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PubMed2002

Isolation of receptor-ligand pairs by capture of long-lived multivalent interaction complexes.

We have combined phage display and array screening for the rapid isolation of pairs of interacting polypeptides. Our strategy, named SAC (selection by avidity capture), is based on the avidity effect, the formation of highly stable complexes formed by multivalent interactions; in our case, between a receptor (multivalently displayed on phage) and a ligand (coexpressed as a multimeric fusion protein). Capture of the long-lived interaction complex allows the isolation of phage bearing cognate interaction pairs, as we demonstrate for a range of interactions, including Ab-antigen pairs and the rapamycin-dependent interaction of FKBP-12 and FRAP. Cognate phage are enriched by SAC up to 1000-fold and interacting pairs can be identified by array screening. Application of SAC to Ab-antigen interactions as a model system yielded over 140 specific Abs to a single antigen and 92 Abs to three different fetal human brain antigens in a single round of SAC each. Our results suggest that SAC should prove useful for the identification and study of receptor-ligand interactions in particular among extracellular proteins, as well as for the rapid generation of specific Abs to multiple antigens.

Antibodies, Monoclonal↗

PubMed2002

Evidence that subcellular localization of a bacterial membrane protein is achieved by diffusion and capture.

Bacteria lack an endoplasmic reticulum, a Golgi apparatus, and transport vesicles and yet are capable of sorting and delivering integral membrane proteins to particular sites within the cell with high precision. What is the pathway by which membrane proteins reach their proper subcellular destination in bacteria? We have addressed this question by using green fluorescent protein (GFP) fused to a polytopic membrane protein (SpoIVFB) that is involved in the process of sporulation in the bacterium Bacillus subtilis. SpoIVFB-GFP localizes to a region of the sporulating cell known as the outer forespore membrane, which is distinct from the cytoplasmic membrane. Experiments are presented that rule out a mechanism in which SpoIVFB-GFP localizes to all membranes but is selectively eliminated from the cytoplasmic membrane by proteolytic degradation and argue against a model in which SpoIVFB-GFP is selectively inserted into the outer forespore membrane. Instead, the results are most easily compatible with a model in which SpoIVFB-GFP achieves proper localization by insertion into the cytoplasmic membrane followed by diffusion to, and capture in, the outer forespore membrane. The possibility that diffusion and capture is a general feature of protein localization in bacteria is discussed.

Bacillus subtilis↗

PubMed2002

Modeling of automatic capture and focusing of visual attention.

An explanation, based on simple analysis of the spatiotemporal variations of the visual environment, is given to the automatic capture and focusing of visual attention. It is assumed that the transmittance for the sensory signals is modulated by separate control circuits that sample input from the same area of the visual field but at a lower resolution. When these circuits detect significant spatial and/or temporal variations, they "open gates" for the more accurate information arising from the same area. If the variations are related to the spatial resolution, which varies within wide limits over the retina, the visual field is "opened" up to a radius where it captures the most salient structures of the image. If the temporal variations of the signals are further emphasized, the high spatial frequencies begin to dominate. If then the gaze is moved by a small amount, the transmittance of the foveal signal paths is activated strongest.

Animals↗

PubMed1990

Molecular epidemiology of human hepatitis A virus defined by an antigen-capture polymerase chain reaction method.

We describe an immunoaffinity-linked nucleic acid amplification system (antigen-capture/polymerase chain reaction, or AC/PCR) for detection of viruses in clinical specimens and its application to the study of the molecular epidemiology of a picornavirus, hepatitis A virus (HAV). Immunoaffinity capture of virus, synthesis of viral cDNA, and amplification of cDNA by a polymerase chain reaction (PCR) were carried out sequentially in a single reaction vessel. This approach simplified sample preparation and enhanced the specificity of conventional PCR. AC/PCR detected less than one cell culture infectious unit of virus in 80 microliters of sample. Sequencing of AC/PCR reaction products from 34 virus strains demonstrated remarkable conservation at the nucleotide level among most strains but revealed hitherto unsuspected genetic diversity among human isolates. Epidemiologically related strains were identical or closely related in sequence. Virus strains recovered from epidemics of hepatitis A in the United States and Germany were identical in sequence, providing evidence for a previously unrecognized epidemiologic link between these outbreaks.

Animals↗

PubMed1992

Diagnosis of hepatitis C virus (HCV) infection using an immunodominant chimeric polyprotein to capture circulating antibodies: reevaluation of the role of HCV in liver disease.

