bluuebunny/crossref_metadata_2025 huggingface dataset:
Phosphorylation of ribosomal proteins by the vaccinia virus B1R protein kinase
Authors: Banham Alison H. , Leader David P. , Smith Geoffrey L.
Published: July 2002

Two proteins of the 40S ribosomal subunit were shown to be phosphorylated in vitro by a vaccinia virus‐encoded serine/threonine protein kinase. These were identified by two‐dimensional gel electrophoresis as ribosomal proteins Sa and S2 and were shown by phosphoamino acid analysis to both be phosphorylated on serine and threonine residues. The three phosphorylated forms of S2 produced by the B1R protein kinase in vitro matched the phosphorylated forms of S2 previously observed in cells infected with vaccinia virus. These data strongly suggest that this enzyme is responsible for the phosphorylation of S2 and Sa which occurs early during vaccinia virus infection.
Endothelin‐receptor interactions
Authors: Spinella Michael J. , Kottke Rebecca , Magazine Harold I. , Healy Matthew S. , Catena John A. , Wilken Philip , Andersen Thomas T.
Published: July 2002

The mechanism of action of endothelin‐receptor interactions was studied, using radioligand binding assays and SDS‐PAGE, to investigate the possibility of disulfide interchange. Electrophoretic analysis suggested involvement of disulfide bond(s) in the receptor‐ligand complex. Treatment of Et receptors with sulfhydryl‐specific alkylating reagents (NEM or others) resulted in decreased ability to bind [125I]Et‐1. [Dpr1‐Asp15]Et‐1, an antagonist homologous to Et but with an amide link replacing one of the disulfides, bound to Et receptors reversibly, but binding of Et‐1 was less reversible. Preincubation of receptors with Et‐L, but not with [Dpr1‐Asp15]Et‐L, protected receptors from alkylation with [14C]NEM. The data suggest that the Et receptor has a sulfhydryl group at or near the Et binding site. A model is proposed in which the role of the putative sulfhydryl group is discussed.
Nucleotide/H+‐dependent change in Mg2+ affinity at the ATPase inhibitory site of the mitochondrial F1‐F0 ATP synthase
Authors: Bulygin V.V. , Syroeshkin A.V. , Vinogradov A.D.
Published: July 2002

The interactions between ADP and Mg2+ that result in the slowly reversible inhibition of the mitochondrial F1‐F0 ATPase were studied. The K
i for the inhibitory Mg2+ is shown to be strongly dependent on the occupation of the nucleotide‐binding sites. The inhibitory binding site for Mg2+ is not seen unless a stoichiometric amount of ADP is added [Biochem. J. 276 (1991) 149‐156]; it appears (K
i = 2.10−6 M) in the presence of stoichiometric ADP and the affinity for inhibitory Mg2+ decreases to a K
i, value of 7.10−5 M when the second nueleotide binding site with k
d = 5.10−6 M is loaded with ADP. The binding of the inhibitory Mg2+ is competitively inhibited by H+ ions within the pH interval 6.8–8.2. The nucleotide‐dependent affinity transition of the Mg2+‐specific site suggests that H+/Mg2+ exchange may play an important role in the catalytic mechanism of ATP synthesis/hydrolysis at the active site(s) of F1‐F0 ATP synthase.
Intracellular protons control the affinity of skeletal muscle ATP‐sensitive K+ channels for potassium‐channel‐openers
Authors: Forestier Cyrille , Depresle Yves , Vivaudou Michel
Published: July 2002

Levcromakalim, a potential antihypertensive agent, is known to activate potassium channels dependent on intracellular ATP (K‐ATP channels). In inside‐out patches excised from frog skeletal muscle, levcromakalim or a related compound, SR 47063, caused a strong and persistent activation of K‐ATP channels at a cytoplasmic pH of 7.1. However, at pH 6, these activators could no longer affect the K‐ATP currents. Conversely, in the continuous presence of activator, lowering pH from 7.1 to 6 returned channel activity to its level in pH 6 alone. After wash‐out of the activator, recovery from activation took minutes at pH 7.1 but only seconds at pH 6, thus ruling out an effect of protons on the activators in solution. These experiments suggest that K‐channel‐activators are unable to bind to their receptor when it is protonated, and more generally, they provide evidence at the microscopic level for proton‐induced allosteric modulation of drug‐receptor interaction.
Loss of α‐tocopherol upon exposure to nitric oxide or the sydnonimine SIN‐1
Authors: de Groot Herbert , Hegi Udo , Sies Helmut
Published: July 2002

SIN‐1 which spontaneously decomposes to yield nitric oxide (NO. and Superoxide anion (O2
.− radicals caused a loss of microsomal α‐tocopherol paralleled by the formation of α‐tocopheryl quinone. The loss was partially prevented by Superoxide dismutase but not by catalase. The SIN‐1‐induced loss of α‐tocopherol also occurred when tocopherol was dissolved in ethanol/potassium phosphate buffer (20/80, v/v). Likewise, addition of authentic NO. to α‐tocopherol dissolved in ethanol resulted in loss of the vitamin and quinone formation. These results suggest that NO. or its products such as peroxynitrite or nitrogen dioxide react with α‐tocopherol, the quinone derivative being a major oxidation product. Depletion of vitamin E by NO. may contribute to tissue injury, e.g. in neuronal tissues.
Interleukin‐1 ‐induced expression of nitric oxide synthase in insulin‐producing cells is preceded by c‐fos induction and depends on gene transcription and protein synthesis
Authors: Eizirik Décio L. , Björklund Andreas , Welsh Nils
Published: July 2002

The cytokine interleukin 1β (IL‐1β) induces the expression of an isoform of nitric oxide synthase (NOS) in insulin‐producing cells which is similar to that expressed in activated macrophages. In the present study we show that IL‐1β‐induced expression of NOS mRNA in these cells is preceded by expression of c‐fos mRNA. Moreover, the stimulatory effects of recombinant IL‐1β on NOS mRNA expression are prevented by co‐incubation with an inhibitor of gene transcription (actinomycin D) or an inhibitor of protein synthesis cycloheximide). These data suggest that IL‐1β‐induced NOS mRNA expression may be mediated by transcription of immediate early response genes, and that c‐fos may be one of these genes.
Characterization of 3':5' cyclic nucleotide phosphodiesterase activities of mouse neuroblastoma N18TG2 cells
Authors: Giorgi Mauro , Caniglia Cristiana , Scarsella Gianfranco , Augusti-Tocco Gabriella
Published: July 2002

