Is platelet-activating factor (PAF-acether) synthesis by murine peritoneal cells (PC) a two-step process?

PAF-acether (1-O-alkyl-2-acetyl-sn-glyceryl-3-phosphorylcholine) is released from several cell sources simultaneously with an inactive non-acylated compound (1-O-alkyl-glyceryl-3-phosphorylcholine) (lyso-PAF-acether). Formation of the latter probably results from the activation of a phospholipase A₂ (PLA₂). Indeed, the PLA₂ inhibitors, bromophenacyl bromide (BPB), mepacrine, 874CB (100 M), and EDTA (5 mM), blocked the zymosan-induced release of PAF-acether from PC. EDTA and BPB also markedly reduced the release of lyso-PAF-acether. PAF-acether formation could involve a metabolic step linking the acetyl moiety to the 2nd carbon of lyso-PAF-acether. To verify this hypothesis, acetyl coenzyme A (acetyl-CoA) was added to stimulated PC. This enhanced the release of PAF-acether in a dose-dependent fashion from 1 M acetyl-CoA to reach a maximal increase –200%—at 100 M. Furthermore, using³H acetyl-CoA, incorporation of labelled acetate into PAF-acether was suggested by (1) identical chromatographic patterns of biological activity and radioactivity; (2) disappearance of these activities after treatment with PLA₂, but not after exposure to lipase fromRhizopus arrhizus. PAF-acether was also obtained when both acetyl-CoA (100 M) and synthetic lyso-PAF-acether (0.2 M) were added to unstimulated PC previously treated with BPB (100 M for 10 min). These results suggest that the release of PAF-acether is the consequence of at least two different steps: (1) hydrolysis of 1-O-alkyl-2-acyl-glyceryl-phosphorylcholine by PLA₂; (2) enzymatic acetylation of the hydrolysis product.     

Hypotensive activity of PAF-acether in rats

PAF-acether caused a dose-related decrease in blood pressure of conscious SHR rats (5–20 g/kg i.p.) and anaesthetized normotensive rats (0.06–0.50 g/kg i.v.). In anaesthetized normotensive rats, PAF-acether-induced hypotension was not associated with tachycardia and not modified by various pretreatments such as atropine, mepyramine, cimetidine, propranolol, sulpiride, ketoprofen (an inhibitor of prostaglandin cyclooxygenase) and teprotide. PAF-acether (0.5 g/kg i.v.) reduced the pressor effect of norepinephrine but not that of angiotensine II.In pithed rats, PAF-acether (0.12–0.50 g/kg i,.v.) shifted the dose-response curve for norepinephrine-induced hypertension to the right in a parallel manner and did not reverse the inhibitory action of clonidine on cardiac nerve stimulation.These results suggest that the hypotensive effect of PAF-acether in anaesthetized rats may be mediated by -adrenergic blockade and PAF-acether looks like a powerful postsynaptic adrenoceptor blocking agent.However,in vitro, up to 100 g/l, PAF-acether did not modify the norepinephrine-induced contractions on non-vascular (vas deferens) and vascular (thoracic aorta) preparations of rats. Moreover, PAF-acether did not inhibitin vitro [³H]WB-4101 and [³H]p-aminoclonidine binding in rat brain preparations.This absence ofin vitro activity suggest that thein vivo adrenolytic activity of PAF-acether is in fact an indirect one either via a directly active metabolite or possibly through the release of an unidentified endogenous adrenolytic factor.     

