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.     

Introduction of nonselectible 2μ plasmid into [cir°] cells of the yeast S. cerevisiae by DNA transformation and in vivo site-specific resolution

Summary The 2 DNA plasmid of the yeast Saccharomyces cerevisiae does not confer any known selectable phenotype to the host cell carrying it. Selection of cells transformed with purified 2 DNA therefore cannot be achieved, and the intracellular presence of 2 can only be assessed by molecular analysis of the DNA complement. In addition, 2 alone does not replicate in bacterial hosts, thus rendering its amplification by conventional methods impossible. We have isolated a shuttle plasmid, pBH-2L, generated by in vivo sites-pecific recombination between the endogenous 2 DNA plasmid and pRL, a pBR322 derivative containing the yeast LEU2 gene and one 2 repeat sequence associated with the origin of replication. This new shuttle plasmid has the property, when transformed into yeast, of undergoing site-specific recombinational resolution between its two direct repeat sequences. This releases 2 plasmid and pRL as individual molecules. The latter can undergo progressive mitotic loss during growth in nonselective medium, ultimately leaving leucine auxotrophic transformants that contain only 2 DNA plasmid. This system can be utilized to introduce 2 DNA alone into cells lacking it, thereby providing a novel means to study the biology and the molecular genetics of the plasmid and its potential practical applications as a vector.     

Susceptibility to beta-lactam antibiotics in septicemia isolates from twenty-nine European laboratories

In 1984 the European Study Group on Antibiotic Resistance (ESGAR) consecutively collected gram-negative bacilli and staphylococci blood isolates and performed susceptibility testing with 11 antibiotics using the microdilution method. In all 2,578 isolates were collected: 68% gram-negative bacilli and 32% staphylococci. The MICs of ampicillin and cefazoline for the susceptible gram-negative bacilli were 1–8g/ml; of piperacillin0.5–4; of Sch 34343, cefotaxime, moxalactam, ceftazidime and aztreonam0.5–2g/ml; of cefoxitin, cefuroxime and cefamandole0.5–8g/ml. For susceptible staphylococci the MICs of cefazoline and cefuroxime were0.5–1g/ml, and of cefoxitin, moxalactam, ceftazidime and cefotaxime,0.5–32 g/ml. The resistance levels varied between laboratories and countries, being lower in Northern Europe. In clinical protocols on patients with gram-negative septicemia from whom cefazoline-resistant strains were isolated, cefotaxime was the beta-lactam most commonly used (12%). In protocols on patients with staphylococcal septicemia from whom gentamicin-resistant or cefazoline-resistant strains were isolated, the most commonly used beta-lactam was cloxacillin (6%).     

Beeinflussung des post-asphyktischen Wiederaufbaues der Adeninnucleotide in Kaninchenherzenin vivo durch Substratangebot

Zusammenfassung Die Möglichkeit einer Beschleunigung der langsamen postanaeroben Erholung im Status der myokardialen Adeninnucleotide durch ein methodisch einfach durchführbares kontinuierliches Angebot von Substraten, die zum Aufbau von Nucleotiden bedeutungsvoll sein könnten, wurde unterin vivo-Bedingungen am Herzen des Kaninchens anhand von Bestimmungen der Gewebsgehalte von Metaboliten und Substraten des Adenylsäure-Phosphokreatin-Systems und des Glykolysecyclus geprüft. Als anaerobe Belastung diente eine Serie von 4 Asphyxien von 1 mal 3 min und 3 mal 2,5 min Dauer mit zwischenzeitlichen Erholungspausen von 10 min Dauer. Nach Abschluß der raschen Erholungsvorgänge im Herzstoffwechsel wurden die Substrate oder physiologische NaCl-Lösung bis zu einer post-asphyktischen Erholungsdauer von 5 Std in die V. cava sup. oder in das linke Herzohr infundiert. Die Infusion von Bausteinen zurde novo-Synthese des Purinkörpers mit Glycin (6 mol/min), Glutamin (6 mol/min), Asparaginsäure (6 mol/min), Folsäure (2 mol/min), Ameisensäure (0.04 mol/min), Oxalessigsäure (6 mol/min) und Ribose (12 mol/min) und die Infusion von Adenin+Ribose (0.6 bzw. 12 mol/min+12 mol/min) und von Inosin (20 mol/min) resultierte nicht in einem beschleunigten Wiederaufbau der myokardialen Adeninnucleotide; die Ursache wird in einem Mangel an aktivierter Ribose und im Fehlen notwendiger Enzyme im Kaninchenherzmuskel gesehen. Das Angebot von Adenosin (7,5 mol/min) resultierte in einer starken Beschleunigung des post-asphyktischen Wiederaufbaus der Adeninnucleotide und in einer Erhöhung des ATP-Gehaltes und der Summe der Adeninnucleotide um 35 bzw. 39% über die Norm.Mit Unterstützung durch die Deutsche Forschungsgemeinschaft.     

