bluuebunny/crossref_metadata_2025 huggingface dataset:
Zonation of fatty acid metabolism in rat liver
Authors: Guzmán M , Castro J
Published: August 2015

Fatty acid metabolism was studied in periportal and perivenous hepatocytes isolated by the method of Chen & Katz [Biochem. J. (1988) 255, 99-104]. The rate of fatty acid synthesis and the activity of acetyl-CoA carboxylase were markedly enhanced in perivenous hepatocytes as compared with periportal cells. However, the response of these two parameters to short-term modulation by cellular effectors such as the hormones insulin and glucagon, the phorbol ester 4 beta-phorbol 12 beta-myristate 13 alpha-acetate and the xenobiotics ethanol and acetaldehyde was similar in the two zones of the liver. In addition, perivenous hepatocytes showed a higher capacity of esterification of exogenous fatty acids into both cellular and very-low-density-lipoprotein lipids. Nevertheless, no difference between the two cell sub-populations seemed to exist in relation to the secretion of very-low-density lipoproteins. On the other hand, the rate of fatty acid oxidation was increased in periportal cells. This could be accounted for by a higher activity of carnitine palmitoyltransferase I and a lower sensitivity of this enzyme to inhibition by malonyl-CoA in the periportal zone. No differences were observed between periportal and perivenous hepatocytes in relation to the short-term response of fatty acid oxidation and carnitine palmitoyltransferase I activity to the cellular modulators mentioned above. In conclusion, our results show that: (i) lipogenesis is achieved at higher rates in the perivenous zone of the liver, whereas the fatty-acid-oxidative process occurs with a certain preference in the periportal area of this organ; (ii) the short-term response of the different fatty-acid-metabolizing pathways to cellular effectors is quantitatively similar in the two zones of the liver.
Effect of 25-hydroxycholesterol and bile acids on the regulation of cholesterol metabolism in Hep G2 cells
Authors: Carlson T L , Kottke B A
Published: August 2015

The effect of 25-hydroxycholesterol (25-OH-cholesterol) and chenodeoxycholic (CDC) acid on apoprotein secretion, low-density lipoprotein receptor activity, and [3H]triacylglycerol secretion in Hep G2 cells was studied. Both 25-OH-cholesterol and CDC acid increased the secretion of apolipoprotein (apo) E by Hep G2 cells. The secretion of apo A-I was slightly lowered (less than 10% disease). The maximal increase in apo E secretion was observed in culture medium containing 2 micrograms of 25-OH-cholesterol/ml or 10 micrograms of CDC acid/ml plus 10% fetal calf serum. Cholesterol, 7-OH-cholesterol and other bile acids were ineffective in inducing increases in apo E secretion. Another cholesterol synthesis inhibitor, mevinolin, was also ineffective in generating an increase in apoprotein secretion. The data indicated a specific interaction between 25-OH-cholesterol or CDC acid and apo E secretion in Hep G2 cells. Cholesterol synthesis, as measured by the incorporation of [14C]acetic acid into sterols, was repressed in Hep G2 cells in the presence of 25-OH-cholesterol (17% of control value). CDC acid, on the other hand, increased [14C]acetic acid incorporation (156% of control value). The number of LDL receptors in Hep G2 cells was decreased after incubation with 25-OH-cholesterol (62% of control value), but increased significantly after incubation with CDC acid (149% of control value). The secretion of [3H]triacylglycerol by Hep G2 cells incubated with 25-OH-cholesterol was greatly increased (248% of control value). On the contrary, CDC acid did not cause any increase in [3H]triacylglycerol secretion. The above results suggest that 25-OH-cholesterol and CDC acid have different effects on lipid metabolism in Hep G2 cells. The mRNA levels of apo E increased in cells preincubated with 25-OH-cholesterol and CDC acid, which suggested that the increase in apo E secretion is at least partly due to an increase in synthesis.
Electrochemical and spectroscopic characterization of the 7Fe form of ferredoxin III from Desulfovibrio africanus
Authors: Armstrong F A , George S J , Cammack R , Hatchikian E C , Thomson A J
Published: August 2015

Desulfovibrio africanus ferredoxin III is a monomeric protein (Mr 6585) containing seven cysteine residues and 7-8 iron atoms and 6-8 atoms of acid-labile sulphur. It is shown that reversible unmediated electrochemistry of the two iron-sulphur clusters can be obtained by using a pyrolytic-graphite-‘edge’ carbon electrode in the presence of an appropriate aminoglycoside, neomycin or tobramycin, as promoter. Cyclic voltammetry reveals two well-defined reversible waves with E0′ = -140 +/- 10 mV and -410 +/- 5 mV (standard hydrogen electrode) at 2 degrees C. Bulk reduction confirms that each of these corresponds to a one-electron process. Low-temperature e.p.r. and magnetic-c.d. spectroscopy identify the higher-potential redox couple with a cluster of core [3Fe-4S]1+.0 and the lower with a [4Fe-4S]2+.1+ centre. The low-temperature magnetic-c.d. spectra and magnetization properties of the three-iron cluster show that it is essentially identical with that in Desulfovibrio gigas ferredoxin II. We assign cysteine-11, -17 and -51 as ligands of the [3Fe-4S] core and cysteine-21, -41, -44 and -47 to the [4Fe-4S] centre.
Regulation of cyclic AMP-dependent protein kinase levels during skeletal myogenesis
Authors: Lorimer I A J , Sanwal B D
Published: August 2015

