bluuebunny/crossref_metadata_2025 huggingface dataset:
The properties of β-galactosidases (Escherichia coli) with halogenated tyrosines
Authors: Ring Mark , Huber Reuben E.
Published: December 2009

An Escherichia coli tyrosine auxotroph (MR1) with an inducible lacZ was generated by mutagenesis. Of several tyrosine derivatives tested, only m-fluorotyrosine supported the growth of this mutant and allowed synthesis of active β-galactosidase. The pH profiles of the β-galactosidase that was obtained when this mutant was grown on m-fluorotyrosine (81.5% of the tyrosine was replaced by m-fluorotyrosine) indicated that a tyrosine may be acting as a general acid–base catalyst and that it (or another tyrosine with the same pKa) may be involved in substrate binding. Inactivation of normal β-galactosidase by treatment with lactoperoxidase in the presence of I− did not affect affinity-column binding, but incubation of this iodinated β-galactosidase with chymotrypsin caused a rapid degradation of a portion of the treated enzyme equal to the portion of the activity that was lost. A study with 125I− showed that the rapid degradation was mainly confined to iodinated molecules of enzyme. These studies indicate that iodination of β-galactosidase does not affect binding ability, but causes the enzyme to lose catalytic activity and become susceptible to chymotryptic action. Chloroperoxidase also caused rapid inactivation of normal β-galactosidase in the presence of Br− or I−, but there was a lag followed by a slow inactivation in the presence of CI−.Key words: β-galactosidase, tyrosine, halogenation, lactoperoxidase, mechanism.
Molecular species of glycerophospholipids and diacylglycerols of cultured SH-SY5Y human neuroblastoma cells
Authors: Liepkalns Vis A. , Myher John J. , Kuksis Arnis , Leli Ubaldo , Freysz Nathalie , Hauser George
Published: December 2009

Total lipid extracts were obtained from SH-SY5Y human neuroblastoma cells grown to confluency in mycoplasma-free 10% fetal calf serum. The major glycerophospholipid classes and free diacylglycerols (DAG) were isolated and quantitated by silicic acid and DEAE-cellulose column and thin-layer chromatography. The choline (CGPL), ethanolamine (EGPL), serine (SGPL), and inositol (IGPL) glycerophospholipids were converted to the corresponding diradylglycerols by phospholipase C. The molecular species of the diradylglycerols were determined by capillary gas–liquid chromatography of the trimethylsilyl or t-butyldimethylsilyl ethers. The CGPL was rich in the oligoenoic species and IGPL was rich in the polyenoic species, especially the 18: 0–20: 4(n–6). The EGPL contained 30–40% diacyl, 60–64% alkenylacyl, and 1–3% alkylacyl species, which were also rich in polyenoic derivatives. Small amounts of alkenylacyl species were detected also in CGPL. The cellular DAG possessed a molecular species composition halfway between those of the DAG moieties of CGPL and IGPL. The cells grown in the presence of 10% calf serum exhibited great variability in the content of 20: 3(n–9) fatty acid, which was found to substitute for the 20: 4(n–6) acid in the molecular species with 18: 0 in both IGPL and DAG. The 20: 3(n–9) was largely absent from the CGPL, but occurred also in EGPL, where it was preferentially associated with 18: 0 in the diacyl but with 18: 1 in the alkylacyl and alkenylacyl species. The detailed documentation of molecular species of glycerophospholipids of the neuroblastoma cells offers new opportunities for identification of the source of free DAG released in transmembrane signalling.Key words: gas–liquid chromatography; alkylacylglycerols; alkenylacylglycerols; choline, ethanolamine, and inositol glycerophospholipids.
The influence of hydrostatic pressure on the distribution of histone mRNA in HeLa cells
Authors: Symington Alison L. , Zimmerman Selma , Zimmerman A. M.
Published: December 2009

Hydrostatic pressure and HeLa S3 cells were used (as a model system) to investigate the relationship of the cytoskeleton and histone gene expression. Exposure of HeLa S3 cells to hydrostatic pressure of 1000 – 10 000 psi (6.89 × 103 – 6.89 × 104 kPa) disrupts the cytoskeleton and reduces H1 and core histone mRNA and actin mRNA levels as determined by hybridization to specific DNA probes. Soluble and insoluble cell fractions were isolated from HeLa cells after lysis in Triton X-100 buffered with PIPES and being subjected to low-speed centrifugation. The insoluble fraction was designated the cytoskeletal fraction. At atmospheric pressure, 76% of H4 histone mRNA is associated with the cytoskeletal fraction and 24% of the H4 histone mRNA is in the soluble fraction. At 6000 and 10 000 psi for a duration of 10 min, H4 mRNA levels in the cytoskeletal fraction were reduced to 52 and 41%, respectively. The reduction of mRNA in the cytoskeletal fraction is accompanied by a corresponding increase of mRNA in the soluble cell fraction. The other core (H2A, H2B, and H3) and H1 histone mRNA transcripts exhibited similar sensitivity to pressure treatment. The effects of pressure on histone gene regulation may be mediated through alteration of mRNA–cytoskeleton association.Key words: cytoskeleton, HeLa cells, histone mRNA, hydrostatic pressure.
The electrical and spectroscopic properties of planar asymmetrical membranes incorporating chlorophyll a and plastoquinone-9. Influence of surface charges on electron transfer
Authors: Robert Sylvain , Tancrède Pierre
Published: December 2009

