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At least 757 records · Page 42Linked to original sources

PubMed2000

Sensitive immunoassay of tissue cell proteins procured by laser capture microdissection.

Coupling laser capture microdissection (LCM) with sensitive quantitative chemiluminescent immunoassays has broad applicability in the field of proteomics applied to normal, diseased, or genetically modified tissue. Quantitation of the number of prostate-specific antigen (PSA) molecules/cell was conducted on human prostate tissue cells procured by LCM from fixed and stained frozen sections. Under direct microscopic visualization, laser shots 30 microm in diameter captured specific cells from the heterogeneous tissue section onto a polymer transfer surface. The cellular macromolecules from the captured cells were solubilized in a microvolume of extraction buffer and directly assayed using an automated (1.5 hour) sandwich chemiluminescent immunoassay. Calibration of the chemiluminescent assay was conducted by developing a standard curve using known concentrations of PSA. After the sensitivity, precision, and linearity of the chemiluminescent assay was verified for known numbers of solubilized microdissected tissue cells, it was then possible to calculate the number of PSA molecules per microdissected tissue cell for case samples. In a study set of 20 cases, using 10 replicate samples of 100 laser shots per sample, the within-run (intraassay) SD was approximately 10% of the mean or less for all cases. In this series the number of PSA molecules per microdissected tissue cell ranged from 2 x 10(4) to 6. 3 x 10(6) in normal epithelium, prostate intraepithelial neoplasia (PIN), and invasive carcinoma. Immunohistochemical staining of human prostate for PSA was compared with the results of the soluble immunoassay for the same prostate tissue section. Independent qualitative scoring of anti-PSA immunohistochemical staining intensity paralleled the LCM quantitative immunoassay for each tissue subpopulation and verified the heterogeneity of PSA content between tissue subpopulations in the same case. Extraction buffers were successfully adapted for both secreted and membrane-bound proteins. This technology has broad applicability for the quantitation of protein molecules in pure populations of tissue cells.

Calibration↗

PubMed2002

Immunomagnetic capture of lens membrane fractions containing steroid binding protein.

This study describes the use of magnetic Dynabeads to purify microsomes from a crude microsomal fraction. A 28 kDa membrane-associated protein is proposed to mediate the binding of progesterone and other steroid hormones to ocular lens membranes and the rapid-nongenomic actions of these steroids. The subcellular location of this membrane steroid binding protein (MSBP) was probed by capture of organelles containing MSBP by magnetic beads displaying an antibody to a cytoplasmic domain of the protein. The beads were exposed to a crude microsomal fraction from lens epithelia. Western blotting was used to identify captured organelles and confirm the presence of MSBP. Microsomes and trace fiber cell plasma membrane were captured. Microsomes contained the 28 kDa MSBP. Lens fiber cell membrane contained a 55 kDa immunoreactive protein. The role of this serendipitously recognized protein in binding of steroids is unknown.

Animals↗

PubMed2002

[Tuberculosis in the Zaragoza province. Estimation by means of the capture-recapture method].

INTRODUCTION: Tuberculosis (TB) is an underreported condition to the Communicable Diseases Control System (CDCS). The objective of this work was to estimate the actual incidence of tuberculosis and the epidemiological characteristics of the diseases in the Zaragoza province. MATERIAL AND METHODS: Retrospective study in which the capture-recapture method was used to estimate the number of tuberculosis cases, by using the microbiology laboratories and the CDSC as data sources. The socio-demographic and clinical characteristics of diagnosed patients throughout three years in this province were analyzed. RESULTS: The mean annual incidence rate of TB was 22.02 per 100,000 inhabitants according to microbiological data, and 48.5 per 100,000 according to the capture-recapture method. A total of 569 patients were studied, and 400 of them were males (70.3%). The mean age was 43.2 years, and the 25-34 year-old group had the highest number of cases (27.9%). Twenty-three percent of cases were co-infected with HIV, and 77.4% were parenteral drug abusers, and 4.4% immigrants. CONCLUSIONS: The capture-recapture method has demonstrated to be useful to know the relevance of TB in our setting. The actual incidence of tuberculosis was higher than that pointed out by the CDCS. Relevant differences were observed regarding incidence rates by age and sex groups. HIV infection and immigration do not appear to explain the frequency of this disease in our setting.

