bluuebunny/crossref_metadata_2025 huggingface dataset:
The chloride channel blocker 5‐nitro‐2‐(3‐phenylpropyl‐amino) benzoic acid (NPPB) uncouples mitochondria and increases the proton permeability of the plasma membrane in phagocytic cells
Authors: Lukacs Gergely L. , Nanda Arvind , Rotstein Ori D. , Grinstein Sergio
Published: July 2002

We present evidence that the potent chloride channel blocker NPPB has protonophoric activity in the mitochondria and across the plasma membrane of phagocytic cells. The resting O2 consumption of murine peritoneal macrophages was stimulated up to 2.5‐fold in the presence of NPPB, with a K
0.5 of 15 μM. The stimulatory effect of NPPB also O2 consumption, like that of the classical protonophore CCCP, was prevented by the mitochondrial respiratory chain inhibitors antimycin A, rotenone or cyanide. NPPB also mediated rheogenic proton transport across the plasma membrane of human neutrophils and macrophages in the direction dictated by the electrochemical proton gradient. As a consequence of its protonophoric activity, NPPB uncoupled mitochondrial ATP synthesis, resulting in partial depletion of cellular ATP. These observations indicate that, at the concentrations frequently used for blockade of anion channels, NPPB acts as an effective protonophore, potentially disturbing cytosolic pH and mitochondrial ATP synthesis.
Calcium influx through ‘L’‐type channels into rat anterior pituitary cells can be modulated in two ways by protein kinase C (PKC‐isoform selectivity of 1,2‐dioctanoyl sn‐glycerol?)
Authors: MacEwan David J. , Mitchell Rory
Published: July 2002

The depolarisation‐induced influx of 45Ca2+ into anterior pituitary tissue and GH3 cells through ‘L’‐type, nimodipine‐sensitive channels was investigated. In anterior pituitary prisms, phorbol esters, activators of protein kinase C, caused an enhancement of K+‐induced 45Ca2+ influx. However, in the GH3 anterior pituitary cell line, phorbol esters inhibited K+‐induced 45Ca2+ influx. The modulation by phorbol esters in both tissues was stereo‐specific and time‐ and concentration‐dependent. The diacylglycerol analogue, 1,2‐dioctanoyl sn‐glycerol was able to mimic the phorbol ester‐induced enhancement of calcium influx into anterior pituitary pieces, but was ineffective in GH3 cell. 1,2‐Diactanoyl sn‐glycerol may selectively activate an isoform of protein kinase C which is responsible for enhanced ‘L’‐type Ca2+‐channel activity.
Phosphoramidon inhibits the generation of endothelin‐1 from exogenously applied big endothelin‐1 in cultured vascular endothelial cells and smooth muscle cells
Authors: Ikegawa Ruriko , Matsumura Yasuo , Tsukahara Yaeko , Takaoka Masanori , Morimoto Shiro
Published: July 2002

When cultured porcine aortic endothelial cells (ECs) were incubated with porcine big endothelin‐1 (bit ET‐I1–39), there was a time‐dependent increase in immunoreactive (IR)‐ET in the culture supernatant, in addition to an endogenous IR‐ET release fron the cells. Reverse‐phase HPLC of the culture supernatant revealed one major IR‐ET component corresponding to the elution position of synthetic ET‐1, thereby indicating that the additional increase in IR‐ET was due to the conversion of big ET‐1 to mature ET‐11–21. Phosphoramidon, a metalloproteinase inhibitor, strongly suppressed this increase in IR‐ET as well as the endogenous IR‐ET release. Cultured vascular smooth muscle cells (VSMCs) also released IR‐ET. The apparent conversion of exogenously applied big ET‐1 to ET‐1 and its inhibition by phosphoramidon were observed using cultured VSMCs, although the enzyme inhibitor did not influence the basal secretion of IR‐ET from VSMCs. These results suggest that both cultured ECs and VSMCs can generate ET‐1 from exogenously applied big ET‐1 via action of the same type of phosphoramidon‐sensitive metalloproteinase, which is also involved in the endogenous ET‐1 generation in ECs.
A novel conformationally restricted protein kinase C inhibitor, Ro 31‐8425, inhibits human neutrophil superoxide generation by soluble, particulate and post‐receptor stimuli
Authors: Muid Robert E. , Dale M.Maureen , Davis Peter D. , Elliott Lucy H. , Hill Christopher H. , Kumar Hari , Lawton Geoffrey , Twomey Breda M. , Wadsworth Julie , Wilkinson Sandra E. , Nixon John S.
Published: July 2002

A novel, bis‐indolylmaleimide, Ro 31‐8425, bearing a conformationally restricted side chain, inhibits protein kinase C isolated from rat brain and human neutrophils with a high degree of selectivity over cAMP‐dependent kinase and Ca2−/calmodulin‐dependent kinase. It also inhibits phorbol ester‐induced intracellular events known to be mediated by protein kinase C(p47 phosphorylation in intact platelets, CD3 and CD4 down‐regulation in T‐cells). Ro 31‐8425 inhibited superoxide generation in human neutrophils activated by both receptor stimuli (formyl‐methionyl‐leucylphenylalanine, opsonized zymosan. IgG and heat aggregated IgG) and post‐rcceptor stimuli (1,2‐dioctanoylglycerol and fluoride). The compound also blocked antigen driven, but not IL‐2 induced, T‐cell proliferation. These results support a central role for protein kinase C in the activation of the respiratory burst and antigen‐driven T‐cell proliferation.
Peptide competition of actin activation of myosin‐subfragment 1 ATPase by an amino terminal actin fragment
Authors: Kögler Harald , Moir Arthur J.G. , Trayer Ian P. , Rüegg J.Caspar
Published: July 2002

