Biomedical subjects

P J Oefner

Publications and source records attributed to P J Oefner.

At least 55 records · Page 3Linked to original sources

PubMed1996

Familial hemiplegic migraine and episodic ataxia type-2 are caused by mutations in the Ca2+ channel gene CACNL1A4.

Genes for familial hemiplegic migraine (FHM) and episodic ataxia type-2 (EA-2) have been mapped to chromosome 19p13. We characterized a brain-specific P/Q-type Ca2+ channel alpha1-subunit gene, CACNL1A4, covering 300 kb with 47 exons. Sequencing of all exons and their surroundings revealed polymorphic variations, including a (CA)n-repeat (D19S1150), a (CAG)n-repeat in the 3'-UTR, and different types of deleterious mutations in FHM and EA-2. In FHM, we found four different missense mutations in conserved functional domains. One mutation has occurred on two different haplotypes in unrelated FHM families. In EA-2, we found two mutations disrupting the reading frame. Thus, FHM and EA-2 can be considered as allelic channelopathies. A similar etiology may be involved in common types of migraine.

Base Sequence↗

PubMed1996

Efficient random subcloning of DNA sheared in a recirculating point-sink flow system.

Based on a high-performance liquid chromatographic pump, we have built a device that allows recirculation of DNA through a 63-microm orifice with ensuing fractionation to a minimum fragment size of approximately 300 base pairs. Residence time of the DNA fragments in the converging flow created by a sudden contraction was found to be sufficiently long to allow extension of the DNA molecules into a highly extended conformation and, hence, breakage to occur at midpoint. In most instances, 30 passages sufficed to obtain a narrow size distribution, with >90% of the fragments lying within a 2-fold size distribution. The shear rate required to achieve breakage was found to be inversely proportional to the 1.0 power of the molecular weight. Compared with a restriction digest, up to 40% of all fragments could be cloned directly, with only marginal improvements in cloning efficiency having been observed upon prior end repair with Klenow, T4 polymerase or T4 polynucleotide kinase. Sequencing revealed a fairly random distribution of the fragments.

Bacteriophage M13↗

PubMed1996

A pre-Columbian Y chromosome-specific transition and its implications for human evolutionary history.

A polymorphic C-->T transition located on the human Y chromosome was found by the systematic comparative sequencing of Y-specific sequence-tagged sites by denaturing high-performance liquid chromatography. The results of genotyping representative global indigenous populations indicate that the locus is polymorphic exclusively within the Western Hemisphere. The pre-Columbian T allele occurs at > 90% frequency within the native South and Central American populations examined, while its occurrence in North America is approximately 50%. Concomitant genotyping at the polymorphic tetranucleotide microsatellite DYS19 locus revealed that the C-->T mutation displayed significant linkage disequilibrium with the 186-bp allele. The data suggest a single origin of linguistically diverse native Americans with subsequent haplotype differentiation within radiating indigenous populations as well as post-Columbian European and African gene flow. The mutation may have originated either in North America at a very early time during the expansion or before it, in the ancestral population(s) from which all Americans may have originated. The analysis of linkage of the DYS199 and the DYS19 tetranucleotide loci suggests that the C-->T mutation may have occurred around 30,000 years ago. We estimate the nucleotide diversity over 4.2 kb of the nonrecombining portion of the Y chromosome to be 0.00014. compared to autosomes, the majority of variation is due to the smaller effective population size of the Y chromosome rather than selective sweeps. There begins to emerge a pattern of pronounced geographical localization of Y-specific nucleotide substitution polymorphisms.

Alleles↗

PubMed1996

Hepatitis C virus detection by single-round PCR specific for the terminal 3' noncoding region.

