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Biomedical subjects

W F Vann

Publications and source records attributed to W F Vann.

At least 37 records · Page 2Linked to original sources
PubMed1995

Nucleotide sequence and genetic analysis of the neuD and neuB genes in region 2 of the polysialic acid gene cluster of Escherichia coli K1.

The K1 capsular polysaccharide, a polymer of sialic acid, is an important virulence determinant of extraintestinal pathogenic Escherichia coli. The genes responsible for the synthesis and expression of the polysialic acid capsule of E. coli K1 are located on the 17-kb kps gene cluster, which is functionally divided into three regions. Central region 2 encodes proteins necessary for the synthesis, activation, and polymerization of sialic acid, while flanking regions 1 and 3 are involved in polymer transport to the cell surface. In this study, we identified two genes at the proximal end of region 2, neuD and neuB, which encode proteins with predicted sizes of 22.7 and 38.7 kDa, respectively. Several observations suggest that the neuB gene encodes sialic acid synthase. EV24, a neuB chromosomal mutant that expresses a capsule when provided exogenous sialic acid, could be complemented in trans by the cloned neuB gene. In addition, NeuB has significant sequence similarity to the product of the cpsB gene of Neisseria meningitidis group B, which is postulated to encode sialic acid synthase. We also present data indicating that neuD has an essential role in K1 polymer production. Cells harboring pSR426, which contains all of region 2 but lacks region 1 and 3 genes, produce an intracellular polymer. In contrast, no polymer accumulated in cells carrying a derivative of pSR426 lacking a functional neuD gene. Unlike strains with mutations in neuB, however, neuD mutants are not complemented by exogenous sialic acid, suggesting that NeuD is not involved in sialic acid synthesis. Additionally, cells harboring a mutation in neuD accumulated sialic acid and CMP-sialic acid. We also found no significant differences between the endogenous and exogenous sialyltransferase activities of a neuD mutant and the wild-type organism. NeuD shows significant similarity to a family of bacterial acetyltransferases, leading to the theory that NeuD is an acetyltransferase which may exert its influences through modification of other region 2 proteins.

Acetyltransferases↗
PubMed1995

Restoration of primary molars using a new "compomer" material.

The primary advantages of glass ionomer restorative materials are adhesion to tooth structure and release of fluoride. A new material has been recently introduced for the restoration of cervical lesions and Class I defects in primary teeth. The material combines the properties of glass ionomers with the advantages of a single-component, light-activated composite resin and is known as a "compomer". This new material contains two resins, instead of the acidic polymers found in conventional glass ionomers. The restorative material releases fluoride and can be infected directly into the cavity. With its own bonding agent, the compomer offers a simple and relatively rapid technique. The learning objective of this paper is to describe the utilization of the new restorative material in conjunction with a bonding agent to restore conservative cavity preparations in primary molars. Two clinical cases are used to illustrate the presentation.

Child↗
PubMed1994

Molecular cloning and analysis of genes for sialic acid synthesis in Neisseria meningitidis group B and purification of the meningococcal CMP-NeuNAc synthetase enzyme.

The gene encoding for the CMP-NeuNAc synthetase enzyme of Neisseria meningitidis group B was cloned by complementation of a mutant of Escherichia coli defective for this enzyme. The gene (neuA) was isolated on a 4.1-kb fragment of meningococcal chromosomal DNA. Determination of the nucleotide sequence of this fragment revealed the presence of three genes, termed neuA, neuB, and neuC, organized in a single operon. The presence of a truncated ctrA gene at one end of the cloned DNA and a truncated gene encoding for the meningococcal sialyltransferase at the other confirmed that the cloned DNA corresponded to region A and part of region C of the meningococcal capsule gene cluster. The predicted amino acid sequence of the meningococcal NeuA protein was 57% homologous to that of NeuA, the CMP-NeuNAc synthetase encoded by E. coli K1. The predicted molecular mass of meningococcal NeuA protein was 24.8 kDa, which was 6 kDa larger than that formerly predicted (U. Edwards and M. Frosch, FEMS Microbiol. Lett. 96:161-166, 1992). Purification of the recombinant meningococcal NeuA protein together with determination of the N-terminal amino acid sequence confirmed that this 24.8-kDa protein was indeed the meningococcal CMP-NeuNAc synthetase. The predicted amino acid sequences of the two other encoded proteins were homologous to those of the NeuC and NeuB proteins of E. coli K1, two proteins involved in the synthesis of NeuNAc. These results indicate that common steps exist in the biosynthesis of NeuNAc in these two microorganisms.

