Rat osseous plate alkaline phosphatase: Effect of neutral protease digestion on the hydrolysis of pyrophosphate and nitrohenylphospate

Collagenase treatment, commonly used to prepare alkaline phosphatase-rich matrix vesicles from epiphyseal cartilage growth plates, seems to affect the integrity of this membrane-bound enzyme. Alkaline phosphatase-rich rat osseous plates were incubated with 1000 U/mL collagenase for 3 h, at 37°C and after purification on Sepharose 4B, kinetic studies were performed using nitrophenylphosphate and pyrophosphate as substrates.The optimum apparent pH for the hydrolysis of p-nitrophenylphosphate and pyrophosphate increased from 9.4 to 10.25 and from 8.0 to 9.0, respectively, as a consequence of collagenase treatment. In the absence of Mg²⁺ ions, the enzyme hydrolyzed PNPP with K_M = 322.5 ± 15.3 M and V = 965.2 ± 45.8 U/mg, while in the presence of 2 mM Mg²⁺ ions, V increased 66%. Cobalt (K_0.5 = 5.3 ± 0.3 M) and manganese (K_0.5 = 0.72 ± 0.03 M) ions stimulated the PNPPase activity of the collagenase-treated enzyme, but with a lower apparent affinity when compared with that of not-treated enzyme. In the absence of Mg²⁺ ions pyrophosphate was hydrolyzed according to Michaelis-Menten kinetics (K_M = 105.1 ± 6.3 M and V = 64.9 ± 3.9 U/mg), but site-site interactions (n_H = 1.2) were observed in the presence of 2 mM Mg²⁺ ions (V = 110.8 ± 5.5 U/mg; K_0.5 = 42.7 ± 2.0 M).To our knowledge this is the first report showing significant alterations on phosphohydrolytic activity and metal binding properties of bone alkaline phosphatase due to associated neutral proteases in collagenase preparations often used for the isolation of matrix vesicles.     

The effects of pH and calcium concentrations on gill potentials in the Brown Trout,Salmo trutta

Summary Measurements of the transepithelial potential (V_int-V_ext) across the gills of Brown Trout,Salmo trutta, were made in solutions of a range of pH and calcium concentrations. The potential was strongly dependent on external pH, being negative in neutral solutions but positive in acid solutions. The addition of calcium to the external medium produced a positive shift in potential in all but very acid media (pH 4.0–3.5), where very little change was seen. The gill membrane appears to act as a hydrogen electrode having a very high permeability to H⁺ ions, and the potential behaves as a diffusion potential. The presence of calcium reduced the permeability to both H⁺ and Na⁺ ions but even at a calcium concentration of 8.0 mM/l the permeability ratio H⁺/Na⁺ was still more than 900. The transepithelial potential is shown to be diffusional in origin and is discussed in terms of the relative permeability of the gill to H⁺, Na⁺ and Cl^– ions. Sodium fluxes across the gills were measured and provide the basis for a theoretical consideration of Na⁺, Cl^– and H⁺ fluxes across the gills in neutral and acid solutions. The positive potential at low pH largely accounts for the increased loss of sodium from fish in these conditions.     