Characterization of ‘low K
m’ 3':5' cyclic nucleotide phosphodiesterase activities (PDE) expressed in mouse N 18TG2 neuroblastoma cells is reported. At least 3 peaks of activity were isolated by DEAE chromatography, none of which was calcium‐calmodulin stimulated and cGMP stimulated or inhibited. A first peak elutes at 200 mM sodium acetate: it specifically hydrolyzes cGMP with a K
m of 4.7 μM and shows sensitivity to zaprinast [M&B 22948] (1.8 μM). A second peak eluting at 410 mM sodium acetate hydrolyzes both cyclic nucleotides. A third peak. specific for cAMP hydrolysis, elutes at 580 mM sodium acetate, has a K
m of 3.2 μM and is sensitive to RO 20 1724 (7.6 μM) and rolipram (2 μM). Hydrodynamic analysis showed for the first peak a Stokes radius of 5.3 nm with a sedimentation coefficient of 8.1 S, a frictional ratio (f/f
0) of 1.41 and a native molecular mass of 182 kDa. The same analysis for peak 3 showed a Stokes radius of 4.1 nm with a sedimentation coefficient of 3.2 S, a frictional ratio of 1.63 andanative molecular mass of 56kDa. The biochemical features reported for the enzyme eluting in the first peak, and its cGMP‐binding activity stimulated by inhibitors of phosphodiesterase activity, demonstrate that it belongs to the PDE V subfamily; on the other hand the cAMP specific enzyme eluting in the third peak can be assigned to the ‘RO 20 1724 inhibited’ form. The significance of these findings is discussed in relation to the functional characteristics of the N18TG2 cell line.
Primary structure of cryX1, the novel δ‐endotoxin‐related gene from Bacillus thuringiensis spp. galleriae
Authors: Shevelev A.B. , Svarinsky M.A. , Karasin A.I. , Kogan Ya.N. , Chestukhina G.G. , Stepanov V.M.
Published: July 2002

A cry‐related sequence, designated cryX (EMBL X75019), was localized upstream of cryIG, the δ‐endotoxin gene cloned from spp. galleriae of Bacillus thuringiensis and sequenced earlier [(1991) FEBS Lett. 293, 25‐28]. Analysis of the cryX complete nucleotide sequence enabled us to explain its virtual crypticity and to reveal the chimeric structure of the genes, cryX and cryIG. The amino acid sequence of 1,151 residues encoded by the continuous reading frame of cryX is similar to the other δ‐endotoxins but differs essentially from them.
Volume‐regulatory taurine release from a human lung cancer cell line
Authors: Kirk Kiaran , Kirk Julie
Published: July 2002

Exposure of a human lung epithelial cancer cell‐line to hypo‐osmotic media led to a marked increase in the rate of efflux from the cells of taurine, a non‐essential sulfonic amino acid. The osmotically‐activated taurine efflux was inhibited by a range of known Cl− channel blockers, the most potent of which were NPPB and 1,9‐dideoxyforskolin. These reagents were similarly effective at inhibiting the osmotically‐activated efflux of I−, a known substrate of volume‐activated Cl− channels. The results are consistent with the hypothesis that volume‐regulatory taurine release from these cells is mediated by a volume‐activated Cl− channel.
Identification of casein kinase II as a major endogeneous caldesmon kinase in sheep aorta smooth muscle
Authors: Vorotniko Alexander V. , Gusev Nikolai B. , Hua Suming , Collins John H. , Redwood Charles S. , Marston Steven B.
Published: July 2002

A caldesmon kinase activity was detected in an ATP extract of the myofibril‐like pellet from sheep aorta. The enzyme was purified 745‐fold and was identified as casein kinase II on the basis of molecular size, substrate specificity, and high sensitivity to heparin inhibition. Casein kinase II phosphorylated isolated caldesmon and caldesmon incorporated into native thin filaments, and transferred about 1 mol of phosphate per mol of caldesmon‐h. Ser‐73 was the main site phosphorylated by casein kinase II in chicken gizzard caldesmon. Phosphorylation of caldesmon reduced its affinity for smooth muscle myosin but had no effect upon the ability of caldesmon to inhibit the ATPase activity of actomyosin.
Engineering resistance to ‘aging’ of phosphylated human acetylcholinesterase Role of hydrogen bond network in the active center
Authors: Ordentlich Arie , Kronman Chanoch , Barak Dov , Stein Dana , Ariel Naomi , Marcus Dino , Velan Baruch , Shafferman Avigdor
Published: July 2002

Recombinant human acetylcholinesterase (HuAChE) and selected mutants (E202Q, Y337A, E450A) were studied with respect to catalytic activity towards charged and noncharged substrates, phosphylation with organophosphorus (OP) inhibitors and subsequent aging of the OP‐conjugates. Amino acid E450, unlike residues E202 and Y337, is not within interaction distance from the active center. Yet, the bimolecular rates of catalysis and phosphylation are 30 100 fold lower for both E450A and E202Q compared to Y337A or the wild type and in both mutants the resulting OP‐conjugates show striking resistance to aging. It is proposed that a hydrogen bond network, that maintains the functional architecture of the active center, involving water molecules and residues E202 and E450, is responsible for the observed behaviour.
Purification and characterization of an endogenous inhibitor specific to the Z‐Leu‐Leu‐Leu‐MCA degrading activity in proteasome and its identification as heat‐shock protein 90
Authors: Tsubuki Satoshi , Saito Yumiko , Kawashima Seiichi
Published: July 2002

