bluuebunny/crossref_metadata_2025 huggingface dataset:
Evidence that the newly cloned low‐density‐lipoprotein receptor related protein (LRP) is the α2‐macroglobulin receptor
Authors: Kristensen Torsten , Moestrup Søren K. , Gliemann Jørgen , Bendtsen Lone , Sand Ole , Sottrup-Jensen Lars
Published: July 2002

The human placental receptor (α2MR) for α2‐macroglobulin‐proteinase complexes contains 3 polypeptides of approx. 500 kDa, 85 kDa, and 40 kDa. N‐terminal sequence analysis of the 500 kDa and 85 kDa polypeptides, analysis of a random selection of peptides covering 536 residues from these polypeptides, and analysis of a 1772 bp cDNA encoding part of the 500 kDa polypeptide provide evidence that the 500 kDa and 85 kDa chains are the α‐ and β‐subunits, respectively, of a recently cloned hepatic membrane protein, termed the low density lipoprotein receptor related protein (LRP) (Herz, J., Hamann, U., Rogne, S., Myklebost, O., Gausepohl, H. and Stanley, K.K. (1988) EMBO J. 7, 4119‐4127; Herz, J., Kowal, R.C., Goldstein, J.L. and Brown, M.S. (1990) EMBO J. 9, 1769‐1776). N‐terminal sequence analysis of the 40 kDa polypeptide shows that it is of distinct genetic origin. It is suggested that LRP is the functional receptor for α2‐macroglobulin‐proteinase complexes (α2MR) and in addition may have as yet unsettled functions in lipoprotein metabolism.
A common mechanism of transcriptional activation by the three positive regulators, VirG, PhoB, and OmpR
Authors: Aoyama Takashi , Oka Atsuhiro
Published: July 2002

VirG, PhoB, and OmpR are positive regulators of the virulence, phosphate, and osmolarity regulons, respectively. Their target genes are attended with one or more significant blocks of similar 6‐bp sequences in their regulatory regions. The distance between the block(s) and transcriptional start site varies with the target genes, but the blocks are located in a similar manner phase‐specific to the promoter sequences. On the basis of these facts, we offer a novel model of transcriptional activation common to these regulators.
Examination of the role of serine phosphorylation in phospholipase C‐γ and its related P47 in cAMP‐mediated depression of epidermal growth factor receptor signal transduction
Authors: Mitsui Ken-ichi , Iwashita Shintaro
Published: July 2002

Forskolin‐pretreatment ofA431 cells reduced both intrinsic and epidermal growth factor (EGF)‐induced EGF receptor phosphorylation, however, phosphorylation of pospholipase c‐γ (PLC‐γ) was stimulated under the same conditions. No significant difference was detected in the amount of phosphotyrosine of PLC‐γ between two cultures with or without forskolin treatment followed by EGF. On the other hand, phosphorylation of a 47 kDa protein (P47) which cross‐reacted with an anti‐PLC‐y monoclonal antibody, was stimulated by both forskolin and EGF. Phosphorylation was exclusively on serine residues in this case. These results indicate that both PLC‐γ and P47 are posphorylated by a cAMP‐dependent protein kinase and the EGF‐stimulated serine kinase, and suggest that serine phosphorylation of PLC‐γ has no effect on ligand‐dependent coupling with the EGF receptor.
Glucose‐stimulated efflux of indo‐1 from pancreatic β‐cells is reduced by probenecid
Authors: Arkhammar Per , Nilsson Thomas , Berggren Per-Olof
Published: July 2002

Indo‐1 loaded pancreatic β‐cells, isolated from obese hyperglycaemic mice, were studied with respect to cytoplasmic free Ca2+ concentration ([Ca2+]i), efflux of indicator and insulin release. In the absence of glucose there was a continuous efflux of indo‐1 which increased upon stimulation with 20 mM of the sugar. The anion exchange inhibitor probenecid reduced both basal efflux of indo‐1 and prevented that promoted by glucose. Measurements of [Ca2+]i and insulin release revealed similar results as previously reported with quin‐2 and fura‐2. Furthermore, probenecid did not influence the [Ca2+]i responses. It is thus possible to reduce efflux of indo‐1 probenecid and thereby improve the measurements of [Ca2+]iin pancreatic β‐cells.
Dinucleoside tetraphosphatase from human blood cells
Authors: Pinto Rosa Maria , Costas Maria Jesús , Fernández Ascensión , Canales José , García-Agundez José Augusto , Cameselle José Carlos
Published: July 2002

Dinucleoside tetraphosphatase (Np4Nase; EC 3.6.1.17) has been purified 170000‐fold from a 30–60% ammonium sulfate fraction of a human blood cell extract. Purification included a dye‐ligand affinity elution step using the inhibitor adenosine 5'‐tetraphosphate. Human blood Np4Nase resembled rat liver Np4Nase, including recognition by anti‐rat Np4Nase, but differed from homogeneous human leukemia Np4Nase in the 1000‐fold lower specific activity of the latter. The results are discussed in relation to the potential role of diadenosine tetraphosphate (Ap4A) in the control of cell division and the turnover of Ap4A in blood.
Dynamics of the excited state of the primary electron donor in reaction centers of Rhodopseudomonas viridis as revealed by hole burning at 1.7K
Authors: Shkuropatov A.Ya. , Ganago A.O. , Shuvalov V.A.
Published: July 2002

