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Biomedical subjects

C E Costello

Publications and source records attributed to C E Costello.

At least 19 recordsLinked to original sources
PubMed2004

Tritrichomonas foetus induces apoptotic cell death in bovine vaginal epithelial cells.

Tritrichomonas foetus is a serious veterinary pathogen, causing bovine trichomoniasis, a sexually transmitted disease leading to infertility and abortion. T. foetus infects the mucosal surfaces of the reproductive tract. Infection with T. foetus leads to apoptotic cell death of bovine vaginal epithelial cells (BVECs) in culture. An affinity-purified cysteine protease (CP) fraction yielding on sodium dodecyl sulfate-polyacrylamide gel electrophoresis a single band with an apparent molecular mass of 30 kDa (CP30) also induces BVEC apoptosis. Treatment of CP30 with the protease inhibitors TLCK (Nalpha-p-tosyl-l-lysine chloromethyl ketone) and E-64 [l-trans-epoxysuccinyl-leucylamide-(4-guanido)-butane] greatly reduces induction of BVEC apoptosis. Matrix-assisted laser desorption ionization-time-of-flight MALDI-TOF mass spectrometry analysis of CP30 reveals a single peak with a molecular mass of 23.7 kDa. Mass spectral peptide sequence analysis of proteolytically digested CP30 reveals homologies to a previously reported cDNA clone, CP8 (D. J. Mallinson, J. Livingstone, K. M. Appleton, S. J. Lees, G. H. Coombs, and M. J. North, Microbiology 141:3077-3085, 1995). Induction of apoptosis is highly species specific, since the related human parasite Trichomonas vaginalis and associated purified CPs did not induce BVEC death. Fluorescence microscopy along with the Cell Death Detection ELISA(PLUS) assay and flow cytometry analyses were used to detect apoptotic nuclear condensation, DNA fragmentation, and changes in plasma membrane asymmetry in host cells undergoing apoptosis in response to T. foetus infection or incubation with CP30. Additionally, the activation of caspase-3 and inhibition of cell death by caspase inhibitors indicates that caspases are involved in BVEC apoptosis. These results imply that apoptosis is involved in the pathogenesis of T. foetus infection in vivo, which may have important implications for therapeutic interference with host cell death that could alter the course of the pathology in vivo.

Animals↗
PubMed2001

Tandem mass spectrometric determination of the 4S/6S sulfation sequence in chondroitin sulfate oligosaccharides.

Chondroitin sulfate (CS) is a glycosaminoglycan consisting of repeating uronic acid, N-acetylgalactosamine sulfate disaccharide units [-UroA(beta1,3)-GalNAcS(beta1,4)]n. Chondroitin sulfate type A (CSA) contains glucuronic acid, and 90% of the GalNAc residues are sulfated at the 4-position with 10% at the 6-position. Chondroitin sulfate type C (CSC) contains glucuronic acid, and 90% of the GalNAc residues are sulfated at the 6-position with 10% sulfated at the 4-position. These molecules are fragile due to their high degree of sulfation and are challenging to analyze as a result. This work presents the first evidence that tandem mass spectrometry can be used for the determination of a CS oligosaccharide sequence with respect to the positions of GalNAc sulfation. Using this technique, it is possible to analyze individual components from mixtures, saving much purification effort. Oligosaccharides produced from CSA and CSC are used in this work to demonstrate that CID MS/MS can be used to distinguish positional sulfation isomers. For charge states where charge equals the number of sulfates, abundant odd-numbered Bn and Yn ions are observed. The percent total ion abundances of these ions indicate the position of sulfation.

Carbohydrate Sequence↗
PubMed2001

Electron capture dissociation and infrared multiphoton dissociation MS/MS of an N-glycosylated tryptic peptic to yield complementary sequence information.

