bluuebunny/crossref_metadata_2025 huggingface dataset:
Modulation by phosphorylation of glycogen phosphorylase‐sarcoplasmic reticulum interaction
Authors: Cuenda Ana , Centeno Francisco , Gutierrez-Merino Carlos
Published: July 2002

Glycosen phosphorylase b at concentrations close to those found in skeletal muscle interacts with sarcoplasmic reticulum membranes, but not with liposomes made of lipids extracted from these membranes, and is inhibited upon binding to the membrane. The interaction of glycogen phosphorylase with the sarcoplasmic reticulum membrane is modulated by phosphorylation, for the a form of this enzyme shows a K
0.5 of interaction about 10‐fold lower than the b form. Upon association to the membrane the fluorescence properties of the coenzymes of glycogen phosphorylase, pyridoxal‐5′‐phosphate, are strongly altered, for the fluorescence at 535 nm is partially quenched and the fluorescence at 415–420 nm increases. Using fluorescein labeled sarcoplasmic reticulum membranes we have found that average conformation of the Ca2+ + Mg2+‐ATPaze is also altered on binding or phosphorylase b. In conclusion, the results reported in this paper suggest that glycogen phosphorylase and Ca2+ + Mg2+‐ATPase directly interact under experimental conditions similar to those found in the sarcoplasm, and that this interaction is modulated by phosphorylation of the phosphorylase.
Keratinization is associated with the expression of a new protein related to the desmosomal cadherins DGII/III
Authors: King Ian A. , Magee Anthony I. , Rees David A. , Buxton Roger S.
Published: July 2002

Amino acid sequencing of a 48/46 kDa glycoprotein from human plantar callus, recognised by antisera raised against the desmosomal cadherins DGII/III has revealed N‐terminal homology to the DNA‐derived sequence of human and bovine DGII/III. However, a tryptic fragment has homology only with a bovine clone. We propose that there are two classes of DGII/III‐like molecule, that represented by the bovine cDNA clone and the 48/46 kDa protein, a monoclonal antibody against which stains mainly the suprabasal layers of human epidermis, and that represented by the human cDNA clone, identified by a monoclonal antibody which stains uniformly the living layers of the epidermis.
Shaw‐like rat brain potassium channel cDNA's with divergent 3′ ends
Authors: Luneau Christopher , Wiedmann Richard , Smith Jeffrey S. , Williams Jacinta B.
Published: July 2002

The complete amino acid sequence of a potassium channel protein of rat brain, Kv3.2b, plus a partial sequence of a related channel, Kv3.2c, are deduced from molecular cloning of the respective cDNA's. Kv3.2b and Kv3.2c share extensive amino acid sequence identity with a previously identified channel, RKShIIIA[1], before diverging to unique carboxy termini. Probes specific for Kv3.2b and RKShIIIA detect similarly sized mRNA's on Northern blots. These two proteins are encoded by a single gene based on genomic Southern blotting, and therefore arise by alternative splicing. In vitro transcribed mRNA for Kv3.2b induces the expression of outward K+ currents in Xenopus oocytes under voltage‐clamp conditions.
Identification of different receptor types for toxic phospholipases A2 in rabbit skeletal muscle
Authors: Lambeau Gérard , Barhanin Jacques , Lazdunski Michel
Published: July 2002

Oxyuranus scutellatus scutellatus toxins I (OS1) and 2 (OS2) are two phospholipase A2s (PLA2) isolated from the venom of the Australian Taipan snake. Their iodinated derivatives have been used to characterize PLA2 binding sites on rabbit skeletal muscle. Competition and cross‐linking experiments indicate that 125I‐labelled OS2 binding sites in rabbit skeletal muscle in vivo are distributed into two classes of receptors. One class binds OS2 and OS1 and is insensitive to the bee venom PLA2. It is composed of a 180 kDa binding protein. This class of PLA2 receptor is expressed at a high level in rabbit myotube membranes. The other class of PLA2 receptor identified with 125I‐OS2 also binds with high affinity the bee venom PLA2 but not OS1 and is composed of major polypeptides of 34.48 and 82 kDa. This second class of receptor is similar to the one found in brain membranes. The density of the two classes of receptors varies during muscle development.
TPA‐induced activation of MAP kinase
Authors: Adams Peter D. , Parker Peter J.
Published: July 2002

Threonine and tyrosine residue phosphorylation of a 42 kDa protein identified as mitogen‐activated protein kinase (MAP kinase) was stimulated in extracts from TPA‐pretreated cells. It is further shown that TPA pretreatment leads to the enhancement of an activity that will induce reactivation of dephosphorylated/inactivated MAP kinase. This TPA‐induced activity induces the threonine and tyrosine phosphorylation of p42 in extracts from unstimulated cells.
Autocrine receptors for endothelins in the primary culture of endothelial cells of human umbilical vein
Authors: Fujitani Yasushi , Oda Kyoko , Takimoto Misato , Inui Takashi , Okada Toshikazu , Urade Yoshihiro
Published: July 2002

Human umbilical vein endothelial cells (HUVECs) in primary culture produced and secreted endothelin I (ET‐1) actively. Specific binding of [125I]ET‐1 to these cells was not detectable because of the saturation of ET receptors with endogenously produced ET‐1. However, addition of phosphoramidon, an inhibitor of ET‐converting enzyme, to the medium reduced the production of ET‐1 and thus the receptors on HUVECs were made available for exogenously added [125I]ET‐1. Binding studies using phosphoramidon‐treated HUVECs indicated the existence of a non‐isopeptide‐selective type (ETB) or ET receptor with a K
d of 17 pM. This receptor is thought to be involved in ET‐induced vasodilation in autocrine manner in vivo.
Herbimycin A blocks IL‐1‐induced NF‐κB DNA‐binding activity in lymphoid cell lines
Authors: Iwasaki Takaaki , Uehara Yoshimasa , Graves Lee , Rachie Nisa , Bomsztyk Karol
Published: July 2002

