The estimated number of employees in the United Stated screened annually for illicit drugs is approximately 20 million, with marijuana being the most frequently abused drug. Urine adulterants provide an opportunity for illicit drug users to obtain a false-negative result on commonly used primary drug screening methods such as the enzyme multiplied immunoassay technique and the fluorescence polarized immunoassay technique (FPIA). Typical chemical adulterants such as nitrites are easily detected or render the urine specimen invalid as defined in the proposed SAMHSA guidelines for specimen validity testing based on creatinine, specific gravity, and pH. Papain is a cysteine protease with intrinsic ester hydrolysis capability. The primary metabolite of the psychoactive chemical in marijuana, 11-norcarboxy-Δ9- tetrahydrocannibinol (THC-COOH), was assayed by FPIA in concentrations ranging from 25 to 500 ng/mL, at pH values ranging from 4.5 to 8, over the course of 3 days with papain concentrations ranging from 0 to 10 mg/mL. FPIA analysis of other frequently abused drugs: amphetamines, barbiturates, benzodiazepines, cocaine, opiates, and phencyclidine, along with gas chromatography-mass spectrometry (GC-MS) of THC-COOH and high-pressure liquid chromatography- ultraviolet detection (HPLC-UV) of nordiazepam was performed in order to determine if the mechanism of urine adulteration by papain was analyte specific. Control and adulterated urine specimens (n = 30) were assayed for creatinine, specific gravity, and pH to determine if papain rendered the specimens invalid based on the proposed SAMHSA guidelines. There was a direct pH, temperature, and time-dependent correlate between the increase in papain concentration and the decrease in THC-COOH concentration from the untreated control groups (p < 0.01). The average 72-h THC-COOH concentration decrease at pH 6.2 with a papain concentration of 10 mg/mL was 50%. Papain did not significantly decrease the concentration of the other drugs analyzed with the exception of nordiazepam. GC-MS of THC-COOH and HPLC-UV of nordiazepam revealed a 66% and 24% decrease in concentration of the respective analyte with 10 mg/mL papain after 24 h at room temperature (∼ 23°C). No adulterated specimens were rendered invalid based on the SAMHSA guidelines. Immediate FPIA analysis is suggested to minimize the interfering effects of papain with regards to primary drug screening.

No description available.

A new streptogramin antibiotic XRP 2868 was compared with quinupristin-dalfopristin for inhibitory activities against antibiotic-resistant Haemophilus influenzae, Staphylococcus aureus, and Streptococcus pneumoniae. In each organism examined, XRP 2868 had an IC50 that was twofold to fivefold lower than quinupristin-dalfopristin, for inhibition of cell viability, protein synthesis, and ribosomal subunit formation.

A prior study examining differences in the activities of erythromycin and azithromycin on cellular functions in the Gram-negative pathogen, Haemophilus influenzae, revealed a marked difference in their inhibitory activities. The study revealed that protein synthesis and 50S ribosomal subunit assembly were equal targets for inhibition by azithromycin while erythromycin was a preferential inhibitor of translation. This contrast in inhibitory activities stimulated a comparative analysis of three additional antibiotics: clarithromycin, flurithromycin and roxithromycin. Each compound was tested over a concentration range for inhibitory effects on cellular processes. Clarithromycin was the most effective inhibitor of protein synthesis with an IC50 of 5.6 μg/mL, followed by flurithromycin at 6 μg/mL, and roxithromycin at 9 μg/mL. IC50 values for antibiotic effects on viable cell counts and growth rates were similar to those obtained for protein synthesis. Flurithromycin had the strongest effect on 50S ribosomal subunit formation with an IC50 of 8 μg/mL, followed by clarithromycin and roxithromycin, at 9.0 μg/mL and 12.5 μg/mL respectively. 30S ribosomal subunit formation in cells treated with flurithromycin and roxithromycin was also reduced to some extent. Pulse-and-chase labeling kinetics examining subunit assembly rates verified the slower synthesis rate of the subunits in the presence of each macrolide. The results are discussed in terms of structural differences of these macrolides and their differential inhibitory effects on both cellular targets.