The effect of glucose on chloride uptake by Chlorella

Active Cl^- uptake by Chlorella fusca was examined by using ³⁶Cl as a label. Under light/air conditions chloride influx from a 2.4·10^-5 M solution was 4.0±0.04 nmol m^-2s^-1. After 70±10 min a stationary 380±40 fold accumulation was reached. In dark/air and dark/argon influx and accumulation were reduced to 25±6%, respectively, 5±1.5% of the light/air control. Cl^- uptake had a broad optimum around pH 7 and showed saturation kinetics with a K _M of 1.25·10^-5 M and a v _max of 7.0 nmol m^-2s^-1 in light/air. Br^- inhibited Cl^- uptake strongly, J^-, ClO ₄ ^- , SO ₄ ^2- , and NO ₃ ^- had no inhibitory effect. Inhibitor studies with carbonyl cyanide m-chlorophenylhydrazone and N,N-dicyclohexylcarbodiimide resulted in a good correlation between Cl^- uptake and ATP level. 3-(3,4-Dichlorophenyl)-1,1-dimethylurea and darkness reduced transport activity without affecting the ATP level.The magnitudes of the pH gradient and the membrane potential across the cell membrane were determined and/or estimated under different conditions. It could be shown that in Chlorella Cl^- transport cannot proceed via secondary active H⁺/Cl^- cotransport. In addition, 2H⁺/Cl^- cotransport seems unlikely for energetic reasons. On the basis of the results of this and the following study, a primary active ATP-driven Cl^-/OH^- exchange pump is proposed.Abbreviations CCCP carbonyl cyanide m-chlorophenylhyd razone - DCCD N,N-dicyclohexylcarbodiimide - DCMU 3-(3.4-dichlorophenyl)-1.1-dimethylurea - DMO 5,5-dimethyloxazolidine-2,4-dione - Hepes N-2-hydroxyethylpiperazine-N ethane-sulfonic acid - POPOP 1.4-bis-2-(4-methyl-5-phenyloxazolyl)-benzene - PPO 2.5-diphenyloxazole To whom correspondence should be addressed     

Continuous and High-Intensity Interval Training: Which Promotes Higher Pleasure?

Objectives

To compare the psychological responses to continuous (CT) and high-intensity interval training (HIT) sessions.

Methods

Fifteen men attended one CT session and one HIT session. During the first visit, the maximum heart rate, VO_2Peak and respiratory compensation point (RCP) were determined through a maximal cardiopulmonary exercise test. The HIT stimulus intensity corresponded to 100% of VO_2Peak, and the average intensity of both sessions was maintained at 15% below the RCP. The order of the sessions was randomized. Psychological and physiological variables were recorded before, during and after each session.

Results

There were no significant differences between the average percentages of VO₂ during the two exercise sessions (HIT: 73.3% vs. CT: 71.8%; p = 0.779). Lower responses on the feeling scale (p≤0.01) and higher responses on the felt arousal scale (p≤0.001) and the rating of perceived exertion were obtained during the HIT session. Despite the more negative feeling scale responses observed during HIT and a greater feeling of fatigue (measured by Profile of Mood States) afterwards (p<0.01), the physical activity enjoyment scale was not significantly different between the two conditions (p = 0.779).

Conclusion

Despite the same average intensity for both conditions, similar psychological responses under HIT and CT conditions were not observed, suggesting that the higher dependence on anaerobic metabolism during HIT negatively influenced the feeling scale responses.     

Purification and characterization of membrane-bound hydrogenase from Methanosarcina barkeri MS

Hydrogenase was solubilized from the membrane of acetate-grown Methanosarcina barkeri MS and purification was carried out under aerobic conditions. The enzyme was reactivated under reducing conditions in the presence of H₂. The enzyme showed a maximal activity of 120±40 mol H₂ oxidized · min^–1 · min^–1 with methyl viologen as an electron acceptor, a maximal hydrogen production rate of 45±4 mol H₂ · min^–1 · mg^–1 with methyl viologen as electron donor, and an apparent K _m for hydrogen oxidation of 5.6±1.7 M. The molecular weight estimated by gel filtration was 98,000. SDS-PAGE showed the enzyme to consist of two polypeptides of 57,000 and 35,000 present in a 1:1 ratio. The native protein contained 8±2 mol Fe, 8±2 mol S^2–, and 0.5 mol Ni/mol enzyme. Cytochrome b was reduced by hydrogen in a solubilized membrane preparation. The hydrogenase did not couple with autologous F₄₂₀ or ferredoxin, nor with FAD, FMN, or NAD(P)⁺. The physiological function of the membrane-bound hydrogenase in hydrogen consumption is discussed.Abbreviation CoM-S-S-HTP the heterodisulfide of 7-mercaptoheptanoylthrconine phosphate and coenzyme M (mercaptoethanesulfonic acid)     

Beta-adrenergic control of trans-cutaneous evaporative cooling mechanisms in birds

