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Biomedical subjects

W F Vann

Publications and source records attributed to W F Vann.

At least 19 recordsLinked to original sources
PubMed2005

Addressing selection bias in dental health services research.

When randomization is not possible, researchers must control for non-random assignment to experimental groups. One technique for statistical adjustment for non-random assignment is through the use of a two-stage analytical technique. The purpose of this study was to demonstrate the use of this technique to control for selection bias in examining the effects of the The Supplemental Program for Women, Infants, and Children's (WIC) on dental visits. From 5 data sources, an analysis file was constructed for 49,512 children ages 1-5 years. The two-stage technique was used to control for selection bias in WIC participation, the potentially endogenous variable. Specification tests showed that WIC participation was not random and that selection bias was present. The effects of the WIC on dental use differed by 36% after adjustment for selection bias by means of the two-stage technique. This technique can be used to control for potential selection bias in dental research when randomization is not possible.

Adult↗
PubMed2001

Cloning and expression of human sialic acid pathway genes to generate CMP-sialic acids in insect cells.

The addition of sialic acid residues to glycoproteins can affect important protein properties including biological activity and in vivo circulatory half-life. For sialylation to occur, the donor sugar nucleotide cytidine monophospho-sialic acid (CMP-SA) must be generated and enzymatically transferred to an acceptor oligosaccharide. However, examination of insect cells grown in serum-free medium revealed negligible native levels of the most common sialic acid nucleotide, CMP-N-acetylneuraminic acid (CMP-Neu5Ac). To increase substrate levels, the enzymes of the metabolic pathway for CMP-SA synthesis have been engineered into insect cells using the baculovirus expression system. In this study, a human CMP-sialic acid synthase cDNA was identified and found to encode a protein with 94% identity to the murine homologue. The human CMP-sialic acid synthase (Cmp-Sas) is ubiquitously expressed in human cells from multiple tissues. When expressed in insect cells using the baculovirus vector, the encoded protein is functional and localizes to the nucleus as in mammalian cells. In addition, co-expression of Cmp-Sas with the recently cloned sialic acid phosphate synthase with N-acetylmannosamine feeding yields intracellular CMP-Neu5Ac levels 30 times higher than those observed in unsupplemented CHO cells. The absence of any one of these three components abolishes CMP-Neu5Ac production in vivo. However, when N-acetylmannosamine feeding is omitted, the sugar nucleotide form of deaminated Neu5Ac, CMP-2-keto-3-deoxy-D-glycero-D-galacto-nononic acid (CMP-KDN), is produced instead, indicating that alternative sialic acid glycoforms may eventually be possible in insect cells. The human CMP-SAS enzyme is also capable of CMP-N-glycolylneuraminic acid (CMP-Neu5Gc) synthesis when provided with the proper substrate. Engineering the CMP-SA metabolic pathway may be beneficial in various cell lines in which CMP-Neu5Ac production limits sialylation of glycoproteins or other glycans.

Amino Acid Sequence↗
PubMed2001

Elongation of alternating alpha 2,8/2,9 polysialic acid by the Escherichia coli K92 polysialyltransferase.

We have chosen E. coli K92, which produces the alternating structure alpha(2-8)neuNAc alpha(2-9)neuNAc as a model system for studying bacterial polysaccharide biosynthesis. We have shown that the polysialyltransferase encoded by the K92 neuS gene can synthesize both alpha(2-8) and alpha(2-9) neuNAc linkages in vivo by 13C-nuclear magnetic resonance analysis of polysaccharide isolated from a heterologous strain containing the K92 neuS gene. The K92 polysialyltransferase is associated with the membrane in lysates of cells harboring the neuS gene in expression vectors. Although the enzyme can transfer sialic acid to the nonreducing end of oligosaccharides with either linkage, it is unable to initiate chain synthesis without exogenously added polysialic acid. Thus, the polysialyltransferase encoded by neuS is not sufficient for de novo synthesis of polysaccharide but requires another membrane component for initiation. The acceptor specificity of this polysialyltransferase was studied using sialic acid oligosaccharides of various structures as exogenous acceptors. The enzyme can transfer to the nonreducing end of all bacteria polysialic acids, but has a definite preference for alpha(2-8) acceptors. Gangliosides containing neuNAc alpha(2-8)neuNAc are elongated, whereas monsialylated gangliosides are not. Disialylgangliosides are better acceptors than short oligosaccharides, suggesting a lipid-linked oligosaccharide may be preferred in the elongation reaction. These studies show that the K92 polysialyltransferase catalyzes an elongation reaction that involves transfer of sialic acid from CMP-sialic acid to the nonreducing end of two different acceptor substrates.

Amino Acid Motifs↗
PubMed2001

Adverse events and outcomes of conscious sedation for pediatric patients: study of an oral sedation regimen.