The spectra of absorbance changes (ΔA) due to the formation of P+Q− (P, primary electron donor. Q, primary quinone acceptor) at 1.7K in Rhodopseudomonas viridis reaction centers (RCs) excited at 1014nm has been shown to include, besides a progression of broad (170 190 cm−1) Gaussian vibronic bands separated by 150 cm−1, a ‘narrow’ structure near 1014 nm which can be simulated by a Lorentian zero‐phonon hole (ZPH) and Lorentian one‐mode (26.8 cm−1) phonon wings. The widths of ZPH of ≈ 17 cm−1 for ΔA reflecting the formation of P+Q− decaying in the ms time domain and of 6.8 ± 0.4 cm−1 for P+Q− decaying in the min time domain at 1.7K, seems to correspond to different conformations of RCs with a relaxation time of P∗ of ≈0.6 ps (in agreement with measurements in this time domain) and 1.6 ± 0.1 ps. respectively. The comparison of the spectra of ΔA in the region of the B1, band for slow (min) and fast (ms) decaying components suggests a different mutual arrangement of P and BL for different conformations of RCs. It is assumed that the broad and narrow structures of the P band reflect the transitions to two configurations with different P‐protein interactions. ‘Narrow’ structure of ΔA spectrum with essentially the same phonon wings and ZPH (width of 3.8 ± 0.4 cm−1) was observed within the P band when HI was photoreduced at 1.7K.
Reversible expression of sm α‐actin protein and sm α‐actin mRNA in cloned cerebral endothelial cells
Authors: Amberger A. , Bauer H. , Tontsch U. , Gabbiani G. , Kocher O. , Bauer H.C.
Published: July 2002

The expression of smooth muscle (sm) α‐actin was studied in cloned capillary cerebral endothelial cells of two phenotypes. Type I cells were cultured in medium containing 10% FCS, heparin and ECGS (or α‐ECGF) and stained positive for a specific endothelial cell marker (Bandeiraea simplicifolia). Depletion of heparin and ECGS resulted in a smooth muscle‐like appearance after 2–3 days. Cells of this phenotype, (type II) stained positive for the endothelial cell marker and for sm α‐actin. In contrast to type I cells, type II cells expressed sm α‐actin protein and mRNA as evidenced by Immunoblots and Northern blots. This phenotypic switch was shown to be reversible and so was the expression of sm α‐actin.
The effects of parathyroid hormone on L‐type voltage‐dependent calcium channel currents in vascular smooth muscle cells and ventricular myocytes are mediated by a cyclic AMP dependent mechanism
Authors: Wang Rui , Wu Lingyun , Karpinski Edward , Pang Peter K.T.
Published: July 2002

The present study demonstrated that L channel currents were decreased in smooth muscle cells, and increased in ventricular myocytes by both bovine parathyroid hormone, (bPTH‐(1–34)), and dibutyryl cyclic AMP (db‐cAMP), using the whole cell version of the patch clamp technique with Ba2+ as the charge carrier. The effects of bPTH‐(1–34) and db‐cAMP on L channel currents were additive but not synergistic. Furthermore, the effects of bPTH‐(1–34) on L channel currents in these 2 cell preparations were abolished in the presence of a cAMP antagonist. These results suggest that the effects of bPTH‐(1–34) on L channel currents in vascular smooth muscle cells and ventricular myocytes are mediated by a cAMP‐dependent mechanism.
cDNA cloning and chromosomal localization of the human α‐adrenergic receptor kinase
Authors: Benovic J.L. , Stone W.C. , Huebner K. , Croce C. , Caron M.G. , Lefkowitz R.J.
Published: July 2002

The β‐adrenergic receptor kinase (βARK) mediates agonist‐dependent phosphorylation of the β2‐adrenergic and related G protein‐coupled receptors. A cDNA encoding bovine βARK has recently been isolated. In this work we have isolated a cDNA encoding human βARK from a retinal cDNA library. The cDNA encodes a protein of 689 amino acids with an overall 98.0% amino acid and 92.5% nucleotide identity with bovine βARK. Chromosomal location of the human βARK gene was determined by correlating the presence of the βARK locus with retention of a specific human chromosome in a rodent‐human hybrid panel. This analysis revealed that the human βARK locus segregated with the long arm of chromosome 11, centromeric to 11q13.
Sub‐picosecond dynamics of excited state of primary electron donor in reaction centers of Rhodopseudomonas viridis as revealed by hole burning at 1.7K broad and narrow holes
Authors: Ganago A.O , Shkuropatov A.Ya , Shuvalov V.A
Published: July 2002

Within the Qγ band of the primary electron donor (P), the spectra of absorbance changes due to the formation of a state P+QA
− (QA is the primary quinone) at 1.7K in Rhodopseudomous viridis reaction centers excited at 1014 nm have been shown to involve two spectral features characterized by: (i) a progression of broad (170–190 cm−1) Gaussian vibronic bands (S‐factor = 1.4) separated by 150 cm−1 and (ii) a ‘narrow’ structure near 1014 nm, characterized by 0‐0 transition at 1014 nm with a width of ∼50 cm−1 and 0–1 transition at 1000 nm with the width of ∼100 cm−1, and S‐factor = 0.9. The width of 50 cm−1 can be related to either zero‐phonon hole (ZPH) width or the structure involving phonon wings and ZPH being unresolved. Since dichroic value (∼0.37) is unvarying over the P band, the vibrations involved are totally symmetric. The ZPH (width of ∼3 cm−1) and phonon wings (frequency of ∼30 cm−1) are resolved within the P band near 1014 nm when the spectrum of ΔA due to the formation of bacteriopheophytinL
− is measured at 1.7K.
The calpain—calpastatin system in vascular smooth muscle
Authors: McClelland Phadungchom , Hathaway David R.
Published: July 2002

Vascular smooth muscle contains large amounts of the Ca2+‐dependent protease calpain II. In this study, we compared bovine aortic muscle (muscle phenotype) to cultured bovine aortic cells of smooth muscle origin (modulated phenotype) with respect to major constituents of the calpain—calpastatin system. Bovine aortic muscle contained only calpain II by activity measurements, Western blot of tissue extracts and Northern blot of poly(A)+ RNA. On the other hand, using the same methodologies, both calpains I and II as well as the 110 kDa inhibitor protein, calpastatin, were identified in cultured bovine aortic cells of smooth muscle origin. We conclude that the phenotypic state of smooth muscle cells is associated with differential expression of major components of the calpain—calpastatin system. Moreover, bovine aortic muscle is the only tissue identified to date that contains calpain II exclusively.
EPR method for the measurement of cellular sulfhydryl groups
Authors: Weiner L.M. , Hu H. , Swartz H.M.
Published: July 2002