Summary The decreasing effect of -adrenergic blockade on skin resistance to vapor diffusion and the onset of cutaneous water evaporation in the pigeon (Columba livia) was investigated. Oral administration of 1, 2.3 and 5 mg propranolol to pigeons (268±53 g) initiated intensive trans-cutaneous water evaporation (CWE) up to 29.1 mg H₂O·cm^–2·h^–1 in resting birds at 30°C air temperature (Ta), but had only a slight effect on CWE of birds exposed to 50 °C Ta.After 7 h of effective -adrenergic blockade (oral administration of 5 mg propranolol), skin and body temperature stabilized at 39.0±0.5 °C and 41.0±0.7 °C, compared to 40.2±0.8 °C and 41.9±0.6 °C in the control group, respectively. A slight hypothermia was accompanied by feather fluffing.Intradermal injection of 0.001, 0.01 and 0.12 mg propranolol also caused intensive CWE. Local -adrenergic blockade in relatively low blocker doses (0.001 and 0.01 mg propranolol) decreased skin resistance from a high value of 44.5 s·cm^–1 to about 6.0 s·cm^–1, and caused a sharp increase in CWE from a control value of about 4 to a high of 26.4 mg H₂O·cm^–2·h^–1 during the first two hours of exposure to 30°C Ta.The possible role of -adrenergic blockade in regulation of trans-cutaneous water evaporation of latent heat dissipation is discussed.     

Long-term biological effects of air ions and D.C. electric fields on Namru mice: Second year report

This report describes the second year of long-term continuous exposures of female NAMRU mice to small air ions and D.C. electric fields in the following conditions: ± high ions ((2×10⁵/cm³), ± low ions (2×10³/cm³), ± field (2 kV/m) only and ground (ion depleted, no field). Using an isolated anesthesized mouse, whole body ion flux values averaged 1.04±0.63×10^–10 A in high ion cages for different positions on the cage floor, with about a hundred-fold reduction for low ion cages.During the second year (sample periods 5–8) of exposure serum chemistry variability increased, due to increased pathology and decreased numbers of animals as our experimental population died off. The fifth sample period yielded results consistent with those seen earlier, but later sample periods had many fewer significant differences between cages than did those of the first year. Nevertheless, MCA statistics for serum glucose for the second year found a pattern remarkably similar to the first, with the low ion cages (LN and LP) having the lowest levels. MCA statistics for both years emphasized this possible window effect of low level ionized conditions. Also, a comparison between the combined values for ionized (HN, LN, HP and LP) and ion depleted cages (NF, PF, G₁ and G₂) showed a highly significant difference (p<10^–6) for serum glucose for both years of exposure, with lower glucose values seen for animals in the ionized cages overall. Animals of all conditions also showed a highly significant decrease in serum glucose with age.Comparison of mice in ionized cages vs. the non-ionized cages also resulted in a significant difference (p<.013) for survival characteristics between groups, with ion exposed animals having a shorter lifespan. These statistics argue strongly for significant effects of long-term exposure of NAMRU mice to the ionized environment.     

Effects of (±)-kavain on inactivation of voltage-operated Na+ channels

Effects of a kava-pyrone (±)-kavain on fast inactivation of Na⁺ channels were studied in experiments on isolated neurons from the rat hippocampus. (±)-Kavain was found to block Na⁺ channels, and its effect was voltage-dependent. At the holding potentials of –100 and –80 mV, IC₅₀ for (±)-kavain was 744.9 and 178.8 µM, respectively. The inactivation characteristic of Na⁺ channels was satisfactorily described with the Boltzmann's equation both in the control and under (±)-kavain application. (±)-Kavain at a 330 µM concentration shifted theV _1/2 toward more negative values by 14.4 mV and concurrently modified the slope factor: the latter was 5.7 mV in the control, while under the influence of 330 µM (±)-kavain it reached 6.7 mV. In agreement with Hille's hypothesis of a modulated receptor, inactivated Na⁺ channels demonstrated an increased sensitivity to kavain. (±)-Kavain effects resulted in an increase in the rate of depolarization-related fast inactivation, while the process of recovery from inactivation became slower when the membrane was hyperpolarized. Our data show that under the (±)-kavain effect the probability of the inactivated state of Na⁺ channels increases, and the state of fast inactivation is stabilized.Neirofiziologiya/Neurophysiology, Vol. 28, No. 4/5, pp. 218–224, July–October, 1996.     

