bluuebunny/crossref_metadata_2025 huggingface dataset:
Quantitative profiling of tissue- and gender-related expression of glutathione S-transferase isoenzymes in the mouse
Authors: MITCHELL Alyson E. , MORIN Dexter , LAKRITZ Jeffrey , JONES A. Daniel
Published: August 2015

Cytosolic glutathione S-transferase (GST) isoenzymes from brain, heart, lung, liver, kidney and gonads of male and female CD-1 mice were identified and quantified with a combination of affinity purification, electrospray ionization MS, Edman microsequencing, Western blot analysis and reverse-phase HPLC. The three principal hepatic GST subunits, mGSTA3 (25271 Da), mGSTP1 (23478 Da), and mGSTM1 (25839 Da), were isolated from liver, lung, kidney, testes and female heart, whereas brain, ovaries and male heart contained mGSTM1 and mGSTP1. Additional isoenzymes were detected in tissues, including mu class subunits mGSTM2 (25580 Da) and mGSTM3 (25570 Da), an N-terminally blocked Alpha subunit (25480 Da) assigned as mGSTA4, and proteins of molecular masses 25490, 22540, 24493, 24378 and 25383 Da. Distinct gender differences in expression of GST subunits were observed for liver, heart, kidney and gonads, whereas GST expression was similar in brain and lung for both genders. In contrast with patterns of expression in liver (high ratio of mGSTA3 to mGSTP1 in females relative to males), mGSTP1 was the most abundant subunit in female gonads, whereas mGSTA3 was not present in detectable quantities. The profile of GST expression in kidney was similar to that in liver; however, male kidneys expressed 30% more soluble GST than female kidneys. A striking gender-related difference in GST expression was found in cardiac tissue, where female animals expressed 50% more soluble GST than male tissues, and the GST isoenzyme with the greatest documented activity towards lipid hydroperoxides, mGSTA3, was present in female tissue yet absent from male tissue. These results point to complex gender- and tissue-dependent expression of individual mouse GST isoenzymes.
Involvement of diacylglycerol production in activation of nuclear factor κB by a CD14-mediated lipopolysaccharide stimulus
Authors: YAMAMOTO Hisanori , HANADA Kentaro , NISHIJIMA Masahiro
Published: August 2015

Exposure of Chinese hamster CHO-K1 transfectant cells expressing mouse CD14 (CHO/CD14 cells) to lipopolysaccharide (LPS) induced rapid elevation of the cellular diacylglycerol (DAG) and choline/phosphocholine levels and nuclear translocation of nuclear factor κB (NFκB). When cells were incubated with short-chain DAG analogues or bacterial phospholipase C, NFκB activation occurred even without the LPS stimulus. Treatment of CHO/CD14 cells with tricyclo[5.2.1.02.6]decyl-(9[8])xanthogenate (D609), an inhibitor of phosphatidylcholine-specific phospholipase C and phospholipase D, almost completely inhibited not only the LPS-dependent production of DAG and choline/phosphocholine but also the LPS-dependent NFκB activation. In contrast, treatment of cells with 1-(6-{[3-methoxyoestra-1,3,5(10)-trien-17β-yl]-1H-pyrrole-2,5-dione (U73122), an inhibitor of phosphatidylinositol-specific phospholipase C in vitro, did not affect the LPS-dependent activation of NFκB. Production of DAG and activation of NFκB after the LPS stimulus were observed in mouse macrophage-like J774.1 cells, and this response to LPS by J774.1 cells was also inhibited by D609. These results suggest that the production of DAG from phosphatidylcholine was upstream of NFκB activation in response to a CD14-mediated LPS stimulus.
Interactions of polyamines with ion channels
Authors: WILLIAMS Keith
Published: August 2015

Endogenous polyamines, in particular spermine, have been found to cause block and modulation of a number of types of ion channel. Intracellular spermine is responsible for intrinsic gating and rectification of strong inward rectifier K+ channels by directly plugging the ion channel pore. These K+ channels control the resting membrane potential in both excitable and non-excitable cells, and control the excitability threshold in neurons and muscle cells. Intracellular spermine causes inward rectification at some subtypes of Ca2+-permeable glutamate receptors in the central nervous system, again by plugging the receptor channel pore, and spermine can even permeate the ion channel of these receptors. Extracellular spermine has multiple effects at the N-methyl-D-aspartate (NMDA) subtype of glutamate receptor, including stimulation that increases the size of NMDA receptor currents, and voltage-dependent block. A number of polyamine-conjugated arthropod toxins and synthetic polyamine analogues are potent antagonists of glutamate receptors, and represent new tools with which to study these receptors. Interactions of polyamines with other types of cation channels have been reported. This area of research represents a new biology and a new pharmacology of polyamines.
Adhesion of fibroblasts to fibronectin stimulates both serine and tyrosine phosphorylation of paxillin
Authors: BELLIS Susan L. , PERROTTA Joseph A. , CURTIS Michael S. , TURNER Christopher E.
Published: August 2015

