Serum carbonic anhydrase III,an enzyme of type I muscle fibres,and the intensity of physical exercise

The effects of 30 min running with stepwise increasing intensity (exhaustive, energy demand approx. 50 100% ofVO_2max), 60 s supramaximal running (anaerobic, 125% ofVO_2max) and 40–60 min low-intensity running (acrobic, 40–60% ofVO_2max) on serum concentration of muscle-derived proteins were studied in 5 male and 5 female elite orienteerers. S-Carbonic anhydrase III (S-CA III) was used as a marker of protein leakage from type I (slow oxidative) muscle fibres and S-myoglobin (S-Mb) as a non-selective (type I+II) muscular marker. The fractional increase in S-CA III (S-Ca III) was 0.37±0.09 (mean±SEM,p<0.001), 0.10±0.05 (N. S.) and 0.46±0.09 (p<0.001) 1 h after exhaustive, anaerobic and aerobic exercise, respectively. The corresponding values for S-Mb were 1.45±0.36 (p<0.001), 0.39±0.13 (p<0.01) and 0.67±0.18 (p<0.001). The value for the S-CA III/S-Mb ratio was 0.68±0.03 after the acrobic exercise, but only 0.25–0.26 (p vs. aerobic exercise <0.001) after the two high-intensity forms of exercise. Since type I fibres of skeletal muscle are known to be responsible for power production during low-intensity exercise, whereas fibres of both type I and type II are active at higher intensities, the S-CA III/S-Mb ratio may depend on the recruitment profile of type I vs. type I+II fibres.     

Candida albicans induces the release of inflammatory mediators from human peripheral blood monocytes

Candida albicans (C. albicans) is a major nosocomial pathogen. We examined arachidonic acid (AA) and cytokine production by monocytes stimulated with C. albicans. [¹⁴C]-AA labeled monocytes released 8.9 ±2.3% of the incorporated AA following stimulation with live C. albicans (C. albicans: monocyte of 161) (P=0.0002). Prior studies indicate that soluble-mannans and-glucans antagonize mannose and-glucan receptors, respectively. Preincubation of monocytes with-mannan (100g/ml) caused 45.8 ±5.7% inhibition of [¹⁴C]-AA release, whereas-glucan (100g/ml) yielded 43.7 ±6.0% inhibition (P<0.05 for each compared to control). Additionally, monocytes stimulated with C. albicans also released interleukin-1 (IL-1), tumor necrosis factor- (TNF), interleukin-6 (IL-6) and interleukin-8 (IL-8). However, a-mannan or-glucan failed to inhibit IL-1 release. These data indicate that C. albicans induces monocytes to release AA and inflammatory cytokines. Furthermore, AA, but not cytokine liberation, is partially mediated by a-mannan and-glucan components of the fungus.     

Endocrine activity of the “silent” adrenocortical adenoma is uncovered by response to corticotropin-releasing hormone

Summary The purpose of this study was to ascertain whether the pituitary-adrenal responses to human corticotropin-releasing hormone (hCRH) in non-functioning adrenocortical adenoma would uncover a functional activity in these adrenal nodules. Eleven patients with incidentally discovered silent adrenocortical adenoma and eleven controls were studied. The initial clinical and laboratory examination, including an overnight 1 mg dexamethasone suppression test, revealed no abnormalities in any of the subjects. IR-ACTH and serum steroids (F, S, P, 17OHP, 18OHB, and aldosterone) were normal in both controls and patients. After pulse IV injection of 100 g hCRH, the cortisol response was significantly exaggerated (P=0.01). Stimulated plasma ACTH levels were, however, significantly lower in patients than in controls (P=0.01), indicating counter-feedback regulation of cortisol. The peak cortisol/peak ACTH ratio (F_max/ACTH_max) in the patients was significantly elevated (26.8±4.37 nmol/ng vs. 14.6±2.16 nmol/ ng,P=0.02). Two further patients with incidentally discovered pre-Cushing's adrenocortical adenoma displayed an even higher ratio (43.5 and 45.5 nmol/ng). In established Cushing's syndrome due to an autonomous adrenocortical adenoma, suppression of ACTH and of the ACTH response to hCRH occurs with a very high basal cortisol/ basal ACTH ratio. Our findings suggest some functional activity even in clinically silent adrenocortical adenoma. Response to hCRH uncovers a continuous spectrum between adrenocortical adenoma, pre-Cushing's, and Cushing's syndrome.Abbreviations ACA adrenocortical adenoma - ACTH adrenocorticotropin - DHEAS Dehydroepiandrosterone sulfate - F cortisol - hCRH human corticotropin releasing hormone - P progesterone - S 11-deoxycortisol - 17OHP 17-hydroxyprogesterone - 18OHB 18-hydroxy-corticosterone     