Several reports in the literature have described a differential sensitivity to ketolide antibiotics in ermB strains of Streptococcus pyogenes and Streptococcus pneumoniae resistant to erythromycin. Strains of S. pyogenes and S. pneumoniae carrying different erm gene alleles were examined for their susceptibility to the ketolide antibiotics cethromycin (ABT-773) and telithromycin. The effect of the antibiotics on cell growth and viability was assessed as were effects on protein synthesis and 50S ribosomal subunit formation. The susceptibility of wild-type strains of both organisms was compared with effects in strains containing the ermA and ermB methyltransferase genes. A wild-type antibiotic-susceptible strain of S. pyogenes was comparable to an ermA strain of the organism in its ketolide sensitivity, with IC 50 values for 50% inhibition of protein synthesis and 50S ribosomal subunit formation of 10 ng/mL for cethromycin and 16 ng/mL for telithromycin. An S. pneumoniae strain with the ermB gene and an S. pyogenes strain with the ermA gene were also similar in their sensitivity to ketolide inhibition. IC 50 values for inhibition of translation and subunit formation in S. pneumoniae (ermB) were 30 ng/mL and 55 ng/mL and for the ermA strain of S. pyogenes they were 15 ng/mL and 35 ng/mL respectively. By contrast, an S. pyogenes ermB strain was significantly more resistant to both ketolides, with IC50 values for inhibition of 50S synthesis of 215 and 380 ng/mL for the two ketolides. Experiments were conducted to examine ribosome synthesis and translational activity in the two ermB strains at intervals during growth in the presence of each antibiotic. Cell viability and 50S subunit formation were dramatically reduced in the S. pneumoniae strain during continued growth with either drug. By contrast, the ketolides had little effect on the S. pyogenes strain growing with the antibiotics. The results indicate that ketolides have a reduced inhibitory effect on translation and 50S subunit synthesis in S. pyogenes with the ermB gene compared with the other strains examined.

Normal lymphoid tissue development and function depend upon chemokine-directed cell migration. Since chemokines signal through heterotrimeric G-protein-coupled receptors, RGS proteins, which act as GTPase-activating proteins for Gα subunits, likely fine tune the cellular responses to chemokines. Here we show that Rgs1-/- mice possess B cells that respond excessively and desensitize improperly to the chemokines CXCL12 and CXCL13. Many of the B-cell follicles in the spleens of Rgs1 -/- mice have germinal centers even in the absence of immune stimulation. Furthermore, immunization of these mice leads to exaggerated germinal center formation; partial disruption of the normal architecture of the spleen and Peyer's patches; and abnormal trafficking of immunoglobulin-secreting cells. These results reveal the importance of a regulatory mechanism that limits and desensitizes chemokine receptor signaling.

HlyC, hemolysin-activating lysine acyltransferase, catalyzes the acylation (from acyl-ACP) of Escherichia coli prohemolysin (proHlyA) on the ε-amino groups of specific lysine residues, Lys564 and Lys690 of the 1024-amino acid primary structure, to form hemolysin (HlyA). The amino acid sequences flanking the two acylation sites are not homologous except that each has a glycine residue immediately preceding the lysine which is acylated; there are, however, numerous GK sequences throughout proHlyA that are not acylation sites. The substrate specificity of acylation was examined. ProHlyA-derived structures, altered by substantial deletions and separation of the acylation sites into two different peptides and site-directed mutation analyses of acylation sites, often served as internal protein acylation substrates, and the kinetics of the acylations were measured. The two sites of acylation of proHlyA functioned independently of one another with HlyC; there did not appear to be a common HlyC binding site or processivity of the enzyme between the sites. Acyl-HlyC was likely the enzyme form that interacted with the final acylation substrate. In a variety of constructs, the two acylation sites had similar Km values, but their Vmax values and catalytic efficiencies as substrates differed. Internal protein acylation was inhibited by specific small peptides mimicking the primary structure of each acylation site except that the crucial lysines were replaced with arginines; similar small peptides containing the crucial lysine, however, were not acylated.

