Capillary zone electrophoresis method for fingerprint of allantoic fluid in normal and infected SPF embryonated chicken eggs

A capillary zone electrophoresis method was developed for fingerprint determination of allantoic fluid in specific pathogen free (SPF) embryonated chicken eggs. The effects of some crucial parameters, such as buffer type, pH, wavelength and running voltage on the separation were studied systematically. The components of the allantoic fluid were well separated using a fused-silica uncoated capillary with an effective length of 50 cm and an internal diameter of 50 microm. One hundred millimolars sodium tetraborate buffer containing 20 mM sodium dihydrogen phosphate with a final pH 9.8 was used as a running buffer. Comparative fingerprints of allantoic fluid in normal and infected with infectious bronchitis virus (IBV) SPF embryonated chicken eggs were also evaluated. The results showed that there were significant differences between composition of normal allantoic fluid and allantoic fluid infected with IBV, which led to different migration behavior. This method was shown to be stable and reproducible with a relative standard deviation of less than 5% for both migration time and peak current.     

SSCP analysis: A blind sensitivity trial

Studies of the sensitivity of SSCP analysis usually have been performed under conditions contrary to the rules of quality control trials and have produced widely different results. We have performed a blind trial of the sensitivity of SSCP analysis for the detection of mutations in fragments up to 500 bp in length under a fixed single set of electrophoretic conditions. The mutation detection rate was 84%. In addition, we have identified a second mutation in nine samples. All these mutations are polymorphisms, including a novel polymorphism 1248+52T/C first reported in the present work. Hum Mutat 10:65–70, 1997. © 1997 Wiley-Liss, Inc.     

Analysis of mutations using PCR and denaturing gradient gel electrophoresis.

Denaturing gradient gel electrophoresis (DGGE) separates DNA molecules based on primary sequence. Under the appropriate conditions, all base pair (bp) substitutions, frame-shifts, and deletions less than about 10 bp can be resolved from the wild type sequence using DGGE. Polymerase chain reaction (PCR) permits facile amplification of a given region of the genome. We have combined PCR and DGGE to: (i) Localize mutations in the X-linked human androgen receptor gene. PCR/DGGE was used to screen the individual exons in the 2757-bp coding region of the gene in afflicted individuals as well as in potential carriers. Inheritance of a mutant allele has been demonstrated in several cases; (ii) Analyze thousands of thioguanine-resistant mutants simultaneously. The in vitro mutational spectra of MNNG, ICR-191, and cisplatin at the human HPRT locus have been examined by this method. The compounds all have mutational hotspots in a GGGGGG sequence in exon 3; however, the particular mutations induced by the agents were different; (iii) Examine the fidelity of several DNA polymerases used in PCR. The fidelity of Thermus aquaticus DNA polymerase (Taq) is 1-2 x 10(-4) misincorporations/bp/replication. Problems with Taq polymerase arise in the analysis of complex mutant populations by DGGE because the Taq-induced errors reduce the sensitivity of the system. To circumvent this, it had been necessary to use Sequenase, a modified T7 DNA polymerase with a higher fidelity. However, Sequenase is not thermostable and must be added every PCR cycle. A thermostable DNA polymerase from Thermococcus litoralis (Vent) is now available, and we have examined the fidelity of Vent, Taq, and Sequenase polymerase in PCR using DGGE. The fidelity of Vent, Taq, and Sequenase polymerase was 2.4 x 10(-5), 8.9 x 10(-5), and 4.4 x 10(-5) errors/bp, respectively. Vent polymerase had the highest fidelity of the three enzymes tested.     

