bluuebunny/crossref_metadata_2025 huggingface dataset:
Ten Challenges Associated with Management of Paracetamol Overdose: An Update on Current Practice and Relevant Evidence from Epidemiological and Clinical Studies
Authors: Fathelrahman Ahmed Ibrahim
Published: April 2021

Paracetamol is a commonly used medication all over the world. Although it is relatively safe, it results in serious toxicities requiring emergency department visits and hospitalisations, and may cause death. Toxicity can be predicted from drug blood concentration using Rumack-Matthew nomogram or from the ingested amount per body weight. Patients with expected toxicities are treated with activated charcoal if presented within two hours and N-acetylcysteine besides symptomatic and supportive measures. The present review represents a compilation of ten common challenging situations associated with management of paracetamol overdose. They include high risk patients, hypersensitivity reactions to the antidote, massive ingestions, late presentations, multiple ingestions of immediate release formulations, modified release ingestions, repeated supratherapeutic ingestions for therapeutic purposes, paediatric exposures, toxicity during pregnancy, and co-administered medications. Medical practitioners, who treat the patients presenting with paracetamol overdose and pharmacists working with drug or poison information centers, would benefit from having all ten challenging scenarios presented together in one place.
The Study of Electrical Energy Consumption in Cascaded Pumps for Pipeline Operations
Authors: Audu Eliazar Elisha
Published: April 2021

Pumps are critical infrastructure in the Oil and Gas industry, and have been widely used in pipeline transportations of petroleum products. The electrical energy needed by a pump to meet the minimum pipeline operational requirement plays an important role in the overall cost and evaluation of pumping systems performance, which has become an important issue in pump energy management and pump station designs. This paper provides a quantitative and analytical method using Bernoulli’s equation for studying energy dependence between two pumps (Booster and Mainline pumps) in series within a pump station as a function of pump’s head, flow-rate, and density. Using actual parameters from a pump station, the derived equations are validated on four different products. The densities of products are 1000 kg/cm3, 835 kg/cm3, 800 kg/cm3 and 660 kg/cm3 for Water, Automotive Gas Oil (AGO), Dual Purpose Kerosene (DPK), and Premium Motor Spirit (PMS) respectively. The results show that the energy requirement of the Booster pump is determined by the energy demand of the Mainline pump as a function of flowrate, density and pump’s head. The study is essential for developing energy saving strategy in pipeline operations and in electrical consideration when selecting the right electric motors for pumps in pump station design.
In vitro and In vivo Anticonvulsant Effect of Hydroalcoholic Extracts of Clutia abyssinica in Mice Model
Authors: Salile Samson Sahile , Abula Teferra
Published: April 2021

Background: Epilepsy is a chronic disorder of the brain that affects people of all ages worldwide. In the search of safe and effective antiepileptics traditional treatment practices are one area of research to obtain novel molecules. Research is also needed to validate and standardize the traditional claim. Clutia abyssinica leaves were one of the medicinal plants claimed for use against epilepsy and evil eye and other diseases in different parts of Ethiopia. But there was no scientific research evidence for the claimed use of the plant. Therefore this work was designed to evaluate the anticonvulsant effect of hydroalcoholic extract of Clutia abyssinica leaves.

Methods: The dry residue of the plant extract was used for the test. In vitro 0Mg2+ mice model at dose 0.7 mg/kg of extract, diazepam (3μM) and untreated brain slice groups were used to compare the presence of seizure like event (SLE). In vivo pentylenetetrazol (PTZ) model with 85 mg/kg subcutaneously was used to compare the seizure onset time with two extract doses and diazepam 5 mg/kg. The data was presented with mean± standard error. In maximum electric shock (MES) model 54 mA was passed for 0.2 second transauricularly in mice. The mean time of hind limb extension was recorded for doses 400 mg/kg and 800mg/kg of the extract and 10 mg/kg phenytoin. The means were compared for statistical significance using one way ANOVA post hoc LSD whereas proportions were compared using Fishers exact test with P-value < .05. 

Results: In vitro anticonvulsant tests C. abyssinica extract effect was not statistically significant compared to negative control (P>0.05).A positive control using the known anticonvulsant diazepam (3μM), showed significant anticonvulsant activity (P<0.05). The in vivo PTZ test showed no statistically significant effect in plant extract at all dose levels (P>0.05). In the in vivo MES test the extract of Clutia abyssinica both low and higher dose didn't show statistically significant effect (P>0.05) compared with the negative control. But the extract improved survival (p<0.05). The qualitative secondary metabolite test evidenced the presence of alkaloids, cardiac glycosides, flavanoids, phenols, saponins, sterols and terpeoids in Clutia abyssinica extract.

Conclusion: The hydroalcoholic crude extract result of the C. abyssinica as anticonvulsant is weak based on the models used in this study. For most of the local preparation are mixes of different plants it may have synergistic action with other plants. Or it may have action with other models of chronic epilepsy.
Identification of putative calcium channels in skeletal muscle microsomes
Authors: Ferry David R. , Glossmann Hartmut
Published: July 2002

Saturable binding sites for the labelled calcium antagonist (±)[3H]nimodipine were found in guinea‐pig hind limb skeletal muscle homogenates. Binding sites were enriched in a microsomal pellet by differential centrifugation of the homogenate. [3H]Nimodipine binding (K
d = 1.5±0.03 nM, B
max = 2.1 ± 0.25 pmol/protein, at 37°C) copurified (6‐fold) in this fraction with [3H]ouabain binding (6.6‐fold) and 125I‐α‐bungarotoxin binding (5‐fold). d‐cis‐Diltiazem (but not 1‐cis‐diltiazem) stimulated (±) [3H]nimodipine binding (ED
50 1 μM) by increasing the B
max. Binding sites discriminated between the optical enantiomers of 1,4‐dihydropyridine calcium antagonists and the optically pure enantiomers of D‐600. The data confirm, with biochemical techniques, the presence of 1,4‐dihydropyridine and (±)D‐600 inhibitable calcium channels in skeletal muscle, previously found with electrophysiological techniques.
Cytoplasmic free Ca2+ in human platelets: Ca2+ thresholds and Ca‐independent activation for shape‐change and secretion
Authors: Rink T.J. , Smith S.W. , Tsien R.Y.
Published: July 2002

