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PubMed1996

Purification of single-stranded M13 DNA by cooperative triple-helix-mediated affinity capture.

A solid-phase triple-helix-mediated affinity capture method is described for the purification of single-stranded M13 DNA for use as template in fluorescence-based DNA sequencing reactions. In this method, a biotinylated polypyrimidine oligonucleotide "loop" bound to streptavidin-coated magnetic beads is used to selectively capture single-stranded M13 DNA from high-titer phage supernatant through the formation of a cooperative triple helix (CTH) complex between the oligonucleotide and a polypurine site previously cloned into the M13 vector. Capture is accomplished at acidic pH to encourage triple-helix formation, while elution is performed at alkaline pH with heating to destroy the CTH complex. The beads can be reused up to three times without probe replenishment. Yields of M13 ssDNA in excess of 1 microgram per milliliter of culture are obtained, sufficient for use as template in fluorescence-based DNA sequencing reactions.

Bacteriophage M13↗

PubMed1999

Development of a carbon dioxide-capture assay in microtiter plate for aspartyl-beta-hydroxylase.

CO2-capture methods have been used for assaying many decarboxylating enzymes including hydroxylation-coupled decarboxylation reactions. The traditional CO2-capture method involves performing the reaction in capped tubes and radiometric measurement of trapped 14CO2 by scintillation counting. In this report, a 14CO2-capture method in a 96-well microtiter plate format has been developed and a phosphor imaging system has been employed for sample measurement. The new assay method has been used successfully to assay aspartyl-beta-hydroxylase activity in microtiter plate format. The results obtained here compare favorably with those obtained from the traditional tube method. The method is sensitive, suitable for high throughput, and generally applicable to many CO2-releasing enzyme assays.

Carbon Dioxide↗

PubMed2002

A rapid method to capture and screen for transcription factors by SELDI mass spectrometry.

A novel method to screen for transcription factors binding to promoter DNA sequences has been developed using DNA chip surfaces and mass spectrometry. This technique was demonstrated with Escherichia coli lac repressor, LacI. The consensus promoter binding sequence for LacI and a scrambled version of the same DNA sequence were prepared on two affinity chip surfaces. Total E. coli protein lysate was applied to the two surfaces. A 38.2 kDa protein, as detected by SELDI-MS, was captured on the chip surface containing the binding sequence for LacI but not on the surface containing the scrambled sequence. The protein was identified following one-step, small-scale affinity capture and peptide mapping. Subsequent database searches identified the 38.2 kDa protein as the lac repressor of E. coli. We discuss application of DNA chip affinity capture to characterize transcription factors and to screen for differences in cellular regulatory networks.

Bacterial Proteins↗

PubMed2001

Rapid, efficient genotyping of clinical tumor samples by laser-capture microdissection/PCR/SSCP.

BACKGROUND: Mutation analysis is becoming increasingly important in clinical practice, since sporadic mutations in tumors often correlate with prognosis and/or therapeutic response. However, the labor-intensive nature of the molecular analyses has limited the routine clinical use of tumor genotyping. Laser-capture microdissection (LCM) allows procurement of relatively pure tumor cell populations. We have investigated the possibility that the use of laser-capture microdissection would allow elimination of time-consuming intermediate steps in tumor genotyping. Design. Archival formalin-fixed, paraffin-embedded tissues from seven cases of colorectal adenocarcinoma were laser- and hand-microdissected and subsequently evaluated by PCR/SSCP/sequencing for Ki-ras exon 1 and p53 exons 5, 7, and 8. Results. Mutations in Ki-ras exon 1 and/or p53 exons 5 and 7 were detected in five of the seven samples. In the hand-microdissected samples, confident identification of mutations was possible in several cases only after band excision, DNA elution, reamplification, and verification of mutant enrichment by a second SSCP analysis prior to sequencing. In the laser-microdissected samples, confident mutation identification was possible in all cases with direct sequencing of the original PCR product, reducing the time required for molecular analysis to 3 days. Conclusion. Using laser-capture microdissection, mutant signals are strong enough to sequence directly from original PCR products. With rapid, efficient genotyping by LCM/PCR/SSCP, results can be incorporated directly into the surgical pathology report.