The mechanism by which interleukin‐1α (IL‐1α) activates NF‐κB DNA‐binding activity is not completely understood. While it is well established that protein kinase C can activate NF‐κB, neither protein kinase C nor protein kinase A appears to be critical in the induction of NF‐κB by IL‐1α. Since a number of growth factors signal via protein tyrosine kinase, in this study we examined a possible involvement of protein tyrosine kinase in the IL‐1α‐induced NF‐κB. The results showed that in the murine pre‐B cell line 7OZ/3 and in the murine T cell line EL‐4 6.1 C10 IL‐1α‐induced NF‐κB was associated with transient increase in protein tyrosine kinase activity. Pre‐treatment of these cell lines with herbimycin A, an inhibitor of tyrosine kinase activity, blocked the IL‐1α‐enhanced protein tyrosine kinase activity and the IL‐1α‐induced NF‐κB DNA‐binding activity. Herbimycin A at concentrations sufficient to block IL‐1α‐induced NF‐κB did not block the phorbol 12‐myristate 13‐acetate(PMA)‐induced NF‐κB. The data suggest that IL‐1α and PMA activate NF‐κB by different pathways and that induction of NF‐κB DNA‐binding activity by IL‐1 might be dependent on protein tyrosine phosphorylation.
Time course of K+ current inhibition by low oxygen in chemoreceptor cells of adult rabbit carotid body Effects of carbon monoxide
Authors: López-López J.R. , González C.
Published: July 2002

K+ currents recorded from adult rabbit chemoreceptor cells are reversibly inhibited on lowering the pO2 in the bathing solution. Bath application of a hypoxic TTX‐containing solution revealed that inhibition of K+ current by low pO2, proceeds faster than TTX inhibition of Na+ currents, the apparent t½ being 3.68 and 7.14 s, respectively. Addition of carbon monoxide to the hypoxic gas mixture used to equilibrate the bathing solution reversed the inhibition of K+ currents by approx. 70%.
Cyclosporin A protects hepatocytes subjected to high Ca2+ and oxidative stress
Authors: Brockemeier Kimberly M. , Carpenter-Deyo Laurie , Reed Donald J. , Pfeiffer Douglas R.
Published: July 2002

Hepatocytes incubated with 0.8 mM t‐butylhydroperoxide are protected by cyclosporin A when the medium Ca2+ concentration is 10 mM, but not when it is 2.5 mM. The highest Ca2+ level is associated with an inhibition of t‐butylhydroperoxide‐dependent malondialdehyde accumulation and with mitochondrial Ca2+ loading within the cells. These findings are new evidence that t‐butylhydroperoxide can kill cells by peroxidation‐dependent and ‐independent mechanisms, and suggest that the mitochondrial permeability transition and the resultant de‐energization are components of the peroxidation‐independent mechanism. Cyclosporin A may have considerable utility for the protection of cells subjected to oxidative stress.
Measurement of GTPγS binding to specific G proteins in membranes using G‐protein antibodies
Authors: Okamoto Takashi , Ikezu Tsuneya , Murayama Yoshitake , Ogata Etsuro , Nishimoto Ikuo
Published: July 2002

We developed a novel method to quantitatively measure GTPγS binding to specific G proteins in crude membranes using G‐protein antibodies. The basic strategy was that the materials were initially incubated with [35S]GTPγS at 37°C. After 4°C incubation in the wells of an ELISA plate precoated with G‐protein antibodies, the radioactivity of each well was counted. This method, using an anti‐Gi antiserum and an anti‐G3 antiserum, quantitatively and specifically detected the binding of GTPγS to purified G12 and G3. In S49 cell membranes, GTPγS binding to immunoreactive G3 was observed in a time‐dependent manner that obeyed first order kinetics, and the rate constant was stimulated ∼tworold in response to isoproterenol. The effect of isoproterenol was not observed in unc mutant membranes. The present method thus makes it possible to quantitatively measure GTPγS binding to specific G proteins in cell membranes.
A voltage‐gated calcium channel is linked to the antigen receptor in Jurkat T lymphocytes
Authors: Densmore John J. , Szabo Gabor , Gray Lloyd S.
Published: July 2002

Activation of T lymphocytes results in an increase in intracellular Ca2+ due in large part to influx of extracellular Ca2+. Using the patch clamp technique, an inward current in Jurkat T lymphocytes was observed upon depolarization from a holding potential of −90 mV but not from −60 mV. This whole‐cell current was insensitive to tetrodotoxin, carried by Ba2+, and blocked by Ni2+. Occupancy of the T lymphocyte antigen receptor increased the current's magnitude. These data suggest that antigen receptor‐induced Ca2+ entry in T lymphocytes may be mediated by a voltage‐regulated Ca channel.
Thrombin‐induced proliferation and expression of platelet‐derived growth factor‐A chain gene in human vascular smooth muscle cells
Authors: Kanthou Chryso , Parry Graham , Wijelath Errol , Kakkar Vijay Vir , Demoliou-Mason Catherine
Published: July 2002

Treatment of human vascular smooth muscle cells (SMC) with human α‐thrombin greatly increased DNA synthesis and cell proliferation. Both the integrity of the catalytic site and that of the anion binding exosite were required for expression of this activity. Experiments employing Northerns indicated induction of c‐ƒos expression as well as a time‐dependent induction of platelet‐derived growth factor‐A (PDGF‐A) gene by thrombin. The thrombin mitogenic activity was potentiated by PDGF‐BB, insulin and the vasoconstrictor peptide endothelin‐1 suggesting synergism by convergence of intracellular growth‐promoting signals. SMC treatment with pertussis toxin and forskolin indicated that the mitogenic activity of thrombin may be induced via signal transduction mechanism(s) involving changes in cAMP levels and activation of a Gi‐like protein. These results suggest that thrombin may play a functional role in the regulation of human vascular SMC proliferation.
Deduced amino acid sequence and E1–E2 equilibrium of the sarcoplasmic reticulum Ca2+‐ATPase of frog skeletal muscle Comparison with the Ca2+‐ATPase of rabbit fast twitch muscle
Authors: Vilsen Bente , Andersen Jens Peter
Published: July 2002