Structural and nonstructural regions of the HCV-encoded polyprotein have been expressed in recombinant yeast, bacteria, or insect cells and used to capture and measure reactive antibodies circulating in different individuals. The putative nucleocapsid protein (C) and nonstructural proteins 3-5 (NS3-NS5) were found to contain the most immunodominant epitopes. The NS3, NS4, and C regions were expressed in yeast in the form of a fused, chimeric polyprotein (C25) and a capture assay for reactive antibody was developed. This anti-C25 assay detects all previously identified HCV-seropositive cases and provides a substantially more sensitive diagnostic for both acute and chronic HCV infections than the current anti-C100-3 (NS4) assay. Anti-C25 was detected more frequently than anti-C100-3 in chronic, transfusion-associated non-A, non-B hepatitis patients from the United States (95% vs. 71%) and Japan (98% vs. 82%), in cryptogenic cirrhosis patients from the United States (62% vs. 28%), and in hepatitis B surface antigen-negative cases of hepatocellular carcinoma from Japan (83% vs. 63%). These data indicate that HCV has a greater role in these liver diseases than was previously thought. In volunteer United States blood donors sampled following the introduction of anti-C100-3 screening, the prevalence of anti-C25 and anti-C100-3 was 0.5% and 0.08%, respectively.

Antigens, Viral↗

PubMed2005

Determination of aberrant O-glycosylation in the IgA1 hinge region by electron capture dissociation fourier transform-ion cyclotron resonance mass spectrometry.

In a number of human diseases of chronic inflammatory or autoimmune character, immunoglobulin molecules display aberrant glycosylation patterns of N- or O-linked glycans. In IgA nephropathy, IgA1 molecules with incompletely galactosylated O-linked glycans in the hinge region (HR) are present in mesangial immunodeposits and in circulating immune complexes. It is not known whether the Gal deficiency in IgA1 proteins occurs randomly or preferentially at specific sites. To develop experimental approaches to address this question, the synthetic IgA1 hinge region and hinge region from a naturally Gal-deficient IgA1 myeloma protein have been analyzed by 9.4 tesla Fourier transform-ion cyclotron resonance mass spectrometry. Fourier transform-ion cyclotron resonance mass spectrometry offers two complementary fragmentation techniques for analysis of protein glycosylation by tandem mass spectrometry. Infrared multiphoton dissociation of isolated myeloma IgA1 hinge region peptides confirms the amino acid sequence of the de-glycosylated peptide and positively identifies a series of fragments differing in O-glycosylation. To localize sites of O-glycan attachment, synthetic IgA1 HR glycopeptides and HR from a naturally Gal-deficient polymeric IgA1 myeloma protein were analyzed by electron capture dissociation and activated ion-electron capture dissociation. Multiple sites of O-glycan attachment (including sites of Gal deficiency) in myeloma IgA1 HR glycoforms were identified (in all but one case uniquely). These results represent the first direct identification of multiple sites of O-glycan attachment in IgA1 hinge region by mass spectrometry, thereby enabling future characterization at the molecular level of aberrant glycosylation of IgA1 in diseases such as IgA nephropathy.

Amino Acid Sequence↗

PubMed2001

Value of morphological criteria in diagnosing cervical HPV lesions confirmed by in situ hybridization and hybrid capture assay.

The present study evaluated the value of morphological criteria (binucleation, multinucleation, koilocytosis, spindle koilocytes, abnormal mitosis and dyskeratosis) in the diagnosis of cervical human papillomavirus (HPV) lesions confirmed by in situ hybridization (ISH) and hybrid capture (HC) assay. Colposcopic punch biopsies from a series of 138 women with abnormal Pap smears were examined on light microscopy and in situ hybridization (DAKO widespectrum cocktail probe) for HPV-induced morphological changes and HPV DNA, respectively. Cervical swabs were analyzed for HPV DNA of the oncogenic types using Hybrid Capture. CIN 2 and CIN 3 were found in 44 biopsies, CIN 1 in 62, and no evidence of HPV in 32 cases. HPV was detected by ISH in 51/138 (37%) cases and by HC in 66/138 (48%) lesions. With both tests, HPV DNA detection increased parallel with lesion severity, up to 70% and 59% in CIN 2/3 by HC and ISH, respectively OR 4.6 (1.7-12.1) and 10.1 (3.0-33.8). Among the histological criteria, multinucleation, binucleation and abnormal mitoses were significantly associated with HPV DNA detection. Multinucleation proved to be the strongest predictor of HPV DNA-positivity. Binucleation, abnormal mitosis, koilocytosis and spindle koilocytes were also reliable criteria of HPV lesions. Minor nuclear atypia, and "mild koilocytosis" were of no value in making this diagnosis.

