Biomedical subjects

R P Oda

Publications and source records attributed to R P Oda.

32 records · Page 2Linked to original sources

PubMed1995

Capillary electrophoresis of DNA potential utility for clinical diagnoses.

The last few years have witnessed a tremendous shift in the use of capillary electrophoresis for clinical applications, particularly with DNA analysis. As a result of the large number of DNA-based clinical assays, there is an intense interest in making DNA analysis faster, less expensive and more automated. We describe the evaluation of CE-based single-strand conformation polymorphism (SSCP) and dideoxy fingerprinting (ddF) analysis for the detection of single-point mutations within a Mycobacterium tuberculosis-specific amplified DNA fragment. Both were found to be capable of detecting the mutation in the resistant isolate but ddF showed the most promise with respect to specificity and ease of implementation. In addition, initial results with a CE-based sizing method is shown to be competitive and, perhaps, superior to a Southern blot analysis for the detection of hepatitis C viral (HCV) infection.

DNA Fingerprinting↗

PubMed1995

Detection of hypoglycemic drugs in human urine using micellar electrokinetic chromatography.

Micellar electrokinetic chromatography (MEKC) is evaluated as a potential analytical method for the separation and detection of a series of sulfonylurea drugs used in the treatment of hyperglycemia. These drugs are often surreptitiously abused, producing extremely low blood glucose levels and symptoms indistinguishable from those associated with an insulin-secreting tumor. Separation buffer containing 50 mM sodium dodecyl sulfate (SDS) was found to be adequate for the MEKC separation of the third generation drugs (glipizide and glyburide) but not the second generation drugs (acetohexamide chlorpropamide, tolazamide, and tolbutamide). At a pH of 8.5 in the presence of 20 mM borate/20 mM phosphate and 150 mM SDS, all seven components were adequately resolved with an analysis time of 17 min. Altering the concentration of the buffering components to either 5 mM borate/5 mM phosphate or 40 mM borate alone reduced the analysis time to less than 10 min with no observable loss in resolution. A series of other micelle-forming surfactants were evaluated, and only sodium cholate provided an improvement over the SDS-based system. Optimal separation was obtained with 75 mM sodium cholate and led to complete analysis with baseline resolution of all seven components in less than 8 min. These conditions were shown to be adequate for the detection of the hypoglycemic drugs spiked into normal urine and in patients taking these drugs. The precision associated with nine consecutive injections of six samples (n = 54) was found to be acceptable with percent coefficient of variance for absolute migration times (MTabs) for all peaks averaging 0.89 with peak area and peak height being 8.49 and 8.26, respectively. The between-sample precision was found to average 0.92% for MTabs and 8.56% and 8.45%, respectively, for the relative peak area and peak height. With a detection limit for the drugs in urine (following extraction) in the 50 ng/mL range, the potential exists for an MEKC-based assay for the detection of sulfonylurea drugs in urine.

Chromatography, High Pressure Liquid↗

PubMed1995

Preliminary investigations of preconcentration-capillary electrophoresis-mass spectrometry.

Analyte preconcentration on-line with capillary electrophoresis-mass spectrometry (PC-CE-MS) is described. Preconcentration cartridges were fabricated from PTFE tubing filled with ca. 1-2 mm bed of reversed-phase C18 HPLC packing or polymeric reversed-phase beads. The particle size of the stationary phase was of larger dimension than the internal diameter of the CE capillary. Therefore, PC-CE capillaries were assembled without frit material and held together by friction. The wide applicability of on-line PC-CE-MS is demonstrated by the analysis of solutions containing peptides, proteins, and synthetic analogues of putative metabolites of the neuroleptic agent haloperidol.

Amino Acid Sequence↗

PubMed1994

Multiple-buffer-additive strategies for enhanced capillary electrophoretic separation of peptides.

