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.     

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.     

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.     

Differential regulation of the activation of human eosinophils,macrophages, and neutrophils: Effect of the allergic mediator release inhibitor CI-949

The allergic mediator release inhibitor CI-949 [5-methoxy-3-(1-methylethoxy)-1-phenyl-N-1H-tetrazol-5-yl-1H-indole-2-carboxamide,l-arginine salt] was evaluated for its effect on the activation of human eosinophils, macrophages, and neutrophils by the phagocytic stimulus serum-opsonized zymosan (SOZ). CI-949 inhibited the SOZ-stimulated respiratory burst of eosinophils, measured as the generation of superoxide anion, with an IC₅₀ of 22.8 M. At concentrations up to 100M, CI-949 had no inhibitory effect against superoxide anion generation by human macrophages, while inhibiting the response of human neutrophils by only 24.8 percent. CI-949 exhibited a different profile of inhibitory activity against lysosomal enzyme release by these cells. At 100 M, CI-949 had no inhibitory effect against release of eosinophil peroxidase while inhibiting release of the macrophage lysosomal enzyme N-acetyl--d-glucosaminidase by only 11.7 percent. In contrast, CI-949 inhibited the release of the neutrophil primary granule enzyme myeloperoxidase with an IC₅₀ of 21.4M, while inhibiting release of lysozyme from secondary granules with an IC₅₀ of 99.3M. These results demonstrate that oxygen radical generation and lysosomal enzyme release by human eosinophils, macrophages, and neutrophils are differentially regulated by CI-949. These results suggest that these inflammatory cells may have distinct stimulus-response coupling mechanisms.     

The long-term cellular response to taxol in peripheral nerve: Schwann cell and endoneurial cell changes

Summary Taxol, an agent known to stabilize and increase the assembly of microtubules, causes long-lasting nerve damage when injected into peripheral nerve. In the present study, the cellular response to taxol in rat sciatic nerve was studied for up to 6 months after a single injection. The initial response of Schwann cells to taxol at the lesion site involved the accumulation of cytoplasmic microtubules which persisted up to 4 months after injection. Some novel microtubule-related cytoplasmic structures were also noted; these included microtubule-lined cytoplasmic crypts and channels. Despite these structural abnormalities, Schwann cells were able to produce myelin sheaths around taxol-induced axonal bulbs. This myelination showed some anomalies up to 4 months consisting of the widening of myelin lamellae, variability in sheath thickness, paranodal myelin infoldings and myelin protrusions. With time the diameter of the axonal bulbs decreased and, concomitant with this, more normal-appearing remyelination occurred. By 5 months, the previously noted myelin abnormalities were rare. By 6 months only a few naked axonal segments occurred at the lesion site. In endoneurial fibroblasts and macrophages cytoplasmic lamellar microtubule formations were frequent at 10 weeks. Needle-like cytoplasmic structures appeared within endoneurial cells at the site of the lesion after 10 weeks. By 3 months these inclusions were numerous and were often surrounded by extended cytoplasmic processes. The needles were up to 50 m long and 3 m wide and probably represented cholesterol. By 4 months the number of cytoplasmic needles decreased and at 5 months onwards none was observed. The present findings confirm and extend previous findings that taxol has a long-lasting effect upon both Schwann cells and endoneurial cells and that this is related to abnormal tubulin synthesis.     

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.     

Acute effects of lysine vasopressin injection (Single and continuous) on urinary composition in the conscious water diuretic rat

Summary ADH (synthetic lysine-vasopressin) was administered to conscious rats undergoing steady, sustained water diuresis. The magnitude and duration of the transient antidiuresis induced by single i. v. ADH injection (0.5–8 mU/100 g body weight) increased with the dose to a maximum urinary osmolality of 630 -osmole/g H₂O (returning towards control values within 1 hr).Both the magnitude of, and the time to attain, a stable antidiuretic effect with continuous i.v. ADH infusion (2.5–30 U/min/100 g body weight) varied with the dose. The maximal osmolality with 15 was almost as high as that with 30 U/min/100 g body weight (1815 and 1928 -osmole/g H₂O), but took longer to attain (4 and 2 hr, respectively).With both single and continuous ADH injection, a variable, inconsistent natriuresis and kaluresis occurred; the peak natriuresis preceded the time of maximal urinary osmolal and Na concentrations.The data are discussed in relation to (a) physiological rate of secretion of endogenous ADH and (b) the mechanisms responsible for the natriuresis.It is concluded that the natriuretic effect of ADH has little physiological significance in salt regulation.     