BACKGROUND: The authors report on adverse events and sedation outcomes for an oral sedation regimen of chloral hydrate, meperidine and hydroxyzine with 100 percent oxygen, or O2, supplementation. METHODS: In a five-year retrospective study, the authors examined 195 records of conscious sedation performed in 111 healthy children aged 24 to 48 months (mean, 47 months). The authors analyzed age, sex, weight, methods of drug delivery, waiting time after drug administration, treatment rendered, treatment time, adverse events, sedation outcomes and the number of visits needed to complete treatment using descriptive statistics, chi 2 tests, t test and analysis of variance. RESULTS: Adverse events--including vomiting, desaturation, prolonged sedation and an apneic event--occurred in 3 percent of all sedations and were minor. Seventy-two percent of sedations had satisfactory behavioral outcomes, 23 percent had unsatisfactory outcomes, and 5 percent of the cases were aborted because of disruptive behavior. Sex was not a significant factor for the success. Patient compliance with drinking medications (P = .013) and a longer waiting time after medication intake (P = .012) yielded better sedation outcomes. CONCLUSIONS: Minimal minor adverse events occurred with this sedation regimen. The success rate was 72 percent. Compliance with taking oral medications and waiting time appeared to be important factors in predicting sedation success. CLINICAL IMPLICATIONS: This oral sedation regimen offers reasonable outcomes with minimal adverse events under a strict protocol and use of O2 supplementation. The results also revealed associations that give guidance for case selection and outcome prediction.

Administration, Oral↗
PubMed2001

Laser Doppler flowmetry for monitoring traumatized teeth.

Laser Doppler Flowmetry (LDF) has been shown to be valuable in monitoring revascularization of immature incisors following severe dental trauma. Several investigators have demonstrated the ability of LDF to record blood flow signals from vital tooth pulps. In this case report, LDF was used for a 7-year-old child patient following a severe luxation of tooth #9. During follow-up examinations the traumatized tooth was unresponsive to traditional vitality testing during the first 6 months; however, LDF indicated that revascularization had occurred much sooner. Until recently, CO2 ice has been the most effective method for sensitivity testing in trauma cases such as presented here. In this case, LDF gave us the assurance that we could defer invasive care during a critical time period when root canal therapy might have been initiated for this child patient.

Child↗
PubMed2001

A cost analysis of treating pediatric dental patients using general anesthesia versus conscious sedation.

The purpose of this pilot study is to report a cost comparison of general anesthesia (GA) versus oral conscious sedation (CS) for pediatric dental patients. The study sample included 22 children whose parents or guardians selected GA care for their child. Selection criteria limited inclusion to healthy children (American Society of Anesthesiologists' classification I) aged 24-60 months. The subjects acted as their own comparison group to an estimation CS model. Models were developed to assess societal costs for treatment under GA and CS. Treatment rendered was equalized using the dental relative based value unit scale.

Anesthesia, Dental↗
PubMed2000

Cloning and expression of the human N-acetylneuraminic acid phosphate synthase gene with 2-keto-3-deoxy-D-glycero- D-galacto-nononic acid biosynthetic ability.

Sialic acids participate in many important biological recognition events, yet eukaryotic sialic acid biosynthetic genes are not well characterized. In this study, we have identified a novel human gene based on homology to the Escherichia coli sialic acid synthase gene (neuB). The human gene is ubiquitously expressed and encodes a 40-kDa enzyme. The gene partially restores sialic acid synthase activity in a neuB-negative mutant of E. coli and results in N-acetylneuraminic acid (Neu5Ac) and 2-keto-3-deoxy-D-glycero-D-galacto-nononic acid (KDN) production in insect cells upon recombinant baculovirus infection. In vitro the human enzyme uses N-acetylmannosamine 6-phosphate and mannose 6-phosphate as substrates to generate phosphorylated forms of Neu5Ac and KDN, respectively, but exhibits much higher activity toward the Neu5Ac phosphate product.

Amino Acid Sequence↗
PubMed1999

Identification of Arg-12 in the active site of Escherichia coli K1 CMP-sialic acid synthetase.

Escherichia coli K1 CMP-sialic acid synthetase catalyses the synthesis of CMP-sialic acid from CTP and sialic acid. The active site of the 418 amino acid E. coli enzyme was localized to its N-terminal half. The bacterial CMP-sialic acid synthetase enzymes have a conserved motif, IAIIPARXXSKGLXXKN, at their N-termini. Several basic residues have been identified at or near the active site of the E. coli enzyme by chemical modification and site-directed mutagenesis. Only one of the lysines in the N-terminal motif, Lys-21, appears to be essential for activity. Mutation of Lys-21 in the N-terminal motif results in an inactive enzyme. Furthermore, Arg-12 of the N-terminal motif appears to be an active-site residue, based on the following evidence. Substituting Arg-12 with glycine or alanine resulted in inactive enzymes, indicating that this residue is required for enzymic activity. The Arg-12-->Lys mutant was partially active, demonstrating that a positive charge is required at this site. Steady-state kinetic analysis reveals changes in k(cat), K(m) and K(s) for CTP, which implicates Arg-12 in catalysis and substrate binding.

