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Biomedical subjects

M V Novotny

Publications and source records attributed to M V Novotny.

At least 19 recordsLinked to original sources
PubMed2005

Putative chemosignals of the ferret (Mustela furo) associated with individual and gender recognition.

Quantitative stir bar sorptive extraction methods, both in the aqueous and headspace modes, followed by thermal desorption gas chromatography-mass spectrometry were used to investigate individual variations in the volatile components of male and female ferret (Mustela furo) urine. The urinary profiles were further compared with volatile profiles of anal gland secretions of breeding male and female ferrets. Thirty volatile compounds were quantified in male and female urine. Among them, 2-methylquinoline was unique to male urine. Four ketones (4-heptanone, 2-heptanone, o-aminoacetophenone, and a dimethoxyacetophenone) and several nitrogen compounds (e.g., 2,5-dimethylpyrazine, quinoline, 4-methylquinazoline) and low levels of three unidentified nonsulfur compounds were significantly more abundant in males than in females. Quantitative comparison of 30 volatile urinary compounds showed several statistically significant differences between the sexes and individuals of the same sex. These findings suggest that ferrets may use urine marking for sex and individual recognitions. Ten of the 26 compounds identified in anal gland secretions from females and males were also found in urine. However, most of the major compounds (thietanes, dithiolanes, and indole) in anal glands were not present in urine. This suggests that urine may convey specific signals that differ from those of anal glands. Additionally, 10 volatiles (two aldehydes, five ketones, benzothiazole, 2-methylquinoline, and 4-methylquinazoline), not previously identified, were found in ferret anal gland secretions. Among the new compounds, o-aminoacetophenone was found only in males, while only traces of this compound were found in females. Similar results were previously obtained in anal glands of three other Mustela species. These findings provide new information about the constituents of urine and volatile components of anal gland secretions in ferrets.

Anal Canal↗
PubMed2003

Pheromones, binding proteins and receptor responses in rodents.

Their well-developed chemical communication systems make rodents popular in research that aims to understand the connections between genes, hormones and behaviour. Structural identification of several pheromones in mice, rats and hamsters now makes it feasible to employ their synthetic analogues in probing olfactory neurons and in the study of various pheromone-protein interactions in intimate detail.

Animals↗
PubMed2001

Microscale nonreductive release of O-linked glycans for subsequent analysis through MALDI mass spectrometry and capillary electrophoresis.

A new beta-elimination-based procedure has been devised for a microscale release of O-linked oligosaccharides from glycoproteins. Unlike the conventional Carlson degradation, which leads to formation of alditols, the procedure reported here renders the reducing end intact. Conversion of the liberated oligosaccharides to glycosylamines in ammonia medium is followed by the production of the reducing oligosaccharides through the addition of boric acid. The quantitatively generated oligosaccharides with the reducing end can subsequently be derivatized with a fluorophoric reagent for capillary electrophoresis or, alternatively, analyzed through MALDI mass spectrometry. The microscale version of these chemical steps permits us to investigate structurally O-linked oligosaccharides at very low levels.

Ammonia↗
PubMed2001

Sugar-lectin interactions investigated through affinity capillary electrophoresis.

The affinity interactions of Concanavalin A (Con A) with various saccharide oligomers (dextrins, dextrans, and selected N-linked glycans from various glycoproteins) have been investigated through a capillary electrophoresis approach. Con A has shown a notable binding discrimination between the alpha-1,6-linked dextran and alpha-1,4-linked dextrin oligomers. Both the binding capacity and binding discrimination appear to decrease with an increase in sugar chainlength. While the core structure of N-linked glycans is deemed to be responsible for the overall binding of various glycans to Con A, the presence of mannose units at the non-reducing ends was found to be very beneficial to the affinity interaction with Con A. Finally, a connection between the glycan-lectin interaction and glycoprotein-lectin interaction has also been suggested.

Carbohydrates↗
PubMed2001

Structural basis of pheromone binding to mouse major urinary protein (MUP-I).

The mouse major urinary proteins are pheromone-binding proteins that function as carriers of volatile effectors of mouse physiology and behavior. Crystal structures of recombinant mouse major urinary protein-I (MUP-I) complexed with the synthetic pheromones, 2-sec-butyl-4,5-dihydrothiazole and 6-hydroxy-6-methyl-3-heptanone, have been determined at high resolution. The purification of MUP-I from mouse liver and a high-resolution structure of the natural isolate are also reported. These results show the binding of 6-hydroxy-6-methyl-3-heptanone to MUP-I, unambiguously define ligand orientations for two pheromones within the MUP-I binding site, and suggest how different chemical classes of pheromones can be accommodated within the MUP-I beta-barrel.