Tyrosine phosphorylation of paxillin by the focal adhesion kinase (FAK) has been implicated as a signal transduction mechanism associated with cell adhesion and cytoskeletal reorganization. The potential role of serine phosphorylation of paxillin in these events has not been well characterized. In this study we have examined the phosphorylation profile of paxillin both invitro and invivo. By using glutathione S-transferase–paxillin fusion proteins in precipitation-kinase assays invitro we observed that a fusion protein spanning amino acid residues 54–313 of paxillin, and containing a FAK-binding site, precipitated substantial serine kinase activity as well as FAK activity from a smooth-muscle lysate. Together these kinases phosphorylated paxillin on tyrosine residue 118, a site that has been identified previously as a target for FAK phosphorylation, and on serine residues 188 and/or 190. The binding site for the serine kinase, the identity of which is currently unknown, was further mapped to residues 168–191 of paxillin. To assess the physiological relevance of these sites phosphorylated invitro, the profile of paxillin phosphorylation invivo stimulated by seeding fibroblasts on fibronectin was characterized. As expected, plating cells on fibronectin enhanced the tyrosine phosphorylation of paxillin. However, 96% of the phosphorylation of paxillin occurred on serine residues. Comparison by two-dimensional phosphopeptide analyses indicated that the major sites of tyrosine and serine phosphorylation detected in the assays invitro co-migrate with phosphopeptides derived from paxillin phosphorylated invivo in response to plating cells on fibronectin. These findings support a role for both tyrosine and serine kinases in the signal transduction pathway linking integrin activation to paxillin phosphorylation.
Angiotensin II type 2 receptors mediate inhibition of mitogen-activated protein kinase cascade and functional activation of SHP-1 tyrosine phosphatase
Authors: BEDECS Katarina , ELBAZ Nathalie , SUTREN Malène , MASSON Maryline , SUSINI Christiane , STROSBERG A. Donny , NAHMIAS Clara
Published: August 2015

Angiotensin II type 2 (AT2) receptors are involved in the inhibition of cell proliferation as well as in apoptosis and neuronal differentiation, through intracellular signalling pathways that remain poorly defined. The present study examines the effect of AT2-receptor stimulation on growth-factor-induced pathways leading to the activation of mitogen-activated protein (MAP) kinases. In N1E-115 neuroblastoma cells, AT2 receptors inhibit the activity of MAP kinases induced by serum as well as by epidermal growth factor. The inhibitory effect of angiotensin II (Ang II) is rapid and transient, and affects both ERK1 and ERK2 (extracellular signal-related protein kinase) isoforms of the enzyme. AT2-mediated MAP kinase inactivation is not sensitive to pertussis toxin or okadaic acid, but involves a vanadate-sensitive protein tyrosine phosphatase (PTP). Expression of MAP kinase phosphatase-1 (MKP-1) is not significantly modified upon AT2-receptor activation, and insensitivity to actinomycin D also rules out transcriptional induction of other MKPs as a possible mechanism for AT2-mediated inactivation of MAP kinases. In addition, we report here that both in N1E-115 cells and in Chinese hamster ovary cells expressing recombinant human AT2 receptors, Ang II rapidly stimulates the catalytic activity of SHP-1, a soluble PTP that has been implicated in termination of signalling by cytokine and growth-factor receptors. These findings thus demonstrate functional negative cross-talk between heptahelical AT2 receptors and receptor tyrosine kinases, and suggest that SHP-1 tyrosine phosphatase is an early transducer of the AT2 receptor signalling pathway.
Subunit diversity and tissue distribution of human glutathione S-transferases: interpretations based on electrospray ionization-MS and peptide sequence-specific antisera
Authors: ROWE Jonathan D. , NIEVES Edward , LISTOWSKY Irving
Published: August 2015

Uncertainties about the composition and identities of glutathione S-transferases (GSTs) in human tissue have impeded studies on their biological functions. A rigorous protocol has therefore been developed to characterize the human proteins. Cytosolic GST subunits were resolved by reverse-phase HPLC methods, individual components were assigned to Alpha, Mu and Pi classes on the basis of their immunoreactivities, and peptide-sequence-specific antisera were used to distinguish among five different Mu-class subunits (GSTM1–GSTM5). Each subunit type was characterized and identified unambiguously by electrospray ionization-MS. Acetylation of N-terminal residues in the GSTA1, GSTA2, GSTM3 and GSTM4 subunits were the only natural post-translational modifications detected. The unique structure of GSTM3, with N- and C-terminal peptide extensions predicted from cDNA sequences, was confirmed. Only testis and brain were rich sources of GSTM3 subunits. Subunit profiles were distinct and characteristic of the particular tissue type, and this tissue specificity in GST expression was evident even in organs from different individuals. For instance, livers had relatively simple GST compositions, consisting of a preponderance of Alpha-class subunits and GSTM1 (when present). By contrast, representation of most subunit types was a characteristic feature of testis, which had the highest levels of GSTs. GSTM4 and GSTM5 subunits, here identified for the first time in human tissue extracts, were minor components, with GSTM5 found only in brain, lung and testis. Specimens devoid of GSTM1 subunits, particularly those from null-genotype individuals, were readily discerned at the protein level. Liver was the only rich source of the GSTM1 subunit (although it also constituted a major fraction of adrenal GSTs), and so the functional consequences of the GSTM1 gene deletion are likely to vary in extrahepatic tissues.
The effects of truncations of the small subunit on m-calpain activity and heterodimer formation
Authors: ELCE John S. , DAVIES Peter L. , HEGADORN Carol , MAURICE Donald H. , ARTHUR J. Simon C.
Published: August 2015