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.     

Scanning electron microscopy of merogonous stages ofEimeria falciformis var.pragensis inMus musculus

Asexual stages ofEimeria falciformis var.pragensis in Swiss-Webster mice were studied by scanning electron microscopy. Sporozoites were present in the cecum and colon 2 h post-inoculation (PI) and measured 11.3×2.1 m (9–13.9×2–2.2 m). Sporozoites penetrated epithelial cells with an extended anterior end and were constricted at the site of entry. Asexual generations were found in the cecum and colon epithelial cells. In meronts found at days 3–6 PI, merozoites matured synchronously, were oriented in the same direction, and were arranged in a helical pattern. Such meronts measured 11.3×6.4 m (8–13.7×5–7.4 m) and contained 8–12 meroxoites, which measured 11.9×1.5 m (7.4–15.7×1.3–1.8 m). Meronts which were present at day 7 PI measured 9.5×8.2 m (9–10.5×7–9.5 m) and contained 20–50 small merozoites which budded asynchronously from a central residuum. At days 3–7 PI, parasitized epithelial cells had shorter and fewer or no microvilli. The lumenal plasmalemma of the host cell was often disrupted or absent in cells containing mature meronts and escaping merozoites. At day 6 PI, phagocytic cells appeared on the epithelial surface, some of which were in contact with merozoites. Small foci of exposed basal lamina were present at day 7 PI in areas where cells had sloughed from the epithelium.     

Fibre types inLimulus telson muscles: morphology and histochemistry

Summary Using a variety of techniques, we have demonstrated the presence of at least two fibre types inLimulus median telson levator muscle. By light and electron microscopy, large (21 56 m² mean cross-sectional area) fibres have A-bands of 4.1 m, one-half I bands of 2.15 m and Z lines 0.5 m in width. Few mitochondria are found in these fibres, which comprise 54% of those present in a given microscope field and which occupy 82% of the total cross-sectional area. Small fibres (484 m² mean cross-sectional area) have A bands of 6.3 m, one-half I bands of 3.1 m and Z lines between 0.5 and 1.0 m in width and are rich in mitochondria. Although small fibres comprise nearly one-half (46%) of the fibres in a field, they occupy only 18% of the total cross-sectional area.Histochemical staining for alkaline-stable myofibrillar ATPase activity and mitochondrial reduced -nicotinamide adenine nucleotide (-NADH) tetrazolium reductase activity confirms the presence of two fibre types. The large fibres react positively for the myofibrillar ATPase activity and negatively for the mitochondrial enzyme activity. The reverse is seen with the small fibres. Some fibres of intermediate size, having intermediate staining characteristics, were also observed. Native gel electrophoresis of both myofibrillar and purified myosin preparations supports the observed differences in myofibrillar ATPase activity in that two myosin isozymes are resolved on pyrophosphate gels. Although the thick filaments isolated from unstimulated small fibres are longer (>6.0 m) than those isolated from unstimulated large fibres (4.26 m), all have a similar appearance with respect to the arrangement of myosin heads on their surfaces, and similar diameters. The implications of the observed heterogeneity of fibre types is discussed with reference to previously reported phenomena inLimulus telson muscle, including changes in length of thick filaments on fibre stimulation and the shape of the length-tension curve obtained from fibre bundles.     

Relation between cell na extrusion and transtubular absorption in the perfused toad kidney: the effect of K,ouabain and ethacrynic acid

Summary Transtubular absorption of Na and Cl, and intracellular ion concentrations were evaluated in toad kidneys perfused with solutions containing K and without K, and in the presence of 1 mM Ouabain and 1 mM Ethacrynic acid. The following values were obtained with 8.5 mM K: Transtubular absorption of Na and Cl68% (percent of filtered load); cell content 294 mole Na, 433 mole K, 100 mole Cl/g solids. Lack of K in the perfusate diminished transtubular absorption to 25% and the cells gain 244 mole Na/g solids, and lose an equimolecular quantity of K. The process is reversible upon raising the K concentration in the perfusate. Ouabain inhibits transtubular absorption to 6%; the cells lose about 110 mole K/g solids, but cellular Na is maintained at the control levels. Ethacrynic acid inhibits transtubular absorption to 3%; the cells approximately double their Na and Cl content, but their K is maintained at the control levels. These observations cannot be explained exclusively in terms of an effect on the distal tubule. Probably proximal as well as distal tubules are involved. A single Na pump seems insufficient to account for all experimental findings. The existence of two separate pumps is therefore proposed.     

