bluuebunny/crossref_metadata_2025 huggingface dataset:
Bovine gall-bladder mucin contains two distinct tandem repeating sequences: evidence for scavenger receptor cysteine-rich repeats
Authors: Nunes D P , Keates A C , Afdhal N H , Offner G D
Published: August 2015

Gall-bladder mucin is a densely glycosylated macromolecule which is the primary secretory product of the gall-bladder epithelium. It has been shown to bind cholesterol and other biliary lipids and to promote cholesterol crystal nucleation in vitro. In order to understand the molecular basis for mucin-lipid interactions, bovine gall-bladder mucin cDNAs were identified by expression cloning and were isolated and sequenced. The nucleotide sequences of these cDNAs revealed two distinct tandem repeating domains. One of these domains contained a 20-amino acid tandem repeating sequence enriched in threonine, serine and proline. This sequence was similar to, but not identical with, the short tandem repeating sequences identified previously in other mammalian mucins. The other domain contained a 127-amino acid tandem repeating sequence enriched in cysteine and glycine. This repeat displayed considerable sequence similarity to a family of receptor- and ligand-binding proteins containing scavenger receptor cysteine-rich repeats. By analogy with other proteins containing these cysteine-rich repeats, it is possible that, in gall-bladder mucin, this domain serves as a binding site for hydrophobic ligands such as bilirubin, cholesterol and other biliary lipids.
The interaction of coatomer with inositol polyphosphates is conserved in Saccharomyces cerevisiae
Authors: Ali N , Duden R , Bembenek M E , Shears S B
Published: August 2015

Coatomer is an oligomeric complex of coat proteins that regulates vesicular traffic through the Golgi complex and from the Golgi to the endoplasmic reticulum [Pelham (1994) Cell 79, 1125-1127]. We have investigated whether the binding of InsP6 to mammalian coatomer [Fleischer, Xie, Mayrleitner, Shears and Fleischer (1994) J. Biol. Chem. 269, 17826-17832] is conserved in the genetically amenable model Saccharomyces cerevisiae. We have isolated coatomer from S. cerevisiae and found it to bind InsP6 at two apparent classes of binding sites (KD1 = 0.8 +/- 0.2 nM; KD2 = 361 +/- 102 nM). Ligand specificity was studied by displacing 4.5 nM [3H]InsP6 from coatomer with various Ins derivatives. The following IC50 values (nM) were obtained: myo-InsP6 = 6; bis(diphospho)inositol tetrakisphosphate = 6; diphosphoinositol pentakisphosphate = 6; scyllo-InsP6 = 12; Ins(1,3,4,5,6)P5 = 13; Ins(1,2,4,5,6)P5 = 22; Ins(1,3,4,5)P4 = 22; 1-O-(1,2-di-O-octanoyl-sn-glycero-3-phospho)-D-Ins(3,4,5)P3 = 290. Less than 10% of the 3H label was displaced by 1 microM of either Ins(1,4,5)P3 or inositol hexakis-sulphate. A cell-free lysate of S. cerevisiae synthesized diphosphoinositol polyphosphates (PP-InsPn) from InsP6, but our binding data, plus measurements of the relative levels of inositol polyphosphates in intact yeast [Hawkins, Stephens and Piggott (1993) J. Biol. Chem. 268, 3374-3383], indicate that InsP6 is the major physiologically relevant ligand. Thus a reconstituted vesicle trafficking system using coatomer and other functionally related components isolated from yeast should be a useful model for elucidating the functional significance of the binding of InsP6 by coatomer.
Long-term reduction of amplified ornithine decarboxylase sequences in human myeloma cells
Authors: Wahlfors J , Myöhänen S , Korhonen V P , Alhonen L , Jänne J
Published: August 2015

(1) Human myeloma cell line Sultan, resistant to 20 mM difluoro-methylornithine (DFMO) owing to ornithine decarboxylase (ODC) gene amplification, was grown in the absence of DFMO for a period of 10 months. The gene copy number and methylation status of the ODC gene were monitored after withdrawal of DFMO. Moreover, levels of ODC mRNA, immunoreactive ODC protein, ODC activity and polyamine levels were recorded recurrently during the course of the study. (2) The results revealed that ODC gene copy number started to decrease after 4 weeks growth without DFMO, to a final level of less than 30% of the original gene dosage. The methylation status of the ODC gene, however, remained almost unaltered, displaying only a modest increase in methylation after 10 months without DFMO. The amount of ODC message dropped very rapidly to 75% of the original value, then started to decrease in a gene copy-number-dependent manner. The amount of ODC protein closely followed the levels of mRNA during the study, whereas the ODC activity, after a transient increase during the first week, decreased to half of the original level after 4 weeks. Between 6 and 16 weeks ODC activity stabilized to a fifth of the original value and no more changes were detected during the subsequent period of observation. (3) Due to the grossly elevated ODC enzyme activity, levels of putrescine and spermidine first peaked and then stabilized at 6 weeks after DFMO withdrawal. The final spermidine level was comparable with that of the parental Sultan cell line with only one copy of active ODC gene. However, putrescine content was strikingly elevated, being stabilized to a level that was 20 times higher than in parental cells. Spermine concentration was practically unchanged during the study. (4) According to the results obtained in this study, the abnormal level of ODC expression in human myeloma cells is suppressed partially at the level of transcription or post-transcriptionally, but it is not due to increased methylation of the gene. The major regulatory mechanism to compensate for a highly elevated ODC expression was modulation of the enzyme activity. After 10 months without DFMO, the cells still displayed about 20 times higher ODC activity and putrescine concentration than the myeloma cell line with a single copy of the ODC gene. They did not, however, show any signs of growth retardation or other features different from the parental cells.(ABSTRACT TRUNCATED AT 400 WORDS)
Inhibitors of signalling identify differential control processes responsible for selective effects of insulin on the expression of phosphoenolpyruvate carboxykinase and gene 33 in rat H4 hepatoma cells
Authors: Yang S H , Dickson A J
Published: August 2015