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.     

Anti-inflammatory and immuno-modulatory effects of novel retinoidlike 2,4,6,8-nonatetraenoic acids (NTA) in adjuvant-induced arthritis

Prophylactic treatment (p.o.) of rats with adjuvant-induced arthritis (AA) with two retinoid-like 2,4,6,8-nonatetraenoic acids (NTA), Ro 23-6457 and Ro 23-2895, significantly reduced hind paw swelling between days 10–23 and the level of plasma fibrinogen (MED 25 moles/kg). When given therapeutically (75 moles/kg between day 21 and 28) either NTA arrested the progression of the disease (MED, 25–75 moles/kg).Unseparated and adherent cell (AC) depleted spleen cells from rats with AA (day 12–15) responded poorly to the T cell mitogen, Con A (2.5 g/ml) and the B cell mitogen, LPS (10 g/ml). The responses were partially restored (30% of normal responses) in AC-depleted (but not unseparated) spleen cells from Ro 23-6457 treated rats (75 and 250 moles/kg/day). These data demonstrate an immunomodulatory effect of Ro 23-6457 in the adjuvant rat which may contribute to its anti-inflammatory activity in AA.     

Possible involvement of microtubules in platelet-activating factor-induced increases in microvascular permeability in vitro

The blood vessels of the rat small intestine were perfused in vitro with a gelatin-containing physiological salt solution (GPSS). The addition of platelet-activating factor (PAF, 5M), podophyllotoxin (50M), colcemid (50M), or nocodazole (50M) to the GPSS for 5 min caused an increase in vascular permeability. This was manifested as an increased trapping of circulating colloidal carbon (CC) within the walls and was assessed using semiautomated image analysis. Pretreatment for 10 min with taxol (5M) in the perfusate significantly reduced the permeability-enhancing effects of all four agonists. Since podophyllotoxin, colcemid, and nocodazole are all microtubule-disrupting agents, and since taxol is a microtubule-stabilizing agent, these results suggest that microtubules are involved in the response of the microvessels to PAF. An explanation based on tensegrity or force-counterbalance is put forward to account for these findings.     