The amino‐terminal region of actin participates in the binding of myosin subfragment 1 (S1) during cross‐bridge cycling, thereby assisting in the activation of the magnesium‐dependent myosin ATPase. Effects of three actin fragments on the magnesium‐dependent S1 and acto‐S1 ATPase activities in solution were studied. One of the peptides, containing residues actin 1–44, mimicked the S1 ATPase‐activating properties of actin and in turn inhibited acto‐S1 ATPase both in a concentration‐dependent manner. This suggests peptide competition for the actin binding site on myosin. The other fragments, residues actin 1–18 and 82–119, respectively, had no detectable effect on S1‐ and acto‐S1 ATPase activity.
Catalytic fragment of protein kinase C exhibits altered substrate specificity toward smooth muscle myosin light chain
Authors: Nakabayashi Hiroki , Sellers James R. , Huang Kuo-Ping
Published: July 2002

Smooth muscle myosin light chain (LC) can be phosphorylated by myosin light chain kinase (MLCK) at Ser19 and Thr18 and by protein kinase C (PKC) at Thr9 and Ser1 or Ser2 under the in vitro assay conditions. Conversion of PKC to the spontaneously active protein kinase M (PKM) by proteolysis resulted in a change in the substrate specificity of the kinase. PKM phosphorylated both sets of sites in LC recognized by MLCK and PKC as analyzed by peptide mapping analysis. The PKM‐catalyzed phosphorylation of these sites was not greatly affected by a MLCK inhibitor, ML‐9, nor by the activators of MLCK, Ca2+ and calmodulin.
Molecular characterisation, expression and localisation of human neurokinin‐3 receptor
Authors: Buell G. , Schulz M.F. , Arkinstall S.J. , Maury K. , Missotten M. , Adami N. , Talabot F. , Kawashima E.
Published: July 2002

The complete amino acid sequence of the human neurokinin‐3 receptor was deduced by DNA sequence analysis of human genomic fragments. Comparison of the predicted primary structure with those for the human neurokinin receptors 1 and 2 shows a highly conserved pattern of seven hydrophobic regions with maximum divergence occuring at the amino‐ and carboxy‐termini. The position of intron‐exon junctions are identical to those in other reported neurokinin genes. Using a chimeric genomic‐cDNA gene, the human NK‐3 receptor was expressed in Xenopus laevis oocytes where it mediates membrane conductance changes in response to its agonist, neurokinin B. More significantly, expression of the gene in mammalian cells resulted in detection of receptor binding as well as neurokinin‐stimulated calcium mobilization and arachidonic acid release, all displaying the pharmacological characteristics expected of a neurokinin‐3 receptor, By using the polymerase chain reaction we have shown that mRNA for the human neurokinin‐3 receptor is expressed predominantly in the central nervous system.
Activation of Na+/Mg2+ antiport in thymocytes by cAMP
Authors: Günther Theodor , Vormann Jürgen
Published: July 2002

Mg2+ efflux from Mg2+‐loaded rat thymocytes was stimulated by 0.1 mM dibutyryl cAMP (db cAMP). The activation of Mg2+ efflux by db cAMP was more expressed at lower Mg2+‐loading, cAMP induced only a very small increase in the concentration of intracellular free Mg2+ which cannot explain the activation of Na+/Mg2+ antiport. From these results it was concluded that cAMP increases the affinity of the Na+/Mg2+ antiporter for intracellular Mg2+, probably by phosphorylation.
Phosphorylation sequences in h‐caldesmon from phorbol ester‐stimulated canine aortas
Authors: Adam Leonard P. , Gapinski Connie J. , Hathaway David R.
Published: July 2002

The high molecular weight form of caldesmon (h‐caldesmon) is phosphorylated in vascular smooth muscle. The stoichiometry of caldesmon phosphorylation increases in response to stimulation of the muscle by several contractile agonists; however, the responsible kinase has not been identified. In this study, we have sequenced the phosphopeptides prepared from h‐caldesmon phosphorylated in vitro by protein kinase C (PKC) as well as the phosphopeptides prepared from caldesmon phosphorylated in intact canine aortas that were stimulated to contract with PDBu. PKC phosphorylated three sites located in the C terminus: GSS*LKIEE, AEFLNKS*VQK and NLWEKQS*VDK, while h‐caldesmon from intact tissue was phosphorylated at two separate sites also in the C terminus: VTS*PTKV and S*PAPK. By comparison to known substrate consensus sequences for various protein kinases these data suggest that h‐caldesmon is directly phosphorylated by a proline‐directed protein kinase and not by PKC.
Brefeldin A and the endocytic pathway Possible implications for membrane traffic and sorting
Authors: Hunziker Walter , Whitney J.Andrew , Mellman Ira
Published: July 2002

A number of recent observations have suggested that the endocytic and biosynthetic pathways may share fundamentally similar transport mechanisms at the molecular level. Some of the more striking of these suggestions have come from a comparison of the effects of the macrocyclic lactone brefeldin A (BFA) on endosomes and the Golgi complex. BFA is thought to affect Golgi‐specific coat proteins that may be involved in maintaining the structural integrity of the organelle and in regulating membrane transport in the secretory pathway. Many of the effects of BFA on the endocytic system, such as the guanine nucleotide and aluminum fluoride (AlF−
4)‐regulated induction of microtubule‐dependent endosomal tubules, are strikingly reminiscent of the action of the drug on the Golgi complex. Therefore, the similar mechanisms of action of the drug on endosomes suggest that organelles of the endocytic pathway may be associated with similar cytoplasmic coats that could regulate endosome function and integrity.
The angiotensin AT2 receptor modulates T‐type calcium current in non‐differentiated NG108‐15 cells
Authors: Buisson Bruno , Bottari Serge P. , de Gasparo Marc , Gallo-Payet Nicole , Payet Marcel D.
Published: July 2002

We report here that angiotensin II (AII) and the AT2 receptor‐selective ligand, CGP 42112, modulate the T‐type calcium current in non‐differentiated NG108‐15 cells, which express only AT2 receptors. Both peptides decrease the T‐type calcium current at membrane potentials above −40 mV and shift the current—voltage curve at lower potentials with maximal effect between 5 and 10 min after application. These data describe a new cellular response to AII and suggest that the AT2 receptor mediates certain neurophysiological actions of this hormone.
Time‐dependoent increases in Na+,K+‐ATPase content of low‐frequency‐stimulated rabbit muscle
Authors: Green H.J. , Ball-Burnett M. , Chin E.R. , Dux L. , Pette D.
Published: July 2002

