Vibrio cholerae O1 El Tor from southern Vietnam in 2010 was molecularly distinct from that present from 1999 to 2004.

The Vibrio cholerae O1 (VCO1) El Tor biotype appeared during the seventh cholera pandemic starting in 1961, and new variants of this biotype have been identified since the early 1990s. This pandemic has affected Vietnam, and a large outbreak was reported in southern Vietnam in 2010. Pulsed-field gel electrophoresis (PFGE) and multilocus variable-number tandem-repeat analyses (MLVA) were used to screen 34 VCO1 isolates from the southern Vietnam 2010 outbreak (23 patients, five contact persons, and six environmental isolates) to determine if it was genetically distinct from 18 isolates from outbreaks in southern Vietnam from 1999 to 2004, and two isolates from northern Vietnam (2008). Twenty-seven MLVA types and seven PFGE patterns were identified. Both analyses showed that the 2008 and 2010 isolates were distinctly clustered and separated from the 1999-2004 isolates.

N1s and O1s double ionization of the NO and N2O molecules.

Single-site N1s and O1s double core ionisation of the NO and N2O molecules has been studied using a magnetic bottle many-electron coincidence time-of-flight spectrometer at photon energies of 1100 eV and 1300 eV. The double core hole energies obtained for NO are 904.8 eV (N1s(-2)) and 1179.4 eV (O1s(-2)). The corresponding energies obtained for N2O are 896.9 eV (terminal N1s(-2)), 906.5 eV (central N1s(-2)), and 1174.1 eV (O1s(-2)). The ratio between the double and single ionisation energies are in all cases close or equal to 2.20. Large chemical shifts are observed in some cases which suggest that reorganisation of the electrons upon the double ionization is significant. Δ-self-consistent field and complete active space self-consistent field (CASSCF) calculations were performed for both molecules and they are in good agreement with these results. Auger spectra of N2O, associated with the decay of the terminal and central N1s(-2) as well as with the O1s(-2) dicationic states, were extracted showing the two electrons emitted as a result of filling the double core holes. The spectra, which are interpreted using CASSCF and complete active space configuration interaction calculations, show atomic-like character. The cross section ratio between double and single core hole creation was estimated as 1.6 × 10(-3) for nitrogen at 1100 eV and as 1.3 × 10(-3) for oxygen at 1300 eV.

Alterations in PNA binding of keratinocytes in oral keratosis.

Changes in the expression of peanut lectin (PNA) were examined in keratinocytes of oral keratosis showing a mixture of hyperortho- and hyperparakeratinized epithelium. In the hyperorthokeratinized epithelium, which was reacted with anti-filaggrin antibody in both granular and cornified cells, PNA bound to the surface of keratinocytes from the spinous layer to the granular layer. Neither anti-filaggrin nor PNA reactions were detected in keratinocytes of the hyperparakeratinized epithelium. After neuraminidase pretreatment, however, PNA staining appeared in all cells, except cornified cells, of both hyperortho- and hyperparakeratinized epithelia. These findings suggest that PNA-binding epitopes in keratinocytes were modified by sialic acid during the hyperparakeratotic process of oral keratosis.

Double core hole creation and subsequent Auger decay in NH3 and CH4 molecules.

Energies of the hollow molecules CH(4)(2+) and NH(3)(2+) with double vacancies in the 1s shells have been measured using an efficient coincidence technique combined with synchrotron radiation. The energies of these states have been determined accurately by high level electronic structure calculations and can be well understood on the basis of a simple theoretical model. Their major decay pathway, successive Auger emissions, leads first to a new form of triply charged ion with a core hole and two valence vacancies; experimental evidence for such a state is presented with its theoretical interpretation. Preedge 2-hole-1-particle (2h-1p) states at energies below the double core-hole states are located in the same experiments and their decay pathways are also identified.

Plaque characterisation by Virtual Histology intravascular ultrasound analysis in patients with type 2 diabetes.