Mycoplasma fermentans—Induced Inflammatory Response of Astrocytes: Selective Modulation by Aminoguanidine, Thalidomide, Pentoxifylline and IL-10

Exposure of primary rat glial cells, mostly astrocytes, to heat-inactivated Mycoplasma fermentans triggers the production of tumor necrosis factor (TNF) nitric oxide (NO) and prostaglandin E₂ (PGE₂). To attenuate the production of these proinflammatory mediators, four agents: aminoguanidine, pentoxifylline, thalidomide and IL-10 were added to astrocyte cultures. Aminoguanidine (1 and 3 mM), an inhibitor of inducible nitric oxide synthase (iNOS), suppressed the production of the three mediators. TNF was the most sensitive to thalidomide, showing dose-response inhibition at concentrations of 20 g/ml, 50 g/ml and 250 g/ml. PGE₂ was affected only by concentrations of 50 g/ml and 250 g/ml, whereas NO responded solely to the highest amount of this inhibitor. The cytokine IL-10, at 10 U and 50 U, inhibited only TNF production. Our results imply that selective suppression of proinflammatory mediators by various agents may prove feasible for amelioration of central nervous system inflammatory diseases.     

2 μm plasmid copy number in different yeast strains and repartition of endogenous and 2 μm chimeric plasmids in transformed strains

By using the renaturation kinetics technique we tried to get informations about the maintenance of the 2 m plasmid in yeast cells. For this purpose we determined the 2 m plasmid copy number: in various yeast strains, in a special set of mutants, in cells treated with ethidium bromide and cycloheximide and in different yeast strains obtained by transformation with 2 m chimeric plasmids.According to the strain used the proportion of 2m DNA varied from 1.1% to 3.9%, which corresponds to 24 to 88 2 m molecules per haploid genome. The particular multiresistant mutant, where the frequent loss of oligomycine resistance is correlated with the loss of extractible covalently closed circular DNA, contained 39 2 m copies per haploid genome. In the partial revertant oligomycine sensitive all the 2 m DNA sequences were lost. (Less than 0.1 copy per haploid genome.)Ethidium bromide did not affect the 2 m copy number while cycloheximide induces an increase of 36%.When a strain containing 88 2 m DNA copies per haploid genome is transformed with 2 m chimeric plasmids there is no significative change in the total number of plasmid: 36 copies of endogenous and 44 of chimeric plasmid per haploid genome. When 2 m chimeric plasmids were introduced in our 2 m-less strain despite the stability of the transformants, there is only 8 copies per haploid genome.     

On the regulation of the expressed L-type calcium channel by cAMP-dependent phosphorylation

The Ca²⁺ channel subunits _1C-a and _1C-b were stably expressed in Chinese hamster ovary (CHO) and human embryonic kidney (HEK) 293 cells. The peak Ba²⁺ current (I _Ba) of these cells was not affected significantly by internal dialysis with 0.1 mM cAMP-dependent protein kinase inhibitor peptide (mPKI), 25 M cAMP-dependent protein kinase catalytic subunit (PKA), or a combination of 25 M PKA and 1 M okadaic acid. The activity of the _1C-b channel subunit expressed stably in HEK 293 cells was depressed by 1 M H 89 and was not increased by superfusion with 5 M forskolin plus 20 M isobutylmethylxanthine (IBMX). The _1C-a·₂·₂/ complex was transiently expressed in HEK 293 cells; it was inhibited by internal dialysis of the cells with 1 M H 89, but was not affected by internal dialysis with mPKI, PKA or microcystin. Internal dialysis of cells expressing the _1C-a·₂·₂/ channel with 10 M PKA did not induce facilitation after a 150-ms prepulse to +50 mV. The Ca²⁺ current (I _Ca) of cardiac myocytes increased threefold during internal dialysis with 5 M PKA or 25 M microcystin and during external superfusion with 0.1 M isoproterenol or 5 M forskolin plus 50 M IBMX. These results indicate that the L-type Ca²⁺ channel expressed is not modulated by cAMP-dependent phosphorylation to the same extent as in native cardiac myocytes.     

