bluuebunny/crossref_metadata_2025 huggingface dataset:
A deazaadenosine‐insensitive methylation of phosphatidylethanolamine is involved in lipoprotein secretion
Authors: Vance Jean E. , Vance Dennis E.
Published: July 2002

We have examined the effect of inhibitors of methylation of phosphatidylethanolamine on lipoprotein secretion from cultured rat hepatocytes. The incorporation of [1‐3H]ethanolamine into phosphatidylcholine of hepatocytes and secreted lipoproteins was inhibited by greater than 90% by the methylation inhibitors 3‐deazaadenosine and Neplanocin. In addition, these compounds strongly inhibited the incorporation of [3‐3H]serine into the choline moiety of phosphatidylcholine of the hepatocytes, but had no effect on incorporation of [3‐3H]serine into secreted phosphatidylcholine. The results suggest that a pool of phosphatidylcholine targeted for lipoprotein secretion originates from phosphatidylethanolamine made from serine and this methylation reaction has the unique property of being insensitive to 3‐deazaadenosine.
Identification of disulphide‐bonded type X procollagen polypeptides in embryonic chick chondrocyte culture
Authors: Kwan A.P.L. , Sear C.H.J. , Grant M.E.
Published: July 2002

A high‐M
r (M
r 120000), disulphide‐bonded collagenous polypeptide was observed to co‐purify with the proα1(X) chain during isolation of cartilage collagens from culture medium of embryonic chick tibiotarsal chondrocytes. This high‐M
r polypeptide was subsequently shown by two‐dimensional SDS‐PAGE and peptide mapping to represent a dimer of the proα1(X) chain of type X collagen linked by disulphide bonding in the non‐collagenous domains.
Rapid effects ofphorbol ester on platelet shape change, cytoskeleton and calcium transient
Authors: Yoshida Ken-ichi , Dubyak George , Nachmias V.T.
Published: July 2002

Platelets are rapidly activated by several agonists. When phorbol 12‐myristate 13‐acetate (PMA) was added to washed platelet suspensions 10 s prior to either thrombin or ADP, it caused a dose‐dependent inhibition of shape change correlated with decreased myosin association with the cytoskeleton and with inhibition of the calcium transient measured in fura‐2‐loaded platelets. PMA added 5–10 s after agonists did not reverse shape change or the association of myosin with the cytoskeleton, but markedly increased the rate at which the calcium signal returned to the baseline. The analogue, 4α‐phorbol didecanoate did not cause these effects. Our results suggest that one effect of C‐kinase activation is to provide negative feedback in sequential responses.
Inhibition of topoisomerase I by antibodies in sera from scleroderma patients
Authors: Scott Samuels D. , Tojo Takeshi , Homma Mitsuo , Shimizu Nobuyoshi
Published: July 2002

Purified type I topoisomerase from calf thymus as well as nuclear and cytoplasmic extracts from EGF‐stimulated human and mouse fibroblasts in cell culture efficiently convert supercoiled plasmid DNA to the relaxed form. The purified IgG fraction from the sera of Japanese patients with the rheumatic disease scleroderma were shown to inhibit this relaxation activity. Thus, these patients likely produce autoantibodies to topoisomerase I. In addition, the human, bovine and murine enzymes share antigenic determinants recognized by the antisera.
Decrease in myosin light chain kinase activity of rabbit fast muscle by chronic stimulation
Authors: Klug G.A. , Houston M.E. , Stullo J.T. , Pette D.
Published: July 2002

Analysis of myosin light chain kinase (MLCK) activity in tibialis anterior muscles of the rabbit revealed that chronic stimulation at a frequency of 10 Hz for 24 h per day reduced the enzyme activity in a timedependent manner. Since fast twitch muscle contains significantly more myosin light chain kinase than slow twitch muscle, the observed reductions are consistent with the type of fast‐to‐slow transformation observed for other type‐specific muscle characteristics. The present data also indicate that the stimulation‐induced decrease in MLCK activity precedes the fast‐to‐slow conversion of the myosin molecule as judged by pyrophosphate‐polyacrylamide gel electrophoresis.
A large conductance, voltage‐ and calcium‐activated K+ channel in the basolateral membrane of rat enterocytes
Authors: Morris A.P. , Gallacher D.V. , Lee J.A.C.
Published: July 2002

The patch‐clamp technique was employed to record single channel currents in patches of basolateral membrane from enterocytes isolated from rat small intestine. We demonstrate the presence of a large conductance (250 pS), voltage‐ and calcium‐activated, the maxi. K+ channel in this membrane. Currents in this K+ channel were blocked by the application of barium (5 mM) to the extracellular membrane face.
cDNA clones coding for α‐actinin of Dictyostelium discoideum
Authors: Noegel A. , Witke W. , Schleicher M.
Published: July 2002

cDNA clones coding for Dictyostelium α‐actinin were isolated from a library in the expression vector λgt11 using a genomic probe that contains α‐actinin‐specific sequences. The recombinant phages harbored inserts with sizes up to 3.0 kb. They hybridized to a 3.0 kb message in Northern blot analysis and some produced fusion proteins that reacted with monoclonal antibodies directed against different epitopes of D. discoideum α‐actinin. The sizes of the inserts and the reaction with the monoclonal antibodies indicated that two of the phages carry a nearly complete copy of the α‐actinin message.
ATP synthesis by the (Na+ + K+)‐ATPase in the absence of an ionic gradient Effects of organic solvent
Authors: de Moraes Vera Lucia Goncalves , de Meis Leopoldo
Published: July 2002

A 200‐fold decrease in the Pi concentration required for half‐maximal phosphorylation of the (Na+ + K+)‐ATPase is observed when 40% (v/v) dimethyl sulfoxide is added to the assay medium. The phosphoenzyme formed in the presence of dimethyl sufoxide is able to transfer its phosphate to ADP to form ATP when 400 mM NaCl is added to the medium. The synthesis of ATP depends on the concentrations of organic solvent and of Na+, and is inhibited by ouabain.
Rat plasma α1‐inhibitor3 binds to receptors for α2‐macroglobulin
Authors: Gliemann J. , Sottrup-Jensen L.
Published: July 2002

