bluuebunny/crossref_metadata_2025_split huggingface dataset:
Determinants in the cytoplasmic domain of P-selectin required for sorting to secretory granules
Authors: MODDERMAN Piet W. , BEULING Esther A. , GOVERS Loeki A. T. , CALAFAT Jero , JANSSEN Hans , BORNE Albert E. G. Kr. VON DEM , SONNENBERG Arnoud 
Published: August 2015

P-selectin is a granule membrane protein of platelets and endothelial cells that is expressed at the plasma membrane after cell activation. To determine which residues in its cytoplasmic tail are important for sorting to storage granules during biosynthesis, we expressed P-selectin mutants in AtT-20, a murine cell line with secretory granules that contain the hormone corticotropin (‘ACTH’). Immunofluorescence microscopy of permeabilized cells revealed that wild-type P-selectin and mutants with alanine substitutions at 14 different positions in the cytoplasmic tail were concentrated in the tips of the cellular processes, which contain the majority of corticotropin granules. However, targeting to the cell tips was greatly decreased for Tyr777 → Ala, Tyr777 → Phe, Gly778 → Ala, Phe780 → Ala and Leu768/Asn769 → Ala/Ala mutants. The reduced presence of these mutants in corticotropin granules was confirmed by immunoelectron microscopy. Stimulation of AtT-20 transfectants with 8-Br-cAMP resulted in a significant increase in membrane expression of wild-type P-selectin, but in only a marginal increase in the surface expression of the five mutants. Antibody binding studies with intact and permeabilized cells demonstrated that the percentage of P-selectin that is expressed on the surface of the cells was considerably higher for these mutants than for wild-type P-selectin (6%), ranging from ∼ 20% for the Gly778 and Phe780 mutants to 63% for the Leu768/Asn769 mutant. Taken together, these results indicate that Tyr777, Gly778 and Phe780 form part of an atypical tyrosine-based motif, which also requires the presence Leu768 and/or Asn769 to mediate sorting of P-selectin to secretory granules.
Insulin-mimetic signalling of synthetic phosphoinositolglycans in isolated rat adipocytes
Authors: FRICK Wendelin , BAUER Andrea , BAUER Jochen , WIED Susanne , MÜLLER Günter 
Published: August 2015

A set of synthetic phosphoinositolglycan (PIG) compounds has been demonstrated to exert insulin-mimetic activity on glucose and lipid metabolism in rat adipocytes differing considerably in potency [compound 41 &gt; 37 &gt; 45 ≫ 7 &gt; 1; W. Frick, A. Bauer, J. Bauer, S. Wied and G. Müller, G. (1998) Biochemistry 37, 13421–13436]. In the present study we examine whether these differences are based on the capability of the PIG compounds to stimulate signalling components which are thought to mediate metabolic insulin action. Studies using a tyrosine kinase inhibitor and introduction into adipocytes of anti-phosphotyrosine or inhibitory anti-insulin receptor β-subunit antibodies demonstrated dependence on tyrosine phosphorylation but independence of insulin receptor kinase activation of the insulin-mimetic signalling and metabolic activity of the PIG compounds. The five compounds elicited in rat adipocytes a significant increase in tyrosine phosphorylation of both insulin receptor substrate 1 (IRS-1) and IRS-3 and, to a minor degree, IRS-2, in IRS-1/3-associated phosphatidylinositol 3-kinase (PI 3-K) protein as well as activity, and in protein kinase B (PKB) activity as well as phosphorylation. This was most pronounced for compound 41, approaching 65–95% of the maximal insulin response (MIR) at 20 µM, and declined in the order of compounds 37, 45, 7 and 1. The same ranking was true for the maximal inhibition of glycogen synthase kinase 3 activity (GSK-3) (41, 75% of MIR; compound 37, 65%; compound 7, 25%; compound 1, 10%) and GSK-3 autophosphorylation. The half-maximal concentrations effective for signalling (compound 41, 2–5 µM; compound 37, 10–20 µM) corresponded well to those stimulating glucose and lipid metabolism. Interestingly, compounds 37 and 41 stimulated mitogen-activated protein kinase (MAPK) and protein synthesis in rat adipocytes to only about 20–30% (at 50 µM) of MIR. We conclude that in rat adipocytes: (i) the potency of PIG compounds to regulate glucose/lipid metabolism depends on the activation of PI 3-K and PKB and inhibition of GSK-3; (ii) initiation of tyrosine phosphorylation of IRS-1/3 is sufficient and activation of the PI 3-K cascade is required for insulin-mimetic metabolic signalling; and (iii) PIG compounds are quite selective for the PI 3-K compared to the MAPK cascade, (iv) PIG compounds seem to use the same signalling components downstream of PI 3-K (including Rab4) for stimulation of glucose transport as does insulin. Thus the early signalling step(s) used by PIG, but not by insulin, may represent a target for the treatment of insulin-resistant states.
Mitochondrial permeability transition during hypothermic to normothermic reperfusion in rat liver demonstrated by the protective effect of cyclosporin A
Authors: LEDUCQ Nathalie , DELMAS-BEAUVIEUX Marie-Christine , BOURDEL-MARCHASSON Isabelle , DUFOUR Sylvie , GALLIS Jean-Louis , CANIONI Paul , DIOLEZ Philippe 
Published: August 2015

