bluuebunny/crossref_metadata_2025 huggingface dataset:
Loss of quin 2 accompanies degranulation of mast cells
Authors: Bibb Philip C. , Cochrane David E. , Morel-Laurens Nicole
Published: July 2002

Stimulation of quin 2 loaded mast cells in the presence of 1 mM extracellular calcium produced a rapid and sustained increase in quin 2 fluorescence. This was accompanied by degranulation and the release of histamine. When Ca was replaced by EGTA or when Mn was present, a decrease in fluorescence accompanied degranulation. The increase in quin 2 fluorescence accompanying stimulation of mast cells appears to be due to the interaction of extracellular Ca with quin 2 associated with the secretory granule matrix released upon exocytosis.
De novo CDP‐choline‐dependent glycerophosphorylcholine synthesis and its involvement as an intermediate in phosphatidylcholine synthesis
Authors: Infante J.P.
Published: July 2002

The activity of CDP‐choline‐dependent glycerophosphorylcholine synthetase (CDP‐choline:sn‐3‐glycerophosphate cholinetransferase), a newly discovered enzyme involved in the recently proposed pathways of acyl‐specific phosphatidylcholine synthesis, is reported in rat liver. Endogenous CDP‐choline, synthesized via the CMP‐driven back reaction of phosphorylcholine transferase, is also shown to be a choline donor for this glycerophosphorylcholine synthetase. The function of glycerophosphorylcholine as an intermediate in phosphatidylcholine synthesis is demonstrated by specific isotope trapping whereby unlabelled glycerophosphorylcholine inhibited label incorporation from sn‐[14C]glycerol‐3‐phosphate into phosphatidylcholine in mouse gastrocnemius, a tissue that is essentially devoid of the cytidine pathway for phosphatidylcholine synthesis and uses a non‐allelic glycerophosphorylcholine synthetase (exogenous PC:sn‐3‐glycerophosphate cholinetransferase) in the synthesis of glycerophosphorylcholine.
Calcium‐activated membrane depolarization via modulation of chloride efflux from parietal cells during gastrin stimulation
Authors: Tsunoda Yasuhiro , Matsumiya Hidemi
Published: July 2002

In the isolated parietal cell the following observations were made: gastrin led to an increase in cytoplasmic free Ca2+ concentration ([Ca2+]i) even in the absence of medium Ca2+ and to transient membrane depolarization in both the absence of the medium Ca2+ and the fura‐2‐loaded cell. The incorporated Ca2+ chelator BAPTA inhibited the gastrin‐induced membrane depolarization. The magnitude of depolarization caused by gastrin was unchanged on removal of medium Na+. Furosemide but not tetraethylammonium inhibited the gastrin‐induced depolarization. The results suggest that the Ca2+ released from the store(s) induces membrane depolarization, possibly via modulation of a Cl− efflux across the luminal surface during gastrin stimulation.
Inositol trisphosphate accumulation by high K+ stimulation in cultured adrenal chromaffin cells
Authors: Sasakawa Nobuyuki , Nakaki Toshio , Yamamoto Satoshi , Kato Ryuichi
Published: July 2002

Stimulation with high K+ (KCl, 56 mM) of myo‐[3H]inositol‐prelabelled cells increased Ca2+ uptake and [3H]inositol trisphosphate (IP3) accumulation in a concentration‐dependent manner. Nifedipine, a Ca2+ channel antagonist, inhibited high K+‐induced [3H]IP3 accumulation and 45Ca2+ uptake with a similar potency. Furthermore, ionomycin (1 μM), a Ca2+ ionophore, also induced 45Ca2+ uptake and [3H]IP3 accumulation. These results indicate the existence of the Ca2+ uptake‐triggered mechanism of IP3 formation in cultured adrenal chromaffin cells.
Recombinant γ‐interferon stimulates iodide uptake and cyclic AMP production by the FTRL5 thyroid cell line
Authors: Weetman A.P.
Published: July 2002

The effect of recombinant rat γ‐interferon (γ‐IFN) on iodide uptake and cAMP production by rat thyroid cells in vitro was studied using the continuously growing, functional FRTL5 cell line. Both functions were stimulated by γ‐IFN at concentrations of 1–10 U/ml. Iodide uptake was dependent on protein synthesis, since it was blocked by cycloheximide treatment, but was not dependent on growth factors in calf serum routinely used for FRTL5 cell culture. These results show that γ‐IFN can stimulate thyroid cell function as well as aberrant Ia expression in vitro.
Effect of atrial natriuretic factor on cytosolic free calcium in rat glomerular mesangial cells
Authors: Appel Richard G. , Dubyak George R. , Dunn Michael J.
Published: July 2002

Since atrial natriuretic factor (ANF) inhibits angiotensin II (ANGII)‐induced mesangial cell contraction, we studied its effect on cytosolic free calcium in fura‐2‐loaded monolayers. ANF lowered basal calcium from 117 ± 5 to 89 ± 2 nM, and also reduced the ANGII‐induced calcium transient. The absolute level of cytosolic calcium after ANGII was lower in cells preincubated with ANF compared to buffer. In a few cell lines, ANF had no effect on cytosolic calcium, while in all cell lines ANF had inhibited contraction. Thus, while ANF has effects on cytosolic calcium in mesangial cells, the vasorelaxant effects of ANF may require additional physiologic interactions.
Molecular cloning and sequencing of cDNA for rat cathepsin L
Authors: Ishidoh Kazumi , Towatari Takae , Imajoh Shinobu , Kawasaki Hiroshi , Kominami Eiki , Katunuma Nobuhiko , Suzuki Koichi
Published: July 2002

A near full‐length cDNA for rat cathepsin L was isolated. The deduced protein comprises 334 amino acid residues (M
r 37 685) containing a typical signal sequence (N‐terminal 17 residues), pro‐peptide (96 residues), and the sequence for mature cathepsin L (221 residues). Rat cathepsin L shows 94% amino acid identity with mouse cysteine proteinase. Amino acid sequence homologies of rat cathepsin L with rat cathepsins H and B are 45 and 25%, respectively. These facts indicate that mouse cysteine proteinase is probably mouse cathepsin L and that cathepsin L is more closely related to cathepsin H than cathepsin B.
Hormonal regulation of pituitary GH3 cell K+ channels by Gk is mediated by its α‐subunit
Authors: Codina Juan , Grenet Dagoberto , Yatani Atsuko , Birnbaumer Lutz , Brown Arthur M.
Published: July 2002