We previously identified a benzyloxycarbonyl(Z)‐Leu‐Leu‐Leu‐4‐methylcoumaryl‐7‐amide (ZLLL‐MCA) degrading activity in proteasome as a candidate for the regulator of neurite outgrowth. As its counterpart, we purified a protein from bovine brain that specifically inhibits the ZLLL‐MCA degrading activity in proteasome. This protein is heat stable and has no effect on the other catalytic activities in proteasome, or on the activities of trypsin, chymotrypsin, or m‐ and μ‐calpains either. The molar ratio of inhibitor‐to‐proteasome that inhibits 50% of the ZLLL‐MCA degrading activity of proteasome is 1:1. The inhibitory mechanism of the inhibitor against proteasome is non‐competitive. Finally, the inhibitor was identified as heat‐shock protein 90 (HSP90) by partial amino acid sequencing and immunodetection. The results suggest that HSP90 initiates neurite outgrowth through the inhibition of the ZLLL‐MCA degrading activity in proteasome.
Cyclic GMP in the perfused rat heart Effect of ischaemia, anoxia and nitric oxide synthase inhibitor
Authors: Depré Christophe , Hue Louis
Published: July 2002

Working rat hearts perfused with 5.5 mM glucose were submitted to a 10‐min period of no‐flow ischaemia or anoxia. Both conditions stimulated glycogenolysis, activated phosphorylase and increased cyclic GMP content, although the time course of these changes differed in anoxia and ischaemia. Changes in cyclic GMP content were not correlated with glycogenolysis or phosphorylase activation. Perfusion with 1 μM l‐nitroarginine methylester, an inhibitor of nitric oxide synthase, decreased cGMP concentration under normoxic conditions and abolished the ischaemia‐induced increase in cGMP. The inhibitor decreased the coronary flow without affecting the overall working performance of the hearts under normoxic conditions.
Tyrosine phosphorylation and stimulation of protein kinase Cδ from porcine spleen by src in vitro
Authors: Gschwendt Michael , Kielbassa Kirsten , Kittstein Walter , Marks Friedrich
Published: July 2002

Native protein kinase Cδ from porcine spleen is phosphorylated in vitro by the tyrosine kinase src and to a much smaller extent by fyn. The tyrosine phosphorylation of PKCδ is restricted to the activated state of the enzyme, i.e. it occurs only in the presence of an activator, such as TPA or bryostatin. Upon phosphorylation at tyrosine, the apparent molecular weight of PKCδ increases by 6 kDa. Phosphorylation by src induces a stimulation of PKCδ activity apparently exhibiting some substrate selectivity. Other PKC isoenzymes, such as cPKC (α,β,γ), are not phosphorylated by src or only to a very small extent. This phosphorylation is not dependent on TPA and does not cause an increase in activity and molecular weight of the enzyme.
NDF/heregulin stimulates the phosphorylation of Her3/erbB3
Authors: Kita Yoshiko A. , Barff Jenny , Luo Yi , Wen Duanzhi , Brankow David , Hu Sylvia , Liu Naili , Prigent Sally A. , Gullick William J. , Nicolson Margery
Published: July 2002

Her3/erbB3 has been identified as a third member of the epidermal growth factor receptor (EGFR) family [(1989) Proc. Natl. Acad. Sci. USA 86, 9193–9197; (1990) Proc. Natl. Acad. Sci. USA 87, 4905–4909]. The natural ligand for Her3 has not been identified. Although recently NDF has been proposed as a specific ligand for Her4 [(1993) Nature 366, 473–475; (1993) J. Biol. Chem. 268, 18407–18410], we report here that Her3 was phosphorylated on tyrosine not only in three breast carcinoma cell lines, MDAMB453, MDAMB468 and SKBR3, but also in Her3‐transfected CHO cells in response to NDF stimulation. In further studies, cells were reacted with 125I‐labeled NDF and then chemically crosslinked. Immunoprecipitation with anti‐Her3 revealed a dense high M
W band, greater than 400 kDa. The results suggest that NDF may be a ligand of Her3 and induces receptor hetero‐oligomerization.
Molecular structure of the cyanobacterial tumor‐promoting microcystins
Authors: Rudolph-Böhner Sabine , Mierke Dale F. , Moroder Luis
Published: July 2002

The three‐dimensional structure of the two hepatotoxic microcystins LR and LY has been determined by two‐dimensional nuclear magnetic resonance (2D NMR) spectroscopy and distance geometry calculations. For the microcystin LY a single family of highly convergent structures was obtained. This family is characterized by a relatively compact boat‐like ring structure with the large side chain of the Adda residue protruding from the concave side, in close proximity to the Tyr side chain. Conversely, for the microcystin LR the calculations result in three conformational families characterized by an even more compact ring structure. The Adda and Arg side chains protrude from the ring distal from one another caused by the repulsion between the guanido function of Arg and the hydrophobic Adda. The lower toxicity of the LY microcystin could result from the restricted access of the Adda side chain, an essential residue for activity, which results from the close proximity of the aromatic Tyr residue. A significant enthalpic cost would be expected for disturbance of this hydrophobic collapse and correspondingly lower binding affinity to receptor molecules would be predicted. From the structures of the two related microcystins, and homology with other known toxins, we propose a working hypothesis of the Adda side chain interacting with a hydrophobic pore of the phosphatases while the rest of the microcystin acts as a scaffold to help stabilize the interdigitation of the Adda with additional intermolecular interactions.
Q‐ and L‐type Ca2+ channels dominate the control of secretion in bovine chromaffin cells
Authors: López Manuela G. , Villarroya Mercedes , Lara Baldomero , Sierra Rafael Martínez , Albillos Almudena , Gar'ia Antonio G. , Gandía Luis
Published: July 2002