In order to study subunit interactions in calpain, the effects of small subunit truncations on m-calpain activity and heterodimer formation have been measured. It has been shown previously that active calpain is formed by co-expression of the large subunit (80 kDa) of rat m-calpain with a δ86 form (21 kDa) of the small subunit. cDNA for the full-length 270 amino acid (28.5 kDa) rat calpain small subunit has now been cloned, both with and without an N-terminal histidine tag (NHis10). The full-length small subunit constructs yielded active calpains on co-expression with the large subunit, and the small subunit was autolysed to 20 kDa on exposure of these calpains to Ca2+. A series of deletion mutants of the small subunit, NHis10-δ86,-δ99, -δ107, and -δ116, gave active heterodimeric calpains with unchanged specific activities, although in decreasing yield, and with a progressive decrease in stability. NHis10-δ125 formed a heterodimer which was inactive and unstable. Removal of 25 C-terminal residues from δ86, leaving residues 87–245, abolished both activity and heterodimer formation. The results show that: (a) generation of active m-calpain in Escherichia coli requires heterodimer formation; (b) small subunit residues between 94 and 116 contribute to the stability of the active heterodimer but do not directly affect the catalytic mechanism; (c) residues in the region 245–270 are essential for subunit binding. Finally, it was shown that an inactive mutant Cys105 → Ser-80k/δ86 calpain, used in order to preclude autolysis, did not dissociate in the presence of Ca2+, a result which does not support the proposal that Ca2+-induced dissociation is involved in calpain activation.
Cysteine residues in human lysosomal acid lipase are involved in selective cholesteryl esterase activity
Authors: PAGANI Franco , PARIYARATH Rajalakshmi , STUANI Cristiana , GARCIA Rodolfo , BARALLE Francisco E.
Published: August 2015

Human lysosomal acid lipase (LAL) catalyses the deacylation of triacylglycerol and cholesteryl esters in the acidic lysosomal compartment. Treatment of LAL with the reducing agent dithiothreitol affected the triacylglycerol and cholesteryl esterase activities differentially, suggesting the involvement of cysteine residues in determining substrate specificity. To identify the residues involved, human LAL cDNA, under the control of the T7 promoter and tagged with a herpes simplex virus coding epitope, was specifically mutated in order to introduce single amino acid substitutions of each of the six cysteine residues of mature LAL. All Cys-227 mutants showed selectively decreased activity towards cholesteryl oleate, while preserving that towards trioleylglycerol. Substitutions of Cys-236, Cys-240 and Cys-244 affected catalysis towards the two substrates to a variable degree, depending on the side chain of the amino acid introduced. The replacement of Cys-41 or Cys-188 did not result in the preferential cleavage of either one of the two substrates. These data indicate that Cys-227, Cys-236, Cys-240 and Cys-244 play a crucial role in determining LAL substrate specificity. We propose that these cysteine residues are involved in the hydrolysis of cholesteryl ester by affecting selectively the access of this substrate to the catalytic active site. In addition, the fact that the catalytic activity is never completely abolished in cysteine mutants demonstrates that LAL is not a thiol enzyme.
A specific distal promoter controls γ-glutamyl transpeptidase gene expression in undifferentiated rat transformed liver cells
Authors: NOMURA Seiji , LAHUNA Olivier , SUZUKI Takanobu , BROUILLET Arthur , CHOBERT Marie Noêle , LAPERCHE Yannick
Published: August 2015

In rat undifferentiated hepatoma cells, the γ-glutamyl transpeptidase (GGT) gene is transcribed into a 2.3 and a 2.6 kb mRNA which, in contrast with other rat GGT transcripts, are not detected in more differentiated liver cells or adult tissues. Analysis of the cDNA sequences obtained from H5 hepatoma cells reveals that these two transcripts differ from other GGT mRNAs by a 312-nt unique untranslated leader sequence; this sequence maps on the gene in a single exon 10 kb upstream from the GGT promoter IV transcription start site. We established that the 2.6 kb mRNA V-1 and the 2.3 kb GGT mRNA V-2 derive, by alternate splicing, from a primary transcript initiated on a distal promoter on the rat GGT gene. This gene appears to be transcribed from five promoters, and the specific expression of this new distal promoter in undifferentiated hepatoma cells requires binding of activator protein-1 and hepatic nuclear factor 3 specific transcription factors to a composite cis-element in the proximal region of the promoter. The distal GGT promoter, specifically expressed in undifferentiated liver cells, might reflect the expression of that gene in liver precursor cells before they differentiate in the hepatocytic or biliary lineage.
Isolation, cloning and characterization of a low-molecular-mass purine nucleoside- and nucleotide-binding protein
Authors: GILMOUR Jeffrey , LIANG Nianci , LOWENSTEIN John M.
Published: August 2015

A purine nucleoside- and nucleotide-binding protein has been isolated from extracts of rat and rabbit heart, calf aortic smooth muscle and rat liver using an affinity column containing adenosine bound through the N6-position. The protein, which was eluted by adenosine, was cloned and expressed in Escherichia coli. The deduced amino acid sequence has a calculated Mr of 13693 (p13.7). The expressed protein has properties identical with the protein isolated from heart and liver, including an anomalous, apparent Mr of 15300, observed on gel electrophoresis. Gel filtration shows it to be a dimer. p13.7 differs by only three amino acids out of 125 from protein kinase C inhibitor 1 [Pearson, DeWald, Mathews, Mozier, Zürcher-Neely, Heinrikson, Morris, McCubbin, McDonald, Fraser et al. (1990) J. Biol. Chem. 265, 4583–4591]. However, we have not been able to demonstrate inhibition of protein kinase C by physiological concentrations of p13.7, regardless of whether it was isolated from tissue extracts or expressed in E. coli. p13.7 is a member of the histidine triad motif family of proteins [Séraphin (1992) J. DNA Sequencing Mapping 3, 177–179]. The affinity of p13.7 for a number of different purine nucleosides and nucleotides, as measured by fluorescence titration and gel filtration, falls within the range 5–50 μM. On the basis of these properties and its crystal structure [Brenner, Garrison, Gilmour, Peisach, Ringe, Petsko and Lowenstein (1997) Nature Struct. Biol. 4, 231–238], we have coined the acronym HINT (histidine triad nucleotide-binding motif) to describe the family of proteins of which p13.7 is a member. Other proteins that bind to the affinity column have been identified as malate and lactate dehydrogenases, cAMP-binding proteins, adenosine kinase and S-adenosylhomocysteine hydrolase.
Glutathione synthetase: similarities of the proteins from Schizosaccharomyces pombe and Arabidopsis thaliana
Authors: WANG Chang-Lin , OLIVER David J.
Published: August 2015