Capillary electrophoretic immunoassays for digoxin and gentamicin with laser-induced fluorescence polarization detection

New immunoassays for therapeutic drugs digoxin and gentamicin have been described, which involved the separation of free and antibody-bound drug by capillary electrophoresis (CE) and the detection by laser-induced fluorescence polarization (LIFP). While the fluorescein-labeled digoxin and gentamicin (tracers) displayed negligible fluorescence polarization in solution, the complex formation between these small molecules and their antibodies resulted in substantial increases in fluorescence polarization due to the increase in molecular size. The LIFP detection, capable of measuring vertically and horizontally polarized fluorescence components simultaneously, provides enhanced capability for the identification of complex in capillary electrophoretic immunoassays. Proper adjustments of the running buffer pH and the ratio of antibody to tracer are essential for optimization of the performance of these assays. The digoxin-antibody complex remained stable during CE separation with running buffer pH ranging from 9.3 to 12. Calibration curves covering a concentration range of 0.05 to 0.5 ng/ml were obtained with a running buffer of pH 12. The concentration and mass detection limits were 0.02 ng/ml and 26 zmol, respectively. For gentamicin assay, the running buffer pH 10 was used to reduce the adsorption of the tracer while minimizing the dissociation of the antibody-tracer complex during the separation. The calibration curves covered a concentration range 0.05-1.0 microg/ml, with a concentration detection limit of 25 ng/ml and a mass detection limit of 52 amol of gentamicin.     

Sensitivity of the polymerase chain reaction for detecting human T-cell leukemia virus type I sequences in paraffin-embedded tissue. Effect of unbuffered formalin fixation.

Recently, the application of the polymerase chain reaction (PCR) to formalin-fixed paraffin-embedded tissue has been reported. But formalin, especially unbuffered formalin, is known to break DNA into small fragments. DNA extracted from MT-2 cells fixed in unbuffered formalin for various periods of time were subjected to the PCR and the effect of unbuffered formalin fixation on the ability of the PCR to detect exogenous sequences; i.e., human T-cell leukemia virus type I (HTLV-I) proviral DNA, was examined. The sensitivity of the PCR decreased as a function of both the duration of fixation and the length of the expected DNA products. When the expected length of the PCR product was about 200 bp, a slight decrease in the sensitivity was observed after 4-day fixation. When it was about 300 bp, a similar decrease was observed following 4-h fixation. In the case of a 500 bp product, the sensitivity began to decrease after 30-min fixation and a 100-fold decrease was observed after 10-day fixation. A decrease was not observed, however, with a 100 bp product. The appropriate design of primers, especially with regard to the length of the amplified product, is essential to keep the sensitivity of the PCR, particularly when the target tissues have been fixed in unbuffered formalin.     

Induction of germline-length mutations at the minisatellites PC-1 and PC-2 in male mice exposed to polychlorinated biphenyls and diesel exhaust emissions

PC-1 and PC-2 are hypervariable mouse minisatellites. The rates of spontaneous germline-length mutation have been shown to vary between different mouse strains. PC-1 is composed of GGCAG repeat units and PC-2 of GGCAGGA. Minisatellites frequently mutate by gaining or losing repeat units. Such length mutations in mini- and microsatellites have been associated with human disease and may therefore be an important endpoint in genetic toxicity testing. Carcinogenic activity of many chemicals is associated with their ability to induce heritable mutations. Since minisatellites are highly prone to mutate to new lengths, which can be assayed by Southern analysis, we used this method to detect heritable genetic effects in mice. Male mice exposed to diesel exhausts and/or polychlorinted biphenyls (PCB) were investigated for effects on the germline mutation frequenallele lengths in parents and offspring. For PC-1 significantly higher mutation frequencies were found in males treated with diesel exhausts + PCB (6 of 35 alleles) and with PCB alone (6 of 51 alleles) as compared to the males in the control group (0 of 43 alleles). The mutation frequency in the diesel exhaust group was not significantly increased (2 of 43 alleles). For PC-2 the only mutation found occurred in the PCB group (1 of 51 alleles). This in vivo study demonstrates—for the first time—chemically induced minisatellite mutations in the germline. Environ. Mol. Mutagen. 30:254–259, 1997 © 1997 Wiley-Liss, Inc.     