We studied the influence of surface charges on the efficiencies of electron transfer between a donor molecule, chlorophyll a (Chla), and an acceptor molecule, plastoquinone-9 (PQ-9), asymmetrically incorporated into a phospholipid matrix built from phosphatidylethanolamine, phosphatidylserine, and dimethyldistearylammonium bromide. Membrane conductance and capacitance measurements, as well as fluorescence emission experiments, were performed on bilayers containing positive or negative surface charges. The conductance of the bilayers showed important increases upon illumination of the Chla, this effect being observed only when both the donor and the acceptor molecules were present within the bilayer. This suggested that an electron transfer between Chla and PQ-9 occurred. The same kind of behaviour was observed with the membrane capacitance, but the amplitude of the effect was smaller. The results showed that an electric field gradient favorably oriented to promote electron transfer from Chla to PQ-9 maximized the electron transfer between these two molecules. However, both the membrane resistance and capacitance were permanently modified when illumination was stopped. On the other hand, the fluorescence results showed that for the range of surface charges covered, the position of the maximum of absorption was found unchanged around 675 nm and the intensity of fluorescence was almost constant, of the order of 7 × 106 – 8 × 106 photons∙s−1. This suggested that Chla was embedded as microdomains within the bilayer. The results presented here were also compared with what is known on the organization of the donor and acceptor molecules within the reaction centres of photosynthetic bacteria.Key words: Montal–Müeller membranes, asymmetrical membranes, electrical properties, resistance, capacitance, electron transfer, fluorescence, chlorophyll a.
Endogenous lipids of the earthworm Lumbricus terrestris
Authors: Albro Phillip W. , Corbett Jean T. , Schroeder Joanna L.
Published: December 2009

Earthworms (Lumbricus terrestris) were given [1-14C]-labeled palmitic acid by gavage on days 0 and 3, and sacrificed on day 7. The distribution of label among lipid classes indicated that glycerides, sterol esters, cerebrosides, sulfatides, phosphatidylethanolamine, phosphatidylserine and (or) phosphatidylinositol, phosphatidylcholine, and sphingomyelin turn over in, or are synthesized by, the earthworm. Free fatty acids still had the highest specific radioactivity of any lipid class at the end of the experiment. Incorporation of label into sterol and hydrocarbon fractions was insignificant and there was no detectable label incorporated into gangliosides. Phosphatidylethanolamine apparently turned over quite slowly compared with other lipid classes, while the cerebroside fraction became highly labeled. Elongation of palmitic acid to stearate and oxidation to CO2 occurred extensively, but there was no evidence for desaturation.Key words: earthworm, Lumbricus, lipids, gangliosides, metabolism.
Inhibition of α-adrenergic responses in the rat liver by lipophilic K+ channel blockers or depolarizing Cl− gradients. Evidence for a potential-sensitive step in the signal transduction path
Authors: Hill Ceredwyn E. , Ajikobi David O.
Published: December 2009

To study the role of K+ channels and membrane potential in α-adrenergic responses of the rat liver, lipophilic K+ channel blockers quinidine and 4-aminopyridine were used or external Cl− was replaced with gluconate, an impermeant ion. Glucose release, O2 uptake, portal pressure, and K+ flux were measured in the isolated perfused liver. The α-agonist phenylephrine caused biphasic changes in each parameter, a fast transient followed by sustained elevated responses. Infusion of 5 mM 4-aminopyridine, 0.1 mM quinidine, or gluconate prior to phenylephrine inhibited each parameter, with the greatest inhibition occurring during the second phase. A similar pattern was seen with 2 mM EGTA. This contrasts with the full inhibition of all responses following exposure to the α-antagonist phentolamine. Infusion of each inhibitor at the peak of the sustained phase inhibited all responses. Phenylephrine-stimulated release of K+ was augmented in the presence of EGTA and was inhibited by 4-aminopyridine or quinidine. In contrast, β-adrenergic stimulation of glucose release and K+ flux were not affected by the K+ channel blockers. Phenylephrine-stimulated glucose release from hepatocyte suspensions decreased by about 50% in the presence of 4-aminopyridine, EGTA, or gluconate. The results are discussed in terms of a potential role for K+ channels in α-adrenergic signal transduction in the liver.Key words: perfusion, hepatocyte, glycogenosis, haemodynamics, pinacidil.
Ontogeny of hepatocyte proliferation inhibitor activity during human liver development and its effect on cell proliferation in in vivo and in vitro studies
Authors: Devi B. Geetha , Habeebullah C. M. , Gupta P. D.
Published: December 2009

Ontogeny of hepatocyte proliferation inhibitory (HPI) activity was studied during human liver development. HPI activity was first noticed in the cytosolic fraction of 20-week-old fetal liver and thereafter it began to increase with the liver maturation. An inverse correlation between the ontogeny of HPI activity and mitotic counting of the hepatocytes during human liver development was established. The crude HPI fraction from fetal and adult human liver inhibited the incorporation of [3H]thymidine into the DNA of 2-day-old rat liver and HepG2 cells and the inhibitory effect was directly proportional to the age of the HPI source. Prolonged exposure of HepG2 cells to the HPI fraction was also found to be cytotoxic. Preliminary characterization of the crude HPI fraction revealed an age-dependent increase in proteins of 18, 28, and 60 kilodaltons following silver chloride staining of sodium dodecyl sulfate - polyacrylamide gel electrophoresis and densitometric scanning.Key words: human liver, hepatocyte proliferation inhibitor, ontogeny, sodium dodecyl sulfate - polyacrylamide gel electrophoresis, HepG2 cells, cytotoxic.
Reconstitution of rabbit sarcoplasmic reticulum calcium ATPase in a series of phosphatidylcholines containing a saturated and an unsaturated chain: suggestion of an optimal lipid environment
Authors: Matthews Paul L. J. , Bartlett Eileen , Ananthanarayanan V. S. , Keough Kevin M. W.
Published: December 2009

