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.     

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.     

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.     

Effects of ryanodine on skinned skeletal muscle fibers of the rabbit

The mechanism(s) of ryanodine-induced contracture of skeletal muscle were studied in skinned fibers from soleus (SL) and adductor magnus (AM) (slow- and fast-twitch skeletal muscles) of rabbits. Pieces of SL or AM were homogenized (sarcolemma disrupted). Single fibers were dissected from the homogenate and mounted on photodiode force transducers. At concentrations 1–50 M, ryanodine slightly but significantly increased the submaximal Ca²⁺-activated tension development of the contractile proteins in skinned fibers of AM but not of SL. Ryanodine in uptake phase or release phase increased caffeine-induced tension transients in the SR of both muscle types; however, no dose-response relation was found. Ryanodine 1 M decreased, however, the second control tension transients in a dose-dependent manner. The depression was nearly irreversible and activity-dependent. The concentrations of ryanodine that inhibited the second control tension transients by 50% were 10 M and 5 M for SL and AM, respectively, following ryanodine administration in the release phase, and 100 M and 30 M, respectively, for these preparations after the drug was present in the uptake phase. The quantity of calcium released from the SR by Triton X-100 and caffeine in the second control tension transient was unchanged by ryanodine at all concentrations tested when compared with that of the absence of ryanodine. The present findings suggest that the ability of ryanodine to increase immediate calcium release from the SR, and in AM but not SL, to increase the sensitivity of the contractile proteins to Ca²⁺ underlies the contracture caused by this agent in intact skeletal muscles. The delayed decreased Ca²⁺ efflux by caffeine, as evidenced by depression of tension transient with no change in the calcium content may be responsible for the decreased twitch tension caused by this agent.     

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%).     

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.     

Antibody response inPlasmodium vinckei malaria after treatment with chloroquine and adjuvant interferon-γ

The antibody response of mice infected withPlasmodium vinckei after treatment with chloroquine either alone or in combination with interferon- (IFN-) was determined. Sequential serum samples were drawn from BALB/c mice receiving either 240 g chloroquine on the day of infection or 120 g chloroquine plus 10⁴ units IFN- daily for 11 days beginning on day 3 prior to infection. Mice treated with additional IFN- showed an early induction of IgG2a response and a reduction in IgG1 antibodies as detected by the immunofluorescence technique at between 10 and 16 days after infection as compared with mice treated with chloroquine alone. Thus, IFN- may partly exert its antimalarial activity via the induction of IgG2a antibody formation. At 4–6 weeks after infection, when mice from both groups resisted homologous re-infection, the predominant antibody isotypes found in both groups were IgG1 and IgG2a. Serum samples obtained from mice in both treatment groups at 6 weeks after infection were used for serum transfer experiments. When parasitised erythrocytes were preincubated with such immune serum, a retardation of the course of parasitaemia by 2 days was observed.     

Interferon-γ for treatment of severe atopic eczema in two children

Two 4- and 5-year-old children suffering from refractory atopic dermatitis were treated with recombinant interferon- (rIFN-). rIFN- was injected at 50 g subcutaneously three times a week in the first child for 3 weeks, followed by three times 25 g in week 4. In the other child two treatment courses of 4 weeks were given after a break of 2 weeks. Therapy was well tolerated. In child one reductions in eczematous body surface and severity of lesions were observed, while no beneficial effect was seen in the other. Clinical chemistry data remained unchanged. Immunological studies performed in parallel showed a decrease in total serum IgE of 50% in child 1, a decrease in spontaneous in vitro IgE production, an increase in in vitro production of interleukin-6, and a normalization of previously decreased in vitro lymphocyte responses to several mitogens. While marked immunological changes were noted during IFN- treatment, clinical benefits were not encouraging. Diminished IFN- production has been claimed to be a major pathogenic factor in atopic eczema. Our results indicate that the pathogenesis is more complex. Clinically, we were unable to confirm previous observations in adults. Further studies are needed before IFN- can be recommended for therapy of pediatric atopic eczema.Abbreviations IFN- interferon- - IL interleukin     

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.     

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.     