Amino Acid Sequence↗
PubMed1993

The role of cysteine residues 129 and 329 in Escherichia coli K1 CMP-NeuAc synthase.

N-Acetylneuraminic acid cytidyltransferase (CMP-NeuAc synthase) of Escherichia coli K1 is sensitive to mercurials and has cysteine residues only at positions 129 and 329. The role of these residues in the catalytic activity and structure of the protein has been investigated by site-directed mutagenesis and chemical modification. The enzyme is inactivated by the thiol-specific reagent dithiodipyridine. Inactivation by this reagent is decreased in the presence of the nucleotide substrate CTP, suggesting that a thiol residue is at or near the active site. Site-directed mutagenesis of either residue Cys-129 to serine or Cys-329 to selected amino acids has minor effects on the specific activity of the enzyme, suggesting that cysteine is not essential for catalysis and that a disulphide bond is not an essential structural component. The limited reactivity of the enzyme to other thiol-blocking reagents suggests that its cysteine residues are partially exposed. The accessibility and role of the cysteine residues in enzyme structure were investigated by fluorescence, c.d. and denaturation studies of wild-type and mutant enzymes. The mutation of Cys-129 to serine makes the enzyme more sensitive to heat and chemical denaturation, but does not cause gross changes in the protein structure as judged by the c.d. spectrum. The mutant containing Ser-129 instead of Cys-129 had a complex denaturation pathway similar to that of wild-type E. coli K1 CMP-NeuAc synthase consisting of several partially denatured states. Cys-329 reacts more readily with N-[14C]ethylmaleimide when the enzyme is in a heat-induced relaxed state. Cys-129 is less reactive and is probably a buried residue.

Base Sequence↗
PubMed1992

13C NMR investigation of the anomeric specificity of CMP-N-acetylneuraminic acid synthetase from Escherichia coli.

The anomeric specificity of Escherichia coli CMP-N-acetylneuraminic acid (CMP-NeuAc) synthetase was investigated by NMR using 13C-labeled N-acetylneuraminic acid (NeuAc). Consumption of the beta-anomer of [2-13C]N-acetylneuraminic acid was observed upon addition of enzyme, with a concomitant appearance of an anomeric resonance for CMP-N-acetylneuraminic acid. Inhibition by substrate analogues the anomeric oxygen was determined in a similar manner using [2-13C,(50 atom %)18O]N-acetylneuraminic acid. An upfield shift of 1.5 Hz in the anomeric resonance of both the [13C]NeuAc substrate and CMP-[13C]NeuAc product was observed due to the 18O substitution. This result implies conservation of the NeuAc oxygen. Results of steady-state kinetic analysis suggest a sequential-type mechanism and therefore no covalent intermediate. Thus, CMP-beta-NeuAc is probably formed by a direct transfer of the anomeric oxygen of beta-NeuAc to the alpha-phosphate of CTP.

Carbohydrate Conformation↗
PubMed1992

Two natal maxillary molars. Report of a case.

A 20-day-old black male infant with two rare posterior natal teeth was referred for examination and care. Oral radiographs suggested that the teeth were the right and left maxillary primary molars. The hypermobile teeth and associated suspicious soft tissue masses were removed. Histologic examination revealed irregular tooth structures and infiltrate consistent with inflamed dental follicles.

Humans↗
PubMed1992

Identification of variable region differences in Neisseria meningitidis class 3 protein sequences among five group B serotypes.

