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At least 199 records · Page 11Linked to original sources

PubMed2004

Characterization of glycopeptides from HIV-I(SF2) gp120 by liquid chromatography mass spectrometry.

Previously, we have characterized the HIV-I(SF2) gp120 glycopeptides using matrix-assisted laser desorption/ionization mass spectrometry (MALDI/MS) and nanospray electrospray ionization (ESI). Although we characterized 25 of 26 consensus glycosylation sites, we could not obtain any information about the extent of sialylation of the complex glycans. Sialylation is known to alter the biological activity of some glycoproteins, e.g., infectivity of some human and nonhuman primate lentiviruses is reduced when the envelope glycoproteins are extensively sialylated, and thus, characterization of the extent of sialylation of complex glycoproteins is of biological interest. Since neither MALDI/MS nor nanospray ESI provided much information about sialylation, probably because of suppression effects inherent in these techniques, we utilized online nanocapillary high performance liquid chromatography (nHPLC) with ESI/MS to characterize the sites and extent of sialylation on gp120. Eight of the known 26 consensus glycosylation sites of HIV-ISF2 gp120 were determined to be sialylated. Two of these sites were previously uncharacterized complex glycans. Thirteen high mannose sites were also determined. The heterogeneity of four of these sites had not been previously characterized. In addition, a peptide containing two consensus glycosylation sites, which had previously been determined to contain complex glycans, was also determined to be high mannose as well.

Animals↗

PubMed2006

Loose-patch-juxtacellular recording in vivo--a method for functional characterization and labeling of neurons in macaque V1.

We describe a method that uses a modified version of juxtacellular labeling [Pinault D. A novel single-cell staining procedure performed in vivo under electrophysiological control: morpho-functional features of juxtacellularly labeled thalamic cells and other central neurons with biocytin or neurobiotin. J Neurosci Meth 1996;65:113-36], which allows us to functionally characterize and subsequently label single neurons in vivo in macaque V1. The method is generally applicable in acute in vivo preparations. Extracellular recording is made with a patch electrode when the electrode is attached to the cell membrane. Initially a 'blind' search method is used as a guide to obtaining a cell attached configuration that we refer to as a loose-patch (LP). The neuron's receptive field properties are functionally characterized, the neuron is labeled and then characterization is confirmed, all in the LP configuration. There are a number of advantages of the method that we describe over other methods. First, we have found that we can obtain stable extracellular recordings for periods of hours that enable us to make a relatively comprehensive visual functional characterization of a neuron's receptive field properties. Second, because the electrode is closely apposed to the cell we obtain excellent isolation of the extracellular spike. Third, the method provides labeling that gives complete dendritic and axonal filling that survives over a number of days, which is an important feature in acute primate experiments. Fourth, the in vivo method of labeling and reconstructing neurons gives complete three-dimensional structure of the neuron including its intra-cortical axonal arbor. These features overcome known limits of the established methods of studying neuronal morphology including the Golgi stain (limited when adult tissue is used) and in vitro whole cell methods (incomplete axonal filling due to limited slice thickness). They also overcome the known limits of the established method of combined function-morphology studies i.e. intracellular recording in vivo. The modified juxtacellular method provides a reliable alternative to the difficult method of characterization by extracellular recording and subsequent intracellular labeling [Anderson JC, Martin KAC, Whitteridge D. Form, function and intracortical projections of neurons in the striate cortex of the monkey Macacus nemestrinus. Cerebral Cortex 1993;3:412-20]. We show the method can be used to record at a range of depths through V1 cortex allowing for sampling of neurons in the different layers and functional subpopulations. Links can then be made with existing knowledge about the anatomical organization of V1, the various morphological classes of neurons found therein, their functional connectivity and visual response properties.

Animals↗

PubMed1999

Characterization of a peptide-loading compartment by monoclonal antibodies.

