal led to the isolation of insertions in pdxA, pdxB, and pdxH but not in pdxJ. This observation suggested that pdxJ, like pdxA, pdxB, and serC, might be in a complex operon. To test this hypothesis, we constructed stable insertion mutations in and around pdxJ in plasmids and forced them into the bacterial chromosome. Physiological properties of the resulting insertion mutants were characterized, and the DNA sequence of pdxJ and adjacent regions was determined. These combined approaches led to the following conclusions: (i) pdxJ is the first gene in a two-gene operon that contains a gene, temporarily designated dpj, essential for Escherichia coli growth; (ii) expression of the rnc-era-recO and pdxJ-dpj operons can occur independently, although the pdxJ-dpj promoter may lie within recO; (iii) pdxJ encodes a 26,384-Da polypeptide whose coding region is preceded by a PDX box, and dpj probably encodes a basic, 14,052-Da polypeptide; (iv) mini-Mud insertions in dpj and pdxJ, which are polar on dpj, severely limit E. coli growth; and (v) three classes of suppressors, including mutations in lon and suppressors of lon, that allow faster growth of pdxJ::mini-Mud mutants can be isolated. A model to account for the action of dpj suppressors is presented, and aspects of this genetic analysis are related to the pyridoxal 5'-phosphate biosynthetic pathway.
arcD, the first gene of the arc operon for anaerobic arginine catabolism in Pseudomonas aeruginosa, encodes an arginine-ornithine exchanger
Authors: Verhoogt H J , Smit H , Abee T , Gamper M , Driessen A J , Haas D , Konings W N
Published: November 2016

In the absence of oxygen and nitrate, Pseudomonas aeruginosa metabolizes arginine via the arginine deiminase pathway, which allows slow growth on rich media. The conversion of arginine to ornithine, CO2, and NH3 is coupled to the production of ATP from ADP. The enzymes of the arginine deiminase pathway are organized in the arcDABC operon. The arcD gene encodes a hydrophobic polytopic membrane protein. Translocation of arginine and ornithine in membrane vesicles derived from an Escherichia coli strain harboring a recombinant plasmid carrying the arcD gene was studied. Arginine and ornithine uptake was coupled to the proton motive force with a bias toward the transmembrane electrical potential. Accumulated ornithine was readily exchangeable for external arginine or lysine. The exchange was several orders of magnitude faster than proton motive force-driven transport. The ArcD protein was reconstituted in proteoliposomes after detergent solubilization of membrane vesicles. These proteoliposomes mediate a stoichiometric exchange between arginine and ornithine. It is concluded that the ArcD protein is a transport system that catalyzes an electroneutral exchange between arginine and ornithine to allow high-efficiency energy conversion in the arginine deiminase pathway.
Premature termination of in vivo transcription of a gene encoding a branched-chain amino acid transport protein in Escherichia coli
Authors: Williamson R M , Oxender D L
Published: November 2016

Previous studies have suggested that control of expression of genes of the LIV-I permease system for the high-affinity transport of branched-chain amino acids in Escherichia coli involves modulation in the frequency of mRNA elongation. Mutation of the Rho transcription termination factor and shortages of charged leucyl-tRNA have been shown to alter LIV-I transport activity. Rho-dependent transcription termination regulated by shortages of charged leucyl-tRNA at sites preceding structural genes has been proposed to account for their role in regulation of LIV-I transport. Transcription of the livJ-binding protein gene, encoding one of the periplasmic components of the LIV-I system, was analyzed in vivo with strains which lack repression of the LIV-I genes and harbor a temperature-sensitive allele for either leucyl-tRNA synthetase or Rho factor. Analysis of mRNA synthesis by DNA-RNA hybridization in the various mutant strains indicated that both shortages of leucyl-tRNA caused by inactivation of the temperature-sensitive leucyl-tRNA synthetase and inactivation of the Rho factor were associated with increased synthesis of livJ mRNA. Nuclease protection and gel electrophoresis studies detected prematurely terminated transcripts corresponding in size to the leader region of livJ mRNA. Accumulations of these short transcripts were suppressed in strains harboring temperature-sensitive alleles for either leucyl-tRNA synthetase or Rho factor. These results provide support for the hypothesis that expression of livJ involves Rho-dependent transcription termination in which antitermination is associated with the intracellular availability of aminoacyl leucyl-tRNA.
In vivo processing of Staphylococcus aureus lipase
Authors: Rollof J , Normark S
Published: November 2016