The cDNA encoding a Ca2+‐transport ATPase of frog (Rana esculenia) skeletal muscle was isolated and characterized. The deduced amino acid sequence, consisting of 994 residues, showed 89% identity to the fast twitch muscle sarcoplasmic reticulum Ca2+‐ATPases of chicken and rabbit. Northern blot analysis using a fragment of this cDNA as probe detected a 5.0 kb message in frog skeletal muscle but did not detect any mRNA encoding sarcoplasmic reticulum Ca2+‐ATPase in frog cardiac muscle. The enzymatic properties of the amphibian skeletal muscle Ca2+‐ATPase were compared with those of the rabbit fast twitch muscle Ca2+‐ATPase by functional expression of the cDNAs in COS‐1 cells. The amphibian Ca2+‐ATPase displayed a reduced apparent affinity for Ca2+ and an increased apparent affinity for the inhibitors, vanadate and thapsigargin, relative to the mammalian enzyme. This may be explained by a mechanism in which relatively more of the E2 conformation accumulated 1n the frog Ca2+‐ATPase than in the mammalian enzyme.
Fast and slow kinetics of porin channels from Escherichia coli reconstituted into giant liposomes and studied by patch‐clamp
Authors: Berrier Catherine , Coulombe Alain , Houssin Christine , Ghazi Alexandre
Published: July 2002

E. coli porins (OmpF and OmpC) were purified and reconstituted into liposomes which were enlarged to giant proteoliposomes by dehydration—rehydration and studied by patch‐clamp. The porins could be closed by voltage pulses under −100mV. The kinetics of closure was slow, with closure events of about 200 pS in 0.1 M KCl. Rapid fluctuations (in the millisecond range) of about one third (60–70 pS) of the large closure steps were also observed. The data are interpreted as follows: an increase in membrane potential favours the cooperative transition of multimers towards an inactivated state, while monomers which have not been inactivated can flicker rapidly between an open and a short‐lived closed state.
Comparative analysis of sodium‐dependent l‐glutamate transport of synaptosomal and astroglial membrane vesicles from mouse cortex
Authors: Rauen Thomas , Jeserich Gunnar , Danbolt Niels C. , Kanner Baruch I.
Published: July 2002

Uptake of [3H]l‐glutamate into membrane vesicles prepared from either mouse cortical astrocyte cultures or synaptosomes was found to be an electrogenic sodium‐ and potassium‐dependent transport process with saturable uptake kinetics. Pharmacological differences were revealed by using a variety of substrate analogues. l‐trans‐PDC inhibited the synaptosomal glutamate transport 2–4‐fold stronger than the astroglial uptake. The substrate analogues dl‐threo‐β‐hydroxy‐aspartate, dl‐aspartate‐β‐hydroxamate, l‐aspartate and d‐aspartate inhibited glutamate transport of astroglial and neuronal membrane vesicles in a distinctive manner, whereas d‐glutamate, quisqualate and dihydrokainate had no effect in either case. Immunoblotting and immunocytochemical labeling with antibodies against the rat brain glutamate transporter revealed the selective reaction of a band at about 75 kDa mol. wt. and a specific pattern of astrocyte immunostaining.
The single‐copy gene psbS codes for a phylogenetically intriguing 22 kDa polypeptide of photosystem II
Authors: Wedell N. , Klein R. , Ljungberg U. , Andersson B. , Herrmann R.G.
Published: July 2002

Recombinant phages that encode the complete precursor polypeptide for the 22 kDa polypeptide associated with photosystem 11 have been serologically selected from two λgt11 expression libraries made from polyadenylated RNA or spinach seedlings. The cDNAs hybridize to a 1.3 kb RNA species. The precursor protein is comprised of 274 amino acid residues and carries an N‐terminal transit peptide of probably 69 amino acid residues. The mature protein exhibits four, predicted transmembrane segments and is shown to be an integral component of photosystom 11 originating in a single‐copy gene. The unique characteristics of this protein are: (1) it is the result of a gene‐internal duplication of an ancestor with two membrane spans. (ii) a striking resemblance to LHC 1/11, CP24/CP29 apoproteins, and ELIPs, although it does not bind chlorophyll and is present in cyanobacteria, and, as these proteins, (iii) it integrates into the membrane with uncleaved routing signals that display remarkable resemblance to patterns found in bipartite transit pepticies.
Tumor necrosis factor α stimulates sphingomyelinase through the 55 kDa receptor in HL‐60 cells
Authors: Yanaga Fumi , Watson Steve P.
Published: July 2002

Tumor necrosis factor α (TNFα) stimulated rapid (seconds) hydrolysis of sphingomyelin in HL‐60 cells, formation of phosphocholine (PCho) and a decrease in choline. The response to TNFα was concentration dependent with a maximal effect at 3–10 nM. The monoclonal antibody (mAb). htr‐9, which behaves as an agonist at the 55 kDa subtype of the TNF receptor, also stimulated sphingomyelin hydrolysis in intact cells. In contrast, the mAb, utr‐1, which behaves as an antagonist at the 75 kDa receptor subtype, had no effect on sphingomyelin hydrolysis either on its own or in the presence of TNFα. In addition, htr‐9 or TNFα stimulated hydrolysis of sphingomyelin in a membrane fraction of HL‐60 cells. These results are consistent with a role of sphinpomyelin hydrolysis as an early event in the signalling mechanism or TNFα, and suggest that this pathway is activated through the 55 kDa subtype of the TNF receptor.
Bacterial expression of an active tyrosine kinase from a protein A/truncated c‐src fusion protein
Authors: Saya Hideyuki , Lee Polly S.Y. , Nishi Toru , Izawa Ichiro , Nakajima Motowo , Gallick Gary E. , Levin Victor A.
Published: July 2002