Biopsy↗

PubMed2002

Detection of enteroviruses, hepatitis A virus and rotaviruses in sewage by means of an immunomagnetic capture reverse transcription-PCR assay.

An immunomagnetic capture reverse transcription-PCR (IMC-RT-PCR) assay was evaluated to recover and detect enteric viruses in sewage and to remove PCR inhibitors. The procedure was applied along with a simple sample processing consisting of an initial separation of solids followed by polyethylen glycol precipitation and solvent extraction. This procedure reduced sample volumes by about 65-fold without eliminating RT-PCR inhibitors. Paramagnetic beads coupled to pooled human immunoglobulins were used to simultaneously capture poliovirus 1 (PV-1) and hepatitis A virus (HAV) from seeded sewage concentrates. The IMC was efficient in removing PCR inhibitors and in further reducing sample volumes by approximately 10-fold allowing the analysis of 6-7 ml of sewage sample per RT-PCR reaction. The detection limits of IMC-RT-PCR from seeded concentrates were 0.1-1 PFU for PV-1 and 1 MPNCU for HAV. The described procedure could be applied successfully for the detection of enteroviruses, HAV and rotaviruses in field sewage samples.

Enterovirus↗

PubMed2004

Prevalence of Parkinson's disease and parkinsonism in a Colombian population using the capture-recapture method.

Our objective was to estimate the prevalence of Parkinson's disease (PD) and Parkinsonism (Ps) in Antioquia (Colombia), using the Capture-Recapture method. The two biggest institutions for attending neurological patients in Antioquia were selected as sources for the use of the Capture-Recapture method. Prevalences of PD (PPD) and Ps (PPs) were estimated according to the following expression: PPD (or PPs) = n/Nl105. The number of cases (n) of PD (or Ps), n = a + b + c + d, where a = cases identified from the two sources, b = cases identified only in the first source, c = cases identified only in the second source, and d = nondetected cases from any source = bc/a. The projected Antioquian population for the year 2000 was used as denominator. Information obtained between January 1, 1996, and December 31, 2000, was reviewed in order to identify the clinical records of all patients that fulfilled the Ps or PD criteria. General prevalence of PD in Antioquia was 30.7/100.000 (C195% = 29.2-32.2), and that of Ps was 42.1/100.000 (CI95% = 40.3-43.8). Prevalence of PD in people older than 50 years was 176.4/100.000 (CI95% = 166.6-186.3) and that of Ps was of 339.6/100.000 (C195% = 326.0-353.2). Ps and PD prevalences in Antioquia were lower than the figures reported by the National Neuroepidemiologic Study (470/100.000) and similar to the estimated prevalence of these diseases in Caucasian populations (80 to 270/100.000). These findings evidence the great variability of PD prevalence in different regions; therefore, a nationwide study is necessary to determine the prevalence of PD and Ps in Colombia.

Adolescent↗

PubMed2002

Analysis of odontoblast gene expression using a novel approach, laser capture microdissection.

Studying the mechanisms of molecular interactions in developing tissues demands sensitive molecular biological in vivo and in vitro techniques. Laser capture microdissection (LCM) allows for the isolation of mRNA in histological sections even from single cells, thus enabling the identification of in vivo gene expression products in closely circumscribed tissue areas. The aims of this study were to assess the optimal fixation, processing, and staining conditions to retrieve RNA from microdissected odontoblasts. Fluorometric assays and RT-PCR analysis of alpha 1(I) collagen, dentin sialophosphoprotein (Dspp), and osteocalcin (OC) confirmed that the total RNA isolated from day 0 and day 3 captured odontoblasts was sufficient in quantity and quality. Our results indicate that individual odontoblasts obtained by LCM are morphologically intact and chemically unaltered, allowing accurate molecular and biochemical analyses.

Animals↗

PubMed1989

Lethal effect and potentially lethal damage recovery in cultured mammalian cells irradiated by neutron-capture beams.