The Staphylococcus aureus lipase gene encodes a 76-kDa protein. Extracellular lipase purified from culture supernatants is only 45 to 46 kDa, however. We show that the lipase is secreted in vivo as an 82-kDa protein with full enzymatic activity. It is then sequentially processed, both in culture and in cell-free supernatants, to a mature, 45- to 46-kDa protein. Protein sequencing demonstrates that the N-terminal region of the 82-kDa prolipase, comprising 295 amino acids, is cleaved from the central and C-terminal moieties, which contain the active site. A metallocysteine protease is probably responsible for initiating this processing. The extremely hydrophobic, mature lipase is resistant to further protease degradation and retains the full catalytic activity of the prolipase.
Surface presentation of Shigella flexneri invasion plasmid antigens requires the products of the spa locus
Authors: Venkatesan M M , Buysse J M , Oaks E V
Published: November 2016

An avirulent, invasion plasmid insertion mutant of Shigella flexneri 5 (pHS1059) was restored to the virulence phenotype by transformation with a partial HindIII library of the wild-type invasion plasmid constructed in pBR322. Western immunoblot analysis of pHS1059 whole-cell lysates revealed that the synthesis of the invasion plasmid antigens VirG, IpaA, IpaB, IpaC, and IpaD was similar to that seen in the corresponding isogenic S. flexneri 5 virulent strain, M90T. IpaB and IpaC, however, were not present on the surface of pHS1059 as was found in M90T, suggesting that the transport or presentation of the IpaB and IpaC proteins onto the bacterial surface was defective in the mutant. pHS1059 was complemented by pWR266, which carried contiguous 1.2- and 4.1-kb HindIII fragments of the invasion plasmid. pHS1059(pWR266) cells were positive in the HeLa cell invasion assay as well as colony immunoblot and enzyme-linked immunosorbent assays, using monoclonal antibodies to IpaB and IpaC. These studies established that the antigens were expressed on the surface of the transformed bacteria. In addition, water extraction of pHS1059 and pHS1059(pWR266) whole cells, which can be used to remove IpaB and IpaC antigens from the surface of wild-type M90T bacteria, yielded significant amounts of these antigens from pHS1059(pWR266) but not from pHS1059. Minicell and DNA sequence analysis indicated that several proteins were encoded by pWR266, comprising the spa loci, which were mapped to a region approximately 18 kb upstream of the ipaBCDAR gene cluster. Subcloning and deletion analysis revealed that more than one protein was involved in complementing the Spa- phenotype in pHS1059. One of these proteins, Spa47, showed striking homology to ORF4 of the Bacillus subtilis flaA locus and the fliI gene sequence of Salmonella typhimurium, both of which bear strong resemblance to the alpha and beta subunits of bacterial, mitochondrial, and chloroplast proton-translocating F0F1 ATPases.
LcrF is the temperature-regulated activator of the yadA gene of Yersinia enterocolitica and Yersinia pseudotuberculosis
Authors: Skurnik M , Toivanen P
Published: November 2016

The virulence plasmid of human pathogenic Yersinia species, pYV, encodes secreted proteins, Yop proteins, and an outer membrane protein, YadA. YadA has been associated with binding to a variety of substrates and with interference with host defense. YadA is regulated by temperature and is expressed only at 37 degrees C. Unlike the yop regulon, the yadA gene is not under Ca2+ regulation. Here, we show that LcrF (VirF), the temperature-regulated activator of the yop regulon, also acts as an activator for yadA.
Evolutionary relationships of a plant-pathogenic mycoplasmalike organism and Acholeplasma laidlawii deduced from two ribosomal protein gene sequences
Authors: Lim P O , Sears B B
Published: November 2016