A dodeca-peptide from the beta-subunit of thyroid stimulating hormone (TSH) was used as a model system for developing "multiple-buffer-additive" strategies to effect the separation of structurally-similar peptides. A series of synthetic peptides included six peptides with identical amino acid composition and two with multiple alanine substitutions at selected positions. Those with identical amino acid composition included the native and reverse sequences of residues 101-112 of the beta-TSH and four "computer-shuffled" amino acid sequences. Buffer additives such as acetonitrile (ACN), hexane sulfonic acid (HSA), and hexamethonium bromide (HxMBr), were shown to alter selectivity dramatically. HSA, an ion-pairing agent, and ACN, known to alter the hydrophobic environment of the solute, and HxMBr, which is thought to negate solute-wall interactions, are shown to independently effect only partial resolution of the mixture. It is shown that only with the proper combination of HSA and ACN are all mixture components resolved. These results re-affirm that CE selectivity may be altered by changes in buffer ionic strength or with the addition of HSA, but also show that further changes in selectivity can be achieved through alteration of buffer hydrophobicity. The observed changes in selectivity accompanying the addition of HSA and ACN may be due to differing electrophoretic mobilities resulting from nearest-neighbor effects or subtle differences in peptide secondary structure or solvation. This emphasizes the importance of employing multiple-buffer-additive strategies for effecting the resolution of peptide mixtures that are difficult to separate.

Amino Acid Sequence↗

PubMed1993

Utility of high resolution capillary electrophoresis for monitoring peptide homo- and hetero-dimer formation.

The monomer and disulfide-linked homo-dimer of two different peptides, one with an amino-terminal cysteine, the other with a cysteine at the carboxy-terminal, are shown to be baseline resolved by capillary electrophoresis in less than 15 min. Time-course for homo-dimer formation with both peptides, either under mild (air) or stronger (hydrogen peroxide oxidizing conditions, was easily monitored. Confirmation that the second peak appearing under oxidizing conditions was indeed the homo-dimer was obtained with mass spectrometry. The possibility that stronger oxidizing conditions led to the production of the sulfonic acid derivative of the monomeric peptide, was ruled out through generation of the derivative by performic acid oxidation. As expected, the negative charge of the sulfonic acid moiety gives the peptide a slower electrophoretic mobility than both the monomer and the dimer. Moreover, as would be expected with a sulfonic acid derivative, oxidation to the dimeric form was not possible. This was consistent with the observation that the homo-dimer peak could be reduced to monomeric form in the presence of dithiothreitol. Co-oxidization of the amino- and carboxy-terminal peptides led to the expected production of both homo-dimers and the hetero-dimer, all of which were resolved.

Amino Acid Sequence↗

PubMed1993

Capillary electrophoresis: a powerful microanalytical technique for biologically active molecules.

Capillary electrophoresis (CE) is a versatile microanalytical technique that has gained much attention, particularly from those working with biologically active molecules. Its appealing characteristics include unprecedented sensitivity and the ability for automating the rapid electrophoretic separation of a number of low-volume samples in a reproducible manner, with relatively short analysis times. The picomole-femtomole (10(-12)-10(-15) mol) sensitivity of UV-CE has been enhanced tremendously by the interfacing of detection systems such as laser-induced fluorescence, which has extended the sensitivity into the attomole-zeptomole (10(-18)-10(-21) mol) range. Fluorescence detection has shown great potential for the CE analysis of a wide range of biomolecules including peptides, proteins and DNA. CE research and development has taken on directions focused primarily on improving detection, understanding and exploiting the basic chemistry of CE and devising new applications.

Biotechnology↗

PubMed1992

High-performance capillary electrophoresis of glycoproteins: the use of modifiers of electroosmotic flow for analysis of microheterogeneity.

High-performance capillary electrophoresis (HPCE) is rapidly gaining acceptance as an analytical tool for the study of biological macromolecules. In the present study, the utility of HPCE for separation of glycoproteins is highlighted using a pure ovalbumin preparation. Ovalbumin, the 43-kDa glycoprotein of avian egg white, is known to be heterogeneous in nature with at least nine different carbohydrate structures having been identified on the single Asn residue. HPCE separation in an 87-cm capillary containing borate buffer and 1 mM putrescine resolves five major protein peaks in less than 30 min under nondenaturing conditions. This effect appears to be specific to glycoproteins since analysis of the nonglycosylated protein carbonic anhydrase under the same conditions showed no enhanced separation. The sodium borate buffer is proposed to play a key role in the separation by preferentially complexing with the diols of specific carbohydrate moieties on ovalbumin. Addition of putrescine enhances resolution by slowing bulk flow through the capillary and allowing electrophoretic separation of what is deduced to be closely related glycoforms of ovalbumin. Dephosphorylation of the ovalbumin with either calf intestinal alkaline phosphatase or potato acid phosphatase results in a shift of all peaks to a more rapid migration time and is consistent with a loss of negative charge. This suggests that all major ovalbumin isoforms are phosphorylated to the same degree and that heterology among ovalbumin isoforms resides solely in the carbohydrate structure. The enhanced resolution obtained with the employment of longer capillaries and modifiers of endo-osmotic flow was not restricted to ovalbumin since partial resolution of pepsin isoforms was observed under the same conditions.

