bluuebunny/crossref_metadata_2025 huggingface dataset:
Intracellular Ca2+, inositol 1,4,5-trisphosphate and additional signalling in the stimulation by platelet-activating factor of prostaglandin E2 formation in P388D1 macrophage-like cells
Authors: Asmis R , Randriamampita C , Tsien R Y , Dennis E A
Published: August 2015

In the P388D1 macrophage-like cell line, phospholipase A2 activity and prostaglandin production are stimulated by platelet-activating factor (PAF) and bacterial lipopolysaccharide (LPS). We have investigated the role of Ins(1,4,5)P3 and Ca2+ in signal transduction of PAF-induced prostaglandin E2 (PGE2) formation in these cells. The role of Ca2+ in the activation mechanism was studied by fluorescence imaging of intracellular Ca2+ in individual adherent cells and by determining the PGE2 production in the same population of cells. This new approach enabled us to correlate directly events on the single-cell level with a physiologically relevant response of the cell population. Priming the cells with LPS was required for PAF to stimulate PGE2 formation, yet LPS affected neither the intracellular free Ca2+ concentration ([Ca2+]i) nor the PAF-induced rise in [Ca2+]i. In addition, basal and PAF-stimulated Ins(1,4,5)P3 levels were not affected by LPS priming. However, the Ca2+ transient, the release of Ins(1,4,5)P3 and the formation of PGE2 induced by PAF were inhibited in cells pretreated with pertussis toxin. Buffering the [Ca2+]i with intracellular BAPTA [bis-(o-aminophenoxy)ethane-NNN'N'-tetra-acetic acid] blocked the PAF-stimulated rise in [Ca2+]i and PGE2 formation. Removal of extracellular Ca2+ during PAF stimulation prevented the influx of Ca2+, but did not affect the initial [Ca2+]i transient, nor did it inhibit PGE2 formation. Under the same conditions, ionomycin stimulated an identical [Ca2+]i transient, but, in contrast with PAF stimulation, no PGE2 formation was observed. PGE2 production could be rescued by prompt subsequent addition of PAF, which caused no further [Ca2+]i change on its own. These results show that the transient initial rise in [Ca2+]i, produced either by PAF via the formation of Ins(1,4,5)P3 or directly by ionomycin, is necessary, but not sufficient for the formation of PGE2 in LPS-primed P388D1 cells. Furthermore, we have demonstrated for the first time that PAF triggers a second signal that is not mediated by a change in [Ca2+]i. However, both signals are required to induce PGE2 formation.
Biochemical and functional analysis of an adenovirus-based ligand complex for gene transfer
Authors: Fisher K J , Wilson J M
Published: August 2015

Ligand-mediated approaches to gene transfer offer an alternative to viral vectors for both in vivo and in vitro applications. Although a significant percentage of the plasmid-based DNA complex is lost to lysosomal degradation following receptor-mediated endocytosis, simultaneous infection with adenovirus has been shown to increase the level of transgene expression [Curiel, Agarwal, Wagner and Cotten (1991) Proc. Natl. Acad. Sci. U.S.A. 88, 8850-8854; Wagner, Zatloukal, Cotten, Kirlappos, Mechtler, Curiel and Birnstiel (1992) Proc. Natl. Acad. Sci. U.S.A. 89, 6099-6103]. In this study we describe an adenovirus-based ligand complex where the plasmid DNA, polycation-ligand conjugate and adenovirus are contained within a single particle structure. At the core of the transfection particle is a replication-defective recombinant adenovirus encoding a cDNA minigene for human placenta alkaline phosphatase that was chemically modified with poly(L-lysine) (Ad-pLys). Electron microscopy of an adenovirus-based ligand complex formed by successively adding plasmid DNA and an asialo-orosomucoid-poly(L-lysine) conjugate to Ad-pLys revealed structures that appeared as intact viral particles coated with a dense biomolecular layer. Adenovirus-based ligand complexes containing either a luciferase or beta-galactosidase reporter plasmid were shown to efficiently deliver the plasmid transgene to cells that express the hepatic asialoglycoprotein receptor. Furthermore, the poly(L-lysine) modification greatly reduced the infectivity potential of the virus without causing a concomitant loss of augmented gene transfer. As an alternative to infectious virions, incomplete products of viral assembly were also considered as a source for endosomalytic activity. However, these defective virions were unable to significantly enhance plasmid transgene delivery.
Synthetic, structural and biological studies of the ubiquitin system: the total chemical synthesis of ubiquitin
Authors: Ramage R , Green J , Muir T W , Ogunjobi O M , Love S , Shaw K
Published: August 2015

The small protein ubiquitin (76 amino acids) has been synthesized under optimized conditions by Merrifield solid-phase methodology using the N alpha-Fmoc protecting group. The crude polypeptide mixture was purified to homogeneity by gel filtration, dialysis and a combination of cation- and anion-exchange chromatography to yield ubiquitin. Amino acid analysis, enzymic digestion and sequencing by automated Edman degradation were used to authenticate the primary structure. Isoelectric focusing and m.s. were used to demonstrate that the final product was greater than 98% pure with a final yield of 93 mg (4.3%) from a single synthesis on a 0.25 nmol scale.
Expression of an enzymically active glycosylphosphatidylinositol-anchored form of neutral endopeptidase (EC 3.4.24.11) in Cos-1 cells
Authors: Howell S , Lanctôt C , Boileau G , Crine P
Published: August 2015

