bluuebunny/crossref_metadata_2025 huggingface dataset:
Inhibition of Ca2+ inflow causes an abrupt cessation of growth-factor-induced repetitive free Ca2+ transients in single NIH-3T3 cells
Authors: Polverino A J , Hughes B P , Barritt G J
Published: August 2015

In single NIH-3T3 fibroblasts loaded with fura-2, bombesin induced one of three patterns of increase in the concentration of intracellular free Ca2+ [( Ca2+]i): a single transient increase, a sustained increase, or repetitive transient increases in [Ca2+]i. Foetal-calf serum and ATP also gave these three patterns of response, although a lower proportion of cells gave repetitive Ca2+ transients in response to ATP. An increase in the concentration of bombesin from 1 to 25 nM increased the proportion of cells which exhibited repetitive Ca2+ transients. At 25 nM-bombesin, the proportion of cells which exhibited repetitive Ca2+ transients increased as the extracellular Ca2+ (Ca2+o) concentration was increased from 1 to 5 mM. Removal of Ca2+o by addition of EGTA, or inhibition of Ca2+ inflow by treatment of cells incubated in the presence of Ca2+o with verapamil or an activator of protein kinase C, abruptly terminated repetitive Ca2+ transients, with only one transient observed after the cessation of Ca2+ inflow. Repetitive Ca2+ transients were not observed in cells incubated in the absence of Ca2+o and in the presence of EGTA. Addition of Ca2+o to cells previously incubated in the presence of EGTA caused a resumption of repetitive Ca2+ transients. Addition of thapsigargin alone induced a large transient increase in [Ca2+]i, whereas much smaller transient increases in [Ca2+]i were induced in about 30% of cells tested by caffeine or carbonyl cyanide m-chlorophenylhydrazone (CCCP) plus oligomycin. Thapsigargin or the combination of CCCP plus oligomycin completely inhibited bombesin-induced repetitive Ca2+ transients, whereas caffeine had no effect. It is concluded from the studies of the role of Ca2+o that NIH-3T3 cells differ from other cell types in the anatomical or chemical links between extracellular Ca2+ and the intracellular stores involved in the generation of Ca2+ transients, whereas the results of the experiments with inhibitors indicate that the generation of repetitive Ca2+ transients in NIH-3T3 cells is unlikely to involve Ca(2+)-induced Ca2+ release from caffeine-sensitive stores.
Levels of ornithine decarboxylase genomic sequences, heterogeneous nuclear RNA and mRNA in human myeloma cells resistant to α-difluoromethylornithine
Authors: Hyttinen J M , Halmekytö M , Alhonen L , Jänne J
Published: August 2015

We previously isolated and characterized a human myeloma cell line overproducing ornithine decarboxylase (ODC) due to gene amplification [Leinonen, Alhonen-Hongisto, Laine, Jänne & Jänne (1987) Biochem. J. 242, 199-203]. We have now employed the PCR combined with reverse transcription to determine semiquantitatively ODC gene dosage and the amounts of heterogeneous nuclear (hn) RNA and of mature mRNA of the enzyme in parental and alpha-difluoromethylornithine-resistant human myeloma cells. Experiments with dilution series revealed that the ODC gene copy number and the amount of both hnRNA and mRNA were increased to the same extent (about 100-fold) in the resistant cells. Similar dot-blot analyses of ODC-specific genomic DNA and total RNA indicated that the ODC gene copy number was increased by a factor of 380 and the amount of ODC mRNA by a factor of 700. Our results indicate that the PCR combined with reverse transcription is at least as useful as blot analyses to give semiquantitative assessment of the amounts of specific DNA or RNA sequences. In addition, the use of the PCR enables the analysis of minute sample amounts in extremely short time.
Minoxidil specifically decreases the expression of lysine hydroxylase in cultured human skin fibroblasts
Authors: Hautala T , Heikkinen J , Kivirikko K I , Myllylä R
Published: August 2015

The levels of lysine hydroxylase protein and the levels of the mRNAs for lysine hydroxylase and the alpha- and beta-subunits of proline 4-hydroxylase were measured in cultured human skin fibroblasts treated with 1 mM-minoxidil. The data demonstrate that minoxidil decreases the amount of lysine hydroxylase protein, this being due to a decrease in the level of lysine hydroxylase mRNA. The effect of minoxidil appears to be highly specific, as no changes were observed in the amounts of mRNAs for the alpha- and beta-subunits of proline 4-hydroxylase.
Phosphatidylethanolamine metabolism in rat liver after partial hepatectomy. Control of biosynthesis of phosphatidylethanolamine by the availability of ethanolamine
Authors: Houweling M , Tijburg L B M , Vaartjes W J , van Golde L M G
Published: August 2015

The effect of partial (70%) hepatectomy on phosphatidylethanolamine (PE) synthesis was studied in rat liver during the first 4 post-operative days. Between 4 and 96 h after partial hepatectomy, the mass of PE increased from 30% to 80% of sham-operation values. In line with the increase in PE mass, the rate of PE synthesis in vivo from [14C]ethanolamine was stimulated 1.6- and 1.3-fold at 22 and 48 h after partial hepatectomy respectively. Surprisingly, the activity of CTP:phosphoethanolamine cytidylyltransferase (EC 2.7.7.14) was virtually unchanged after partial hepatectomy. In addition, neither ethanolamine kinase (EC 2.7.1.82) nor ethanolaminephosphotransferase (EC 2.7.8.1) showed any changes in activity over the time period studied. Hepatic levels of ethanolamine and phosphoethanolamine were drastically increased after partial hepatectomy, as compared with sham operation, whereas levels of CDP-ethanolamine and microsomal diacylglycerol were not affected. Interestingly, partial hepatectomy caused the concentration of free ethanolamine in serum to increase from 29 microM to approx. 50 microM during the first day after surgery. In hepatocytes isolated from non-operated animals, incorporation of [3H]ethanolamine into PE was stimulated by increasing the ethanolamine concentration from 10 up to 50 microM, whereas the radioactivity associated with phosphoethanolamine only increased at ethanolamine concentrations higher than 30 microM. Taken together, our results indicate that the observed increase in serum ethanolamine concentration after partial hepatectomy is probably responsible for both the increase in PE biosynthesis and the accumulation of ethanolamine and phosphoethanolamine in regenerating liver.
Purification and characterization of a new endoglucanase from Clostridium thermocellum
Authors: Romaniec M P M , Fauth U , Kobayashi T , Huskisson N S , Barker P J , Demain A L
Published: August 2015