Inhibition of the M current in NG108-15 neuroblastoma × glioma hybrid cells

The M current, I _M, a voltage-dependent non-inactivating K current, was recorded in NG108-15 neuroblastoma × glioma hybrid cells, using the whole-cell mode of the patch-clamp technique. We studied inhibition of the M current by bradykinin, phorbol dibutyrate (PDBu), an activator of protein kinase C (PKC), and methylxanthines. Focal application of 0.1–5 M bradykinin inhibited I _M by about 60%; 5 nM bradykinin inhibited by about 40%. Bath application of 0.1 M and 1 M PDBu diminished I _M to about half of the control value. Staurosporine, a PKC inhibitor, applied for 35–43 min in a concentration of 0.3 M significantly reduced the effect of 1 M PDBu. M current blockage by PDBu could be partly reversed by bath application of H-7 (51–64 M), another PKC inhibitor. These observations suggest that the PDBu effect is really due to activation of PKC. The findings are compatible with the view [Brown DA, Higashida H (1988) J Physiol (Lond) 397:185–207] that the bradykinin effect on I _M is mediated by PKC. However, three further observations suggest that this is only true for part of the bradykinin effect. When the suppression of I _M by 1 M PDBu was fully developed, 0.1 M bradykinin produced a further inhibition of I _M. Down-regulation of PKC by long-term treatment with PDBu reduced the effect of 0.1 M bradykinin significantly but did not abolish it. Staurosporine (0.3 M, applied for 31–46 min) failed to reduce the effect of 5 nM bradykinin significantly. The M current could be reversibly blocked by methylxanthines (caffeine, isobutylmethylxanthine, theophylline) in the millimolar range, probably because of a direct action on the M channels.     

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.     

Effect of lysolecithin on blood vessel reactivity and of some ethers and esters of glycerophosphocholine and phosphocholine,respectively, on aggregation of rat platelets

Infusion of lysolecithin (LPC; e.g. 88 g/ml for 0.5–1.0 min) did not significantly impair the vasopressor action of norepinephrine (NE), prostaglandin F₂ (PGF₂) and extract of posterior pituitary (EPP) in the isolated perfused hind legs of rats. In other words, vascular smooth muscle behaves differently from the smooth muscle of the guinea-pig small intestine, since, in the latter, contractions evoked by acetylcholine, prostaglandins etc., are inhibited by LPC. Triton X 100 which, by comparison, was used as a detergent effective on the guinea-pig small intestine, depressed the vasopressor effect of NE, PGF₂ and EPP.LPC, at low concentrations (40 mol/l), potentiated (15% max.) ADP-induced platelet aggregation (PA) in rat PRP but, at high concentrations, inhibited PA (IC₅₀=390 mol/l). 2-Hexadecylglycerophosphocholine and its short-chain 1-alkyl ethers, which are structurally related to platelet-activating factor, as well as some long-chain alkanol phosphocholine esters, were somewhat more active than LPC. Dipalmitoyllecithin (4–700 mol/l) was without any effect.     

A simple method for measurement of inulin in nanoliter samples using glass capillaries

Summary A simple method using glass capillaries instead of microcuvettes for measurement of inulin in nanoliter samples is given. Inulin was determined with anthron reagent (5 or 10 nl samples +3 l anthron reagent). Glass capillary tubes (o.d.=1 mm, i.d.=0.68 mm, length=150 mm) in which the chemical reaction took place during incubation at 56°C were directly introduced into the optical system of a Zeiss spectrophotometer PMQ II with sphere attachment and objective.Extinction was measured vertically to the axis of the capillary. The changes of extinction of 20 different capillaries with the blank at different positions was only 1.13×10^–3. The exactness of measurement in the concentration range of 100 200 400 750 1500 3000 mg-% inulin was for 5nl/3 l: 19.8 11.0 6.7 4.7 3.0 2.2%. 10nl/3 l: 13.0 8.4 5.1 3.9%.This method of measurement may also be applicable for other colorimetric reactions with nanoliter samples.This work was supported by Fonds zur Förderung der wissenschaftlichen Forschung.     

Transformation of Saccharomyces cerevisiae with plasmids containing fragments of yeast 2 μ DNA and a suppressor tRNA gene

Hybrid plasmids have been constructed containing segments of the yeast plasmid 2 DNA, the yeast ochre-suppressing SUP4.0 gene and the bacterial plasmid pBR322. Yeast transformation is detected with a host containing multiple ochre auxotrophic mutations. The transformed SUP4.0 gene is active and can promote growth in the absence of all the requirements. Plasmids containing different fragments of 2 DNA all appear to be active in high frequency transformation of yeast containing 2 DNA, except those containing the HindlII-D fragment. The transforming plasmids undergo recombination with the indigenous 2 DNA. Integration of the transforming plasmid into the host chromosome has been detected by hybridization of restriction enzyme cleaved DNA with labelled pBR322. The plasmids contain restriction enzyme sites which can be used for cloning other genes into yeast.Abbreviations kb kilobase pair - 2 the yeast plasmid of 6.2 kb size     

In vitro activity of nonsteroidal antiinflammatory agents,phenotiazines, and antidepressants againstBrucella species

