bluuebunny/crossref_metadata_2025 huggingface dataset:
Identification of a brain-specific protein kinase Cζ pseudogene (ΨPKCζ) transcript
Authors: Andrea J E , Walsh M P
Published: August 2015

Protein kinase C (PKC), a widely-distributed enzyme implicated in the regulation of many physiological processes, consists of a family of at least twelve isoenzymes which differ in tissue distribution, subcellular localization, regulatory properties, etc. In addition to this heterogeneity at the protein level, we identify here for the first time a PKC zeta pseudogene (psi PKC zeta) transcript, specifically expressed in the brain, which is identical with PKC zeta except for sequence divergence within the first variable domain (V1). The authenticity of this unique V1 sequence (V1′) in mRNA was confirmed by RNase protection and reverse transcriptase PCR (RT-PCR) analysis. When translated in-frame with PKC zeta, a stop codon is located 28 amino acids towards the N-terminus of the divergence point and the intervening sequence lacks an expected initiating methionine. psi PKC zeta is non-functional in terms of protein synthesis since Western blotting with an antibody directed against the C-terminus of PKC zeta failed to reveal a protein smaller than PKC zeta, and synthetic psi PKC zeta RNA failed to support protein synthesis in a translation system in vitro. PCR amplification of rat genomic DNA demonstrated lack of an intron at the junction between V1′ and the first constant domain (the V1′-C1 border), and genomic DNA Southern blot analysis using PKC zeta and psi PKC zeta-specific probes indicated that they have different loci. psi PKC zeta, therefore, is not derived from the PKC zeta gene by alternative splicing, but rather is the product of a distinct gene. In Northern blot analysis, brain PKC zeta mRNA was identified as a low-abundance 3.1 kb transcript, while the abundant 2.5 and 4.7 kb mRNAs previously reported to encode PKC zeta are, in fact, psi PKC zeta transcripts. Analysis of rat brain, heart, lung, liver, kidney and skeletal muscle revealed psi PKC zeta mRNA only in brain. PKC zeta transcripts were most abundant in lung and kidney (2.7 and 4.7 kb mRNAs), correlating with the tissue profile of PKC zeta immunoreactivity in Western blots. Probes complementary to the common V5 and C1 domains detected both PKC zeta and psi PKC zeta transcripts. Interestingly, the C1 probe also detected an abundant novel 1.75 kb mRNA in brain and heart, suggesting the existence of an additional PKC zeta-related species. This work, therefore, also emphasizes the importance of careful choice of oligonucleotide and cDNA probes to study PKC zeta mRNA.
Binding of sesquiterpene lactone inhibitors to the Ca2+-ATPase
Authors: Wictome M , Khan Y M , East J M , Lee A G
Published: August 2015

The mechanism of inhibition of the Ca(2+)-ATPase from sarcoplasmic reticulum by the sesquiterpene lactones thapsigargin, trilobolide and thapsivillosin A (TvA) has been determined. A decrease in the affinity of the ATPase for Ca2+ is observed in the presence of the inhibitors (I), consistent with a shift in the E1/E2 equilibrium for the ATPase towards E2 forms. Amounts of inhibitor beyond a 1:1 molar ratio with ATPase produce no further decrease in affinity for Ca2+, inconsistent with the formation of a dead-end complex. Measurements of the rate of quenching of the tryptophan fluorescence of the ATPase by TvA are consistent with an association step to give E2I followed by an isomerization to a modified state E2AI. The kinetics of the reversal of the effects of TvA by Ca2+ at sub-stoichiometric amounts of TvA are bi-exponential, with a fast component whose rate is independent of TvA concentration and equal to the rate observed in the absence of TvA, and a slow component whose rate decreases with increasing TvA concentration. These observations are also consistent with the formation of a modified state E2AI following the initial binding of I to E2. The equilibrium constant E2AI/E2I increases in the order TvA < trilobolide < thapsigargin. The results suggest that the effects of the inhibitors on the overall ratio of E2 to E1 forms of the ATPase follow largely from the formation of E2AI from E2I, and that binding constants are very similar for E1Ca2, E1 and E2.
Extracellular Ca2+ stimulates the activation of mitogen-activated protein kinase and cell growth in human fibroblasts
Authors: Huang S , Maher V M , McCormick J J
Published: August 2015

In serum-free medium containing serum replacements but totally lacking in protein growth factors, diploid human fibroblasts remained quiescent if the extracellular Ca2+ concentration was only 0.1 mM. However, when the Ca2+ concentration in this medium was increased to 1 mM, the cells replicated as rapidly as they do in medium supplemented with protein growth factors. When quiescent cells in medium with only 0.1 mM Ca2+ were exposed to 1 or 10 mM Ca2+ or 100 ng/ml epidermal growth factor (EGF), the 42 kDa and 44 kDa forms of mitogen-activated protein kinase (MAPK) were rapidly activated, as demonstrated by a characteristic electrophoretic mobility shift of these proteins and by their enhanced ability to phosphorylate myelin basic protein (MBP). Analysis of fractions from Mono Q anion-exchange chromatography of lysates of cells exposed to 10 mM Ca2+ or 100 ng/ml EGF revealed a peak of MBP phosphorylation activity that was coeluted with p42 and p44 MAPK as shown by immunoblot analysis. Activation of MAPK by extracellular Ca2+ was dose-dependent and biphasic, with a peak of activation at 5-10 min after exposure, followed by a period of sustained activation of MAPK at a lower level. This pattern has been shown [Vouret-Craviari, Van Obberghen-Schilling, Scimeca, Van Obberghen and Pouysségur (1993) Biochem J. 289, 209-214] to correlate with the re-entry of mammalian cells into the cell cycle.
Physiological control of metabolic flux: the requirement for multisite modulation
Authors: Fell D A , Thomas S
Published: August 2015