We showed previously that the levels of type I regulatory subunit of cyclic AMP-dependent protein kinase increase during differentiation of L6 skeletal myoblasts as a result of a specific decrease in its rate of degradation. Studies on the rates of degradation of the catalytic subunit show that unlike the type I regulatory subunit, catalytic subunit is degraded very slowly in myoblasts (t1/2 = 29 h) and more rapidly in myotubes (t1/2 = 14 h). As with the regulatory subunit, the degradation of catalytic subunit is increased by treatment of myoblasts with cyclic AMP analogues. These results suggest that the overall increase in the amount of type I cyclic AMP-dependent protein kinase holoenzyme during myogenesis is due to the increase in levels of mRNA for the catalytic subunit. This probably leads to an increase in the amount of catalytic subunit, which then stabilizes the regulatory subunit, thereby causing an increase in the levels of this protein also.
Time-dependent activity and expression of glutathione S-transferases in the human colon adenocarcinoma cell line Caco-2
Authors: Peters W H N , Roelofs H M J
Published: August 2015

The human colon carcinoma cell line Caco-2 was examined for glutathione S-transferase (GST) composition and activity. Freshly seeded cells and cells until 4 days after confluency contain only the placental (Pi) form of glutathione transferase. Cells in culture for longer periods start to express class-Alpha GST isoenzymes. Confluent cells in culture for 20 days or longer contain up to 90% class-Alpha GST. Class-Mu GSTs are not detectable. GST activity gradually increases from 564 +/- 28 to 5381 +/- 165 nmol/min per mg of protein at day 0 and 32 after confluency respectively. With regard to GST composition, Caco-2 cells in culture for longer periods most resemble small-intestinal cells, whereas short-time cultures have characteristics of colonic cells. This cell line is very well suited for the study of both the in vitro properties and the expression of class Alpha and Pi GSTs.
Binding of two spin-labelled derivatives of chlorpromazine to human erythrocytes
Authors: Olivier J L , Chachaty C , Wolf C , Daveloose D , Bereziat G
Published: August 2015

The binding to human intact erythrocytes of two different spin-labelled derivatives of chlorpromazine has been studied. The influence of the positively charged side chain of the drug has been the focus of our attention. The positively charged amphiphilic compound (spin derivative I) is water-soluble up to 80 microM at pH values below 5.9. The apolar analogue (spin derivative II) aggregates in aqueous buffer from the lowest concentration tested. Both spin derivatives undergo a slow reduction inside the erythrocyte. The reduced nitroxides are readily reoxidized by adding a low, non-quenching, concentration of potassium ferricyanide to the intact erythrocytes. The fractions of spin label I and II bound to the erythrocyte membrane or to the erythrocyte-extracted lipids remain constant as a function of the temperature (3-42 degrees C) and as a function of the concentration of the spin label up to 150 microM. E.s.r. spectra of both spin labels show a two-component lineshape when they are bound to intact erythrocytes. Below 35 degrees C for the positively charged spin probe, and below 32 degrees C for the apolar spin probe, the simulation of the lineshape shows that more than 50% of the spectrum originates from a slow-motion component. This slow-motion component is also found in erythrocyte-extracted lipids probed by the positively charged spin label below 25 degrees C. In contrast, no slow-motion component is detected in the range 4-40 degrees C for the apolar spin label in erythrocyte-extracted lipids. In this environment the apolar probe experiences a single fast anisotropic motion with an exponential dependence on 1/temperature. Detailed lineshape simulations take into account the exchange frequency between binding sites where the probe experiences a fast motion and binding sites where it experiences a slow motion. The exchange frequency is strongly temperature-dependent. Characterization of the different motions experienced inside the different locations has been achieved and compared for whole erythrocytes and for the extracted lipids. The biochemical nature of the binding sites (membrane protein/acidic phospholipid) giving rise to the slow-motion component is discussed as a function of the polarity of the spin-labelled drug and as a function of the temperature controlling the fluidity of the lipid bulk and influencing the distribution of the drug inside the membrane.
Hydrolysis of inositol phosphates by plant cell extracts
Authors: Joseph S K , Esch T , Bonner W D
Published: August 2015

A gel-filtered soluble fraction prepared from suspension-cultured Nicotiana tabacum cells hydrolysed inositol mono-, bis- and tris-phosphates. At a concentration of 7.5 microM the rates of hydrolysis followed the sequence Ins(1,4,5)P3 greater than Ins(1,4)P2 greater than Ins(4)P congruent to Ins(1)P. The major products of Ins(1,4,5)P3 hydrolysis identified by h.p.l.c. were Ins(1,4)P2 and Ins(4,5)P2. Ins(1,4)P2 was hydrolysed exclusively to Ins(4)P. The inclusion of Ca2+ in the incubation buffer markedly stimulated the hydrolysis of all the inositol phosphate substrates. Under identical conditions, Ca2+ inhibited the hydrolysis of inositol phosphates by soluble extracts prepared from rat brain. Half-maximal stimulation of Ins(1,4)P2 hydrolysis was obtained at free [Ca2+] of 0.6 and 1.2 microM when the Mg2+ concentration in the incubations was 0.3 and 1.0 mM respectively. This effect of Ca2+ was exerted solely by increasing the Vmax. of hydrolysis without affecting the Km for Ins(1,4)P2. Again, in contrast with brain, the hydrolysis of inositol bis- or mono-phosphates was insensitive to high concentrations of Li+. We conclude that plants contain specific Li+-insensitive inositol phosphate phosphatases that are regulated by low concentrations of Ca2+ in a manner which is different from that observed in mammalian tissues.
The cDNA cloning of a pea (Pisum sativum) seed lipoxygenase. Sequence comparisons of the two major pea seed lipoxygenase isoforms
Authors: Ealing P M , Casey R
Published: August 2015

Cloning and sequencing of two cDNAs from mRNA of maturing pea (Pisum sativum) seeds allowed the deduction of the complete amino acid sequence of a lipoxygenase polypeptide which is most similar to that of soya-bean lipoxygenase 2. The predicted Mr of this polypeptide is 97134, and its sequence permits comparisons between the lox2-type and the lox3-type lipoxygenase isoforms from pea and soya bean (Glycine max).
Purification and assembly in vitro of tubulin from Trypanosoma brucei brucei
Authors: MacRae T H , Gull K
Published: August 2015