Potassium‐stimulated catecholamine release from superfused bovine adrenal chromaffin cells (70 mM K+ in the presence of 2 mM Ca2+ for 10 s, applied at 5‐min intervals) was inhibited by the dihydropyridine furnidipine (3 μM) by 50%. ω‐Conotoxin MVIIC (CTx‐MVIIC, 3 μM) also reduced the secretory response by about half. Combined CTx‐MVIIC plus furnidipine blocked 100% catecholamine release. 45Ca2+ uptake and cytosolic Ca2+ concentrations ([Ca2+]i) in K+‐depolarized cells were partially blocked by furnidipine or CTx‐MVIIC, and completely inhibited by both agents. The whole cell current through Ca2+ channels carried by Ba2+ (I
Ba) was partially blocked by CTx‐MVIIC. Although ω‐conotoxin GVIA (CTx‐GVIA, 1 μM) and ω‐agatoxin IVA (Aga‐IVA, 0.2 μM) partially inhibited 45Ca2+ entry, I
Ba and the increase in [Ca2+]i, the combination of both toxins did not affect the K+‐evoked secretory response. The results are compatible with the presence in bovine chromaffin cells of a Q‐like Ca2+ channel which has a prominent role in controlling exocytosis. They also suggest that Q‐ and L‐type Ca2+ channels, but not N‐ or P‐types are localized near exocytotic active sites in the plasmalemma.
Identification of a fetal exon in the human fast Troponin T gene
Authors: Briggs Margaret M. , Maready Melinda , Schmidt James M. , Schachat Fred
Published: July 2002

A developmentally regulated exon has been identified in the 5′‐alternatively spliced region of the human fast Troponin T (TnT) gene. Expressed in fetal (but not adult) muscle, this exon is homologous with the fetal exons recently described in the rabbit and rat fast TnT genes. They all exhibit a split codon organization and encode a highly acidic peptide. To determine if the splicing pathways, including the human fetal exon, are also conserved, we defined the major TnT splicing patterns in fetal muscle. They generate fetal TnT 1, fetal TnT 3, and TnT1f, and TnT3f, species previously described in rabbit and rat skeletal muscles.
A C‐terminally truncated human parathyroid hormone receptor is functional and activates multiple G proteins
Authors: Schneider Helmut , Feyen Jean H.M. , Seuwen Klaus
Published: July 2002

We have investigated the role of the C‐terminal cytoplasmic domain of the human PTH receptor in effector coupling. Following transient expression in COS‐1 cells, coupling to both AC and PI‐PLC was observed with the full‐length receptor. Progressive C‐terminal truncations did not dissociate activation of the two signalling systems. In stably transfected 293 cells, however, the full‐length receptor as well as the majority of truncated constructs stimulated AC exclusively but failed to activate PI‐PLC. Activation of both signalling systems was again observed following stable expression of a severely truncated receptor (R483) in 293 cells. In this case, pertussis toxin was also found to potentiate the cAMP response to hPTH‐(1–38) significantly, indicating functional coupling of R483 to Gi proteins. Our results suggest that a core region of the human PTH receptor (first, second, third intracellular loop) can interact promiscuously with different G proteins and that the C‐terminus of the full‐length receptor directs the receptor towards an interaction with Gs
Functional expression of nicotinic acetylcholine receptors containing rat α7 subunits in human SH‐SY5Y neuroblastoma cells
Authors: Puchacz Elzbieta , Buisson Bruno , Bertrand Daniel , Lukas Ronald J.
Published: July 2002

Neuronal nicotinic acetylcholine receptors (nAChR) are made from different combinations of subunits encoded by a diverse family of genes. However, the recently cloned α7 gene codes for subunits that can form homooligomeric nAChR complexes when expressed in Xenopus oocytes. Electrophysiological studies reveal that these α7‐nAChR function as α‐bungarotoxin (Bgt)‐sensitive, quickly activating/inactivating ion channels with a unique pharmacological profile and an unusually high permeability to calcium ions. Although similar observations have been made in studies of Bgt‐sensitive, functional nAChR subtypes that are naturally expressed in neuronal cells, all attempts until now to reconstitute functional α7‐nAChR in cell lines have failed. Here we report the successful use of SH‐SY5Y human neuroblastoma cells, which naturally express low levels of endogenous α7 transcripts, to stably overexpress heterologous rat nAChR α7 transgenes. These transgenes are expressed as the appropriately‐sized α7 messages and protein, and stably transfected SH‐SY5Y cells have over 30‐times higher levels of specific Bgt binding sites than do wild‐type cells. Whole cell current recordings confirm that transfected cells express functional nAChR that are sensitive to blockade by Bgt and display the typical physiological and pharmacological profiles of α7‐nAChR. We conclude that stable, functional expression of α7 transgenes in a mammalian cell line has been achieved for the first time.
Roles of Arg231 and Tyr284 of Thermus thermophilus isocitrate dehydrogenase in the coenzyme specificity
Authors: Yaoi Takuro , Miyazaki Kentaro , Oshima Tairo
Published: July 2002

The coenzyme binding site of isocitrate dehydrogenase from Thermus thermophilus was analyzed by site‐directed mutagenesis. The mutation analysis revealed that Arg231 and Tyr284 are involved in the discrimination between NAD and NADP, suggesting that these two residues interact with 2′‐phosphate group of NADP.
APS‐sulfotransferase activity is identical to higher plant APS‐kinase (EC 2.7.1.25)
Authors: Schiffmann Sandra , Schwenn Jens D.
Published: July 2002

A cDNA from Arabidopsis thaliana L. Heynh encoding the APS‐kinase (EC 2.7.1.25) was modified by deletion of a plastidic transit peptide to enable its expression in Escherichia coli. The resultant protein (MW 25,761) is enzymatically active as APS‐kinase and restores prototrophic growth in an APS‐kinase mutant. All transformants harbouring the modified plant DNA also acquired APS‐sulfotransferase activity. In the absence of ATP but provided with DTT, a tetrameric form of recombinant APS‐kinase exhibits APS‐sulfotransferase activity. Monospecific polyclonal antibodies raised against the APS‐kinase as immunogen also reacted against APS‐sulfotransferase. We propose that APS‐sulfotransferase activity is a non‐physiological side reaction of APS‐kinase.
Interactions of eukaryotic elongation factor 2 with actin: A possible link between protein synthetic machinery and cytoskeleton
Authors: Bektaş M. , Nurten R. , Gürel Z. , Sayers Z. , Bermek E.
Published: July 2002