Cytoplasmic free [Ca2+], [Ca2+]i, was measured in human platelets using the intracellularly‐trapped, fluorescent indicator quin2. Basal [Ca2+]i with the Ca2+‐ionophore ionomycin revealed apparent thresholds for shape‐change, 5‐HT release and aggregation of approx. 0.5μM, 0.8μM and 2 μM. Thrombin raised [Ca2+]i to 3 μM fast enough for the Ca2+ to have triggered the cell activation. However, thrombin released more 5‐HT than ionomycin could, and in Ca2+‐free medium thrombin evoked shape‐change and secretion even when [Ca2+]i remained near basal levels throughout, suggesting the existence of alternative triggers for shape‐change and secretory exocytosis.
Purification of three rat atrial natriuretic factors and their amino acid composition
Authors: Thibault G. , Garcia R. , Seidah N.G. , Lazure C. , Cantin M. , Chrétien M. , Genest J.
Published: July 2002

A natriuretic factor has been described in the specific granules of rat atria. We have purified three factors which seem to be low‐M
r peptides. They have been purified by means of acid extraction, octadecyl Sep‐Pak cartridges, and chromatography on Bio‐Gel P‐10, CM Bio‐Gel A, Mono S and reverse‐phase high‐performance liquid chromatography columns. The factors contain 26, 31 and 33 amino acids and may have been partially degraded during isolation. They are all 3 biologically active and the shorter one is the most active with a specific activity of 450000 units/mg.
Phosphorylation of chicken gizzard myosin and the Ca2+‐sensitivity of the actin‐activated Mg2+‐ATPase
Authors: Cole Heather A. , Patchell Valerie B. , Perry S.Victory
Published: July 2002

A method is described for the preparation of partially and fully phosphorylated chicken gizzard myosin. When fully phosphorylated it possessed an actin‐activated Mg2+‐ATPase of similar specific activity to that of mammalian skeletal muscle myosin. The Mg2+‐ATPase activity of these preparations was related in a non‐linear fashion to increasing phosphorylation of the P light chain. When P light chain phosphorylation occurred during enzymic assay the Mg2+‐ATPase activity remained constant. Fully phosphorylated preparations of gizzard myosin possessed an actin‐activated Mg2+‐ATPase that was not Ca2+‐sensitive, whereas the Mg2+‐ATPase of partially phosphorylated myosin preparations was Ca2+‐sensitive.
Adenylate cyclase inhibition and GTPase stimulation by somatostatin in S49 lymphoma cyc− variants are prevented by islet‐activating protein
Authors: Aktories Klaus , Schultz Günter , Jakobs Karl H.
Published: July 2002

cyc−‐Variants of S49 lymphoma cells are defective in the stimulatory guanine nucleotide site of the adenylate cyclase but contain an inhibitory site. Treatment of cyc− cells with islet‐activating protein (IAP), which causes ADP‐ribosylation of an M
r 40 000 polypeptide in cyc− membranes, abolishes adenylate cyclase inhibition by GTP and the peptide hormone, somatostatin, but not that induced by GTPγS. Furthermore, somatostatin‐induced stimulation of GTP hydrolysis is lost. Thus, the data indicate that IAP interferes with the adenylate cyclase system by an action at the inhibitory guanine nucleotide site.
Induction by chemically modified actin derivatives of antibody specificity
Authors: Benyamin Y. , Roustan C. , Boyer M.
Published: July 2002

A comparison of specific antibodies induced by unfolded actins modified either by oxidation or by arylation of lysine residues was reported. We have focused our work on binding properties with filamentous actin and located its preferential antigenic sites for the anti‐arylated‐actin antibodies in the C‐part of the molecule. An interference of anti‐oxidized actin antibodies upon actin polymerisation has also been reported.
Effect of pertussis toxin on hormonal responsiveness of rat hepatocytes
Authors: Pushpendran Choliparambil K. , Corvera Silvia , Adolfo García-Sáinz J.
Published: July 2002

The ureogenic action of epinephrine in hepatocytes from normal adult rats is mediated through activation of α1‐adrenoceptors. β‐Adrenoceptors in addition to α1‐adrenoceptors, became involved in mediating this effect in cells from animals treated with pertussis toxin. The accumulation of cyclic‐AMP in response to epinephrine or isoproterenol was markedly increased in hepatocytes from pertussis‐treated rats as compared to that observed in control cells. The accumulation of cyclic‐AMP due to glucagon was also increased. It is suggested that pertussis toxin may release a constraint on adenylate cyclase activity by blocking the inhibitory coupling mechanism (N
i) or some other entity involved in the regulation of the activity of this enzyme.
Metabolic properties of low ATP erythrocytes of the monotremes
Authors: Kim Hyun Dju , Zeidler Robert B. , Sallis John , Nicol Stewart , Isaacks Russel E.
Published: July 2002

The erythrocytes of the monotrenes, having a trace amount of ATP, can metabolize glucose to lactate at a rate comparable to human and other mammalian erythrocytes. The echidna energy metabolism is unique in that adenosine can stimulate glycolytic carbon flow, resulting in a nearly 20‐fold net synthesis of ATP.
Molecular mechanism of uncoupling in brown adipose tissue mitochondria
Authors: Kopecký Jan , Guerrieri Ferruccio , Ježek Petr , Drahota Zdeněk , Houštěk Josef
Published: July 2002