Neutral endopeptidase (EC 3.4.24.11, NEP) is a type-II integral membrane protein found in a wide variety of cell types. We previously produced a secreted form of the enzyme by deletion of the cytoplasmic and transmembrane domains and in-frame fusion of the cleavable signal peptide of pro-opiomelanocortin [Lemay, Waksman, Roques, Crine and Boileau (1989) J. Biol. Chem. 264, 15620-15623]. Here we have used this secreted form of NEP and fused to it the glycosylphosphatidylinositol (GPI)-anchor attachment signal of decay-accelerating factor to produce a GPI-anchored form. Expression of this chimeric form in Cos-1 cells resulted in cell-surface activity. This activity could be released from the cell surface by phosphatidylinositol-specific phospholipase C and radiolabelling studies showed that the protein could incorporate [3H]ethanolamine, indicating that the enzyme was GPI-anchored. The Km value, using [D-Ala2,Leu5]enkephalin as substrate, of GPI-anchored NEP (62 +/- 5 microM) was comparable with that of wild-type NEP (70 +/- 4 microM), as were the sensitivities to the inhibitors phosphoramidon and thiorphan. However, pulse-chase studies showed that the biosynthesis and cell-surface delivery of GPI-anchored NEP was delayed compared with that of the wild-type transmembrane form of NEP. These results suggest a lower rate of biosynthesis and/or cellular transport for GPI-anchored NEP compared with its transmembrane counterpart.
Baculovirus-directed expression of the γ-subunit of phosphorylase kinase: purification and calmodulin dependence
Authors: Lanciotti R A , Bender P K
Published: August 2015

A recombinant baculovirus containing a cDNA encoding the gamma-subunit of phosphorylase kinase from mouse skeletal muscle was constructed. Cultures of Sf-9 insect cells infected with the gamma-baculovirus produce an intact and soluble gamma-protein. A purification procedure is presented that yields a sample of gamma-protein which is devoid of interfering enzyme activity and which is not associated with calmodulin from the insect cells. The isolated gamma sample has a Km for phosphorylase b of 36 (+/- 6, S.E.M) microM at pH 8.2 and 140 (+/- 25) microM at pH 6.8. These values are similar to those reported for the activated phosphorylase kinase holoenzyme isolated from skeletal muscle tissue. However, the Vmax. of the baculovirus-expressed gamma is 65 and 80% of that of the activated holoenzyme at pH 6.8 and 8.2 respectively. These results indicate that one or more of the regulatory subunits alpha, beta, or calmodulin stimulate the activity of the catalytic subunit gamma in the activated holoenzyme. Addition of calmodulin to the baculovirus-expressed gamma stimulates its activity 1.5-2.0 fold at pH 6.8 in both the presence and absence of calcium. At pH 8.2, calmodulin has only minor stimulatory affects. The stimulation by calmodulin at pH 6.8 results from an increase in the Vmax of gamma with little effect on its Km. This result is unlike that for most calmodulin-stimulated kinases which bind calmodulin only in the presence of calcium and exhibit a decrease in their Km upon binding calmodulin. The change in Vmax. of gamma in the presence of calmodulin and in the absence of calcium presents a novel mechanism for the regulation of a calmodulin-stimulated kinase.
Neutrophil lysosomal dysfunctions in mutant C57 Bl/6J mice: interstrain variations in content of lysosomal elastase, cathepsin G and their inhibitors
Authors: Gardi C , Cavarra E , Calzoni P , Marcolongo P , de Santi M , Martorana P A , Lungarella G
Published: August 2015

In this paper we report the serum antiprotease screening and the biochemical and functional characteristics of neutrophils in a variety of mouse strains with different susceptibilities for developing a protease-mediated injury. C57Bl/6J mice and their mutants tight-skin and pallid have a lower serum elastase inhibitory capacity (-30, -65 and -70% respectively) than other inbred strains (i.e. NMRI and Balb/c, which both have similar values). We demonstrate that these values are a consequence of a decreased concentration of the alpha 1-protease inhibitor for elastase [PI(E)], which is the major serum inhibitor of elastase and cathepsin G. In addition, neutrophil lysosomal dysfunctions characterized by abnormally high contents of elastase and cathepsin G, or defective lysosomal secretion are observed in tight-skin and pallid mice respectively. Another C57Bl/6J mutant with lysosomal abnormalities is the beige mouse. Negligible amounts of elastase and cathepsin G, as well as defective neutrophil degranulation, have been described previously in this strain. We found, however, discrete amounts of a latent form of neutrophil elastase that undergoes a spontaneous activation by a protease-dependent mechanism. We also report that neutrophil cathepsin G in this mouse is tightly bound to lysosomal membranes, but is released in near normal quantities during exocytosis. Cytosolic elastase and cathepsin G inhibitors, which were previously reported as being specific for the beige neutrophils, have also been detected in all the examined strains. Neutrophil functions, lysosomal enzyme content and serum antiprotease screening may represent key elements in the protease-antiprotease balance and may explain the different interstrain susceptibility to developing lesions in which an elastolytic activity has been implicated.
A meiotic DNA polymerase from a mushroom, Agaricus bisporus
Authors: Takami K , Matsuda S , Sono A , Sakaguchi K
Published: August 2015

A meiotic DNA polymerase [DNA nucleotidyltransferase (DNA-directed), EC 2.7.7.7], which likely has a role in meiotic DNA repair, was isolated from a mushroom, Agaricus bisporus. The purified fraction displays three bands in SDS/PAGE, at molecular masses of 72 kDa, 65 kDa and 36 kDa. Optimal activity is at pH 7.0-8.0 in the presence of 5 mM Mg2+ and 50 mM KCl and at 28-30 degrees C, which is the temperature for meiosis. This enzyme is resistant to N-ethylmaleimide and sensitive to 2′,3′-dideoxythymidine 5′-triphosphate, suggesting that it is a beta-like DNA polymerase. These characteristics are similar to those of Coprinus DNA polymerase beta [Sakaguchi and Lu (1982) Mol. Cell. Biol. 2, 752-757]. In Western-blot analysis, the antiserum against the Coprinus polymerase reacts only with the 65 kDa band, which coincides with the molecular mass of the Coprinus polymerase. Western-blot analysis also showed that the antiserum could react with crude extracts not only from the Agaricales family, to which Agaricus and Coprinus belong, but also from different mushroom families and Saccharomyces. The Agaricus polymerase activity can be found only in the meiotic-cell-rich fraction, but the enzyme is also present in the somatic cells in an inactive state.
Purification and characterization of a sialic acid-specific lectin from Tritrichomonas mobilensis
Authors: Babál P , Pindak F F , Wells D J , Gardner W A
Published: August 2015