Strains of Neisseria meningitidis express one of two porin proteins. These porins have been identified as the class 2 and class 3 proteins, and express serotype-specific epitopes. The gene for the class 3 protein was amplified by the polymerase chain reaction from the DNA of a serotype 4 strain as a 1025 bp fragment. The nucleotide sequence of this gene was determined and compared with two recently published sequences. On the basis of this comparison we have identified two major variable regions in the translated protein sequence, VR1 and VR2, that may be associated with serotype specificity. Three other variable regions were also identified. The sequences in the VR1 and VR2 regions from five additional group B N. meningitidis strains of serotypes 1, 4, 8, 12, and 15, all expressing class 3 proteins, were determined. The VR1 and VR2 regions were variable and were flanked by highly conserved regions among eight different class 3 sequences. These two variable regions of 15 and 9 amino acids are predicted to be in surface-exposed loops.

Amino Acid Sequence↗
PubMed1992

Sequence and expression of the Escherichia coli K1 neuC gene product.

The nucleotide sequence of the neuC gene of the Escherichia coli K1 capsule gene cluster encodes a protein with a predicted molecular weight of 44,210 containing 391 amino acids. A chimeric protein with beta-galactosidase fused to the carboxy terminus of the neuC gene product (P7) was constructed and purified. Its amino-terminal sequence confirmed the prediction from the nucleotide sequence that the neuC gene overlaps the distal end of the neuA gene by a single base pair. Both the neuA and neuC genes are coexpressed under the control of a single upstream T7 or tac promoter, suggesting that neuA and neuC are part of an operon.

Amino Acid Sequence↗
PubMed1991

Prevention of nonspecific binding of immunoglobulin to Staphylococcus aureus protein A in ELISA assays.

The Fc region of IgG of most mammals binds protein A on S. aureus resulting in high backgrounds when measuring specific antibodies to S. aureus in the ELISA. Removal of protein A from S. aureus or modification of the Ig Fc to prevent binding to protein A could affect specific antibody binding. We compared effects of blockage of Fc binding to protein A with purified protein A to trypsin removal of protein A from S. aureus, on specific antibody binding. When NMS was incubated without and with protein A (0 microgram, 50 micrograms, 200 micrograms and 400 micrograms) and high protein A Cowan I was the bound S. aureus antigen in the ELISA, absorbance OD405 was 0.769, 0.240, 0.224 and 0.210 +/- SE 0.026. When mouse Mab (IgG1, kappa) to bovine IgA was incubated without and with protein A (400 micrograms) prior to reaction with bovine IgA in the ELISA, absorbance was 0.645 and 0.639, indicating protein A had no effect on specific antibody binding. To determine the effect of trypsin on specific binding, Becker S. aureus was trypsin treated before linking it to microtiter wells. When Mab (IgM) to Becker (Nelles et al., Infect. Immun. (1985) 49, 14) was incubated with protein A (400 micrograms) before use in the ELISA, trypsin treatment of Becker resulted in reduced specific antibody activity (untreated Becker = 1.306, trypsin treated Becker = 0.331). These results suggest that purified protein A can be used to block nonspecific binding via Fc of Ig to S. aureus, thus avoiding trypsin denaturation of surface antigens.

Animals↗
PubMed1991

Identity between polysaccharide antigens of Moraxella nonliquefaciens, group B Neisseria meningitidis, and Escherichia coli K1 (non-O acetylated).

A surface polysaccharide antigen of Moraxella nonliquefaciens, reported to be cross-reactive with the capsular polysaccharides of group B Neisseria meningitidis and Escherichia coli K1 (K. Błvre, K. Bryn, O. Closs, N. Hagen, and L. O. Froholm, NIPH Ann. 6:65-73, 1983), was isolated, purified, and characterized chemically, immunologically, and by nuclear magnetic resonance. This polysaccharide was shown to be a linear homopolymer of alpha (2----8)-linked N-acetylneuraminic acid, identical to the capsular polysaccharide of group B N. meningitidis and O-acetyl-negative variants of E. coli K1.