The resolved α‐GTPγS (α*) and βγ‐subunits of human erythrocyte Gk, the stimulatory regulatory component of hormone‐responsive K+ channels, were tested for their potential stimulatory activities on the K+ channel of the endocrine GH3 cell. Concentrations as low as 0.5 pM αk* consistently activated K+ channels in isolated membrane patches, and saturating effects were obtained with 50 pM αk*. In contrast 2000–4000 pM βγ was without effect. We conclude that Gk acts on K+ channels through its α‐subunit in a manner akin to that of Gs acting on adenylyl cyclase and transducin acting on cGMP‐specific phosphodiesterase of photo‐receptor cells.
Molecular cloning of cDNA coding for human PGP 9.5 protein
Authors: Day Ian N.M. , Thompson R.J.
Published: July 2002

The co‐ordinate sequencing of the human neuronal and neuroendocrine marker protein PGP 9.5 and its cDNA is described. The cDNA encodes the complete protein (212 amino acids), and the 340 nucleotide 3'‐noncoding region including the polyadenylation signal, indicating an mRNA slightly larger than 1 kb in size. Protein sequencing of 50% of PGP 9.5 confirms the deduced protein sequence.
Activation of an S6 kinase from rat astroglial cells by cAMP
Authors: Pierre Michel , Gavaret Jean-Michel , Matricon Carole , Pomerance Martine , Jacquemin Claude , Toru-Delbauffe Danièle
Published: July 2002

Forskolin and isoproterenol, agonists of adenylate cyclase activity, and dibutyryl cyclic AMP, stimulated an S6 kinase activity in astroglial cells. This activity was insensitive to the thermostable inhibitor of cyclic AMP‐dependent protein kinase and had the same behaviour on a DEAE‐Sephacel column as the mitogen stimulated S6 kinase. These observations support the idea that the cyclic AMP cascade, as well as various growth factors, can activate S6 kinase.
Radioligand binding of antagonists of platelet‐activating factor to intact human platelets
Authors: Ukena Dieter , Dent Gordon , Birke Frank W. , Robaut Christine , Sybrecht Gerhard W. , Barnes Peter J.
Published: July 2002

Two new antagonists of platelet‐activating factor (PAF), the pyrrolothiazole derivative 52770 RP and the triazolodiazepine WEB 2086, have been studied as radioligands in intact human platelets. [3H]52770 RP and [3H]WEB 2086 bound specifically to high‐affinity sites with dissociation constants (K
d) of 14.8 and 6.1 nM, respectively. The maximal number of sites for [3H]52770 RP binding was approx. 15‐fold higher than for [3H]PAF and [3H]WEB 2086. In addition, C16‐PAF, lyso‐PAF, WEB 2086 and 52770 RP had K
i values which were nearly identical for both [3H]PAF and [3H]WEB 2086, whereas only 52770 RP competed for [3H]52770 RP‐binding sites. These results demonstrate that in human platelets the sites of [3H]WEB 2086 binding are identical to [3H]PAF‐binding sites, whereas those of [3H]52770 RP are not. [3H]WEB 2086 appears, therefore, to be a suitable antagonist radioligand for labelling PAF receptors.
C‐reactive protein decreases protein phosphorylation in stimulated human neutrophils
Authors: Buchta Richard , Gennaro Renato , Pontet Michel , Fridkin Mati , Romeo Domenico
Published: July 2002

Treatment of human neutrophils with C‐reactive protein (CRP) causes a concentration‐dependent decrease in the extent of activation of superoxide production and of granule secretion, induced by phorbol‐12‐myristate‐13‐acetate (PMA) or N‐formyl‐L‐methionyl‐L‐leucyl‐L‐phenylalanine (fMLF). The same treatment also causes a significant reduction in the degree of PMA‐ and fMLF‐stimulated phosphorylation of several cell proteins. These include the proteins of 43–47 kDa, whose extent of phosphorylation correlates with the activation of superoxide production and of secretion. Contrary to the effects exerted on protein phosphorylation, CRP does not affect the fMLF‐elicited increase in neutrophil cytosolic Ca2+.
Long‐range 15N‐1H correlation as an aid to sequential proton resonance assignment of proteins Application to the DNA‐binding protein ner from phage Mu
Authors: Clore G.Marius , Bax Ad , Wingfield Paul , Gronenborn Angela M.
Published: July 2002

A method is described for sequential resonance assignment of protein 1H‐NMR spectra relying on the detection of long‐range correlations between 15N and CαH atoms using 1H‐detected heteronuclear multiple‐bond correlation spectroscopy. In particular, the observation of the two‐bond 15N(i)‐CαH(i) and three‐bond 15N(i)‐CαH(i−1) correlations enables one to connect one residue with the next. Because the magnitude of the long‐range couplings is small (<6 Hz), the sensitivity of this experiment is necessarily low and requires the use of 15N‐enriched protein samples. Further, because the size of the 15N(i)‐CαH(i−1) coupling is very sensitive to the ψ backbone torsion angle, structural information can be derived. The application of this experiment is illustrated with the 75‐residue DNA‐binding protein ner from phage Mu.
Inhibition by amiloride and by Na+‐depletion of A23187‐stimulated arachidonic acid and histamine release from rat mast cells
Authors: Linnebjerg Helle , Hansen Harald S. , Jensen Benny
Published: July 2002