The carboxy‐terminal half of the c‐src protein fused to the protein A moiety was expressed in bacteria. The protein A/truncated c‐src fusion protein, which does not have SH2 and SH3 domains, is found in the periplasmic space allowing for a simple one‐step purification and demonstrated high efficiency in autophosphorylation and exogeneous substrate phosphorylation. The missense mutation at codon 294 (Ile → Thr), which is located in the ATP‐binding domain of the c‐src, resulted in dramatic reduction of tyrosine kinase activity of the fusion protein. Using the fusion protein. we also revealed that staurosporin, a well‐known kinase inhibitor, directly affects autophosphorylation of the C‐terminal half of the c‐src protein. This truncated c‐src expression system provides a good source of enzyme for diverse experiments and is an ideal model for understanding the implication of structural alterations in the catalytic activity of the c‐src kinase by site‐directed mutagenesis experiments.
The expression of the abnormal human red cell anion transporter from South‐East Asian ovalocytes (band 3 SAO) in Xenopus oocytes
Authors: Groves Jonathan D. , Ring Susan M. , Schofield Ann E. , Tanner Michael J.A.
Published: July 2002

South‐East Asian ovalocytosis (SAO) is caused by the heterozygous presence of a variant form of the human erythrocyte anion transporter (band 3; AE1). The expression of band 3 SAO has been studied in Xenopus oocytes. Band 3 SAO is not functional as an anion transporter but is inserted stably into the plasma membrane of oocytes. Band 3 SAO translocation to the cell surface does not require co‐expression of normal band 3. Co‐expression of glycophorin A (GPA) increases the rate of translocation of band 3 SAO to the oocyte membrane but is not essential for this process. We suggest that the increased tendency of band 3 SAO to form oligomers may facilitate its translocation to the cell surface.
A complex of rab3A, SNAP‐25, VAMP/synaptobrevin‐2 and syntaxins in brain presynaptic terminals
Authors: Horikawa Hiroshi P.M. , Saisu Hideo , Ishizuka Toru , Sekine Yoko , Tsugita Akira , Odani Shoji , Abe Teruo
Published: July 2002

Two monoclonal antibodies (SPM‐1 and SPM‐2) immunoprecipitate brain N‐type calcium channels. On immunoaffinity chromatography of digitonin extracts of bovine brain membranes on SPM‐1‐ and SPM‐2‐Sepharose, proteins of 36 (syntaxins A and B), 28 and 19 kDa are specifically retained by both columns. Here we show that the 19 and 28 kDa bands contain VAMP/synaptobrevin‐2, and rab3A/smg25A and SNAP‐25, respectively. Since SPM‐1 and SPM‐2 recognize only syntaxins and the 28 kDa band (rab3A/smg25A and SNAP‐25), respectively, the results indicate that all these proteins form a complex. Our results suggest tight linkage between the components involved in neurotransmitter release.
Effects of dexamethasone on L‐type calcium currents in the A7r5 smooth muscle‐derived cell line
Authors: Obejero-Paz Carlos A. , Lakshmanan Mark , Jones Stephen W. , Scarpa Antonio
Published: July 2002

Patch clamp experiments were used to characterize the effect of dexamethasone on calcium currents in A7r5 cells. Pretreatment for 48 h with 200 nM dexamethasone did not affect the single channel conductance, the voltage dependence of channel opening, or the voltage‐dependent inactivation of L‐type channels. However, dexamethasone caused an approximately 2‐fold increase in the amplitude of L‐type calcium currents in 5 out of 9 experiments, suggesting an increase in the number of active channels. The effect of dexamethasone appeared to be greatest on batches of cells with low control current density. The amplitude of T‐type calcium current was not affected by dexamethasone.
Variant plasma gelsolin responsible for familial amyloidosis (Finnish type) has defective actin severing activity
Authors: Weeds A.G. , Gooch John , McLaughlin P. , Maury C.P.J.
Published: July 2002

Familial amyloidosis, Finnish type is caused by a single base mutation in gelsolin, an actin filament severing and capping protein that is present in most tissues and in blood plasma. The mutation replaces aspartic acid with asparagine at residue 187 of the plasma sequence. This renders the gelsolin susceptible to proteolysis as a consequence of which amyloid protein is formed. Here it is shown that the mutant protein in plasma from a patient homozygous for this mutation lacks both actin severing and nucleating activities. Evidence is presented that the cleaved mutant gelsolin has dissociated under non‐denaturing conditions and that the resultant 65,000 and 55,000 M
r C‐terminal fragments aggregate.
Non‐radioactive reverse transcriptase/polymerase chain reaction for quantification of myosin heavy chain mRNA isoforms in various rabbit muscles
Authors: Peuker Heidemarie , Pette Dirk
Published: July 2002

A method was established for measuring molecule numbers of three different myosin heavy chain (MHC) mRNA isoforms in total RNA preparations. The quantification was based on a combination of primer‐directed reverse transcriptase and polymerase chain reactions with 5'‐digoxigenin‐labeled oligonucleotides, using external standards. The sensitivity of the method allowed the quantitation of mRNA amounts down to the range of 1,000 molecules (detection limit 50 molecules). The numbers determined for eight different rabbit muscles are in the range of 103–109/μg total RNA. In soleus muscle, the value of 1.11 × 109 MHCI mRNA molecules corresponds to approximately 8% of the total mRNA. With reference to myonuclei, this amount corresponds to 1–2 × 104 molecules/nucleus. A quantitative comparison of the two fast MHC mRNA isoforms with the distribution of different MHC isoforms at the protein level indicates that one of these two fast sequences is specific to MHCIIb and the other to MHCIId. However, our data point to the existence of additional MHCIId mRNA subtypes.
What is hemimethylated DNA?
Authors: Adams R.L.P. , Lindsay H.
Published: July 2002

De novo methylation of a CG dinucleotide pair by murine DNA methyltransferase is stimulated by the presence of a single methylcytosine positioned close to the target site.
Identification of an actin binding region in aldolase
Authors: O'Reilly Gabrielle , Clarke Frank
Published: July 2002

