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PubMed2000

Estimation of the amyotrophic lateral sclerosis incidence by capture-recapture method in the Limousin region of France.

Amyotrophic lateral sclerosis (ALS) is a rapidly fatal degenerative neurological disease categorized among motor neuron diseases. In the literature, the incidence of ALS varies between 0.7 and 2.4 per 100, 000 inhabitants. A study using the capture-recapture method (multiple records system analysis) was undertaken in Limousin (France) ascertaining all patients having onset of definite or probable ALS during the period 1994-1995. Three information sources able to identify these new ALS cases were selected: the first source was a computerized database of the Neurology Department of the University Hospital of Limoges; the second source consisted of the neurologists of the Limousin region and neighboring provinces (county-sized regions); the third source grouped the hospitals of the Limousin region and neighboring provinces (county-sized regions). During this period, 46 new cases of ALS were seen, corresponding to an observed mean annual incidence of 3.2 (+/-0.6) per 100,000 inhabitants. After standardization for age, the annual incidence was 2.5 per 100,000 inhabitants. The number of new cases estimated by the capture-recapture method was 70, corresponding to an estimated mean annual incidence of 4.9 (+/-1.0) for 100,000 inhabitants. Hence, statistical modeling utilizing partially overlapping information sources permitted a more exhaustive compendium of the new cases of ALS and may be a truer reflection of actual disease incidence than has been previously reported.

Amyotrophic Lateral Sclerosis↗

PubMedDate not supplied

The electric sense of the paddlefish: a passive system for the detection and capture of zooplankton prey.

Behavioral and electrophysiological experiments have shown that the elongated paddlefish rostrum, with its extensive population of ampullae of Lorenzini, constitutes a passive electrosensory antenna of great sensitivity and spatial resolution. As demonstrated in juvenile paddlefish, the passive electrosense serves a novel function in feeding serving as the primary, if not exclusive sensory modality for the detection and capture of zooplanktonic prey. Ampullary receptors are sensitive to the weak electrical fields of plankton from distances up to 9 cm, and juvenile paddlefish capture plankton individually with great swimming dexterity in the absence of vision or other stimulus signals. Paddlefish also detect and avoid metal obstacles, the electrical signatures of which are a potential hindrance to their feeding and reproductive migrations. The ampullary receptors, their peripheral innervation and central targets in the dorsal octavolateral nucleus, are described. We also describe the ascending and descending neuronal circuitry of the electrosensory system in the brain based on tracer studies using dextran amines.

Action Potentials↗

PubMed1992

Monoclonal antibody based capture ELISA/ELIFA for the detection of Chlamydia psittaci in veterinary clinical specimens.

A capture ELISA/ELIFA system based on monoclonal capture and biotinylated monoclonal detection antibody with specificity for an epitope on chlamydial lipopolysaccharide (LPS) is described. The assay is fast, specific and detects a minimum dose of approximately 2000 chlamydial particles (C. psittaci). Unlike other commercially available test kits, it comprises a proteinase K treatment to exclude non-specific binding of antibodies to Fc receptors. In contrast to the sophisticated and cumbersome isolation procedures, even non-specialized laboratories may use this assay system for the investigation of clinical samples of different origin for the presence of C. psittaci within a short period of time. Due to the genus specificity of the monoclonal antibody used C. trachomatis and C. pneumoniae can be detected as well.

Animals↗

PubMed2002

Microtubule capture: IQGAP and CLIP-170 expand the repertoire.

Rho GTPases regulate microtubule capture near the cell cortex to polarize cells. What is surprising is the repertoire of interactions between proteins at the ends of microtubules and their cortical targets. The microtubule tip protein CLIP-170 has now been found to interact with the Cdc42/Rac effector IQGAP and mediate transient capture of microtubules.

Microfilament Proteins↗

PubMed2002

Optimization of experimental parameters for electron capture dissociation of peptides in a Fourier transform mass spectrometer.