A single-round PCR method with primers specific for the 3' noncoding region (NCR) of hepatitis C virus (HCV) has been developed. Using a double RNAzol-B extraction, a high-temperature reverse-transcription step with SuperScript II reverse transcriptase, and a 40-cycle two-temperature PCR with a TaqStart antibody hot-start procedure, we were able to detect a 92-nucleotide fragment of the recently discovered 98-nucleotide highly conserved sequence at the 3' terminus of the HCV genome. Direct sequencing of the PCR products confirmed the specificity of the PCR and demonstrated conservation in this region. Only one nucleotide change in 14 specimens was found. End point dilution titration of sera with known viral RNA titers showed the sensitivity of the single-round 3' NCR PCR to be comparable to those of the established nested 5' NCR assays (fewer than 25 HCV genome equivalents). To evaluate specificity and sensitivity, a panel of 116 serum samples characterized by nested 5'-end PCR, genotyping, and quantitative assays was tested. A high degree of concordance (96%) between the 3' NCR and 5' NCR PCR results was found. The sequence conservation at the 3' end of the HCV genome among common genotypes and the savings in time, labor, and reagents from a single-round PCR make this assay a useful addition to the detection systems available to identify and monitor HCV infection.

DNA Primers↗

PubMed1996

Cloning, characterization, and steroid-dependent posttranscriptional processing of RUSH-1 alpha and beta, two uteroglobin promoter-binding proteins.

We previously used gel shift assays, Southwestern blots, and UV cross-linking to identify four proteins that bind to the 203-bp 5'-flanking region (-194/ +9) of the rabbit uteroglobin gene. Here we report cloning, by recognition site screening, the cDNAs for two of the uteroglobin promoter-binding proteins (95 kDa and 113 kDa). Their presumptive nucleotide-binding motifs share 61% identity with the SWI2/SNF2 helicase superfamily, and each protein has the novel C3HC4 (RING) zinc-finger signature near its C terminus. RUSH-1 alpha, the 113-kDa protein, is the rabbit homolog of human HIP116, a protein that binds to the human immunodeficiency virus-1 promoter. RUSH-1 beta is a 95-kDa truncated version of RUSH-1 alpha that results from alternative splicing of a 57-bp exon as confirmed by genomic cloning. Northern analysis showed mRNA expression (5.2 kb) was induced by progesterone +/- PRL and antagonized by estrogen. However, because the two proteins result from alternative splicing of a 57-bp exon, the small difference in their mRNA sizes could not be detected by Northern analysis. Therefore, competitive RT-PCR and HPLC were used to quantify differences in the ratios of their mRNAs. Progesterone +/- PRL treatment increased (P < 0.005) the ratio of message for RUSH-1 alpha compared with RUSH-1 beta. Western analysis showed the RUSH-1 alpha protein is increased in response to progesterone +/- PRL and decreased in response to estrogen. The antiserum used for immunoblotting specifically supershifts uteroglobin promoter-protein complexes in gel shift experiments. Because RUSH-1 alpha and beta messages were detected in lung, liver, and HRE-H9 cells, these proteins may regulate genes in numerous cell types.

Alternative Splicing↗

PubMed1996

Rapid quantification of gene expression by competitive RT-PCR and ion-pair reversed-phase HPLC.

Competitive reverse-transcription PCR (RT-PCR) techniques for quantification of gene expression employ titrations in which the products of multiple PCRs must be separated, analyzed and quantified to compute gene expression in a single sample. We have employed a novel, ion-pair reversed-phase HPLC (IP-RP-HPLC) system to analyze and quantify RT-PCRs performed with mutant RNA internal standards. PCR products could be separated and quantified in 6 minutes per reaction using the absorbance signal from an on-line UV detector. Crude PCR products can be analyzed without further processing and without the addition of radioactive or fluorescent markers to reactions. Analysis of titration regression and slope values approached mathematical ideals indicating that amplification of native and competitor RNA occurred with equal efficiency. Further, serial dilution of input RNA over three orders of magnitude did not affect the calculated level of gene expression or the slope of the titration. IP-RP-HPLC appears to offer important advantages to quantitative measurements of gene expression. These include rapid sample analysis and column re-equilibration, reduced sample handling and opportunity for introduction of quantification error, avoidance of fluorescent or radioactive tracers, high detector sensitivity and linearity and excellent quantitative reliability.