Adolescent↗

PubMed1986

A factor inhibiting ovum capture by the oviductal fimbriae present in endometriosis peritoneal fluid.

The cumulus-fimbria interaction was investigated in vitro with endometriosis peritoneal fluid (PF). On the oviductal fimbria of the golden hamster, incubated with endometriosis and nonendometriosis PF and culture medium 199 contained 4% bovine serum albumin (control), was placed the mouse oocyte-cumulus complex at 5-minute intervals for 60 to 70 minutes. Incubation periods until the loss of fimbrial capability of ovum capture (ovum capturability disappearance time [OCDT] ) were 23.3 +/- 2.7 (standard error of the mean), 51.7 +/- 2.9, and 61.3 +/- 0.9 minutes with endometriosis, nonendometriosis PF, and control, respectively. OCDT was significantly decreased with cell-free and ultrafiltrated endometriosis PF containing a molecular size greater than 100,000 (26.0 +/- 2.8 and 26.1 +/- 2.5 minutes, respectively). A factor inhibiting fimbrial capability of ovum capture is present in endometriosis PF.

Adult↗

PubMed2002

Strategy for highly selective ion-exchange capture using a charge-polarizing fusion partner.

To achieve efficient recovery of recombinantly produced target proteins using cation-exchange chromatography, a novel basic protein domain is used as a purification handle. The proteolytic instability usually encountered for basic peptide tags is avoided by the use of a highly constrained alpha-helical domain based on staphylococcal protein A into which positively charged amino acids have been introduced. Here we show that this domain, consisting of 58 amino acids with a calculated isoelectric point (pI) of 10.5, can be used to efficiently capture different fused target proteins, such as a bacterial DNA polymerase (Klenow fragment), a viral protease (3C) and a fungal lipase (Cutinase). In contrast to standard cation-exchange chromatography, efficient capture can be achieved also at a pH value higher than the pI of the fusion protein, demonstrated here by Zbasic-Klenow polymerase (pI approximately/= 5.8) and ZZ-Cutinase-Zbasic (pI approximately/= 7.2) both purified at a pH of 7.5. These results show that the Zbasic domain is able to confer a regional concentration of positive charge on the fusion protein even at a relatively high pH. Hence, the data suggest that this domain could be used for highly efficient and selective capture of target proteins at conditions where most host-cell proteins do not bind to the chromatographic resin. The obtained purity after this one-step procedure suggests that the strategy could be an alternative to standard affinity chromatography. Methods for site-specific proteolysis of the fusion proteins to release native target proteins are also discussed.

Base Sequence↗

PubMed1988

Femtogram detection of perfluorocarbon tracers using capillary gas chromatography-electron-capture negative ion chemical ionisation mass spectrometry.

An ultrasensitive and selective method has been developed for the detection of a range of perfluorocarbon tracers suitable for long-range atmospheric studies. Following direct injection onto a capillary column the perfluorocarbons are separated and detected using mass spectrometry with electron-capture negative ion chemical ionisation (ECNICI-MS) with methane as reagent gas and selected-ion monitoring. All nine perfluorocarbons tested underwent non-dissociative resonance electron capture to form the molecular anion which was the base peak in all cases. Using this technique, detection limits (signal-to-noise ratio of 2:1) of 3 and 2 fg were obtained for perfluoromethylcyclohexane and perfluoro-1,3-dimethylcyclohexane respectively. The corresponding detection limits using a conventional electron-capture detector were 8 and 9 fg respectively. In addition to increased sensitivity, the ECNICI-MS technique allows temperature programming for the simultaneous analysis of a range of perfluorocarbons and has superior selectivity being less prone to coeluting interferences. Furthermore the method is potentially one to two orders of magnitude more sensitive than the current gas chromatography-MS methods using 13C2H4 tracers.

Air Pollutants, Occupational↗

PubMed2003

Preparation and characterization of prototypes for multi-modal separation aimed for capture of positively charged biomolecules at high-salt conditions.

