bluuebunny/crossref_metadata_2025 huggingface dataset:
Ethanol increases superoxide anion production stimulated with 4β‐phorbol 12‐myristate 13‐acetate in human polymorphonuclear leukocytes
Authors: Raddassi Khadir , Murray John J.
Published: March 2003

Stimulation of human polymorphonuclear leukocytes (PMNs) with PMA initiates a cascade of events leading to the production and release of superoxide anion (O−2), a major component in anti‐bacterial defense. Generation of O−2 by PMA‐stimulated PMNs occurs through the translocation and activation of protein kinase C (PKC). In this study, using freshly isolated PMNs, we examined the effect of ethanol on this response to PMA. Our results show that the basal production of O−2 was not affected by ethanol. In contrast, the response induced by PMA was potentiated by ethanol. This potentiation was observed even at high doses of PMA (200 nm) which alone had stimulated the O−2 response maximally. This enhanced response was not due to an increase of PMA uptake by PMNs. The maximal effect was obtained when the cells were preincubated with 80 mm of ethanol before PMA stimulation. Measurement of PKC activity in the cytosolic and membrane fractions showed that pretreatment of PMNs with ethanol increased twofold the PMA‐stimulated PKC activity in the membrane fraction. Furthermore, Western blot analysis verified that this increase in PKC activity in the membrane fraction was linked to an increase in the translocation of PKC‐α and ‐β isoforms to the membrane. These results suggest that ethanol potentiates PMA‐induced O−2 production through increasing PKC translocation and activity in PMNs.
Molecular cloning and characterization of calreticulin, a calcium‐binding protein involved in the regeneration of rice cultured suspension cells
Authors: Li Zhijun , Komatsu Setsuko
Published: March 2003

A full‐length cDNA clone encoding a phosphoprotein (pp56) involved in the regeneration of rice (Oryza sativa L.)‐cultured suspension cells was isolated by screening a rice cultured suspension cell cDNA library. The 1558‐bp cDNA sequence contains an ORF encoding an acidic (pI 4.38) protein of 424 amino acids (47.9 kDa), sharing 70–93% and 50–53% homology with other plant and mammalian calreticulins, respectively. Sequence analysis of the cDNA clone revealed several significant conserved motifs, including a calreticulin family repeat motif in the central domain and two calreticulin family motifs in the N‐domain, indicating that this gene is a rice calreticulin (CRO1). The CRO1 gene in long‐term rice cultured suspension cells shows constitutive expression in both suspension culture and regeneration media. In contrast, expression of the CRO1 gene in short‐term rice cultured suspension cells, which possess regeneration potential, is increased dramatically when these cells are transferred to the regeneration medium. After ≈ 2 weeks in the regeneration medium, the expression of the CRO1 gene reverts to constitutive levels. These results demonstrate the presence of calreticulin in rice cultured suspension cells and its developmental regulation during the regeneration of rice cultured suspension cells.
Site‐directed mutagenesis of the active site serine290 in flavanone 3β‐hydroxylase from Petunia hybrida
Authors: Lukačin Richard , Gröning Inga , Pieper Uwe , Matern Ulrich
Published: March 2003

Flavanone 3β‐hydroxylase (FHT) catalyzes a pivotal reaction in the formation of flavonoids, catechins, proanthocyanidins and anthocyanidins. In the presence of oxygen and ferrous ions the enzyme couples the oxidative decarboxylation of 2‐oxoglutarate, releasing carbon dioxide and succinate, with the oxidation of flavanones to produce dihydroflavonols. The hydroxylase had been cloned from Petunia hybrida and expressed in Escherichia coli, and a rapid isolation method for the highly active, recombinant enzyme had been developed. Sequence alignments of the Petunia hydroxylase with various hydroxylating 2‐oxoglutarate‐dependent dioxygenases revealed few conserved amino acids, including a strictly conserved serine residue (Ser290). This serine was mutated to threonine, alanine or valine, which represent amino acids found at the corresponding sequence position in other 2‐oxoglutarate‐dependent enzymes. The mutant enzymes were expressed in E. coli and purified to homogeneity. The catalytic activities of [Thr290]FHT and [Ala290]FHT were still significant, albeit greatly reduced to 20 and 8%, respectively, in comparison to the wild‐type enzyme, whereas the activity of [Val290]FHT was negligible (about 1%). Kinetic analyses of purified wild‐type and mutant enzymes revealed the functional significance of Ser290 for 2‐oxoglutarate‐binding. The spatial configurations of the related Fe(II)‐dependent isopenicillin N and deacetoxycephalosporin C synthases have been reported recently and provide the lead structures for the conformation of other dioxygenases. Circular dichroism spectroscopy was employed to compare the conformation of pure flavanone 3β‐hydroxylase with that of isopenicillin N synthase. A double minimum in the far ultraviolet region at 222 nm and 208–210 nm and a maximum at 191–193 nm which are characteristic for α‐helical regions were observed, and the spectra of the two dioxygenases fully matched revealing their close structural relationship. Furthermore, the spectrum remained unchanged after addition of either ferrous ions, 2‐oxoglutarate or both of these cofactors, ruling out a significant conformational change of the enzyme on cofactor‐binding.
Mouse brain and muscle tissues constitutively express high levels of Homer proteins
Authors: Soloviev Mikhail M. , Ciruela Francisco , Chan Wai‐Yee , McIlhinney R. A. Jeffrey
Published: March 2003