Localization and elasticity of connectin (titin) filaments in skinned frog muscle fibres subjected to partial depolymerization of thick filaments

Summary The localization and elasticity of connectin (titin) filaments in skinned fibres of frog skeletal muscle were examined for changes in the localization of connectin and in resting tension during partial depolymerization of thick filaments with a relaxing solution containing increased KCl concentrations. Immunoelectron microscopic studies revealed that deposites of antibodies against connectin at a sarcomere length of 3.0 m remained at about 0.8 m from the M-line, until the thick filament was depolymerized to the length of approximately 0.4 m. On further depolymerization, the bound antibodies were found to move towards the Z-line and, on complete depolymerization, were observed to be within 0.3 m of the Z-line; a marked decrease in resting tension accompanied this further depolymerization. These results suggest that connectin filament starts from the Z-line, extends to the M-line, and contributes to resting tension. After partial depolymerization of thick filaments, the distances between the anti-connectin deposits and the Z-line and between anti-connectin deposits and the M-line increased with sarcomere length, suggesting that connectin filaments are elastic along their entire length.     

Flow cytometric analysis ofEimeria tenella sporozoite populations exposed to salinomycin sodium in vitro: A comparative study using light and electron microscopy and an in vitro sporozoite invasion-inhibition test

Eimeria tenella sporozoites exposed to 100, 70, 60 and 50 g salinomycin sodium (SAL)/ml medium 199 at 41° C and then stained with propidium iodide/fluorescein diacetate were analysed by means of flow cytometry (FCM). After 20 min exposure, they showed dose-dependent alterations in their size and shape, i.e. ballooning of most cells, and enhanced intracellular esterase activity as compared with untreated controls. After longer exposure periods (40 and 70 min), inflated cells gradually changed into shrivelled or crumpled, nonviable ones, thereby showing a gradual decrease in esterase activity and a gradual loss of membrane integrity (RFA⁺). As compared with untreated controls, sporozoites treated with 10 g SAL/ml showed negligible RFA⁺ values (0.4%–2%), whereas those exposed to 1 and 0.1 g SAL/ml and to the solvent dimethylsulfoxide (DMSO, 1%) did not, even after 70 min exposure. Slight to severe structural changes manifesting as an extremely wavy surface (1 g SAL/ml), vacuolization of the cytoplasm, distension or destruction of the mitochondrion and rupture of cell membranes (10 g SAL/ml) were seen not only at higher SAL concentrations but also (rarely) at lower ones. The ability of sporozoites to invade primary chick-kidney cells was significantly inhibited by 70, 60 and 50 g SAL/ml. In general, there were close relationships between findings obtained using FCM, electron microscopy and an invasion-inhibition test. The results indicate that FCM is a reliable and sensitive technique for characterizing the parasiticidal effects on and the possible mode of action of drugs in free coccidian sporozoites.     

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.     

Naegleria fowleri andN. lovaniensis: Differences in sensitivity to trimethoprim and other antifolates

The growth ofNaegleria fowleri cultures in a BCS medium was not affected either by trimethoprim at 400 g/ml or by aminopterine, 3,5-diaminopterine and methotrexate at 500 g/ml.N. lovaniensis propagation in the same medium was inhibited with 10 g/ml of trimethoprim, 50 g/ml methotrexate and 100 g/ml 3,5-diaminopteridine. Aminopterine was ineffective at a concentration of 500 g/ml. The inhibitory effect of trimethoprim onN. lovaniensis cultures depended on the medium composition and could be neutralized by an addition of folic or tetrahydrofolic acids and a suspension of heat-killedEnterobacter aerogenes. Thymine, thymidine, hypoxantine and 2-amino-4-hydroxy-6-(tatrahydroxybutyl)-pteridine did not have an adverse effect. Trimethoprim activity inN. fowleri cultures could not be enhanced by the addition of Triton X-100 and Polymyxine B. Cryolyzate ofN. fowleri amoebae did not influence the trimethoprim inhibition ofN. lovaniensis cultures. Deviation in dihydrofolatereductase chemical structure or thymine dependency seems to be the probable explanation forN. fowleri antifolate resistance.     