Several prototypes of aromatic (Ar) and non-aromatic (NoAr) cation-exchange ligands suitable for capture of proteins from high conductivity (ca. 30 mS/cm) mobile phases were coupled to Sepharose 6 Fast Flow. These new prototypes of multi-modal cation-exchangers were found by screening a diverse library of multi-modal ligands and selecting cation-exchangers resulting in elution of test proteins at high ionic-strength. Candidates were then tested with respect to breakthrough capacity of bovine serum albumin (BSA), human IgG and lysozyme in buffers adjusted to a high conductivity. By applying a salt-step or a pH-step the recoveries were also tested. We have found that aromatic multi-modal cation-exchanger ligands based on carboxylic acids seem to be optimal for the capture of proteins at high-salt conditions. Experimental evidence on the importance of the relative position of the aromatic group in order to improve the breakthrough capacity at high-salt conditions has been found. It was also found that an amide group on the alpha-carbon was essential for capture of proteins at high-salt conditions. Compared to a strong cation-exchanger such as SP Sepharose Fast Flow the best new multi-modal weak cation-exchangers have breakthrough capacities of BSA, human IgG and lysozyme that are 10-30 times higher at high-salt conditions. The new multi-modal cation-exchangers can also be used at normal cation-exchange conditions and with either a salt-step or a pH-step (to pH-values where the proteins are negatively charged) to accomplish elution of proteins. In addition, the functional performance of the new cation-exchangers was found to be intact after treatment in 1.0 M sodium hydroxide solution for 10 days. For BSA it was also possible to design cation-exchangers based on non-aromatic carboxyl acid ligands with high capacities at high-salt conditions. A common feature of these ligands is that they contain hydrogen acceptor groups close to the carboxylic group. Furthermore, it was also possible to obtain high breakthrough capacities for lysozyme and BSA of a strong cation-exchanger (SP Sepharose Fast Flow) if phenyl groups were attached to the beads. Varying the ligand ratio (SP/Phenyl) could be used for optimizing the function of mixed-ligand ion-exchange media.

Cation Exchange Resins↗

PubMed1982

Unusual capture phenomena during interaction of dissociated sinus and junctional rhythms: spontaneous expression of intranodal duality and gap phenomenon.

A ten year old boy presented with an irregular pulse which was caused by AV dissociation with capture beats and pauses. Electrophysiologic studies revealed interaction between sinus rhythm and junctional rhythm. Capture beats were of several types. Early captures blocked below the His bundle but reset the junctional pacemaker causing pauses. Earlier sinus beats conducted with marked atrio His delay. Measurement of intervals during spontaneous rhythm demonstrated a type I gap phenomenon due to dual AH pathways. This case illustrates the value of electrophysiologic studies in analysing complex arrhythmias.

Arrhythmia, Sinus↗

PubMed2002

Optimizing the hybrid capture II human papillomavirus test to detect cervical intraepithelial neoplasia.

OBJECTIVE: To estimate the optimal relative light unit ratio, and correspondingly viral load, of the hybrid capture II oncogenic human papillomavirus deoxyribonucleic acid test for detecting cervical intraepithelial neoplasia (CIN). METHODS: Women with abnormal cytology were referred for colposcopy, and a cervical swab or brush specimen was obtained for human papillomavirus testing. Sensitivities, specificities, and likelihood ratios of different relative light unit ratio cutoffs were calculated using a reference standard of colposcopy or biopsy of either CIN II+ (CIN II, III, or carcinoma), or CIN I+ (CIN I, CIN II+). The receiver operating characteristic curve was used to estimate optimal test-positive cutoff points for the hybrid capture II test. RESULTS: CIN II+ was found in 18.7% (98 of 524) and CIN I in 10.5% (55 of 524) of the women. The optimal relative light unit ratio was 15.56, giving a sensitivity and specificity of 82.7% and 73.2% for CIN II+, and 74.2% and 77.8% for CIN I+. In a stratified analysis, a higher relative light unit cutoff (15.19) optimized sensitivity and specificity for CIN II+ (sensitivity 81.8%, specificity 51.5%) for women with low-grade squamous intraepithelial lesions cytology, whereas the optimal cutoff was 2.36 (sensitivity 100%, specificity 73.0%) for women with atypical squamous cells of undetermined significance, yielding referral rates of 53.3% and 28.7%, respectively. Use of a swab or brush was not associated with the level of human papillomavirus detected (P >.05). CONCLUSION: Use of a higher cutoff for the relative light unit ratio (higher viral load) of the hybrid capture II test may improve the management of women, especially those with low-grade squamous intraepithelial lesions cytology.