Angiotensinogen↗

PubMed1995

Surface-charge reversed capillary zone electrophoresis of inorganic and organic anions.

Anions commonly found in biological fluids such as chloride, citrate, phosphate, bicarbonate, lactate, aspartate and glutamate, were determined within 60 s by means of capillary zone electrophoresis and indirect UV detection using 10 mM of chromate, pH 8.0, as both carrier electrolyte and chromophore. The lower mass detection limit was 0.5 pmol. In order to avoid a gradual shift of the pH and, in consequence, drifting ionic mobilities due to the formation of hydroxyl ions at the cathode, 1 mM of 5,5-diethylbarbiturate was added to the background electrolyte. Moreover, fused silica capillaries were coated with a 0.0003% solution of hexadimethrine bromide for two minutes between runs to reverse electroendosmotic flow which otherwise would counteract the electrophoretic migration of the anions and, hence, increase time of analysis. It is concluded that charge-reversed capillary zone electrophoresis in concert with indirect UV detection is superior to ion chromatography and conductivity detection in the determination of inorganic and organic anions in biological fluids both with regard to selectivity, sensitivity and speed of analysis.

Anions↗

PubMed1995

Accurate and absolute quantitative measurement of gene expression by single-tube RT-PCR and HPLC.

We report a method that allows accurate, absolute quantification of gene expression in a single reverse transcriptase (RT)-PCR reaction. This method makes use of novel high-performance liquid chromatography (HPLC) technology to resolve and quantify the products of competitive, mutant RNA PCRs. The HPLC technique allows rapid, high resolution of reaction products. On-line UV detection eliminates the need for radiolabel or other tracers. The HPLC technique also demonstrates that these competition reactions readily generate heteroduplex products. The ability of HPLC to resolve and quantify heteroduplex products is fundamental to the accuracy of the technique. Accurate measurements of gene expression have been obtained over four orders of magnitude and experiments employing predetermined quantities of specific native RNA input have demonstrated the ability of the system to provide absolute estimates of gene expression. Large size differences between native and mutant RNA inputs affected reverse transcriptase (RT) efficiency, but not PCR amplification efficiency. However, the magnitude of the RT efficiency effect can be estimated, is reproducible, and can therefore be adjusted by a calculated correction factor. The RT efficiency difference can been eliminated by reduction in the magnitude of the sequence difference between native and mutant RNA so that no correction factor is required. The application of the technique to quantification of expression of the alpha 1 subunit of sodium, potassium-ATPase in microdissected nephron segments is demonstrated.

Animals↗

PubMed1994

High-resolution liquid chromatography of fluorescent dye-labeled nucleic acids.

Using 100 mM of triethylammonium acetate as ion-pairing reagent, phosphodiester oligonucleotides labeled fluorescently at their 5' terminus could be separated successfully on alkylated nonporous 2.3-microns poly(styrene-divinylbenzene) particles by means of high-resolution liquid chromatography. Applying excitation wavelengths of 490, 520, 550, and 575 nm, respectively, optimum sensitivity was achieved for the fluorophores 5-carboxyfluorescein, 2',7'-dimethoxy-4',5'-dichloro-6-carboxyfluorescein, N,N,N',N'-tetramethyl-6-carboxyrhodamine, and 6-carboxy-X-rhodamine (FAM, JOE, TAMRA, and ROX, respectively) at emission wavelengths of 520, 550, 580, and 605 nm, respectively. With calibration curves being linear over at least three orders of magnitude, the lower detection limits were 0.5, 2, 2, and 3 fmol, respectively. Depending on the type of fluorescent dye attached, retention times increased in the order JOE < FAM < TAMRA < ROX. Subsequently, fluorescent oligonucleotides were employed to prime polymerase chain reactions (PCR). Again the fluorophores were found to increase the retention times of double-stranded nucleic acids, but to a lesser degree than those of single-stranded oligonucleotides. Using a single FAM label attached to one of the two PCR primers, the sensitivity of fluorescence detection was found to be approximately 1 fmol or 30-70 times higher than that of uv absorbance detection depending on the length of the PCR product. Since the technique allows the separation of PCR products differing only 4 to 8 base pairs in length within a size range of 50 to 200 base pairs, it may be employed for the quantitative assessment of competitive PCR.

