bluuebunny/crossref_metadata_2025 huggingface dataset:
Transformation of subcutaneous nitric oxide into nitrate in the rat
Authors: BENTHIN Günther , BJÖRKHEM Ingemar , BREUER Olof , SAKINIS Augustinas , WENNMALM Åke
Published: August 2015

Following its addition to arterialized blood in vitro, nitric oxide (NO) is transformed into nitrate in the erythrocytes. Inhaled NO is similarly transformed into nitrate in the blood in vivo. These observations suggest that nitrate is a universal end-metabolite of NO, i.e. of endogenously formed NO as well. However, endogenous NO may also be inactivated in tissues, i.e. outside the vascular lumen. To study the fate of NO metabolized with delayed access to the blood, rats were given subcutaneous injections of 15NO or K15NO3, and the plasma concentrations of 15NO3- were followed for 450 min after injection. The values for the distribution volume and plasma decay (t½) of 15NO3- did not differ between rats given 15N-labelled NO and NO3-. The area under the plasma decay curve for rats given 15NO amounted to 89% of the corresponding area for animals given K15NO3. This demonstrates that 15NO, when given extravascularly in millimolar concentrations, is mainly transformed into 15N-labelled nitrate. Other rats were kept in an atmosphere containing a mixture of 16O2 and 18O2. Nitrate residues containing either one or two 18O atoms were isolated from the blood, indicating that inhaled oxygen was incorporated during both the formation of NO and the subsequent transformation of NO into nitrate. The fraction of nitrate residues containing two 18O atoms was larger than that containing one 18O atom. We propose that nitrate is a major stable metabolite of endogenous NO that does not primarily diffuse into the vascular lumen following formation. Hence nitrate seems to be the quantitatively most important end-product of the metabolism of endogenous NO. The transformation of endogenous NO into nitrate involves the incorporation of inhaled oxygen.
Deletion of 24 amino acids from the C-terminus of phosphatidylinositol transfer protein causes loss of phospholipase C-mediated inositol lipid signalling
Authors: PROSSER Simon , SARRA Robert , SWIGART Philip , BALL Andrew , COCKCROFT Shamshad
Published: August 2015

Phosphatidylinositol transfer protein α (PITPα) is a 32 kDa protein of 270 amino acids that is essential for phospholipase C-mediated phosphatidylinositol bisphosphate hydrolysis. In addition, it binds and transfers phosphatidylinositol and phosphatidylcholine between membrane compartments in vitro. Here we have used limited proteolysis of PITPα by subtilisin to identify the structural requirements for function. Digestion by subtilisin results in the generation of a number of slightly smaller peptide fragments, the major fragment being identified as a 29 kDa protein. The fragments were resolved by size-exclusion chromatography and were found to be totally inactive in both in vivo PLC reconstitution assays and in vitro phosphatidylinositol transfer assays. N-terminal sequencing and MS of the major 29 kDa fragment shows that cleavage occurs at the C-terminus of PITP at Met246, leading to a deletion of 24 amino acid residues. We conclude that the C-terminus plays an important role in mediating PLC signalling in vivo and lipid transfer in vitro, supporting the notion that lipid transfer may be a facet of PITP function in vivo.
Regulatory elements in the first intron of the rat fatty acid synthase gene
Authors: OSKOUIAN Babak , RANGAN Vangipuram S. , SMITH Stuart
Published: August 2015

Sequence elements have been identified within the 1.2 kb-long first intron of the fatty acid synthase (FAS) gene that mediate both positive and negative effects on transcription. The negative regulatory element, when positioned downstream of either the FAS or simian virus 40 promoter, down-regulates the expression of a coupled reporter gene in an orientation-dependent manner. Sequences mediating this effect have been mapped, by deletion mutagenesis, to two regions approximately within nucleotides +405 to +768 and +924 to +1083. Both regions contain sequence elements that are strongly protected from DNase I digestion by nuclear extracts prepared from liver, but not by those prepared from spleen. The results of run-on assays performed with nuclei derived from tissues that express FAS at either high or low levels indicate that the different rates of transcription of the endogenous FAS gene result from differences in the extent of initiation, so it is unlikely that the negative effect is caused by transcriptional pausing in the first intron. The positive element maps to nt +292 to +297 and corresponds to an authentic binding site for upstream stimulatory factor (USF). This USF-binding element can up-regulate transcription from a heterologous promoter in a position- and orientation-independent manner. However, in the context of the entire FAS first intron, the effect of the USF-binding site is masked unless the effect of the negative elements is ablated by mutagenesis. These results suggest that the dominant negative element of the first intron may play a role in determining the tissue-specific expression of the FAS gene.
Identification of membrane dipeptidase as a major glycosyl-phosphatidylinositol-anchored protein of the pancreatic zymogen granule membrane, and evidence for its release by phospholipase A
Authors: HOOPER Nigel M. , COOK Sarah , LAINÉ Jean , LeBEL Denis
Published: August 2015