Adolescent↗

PubMed2002

Fixational eye movements are not affected by abrupt onsets that capture attention.

Recent work shows that abrupt onsets reflexively capture attention and trigger saccades that compete with voluntary saccades. To test whether oculomotor capture occurs when no saccade is being planned, we measured fixational eye movements in the absence or presence of an abrupt onset at peripheral locations. We found no effect of abrupt onset location on the average pattern of eye movements during fixation. We conclude that the capture of eye movements by an abrupt onset only happens when the oculomotor system has been preset to make a saccade. This implies that the oculomotor system is not obligatorily driven by events in the visual array.

Adult↗

PubMed1997

Capture of visual direction: an unexpected phenomenon in binocular vision.

Binocular perception of visual direction is based on laws which were formulated more than 100 years ago. These laws govern the directions in which human beings perceive objects visible to both eyes (binocular objects) and objects visible to only one eye (monocular objects). We report here that the laws do not hold for monocular objects adjacent to binocular objects. The perceived directions of these monocular objects are captured by those of nearby binocular objects. Capture of binocular visual direction is an unexpected phenomenon because it refutes the generally accepted notion that a particular retinal location gives rise to a particular subjective visual direction. The practical consequence is that the subjective techniques for measuring eye position which are widely used in fundamental research and clinical practice are unreliable if they are used in densely structured stimuli. We suggest that capture results from a mechanism of lateral interaction between adjacent visual directions. This mechanism ensures that, despite eye movements, objects have the same spatial order in monocular and binocular vision. This conservation of spatial order also explains why retinal blind spots are not manifest in binocular vision.

Convergence, Ocular↗

PubMed1997

Capture of the visual direction of monocular objects by adjacent binocular objects.

Investigations of binocular visual direction have concentrated mainly on stationary objects. Eye positions were generally not measured and binocular fixation was assumed to be perfect. During the viewing of stationary objects, vergence errors are not negligible but small. During the viewing of moving objects, however, errors in binocular fixation are much larger. Existing rules for binocular visual direction were examined under the latter, more demanding viewing conditions. Eye movements were measured objectively by the scleral coil technique. Subjects viewed a large stereogram in which the half-images oscillated in counterphase. The stereogram contained two square random-dot patterns placed side by side with a gap in between. A vertical line, visible only to one eye, oscillated in the gap. Subjects were asked to adjust the amplitude of line motion until the line was perceived to be stationary. In so doing, they set amplitudes equal to the amplitudes of half-image motion if the gap between the patterns was narrow. They set amplitudes significantly smaller in wider gaps. Subjects made considerable fixational errors in following the oscillations of the line and the random-dot patterns. The results of the settings and of the retinal errors together refute existing rules for binocular visual direction of monocular objects. Perceived directions of monocular objects cannot be specified by geometrical rules that include only the positions of the objects and of the two eyes. The results suggest that perceived directions of monocular objects are captured by the binocular visual directions of adjacent binocular objects. Capture of binocular visual direction was found to be effective for gaps as wide as 8 deg between the binocular objects. The phenomenon of binocular capture has negative consequences for the general use of nonius lines as indicators of eye position.

Depth Perception↗

PubMed1997

Capture and transparency in coarse quantized images.