An EPR method that can measure the concentration of sulfhydryl groups in intact cells has been developed using a specially designed stable nitroxyl biradical. The biradical, ·RS—S·R, contains a disulfide bond and readily undergoes thiol—disulfide exchange reactions with thiols resulting in a characteristic EPR spectrum which can be analyzed to provide a quantitative measure of sulfhydryl groups. The data obtained from the EPR method are in good agreement with those obtained from the conventional optical method using Ellman's reagent. The advantages of the EPR method are that the measurement can be carried out on intact cells or any other highly colored, absorbing and/or scattering solutions and the sensitivity is such that only a few cells (∼ 100) are needed for each quantitative measurement.
Molecular mechanism of protein kinase C modulation of sodium channel α‐subunits expressed in Xenopus oocytes
Authors: Schreibmayer W. , Dascal N. , Lotan I. , Wallner M. , Weigl L.
Published: July 2002

The mechanism of modulation of sodium channel α‐subunits (Type IIA) by a protein kinase C (PKC) activator was studied on single channel level. It was found that: (i) time constants for channel activation were prolonged; (ii) inactivation remained virtually unchanged; (iii) peak sodium inward current was reduced as evidenced by calculation of average sodium currents; and (iv) time constants for current activation and decay were prolonged. (i), (iii) and (iv) were voltage dependent, being most prominent at threshold potentials. The data show that a voltage dependent action on the activation gate can account for the observed reduction of peak inward sodium current and prolongation of current decay in macroscopic experiments.
Regulation of Mg2+ uptake in isolated rat myocytes and hepatocytes by protein kinase C
Authors: Romani A. , Marfella C. , Scarpa A.
Published: July 2002

A large Mg2+ cell uptake against concentration gradients is stimulated in collagenase‐dispersed rut myocytes by carbachol and in hepatocytes by carbachol or vasopressin. The signalling pathway(s) responsible for this stimulation of Mg2+ uptake was investigated by using various activators or inhibitors of protein kinase C (PKC) and by correlating Mg2+ uptake with cell PKC activity and cAMP content. In both cell preparations, the direct stimulation or PKC by diacylglycerol analogs or phorbol esters reproduce the same pattern of Mg2+ uptake as that induced by carbachol or vasopressin, These data indicate that the activation of PKC is responsible for a stimulation of Mg2+ uptake by myocytes or hepatocytes, whereas increase in cAMP in these cells stimulates Mg2+ release.
Conformational study of cyclo[D‐Trp‐D‐Asp‐Pro‐D‐Val‐Leu], an endothelin‐A receptor‐selective antagonist
Authors: Atkinson R.Andrew , Pelton John T.
Published: July 2002

The conformation of cyclo[D‐Trp‐D‐Asp‐Pro‐D‐Val‐Leu], (BQ123), an endothelin‐A receptor‐selective antagonist, has been studied in 20% acetonitrile in water by CD and NMR spectroscopy. CD studies showed the peptide adopted a similar, constrained conformation in both water alone and 20% acetonitrile in water. NMR spectra showed the proline residue to be in the trans conformation and 2 of the NH protons to exchange slowly with the solvent, indicating hydrogen bonding. Structural constraints derived from the NMR spectra were used to define the conformation in molecular dynamics simulations. A single backbone conformation is observed for the cycle, comprising a β type II turn and a γ′ turn.
Identification of the active site serine of the X‐prolyl dipeptidyl aminopeptidase from Lactococcus lactis
Authors: Chich J.-F. , Chapot-Chartier M.-P. , Ribadeau-Dumas B. , Gripon J.-C.
Published: July 2002

The active site serine of the X‐prolyl dipeptidyl aminopeptidase from Lactococcus lactis (PepX) was identified. The enzyme was labeled by [3H]DFP, treated by CNBr and the resulting peptides were separated by reverse‐phase‐HPLC. The main radiolabeled peptide was sequenced. Ser‐348, in the following sequence, Gly‐Lys‐Ser‐Tyr‐Leu‐Gly, was identified as the active site serine. A sequence comparison between the active site of PepX and other serine proteases was made, showing only limited sequence homologies in this area. The consensus sequence surrounding the active site serine in the three known X‐prolyl dipeptidyl aminopeptidases (mammalian DPPIV, yeast DPAB and PepX) is G‐X‐S‐Y‐X‐G, where X is a non‐conserved amino acid.
Induction of Ca2+/calmodulin‐dependent NO synthase in various organs of rats by Propionibacterium acnes and lipopolysaccharide treatment
Authors: Oguchi Shinobu , Iida Sachio , Adachi Hiroko , Ohshima Hiroshi , Esumi Hiroyasu
Published: July 2002

Ca2+/calmodulin‐dependent nitric oxide synthase was found to be induced during rat liver necrosis caused by administration of Propionibacterium acnes and E. coli lipopolysaccharide to rats. Examination of the specific induction of Ca2+/calmodulin‐dependent NO synthase showed that the enzyme was induced in the lung, spleen and colon as well as the liver. Northern blot analysis revealed that the induction occurred at the transcriptional level.
Angiotensin‐II‐induced expression of proto‐oncogene (c‐fos, jun‐B and c‐jun) mRNA in bovine adrenocortical fasciculata cells (BAC) is mediated by AT‐1 receptors
Authors: Viard I. , Jaillard C. , Ouali R. , Saez J.M.
Published: July 2002

We have shown previously that angiotensin‐II (A‐II) controls proto‐oncogene (c‐fos, jun‐B and c‐jun) mRNA accumulation in bovine adrenal fasciculata cells (BAC). Since BAC contain both subtypes (AT‐1 and AT‐2) of the A‐II receptor, we have investigated which subtype was involved in the effect of A‐II on proto‐oncogene mRNA by using a selective antagonist for AT‐1 (DUP 753) and for AT‐2 (CGP 42112A). DUP 753, but not CGP 421 12A, inhibited the stimulatory effect of A‐II on proto‐oncogene mRNA, with ID3ds of 4 × 10−7 M, 7 × 10−7 M and 2 × 10−6 M for c‐fos, jun‐B and c‐jun, respectively. Neither of the two antagonists by themselves had a direct effect on proto‐oncogene mRNA. As the A‐II AT‐1 receptors are coupled to the phospholipase C system in BAC, we have investigated whether the A‐II effects on the proto‐oncogenes were mediated by protein kinase C (PKC) or by Ca2+ calmodulin. First, activation of PKC by the phorbol ester, PMA, increased the level of the three proto‐oncogene mRNAs, whereas calcium ionophore had no effect. Second, staurosporine, a specific inhibitor of PKC, reduced the stimulatory action of A‐II on proto‐oncogene mRNA by 80–90%, whereas trifluoroperazine, an inhibitor of calmodulin, had no significant effect. These results demonstrate that the effects of A‐II on proto‐oncogene mRNA are mediated by AT1 receptor subtypes, mainly through activation of the PKC pathway.
Induction of vascular smooth muscle cell growth by selective activation of the thrombin receptor Effect of heparin
Authors: Herbert J.M. , Lamarche I. , Dol F.
Published: July 2002

