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Biomedical subjects

L Baczynskyj

Publications and source records attributed to L Baczynskyj.

At least 19 recordsLinked to original sources
PubMed1992

Capillary electrophoresis-electrospray mass spectrometry for the analysis of recombinant bovine and porcine somatotropins.

A Beckman P/ACE 2050 high-performance capillary electrophoresis (HPCE) instrument has been interfaced with a Vestec electrospray ionization (ESI) mass spectrometer for the analysis of recombinant proteins. Peak resolution is not compromised by coupling HPCE to an ESI mass spectrometer. Recombinant bovine and porcine somatotropins (rbSt and rpSt) were used as model proteins. The standard curve of the capillary zone electrophoresis (CZE) method with UV detection for the determination of rpSt is linear in the range of 7-300 fmol with theoretical plates of approximately 410,000 m-1. The relative standard deviation for the rpSt peak migration time is less than 1%. The multiply-charged ion clusters obtained in the CZE-ESI mass spectrum for a sample of rpSt ranged from mlz 1363.2 (the cluster with 16 charges) to 1982.5 (the cluster with 11 charges). The average molecular weights of 21,812.6 and 21,798.3 for a sample of rbSt and rpSt determined in this study were nearly identical to the theoretical values of 21,812.0 and 21,797.9, respectively. Detection limit of the CZE-ESI mass spectrometer is approximately 100 fmol. The CZE method separated mono- and dideamidated species and monoacetylated compounds while the ESI mass spectrometer detected an analogue and a truncated homologue of rpSt comigrating with the major peak. The presence of mono- and dioxidized homologues was also detected in the major peak of some rbSt and rpSt samples. These data clearly indicated that, individually, both CZE and ESI mass spectrometric methods could not detect all impurities. Coupling of the HPCE Instrument and the ESI mass spectrometer enhances analytical capabilities of both tools for rapid characterization of recombinant proteins.

Animals↗
PubMed1992

Utility of the parent-neutral loss scan screening technique: partial characterization of urinary metabolites of U-78875 in monkey urine.

Metabolites of an antianxiety-sedative drug candidate (U-78875; 3-(5-cyclopropyl-1,2,4-oxadiazol-3-yl)-5-(1-methylethyl)-imidazo[1 ,5-alpha] quinoxalin-4(5H)-one (I)) present in the urine of monkeys were detected using tandem mass spectrometry (MS/MS) by application of parent ion scans and characterized or partially characterized by performing daughter ion scans of the pseudo-molecular ions of suspected metabolites. The use of liquid secondary ion mass spectrometry ionization of crude urinary extracts in combination with tandem quadrupole MS/MS analyses using parent ion scans of m/z 69 and subsequent daughter ion scans characterized unmetabolized I and N-dealkyl I (U-85466) and partially characterized aryl hydroxyl, aryl hydroxyl-N-dealkyl, aryl O-glucuronide, aryl O-glucuronide-N-dealkyl, aryl O-sulfate and aryl O-sulfate-N-dealkyl metabolites. From these data it was concluded that some of the metabolic pathways involved in the biotransformation of U-78875 include N-dealkylation, aryl hydroxylation and conjugation of aryl hydroxides. Several other metabolites of U-78875 not detected using this analytical approach were subsequently identified by alternative mass spectrometric approaches. These data clearly demonstrated both the utility and, just as important, the limitations of the parent-neutral loss scan screening technique in detecting drug metabolites in complex biological milieux.

Animals↗
PubMed1991

Particle beam liquid chromatography-mass spectrometry on a double-focusing sector instrument.

A commercially available particle beam interface developed by the Vestec Corporation has been coupled to a double-focusing high-resolution mass spectrometer (VG ZAB 2F) via a momentum separator. An ultraviolet detector was in-line with the interface and allowed monitoring of the eluting materials. With this instrumentation, complete electron-impact (EI) mass spectra on synthetic mixtures of steroids, nucleosides, and several derivatives of amino acids were recorded. Amongst these the EI mass spectra of 2,4-dinitrophenyl, 9-fluorenylmethoxycarbonyl and phenylthiohydantoin derivatives were obtained. The standard LC conditions used here (250 mm x 4.6 mm I.D., reversed-phase C18 column, 1.0 or 1.5 ml/min flow-rate, isocratic or gradient programming) allowed separation of the mixtures. The resulting mass spectra were compared with those obtained using a direct insertion probe. The agreement was excellent in most cases. Sensitivity measurements were performed on cholesterol and caffeine. A complete low-resolution mass spectrum can be obtained on 100 ng of cholesterol. Single-ion monitoring of the M+.of 200 pg of caffeine gave a 4:1 signal-to-noise ratio at m/z 194. Complete high-resolution mass spectra were obtained on 5 micrograms of cholesterol (loop injection) and on every peak of a five-steroid mixture of 5 micrograms each. The accuracy of the mass measurements were better than 5 m.m.u. for most cases. Three to four scans could be obtained for each liquid chromatographic peak. Mass-analyzed ion kinetic energy spectra were recorded on the M+.of 2.5 micrograms of cholesterol at m/z 386. Similarly the B/E linked scan of the same ion was recorded on 2.5 micrograms and 500 ng.