Chronic low‐frequency stimulation of rabbit fast‐twitch muscle induced time‐dependent increases in the concentration of the sarcoleminal Na+,K+‐ATPase and in mitochondrial citrate synthase activity. The almost twofold increase in Na+,K+‐ATPase preceded the rise in citrate synthase and was complete after 10 days of stimulation, We suggest that the increase in Na+,K+‐ATPase enhances resistance to fatigue of low‐frequency‐stimulated muscle prior to elevations in aerobic‐oxidative capacity.
Amino acids 356–372 constitute a Gi‐activator sequence of the α2‐adrenergic receptor and have a Phe substitute in the G protein‐activator sequence motif
Authors: Ikezu Tsuneya , Okamoto Takashi , Ogata Etsuro , Nishimoto Ikuo
Published: July 2002

The human α2‐adrenergic receptor contains the sequence KASRWRGRQNREKRFTF (amino acids 356–372) at the C‐terminal end of its third intracellular loop. This sequence satisfies the structural criteria for G protein‐activating sequences [(1992) J. Biol. Chem. 267, 8342–8346] except that the C‐terminal sequence is B‐B‐X‐X‐Phe instead of B‐B‐X‐B or B‐B‐X‐X‐B (B: basic residue, X: non‐basic residue). Nevertheless, the synthetic peptide corresponding to this sequence (peptide α2‐F) was found to activate Gi and Go strongly with a saturated effect at 1–3 μM. Furthermore, the substitution of the C‐terminal Phe of peptide α2‐F with Arg, Trp, and Tyr (but not Ala or Asp) did not appreciably affect the Gi‐activating potency. It is suggested that the C‐terminal basic residue of the B‐B‐X‐X‐B motif in Gi‐activating sequences can be replaced by an aromatic residue.
Transforming growth factor‐β1 rapidly activates phosphorylase in a calcium‐dependent manner in rat hepatocytes
Authors: Hahm Sung Ho , Cooper Ronald H.
Published: July 2002

Transforming growth factor‐β1 (TGF‐β1) rapidly activated phosphorylase in isolated rat hepatocytes (half‐maximal rate of activation with approximately 0.1 ng/ml). Removal of Ca2+ from the external medium just before TGF‐β1 addition markedly attenuated phosphorylase activation. TGF‐β1 (1 ng/ml) produced a small increase in [Ca2+]i (approximately 10% increase after 30 s), which appears sufficient to account for phosphorylase activation. These observations indicate that activation of the TGF‐β1 signal transduction system in hepatocytes is linked with a small increase in [Ca2+]i, and external Ca2+ may contribute in part to this increase.
Na+‐dependent AIB transport by neuroblastoma cells
Authors: del Castillo-Olivares Antonio , Medina Miguel Angel
Published: July 2002

Na+‐dependent amino isobutyric acid transport by two neuroblastoma cell lines with and without amplification of the oncogene N‐myc is studied. Surprisingly, the contribution of system A is greater in the cell line showing no N‐myc amplification. Preliminary data support a role for essential tyrosine and cysteine residues in the active center of the carriers, mainly in system A.
Diabetes mellitus is one of the heterogeneous phenotypic features of a mitochondrial DNA point mutation within the tRNALeu(UUR) gene
Authors: Gerbitz K.-D. , Paprotta A. , Jaksch M. , Zierz S. , Drechsel J.
Published: July 2002

A heteroplasmic point mutation (transition A‐to‐G at nucleotide position 3,243 in the mitochondrial tRNALeu(UUR) gene) is found in a family suffering from a syndrome with diabetes, deafness and cardiomyopathy as the predominant clinical features.
PCR‐based assignment of two myosin heavy chain cDNA clones to biochemically and histochemically defined single type IIB and IID fibers of rabbit muscle
Authors: Uber Andreas , Pette Dirk
Published: July 2002

The present study assigns two as yet unidentified fast myosin cDNA clones to specific myosin heavy chain (MHC) isoforms and their mRNAs in different fiber types of rabbit skeletal muscle. Specific oligonucleotide primers were used for reverse transcription and polymerase chain reaction (PCR) to yield products of defined lengths. The method was sensitive enough to detect specific mRNA sequences in total RNA extracts from microdissected, freeze‐dried, single‐fiber fragments down to 16 ng dry weight. The fibers were typed histochemically and biochemically by their electrophoretically assessed MHC complement. The following results were obtained: clone pMHC20‐40 was assigned to type IIB fibers and clone pMHC24‐79 to type IID fibers.
Characterization of cAMP dependent CFTR‐chloride channels in human trache gland cells
Authors: Becq F. , Merten M.D. , Voelckel M.A. , Gola M. , Figarella C.
Published: July 2002

Human tracheal gland cells are believed to be a major site at the origin of cystic fibrosis. Since this disease is due to mutations in a protein called CFTR, we looked for the activity of CFTR in human tracheal gland cells in culture. We have identified CFTR‐like chloride‐selective channels as having a linear current‐voltage relationship and unitary conductance of 7 pS in these cells. In cell‐attached patches, theophylline (1 mM), IBMX (1 mM), or a cocktail of dibutyryl cAMP (1 mM) and IBMX (0.1 mM) promoted the opening of channels. The unitary current had a reversal potential close to the cell resting potential. Replacement of choline by K+ or Na+ in the pipette solution was without effect on the current‐voltage relationship, the reversal potential or the unitary conductance, which is consistent with the chloride selectivity of the channel. Channels were always found clustered and their opening probability was not noticeably dependent on membrane potential. This work therefore represents the first observation of a CFTR‐like channel activity in submucosal gland cells.
Fragmentation of DNA in P388D1 macrophages exposed to oxidised low‐density lipoprotein
Authors: Reid Vanessa C. , Hardwick Simon J. , Mitchinson Malcolm J.
Published: July 2002