Osteopontin (OPN), also called cytokine Eta-1, expressed in the myocardium co-incident with heart failure plays an important role in post myocardial infarction (MI) remodeling by promoting collagen synthesis and accumulation. Angiotensin II (Ang II) and inflammatory cytokines are increased in the heart following MI. We studied the involvement of mitogen-activated protein kinases (ERK1/2, JNKs, p38 kinase) and reactive oxygen species (ROS) in Ang II- and cytokine-induced OPN gene expression in adult rat cardiac fibroblasts. Ang II alone increased OPN mRNA (3.3 ± 0.3-folds; P < 0.05; n = 7), while interleukin-1β (IL-1β), tumor necrosis factor (TNF-α), and interferon-γ (IFN-γ) had no effect. A combination of Ang II with IL-1β or TNF-α, not IFN-γ, increased OPN mRNA more than Ang II alone. Nitric oxide donor, S-nitrosoacetylpenicillamine (SNAP), alone or in combination with Ang II had no effect. Diphenylene iodonium (DPI), inhibitor of NAD(P)H oxidase, and tiron, superoxide scavenger, inhibited Ang II- and Ang II+ IL-1β-stimulated inzcreases in OPN mRNA. Ang II activated ERK1/2 within 5 min of treatment, not JNKs. IL-1β activated ERK1/2 and JNKs within 15 min of treatment. A combination of Ang II and IL-1β activated ERK1/2 within 5 min of treatment. None of these stimuli activated p38 kinase. DPI almost completely inhibited Ang II + IL-1β-stimulated activation of ERK1/2, while partially inhibiting JNKs. PD98059, ERK1/2 pathway inhibitor, and SP600125, JNKs inhibitor, partially inhibited Ang II + IL-1β-stimulated increases in OPN mRNA. A combination of PD98059 and SP600125 almost completely inhibited Ang II + IL-1β-stimulated increases in OPN mRNA. Thus, Ang II alone increases OPN expression, while IL-1β and TNF-α act synergistically with Ang II to increase OPN mRNA possibly via NO independent mechanisms. The synergistic increase in OPN mRNA involves ROS-mediated activation of ERK1/2 and JNKs, not P38 kinase, pathways in cardiac fibroblasts.

Sevoflurane is a nonflammable general anesthetic administered by inhalation of vaporized liquid that rapidly partitions out of aqueous biological matrices into a gaseous phase because of its volatility and hydrophobicity. We describe a headspace analysis of sevoflurane that can be performed without the use of an expensive automated headspace analyzer. Sevoflurane standards (0-109 mg/L) and quality control specimens (12.2 and 72.9 mg/L) were prepared and quantitated on an intraday and interday basis. Headspace gas was manually injected (150°C) with a 2.5-mL gas-tight syringe into a Perkin-Elmer model 8500 gas Chromatograph equipped with a 6-ft × 2-mm i.d. glass column (100°C) containing 0.2% Carbowax 1500 on Carbopak C packing with a flame-ionization detector (200°C), which allowed for elution of the internal standard, 1 -propanol (1.56 min), and sevoflurane (2.92 min). Linear regression of the peak-area ratios of sevoflurane to 1-propanol (6.38 g/L), versus the sevoflurane concentrations yielded an average intraday correlation coefficient of 0.989 (S.D. = 0.003, n = 5) and mean quality control specimen values of 14.19 mg/L (C.V. = 5.1 %, n = 5) and 66.72 mg/L (C.V. = 3.3%, n = 5). The average interday standard curve correlation coefficient was 0.987 (S.D. = 0.01, n = 5), and the mean quality control specimen values were 12.22 mg/L (C.V. = 13.7%, n = 5) and 74.27 mg/L (C.V. = 8.7%, n = 5). The Chromatographic method described produced accurate and reproducible results with a simple on-column headspace gas injection. This method allows for quantitation of sevoflurane in various biological matrices by Chromatographic separation of the headspace gas in a sealed specimen container.

No description available.