Gene 33 and phosphoenolpyruvate carboxykinase (PEPCK) present excellent model systems for the analysis of the differential control of hepatic gene expression by the insulin-regulated signal-transduction pathway(s). We have analysed the importance of specific components in the insulin-regulated transduction pathway(s) towards enhanced gene expression (gene 33) and inhibited gene expression (PEPCK) by examination of the influence of selective inhibitors. Rapamycin, which inhibits the 70 kDa S6 kinase (p70rsk) does not influence the actions of insulin on gene 33 or PEPCK; thus the kinase p70rsk appears to play no direct role in the regulation of expression of these two hepatic genes. Wortmannin, an inhibitor of phosphatidylinositol 3-kinase, differentiates between processes involved in insulin regulation of gene 33 and PEPCK mRNA expression. Although the actions of insulin on gene 33 expression are abolished by wortmannin, the actions of insulin on PEPCK expression are insensitive to wortmannin. The existence of wortmannin-sensitive and rapamycin/wortmannin-insensitive pathways for transducing insulin signals to factors controlling gene expression, and the differential actions on specific genes, presents an initial step towards deciphering the linkage between signalling components and selective control of gene expression.
Sulphate-activated phosphorylase b: the pH-dependence of catalytic activity
Authors: Zographos S E , Oikonomakos N G , Dixon H B F , Griffin W G , Johnson L N , Leonidas D D
Published: August 2015

The pH-dependence of sulphate-activated phosphorylase b has been studied in the direction of glycogen synthesis. The bell-shaped curve of the pH-dependence of the catalytic constant for the AMP-activated enzyme showed pK values of 6.1 and 7.3, but the curve for the enzyme activated by 0.9 M ammonium sulphate showed a drop of activity on the acid side at much higher pH values. Its bell was centred at pH 7.8 but it was too narrow to be characterized by only two pK values. The narrowness of the curve could be explained by positive co-operativity, but not its unusually steep acid side. We suggest that the fall on the acid side is due to more than one hydronation (addition of H+). The points can be fitted by a curve with two de-activating hydronations and a de-activating dehydronation having identical titration pK values of 7.5, and hence molecular values of 7.0, 7.5 and 8.0. If both 0.9 M ammonium sulphate and 5 mM AMP are added, the bell is as broad as with AMP alone, but is somewhat raised in pH optimum. The results are discussed in the light of new structural data from crystallographic studies on binary complexes of the enzyme.
Substrate-dependent utilization of the glycerol 3-phosphate or malate/aspartate redox shuttles by Ehrlich ascites cells
Authors: Grivell A R , Korpelainen E I , Williams C J , Berry M N
Published: August 2015

The rate of transfer of reducing equivalents from cytoplasm to mitochondria has been examined in Ehrlich ascites tumour cells incubated in the presence of lactate. The flux of reducing equivalents was determined from the rate of metabolism of reduced intermediates that are oxidized within the cytosol. The magnitude of the flux of reducing equivalents was dependent on both the concentration of added lactate and the presence of carbohydrate. The rate of flux was twice as great in the presence of glucose and four times as high when glucose and lactate were added together as when lactate was the only added substrate. Fructose was less effective than glucose in stimulating reducing equivalent flux. In the presence of glucose or fructose, there was a substantial accumulation of hexose phosphates, dihydroxyacetone phosphate and glycerol 3-phosphate. Rotenone, an inhibitor of NADH dehydrogenase, and amino-oxyacetate, which inhibits the malate/aspartate shuttle, were powerful suppressors of reducing equivalent flux from lactate as sole substrate, but were much less potent in the presence of carbohydrate. Antimycin substantially inhibited reducing equivalent flux from all combinations of added substrates, consistent with its ability to block oxidation of reducing equivalents transferred by both the malate/aspartate and glycerol 3-phosphate shuttles. The glycerol 3-phosphate shuttle represents around 80% of the maximum total observed activity but is active only while glycolytic intermediates are present to provide the necessary substrates of the shuttle. This Ehrlich ascites cell line has an essentially similar total reducing equivalent shuttle capacity to that of isolated hepatocytes.
Arsenite release on enzymic transformation of arsonomethyl substrate analogues: a potentially lethal synthesis by glycerol-3-phosphate dehydrogenase
Authors: Mutenda E K , Sparkes M J , Dixon H B F
Published: August 2015

The isosteric arsenical analogue of glycerol 3-phosphate, 3,4-dihydroxybutylarsonic acid, is a good substrate for rabbit muscle glycerol-3-phosphate dehydrogenase. Its oxidation is accompanied by release of arsenite. This release seems to be due to a spontaneous elimination of arsenite by 3-oxoalkylarsonic acids, as it is also observed in (1) the oxidation of 3-hydroxypropylarsonic acid by yeast alcohol dehydrogenase, (2) treatment of 3,4-dihydroxybutylarsonic acid with periodate and (3) nonenzymic transamination of the glutamate analogue 2-amino-4-arsonobutyric acid. Enzymic formation of 3-oxoalkylarsonic acids in cells can therefore be lethal, as arsenite is poisonous to most organisms because of its high affinity for dithiols such as dihydrolipoyl groups.
Phosphatidylinositol 3-kinase activity is required for early endosome fusion
Authors: Jones A T , Clague M J
Published: August 2015