Molecular evolution of noncoding regions of the chloroplast genome in the Crassulaceae and related species

Universal primers were used for PCR amplification of three noncoding regions of chloroplast DNA (cpDNA) in order to study sequence-length variation in the Crassulaceae and in related species. Several length mutations were observed that are of diagnostic value for evolutionary relationships in the Crassulaceae and the Saxifragaceae. Length variation and sequence divergence in the intergenic spacer between the trnL (UAA) 3 exon and the trnF (GAA) gene among 15 species were studied in detail by nucleotide-sequence analysis. A total of 50 insertion/deletion mutations were observed, accounting for a spacer-length variation in the range of 228–360 bp. Eighteen short direct repeat motifs (4–11 bp) and two inverted repeat motifs (7–11 bp) were found to be associated with length variation. Phylogenetic analysis of the sequence data indicated a pattern of relationships that was largely consistent with a previous analysis of cpDNA restriction-site variation. Evaluation of the level of homoplasy in insertion/deletion mutations within a phylogenetic framework revealed that only 1 out of 34 length mutations longer than 2 bp must have had multiple origins. The feasibility of the noncoding chloroplast DNA regions for molecular evolutionary studies is discussed.     

APC germline mutations identified in Czech patients with familial adenomatous polyposis

Familial adenomatous polyposis (FAP) is an autosomal dominantly inherited predisposition to colorectal cancer, which is caused by germline mutations in the adenomatous polyposis coli (APC) gene. The APC mutations have been investigated in 46 Czech unrelated FAP families and 9 suspected FAP families using DGGE analysis and direct DNA sequencing. We found 25 germline APC mutations and identified 11 which were not previously reported. Of the identified mutations, 10 were 1 to 5 bp deletions, four were 1 bp insertions and six were nonsense, all leading to the formation of premature stop codon. In addition, we detected two mutations in the splice-donor region of APC intron 11, one missense and two samesense mutations. Phenotypes of patients with known and novel types of mutations are presented and discussed.     

Separation and simultaneous determination of nalidixic acid, hydroxynalidixic acid and carboxynalidixic acid in serum and urine by micellar electrokinetic capillary chromatography

Separation in capillary electrophoresis is governed by various factors, including buffer type, buffer concentration, pH, temperature, voltage and micelles. Through proper adjustment of these parameters, nalidixic acid and its two major metabolites, 7-hydroxynalidixic and 7-carboxynalidixic, could be separated by micellar electrokinetic capillary chromatography using an electrophoretic electrolyte consisting of 50 mM borate buffer (pH 9) containing 25 mM sodium dodecyl sulphate and 10% acetonitrile. A linear relationship between concentration and peak area for each compound was obtained in the concentration range 0.15-100 micrograms ml-1, with a correlation coefficient greater than 0.999 and detection limits in the 0.2-0.7 ng ml-1 range. Intra- and inter-day precision values of about 0.8-1.2% RSD (n = 11) and 1.3-2.0% RSD (n = 30), respectively, were obtained. The method has been applied to the analysis of nalidixic acid and its two major metabolites in serum and urine with limits of sensitivity lower than 0.8 ng ml-1.     

Cold single-strand conformation polymorphism analysis: optimization for detection of APC gene mutations in patients with familial adenomatous polyposis

Over 200 adenomatous polyposis coli (APC) gene mutations have been described in familial adenomatous polyposis (FAP) patients. Recent single-strand conformation polymorphism (SSCP) screening methods have introduced minigel runs, simple ethidium bromide staining and external temperature control without any loss of sensitivity (cold-SSCP). In order to test the effectiveness in APC mutation detection, cold-SSCP was employed following polymerase chain reaction (PCR) amplification in three patients with FAP. Different running parameter combinations were compared. The three mutations already known were all diagnosed by cold-SSCP. The gel concentration was found to be essential in detecting the single-base substitution in fragments of different lengths. The observation of deletions was not affected by gel concentrations and heteroduplex bands were always produced. The temperature or glycerol addition did not significantly affect sensitivity. This modified cold-SSCP method provides a simple and effective way for detecting several known Apc gene mutations without any loss of sensitivity and could be useful for large-scale molecular diagnosis of FAP.     