Exposing a macrophage‐like murine cell line to copper‐oxidised low‐density lipoprotein led to DNA fragmentation which was inhibited by the putative Ca2+/Mg2+ endonuclease inhibitor, zinc sulphate. DNA fragmentation preceded loss of membrane impermeability. These results suggest that apoptosis may be a mechanism of macrophage foam cell death in atherosclerotic lesions in the arterial wall.
Raf‐1 is not a major upstream regulator of MAP kinases in rat fibroblasts
Authors: Kizaka-Kondoh Shinae , Okayama Hiroto
Published: July 2002

RCR cells are NRK clones in which Raf‐1 production is blocked by the expression of an antisense RNA, and consequently they are refractory to transformation by various oncogenes. In RCR cells, MAP kinases (ERK1 and ERK2) were activated to an extent and in a time course similar to those of the original NRK cells, irrespective of whether the stimulus was oncogenic or non‐oncogenic. Moreover, there was no significant elevation of ERK activities in oncogene‐transfonned NRK cells. These results indicate that Raf‐1 kinase is not the major upstream activator of ERK's in NRK cells and that neither ERK1 nor ERK2 are likely to mediate oncogenic signals from Raf‐1 kinase.
Molecular cloning, functional expression and localization of an inward rectifier potassium channel in the mouse brain
Authors: Morishige Ken-Ichirou , Takahashi Naohiko , Findlay Ian , Koyama Hidekazu , Zanelli Jill S. , Peterson Christine , Jenkins Nancy A. , Copeland Neal G. , Mori Nozomu , Kurach Yoshihisa
Published: July 2002

We have cloned an inward‐rectifier potassium channel from a mouse brain cDNA library, studied its distribution in the brain by in situ hybridization and determined the chromosomal localization of the gene. A mouse brain cDNA library was screened using a fragment of the mouse macrophage IRK1 cDNA as a probe. Two duplicate clones of ~5.5 kb were obtained. Xenopus ococytes injected with cRNA derived from the clone expressed a potassium channel with inwardly rectifying channel characteristics. The amino acid sequence of the clone was identical to that of IRK1 recently cloned from a mouse macrophage cell line. In situ hybridization study showed the mouse brain IRK1 to be generally distributed throughout the brain, but in particular subsets of neurons at high levels. The gene was placed in the distal region of mouse chromosome 11, which contains several uncloned neurological mutations. These results provide the first demonstration of the cloning and distribution of an inward rectifier potassium channel from the nervous system.
Stimulation‐induced expression of slow muscle myosin in a fast muscle of the rat
Authors: Mayne Caroline N. , Mokrusch Thomas , Jarvis Jonathan C. , Gilroy Stephen J. , Salmons Stanley
Published: July 2002

Fast muscles of the rat hind limb were stimulated continuously at 10 or 20 Hz for periods of 55–61 days by means of an implantable neuromuscular stimulator. Gel electrophoresis clearly demonstrated the presence in stimulated muscles of slow myosin light and heavy chains, although fast isoforms were still present in all cases. Thus, contrary to previous reports, induction of slow myosin isoforms does occur in this, as in other, mammalian species. The time course of the response to stimulation appears to be more extended than that seen in the rabbit.
Rapid uptake of calcium, ATP, and inositol 1,4,5‐trisphosphate via cation and anion channels into surface‐derived vesicles from HIT cells containing the inositol 1,4,5‐trisphosphate‐sensitive calcium store
Authors: Lange Klaus , Brandt Ursula
Published: July 2002

In a previous study [K. Lange and U. Brandt (1993) FEBS Lett. 320, 183‐188], we showed that the bulk of the ATP‐dependent IP3‐sensitive Ca2+ store of the hamster insulinoma cell line, HIT‐T15, resides in cell surface‐derived vesicles most likely of microvillar origin. The origin and orientation of these vesicles suggested that Ca2+ storage is not due to a membrane‐located Ca2+ pumping ATPase but rather to ATP‐dependent Ca2+‐binding within the vesicles. In this case, Ca2+, ATP and IP3 should have free access to the vesicle lumen. This hypothesis was tested. ATP‐independent Ca2+ uptake occurred with biphasic kinetics. An initial rapid uptake, which was complete within 30 s, was followed by a slow linear uptake lasting about 10 min. The rapid component was shown by efflux experiments to have an equilibration half‐time of about 4 s. This rapid Ca2+ efflux pathway was inhibited by externally applied La3+ (0.1 mM). A similar rapidly equilibrating La3+‐sensitive Ca2+ pool was also present in vesicles which had been actively loaded with Ca2+ in the presence of ATP. The intravesicular distribution space of this labile Ca2+ pool was identical with that of the non‐metabolizable hexose analogue 3‐O‐methyl‐D‐glucose, demonstrating that rapid Ca2+ uptake occurs into a true vesicular water space and is not due to binding. ATP and IP3 were also shown to enter the vesicles by an energy‐independent pathway which is inhibited by the anion channel inhibitor, 4,4'‐diisothiocyanostilbene‐2,2'‐disulfonic acid (DIDS; 0.5 mM). Both ATP‐dependent Ca2+ uptake and IP3‐induced Ca2+ release from preloaded vesicles were inhibited by DIDS. These findings clearly demonstrate that (1) the vesicle membrane is permeable to ATP and IP3 via anion channels, and (2) Ca2+ uptake into as well as IP3‐induced Ca2+ release from the vesicles occur by passive diffusion through a cation channel which is not regulated by IP3. Consequently, the mechanisms for Ca2+ storage and IP3‐induced Ca2+ release must be located in the vesicle lumen. Moreover, the microvillar diffusion‐barrier concept, originally proposed for the regulation of hexose transport may also be valid for the receptor‐operated regulation of cation and anion influx pathways. Functional coupling of the microvillar Ca2+ stores with the associated cation influx pathway is also strongly supported by the previously demonstrated microvillar shape changes accompanying depletion of the Ca2+ stores by bombesin or thapsigargin in HIT cells [K. Lange and U. Brandt (1992) FEBS Lett. 320, 183‐188].
Staurosporine stimulates phospholipase D activation in human polymorphonuclear leukocytes
Authors: Périanin Axel , Combadière Christophe , Pedruzzi Eric , Djerdjouri Bahia , Hakim Jacques
Published: July 2002