A comparison of physiological responses and rating of perceived exertion between high-impact and low-impact aerobic dance sessions

The aim of this study was to compare the exercise intensity and rating of perceived exertion (RPE) of a high-impact (HIP) and a low-impact (LIP) university aerobic dance session. Ten women [mean (SD) age 22.9 (2.6) years] took part in the study. An incremental treadmill test was performed by each subject to determine maximum oxygen consumption (V˙O_{2 max} ) and maximum heart rate (HR _max ). The measured V˙O_{2 max} [mean (SD)] was 49.0 (7.5) ml · kg⁻¹ · min⁻¹. The subjects were randomly assigned to LIP and HIP sessions (i.e. five of the subjects participated in the HIP session first, and the other five participated in the LIP session first). In a laboratory, heart rate, oxygen uptake and RPE were measured throughout each session for each subject. Expired air was collected continuously throughout the sessions using Douglas bags (ten bags over a 30-min period). The sessions consisted of 20 min of aerobic exercise (bags 1–7) followed by 5 min of local muscular endurance exercise (bags 8 and 9) and 5 min of flexibility exercises (bag 10). The mean intensity of the aerobic section of the LIP and HIP sessions was 51.6% and 64.7% V˙O_{2 max} , respectively. Ninety-five percent confidence intervals for the average difference between the HIP and LIP sessions demonstrate that the %V˙O_{2 max} was between 12% and 14% higher for the HIP session. The mean %HR _max for the LIP and HIP sessions was 71.4% and 76.7%, respectively, with the %HR _max in the HIP session being between 5.4% and 7.2% higher on average than that of the LIP session. On average, the RPE for the aerobic section of the HIP session (12.1) was consistently higher than that of the LIP session (11.1). HIP activity has the potential to maintain/improve the aerobic fitness of its participants. According to the literature, the exercise intensity elicited by LIP activity may have a limited training effect for the population utilised in this study, and for some individuals may result in detraining. Conversely, LIP activities may be an appropriate mode of exercise for overweight and unfit individuals. Accepted: 5 January 1998     

Interrelationships Between Nitrogen Supply and Photosynthetic Parameters in Vicia faba L.

We determined for Vicia faba L the influence of nitrogen uptake and accumulation on the values of photon saturated net photosynthetic rate (P _Nmax), quantum yield efficiency (), intercellular CO₂ concentration (C _i), and carboxylation efficiency (C _e). As leaf nitrogen content (N_L) increased, the converged onto a maximum asymptotic value of 0.0664±0.0049 mol(CO₂) mol(quantum)^–1. Also, as N_L increased the C _i value fell to an asymptotic minimum of 115.80±1.59 mol mol^–1, and C _e converged onto a maximum asymptotic value of 1.645±0.054 mol(CO₂) m^–2 s^–1 Pa^–1 and declined to zero at a N_L-intercept equal to 0.596±0.096 g(N) m^–2. fell to zero for an N_L-intercept of 0.660±0.052 g(N) m^–2. As N_L increased, the value of P _Nmax converged onto a maximum asymptotic value of 33.400±2.563 mol(CO₂) m^–2 s^–1. P _N fell to zero for an N_L-intercept of 0.710±0.035 g(N) m^–2. Under variable daily meteorological conditions the values for N_L, specific leaf area (_L), root mass fraction (R_f), P _Nmax, and remained constant for a given N supply. A monotonic decline in the steady-state value of R_f occurred with increasing N supply. _L increased with increasing N supply or with increasing N_L.     