Glutathione synthetase predicted from the reported gene sequence from Schizosaccharomyces pombe is substantially smaller than the equivalent protein predicted from the cDNAs sequenced fromArabidopsis thaliana, Saccharomyces cerevisiae and other eukaryotes. Sequence alignments of the proteins encoded by the cDNA clones for glutathione synthetase from Arabidopsis and S. pombe show that the Arabidopsis protein contains 200 extra amino acids at the N-terminus. In order to test if this sequence is essential in the function of the protein, the full-length Arabidopsis protein and as two N-terminal deletions (Δ67–71 and Δ67–200) were expressed in S. pombemutant MN101, which lacks endogenous glutathione synthetase activity. Although the wild-type plant cDNA could complement the yeast mutation, neither deletion mutant was able to restore glutathione-dependent cadmium resistance. When the three proteins were expressed as fusion proteins in Escherichia coli, they accumulated to the same level, but only the plasmid containing the full-length cDNA, pFLAG222, produced detectable enzyme activity in vitro. These results suggested that the N-terminus of the Arabidopsisglutathione synthetase is essential for its function and opened up the possibility that there was a sequencing error in the reported S. pombe sequence. Therefore the gsh2 sequence from wild-type S. pombeand the mutant strain MN101 were determined. The wild-type S. pombe gsh2 encodes a protein that is about the same length as that found in Arabidopsis, and the MN101 mutation involves a frameshift mutation early in the glutathione synthetase reading frame.
Comparative anatomy of the aldo–keto reductase superfamily
Authors: JEZ Joseph M. , BENNETT Melanie J. , SCHLEGEL Brian P. , LEWIS Mitchell , PENNING Trevor M.
Published: August 2015

The aldo–keto reductases metabolize a wide range of substrates and are potential drug targets. This protein superfamily includes aldose reductases, aldehyde reductases, hydroxysteroid dehydrogenases and dihydrodiol dehydrogenases. By combining multiple sequence alignments with known three-dimensional structures and the results of site-directed mutagenesis studies, we have developed a structure/function analysis of this superfamily. Our studies suggest that the (α/β)8-barrel fold provides a common scaffold for an NAD(P)(H)-dependent catalytic activity, with substrate specificity determined by variation of loops on the C-terminal side of the barrel. All the aldo–keto reductases are dependent on nicotinamide cofactors for catalysis and retain a similar cofactor binding site, even among proteins with less than 30% amino acid sequence identity. Likewise, the aldo–keto reductase active site is highly conserved. However, our alignments indicate that variation of a single residue in the active site may alter the reaction mechanism from carbonyl oxidoreduction to carbon–carbon double-bond reduction, as in the 3-oxo-5β-steroid 4-dehydrogenases (Δ4-3-ketosteroid 5β-reductases) of the superfamily. Comparison of the proposed substrate binding pocket suggests residues 54 and 118, near the active site, as possible discriminators between sugar and steroid substrates. In addition, sequence alignment and subsequent homology modelling of mouse liver 17β-hydroxysteroid dehydrogenase and rat ovary 20α-hydroxysteroid dehydrogenase indicate that three loops on the C-terminal side of the barrel play potential roles in determining the positional and stereo-specificity of the hydroxysteroid dehydrogenases. Finally, we propose that the aldo–keto reductase superfamily may represent an example of divergent evolution from an ancestral multifunctional oxidoreductase and an example of convergent evolution to the same active-site constellation as the short-chain dehydrogenase/reductase superfamily.
Association of purified skeletal-muscle AMP deaminase with a histidine–proline-rich-glycoprotein-like molecule
Authors: RANIERI-RAGGI Maria , MONTALI Umberto , RONCA Francesca , SABBATINI Antonietta , BROWN Paul E. , MOIR Arthur J. G. , RAGGI Antonio
Published: August 2015

Denaturation of rabbit skeletal-muscle AMP deaminase in acidic medium followed by chromatography on DEAE-cellulose in 8 M urea at pH 8.0 allows separation of two main peptide components of similar apparent molecular mass (75–80 kDa) that we tentatively assume correspond to two different enzyme subunits. Whereas the amino acid composition of one of the two peptides is in good agreement with that derived from the nucleotide sequence of the known rat and human AMPD1 cDNAs, the second component shows much higher contents of proline, glycine and histidine. N-Terminal sequence analysis of the fragments liberated by limited proteolysis with trypsin of the novel peptide reveals a striking similarity to the fragments produced by plasmin cleavage of the rabbit plasma protein called histidine–proline-rich glycoprotein (HPRG). However, some divergence is observed between the sequence of one of the fragments liberated from AMP deaminase by a more extensive trypsinization and rabbit plasma HPRG in the region containing residues 472–477. A fragment with a blocked N-terminus, which was found among those liberated by proteolysis with pepsin of either whole AMP deaminase or the novel component of the enzyme, shows an amino acid composition quite different from that of the N-terminus of the known subunit of AMP deaminase. By coupling this observation with the detection in freshly prepared AMP deaminase of a low yield of the sequence (LTPTDX) corresponding to that of HPRG N-terminus, it can be deduced that in comparison with HPRG, the putative HPRG-like component of AMP deaminase contains an additional fragment with a blocked N-terminus, which is liberated by a proteolytic process during purification of the enzyme. The implications of the association to rabbit skeletal-muscle AMP deaminase of a HPRG-like protein species are discussed.
NAD(P)H-dependent aldose reductase from the xylose-assimilating yeast Candida tenuis: Isolation, characterization and biochemical properties of the enzyme
Authors: NEUHAUSER Wilfried , HALTRICH Dietmar , KULBE Klaus D. , NIDETZKY Bernd
Published: August 2015