This paper describes our effort in optimizing the experimental parameters for electron capture dissociation (ECD) of peptides in a commercially available Fourier-transform mass spectrometer. Using a built-in electrically heated filament electron gun, it was demonstrated that good quality ECD spectra of peptides (MW < 2500) could be obtained by irradiating the isolated peptide molecule-ions with a short pulse (50 ms) of low-energy (3-6 eV) electrons. In addition, we have also demonstrated that pulsing of inert cooling gas (argon) could further improve the intensity of the ECD-induced fragment ions. Due presumably to the influence of the strong magnetic field on the trajectories of electrons, the distance between the electron gun and the trapped-ion cell (i.e., 108 mm versus 20 mm) was found to have little influence on the efficiency of the ECD process(es). From a systematic study on the impact of the filament heating current, filament bias voltage, and electron irradiation time on the intensities of precursor ions and various fragment ions, it was postulated that subsequent capture of electrons by the fragment ions, i.e., neutralization of the fragment ions, might be a significant event for limiting the intensity of the fragment ions.

Amino Acid Sequence↗

PubMed2002

Identification and localization of the fatty acid modification in ghrelin by electron capture dissociation.

Electron capture dissociation (ECD) has been demonstrated to be an effective fragmentation technique for characterizing the site and structure of the fatty acid modification in ghrelin, a 28-residue growth-hormone-releasing peptide that has an unusual ester-linked n-octanoyl (C8:0) modification at Ser-3. ECD cleaves 21 of 23 possible backbone amine bonds, with the product ions (c and z* ions) covering a greater amino acid sequence than those obtained by collisionally activated dissociation (CAD). Consistent with the ECD nonergodic mechanism, the ester-linked octanoyl group is retained on all backbone cleavage product ions, allowing for direct localization of this labile modification. In addition, ECD also induces the ester bond cleavage to cause the loss of octanoic acid from the ghrelin molecular ion; the elimination process is initiated by the capture of an electron at the protonated ester group, which is followed by the radical-site-initiated reaction known as alpha-cleavage. The chemical composition of the attached fatty acid can be directly obtained from the accurate Fourier transform ion cyclotron resonance (FTICR) mass measurement of the ester bond cleavage product ions.

Acylation↗

PubMed2003

Electron capture dissociation and infrared multiphoton dissociation of oligodeoxynucleotide dications.

We report electron capture dissociation (ECD) and infrared multiphoton dissociation (IRMPD) of doubly protonated and protonated/alkali metal ionized oligodeoxynucleotides. Mass spectra following ECD of the homodeoxynucleotides polydC, polydG, and polydA contain w or d "sequence" ions. For polydC and polydA, the observed fragments are even-electron ions, whereas radical w/d ions are observed for polydG. Base loss is seen for polydG and polydA but is a minor fragmentation pathway in ECD of polydC. We also observe fragment ions corresponding to w/d plus water in the spectra of polydC and d(GCATGC). Although the structure of these ions is not clear, they are suggested to proceed through a pentavalent phosphorane intermediate. The major fragment in ECD of d(GCATGC) is a d ion. Radical a- or z-type fragment ions are observed in most cases. IRMPD primarily results in base loss, but backbone fragmentation is also observed. IRMPD provides more sequence information than ECD, but the spectra are more complex due to extensive base and water losses. It is proposed that the smaller degree of sequence coverage in ECD, with fragmentation mostly occurring close to the ends of the molecules, is a consequence of a mechanism in which the electron is captured at a P=O bond, resulting in a negatively charged phosphate group. Consequently, at least two protons (or alkali metal cations) must be present to observe a w or d fragment ion, a requirement that is less likely for small fragments.

Calibration↗

PubMed2003

Electron capture and collisionally activated dissociation mass spectrometry of doubly charged hyperbranched polyesteramides.

