bluuebunny/crossref_metadata_2025 huggingface dataset:
Developmental changes in the lateral diffusion of Leydig cell membranes measured by the FRAP method
Authors: Zlatanov I.V. , Foley M. , Birmingham J. , Garland P.B.
Published: July 2002

A simple method for isolation and fluorescence labelling of Leydig cells (L‐cells) from rat testes was developed. Lateral diffusion coefficients of both lipid and protein membrane fluorescent probes were measured by the method of fluorescence recovery after photobleaching (FRAP). Age‐dependent changes in diffusibility of membrane lipids and proteins were discovered.
Control of proteolysis in perifused rat hepatocytes
Authors: Leverve X.M. , Caro L.H.P. , Plomp P.J.A.M. , Meijer A.J.
Published: July 2002

The mechanism by means of which amino acids inhibit intrahepatic protein degradation has been studied in perifused rat hepatocytes. Proteolysis was extremely sensitive to inhibition by low concentrations of amino acids. A mixture of 0.5 mM leucine and 1–2 mM alanine, concentrations found in the portal vein of the rat after feeding, inhibited proteolysis to the same extent as a complete physiological mixture of amino acids. Inhibition by these two amino acids was accompanied by a rise in the intracellular concentrations of glutamate and aspartate, and was largely prevented by addition of glucagon, by addition of the transaminase inhibitor aminooxyacetate, or by omission of K+. Acceleration of proteolysis by K+ depletion was accompanied by a fall in intracellular glutamate caused by an increased rate of transport of this amino acid to the extracellular fluid. It is concluded that intracellular leucine, glutamate and aspartate are important elements in the control of hepatic protein degradation.
Are Ca2+ channels in neutrophils activated by a rise in cytosolic free Ca2+?
Authors: Nasmith Patricia E. , Grinstein Sergio
Published: July 2002

It was recently suggested that the opening of neutrophil plasma membrane Ca2+ channels by chemotactic agents is mediated by a rise in free cytosolic Ca2+ concentration ([Ca2+]i). This hypothesis was tested in human cells monitoring [Ca2+]i, with the indicator indo‐1. In cells loaded with the Ca2+‐chelating agent bis(o‐aminophenoxy)ethane‐N,N,N′,N′‐tetraacetate, transmembrane Ca2+ uptake could be stimulated by formyl‐methionyl‐leucyl‐phenylalanine (fMLP) even when [Ca2+]i was at or below the resting level. In contrast, simply elevating [Ca2+]i in unstimulated cells failed to increase transmembrane uptake. It was concluded either that Ca2+ uptake across the plasma membrane is activated directly by the formation of the chemotactic factor‐receptor complex or, more likely, that a transduction mechanism distinct from changes in [Ca2+]i is involved.
Sulfur oxidation of free methionine by oxygen free radicals
Authors: Scislowski Piotr W.D. , Davis E.Jack
Published: July 2002

The oxidation of free methionine to methionine sulfoxide by chemically or enzymatically generated oxygen free radicals is presented. The physiological significance of this process in living cells is suggested.
Complete nucleotide sequence coding for rat liver 6‐phosphofructo‐2‐kinase/fructose‐2,6‐bisphosphatase derived from a cDNA clone
Authors: Darville M.I. , Crepin K.M. , Vandekerckhove J. , Van Damme J. , Octave J.N. , Rider M.H. , Marchand M.J. , Hue L. , Rousseau G.G.
Published: July 2002

cDNA clones for 6‐phosphofructo‐2‐kinase/fructose‐2,6‐bisphosphatase were isolated from rat liver expression libraries in λgt11 by antibody, oligonucleotide, and cDNA screening. One 1860 bp long clone contained a full‐length nucleotide sequence coding for the 470 amino acids of each of the two identical subunits of the bifunctional enzyme. This clone also contained untranslated sequences, one 173 bp long upstream from the ATG start codon and one 271 bp long downstream from the TGA stop codon. The clone was terminated by a poly(A) tail of 29 nucleotides.
Purification of Ca2+‐activated K+ channel protein on calmodulin affinity columns after detergent solubilization of luminal membranes from outer renal medulla
Authors: Klærke Dan A. , Petersen Janne , Jørgensen Peter L.
Published: July 2002

A method is developed for purification of the protein of the Ca2+‐activated K+ channel from outer renal medulla of pig kidney. The response of this K+ channel to physiological concentrations of Ca2+ is important for regulation of transtubular NaCl transport. In reconstituted vesicles direct addition of calmodulin doubles Ca2+ activation with sufficient affinity (K
0.1 nM) for chromatographic purification of the protein. For purification luminal plasma membrane vesicles are isolated on metrizamide density gradients and solubilized in CHAPS. The fraction of soluble protein retained on calmodulin‐Sepharose 4B columns in the presence of Ca2+ and eluted by EGTA is 0.7%. The purified protein has high Ca2+‐activated K+ channel activity after reconstitution into phospholipid vesicles. It distributes on two bands of 51 and 36 kDa after gel electrophoresis in SDS. The 36 kDa band is rapidly cleaved by trypsin and may be involved in Ca2+ stimulation of the channel. Phosphorylation from cAMP‐dependent protein kinase strongly stimulates Ca2+‐activated K+ channel activity and labels the 51 kDa band suggesting that this protein is involved in regulation of K+ channel opening.
Parathyroid hormone regulates phosphate transport in OK cells via an irreversible inactivation of a membrane protein
Authors: Malmström Kerstin , Murer Heini
Published: July 2002