Membrane dipeptidase (EC 3.4.13.19) enzyme activity that is inhibited by cilastatin has been detected in pancreatic zymogen granule membranes of human, porcine and rat origin. Immunoelectrophoretic blot analysis of human and porcine pancreatic zymogen granule membranes with polyclonal antisera raised against the corresponding kidney membrane dipeptidase revealed that the enzyme is a disulphide-linked homodimer of subunit mass 61 kDa in the human and 45 kDa in the pig. Although membrane dipeptidase was, along with glycoprotein-2, one of the only two major components of carbonate high pH-washed membranes, no enzyme activity or immunoreactivity was detected in the zymogen granule contents. Digestion with bacterial phosphatidylinositol-specific phospholipase C (PI-PLC), and subsequent recognition by antibodies specific for the cross-reacting determinant, revealed that membrane dipeptidase in human and porcine pancreatic zymogen granule membranes is glycosyl-phosphatidylinositol-anchored. Membrane dipeptidase was released from the pancreatic zymogen granule membranes by an endogenous hydrolase, and the released form migrated as a disulphide-linked dimer on SDS/PAGE under non-reducing conditions. Under reducing conditions it migrated with the same apparent molecular mass as the membrane-bound form, and was still a substrate for bacterial PI-PLC. Treatment of kidney microvillar membranes with phospholipase A2 resulted in the release of membrane dipeptidase in a form that demonstrated electrophoretic and cilastatin–Sepharose binding properties identical to those of the endogenously released form of the enzyme from zymogen granule membranes. These results indicate that the glycosyl-phosphatidylinositol anchor on the pancreatic membrane dipeptidase is cleaved by an endogenous hydrolase, probably a phospholipase A, and that this cleavage may promote the release of the protein from the membrane.
Expression of the calcium-independent cytokine-inducible (iNOS) isoform of nitric oxide synthase in rat placenta
Authors: CASADO Marta , DÍAZ-GUERRA María J. M. , RODRIGO José , FERNÁNDEZ Ana P. , BOSCÁ Lisardo , MARTÍN-SANZ Paloma
Published: August 2015

The presence of the calcium-independent cytokine-inducible nitric oxide synthase (iNOS) has been investigated in rat placenta from day 19 of gestation till delivery. iNOS has been detected at the mRNA, enzyme activity and protein levels in complete placenta. Immunocytochemical detection of iNOS was heterogeneously distributed in control placenta. Intraperitoneal injection of pregnant rats at 21 days of gestation with lipopolysaccharide (LPS) increased the iNOS immunoreactivity in the decidua basalis of the placenta, and, when the mRNA levels and enzyme activity were measured in total tissue, a moderate increase (approx. 160%) was observed. A constitutive nuclear factor κB activity was observed in placenta from both control and LPS-treated animals. These results indicate constitutive expression of iNOS in rat placenta.
Two genes encoding an endoglucanase and a cellulose-binding protein are clustered and co-regulated by a TTA codon in Streptomyces halstedii JM8
Authors: GARDA Ana Lila , FERNÁNDEZ-ABALOS José M. , SÁNCHEZ Pilar , RUIZ-ARRIBAS Alberto , SANTAMARÍA Ramón I.
Published: August 2015

Streptomyces halstediiJM8 Cel2 is an endoglucanase of 28 kDa that is first produced as a protein of 42 kDa (p42) and is later processed at its C-terminus. Cel2 displays optimal activity towards CM-cellulose at pH 6 and 50 °C and shows no activity against crystalline cellulose or xylan. The N-terminus of p42 shares similarity with cellulases included in family 12 of the β-glycanases and the C-terminus shares similarity with bacterial cellulose-binding domains included in family II. This latter domain enables the precursor to bind so tightly to Avicel that it can only be eluted by boiling in 10% (w/v) SDS. Another open reading frame (ORF) situated 216 bp downstream from the p42 ORF encodes a protein of 40 kDa (p40) that does not have any clear hydrolytic activity against cellulosic or xylanosic compounds, but shows high affinity for Avicel (crystalline cellulose). The p40 protein is processed in old cultures to give a protein of 35 kDa that does not bind to Avicel. Translation of both ORFs is impaired in Streptomyces coelicolor bldA mutants, suggesting that a TTA codon situated at the fourth position of the first ORF is responsible for this regulation. S1 nuclease protection experiments demonstrate that both ORFs are co-transcribed.
Latent transforming growth factor-β complex in Chinese hamster ovary cells contains the multifunctional cysteine-rich fibroblast growth factor receptor, also termed E-selectin-ligand or MG-160
Authors: OLOFSSON Anders , HELLMAN Ulf , DIJKE Peter TEN , GRIMSBY Susanne , ICHIJO Hidenori , MORÉN Anita , MIYAZONO Kohei , HELDIN Carl-Henrik
Published: August 2015

Transforming growth factor-β (TGF-β) is secreted as latent high molecular mass complexes from producer cells. The N-terminal precursor remnant, also called latency-associated peptide (LAP), forms a non-covalently linked complex with TGF-β and confers the latency to TGF-β. In human platelets and certain other cell types, latent TGF-β binding protein-1 (LTBP-1) is disulphide-linked to LAP, and forms complexes of more than 230 kDa. In addition, LTBP-2 and -3, which are structurally similar to LTBP-1, can be part of latent TGF-β complexes. In Chinese hamster ovary (CHO) cells transfected with the TGF-β1 cDNA, a major part of the latent TGF-β secreted into the medium is a 100-kDa small latent complex containing TGF-β and LAP. In addition, we found two other forms of latent TGF-β complexes, i.e. a 220-kDa complex containing LTBP-1, and a 220-kDa complex containing a 140-kDa protein. Purification of the 140-kDa component, termed latent TGF-β complexed protein-1 (LTCP-1), followed by amino acid sequencing and cDNA cloning from a CHO cell cDNA library, revealed that it is a hamster counterpart of a previously identified, multifunctional protein known as chicken cysteine-rich fibroblast growth factor (FGF) receptor, mouse E-selectin-ligand and rat MG-160 (a 160-kDa membrane sialoglycoprotein of the Golgi apparatus). Immunoprecipitation of LTCP-1 and TGF-β1 from CHO cells stably transfected with TGF-β1 precursor cDNA revealed that the expressed protein forms a complex with LAP, and that a major part of the complex is secreted. Northern blot analysis showed that mRNA for LTCP-1 was expressed in large amounts in testis, ovary and placenta, but less abundantly in other tissues. These results suggest that TGF-β, produced in certain cell types, may form a complex with LTCP-1, which may have different properties compared with other latent TGF-β complexes. It remains to be investigated whether the complex formation between LTCP-1 and TGF-β1 also occurs in other cells, whether the association between them occurs in the Golgi complex, and whether it affects the interaction of LTCP-1 with FGF or E-selectin.
Sphingosine 1-phosphate stimulates Rho-mediated tyrosine phosphorylation of focal adhesion kinase and paxillin in Swiss 3T3 fibroblasts
Authors: WANG Fang , NOBES Catherine D. , HALL Alan , SPIEGEL Sarah
Published: August 2015