Amino Acid Motifs↗
PubMed1999

Purification of CMP-N-acetylneuraminic acid synthetase from bovine anterior pituitary glands.

CMP-beta-N-acetylneuraminic acid (CMP-neuNAc) is the substrate for the sialylation of glycoconjugates by sialyltransferases in microbes and higher eukaryotes. CMP-neuNAc synthetase catalyzes the formation of this substrate, CMP-neuNAc, from CTP and neuNAc. In this report we describe the purification of CMP-neuNAc synthetase from bovine anterior pituitary glands. The enzyme was purified by ion exchange, gel filtration, and affinity chromatography. The protein was homogeneous on SDS-PAGE with a molecular weight of 52 kDa, a subunit size similar to that of the E.coli K1 (48.6 kDa). The identity of the 52 kDa protein band was confirmed by native gel electrophoresis in that the position of the enzyme activity in gel slices coincided with the position of major bands in the stained gel. Photoaffinity labeling with 125I-ASA-CDP ethanolamine resulted in the modification of a 52 kDa polypeptide that was partially protected against modification by the substrate CTP. Enzyme activity in crude fractions could be adsorbed onto an immunoadsorbent prepared from antibody against the purified 52 kDa protein. Taken together these data suggest that the 52 kDa polypeptide purified by this procedure described in this report is indeed CMP-neuNAc synthetase. The active enzyme chromatographed on a gel filtration column at 158 kDa suggesting it exists in its native form as an oligomer.

Animals↗
PubMed1999

Covalent modification of Lys19 in the CTP binding site of cytidine 5'-monophosphate N-acetylneuraminic acid synthetase.

Periodate oxidized CTP (oCTP) was used to investigate the importance of lysine residues in the CTP binding site of the cytidine 5'-monophosphate N-acetylneuraminic acid (CMP-NeuAc) synthetase (EC 2.7.7.43) from Haemophilus ducreyi. The reaction of oCTP with the enzyme follows pseudo-first-order saturation kinetics, giving a maximum rate of inactivation of 0.6 min(-1) and a K(I) of 6.0 mM at pH 7.1. Mass spectrometric analysis of the modified enzyme provided data that was consistent with beta-elimination of triphosphate after the reaction of oCTP with the enzyme. A fully reduced enzyme-oCTP conjugate, retaining the triphosphate moiety, was obtained by inclusion of NaBH3CN in the reaction solution. The beta-elimination product of oCTP reacted several times more rapidly with the enzyme compared to equivalent concentrations of oCTP. This compound also formed a stable reduced morpholino adduct with CMP-NeuAc synthetase when the reaction was conducted in the presence of NaBH3CN, and was found to be a useful lysine modifying reagent. The substrate CTP was capable of protecting the enzyme to a large degree from inactivation by oCTP and its beta-elimination product. Lys19, a residue conserved in CMP-NeuAc synthetases, was identified as being labeled with the beta-elimination product of oCTP.

Binding Sites↗
PubMed1997

Purification and characterization of the Escherichia coli K1 neuB gene product N-acetylneuraminic acid synthetase.

Escherichia coli K1 produces a capsular polysaccharide of alpha(2-8) poly-N-acetylneuraminic acid. This polysaccharide is an essential virulence factor of these neuropathogenic bacteria. The genes necessary for the synthesis of neuNAc were localized to a plasmid containing the neuBAC genes of the K1 gene cluster. Cells harboring the neuB+ allele in an aldolase (nanA-) negative background produce neuNAc in vivo. Enzymatic synthesis of neuNAc could be demonstrated in extracts of cells harboring an expression plasmid (pNEUB) containing the neuB gene alone. NeuNAc synthetase was purified to homogeneity from extracts of cells harboring pNEUB. The molecular weight of the purified enzyme is 40 kDa, similar to that predicted by the nucleotide sequence of the neuB gene. The amino terminal sequence of the purified protein matches that predicted by the nucleotide sequence of the neuB gene. NeuNAc synthetase catalyzes the formation of neuNAc as indicated by its coupling to the CMP-neuNAc synthetase reaction. The enzyme condenses manNAc and PEP with the release of phosphate. The E. coli neuNAc synthetase is specific for manNAc and PEP, unlike rat liver enzyme that utilizes N-acetylmannosamine-6-phosphate to form neuNAc-9-PO4. This represents the first report of a purification of a sialic acid synthetase from either a eukaryotic or prokaryotic source to homogeneity. These experiments clearly demonstrate an aldolase-independent sialic acid synthetase activity in E. coli K1.

Amino Acid Sequence↗