Emerging multiple mutations and high-level fluoroquinolone resistance in methicillin-resistant Staphylococcus aureus isolated from ocular infections.

We investigated the prevalence of mutations in the gyrA, gyrB, grlA, and grlB genes in 21 clinical isolates of methicillin-resistant Staphylococcus aureus recovered from ocular infections. These isolates were classified into 16 groups on the basis of pulsed-field gel electrophoresis (PFGE) patterns. Among these nonidentical strains, 9 different combinations of mutations were observed. We observed an unusually high frequency of mutations: 10 (62.5%) of the isolates had four or five mutations, 5 (31.3%) of the isolates had double mutations, and 1 isolate had a single mutation. Different mutations were detected in strains of the same PFGE type, and this is consistent with the independent acquisition of mutations. The minimun inhibitory concentrations values ranged from 0.5 to 512 microg/mL for levofloxacin and 0.125 to 128 microg/mL for gatifloxacin. Only 1 isolate was found to be susceptible to both agents. These results suggest that application of high concentration fluoroquinolone ophthalmic solutions selects for isolates presenting such mutations that may result in a high level of resistance to fluoroquinolones.

Modulation by the steroid/thyroid hormone superfamily of TGF-beta-stimulated VEGF release from vascular smooth muscle cells.

We previously reported that transforming growth factor-beta (TGF-beta) stimulates the release of vascular endothelial growth factor (VEGF) from aortic smooth muscle A10 cells via activation of p38 mitogen-activated protein (MAP) kinase. In the present study, we investigated whether nuclear hormone receptor superfamily members affect TGF-beta-stimulated VEGF release from A10 cells. Retinoic acid or 1,25-dihydroxyvitamin D3 enhanced TGF-beta-induced VEGF release in a concentration-dependent manner, whereas dexamethasone or corticosterone suppressed TGF-beta-induced VEGF release. 1,25-Dihydroxyvitamin D3 and TGF-beta stimulated phosphorylation of p38 MAP kinase in an additive manner. SB203580, an inhibitor of p38 MAP kinase, decreased the VEGF release induced by TGF-beta or 1,25-dihydroxyvitamin D3. However, retinoic acid, dexamethasone, or corticosterone did not affect phosphorylation of p38 MAP kinase. These results indicate that retinoic acid, 1,25-dihydroxyvitamin D3, and glucocorticoids affect TGF-beta-stimulated VEGF release from aortic smooth muscle cells. The stimulatory effect of 1,25-dihydroxyvitamin D3 occurs, in part, via modification of TGF-beta-induced activation of p38 MAP kinase.

Methotrexate suppresses inflammatory agonist induced interleukin 6 synthesis in osteoblasts.

OBJECTIVE: Interleukin 6 (IL-6) is a pleiotropic cytokine that plays a crucial role in the pathogenesis of rheumatoid arthritis (RA). In bone metabolism, it is known that IL-6 is produced and secreted by osteoblasts, and that IL-6 induces osteoclast formation and stimulates bone resorption. Various bone inflammatory agonists such as tumor necrosis factor-alpha (TNF-alpha), IL-1alpha, prostaglandin D2 (PGD2), PGE2, and PGF2alpha, which play important roles in the pathogenesis of RA, induce IL-6 synthesis in osteoblast-like MC3T3-E1 cells. Low dose methotrexate (MTX) is currently used for treatment of patients with RA. We investigated the effect of MTX on IL-6 synthesis induced by these agents in MC3T3-E1 cells. METHODS: Cultured cells were pretreated with various doses of MTX, and then stimulated by these inflammatory agonists. The IL-6 in the conditioned medium was measured by IL-6 enzyme immunoassay. RESULTS: MTX significantly suppressed IL-6 synthesis stimulated by these agonists in a dose-dependent manner, although MTX alone had no effect on the levels of IL-6. In addition, MTX significantly inhibited the enhancement by IL-17 of TNF-alpha-stimulated IL-6 synthesis. MTX reduced the levels of IL-6 induced by 12-O-tetradecanoylphorbol 13-acetate, a direct activator of protein kinase C (PKC), suggesting that MTX inhibits PKC signals for IL-6 synthesis. CONCLUSION: MTX suppresses IL-6 synthesis stimulated by various inflammatory agonists in osteoblasts.