In a cultured, renal epithelial cell line, OK, parathyroid hormone (PTH) reduces Na/phosphate cotransport and upon removal of the hormone the activity is regained. Cycloheximide, an inhibitor of protein synthesis, did not interfere with the PTH action, but prevented the reduced phosphate transport from regaining its original activity. Drugs, such as colchicine, that disrupt the microtubular network, lessened both the action of PTH and prevented the recovery of activity. The results are consistent with the irreversible inactivation of a plasma membrane protein necessary for full activity of the Na/phosphate cotransport.
The structure of the lipopolysaccharide O‐chain (M antigen) and polysaccharide B produced by Brucella melitensis 16M
Authors: Bundle David R. , Cherwonogrodzky John W. , Perry Malcolm B.
Published: July 2002

The surface M antigen of Brucella species has been identified as the lipopolysaccharide O‐polysaccharide component composed of a repeating pentasaccharide unit containing a sequence of one 1,3‐ and four 1, 2‐linked, 4,6‐dideoxy‐4‐formamido‐α‐D‐mannopyranosyl units. A neutral polysaccharide produced by Brucella species and referred to as polysaccharide B (poly B) has been identified as a family of circular 1,2‐linked polymers of β‐D‐glucopyranosyl units ranging in ring size from 17 to 24 glucosyl units.
Human T lymphocyte mitogenesis in response to the B oligomer of pertussis toxin is associated with an early elevation in cytosolic calcium concentrations
Authors: Strnad Colette F. , Carchman Richard A.
Published: July 2002

Pertussis toxin was found to serve as a mitogen in the human T lymphocyte, an effect which could be mimicked by its resolved binding component, the B oligomer. The mechanism of action of this component appeared to involve a rapid and sustained elevation of cytosolic calcium levels, as monitored by fura‐2 fluorescence. The source of mobilized calcium was predominantly extracellular, suggesting that the binding of the B oligomer to the T cell plasma membrane in some way elicited calcium channel activation. Notably, the influx of calcium was not observed with cholera toxin, an AB toxin lacking mitogenic effects on the human T lymphocyte.
Glucose transport in human erythrocytes measured using 13C NMR spin transfer
Authors: Kuchel Philip W. , Chapman Bogdan E. , Potts Jennifer R.
Published: July 2002

We present the results of a new NMR‐based procedure for measuring the fast transmembrane exchange of D‐[1‐13C]glucose in human erythrocytes. The method relies on different rates of exchange between the α‐ and β‐anomers of glucose inside and outside the cells; the rate outside the cells is greatly increased by the addition of mutarotase to the suspension. Theory is developed to describe nuclear‐spin transfer in the present system and is used to analyse the data to yield estimates of transmembrane‐exchange rate constants and their statistical uncertainties. For a total glucose concentration of 25.5 mmol/1 at 40°C the first order efflux rate constants for the α‐ and β‐anomers were 1.20±0.40 s−1 and 0.71±0.30 s−1, respectively.
Affinity modification of E1‐form of Na+, K+ ‐ATPase revealed Asp‐710 in the catalytic site
Authors: Ovchinnikov Yu.A. , Dzhandzugazyan K.N. , Lutsenko S.V. , Mustayev A.A. , Modyanov N.N.
Published: July 2002

An alkylating ATP analogue, γ‐[4‐(N‐2‐chlorethyl‐N‐methylamino)]benzylamide ATP (ClRATP), covalently binds to the catalytic α‐subunit of Na+, K+ ‐ATPase yielding a product resistant to hydrolysis by the enzyme and inhibiting the ATP‐hydrolysing activity. The Na+ ‐form of the membrane‐bound Na+, K+ ‐ATPase modified with ClRATP was hydrolysed by pepsin under conditions providing maximum stability of the modification product (4°C, pH 1.5). The modified peptide was isolated by HPLC and its amino acid sequence was found to involve residues 706–713 of the α‐subunit polypeptide chain. This fragment located near the γ‐phosphate of ATP is a component of the active site. It is highly homologous with corresponding regions of the catalytic subunits of all the known E1‐E2 ATPases. In the Na+ ‐(or E1‐)enzyme form Asp‐710 is the target of modification. Evidently E1‐ and E2‐enzymes have different targets in CIRATP modification, i.e. the polypeptide chain regions near the ATP γ‐phosphate in the enzyme active site differ somewhat in their conformations.
Cholinergic receptors regulate a voltage‐insensitive but Na+‐dependent calcium influx pathway in salivary acinar cells
Authors: Morris A.P. , Fuller C.M. , Gallacher D.V.
Published: July 2002

The calcium‐sensitive fluorescent probe, quin 2, was employed to investigate the cholinergic receptor regulation of cytosolic free calcium concentration ([Ca2+]i) in isolated rat submandibular acinar cells. Cholinergic receptor stimulation results in a marked and sustained elevation [Ca2+]i. The major component of this response is shown to be due to activation of a calcium influx pathway. There is no evidence in K+‐depolarized cells of any voltage‐gated calcium influx. Acetylcholine failed to activate the calcium influx pathway in cells bathed in Na+‐free solutions. The cholinergic receptors thus regulate a voltage‐insensitive but Na+ dependent transport system for calcium entry in submandibular acinar cells.
Catalytic subunit of cAMP‐dependent protein kinase is essential for cAMP‐mediated mammalian gene expression
Authors: Büchler W. , Walter U. , Jastorff B. , Lohmann S.M.
Published: July 2002

Cyclic AMP‐stimulated mRNA levels in cultured rat hepatocytes were inhibited by three different inhibitors of cAMP‐dependent protein kinase activity: (i) Rp‐cAMPS, a cAMP analog with a sulfur substitution at the equatorial oxygen of the cyclic monophosphate; (ii) H8, an isoquinoline sulfonamide derivative; and (iii) PKI, a 20‐amino acid synthetic peptide of the Walsh protein kinase inhibitor. These inhibitors specifically blocked the cAMP‐stimulated increase in mRNA for tyrosine aminotransferase and phosphoenolpyruvate carboxykinase; they had no effect on the level of albumin mRNA which is not cAMP regulated. These results provide functional evidence that kinase activity involving protein phosphorylation is required in cAMP‐mediated gene expression in mammalian cells.
Yeast adenylate kinase is transcribed constitutively from a promoter in the short intergenic region to the histone H2A‐1 gene
Authors: Oechsner Ulrich , Magdolen Viktor , Zoglowek Cornelia , Häcker Udo , Bandlow Wolfhard
Published: July 2002