Specific permeability properties of the inner membrane of brown adipose tissue mitochondria were analysed with the aid of simultaneous pH measurements outside mitochondria and of mitochondria swelling. It was shown that valinomycin‐induced potassium diffusion potential drives a parallel passive uptake of chlorides and extrusion of proton. Electrogenic H+‐extrusion was independent on anion transport, no competition was found between the two processes and the former process exerted a lower sensitivity to the inhibitory effect of GDP. The existence of two distinct, independent pathways for translocation of protons and halide anions across the membrane is suggested.
Estimation of the rate of photochemical charge separation in Rhodopseudomonas sphaeroides reaction centers by fluorescence and absorption picosecond spectroscopy
Authors: Paschenko Vladimir Z. , Korvatovskii Boris N. , Kononenko Alexander A. , Chamorovsky Sergei K. , Rubin Andrew B.
Published: July 2002

Time‐resolved fluorometry of reaction center (RC) preparations from Rhodopseudomonas sphaeroides, wild strain 1760‐1, shows that the lifetime of the excited state of bacteriochlorophyll P870∗ is τ = 6± 1.5 ps and independent of temperature within the range 293 – 77 K. This value was found to coincide well with the time (7 ± 3 ps) of the RC porphyrin pigment transition into the ion‐radical pair state PF, as measured by picosecond absorption spectroscopy of the same preparations.
Evidence that a chlorophyll a' dimer constitutes the photochemical reaction centre 1 (P700) in photosynthetic apparatus
Authors: Watanabe Tadashi , Kobayashi Masami , Hongu Akinori , Nakazato Masataka , Hiyama Tetsuo , Murata Norio
Published: July 2002

Spinach chloroplasts and their various fractions enriched in either photochemical reaction centre 1 (P700) or reaction centre 2 (P680) have been analyzed for pigment composition by high‐performance liquid chromatography (HPLC). The results clearly establish a stoichiometry of chlorophyll (Chl) a'/P700 = 2, where Chl a' represents the C 10 epimer of Chl a. This is the first demonstration of the association of Chl a' with any functional site in vivo. It is further shown that a Chl a' dimer prepared in vitro reproduces fairly well the redox and spectroscopic behaviour of P700. These findings provide strong evidence that a Chl a dimer constitutes P700.
A Ca2+‐sensitive actin regulatory protein from smooth muscle
Authors: Kanno Kimiyoshi , Sasaki Yasuharu , Hidaka Hiroyoshi
Published: July 2002

Using a procedure involving DNase I affinity chromatography and Sephadex G‐200 gel filtration, we partially purified a Ca2+‐sensitive actin regulatory 90‐kDa protein from bovine aorta. The 90‐kDa protein existed in the form of a complex with actin on a DNase I column even in the presence of 5 mM EGTA, indicating that the 90‐kDa protein binds tightly to actin in a Ca2+‐insensitive manner. The isolation procedure described above indicates that the 90‐kDa protein is present in smooth muscles including aorta, uterus and bladder, but not in skeletal and heart muscles. When added to G‐actin before polymerization, the 90‐kDa protein increases the initial rate of actin polymerization and lowers the final viscosity at Ca2+ concentrations greater than 10−7M. This decrease in viscosity is due to the generation of filaments which cannot be readily sedimented.
Ca2+ channel agonist BAY‐k 8644 does not elicit Ca2+ release from skeletal muscle sarcoplasmic reticulum
Authors: Zorzato Francesco , Volpe Pompeo , Salviati Giovanni , Margreth Alfredo
Published: July 2002

BAY‐k 8644, a nifedipine analogue, promotes Ca2+ influx into excitable cells via plasma membrane voltage‐sensitive Ca2+ channels. We report here that sarcoplasmic reticulum (SR) Ca2+ release channels are insensitive to BAY‐k 8644, as studied in highly purified isolated fractions and in chemically skinned fibers of rabbit skeletal muscle. This result suggests that a subcellular heterogeneity exists among Ca2+ channels, at least with respect to drug‐receptor sites. In the course of this study, however we found that BAY‐k 8644 reversibly inhibits the SR Ca2+ pump, i.e., it decreases Ca2+ influx into the SR lumen, although at concentrations (IC50 = 3‐5 × 10−5M) much higher than those effective on voltage‐sensitive Ca2+ channels.
Insulin release independent of a rise in cytosolic free Ca2+ by forskolin and phorbol ester
Authors: Tamagawa Tatsuo , Niki Hatsumi , Niki Atsushi
Published: July 2002

The role of cytosolic free Ca2+ in insulin release was evaluated using isolated rat pancreatic islets permeabilized with digitonin and incubated in Ca‐EGTA buffers to fix free Ca2+ concentration at arbitrary levels. Ca2+ induced insulin release in a concentration‐dependent manner with the threshold being between 0.1 and 1 μM. The hormone release was increased by forskolin and 12‐O‐tetradecanoyl phorbol‐13‐acetate (TPA), a potent activator of adenylate cyclase and that of protein kinase C, respectively. The findings suggest that activation of both protein kinase A and protein kinase C modulate insulin release without a concomitant increase in cytosolic free Ca2+.
Voltage‐activated Ca2+ currents in insulin‐secreting cells
Authors: Findlay Ian , Dunne Mark J.
Published: July 2002

Membrane voltage and voltage‐clamped membrane currents have been investigated with the whole‐cell patch clamp method in the insulin‐secreting cell line RINm5F. The mean resting membrane potential of RINm5F cells was found to be −52 mV. Overshooting spike potentials could be evoked by membrane depolarisation in the absence of a secretagogue. Inward membrane currents evoked by depolarising voltage steps were dependent upon extracellular Ca2+ and blocked by Co2+, nifedipine and verapamil. Outward membrane currents which were evoked by depolarising voltage steps to positive membrane potentials were reduced when Ca2+ entry was prevented. It is concluded that the voltage‐activated Ca2+ currents underlie the voltage‐activated spike potentials recorded from insulin‐secreting cells.
Treatment of rabbit neutrophils with phorbol esters results in increased ADP‐ribosylation catalyzed by pertussis toxin and inhibition of the GTPase stimulated by fMet‐Leu‐Phe
Authors: Matsumoto T. , Molski T.F.P. , Volpi M. , Pelz C. , Kanaho Y. , Becker E.L. , Feinstein M.B. , Naccache P.H. , Sha'afi R.I.
Published: July 2002

