bluuebunny/crossref_metadata_2025 huggingface dataset:
Human extracellular superoxide dismutase is a tetramer composed of two disulphide-linked dimers: a simplified, high-yield purification of extracellular superoxide dismutase
Authors: OURY Tim D. , CRAPO James D. , VALNICKOVA Zuzana , ENGHILD Jan J.
Published: August 2015

Studies examining the biochemical characteristics and pharmacological properties of extracellular superoxide dismutase (EC SOD) have been severely limited because of difficulties in purifying the enzyme. Recently EC SOD was found to exist in high concentrations in the arteries of most mammals examined and it is the predominant form of SOD activity in many arteries. We now describe a three-step, high-yield protocol for the purification of EC SOD from human aorta. In the first step, the high affinity of EC SOD for heparin is utilized to obtain a fraction in which EC SOD constitutes roughly 13% of the total protein compared with only 0.3% of that of the starting material. In addition, over 80% of the original EC SOD activity present in the aortic homogenate was retained after the first step of purification. EC SOD was further purified using a combination of cation- and anion-exchange chromatography. The overall yield of EC SOD from this purification procedure was 46%, with over 4 mg of EC SOD obtained from 230 g of aorta. Purified EC SOD was found to exist predominantly as a homotetramer composed of two disulphide-linked dimers. However, EC SOD was also found to form larger multimers when analysed by native PAGE. It was shown by urea denaturation that the formation of multimers increased the thermodynamic stability of the protein. Limited proteolysis of EC SOD suggested that there is one interchain disulphide bond covalently linking two subunits. This disulphide bond involves cysteine-219 and appears to link the heparin-binding domains of the two subunits.
Protection by chlorpromazine, albumin and bivalent cations against haemolysis induced by melittin, [Ala-14]melittin and whole bee venom
Authors: RUDENKO S. V. , NIPOT E. E.
Published: August 2015

The ability of the peptides melittin, [Ala-14]melittin (P14A) and whole bee venom to lyse red blood cells (RBC) and to cause shape transformation, binding, partitioning and changes in volume of the cells during haemolysis, as well as the action of the bivalent cations Zn2+ and Ca2+, chlorpromazine, albumin and plasma on the peptide-induced haemolysis of RBC in high ionic-strength solution, have been investigated. The protective effect of all inhibitors depends on whether they have been added to the media before or after the cells. When added before the cells they reduced significantly the rate of peptide-induced haemolysis and shape transformation. The effect was maximal when agents acted simultaneously after introduction of the cells into the media containing both inhibitors and peptides. Incubation of the cells in isotonic solution before the addition of peptides enhanced 2–3-fold the RBC susceptibility (i.e. rate of haemolysis) to lytic action of the same amount of peptides, and increased the order of the haemolytic reaction, although the power law coefficient did not exceed a value of 2 for all peptides, suggesting that haemolysis is attributable to the monomeric or dimeric forms of the peptides. Partition coefficients were of the order of ∼106 M-1, and P14A possessed a value 3-fold larger compared with melittin and bee venom, which correlated with its enhanced haemolytic activity. The protective action of inhibitors against peptide-induced haemolysis has been explained on the basis of their ability to compete with peptide binding at an early stage of peptide–membrane interaction, and not as a result of inhibition of a pre-existing peptide-induced pore. Whereas melittin increased the volume of RBC during haemolysis, P14A, melittin in the presence of phospholipase A2 or bee venom, reduced the volume in a concentration-dependent manner. The present data reveal the significant role of the initial stage of peptide–membrane interaction and peptide structure in the mechanism of haemolysis. These data are not consistent with a lipid-based mechanism of peptide-induced haemolysis, indicating that the mode of peptide–protein interaction is an important and decisive step in the haemolytic mechanism. It should be noted that data (in the form of three additional Tables) on the ability of inhibitors to protect cells from haemolysis when inhibitor and peptide act simultaneously are available. They are reported in Supplementary Publication SUP 50178, which has been deposited at the British Library Document Supply Centre, Boston Spa, Wetherby, West Yorkshire LS23 7BQ, U.K., from whom copies can be obtained on the terms indicated in Biochem. J. (1996) 313, 9.
Identification of the transcription factors NF-YA and NF-YB as factors A and B that bound to the promoter of the major histocompatibility complex class II gene I-A β
Authors: CELADA Antonio , McKERCHER Scott R. , MAKI Richard A.
Published: August 2015

The Y box is a conserved sequence in the promoter of major histocompatibility complex (MHC) class II genes, which contains a CCAAT sequence (CCAAT box). Previously, we partially purified the DNA-binding protein that recognizes the Y box of the I-Aβ gene and showed that it consisted of two components (factors A and B) both of which were necessary for optimal DNA binding. The genes for the heteromeric protein NF-Y (NF-YA and NF-YB), which binds to the I-Eα Y box have been cloned. We subsequently isolated the genes for NF-YA and NF-YB using oligonucleotides designed from the published sequences. NF-YA and NF-YB were tested for binding to the I-Aβ and I-Eα Y boxes. While neither NF-YA or NF-YB alone bound to the Y box, when the components were mixed the complex bound to the I-Aβ Y box with high affinity. Moreover, NF-YA and NF-YB could be complemented for binding to DNA by factor B or factor A, respectively. These results suggest that the active binding protein is NF-YA in factor A extracts and NF-YB in factor B extracts. Finally, antibodies against NF-YA and NF-YB were shown to induce a supershift when nuclear extracts were added to the double-stranded oligodeoxynucleotide covering the Y box of the I-Aβ gene. Antisense expression constructs of both NF-YA and NF-YB were made and their effect on expression from the I-Aβ promoter was tested. Either antisense construction, when transfected into cells, lowered the expression of a reporter gene linked to the I-Aβ promoter. This study provides direct evidence of the identification of NF-YA and NF-YB as the previously described factors A and B. Moreover, these results strongly implicate NF-Y in the expression of the MHC class II gene I-Aβ.
Hypoxanthine enters human vascular endothelial cells (ECV 304) via the nitrobenzylthioinosine-insensitive equilibrative nucleoside transporter
Authors: OSSES Nelson , PEARSON Jeremy D. , YUDILEVICH David L. , JARVIS Simon M.
Published: August 2015