The synthetic peptide, SFLLRNPNDKYEPF, has been recently described as a peptide mimicking the new amino‐terminus created by cleavage of the thrombin receptor, therefore acting as an agonist of the thrombin receptor. This peptide was a potent mitogen for rabbit arterial smooth muscle cells (SMC) and exhibited the same activity as that of native α‐thrombin. Both compounds stimulated the proliferation of growth‐arrested SMCs with half‐maximum mitogenic responses at 1 nM. NAPAP, a synthetic inhibitor of the enzymatic activity of thrombin, specifically inhibited thrombin‐induced SMC growth (IC50 = 0.35 ± 0.04 μM) but was without effect on the mitogenic effect of the agonist peptide. These results therefore demonstrate that the mitogenic effect of α‐thrombin for SMCs is intimately linked to its esterolytic activity. Heparin, which inhibited fetal calf serum‐induced SMC growth, was without effect on thrombin‐induced SMC growth but strongly reduced the mitogenic effect of the agonist peptide (IC50 = 32 ± 5μ/ml). This effect was not related to the anti‐coagulant activity of heparin but was highly dependent on molecular mass and on the global charge of the molecule and was also observed for other sulphated polysaccharides such as pentosan polysulphate.
ATP‐sensitive K+‐channels in the human adult ventricular cardiomyocytes membrane
Authors: Babenko Andrew P. , Samoilov Vladimir O. , Kazantseva Svetlana T. , Shevchenko Yuri L.
Published: July 2002

Using the patch‐clamp method in 27 different inside‐out patches it was shown for the first time that they defined the basic parameters of the functioning of single ATP‐sensitive K+ channels in the human adult ventricular cardiomyocytes membrane. At [K+]o = 140 mM single channel conductance (over the linear part of the I—V relation) reaches 100 pS. The possibility of the existence of these channels' conductance sublevels as well as the cluster character of their localization in sarcolemma is shown. The channel activity demonstrates an obvious run‐down with τ at about a minute. The analysed channels possess one open and two closed states.
Farnesylcysteine analogues inhibit chemotactic peptide receptor‐mediated G‐protein activation in human HL‐60 granulocyte membranes
Authors: Scheer Alexander , Gierschik Peter
Published: July 2002

Analogues of S‐prenylated cysteine like N‐acetyl‐S‐trans,trans‐farnesyl‐l‐cysteine (AFC) have previously been shown to inhibit the carboxyl methylation of proteins carrying a C‐terminal S‐prenylated cysteine residue and to block the endotoxin‐activated serum‐elicited ehemotactic response of mouse macrophages. Here, we show that AFC inhibits both basal and fonnyl peptide receptor‐stimulated binding of guanosine 5'‐O‐(3‐thiotriphosphate) (GTP[S]) to and hydrolysis of GTP by membranes of myeloid differentiated HL‐60 granulocytes. Receptor‐stimulated GTP[S] binding and GTP hydrolysis are more sensitive to AFC inhibition than basal G‐protein functions. Inhibition of fonnyl peptide receptor‐mediated G‐protein activation is also observed for S‐trans,trans‐famesyl‐3‐thiopropionic acid, but not for N‐acetyl‐S‐tarns‐geranyl‐l‐cysteine, N‐acetyl‐l‐cysteine, or the methyl ester of AFC, suggesting that the farnesyl moiety and the carboxyl group, but not the peptide bond of AFC are required for inhibition. The observations that exogeneous S‐adenosyl‐l‐methionine is apparently not required for and S‐adenosyl‐l‐homocysteine does not attenuate the inhibitory action of AFC raise the distinct possibility that AFC inhibits receptor‐mediated G‐protein interaction by a mechanism other than inhibition of protein carboxyl methylation.
Reduced influx is a factor in accounting for reduced vincristine accumulation in certain verapamil‐hypersensitive multidrug‐resistant CHO cell lines
Authors: Stow M.W. , Warr J.R.
Published: July 2002

The rates of accumulation, influx and efflux of vincristine have been examined in a series of multidrug‐resistant Chinese hamster ovary cell lines which show exceptionally high levels of hypersensitivity (collateral sensitivity) to several resistance modifiers. The more highly resistant members of the series show significantly reduced levels of vincristine influx compared to the control cell line from which they were derived. It is possible that resistance modifier hypersensitivity and reduced vincristine influx may be due to a common change in membrane composition which has arisen during prolonged selection for vincristine resistance in these cell lines.
Endothelin‐converting enzyme activity in human serum lipoprotein fraction
Authors: Ohwaki Tatsuya , Sakai Hiroshi , Hirata Yukio
Published: July 2002

Endothelin‐1 (ET‐1)‐converting enzyme (ECE) activity in the human serum lipoprotein fraction was studied using a sensitive enzyme immunoassay and reverse‐phase high performance liquid chromatography. The ECE activity of cleaving synthetic human big ET‐1 into ET‐1 by the serum lipoprotein fraction was about 14‐times greater than that by whole serum, and the activity was closely associated with lipoprotein itself. The lipoprotein ECE activity, which was optimal at pH 7.0, was inhibited by EDTA, o‐phenanthroline, phosphoramidon, thiorphan, phenylmethane‐sulfonyl fluoride and chymostatin, but not by cysteine or aspartic proteinase inhibitors, suggesting metalloproteinase‐ and chymotrypsin‐like properties. These results suggest that the serum lipoprotein ECE may be involved in the processing of big ET‐1 to ET‐1 in the circulatory system.
Control of muscle pyruvate oxidation during late pregnancy
Authors: Sugden Mary C. , Holness Mark J.
Published: July 2002