This study examines the effect of coarse quantization (blocking) on image recognition, and explores possible mechanisms. Thresholds for noise corruption showed that coarse quantization reduces drastically the recognizability of both faces and letters, well beyond the levels expected by equivalent blurring. Phase-shifting the spurious high frequencies introduced by the blocking (with an operation designed to leave both overall and local contrast unaffected, and feature localization) greatly improved recognizability of both faces and letters. For large phase shifts, the low spatial frequencies appear in transparency behind a grid structure of checks or lines. We also studied a more simple example of blocking, the checkerboard, that can be considered as a coarse quantized diagonal sinusoidal plaid. When one component of the plaid was contrast-inverted, it was seen in transparency against the checkerboard, while the other remained "captured" within the block structure. If the higher harmonics are then phase-shifted by pi, the contrast-reversed fundamental becomes captured and the other seen in transparency. Intermediate phase shifts of the higher harmonics cause intermediate effects, which we measured by adjusting the relative contrast of the fundamentals until neither orientation dominated. The contrast match varied considerably with the phase of the higher harmonics, over a range of about 1.5 log units. Simulations with the local energy model predicted qualitatively the results of the recognizability of both faces and letters, and quantitatively the apparent orientation of the modified checkerboard pattern. More generally, the model predicts the conditions under which an image will be "captured" by coarse quantization, or seen in transparency.

Contrast Sensitivity↗

PubMed2002

Alumina-alumina artificial hip joints. Part I: a histological analysis and characterisation of wear debris by laser capture microdissection of tissues retrieved at revision.

The aims of this study were to investigate the tissues from uncemented Mittelmeier alumina ceramic-on-ceramic total hip replacements using histological methods and to isolate and characterise the ceramic wear debris using laser capture microdissection and electron microscopy. Tissues from around 10 non-cemented Mittelmeier alumina ceramic on ceramic THRs were obtained from patients undergoing revision surgery. Tissues were also obtained from six patients who were undergoing revisions for aseptic loosening of Charnley, metal-on-polyethylene prostheses. Tissue sections were analysed using light microscopy to determine histological reactions and also the location and content of alumina ceramic wear debris. Tissue samples were extracted from sections using laser capture microdissection and the characteristics of the particles subsequently analysed by TEM and SEM. The tissues from around the ceramic-on-ceramic prostheses all demonstrated the presence of particles, which could be seen as agglomerates inside cells or in distinct channels in the tissues. The tissues from the ceramic-on-ceramic retrievals had a mixed pathology with areas that had no obvious pathology, areas that were relatively rich in macrophages and over half of the tissues had in the region of 60% necrosis/necrobiosis. In comparison, the Charnley tissues showed a granulomatous cellular reaction involving a dense macrophage infiltrate and the presence of giant cells and < 30% necrosis/necrobiosis. The tissues from the ceramic prostheses also showed the presence of neutrophils and lymphocytes, which were not evident in the tissues from the Charnley retrievals. There were significantly more macrophages (p < 0.05), and giant cells (p < 0.01) in the Charnley tissues and significantly more neutrophils (p < 0.01) in the ceramic-on-ceramic tissues. TEM of the laser captured tissue revealed the presence of very small alumina wear debris in the size range 5-90 nm, mean size + SD of 24 +/- 19nm whereas SEM (lower resolution) revealed particles in the 0.05-3.2 microm size range. This is the first description of nanometre sized ceramic wear particles in retrieval tissues. The bi-modal size range of alumina ceramic wear debris overlapped with the size ranges commonly observed with metal particles (10-30 nm) and particles of ultra-high molecular weight polyethylene (0.1-1,000 microm). It is possible that the two size ranges of contributed to the mixed tissue pathology observed. It is speculated that the two types of ceramic wear debris are generated by two different wear mechanisms in vivo, under normal articulating conditions, relief polishing wear and very small wear debris is produced. while under conditions of microseparation of the head and cup and rim contact, intergranular and intragranular fracture and larger wear particles are generated.

Aluminum Oxide↗

PubMed1983

Pupil capture: prevention and management.

Pupil capture occurs in a small percentage of patients following posterior chamber lens implantation. We noted a 3% incidence in 100 patients using sulcus fixated planar J-loop lenses, a 1% incidence using sulcus fixated angulated J-loop lenses, and a 1% incidence in capsular bag fixated flexible Y-loop lenses. No patients lost vision from pupil capture, and no clinically significant late complications were observed. Pupil capture may be successfully treated in the early postoperative period in some patients by mydriasis and a noninvasive loop compression technique using Q-tips.