The transport properties of the nucleobase hypoxanthine were examined in the human umbilical vein endothelial cell line ECV 304. Initial rates of hypoxanthine influx were independent of extracellular cations: replacement of Na+ with Li+, Rb+, N-methyl-d-glucamine or choline had no significant effect on hypoxanthine uptake by ECV 304 cells. Kinetic analysis demonstrated the presence of a single saturable system for the transport of hypoxanthine in ECV 304 cells with an apparent Km of 320±10 μM and a Vmax of 5.6±0.9 pmol/106 cells per s. Hypoxanthine uptake was inhibited by the nucleosides adenosine, uridine and thymidine (apparent Ki 41±6, 240±27 and 59±8 μM respectively) and the nucleoside transport inhibitors nitrobenzylthioinosine (NBMPR), dilazep and dipyridamole (apparent Ki 2.5±0.3, 11±3 and 0.16±0.006 μM respectively), whereas the nucleobases adenine, guanine and thymine had little effect (50% inhibition at > 1 mM). ECV 304 cells were also shown to transport adenosine via both the NBMPR-sensitive and -insensitive nucleoside carriers. Hypoxanthine specifically inhibited adenosine transport via the NBMPR-insensitive system in a competitive manner (apparent Ki 290±14 μM). These results indicate that hypoxanthine entry into ECV 304 endothelial cells is mediated by the NBMPR-insensitive nucleoside carrier present in these cells.
Cytoplasmic tail length influences fatty acid selection for acylation of viral glycoproteins
Authors: VEIT Michael , REVEREY Helmut , SCHMIDT Michael F. G.
Published: August 2015

We report remarkable differences in the fatty acid content of thioester-type acylated glycoproteins of enveloped viruses from mammalian cells. The E2 glycoprotein of Semliki Forest virus contains mainly palmitic acid like most other palmitoylated proteins analysed so far. However, the other glycoprotein (E1) of the same virus, as well as the HEF (haemagglutinin esterase fusion) glycoprotein of influenza C virus, are unique in this respect because they are acylated primarily with stearic acid. Comparative radiolabelling of uninfected cells with different fatty acids suggests that stearate may also be the prevailing fatty acid in some cellular acylproteins. To look for further differences between palmitoylated and stearoylated glycoproteins we characterized stearoylation in more detail. We identified the acylation site of HEF as a cysteine residue located at the boundary between the transmembrane region and the cytoplasmic tail. The attachment of stearate to HEF and E1 occurs post-translationally in a pre-Golgi compartment. Thus, stearoylated and palmitoylated proteins cannot be discriminated on the basis of the fatty acid linkage site or the intracellular compartment, where acylation occurs. However, stearoylated acylproteins contain a very short, positively charged cytoplasmic tail, whereas in palmitoylated proteins this molecular region is longer. Replacing the short cytoplasmic tail of stearoylated HEF with the long influenza A virus haemagglutinin (HA) tail in an HEF–HA chimera, and subsequent vaccinia T7 expression in CV-1 cells, yielded proteins with largely palmitic acid bound. The reverse chimera, HA–HEF with a short cytoplasmic tail was not fatty acylated at all during expression, indicating that conformational or topological constraints control fatty acid transfer.
Different mucins are produced by the surface epithelium and the submucosa in human trachea: identification of MUC5AC as a major mucin from the goblet cells
Authors: HOVENBERG Hans W , DAVIES Julia R , CARLSTEDT Ingemar
Published: August 2015

Mucins were extracted from the epithelial surface and the submucosal tissue of human trachea in order to enrich glycoproteins from the goblet cells and the submucosal glands respectively. The macromolecules were purified using density-gradient centrifugation, and the presence of the MUC5AC mucin was investigated using an antiserum raised against a synthetic peptide based on the sequence of the MUC5AC apoprotein. Mucins from the surface epithelium showed a higher reactivity with the antiserum relative to carbohydrate than those from the submucosa, and ion-exchange HPLC of reduced subunits revealed the presence of two distinct mucin populations in the samples. The predominant species from the surface epithelium was more acidic than the major population from the submucosa and showed a strong reactivity with the anti-MUC5AC antiserum. In contrast, the major portion of the submucosal mucins were less acidic and showed no MUC5AC reactivity, although a more acidic population did react with the antibody. Rate-zonal centrifugation showed that the MUC5AC mucin from the surface epithelium is smaller than the major submucosal mucin, and that both are composed of subunits. Immunolocalization confirmed that the MUC5AC mucin from human trachea originates from the goblet cells and that this glycoprotein is not a major product of the submucosal glands.
CD28: a signalling perspective
Authors: WARD Stephen G
Published: August 2015