New sialic acid-specific lectin has been isolated from culture supernatant of the protozoan Tritrichomonas mobilensis. It was purified by adsorption by erythrocytes or bovine submaxillary gland mucin (BSM)-Sepharose affinity chromatography. The T. mobilensis lectin (TML) does not require bivalent cations for activity and agglutinates all human erythrocytes. The lectin forms multimeric complexes with molecular mass 556 and 491 kDa as determined by size-exclusion chromatography. SDS/PAGE under reducing conditions disclosed a large band of 343 kDa and three bands of 246, 265 and 286 kDa which, after denaturation with urea, were split into three subunits of 56, 61 and 66 kDa; under non-reducing conditions there were two bands, of 360 and 260 kDa. Western blots performed with anti-TML monoclonal antibodies revealed bands identical with those in the silver-stained gels, suggesting homogeneity of the BSM -Sepharose-purified lectin. TML is a highly glycosylated protein with approx. 8% of N-linked glycosides found by protein-N-glycanase F treatment; the total amount of saccharides revealed by chemical deglycosylation was 20%. Haemagglutination-inhibition studies documented exclusive specificity for sialic acid (NeuAc). Both (alpha 2->6)- and (alpha 2->3)-linked and free NeuAc were eight times more potent inhibitors than N-glycolylneuraminic acid. The lectin does not require O-acetyl groups on NeuAc for recognition. A spectrum of mono- and oligo-saccharides other than sialic acid had no inhibitory effect at 200 mM. Anti-TML monoclonal antibodies strongly inhibited the lectin activity. TML was stable at temperatures below 4 degrees C and lyophilized with 3% (w/w) glycerol.
Molecular cloning, functional expression and pharmacological characterization of a mouse melanocortin receptor gene
Authors: Desarnaud F , Labbe O , Eggerickx D , Vassart G , Parmentier M
Published: August 2015

We describe the cloning of the mouse HGMP01A gene that encodes a melanocortin receptor functionally distinct from the adrenal cortex corticotropin (adrenocorticotrophic hormone; ACTH) receptor and the melanocyte-stimulating hormone (MSH) receptor expressed in melanoma. The gene encodes a protein of 323 amino acids with a calculated molecular mass of 35,800 Da, displaying potential sites for N-linked glycosylation and phosphorylation by protein kinase C. An RNAase protection assay detected weak expression in the brain, but not in adrenal gland, skin, or any of the other tissues tested. Stable CHO cell lines expressing over 100,000 receptors per cell were generated. The recombinant receptor binds iodinated [Nle4,D-Phe7]alpha-MSH (NDP-MSH) with an apparent Kd of 700 pM. Displacement of the ligand by a variety of pro-opiomelanocortin-derived peptides revealed a pharmacological profile distinct from that of the classical ACTH and MSH receptors. NDP-MSH was the most powerful competitor (IC50 1.4 nM), followed by gamma-MSH (IC50 7 nM). alpha-MSH, beta-MSH and ACTH-(1-39) were significantly less potent, with IC50 values of 30, 19 and 21 nM respectively. ACTH-(4-10) was poorly active (IC50 2.4 microM), while corticotropin-like intermediate lobe peptide (CLIP) and beta-endorphin were totally ineffective. The recombinant receptor was found to stimulate adenylate cyclase. The potency order of the agonists in this assay was consistent with that of the binding displacement assays. This receptor represents the orthologue of the human melanocortin 3 receptor reported recently. The growing family of melanocortin receptors constitute the molecular basis for the variety of actions of melanocortins that have been described over the years. The availability of functionally expressed receptors from the melanocortin family will allow the development of a specific pharmacology, and a better understanding of the function of the pro-opiomelanocortin-derived peptides.
Modulation of phosphatidylserine synthesis by a muscarinic receptor occupancy in human neuroblastoma cell line LA-N-1
Authors: Mikhaevitch I S , Singh I N , Sorrentino G , Massarelli R , Kanfer J N
Published: August 2015

The incorporation of [3H]serine into lipids, water-soluble metabolites and proteins by the human neuroblastoma cell line LA-N-1 exposed to oxotremorine-M, a muscarinic agonist, was investigated. Oxotremorine-M increased the incorporation of this labelled precursor into phosphatidylserine and proteins in a concentration-dependent manner, with the maximal stimulation at 250 microM. This activation was blunted by 100 microM atropine. There were no detectable changes of the radioactivity in the water-soluble metabolites. Acetylcholine, another muscarinic agonist, slightly decreased the serine incorporation into lipids, but did not affect the protein or water-soluble compartments. Several other muscarinic agonists, including 250 microM pilocarpine, 100 microM McN-A-343 and 1 mM carbachol, did not effect these [3H]serine incorporations. Preincubation of cells with 1 mM oxotremorine M, or 1 mM carbachol, or 1 mM McN-A-343, for 4 h prevented the oxotremorine-M-induced increase of serine incorporation. These observations are consistent with the oxotremorine-M action being mediated by muscarinic-receptor occupancy. The G-protein inhibitor guanosine 5′-[beta-thio]diphosphate (1 mM) and the G-protein activators, guanosine 5′-[gamma-thio]triphosphate (100 microM) and A1F3, prevented the oxotremorine stimulation. The muscarinic agonists, 250 microM oxotremorine-M, 1 mM carbamoylcholine and 500 microM acetylcholine, triggered the accumulation of inositol mono- and di-phosphates by cells that had been prelabelled with myo-[3H]inositol, and this phospholipase C activation was blunted by 100 microM atropine. The protein kinase C inhibitor H7 prevented the oxotremorine-M stimulation of serine incorporation. Over-night exposure of LA-N-1 cells to 100 nM phorbol 12-myristate 13-acetate resulted in a decrease of cytosolic protein kinase C activity, and prevented the oxotremorine-M stimulation of serine incorporation. Neither oxotremorine-M nor acetylcholine caused a redistribution of protein kinase C activity between the cytosol and membrane compartments. In addition, oxotremorine-M did not activate phospholipase D of the LA-N-1 cells.
Tissue specificity of rat mitochondrial dimethylglycine dehydrogenase expression
Authors: Lang H , Minaian K , Freudenberg N , Hoffmann R , Brandsch R
Published: August 2015