Biochemists have long assumed that the flux through a metabolic pathway can be controlled by the activity of a key regulatory enzyme near the beginning of the pathway. We present the accumulating evidence that every step in this assumption is flawed. Instead, effective physiological control of metabolism is shown to involve simultaneous multisite modulation through action on a number of enzymes.
The protein phosphatase inhibitor calyculin A stimulates chemokine production by human synovial cells
Authors: Jordan N J , Watson M L , Westwick J
Published: August 2015

Cultured human synovial fibroblasts express mRNA for the chemotactic cytokines (chemokines) interleukin-8 (IL-8), monocyte chemotactic protein 1 (MCP-1) and regulated upon activation normal T-cell expressed and presumably secreted (RANTES), when stimulated with IL-1 or tumour necrosis factor alpha (TNF alpha). Calyculin A, a potent type 1/2A protein serine/threonine phosphatase inhibitor, was used to examine the role of protein phosphatases in the regulation of chemokine gene expression. Calyculin A (1 nM) mimicked IL-1 by inducing IL-8 and MCP-1 mRNA expression in synovial cells. IL-8 mRNA was induced over a similar time period (1-6 h) in response to IL-1 or calyculin A, whereas MCP-1 mRNA was induced more rapidly (1-2 h) by calyculin A than by IL-1 (4-6 h). Expression of RANTES mRNA occurred in response to TNF alpha, but could not be induced by stimulation with calyculin A alone. These results suggest that inhibition of protein phosphatase type 1/2A may have a differential role in the regulation of the expression of each of the chemokine genes. Synovial fibroblasts also secreted IL-8 and IL-6 peptide when stimulated with either IL-1/TNF alpha or calyculin A. The amount of IL-8 and IL-6 peptide produced in response to calyculin A was significantly increased above that produced by untreated synovial cells, though it was much less than the amount induced by IL-1 or TNF alpha. Calyculin A also acted synergistically with IL-1 or TNF alpha to cause a 2-fold potentiation of IL-1- or TNF alpha-induced IL-8 mRNA and peptide and RANTES mRNA expression. These results suggest that although inhibition of a protein phosphatase may be able to regulate the magnitude of IL-1-induced chemokine gene expression, the IL-1 signal transduction pathway involves components in addition to phosphatase inhibition, possibly including the activation of a protein kinase, the action of which may be opposed by a protein phosphatase inhibited by calyculin A.
Regulation of the β-lactamase BlaL of Streptomyces cacaoi: the product of the blaB regulatory gene is an internal membrane-bound protein
Authors: Magdalena J , Joris B , Van Beeumen J , Brasseur R , Dusart J
Published: August 2015

The beta-lactamase-encoding gene blaL, cloned from Streptomyces cacaoi in Streptomyces lividans, is inducible by beta-lactam compounds. This regulation has been shown to depend on the products of two open reading frames, ORF1 (blaA) and ORF2 (blaB) [Lenzini, Magdalena, Fraipont, Joris, Matagne and Dusart (1992) Mol. Gen. Genet. 235, 41-48]. BlaA belongs to the LysR family of transcription activators, whereas BlaB shares some features with the penicillin-recognizing proteins. BlaB has now been overexpressed in Escherichia coli, purified and used for antibody preparation. Immunoblotting of cell-fractionated materials from S. cacaoi showed that BlaB is attached to the internal face of the cytoplasmic membrane. It could not be released by high salt concentrations or EDTA, but only by protease treatment. Under the assay conditions, BlaB did not act as a penicillin-binding protein, a beta-lactamase, a D-amino-peptidase or a target in a phosphorylation step.
Heparin proteoglycans synthesized by mouse mastocytoma contain chondroitin sulphate
Authors: Lidholt K , Eriksson I , Kjellén L
Published: August 2015

Proteoglycans (PGs), biosynthetically labelled with [35S]sulphate, were isolated from mouse mastocytoma tissue. Chromatography on antithrombin (AT)-Sepharose resulted in the separation of the 35S-labelled PGs into three fractions: PGs with no affinity for the gel (NA-PGs), PGs with low affinity (LA-PGs), and PGs with high affinity (HA-PGs) for antithrombin. Whereas NA-PGs contained almost exclusively chondroitin sulphate (CS), the AT-binding PGs contained 80-85% heparin and 15-20% CS. [35S]CS-containing macromolecules obtained from the HA-PG fraction after removal of the heparin polysaccharide chains were rechromatographed on AT-Sepharose. A majority of these 35S-labelled macromolecules no longer showed affinity for AT. These experiments indicate that the [35S]CS recovered in the AT-binding PGs is present in hybrid PGs. Polysaccharide chain-length determination demonstrated that the heparin chains were somewhat larger (M(r) approximately 30,000) than the CS chains in the NA-PGs (M(r) approximately 25,000). CS chains in the hybrid PGs were slightly smaller (M(r) approximately 20,000). Characterization of the sulphated CS disaccharides from NA- and HA-PGs showed that they contained similar amounts (20%) of disulphated disaccharides of [GlcA-GalNAc(4,6-di-OSO3)] type. The monosulphated CS-disaccharides were O-sulphated at C-4 of the galactosamine units. Analysis by gel chromatography of the [35S]CS components isolated from HA-PGs after heparinase treatment showed that a major portion of these contained one CS chain only. Calculations of the number of CS and heparin chains in AT-binding PGs, based on polysaccharide composition and polysaccharide chain length, indicate that all heparin-containing PGs are hybrids.
Evidence for an essential serine residue in the active site of the Theta class glutathione transferases
Authors: Board P G , Coggan M , Wilce M C J , Parker M W
Published: August 2015