Electron capture dissociation (ECD) of doubly protonated hyperbranched polyesteramide oligomers (1100-1900 Da) was examined and compared with the structural information obtained by low energy collisionally activated dissociation (CAD). Both the ester and amide bonds of the protonated species were cleaved easily upon ECD with the formation of odd electron (OE(.+)) or even electron (EE(+)) fragment ions. Several mechanistic schemes are proposed that describe the complex ECD fragmentation behavior of the multiply charged oligomers. In contrast to studies of biomolecules, the present results indicate that consecutive cleavages induced by intramolecular H-shifts are significant for ECD and of less importance for low energy CAD. The capture of an electron by the ionized species results in fragmentation associated with a redistribution of the excess internal energy over the products and the subsequent bond cleavage. Low energy, multiple collision CAD is found to be a more selective dissociation method than ECD in view of the observation that only amide bonds are cleaved for most of the hyperbranched polymers examined with CAD in this study. ECD appears not to provide complementary structural information compared to CAD in the study of hyperbranched polymers, even though a significantly more complex ECD fragmentation behavior is observed. ECD is shown to be of use for the structural characterization of large oligomers that may not dissociate upon low energy CAD. This is a direct result of the fact that ECD produces ionized hyperbranched oligomers with a relatively high internal energy.

Journal Article↗

PubMed2003

An antibiotic linked to peptides and proteins is released by electron capture dissociation fourier transform ion cyclotron resonance mass spectrometry.

Desfuroylceftiofur (DFC) is a bioactive beta-lactam antibiotic metabolite that has a free thiol group. Previous experiments have shown release of DFC from plasma extracts after addition of a disulfide reducing agent, suggesting that DFC may be bound to plasma and tissue proteins through disulfide bonds. We have reacted DFC with [Arg(8)]-vasopressin (which has one disulfide bond) and bovine insulin (which has three disulfide bonds) and analyzed the reaction products by use of electron capture dissociation Fourier transform ion cyclotron resonance mass spectrometry (ECD FT-ICR MS), which has previously shown preferential cleavage of disulfide bonds. We observe cleavage of DFC from vasopressin and insulin during ECD, suggesting that DFC is indeed bound to peptides and proteins through disulfide bonds. Specifically, we observed dissociative loss of one, as well as two, DFC species during ECD of [vasopressin + 2(DFC-H) + 2H](2+) from a single electron capture event. Loss of two DFCs could arise from either consecutive or simultaneous loss, but in any case implies a gas phase disulfide exchange step. ECD of [insulin + DFC + 4H](4+) shows preferential dissociative loss of DFC. Combined with HPLC, ECD FT-ICR-MS may be an efficient screening method for detection of drug-biomolecule binding.

Anti-Bacterial Agents↗

PubMedDate not supplied

A rapid electron-capture gas chromatographic procedure for the analysis of p-hydroxymephenytoin.

An electron-capture gas chromatographic procedure was developed for detection and quantification of p-hydroxymephenytoin (OHMEP), a metabolite of S-mephenytoin, in human liver microsomal preparations. OHMEP was derivatized with pentafluorobenzoyl chloride (PFBC) under basic aqueous conditions prior to analysis on a gas chromatograph equipped with a capillary column and an electron-capture detector. Dextrorophan was carried through the procedure as internal standard. The structure of the PFB derivative was confirmed using combined gas chromatography-mass spectrometry (GC-MS). The procedure is rapid and reproducible and produces a stable derivative that has excellent chromatographic properties. The limit of detection was less than 5 ng/ml, and the method was applied to extracts of human liver microsomes, which had been incubated with S-mephenytoin [a probe substrate for cytochrome P450 (CYP) 2C19].

Anticonvulsants↗

PubMedDate not supplied

A rapid method of determining amphetamine in plasma samples using pentafluorobenzenesulfonyl chloride and electron-capture gas chromatography.