Efficient selection of μ m − mutants from μm-expressing myeloma cells by treatment with ricin A-conjugated anti-μ antibody

We have developed an efficient system for obtaining myeloma mutants defective intrans-acting factors required for immunoglobulin (Ig) gene expression. The system consists of a myeloma cell line designed for this purpose and an efficient method for selecting mutants from it. The cell line is X63.653 transfected with the gene, whose tailpiece sequence was replaced with the transmembrane sequence of human EGF receptor to hold on the cell surface and whose CH1 sequence was removed to prevent from being retained in the endoplasmic reticulum. It efficiently and stably expressed chains of IgM on the cell surface ( _m ⁺ ) without light chains. To obtain mutants lacking _m ( _m ^– ) from the _m ⁺ cell line by selectively killing _m ⁺ cells, a method with ricin A-conjugated anti- antibody was more reliable than complement lysis mediated by anti- antibody. Applying the system, we obtained a variety of _m ^– mutants.     

Different inhibitory effect of etomidate and ketoconazole on the human adrenal steroid biosynthesis

Summary The narcotic agent etomidate and the antimycotic drug ketoconazole are known to block steroid biosynthesis in man. To study the different effects of these imidazole derivatives on human adrenal steroid biosynthesis we incubated slices of human adrenal glands with 3H-labeled precursors and increasing concentrations of etomidate or ketoconazole (0-2000 M). After extraction the labeled metabolites were separated by thin-layer chromatography and quantified by scintillation counting. Etomidate inhibited most potently 11-hydroxylase activity by suppressing the formation of corticosterone from 11-deoxycorticosterone to 1 % of control [50% inhibitory concentration (IC₅₀) 0.03 M] while ketoconazole suppressed 11-hy-droxylase to only 39% of control activity (IC₅₀ 15 M). Ketoconazole however, most potently blocked the conversion of 17-hydroxy-proges-terone to androstenedione by C17,20-desmolase to about 15% of control activity (IC₅₀ 1 M) while etomidate showed a much weaker effect on this enzyme with a suppression to 50% of C17,20-desmolase control activity at a concentration of 380 M. Both imidazole drugs showed a similar strong inhibitory effect on the activity of 17-hy-droxylase (IC₅₀ 6-18 M) and 16-hydroxylase (IC₅₀ 4–8 M) and did not affect 21-hydroxylase. These in vitro data indicate a predominant inhibitory effect of etomidate on corticosteroid biosynthesis by relative selective inhibition of 11-hydroxylase and of ketoconazole on the adrenal androgen biosynthesis by a predominant inhibition of C17,20-desmolase. This differential inhibitory effect of etomidate and ketoconazole on human steroid biosynthesis may be of clinical importance for a possible therapeutic use of these imidazole derivatives in endocrine disorders.Abbreviations IC₅₀ 50% inhibitory concentration Dedicated to Prof. Dr. G. Paumgartner on the occasion of his 60th birthday     

DSLET and ACTH(4-10) increase mitotic activity of hepatocytes and suppress antibody production

The mitotic index of hepatocytes remained unchanged after 10 intraperitoneal injections of DSLET and ACTH_4-10 in doses of 0.5, 1.5, and 5 g/kg, but increased after injection of these substances in doses of 50 and 150 g/kg. DSLET in doses of 5, 50, and 150 g/kg decreased the number of antibody-producing cells in the spleen. ACTH_4-10 possessed immunosuppressive activity not only in these doses, but also in a dose of 1.5 g/kg. As differentiated from mitotic activity of hepatocytes, the degree of immunosuppression increased with increasing the dose of test peptides.     

The effects of TMB-8 on the shape changes of vascular endothelial cells resulting from exposure to various inflammatory agents

Pre-treatment of the endothelial cells lining the guinea pig inferior vena cava with 8-(diethylamino) octyl 3,4,5-trimethoxybenzoate (TMB-8) (10 M)in vitro significantly reduced the shape changes resulting from subsequent exposure to platelet activating factor (PAF) (0.1 M), calcium ionophore A23187 (10 M), histamine (300 M), bradykinin (2 M), prostaglandin E₂ (PGE₂) (30 M), or leukotrienes (LT) C₄, D₄ or E₄ (1 M). Since TMB-8 is an intracellular calcium antagonist, this provides evidence to support the suggestion that these inflammatory agents increase the concentration of intracellular calcium which brings about a contraction of the actin-myosin complex resulting in endothelial cell shape changes, and the formation of interendothelial cell gaps.     