OBJECTIVES: To evaluate the in-vivo plaque composition and characteristics in patients with type 2 diabetes mellitus (DM) using Virtual Histology intravascular ultrasound (VH IVUS). METHODS: In 90 patients with stable angina pectoris, de novo target vessels were studied and plaque components were analysed. Patients were divided into two groups: a diabetic group (36 vessels) and a non-diabetic group (54 vessels). RESULTS: The percentage area of necrotic core and dense calcium were significantly larger in the DM group than the non-DM group (necrotic core: 11.0% (interquartile range (IQR): 7.2-15.2%) vs 7.6% (IQR 5.6-13.2%), p = 0.03; dense calcium: 5.6% (IQR: 2.3-7.3%) vs 2.9% (IQR: 1.7-4.9%), p = 0.01). The DM group presented with a significantly higher presence of at least one VH IVUS-derived thin-cap fibroatheroma (VHD-TCFA) (75% vs 41%, p = 0.001) and VH IVUS-derived fibrocalcific atheroma (VHD-FCA) (75% vs 40%, p = 0.001). In the DM group, 53% of the vessels had both VHD-TCFA and VHD-FCA, which was significantly higher than non-DM group (17%, p = 0.0004). CONCLUSIONS: Coronary plaque characteristics in DM patients showed an increased amount of dense calcium and necrotic core, as well as a higher frequency of VHD-TCFA and VHD-FCA. Atherosclerosis of the target vessel was more advanced in diabetic patients.

C1s and O1s photoelectron satellite spectra of CO with symmetry-dependent vibrational excitations.

The photoelectron shake-up satellite spectra that accompany the C1s and O1s main lines of carbon monoxide have been studied by a combination of high-resolution x-ray photoelectron spectroscopy and accurate ab initio calculations. The symmetry-adapted cluster-expansion configuration-interaction general-R method satisfactorily reproduces the satellite spectra over a wide energy region, and the quantitative assignments are proposed for the 16 and 12 satellite bands for C1s and O1s spectra, respectively. Satellite peaks above the pi(-1)pi(*) transitions are mainly assigned to the Rydberg excitations accompanying the inner-shell ionization. Many shake-up states, which interact strongly with three-electron processes such as pi(-2)pi(*2) and n(-2)pi(*2), are calculated in the low-energy region, while the continuous Rydberg excitations are obtained with small intensities in the higher-energy region. The vibrational structures of low-lying shake-up states have been examined for both C1s and O1s ionizations. The vibrational structures appear in the low-lying C1s satellite states, and the symmetry-dependent angular distributions for the satellite emission have enabled the Sigma and Pi symmetries to be resolved. On the other hand, the potential curves of the low-lying O1s shake-up states are predicted to be weakly bound or repulsive.

Symmetry-dependent vibrational excitation in N 1s photoionization of N2: experiment and theory.

We have measured the vibrational structures of the N 1s photoelectron mainline and satellites of the gaseous N2 molecule with the resolution better than 75 meV. The gerade and ungerade symmetries of the core-ionized (mainline) states are resolved energetically, and symmetry-dependent angular distributions for the satellite emission allow us to resolve the Sigma and Pi symmetries of the shake-up (satellite) states. Symmetry-adapted cluster-expansion configuration-interaction calculations of the potential energy curves for the mainline and satellite states along with a Franck-Condon analysis well reproduce the observed vibrational excitation of the bands, illustrating that the theoretical calculations well predict the symmetry-dependent geometry relaxation effects. The energies of both mainline states and satellite states, as well as the splitting between the mainline gerade and ungerade states, are also well reproduced by the calculation: the splitting between the satellite gerade and ungerade states is calculated to be smaller than the experimental detection limit.

Theoretical studies on magnetic circular dichroism by the finite perturbation method with relativistic corrections.

A theoretical method for calculating magnetic circular dichroism (MCD) of molecules is presented. We examined the numerical accuracy and the stability of the finite perturbation (FP) method and the sum-over-state (SOS) perturbation method. The relativistic effects are shown to be important for the MCD spectra of molecules containing heavy elements. Calculations using the FP and the SOS methods were carried out for ethylene, para- and ortho-benzoquinone, showing that the FP method is superior to the SOS method, as expected. The relativistic effect was examined using the second-order Douglas-Kroll Hamiltonians for the halogen molecules F2, Cl2, Br2, and I2. The Faraday terms of I2 and Br2 were strongly affected by the relativistic effects, while the effect was negligible for Cl2 and F2.