CD28 and the related molecule cytotoxic T lymphocyte-associated molecule-4 (CTLA-4), together with their natural ligands B7.1 and B7.2, have been implicated in the differential regulation of several immune responses. CD28 provides signals during T cell activation which are required for the production of interleukin 2 and other cytokines and chemokines, and it has also been implicated in the regulation of T cell anergy and programmed T cell death. The biochemical signals provided by CD28 are cyclosporin A-resistant and complement those provided by the T cell antigen receptor to allow full activation of T cells. Multiple signalling cascades which may be independent of, or dependent on, protein tyrosine kinase activation have been demonstrated to be activated by CD28, including activation of phospholipase C, p21ras, phosphoinositide 3-kinase, sphingomyelinase/ceramide and 5-lipoxygenase. The relative contributions of these cascades to overall CD28 signalling are still unknown, but probably depend on the state of activation of the T cell and the level of CD28 activation. The importance of these signalling cascades (in particular the phosphoinositide 3-kinase-mediated cascade) to functional indications of CD28 activation, such as interleukin 2 gene regulation, has been investigated using pharmacological and genetic manipulations. These approaches have demonstrated that CD28-activated signalling cascades regulate several transcription factors involved in interleukin 2 transcriptional activation. This review describes in detail the structure and expression of the CD28 and B7 families, the functional outcomes of CD28 ligation and the signalling events that are thought to mediate these functions.
Recombinant apoaequorin acting as a pseudo-luciferase reports micromolar changes in the endoplasmic reticulum free Ca2+ of intact cells
Authors: KENDALL Jonathan M. , BADMINTON Michael N , SALA-NEWBY Graciela B. , CAMPBELL Anthony K , REMBOLD ChristopherM
Published: August 2015

We describe a novel method to monitor the endoplasmic reticulum (ER) free Ca2+ in intact cells. Continuous perfusion of HeLa cells, expressing ER-targeted apoaequorin, with coelenterazine allowed the apoprotein to act as a pseudo-luciferase capable of reporting free Ca2+ from 0.1–100 µM. In intact HeLa cells, addition of ionomycin increased apoaequorin-generated light by 91%, indicating that resting ER free Ca2+ was approx. 2 µM. Agonist stimulation decreased the ER apoaequorin signal and proportionally increased cytosolic free Ca2+ consistent with agonist-induced release of Ca2+ from the ER.
Peripheral α-linked N-acetylglucosamine on the carbohydrate moiety of mucin derived from mammalian gastric gland mucous cells: epitope recognized by a newly characterized monoclonal antibody
Authors: ISHIHARA Kazuhiko , KURIHARA Makoto , GOSO Yukinobu , URATA Tsukiko , OTA Hiroyoshi , KATSUYAMA Tsutomu , HOTTA Kyoko
Published: August 2015

To obtain a tool to study the structural characterization and the detection of mucin derived from the gastric gland mucous cells, we developed a monoclonal antibody, designated HIK1083, against mucin purified from rat gastric mucosa. In an ELISA, HIK1083 reacted strongly with the mucin purified from a deep layer of the corpus and antrum but only slightly reacted with that obtained from the surface mucosal layer. The reaction of mucin and HIK1083 was inhibited by the oligosaccharides obtained by the alkaline borohydride reduction of antigenic mucin. Two purified oligosaccharide alditols reacting with the monoclonal antibody obtained from the antigenic mucin had one and two peripheral α-linked N-acetylglucosamine residues, respectively, according to the evidence from NMR spectroscopy. Moreover, among the commercially available p-nitrophenyl derivatives of monosaccharides, only p-nitrophenyl-N-acetyl-α-d-glucosaminide inhibited the reaction of this monoclonal antibody and the antigenic mucin in a concentration-dependent manner. These results, as well as the immunohistochemical observations, indicate that α-linked N-acetylglucosamine residues are specifically attached to the peripheral region of the carbohydrate moiety of the mucin synthesized in and secreted from the gastric-gland-type cells, and indicate that the monoclonal antibody HIK1083 recognizes this structure.
Induction of calreticulin expression in HeLa cells by depletion of the endoplasmic reticulum Ca2+ store and inhibition of N-linked glycosylation
Authors: LLEWELLYN David H , KENDALL Jonathan M. , Naureen SHEIKH , CAMPBELL Anthony K. F.
Published: August 2015

Calreticulin is now considered to be a multifunctional Ca2+-binding protein. Its primary role is as a Ca2+ storage protein within the lumen of the endoplasmic reticulum (ER), where it also seems to assist in the correct folding and assembly of proteins. We have investigated whether agents that affect these processes can alter calreticulin expression in HeLa cells. Perturbation of intracellular Ca2+ levels by prolonged exposure to either thapsigargin or ionomycin induced calreticulin mRNA, both in the presence and absence of extracellular Ca2+, consistent with the proposal that sustained depletion of the ER Ca2+ store can trigger these increases. The mechanism underlying the induction seems to be transcriptional up-regulation as both agents increased calreticulin promoter-driven firefly luciferase expression in transfected cells to the same degree as the observed increases in calreticulin mRNA. Experiments with a truncated promoter construct showed that the sequences that confer this inducibility reside within the 225 bp immediately upstream of the putative major transcriptional start site. We also examined the effect of tunicamycin, which inhibits N-linked glycosylation in the ER thereby interfering with protein processing. This caused increases in calreticulin mRNA greater than those with either thapsigargin or ionomycin, but failed to transactivate the calreticulin promoter. Thus either additional cis sequences that reside outside our promoter region are necessary for transcriptional activation by tunicamycin, or the increases in calreticulin mRNA occur post-transcriptionally. This suggests that there are probably different mechanisms by which calreticulin expression can be induced in response to agents that affect normal ER functioning.
Signal-transducing protein phosphorylation cascades mediated by Ras/Rho proteins in the mammalian cell: the potential for multiplex signalling
Authors: DENHARDT David T
Published: August 2015