The cellular binding and uptake was studied for α1‐inhibitor3, a monomeric 200 kDa proteinase inhibitor present in rat plasma. After intravenous injection in the rat the inhibitor disappeared from the circulation with a half‐time of 2.5 min when complexed with chymotrypsin, whereas the half‐time for uncomplexed inhibitor was more than 60 min. 6 min after the injection of labelled complex, 83% was in the liver and 2.5% in the spleen. In vitro experiments at 4°C with isolated hepatocytes and peritoneal macrophages showed binding to the previously described receptors which bind and internalize the tetrameric rat and human α2‐macroglobulin‐proteinase complexes. The binding affinities were similar for the two types of complexes and binding was followed by uptake and degradation of the labelled complex when the cells were warmed to 37°C. The binding of uncomplexed α1‐inhibitor3 was low and did not increase following treatment with methylamine in spite of cleavage of the internal thiol ester. α1‐Inhibitor3‐methylamine was changed to the receptor binding form when treated with chymotrypsin which caused the cleavage of at least one peptide bond in the bait region.
Activation and inhibition of the calcium gate of sarcoplasmic reticulum by high‐affinity ryanodine binding
Authors: Hasselbach Wilhelm , Migala Andrea
Published: July 2002

The occupancy of high‐affinity ryanodine‐binding sites of isolated heavy sarcoplasmic reticulum vesicles occurring in concentrated salt solutions affects ATP‐dependent calcium accumulation and caffeine‐induced calcium release. The initial suppression of calcium uptake is followed by a marked uptake activation resulting in a reduction of the final calcium level in the medium. Simultaneously, caffeine‐induced calcium release is blocked. The dependence of inhibition of calcium uptake and caffeine‐induced calcium release observed in assay media containing physiological concentrations of magnesium and ATP on the concentration of ryanodine corresponds to the drug's effectiveness in living muscles.
Pseudo‐homology of protein standard structures formed by two consecutive β‐strands
Authors: Efimov A.V.
Published: July 2002

Protein standard structures formed by two consecutive β‐strands connected by short loops are considered in this paper. A stereochemical analysis of each standard structure has been performed to determine the necessary conditions which must be fulfilled in the amino acid sequence encoding the standard structure. It is shown that amino acid sequences coding for the same standard structure in ditterent proteins have practically the same order of hydrophobic, hydrophilic and glycine residues. The results of the stereochemical analysis are confirmed by a large number of examples from known protein structures.
Sequence and expression of the chicken calcitonin gene
Authors: Minvielle S. , Cressent M. , Delehaye M.C. , Segond N. , Milhaud G. , Jullienne A. , Moukhtar M.S. , Lasmoles F.
Published: July 2002

The avian calcitonin gene was isolated and sequenced; two mRNAs are expressed by tissue‐specific alternate splicing. The peptides encoded by the mRNAs are the protein precursors of either calcitonin or calcitonin gene‐related peptide (CGRP). Calcitonin is expressed predominantly in ultimobranchial bodies and CGRP in brain.
Interaction of the human insulin receptor with the ras oncogene product p21
Authors: O'Brien Richard M. , Siddle Kenneth , Houslay Miles D. , Hall Alan
Published: July 2002

Autophosphorylation of the purified human insulin receptor tyrosyl kinase was found to be inhibited by the ras oncogene product p21 in a concentration‐ and GDP‐dependent manner. GDP‐β‐S but not Gpp(NH)p could substitute for GDP in eliciting the ras‐dependent inhibition. The inhibition was seen with both normal or mutant (Lys‐61) p21N‐ras and normal or mutant (Val‐12) p21Ha‐ras. Inhibition occurred at 23°C but not 4°C and was unaffected by the presence or absence of insulin although insulin stimulated the autophosphorylation rate of the receptor β‐subunit some 2‐fold. The insulin receptor did not phosphorylate native p21Ha‐ras in the presence or absence of added guanine nucleotide. After denaturation of p21Ha‐ras with urea it became a substrate, but then failed to inhibit receptor autophosphorylation even in the presence of added GDP.
Respiratory control in the glucose perfused heart
Authors: Katz Lawrence A. , Koretsky Alan P. , Balaban Robert S.
Published: July 2002

The phosphate metabolises, adenosine diphosphate (ADP), inorganic phosphate (Pi), and adenosine triphosphate (ATP), are potentially important regulators of mitochondrial respiration in vivo. However, previous studies on the heart in vivo and in vitro have not consistently demonstrated an appropriate correlation between the concentration of these phosphate metabolites and moderate changes in work and respiration. Recently, mitochondrial NAD(P)H levels have been proposed as a potential regulator of cardiac respiration during alterations in work output. In order to understand better the mechanism of respiratory control under these conditions, we investigated the relationship between the phosphate metabolites, the NAD(P)H levels, and oxygen consumption (Q02) in the isovolumic perfused rat heart during alterations in work output with pacing. ATP, creatine phosphate (CrP), Pi and intracellular pH were measured using 31P NMR. Mitochondrial NAD(P)H levels were monitored using spectrofluorometric techniques. Utilizing glucose as the sole substrate, an increase in paced heart rate led to an increase in Q02 from 1.73 ± 0.09 to 2.29 ± 0.12 mmol Q2/h per g dry wt. No significant changes in the levels of Pi, PCr, ATP, or the calculated ADP levels were detected. Under identical conditions, an increase in heart rate was associated with a 23±3% increase in NAD(P)H fluorescence. Thus, under the conditions of these studies, an increase in Q02 was not associated with an increase in ADP or Pi. In contrast, increases in Q02 were associated with an increase in NAD(P)H. These data are consistent with the notion that increases in the mitochondrial NADH redox state regulate steady‐state levels of respiration when myocardial work is increased.
α2‐Adrenergic inhibition of pancreatic islet glucose utilization is mediated by an inhibitory guanine nucleotide regulatory protein
Authors: Laychock Suzanne G. , Bilgin Suna
Published: July 2002

