Calcium release from frog sarcoplasmic reticulum by an imidazolyl reagent

Calcium is released from the isolated heavy sarcoplasmic reticulum (SR) of frog skeletal muscle upon application of 0.1-1 mM diethylpyrocarbonate (DEP, an imidazolyl reagent). The Ca-ATPase activity of SR was suppressed by 20% in the presence of 1 mM DEP. More than 1 mM of free magnesium ion or 5 microM ruthenium red eliminated the effect of DEP on calcium release but not on Ca-ATPase activity. A plausible site of DEP action is on the calcium channel.     

Effects of several preoperative medications on fat cell lipolysis,and activity of adipose tissue cyclic AMP phosphodiesterase

Summary Effects were examined of atropine, diazepam, pethidene, promethazine, scopolamine, omnopon and papaverine on basal and noradrenaline-stimulated lipolysis in rat isolated fat cells and on rat adipose tissue cyclic AMP phosphodiesterase activity. Papaverine at high concentration (1 mM) inhibited both basal and hormone-stimulated lipolysis, whereas diazepam enhanced basal lipolysis. At a clinical dose, omnopon increased both basal and noradrenaline-stimulated lipolysis. Adipose tissue cAMP phosphodiesterase activity was strongly inhibited by 1 mM diazepam, papaverine, promethazine and omnopon (280 g ml^–1). Lack of enhancement of lipolysis by the established cAMP phosphodiesterase antagonist papaverine, is compartible with simultaneous inhibition also of adipose adenyl cyclase. Diazepam-stimulated lipolysis is compatible with its phosphodiesterase inhibitory activity. It is proposed that papaverine-containing omnopon may offer some survival advantages during surgical stress by facilitating a caloric supply.The authours are grateful to Dr D. C. Williams for his continued support and encouragement.     

Effect of theophylline on myocardial adenylate cyclase activity

Summary Theophylline (0.01–10.0 mM) did not incease but rather decreased adenylate cyclase activity (AC) of guineapig auricles. Isoprenaline (1–100 M) and sodium fluoride (0.3–10.0 mM)stimulated AC in aconcentration-dependent manner.This work was supported by the Deutsche Forschungsgemeinschaft. The continous advice of Dr U. Schwabe is gratefully acknowledged.     

An in vitro kinetic study of the mixed inhibition of honeybee hemolymph PNP-α-D-glucosidase by sucrose

Summary Sucrose acts in vitro as a mixed inhibitor of (V+K+n) type towards honeybee hemolymph PNP--D-glucosidase activity. Between the stages of emergence and foraging, there is little change in the effect of the inhibitor on V_M (f_i=from 1.31 to 1.35 respectively) or n (f_i=from 1.09 to 1.07) but K is more markedly affected (f_i ^–1=from 1.17 to 1.87)^M. These observations reflect the decrease of K_i from 277 to 98 mM and of I₅₀ from 154 to 111 mM, but K_i scarcely alters during development (from 477 to 425 mM). These inhibitory effects of sucrose are intermediate between those previously reported for trehalose and glucose.Acknowledgments. Particular appreciation is due to Mrs C. Joliet for her meticulous technical assistance, and to Mr R. Paris for his care as beekeeper. Dr M.R.J. Morgan is grateful for financial support from The Leverhulme Trust. The Royal Society, I.C.I. Plant Protection and Shell International Petroleum.     

Reactive oxygen species are crucial for hydroxychavicol toxicity toward KB epithelial cells

Betel quid (BQ) chewing shows a strong correlation to the incidence of oral submucous fibrosis (OSF), leukoplakia and oral cancer. BQ contains mainly areca nut, lime, Piper betle leaf (PBL) and the inflorescence of P. betle (IPB). Hydroxychavicol (4-allyl-catechol, HC), as a major phenolic compound in PBL and IPB, is shown to induce oxidative stress, glutathione (GSH) depletion and cell cycle deregulation. Using bivariate BrdU/PI flow cytometry, KB cells in DNA synthesis (S phase) are shown to be sensitive to the toxic effect of HC and show cell cycle arrest and apoptosis following exposure to 0.1 and 0.3 mM HC. HC-induced apoptosis and cell cycle arrest are associated with mitochondrial membrane potential (m) depolarization as revealed by a decrease in rhodamine fluorescence. N-acetyl-L-cysteine (1 mM), superoxide dismutase (100 U/ml) and catalase (1000 U/ml) were effective in prevention of HC-induced GSH depletion (as indicated by chloromethylfluorescein fluorescence), reactive oxygen species (ROS) production (by dichlorofluorescein fluorescence), cell cycle arrest and apoptosis. However, dimethylthiourea (2 mM), neocuproine (1 mM), 1,10-phenanthroline (200 M) and desferrioxamine (0.5 mM) showed little effect on HC-induced cell changes. HC elevated the cellular and mitochondrial GSH levels at moderate concentrations (0.05–0.1 mM), whereas at a concentration of 0.3 mM, inhibitory effects were noted. These results indicate that HC consumption may be associated with BQ-chewing-related oral mucosal diseases via GSH depletion, ROS production, mitochondrial dysfunction, cell cycle disturbance and the induction of apoptosis. These events are related to the production of superoxide radicals and hydrogen peroxide.Received 9 July 2003; received after revision 28 September 2003; accepted 24 October 2003     

