High-molecular-weight (> 300 daltons) polycyclic aromatic compounds which were extracted from ... more High-molecular-weight (> 300 daltons) polycyclic aromatic compounds which were extracted from a carbon black were separated by reversed-phase high-performance liquid chromatography using gradient elution. Fractions were collected and analyzed by mass spectrometry and spectrofluorimetry. While only 15 compounds could be positively identified, 38 others were given tentative structural assignments, among which were several large sulfur heterocycles.
The vomeronasal organ (VNO) is a chemoreceptive organ that is thought to transduce pheromones int... more 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
Enhancement of adult neurogenesis in female mice was previously demonstrated through exposure to ... more Enhancement of adult neurogenesis in female mice was previously demonstrated through exposure to soiled bedding from males, although the identity of relevant chemosignals has remained unknown. The farnesenes and SBT (2-sec-butyl-4,5-dihydrothiazole) are male murine pheromones that dominant males secrete at higher levels. Previous studies have shown that they induce oestrus in female mice. We have recently shown that these pheromones strongly increase cell proliferation in the SVZ (subventricular zone) of adult female mice. In addition, we found that a female murine pheromone, 2,5-dimethylpyrazine, facilitates similar changes in males. 2,5-dimethylpyrazine is a female pheromone that is secreted when females are housed in large groups and it was originally found to suppress oestrus in females. We found that it does not have suppressive effect on the cell proliferation in the SVZ of females. Similarly, male murine pheromones, SBT and the farnesenes, do not show a suppressive effect on ...
Methods in molecular biology (Clifton, N.J.), 2013
High-precision quantitative profiling of volatile organic constituents in rodent physiological fl... more High-precision quantitative profiling of volatile organic constituents in rodent physiological fluids and glandular secretions is needed to relate olfactory signals to physiology and behavior. Whereas capillary gas chromatography-mass spectrometry (GC-MS) analysis has become the most widely applied in such investigations, the extraction and preconcentration of volatile organics is arguably the most critical step in the overall analytical task. In this chapter, we describe technical details of two main sample extraction procedures used in our laboratory: dynamic headspace trapping, and stir bar sorptive extraction (SBSE). They have been demonstrated here for the chromatographic analysis of mouse urine, serum, saliva, and preputial gland specimens.
For complexes between proteins and very small hydrophobic ligands, hydrophobic effects alone may ... more For complexes between proteins and very small hydrophobic ligands, hydrophobic effects alone may be insufficient to outweigh the unfavorable entropic terms resulting from bimolecular association. NMR relaxation experiments indicate that the backbone flexibility of mouse major urinary protein increases upon binding the hydrophobic mouse pheromone 2-sec-butyl-4,5-dihydrothiazole. The associated increase in backbone conformational entropy of the protein appears to make a substantial contribution toward stabilization of the protein-pheromone complex. This term is likely comparable in magnitude to other important free energy contributions to binding and may represent a general mechanism to promote binding of very small ligands to macromolecules.
A submicroliter electrochemical detector for liquid chromatography has been designed, using press... more A submicroliter electrochemical detector for liquid chromatography has been designed, using pressure-annealed pyrolytic graphite technology. The analytical performance of this detector was studied in connection with a reversed-phase packed microcapillary column at very low flow-rates. Although the miniaturized version of the electrochemical detector is less sensitive, a direct analysis of a number of urinary metabolites in 0.1--1.0 microliters samples is feasible.
... Jinping Liu, J. Fred Banks, Jr., and Milos Novotny* Department of Chemistry, Indiana Universi... more ... Jinping Liu, J. Fred Banks, Jr., and Milos Novotny* Department of Chemistry, Indiana University Bloomington, Indiana 47405 Abstract. A high-speed separation of the mono-, di-, and triphosphorylated common ribonucleosides has been explored using micellar electrokinetic ...
Pacific Symposium on Biocomputing. Pacific Symposium on Biocomputing, 2007
A major challenge in shotgun proteomics has been the assignment of identified peptides to the pro... more A major challenge in shotgun proteomics has been the assignment of identified peptides to the proteins from which they originate, referred to as the protein inference problem. Redundant and homologous protein sequences present a challenge in being correctly identified, as a set of peptides may in many cases represent multiple proteins. One simple solution to this problem is the assignment of the smallest number of proteins that explains the identified peptides. However, it is not certain that a natural system should be accurately represented using this minimalist approach. In this paper, we propose a reformulation of the protein inference problem by utilizing the recently introduced concept of peptide detectability. We also propose a heuristic algorithm to solve this problem and evaluate its performance on synthetic and real proteomics data. In comparison to a greedy implementation of the minimum protein set algorithm, our solution that incorporates peptide detectability performs fa...