Trypanosome tubulin was purified to near homogeneity by chromatography on DEAE-Sephadex, Amicon filtration and assembly-disassembly in vitro. Polymerization of the tubulin in vitro yielded long, structurally normal, microtubules and some sheet structures on addition of GTP and incubation at 37 degrees C, in either the presence or the absence of Mg2+. Tubulin assembly was disrupted by glycerol and a selection of microtubule-reactive drugs. Immunological analysis of the purified tubulin revealed tyrosinated and acetylated alpha-tubulin, in addition to defining the migration characteristics of the alpha- and beta-tubulin on one-dimensional SDS/polyacrylamide gels. This is the first isolation of trypanosome tubulin with the ability to form structurally normal microtubules independent of the addition of taxol or nucleating microtubule fragments. The development of the purification procedure thus provides an important step for subsequent study of microtubule-associated protein-tubulin and plasma-membrane-microtubule cytoskeleton interactions of trypanosomes, and increases the potential for development of tubulin-based anti-trypanosome drugs.
Isolation and partial characterization of ascites sialoglycoprotein-2 of the cell surface sialomucin complex of 13762 rat mammary adenocarcinoma cells
Authors: Hull S R , Sheng Z , Vanderpuye O , David C , Carraway K L
Published: August 2015

Sialomucins are the dominant components of the cell surfaces of some carcinoma ascites cells and have been postulated to inhibit recognition of tumours by the immune system. The sialomucin ASGP-1 (ascites sialoglycoprotein-1) of the 13762 rat mammary adenocarcinoma is associated with the cell surface as a complex with a concanavalin-A-binding glycoprotein called ASGP-2. This sialomucin complex has been purified from ascites cell microvilli by extraction with Triton X-100 and CsCl density-gradient centrifugation. ASGP-1 (which has been purified previously) and ASGP-2 were dissociated in 6 M-guanidine hydrochloride and separated by gel filtration. The molecular mass of the undenatured detergent complex of ASGP-2, estimated by gel filtration and velocity sedimentation in Triton X-100, was 148 kDa. Since the apparent molecular mass by SDS/polyacrylamide-gel electrophoresis was about 120 kDa, ASGP-2 must be a monomer as extracted from the membrane. Studies of its chemical composition indicate that it contains about 45% carbohydrate by weight, including both mannose and galactosamine. Alkaline borohydride treatment of ASGP-2 converted approx. half of the N-acetylgalactosamine to N-acetylgalactosaminitol, demonstrating the presence of O-linked oligosaccharides. Analyses of mannose-labelled Pronase glycopeptides from ASGP-2 by lectin-affinity chromatography on concanavalin A and leucocyte-agglutinating phytohaemagglutinin suggested that 40% of the label was present in high-mannose/hybrid oligosaccharides, 20% in triantennary oligosaccharides substituted on the C-2 and C-4 mannose positions and 40% in tri- or tetra-antennary oligosaccharides substituted on C-2 and C-6. The presence of polylactosamine sequences on these oligosaccharides was suggested by lectin blots and by precipitation from detergent extracts with tomato lectin. From chemical analyses and lectin-affinity studies, we estimate that ASGP-2 contains four high-mannose and 13 complex N-glycosylated oligosaccharides, plus small amounts of polylactosamine and O-linked oligosaccharides. The presence of four different classes of oligosaccharides on this glycoprotein suggests that it will be an interesting model system for biosynthetic comparisons of the different glycosylation pathways.
Regulation of platelet AMP deaminase activity in situ
Authors: Verhoeven A J M , Marszalek J , Holmsen H
Published: August 2015

The regulation of platelet AMP deaminase activity by ATP, GTP and phosphate was studied in human platelets in situ, and in vitro after partial purification. In intact platelets, a similar 50% decrease in cytosolic ATP was induced by either glucose starvation or treatment with H2O2. During starvation, AMP deaminase was in the inhibited state, as ATP consumption was mostly balanced by the accumulation of AMP. During H2O2 treatment, however, the enzyme was in the stimulated state, as the AMP formed was almost completely deaminated to IMP. Cytosolic GTP fell by 40-50% in both starvation and H2O2 treatment. In contrast, intracellular phosphate was 4-5-fold higher in starved than in H2O2-treated cells. These data point to phosphate as the main regulator of AMP deaminase activity in situ. This conclusion was verified by kinetic analysis of partially purified AMP deaminase. At near-physiological concentrations of MgATP, MgGTP and phosphate, the S0.5 (substrate half-saturation constant) for AMP was 0.35 mM. Half-maximal stimulation by MgATP occurred at a concn. between 2 and 3 mM. This stimulation was antagonized by the inhibitory effects of phosphate (IC50 = 2.0 mM) and MgGTP (IC50 = 0.2-0.3 mM), which acted in synergism (IC50 is the concentration causing 50% inhibition). We conclude that the difference in adenylate catabolism between starved and H2O2-treated platelets is due to the distinct phosphate concentrations. During starvation, refeeding and H2O2 treatment, the values of the adenylate charge and the phosphorylation potential were kept closely co-ordinated, which may be effected by AMP deaminase.
Granulocyte-macrophage colony-stimulating factor can stimulate macrophage proliferation via persistent activation of Na+/H+ antiport. Evidence for two distinct roles for Na+/H+ antiport activation
Authors: Vallance S J , Downes C P , Cragoe E J , Whetton A D
Published: August 2015