The calcium-dependent ATPase from sarcoplasmic reticulum of rabbit has been purified and reconstituted in dispersions containing pure phosphatidylcholines. Each phosphatidylcholine (PC) had palmitate (16:0) at the sn-1 position of glycerol and stearate (18:0), oleate (18:1), linoleate (18:2), arachidonate (20:4), or docosahexaenoate (22:6) at the sn-2 position. The activities and activation energies of the enzyme indicated that the best enzyme function occurred when 16:0–18:1 PC or 16:0–18:2 PC was the lipid in which the ATPase was embedded. Circular dichroism measurements made as a function of temperature suggested that the protein in 16:0–18:0 and 16:0–18:1 PC behaved most like sarcoplasmic reticulum or purified ATPase. The results suggest that there may be an optimal lipid environment for the ATPase which is provided by 16:0–18:1 PC and 16:0–18:2 PC, the two most common lipids of the sarcoplasmic reticulum.Key words: lipid–protein interactions, adaptation, mixed acid lipids.
Effect of chicken lysozyme signal peptide alterations on secretion of human lysozyme in Saccharomyces cerevisiae
Authors: Tsuchiya Yoshinori , Fujisawa Hirofumi , Nakayama Ken-Ichi , Nagahora Hitoshi , Jigami Yoshifumi
Published: December 2009

To investigate the structural requirements and functions of the N-terminal and the C-terminal regions of the chicken lysozyme signal peptide, the amino acids of each region were altered. The replacement of Gly(−1) and Leu(−2) with Pro(−1) and Ala(−2) or Val(−2), respectively, resulted in the complete shift of the cleavage site from position −1 to −2 in yeast (Saccharomyces cerevisiae). This shows that the introduction of a turn-promoting residue like Pro makes it possible to control the cleavage site of the signal peptide. Deletion of the positive charge and introduction of a negative charge in the N-terminal region decreased the lytic activity of secreted human lysozyme (HLY) and processing efficiency of preHLY, but the length and additional positive charge in this region had little influence. This suggests that the length of the N-terminal region scarcely influences the function of the signal peptide and that this region possibly interacts with the endoplasmic reticulum membrane to initiate the translocation of preprotein, similar to prokaryotic signal peptide. However, it needs only minimum positive charge for its function.Key words: N-terminal region, signal peptide, cleavage site, human lysozyme, yeast.
Increased phosphatidic acid and decreased lysophosphatidic acid in response to thrombin is associated with inhibition of platelet aggregation
Authors: Gerrard Jon M. , Robinson Pauline , Narvey Michael , McNicol Archibald
Published: December 2009

Thromboxane A2, produced from the arachidonic acid released from platelet phospholipids by phospholipase A2, stimulates platelet aggregation. It remains unresolved whether additional products of platelet phospholipase A2 might promote aggregation. To address this question, we have used aspirin-treated platelets to block thromboxane A2 formation and studied the influence of the phospholipase A2 inhibitor U10029A on platelet aggregation and secretion in response to thrombin. U10029A at 100 μM markedly inhibited platelet aggregation, but had no effect on platelet secretion. Since this concentration of U10029A effectively blocked lysophosphatidic acid (LPA) formation, LPA was added and found to substantially reverse the inhibitory effect of U10029A in these platelets. Furthermore, the action of U10029A was not due to inhibition of phosphatidate phosphohydrolase because U10029A, unlike propranolol, did not inhibit this enzyme. Although it is not possible to conclusively rule out an effect of U10029A in addition to its inhibition of phospholipase A2, our results reveal that a product of phospholipase A2 other than thromboxane A2 is important for platelet aggregation, but not for secretion in response to thrombin. Our data suggest that this product is LPA. Since the amount of phosphatidic acid (PA) increased dramatically concurrent with inhibition of platelet aggregation, it is safe to conclude that PA has no direct role to promote platelet aggregation in response to thrombin.Key words: lysophosphatidic acid, phosphatidic acid, phospholipase A2, human platelet.
Lithium preserves F-actin from the disarrangement induced by either DNase I or cytochalasin D
Authors: DalleDonne Isabella , Milzani Aldo , Fascio Umberto , Ratti Antonia , Colombo Roberto
Published: December 2009

Light scattering at 546 nm, which is mainly related to the presence of rodlike particles longer than 50 nm, showed that Li+ accelerates the formation of actin filaments. Intermolecular cross-linking with N,N′-1,4-phenylene-bismaleimide proved that the observed enhancement in the light-scattering intensity is caused by the increase in the concentration of actin oligomers, which gradually elongate to form longer filaments. DNase-I-related F-actin disassembly was reduced in the presence of lithium ions, as demonstrated by fluorimetric and viscometric experiments. Li+–F-actin showed an apparently similar behaviour when exposed to cytochalasin D. We confirm that Li+ acts on actin polymerization by stabilizing actin nuclei and polymers. The stabilization of cytoskeletal polymers really appears as one of the mechanisms by which lithium ions influence some of the cell activities.Key words: lithium, F-actin stability, cytochalasin D, DNase I.
Glutathione synthetase from the fission yeast. Purification and its unique heteromeric subunit structure
Authors: Nakagawa Chiaki W. , Mutoh Norihiro , Hayashi Yukimasa
Published: December 2009