Glycoproteins are a functionally important class of biomolecules for which structural elucidation presents a challenge. Fragmentation of N-glycosylated peptides, employing collisionally activated dissociation, typically yields product ions that result from dissociation at glycosidic bonds, with little occurrence of dissociation at peptide backbone sites. We have applied two dissociation techniques, electron capture dissociation (ECD) and infrared multiphoton dissociation (IRMPD), in a 7-T Fourier transform ion cyclotron resonance mass spectrometer, in the investigation of an N-glycosylated peptide from an unfractionated tryptic digest of the lectin of the coral tree, Erythrina corallodendron. ECD provided c and z. ions derived from the peptide backbone, with no observed loss of sugars. Cleavage at 11 of 15 backbone amine bonds was observed. The lack of cleavage at sites located close to the glycosylated asparagine residue may result from steric blocking by the glycan. IRMPD provided abundant fragment ions, primarily through dissociation at glycosidic linkages. The monosaccharide composition and the presence of three glycan branch sites could be determined from the IRMPD fragments. The two types of spectra, obtained with the same instrument, thus provide complementary structural information about the glycopeptide. The current result extends the applicability of ECD for glycopeptide analysis to N-glycosylated peptides and to peptides containing branched, highly substituted glycans.

Electrons↗
PubMed2001

Identification and location of a cysteinyl posttranslational modification in an amyloidogenic kappa1 light chain protein by electrospray ionization and matrix-assisted laser desorption/ionization mass spectrometry.

Amyloid-deposited light chain (AL) amyloidosis is correlated with the overproduction of a monoclonal immunoglobulin light chain protein by a B-lymphocyte clone. Since the amyloid fibril deposits in AL amyloidosis most often consist of the N-terminal fragments of the light chain, the majority of studies have focused on the determination of the primary structure of the protein, and reducing agents have been used routinely in the initial purification process. In this study, two light chain proteins were isolated and purified, without reduction, from the urine of a patient diagnosed with kappa 1 (kappa1) AL amyloidosis. One protein had a relative molecular mass of 12,000 and the other 24,000. Electrospray ionization and matrix-assisted laser desorption/ionization mass spectrometry, in combination with enzymatic digestions, were used to verify the amino acid sequences and identify and locate posttranslational modifications in these proteins. The 12-kDa protein was confirmed to be the N-terminal kappa1 light chain fragment (variable region) consisting of residues 1-108 or 1-109 and having one disulfide bond. The 24-kDa protein was determined to be the intact kappa1 light chain containing a cysteinyl posttranslational modification at Cys214 and disulfide bonds located at Cys23-Cys88, Cys134-Cys194, and Cys214-Cys. The methods used in this report enable high-sensitivity determination of amino acid sequence and variation in intact and truncated light chains as well as posttranslational modifications. This approach facilitates consideration of the effect of cysteinylation on the native protein structure and the potential involvement of this modification in AL amyloidosis.

Amino Acid Sequence↗
PubMed2001

Compositional analysis of glycosaminoglycans by electrospray mass spectrometry.

The purpose of this work is to analyze glycosaminoglycans (GAGs) directly from complex mixtures without the need to purify individual components. Novel conditions for negative ion electrospray MS of chondroitin sulfate (CS) oligosaccharides are described in which sodium adduction and fragmentation are avoided. Differentiation between positional sulfation isomers is demonstrated for CS disaccharides, and a selected reaction monitoring scheme is used to quantify sulfation isomers in disaccharides liberated from decorin and biglycan. A size exclusion chromatography LC/MS method is shown to be effective for compositional analysis of longer CS oligosaccharides. The SEC step serves to simplify the composition of GAGs entering the mass spectrometer at any time, thus allowing the masses of the constituent molecules to be extracted. Mass spectrometric detection produces far more information than conventional UV or fluorescent detectors and allows the monosaccharide composition of individual components to be determined.

Chromatography, Gel↗
PubMed2001

A high pressure matrix-assisted laser desorption/ionization Fourier transform mass spectrometry ion source for thermal stabilization of labile biomolecules.