The purpose of this study was to test the hypothesis that mitochondrial permeability transition might be implicated in mitochondrial and intact organ dysfunctions associated with damage induced by reperfusion after cold ischaemia. Energetic metabolism was assessed continuously by 31P-NMR on a model system of isolated perfused rat liver; mitochondria were extracted from the livers and studied by using top-down control analysis. During the temperature transition from hypothermic to normothermic perfusion (from 4 to 37 °C) the ATP content of the perfused organ fell rapidly, and top-down metabolic control analysis of damaged mitochondria revealed a specific control pattern characterized by a dysfunction of the phosphorylation subsystem leading to a decreased response to cellular ATP demand. Both dysfunctions were fully prevented by cyclosporin A, a specific inhibitor of the mitochondrial transition pore (MTP). These results strongly suggest the involvement of the opening of MTP in vivo during the transition to normothermia on rat liver mitochondrial function and organ energetics.
Genetic defect in N-acetylglucosaminyltransferase I gene of a ricin-resistant baby hamster kidney mutant
Authors: OPAT Andrew S. , PUTHALAKATH Hamsa , BURKE Jo , GLEESON Paul A. 
Published: August 2015

The analysis of mutations associated with glycosylation-defective cell lines has the potential for identifying critical residues associated with the activities of enzymes involved in the biosynthesis of glycoconjugates. A ricin-resistant (RicR) baby hamster kidney (BHK) cell mutant, clone RicR14, has a deficiency in N-acetylglucosaminyltransferase I (GlcNAc-TI) activity and as a consequence is unable to synthesize complex and hybrid N-glycans. Here we show that RicR14 cells transfected with wild-type GlcNAc-TI regained the ability to synthesize complex N-glycans, demonstrating that the glycosylation defect of RicR14 cells is due solely to the lack of GlcNAc-TI activity. With the use of specific antibodies to GlcNAc-TI, RicR14 cells were shown to synthesize an inactive GlcNAc-TI protein that is correctly localized to the Golgi apparatus. We have cloned and sequenced the open reading frame of GlcNAc-TI from parental BHK and RicR14 cells. A comparison of several RicR14 cDNA clones with the parental BHK GlcNAc-TI sequence indicated the presence of two different RicR14 cDNA species. One contained a premature stop codon at position +81, whereas the second contained a point mutation in the catalytic domain of GlcNAc-TI resulting in the amino acid substitution Gly320 → Asp. The introduction of a Gly320 → Asp mutation into wild-type rabbit GlcNAc-TI resulted in a complete loss of activity; the GlcNAc-TI mutant was correctly localized to the Golgi, indicating that the inactive GlcNAc-TI protein was transport-competent. Gly320 is conserved in GlcNAc-TI from all species so far examined. Overall these results demonstrate that Gly320 is a critical residue for GlcNAc-TI activity.
The nucleotide sequence data reported will appear in DDBJ, EMBL and GenBank Nucleotide Sequence Databases under the accession numbers AF087456 and AF087457.
Calcium entry in Trypanosoma brucei is regulated by phospholipase A2 and arachidonic acid
Authors: EINTRACHT Jason , MAATHAI Ronald , MELLORS Alan , RUBEN Larry 
Published: August 2015