INTRODUCTION: Acute administration of (+)-amphetamine has been used as a model for mania in humans since it mimics the physiological, biochemical, and cognitive effects seen in mania. A rapid and sensitive method for the determination of amphetamine in human plasma samples using gas chromatography with electron-capture detection was developed in our laboratory to follow the time course of amphetamine levels in patients receiving this drug as part of a study using amphetamine as a model for mania. METHODS: Blood samples were taken from healthy male volunteers at 30, 60, 90, 150, 210, 240, and 480 min after administration of 25 mg of (+)-amphetamine. Plasma was isolated by centrifugation and used for the analysis. This method is a modification of the procedure described by Paetsch et al. [J. Chromatogr. 573 (1992) 313] for the determination of amphetamine in rat brain tissue. Amphetamine was derivatized under basic conditions using pentafluorobenzenesulfonyl chloride (PFBSC) prior to analysis on a gas chromatograph equipped with a capillary column and an electron-capture detector. The internal standard used was benzylamine. The structure of the amphetamine derivative was confirmed using combined gas chromatography-mass spectrometry (GC-MS). RESULTS: The limit of detection was <1 ng/ml, and the method was linear in the 1- to 100-ng range used. Mean amphetamine levels peaked at 3.5 h after drug administration, and were 40.8 +/- 1.5 ng/ml at that time. DISCUSSION: This procedure produces a stable derivative with excellent chromatographic properties and is both simple and reproducible.

Adolescent↗

PubMed2001

Covalent capture: a new tool for the purification of synthetic and recombinant polypeptides.

BACKGROUND: Purification of polypeptides and proteins derived from recombinant DNA techniques and of long synthetic polypeptides often represents a challenge. Affinity methods exist, but generally require addition of a large recognition unit to the target protein and use of expensive purification media. Use of large units is dictated by the characteristics of non-covalent complexes, where the energy necessary to form the complex derives from the sum of multiple weak energy interactions. Covalent interactions in contrast are of high energy, even when only a few bonds are formed. We decided to explore the use of the reversible covalent bond formed between N-terminal cysteine and threonine residues with an aldehyde as a method of protein purification. RESULTS: A series of test peptides with N-terminal cysteine and threonine were captured by a polyethyleneglycol-polyacrylamide resin to which an aldehyde function had been grafted. Peptides with other amino acids at the N-terminus did not interact with the resin. A recombinant polypeptide with N-terminal cysteine was purified to 90% purity in one step. Polypeptides were eluted from the resin simply by adding a hydroxylamine derivative, which reacts with aldehyde functions to form an oxime. CONCLUSIONS: Polypeptides possessing N-terminal cysteine or threonine can be easily purified using this 'covalent capture' approach.

Aldehydes↗

PubMed2003

Plasma glucocorticoid concentrations in free-ranging platypuses (Ornithorhynchus anatinus): response to capture and patterns in relation to reproduction.

Energy demands in the platypus are likely to increase in the breeding season, which occurs from winter to early spring. Glucocorticoids, which play a major role in energy mobilisation, were measured in consecutive blood samples from free-ranging adults at approximately monthly intervals throughout the year. Glucose and free fatty acids were also measured in some samples. Plasma concentrations of glucocorticoids rose significantly during the first 30 min after capture, accompanied by a rise in free fatty acids, but no corresponding increase in glucose concentrations. We observed a strong pattern in plasma glucocorticoids in samples collected within 15 min of capture (indicative of pre-disturbance concentrations) in different phases of the annual reproductive cycle, with significantly higher levels in both males and females in the breeding season compared to the non-breeding season. These data, and the decline in tail fat stores that occur towards the end of the mating period (around October), suggest that platypus experience high-energy demands during this phase of reproduction. Plasma glucocorticoid concentrations in females sampled during the lactation period (October-February) were relatively low, and similar to those in females sampled in the non-breeding, non-lactation period (March-June). The latter requires further investigation as results may have been influenced by sampling limitations.

Animals↗

PubMed2002

Identification of Tula hantavirus in Pitymys subterraneus captured in the Cacak region of Serbia-Yugoslavia.

