In vitro activity of the aryl-fluoroquinolones A-56619 and A-56620 and evaluation of disk susceptibility tests

The activity of two new quinolones, A-56619 and A-56620, was compared in vitro to that of norfloxacin and ciprofloxacin against 6,699 bacterial isolates in four separate clinical laboratories. The overall percentage of strains susceptible to designated concentrations were as follows: 99.1% for norfloxacin (MIC4.0 g/ml), 96.1% for ciprofloxacin (MIC1.0 g/ml), 96.8% for A-56620 (MIC 2.0 g/ml) and 96.1% for A-56619 (MIC 4.0 g/ml). For disk diffusion susceptibility tests 10 g A-56619 disks are tentatively recommended with interpretive standards of 18mm for susceptibility and 13mm for resistance; 5 g A-56620 disks may be used with tentative standards of 19mm for susceptibility and 14mm for resistance.     

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.     

Disk diffusion and disk elution tests with A-56268 and erythromycin

Zones of inhibition around 15g A-56268 disks were essentially the same size as those around 15g erythromycin disks. If the same MIC breakpoints are to be used for defining susceptible categories for both macrolides, interpretive zone size standards for erythromycin disk tests may also be used for A-56268 disk tests. Against anaerobic bacteria, the two macrolides were only marginally effective when broth dilution tests were incubated in anaerobic jars. The aerobically incubated thioglycolate broth disk elution test indicated that both macrolides were much more effective against anaerobes. Three 15g disks eluted in 5 ml thioglycolate provided satisfactory results.     

Effects of inhibitors and ion substitutions on oscillations of cell membrane potential in cells expressing the RAS oncogene

Previous studies revealed that in NIH fibroblasts expressing the ras oncogene but not in other NIH fibroblasts, bradykinin leads to sustained, calcium dependent oscillations of cell membrane potential by repetitive activation of calcium-sensitive K⁺ channels. The present study has been performed to test for ion and inhibitor sensitivity of these oscillations. Both, Lys-bradykinin (kallidin) and bradykinin, but not any shorter peptide tested, maintained the oscillations. The oscillations are abolished in the presence of the K⁺ channel blocker barium (10 nmol/l). The amplitude but not the frequency of the oscillations is dependent on the extracellular potassium concentration. The oscillations are not dependent on the presence of extracellular sodium, bicarbonate or chloride. The oscillations are abolished in the absence of extracellular calcium and their frequency is significantly decreased at reduced extracellular calcium (to 0.2 mmol/l). The oscillations are not inhibited by acute administration of ouabain (0.1 mmol/l), by dimethylamiloride (100 mol/l), furosemide (1 mmol/l) and hydrochlorothiazide (100 mol/l), by cobalt (100 mol/l), zinc (100 mol/l), gadolinium (100 mol/l), verapamil (10 mol/l) and diltiazem (10 mol/l), but are abolished in the presence of 100 mol/l lanthanum, 1 mmol/l cadmium, 10 mol/l nifedipine, 25 mol/l SK & F 96365 and 200 mol/l TMB-8. Stimulation of calcium entry by 10 mol/l ionomycin is frequently followed by oscillations of cell membrane potential even in the absence of bradykinin. In conclusion, in cells expressing the ras oncogene bradykinin leads to sustained activation of calcium channels at the cell membrane, which cause oscillations of the cell membrane potential by triggering intracellular calcium release.     

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.     

Facilitation by 5-hydroxytryptamine of ATP-activated current in rat pheochromocytoma cells