Base Sequence↗

PubMed1994

Capillary zone electrophoresis and micellar electrokinetic chromatography of 4-aminobenzonitrile carbohydrate derivatives.

Aldoses, ketoses and uronic acids were derivatized successfully within 15 min at a temperature of 90 degrees C by reductive amination with 4-aminobenzonitrile. Subsequently, the derivatives were separated as their borate complexes by capillary zone electrophoresis, using 175 mM borate buffer, pH 10.5, as carrier. The electrophoretic mobilities were determined by the complex stability, which was found to depend on the number of hydroxyl groups on any given carbohydrate derivative, the presence of substituents, and most strongly on the configuration of the vicinal hydroxyl groups at C-3 and C-4 in aldoses and uronic acids, and with regard to ketoses on those at C-4 and C-5. Time of analysis could be reduced considerably by the use of micellar electrokinetic chromatography, which separated 4-aminobenzonitrile sugar derivatives on the basis of their differential partitioning into an electroendosmotically driven aqueous phase and into sodium dodecyl sulfate micelles. Optimum resolution was achieved with a Tris-phosphate buffer, pH 7.5, containing 100 mM of sodium dodecyl sulfate. The method made it possible to resolve several carbohydrates which had not been resolved successfully by means of capillary zone electrophoresis, such as glucose and fructose. Moreover, separation selectivity could be adjusted by varying the capillary temperature. Finally, on-column UV monitoring at 285 nm allowed the detection of glucose with a lower mass detection limit of 1 fmol and a concentration sensitivity of 0.3 microM.

Borates↗

PubMed1994

Capillary electrophoresis of carbohydrates.

Capillary electrophoresis has emerged as a highly promising technique for the analysis of mono- and oligosaccharides. The approaches developed for overcoming the lack of chromophoric and fluorophoric functions in most carbohydrates involve the use of indirect photometric detection, amperometry, mass spectrometry, and precolumn derivatization with various tags. The merits and drawbacks of the derivatizing agents, including 2-aminopyridine, 4-amino-benzoic acid and its analogues, which for the first time permitted the reproducible determination of aldoses, uronic acids and even ketoses in the low femtomole range by means of readily available UV detection, and other agents such as 8-aminonaphthalene-1,3,6-trisulphonic acid, 1-phenyl-3-methyl-5-pyrazolone and 3-(4-carboxybenzoyl)-2-quinoline-carboxaldehyde, are discussed in detail. Means to secure electromigration of the usually neutral carbohydrates are: (i) ionization of hydroxyl groups at high pH; (ii) complexation of vicinal or alternate hydroxyl groups with borate or other charged compounds such as alkaline earth metal ions; (iii) derivatization with a reagent possessing ionizable functions; and (iv) partitioning into a pseudostationary phase such as sodium dodecyl sulphate micelles. Each alternative has its own analytical rewards, and combinations of the above mechanisms allow the two-dimensional and perhaps even three-dimensional mapping of oligosaccharides. Pyridylaminated oligosaccharides, for instance, have been separated both according to size by exploiting differences in the charge-to-mass ratio, with the charge being identical for each oligomer under acidic conditions due to protonation of the imino group incorporated by precolumn derivatization, as well as on the basis of structural differences, as a consequence of differences in the ease of borate complexation of the peripheral monosaccharide residues. It is also shown that the 4-aminobenzonitrile derivatives of mono- and disaccharides can be separated by micellar electrokinetic chromatography with a resolving power superior to that achieved by capillary zone electrophoresis of sugar-borate complexes. Based on the progress made, it can be concluded that capillary electrophoresis represents a powerful alternative and complement to existing methodology in the area of carbohydrate analysis.