Treatment of 1‐O‐[3H]alkyl‐2‐acyl‐phosphatidylcholine‐prelabeled human polymorphonuclear leukocytes (PMNs) with Staurosporine (50 nM to 1 μM) induced a time‐ and concentration‐dependent generation of tritiated phosphatidic acid (PA), reaching approximately 225% of the control value at 15–20 min. In the presence of ethanol, Staurosporine induced a production of phosphatidylethanol (PEt) reaching, 250% of control values, and partial inhibition of PA production, consistent with PLD activation. The amount of ether‐linked acylglycerol (EAG) was weakly enhanced (29%) after 5 min of PMN treatment; longer treatment resulted in no significant EAG production, suggesting a possible late inhibition of PA hydrolase activity. Staurosporine concentrations that induced an elevation in PA completely depressed protein kinase C (PKC) activity in both soluble and particulate cell fractions, suggesting that PLD activation may occur independently from PKC activation. PLD may thus represent a potential cellular target for Staurosporine action.
Expression of a cDNA encoding the glycolipid‐anchored form of rat acetylcholinesterase
Authors: Legay Claire , Bon Suzanne , Massoulié Jean
Published: July 2002

We amplified by PCR and characterized a fragment of cDNA from rat spleen, encoding the distinctive C‐terminal region of the acetylcholinesterase (AChE) H subunit. A recombinant vector encoding this subunit was constructed and expressed in COS cells: the H subunits produced glycophosphatidylinositol (GPI)‐anchored dimers, showing that the spleen cDNA fragment contained a functional GPI cleavage/attachment site. Using PCR, we did not detect mRNAs encoding AChE H in rat muscle or hypothalamus. In the liver of 16‐day rat embryos, we found both H and T transcripts, in agreement with the presence of both GPI‐anchored dimers and amphiphilic monomers of type II. In addition, we detected ‘read‐through’ (R) transcripts, in which regular introns are spliced, but the intervening sequence between the common exon 4 and the alternative exon 5 (H) is maintained.
Characterization of β subunit modulation of a rabbit cardiac L‐type Ca2+ channel α1 subunit as expressed in mouse L cells
Authors: Lory Philippe , Varadi Gyula , Slish Donald F. , Varadi Marie , Schwartz Arnold
Published: July 2002

Functional properties of a rabbit cardiac α1 Ca2+ channel subunit (CARDα1) were investigated using the patch‐clamp technique in mouse L cells, a recipient cell line which is devoid of any Ca2+ channel subunits. Cell lines resulting from stable transfection of the CARDα1 subunit as well as in coexpression with a β subunit (cardα1β) derived from skeletal muscle (SKMβ) were characterized. The results show that while the CARDα1‐Ca2+ channel activity is negligible, the Ba2+ current density is dramatically increased in the presence of β subunit (2̃0‐fold). CARDα1‐ and CARDα1β‐Ba2+ currents were both sensitive to the 1,4‐dihydropyridine (DHP) agonist, Bay K 8644 (5‐ to 8‐fold increase). Activation kinetics of CARDα1‐ and CARDα1β‐Ba2+ currents were comparable. The inactivation time‐course was faster (3‐ to 4‐fold) for CARDα1β)‐Ba2+ currents. We conclude that the main role of the β subunit in heart is to modulate the L‐type current density and present several lines of evidence that SKMα1 and CARDα1 are differentially regulated by the β subunit.
GTPγS and phorbol ester act synergistically to stimulate both Ca2+‐independent secretion and phospholipase D activity in permeabilized human platelets
Authors: Coorssen Jens R. , Haslam Richard J.
Published: July 2002

We have tested the hypothesis that phospholipase D (PLD) is the effector of the unidentified G protein (GE) mediating Ca2+‐independent exocytosis in platelets. Although GTPγS, and to a lesser extent phorbol 12‐myristate 13‐acetate (PMA), caused some secretion of 5‐HT from electropermeabilized human platelets in the effective absence of Ca2+ (pCa > 9), these stimuli had much more potent synergistic effects when added together. In all cases, secretion of 5‐HT was closely correlated to the stimulus‐induced formation of [3H]phosphatidic acid ([3H]PA) from [3H]arachidonatelabelled phospholipids. Addition of ethanol inhibited both secretion and [3H]PA formation and led to the accumulation of [3H]phosphatidylethanol ([3H]PEt), indicating that [3H]PA was formed largely by activation of PLD. BAPTA and analogues caused dose‐dependent inhibitions of both GTPγS‐induced secretion and PLD activity in the permeabilized platelets. This action of BAPTA did not appear to be mediated by chelation of Ca2+ or by direct inhibition of protein kinase C (PKC). The results suggest that PLD is the target of GE in platelets and that BAPTA can block PLD activation.
The sarco(endo)plasmic reticulum Ca2+‐ATPase mRNA isoform, SERCA 3, is expressed in endothelial and epithelial cells in various organs
Authors: Anger Marielle , Samuel Jane-Lyse , Marotte Françoise , Wuytack Frank , Rappaport Lydie , Lompré Anne-Marie
Published: July 2002

The sarco(endo)plasmic reticulum Ca2+‐ATPase mRNA isoform, SERCA 3, was previously shown to be expressed in a great variety of muscle and non‐muscle tissues [(1989) J. Biol. Chem. 264, 18561–18568] but its cellular localization within these organs was unknown. We have used in situ hybridization and RNase protection techniques to demonstrate that SERCA 3 mRNA is expressed in specific cell types, namely the endothelial and epithelial cells.
Serum immunoglobulins interact with oligonucleotides
Authors: Rykova E.Yu. , Pautova L.V. , Yakubov L.A. , Karamyshev V.N. , Vlassov V.V.
Published: July 2002