The features of three distinct protein phosphorylation cascades in mammalian cells are becoming clear. These signalling pathways link receptor-mediated events at the cell surface or intracellular perturbations such as DNA damage to changes in cytoskeletal structure, vesicle transport and altered transcription factor activity. The best known pathway, the Ras → Raf → MEK → ERK cascade [where ERK is extracellular-signal-regulated kinase and MEK is mitogen-activated protein (MAP) kinase/ERK kinase], is typically stimulated strongly by mitogens and growth factors. The other two pathways, stimulated primarily by assorted cytokines, hormones and various forms of stress, predominantly utilize p21 proteins of the Rho family (Rho, Rac and CDC42), although Ras can also participate. Diagnostic of each pathway is the MAP kinase component, which is phosphorylated by a unique dual-specificity kinase on both tyrosine and threonine in one of three motifs (Thr-Glu-Tyr, Thr-Phe-Tyr or Thr-Gly-Tyr), depending upon the pathway. In addition to activating one or more protein phosphorylation cascades, the initiating stimulus may also mobilize a variety of other signalling molecules (e.g. protein kinase C isoforms, phospholipid kinases, G-protein α and βγ subunits, phospholipases, intracellular Ca2+). These various signals impact to a greater or lesser extent on multiple downstream effectors. Important concepts are that signal transmission often entails the targeted relocation of specific proteins in the cell, and the reversible formation of protein complexes by means of regulated protein phosphorylation. The signalling circuits may be completed by the phosphorylation of upstream effectors by downstream kinases, resulting in a modulation of the signal. Signalling is terminated and the components returned to the ground state largely by dephosphorylation. There is an indeterminant amount of cross-talk among the pathways, and many of the proteins in the pathways belong to families of closely related proteins. The potential for more than one signal to be conveyed down a pathway simultaneously (multiplex signalling) is discussed. The net effect of a given stimulus on the cell is the result of a complex intracellular integration of the intensity and duration of activation of the individual pathways. The specific outcome depends on the particular signalling molecules expressed by the target cells and on the dynamic balance among the pathways.
The role of changes in the sensitivity of hepatic mitochondrial overt carnitine palmitoyltransferase in determining the onset of the ketosis of starvation in the rat
Authors: DRYNAN Lesley , QUANT Patti A. , ZAMMIT Victor A.
Published: August 2015

The relationships between the increase in blood ketone-body concentrations and several parameters that can potentially influence the rate of hepatic fatty acid oxidation were studied during progressive starvation (up to 24 h) in the rat in order to discover whether the sensitivity of mitochondrial overt carnitine palmitoyltransferase (CPT I) to malonyl-CoA plays an important part in determining the intrahepatic potential for fatty acid oxidation during the onset of ketogenic conditions. A rapid increase in blood ketone-body concentration occurred between 12 and 16 h of starvation, several hours after the marked fall in hepatic malonyl-CoA and in serum insulin concentrations and doubling of plasma non-esterfied fatty acid (NEFA) concentration. Consequently, both the changes in hepatic malonyl-CoA and serum NEFA preceded the increase in blood ketone-body concentration by several hours. The maximal activity of CPT I increased gradually throughout the 24 h period of starvation, but the increases did not become significant before 18 h of starvation. By contrast, the sensitivity of CPT I to malonyl-CoA and the increase in blood ketone-body concentration followed an identical time course, demonstrating the central importance of this parameter in determining the ketogenic response of the liver to the onset of the starved state.
Immunocytochemical localization and biochemical characterization of a novel plasma membrane-associated, neutral pH optimum α-l-fucosidase from rat testis and epididymal spermatozoa
Authors: AVILÉS Manuel , ABASCAL Irene , MARTÍNEZ-MENÁRGUEZ José Angel , CASTELLS María Teresa , SKALABAN Sheri R. , BALLESTA José , ALHADEFF Jack A.
Published: August 2015

1. Immunocytochemical and biochemical techniques have been used to localize and characterize a novel plasma membrane-associated, neutral-pH-optimum α-l-fucosidase from rat spermatozoa. Light and electron microscopy specifically localized the fucosidase on the plasma membrane of the convex region of the principal segment of testicular and cauda epididymal sperm heads. Immunoreactivity for α-l-fucosidase was also detected in the Golgi apparatus of spermatocytes and spermatids but no immunoreactivity was observed in the acrosome. 2. Fractionation of epididymal sperm homogenates indicated that over 90% of the α-l-fucosidase activity was associated with the 48000 g pellet. This pellet-associated activity could be solubilized with 0.5 M NaCl but not with 0.5% Triton X-100, suggesting that fucosidase is peripherally associated with membranes. Sucrose-density-gradient centrifugation of sperm homogenates indicated that fucosidase was enriched in the plasma membrane-enriched fraction. Analysis of α-l-fucosidase on intact epididymal sperm indicated that the enzyme was active, displayed linear kinetics and had a pH–activity curve (with an optimum near 7) which was comparable to that of fucosidase from epididymal sperm extracts. These results further suggest that fucosidase is associated with plasma membranes, and that its active site is accessible to fucoconjugates. Evidence that most of the fucosidase is associated with the exterior of the plasma membrane came from studies in which intact sperm had fucosidase activity comparable to that of sperm sonicates, and from studies in which approx. 90% of the fucosidase activity on intact sperm could be released from the sperm by gentle shaking with 0.5 M NaCl. Isoelectric focusing indicated that the NaCl-solubilized epididymal sperm fucosidase appears to have one major and one minor isoform with pIs near 7.2 and 5.2, respectively. SDS/PAGE and Western blotting indicated that the NaCl-solubilized extract of epididymal sperm contains two protein bands of 54 and 50 kDa which were highly immunoreactive with the IgG fraction of anti-fucosidase antibodies. Although the function of the novel sperm fucosidase is not known, its specific localization to the plasma membrane of the region of the rat sperm head involved in sperm–egg binding and its high enzymic activity at neutral pH on intact sperm suggest that this enzyme may have a role in sperm–egg interactions.
Autolysis parallels activation of μ-calpain
Authors: BAKI Andrea , TOMPA Péter , ALEXA Anita , MOLNÁR Orsolya , FRIEDRICH Peter
Published: August 2015