Despite significant increases in circulating concentrations of lipid fuels (triacylglycerol, non‐esterified fatty acids (NEFA) and ketone bodies) in late‐pregnant rats sampled in the fed (absorptive) state, cardiac and skeletal muscle active pyruvate dehydrogenase (PDHa) activities remained comparable with those observed in fed, age‐matched virgin controls. Cardiac PDHa activity was suppressed in response to acute (6 h) starvation in late‐pregnant (as well as virgin) rats: this inactivation was opposed by inhibition of mitochondrial long‐chain FA oxidation. Starvation (6 h) also led to PDH inactivation in skeletal muscles of late‐pregnant, but not virgin, rats. Starvation for 24 h led to further suppression of cardiac PDHa activity and was associated with significant increases in PDH kinase activities in both virgin and late‐pregnant rats. Late pregnancy did not itself influence cardiac PDH kinase activity.
On the origin of a sustained increase in cytosolic Ca2+ concentration after a toxic glutamate treatment of the nerve cell culture
Authors: Khodorov B. , Pinelis V. , Golovina V. , Fajuk D. , Andreeva N. , Uvarova T. , Khaspekov L. , Victorov I.
Published: July 2002

A sustained increase of cytosolic Ca2+ concentration, [Ca2+]i, (Ca2+ plateau) was induced by a 15‐min treatment with 50 μM glutamate of cultured cerebellar granule cells and hippocampal neurons in a Mg2+‐free solution. Plateau proved to be insensitive to inhibition of Na+
o/Ca2+
i exchange caused by removal external Na+ in the post‐glutamate period. A ~ 105‐fold reduction of [Ca2+]o (from 1.5 mM to 20 nM) in the post‐glutamate period caused in most cells only a slow and small decrease in [Ca2+]i, although the same low‐Ca2+ trial before glutamate treatment caused in hippocampal cells very quick blockade of spontaneous [Ca2+]i oscillation and a decrease in the basal [Ca2+]i. The results suggest that the Ca2+ plateau is due to a suppression of the Ca2+ extrusion from the cell (in particular via Na+/Ca2+ exchange) rather than from a persistent increase in Ca2+ permeability of neuronal membrane.
GTP/ggS‐Induced phosphorylation of myosin light chain kinase in smooth muscle
Authors: Tang Da-Chun , Kubota Yasutaka , Kamm Kristine E. , Stull James T.
Published: July 2002

Phosphorylation of myosin light chain kinase by a Ca2+‐dependent protein kinase increases the concentration of Ca2+/calmodulin required for half‐maximal activation. The Ca2+ concentrations required for myosin light chain kinase phosphorylation in permeable smooth muscle are similar to those required for myosin light chain phosphorylation. Both GTPγS and carbachol increase the Ca2+ sensitivity of myosin light chain kinase phosphorylation as well as light chain phosphorylation. It is proposed that a similar G‐protein mediated mechanism regulates the Ca2+‐dependent phosphorylation of these two contractile proteins in smooth muscle.
Molecular analysis of SAR1 ‐related cDNAs from a mouse pituitary cell line
Authors: Shen Katherine A. , Hammond Connie M. , Moore Hsiao-Ping H.
Published: July 2002

Vesicular transport between the endoplasmic reticulum (ER) and the Golgi in the yeast Saccharomyces cerevisiae requires a Ras‐like, small GTP‐binding protein, Sarip [1–3]. Whether a functional homologue operates in export from the ER in mammalian cells is unknown, nor is it clear if transport in other branches of the secretory pathway requires member(s) of a gene family. In this study, we used a PCR approach to examine the complexity of SAR1‐related sequences expressed in mammalian cells that possess multiple secretory pathways. Amplification of cDNA sequences from rodent pituitary cells with primers corresponding to two conserved GTP binding domains of Sarlp yielded several clones with sequences homologous to Sarl and/or the closely related ADP‐ribosylation factor (ARF) family. Of these, only two showed closer homologies to S. cerevisiae Sar1 than members of the ARF family and are designated as mSARa and mSARb. Northern blot analysis shows that mSARa is expressed in most tissues including liver, heart, brain, skeletal muscle and kidney. In contrast, mSARb is preferentially expressed in skeletal muscle and liver. The full‐length cDNA of mSARa isolated from a mouse pituitary AtT‐20 cDNA library encodes a protein of 198 amino acids, and is 61.6% identical to Sarlp from S. cerevisiae. Thus in contrast to the large rab family of GTP‐binding proteins, vesicular transport in mammalian cells appears to be mediated by a relatively small number of Sar1‐related proteins.
Differential expression of AMPA glutamate receptor mRNAs in the rat adrenal gland
Authors: Kristensen Peter
Published: July 2002

Localisation of mRNA encoding the four AMPA glutamate receptor subunits in the rat adrenal gland was investigated using in situ hybridisation. GluR1 is found in the zona glomerulosa of the cortex, GluR3 in the remaining parts of the cortex, GluR2 in adrenal medullary cells and GluR4 at a very low level in the zona glomerulosa. All four receptor mRNAs are found in medullary ganglion cells. The flip form of GluR2 and GluR3 dominate and the GluR2 mRNA is present in the arginine encoding form. Different cell populations of the adrenal gland may express homomeric forms of different receptor subtypes.
Regulation of endothelin‐1 action on the perfused rat liver
Authors: Tran-Thi Thuy-Anh , Kawada Norifumi , Decker Karl
Published: July 2002

Endothelin‐1 (ET‐1) was found to be a very potent stimulus for contraction and glycogenolysis in the perfused rat liver. At 1 nM it caused a dramatic increase in portal pressure of 22.1 ± 2.7 cm water and enhanced the glucose output up to 3‐fold. Extracellular Ca2+ and protein kinase C were involved in the signal transduction of ET‐1. ET‐1 action does not seem to be mediated by endogenous eicosanoids. The effects of ET‐1 were significantly reduced in the presence of 1 μM Iloprost, a prostaglandin I2 analogue, or by 100 μM sin‐1, a nitric oxide donor. In cultured hepatocytes, glycogenolysis was also stimulated by ET‐1 although to an extent too small to explain the high glucose output found in the perfused liver.
Purified yeast aspartic protease 3 cleaves anglerfish pro‐somatostatin I and II at di‐ and monobasic sites to generate somatostatin‐14 and ‐28
Authors: Cawley Niamh X. , Noe Bryan D. , Loh Y.Peng
Published: July 2002