The homotypic fusion between early endosomes from baby-hamster kidney cells is blocked by addition of the fungal metabolite wortmannin with an IC50 of approx. 15 nM. Over this concentration range, wortmannin has been regarded as a specific inhibitor of phosphatidylinositol (PI) 3-kinase. Further confirmation of the participation of a PI 3-kinase in the fusion reaction has been obtained by demonstrating a sensitivity to an additional, structurally unrelated, PI 3-kinase inhibitor, LY294002 [2-(4-morpholinyl)-8-phenyl-4H-1-benzopyran-4-one]. Assays constructed such that only the membranous component has been incubated with wortmannin show in vitro fusion to be sensitive to treatment with the drug. Assays in which only the cytosolic component has been treated with wortmannin also showed inhibition of in vitro fusion, but to a lesser extent. PI 3-kinase action almost certainly involves direct regulation of membrane fusion, as no vesicular intermediate has been identified, despite previous extensive morphological examination of in vitro endosome fusions.
The refolding of hen egg white riboflavin-binding protein: effect of protein disulphide isomerase on the reoxidation of the reduced protein
Authors: McClelland D A , McLaughlin S H , Freedman R B , Price N C
Published: August 2015

Hen egg white riboflavin-binding protein (RfBP) contains nine disulphide bonds. Provided these remain intact, the refolding of RfBP after incubation in 6 M guanidinium chloride is highly efficient with at least 95% of the binding activity regained within 3 min. Kinetic studies indicate that this regain consists of at least two phases. When the disulphide bonds of RfBP are reduced, reoxidation using a mixture of oxidized and reduced glutathione leads to less than 5% recovery of activity. However, if protein disulphide isomerase (PDI; EC 5.3.4.1) is present during the reoxidation nearly 50% activity can be regained, suggesting that PDI may play an important role in the maturation of RfBP in vivo.
On the substrate specificity of α-crystallin as a molecular chaperone
Authors: Das K P , Surewicz W K
Published: August 2015

alpha-Crystallin, the major protein of the ocular lens, acts as a molecular chaperone by preventing the thermal aggregation of proteins. However, in contrast with many other heat shock proteins, alpha-crystallin fails to protect proteins from aggregation during refolding reactions. Our results indicate that alpha-crystallin has substrate specificity different from other chaperones and recognizes specific non-native intermediates formed on the denaturation pathway only, with no affinity for intermediates formed on the refolding pathway.
Stimulatory antibody-induced activation and selective translocation of protein kinase C isoenzymes in human platelets
Authors: Wang F , Naik U P , Ehrlich Y H , Osada S , Ohno S , Kornecki E
Published: August 2015

A novel stimulatory monoclonal antibody (Mab) termed Mab.F11 induces granular secretion and subsequent aggregation of human platelets. Mab.F11 recognizes a unique 32 and 35 kDa protein duplex on the platelet membrane surface, called the F11 receptor; binding of Mab.F11 to its receptor results in increased intracellular phosphorylation of P47, the known protein kinase C (PKC) substrate pleckstrin. In order to determine whether the mechanism of action of Mab.F11 involves direct activation of PKC, two types of functional assays for measuring PKC activity were performed. Measurement of PKC activity in digitonin-permeabilized platelets revealed that Mab.F11 produced a rapid, 2-3 fold increase in the control value in the phosphorylation of the PKC peptide substrate, PKC(19-31) Ser25. The increase in PKC activity induced by Mab.F11 was found to be associated with the platelet membrane; a 1.6-fold control value increase in membrane PKC activity occurred rapidly, within 10 s of the addition of Mab.F11. The translocation from the cytoplasm to the membrane induced by Mab.F11 in PKC isoenzymes alpha and zeta was reversible, whereas translocation of the PKC isoenzymes delta, beta, eta' and theta was irreversible, with PKC levels remaining elevated in the membrane for at least 15 min. Taken together, our results demonstrate that in the initial stages of platelet activation by this stimulatory antibody, the enhanced membrane PKC activity reflects the presence of all six isoenzymes. At later stages, PKC activity is reflective of four isoenzymes. These results demonstrate that separate groups of PKC isoenzymes must be involved in different aspects of platelet activation. The long lag period and prolonged activation time of platelets by Mab.F11 renders this agonist most suitable for identifying the isoenzymes and their specific endogenous protein substrates involved in platelet secretion and aggregation induced by platelet membrane protein antibodies.
Characterization of rat spleen prostaglandin H d-isomerase as a sigma-class GSH transferase
Authors: Meyer D J , Thomas M
Published: August 2015

The prostaglandin H D-isomerase of rat immune accessory cells has been purified from spleen by a simple procedure, and its high specificity and activity [Urade, Fujimoto, Ujihara and Hayaishi (1987) J. Biol. Chem. 262, 3820-3825] have been confirmed in an assay coupled to prostaglandin H synthase. The enzyme also decreases the formation of 12[S]-hydroxy-5,8,10-heptadecatrienoic acid formed by the synthase in the presence of GSH and increases the overall rate of arachidonate oxidation. A partial amino acid sequence shows a strong relationship to GSH transferases of parasitic helminths and molluscs, indicating that it is the first example of a vertebrate sigma-class GSH transferase, and suggesting that certain helminth GSH transferases may be involved in prostaglandin synthesis.
Identification of the integrin α3β1 as a component of a partially purified A-system amino acid transporter from Ehrlich cell plasma membranes
Authors: McCormick J I , Johnstone R M
Published: August 2015