A high pressure matrix-assisted laser desorption/ionization (MALDI) Fourier transform mass spectrometry (FTMS) ion source was designed and tested. With this design, pressure is pulsed to an estimated 1-10 mbar in the region of the MALDI sample during desorption with the result of significantly decreased fragmentation compared to similar systems operating with pressures of <0.1 mbar. The thermal stabilization of vibrationally excited ions under these conditions is shown with small peptides desorbed from the "hot" matrix alpha-cyano-4-hydroxycinnamic acid, and with the highly labile oxidized beta-chain of insulin. Fragile gangliosides with several sialic acid residues are desorbed under high pressure and remain intact without the typical losses of sialic acid, and a protein standard, ubiquitin (8565.64 Da), is desorbed with minimal dehydration. Under high pressure collisional cooling conditions, non-covalent matrix adduction to the molecular ions becomes prominent, but with the trapped ions in an FT mass spectrometer, the ions can be mildly activated to detach the matrix adducts. The new source, additionally, generates significant levels of the multiply charged ions which are commonly seen in MALDI-TOFMS, but are rarely observed in MALDI-FTMS. This effect is more likely due to the elimination of a mass filtering effect in the previous FTMS ion source than to collisional cooling of the ions.

Animals↗
PubMed2001

Compositional analysis of human acquired enamel pellicle by mass spectrometry.

Relatively little is known about the formation of the acquired enamel pellicle other than that it involves the selective adsorption of specific proteins from oral fluids. Previous studies on the identification of pellicle components have relied largely on immunological or enzymatic detection and have been hampered by the fact that only minute quantities of pellicle can be removed from tooth surfaces. The present work describes an improved method of harvesting pellicle that combines mechanical and chemical removal; this approach was used to investigate systematically the desorption of in vitro pellicle components with different solutions. Eleven major in vitro pellicle proteins were identified by using a combination of electrophoretic separation and matrix-assisted laser desorption/ionization-reflectron time-of-flight mass spectrometry. A similar analysis of in vivo-formed pellicle revealed the presence of intact statherin, lysozyme, albumin and amylase. Further analysis of in vivo pellicle by liquid chromatography-electrospray ionization mass spectrometry suggested the presence of numerous low molecular-weight fragments of precursor proteins. The protein composition of in vitro whole-salivary pellicle adsorbed to hydroxyapatite and that of in vivo enamel pellicle differed for proline, the result of a reduction in the content of acidic proline-rich proteins in the in vivo samples. Unique features of the oral environment such as enzymatic activities or mineral surface properties may account for these differences between in vivo and in vitro pellicle formation.

Adult↗
PubMed2001

Heparin reacts with and inactivates nitric oxide.

Although heparin is a well-known anticoagulant, in some cases it promotes a prothrombotic state and does so through both antibody-dependent and antibody-independent platelet activation. In this study, heparin was found to reverse the antiplatelet effect of an NO donor. S-nitroso-glutathione (SNO-Glu), with an EC(50) of 1.8 U/mL. Ultraviolet/visible spectral analysis and the Griess assay showed that increasing heparin concentrations on a dose-dependent basis eliminated acidified NO(x) species. Since heparin is a heterogeneous mixture of glycosaminoglycans, the effects of six different heparin disaccharides were compared with various substitutions on the hexose rings to determine which functional group(s) of the polysaccharide interact with acidified NO(x). Among the six disaccharides tested, only types I-S and II-S had the effect, suggesting that the sulfamino-group at the C2 position of the glucosamine moiety was critical for the elimination of acidified NO(x) species. Mass spectrometry experiments gave results consistent with these observations, indicating that only the I-S and II-S heparin disaccharides were modified upon treatment with NaNO(2)/HCl. Negative-ion electro-spray ionization MS and tandem MS analyses of the native compounds and their deuterium-labeled analogs confirmed that the reaction products from nitrosation of these N-sulfated disaccharides had eliminated the C2-sulfamino-moiety and replaced it with methoxide derived from the solvent. Participation of the 6-sulfato-substituent appears to facilitate the elimination reaction. These data show that heparin can impair the antiplatelet properties of nitric oxide by interacting with the nitrosating species, and suggest that heparin-like glycosamino-glycans may interact with endothelium-derived nitric oxide in vivo to regulate the bioactivity of this important antiplatelet and vasorelaxant substance.