Rat peritoneal mast cells, labelled with [14C]arachidonic acid, released histamine and [14C]arachidonic acid upon the addition of A23187. The release induced by low concentrations of A23187 was suppressed by removal of extracellular Na+ and by addition of the Na+/H+ exchange inhibitor, amiloride. Addition of protein kinase C inhibitors resulted in only a modest decrease of [14C]arachidonic acid and histamine release. [14C]Arachidonic acid was hydrolyzed from both phosphatidylcholine and triacylglycerol. Amiloride inhibited only the hydrolysis from phosphatidylcholine. It is suggested that an Na+/H+ exchange is stimulated secondary to an A23187‐induced increase in intracellular calcium concentration. Increased Na+/H+ exchange seems to facilitate the activation by intracellular calcium ions of phospholipase A2 as well as of histamine secretion.
The ATP‐driven primary Na+ pump in subcellular vesicles of Vibrio alginolyticus
Authors: Dibrov P.A. , Skulachev V.P. , Sokolov M.V. , Verkhovskaya M.L.
Published: July 2002

Subcellular vesicles of Vibrio alginolyticus hydrolyse ATP and accumulate Na+ in an ATP‐dependent fashion. The Na+ uptake is (i) strongly stimulated by Δψ‐discharging agents, i.e., the protonophorous uncoupler CCCP or valinomycin +K+ and (ii) arrested by DCCD at a concentration strongly inhibiting ATP hydrolysis. Lower concentrations of DCCD stimulate the Na+ accumulation supported by ATP hydrolysis as well as by NADH oxidation. It is concluded that there is an electrogenic DCCD‐sensitive Na+‐ATPase in the cytoplasmic membrane of V. alginolyticus.
Low‐density lipoproteins increase intracellular calcium in aequorin‐loaded platelets
Authors: Dunn R.C. , Schachter M. , Miles C.M.M. , Feher M.D. , Tranter P.R. , Bruckdorfer K.R. , Sever P.S.
Published: July 2002

Low‐density lipoproteins activate isolated human platelets. The mechanism of this activation is unknown, but may involve increased phosphoinositide turnover. We have examined the effect of low‐density lipoproteins on intracellular calcium concentrations in platelets loaded with the photoprotein aequorin. The lipoproteins induced concentration‐dependent increases in intracellular calcium, associated with shape change and aggregation. These responses could be partially inhibited by the removal of extracellular calcium and by pre‐incubation with acetylsalicylic acid. They were also antagonised by agents which increase cellular concentrations of cyclic adenosine and guanosine monophosphates. It is not clear whether the platelet‐lipoprotein interaction involves a ‘classical’ lipoprotein receptor.
Acetylcholine and cholecystokinin receptors functionally couple by different G‐proteins to phospholipase C in pancreatic acinar cells
Authors: Schnefel S. , Banfic H. , Eckhardt L. , Schultz G. , Schulz I.
Published: July 2002

We have studied the involvement of GTP‐binding proteins in the stimulation of phospholipase C from rat pancreatic acinar cells. Pretreatment of permeabilized cells with activated cholera toxin inhibited both cholecystokinin‐octapeptide (CCK‐OP) and GTPγS but not carbachol (CCh)‐induced production of inositol trisphosphate. Pertussis toxin had no effect. Neither vasoactive intestinal polypeptide, a stimulator of adenylyl cyclase, nor the cAMP‐analogue, 8‐bromo cAMP, mimicked the inhibitory effect of cholera toxin on agonist‐induced phospholipase C activation. This indicates that inhibition by cholera toxin could not be attributed to a direct interaction of cholera toxin activated Gs with phospholipase C or to an elevation of cAMP. In isolated rat pancreatic plasma membranes cholera toxin ADP‐ribosylated a 40 kDa protein, which was inhibited by CCK‐OP but not by CCh. We conclude from these data that both CCK‐ and muscarinic acetylcholine receptors functionally couple to phospholipase C by two different GTP‐binding proteins.
CCK and gastrin inhibit adenylate cyclase activity through a pertussis toxin‐sensitive mechanism in the tumoral rat pancreatic acinar cell line AR 4‐2J
Authors: Scemama Jean Luc , Robberecht Patrick , Waelbroeck Magali , De Neef Philippe , Pradayrol Lucien , Vaysse Nicole , Christophe Jean
Published: July 2002

(Thr28,Nle31)CCK(23–33) (CCK‐9) and gastrin(1–17)I (gastrin) inhibited adenylate cyclase activity in membranes from the tumoral rat pancreatic acinar cell line AR 4‐2J through a Bordetella pertussis toxin‐sensitive mechanism. This contrasted with the stimulatory effect exerted by CCK‐9 on adenylate cyclase activity in membranes from normal rat pancreas. The relative potency of CCK‐9, gastrin, and related peptides in inhibiting adenylate cyclase, when confronted with previous evidence, suggests that ‘non‐selective CCK‐gastrin CCK‐B receptors’ predominating over ‘selective CCK‐A receptors’ in the AR 4‐2J cell line, favored the coupling of the first receptors to adenylate cyclase through Gi, while CCK‐A receptors capable of stimulating the enzyme through Gs were detected only after Bordetella pertussis toxin pretreatment.
The Saccharomyces cerevisiae ARO1 gene An example of the co‐ordinate regulation of five enzymes on a single biosynthetic pathway
Authors: Duncan Kenneth , Edwards R.Mark , Coggins John R.
Published: July 2002

The ARO1 gene of Saccharomyces cerevisiae encodes the arom multifunctional enzyme. Specific inhibitors of amino acid biosynthesis have been used to obtain evidence that expression of a cloned ARO1 gene is regulated in response to amino acid limitation. Northern blot analysis and sequence studies indicate that ARO1 is regulated by the well characterised S. cerevisiae ‘general control’ mechanism. This provides a very economical means of simultaneously tailoring the synthesis of five shikimate pathway enzymes to the needs of the cell.
Ionic currents in avian granulosa cells
Authors: Schwartz Jean-Louis , Mealing Geoffrey A.R. , Asem Elikplimi K. , Whitfield James F. , Tsang Benjamin K.
Published: July 2002