The in vitro antimicrobial activity of streptomycin, rifampicin, tetracycline, seven nonsteroidal antiinflammatory agents (acetyl-salicylic acid, piroxicam, indomethacin, ibuprofen, ketoprofen, sulindac, and diclofenac), and eight phenotiazine derivatives and antidepressant agents (clorpromazine, fluphenazine, amitryptiline, clomipramine, imipramine, maprotiline, sertraline, and diazepam) against 62 strains ofBrucella spp. was tested. Diclofenac was the most active of the anti-inflammatory agents (MIC90 = 16 g/ml). The activity of the phenotiazines and antidepressants was heterogeneous, with MIC90S ranging from 16 g/ml for sertraline and 32 g/ml for fluphenazine and clomipramine to > 512 g/ml for diazepam. When the six most active anti-inflammatory agents and the six most active psychiatric drugs were tested at pH 5 and pH 4, the MICs remained unchanged except for those of fluphenazine; the MIC50 and MIC90 of this agent increased by one dilution.     

The effect of praziquantel on the pseudophyllidean cestodeBothriocephalus acheilognathi in vitro

AdultBothriocephalus acheilognathi were incubated in solutions containing 0 (control), 0.1, 1.0, 10.0 and 100 g praziquantel per ml (0, 10², 10³, 10⁴ and 10⁵ gl^–1) of 0.9% saline for 5, 15 and 60 min at a temperature of 18°C. The worms contracted immediately upon being placed in the drug. Scanning and transmission electron microscopy revealed considerable tegumental damage particularly in the neck region. Vacuolization and bubbling to the tegument occurred in all of the drug solutions tested. Exposure to drug concentrations of more than 1.0 gml^–1 (10³ gl^–1) praziquantel for 15 min or greater resulted in many of the bubbles bursting and releasing their contents to the exterior. Mature proglottides were distorted and had occasional large swellings resulting in the mass expulsion of eggs. Praziquantel had no ovicidal activity. Exposure to drug concentrations of 100 g (10⁵ gl^–1) praziquantel per ml saline for 24 h was not lethal to the worms.     

Sodium salicylate: alternate mechanism of central antipyretic action in the rat

Infusion of sodium salicylate (50.0 or 100.0 g/l) into the ventral septal area (VSA) of the rat brain suppressed Prostaglandin-E₁-induced hyperthermia. Infusion of artificial cerebrospinal fluid (aCSF) or 10.0 g doses of salicylate did not. The suppression of intracerebroventricularly-induced (icv) Prostaglandin E₁ (PGE₁) hyperthermia was not due to a hypothermic action of salicylate since salicylate infusions given during cold exposure (10.0°C) did not lower core body temperatures. A possible interaction between salicylate and endogenous arginine vasopressin (AVP) was investigated. Infusion of both salicylate (50.0 g/l) and either AVP antiserum or AVP antagonist into the VSA resulted in PGE hyperthermias occurring at levels which were not different from control levels as opposed to enhanced hyperthermia (antiserum or antagonist alone) or suppressed hyperthermia (salicylate alone). These results are consistent with the notion that sodium salicylate infusions within the VSA enhance AVP action and thus bring about the attenuation of PGE-induced hyperthermia.     

Lack of additive effect between mechanisms of resistance to carbapenems and other beta-lactam agents inPseudomonas aeruginosa

Eighty-nine clinical isolates resistant (n=61) or susceptible (n=28) to imipenem and exhibiting the main patterns of susceptibility to other -lactam agents (wild type pattern, penicillinase pattern, constitutive cephalosporinase pattern) were studied in order to investigate (i) the mechanism of resistance involved and (ii) whether resistance to carbapenems affects the level of resistance to other -lactam agents and, conversely, if resistance to other -lactam agents affects the level of resistance to carbapenems. For this purpose, the presence of OprD protein in the cell wall was detected by Western blot and -lactamase activity by spectrophotometric assay and isoelectric focusing. OprD expression was not detectable in the imipenem-resistant (MIC16 g/ml) strains. It was decreased in half the strains for which MICs of imipenem were 2 to 8 g/ml and was close to a normal level in the most susceptible strains (MIC 1 g/ml), thus demonstrating a direct correlation between the level of susceptibility to imipenem and the level of OprD expression. No imipenemase activity was detected in imipenem-resistant strains. Synergy between imipenem or meropenem and BRL42715 was observed for all of the strains, demonstrating the role of cephalosporinase in carbapenem resistance. Within each pattern of susceptibility, the mean MICs of -lactam agents other than carbapenems were similar, whether the strains were susceptible or resistant to imipenem. Conversely, the mean MICs of imipenem or meropenem for either the imipenem-resistant or the imipenem-susceptible strains were similar, regardless of the susceptibility of these strains to the other -lactam agents. Thus, when several mechanisms of resistance to -lactam agents are present in the same strain ofPseudomonas aeruginosa, there is no additive effect between these mechanisms.     

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.