Borates↗

PubMed1987

Choline and acetylcholine concentration in transplanted rat heart.

A liquid chromatographic assay was used to determine the choline and acetylcholine concentrations in the four chambers of rat hearts 2, 4, and 8 days after transplantation into an abdominal site. Corresponding measurements were made in the hearts of host rats. We found regional cardiac acetylcholine concentrations in controls follow the nonuniform pattern seen with choline acetyltransferase (CAT) activity, being highest in the atria (8-12 nmol/g) and lower in the ventricles (0.7-1.6 nmol/g). Following transplantation, acetylcholine levels decreased significantly only in the right ventricle after 8 days. Following a unilateral vagotomy (random, left or right), acetylcholine concentrations in the distal portion of the severed nerve decreased to half the value of the intact contralateral side by 4 days. The continued presence of acetylcholine, despite significantly reduced CAT activity in the severed nerve and transplanted heart, suggests that acetylcholine is preserved, perhaps by vesiculation in nonstimulated postganglionic terminals. The localized decrease in acetylcholine in the right ventricle after 8 days suggests that transplantation may interrupt the postganglionic fibers to this area.

Acetylcholine↗

PubMed1987

Select cardiovascular and metabolic responses of diabetic rats to moderate exercise training.

The combined influence of diabetes and moderate treadmill exercise training on select metabolic and cardiovascular parameters was investigated with mature male Sprague-Dawley rats assigned to either control diabetic or diabetic groups receiving exogenous insulin. Experimental diabetes was induced with streptozotocin (80 mg.kg-1, i.v.) and verified by blood glucose concentrations greater than 16 mmol. The animals were designated as control, insulin-injected (5 U.kg-1, twice daily), or saline-injected (twice daily), and assigned to either non-trained or trained sub-groups. Insulin treatment partially restored the measured physiological functions to within normal limits. All animals were trained at 60 to 70% maximal oxygen consumption for 9 wk and exhibited higher maximal oxygen consumption values and cytochrome oxidase activity of the soleus muscles. Diabetes caused lower (P less than 0.05) reductions in resting heart rate but training-induced bradycardia did not occur in any group. Heart rate response to atropine sulfate (1 mg.kg-1, atrial choline acetyltransferase activity, atrial acetylcholine concentration, and quinuclidinyl benzilate binding was measured to evaluate changes in the parasympathetic nervous system. Atropine-induced cardiac acceleration was most pronounced in control and least effective in diabetic animals. Endurance training had no meaningful influence on this response to cholinergic inhibition. Quinuclidinyl benzilate binding for the diabetic and the diabetic groups receiving insulin revealed no change in receptor number, receptor affinity, or training effects. These findings indicated that 9 wk of exercise training improves the aerobic capability of insulin-deficient rats without changing cardiovascular characteristics associated with the parasympathetic nervous system.

Acetylcholine↗

PubMed1986

A microcomputer program for determining turnover rates before and after intervention in a single animal.

A set of three programs to calculate the turnover of biomolecules whose metabolism follows a steady-state precursor-product relationship and follows the open, single-compartment kinetics model has been written in MS-BASIC. The programs comprise a system for determining two values of turnover, before and after an intervention which may alter the turnover rate, in a single animal. The programs have been extensively tested in our laboratory for the determination of norepinephrine turnover under differing physiological and pharmacological conditions. The utility of the programs lies in their ability to readily adapt to turnover determinations for any substance whose metabolic pathway conforms to the model constraints. This includes the biogenic amine neurotransmitters, peptides and proteins, and many small biological molecules or pharmacological agents.

Animals↗

PubMed1986

Vagus nerve stimulation alters regional acetylcholine turnover in rat heart.