Bradykinin Antagonizes the Effects of α-Thrombin

-Thrombin (AT) and bradykinin (BK) are endogenous mediators that are released during an inflammatory response, and could have a synergistic effect on endothelial permeability. Human umbilical vein endothelial cells (HUVEC) were grown on Transwell membranes and then tested for alterations in permeability to fluorescein isothiocyanate-labeled human serum albumin. Addition of 1M AT produced a significant increase in the permeability coefficient at 30 minutes from control levels of 1.59 × 10^–6 cm/sec to 4.92 × 10^–6 cm/sec. BK (1M) produced a similar increase to 4.46 × 10^–6 cm/sec. For both compounds, permeability remained elevated for 90 minutes. Pre-treatment of the HUVEC with the bradykinin receptor antagonist, Na-adamantaneacetyl-bradykinin (NA-BK) (1M), prior to addition of AT, reduced the AT permeability coefficient to 2.69 × 10^–6 cm/sec. Addition of NA-BK (1 M) for 5 minutes, then BK (1 M) for 5 minutes, inhibited the effect of BK and of AT (1 M) on permeability, decreasing the permeability coefficient of the endothelial monolayer to control levels (1.62 × 10^–6 cm/sec). AT (1 M) increased HUVEC intracellular calcium mobilization, as monitored by FURA-2, to 245 nM from control (70 nM), however, pre-treatment with either BK or the bradykinin receptor antagonist decreased the AT induced intracellular calcium mobilization compared to AT alone. Pre-treatment of the HUVEC with bradykinin (1 M) for 2 minutes also inhibited the effects of -thrombin (1 M) on f-actin distribution examined by BODIPY-phallodin staining and increased the clotting times for an -thrombin dependent fibrinogen to fibrin clotting assay. However, incubation of bradykinin (1 M) with -thrombin (1 M) for either 10 minutes or 100 minutes produced no detectable hydrolysis products. These data strongly suggest that the inflammatory mediators -thrombin and bradykinin when released together, rather than being synergistic, are antagonistic.     

Fast K channels are more sensitive to riluzole than slow K channels in myelinated nerve fibre

The effects of 1–500 M riluzole, a novel psychotropic agent, were studied on the nodal K current of isolated nerve fibres of the frog. When added to the external solution, the substance rapidly and reversibly inhibited slow, fast 1 and fast 2 K components of the tail K current. The concentrations of riluzole inducing half maximum reduction of slow, fast 1 and fast 2 K conductances were 413 M, 24 M and 21 M respectively. It is concluded that the substance is about 20 times more effective in blocking fast than slow K channels.     

Potentiation of antifungal activity of amphotericin B by azithromycin againstAspergillus species

The potential role of azithromycin in combination with amphotericin B against 25 clinical isolates ofAspergillus was assessed. The MIC of amphotericin B was 1 g/ml for 44% of the isolates, 0.5 g/ml for 48%, and 0.25 g/ml for 8%. All isolates were resistant to azithromycin. Synergism, defined as a twofold reduction in the MIC of both drugs upon combination, was demonstrated between amphotericin B and azithromycin for all 25 isolates. To prove that azithromycin exerts its antifungal effect by inhibiting protein synthesis, we studied [³⁵S]-methionine incorporation into protein inone Aspergillus isolate. Neither amphotericin B at 0.125 g/ml (fourfold below its MIC) nor azithromycin at 16 g/ml ( 16-fold below its MIC) had any effect on protein synthesis when tested alone. Upon combination, however, a 68% inhibition in protein synthesis was evident by the inhibition of [³⁵S]-methionine incorporation.     

Effect of natural inhibitors of free radical reactions on adrenalin autoxidation

Water-soluble antioxidants (1 M reduced glutathione, 3 M cysteine, 1 M ascorbate), in the presence of EDTA, inhibit the adrenalin autoxidation reaction at pH 10.2, whereas their oxidized forms, and also -tocopherol (40 M), have no such effect. The inhibiting power of superoxide dismutase is many times greater than that of the substances tested. Adrenochrome formation during free-radical oxidation of adrenalin takes place without the participation of the hydroxyl radical.Department of Biochemistry, Second Moscow Medical Institute. (Presented by Academician of the Academy of Medical Sciences of the USSR V. N. Orekhovich.) Translated from Byulleten' Éksperimental'noi Biologii i Meditsiny, Vol. 81, No. 1, pp. 33–35, January, 1976.     

Histamine H3-receptor activation inhibits acetylcholine release from the guinea pig myenteric plexus

The role of histamine H₃-receptors in the control of acetylcholine release from peripheral cholinergic neurons was evaluated in the isolated guinea pig ileum, previously loaded with³H-choline. When tested in the presence of H₁- and H₂-blockade, histamine (0.1–100 mol/l) and (R)-methylhistamine (0.01–1 mol/l) dose-dependently reduced the electrically-evoked choline outflow, with (R)-methylhistamine being a partial agonist. Selective H₃-receptor blocking drugs, thioperamide (0.1 mol/l) and impromidine (0.1 mol/l) reversed the histamine-induced inhibitory, effect. These data suggest that intestinal cholinergic nerves are endowed with histamine H₃-receptors whose activation produces an inhibitory effect upon acetylcholine release. The practical implications of these findings are obvious.     