Identification of 54 mycobacterial species by PCR-restriction fragment length polymorphism analysis of the hsp65 gene

A total of 121 reference and clinical strains of both slowly and rapidly growing mycobacteria belonging to 54 species were studied for restriction fragment length polymorphism of a PCR-amplified 439-bp segment of the gene encoding the 65-kDa heat shock protein. Restriction digests were separated by 10% polyacrylamide gel electrophoresis (PAGE). By including a size standard in each sample, the restriction fragment profile was calculated using a computer-aided comparison program. An algorithm describing these 54 species (including 22 species not previously described) is proposed. We found that this assay based on 10% PAGE provided a more precise estimate than that based on agarose gel electrophoresis of the real size of restriction fragments as deduced from the sequence analysis and allowed identification of mycobacteria whose PCR-restriction fragment length polymorphism analysis patterns were unequivocally identified by fragments shorter than 60 bp.     

Conditions for binding bovine IgG1 to protein A-Sepharose

Conditions were established so that both subclasses of bovine IgG were bound to Protein A-Sepharose. Increasing the pH of the starting buffer to pH 8.0 from pH 7.0 and increasing the starting phosphate concentration of the buffer to 0.5 M from 0.2 M enhanced the separation. Using these modifications in the buffer system, IgG1 was eluted from pH 7.0 to 7.8 and IgG2 at pH 5.0. Two major peaks were associated with IgG1 activity indicating heterogeneity of binding to protein A-Sepharose. One peak was found for IgG2. The molecular weights of the fractions were determined to be that of IgG by sodium dodecyl sulfate polyacrylamide gel electrophoresis.     

Rapid detection of deletion, insertion, and substitution mutations via heteroduplex analysis using capillary- and microchip-based electrophoresis

In this report, we explore the potential of capillary and microchip electrophoresis for heteroduplex analysis- (HDA) based mutation detection. Fluorescent dye-labeled primers (6-FAM-tagged) were used to amplify the DNA fragments ranging from 130 to 400 bp. The effects of DNA fragment length, matrix additives, pH, and salt were evaluated for capillary electrophoresis- (CE) and/or microchip electrophoresis-based HDA, using six heterozygous mutations, 185delAG, E1250X (3867GT), R1443G (4446CG), 5382insC, 5677insA in BRCA1, and 6174delT in BRCA2. For this system, the effective fragment size for CE-based HDA was found in the range of 200-300 bp, however, the effective range was 150-260 bp for microchip-based HDA. Sensitivity studies show CE-based HDA could detect a mutated DNA present at only 1%-10% of the total DNA. Discrimination between wild-type and deletion or insertion mutations in BRCA1 and BRCA2 with CE-based HDA could be achieved in <8 min, while the substitution mutations required 14 min of analysis time. For each mutation region, 15 samples were run to confirm the accuracy and reproducibility of the method. Using the method described, two previously reported mutations, E1038G (3232AG, missense) and 4427 C/T (4427CT, polymorphism), were detected in the tested samples and confirmed by DNA sequencing. Translation of the CE-based methodology to the microchip format allowed the analysis time for each mutation to be decreased to 130 sec. Based on the results obtained with this model system, it is possible that CE-based HDA methodologies can be developed and used effectively in genetic testing. The fast separation time and automated operation afforded with CE instrumentation provide a powerful system for screening mutations that include small deletions, insertions, and point mutations. Translation to the microchip platform, especially to a multichannel microchip system, would allow for screening mutations with high throughput.     