The interaction of reactive derivatives of oligonucleotides bearing a 4‐[(N‐2‐chloroethyl‐N‐methyl)amino]benzylamin residue at the 5′‐terminal phosphate with serum blood proteins has been investigated. It was found that the compounds react with serum albumin and immunoglobulins M and G, the reactivity increasing in the order: albumin < IgG < IgM. The reactions with immunoglobulins were inhibited in the presence of different oligonucleotides, DNA and heparin, suggesting the oligonucleotide binding to some cationic region of the proteins. Myoglobin inhibited the interaction of oligonucleotide derivatives with myoglobin‐specific monoclonal antibodies which indicates that the derivatives interact with the proteins within or near the antigen binding site.
The disulfide bridges of toxin 2 from the scorpion Centruroides noxius Hoffmann and its three‐dimensional structure calculated using the coordinates of variant 3 from Centruroides sculpturatus
Authors: Gurrola G.B. , Moreno-Hagelsieb G. , Zamudio F.Z. , García M. , Soberon X. , Possani L.D.
Published: July 2002

The disulfide bridges of toxin 2 from the venom of the scorpion Centruroides noxius Hoffmann were found by amino acid sequence determination of fragments of native toxin, produced by enzymatic cleavage and separated by high‐performance liquid chromatography (HPLC). They are: Cys12‐Cys65, Cys16‐Cys41, Cys25‐Cys46 and Cys29‐Cys48. The coordinates of the X‐ray diffraction structure of toxin variant 3 of C. sculpturatus [(1980) Proc. Natl. Acad. Sci. USA 77, 6496–6500] were used to construct a three dimensional model of toxin 2. All the amino acid replacements were easily accommodated, and the modeled structure reveals a clustered pattern of sequence variation, which may help to identify residues responsible for functional differences among toxins of mammals and insects.
Effects of cobalt on haem proteins of erythropoietin‐producing HepG2 cells in multicellular spheroid culture
Authors: Görlach A. , Fandrey J. , Holtermann G. , Acker H.
Published: July 2002

The hypoxia‐induced increase of spectrophotometrically measured light absorption at 560 nm, considered as reduced cytochrome b, in HepG2 cells is diminished after exposure to cobalt chloride (50 or 100 μM) for 18–36h. The redox state of cytochrome c and cytochrome aa
3, however, remains stable, indicating a particular affinity of cytochrome b for cobalt. Erythropoietin production of HepG2 cells increases after application of cobalt chloride, whereas H2O2 production, as measured by the dihydrorhodamine technique, decreases. It is concluded that cobalt stimulates a signal cascade with cytochrome b as receptor and H2O2 as second messenger for regulating erythropoietin production.
Conditioned media of glial cell lines induce alkaline phosphatase activity in cultured artery endothelial cells Identification of interleukin‐6 as an induction factor
Authors: Takemoto Hiroshi , Kaneda Kazue , Hosokawa Mamiko , Ide Misao , Fukushima Hisashi
Published: July 2002

Conditioned media of human glial cell lines induced alkaline phosphatase activity in cultured calf artery endothelial cells. The maximal alkaline phosphatase activity in the culture was comparable to the level in isolated brain capillary endothelial cells. An induction factor in the conditioned media was purified and identified as interleukin‐6 from its amino‐terminal sequence, molecular weight, amino acid composition and immunoreactivity. Recombinant interleukin‐6 had similar induction activity. Our findings raise the possibility that interleukin‐6 induces and modulates alkaline phosphatase activity in endothelial cells during normal development of the blood—brain barrier and under certain pathological conditions.
Interleukin‐1 stimulates de novo synthesis of mitogen‐activated protein kinase in glomerular mesangial cells
Authors: Huwiler Andrea , Pfeilschifter Josef
Published: July 2002

Interleukin‐1 (IL‐1) stimulates a lime‐ and concentration‐dependent mitogen‐activated protein (MAP) kinase activation in rat mesangial cells. A rapid increase in activity (maximal at 10 min) is followed by a second persistent level of activity which steadily increases over 24 h. The second peak of MAP kinase activity is paralleled by a marked de novo synthesis of p42 MAP kinase as measured by immunoprecipitation of [35S]methionine‐labelled mesangial cells and by a 60% increase in total p42 MAP kinase protein as detected by Western blot analysis. We propose that IL‐1 induced de novo synthesis of p42 MAP kinase is important for the multiplicity of long‐term actions of this cytokine in renal mesangial cells.
The disulfide bond in chromogranin B, which is essential for its sorting to secretory granules, is not required for its aggregation in the trans‐Golgi network
Authors: Chanat Eric , Weiß Ursula , Huttner Wieland B.
Published: July 2002

Chromogranin B (secretogranin I), a protein sorted to secretory granules in many endocrine cells and neurons, undergoes selective aggregation during the sorting process in the trans‐Golgi network. Reduction of the single, highly conserved intramolecular disulfide bond of chromogranin B by exposure of intact PC12 cells to the thiol reducing agent dithiothreitol has previously been shown to cause its missorting to the constitutive pathway of secretion. Using saponin perforation of membrane vesicles in aggregative buffer mimicking the milieu in the lumen of the trans‐Golgi network (pH 6.4, 10 mM calcium), we show here that treatment with dithiothreitol does not prevent the aggregation of chromogranin B in this compartment. This implies that the loop in the chromogranin B polypeptide that is formed by the disulfide bond has a critical role in the membrane recognition of aggregated chromogranin B during secretory granule formation.
Functional and structural similarity between the X protein of hepatitis B virus and nucleoside diphosphate kinases
Authors: De-Medina Tali , Shaul Yosef
Published: July 2002

One of the four genes encoded by hepatitis B virus (HBV) is the regulatory 17 kDa protein called HBx (or pX). HBx is a transcription transactivator of many cellular and viral regulatory elements. We report here that recombinant HBx supports transcription in vitro and has phosphotransfer enzymatic activity. In the presence of EDTA, a phosphoryl‐HBx is formed that releases the phosphate residue upon the addition of Mg2+. This two‐step NTP hydrolysis reaction is characteristic of a group of enzymes termed nucleoside diphosphate kinases (NDPKs). Remarkably, structural similarity between HBx and NDPKs is also evident. Our findings suggest that HBx has evolved from this group of enzymes but acquired additional activities that satisfy the viral needs.
Iron released from transferrin at acidic pH can catalyse the oxidation of low density lipoprotein
Authors: Lamb David J. , Leake David S.
Published: July 2002