Z-line structural diversity in frog single muscle fiber in the passive state

The structural changes of the Z-line between small square net (ss) and basket weave (bw) cross-sectional patterns were examined using intact single fibers and mechanically skinned fibers in the passive state to determine if the pattern is related to the sarcomere length (SL) and if the pattern undergoes a reversible transition in low- and high-osmotic medium.Frog single fibers were isolated from the anterior tibial muscle in Ringer's solution. Entirely or partially skinned single fibers were prepared in relaxing solution (also called low-osmotic medium).The high osmotic medium contained 10% polyvinylpyrrolidone (PVP) in relaxing solution.The sarcomere length (SL) of each fiber was measured directly by use of a laser beam or indirectly from electron micrographs with use of a correction factor. The ss and bw forms in cross sections were quantified by analysis of electron micrographs. The results show that the structural change of Z-line occurs around bw 2.3–2.4m ss (n = 25) and bw 3.1–3.2m ss (n = 13) in intact single fibers and skinned fibers, respectively. With the quick freeze-freeze substitution method, an intact single fiber with a SL of 2.35m showed almost 100% of ss form. The structural transition in cross section was also confirmed in four partially skinned fibers, where patterns went from mostly ss form (intact portion) to mostly bw form (skinned portion) at the SL between 2.40 to 3.20m.The reversibility of the change between ss and bw was proved by using low- and high-osmotic medium. The transition and reversion of cross-sectional patterns both occur in the passive state.     

β-Adrenoceptor stimulation activates large-conductance Ca2+-activated K+ channels in smooth muscle cells from basilar artery of guinea pig

We studied the effect of isoproterenol on the Ca²⁺-activated K⁺(BK) channel in smooth muscle cells isolated from the basilar artery of the guinea pig. Cells were studied in a whole-cell configuration to allow the clamping of intracellular Ca²⁺ concentration, [Ca²⁺]_i. Macroscopic BK channel currents were recorded during depolarizing test pulses from a holding potential (V _H) of 0 mV, which was used to inactivate the outward rectifier. The outward macroscopic current available from aV _H of 0 mV was highly sensitive to block by external tetraethylammonium·Cl (TEA) and charybdotoxin, and was greatly augmented by increasing [Ca²⁺]_i from 0.01 to 1.0 M. With [Ca²⁺]_i between 0.1 and 1.0 M, 0.4 M isoproterenol increased this current by 58.6±17.1%, whereas with [Ca²⁺]_i at 0.01 M a sixfold smaller increase was observed. With [Ca²⁺]_i0.1 M, 100 M dibutyryl-adenosine 3:5: cyclic monophosphate (cAMP) and 1 M forskolin increased this current by 58.5±24.1% and 59.7±10.3%, respectively. The increase with isoproterenol was blocked by 4.0 M propranolol extracellularly, and by 10 U/ml protein kinase inhibitor intracellularly. Single-channel openings during depolarizing test pulses from aV _H of 0 mV recorded in the whole-cell configuration under the same conditions (outside-outwhole-cell recording) indicated a slope conductance of 260 pS. In conventional outside-out patches, this 260-pS channel was highly sensitive to block by external TEA, and in inside-out patches, its probability of opening was greatly augmented by increasing [Ca²⁺]_i from 0.01 to 1.0 M. Outside-out-whole-cell recordings with [Ca²⁺]_i0.1 M indicated that 100 M dibutyryl-cAMP increased the probability of opening of the 260-pS channel by 152±115%. In inside-out patches, the catalytic subunit of protein kinase A increased the probability of opening, and this effect also depended on [Ca²⁺]_i, with a 35-fold larger effect observed with 0.1–0.5 M Ca²⁺ compared to 0.01 M Ca²⁺. We conclude that the BK channel in cerebrovascular smooth muscle cells can be activated by-adrenoceptor stimulation, that the effect depends strongly on [Ca²⁺]_i, and that the effect is mediated by cAMP-dependent protein kinase A with no important contribution from a direct G-protein or phosphorylation-independent mechanism. Our data indicate that the BK channel may participate in-adrenoceptor-mediated relaxation of cerebral vessels, although the importance of this pathway in obtaining vasorelaxation remains to be determined.     