Microcolumn liquid chromatography with slurry-packed capillary columns has been used to resolve m... more Microcolumn liquid chromatography with slurry-packed capillary columns has been used to resolve mixtures of biologically important steroids and prostaglandins. In order to enhance detection sensitivity, the samples are first derivatized with a suitable chromophore-yielding agent. Hydroxy steroids were derivatized with either benzoyl chloride or 7-(chlorocarbonylmethoxy)-4-methylcoumarin, while Dns hydrazine was employed to react with the ketonic groups of certain steroidal conjugates. Bile acids can also be derivatized with bromomethylcoumarin to yield fluorescent products. The use of microcolumns permits both a high degree of component resolution and enhanced detection sensitivity.
ABSTRACT Aminodextran (AD) is shown here as an effective electrolyte additive for a size-dependen... more ABSTRACT Aminodextran (AD) is shown here as an effective electrolyte additive for a size-dependent separation of hyaluronate (HA) oligomers in capillary gel electrophoresis. AD interacts mainly with the negatively charged HA through an ion-exchange mechanism, while its participation is dependent on the polymer chain size. The degree of complexation and, consequently, electrophoretic mobility can be adjusted through charge density in the analyte or additive molecules through a choice of pH or addition of competing ions to the separation buffer. This approach enables adequate separation, in a large molecular-size range, without the need for highly viscous polymer solutions, thus simplifying a buffer replacement between the runs. Through a proper choice of buffer components, resolution of the individual HA polymer chains up to molecular masses of about 70 000 Da was achieved in less than 45 min. The migration behavior is discussed and presented in combination with its descriptive models.
Mice lacking the melanocortin-5 receptor (MC5R) exhibit decreased sensitivity to the stimulatory ... more Mice lacking the melanocortin-5 receptor (MC5R) exhibit decreased sensitivity to the stimulatory effects of systemic melano- cortin injections on aggressive behavior. Because the pheromone-producing preputial gland expresses the MC5R, we tested the hypothesis that decreases in preputial pheromones underlie the behavioral deficit. Here we show that MC5R deficiency decreases preputial and urine levels of the sex pheromones, alpha- and beta-farnesene,
Suitable buffer additives can either induce or reduce the electrophoretic mobility of certain pol... more Suitable buffer additives can either induce or reduce the electrophoretic mobility of certain polysaccharides during capillary electrophoresis. With water-soluble cellulose derivatives, adsorption of detergents on the analyte molecules governs the migration rates. Different types of adsorption sites, including multilayer formation, were found to be present on the cellulose-derived polymers. For highly charged polysaccharides (heparins), the electrophoretic mobility can be controlled by the use of ion-pairing reagents. The concentration and number of cationic charges appear useful in optimizing the separation selectivity. Migration models based on secondary thermodynamic equilibria are presented and evaluated for different polysaccharides.
The heterogeneous nature of most polysaccharides found in nature includes distribution in molecul... more The heterogeneous nature of most polysaccharides found in nature includes distribution in molecular weight, primary sequence, and branching. The analytical methodology used in the characterization of these structural aspects must ensure high separation efficiency and selectivity. This paper reports on the high-performance capillary electrophoresis (HPCE) separation of branched forms of oligosaccharides as well as some variants in the primary structure. Oligosaccharide maps were obtained after selective debranching using isoamylase, laminarinase, and cellulase enzymes. The samples investigated were a-D-glucans (amylose, amylopectin, and pullulan) and p-D-glucans (exemplified by lichenan). The solutes were separated as fluorescent derivatives with &aminonaphthalene-1,3,6-trisulfonate (ANTS) and detected by laser-induced fluorescence at 514 nm using a He/Cd laser (excitation at 325 nm). The number of theoretical plates was in excess of one million per meter. Baseline resolution of oligosaccharides with a degree of polymerization N 70 was obtained within 15 min using borate buffer as the electrolyte. * Corresponding author. 0008-6215/94/$07.00 Q 1994 Elsevier Science B.V. AU rights reserved SSDI 0008-6215(94)00003-X
The fluorescent derivatives of complex oligosaccharide mixtures from different origins are separa... more The fluorescent derivatives of complex oligosaccharide mixtures from different origins are separated in open capillaries. The number of negatively charged sulfonate groups in the tag molecule strongly affects separation efficiency and selectivity. Coating of the capillary surface with linear polyacrylamide has been essential to ensure fast and stable migration velocities. Efficiencies in excess of 1 million plates/m have been achieved, facilitating resolution of branched oligosaccharides. The effects of field strength and buffer composition on the apparent electrophoretic mobility of dextran and dextrin oligomers are discussed, with relation to borate complexation. Preliminary examples of applications to monitoring the action of hydrolytic and synthesizing enzymes are also described.