During growth on D-xylose the yeast Candida tenuis produces one aldose reductase that is active with both NADPH and NADH as coenzyme. This enzyme has been isolated by dye ligand and anion-exchange chromatography in yields of 76%. Aldose reductase consists of a single 43 kDa polypeptide with an isoelectric point of 4.70. Initial velocity, product inhibition and binding studies are consistent with a compulsory-ordered, ternary-complex mechanism with coenzyme binding first and leaving last. The catalytic efficiency (kcat/Km) in D-xylose reduction at pH 7 is more than 60-fold higher than that in xylitol oxidation and reflects significant differences in the corresponding catalytic centre activities as well as apparent substrate-binding constants. The enzyme prefers NADP(H) approx. 2-fold to NAD(H), which is largely due to better apparent binding of the phosphorylated form of the coenzyme. NADP+ is a potent competitive inhibitor of the NADH-linked aldehyde reduction (Ki 1.5 μM), whereas NAD+ is not. Unlike mammalian aldose reductase, the enzyme from C. tenuisis not subject to oxidation-induced activation. Evidence of an essential lysine residue located in or near the coenzyme binding site has been obtained from chemical modification of aldose reductase with pyridoxal 5′-phosphate. The results are discussed in the context of a comparison of the enzymic properties of yeast and mammalian aldose reductase.
Interaction of Ras with phosphoinositide 3-kinase γ
Authors: RUBIO Ignacio , RODRIGUEZ-VICIANA Pablo , DOWNWARD Julian , WETZKER Reinhard
Published: August 2015

Phosphoinositide 3-kinase γ (PI3Kγ) can be activated in vitro by both α and βγ subunits of heterotrimeric G-proteins and does not interact with p85, the regulatory subunit of PI3Kα. Here we demonstrate the binding of Ras to PI3Kγ in vitro. An N-terminal region of PI3Kγ was identified as a binding site for Ras. After co-expression with PI3Kγ in COS-7 cells, Ras induced only a modest increase in PI3K activity compared with the stimulation of PI3Kα by Ras in the same cells.
Syndecans: multifunctional cell-surface co-receptors
Authors: CAREY David J.
Published: August 2015

This review will summarize our current state of knowledge of the structure, biochemical properties and functions of syndecans, a family of transmembrane heparan sulphate proteoglycans. Syndecans bind a variety of extracellular ligands via their covalently attached heparan sulphate chains. Syndecans have been proposed to play a role in a variety of cellular functions, including cell proliferation and cell–matrix and cell–cell adhesion. Syndecan expression is highly regulated and is cell-type- and developmental-stage-specific. The main function of syndecans appears to be to modulate the ligand-dependent activation of primary signalling receptors at the cell surface. Principal functions of the syndecan core proteins are to target the heparan sulphate chains to the appropriate plasma-membrane compartment and to interact with components of the actin-based cytoskeleton. Several functions of the syndecans, including syndecan oligomerization and actin cytoskeleton association, have been localized to specific structural domains of syndecan core proteins.
Lysosomal α-mannosidases of mouse tissues: characteristics of the isoenzymes, and cloning and expression of a full-length cDNA
Authors: BECCARI T. , APPOLLONI M. G. , COSTANZI E. , STINCHI S. , STIRLING J. L. , DELLA FAZIA M. A. , SERVILLO G. , VIOLA M. P. , ORLACCHIO A.
Published: August 2015

Lysosomal α-D-mannosidase from mouse tissues was separated into its constituent isoenzymes by DEAE-cellulose chromatography. Forms corresponding to the human isoenzymes B and A were present in testis, brain, spleen and kidney, whereas in epididymis and liver only the B form was present. Murine α-mannosidases A and B are glycoproteins and have pH optima, thermal stabilities and molecular masses similar to those of the human isoenzymes. A full-length cDNA (3.1 kb) containing the complete coding sequence for α-mannosidase was isolated from a mouse macrophage cDNA library. Comparison of the deduced amino acid sequences of human and mouse α-mannosidases showed that they had 75% identity and 83% similarity. Expression of this cDNA in COS cells showed that both the A and the B isoenzymes can arise from a single transcript. Northern blotting analysis showed a 10-fold range in the abundance of α-mannosidase mRNA in mouse tissues, with the highest levels found in epididymis, and the lowest in liver.
Influence of thyroid hormone on the tissue-specific expression of cytochrome c oxidase isoforms during cardiac development
Authors: MEEHAN Julia , KENNEDY John M.
Published: August 2015