Anglerfish somatostatin‐14 (SS‐14) and somatostatin‐28 (aSS‐28) are derived from pro‐somatostatin I (aPSS‐I) and pro‐somatostatin II (PSS‐II), respectively. Purified yeast aspartic protease 3 (YAP3), was shown to cleave aPSS‐I at the Arg18‐Lys82 to yield SS‐14 and Lys−1SS‐H. In contrast, YAP3 cleaved aPSS‐II only at the monobasic residue. Arg73 to yield aSS‐28. Since the paired basic and monobasic sites are present in both precursors, the results indicate that the structure and conformation of these substrates dictate where cleavage occurs. Furthermore, the data show that YAP3 has specificity for both monobasic and paired basic residues.
Calcium ionophore A23187 induces interleukin‐8 gene expression and protein secretion in human monocytic cells
Authors: Wilson Lynn , Butcher Christopher J. , Kellie Stuart
Published: July 2002

The regulation of the expression of the interleukin‐8 (IL‐8) gene in human monocytic cell lines has been investigated. Agents such as interleukin‐1 (IL‐1) or interferon‐γ (IFNγ) did not induce increased IL‐8 expression in THP‐1 or U937 cells. Bacterial lipopolysaccharide endotoxin (LPS) or phorbol myristate acetate (PMA) alone induced suboptimal expression as assessed by Northern blotting; however, preincubation of cells with PMA followed by endotoxin induced much higher levels of IL‐8 mRNA. Incubation of the cells with the calcium ionophore A23187 resulted in consistent increased IL‐8 gene expression comparable to that of cells treated with endotoxin alone. In addition to inducing IL‐8 mRNA this calcium ionophore also induced IL‐8 protein synthesis as assessed by immunofluorescence and secretion as detected by ELISA. These results indicate that increases in intracellular calcium result in IL‐8 gene expression and protein secretion.
Nitric oxide preferentially stimulates auto‐ADP‐ribosylation of glyceraldehyde‐3‐phosphate dehydrogenase compared to alcohol or lactate dehydrogenase
Authors: Dimmeler Stefanie , Brüne Bernhard
Published: July 2002

Recently we demonstrated that the radical nitric oxide (NO) stimulates the auto‐ADP‐ribosylation of the glycolytic enzyme glyceraldehyde‐3‐phosphate dehydrogenase (GAPDH) resulting in enzyme inhibition. To further characterize this auto‐ADP‐ribosylation reaction we studied alcohol dehydrogenase (ADH) and lactate dehydrogenase (LDH) for comparison. Whereas auto‐ADP‐ribosylation of ADH was stimulated to a minor extent by the NO‐liberating agent 3‐morpholinosydnonimine (SIN‐1), LDH was unaffected. The susceptibility of dehydrogenases towards autoADP‐ribosylation correlated with the potency of NO to decrease enzyme activity. Again, GAPDH was much more sensitive compared to ADH, whereas LDH again was unaffected. Interestingly, the efficiency of the SH‐alkylating agent N‐ethylmaleimide (NEM) to inhibit the enzymatic activity of the chosen dehydrogenases correlates with the sensitivity of dehydrogenases towards NO. These studies demonstrate the requirement of a reactive SH‐group besides the NAD+ binding site as a prerequisite for NO‐stimulated auto‐ADP‐ribosylation reactions. Furthermore, we establish that under physiological conditions and among the dehydrognases tested, only GAPDH is a potential target for this post‐translational protein modification mechanism.
A brain‐specific transcript from the 3′‐terminal region of the skeletal muscle ryanodine receptor gene
Authors: Takeshima Hiroshi , Nishimura Seiichiro , Nishi Miyuki , Ikeda Michiko , Sugimoto Tetsuo
Published: July 2002

We have shown previously that the skeletal muscle ryanodine receptor mRNA of ∼16,000 nucleotides codes 5,037 amino acid residues constituting the calcium release channel in skeletal muscle. In this study, RNA blot hybridization analysis shows that the brain contains an RNA species with an estimated size of ∼2,400 nucleotides hybridizable with the 3′‐terminal region of the skeletal muscle ryanodine receptor cDNA. cDNA cloning and genome analysis indicated that two transcripts differing in their start sites are produced from the skeletal muscle ryanodine receptor gene in a tissue‐specific fashion, and that the mRNA in brain may code the carboxyl‐terminal region of the ryanodine receptor molecule. cDNA expression experiments suggested that the ATG triplet encoding Met4382 of the skeletal muscle ryanodine receptor can function as a translation initiation codon, and that the expressed protein composed of the carboxy terminal 656 amino acid residues of the receptor is located on the endoplasmic reticulum membrane.
Formation of F2‐isoprostanes during aortic endothelial cell‐mediated oxidation of low density lipoprotein
Authors: Gopaul N.K. , Nourooz-Zadeh J. , Mallet A.I. , Änggård E.E.
Published: July 2002

We investigated the formation of F2‐isoprostanes produced by non‐enzymatic peroxidation of arachidonic acid during rabbit aortic endothelial cell‐mediated oxidation of low density lipoprotein (LDL). Free and total (sum of free and esterified) levels of F2‐isoprostanes were measured using a solid‐phase extraction procedure and gas chromatography‐mass spectrometry. Free levels of F2‐isoprostanes in native LDL were 0.06 ± 0.03 ng/mg protein (n = 4), whereas total levels were 0.28 ± 0.09 ng/mg protein (n = 4). Both free and total levels of the isoprostanes were found to increase during the oxidation. 8‐epi‐PGF2α was the major isoprostane formed (free and total concentrations after 24 h, 2.50 ± 0.24 and 6.42 ± 1.36 ng/mg protein (n = 4), respectively). The release of F2‐isoprostanes during aortic endothelial cell‐induced oxidation of LDL could be a contributory factor in the development of atherosclerosis.
Exposure to galactose oxidase of GM1 ganglioside molecular species embedded into phospholipid vesicles
Authors: Palestini Paola , Masserini Massimo , Tettamanti Guido
Published: July 2002