Fragmentation of the actin binding glycolytic enzyme, aldolase, with cyanogen bromide yields an 18K actin binding fragment which corresponds to residues 1–164 of the aldolase sequence. Within this fragment there is a region of sequence (residues 32–52) which is highly homologous to a region of sequence near the C‐terminus of actin itself and which is also found in the actin binding domains of a number of other actin binding proteins. A synthetic peptide corresponding to the aldolase sequence 32–52 encompassing this region of homology binds to F‐actin and specifically competes with native aldolase for binding to this cytoskeletal protein.
Super‐secondary structures involving triple‐strand β‐sheets
Authors: Efimov Alexander V.
Published: July 2002

Triple‐strand β‐sheets having up‐and‐down topology are widespread in proteins and occur in two forms denoted here as S‐like and Z‐like β‐sheets. In many cases they are included in super‐secondary structures of higher order. A number of such structures is described in this paper. An important feature of these super‐secondary structures is that they have a unique handedness. Another feature is that some of them only involve S‐like β‐sheets and others only Z‐like β‐sheets.
The affinity‐labelling of cathepsin s with peptidyl diazomethyl ketones
Authors: Shaw Elliott , Mohanty Sasank , Colic Adrijana , Stoka Veronika , Turk Vito
Published: July 2002

Since peptidyl diazomethyl ketones are useful irreversible inhibitors for inactivating cysteinyl proteinases in vitro and in vivo and in order to reveal their role, we set out to obtain selective and effective reagents for cathepsin S. A number of such derivatives with hydrophobic amino acid residues, such as valine, leucine and tryptophane in positions adjacent to the primary specificity site were synthesized and these provided inhibitors rapidly acting at high dilution. For example, 1 nM Z‐Leu‐Leu‐Nle‐CHN2 inactivates cathepsin S with k
2nd = 4.6 × 106 M−1.s−1 at pH 6.5,25°C. Similarities to the specificities of cathepsin L and calpain were evident. However, Z‐Val‐Val‐NleCHN2 is over 300 times more effective in inactivating S than L. On the other hand, Z‐Phe‐Tyr(t‐Bu)CHN2 is about 104 more effective against L than S. Reagents are thus now available for a clear discrimination between these proteases.
Mammalian calponin
Authors: Strasser Peter , Gimona Mario , Moessler Herbert , Herzog Monika , Small J.Victor
Published: July 2002

Calponin is a smooth muscle specific, actin‐, tropomyosin‐ and calmodulin‐binding protein thought to be involved in some way in the regulation or modulation of contraction. Here we describe the cloning and bacterial expression of two calponin species from murine and porcine smooth muscle tissues. Primary and secondary structural analyses of the deduced amino acid sequences revealed a high degree of homology to avian calponin with the exception of a short and variable C‐terminal segment. The sequence data demonstrate that the two mammalian calponin variants do not arise via alternative splicing but are encoded by different genes.
Osteoblast‐like cells have a variable mixed population of purino/nucleotide receptors
Authors: Yu Hesheng , Ferrier Jack
Published: July 2002

Osteoblast‐like UMR 106.06 cells respond to extracellular application of nucleotides with a fast intracellular calcium pulse (latency of about 20 s, half‐width of about 10 s), as measured with fluo‐3 on a confocal laser scanning system. Cross‐inhibition experiments at 50 μM show that, on a cell population basis, adenosine triphosphate (ATP) strongly inhibits the effect of uridine triphosphate (UTP) or 2‐methylthio‐ATP (2‐MeSATP) applied within 2 min after the end of the ATP‐induced pulse, while prior application of UTP or 2‐MeSATP only weakly inhibits the ATP effect, and UTP and 2‐MeSATP weakly inhibit each other. Furthermore, there are clear differences in cross‐inhibition responses between individual cells. Our measurements provide strong evidence that these cells have at least two types of purino/nucleotide receptors, probably P2y and P2u, with a proportion that varies between individual cells.
The human rod photoreceptor cGMP phosphodiesterase β‐subunit
Authors: Khramtsov N.V. , Feshchenko E.A. , Suslova V.A. , Shmukler B.E. , Terpugov B.E. , Rakitina T.V. , Atabekova N.V. , Lipkin V.M.
Published: July 2002

cDNA clones encoding the β‐subunit of the photoreceptor cGMP phosphodiesterase (PDE) were isolated from a human retina library and their sequence was determined. The encoded polypeptide consists of 854 amino acid residues with a calculated molecular mass of 98,416 Da. Alignment of the deduced amino acid sequence with the earlier analysed α‐, β‐ and α'‐subunits of bovine and mouse PDEs demonstrates a high homology. Two overlapping recombinant λ phage clones containing 26 kb of the human PDE β‐subunit gene were isolated from the genomic library. A total nucleotide sequence of exons 4–22 of the PDE β‐subunit gene was established which completely corresponded to the cDNA structure. According to sequence analysis no potential possibility for alternative splicing of the β‐subunit gene was observed between exons 20 and 21 which led to the formation of the β'‐subunit as described for mouse PDE. Polymerase chain reaction (PCR) experiments also confirm the absence of the PDE β'‐subunit in human retina.
Cloning and sequencing of the cyclodiene insecticide resistance gene from the yellow fever mosquito Aedes aegypti
Authors: Thompson Michael , Shotkoski Frank , ffrench-Constant Richard
Published: July 2002

In order to examine the conservation of the mechanism of cyclodiene insecticide resistance between species we cloned a cDNA from the yellow fever mosquito Aedes aegypti homologous to the resistance gene Rdl in Drosophila. In D. melanogaster, resistance to cyclodienes and picrotoxinin is caused by a single amino acid substitution (alanine to serine) in the putative channel lining of a γ‐aminobutyic acid gated chloride ion channel. We report that the mosquito gene not only shows high homology to that of Drosophila but also that resistant strains display substitution of the same amino acid. The significance of this result in relation to the evolution of pesticide resistance, the use of Drosophila as a model insect for resistance studies and the potential use of this gene as a selectable marker in the genetic transformation of non‐Drosophilids is discussed.
Hepatic apolipoprotein B synthesis in copper‐deficient rats
Authors: Nassir Fatiha , Mazur Andrzej , Sérougne Colette , Gueux Elyett , Rayssiguier Yves
Published: July 2002