The families within the class Mollicutes are distinguished by their morphologies, nutritional requirements, and abilities to metabolize certain compounds. Biosystematic classification of the plant-pathogenic mycoplasmalike organisms (MLOs) has been difficult because these organisms have not been cultured in vitro, and hence their nutritional requirements have not been determined nor have physiological characterizations been possible. To investigate the evolutionary relationship of the MLOs to other members of the class Mollicutes, a segment of a ribosomal protein operon was cloned and sequenced from an aster yellows-type MLO which is pathogenic for members of the genus Oenothera and from Acholeplasma laidlawii. The deduced amino acid sequence data from the rpl22 and rps3 genes indicate that the MLOs are more closely related to A. laidlawii than to animal mycoplasmas, confirming previous results from 16S rRNA sequence comparisons. This conclusion is also supported by the finding that the UGA codon is not read as a tryptophan codon in the MLO and A. laidlawii, in contrast to its usage in Mycoplasma capricolum.
Purification and determination of the structure of capsular polysaccharide of Vibrio vulnificus M06-24
Authors: Reddy G P , Hayat U , Abeygunawardana C , Fox C , Wright A C , Maneval D R , Bush C A , Morris J G
Published: November 2016

Virulence of Vibrio vulnificus has been strongly associated with encapsulation and an opaque colony morphology. Capsular polysaccharide was purified from a whole-cell, phosphate-buffered saline-extracted preparation of the opaque, virulent phase of V. vulnificus M06-24 (M06-24/O) by dialysis, centrifugation, enzymatic digestion, and phenol-chloroform extraction. Nuclear magnetic resonance spectroscopic analysis of the purified polysaccharide showed that the polymer was composed of a repeating structure with four sugar residues per repeating subunit: three residues of 2-acetamido-2,6-dideoxyhexopyranose in the alpha-gluco configuration (QuiNAc) and an additional residue of 2-acetamido hexouronate in the alpha-galactopyranose configuration (GalNAcA). The complete carbohydrate structure of the polysaccharide was determined by heteronuclear nuclear magnetic resonance spectroscopy and by high-performance anion-exchange chromatography. The 1H and 13C nuclear magnetic resonance spectra were completely assigned, and vicinal coupling relationships were used to establish the stereochemistry of each sugar residue, its anomeric configuration, and the positions of the glycosidic linkages. The complete structure is: [----3) QuipNAc alpha-(1----3)-GalpNAcA alpha-(1----3)-QuipNAc alpha-(1----]n QuipNAc alpha-(1----4)-increases The polysaccharide was produced by a translucent phase variant of M06-24 (M06-24/T) but not by a translucent, acapsular transposon mutant (CVD752). Antibodies to the polysaccharide were demonstrable in serum from rabbits inoculated with M06-24/O.
Characterization of an autonomously replicating region from the Streptomyces lividans chromosome
Authors: Zakrzewska-Czerwińska J , Schrempf H
Published: November 2016

The chromosomal replication origin of the plasmidless derivative (TK21) from Streptomyces lividans 66 has been cloned as an autonomously replicating minichromosome (pSOR1) by using the thiostrepton resistance gene as a selectable marker. pSOR1 could be recovered as a closed circular plasmid which shows high segregational instability. pSOR1 was shown to replicate in Streptomyces coelicolor A3(2) and in S. lividans 66 and hybridized with DNA from several different Streptomyces strains. Physical mapping revealed that oriC is located on a 330-kb AseI fragment of the S. coelicolor A3(2) chromosome. DNA sequence analyses showed that the cloned chromosomal oriC region contains numerous DnaA boxes which are arranged in two clusters. The preferred sequence identified in the oriC region of Escherichia coli and several other bacteria is TTATCCACA. In contrast, in S. lividans, which has a high GC content, the preferred sequence for DnaA boxes appears to be TTGTCCACA.
Contrasting geochemical signatures of fluid-absent versus fluid-fluxed melting of muscovite in metasedimentary sources: The Himalayan leucogranites
Authors: Gao Li-E. , Zeng Lingsen , Asimow Paul D.
Published: November 2016