Sphingosine 1-phosphate (SPP), a sphingolipid second messenger implicated in the mitogenic action of platelet-derived growth factor [Olivera, A. and Spiegel, S. (1993) Nature (London) 365, 557–560], induced rapid reorganization of the actin cytoskeleton resulting in stress-fibre formation. SPP also induced transient tyrosine phosphorylation of focal adhesion kinase (p125FAK), a cytosolic tyrosine kinase that localizes in focal adhesions, and of the cytoskeleton-associated protein paxillin. Exoenzyme C3 transferase, which ADP-ribosylates Rho (a Ras-related small GTP binding protein) on asparagine-41 and renders it biologically inactive, inhibited both stress-fibre formation and protein tyrosine phosphorylation induced by SPP. Thus Rho may be an upstream regulator of both stress-fibre formation and tyrosine phosphorylation of p125FAK and paxillin. Pretreatment with PMA, an activator of protein kinase C (PKC), inhibited the stimulation of stress-fibre formation induced by 1-oleoyl-lysophosphatidic acid (LPA) but not that by SPP. Similarly, PMA also decreased LPA-induced tyrosine phosphorylation of p125FAK and paxillin without abrogating the response to SPP. Thus PKC is involved in LPA- but not SPP-dependent signalling. The polyanionic drug suramin, a broad-specificity inhibitor of ligand–receptor interactions, did not inhibit either the mitogenic effect of SPP or its stimulation of tyrosine phosphorylation of p125FAK. However, suramin markedly inhibited these responses induced by LPA. These results suggest that in contrast with LPA, SPP may be acting intracellularly in Swiss 3T3 fibroblasts to stimulate tyrosine phosphorylation of p125FAK and paxillin and cell growth.
A soluble 3-hydroxy-3-methylglutaryl-CoA reductase in the protozoan Trypanosoma cruzi
Authors: PEÑA-DÍAZ Javier , MONTALVETTI Andrea , CAMACHO Ana , GALLEGO Claribel , RUIZ-PEREZ Luis M. , GONZALEZ-PACANOWSKA Dolores
Published: August 2015

We report the isolation and characterization of a genomic clone containing the open reading frame sequence for 3-hydroxy-3-methylglutaryl-CoA (HMG-CoA) reductase from Trypanosoma cruzi, the causative agent of Chagas' disease. The protozoan gene encoded for a smaller polypeptide than the rest of the genes described from eukaryotic organisms and the deduced amino acid sequence could be aligned with the C-terminal half of animal and plant reductases exhibiting pronounced similarity to other eukaryotic counterparts. Further examination of the 5′ flanking region by cDNA analysis and establishment of the splice acceptor sites clearly indicated that the corresponding mRNA apparently lacks sequences encoding a membrane N-terminal domain. The reductase gene is a single copy and is located on a chromosome of 1.36 Mb as determined by contour-clamped homogeneous electric field electrophoresis. The overall cellular distribution of enzymic activity was investigated after differential centrifugation of Trypanosoma cell extracts. Reductase activity was primarily associated with the cellular soluble fraction because 95% of the total cellular activity was recovered in the supernatant and was particularly sensitive to proteolytic inactivation. Furthermore the enzyme can be efficiently overexpressed in a highly active form by using the expression vector pET-11c. Thus Trypanosoma cruziHMG-CoA reductase is unique in the sense that it totally lacks the membrane-spanning sequences present in all eukaryotic HMG-CoA reductases so far characterized.
Construction, expression and characterization of chimaeric toxins containing the ribonucleolytic toxin restrictocin: intracellular mechanism of action
Authors: RATHORE Dharmendar , BATRA Janendra K.
Published: August 2015

Restrictocin is a ribonucleolytic toxin produced by the fungus Aspergillus restrictus. Two chimaeric toxins containing restrictocin directed at the human transferrin receptor have been constructed. Anti-TFR(scFv)–restrictocin is encoded by a gene produced by fusing the DNA encoding a single-chain antigen-combining region (scFv) of a monoclonal antibody, directed at the human transferrin receptor, at the 5′ end of that encoding restrictocin. The other chimaeric toxin, restrictocin–anti-TFR(scFv), is encoded by a gene fusion containing the DNA encoding the single-chain antigen-combining region of antibody to human transferrin receptor at the 3′ end of the DNA encoding restrictocin. These gene fusions were expressed in Escherichia coli, and fusion proteins purified from the inclusion bodies by simple chromatography techniques to near-homogeneity. The two chimaeric toxins were found to be equally active in inhibiting protein synthesis in a cell-free in vitrotranslation assay system. The chimaeric toxins were selectively toxic to the target cells in culture with potent cytotoxic activities. However, restrictocin–anti-TFR(scFv) was more active than anti-TFR(scFv)–restrictocin on all cell lines studied. By using protease and metabolic inhibitors, it can be shown that, to manifest their cytotoxic activity, the restrictocin-containing chimaeric toxins need to be proteolytically processed intracellularly and the free toxin or a fragment thereof thus generated is translocated to the target via a route involving the Golgi apparatus.
Gene structure and functional analysis of the human Na+/phosphate co-transporter
Authors: TAKETANI Yutaka , MIYAMOTO Ken-ichi , TANAKA Keiko , KATAI Kanako , CHIKAMORI Mika , TATSUMI Sawako , SEGAWA Hiroko , YAMAMOTO Hironori , MORITA Kyoko , TAKEDA Eiji
Published: August 2015