Characterization and localization of α-connectin (titin 1): An elastic protein isolated from rabbit skeletal muscle

Summary A simplified procedure to isolate-connectin (titin 1, TI), a gigantic elastic protein, from rabbit skeletal muscle is described. A rapid column chromatography step to concentrate-connectin is introduced. Separation of-connectin from-connectin is introduced. Separation of-connectin from-connectin (titin 2, TII) in the presence of 4 M urea at pH 7.0 did not cause any change in the secondary structure of-connectin as judged by circular dichroic spectra. Ultraviolet absorption spectra and the amino acid composition of-connectin (MW, approximately 3×10⁶) were similar to those of its proteolytic product,-connectin (MW, approximately 2×10⁶). Circular dichroic spectra suggested that both- and-connectin consist of 60%-sheet and 30%-turn. It thus appears that the whole elastic filament of connectin has a folded-strand structure. Proteolysis of-connectin by calpain resulted in formation of-connectin and smaller peptides. The-connectin interacted with both myosin and actin filaments similarly to-connectin. Polyclonal antibodies raised against 1200 kDa peptides obtained from aged rabbit skeletal myofibrils reacted with-connectin (titin 1, TI) but only weakly with-connectin (titin 2, TII) in rabbit skeletal muscle. Immunoelectron microscopy and indirect immunofluorescence microscopy revealed that the antibodies bound at the Z-line and at the epitope regions in the I-band near the binding site of a monoclonal antibody SMI whose position depends on sarcomere length. It thus appears that-connectin extends from the edge of M-line to the above epitope region in the I-band.     

Effects of aerobic endurance training status and specificity on oxygen uptake kinetics during maximal exercise

The main purpose of this study was to analyze the effects of exercise mode, training status and specificity on the oxygen uptake (O₂) kinetics during maximal exercise performed in treadmill running and cycle ergometry. Seven runners (R), nine cyclists (C), nine triathletes (T) and eleven untrained subjects (U), performed the following tests on different days on a motorized treadmill and on a cycle ergometer: (1) incremental tests in order to determine the maximal oxygen uptake (O_2max) and the intensity associated with the achievement of O_2max (IO_2max); and (2) constant work-rate running and cycling exercises to exhaustion at IO_2max to determine the effective time constant of the O₂ response (O₂). Values for O_2max obtained on the treadmill and cycle ergometer [R=68.8 (6.3) and 62.0 (5.0); C=60.5 (8.0) and 67.6 (7.6); T=64.5 (4.8) and 61.0 (4.1); U=43.5 (7.0) and 36.7 (5.6); respectively] were higher for the group with specific training in the modality. The U group showed the lowest values for O_2max, regardless of exercise mode. Differences in O₂ (seconds) were found only for the U group in relation to the trained groups [R=31.6 (10.5) and 40.9 (13.6); C=28.5 (5.8) and 32.7 (5.7); T=32.5 (5.6) and 40.7 (7.5); U=52.7 (8.5) and 62.2 (15.3); for the treadmill and cycle ergometer, respectively]; no effects of exercise mode were found in any of the groups. It is concluded that O₂ during the exercise performed at IO_2max is dependent on the training status, but not dependent on the exercise mode and specificity of training. Moreover, the transfer of the training effects on O₂ between both exercise modes may be higher compared with O_2max.     