The effects of pretreatment of rabbit neutrophils with phorbol 12‐myristate 13‐acetate on the ability of pertussis toxin to catalyze ADP‐ribosylation and of fMet‐Leu‐Phe to activate a high‐affinity GTPase in these cell homogenates were examined. The addition of phorbol 12‐myristate 13‐acetate, but not 4α‐phorbol 12,13‐didecanoate, to intact cells was found to stimulate by more than 100% the pertussis toxin‐dependent ribosylation of a 41 kDa protein (either the α‐subunit of the ‘inhibitory’ guanine nucleotide‐binding protein Ni or a closely analogous protein) and to inhibit by more than 60% the activation by fMet‐Leu‐Phe of the GTPase of the neutrophil homogenates. The addition of fMet‐Leu‐Phe to intact cells increases the ADPribosylation catalyzed by pertussis toxin of the 41 kDa protein. On the other hand, the exposure of neutrophil homogenates to fMet‐Leu‐Phe results in a decreased level of ADP‐ribosylation. This decreased ribosylation reflects a dissociation of the GTP‐binding protein oligomer that is not followed by association, possibly because of the release of the α‐subunit into the suspending media. The implications of these results for the understanding of the mechanism of inhibition of cell responsiveness by phorbol esters and the heterologous desensitization phenomenon are discussed. Prominent among these are the possibilities that (i) the rate of dissociation of the Ni oligomer is affected by the degree of its phosphorylation by protein kinase C, and/or (ii) the dissociated phosphorylated α‐subunit (the 41 kDa protein) is functionally less active than its dephosphorylated couterpart.
Volume‐dependent and NEM‐stimulated K+Cl−1 transport is elevated in oxygenated SS, SC and CC human red cells
Authors: Canessa Mitzy , Spalvins Anda , Nagel Ronald L.
Published: July 2002

Mechanisms involved in cell volume regulation are important in SS, SC cells as they might be involved in determining the extent of sickling and the generation of dense cells and irreversibly sickled cells. We have studied in these cells the response to cell swelling of the K+Cl− transporter. We found that Hb SS, SC and CC red cells have higher values of a ouabain‐resistant, chloride‐dependent and NEM‐stimulated K+ efflux than AA red cells. In contrast, the Na+K+Cl−cotransport estimated from the bumetanide‐sensitive component of K+ efflux was not significantly different in SS, SC and CC red cells. The (ouabain + bumetanide)‐resistant K
+ efflux from SS, SC and CC red cells was stimulated by cell swelling induced by reduction of the osmotic pressure (300 to 220 ) and pH (8 to 7) of the flux media (140 mM NaCl). The Cl−‐dependent K+ efflux stimulated by osmotic swelling highly correlated with the NEM‐stimulated component (r = 0.8, p< 0.001, n = 22) and the acid‐pH‐induced swelling (r = 0.969,p< 0.001, n = 22), indicating that it is driven by the K+Cl− transporter.
Solubilization of trehalase from rabbit renal and intestinal brush‐border membranes by a phosphatidylinositol‐specific phospholipase C
Authors: Takesue Yoshiki , Yokota Kunio , Nishi Yoshimi , Taguchi Ryo , Ikezawa Hiroh
Published: July 2002

Trehalase (EC 3.2.1.28) associated with renal and intestinal brush‐border membranes was solubilized by highly purified phosphatidylinositol‐specific phospholipase C (EC 3.1.4.10) from Bacillus thuringiensis, but not by phosphatidylcholine‐hydrolyzing phospholipase C (EC 3.1.4.3) from Clostridium welchii or phospholipase D (EC 3.1.4.4) from cabbage. The solubilized trehalase was not adsorbed on phenyl‐sepharose, indicating that it was hydrophilic. Phosphatidylinositol‐specific phospholipase C also converted Triton X‐100‐solubilized amphipathic trehalase into a hydrophilic form. These results suggest that trehalase is bound to the membrane through a direct and specific interaction with phosphatidylinositol.
Evidence that (a) serine specific protein kinase(s) different from protein kinase C is responsible for the insulin‐stimulated actin phosphorylation by placental membrane
Authors: Carrascosa J.M. , Wieland O.H.
Published: July 2002

Partially purified phospholipid‐ and Ca2+‐dependent protein kinase C from human placenta catalyzes the Mg‐ATP‐dependent phosphorylation of serine residues of purified rabbit muscle actin. Two tryptic [32P]‐phosphopeptides were found on HPLC separation. Confirming the previous report by Machicao and Wieland [(1985) Curr. Top. Cell. Regul. 27, 95‐105], actin is phosphorylated at serine residues by human placental membranes, and this is stimulated by insulin. In the absence of insulin trypsin treatment yielded eight [32P]phosphopeptides, two of which coincided with the ones due to protein kinase C. Insulin led to the appearance of three new [32P]phosphopeptides. These results suggest that insulin stimulates (a) serine protein kinase(s) which, like protein kinase C, is present in placental membranes.
Pig kidney Na+,K+‐ATPase
Authors: Ovchinnikov Yu.A. , Modyanov N.N. , Broude N.E. , Petrukhin K.E. , Grishin A.V. , Arzamazova N.M. , Aldanova N.A. , Monastyrskaya G.S. , Sverdlov E.D.
Published: July 2002

(Na+ + K+)‐ATPase α‐Subunit β‐Subunit
cDNA nucleotide sequence
Primary structure
Glycopeptide
Transmembrane arrangement
Cell alkalinization is not necessary and increased sodium influx is not sufficient for stimulated superoxide production
Authors: Molski T.F.P. , Ford C. , Weisman S.J. , Sha'afi R.I.
Published: July 2002