A consistent feature of the Alpha-, Mu- and Pi-class glutathione transferases (GSTs) is the presence near the N-terminus of a tyrosine residue that contributes to the activation of glutathione. While this residue appears to be conserved in many Theta-class GSTs, its absence in some suggested that the Theta-class GSTs may have a significantly different structure or catalytic mechanism. The elucidation of the crystal structure of the Theta-class GST from the Australian sheep blowfly, Lucilia cuprina, has indicated that a serine residue rather than a tyrosine residue can form a hydrogen bond with the glutathionyl sulphur atom. The present studies show that mutation of Ser-9 to alanine substantially inactivates the L. cuprina GST, confirming its importance in the reaction mechanism. As this serine is conserved in all Theta-class enzymes reported so far, it seems that an active-site serine is a significant factor that distinguishes the Theta-class GSTs from members of the Alpha-, Mu- and Pi-class isoenzymes.
Inhibition of asialoglycoprotein endocytosis and degradation in rat hepatocytes by protein phosphatase inhibitors
Authors: Holen I , Gordon P B , Strømhaug P E , Berg T O , Fengsrud M , Brech A , Roos N , Berg T , Seglen P O
Published: August 2015

In isolated rat hepatocytes, a radiolabelled tyramine-cellobiose conjugate of asialo-orosomucoid, 125I-TC-AOM, was rapidly taken up by receptor-mediated endocytosis and proteolytically degraded in the lysosomes, where radioactive degradation products accumulated. Okadaic acid and other protein phosphatase inhibitors (microcystin-LR, calyculin A) strongly reduced the fraction of asialoglycoprotein (ASGP) receptors localized to the cell surface, and correspondingly inhibited the uptake of 125I-TC-AOM. In addition, the inhibitors suppressed 125I-TC-AOM degradation strongly (90% at 150 nM) and potently (half-maximal effect at 20 nM okadaic acid), indicating an involvement of protein phosphorylation, and of a protein phosphatase of type 2A, in the regulation of intracellular endocytic flux. The effects of okadaic acid on 125I-TC-AOM accumulation, as well as on degradation, could be eliminated by the protein kinase inhibitor genistein. Okadaic acid prevented the transfer of 125I-TC-AOM to a non-recycling endocytic compartment, causing its retention in a recycling compartment from which about one-third of the endocytosed 125I-TC-AOM could be returned to the cell surface and detached from its receptor in the presence of EGTA. ASGP receptors recycled extensively both in the presence and absence of okadaic acid, as indicated by a sustained uptake of 125I-TC-AOM. Sucrose density gradient analysis and sedimentation studies indicated that okadaic acid caused accumulation of 125I-TC-AOM in light endosomes (1.11 g/ml), preventing its transfer to dense endosomes (1.14 g/ml) and lysosomes (1.18 g/ml). The lysosomes could be identified in density gradients by their contents of lysosomal marker enzymes and acid-soluble radioactivity, and by their sensitivity towards the lysosome-disrupting agent glycyl-L-phenylalanine-2-naphthylamide. By using endocytosed AOM-gold particles as an ultrastructural endocytic marker, it could be shown that the light endosomes accumulating ASGP in the presence of okadaic acid had the morphological appearance of small endocytic vesicles/tubules and multivesicular endosomes. Whereas in control cells 4% of the AOM-gold was in small vesicles/tubules, 55% in multivesicular endosomes and 41% in lysosomes, the corresponding figures for okadaic acid-treated cells were 17%, 73% and 11%. Our results thus indicate that protein phosphatase inhibitors have two effects on ASGP endocytosis: (1) an early inhibition of ligand uptake, due to a reduction in the fraction of ASGP receptors at the cell surface, and (2) an inhibition of ASGP transfer from a recycling compartment consisting of light, small endocytic vesicles and multivesicular endosomes, to a non-recycling compartment consisting of dense multivesicular endosomes.(ABSTRACT TRUNCATED AT 400 WORDS)
Expression, purification and characterization of recombinant human N-acetylgalactosamine-6-sulphatase
Authors: Bielicki J , Fuller M , Guo X H , Morris C P , Hopewood J J , Anson D S
Published: August 2015

Full-length cDNA sequences encoding human N-acetylgalactosamine-6-sulphatase were stably expressed in Chinese hamster ovary cells under the transcriptional control of the human polypeptide chain elongation factor 1 alpha gene promoter. A clonal cell line overexpressing recombinant N-acetylgalactosamine-6-sulphatase to a level of approx. 3 mg/l of culture medium was isolated. The secreted precursor enzyme was purified to homogeneity by a two-column procedure with an overall yield of 53% of the activity. The physical and catalytic parameters of the recombinant enzyme were similar to those of the mature form isolated from liver. On SDS/PAGE and gel filtration, recombinant N-acetylgalactosamine-6-sulphatase had a native molecular mass of 58-60 kDa. Recombinant N-acetylgalactosamine-6-sulphatase was endocytosed by mucopolysaccharidosis IVA fibroblasts via the mannose-6-phosphate receptor-mediated pathway and was efficiently localized to lysosomes.
Identification of histatins as tannin-binding proteins in human saliva
Authors: Yan Q , Bennick A
Published: August 2015