Eukaryotic elongation factor 2 (EF‐2) was shown to bind to F‐actin as assayed by co‐sedimentation. In the presence of guanosine‐5′‐O‐(3‐thiotriphosphate) (GTPγS) binding was increased fourfold. At saturation level a molar ratio of about 0. 12 EF‐2 per F‐actin (subunit) was observed. Our results suggest a single type of binding site with an apparent dissociation constant of 0.85 μM. The stoichiometry was independent of the filament length, and ADP‐ribosylation had no effect on the binding. Experimental data indicated the involvement of SH‐groups of both EF‐2 and actin in the binding. The interaction EF‐2 with F‐actin appeared to be inhibited competitively by EF‐1α and non‐competitively by G‐actin.
Yeast GAL11 protein stimulates basal transcription in a gene‐specific manner by a mechanism distinct from that by DNA‐bound activators
Authors: Sakurai Hiroshi , Ohishi Tomoko , Amakasu Hiroko , Fukasawa Toshio
Published: July 2002

The GAL11 gene encodes an auxiliary transcription factor required for full expression of many, if not all, genes of the yeast Saccharomycescerevisiae. We have recently shown that GAL11/‐encoded protein (GAL111p) enhances basal transcription from the CYC1 promoter in a cell‐free transcription system [(1993) Proc. Natl. Acad. Sci. USA 90, 8382‐8386]. Here we indicate that Ga111p stimulates basal transcription in a gene‐specific manner in vitro. We further suggest that the mechanism underlying the transcriptional stimulation by Ga111p is distinct from that by DNA‐bound activators, since Ga111p stimulated transcription in a reaction system where activators were unable to enhance transcription due to the lack of intermediary factors.
Alkalinization of acidic cellular compartments following cell swelling
Authors: Völkl Harald , Busch Gillian L. , Häussinger Dieter , Lang Florian
Published: July 2002

Osmotic swelling of rat hepatocytes increases fluorescence of Acridine orange and of fluorescein isothiocyanate (FITC)‐dextran, both indicative of alkalinization of acidic intracellular vesicles. Similar to osmotic cell swelling, insulin and glutamine lead to an increase in Acridine orange fluorescence, an effect virtually abolished upon osmotic reversal of glutamine‐induced cell swelling. Barium, which blocks K+ channels in the plasma membrane, similarly leads to cell swelling and increase of Acridine orange fluorescence. Since proteolysis is governed by lysosomal pH, these observations indicate that the anti‐proteolytic action of osmotic cell swelling is mediated by lysosomal alkalinization. Thereby, insulin, glutamine and barium probably exert their anti‐proteolytic action by cell swelling and subsequent lysosomal alkalinization.
Evidence for the involvement of protein phosphorylation in cyclic AMP‐mediated amylase exocytosis from parotid acinar cells
Authors: Takuma Taishin , Ichida Tokuro
Published: July 2002

We evaluated the role of protein phosphorylation in cAMP‐mediated amylase exocytosis from parotid acinar cells by using H89, a new protein kinase A (PKA) inhibitor, which is more lipophilic and 25 times more potent than H8. In our previous studies, H8 markedly inhibited protein phosphorylation without decreasing amylase release [Takuma T. (1988) Biochem. J. 256, 867‐871]. These findings were completely reproduced even in the small acini that were prepared by trypsin treatment before collagenase digestion. In the present study, however, H89 strongly inhibited both amylase release and protein phosphorylation in a dose‐dependent manner. The inhibibory effect was specific for PKA at least up to 33 (μM since 33 μM H89 did not block amylase release stimulated by PMA. H85, a closely related compound of H89 without inhibitory effect on PKA, did not prevent amylase release or protein phosphorylation at least up to 33 μM. These results suggest that protein phosphorylation by PKA is involved in cAMP‐mediated amylase exocytosis. The inhibition of protein phosphorylation by H8 might be insufficient or inadequate for blocking of amylase release.
Affinity purification of GTPase proteins from oat root plasma membranes using biotinylated GTP
Authors: de Boer Albertus H. , van Hunnik Eddy , Korthout Henrie A.A.J. , Sedee Norbert J.A. , Wang Mei
Published: July 2002

Biotinylated GTP was synthesized and it was demonstrated that this ligand was bi‐functional: it competed with [3H]Gpp(NH)p for binding to membrane proteins and it bound to immobilized avidin. Peripheral plasma membrane proteins were solubilized in a low‐salt wash, incubated with GTP‐biotin and biotinylated proteins were coupled to an avidin column. Elution with excess biotin yielded 10 polypeptides as seen with a silver stained SDS‐PAGE gel. Antisera raised against Ras, a small GTPase, strongly interacted with three proteins with MW of 38, 27 and 25 kDa and also with 6 other proteins. Gα‐common antibodies interacted with proteins of MW = 66 and 38 kDa. This method enables the rapid purification of GTP‐binding proteins and opens the possibility to assign a role to specific GTPases in signal transduction pathways.
Molecular cloning and expression of a novel type V adenylyl cyclase from rabbit myocardium
Authors: Wallach J. , Droste M. , Kluxen F.W. , Pfeuffer T. , Frank R.
Published: July 2002

A cDNA of a novel form of type V adenylyl cyclase has been cloned from rabbit myocardium using oligonucleotide probes derived from peptides that were produced by enzymatic cleavage of purified heart cyclase. A corresponding mRNA (6 kb) has been detected in rabbit myocardial tissue by Northern blot analysis. The cDNA encodes a protein of 1,264 amino acids exhibiting 12 putative membrane‐spanning regions in its hydrophilicity profile. Sequence comparison to two other previously published type V adenylyl cyclases reveals amino‐terminal domains of different length and low correlative homology, whereas the rest of the sequences is almost identical. The nonconserved amino‐terminal region of the subtype consists of 214 amino acids and exceeds the length of the others by 40 and 80 residues, respectively. Its presence in membrane preparations from different tissues has been confirmed immunologically using an antibody directed against a synthetic peptide. The cloned adenylyl cyclase was functionally expressed in COS‐1 cells to attain an enzymatic activity 3.5‐ to 14‐fold above control in the presence of forskolin.
Effects of morphine on prolactin receptors in the rat brain
Authors: Lai Zhennan , Zhai Qingzhu , Yukhananov Rustam , Larsson Christer , Roos Paul , Kreek Mary Jeanne , Nyberg Fred
Published: July 2002