The kinetics of autolysis and activation of µ-calpain were measured with microtubule-associated protein 2 (MAP2) as a very sensitive substrate. The initial rate of MAP2 hydrolysis was found to be a linear function of the autolysed 76 kDa form of µ-calpain large subunit at both 10 and 300 µM Ca2+, and both straight lines intersected the origin. This finding supports the view that native µ-calpain is an inactive proenzyme and that activation is accompanied by autolysis. The first-order rate constant of autolysis, kI(aut), was determined at different Ca2+ concentrations: the half-maximal value was at pCa2+ = 3.7 (197 µM Ca2+), whereas the maximal value was 1.52 s-1, at 30 °C. The Ca2+-induced activation process was then monitored by using our novel, continuous fluorimetric assay with labelled MAP2 as substrate. The first-order rate constant of activation, kI(act), was derived as the reciprocal of the lag phase (‘transit time’) at the initial part of the progress curve: half-maximum was at pCa2+ = 3.8 (158 µM Ca2+) and the maximum value was 2.15 s-1. The good agreement between the kinetic parameters of µ-calpain autolysis and activation is remarkable. We claim that this is the first kinetically correct determination of the rate constant of autolysis of µ-calpain. Pre-activated µ-calpain has a Ca2+ requirement that is almost three orders of magnitude smaller [half-maximal activation at pCa2+ = 6.22 (0.6 µM Ca2+)]. We cannot exclude the possibility that the activation process involves other mechanistic steps, e.g. the rapid dissociation of the µ-calpain heterodimer, but we state that in our conditions in vitro autolysis and activation run in close parallel.
Occurrence of PG-Lb, a leucine-rich small chondroitin/dermatan sulphate proteoglycan in mammalian epiphyseal cartilage: molecular cloning and sequence analysis of the mouse cDNA
Authors: KURITA Kazuhiro , SHINOMURA Tamayuki , UJITA Minoru , ZAKO Masahiro , KIDA Daihei , IWATA Hisashi , KIMATA Koji
Published: August 2015

PG-Lb is a chondroitin/dermatan sulphate proteoglycan first isolated from chick embryo limb cartilage. It had been assumed that osteoglycin represents its mammalian homologue. However, partial amino acid sequences of a novel proteoglycan from bovine epiphyseal cartilage showed high identity with those of chick PG-Lb (P. Neame, L. Rosenberg and M. Höök, personal communication). Reverse transcriptase PCR using degenerate oligonucleotide primers gave a cDNA fragment that might correspond to mouse PG-Lb. We isolated a clone from a cDNA library of newborn mouse epiphyseal cartilage using the cDNA fragment as a probe. The cloned cDNA was 1430 bp long and contained a 966 bp open reading frame which encoded the core protein consisting of 322 amino acid residues. The deduced amino acid sequence showed a high overall identity with chick PG-Lb (about 62%, reaching about 80% over the carboxyl two-thirds). In addition, the amino acid sequence contained a signal peptide, six cysteine residues at the invariant relative position to chick PG-Lb, six leucine-rich repeats at the carboxyl two-thirds, three possible glycosaminoglycan-attachment sites (two sites at the N-terminal side and one site at the C-terminus) and two possible Asn-glycosylation sites near the C-terminus. Northern-blot analysis demonstrated the specific expression of a 1.5 kb message in cartilage and testis. These structural features and the characteristic expression suggest that the cloned molecule is mouse PG-Lb.
Multiple components of arginine and phenylalanine transport induced in neutral and basic amino acid transporter-cRNA-injected Xenopus oocytes
Authors: PETER George J , DAVIDSON Iain G. , AHMED Aamir , McILROY Lynn , FORRESTER Alexander R. , TAYLOR Peter M.
Published: August 2015

The induced uptakes of l-[3H]phenylalanine and l-[3H]arginine in oocytes injected with clonal NBAT (neutral and basic amino acid transporter) cRNA show differential inactivation by pre-treatment with N-ethylmaleimide (NEM), revealing at least two distinct transport processes. NEM-resistant arginine transport is inhibited by leucine and phenylalanine but not by alanine or valine; mutual competitive inhibition of NEM-resistant uptake of arginine and phenylalanine indicates that the two amino acids share a single transporter. NEM-senstive arginine transport is inhibited by leucine, phenylalanine, alanine and valine. At least two NEM-sensitive transporters may be expressed because we have been unable to confirm mutual competitive inhibition between arginine and phenylalanine transport. The NEM-resistant transport mechanism appears to involve distinct but overlapping binding sites for cationic and zwitterionic substrates. NBAT is known to form oligomeric protein complexes in cell membranes, and its functional roles when expressed in Xenopus oocytes may include interaction with oocyte proteins, leading to increased native amino acid transport activities; these resemble NBAT-expressed activities in terms of NEM-sensitivity and apparent substrate range (including an unusual inhibition by β-phenylalanine).
The unusual structures of the hot-regions flanking large-scale deletions in human mitochondrial DNA
Authors: HOU Jeng-Horng , WEI Yau-Huei
Published: August 2015