Anticoagulants↗
PubMed2001

Acidic glycerol lipids of Trichomonas vaginalis and Tritrichomonas foetus.

The isolation and characterization of acidic lipids from both Trichomonas vaginalis and Tritrichomonas foetus have been carried out using radiolabeling, a combination of high performance liquid and thin layer chromatographic techniques, and mass spectrometry. Unique among the eukaryotes, these organisms produce phosphatidylglycerols and O-acyl phosphatidylglycerol-like compounds. In this study, the molecular weight distributions of the phosphatidylglycerols and acyl phosphatidylglycerols were determined by negative-ion liquid secondary ionization mass spectrometry (LSIMS) and the fatty acyl groups within each molecular species were assessed by collision-induced decomposition tandem mass spectrometry (CID MS/MS). Both species were found to contain primarily oleic acid in the sn-2 position. The lipids of T. vaginalis had approximately equal amounts of C16 and C18 in the sn-1 position, with varying degrees of unsaturation, especially in the C18 species. The T. foetus lipids had C18 almost exclusively, but also varied in the unsaturation. Other acidic lipids included inositol phosphosphingolipids and inositol diphosphosphingolipids.

Animals↗
PubMed2000

Internal calibration on adjacent samples (InCAS) with Fourier transform mass spectrometry.

Using matrix-assisted laser desorption/ionization (MAL DI) on a trapped ion mass spectrometer such as a Fourier transform mass spectrometer (FTMS) allows accumulation of ions in the cell from multiple laser shots prior to detection. If ions from separate MALDI samples are accumulated simultaneously in the cell, ions from one sample can be used to calibrate ions from the other sample. Since the ions are detected simultaneously in the cell, this is, in effect, internal calibration, but there are no selective desorption effects in the MALDI source. This method of internal calibration with adjacent samples is demonstrated here on cesium iodide clusters, peptides, oligosaccharides, poly(propylene glycol), and fullerenes and provides typical FTMS internal calibration mass accuracy of < 1 ppm.

Calibration↗
PubMed2000

Further definition of the substance P (SP)/neurokinin-1 receptor complex. MET-174 is the site of photoinsertion p-benzoylphenylalanine4 SP.

The covalent attachment site of a substance P (SP) analogue containing the photoreactive amino acid p-benzoyl-l-phenylalanine (Bpa) in position 8 of the C-terminal portion of the peptide was identified previously as Met-181 on the neurokinin-1 (NK-1) receptor. In this study, a second photoreactive SP analogue, Bpa(4)-SP, in which the Bpa residue is located in the N-terminal portion of the peptide, was used to define further the peptide-receptor interface. The NK-1 receptor expressed in Chinese hamster ovary cells was specifically and efficiently photolabeled with a radioiodinated derivative of Bpa(4)-SP. Fragmentation analysis of the photolabeled receptor restricted the site of photoincorporation of Bpa(4)-SP to an amino acid within the sequence Thr-173 to Arg-177 located on the N-terminal side of the E2 loop. To identify the specific amino acid in this sequence that serves as the covalent attachment site for Bpa(4)-SP, a small photolabeled receptor fragment was generated by chemical cleavage with cyanogen bromide. Matrix-assisted laser desorption/ionization time of flight mass spectrometric analysis of the purified fragment identified a single protonated molecular ion with a molecular mass of 1801.3 +/- 1.8, indicating that upon irradiation, the bound photoligand covalently attaches to the terminal methyl group of a methionine residue. This result, taken together with the results of the peptide mapping studies, establishes that the site of Bpa(4)-SP covalent attachment to the NK-1 receptor is Met-174.

Animals↗
PubMed2000

Glycosylphosphatidylinositol anchors of Plasmodium falciparum: molecular characterization and naturally elicited antibody response that may provide immunity to malaria pathogenesis.