Whether or not peptide-loading compartments are classical or specialized compartments of the endocytic pathway of antigen presenting cells is still a matter of debate. One way to solve this discrepancy would be to characterize specific markers for the peptide-loading compartment. We chose to generate monoclonal antibodies against the peptide-loading compartment that we previously characterized as lysozyme loading compartment (LLC) [Escola, J.M., Grivel, J.C., Chavrier, P., Gorvel, J.P., 1995. Different endocytic compartments are involved in the tight association of class II molecules with processed hen egg lysozyme and ribonuclease A in B cells. J. Cell Sci. 108, 2337; Escola, J.M., Deleuil, F., Stang, E., Boretto, J., Chavrier, P., Gorvel, J.P., 1996. Characterization of a lysozyme-major histocompatibility complex class II molecule-loading compartment as a specialized recycling endosome in murine B lymphocytes. J. Biol Chem. 271, 27360]. A preliminary screening by dot blot enabled us to identify several monoclonal antibodies recognizing the LLC and not early and late endosomes. One of these antibodies, the 20C4, was then characterized. It is directed against mature class II molecules of all murine haplotypes. By electron microscopy, 20C4 labeling was restricted to both the plasma membrane and the LLC. These reagents may be useful in the further characterization of the specialized function of these intracellular organelles.

Animals↗

PubMed1999

Kinematic characterization of standing reach: comparison of younger vs. older subjects.

OBJECTIVE: To characterize typical spinal motions that occur during standing reach and to describe differences in spinal motions and center of pressure displacements during reach between younger and older healthy adults. DESIGN: Exploratory, cross sectional investigation utilizing video motion and biomechanics force platform analysis. BACKGROUND: Standing reach provides a means for assessing both arm function and balance control in the context of a common functional activity. The interaction between age-related declines in spinal mobility and the spinal motion occurring during reach is poorly understood. The characterization of spinal motions during task performance for healthy subjects of different age groups is an important first step for understanding the relationship between impairments and physical performance in disabled populations. METHODS: Thirty-four subjects ages 20-36 and 33 subjects ages 60-76 participated. Video motion and force plate analysis were used to characterize spinal motion and center of pressure displacements during the functional reach test for younger and older subjects. RESULTS: Spinal motion during standing reach was characterized by forward trunk flexion, lateral trunk flexion, thoracolumbar rotation, and lower body rotation. Younger and older subjects differed (P = 0.05) in the amount of forward trunk flexion and thoracolumbar rotation which occurred but not lower body rotation. Younger subjects displaced their center of pressure further forward (P = 0.0001) and through a greater percentage of their initial base of support (P = 0.0001) than older subjects. CONCLUSION: This study provides the first multiplanar characterization of spinal motion used during standing reach. Significant differences for a number of variables existed between younger and older subjects.

Adult↗

PubMed2003

Phenotypic and molecular characterization of bovine Campylobacter fetus strains isolated in Brazil.

The objective of the present study was to characterize the phenotypic and molecular aspects of Campylobacter fetus strains isolated from bovine herds with reproductive problems. Thirty-one Brazilian field isolates, together with one reference strain of each subspecies of C. fetus, were analyzed. The strains were submitted to phenotypic identification followed by subspecies characterization using the polymerase chain reaction (PCR) and numeric evaluation of restriction fragment length polymorphism (RFLP) separated by pulsed-field gel electrophoresis (PFGE). Phenotypically, 4 isolates (12.1%) were classified as C. fetus subsp. fetus, and 29 isolates (87.9%) were classified as C. fetus subsp. venerealis. However, according to molecular analysis, only 1 isolate (3.0%) was classified as C. fetus subsp. fetus (the reference strain), whereas 32 isolates (97.0%) were considered C. fetus subsp. venerealis. SalI digestion of C. fetus genomic DNA, obtained from the 33 strains, yielded 7-10 DNA fragments ranging in size from 40 to 373kb, with 12 distinct patterns. Furthermore, the numeric analysis by neighbor-joining of the DNA from the 33 strains resulted in a dendrogram in which 2 distinct groups were identified. It was concluded that phenotypic characterization of C. fetus subspecies might lead to erroneous classification of field isolates. Although RFLP-PFGE is a powerful and reliable technique to characterize C. fetus, it has the inconvenience of being time consuming and laborious. Whereas PCR, besides providing rapid results, was found to be reliable and convenient for the characterization of field isolates of C. fetus.

Animals↗

PubMed1996

Comparison of gadolinium chelates with manganese-DPDP for liver lesion detection and characterization: preliminary results.