Large-scale deletions of mitochondrial DNA (mtDNA) are common events that have been found to occur in human ageing and in patients with mitochondrial myopathies. The mechanisms by which these deletions occur remain unclear, but several mechanisms have been proposed, such as slipped-mispairing, illegitimate recombination, and oxidative reactions elicited by free radicals. In addition, the DNA topological stress and local DNA structures have been suggested as the important factors in eliciting the recombinational events. Upon examination of 128 breakpoints of human mtDNA deletions that have been published in the past 8 years, we found that these large-scale deletions often occur at some ‘hot-regions’. We thus hypothesized that there exist unusual structures in these regions of human mtDNA that are important for eliciting the deletions. To test this hypothesis, we used PCR techniques to amplify the sequences of the so-called hot-regions and analysed the PCR products by two-dimensional gel electrophoresis. We found that the sequences of nucleotide position (np) 5221–5988, np 6928–7493, np 7901–8732 and np 15327–16228 exhibited retarded mobilities like bent DNA structures; np 5989–6750, np 13282–13653 and np 13282–14850 showed increased mobilities like anti-bent DNA structures. Moreover, except for the sequences of np 1175–1766 found in 12 S and 16 S rRNA genes exhibiting abnormal mobility like bent DNA structures, we did not observe significant mobility abnormalities in the np 499–5545 region where deletions rarely occurred. We thus conclude that these hot-regions assume some kinds of unusual DNA structures, which may render these regions more sensitive to the attack of free radicals or serve as recognition motifs for certain recombination machinery that is involved in the large-scale deletions of human mtDNA.
Pervanadate activation of intracellular kinases leads to tyrosine phosphorylation and shedding of syndecan-1
Authors: REILAND Jane , OTT Vanessa L. , LEBAKKEN Connie S. , YEAMAN Charles , McCARTHY James , RAPRAEGER Alan C.
Published: August 2015

Syndecan-1 is a transmembrane haparan sulphate proteoglycan that binds extracellular matrices and growth factors, making it a candidate to act between these regulatory molecules and intracellular signalling pathways. It has a highly conserved transmembrane/cytoplasmic domain that contains four conserved tyrosines. One of these is in a consensus sequence for tyrosine kinase phosphorylation. As an initial step to investigating whether or not phosphorylation of these tyrosines is part of a signal-transduction pathway, we have monitored the tyrosine phosphorylation of syndecan-1 by cytoplasmic tyrosine kinases in intact cells. Tyrosine phosphorylation of syndecan-1 is observed when NMuMG cells are treated with sodium orthovanadate or pervanadate, which have been shown to activate intracellular tyrosine kinases. Initial studies with sodium orthovanadate demonstrate a slow accumulation of phosphotyrosine on syndecan-1 over the course of several hours. Pervanadate, a more effective inhibitor of phosphatases, allows detection of phosphotyrosine on syndecan-1 within 5 min, with peak phosphorylation seen by 15 min. Concurrently, in a second process activated by pervanadate, syndecan-1 ectodomain is cleaved and released into the culture medium. Two phosphorylated fragments of syndecan-1 of apparent sizes 6 and 8 kDa remain with the cell after shedding of the ectodomain. The 8 kDa size class appears to be a highly phosphorylated form of the 6 kDa product, as it disappears if samples are dephosphorylated. These fragments contain the C-terminus of syndecan-1 and also retain at least a portion of the transmembrane domain, suggesting that they are produced by a cell surface cleavage event. Thus pervanadate treatment of cells results in two effects of syndecan-1: (i) phosphorylation of one or more of its tyrosines via the action of a cytoplasmic kinase(s) and (ii) cleavage and release of the ectodomain into the medium, producing a C-terminal fragment containing the transmembrane/cytoplasmic domain.
Improved thermostability of the North American firefly luciferase: saturation mutagenesis at position 354
Authors: WHITE Peter J. , SQUIRRELL David J. , ARNAUD Phillipe , LOWE Christopher R , MURRAY James A. H.
Published: August 2015

We have used random chemical mutagenesis and a simple genetic screen to generate and isolate a thermostable mutant of luciferase from the North American firefly (Photinus pyralis). A single G-to-A transition mutation, resulting in the substitution of a glutamate for a lysine residue at position 354 in the protein sequence, was shown to be responsible for this enhanced thermostability. Replacement of Glu-354 with all possible amino acid residues was achieved using directed mutagenesis, and produced mutant enzymes with a range of thermostabilities. The mutations E354K and E354R conferred the largest increases in thermostability, suggesting that side-chain size and hydrophobicity, as well as charge, may also be important contributors to the overall thermostability of the polypeptide chain at this position. Unusually for such mutations, biochemical studies suggest that this position is on the surface of the protein and exposed to solvent.
Association of the two lobes of ovotransferrin is a prerequisite for receptor recognition. Studies with recombinant ovotransferrins
Authors: MASON Anne B. , WOODWORTH Robert C. , OLIVER Ronald W. A. , GREEN Brian N. , LIN Lung-Nan , BRANDTS John F. , SAVAGE Kerry J. , TAM Beatrice M. , MACGILLIVRAY Ross T. A.
Published: August 2015

