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Biomedical subjects

A Guttman

Publications and source records attributed to A Guttman.

At least 37 records · Page 2Linked to original sources
PubMed1999

High-throughput genotyping of factor V Leiden mutation by ultrathin-layer agarose gel electrophoresis.

Ultrathin-layer agarose gel electrophoresis is a novel combination of the established methodologies of slab gel electrophoresis and capillary gel electrophoresis. This new format provides a multilane separation platform with rapid analysis time and excellent sensitivity by using laser-induced fluorescence scanning detection system. Sample injection onto the ultrathin-layer separation platform is easily accomplished by membrane mediated loading technology. In this paper, we demonstrate the sensitivity and high-throughput fashion of this novel separation and detection system for rapid genotyping of the coagulation factor V Leiden mutation by polymerase chain reaction (PCR)-restriction fragment length polymorphism (RFLP) analysis. The PCR amplified fragment from exon 10 of the factor V gene was digested by the Mnl I restriction enzyme, followed by automated ultrathin-layer agarose gel electrophoresis analysis with "in migratio" fluorescent labeling during the separation process. Due to its speed and automation, this method should be considered for large scale screening of factor V Leiden mutation.

DNA Mutational Analysis↗
PubMed1999

Effect of linear polymer additives on the electroosmotic characteristics of agarose gels in ultrathin-layer electrophoresis.

Electroosmotic properties of agarose gels with low, medium, high and super high electroendosmosis (EEO) were evaluated based on the apparent electric field mediated mobility of a neutral, fluorescent marker under constant field strength using ultrathin-layer separation configuration. Electroosmotic flow mobility values were measured in different gel concentrations and also in the absence and the presence of various linear polymer additives. Under ultrathin-layer separation conditions, a slight decrease in electroosmotic flow mobility was observed with increasing agarose gel concentration of 1 to 3% for all agarose gels investigated. When linear polymer additives, such as linear polyacrylamide, hydroxyethyl cellulose or polyethylene oxide were added to 1% low electroendosmosis agarose gel, significant reduction of the electroosmotic flow properties were observed with increasing additive concentration. Effect of the intrinsic electroosmotic properties of the various electroendosmosis agaroses on the apparent mobilities and separation performance of double-stranded DNA fragments during automated ultrathin-layer agarose gel electrophoresis was also investigated.

DNA↗
PubMed1999

Sample stacking during membrane-mediated loading in automated DNA sequencing.

Microporous membrane-mediated loading is a novel and efficient sample injection technique for ultrathin slab gel electrophoresis-based automated DNA sequence analysis. The sequencing reaction mixture is spotted directly onto the tabs of the membrane loader, which is then inserted to close proximity of the straight edge of the separation gel. The use of a higher viscosity (> 60 cSt), low ionic strength (conductivity < 320 microS) well solution with a pH of more than one unit lower that of the separation and gel buffer system makes possible efficient stacking of the DNA sequencing fragments at the interface of the gel matrix. When the injection/separation process is initiated by the application of the electric field, a high local electric potential drop is formed across the low ionic strength well solution zone. This results in very fast migration of the DNA sequencing fragments toward the interface of the separation gel, where they stack up against the higher conductivity sieving matrix. During this stacking process, primarily the local pH mediates the actual mobility of the buffer co-ions (borate), forming the leading and terminating zones in the well solution and separation gel, respectively.

Base Sequence↗
PubMed1999

Ultra-thin-layer agarose gel electrophoresis. I. Effect of the gel concentration and temperature on the separation of DNA fragments.

A novel, rapid and efficient separation method is described for the analysis of double stranded (ds) DNA fragments in the form of horizontal ultra-thin-layer agarose gel electrophoresis. This separation technique combines the multilane, high-throughput separation format of agarose slab gel electrophoresis with the excellent performance of capillary electrophoresis. The electrophoretic separation of the fluorophore (Cy5)-labeled dsDNA molecules were imaged in real time by a scanning laser-induced fluorescence/avalanche photodiode detection system. Effects of the gel concentration (Ferguson plot) and separation temperature (Arrhenius plot) on the migration characteristics of the DNA fragments are discussed. An important genotyping application is also shown by characterizing the polymorphic region (2 X or 4 X 48 base pair repeats) of the dopamine D4 receptor gene (D4DR, exon III region) for ten individuals, using PCR technology with Cy5-labeled primers and ultra-thin-layer agarose gel electrophoresis.

DNA↗
PubMed1999

Human dopamine D4 receptor allele genotyping by ultrathin agarose gel electrophoresis with To-Pro-3 complexation.