Glutathione (GSH) synthetase (EC 6.3.2.3) was purified from the fission yeast Schizosaccharomyces pombe L972h− and from the GSH synthetase deficient mutant MN101/pYS41, which harbors a plasmid containing the GSH synthetase gene of the fission yeast. GSH synthetase is expressed at 10 times higher the amount in MN101/pYS41 than in wild-type L972 h−. The purified enzyme gave a single band on polyacrylamide gel electrophoresis in the absence of sodium dodecyl sulfate (native PAGE). The molecular weight of this enzyme was determined to be 1.2 × 105 by Sepharose CL-6B gel filtration. Polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate (SDS-PAGE) revealed that this enzyme was composed of two kinds of subunits, A (Mr = 33 × 103) and B (Mr = 26 × 103), and existed as a heterotetramer (A2B2). The enzyme purified from the wild-type fission yeast, which did not harbor the plasmid, showed the same electrophoretic mobilities on both native PAGE and SDS–PAGE and similar catalytic properties under standard conditions. This enzyme is most active at 45 °C and pH 8.0–8.5 with 20 mM Mg2+ + 10 mM ATP and 50 mM K+. The strict requirement for the monovalent cation is rather specific for the enzymes from yeasts. The presence of sugar components in the enzyme is also observed, similar to that in the rat kidney enzyme.Key words: Schizosaccharomyces pombe, glutathione synthetase, heteromeric subunit structure.
Identification and localization of Giα3 in the clonal adipocyte cell lines HGFu and Obl7
Authors: Cadrin Monique , McFarlane-Anderson Norma , Bégin-Heick Nicole
Published: December 2009

The HGFu and Ob17 cell lines, derived from adipose tissue of lean (+/?) and ob/ob mice, respectively, express several G-protein peptides. Investigation of the expression and subcellular localization of the Giα3 subunit showed that this peptide is associated with the Golgi apparatus. These findings indicate a role for this subunit in vesicular traffic and are in agreement with the view of the adipocyte as a secretory cell.Key words: preadipocytes, differentiation, immunofluorescence, confocal microscopy, obesity, Golgi system.
MAP kinases from bovine brain: purification and characterization
Authors: Childs Timothy J. , Mak Alan S.
Published: December 2009

We have purified 42- and 44-kilodalton (kDa) isoforms of the mitogen-activated protein (MAP) kinase family from bovine brain. The kinases were assayed with myelin basic protein as the substrate and detected by anti-sea star p44mpk antibody. Purification was achieved using phenyl-Sepharose, polylysine–agarose, hydroxylapatite, and Mono-Q column chromatography. Both myelin basic protein and smooth muscle caldesmon, but not histone H1, served as good substrates. Based on chromatographic behaviors and specific activities toward myelin basic protein, it is likely that the 42-kDa brain isoform is similar to that of brain tau kinase. The 44-kDa enzyme, however, is a novel brain MAP kinase isoform not reported previously. Although it has been demonstrated that p44mpk can be activated in vitro through phosphorylation by the tyrosine kinase p56lck, neither of the brain kinases were significantly stimulated by the tyrosine kinases p56lck, p56lyn, or p59fyn. However, based on antibody cross-reactivity, a MAP kinase kinase is present in the crude brain extract. Both brain MAP kinases were capable of autophosphorylation which occurred, at least in part, on tyrosine residues. However, only the 44-kDa isoform showed a significant degree of coincident activation.Key words: brain, MAP kinase, purification.
Temperature and phenylmethylsulfonyl fluoride sensitive loss of uncoupling protein in isolated brown adipose tissue mitochondrial membranes
Authors: Desautels M. , Dulos R. A.
Published: December 2009

When a membrane suspension prepared from isolated rat brown fat mitochondria was incubated at 37 °C for 4 h, a loss of uncoupling protein (UCP) immunoreactivity was observed on Western blots. Analysis of [3H]GDP-binding characteristics to UCP in isolated membranes also showed a significant reduction in Bmax without significant effect on Kd. The loss of UCP was not due to protease contamination from lysosomes or mast cell granules, since loss of UCP was still observed when mitochondria were treated with digitonin to lyse lysosomes prior to membrane preparation and when mitochondria were isolated from rats injected with compound 48/80 to degranulate mast cells. Furthermore, loss of UCP was observed at alkaline pH and was not affected by inhibitors of lysosomal enzymes. Loss of UCP immunoreactivity was markedly reduced when membranes were incubated at 4 °C or in the presence of phenylmethylsulfonyl fluoride, but was not influenced by the addition of GDP. Overall, these results indicate the presence of a serine protease within brown fat mitochondrial membranes that may be involved in the breakdown of UCP.Key words: thermoregulation, body weight, mitochondria, uncoupling protein, protein degradation, protease.
Distribution of MAP kinase, S6 kinase, and casein kinase II in rat tissues: activation by insulin in spleen
Authors: Hei Yong-Jiang , Chen Xunsheng , Diamond Jack , McNeill John H.
Published: December 2009

We examined the distribution of mitogen-activated protein (MAP) kinase, S6 kinase, and casein kinase II (CK-II) in the muscle, spleen, brain, and testes of Wistar rats. It was observed that spleen extracts contained the highest activity of all the kinases. Anion-exchange chromatography of spleen extracts by a MonoQ column resolved a single peak of myelin basic protein phosphotransferase activity that eluted after the usual position of the previously described p42 and p44 MAP kinases. Immunoblotting of the peak fractions with anti-MAP kinase antibody did not detect any immunoreactive bands that coincided with the activity peak, suggesting that the activity may represent a potentially novel MAP kinase. The MonoQ fractionation also resolved a single peak of phosvitin phosphotransferase activity which coincided with the intensity of two immunoreactive bands of 39 and 43 kilodaltons that were detected with antibodies against CK-II. The chromatographic behaviour and immunoblotting data indicate that the phosvitin kinase peak represented CK-II and suggested that the rat spleen CK-II had a molecular structure of αα′β2. Furthermore, using an intact rat model, we showed that the potentially novel spleen MAP kinase and CK-II were markedly activated following intravenous injection of insulin. The significance of these findings remains to be determined.Key words: mitogen-activated protein kinase, S6 kinase, casein kinase II, insulin, spleen.
Lack of excision of introns from primary transcripts of certain chicken vitellogenin II minigenes
Authors: Millan Cynthia L. Brazolot , Gibbins Ann M. Verrinder
Published: December 2009