In contrast with mammalian cells, little is known about the control of Ca2+ entry into primitive protozoans. Here we report that Ca2+ influx in pathogenic Trypanosoma brucei can be regulated by phospholipase A2 (PLA2) and the subsequent release of arachidonic acid (AA). Several PLA2 inhibitors blocked Ca2+ entry; 3-(4-octadecyl)-benzoylacrylic acid (OBAA; IC50 0.4±0.1 µM) was the most potent. We identified in live trypanosomes PLA2 activity that was sensitive to OBAA and could be stimulated by Ca2+, suggesting the presence of positive feedback control. The cell-associated PLA2 activity was able to release [14C]AA from labelled phospholipid substrates. Exogenous AA (5–50 µM) also initiated Ca2+ entry in a manner that was inhibited by the Ca2+ antagonist La3+ (100 µM). Ca2+ entry did not depend on AA metabolism or protein kinase activation. The cell response was specific for AA, and fatty acids with greater saturation than tetraeicosanoic acid (AA) or with chain lengths less than C20 exhibited greatly diminished ability to initiate Ca2+ influx. Myristate and palmitate inhibited PLA2 activity and also inhibited Ca2+ influx. Overall, these results demonstrate that Ca2+ entry into T. bruceican result from phospholipid hydrolysis and the release of eicosanoic acids.
Calsequestrin: more than ‘only’ a luminal Ca2+ buffer inside the sarcoplasmic reticulum
Authors: SZEGEDI Csaba , SÁRKÖZI Sándor , HERZOG Anke , JÓNA István , VARSÁNYI Magdolna 
Published: August 2015

In striated muscle, the sarcoplasmic reticulum (SR) Ca2+ release/ryanodine receptor (RyR) channel provides the pathway through which stored Ca2+ is released into the myoplasm during excitation–contraction coupling. Various luminal Ca2+-binding proteins are responsible for maintaining the free [Ca2+] at 10-3–10-4 M in the SR lumen; in skeletal-muscle SR, it is mainly calsequestrin. Here we show that, depending on its phosphorylation state, calsequestrin selectively controls the RyR channel activity at 1 mM free luminal [Ca2+]. Calsequestrin exclusively in the dephosphorylated state enhanced the open probability by approx. 5-fold with a Hill coefficient (h) of 3.3, and increased the mean open time by about 2-fold, i.e. solely dephosphorylated calsequestrin regulates Ca2+ release from the SR. Because calsequestrin has been found to occur mainly in the phosphorylated state in the skeletal-muscle SR for the regulation of RyR channel activity, the dephosphorylation of calsequestrin would appear to be a quintessential physiological event.
N-lobe versus C-lobe complexation of bismuth by human transferrin
Authors: SUN Hongzhe , LI Hongyan , MASON Anne B. , WOODWORTH Robert C. , SADLER Peter J. 
Published: August 2015