Most of the Himalayan Cenozoic leucogranites are products of partial melting of metapelite sources. In the Malashan-Gyirong area (southern Tibet), the geochemical compositions of leucogranites define two groups with distinct whole-rock major elements, large ion lithophile elements, rare earth elements, high field strength elements, and Sr and Hf isotope ratios. Based on published experimental results that define generalized melting reactions of metapelitic sources, we infer that these leucogranites are the products of two different types of crustal anatexis: fluid-fluxed melting and fluid-absent melting of muscovite in metasedimentary sources. As compared to the leucogranites derived from fluid-absent melting, those from fluid-fluxed melting have relatively higher Ca, Sr, Ba, Zr, Hf, Th, and light rare earth element concentrations, and Zr/Hf, Eu/Eu*, and Nd/Nd*, but lower Rb, Nb, Ta, and U concentrations, Rb/Sr and 87Sr/86Sr ratios, and εHf(t). The geochemical differences can be explained by melting behaviors of major (muscovite, feldspar) and accessory minerals (zircon and monazite) during different modes of crustal anatexis. The systematic elemental and isotopic signatures of different types of crustal anatexis and, in particular, the coupling of major and trace elements that results from common influences on rock-forming and accessory mineral behaviors provide tools with which to refine our understanding of the nature of crustal anatexis.
Demise of Ediacaran dolomitic seas marks widespread biomineralization on the Siberian Platform
Authors: Wood R.A. , Zhuravlev A.Yu. , Sukhov S.S. , Zhu M. , Zhao F.
Published: November 2016

The trigger for biomineralization of metazoans in the terminal Ediacaran, ca. 550 Ma, has been suggested to be the rise of oxygenation or an increase in seawater Ca concentration, but geochemical and fossil data have not been fully integrated to demonstrate cause and effect. Here we combine the record of macrofossils with early marine carbonate cement distribution within a relative depth framework for terminal Ediacaran to Cambrian successions on the eastern Siberian Platform, Russia, to interrogate the evolution of seawater chemistry and biotic response. Prior to ca. 545 Ma, the presence of early marine ferroan dolomite cement suggests dominantly ferruginous anoxic “aragonite-dolomite seas”, with a very shallow oxic chemocline that supported mainly soft-bodied macrobiota. After ca. 545 Ma, marine cements changed to aragonite and/or high-Mg calcite, and this coincides with the appearance of widespread aragonite and high-Mg calcite skeletal metazoans, suggesting a profound change in seawater chemistry to “aragonite seas” with a deeper chemocline. By early Cambrian Stage 3, the first marine low-Mg calcite cements appear, coincident with the first low-Mg calcite metazoan skeletons, suggesting a further shift to “calcite seas”. We suggest that this evolution of seawater chemistry was caused by enhanced continental denudation that increased the input of Ca into oceans so progressively lowering Mg/Ca, which, combined with more widespread oxic conditions, facilitated the rise of skeletal animals and in turn influenced the evolution of skeletal mineralogy.
Health-related quality of life after pulmonary embolism: a cross-sectional study
Authors: Tavoly Mazdak , Utne Kristin Kornelia , Jelsness-Jørgensen Lars-Petter , Wik Hilde Skuterud , Klok Frederikus A , Sandset Per Morten , Ghanima Waleed
Published: November 2016

Objectives

The psychological effects of acute pulmonary embolism (PE) have scarcely been studied. The aims of this study were to evaluate health-related quality of life (HRQoL) in patients with a history of PE compared with that of the general population and buddy controls, and to explore factors that may predict impaired HRQoL.

Design

Cross-sectional.

Setting

Haematology and thrombosis unit in Fredrikstad, Norway.

Participants

213 consecutive patients treated for PE were identified from hospital registries. Eligible patients were scheduled for a single study visit, including a functional capacity test (6 min walking test). HRQoL was assessed using the EuroQol 5D dimensions 3-level (EQ-5D-3L) questionnaire, of which the results were compared with Danish population norms and age-matched and sex-matched buddy controls. The buddy controls were recruited by asking every patient to hand over the EQ-5D questionnaire to 2 age-matched and sex-matched friends or relatives. Multivariable regression analyses were used to examine possible determinants of reduced HRQoL.

Results

Mean age was 61 years (SD 15), 117 (55%) were males, and median time since diagnosis was 3.8 years (range 0.3–9.5). Mean EuroQol visual analogue scale (EQ VAS) was 67 in PE as compared with 81 in the general population (p<0.005) and corresponding EQ-5D index values were 0.80 and 0.86 (p<0.005). Patients reported more problems in all 5 EQ-5D compared with both the buddy controls and the general population, p<0.05. Shorter 6 min walking distance (β=0.09, p<0.005) and patient-reported dyspnoea (β=11.27, p<0.005) were independent predictors of lower EQ VAS scores.

