ISSN:0173-0835    

DOI:10.1002/elps.1150181002

出版商:Wiley Subscription Services, Inc., A Wiley Company    

年代:1997

数据来源: WILEY

摘要:

AbstractA number of applications of capillary zone electrophoresis (CZE) in sieving liquid polymers (notably linear polyacrylamides and cellulose) for the analysis of polymerase chain reaction (PCR) products of clinically relevant, diagnostic DNA, are reviewed. The fields covered are: human genetics, quantitative gene dosage, microbiology and virology, forensic medicine and therapeutic DNA (notably, antisense nucleotides). Some unique, novel developments are highlighted, such as: (i) nonisocratic CZE,i.e., temperature‐programmed CZE for detection of DNA point mutations; (ii) the synthesis of novelN‐substituted acrylamides, offering extreme resistance to alkaline hydrolysis coupled to high hydrophilicity. In the field of denaturing gradient gel electrophoresis (DGGE), as routinely performed in gel slabs, a novel methodology is described in CZE: double‐gradient DGGE. In this technique, two gradients are simultaneously applied along the migration direction: a chemical (or thermal) denaturing gradient, for partially unwinding homo‐ and hetero‐duplexes of DNA, and a porosity gradient, for recompacting diffuse bands melting over a broader range of denaturing conditions. It is thus demonstrated that chemical gradients, in addition to temperature gradients, can be easily implemented even in a capilla

ISSN:0173-0835    

DOI:10.1002/elps.1150181003

出版商:Wiley Subscription Services, Inc., A Wiley Company    

年代:1997

数据来源: WILEY

摘要:

AbstractMost electrophoretic analyses of proteins in the clinical laboratory are currently carried out by electrophoresis in acrylamide or agarose gels. This labor‐intensive method is now being challenged by capillary electrophoresis (CE) because of its potential for automated, rapid, high efficiency separations. The automatability of CE itself, combined with its amenability to interfacing with other robotized functions, positions this technology perfectly for the analysis of proteins in physiological matrices, such as serum, urine, and cerebrospinal fluid. The focus of this overview is to familiarize the clinical scientist with the research demonstrating the applicability of the technology to the clinical protein laborator

ISSN:0173-0835    

DOI:10.1002/elps.1150181004

出版商:Wiley Subscription Services, Inc., A Wiley Company    

年代:1997

数据来源: WILEY

摘要:

AbstractThe application of capillary electrophoresis (CE) for the analysis of small molecules in clinical research is growing steadily. Initial studies have dealt with separations of standards or compounds in clean matrices. However, later studies dealt with analysis of those compounds in serum, urine or tissues. Great progress has been accomplished in three areas of clinical interest: organic acids, amino acids and drug analysis. The analysis of these compounds by capillary electrophoresis has several distinct advantages: high resolution, simplicity, versatility and especially low operating costs. In many cases, the sample can be injected directly without complex pretreatment. Most of the described methods have been validated for their precision, linearity and accuracy. In forensic toxicology, the CE has been used for drug identification and as a complementary analytical method.

ISSN:0173-0835    

DOI:10.1002/elps.1150181005

出版商:Wiley Subscription Services, Inc., A Wiley Company    

年代:1997

数据来源: WILEY

摘要:

AbstractClinical interest in the use of capillary electrophoresis (CE) has recently been extended to the microchip environment. Clinical analyses demand careful handling of complex samples that are often limited in quantity and in concentration. The integrated sample handling and analysis capabilities of microchip substrates thus seem ideally suited to clinical applications. This review surveys the development of sample handling (injection, mixing, and reaction) and separation elements on‐chip. The integration of these elements to create a variety of clinical analyzers has been demonstrated. The application of microchip CE systems to human serum protein analysis, immunoassay, and DNA studies is reviewed, along with various other clinical applications. In addition, the clinical potential of the lab‐on‐a‐chip concept is di

ISSN:0173-0835    

DOI:10.1002/elps.1150181006

出版商:Wiley Subscription Services, Inc., A Wiley Company    

年代:1997

数据来源: WILEY

摘要:

AbstractThe development of sensitive, rapid, and accurate methods and apparatus for high‐throughput short tandem repeat (STR) analysis will be critical for the use of microsatellite alteration in cancer screening. Here we show that STR‐based bladder cancer diagnosis can be performed using capillary array electrophoresis and two‐color labeling with energy‐transfer (ET) fluorescent primers. Rapid (≤35 min) separations are achieved on capillary arrays using replaceable separation matrices and the allelic ratios are quantitatively determined with a precision of ± 10%. With this precision, a variation of 20% was considered diagnostically significant. These methods provide a significant improvement in the speed, ease, and precision of STR analyses compared to slab gel elect

ISSN:0173-0835    

DOI:10.1002/elps.1150181007

出版商:Wiley Subscription Services, Inc., A Wiley Company    

年代:1997

数据来源: WILEY

摘要:

AbstractProgressive and selective degeneration of specific classes of neurons occurs in the Alzheimer's disease (AD) brain. Differential vulnerability in this disease is evident even within supopulations that synthesize and release acetylcholine as a transmitter;i.e., basal forebrain cholinergic neurons degenerate but other classes of cholinergic neurons are relatively preserved. The basis for this selective vulnerability is unknown. Studies of differential neuronal vulnerability in AD would be facilitated if cell lines expressing neurotransmitter‐specific phenotypes could be cloned from the brain. Capillary electrophoresis (CE) with laser‐induced fluorescence (LIF) has been shown to be a sensitive method of detection and quantitation of the DNA products of the polymerase chain reaction (PCR). CE/LIF was combined with the PCR to detect phenotypic messenger RNA (mRNA) molecules, converted to cDNA using reverse transcriptase (RT), in cultures of virally immortalized brainstem progenitor cells produced during establishment of a cloning strategy. RT/PCR methods were developed for detection of the mRNAs for choline acetyltransferase (ChAT), the neuronal, constitutive isoform of nitric oxide synthase (c‐NOS), and the growth‐associated protein GAP‐43, three genes known to be expressed in central cholinergic neurons. A “nondestructive” method of screening cultured cells for their expression of c‐NOS was established using depolarization with medium containing 50 mMpotassium ion. These approaches were first validated using cultured SN56 (cholinergic) and N1E‐115 (c‐NOS‐positive) neuroblastoma cells, and with primary brainstem cultures. For the cloning of novel cell lines, progenitor cells were isolated from the embryonic day 13 fetal brainstem and were immortalized by transfection with a retroviral vector that confers a temperature‐sensitive SV‐40 transforming activity and neomycin resistance. Cell colonies surviving in G418‐containing media were isolated and cloned by dilution. Clonal cultures were expanded by growth at 33°C, differentiated by switching to a low‐serum medium and growth at 39°C, and screened for depolarization‐induced accumulation of nitrite in the medium. The subset of putative c‐NOS‐positive clones (about 4%) were then screened for their expression of mRNAs using RT/PCR in combination with CE/LIF. This screening protocol proved to be powerful in the rapid isolation and phenotypic characterization of immortalized progenitor cell

ISSN:0173-0835    

DOI:10.1002/elps.1150181008

出版商:Wiley Subscription Services, Inc., A Wiley Company    

年代:1997

数据来源: WILEY

摘要:

AbstractSingle cell analysis with capillary electrophoresis, a technique capable of detecting zeptomole quantities (10−21mole) of neurochemical species, has been used to demonstrate that lymphocytes are capable of active synthesis of dopamine and norepinephrine. Exposure of lymphocytes to catecholamines at concentrations as low as 10 nMleads to decreased proliferation and differentiation,e.g. interferon‐γ (IFN‐γ), interleukin‐4 (IL‐4) and immunoglobulin (Ig). In addition, both inhibition of dopamine uptake with nomifensine and inhibition of packing of catecholamines into vesicles with tetrabenazine, results in sinificantly lower levels of dopamine and norepinephrine (p<0.01 andp<0.05, respectively). The catecholamine‐dependent inhibition of T‐ and B‐lymphocyte activity is mediated via an induction of a Bcl‐2/Bax and Fas/FasL involved apoptosis. These findings indicate a novel mechanism for regulation of lymphocyte activity in the central nervous system, whereby elevated regional levels of catecholamines might lead to the immunopri

ISSN:0173-0835    

DOI:10.1002/elps.1150181009

出版商:Wiley Subscription Services, Inc., A Wiley Company    

年代:1997

数据来源: WILEY

摘要:

AbstractThe structural investigation of G protein‐coupled receptors has been hindered by the lack of techniques to effectively resolve the hydrophobic peptides obtained by chemical or proteolytic cleavage, as well as the minute amounts of protein typically isolated. We have developed a capillary electrophoresis method for efficient separation of hydrophobic peptides using a cyanogen bromide digest of bacteriorhodopsin as a model for these clinically important membrane proteins. This procedure includes (i) solubilization of the protein digest in acetic acid; and (ii) electrophoresis using an acetic acid‐based buffer system augmented by acetonitrile and hexane sulfonic acid, in a Polybrenecoated fused silica capillary. The potential for detection sensitivity to be increased at least 100‐fold by use of on‐line solid‐phase extraction on C18‐silica is shown. This approach is potentially useful for peptide fingerprinting of sparse and extremely hydrophobic membran

ISSN:0173-0835    

DOI:10.1002/elps.1150181010

出版商:Wiley Subscription Services, Inc., A Wiley Company    

年代:1997

数据来源: WILEY

摘要:

AbstractA selected group of 308 sera were analyzed by capillary electrophoresis (CE), agarose gel electrophoresis (AGE), and cellulose acetate electrophoresis (CAE) and evaluated for abnormalities that would suggest the presence of a monoclonal protein. The sensitivity (an electrophoretic abnormality in sera that contained a monoclonal protein) and specificity (a normal electrophoretic pattern in sera that did not contain a monoclonal protein) was determined for each electrophoretic procedure. CAE was the most specific procedure and CE was the most sensitive. The increase in sensitivity of CE was primarily due to increased detection of cryoglobulins and free light chains. The quantitation of the gamma region and/or monoclonal antibody peaks by CE was similar to results obtained by AGE. Quantitation of very large monoclonal protein peaks (>3.0 g/dL) by on‐line absorption detection (CE) yielded higher results than quantitation by dye‐binding (A

ISSN:0173-0835    

DOI:10.1002/elps.1150181011

出版商:Wiley Subscription Services, Inc., A Wiley Company    

年代:1997

数据来源: WILEY