Interactions of recombinant N-lobe of human serum transferrin (hTF/2N) with Bi3+, a metal ion widely used in medicine, have been investigated by both UV and NMR spectroscopy. The bicarbonate-independent stability constant for Bi3+ binding (K*) to hTF/2N was determined to be log K* 18.9±0.2 in 5 mM bicarbonate/10 mM Hepes buffer at 310 K, pH 7.4. The presence of Fe3+ in the C-lobe of intact hTF perturbed Bi3+ binding to the N-lobe, whereas binding of Bi3+ to the C-lobe was unaffected by the presence of Fe3+ in the N-lobe. Reactions of Bi3+ (as bismuth nitrilotriacetate or ranitidine bismuth citrate) with hTF/2N in solutions containing 10 mM bicarbonate induced specific changes to high-field 1H-NMR peaks. The 1H co-ordination shifts induced by Bi3+ were similar to those induced by Fe3+ and Ga3+, suggesting that Bi3+ binding causes similar structural changes to those induced by hTF/2N. 13C-NMR data showed that carbonate binds to hTF/2N concomitantly with Bi3+.
Molecular characterization of the Acremonium chrysogenum cefG gene product: the native deacetylcephalosporin C acetyltransferase is not processed into subunits
Authors: VELASCO Javier , GUTIERREZ Santiago , CAMPOY Sonia , MARTIN Juan F. 
Published: August 2015

Constructions starting at each of the three in-frame ATG codons of the Acremonium chrysogenum cefG gene (Met1, Met46 and Met60) were expressed in Escherichia coli, obtaining proteins of 49, 44 and 43 kDa, respectively. All three proteins showed deacetylcephalosporin C (DAC) acetyltransferase activity. The native A. chrysogenum DAC acetyltransferase was purified to electrophoretic homogeneity by immunoaffinity chromatography. It showed a molecular mass of 50 kDa by filtration in calibrated Sephadex G-75 SF or Superose 12 (FPLC) columns. The N-terminal end of the pure DAC acetyltransferase was Met-Leu-Pro-Ser-Ala-Gln-Val-Ala-Arg-Leu, which matched perfectly the deduced amino acid sequence starting at Met1. The putative α- and β-subunits of DAC acetyltransferase were also obtained in E. coli but showed no enzymic activity either separately or in combination. Immunoblotting (Western) analysis revealed that the 50 kDa DAC acetyltransferase showed high protein levels in A. chrysogenum cultures at 72 and 96 h and decreased sharply thereafter, but in all cases no detectable processing of the enzyme into subunits was found. Three different A. chrysogenum strains (including the wild-type Brotzu strain and two high-cephalosporin-producing mutants) showed the same unprocessed 50 kDa DAC acetyltransferase. The non-producer mutant ATCC 20371 showed no DAC acetyltransferase protein band but formed a normal transcript of 1.4 kb.
A sensitive immunoassay for rat fatty acid translocase (CD36) using phage antibodies selected on cell transfectants: abundant presence of fatty acid translocase/CD36 in cardiac and red skeletal muscle and up-regulation in diabetes
Authors: PELSERS Maurice M. A. L. , LUTGERINK Jan T. , NIEUWENHOVEN Frans A. van , TANDON Narendra N. , VUSSE Ger J. van der , ARENDS Jan-Willem , HOOGENBOOM Hennie R. , GLATZ Jan F. C. 
Published: August 2015