Low density lipoprotein (LDL) oxidation within the arterial wall may contribute to the disease of atherosclerosis. We have investigated the conditions under which transferrin (the major iron‐carrying protein in plasma) may release iron ions to catalyse the oxidation of LDL. Transferrin that had been incubated at pH 5.5 released approximately 10% of its bound iron in 24 h, as measured by ultrafiltration and atomic absorption spectroscopy. Furthermore, transferrin co‐incubated with LDL and l‐cysteine at pH 5.5 resulted in the oxidation of the LDL as measured by thiobarbituric acid‐reactive substances and electrophoretic mobility. This effect was observed at transferrin concentrations as low as 40% of its average plasma concentration. The release of iron from transferrin in atherosclerotic lesions due to a localised acidic pH may help to explain why LDL oxidation occurs in these lesions.
Phosphorylation of JAK2 in thrombin‐stimulated human platelets
Authors: Rodríguez-Liñares Belén , Watson Steve P.
Published: July 2002

We show the presence of the tyrosine kinase JAK2 in human platelets and demonstrate that it undergoes phosphorylation on tyrosine residues on challenge with the G protein receptor stimulus, thrombin, or the tyrosine phosphatase inhibitor, peroxovanadate. Thrombin‐induced phosphorylation of JAK2 is inhibited by two structurally distinct inhibitors of tyrosine kinases, staurosporine and the tyrphostin ST271. The protein kinase C (PKC) inhibitor, Ro 31‐8220, and intracellular Ca2+ chelator, BAPTA‐AM, also inhibit thrombin‐induced phosphorylation of JAK2, while the phorbol ester, phorbol dibutyrate (PDBu), and Ca2+ ionophore, A23187, induce tyrosine phosphorylation of JAK2. These results suggest that tyrosine phosphorylation of JAK2 stimulated by thrombin may be mediated downstream of phosphoinositide metabolism.
Nucleotide receptors in hepatic stellate cells of the rat
Authors: Takemura Shigekazu , Kawada Norifumi , Hirohashi Kazuhiro , Kinoshita Hiroaki , Inoue Masayasu
Published: July 2002

When hepatic stellate cells were stimulated by UTP, ATP, or ADP, cellular levels of inositol phosphates significantly increased (UTP > ATP > ADP > 5′‐O‐(3‐thiotriphosphate). Thirty min after incubation with 100 μM of UTP, ATP, or ADP, levels of inositol monophosphate increased to 1318 ± 116, 616 ± 87 and 591 ± 234% of control levels, respectively, with concomitant increase in the production of inositol trisphosphate and bisphosphate. These nucleotides transiently increased the [Ca2+]i of fura‐2‐loaded stellate cells. Moreover, UTP, ATP, ADP and adenosine 5′‐O‐(3‐thiotriphosphate) were able to induce contraction of stellate cells as detected using the silicone‐rubber membrane method. These results suggested that hepatic stellate cells have nucleotide receptors which react predominantly with extracellular UTP and ATP and trigger the receptormediated contraction of the cells.
Effect of adrenomedullin on hepatic pericytes (stellate cells) of the rat
Authors: Kawada Norifumi , Inoue Masayasu
Published: July 2002

When hepatic stellate cells (Ito cells, fat‐storing cells) were incubated with adrenomedullin, they underwent relaxation as monitored by the silicone‐rubber membrane method; 43%, 65% and 87% of stellate cells relaxed 5, 10 and 20 min, respectively, after addition of 10−6 M adrenomedullin. Adrenomedullin also triggered the dissociation of F‐actin and induced transformation of stellate cells to dendritic cell‐like structure. When incubated with 10−6 M of adrenomedullin for 30 min, cellular levels of cAMP increased from the basal value of 10.2 ± 1.4 to 107 ± 2.8 pmol/2 × 105 cells without affecting cGMP levels. The reaction occurred dose‐dependently and was inhibited by an antagonist of calcitonin gene‐related peptide. Adrenomedullin had negligible effects on DNA and protein synthesis in proliferating stellate cells. Thus, adrenomedullin is a potent relaxing peptide to hepatic stellate cells and may contribute to the regulation of sinusoidal microcirculation.
Purification and crystallization of the ternary complex of elongation factor Tu:GTP and Phe‐tRNAPhe
Authors: Nissen Poul , Reshetnikova Ludmila , Siboska Gunhild , Polekhina Galina , Thirup Søren , Kjeldgaard Morten , Clark Brian F.C. , Nyborg Jens
Published: July 2002

Elongation factor Tu (EF‐Tu) is the most abundant protein in prokaryotic cells. Its general function in protein biosynthesis is well established. It is a member of the large family of G‐proteins, all of which bind guanosine phosphates (GDP or GTP) as cofactors. In its active GTP bound state EF‐TU binds aminoacylated tRNA (aa‐tRNA) forming the ternary complex EF‐TU: GTP: aa‐tRNA. The ternary complex interacts with the ribosome where the anticodon on tRNA recognises a codon on mRNA, GTPase activity is induced and inactive EF‐TU: GDP is released. Here we report the successful crystallization of a ternary complex of Thermus aquaticus EF‐TU: GDPNP and yeast Phe‐tRNAphe after its purification by HPLC.
Okadaic acid stimulates ouabain‐sensitive 86Rb+‐uptake and phosphorylation of the Na+/K+‐ATPase α‐subunit in rat hepatocytes
Authors: Lynch Christopher J. , Mader Annie Cielo L. , McCall Kenneth M. , Ng Yuk-Chow , Hazen Stacy A.
Published: July 2002