Development of pump electrogenesis in hypokalaemic rat muscle

In hypokalaemic rats maintained on a potassium deficient diets for 10–50 days, the isolated Na-loaded and K-depleted (Na-rich) muscle fibers showed the membrane potential less than –115 mV in fresh muscles of normal rats in K⁺-free Krebs solution. Upon adding 5 mM K⁺ to the K⁺-free medium bathing the soleus muscles, the measured potentials of Na-rich muscles always exceeded the membrane potentials of fresh muscles in 5 mM K⁺. The hyperpolarization was dependent on the amount of intracellular Na⁺ concentration ([Na]_i) accumulated during the potassium deficiency. The electrogenic Na-pump was activated by an increase of [Na]_i of less than 5 mM. Further increases in [Na]_i resulted in increases in membrane potential which appeared to approach a limit at [Na]_i levels higher than 65 mM.     

Binding of human fibrinogen and its polypeptide chains to group B streptococci

Of 51 group-B streptococcal cultures, 20 bound¹²⁵I-labelled human fibrinogen. In contrast to the streptococci of groups A, C and G, the group-B streptococci interacted more with the, -chain of fibrinogen than with whole fibrinogen. None of the streptococcal cultures reacted with-chain. The fibrinogen-negative group-B streptococci still bound the, -chain. The binding of¹²⁵I-labelled,, -chain could be inhibited by unlabelled fibrinogen with fibrinogen-positive, but not with fibrinogen-negative streptococci of group B.     

Differential effects of halothane on adult and juvenile sodium channels in human muscle

Myoballs were cultured from biopsies of adult human skeletal muscle. Transient currents through the sodium channels were elicited by depolarizing a myoball membrane with the whole-cell patch-clamp technique. The properties of the sodium channels were determined from the Hodgkin-Huxley parameters (I _{Na max}, _m, _h,h -curve) derived from these transients. Halothane, when applied at 3.4 mmol/l (15 kPa), blocked about 50% of the current through the adult, TTX-sensitive sodium channels but had little effect on the current through the juvenile, TTX-insensitive sodium channels. At >12 mmol/l, halothane blocked both channel types completely. The time constants of activation and inactivation were decreased in the presence of 3.4 mmol/l halothane but not enough to account for the decrease of the current amplitude. Halothane shifted the h-curves of both channel types towards more negative potentials by an amount that was roughly proportional to its concentration. Myoballs from a man susceptible to malignant hyperthermia (MH) gave the same results as the controls indicating that the halothane effects on the action potential of MH-susceptible muscle are not mediated by a specific effect on the sodium channels.     

Adaptation and habituation of the vestibulo-ocular reflex in intact and inferior olive-lesioned rats