An endoglucanase (1,4-beta-D-glucan glucanohydrolase, EC 3.2.1.4) from the thermophilic anaerobe Clostridium thermocellum was purified to apparent homogeneity without the use of denaturants. No carbohydrate is associated with the endoglucanase. A molecular mass of 76,000 Da was determined by SDS/PAGE. The optimal pH is 7.0 and the enzyme is isoelectric at pH 5.05. The enzyme has a temperature optimum of 70 degrees C and retains approx. 50% of its activity after 48 h at 60 degrees C. Hydrolysis of CM-cellulose takes place with a rapid decrease in viscosity but a slow liberation of reducing sugars, indicating an endoglucanase type of activity. The endoglucanase shows little ability to hydrolyse highly ordered cellulose. Cellobiose inhibits whereas Mg2+ and Ca2+ stimulate the activity. The enzyme is completely inactivated by 1 mM-Hg2+ and is inhibited by a thiol-blocking reagent.
Effect of intestinal chylomicron secretory blockade on apolipoprotein synthesis in the newborn piglet
Authors: Black D D
Published: August 2015

Pluronic L-81 is a hydrophobic surfactant which blocks intestinal chylomicron secretion at the pre-Golgi level without affecting triacylglycerol uptake and re-esterification. To study the effects of such blockade on apolipoprotein synthesis, newborn female piglets received 24 h intraduodenal infusions of low-triacylglycerol, or high-triacylglycerol with or without Pluronic L-81, diets, followed by determination of apolipoprotein (apo) B-48, A-I and A-IV synthesis and content and apo B and A-IV mRNA levels in the small intestine. Jejunal apo B-48 content, synthesis and mRNA levels were down-regulated below basal levels by the addition of Pluronic to the high-triacylglycerol infusion. The normal increase in apo A-I synthesis induced by triacylglycerol absorption was ablated in both jejunum and ileum, even though the expected increase in apo A-I content in jejunum still occurred. Although attenuated, the expected increase in jejunal apo A-IV synthesis and mRNA levels with triacylglycerol absorption was still present with Pluronic treatment. These results suggest very different mechanisms of cellular regulation and trafficking for the various apolipoproteins incorporated into nascent intestinal chylomicrons. Apo B may be specifically down-regulated by the chylomicron secretory blockade induced by Pluronic L-81.
Point mutations of two arginine residues in the Streptomyces R61 dd-peptidase
Authors: Bourguignon-Bellefroid C , Joris B , Van Beeumen J , Ghuysen J M , Frère J M
Published: August 2015

Incubation of the exocellular DD-carboxypeptidase/transpeptidase of Streptomyces R61 with phenylglyoxal resulted in a time-dependent decrease in the enzyme activity. This inactivation was demonstrated to be due to modification of the Arg-99 side chain. In consequence, the role of that residue was investigated by site-directed mutagenesis. Mutation of Arg-99 into leucine appeared to be highly detrimental to enzyme stability, reflecting a determining structural role for this residue. The conserved Arg-103 residue was also substituted by using site-directed mutagenesis. The modification to a serine residue yielded a stable enzyme, the catalytic properties of which were similar to those of the wild-type enzyme. Thus Arg-103, although strictly conserved or replaced by a lysine residue in most of the active-site penicillin-recognizing proteins, did not appear to fulfil any essential role in either the enzyme activity or structure.
Characterization of a family of structurally related glycoproteins expressing β1-6-branched asparagine-linked oligosaccharides in human colon carcinoma cells
Authors: Laferté S , Loh L C
Published: August 2015

Previous studies have established that metastatic tumour cells express high levels of beta 1-6-branched Asn-linked oligosaccharides which can be detected with the lectin leucoagglutinin (L-PHA) [Dennis, Laferté, Waghorne, Breitman & Kerbel (1987) Science 236, 582-585]. In order to identify L-PHA-binding glycoproteins which may play a role specifically in colon cancer, we have prepared monoclonal antibodies (MAbs) to the moderately well-differentiated human colon carcinoma cell line HT29. In this paper we present the initial characterization of a family of structurally related L-PHA-binding glycoproteins detected by MAb 1H9 which are differentially expressed and processed by HT29 cells and by two other human colon carcinoma cell lines, SW480 and SW620. In contrast to HT29, the SW480 and SW620 cell lines were established from a poorly differentiated grade III/IV primary tumour and one of its lymph node metastases respectively. MAb 1H9 detects in HT29 cells a conformational determinant present on three L-PHA-binding glycoproteins of 100, 70 and 25kDa, as well as a 74 kDa glycoprotein with high-mannose-type Asn-linked oligosaccharides. Pulse-chase experiments and peptide mapping analyses revealed that the 74 kDa and 100 kDa species are related by carbohydrate processing and are probably derived from a common 76 kDa precursor. On the other hand, the 70 kDa glycoprotein is synthesized from an endoglycosidase H-sensitive precursor of 56 kDa which is structurally related to, but distinct from, the aforementioned 76 kDa precursor. In addition, the 100 kDa species is secreted into the culture medium, whereas the 70 kDa glycoprotein is retained intracellularly. SW480 and SW620 cells showed qualitative and quantitative differences from HT29 cells, including increased secretion of a smaller L-PHA-binding glycoprotein of 92 kDa into the culture medium, as well as apparent differences in glycosylation of the intracellular 66 kDa glycoprotein. These results suggested that the expression, glycosylation and subcellular localization of this family of L-PHA-binding glycoproteins may correlate with the differentiation status of colon cancer cells and/or reflect biochemical changes. characteristic of more progressive metastatic tumours.
Regulation of cardiac adenylate cyclase activity in rodent models of obesity
Authors: Strassheim D , Houslay M D , Milligan G
Published: August 2015