Animals↗
PubMed2000

Steroid profiles determined by capillary electrochromatography, laser-induced fluorescence detection and electrospray-mass spectrometry.

Macroporous, monolithic capillary electrochromatography (CEC) columns, featuring a hydrophobic stationary phase, have been applied to the separations of steroids with good column efficiency. Using isocratic and gradient elution runs, mixtures of neutral or conjugated steroids could be resolved. While dansylated ketosteroids were detectable through laser-induced fluorescence at attomole levels, the CEC columns coupled to electrospray-ion-trap mass spectrometry featured femtomole detection limits.

Electrophoresis, Capillary↗
PubMed2000

Analysis of bile acids and their conjugates by capillary electrochromatography/electrospray ion trap mass spectrometry.

Different macroporous, monolithic capillary columns were prepared to separate various bile acid mixtures through capillary electrochromatography (CEC) at high efficiency. These columns are shown to be ideally suitable for coupling to an electrospray ionization/ion trap mass spectrometer. Detection and structural identification of different bile acid derivatives in either the positive- or negative-ion mode necessitated column technologies with different polarities and the capabilities of a reversed electroosmotic flow. High column efficiencies (610,000 theoretical plates/meter for glycocholic acid in normal-phase separation) were preserved in the coupling to mass spectrometry (MS), with the detection limits of approximately 40 femtomole (for cholic acid) and identification through CEC/MS/MS.

Bile Acids and Salts↗
PubMed2000

Ultrasensitive pheromone detection by mammalian vomeronasal neurons.

The vomeronasal organ (VNO) is a chemoreceptive organ that is thought to transduce pheromones into electrical responses that regulate sexual, hormonal and reproductive function in mammals. The characteristics of pheromone signal detection by vomeronasal neurons remain unclear. Here we use a mouse VNO slice preparation to show that six putative pheromones evoke excitatory responses in single vomeronasal neurons, leading to action potential generation and elevated calcium entry. The detection threshold for some of these chemicals is remarkably low, near 10(-11) M, placing these neurons among the most sensitive chemodetectors in mammals. Using confocal calcium imaging, we map the epithelial representation of the pheromones to show that each of the ligands activates a unique, nonoverlapping subset of vomeronasal neurons located in apical zones of the epithelium. These neurons show highly selective tuning properties and their tuning curves do not broaden with increasing concentrations of ligand, unlike those of receptor neurons in the main olfactory epithelium. These findings provide a basis for understanding chemical signals that regulate mammalian communication and sexual behaviour.

Animals↗
PubMed2000

Changes in glycosylation of human bile-salt-stimulated lipase during lactation.

Bile-salt-stimulated lipase (BSSL) is an enzyme in human milk, which is important for the fat digestion in the newborn infant. BSSL is highly glycosylated and includes one site for N-glycosylation and several sites for O-glycosylation. BSSL has previously been found to express Lewis a, Lewis b, and Lewis x carbohydrate antigens. In this study, glycosylation of BSSL was studied at different times during lactation. BSSL was purified from milk collected individually from four donors at several different times during the first 6 months of lactation. The BSSL glycans were characterized through monosaccharide analysis, high-pH anion-exchange chromatography, matrix-assisted laser desorption-ionization mass spectrometry, and ELISA. Both total carbohydrate content and relative amount of sialic acid were higher in BSSL from the first lactation month as compared to BSSL from milk collected later in lactation. BSSL from the first lactation month also showed a different composition of sialylated O-linked glycans and the N-linked oligosaccharides consisted of lower amounts of fucosylated structures compared to later in lactation. We also found a gradual increase in the expression of the carbohydrate epitope Lewis x on BSSL throughout the lactation period. This study shows that glycosylation of BSSL is dependent on blood group phenotype of the donor and changes substantially during the lactation period.

Amidohydrolases↗
PubMed2000

N-linked oligosaccharide structures in the diamine oxidase from porcine kidney.

Structures of the N-linked glycans released from porcine kidney diamine oxidase (DAO) were characterized utilizing various analytical techniques, including matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI/TOF-MS), high-performance capillary electrophoresis (HPCE), and high-pH anion-exchange chromatography with pulsed amperometric detection (HPAEC-PAD). The oligosaccharide sequences present in DAO were conclusively determined using specific exoglycosidases in conjunction with MALDI/TOF-MS. The structures found in the glycoprotein are primarily linear, di-, or tribranched fucosylated complex type. MS analysis of the esterified N-glycan pool derived from DAO indicated the presence of several di- and trisialylated structures.