Inhibition of respiratory oxidation of reduced sulfur compounds by intact cells ofThiobacillus denitrificans (strain RT) grown on thiosulfate

Thiobacillus denitrificans strain RT, an obligate sulfur-oxidizing chemolithoautotroph, was grown under microaerophilic conditions with thiosulfate as the only energy source. The rates of tetrathionate, thiosulfate, elemental sulfur (S^o) and sulfite oxidation were measured respirometrically with an oxygen electrode, using actively growing cells. Cells oxidized thiosulfate, elemental sulfur (S^o) and sulfite, but not tetrathionate. The thiosulfateoxidizing activity and elemental sulfur-oxidizing activity (SOA) were almost totally inhibited by 50 M myxothiazol (>80%), an inhibitor of the quinone-cytochrome b region, and by 10 M of the uncoupler carbonyl cyanide m-chlorophenylhydrazone (CCCP) (>82%). Sulfite-oxidizing activity was also significantly inhibited (>60%) by 50 M myxothiazol and 10 M CCCP. 1 mM KCN totally inhibited (>90%) all respiratory activities. This study confirms that a sulfur-oxidizing activity appears during microaerophilic growth ofThiobacillus denitrificans strain RT on thiosulfate. The SOA is linked to the respiratory chain, probably releasing electrons in the quinone-cytochrome b region.To whom correspondence should be addressed. Submitted by R. Bachofen.     

Extraction of two different protein kinase activities from bovine rod outer segments

Summary An optimization of the rod outer segment (ROS) preparation technique is described. The protein responsible for ATP-³²P binding to bovine ROS was separated from the protein active with protamine on a DEAE Sephadex column. Molecular weight evaluation on a G 100 Sephadex column gave a value of 75,000 for the protein active with ROS, and 42,000 for that active with protamine. 1.25 mM c-AMP or c-GMP reduced the activity to 0.7 or 0.8 of the control respectively. 10 mM c-GMP doubled the yield of the active protein extracted from ROS.The experiments were performed at the Institut für Neurobiologie of the Kernforschungsanlage Jülich GmbH, FRG. The author was supported by a grant from SFB 160 Biologische Membranen of Deutsche Forschungsgemeinschaft. The expert technical assistance of Miss Regina Augustin is gratefully acknowledged.     

Identification and characterization of a highly thermostable bacteriophage lysozyme

Pseudomonas aeruginosa bacteriophage KMV is a T7-like lytic phage. Liquid chromatography-mass spectrometry of the structural proteins revealed gene product 36 (gp36) as part of the KMV phage particle. The presence of a lysozyme domain in the C terminal of this protein (gp36C) was verified by turbidimetric assays on chloroform-treated P. aeruginosa PAO1 and Escherichia coli WK6 cells. The molecular mass (20,884 Da) and pI (6.4) of recombinant gp36C were determined, as were the optimal enzymatic conditions (pH 6.0 in 16.7 mM phosphate buffer) and activity (4800 U/mg). Recombinant gp36C is a highly thermostable lysozyme, retaining 26% of its activity after 2 h at 100°C and 21% after autoclaving. This thermostability could prove an interesting characteristic for food conservation technology.Received 13 July 2004; received after revision 31 August 2004; accepted 6 September 2004     

Unusual high volume of sarcoplasmic reticulum in a wasp leg muscle

Summary Ultrastructural and morphometric examination of a wasp leg muscle showed that it contained a high volume of sarcoplasmic reticulum (volume fraction=0.35, surface density=21.4 m²/m³). As well as being arranged in double or triple layers between the myofibrils, the SR was found in large multilayered accumulations around the nuclei and in the subsarcolemmal space. Fibres of adjacent muscles had the normal volume and arrangement of SR.     

Analysis of translation products synthesized in isolated rat hepatocytes treated with diethylnitrosamine

Summary Isolated rat hepatocytes were labeled with³⁵S-methionine in the presence of 25 mM diethylnitrosamine (DENA). The intrinsically labeled proteins were analyzed by one-and two-dimensional gel electrophoresis and the fluorographic patterns were compared with those obtained from untreated hepatocytes. The results of short term experiments (2 h) show that, in the presence of 25 mM DENA, protein synthesis is inhibited by 50%. This reduction encompasses all protein species without selective inhibition of certain proteins.This work was supported by CNR (Project Control of Neoplastic Growth) grant No. 810132696 and partially by AIRC.     