Expression of mitochondrial dimethylglycine dehydrogenase (Me2GlyDH) was analysed in various tissues, liver cell types and developmental stages of the rat. Total RNA extracted from liver, spleen, brain, kidney, lung and heart was reverse-transcribed into cDNA and amplified with Me2GlyDH cDNA-specific oligonucleotides by PCR. Expression of the enzyme was observed mainly in liver and kidney. In addition, Me2GlyDH mRNA could be demonstrated in total RNA samples of lung, heart and brain but was barely detectable in spleen total RNA. In RNA prepared from 14-day rat embryos, Me2GlyDH-specific mRNA was clearly present. Among various liver cell types, besides hepatocytes, endothelial cells showed a high level of Me2GlyDH mRNA expression. There was no amplification product detectable in liver macrophages (Kupffer cells) and only a very faint one in fat-storing cells (Ito cells). Western blots confirmed at the protein level the predominant expression of the enzyme in liver and kidney, but Me2GlyDH protein was also present in the protein extract of lung, heart, spleen and brain. Immunohistochemical staining of liver slices with Me2GlyDH-specific antiserum revealed that expression of this enzyme is evenly distributed throughout the liver tissue. In the kidney, expression of the enzyme was located in the proximal tubule cells. Our results demonstrate that, contrary to the previously assumed liver-restricted expression, this enzyme is specifically expressed predominantly in the liver and kidney, but, in addition, it is detectable in many other tissues of the rat.
Purification, molecular cloning and heterologous expression of a glutathione S-transferase from the Australian sheep blowfly (Lucilia cuprina)
Authors: Board P , Russell R J , Marano R J , Oakeshott J G
Published: August 2015

Three glutathione S-transferases from Lucilia cuprina (Australian sheep blowfly) pupae were purified by affinity chromatography and anion-exchange chromatography. One isoenzyme was composed of M(r)-24,800 subunits, and two isoenzymes had subunits of M(r) 23,900. The M(r)-23,900 subunits showed immunological identity and were immunologically distinct from the M(r)-24,800 subunits. All three enzymes were active with the substrate 1-chloro-2,4-dinitrobenzene and had low activity with 1,2-dichloro-4-nitrobenzene. A cDNA clone encoding a M(r)-23,900 subunit (LuGST1) was isolated and sequenced. The sequence has close similarities (> 81%) to that of GSTs from the fruitfly Drosophila melanogaster and Musca domestica (housefly). The deduced amino acid sequence of the Lu GST1 subunit showed no significant similarity to that of the mammalian GSTs to the Alpha, Mu and Pi classes, but shows some similarity (33%) over the first 100 residues with the rat subunit 12 Theta-class GST. Southern blots of genomic DNA hybridized with the LuGST1 cDNA identified many hybridizing fragments. Taken together, these data indicated that the L. cuprina genome contains multiple glutathione S-transferase genes.
Sequence-specific recognition of the HIV-1 long terminal repeat by distamycin: a DNAase I footprinting study
Authors: Feriotto G , Mischiati C , Gambari R
Published: August 2015

Pharmacological modulation of the interaction between transcription factors and target DNA sequences of cellular and viral genes could have important effects in the experimental therapy of a large variety of human pathologies. For instance, alteration of the DNA/protein interaction might be among the molecular mechanisms of action of DNA-binding drugs, leading to an inhibition of the expression of genes involved in the control of in vitro and in vivo growth of neoplastic cells and virus DNA replication. Natural oligopeptides, such as distamycin, are powerful inhibitors of the interaction between nuclear factors and target DNA sequences and, therefore, have been proposed as compounds retaining antibiotic, antineoplastic and antiviral properties. In this study we performed DNAase I footprinting analysis using a PCR product mimicking a region of the long terminal repeat (LTR) of the human immunodeficiency type 1 (HIV-1) retrovirus. The data obtained suggest that distamycin binds to different regions of the HIV-1 LTR depending on the DNA sequence. Electrophoretic mobility shift assays using both crude nuclear extracts from the Jurkat T-lymphoid cell line and the recombinant proteins transcription factor IID and Sp1 suggest that distamycin differentially inhibits the interaction of these two proteins with their specific DNA target sequences, in good agreement with the results obtained by DNAase I footprinting analysis.
New isoforms of Ca2+/calmodulin-dependent protein kinase II in smooth muscle
Authors: Zhou Z L , Ikebe M
Published: August 2015