The effects of 5-hydroxytryptamine (5-HT) on an inward current activated by extracellular ATP were investigated in rat pheochromocytoma PC12 cells. Under whole-cell voltage-clamp conditions 5-HT (10 M) reversibly enhanced the amplitude of the current activated by 30 M ATP. The enhancement may not be due to an increase in the number of functional channels because the current activated by 300 M ATP was not remarkably augmented compared with the current activated by 30 M ATP. The current enhancement by 100 M 5-HT was less obvious than that by 10 M 5-HT. When the current kinetics were compared, activation of the ATP-evoked current was accelerated to the same extent by either 10 or 100 M 5-HT, whereas deactivation was largely more accelerated by 100 M 5-HT. Propranolol (10 M), a 5-HT₁ receptor antagonist, or LY53857 (10 M), a 5-HT₂ receptor antagonist, exerted an agonistic effect: the ATP-activated current was facilitated by these compounds. Metoclopramide (10 M), a 5-HT₃ receptor antagonist, neither facilitated the ATP-activated current, nor blocked the current facilitation by 5-HT. Guanosine 5-O-(2-thiodiphosphate) (GDP[S]) (2 mM), the non-hydrolysable analog of guanosine 5-triphosphate (GTP), or K-252a (2 M), a protein kinase inhibitor, did not affect the facilitation by 5-HT of the ATP-activated current when they were included in the intracellular solution. The ATP-activated current pre-facilitated by 10 M dopamine was not enhanced by 10 M 5-HT. Similarly, the pre-facilitation by 5-HT attenuated the current enhancement by dopamine. The results suggest that 5-HT facilitates the ATP-activated channels through receptors that are not readily classified into conventional subclasses of 5-HT receptors. The reciprocal masking between the current facilitation by 5-HT and that by dopamine, combined with their sensitivities to the compounds involved in the intracellular solution, indicates that the facilitation by 5-HT may share not all, but some, common cellular mechanism with that by dopamine.     

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.     

Enhanced stability of YEp plasmids in lager brewing yeasts is related to lager brewing yeast 2-μm DNA

Summary YEp plasmid stability in the presence of either Saccharomyces cerevisiae laboratory strain 2-m DNA, or lager brewing yeast 2-m DNA in the same genetic background, was compared under non-selective culture conditions. It was found that YEp plasmids were more stably maintained in the presence of lager 2-m DNA under these conditions. By construction of laboratory-lager 2-m DNA hybrid plasmids, an 867 bp StuI fragment of lager 2-m DNA was shown to be responsible for the enhanced stability of the YEp plasmid. Nucleotide substitutions at two sites were found by sequencing this region. It was also confirmed that increasing cell ploidy enhanced YEp stability under non-selective conditions.     

Effects of PAF and PAF antagonists on the shape of venous endothelial cellsin vitro

Three platelet-activating factor (PAF) antagonists were tested for their ability to prevent or reduce PAF-induced shape changes of large vein endothelial cellsin vitro. BN52021 had a significant protective action at concentrations of 1 M and 0.1 M, but at 100 M had a damaging effect of its own. CV3988 (0.1 M and 1 M) and L652, 731 (20 M) did not reduce the responses to PAF, and at higher concentrations (CV3988 10 M and 100 M, L652, 731 100 M) both compounds alone caused significant changes of shape. BN52021 (0.1 M) was also effective against leukotriene (LT) C₄, at 1 M against bradykinin and LTE₄, and at 10 M against LTD₄ and the calcium ionophore A23187. BN52021 (10 M) was ineffective against shape changes induced by histamine, prostaglandin (PG) E₂ and lysophosphatidylcholine (LPC). Neither indomethacin (100 M) nor verapamil (20 M) altered the response to PAF.Using electron spin resonance (ESR) spectrometry it was shown that the damaging effects of LPC and CV3988 may be due partly to their detergent properties. It is suggested that the mechanism by which PAF alters the shape of large vein endothelial cells is primarily receptor mediated.     

Effects of ketamine on GABA-evoked excitability of peripheral nerve

Summary The effects of the dissociative anaesthetic, ketamine on GABA-evoked changes in the excitability of myelinated fibers of dorsal and ventral roots of isolated bullfrog sciatic nerves were examined. Ketamine alone (0.01–1000 M) evoked small increases (<5%) in dorsal root fiber excitability, as reflected in the half-maximal A-fiber compound action potential when concentrations were >10 M; with 0.1 M even larger increases (10%) were elicited in the ventral root fibers. As well, the increases evoked by 10 M ketamine were followed by graded decreases. 0.1 and 10 M ketamine, concentrations which by themselves had small or no effect, produced a dose-dependent depression of the large increases in excitability which are induced by activation of GABA receptors. In the presence of ketamine GABA concentration-response curves of the dorsal root fibers showed depression of the maximal response, while those of the ventral root fibers were shifted to the right. This apparent antagonism of GABA responses by ketamine may arise from blockade of receptor-mediated effects (e.g. K⁺/Cl^- currents and/or secondary depolarization from K⁺ accumulation), but could also be caused by a selective potentiation of hyperpolarizing receptors.     