Drug susceptibility and its genetic basis in epidemic Vibrio cholerae O1 in Vietnam.

The drug susceptibility and genes responsible for the drug resistance of Vibrio cholerae O1 isolated in Vietnam in 1995, 2000 and 2002 were studied. The strains isolated in 1995 were resistant to streptomycin and harboured the class I integron which contained the aadA1 gene responsible for streptomycin resistance. The strains isolated in 2000 were devoid of a class I integron but were multiple-drug resistant and harboured SXT constin, with several drug-resistant genes. The genes responsible for streptomycin resistance were strA and strB. The strains isolated in 2002 were sensitive to all drugs examined, and the organisms were devoid of both class I integron and SXT constin. Cholera outbreaks in the three periods examined (1995, 2000 and 2002) were apparently due to different categories of V. cholerae O1.

[The characterization of Vibrio cholerae non-O1 strain causing perforative pan-peritonitis].

We report a case of a 53-year-old male with Vibrio cholerae non-O1 (serotype O19) infection, resulting in perforative pan-peritonitis. The patient had a history of gastric cancer and a gastrectomy was performed one year prior. The patient had previously been admitted with nausea and vomiting and was diagnosed with a sub-ileus condition. He was provisionally discharged when his condition improved and during that period he ate raw fish caught locally in Nagasaki Prefecture, and several hours later he experienced a sudden onset of severe abdominal pain and nausea and on diagnosis of pan-peritonitis an emergency resection of the transverse colon was performed. We subsequently isolated Vibrio cholerae non-O1 from the patient's peritoneal fluid and stool. He died of multiple organ failure three weeks later despite intensive chemotherapeutic care and treatment for shock and disseminated intravascular coagulation. The strain of Vibrio cholerae non-O1 isolated was non-toxigenic but hemolytic with hyper-producing of metalloprotease.

Distribution of cognates of group II introns detected in mitochondrial cox1 genes of a diatom and a haptophyte.

We identified group IIA introns that contain an open reading frame (ORF) in the mitochondrial cytochrome oxidase subunit I (cox1) genes of yellow algae, a diatom Thalassiosira (Th.) nordenskioeldii CCMP 992 collected from the east coast of USA, and a haptophyte Pavlova (Pa.) lutheri CCMP 1325 collected from Finland. Cognate introns of CCMP 1325 were detected in all Pa. lutheri strains investigated, which were collected from various oceans. In contrast, the intron was absent from closely related species belonging to the same genus Pavlova. This was also the case for the group II intron detected in a diatom Th. nordenskioeldii CCMP 992. The group II intron of CCMP 992 was located at the corresponding site to the group IIA intron found in Pylaiella (synonym, Pilayella) littoralis. The deduced secondary structures of these introns, one of which is from a diatom and the other from a brown alga, were virtually identical. In contrast, the haptophyte group II intron was inserted at a novel locus, and shares no particularly high sequence homology with any intron known to date. The phylogenetic tree based on the intronic ORF domain was not congruent with that based on the cox1 exon. The most prominent property of the intronic ORF tree was that introns located at homologous sites made robust pair clades irrespective of the phylogenetic relationships of the organisms. This suggests that mitochondrial group II introns often invade intronless alleles across the species barrier with site specificity. Homology analysis of the haptophyte intronic ORF suggested that it comprises three domains: reverse transcriptase (RT), RNA maturase (Ma), and H-N-H endonuclease. However, the intronic ORF of the diatom contains the Ma domain but is apparently missing the H-N-H domain, and its RT domain is most probably partly or completely lacking in function.

Development of hyperfimbriated strains of Vibrio cholerae O1.