Three λ phage clones encompassing the Na+/phosphate co-transporter (NaPi-3) gene and its 5′ flanking region were isolated from a human genomic DNA library. The gene comprises 13 exons and 12 introns and spans approx. 14 kb. All exon–intron junctions conform to the GT/AG rule. The major transcription-initiation site was determined by primer-extension analysis and is an adenosine residue 57 bp upstream of the 3′ end of the first exon. There is a typical TATA box 28 bp upstream of the major transcription-initiation site and various cis-acting elements, including a cAMP-responsive element, AP-1, AP-2 and SP-1 sites in the 5′ flanking region. This region also contains three direct-repeat-like sequences that resemble the consensus binding sequence for members of the steroid–thyroid hormone receptor superfamily, including vitamin D. Deletion analysis suggests that the region from nt-2409 to nt-1259 in the 5′ flanking region may be involved in kidney-specific gene expression. Vitamin D responsiveness of the NaPi-3 promoter was also detected in COS-7 cells co-transfected with a human vitamin D receptor expression vector. The presence of the three vitamin D receptor-responsive elements in the NaPi-3 promoter may be important in mediating the enhanced expression of the gene by 1,25-dihydroxyvitamin D3.
Identification of an 11-residue portion of CTP–phosphocholine cytidylyltransferase that is required for enzyme–membrane interactions
Authors: YANG Jilin , WANG Jinxia , TSEU Irene , KULISZEWSKI Maciej , LEE Wensu , POST Martin
Published: August 2015

CTP–phosphocholine cytidylyltransferase (CT) is a key regulatory enzyme in the biosynthesis of phosphatidylcholine (PC) in many cells. Enzyme–membrane interactions appear to play an important role in CT activation. A putative membrane-binding domain appears to be located between residues 236 and 293 from the N-terminus. To map the membrane-binding domain more precisely, glutathione S-transferase fusion proteins were prepared that contained deletions of various domains in this putative lipid-binding region. The fusion proteins were assessed for their binding of [3H]PC/oleic acid vesicles. Fusion proteins encompassing residues 267–277 bound to PC/oleic acid vesicles, whereas fragments lacking this region exhibited no specific binding to the lipid vesicles. The membrane-binding characteristics of the CT fusion proteins were also examined using intact lung microsomes. Only fragments encompassing residues 267–277 competed with full-length 125I-labelled CT, expressed in recombinant Sf9 insect cells, for microsomal membrane binding. To investigate the role of this region in PC biosynthesis, A549 and L2 cells were transfected with cDNA for CT mutants under the control of a glucocorticoid-inducible long terminal repeat (LTR) promoter. Induction of CT mutants containing residues 267–277 in transfectants resulted in reduced PC synthesis. The decrease in PC synthesis was accompanied by a shift in endogenous CT activity from the particulate to the soluble fraction. Expression of CT mutants lacking this region in A549 and L2 cells did not affect PC formation and subcellular distribution of CT activity. These results suggest that the CT region located between residues 267 and 277 from the N-terminus is required for the interaction of CT with membranes.
Platelet-derived growth factor activation of mitogen-activated protein kinase depends on the sequential activation of phosphatidylcholine-specific phospholipase C, protein kinase C-ζ and Raf-1
Authors: VAN DIJK Marc C. M. , HILKMANN Henk , VAN BLITTERSWIJK Wim J.
Published: August 2015

The mechanism of Raf-1 activation by platelet-derived growth factor (PDGF) is poorly defined. We previously reported that, in Rat-1 fibroblasts, PDGF activates a phosphatidylcholine-specific phospholipase C (PC-PLC) and that the product, diacylglycerol, somehow activates protein kinase C-ζ (PKC-ζ). Both PC-PLC and PKC-ζ activities were required for PDGF activation of mitogen-activated protein kinase (MAPK). Now we report that MAPK activation by exogenous PC-PLC depends on Raf-1 activation. PKC-ζ co-immunoprecipitates with, phoshorylates and activates Raf-1, suggesting that in the PDGF- and PC-PLC-activated MAPK pathway, PKC-ζ operates in a signalling complex as a direct activator of Raf-1.
n 6 and n 3 Polyunsaturated fatty acids stimulate translocation of protein kinase Cα, -βI, -βII and -ε and enhance agonist-induced NADPH oxidase in macrophages
Authors: HUANG Zhi Hua , HII Charles S. T. , RATHJEN Deborah A. , POULOS Alf , MURRAY Andrew W. , FERRANTE Antonio
Published: August 2015

The polyunsaturated fatty acids (PUFA), arachidonic acid (AA), eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA) were poor inducers of oxygen-dependent respiratory activity (chemiluminescence) in human monocytes and macrophages, but markedly enhanced the response to the tripeptide, N-formylmethionyl-leucyl-phenylalanine. The effects of these fatty acids were seen at concentrations of 1 μg/ml. A similar enhancement was seen with PMA, a stimulus that acts on protein kinase C (PKC), or calcium ionophore (A23187), which increases intracellular calcium, suggesting that the effect of the fatty acids was post-surface receptor binding. HL-60 cells, differentiated to macrophage-like cells by culture in the presence of vitamin D3, were similarly affected by the fatty acids. In experiments in which the time of pre-exposure of the monocytes to PUFA was varied, it was found that the priming effect induced by AA, EPA and DHA was maximal at 5 min. The ability of these fatty acids to synergize with other agonists was completely lost if the fatty acids were either methylated or oxidized to the hydro and hydroperoxy derivatives. Saturated fatty acids were inactive. Western blot analysis demonstrated that the PUFA induced the translocation of PKCα, -βI, -βII and -ϵ isoenzymes to a particulate fraction. The synergistic response between fatty acids and A23187 was completely inhibited by pretreating the cells with a PKC inhibitor, GF-109203X, or by pretreatment of monocytes with PMA for 18 h, to deplete PKC levels. From these investigations it is evident that PUFA prime macrophages, causing increased/synergistic oxidative respiratory burst activity to other stimuli and that this priming is dependent on PKC translocation and activation.
Analysis of the endoplasmic reticular Ca2+ requirement for α1-antitrypsin processing and transport competence
Authors: COOPER Gary R. , BROSTROM Charles O. , BROSTROM Margaret A.
Published: August 2015