The exposure of GM1 molecular species present in the native ganglioside, carrying C18:1 or C20:1 long‐chain bases (LCB), to Dactylium dendroides galactose oxidase was studied. When native GM1 (49.3% C18:1 and 50.7% C20:1 LCB, respectively), was inserted in dipalmitoylphosphatidylcholine vesicles and partially oxidized (10%), the proportion of C18:1 and C20:1 species in the oxidized GM1 was 59.6% and 40.4%, respectively, suggesting a preferential action of the enzyme on the shorter species. The V
max of the enzyme was higher on C18:1 GM1 than on C20:1 GM1. The molecular species were affected without any preference after partial (10%) oxidation of GM1 incorporated in egg phosphatidylcholine vesicles or in micellar form. These data indicate that the exposure of the terminal galactose moiety of GM1 ganglioside to galactose oxidase is affected by the ganglioside ceramide composition as well as the phospholipid environment, that presumably determine the distribution (molecular dispersion, segregation) of the ganglioside within the membrane.
Rapid deactivation of MAP kinase in PC12 cells occurs independently of induction of phosphatase MKP‐1
Authors: Wu Jie , Lau Lester F. , Sturgill Thomas W.
Published: July 2002

Growth factors or serum can induce transcription and translation of a dual specificity MAP (mitogen‐activated protein) kinase phosphatase, MKP‐1 (MAP kinase phosphatase‐1). The role of induction of MKP‐1 (formerly 3CH134) in the rapid phase of MAP kinase deactivation was studied in rat pheochromocytoma (PC12) cells. MAP kinase was nearly completely deactivated in PC12 cells by 10 min after stimulation with epidermal growth factor (EGF) whereas MAP kinase activity remained elevated at 30% of the maximal response after stimulation with nerve growth factor. Protocols for treating cells with actinomycin D and cycloheximide were established that eliminate detection of MKP‐1 mRNA and protein in PC 12 cells. Treatment of PC12 cells with actinomycin D and cycloheximide did not affect the rapid deactivation of MAP kinase. Thus, the rapid phase of MAP kinase deactivation in PC12 cells is not dependent on the induction of the MAP kinase phosphatase MKP‐1.
Cysteines of chloroplast NADP‐malate dehydrogenase form mixed disulfides
Authors: Ocheretina Oksana , Scheibe Renate
Published: July 2002

Chloroplast NADP‐malate dehydrogenase (NADP‐MDH) from pea and from spinach was N‐terminally truncated by limited proteolysis with Staphylococcus aureus protease V8. The resulting monomeric enzymes lacking, respectively, the 37 and 38 N‐terminal amino acids were inactive. Reduction and addition of low concentrations of guanidine‐HCl (50–100 mM) resulted in a highly active enzyme of 850 units per mg protein. Equilibration of the truncated enzyme with various glutathione (GSH) redox buffers and assaying its activity in the presence of guanidine‐HCl was used to establish the existence of protein—GSH mixed disulfides. This finding was further confirmed using incorporation of radioactively labelled thiol. The possible function of such cysteine modifications under oxidative stress and their regeneration by the thioredoxin system in the light is discussed.
Molecular cloning and characterization of Byp, a murine receptor‐type tyrosine phosphatase similar to human DEP‐1
Authors: Kuramochi Satomi , Matsuda Satoru , Matsuda Yoichi , Saitoh Toshiyuki , Ohsugi Miho , Yamamoto Tadashi
Published: July 2002

Novel murine cDNAs encoding a receptor‐like protein tyrosine phosphatase, termed Byp (HPTP beta‐like tyrosine phosphatase) were cloned. The putative Byp protein consists of 1238 amino acids, which possesses a single catalytic domain in the cytoplasmic region. The extracellular region comprises eight repeats of a fibronectin type III module and contains multiple N‐glycosylation sites. The byp mRNA was 7.7‐kb long and expressed in every tissue examined, its level being high in the brain and kidney. Transfection of the byp cDNA expression plasmid into COS7 cells resulted in the expression of a 220‐kDa tyrosine phosphorylated protein. Furthermore, co‐expression of Byp and the Src family kinase Fyn increased the level of tyrosine phosphorylation of Byp, suggesting that Byp was tyrosine‐phosphorylated by Fyn. Finally, the byp gene was located to mouse chromosome 2E1–2E2 and rat chromosome 3832–3833.
Ceramide induces apoptosis via CPP32 activation
Authors: Mizushima Noboru , Koike Ryuji , Kohsaka Hitoshi , Kushi Yasunori , Handa Shizuo , Yagita Hideo , Miyasaka Nobuyuki
Published: July 2002

Although both ceramide and interleukin‐1β converting enzyme (ICE) family proteases are key molecules during apoptosis, their relationship remains to be elucidated. We report here that cell‐permeable ceramide induced cleavage and activation of CPP32, a Ced‐3/ICE‐like protease, but not ICE. Ceramide‐induced apoptosis of Jurkat cells was blocked by the CPP32‐specific tetrapeptide inhibitor DEVD‐CHO, but not by the ICE inhibitor YVAD‐CHO. Furthermore, variant Jurkat cells with defective CPP32 activation were resistant to both antiFas‐ and ceramide‐induced apoptosis. These results indicate that CPP32 activation is required for ceramide‐induced apoptosis, and suggest sphingomyelin‐ceramide pathway functions upstream of CPP32.
Regulation of Mg2+ homeostasis by insulin in perfused rat livers and isolated hepatocytes
Authors: Keenan Donald , Romani Andrea , Scarpa Antonio
Published: July 2002