Growing evidence shows the correlation between the allelic type of the dopamine D4 receptor and the human novelty-seeking personality trait. A sensitive, ultrathin agarose gel electrophoresis-based, high-throughput screening method was developed for genotyping the dopamine D4 receptor (D4DR) exon III 48 base pair repeat polymorphism. The efficiency of the method was increased by reamplification nested polymerase chain reaction (PCR) - of the 48 base pair repeat containing the PCR product with internal primers. The nested PCR fragments were analyzed by ultrathin layer agarose gel electrophoresis with an automated real-time laser-induced fluorescent detection system. Noncovalent affinity complexation was accomplished during the separation process by the addition of a very low concentration of intercalation dye, To-Pro-3 (2 nM) to the gel buffer system. This resulted in instant fluorescent labeling of the migrating PCR fragments. This method can readily facilitate genetic association studies between dopamine receptor genotypes and some human behavioral and neuropsychiatric disorders.

Alleles↗
PubMed1999

Efficacy of inhaled steroids (beclomethasone dipropionate) for treatment of mild to moderately severe asthma in the emergency department: a randomized clinical trial.

STUDY OBJECTIVE: To examine the efficacy of an inhaled steroid, when added to a standard regimen of beta-agonist therapy, in the treatment of patients with mild to moderately severe asthma in the emergency department. METHODS: A convenience sample of adult patients with asthma (FEV1 % predicted 40% to 69%) presenting to the ED was randomly assigned in a double-blind fashion into 2 treatment groups. The first group received 2.5 mg nebulized salbutamol plus 1 mg (4 puffs) of beclomethasone dipropionate (BDP) at baseline, 30 minutes, and at 1, 2, and 4 hours, delivered by a metered-dose inhaler (MDI) attached to a spacer device (Vent-AH-aler, Glaxo). The second group was given the same salbutamol regimen plus MDI placebo through the Vent-AH-aler. The primary endpoint was improvement in FEV1 %predicted at 6 hours. RESULTS: Of 54 patients enrolled, 28 were assigned to the BDP group and 26 to the placebo group. Spirometry improved significantly in both groups over the 6 hours compared with baseline (ANOVA, P <.001). At 6 hours, the mean absolute improvement in FEV1 % predicted for BDP was 18% versus 17% for placebo (95% confidence interval for the absolute difference of 1% [-8% to 10%]). The proportion of patients in the BDP group who were hospitalized was 7% compared with 19% for patients in the placebo group (95% confidence interval for the difference of 12% [-6%, 30%]). CONCLUSION: In this group of patients with mild to moderately severe asthma, 5 mg BDP delivered by MDI during the initial 4 hours of an emergency visit was of no added benefit over standard therapy, as measured by improvement in FEV1 % predicted at 6 hours. However, a trend toward a difference in admission favoring BDP was observed. [Afilalo M, Guttman A, Colacone A, Dankoff J, Tselios C, Stern E, Wolkove N, Kreisman H: Efficacy of inhaled steroids (beclomethasone dipropionate) for treatment of mild to moderately severe asthma in the emergency department: A randomized clinical trial.

Administration, Inhalation↗
PubMed1999

A comparative study of the chemical stability of various mitomycin C solutions used in glaucoma filtering surgery.

OBJECTIVE: To determine the chemical stability of various mitomycin C (MMC) solutions used in glaucoma filtering surgery. METHODS: A survey of the MMC solutions currently in use in 21 hospitals (11 in Canada, 10 in the United States) was conducted. A comparative study of the chemical stability of five different representative solutions was performed. The effects of buffer and storage variables on the chemical breakdown of MMC in the solutions were studied by means of high-performance liquid chromatography (HPLC). RESULTS: The survey revealed 33 different variations (including recipes and storage conditions) in the preparation of MMC solutions. Although the majority of the hospitals (15 of 21; 72%) were preparing stable solutions, six of the hospitals (28%) were preparing potentially unstable solutions. The stability of the solutions varied in a nonuniform manner when stored at different temperatures in different buffers. CONCLUSION: The lack of standardization and quality control of MMC solutions used in filtering surgery allows for the possibility of hospitals preparing unstable solutions.

Buffers↗
PubMed1998

Real-time detection of allele-specific polymerase chain reaction products by automated ultra-thin-layer agarose gel electrophoresis.