Summary The gain of the vestibulo-ocular reflex (VOR) of intact pigmented rats was adaptively modified by training protocols that created a visual-vestibular conflict. For training, head restrained animals were oscillated on a turntable in front of an optokinetic pattern projected onto a cylindrical wall. The optokinetic pattern either moved the same amplitude with the animal (in-phase: 0.05 Hz ± 20°/s) or opposite in direction (out-of-phase: turntable and pattern 0.05 Hz ± 10°/s each). VOR responses were tested in darkness before and after each 8 min training period for a duration of 40 min. During out-of-phase training the gain of compensatory eye movements measured in light was close to 2 from the beginning on and the VOR tested in darkness increased in gain progressively from 0.48 (±0.12) to 0.9 (±0.3; P < 0.05) in 5 out of 7 rats. Two rats did not adapt their VOR gain. Phase values decreased slightly by about 10°. During in-phase stimulation compensatory eye movements were almost completely suppressed (gain close to 0) from the beginning on and the VOR tested in darkness decreased gradually in gain from 0.62 (±0.17) to 0.13 (±0.1; P<0.001) in all 6 trained rats. Phase values decreased in parallel from 151° to 119° (P< 0.01). The effectiveness of the in-phase training paradigm in the absence of compensatory eye movements indicates that retinal image slip is the relevant signal for adaptation. In seven rats with histologically verified almost complete inferior olive (IO) lesions (chemically induced at least 45 days prior to training), out-of-phase and in-phase stimulation evoked compensatory eye movements with gains comparable to those in intact rats. VOR parameters measured in darkness were altered with respect to those of control rats. Gain differed extremely between individuals and phase lag re acceleration was in all IO-lesioned rats larger than in intact rats. The time constant of the VOR in response to table velocity steps was significantly longer (17 s ±4) than in intact rats (11 s ± 3). Training did not alter the gain of the VOR in 5 out of 7 IO-lesioned rats. One rat increased its gain during out-of-phase training in the first, but not during a second training session (and not during in-phase training) and another rat decreased its gain during in-phase training (but not during out-of-phase training). These changes in VOR gain might have occurred by chance rather than by learning. The absence of adaptation in IO-lesioned rats can be explained either by the absence of climbing fiber mediated slip signals in the cerebellar cortex or by lesion-induced secondary changes which result in a long-term reduction of the inhibitory efficacy of Purkinje-cells. In the absence of arousing stimuli VOR responses of intact rats exhibit a strong decrement during table oscillations in darkness. Between trials, with the rat at rest, response magnitude recovered spontaneously. Six out of 8 IO-lesioned rats expressed a very similar modification of their VOR gain. These results indicate that the neural mechanisms responsible for adaptive gain decrease during in-phase training and those responsible for a gain decrease during short-term habituation are different.     

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

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

Glucocorticoids modulate TGF-beta production by human fetal lung fibroblasts

TGF- is thought to play a central role in pulmonary fibrosis inducing fibroblast differentiation and extracellular matrix synthesis. In human lung fibroblasts, it is still unclear how various TGF- isoforms affect TGF- production and whether glucocorticoids, commonly used agents to treat fibrotic lung disease, modulate these processes. To this end, human fetal lung fibroblasts (HFLF) were cultured with various concentrations of glucocorticoids (budesonide, dexamethasone or hydrocortisone) with and without TGF-1, -2, or -3. Post-culture media were collected for ELISA assays of TGF-1, -2, and -3 . TGF- mRNA was assessed by real time RT-PCR. Smad 2, 3, and 4 and AP-1 complex (c-fos and c-Jun) cellular localization were evaluated by immunostaining. TFG-2 and -3 stimulated TGF-1 production significantly (p < 0.01 relative to control). TGF-1 stimulated TGF-2 production (p < 0.01 relative to control). TGF-3 was undetectable. Glucocorticoids significantly inhibited TGF-1 and TGF-2 production and reduced expression of the up-regulated TGF-1 and TGF-2 mRNA induced by exogenous TGF-1, -2, or -3 (p < 0.01 for each) but had no effect on Smads. Although c-jun-related nuclear staining was not intensified in TGF--stimulated cells, it was reduced by glucocorticoids. Thus, TGF- isoforms may stimulate production of various TGF- isoforms in the lung. Glucocorticoids then may block TGF- production by modulating mRNA levels and c-Jun.     

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.     

Bioactive 5-Reduced 16α,17α-Cyclohexanoprogesterone Derivatives Weakly Interact with Proteins of the Soluble Uterine Fraction

We studied competitive activities of 16,17-cyclohexano-5- and 5-dihydroprogesterone in replacing ₃H-progesterone and ₃H-16,17-cyclohexano-6-methylprogesterone from protein complexes. Direct binding of ₃H-5-reduced derivatives with proteins of soluble fractions from rat and rabbit uteri was also assayed. Cd values for 5-reduced derivatives were in the micro- or submicromolar range. The data suggest that biological effects of these analogues are not mediated via soluble uterine receptors.     