Several recent studies demonstrate that adrenergic receptor stimulation evokes marked changes in Mg2+ homeostasis. As insulin counter‐regulates many of the metabolic consequences of adrenergic receptor stimulation, we evaluated the potential influence of insulin on Mg2+ movements in response to adrenergic stimulation. The data demonstrate that insulin is able to block the Mg2+ efflux from perfused rat livers stimulated by isoproterenol or 8‐Br‐cAMP, but has little or no effect on epinephrine or phenylephrine induced Mg2+ efflux. Thus, evidence is provided demonstrating that there are redundant adrenergic pathways regulating Mg2+ efflux from liver tissue. One of these pathways, the β‐adrenergic component, is selectively blocked by insulin. Furthermore, these findings may provide a cellular explanation for hypomagnesemia associated with diabetes.
Nerve growth factor treatment of adult rats selectively enhances innervation of urinogenital tract rather than vascular smooth muscle
Authors: Milner P. , Crowe R. , Fernyhough P. , Diemel L.T. , Tomlinson D.R. , Burnstock G.
Published: July 2002

Following treatment of adult rats with nerve growth factor (0.5 mg/rat, three times a week for 3 weeks), the innervation of cardiovascular and urinogenital tract smooth muscle was investigated using immunoassay and immunohistochemical techniques. Substance P and calcitonin gene‐related peptide levels were increased in the vas deferens, but not in the atria or femoral artery. Neuropeptide Y and vasoactive intestinal polypeptide levels were unchanged. In penile tissues, there was a marked increase in the density of substance P‐, calcitonin gene‐related peptide‐, neuropeptide Y‐, tyrosine hydroxylase‐ and vasoactive intestinal polypeptide‐containing nerves innervating the urethra and in SP‐containing nerves in the tunica with little change in the innervation of the deep dorsal vein and artery and corpus cavernosum. In the bladder, there was increased innervation of the detrusor by neuropeptide Y‐ and vasoactive intestinal polypeptide‐containing nerves, but a decrease in innervation by substance P‐containing nerves in the trigone. There were no changes in the density of innervation of the femoral artery after nerve growth factor treatment. Thus, in the mature rat, sensory and sympathetic nerves innervating urinogenital tract smooth muscle appear to be more responsive to exogenous nerve growth factor than those innervating cardiovascular smooth muscle. This may reflect an ongoing requirement of plasticity of innervation in the urinogenital tract of the sexually mature animal.
A Raf‐induced, MEK‐independent signaling pathway regulates atrial natriuretic factor gene expression in cardiac muscle cells
Authors: Jette Cicely , Thorburn Andrew
Published: July 2002

The atrial natriuretic factor (ANF) gene is activated in cardiac myocytes by Ras and its effector Raf. However, MEK, the best‐characterized Raf substrate, cannot efficiently activate ANF suggesting that Raf uses a MEK‐independent pathway to activate ANF. By manipulating MEK and Raf activities so that they are equally effective at activating ERK, we now demonstrate that Raf activates at least two signaling pathways in cardiac myocytes that regulate the ANF promoter; the MEK→ERK pathway inhibits ANF gene expression while a Raf‐induced, MEK‐independent pathway activates expression. This mechanism may provide increased ability to regulate ANF expression in response to hypertrophic stimuli.
Molecular cloning and functional characterization of the zebrafish ATP‐gated ionotropic receptor P2X3 subunit
Authors: Egan Terrance M. , Cox Jane A. , Voigt Mark M.
Published: July 2002

We describe a P2X subunit cloned from the zebrafish (Danio rerio) that is an orthologue of the mammalian P2X3 subunit. Like the mammalian P2X3, this receptor desensitizes rapidly in the presence of agonist. However, it differs in that αβ‐meATP is a much less potent agonist than ATP and the antagonist TNP‐ATP is not active at low nanomolar concentrations. Similar to the rat P2X3 subunit, the zebrafish subunit forms hetero‐oligomeric assemblies with the rat P2X2 that possesses a phenotype distinct from either parent. This novel clone will provide an important basis for future experiments investigating the structure/function relationships of P2X subunit domains.
The direct association of the multiple PDZ domain containing proteins (MUPP‐1) with the human c‐Kit C‐terminus is regulated by tyrosine kinase activity
Authors: Mancini Annalisa , Koch Alexandra , Stefan Monica , Niemann Heiner , Tamura Teruko
Published: July 2002

We have identified the multiple PDZ domain containing protein (MUPP‐1 or MPDZ) as a novel binding partner of the human c‐Kit. c‐Kit binds specifically to the 10th PDZ domain of MUPP‐1 via its C‐terminal sequence. Furthermore, a kinase negative‐mutant receptor interacted more strongly with MUPP‐1 than the wild‐type c‐Kit. Strikingly, a constitutively activated c‐Kit (D816V‐Kit) did not bind to MUPP‐1, although this oncogenic form retains the PDZ binding motif ‘HDDV’ at the C‐terminal end. Deletion of V967 of c‐Kit abolished binding to MUPP‐1 and drastically reduced its tyrosine kinase activity, suggesting that the structure of the C‐terminal tail of c‐Kit influences its enzymatic activity.
Phosphorylation site specificity of the Pak‐mediated regulation of Raf‐1 and cooperativity with Src
Authors: King Alastair J. , Wireman Randall S. , Hamilton Mark , Marshall Mark S.
Published: July 2002

The p21‐activated kinase, Pak, has recently been shown to phosphorylate Raf‐1 on serine 338 (S338), a critical regulatory residue. The specificity requirements for Pak‐mediated phosphorylation of S338 were examined by substitution analysis of Raf‐1 peptides and conserved region 3 (CR3) proteins. Phosphorylation was found to be very sensitive to alterations in amino acid side chains proximal to S338. Loss of N‐terminal arginines resulted in decreased peptide phosphorylation while loss of these residues, as well as C‐terminal glutamates and bulky C‐terminal hydrophobic residues, decreased phosphorylation of the CR3 protein. Phosphorylation of Raf‐1 on tyrosine 341 is significant in epidermal growth factor‐ and Src‐mediated signaling, suggesting that cooperativity may exist between Pak and Src phosphorylation of Raf‐1. Purified Pak and Src were found not to be cooperative in phosphorylating peptides or purified CR3 protein. However, the phosphorylation of Raf‐1 S338 by Pak was increased in the presence of Src. The complexity of this signaling module could thus account for the different levels of Raf‐1 activation required for fulfillment of different biological roles within the cell.