Transmembrane ionic currents have been recorded in single granulosa cells from the laying hen using the whole‐cell patch‐clamp technique. Under voltage‐clamp conditions, depolarizing voltage steps evoked currents composed of a fast inactivating inward component and a delayed outward component. The former was activated at voltages more positive than −50 mV and was fully inactivated within 500 ms. It was blocked by D600 (methoxyverapamil) and by cobalt, suggesting that it is a calcium current. The latter displayed inward rectification and did not inactivate during long duration pulses. It was blocked by tetraethylammonium indicating that it is a potassium current. This is the first evidence of the existence of potassium and calcium transmembrane currents in granulosa cells.
Monensin inhibits the expression of sucrase‐isomaltase in Caco‐2 cells at the mRNA level
Authors: Chantret Isabelle , Trugnan Germain , Dussaulx Elisabeth , Zweibaum Alain , Rousset Monique
Published: July 2002

Using L‐[35S]methionine labeling, SDS‐PAGE and Northern blot analysis of sucrase‐isomaltase mRNA, two different concentrations of monensin were used to delineate in Caco‐2 cells the effect of the drug on the conversion of the high mannose to the complex form of sucrase‐isomaltase from its dual effect on the biosynthesis of the enzyme and on the rate of glucose consumption. At 0.1 μM the drug has no effect on the rate of glucose consumption and, although it inhibits the conversion of the high mannose to the complex form of the enzyme, it has no effect on the level of sucrase‐isomaltase mRNA and on the amount of neosynthesized enzyme. At 1 μM, in addition to its inhibiting effect on the maturation of the enzyme, monensin provokes concomitantly an increase in the rate of glucose consumption and a decrease in the level of sucrase‐isomaltase mRNA and in the amount of neosynthesized enzyme. All these effects are reversible within 48 h after removal of the drug.
Nerve growth factor induces rapid redistribution of F‐actin in PC12 cells
Authors: Paves Heiti , Neuman Toomas , Metsis Madis , Saarma Mart
Published: July 2002

Nerve growth factor (NGF) induces the redistribution of F‐actin in rat pheochromocytoma PC12 cells within 2–10 min, whereas epidermal growth factor (EGF) has no effect on microfilament organization. This redistribution of F‐actin in PC12 cells is not protein synthesis dependent, but can be blocked by methyltransferase inhibitors.
The nature of the Fe(III) EPR signal from the acceptor‐side iron in photosystem II
Authors: Aasa Roland , Andréasson Lars-Erik , Styring Stenbjörn , Vänngård Tore
Published: July 2002

The EPR spectrum at both X‐ and S‐band (3.94 GHz) of the oxidized acceptor‐side iron in photosystem II from spinach shows two absorption‐type peaks at g = 8.0 and 5.6. The intensities of these peaks have been measured at X‐band in the temperature range 2–10 K. All results can be fully described assuming that the EPR spectrum arises from high‐spin Fe(III) with D = 1.0±0.3 cm−1 and E/D = 0.10 ±0.01. Quantifications show that the spectrum in our case represents 0.4–0.5 Fe(III) per reaction center. The EPR parameters are consistent with the iron having bicarbonate and/or tyrosine as ligands in addition to four imidazoles.
Protein kinase C is involved in regulation of Ca2+ channels in plasmalemma of Nitella syncarpa
Authors: Zherelova O.M.
Published: July 2002

Ca2+ current recordings have been made on Nitella syncarpa cells using the intracellular perfusion and the voltage‐clamp technique. TPA (12‐O‐tetradecanoylphorbol‐13‐acetate), a substance capable of activating protein kinase C from plasmalemma of Nitella cells, modulates voltage‐dependent Ca2+ channels. Polymixin B, inhibitor of protein kinase C, blocks the Nitella plasmalemma Ca2+ channels; the rate of channel blockage depends on the concentration and exposure time of the substance.
Biochemical and antigenic characterization of CD45 polypeptides expressed on plasma membrane and internal granules of human neutrophils
Authors: Pulido Rafael , Lacal Pedro , Mollinedo Faustino , Sánchez-Madrid Francisco
Published: July 2002

The expression of CD45 polypeptides, a phosphotyrosine phosphatase complex specific of leukocytes, has been investigated in both resting and activated neutrophils by using anti‐CD45 monoclonal antibodies (MAb) which specifically recognize different polypeptide components of the CD45 molecular complex. Polypeptides of 180 and 130–150 kDa were equally precipitated by either a conventional CD45 MAb recognizing an antigenic determinant shared by the four CD45 glycoproteins (220, 205, 190 and 180 kDa) or by the anti‐180 kDa UCHL1 MAb. These polypeptides were overexpressed on neutrophil plasma membranes after degranulatory stimulation. Conversely, neither the anti‐220 kDa CD45R nor anti‐220/205/190 kDa MAb reacted with CD45 molecules from resting or activated neutrophils. Furthermore, permeabilization analysis and comparative immunoprecipitation studies with different anti‐CD45 MAb from fractions enriched in various neutrophil granules revealed that CD45 polypeptides (180 and 130–150 kDa) from internal granules are antigenic and biochemically identical to those expressed on plasma membrane.
Work overload induced changes in fast and slow skeletal muscle myosin heavy chain gene expression
Authors: Morgan Matthew J. , Loughna Paul T.
Published: July 2002

Work induced hypertrophy of the slow postural soleus and the fast phasic plantaris muscles was produced by tenotomy of the synergistic gastrocnemius muscle. Increases in weight of both muscles were associated with proportionately even larger increases in total RNA and mRNA levels. Alterations in levels of specific myosin heavy chain (MHC) isoform mRNAs were measured using the slot blot procedure with radioactively labelled oligonucleotides as probes. Type 1 MHC gene expression was unaffected in both muscles by work overload, whereas type 2a was deinduced in the soleus and type 2b was deinduced in the plantaris. The neonatal MHC gene was transiently reinduced in the plantaris.
A ‘branched’ mechanism of the reverse reaction of yeast glutathione reductase An estimation of the enzyme standard potential values from the steady‐state kinetics data
Authors: Rakauskien≐ Gelm≐ A. , Č≐nas Narimantas K. , Kulys Juozas J.
Published: July 2002