Amino Acids↗
PubMed1991

Naphtho and benzo analogues of the kappa opioid agonist trans-(+/-)-3,4-dichloro-N-methyl-N-[2-(1-pyrrolidinyl)cyclohexyl] benzeneacetamide.

Further elaboration on the structure-activity relationships in our U-50,488 series has revealed that benzologation of this cyclohexane-1,2-diamine derivative provides compounds which either maintain the interaction with the kappa receptor (e.g. compounds 3a and 5a in the phenylacetamido series) or eliminate the mu receptor mediated analgesia (e.g. compounds 3-6 in the benzamido series). Naphthologation also caused the elimination of mu receptor mediated analgesia (e.g. compounds 17a and 17b).

3,4-Dichloro-N-methyl-N-(2-(1-pyrrolidinyl)-cycloh↗
PubMed1991

Purification of lincosaminide O-nucleotidyltransferase from Streptomyces coelicolor Müller.

An enzyme (lincosaminide O-nucleotidyltransferase) that catalyzes 3-(5'-ribonucleotidylation) of pirlimycin and several other lincosaminide antibodies has been purified approximately 35-fold from cell-free extracts of Streptomyces coelicolor Müller NRRL 3532 (UC 5240). The crude enzyme was prepared using lysozyme and was treated with MnCl2 and (NH4)2SO4. Final purification was achieved by anion exchange chromatography. The pirlimycin reaction product was verified as being pirlimycin-3-(5'-adenylate) by NMR spectroscopy and MS. As a result of purification, this lincosaminide nucleotidylating and inactivating enzyme was separated from the macrolide phosphorylating enzyme also present in the cell-free extract.

Clindamycin↗
PubMed1990

Electrospray mass spectrometry of recombinant growth hormones.

The electrospray mass spectra of several standard proteins were recorded and their molecular weights determined. These were compared to Literature values obtained by other laboratories. The agreement was quite good. Seven recombinant growth hormones were then investigated by this technique. The determined molecular weights were in agreement with the theoretical values. Differentiation between six of the seven analogs could be made on the basis of the molecular weight determinations. Two of these analogs differed in molecular weight by only 1 Da and the accuracy of the mass measurements was not sufficient for distinguishing these two homologous proteins. The influence of solvent on the mass accuracy determinations was studied. It appears that solvents form clusters with the proteins in the electrospray ionization method. These clusters broaden the peaks corresponding to the multiply charged ions of the proteins and make the determination of centroids more difficult.

Animals↗
PubMed1988

Structural relationships between senfolomycins and paulomycins.

Senfolomycins A and B (Antimicrob. Agents Chemother.-1965: 828-831, 1966) are two antibacterial agents with physico-chemical and biological properties similar to those of paulomycin. Recent studies indicate that senfolomycin A (C29H36N2O16S, MW 700) has molecular composition and fast atom bombardment MS fragmentation pattern identical to those of paulomycin E. Extensive NMR work indicates that the two antibiotics, which have been separated by HPLC and TLC, differ only in the stereochemistry of the OCH3 group present in their respective sugar moieties. Indirect evident suggests that senfolomycin B is dihydrosenfolomycin A (C29H38N2O16S, MW 702) and in this respect it is related to paulomycin F. The proposed structures for senfolomycins A and B are discussed.

Anti-Bacterial Agents↗
PubMed1988

O-demethylpaulomycins A and B, U-77,802 and U-77,803, paulomenols A and B, new metabolites produced by Streptomyces paulus.

O-Demethylpaulomycin A (C33H44N2O17S), O-demethylpaulomycin B (C32H42N2O17S), paulomenol A (C29H43NO16), paulomenol B (C28H41NO16), and the hydrogen sulfide adducts of paulomycin A (U-77,802, C34H48N2O17S2), and paulomycin B (U-77,803, C33H46N2O17S2) have been isolated from fermentations of Streptomyces paulus strain 273. The structure of these compounds was determined by 1H and 13C NMR and fast atom bombardment mass spectrum spectroscopic techniques and degradative studies. The antibacterial properties of these new metabolites, which are related to paulomycins A and B (J. Antibiotics 35: 285-294, 1982), are briefly discussed.

Anti-Bacterial Agents↗
PubMed1988

Adenylylation of trospectomycin by crude enzyme preparations from Escherichia coli.

Employing osmotically shocked lysate of a spectinomycin resistant strain of Escherichia coli, trospectomycin, a new alkylspectinomycin, was adenylylated in the presence of adenosine 5'-triphosphate and magnesium ion. A highly resistant strain of E. coli was obtained by transforming a laboratory strain with a newly constructed plasmid consisting of pBR322 and a determinant for spectinomycin resistance originally found on a low copy number plasmid in E. coli strain NR79. The biologically inactive adenylylated trospectomycin was found to be trospectomycin 6-(5'-adenylate).

