Development and pharmacological characterization of a model of sleep disruption-induced hypersensitivity in the rat.

BACKGROUND: Sleep disturbance is a commonly reported co-morbidity in chronic pain patients, and conversely, disruption of sleep can cause acute and long-lasting hypersensitivity to painful stimuli. The underlying mechanisms of sleep disruption-induced pain hypersensitivity are poorly understood. Confounding factors of previous studies have been the sleep disruption protocols, such as the 'pedestal over water' or 'inverted flower pot' methods, that can cause large stress responses and therefore may significantly affect pain outcome measures. METHODS: Sleep disruption was induced by placing rats for 8 h in a slowly rotating cylindrical cage causing arousal via the righting reflex. Mechanical (Von Frey filaments) and thermal (Hargreaves) nociceptive thresholds were assessed, and plasma corticosterone levels were measured (mass spectroscopy). Sleep disruption-induced hypersensitivity was pharmacologically characterized with drugs relevant for pain treatment, including gabapentin (30 mg/kg and 50 mg/kg), Ica-6p (Kv7.2/7.3 potassium channel opener; 10 mg/kg), ibuprofen (30 mg/kg and 100 mg/kg) and amitriptyline (10 mg/kg). RESULTS: Eight hours of sleep disruption caused robust mechanical and heat hypersensitivity in the absence of a measurable change in plasma corticosterone levels. Gabapentin had no effect on reduced nociceptive thresholds. Ibuprofen attenuated mechanical thresholds, while Ica-6p and amitriptyline attenuated only reduced thermal nociceptive thresholds. CONCLUSIONS: These results show that acute and low-stress sleep disruption causes mechanical and heat hypersensitivity in rats. Mechanical and heat hypersensitivity exhibited differential sensitivity to pharmacological agents, thus suggesting dissociable mechanisms for those two modalities. Ultimately, this model could help identify underlying mechanisms linking sleep disruption and hypersensitivity.

Chromatographic analysis of compounds of pharmaceutical interest.

A set of 17,600 samples belonging to our compound collection is selectively examined by liquid chromatography with UV, evaporative light scattering, and mass spectrometric detection methods. At least 70% of this set consists of pure samples with the expected structures. Subsequent studies by flow injection mass spectrometry show that this value is a conservative estimate and that the actual percentage of pure and correct compounds is close to 80%. Because this is the first time that sample quality information becomes available on such a large scale for the compound collection, it offers an opportunity to perform chemi-informatic studies for which structural integrity is essential. Results of these studies can be used to improve the selection of compounds for screening and evaluate the quality of compounds from particular sources.

Isolation and identification of a major metabolite of PNU-107859, an MMP inhibitor from the biliary fluid of rats.

PNU-107859, an important representative structure in a novel class of matrix metalloproteinases (MMP) inhibitors known as thiadiazoles, was found to be quickly eliminated from rats. A major metabolite (approximately 10% of total dose) was found to be present in the bile of rats. The metabolite in question was isolated and purified from the bile fluids collected from six cannulated rats. From a total of approximately 75 mg of PNU-107859 administered to rats, 3.3 mg of the metabolite was recovered. The NMR and mass spectrometry results indicated that the metabolite is a glucuronide conjugate (1-deoxy-1beta-substituted D-glucopyranosiduronic acid) of the intact drug. Furthermore, the UV, MS, and NMR data established that the conjugate is located at the nitrogen alpha to the thiocarbonyl of the thiadiazole ring.

A new resistance gene, linB, conferring resistance to lincosamides by nucleotidylation in Enterococcus faecium HM1025.

Resistance to lincomycin and clindamycin in the clinical isolate Enterococcus faecium HM1025 is due to a ribosomal methylase encoded by an ermAM-like gene and the plasmid-mediated inactivation of these antibiotics. We have cloned and determined the nucleotide sequence of the gene responsible for the inactivation of lincosamides, linB. This gene encodes a 267-amino-acid lincosamide nucleotidyltransferase. The enzyme catalyzes 3(5'-adenylation) (the adenylation of the hydroxyl group in position 3 of the molecules) of lincomycin and clindamycin. Expression of linB was observed in both Escherichia coli and Staphylococcus aureus. The deduced amino acid sequence of the enzyme did not display any significant homology with staphylococcal nucleotidyltransferases encoded by linA and linA' genes. Sequences homologous to linB were found in 14 other clinical isolates of E. faecium, indicating the spread of the resistance trait in this species.

Biosynthesis of marcfortine A.

This report describes the results of a biosynthesis study of marcfortine A (MA). We report here that MA is derived from methionine, tryptophan, lysine and two isoprene units, the latter two being derived from acetic acid. From the 13C enrichment pattern of the pipecolic acid moeity we further conclude that this unit is derived from lysine via alpha-ketoglutarate. Therefore, we have accounted for the biogenesis of every carbon atom of MA and established the biosynthetic pathway for the pipecolic acid moiety of MA.

Isolation and identification of 3-propylidene-delta 1-pyrroline-5-carboxylic acid, a biosynthetic precursor of lincomycin.

An accumulated lincomycin intermediate in UC 8292, a lincomycin nonproducing strain of Streptomyces lincolnensis, has been isolated and purified by employing an assay system based on complementation of UC 11066, another lincomycin nonproducing strain of S. lincolnensis. The structure of the purified intermediate is shown to be 3-propylidene-delta 1-pyrroline-5-carboxylic acid, or 1, 2, 3, 6-tetradehydro-propylproline by mass spectrometry and NMR spectroscopic studies. Based on the structure of this newly found intermediate, a biosynthetic pathway for propylproline is proposed as tyrosine-->L-3-hydroxytyrosine (Dopa)-->-->-->-->3-propylidene-delta 1-pyrroline-5-carboxylic acid-->3-propyl-delta 2-pyrroline-5-carboxylic acid-->propylproline.

Microbial O-carbamylation of novobiocin.

Novobiocin was inactivated by Streptomyces niveus US 2094 in fermentation. The inactivation product was isolated and characterized by NMR and MS as 2"-O-carbamylnovobiocin. The MICs of novobiocin and 2"-O-carbamylnovobiocin were determined for S. niveus strains.

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.

Structure elucidation of ficellomycin.

The structure of ficellomycin, an antibiotic previously discovered by Argoudelis et al., is elucidated as valyl-2-[4-guanidyl-1-azabicyclo[3.1.0]hexan-2-yl]glycine (1) by NMR, MS, and derivatization studies. The 1-azabicyclo[3.1.0]hexane moiety in 1 represents an unusual ring system making ficellomycin a unique natural product compound.