Different recombinant N-lobes of chicken ovotransferrin (oTF/2N) have been isolated from the tissue-culture medium of baby hamster kidney cells transfected with the plasmid pNUT containing the relevant DNA coding sequence. Levels of up to 40, 55 and 30 mg/l oTF/2N were obtained for constructs defining residues 1–319, 1–332 and 1–337-(Ala)3 respectively. In addition, a full-length non-glycosylated oTF was expressed at a maximum of 80 mg/l and a foreshortened oTF consisting of residues 1–682 was expressed at a level of 95 mg/l. These preparations were then used to produce, proteolytically, two different C-lobes (oTF/2C) comprising residues 342–686 and 342–682. The purified recombinant N-lobes (oTF/2N) are similar to the proteolytically derived half-molecule with regard to immunoreactivity and spectral properties; they show some interesting differences in thermal stability. A sequence analysis of the cDNA revealed six changes at the nucleotide level that led to six differences in the amino acid sequence compared with that reported by Jeltsch and Chambon [(1982) Eur. J. Biochem. 122, 291–295]. Electrospray mass spectrometry gives results consistent with these six changes. Interaction between the various N- and C-lobes was measured by titration calorimetry. Studies show that only those lobes that associate in solution are able to bind to the receptors on chick embryo red blood cells. These findings do not support a previous report by Oratore et al. [(1989) Biochem. J. 257, 301–304].
Quantification of aggrecan and link-protein mRNA in human articular cartilage of different ages by competitive reverse transcriptase-PCR
Authors: BOLTON Mark C , DUDHIA Jayesh , BAYLISS Michael T
Published: August 2015

A competitive reverse transcriptase–PCR (RT-PCR) assay has been developed for the quantification of particular mRNA species in human articular cartilage. Competitor RNA species were synthesized that differed from the amplified target sequence only by the central insertion of an EcoRI restriction site. By using known amounts of synthetic target and competitor RNA, it was shown that competitor RNA molecules designed in this way are reverse-transcribed and amplified with equal efficiency to the target of interest. Furthermore quantification could be performed during the plateau phase of the PCR, which was necessary when using ethidium bromide fluorescence as a detection system. The inhibition of aggrecan and link-protein mRNA expression by interleukin 1 or tumour necrosis factor in monolayers of human articular chondrocytes quantified by this competitive RT-PCR method compared favourably with Northern hybridization studies. The main advantage of this technique is that it can be used to quantify levels of mRNA with RNA extracted directly from 100 mg wet weight of human articular cartilage. Age-related changes in aggrecan and link-protein mRNA were therefore quantified in human articular cartilage directly after dissection from the joint. The concentration of link-protein mRNA was higher in immature cartilage than in mature cartilage when expressed relative to the amount of glyceraldehyde-3-phosphate dehydrogenase mRNA, but no age-related changes were observed in aggrecan mRNA expression. The ratio of aggrecan to link-protein mRNA was higher in mature cartilage than in immature tissue. These age-related differences in the molecular stoichiometry of aggrecan and link-protein mRNA might have implications with respect to the regulation of the formation and the stability of the proteoglycan aggregates in cartilage.
Cloning, sequencing, expression, purification and preliminary characterization of a type II dehydroquinase from Helicobacter pylori
Authors: BOTTOMLEY Joanna R , CLAYTON Christopher L. , CHALK Peter A. , KLEANTHOUS Colin
Published: August 2015

A heat-stable dehydroquinase was purified to near homogeneity from a plate-grown suspension of the Gram-negative stomach pathogen Helicobacter pylori, and shown from both its subunit and native molecular masses to be a member of the type II family of dehydroquinases. This was confirmed by N-terminal amino acid sequence data. The gene encoding this activity was isolated following initial identification, by random sequencing of the H. pylori genome, of a 96 bp fragment, the translated sequence of which showed strong identity to a C-terminal region of other type II enzymes. Southern blot analysis of a cosmid library identified several potential clones, one of which complemented an Escherichia coliaroD point mutant strain deficient in host dehydroquinase. The gene encoding the H. pylori type II dehydroquinase (designated aroQ) was sequenced. The translated sequence was identical to the N-terminal sequence obtained directly from the purified protein, and showed strong identity to other members of the type II family of dehydroquinases. The enzyme was readily expressed in E. coli from a plasmid construct from which several milligrams of protein could be isolated, and the molecular mass of the protein was confirmed by electrospray MS. The aroQ gene in H. pylori may function in the central biosynthetic shikimate pathway of this bacterium, thus opening the way for the construction of attenuated strains as potential vaccines as well as offering a new target for selective enzyme inhibition.
Circadian regulation of hydroxyindole-O-methyltransferase mRNA in the chicken pineal gland in vivo and in vitro
Authors: GRÈVE Pierre , VOISIN Pierre , GRECHEZ-CASSIAU Aline , BERNARD Marianne , COLLIN Jean-Pierre , GUERLOTTÉ Jérôme
Published: August 2015

The production of the pineal hormone melatonin displays circadian variations with high levels at night. The last enzyme involved in melatonin biosynthesis is hydroxyindole-O-methyltransferase (HIOMT, EC 2.1.1.4). The expression of the mRNA encoding chicken HIOMT was investigated in vivo and in vitro throughout the light/dark cycle, in constant darkness and with light interruption of the dark phase. The stability of HIOMT mRNA was also examined. A day/night rhythm of HIOMT mRNA levels, with a peak at the midlight phase, was observed in vivo as well as in vitro. Constant darkness did not abolish this rhythm in vivo. One cycle of the HIOMT mRNA rhythm could be observed in constant darkness in vitro. In addition, a stimulatory effect of light on HIOMT mRNA levels during the dark phase could be observed in vivo as well as in vitro. HIOMT mRNA stability was not affected by light or dark conditions, as demonstrated by chase experiments with actinomycin D. The results indicate that the daily changes in HIOMT mRNA concentration reflect transcriptional regulation by circadian oscillators and photosensory mechanisms that are endogenous to the pineal gland.
Purification and characterization of an acetyl xylan esterase produced by Streptomyces lividans
Authors: DUPONT Claude , DAIGNEAULT Nicole , SHARECK François , MOROSOLI Rolf , KLUEPFEL Dieter
Published: August 2015