The rat membrane protein fatty acid translocase (FAT), which shows sequence similarity to human CD36 (a membrane protein supposedly involved in a variety of membrane processes), is implicated in the transport of long-chain fatty acids across cellular membranes. To set up an immunoassay for quantification of FAT in different tissues, we isolated a series of anti-FAT antibodies by panning a large naive phage antibody library on FAT-transfected H9c2 cells. All seven different phage antibody fragments isolated reacted specifically with FAT, and most likely recognize the same or closely located immunodominant sites on FAT, as a competitive monoclonal antibody (mAb) (CLB-IV7) completely blocked the binding of all these phage antibodies to cells. A sandwich ELISA was set up using mAb 131.4 (directed against purified CD36 from human platelets) as capture antibody and phage antibodies and anti-phage sera as detector. With this ELISA (sensitivity 0.05 µg/ml), the FAT content in isolated cardiomyocytes was found to be comparable with that of total heart (≈ 3 mg/g of protein), while liver tissue and endothelial cells were below the detection limit (&lt; 0.1 mg of FAT/g of protein). During rat heart development, protein levels of FAT rose from 1.7±0.7 mg/g of protein on the day before birth to 3.6±0.4 mg/g of protein on day 70. Comparing control with streptozotocin-induced diabetic rats, a statistically significant (P&lt; 0.05) 2–4-fold increase of FAT was seen in heart (from 4.2±2.3 to 11.0±5.7 mg/g of protein), soleus (from 0.6±0.1 to 1.4±0.5 mg/g of protein) and extensor digitorum longus (EDL) muscle (from 0.3±0.1 to 1.2±0.8 mg/g of protein). In addition, the FAT contents of each of these muscles were found to be of similar magnitude to the contents of cytoplasmic heart-type fatty-acid-binding protein in both diabetic rats and controls, supporting the suggested roles of these two proteins in cellular fatty acid metabolism. This is the first time phage display technology has been succesfully applied for direct selection, from a large naive antibody library, of antibodies that recognize selected membrane proteins in their natural context.
Inhibition of proprotein convertases-1, -7 and furin by diterpines of Andrographis paniculata and their succinoyl esters
Authors: BASAK Ajoy , COOPER Sam , ROBERGE Andreé G. , BANIK Upen K. , CHRÉTIEN Michel , SEIDAH Nabil G. 
Published: August 2015

Studies were performed to investigate the prohormone/proprotein convertase (PC)-inhibitory properties of chemical constituents of the medicinally active plant Andrographis paniculata (AP; from the family Acanthaceae), also known as ‘King of Bitters ’. Among the individual components tested against the clinically important convertases, furin and PC1, neoandrographolide (a C3 O-glucoside derivative of the major constituent andrographolide) exhibited the highest inhibitory action with an IC50 of 53.5 µM against furin. The data further revealed that although andrographolide, the major bitter principle of AP, exhibited a relatively small enzyme inhibition (IC50 = 1.0 mM and Ki = 200 µM against furin), upon succinoylation, its inhibitory action against the above convertases was enhanced significantly with a Ki in the low micromolar range (&lt; 30 µM), suggesting that a specific structural modification of the andrographolide skeleton may be exploited to develop a new class of non-peptide inhibitors of PCs. When tested against PC7, these succinoylated derivatives of andrographolide also displayed strong inhibitory action, with Ki values again in the low micromolar range. This potentially interesting observation may be attributed to the reported anti-HIV property of 14-dehydroandrographolide succinic acid monoester (DASM). It is suggested here that DASM, by virtue of this protease inhibitory property, possibly acts by suppressing the proteolytic cleavage of envelope glycoprotein gp160 of HIV, which is known to be PC-mediated, particularly by furin and PC7.
Upstream region of rat serum albumin gene promoter contributes to promoter activity: presence of functional binding site for hepatocyte nuclear factor-3
Authors: HSIANG Chin-Hui , MARTEN Norman W. , STRAUS Daniel S. 
Published: August 2015