The rate of glucose utilization in isolated pancreatic islets of the rat was inhibited by the α2‐adrenoceptor agonists clonidine and epinephrine. Yohimbine reversed the inhibition. α1 or β‐adrenoceptor agonists had little or no effect on glucose utilization. Stimulation of muscarinic receptors by carbamylcholine reversed the effect of clonidine. Pertussis toxin blocked the effect of clonidine on glucose utilization, and potentiated the response to carbamylcholine. 8‐Bromo‐cAMP did not affect glucose utilization in the presence of clonidine. Thus, α2‐adrenoceptors negatively modulate glucose utilization, and the effect is mediated by an inhibitory guanine nucleotide regulatory protein, but not by cAMP.
ß‐Subunit of Escherichia coli F1‐ATPase
Authors: Hsu Shih-Yuan , Noumi Takato , Takeyama Michiyasu , Maeda Masatomo , Ishibashi Sadahiko , Futai Masamitsu
Published: July 2002

A mutant strain KF87 of E. coli with a defective ß‐subunit (Ala‐151 → Val) of F1‐ATPase was isolated. The mutation is within the conserved sequence (G‐X‐X‐X‐X‐G‐K‐T/S) of nucleotide‐binding proteins. The mutant F1‐ATPase had a much higher rate of uni‐site hydrolysis of ATP than the wild type, and about 6% of the wild‐type multi‐site activity. The mutant enzyme showed defective transmission of conformational change(s) between the ligand‐ and aurovertin‐binding sites.
L6 skeletal muscle cells have functional V1‐vasopressin receptors coupled to stimulated inositol phospholipid metabolism
Authors: Wakelam Michael J.O. , Patterson Scan , Hanley Michael R.
Published: July 2002

The effects of vasopressin and related peptides upon the rat skeletal muscle cell line, L6, have been examined. No effects upon cellular cyclic AMP levels were found indicating that L6 cells possess no functional V2‐vasopressin receptors. Vasopressin and its analogues did, however, stimulate the rapid and dose‐dependent accumulation of inositol phosphates. This effect and the rank order of potency of vasopressin analogues demonstrate the presence of functional V1‐vasopressin receptors upon L6 cells. These results suggest that the L6 line may be a useful model for vasopressin effects upon skeletal muscle metabolism.
Amino acid neurotransmitters in the CNS
Authors: Erecińska Maria , Nelson David
Published: July 2002

Carefully isolated, metabolically competent rat brain synaptosomes accumulate acidic amino acid neuro‐transmitters down to very low external levels. This supports the suggestion that nerve endings are involved in terminating transmission at the synapses and in maintaining low levels of these molecules in the external environment in the brain. At saturating levels of acidic amino acids, the rate of inward and outward movements of the Na+‐amino acid complex (exchange) is much faster than the net uptake. The transmembrane gradients of aspartate and glutamate approach each other under all conditions explored which indicates that these two amino acids share the same transport system.
Three adenine nucleotide binding sites in F1‐F0 mitochondrial ATPase as revealed by presteady‐state and steady‐state kinetics of ATP hydrolysis Evidence for two inhibitory ADP‐specific noncatalytic sites
Authors: Bulygin V.V. , Vinogradov A.D.
Published: July 2002

Preincubation of submitochondrial particles with ADP in the presence of Mg2+ results in the complete inhibition of ATPase which is slowly reactivated in the assay mixture containing ATP and the ATP regenerating system. Significantly, the rate of activation increases as the concentration of ADP in the preincubation mixture rises from 1 μM to 20 μM and reaches a constant value at higher ADP concentrations. The first‐order rate constant for the activation process in the assay mixture is ATP‐dependent at any level of inhibitory ADP. The data obtained strongly suggest that two ADP‐specific inhibitory sites and one ATP‐specific hydrolytic site are present in F1‐F0 ATPase. Taking into account the (3α·3β)·γ·δ·ϵ structure of F1, it is concluded that the synchronous discharge of ADP from two inhibitory sites during the activation occurs after ATP binds to the ATPase catalytic site.
K+ channels activated by L‐alanine transport in isolated Necturus enterocytes
Authors: Sheppard D.N. , Giraldez F. , Sepúlveda F.V.
Published: July 2002

Using the patch‐clamp technique, we demonstrate here the opening of K+ channels evoked by the actively transported amino acid L‐alanine in isolated Necturus enterocytes. These channels had a conductance of about 30 pS and their activation was dependent on transmembrane electrical potential and cytosolic Ca2+.
NMR studies identify four intermediate states of ATPase and the ion transport cycle of sarcoplasmic reticulum Ca2+‐ATPase
Authors: Klemens Mary R. , Grisham Charles M.
Published: July 2002

Water proton nuclear relaxation measurements are used to detect and characterize four distinct intermediate states for Gd3+ bound to Ca2+ sites of sarcoplasmic reticulum Ca2+‐ATPase in complexes with ATP analogues. In the absence of nucleotides, Gd3+ binds to two occluded Ca2+ transport sites on Ca2+‐ATPase which have a low accessibility to solvent water. In the presence of the nonhydrolyzable ATP analogue, Co(NH3)4AMPPCP, a new state for bound Gd3+ (still occluded and with fewer waters of hydration) is observed. In the presence of Co(NH3)4ATP or ATP, two additional states for bound Gd3+ are detected in the NMR studies. The first of these probably represents an intermediate state for bound Gd3+ during ATP hydrolysis. The latter is the most occluded Gd3+ site yet observed in these studies and is probably analogous to the highly occluded E1‐P state observed with CrATP [(1987) Biochim. Biophys. Acta 898, 313–322].
Crystal structure of a complex between thermitase from Thermoactinomyces vulgaris and the leech inhibitor eglin
Authors: Dauter Zbigniew , Betzel Christian , Höhne Wolfgang-Ernst , Ingelman Margareta , Wilson Keith S.
Published: July 2002