Base Sequence↗

PubMed2000

Contribution of human papillomavirus testing by hybrid capture in the triage of women with repeated abnormal pap smears before colposcopy referral.

OBJECTIVE: The purpose of this work was to evaluate the ability of testing for high-risk human papillomavirus (HPV) types using the hybrid capture technique to predict the presence of cervical intraepithelial neoplasia (CIN) II,III in patients with repeated atypical squamous cells of undetermined significance (ASCUS) and low-grade squamous intraepithelial lesion (LGSIL) on Pap smears. METHODS: Hybrid capture testing and tissue biopsy were performed on 503 consecutive women with ASCUS or LGSIL on repeated Pap smears who were referred for colposcopy. RESULTS: A highly significant association (P < 0.0001) was found between a positive test for high-risk HPV types and CIN II,III, with an 87.0% positive predictive value and a 95.7% negative predictive value. In 226 women with ASCUS on repeated Pap smears, a positive test for high-risk HPV types had a 85.7% sensitivity and a 97% specificity for CIN II,III. In 277 patients with LGSIL on repeated Pap smears, a positive test for high-risk HPV types had an 88.2% sensitivity and a 94.7% specificity for CIN I,II. Reserving colposcopy examination for women who were positive for high-risk HPV types would have reduced the number of referrals for colposcopy to 24.6% and maintained a sensitivity of 87.0% for CIN II,III. CONCLUSIONS: A positive hybrid capture test for high-risk HPV types was highly sensitive and specific for the presence of CIN II,III in patients with ASCUS and LGSIL on repeated Pap smears. We believe that improved methodology will eventually enable more selective colposcopy referrals without affecting patient safety among these women.

Adolescent↗

PubMed2002

Blockerette-ligated capture T7-amplified RT-PCR, a new method for determining flanking sequences.

We have developed a highly sensitive PCR-based technique termed blockerette-ligated capture T7-amplified reverse-transcription PCR (BCT-RT-PCR), which can be used to characterize unknown proviral flanking sequences from a broad range of samples and depends only on knowing the retroviral sequence. This method incorporates several essential elements to make it both sensitive and specific, including a "blockerette" linker, magnetic capture of target sequences, and exponential replication of potentially rare sequences using a nested promoter for T7 RNA polymerase, followed by nested RT-PCR. This linkage of methods was designed to increase sensitivity by decreasing DNA complexity in favor of specific amplification. The resulting PCR products can be directly sequenced to determine integration sequences. We have successfully determined the integration sequences from as little as 30 pg of provirus-containing DNA in the background of 30 ng of untransduced DNA, representing a 0.1% transduction rate. We also show this technique to have single-cell resolution even in the background of 5000 cells. We describe here for the first time the combined use of BCT-RT-PCR and laser-capture microscopy (LCM) for precise isolation of retrovirally transduced cells followed by determination of the 3' retroviral flanking sequence at the single-cell level. This method will aid significantly in determining clonality both in transplant experiments and in the generation of clonal cell populations.

3' Flanking Region↗

PubMedDate not supplied

New device for wire and catheter capturing: in vivo experiments and first clinical experience.

PURPOSE: A new self-expanding tulip sheath was tested for percutaneous wife capturing in a femoro-femoral pull-through maneuver. METHODS: In six sheep, a self-expanding tulip sheath was introduced into the right iliac artery. A 5 Fr catheter and an 0.035-inch guidewire were guided into the iliac artery from a left femoral approach. Thirty-six tests were performed to guide the guidewire out of the right groin using the tulip sheath, by catching the catheter first in 18 tests and just by catching the guidewire in the other 18. As a control, six tests were performed using a conventional sheath. We performed one clinical arterial application. RESULTS: There was prompt capture of the wire in 35 of 36 tests using a self-expanding sheath. Five of six tests using a conventional sheath failed. The clinical application of the tulip principle was successful. CONCLUSION: Using the self-expanding tulip sheath to facilitate wire capture during pull-through maneuvers, even in difficult situations, is promising.