Preincubation of rabbit neutrophils for 5 min with the protein kinase C inhibitor H7 causes inhibition of the rise in intracellular pH but not the increase in Na+ influx or stimulated oxidative burst produced by the chemotactic factor formyl‐methionyl‐leucyl‐phenylalanine. On the other hand, the stimulated superoxide production, but not the increase in Na+ influx produced by phorbol 12‐myristate 13‐acetate, is inhibited by H7. The effect is more pronounced on the rate than the extent of the stimulated superoxide release. Furthermore, cell acidification produced by the phorbol ester but not by the chemotactic factor is decreased in the presence of H7. These results suggest that (i) most of the stimulated Na+ influx is not coupled to H+ efflux, (ii) in the case of the chemoattractant, the rise in intracellular pH is not necessary for stimulated superoxide production, (iii) the increase in Na+influx, in the case of the phorbol ester, is not sufficient for the stimulation of the oxidative burst, and (iv) the sources of the H+ responsible for the stimulated pH drop are the various metabolic activities of the cell, including NADPH oxidation and activation of the hexose monophosphate shunt.
N‐terminal amino acid sequence of the Rieske iron‐sulfur protein from the cytochrome b6/f‐complex of spinach thylakoids
Authors: Pfefferkorn Bernhard , Meyer Helmut E.
Published: July 2002

The 20 kDa subunit of the cytochrome b
6/f‐complex, the Rieske iron‐sulfur protein, was isolated by preparative SDS‐PAGE. For automatic Edman degradation the protein was recovered by electrophoretic elution. Several peptides were purified after tryptic, CNBr or Arg C endopeptidase digestion or cleavage at Trp‐residues. The determination of the amino acid sequence of these peptides yielded overlapping sequences for a total of 96 amino acids starting at the N‐terminus. This sequence shows no homology to the primary structure of the two Rieske FeS proteins already published by other authors. But like these it contains a hydrophobic sequence of 30 amino acids close to the N‐terminal indicating the sequence that spans the membrane. Two polypeptides, closer to the C‐terminus, contained sequences which are also present in Neurospora crassa and Rhodopseudomonas sphaeroides including a Cys‐Pro‐Cys containing sequence involved in iron binding.
Protein kinase C translocation in relation to proliferative state of C3H 10T1/2 cells
Authors: Miłoszewska Joanna , Trawicki Witold , Janik Przemysław , Moraczewsk Jerzy , Przybyszewska Małgorzata , Szaniawska Bożena
Published: July 2002

Protein kinase C (PKC) activity of cytosol and membrane fractions of 10T cells was studied. In cytosol of fast growing cells PKC activity was found in material eluted with 0.150 M NaCl whereas in membrane fractions activity was eluted with 0.065 and 0.150 M NaCl. In the membrane fraction of confluent cells, in contrast to cytosol, very low PKC activity was observed. The translocation pattern of PKC activity eluted with 0.065 M NaCl may be associated with proliferation.
Fluorescence studies on the Ca2+ and Zn2+ binding properties of the α‐subunit of bovine brain S‐100a protein
Authors: Leung Iris K.M. , Mani Rajam S. , Kay Cyril M.
Published: July 2002

The single cysteine on the α‐subunit of bovine brain S‐100a protein has been modified with the thiol specific probe, Acrylodan. When the labelled apoprotein was excited at 380 nm the fluorescence emission maximum was centered at 484 ± 2 nm, suggesting that the probe is in a fairly hydrophobic environment. Addition of Ca2+ to the protein caused the emission maximum to undergo a red shift to 504 ± 2 nm, implying that the fluorophore is now more exposed to the solvent. Zn2+, when added to the protein, induced only a small perturbation and the emission maximum shifted to 481 ± 2 nm. Ca2+ was able to perturb the fluorophore in the presence of Zn2+. 2‐p‐Toluidinylnaphthalene‐6‐sulfonate (TNS)‐labelled α‐subunit when excited at 345 nm exhibited very little fluorescence in the absence of Ca2+. Addition of Ca2+ resulted in an increase in TNS fluorescence accompanied by a blue shift of the emission maximum to 445 ± 1 nm indicating that the probe in the presence of Ca2+ moves to a hydrophobic domain. The fact that Ca2+ and Zn2+ can perturb the labelled sulfhydryl group in the presence of each other clearly demonstrates that the binding sites for the two metal ions must be different on the α‐subunit as well as on the S‐100a protein.
Expression of soybean glycinin subunit precursor cDNAs in Escherichia coli
Authors: Fukazawa Chikafusa , Udaka Kyoko , Murayama Akiko , Higuchi Wakako , Totsuka Atsushi
Published: July 2002

As the cDNAs encoding A1aB1b and A2B1a subunit precursors of the glycinin A2 subfamily contain a unique NcoI site sequence, (A)CCATGG, occurring at their translation initiation sites, plasmids were constructed to direct the synthesis of those precursor proteins by inserting NcoI/PstI fragments derived from those cDNA clones into the Nco/PstI‐pKK233‐2 expression vector in Escherichia coli MV 1190, respectively. The resultant plasmids directed the expression of 57‐kDa protein components that have molecular masses in agreement with those of the in vitro translation products directed by glycinin A2 subfamily mRNAs, by the addition of isopropyl β‐D‐thiogalactoside. These proteins, which comprised as much as 1% of the total bacterial protein, are immunoprecipitable with rabbit antibodies specific for glycinin subunits. This procedure makes glycinin subunits available as a model for studying structure‐function relationships in seed proteins using site‐directed mutagenesis. This is the first expression of glycinin‐like storage protein in E. coli.
ATP release from pure cholinergic synaptosomes is not blocked by tetanus toxin
Authors: Rabasseda Xavier , Solsona Carles , Marsal Jordi , Egea Gustau , Bizzini Bernard
Published: July 2002