Yeast mitochondrial adenylate kinase (high molecular mass form, gene locus: AKY2) is encoded on chromosome IV of the same DNA strand as histone H2A‐1. The nontranslated intergenic region spans 560 bp, the nontranscribed spacer can be estimated to comprise at most 300 bp. The TATA‐box sequence is contained in a striking environment consisting of 20 alternating pyrimidines and purines. The AKY2 transcript is made constitutively: (i) the cellular mRNA concentration does not vary significantly with either growth conditions or elapse of the cell cycle; (ii) β‐galactosidase activity is about constant in yeast cells grown on various carbon sources after transformation with AKY2‐promoter/lacZ fusions; (iii) primer elongation analysis shows that utilization of 5 initiation sites is qualitatively and quantitatively independent of the growth conditions and the carbon source used; (iv) Western blot analysis and adenylate kinase activity measurements indicate the absence of post‐transciptional controls as well.
Broad‐host range expression vectors containing manipulated meta‐cleavage pathway regulatory elements of the TOL plasmid
Authors: Ramos Juan L. , Gonzalez-Carrero Manuel , Timmis Kenneth N.
Published: July 2002

The construction of pERD20 and pERD21, two broad‐host range expression vectors, is described. The vectors contain the Pm promoter of the meta‐cleavage pathway operon of the TOL plasmid pWWO; this promoter is present within a polylinker which provides a number of downstream cloning sites close to the transcription initiation site. Transcription from the Pm promoter in these vectors is controlled not by the natural positive regulator of Pm, the Xy1S protein, but by an Xy1S mutant analogue, Xy1S2tr6, which ??hibits an altered effector specificity and can mediate a 3–8‐fold higher level of transcription than can Xy1S in a wide range of temperatures. Controlled expression of cloned genes can be achieved in a broad spectrum of Gram negative bacteria grown at a wide range of temperatures.
Nucleoside diphosphate kinase from human erythrocytes: Purification, molecular mass and subunit structure
Authors: Presecan Elena , Vonica Alin , Lascu Ioan
Published: July 2002

A new procedure for the purification of nucleoside diphosphate kinase from human erythrocytes is described. The enzyme (105 kDa by gel filtration) is made up of two different kinds of subunits (19.0 and 20.5 kDa), both displaying enzymatic activity. The probable subunit structure of the enzyme is hexameric. The discrepancies related to earlier work are discussed.
Phosphorylation of caldesmon
Authors: Abougou J.-C. , Hagiwara M. , Hachiya T. , Terasawa M. , Hidaka H. , Hartshorne D.J.
Published: July 2002

The phosphorylation of caldesmon was studied to determine if kinase activity reflected either an endogenous kinase or caldesmon itself. Titration of kinase activity with calmodulin yielded maximum activity at substoichiometric ratios of calmodulin/caldesmon. The sites of phosphorylation on caldesmon for calcium/calmodulin‐dependent protein kinase II and endogenous kinase were the same, but distinct from protein kinase C sites. Phosphorylation in the presence of Ca2+ and calmodulin resulted in a subsequent increase of endogenous kinase activity in the absence of Ca2+. These results suggest that caldesmon is not a protein kinase and that kinase activity in caldesmon preparations is due to calcium/calmodulindependent protein kinase II.
The psbC start codon in Synechocystis sp. PCC 6803
Authors: Carpenter Shelly D. , Charite Jeroen , Eggers Beth , Vermaas Wim F.J.
Published: July 2002

The translation start codon for psbC, the gene encoding CP43, a chlorophyll‐binding protein of photosystem II, has been identified for the cyanobacterium Synechosystis sp. PCC 6803 using site‐directed mutagenesis. An AUG codon, about 50 bases upstream from the end of psbD‐I had previously been assumed to be the translation start site of psbC. However, the fact that the AUG codon is not present in psbC from several other organisms, whereas a GUG codon 14 bases upstream from the end of psbD‐I is strictly conserved suggests that CP43 translation starts at the latter codon. Mutation of GUG, but not of AUG, led to a loss of CP43 and photoautotrophic growth, indicating that the GUG codon is the sole initiation site for translation of the CP43 protein in Synechocystis sp. PCC 6803.
Restricted localization of the adipocyte/muscle glucose transporter species to a cell surface‐derived vesicle fraction of 3T3‐L1 adipocytes
Authors: Lange Klaus , Brandt Ursula
Published: July 2002

The recent demonstration of a large cell surface‐derived pool of insulin‐sensitive glucose transporters, presumably concentrated in the microvilli of 3T3‐L1 adipocytes, induced the assumption that in differentiated adipocytes, newly inserted plasma membrane areas may display restricted lateral mobility, thereby preventing diffusion of integral membrane proteins out of these areas into the adjoining plasma membrane. In order to test this assumption, the cell surface distributions of the two glucose transporter species expressed by 3T3‐L1 cells were determined using specific antisera against the HepG2/erythrocyte transporter, GluT1, which is synthesized in both fibroblasts and adipocytes, and the adipocyte/muscle‐specific transporter, GluT4, expressed for the first time 3–4 days after induction of adipose conversion. GluT1 was shown to be localized in the plasma membrane of both 3T3‐L1 preadipocytes and adipocytes, whereas GluT4 was almost entirely restricted to the low density surface‐derived vesicle (LDSV) fraction of 3T3‐L1 adipocytes most likely consisting of microvilli‐derived vesicles. In contrast to the minor portion of GluT4 found in the adipocyte plasma membrane fraction, equal amounts of the GluT1 protein were detected in both the plasma membrane and the LDSV fractions of adipocytes. Both transporter species were present in the microsomal and the LDSV fractions of adipocytes. The observed distribution of the two transporter species is in accordance with the postulated restriction of the lateral mobility in plasma membrane areas formed by newly inserted transgolgi vesicles of differentiated adipocytes.
Mapping of a dominant immunogenic region of synaptophysin, a major membrane protein of synaptic vesicles
Authors: Knaus Petra , Betz Heinrich
Published: July 2002