Cell killing and potentially lethal damage (PLD) recovery in Hela cells were examined after irradiation in suspension using a 10B neutron-capture beam. Thermal-neutron irradiation in the medium containing from 0.09 to 9 mM boron-10 (0.9-90 micrograms/g 10B, 90 per cent enriched boric acid) resulted in steeper survival curves than in the boron-free medium. The relative biological effectiveness (r.b.e.), expressed as the Do ratio, increased from 2.2 +/- 0.1 for the control without boron-10 to 5.4 +/- 0.3 in the medium containing 0.9 mM boron-10. The r.b.e. value was 5.2 +/- 0.5 at a boron-10 concentration of 9 mM, and did not increase when the boron-10 concentration was increased from 0.9 to 9 mM. It was suggested that the observed plateau of r.b.e. at boron-10 concentrations higher than 0.9 mM was due to the dominancy of the 10B(n, alpha)7Li reaction for dose accumulation. PLD recovery after the neutron irradiation containing 0.9 mM boron-10 was not observed, while a small effect was observed after irradiation in the thermal neutron beam, although much less than after gamma rays. The results show that the neutron-capture beam is an excellent beam for radiotherapy if the boron-10 concentration in tumour cells could be elevated to about 1 mM.

Boron↗

PubMed2004

A mechanistic model for mercury capture with in situ-generated titania particles: role of water vapor.

A mechanistic model to predict the capture of gas-phase mercury (Hg) species using in situ-generated titania nanosize particles activated by UV irradiation is developed. The model is an extension of a recently reported model for photochemical reactions by Almquist and Biswas that accounts for the rates of electron-hole pair generation, the adsorption of the compound to be oxidized, and the adsorption of water vapor. The role of water vapor in the removal efficiency of Hg was investigated to evaluate the rates of Hg oxidation at different water vapor concentrations. As the water vapor concentration is increased, more hydroxy radical species are generated on the surface of the titania particle, increasing the number of active sites for the photooxidation and capture of Hg. At very high water vapor concentrations, competitive adsorption is expected to be important and reduce the number of sites available for photooxidation of Hg. The predictions of the developed phenomenological model agreed well with the measured Hg oxidation rates in this study and with the data on oxidation of organic compounds reported in the literature.

Adsorption↗

PubMed1992

Rapid antibody capture assay for detection of group-A streptococci using monoclonal antibody and colloidal gold-monospecific polyvalent antibody conjugate.

A rapid one step, sensitive and specific antibody capture assay for detection of group-A streptococci from the throat swabs of children is described. Monoclonal antibody either MA-106 or MA-107 specific for group-A streptococci polysaccharide (APS) was used as the capture antibody on nitrocellulose paper and rabbit monospecific polyvalent antibody conjugated with colloidal gold to detect the presence of antigen. The lower detection limit of this assay is 15.6ng APS/ml. The assay is specific for APS and failed to recognize polysaccharides obtained from group-B,-C,-G streptococci as well as Staphylococcus aureus. Antigen extracted from throat swabs of children who were positive for beta-hemolytic plaques (other than group-A streptococci) as seen on blood agar culture gave negative readings, thereby confirming the specificity of the assay for APS.

Adolescent↗

PubMed2004

Particle formation and capture during spray drying of inhalable particles.

An investigation of the spray drying process is made in great detail regarding particle formation and capture efficiency with focus on the production of inhalable particles. Mannitol was spray dried as model substance and the spray-dried products were characterized. The resulting products consisted of smooth spheres with a volume median diameter of 2.2-5.5 microm, and narrow size distributions. The investigation was performed in pilot scale of sufficient size to draw general conclusions and make some recommendations. It has been shown that the size of particles is decreased when the feed concentration is decreased, the nozzle gas/feed flow mass ratio increased, and the droplet size decreased. The collection efficiency of the cyclone device used in this study was shown to have a cut-off of 2 microm, i.e., 50% of the particles less than 2 microm are not captured. The data reported indicate that the majority of the single particles formed here, <5 microm, arise from single droplets (of about 10 microm) and are solid, nonporous particles.

Administration, Inhalation↗

PubMed1985

Properties of the kinetochore in vitro. II. Microtubule capture and ATP-dependent translocation.

We have studied the interaction of preformed microtubules (MTs) with the kinetochores of isolated chromosomes. This reaction, which we call MT capture, results in MTs becoming tightly bound to the kinetochore, with their ends capped against depolymerization. These observations, combined with MT dynamic instability, suggest a model for spindle morphogenesis. In addition, ATP appears to mobilize dynamic processes at captured MT ends. We used biotin-labeled MT seeds to follow assembly dynamics at the kinetochore. In the presence of ATP and unlabeled tubulin, labeled MT segments translocate away from the kinetochore by polymerization of subunits at the attached end. We have termed this reaction proximal assembly. Further studies demonstrated that translocation could be uncoupled from MT assembly. We suggest that the kinetochore contains an ATPase activity that walks along the MT lattice toward the plus end. This activity may be responsible for the movement of chromosomes away from the pole in prometaphase.

