bluuebunny/crossref_metadata_2025 huggingface dataset:
La psicosociología radical de lo moderno de Simmel: intersubjetividad y emancipación
Authors: Ribes Alberto J.
Published: March 2020

The purpose of this article is to explore Simmel’s radical psychosociology of modernity. To this end, we address the intersubjective constitution of individuals in Simmel and deal with the concept of the psychological expansion of roles, qualities and individuality. We then address the tension between the modern social and the modern individual, mask and shame, and the question of sociability and “alienated sociability.” The analysis and the integral interpretation of the psycho-sociological foundations of Simmel’s work allow us to conclude the article by stressing the potentialities of this approach to analyze the relationships between intersubjectivity and emancipation.
Reinterpretación del pasado fotográfico de la Misión Josefina con la Asociación de Mujeres Parteras Kichwas del Alto Napo (AMUPAKIN)
Authors: Uribe Taborda Saúl , Bermúdez Arboleda Patricia , Ángel Penagos Alexandra
Published: March 2020

In 2015, a corpus of 70 photographs registered between 1922 and 1932 by the Josephites of Murialdo was found in the Ecuadorian Library Aurelio Espinosa Polit (BEAEP), in the city of Quito. From the analysis of this corpus, a proposal was made to understand the configuration of the processes of visual representation and subjectivation of the Napo Runa Amazon Indians framed in that historical past, as well as in the ethnographic present. The article proposes two objectives: the first, to analyze the historical context of production of the photographic corpus. And the second, to investigate how the processes of visual representation and subjectivation are currently reinterpreted by the Association of Kichwa Midwives of the Upper Napo (AMUPAKIN).
Approaching boiling point stability of an alcohol dehydrogenase through computationally-guided enzyme engineering
Authors: Aalbers Friso S , Fürst Maximilian JLJ , Rovida Stefano , Trajkovic Milos , Gómez Castellanos J Rubén , Bartsch Sebastian , Vogel Andreas , Mattevi Andrea , Fraaije Marco W
Published: March 2020

Enzyme instability is an important limitation for the investigation and application of enzymes. Therefore, methods to rapidly and effectively improve enzyme stability are highly appealing. In this study we applied a computational method (FRESCO) to guide the engineering of an alcohol dehydrogenase. Of the 177 selected mutations, 25 mutations brought about a significant increase in apparent melting temperature (ΔTm ≥ +3 °C). By combining mutations, a 10-fold mutant was generated with a Tm of 94 °C (+51 °C relative to wild type), almost reaching water’s boiling point, and the highest increase with FRESCO to date. The 10-fold mutant’s structure was elucidated, which enabled the identification of an activity-impairing mutation. After reverting this mutation, the enzyme showed no loss in activity compared to wild type, while displaying a Tm of 88 °C (+45 °C relative to wild type). This work demonstrates the value of enzyme stabilization through computational library design.
SARS-CoV-2 (COVID-19) by the numbers
Authors: Bar-On Yinon M , Flamholz Avi , Phillips Rob , Milo Ron
Published: March 2020

The COVID-19 pandemic is a harsh reminder of the fact that, whether in a single human host or a wave of infection across continents, viral dynamics is often a story about the numbers. In this article we provide a one-stop, curated graphical source for the key numbers (based mostly on the peer-reviewed literature) about the SARS-CoV-2 virus that is responsible for the pandemic. The discussion is framed around two broad themes: i) the biology of the virus itself; ii) the characteristics of the infection of a single human host.
Effects of CO2 concentration and UV-B radiation on date palm (Phoenix dactylifera) grown in open-top chambers
Authors: Karthishwaran Kandhan , Senthilkumar Annadurai , Alzayadneh Wasef Ayed , Mohsen Alyafei Mohammed Abdul
Published: March 2020

Date palm (Phoenix dactylifera L.) is a major plant grown under natural conditions in the Middle East and is subject to multiple environmental stresses. Increased concentration of atmospheric carbon dioxide (CO2) and ultraviolet-B (UV-B) irradiation in the growth environment can have a high impact on plant carbon accumulation, and the various factors can function in opposite directions or cause additive effects. The objective of the present investigation was to screen UAE date palm for susceptibility to elevated level of CO2, UVB and their combined effect on a date palm variety was assessed in transparent open - top chambers (OTC) conditions in the hot climate of UAE. After the screening of the cultivars, experiment was conducted in an OTC facility and the treatments were given for 120 days. After the treatment of the selected cultivar, content of chlorophyll a, b and total, carotenoids, protein, amino acids, phenol and activities including γ-glutamyl kinase, proline oxidase, a-tocopherol and peroxidases activity were determined. The results revealed that the high concentration of CO2 alone increased the growth parameters, whereas the treatment with UV-B significantly affected the growth of the plant relative to regulation. Enzyme observations have shown that an increase in antioxidant enzymes can affect a defense response to the abiotic stress-induced cellular damage. Further extension of this study with other cultivated varieties, other stress parameters and determination of yield parameters will give scope to identify new stress tolerant cultivars of date palm trees.
Structure of a heat‐stable enterotoxin produced by a human strain of Escherichia coli
Authors: Rönnberg Bengt , Wadström Torkel , Jörnvall Hans
Published: July 2002

The amino acid sequence of a heat‐stable enterotoxin produced by a human strain of enterotoxinogenic Escherichia coli was determined to be (disregarding the sulfide bridges): Asn‐Thr‐Phe‐Tyr‐Cys‐Cys‐Glu‐Leu‐Cys‐Cys‐Asn‐Pro‐Ala‐Cys‐Ala‐Gly‐Cys‐Tyr Compensated amino acid exchanges have occurred in positions 11 and 18 in relation to the structure of a heat‐stable enterotoxin from a different human strain of Escherichia coli
Glucose inhibits insulin release induced by Na+ mobilization of intracellular calcium
Authors: Hellman Bo , Honkanen Tapio , Gylfe Erik
Published: July 2002