The reduced glutathione‐linked NADP+ reduction, catalyzed by yeast glutathione reductase, follows a ‘sequential’ or ‘ping‐pong’ mechanism at high or low NADP+ concentrations, respectively. The pattern of the NADPH and NADP+ cross‐inhibition reflects not only the competition for the binding site, but the shift of the reaction equilibrium as well. A ‘branched’ scheme of the glutathione reductase reaction is presented. The enzyme standard potential (−255 mV, pH 7.0) was estimated from the ratio of the NADPH and NADP+ rate constants corresponding to the ping‐pong mechanism.
Defect in phorbol acetate‐induced translocation of diacylglycerol kinase in erbB‐transformed fibroblast cells
Authors: Kato Miwako , Kawai Sadaaki , Takenawa Tadaomi
Published: July 2002

We here show that tetradecanoyl phorbol acetate (TPA) and 1‐oleoyl 2‐acetyl glycerol (OAG) cause the translocation of diacylglycerol (DG) kinase from the cytosol to the membrane fractions in chick embryo fibroblast (CEF) cells. However, this translocation is not marked in erbB‐transformed chick embryo fibroblast (GEV) cells. The activities of phosphatidylinositol (PI) and phosphatidylinositol 4‐phosphate (PIP) kinases in membrane fractions are not altered by TPA treatment in either CEF or GEV cells. Such reduced translocation of DG kinase by TPA is also observed in src‐transformed cells, but not in myc‐transformed cells. These results suggest that the defect in DG kinase translocation may result in failure to suppress the overactivation of protein kinase C in erbB‐2 and src‐transformed cells, which may lead to cell growth and transformation.
Spatial localization of the stimulus‐induced rise in cytosolic Ca2+ in bovine adrenal chromaffin cells
Authors: Cheek Timothy R. , O'Sullivan Antony J. , Moreton Roger B. , Berridge Michael J. , Burgoyne Robert D.
Published: July 2002

The spatial distribution of the intracellular free Ca2+ (Ca2+
i) rise elicited by different stimuli in bovine adrenal chromaffin cells was examined in single fura‐2‐loaded cells. In response to the potent secretagogues nicotine and high K+, Ca2+
i was initially localized exclusively to the entire subplasmalemmal area of the cell. In response to the ineffective secretagogues, methacholine and muscarine, the rise in Ca2+
i originated only in one pole of the cell and even at the peak of the response Ca2+ was still generally restricted to this same area of the cell. These results suggest that the triggering of exocytosis from these cells requires a specific spatial distribution of Ca2+
i.
Chronic long‐term electrostimulation creates a unique metabolic enzyme profile in rabbit fast‐twitch muscle
Authors: Hood David A. , Pette Dirk
Published: July 2002

Long‐term low‐frequency stimulation (up to 120 days) of rabbit fast‐twitch tibialis anterior muscle led, in a first‐order‐like time course, to changes in enzyme activities of energy metabolism which became stable with ongoing stimulation after 50 days. The glycolytic enzymes decreased to 30–40% of their normal values, but remained 2–3‐fold higher than in heart or soleus muscle. The LDH isozyme pattern ultimately resembled that of the slow‐twitch soleus muscle. Citrate synthase activity increased 3.7‐fold which brought this enzyme to a value 45% above that of heart. These results indicate that chronic stimulation does not simply convert the fast‐twitch muscle into a soleus‐like slow‐twitch muscle, but creates a tissue of unique metabolic properties.
Voltage‐activated and stretch‐activated Ba2+ conducting channels in an osteoblast‐like cell line (UMR 106)
Authors: Duncan Randall , Misler Stanley
Published: July 2002

Calcium entry may mediate osteoblast activation by calciotropic hormones or changes in electrical fields or mechanical stress in bone. Using cell‐attached patch clamping techniques, we have seen two Ba2+ conducting channels in UMR‐106 osteoblast‐like cells: (i) a voltage‐dependent, dihydropyridine‐sensitive channel resembling L‐type Ca2+ channels in other cells, and (ii) a stretch‐activated non‐selective channel resembling those involved in mechanoreception and triggering of volume regulation in other cells.
Glucose‐induced oscillations of intracellular Ca2+ concentration resembling bursting electrical activity in single mouse islets of Langerhans
Authors: Valdeolmillos Miguel , Santos Rosa M. , Contreras Diego , Soria Bernat , Rosario Luis M.
Published: July 2002

Intracellular Ca2+ levels were monitored in single, acutely isolated mouse islets of Langerhans by dual emission Indo‐1 fluorometry. High‐frequency (3.1 min−1) [Ca2+]i, oscillations with a brief rising time (1–2 s) and 10 s half‐width (‘fast’ oscillations) were detected in 11 mM glucose. Raising the glucose concentration to 16.7 mM increased the duration of these oscillations, which were otherwise absent in 5.5 mM glucose. [Ca2+]i waves of lower frequency (0.5 min−1) and longer rising time (‘slow’ oscillations) were also recorded. The data indicate that “fast” oscillations are directly related to β‐cell bursting electrical activity, and suggest the existence of extensive networks of electrically coupled cells in the islet.
Onset of transcription of the aminopeptidase N (leukemia antigen CD 13) gene at the crypt/villus transition zone during rabbit enterocyte differentiation
Authors: Norén O. , Dabelsteen E. , Høyer P.E. , Olsen J. , Sjöström H. , Hansen G.H.
Published: July 2002

The sequence of a cDNA clone (2.82 kbp) of rabbit intestinal aminopeptidase N (CD 13) is reported. Using the corresponding anti‐sense RNA probe, the distribution of aminopeptidase N mRNA along the crypt/villus axis of the rabbit small intestine was studied by in situ hybridization. The aminopeptidase N gene is expressed along the whole length of the villus with a maximum at its base. Expression was not detected in the crypt cells. The distribution of aminopeptidase N mRNA correlates with the presence of active enzyme as monitored by histochemical staining. The results are compatible with onset of transcription of the aminopeptidase N gene at the crypt/villus transition zone during the enterocyte differentiation.
GPC phosphodiesterase and phosphomonoesterase activities of renal cortex and medulla of control, antidiuresis and diuresis rats
Authors: Kanfer Julian N. , McCartney Douglas G.
Published: July 2002

