Biomedical subjects

M Novotny

Publications and source records attributed to M Novotny.

At least 19 recordsLinked to original sources

PubMed1992

Experimental evaluation of the separation efficiency in capillary electrophoresis using open tubular and gel-filled columns.

The band dispersion phenomena in capillary zone electrophoresis (CZE) using untreated and surface-treated open tubular and gel-filled capillaries were experimentally evaluated, with emphasis on small capillary diameters (10-100 microns). Laser-induced fluorescence detection was used for high-sensitivity detection of the isoindoles originated from model amino acids. The plots of plate height vs electric field strength were generated for different column radii and compared with a theoretical model for CZE. In addition to the diffusion-controlled band dispersion in the relatively low electric field range, adsorptive interactions between a solute and the capillary wall may play a certain role in band-broadening. The sorption-desorption kinetics become important with increasing electric field strength. Thermal effects appear to contribute little to band-broadening in relatively small capillaries (less than 50-microns i.d.) within normal operating voltages (less than 30 kV), but could become significant in capillaries with larger bores (greater than 75-microns i.d.). With gel-filled capillaries of small diameters (less than 50-microns i.d.), diffusion processes can be minimized. In addition, thermal effects do not appear critical in such columns at reasonable voltages.

Animals↗

PubMed1992

High-sensitivity analysis of sialyl-oligosaccharide glycosylation sites in glycoproteins by miniaturized tryptic digestion and microcolumn liquid chromatography.

A highly sensitive analytical procedure was developed to assess the site of glycosylation in a model glycoprotein, bovine fetuin. First, sample cleavage with immobilized trypsin and the peptide map development are accomplished by microcolumn liquid chromatography. Second, the sialic acids content is measured fluorometrically using their precolumn conversion to quinoxaline derivatives. A unique preconcentration system was developed to secure sensitivity of the second measurement.

Amino Acid Sequence↗

PubMed1992

Urine marking in male mice: responses to natural and synthetic chemosignals.

The effects of natural chemosignals and their synthetic terpenic analogs on urinary marking in mice were investigated. The number and size of marking spots, and percentage of response and avoidance/counter-marking behavior, were measured for dominant and subordinate males. The males' marking behavior was found to differ significantly based on the type of stimulus sample used. Marking behavior in response to female urine was significantly altered by addition of synthetic analogs (farnesene isomers) to it. Both dominant and subordinate males avoided marking the section of the test area soiled with stimulus samples containing either natural or synthetic farnesenes. Differential responses of the dominant and subordinate male mice were observed.

Animal Communication↗

PubMed1991

Selected applications of cyclodextrin selectors in capillary electrophoresis.

Through the use of alpha-, beta-, gamma- and heptakis(2,6-di-O-methyl)-beta-cyclodextrin as stereospecific selectors or electrolyte modifiers, both in capillary zone electrophoresis and isotachophoresis, selected model isomeric compounds (including optical isomers) were resolved. Soluble alkylhydroxyalkylcellulose derivatives were further added to the cyclodextrin-modified background electrolytes under study. Their presence was found to be essential, as demonstrated by improvements in both enantioselectivity and separation efficiency. The results obtained in both electrophoretic modes, under optimized conditions, are compared and discussed.

Cyclodextrins↗

PubMed1991

Separation of fluorescent oligosaccharide derivatives by microcolumn techniques based on electrophoresis and liquid chromatography.

Various aldose oligosaccharides can be quantitatively derivatized into primary amines for subsequent reaction with fluorogenic reagents, such as 3-(4-carboxybenzoyl)-2-quinolinecarboxaldehyde or 3-benzoyl-2-naphthaldehyde. Capillary electrophoresis (CE) and microcolumn liquid chromatography (LC), coupled with laser-induced fluorescence detection, were evaluated as a means of separating complex oligosaccharide mixtures. Whereas microcolumn LC and open-tubular CE appear confined in their utility to relatively small oligosaccharides, unprecedented results were obtained with polyacrylamide gel-filled capillaries on hydrolyzed malto-oligosaccharides and enzymatically degraded samples of chondroitin sulfate and hyaluronic acid.

Chromatography, Liquid↗

PubMed1991

Ultrasensitive fluorometric detection of carbohydrates as derivatives in mixtures separated by capillary electrophoresis.

Reducing monosaccharides and oligosaccharides, after reductive amination, were separated and detected as their 3-(4-carboxybenzoyl)-2-quinolinecarboxaldehyde (CBQCA) derivatives by capillary electrophoresis/laser-induced fluorescence. Under optimized conditions, the minimum detectable quantities for monosaccharide solutes were assessed at low attomole levels (0.5 amol for the CBQCA derivative of galactose). The system has shown considerable promise for high-sensitivity analysis of both neutral and amino sugars in glycoproteins. Complex oligosaccharides, isolated from bovine fetuin by hydrazinolysis, were also successfully "mapped."