Complexation between the linear maltodextrin oligosaccharides and certain enantiomeric compounds ... more Complexation between the linear maltodextrin oligosaccharides and certain enantiomeric compounds of pharmaceutical interest in buffered solutions can lead to an analytically desirable chiral recognition. Different maltodextrins were assessed in their capacity to cause enantiomeric separations under various conditions of capillary electrophoresis. The mechanism of chiral recognition has been probed through electrophoretic mobility and selectivity measurements for different buffer solutions and organic solvent additives. A differential interaction of chiral solutes with the maltodextrin helical entities emerges as the basis of such enantioselectivity. This notion is further supported by 1H- and 13C-NMR experiments. Optimized separations of simendan, ibuprofen, warfarin, and ketoprofen enantiomers are demonstrated together with a chiral determination of ibuprofen in a blood serum sample at the therapeutic level.
Proceedings of The National Academy of Sciences, 1986
Two volatile constituents of male mouse urine, 2-(sec-butyl)-4,5-dihydrothiazole and dehydro-exo-... more Two volatile constituents of male mouse urine, 2-(sec-butyl)-4,5-dihydrothiazole and dehydro-exo-brevicomin, were synthesized and tested for their ability to induce estrous cycle in female mice (the Whitten effect). The suppression of ovarian cycling activity that resulted from grouping the females was abolished by exposure to normal male urine. The synthetic compounds, when added together in appropriate concentrations to the (previously inactive)
Proceedings of The National Academy of Sciences, 1985
Two volatile constituents of male mouse urine, dehydro-exo-brevicomin and 2-(sec-butyl)-dihydroth... more Two volatile constituents of male mouse urine, dehydro-exo-brevicomin and 2-(sec-butyl)-dihydrothiazole, have been found active in bioassays of inter-male aggressive behavior. The two synthetic compounds act synergistically when added to castrated male urine but not when added to water, and they provoke fighting that is quantitatively and qualitatively comparable to that elicited by intact male urine.
High-molecular-weight (> 300 daltons) polycyclic aromatic compounds which were extracted from ... more High-molecular-weight (> 300 daltons) polycyclic aromatic compounds which were extracted from a carbon black were separated by reversed-phase high-performance liquid chromatography using gradient elution. Fractions were collected and analyzed by mass spectrometry and spectrofluorimetry. While only 15 compounds could be positively identified, 38 others were given tentative structural assignments, among which were several large sulfur heterocycles.
The vomeronasal organ (VNO) is a chemoreceptive organ that is thought to transduce pheromones int... more 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
Enhancement of adult neurogenesis in female mice was previously demonstrated through exposure to ... more Enhancement of adult neurogenesis in female mice was previously demonstrated through exposure to soiled bedding from males, although the identity of relevant chemosignals has remained unknown. The farnesenes and SBT (2-sec-butyl-4,5-dihydrothiazole) are male murine pheromones that dominant males secrete at higher levels. Previous studies have shown that they induce oestrus in female mice. We have recently shown that these pheromones strongly increase cell proliferation in the SVZ (subventricular zone) of adult female mice. In addition, we found that a female murine pheromone, 2,5-dimethylpyrazine, facilitates similar changes in males. 2,5-dimethylpyrazine is a female pheromone that is secreted when females are housed in large groups and it was originally found to suppress oestrus in females. We found that it does not have suppressive effect on the cell proliferation in the SVZ of females. Similarly, male murine pheromones, SBT and the farnesenes, do not show a suppressive effect on ...
Methods in molecular biology (Clifton, N.J.), 2013
High-precision quantitative profiling of volatile organic constituents in rodent physiological fl... more High-precision quantitative profiling of volatile organic constituents in rodent physiological fluids and glandular secretions is needed to relate olfactory signals to physiology and behavior. Whereas capillary gas chromatography-mass spectrometry (GC-MS) analysis has become the most widely applied in such investigations, the extraction and preconcentration of volatile organics is arguably the most critical step in the overall analytical task. In this chapter, we describe technical details of two main sample extraction procedures used in our laboratory: dynamic headspace trapping, and stir bar sorptive extraction (SBSE). They have been demonstrated here for the chromatographic analysis of mouse urine, serum, saliva, and preputial gland specimens.
