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At least 1,513 records · Page 84Linked to original sources
PubMed2005

Kinetochore capture and bi-orientation on the mitotic spindle.

Kinetochores are large protein complexes that are formed on chromosome regions known as centromeres. For high-fidelity chromosome segregation, kinetochores must be correctly captured on the mitotic spindle before anaphase onset. During prometaphase, kinetochores are initially captured by a single microtubule that extends from a spindle pole and are then transported poleward along the microtubule. Subsequently, microtubules that extend from the other spindle pole also interact with kinetochores and, eventually, each sister kinetochore attaches to microtubules that extend from opposite poles - this is known as bi-orientation. Here we discuss the molecular mechanisms of these processes, by focusing on budding yeast and drawing comparisons with other organisms.

Cell Cycle↗
PubMed2001

Evaluation of the Murex CMV DNA Hybrid Capture assay (version 2.0) for early diagnosis of cytomegalovirus infection in recipients of an allogeneic stem cell transplant.

Early diagnosis of CMV infection based on sensitive diagnostic assays has helped to reduce CMV-related mortality after allogeneic stem cell transplantation (SCT). In this study, the commercialized Murex CMV DNA Hybrid Capture assay (version 2.0) (HCS) was prospectively compared to an in-house CMV-DNA PCR assay from whole blood in patients after allogeneic stem cell transplantation. Overall, a high concordance between HCS and PCR was documented (kappa = 0.686; n = 385). The HCS assay was found to be as sensitive as the PCR indicating active CMV infection at a median of 35 and 34 days after transplantation, respectively. None of the HCS-negative patients developed CMV-related symptoms (negative predictive value 100%). Declining CMV DNA load in the blood was found to be an indicator for effective antiviral therapy, whereas persistence of a high viral load was associated with fatal CMV disease. In conclusion, the Hybrid Capture CMV DNA assay (v 2.0) allows early diagnosis of CMV infection after allogeneic SCT and assessment of the efficacy of antiviral therapy.

Adolescent↗
PubMed2001

Detection of immunomagnetically captured Escherichia coli O157:H7 by antibody-conjugated alkaline phosphatase.

A rapid and sensitive detection process for Escherichia coli O157:H7 was developed using alkaline phosphatase (APase)-labeled anti-E. coli O157 antibodies to tag the targeted bacteria. Immunomagnetic beads or antibody-labeled streptavidin-coated magnetic beads were then used to capture the APase-tagged E. coli. Immunomagnetically captured bacteria were washed and distributed into microplates or optical cuvettes. The enzyme-catalyzed hydrolysis of p-nitro-phenol phosphate in alkaline solutions was then followed. Less than 1000 cfu/ml of E. coli O157:H7 could be detected. This approach was applied to detect the bacteria artificially spiked in beef hamburgers. Less than 1 cfu/g of E. coli O157:H7 produced a significant response after cultural enrichment for 4-6 h at 37 degrees C.

Alkaline Phosphatase↗
PubMed2003

Laser capture microdissection-based in vivo genomic profiling of wound keratinocytes identifies similarities and differences to squamous cell carcinoma.

Keratinocytes undergo a dramatic phenotypic conversion during reepithelialization of skin wounds to become hyperproliferative, migratory, and invasive. This transient healing response phenotypically resembles malignant transformation of keratinocytes during squamous cell carcinoma progression. Here we present the first analysis of global changes in keratinocyte gene expression during skin wound healing in vivo, and compare these changes to changes in gene expression during malignant conversion of keratinized epithelium. Laser capture microdissection was used to isolate RNA from wound keratinocytes from incisional mouse skin wounds and adjacent normal skin keratinocytes. Changes in gene expression were determined by comparative cDNA array analyses, and the approach was validated by in situ hybridization. The analyses identified 48 candidate genes not previously associated with wound reepithelialization. Furthermore, the analyses revealed that the phenotypic resemblance of wound keratinocytes to squamous cell carcinoma is mimicked at the level of gene expression, but notable differences between the two tissue-remodeling processes were also observed. The combination of laser capture microdissection and cDNA array analysis provides a powerful new tool to unravel the complex changes in gene expression that underlie physiological and pathological remodeling of keratinized epithelium.