Induction of proinflammatory cytokine responses by glycosylphosphatidylinositols (GPIs) of intraerythrocytic Plasmodium falciparum is believed to contribute to malaria pathogenesis. In this study, we purified the GPIs of P. falciparum to homogeneity and determined their structures by biochemical degradations and mass spectrometry. The parasite GPIs differ from those of the host in that they contain palmitic (major) and myristic (minor) acids at C-2 of inositol, predominantly C18:0 and C18:1 at sn-1 and sn-2, respectively, and do not contain additional phosphoethanolamine substitution in their core glycan structures. The purified parasite GPIs can induce tumor necrosis factor alpha release from macrophages. We also report a new finding that adults who have resistance to clinical malaria contain high levels of persistent anti-GPI antibodies, whereas susceptible children lack or have low levels of short-lived antibody response. Individuals who were not exposed to the malaria parasite completely lack anti-GPI antibodies. Absence of a persistent anti-GPI antibody response correlated with malaria-specific anemia and fever, suggesting that anti-GPI antibodies provide protection against clinical malaria. The antibodies are mainly directed against the acylated phosphoinositol portion of GPIs. These results are likely to be valuable in studies aimed at the evaluation of chemically defined structures for toxicity versus immunogenicity with implications for the development of GPI-based therapies or vaccines.

Adult↗
PubMed2000

Application of multishot acquisition in Fourier transform mass spectrometry.

A new method of ion injection and trapping is discussed wherein ions are accumulated over several laser shots in the FT-ICR cell prior to detection. This allows accumulation of ion signal without accumulating noise so that the signal/noise ratio is much improved provided that the "space-charge" limit of the total number of ions in the cell is not exceeded. "In-cell" ion accumulation allows selected ion accumulation by simply sweeping unwanted ions out of the cell prior to subsequent ion trapping events and also allows shifted ion accumulations to correct for time-of-flight distortions in the ion abundance distributions.

Mass Spectrometry↗
PubMed2000

CD1c-mediated T-cell recognition of isoprenoid glycolipids in Mycobacterium tuberculosis infection.

The discovery of the CD1 antigen presentation pathway has expanded the spectrum of T-cell antigens to include lipids, but the range of natural lipid antigens and functions of CD1-restricted T cells in vivo remain poorly understood. Here we show that the T-cell antigen receptor and the CD1c protein mediate recognition of an evolutionarily conserved family of isoprenoid glycolipids whose members include essential components of protein glycosylation and cell-wall synthesis pathways. A CD1c-restricted, mycobacteria-specific T-cell line recognized two previously unknown mycobacterial hexosyl-1-phosphoisoprenoids and structurally related mannosyl-beta1-phosphodolichols. Responses to mannosyl-beta1-phosphodolichols were common among CD1c-restricted T-cell lines and peripheral blood T lymphocytes of human subjects recently infected with M. tuberculosis, but were not seen in naive control subjects. These results define a new class of broadly distributed lipid antigens presented by the CD1 system during infection in vivo and suggest an immune mechanism for recognition of senescent or transformed cells that are known to have altered dolichol lipids.

Adult↗
PubMed2000

Betabellins 15D and 16D, de Novo designed beta-sandwich proteins that have amyloidogenic properties.

The betabellin structure is a de novo designed beta-sandwich protein consisting of two 32-residue beta-sheets packed against one another by hydrophobic interactions. d-Amino acid residues are used to energetically favor formation of type-I' beta turns. Air oxidation of betabellin 15S (B15S) (HSLTAKIpkLTFSIAphTYTCAVpkYTAKVSH, where p denotes d-Pro, h denotes d-His, and k denotes d-Lys) yields betabellin 15D (B15D), a 64-residue disulfide-bridged protein. The amino acid sequence of B15D contains a conformationally constrained d-Pro residue at the i + 1 position of each type-I' beta turn. To test whether d-Pro residues are necessary for folding at these positions, the six d-Pro residues of B15D are replaced by d-Ala residues in betabellin 16D (B16D). Previously, transmission electron microscopy showed that B15D forms unbranched, 35-A wide fibrils that associate into bundles in 5.0 mM 3-(N-morpholino)propanesulfonate and 250 mM NaCl at pH 7; under these conditions, B16D forms ribbon-like assemblies. The B15D fibrils resemble the protofilaments that constitute amyloid fibrils. The present studies show that both B15D and B16D have characteristics of amyloidogenic proteins: the unbranched fibrils and ribbons stained with Congo red and displayed a green birefringence, exhibited a cross-beta structure, and bound 1-anilino-8-naphthalenesulfonate. Thus, these de novo designed beta-sandwich proteins should provide useful models for studying the mechanism of amyloid protofilament formation and assembly into amyloid fibrils and for designing potential inhibitors of amyloidogenesis.