We have previously reported [McCormick and Johnstone (1988) Proc. Natl. Acad. Sci. U.S.A. 85, 7877-7881] the partial purification of the Na(+)-dependent A-system amino acid transporter from Ehrlich cell plasma membranes and have suggested that a 120-130 kDa peptide, a major component of the purified fraction [octyl glucoside (OG) extract], is involved in Na(+)-dependent amino acid transport. In the present study, N-terminal sequence analysis of the 120-130 kDa peptide revealed a sequence similar to that of the alpha 3 subunit of the integrin alpha 3 beta 1. The presence of alpha 3 beta 1 was confirmed by Western blots of the OG extract probed with anti-alpha 3 or -beta 1 antibodies. Western blots also showed that an antibody originally raised against the 120-130 kDa peptide crossreacts with both the alpha 3 and beta 1 integrin subunits. Co-purification of alpha 3 beta 1 and Na(+)-dependent transport activity suggested that the two activities might be associated. Evidence that alpha 3 plays a role in transport is shown by the fact that an antibody against human alpha 3, but not beta 1, removed transport activity (approximately 25% loss) from cholate-solubilized Ehrlich membranes. Further purification of OG extracts using concanavalin A and wheat-germ lectin columns resulted in the separation of transport activity from the bulk (but not all) of alpha 3 beta 1 integrin without loss of the transport activity. These results indicate that the integrin itself is not essential for amino acid transport. Reconstitution of a purified alpha 3 beta 1-depleted protein fraction showed high levels of Na(+)-dependent, alpha-methylaminoisobutyric-acid-inhibitable amino acid transport in proteoliposomes, whereas reconstituted integrin alone showed little transport activity. However, in the integrin-depleted fractions, high amino acid uptake occurred in K+ which compromised the accurate measurement of the Na(+)-dependent component of uptake. The data suggest that alpha 3 may be associated with the A-system transporter and may modulate the activity of this carrier. Moreover, transfection of K562 and RD cells with human alpha 3 and alpha 2 cDNA showed that the former but not the latter increased A-system transport, thus providing more direct evidence that alpha 3 may modulate A-system transport activity.
Mouse gastric mucin: cloning and chromosomal localization
Authors: Shekels L L , Lyftogt C , Kieliszewski M , Filie J D , Kozak C A , Ho S B
Published: August 2015

Mucins protect gastric epithelium by maintaining a favourable pH gradient and preventing autodigestion. The purpose of this study was to clone a mouse gastric mucin which would provide a foundation for analysis of mucin gene regulation. Mucin was purified from the glandular portion of gastric specimens and deglycosylated by HF solvolysis. Antibodies against native and deglycosylated mouse gastric mucin (MGM) were raised in chickens. Screening of a mouse stomach cDNA library with the anti-(deglycosylated MGM) antibody yielded partial clones containing a 48 bp tandem repeat and 768 bp of non-repetitive sequence. The 16-amino-acid tandem repeat has a consensus sequence of QTSSPNTGKTSTISTT with 25% serine and 38% threonine. The MGM tandem repeat sequence bears no similarity to previously identified mucins. The MGM non-repetitive region shares sequence similarity with human MUC5AC and, to a lesser extent, human MUC2 and rat intestinal mucin. Northern blot analysis reveals a polydisperse message beginning at 13.5 kb in mouse stomach with no expression in oesophagus, trachea, small intestine, large intestine, caecum, lung or kidney. Immunoreactivity of antibodies against deglycosylated MGM and against a synthetic MGM tandem repeat peptide was restricted to superficial mucous cells, antral glands and Brunner's glands in the pyloric-duodenal region. DNA analysis shows that MGM recognizes mouse and rat DNA but not hamster, rabbit or human DNA. The MGM gene maps to a site on mouse chromosome 7 homologous to the location of a human secretory mucin gene cluster on human chromosome 11p15. Due to sequence similarity and predominant expression in the stomach, the MGM gene may be considered a MUC5AC homologue and named Muc5ac.
Human dipeptidyl peptidase IV gene promoter: tissue-specific regulation from a TATA-less GC-rich sequence characteristic of a housekeeping gene promoter
Authors: Böhm S K , Gum J R , Erickson R H , Hicks J W , Kim Y S
Published: August 2015

The dipeptidyl peptidase IV gene encodes a plasma-membrane exopeptidase that is highly expressed in small intestine, lung and kidney. In order to better understand the mechanisms responsible for this tissue-specific expression we cloned, sequenced and functionally characterized the 5′-flanking region of the human dipeptidyl peptidase IV gene. The first 500 bases of the 5′-flanking sequence constituted an unmethylated CpG island, contained several Sp1-binding sites and lacked a consensus TATA box, all characteristics of gene promoters lacking tissue-specific expression. RNase-protection analysis using both small intestinal and Caco2 cell RNA indicated that the dipeptidyl peptidase IV transcript was initiated from no fewer than six major and 12 minor start sites. The 5′-flanking sequence also exhibited functional promoter activity in transient transfection experiments. Here, various lengths of the sequence were cloned upstream of a luciferase gene and introduced into cultured cells using lipofectin. A region located between bases -150 and -109 relative to the start of translation was found to be important for high-level promoter activity in both Caco2 and HepG2 cells. Moreover, Caco2 cells and HepG2 cells, which express high levels of dipeptidyl peptidase IV activity, exhibited much higher normalized luciferase activity after transfection than did 3T3, Jurkat or COS-7 cells, which have low enzyme levels. Sodium butyrate was found to increase both enzyme activity and normalized luciferase in HepG2 cells. Thus the dipeptidyl peptidase IV promoter possesses the ability to initiate transcription in a tissue-specific fashion in spite of having the sequence characteristics of a housekeeping gene promoter.
Proteoglycans in polarized epithelial Madin-Darby canine kidney cells
Authors: Svennevig K , Prydz K , Kolset S O
Published: August 2015