Tetanus toxin (TeTx) is a neurotransmission impairing toxin that acts on several neurotransmitter systems. TeTx also inhibits the K+‐induced release of acetylcholine (ACh) from synaptosomes isolated from the electric organ of Torpedo. Neither the membrane potential and depolarization, nor the depolarization‐induced calcium uptake into cholinergic nerve terminals is modified after TeTx poisoning. On the other hand, it is known that, when cholinergic nerve terminals are stimulated, there is a release of ATP associated with the release of ACh. We have explored the action of TeTx on this co‐release, and have found that there is no action of TeTx on the nucleotide release. Thus, TeTx blocks ACh release without modifying ATP release.
Inhibition of the H+‐ATPase in bovine adrenal chromaffin granule ghosts by diethylstilbestrol Evidence for a tight coupling between ATP hydrolysis and proton translocation
Authors: Grønberg Martin , Flatmark Torgeir
Published: July 2002

Diethylstilbestrol (DES) was found to inhibit reversibly the hydrolysis of MgATP (80% at 100μM) and proton pump activity (I
50⋍15 μM, complete at 100 μM) in chromaffin granule ghosts. The parallel inhibition suggests a tight kinetic coupling between the two activities. The Mg2+‐ATPase activity, but not proton pumping, was partially restored by N,N′‐dicyclohexylcarbodiimide,indicating that the two inhibitors in combination cause a partial uncoupling. The non‐competitive type of inhibition shows that the action of DES is distal to the site of ATP binding and hydrolysis. Although unspecific, the interaction of DES with the chromaffin granule membrane seems primarily to affect the H+‐ATPase.
The mitochondrial outer membrane channel, VDAC, is modulated by a soluble protein
Authors: Holden Marcia J. , Colombini Marco
Published: July 2002

The mitochondrial outer membrane channel, VDAC, serves as the primary permeability pathway for metabolite flux between cytoplasmic and mitochondrial compartments. VDAC can occupy several conformational states differing in ion conductivity. Small transmembrane potentials cause transitions from open‐ to closed‐channel conformations. A soluble mitochondrial protein enhances the channel's response to voltage by increasing the rate of channel closing; inducing the occupation of lower conductance states; and decreasing the rate of channel reopening. This protein modulator acts at very low concentrations and its role in the cell may be to regulate the permeability of the mitochondrial outer membrane by inducing channel closure.
Phospholipase activation, free fatty acids and the proton permeability of a biological membrane
Authors: Lachowiez Robert M. , Hammoud Nasser M. , Teibel Jeffrey L. , Dix James A.
Published: July 2002

The rate of collapse of a proton gradient across the apical membrane of rat kidney proximal tubule increases upon treatment with calcium, mercuric chloride and mellitin, substances which activate phospholipase A2. Treatment with phospholipase A2 or oleic acid also enhances the rate of proton gradient dissipation. Membrane water permeability is not affected. This phenomenon may have implications in pathological states arising from ischemia or toxic exposure.
Inhibition of dihydropyridine‐sensitive calcium channels by the plant alkaloid ryanodine
Authors: Valdivia Hector H. , Coronado Roberto
Published: July 2002

At micromolar concentrations, ryanodine interacts with the dihydropyridine receptor of rabbit skeletal muscle transverse tubules. Ryanodine displaces specifically bound [3H]PN200‐110 with an apparent inhibition constant of approx. 95 μM and inhibits dihydropyridine‐sensitive calcium channels in the same preparation with an IC50 of approx. 45 μM. These concentrations of ryanodine are approximately three orders of magnitude higher than those required to saturate binding of the alkaloid to the ryanodine receptor of sarcoplasmic reticulum and to open the calcium release channel of sarcoplasmic reticulum (i.e. 20 nM (1988) J. Gen. Physiol. 92, 1–26). Thus at sufficiently high dose, ryanodine may affect SR as well as plasma membrane Ca permeabilities.
Treatment of intact hepatocytes with either the phorbol ester TPA or glucagon elicits the phosphorylation and functional inactivation of the inhibitory guanine nucleotide regulatory protein Gi
Authors: Pyne Nigel J. , Murphy Gregory J. , Milligan Graeme , Houslay Miles D.
Published: July 2002

The antiserum AS7 can specifically immunoprecipitate α‐Gi from membrane extracts as well as from a mixture of purified α‐Gi and α‐Go. as ascertained using [32P]ADP‐ribosylated G‐proteins. Using this antiserum to immunoprecipitate α‐Gi from hepatocytes labelled with 32P it was evident that α‐Gi was phosphorylated under basal (resting) conditions. Challenge of hepatocytes with the tumour promoting phorbol ester TPA, however, elicited a marked enhancement of the phosphorylation state of α‐Gi. This was accompanied by the loss of inhibitory effect of Gi on adenylate cyclase, as judged by the inability of low concentrations of p[NH]ppG to inhibit forskolin‐stimulated adenylate cyclase activity. Such actions were mimicked by treatment of hepatocytes with either glucagon or TH‐glucagon, an analogue of glucagon which is incapable of activating adenylate cyclase and elevating intracellular cyclic AMP concentrations. Pre‐treatment of hepatocytes with either glucagon, TPA or insulin did not affect the ability of pertussis toxin to cause the NAD+‐dependent, [32P]ADP‐ribosylation of α‐Gi in membrane fractions isolated from such pre‐treated hepatocytes. We suggest that protein kinase C can elicit the phosphorylation and functional inactivation of α‐Gi in intact hepatocytes. As pertussis toxin only causes the ADP‐ribosylation of the holomeric form of Gi, it may be that phosphorylation leaves α‐Gi in its holomeric state.
Electric field‐induced fusion of mitochondria
Authors: Reynaud J.A. , Labbe H. , Lequoc K. , Lequoc D. , Nicolau C.
Published: July 2002

Fusion of mitochondria in H‐medium from rat liver was induced by the application of square‐wave voltage with electric field strengths of 1–2.5 kV/cm and duration 100 μs. Electron micrographs showed that the newly fused mitochondria could contain up to five mitoplasts. The fusion yield was close to 12% and respiratory activity was enhanced. The electric field lines did not go through the inner membrane, however, when the electric field strength was greater than 3 kV/cm they did so, resulting in total destruction of the mitochondria.
Effect of adenosine on fructose 2,6‐bisphosphate levels and glucose metabolization by chicken erythrocytes
Authors: Espinet Carme , Bartrons Ramon , Carreras José
Published: July 2002