Development of hen's egg with low antigenicity for allergic patients]

We tried to prepare hen's egg with low antigenicity for allergy patients. First, we examined the effects of pH, heating temperature and time on antigenicity of egg. Dried whole egg solid (DES) was dissolved in the buffer solution adjusted at various pH and heated at 120 degrees C for 10, 20 and 40 min. As a result the antigenicity didn't significantly decrease. Next, DES was treated with succinic anhydride, sodium hydroxide and was heated. The antigenicity of the resultant modified whole egg solid (MES) was much lower than that of heated whole egg solid. Antigenicity was measured precisely by enzyme immunoassay, inhibition ELISA, RAST inhibition and PCA. It was found that the antigenicity of MES was decreased less than 1/1000 of that of DES. Then SDS-polyacryl-amide gel electrophoresis and gel filtration were performed to clarify the degree of the change of egg proteins. The bands and peaks which corresponded to ovalbumin and ovomucoid known to be major allergenic proteins responsible for egg allergy disappeared and new bands and peaks were detected.     

First isolation and characterization in humans of Entamoeba histolytica (laboratory-made) zymodeme XX

Isoenzyme analysis by starch-gel electrophoresis has proved to be a useful method for the biochemical differentiation of pathogenic Entamoeba histolytica and non-pathogenic E. dispar isolates. Of the 24 known zymodemes, 3 are laboratory-made and have not previously been identified in humans. Parasitology screening was carried out in a psychiatric institution. Two amebic stocks were isolated and characterized that had never previously been found in humans and that have protein patterns identical to that of the laboratory-made zymodeme XX. Received: 15 February 1997 / Accepted: 10 March 1997     

pH regulation and buffering power in gastric smooth muscle

Intracellular pH can have profound effects on tissue function, but little is known about how pH is regulated, buffered or affects the function of gastric smooth muscle. As the pH of gastric myocytes may alter with pathophysiological disturbance of the gastric lining, or reduction in blood flow to the stomach, these parameters were investigated. Intracellular pH was measured in strips of corpus from rats and guinea-pigs and pH perturbed by the addition of Na butyrate. pH regulation was investigated using pharmacological inhibitors and ionic substitutions. Resting pH was found to be around 7.0, and buffering power relatively high, compared to other muscles in both species. In the guinea-pig amiloride, EIPA and HOE694 prevented pH regulation from an acid load, but amiloride- and EIPA-insensitive pH-regulating mechanisms were found in the rat. The pH-regulatory mechanism present in the rat was also insensitive to DIDS, SITS and removal of external Cl-, but inhibited by Na+ substitution and HOE694. Acidification reduced gastric tone in both species. We conclude that pH alteration will significantly affect gastric contractility, despite a high capacity of the tissue to buffer and regulate pH change. The sensitivity to NHE inhibitors differs between rat and guinea-pig, suggesting that Na+/H+ exchanger isoform expression differs between gastric tissue.     

Isolation and immunochemical characterization of the group-specific antigen of Streptococcus mutants 6715.

The group d antigen of Streptococcus mutans 6515 was isolated from a buffer (pH 7.3)-boiled extract of whole cells and analyzed immunochemically. Rabbits immunized in three different fashions with whole S. mutans 6715 each responded to the same antigenic cell surface component. This presumptive major antigen was found in culture supernatant, sonically treated supernatant, acid and buffer extracts of whole cells, and trichloroacetic acid extract of cell membranes. A crude preparation of this antigen could completely inhibit antibody-mediated cell (S. mutans 6715) agglutination in a spectrophotometric analysis. The antigen was purified from buffer-boiled extracts by gel filtration on columns of Sepharose 4B. The antigen did not migrate to the anode on electrophoresis nor did it contain appreciable quantities of phosphorus, glycerol, or ribitol. This suggested that the d antigenicity did not reside in a teichoic acid. The d antigen contained galactose and glucose as the sole saccharides, in a ratio of 5.9:1.0. Protein (9.5%) appeared to be a portion of the antigen, although Pronase-digested antigen retained the same electrophoretic mobility and could precipitate virtually all (98.6%) purified antibody directed to the intact antigen. The data obtained from hapten innvolved. Glucose also contributed to the immunodominant region. Antibody directed to the d antigen may be of importance in the inhibition of adherence phenomena manifested by S. mutans organisms of the d group.     