Circannual variation in the expression of β2-adrenoceptors on human peripheral mononuclear leukocytes (MNLs)

Summary Peripheral mononuclear leukocytes (MNLs) are widely used as a tissue model in studies of -adrenoceptor disturbances in hypertension and asthmatic diseases. The ₂-adrenoceptor density (B_max), however, depends not only on the gender of the person under study and on the time of day the blood specimens are obtained. Evidence is now reported for a circannual variation in the expression of ₂-adrenoceptor sites on peripheral MNLs. In male volunteers the 24-h mean was found to be highest in the men studied in April/May (1135±10 sites/cell) and decreased to 891±16 sites/cell in August and to 712±90 sites/cell in December (±SE,P<0.01 April/May compared to December). Concomitantly the circadian amplitude increased from 17.3%±6.4% of 24-h mean in April/May to 28.2%±1.4% of 24-h mean in August and to 34.2%±4.2% of 24-h mean in December (±SE,P<0.05, April/May compared to December). The circadian acrophase remained constant (190°±30° equivalent to 12 h 40 min±2 h 00 min, ±SE).Abbreviations MNLs Peripheral mononuclear leucocytes - B_{max 2}-Adrenoceptor density - ¹²⁵ICYP ¹²⁵Iodo-cyanopindolol - PEF Peak expiratory flow - SE Standard error - ANOVA Analysis of variance - M Circadian mesor - A Circadian amplitude - Circadian acrophase     

Suppression of Adjuvant Arthritis and Synovial Macrophage Inducible Nitric Oxide by N-iminoethyl-L-Ornithine, A Nitric Oxide Synthase Inhibitor

Nitric oxide NO is a pro-inflammatory effector molecule in certain inflammatory diseases, including arthritis. We investigated the production of NO by adjuvant arthritis (AA) synovial macrophages, and studied the effects of a NO synthase inhibitor, N-iminoethyl-L-ornithine (L-NIO). Compared to control rats, rats treated with L-NIO in vivo exhibited significantly lower articular index (p < 0.05), paw volume (p < 0.05), and synovial fluid cell count (p < 0.05). No effect on cutaneous delayed-type hypersensitivity to the disease-initiating antigen was observed. Inducible NO synthase (iNOS) was detected in AA synovial macrophages, and cultured AA synovial macrophage iNOS levels were increased by a factor of 138 ± 17% (p < 0.01) by 1 g/ml LPS in vitro. Constitutive NO production by AA synovial macrophages (43 ± 1 nmol/10⁵ cells/24 h) was significantly inhibited by 10 mM L-NIO in vitro (32 ± 0.5, p<0.01). NO production induced by 1 g/ml LPS (48 ± 2) was also decreased by L-NIO (39 ± 2, p<0.05). In vivo L-NIO treatment also inhibited alveolar macrophage NO production (p < 0.05). The ability of L-NIO to decrease iNOS-mediated synovial macrophage NO production and inhibit the clinical parameters of AA implicate macrophage-derived NO in the pathogenesis of this disease.     

Properties of two calcium transport systems of isolated rat ileal epithelial cells: effects of Ca2+ channel modulators and membrane potential examined with fluorescent dye,fura-2