Thermitase, the thermostable alkaline protease from Thermoactinomyces vulgaris, has been crystallised in a 1:1 complex with eglin, the inhibitor from the medical leech. Two large crystals were grown, with cell dimensions of a = 49.3 Å, b = 67.3 Å, c = 90.5 Å and space group P212121. The crystals are relatively tightly packed with V
m = 2.1 Å3/Da. Three‐dimensional data to 1.9 Å have been recorded from one of these crystals. The orientation and position of the complex in the unit cell have been established using the subtilisin Carlsberg‐eglin structure as a model. The structure of the complex is being refined by restrained least‐squares. The present crystallographic R factor (= Σ|F
o−F
c|/Σ|F
o) is 26% at 2.5 Å resolution.
Fractionation of synaptophysin‐containing vesicles from rat brain and cultured PC12 pheochromocytoma cells
Authors: Wiedenmann Bertram , Rehm Hubert , Knierim Martin , Becker Cord-Michael
Published: July 2002

Synaptophysin is a transmembrane glycoprotein of neuroendocrine vesicles. Its content and distribution in subcellular fractions from cultured PC12 cells, rat brain and bovine adrenal medulla were determined by a sensitive dot immunoassay. Synaptophysin‐containing fractions appeared as monodispersed populations similar to synaptic vesicles in density and size distribution. Membranes from synaptic vesicles contained ≈ 100‐times more synaptophysin than chromaffin granules. In conclusion, synaptophysin is located almost exclusively in vesicles of brain and PC12 cells which are distinct from dense core granules.
Effect of Graves' IgG on gene transcription in human thyroid cell cultures Thyroglobulin gene activation
Authors: Kung Annie W.C. , Collison Kate , Banga J.Paul , McGregor Alan M.
Published: July 2002

In Graves' disease (GD) the presence of antibodies to the thyroid stimulating hormone (TSH) receptor leads to stimulation of the thyroid gland. The thyroid stimulating activity of Graves' IgG is normally ascertained by bioassays measuring cAMP production. We have investigated the effect of Graves' IgG on the quantitative activation of thyroglobulin (TG) gene in cultured human thyroid cells by RNA hybridisation. TG mRNA expression was activated by TSH and Graves' IgG. Nuclear transcription assays showed that the increase in cytoplasmic mRNA levels was due to increased transcription of TG specific mRNA in nuclei of thyroid cells. Whilst TSH led to a dose dependent increase in TG mRNA levels, Graves' IgG led to a variable activation of TG gene. A significant correlation between the increased TG mRNA transcription and cAMP production was observed with Graves' IgG. Thus the activation of the TG gene by Graves' IgG occurs in parallel with elevation of cAMP.
Extracellular ‘communicator RNA’
Authors: Benner Steven A.
Published: July 2002

Evidence is presented that RNA molecules exist that act as extracellular messages important in the development of higher organisms.
Topology of Na+,K+‐ATPase Identification of the extra‐ and intracellular hydrophilic loops of the catalytic subunit by specific antibodies
Authors: Ovchinnikov Yu.A. , Luneva N.M. , Arystarkhova E.A. , Gevondyan N.M. , Arzamazova N.M. , Kozhich A.T. , Nesmeyanov V.A. , Modyanov N.N.
Published: July 2002

To study the topology of Na+,K+‐ATPase monoclonal antibodies (MAbs) specific for membrane‐bound enzyme were produced. Using immunofluorescence staining of viable cells or smears of a pig kidney embryonic (PKE) cell line, two groups of MAbs were selected, namely those binding to extra‐ or intracellular portions of the α‐subunit. The extracellular location of peptide loop 804–841 linking the Vth and VIth intramembrane hydrophobic segments was proved using MAb VG2. Another MAb, IIC9, interacting with PKE cells only after membrane perforation (4% formaldehyde and 0.1% Tween‐20), was shown to bind to the hydrophilic loop 868–945. The antigenic determinants recognized by MAb IIC9 and VG2 are located in peptides 887–904 and 810–825, respectively. The C‐terminus of the α‐subunit molecule was positioned on the outer side of the cytoplasmic membrane utilizing affinity‐purified antibodies to the synthetic peptide corresponding to fragment 999–1008.
Measurement of cytosolic free Ca2+ in individual pancreatic acini
Authors: Pralong William F. , Wollheim Claes B. , Bruzzone Roberto
Published: July 2002

The kinetics of changes in cytosolic free Ca2+ ([Ca2+]i) were determined in individual rat pancreatic acini by microfluorimetry. Three major findings are reported. First, at maximal stimulatory concentrations for amylase release, both caerulein and bombesin induced an initial rise in [Ca2+]i followed by prolonged secondary oscillations of smaller amplitude. The latter effect was not observed with supramaximal doses of caerulein. Second, these cyclic changes were dependent, at least in part, on extracellular Ca2+. Finally, comparison of the threshold doses for [Ca2+]i mobilization and enzyme discharge demonstrated that pathways independent of an elevation of [Ca2+]i control the secretory activity of pancreatic acini at low, picomolar agonist concentrations.
Secondary structure of the promastigote surface protease of Leishmania
Authors: Jähnig Fritz , Etges Robert
Published: July 2002

By Raman spectroscopic analysis we have determined the secondary structure of the promastigote surface protease, named PSP or gp63, of Leishmania major. It consist of nearly 50% antiparallel β‐strand, and less than 20% α‐helix. These results are contrasted with the predominantly α‐helical VSGs of the African trypanosomes and the α‐helical metalloprotease thermolysin. The PSP of Leishmania thus represents a novel kind of membrane‐anchored protease.
Serum induces the immediate opening of Ca2+‐activated channels in quiescent human fibroblasts
Authors: Peres Antonio , Zippel Renata , Sturani Emmapaola
Published: July 2002