Animals↗
PubMed2004

Genomic DNA is captured and amplified during double-strand break (DSB) repair in human cells.

Genomic stability is maintained by the surveillance and repair of DNA damage. Here, we describe a mechanism whereby repair of extrachromosomal DNA double-strand breaks (DSBs) in human cells can be accompanied by capture of genomic DNA fragments. The availability of the human genome sequence enabled us to characterize these inserts in cells from a normal individual and from a patient with ataxia telangiectasia (AT), deficient for the damage response kinase ATM and prone to genomic instability. We find AT cells exhibit insertions of human chromosomal DNA fragments in excess of 17 kb during DSB repair, whereas we detected no such genomic inserts in normal cells. However, the presence of simian virus 40 (SV40), used to transform these cell lines, resulted in capture of genomic DNA associated with sites of viral integration in both cell types. The genomic instability at sites of SV40 integration was exported to other sites of DNA damage, and acquisition of the viral origin of replication resulted in gene amplification through autonomous replication of the plasmid harbouring the repaired extrachromosomal DSB. Should this same phenomenon apply to the repair of chromosomal DSBs, genome rearrangements made possible via this DSB insertional repair pose risks to genomic integrity, and may contribute to tumorigenic progression.

DNA↗
PubMed2004

Development of a solid-phase 'asymmetric resin-capture-release' process: application of an ephedrine chiral resin in an approach to gamma-butyrolactones.

The potential of a solid-phase asymmetric resin-capture-release strategy for high-throughput synthesis has been evaluated. Fukuzawa's Sm(ii)-mediated, asymmetric approach to gamma-butyrolactones was selected to illustrate the feasibility of such a process. Alpha,beta-unsaturated esters immobilised on an ephedrine chiral resin have been applied in an asymmetric approach to gamma-butyrolactones. Lactone products are obtained in moderate isolated yields with selectivities up to 96% ee. In addition, we have shown that the ephedrine resin can be conveniently recovered and recycled although in some cases lower yields were obtained on reuse of the chiral resin. A short synthesis of a moderate DNA-binding microbial metabolite using asymmetric resin-capture-release is also described.

4-Butyrolactone↗
PubMed1968

Ultramicro determination of plasma testosterone by electron-capture detection of testosterone diheptafluorobutyrate.

1. A new highly sensitive and accurate ultramicro method for the estimation of testosterone in human peripheral plasma is described. The method uses paper-and thin-layer-chromatographic separation of plasma testosterone, which is determined as testosterone diheptafluorobutyrate by electron-capture detection after gas-liquid chromatography. 2. The average difference between duplicates is +/-2% (range 1-5%) with as little as 2.5ml. of human male peripheral plasma. With 10ml. of plasma the method is sensitive enough for the accurate determination of testosterone in human female plasma. The high order of accuracy is achieved by the use of a radioactive label and an internal standard for gas chromatography, and by obtaining several gas chromatograms from the same plasma sample. 3. As little as 40mumug. of peripheral plasma testosterone can be detected. The method is 20 times as sensitive as electron-capture techniques with the monochloroacetate derivative. 4. The method is simpler and quicker than double-isotope-derivative methods, and slightly more sensitive. The advantages of the method, which is specific for testosterone, are its high sensitivity and accuracy, which are achieved with relative convenience.

Chemical Phenomena↗
PubMed1979

Rate of pinocytic capture of macromolecular substrates by rat yolk sac incubated in serum-free culture medium.