Animals↗

PubMed1994

High-performance liquid chromatography for routine analysis of hepatitis C virus cDNA/PCR products.

Ion-pair reversed-phase high-performance liquid chromatography on alkylated nonporous polystyrene-divinylbenzene particles with a mean diameter of 2.1 microns was used to analyze PCR products according to their chain length within a few minutes. The simple and reliable procedure allows the simultaneous separation and isolation of DNA fragments differing in chain length by 1%-5% up to a size of 500 base pairs with recovery rates exceeding 97%. A greater than 70-fold increase in sensitivity could be achieved through the use of a fluorescein-labeled primer, which allowed the determination of a 127-bp hepatitis C virus cDNA/PCR product with a lower mass detection limit of 2 fmol. Calibration curves showed excellent linearity over a range of at least 4 magnitudes. Finally, the stationary phase allowed the routine analysis of hundreds of PCR products with high reproducibility of both retention times and peak areas.

Base Sequence↗

PubMed1993

High-resolution liquid chromatography of oligonucleotides on nonporous alkylated styrene-divinylbenzene copolymers.

Phosphodiester-oligonucleotides were separated by ion-pair reversed-phase HPLC on nonporous polystyrene-based particles having a diameter of 2.1 +/- 0.12 microns. With unmodified poly(styrene-divinylbenzene) beads it was not possible to resolve oligonucleotides efficiently. However, upon addition of polyvinyl alcohol during polymerization baseline resolution of phosphorylated oligodeoxyadenylic acids with a chain length of up to 30 bases was obtained, with triethylammonium acetate serving as ion-pairing reagent. An even higher separation efficiency was achieved by Friedel-Crafts alkylation of poly(styrene-divinylbenzene) particles. At a column temperature of 50 degrees C, the number of theoretical plates exceeded 6 x 10(5) per meter. The maximum loading capacity for a 50 x 4.6-mm i.d. column still resulting in the highest resolution was 0.1 microgram. Calibration curves showed excellent linearity over a range of at least 4 magnitudes, with a lower mass detection limit of 0.16 ng. Recoveries ranged from 96.7 to 100.8%. The same stationary phase also allowed the separation of phosphorylated from dephosphorylated oligonucleotides, the former ones being eluted earlier.

Alkylation↗

PubMed1993

High-resolution liquid chromatography of DNA fragments on non-porous poly(styrene-divinylbenzene) particles.

DNA restriction fragments and PCR products were separated by means of ion-pair reversed-phase high-performance liquid chromatography on alkylated non-porous poly(styrene-divinylbenzene) particles with a mean diameter of 2.1 microns. Optimum resolution was obtained by using an acetonitrile gradient in 100 mM of triethylammonium acetate and a column temperature of 50 degrees C. This allowed the separation of DNA fragments differing in chain length by 1-5% up to a size of 500 base pairs. PCR products could be analyzed directly in less than two minutes with a concentration sensitivity of at least 300 ng/ml. Compared with anion-exchange chromatography or gel electrophoresis no desaltation of the purified DNA molecules is required because the volatile buffer system can be readily evaporated. Subsequently, the method was used for the semiquantitative evaluation of the expression of multidrug resistance genes in mononuclear white blood cells.

Base Sequence↗

PubMed1992

Comparative study of capillary zone electrophoresis and high-performance liquid chromatography in the analysis of oligonucleotides and DNA.