45Ca2+ incorporated in response to glucose was selectively mobilized from the β‐cell‐rich pancreatic islets of ob/ob‐mice after raising the intracellular Na+ by removal of K+ or addition of ouabain or veratridine. Also studies of insulin release indicated opposite effects of glucose and Na+ on the intracellular sequestration of calcium. The fact that glucose inhibits insulin release induced by raised intracellular Na+ indicates that this sugar can lower the cytoplasmic [Ca2+]. The concept of a dual action of glucose on the cytoplasmic [Ca2+]. The concept of a dual action of glucose on the cytoplasmic [Ca2+] might well explain previous observations of an inhibitory component in the glucose action on the 45Ca2+ efflux.
Reassessment of pathways of electron flow to nitrate reductase that are coupled to energy conservation in Paracoccus denitrificans
Authors: Parsonage Derek , Ferguson Stuart J.
Published: July 2002

Electron flow from succinate dehydrogenase, or from non‐physiological reductants including ascorbate plus phenazine methosulphate, to nitrate reductase in Paracoccus denitrificans is shown to be linked to energy conservation, contrary to previously accepted schemes for electron transport in this organism. The function of the P. denitrificans nitrate reductase is compared with its counterpart in Escherichia coli.
The (Na+,K+)ATPase exhibits enzymic activity in the absence of the glycoprotein subunit
Authors: Freytag J.William
Published: July 2002

Membrane‐bound (Na+,K+)ATPase from avian nasal salt glands was exposed to limited papain digestion. Such treatment results in the selective removal of the β‐subunit rendering the α‐subunit still membrane‐bound and expressing full enzymic activity. With further exposure to papain the α‐chain becomes fragmented into two major polypeptide components. The fragmented membrane‐bound catalytic chain is extremely sensitive to detergent treatment and cannot be solubilized in an active state.
Liposome‐mediated labeling of adrenocorticotropin fragments parallels their biological activity
Authors: Gysin Beat , Schwyzer Robert
Published: July 2002

To test our hypothesis that specific interactions of ACTH peptides with model lipid membranes reflect the biological importance of similar interactions on target cells, we investigated the liposome‐mediated labeling of ACTH fragments with the extremely hydrophobic photolabel, 3‐trifluoromethyl‐3‐(m‐[125I]iodophenyl)diazirine. Correlations were found between the labeling rates and the agonistic and antagonistic potencies of the peptides for in vitro steroidogenesis and inhibition of a synaptosomal protein kinase. A model for the cross‐reactivity between ACTH and opioid peptides is discussed.
Calcium‐induced Ca2+ release from sarcoplasmic reticulum of pigs susceptible to malignant hyperthermia
Authors: Tsuyoshi Ohnishi S. , Taylor Stuart , Gronert Gerald A.
Published: July 2002

Calcium‐induced calcium release and halothane‐induced calcium release from pig sarcoplasmic reticulum (SR) were studied. The SR prepared from pig susceptible to malignant hyperthermia (MH) was shown to release calcium at a much lower level of calcium content than in normal pig SR. The concentration above which halothane can release calcium is 40 μM for both MH‐SR and normal SR, although the latter required a high calcium content to demonstrate the calcium release. Dantrolene was shown to inhibit the halothane‐induced calcium release. Results suggests that SR plays an importnat role in pathogenesis of MH.
Phosphorylation of a 16‐kDa protein by diacylglycerol‐activated protein kinase C in vitro and by vasopressin in intact hepatocytes
Authors: Cooper Ronald H. , Kobayashi Koichi , Williamson John R.
Published: July 2002

A 16‐kDa protein present in a purified rat liver plasma membrane fraction and also in cytosol can be phosphorylated by endogenous diacylglycerol‐activated protein kinase C. In intact hepatocytes prelabeled with 32P, vasopressin causes a rapid increase in the phosphorylation of a 16‐kDa protein having a similar pI value to that observed in in vitro studies. These findings suggest that vasopressin‐induced phosphorylation of the 16‐kDa in the intact hepatocyte may reflect increased activity of protein kinase C, secondary to membrane polyphosphoinositide breakdown. Phosphorylation of the 16‐kDa protein may thus be part of the coordinated mechanism associated with hormonal regulation of cellular Ca2+ fluxes.
Kinetic evidence for the re‐definition of electron transfer pathways from cytochrome c to O2 within cytochrome oxidase
Authors: Hill Bruce C. , Greenwood Colin
Published: July 2002

The reaction with O2 of equimolar mixtures of cytochrome c and cytochrome c oxidase in high and low ionic strength buffers has been examined by flow‐flash spectrophotometry at room temperature. In low ionic strength media where cytochrome c and the oxidase are bound in an electrostatic, 1:1 complex some of the cytochrome c is oxidised at a faster rate than a metal centre of the oxidase. In contrast, when cytochrome c and cytochrome c oxidase are predominantly dissociated at high ionic strength cytochrome c oxidation occurs only slowly (t

=5 s) following the complete oxidation of the oxidase. These results demonstrate that maximal rates of electron transfer from cytochrome c to O2 occur when both substrates are present on the enzyme. The heterogeneous oxidation of cytochrome c observed in the complex implies more than one route for electron transfer within the enzyme. Possibilities for new electron transfer pathways from cytochrome c to O2 are proposed.
Guanine nucleotides and adenosine ‘Ri’‐site analogues stimulate the membrane‐bound low‐Km cyclic AMP phosphodiesterase of rat adipocytes
Authors: de Mazancourt Philippe , Giudicelli Yves
Published: July 2002

Guanine nucleotide
Adenosine analog
Phosphodiesterase
Adipocyte
Rat
Pertussis toxin catalyzes the ADP‐ribosylation of two distinct peptides, 40 and 41 kDa, in rat fat cell membranes
Authors: Malbon Craig C. , Rapiejko Peter J. , Garciá-Sáinz J.Adolfo
Published: July 2002