Anterior Chamber↗

PubMed2001

Sensitive detection of the Egyptian species of sugarcane streak virus by PCR-probe capture hybridization (PCR-ELISA) and its complete nucleotide sequence.

A rapid and sensitive assay for the specific detection of Sugarcane streak virus (SSV) using PCR-probe capture hybridization (PCR-ELISA) was developed. Nucleic acids suitable for PCR were extracted from SSV-infected tissue using organic solvents or Fast DNA kit. SSV cDNA was amplified using viral specific primers and the amplified SSV cDNA (amplicon) was DIG-labelled during the amplification process. The amplicon was then detected in a colorimetric hybridization system by a microtiter plate using a biotinylated cDNA (22 nt), cDNA (789 nt) or cRNA (789 nt) capture probe. This system combines the specificity of molecular hybridization, the ease of the colorimetric protocol, and is 10-100 fold more sensitive than agarose gel electrophoretic analysis in detecting the amplified product. Long cDNA or cRNA capture probe was 2-7 fold more sensitive than the oligo cDNA probe for the detection. Complete nucleotide sequence of SSV from Naga Hammady, Egypt, revealed that SSV-EG is a new species of SSV that shares 66% nucleotide identity with the virus species from Natal, South Africa.

Cloning, Molecular↗

PubMed1997

Development of a nucleic acid capture probe with reverse transcriptase-polymerase chain reaction to detect poliovirus in groundwater.

There is a need to develop a practical method for the detection of viral contaminates in water supplies. In this study, poliovirus was used as a model to develop a nucleic acid capture technique. This technique was used to recover viral RNA from concentrated groundwater samples. Poliovirus RNA was isolated using magnetic bead technology. A biotinylated oligonucleotide probe was hybridized to poliovirus-RNA in solution. Streptavidin coated magnetic beads were then added to isolate the RNA-oligonucleotide hybrid. The procedure allows for the recovery of viral RNA suitable for amplification by reverse transcription-polymerase chain reaction (RT-PCR). This nucleic acid capture system was effective in both concentrating, and purifying poliovirus RNA while removing environmental RT-PCR inhibitors. A detection sensitivity of one plaque forming unit (PFU) in 250 microliters of a concentrated environmental sample was routinely attained. This was the same detection level found with seeded purified water. It was shown that the sensitivity of nucleic acid capture RT-PCR was significantly greater than direct RT-PCR, when applied to environmental samples. The amplified product was sequenced to ensure specificity. Furthermore, this technique is rapid, reliable and can be readily adapted to detect other viral pathogens.

DNA, Viral↗

PubMed1997

Rapid direct diagnosis of mumps meningitis by ELISA capture technique.

ELISA capture technique (ELISAc) was carried out using a rabbit hyperimmune serum attached to a solid phase for capturing mumps antigens in cerebrospinal fluid (CSF) in patients with meningitis and/or in supernatants of infected Vero cells. A biotin-labelled rabbit serum prepared from the previous serum was added and the reaction was read by an enzymatic (avidine-peroxidase) reaction by automated reading. The cut-off was calculated in 100 CSFs negative for viruses by conventional diagnosis. The specificity was evaluated in Vero cells infected with 22 CSFs collected from vaccinated children (URABE AM9 attenuated vaccine) who developed meningitis. A guinea pig hyperimmune serum confirmed the specificity. Results in culture correlated with the ELISA capture technique (ELISAc). No cross-reactivity was observed with parainfluenza 1, 2, 3 human reference strains. At least 2.5 ngs of purified mumps proteins were detected corresponding to 10(1.5) infectious particles per ml. ELISAc applied directly to 14 CSFs collected from unvaccinated children with meningitis diagnosed five positive cases, whereas in four cases conventional diagnosis had to be undertaken twice. ELISAc permitted the diagnosis of one additional patient. The test can be carried out in 3 h.

Animals↗