β-Adrenoreceptors of multiple affinities in a clonal capillary endothelial cell line and its functional implication

-Adrenoreceptor has been studied in a clonal capillary endothelial cell line established from the vascular bed of the bovine adrenal medulla. [³H]Dihydroalprenolol ([³H]DHA) binding to the isolated plasma membranes from these cells has demonstrated the presence of -adrenoreceptors with two different affinities. the dissociation constants (K_d) have been found to be 0.27±0.09×10^–9 M and 2.96±0.31×10^–9 M, respectively with the corresponding B_max of 5.1±0.05 and 70.0±0.2 pmol/mg protein, respectively. Inhibition of [³H]DHA binding to the -receptor by atenolol (a ₁-antagonist) and ICI 118,551 (a ₁-antagonist) has suggested that the IC_50cor (=K_i) for atenolol and ICI 118,551 for high affinity site are 0.08±0.03×10^–12 M and 0.25±0.08×10^–12 M, respectively. This, therefore, indicates that both atenolol and ICI 118,551 are able to displace the bound ligand effectively but the ₁-selective antagonist atenolol is 3 times more potent than its ₂ counterpart, ICI 118,551. Displacement of [³H]DHA binding to the endothelial cell plasma membrane by the agonists isoproterenol, epinephrine and norephinephrine has established a relative order of K_i for these agents as isoproterenol (0.56±0.19×10^–9 M)–9 M)>-norepinephrine (0.71±0.24×10^–9 M) for the high affinity site. The corresponding values for the low affinity site, however, are 4.62±0.64×10^–9 M, 6.21±0.86×10^–9 M and 5.90±0.82×10^–9 M, respectively for the same agonists. Increased intracellular cAMP accompanied with cellular proliferation in the presence of isoproterenol has suggested not only the coupling of -adrenoreceptors to the adenylate cyclase system but also its involvement in endothelial cell proliferation.Abbreviations DHA Dihydroalprenolol - cAMP 3:5 cyclic adenosine monophosphate - DTT dithiothreitol - MEM minimal essential medium - 8Br-cAMP 8-bromo-adenosine 3:5 cyclic monophosphate     

Investigation of ion binding to the cytoplasmic binding sites of the Na,K-pump

A dual-wavelength fluorimeter was constructed, which used two light emitting diodes (LEDs) to excite the fluorescence dye RH 421 alternately with two different wavelengths. The ratio of the emissions at the two excitation wavelengths provided a drift-insensitive signal, which allowed detection of very small changes of the fluorescence intensity. Those small changes were induced by ion binding and release in conformation E₁ of the Na,K-ATPase. Titration experiments were performed to determine equilibrium dissociation constants (± standard deviation) for each step in the complete binding and release sequence: 0.12 ± 0.01 mM (E₂(K₂) KE₁), 0.08 ± 0.01 mM (KE₁ E₁), 3.0 ± 0.2 mM (NaE₁ E₁), 5.2 ± 0.4 mM (Na₂E₁ NaE₁) and 6.5 ± 0.4 m_M (Na₃E₁ Na₂E₁) at pH 7.2 and T=16°C. These numbers show that the affinities of the binding sites exposed to the cytoplasm, are higher for K⁺ than for Na⁺ ions, similar to what was found on the extracellular side. The physiological requirement for extrusion of Na⁺ from the cytoplasm, and for import of K⁺ from the extracellular medium seems to be facilitated not by favorable binding affinities in state E₁ but by the two ATP-driven reaction steps of the cycle, E₂(K₂) + ATP K₂E₁ · ATP and Na₃E₁ · ATP (Na₃) E_l-P, which border the ion exchange reactions at the binding sites in conformation E₁. Correspondence to: H.-J. Apell     

A quantitative histochemical technique for the characterisation of α-glucosidases in the brush-border membrane of rat jejunum