We have investigated beta-adrenergic regulation of adenylate cyclase activity in heart tissue membranes from the genetically obese Zucker rat, the genetically obese CBA mouse and the genetically obese diabetic (db/db) mouse. Responsiveness to beta-adrenergic stimulation was impaired in membranes from the obese Zucker rat, but not in the other models. The membranes from obese Zucker rats showed both decreased beta-adrenergic-receptor number and altered coupling between beta-adrenergic receptors and the stimulatory guanine-nucleotide-binding protein, Gs. In contrast, no alterations in either the levels of Gs or the functional interaction between this protein and the catalytic moiety of adenylate cyclase were observed. In these three genetic models of obesity we observe dissimilar alterations in the control of adenylate cyclase.
Lipid remodelling during epididymal maturation of rat spermatozoa. Enrichment in plasmenylcholines containing long-chain polyenoic fatty acids of the n-9 series
Authors: Aveldaño M I , Rotstein N P , Vermouth N T
Published: August 2015

In their transit from the caput to the cauda segments of the epididymis, rat spermatozoa undergo significant modifications in lipid content and composition. The amount of lipid phosphorus per cell decreases, and most lipid classes show specific changes in their constituent fatty acids. A depletion of phosphatidylcholine and phosphatidylethanolamine, concomitant with a virtually unchanged amount of the corresponding plasmalogens, are the major alterations, plasmenylcholine thereby becoming the major phospholipid. Diphosphatidylglycerol, sphingomyelin and the phosphoinositides decrease to a lesser extent or do not change at all, also resulting in relative increases with sperm maturation. Concerning the fatty acids, the proportions of oleate (C18:1, n-9) and linoleate (C18:2, n-6) in most lipids decrease on movement of sperm from caput to cauda, augmenting in turn the proportions of longer-chain (C20 to C24) and more unsaturated fatty acids. Docosapentaenoate (C22:5, n-6) is a major acyl chain present in all lipids at both stages, but uncommon long-chain polyenoic fatty acids of the n-9 series are also present, being almost exclusively found in the choline glycerophospholipids. These fatty acids are found to undergo the most significant changes during sperm maturation. They are minor components of plasmenylcholine in immature spermatozoa, but increase severalfold on maturation, representing more than half of the acyl chains of this major lipid in cells from the cauda. The high concentration of n-9 polyenes in mature sperm plasmenylcholine raises intriguing questions on the possible role epididymal cells may play in providing spermatozoa with such an unusual phospholipid. These plasmenylcholines could contribute to the characteristic lipid domain organization of the mature spermatozoa plasma membrane.
Gluconeogenesis stimulated by extracellular ATP is triggered by the initial increase in the intracellular Ca2+ concentration of the periphery of hepatocytes
Authors: Koike M , Kashiwagura T , Takeguchi N
Published: August 2015

Extracellular ATP, ADP and GTP increased the intracellular free Ca2+ concentration ([Ca2+]i) in a suspension of isolated rat hepatocytes. The [Ca2+]i was determined by measuring fura-2 fluorescence, and its increase was biphasic. The initial transient rise was followed by a longer-lasting plateau. The peak of the early component preceded the plateau level of the second component. A time course of change in [Ca2+]i in single cells at 100 microM-ATP was very similar to that observed in the suspension system. Preincubation of hepatocytes with 40 mM-caffeine, 2 mM-oxalate or 60 microM-dantrolene sodium inhibited the P2 purinergic response. The plateau phase was not observed when measured in the presence of extracellular 100 microM-LaCl3 or in the absence of extracellular Ca2+. The distribution of [Ca2+]i in single hepatocytes was also determined by fluorescence image analysis. In the initial phase, the increase in [Ca2+]i is greater in the peripheral region than the central region of the cell. Degradation of extracellular ATP by ecto-ATPase in the hepatocyte suspension was measured; the amount of ATP degradation was less than 10-15% of the initial amount (100 microM) during the measurement of the intracellular [Ca2+]i in the cell suspension. Extracellular ATP stimulated glucose synthesis. The rate of glucose production also showed two components, the initial fast component within 1 min and the subsequent slower component. The rate of the initial fast component did not depend on the presence or absence of extracellular Ca2+, whereas the rate of the subsequent component depended on it. The present study shows that the initial transient rise in [Ca2+]i plays an important role in triggering the gluconeogenesis.
Kinetic studies of the regulation of mitochondrial malate dehydrogenase by citrate
Authors: Gelpí J L , Dordal A , Montserrat J , Mazo A , Cortés A
Published: August 2015

Mitochondrial malate dehydrogenase shows a complex regulation pattern in the presence of citrate. Previously published results indicate that this enzyme is activated by citrate in the NAD(+)----NADH direction and inhibited in the opposite direction. Moreover, high concentrations of L-malate or oxaloacetate produce deviations from the Michaelis-Menten behaviour. Results reported in this paper clearly show that citrate both activates and inhibits mitochondrial malate dehydrogenase in the same direction (NAD(+)----NADH), and in the same reaction medium, depending on substrate concentration. This surprising effect has made it necessary to propose a new kinetic mechanism that extends those previously suggested and allows us to explain both the citrate effect (activating or inhibitory) and the effect of high concentrations of L-malate and oxaloacetate.
Polypeptide N-acetylgalactosaminyltransferase activity in tracheal epithelial microsomes
Authors: Cottrell J M , Hall R L , Sturton R G , Kent P W
Published: August 2015