Tannins have a number of detrimental biological effects and these include interference with normal growth and metabolism if they are present in the feed of various animals. Proline-rich proteins (PRPs) in saliva have been shown to provide protection against tannin, but little is known about the mechanism of protection and interaction of other salivary proteins with tannin. To identify tannin-binding human salivary proteins, parotid and submandibular/sublingual saliva samples were adsorbed with tannin. PRPs, and in particular a group of low-M(r) proteins, were readily precipitated by tannin. The low-M(r) proteins were purified from parotid saliva and demonstrated to be histatins, a family of well-characterized histidine-rich salivary proteins. The ability of synthetic histatin 5, as well as an acidic PRP (PRP-1) and gelatin to precipitate quebracho condensed tannin and tannic acid was determined. At pH 7.4 histatin 5 was the most effective precipitant of both condensed tannin and tannic acid and it also precipitated the largest amount of condensed tannin at pH 3.0, but the smallest amount of tannic acid at that pH. In contrast PRP-1 showed a greater ability to precipitate both condensed tannin and tannic acid at pH 3.0 than at pH 7.4. Under most circumstances histatin 5 was therefore more effective in precipitating tannins than proteins with high proline content which generally have been recognized as strong precipitants of tannin. Pre-incubation of tannic acid with alpha-amylase inhibited the enzyme, but addition of histatin 5 or the acidic PRP PIF-s protected amylase from inhibition by tannin. Similarly salivary proteins may protect other biological activities in the digestive tract from inhibition by dietary tannin.
Reaction mechanism of chitosanase from Streptomyces sp. N174
Authors: Fukamizo T , Honda Y , Goto S , Boucher I , Brzezinski R
Published: August 2015

Chitosanase was produced by the strain of Streptomyces lividans TK24 bearing the csn gene from Streptomyces sp. N174, and purified by S-Sepharose and Bio-Gel A column chromatography. Partially (25-35%) N-acetylated chitosan was digested by the purified chitosanase, and structures of the products were analysed by NMR spectroscopy. The chitosanase produced heterooligosaccharides consisting of D-GlcN and GlcNAc in addition to glucosamine oligosaccharides [(GlcN)n, n = 1, 2 and 3]. The reducing- and non-reducing-end residues of the heterooligosaccharide products were GlcNAc and GlcN respectively, indicating that the chitosanase can split the GlcNAc-GlcN linkage in addition to that of GlcN-GlcN. Time-dependent 1H-NMR spectra showing hydrolysis of (GlcN)6 by the chitosanase were obtained in order to determine the anomeric form of the reaction products. The chitosanase was found to produce only the alpha-form; therefore it is an inverting enzyme. Separation and quantification of (GlcN)n was achieved by HPLC, and the time course of the reaction catalysed by the chitosanase was studied using (GlcN)n (n = 4, 5 and 6) as the substrate. The chitosanase hydrolysed (GlcN)6 in an endo-splitting manner producing (GlcN)2, (GlcN)3 and (GlcN)4, and did not catalyse transglycosylation. Product distribution was (GlcN)3 >> (GlcN)2 > (GlcN)4. Cleavage to (GlcN)3 + (GlcN)3 predominated over that to (GlcN)2 + (GlcN)4. Time courses showed a decrease in rate of substrate degradation from (GlcN)6 to (GlcN)5 to (GlcN)4. It is most likely that the substrate-binding cleft of the chitosanase can accommodate at least six GlcN residues, and that the cleavage point is located at the midpoint of the binding cleft.
Uptake of apolipoprotein E-rich and apolipoprotein E-poor subfractions of high-density lipoprotein by liver membranes and HepG2 cells
Authors: Fragoso Y D , Skinner E R
Published: August 2015

Apolipoprotein (apo) E plays an important role in mediating high-density lipoprotein (HDL) cholesterol transport and uptake by the liver. Evidence for and against the existence of conventional liver receptors for HDL containing apoE have been reported, although the selective uptake of the cholesterol moiety of HDL has been demonstrated. The present study investigated the hepatic uptake of subfractions of HDL separated on the basis of their apoE content. Rabbit HDL and its apoE-rich and apoE-poor subfractions, separated by heparin-Sepharose affinity chromatography, were labelled in their apoprotein moieties with [14C]sucrose and in their cholesteryl ester moiety with 3H. No binding of either subfraction to rabbit liver membranes could be detected. With cultured HepG2 cells, however, there was a high uptake of 3H but a very low uptake of 14C from both HDL subfractions, demonstrating that selective uptake was operating. Addition of unlabelled apoE-poor HDL inhibited the uptake of both labels from the two subfractions to the same extent. These studies, which differed from previously reported investigations by employing native homologous HDL subfractions of known apolipoprotein composition, demonstrated that apoE is not directly involved in the selective uptake of HDL cholesterol by the liver. In the absence of specific binding sites on liver membranes, it is suggested that an alternative mechanism might exist for the clearance of HDL cholesterol from the plasma.
Bradykinin-stimulated phosphatidylcholine hydrolysis in airway smooth muscle: the role of Ca2+ and protein kinase C
Authors: Pyne S , Pyne N J
Published: August 2015

The regulation of phosphatidylcholine (PtdCho) hydrolysis by Ca2+ and protein kinase C (PKC) was measured in [3H]palmitate-labelled cultured guinea-pig airway smooth-muscle cells as phosphatidylbutanol ([3H]PtdBut) and phosphatidate ([3H]PtdOH) formation in the presence of butanol. The former is a direct measure of phospholipase D (PLD) activity, whereas the latter, in airway smooth muscle, is indicative of net PtdCho-specific phospholipase C (PLC)-like/diacylglycerol (DG) kinase activity. Bradykinin-stimulated responses exhibited a requirement for extracellular Ca2+ influx, since they were inhibited in the presence of EGTA. This influx was independent of voltage-operated channels, since the L-type channel blocker nifedipine (up to 10 microM) was without effect on bradykinin-stimulated responses. In support of this, membrane depolarization with KCl (30 mM) failed to elicit either response. However, bradykinin-stimulated formation of both [3H]PtdBut and [3H]PtdOH was partially inhibited by 100 microM SKF96365. Ionomycin, a Ca2+ ionophore, induced PtdCho hydrolysis to a greater extent than bradykinin, also in an extracellular-Ca(2+)-dependent manner. Thapsigargin-induced emptying of intracellular Ca2+ pools elicited the formation of both [3H]PtdBut and [3H]PtdOH and displayed a requirement for extracellular Ca2+. Bradykinin-stimulated PtdCho-specific PLC-like/DG kinase pathway and PLD responses were unaffected by thapsigargin pretreatment, thereby questioning the role of Ins(1,4,5)P3/Ins(1,3,4,5)P4-dependent Ca2+ stores in the receptor stimulation of these activities in airway smooth-muscle cells. In this regard, we have previously demonstrated that the bradykinin-stimulated PtdCho-specific PLD and PLC-like activities can occur under conditions of apparent complete blockade of bradykinin-stimulated Ins(1,4,5)P3 formation by receptor antagonist in guinea-pig airway smooth muscle. The PKC inhibitor, Ro31-8220, selectively blocked both bradykinin- and ionomycin-stimulated PLD activity in a concentration-dependent manner (IC50 approx. 1 microM), but was without effect on bradykinin-stimulated PtdCho-PLC-like/DG kinase-derived PtdOH formation. In contrast, an inhibitor of PtdCho-PLC, D609, selectively blocked the formation of [3H]PtdOH in the presence of butanol (PtdCho-PLC-like/DG kinase activity), but not [3H]PtdBut formation. In conclusion, PtdCho hydrolysis appears to occur via two distinguishable routes which both require extracellular Ca2+, whereas only the PLD route is regulated by PKC.
The lysosomal membrane glycoproteins Lamp-1 and Lamp-2 are present in mobilizable organelles, but are absent from the azurophil granules of human neutrophils
Authors: Dahlgren C , Carlsson S R , Karlsson A , Lundqvist H , Sjölin C
Published: August 2015

