The metabolic responses of equine articular cartilage to incubation with bacterial lipopolysaccharide (LPS) were studied, using explant cultures of articular cartilage obtained from the metatarsophalangeal joints of 15 horses, age of which ranged from 3 months to 20 years. For comparison, explants were also established from the metatarsophalangeal joints of 3 calves. Explants were cultured for 3 days in medium containing various concentrations of LPS from 0 (control) to 100 microgram/ml. Glycosaminoglycan (GAG) released during the 3-day incubation was determined by a spectrophotometric assay, using the dye 1,9-dimethylmethylene blue. Newly synthesized GAG content was assayed by measuring [35S]sulfate incorporation during a 3-hour pulse labeling period. In addition, prostaglandin E2 (PGE2) synthesis was quantified, using a [3H]PGE2 radioimmunoassay kit and magnetic separation. Finally, explants from 3 animals were used to evaluate the effect of supplementing culture medium with 5% serum on the response of explants to LPS, and explants from 1 horse were used to compare responses to stimulation with LPS derived from 2 bacterial sources. Equine explants cultured with bacterial LPS had a dose-dependent decrease in synthesis and increase in release of GAG, and these responses were significantly (P < 0.0001) greater in explants from younger horses. In addition, equine explants had a significant (P = 0.0001) dose-dependent increase in concentration of PGE2 released into the culture medium in response to incubation with LPS. Comparison of data for GAG synthesis from equine and bovine explants revealed a significant (P = 0.025) difference in responsiveness to LPS between the 2 species. Equine explants tended to have a greater suppression of GAG synthesis in response to incubation with increasing concentrations of LPS than did age-corrected bovine samples.

Middle carpal cartilage explants from 4 horses with mild osteoarthritis involving that joint were maintained in tissue culture to test the effects of a polysulfated glycosaminoglycan (PSGAG) on proteoglycan synthesis and degradation. Cultures were exposed to 0.025 or 25 mg of PSGAG/ml for 48 hours, after which the medium was replaced with medium containing similar doses of PSGAG and 35S. Subsequently, the sulfated proteoglycan content of the medium and extracts of the explants was measured. Gel filtration chromatography was used to estimate the size and to purify the principal, large proteoglycan monomer, which was further characterized by digestion, using glycosidic enzymes. In a second experiment, explants were incubated with 35S for 48 hours, and were subsequently exposed to the same concentrations of the PSGAG for an additional 48 hours. The amount of remaining labeled proteoglycan was determined for culture medium and cartilage extracts. Gel filtration chromatography was used to assess the hydrodynamic size of the large proteoglycan monomer. Aliquots of proteoglycans from the second experiment were incubated in high-molecular weight hyaluronate and chromatographed to assess reaggregation. Polysulfated glycosaminoglycan caused a significant (P < 0.04) decrease in sulfated proteoglycan synthesis by cartilage explants. Radioactive proteoglycan content in explants labeled prior to exposure to PSGAG were similar. Large proteoglycan monomer size was similar in both experiments (median partition coefficient [K(AV)] = 0.40), and was not influenced by PSGAG treatment. Prelabeled explants exposed to hyaluronate and chromatographed under associative conditions had similar proportions of the radiolabel eluting as proteoglycan aggregate. Enzymatic digestion of newly synthesized large monomer revealed a mild dose-dependent increase in the proportion of keratan sulfate substitution on core protein. It was concluded that PSGAG in vitro, at the dosages evaluated, caused a decre.

The antibiotic polymyxin B sulfate is a cationic polypeptide with a unique cyclical configuration and distinct cationic characteristics. In this investigation, polymyxin B was evaluated to determine its ability to prevent synthesis of lactic acid and tumor necrosis factor-alpha (TNF-alpha) by lipopolysaccharide-stimulated strain RAW 2647 macrophage-like cell populations. In this context, gradient concentrations of polymyxin B were formulated in the presence of fixed concentrations of lipopolysaccharide fractions from Escherichia coli (B4:0111), E. coli (J5), Klebsiella pneumoniae, Pseudomonas aeruginosa, Salmonella minnesota, and S. typhimurium (Re). Quantitation of TNF-alpha was established by the application of a tissue culture-based biological assay system, using the WEHI 164 clone 13 indicator cell line. Investigations also included evaluation of the ability of gradient concentrations of lipopolysaccharide fractions from E. coli (B4:0111), E. coli (J5), K. pneumoniae, P. aeruginosa, S. minnesota, and S. typhimurium (Re) to form a complex with polymyxin B. This was established through application of high-performance thin-layer chromatography techniques. On the basis of the known molecular characteristics of lipopolysaccharide, its lipid A-core subfractions, and polymyxin B, these results imply that cytoprotective properties of polymyxin B are attributable to direct interaction and subsequent complex formation. More specifically, the mechanism by which polymyxin B exerts affinity for lipopolysaccharide fractions is proposed to occur through attractive ionic interactions established between the cationic diaminobutyric acid residues of polymyxin B and the mono- or diphosphate group(s) of the lipid A-core moiety. It is highly probable that this molecular phenomenon is accompanied by hydrophobic interactions established between the terminal methyloctanoyl or methylheptanoyl groups of polymyxin B and the saturated carbon chains of the lipid A-core subfraction of lipopolysaccharide fractions.

In the present study we have analyzed the characteristics and distribution of the mu-opioid receptor(MOR) by raising anti-peptide antisera to the C-terminal peptide of MOR. The antisera against MOR was produced in New Zealand White rabbit against 15 residue corresponding to amino acids, 384-398of the cloned rat MOR. The antigenic peptide was synthesized using an Applied Biosystems 432 solid-phase peptide aynthesizer. The specificity and identification fo the antisera were tested by analysisi fo transfected cells, epitope mapping and immunohistochemical method. COS-7 cells electroporated with MOR cDNA were used to evaluate the characteristics and subcellular distribution of MOR.MOR immunoreactivity was prodominent in the plasmalemma and subcellular compartments such as encoplasmicreticulum, Golgi apparatus and vesicle like structure. Furthermore, both tissue sections and transfected cell lines could be immunostained with these antisera and the immunoreactivity ws abolished when anti-MOR sera were preincubated with the peptide against which they wer raised. Based on epitope mapping analysis, all antisera appeared to have a similar epitope, which ws detemined to be within the last amino acid,391-398. Moreover, immunohistochemistry showed that MOR immunoreactivity was ovserved in many brain areas including cerebral cortex, striatum, hippocampus, locus coeruleus and the superficial laminae of the dorsal horn. These stained spinal cord and brain aras showed themirrored pattern observed in autoradiographic studies of mu-opioid binding as well as a pattern similar to that seen by in situ hybridization for MOR. Thus, several lines of evidence support the conclusion that the antisera produced in the present study most likely recognize mu-opioid receptor. These results suggest that MOR antisera may be utilized as useful tool to analyze the physiological and pharmacological studies for mu-opioid receptor in the future