Conclusions

Our findings show that patients with a history of PE have impaired HRQoL when compared with the general population and buddy controls. Reduced functional capacity and persistent dyspnoea were the main predictors of this impairment.
Endoscopic resection of high-risk T1 colorectal carcinoma prior to surgical resection has no adverse effect on long-term outcomes
Authors: Overwater A , Kessels K , Elias S G , Backes Y , Spanier B W M , Seerden T C J , Pullens H J M , de Vos tot Nederveen Cappel W H , van den Blink A , Offerhaus G J A , van Bergeijk J , Kerkhof M , Geesing J M J , Groen J N , van Lelyveld N , ter Borg F , Wolfhagen F , Siersema P D , Lacle M M , Moons L M G
Published: November 2016

Objective

It is difficult to predict the presence of histological risk factors for lymph node metastasis (LNM) before endoscopic treatment of T1 colorectal cancer (CRC). Therefore, endoscopic therapy is propagated to obtain adequate histological staging. We examined whether secondary surgery following endoscopic resection of high-risk T1 CRC does not have a negative effect on patients' outcomes compared with primary surgery.

Design

Patients with T1 CRC with one or more histological risk factors for LNM (high risk) and treated with primary or secondary surgery between 2000 and 2014 in 13 hospitals were identified in the Netherlands Cancer Registry. Additional data were collected from hospital records, endoscopy, radiology and pathology reports. A propensity score analysis was performed using inverse probability weighting (IPW) to correct for confounding by indication.

Results

602 patients were eligible for analysis (263 primary; 339 secondary surgery). Overall, 34 recurrences were observed (5.6%). After adjusting with IPW, no differences were observed between primary and secondary surgery for the presence of LNM (OR 0.97; 95% CI 0.49 to 1.93; p=0.940) and recurrence during follow-up (HR 0.97; 95% CI 0.41 to 2.34; p=0.954). Further adjusting for lymphovascular invasion, depth of invasion and number of retrieved lymph nodes did not alter this outcome.

Conclusions

Our data do not support an increased risk of LNM or recurrence after secondary surgery compared with primary surgery. Therefore, an attempt for an en-bloc resection of a possible T1 CRC without evident signs of deep invasion seems justified in order to prevent surgery of low-risk T1 CRC in a significant proportion of patients.
Efficacy and tolerability of repository corticotropin injection in patients with persistently active SLE: results of a phase 4, randomised, controlled pilot study
Authors: Furie Richard , Mitrane Margaret , Zhao Enxu , Das Maya , Li Daner , Becker Patrice M
Published: November 2016

Objective

To evaluate the efficacy of a prolonged-release formulation of a porcine adrenocorticotropic hormone analogue (repository corticotropin injection (RCI)) added to standard of care in patients requiring moderate-dose corticosteroids for symptomatic SLE.

Methods

This prospective, randomised, double-blind, phase 4, pilot study (NCT01753401) enrolled 38 patients with persistently active SLE involving skin and/or joints. Enrolled patients received RCI, 40 U daily or 80 U every other day, or volume-matched placebo gel, for 8 weeks, with dose tapering to twice weekly during weeks 5–8. Efficacy endpoints included proportion of responders at week 4 based on a novel composite measure that included resolution of rash or arthritis measured using the hybrid SLE Disease Activity Index (hSLEDAI) without worsening British Isles Lupus Assessment Group (BILAG) scores in other organ systems at week 4 (primary), as well as improvements in total hSLEDAI and BILAG scores and other measures of skin and joint disease activity over the 8-week treatment period.

Results

Response, as defined for the primary endpoint, did not differ significantly between the combined placebo and RCI-treated groups at week 4. At week 8, the proportion of responders was higher in RCI-treated patients but did not statistically differ between groups (RCI 40 U (53.8%), RCI 80 U (33.3%), combined placebo (27.3%)). However, RCI treatment was associated with statistically significant improvements in several secondary endpoints, including total hSLEDAI, total BILAG and Cutaneous Lupus Erythematosus Disease Area and Severity Index Activity scores within 8 weeks. Treatment was well tolerated.