Pertussis toxin catalyzes the ADP‐ribosylation of a single 41‐kDa peptide of membranes prepared from rat hepatocytes, S49 mouse lymphoma wild‐type and cyc‐mutant cells. This 41‐kDa peptide has been shown to be the α‐subunit of the inhibitory, guanine nucleotide binding regulatory component of adenylate cyclase (Ni). Incubating membranes of rat fat cells with pertussis toxin and [32P]NAD+ radiolabels a 41‐ and a 40‐kDa peptide. Possible homologies between these peptides were investigated by comparing the electrophoretic patterns of proteolytic fragments derived from each of them that are radiolabeled by [32P]NAD+ and pertussis toxin. The 40‐kDa substrate for pertussis toxin‐catalyzed ADP‐ribosylation and the α‐subunit of Ni in rat fat cells appear to be homologous, but non‐identical peptides.
Interaction of electron acceptors with the excited triplet state of Zn cytochrome c
Authors: Horie Toshiharu , Maniara G. , Vanderkooi J.M.
Published: July 2002

Cytochrome c
Excited triplet state
Electron acceptor
Phosphorescence lifetime
The phorbol ester, TPA inhibits glucagon‐stimulated adenylate cyclase activity
Authors: Heyworth Clare M. , Whetton Anthony D. , Kinsella Anne R. , Houslay Miles D.
Published: July 2002

The ability of glucagon (10 nM) to increase hepatocyte intracellular cyclic AMP concentrations was reduced markedly by the tumour‐promoting phorbol ester TPA (12‐O‐tetradecanoyl phorbol‐13‐acetate). The half‐maximal inhibitory effect occurred at 0.14 ng/ml TPA. This action occurred in the presence of the cyclic AMP phosphodiesterase inhibitor isobutylmethylxanthine (1 mM) indicating that TPA inhibited glucagon‐stimulated adenylate cyclase activity. TPA did not affect either the binding of glucagon to its receptor or ATP concentrations within the cell. TPA did inhibit the increase in intracellular cyclic AMP initiated by the action of cholera toxin (1 μg/ml) under conditions where phosphodiesterase activity was blocked. TPA did not inhibit glucagon‐stimulated adenylate cyclase activity in a broken plasma membrane preparation unless Ca2+, phosphatidylserine and ATP were also present. It is suggested that TPA exerts its inhibitory effect on adenylate cyclase through the action of protein kinase C. This action is presumed to be exerted at the point of regulation of adenylate cyclase by guanine nucleotides.
Induction of protein kinase C activation and Ca2+ mobilization by fibroblast growth factor in Swiss 3T3 cells
Authors: Tsuda Terutaka , Kaibuchi Kozo , Kawahara Yasuhiro , Fukuzaki Hisashi , Takai Yoshimi
Published: July 2002

Addition of fibroblast growth factor (FGF) to quiescent cultures of Swiss 3T3 cells rapidly induced diacylglycerol formation, protein kinase C activation and Ca2+ mobilization. Protein kinase C‐activating agents such as 12‐O‐tetradecanoylphorbol‐13‐acetate (TPA) and 1‐oleoyl‐2‐acetylglycerol (OAG) mimicked the action of FGF and stimulated DNA synthesis in the presence of insulin. Prolonged treatment of the cells with phorbol‐12,13‐dibutyrate (PDBu) led to the down‐regulation and complete disappearance of protein kinase C. In these cells, TPA and OAG did not induce DNA synthesis any more. FGF still elicited Ca2+ mobilization and DNA synthesis, but the magnitude of DNA synthesis was reduced to almost half as compared with that in the control cells. These results clearly indicate that both diacylglycerol and Ca2+ may serve as second messengers for FGF and suggest that these messengers may be involved in the mitogenic action of this growth factor.
Sequence homology between barley endosperm protein Z and protease inhibitors of the α1‐antitrypsin family
Authors: Hejgaard J. , Rasmussen S.K. , Brandt A. , Svendsen I.
Published: July 2002

Six cDNA clones encoding parts of protein Z, a major barley endosperm albumin, have been identified. Nucleotide and amino acid sequencing have established a 180 residues long C‐terminal amino acid sequence of protein Z as well as two minor amino acid sequences (14 and 7 residues). These sequences show that barley protein Z is homologous with human α1‐antitrypsin, human otj‐antichymotrypsin, human antithrombin III, mouse contrapsin and chicken ovalbumin (26–32% of the 180 residues in the C‐terminal sequence in identical positions). The sequence homology and specific cleavage of protein Z at a bond corresponding to the reactive site of the inhibitors indicate a possible inhibitory function. Inhibition of microbial or pancreatic serine proteases could, however, not be associated with protein Z.
Bradykinin‐induced transient accumulation of inositol trisphosphate in neuron‐like cell line NG 108‐15 cells
Authors: Yano Koh , Higashida Haruhiro , Hattori Hiroaki , Nozawa Yoshinori
Published: July 2002

Studies were undertaken to further elucidate the mechanism(s) by which bradykinin‐dependent phosphoinositide metabolism takes place in neuroblastoma × glioma hybrid NG108‐15 cells [(1984) J. Biol. Chem. 259, 10201–10207] using [3H]inositol‐labelled cells. Bradykinin produced net increases in the level of [3H]inositol phosphates, especially of [3H]inositol trisphosphate which is formed transiently and most rapidly. The results indicate that bradykinin activates a phosphodiesterase to break down phosphatidylinositol 4,5‐bisphosphate, generating two recently recognized intracellular messengers, 1,2‐diacylglycerol and inositol trisphosphate.
Ca2+‐dependent inhibition by trifluoperazine of the Na+‐Ca2+ carrier in mitoplasts derived from heart mitochondria
Authors: Hayat L.H. , Crompton M.
Published: July 2002

The interaction of trifluoperazine and extramitochondrial Ca2+ with the heart mitochondrial Na+‐Ca2+ carrier has been investigated. External Ca2+ inhibits the carrier equally in mitochondria and mitoplasts in which the outer membrane is lysed. Sensitivity to Ca2+ is not removed by washing mitoplasts under varied conditions. Trifluoperazine is a potent inhibitor of the carrier in mitoplasts but not in mitochondria. Trifluoperazine inhibition in mitoplasts depends markedly on the presence of extramitochondrial Ca2+ (2 μM)
Phorbol myristate acetate causes in guinea‐pig lung parenchymal strip a maintained spasm which is relatively resistant to isoprenaline
Authors: Dale M.Maureen , Obianime W.
Published: July 2002