Pinocytic activity was quantified for rat yolk sacs incubated in a medium that was either serum-free or contained 10% (v/v) of calf serum. Absence of serum from the medium caused a small increase in the rate of pinosome formation, as determined by the rates of capture of both 125I-labelled poly(vinylpyrrolidone) and [14C]sucrose. In contrast, the rates of uptake of substrates ingested by adsorptive pinocytosis were greatly enhanced when serum proteins, which compete for the same binding sites on the plasma membrane as used by adsorbing substrates, were absent. Elimination of such competition greatly simplifies the quantitative analysis of the binding process, and permitted a detailed study of the binding to the plasma membrane of formaldehyde-denatured bovine serum albumin, a protein that is rapidly digested within the lysosomal system after its pinocytic capture. Binding obeyed Michaelis-Menten kinetics and showed a dissociation constant of approx. 1 micron, indicating the high affinity of this protein for binding sites on the surface of actively pinocytosing yolk-sac cells.

Animals↗
PubMed1981

Rates of pinocytic capture of simple proteins by rat yolk sacs incubated in vitro.

The rates of pinocytic uptake of a number of small 125I-labelled simple proteins (insulin, ribonuclease A and lysozyme) by rat yolk sacs incubated in vitro were determined both before and after treating these proteins with reagents that are known to increase the rate of capture of 125I-labelled bovine serum albumin. Uptake of the untreated forms of all three proteins was extremely rapid, indicating that adsorptive pinocytosis is the principal mechanism by which yolk-sac cells capture these simple proteins, but these rates show no simple correlation with molecular charge. In contrast with albumin, the rates of uptake of treated proteins were either unchanged or lower than that of the corresponding untreated protein preparations; polymeric forms of 125I-labelled lysozyme larger than dimers were ingested at rates significantly lower than that of the monomer.

Animals↗
PubMed2001

The thymidylate synthase gene of Hz-1 virus: a gene captured from its lepidopteran host.

The sequence analysis of a thymidylate synthase gene was identified in the Hz-1 virus HindIII-D fragment. The viral thymidylate synthase gene encodes a protein of 295 amino acids, and is closely related to that of insect, mammals and herpesvirus. The thymidylate synthase gene identified was a genuine viral gene in that it was only detected in cells infected with Hz-1 virus but not in the mock infected cells, by Southern blot analysis and by RT-PCR. Results of phylogenetic analysis based on non-synonymous and amino acid distances suggested that the TS gene of Hz-1 virus was grouped closely with that of Bombyx mori. High bootstrap values confirmed that the thymidylate synthase of Hz-1 virus was acquired by a capture event from its lepidopteran host. Results of both sequence divergences and phylogenetic analysis suggested that TS genes in insect viruses, Hz-1, CIV, and MsEPV may have a different history or originated from different capture events.

Amino Acid Sequence↗
PubMed1999

Estimating the prevalence of problem drug use in inner London: a discussion of three capture-recapture studies.

AIMS: To provide an evidence base of estimates of the prevalence of problem drug use in inner London. DESIGN: Re-analysis of three capture-recapture studies using subjects aged 15-49 years, that aim to estimate the hidden population from analysing the overlaps between three data sources. SETTING: Newham (1995) Camden and Islington (C&I) (1993/4) and Lambeth, Southwark and Lewisham (LSL) (1992). PARTICIPANTS: Each study collected data from three sources of problem drug users including: the Regional Drug Misuse Database, specialist drug agencies, HIV tests, social services, police arrests and court records. In LSL opiate users were analysed separately. The studies identified 1832 individuals in LSL, 543 in Newham, and 1321 in C&I. MEASUREMENTS: Poisson models were fitted to the data testing different interactions between the data sources representing potential dependencies. The simplest model was selected on the basis of its AIC score and log-likelihood ratio tests. FINDINGS: The number of hidden problem drug users were estimated to be 12,500 (95% CI 9600-16,100) in LSL with 4400 (3200-6100) opiate users; 7000 (5000-10,000) in C&I and 3800 (2000-7200) in Newham. The prevalence of problem drug use in those aged 15-49 was estimated to be 3.1% (2.5-3.9%) in LSL with 1.3% (1.0-1.6%) opiate users; and 3.6% (2.7-4.9%) and 3.3% (1.9-5.7%) in C&I and Newham, respectively. CONCLUSIONS: Despite the inherent problems with capture-recapture methods, our three studies establish an evidence base for estimates of problem drug use in London. It is important that a larger study is carried out in London.