Inhibitory effects of E-5110 on interleukin-1 generation from human monocytes

Effects of E-5110, a novel non-steroidal antiinflammatory drug, on interleukin-1 (IL-1) generation from human monocytes were studiedin vitro. E-5110 reduced the amounts of extra- and intracellular IL-1 activity induced by lipopolysaccharide (LPS, 1 g/ml) in a dose-dependent manner (1–10M). E-5110 also inhibited the IL-1 generation induced by antigen-antibody complexes, opsonized zymosan and silica particles. It was suggested that the inhibition of IL-1 generation by E-5110 was independent of the inhibitory effects on arachidonate cyclooxygenase and/or lipoxygenase because indomethacin, piroxicam, BW755C and AA861 had no effects on IL-1 generation. Hydrocortisone (IC₅₀:0.084 M), aurothioglucose (11.5 M) and lobenzarit (75.0 M), which are clinically effective antirheumatic drugs, also inhibited IL-1 generation, like E-5110 (1.21 M). It is expected that E-5110 will be superior to classical non-steroidal antiinflammatory drugs in medical treatment of rheumatoid arthritis.     

Activity of trimethoprim/ sulfamethoxazole plus polymyxin B against multiresistantStenotrophomonas maltophilia

The inhibitory activity of eight antibiotics and the inhibitory and bactericidal activities of combinations of trimethoprim/sulfamethoxazole (TMP/SMX) plus three fixed concentrations of polymyxin B (0.01 g/ml, 0.1 g/ml and 0.5 g/ml) against 30 multiresistant strains ofStenotrophomonas maltophilia were tested. Polymyxin B at 0.01 g/ml modified the inhibitory activity of TMP/SMX against only 40% of strains. At 0.1 g/ml and 0.5 g/ml, polymyxin B enhanced the inhibitory activity of TMP/SMX activity against all strains. Polymyxin B enhanced the bactericidal activity of TMP/SMX only at concentrations near the minimum inhibitory concentration of polymyxin B alone.     

Inhibition of cysteinyl-leukotriene production by azelastine and its biological significance

Azelastine is a phthalazinone derivative with a wide spectrum of pharmacological activities. Actively sensitized guinea pigs were used to examine the broncholytic effect of azelastinein vivo. Furthermore, the influence of azelastine on the production of arachidonic acid (AA) metabolites was investigatedin vitro and compared to the effects of nordihydroguaiaretic acid (NDGA), indomethacin and ketotifen.In vivo, azelastine protected actively sensitized guinea-pigs against ovalbumin-induced bronchospasm with an ID₅₀ of 0.08 mg/kg orally. Ketotifen was similarly active (ID₅₀=0.05 mg/kg). Antigen-induced contraction of isolated tracheal rings of sensitized guinea-pigs was concentration-dependently inhibited by azelastine and NDGA with IC₅₀-values of 94.1 and 34.2 mol/l, respectively. Ketotifen exerted only weak inhibitory activity (18% at 100 mol/l). The arachidonic acid-induced contraction of isolated guinea-pig tracheal rings was also inhibited both by azelastine (IC₅₀=92.6 mol/l) and NDGA (IC₅₀=20.4 mol/l). Ketotifen was inactive on this model. Antigen challenge of chopped lung tissue from sensitized guinea-pigs resulted in the release of cysteinyl-leukotrienes (LT) which were identified by reversed phase high pressure liquid chromatography (HPLC) as LTD₄ and LTE₄. The release of cysteinyl-LT from sensitized guinea-pig lung tissue induced by antigen challenge was concentration-dependently inhibited by azelastine (IC₅₀=35.2 mol/l) and NDGA (IC₅₀=8.4 mol/l) but not by ketotifen and indomethacin. By contrast, indomethacin caused a pronounced augmentation of cysteinyl-LT release. The concentration of indomethacin, which augmented cysteinyl-LT release by 50% was 0.19 mol/l. At the same concentration, indomethacin inhibited the release of 6-keto-PGF₁ and TXB₂ by about 50%. Azelastine negligible influenced 6-keto-PGF₁ and slightly diminished TXB₂ release from the chopped lung tissue after challenge. Its IC₅₀-values were >2 mmol/l and 443 mol/l, respectively. NDGA inhibited the release of 6-keto-PGF₁ and TXB₂ with IC₅₀-values of 47.3 and 38.3 mol/l, respectively. Ketotifen was ineffective in inhibiting the release of cyclo-oxygenase products of AA metabolism. It seems likely that inhibition of release of 5-lipoxygenase-derived products of AA metabolism by azelastine contributes to its antiallergic and antiasthmatic activity.Author for correspondence.     