The present study was designed to examine if induction of apolipoprotein B synthesis is associated with hypercholesterolemia in copper‐deficient rats. This hypercholesterolemia mainly resides in an increase in the HDL‐1 and LDL and is associated with a significant increase in plasma apoB concentration. Liver apoB mRNA levels were not significantly modified in deficient animals as compared to control rats. Studies on liver apolipoprotein synthesis indicated that apoB100 synthesis was increased in deficient animals whereas apoB48 synthesis was unchanged. Thus, it appears that the increase in apoB synthesis in the liver of copper‐deficient rats occurs at the posttranscriptional level. The selective increase in apoB100 synthesis indicates the possible impact of this deficiency on the editing of apoB. An increase in apoB100 synthesis by the liver in copper‐deficient rats may significantly contribute to the increase in plasma concentration of LDL.
Cloning and sequence analysis of cDNA for the Ca2+‐activated photoprotein, clytin
Authors: Inouye Satoshi , Tsuji Frederick I.
Published: July 2002

Clytin is a member of the aequorin family of photoproteins. It is made up of 189 amino acid residues, contains 3 Ca2+‐binding sites, and shows 62% homology in amino acid residues to those in aequorin. The cysteine, tryptophan, and histidine residues, and the C‐terminal proline, that are conserved in aequorin and clytin may be involved in the Ca2+‐activated bioluminescence of the two proteins. Clytin may also prove useful in the determination of Ca2+.
Ras proteins increase Ca2+‐responsiveness of smooth muscle contraction
Authors: Satoh S. , Rensland H. , Pfitzer G.
Published: July 2002

G‐proteins may be involved in receptor‐mediated Ca2+‐sensitization of smooth muscle contraction, but the responsible G‐proteins are not yet known. Here we show that in β‐escin skinned mesenteric microarteries, H‐ras p21 proteins, preactivated with GTP or GTP γ S, increase force at constant submaximal Ca2+ (pCa 6.3) concentration dependently. The GTP‐bound form of the wild‐type H‐ras p21 and the oncogenic mutant (p21[G12V]) were equally effective. The nucleotide‐free and the inactive GDP‐bound form of ras p21 had no effect on force. The tyrosine kinase inhibitor, tyrphostin, partially reversed the effect of the ras proteins in the GTP‐bound form on force. Thus, ras proteins mimic the Ca2+‐sensitizing effect of GTPγS and vasoconstrictors in mesenteric microarteries which may involve tyrosine phosphorylation.
Protein kinase C isoenzymes in human neuroblasts involvement of PKCε in cell differentiation
Authors: Ponzoni M. , Lucarelli E. , Corrias M.V. , Cornaglia-Ferraris P.
Published: July 2002

Although neuronal cells are a major target of phorbol ester action, the activity of the various protein kinase C (PKC) isoenzymes have not been studied in detail in human neuroblasts. Differentiation of the LAN‐5 human neuroblastoma cell line by interferon‐γ (IFN‐γ) is accompanied by a twofold increase in PKC activity. Since PKC is a multigene family, we investigated which isoforms were expressed in control and differentiated cells, and which of these isoenzymes is involved in neuronal differentiation. We found that: (1) PKC activity is higher in differentiated than in undifferentiated cells; (2) RT‐PCR analysis showed the expression of mRNA for PKCα, ‐γ, ‐δ ‐ε and‐ζ and the absence of mRNA for β in untreated LAN‐5 cells; (3) Western blot evaluation with PKC isoform‐specific antibodies showed the same pattern of PKC expression in non‐differentiated cells; (4) Expression of PKCε mRNA was significantly enhanced by IFN‐γ‐induced differentiation, while the other isoforms were not affected; (5) Differentiation of LAN‐5 cells with IFN‐γ or retinoic acid induced overexpression of the PKCε protein, while inhibition of cell proliferation by fetal calf serum starvation was without effect. These findings suggest that expression of PKCε isoform is tightly coupled with neuronal differentiation and may play a role in the maintenance of the differentiated state.
Adenine dinucleotide‐mediated cytosolic free Ca2+oscillations in single hepatocytes
Authors: Green Anne K. , Dixon C.Jane , McLennan Alexander G. , Cobbold Peter H. , Fisher Michael J.
Published: July 2002

Single rat hepatocytes microinjected with aequorin respond to Ca2+‐mobilizing agonists, including ADP and ATP, with oscillations in cytosolic free Ca2+. We show here that single rat hepatocytes also respond to the adenine dinucleotides Ap3A and Ap4A with Ca2+oscillations which resemble those induced by ADP and ATP.
Identification and characterisation of a type‐1 protein phosphatase from the okadaic acid‐producing marine dinoflagellate Prorocentrum lima
Authors: Boland Marion P. , Taylor Max F.J.R. , Holmes Charles F.B.
Published: July 2002

The unicellular marine dinoflagellate, Prorocentrum lima, an established producer of okadaic acid (OA), was shown to contain a type‐1 protein phosphatase (PP‐1) the biochemical profile of which on Mono‐Q and Superdex‐75 fast protein liquid chromatography was identical to the catalytic subunit of PP‐1 from rabbit skeletal muscle. Purified P. lima PP‐1 (apparent molecular mass 37.5 kDa) was highly sensitive to inhibition by mammalian protein phosphatase inhibitor‐1 and inhibitor‐2, and to OA itself. A 6–7‐fold increase in OA production by P. lima, when grown under controlled conditions, correlated with an up to 300‐fold increase in P. lima PP‐1 activity. Furthermore, P. lima did not contain any detectable type‐2A protein phosphatase activity. This study represents the first identification of a serine/threonine protein phosphatase in a dinoflagellate.
Interleukin 1 and tumour necrosis factor activate the mitogen‐activated protein (MAP) kinase kinase in cultured cells
Authors: Saklatvala Jeremy , Rawlinson Lesley M. , Marshall Christopher J. , Kracht Michael
Published: July 2002