BACKGROUND: Atypical serum neutralizing antibody responses to prototype strains of Puumala viruses in some patients with hemorrhagic fever with renal syndrome (HFRS) have long suggested the existence of other hantaviruses in the Balkans. OBJECTIVE: To determine the presence of arvicolid rodent-borne Puumala-like hantaviruses in Yugoslavia. MATERIALS AND METHODS: Using reverse transcript-polymerase chain reaction, Tula virus RNA was amplified from lung tissues of a European pine vole (Pitymys subterraneus) captured in 1987, following an outbreak of HFRS in the Cacak region of Serbia-Yugoslavia. RESULTS: Sequence analysis of the entire coding region of the S segment and a 948-nucleotide region of the G2 glycoprotein-encoding M segment revealed divergence of approximately 14% from Tula virus strains harbored by European common voles (Microtus arvalis) captured in Central Russia and the Czech Republic. However, nearly complete identity was found in the corresponding deduced amino acid sequences. Moreover, phylogenetic trees constructed by the maximum parsimony and neighbor-joining methods indicated that this Pitymys-borne hantavirus shared a common ancestry with other Tula virus strains. CONCLUSIONS: The data demonstrate that Pitymys subterraneus also serves as a rodent reservoir of Tula virus in Serbia-Yugoslavia. To what extent this represents virus spillover from Microtus arvalis warrants further investigation.

Animals↗

PubMed2002

A mu-capture immunoassay for detection of human herpes virus-6 (HHV-6) IgM antibodies in human serum.

BACKGROUND: Human herpes virus-6 (HHV-6) was first isolated in 1986. It has been shown to cause exanthema subitum and has been associated with various other diseases. HHV-6 infection is widespread, and more than 90% of the population have antibodies against HHV-6 at the age of 2 years. Once acquired, the virus remains latent in the body. This makes it difficult to draw any conclusions about a causal relationship between the demonstration of HHV-6 and a specific disease. OBJECTIVES: This work was to develop a mu-capture HHV-6 IgM enzyme linked immuno sorbent assay (ELISA) for use in routine diagnosis and for wide scale patient population analysis. STUDY DESIGN: A mu-capture HHV-6 IgM ELISA was established. A total of 682 sera consisting of 585 sera from Danish blood donors and 97 sera from patients with autoimmune antibodies were analysed in the HHV-6 IGM ELISA. One hundred and ninety-two sera had earlier been analysed for total HHV-6 antibody content in a competitive ELISA, 94 sera were analysed for cytomegalovirus (CMV) IgM and 57 sera for Epstein Barr virus (EBV) antibodies, using different ELISA assays. The results for 12 primary infections with HHV-6 are also reported. RESULTS: A HHV-6 IgM optical density (OD)-ratio was calculated according to a constant positive control. An empirical cut off of 0.5 HHV-6 IgM OD-ratio was chosen (with regard to the 10 HHV-6 seroconverters), which resulted in a specificity of 97.5% of the HHV-6 IgM ELISA. Two of the three donor sera with HHV-6 IgM OD-ratios more than 1.05 had total HHV-6 antibody titers significantly above the group with IgM OD-ratios below 0.7 consisting with HHV-6 reactivation. There was no cross reactions to EBV or CMV IgM positive sera. CONCLUSION: The HHV-6 IgM ELISA seems valid to diagnose primary HHV-6 infection in particular in combination with the HHV-6 total antibody assay.

Antibodies, Viral↗

PubMed1999

Problems with biotin-labelled virions as probes in poliovirus-specific mu-capture-IgM assays.