Macrophages respond to a variety of extracellular stimuli which can modulate the proliferation, development, activation and functional activity of these cells. The effects of two such agents, granulocytemacrophage colony-stimulating factor (GM-CSF, which stimulates proliferation) and platelet-activating factor (PAF, which stimulates chemotaxis and bactericidal activity), on cellular signal transduction mechanisms were compared. PAF can stimulate inositol lipid hydrolysis leading to Ca2+ mobilization. GM-CSF on the other hand has no effect on these events. Both agonists do, however, share an ability to activate an amiloride-sensitive Na+/H+ antiport and, furthermore, amiloride analogues are shown to inhibit the proliferative effects of GM-CSF on these cells. Long-term incubations with either PAF or GM-CSF demonstrate that it is only those cells pretreated with the latter which show a persistent activation of the antiport together with a sustained increase in intracellular pH. PAF-treated cells exhibit only a transitory increase in antiport activity, their intracellular pH levels returning to resting levels in spite of the continuous presence of the agonist in the medium. These effects of GM-CSF and PAF on Na+/H+ exchange are observed in both bicarbonate-free and bicarbonate-containing medium. These results lead us to suggest that the Na+/H+ antiport has a role in macrophage proliferation and in the regulation of intracellular pH during the oxidative burst stimulated by PAF and other agonists, and that differential mechanisms whereby this antiport is regulated exist in macrophages.
Bombesin receptor in membranes from Swiss 3T3 cells. Binding characteristics, affinity labelling and modulation by guanine nucleotides
Authors: Sinnett-Smith J , Lehmann W , Rozengurt E
Published: August 2015

Bombesin-like neuropeptides, including mammalian gastrin-releasing peptide (GRP), are potent mitogens for Swiss 3T3 cells. In this study, we have characterized the bombesin receptor in membrane preparations from these cells. Addition of Mg2+ during cell homogenization was essential to preserve 125I-GRP binding activity in the resulting membrane preparation. The effect of Mg2+ was concentration dependent, with a maximum at 5 mM. Specific binding of 125I-GRP was saturable; Scatchard analysis indicated a single class of high-affinity sites of Kd = (2.1 +/- 0.3) x 10(-10) M at 15 degrees C and Kd = (1.9 +/- 0.4) x 10(-10) M at 37 degrees C, and a maximum binding capacity of 580 +/- 50 fmol/mg of protein (15 degrees C) or 604 +/- 40 fmol/mg of protein (37 degrees C). The kinetically derived dissociation constant was 1.5 x 10(-10) M. 125I-GRP binding was inhibited in a concentration-dependent manner by various peptides containing the highly conserved C-terminal heptapeptide of the bombesin family, including bombesin, GRP, neuromedin B and the 8-14 fragment of bombesin. In contrast, a variety of structurally unrelated mitogens and neuropeptides had no effect. The cross-linking agent ethyleneglycolbis(succinimidylsuccinate) covalently linked 125I-GRP to a single Mr 75 000-85 000 protein in membrane preparations of 3T3 cells. Affinity labelling of this molecule was specific and dependent on the presence of Mg2+ during membrane preparation. Finally, the non-hydrolysable GTP analogue guanosine-5′-[gamma-thio]triphosphate (GTP[S]) caused a concentration-dependent inhibition of 125I-GRP binding and cross-linking to 3T3 cell membranes [concentration giving half-maximal inhibition (IC50) approximately 0.2 microM]. The inhibitory effect was specific (GMP, ATP or ATP[S] had no effect at 10 microM) and was due to an increase in Kd from (1.7 +/- 0.2) x 10(-10) M to (4.3 +/- 0.6) x 10(-10) M in the presence of 10 microM-GTP[S]. This modulation of ligand affinity and cross-linking implies that the bombesin receptors that mediate mitogenesis in Swiss 3T3 cells are coupled to a guanine-nucleotide-binding-protein signal-transduction pathway.
The complement component C4 of mammals
Authors: Dodds A W , Law S K A
Published: August 2015

Human complement component C4 is coded by tandem genes located in the HLA class III region. The products of the two genes, C4A and C4B, are different in their activity. This difference is due to a degree of ‘substrate’ specificity in the covalent binding reactions of the two isotypes. Mouse also has a duplicated locus, but only one gene produces active C4, while the other codes for the closely related sex-limited protein (Slp). In order to gain some insight into the evolutionary history of the duplicated C4 locus, we have purified C4 from a number of other mammalian species, and tested their binding specificities. Like man, chimpanzee and rhesus monkey appear to produce two C4 types with reactivities similar to C4A and C4B. Rat, guinea pig, whale, rabbit, dog and pig each expresses C4 with a single binding specificity, which is C4B-like. Sheep and cattle express two C4 types, one C4B-like, the other C4A-like, in their binding properties. These results suggest that more than one locus may be present in these species. If this is so, then the duplication of the C4 locus is either very ancient, having occurred before the divergence of the modern mammals, or there have been three separate duplication events in the lines leading to the primates, rodents and ungulates.
Phosphoglucoisomerase-catalysed interconversion of hexose phosphates. Kinetic study by 13C n.m.r. of the phosphoglucoisomerase reaction in 2H2O
Authors: Willem R , Malaisse-Lagae F , Ottinger R , Malaisse W J
Published: August 2015

The fate of D-[2-13C]glucose 6-phosphate exposed to phosphoglucoisomerase (glucose-6-phosphate isomerase, EC 5.3.1.9) in 2H2O was monitored by 13C-n.m.r. spectroscopy. The generation of the anomers of both D-[2-13C]fructose 6-phosphate and D-[2-13C,2-2H]glucose 6-phosphate followed a single-exponential pattern. The rate constant, which was proportional to the enzyme concentration, was about 14 times higher, however, in the former than in the latter case. The disappearance of D-[2-13C,2-1H]glucose 6-phosphate occurred in a bi-exponential manner, the rate constants for the fast and the slow processes being in fair agreement with those obtained for the generation of D-[2-13C]fructose 6-phosphate and D-[2-13C,2-2H]glucose 6-phosphate respectively. These findings indicate that the process of equilibration of D-[2-13C]glucose 6-phosphate and D-[2-13C]fructose 6-phosphate is at least one order of magnitude faster than the intermolecular proton transfer involving the deuterons from the solvent. Such a difference provides strong support to the view that the inverconversion of hexose phosphates in the reaction catalysed by phosphoglucoisomerase proceeds in two distinct steps, the second of which occurs according to two competing modalities with either an intramolecular or an intermolecular proton transfer.
Muscarinic-receptor-mediated changes in intracellular Ca2+ and inositol 1,4,5-trisphosphate mass in a human neuroblastoma cell line, SH-SY5Y
Authors: Lambert D G , Nahorski S R
Published: August 2015