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.     

Roles of inositol trisphosphate and protein kinase C in the spontaneous outward current modulated by calcium release in rabbit portal vein

We examined the effects of heparin, guanosine nucleotides, protein kinase C (PKC) modulators, such as phorbol 12,13-dibutylate (PDBu) and H-7 on Ca²⁺-dependent K⁺ currents in smooth muscle cells of the rabbit portal vein using the whole-cell patch-clamp technique, to explore the effects of PKC on the oscillatory outward current (I _oo). Neomycin (30 M), an inhibitor of phospholipase C, and intracellular applications of heparin (10 g/ml) and guanosine 5-O-(2-thiodiphosphate) (GDP[S]; 1 mM) partly but consistently inhibited the generation of I _oo, whereas a higher concentration of heparin (100 g/ml) transiently enhanced then suppressed the generation of I _oo. Inhibition of I _oo generation by heparin was more powerful at the holding potential of + 20 mV than at –20 mV. Inositol 1,4,5-trisphosphate (InsP ₃; 30 M) continuously generated I _oo at holding potentials more positive than –60 mV. Noradrenaline (10 M) and caffeine (3–20 mM) transiently augmented, then reduced the generation of I _oo. Heparin (10 g/ml) completely inhibited responses induced by InsP ₃ and noradrenaline, but not those induced by caffeine. Intracellular application of guanosine 5-triphosphate (GTP; 200 M) or low concentrations of guanosine 5-O-(3-thiotriphosphate) (GTP[S]; 3 M) continuously augmented the generation of I _oo. High concentrations of GTP[S] (10 M) transiently augmented, then inhibited I _oo. Neither GTP[S] nor noradrenaline induced the transient augmentation or the subsequent inhibition of I _oo when applied in the presence of GDP[S] (1 mM), neomycin (30 M) or heparin (10 g/ml). PDBu (0.1 M) reduced the generation of I _oo but failed to produce an outward current following application of caffeine (3–5 mM). This action of PDBu was inhibited by pretreatment with H-7 (20 M). In the presence of H-7, GTP[S] continuously enhanced the generation of I _oo. The suppression of the generation of I _oo during application of noradrenaline (10 M) was reduced by pretreatment with H-7. Thus both InsP₃ and protein kinase C contribute to the generation of I _oo in smooth muscle cells of the rabbit portal vein and heparin is not a specific InsP ₃ antagonist on the InsP ₃-induced Ca²⁺-release channel (PIRC). InsP ₃ opens PIRC and protein kinase C may deplete the stored Ca²⁺ by either inhibiting the reuptake of Ca²⁺ or by enhancement of the releasing actions of InsP ₃.     

Provisional quality control parameters and interpretive criteria for testing susceptibility ofStreptococcus pneumoniae andHaemophilus influenzae to quinupristin/dalfopristin (RP59500)

Studies were undertaken to select tentative criteria for susceptibility testing of quinupristin/dalfopristin againstStreptococcus pneumoniae andHaemophilus influenzae. Against 612 isolates ofStreptococcus pneumoniae, MICs of quinupristin/dalfopristin were 1.0 g/ml for all but one strain. With a tentative MIC breakpoint of either 1.0 g/ml or 2.0 g/ml for susceptible, a disk diffusion zone diameter breakpoint of 19 mm embraced all but two of the susceptible pneumococci; 16 mm included all strains. ForHaemophilus influenzae, MICs of quinupristin/dalfopristin clustered near the tentative breakpoints; 91.5% of the MICs were 2.0 to 8.0 g/ml. This precluded satisfactory performance of the disk diffusion test in discriminating between resistant and susceptible isolates unless MIC breakpoints are modified for this species: clinical experience will be needed before that can be justified. Based on data from a multilaboratory study, the following quality control limits are proposed forStreptococcus pneumoniae ATCC 49619 when testing quinupristin/dalfopristin: 0.25 to 1.0 g/ml for broth microdilution tests and 19 to 24 mm for disk diffusion tests. For tests ofHaemophilus influenzae ATCC 29247, MIC limits are 2.0 to 16 g/ml; disk tests were very reproducible but are not yet recommended.     