Neurosteroid enhances glutamate release in rat prelimbic cortex via activation of α1-adrenergic and σ1 receptors

The present paper studied the effect and mechanism of neurosteroid pregnenolone sulfate (PREGS) on spontaneous glutamate release using electrophysiological and biochemical methods combined with a pharmacological approach. The results suggested that PREGS had a selective enhancing effect on spontaneous glutamate release in the prelimbic cortex and the hippocampus but not in the striatum. The effect of PREGS in the prelimbic cortex appeared to be via modulation of ₁-adrenergic and ₁ receptors, but in the hippocampus it might be dependent on ₁ receptors only. The activation of ₁-adrenergic receptors synergized ₁ receptor activation in the prelimbic cortex. Intracellular calcium released from the endoplasmic reticulum, protein kinase C, adenylyl cyclase and protein kinase A played a key role in the effect of PREGS. Intracellular calcium, protein kinase C and adenylyl cyclase might be upstream events in the activation of protein kinase A after PREGS.Received 7 January 2005; received after revision 19 February 2005; accepted 22 February 2005 Available online 29 March 2005     

Adenosine uptake in pyrimidine 5′-nucleotidase deficient human erythrocytes via a high affinity transport system

Summary Using a pulse-labeling technique,¹⁴C-adenosine uptake into pyrimidine 5-nucleotidase (P5N) deficient erythrocytes (RBC) was found to be impaired. The Lineweaver-Burk plot showed K_m values of 2.0×10^–3 mM and 0.2 ×10^–3 mM for normal RBC and P5N deficient RBC, respectively. These results indicate that P5N is one of regulators of the adenosine transport system and/or is associated with adenosine carrier protein.     

Green tea epigallocatechin-3-gallate is an inhibitor of mammalian histidine decarboxylase

(–)-Epigallocatechin-3-gallate, an antiproliferative and antiangiogenic component of green tea, has been reported to inhibit dopa decarboxylase. In this report, we show that this compound also inhibits histidine decarboxylase, the enzymic activity responsible for histamine biosynthesis. This inhibition was proved by a double approach, activity measurements and UV-Vis spectra of enzyme-bound pyridoxal-5-phosphate. At 0.1mM (–)-epigallocatechin-3-gallate, histidine decarboxylase activity was inhibited by more than 60% and the typical spectrum of the internal aldimine form shifted to a stable major maximum at 345nm, suggesting that the compound causes a stable change in the structure of the holoenzyme. Since histamine release is one of the primary events in many inflammatory responses, a new potential application of (–)-epigallocatechin-3-gallate in prevention or treatment of inflammatory processes is suggested by these data.Received 8 April 2003; received after revision 20 May 2003; accepted 3 June 2003     

Structural variations in wheat HKT1;5 underpin differences in Na<Superscript>+</Superscript> transport capacity

An important trait associated with the salt tolerance of wheat is the exclusion of sodium ions (Na⁺) from the shoot. We have previously shown that the sodium transporters TmHKT1;5-A and TaHKT1;5-D, from Triticum monoccocum (Tm) and Triticum aestivum (Ta), are encoded by genes underlying the major shoot Na⁺-exclusion loci Nax1 and Kna1, respectively. Here, using heterologous expression, we show that the affinity (K _m) for the Na⁺ transport of TmHKT1;5-A, at 2.66 mM, is higher than that of TaHKT1;5-D at 7.50 mM. Through 3D structural modelling, we identify residues D⁴⁷¹/a gap and D⁴⁷⁴/G⁴⁷³ that contribute to this property. We identify four additional mutations in amino acid residues that inhibit the transport activity of TmHKT1;5-A, which are predicted to be the result of an occlusion of the pore. We propose that the underlying transport properties of TmHKT1;5-A and TaHKT1;5-D contribute to their unique ability to improve Na⁺ exclusion in wheat that leads to an improved salinity tolerance in the field.     

Effect of hormones and cyclic AMP on γ-glutamyltranspeptidase activity of rat mammary gland explants

Summary -Glutamyl transpeptidase activity was assayed in midpregnant rat mammary gland explants at 14, 22 and 38 h, in the presence and absence of insulin, prolactin and corticosterone. With these 3 hormones the explants attained the characteristics of a secretory gland after 22 h of tissue culture, at which time the enzyme exhibited its maximal activity. The addition of dibutyryl cyclic AMP in the presence of the 3 hormones produced a significant increase in enzyme activity, which was maximal with a 1 mM concentration of the cyclic nucleotide. A similar effect was observed when theophylline or theophylline plus dibutyryl cyclic AMP were added to the culture medium.This work was supported by grant B1138-8333 from the Departamento de Desarrollo de la Investigación, Universidad de Chile.     