In mammals, cytochrome coxidase (COX) is composed of 13 different protein subunits. In the rat, two nuclear-encoded subunits, COX VIa and VIII, exist as tissue-specific isoforms: heart and liver. Using Northern-blot analysis, the levels of transcripts for the heart and liver isoforms of VIa and VIII were examined in developing rat hearts. The liver isoform was found to be the predominant form of subunit VIa and the exclusive form of VIII in the 18-day fetal hearts. The mRNA levels of the heart isoform of both subunits increased dramatically to reach adult levels by 14 days. Although the levels of the VIa- and VIII-liver isoform mRNAs remained stable throughout early development, their levels decreased by 40 and 36% respectively between the 18-day fetal stage and 18-day neonatal stage. Therefore the up-regulation of the heart isoforms and down-regulation of the liver isoforms appear to be regulated in a co-ordinated manner during development. To determine if thyroid hormone influences the expression of these developmentally regulated isoforms, the RNA was also extracted from the hearts of 2-week-old hypothyroid rats. The results showed that the levels of VIII-heart and VIa-liver COX mRNAs were approx. 40% lower in the hypothyroid hearts, while VIII-liver and VIa-heart COX isoform expression remained unchanged. These data demonstrate that the isoforms of COX subunits VIa and VIII are not co-ordinately regulated by changes in thyroid hormone levels. Therefore we conclude that, although thyroid hormone influences the expression of isoforms, it appears to do so via a different mechanism from that which regulates the developmental transition.
Purification and characterization of δ-(l-α-aminoadipyl)-l-cysteinyl-d-valine synthetase from Penicillium chrysogenum
Authors: THEILGAARD Hanne B.Aa. , KRISTIANSEN Klaus N. , HENRIKSEN Claus M. , NIELSEN Jens
Published: August 2015

δ-(L-α-Aminoadipyl)-L-cysteinyl-d-valine synthetase (ACVS) from Penicillium chrysogenum was purified to homogeneity by a combination of (NH4)2SO4 precipitation, protamine sulphate treatment, ion-exchange chromatography, gel filtration and hydrophobic interaction chromatography. The molecular mass of ACVS was estimated with native gradient gel electrophoresis and SDS/PAGE. The native enzyme consisted of a single polymer chain with an estimated molecular mass of 470 kDa. The denatured enzyme had an estimated molecular mass of 440 kDa. The influence of different reaction parameters such as substrates, cofactors and pH on the activity of the purified ACVS was investigated. The Km values for the three precursor substrates L-α-aminoadipic acid, L-cysteine and L-valine were determined as 45, 80 and 80 μM respectively, and the optimal assay concentration of ATP was found to be 5 mM (with 20 mM MgCl2). The dimer of the reaction product bis-δ-(L-α-aminoadipyl)-l-cysteinyl-D-valine (bisACV) gave feedback inhibition of the purified ACVS; the inhibition parameter KbisACV was determined as 1.4 mM. Furthermore dithiothreitol was shown to inhibit the purified ACVS. From the addition of a glucose pulse to a steady-state glucose-limited continuous culture of P. chrysogenum it was found that there is glucose repression of the synthesis of ACVS and that there must be a constant turnover of ACVS owing to synthesis and degradation.
Mouse white adipocytes and 3T3-L1 cells display an anomalous pattern of carnitine palmitoyltransferase (CPT) I isoform expression during differentiation: Inter-tissue and inter-species expression of CPT I and CPT II enzymes
Authors: BROWN Nicholas F. , HILL Jennifer K. , ESSER Victoria , KIRKLAND James L. , CORKEY Barbara E. , FOSTER Daniel W. , MCGARRY J. Denis
Published: August 2015

The outer mitochondrial membrane enzyme carnitine palmitoyltransferase I (CPT I) represents the initial and regulated step in the β-oxidation of fatty acids. It exists in at least two isoforms, denoted L (liver) and M (muscle) types, with very different kinetic properties and sensitivities to malonyl-CoA. Here we have examined the relative expression of the CPT I isoforms in two different models of adipocyte differentiation and in a number of rat tissues. Adipocytes from mice, hamsters and humans were also evaluated. Primary monolayer cultures of undifferentiated rat preadipocytes expressed solely L-CPT I, but significant levels of M-CPT I emerged after only 3 days of differentiation in vitro; in the mature cell M-CPT I predominated. In sharp contrast, the murine 3T3-L1 preadipocyte expressed essentially exclusively L-CPT I, both in the undifferentiated state and throughout the differentiation process in vitro. This was also true of the mature mouse white fat cell. Fully developed adipocytes from the hamster and human behaved similarly to those of the rat. Thus the mouse white fat cell differs fundamentally from those of the other species examined in terms of its choice of a key regulatory enzyme in fatty acid metabolism. In contrast, brown adipose tissue from all three rodents displayed the same isoform profiles, each expressing overwhelmingly M-CPT I. Northern blot analysis of other rat tissues established L-CPT I as the dominant isoform not only in liver but also in kidney, lung, ovary, spleen, brain, intestine and pancreatic islets. In addition to its primacy in skeletal muscle, heart and fat, M-CPT I was also found to dominate in the testis. The same inter-tissue isoform pattern (with the exception of white fat) was found in the mouse. Taken together, the data bring to light an intriguing divergence between white adipocytes of the mouse and other mammalian species. They also raise a cautionary note that should be considered in the choice of animal model used in further studies of fat cell physiology.
Prolonged activation of phospholipase D in Chinese hamster ovary cells expressing platelet-activating-factor receptor lacking cytoplasmic C-terminal tail
Authors: LIU Bo , NAKASHIMA Shigeru , ADACHI Takahito , ITO Yuzuru , TAKANO Tomoko , SHIMIZU Takao , NOZAWA Yoshinori
Published: August 2015