For complexes between proteins and very small hydrophobic ligands, hydrophobic effects alone may ... more For complexes between proteins and very small hydrophobic ligands, hydrophobic effects alone may be insufficient to outweigh the unfavorable entropic terms resulting from bimolecular association. NMR relaxation experiments indicate that the backbone flexibility of mouse major urinary protein increases upon binding the hydrophobic mouse pheromone 2-sec-butyl-4,5-dihydrothiazole. The associated increase in backbone conformational entropy of the protein appears to make a substantial contribution toward stabilization of the protein-pheromone complex. This term is likely comparable in magnitude to other important free energy contributions to binding and may represent a general mechanism to promote binding of very small ligands to macromolecules.
A submicroliter electrochemical detector for liquid chromatography has been designed, using press... more A submicroliter electrochemical detector for liquid chromatography has been designed, using pressure-annealed pyrolytic graphite technology. The analytical performance of this detector was studied in connection with a reversed-phase packed microcapillary column at very low flow-rates. Although the miniaturized version of the electrochemical detector is less sensitive, a direct analysis of a number of urinary metabolites in 0.1--1.0 microliters samples is feasible.
... Jinping Liu, J. Fred Banks, Jr., and Milos Novotny* Department of Chemistry, Indiana Universi... more ... Jinping Liu, J. Fred Banks, Jr., and Milos Novotny* Department of Chemistry, Indiana University Bloomington, Indiana 47405 Abstract. A high-speed separation of the mono-, di-, and triphosphorylated common ribonucleosides has been explored using micellar electrokinetic ...
Pacific Symposium on Biocomputing. Pacific Symposium on Biocomputing, 2007
A major challenge in shotgun proteomics has been the assignment of identified peptides to the pro... more A major challenge in shotgun proteomics has been the assignment of identified peptides to the proteins from which they originate, referred to as the protein inference problem. Redundant and homologous protein sequences present a challenge in being correctly identified, as a set of peptides may in many cases represent multiple proteins. One simple solution to this problem is the assignment of the smallest number of proteins that explains the identified peptides. However, it is not certain that a natural system should be accurately represented using this minimalist approach. In this paper, we propose a reformulation of the protein inference problem by utilizing the recently introduced concept of peptide detectability. We also propose a heuristic algorithm to solve this problem and evaluate its performance on synthetic and real proteomics data. In comparison to a greedy implementation of the minimum protein set algorithm, our solution that incorporates peptide detectability performs fa...
Microcolumn liquid chromatography with slurry-packed capillary columns has been used to resolve m... more Microcolumn liquid chromatography with slurry-packed capillary columns has been used to resolve mixtures of biologically important steroids and prostaglandins. In order to enhance detection sensitivity, the samples are first derivatized with a suitable chromophore-yielding agent. Hydroxy steroids were derivatized with either benzoyl chloride or 7-(chlorocarbonylmethoxy)-4-methylcoumarin, while Dns hydrazine was employed to react with the ketonic groups of certain steroidal conjugates. Bile acids can also be derivatized with bromomethylcoumarin to yield fluorescent products. The use of microcolumns permits both a high degree of component resolution and enhanced detection sensitivity.
ABSTRACT Aminodextran (AD) is shown here as an effective electrolyte additive for a size-dependen... more ABSTRACT Aminodextran (AD) is shown here as an effective electrolyte additive for a size-dependent separation of hyaluronate (HA) oligomers in capillary gel electrophoresis. AD interacts mainly with the negatively charged HA through an ion-exchange mechanism, while its participation is dependent on the polymer chain size. The degree of complexation and, consequently, electrophoretic mobility can be adjusted through charge density in the analyte or additive molecules through a choice of pH or addition of competing ions to the separation buffer. This approach enables adequate separation, in a large molecular-size range, without the need for highly viscous polymer solutions, thus simplifying a buffer replacement between the runs. Through a proper choice of buffer components, resolution of the individual HA polymer chains up to molecular masses of about 70 000 Da was achieved in less than 45 min. The migration behavior is discussed and presented in combination with its descriptive models.