Adolescent↗
PubMed2003

All-digital image capture and whole-field analysis of ciliary beat frequency.

We hypothesized that a high-speed all-digital video imaging system, with computerized analysis, would precisely capture and measure ciliary beat frequency (CBF) and would shorten the time from data capture to data analysis. We compared a conventional analog video system with a new high-speed digital system we developed for CBF analysis. Using ciliated primary bovine bronchial epithelial cells we made simultaneous analog and digital CBF measurements of the same region of interest (ROI) while temperature was varied. This yielded nearly identical data over a wide range of frequencies (7-15 Hz) using either system. Unlike the digital system however, the analog system did not accurately detect CBF above 15 Hz (temperatures higher than 30 degrees C). We also compared ROI analysis with a new analysis algorithm we have named whole-field analysis (WFA). WFA measurement of CBF agreed with ROI and reduced operator time required to analyse data by more than 90% compared with the analog system. We conclude that all-digital computerized CBF analysis correlates closely with standard video methods, markedly speeds up data analysis and provides new ways, including WFA, to analyse entire fields of motile cilia simultaneously. We have termed this system 'Sisson-Ammons Video Analysis' (SAVA).

Animals↗
PubMed2004

Detection and quantitation of HCV core protein in single hepatocytes by means of laser capture microdissection and enzyme-linked immunosorbent assay.

Immunohistochemistry provides valuable information concerning the localization and distribution of hepatitis C virus (HCV)-related proteins in histological sections of liver tissue, but does not readily permit their quantitation in individual cells and the staining intensity of cell immunodeposits cannot be calibrated with the current number of antigen molecules. We specifically detected and quantitated HCV core protein in single hepatocytes by coupling laser capture microdissection (LCM) with a sensitive enzyme-linked immunosorbent assay (ELISA). Quantitation of HCV core protein per cell was carried out on liver tissue cells obtained by LCM from fixed and stained frozen sections of 10 HCV-positive patients with chronic active hepatitis (CAH). Macromolecules from captured cells were solubilized in an extraction buffer and directly assayed for core protein using a sandwich ELISA. Calibration was achieved by developing a standard curve based on known concentrations of HCV core protein. Precision, linearity and sensitivity were verified for known numbers of microdissected tissue cells. In this study, the concentration of HCV core protein in single hepatocytes ranged from 7 to 56 pg/cell. Specificity was verified on 10 replicates of 10 HCV-negative liver tissues. Immunohistochemical staining of HCV core protein was compared with the results of the soluble immunoassay for the adjacent liver tissue sections. Independent scoring of HCV immunostaining failed to parallel the LCM quantitative immunoassay. LCM-based immunoassay significantly expands our ability to investigate function-related antigens in apparently pure cell populations in HCV infection.

Aged↗
PubMed1998

Adaptation to high CO2 concentration in an optimal environment: radiation capture, canopy quantum yield and carbon use efficiency.

The effect of elevated [CO2] on wheat (Triticum aestivum L. Veery 10) productivity was examined by analysing radiation capture, canopy quantum yield, canopy carbon use efficiency, harvest index and daily C gain. Canopies were grown at either 330 or 1200 micromoles mol-1 [CO2] in controlled environments, where root and shoot C fluxes were monitored continuously from emergence to harvest. A rapidly circulating hydroponic solution supplied nutrients, water and root zone oxygen. At harvest, dry mass predicted from gas exchange data was 102.8 +/- 4.7% of the observed dry mass in six trials. Neither radiation capture efficiency nor carbon use efficiency were affected by elevated [CO2], but yield increased by 13% due to a sustained increase in canopy quantum yield. CO2 enrichment increased root mass, tiller number and seed mass. Harvest index and chlorophyll concentration were unchanged, but CO2 enrichment increased average life cycle net photosynthesis (13%, P < 0.05) and root respiration (24%, P < 0.05). These data indicate that plant communities adapt to CO2 enrichment through changes in C allocation. Elevated [CO2] increases sink strength in optimal environments, resulting in sustained increases in photosynthetic capacity, canopy quantum yield and daily C gain throughout the life cycle.