Synaptophysin is a major integral membrane protein of synaptic vesicles. Its transmembrane topology deduced from the cDNA sequence predicts 4 transmembrane regions and a carboxy‐terminal cytoplasmic tail containing a characteristic pentapeptide repeat structure. The monoclonal antibody (mAb), SY38, binds to a cytoplasmic domain of synaptophysin. By using fusion proteins corresponding to truncated forms of the cytoplasmic tail, its epitope was located to a flexible segment in the center of the repeat structure. Four other mAbs (c7.1, c7.2, c7.3, c7.4) share the same epitope, which thus emerges as the major immunogenic region of this membrane protein.
The bulk of Ca2+ released to the myoplasm is free in the sarcoplasmic reticulum and does not unbind from calsequestrin
Authors: Volpe Pompco , Simon Bruce J.
Published: July 2002

Calsequestrin (CS) is the major Ca2+ binding protein contained in the lumen of sarcoplasmic reticulum (SR). Ca2+ binding properties and tissue concentration of CS of frog skeletal muscle were measured. At equilibrium, maximal Ca2+ binding capacity of purified CS was about 1.2 μmol Ca2+/mg protein. Apparent K
as for Ca2+ were around 50 μM in the absence of salts, around 0.9 mM in the presence of 100 mM KCl, and around 1.1 mM under ‘physiological’ conditions. Quantitation of CS in homogenates was accomplished by three methods (Stains‐all staining, immunobiolting and 45Ca ligand overlay). Frog muscle contained about 0.5 mg of CS/g wet weight, that is 6.1 mM CS inside the SR. At rest the in situ free [Ca2+] of SR was calculated to be 3.6 mM, and, thus, CS is largely saturated with Ca2+. Moreover, computer simulations of Ca2+ release indicated that about 75% of Ca2+ released during a twitch is free in the SR and does not unbind from CS.
Methotrexate binds in a non‐productive orientation to human dihydrofolate reductase in solution, based on NMR spectroscopy
Authors: Stockman Brian J. , Nirmala N.R. , Wagner Gerhard , Delcamp Tavner J. , DeYarman Michael T. , Freisheim James H.
Published: July 2002

Dihydrofolate reductase (DHFR) is an intracellular target enzyme for folate antagonist drugs, including methotrexatte. In order to compare the binding of methotrexate to human DHFR in solution with that observed in the crystalline state, NMR spectroscopy has been used to determine the conformation of the drug bound to human DHFR in solution. In agreement with what has been observed in the crystalline state, NOE's identified protein and methotrexate protons indicate that methotrexate binds in a non‐productive orientation. In contrast to what has been reported for E. coli DHFR in solution, only one bound conformation of methotrexate is observed.
Translocation of the α‐ and β‐isoforms of protein kinase C following activation of human T‐lymphocytes
Authors: Kvanta Anders , Jondal Mikael , Fredholm Bertil B.
Published: July 2002

We have analyzed how activation of human Jurkat T‐cells by the mitogenic lectin, concanavalin A (Con A), may affect the cellular distribution of the α‐ and β‐isoforms of protein kinase C (PKC) in T‐cells. In non‐stimulated cells almost all of the α‐ and β‐PKC was localized to the cytoplasmic compartment. Stimulation with Con A caused a transient translocation of both α‐ and β‐PKC from the cytoplasm to the cell membrane. The α‐ isoform appeared to be translocated to a somewhat greater extent and for a longer period of time that the β‐form. Translocation was maximal between 1 and 5 min for both of the isoforms. 30 min after stimulation, β‐PKC had returned to basal levels, whereas a substantial amount of α‐PKC remained associated with the particulate fraction. We conclude that activation of human T‐cells causes the translocation of at least two different isoforms of PKC, α‐PKC and β‐PKC.
Lipid—peptide interactions between fragments of the transit peptide of ribulose‐1,5‐bisphosphate carboxylase/oxygenase and chloroplast membrane lipids
Authors: van't Hof Ron , Demel Rudy A. , Keegstra Kenneth , de Kruijff Ben
Published: July 2002

The interactions of fragments of the transit peptide of ribulose‐1,5‐bisphosphate carboxylase/oxygenase with lipid monolayers was studied in order to investigate the possible involvement of the membrane lipids in the protein import process. The fragments are surface active and have a differential ability to insert in lipid monolayers. The fragments have a preference for the chloroplast galacto‐ and sulpholipids and phosphatidylglycerol and interact with envelope membrane lipid extracts. These results suggest that probably transit peptide—lipid interactions are involved in the chloroplast protein import process.
Radioligand binding studies of the atypical β3‐adrenergic receptor in rat brown adipose tissue using [3H]CGP 12177
Authors: Muzzin P. , Revelli J.-P. , Fraser C.M. , Giacobino J.-P.
Published: July 2002

Two populations of [3H]CGP 12177 binding sites exist in rat interscapular brown adipose tissue (IBAT) plasma membrane. The majority of binding sites are of low affinity with a K
d of 31 nM, a value in close agreement with that for the K
d or [3H]CGP 12177 binding to a cloned rat β3‐adrenergic receptor (AR) expressed in CHO cells (44 nM). Competition binding studies demonstrate that the K
i values of the cloned rat β3‐AR and of the low affinity sites in IBAT are 45 and 29 nM, respectively, for BRL 37344 and 1.4 and 1.0 μM, for (−)‐propranolol. These findings strongly suggest that the low affinity [3H]CGP 12177 binding site measured in IBAT plasma membranes represents the atypical β3‐AR in this tissue.
Peroxisomal protein import In vivo evidence for a novel ranslocation competent compartment
Authors: Heinemann Petra , Just Wilhelm W.
Published: July 2002