Ca2+‐mobilizing and cAMP‐dependent hormones rapidly increase sodium, potassium‐dependent adenosine triphosphatase (Na+/K+‐ATPase)‐mediated transport in rat hepatocytes. To explore the possible role of protein phosphatases in these responses we used a protein phosphatase inhibitor, okadaic acid. Okadaic acid stimulation of ouabain‐sensitive 86Rb+‐uptake was maximal between two and three minutes and displayed an EC50 of 41 ± 1 nM. Inhibition of Na+/H+ exchange with an amiloride analog abolished the response to insulin, but had no effect on okadaic acid‐mediated stimulation of Na+/K+‐ATPase transport. In hepatocytes metabolically‐radiolabeled with 32Pi, okadaic acid stimulated the incorporation of radioactivity into several 95 kDa peptides, one of which reacted with anti‐LEAVE peptide antisera, that recognizes Na+/K+‐ATPase α‐subunits. In other experiments Na+/K+‐ATPase was immunoprecipitated from detergent‐solubilized membrane fractions of metabolically‐radiolabeled cells with an antisera to purified rat kidney Na+/K+‐ATPase. A 95 kDa phosphoprotein was immunoprecipitated using anti‐Na+/K+‐ATPase antisera, but not by preimmune serum. Okadaic acid stimulated incorporation of radioactivity into this band by 220 ± 28%. These findings provide support for the hypothesis that rapid stimulation of hepatic Na+/K+‐ATPase by hormones may be related to protein kinase/phosphatase‐mediated changes in the phosphorylation state of the Na+/K+‐ATPase α‐subunit.
Cloning and functional expression of a cardiac inward rectifier K+ channel
Authors: Ishii Kuniaki , Yamagishi Toshio , Taira Norio
Published: July 2002

We have isolated a cDNA coding for an inward rectifier K+ channel (RBHIK1) from rabbit heart. The cloned cDNA encodes a protein of 427 amino acids with two putative transmembrane segments. The primary structure of RBHIK1 is highly homologous to that of IRK1 which is an inward rectifier K+ channel recently cloned from mouse macrophage by expression cloning. When expressed in Xenopus oocytes, RBHIK1 current showed strong inward rectification and was inhibited by extracellular Ba2+ and Cs+. RNA blot analysis revealed the expression of RBHIKl mRNA in various rabbit tissues, especially high level in the ventricular muscle.
Glycogen synthase kinase 3 phosphorylates recombinant human tau protein at serine‐262 in the presence of heparin (or tubulin)
Authors: Moreno Francisco J. , Medina Miguel , Pérez Mar , Montejo de Garcini Esteban , Avila Jesús
Published: July 2002

Tau protein, the major component of the aberrant structures termed paired helical filaments (PHFs) present in the brain of Alzheimer's disease patients, is pathologically phosphorylated in sites in and around the tubulin‐binding sites. A single protein kinase, glycogen synthase kinase 3 (GSK 3), is able to phosphorylate tau at the flanking regions and, additionally, at the tubulin‐binding motifs if heparin or tubulin is present. Serines‐262 and ‐324 have been found to be modified at the tubulin‐binding region of tau protein by GSK 3 in the presence of heparin or tubulin.
Photolabeling acyl CoA binding proteins in microsome preparations
Authors: Hare James F
Published: July 2002

To identify key enzymes that participate in acylglycerol metabolism, 12‐[(5‐iodo‐4‐azidosalicyl)amino]dodecanoylcoenzyme A (ASACoA) was emploued as a photoaffinity label for those enzymes that use fatty acyl CoA as a co‐substrate. ASACoA inhibited diacylglycerol acyltransferase activity in liver microsomes and was incorporated into triacylglycerol in a microsome dependent reaction. When photoactivated, ASACoA labeled four proteins in rat liver (75, 54, 50 and 37 kDa) and in epididymal fat cell (75, 64, 54 and 37 kDa) microsomes. Photolabeling was sensitive to palmitoyl CoA. After solubilization in Triton X‐114, all four proteins were concentrated into the detergent phase, indicating that they are integral membrane proteins.
Tumor necrosis factor (TNF) stimulates the production of nerve growth factor in fibroblasts via the 55‐kDa type 1 TNF receptor
Authors: Hattori Akira , Hayashi Kyozo , Kohno Michiaki
Published: July 2002

The role of the two types of tumor necrosis factor (TNF) receptors, TNF‐RI and TNF‐R2, in mediating the capacity of TNF to stimulate nerve growth factor (NGF) production in fibroblasts has been investigated. Although Swiss 3T3 cells express both the TNF‐R1 and TNF‐R2, an agonistic anti‐TNF R1 antibody, but not an agonistic anti‐TNF‐R2 antibody, increases the NGF mRNA level and stimulates the production of NGF protein in the cells. Treatment of the cells with a combination of anti‐TNF‐R2 antibody and interleukin‐1β (IL‐1β) does not increase the NGF production induced by IL‐1β alone, although TNF's activity to stimulate NGF production is markedly enhanced by IL‐1β. Furthermore, simultaneous addition of the both antibodies does not increase the NGF production above that observed with the anti‐TNF‐RI antibody, indicating that TNF‐R1 alone mediates the TNF's activity to stimulate NGF production in fibroblasts.
Calcium store depletion potentiates a phosphodiesterase inhibitor‐ and dibutyryl cGMP‐evoked calcium influx in rat pituitary GH3 cells
Authors: Willmott Nicholas J. , Asselin Judith , Galione Antony
Published: July 2002

A role for cGMP in the control of capacitative Ca2+ influx was identified in rat pituitary GH3 cells. Application of 50 μM‐1 mM of the non‐specific phosphodiesterase inhibitor, 3‐isobutyl‐1‐methylxanthine (IBMX), or the specific cGMP‐phosphodiesterase inhibitor, zaprinast, induced a dose‐dependent increase in the intracellular free Ca2+ concentration [Ca2+]i of the pituitary cell line, as assessed by video ratio imaging using fura‐2. Response onset times were identical and response profiles were similar in all cells analysed. Application of 50 μM dibutyryl cGMP to GH3 cells resulted in heterogeneous Ca2+ responses, consisting of single or multiple transients with varying onset times. In all cases, increases in [Ca2+]i were predominantly due to Ca2+ influx, since no responses were detected in low Ca2+ medium, or following pre‐incubation of cells with 1 μM verapamil, or nicardipine. Depleting intracellular Ca2+ stores by prior treatment of cells with 1 μM thapsigargin resulted in a dramatic potentiation in the Ca2+ influx mediated by both phosphodiesterase inhibitors and dibutyryl cGMP, suggesting that cGMP modulates a dihydropyridine‐sensitive Ca2+ entry mechanism in GH3 cells which is possibly regulated by the state of filling of Ca2+ stores.