Morphology of the lamina propria in the human esophagus with special reference to the proprial papillae

The three-dimensional configurations of the proprial papillae in the human esophagus were observed by light microscopy, routine transmission electron microscopy, and scanning electron microscopy combined with NaOH maceration. Numerous finger-like or filiform papillae with a height of about 100m and a width at the base of approximately 30m were clearly distributed in the uppermost proprial layer at approximately equal intervals. The adepithelial surface of the proprial papillae was bordered by a reticular fiber sheet that was stained a deep black color by silver staining. The papillae possessed blood capillaries with fenestration, nerve fibers, and free cells such as lymphocytes, eosinophils, mast cells, and Langerhans-like cells. These findings clearly demonstrate characteristic three-dimensional features of proprial papillae, and their constituent cellular and structural elements in human esophagus.     

Riluzole specifically blocks inactivated Na channels in myelinated nerve fibre

The effects of 0.15–250 M riluzole, a novel psychotropic agent with anticonvulsant properties, were studied on voltage-clamped nodes of Ranvier of isolated nerve fibres of the frog. When added to the external solution, the drug rapidly and reversibly inhibited both K and Na currents with an apparent dissociation constant of 0.09 mM. The riluzole-induced decrease of these currents was not use-dependent. At concentrations up to 100M, the drug had no noticeable effect on the time course of Na current inactivation nor on the shape and the position along voltage axis of the Na conductance/voltage relationship. On the other hand, it induced substantial shifts towards negative voltages of the steady-state Na inactivation/voltage curve. From these results, according to the modulated-receptor model, an apparent dissociation constant of 0.29 M could be calculated for riluzole-induced blockage of inactivated Na channels. The recovery from Na current inactivation was also affected by the drug. It is concluded that riluzole is a highly specific blocker of inactivated Na channels, which is more than 300 times more effective on these channels than on K or resting Na channels.     

Der Phosphocreatingehalt des unbelasteten und des ischämischen Gehirns

Zusammenfassung Die mit unterschiedlichen Methoden gefundenen PC-Werte des Gehirns ergaben bisher Mittelwerte von 2,0–3,5 mol/g Frischgewicht. Es wird an in situ eingefrorenen Gehirnen unter milden Enteiweißungsbedingungen ein mittlerer Gehalt von 4,6 mol/g (Maximalwert 4,98 mol/g) gemessen und damit bewiesen, daß auch im Gehirn 50–60% des Gesamtcreatins als Phosphocreatin vorliegen.Mit 1 TextabbildungDurchgeführt mit Mitteln der Deutschen Forschungsgemeinschaft.     

Macrophage-mediated killing of splenic lymphocytes in adjuvant-induced arthritis

After 72 hrs in culture, unseparated spleen cells from rats with adjuvant-induced arthritis (AA) stimulated with high concentrations of Con A (>0.63 g/ml) leaked more lactate dehydrogenase (LDH) than did either unstimulated cultures or cultures stimulated with lower concentrations of Con A. This reflects and increase in dead or dying cells at concentrations of Con A >0.63 g/ml. Since Con A did not increase LDHlleakage in macrophage-depleted cultures of AA splenocytes, the decreased viability in unseparated, Con A-stimulated cultures appears to be a macrophage-dependent phenomenon. Since the increase in LDH release at high concentrations of Con A paralleled the decrease in [³H]-thymidine incorporation, these results suggest that Con A (>0.63 g/ml) induces macrophages from AA rats to kill splenic lymphocytes in culture.     

Inhibition of extensorγ motoneurons by antagonistic primary and secondary spindle afferents

Summary About 2/3 of the efferents isolated from the medial gastrocnemius nerve were inhibited by longitudinal high-frequency vibration applied to the tendons of the non-contracting pretibial flexors (decerebrate cats). The inhibition appeared at 15–25 m amplitude of vibration and increased up to a maximum at nearly 100 m. Increasing the frequency of vibration from 100 to 300 Hz increased the inhibition. The reflex effects elicited by muscle vibration corresponded well in incidence and magnitude with those evoked by tetanization of the deep peroneal nerve at group I stimulus strength. The reflex disappeared when the nerve supply of the vibrated muscles was cut. The sensitivity of some pretibial proprioceptors to vibration was also tested. It is concluded that primary spindle endings of the pretibial flexors inhibit the extensor motoneurons. Some findings hint at a spinal pathway involving I a inhibitory interneurons.In addition, an inhibitory action of pretibial group II afferents, probably secondary spindle endings, on extensor efferents was demonstrated.The described fusimotor inhibition by antagonistic muscle spindle afferents is a further example of --linkage.