Adaptation, Physiological↗
PubMed2001

Analysis of HCV-IG isotype complexes by a novel immuno-capture RT-PCR method.

HCV can be present in the circulating blood either as a free virus or as a virion-immunoglobulin (Ig) complex. All isotypes of Igs may form the virus complexes, but it remains unclear what specific role of each Ig plays in the clearance of HCV. In the present study, we have combined immuno-capture and RT-PCR, and developed a quick double-specificity method for detecting and distinguishing different HCV-Ig complexes. We compared our new method, the immuno-capture RT-PCR (iRT-PCR), with the conventional RT-PCR (cRT-PCR) for the sensitivity of detecting HCV in 35 clinically diagnosed patients with HCV infection. The results showed that 31 patients were detected to be positive by using iRT-PCR, whereas 16 patients were positive with the use of cRT-PCR. HCV-IgM, HCV-IgG, HCV-IgA could separately be detected by iRT-PCR and their positive rates were 66.7%, 51.0%, 62.7%, respectively. HCV bound to antibody was a common feature of hepatitis C (HC) and 86.3% of patients were positive at least by one of the HCV-Ig tests. The patterns of HCV RNA constituents varied according to disease categories. In summary, iRT-PCR is a valuable method for analysis of the composition of the immune complexes, which may provide new and valuable insights into HCV pathogenesis.

Adolescent↗
PubMed2002

Analysis of complement-bound HCV complexes using a novel immuno-capture RT-PCR method.

Recently, more and more evidence has supported the hypothesis that liver cell injury was immune-mediated in patients with hepatitis C virus (HCV) infection, and that circulating immune complexes (CICs) might play a role in the pathogenesis of chronic hepatitis C (HC). In the present study, we have combined immuno-capture and reverse transcriptase-polymerase chain reaction (RT-PCR), and developed a quick method of high specificity for the detection of complement-bound HCV-CIC. We found that there were higher frequencies of HCV-C1q CIC than that of HCV-factor B, and there was a deviation of complement from immunoglobulin (Ig) in HCV-CIC. These findings suggest that immuno-capture RT-PCR (iRT-PCR) for the detection of HCV-bound CIC is a valuable method for the analysis of the composition of the immune complexes, and for the understanding of host immune response and immune pathogenesis in HCV-infected individuals.

Adolescent↗
PubMed2003

Analysis of hepatitis B virus-immunoglobulin isotype complexes by a novel immuno-capture polymerase chain reaction method.

Hepatitis B virus (HBV) can be present in the circulating blood either as free virus or as a virion-immunoglobulin (Ig) complex. Presently, it remains unclear what specific role each Ig plays in the clearance of HBV. In this study, a novel method that combined immuno-capture and polymerase chain reaction (PCR) amplification was used for detecting and distinguishing different HBV-Ig complexes. Three isotypes of Ig (IgM, IgG and IgA) bound to HBV were detected in the four clinically defined stages of HBV infection in 108 patients. The results showed that all the three isotypes of Ig could bind to HBV, and the patterns of HBV-Ig complexes varied according to disease categories. Interestingly, the frequency of HBV DNA-Ig complexes in hepatitis B e antigen (HBeAg)-positive patients was significantly lower than that in HBeAg-negative patients. All the data suggest that the three isotypes of HBV DNA-Ig circulating immune complex (CIC) may have different biological meanings. In summary, HBV bound to an antibody is a common feature of hepatitis B, and immuno-capture PCR is a valuable method for the analysis of the composition of the immune complexes. The detection of HBV-Ig complexes may provide new and valuable insights into HBV pathogenesis.

Adolescent↗