Nonspecific extracellular gadolinium chelate (NEGd) was prospectively compared with managanese (Mn)-DPDP (Mn) for the detection and characterization of focal liver lesions of various histology. Seventeen patients with known or suspected focal liver lesions underwent NEGd and Mn-enhanced studies at 1.5 T. Study findings were correlated with histology (five patients), computed tomography (CT) examinations (17 patients), and 4- to 13-month imaging follow-up by CT and/or MR (five patients). NEGd studies were performed as serial postcontrast spoiled gradient echo (SGE) sequences, and Mn studies were performed as SGE sequences 15 and 30 min postocontrast and T1-weighted, fat-suppressed spin echo at 16 min. NEGd and Mn images were prospectively interpreted in a separate blinded fashion. Lesion detection and characterization were determined. NEGd and Mn-enhanced images demonstrated 61 and 49 lesions, respectively (p = .1, NS). A total of 60 and 33 lesions were characterized on NEGd and Mn images, respectively, which was significantly different (p = .008). No differences were observed for the detection and characterization of liver metastases; whereas there was a trend for superior detection and characterization for hepatocellular carcinoma with NEGA.

Adult↗

PubMed2001

NMR characterization of a novel bile acid sequestrant, DMP 504.

DMP 504, a potential bile acid sequestrant for the treatment of hypercholesterolemia, is a highly insoluble, cross-linked polymer which does not lend itself to ordinary means of characterization used for drug substances in the pharmaceutical industry. Therefore, alternative characterization techniques have been sought. As part of an effort into extensive characterization of DMP 504 drug substance, nuclear magnetic resonance (NMR) was employed to provide insight into details of the DMP 504 polymer structure. The primary motivation for determining the structure of the polymer chain is to relate the DMP 504 structure to its performance properties as a bile acid sequestrant. Characterization of the polymer chain and understanding of the structural basis of its properties is essential in optimizing and controlling the manufacture of reproducible drug substance. NMR has proven a versatile tool for the description of polymer structure and dynamics because of the wide range of nuclear interactions affecting the NMR signal. This allows the design of experiments to elicit information about specific polymer interactions or properties. The methods of sample preparation utilized to obtain NMR spectra of the insoluble polymer, as well as a discussion and comparison of results for the characterization of DMP 504 obtained using several different NMR techniques will be presented.

Bile Acids and Salts↗

PubMed2001

Characterization of asphalt fume composition under simulated road paving conditions by GC/MS and microflow LC/quadrupole time-of-flight MS.

A highly sensitive, selective, and reliable analytical method has been developed and validated for characterization of asphalt fume generated under simulated road paving conditions. A dynamic asphalt fume generation system was modified to provide consistent test atmospheres at simulated asphalt road paving conditions. In the process of fume generation, asphalt was initially preheated in an oven to 170 degrees C, pumped to a large kettle, which maintained the asphalt temperature between 150 and 170 degrees C, and then transferred to the generator. The fume was conducted from the generator to an exposure chamber through a heated transfer line. Characterization of the asphalt fume test atmospheres included the following: (1) determination of the consistency of the asphalt aerosol composition within the generation system; (2) quantification of total organic matter of the asphalt fume by electron impact ionization of isotope dilution gas chromatography/ mass spectrometry); and (3) identification of individual priority polycyclic aromatic hydrocarbons (PAHs) in asphalt fume by selected ion monitoring. With the developed method, asphalt fumes could be characterized into three fractions: (1) filter collection of a large molecular size fraction over a range of mass-to-charge (m/z) ratios of 173-309; (2) XAD-2 trapping of a medium molecular size fraction over a range of m/z ratios of 121-197; and (3) charcoal trapping of a small molecular size fraction that contained mainly the volatile vapor fraction over a range of m/z ratios of 57-141. Total organic matter of the asphalt fume was quantified over the 5 exposure days. Sixteen specific priority PAHs were monitored and identified. These PAHs were determined at trace levels on the filter fraction. A novel approach, which utilizes collision-induced dissociation of fragmentation pathway leading to a characteristic fragmentation pattern by coupling microflow liquid chromatography to atmospheric pressure chemical ionization of quadrupole time-of-flight mass spectrometry, was used to further clarify the trace amount of key components present in simulated road paving asphalt fumes. These results demonstrate that asphalt fume composition could be characterized and specific priority PAHs could be identified by this method. The major advantages of this method are its highly sensitivity, selectivity, and reliability for chemical hazard characterization in a complex mixture. This method is suitable for support toxicity studies using simulated occupational exposure to asphalt fumes.