This study reports increased intracellular Ca2+ and inositol 1,4,5-trisphosphate [Ins(1,4,5)P3] in response to muscarinic-cholinergic stimulation of human neuroblastoma (SH-SY5Y) cells. Carbachol stimulation leads to a rapid increase in intracellular Ca2+ and Ins(1,4,5)P3 mass, both reaching a peak at around 10 s and then declining to a new maintained phase significantly above basal. Dose-response analysis of peak and plateau phases of intracellular Ca2+ shows different agonist potencies for both phases, carbachol being more potent for the plateau phase. The plateau-phase intracellular Ca2+ was dependent on extracellular Ca2+, which is admitted to the cell through a non-voltage-sensitive Ni2(+)-blockable Ca2+ channel. Using a Mn2+ quench protocol, we have shown that Ca2+ entry occurs early during the discharge of the internal stores. The plateau phase (Ca2(+)-channel opening) is dependent on the continued presence of agonist, since addition of atropine closes the Ca2+ channel and intracellular Ca2+ declines rapidly back to basal. We also failed to detect a refilling transient when we added back Ca2+ after intracellular Ca2+ had reached a peak and then declined in Ca2(+)-free conditions. These data strongly suggest that muscarinic stimulation of SH-SY5Y cells leads to a rapid release of Ca2+ from an Ins(1,4,5)P3-sensitive internal store and a parallel early entry of Ca2+ across the plasma membrane.
Delivery of iron to human cells by bovine transferrin. Implications for the growth of human cells in vitro
Authors: Young S P , Garner C
Published: August 2015

Following suggestions that transferrin present in fetal-bovine serum, a common supplement used in tissue-culture media, may not bind well to human cells, we have isolated the protein and investigated its interaction with both human and bovine cells. Bovine transferrin bound to a human cell line, K562, at 4 degrees C with a kd of 590 nM, whereas human transferrin bound with a kd of 3.57 nM, a 165-fold difference. With a bovine cell line, NBL4, bovine transferrin bound with the higher affinity, kd 9.09 nM, whereas human transferrin bound with a kd of 41.7 nM, only a 5-fold difference. These values were reflected in an 8.6-fold difference in the rate of iron delivery by the two proteins to human cells, whereas delivery to bovine cells was the same. Nevertheless, the bovine transferrin was taken up by the human cells by a specific receptor-mediated process. Human cells cultured in bovine diferric transferrin at 40 micrograms/ml, the concentration expected in the presence of 10% fetal-bovine serum, failed to thrive, whereas cells cultured in the presence of human transferrin proliferated normally. These results suggest that growth of human cells in bovine serum could give rise to a cellular iron deficiency, which may in turn lead to the selection of clones of cells adapted for survival with less iron. This has important consequences for the use of such cells as models, since they may have aberrant iron-dependent pathways and perhaps other unknown alterations in cell function.
ADP evokes biphasic Ca2+ influx in fura-2-loaded human platelets. Evidence for Ca2+ entry regulated by the intracellular Ca2+ store
Authors: Sage S O , Reast R , Rink T J
Published: August 2015

Stopped-flow fluorimetric studies at 37 degrees C have shown that ADP, at optimal concentrations, can evoke Ca2+ or Mn2+ influx in fura-2-loaded human platelets without measurable delay. In contrast, the release of Ca2+ from intracellular stores is delayed in onset by about 200 ms. By working at a lower temperature, 17 degrees C, we have now shown that the rise in cytosolic calcium concentration ([Ca2+]i) evoked by ADP in the presence of external Ca2+ is biphasic. The use of Mn2+ as a tracer for bivalent-cation entry indicates that both phases of the ADP-evoked response are associated with influx. The fast phase of the ADP-evoked rise in [Ca2+]i, which occurs without measurable delay at both 17 degrees C and 37 degrees C, is consistent with Ca2+ entry mediated by receptor-operated channels in the plasma membrane. The delayed phase, indicated by Mn2+ quench, is coincident with the discharge of the intracellular Ca2+ stores. Forskolin did not inhibit the fast phases of ADP-evoked rise in [Ca2+]i or Mn2+ quench, but completely abolished ADP-evoked discharge of the intracellular stores, the delayed phase of the rise in [Ca2+]i observed in the presence of external Ca2+ and the second phase of Mn2+ quench. The timing of the delayed event appears to be modulated by [Ca2+]i: the delayed phase of Mn2+ quench coincides with discharge of the intracellular stores in the absence of added Ca2+, but with the second phase of the ADP-evoked rise in [Ca2+]i in the presence of extracellular Ca2+. Similarly, blockade of the early phase of Ca2+ entry by SK&F 96365 further delays the second phase. It is suggested that a pathway for Ca2+ entry which is regulated by the intracellular Ca2+ store exists in platelets. This pathway operates alongside, and appears to be modulated by the activity of other routes for Ca2+ entry into the cytosol.
Binding characteristics of reduced hepatic receptors for acetylated low-density lipoprotein and maleylated bovine serum albumin
Authors: Ottnad E , Via D P , Sinn H , Friedrich E , Ziegler R , Dresel H A
Published: August 2015