Pig tracheal epithelium, a site of extensive mucin biosynthesis, contained polypeptide N-acetylgalactosaminyltransferase activity directed towards L-threonine residues. The enzyme preparation was broadly similar in properties to preparations from other tissues, e.g. pig and bovine submaxillary glands, bovine colostrum, BW5147 mouse lymphoma and baby-hamster kidney cells. Enzyme was membrane-bound and was released from microsomal preparations by extraction with Triton X-100. Extracted enzyme had a pH optimum of 7.5, had a requirement for Mn2+ (10 mM) and was inhibited by Na2EDTA. The Km for UDP-N-acetylgalactosamine was 110 microM and that for an octapeptide acceptor (VTPRTPPP) was 3.0 mM at 37 degrees C. Using a range of synthetic peptides of known structure related to TPPP it was established that L-threonine residues were specifically O-glycosylated probably in the alpha-configuration. Synthetic peptides containing the TPPP sequence required a peptide length of five or more for significant acceptor activity. In VTPRTPPP the two threonine residues were similarly glycosylated, as revealed by tryptic cleavage of the glycosylated product and separation of the 3H-labelled fragments. The enzyme preparation also specifically catalysed the transfer of N-acetylgalactosaminyl residues from UDP-N-acetyl[1-3H]galactosamine to bovine submaxillary mucin core protein and to myelin basic protein.
Molecular cloning of higher-plant 3-oxoacyl-(acyl carrier protein) reductase. Sequence identities with the nodG-gene product of the nitrogen-fixing soil bacterium Rhizobium meliloti
Authors: Slabas A R , Chase D , Nishida I , Murata N , Sidebottom C , Safford R , Sheldon P S , Kekwick R G , Hardie D G , Mackintosh R W
Published: August 2015

cDNA clones encoding the fatty-acid- biosynthetic enzyme NADPH-linked 3-oxoacyl-(acyl carrier protein) (ACP) reductase were isolated from a Brassica napus (rape) developing seed library and from an Arabidopsis thaliana (thale cress) leaf library. The N-terminal end of the coding region shows features typical of a stromal-targeting plastid-transit peptide. The deduced amino acid sequences have 41% and 55% identity respectively with the nodG-gene product of Rhizobium meliloti, one of the host-specific genes that restrict infectivity of this bacterium to a small range of host plants. The probability that the nodG-gene product is a oxoreductase strengthens the hypothesis that some of the host-specific nod-gene products are enzymes which synthesize polyketides that uniquely modify the Rhizobium nodulation signal molecule.
The 8-amino-7-oxopelargonate synthase from Bacillus sphaericus. Purification and preliminary characterization of the cloned enzyme overproduced in Escherichia coli
Authors: Ploux O , Marquet A
Published: August 2015

The 8-amino-7-oxopelargonate synthase [6-carboxyhexanoyl-CoA:L-alanine carboxyhexanoyltransferase (decarboxylating); EC 2.3.1.47] from Bacillus sphaericus involved in biotin biosynthesis was purified from an Escherichia coli overproducing strain. The purification afforded an electrophoretically homogeneous enzyme with a specific activity of 0.67 unit/mg. The purified enzyme is a monomer of 41 kDa. N-Terminal sequencing of the first 14 amino acid residues showed complete agreement with the predicted sequence from the bioF gene. The pure enzyme showed the characteristic absorption band (425 nm) of pyridoxal 5′-phosphate-dependent enzymes. Furthermore, the holoenzyme was resolved during an affinity step yielding the inactive apoenzyme, which recovered activity and the 425 nm-absorption band on dialysis against pyridoxal 5′-phosphate. Km values for L-alanine and pimeloyl-CoA were respectively 3 mM and 1 microM.
Regulation of glycine catabolism in rat liver mitochondria
Authors: Jois M , Ewart H S , Brosnan J T
Published: August 2015

1. The catabolism of glycine was studied in isolated rat liver mitochondria by measuring release of 14CO2 from [1-14C]-glycine. Incubation of mitochondria in a medium containing 0.5 microM free Ca2+ resulted in an 8-fold increase in the rate of degradation of glycine. Intraperitoneal injection of glucagon (33 or 100 micrograms/100 g body wt.) 25 min before killing of rats also resulted in a 3-fold or 10-fold (depending on dosage) increase in the rate of catabolism of glycine. 2. Both the stimulation by free Ca2+ and that by injection of glucagon in vivo were dependent on phosphate in the incubation medium. This requirement for phosphate was specific, as replacement of phosphate by other permeant anions such as thiocyanate and acetate did not permit the stimulation. The phosphate-dependent stimulation of glycine catabolism by Ca2+ was also evident when mitochondria were incubated in the absence of K+. 3. Mitochondria isolated from rats previously injected with glucagon showed elevated rates of degradation of glycine even in the presence of rotenone, provided that regeneration of NAD+ was affected by providing acetoacetate. 4. Hypo-osmolarity of the medium markedly stimulated the rate of degradation of glycine by mitochondria. Although hypo-osmolarity-induced stimulation of glycine degradation was accompanied by parallel changes in mitochondrial matrix volume, no measurable changes in matrix volume were observed in mitochondria stimulated either by free Ca2+ (0.5 microM) or by injection of glucagon in vivo. Furthermore, Ca2+ stimulated glycine decarboxylation in mitochondria exposed to either hyper-osmolar (410 mosmol) or hypo-osmolar (210 mosmol) conditions. Although hyper-osmolarity decreased and hypo-osmolarity increased matrix volume, stimulation of glycine degradation by Ca2+ was not associated with any further changes in matrix volume. 5. These data demonstrate that the regulation of hepatic glycine oxidation by glucagon and by free Ca2+ is largely independent of changes in mitochondrial matrix volume.
The synthesis, kinetic characterization and application of biotinylated aminoacylchloromethanes for the detection of chymotrypsin and trypsin-like serine proteinases
Authors: Kay G , Bailie J R , Halliday I M , Nelson J , Walker B
Published: August 2015