Depletion of Ca2+ sequestered within the endoplasmic reticulum (ER) of HepG2 hepatoma cells results in the luminal accumulation of immature α1-antitrypsin possessing Man8–9 GlcNAc2 oligosaccharide side chains. This study explores the basis for this arrest and describes consequent alterations in the size and rate of secretion of the complex endoglycosidase H-resistant form of the protein. Inhibition of glucosidase I and II with castanospermine or α-1,2-mannosidase with 1-deoxymannojirimycin produced altered ER processing intermediates that were rapidly secreted. Subsequent mobilization of ER Ca2+ stores resulted in the appearance and retention of slightly larger related forms of these intermediates. Retention of glycosylated intermediates was not ascribable to an association with α1,2-mannosidase or lectin-like chaperones, the intermediates were not degraded and all evidence of ER retention or size alterations produced by Ca2+ depletion was quickly reversed by Ca2+ restoration. Cells that were Ca2+ depleted for 2 h slowly secreted an abnormal slightly smaller complex oligosaccharide form of α1-antitrypsin at approximately the same rate as the non-glycosylated protein generated by treatment with tunicamycin. The hypothesis that Ca2+ affects the folding and ER transport competence of glycosylated forms of α1-antitrypsin is discussed.
Peptidyl inverse esters of p-methoxybenzoic acid: a novel class of potent inactivator of the serine proteases
Authors: LYNAS John , WALKER Brian
Published: August 2015

A series of novel synthetic peptides, containing a C-terminal β-amino alcohol linked to p-methoxybenzoic acid via an ester linkage, have been prepared and tested as inhibitors against typical members of the serine protease family. For example, the sequences Ac-Val-Pro-NH-CH-(CH2-C6H5)-CH2O-CO-C6H4-OCH3 (I) and Ac-Val-Pro-NH-CH-[CH-(CH3)2]-CH2O-CO-C6H4-OCH3 (II), which fulfil the known primary and secondary specificity requirements of chymotrypsin and elastase respectively, have been found to behave as exceptionally potent irreversible inactivators of their respective target protease. Thus I was found to inactivate chymotrypsin with an overall second-order rate constant (k2/Ki) of approx. 6.6×106 M-1·s-1, whereas II is an even more potent inactivator of human neutrophil elastase, exhibiting a second-order rate constant of inactivation of approx. 1.3×107 M-1·s-1. These values represent the largest rate constants ever reported for the inactivation of these proteases with synthetic peptide-based inactivators. On prolonged incubation in substrate-containing buffers, samples of the inactivated proteases were found to regain activity slowly. The first-order rate constants for the regeneration of enzymic activity from chymotrypsin and human neutrophil elastase inactivated by I and II respectively were determined to be approx. 5.8×10-5 s-1 and approx. 4.3×10-4 s-1. We believe that the most likely mechanism for the inactivation and regeneration of enzymic activity involves the formation and subsequent slow hydrolysis of long-lived acyl enzyme intermediates.
Rac1 is required for cell proliferation and G2/M progression
Authors: MOORE Kimberly A. , SETHI Rachna , DOANES A. Masharn , JOHNSON Thomas M. , PRACYK John B. , KIRBY Martha , IRANI Kaikobad , GOLDSCHMIDT-CLERMONT Pascal J. , FINKEL Toren
Published: August 2015

We have transiently expressed a dominant negative form of rac1 (N17rac1) using adenoviral-mediated gene transfer. The level of N17rac1 expression is demonstrated to be proportional to the multiplicity of infection. Expression of N17rac1 in Rat 2 fibroblasts results in cytostatic growth arrest. Cell-cycle analysis demonstrates that cells expressing N17rac1 accumulate in G2/M. These results suggest that rac1 is required for cell proliferation and provide the first demonstration in mammalian cells of a role for small GTP-binding proteins in the G2/M transition.
Identification by site-directed mutagenesis of three essential histidine residues in membrane dipeptidase, a novel mammalian zinc peptidase
Authors: KEYNAN Shoshana , HOOPER Nigel M. , TURNER Anthony J.
Published: August 2015

Membrane dipeptidase (EC 3.4.13.19) is a plasma membrane zinc peptidase that is involved in the renal metabolism of glutathione and its conjugates, such as leukotriene D4. The enzyme lacks the classical signatures of other zinc-dependent hydrolases and shows no homology with any other mammalian protein. We have used site-directed mutagenesis to explore the roles of five histidine residues in pig membrane dipeptidase that are conserved among mammalian species. When expressed in COS-1 cells, the mutants H49K and H128L exhibited a specific activity and Km for the substrate Gly-D-Phe comparable with those of the wild-type enzyme. However, the mutants H20L, H152L and H198K were inactive, but were expressed at the cell surface at equivalent levels to the wild-type, as assessed by immunoblotting and immunofluorescence. These three mutants were compared with regard to their ability to bind to the competitive inhibitor cilastatin, which binds with equal efficacy to native and EDTA-treated pig kidney membrane dipeptidase. Expressed wild-type enzyme and mutants H20L and H198K were efficiently bound by cilastatin–Sepharose, but H152L failed to bind. Thus His-152 appears to be involved in the binding of substrate or inhibitor, whereas His-20 and His-198 appear to be involved in catalysis. Membrane dipeptidase shares some similarity with a dipeptidase recently cloned from Acinetobacter calcoaceticus. In particular, His-20 and His-198 of membrane dipeptidase are conserved in the bacterial enzyme, as are Glu-125 and His-219, previously shown to be required for catalytic activity.
Human phosphodiesterase 4A: characterization of full-length and truncated enzymes expressed in COS cells
Authors: OWENS Raymond J. , CATTERALL Cath , BATTY Dawn , JAPPY John , RUSSELL Annette , SMITH Bryan , O'CONNELL Jimi , PERRY Martin J.
Published: August 2015