Application of fetal calf serum to quiescent human fibroblasts produces an immediate (3 – 20 s delay) increase in membrane conductance which lasts about 20 – 30 s. This conductance is strongly outwardly‐rectifying and has a reversal potential between −45 and −10 mV. The conductance increase may also be induced by application of the Ca2+ ionophore A23187 while it does not occur when intracellular K+ is replaced by Cs+. It is concluded that this early effect of serum is due to the opening of Ca2+‐activated channels. This permeability change will alter the membrane potential and thus possibly interact with other voltage‐sensitive processes induced by serum growth factors.
Identification and characterization of glucagon‐like peptide‐1 7–36 amide‐binding sites in the rat brain and lung
Authors: Kanse Sandip M. , Kreymann Bernhard , Ghatei Mohammad A. , Bloom Stephen R.
Published: July 2002

High‐affinity binding sites for glucagon‐like peptide‐1 7–36 amide (GLP‐1 7–36 NH2) were identified in rat brain and lung membranes. Binding of [125]GLP‐1 7–36 NH2 was rapid, reversible, specific, saturable and pH dependent. Specific binding in the central nervous system was particularly high in the hypothalamus and the brain stem. Oxyntomodulin, glucagon‐like peptide‐1, glucagon‐like peptide‐2 and glucagon were 100–1000‐fold less potent than GLP‐1 7–36 NH2 in competition for this binding site.
Isolation and characterization of complementary DNAs encoding human manganese‐containing superoxide dismutase
Authors: Ho Ye-Shih , Crapo James D.
Published: July 2002

cDNAs coding for human manganese‐containing superoxide dismutase (Mn SOD) have been isolated from a human liver and a dibutyryl cyclic AMP differentiated U937 cDNA library constructed in vector λgtll. The nucleotide sequences of the insert cDNAs had an opening reading frame coding for 222 amino acid residues. The first 24 amino acids of the primarily translated polypeptide might constitute the leader peptide for transport of the precursors to the mitochondria. Differentiation of the U937 cells with dibutyryl cyclic AMP resulted in a 70% decrease in Mn SOD mRNA. The amino acid sequences of the mature Mn SODs of human, rat and mouse are highly conserved, while the sequences of the leader peptides of these species are moderately conserved.
Enhanced transcription of mitochondrial genes after growth stimulation and glucocorticoid treatment of Reuber hepatoma H‐35
Authors: Kadowaki Tatsuhiko , Kitagawa Yasuo
Published: July 2002

When a serum‐deprived culture of Reuber hepatoma H‐35 cells was stimulated by serum, cells proliferated synchronously showing a peak of [3H]thymidine incorporation within 16 h and doubling of cell number within 24 h. A 5–10‐fold increase of mRNAs of mitochondrial genes was observed prior to S‐phase by Northern hybridization of total cellular RNA with cloned fragments of rat mitochondrial DNA as probes. In organelle transcription by mitochondria isolated from growth‐arrested or growth‐stimulated cells suggested that the increase of mitochondrial mRNAs was mainly due to enhanced transcription. Less enhanced transcription was observed by treating the cells with dexamethasone, which causes translocation of large amount of protein into mitochondria. The latter effect was counteracted by insulin.
Selective terminal α2–3 and α2–6 sialylation of glycosphingolipids with lacto‐series type 1 and 2 chains in human meconium
Authors: Iwamori M. , Noguchi M. , Yamamoto T. , Yago M. , Nozawa S. , Nagai Y.
Published: July 2002

Human meconium was found to contain two kinds of gangliosides with the same carbohydrate sequences belonging to the lacto‐series. They were detected by TLC‐immunostaining with monoclonal antibodies directed to the NeuAcα2–Gal and Lc4Cer structures. One of these two gangliosides, a major one, which migrated on TLC to a position below that of standard IV3NeuAcnLc4Cer from human erythrocytes, reacted with the antibody to NeuAcα2–6Gal. The other minor one, which migrated on TLC to a position corresponding to standard IV3NeuAcnLc4Cer, was detected with the antibody to Lc4Cer only when the plate, on which the individual gangliosides were separated, was subjected to prior treatment with Vibrio cholerae sialidase. The structures of the gangliosides, each identified by means of permethylation analysis and enzyme treatment after isolation with antibody monitoring, were shown to be IV6NeuAcnLc4Cer for the former and IV3NeuAcLc4Cer for the latter, indicating that the lacto‐series type 2 (nLc4Cer) and 1 (Lc4Cer) chains are sialylated at different linkages, α2–6 and α2–3, respectively. IV6NeuAcLc4Cer and IV3NeuAcnLc4Cer were not detected, even in trace amounts, on TLC‐immunostaining with the monoclonal antibodies. The concentrations of IV6NeuAcnLc4Cer and IV3NeuAcLc4Cer were 448 and 18 nmol/g dry wt of human meconium.
The phosphatidylcholine pathway of diacylglycerol formation stimulated by phorbol diesters occurs via phospholipase D activation
Authors: Cabot Myles C. , Welsh Clement J. , Cao Hui-ting , Chabbott Holly
Published: July 2002

Agonist‐induced degradation of phosphatidylcholine (PC) is of interest as this pathway of diacylglycerol (DG) generation may provide added opportunities for the regulation of protein kinase C (PKC). In REF52 cells [3H]myristic acid is preferentially incorporated into PC; this, coupled with the use of [3H]choline, allows for quantitation of both the water‐soluble and the lipid products generated when PC is degraded. In cells prelabeled with [3H]choline, TPA stimulated a time‐dependent release, into the medium, of choline and not phosphocholine or glycerophosphocholine. Treatment of [3H]myristic acid‐labeled cells with either phorbol diesters, sn‐1,2‐dioctanoylglycerol, or vasopressin elicited the formation of labeled phosphatidate (PA) and DG. The temporal pattern of PC hydrolysis in cells treated with TPA is indicative of a precursor (PA)‐product (DG) relationship for an enzymatic sequence initiated by phospholipase D. Adding propranolol, a phosphatidate phosphohydrolase inhibitor, eliminated TPA‐induced DG formation, whereas PA generation was unaffected. From these data we conclude that TPA elicits DG formation from PC by the sequential actions of phospholipase D and phosphatidate phosphohydrolase.
Tissue‐specific expression of profilin
Authors: Buß Folma , Jockusch Brigitte M.
Published: July 2002