Akt regulates thrombin-induced HSP27 phosphorylation in aortic smooth muscle cells: function at a point downstream from p38 MAP kinase.

We previously reported that p38 MAP kinase takes part in thrombin-induced HSP27 phosphorylation in aortic smooth muscle A10 cells. In the present study, we investigated whether Akt is involved in the phosphorylation of HSP27 and the role of adenylyl cyclase-cAMP system. Thrombin time-dependently induced the phosphorylation of heat shock protein 27 (HSP27) and Akt in aortic smooth muscle A10 cells. SB203580, a p38 MAP kinase inhibitor, significantly suppressed the thrombin-induced phosphorylation of Akt and the Akt inhibitor suppressed the phosphorylation of HSP27. Furthermore, the thrombin-induced phosphorylation of HSP27, p38 MAP kinase and Akt were decreased by dibutyryl-cAMP (DBcAMP). These results strongly suggest that Akt functions the thrombin-induced phosphorylation of HSP27 at a point downstream from p38 MAP kinase in aortic smooth muscle cells and the adenylyl cyclase-cAMP system is upstream regulator of the HSP27 phosphorylation in these cells.

Lytic enzyme, labiase for a broad range of Gram-positive bacteria and its application to analyze functional DNA/RNA.

The lytic activity of labiase and achromopeptidase for bacterial DNA/RNA extraction were compared. Rapid lysis of many bacterial strains was observed with labiase followed by SDS treatment. Both labiase and achromopeptidase showed high lytic activity against bacterial strains with the A1alpha chemotype (e.g., Aerococcus viridans) and the A3alpha chemotype (e.g., Staphylococcus epidermidis) for cell wall peptidoglycan structures. The lytic activity of labiase was higher than that of achromopeptidase against strains with the A1gamma chemotype (e.g., Bacillus subtilis). The activity of labiase was not detrimentally affected with increasing NaCl concentration. Labiase lysates were successfully used for rapid extraction of DNA and RNA, whereas achromopeptidase lysates degraded RNA. The DNA and RNA obtained were successfully used for 16S rRNA amplification and real-time RT-PCR detection. It is concluded that labiase is useful for rapid lysis of a wide variety of Gram-positive bacteria and can be used for DNA/RNA isolation protocols.

PPAR-gamma ligands up-regulate basic fibroblast growth factor-induced VEGF release through amplifying SAPK/JNK activation in osteoblasts.

We previously reported that basic fibroblast growth factor (FGF-2) activates stress-activated protein kinase/c-Jun N-terminal kinase (SAPK/JNK) and p44/p42 mitogen-activated protein (MAP) kinase resulting in the stimulation of vascular endothelial growth factor (VEGF) release in osteoblast-like MC3T3-E1 cells and that FGF-2-activated p38 MAP kinase negatively regulates the VEGF release. In the present study, we investigated the effects of ciglitazone and pioglitazone, peroxisome proliferator-activated receptor-gamma (PPAR-gamma) ligands, on the VEGF release by FGF-2 in MC3T3-E1 cells. The FGF-2-induced VEGF release was significantly enhanced by ciglitazone. The amplifying effect of ciglitazone was dose-dependent between 0.1 and 10 microM. Pioglitazone had a similar effect on the VEGF release. GW9662, an antagonist of PPAR-gamma, reduced the effects of ciglitazone and pioglitazone. Ciglitazone or pioglitazone markedly enhanced the phosphorylation of SAPK/JNK induced by FGF-2 without affecting both the FGF-2-induced phosphorylation of p44/p42 MAP kinase and p38 MAP kinase. GW9662 markedly reduced the amplification by ciglitazone of the SAPK/JNK phosphorylation. Taken together, these results strongly suggest that PPAR-gamma ligands up-regulate FGF-2-stimulated VEGF release resulting from amplifying activation of SAPK/JNK in osteoblasts.

Adenylyl cyclase-cAMP system inhibits thrombin-induced HSP27 in vascular smooth muscle cells.