The subcellular localization of two members of a highly glycosylated protein group present in lysosomal membranes in most cells, the lysosome-associated membrane proteins 1 and 2 (Lamp-1 and Lamp-2), was examined in human neutrophil granulocytes. Antibodies that were raised against purified Lamp-1 adn Lamp-2 gave a distinct granular staining of the cytoplasm upon immunostaining of neutrophils. Subcellular fractionation was used to separate the azurophil and specific granules from a light-membrane fraction containing plasma membranes and secretory vesicles, and Western blotting was used to determine the presence of the Lamps in these fractions. The results show that Lamp-1 and Lamp-2 are present in the specific-granule-enriched fraction and in the light-membrane fraction, but not in the azurophil granules. Separation of secretory vesicles from plasma membranes disclosed that the light-membrane Lamps were present primarily in the secretory-vesicle-enriched fraction. During phagocytosis both Lamp-1 and Lamp-2 became markedly concentrated around the ingested particle and they both appear on the cell surface when the secretory organelles are mobilized.
In search of a selective inhibitor of the induced transport of small solutes in Plasmodium falciparum-infected erythrocytes: effects of arylaminobenzoates
Authors: Kirk K , Horner H A
Published: August 2015

Following invasion of the human erythrocyte by the malaria parasite, Plasmodium falciparum, there appear in the parasitized cell new, high-capacity permeation pathways that transport a diverse range of low-molecular-mass solutes. In this study a series of 16 arylaminobenzoates, analogues of the Cl- channel blocker 5-nitro-2-(3-phenylpropylamino)benzoic acid (NPPB), were tested for their effects on the transport of choline, a univalent cation, into malaria-infected cells. A number of the arylaminobenzoates were found to be potent inhibitors of malaria-induced choline transport and to be similarly effective at blocking the induced transport of the uncharged pyrimidine nucleoside thymidine and the univalent anion lactate. The data are consistent with the hypothesis that much of the induced transport of cations, anions and non-electrolytes into parasitized cells is via broad-specificity, anion-selective pathways of a single type. A comparison of the effects of the arylaminobenzoates on malaria-induced transport with their effects on a number of representative anion transport systems in normal mammalian cells suggests that it is possible to identify pharmacological agents that block the malaria-induced pathway while not significantly affecting important transport mechanisms in host tissues. The most potent of the induced-transport inhibitors identified were shown to inhibit [3H]hypoxanthine incorporation in in vitro parasite growth assays. These data support the view that the induced-transport pathway may be a viable pharmacological target.
Pseudopregnancy induces the expression of hepatocyte nuclear factor-1β and its target gene aminopeptidase N in rabbit endometrium via the epithelial promoter
Authors: Olsen J , Classen-Linke I , Sjöström H , Norén O
Published: August 2015

The rabbit endometrium is an excellent model system allowing experimental manipulation of aminopeptidase N (APN) mRNA expression in vivo. By RNase mapping and sequencing of cloned PCR-amplified primer-extended RNA, it was demonstrated that endometrial APN expression is directed by the epithelial APN promoter and is increased in human-choriogonadotropin-induced pseudopregnancy. Cloning and sequencing of the rabbit APN epithelial promoter revealed conservation of the upstream footprint (UF), hepatocyte nuclear factor-1 (HNF1) and Sp1 elements known to be present in the pig and human promoters as well. The pseudopregnancy-induced APN expression was found to be accompanied by a parallel increase in the level of the transcription factor HNF1 beta, whereas a much smaller increase in Sp1 and UF-binding proteins was observed. This indicates that HNF1 beta acts as a switch triggering the pregnancy-induced APN expression. The sequence of the UF element suggests members of the nuclear hormone-receptor superfamily as possible UF-binding proteins, and competition experiments suggest that the chicken ovalbumin upstream promoter transcription factor functions as such in the rabbit endometrium.
The nature of topogenic sequences determines the transport competence of topological mutants of neutral endopeptidase-24.11
Authors: Yang X F , Crine P , Boileau G
Published: August 2015