Expression of profilin and profilin:actin ratios in vertebrates were determined with polyclonal antibodies against profilin and actin. Profilin was detected in a wide variety of bovine tissues and was especially enriched in smooth muscle of bovine, porcine and avian origin. The protein was purified from pig stomach muscle tissue. Smooth muscle profilin was found to be more effective in inhibiting the polymerization of skeletal muscle actin than thymus profilin purified by the same method.
Predicted effects on herbicide binding of amino acid substitutions in the D1 protein of photosystem II
Authors: Horovitz Amnon , Ohad Nir , Hirschberg Joseph
Published: July 2002

The effects of an amino acid substitution in the D1 protein of photosystem II on its binding to different herbicides are here successfully predicted. This is done by assuming additivity in the contributions to the binding energy of the amino acids at positions 255 and 264 of this protein which are part of the herbicide binding niche. Additivity in D1 protein‐herbicide interactions is demonstrated, thus indicating an apparent lack of interactions between the amino acids at these two positions.
87Rb NMR studies of the perfused rat heart
Authors: Allis Jonathan L. , Snaith Christine D. , Seymour Anne-Marie L. , Radda George K.
Published: July 2002

We have studied 87Rb+ fluxes in the perfused rat heart using nuclear magnetic resonance spectroscopy (NMR). A simple model for the interpretation of the data is presented. A comparison of radioactively measured K+ fluxes and the K+ fluxes deduced from the 87Rb+ measurements shows them to be very similar. This method provides a means of noninvasively measuring uni‐directional K+ fluxes in the heart.
The [Ca2+]i increase induced in murine thymocytes by extracellular ATP does not involve ATP hydrolysis and is not related to phosphoinositide metabolism
Authors: El-Moatassim Chakib , Maurice Tangui , Mani Jean-Claude , Dornand Jacques
Published: July 2002

We have previously demonstrated that exogenous ATP can give medullary thymocytes the calcium message required for the induction of their blastogenesis. In the present study, using the highly sensitive calcium indicator Indo‐1, we have measured the effect of exogenous nucleotides on the cytosolic‐free calcium concentration [Ca2+]i of thymocytes, and determined inositol phosphate IP formation in the same cells, in parallel assays. The results were compared to those obtained with the mitogenic lectin concanavalin A ConA in similar experiments. They show that ATP does not mobilize calcium from its internal stores but stimulates its influx from the extracellular medium. Nevertheless, these data do not rule out the possibility that the nucleotide acts through specific P2 purinergic sites.
The inotropic effect of endothelin‐1 on rat atria involves hydrolysis of phosphatidylinositol
Authors: Vigne Paul , Lazdunski Michel , Frelin Christian
Published: July 2002

Endothelin‐1 induces a positive inotropic response in isolated left atria of the rat with an IC50 value of 20 nM. The contractile effect of endothelin is larger than that of other inotropic hormones such as phenylephrine and epinephrine and smaller than that of Bay K8644. In the spontaneously active right atria, endothelin induces a positive inotropic effect with no chronotropic effect. Endothelin does not modify intracellular levels of cAMP under basal conditions or after stimulation with isoproterenol but stimulates the formation of inositol phosphates. Mobilization of inositol phospholipids is observed in the same range of concentrations as for the contractile action of endothelin. The contractile action of endothelin is not mediated by protein kinase C. It is antagonized by blockers of L‐type Ca2+ channels, low external Ca2+ concentrations and drugs such as caffeine and ryanodine that interfere with Ca2+ release by the sarcoplasmic reticulum.
Synthesis of an affinity ligand (‘UPHIT’) for in vivo acylation of the κ‐opioid receptor
Authors: de Costa Brian R. , Band Linda , Rothman Richard B. , Jacobson Arthur E. , Bykov Victor , Pert Agu , Rice Kenner C.
Published: July 2002

The isothiocyanate analog (1S,2S‐trans‐2‐isothiocyanato‐4,5‐dichloro‐N‐methyl‐N‐[2‐(1‐pyrrolidinyl)cyclohexyl]benzeneacetamide, 3a) of the highly selective κ‐opioid receptor agonist, U50,488, was prepared as a potential site‐directed affinity ligand for acylation of κ‐opioid receptors in vivo. The isothiocyanate (3a) which we have designated UPHIT and its enantiomer (3b) were synthesized in 3 steps starting from optically pure (1S,2S)‐(+)‐trans‐2‐pyrrolidinyl‐N‐methyl‐cyclohexylamine (4a) and its enantiomer (4b), respectively, thus defining their absolute stereochemistry. Binding in vitro of the 1S,2S enantiomer 3a to κ receptors labelled by [3H]U69,593 was shown to occur with an IC50 value of 25.92 ± 0.36 nM, whereas 827.42 ± 5.88 and 115.10 ± 1.23 nM were obtained for the IC50 value of the 1R,2R enantiomer (3b) and (±)‐3 respectively. Intracerebroventricular (ICV) injection of 100 μg of (±)‐3 into guinea‐pig brain followed by analysis of remaining κ‐binding sites 24 h later revealed that (±)‐3 depleted 98% of the κ receptors that bind [3H]U69,593 and 40% of those that bind [3H]bremazocine. These preliminary data suggest exciting uses for these compounds in furthering our knowledge of the κ‐opioid receptor.
A synthetic peptide analog of the putative substrate‐binding motif activates protein kinase C
Authors: House Colin , Robinson Phillip J. , Kemp Bruce E.
Published: July 2002