Glycerylphosphorylcholine (GPC) concentration was reported to be elevated in renal medulla of experimental animals deprived of water. The activities of GPC phosphodiesterases were similar in homogenates and membrane subfractions of renal cortex prepared from control, diuresis and antidiuresis rats. There were no differences in these preparations' ability to hydrolyze phosphorylcholine. In contrast, there was a nearly 50% reduction of non‐specific phosphomonoesterase activity, using p‐nitrophenylphosphate as substrate and membrane subfractions prepared from the antidiuresis animals. It is suggested that as a consequence, a pathway for the formation from L‐α‐glycerylphosphate is activated.
Calcium mobilization and Na+/H+ antiport activation by endothelin in human skin fibroblasts
Authors: Gardner Jeffrey P. , Maher Elizabeth , Aviv Abraham
Published: July 2002

Endothelin; Growth factor; (Human skin fibroblast)
Molecular cloning of the Na,K‐ATPase α‐subunit in developing brine shrimp and sequence comparison with higher organisms
Authors: Baxter-Lowe Lee Ann , Guo Jian Zhong , Bergstrom Ellen E. , Hokin Lowell E.
Published: July 2002

Na,K‐ATPase; Molecular cloning; DNA, complementary; Amino acid sequence; (Artemia)
Phosphorylation of the skeletal muscle AMP‐deaminase by protein kinase C
Authors: Tovmasian E.K. , Hairapetian R.L. , Bykova E.V. , Severin S.E. , Haroutunian A.V.
Published: July 2002

Protein kinase C catalyzes phosphorylation of the rat skeletal muscle AMP‐deaminase in the presence of calcium ions and phosphatidylserine. At the same time, the catalytic subunit of cAMP‐dependent protein kinase fails to phosphorylate AMP‐deaminase. Ca2+ phosphatidylserine‐dependent phosphorylation decreases three‐fold (from 0.6 to 0.2 mM) the K
m value and does not affect V
max Protein kinase C‐induced phosphorylation of AMP‐deaminase, besides ADP‐ribosylation, is suggested to be involved in regulating the AMP‐deaminase activity in vivo.
Y1 receptors for neuropeptide Y are coupled to mobilization of intracellular calcium and inhibition of adenylate cyclase
Authors: Aakerlund Lars , Gether Ulrik , Fuhlendorff Jannie , Schwartz Thue W. , Thastrup Ole
Published: July 2002

Two types of binding sites have previously been described for neuropeptide Y (NPY), called Y1 and Y2 receptors. The intracellular events following Y1, receptor activation was studied in the human neuroblastoma cell line SK‐N‐MC. Both NPY and the specific Y1 receptor ligand, [Leu31,Pro34]‐NPY, caused a rapid and transient increase in the concentration of free calcium in the cytoplasm as measured by the fluorescent probe, Fura‐2. The effect of both peptides was independent of extracellular calcium as addition of EGTA or manganese neither changed the size nor the shape of the calcium response. The calcium response to NPY was abolished by pretreatment with thapsigargin, which can selectively deplete a calcium store in the endoplasmic reticulum. Y1 receptor stimulation, by both NPY and [Leu31,Pro34]NPY, also inhibited the forskolin‐stimulated cAMP production with an EC
50 of 3.5 nM. There was a close relation between the receptor binding and the cellular effects as half‐maximal displacement of [125I‐Tyr36] monoiodoNPY from the receptor was obtained with 2.1 nM NPY. The Y2‐specific ligand NPY(16–36)peptide had no effect on either intracellular calcium or cAMP levels in the SK‐N‐MC cells. It is concluded that Y1 receptor stimulation is associated with both mobilization of intracellular calcium and inhibition of adenylate cyclase activity.
Video enhanced imaging of the fluorescent Na+ probe SBFI indicates that colonic crypts absorb fluid by generating a hypertonic interstitial fluid
Authors: Naftalin R.J. , Pedley K.C.
Published: July 2002

Extracellular accumulation of Na7+ detected by video‐enhanced microscopic imaging of the impenneant fluorescent probe SBFI[1,2] confirms the view that colonic crypts produce a hypertonic absorbate ca 1000 mOsm·kg−1, thereby generating a large osmotic pressure across the crypt wall. This creates a high fluid tension within the crypt lumen, sufficient to dehydrate faeces [3,4]. When bathed in Tyrode the SBFI·Na+ fluorescence indicates a [Na+) ca 750 mM within the interstitial space of metabolizing rat descending colon. There is no evidence of interstitial Na+ accumulation in octanol (2 mM) or in rabbit colon incubated with 1.0 mM ouabain and no evidence of Na+ secretion via the crypt lumen during absorption [5].
α1‐Adrenergic stimulation of Cl− efflux in isolated brown adipocytes
Authors: Dasso Leonardo , Connolly Eamonn , Nedergaard Jan
Published: July 2002

Unidirectional 36Cl− efflux from preloaded isolated brown adipocytes was studied. A norepinephrine‐stimulated 36Cl− efflux pathway was found which approximately doubled the rate of 36Cl− efflux from the cells. The response to norepinephrine was fully inhibited by the α1‐adrenergic antagonist prazosin, but was unaffected by the β‐adrenergic antagonist propranolol, showing that norepinephrine stimulated the 36Cl− efflux pathway via the α1‐adrenoceptor. The stimulation of 36Cl− efflux could not be mimicked by the Ca2+ ionophore A23187, indicating that the effect was not mediated by elevation of the intracellular Ca2+ level. It is concluded that brown fat cells possess a specific mechanism for α1‐adrenergic stimulation of Cl− efflux. The possibility is discussed that this Cl− efflux pathway could be the basis for the early α‐adrenergic depolarization seen in brown fat cells.
Simultaneous recordings of glucose dependent electrical activity and ATP‐regulated K+‐currents in isolated mouse pancreatic β‐cells
Authors: Smith Paul A. , Ashcroft Frances M. , Rorsman Patrik
Published: July 2002