Phagocytosis and Chemiluminescence Response of Granulocytes to Monodisperse Latex Particles of Varying Sizes and Surface Coats

The response of human granulocytes to polystyrene latex beads of diameter 0.1–7 m was measured by luminol-dependent chemiluminescence. In all instances, the response to beads of 3–7 m was definitely higher than with smaller beads. In protein-free medium, the chemiluminescence response was slow compared to that of opsonized zymosan, and the highest response was only 9% of the response to opsonized zymosan. Scanning electron microscopy showed that granulocytes in suspension bound the particles, occasionally by extending rope-like protrusions. When the beads were coated with albumin, the chemiluminescence diminished to about 1/3 of that seen with uncoated beads; however, preincubating the beads in serum led to a large increase with beads of 1.1 m (to 25% of the maximal response to opsonized zymosan) and 3.19 m (to 42%), but with the smallest beads, no increase was noted. "Priming" of the cells with tumor necrosis factor- caused a further increase with serum-coated beads. When uncoated beads of 1.1 m were tested with "primed" cells, there was an increase of 6 times in the chemiluminescence compared to un-"primed" cells.     

A comparison of isometric force,maximum power and isometric heat rate as a function of sarcomere length in mouse skeletal muscle

The isometric force, maximum power and isometric heat rate have been measured at different sarcomere lengths (SL) between 1.40 and 3.63 m in two types of mouse muscle, soleus and omohyoideus, at 25°C. The SL force relationship is different in the two muscles. At a SL above optimum filament overlap, 2.44 m in omohyoideus muscles, maximum power declined while isometric force remained high. In soleus muscles this occurred above a SL of 2.33 m. In parallel experiments, the isometric heat rate declined linearly with increasing SL above 2.33 m in soleus muscles, while isometric force remained closer to its maximum. At short SL, between 2.33–1.75 m in soleus and 2.44–2.15 m in omohyoideus, maximum power remained at or near its maximum value as did heat rate (soleus) while isometric force fell. In both muscles at SL greater than optimum for force development maximum power output (unlike force) is proportional to filament overlap. The variation in heat rate over this SL range can be described as the sum of a constant rate and a rate proportional to filament overlap. These observations are compatible with the idea that maximum power and heat rate are less affected by non-uniformities in SL than is force.     

Evidence for Na+/Ca2+ exchange in isolated smooth muscle cells: a fura-2 study

Isolated smooth muscle cells (SMC) from guinea pig taenia coli were employed. Suspension of cells were externally loaded in saline with the fluorescent calcium indicators quin-2/AM or fura-2/AM at 20–40 M or 4 M respectively, resulting in an estimated intracellular concentration of 100–200 M for quin-2 or 10–20 M fura-2 (free acid). On addition of 100 M carbachol or high K _o ⁺ (80 mM) depolarization, fura-2 loaded cells contracted (104±47 m,n=121 rest: 39±13 m,n=59 contracted) identically to control (103±35 m,n=232 rest: 39±16 m,n=89 contracted) cells, whereas quin-2 loaded cells were unresponsive to these protocols and there was no significant length change. The Ca _i ²⁺ of fura-2 loaded cells was 100±18 nM (mean±SD,n=15) and was not significantly different from quin-2 loaded cells 107±26 nM (n=13). Treatment of fura-2 loaded cells with 100 M ouabain saline for 10–60 min progressively elevated the Ca _i ²⁺ to a mean of 266±83 nM (n=15). Reduction of Na _p ⁺ (96% Li⁺ replaced) significantly increased Ca _i ²⁺ to 317±77 nM (n=8). After pretreatment with ouabain (100 M), Na _o ⁺ replacement (Li⁺) increased Ca _i ²⁺ at a significantly faster rate [3.6 nM min^–1 (control) cf. 19.8 nM min^–1 (ouabain)].     