Mechanisms of antagonistic action of internal Ca2+ on serotonin-induced potentiation of Ca2+ currents in Helix neurones

The influence of internal Ca²⁺ ions has been investigated during intracellular perfusion of isolated neurones from pedal ganglia of Helix pomatia in which serotonin (5-HT) induces a cyclic-adenosine-monophosphate-(cAMP)-dependent enhancement of high-threshold Ca²⁺ current (I _Ca). Internal free Ca²⁺ ([Ca²⁺]_i) was varied between 0.01 and 10 M by addition of Ca²⁺-EGTA [ethylenebis(oxonitrilo)tetraacetate] buffer. Elevation of [Ca²⁺]_i depressed the 5-HT effect. The dose/ effect curve for the Ca²⁺ blockade had a biphasic character and could be described by the sum of two Langmuir's isotherms for tetramolecular binding with dissociation constants K _d1=0.063 M and K _d2=1 M. Addition of calmodulin (CM) antagonists (50 M trifluoperazine or 50 M chlorpromazine), phosphodiesterase (PDE) antagonists [100 M isobutylmethylxanthine (IBMX) or 5 mM theophylline] and protein phosphatase antagonists [2 M okadaic acid (OA)] in the perfusion solution caused anticalcium action and modified the Ca²⁺ binding isotherm. Using the effect of OA and IBMX, two components of the total Ca²⁺ inhibition were separated and evaluated. In the presence of one of these blockers tetramolecular curves with K _d1=0.04 M and K _d2=0.69 M were obtained describing the activation of the retained unblocked enzyme — PDE or calcineurin (CN) correspondingly. The sum of these isotherms gave a biphasic curve similar to that in control. Leupeptin (100 M), a blocker of Ca²⁺-dependent proteases did not influence the amplitude of 5-HT effect, indicating that channel proteolysis is not involved in the depression. Our findings show that the molecular mechanism of Ca²⁺-induced suppression of the cAMP-dependent upregulation of Ca²⁺ channels is due to involvement of two Ca²⁺-CM-dependent enzymes: PDE reducing the cAMP level, and CN causing channel dephosphorylation. No other processes are involved in the investigated phenomenon at a Ca²⁺ concentration of less than or equal to 10 M.     

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.     

Characterization of the in vitro anti-inflammatory activity of AL-5898 and related benzopyranyl esters and amides

Selected ester- (AL-5898 and AL-8417) and amide-linked benzopyran analogues (AL-7538 and AL-12615) were evaluated in vitro for their ability to inhibit key enzymes/processes of the inflammatory response. AL-7538 and AL-12615 exhibited weak intrinsic cyclooxygenase inhibitory activity (IC₅₀ = 13 M, 37 M). In contrast, 5-HETE and LTB₄ synthesis in A₂₃₁₈₇-stimulated neutrophils was effectively inhibited by both ester and amide analogs (IC₅₀ = 2–3 M). While there was some indication for differing sensitivities among benzopyran esters and amides in the suppression of cytokine synthesis in stimulated U-937 cells, there appeared to be no great discrimination when assessing their effect on U-937 cell adhesion to IL-1 activated HMVEC-L cells. Inhibition of cell adhesion was concentration-dependent, with IC₅₀ values ranging between 18 M and 30 M for AL-5898. Concentration-dependent inhibition of inflammatory cytokine production (i.e., IL-1, TNF-, GM-CSF and IL-6) was also apparent in LPS-stimulated, cultured PBMC as well as in PMA/A₂₃₁₈₇ activated U-937 cells monitoring the synthesis of IL-1, IL-8, TNF-, and MCP-1. Notably, the hydrolysis products of the benzopyranyl ester, AL-5692 and (S)-6-methoxy--methyl-2-naphthaleneacetic acid, were devoid of pharmacological activity when assessed for inhibition of monocyte adhesion or IL-1 synthesis. Collectively, our data demonstrate the unique in vitro polypharmacology of a novel series of benzopyran analogs that suppress pivotal enzymes and processes in the inflammatory response.     

