Rapid Determination of Cocaine and Its Metabolite Benzoylecgonine in Hair by LC-MS/MS.

OBJECTIVES: To establish a rapid determination method with LC-MS/MS for cocaine and its metabolite benzoylecgonine in hair. METHODS: Deuterated internal standards （cocaine-D3 and benzoylecgonine-D8） were added to the decontaminated hair. After the extraction by ultrasonication with methanol, the compounds were separated by the Restek Allure PFP propyl column, and cocaine and benzoylecgonine were simultaneously analysed in multiple reaction monitoring mode. RESULTS: The cocaine and benzoylecgonine in hair showed a good linearity in the range of mass fraction between 0.02 and 10.00 ng/mg with the limits of detection of 0.01 ng/mg. CONCLUSIONS: The developed method is simple and rapid with a good selectivity, which is suitable for the determination of cocaine and its metabolite benzoylecgonine in hair.

Human cytomegalovirus pp65 lower matrix protein: a humoral immunogen for systemic lupus erythematosus patients and autoantibody accelerator for NZB/W F1 mice.

Both the infection of human cytomegalovirus (HCMV) and the immunization of its recombinant glycoprotein (gB) in mice have been known to induce autoimmunity, resulting in symptoms similar to those of human systemic lupus erythematosus (SLE). Research has also found that the murine cytomegalovirus (MCMV)-specific monoclonal antibody (mAb) is able to react with a human U1-70K-like autoantigen. To investigate HCMV involvement in autoimmunity, we analysed the humoral responses to HCMV by autoimmune patients and normal adults. Our studies show unambiguously that sera from SLE patients exhibited an elevated IgG titre to HCMV when compared with those observed in controls and other connective tissue disease (CTD) patients (P < 0.001). The IgM titres to HCMV and IgG to HBV were evaluated, and no significant differences were noted among all testing groups. In addition to initiating T cell activity, as reported by many investigators, we found that the HCMV pp65 antigen (also known as lower matrix protein) was able to induce humoral responses in SLE patients. Immunoblot assays showed that 82.56% of sera from SLE patients reacted with the HCMV pp65 antigen, but only 11.11%, 23.53% and 31.17% of patients from normal control, rheumatoid arthritis (RA) and CTD patients, respectively, reacted to it. Unlike HCMV pp65, HCMV pp150 induced B cell activity in most collected sera (92.22%-98.04%). Finally, female NZB/W F1 mice immunized with plasmids encoding HCMV pp65 open reading frame (pcDNApp65) developed an early onset of autoantibody activity and more severe glomerulonephritis. Thus, we conclude that the HCMV pp65 antigen triggers humoral immunity in SLE patients and autoimmune-prone mice and that it could very well exacerbate the autoimmune responses in susceptible animals.

Non-steroidal anti-inflammatory drugs inhibit matrix metalloproteinase-2 expression via repression of transcription in lung cancer cells.

Recent studies show that up-regulation of cyclooxygenase-2 (COX-2) in human cancer cells induces activation of matrix metalloproteinases (MMPs) and increase of metastatic potential. In this study, we investigate the effect of a COX-2 selective inhibitor, NS398, on the expression and enzymatic activity of MMPs in human lung cancer cells. We found that NS398 inhibited MMP-2, not MMP-9, mRNA expression. NS398 also reduced the amount of MMP-2, not MMP-9, released into the medium. Additionally, this COX-2 inhibitor attenuated the degrading activity of MMP-2 as demonstrated by gelatin zymography. Investigation of cellular MMP-2 by Western blotting indicated that synthesis and processing of MMP-2 was significantly suppressed by NS398. We performed promoter activity assay to address whether NS398 might affect MMP-2 gene transcription. Our results indicated that NS398 directly inhibited MMP-2 promoter activity. However, the inhibitory effect of NS398 is not fully dependent on inhibition of COX-2 because a high concentration of NS398 was needed to suppress MMP-2 expression and addition of prostaglandin E2 only partially reversed the action of NS398. Moreover, a non-selective COX inhibitor indomethacin also suppressed the expression of MMP-2. Taken together, these results indicate that non-steroidal anti-inflammatory drugs suppress MMP-2 expression via repression of transcription and support the notion that COX inhibitors may be useful in inhibition and/or prevention of metastasis.

Induction of p27(KIP1) as a mechanism underlying NS398-induced growth inhibition in human lung cancer cells.

Increased expression of cyclooxygenase-2 (COX-2) causes enhanced production of prostaglandins, which are emerging as important mediators of growth stimulation of cancer cells. Overexpression of COX-2 has been found in human non-small cell lung cancer tissues and cell lines. In vitro and in vivo studies showed that nonselective cyclooxygenase inhibitors (like aspirin and indomethacin) may suppress growth of lung cancer cells and may prevent lung tumorigenesis induced by the tobacco-specific carcinogens. However, the molecular mechanisms that mediated the anticancer action of these inhibitors are not well defined. In this study, we examined the effect of a specific COX-2 inhibitor, N-(2-cyclohexyloxy-4-nitrophenyl)methanesulfonamide (NS398), on high COX-2-expressing A549 lung cancer cells. Our results indicated that NS398 inhibited prostaglandin E(2) synthesis and induced G(1) growth arrest in these cells. NS398 specifically up-regulated cyclin-dependent kinase inhibitor p27(KIP1), whereas the expressions of G(1)-acting cyclins and cyclin-dependent kinases were not changed. Additionally, NS398 effectively suppressed cyclin E-associated kinase activity in A549 cells. The molecular mechanism responsible for the induction of p27(KIP1) by NS398 was characterized. We found that NS398 did not induce p27(KIP1) through transcriptional activation because this drug could not stimulate the p27(KIP1) promoter. Metabolic labeling experiments showed that the synthesis rate of p27(KIP1) protein was not altered by NS398. Conversely, pulse-chase assays demonstrated that degradation of p27(KIP1) protein was obviously reduced in NS398-treated cells. We conclude that NS398 enhances p27(KIP1) expression via post-translational regulation, and our results provide a new mechanism by which specific COX-2 inhibitors suppress proliferation of cancer cells.