The effect of phorbol myristate acetate (PMA) was compared with that of histamine on the guinea‐pig lung parenchymal strip. PMA, 10−5 M, caused a slowly developing sustained contraction which had approximately the same magnitude as the maximal histamine contraction. Isoprenaline, at 10−5 M, caused 86% relaxation of the histamine contraction but only 22% relaxation of the PMA contraction. Forskolin, at 10−5 M had a similar action to isoprenaline on the effects of both spasmogens while aminophylline, 5 × 10−4 M, was considerably less effective. Sodium nitroprusside had little effect on the histamine contraction and actually increased the PMA spasm. It is suggested that protein kinase C may have a role in the tonic phase of the contraction of bronchiolar smooth muscle. These findings could have relevance for the delayed phase of asthma, which is known to be insensitive to β‐agonists.
Intrinsic regulation of substrate fluxes and energy conservation in Ca2+‐ATPase
Authors: de Meis Leopolde , Inesi Giuseppe
Published: July 2002

The Ca2+‐ATPase of sarcoplasmic reticulum was utilized to demonstrate an intrinsic regulation of enzyme catalysis, whereby the ratio of forward and reverse flow is altered by the binding of Ca2+ and Pi to the enzyme. This is related to displacement of internal equilibria among intermediate enzyme ligand complexes, independent of the overall equilibrium of the ATP ⇌ ADP + Pi transformation. A very high energy conservation with a velocity of reverse flow approaching that of forward flow, was obtained by increasing the enzyme affinity for Pi in the presence of Me2SO.
Heparin induces Ca2+ release from the terminal cysterns of skeletal muscle sarcoplasmic reticulum
Authors: Ritov Vladimir B. , Men'shikova Elizaveta V. , Kozlov Yuri P.
Published: July 2002

Using a Ca2+‐selective electrode and the chlorotetracycline fluorescence technique, the effects of heparin on Ca2+ transport in the sarcoplasmic reticulum (SR) of skeletal muscles in the absence of oxalate were investigated. It was shown that heparin (0.5–10 ) causes a rapid release of 40–50 nmol protein from the terminal cystern SR vesicles bound to 130–150 protein of Ca2+ in the presence of ATP. However, heparin has practically no effect on the longitudinal cystern fraction of SR. The effects of heparin can be prevented by ruthenium red. No influence of heparin is observed in the case of the Ca2+‐induced release of Ca2+ from the terminal cysterns. When the Ca2+ release is induced by heparin, no Ca2+‐induced release of Ca2+ takes place.
Energy expenditure for cyclic retention of NH3/NH4+ during N2 fixation by Klebsiella pneumoniae
Authors: Kleiner D.
Published: July 2002

The permeability coefficeint for NH3 through bacterial membranes was determined to be around 2 × 10−3 cm·s−1. This value was used to calculate the outward diffusion of NH3 from intracellular pools and the energy costs for reabsorption as NH4
+. For an N2‐fixing continuous culture of Klebsiella pneumoniae an energy expenditure of around 4 ATP per NH3 produced was calculated, thus increasing significantly the energy requirement for N2 fixation in vivo.
Primary structure of the β‐subunit of Torpedo californica (Na+ + K+)‐ATPase deduced from the cDNA sequence
Authors: Noguchi Shunsuke , Noda Masaharu , Takahashi Hideo , Kawakami Kiyoshi , Ohta Toshiko , Nagano Kei , Hirose Tadaaki , Inayama Seiichi , Kawamura Masaru , Numa Shosaku
Published: July 2002

DNA complementary to the Torpedo californica electroplax mRNA coding for the β‐subunit of (Na+ + K+)‐ATPase has been cloned by screening a cDNA library with an oligodeoxyribonucleotide probe. Nucleotide sequence analysis of the cloned cDNA has revealed that this polypeptide consists of 305 amino acid residues (including the initiating methionine). The transmembrane topology and the potential N‐glycosylation sites of this polypeptide are discussed.
Control of energy metabolism by glucagon and adrenaline in perfused rat liver
Authors: Soboll Sibylle , Scholz Roland
Published: July 2002

Changes in subcellular distribution of adenine nucleotides, mitochondrial/cytosolic proton gradients, rates of respiration, gluconeogenesis (fasted state) and glycogenolysis (fed state) were studied in isolated perfused rat livers following addition of glucagon (10−8 M) or adrenaline (10−7 M). Glucagon increased the gradient in all states. The cytosolic ATP/ADP ratio was increased in the fasted but decreased in the fed state which is consistent with a diminished futile cycling in gluconeogenesis (fasted state) or a decreased glycolytic rate (fed state). Adrenaline caused an increase in the proton gradient and the mitochondrial ATP/ADP ratio. The two effects are attributed to increased calcium entry into the matrix space.
Calpain‐calpastatin and fusion
Authors: Glaser Tova , Kosower Nechama S.
Published: July 2002

Rat erythrocytes fuse when treated with the membrane mobility agent, 2‐(2‐methoxyethoxy)ethyl‐cis‐8‐(2‐octylcyclopropyl) octanoate (A2C) and Ca2+, whereas human cells do not. Membrane proteolysis promoted by calpain is required for rat cell fusion [(1986) Eur. J. Biochem., in press]. Human calpain induced a selective proteolysis in both the human and rat erythrocyte ghosts (mainly band 4.1 in the human, band 4.1 and band 3 in the rat cell) and rendered them fusible. Calpastatin (calpain inhibitor) prevented A2C‐induced fusion in both ghosts, via inhibition of proteolysis. The human erythrocyte has excess calpastatin and resists A2C‐promoted fusion. A regulatory role of calpastatin in membrane fusion is thus indicated.
Assignment of hyperfine shifted haem methyl carbon resonances in paramagnetic low‐spin met‐cyano complex of sperm whale myoglobin
Authors: Yamamoto Yasuhiko
Published: July 2002