Two truncated versions of the chicken vitellogenin II gene VTGII were designed and constructed to include all known essential regulatory elements of the complete gene. Both pCB123 and pCB123/4 contain 945 base pairs (bp) of the 5′-flanking sequence, introns and exons 1–3, and a subset of the remaining 32 exons of VTGII, inserted into a pBluescript SK (+/−)™ vector. pCB123/4 contains 752 bp of legitimate VTGII 3′-flanking sequences, while the 3′ end of pCB123 terminates at the VTGII cDNA end, followed by AT-tailing and vector sequences carried over during cloning. Expression of these plasmids was tested following their lipofection into primary cultures of chicken hepatocytes established from day 14 embryos. Poly(A)+ RNA derived from pCB123 was detected by Northern blotting and reverse transcription – polyacrylamide chain reaction. No evidence was observed for appropriate hormonal control of expression, despite the presence of 17β-estradiol or colipofection with the estrogen receptor clone pHEO. VTGII sequences at the 3′ end of pCB123/4 led to an apparent destabilization of the RNA transcript. Unexpectedly, unprocessed pCB123 transcripts of varying lengths accumulated in the cells. These experiments constitute the first reported attempts to express authentic VTGII coding sequences in cultured cells and highlight the dilemma of which introns to include in a minigene. Despite reports that some minigenes are expressed more efficiently if one or two introns are included, other minigenes may be expressed more effectively in the absence of introns. In the case of a complex gene with many introns, such as VTGII, there may be a preferential order in which introns are removed from the primary construct. The truncation of complex genes to give functional minigenes for transgenic studies may require considerable experimentation.Key words: chicken, vitellogenin, minigenes, introns, splicing.
Interaction of tissue plasminogen activator with a human endothelial cell 45-kilodalton plasminogen receptor
Authors: Dudani Anil K. , Pluskota Agatha , Ganz Peter R.
Published: December 2009

We have investigated the interaction of tissue plasminogen activator (tPA) with endothelial cell proteins of the human umbilical vein using the technique of ligand blotting. It was observed that tPA interacted with a 45-kilodalton (kDa) endothelial cell protein which appeared to be similar to the 45-kDa plasminogen receptor. Binding of tPA to the 45-kDa protein could be inhibited by excess cold tPA. Morever, excess lysine could inhibit the binding of tPA to the 45-kDa protein in both coincubation and reversibility experiments. These studies indicated that like plasminogen, tPA interacts with the 45-kDa protein in a kringle-dependent and specific manner. To confirm that tPA and plasminogen are interacting with the same protein, we investigated the effect of excess cold plasminogen on tPA binding and excess cold tPA on plasminogen binding in reversibility experiments. It was observed that binding of tPA to the 45-kDa protein was reduced by plasminogen and vice versa. In addition, the 45-kDa protein did not cross-react with antibodies to annexin II, a 40-kDa protein that binds plasminogen and tPA. These latter properties distinguish the 45-kDa receptor from plasminogen/tPA-binding proteins described by others. Therefore, the above studies suggest that the 45-kDa protein represents a unique plasminogen/tPA receptor on human venous endothelial cells.Key words: plasminogen, tissue plasminogen activator, endothelial cells, receptors.
Analysis of the lipoprotein binding site of rat liver membranes
Authors: Adam Lynda , Brissette Louise
Published: December 2009

Intermediate density lipoproteins (IDL) were shown to bind to high- and low-affinity binding sites on rat liver membranes. The low-affinity sites were named lipoprotein binding sites (LBS), since they bind all classes of lipoproteins. This study was undertaken to further characterize the interaction of 125I-labelled IDL with the LBS of rat liver membranes to determine the chemical nature of the LBS. We found that the binding of IDL to the LBS is insensitive to EDTA and sensitive to heparin and that it is present on plasma membranes. Also, membranes were pretreated with various enzymes that have an effect on the membrane constituents, and the activity of the LBS on these treated membranes was determined. Our results reveal that the LBS of rat liver membranes is insensitive to heparinase I, chondroitinase ABC, and phospholipase C, while it is partially sensitive to phospholipase A2 and sensitive to proteases and heat. Rat liver membrane proteins were solubilized with Triton X-100, reconstituted in liposomes, and analyzed for their ability to bind lipoproteins. 125I-labelled IDL were shown to bind to high- and low-affinity sites that are similar, in affinity and specificity, to the ones observed with intact rat liver membranes, indicating that a LBS activity is detectable on these liposomes. We found that the binding capacity of low-affinity sites in liposomes containing either no protein or containing proteins solubilized from Escherichia coli membranes is five times weaker than low-affinity sites in liposomes containing liver membrane proteins. Thus, a protein solubilized from rat liver membranes has LBS activity when reconstituted in liposomes. Taken altogether our results provide new information on the binding of IDL to the LBS and indicate that the LBS activity is in part mediated by a protein. Thus, the LBS appears to be a bona fide receptor.Key words: lipoprotein, receptor, binding, metabolism.
Cyclosporin treatment alters protein phosphorylation in kidney membranes
Authors: Demeule Michel , Giroux Sylvie , Murphy Gérard F. , Béliveau Richard
Published: December 2009