The mechanism and role of phospholipase D (PLD) activation by platelet-activating factor (PAF) were examined with Chinese hamster ovary cells stably expressing wild-type PAF receptor (WT-H cells) and truncated PAF receptor lacking the C-terminal cytoplasmic tail (D-H cells). Treatment of D-H cells with PAF resulted in the rapid formation of Ins(1,4,5)P3, which was followed by a sustained phase for more than 10 min. In these cells, PAF-induced PLD activation lasted for more than 20 min. In contrast, PLD activation in WT-H cells was transient. PAF stimulation caused the biphasic formation of 1,2-diacylglycerol (DG) in both types of cell. The first phase was rapid and transient, coinciding with the Ins(1,4,5)P3 peak. The second sustained phase of DG formation was attenuated by butanol, which produces phosphatidylbutanol at the expense of phosphatidic acid (PA) by transphosphatidylation activity of PLD, and by propranolol, a selective inhibitor for PA phosphohydrolase catalysing the conversion of PA into DG. The DG level returned nearly to basal at 20 min after PAF stimulation in WT-H cells, whereas in D-H cells the elevated DG level was sustained for more than 20 min. The profile of translocation of protein kinase Cα (PKCα) to membrane was similar to that of DG formation. In WT-H cells, PKCα was transiently associated with membranes and then returned to the cytosol. However, in D-H cells PKCα was rapidly translocated to and remained in membranes for more than 20 min. Butanol suppressed this sustained translocation of PKCα. Furthermore the mRNA levels of c-fos and c-jun by PAF in WT-H cells were much lower than those in D-H cells. Propranolol and butanol at concentrations that inhibited the formation of DG suppressed the PAF-induced mRNA expression of c-fos and c-jun. Taken together, the prolonged PLD activation in D-H cells confirmed a primary role for phospholipase C/PKC in PLD activation by PAF. Furthermore the results obtained here suggest that sustained PLD activation in turn leads to chronic activation and membrane translocation of PKCα, which might play an important role in the expression of c-fos and c-jun.
Rapid stimulation of amyloid precursor protein release by epidermal growth factor: role of protein kinase C
Authors: SLACK Barbara E. , BREU Jeffrey , MUCHNICKI Lisa , WURTMAN Richard J.
Published: August 2015

The amyloid precursor protein (APP) of Alzheimer's disease is a transmembrane protein that is cleaved by an uncharacterized enzyme known as α-secretase within its extracellular/intraluminal domain after the activation of guanine nucleotide-binding protein-coupled receptors linked to phosphoinositide hydrolysis. The secretory process results in the release of large soluble derivatives of APP (APPs), and, when elicited by muscarinic receptor activation, exhibits both protein kinase C (PKC)-dependent and tyrosine phosphorylation-dependent components [Slack, Breu, Petryniak, Srivastava and Wurtman (1995) J. Biol. Chem. 270, 8337–8344]. In this report we examine the regulation of the release of APPs by epidermal growth factor (EGF) receptors, which possess intrinsic tyrosine kinase activity, and are coupled to a variety of effectors including phosphoinositide-specific phospholipase Cγ. In A431 cells, EGF caused time-dependent and dose-dependent increases in the formation of inositol phosphates in cultures prelabelled with myo-[3H]inositol, and in the release of APPs into the culture medium; the two responses exhibited similar time courses and EC50 values for EGF. Concomitant with these effects, there were concentration-dependent (3–300 ng/ml) increases in the phosphorylation of tyrosine residues in several proteins, including the EGF receptor itself. The specific PKC antagonist GF 109203X decreased the effect of EGF by approx. 35% at a concentration that abolished the stimulation of the release of APPs by the PKC activator PMA. Tyrphostin AG 1478, an inhibitor of EGF receptor tyrosine kinase, abolished the EGF-induced release of APPs. These results demonstrate that in A431 cells, activation of the EGF receptor stimulates α-secretase activity by a mechanism that is partly dependent on PKC activity.
Human group II 14 kDa phospholipase A2 activates human platelets
Authors: POLGÁR János , KRAMER Ruth M. , UM Suzane L. , JAKUBOWSKI Joseph A. , CLEMETSON Kenneth J.
Published: August 2015

Recombinant human group II phospholipase A2 (sPLA2) added to human platelets in the low μg/ml range induced platelet activation, as demonstrated by measurement of platelet aggregation, thromboxane A2 generation and influx of intracellular free Ca2+ concentration and by detection of time-dependent tyrosine phosphorylation of platelet proteins. The presence of Ca2+ at low millimolar concentrations is a prerequisite for the activation of platelets by sPLA2. Mg2+ cannot replace Ca2+. Mg2+, given in addition to the necessary Ca2+, inhibits sPLA2-induced platelet activation. Pre-exposure to sPLA2 completely blocked the aggregating effect of a second dose of sPLA2. Albumin or indomethacin inhibited sPLA2-induced aggregation, similarly to the inhibition of arachidonic acid-induced aggregation. Platelets pre-treated with heparitinase or phosphatidylinositol-specific phospholipase C lost their ability to aggregate in response to sPLA2, although they still responded to other agonists. This suggests that a glycophosphatidylinositol-anchored platelet-membrane heparan sulphate proteoglycan is the binding site for sPLA2 on platelets. Previous reports have stated that sPLA2 is unable to activate platelets. The inhibitory effect of albumin and Mg2+, frequently used in aggregation studies, and the fact that isolated platelets lose their responsiveness to sPLA2 relatively quickly, may explain why the platelet-activating effects of sPLA2 have not been reported earlier.
α-Adrenergic stimulation induces phosphorylation of retinoblastoma protein in neonatal rat ventricular myocytes
Authors: LIU Qingquan , DAWES Nicola J. , LU Yujuan , SHUBEITA Huda S. , ZHU Hong
Published: August 2015