Amino Acid Sequence↗
PubMed2000

Detection of transthyretin variants using immunoprecipitation and matrix-assisted laser desorption/ionization bioreactive probes: a clinical application of mass spectrometry.

In our continuing efforts to develop mass spectrometry-based methods for transthyretin (TTR) variant detection and characterization, we have sought to use matrix-assisted laser desorption/ionization (MALDI) bioreactive probes incorporating immobilized trypsin for screening purposes. These devices show good diagnostic potential as a clinical screening tool to detect amino acid substitutions in TTR. MALDI probes allow the on-probe generation of tryptic digests. The subsequent mass analysis of the on-probe digest yields the peptide map. The inherent advantages of this method include considerably reduced digestion times (minutes vs. hours), absence of autolysis products, minimized sample handling, and hence minimal sample loss. A further advantage is that the opportunity for loss of hydrophobic peptides is reduced because no sample transfer occurs. The method can be applied as a preliminary screen for TTR variants where TTR is isolated from patient serum through immunoprecipitation. This method should also be applicable to other proteins and suitable for automation.

Hemoglobins↗
PubMed1999

Plasmin desensitization of the PAR1 thrombin receptor: kinetics, sites of truncation, and implications for thrombolytic therapy.

It has been hypothesized that protease-activated receptors may be activated and attenuated by more than one protease. Here, we explore a desensitization mechanism of the PAR1 thrombin receptor by anticoagulant proteases and provide an explanation to the enigma of why plasmin/tissue plasminogen activator (t-PA) can both activate and deactivate platelets prior to thrombin treatment. By using a soluble N-terminal exodomain (TR78) as a model for the full-length receptor, we were able to unambiguously compare cleavage rates and specificities among the serum proteases. Thrombin cleaves TR78 at the R41-S42 peptide bond with a kcat of 120 s-1 and a KM of 16 microM to produce TR62 (residues 42-103). We found that, of the anticoagulant proteases, only plasmin can rapidly truncate the soluble exodomain at the R70/K76/K82 sites located on a linker region that tethers the ligand to the body of the receptor. Plasmin cleavage of the TR78 exodomain is nearly equivalent to that of thrombin cleavage at R41 with similar rates (kcat = 30 s-1) and affinity (KM = 18 microM). Specificity was demonstrated since there is no observed cleavage at the five other potential plasmin-cleavage sites. Plasmin also cleaves the TR78 exodomain at the R41 thrombin-cleavage site generating transiently activated exodomain. We directly demonstrated that plasmin cleaves these same sites in full-length membrane-embedded receptor expressed in yeast and COS7 fibroblasts. The rate of plasmin truncation is similar between the extensively glycosylated COS7-expressed receptor and the nonglycosylated yeast-produced receptor. Mutation of the R70/K76/K82 sites to A70/A76/A82 eliminates plasmin truncation and desensitization of thrombin-dependent Ca2+ signaling and converts PAR1 into a plasmin-activated receptor with full agonist activity for plasmin. Plasmin does not desensitize the Ca2+ response of platelets or COS7 cells to SFLLRN consistent with intermolecular ligand-binding sites being located to the C-terminal side of K82. Truncation of the wild-type receptor at the C-terminal plasmin-cleavage sites removes the N-terminal tethered ligand or preligand, thereby providing an effective pathway for PAR1 desensitization in vivo.

Amino Acid Sequence↗