A pharmacologic study on the histamine releasing effect of atracurium and other muscle relaxants in rat isolated ileum

In this study, the effects of histamine, antihistamines (terfenadine and mepyramine), 5-hydroxytryptamine, and muscle relaxants, atracurium, vecuronium and gallamine, on the tone and contractility of rat ileum were studied and compared in vitro.The aim of the present investigation was to measure, pharmacologically, the histamine releasing effect of muscle relaxants, e.g. atracurium, vecuronium and gallamine, by comparing their contractile response in the absence and presence of antihistamines and comparing their mechanical responses with those produced by histamine and 5-hydroxytryptamine (5-HT).The results showed that the antihistamines, triludan (terfenadine) and mepyramine produced opposite effects in rat ileum. Terfenadine (0.1–20 M) produced concentration-dependent contractions in the rat ileum, whereas mepyramine (0.1–10 M) relaxed the muscle, e.g. by 1.2 g tension. Atracurium (0.5–500 M), vecuronium (0.2–200 M), and gallamine (0.1–7.0 M) produced marked contractions (1.5–4.0 g tension) in rat ileum, and these contractions were markedly reduced by mepyramine (1.3 M) or terfenadine (5 M), implicating histamine release in the generation of these contractions. However, there was some residual contraction which was not blocked by mepyramine, but by 5-HT antagonist, methysergide (1 M), indicating that a mechanism other than histamine release may be responsible for the residual contraction, i.e. release of other mediators such as 5-HT, prostaglandins, or calcium. 5-HT (0.5–500 M) and histamine (0.5–500 M) produced contractions in the rat ileum, but 5-HT was more effective than histamine in producing these contractions. Similarly, gall amine was more effective than atracurium and vecuronium in contracting the rat ileum. Since very high concentrations of muscle relaxants were used, it is suggested that in clinical concentrations, the histamine releasing effect of muscle relaxants was minimal, except that of gallamine, which may release histamine event at very low concentrations. The results are discussed in terms of pharmacologic and immunologic implications of drug reactions at the rat intestinal smooth muscle.     

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.     

Pyramidal tract of the cat: axon size and morphology

Summary The purpose of this work was to determine the number and morphology of pyramidal tract (PT) axons in the cat, using electron microscopy, modern methods of fixation, and computer-assisted morphometric analysis. Sections taken at the level of the medullary pyramids in three animals were fixed and magnified up to 10,000 x to produce photomicrographs. Morphological data were entered into computer files for analysis by tracing axon perimeters on micrographs mounted on a digitizer tablet. The number of axons per PT averaged 415,000, of which 88% were myelinated and 12% were unmyelinated. 90% of the myelinated axons fell in the diameter range 0.5–4.5 m. Axons larger than 9 m diameter accounted for 1% of the total; the largest were 20–23 m. Myelinated axon mean diameter was 1.98 m; because of the skewed distribution, with many small axons and a few very large axons, median diameter was 1.60 m. Size distribution was relatively uniform throughout the PT cross section, with all sizes represented in all regions. However, the more medial regions had a higher proportion of small fibers than the more lateral regions: mean medial diameter was 1.85 m while mean lateral diameter was 2.09 m. Myelin sheath thickness averaged 7.9% of fiber diameter for axons up to 11 m, but was constant at 0.9 m for larger fibers. Myelinated fibers were distorted from the circular shape in cross section, with a mean circularity index (or form factor) of 0.85, which implies that the fibers could swell about 15% without rupture of the cell membrane. Unmyelinated fibers averaged 0.18 m diameter (range 0.05–0.6 m); the largest unmyelinated axons were larger than the smallest myelinated axons. It is concluded that previous work greatly underestimated the number of axons in the cat pyramidal tract.     

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.