Aortic Dissection↗

PubMed1995

Prey capture phase of feeding behavior in the pteropod mollusc Clione limacina: neuronal mechanisms.

The prey capture phase of feeding behavior in the pteropod mollusc Clione limacina consists of an explosive extrusion of buccal cones, specialized structures which are used to catch the prey, and acceleration of swimming with frequent turning and looping produced by tail bend. A system of neurons which control different components of prey capture behavior in Clione has been identified in the cerebral ganglia. Cerebral B and L neurons produce retraction of buccal cones and tightening of the lips over them--their spontaneous spike activities maintain buccal cones in the withdrawn position. Cerebral A neurons inhibit B and L cells and produce opening of the lips and extrusion of buccal cones. A pair of cerebral interneurons C-BM activates cerebral A neurons and synchronously initiates the feeding motor program in the buccal ganglia. Cerebral T neurons initiate acceleration of swimming and produce tail bending which underlies turning and looping during the prey capture. Both tactile and chemical inputs from the prey produce activation of cerebral A and T neurons. This reaction appears to be specific, since objects other than alive Limacina or Limacina juice do not initiate activities of A and T neurons.

Action Potentials↗

PubMed1975

The demonstration of arylsulfatases with 4-nitro-1,2-benzenediol mono(hydrogen sulfate) by the formation of osmium blacks at the sites of copper capture.

A new method is described for the direct cytochemical demonstration of lysosomal arylsulfatases utilizing a synthetic substrate, 4-nitro-1,2-benzenediol mono(hydrogen sulfate), and a copper capture reaction. A small amount of Hatchett's brown (cupric ferrocyanide, Cu2Fe(CN)6-7 H2O) formed at the subcellular sites of copper capture is then utilized as a heterogeneous catalyst to effect the oxidative polymerization of 3,3'-diaminobenzidine which results in the formation of an insoluble, highly colored osmiophilic indamine polymer at the sites of enzymatic activity. The reaction product even at this stage prior to osmication is highly visible. It is readily seen with a light microscope in 50 mum sections of fixed tissues prepared with a mechanical chopper or in 10 micron cryostat sections treated for arylsulfatase activity. Upon osmication, an electron-opaque osmium black is formed which is much less soluble than the products of either the lead or barium capture reactions currently used for the demonstration of arylsulfatase with the electron microscope. The selection of areas of plastic-embedded tissues for ultrathin sectioning is facilitated by the ready visibility of these osmium black end products on 1-2 mum plastic sections which can be studied with the light microscope. This method gives permanent specimens demonstrating arylsulfatases A or B in lysosomes and autophagic vacuoles. In addition, enzyme activity is seen occasionally in the Golgi region or lamellae of certain cells believed to be elaborating sulfated products. In these instances, it may be demonstrating sulfotransferase activity.

Animals↗

PubMed1995

Microparticle agglutination versus antibody-capture enzyme immunoassay for diagnosis of community-acquired Mycoplasma pneumoniae pneumonia.

Community-acquired Mycoplasma pneumoniae pneumonia is a common disease which is usually diagnosed by serological methods. The objective of the present study was to understand the diagnostic significance and test characteristics of two different serological tests used to identify current Mycoplasma pneumoniae infection. Three hundred sixty-six patients who suffered from community-acquired pneumonia served as the study population. Six hundred ninety-four (328 paired and 38 unpaired) sera were examined for the presence of antibodies to Mycoplasma pneumoniae with commercial kits based on two serological methods, microparticle agglutination and antibody-capture EIA. Agreement between the two kits was 85.2% when individual sera were compared (kappa = 0.62) and 88.5% when patients were compared (Kappa = 0.69). The positive predictive value and the specificity for the identification of current Mycoplasma pneumoniae infection using a single acute-phase serum were 49.3% and 86.9%, respectively, for the microparticle agglutination method, compared to 91.3% and 97.7% for the antibody-capture EIA method (p < 0.001). The negative predictive value and the sensitivity were 86.3% and 48.1% for the microparticle agglutination, not significantly different from the corresponding values of 86.5% and 61.2% for the antibody-capture EIA. It is concluded that the overall agreement between the two methods tested is good, but not perfect. The methods complement each other in the identification of Mycoplasma pneumoniae as the causative agent in patients with community-acquired pneumonia.