In homogenates of isolated hepatocytes separated by Nycodenz density gradient centrifugation, two peroxisomal populations are identified that differ in buoyant density. Organelles present in a high density fraction (1.22–1.23 g/cm3) represent mature peroxisomes. Vesicles of intermediate density (1.16–1.17 gm̧3) represent mature peroxisomes. Vesicles of intermediate density (1.16–1.17 g/cm3) are present in much lower concentration and seem to play a particular role in the import and distribution or newly synthesized peroxisomal proteins. In a typical pulse‐chase experiment with a 7.5 min pulse, peroxisomal acyl‐CoA oxidase is first imported into the peroxisomal fraction of intermediate density. After a chase of up to 30 min, the enzyme is found in mature peroxisomes.
Intracellular Ca2+ release by flufenamic acid and other blockers of the non‐selective cation channel
Authors: Poronnik P. , Ward M.C. , Cook D.I.
Published: July 2002

We report in this paper using measurement of intracellular free Ca2+ with fura‐2, that flufenamic acid and several related blockers of the 25 pS Ca2+‐activated nonselective cation channel cause release of Ca2+ from an intracellular store other than the endoplasmic reticulum, Possibly from mitochondria. A new compound, 4′‐methyl‐DPC, is found to be as effective in blocking non‐selective cation channels as other flufenamate analogs but, like the parent compound, the non‐selective cation channel blocker DPC, it does not cause release of Ca2+ from intracellular stores. DPC and 4′‐methyl‐DPC are thus the most suitable of the available blockers of non‐selective cation channels for use in studies on the role of these channels in normal cell function.
Phosphorylation of vascular smooth muscle caldesmon by endogenous kinase
Authors: Pinter Katalin , Marston Steven B.
Published: July 2002

Caldesmon was phosphorylated up to 1.2 molPi/mol using a partially purified endogenous kinase fraction. The phosphorylation site was within the C‐terminal 99 amino acids. We were also able to phosphorylate caldesmon incorporated into native and synthetic smooth muscle thin filaments. Phosphorylation did not alter caldesmon binding to actin or inhibition or actomyosin ATPase. It also did not change Ca2+ sensitivity in native thin filaments. Phosphorylated caldesmon bound to myosin less than unphosphorylated caldesmon, especially when the myosin was also not phosphorylated. This work did not support the hypothesis that caldesmon function is modulated by phosphorylation.
Immunocharacterization of δ‐ and ζ‐isoenzymes of protein kinase C in rat renal mesangial cells
Authors: Huwiler Andrea , Fabbro Doriano , Stabel Silvia , Pfeilschifter Josef
Published: July 2002

The isoforms of protein kinase C (PKC) present in rat mesangial cells were identified by immunoblot analysis with antibody raised against isotype‐specific peptides. In addition to the previously observed α‐ and ε‐subspecies, mesangial cells also express the δ‐ and ζ‐isoenzymes of PKC. On exposure to phorbol 12,13‐dibutyrate (PDB) a complete depletion of PKC‐δ is observed within 8 h. Removal of PDB results in a recovery of PKC‐δ. In contrast, PKC‐ζ is unaffected by addition or removal of PDB.
Biochemical properties of rat protein kinase C‐η expressed in COS cells
Authors: Dekker Lodewijk V. , Parker Peter J. , McIntyre Peter
Published: July 2002

Using a PKC‐ε cDNA probe a cDNA for PKC‐η has been cloned from a rat lung cDNA library. When expressed in COS cells, rat PKC‐η appeared as an 84 kDa protein. PKC‐η expressed in COS cells. was solubilized by 1% Triton X‐100 and purified away from the endogenous PKC‐α by ammonium sulphate fractionation. The activity of this PKC‐η preparation was characterized with respect to cofactor dependence and substrate specificity, Various PKC pseudosubstrate peptides are phosphorylated by PKC‐η in a phospholipid and TPA‐dependent but calcium‐independent manner. The polypeptide histone IIIS is a poor substrate.
Primary structure and distribution of a novel ryanodine receptor/calcium release channel from rabbit brain
Authors: Hakamata Yasuhiro , Nakai Junichi , Takeshima Hiroshi , Imoto Keiji
Published: July 2002

The complete amino acid sequence of a novel ryanodine receptor/calcium release channel from rabbit brain has been deduced by cloning and sequence analysis of the EDNA. This protein is composed of 4872 amino acids and shares characteristic structural features with the skeletal muscle and cardiac ryanodine receptors. RNA blot hybridization analysis shows that the brain ryanodine receptor is abundantly expressed in corpus striatum, thalamus and hippocampus, whereas the cardiac ryanodine receptor is more uniformly expressed in the brain. The brain ryanodine receptor gene is transcribed also in smooth muscle.
Regulation of arachidonate‐mobilizing phospholipase A2 by phosphorylation via protein kinase C in macrophages
Authors: Wijkander Jonny , Sundler Roger
Published: July 2002

Stimulation of 32P‐labeled macrophages with phorbol ester caused an increase in phosphorylation of the intracellular, high molecular weight phospholipase A2. This increase in phosphorylation was accompanied by an increase in enzyme activity, but led to no detectable shift in the concentration dependence for Ca2+‐induced activation. The phosphorylated phospholipase A2 could be dephosphorylated by treatment with acid phosphatase, and such treatment also reduced its catalytic activity. Together with previous data, these results indicate that the arachidonate‐mobilizing phospholipase A2 is dually regulated by Ca2'+ (membrane interaction) and by phosphorylation (catalytic activity).
Presence of functionally different compartments of the Ca2+ store in single intestinal smooth muscle cells
Authors: Yamazawa Toshiko , Iino Masamitsu , Endo Makoto
Published: July 2002