The binding characteristics of reduced hepatic membrane proteins for acetylated low-density lipoprotein (acetyl-LDL) and maleylated bovine serum albumin (Mal-BSA) have been examined. Two receptor activities were extracted from hepatic membranes in the presence of octyl beta-D-glucoside and beta-mercaptoethanol, and were separated by chromatography on Mal-BSA-Sepharose 4B. The receptors were revealed by ligand blotting. The active binding proteins had apparent molecular masses of 35 and 15 kDa in SDS/polyacrylamide gels. Equilibrium studies with protein-phosphatidylcholine complexes indicated that the reduced 35 kDa protein expresses two binding sites for Mal-BSA and one for acetyl-LDL, whereas the 15 kDa protein-phosphatidylcholine complex binds 131I-Mal-BSA and 131I-acetyl-LDL with a 4:1 stoichiometry. 131I-Mal-BSA binding was linear with both proteins, with a Kd of 4.8 nM at the 35 kDa protein and a Kd of 5.6 nM at the 15 kDa protein. The 35 kDa protein displayed saturable binding of 131I-acetyl-LDL with a Kd of 5 nM; the 15 kDa binding protein bound 131I-acetyl-LDL with a Kd of 2.3 nM. A 85 kDa protein was obtained by Mal-BSA-Sepharose chromatography when the hepatic membranes had been solubilized with Triton X-100 in presence of GSH/GSSG. This protein displayed saturable 131I-Mal-BSA binding with a Kd of 30 nM and 131I-acetyl-LDL binding with a Kd of 6.5 nM. The 131I-Mal-BSA binding capacity was four times higher than that of 131I-acetyl-LDL. Competition studies with the 35 kDa, 15 kDa and 85 kDa proteins binding Mal-BSA, acetyl-LDL, formylated albumin and polyanionic competitors provide evidence for the existence of more than one class of binding sites at the reduced binding proteins.
Fusion of phospholipid vesicles produced by the anti-tumour protein α-sarcin
Authors: Gasset M , Oñaderra M , Thomas P G , Gavilanes J G
Published: August 2015

The anti-tumour protein alpha-sarcin causes fusion of bilayers of phospholipid vesicles at neutral pH. This is demonstrated by measuring the decrease in the efficiency of the fluorescence energy transfer between N-(7-nitro-2-1,3-benzoxadiazol-4-yl)-dimyristoylphosphatidylethano lamine (NDB-PE) (donor) and N-(lissamine rhodamine B sulphonyl)-diacylphosphatidylethanolamine (Rh-PE) (acceptor) incorporated in dimyristoylphosphatidylcholine (DMPG) vesicles. The effect of alpha-sarcin is a maximum at 0.15 M ionic strength and is abolished at basic pH. alpha-Sarcin promotes fusion between 1,6-diphenylhexa-1,3,5-triene (DPH)-labelled DMPG and dipalmitoyl-PG (DPPG) vesicles, resulting in a single thermotropic transition for the population of fused phospholipid vesicles. Bilayers composed of DMPC and DMPG, at different molar ratios in the range 1:1 to 1:10 PC/PG, are also fused by alpha-sarcin. Freeze-fracture electron micrographs corroborate the occurrence of fusion induced by the protein. alpha-Sarcin also modifies the permeability of the bilayers, causing the leakage of calcein in dye-trapped PG vesicles. All of the observed effects reach saturation at a 50:1 phospholipid/protein molar ratio, which is coincident with the binding stoichiometry previously described.
Regulation of glycogen synthesis from glucose and gluconeogenic precursors by insulin in periportal and perivenous rat hepatocytes
Authors: Agius L , Peak M , Alberti K G M M
Published: August 2015

Glycogen synthesis in hepatocyte cultures is dependent on: (1) the nutritional state of the donor rat, (2) the acinar origin of the hepatocytes, (3) the concentrations of glucose and gluconeogenic precursors, and (4) insulin. High concentrations of glucose (15-25 mM) and gluconeogenic precursors (10 mM-lactate and 1 mM-pyruvate) had a synergistic effect on glycogen deposition in both periportal and perivenous hepatocytes. When hepatocytes were challenged with glucose, lactate and pyruvate in the absence of insulin, glycogen was deposited at a linear rate for 2 h and then reached a plateau. However, in the presence of insulin, the initial rate of glycogen deposition was increased (20-40%) and glycogen deposition continued for more than 4 h. Consequently, insulin had a more marked effect on the glycogen accumulated in the cell after 4 h (100-200% increase) than on the initial rate of glycogen deposition. Glycogen accumulation in hepatocyte cultures prepared from rats that were fasted for 24 h and then re-fed for 3 h before liver perfusion was 2-fold higher than in hepatocytes from rats fed ad libitum and 4-fold higher than in hepatocytes from fasted rats. The incorporation of [14C]lactate into glycogen was 2-4-fold higher in periportal than in perivenous hepatocytes in both the absence and the presence of insulin, whereas the incorporation of [14C]glucose into glycogen was similar in periportal and perivenous hepatocytes in the absence of insulin, but higher in perivenous hepatocytes in the presence of insulin. Rates of glycogen deposition in the combined presence of glucose and gluconeogenic precursors were similar in periportal and perivenous hepatocytes, whereas in the presence of glucose alone, rates of glycogen deposition paralleled the incorporation of [14C]glucose into glycogen and were higher in perivenous hepatocytes in the presence of insulin. It is concluded that periportal and perivenous hepatocytes utilize different substrates for glycogen synthesis, but differences between the two cell populations in the relative utilization of glucose and gluconeogenic precursors are dependent on the presence of insulin and on the nutritional state of the rat.
A new method to monitor Kupffer-cell function continuously in the perfused rat liver. Dissociation of glycogenolysis from particle phagocytosis
Authors: Cowper K B , Currin R T , Dawson T L , Lindert K A , Lemasters J J , Thurman R G
Published: August 2015