Adolescent↗

PubMed1987

Ultrasensitive analysis of microbial fatty acids using gas chromatography with electron capture detection.

Pentafluorobenzyl and pentafluoropropionyl/pentafluorobenzyl esters of bacterial fatty acids were analysed by capillary gas chromatography using both flame ionization and electron capture detection. No differences between the relative peak areas of the various fatty acids were observed as regards the two detectors used except that response of the electron capture dectector to the hydroxy acid derivatives exceeded that to the non-hydroxy acid derivatives by 20%. Use of the described derivatives in combination with electron capture detection yields chromatograms comparable with those obtained by analysis of methyl esters using flame ionization detection but with superior sensitivity.

Bacteria↗

PubMed1985

An electron-microscopical analysis of capture and initial stages of penetration of nematodes by Arthrobotrys oligospora.

A detailed analysis was made of the capture and subsequent penetration of nematodes by the nematophagous fungus Arthrobotrys oligospora using different electron-microscopical techniques. Capture of nematodes by this fungus occurred on complex hyphal structures (traps) and was effectuated by an adhesive coating, present on these trap cells. The adhesive layer was largely fibrillar in nature and was absent on cells of normal hyphae. Following capture, penetration hyphae were formed at those sites where the trap cell wall was anchored to the nematode cuticle by the adhesive. New walls of these hyphae were formed underneath the original trap cell walls, which were partly hydrolysed to allow growth and development of the penetration tubes through the adhesive coating towards the cuticle. Our observations indicated that the cuticle of the nematode was subsequently penetrated by the penetration tubes by mechanical means. After penetration a large infection bulb was formed from which trophic hyphae arose. Cytochemical experiments indicated that the sites of penetration of the cuticle were intensely stained for acid phosphatase activity. At later stages of infection activity of this enzyme was present throughout the nematode contents; the enzyme was most probably secreted by complex membranous structures associated with the cytoplasmic membrane of the infection bulb and the trophic hyphae.

Acid Phosphatase↗

PubMed1980

The determination of steroids with and without natural electrophores by gas chromatography and electron-capture detection.

The response of the electron-capture detector to organic compounds is poorly defined, and the steroids are no exception to this observation. For those steroids which are naturally electron-capturing, the structures of the electrophores will be defined. Other steroids can be made electron-capturing by the formation of appropriate derivatives. Some new or infrequently used reagents for this purpose (flophemesyl ethers, t-bulflophemesyl ethers, pentafluorophenylhydrazone derivatives and halogen-substituted aromatic boronic acids) are described.

Animals↗

PubMed2003

Translocation breakpoint mapping and sequence analysis in three monosomy 1p36 subjects with der(1)t(1;1)(p36;q44) suggest mechanisms for telomere capture in stabilizing de novo terminal rearrangements.

Monosomy 1p36 results from a variety of chromosome rearrangements, including terminal deletions, interstitial deletions, derivative chromosomes, and complex rearrangements. Our previous molecular studies on a large cohort of monosomy 1p36 subjects suggest that a significant percentage of terminal deletions of 1p36 are stabilized by the acquisition of telomeric sequences from other chromosome ends, forming derivative chromosomes (i.e., "telomere capture"). However, the molecular mechanism(s) that results in and/or stabilizes terminal deletions of 1p36 by telomere capture is poorly understood. In this report, we have mapped the translocation breakpoints in three subjects with der(1)t(1;1)(p36;q44) chromosomes by fluorescence in situ hybridization (FISH). These results indicate that the breakpoint locations are variable in all three subjects, with no common 1p deletion or 1q translocation breakpoints. In addition, sequence analysis of the 1p and 1q breakpoint-containing clones did not identify homologous sequences or low-copy repeats in the breakpoint regions, suggesting that nonallelic homologous recombination did not play a role in mediating these rearrangements. Microsatellite marker analysis indicates that two of the three derivative chromosomes were formed by intra-chromosomal rearrangements. These data are consistent with a number of recent reports in other model organisms that suggest break-induced replication at the site of a double-strand break may act as a mechanism of telomere capture by generating nonreciprocal translocations from terminally deleted chromosomes. Alternative models are also discussed.