Studies in smooth muscle bundles have shown the presence of functionally different compartments of Ca2+ store, one (Sα) sensitive to both caffeine and inositol 1,4,5‐trisphosphate (IP3), and the other (Sβ) sensitive only to IP3, Ca2+ release in isolated single smooth muscle cells from guinea pig taenia caeci was studied to see if both compartments exist within a cell. Responses to caffeine and carbachol were consistently observed but were abolished after treatment with ryanodine, while intracellular application of IP3 induced Ca2+ release after the treatment, albeit smaller in size than control. Thus Sα and Sβ coexist in a single smooth muscle cell and agonist‐induced Ca2+ release requires whole store to be loaded with Ca2+.
BisG10, a K+ channel blocker, affects the calcium release channel from skeletal muscle sarcoplasmic reticulum
Authors: Allard Bruno , Moutin Marie-Jo , Ronjat Michel
Published: July 2002

The action of bisG10, a potent K+ channel inhibitor, was tested on the Ca2+ release from isolated sarcoplasmic reticulum vesicles of rabbit skeletal muscle. Using a rapid filtration technique, we found that the drug inhibited Ca2+‐induced Ca2+ release elicited in the presence of extravesicular K+ as counter‐ion. This inhibition was not reversed by the addition of valinomycin and still occurred when Cl− was used as co‐ion, indicating that not only K+ channels are involved in the inhibiting effect. We found that bisG10 decreased the binding of ryanodine to sarcoplasmic reticulum vesicles, showing that bisG10 is able to block the sarcoplasmic reticulum Ca2+ release channel.
Protein kinase C α, δ, ϵ and ζ in C6 glioma cells
Authors: Chen Ching-Chow
Published: July 2002

Both the cytosol and membrane in C6 glioma cells express abundance of PKCα, δ, ζ and trace amount of PKCϵ by Western blot analysis with isozyme‐specific antibodies. These characteristics make this cell line a good model to study the properties of different classes of PKC isoforms in one cell type. Exposure of the cells to 100 nM TPA for 10 min resulted in the translocation of conventional PKCα (cPKCα) and new PKCδ(nPKCδ) and ‐ϵ from the cytosolic to the membrane fraction, while left atypical PKCζ (aPKCζ) unaffected. The extent of translocation of cPKCα induced by TPA was more prominent than that of nPKCδ and nPKCϵ. α‐TPA, the inactive phorbol ester, did not induce translocation of these isozymes. After treatment of the cells with 1 μM TPA for 17 h, cPKCα, nPKGδ and nPKCϵ were almost completely down‐regulated, whereas aPKCζ was still unaffected. The natural activators of this cell line, endothelin‐1 and ATP also translocated cPKCa and nPKCδ. However, the extent of translocation induced by these two agonists was much less than that of TPA.
Role of isoprenylation in intracellular pH regulation of granulocytes
Authors: Arellano Ramiro , Grinstein Sergio
Published: July 2002

Lovastatin was used to study the role of isoprenylated GTP‐binding proteins in the regulation of intracellular pH in granulocytic HL‐60 cells. The cytosolic acidification that accompanies stimulation by chemoattractants was completely eliminated by lovastatin. The subsequent activation of Na+/H+ exchange was partially inhibited by the isoprenylation antagonist. In contrast, the osmotic activation of the Na+/H+ antiport was unaffected. The data indicate that the osmotically‐ and receptor‐induced activation of the antiport utilize divergent signalling pathways. The results also provide evidence supporting the notion that cytosolic acidification by chemoattractants results from accumulation of metabolic acid generated during the respiratory burst.
Rise in heat‐shock protein level confers tolerance to energy deprivation
Authors: Gabai Vladimir L. , Kabakov Alexander E.
Published: July 2002

Heat shock (44°C for 10 min) or ATP depletion by an uncoupler (CCCP for 20 min) is shown to result in stimulation of hsp68/70 synthesis in Ehrlich tumor cells. After 3 h of recovery, the cells become thermotolerant. Surprisingly, repeated ATP depletion caused by CCCP or rotenone (a respiratory inhibitor) treatment, had a much lower effect on cell viability. Both induction of tolerance to energy deprivation and hsp68/70 synthesis were totally suppressed by cycloheximide, an inhibitor of cytosolic protein synthesis. In tolerant cells, rotenone still induced ATP depletion; however, protein aggregation (the rise in Triton‐insoluble proteins) was inhibited in these cells. It is suggested that cellular chaperones (e.g. hsp70) are involved in the protection of ischemie cells from necrosis, preventing protein aggregation under ATP deficiency.
α‐Helical distorting substitutions disrupt coupling between m3 muscarinic receptor and G proteins
Authors: Duerson Kevin , Carroll Reed , Clapham David
Published: July 2002

Acetylcholine stimulation of the m3 or m2 muscarinic receptor expressed in Xenopus laevis oocytes induces either a fast transient or slowly oscillating calcium‐sensitive chloride current. The speed of these currents reflects the efficiency of receptor coupling to guanine nucleotide‐binding proteins and phosphatidylinositol (PI) turnover. Point mutations of the m3 receptor were made in a region of the third cytoplasmic loop to test whether receptor function relied on an α‐helical structure of the G protein‐coupling domain. Proline substitution for glutamate at position 257 disrupted the m3 response. Also, single alanine insertions between residues 259 and 260 disrupted the m3 receptor‐stimulated response while double alanine insertions at this site had no effect. Based on these results, we suggest that a region of the third cytoplasmic loop of the m3 receptor possesses an amphipathic α‐helical conformation.
Prolyl oligopeptidase catalysis Reactions with thiono substrates reveal substrate‐induced conformational change to be the rate‐limiting step
Authors: Polgár László , Kollát Emma , Hollósi Miklós
Published: July 2002