We previously reported that thrombin stimulates the induction of heat shock protein (HSP) 27 via p38 mitogen-activated protein (MAP) kinase activation in aortic smooth muscle A10 cells. In the present study, we investigated the effect of the adenylyl cyclase-cAMP system on the thrombin-stimulated induction of HSP27 in A10 cells. Forskolin, a direct activator of adenylyl cyclase, reduced the thrombin-induced p38 MAP kinase phosphorylation, and significantly suppressed the thrombin-stimulated accumulation of HSP27. However, dideoxyforskolin, a forskolin derivative that does not activate cAMP, failed to suppress the HSP27 accumulation. Furthermore, dibutyryl-cAMP (DBcAMP), a permeable analog of cAMP, significantly suppressed the accumulation of HSP27. On the other hand, calphostin C, an inhibitor of protein kinase C (PKC), reduced the thrombin-induced p38 MAP kinase phosphorylation, and significantly suppressed the thrombin-stimulated accumulation of HSP27. Moreover, forskolin reduced the p38 MAP kinase phosphorylation induced by the 12-O-tetradecanoylphorbol-13-acetate (TPA), a PKC-activating phorbol ester, and significantly suppressed the TPA-stimulated accumulation of HSP27. These results indicate that adenylyl cyclase-cAMP system has an inhibitory role in thrombin-stimulated HSP27 induction in aortic smooth muscle cells, and the effect seems to be exerted on the thrombin-induced PKC- p38 MAP kinase signaling pathway.

Possible involvement of p44/p42 MAP kinase in retinoic acid-stimulated vascular endothelial growth factor release in aortic smooth muscle cells.

Retinoic acid modulates cell growth and differentiation of the vascular system. Vascular endothelial growth factor (VEGF) is known as a vascular permeability factor and a potent mitogen for vascular endothelial cells. In the present study, we investigated whether retinoic acid induces VEGF release in aortic smooth muscle A10 cells and if so, the mechanism of VEGF release. Retinoic acid stimulated VEGF release dose-dependently over the range 0.1 nM-0.1 microM. The retinoic acid-stimulated VEGF release was significantly reduced by actinomycin D. Retinoic acid induced the phosphorylation of p44/p42 mitogen-activated protein (MAP) kinase but not p38 MAP kinase or stress-activated protein kinase/c-Jun N-terminal kinase among the MAP kinase superfamily. This effect of retinoic acid was dose-dependent (30 nM-5 microM) and the maximum effect was observed at 0.3 microM. The retinoic acid-stimulated release of VEGF was significantly reduced by PD98059 and U0126, specific MEK inhibitors, which attenuated the retinoic acid-induced phosphorylation of p44/p42 MAP kinase. These results strongly suggest that retinoic acid stimulates the release of VEGF in a p44/p42 MAP kinase-dependent manner in aortic smooth muscle cells.

High-performance liquid chromatographic assay of clonazepam in human plasma using a non-porous silica column.

A new HPLC method has been developed for measuring clonazepam (CZP) in plasma, using a reversed-phase non-porous silica column packed with 2 microm particles. CZP in plasma was first purified with a column extraction technique and injected onto a non-porous silica column. The calibration curve was linear from 5-200 ng/ml. The recoveries of CZP added to plasma were more than 94.0%, with a coefficient of variation in the range of 5.1-13.8%. We developed a rapid routine method using a non-porous silica column that was accurate and improved solvent consumption in the measurement of CZP.

Sialic acid 9-O-acetylesterase catalyzes the hydrolyzing reaction from alacepril to deacetylalacepril.