The hyperfine shifted resonances arising from all four individual haem carbons of the paramagnetic low‐spin met‐cyano complex of sperm whale myoglobin have been clearly identified and assigned for the first time with the aid of 1H‐13C heteronuclear chemical shift correlated spectroscopy. Alteration of the in‐plane symmetry of the electronic structure of haem induced by the ligation of proximal histidyl imidazole spreads the haem carbon resonances to 32 ppm at 22°C, indicating the sensitivity of those resonances to the haem electronic/molecular structure. Those resonances are potentially powerful probes in characterizing the nature of haem electronic structure.
Elevation of pHi is not an essential step in calcium mobilisation in fura‐2‐loaded human platelets
Authors: Simpson A.W.M. , Rink T.J.
Published: July 2002

Human platelets were co‐loaded with the fluorescent indicators BCECF and fura‐2 to mesure pHi and [Ca2+]i and incubated with aspirin to block cyclooxygenase. Either pHi and shape change and aggregation or pHi and [Ca2+]i were measured simultaneously in the same stirred cuvette, at 37°C. In Hepes‐buffered saline containing 1 mM Ca2+, mean resting pHi was 6.98±0.01 (SE, n = 59). Changes of pHi up to ±0.35 units, imposed by additions of NH4Cl, CO2 or nigericin, produced no shape change or aggregation and only insignificant changes in [Ca2+]i. Sufficient thrombin to raise [Ca2+]i over 1 μM and cause rapid shape change and aggregation increased pHi by no more than 0.05 units, and the increase in pHi lagged behind the elevation of [Ca2+]i. We conclude that changes in pHi do not form a necessary or sufficient component of the pathways leading to receptor‐mediated Ca2+ mobilisation or the stimulation of shape change or aggregation.
[4,4′‐(Z)‐Dehydrophenylalanine]gramicidin S with stabilized bioactive conformation and strong antimicrobial activity
Authors: Shimohigashi Yasuyuki , Kodama Hiroaki , Imazu Sachiko , Horimoto Hideaki , Sakaguchi Kazuyasu , Waki Michinori , Uchida Hiroaki , Kondo Michio , Kato Tetsuo , Izumiya Nobuo
Published: July 2002

Dehydrophenylalanine (ΔPhe) was incorporated into an antibiotic peptide gramicidin S (GS) in place of D‐Phe4,4′ to prepare an unsaturated analog. Conformational analysis with 1H‐NMR indicated that the unsaturated analog has much the same backbone conformation as that of natural gramicidin S as shown by NOE experiments. Studies on temperature dependences and on the chemical shift differences showed that the hydrogen bonds between Val‐NH and Leu‐CO in the unsaturated analog are strengthened by the incorporation of ΔPhe4,4′. This resulted in the reinforcement of the β‐sheet structure which is the most important structural element for GS bioactivity. [ΔPhe4,4′]gramicidin S exhibited indeed very strong antimicrobial activities against Gram‐positive bacteria as well as the natural peptide.
Internal perfusion of guinea‐pig hepatocytes with buffered Ca2+ or inositol 1,4,5‐trisphosphate mimics noradrenaline activation of K+ and Cl− conductances
Authors: Capiod T. , Field Anne C. , Ogden D.C. , Sandford Christine A.
Published: July 2002

External application of noradrenaline to voltage‐clamped guinea‐pig isolated hepatocytes evoked membrane conductance increases to K+ and Cl−. This effect was reproduced by internal perfusion of the cells with 2 μM buffered Ca2+ and with 20 μM inositol 1,4,5‐trisphosphate (IP3). The kinetic properties of the K+ conductance and its selective block by the toxin apamin were the same in each case. Cyclical fluctuations of conductance observed with noradrenaline were reproduced by internal IP3 but not by Ca2+ perfusion, indicating that oscillations of intracellular free Ca2+ may arise from properties of the Ca2+ sequestration mechanism at constant IP3 concentration.
Detailed structural analysis of exposed domains of membrane‐bound Na+, K+‐ATPase A model of transmembrane arrangement
Authors: Ovchinnikov Yu.A. , Arzamazova N.M. , Arystarkhova E.A. , Gevondyan N.M. , Aldanova N.A. , Modyanov N.N.
Published: July 2002

Exposed regions of the α‐ and β‐subunits of membrane‐bound Na+,K+‐ATPase were in turn hydrolyzed with trypsin. Resistance of the β‐subunit to proteolysis was shown to be due mainly to the presence of disulfide bridge(s) in the molecule. A model for the spatial organisation of the enzyme in the membrane was proposed on the basis of detailed structural analysis of extramembrane regions of both subunits.
Insulin stimulates a novel GTPase activity in human platelets
Authors: Gawler Debra , Houslay Miles D.
Published: July 2002

Insulin stimulated the activity of a high‐affinity GTPase activity in human platelet membranes some 62% over that of the basal activity. Half‐maximal stimulation (K
a) was achieved with 3.1 nM insulin. The K
m for GTP of the insulin‐stimulated GTPase was 0.6 μM GTP. Treatment of isolated platelet membranes with cholera toxin, but not pertussis toxin, blocked insulin's ability to stimulate GTPase activity. Cholera toxin acted as a more potent inhibitor of the insulin‐stimulated GTPase activity than that of the GTPase activity of the stimulatory guanine nucleotide regulatory protein, Gs, as monitored by stimulation using prostaglandin E1 (PGE1). Mixed ligand experiments showed that insulin stimulated GTPase activity in an additive fashion to GTPase activity stimulated by PGE1, due to Gs; by adrenaline (+ propranolol), due to the inhibitory guanine nucleotide regulatory protein, Gi and by vasopressin, which stimulates the putative ‘Gp’, a G‐protein suggested to control the stimulation of inositol phospholipid metabolism. Insulin thus appears to stimulate a novel high‐affinity GTPase activity in human platelet membranes. This may reflect the functioning of the putative Gins, a guanine nucleotide regulatory protein which has been suggested to mediate certain of insulin's actions on target tissues.
Cyclic AMP induces a transient alkalinization in Dictyostelium
Authors: Aerts Rob J. , De Wit René J.W. , Van Lookeren Campagne Michiel M.
Published: July 2002

