bluuebunny/crossref_metadata_2025_split huggingface dataset:
Metabolism of neutral lipids in cultured fibroblasts from multisystemic (or type 3) lipid storage myopathy
Authors: RADOM Jean , SALVAYRE Robert , NEGRE Anne , MARET Arlette , DOUSTE‐BLAZY Louis 
Published: March 2005

The lipid metabolism in cultured fibroblasts from multisystemic (type 3) lipid storage myopathy (MLSM) and controls has been studied through pulse‐chase experiments using radiolabelled oleic acid and acetate precursors.1. The uptake of radiolabelled oleic acid by MLSM fibroblasts was slightly higher than in controls but did not seem to be the primary defect of the multisystemic lipid storage myopathy. The uptake of radiolabelled acetate was quite similar in MLSM and in control cells.2. During short‐time pulse periods, using either radiolabelled oleic acid or acetate as precursors, we observed no significant difference in lipid composition between MLSM and controls. In contrast, pulse experiments using radiolabelled oleic acid as precursor showed a major accumulation of radiolabelled triacylglycerols in MLSM (around 1000% of controls); no significant increase of other neutral or polar lipids was noticed. A similar triacylglycerol storage was observed by using radiolabelled acetate as precursor, but in this case the difference between MLSM and controls was more pronounced; we also observed in MLSM cells a higher amount of polar lipids which can be due to an increased rate of fatty acid biosynthesis (from radiolabelled acetate).3. Chase experiments, after pulse by low concentration of exogenous radiolabelled oleic acid or acetate, showed similar features: the biosynthesized triacylglycerols were not at all degraded in MLSM, but on the contrary increased, probably by accumulation of radiolabelled triacylglycerols newly synthesized from radiolabelled fatty acids liberated during the phospholipid turnover. Similarly, the triacylglycerol storage induced by high doses of fatty acids was not degraded in MLSM cells, in contrast to control cells. This suggested that the triacylglycerols synthesized in the presence of low and high levels of fatty acids were accumulated in only one subcellular cytoplasmic compartment without relationship with the lysosomal compartment, since these cells were not deficient in acid lysosomal lipase. The more probable hypothesis is a deficiency of the cytoplasmic catabolism of triacylglycerols in MLSM cells.
The reaction of human α2‐macroglobulin with α‐chymotrypsin
Authors: FAVAUDON Vincent , TOURBEZ Martine , POCHON François , MAREIX Robert , TOURBEZ Hervé 
Published: March 2005

The kinetics of the conformational changes of human α2‐macroglobulin (α2M) induced by reaction with pure α‐chymotrypsin, have been analyzed using three fluorescent probes, namely protein tryptophan groups and the dye 6‐(4‐toluidino)‐2‐naphthalenesulfonate, to monitor alterations of the α2M structure, and a covalent conjugate of chymotrypsin and fluorescein isothiocyanate (Chy‐FITC). The main reaction sequence exhibits a triphasic time course with any of the labels used. Each phase is first‐order. The fixation of a single molecule of chymotrypsin to one protease‐binding site of α2M (site A) initiates the whole process and determines the access to the second site (site B). Of the three exponential phases of the reaction (20°C), phase I (k1∼ 19.6 min−1) and phase II (k2∼ 5.3 min−1) belong to site A. Phase III is related to site B transformation. It contains two steps with different responses from tryptophan (k3∼ 0.77 min−1) and Chy‐FITC (k3∼ 0.19 min−1) fluorescence measurements. The point to be stressed is that site A and site B, while presumably identical in the native form, are not equivalent with regard to their fluorescence and kinetic properties. However, the activation energy (E= 30.1 ± 2.7 kJ mol−1) is the same for the three phases of the reaction.When present in sufficient excess, free chymotrypsin or native α2M is able to form reversible complexes with the above‐related chymotrypsin‐α2M adducts. Only the α2M site A core seems to be involved in this parallel process. In addition the conformational state of the chymotrypsin‐α2M complexes is shown to depend on the pH, with a pKa of 6.4.
Characteristics of thyroid protein kinase C
Authors: OMRI Boubaker , BRETON Marie France , PAVLOVIC‐HOURNAC Miroslava 
Published: March 2005