Madin-Darby canine kidney (MDCK) cells were cultured on polycarbonate filters to study the synthesis and sorting of proteoglycans in polarized epithelial cells. Two strains of MDCK cells were used. MDCK I cells resemble distal tubule epithelial cells, and MDCK II cells share some characteristics with proximal tubule cells. Both strains were grown to confluency and labelled with [35S]sulphate for 24 h. The apical and basolateral media and the cell fractions were harvested and analysed by DEAE ion-exchange chromatography. A large portion of the [35S]sulphate-labelled macromolecules bound strongly to the ion-exchange columns, and could be eluted in three distinct peaks. The latest eluting peak was demonstrated to contain almost exclusively chondroitin sulphate, whereas peak 2 contained mostly heparan sulphate, demonstrated by using chondroitinase ABC and nitrous acid (pH 1.5) respectively to depolymerize the [35S]glycosaminoglycan chains. Peak 1 contained negligible amounts of proteoglycans. Large differences could be observed in proteoglycan sorting in MDCK I and II cells. Strain I secreted approx. 67% of the proteoglycans to the apical side and 17% to the basolateral side. The cell fraction contained 17% of the proteoglycans after 24 h of labelling. In contrast, 19% of the proteoglycans were sorted to the apical side of MDCK II cells and 61% to the basolateral side, whereas the cell fraction contained 20%. Furthermore, the level of [35S]proteoglycan biosynthesis (apical and basolateral media and cell fraction total) was higher in MDCK I cells than in strain II. Based on the amount of material degraded by chondroitinase ABC and nitrous acid respectively, and the total amounts of [35S]proteoglycans recovered from the cells, it was calculated that the MDCK I strain synthesized approx. 56% chondroitin sulphate and 44% heparan sulphate. In contrast, the MDCK II strain synthesized 69% heparan sulphate and 31% chondroitin sulphate. To further identify the [35S]proteoglycans synthesized by MDCK I and II cells, antibodies against perlecan, versican and syndecan were used. The antibody against mouse syndecan did not cross-react with any of the proteoglycans produced in MDCK I or II cells. Both MDCK I and II cells expressed perlecan; 57-61% could be recovered from the basolateral fractions and 18-34% from the apical medium. Versican was also found in both MDCK I and II cells. Compared with perlecan, a larger percentage of versican (43-53%) was found in the cell fractions.
Relationship between the microsomal epoxide hydrolase and the hepatocellular transport of bile acids and xenobiotics
Authors: Honscha W , Platte H D , Oesch F , Friedberg T
Published: August 2015

Recently two different bile-acid carriers for the hepatocellular sodium-dependent uptake of taurocholate have been described. The first transport system was isolated and characterized by functional expression cloning in Xenopus laevis oocytes. The corresponding cDNA clone, named Ntcp for Na+/taurocholate co-transporting polypeptide, codes for a protein of 362 amino acids and shows no similarity to previously known sequences. The transport function of this carrier system is well documented by expression in Xenopus laevis oocytes and by transient and stably transfected cell lines. In addition, several lines of evidence implied that the well-known xenobiotic-metabolizing enzyme microsomal epoxide hydrolase (mEH, EC 3.3.2.3) is also able to mediate sinusoidal uptake of taurocholate. Furthermore, it was claimed that the same enzyme also mediates the uptake of the conjugated bile acid into the smooth endoplasmic reticulum (ER). No direct proof of the transport function of mEH by its heterologous expression has yet been published. In the present work we used a stable transfected cell line that expressed high levels of heterologous mEH for uptake studies of various bile acids and the loop diuretic bumetanide. The uptake of the conjugated bile acid taurocholate, of the non-conjugated bile acid cholate and of the organic anion bumetanide was measured in the transfected as well as in the non-transfected parental cell line. These organic anions represent the main substrates of the known transport systems for organic anions in the rat liver. The results show that the microsomal epoxide hydrolase is unable to transport taurocholate, cholate or bumetanide. Furthermore, Western-blot analysis revealed the expression of mEH in hepatoma tumor cell lines, which show no transport activity for these organic anions. These results show that it is unlikely that mEH can mediate the transport of these substrates.
Effects of hydration state on the synthesis and secretion of triacylglycerol by isolated rat hepatocytes. Implications for the actions of insulin and glucagon on hepatic secretion
Authors: Zammit V A
Published: August 2015