In a wide range of cell types, stimulus‐response coupling is accompanied by a rise in cytoplasmic pH (pHi). It is shown that stimulation of developing Dictyostelium discoideum cells with the chemoattractant cAMP also results in a rise in pHi. About 1.5 min after stimulation, pHi starts increasing from pHi∼7.45 to pHi∼7.60, as is revealed independently by two different pH null‐point methods. The rise in pHi is transient, also with a persistent stimulus, and effectively inhibited by diethylstilbestrol (DES), strongly suggesting that the rise in pHi is accomplished by the DES‐sensitive plasma membrane proton pump which has been demonstrated in D. discoideum.
The phorbol ester, TPA, increases transepithelial epidermal growth factor flux
Authors: Mullin James M. , McGinn Mary T.
Published: July 2002

Exposure of cultured kidney epithelial (LLC‐PK1) cell sheets to 10−7 M TPA, a potent tumor promoter and activator of protein kinase C, initiates within minutes a drop in the transepithelial voltage across these sheets. This fall in potential difference correlates with an over 40‐fold increase in the transepithelial flux of 1 mM D‐mannitol, suggesting that the intercellular junctions have become leaky. Dual labeling experiments with 1 mM D‐[14C]mannitol and 10 nM 125I‐EGF show that after promoter treatment, a 7‐fold increase in net 125I flux accompanies the increase in mannitol flux. Gel filtration and gel electrophoresis indicate that for control cell sheets only 15% of the transited 125I is actually EGF, whereas with TPA‐treated cell sheets, 60% of the 125I which passed across is EGF. These percentages permitted determination of actual EGF flux values, and show that TPA treatment engenders a 35‐fold increase in transepithelial EGF flux. Diacylglycerols also increase the junctional permeability of these cells, thereby suggesting the involvement of protein kinase C.
Phosphoinositide metabolism in resting and thrombin‐stimulated human platelets Evidence against metabolic heterogeneity
Authors: Tysnes Ole-Bjørn , Verhoeven Adrie J.M. , Aarbakke Grethe M. , Holmsen Holm
Published: July 2002

The specific radioactivity of the phosphodiester and phosphomonoester moieties of the phosphoinositides was determined in resting and thrombin‐stimulated platelets that were prelabelled with [32P] Pi. In the unstimulated cells, the specific radioactivity of the monoester phosphates of PIP and PIP2 was similar. Both prolonged incubation at 37°C and upon stimulation with 0.5 U/ml of thrombin, the specific radioactivity of the monoester phosphates decreased 10–15% in both polyphosphoinositides. In the unstimulated cells, the specific radioactivity of the diester phosphates was similar in PI, PIP and PIP2 but amounted only to 3% of the activity of the monoester groups. Prolonged incubation of the unstimulated cells as well as stimulation with thrombin induced a similar 5–6 fold increase in specific radioactivity of the diester phosphate of PI, PIP and PIP2. The results indicate that the phosphoinositides in both resting and thrombin‐stimulated platelets exist in a metabolically homogeneous pool.
Inhibition of DNA synthesis by phorbol esters through protein kinase C in cultured rabbit aortic smooth muscle cells
Authors: Kariya Ken-ichi , Fukumoto Yasuo , Tsuda Terutaka , Kawahara Yasuhiro , Fukuzaki Hisashi , Yamamoto Takeshi , Takai Yoshimi
Published: July 2002

In cultured rabbit aortic smooth muscle cells (SMC), 12‐O‐tetradecanoylphorbol‐13‐acetate (TPA) induced DNA synthesis in the presence of plasma‐derived serum to a small extent, but inhibited markedly the rabbit whole blood serum (WBS)‐, platelet‐derived growth factor (PDGF)‐ and epidermal growth factor‐induced DNA synthesis. Phorbol‐12,13‐dibutyrate (PDBu) mimicked this antiproliferative action of TPA, but 4α‐phorbol‐12, 13‐didecanoate was inactive in this capacity. Prolonged treatment of the cells with PDBu caused the partial down‐regulation of protein kinase C. In these protein kinase C‐reduced cells, WBS still induced DNA synthesis, but TPA did not inhibit the WBS‐induced DNA synthesis. We have previously shown that protein kinase C is involved at least partially in the PDGF‐induced DNA synthesis in rabbit aortic SMC. The present results together with this earlier observation suggest that protein kinase C has not only a proliferative but also an antiproliferative action in rabbit aortic SMC.
Differential down‐regulation of protein kinase C subspecies in KM3 cells
Authors: Ase Katsuhiko , Berry Nicola , Kikkawa Ushio , Kishimoto Akira , Nishizuka Yasutomi
Published: July 2002

The down‐regulation of protein kinase C (PKC) subspecies in KM3 cells (a pre‐B, pre‐T cell line) has been examined. The PKC from KM3 cells was resolved into two subspecies, type II (mainly βII) and type III (α), upon hydroxyapatite column chromatography. Biochemical and immunocytochemical analysis revealed that, when these cells were treated with 12‐O‐tetradecanoylphorbol 13‐acetate (TPA), the time course of down‐regulation of the PKC subspecies was different; type II PKC was translocated and depleted from the cell more quickly than type III enzyme. The results suggest that each PKC subspecies plays a different role in the cellular response to TPA and probably to other external stimuli.
Guanine nucleotides inhibit agonist‐stimulated arachidonic acid release in both intact and saponin‐permeabilized human platelets
Authors: Rehm Alan G. , Wu Hung , Halenda Stephen P.
Published: July 2002