Conclusions

Although the primary endpoint was not met in this pilot study, secondary and post hoc analyses suggested that RCI was associated with improvements in SLE disease activity in a select patient population with steroid-dependent persistent disease.

Trial registration number

NCT01753401; results.
Supersize Cp! Tetrabenzo[a,c,g,i]fluorenyl complexes of yttrium
Authors: Sun Jianlong , Berg David J. , Twamley Brendan
Published: November 2016

The synthesis of tetrabenzo[a,c,g,i]fluorenyl (Tbf) yttrium dialkyl complexes, (Tbf)Y(CH2SiMe3)2(L) (L = tetrahydrofuran (THF), 1; L = bipy, 2), by direct protonolysis of the tris(alkyl) complex, Y(CH2SiMe3)3(THF)2, are reported. The X-ray crystal structures of 1 and 2 display the helical twisting typically observed for the Tbf ligand. Dynamic nuclear magnetic resonance (NMR) studies on 1 show a barrier to Tbf helical inversion (epimerization or “wagging”) of 38.1 ± 0.5 kJ mol−1. The reaction of 1 with acidic hydrocarbons such as 1,3-bis(trimethylsilyl)cyclopentadiene or trimethylsilylacetylene results in protonolysis to form the mixed Cp derivative [(Tbf){C5H3(SiMe3)2}Y(CH2SiMe3)(THF)] (3) or [(Tbf)Y(CCSiMe3)2(THF)]n (4), respectively. In the case of 4, a small amount of the trinuclear cluster (Tbf)Y3(μ3-CCSiMe3)2(μ2-CCSiMe3)3(CCSiMe3)3(THF)2 (5) was isolated and characterized by X-ray crystallography. Dialkyl 1 undergoes smooth insertion of trimethylsilyl isocyanate to afford [(Tbf)Y{κ2-(N,O)-Me3SiN(Me3SiCH2)CO}2(THF)] (6) but it does not react with alkenes. Treating 1 with [Ph3C]+[B(C6F5)4]− in bromobenzene generates a moderately active ethylene polymerization catalyst (36 kg mol−1 h−1 bar−1).
Subchronic metformin pretreatment enhances novel object recognition memory task in forebrain ischemia: behavioural, molecular, and electrophysiological studies
Authors: Ashabi Ghorbangol , Sarkaki Alireza , Khodagholi Fariba , Zareh Shahamati Shima , Goudarzvand Mahdi , Farbood Yaghoob , Badavi Mohammad , Khalaj Leila
Published: November 2016

Metformin exerts its effect via AMP-activated protein kinase (AMPK), which is a key sensor for energy homeostasis that regulates different intracellular pathways. Metformin attenuates oxidative stress and cognitive impairment. In our experiment, rats were divided into 8 groups; some were pretreated with metformin (Met, 200 mg/kg) and (or) the AMPK inhibitor Compound C (CC) for 14 days. On day 14, rats underwent transient forebrain global ischemia. Data indicated that pretreatment of ischemic rats with metformin reduced working memory deficits in a novel object recognition test compared to group with ischemia–reperfusion (I–R) (P < 0.01). Pretreatment of the I–R animals with metformin increased phosphorylated cyclic-AMP response element-binding protein (pCREB) and c-fos levels compared to the I–R group (P < 0.001 for both). The level of CREB and c-fos was significantly lower in ischemic rats pretreated with Met + CC compared to the Met + I–R group. Field excitatory postsynaptic potential (fEPSP) amplitude and slope was significantly lower in the I–R group compared to the sham operation group (P < 0.001). Data showed that fEPSP amplitude and slope was significantly higher in the Met + I–R group compared to the I–R group (P < 0.001). Treatment of ischemic animals with Met + CC increased fEPSP amplitude and slope compared to the Met + I–R group (P < 0.01). We unravelled new aspects of the protective role of AMPK activation by metformin, further emphasizing the potency of metformin pretreatment against cerebral ischemia.
Nonlinear Equilibrium and Buckling of Fixed Shallow Arches Subjected to an Arbitrary Radial Concentrated Load
Authors: Pi Yong-Lin , Bradford Mark Andrew , Liu Airong
Published: November 2016