Four novel isoforms of Ca2+/calmodulin-dependent protein kinase II (CaM kinase II) were found in rat aorta smooth muscle. Two of them were related to gamma-isoform of brain CaM kinase II (gamma-a). Differences in the primary structure of these isoforms were located in the variable region. One of them (gamma-b) contained 23 unique amino acid residues, whereas the other (gamma-c) did not contain this sequence. Both isoforms lacked the two segments (Val-316 to Gln-337 and Lys-353 to Leu-362) present in gamma-a. The DNA sequence of these gamma-isoforms except the variable region was exactly the same, suggesting that they are produced by alternative splicing. Another two isoforms were related to the delta-isoform of brain CaM kinase II (delta-a). delta-b contained a unique 11-residue sequence in the variable region whereas delta-c did not. As found for gamma-isoforms, the sequence analysis suggested that the three delta-isoforms are also produced by alternative splicing. Analysis of RNA by reverse transcription PCR confirmed the existence of specific messages for gamma-b, delta-a and delta-b. The variety of isoforms of CaM kinase II suggest that each isoform may play a specialized role in cell regulation.
Extraction and characterization of the tissue forms of collagen types II and IX from bovine vitreous
Authors: Bishop P N , Crossman M V , McLeod D , Ayad S
Published: August 2015

We report for the first time that, after centrifugation of adult bovine vitreous, the hyaluronan-rich supernatant contains collagens which can be isolated in their intact forms by precipitation with 4.5 M NaCl. This precipitate constituted approx. 4% of the total vitreous collagen and comprised collagen types IX and II (in the approximate ratio of 4:1) with negligible amounts of type-V/XI collagen. Type-II collagen was present partly in a pro-alpha 1(II) form, suggesting that there is active synthesis of type-II collagen into the matrix of adult bovine vitreous. Type-IX collagen was purified (2-2.5 mg/l of vitreous) and its glycosaminoglycan chain composition was analysed. Bovine vitreous type-IX collagen always possessed a glycosaminoglycan chain of comparatively low M(r) that was predominantly 4-sulphated, with chondroitin 6-sulphate representing a more minor component. By contrast, chick vitreous has been shown to contain type-IX collagen which always possesses a high-M(r) chondroitin sulphate chain that is predominantly 6-sulphated. The functional significance of these different glycosaminoglycan chain lengths and sulphation patterns is discussed.
Mitochondrial K+ as modulator of Ca2+-dependent cytotoxicity in hepatocytes. Novel application of the K+-sensitive dye PBFI (K+-binding benzofuran isophthalate) to assess free mitochondrial K+ concentrations
Authors: Zoeteweij J P , van de Water B , de Bont H J , Nagelkerke J F
Published: August 2015

In isolated rat hepatocytes a sustained high increase in intracellular free Ca2+ ([Ca2+]i), induced by extracellular ATP, is associated with mitochondrial dysfunction and cell death. The Ca(2+)-induced effects are Pi-dependent and less severe when the intracellular K+ content is low. In this study, the involvement of mitochondrial K+ processing in Ca(2+)-induced loss of mitochondrial membrane potential (MMP) and viability was investigated. The recently introduced K(+)-sensitive dye PBFI (K(+)-binding benzofuran isophthalate) has been used in combination with video-microscopy to assess intramitochondrial free K+ concentration ([K+]mito) in rat liver mitochondria in situ. After rapid permeabilization of the plasma membrane to remove cytosolic PBFI, the remaining PBFI was localized in mitochondria, and a ‘resting’ [K+]mito of approx. 15 mM could be measured. Increased [K+]mito levels were measured after induction of a prolonged increase in [Ca2+]i by ATP. Much lower [K+]mito, more comparable with control levels, were observed when intracellular K+ was depleted by omission of extracellular K+. In permeabilized cells the Ca(2+)-induced, Pi-dependent, dissipation of the MMP was markedly delayed in the absence of K+. These observations suggest involvement of [K+]mito as modulating agent in Ca(2+)-induced cytotoxicity in hepatocytes.
Serine/threonine phosphorylation of the γ-subunit after activation of the high-affinity Fc receptor for immunoglobulin G
Authors: Durden D L , Rosen H , Cooper J A
Published: August 2015

In this report we show that interferon gamma treatment of U937 cells induces increased expression of the gamma-subunit of the high-affinity Fc receptor for IgG (Fc gamma RI). Interferon treatment results in a 10-fold increased expression of the gamma-subunit and induces expression of a phosphorylated form (gamma 1). The increased expression of the gamma-subunit correlates with its ability to transmit a signal via Fc gamma R, as measured by activation of the respiratory burst using insoluble immune complexes. During Fc gamma R activation, a mobility shift occurs in the phosphorylated form of this gamma 1-subunit. Phosphoamino acid analysis demonstrates that this gamma 1 subunit is threonine phosphorylated in resting differentiated U937 cells and becomes predominantly serine phosphorylated on Fc receptor activation. The mobility shift in the gamma-subunit can be induced by treating U937 cells with phorbol 12-myristate 13-acetate or by monoclonal antibody cross-linking of Fc gamma RI. Hence the gamma-subunit is serine phosphorylated in response to Fc gamma RI and protein kinase C activation. Therefore the gamma-subunit, initially described as a subunit of Fc epsilon RI, now appears to be involved in signal transduction via Fc gamma RI. The data also suggest that the gamma-subunit, in contrast with the zeta-subunit of the T-cell receptor-CD3 complex, is a substrate for serine/threonine kinase(s) in the cell. The serine phosphorylation of the gamma-subunit suggests a divergence of structure and function between the gamma-subunit and its homologue, the zeta-subunit of the T-cell receptor. Phosphorylation of the gamma-subunit on serine may play some regulatory role in Fc gamma RI signal transduction in myeloid cells.
Topoisomerase I action on the heterochromatic DNA from the brine shrimp Artemia franciscana: studies in vivo and in vitro
Authors: Carettoni D , Landsberger N , Zagni E , Benfante R , Badaracco G
Published: August 2015