The synthesis of two biotinylated affinity labels for chymotrypsin and trypsin-like serine proteinases is described, along with their kinetic characterization and application to the detection of these proteinases after PAGE and Western blotting. Thus the chloromethane analogues biotinylphenylalanylchloromethane (Bio-Phe-CH2Cl; reagent 1) and biotinylarginylchloromethane (Bio-Arg-CH2Cl, reagent 2), have been shown to be potent active-site-directed inactivators of chymotrypsin and trypsin respectively. The apparent overall second-order rate constants (kobs./[I]) for the inactivation of chymotrypsin and trypsin by reagent 1 (approximately 4.9 x 10(3) M-1.min-1) and reagent 2 (approximately 1.0 x 10(5) M-1.min-1) respectively are comparable with those obtained by other workers with simple urethane-protected analogues and demonstrates that the presence of the bulky biotinyl moiety is compatible with inhibitor effectiveness. Samples of chymotrypsin and trypsin that have been inactivated by reagents 1 and 2 respectively and which have been subjected to SDS/PAGE and Western blotting can be revealed with a streptavidin/alkaline phosphatase label. We can presently detect down to 20 ng of inactivated proteinase by using this system. The utility of the arginine derivative for the detection of the plasma trypsin-like proteinases plasmin and thrombin has also been demonstrated, thus holding out the possibility that this reagent may find general application as an active-site-directed label for this class of proteinase.
Critical evaluation of a theory of molecular recognition using human insulin-like-growth-factor-I fragment 21–40 and its complementary peptide
Authors: Beattie J , Flint D J
Published: August 2015

Using solid-phase methods we have synthesized human insulin-like-growth-factor-I (IGF-I) fragment 21-40 (IGF-I 21-40) and the peptide derived from the 5′----3′ translation of the complementary nucleic acid of this peptide, ‘I-FGI 20-40’ (the complementary peptide). According to a recently proposed theory of molecular recognition, these two peptides should bind specifically to each other. We have tested this theory by using both solid- and solution-phase direct-binding assays for this complementary-peptide pair. We have also investigated the ability of I-FGI 20-40 to interfere with native IGF-I binding during radioimmunoassay (r.i.a.), radio-receptor (r.r.a.) assay and ligand-blot analysis of IGF-binding proteins. We have obtained no evidence of any interaction between IGF-I 21-40 and I-FGI 20-40 in either solid- or solution-phase assays. In addition, I-FGI 20-40 does not interfere in the assays used to detect IGF-I binding antibodies (r.i.a.), receptors (r.r.a.) or binding proteins (ligand blots). Our data therefore question the universality of this particular theory of molecular recognition.
Identification of two distinct galactosyltransferase activities acting on the variant surface glycoprotein of Trypanosoma brucei
Authors: Pingel S , Duszenko M
Published: August 2015

Variant surface glycoproteins (VSGs) of Trypanosoma brucei contain two distinct glycosylation sites: (1) N-linked glycans within the protein portion of the molecules, and (2) the glycosyl-phosphatidylinositol (GPI) membrane anchor. Since galactose residues show uncommon alpha-glycosidic linkages in the GPI membrane anchor, we were prompted to investigate galactosylation of the GPI anchor. On comparing a trypanosome clone galactosylated exclusively in N-glycans (clone MITat 1.5) with clones galactosylated predominantly in the glypiated membrane anchor (clones MITat 1.4, MITat 1.6 and AnTat 1.8), clone MITat 1.5 showed a 10-fold increased enzyme activity when using a protocol including Triton X-100 to assay UDPgalactose:N-acetylglucosaminyl glycopeptide beta 1,4-galactosyltransferase (EC 2.4.1.38). Only the VSG of clone MITat 1.5 could be radiochemically labelled with UDP[14C]galactose, and galactosylation of N-glycans was confirmed by digestion with peptide-N4-(N-acetylglucosaminyl)asparagine amidase (PNGase F). However, in a modified enzyme assay without detergent, galactosyltransferase activity was increased considerably (15-fold) in clone MITat 1.4. VSG galactosylation of clones MITat 1.4, MITat 1.6 and AnTat 1.8 was readily detected by fluorography of the respective SDS/polyacrylamide gels, suggesting that galactosyltransferase activity modifies the VSG membrane anchor in these clones. In this case, [14C]galactose labelling of immunoprecipitated VSG (clone MITat 1.4) was resistant to the release of N-glycans by PNGase F treatment, and thus revealed galactosylation in vitro of a VSG membrane anchor. Exoglycosidase digestions of VSG MITat 1.4 confirmed the presence of alpha-linked galactose residues. We suggest that these specific alpha-galactosyltransferases are inhibited by the action of detergent, but can be activated in a detergent-free buffer system.
Staurosporine clamps cytosolic free Ca2+ concentrations of human neutrophils
Authors: Wong K , Kwan-Yeung L , Turkson J
Published: August 2015