A 29‐residue synthetic peptide, Leu530‐Leu‐Tyr‐Glu‐Met‐Leu‐Ala‐Gly‐Gln‐Ala‐Pro‐Phe‐Glu‐Gly‐Glu‐Asp‐Glu‐Asp‐Glu‐Leu‐Phe‐Gln‐Ser‐Ile‐Met‐Glu‐His‐Asn‐Val‐NH2, 558, corresponding to part of the catalytic domain of protein kinase C, is a potent activator of the enzyme, with a K
a, of approx. 10 μM. Activation was 59 ± 4% of that observed with phosphatidylserine, predominantly due to an increased V
max, partially calcium‐dependent, observed with all three isoenzymes (α, β, γ), and resulted in autophosphorylation. It is proposed that the region between Gly528 and Arg583 is part of the protein substrate binding region of protein kinase C and synthetic peptide analogs of this region activate the enzyme by blocking the action of the enzyme's basic pseudosubstrate autoregulatory region.
Direct activation of a protein kinase activity from rat lacrimal gland by PMA in a phospholipid‐free system
Authors: Mauduit Philippe , Zoukhri Driss , Rossignol Bernard
Published: July 2002

The analysis of the cytosolic fraction from rat exorbital lacrimal gland with DEAE‐cellulose ion‐exchange chromatography showed the presence of a peak of protein kinase activity which was dependent on the presence of phosphatidylserine and diolein as well as calcium. This activity showed the same properties as the previously reported protein kinase C (PKC). Moreover, we have shown for the first time that this kinase or a kinase that coeluted from the column with PKC could be activated by a phorbol ester, PMA, in a phospholipid‐free system, i.e. in the absence of any cofactor of PKC. These findings emphasize the need for caution in the interpretation of experimental results obtained when using phorbol esters to probe for a role of PKC in many regulatory processes.
Differentiation of intestinal epithelial cell line (IEC‐18) by an acid extract of rat small intestine
Authors: Shintani Takafumi , Fushiki Tohru , Fukuoka Shin-ichi , Takahashi-Iwanaga Hiromi , Sugimoto Etsuro
Published: July 2002

A factor which may induce differentiation of intestinal epithelial cell lines in vitro was found in an acid extract of adult rat small intestine. The addition of a partially purified acetic acid extract of rat small intestine to IEC‐18 cell culture dishes increased sucrase activity within 48 h. Thymidine incorporation markedly decreased within 24 h. Significant development of microvilli‐like structures was observed on the acid extract‐treated IEC‐18 cells, compared with controls. This activity of rat acid extract was heat‐stable and the apparent molecular weight of the factor was 400–800. These findings suggested that the factor may be related to the epithelial differentiation of rat small intestinal crypt cells.
Stimulation of polyphosphoinositide hydrolysis in Swiss 3T3 cells by recombinant fibroblast growth factors
Authors: Brown Kenneth D. , Blakeley Diana M. , Brigstock David R.
Published: July 2002

Mitogenic concentrations of recombinant acidic or basic fibroblast growth factor (FGF) stimulated the accumulation of [3H]inositol phosphates ([3H]IPs) in Swiss 3T3 cells pre‐labelled for 48 h with [3H]inositol. Maximal effects were obtained at 0.3 ng/ml and 3 ng/ml for basic and acidic FGF, respectively. Higher doses of either factor led to a diminished stimulation. FGF also stimulated 45Ca2+ release from cells pre‐labelled with the isotope. However, FGF‐stimulated production of [3H]IPs and release of 45Ca2+ exhibited marked differences when compared with the responses to the peptide mitogen bombesin; the FGF responses were markedly slower and were not inhibited by phorbol esters.
Effects of pretreatment with tetradecanoyl phorbol acetate on regulation of growth hormone and prolactin secretion from ovine anterior pituitary cells
Authors: Boyd Robert S. , Wallis Michael
Published: July 2002

Tetradecanoyl phorbol acetate (TPA) stimulates growth hormone (GH) and prolactin secretion from ovine anterior pituitary cells. Pretreatment of the cells with TPA abolishes this effect, presumably due to down‐regulation of protein kinase C. Such pretreatment did not alter effects of thyrotropin‐releasing hormone or dopamine on prolaction secretion, suggesting no involvement of protein kinase C. Pretreatment with TPA attenuated actions of GH‐releasing hormone on GH release (but not actions on cyclic AMP levels) possibly due to depletion of cellular stores of GH. Such pretreatment also attenuated inhibition of GH release by somatostatin, possibly due to phosphorylation of receptors or associated proteins by protein kinase C.
Protein kinase C phosphorylates DNA topoisomerase I
Authors: Samuels D.Scott , Shimizu Yoshiko , Shimizi Nobuyoshi
Published: July 2002

The induction of mammalian cell proliferation requires the expression of a specific set of genes. Tumor promoters stimulate cell growth by activating the Ca2+ and phospholipid‐dependent protein kinase, protein kinase C (PKC). DNA topoisomerase I, a nuclear enzyme involved in transcription, was phosphorylated by activated PKC in vitro. Phosphorylation by PKC stimulated the DNA relaxation activity of topoisomerase I two‐ to three‐fold. Therefore, DNA topoisomerase I is a substrate for PKC‐mediated activation by phosphorylation and may serve as a nuclear target of mitogenic signals generated by tumor promoters in vivo.
Glycosylated forms of nuclear lamins
Authors: Ferrare A. , Eufemi M. , Cervoni L. , Marinetti R. , Turano C.
Published: July 2002

Chromatin and pore complex‐lamina preparations were obtained from pig and chicken tissues, and their proteins were analysed by mono‐ and bidimensional electrophoresis. A glycosylated form of lamin A, recognized by concanavalin A, was shown to be present in at least 3 of the tissues examined. Glycosylation is suggested to be a further postsynthetic modification, besides phosphorylation and methylation, which can modify the properties of lamins.
Sequence and expression of a novel GABAA receptor α subunit
Authors: Ymer Sanie , Draguhn Andreas , Köhler Martin , Schoneid Peter R. , Seeburg Peter H.
Published: July 2002