Animals↗

PubMed1991

Design of 3-(4-carboxybenzoyl)-2-quinolinecarboxaldehyde as a reagent for ultrasensitive determination of primary amines by capillary electrophoresis using laser fluorescence detection.

Amino acids and peptides, from both standard solutions and biological samples, were successfully reacted with 3-(4-carboxybenzoyl)-2-quinolinecarboxaldehyde, at low concentration, to form highly fluorescent isoindole derivatives. The formed mixtures are effectively separated by high-performance capillary electrophoresis and their constituents detected by their laser-induced fluorescence signals. The minimum detectable quantities in the low attomole (10(-18) mol) range are encountered.

Amines↗

PubMed1991

Capillary electrophoresis of amino sugars with laser-induced fluorescence detection.

3-(4-Carboxybenzoyl)-2-quinolinecarboxaldehyde has been utilized as a precolumn derivatization agent for various amino sugars. Constituents of various biological mixtures can be converted to highly fluorescent isoindole derivatives, separated by high-performance capillary electrophoresis and determined at attomole (10(-18) mol) levels by a laser-induced fluorescence detector. This method has been applied to the analysis of monosaccharides and acid-hydrolyzed polysaccharides. Carbohydrate moieties derived from a glycoprotein were also tagged and determined.

Amino Sugars↗

PubMed1991

Socio-sexual olfactory preference in female mice: attractiveness of synthetic chemosignals.

Two sesquiterpenic compounds, E,E,-alpha-farnesene and E-beta-farnesene, which were previously found as major constituents of the male mouse preputial glands, were tested for their attractiveness to female mice. Sexually naive and sexually experienced females were given the opportunity to choose between natural stimuli and synthetic analogs of preputial chemosignals. Naive females preferred investigating the odors of intact males' urine and synthetic farnesenes when spiked in high concentration in bladder urine or water over control stimulus (water or bladder urine alone). Investigatory preference was not observed when synthetic farnesenes were presented to naive females in low concentration, i.e., only twice the natural content in the dominant male urine. However, sexually experienced females were clearly able to recognize and prefer samples with synthetic farnesenes, even in low concentration. We suggest that those sesquiterpenic compounds may play a wide-ranging role in the female recognition of sexually mature and socially dominant males.

Aggression↗

PubMed1990

Electrophoretic separations of proteins in capillaries with hydrolytically stable surface structures.

A procedure for obtaining highly stable coated capillaries for use in capillary electrophoresis (CE) is described. Reaction of surface-chlorinated fused silica capillaries with the Grignard reagent, vinyl magnesium bromide, followed by reaction of the vinyl group with acrylamide, results in an immobilized layer of polyacrylamide attached through hydrolytically stable Si-C bonds. This method is an extension of the capillary coating procedure described previously by Hjerten, differing in the means by which the polyacrylamide layer is bonded to the capillary walls. Capillaries treated in the manner described here can be used over a pH range of 2-10.5, without noticeable decomposition of the coating. In comparison to uncoated capillaries, separations of proteins using such coated capillaries are improved due to a reduction in protein adsorption to the capillary walls, although interaction is still present to some degree as evidenced by an inability to obtain plate counts as high as those predicted by theory. Electroosmotic flow is virtually eliminated in the coated capillaries, resulting in improved reproducibilities of protein migration times in comparison to uncoated capillaries. Additionally, peak skew is evaluated for model proteins and improvements are noted for the coated capillaries. Results are presented for separations of model protein mixtures, comparing the performance of the vinyl-bound polyacrylamide coated capillaries and uncoated capillaries at both high and low pH extremes.

Chemical Phenomena↗

PubMed1990

Capillary electrophoretic separations of peptides using micelle-forming compounds and cyclodextrins as additives.

The value of electrokinetic capillary chromatography for separating structurally similar model peptides and tryptic digests is demonstrated. The behavior of model peptides in buffer systems containing dodecyltrimethylammonium bromide, hexadecyltrimethylammonium bromide, sodium dodecyl sulfate and two cyclodextrins as additives is described. These additives, under different analytical circumstances, exhibit certain beneficial effects for peptides with similar net charges but different hydrophobicities. Separations of underivatized peptides, utilizing UV detection, are presented. In addition, separation of fluorescent products of peptides derivatized with o-phthalaldehyde, fluorescamine, and a new reagent, 3-(4-carboxybenzoyl)-2-quinolinecarboxaldehyde, are demonstrated and discussed. Beneficial spectroscopic detection effects with cyclodextrin are also noted.

Chromatography↗

PubMed1990

Application of 3-(2-furoyl)quinoline-2-carbaldehyde as a fluorogenic reagent for the analysis of primary amines by liquid chromatography with laser-induced fluorescence detection.

3-(2-Furoyl)quinoline-2-carbaldehyde (FQCA) has been synthesized and characterized as a fluorogenic derivatizing reagent for liquid chromatography. The reagent forms highly fluorescent isoindoles upon reaction with primary amines. The spectral properties and optimum reaction conditions for the formation of isoindoles have been investigated. The utility of FQCA for the high-sensitivity analysis of amino acids by laser-induced fluorescence with an argon-ion laser is demonstrated.