Chromosome Aberrations↗

PubMed2006

Characterization of product capture resin during microbial cultivations.

Various bioactive small molecules produced by microbial cultivation are degraded in the culture broth or may repress the formation of additional product. The inclusion of hydrophobic adsorber resin beads to capture these products in situ and remove them from the culture broth can reduce or prevent this degradation and repression. These product capture beads are often subjected to a dynamic and stressful microenvironment for a long cultivation time, affecting their physical structure and performance. Impact and collision forces can result in the fracturing of these beads into smaller pieces, which are difficult to recover at the end of a cultivation run. Various contaminating compounds may also bind in a non-specific manner to these beads, reducing the binding capacity of the resin for the product of interest (fouling). This study characterizes resin bead binding capacity (to monitor bead fouling), and resin bead volume distributions (to monitor bead fracture) for an XAD-16 adsorber resin used to capture epothilone produced during myxobacterial cultivations. Resin fouling was found to reduce the product binding capacity of the adsorber resin by 25-50%. Additionally, the degree of resin bead fracture was found to be dependent on the cultivation length and the impeller rotation rate. Microbial cultivations and harvesting processes should be designed in such a way to minimize bead fragmentation and fouling during cultivation to maximize the amount of resin and associated product harvested at the end of a run.

Adsorption↗

PubMed2006

Delivery of antibody-captured proteins into living cells using PTD-fused protein A.

Protein transduction domain (PTD)-mediated protein delivery into animal cells is a useful technique for regulating cellular functions. Proteins captured by antibodies were delivered into living cells using an antibody/PTD-fused protein A complex. As a model protein, fluorescent-modified antibodies, captured by their respective primary antibody, were analyzed by fluorescence-activated cell sorting (FACS) which showed that the fluorescent-modified antibodies were directly delivered into cells. Peroxidase, captured by its specific antibody, was also delivered into cells and retained its activity.

Animals↗

PubMed1983

Ultrarapid single-capture train stimulation for termination of ventricular tachycardia.

Termination of ventricular tachycardia (VT) with single programmed extrastimuli or slow underdrive competitive pacing is often possible. The usefulness of these techniques is limited because the zone in the cardiac cycle during which programmed stimuli are effective may be narrow, with significant time elapsing until termination of the VT, and the zone may vary markedly between episodes, rendering preset extrastimuli ineffective. Ultrarapid trains of stimuli beginning during the refractory period and of a duration sufficient to cause only a single capture should terminate all tachycardias whose termination zone begins just after the refractory period. To test this hypothesis, 21 patients with 371 well-tolerated episodes of VT had single programmed extrastimuli and trains of 10 bipolar stimuli at 25, 50, and 100 Hz. VT was terminated 1 or more times in 18 of the 21 patients with programmed extrastimuli or trains, or both. Forty-eight percent of 290 episodes were terminated with programmed stimulation, compared with 56% of 120 episodes with trains (difference not significant). In 98 episodes, the effects of trains and programmed stimulation could be matched (same patient, date, tachycardia morphology, rate, and drug therapy); the results were concordant (both techniques either terminated the VT or failed) in 92 and discordant in 6. With only 2 possible exceptions, the termination zone always began just after the refractory period; thus, for tachycardias terminating with single captures, the first train to achieve capture was effective, minimizing the duration of the VT.

Adolescent↗

PubMed1988

The captured volume of multilamellar vesicles.

Characterization of classical 'hand-shaken' multilamellar lipid vesicles (MLVs) confirmed that these systems exclude solute during formation thus confounding previous captured volume measurements which typically have utilized solute as a merker of the occluded aqueous space. We used solvent rather than solute to determine the captured volume of these systems and obtained values at least twice those previously reported. We present here a captured volume and lamellarity profile of 'hand-shaken' MLVs and suggest that these parameters are dependent on the lipid concentration present during hydration.

Electron Spin Resonance Spectroscopy↗