The genomes of higher eukaryotes contain various amounts of tandem repeated DNA sequences (satellite DNA) typically located in the constitutive heterochromatin, the most highly condensed region of interphase chromosomes. We have previously demonstrated that an AluI DNA family of repeats is the major component of constitutive heterochromatin in the brine shrimp Artemia franciscana. The analysis of cloned heterochromatic fragments revealed that this repetitive DNA shows a stable curvature conferring a solenoidal geometry to the double helix. In this paper we provide evidence, using the antitumour drug camptothecin, that, in vivo, topoisomerase I cleaves heterochromatin with a frequency comparable with that observed in the whole genome. The analysis of the break sites shows that the enzyme cleaves heterochromatic DNA at specific sites characterized by a degenerate consensus sequence. Moreover the enzyme-mediated breaks have, in vitro, a degenerate consensus sequence similar to, but not identical with, the in vivo one. Some of these sites are influenced by the DNA flanking the heterochromatic insert, suggesting that structural variations could modify the enzyme specificity.
The secreted cathepsin L-like proteinases of the trematode, Fasciola hepatica, contain 3-hydroxyproline residues
Authors: Wijffels G L , Panaccio M , Salvatore L , Wilson L , Walker I D , Spithill T W
Published: August 2015

The cysteine proteinases synthesized by the adult stage of the trematode Fasciola hepatica were found to be a very heterogeneous group of proteins as demonstrated by one- and two-dimensional gel analyses. N-terminal amino acid sequencing indicated the presence of at least two distinct gene products among the secreted cysteine proteinases. Enzymic studies and peptide sequence analysis of the excreted/secreted cysteine proteinases suggested a close relationship to the plant thiol cathepsins and the mammalian cathepsin L subfamily. The cloning of a representative cDNA for a putative Fasciola cathepsin confirmed similarities to the cathepsin L subfamily but revealed low identity with the cathepsin-like proteinases of the related trematode, Schistosoma, nematode cathepsins and the mammalian cathepsin B subfamily. Furthermore, peptide and protein sequencing revealed the modification of certain highly conserved prolines to unusual 3-hydroxyproline derivatives. This is the first report of modified prolines in any proteinase. This finding, as well as the high activities of these cathepsins at neutral to alkaline pH values, raises a number of questions as to the physiological function of these thiol cathepsins and their interaction with host tissues.
A rapid and simple method for the purification of transglutaminase from Streptoverticillium mobaraense.
Authors: Gerber U , Jucknischke U , Putzien S , Fuchsbauer H L
Published: August 2015

Transglutaminase from Streptoverticillium mobaraense was partially purified by ion-exchange chromatography on a weak acid material and hydrophobic chromatography. The separation with a strong acid ion-exchanger produces homogeneous transglutaminase, in a single step and with high yields, directly from the centrifuged and filtered culture fluid of the micro-organism. The procedure reproduced several times could be also carried out on a larger scale with the optimized parameters of the laboratory isolations. The purified enzyme demonstrated good storage stability.
A new dopachrome-rearranging enzyme from the ejected ink of the cuttlefish Sepia officinalis.
Authors: Palumbo A , d'Ischia M , Misuraca G , De Martino L , Prota G
Published: August 2015

A melanogenic enzyme catalysing the rearrangement of dopachrome has been identified in the ejected ink of the cuttlefish Sepia officinalis. This enzyme occurs as a heat-labile protein which co-migrates with tyrosinase under a variety of chromatographic and electrophoretic conditions. On SDS/PAGE it shows like a single band with an approx. molecular mass of 85 kDa. The enzyme possesses high substrate specificity, acting on L-dopachrome (Km = 1 mM at pH 6.8) and on L-alpha-methyl-dopachrome, but not on D-dopachrome, L-dopachrome methyl ester, dopaminochrome and adrenochrome. Significant inhibition of the catalytic activity was observed with tropolone and L-mimosine. H.p.1.c. analysis of the enzyme-catalysed rearrangement of L-dopachrome revealed the quantitative formation of the decarboxylated product, 5,6-dihydroxyindole. These results point to marked differences between melanogenesis in cephalopod pigment cells and in melanocytes, which may have important implications in relation to the use of sepiomelanin as a model for studies of mammalian melanins.
Mechanism of inhibition of protein kinase C by 14-3-3 isoforms. 14-3-3 isoforms do not have phospholipase A2 activity.
Authors: Robinson K , Jones D , Patel Y , Martin H , Madrazo J , Martin S , Howell S , Elmore M , Finnen M J , Aitken A
Published: August 2015

The ability of individual members of the 14-3-3 protein family to inhibit protein kinase C (PKC) has been studied by using a synthetic peptide based on the specific 80 kDa substrate for PKC (MARCKS protein) in two different assay systems. Recombinant 14-3-3 and isoforms renatured by a novel method after separation by reverse-phase h.p.l.c. were studied. The detailed effects of diacylglycerol and the phorbol ester phorbol 12-myristate 13-acetate on the inhibition were also investigated. This suggests that one of the sites of interaction of 14-3-3 may be the cysteine-rich (C1) domain in PKC. Since a region in secreted phospholipase A2 (PLA2) shares similarity with this domain, the ability of 14-3-3 to interact with mammalian PLA2 was studied. Cytosolic PLA2 has some similarity to the C2 region of PKC, and the effect of 14-3-3 on this class of PLA2 was also analysed. In contrast with a previous report, no PLA2 activity was found in brain 14-3-3, nor in any of the recombinant proteins tested. These include zeta 14-3-3 isoform, on which the original observation was made.
Characterization of insulins and proglucagon-derived peptides from a phylogenetically ancient fish, the paddlefish (Polyodon spathula)
Authors: Nguyen T M , Mommsen T P , Mims S M , Conlon J M
Published: August 2015