The acetyl xylan esterase cloned homologously from Streptomyces lividans [Shareck, Biely, Morosoli and Kluepfel (1995) Gene 153, 105–109] was purified from culture filtrate of the overproducing strain S. lividans IAF43. The secreted enzyme had a molecular mass of 34 kDa and a pI of 9.0. Under the assay conditions with chemically acetylated birchwood xylan the kinetic constants of the enzyme were: specific activity, 715 units/mg, Km 7.94 mg/ml and Vmax 1977 units/mg. Optimal enzyme activity was obtained at 70 °C and pH 7.5. Hydrolysis assays with different acetylated substrates showed that the enzyme is specific for deacetylating the O-acetyl group of polysaccharides and is devoid of N-deacetylation activity. Sequential hydrolysis shows that its action is essential for the complete degradation of acetylated xylan by the xylanases of S. lividans.
Asp-49 is not an absolute prerequisite for the enzymic activity of low-Mr phospholipases A2: purification, characterization and computer modelling of an enzymically active Ser-49 phospholipase A2, ecarpholin S, from the venom of Echis carinatus sochureki (saw-scaled viper)
Authors: POLGÁR János , MAGNENAT Edith M. , PEITSCH Manuel C. , WELLS Timothy N. C. , CLEMETSON Kenneth J.
Published: August 2015

Several studies have shown that Asp-49 is the residue that controls calcium binding in, and so plays a critical role in the calcium-mediated activation of, low-Mr group I-III phospholipases A2 (PLA2s). The present paper provides experimental evidence that Asp-49 is not an absolute prerequisite for the enzymic activity of PLA2s, and that proteins with amino acid(s) other than Asp at position 49 can exhibit significant phospholipase activity. The purification, complete amino acid sequence and characterization of ecarpholin S, a PLA2 from Echis carinatus sochureki (saw-scaled viper) venom, is described. This single-chain, 122-amino-acid, basic (pI 7.9) protein is a group II PLA2. Although Asp-49 is replaced by Ser and Tyr-28 by Phe (both of these positions being involved in the Ca2+-binding site of PLA2s), the lipolysis of soybean phosphatidylcholine and egg yolk in the presence of 10 mM CaCl2 was 1.5 times and 2.9 times greater respectively with ecarpholin S than with recombinant human group II PLA2. The Ca2+-dependencies of the enzymic activities of ecarpholin S and rPLA2 were found to be similar. Ecarpholin S added to washed platelets induced aggregation; the presence of Ca2+ was a prerequisite for this platelet-aggregating effect. Computer modelling of the Ca2+-binding site of Ser-49 PLA2 compared with the Asp-49 and Lys-49 forms, for which crystallographic data exist, shows that the Ca2+-binding site is sterically blocked by Lys-49 but not by Ser-49; in the latter, the Ser hydroxy group may replace the Asp carboxylate in stabilization of Ca2+ binding. Sequence comparisons of ecarpholin S and other low-Mr PLA2s predicts the presence of a Ser-49 group in the protein family of low-Mr PLA2s that is distinct from the Asp-49 and Lys-49 groups.
Inhibition of insulin release by synthetic peptides shows that the H3 region at the C-terminal domain of syntaxin-1 is crucial for Ca2+- but not for guanosine 5′-[γ-thio]triphosphate-induced secretion
Authors: MARTIN Franz , SALINAS Eva , VAZQUEZ Jesús , SORIA Bernat , REIG Juan A.
Published: August 2015

Recently, we have described the presence and possible role of syntaxin in pancreatic β-cells by using monoclonal antibodies [F. Martin, F. Moya, L. M. Gutierrez, J. A. Reig, B. Soria (1995) Diabetologia 38, 860–863]. In order to characterize further the importance of specific domains of this protein, the functional role of a particular region of the syntaxin-1 molecule has now been investigated by using two synthetic peptides, SynA and SynB, corresponding to two portions of the H3 region at the C-terminal domain of the protein, residues 229–251 and 197–219 respectively. Functional experiments carried out in permeabilized pancreatic β-cells demonstrate that these peptides inhibit Ca2+- dependent insulin release in a dose-dependent manner. This effect is specific because peptides of the same composition but random sequence do not show the same effect. In contrast with this inhibitory effect on Ca2+-induced secretion, both peptides increase basal release. However, under the same conditions, SynA and SynB do not affect guanosine 5´-[γ-thio]triphosphate-induced insulin release. These results demonstrate that specific portions of the H3 region of syntaxin-1 are involved in critical protein–protein interactions specifically during Ca2+-induced insulin secretion.
Immunochemical detection of CoA-modified mitochondrial matrix proteins
Authors: HUTH Walter , PAULI Christian , MÖLLER Ulrike
Published: August 2015

An in vitro assay mixture consisting of mitochondrial matrix proteins from rat liver and CoA resulted in the formation of CoA-modified proteins. CoA-modified proteins were demonstrated by detection of CoA. CoA was released from the proteins by dithioerythritol treatment under denaturing conditions and was identified by (a) its retention time on HPLC, (b) its absorption spectrum and (c) its activity in a CoA-specific assay. This CoA represents protein-bound CoA and, in addition, protein-bound palmitoyl-CoA when MgATP was also present in the in vitro assay. The detection of immunoreactive proteins using monospecific anti-CoA antibodies exclusively identifies CoA-modified proteins. The specificity of these antibodies can be used to identify both endogenously occurring CoA-modified proteins and proteins that have been modified in the in vitro assay. An intact thiol group of CoA is an essential precondition for the modification to occur.