This paper is concerned with an analytical study of the nonlinear in-plane equilibrium and buckling of fixed shallow circular arches that are subjected to an arbitrary radial concentrated load. The structural behavior of an arch under an arbitrary radial concentrated load is quite different from that of an arch under a central concentrated load. It is shown that a fixed arch under an arbitrary radial concentrated load can buckle in a limit point instability mode, but cannot buckle in a bifurcation mode, which is different from that of a fixed arch under a central concentrated load that can buckle in a bifurcation mode or in a limit point instability mode. Analytical solutions for the nonlinear equilibrium path and limit point buckling load of shallow circular arches under an arbitrary radial concentrated load are derived. It is found that the load position influences the buckling load significantly and the influence is much related to the modified slenderness of the arch defined in the paper. It is also found that when the modified slenderness of an arch is smaller than a specific value, the arch has no typical buckling behavior. The analytical solution for the relationship of the specific modified slenderness with the load position is also derived. Comparisons with finite element (FE) results show that the analytical solutions can accurately predict the nonlinear equilibrium and buckling load of shallow fixed arches under an arbitrary radial concentrated load.
Analyzing nonlinear large deformation with an improved element-free Galerkin method via the interpolating moving least-squares method
Authors: Cheng Yumin , Bai Funong , Liu Chao , Peng Miaojuan
Published: November 2016

Using the interpolating moving least-squares (IMLS) method to form the shape function, a novel improved element-free Galerkin (IEFG) method is presented for solving nonlinear elastic large deformation problems. To obtain the formulae of the IEFG method for elastic large deformation problems, we use the Galerkin weak form to obtain the discretized system equation, and use the penalty method to apply the displacement boundary conditions. Some selected numerical examples of two-dimensional elastic large deformation problems are given, and the numerical results are analyzed. From the examples, it is shown that the IEFG method in this paper has higher computational precision than the element-free Galerkin (EFG) method presented before.
Early Jurassic magmatism on the Antarctic Peninsula and potential correlation with the Subcordilleran plutonic belt of Patagonia
Authors: Riley Teal R. , Flowerdew Michael J. , Pankhurst Robert J. , Curtis Mike L. , Millar Ian L. , Fanning C. Mark , Whitehouse Martin J.
Published: November 2016

Early Jurassic silicic volcanic rocks of the Chon Aike Province (V1: 187 – 182 Ma) are recognized from many localities in the southern Antarctic Peninsula and NE Patagonia and are essentially coeval with the extensive Karoo (182 Ma) and Ferrar (183 Ma) large igneous provinces of pre-breakup Gondwana. Until recently, plutonic rocks of this age were considered either rare in or absent from the Antarctic Peninsula batholith, which was thought to have been mainly constructed during the Middle Jurassic and the mid-Cretaceous. New U–Pb zircon geochronology from the Antarctic Peninsula and recently published U–Pb ages from elsewhere in the Peninsula and Patagonia are used to demonstrate the more widespread nature of Early Jurassic plutonism. Eight samples are dated here from the central and southern Antarctic Peninsula. They are all moderately to strongly foliated granitoids (tonalite, granite, granodiorite) and locally represent the crystalline basement. They yield ages in the range 188 – 181 Ma, which overlap with published ages of 185 – 180 Ma from granitoids from elsewhere on the Antarctic Peninsula and from the Subcordilleran plutonic belt of Patagonia (185 – 181 Ma). Whereas Early Jurassic plutons of the Subcordilleran plutonic belt of Patagonia are directly related to subduction processes along the proto-Pacific margin of Gondwana, coeval volcanic rocks of the Chon Aike Province are interpreted to be directly associated with extension and plume activity during the initial stages of Gondwana breakup. This indicates that subduction was continuing when Chon Aike Province volcanism started. The Early Jurassic plutonism on the Antarctic Peninsula is transitional between subduction-related and breakup-related magmatism.

Supplementary material
: Cathodoluminescence images of analysed zircon grains from sites on the Antarctic Peninsula are available at
https://doi.org/10.6084/m9.figshare.c.3521973