Expression of heterologous aquaporins for functional analysis in Saccharomyces cerevisiae

In this study the yeast Saccharomyces cerevisiae, which is a genetically tractable model for analysis of osmoregulation, has been used for analysis of heterologous aquaporins. Aquaporin water channels play important roles in the control of water homeostasis in individual cells and multicellular organisms. We have investigated the effects of functional expression of the mammalian aquaporins AQP1 and AQP5 and the aquaglyceroporins AQP3 and AQP9. Expression of aquaporins caused moderate growth inhibition under hyperosmotic stress, while expression of aquaglyceroporins mediated strong growth inhibition due to glycerol loss. Water transport was monitored in protoplasts, where the kinetics of bursting was influenced by presence of aquaporins but not aquaglyceroporins. We observed glycerol transport through aquaglyceroporins, but not aquaporins, in a yeast strain deficient in glycerol production, whose growth depends on glycerol inflow. In addition, a gene reporter assay allowed to indirectly monitor the effect of AQP9-mediated enhanced glycerol loss on osmoadaptation. Transport activity of certain aqua(glycero)porins was diminished by low pH or CuSO₄, suggesting that yeast can potentially be used for screening of putative aquaporin inhibitors. We conclude that yeast is a versatile system for functional studies of aquaporins, and it can be developed to screen for compounds of potential pharmacological use.     

Sensitivity of the denaturing gradient gel electrophoresis technique in detection of known mutations and novel Asian mutations in the CFTR gene

More than 500 mutations have been identified in the CFTR gene, making it an excellent system for testing mutation scanning techniques. To assess the sensitivity of denaturing gradient gel electrophoresis (DGGE), we collected a representative group of 202 CFTR mutations. All mutations analyzed were detected by scanning methods other than the DGGE approach evaluated in this study. DGGE analysis was performed on 24 of the 27 exons and their flanking splice site sequences. After optimization, 201 of the 202 control samples produced an altered migration pattern in the region in which an alteration occurred. The remaining sample was sequenced and found not to have the reported mutation. The ability of DGGE to identify novel mutations was evaluated in three Asian CF patients with four unknown CF alleles. Three novel Asian mutations were detected—K166E, L568X, and 3121-2 A→G (in homozygosity)—accounting for all CF alleles. These results indicate that an optimized DGGE scanning strategy is highly sensitive and specific and can detect 100% of mutations. Hum Mutat 9:136–147, l997. © 1997 Wiley-Liss, Inc.     

Stereoselective determination of ofloxacin and its metabolites in human urine by capillary electrophoresis using laser-induced fluorescence detection

A capillary electrophoresis method for the simultaneous separation and enantioseparation of the antibacterial drug ofloxacin and its metabolites desmethyl ofloxacin and ofloxacin N-oxide in human urine has been developed and validated. Enantioseparation was achieved by adding sulfobutyl beta-cyclodextrin to the running buffer. The detection of the analytes was performed by laser-induced fluorescence (LIF) detection using a HeCd-laser with an excitation wavelength of 325 nm. In comparison with conventional UV detection, LIF detection provides higher sensitivity and selectivity. The separation can be performed after direct injection of urine into the capillary without any sample preparation, because no matrix compounds interfere with the assay. Additionally, the high sensitivity of this method allows the quantification of the very low concentrations of enantiomers of both metabolites. The limit of quantification was 250 ng/ml for ofloxacin enantiomers and 100 ng/ml for each metabolites' enantiomers. This method was applied to the analysis of human urine samples collected from a volunteer after oral administration of 200 mg of (+/-)-ofloxacin to elucidate stereoselective differences in the formation and excretion of the metabolites. It could be demonstrated that the renal excretion of the S-configured metabolites, especially S-desmethyl ofloxacin, within the first 20 h after dosage, is significantly lower than that of the R-enantiomers.