Thyroid protein kinase C (PKc) from cytosols of porcine and rat thyroid glands has been characterized using histone H1 or endogenous proteins as substrates. As in many other tissues histone H1 is by far the preferred exogenous substrate of thyroid PKc. Kinetic studies with H1 showed that, compared to rat thyroids, porcine glands are particularly rich in PKc, the predominant kinase activity in this tissue. The cAMP‐dependent protein kinase (PKa) level, on the contrary, is very similar in both rat and porcine thyroids. Consequently, for the same type of tissue, there may be great species differences in the PKc level and the ratios between PKc and PKa kinase activities.Chromatographic properties of thyroid PKc are similar to those described in other tissues (one major peak followed by a small shoulder) except that elution of the main peak can vary depending on the nature of the salt gradient (∼ 55 mM for NaCl and 15 mM for sodium phosphate). In the first case PKc is completely separated from the PKa activity, in the second it is coeluted with the peak of PKa type I.The one‐dimensional PAGE pattern of proteins phosphorylated by porcine PKc is very similar to the pattern obtained by rat enzyme. Protein bands of 18 kDa, 22–25 kDa and 32–36 kDa are specific substrates of the thyroid PKc, after in vitro phosphorylation of cytosol proteins.A great difference in Ca2+ requirement for PKc activation was noted, depending whether histone H1 or endogenous proteins were substrates. As in other tissues, calcium was absolutely necessary for phosphorylation of histone H1 by PKc. The addition of calcium was not absolutely necessary when endogenous proteins were the substrates, either for the activation of the enzyme or for phosphorylation of the PKc‐specific substrates. Almost the same rate of phosphorylation was obtained with or without calcium in the incubation medium. However the one‐dimensional PAGE pattern of phosphorylated proteins was different in the presence or absence of calcium. While addition of calcium was not absolutely necessary for the phosphorylation of a great number of proteins by the PKc, its presence was indispensable for the phosphorylation of certain endogenous substrates. However, calcium alone, in the absence of phospholipids had no effect on the phosphorylation of these proteins.Endogenous proteins, phosphorylated by the PKc only when calcium was present, were resolved by the two‐dimensional PAGE into several distinct spots with molecular masses of 32–35 kDa and pI range of 5–7.5.Our results suggest that only very low concentrations of Ca2+, if any, are sufficient for the phosphorylation of many endogenous substrates. Higher Ca2+ concentrations are necessary for the phosphorylation of specific proteins. It could be that modulation of Ca2+ concentration plays a role in the selection of protein substrates phosphorylated by PKc.
Differential binding of mannose‐specific lectins to the carbohydrate chains of fibrinogen domains D and E
Authors: SOLIS Dolores , ESTREMERA Dolores , USOBIAGA Pilar , DÍAZ‐MAURIÑO Teresa 
Published: March 2005

The interaction of fibrinogen with the mannose‐specific lectins concanavalin A (ConA), its acetyl derivative (Ac‐ConA) and Lens culinaris agglutinin (LcH) was studied. Both ConA and LcH interact specifically with individual fibrinogen Bβ and γ chains and with denatured fragments D and E. However, analysis of the binding data shows that four moles of Ac‐ConA are bound per mole of fibrinogen with two sets of binding sites (Kd1= 2.4 μM and Kd2= 16.6 μM; n1=n2= 2) while only two moles of LcH are bound per mole of fibrinogen (Kd= 2.6 μM). Ultracentrifugation studies are also in agreement with the presence in the fibrinogen molecule of two and four binding sites for LcH and Ac‐ConA, respectively. No aggregates of fibrinogen formed through LcH or Ac‐ConA linkages are observed. The use of a crosslinking reagent and ultracentrifugal analysis of the lectinfibrinogen fragments D1 and E complexes indicated that ConA, as well as Ac‐ConA, interact with both fragments D and E while LcH interacts only with fragment D. Furthermore, the binding of ConA to both D and E domains in the intact fibrinogen molecule is clearly demonstrated by using a bifunctional reagent. The bivalent character of ConA tetramers may be misinterpreted as a lack of accessibility of the lectin to two of the four carbohydrate chains of fibrinogen.The differential binding of LcH and ConA to the carbohydrate chains of fibrinogen can be related to a different exposure of the oligosaccharide in D and E fragments and domains and to the different requirements of both lectins for their binding to glycoproteins.
Metabolic control and its analysis
Authors: SAURO Herbert M. , SMALL J. Rankin , FELL David A. 
Published: March 2005