Cloned cDNA encoding the bovine α4 subunit of the GABAa receptor has been isolated. The predicted 521 amino acid long mature protein contains an exceptionally long intracellular domain and shares 53–56% sequence similarity to the previously characterized α1, α2 and α3, subunits. Co‐expression of α4 and β1 in Xenopus oocytes resulted in the formation of GABA‐gated chloride channels with expected pharmacology, although no benzodiazepine potentiation was observed. Northern analysis indicates that a 4 kb α4 mRNA is expressed in the calf cerebellum, cortex and hippocampus but is barely detectable in the rat brain.
Insulin induces a similar reduction in the concentrations of its own receptor and of an insulin‐sensitive glycosyl‐phosphatidylinositol in isolated rat hepatocytes
Authors: Ruiz-Albusac Juan Miguel , Zueco José Antonio , Velázquez Esther , Alvarez José Félix , Mato José Maria , Blázquez Enrique
Published: July 2002

We have used isolated rat hepatocytes to study whether the insulin‐induced reduction of its own receptors may modify the transduction of hormone signals by changes in the content of a glycosyl‐phosphatidylinositol. Both subsequent insulin binding and glycosyl‐phosphatidylinositol concentrations markedly decreased as a function of time and insulin concentration during preincubation of hepatocytes with insulin. The modifications observed in insulin binding were due to changes in receptor concentration. These results show that insulin regulates both the number of its own receptors and glycosyl‐phosphatidylinositol concentrations in target cells, which may be of interest in many pathophysiological situations.
Ca‐ATPase isozyme expression in sarcoplasmic reticulum is altered by chronic stimulation of skeletal muscle
Authors: Briggs F.Norman , Lee K.Francis , Feher Joseph J. , Wechsler Andrew S. , Ohiendieck Kay , Campbell Kevin
Published: July 2002

Chronic stimulation of a predominantly fast skeletal muscle enhanced the expression of type I (slow muscle) Ca‐ATPase and suppressed the expression of the type II (fast muscle) Ca‐ATPase. Monoclonal antibodies IID8 and IIH11 against type I (slow) and type II (fast) isozymes respectively, were used to type the Ca‐ATPases of the isolated SR (sarcoplasmic reticulum) by Western blots, and the Ca‐ATPases of the muscle fibers by immunohistochemistry. Of the fibers from control muscles 80% stained for the type II isozyme and 20% for the type I isozyme. Following chronic stimulation all fibers stained for type I isozyme and none stained for type II isozyme. Ca‐ATPase isozyme distribution in isolated SR confirmed this effect of chronic stimulation. The calcium uptake activities of homogenates of stimulated muscles were 22% of the control muscles. The Ca‐ATPase and calcium‐uptake activities of the isolated SR from stimulated muscles were, respectively, 32 and 45% of the control muscles.
Polyphosphoinositide synthesis in platelets stimulated with low concentrations of thrombin is enhanced before the activation of phospholipase C
Authors: Lassing Ingrid , Lindberg Uno
Published: July 2002

When platelets, prelabelled with [32P]orthophosphate, were stimulated with thrombin (0.5 U·ml−1) there was an immediate increase in the radioactivity associated with the pools of polyphosphoinositides. Only subsequent to this increase, did the radioactivity of these phospholipid pools decrease as expected from a receptor‐mediated activation of phospholipase C (phosphoinositidase). Phosphorylation of diacylglycerol (one of the second messengers formed in the hydrolysis of phosphatidylinositol‐bisphosphate) to phosphatidic acid took place with a lag phase of about 3–5 s. Together these experiments suggest that stimulation of kinases phosphorylating phosphatidylinositol and phosphatidylinositol‐phosphate may precede or occur in parallel with activation of receptor‐linked phosphoinositidase.
Guanine nucleotide dependent and independent reconstitution of G‐proteins with adenylate cyclase: stimulation or attenuation of the enzyme by Gi α subunits
Authors: Newton Dianne L. , Klee Werner A.
Published: July 2002

The α subunits of five members of the Gi family of bovine brain proteins were reconstituted with adenylate cyclase in phospholipid vesicles. Our results support both the views that much of the inhibition of the enzyme by Gi is due to the action of its liberated βγ subunits, and that the α subunits themselves interact with the enzyme. Inhibition of basal or Gs‐stimulated adenylate cyclase activity is small or undetectable by a subunits of Go, Go∗ and Gi‐2B. On the other hand, adenylate cyclase activity is stimulated by the α subunits of Gi‐1 and Gi‐2A. The G proteins act in the absence of added GTP when reconstituted with phospholipids of low but not high fluidity.
Differential inactivation of vasopressin receptor subtypes in isolated membranes and intact cells by N‐ethylmaleimide
Authors: Pávó Imre , Fahrenholz Falk
Published: July 2002

Vasopressin receptors in plasma membranes and on cell monolayers were treated with sulfhydryl reagents. Specific binding of [3H]AVP to renal V2 receptors in membranes from bovine and porcine kidney and on LLC‐PK1 cells was markedly (80–90%) reduced after treatment with NEM but that to V1 receptors on rat liver membranes and A7r5 smooth muscle cells only slightly (10–30%). Inactivation of receptors by NEM reduced the number of binding sites without altering the affinity of unmodified receptor molecules. High affinity ligands (agonists and antagonists), in complex with the V2 receptor, protected against its inactivation. The results suggest that one or more cysteine residues are located in the ligand‐binding site of the V2 receptor, and are essential for hormone binding. Furthermore, it is possible to use NEM to differentiate between vasopressin isoreceptors.
Proteolytic activation of a bovine brain protein with phosphatidylinositol transfer activity
Authors: van den Akker Willem M.R. , Westerman Jan , Gadella Theodorus W.J. , Wirtz Karel W.A. , Snoek Gerry T.
Published: July 2002

We have purified a 38 kDa protein from bovine brain which is cross‐reactive with an affinity purified antibody against the 35 kDa phosphatidylinositol transfer protein from the same source. Controlled trypsinization of the 38 kDa protein yielded an immunoreactive protein of 35 kDa which displayed a 6‐fold increase in phosphatidylinositol transfer activity and a 10‐fold higher affinity for this phospholipid. The possibility that the 38 kDa protein is a precursor of the phosphatidylinositol transfer protein is discussed.