Ultra-thin-layer agarose gel electrophoresis, a novel combination of agarose slab gel electrophoresis and capillary gel electrophoresis was introduced in conjunction with laser-induced fluorescence (LIF) scanning detection for the analysis of polymerase chain reaction (PCR) products. Allele-specific fragments, amplified from genomic DNA of patients with congenital adrenal hyperplasia (most often caused by mutations of 21-hydroxylase gene, CYP-21), were used as a model system to investigate the applicability, sensitivity and resolving power of the method. The allele-specific products were generated by PCR and separated by ultra-thin-layer agarose gel electrophoresis. The double-stranded DNA fragments were easily visualized in real-time via complexation during the separation process by the intercalator dye TO-PRO-3 which was part of the separation gel-buffer system. In this way, the migrating dsDNA-dye complexes were detected in real-time by a scanning LIF detection system with sub-nanogram sensitivity. The system employs a 632-nm solid-state laser and an avalanche photodiode detector scanning to the separation platform by means of a fiber bundle system. Automated ultra-thin-layer agarose gel electrophoresis with 'on the fly' TO-PRO-3 staining of dsDNA fragments and LIF detection system proved to be a very fast, high-throughput separation method for individual or multiplexed PCR products, with excellent sensitivity.

Alleles↗
PubMed1998

Fiber bundle based scanning detection system for automated DNA sequencing.

High-throughput DNA sequencing techniques are under rapid development currently, mainly triggered by the Human Genome Project. At the present time, slab gel based automated DNA sequencing is the standard procedure, utilizing fluorophore labeling and laser-induced fluorescence detection with scanning technology. In this paper, a novel, fiber-optic bundle based detection system is introduced, where a central illuminating fiber is used for the excitation of the electrophoretically separated fluorophore-labeled DNA sequencing fragments, along with several collecting fibers disposed around the illuminating fiber to collect the emitted fluorescent signal. As a model system, Cy5-labeled DNA sequencing fragments were separated on an ultrathin polyacrylamide slab gel and detected by the fiber bundle based laser-induced fluorescence detection system. A 640-nm diode laser was used to generate the illumination beam, and the emitted light collected by the fiber bundle was detected by a solid-state avalanche photodiode.

Autoanalysis↗
PubMed1998

Simultaneous analysis of various mutations on the 21-hydroxylase gene by multi-allele specific amplification and capillary gel electrophoresis.

A detailed study is presented on the detection of various known point mutations using polymerase chain reaction (PCR) based multi-allele specific amplification (MASA) in conjunction with capillary gel electrophoresis (CGE) separation. The resulting PCR products, corresponding to the individual mutations, are labeled with ethidium bromide during CGE separation, and detected by laser-induced fluorescence. MASA proved to be a novel, fast and cost-effective method for simultaneous analysis of multiple known mutation sites, employing more than one allele specific primers in a single PCR reaction. It results in coexisting amplification of numerous DNA fragments differing in size, which are subsequently separated by CGE. In the present study, several point mutations were analyzed simultaneously by MASA-CGE on the 21-hydroxylase gene of a patient with congenital adrenal hyperplasia.

Alleles↗
PubMed1998

Membrane-mediated sample loading for automated DNA sequencing.

A new and improved sample loading method for DNA sequencing gels using oligo/polynucleotide binding membranes is described. The labeled DNA sequencing fragments were spotted onto a membrane sample loader, which was then placed in contact with the precast polyacrylamide separation gel. In this way, the time-consuming sample well loading by the tedious pipetting procedure was avoided. The spotted DNA fragments remain immobilized on the membrane until the separation process is initiated by the application of the electric field (an active DNA release mechanism). This novel technique enables sample loading outside of the separation platform, thereby allowing full utilization of various automated sample preparation and liquid handling (robotics) systems, resulting in "real" automated DNA sequencing. The loaded membranes can be stored for more than 24 hours for later use.

Automation↗
PubMed1997

Analysis of 1-aminopyrene-3,6,8-trisulfonate-derivatized oligosaccharides by capillary electrophoresis with matrix-assisted laser desorption/ionization time-of-flight mass spectrometry.

Matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS), has been used for structure characterization of 1-aminopyrene-3,6,8-trisulfonate (APTS)-derivatized oligosaccharides previously separated by capillary electrophoresis (CE). The resolved components were first isolated by employing an automated high-resolution fraction collector. Using on-probe sample cleanup with a cation-exchange resin and a matrix mixture of (1:1) 6-hydroxypicolinic acid and 3-hydroxypicolinic acid, APTS-labeled oligosaccharides were successfully detected by MALDI-TOF MS in the negative ionization mode. Each APTS-labeled oligosaccharide produced single [M-H]-1 peaks. Detection limits for standard APTS-derivatized oligosaccharide (maltoheptaose) were down to 30 fmol. APTS labeled maltooligosaccharides and various standard carbohydrates were separated and collected by CE, followed by molecular weight determination with MALDI-TOF MS. The mass spectral characterization enhances the power of the CE analysis of oligosaccharides.

Carbohydrate Sequence↗
PubMed1997

Exoglycosidase matrix-mediated sequencing of a complex glycan pool by capillary electrophoresis.