Animals↗
PubMed1990

Structure of the type 5 capsular polysaccharide of Staphylococcus aureus.

The Staphylococcus aureus type 5 capsular polysaccharide is composed of 2-acetamido-2-deoxy-L-fucose (1 part), 2-acetamido-2-deoxy-D-fucose (1 part), and 2-acetamido-2-deoxy-D-mannuronic acid (1 part). On the basis of methylation analysis, optical rotation, high-field one- and two-dimensional 1H- and 13C-n.m.r. experiments, and selective cleavage with 70% aqueous hydrogen fluoride, the polysaccharide was found to be a partially O-acetylated (50%) polymer of the repeating trisaccharide unit, [----4)-3-O-Ac-beta-D-ManpNAcA-(1----4)-a-L-FucpNAc-(1----3) -beta-D-FucpNAc-(1----]n.

Carbohydrate Sequence↗
PubMed1990

Serum antibody response in adult volunteers elicited by injection of Streptococcus pneumoniae type 12F polysaccharide alone or conjugated to diphtheria toxoid.

Conjugates of an uronic acid-containing capsular polysaccharide (CP), pneumococcous type 12F (Pn12F) bound to diphtheria toxoid (DT), were studied for safety and immunogenicity in adult volunteers. In mice, these conjugates, prepared with the same lot of DT and Pn12F-40234-006, a homogenous CP of high molecular weight, or Pn12-812408, a polydisperse CP with lower-molecular-weight material, were more immunogenic than the Pn12F alone and had T-cell dependent properties (A. Fattom, W. F. Vann, S.C. Szu, A. Sutton, X. Li, B. Bryla, G. Schiffman, J. B. Robbins, and R. Schneerson, Infect. Immun. 56:2292-2298, 1988). Adult volunteers, randomized into three groups, were injected either with one of these two conjugates or with Pnu-Imune, the 23 valent pneumococcus vaccine containing 25 micrograms of Pn12F as one of its components. Volunteers were injected two times, 4 weeks apart, with the Pn12F-DT conjugates and once with the Pnu-Imune. Side reactions following injection of the conjugates of Pnu-Imune were mild and short-lived. At 4 weeks and at 7 months after the first injection, higher levels of Pn12F antibodies were found in the volunteers injected with the conjugates than in the Pnu-Imune group (P less than 0.001). The conjugate prepared with the higher-molecular-weight Pn12F elicited higher levels of antibodies than the conjugate prepared with a lower-molecular-weight Pn12F preparation (P = 0.05). Both conjugates elicited about a 13-fold rise in DT antibodies.

Adolescent↗
PubMed1990

Synthesis and immunologic properties in mice of vaccines composed of Staphylococcus aureus type 5 and type 8 capsular polysaccharides conjugated to Pseudomonas aeruginosa exotoxin A.

Epidemiological, serological and in vitro phagocytosis experiments provide evidence that the newly discovered type 5 and type 8 capsular polysaccharides (CPs) are both virulence factors and protective antigens for bacteremia caused by Staphylococcus aureus. Neither type 5 nor type 8 CP elicited serum antibodies when injected into mice. These two CPs were bound to Pseudomonas aeruginosa exotoxin A (ETA) to form conjugates by using the synthetic scheme devised for the CP (Vi) of Salmonella typhi and of pneumococcus type 12F (A. Fattom, W. F. Vann, S. C. Szu, A. Sutton, X. Li, D. Bryla, G. Schiffman, J. B. Robbins, and R. Schneerson, Infect. Immun. 56:2292-2298, 1988; S. C. Szu, A. L. Stone, J. D. Robbins, R. Schneerson, and J. B. Robbins, J. Exp. Med. 166:1510-1524, 1987). Both S. aureus CP-ETA conjugates elicited a rise in CP antibodies. As components of conjugates, both S. aureus CPs acquired T-cell-dependent properties, as shown by their ability to respond to carrier priming and to stimulate booster responses. The conjugate-induced antibodies facilitated type-specific opsonization of S. aureus by human polymorphonuclear leukocytes. The conjugates also induced ETA antibodies which neutralized the native toxin in vitro. Clinical studies of these two conjugates for active or passive immunization of patients at risk for S. aureus bacteremia are planned.