Summary A quantitative histochemical method to determine the K_m and V_max of -glucosidases in the intestinal epithelium without disruption of the cellular structure is described. 2-Naphthyl--D-glucoside was used as substrate and hexazonium-p-rosaniline as coupling agent. Using a Leitz MPV2 microdensitometer and a field measuring 4×4 m, and reading the test samples against a blank focused on the lamina propria, we observed that the intensity of the colour was a linear function of both the incubation time up to 20 min, and the thickness of the slice up to 20 m. The ratio between the extinction at the absorption maximum and at a second wavelength was constant, whatever the intensity of the colour.By determining the relationship between the extinction and the substrate concentration under standard conditions (slice thickness of 10 m and incubation time of 10 min), we obtained a saturation curve described by a K_m of 0.68±0.038 mM and a V_max of 1.41±0.039 A₄₈₀·10^–2·m^–1·min^–1. When the hydrolysis of the same substrate by a homogenate of jejunal mucosa was examined biochemically under comparable conditions, a K_m of 0.64±0.012 mM and a V_max of 57.3±0.70 mU/mg protein were obtained. When the natural substrate, sucrose, was used in the biochemical study, a K_m of 15±3.5 mM and a V_max of 149±24.7 mU/mg protein were obtained.These experiments demonstrate that the kinetic constants of enzyme reactions can be assessed with equal accuracy on histochemical sections as in tissue homogenates.     

A new (μ3-carbonato)tricopper(II) complex with symmetry related equilateral triangular array of metal centers; structure and magnetism

Using an unsymmetrical N-tridentate (2-pyridyl)alkylamine ligand methyl[2-(2-pyridyl)ethyl](2-pyridylmethyl)amine (mpepma), a new tricopper(II) complex [{(mpepma)Cu(OClO₃)}₃(μ₃-OCO₂)][ClO₄] (1), having triply bridging μ₃-carbonate group, with three symmetry related triangular array of copper(II) centers has been synthesized. In 1, each copper ion is five-coordinate (almost perfect square pyramidal) by three nitrogens from the ligand (two pyridyl and an amine) and an oxygen atom of the carbonate group in the equatorial plane and by an oxygen atom of the perchlorate ion in the apical position. Magnetic susceptibility data (2-300 K) were fitted using the Hamiltonian: , which corresponds to a triangular array of spins, corrected with a zJ′ intertrimer interaction or zero-field splitting within the ground state, to fit low temperature data. It is concluded that the copper(II) ions in 1 are ferromagnetically coupled. The best parameters obtained are g = 2.11, J = 18 ± 1 cm⁻¹, zJ′ = −0.36 ± 0.02 cm⁻¹ or D = 0.50 ± 0.05 cm⁻¹.     

Fusion of cultured dog kidney (MDCK) cells: II. Relationship between cell pH and K+ conductance in response to aldosterone

Summary We have chosen the MDCK cell line to investigate aldosterone action on H⁺ transport and its role in regulating cell membrane K⁺ conductance (G _m ^K ). Cells grown in a monolayer respond to aldosterone indicated by the dose-dependent formation of domes and by the alkalinization of the dome fluid. The pH sensitivity of the plasma membrane K⁺ channels was tested in giant cells fused from individual MDCK cells. Cytoplasmic pH (pH _i ) andG _m ^K were measured simultaneously while the cell interior was acidified gradually by an extracellular acid load. We found a steep signoidal relationship between pH _i andG _m ^K (Hill coefficient 4.4±0.4), indicating multiple H⁺ binding sites at a single K⁺ channel. Application of aldosterone increased pH _i within 120 min from 7.22±0.04 to 7.45±0.02 and from 7.15±0.03 to 7.28±0.02 in the absence and presence of the CO₂/HCO ₃ ^– buffer system, respectively. We conclude that the hormone-induced cytoplasmic alkalinization in the presence of CO₂/ HCO ₃ ^– is limited by the increased activity of a pH _i -regulating HCO ₃ ^– extrusion system. SinceG _m ^K is stimulated half-maximally at the pH _i of 7.18±0.04, internal H⁺ ions could serve as an effective intracellular signal for the regulation of transepithelial K⁺ flux.     

Change in substrate metabolism in isolated mouse hearts following ischemia-reperfusion