A novel pattern of fast calcium oscillations points to calcium and electrical activity cross-talk in rat chromaffin cells

Slow oscillations of cytosolic calcium ion concentration – – typically originate from release by intracellular stores, but in some cell types can be triggered and sustained by Ca²⁺ influx as well. In this study we simultaneously monitored changes in and in the electrical activity of the cell membrane by combining indo-1 and patch-clamp measurements in single rat chromaffin cells. By this approach we observed a novel type of spontaneous oscillations, much faster than those previously described in these cells. These oscillations are triggered and sustained by complex electrical activity (slow action potentials and spike bursts), require Ca²⁺ influx and do not involve release from intracellular stores. The possible physiological implications of this new pathway of intracellular signalling are discussed.Received 30 July 2004; received after revision 14 October 2004; accepted 1 November 2004     

Partial purification and characterization of acid phosphatase from sporulated oocysts ofEimeria tenella

Summary Acid phosphatase ofEimeria tenella oocysts (Peak II) was purified 77-fold with a recovery of 26% using protamine sulfate precipitation, DEAE-cellulose chromatography and Sephadex G-200 gel filtration. This enzyme occurs in multiple forms as indicated by two peaks which can be separated by DEAE-cellulose chromatography and polyacrylamide gel electrophoresis. The partially purified enzyme has optimal activity at pH 4.5. With p-nitrophenyl phosphate the Km and V_max values for (Peak II) were 25 mM and 1.57 mol/min/mg protein, respectively. The enzyme (Peak II) ist strongly inhibited by Hg⁺⁺, Cu⁺⁺, iodoacetamide, fluoride and molybdate. Tartrate and other divalent metal ions have no effect on enzyme activity. The partially purified Peak II phosphatase is not a glycoprotein as it is not absorbed on concanavalin-A Sepharose and its treatment with bacterial neuraminidase does not alter its elution profile through DEAE cellulose.     

Equilibrium constant for calcium ion and ascorbate ion

Summary The combination of calcium and ascorbic acid in water at 25°C has been examined by measuring the change of free calcium ion concentration as ascorbate was added in small increment to a solution of calcium. The data show clearly that complex formation between calcium ion and ascorbate ion occurred. At ionic strength =0.1–0.2, the equilibrium constant of Ca⁺⁺ and the singly-charged ascorbate ion has been measured to be 2.1 M^–1. The precision of the result is better than 5% and the accuracy is estimated to be better than 20%. The application of the equilibrium constants is discussed.     

HCN channels contribute to the intrinsic activity of cochlear pyramidal cells

A hyperpolarization-activated current recorded from the pyramidal cells of the dorsal cochlear nucleus was investigated in the present study by using 150- to 200-m-thick brain slices prepared from 6- to 14-day-old Wistar rats. The pyramidal neurones exhibited a slowly activating inward current on hyperpolarization. The reversal potential of this component was –32 ± 3 mV (mean ± SE, n = 6), while its half-activation voltage was –99 ± 1 mV with a slope factor of 10.9 ± 0.4 mV (n = 27). This current was highly sensitive to the extracellular application of both 1 mM Cs⁺ and 10 M ZD7288. The electrophysiological properties and the pharmacological sensitivity of this current indicated that it corresponded to a hyperpolarization-activated non-specific cationic current (I_h). Our experiments showed that there was a correlation between the availability of the h-current and the spontaneous activity of the pyramidal cells, suggesting that this conductance acts as a pacemaker current in these neurones. Immunocytochemical experiments were also conducted on freshly isolated pyramidal cells to demonstrate the possible subunit composition of the channels responsible for the genesis of the pyramidal h-current. These investigations indicated the presence of HCN1, HCN2 and HCN4 subunits in the pyramidal cells.Received 15 May 2003; received after revision 26 June 2003; accepted 21 July 2003     

Plasma testosterone and dihydrotestosterone in normal and abnormal pregnancy

Summary The concentration of plasma testosterone (T) and dihydrotestosterone (DHT) was determined in 2 groups of nonpregnant and pregnant women. The 1st group consisted of normal women and the 2nd of women with recurrent pregnancy disorders of unknown etiology. Significantly higher concentration of plasma DHT in nonpregnant women from the 2nd group was found (44.9±22 ng/100 ml) as compared to nonpregnant normals (24.2±5.2 ng/100 ml), p<0.01. There was no difference in the concentration of plasma T between the groups studied (p=0.165).Acknowledgment. The authors thank Prof. A. Draani, Mrs Lj. Vukeli and Mrs Z. Tomakovi for their collaboration in this study. This study was supported by the Scientific Fund of SR Croatia (Zagreb), SIZ V.