Amines↗

PubMed1990

Chemistry of male dominance in the house mouse, Mus domesticus.

Two terpenic constituents, E,E,-alpha-farnesene and E-beta-farnesene, were found to be elevated in dominant male urine when compared to subordinate or control males. These two urinary compounds were absent in the bladder urine of males; however, they were the most prominent constituents of the perputial gland's aliquots. The results of a two-choice preference test, conducted on ICR/Alb subordinate males, gave a strong indication that these two terpenic constituents introduced into the previously attractive stimulus significantly discouraged prolonged investigations by male mice. The compounds, whether present in the urine matrix or water, rendered the stimulus with a quality behaviorally similar to the urine of dominant males. It appears that they may be synonymous with the previously described aversion signal produced by dominant males. We suggest that these compounds may play a wide-ranging role in the territorial marking behavior of male mice.

Animals↗

PubMed1989

High-sensitivity peptide mapping by capillary zone electrophoresis and microcolumn liquid chromatography, using immobilized trypsin for protein digestion.

Procedures for the reduced-scale analysis of proteins by peptide mapping have been developed, allowing peptide maps to be obtained from picomole to femtomole quantities of protein. The use of trypsin immobilized on agarose gel and placed in a small reactor column has made it possible to reproducibility digest as little as 50 ng of protein. This represents a decrease in sample size of approximately 3 orders of magnitude from conventional tryptic digestion schemes. Separations of tryptic digests were accomplished by using either microcolumn high-performance liquid chromatography (HPLC) or capillary zone electrophoresis (CZE). Separations of 100 ng (4 pmol) of tryptic digest samples of beta-casein were achieved with microcolumn HPLC, while separations of approximately 2 ng (80 fmol) of beta-casein tryptic digest (from a total sample size of 50 ng) were possible with CZE. Peptide maps from phosphorylated and dephosphorylated forms of beta-casein were readily distinguishable using both separation methods, demonstrating an ability to detect a single amino acid modification in a protein. Relative standard deviations of peak retention or migration times were less than 3% for microcolumn HPLC and less than 1% for CZE.

Amino Acid Sequence↗

PubMed1989

Formation of cyclic enol ethers from a labile biological precursor: an example of analytical artifacts.

Three isomeric enol ethers are among those constituents apparently unique to mouse urine as identified by gas chromatographic analysis. These compounds appear to be artifacts arising from the cyclization and dehydration of 6-hydroxy-6-methyl-3-heptanone. Identification of the trimethylsilyl ether of 6-hydroxy-6-methyl-3-heptanone in the silylated ether extract of mouse urine indicates that the precursor keto alcohol is indeed present in the urine. Since similar heterocyclic compounds are often identified in urine samples analyzed by gas chromatography, formation of various analysis artifacts arising from analogous cyclization and dehydration reactions is likely.

Animals↗

PubMed1989

Separation of precolumn ortho-phthalaldehyde-derivatized amino acids by capillary zone electrophoresis with normal and micellar solutions in the presence of organic modifiers.

Isoindole derivatives of amino acids, formed through a well-established reaction with o-phthalaldehyde, have been effectively resolved by capillary zone electrophoresis and detected fluorimetrically. Relative retention of the formed derivatives is strongly influenced by added detergents and organic modifiers. Analytical reproducibility has also been assessed.

Amino Acids↗

PubMed1989

Analysis of steroids by capillary supercritical fluid chromatography with phosphorus-selective detection.

Steroids were derivatized with dimethylthiophosphinic chloride to produce steroidal thiophosphinic esters. A catalyst, 4-dimethylaminopyridine, was used to promote quantitative and reproducible thiophosphinic ester formation at low reaction temperatures. The derivatives were analyzed by capillary supercritical fluid chromatography (SFC) with phosphorus thermionic detection. The phosphorus thermionic detector exhibited linearity over 3-4 orders of magnitude. A sensitivity of 120 fg P/s was obtained for the dithiophosphinic ester of pregnanediol at a signal-to-noise ratio of 3. The efficiency of capillary SFC and the excellent sensitivity of the phosphorus thermionic detector were demonstrated by the analysis of steroids isolated from both human urine and plasma.

Androsterone↗

PubMed1989

Puberty-affecting synthetic analogs of urinary chemosignals in the house mouse, Mus domesticus.

Endocrinologically- and socially-dependent volatile constituents of female mouse urine, identified in a previous study, were tested for their capability to accelerate puberty and extend the estrous period in young females. Several volatile ketones advanced puberty by approximately three days and extended the period of vaginal cornification in 55-75% of exposed females. High High concentrations of these substances were capable of overriding the known puberty-delaying chemosignals. Volatile cyclic enol ethers were also effective in extending estrus, but not puberty acceleration.

Animals↗