Type II integral membrane proteins are anchored by a signal-peptide/membrane-anchor domain (SA domain) located near their N-terminus, whereas type I membrane proteins are anchored by stop-transfer sequences usually located near the C-terminus. In this study we have attempted to transform neutral endopeptidase-24.11 (EC 3.4.24.11; NEP), a type II membrane protein, into a type I membrane protein. Three type I mutant proteins were constructed by fusion of topogenic sequences to the C-terminus of SecNEP, a soluble form of NEP. The first two type I mutants, SecNEP-TMC and SecNEP-TMIC, were constructed by fusing in frame the cytosolic and SA domains of NEP to the C-terminus of SecNEP. These two fusion proteins differ only in the orientation of the cytosolic tail. The third type I mutant, SecNEP-ACE, was constructed by fusing in frame the stop-transfer and cytosolic domains of angiotensin I-converting enzyme (EC 3.4.15.1; ACE) to the C-terminus of SecNEP. Our results suggest that: (1) the NEP ectodomain can be anchored with a type I topology in the endoplasmic reticulum (ER) membrane by both NEP and ACE topogenic sequences; (2) SecNEP-TMC and SecNEP-TMIC were transport-incompetent and needed proteolytic cleavage in the C-terminal region to leave the ER, whereas SecNEP-ACE was transported out of the ER as a type I membrane protein. Therefore we concluded that the nature of topogenic sequences determines the transport-competence of topological mutants of neutral endopeptidase-24.11.
A hierarchy of ATP-consuming processes in mammalian cells
Authors: Buttgereit F , Brand M D
Published: August 2015

The rates of different ATP-consuming reactions were measured in concanavalin A-stimulated thymocytes, a model system in which more than 80% of the ATP consumption can be accounted for. There was a clear hierarchy of the responses of different energy-consuming reactions to changes in energy supply: pathways of macromolecule biosynthesis (protein synthesis and RNA/DNA synthesis) were most sensitive to energy supply, followed by sodium cycling and then calcium cycling across the plasma membrane. Mitochondrial proton leak was the least sensitive to energy supply. Control analysis was used to quantify the relative control over ATP production exerted by the individual groups of ATP-consuming reactions. Control was widely shared; no block of reactions had more than one-third of the control. A fuller control analysis showed that there appeared to be a hierarchy of control over the flux through ATP: protein synthesis > RNA/DNA synthesis and substrate oxidation > Na+ cycling and Ca2+ cycling > other ATP consumers and mitochondrial proton leak. Control analysis also indicated that there was significant control over the rates of individual ATP consumers by energy supply. Each ATP consumer had strong control over its own rate but very little control over the rates of the other ATP consumers.
Re-evaluation of the distributions of dystrophin and utrophin in sciatic nerve
Authors: Fabbrizio E , Latouche J , Rivier F , Hugon G , Mornet D
Published: August 2015

Differential expression of proteins belonging to the dystrophin family was analysed in peripheral nerves. In agreement with previous reports, no full-size dystrophin was detectable, only Dp116, one of the short dystrophin products of the Duchenne muscular dystrophy (DMD) gene. We used specific monoclonal antibodies to fully investigate the presence of utrophin, a dystrophin homologue encoded by a gene located on chromosome 6q24. Evidence is presented here of the presence of two potential isoforms of full-length utrophin in different nerve structures, which may differ by alternative splicing of the 3′-terminal part of the utrophin gene according to the specificities of the monoclonal antiobodies used. One full-length utrophin was co-localized with Dp116 in the sheath around each separate Schwann cell-axon unit, but the other utrophin isoform was found to be perineurium-specific. We also highlighted a potential 80 kDa utrophin-related protein. The utrophin distribution in peripheral nerves was re-evaluated and utrophin isoforms were detected at the protein level. This preliminary indication will require more concrete molecular evidence to confirm the presence of these two utrophin isoforms as well as the potential 80 kDa utrophin isoform, but the results strongly suggest that each isoform must have a specialized role and function within each specific nervous structure.
Correlation of a missense mutation in the human Secretor
α1,2-fucosyltransferase gene with the Lewis(a+b+) phenotype: a potential molecular basis for the weak Secretor allele (Se
w)
Authors: Yu L C , Yang Y H , Broadberry R E , Chen Y H , Chan Y S , Lin M
Published: August 2015

A missense mutation (A385 to T), predicting an Ile129 to Phe substitution, in the human Secretor alpha 1,2-fucosyltransferase gene was present in double dose in Lewis(a+b+) individuals, but not in Lewis(a-b+) individuals. Co-segregation of the Lewis(a+b+) phenotype with homozygosity for the mutation was also verified. These results yield a potential molecular basis for the weak Secretor allele (Sew) accounting for the Lewis(a+b+) phenotype.
Characterization of human AMP deaminase 2 (AMPD2) gene expression reveals alternative transcripts encoding variable N-terminal extensions of isoform L
Authors: Van den Bergh F , Sabina R L
Published: August 2015

AMP deaminase (AMPD) is a highly regulated enzymic activity and multiple isoforms of this enzyme are coded for by a multigene family in mammalian species, including man. Isoform L (liver) is the main activity present in adult human liver and is the protein product of the AMPD2 gene, which is widely expressed in non-muscle tissues and cells. A previous report described almost the full-length cDNA sequence and part of the human AMPD2 gene and also presented Northern blot evidence for multiple transcripts in brain. This study was performed to further characterize the AMPD2 gene and its expression in human tissues. AMPD2 genomic and human cerebellum cDNA clones were isolated, sequenced and used as probes in RNase protection analyses which together demonstrated the following: (1) an intervening sequence near the 5′-end of the published AMPD2 cDNA, which affects the predicted N-terminal amino acid sequence of isoform L; (2) alternative transcripts resulting from exon shuffling at, or near, the 5′-end of the AMPD2 gene that exhibit tissue-specific patterns of relative abundance; (3) predicted usage of three different initiation codons to confer variable N-terminal extensions on isoform L polypeptides; and (4) an extension of a 3′ untranslated sequence in some AMPD2 transcripts. In addition, reverse transcriptase PCR and additional RNase protection analyses were used to map the 5′-ends of two mutually-exclusive exon 1 sequences, both of which contain multiple transcription-initiation sites. These results are discussed in relation to predicted isoform L diversity across human tissues and cells.
Decrease in free-radical production with age in rat peritoneal macrophages
Authors: Alvarez E , Conde M , Machado A , Sobrino F , Santa Maria C
Published: August 2015