Membrane potential and membrane currents were recorded from single mouse pancreatic β‐cells using the perforated patch whole‐cell recording technique at 30 °C. Single β‐cells maintained in primary tissue culture exhibited glucose‐dependent electrical activity similar to that reported for freshly isolated intact islets. The resting input conductance (5.1±0.9 nS) was determined by ATP‐regulated K+ (KATP) channels as it was blocked by 1 mM tolbutamide. 8 mM glucose decreased the input conductance by 80%. The input conductance at −70 mV was of a similar value during the plateau phase and during the silent phase of electrical activity in 8 mM glucose. This suggests that oscillations of KATP channel activity do not underlie the slow waves.
High concentrations of low density lipoprotein decrease basement membrane‐associated heparan sulfate proteoglycan in cultured endothelial cells
Authors: Olgemöller Bernhard , Schleicher Erwin D. , Schwaabe Susanne , Guretzki Hans-Joachim , Gerbitz Klaus D.
Published: July 2002

The effect of increasing low density lipoprotein (LDL) concentrations on the synthesis of basement membrane components was investigated in proliferating porcine aortic endothelial cells (PAEC) in culture. Basement membrane‐associated heparan sulfate proteoglycan (HSPG) and fibronectin were determined by enzyme immunoassay. Low extracellular LDL‐levels increase, high extracellular LDL‐levels decrease the HSPG content of PAEC. Fibronectin synthesis was only slightly affected while proliferation and metabolic activity as assessed by lactate production were constant. Insulin or high extracellular glucose did not influence the effect of LDL on basement membrane components.
Rapid and reversible changes in myosin heavy chain expression in response to increased neuromuscular activity of rat fast‐twitch muscle
Authors: Kirschbaum Bernhard J. , Schneider Susanne , Izumo Seigo , Mahdavi Vijak , Nadal-Ginard Bernardo , Pette Dirk
Published: July 2002

Chronic 10 Hz stimulation of rat fast‐twitch muscle induced rapid and reversible changes in the tissue levels offast myosin heavy chain (HC) mRNA isoforms. These changes consisted of a rapid decrease in HCIIb mRNA and a progressive increase in HCIIa mRNA. After 15 days, the HCIIb mRNA normally amounting to approximately 80%, had decreased to less than 5% of the sum of the two HC mRNA isoforms. HCIIb mRNA was again detectable one day after cessation of stimulation and progressively increased at the expense of HCIIa mRNA with ongoing recovery. These results point to a down‐regulation of the HCIIb gene by the applied stimulus pattern which, conversely, enhances the expression of the HCIIa gene.
Synergy between zinc and phorbol ester in translocation of protein kinase C to cytoskeleton
Authors: Zalewski P.D. , Forbes I.J. , Giannakis C. , Cowled P.A. , Betts W.H.
Published: July 2002

Protein kinase C was measured in the cytoskeletal fraction of lymphocytes, platelets and HL60 cells, by specific binding of [3H]phorbol dibutyrate and by immunoblotting with antibody to a consensus sequence in the regulatory domain of α‐, β‐ and γ‐isozymes of protein kinase C. Treatment of cells for 40 min with a combination of zinc (2–50 μM), zinc ionophore pyrithione and unlabelled phorbol dibutyrate (200 nM) caused up to a ten‐fold increase in cytoskeletal protein kinase C and a corresponding decrease in other cellular compartments. Omission of any of the reagents resulted in much less or no translocation. These effects were inhibited by 1,10‐phenanthroline, which chelates zinc, and were not seen with calcium. Increase in cytoskeletal protein kinase C persisted for several hours and appeared to involve attachment of the enzyme to actin microfilaments. We propose that zinc, like calcium, regulates the distribution of PKC in cells. However, unlike calcium which controls the binding of PKC to the lipid component on cell membranes, zinc controls the distribution of PKC to membrane cytoskeleton, possibly actin.
Purification and characterization of the RNase H domain of HIV‐1 reverse transcriptase expressed in recombinant Escherichia coli
Authors: Becerra S.Patricia , Clore G.Marius , Gronenborn Angela M. , Karlström Anders R. , Stahl Stephen J. , Wilson Samuel H. , Wingfield Paul T.
Published: July 2002

The ribonuclease H (RNase H) domain of human immune‐deficiency virus (HIV‐1) reverse transcriptase has been produced with the aim of providing sufficient amounts of protein for biophysical studies. A plasmid vector is described which directs high level expression of the RNase H domain under the control of the λ pl promoter. The domain corresponds to residues 427‐560 of the 66 kDa reverse transcriptase. The protein was expressed in Escherichia coli and was purified using ion‐exchange and size exclusion chromatography. The purified protein appears to be in a native‐like homogeneous conformational state as determined by 1H‐NMR spectroscopy and circular dichroism measurements. HIV‐protease treatment of the RNase H domain resulted in cleavage between Phe‐440 and Tyr‐441.
Protein kinase C in transmembrane signalling
Authors: Farago Anna , Nishizuka Yasutomi
Published: July 2002

Protein kinase C functions as the transducer of a second messenger, diacylglycerol, and is the major receptor for tumour‐promoting phorbol esters. The enzyme is a family of proteins with closely but distinct structures and individual enzymological properties. Members of the family are differently distributed in particular cell types and limited intracellular locations from lower organisms to mammalian tissues. The enzyme appears to interact with many signalling pathways, and display functions in the processing and modulation of cellular responses to external stimuli. Presumably, each member of the family plays discrete roles in the control of a variety of membrane functions and activation of gene transcription.
Specific receptors for endothelin‐3 in cultured bovine endothelial cells and its cellular mechanism of action
Authors: Emori Toshiaki , Hirata Yukio , Marumo Fumiaki
Published: July 2002