The North American paddlefish, Polyodon spathula (Order Acipenseriformes) is an extant representative of a group of primitive Actinopterygian (ray-finned) fish that probably shared a common ancestor with present-day teleosts. Two molecular forms of insulin which differ by a single amino acid substitution, His or Asp at position 15 of the A chain, were isolated from the pancreas of the paddlefish. Paddlefish insulins show greatest structural similarity to insulin from the garfish (order Lepisosteiformes) and resemble mammalian insulins more strongly than do insulins from teleost fish. The primary structures of several proglucagon-derived peptides, two molecular forms of glucagon which differ by the single amino acid substitution Arg18-->Ser, and glucagon-like peptide, have been less well conserved during evolution. The paddlefish glucagons contain 31 amino acid residues, rather than the usual 29, and show several structural features, such as Met5, Glu24 and Gly29, not previously observed in glucagons from other species. In spite of considerable differences in structure between paddlefish and mammalian glucagons (10 or 11 amino acid substitutions), both paddlefish glucagons are equally as effective as bovine glucagon in stimulating glycogenolysis in dispersed hepatocytes from the teleost fish Sebastes caurinus (rockfish). However, the substitution Arg18-->Ser in the paddlefish glucagon results in a 6-fold decrease in potency in this system.
Cardiac adenylate deaminase: molecular, kinetic and regulatory properties under phosphate-free conditions
Authors: Thakkar J K , Janero D R , Sharif H M , Hreniuk D , Yarwood C
Published: August 2015

Adenylate deaminase (EC 3.5.4.6) may help to regulate the adenine nucleotide catabolism characteristic of such disease states as myocardial ischaemia. We report analysis of the molecular, kinetic and allosteric properties of rabbit heart adenylate deaminase when extracted and purified under phosphate-free conditions (i.e., with Hepes/KOH). The enzyme's subunit molecular mass (approximately 81 kDa), pI (6.5), substrate specificity for 5′-AMP, and activation by K+ were identical in the absence or presence of phosphate. At each chromatographic step during isolation without phosphate, cardiac adenylate deaminase showed a lower apparent activity as compared with the enzyme prepared with phosphate present. Kinetic constants for the phosphate-free rabbit heart adenylate deaminase preparation (Km 0.54 mM AMP; Vmax. 1.4 mumol/min per mg of protein) were approximately 10-fold lower than those of the enzyme isolated with phosphate. The same irreversible decrease in kinetic constants could be achieved by dialysing phosphate from the phosphate-containing enzyme preparation. The relationship between enzyme activity and substrate concentration was sigmoidal in the presence of phosphate, but hyperbolic in its absence. Cardiac adenylate deaminase under phosphate-free conditions was no longer allosterically activated by ATP and ADP, yet remained inhibitable by GTP. Enzyme inhibition by the transition-state mimic coformycin was not influenced by phosphate status. The phosphate-free preparation of rabbit heart adenylate deaminase was markedly labile and extremely susceptible to proteolysis by trypsin or chymotrypsin. The inactivation kinetics and fragmentation pattern in response to controlled proteolysis depended on whether the enzyme had been isolated with or without phosphate present, suggesting a conformational difference between the two enzyme preparations. These data constitute direct evidence that the absence of phosphate irreversibly converts cardiac adenylate deaminase into a pseudo-isoenzyme with distinct kinetic, regulatory and stability properties.
Structure-function studies on human retinol-binding protein using site-directed mutagenesis
Authors: Sivaprasadarao A , Findlay J B
Published: August 2015

Retinol-binding protein (RBP) transports vitamin A in the plasma. It consists of eight anti-parallel beta-strands (A to H) that fold to form an orthogonal barrel. The loops connecting the strands A and B, C and D, and E and F form the entrance to the binding site in the barrel. The retinol molecule is found deep inside this barrel. Apart from its specific interaction with retinol, RBP is involved in two other molecular-recognition properties, that is it binds to transthyretin (TTR), another serum protein, and to a cell-surface receptor. Using site-directed mutagenesis, specific changes were made to the loop regions of human RBP and the resultant mutant proteins were tested for their ability to bind to retinol, to TTR and to the RBP receptor. While all the variants retained their ability to bind retinol, that in which residues 92 to 98 of the loop E-F were deleted completely lost its ability to interact with TTR, but retained some binding activity for the receptor. In contrast, the double mutant in which leucine residues at positions 63 and 64 of the loop C-D were changed to arginine and serine respectively partially retained its TTR-binding ability, but completely lost its affinity for the RBP receptor. Mutation of Leu-35 of loop A-B to valine revealed no apparent effect on any of the binding activities of RBP. However, substitution of leucine for proline at position 35 markedly reduced the affinity of the protein for TTR, but showed no apparent change in its receptor-binding activity. These results demonstrate that RBP interacts with both TTR and the receptor via loops C-D and E-F. The binding sites, however, are overlapping rather than identical. RBP also appears to make an additional contact with TTR via its loop A-B. A further implication of these results is that RBP, when bound to TTR, cannot bind simultaneously to the receptor. This observation is consistent with our previously proposed mechanism for delivery of retinol to target tissues [Sivaprasadarao and Findlay (1988) Biochem. J. 255, 571-579], according to which retinol delivery involves specific binding of RBP to the cell-surface receptor, an interaction that triggers release of retinol from RBP to the bound cell rather than internalization of retinol-RBP complex.
Purification and characterization of S-adenosylmethionine-protein-arginine N-methyltransferase from rat liver
Authors: Rawal N , Rajpurohit R , Paik W K , Kim S
Published: August 2015