The effect of chronically given morphine on the binding of ovine prolactin (oPRL) to specific areas in the male rat brain was studied. The drug was delivered through subcutaneously implanted miniosmotic pumps. The results indicated that the density of prolactin binding sites in the hypothalamus and the choroid plexus was significantly decreased in the acute phase of morphine administration but restored to control levels when tolerance to morphine was developed. The decrease in prolactin binding was contrasted by elevated plasma levels of the hormone. A negative correlation was found between the hormone concentration in plasma and the density of its binding sites in the hypothalamus and choroid plexus. The hormone‐binding sites in these two regions were further characterized with regard to binding constants and molecular sizes. The relevance of the present results with respect to the hypothalamic control of prolactin secretion is discussed.
Changes in α1‐adrenoceptor coupling to Ca2+ channels during development in rat heart
Authors: Liu Qin-Yue , Karpinski Edward , Pang Peter K.T.
Published: July 2002

It has been reported in the literature that α1,‐adrenoceptor activation in adult rat heart does not cause an increase in Ca2+ current but involves a decrease in I
t. This may explain in part the positive inotropic effect of α1‐adrenoceptor activation. In this study, the effect of phenylephrine, an α‐adrenergic agonist, on L‐type Ca2+ channel current was compared in young and neonatal rat myocytes. In the presence of propranolol, phenylephrine increased the Ca+ current (reversed by prazosin) in neonatal but not in young rat myocytes suggesting that the coupling of the α1‐adrenoceptor to Ca2+ channels may switch during development.
α2A‐Adrenergic receptors activate protein kinase C in human platelets via a pertussis toxin‐sensitive G‐protein
Authors: Nieuwiand Rienk , Wijburg Odilia L.C. , van Willigen Gijsbert , Akkerman Jan-Willem N.
Published: July 2002

4,4'‐Diisothiocyanato‐stilbene‐2,2'‐disulfonic acid (DIDS) stimulates human platelets via α2A‐adrenergic receptor‐mediated activation of protein kinase C (PKC) independent of the phospholipase C pathway. Here we show, that in permeabilized platelets activation of PKC by DIDS (20 μM), measured as 32P incorporation in pleckstrin, is completely inhibited by guanosine 5'‐(2‐O‐thio)diphosphate (200 μM), an inhibitor of heterotrimeric G‐proteins. Also pertussin toxin (4 ), which ADP‐ribosylates the α‐subunits of Gi's and Go, prevents pleckstrin phosphorylation by DIDS. N‐Ethylmaleimide (50 μM), which uncouples Gi from α2A‐adrenoceptors, inhibits pleckstrin phosphorylation by DIDS in intact platelets. Activation of PKC by 55 nM phorbol 12‐myristate 13‐acetate and 500 nM platelet‐activating factor are not disturbed by NEM. DIDS inhibits by 40 ± 5% (n = 4) the pertussis toxin‐catalyzed [3P]ADP‐ribosylation of a 41 kDa protein fraction previously shown to contain the α‐subunits ofGiα‐1, Giα‐2 and Giα‐3. Thus, the α2A‐adrenergic receptor activates PKC via a G‐protein of the Gi‐family.
Kinetics of inhibition of bovine cathepsin S by bovine stefin B
Authors: Turk Boris , C̆olić Adrijana , Stoka Veronika , Turk Vito
Published: July 2002

The kinetics of the complex formation between bovine cathepsin S and bovine stefin B was studied by conventional and stopped‐flow techniques. The inhibition at low inhibitor concentrations was tight and reversible (k
ass = 5.8 × 107 M−1 · s−1, k
diss = 4.9 × 10−4 s−1 at pH 6.0 and 25°C), whereas at higher inhibitor concentrations it was pseudo‐irreversible (k
ass = 6.14 × 107 M−1 · s−1). The complex was formed directly lacking the fast preequilibrium step with the dissociation equilibrium constant of ~ 8 pM. The competitive nature of inhibition was confirmed. The k
ass was found to be pH‐independent between pH 6.0 and 7.5 and decreased at lower or higher pH values in a way that strongly suggests involvement of two ionizable groups in the interaction (pK
1 = 5.2, pK
2 = 8.3). The enzyme‐substrate interaction seems to be influenced by different ionizable groups (pK
1 = 4.4, pK
2 = 7.8).
Exogenous NG‐hydroxy‐l‐arginine causes nitrite production in vascular smooth muscle cells in the absence of nitric oxide synthase activity
Authors: Schott C.A. , Bogen C.M. , Vetrovsky P. , Berton C.C. , Stoclet J.C.
Published: July 2002

Nitric oxide (NO) production from exogenous N
G‐hydroxy‐l‐arginine (OH‐l‐Arg) was investigated in rat aortic smooth muscle cells in culture by measuring nitrite accumulation in the culture medium. As well, the interaction between OH‐l‐Arg and l‐arginine uptake via the y+ cationic amino acid transporter was studied. In cells without NO‐synthase activity, OH‐l‐Arg (1–1000 μM) induced a dose‐dependent nitrite production with a half‐maximal effective concentration (EC50) of 18.0 ± 1.5 μM (n = 4–7). This nitrite accumulation was not inhibited by the NO‐synthase inhibitor N
G‐nitro‐l‐arginine methyl ester, l‐NAME (300 μM). In contrast, it was abolished by miconazole (100 μM), an inhibitor of cytochrome P450. Incubation of vascular smooth muscle cells with LPS (10 ) induced an l‐name inhibited nitrite accumulation, but did not enhance the OH‐l‐Arg induced nitrite production. OH‐l‐Arg and other cationic amino acids, L‐lysine and l‐ornithine, competitively inhibited [3H]‐l‐arginine uptake m rat aortic smooth muscle cells, with inhibition constants of 195 ± 23 μM(n = 12), 260 ± 40 μM(n= 5) and 330 ± 10 μM(n = 5), respectively. These results show that OH‐l‐Arg is recognized by the cationic l‐amino acid carrier present in vascular smooth muscle cells and can be oxidized to NO and nitrite in these cells in the absence of NO‐synthase, probably by cytochrome P450 or by a reaction involving a cytochrome P450 byproduct.
Synthetic melanin and ferric ions promote superoxide anion‐mediated lipid peroxidation
Authors: Sotomatsu A. , Tanaka M. , Hirai S.
Published: July 2002