Carcinogens, Environmental↗

PubMed2006

Glycoprotein characterization combining intact protein and glycan analysis by capillary electrophoresis-electrospray ionization-mass spectrometry.

Glycosylated proteins play important roles in a large number of biological processes. Therefore, a complete characterization in terms of glycan structures and glycoform heterogeneity is needed. In this paper, a combined approach based on glycan and intact glycoprotein analysis by capillary zone electrophoresis-electrospray-mass spectrometry (CZE-ESI-MS) is presented. Based on a new capillary coating, a CZE-ESI-MS method for the separation and characterization of intact glycoproteins has been developed and compared to a method recently introduced for the characterization of erythropoietin. The excellent glycoform separation results in high-quality mass spectra, high dynamic range, and good sensitivity, allowing the correct characterization of minor glycan modifications. Additionally, a CZE-ESI-MS separation method for underivatized N-glycans has been developed. The separation of glycans differing in the degree of sialic acids and repeats of noncharged carbohydrates is achieved. The separation power of the method is demonstrated by obtaining mobility differences in glycans differing only by 16 Da. A time-of-flight mass spectrometer allowed the correct identification of the glycan composition based on high mass accuracy and resolution, identifying even minor modifications such as the exchange of "O" by "NH". An ion trap mass spectrometer provided structural information of the underivatized glycans from fragmentation spectra. The general applicability of both methods to glycoprotein analysis is illustrated for erythropoietin, fetuin, and alpha1-acid glycoprotein. The results obtained by the glycan analysis allowed an unequivocal glyco-assignment to the masses obtained for the intact proteins as long as the protein backbone is well characterized. Furthermore, modifications found for intact proteins can be attributed to differences in the glycostructure.

Electrophoresis, Capillary↗

PubMedDate not supplied

Molecular characterization of a conformational epitope of hen egg white lysozyme by differential chemical modification of immune complexes and mass spectrometric peptide mapping.

A new approach for the characterization of conformationally dependent epitope structures in protein antigens is described using differential chemical modification of immune complexes in combination with mass spectrometric peptide mapping analysis. Well-established methods for epitope characterization are frequently not applicable to conformationally dependent epitopes, and direct methods of structure analysis such as X-ray crystallography of immune complexes have been successful only in a few cases. Our approach combines tertiary structure-selective chemical modification of immune complexes with the molecular characterization of reaction products by mass spectrometric peptide mapping. The comparison of the modification pattern of free and antibody-bound antigen provides the identification of residues protected from modification by the antibody. These residues hence are characterized as part of the epitope structure. The well-characterized hen egg white lysozyme and a corresponding monoclonal IgM-type antibody were investigated as a model system. Specific modification reactions for arginine, lysine, and tyrosine residues were performed, and the modification sites in free and antibody-bound antigen were determined by mass spectrometric peptide mapping. The R14 residue and residues K13 and K96 in the antibody-bound lysozyme were found to be protected from modification, comprising a surface of spatially adjacent residues by folding of the native protein. In contrast, other K and R residues as well as Y20 and Y23 showed no significant shielding from modification in the immune complex. These results provided an estimation of the molecular epitope surface area of native lysozyme.

Animals↗

PubMedDate not supplied

A systematic approach for scale-down model development and characterization of commercial cell culture processes.

The objective of process characterization is to demonstrate robustness of manufacturing processes by understanding the relationship between key operating parameters and final performance. Technical information from the characterization study is important for subsequent process validation, and this has become a regulatory expectation in recent years. Since performing the study at the manufacturing scale is not practically feasible, development of scale-down models that represent the performance of the commercial process is essential to achieve reliable process characterization. In this study, we describe a systematic approach to develop a bioreactor scale-down model and to characterize a cell culture process for recombinant protein production in CHO cells. First, a scale-down model using 2-L bioreactors was developed on the basis of the 2000-L commercial scale process. Profiles of cell growth, productivity, product quality, culture environments (pH, DO, pCO2), and level of metabolites (glucose, glutamine, lactate, ammonia) were compared between the two scales to qualify the scale-down model. The key operating parameters were then characterized in single-parameter ranging studies and an interaction study using this scale-down model. Appropriate operation ranges and acceptance criteria for certain key parameters were determined to ensure the success of process validation and the process performance consistency. The process worst-case condition was also identified through the interaction study.