Interleukin 1 (IL1) activated mitogen‐activated protein (MAP) kinase kinase in human gingival and foreskin fibroblasts and KB cells. Maximal activity was found in cytosolic extracts made after stimulating cells for 15 min. On anion‐exchange chromatography two differently charged forms of MAP kinase kinase were identified, both phosphorylated a kinase‐defective mutant MAP kinase, and activated recombinant wild type MAP kinase to phosphorylate MBP. Both were inhibited by an antiserum to recombinant MAP kinase kinase and the less acidic form was identified on Western blotting as an antigen of ∼43 kDa. Indistinguishable forms were very much more strongly induced by phorbol myristate acetate (PMA). TNF had a similar effect to that of IL1.
Effects of [Ca2+]i and temperature on minK channels expressed in Xenopus oocytes
Authors: Busch Andreas E. , Lang Florian
Published: July 2002

Slowly activating, voltage‐dependent minK channels cloned from rat kidney were expressed in Xenopus oocytes. Increase in the bath temperature from 22 to 32°C resulted in a dramatic acceleration of minK channel activation. The extraordinarily high Q10 of minK channel activation was voltage‐dependent, being higher at more negative potentials (Q10 at −20 mV: 7.02; at 20 mV: 4.0). While activation of minK channels was highly voltage‐dependent at 22°C, voltage had only little effect on minK channel activation at 32°C. Increase in [Ca2+]i, which has recently been shown to increase the maximal conductance g
max at room temperature, did not affect g
max at 32°C. However, increase of [Ca2+]i caused acceleration of minK channel activation at both temperatures. The interaction of [Ca2+]i and temperature on g
max and activation rate of minK channels described here is very similar to recent findings on Ca‐ and temperature‐effects on the slowly activating potassium conductance I
Ks in guinea pig cardiac myocytes.
Antipeptide antibodies directed against the C‐terminus of protein kinase Cζ (PKCζ) react with a Ca2+‐ and TPA‐sensitive PKC in HT‐29 human intestinal epithelial cells
Authors: Batlle Eduard , Fabre Myriam , de Herreros Antonio García
Published: July 2002

We have studied the PKC isoforms present in HT‐29 M6 colon cancer cells, the differentiation of which to mucus‐secreting cells is blocked by TPA. In addition to a major 72 kDa band, a 77 kDa PKC isoform was recognized by two different antibodies raised against a C‐terminus‐specific peptide for the TPA‐insensitive isoform, PKCζ. By different criteria (association to the membrane, down‐regulation, PKC activity in immunoprecipitates) we conclude that, contrary to the 72 kDa band, the 77 kDa band corresponds to a Ca2+‐ and TPA‐sensitive PKC. These results suggest that antipeptide antibodies directed against the C‐terminus of PKCζ react in human cells with a member of the conventional PKC subfamily besides PKCζ. Therefore, the data indicating that PKCζ is sensitive to different agents in various cell lines should be carefully re‐evaluated.
The primary oxygen sensor of the cat carotid body is cytochrome a3 of the mitochondrial respiratory chain
Authors: Wilson David F. , Mokashi Anil , Chugh Deepak , Vinogradov Sergei , Osanai Shinobu , Lahiri Sukhamay
Published: July 2002

Carbon monoxide was shown to be competitive with O2 in oxygen sensing by perfused carotid bodies isolated from cats, afferent electrical activity increasing with either decreasing O2 or increasing CO. The CO‐induced increase in afferent activity was fully reversed by bright light. At submaximal light intensities the extent of reversal, after correcting to equal light intensity of light quanta at each wavelength, was maximal for light of 432 ± 2 and 590 ± 2 nm, with a ratio (432/590) of approximately 6. This spectrum is characteristic of the CO compound of mitochondrial cytochrome a
3. The photo‐reversible inhibition of oxygen sensing activity by CO accounts for at least 80% of the oxygen chemosensory activity of the carotid body.
A role for metals and free radicals in the induction of apoptosis in thymocytes
Authors: Wolfe James T. , Ross David , Cohen Gerald M.
Published: July 2002

Recent reports have implicated a possible but undefined role for reactive oxygen species in the induction and mediation of apoptosis. In the present study, the role of free radicals and metal ions in apoptosis induced in rat thymocytes by dexamethasone and etoposide was examined. Copper chelators, but not iron specific chelators, inhibited apoptosis induced by both these stimuli. Several antioxidants also possessed potent inhibitory effects. We therefore propose that diverse agents may induce apoptosis in thymocytes by a common mechanism involving a copper mediated Fenton reaction, generating site specific hydroxyl radicals, possibly as a result of activation of the redox sensitive transcription factor NF‐ϰB.
Cyclic ADP‐ribose does not affect cardiac or skeletal muscle ryanodine receptors
Authors: Fruen Bradley R. , Mickelson James R. , Shomer Nirah H. , Velez Patricio , Louis Charles F.
Published: July 2002

The cardiac muscle isoform of the ryanodine receptor/Ca2+ release channel (RYR) has been proposed to be an important target of cyclic ADP‐ribose (cADPR) action in mammalian cells. However, we now demonstrate that neither cADPR (0.1–5 μM), nor the related metabolites, β‐NAD+ (0.1–30 mM) and ADP‐ribose (0.1–5 μM), affected cardiac RYR activity as determined by [3H]ryanodine binding to cardiac sarcoplasmic reticulum (SR) vesicles. Similarly, cADPR (1 μM) failed to activate single cardiac RYR channels in planar lipid bilayers. Skeletal muscle SR [3H]ryanodine binding was also unaffected by cADPR (up to 30 μM). These results argue against a direct role for the well‐characterized RYRs of cardiac or skeletal muscle in mediating cADPR‐activated Ca2+ release.
A herbicide antidote (safener) induces the activity of both the herbicide detoxifying enzyme and of a vacuolar transporter for the detoxified herbicide
Authors: Gaillard Cécile , Dufaud Annie , Tommasini Roberto , Kreuz Klaus , Amrhein Nikolaus , Martinoia Enrico
Published: July 2002