Adenosine Monophosphate↗
PubMed1988

New paulomycins produced by Streptomyces paulus.

Paulomycin A2 (C34H46N2O17S), paulomycin C (C32H42N2O17S), paulomycin D (C31H40N2O17S), paulomycin E (C29H36N2O16S) and paulomycin F (C29H38N2O16S) have been isolated from fermentations of Streptomyces paulus strain 273. The structure of these compounds was determined using NMR and mass spectroscopic techniques. The new paulomycins, like paulomycins A and B (J. Antibiotics 35: 285-294, 1982) are highly active mainly against Gram-positive organisms.

Anti-Bacterial Agents↗
PubMed1987

Purification and characterization of digitalislike factors from human plasma.

Increased levels of a humoral inhibitor of active sodium transport have been associated with the response to acute and chronic hypervolemia and various forms of experimental as well as human essential hypertension. In this report, we describe the purification of inhibitors of Na+, K+-adenosine triphosphatase (ATPase) from the plasma of volume-expanded individuals. Of the two amphipathic materials obtained, only one of the factors when present in high concentrations showed the slow time-dependent component of inactivation similar to that of the cardiac glycosides. Inhibition was reduced in the presence of plasma proteins and was freely reversible. Both factors inhibited potassium-dependent p-nitrophenylphosphatase activity and specific [3H]ouabain binding in a manner similar to the cardiac glycosides. In contrast to ouabain and vanadate, however, high concentrations of potassium or norepinephrine, respectively, did not affect the magnitude or kinetic characteristics of inhibition. Structural analysis by mass spectroscopy showed a mass of 444 for factor 1, whereas factor 2 was conclusively identified as lysophosphatidylcholine-gamma-palmitoyl. These factors probably inhibit Na+, K+-ATPase by a nonspecific mechanism involving reversible perturbation of lipid-enzyme interactions required for normal catalytic activity. The significance of these factors as modulators of sodium transport may be limited to pathological states associated with abnormalities in plasma protein binding or lipid metabolism. They do not appear to be directly related to the humorally mediated disturbance of cellular sodium transport in hypertension.

4-Nitrophenylphosphatase↗
PubMed1987

Enzymatic phosphorylation of macrolide antibiotics.

Five macrolide antibiotics (erythromycin A, 1; oleandomycin, 3a; tylosin, 4a; spiramycins, 5a; leucomycin A3, 6a) have been phosphorylated enzymatically using cell-free extracts derived from Streptomyces coelicolor UC 5240. The necessary cofactors and the rates of the conversion have been determined.

Anti-Bacterial Agents↗
PubMed1987

Paulomycin-related antibiotics: paldimycins and antibiotics 273a2. Isolation and characterization.

The isolation of paulomycins A and B from fermentations of Streptomyces paulus has been reported earlier [J. Antibiotics 35: 285-294, 1982]. Further work on the antibiotics produced by S. paulus revealed the production of two paulomycin-related compounds, antibiotics 273a1 and 273a2 which were isolated by procedures involving extractions and chromatography over buffered silica gel. Antibiotic 273a1 which has been named paldimycin, was found to be a mixture of two materials, paldimycins A and B (antibiotics 273a1 alpha, and 273a1 beta). Similarly, antibiotic 273a2 was found to consist of antibiotic 273a2 alpha and antibiotic 273a2 beta. Paldimycin and antibiotic 273a2, which are produced by addition of two or one molecules of N-acetyl-L-cysteine, respectively, to paulomycins A and B, are active vs. Gram-positive bacteria.

Anti-Bacterial Agents↗
PubMed1987

Paldimycins A and B and antibiotics 273a2 alpha and 273a2 beta. Synthesis and characterization.

Paldimycin (antibiotic 273a1) and antibiotic 273a2 as well as their individual components, paldimycins A (273a1 alpha) and B (273a1 beta) and antibiotics 273a2 alpha and 273a2 beta were synthesized from paulomycin, paulomycin A and paulomycin B, respectively, by reacting with N-acetyl-L-cysteine. The semisynthetic antibiotics had chromatographic behavior (TLC, HPLC) and physical and chemical properties identical to the properties of the corresponding antibiotics produced by Streptomyces paulus.

Acetylcysteine↗
PubMed1987

Isolation, identification and synthesis of a metabolite of tazadolene succinate.

Metabolism of tazadolene (1), a novel non-opioid analgesic with antidepressant properties, affords the 4-hydroxy and 3-methoxy-4-hydroxy derivatives (phenyl ring) of the drug, and N-[2-(phenylmethylene)cyclohexyl]-beta-alanine (4). The isolation, identification and synthesis of the latter metabolite is described.

Analgesics↗