Phosphorylation, protein carboxyl methylation, and ADP-ribosylation were assayed in renal basolateral membranes and brush border membranes isolated from rats treated by subcutaneous administration of 5 or 10 mg/(kg∙day) of cyclosporin A (CsA) for 10 days to investigate potential alterations in signal transduction in kidney cortex. Protein carboxyl methylation of class II measured in membranes and in cytosolic fraction was not affected by CsA treatment. ADP-ribosylation performed in the presence of pertussis or cholera toxin was also similar in control and treated rats. However, changes in phosphorylation of endogenous substrates were observed in membranes and cytosol isolated from rats treated with 10 mg/(kg∙day) of CsA. Phosphorylation was increased for two brush border membrane proteins (56 and 77 kilodaltons (kDa)) by 47 and 24% and for two basolateral membrane proteins (51 and 80 kDa) by 28 and 29%, respectively. In the cytosolic fraction, phosphorylation of two proteins (31 and 65 kDa) was increased by 37% and that of 25- and 43-kDa proteins was reduced by 29%. Protein kinase A, protein kinase C, and tyrosine protein kinase activities were also determined in membranes. Increases in protein kinase C and tyrosine protein kinase activities were observed in basolateral membranes, but not in brush border membranes after cyclosporin A administration. Endogenous substrates for tyrosine kinase were also detected with an antiphosphotyrosine (PY20) monoclonal antibody. Densitometric analysis indicated that the phosphorylation of three proteins of high molecular masses (61, 132, and 183 kDa) was stimulated by CsA in basolateral membranes. Immunodetection of calcineurin with a monoclonal antibody directed against the β-subunit was similar in control and treated rats. These results suggest that cyclosporin A treatment may bring about changes in covalent modifications as an adaptative response in signal transduction in the kidney proximal tubule.Key words: cyclosporin A, kidney, membranes, carboxyl methylation, ADP-ribosylation, phosphorylation.
Serological and structural relationships between Escherichia coli O: 98 and Yersinia enterocolitica O: 11,23 and O: 11,24 lipopolysaccharide O-antigens
Authors: Marsden Brian J. , Bundle David R. , Perry Malcolm B.
Published: December 2009

The serologically related lipopolysaccharide O-antigens of Yersinia enterocolitica serotypes 11,23 and 11,24 and of Escherichia coli O: 98 were analysed by composition analysis, methylation, and the use of one- and two-dimensional nuclear magnetic reasonance methods. They were found to be composed of the same basic linear unbranched polysaccharide of repeating tetrasaccharide units containing 2-acetamido-2-deoxy-D-glucose (D-GlcNAc), 2-acetamido-2-deoxy-D-galacturonic acid (D-GalNAcA), and 2-acetamido-2,6-dideoxy-L-glucose (L-quinovosamine, L-QuiNAc), having the structure[Formula: see text]The lipopolysaccharide O-antigens of E. coli O: 98 and Y. enterocolitica O: 11,24 were polymers of the above repeating unit, whereas that of Y. enterocolitica O: 11,23 was a linear polymer of the same repeating units in which the α-D-GalpNAcA residues were stoichiometrically substituted by acetyl groups at O-3.Key words: lipopolysaccharide, polysaccharide, Escherichia coli, antigen, nuclear magnetic resonance.
Among a range of transition metals and ligands vanadium∙desferroxamine excels in accelerating reactivity of ferrocytochrome c toward molecular oxygen
Authors: Davison Allan J. , Wu Qizhuan , Moon Jim , Stern Arnold
Published: December 2009

Despite early knowledge of the requirement for metals in the reactions of ferrocytochrome c with oxygen, the relative effectiveness of metals and the factors that modulate effectiveness remain unknown. We have compared the catalytic power of five metals and report the effects of pH and ligand on their effectiveness as catalysts. Catalysis by metal ions was greatest at higher pH, where the rate of aerobic oxidation was lowest. Iron (Fe2+), copper (Cu2+), vanadium(V) (V(V)), manganese (Mn2+), and aluminum (Al3+) were tested in combination with EDTA, ADP, histidine, or desferrioxamine (Des) at pH 2.6, 3.2, and 4.0. At pH 2.6, only vanadium(V) increased the initial rate of the oxidation of ferrocytochrome c (by 6.2-fold). At pH 4.0, however, all the metals markedly stimulated the oxidation of cytochrome c. The order of effectiveness was V(V)∙Des >> Cu∙ADP2+ > Fe∙EDTA2+ > Mn∙Des2+ > Al∙EDTA3+ (where the stated ligand represents the most stimulating one for a given metal). At pH 3.2 the metal complexes had intermediate effects, with vanadium again being the most effective. The preeminence of vanadium among the metals is novel. Where the heme crevice is closed (pH 4), transition metal ions mediated almost all of the reduction of oxygen, while at the lowest pH (2.6) transition metal ions were largely unnecessary. Vanadium(V) was the most active of the metals at all values of pH and the only metal to accelerate the oxidation of ferrocytochrome c at pH 2.6. Understanding of the range of biological actions of vanadium will not be complete without a knowledge of its redox reactivity within the components of biological systems.Key words: aerobic oxidation, ferrocytochrome c, transition metals, ligands, vanadium, iron, manganese, copper, aluminium.
Translocation of neutral endopeptidase 24.11 mutants with deletions of the NH2- terminal cytosolic domain
Authors: Lemire Isabelle , Roy Patrice , Boileau Guy
Published: December 2009