Animals↗

PubMed2005

Synthesis, structural and computational investigations of UO2I42-: a structurally characterized U(VI)-I anion.

The synthesis and characterization of (Ph(4)P)(2)UO(2)I(4).2NCCH(3) is reported. The UO(2)I(4)(2-) anion is not only the first unambiguously characterized anion containing a uranium(VI)-iodine bond, but is also the last member of the UO(2)X(4)(2-) (X = Cl, Br, I) series to be unambiguously identified and structurally characterized, in contrast to salts of the UO(2)Cl(4)(2-) and UO(2)Br(4)(2-) anions, which have all been thoroughly investigated and structurally characterized. (Ph(4)P)(2)UO(2)I(4).2NCCH(3) was characterized using IR, Raman, (1)H, (13)C, and (31)P NMR spectroscopy as well as X-ray diffraction. In addition, a computational investigation of the UO(2)I(4)(2-) anion was undertaken and compared with the experimentally observed structure.

Journal Article↗

PubMed2004

Raman spectroscopic characterization of secondary structure in natively unfolded proteins: alpha-synuclein.

The application of Raman spectroscopy to characterize natively unfolded proteins has been underdeveloped, even though it has significant technical advantages. We propose that a simple three-component band fitting of the amide I region can assist in the conformational characterization of the ensemble of structures present in natively unfolded proteins. The Raman spectra of alpha-synuclein, a prototypical natively unfolded protein, were obtained in the presence and absence of methanol, sodium dodecyl sulfate (SDS), and hexafluoro-2-propanol (HFIP). Consistent with previous CD studies, the secondary structure becomes largely alpha-helical in HFIP and SDS and predominantly beta-sheet in 25% methanol in water. In SDS, an increase in alpha-helical conformation is indicated by the predominant Raman amide I marker band at 1654 cm(-1) and the typical double minimum in the CD spectrum. In 25% HFIP the amide I Raman marker band appears at 1653 cm(-1) with a peak width at half-height of approximately 33 cm(-1), and in 25% methanol the amide I Raman band shifts to 1667 cm(-1) with a peak width at half-height of approximately 26 cm(-1). These well-characterized structural states provide the unequivocal assignment of amide I marker bands in the Raman spectrum of alpha-synuclein and by extrapolation to other natively unfolded proteins. The Raman spectrum of monomeric alpha-synuclein in aqueous solution suggests that the peptide bonds are distributed in both the alpha-helical and extended beta-regions of Ramachandran space. A higher frequency feature of the alpha-synuclein Raman amide I band resembles the Raman amide I band of ionized polyglutamate and polylysine, peptides which adopt a polyproline II helical conformation. Thus, a three-component band fitting is used to characterize the Raman amide I band of alpha-synuclein, phosvitin, alpha-casein, beta-casein, and the non-A beta component (NAC) of Alzheimer's plaque. These analyses demonstrate the ability of Raman spectroscopy to characterize the ensemble of secondary structures present in natively unfolded proteins.

Amides↗

PubMed2006

Direct and high resolution characterization of cytochrome c equilibrium folding.

Protein folding has emerged as a central problem in biophysics, and the equilibrium folding mechanism of cytochrome c (cyt c) has served as a model system. Unfortunately, the detailed characterization of both the folding process and of any intermediate that might be populated has been limited by the low structural and/or temporal resolution of the available techniques. Here, we report the use of a recently developed technique to study folding that is based on the site-selective incorporation of carbon-deuterium (C-D) bonds and their characterization by IR spectroscopy. Specifically, we synthesize and characterize the protein with deuterated residues spread throughout four structural motifs: (d3)Leu68 in the 60's helix, (d8)Lys72 and (d8)Lys73 in the 70's helix, (d8)Lys79, (d3)Met80, and (d3)Ala83 in the D-loop, and (d3)Leu94, (d3)Leu98, and (d3)Ala101 in the C-terminal helix. The data reveal correlated behavior of the residues within each structural motif, as well as between the residues of the 60's and C-terminal helices and between residues of the 70's helix and D-loop. Residues of the 70's helix and the D-loop are more stable than those within the 60's and C-terminal helices, although the former are more sensitive to added denaturant. The data also suggest that the hydrophobicity of the heme cofactor plays a central role in folding. These results contrast with those from previous H/D exchange studies and suggest that the low denaturant fluctuations observed in the H/D exchange experiments are not similar to those through which the protein actually unfolds. The inherently fast time scale of IR also allows us to characterize the folding intermediate, long thought to be present, but which has proven difficult to characterize by other techniques.