The Vibrio cholerae O1 and O139 fimbrillin genes (fimA or mshA) were amplified by polymerase chain reaction and cloned into an Escherichia coli pCR vector. These clones were sequenced. The fimA sequences were found to be identical between V cholerae O1 and O139. One of the plasmids was digested with EcoR I and inserted into the EcoR I site of pGEX-3X. The plasmid pVPP thus obtained was transferred into strains of wild-type V cholerae O1 Bgd17 (classical in biotype) and its fimbriated strain by electroporation. The recombinant plasmid pVPP overexpressed mature fimbriae following induction of the tac promoter with isopropyl-beta-D-thiogalactopyranoside. The cloned gene product was purified to homogeneity by sucrose-linear gradient centrifugation (7.8 mg of fimbriae/L-culture). All the properties of the recombinant fimbriae (e.g., subunit structure, hydrophobicity, hemagglutinating activity sensitive to D-mannose and D-glucose and immunogenicity) were identical to those of the wild-type fimbriae. This overexpression system will be extremely useful for rapid, inexpensive preparation of large amounts of fimbriae for vaccine design and development.

Cloning, overexpression and mutagenesis of cDNA encoding dihydrolipoamide succinyltransferase component of the porcine 2-oxoglutarate dehydrogenase complex.

Dihydrolipoamide succinyltransferase (E2o) is the structural and catalytic core of the 2-oxoglutarate dehydrogenase (OGDH) complex. The cDNA encoding porcine E2o (PE2o) has been cloned. The PE2o cDNA spans 2547 bases encoding a presequence (68 amino-acid residues) and a mature protein (387 residues, Mr = 41 534). Recombinant porcine E2o (rPE2o) (residues 1-387), C- and N-terminal truncated PE2os, and site-directed mutant PE2os were overexpressed in Escherichia coli via the expression vector pET-11d and purified. The succinyltransferase activity of the rPE2o was about 2.2-fold higher than that of the native PE2o. Electron micrographs of the rPE2o negatively stained showed a cube-like structure very similar to that of the native PE2o. Deletion of five amino-acid residues from the C-terminus resulted in a complete loss of both enzymatic activity and formation of the cube-like structure, but the deletion of only the last two residues had no effect on either function, suggesting the important roles of the C-terminal leucine triplet (Leu383-384-385). Substitution of Ser306 with Ala, and Asp362 with Asn, Glu or Ala in the putative active site, and Leu383-384-385 with Ala or Asp abolished both functions. Substitution of His358 with Cys resulted in an 8.5-fold reduction in kcat, with little change in Km values for dihydrolipoamide and succinyl-CoA. However, self-assembly was not affected. These data indicate that Ser306, Asp362 and the Leu383-384-385 triplet are important residues in both the self-assembly and catalytic mechanism of PE2o.

Human anti-murine antibodies interfere with CPR assays performed with commercial kits.

Quantitation of C-peptide is important for the assessment of insulin secretion, in particular in patients receiving insulin therapy. Since the CPR levels become much higher than the concentration of C-peptide for several reasons, such as the high concentration of proinsulin, CPR values sometimes need to be assessed carefully. We have had two diabetic patients whose CPR values were abnormally high when determined with a Daiichi C-peptide kit III (method 1). CPR values determined by other methods were from two to ten times lower, indicating considerable interference when method 1 was used. Since method 1 uses mouse monoclonal antibodies (mmab) for detection antibodies, we suspected that human anti-murine antibodies (HAMA) were responsible for the interference. HAMA were detected in serum from both patients (45 and 460 ng/ml in case 1 and case 2 (at peak), respectively). Removal of HAMA from serum eliminated the interference. Modification of method 1 to exclude mmab from the assay system removed all interference. HAMA were, therefore, considered to be the cause of the interference. In case 2, the peak concentration of HAMA was recorded 16 months earlier than the maximum of interference. Further analysis revealed that HAMA with high affinities were responsible for the interference.

Phylogenetic analysis of diatom coxI genes and implications of a fluctuating GC content on mitochondrial genetic code evolution.