BACKGROUND: We have previously developed a mu-capture-based radioimmunoassay (RIA) for detecting virus-specific IgM for the diagnosis of poliomyelitis. To probe captured IgM we used radiolabelled, purified preparations of representatives of each poliovirus serotype (Roivainen M, Agboatwalla M, Stenvik M, Rysa T, Akram DS, Hovi T. J Clin Microbiol 1993;31:2427-32). However, this assay is not directly applicable for wider use because preparation and handling of radioactive reagents is cumbersome and potentially hazardous. OBJECTIVES: To develop a non-radioactive modification of the assay retaining the number of steps and reagents to a minimum. STUDY DESIGN: Replacement of radioactive labelling by in vitro biotinylation of purified virions, and detection of bound virions with horseradish peroxidase-conjugated streptavidin. To study sensitivity and poliovirus serotype-specificity, 129 sera and 115 CSF specimens from children with acute poliomyelitis were used in comparative tests with the in-house RIA. In addition, sera from 40 healthy adults and 11 paired sera from patients with non-polio enterovirus infection were used to assess specificity. RESULTS: While results with the new test on specimens from clinically confirmed polio patients revealed some correlation with those obtained in the in-house RIA, studies on sera from healthy adults indicated, that non-specific binding of biotinylated virions is difficult to control. Moreover, examination of sera from patients with non-polio enterovirus infection suggested frequently occurring cross-reactivity between immune responses induced by polio- and other enterovirus infections. The latter were also seen in the RIA. CONCLUSION: Cross-reactive epitopes between poliovirus serotypes and between polioviruses and other enteroviruses may compromise the use of an assay for virus-specific IgM for poliovirus diagnosis. Biotinylation of the virions seemed to aggravate these problems.

Adult↗

PubMed2002

Simultaneous determination of fluoxetine and its metabolite p-trifluoromethylphenol in human liver microsomes using a gas chromatographic-electron-capture detection procedure.

An gas chromatography-electron-capture detection method has been developed for simultaneous determination of fluoxetine and p-trifluoromethylphenol (TFMP), an O-dealkylated metabolite of fluoxetine in human liver microsomes. Prior to the analysis, aliquots of alkalinized microsomal mixture were extracted with ethyl acetate solvent containing acetonitrile (10%, v/v) and the derivatizing reagent, pentafluorobenzenesulfonyl chloride (0.1%, v/v). The organ phase was retained and taken to dryness, the residue was reconstituted in methanol, and the aliquot of extracts was injected directly into a gas chromatograph equipped with an electron-capture detector. 2,4-Dichlorophenol was added to the initial incubation mixture and carried through the procedure as the internal standard. The method provided the mean recoveries of up to 103% for fluoxetine and 104% for TFMP. Acceptable relative standard deviations were found for both within-run and day-to-day assays. The practical limit of detection (signal-to-noise ratio=3) was 1.62 ng/ml for TFMP and 6.92 ng/ml for fluoxetine in human liver microsomes, and the limit of quantitation was 8.1 pg for TFMP and 34.6 pg for fluoxetine. The assay is rapid and sensitive and has been applied successfully to simultaneous quantification of fluoxetine and TFMP in human liver microsomes with different CYP2C19 genotypes.

Chromatography, Gas↗

PubMed1981

Antibody capture radioimmunoassay for anti-rubella IgM.

An M-antibody capture radioimmunoassay (MACRIA) for anti-rubella IgM was developed. Under optimum conditions positive serum specimens bound up to 20 times as much radioactivity as negative specimens. Positive reactions were expressed in arbitrary units/ml by comparison with a calibration curve derived from results obtained with dilutions of a standard serum. The specificity of the assay was confirmed by testing IgM and IgG rich fractions of positive sera. One hundred and forty specimens from blood donors, patients whose sera contained rheumatoid factor and patients with acute, non-rubella, virus infections were tested by MACRIA. No significant non-specific reactions were detected. Paired sera from acute rubella (25 patients) and individual sera from suspected rubella (69 patients) were tested for anti-rubella IgM by MACRIA and by haemagglutination inhibition following sucrose-density-gradient fractionation. There was close agreement between the two methods. The capture assay was more sensitive and could be used to detect the weak IgM response in women given RA 27/3 vaccine. After the natural infection, the MACRIA was strongly positive for two months and remained weakly so for a further two months. Repeat testing of sera demonstrated good reproducibility of the assay. MACRIA proved a simple, sensitive and specific test for anti-rubella IgM and compared favourably with currently used techniques.

Animals↗