Cytochromes c↗

PubMed2006

Isotopic criteria in the characterization of aromatic molecules. 2. Influence of the chemical elaboration process.

Most valued natural aromatic molecules can be substituted by their low-cost chemical counterparts. Isotopic methods, which offer the most powerful tool to infer the origin of a molecule, are applied to the characterization of a large number of chemical aromatic species. Isotopic affiliation between precursors and products is investigated in several types of reactions: oxidation of benzyl chloride and benzyl alcohol and hydrolysis of benzylidene chloride and cinnamaldehyde. The isotopic parameters strongly depend not only on the type of process but, for a given process, on the experimental conditions of the reaction. Kinetic isotope effects occurring in several formylation reactions are estimated. It is shown that, in the drastic experimental conditions of many industrial processes, the benzenic hydrogen atoms may be affected by exchange phenomena. Consequently, the site-specific isotopic parameters of the ring fragment of chemical species are usually much less stable than those of the corresponding natural molecules biosynthesized in mild environments. The isotope ratios of substituents such as CH3, CH2Cl, and CHO are more resistant to exchange and provide useful criteria for characterizing both the raw materials and the process. It is shown in particular that radical hydrogen abstraction in toluene to produce benzyl chloride induces relatively moderate fractionation effects. In contrast, oxidation reactions frequently produce strong fractionation effects. In particular, industrial direct oxidation of toluene into benzaldehyde is characterized by deuterium enrichments at the formyl site, which may exceed 900 ppm. Taking into account the large magnitude and high variability of many fractionation effects occurring in chemical reactions, the isotopic fingerprint may provide unambiguous criteria, not only for excluding a natural origin and characterizing the type of process, but also for differentiating molecules synthesized by a given process in different industrial contexts. The isotopic fingerprint may therefore be used by manufacturers as a powerful label for characterizing their production batches.

Acrolein↗

PubMedDate not supplied

Characterization of N1- and N6-adenosine adducts and N1-inosine adducts formed by the reaction of butadiene monoxide with adenosine: evidence for the N1-adenosine adducts as major initial products.

1,3-Butadiene is a known human mutagen and possible human carcinogen; however, the molecular mechanisms of its activity are poorly understood. We have previously shown that the primary metabolite, butadiene monoxide (BM), reacts with guanosine to form N1-, N2-, and N7-guanosine adducts. In this study we characterize the reaction of BM with adenosine; ten adducts identified as diastereomeric pairs of N1-(1-hydroxy-3-buten-2-yl)adenosine,N1-(2-hydroxy-3-buten-1 -yl)adenosine, N6-(1-hydroxy-3-buten-2-yl)adenosine,N6-(2-hydroxy-3-buten-+ ++1yl)adenosine, and N1-(1-hydroxy-3-buten-2-yl)inosine are characterized. The N6-adenosine and N1-inosine adducts were characterized by their UV spectra, 1H NMR, FAB/MS, and stability studies. The N6-adenosine and N1-inosine adducts were stable for up to 168 h at 37 degrees C in phosphate buffer (pH 7.4). The N1-adenosine adducts, which were unstable at pH 7.4 at 37 degrees C (half-life of 7 and 9.5 h for the two regioisomers), were characterized by their UV spectra and their ability to undergo the Dimroth rearrangement to yield the corresponding N6-adenosine adducts, or undergo deamination to yield the corresponding N1-inosine adducts. Upon the reaction of BM with adenosine in phosphate buffer (pH 7.4) at 37 degrees C, the N1-adenosine adducts were the first to be detected, with the N6-adenosine and N1-inosine adducts. showing a lag in formation possibly due to the time needed for rearrangement/deamination. Reaction of adenosine with an excess of BM in phosphate buffer (pH 7.4) at 37 degrees C, followed by extraction of the reaction mixture with ethyl ether to remove excess unreacted BM and incubation at 80 degrees C for 1 h, resulted in complete conversion of N1-adenosine adducts to the corresponding N6-adenosine and N1-inosine adducts. Under these conditions, adduct formation exhibited pseudo-first-order kinetics, with the combined N6-adenosine adducts being formed 3-fold more favorably than the combined N1-inosine adducts. When incubations were carried out at lower BM concentrations, the N6-adenosine adducts remained the major detectable adducts at all concentrations. These results show that adenosine, in addition to guanosine, can lead to multiple adducts when incubated with BM, and may be useful in development of biomarkers for exposure to 1,3-butadiene. Characterization of the N1-adenosine adducts and their rearrangement/deamination products may also contribute to the understanding of mutagenic and carcinogenic mechanisms of 1,3-butadiene.