In vitro efficacy and fungicidal activity of voriconazole againstAspergillus andFusarium species

Twenty-nineAspergillus isolates and 25Fusarium isolates underwent in vitro antifungal susceptibility testing by a broth macrodilution procedure adapted from the National Committee for Clinical Laboratory Standards guidelines. The MIC50s of both voriconazole and amphotericin B were 0.5 g/ml and 1 g/ml against species ofAspergillus andFusarium, respectively, while the MIC90s of both agents were 1 and 2 g/ml. Voriconazole was more active in vitro than amphotericin B: the geometric mean MICs of voriconazole and amphotericin B againstAspergillus spp. were 0.36 g/ml and 0.64 g/ml, respectively. Voriconazole also demonstrated fungicidal activity againstAspergillus spp., with 86% (24/29) of isolates exhibiting minimum lethal concentrations of 4 g/ml.     

LG 30435, a new bronchodilator/antiallergic agent,inhibits PAF-acether induced platelet aggregation and bronchoconstriction

LG 30435 is a quaternary phenothiazinic antihistamine, endowed with bronchodilator and antiallergic activity. Since PAF-acether (PAF) is a potential mediator of asthma, LG 30435 was assayed for its ability to counteract PAF-induced platelet aggregation (PA) in rabbit platelet rich plasma and bronchoconstriction (BC) in anaesthetized guinea-pigs, in comparison with other antihistamines. LG 30435 was the most potent and selective inhibitor of PAF-induced PA (IC₅₀66 M), concentrations three and more than fifteen fold higher being needed to inhibit PA induced by collagen and arachidonic acid respectively. The other antihistamines, namely mepyramine, promethazine, mequitazine, thiazinamium methyl sulfate and ketotifen were less potent inhibitors of PAF-induced PA, while interfered at lower concentrations with collagen-induced PA. LG 30435 and thiazinamium, administered intravenously, inhibited dose-dependently PAF-induced BC, starting from the dose of 0.1 mol/kg. The travenously, inhibited dose-dependently PAF-induced BC, starting from the dose of 0.1 mol/kg. The ED₅₀ of LG 30435 was 0.28 mol/kg, while the inhibition obtained with thiazinamium did not reach 50% even at 3 mol/kg. Ketotifen and promethazine were partially active only at 3 mol/kg, while mequitazine and mepyramine were inactive up to this dose. These results show that LG 30435 is endowed with a peculiar anti-PAF action, which may be advantageous in the treatment of asthma.A preliminary report on some of these results was presented at the 2nd World Conference on Inflammation, Montecarlo, March 1986.     