The effects of hepatocyte volume on the secretion of triacylglycerol were studied in order to test the suggestion that increases in the portal concentrations of osmolyte amino acids and metal ions during the prandial/early-absorptive phase may be involved in mediating the acute changes in glycerolipid metabolism observed in vivo [Zammit (1995) Biochem Soc. Trans. 23, 506-511]. Incubation of isolated rat hepatocytes with hypo-osmotic medium or in the presence of glutamine (in the presence or absence of leucine), conditions which gave an increase in cell water content of between 8 and 27%, resulted in a decrease in the rate of [14C]triacylglycerol (TAG) secretion when [14C]palmitate was used as substrate. The inhibition was proportional to the increase in cell water content. At low exogenous palmitate concentration (0.05 mM), the inhibition of [14C]TAG secretion was accompanied by a marked shift in the incorporation of label from TAG to phospholipid. In the presence of 0.5 mM palmitate this effect was attenuated, and in the presence of 1 mM palmitate it was abolished. Increased cell volume associated with incubation of hepatocytes with glutamine (in the presence or absence of leucine) also resulted in a decrease in the fraction of newly labelled TAG that was secreted into the medium. Decreased cell volume, achieved by incubation of hepatocytes with hyperosmotic medium (sufficient to decrease cell water content by approx. 9%) decreased overall [14C]TAG secretion, but did not affect the amount of label that was incorporated into phospholipid as a fraction of that incorporated into total glycerolipids. Cell shrinkage, however, diminished the fraction of newly labelled [14C]TAG that was secreted. When intracellular TAG was prelabelled with [3H]glycerol, it was found that cell shrinkage markedly inhibited (preformed) [3H]TAG secretion, whereas cell swelling did not affect this route of TAG secretion. The data are discussed in terms of the possible action of changes in cell hydration at the different loci at which hepatocyte TAG secretion is controlled, with reference to previous observations that both insulin and glucagon are able to inhibit TAG secretion in cultured rat hepatocytes and HepG2 cells.
Amino acid sequencing, molecular cloning and modelling of the chick liver class-theta glutathione S-transferase CL1
Authors: Hsiao C D , Martsen E O , Lee J Y , Tsai S P , Tam M F
Published: August 2015

Glutathione S-transferase CL1-2 heterodimers purified from 1-day-old chick livers were digested with Achromobacter proteinase I. The resulting fragments were separated for amino acid sequence analysis. Oligonucleotide probes were constructed based on sequence similarity to class-Theta glutathione S-transferases for PCR using a chicken liver cDNA library as template. A full-length clone (1725 bp) encoding a polypeptide comprising 261 amino acids was isolated. Including conservative substitutions, this protein has 70-73% sequence similarity with other mammalian class-Theta glutathione S-transferases. Based on known X-ray crystal structures of class-Alpha, -Mu and -Pi glutathione S-transferases, a model is constructed for the N-terminal 232 residues of CL1.
Evaluation of the extent of the binding site in human tissue kallikrein by synthetic substrates with sequences of human kininogen fragments
Authors: Del Nery E , Chagas J R , Juliano M A , Prado E S , Juliano L
Published: August 2015

We have synthesized internally quenched peptides spanning the Met379-Lys380 or Arg389-Ser390 bonds of human kininogen (hkng) that flank lysyl-bradykinin and have studied the kinetics of their hydrolysis by human tissue kallikrein. The kinetic data for the hydrolysis of the Met-Lys bond in substrates with an N-terminal extension showed that interactions up to position residue P10 contribute to the efficiency of cleavage. In contrast, there were no significant variations in the kinetic data for the hydrolysis of substrates with C-terminal extensions at sites P′4 to P′11. A similar pattern was observed for the cleavage of substrates containing an Arg-Ser bond because substrates extended up to residue P6 were hydrolysed with the highest kcat/Km values in the series, whereas those extended to P′11 on the C-terminal side had a lower susceptibility to hydrolysis. Time-course studies of hydrolysis by human and porcine tissue kallikreins of a Leu373 to Ile393 human kininogen fragment containing omicron-aminobenzoic acid (Abz) at the N-terminus and an amidated C-terminal carboxyl group Abz-Leu-Gly-Met-Ile-Ser-Leu-Met-Lys-Arg- Pro-Pro-Gly-Phe-Ser-Pro-Phe-Arg-Ser-Ser-Arg-Ile-NH2 (Abz-[Leu373-Ile393]-hkng-NH2) indicated that the cleavage of Met-Lys and Arg-Ser bonds in the same molecule occurs via the formation of independent enzyme-substrate complexes. The hydrolysis of Abz-F-R-S-S-R-Q-EDDnp [where EDDnp is N-(2,4-dinitrophenyl)ethylenediamine] and Abz-M-I-S-L-M-K-R-P-Q-EDDnp by human tissue kallikrein had maximal kcat/Km values at pH 9-9.5 for both substrates. The pH-dependent variations in this kinetic parameter were almost exclusively due to variations in kcat. A significant decrease in kcat/Km values was observed for the hydrolysis of Arg-Ser and Met-Lys bonds in the presence of 0.1 M NaCl. Because this effect was closely related to an increase in Km, it is likely that sodium competes with the positive charges of the substrate side chains for the same enzyme subsites.
Effects of aging on the synthesis of antithrombin-binding sites on heparin chains and heparan sulphate chains in the rat
Authors: Horner A A
Published: August 2015