This paper discusses oligosaccharide sequencing by consecutive enzymatic digestion of carbohydrates using an exoglycosidase array, followed by capillary electrophoresis separation of the digests. Because of the high resolving power and good reproducibility of capillary electrophoresis, multistructure sequencing of a complex glycan pool can be performed in most instances requiring no prior isolation of the individual oligosaccharides. High sensitivity laser-induced fluorescence detection enables acquisition of complete sequence information from several picomoles of glycoproteins. Comparison of the migration times of the exoglycosidase digest fragments to the maltooligosaccharide ladder, enables calculation of migration shifts, due to cleavage based on the actual exoglycosidases used. The particular sequence of each oligosaccharide in a glycan pool can be proposed with high confidence based on the migration time shifts of the various oligosaccharide structures. However, possible combinations of various sequence fragments may have very similar charge to hydrodynamic volume ratios, resulting in electrophoretic co-migration when a mixture of different oligosaccharides is sequenced together. Then, capillary electrophoresis separations of the resulting fragments should be evaluated after each digestion step. In the instances of complex separation profiles when multiple peaks are present, the evaluation of peak shifts can get very complicated and solved only with the aid of a software program. Data about the monosaccharide composition of the glycan pool provides useful information in designing the digestion enzyme matrix.

Carbohydrate Sequence↗
PubMed1997

Analysis of monosaccharide composition by capillary electrophoresis.

The monosaccharide composition analysis described in this paper employs capillary electrophoretic separation of sugar monomers liberated from glycoproteins or oligosaccharides, by high temperature acidic hydrolysis. Trifluoroacetic acid was used for sialo- and neutral-sugar hydrolysis, and hydrochloric acid was used for amino-sugar hydrolysis. The neutral- and amino-sugars in the hydrolyzates were then labeled with a charged fluorophore, 8-aminopyrene-1,3,6-trisulfonate, while sialic acids were labeled with 9-aminoacridone. The stoichiometry of labeling was such that only one fluorophore molecule was attached to each monosaccharide molecule. The labeled monosaccharides were then separated by high-performance capillary electrophoresis with laser induced fluorescence detection. The acidic hydrolysis and fluorophore labeling conditions described in this paper are suitable for monosaccharide composition analysis of a wide variety of complex carbohydrates from glycoprotein and/or oligosaccharide samples using capillary electrophoresis.

Acetylglucosamine↗
PubMed1997

Multistructure sequencing of N-linked fetuin glycans by capillary gel electrophoresis and enzyme matrix digestion.

Oligosaccharide sequencing by specific enzymatic digestion of N-linked fetuin glycans using a carefully designed exoglycosidase matrix in conjunction with separation of the combined digests by high performance capillary gel electrophoresis is described. Due to the extremely high separation power and excellent reproducibility of capillary gel electrophoresis, in most instances no isolation of the individual oligosaccharides is necessary, enabling multistructure sequencing from a released glycan pool. By comparing the positions of the separated exoglycosidase digest fragments to maltooligosaccharides of known size, relative migration times and migration shifts are easily calculated. Hence, the particular sequence of each oligosaccharide in a released glycan pool can be proposed with high confidence. Additionally, with the use of high sensitivity laser-induced fluorescence detection, complete sequence information can be attained from picomolar amounts of purified glycoproteins.

Animals↗
PubMed1997

The effects of combined intravenous and inhaled steroids (beclomethasone dipropionate) for the emergency treatment of acute asthma. The Asthma ED Study Group.

OBJECTIVE: To compare the efficacy of high-dose inhaled steroids in conjunction with IV steroids with that of IV steroids alone in the emergency treatment for acute asthma. METHODS: A double-blind, placebo-controlled, randomized trial was conducted on 60 ED patients presenting with acute asthma. All patients received nebulized salbutamol, and IV methylprednisolone, 80 mg at baseline and 40 mg at 6 hours. In addition to the above therapy, the experimental group received beclomethasone dipropionate (BDP) 7 mg over 8 hours via a metered-dose inhaler (MDI) attached to a holding chamber, while the control group received a placebo administered in the same fashion. Patients were treated on the protocol for 12 hours with the primary outcome measure being the change in % predicted FEV1. RESULTS: Of 60 patients, 30 were randomized to BDP (age: 42 +/- 16 years; FEV1: 0.97 +/- 0.42 L) and 30 were randomized to placebo (age: 37 +/- 18 years; FEV1: 0.98 +/- 0.35 L). Spirometry and dyspnea measured by the Borg Scale improved significantly in both groups compared with baseline (p < 0.001). Changes in spirometry measures, dyspnea, and vital signs did not differ between treatment groups over the 12 hours of study (p > 0.05). CONCLUSION: Inhaled BDP added to the standard regimen of IV methylprednisolone, and beta-agonist did not further improve flow rates or dyspnea scores measured for up to 12 hours after presentation to the ED.

Administration, Inhalation↗