Rabbit neutral endopeptidase 24.11 (NEP) is a type II membrane protein with a positively charged 27 amino acid residue NH2-terminal cytoplasmic domain, a 20 amino acid residue hydrophobic signal peptide/membrane anchor domain, and a large catalytic COOH-terminal domain exposed on the exoplasmic side of the membrane. To study the role of the cytosolic domain in anchoring NEP in the plasma membrane, we constructed two mutants in which this cytosolic domain was deleted. In the first mutant (NEPΔcyto), a Glu residue was present in NH2-terminus, while a Lys residue was substituted at the same position in the second mutant (NEPΔcyto(K)). To better understand the interaction of these mutants with the rough endoplasmic reticulum membrane, the mutated NEP cDNAs were transcribed and translated in vitro in the presence of microsomal membranes. Our studies showed that deletion of the hydrophillic cytosolic domain affects translocation of the NEP polypeptide chain. Substitution of a positively charged Lys residue for the Glu residue at the NH2-terminus of the deletion mutant only partly restored translocation of the polypeptide chain. Furthermore, carbonate extraction and trypsin digestion of the microsomal membranes indicated that the deletion mutants are inserted in the microsomal membranes as type III membrane proteins with their COOH-terminal domain exposed on the exterior of the microsomes. Thus, efficient translocation is dependent on the presence of a charged cytoplasmic domain.Key words: membrane proteins, translocation, cytosolic domain, neutral endopeptidase 24.11.
Phosphorylation of the precursor sequence of rat B-type natriuretic peptide by p34cdc2 and MAP kinase
Authors: Mezl Vasek A. , Watson Mark H. , Flynn T. Geoffrey , Mak Alan S.
Published: December 2009

Atrial natriuretic peptide (ANP) and B-type natriuretic peptide (BNP), two distinct members of the natriuretic peptide family, share many features in common. However, differences in expression indicate that the processing mechanisms must be different. The leader sequence of rat BNP contains three potential phosphorylation sites for proline-directed kinases that are not present in the leader sequence of ANP. This study has examined how these sites are used by two somewhat different proline-directed kinases. A peptide containing these sites was phosphorylated in vitro by HeLa p34cdc2 kinase and by sea star p44mpk kinase at rates that were comparable to the rates with peptide substrates that are used to assay these enzymes. Sequence analysis of the phosphopeptide shows that both kinases phosphorylate only the two potential phosphorylation sites surrounding the cleavage site of the BNP precursor. The enzymatic potential for such a phosphorylation of BNP in cardiac tissue is demonstrated by immunoblots and kinase assays, showing that in fetal and in adult rat heart both the atria and the ventricles contain a mitogen-activated protein kinase homologue that can phosphorylate this preproBNP sequence.Key words: atrial natriuretic peptide, cdc2 kinase, mitogen-activated protein kinase, phosphorylation.
A 56- to 54-kilodalton non grata signal in immunoblot analysis using the horseradish peroxidase chemiluminescence system
Authors: Fortin A. , Berkova N. , Tremblay S. , Côté F. , Rousseau F. , Khandjian E.W.
Published: December 2009

The major advantages of the horseradish peroxidase chemiluminescence (HRP-CL) immunodetection method in Western blot analysis are its high sensitivity, nonradioactive detection, economy of the primary antibody, and speed of detecting the signal. However, we observed a strong and reproducible signal that was detected regardless of the primary antibody and of the cell type used. This signal, present at 56–54 kilodaltons (kDa), is generated in absence of any primary antibody and seems to be an intrinsic reaction of the HRP-labeled second antibody anti-immunoglobulin with an unidentified cellular protein. The use of dry milk throughout all the steps of the procedure abolishes this signal. For those interested in one of the numerous proteins migrating at or close to 56–54 kDa, the question therefore arises as to which signals generated by the HRP-CL in this region are bona fide and which are non grata pseudo signals.Key words: p53, Western blot analysis, chemiluminescence, horseradish peroxidase.
Cell shape, intracellular pH, and fibroblast growth factor responsiveness during transdifferentiation of retinal pigment epithelium into neuroepithelium in vitro
Authors: Zhou You , Opas Michal
Published: December 2009

In this report we show that some retinal pigment epithelial (RPE) cells, with no expression of neural cell adhesion molecule (N-CAM) [Formula: see text], spontaneously lose pigment and start to express N-CAM in culture. Chick RPE cells normally do not express N-CAM, while the protein is present in chick neural retina. Thus some of the RPE cells in culture started to transdifferentiate into a neuroepithelium[Formula: see text]. We have measured intracellular pH (pHi) in the RPE cultures and followed its changes in response to basic fibroblast growth factor (bFGF). The depigmented cells protrude above the RPE cell sheet and have a lower resting pHi (≈7.05) than the pigmented RPE cells (≈7.15). The majority of cells with low resting pHi express N-CAM. The difference in the resting pHi between [Formula: see text]and [Formula: see text] cells is not due to the N-CAM expression by [Formula: see text] cells, as their pHi is the same as the pHi of freshly plated single "round" [Formula: see text] cells that have not spread yet. [Formula: see text] cells respond to bFGF with a quick and sustained pHi rise. In contrast, neither the cuboidal [Formula: see text] cells in a colony centre nor single round [Formula: see text] cells respond to bFGF with cytoplasmic alkalinization. RPE cells do not proliferate in response to bFGF, while NE cells respond to bFGF with a stimulation of growth. We conclude that bFGF acts not on the fully differentiated [Formula: see text], but only on those cells which have already started to transdifferentiate and changed their shape and (or) adhesive status. bFGF provides [Formula: see text] cells that transdifferentiated from [Formula: see text] cells with a signal necessary to transdifferentiate and (or) histodifferentiate further.Key words: retinal pigment epithelium, retina, transdifferentiation, intracellular pH, basic fibroblast growth factor.
Expression and regulation of three lysosomal cysteine protease activities during growth of a differentiating L6 rat myoblast cell line and its nonfusing variant
Authors: Jane Derek T. , Dufresne Michael J.
Published: December 2009