The type 4 phosphodiesterase (PDE) family comprises four enzymes (4A, 4B, 4C and 4D) that are characterized by their specificity for cAMP and selective inhibition by the antidepressant drug rolipram {4-[3-(cyclopentoxyl)-4-methoxyphenyl]2-pyrrolidone}. In common with other PDEs, they consist of a central conserved domain associated with catalytic activity in addition to two N-terminal upstream conserved regions (UCR1 and UCR2) that are unique to the type 4 enzymes. We have isolated a 2 kb cDNA encoding a full-length type 4A PDE {HS PDE4A4B [Bolger, Michaeli, Martins, St.John, Steiner, Rodgers, Riggs, Wigler and Ferguson (1993) Mol. Cell Biol. 13, 6558–6571]} from a human frontal cortex cDNA library. Northern blot analysis showed that the major PDE4A mRNA of 4.5 kb was widely distributed in different human tissues. The recombinant PDE4A expressed in COS cells had a molecular mass of approx. 117 kDa as revealed by SDS/PAGE/Western blotting with a PDE4A-specific antibody and was specific for cAMP with a Km of 4.8 μM. The enzyme activity was potently inhibited by R-rolipram (IC50 204 nM) and showed a 2.7-fold stereoselectivity over the S enantiomer. Analysis of the kinetics of inhibition indicated that R-rolipram did not behave as a simple competitive inhibitor. Dixon replots suggested that there was more than one mode of interaction consistent with the detection in the enzyme of a high-affinity binding site for R-rolipram with a Kd of 2.3 nM. Truncation of the PDE4A enzyme by deletion mutagenesis showed that neither of the UCRs was required for catalytic activity and identified an approx. 71 kDa core enzyme with a Km for cAMP of 3.3 μM. In contrast with the full-length PDE4A, R-rolipram behaved as a simple competitive inhibitor of this form of the enzyme with decreased potency (IC50 1022 nM) and no stereoselectivity. In addition, no high-affinity rolipram-binding site was detected in the truncated enzyme, indicating that this interaction involves sequences upstream of the catalytic domain of the enzyme.
Incremental Ca2+ mobilization by inositol trisphosphate receptors is unlikely to be mediated by their desensitization or regulation by luminal or cytosolic Ca2+
Authors: BEECROFT Mike D. , TAYLOR Colin W.
Published: August 2015

The kinetics of Ins(1,4,5)P3 (InsP3)-stimulated Ca2+ release from intracellular stores are unusual in that submaximal concentrations of InsP3 rapidly release only a fraction of the InsP3-sensitive Ca2+ stores. By measuring unidirectional 45Ca2+ efflux from permeabilized rat hepatocytes, we demonstrate that such quantal responses to InsP3 occur at all temperatures between 2 and 37 °C, but at much lower rates at the lower temperatures. Preincubation with submaximal concentrations of InsP3, which themselves evoked quantal Ca2+ release, had no effect on the sensitivity of the stores to further additions of InsP3. The final Ca2+ content of the stores was the same whether they were stimulated with two submaximal doses of InsP3 or a single addition of the sum of these doses. Such incremental responses and the persistence of quantal behaviour at 2 °C indicate that InsP3-evoked receptor inactivation is unlikely to be the cause of quantal Ca2+ mobilization. Reducing the Ca2+ content of the intracellular stores by up to 45% did not affect their sensitivity to InsP3, but substantially reduced the time taken for each submaximal InsP3 concentration to exert its full effect. These results suggest that neither luminal nor cytosolic Ca2+ regulation of InsP3 receptors are the determinants of quantal behaviour. Our results are not therefore consistent with incremental responses to InsP3 depending on mechanisms involving attenuation of InsP3 receptor function by cytosolic or luminal Ca2+ or by InsP3 binding itself. We conclude that incremental activation of Ca2+ release results from all-or-nothing emptying of stores with heterogeneous sensitivities to InsP3. These characteristics allow rapid graded recruitment of InsP3-sensitive Ca2+ stores as the cytosolic InsP3 concentration increases.
Phosphotyrosine phosphatase activity associated with c-Src in large multimeric complexes isolated from adrenal medullary chromaffin cells
Authors: VAN HOEK Monique L. , ALLEN Colleen S. , PARSONS Sarah J.
Published: August 2015

Chromaffin cells, which secrete catecholamines in response to acetylcholine, express high levels of the Src-family tyrosine kinases. These kinases contain protein–protein interaction domains which bind signal transduction proteins that participate in a variety of cellular processes. To determine if signalling proteins bind c-Src in chromaffin cells, we examined c-Src immunocomplexes for co-precipitating proteins. We discovered a phosphotyrosine phosphatase (PTPase; EC 3.1.3.48) activity which associates with specific subcellular pools of c-Src in vivo and which preferentially binds the SH2 (Src homology 2) domain of c-Src in vitro. Known PTPases were not identified by blotting of c-Src immunocomplexes with a panel of anti-PTPase antibodies, suggesting that the PTPase may be a novel family member. The c-Src–PTPase complex is enriched in the plasma membrane fraction and exists in several large complexes, as revealed by gel-filtration analysis. This PTPase activity is altered rapidly following stimulation by secretagogues, decreasing within 30 s and returning to basal levels by 60 s of stimulation. Both the subcellular localization and rapid activity changes suggest that the c-Src-associated PTPase may function in early signalling events emanating from the nicotinic acetylcholine receptor. In support of this is the co-precipitation of a PTPase activity with the nicotinic acetylcholine receptor and co-chromatography of this receptor with one of the c-Src–PTPase complexes.
Preparation of a recombinant chimaera of insulin-like growth factor II and interleukin 3 with high proliferative potency for haemopoietic cells
Authors: DIFALCO Marcos R. , CONGOTE L. Fernando
Published: August 2015