ADP Ribose Transferases↗
PubMed1989

Sequence of the cloned Escherichia coli K1 CMP-N-acetylneuraminic acid synthetase gene.

The Escherichia coli CMP-N-acetylneuraminic acid (CMP-NeuAc) synthetase gene is located on a 3.3-kilobase (kb) HindIII fragment of the plasmid pSR23 which contains the genes for K1 capsule production (Vann, W. F., Silver, R. P., Abeijon, C., Chang, K., Aaronson, W., Sutton, A., Finn, C. W., Lindner, W., and Kotsatos, M. (1987) J. Biol. Chem. 262, 17556-17562). The CMP-NeuAc synthetase gene expression was increased 10-30-fold by cloning of a 2.7-kb EcoRI-HindIII fragment onto the vector pKK223-3 containing the tac promoter. The complete nucleotide sequence of the gene encoding CMP-NeuAc synthetase was determined from progressive deletions generated by selective digestion of M13 clones containing the 2.7-kb fragment. CMP-NeuAc synthetase is located near the EcoRI site on this fragment as indicated by the detection of an open reading frame encoding a 49,000-dalton polypeptide. The amino- and carboxyl-terminal sequences of the encoded protein were confirmed by sequencing of peptides cleaved from both ends of the purified enzyme. The nucleotide deduced amino acid sequence was confirmed by sequencing several tryptic peptides of purified enzyme. The molecular weight is consistent with that determined from sodium dodecyl sulfate-gel electrophoresis. Gel filtration and ultracentrifugation experiments under nondenaturing conditions suggest that the enzyme is active as a 49,000-dalton monomer but may form aggregates.

Amino Acid Sequence↗
PubMed1989

An investigation of capnography and pulse oximetry as monitors of pediatric patients sedated for dental treatment.

Traditional methods of monitoring sedated pediatric dental patients have major shortcomings. This study evaluated the use of capnography in conjunction with pulse oximetry for monitoring children during conscious sedation for dental treatment. The specific purposes of the study were to determine if capnography would: (1) detect ventilatory changes that subsequently cause an oxyhemoglobin desaturation as detected by pulse oximetry; and (2) detect an airway obstruction. Ten pediatric dental patients (mean age 2 years, 10 months) were sedated with 75 mg/kg of chloral hydrate in strict accordance with the Guidelines for the Elective Use of Conscious Sedation, Deep Sedation, and General Anesthesia in Pediatric Patients of the American Academy of Pediatric Dentistry and the American Academy of Pediatrics (1985). All patients were monitored continuously using both capnography and pulse oximetry. Analysis of data obtained using these monitors revealed that specific end-tidal CO2 values were not predictive for subsequent oxyhemoglobin desaturations and that capnography was very accurate in detecting complete obstruction of the airway. Pulse oximetry revealed that all patients had mild oxyhemoglobin desaturations and that 50% had moderate desaturations.

Anesthesia, Dental↗
PubMed1989

Screening panoramic radiographs in children: prevalence data and implications.

The purpose of this paper was to review the rationale for the radiographic screening of asymptomatic pediatric patients and to report the prevalence of selected pathologic and developmental conditions using panoramic radiographs. Three observers participated in this retrospective study that utilized panoramic radiographs from 849 subjects, aged 3-9 years, chosen randomly from the School of Dentistry treatment records of the University of North Carolina at Chapel Hill. Findings indicated that 2.4% of the subjects had supernumerary teeth, 7.8% were missing permanent teeth, 9.1% had ectopic eruption, 0.1% had radiolucencies of the jaws, and 0.1% had radiopacities of the jaws. These prevalences are discussed in light of recent evidence concerning the risk/benefit ratio of the panoramic radiograph. We conclude that the panoramic radiograph is a poor projection for screening the dental needs of asymptomatic healthy children; alternative screening protocols should be examined.

Child↗