The present studies indicate that 50 nM-10 microM-staurosporine increased cytosolic free Ca2+ concentrations ([Ca2+]i) of fura-2-loaded neutrophils in a non-linear manner. The rise in [Ca2+]i was rapid, reaching a plateau (e.g. to 0.4 microM with 1 microM-staurosporine) within 30 s, and was maintained for more than 20 min. Pretreating cells with pertussis toxin had no effect on this reaction. The elevation of [Ca2+]i was insensitive to extracellular Ca2+ concentrations and was due entirely to mobilization of intracellular Ca2+ stores. Mn(2+)-quench studies confirmed the absence of Ca2+ influx. No Ca2+ efflux occurred in staurosporine-treated cells. In combination studies, staurosporine potentiated Ca2+ influx induced by N-formylmethionyl-leucyl-phenylalanine (FMLP) and did not block Ca2+ efflux associated with peptide stimulation of neutrophils. Studies with permeabilized cells showed that staurosporine did not directly release intracellular Ca2+ stores, nor did it affect the sequestration of Ca2+ by a Ca2+/ATPase pump. A radioligand-binding assay failed to detect changes in the level of inositol 1,4,5-trisphosphate in neutrophils incubated with less than or equal to 1 microM-staurosporine, but in cells treated with 10 microM-staurosporine the assay recorded a transient increase in this second messenger similar to that induced by FMLP. Finally, lysozyme, but not beta-glucuronidase, was released from staurosporine-treated cells. The present results suggest that staurosporine increased [Ca2+]i by indirectly mobilizing internal Ca2+ stores. Staurosporine suppression of Ca2+ efflux and generation of a persistent signal may account for the maintained elevation of [Ca2+]i.
Thyrotropin-releasing hormone-mediated Mn2+ entry in perifused rat anterior pituitary cells
Authors: Cui Z J , Dannies P S
Published: August 2015

Receptor-mediated Ca2+ influx has been shown to exist in several cell types. Thyrotropin-releasing-hormone (TRH)-stimulated Ca2+ entry has also been postulated to exist in rat anterior pituitary cells, but direct evidence has been lacking. We have measured the fluorescence quenching of indo-1 caused by Mn2+ at a Ca(2+)-insensitive wavelength to investigate the actions of TRH on cation entry in dispersed perifused anterior pituitary cells. In indo-1-loaded cells perifused with Ca(2+)-free medium, Mn2+ caused fluorescence quenching in unstimulated cells; TRH caused further quenching. TRH-stimulated Mn2+ entry was transient, and levelled off within a few minutes in the presence of continuous TRH infusion. TRH-stimulated Mn2+ entry was dependent on the concentration of Mn2+ (50 microM-1 mM). TRH (1 microM) caused a larger effect than TRH (10 nM). La3+ and Ni2+ blocked the quenching stimulated by TRH. The rate of basal quenching was not blocked by dopamine, but TRH-stimulated Mn2+ entry was partially blocked by 1 microM-dopamine and almost completely abolished by 10 microM-dopamine. Thapsigargin (1-5 microM), a tumour promotor which depleted intracellular Ca2+ stores, had little effect on Mn2+. F- (20 mM), which activates G-proteins, also had little effect on Mn2+ entry. We conclude that TRH can transiently stimulate Ca2+ entry through a channel than can pass Mn2+ and be inhibited by dopamine. Depleting Ca2+ stores alone is not sufficient to stimulate Ca2+ entry, and so TRH must do so by other mechanisms.
New kinetic parameters for rat liver arginase measured at near-physiological steady-state concentrations of arginine and Mn2+
Authors: Maggini S , Stoecklin-Tschan F B , Mörikofer-Zwez S , Walter P
Published: August 2015

A cytosolic cell-free system from rat liver containing the last three enzymes of the urea cycle, a number of cofactors and the substrates aspartate and citrulline was shown to synthesize urea at near-physiological rates ranging between 0.40 and 1.25 mumol/min per g of liver. This system was used to determine the kinetic parameters for arginase. With saturating amounts of Mn2+ (30 microM), arginine remained at a steady-state concentration of 5-35 microM depending on the aspartate and citrulline supply. Vmax. at micromolar arginine concentrations was between 1.10 and 1.25 mumol/min per g of liver, the K0.5 (arginine) between 6.0 and 6.5 microM and positive co-operativity was observed (Hill coefficient 2). Omission of Mn2+ caused a significant accumulation of arginine during the incubation, suggesting a regulatory effect of arginase. Under these conditions, Vmax. was 1.10-1.65 mumol/min per g of liver and the Km (arginine) increased up to 14.4-21.1 microM. The apparent Ka for Mn2+ in the presence of physiological concentrations of ATP, Mg2+ and arginine was calculated to be maximally 8 microM. Initial-velocity experiments with millimolar arginine concentrations as the direct substrate gave the following results, which are in good agreement with literature data. In the absence of Mn2+, Vmax. was 71.3 mumol/min per g of liver and the Km (arginine) 1.58 mM. With 30 microM-Mn2+, Vmax. was 69.4 mumol/min per g of liver and the Km (arginine) decreased to 0.94 mM. On the basis of our results, we propose the presence of high-affinity and low-affinity sites for arginine on rat liver arginase and postulate that alterations in arginase activity arising from changes in the concentration of arginine and of the cofactor Mn2+ may contribute to the regulation of ureagenesis in vivo.
Antizyme, a protein induced by polyamines, accelerates the degradation of ornithine decarboxylase in Chinese-hamster ovary-cell extracts
Authors: Murakami Y , Tanaka K , Matsufuji S , Miyazaki Y , Hayashi S I
Published: August 2015

Ornithine decarboxylase (ODC), the key regulatory enzyme for polyamine biosynthesis, is known to have a short intracellular half-life, and antizyme, an ODC-binding protein induced by polyamines, has been suggested to be involved in the process of ODC degradation. In the present study we demonstrated that antizyme markedly accelerated ATP-dependent degradation of ODC in vitro in an extract from ODC-overproducing Chinese-hamster ovary cells.
Calmodulin binding distinguishes between βγ subunits of activated G proteins and transducin
Authors: Mangels L A , Neubig R R , Hamm H E , Gnegy M E
Published: August 2015