The matrix algebra procedure for determining the flux control coefficients of enzymes in metabolic pathways has been extended to allow determination of the concentration control coefficients. Although it is shown that the procedure is essentially unchanged in most cases, the presence of moiety‐conserved cycles in a pathway places additional limitations on the form of the equations that can be used in the matrix formulation for concentration control coefficients. In the case of branched pathways, a new coefficient has been defined, the branch distribution control coefficient, which can be obtained via the matrix procedure. Thus a single matrix equation permits calculation or algebraic evaluation of the control coefficients for flux, concentration and distribution of flux at branches, so that the complete response of a pathway to alteration of enzyme content, or to modulation by an effector, can be determined.The relationships have been determined between flux control coefficients in isolated sections of metabolic pathways and the coefficients for the same enzymes when part of a larger metabolic system. It is shown that the control analysis of the isolated system provides useful information towards determining the control properties of the extended system.
[d‐Phe4]Peptide histidine‐isoleucinamide ([d‐Phe4]PHI), a highly selective vasoactive‐intestinal‐peptide (VIP) agonist, discriminates VIP‐preferring from secretin‐preferring receptors in rat pancreatic membranes
Authors: ROBBERECHT Patrick , COY David H. , De NEEF Philippe , CAMUS Jean‐Claude , CAUVIN Annick , WAELBROECK Magali , CHRISTOPHE Jean 
Published: March 2005

The capacity of vasoactive intestinal peptide (VIP), peptide histidine‐isoleucinamide (PHI), secretin, and a series of analogs to discriminate between VIP‐preferring and secretin‐preferring receptors that coexist in rat pancreatic plasma membranes was evaluated by their ability to inhibit [125I]iodo‐VIP and [125I]iodo‐secretin binding and to activate adenylate cyclase. VIP, the VIP analogs [d‐His1]VIP, [d‐Ser2]VIP, [d‐Asp3]VIP and [d‐Ala4]VIP, PHI, [d‐Phe4]PHI, and secretin inhibited the binding of both ligands in a concentration range of 10−11 M to 10−5 M and with a selectivity factor varying from 18000 to 0.1. The only exception was [d‐Phe4]PHI that inhibited 125I‐VIP binding only, with an IC50 of 7 nM, and with no inhibition of 125I‐secretin binding at 10 μM.
The peptides tested stimulated adenylate cyclase in the same membranes and the slope of the dose‐effect curves indicated that all peptides, except [d‐Phe4]PHI, interacted with at least two classes of receptors: VIP‐preferring and secretin‐preferring receptors.
By contrast, the dose‐effect curve of [d‐Phe4]PHI activation of adenylate cyclase was monophasic and competitively modified by [d‐Phe2]VIP (a VIP antagonist) but not by secretin(7–27) (a secretin antagonist), indicating an interaction with VIP‐preferring receptors only. Thus, [d‐Phe4]PHI appears to be a highly selective tool to characterize these receptors.
Cobra cardiotoxins
Authors: HODGES Stephen J. , AGBAJI Abel S. , HARVEY Alan L. , HIDER Robert C. 
Published: March 2005

A new preparative method for isolating homogeneous cardiotoxins from cobra venoms is described. The technique, based on reverse‐phase high‐pressure liquid chromatography, was used to isolate eight cardiotoxins of known sequence from four different venoms. In each case the method was found to be particularly efficient at removing trace quantities of contaminating phospholipase. Cardiotoxins isolated in this manner were found to retain their full biological activity.Without exception the purified cardiotoxins lacked powerful haemolytic activity at concentrations up to 0.01 mM (about 100 μg ml−1), although some lysis of human erythrocytes was induced at higher concentrations. The cardiotoxins displayed a wide range of depolarizing activity on cultured skeletal muscle, the lowest activity being associated with the highest LD50 value.Correlating variations in amino acid sequence and variations in depolarization potency revealed the importance of residues in the second and third loops, especially lysine‐46, serine‐48 and lysine‐52, together with a number of hydrophobic residues. Further modifications of pharmacological activity were associated with the presence of additional basic residues in the first and second loops and to minor differences in secondary structure.
Nitrate starvation induces homeoviscous regulation of lipids in the cell envelope of the blue‐green alga, Anacystis nidulans
Authors: GOMBOS Zoltán , KIS Mihály , PÁLI Tibor , VIGH László 
Published: March 2005