Among three endothelin (ET) isopeptides, ET‐3 shows the most potent initial depressor response through the endothelium‐dependent mechanism. We studied the presence of specific binding sites for ET‐3 in cultured bovine endothelial cells (EC) and its cellular mechanism of action. Binding studies revealed the presence of two distinct subclasses of ET‐3 receptors with high and low affinities. ET‐3 dose‐dependently (10−10−10−7 M) increased both intracellular Ca2+ levels ([Ca−2]) and inositol trisphosphate (IP3) formation. The ET‐3‐induced increase in [Ca2+]i was not affected by either removal of extracellular Ca2+ or Ca2+‐channel blockers. These data suggest that ET‐3 induces phosphoinositide breakdown and increase in [Ca2+]i in ECs, possibly resulting from intracellular Ca2+ mobilization, thereby leading to vasodilatation.
The structure of protein phosphatase 2A is as highly conserved as that of protein phosphatase I
Authors: Orgad Sara , Brewis Neil D. , Alphey Luke , Axton J.Myles , Dudai Yadin , Cohen Patricia T.W.
Published: July 2002

cDNA coding for protein phosphatase 2A (PP2A) has been isolated from Drosophila head and eye imaginal disc libraries. Drosophila PP2A mRNA is expressed throughout development, but is most abundant in the early embryo. The cDNA hybridises to a single site on the left area of the second chromosomes at position 28D2‐4. The deduced amino acid sequence (309 residues) of Drosophila PP2A shows 94% identity with either rabbit PP2Aα or PP2Aβ, indicating that PP2A maybe the most conserved of all known enzymes.
Synchronized repetitive spikes in cytoplasmic calcium in confluent monolayers of human umbilical vein endothelial cells
Authors: Neylon Craig B. , Irvine Robin F.
Published: July 2002

Synchronized repetitive spikes in cytoplasmic free calcium concentration, [Ca2+]i, are evoked by histamine in confluent monolayers of human endothelial cells. The repetitive spikes, which are apparently dependent upon the establishment of cell coupling, are also induced by caffeine, indicating that they may be due to an oscillatory release of Ca2+ from the endoplasmic reticulum, and may not involve oscillations in inositol phosphates. It is suggested that sunchronized repetitive spikes in [Ca2+]i might lead to oscillatory release of endothelial‐derived substances such as prostacyclin, nitric oxide and endothelin, which have potent effects on the vascular system.
Epidermal growth factor stimulates the phosphorylation of pyruvate kinase in fleshly isolated rat hepatocytes
Authors: Moule S.K. , McGivan J.D.
Published: July 2002

Epidermal growth factor (EGF) has previously been shown to stimulate gluconeogenesis in rat liver by decreasing the activity of pyruvate kinase [(1988) Biochem. J. 255, 361–364]. Here we investigate the mechanism underlying the inactivation of the enzyme. EGF was found to increase the incorporation of phosphate into pyruvate kinase, with maximal phosphorylation achieved only after 10 min in the presence of the growth factor. The increase in phosphorylation was not additive with the caused by cyclic AMP. Phosphoamino acid analysis of pyruvate kinase isolated from cells treated with EGF indicated that EGF phosphorylation solely on serine residues. The exact site of EGF‐mediated phosphorylation has yet to be identified.
Absence of calponin phosphorylation in contracting or resting arterial smooth muscle
Authors: Bárány M. , Rokolya A. , Bárány K.
Published: July 2002

We have tested the hypothesis of Winder and Walsh [(1990) J. Biol. Chem. 256, 10148] that the contractile state of smooth muscle is regulated by calponin phosphorylation. Porcine carotid arterial muscles were highly labeled with 33P, then contracted with four different agents for various times. No radioactivity was detected in calponin isolated by 2D or 1D gel electrophoresis from the muscles. Similarly, resting muscles showed no [33P]phosphate in calponin. Apparently the sites of calponin available for phosphorylation in vitro are rendered unavailable in the intact muscle.
Control region and gastric specific transcription of the rat H+,K+‐ATPase α subunit gene
Authors: Oshiman Ko-Ichi , Motojima Kiyoto , Mahmood Saleh , Shimada Akira , Tamura Shigehiko , Maeda Masatomo , Futai Masamitsu
Published: July 2002

The rat gastric H+,K+‐ATPase α subunit gene was cloned and the nucleotide sequence of its 5′‐upstream region was determined. Sequence comparison with the corresponding part of the human gene indicated the presence of highly conserved regions which may be important for specific transcription of the α subunit in gastric parietal cells. The amino‐terminal sequence (Met‐Gly‐Lys‐Ala‐Glu‐) of the rat enzyme was similar to those of the pig and human enzymes. The gene organization of the rat enzyme was also similar to that of the human gene: introns 1, 2 and 9 were located in exactly the same positions as those in the human gene, and, as in the latter, exon 6 was not separated by an intron. The sequences of introns 1 and 2 were highly conserved among the rat, human and pig genes, but were entirely different from those of Na+,K+‐ATPase catalytic subunit genes. Northern blot hybridization indicated that the gene was transcribed only in gastric mucosa.
Phorbol acetate enhances the phosphorylation of cytokeratins 8 and 18 in human colonic epithelial cells
Authors: Chou C.-F. , Omury M.Bishr
Published: July 2002

The phosphorylation of epithelial‐specific cytokeratin (CK) 8 and 18 was studied in the human colonic cell line HT29. Metabolic labelling of cells with orthophosphate resulted in phosphorylation of cytokeratins 8/18 on serine residues. When phorbol acetate was added to labelled cells, a 2.2‐fold increase in CK8/18 phosphate labelling was noted, whereas increasing intracellular cAMP levels using forskolin or 8‐Br‐cAMP showed no significant change in CK phosphorylation. CKs8/18 were also phosphorylated by added PKC in the presence of [γ‐31P]ATP. Tryptic peptide map analysis of the phosphorylated CK8 species showed that treatment of cells with 8‐Br‐cAMP or phorbol acetate generated a phosphopeptide not seen in control cells. In contrast, tryptic peptide maps of phosphorylated CK 18 showed no discernable differences. Our results support a role for PKC in the phosphorylation of epithelial cytokeratins, with some phosphorylation sites being modulated by cAMP dependent protein kinase.