Intrinsic cholinergic excitation in the rat neostriatum: Nicotinic and muscarinic receptors

Summary An in vitro slice technique was employed to study the receptors involved in intrinsic cholinergic excitation in the rat neostriatum. The locally evoked synaptic potentials were suppressed by antinicotinic agents, mecamylamine (10 M), d-tubocurarine (3 M) or hexamethonium (100 M), but not by the antimuscarinic agent atropine (100 M). If the slices were exposed to an acetylcholinesterase (AChE)-inhibitor (paraoxon 1–20 M, physostigmine 0.1–0.5 M), the synaptic potentials were potentiated. The amplitude of the orthodromic population spike increased, and it was further facilitated when the stimulus frequencies were raised from 1–3 Hz to 10–30 Hz. The frequency facilitation following exposure to an AChE-inhibitor was blocked by atropine (1–100 M). Intracellular recording indicated that a slow depolarizing potential caused the frequency potentiation of the orthodromic discharges. Apparently rat neostriatum is similar to cholinergic systems in sympathetic ganglia and spinal Renshaw cells, in that nicotinic receptors mediate fast excitation and muscarinic receptors mediate slow excitation.     

Effect of Topically Applied Cyclosporin A on Arachidonic Acid (AA)- and Tetradecanoylphorbol Acetate (TPA)-Induced Dermal Inflammation in Mouse Ear

Topical cyclosporin A (CsA) was compared with dexamethasone, indomethacin and phenidone in edema, increases in vascular permeability, eicosanoids and cell-influx induced by arachidonic acid (AA) and tetradecanoylphorbol acetate (TPA) in mouse ears. CsA ED₅₀ on AA-edema (7.7 g/ear) was similar to dexamethasone and lower than indomethacin and phenidone. CsA ED₅₀ in TPA edema (21 g/ear) was higher than dexamethasone and lower than indomethacin or phenidone. All drugs equally reduce the AA-induced increase in vascular permeability, but CsA and dexamethasone had more activity on TPA. AA-increase in 6-keto-PGF₁ was reduced by dexamethasone, indomethacin and phenidone but not by CsA; only phenidone reduced LTB₄. TPA-increase in 6-keto-PGF₁ was reduced by CsA and indomethacin while CsA, dexamethasone and phenidone decreased LTB₄. CsA, indomethacin and phenidone, but not dexamethasone, suppressed AA-neutrophil influx. In TPA-ears all drugs produced similar reduction in neutrophil influx. CsA was shown to be a good topical anti-inflammatory drug.     

Pharmacological evaluation of rat paw oedema induced byBothrops jararaca venom

The mechanism involved in the genesis of the rat paw oedema caused by intraplantar (IPL) injection ofBothrops jararaca venom (BJV) has been investigated. IPL injection of BJV (1 to 30 g/paw) caused a dose- and time-related oedematogenic effect. Oedema was maximal within 1 h after BJV injection, was partially reduced at 6 h and disappeared completely within 24 h. No systemic effect was observed. Previous heating of BJV at 100°C for 3 to 30 min caused a significant inhibition (25%) of its oedematogenic activity. Daily IPL injections of BJV (10 g/paw) for 4 days attenuated BJV-induced oedema (26%), but did not influence oedema-induced by PAF-acether, serotonin (5-HT) and histamine (His), indicating the absence of cross desensitization. In the paw desensitized by daily IPL injections of PAF-acether, BJV induced a full oedematogenic response also indicating absence of cross desensitization. Different groups of drugs including ₁- and ₂-adrenoceptor antagonists (prazosin and yohimbine), inhibitors of both cyclo- and lipo-oxygenase (indomethacin, nordihydroguaiaretic acid), inhibitors of phospholiase A₂ (dexamethasone and mepacrine) caused marked inhibition of BJV-induced rat paw oedema, whereas antagonists of 5-HT, PAF-acether and H₁-histamine receptors were less effective. Pre-treatment with a -adrenoceptor antagonist, a Ca²⁺ channel blocker and a H₂-histamine antagonist failed to affect BJV-induced oedema. Pre-treatment of the animals with captopril did not interfere with BJV-induce oedema, suggesting that kinins are not insolved in the genesis of oedema. Association of BJV with 5-HT and PAF did not potentiate the BJV-induced oedema. It is concluded that BJV-induced rat paw oedema appears to be mediated primarily by cycloxygenase and lipoxygenase eicosanoid products and activation of ₁- and ₂-adrenoceptors, while HIS, 5-HT and PAF-acether appear to exert minor roles.To whom all correspondence should be addressed.     