Replacement of the normal culture liquid to a nitrate‐free medium resulted in an immediate drop in the ratio of protein to lipid in isolated cell envelopes of Anacystis nidulans cells. The relative fluidity of the envelope membranes or liposomes, made from the extracted lipids of the envelope, was estimated by measuring the steady‐state fluorescence polarization of 1,6‐diphenyl‐1,3,5‐hexatriene. A thermotrophic phase transition of lipids within the cytoplasmic membrane of intact cells was also revealed by detecting the temperature‐dependent absorption changes in the proportion of zeaxanthin at 390 nm. It became evident that a decrease in the proportion of protein to lipid within the cell envelope was accompanied neither by changes in the microviscosity level, nor by shifting of characteristic temperatures of the liquid‐crystalline‐to‐gel transition of lipids. In parallel with nitrate starvation, however, the proportion of saturated fatty acids of the envelope lipids increased markedly. Accumulation of saturated, longer‐chain (C18) fatty acids at the cost of C16 counterparts upon nitrate deprivation occurred in all of the complex lipids. In accordance with these findings, a pronounced decrease in the fluidity was demonstrated for the liposomes prepared from the envelope polar lipids of nitrate‐starved cells compared with the corresponding control, throughout the temperature range (45–5°C) studied.We propose that the fluidizing effect due to a fall in the ratio of protein to lipid was compensated by a rapidly triggering regulatory process which enables the preservation of the fluidity characteristics at an optimal level within the cell envelope of A. nidulans.
Purealin, a novel activator of skeletal muscle actomyosin ATPase and myosin EDTA‐ATPase that enhanced the superprecipitation of actomyosin
Authors: NAKAMURA Yoichi , KOBAYASHI Masaki , NAKAMURA Hideshi , WU Houming , KOBAYASHI Jun'ichi , OHIZUMI Yasushi 
Published: March 2005

Purealin, a novel bioactive principle of a sea sponge Psammaplysilla purea, activated the superprecipitation of myosin B (natural actomyosin) from rabbit skeletal muscle. The maximum change in the turbidity increased with increasing purealin concentrations and was three times the control value in the presence of 50 μM purealin.
The ATPase activity of myosin B was also elevated to 160% of the control value by 10 μM purealin. On the other hand, purealin inhibited the myosin ATPase in the presence of 10 mM CaCl2 and 0.5 M KCl (Ca2+‐ATPase), and the concentration for the half inhibition was 4 μM.
On the other hand, purealin activated the myosin ATPase in the presence of 5 mM EDTA and 0.5 M KCl (EDTA‐ATPase). The maximum activation by 10 μM purealin was 160% of the control value.
Furthermore, similar results concerning the modification of ATPase activities by purealin were obtained in myosin subfragment‐1 instead of myosin.
These results suggest that purealin activates the superprecipitation of myosin B by affecting the myosin heads directly. It is also an interesting observation that there is a correlation between the activities of the myosin EDTA‐ATPase and actomyosin ATPase of myosin B.
Isolation of a cDNA and characterization of the 5′ flanking region of the gene encoding the type I regulatory subunit of the cAMP‐dependent protein kinase
Authors: NOWAK Ines , SEIPEL Katja , SCHWARZ Manuela , JANS David A. , HEMMINGS Brian A. 
Published: March 2005

A bovine cDNA probe for the type I regulatory subunit of the cAMP‐dependent protein kinase [Lee et al. (1983) Proc. Natl Acad. Sci. USA 80, 3608–3612] was used to screen two λgt11 libraries constructed from poly(A)‐rich RNA from the porcine kidney cell line, LLC‐PK1. A series of overlapping clones were isolated and characterized. The largest clone, λR115, of 1426 bp was found to code for the entire R1 protein but was apparently missing the 3′ end of the mRNA. The porcine cDNA codes for a protein of 389 amino acids that shows 99% homology to bovine R1 and hybridizes to two major mRNA transcripts of approximately 2.0 kb and 4.5 kb from LLC‐PK1 cells.The porcine cDNA for R1 was used to screen a genomic library of LLC‐PK1 DNA constructed in the EMBL‐3 vector and several clones were isolated and characterized. By using a probe from the 5′ end of the R1 cDNA we isolated the 5′ end of the gene and 700 bp of the promoter region of the gene were sequenced. The promoter region lacks a characteristic TATA box but contains two inverted CAAT boxes and is rich in G + C residues. Several sequence motifs were identified in the 5′ promoter region which could be responsible for the regulation of synthesis of this gene. Multiple transcription initiation sites were identified by S1 nuclease mapping.
Actions of extracellular UTP and ATP in perfused rat liver
Authors: HÄSSINGER Dieter , STEHLE Thomas , GEROK Wolfgang 
Published: March 2005