[35S]Heparin proteoglycans were isolated from the skins and peritoneal mast cells of male rats aged 2 to 22 months. Their [35S]heparin chains were separated on antithrombin-agarose into fractions with high and low affinities for antithrombin. In skin, the proportion of 35S-labelled high-affinity heparin chains declined from 23% at 2 months to 8% at 12 months and did not change significantly between 12 and 22 months. In peritoneal mast cells, the proportion of 35S-labelled high-affinity heparin chains increased from 14% at 2 months to 21% at 4 months and then did not vary significantly until 15 months of age. By 21 months a consistent and significant decline to 8% occurred. The structures of high-affinity heparin proteoglycans did not change with age. Their decreased proportions, without change in their structure, may indicate that they are produced by a unique subset of mast cells, the proportion of which declines with age. [35S]heparan sulphate chains were isolated from skins and brains of rats in the same age range and fractionated on antithrombin-agarose. There were no significant variations in the proportions of 35S-labelled high-affinity heparan sulphate chains in skin (10%) or brain (24%) between 4 and 22 months of age.
The accumulation and compartmentalization of isometamidium chloride in Trypanosoma congolense, monitored by its intrinsic fluorescence
Authors: Wilkes J M , Peregrine A S , Zilberstein D
Published: August 2015

Interaction of the trypanocide isometamidium chloride with components of Trypanosoma congolense results in characteristic shifts in the intrinsic fluorescence of the drug. The specificity of this interaction was investigated by analysing the effects of various physicochemical manipulations on its fluorescence properties. The characteristic shifts involved a preferential increase in the intensity of one emission peak over the other, resulting in a systematic increase in the ratio of fluorescence intensities. These effects were apparently due to constraints on fluorophore free rotation in the solution (that is, viscosity). Purified DNA produced similar effects in a saturable manner displaying high affinity for the drug, indicating that the constraint involves binding of the drug to high-affinity binding sites within the DNA. Such binding sites were demonstrated in lysates derived from trypanosomal cells. The binding sites were associated with macromolecular species (M(r) > 12000), and were partly disrupted by thermal denaturation and proteolysis. Treatment with DNase 1 produced high levels of disruption of the binding sites (> 85%), indicating an involvement of DNA in the binding. BSA demonstrated weak non-specific binding of the drug. Entry of drug into live trypanosomal cells (monitored by 14C-labelled drug uptake) was paralleled by fluorescence shifts observed under comparable conditions of drug concentration and buffer conditions. Both systems (fluorescence shifts and accumulation of labelled drug) indicated the presence of a saturable membrane transporter with high affinity for the drug. We conclude that monitoring the fluorescence shifts of isometamidium constitutes a sensitive and highly specific probe for entry of the drug into trypanosomal cells, thereby enabling resolution of the transport events involved.
Methodology for assaying iodide conductance in proteoliposomes: specific induction by thyroid membrane protein
Authors: Golstein P E , Sener A , Beauwens R
Published: August 2015

A sensitive assay is developed to assess the existence of an iodide channel in a fraction of solubilized membrane proteins. This step is critical when considering various procedures for purification of this channel. Sodium cholate is used as a detergent as it does not denature the iodide channel. A simple and rapid method involving gel-filtration chromatography is used simultaneously to remove the detergent and to adjust the buffer composition, before protein insertion into liposomes. The presence of an iodide channel is investigated by measuring the iodide conductance of these proteoliposomes at 4 degrees C. An outward iodide gradient is set up across the proteoliposomal membrane by anion-exchange chromatography, allowing uptake of radiolabelled iodide. This uptake is conductive as it is abolished by valinomycin in the presence of potassium. It is specifically mediated by a thyroid plasma-membrane protein inserted into liposomes, as its denaturation before insertion totally abolished uptake. It was observed only within a well-defined fraction of thyroid membrane proteins collected by size-exclusion chromatography (molecular mass between 100 and 200 kDa). Furthermore, it was not observed with other membrane proteins such as ileal brush-border-membrane proteins or bacteriorhodopsin. Like many anion channels, this conductance was also inhibited by N-phenylanthranilic acid. Optimization of the assay is described, validating the measurement of conductive iodide uptake at 30 s by proteoliposomes reconstituted in a ratio of 10 micrograms of protein to 90 micrograms of lipid, with an outward iodide gradient (KI 15 mM inside and 1 microM outside). This assay provides a test of the biological activity of the iodide channel at each step of the purification; it can be applied to any anionic channel.
Antisense oligodeoxynucleotide inhibition of δ protein kinase C expression accelerates induced differentiation of murine erythroleukaemia cells
Authors: Pessino A , Passalacqua M , Sparatore B , Patrone M , Melloni E , Pontremoli S
Published: August 2015

The potential regulatory role of delta protein kinase C (delta PKC) in murine erythroleukaemia cell differentiation was studied by using antisense oligodeoxynucleotides targeting the translation initiation region of mouse delta PKC mRNA. Cell treatment with antisense oligonucleotides, at a concentration of 20 microM, followed by hexamethylenebisacetamide induction, produced a specific 2-fold increase in the differentiation rate of both slowly and rapidly differentiating murine erythroleukaemia cell clones. Cell permeabilization by a cationic lipid resulted in a decrease of one order of magnitude in the amounts of antisense oligonucleotides necessary to elicit the maximal response, and accelerated the kinetics of the stimulatory effect. These changes in murine erythroleukaemia cell differentiation rates, observed in both cell clones, were associated with 60% and 50% decreases, respectively, in delta PKC immunoreactive protein in slowly and rapidly differentiating cells. The present results indicate strongly that basal levels of delta PKC in murine erythroleukaemia cells are essential in regulating the initial differentiation rate of these cells in response to chemical induction, and provide further evidence that this PKC isoform plays a fundamental role in maintaining the undifferentiated phenotype of murine erythroleukaemia cells.
Differential regulation of cyclin A, cyclin B and p21 concentrations in a growth-restricted human fibroblast cell line
Authors: Tsao Y P , Kuo S W , Li S F , Liu J C , Lin S Z , Chen K Y , Chen S L
Published: August 2015