The interactions between guanine nucleotide regulatory proteins and the Ca(2+)-binding protein calmodulin were studied using calmodulin-Sepharose affinity chromatography. Purified bovine brain beta gamma subunits bound to calmodulin-Sepharose in a Ca(2+)-dependent manner. On the contrary, beta gamma subunits produced in an activated Go/Gi preparation did not bind to calmodulin-Sepharose. The effect was independent of the type of bovine brain G protein (Go/Gi, Gs), method of activation and the presence of magnesium. To distinguish whether the binding of purified beta gamma subunits to calmodulin was unique to brain beta gamma or to the method of purification, similar experiments were performed using transducin. In contrast to bovine brain G proteins, both purified transducin beta gamma subunits and beta gamma released from rhodopsin-activated transducin bound to calmodulin-Sepharose in a Ca(2+)-dependent manner. To assess the functional significance of the binding of bovine brain beta gamma subunits to calmodulin, the ability of purified beta gamma and of beta gamma in unactivated and activated Go/Gi to inhibit partially purified calmodulin-sensitive adenylate cyclase was determined. Purified beta gamma was highly effective in inhibiting calmodulin-stimulated adenylate cyclase activity. However, unactivated Go/Gi and preactivated Go/Gi inhibited calmodulin-stimulated adenylate cyclase activity to the same extent. This Go/Gi-mediated inhibition also occurred in the presence of a 500-fold molar excess of calmodulin over added G protein. These results demonstrate: (1) that beta gamma subunits may not be completely released upon G protein activation, and (2) that inhibition of calmodulin-stimulated adenylate cyclase by beta gamma subunits does not appear to be mediated by a direct beta gamma-calmodulin interaction. Differences in the binding properties of activated bovine brain G proteins versus those of transducin could be explained by differences in the gamma subunit between the proteins, or by differences in affinities of the alpha and beta gamma subunits for each other and for calmodulin. The different functional properties of purified beta gamma subunits and beta gamma subunits produced in situ by activation of G proteins indicates that extrapolation from the effects of purified subunits to events occurring in membranes should be done with caution.
Cis- and trans-acting elements required for constitutive and cytokine-regulated expression of the mouse complement C3 gene
Authors: Kawamura N , Singer L , Wetsel R A , Colten H R
Published: August 2015

The third component of complement (C3) is an important mediator of inflammation. Murine and human genomic cosmid clones were isolated, characterized and sequenced 5′ to the complement C3 gene transcriptional initiation sites to determine cis elements that participate in constitutive and regulated C3 gene expression. The murine and human 5′ flanking regions are 51% identical overall, with positions -36 to -1 and -146 to -68 showing 80% identity. Four TATA boxes were identified upstream of the murine transcriptional initiation site, but deletion and transfection analysis using reporter gene constructs in HepG2 cells indicated that only the TATA element at position -30, together with sequences -395 to -111, are essential for constitutive expression of murine C3 in hepatocytes. Deletion analysis also suggested that sequences between -1457 and -800 contain regulatory elements that are involved in suppressing basal expression. Sequences between -90 to -41 confer both enhancer activity and interleukin-1/-6 (IL-1/IL-6)-responsiveness. Mutation analyses showed that both sequences between -88 and -83 and -77 to -72 are essential for enhancer activity and responsiveness to IL-1, but only sequences between -88 and -83 are necessary for IL-6-responsiveness. A gel-retardation assay showed that several nucleoproteins, perhaps of the C/EBP family, from HepG2 cells bound to sequences between -88 to -83. Collectively, these results localize cis-acting elements involved in constitutive and IL-1/IL-6-regulated murine C3 gene expression and provide evidence for specific transacting factors.
Nucleotide regulation of heat-stable enterotoxin receptor binding and of guanylate cyclase activation
Authors: Katwa L C , Parker C D , Dybing J K , White A A
Published: August 2015

Certain nucleotides were found to regulate the binding of the Escherichia coli heat-stable enterotoxin (STa) to its receptor in pig intestinal brush border membranes. ATP and adenine nucleotide analogues inhibited 125I-STa binding, while guanine nucleotide analogues stimulated binding, with maximal effects at 0.5-1.0 mM. The strongest inhibitors were adenosine 5′-[beta gamma-imido]triphosphate (App[NH]p) (36%) and adenosine 5′-[beta-thio]diphosphate (ADP[S]) (41%). Inhibition did not require Mg2+, and was blocked by p-chloromercuribenzenesulphonate (PCMBS). Stimulation of binding required Mg2+, was not prevented by PCMBS and was maximal with GDP[S] (41%). While App[NH]p and MgGDP[S] appeared to be acting at different sites, they also interfered with each other. These nucleotides exerted only inhibitory effects on STa-stimulated guanylate cyclase activity, in contrast with the stimulatory effects of adenine nucleotides on atrial natriuretic peptide (ANP)-stimulated guanylate cyclase. Inhibition by low concentrations of MgApp[NH]p and MgATP was weaker above 0.1 mM, while MgGDP[S] and magnesium guanosine 5′-[gamma-thio]triphosphate (MgGTP[S]) inhibited in a single phase. Inhibition by MgApp[NH]p, at all concentrations, was competitive with the substrate (MgGTP), as was that by MgGDP[S] and MgGTP[S]. Whereas membrane guanylate cyclases usually show positively co-operative kinetics with respect to the substrate, STa-stimulated activity exhibited Michaelis-Menten kinetics with respect to MgGTP. This changed to positive co-operativity when Lubrol PX was the activator, or when the substrate was MnGTP. These results suggest the presence of both a regulatory and a catalytic nucleotide-binding site, which do not interact co-operatively with STa activation.
Uptake of the antitrypanosomal drug 5′-{[(Z)-4-amino-2-butenyl]methylamino}-5′-deoxyadenosine (MDL 73811) by the purine transport system of Trypanosoma brucei brucei
Authors: Byers T L , Casara P , Bitonti A J
Published: August 2015