1. In perfused rat liver infusion of UTP and ATP in micromolar concentrations increased the portal pressure, with UTP being three times more effective than ATP at concentrations below 50μM. Whereas ATP (up to 100 μM) increased oxygen consumption, there was a dose‐dependent inhibition of oxygen uptake by UTP.2. Both nucleotides stimulated hepatic glucose output; however, the time‐courses were different. Withdrawal of UTP, but not of ATP (up to 100 μM) caused a further transient, but substantial stimulation of glucose output.3. ATP led to a transient net K+ uptake by the liver being followed by a K+‐release phase. Similar changes were observed with UTP; however, the initial K+ uptake was prolonged compared to ATP (1.9 min versus 3.5 min) and withdrawal of UTP, but not of ATP, stimulated hepatic K+ release markedly.4. Metabolic and hemodynamic effects comparable to those induced by ATP were obtained with β‐and γ‐thio substituted ATP, whereas β, γ‐methylene‐substituted ATP was much less effective. The characteristic effects of UTP on glucose output, portal pressure and K+ fluxes were preserved during constant infusion of ATP or its β,γ‐methylene derivative, pointing to additive effects.5. ATP (20 μM) led to a net Ca2+ release (50–60 nmol/g liver) within 2–3 min. When the extracellular Ca2+ concentration was lowered from 1.25 mM to 0.3 mM, this Ca2+ release was increased to about 110 nmol/g liver whereby its time course remained largely unchanged. With 1.25 mM Ca2+, UTP induced Ca2+ movements only near the detection level (i.e. below 10–20 nmol/g liver); however, with 0.3 mM Ca2+ in influent perfusate, there was a slow Ca2+ release (not completed within 5–6 min). The maximal rates of Ca2+ efflux following ATP and UTP (20 μM each) were 70 nmol and 30 nmol g−1 min−1. Withdrawal of UTP led to a short Ca2+ release supcrimposing a phase of net Ca2+ uptake.6. The data show that extracellular UTP is a potential and effective regulator of hepatic metabolism, ion fluxes across the hepatocyte membrane and hemodynamics. Compared to ATP, UTP seems to be more effective and the responses to both nucleotides are different. The data suggest that the action of UTP could involve a receptor distinct from the purinergic P2 receptor, whereas the ATP action involves predominantly the P2Y purinoceptor subtype.
Identification of protein phosphatases‐1 and 2A and inhibitor‐2 in oocytes of the starfish Asterias rubens and Marthasterias glacialis
Authors: PONDAVEN Pierre , COHEN Philip 
Published: March 2005

Protein phosphatases present in the particulate and soluble fractions of oocytes of the starfish Asterias rubens and Marthasterias glacialis have been classified according to the criteria used for these enzymes from mammalian cells.The major protein phosphatase activity in the particulate fraction had very similar properties to protein phosphatase‐1 from mammalian tissues, including preferential dephosphorylation of the β subunit of phosphorylase kinase, sensitivity to inhibitor‐1 and inhibitor‐2, inhibition of phosphorylase phosphatase activity by protamine and heparin, and retention by heparin‐Sepharose. The major protein phosphatase in the soluble fraction had very similar properties to mammalian protein phosphatase‐2A, including preferential dephosphorylation of the α subunit of phosphorylase kinase, insensitivity to inhibitors‐1 and 2, activation by protamine and heparin, and exclusion from heparin‐Sepharose.An acid‐stable and heat‐stable protein was detected in the soluble fraction of starfish oocytes, whose properties were indistinguishable from those of inhibitor‐2 from mammalian tissues. It inhibited protein phosphatase‐1 specifically, and its apparent molecular mass on SDS polyacrylamide gels was 31 kDa. Furthermore, an inactive hybrid formed between the starfish oocyte inhibitor and the catalytic subunit of mammalian protein phosphatase‐1 could be reactivated by preincubation with MgATP and mammalian glycogen synthase kinase‐3.The remarkable similarities between starfish oocyte protein phosphatases and their mammalian counterparts are indicative of strict phylogenetic conservation of these enzymes. The results will facilitate further analysis of the role of protein phosphorylation in the control of starfish oocyte maturation by the hormone 1‐methyladenine.