Mammalian cardiac myocytes become postmitotic shortly after birth, and the subsequent myocardial growth in adaptation to increasing workloads becomes primarily dependent on hypertrophy of existing myocytes. Although hypertrophic growth of cardiac myocytes has been extensively studied by using both in vitro and in vivo models, the molecular mechanism controlling the switch from hyperplastic to hypertrophic growth of cardiac myocytes is largely unknown. Since the majority of terminally differentiated cardiac myocytes are growth-arrested in G1/G0 phase, it has been hypothesized that the retinoblastoma protein (Rb) or its related pocket proteins which block G1/S transition becomes constitutively active during myocardial terminal differentiation. To test this hypothesis, we studied the regulation of Rb activity by α-adrenergic stimulation in neonatal rat ventricular myocytes which are mostly postmitotic in culture. Our results demonstrate that Rb is predominantly in the active hypophosphorylated state in control neonatal ventricular myocytes. α-Adrenergic stimulation activates G1/S transition in foetal but not neonatal rat ventricular myocytes. Although α-adrenergic stimulation does not activate G1/S transition in neonatal myocytes, it induces hyperphosphorylation of Rb to the same extent as in proliferating skeletal-muscle myoblasts or foetal ventricles. Hyper- but not hypo-phosphorylated Rb in stimulated neonatal myocytes or proliferating skeletal-muscle myoblasts fails to bind to the transcription factor, E2F, suggesting that hyperphosphorylated Rb is inactive. Therefore G1/S transition could also be blocked at steps in addition to Rb inactivation during terminal differentiation and these blockades are refractory to α-adrenergic stimulation.
Mechanism and regulation of Mg-chelatase
Authors: WALKER J. Caroline , WILLOWS D. Robert
Published: August 2015

Mg-chelatase catalyses the insertion of Mg into protoporphyrin IX (Proto). This seemingly simple reaction also is potentially one of the most interesting and crucial steps in the (bacterio)chlorophyll (Bchl/Chl)-synthesis pathway, owing to its position at the branch-point between haem and Bchl/Chl synthesis. Up until the level of Proto, haem and Bchl/Chl synthesis share a common pathway. However, at the point of metal-ion insertion there are two choices: Mg2+ insertion to make Bchl/Chl (catalysed by Mg-chelatase) or Fe2+ insertion to make haem (catalysed by ferrochelatase). Thus the relative activities of Mg-chelatase and ferrochelatase must be regulated with respect to the organism's requirements for these end products . How is this regulation achieved? For Mg-chelatase, the recent design of an in vitro assay combined with the identification of Bchl-biosynthetic enzyme genes has now made it possible to address this question. In all photosynthetic organisms studied to date, Mg-chelatase is a three-component enzyme, and in several species these proteins have been cloned and expressed in an active form. The reaction takes place in two steps, with an ATP-dependent activation followed by an ATP-dependent chelation step. The activation step may be the key to regulation, although variations in subunit levels during diurnal growth may also play a role in determining the flux through the Bchl/Chl and haem branches of the pathway.
Interleukin-6-dependent and -independent regulation of the human C-reactive protein gene
Authors: WEINHOLD Birgit , RÜTHER Ulrich
Published: August 2015

We have investigated the function of different mediators of the regulation of the human C-reactive protein (hCRP) gene in transgenic mice. hCRP was induced by lipopolysaccharide and wounding in interleukin-6 (IL-6) +/+ mice, but not in IL-6 -/- mice. This finding suggested that IL-6 is necessary for the induction of hCRP. However, injection of IL-6 did not induce the hCRP gene. Thus, the induction of hCRP by IL-6 seems to require an additional cofactor. Therefore, we screened different cytokines for their activity in IL-6 +/+ and IL-6 -/- mice. Surprisingly, interleukin-1β, as well as oncostatin M or leukaemia inhibitory factor, led to an induction of hCRP in both genetic backgrounds. These results indicate an IL-6-dependent and -independent regulation of hCRP. These hCRP transgenic mice therefore represent a novel model system for defining the cytokine network involved in the regulation of acute-phase genes during the course of inflammation.
Membrane-binding properties of phospholipase C-β1 and phospholipaseC-β2: role of the C-terminus and effects of polyphosphoinositides, G-proteins and Ca2+
Authors: JENCO M. John , BECKER P. Kevin , MORRIS J. Andrew
Published: August 2015

We have studied the binding of two G-protein-regulated phospholipase C (PLC) enzymes, PLCs-β1 and -β2, to membrane surfaces using sucrose-loaded bilayer phospholipid vesicles of varying compositions. Neither enzyme binds appreciably to pure phosphatidylcholine vesicles at lipid concentrations up to 10-3 M. PLC-β1 and PLC-β2 bind vesicles composed of phosphatidylcholine, phosphatidylserine and phosphatidylethanolamine (molar ratio 1:1:1) with an approximate Kd of 10-5 M. Inclusion of 2% PtdIns(4,5)P2 in these vesicles had no effect on the affinity of this interaction. As reported by others, removal of the C-terminus of PLC-β1 and PLC-β2 produces catalytically active fragments. The affinity of these truncated proteins for phospholipid vesicles is dramatically reduced suggesting that this region of the proteins contains residues important for membrane binding. Inclusion of G-protein α- and βγ-subunit activators in the phospholipid vesicles does not increase the binding of PLC-β1 or PLC-β2, and the magnitude of G-protein-mediated PLC activation observed at low phospholipid concentrations (10-6 M) is comparable to that observed at concentrations at which the enzymes are predominantly membrane-bound (10-3 M). PLC-β1 and -β2 contain C2 domains but Ca2+ does not enhance binding to the vesicles. Our results indicate that binding of these enzymes to membranes involves the C-temini of the proteins and suggest that activation of these enzymes by G-proteins results from a regulated interaction between the membrane-bound proteins rather than G-protein-dependent recruitment of soluble enzymes to a substrate-containing phospholipid surface.