Several genetic and transgenic mouse models are currently being used for studying the regulation of myocardial contractility under normal conditions and in disease states. Little information has been provided, however, about myocardial energy metabolism in mouse hearts. We measured glycolysis, glucose oxidation and palmitate oxidation (using ³H-glucose, ¹⁴C-glucose and ³H-palmitate) in isolated working mouse hearts during normoxic conditions (control group) and following a 15 min global no-flow ischemic period (reperfusion group). Fifty min following reperfusion (10 min Langendorff perfusion + 40 min working heart perfusion) aortic flow, coronary flow, cardiac output, peak systolic pressure and heart rate were 44 ± 4, 88 ± 4, 57 ± 4, 94 ± 2 and 81 ± 4% of pre-ischemic values. Rates of glycolysis and glucose oxidation in the reperfusion group (13.6 ± 0.8 and 2.8 ± 0.2 mol/min/g dry wt) were not different from the control group (12.3 ± 0.6 and 2.5 ± 0.2 mol/min/g dry wt). Palmitate oxidation, however, was markedly elevated in the reperfusion group as compared to the control group (576 ± 37 vs. 357 ± 21 nmol/min/g dry wt, p < 0.05). This change in myocardial substrate utilization was accompanied by a marked fall in cardiac efficiency measured as cardiac output/oxidative ATP production (136 ± 10 vs. 54 ± 5 ml/mol ATP, p < 0.05, control and reperfusion group, respectively). We conclude that ischemia-reperfusion in isolated working mouse hearts is associated with a shift in myocardial substrate utilization in favour of fatty acids, in line with previous observations in rat.     

Sialic acid and the surface charge associated with hyperpolarization-activated,inward rectifying channels

Summary The whole-cell configuration of the patch-clamp technique was used with cultured pacemaker cells from the rabbit sinoatrial node to test the hypothesis that sialic acid residues (NANA) constitute much of the negative surface charge associated with hyperpolarization-activated, inward rectifying channels. Activation-voltage relationships (between –70 and –140 mV) were determined for hyperpolarization-activated (inward rectifying) current (i _f). Addition of 10mm Ca²⁺ shifted the half-activation potential (V _1/2) from –89.5±0.9 mV to –77.9±2.6 mV (P<0.01), confirming the presence of negative fixed charges on the myocytes after 3 to 5 days in culture. Addition of 20mm dimethonium, an organic divalent cation that screens but does not bind to negative surface charge, shiftedV _1/2 from –86.8±1.4 mV to –75.0±1.7 mV (P<0.001) without affecting the amplitude of the current. In contrast, 10mm Ca²⁺ reduced the amplitude ofi _f significantly. Incubation of cells with a highly purified preparation of neuraminidase (0.1–2.0 U/ml, 1 hr, 37°C), an enzyme that selectively removes NANA from glycoproteins and glycolipids, failed to alterV _1/2 or the amplitude ofi _f significantly. Pretreatment of cells with neuraminidase (1.0 U/ml, 1 hr, 37°C) failed to alter the positive shift ofV _1/2 produced by dimethonium. The results suggest that NANA does not constitute the negative surface charge associated with hyperpolarization-activated, inward rectifying channels.     

Opioid receptor binding in rat spinal cord

The interaction of various radioligands with spinal opioid receptors has been characterized under variable experimental conditions. Binding to , , and sites was measured in all (cervical, thoracic, lumbar) segments. The apparent affinity constant (K) of [³H]Ethylketocyclazocine (EKC) was similar in Tris, 2.09 (±1.06)×10⁸ M^–1, and phosphate buffer, 2.16 (±0.02)×10⁸ M^–1, when its interaction with and sites was blocked. Without blocking ligands, EKC binding was resolved in two components:K ₁=1.01 (±0.21)×10⁹ M^–1 andK ₂=0.95 (±0.61)×10⁷ M^–1. Likewise, the binding of [D-Ala², MePhe⁴, Gly(ol)⁵]enkephalin (DAGO) or [D-Ala², D-Leu⁵]-enkephalin (DADLE) alone was represented by a 2-site model. By adjusting the radioligand and receptor concentration or by the addition of blocking ligands, binding was represented by a 1-site model for DAGO,K=4.35 (±1.41)×10⁸ M^–1, and DADLE,K=2.44 (±0.08)×10⁸ M^–1.The abbreviations used are DADLE [D-Ala², D-Leu⁵]enkephalin - DAGO [D-Ala², MePhe⁴, Gly(ol)⁵]enkephalin - EKC ethylketocyclazocine - DYN dynorphin (1–17)     

Biosorption and solubilization of copper oxychloride fungicide by Aspergillus niger and the influence of calcium