In plants potentially toxic compounds are ultimately deposited in the large central vacuole. In this report we show that isolated barley mesophyll vacuoles take up the glucoside conjugate of the herbicide derivate [5‐hydroxyphenyl]primisulfuron. Transport is stimulated by Mg‐ATP and is distinct from that previously described for glutathione conjugates. Treatment of barley with different herbicide antidotes (safeners) revealed that the safener cloquintocet‐mexyl doubles the vacuolar transport activities for both the glutathione and glucoside conjugates. Stimulation of the uptake of the metolachlor—glutathione conjugate was the result of an increased uptake velocity whereas the K
m remained unaltered, suggesting that the higher activity was due to a higher expression of the transporter. These results indicate that modulation of vacuolar transport activities are an integral part of the detoxification mechanism of plants.
Single channel activity of the ryanodine receptor calcium release channel is modulated by FK‐506
Authors: Ahern Gerard P. , Junankar Pauline R. , Dulhunty Angela F.
Published: July 2002

The immunosuppressant drug FK‐506 (3–20 μM) increased the open probability of ryanodine receptor calcium release channels, formed by incorporation of terminal cisternae vesicles from rabbit skeletal muscle into lipid bilayers, with cis (cytoplasmic) calcium concentrations between 10−7 M and 10−3 M. FK‐506 increased mean current and channel open time and induced long sojourns at subconductance levels that were between 28% and 38% of the maximum conductance and were distinct from the ryanodine‐induced subconductance level at about 45% of the maximum conductance. FK‐506 relieved the Ca2+ inactivation of the ryanodine receptor seen at 10−3 M Ca2+. The results are consistent with FK‐506 removal of FK‐506 binding protein from the ryanodine receptor [15].
Ageing‐associated tandem duplications in the D‐loop of mitochondrial DNA of human muscle
Authors: Lee Hsin-Chen , Pang Cheng-Yoong , Hsu Hueih-Shing , Weia Yau-Huei
Published: July 2002

Using PCR techniques, we detected a ∼260 bp (type I) and a ∼200 bp (type II) tandem duplications in the mtDNA of muscle biopsies from aged individuals. Only one 70‐year‐old subject was found to harbour the type I and 28 out of 58 subjects had type II duplication. About 90% of the subjects harbouring the duplicated mtDNAs also had the 4,977 bp deletion. Moreover, the incidence and quantity of the type II duplication were found to increase with age. The proportion of the type II duplicated mtDNA in the muscle of a 71‐year‐old subject was 3.1% while that of a 55‐year‐old individual was only 0.78%. We suggest that the tandem duplications occur alone or with mtDNA deletions in human tissues in an age‐dependent manner, and thereby cause synergistic deleterious effects on mitochondrial respiratory functions in human ageing.
Synergistic activation of intercellular adhesion molecule 1 (ICAM‐1) by TNF‐α and IFN‐γ is mediated by p65/p50 and p65/c‐Rel and interferon‐responsive factor Statlα (p91) that can be activated by both IFN‐γ and IFN‐α
Authors: Jahnke Andreas , P. Johnson Judith
Published: July 2002

Human ICAM‐1 expression is up‐regulated by IFN‐γ and TNF‐α, and synergistically increased by a combination of both. Transient expression of ICAM‐1/luciferase constructs led to definition of the regulatory regions mediating the cytokine response and showed that both are necessary for synergism. Immunochemical electromobility shift assays identified the TNF‐α‐dependent complexes that bound to the NF‐κB like sequence at −187 as p65/p50 and p65/c‐Rel. The interferon responsive region at −75 was bound by a Statlα (p91) containing complex that was activated by both IFN‐γ and IFN‐α. Although both regions were required for synergism, no additional or enhanced binding complexes were observed.
Cloning and characterisation of the human 5‐HT5A serotonin receptor
Authors: Rees Stephen , den Daas Izaak , Foord Steven , Goodson Susannah , Bull David , Kilpatrick Gavin , Lee Melanie
Published: July 2002

The human 5‐HT5A serotonin receptor has been cloned. As with the mouse and rat 5‐HT5A receptors, the gene consists of two coding exons separated by a large intron. The deduced amino acid sequence of the gene reveals a protein of 357 residues which shares 93% (nucleotide) and 84% (amino acid) identity to the cloned mouse 5‐HT5A receptor. We have determined the tissue distribution of the receptor by reverse transcriptase‐PCR and found expression in all regions of the brain examined with little or no expression in peripheral tissues. The receptor has been transiently expressed in Cos M6 cells and exhibits a pharmacological profile closely resembling the mouse and rat 5‐HT5A receptors with high, specific binding for ergotamine and methiothepin.
Intramolecular interactions in vinculin control α‐actinin binding to the vinculin head
Authors: Kroemker Martina , Rüdiger Angelika-H. , Jockusch Brigitte M. , Rüdiger Manfred
Published: July 2002

Using blot overlay techniques we have investigated the interaction of vinculin with α‐actinin. We show that an α‐actinin binding site is located in the 90 kDa vinculin head and confirm a vinculin binding site in the C‐terminal rod of α‐actinin, as recently reported by McGregor et al. [(1994) Biochem. J. 310, 225–233]. The isolated vinculin head binds much more strongly to α‐actinin than intact vinculin. Using a proteolytic 81 kDa head fragment, we show that vinculin residues 1–107 are required for α‐actinin binding. Antibodies directed against vinculin residues 808–850 inhibit the vinculin‐α‐actinin binding, suggesting that this sequence is directly involved in, or topographically related to, the α‐actinin binding site.