Adenosine↗

PubMed2002

Characterization of surface oxide films on titanium and bioactivity.

Biological properties of titanium implant depend on its surface oxide film. In the present study, the surface oxide films on titanium were characterized and the relationship between the characterization and bioactivity of titanium was studied. The surface oxide films on titanium were obtained by heat-treatment in different oxidation atmospheres, such as air, oxygen and water vapor. The bioactivity of heat-treated titanium plates was investigated by immersion test in a supersaturated calcium phosphate solution. The surface roughness, energy morphology, chemical composition and crystal structure were used to characterize the titanium surfaces. The characterization was performed using profilometer, scanning electronic microscopy, ssesile drop method, X-ray photoelectron spectroscopy, common Bragg X-ray diffraction and sample tilting X-ray diffraction. Percentage of surface hydroxyl groups was determined by X-ray photoelectron spectroscopy analysis for titanium plates and density of surface hydroxyl groups was measured by chemical method for titanium powders. The results indicated that heat-treatment uniformly roughened the titanium surface and increased surface energy. After heat-treatment the surface titanium oxide was predominantly rutile TiO(2), and crystal planes in the rutile films preferentially orientated in (1 1 0) plane with the highest density of titanium ions. Heat-treatment increased the amount of surface hydroxyl groups on titanium. The different oxidation atmospheres resulted in different percentages of oxygen species in TiO(2), in physisorbed water and acidic hydroxyl groups, and in basic hydroxyl groups on the titanium surfaces. The immersion test in the supersaturated calcium phosphate solution showed that apatite spontaneously formed on to the rutile films. This revealed that rutile could be bioactivated. The analyses for the apatite coatings confirmed that the surface characterization of titanium has strong effect on bioactivity of titanium. The bioactivity of the rutile films on titanium was related not only to their surface basic hydroxyl groups, but also to acidic hydroxyl groups, and surface energy. Heat-treatment endowed titanium with bioactivity by increasing the amount of surface hydroxyl groups on titanium and its surface energy.

Journal Article↗

PubMed2000

Human alpha 1,3/4 fucosyltransferases. Characterization of highly conserved cysteine residues and N-linked glycosylation sites.

Human alpha1,3 fucosyltransferases (FucTs) contain four highly conserved cysteine (Cys) residues, in addition to a free Cys residue that lies near the binding site for GDP-fucose (Holmes, E. H., Xu, Z. , Sherwood, A. L., and Macher, B. A. (1995) J. Biol. Chem. 270, 8145-8151). The participation of the highly conserved Cys residues in disulfide bonds and their functional significance were characterized by mass spectrometry (MS) analyses and site-directed mutagenesis, respectively. Among the human FucTs is a subset of enzymes (FucT III, V, and VI) having highly homologous sequences, especially in the catalytic domain, and Cys residues in FucT III and V were characterized. The amino acid sequence of FucT III was characterized. Peptides containing the four conserved Cys residues were detected after reduction and alkylation, and found to be involved in disulfide bonds. The disulfide bond pattern was characterized by multiple stage MS analysis and the use of Glu-C protease and MS/MS analysis. Disulfide bonds in FucT III occur between Cys residues (Cys(81) to Cys(338) and Cys(91) to Cys(341)) at the N and C termini of the catalytic domain, bringing these ends close together in space. Mutagenesis of highly conserved Cys residues to Ser in FucT V resulted in proteins lacking enzymatic activity. Three of the four mutants have molecular weights similar to wild type enzyme and maintained an ability to bind GDP, whereas the other (Cys(104)) produced a series of lower molecular weight bands when characterized by Western blot analysis, and did not bind GDP. FucTs have highly conserved, potential N-linked sites, and our mass spectrometry analyses demonstrated that both N-linked sites are modified with oligosaccharides.

Amino Acid Sequence↗