The biosorption of copper oxychloride fungicide particulates(1 m diameter), at concentrations ranging from 25 to 500 ppm active ingredient (ai), by pelleted mycelium of Aspergillus niger grown on Czapek Dox medium was evaluated. The concentration of the fungicide adsorbed to the mycelium, remaining suspended or solubilized in the medium, was determined by analysis of its copper content (CuF)using atomic absorption spectrophotometry (AAS). 2-day-old pellets exhibited highbiosorption efficiency ranging from 97 ± 1.0 to 88 ± 1.2% of the initially added fungicide concentrations, respectively, within 10 min. However, underthe same conditions, amounts of the removed fungicide by 6-day-old mycelial pellets were significantly lower and ranged from 0.5 ± 0.03 to 0.15 ± 0.01%. Scanning electron microscopy studies of 2-day-old pellets supplemented with thefungicide revealed predominant aggregations of clumps and dense particulates on the hyphal tips. The adsorbed CuF of 125 ppm ai fungicide subsequently decreased from 7.5 ± 0.5 to 2.1 ± 0.1 mol Cu (mg dry wt)^-1 after 12 h incubation. Simultaneously, the soluble portion of CuF remaining in the medium increased from 0.9 ± 0.6 to4.9 ± 0.2 mol Cu ml^-1. The presence of 50 mM CaCl₂ resulted in a decrease of the adsorbed CuF to 3.5 ± 0.5 mol Cu (mg dry wt)^-1 and solubilizedcopper in the medium increased to 5.9 ± 0.8 mol Cu ml^-1. Additionally, the cellular copper contents attained after 2 h were 0.08 ± 0.01 and 0.16 ± 0.007 mol Cu (mg dry wt)^-1 in absence and presence of calcium, respectively. The addition of calcium to glucose-starved pellets greatly increased the medium [H⁺] which was conclusively discussed in relation to Ca²⁺/H⁺ exchangecapacity of the fungal cells. These results are of potential environmental,biotechnological and agricultural importance.     

Components of flow-induced dilation in rat perfused coronary artery

This study was undertaken to determine the endothelial factors involved in the flow-induced dilation of a rat perfused coronary artery. Segments of the right interventricular coronary artery were taken from 10–15-week-old male Wistar rats. Vessels were mounted in an arteriograph where internal diameter was continuously monitored while intraluminal pressure was controlled. Vessels were preconstricted with serotonin (10 mol/L), and the dilation induced by flow (0–800 l/min) was quantified. This dilator effect was measured in control conditions, after incubation with L-NAME (100 mol/L), with indomethacin (100µmol/L), and after mechanical destruction of the endothelium (–E). Dilations were expressed as percentage of the serotonin-induced constriction, and wall shear stress due to the physical forces exerted on the wall of the vessel was calculated and expressed in dyn/cm².In control conditions, raising the flow up to 800 l/min led to an increase in dilation (maximal dilation 63% ± 4%) and in sheer stress (maximal shear stress 76 ±4dyn/cm²). With indomethacin, maximal dilation was 69% ± 4% and maximal shear stress was 81 ± 6 dyn/cm². With L-NAME or after destruction of endothelium, dilation was greatly reduced (39% ± 3% and 40% ± 2%, respectively) whereas shear stress values were greatly increased (173 ± 14 and 150 ± 13 dyn/cm², respectively).These results confirm the viability of this model for the study of flow-dependent dilation. This dilation seems to be greatly dependent on NO release. In contrast, results do not favor a significant involvement of prostanoid vasodilating substance. Without endothelium, a dilation was still observed and showed the persistence of an endothelium-independent component of flow-induced dilation in this preparation that remains to be determined.Abbreviations Ach acetylcholine - BSA bovine serum albumin - EDRF endothelium-derived relaxing factor - EDHF endothelium-derived hyperpolarizing factor - L-NAME N -nitro-L-arginine-methyl ester - NO nitric oxide - PSS physiological salt solution - PGI₂ prostacyclin - 5-HT serotonin - SNP sodium nitroprusside - TXA₂ thromboxane A₂