Adenosine can be metabolized by chicken erythrocytes as a carbon source maintaining ATP levels. In addition, it stimulates glucose uptake and lactate production. Adenosine effects do not involve binding to membrane receptors. They are dependent on the provision of a carbon source to glycolysis and correlate with the increase of fructose 2,6‐bisphosphate levels that activating phosphofructokinase would increase the glycolytic flux.
Enzymatic synthesis of nucleoside 5′‐mono and ‐triphosphates
Authors: Zinchenko Anatoli I. , Barai Vladimir N. , Zaiashko Larisa M. , Poopeiko Nikolai E. , Pricota Tamara I. , Sivets Grigorii G. , Mikhailopulo Igor A.
Published: July 2002

The transformation of fluorodeoxy‐nucleosides into 5'‐monophosphates with the use of whole bacterial cells of Erwinia herbicola as a biocatalyst and PNP as a phosphate donor and next into 5'‐triphosphates by using an extract of the Saccharomyces cerevisiae cells has been demonstrated.
Modulation of vertebrate brain Na+ and K+ channels by subtypes of protein kinase C
Authors: Lotan Ilana , Dascal Nathan , Naor Zvi , Boton Rony
Published: July 2002

Effects of purified subtypes I, II and III of protein kinase C (PKC) on voltage‐dependent transient K+ (A) and Na+ channels were studied in Xenopus oocytes injected with chick brain RNA. The experiments were performed in the constant presence of 10 nM β‐phorbol 12‐myristate‐13‐acetate (PMA). Intracellular injection of subtype I (τ) reduced the A‐current (I
A), with no effect on Na+ current (I
Na). PKC subtype II (β1, + β2) and III (α) reduced both currents. PKC did not affect the response to kainate. Inactivated (heated) or unactivated (injected in the absence of PMA) enzyme and vehicle alone had no effect. Our results strongly suggest that I
Na, and I
A in vertebrate neurons are modulated by PKC; all PKC subtypes exert a similar effect on the A‐channel while only subtypes II and III modulate the Na+ channel.
Detection of a large cation‐selective channel in nuclear envelopes of avian erythrocytes
Authors: Matzke A.J.M. , Weiger T.M. , Matzke M.A.
Published: July 2002

To determine whether the nuclear envelope of eukaryotic cells has the capability to regulate ion fluxes, we have used the patch‐clamp technique to detect ion channels in this membrane system. Since possible sites for ion channels in the nuclear envelope include not only the nuclear pores, but also both the inner and outer nuclear membranes, we have patched giant liposomes composed of phosphatidylcholine and nuclear envelope fragments isolated from mature avian erythrocytes. A large, cation‐selective channel with a maximum conductance of approximately 800 pS in symmetrical 100 mM KCl was detected. This channel is a possible candidate for a nuclear pore.
A new class of calcium channels activated by glucose in human pancreatic β‐cells
Authors: Rojas Eduardo , Hidalgo Jorge , Carroll Patricia B. , Li Min Xu , Atwater Illani
Published: July 2002

Single calcium‐channel currents were recorded from membrane patches of cultured β‐cells dissociated from human islets of Langerhans. In the absence of exogenous glucose, low frequency spontaneous calcium‐channel openings of small amplitude (−0.34 ± 0.02 pA at 0 m V pipet potential) were observed in all membrane patches examined (25 mM Ca2+ in the patch pipet). The frequency of channel openings was rather insensitive to the membrane potential across the patch (range from ca 0 to 60 m V pipet potential; chord conductance 4.9 ± 0.2 pS). Addition of glucose induced a dose‐dependent increase in the frequency of openings of the Ca2+‐channel (from now on referred to as the CaG‐channel). A few minutes after the addition of glucose (⩾ 11 mM), bursts of action potentials were often observed which were elicited only if Ca2+ was present in the solution bathing the β‐cells. Application of glucose in the presence of mannoheptulose (11 mM), a blocker of the hexokinase controlling the first stage of glycolysis, had no effect and the activity of the CaG‐channel remained at its resting level. The readily permeant mitochondrial substrate 2‐ketoisocaproate (KIC, 10 mM) was as effective as glucose in eliciting action potentials from cells forming part of cell aggregates. The activity of the CaG‐channel was significantly increased by KIC (11 mM). Although spike and Ca2+‐channel activity were markedly stimulated by glucose or KIC in all cells examined, regular bursts of action potentials were seen only if the patch was formed on β‐cells which were part of a cell aggregate. Mannoheptulose (11 mM) prevented the activation of the CaG‐channel by glucose (11 mM) but not by KIC (11 mM). Once activated, the CaG‐channel remained active even after excision of the patch. We propose that the physiological control of this Ca2+‐channel is mediated by one or more products of glucose metabolism.
Functional modes of retinoic acid in mouse osteoblastic clone MC3T3‐E1, proved as a target cell for retinoic acid
Authors: Nakayama Yasuhiro , Takahashi Kojiro , Nqji Sumihare , Muto Kohki , Nishijima Katsumi , Taniguchi Shigehiko
Published: July 2002

Mouse osteoblastic clone MC3T3‐E1 was proved as a target cell for retinoic acid (RA) in bone tissues through the demonstration of RA‐receptor gene expression by the northern blot analysis. The effect of RA on the cell growth of MC3T3‐E1 was repressive for both subconfluent and confluent growth, whereas RA enhancement of alkaline phosphatase expression was observed at the confluent stage. This implies that RA is a regulatory factor leading osteogenesis of the cells after the confluent stage. RA exhibited simultaneously the stage‐dependent effects on EGF‐dependent mitogenesis: promotive at the subconfluent, but repressive at the confluent stage.
Receptor‐mediated calcium entry
Authors: Rink Timothy J.
Published: July 2002