The turnover of neurotransmitter is a direct measure of neuronal function, varying with the impulse activity of the nerve. It is not known if vagal stimulation increases acetylcholine release uniformly throughout the heart, or if modification of neural signals occurs between the vagal nerve trunks and postganglionic synaptic terminals. The rate constant of acetylcholine turnover was measured in conduction and contractile regions of heart by quantifying the incorporation of [3H]choline into acetylcholine after labeling of the blood choline pool in urethane-anesthetized rats during two levels of vagal activity. Choline and acetylcholine were assayed by high pressure liquid chromatography with electrochemical detection of post-column enzymic reaction product, peroxide. The specific activities of choline and acetylcholine in the tissues at sacrifice were used to calculate the fractional turnover rates in cardiac regions. Supramaximal bilateral vagal stimulation for 20 minutes decreased heart rate (P less than 0.05), while mean arterial blood pressure remained constant. The rate constants for acetylcholine turnover in right atrial regions containing the sinoatrial node, left atrial tissues, and interatrial septum doubled from control values during vagal stimulation. In contrast, the fractional rate constants of acetylcholine turnover did not change in the right and left ventricles during vagal stimulation. We interpret these results to indicate general activation of postganglionic parasympathetic fibers to the atria and selective modulation of postganglionic parasympathetic neural function to the ventricles.

Acetylcholine↗

PubMed1986

Presynaptic regulation of cardiac sympathetic function in guinea pigs.

Cold stress (4 degrees C) induces a pressor response and variable increases in an index of sympathetic neural function, the rate constant of norepinephrine turnover, kNE. In heart, presynaptic cholinergic muscarinic and alpha-2 adrenergic influences may contribute to regional variation in responses of kNE to cold stress. Animals were pretreated with vehicle, a muscarinic cholinergic antagonist, quinuclidinyl benzilate (QNB), an alpha-2 adrenergic antagonist, yohimbine (YOH) or combined QNB + YOH. An increase in kNE was determined from incorporation of radiolabeled tyrosine into norepinephrine in a control period at 24 degrees C and again at 4 degrees C. The increment in kNE factored by the increment in blood pressure indicated the extent of increased sympathetic function in each cardiac region. In sino-atrial node, sympathetic function was increased significantly (P less than .05) by QNB + YOH compared to other treatments, suggesting that both cholinergic and alpha-2 adrenergic presynaptic influences were important. In contrast, in right and left ventricles, YOH or QNB + YOH, but not QNB alone, increased sympathetic function significantly, suggesting that only alpha-2 adrenergic influences were important. These data support the concept that presynaptic regulation of cardiac sympathetic function differs in sino-atrial node and ventricles of guinea pigs during activation with cold stress.

Animals↗

PubMed1979

Evidence of nonuniform sympathetic neural activity to heart regions in guinea pigs.

Central neutral activity may selectively influence cardiac regions. As an index of this, rate constants of norepinephrine turnover, KNE, in regions of guinea pig heart were determined by 1) disappearance of [3H]NE from tissues, and 2) conversion of [3H]tyrosine to [3H]NE. In sinoatrial (SA) node and right atrial appendage, KNE averaged 0.084 +/- 0.014 and 0.066 +/- 0.004 (SE) h-1, respectively (P greater than 0.05). In other specialized regions, KNE was lower than in SA node (P less than 0.05). In other contractile regions, KNE was lower than in right atrial appendage (P less than 0.05). Ganglionic blockade reduced KNE to uniform values in all heart regions. Cold stress increased KNE markedly (P less than 0.05) throughout the heart, but selectively more in SA node, AV node, proximal conduction bundles, and right atrial appendage (P less than 0.05). At room temperature, neural activity is greater to the right atrium including SA node than to other cardiac regions. At 4 degrees C, neural activity increases selectively in the right atrium and the conduction system. This suggests that central neural mechanisms contribute significantly to nonuniform cardiac regulation under conditions of progressive sympathetic activation.

Animals↗

PubMedDate not supplied

Capillary electrophoresis in biotechnology.

Capillary electrophoresis (CE) is a versatile micro/macroanalytical technique gaining widespread usage for the separation and analysis of ionic substances. It has captured the attention of those working in a variety of arenas focused on biologically-active molecules. Its appealing characteristics include unprecedented mass sensitivity and the ability for precise, automated electrophoretic separation of microvolume samples with relatively short analysis times. Such versatility in bioanalysis makes it an inviting replacement for some of the labor-intensive, time-consuming methodologies performed via electrophoretic gels. Moreover, CE compliments the ease and speed of HPLC while eliminating the problem of excessive solvent volume usage and hazardous waste disposal. Further attractive characteristics of this technology include the analyses of a diverse spectrum of analytes, ranging from small organic ions to macromolecular protein complexes and DNA. While combining some of the most robust aspects of traditional electrophoresis, chromatography, and capillary technology, recent CE research and development has focused on avenues leading to improving detection and understanding and employing the basic chemistry of CE vis-a-vis new applications.

Animals↗