An irreversible inhibitor of S-adenosyl-L-methionine decarboxylase (AdoMetDC), 5′-([(Z)-4-amino-2-butenyl]methylamino)-5′-deoxyadenosine (MDL 73811), was found to cure Trypanosoma brucei brucei and multidrug-resistant T. b. rhodesiense infections in mice [Bitonti, Byers, Bush, Casara, Bacchi, Clarkson, McCann & Sjoerdsma (1990) Antimicrob. Agents Chemother. 34, 1485-1490]. Doses of this drug which resulted in a rapid clearance of parasites from T. b. brucei-infected rats resulted in plasma levels of 50-60 microM-MDL 73811 and an intratrypanosomal MDL 73811 concentration of 1.9 mM within 10 min of administration [Byers, Bush, McCann & Bitonti (1991) Biochem. J. 274, 527-533[. Based on this finding we speculated that MDL 73811, which is an adenosine analogue, is a substrate for the trypanosome active purine transport system. We now report evidence that supports this hypothesis. MDL 73811 uptake by T. b. brucei in vitro was time- and temperature-dependent and was saturable over a time course in which MDL 73811 metabolism was undetectable, suggesting that MDL 73811 uptake is a transport-mediated phenomenon. Inhibition of MDL 73811 uptake by purine nucleosides is consistent with the drug being a substrate for the trypanosome purine transport system. The accumulation of MDL 73811 by cultured L1210 mouse leukaemia cells was significantly less than by trypanosomes exposed to the same pharmacologically relevant concentrations of MDL 73811. Given that the half-life of MDL 73811 in the plasma of rats and mice is approx. 10 min, it seems likely that the existence of a highly active parasite transport system for MDL 73811 is crucial for the sensitivity of trypanosomes towards MDL 73811 in vivo, and that the absence of active transport of MDL 73811 by the host's cells may play a role in the selectivity of this drug.
Different preparations of zymosan induce glycogenolysis independently in the perfused rat liver. Involvement of mannose receptors, peptide-leukotrienes and prostaglandins
Authors: Kimura K , Shiota M , Mochizuki K , Ohta M , Sugano T
Published: August 2015

Zymosan (non-boiled) induced glycogenolysis biphasically, with no lag time, in the perfused rat liver. After the zymosan was boiled, it could be separated into two fractions, both of which stimulated glycogenolysis independently. The soluble fraction of boiled zymosan (zymosan sup) showed homologous desensitization, indicating that zymosan sup-induced glycogenolysis is a receptor-mediated event. Mannan (polymannose), which is known to be a biologically active component of zymosan, induced a glycogenolytic response similar to that produced by zymosan sup, and desensitized the response to the latter. Preinfusion of platelet-activating factor (PAF, 20 nM) or isoprenaline (10 microM) did not extinguish the glycogenolytic response to zymosan sup, while the response to a secondary infusion of PAF was blocked. The glycogenolytic response to zymosan sup was completely inhibited by nordihydroguaiaretic acid (NDGA, 10 microM), a lipoxygenase inhibitor, and by ONO-1078 (100 ng/ml), a leukotriene (LT) D4 receptor antagonist. On the other hand, the glycogenolytic effect of zymosan pellet (the particulate fraction of boiled zymosan) was not affected by preinfusion of zymosan sup, and was inhibited by ibuprofen (20 microM), a cyclo-oxygenase inhibitor. Prostaglandins (PGs) detected in the perfusate were augmented with infusion of zymosan pellet. Opsonization of the zymosan pellet by serum (complement) enhanced the glycogenolytic response without a lag period, and with a concomitant enhancement of PG output. Correlations between glucose production and PGs were r = 0.832 (PGD2), r = 0.872 (PGF2 alpha), r = 0.752 (PGE2) and r = 0.349 (6-oxo-PGF1 alpha). The glycogenolytic response to non-boiled zymosan was delayed and the biphasic glycogenolytic response was not observed when mannan was infused first. NDGA mimicked the effects of the preinfusion of mannan, while ibuprofen had no effect on the non-boiled-zymosan-induced glycogenolysis. These results suggest: (1) that non-boiled zymosan stimulates glycogenolysis through a mannose receptor-dependent, but unidentified, pathway, (2) that zymosan sup induces glycogenolysis via mannose receptor activation through the production of peptide-LTs but not PAF, and (3) that zymosan pellet causes glycogenolysis through the production of prostanoids, which is enhanced in the presence of complement.
Transglutaminase catalyses the modification of glutamine side chains in the C-terminal region of bovine β-lactoglobulin
Authors: Coussons P J , Price N C , Kelly S M , Smith B , Sawyer L
Published: August 2015

The transglutaminase-catalysed incorporation of primary amines (putrescine and monodansylcadaverine) into bovine beta-lactoglobulin has been studied. In the presence of 1 mM-dithiothreitol between 1 and 2 mol of amine can be incorporated per mol of beta-lactoglobulin subunit. There is very little incorporation of amines in the absence of reducing agent. By isolating and sequencing the modified peptides, the sites of modification have been identified as Gln-159 (preferred) and Gln-155. C.d. has been used to study the structure of beta-lactoglobulin over a range of pH values and in the presence or absence of dithiothreitol. The results are discussed in terms of the X-ray-crystallographically determined structure of beta-lactoglobulin.