The expression of three lysosomal cysteine protease activities, cathepsins B, H, and L, was examined during differentiation of L6 rat myoblasts. Analyses of intracellular levels of these proteases in unfractionated homogenates prepared from cells at different stages of growth and in parallel HPLC-fractionated samples demonstrated a fusion-related increase in all three cathepsins. Analyses of total levels of endogenous inhibitor activity against purified cathepsin B demonstrated a threefold increase in the ratio of protease to inhibitor during myoblast-myotube formation; however, levels of inhibitor activity remained constant. Extracellular levels of cathepsin B, H, and L activities were also examined in the serum-free defined media of differentiating L6 cells. These studies demonstrated a fusion-related increase in extracellular levels of acid/pepsin-activated (i.e., latent) cathepsin L. While increases in intracellular and extracellular levels of cathepsin activities were temporally related to the fusion process, fusion may not be a prerequisite for increased expression, since the nonfusing L6 variant L6-D3 demonstrated high levels of intracellular cathepsins B and L and extracellular latent cathepsin L activities throughout growth. Taken together, these results support the hypotheses that fusion or fusion-related processes play an important role in the controlled expression of cathepsins in L6 myoblasts and that cathepsins, in turn, play an important role in myoblast-myotube differentiation.Key words: L6 myoblasts, differentiation, lysosomal cysteine proteases.
Generation of poly- and mono-clonal antibodies against trout fibronectin (FN) and their use in FN immunodetection in teleost fishes
Authors: Sadaghiani Bahram , Crawford Bruce J. , Vielkind Juergen R.
Published: December 2009

Immunization of rats with gelatin-affinity column purified fibronectin (FN) from rainbow trout (Oncorhynchus mykiss) plasma produced a polyclonal antiserum that reacts specifically with FN in immunoblotted protein extracts and cultured cells, not only from trout but also from swordtails (Xiphophorus helleri). Most importantly, this antiserum specifically stains FN-containing structures in sections from embryos, as well as skin and dorsal fin of swordtails and platyfish (Xiphophorus maculatus), allowing, e.g., correlation of the distribution of FN with neural crest cell development in Xiphophorus. The antiserum also cross-reacts with FN in sections from embroys of the Japanese medaka (Oryzias latipes) and coho salmon (Oncorhynchus kisutch). In addition to the polyclonal antibodies, monoclonal anti-trout FN antibodies were produced in rats. These did not exhibit reactivity on sections, but stained the cultured fish cells and FN in immunoblots. Both types of antibodies may be of interest to the fish industry for marking the level of FN as an indicator, not only for infectious diseases but also for certain developmental stages such as smoltification and spawning.Key words: antibodies, extracellular matrix, neural crest, Xiphophorus.
Long-term exposure to epidermal growth factor results in apoptosis in T51B cells
Authors: Brabyn Caroline J. , Franks Douglas J. , Kleine Leonard P.
Published: December 2009

The major objective of this research was the development of an in vitro model for liver homeostasis that would allow the future study of early events in cell proliferation and cell death. The model that was set up involves growing T51B rat liver epithelial cells with a single dose of 1 nM epidermal growth factor (EGF). This results in a period of hyperplasia where the cells reach double the control cell numbers 2 days after EGF addition. This is then followed by a decrease in cell numbers and the cell density returns to around the confluent control level 5 days after EGF addition. The model was investigated to ascertain whether the decrease in cell numbers 3–5 days after EGF addition was due to an increase in apoptosis. The results from light and electron microscopy studies, electrophoresis of T51B cell DNA, and quantification of nuclear fragmentation indicated that the cells do die via an increase in apoptosis. The electron microscopy studies also show that healthy T51B cells can phagocytose apoptotic bodies. This suggests that the model is more physiological than other in vitro models of apoptosis. This work describes the development and characterization an in vitro model of liver homeostasis that closely parallels in vivo systems where animals are given mitogenic stimuli.Key words: apoptosis, epidermal growth factor, T51B liver epithelial cell line, tissue homeostasis.
Increased TRPM-2/clusterin mRNA levels during the time of retinal degeneration in mouse models of retinitis pigmentosa
Authors: Wong Paul , Borst Diane E. , Farber Debora , Danciger Janet S. , Tenniswood Martin , Chader Gerald J. , Veen Theo van
Published: December 2009

Retinitis pigmentosa (RP) is a genetically and clinically heterogeneous group of human disorders that is characterized by diminished retinal function, visual cell loss, and blindness. Elevated levels of TRPM-2/clusterin mRNA, a marker for the apoptotic process, have been reported in retinas from patients with advanced stage RP. In the present study we examine TRPM-2/clusterin expression in two genetically distinct mouse models of RP, the rd (retinal degeneration) and rds (retinal degeneration slow) mice. We establish that in advanced postretinal degenerative stages of the rd mutant the retinal TRPM-2/clusterin mRNA levels are highly elevated, as is seen in the case of human RP. Examination of TRPM-2/clusterin mRNA levels in retina and whole eyes from the rd mouse and morphologically normal controls during the period of retinal degeneration (postnatal days 8–21) in the rd phenotype shows that TRPM-2/clusterin mRNA levels are elevated in the rd animal, and this increase begins just after postnatal day 10 and remains high for the remainder of the time course examined. Northern analysis of rds retina and whole eyes shows a delayed increase in TRPM-2/clusterin mRNA levels relative to the rd profile, coinciding with the known period of rds retinal degeneration (postnatal day 14 to 1 year). In each case, the onset of increased TRPM-2/clusterin mRNA levels coincides with the time of photoreceptor cell death.Key words: retinal degeneration, TRPM-2, clusterin, apoptosis, retinitis pigmentosa.