The effects of guanine nucleotides on arachidonic acid (AA) release were studied in intact and saponin‐permeabilized human platelets. While GTP[S] itself caused a stimulation of AA release in permeabilized cells, GTP[S], GDP[S], GTP, ATP and other nucleotides inhibited AA release in response to thrombin and other agonists in intact, as well as permeabilized platelets. Inhibition of agonist‐stimulated AA release by nucleotides was partially attenuated by addition of ADP, and was abolished by prior stimulation of platelets to discharge the ADP‐containing dense granules. These results suggest: (i) that released ADP plays an important contributory role in agonist‐stimulated platelet AA release, and (ii) that guanine nucleotides can modulate platelet activation through an extracellular action which is distinct from their effects on G‐proteins.
Phosphorylation‐activated chloride channels in human skin fibroblasts
Authors: Bear Christine E.
Published: July 2002

A chloride‐selective channel has been found using patch‐clamp electrophysiology in human skin fibroblasts and it exhibits many of the biophysical properties of the Cl− channel found in airway epithelia. As in the case of epithelial Cl− channels, Cl− channels in fibroblasts are activated at depolarized membrane potentials in excised patches, rectifying in an outward direction with a unit conductance of 33 pS at 0 mV. Furthermore, the agonists forskolin and prostaglandin E2 evoke Cl− channel activity in cell‐attached patches. The effect of these agonists can be mimicked by direct application of catalytic subunit of protein kinase A with ATP and Mg2+ to the internal membrane surface of excised, inside‐out patches. The Cl− channel is also sensitive to inhibition by the stilbene derivative, DIDS. These results indicate that fibroblasts may provide a convenient and available model for the study of epithelial Cl− channel regulation and accelerate efforts to determine the regulatory defect expressed in cystic fibrosis.
ATP stimulates the uptake of S‐dinitrophenylglutathione by rat liver plasma membrane vesicles
Authors: Kobayashi Kazuo , Sogame Yoshihisa , Hayashi Koichiro , Nicotera Pierluigi , Orrenius Sten
Published: July 2002

Incubation of inverted plasma membrane vesicles from rat liver with micromolar concentrations of S‐dinitrophenylglutathione (DNP‐SG) in the presence of ATP resulted in the uptake of DNP‐SG into the vesicles. ATP‐dependent DNP‐SG accumulation was half‐maximal with 9 μM DNP‐SG, while the K
m for ATP was 320 μM. Glutathione disulfide (GSSG), but not reduced glutathione, inhibited the ATP‐dependent accumulation of DNP‐SG by the vesicles, suggesting that the same, ATP‐dependent transport system is responsible for the extrusion of glutathione conjugates and GSSG from liver cells.
Different sensitivities to agonist of muscarinic acetylcholine receptor subtypes
Authors: Bujo Hideaki , Nakai Junichi , Kubo Tai , Fukuda Kazuhiko , Akiba Isamu , Maeda Akito , Mishina Masayoshi , Numa Shosaku
Published: July 2002

Muscarinic acetylcholine receptor (mAChR) III expressed in Xenopus oocytes, like mAChR I, mediates activation of a Ca2+‐dependent Cl− current, whereas mAChR IV, like mAChR II, principally induces activation of Na+ and K+ currents in a Ca2+‐independent manner. mAChR III has a sensitivity to agonist of about one order of magnitude higher than that of mAChR I in mediating the Ca2+‐dependent current response in Xenopus oocytes and in stimulating phosphoinositide hydrolysis in NG108‐15 neuroblastoma‐glioma hybrid cells. The agonist‐binding affinity of mAChR III is also about one order of magnitude higher than that of mAChR I.
Changes in myosin heavy chain stoichiometry in pig tracheal smooth muscle during development
Authors: Mohammad Mukhallad A. , Sparrow Malcolm P.
Published: July 2002

The stoichiometry of the myosin heavy chains (MHCs) has been measured in the tracheal smooth muscle of the pig after electrophoresis on SDS 4% polyacrylamide gel. The ratio of slower migrating MHC to the faster migrating MHC was 2.1 in neonates, 1.5 in young and 0.95 in old pigs (P<0.01) showing that MHC composition changes with development. The unequal proportion of MHCs was not compatible with a heterodimeric arrangement of the MHCs in the native molecule as proposed earlier by Rovner et al. [(1986) Am. J. Physiol. 250, C861–870] and it is suggested that native molecules may be composed of homodimer heavy chains.
Evidence that stimulation of gluconeogenesis by fatty acid is mediated through thermodynamic mechanisms
Authors: Berry M.N. , Gregory R.B. , Grivell A.R. , Henly D.C. , Phillips J.W. , Wallace P.G. , Welch G.R.
Published: July 2002

We have studied the stimulatory effects of palmitate on the rate of glucose synthesis from lactate in isolated hepatocytes. Control of the metabolic flow was achieved by modulating the activity of enolase using graded concentrations of fluoride. Unexpectedly, palmitate stimulated gluconeogenesis even when enolase was rate‐limiting. This stimulation was also observed when the activities of phosphoenolpyruvate carboxykinase and aspartate aminotransferase were modulated using graded concentrations of quinolinate and aminooxyacetate, respectively. Linear force‐flow relationships were found between the rate of gluconeogenesis and indicators of cellular energy status (i.e. mitochondrial membrane and redox potentials and cellular phosphorylation potential). These findings suggest that the fatty acid stimulation of glucose synthesis is in part mediated through thermodynamic mechanisms.
Phorbol ester‐induced protein kinase C translocation and lysosomal enzyme release in normal and cystic fibrosis fibroblasts
Authors: Hermelin B. , Cherqui G. , Bertrand F. , Wicek D. , Paul A. , Garcia I. , Picard J.
Published: July 2002

The ability of the tumor‐promoting phorbol ester 4β‐phorbol 12β‐myristate 13α‐acetate (PMA) to induce protein kinase C (PKC) translocation and lysosomal enzyme release was examined in skin fibroblasts from normal subjects and from patients with cystic fibrosis (CF). As compared to normal fibroblasts, those CF exhibited: (i) an increased sensitivity to the effect of PMA on the disappearance of PKC from cytosolic fractions as well as a greater and earlier recovery, in the membrane fraction, of the PKC activity lost in the cytosolic fraction; (ii) an earlier response to PMA for its effect on β‐N‐acetylglucosaminidase release. In contrast, the inactive phorbol ester 4α‐phorbol 12,13‐didecanoate (4αPDD) proved ineffective in inducing PKC translocation and β‐N‐acetylglucosaminidase release in both normal and CF fibroblasts. The data suggest a defect in the regulation of PKC activity in CF fibroblasts, which may lead to altered secretion.
Storage of phosphorylated desmin in a familial myopathy
Authors: Rappaport L. , Contard F. , Samuel J.L. , Delcayre C. , Marotte F. , Tomè F. , Fardeau M.
Published: July 2002