PURPOSE: In this work, the alacepril thiolesterase, which catalyzes the hydrolyzing reaction of the thiolester linkage in alacepril and the conversion from alacepril to deacetylalacepril, was purified from rat liver cytosol and characterized. METHODS: A purification procedure for the thiolesterase consisted of ammonium sulfate fractionation and chromatographies with phenyl Sepharose CL-4B, Q Sepharose FF, ceramic hydroxylapatite, and phenyl Sepharose HP. The thiolesterase activity was assayed for alacepril as a substrate and the reaction product, deacetylalacepril, was measured using high-performance liquid chromatography. RESULTS: The purified thiolesterase is heterodimeric with a molecular mass of 29 and 36 kDa subunits as estimated by sodium dodecyl sulfate -polyacrylamide gel electrophoresis. N-terminal amino acid sequence of these subunits reveals that the thiolesterase is identical to sialic acid 9-O-acetylesterase. The thiolesterase hydrolyzes not only the thiolester bond in alacepril, spironolactone, and acetyl coenzyme A but also the carboxylester bond in alpha-naphtyl acetate. The alacepril thiolestrase activity is competitively inhibited by alpha-naphtyl acetate. CONCLUSION: The thiolesterase, i.e., sialic acid 9-O-acetylesterase, seems to be involved in the metabolism of certain drugs such as alacepril and spironolactone. However, drugs having ester-type and amide-type linkages, for example dilazep. aniracetam, and benazepril, are not substrates for the thiolestrase.

Thrombin stimulates dissociation and induction of HSP27 via p38 MAPK in vascular smooth muscle cells.

We investigated the effects of thrombin on the induction of heat shock proteins (HSP) 70 and 27, and the mechanism behind the induction in aortic smooth muscle A10 cells. Thrombin increased the level of HSP27 but had little effect on the level of HSP70. Thrombin stimulated the accumulation of HSP27 dose dependently between 0.01 and 1 U/ml and cycloheximide reduced the accumulation. Thrombin stimulated an increase in the level of HSP27 mRNA and actinomycin D suppressed the thrombin-increased mRNA level. Thrombin induced the phosphorylation of p38 mitogen-activated protein kinase (MAPK). The HSP27 accumulation by thrombin was reduced by SB-203580 and PD-169316 but not by SB-202474. SB-203580 and PD-169316 suppressed the thrombin-induced phosphorylation of p38 MAPK. SB-203580 reduced the thrombin-increased level of HSP27 mRNA. Dissociation of the aggregated HSP27 to the dissociated HSP27 was induced by thrombin. Dissociation was inhibited by SB-203580. Thrombin induced the phosphorylation of HSP27 and the phosphorylation was suppressed by SB-203580. These results indicate that thrombin stimulates not only the dissociation of HSP27 but also the induction of HSP27 via p38 MAPK activation in aortic smooth muscle cells.

Substrate specificity of human 3(20)alpha-hydroxysteroid dehydrogenase for neurosteroids and its inhibition by benzodiazepines.

In this report, we compared kinetic constants and products in the reduction of the neurosteroids, 3alpha,5alpha-tetrahydroprogesterone (3alpha,5alpha THP) and 3alpha,5alpha-tetrahydrodeoxycorticosterone (3alpha,5alpha-THDOC), and their precursors, 5alpha-dihydroprogesterone (5alpha-DHP), 5alpha-dihydrodeoxycorticosterone (5alpha-DHDOC) and progesterone, by three isoenzymes (AKR1C1, AKR1C2 and AKR1C3) of human 3alpha-hydroxysteroid dehydrogenase. AKR1C1 efficiently reduced 3alpha,5alpha-THP, 5alpha-DHP and progesterone to their 20alpha-hydroxy metabolites, and slowly converted 5alpha-DHDOC to 3alpha,5alpha-THDOC. AKR1C2 exhibited low 20-ketoreductase activity for 3alpha,5alpha-THP and moderate 3-ketoreductase activity for 5alpha-DHP and 5alpha-DHDOC. 3alpha,5alpha-THDOC was not reduced by the two isoenzymes. No significant activity for the steroids was detected with AKR1C3. The results suggest that AKR1C2 is involved in the neurosteroid synthesis, but AKR1C1 decreases the neurosteroid concentrations in human brain by inactivating 3alpha,5alpha-THP and eliminating the precursors from the synthetic pathways. In addition, we found that the several benzodiazepines inhibited the three isoenzymes noncompetitively with respect to the substrate. Although cloxazolam was a potent and specific inhibitor of AKR1C3, diazepam, estazolam, flunitrazepam, medazepam and nitrazepam, that inhibited AKR1C1 and AKR1C2, may influence the neurosteroid metabolism.