In this study, we demonstrate that synthetic dopa‐melanin produced superoxide anions and promoted the peroxidative cleavage of phospholipids in the presence of Fe3+‐ADP complexes. SOD significantly suppressed this lipid peroxidation, while catalase or sodium benzoate did not. During the reaction, MCLA‐dependent chemiluminescence was detected, and this was suppressed to the control level by the addition of SOD. Melanin has been postulated to be toxic to tissues because of its interaction with redox‐active paramagnetic metal ions, and these findings suggest that superoxide anion‐mediated lipid peroxidation is induced by melanin in the presence of iron.
A novel peptidyl‐prolyl cisltrans isomerase from Escherichia coli
Authors: Rahfeld Jens-U. , Schierhorn Angelika , Mann Karlheinz , Fischer Gunter
Published: July 2002

A novel peptidyl‐prolyl cis/trans isomerase was isolated from Escherichia coli cell extract and characterized partially. Determination of the molecular mass by electrospray mass spectrometry indicated a protein of 10102 ± 2 Da, smaller than cyclophilins or FK506 binding proteins currently known. The specificity constant k
cat/K
m determined with Succinyl‐Ala‐Xaa‐Pro‐Phe‐4‐nitroanilide (Xaa = Leu) had a value comparable to those from cyclophilins from the same organism. However, the pattern of subsite specificity (Xaa = Gly, Ala, Val, Ile, Leu, Phe, Trp, His, Lys and Glu) was reminiscent of FK506 binding peptidyl‐prolyl cis/trans isomerases. The enzyme activity was not inhibited by cyclosporin A or FK506 at inhibitor concentrations of < 5 μM, concentrations that affect most bacterial peptidyl‐prolyl cis/trans isomerases. Computer‐assisted analysis of 21 amino acid residues of the N‐terminus determined by Edman degradation revealed no homology to known peptidyl‐prolyl cis/trans isomerases.
Cloning and expression in Escherichia coli of a cDNA encoding a developmentally regulated Ca2+‐binding protein from Dictyostelium discoideum
Authors: Coukell Barrie , Moniakis John , Grinberg Avie
Published: July 2002

We have cloned a full‐length cDNA from Dictyostelium discoideum which encodes a new Ca2+‐binding protein. The deduced protein (termed CBP1) is composed of 156 amino acids and contains four consensus metal‐ligating loop sequences found in helix‐loop‐helix motifs of many Ca2+‐binding proteins. When expressed in bacteria as a GST fusion protein, CBP1 binds Ca2+ in a 45Ca2+ overlay assay. CBP1 exhibits little amino acid sequence homology with Dictyostelium calmodulin or calfumirin‐1 (CAF‐1) except in the putative Ca2+‐binding regions. Moreover, unlike calmodulin and CAF‐1 expression, CBP1 mRNA is expressed preferentially during the multicellular stages of development.
Nature of respiratory stimulation in hyperthyroidism: the redox behaviour of cytochrome c
Authors: Schmehl I , Luvisetto S , Canton M , Gennari F , Azzone G.F
Published: July 2002

Hyperthyroid mitochondria show an increased K
m and V
max in the high affinity phase of cytochrome oxidase kinetics. During inhibitor titrations, cytochrome c shows a different redox behaviour in hyperthyroid with respect to protonophore‐treated euthyroid mitochondria. The observations are discussed in terms of a different regulation of electron input and output into the respiratory chain during slip and leak types of uncoupling. In hyperthyroid mitochondria during inhibitor titrations, the pattern of the relationship between uncoupler‐induced extra‐respiration and membrane potential is highly non‐linear. The complex nature of the respiratory stimulation in hyperthyroid mitochondria is discussed.
M‐caveolin, a muscle‐specific caveolin‐related protein
Authors: Way Michael , Parton Robert G.
Published: July 2002

Caveolae, small invaginations of the plasma membrane, are a characteristic feature of many mammalian cells. The best‐characterised caveolar protein is the integral membrane protein, VIP21‐caveolin. We now describe a novel homologue of VIP21‐caveolin, M‐caveolin, which is expressed exclusively in muscle. M‐caveolin was shown to be expressed in differentiated myotubes but not myoblasts. Epitope‐tagged M‐caveolin expressed in non‐muscle cells was targetted to surface caveolae where it colocalized with endogenous VIP21‐caveolin. M‐caveolin may play a specialised role in the caveolae of muscle cells.
Prevention of growth arrest‐induced cell death of vascular smooth muscle cells by a product of growth arrest‐specific gene, gas6
Authors: Nakano Toru , Kawamoto Keiko , Higashino Ken-ichi , Arita Hitoshi
Published: July 2002

We have purified Gas6 as a growth‐potentiating factor for vascular smooth muscle cells (VSMCs) [Nakano, T. et al. (1995) J. Biol. Chem. 270, 5702‐57051. However, specific production of Gas6 in growth‐arrested cells raises an intriguing question as to the physiological function of Gas6. In this study, we found that serum‐starved VSMCs secreted some survival factors and depletion of the factors induced cell death of VSMCs. Finally, we demonstrated that cell death was prevented by the addition of Gas6, suggesting that one of the major biological activity of Gas6 is protection of growth‐arrested VSMCs from death.
Upregulation of PKC δ‐ and downregulation of PKC α‐mRNA and protein by a phorbol ester in human T84 cells
Authors: Assert R. , Schatz H. , Pfeiffer A.
Published: July 2002