Calcium transport systems of isolated ileal epithelial cells were investigated. The concentration of cytosolic free calcium ions, [Ca²⁺]_i, was monitored with a fluorescent Ca²⁺ dye, fura-2. The fluorescence intensity ratio (I ₃₄₀/I ₃₈₀) was used as an index of [Ca²⁺]_i. [Ca²⁺]_i of the cells suspended in the nominally Ca²⁺-free solution was estimated at 52±3 nM. Ca²⁺ uptake was followed for as long as 5 min in the presence of 100–1000 M added CaCl₂. Most of the experiments were performed at 200 M CaCl₂. The Ca²⁺ uptake was abolished by 0.8 mM Ni²⁺ and 50 M Mn²⁺ and partitally antagonized by 50 M verapamil and 50 M diltiazem but not affected by 20 M nifedipine. The Ca²⁺ entry was reduced by increasing concentrations of extracellular K⁺ in the presence of valinomycin, suggesting a voltage-dependent nature of the uptake. On the other hand, the Ca²⁺ transport doubled in the presence of Bay K8644 (8 M), a Ca²⁺ channel agonist. The Bay-K-8644-induced uptake was inhibited by either 10 M nifedipine, 10 M verapamil or 10 M diltiazem and was relatively independent of extracellular K⁺ concentration. These results suggest that there are at least two distinct Ca²⁺ transport systems in the rat ileal epithelial cells, one resistant to organic Ca²⁺ channel blockers but relatively sensitive to membrane potential (basal uptake) and another inducible by Bay K 8644 and sensitive to the channel blockers but relatively independent of membrane potential.     

Effect of a series of 1-alkyl ether lipids on inhibition of phospholipase A2 activity and PAF responses

Several 1-alkyl ether lipids were studied for their ability to inhibit PLA₂ and antagonize PAF responses. Studies with synthetic micellar substrate (1-stearyl-2-arachidonyl phosphocholine), at concentrations ranging from 0.02 to 1000M, demonstrate that CL 118326 inhibits porcine pancreatic PLA₂ in vitro. As the substrate concentration increases, there is a dose-dependent increase in the IC₅₀ value (IC₅₀ ranges: 1.6–84.6g/ml or 2.6–137M). CL 118326 inhibits mammalian pancreatic PLA₂, but not snake or bee venom PLA₂. CL 118326 inhibits thrombin (IC₅₀ =7.9M), but not Na arachidonate- (IC₅₀ > 100M) induced platelet aggregation, indicative of inhibition of cellular PLA₂. CL 118326 inhibits other PLA₂-dependent processes such as antigen-induced leukotriene (LTC₄) release (IC₅₀=2.3g/ml or 3.8M) and histamine release (IC₅₀=1.4g/ml or 2.2M) in basophil-enriched WBCs. Intradermal coinjection of CL 118326 (10g) with PLA₂ into guinea pig skin inhibits pancreatic PLA₂-induced increase in vascular permeability and leakage, but not snake or bee venom PLA₂-induced leakage. CL 118326 shows no PAF-like agonist activity in stimulating rabbit platelet-rich plasma. It inhibits PAF-induced aggregation (IC₅₀=5.8M), but not ADP-induced aggregation. CL 118326 has greater efficacy as a PLA₂ inhibitor than as a PAF antagonist since the IC₅₀-substrate concentration ratio for PLA₂ inhibition is <- 1.0 at substrate concentrations of 10–1000M while the IC₅₀-agonist ratio for PAF antagonism is > 100. Results for four other compounds related to CL 118326 are also presented.     

Phenotypic differences between induced and spontaneous 2μ-plasmid-free segregants of Saccharomyces cerevisiae

Summary The maximum specific growth rates (_max) of 2 -plasmid-free ([cir°]) segregants of three haploid and one diploid strain of Saccharomyces cerevisiae have been determined and compared with the _max of their 2 -plasmid-containing ([cir ⁺]) progenitors. Two classes of [cir°] strains have been examined: those induced by transformation with a 2 -based recombinant plasmid according to the method of Dobson et al. (1980) and those isolated as spontaneous [cir°] segregants from glucose-limited continuous cultures. The _max of the spontaneous [cir°] segregants was not found to differ significantly from that of their [cir ⁺] parents. In all cases, however, the induced [cir°] strains had a _max which was significantly less than that of their [cir ⁺] counterparts. This effect was particularly marked in the case of the diploid strain where a 34% reduction in _max was observed. The implications of these results are discussed in terms of the effect of the transformation process on host yeast cells.     