The quantity and the electrophoretic characteristics of desmin were analyzed in a familial skeletal muscle disorder, characterized by the intra‐sarcoplasmic accumulation of an electron‐dense granulo‐filamentous material facing the Z‐lines and reacting strongly with polyclonal anti‐desmin antibodies. The analysis was performed on biopsies from the deltoid muscles of 4 patients, members of 2 families. In the 4 biopsies, an increase in the relative amount of desmin compared to that of actin or insoluble proteins (3 fold) and in the number of isovariants (6 instead of 3) was observed. The isovariants of desmin were similar to those described in Purkinje fibres of the heart as a phosphorylated form of the protein [(1987) Eur. J. Cell Biol. 44, 68–781]. Therefore, post‐translational events could affect both the polymerization and the amount of desmin filaments in this autosomal dominant familial myopathy.
Short‐chain alkanols and the functional efficiency of the Ca pump in the sarcoplasmic reticulum of rabbit skeletal muscles
Authors: Melgunov Vladimir I. , Jindal Satish , Belikova Marina P.
Published: July 2002

The effect of 10 low molecular mass alkanols on the activity of Ca‐ATPase (EC 3.6.1.38), Ca uptake and Ca efflux as well as the functional efficiency of the Ca pump in the fragmented sarcoplasmic reticulum of rabbit skeletal muscles has been studied. Some alkanols, especially when taken at low concentration, have been found to stimulate the activity of the Ca pump and Ca‐ATPase, namely tert‐butanol, isopropanol and ethanol (from the group of hydrophilic alkanols), and pentanol, isopentanol and hexanol (from the more hydrophobic alkanols). Methanol (from the first group) and isobutanol, butanol and propanol (from the second) do not stimulate the Ca pump compared with the control. The specific effect of different alkanols cannot be explained in terms of a unitary mechanism based on ‘fluidity’ changes of the membrane. It is assumed that, at low concentrations, certain alkanols (or groups of related alkanols) are able to promote the specific transition of membrane proteins into the active state, whereas at higher concentrations all alkanols provide for the non‐functional state of the proteins.
Cyclic AMP‐dependent protein kinase‐induced vimentin filament disassembly involves modification of the N‐terminal domain of intermediate filament subunits
Authors: Evans Robert M.
Published: July 2002

The intermediate filament protein vimentin was phosphorylated with cAMP‐dependent protein kinase under conditions that induce filament disassembly. Digestion of phosphorylated vimentin with lysine‐specific endoprotease and subsequent tryptic peptide mapping indicated that a 12 kDa N‐terminal fragment contained all the phosphorylation sites found in the intact molecule. Analysis of cyanogen bromide digests indicated that two phosphorylated peptides were produced, with the major 32P‐labeled species representing amino acid position 14–72, and a minor 32P‐labeled peptide representing amino acid positions 1–13. These results demonstrate that phosphorylation of sites within the N‐terminal head domain of vimentin are associated with phosphorylation induced filament disassembly.
The location of CuA in mammalian cytochrome c oxidase
Authors: Rich Peter R. , West Ian C. , Mitchell Peter
Published: July 2002

Imposition of a protonmotive force across the inner membrane of coupled cyanide‐inhibited, beef heart mitochondria by addition of ATP causes reduction of cytochrome c and CuA with concomitant oxidation of haem a
A. The data are consistent with previous demonstrations of an intramembrane location of haem a
A but further indicate that CuA is very close to the cytosolic surface of the membrane. The implications of this finding for electron transfer route and the site of the proton pumping chemistry are discussed.
Predominant localization of phosphoenolpyruvate carboxykinase mRNA in the periportal zone of rat liver parenchyma demonstrated by in situ hybridization
Authors: Bartels Heidrun , Linnemann Helga , Jungermann Kurt
Published: July 2002

In rat liver parenchyma, expression of the phosphoenolpyruvate carboxykinase (PEPCK) gene was studied by Northern blot analysis with a biotinylated cRNA probe and the zonal localization of PEPCK mRNA was demonstrated by in situ hybridization with a radiolabelled cRNA probe. During the feeding period at night, overall PEPCK mRNA levels were low and PEPCK mRNA was detected only in small areas of the periportal zone. At the beginning of the light period (7 am) the overall PEPCK mRNA level began increasing and the periportal areas containing PEPCK mRNA broadened. The maximum of the total abundance and of the area with high levels of PEPCK mRNA was reached at noon. Fasting for 24–72 h did not cause further significant alterations in the level or localization of PEPCK mRNA. The present data are in line with previous findings of the predominant localization of PEPCK activity and enzyme protein in periportal hepatocytes. They suggest that the heterogeneous expression of the PEPCK gene in rat liver is regulated at the pretransiational level.
Three apparent receptor subtypes for the endothelin/sarafotoxin family
Authors: Kloog Y. , Bousso-Mittler D. , Bdolah A. , Sokolovsky M.
Published: July 2002

Competition binding experiments performed with 125I‐sarafotoxin (SRTX)‐b and SRTX‐b, SRTX‐c and endothelin (ET‐1 and ET‐3) using homogenates of rat right and left atria, aorta, uterus, cerebellum and caudate putamen indicated heterogeneity of the ET/SRTX receptor. The evidence pointed to the existence of three receptor subtypes: a high‐affinity ET‐1/SRTX‐b subtype typical of smooth muscle (E‐Sα receptor), a high‐affinity SRTX‐c subtype typical of the cerebellum (E‐Sβ receptor), and a less selective subtype typical of the caudate putamen that binds all of these peptides with high affinity (E‐Sγ receptor).