In order to address the relationships among diatom groups and to investigate possible changes in their mitochondrial (mt) genetic codes, we have analyzed a 1.1-kb region of the cytochrome c oxidase subunit I (coxI) gene from eight diverse diatom species. A phylogenetic analysis of these coxI sequences including representative species of the Phaeophyta, Xanthophyta, Eustigmatophyta and Haptophyta showed that the diatoms (Bacillariophyta) formed a well-supported monophyletic group. Of the eight species investigated, four have been classified together as radial centric diatoms based on morphology. However, in our coxI tree, the two radial centrics belonging to the order Thlassiosirales (Skeletonema costatum and Thalassiosira nordenskioldii) were placed as the sister group to the multipolar centric diatoms, while the other two radial centrics (Melosira ambigua and Rhizosolenia setigera) were in another clade. Also, in two species of the Tharassiosirales we found UGA codons that occur at conserved tryptophan (Trp) sites in the coxI sequences, strongly indicating that UGA codes for Trp in these diatoms. No evidence of a deviant genetic code was detected in the other analyzed diatom species. There was no apparent relationship between the nucleotide third-position GC content of mtDNA (based on the sequenced coxI region) and the presence of a deviant genetic code.

Directionally evolving genetic code: the UGA codon from stop to tryptophan in mitochondria.

For the comprehensive analyses of deviant codes in protistan mitochondria (mt), we sequenced about a 1.1-kb region of a mitochondrial (mt) gene, the cytochrome c oxidase subunit I (coxI) in two chlorarachniophytes, the filose amoeba Euglypha rotunda, the cryptomonad Cryptomonas ovata, the prymnesiophyte (haptophyte) Diacronema vlkianum (Pavlovales), and the diatom Melosira ambigua. As a result of this analysis, we noticed that the UGA codon is assigned to tryptophan (Trp) instead of being a signal for translational termination in two chlorarachniophytes and in E. rotunda. The same type of deviant code was reported previously in animals, fungi, ciliates, kinetoplastids, Chondrus crispus (a red alga), Acanthamoeba castellanii (an amoeboid protozoon), and three of the four prymnesiophyte orders with the exception of the Pavlovales. A phylogenetic analysis based on the COXI sequences of 56 eukaryotes indicated that the organisms bearing the modified code, UGA for Trp, are not monophyletic. Based on these studies, we propose that the ancestral mitochondrion was bearing the universal genetic code and subsequently reassigned the codon to Trp independently, at least in the lineage of ciliates, kinetoplastids, rhodophytes, prymnesiophytes, and fungi. We also discuss how this codon was directionally captured by Trp tRNA.

Distinctive origins of group I introns found in the COXI genes of three gree algae.

Upon surveying the cytochrome c oxidase subunit I (COXI) gene of green algae, we found group I introns in three species of algae, Chlorella vulgaris (Cv), Scenedesmus quadricauda (Sq) and Protosiphon botryoides (Pb). The comparative analysis of these nucleotide sequences and their secondary structures revealed that the introns of Cv, Sq, and Pb belong to groups IB1, ID, and IB2, respectively. Each of the three introns contained an open reading frame (ORF) that showed a similarity to the sequence of the LAGLIDADG endonuclease family. However, each of the intronic ORFs in Sq and Pb had a discontinuity in the middle of' the sequences coding for the LAGLIDADG endonuclease. Either of the two ORFs could be restored to a sequence homologous to the LAGLIDADG endonuclease by the insertion of a nucleotide in the appropriate position. In Sq, a putative pseudo-knot structure was detected in the intronic ORF This suggests the occurrence of a ribosomal frameshift in the translation of the ORF. because such pseudo-knot structures are common in viral ORFs employing a (-1) ribosomal frameshift. In the phylogenetic tree that was inferred from the amino acid sequences of algal and non-algal intronic ORFs, the three algal ORFs did not make a cluster, but were scattered throughout the tree. In addition. each of the three algal ORFs showed a close relationship to the ORFs of non-algal introns that were inserted at the corresponding site of the COX] gene, suggesting distinctive origins of the three algal introns via independent horizontal transfers.

Induction of fimbriated Vibrio cholerae O139.

Several fimbriated phases of Vibrio cholerae O139 strains were selectively induced and compared immunologically and biochemically with those of V. cholerae O1. Fimbrial antigens were detected on the surfaces of vibrio cells colonizing the epithelial cells of a rabbit small intestine. Convalescent-phase sera from six individuals infected with V. cholerae O139 revealed the development of antibody against the fimbrillin. These findings suggest that the fimbriae of V. cholerae O1 and O139 are expressed in vivo during infection and that consideration must be given to the use of fimbrial antigens as components of vaccines against cholera.