Transcription of the serum albumin gene occurs almost exclusively in the liver and is controlled in part by a strong liver-specific promoter. The upstream region of the serum albumin gene promoter is highly conserved among species and is footprinted in vitro by a number of nuclear proteins. However, the role of the upstream promoter region in regulating transcription and the identity of the transcription factors that bind to this region have not been established. In the present study, deletion analysis of the rat serum albumin promoter in transiently transfected HepG2 cells demonstrated that elimination of the region between -207 and -153 bp caused a two-fold decrease in promoter activity (P&lt; 0.05). Additional analysis of the -207 to -124 bp promoter interval led to the identification of two potential binding sites for hepatocyte nuclear factor-3 (HNF-3) located at -168 to -157 bp (site X) and -145 to -134 bp (site Y). Electrophoretic mobility-shift assays performed with the HNF-3 X and Y sites demonstrated that both sites are capable of binding HNF-3α and HNF-3β. Placement of a single copy of the HNF-3 X site upstream from a minimal promoter increased promoter activity by about four-fold in HepG2 cells, and the reporter construct containing this site could be transactivated if co-transfected with an HNF-3 expression construct. Furthermore, inactivation of the HNF-3 X site by site-directed mutagenesis within the context of the -261 bp albumin promoter construct resulted in a 40% decrease in transcription (P&lt; 0.05). These results indicate that the positive effect of the -207 to -153 bp promoter interval is attributable to the presence of the HNF-3 X site within this interval. Additional results obtained with transfected HepG2 cells suggest that the HNF-3 Y site plays a lesser role in activation of transcription than the X site.
Baculovirus expression and biochemical characterization of the human microsomal triglyceride transfer protein
Authors: RITCHIE Penelope J. , DECOUT Anne , AMEY Joanna , MANN Christopher J. , READ Jacqueline , ROSSENEU Maryvonne , SCOTT James , SHOULDERS Carol C. 
Published: August 2015

The microsomal triglyceride transfer protein (MTP) complexed to protein disulphide isomerase (PDI) is obligatory for the assembly of chylomicrons and very-low-density lipoproteins. The determination of the atomic structure of the MTP–PDI heterodimer has important implications for the treatment of those forms of hyperlipidaemia associated with the overproduction of very-low-density lipoproteins, which predispose to premature coronary heart disease. To perform structural studies of the human MTP–PDI complex it was necessary to produce milligram quantities of pure protein. We chose the baculovirus expression system for this purpose. Insects cells were co-infected with recombinant viruses encoding FLAG-tagged MTP and His-tagged PDI; the resulting heterodimer was purified by affinity chromatography. From 5 litres of insect cells, 4–6 mg of more than 95% pure recombinant protein was obtained. CD and attenuated total reflection Fourier-transform infrared spectroscopy indicate that the purified protein has around 34% α-helical and 33% β-structure content. The recombinant protein had a comparable triglyceride transfer activity to that of bovine MTP–PDI. The production of polyclonal antibodies raised against the MTP and PDI subunits of the purified protein is described. The present study demonstrates the feasibility of expressing two proteins at high levels in insect cells and describes a transferable methodology for the purification of the resulting protein complex.
The endosome fusion regulator early-endosomal autoantigen 1 (EEA1) is a dimer
Authors: CALLAGHAN Judy , SIMONSEN Anne , GAULLIER Jean-Michel , TOH Ban-Hock , STENMARK Harald 
Published: August 2015

EEA1, an early-endosomal protein originally identified as an autoantigen, is essential for endocytic membrane fusion. It interacts with early endosomes via binding to the membrane lipid phosphatidylinositol 3-phosphate (PtdIns3P) and the active form of the small GTPase Rab5. Most of the EEA1 sequence contains heptad repeats characteristic of proteins involved in coiled-coil protein–protein interactions. Here we have investigated the ability of EEA1 to self-interact. Crosslinking of cytosolic and recombinant EEA1 resulted in the disappearance of the 180-kDa monomer in SDS/PAGE and the strong appearance of a ∼ 350-kDa crosslinked product. Glycerol gradient centrifugation experiments indicated that native EEA1 had the same hydrodynamic properties as the ∼ 350-kDa crosslinked complex. Two-hybrid analysis indicated that N- and C-terminal fragments of EEA1 can interact with themselves, but not with each other, suggesting that EEA1 forms parallel coiled-coil dimers. The ability of the C-terminus of EEA1 to dimerize correlates with its ability to bind to Rab5 and early endosomes, whereas its binding to PtdIns3P is independent of dimerization. These data enable us to propose a model for the quaternary structure of EEA1.