Prolyl oligopeptidase, a member of the new family of serine proteases, exhibits significant mechanistic differences compared with the enzymes of the chymotrypsin and subtilisin families. Our kinetic study using the thiono substrate, benzyloxycarbonyl‐Gly‐Pro[CS‐NH]‐2‐naphthylamide suggests that the putative oxyanion binding site is important in prolyl oligopeptidase catalysis, although to a lesser extent than in the chymotrypsin‐and subtilisin‐catalyzed reactions. By using another thiono substrate, benzyloxycarbonyl‐Gly[CS‐NH]Pro‐2‐naphthylamide, it is demonstrated that the distant S2P2 hydrogen bond (formed between the S2 subsite and P2 peptide residue) makes a greater contribution to catalysis than does stabilization by the oxyanion binding site involved directly in the bond cleavage. In contrast to the reactions catalyzed by chymotrypsin and subtilisin, no kinetic deuterium isotope effect is apparent in the acylation of prolyl oligopeptidase measured either with the specific benzyloxycarbonyl‐Gly‐Pro‐2‐naphthylamide, or with the very poor substrate, benzyloxycarbonyl‐Gly‐Pro[CS‐NH]‐2‐naphthylamide. This indicates that the rate‐limiting conformational change is induced by the substrate.
Carcinogens stimulate phosphorylation of ethanolamine derived from increased hydrolysis of phosphatidylethanolamine in, C3H/101/2 fibroblasts
Authors: Kiss Zoltan , Crilly Karan S. , Anderson Wayne H.
Published: July 2002

Many human tumors contain high concentrations of ethanolamine phosphate (EtnP). An important question is whether increased formation of EtnP is merely the consequence of cell transformation, or is it associated with the process of carcinogenesis. Here we show that in C3H/10Tl/2 embryonic fibroblasts, an established cellular model for the study of carcinogenesis, the environmental carcinogens, 7,12‐dimethylbenz[a]anthracene (DMBA) and benzo[a]pyrene (B[a]P) (0.1–1 concentration; 24 h treatment), stimulate phosphorylation of ethanolamine derived from increased hydrolysis of phosphatidylethanolamine. The results suggest that increased formation of EtnP is associated with the early stages of carcinogenesis. This observation may have prognostic value.
Characterization of cAMP‐dependent protein kinase activation by CCK in rat pancreas
Authors: Marino Christopher R. , Leach Steven D. , Schaefer Jean F. , Miller Laurence J. , Gorelick Fred S.
Published: July 2002

This study reports on the use of a new sensitive assay of cAMP‐dependent protein kinase activity to examine the effect of cholecystokinin (CCK) on the cAMP second messenger cascade in rat pancreatic acini. Treatment of acini with both low (pM) and high (nM) concentrations of CCK was associated with an increase in cAMP‐dependent protein kinase activity. The increases in kinase activity were detected in the absence of phosphodiesterase inhibition, a condition required to detect a measurable increase in cellular cAMP in these cells. Furthermore, the cAMP cascade was dissociated from the secretory effects of CCK, since the CCK analogue, OPE, mediates enzyme secretion but does not increase cellular cAMP levels or kinase activity.
Expression of the Menkes gene homologue in mouse tissues lack of effect of copper on the mRNA levels
Authors: Paynter Jennifer A. , Grimes Andrew , Lockhart Paul , Mercer Julian F.B.
Published: July 2002

The expression of the homologue of the Menkes disease gene (Mnk) in mice was studied using RNA blots. The highest level of expression of the 8.0 kb mRNA was found in placenta, substantial expression was noted in lung, heart, brain, testis and kidney and gut mucosa, but very low levels were found in spleen and adult liver. In fetal liver, the amount of Mnk mRNA is similar to that found in kidney, however, it declines soon after birth. Results with copper‐loaded normal mice and mutant mice with genetic defects in copper transport suggested that Mnk mRNA levels are not regulated by tissue copper concentrations.
Electrogenic amino acid exchange via the rBAT transporter
Authors: Coady Michael J. , Jalal Fairouze , Chen Xingzhen , Lemay Guy , Berteloot Alfred , Lapointe Jean-Yves
Published: July 2002

A cDNA clone was isolated from rabbit renal cortex using DNA‐mediated expression cloning, which caused alanine‐dependent outward currents when expressed in Xenopus oocytes. The cDNA encodes rBAT, a Na‐independent amino acid transporter previously cloned elsewhere. Exposure of cDNA‐injected oocytes to neutral amino acids led to voltage‐dependent outward currents, but inward currents were seen upon exposure to basic amino acids. Assuming one charge/alanine, the outward current represented 38% of the rate of uptake of radiolabelled alanine, and was significantly reduced by prolonged preincubation of oocytes in 5 mM alanine. The currents were shown to be due to countertransport of basic amino acids for external amino acids using the cut‐open oocyte system. This transport represents a major mode of action of this protein, and may help in defining a physiological role for rBAT in the apical membrane of renal and intestinal cells.
Adhesion molecules: a new target for immunoliposome‐mediated drug delivery
Authors: Bloemen P.G.M. , Henricks P.A.J. , van Bloois L. , van den Tweel M.C. , Bloem A.C. , Nijkamp F.P. , Crommelin D.J.A. , Storm G.
Published: July 2002

The anti‐ICAM‐1 monoclonal antibody F10.2 was conjugated to liposomes to target to cells expressing the cell adhesion molecule ICAM‐1. We demonstrate that F10.2 immunoliposomes bind to human bronchial epithelial cells (BEAS‐2B) and human umbilical vein endothelial cells (HUVEC) in a specific, dose‐ and time‐dependent manner. It appears that the degree of ICAM‐1 expression is the limiting factor in the degree of immunoliposome binding to the cells. These results are a first step in the strategy for specific drug delivery to target sites characterised by increased expression of adhesion molecules.
Activation of mitogen activated protein (MAP) kinases by vanadate is independent of insulin receptor autophosphorylation
Authors: D'Onofrio Franco , Le Minh Quynh Uyen , Chiasson Jean-Louis , Srivastava Ashok K.
Published: July 2002