When the culture temperature was shifted from 35 degrees C to 39 degrees C, human fibroblasts immortalized by the temperature-sensitive simian virus 40 T antigen became larger and acquired the morphological characteristics of senescent fibroblasts. After culture at 39 degrees C for 48 h, most cells had ceased to proliferate. A rapid depletion of cells with S-phase DNA content was observed after the temperature shift. To elucidate the mechanism governing this rapid arrest of proliferation, we studied the expression of genes involved in the regulation of cell cycle progression. Cyclin A, cyclin B and p34cdc2 concentrations were not changed during growth restriction, whereas p21 was rapidly induced in these growth-restricted cells. Transient expression of exogenous p21 in cells cultured at 35 degrees C led to growth restriction and morphological changes characteristic of senescence. Furthermore, we studied the reversibility of growth restriction induced by the temperature increase. The results showed that senescent morphology and growth arrest were not reversible. In these cells the p21 concentration remained high and p34cdc2 remained undetectable. This indicates that p21 accumulation might be responsible for the maintenance of senescence. Our findings provide information on the use of growth restriction of immortalized fibroblasts induced by a temperature shift as a model system to study senescence.
Early events in erythroid differentiation: accumulation of the acidic peroxidoxin (PRP/TSA/NKEF-B)
Authors: Rabilloud T , Berthier R , Vinçon M , Ferbus D , Goubin G , Lawrence J J
Published: August 2015

The acidic peroxidoxin [also named thiol-specific antioxidant protein (TSA) or protector protein (PRP)], which plays a role in the response against oxidative stress, is one of the major proteins of red blood cells. In this work, we show that this protein is induced at early stages of erythroid differentiation prior to haemoglobin accumulation, which suggests that it may play a role at the erythroblast stage, where haemoglobinized, nucleated and genetically active cells are submitted to a maximally dangerous oxidative stress. The early accumulation of this protein has been demonstrated both on transformed cell systems and on normal differentiating human erythroid cells. This suggests that this protein may play an important role in the differentiation of the erythroid cells.
Potentiated bradykinin-induced increase of 1,2-diacylglycerol generation and phospholipase D activity in human senescent fibroblasts
Authors: Meacci E , Vasta V , Faraoni P , Farnararo M , Bruni P
Published: August 2015

1. The comparative study of the effect of bradykinin (BK) in young and old IMR-90 human fibroblasts shows that old cells are characterized by a reduced increase in 1,2-diacylglycerol (1,2-DAG) generation upon stimulation after short-term treatment and a significant higher increase after long-term agonist treatment. BK-induced activation of phospholipase D (PLD), the major enzyme involved in sustained 1,2-DAG generation, was 2.5-fold higher in old cells, strongly suggesting that it is involved in the potentiated increase of 1,2-DAG formation. The increased activation of PLD by BK in old cells was specific, since in parallel experiments the effect of thrombin was not significantly different in young and old cells. PLD activity in old cells was only reduced by down-regulation of protein kinase C (PKC) activity, in contrast to what was observed in young cells where it was completely abolished. This indicates that the enzyme activity in old cells was partially PKC-independent. BK was also able to increase the release of [14C]ethanolamine, a water-soluble product of hydrolysis of phosphatidylethanolamine (PtdEtn), through PLD activation in young and old cells. The BK effect was significantly higher in old cells and, very likely, PKC-independent, since phorbol 12-myristate 13-acetate failed to induce PtdEtn hydrolysis. 2. The present results indicate that the PLD/1,2-DAG pathway is specifically potentiated by BK in old fibroblasts, demonstrating that the formation of positive effectors of PKC activation is not necessarily decreased in cellular senescence. It remains to be established whether the increased generation of DAG upon BK stimulation plays any role in the altered PKC signalling pathway which characterizes old fibroblasts.
Constitutive nuclear NFκB/rel DNA-binding activity of rat thymocytes is increased by stimuli that promote apoptosis, but not inhibited by pyrrolidine dithiocarbamate
Authors: Slater A F G , Kimland M , Jiang S A , Orrenius S
Published: August 2015

Rat thymocytes spontaneously undergo apoptotic death in cell culture, and are also sensitive to the induction of apoptosis by various stimuli. We show that unstimulated thymocytes constitutively express a p50-containing nuclear factor kappa B (NF kappa B)/rel DNA-binding activity in their nuclei. When the cells were fractionated by density-gradient centrifugation this activity was found to be most pronounced in immature CD4+8+ thymocytes, the cell population that undergoes selection by apoptosis in vivo and that is most sensitive to external inducers of apoptosis in vitro. The intensity of the NF kappa B/rel protein-DNA complex was significantly enhanced 30 min after exposing thymocytes to methylprednisolone or etoposide, two agents well known to induce apoptosis in these cells. Expression of this DNA-binding activity therefore correlates with the subsequent occurrence of apoptosis. By analogy to other systems, it has been suggested that antioxidants such as pyrrolidine dithiocarbamate (PDTC) inhibit thymocyte apoptosis by preventing the activation of an NF kappa B/rel transcription factor. However, we have found that etoposide induces a very similar enhancement of the NF kappa B/rel DNA-binding activity in the presence or absence of PDTC, despite a pronounced inhibition of apoptotic DNA fragmentation in the former situation. Dithiocarbamates therefore do not exert their anti-apoptotic activity in thymocytes by inhibiting the activation of this transcription factor.