Soluble β2-μ-Associated and β2-μ-Free HLA Class I Heavy Chain Serum Levels in Interferon-α Nonresponder Chronic Hepatitis C Patients. Markers of Immune Activation, and Response to Antiviral Retreatment

The serum levels of soluble ₂--associated and ₂--free HLA class I heavy chains were determined in 28 interferon- nonresponder chronic hepatitis C patients retreated with interferon- plus ribavirin and in 70 healthy subjects. The baseline levels of ₂--associated and ₂--free HLA class I heavy chains were significantly higher in patients than in healthy controls(P = 0.001). The levels of ₂--associated HLA class I heavy chains significantly increased in responder patients with respect to nonresponders at the third month of treatment(P = 0.03). At the sixth month of treatment and after 6 months of follow up the levels of ₂--associated HLA class I heavy chains decreased in responder patients and increased in nonresponders. The levels of ₂--free HLA class I heavy chains showed only minor changes during and after treatment. We suggest that the determination of hepatitis C virus RNA levels combined with soluble ₂--associated HLA class I heavy chains, as a marker of immune activation, could identify interferon- non responder chronic hepatitis C patients most likely to respond to a retreatment with interferon- plus ribavirin.     

Comparison of disk diffusion,the E test,and detection ofmecA for determination of methicillin resistance in coagulase-negative staphylococci

The aim of this study was to find a reliable, fast, and simple alternative to the methicillin disk method for determination of methicillin resistance in coagulase-negative staphylococci, since results of this method are often difficult to read due to growth within the zone of inhibition. The sensitivity of 319 strains of coagulase-negative Staphylococci to a 5 g methicillin disk on Mueller-Hinton agar using an incubation period of 48 h was compared with that of 1 (1 g and 5 g oxacillin disks on Mueller-Hinton agar with or without 2% NaCl, using an incubation period of 24 h. The detection ofmecA (MecAgen) by the polymerase chain reaction was used as a standard. Minimum inhibitory concentrations were determined by means of the E test. Of the 225mecA-positive strains, 190, 215, and 193 were resistant to 5 g methicillin, 1 g oxacillin and 5 g oxacillin disks on Mueller-Hinton agar, respectively, and 216, 218, and 223 were resistant on Mueller-Hinton agar with 2% NaCl. Of the 94mecA-negative strains, 89, 93, and 94 were susceptible to 5 g methicillin, 1 g oxacillin, and 5 g oxacillin disks on Mueller-Hinton agar, respectively, and 92, 93, and 94 were susceptible on Mueller-Hinton agar with 2% NaCl. Using breakpoints of 2 g/ml for oxacillin resistance and 8 g/ml for methicillin resistance, the E test yielded sensitivities of 99.6 and 99.1% and specificities of 97.9 and 98.9% after 48 h of incubation. The 5 g oxacillin disk was faster and easier to read than the methicillin disk and correlated better with detection ofmecA than the methicillin disk or the 1 g oxacillin disk.     

The effects of indomethacin and verapamil on the shape changes of vascular endothelial cells resulting from exposure to various inflammatory agents

Histamine (300 M), bradykinin (2 M), prostaglandin E₂ (PGE₂) (30 M), or the leukotrienes (LT) C₄ and E₄ (1 M) but not D₄ (1 M) appliedin vitro have been shown to change the shape of endothelial cells lining the guinea pig isolated thoracic inferior vena cava. All caused the formation of inter-endothelial cell gaps. Pre-treatment with either indomethacin (100 M) or verapamil (20 M) reduced the effects of these compounds. It is suggested that indomethacin and verapamil act by reducing the amount of intracellular calcium available for the shortening of contractile protein filaments within endothelial cells.