Mice lacking the melanocortin-5 receptor (MC5R) exhibit decreased sensitivity to the stimulatory ... more Mice lacking the melanocortin-5 receptor (MC5R) exhibit decreased sensitivity to the stimulatory effects of systemic melano- cortin injections on aggressive behavior. Because the pheromone-producing preputial gland expresses the MC5R, we tested the hypothesis that decreases in preputial pheromones underlie the behavioral deficit. Here we show that MC5R deficiency decreases preputial and urine levels of the sex pheromones, alpha- and beta-farnesene,
Suitable buffer additives can either induce or reduce the electrophoretic mobility of certain pol... more Suitable buffer additives can either induce or reduce the electrophoretic mobility of certain polysaccharides during capillary electrophoresis. With water-soluble cellulose derivatives, adsorption of detergents on the analyte molecules governs the migration rates. Different types of adsorption sites, including multilayer formation, were found to be present on the cellulose-derived polymers. For highly charged polysaccharides (heparins), the electrophoretic mobility can be controlled by the use of ion-pairing reagents. The concentration and number of cationic charges appear useful in optimizing the separation selectivity. Migration models based on secondary thermodynamic equilibria are presented and evaluated for different polysaccharides.
The heterogeneous nature of most polysaccharides found in nature includes distribution in molecul... more The heterogeneous nature of most polysaccharides found in nature includes distribution in molecular weight, primary sequence, and branching. The analytical methodology used in the characterization of these structural aspects must ensure high separation efficiency and selectivity. This paper reports on the high-performance capillary electrophoresis (HPCE) separation of branched forms of oligosaccharides as well as some variants in the primary structure. Oligosaccharide maps were obtained after selective debranching using isoamylase, laminarinase, and cellulase enzymes. The samples investigated were a-D-glucans (amylose, amylopectin, and pullulan) and p-D-glucans (exemplified by lichenan). The solutes were separated as fluorescent derivatives with &aminonaphthalene-1,3,6-trisulfonate (ANTS) and detected by laser-induced fluorescence at 514 nm using a He/Cd laser (excitation at 325 nm). The number of theoretical plates was in excess of one million per meter. Baseline resolution of oligosaccharides with a degree of polymerization N 70 was obtained within 15 min using borate buffer as the electrolyte. * Corresponding author. 0008-6215/94/$07.00 Q 1994 Elsevier Science B.V. AU rights reserved SSDI 0008-6215(94)00003-X
The fluorescent derivatives of complex oligosaccharide mixtures from different origins are separa... more The fluorescent derivatives of complex oligosaccharide mixtures from different origins are separated in open capillaries. The number of negatively charged sulfonate groups in the tag molecule strongly affects separation efficiency and selectivity. Coating of the capillary surface with linear polyacrylamide has been essential to ensure fast and stable migration velocities. Efficiencies in excess of 1 million plates/m have been achieved, facilitating resolution of branched oligosaccharides. The effects of field strength and buffer composition on the apparent electrophoretic mobility of dextran and dextrin oligomers are discussed, with relation to borate complexation. Preliminary examples of applications to monitoring the action of hydrolytic and synthesizing enzymes are also described.
Complexation between the linear maltodextrin oligosaccharides and certain enantiomeric compounds ... more Complexation between the linear maltodextrin oligosaccharides and certain enantiomeric compounds of pharmaceutical interest in buffered solutions can lead to an analytically desirable chiral recognition. Different maltodextrins were assessed in their capacity to cause enantiomeric separations under various conditions of capillary electrophoresis. The mechanism of chiral recognition has been probed through electrophoretic mobility and selectivity measurements for different buffer solutions and organic solvent additives. A differential interaction of chiral solutes with the maltodextrin helical entities emerges as the basis of such enantioselectivity. This notion is further supported by 1H- and 13C-NMR experiments. Optimized separations of simendan, ibuprofen, warfarin, and ketoprofen enantiomers are demonstrated together with a chiral determination of ibuprofen in a blood serum sample at the therapeutic level.
Proceedings of The National Academy of Sciences, 1986
Two volatile constituents of male mouse urine, 2-(sec-butyl)-4,5-dihydrothiazole and dehydro-exo-... more Two volatile constituents of male mouse urine, 2-(sec-butyl)-4,5-dihydrothiazole and dehydro-exo-brevicomin, were synthesized and tested for their ability to induce estrous cycle in female mice (the Whitten effect). The suppression of ovarian cycling activity that resulted from grouping the females was abolished by exposure to normal male urine. The synthetic compounds, when added together in appropriate concentrations to the (previously inactive)
Proceedings of The National Academy of Sciences, 1985
Two volatile constituents of male mouse urine, dehydro-exo-brevicomin and 2-(sec-butyl)-dihydroth... more Two volatile constituents of male mouse urine, dehydro-exo-brevicomin and 2-(sec-butyl)-dihydrothiazole, have been found active in bioassays of inter-male aggressive behavior. The two synthetic compounds act synergistically when added to castrated male urine but not when added to water, and they provoke fighting that is quantitatively and qualitatively comparable to that elicited by intact male urine.
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Papers by Milos Novotny