Capillary zone electrophoresis and high-performance liquid chromatography were compared with regard to the separation of oligonucleotides and double-stranded DNA. Both anion-exchange and reversed-phase high-performance liquid chromatography on non-porous particles are considered to be superior to capillary electrophoresis in terms of speed and selectivity in the analysis of oligonucleotides up to 30 bases in length. Moreover, reversed-phase chromatography allows the simultaneous purification of detritylated oligonucleotides with recoveries > 90%. Compared with anion-exchange chromatography, there is no need for a subsequent desaltation step because the volatile buffer system can be readily evaporated. With regard to dsDNA, however, the resolving power of capillary electrophoresis cannot be matched by anion-exchange chromatography at present. Moreover, the combined use of hydroxyethylcellulose and ethidium bromide not only yielded a separation efficiency equal to that achieved by means of gel-filled capillaries but also avoids some of their limitations such as the destruction of the gel matrix at high current densities and the bias involved in electrokinetic injection.

Base Sequence↗

PubMed1992

High-performance liquid chromatographic separation of detritylated oligonucleotides on highly cross-linked poly-(styrene-divinylbenzene) particles.

Detritylated oligonucleotides were separated by reversed-phase high-performance liquid chromatography on highly cross-linked polystyrene-based particles having a mean particle diameter of 2.3 microns. The addition of poly(vinyl alcohol) during polymerization, which resulted in the presence of hydroxyl groups on the surface of the poly(styrene-divinylbenzene) beads, was necessary to obtain baseline resolution of phosphorylated oligodeoxyadenylic acids with a chain length of up to 30 bases. The impact of temperature was investigated and optimum resolution was achieved at 40 degrees C. At pH 7.0, the retention times of oligonucleotides were found to depend on the ratio of bases and to increase in the order of C less than G less than A less than T. Under the same conditions, it was possible to separate phosphorylated from dephosphorylated oligonucleotides, the former being eluted earlier. Recoveries ranged from 92 to 100%.

Base Sequence↗

PubMed1992

High-performance liquid chromatographic determination of free polyamines in human seminal plasma.

Putrescine, spermidine and spermine were determined quantitatively in 192 ultrafiltered samples of human seminal plasma with a precision of +/- 1.9% by pre-column derivatization with benzoyl chloride and reversed-phase chromatography. The concentrations of free putrescine, spermidine and spermine ranged from 0 to 1.96 mmol/l (mean = 0.10), 0.017-0.96 mmol/l (mean = 0.16) and 0.13 to 20.80 mmol/l (mean = 2.43), respectively. A significant correlation between the amounts of spermidine and spermine was found (r2 = 0.806, P less than 0.0005). Otherwise, no significant correlations between free-polyamine levels and routine spermiogram parameters such as volume, sperm count, motility, vitality and morphology were obtained.

Adolescent↗

PubMed1992

Impact of separation capacity on the isotachophoretic analysis of organic acids in human seminal plasma and prostatic fluid.

In the present study, the impact of pH of both the leading electrolyte and the sample on the separation capacity of an isotachophoretic system, used for the analysis of organic acids in human seminal plasma and prostatic fluid, was assessed. Generally, maximum separable amounts of eleven equimolar pairs of anions decreased within a pH range of 2.90-4.25 of the leading electrolyte due to increasing net mobilities of the analytes with rising pH. The values obtained under equimolar conditions were also valid for nonequimolar mixtures as long as the buffer capacity of the leading electrolyte was sufficient to maintain constant mobilities of the ionic species to be separated throughout the capillary. However, when the sample injected has a buffer capacity of its own and a pH higher than that of the leading electrolyte, separation capacity will vary. For that reason, the maximum separable amount of an aqueous solution of phosphate and citrate was higher than that obtained for seminal plasma. Hence, the injection volume had to be reduced in order to ensure complete resolution. Besides phosphate and citrate, which was the prime component of prostatic fluid, lactate, aspartate and glutamate could be quantitatively determined in seminal plasma with a precision of +/- 1.5%.

Acids↗