A protein methylase I (S-adenosylmethionine-protein-arginine N-methyltransferase; EC 2.1.1.23), with a high specificity for recombinant heterogeneous nuclear ribonucleoprotein particle (hnRNP) protein A1, was purified from rat liver. The purification method is simple and rapid; a single initial step of DEAE-cellulose DE-52 chromatography resulted in a 114-fold enrichment from the cytosol, and subsequent Sephadex G-200 chromatography and f.p.l.c. yielded a homogeneous preparation. Ouchterlony double-immunodiffusion analysis indicated that the rat liver enzyme is immunologically different from an analogous enzyme from the calf brain, nuclear protein/histone-specific protein methylase I [Ghosh, Paik and Kim (1988) J. Biol. Chem. 263, 19024-19033; Rajpurohit, Lee, Park, Paik and Kim (1994) J. Biol. Chem. 269, 1075-1082]. The purified enzyme has a molecular mass of 450 kDa on Superose chromatography and 110 kDa on SDS/PAGE, indicating that it is composed of four identical-size subunits. The Km values for protein A1 and S-adenosyl-L-methionine were 0.54 x 10(-6) and 6.3 x 10(-6) M respectively. S-Adenosyl-L-homocysteine and sinefungin were effective inhibitors of the enzyme with Ki values of 8.4 x 10(-6) M and 0.65 x 10(-6) M respectively. Bivalent metal ions such as Zn2+, Mn2+ and Ni2+ were particularly toxic to the enzyme; at 1 mM Zn2+, 99% of the activity was inhibited. In addition, 50% of the enzyme activity was lost by treatment with 0.12 mM p-chloromercuribenzoate, indicating a requirement for a thiol group for enzyme activity. Glycerol, a compound often used to prevent enzyme inactivation, inhibited over 80% of the activity when present in the reaction mixture at a concentration of 20%.
Decreased arachidonic acid metabolism in human platelets by autologous neutrophils: possible role of cell adhesion
Authors: Chabannes B , Molière P , Pacheco Y , Lagarde M
Published: August 2015

The amount of the 12-lipoxygenase and cyclo-oxygenase products, 12(S)-hydroxy-(Z,Z,E,Z)-5,8,10,14-eicosatetraenoic acid (12-HETE) and 12(S)-hydroxy-(E,E,Z)-5,8,10-heptadecatrienoic acid (HHT), in human platelets stimulated by thrombin (0.1 and 2.5 units/ml), was studied in the presence of autologous neutrophils. A decreased formation of both products was induced by unstimulated neutrophils or neutrophils challenged with N-formylmethionyl- leucyl-phenylalanine (0.1 microM) or Ca2+ ionophore A23187 (0.15 microM). The effect of neutrophils was observed only in the presence of Ca2+. 12-HETE and HHT were also produced in platelets stimulated with thrombin in the absence of Ca2+ and/or Mg2+, but their level was not altered by neutrophils. 12(S),20-Dihydroxy-(Z,Z,E,Z)-5,8,10,14-eicosatetraenoic acid (12,20-DHETE), the cytochrome P-450 product from 12-HETE in neutrophils, was hardly detected, and its level did not compensate for the decrease in 12-HETE observed after platelet and neutrophil co-incubation. 5(S),12(S)-Dihydroxy-(E,Z,E,Z)- 6,8,10,14-eicosatetraenoic acid (5(S),12(S)-DHETE), the 5-lipoxygenase product of 12-HETE in neutrophils, was never detectable. In addition, the inhibition of 12-HETE and HHT formations appeared not to be due to degradation or thrombin uptake by neutrophils, nor was the decrease observed when the two cell populations were physically separated. A monoclonal antibody against the human platelet glycoprotein GMP140 (CD62), mediating Ca(2+)-dependent platelet-neutrophil adhesion, mimicked the inhibitory effect of neutrophils in a dose-dependent fashion. Furthermore, the 12-HETE and HHT productions were not affected when platelets were stimulated in the presence of neutrophils previously incubated with sialidase, which removes the sialic acid from a sialyl Lewis(x) structure assumed to be the neutrophil receptor for platelet GMP140. We conclude that the decrease in thrombin-stimulated 12-HETE and HHT formation observed when platelets were co-incubated with autologous neutrophils might be the consequence of platelet-neutrophil adherence, presumably through platelet GMP140.
The Ca2+/NADPH-dependent H2O2 generator in thyroid plasma membrane: inhibition by diphenyleneiodonium
Authors: Dème D , Doussiere J , De Sandro V , Dupuy C , Pommier J , Virion A
Published: August 2015

The thyroid plasma membrane contains a Ca(2+)-regulated NADPH-dependent H2O2-generating system which provides H2O2 for the peroxidase-catalysed biosynthesis of thyroid hormones. The electron transfer from NADPH to O2 catalysed by this system was studied by using diphenyleneiodonium (DPI), an inhibitor of flavo- and haemo-proteins. The prosthetic group of the H2O2 generator was removed by incubation with 5 mM CHAPS at 40 degrees C, and an active holoenzyme was reconstituted with FAD, but not with FMN. The H2O2-generating system also had an intrinsic Ca(2+)-dependent NADPH:ferricyanide reductase activity which is probably linked to its flavodehydrogenase component (or domain). Both activities, H2O2 production and ferricyanide reductase activity, were inhibited by DPI, with similar K1/2 (2.5 nmol/mg of protein). DPI only inhibited a system reduced with NADPH in the presence of Ca2+. NADPH could not be replaced by NADP+, NADH or sodium dithionite, suggesting the need for specific mild reduction of a redox centre in a particular conformation. Ferricyanide protected both activities against inhibition by DPI; the NADPH:ferricyanide reductase activity was completely protected at a ferricyanide concentration 20 times lower than that needed to protect the H2O2 formation, implying at least two target sites for DPI. One might be the flavodehydrogenase component; the other was beyond, on the entity which transfers the electrons to O2. This second site has not been identified.