ATP-Dependent inhibition of Ca2+-activated K+ channels in vascular smooth muscle cells by neuropeptide Y

Neuropeptide Y(NPY) inhibits Ca²⁺-activated K⁺ channels reversibly in vascular smooth muscle cells from the rat tail artery. NPY (200 M) had no effect in the absence of intracellular adenosine 5triphosphate (ATP) and when the metabolic poison cyanide-M-chlorophenyl hydrozone (10 M) was included in the intracellular pipette solution. NPY was also not effective when ATP was substituted by the non-hydrolysable ATP analogue adenosine 5-[, -methylene]-triphosphate (AMP-PCP). NPY inhibited Ca²⁺-activated K⁺ channel activity when ATP was replaced by adenosine 5-O-(3-thiotriphosphate) (ATP [-S]) and the inhibition was not readily reversed upon washing. Protein kinase inhibitor (1 M), a specific inhibitor of adenosine 3, 5-cyclic monophosphatedependent protein kinase, had no significant effect on the inhibitory action of NPY. The effect of NPY on single-channel activity was inhibited by the tyrosine kinase inhibitor genistein (10 M) but not by daidzein, an inactive analogue of genistein. These observations suggest that the inhibition by NPY of Ca²⁺-activated K⁺ channels is mediated by ATP-dependent phosphorylation. The inhibitory effect of NPY was antagonized by the tyrosine kinase inhibitor genistein.     

Central effects of histamine H2-receptor agonists and antagonists on nociception in the rat

The effects of intracerebroventricular injection of histamine H₂-receptor agonists (4-methylhistamine, 4-MeH; dimaprit, DIM), H₂-antagonists (cimetidine, CIM; ranitidine, RAN; famotidine, FAM) and of the DIM chemical analogue SK&F 91487 on hot-plate latency in rats were examined. Both DIM (0.4–0.8 mol/rat) and 4-MeH (0.4–0.8 mol/rat) significantly enhanced the pain threshold, whereas SF&F 91487 (0.8 mol/rat) had no effect, indicating that DIM antinociception is specifically due to its activity on histamine (HA) receptors. The H₂-antagonists CIM (0.8 mol/rat) and RAN (0.6 mol/rat) also enhanced the pain threshold, while FAM (0.03 mol/rat) did not modify pain latency. When injected before 4-MeH, FAM reduced the antinociceptive effect of 4-MeH. These findings suggest that the antinociceptive activity of CIM and RAN is not related to specific blockade of H₂-receptors and that the activation of HA-H₂-receptors is inhibitory to nociception.     

Variable antifungal susceptibility of wild-typeCandida albicans phenotypes from neutropenic hosts

The aim of the present study was to investigate the relationship between phenotypes ofCandida albicans strains isolated from clinical specimens and susceptibility of the strains to two antifungal agents, itraconazole and fluconazole. Oropharyngeal, gastrointestinal tract, and urogenital tract specimens were collected from 131 neutropenic patients withCandida infection who had received no previous prophylactic treatment. The most frequent species isolated wasCandida albicans, followed byCandida glabrata,Candida tropicalis, Candida krusei, andCandida parapsilosis. Each of the 44Candida albicans strains recovered was found to express one of four phenotypes: smooth, irregular, fuzzy or stipple. Mean minimum inhibitory concentrations (MICs) of itraconazole and fluconazole as determined by the microdilution method and the E-test were consistently higher forCandida albicans strains expressing the stipple phenotype. The mean MICs for the four phenotypes of theCandida albicans strains ranged between 0.35 g/ml and 2.41 g/ml for itraconazole and 2.78 g/ml and 5.2 g/ml for fluconazole. Antifungal susceptibility of the stipple phenotype requires careful appraisal, especially in patients clinically unresponsive to azole chemotherapy or in cases of life-threatening, deepseatedCandida infections.