Modification of electrophysiological and pharmacological properties of K channels in neuroblastoma cells induced by the oxidant chloramine-T

The effects of chloramine-T (CL-T) on voltage-dependent potassium channels in neuroblastoma cells were analysed using the whole-cell current recording technique. CL-T irreversibly decreased the peak whole — cell K current, considerably slowed its inactivation and shifted its activation-voltage curve towards positive voltages by 6 mV. Under control conditions, the inactivation of the whole-cell K current could be described by the sum of two exponentials, F and S, whose time constants at +50 mV were _F=1.00±0.15 s and _S=5.72±0.47 s respectively. After CL-T, it could be described by the sum of two (S₁ and S₂) or three (F, S₁ and S₂) exponentials whose time constants at +50 mV were: _F=0.81±0.22 s, _S1=6.46±0.60 s and _S2=48.56±3.64 s. Under control conditions, F and S inactivating components of the whole-cell K current were blocked by 4-aminopyridine, with a Hill coefficient of 1 and apparent dissociation constants of 0.04 and 0.7 mM respectively. After CL-T, both S₁ and S₂ components were equally blocked by 4-aminopyridine with a Hill coefficient of 0.25, being reduced to 64% of their control values by 10 mM. CL-T is known to slow the inactivation of sodium channels and to oxidize sulphydryl amino acids and unsaturated lipids. It is concluded that the inactivation gates of voltage-dependent sodium and potassium channels are either constituted of the same amino acid residues or are controlled by unsaturated lipids surrounding or bound to the channel proteins.     

Interferon-α activates cytotoxic function but inhibits interleukin-2-mediated proliferation and tumor necrosis factor-α secretion by immature human natural killer cells

Natural killer (NK) cells play an important role in host defense mechanisms against infection and neoplasia. Interferon- (IFN-) has been shown to activate NK cells and to augment their cytotoxic activity, albeit its role in the maturation pathway of NK cells has not been elucidated. The present study examined whether IFN- activates the immature NK subset (Free cells) to become cytotoxic and also ascertained whether IFN- uses the same pathway of activation as that mediated by interleukin-2 (IL-2). Incubation of sorted Free cells overnight with IFN- resulted in augmentation of their cytotoxic function against NK sensitive target cells. The enhanced cytotoxic activity was not accompanied by a new recruitment of NK-target binder cells but by an increase in the frequency of killer cells in the conjugate fraction. Activation of the Free subset by IFN- resulted in upregulation of CD69, CD11b, and CD2 surface expression and stimulated secretion of IFN-. Unlike IL-2, IFN- did not stimulate the Free cells to proliferate or secrete TNF- and activation of cytotoxicity and modulation of surface antigens by IFN- were independent of TNF-. The failure of IFN- to stimulate secretion and proliferation by Free cells appeared to be mediated by negative signals. This was corroborated in experiments demonstrating that when Free cells were cultured with both IFN- and IL-2, a significant inhibition was observed for both the IL-2 dependent secretion of TNF- and proliferation. These results demonstrate that IFN- serves as both an activator and a regulator of NK function. Further, activation of the immature Free NK cells by IL-2 and IFN- proceeds by TNF--dependent and independent pathways, respectively. The findings also support our contention that the mechanism of activation of the cytotoxic machinery of NK cells is not linked to the mechanism of activation of cytokine secretion and/or proliferation.Abbreviations used IFN interferon - IL interleukin - PBL peripheral blood leukocytes - PE phycoerythrin - PE-GAM PE-conjugated Fab2 goat anti-mouse IgG - NK natural killer - NRS normal rabbit serum - TNF tumor necrosis factor - FCS fetal calf serum - FITC fluorescein isothiocyanate - PBS phosphate-buffered saline - MACS magnetic cell sorting - ELISA enzyme-linked immunosorbent assay - BSA bovine serum albumin - PKC protein kinase C - mAb monoclonal antibody - PBMC peripheral blood mononuclear cells - BCLL B-chronic lymphocytic leukemia - E effector - T target