In order to study particle phagocytosis and glycogenolysis simultaneously, this study was designed to develop a direct-read-out method to monitor Kupffer-cell function continuously, based on the uptake of colloidal carbon by the isolated perfused rat liver. Livers were perfused for 20 min with Krebs-Henseleit buffer saturated with O2/CO2 (19:1). Colloidal carbon (1-2 mg/ml) was added to the buffer, and absorbance of carbon was monitored continuously at 623 nm in the effluent perfusate. Since colloidal-carbon uptake was proportional to A623, rates of uptake were determined from the influent minus effluent concentration difference, the flow rate and the liver wet weight. Rates of colloidal-carbon uptake were 50-200 mg/h per g and were proportional to the concentration of carbon infused. Data from light-microscopy and cell-separation studies demonstrated that carbon was taken up exclusively by non-parenchymal cells and predominantly by Kupffer cells. Further, the amount of colloidal carbon detected histologically in non-parenchymal cells increased as the concentration of colloidal carbon in the perfusate was elevated. When Kupffer cells were activated or inhibited by treatment with endotoxin or methyl palmitate, carbon uptake was increased or decreased respectively. Taken together, these results indicate that Kupffer-cell function can be monitored continuously in a living organ. This new method was utilized to compare the time course of phagocytosis of carbon by Kupffer cells and carbohydrate output by parenchymal cells. Carbohydrate output increased rapidly by 69 +/- 9 mumol per g within 2-4 min after addition of carbon and returned to basal values within 12-16 min. However, carbon uptake by the liver did not reach maximal rates until about 15 min. Infusion of a cyclo-oxygenase inhibitor, aspirin (10 mM), caused a progressive decrease in carbohydrate output and blocked the stimulation by carbon completely. Aspirin neither altered rates of carbon uptake nor prevented stimulation of carbohydrate release by addition of N2-saturated buffer. The data from these experiments are consistent with the hypothesis that output of mediators by Kupffer cells, presumably prostaglandin D2 and E2, occurs transiently as Kupffer cells begin to phagocytose foreign particles in the intact organ, a process which continues at high rates for hours.
Histamine-H1-receptor-mediated phosphoinositide hydrolysis, Ca2+ signalling and membrane-potential oscillations in human HeLa carcinoma cells
Authors: Tilly B C , Tertoolen L G J , Lambrechts A C , Remorie R , de Laat S W , Moolenaar W H
Published: August 2015

In human HeLa carcinoma cells, histamine causes a dose-dependent formation of inositol phosphates, production of diacylglycerol and a transient rise in intracellular [Ca2+]. These responses are completely blocked by the H1-receptor antagonist pyrilamine. In streptolysin-O-permeabilized cells, formation of inositol phosphates by histamine is strongly potentiated by guanosine 5′-[gamma-thio]triphosphate and inhibited by guanosine 5′-[beta-thio]diphosphate, suggesting the involvement of a GTP-binding protein. Histamine stimulates the rapid but transient formation of Ins(1,4,5)P3, Ins(1,3,4)P3 and InsP4. InsP accumulates in a much more persistent manner, lasting for at least 30 min. Studies with streptolysin-O-permeabilized cells indicate that InsP accumulation results from dephosphorylation of Ins(1,4,5)P3, rather than direct hydrolysis of PtdIns. The rise in intracellular [Ca2+] is biphasic, with a very fast release of Ca2+ from intracellular stores, that parallels the Ins(1,4,5)P3 time course, followed by a more prolonged phase of Ca2+ influx. In individual cells, histamine causes a rapid initial hyperpolarization of the plasma membrane, which can be mimicked by microinjected Ins(1,4,5)P3. Histamine-induced hyperpolarization is followed by long-lasting oscillations in membrane potential, apparently owing to periodic activation of Ca2+-dependent K+ channels. These membrane-potential oscillations can be mimicked by microinjection of guanosine 5′-[gamma-thio]triphosphate, but are not observed after microinjection of Ins(1,4,5)P3. We conclude that H1-receptors in HeLa cells activate a PtdInsP2-specific phospholipase C through participation of a specific G-protein, resulting in long-lasting oscillations of cytoplasmic free Ca2+.
Distribution of distinct arachidonoyl-specific and non-specific isoenzymes of diacylglycerol kinase in baboon (Papio cynocephalus) tissues
Authors: Lemaitre R N , King W C , MacDonald M L , Glomset J A
Published: August 2015

We investigated the diacyglycerol kinase species present in several baboon tissues using the substrates sn-1-stearoyl-2-arachidonoyl diacylglycerol and sn-1,2-didecanoyl diacylglycerol. Chromatography of octyl glucoside extracts of the baboon (Papio cynocephalus papio) tissues on hydroxyapatite columns revealed the presence of three diacylglycerol kinase species with different substrate preferences. One species markedly ‘preferred’ the substrate sn-1-stearoyl-2-arachidonoylglycerol, the two other species preferred sn-1,2-didecanoylglycerol. Measurement of the activity of the baboon brain diacylglycerol kinases toward diacylglycerols with a range of different fatty acid chains revealed a strict preference of the arachidonoyl diacylglycerol kinase for sn-1-acyl-2-arachidonoyl diacylglycerol, whereas the other enzymes showed no preference toward several long-chain-fatty-acid-containing diacylglycerols. The arachidonoyl diacylglycerol kinase was particularly abundant in brain and testis, whereas liver was practically devoid of this enzyme. The arachidonoyl diacylglycerol kinase from baboon brain was found to be predominantly associated with the particulate fraction and exhibited an apparent molecular mass of 130 kDa.
Inactivation of pyruvate decarboxylase by 3-hydroxypyruvate
Authors: Thomas G , Diefenbach R , Duggleby R G
Published: August 2015