Amine Oxidase (Copper-Containing)↗
PubMed2000

Affinity capillary electrophoretic studies of complexation between dextrin oligomers and polyiodides.

Capillary electrophoresis (CE) has been applied to the study of complexation between dextrins and polyiodides. A baseline separation of fluorescently labeled dextrin oligomers has provided a unique platform for the observation of a contribution of single oligomers to the complexation process that could previously be measured only in bulk. The complex formation was easily recognized through comparison of peak migration times and peak shapes in the presence and absence of polyiodides. The degree of polymerization (DP) number was found crucial in the binding process, but the I2/I- ratio in a solution also appeared to determine the nature of complexation. The effects of buffer pH and ionic strength upon complexation were also briefly investigated. Diodearray spectra in the visible wavelength range confirmed the differential complexation of unlabeled maltodextrins with different DP values after a CE iodine affinity separation. 13C-nuclear magnetic resonance (NMR) spectral data on differently sized dextrin fractions were found to be in good agreement with the results from CE measurements.

Dextrins↗
PubMed2000

A simple sample preparation for enhancing the sensitivity of mass spectrometric oligosaccharide determinations through the use of an adsorptive hydrophobic resin.

A simple microadsorption technique is described to remove detergent additives from oligosaccharide samples before their mass spectrometric analysis. The described methodology has been validated with submicrogram quantities of contaminated glycoproteins. This procedure is applicable to investigating minute quantities of glycans in both the positive- and negative-ion mode of matrix-assisted laser desorption/ionization mass spectrometry.

Animals↗
PubMed2000

Electrophoretic studies of polygalacturonate oligomers and their interactions with metal ions.

Polygalacturonic acid, a linear homopolysaccharide, was investigated by capillary electrophoresis (CE) using linear polyacrylamide-coated capillaries and laser-induced fluorescence (LIF) detection. A successful separation of its fluorescently labeled oligomers was achieved through sieving in polyacrylamide entangled matrices. The reaction conditions for the derivatization of polygalacturonic acid were optimized. In studying the interactions between polygalacturonic acid and various metal ions, the end-label, free-solution electrophoretic (ELFSE) technique, developed earlier in our laboratory (Sudor, J., Novotny, M. V., Anal. Chem. 1995, 67, 4205-4209) was found preferable to the sieving method. ELFSE is fast and convenient in that no polymer solutions are needed for the separation. The investigation showed that for the moderately large oligomers, the strongest binding occurred with calcium and cadmium ions, while the smallest interaction was observed with magnesium ions.

Cadmium↗
PubMed2000

Glycosylated major urinary protein of the house mouse: characterization of its N-linked oligosaccharides.

A minor component of the major urinary protein complex of the house mouse was chromatographically isolated and ascertained to be a previously suspected glycoprotein. Using highly sensitive mass-spectrometric techniques for sequencing and linkage analysis, the N-linked oligosaccharides of this glycoprotein were characterized. They were determined to be of the complex type with a wide heterogeneity. The heterogeneity was due to both the degree of sialylation and the presence of galactose residues in either beta(1-3) or beta(1-4) linkages. The biantennary structures were the most pronounced glycans, while tri- and tetraantennary entities were minor.

Animals↗
PubMed1999

Mass spectrometric analysis of benzoylated sialooligosaccharides and differentiation of terminal alpha 2-->3 and alpha 2-->6 sialogalactosylated linkages at subpicomole levels.

Perbenzoylated sialooligosaccharides were found to be stable derivatives, giving intense signals during the matrix-assisted laser desorption/ionization (MALDI) mass spectrometric analysis in the positive-ion mode. Terminal Neu5NAc alpha 2-->3 and alpha 2-->6Gal units of oligosaccharides undergo characteristic structural changes during benzoylation, yielding easily recognizable mass spectral patterns. Subpicomole carbohydrate samples were successfully benzoylated and analyzed through MALDI mass spectrometry.

Benzoates↗
PubMed1999

Positive identification of the puberty-accelerating pheromone of the house mouse: the volatile ligands associating with the major urinary protein.

Five structurally diverse small ligands, all binding to the major urinary protein (MUP) of the male house mouse, show individually puberty-accelerating pheromonal activity in the recipient females. A recombinant MUP (identical structurally to the natural protein) has shown no biological activity. While four of these ligands were previously implicated in oestrus synchronization (Whitten effect), the same chemosignals now appear responsible for both sexual maturation and cycling in adult females.

Animals↗