Protein kinase C (PKC) family members are downregulated by chronic activation at the protein level in most cell types. In T84 human epithelial cells 12‐O‐tetradecanoyl phorbol 13‐acetate (TPA) caused persistent translocation of PKC δ to the membrane compartment and a 400% increase of PKC δ‐mRNA after 24 h. In contrast, PKC α protein was completely downregulated and its mRNA was decreased to 60% of control levels after 24 h. This is the first report of PKC δ‐mRNA upregulation by TPA which was previously only shown for PKCβ. In view of the antimitogenic actions of PKC δ this pattern of regulation may serve to preserve growth control even in the presence of chronic cell activation.
Heteromeric channel formation and Ca2+‐free media reduce the toxic effect of the weaver Kir 3.2 allele
Authors: Tucker Stephen J. , Pessia Mauro , Moorhouse Andrew J. , Gribble Fiona , Ashcroft Frances M. , Maylie James , Adelman John P.
Published: July 2002

weaver mice have a severe hypoplasia of the cerebellum with an almost complete loss of the midline granule cells. Recent genetic studies of weaver mice have identified a mutation resulting in an amino acid substitution (G156S) in the pore of the inwardly rectifying potassium channel subunit Kir 3.2. When expressed in Xenopus oocytes the weaver mutation alters channel selectivity from a potassium‐selective to a nonspecific cation‐selective pore. In this study we confirm by cell‐attached patch‐clamp recording that the mutation produces a non‐selective cation channel. We also demonstrate that the cell death induced by weaver expression may be prevented by elimination of calcium from the extracellular solution as well as by coexpression with the wild‐type Kir 3.2 allele, or other members of the Kir 3.0 subfamily. These results suggest that the weaver defect in Kir 3.2 may cause cerebellar cell death by cell swelling and calcium overload. Cells which express the weaver subunit, but which normally survive, may do so because of heteromeric subunit assembly with wild‐type subunits of the Kir 3.0 subfamily.
Treatment of vaginitis
Authors: Kükner S. , Ergin T. , Ciçek N. , Uǧur M. , Yeşilyurt H. , Gökmen O.
Published: July 2002

Objective: The purpose of this study was to evaluate the efficacy of metronidazole 500 mg and miconazole nitrate 100 mg (Neo‐Penotran®, Embil Pharmacy Company, Istanbul, Turkey) in the treatment of vaginitis. Method: One hundred patients (mean age 26.5 years, range 18–50) with a clinical diagnosis of vaginitis entered this open, noncomparative study. Each patient inserted a Neo‐Penotran® pessary twice daily for 14 days. Assessments were carried out at the beginning of the study and 15 and 22 days after the start of therapy. Sudent's t‐test was used for the statistical analysis. Results: Out of 100 patients who were initially recruited to the study, 20 were subsequently excluded, leaving a total of 80 patients (80%) who completed the full course of treatment. Vaginitis was resolved in 75% of cases, it improved in 18% and was unchanged in 7%. The success rate for the treatment of trichomoniasis was 80%, 93.4% for bacterial vaginosis and 84.4% for candidal vaginitis. Conclusion: Neo‐Penotran® pessaries represent a novel and effective formulation for the treatment of common types of vaginitis and this pessary may also be of particular value in the treatment of resistant or recurrent vaginitis.
Development of ependyma in neural transplants
Authors: Kálmán M. , Tuba A. , Ajtai B.
Published: July 2002

Under in situ conditions, the innermost (juxtaventricular) neuroepithelial layer of the embryonic brain wall develops into ependyma. No development of ependyma was usually observed, however, in transplanted embryonic brain wall. In our telencephalic transplants, however, cysts lined by epithelium resembling ependyma were observed, although only sporadically. We supposed that occasional foldings of the transplanted telencephalic wall enclosed the aforementioned cysts and so induced the formation of ependyma. This hypothesis was supported by the observation that ependyma developed frequently in a model system in which the telencephalic wall was folded artificially prior to transplantation.
Mechanisms of action of clozapine in the treatment of neuroleptic-resistant and neuroleptic-intolerant schizophrenia
Authors: Deutch AY
Published: July 2002

The mechanisms of action which account for the effectiveness of clozapine as a pharmacotherapy for the treatment of neuroleptic non-responders and neuroleptic intolerant schizophrenic subjects remain elusive. We review recent data concerning the actions of clozapine in laboratory animals, and discuss the likely sites of action of clozapine and the receptors through which clozapine acts. We suggest that actions at dopamine D2 receptors in the caudate nucleus and putamen underlie the extrapyramidal side effects of conventional neuroleptics. In contrast, we propose that clozapine acts in the prefrontal cortex, specifically targeting an as yet unidentified DA receptor of the D2 family, to exert therapeutic actions in neuroleptic non-responders. We suggest that the ability of clozapine to augment extracellular dopamine levels in the prefrontal cortex may represent a key mechanism contributing to the therapeutic effects of this drug, and suggest some alternative approaches which might be expected to result in effects similar to those of clozapine.
Emotional deficit: an adaptative and evolutive process in HIV infection
Authors: Bungener C , Lefrère JJ , Widlöcher D , Jouvent R
Published: July 2002

The objective of the present study was to evaluate emotional disturbances and psychopathological symptoms in early stages of human immunodeficiency virus (HIV) infection. Seventy-one homosexual subjects, positive to HIV and two groups of HIV-negative subjects (32 homosexuals and 26 heterosexuals) were evaluated in a semi-structured interview by two trained raters. The results showed the presence of emotional perturbations already in asymptomatic HIV-positive individuals even in the absence of caracterized depression and/or anxiety. This emotional deficit seemed to be more important in more advanced stages of the disease. Depressive and anxious symptoms appeared to be slightly but significantly present in both groups of homosexual men. This emotional deficit could be the reflect of an adaptative process to the threatening consequences of HIV-infection. Emotional perturbations, even mild should not be neglected, because their reduction contributes to the psychological well being of HIV-positive subjects.