Adenosine Triphosphate↗

PubMed1989

Microtubule polarities indicate that nucleation and capture of microtubules occurs at cell surfaces in Drosophila.

Hook decoration with pig brain tubulin was used to assess the polarity of microtubules which mainly have 15 protofilaments in the transcellular bundles of late pupal Drosophila wing epidermal cells. The microtubules make end-on contact with cell surfaces. Most microtubules in each bundle exhibited a uniform polarity. They were oriented with their minus ends associated with their hemidesmosomal anchorage points at the apical cuticle-secreting surfaces of the cells. Plus ends were directed towards, and were sometimes connected to, basal attachment desmosomes at the opposite ends of the cells. The orientation of microtubules at cell apices, with minus ends directed towards the cell surface, is opposite to the polarity anticipated for microtubules which have elongated centrifugally from centrosomes. It is consistent, however, with evidence that microtubule assembly is nucleated by plasma membrane-associated sites at the apical surfaces of the cells (Mogensen, M. M., and J. B. Tucker. 1987. J. Cell Sci. 88:95-107) after these cells have lost their centriole-containing, centrosomal, microtubule-organizing centers (Tucker, J. B., M. J. Milner, D. A. Currie, J. W. Muir, D. A. Forrest, and M.-J. Spencer. 1986. Eur. J. Cell Biol. 41:279-289). Our findings indicate that the plus ends of many of these apically nucleated microtubules are captured by the basal desmosomes. Hence, the situation may be analogous to the polar-nucleation/chromosomal-capture scheme for kinetochore microtubule assembly in mitotic and meiotic spindles. The cell surface-associated nucleation-elongation-capture mechanism proposed here may also apply during assembly of transcellular microtubule arrays in certain other animal tissue cell types.

Animals↗

PubMed2001

Cytoplasmic anchorage of L-selectin controls leukocyte capture and rolling by increasing the mechanical stability of the selectin tether.

L-selectin is a leukocyte lectin that mediates leukocyte capture and rolling in the vasculature. The cytoplasmic domain of L-selectin has been shown to regulate leukocyte rolling. In this study, the regulatory mechanisms by which this domain controls L-selectin adhesiveness were investigated. We report that an L-selectin mutant generated by truncation of the COOH-terminal 11 residues of L-selectin tail, which impairs association with the cytoskeletal protein alpha-actinin, could capture leukocytes to glycoprotein L-selectin ligands under physiological shear flow. However, the conversion of initial tethers into rolling was impaired by this partial tail truncation, and was completely abolished by a further four-residue truncation of the L-selectin tail. Physical anchorage of both cell-free tail-truncated mutants within a substrate fully rescued their adhesive deficiencies. Microkinetic analysis of full-length and truncated L-selectin-mediated rolling at millisecond temporal resolution suggests that the lifetime of unstressed L-selectin tethers is unaffected by cytoplasmic tail truncation. However, cytoskeletal anchorage of L-selectin stabilizes the selectin tether by reducing the sensitivity of its dissociation rate to increasing shear forces. Low force sensitivity (reactive compliance) of tether lifetime is crucial for selectins to mediate leukocyte rolling under physiological shear stresses. This is the first demonstration that reduced reactive compliance of L-selectin tethers is regulated by cytoskeletal anchorage, in addition to intrinsic mechanical properties of the selectin-carbohydrate bond.

Amino Acid Sequence↗

PubMed2003

Minus-end capture of preformed kinetochore fibers contributes to spindle morphogenesis.

Near-simultaneous three-dimensional fluorescence/differential interference contrast microscopy was used to follow the behavior of microtubules and chromosomes in living alpha-tubulin/GFP-expressing cells after inhibition of the mitotic kinesin Eg5 with monastrol. Kinetochore fibers (K-fibers) were frequently observed forming in association with chromosomes both during monastrol treatment and after monastrol removal. Surprisingly, these K-fibers were oriented away from, and not directly connected to, centrosomes and incorporated into the spindle by the sliding of their distal ends toward centrosomes via a NuMA-dependent mechanism. Similar preformed K-fibers were also observed during spindle formation in untreated cells. In addition, upon monastrol removal, centrosomes established a transient chromosome-free bipolar array whose orientation specified the axis along which chromosomes segregated. We propose that the capture and incorporation of preformed K-fibers complements the microtubule plus-end capture mechanism and contributes to spindle formation in vertebrates.

Animals↗