Pyruvate decarboxylase from Zymomonas mobilis is inhibited by 3-hydroxypyruvate, which can also act as a poor substrate. While catalysing the decarboxylation of this alternative substrate, the enzyme undergoes a progressive but partial inactivation over several hours. The extent of inactivation depends upon the pH and upon the concentration of 3-hydroxypyruvate. After partial inactivation and removal of unchanged 3-hydroxypyruvate, enzymic activity recovers slowly. We suggest that inactivation results from accumulation of enzyme-bound glycollaldehyde, which is relatively stable, possibly because it is dehydrated to form an acetyl group.
Inhibition of cytochalasin D-stimulated G-actin ATPase by ADP-ribosylation with Clostridium perfringens iota toxin
Authors: Geipel U , Just I , Aktories K
Published: August 2015

Clostridium perfringens iota toxin belongs to a novel family of actin-ADP-ribosylating toxins. The effects of ADP-ribosylation of skeletal muscle actin by Clostridium perfringens iota toxin on cytochalasin D-stimulated actin ATPase activity was studied. Cytochalasin D stimulated actin-catalysed ATP hydrolysis maximally by about 30-fold. ADP-ribosylation of actin completely inhibited cytochalasin D-stimulated ATP hydrolysis. Inhibition of ATPase activity occurred at actin concentrations below the critical concentration (0.1 microM), at low concentrations of Mg2+ (50 microM) and even in the actin-DNAase I complex, indicating that ADP-ribosylation of actin blocks the ATPase activity of monomeric actin and that the inhibitory effect is not due to inhibition of the polymerization of actin.
Reciprocal post-translational regulation of renal 1α- and 24-hydroxylases of 25-hydroxyvitamin D3 by phosphorylation of ferredoxin. mRNA-directed cell-free synthesis and immunoisolation of ferredoxin
Authors: Mandel M L , Moorthy B , Ghazarian J G
Published: August 2015

We have used a cell-free rabbit reticulocyte translational system programmed with polyadenylated [poly(A)+] RNA prepared from chick kidney tissue to study the synthesis of nascent ferredoxin, a class of iron-sulphur-containing proteins functional in the renal mitochondrial 1 alpha- and 24-hydroxylases of 25-hydroxyvitamin D3. The synthesis of ferredoxin was monitored by determining [35S]methionine incorporation into ferredoxin and quantified by SDS/PAGE and autoradiography after immunoprecipitation from the total translation products. Compared with normal controls, vitamin D deprivation caused a significant increase in the net synthesis of nascent ferredoxin with an Mr of 12,000-13,000. [3H]Orotate incorporation as uridine into kidney poly(A)+ RNA was stimulated by aminophylline, a potent inducer of 25-hydroxyvitamin D3 24-hydroxylase; however, the amount of nascent ferredoxin synthesis was the same as in normal controls. Also, partially purified chick kidney mitochondrial cyclic AMP-stimulated protein kinase catalysed the phosphorylation of ferredoxin in vitro. The catalytic activity of the ferredoxin in 1 alpha- and 24-hydroxylations of 25-hydroxyvitamin D3 in reconstituted systems consisting of cytochrome P-450 and ferredoxin reductase was altered with ferredoxin phosphorylation. The phosphorylation caused inhibition of the 1 alpha-hydroxylase activity while at the same time it stimulated the 24-hydroxylase. Authentic 1 alpha,25- and 24,25-dihydroxyvitamin D3 and 25-hydroxyvitamin D3 were used as standards to monitor the separation of the enzymic products by h.p.l.c. using methanol/water (4:1, v/v) as solvent. These results indicate that, in the absence of vitamin D or its metabolites in the deficient state, the synthesis of ferredoxin necessary for the 1 alpha-hydroxylase is accentuated, whereas the stimulation of the 24-hydroxylase requires the phosphorylation of existing ferredoxin without a net gain in its synthesis. This would suggest a post-translational regulation of the 1 alpha- and 24-hydroxylases. A model delineating the various aspects of this study is presented.
Effects of insulin, biguanide antihyperglycaemic agents and β-adrenergic agonists on pathways of myocardial proteolysis
Authors: Thorne D P , Lockwood T D
Published: August 2015

Pathways of bulk protein degradation controlled by insulin and isoprenaline (isoproterenol) were distinguished in Langendorff-perfused rat hearts. Proteins were biosynthetically labelled in vitro with [3H]leucine, followed by addition of 2 mM non-radioactive leucine to competitively prevent reincorporation. Rapidly degraded proteins were eliminated during a 3 h preliminary perfusion period without insulin. One third of bulk myocardial protein degradation was inhibited by isoprenaline as described previously. An insulin concentration of 5 nM maximally inhibited proteolysis, beginning within 2 min. Inhibition reached 32% within 1.25 h and 35% after 1.5 h. The minimum effective insulin concentration was approx. 10-50 pM, which caused 10-20% inhibition. Following 3 h of perfusion without insulin, the lysosomal inhibitor, chloroquine (30 microM), inhibited 38% of bulk degradation. The 35% proteolytic inhibition caused by insulin was followed by very little further inhibition on subsequent concurrent infusion of chloroquine, i.e. the inhibitory effects of insulin and chloroquine were not additive. In contrast, prior inhibition of lysosomal proteolysis by insulin or chloroquine did not prevent the subsequent additive inhibition caused by isoprenaline. Insulin and beta-agonists additively inhibited approx. two-thirds of bulk degradation. The biguanide antihyperglycaemic agent phenformin (2 microM) inhibited 35% of bulk degradation, beginning at 2 min and reaching a near maximum at approx. 1.25-1.5 h. Following inhibition of proteolysis with phenformin (20 microM), subsequent infusion of chloroquine (30 microM) produced only a slight additional inhibition. Following inhibition of 35% of degradation by 1.5 h of perfusion with insulin (5 nM), subsequent exposure to phenformin (2 microM) produced only a slight additional inhibition which did not exceed 38% of basal proteolysis. Thus insulin and phenformin both inhibit lysosomal proteolysis; however, the adrenergic-responsive pathway is distinct.