The respiratory-burst reaction has been studied in rat peritoneal macrophages of different ages (3, 12 and 24 months) using phorbol 12-myristate 13-acetate (PMA) to stimulate NADPH oxidase. Production of O2-. and H2O2 decreased with age (about 50 and 75% respectively); however, no difference in NADPH oxidase activity was found. NO. production was also reduced with age (40%). Furthermore, a progressive and significant decrease in the pentose phosphate flux was detected as a function of age in control and PMA-stimulated macrophages. The NADPH/NADP+ ratio decreased with age in control and PMA-stimulated macrophages. Glucose uptake was lower in middle-aged (12 months) and old (24 months) animals but no differences were found between these groups.
Enzymes of ecdysteroid transformation and inactivation in the midgut of the cotton leafworm, Spodoptera littoralis: properties and developmental profiles
Authors: Webb T J , Powls R , Rees H H
Published: August 2015

In the midgut cytosol of Lepidoptera, ecdysteroids undergo inactivation by transformation via the 3-dehydro derivative to the corresponding 3-epiecdysteroid (3 alpha-hydroxy) and by phosphate conjugation. The oxygen-dependent oxidase catalyses formation of 3-dehydroecdysteroid, which can be reduced either irreversibly by 3-dehydroecdysone 3 alpha-reductase to 3-epiecdysteroid, or by 3-dehydroecdysone 3 beta-reductase back to the initial ecdysteroid. Furthermore, these ecdysteroids undergo further inactivation by phosphorylation. These ecdysteroid transformations have been investigated in last instar larvae of the cotton leafworm, Spodoptera littoralis. The products of the phosphorylation have been characterized as predominantly ecdysteroid 2-phosphate accompanied by smaller amounts of the corresponding 22-phosphate. The phosphotransferases require Mg2+ and ATP. Whereas the 3-dehydroecdysone 3 alpha-reductase has a clear preference for NADPH rather than NADH, the corresponding 3 beta-reductase markedly favours NADH. The physiological significance of the latter enzyme is unclear. The profiles of the various enzymic activities in dialysed midgut cytosol supplemented with appropriate cofactors were determined throughout the last larval instar. All activities were detectable throughout the instar, but the respective enzymes exhibited maxima at different times. Ecdysone oxidase showed a peak early in the instar, with 3-dehydroecdysone 3 alpha-reductase increasing to a peak as the former activity declined. The 3-dehydroecdysone 3 beta-reductase exhibited peak activity late in the instar, a profile similar to that observed for the corresponding haemolymph enzyme involved in reduction of the 3-dehydroecdysone product of the prothoracic glands to ecdysone. Thus, the significance of the midgut 3 beta-reductase may be related to production of active hormone. Both ecydsteroid 22- and 2-phosphotransferases showed high activities early in the instar and then declined. The physiological significance of the profiles for the ecdysone oxidase, the 3-dehydroecdysone 3 alpha-reductase and phosphotransferases is unclear.
The first putative transmembrane helix of the 16 kDa proteolipid lines a pore in the Vo sector of the vacuolar H+-ATPase
Authors: Jones P C , Harrison M A , Kim Y I , Finbow M E , Findlay J B C
Published: August 2015

The 16 kDa proteolipid is the major component of the vacuolar H(+)-ATPase membrane sector, responsible for proton translocation. Expression of a related proteolipid from the arythropod Nephrops norvegicus in a Saccharomyces strain in which the VMA3 gene for the endogenous proteolipid has been disrupted results in restored vacuolar H(+)-ATPase function. We have used this complementation system, coupled to cysteine substitution mutagenesis and protein chemistry, to investigate structural features of the proteolipid. Consecutive cysteines were introduced individually into putative transmembrane segment 1 of the proteolipid, and at selected sites in extramembranous regions and in segment 3 and 4. Analysis of restored vacuolar H(+)-ATPase function showed that segment 1 residues sensitive to mutation to cysteine were clustered on a single face, but only if the segment was helical. Only residues insensitive to mutation could be covalently modified by the cysteine-specific reagent fluorescein 5-maleimide. A cysteine introduced into segment 3 was the only residue accessible to a relatively hydrophobic reagent, suggesting accessibility to the lipid phase. Analysis of disulphide bond formation between introduced cysteines indicates that the first transmembrane alpha-helices of each monomer are adjacent to each other at the centre of the proteolipid multimeric complex. The data are consistent with a model in which the fluorescein maleimide-accessible face of helix I lines a pore at the centre of a hexameric complex formed by the proteolipid, with the mutationally sensitive face oriented into the protein core. The implications for ion-transport function in this family of proteins are discussed in the context of this structural model.
Early alterations in the brown adipose tissue adenylate cyclase system of pre-obese Zucker rat fa/fa pups: decreased G-proteins and β3-adrenoceptor activities
Authors: Charon C , Krief S , Diot-Dupuy F , Strosberg A D , Emorine L J , Bazin R
Published: August 2015