We have found that a slightly modified insulin-like growth factor II (IGF II) consisting of a chimaera of bombyxin and human IGF II (BOMIGF) is properly secreted in insect cells by using the baculovirus expression system. Human interleukin 3 (IL-3) was attached to the C-terminal amino acid residue of BOMIGF with peptide linkers containing five or twelve residues. Only the chimaera with the 12-residue linker had biological activities of both IGF II and IL-3. The chimaera had a significantly higher mitogenic activity than IL-3 in cell cultures of the human haemopoietic cell line TF-1 and its effect could be observed even at femtomolar concentrations. It was also able to stimulate thymidine incorporation in IGF II-dependent bovine fetal erythroid cells. The chimaera significantly increased the number of macroscopic haemopoietic colonies in cultures of human peripheral blood in comparison with IL-3 or mixtures of IL-3 and BOMIGF in vitro. Subcutaneous injection of a BOMIGF–mouse IL-3 chimaera in normal C57BL/6 mice resulted in a significant increase of the number of spleen stem cells producing macroscopic haemopoietic colonies. This new system for the biosynthesis of IGF–cytokine fusion proteins in insect cells might prove advantageous for the low-cost and high-yield production of molecules with complementary or synergistic biological activities.
P-glycoprotein is strongly expressed in the luminal membranes of the endothelium of blood vessels in the brain
Authors: BEAULIEU Édith , DEMEULE Michel , GHITESCU Lucian , BÉLIVEAU Richard
Published: August 2015

Luminal membranes of the vascular endothelium were isolated from brain, heart and lungs by modification of their density. The presence of P-glycoprotein (P-gp) was detected by Western blotting in luminal membranes from the endothelium of the three tissues. Strong enrichment in brain capillary luminal membranes, compared with brain capillaries (17-fold) and whole membranes (400–500-fold), indicates that P-gp is mainly located on the luminal side of the brain endothelium. Western blotting was also performed with antibodies directed against GLUT1, glial fibrillary acidic protein, adaptin, IP3R-3, integrins αv and collagen IV as controls to determine whether the preparations were contaminated by other membranes. Strong enrichment of GLUT1 in brain capillary luminal membranes (9.9-fold) showed that the preparation consisted mainly of endothelial cell plasma membranes. Poor enrichment of glial fibrillary acidic protein (1.4-fold) and adaptin (2.4-fold) and a decreased level of IP3R-3, integrins αv and collagen IV excludes the possibility of major contamination by astrocytes or internal and anti-luminal membranes. High levels of P-gp in the luminal membranes of brain capillary endothelial cells suggests that it may play an important role in limiting the access of anti-cancer drugs to the brain.
Co-transfection with protein kinase D confers phorbol-ester-mediated inhibition on glucagon-stimulated cAMP accumulation in COS cells transfected to overexpress glucagon receptors
Authors: TOBIAS Edward S. , ROZENGURT Enrique , CONNELL John M. C. , HOUSLAY Miles D.
Published: August 2015

Glucagon elicited a profound increase in the intracellular cAMP concentration of COS-7 cells which had been transiently transfected with a cDNA encoding the rat glucagon receptor and under conditions where cAMP phosphodiesterase activity was fully inhibited. This was achieved in a dose-dependent fashion with an EC50 of 1.8±0.4 nM glucagon. In contrast with previous observations made using hepatocytes [Heyworth, Whetton, Kinsella and Houslay (1984) FEBS Lett. 170, 38–42], treatment of transfected COS-7 cells with PMA did not inhibit the ability of glucagon to increase intracellular cAMP levels. PMA-mediated inhibition was not conferred by treatment with okadaic acid, nor by co-transfecting cells with cDNAs encoding various protein kinase C isoforms (PKC-α, PKC-βII and PKC-ϵ) or with the PMA-activated G-protein-receptor kinases GRK2 and GRK3. In contrast, PMA induced the marked inhibition of glucagon-stimulated cAMP production in COS-7 cells that had been co-transfected with a cDNA encoding protein kinase D (PKD). Such inhibition was not due to an action on the catalytic unit of adenylate cyclase, as forskolin-stimulated cAMP production was unchanged by PMA treatment of COS cells that had been co-transfected with both the glucagon receptor and PKD. PKD transcripts were detected in RNA isolated from hepatocytes but not from COS-7 cells. Transcripts for GRK2 were present in hepatocytes but not in COS cells, whereas transcripts for GRK3 were not found in either cell type. It is suggested that PKD may play a role in the regulation of glucagon-stimulated adenylate cyclase.
G-protein βγ subunits antagonize protein kinase C-dependent phosphorylation and inhibition of phospholipase C-β1
Authors: LITOSCH Irene
Published: August 2015