Occupation of membrane receptors can evoke calcium signals by causing depolarisation and activating voltage‐operated calcium channels, by triggering internal release, or by stimulating calcium influx processes not gated by membrane potential, receptor‐mediated calcium entry, RCME. This brief review considers different possible coupling mechanisms and the proposal that entry can occur from external medium to intracellular store, by‐passing the cytosol, and regulated by the state of filling of the store. Recent studies using Mn2+ as a probe for RCME are outlined, as are some new electrophysiologic measurements with human platelets and investigations of a novel blocker of RMCE, SK&F 96365.
The signal transduction induced by thrombin in human platelets
Authors: Lapetina Eduarde G.
Published: July 2002

The Stimulation of human platelets by thrombin leads to the activation of phospholipases C and A2, protein kinases, formation of 3‐inositol phos‐ pholipids and mobilization of Ca2+ . These biochemical reactions closely parallel platelet shape change, granular secretion and aggregation. The membrane‐bound transducers for the thrombin receptor seem to be the heterotrimeric G protein G12 and the ras‐related G protein rap 1‐b. Phosphorylation of rap 1‐b by the action of the cyclic AMP‐dependent protein kinase seems to uncouple the thrombin receptor from phospholipases. This causes inhibition of the formation of second messenger molecules and the onset of physiological responses.
Mobilization of the hormone‐sensitive calcium pool increases hepatocyte tight junctional permeability in the perfused rat liver
Authors: Llopis Juan , Kass George E.N. , Duddy Steven K. , Farell Geoffrey C. , Gahm Annie , Orrenius Sten
Published: July 2002

Hepatocyte tight junctional permeability has been shown to be regulated by hormones that exert their effects via phospholipase C activation. However, the precise transduction pathway involved in this effect is not known. The present study has employed the selective inhibitor of microsomal Ca2+ sequestration, 2,5‐di(tert‐butyl)‐1,4‐benzohydroquinone (tBuBHQ), to examine the effect of the mobilization of the endoplasmic reticular Ca2+ pool on tight junctional permeability in the perfused rat liver. Infusion of tBuBHQ followed by a bolus infusion of horseradish peroxidase (HRP) resulted in a significant increase in the first peak of biliary HRP, a measure of junctional permeability, whereas transcellular (vesicular) transport of HRP was not affected. Therefore, we conclude that the effect of hormones on tight junctional permeability is mediated, at least in part, by the mobilization of intracellular Ca2+.
Dual regulation of atrial natriuretic factor‐dependent guanylate cyclase activity by ATP
Authors: Marala Ravi B. , Sitaramayya Ari , Sharma Rameshwar K.
Published: July 2002

The ‘second messenger’ of certain atrial natriuretic factor (ANF) signals is cyclic GMP. One type of ANF receptor linked to the synthesis of cyclic GMP is a transmembrane protein which contains both the ANF‐binding and guanylate cyclase activities. The consensus is that the maximal activity or this guanylate cyclase is observed in the presence of ATP. We now show that depending upon the cofactors Mg2+ or Mn2+, ATP stimulates or inhibits the ANF‐dependent guanylate cyclase activity in the testicular plasma membranes; stimulation in the presence of Mg2+ and inhibition in the presence of Mn2+. With Mg2+ as cofactor neither ATP nor ANF stimulate the cyclase activity — it is only when the two are together that the enzyme is activated. Furthermore, this investigation for the first time demonstrates binding of ATP to the ANF receptor guanylate cyclase, suggesting that ATP‐mediated responses could occur by direct ATP binding to the cyclase.
Activation of the PKC‐isotypes α, β1, γ, δ, and ε by phorbol esters of different biological activities
Authors: Ryves W.J. , Evans A.T. , Olivier A.R. , Parker P.J. , Evans F.J.
Published: July 2002

Phorbol esters, tetradecanoylphorbolacetate, sapintoxin‐A, 12‐deoxyphorbol‐phenylacetate, 12‐deoxyphorbol‐phenylacetate‐20‐acetate, thymeleatoxin and resiniferatoxin were investigated for their abilities to activate the PKC‐isotypes α, β1, γ, δ and ε. PKC‐isotypes were grouped into two classes on the basis of Ca2+ requirements for activation by phorbol esters; α, β1, and γ being Ca2+‐dependent forms and δ and ε being Ca2+‐independent. PKC‐isotype selective activation by phorbol esters was observed in that SAPA failed to activate PKC‐δ up to a concentration of 1000 ng ml−1 and DOPPA only activated PKC‐β1, over the same range of concentrations.
Detection of Na+/H+ exchanger mRNA in human neutrophils and lymphocytes using the polymerase chain reaction
Authors: Quednau Beate , Rosskopf Dieter , Weiler-Güttler Hartmut , Siffert Winfried
Published: July 2002

Using the reverse polymerase chain reaction (RT‐PCR), we have examined the expression of Na+/H+ exchanger mRNA in human buffy coat preparations, lymphocytes and neutrophils. Total RNA from all cell types was reverse transcribed specifically and then amplified by PCR. The identity of the PCR products was confirmed by restriction enzyme analysis and hybridization with a specific oligonucleotide probe. The detection or low abundance Na+/H+ antiporter specific transcripts by RT‐PCR in different human blood cells ex vivo should facilitate future studies on regulatory and pathophysiological aspects of Na+/H+ exchanger mRNA expression in human cells and tissue samples.
Structural and functional analysis of Trichoderma reesei endoglucanase I expressed in yeast Saccaromyces cerevisiae
Authors: Aho Sirpa
Published: July 2002

The function of the domains of Trichoderma reesei endoglucanase I (EGI) has been studied. Truncated EGI proteins were expressed from the 3′‐end deleted cDNAs in the yeast Saccharomyces cerevisiae under the control of the ADCI expression cassette. EGI protein was detected by monoclonal antibody EI‐2 and EGI activity as cleared zones around growing colonies on agar plates containing hydroxyethylcellulose (HEC) covalently stained with Ostazin brilliant red (OBR). The results showed that the Thr—Ser‐rich hinge region and the conserved ‘tail’ are not necessary for the efficient synthesis and secretion of EGI in yeast, but the intact core region is necessary for the enzymatic activity.