This study was undertaken to determine whether receptor and non-receptor components of the adenylate cyclase (AC) cascade were altered in brown adipose tissue (BAT) of 14-day-old pre-obese (fa/fa) rats, before endocrine status is strongly modified by fa gene expression. Activity of the AC catalytic subunit did not differ between the two genotypes. In fa/fa rats compared with control Fa/fa rats, there was a 50% decrease in the activity of alpha Gs (stimulated by NaF or guanosine 5′-[gamma-thio]triphosphate) but no change in protein content (Western blotting). alpha Gi function, assessed by the inhibitory action of low concentrations of guanosine 5′-[beta gamma-imido]triphosphate upon 10(-4) M forskolin-stimulated AC activity, was equally low in both genotypes. Analysis of dose-response curves for different beta-agonists revealed that (i) both the basal and the maximally stimulated activity of AC were 2-fold lower in fa/fa rats than in Fa/fa rats; (ii) BRL37344 and CGP12177 (beta 3 agonists) were less potent in fa/fa than in Fa/fa rats (Kact. multiplied by 2); (iii) noradrenaline and isoprenaline (Iso), at the low-affinity site (beta 3-AR), were less potent in fa/fa than in Fa/fa pups (Kact. increased by 30 and 20% respectively). At the high-affinity site (mainly beta 1) these two agonists were more potent in fa/fa than in Fa/fa rats (Kact. decreased by 40 and 80% respectively). In good agreement with the latter result, the beta 1-adrenergic receptor (beta 1-AR)-selective antagonist CGP20712A had more effect on the Iso-stimulated AC activity in pre-obese than in lean pups (2-fold decreased in IC50). Binding experiments with [3H]CGP12177 show that in BAT of suckling rats, beta 3-ARs represent 80% of the total beta-ARs. Bmax values for the two sites were not affected by the genotype, although the beta 3-AR mRNA concentration in BAT (quantitative reverse-transcriptase PCR) was 3-fold lower in fa/fa rats than in Fa/fa pups. In conclusion, these results provide evidence for alterations in beta 1- and beta 3-AR signalling in BAT of 14-day-old suckling pre-obese Zucker rats with a decreased activity of alpha Gs. The impaired AC responsiveness to catecholamines might be a primary contributor to the development of this genetic obesity.
Quantal responses to inositol 1,4,5-trisphosphate are not a consequence of Ca2+ regulation of inositol 1,4,5-trisphosphate receptors
Authors: Patel S , Taylor C W
Published: August 2015

Submaximal concentrations of inositol 1,4,5-trisphosphate (InsP3) rapidly release only a fraction of the InsP3-sensitive intracellular Ca2+ stores, despite the ability of further increases in InsP3 concentration to evoke further Ca2+ release. The mechanisms underlying such quantal Ca2+ mobilization are not understood, but have been proposed to involve regulatory effects of cytosolic Ca2+ on InsP3 receptors. By examining complete concentration-effect relationships for InsP3-stimulated 45Ca2+ efflux from the intracellular stores of permeabilized hepatocytes, we demonstrate that, at 37 degrees C, responses to InsP3 are quantal in Ca(2+)-free media heavily buffered with either EGTA or BAPTA [1,2-bis-(2-aminophenoxy)ethane-N,N,N′,N′-tetra-acetic acid]. Lower concentrations of InsP3 were used to examine the kinetics of Ca2+ mobilization at 2 degrees C, because at the lower temperature the stores were more sensitive to InsP3: the concentration of InsP3 causing half-maximal Ca2+ release (EC50) after a 30 s incubation decreased from 281 +/- 37 nM at 37 degrees C to 68 +/- 3 nM at 2 degrees C. At 2 degrees C, the EC50 for InsP3-stimulated Ca2+ mobilization decreased as the duration of exposure to InsP3 was increased: the EC50 was 68 +/- 3 nM after 30 s, and 29 +/- 2 nM after 420 s. InsP3-stimulated Ca2+ mobilization is therefore non-quantal at 2 degrees C: InsP3 concentration determines the rate, but not the extent, of Ca2+ release. By initiating quantal responses to InsP3 at 37 degrees C and then simultaneously diluting and chilling cells to 2 degrees C, we demonstrated that the changes that underlie quantal responses do not rapidly reverse at 2 degrees C. At both 37 degrees C and 2 degrees C, modest increases in cytosolic Ca2+ increased the sensitivity of the stores to InsP3, whereas further increases were inhibitory; both Ca2+ effects persisted after prior removal of ATP. We conclude that the effects of Ca2+ on InsP3 receptors are unlikely either to be enzyme-mediated or to underlie the quantal pattern of Ca2+ release evoked by InsP3.
Metabolization of iron by plant cells using O-Trensox, a high-affinity abiotic iron-chelating agent
Authors: Caris C , Baret P , Beguin C , Serratrice G , Pierre J L , Laulhère J P
Published: August 2015

A synthetic siderophore, O-Trensox (L), has been designed and synthesized to improve iron nutrition of plants. The affinity for iron of this ligand [pFe(III) = 29.5 and pFe(II) = 17.9] is very high compared with EDTA. In spite of its high and specific affinity for iron, O-Trensox was found to be able to prevent, and to reverse, iron chlorosis in several plant species grown in axenic conditions. It also allows the iron nutrition and growth of Acer pseudoplatanus L. cell suspensions. The rate of iron metabolization was monitored by 59Fe radioiron. Ferritins, the iron storage proteins, are shown to be the first iron-labelled proteins during iron metabolization and to be able to further dispatch the metal. Using Fe(III)-Trensox, the rate of iron incorporation into ferritin was found to be higher than when using Fe-EDTA, but slower than with Fe-citrate, the natural iron carrier in xylem. During a plant cell culture, the extracellular concentrations of iron complex and free ligand were measured; changes in their relative amounts showed that the iron complex is dissociated extracellularly and that only iron is internalized. This suggests a high affinity for iron of a putative carrier on the plasmalemma. In contrast with Fe-citrate and Fe-EDTA complexes, Fe(III)-Trensox is not photoreducible. Its ability to induce radical damage as a Fenton reagent was tested using supercoiled DNA as target molecule. Unlike Fe-citrate and Fe-EDTA, Fe(II)-Trensox and Fe(III)-Trensox were proven to be harmless even during ascorbate-driven reduction, while Fe-EDTA and Fe-citrate generate heavy damage to DNA.