Protein kinase C (PKC) isoforms phosphorylated phospholipase C-β1 (PLC-β1) in vitro as follows: PKCα ≫ PKCϵ; not PKCζ. PLC-β3 was not phosphorylated by PKCα. G-protein βγ subunits inhibited the PKCα phosphorylation of PLC-β1 in a concentration-dependent manner. Half-maximal inhibition occurred with 500 nM βγ. G-protein βγ subunits also antagonized the PKCα-mediated inhibition of PLC-β1 enzymic activity. PKCα, in turn, inhibited the stimulation of PLC-β1 activity by βγ. There was little effect of PKCα on the stimulation of PLC-β1 by αq/11–guanosine 5′[γ-thio]triphosphate (GTP[S]). These findings demonstrate that G protein βγ subunits antagonize PKCα regulation of PLC-β1. Thus βγ subunits might have a role in modulating the negative feedback regulation of this signalling system by PKC.
A novel procedure for the efficient purification of the cystic fibrosis transmembrane conductance regulator (CFTR)
Authors: RAMJEESINGH Mohabir , LI Canhui , GARAMI Elizabeth , HUAN Ling-Jun , HEWRYK Marek , WANG Yanchun , GALLEY Kevin , BEAR Christine E.
Published: August 2015

This report describes a novel, single-step strategy for the purification of the cystic fibrosis transmembrane conductance regulator from Sf9 cells, which will facilitate studies of the structure–function relationships of this clinically important molecule. The new method combines the use of the novel detergent sodium pentadecafluoro-octanoate with metal-affinity chromatography to produce a high yield of purified protein which can be functionally reconstituted as a chloride channel and an ATPase.
Novel bradykinin signalling events in PC-12 cells: stimulation of the cAMP pathway leads to cAMP-mediated translocation of protein kinase Cε
Authors: GRANESS Angela , ADOMEIT Antje , LUDWIG Beate , MÜLLER Wolf-Dieter , KAUFMANN Roland , LIEBMANN Claus
Published: August 2015

In the rat pheochromocytoma cell line PC-12, bradykinin (BK) stimulated phosphatidylinositol hydrolysis by 4–5-fold and, additionally, intracellular cAMP accumulation by approx. 1.6-fold. EC50 values for BK were 3 nM and 2 nM respectively. The BK-induced increase in cAMP accumulation was paralleled by a 1.6-fold increase in protein kinase A (PKA) activity. The time course of BK-stimulated inositol phosphate formation was rapid (t1/2 < 1 min), whereas the BK-induced cAMP accumulation was lagging (t1/2 approx. 6 min). The effect of BK on the cAMP pathway was independent of pertussis toxin, excluding an indirect stimulation of adenylate cyclase via βγ-complexes from Gi or Go proteins. Two different protein kinase C (PKC) inhibitors, bisindolylmaleimide and Ro 31-820, failed to prevent BK-induced cAMP accumulation, and exclude PKC as mediator of BK action on adenylate cyclase. In contrast, the stimulatory effect of BK on cAMP accumulation was completely abolished by two calmodulin antagonists, chlorpromazine and ophiobolin, suggesting an indirect, Ca2+/calmodulin-mediated effect of BK on the cAMP pathway. In addition, exposure of PC-12 cells to BK resulted in a translocation of the PKC isoforms α, δ, ϵ and ζ displaying different kinetics. The BK-induced translocations of the PKCs α and δ were rapid and biphasic, whereas the PKCs ϵ and ζ revealed a slower and slightly transient translocation in response to BK. The BK-elicited translocation of PKCϵ, but not that of the PKCs α, δ and ζ, was prevented by two different inhibitors of adenylate cyclase, 2′,5′-dideoxyadenosine and MDL-12,330A, as well as the PKA inhibitor adenosine 3′:5′-monophosphothioate. These findings suggest that the BK-induced translocation of novel (n)PKCϵ is mediated via the cAMP pathway. Since nPKCϵ appears to regulate neurite outgrowth in PC-12 cells [Hundke, McMahon, Dadgar and Messing (1995) J. Biol. Chem. 270, 30134–30140] our results provide evidence for a novel signalling mechanism that might be involved in BK-induced neuronal differentiation of PC-12 cells.
The heat-shock transcription factor HSF1 is rapidly activated by either hyper- or hypo-osmotic stress in mammalian cells
Authors: CARUCCIO Lorraine , BAE Sangwoo , LIU Alice Yee-Chang , CHEN Kuang Yu
Published: August 2015

Osmoregulation, the cellular response to environmental changes of osmolarity and ionic strength, is important for the survival of living organisms. We have demonstrated previously that an exposure of mammalian cells to hypo-osmotic stress, either in growth medium (30% growth medium and 70% water) or in binary solution containing sorbitol and water, prominently induced the DNA-binding activity of the heat-shock transcription factor (HSF1) [Huang, Caruccio, Liu and Chen (1995) Biochem. J. 307, 347-352]. Since hyperosmotic and hypo-osmotic stress usually elicit opposite biological responses, we wondered what would be the effect of hyperosmotic stress on HSF activation. In this study we have examined the HSF DNA-binding activity in HeLa cells maintained in the sorbitol/water binary solution over a wide concentration range (0.1-0.9 M) and in Dulbecco's medium supplemented with sorbitol or NaCl. We found that HSF-binding activity could be induced prominently under both hypo-osmotic (0.1-0.25 M) and hyperosmotic conditions (0.50-0.90 M). In both cases, HSF activation was observed within 5 min after changing the osmotic pressure. The activation was accompanied by both HSF trimerization and nuclear translocation, and appeared to be independent of protein synthesis. The effects of hypo- or hyper-osmotic stress on HSF activation could be reversed once the cells were returned to iso-osmotic conditions (0.30 M) with a half-life () of 25 min or less. This rapid turnover of the osmotic-stress-induced HSF-binding activity was inhibited by cycloheximide, a potent inhibitor of protein synthesis. Unlike heat shock, activation of HSF by either hypo- or hyper-osmotic stress did not lead to an accumulation of heat-shock protein 70 (HSP70) mRNA in HeLa cells. We propose that HSF activation during osmotic stress may serve physiological functions independent of the synthesis of heat-shock proteins.