Treatment of Chinese hamster ovary (CHO) cells over‐expressing the human insulin receptor (CHO‐HIRc) with the insulin mimetic agent, vanadate, resulted in a dose‐ and time‐dependent tyrosine phosphorylation of two proteins with apparent molecular sizes of 42 kDa (p42) and 44 kDa (p44). However, vanadate was unable to stimulate the tyrosyi phosphorylation of theβ‐subunit of the insulin receptor. By using myelin basic protein (MBP) as the substrate to measure mitogen‐activated protein (MAP) kinase activity in whole cell lysates, vanadate‐stimulated tyrosyl phosphorylation of p42 and p44 was associated with a dose‐ and time‐dependent activation of MAP kinase activity. Furthermore, affinity purification of cell lysates on anti‐phosphotyrosine agarose column followed by immunoblotting with a specific antibody to MAP kinases demonstrated that vanadate treatment increased the tyrosyl phosphorylation of both p44mapk and p42mapk by several folds, as compared to controls, in concert with MAP kinase activation. In addition, retardation in gel mobility further confirmed that vanadate treatment increased the phosphorylation of p44mapk and p42mapk in CHO‐HIRc. A similar effect of vanadate on MAP kinase tyrosyl phosphorylation and activation was also observed in CHO cells over‐expressing a protein tyrosine kinase‐deficient insulin receptor (CHO‐1018). These results demonstrate that the protein tyrosine kinase activity of the insulin receptor may not be required in the signaling pathways leading to the vanadate‐mediated tyrosyl phosphorylation and activation of MAP kinases.
Structural determination and characterization of a 40 kDa protein isolated from rat 40 S ribosomal subunit
Authors: Tohgo Akira , Takasawa Shin , Munakata Hiroshi , Yonekura Hideto , Hayashi Norio , Okamoto Hiroshi
Published: July 2002

We have purified a 40 kDa protein from the rat 40 S ribosomal subunit and determined its primary structure by amino acid and cDNA sequencing. The amino acid sequence of the 40 kDa protein shared 29–37% homology with prokaryotic ribosomal protein S2 of eubacteria and chloroplasts, indicating that the protein is a eukaryotic counterpart to prokaryotic S2. Moreover, the amino acid sequence shared 99% identity with those deduced from cDNAs for 68 kDa laminin binding proteins of human, murine and bovine origins. The cDNAs are capable of encoding polypeptides with predicted molecular mass of 33,000 which lacked typical signal sequences, N‐linked glycosylation sites and putative transmembrane domains. These results indicate that the eDNAs for 68 kDa laminin binding proteins actually code for the 40 kDa ribosomal protein.
Strong affinity of Maackia amurensis hemagglutinin (MAH) for sialic acid‐containing Ser/Thr‐linked carbohydrate chains of N‐terminal octapeptides from human glycophorin A
Authors: Konami Yukiko , Yamamoto Kazuo , Osawa Toshiaki , Irimura Taturo
Published: July 2002

The interaction of the Maackia amurensis hemagglutinin (MAH) with various glycopeptides and oligosaccharides was investigated by means of immobilized lectin affinity chromatography. An amino terminal octapeptide obtained from human glycophorin A having three Neu5Acα→3Galβ1→3(Neu5Acα2→6)GalNAc tetrasaccharide chains, designated as CB‐II, was found to have an extremely strong affinity for MAH. Therefore, it is strongly suggested that hemagglutination by MAH was caused by its interaction with Ser/Thr‐linked carbohydrate chains of human glycophorin A on erythrocyte membranes.
Collagen‐binding recombinant fibronectin fragments containing type II domains
Authors: Skorstengaard Karua , Holtet Thor L. , Etzerodt Michael , Thøgersen Hans C.
Published: July 2002

Each of the two type II domains and four larger fragments, containing one or two type II domains of fibronectin, have been expressed in Escherichia coli. A special vector, containing a fragment encoding the cleavage site for Factor Xa, Ile‐Glu‐Gly‐Arg, inserted immediately before the protein fragment of interest, was used. After treatment of the purified fusion proteins with reduced/oxidized glutathione, the correctly folded fibronectin fragments were released by proteolytic digestion with Factor Xa. The largest fragment, consisting of two type II and two type I domains, was the only fragment able to bind to immobilized gelatin.
Dominant negative chimeras provide evidence for homo and heteromultimeric assembly of inward rectifier K+ channel proteins via their N‐terminal end
Authors: Fink Michel , Duprat Fabrice , Heurteaux Catherine , Lesage Florian , Romey Georges , Barhanin Jacques , Lazdunski Michel
Published: July 2002

Chimeras have been constructed using three different fragments (N‐terminal, central and C‐terminal) of IRK3, a constitutive inward rectifier K+ channel subunit, and GIRK2, a G‐protein activated inward rectifier K+ channel subunit and have been coinjected into Xenopus oocytes together with IRK3 or IRKI (another constitutive inward rectifier) cRNA. Both IRK1 and IRK3 expression was inhibited by coinjection with chimeras containing a N‐terminal fragment of IRK3 suggesting that subunits of K+ channels in the IRK family form a functional multimeric assembly where the N‐terminal end has an important role. In situ hybridization shows that IRK1 and IRK3 are coexpressed in the same areas of the brain and probably in the same cells. Taken together both the localization and the oocyte expression results suggest that not only homomultimeric IRKI or homomultimeric IRK3 assemblies take place but that heteromultimeric assemblies are also formed.