In order to characterize expression of Homers in mouse brain and peripheral tissues we have developed a coupled reverse transcription (RT)‐PCR/restriction digestion approach. This has allowed us to determine the molecular composition and relative levels of the constitutive expression of the Homer‐1, ‐2 and ‐3 mRNAs across mouse tissues. We report here that mammalian brain constitutively expresses high levels of the Homer‐1, ‐2 and ‐3 mRNAs. Expression of the Homer‐1 mRNAs reaches 66% of the brain total Homer mRNAs expression, followed by Homer‐3 mRNA (22%) and Homer‐2 mRNAs (12%). Quantitative RT‐PCR analysis and the Western blotting using pan‐Homer antibody revealed that mouse heart, skeletal muscle and diaphragm constitutively express high levels of the Homer proteins and their mRNAs. We have shown that the molecular profile of expression of Homer‐1, ‐2 and ‐3 mRNAs in muscle containing tissues resembles that obtained for mammalian brain.
Dual regulatory effects of nitric oxide on plasminogen activator inhibitor type 1 expression in endothelial cells
Authors: Swiatkowska Maria , Cierniewska‐Cieslak Aleksandra , Pawlowska Zofia , Cierniewski Czeslaw S.
Published: March 2003

In this report we compared the mechanism by which nitric oxide (NO), generated exogenously and endogenously, affects the plasminogen activator inhibitor type 1 (PAI‐1) expression in endothelial cells. For this purpose, we stimulated the endothelial cell line EA.hy 926 with tumour necrosis factor α (TNFα) in the presence of the exogenously NO‐releasing donors, sodium nitroprusside (SNP) and S‐nitroso‐N‐acetylpenicillamine, or regulators of nitric oxide synthase (NOS) inhibitor N‐nitro‐l‐arginine‐methyl ester hydrochloride and substrate l‐Arg. Expression of PAI‐1 in EA.hy 926 cells was determined by measuring the level of mRNA, using relative quantitative reverse transcriptase PCR, and protein, using ELISA. In addition, we estimated the level of activation of two mitogen‐activated protein kinases (MAPKs), extracellular signal‐regulated kinase (ERK1/2) and c‐Jun N‐terminal kinase (JNK1/2), in the cells before and after treatment with TNFα, in the presence or absence of NO donors and inhibitors. In contrast to exogenously released NO that significantly reduced mostly basal PAI‐1 expression, endogenously generated NO by NOS potentiated TNFα‐induced upregulation of PAI‐1 expression. Exogenously and endogenously generated NO causes different effects on activation of the MAPKs ERK1/2 and JNK1/2. Specifically, the SNP‐released NO activates only ERK1/2, while endogenously generated NO in a pathway induced by TNFα activates both MAPKs. Thus our data indicate that due to different cellular locations and mechanisms of generation, NO may participate in various signalling pathways leading to opposite effects on PAI‐1 expression in endothelial cells.
N‐Linked glycans of proteins from mitral valves of normal pigs and pigs affected by endocardiosis
Authors: Amoresano Angela , Amedeo Stefano , D’Andrea Gabriele , Siciliano Rosa , Gagna Carla , Castagnaro Massimo , Marino Gennaro , Guarda Franco
Published: March 2003

Endocardiosis, a degenerative and dystrophic process affecting cardiac valves and described in many mammalian species, is characterized by the accumulation of glycosaminoglycans, in particular hyaluronic acid, in the extracellular matrix. The glycoprotein patterns of pig mitral valves in normal animals and animals affected by endocardiosis were investigated. A different N‐linked glycosylation pattern of glycoproteins was detected in affected valves compared with normal ones. In either normal or pathological species, the detected N‐linked glycans were of the complex type. However, in samples from affected valves, sialic acid showed a prevalence of the α2,6 linkage to the galactosyl residue, whereas in normal samples the most frequent linkage was of the α2,3 type. In normal valves, the majority of complex oligosaccharides presented two outer branches with different degrees of fucosylation and sialylation, whereas in pathological samples we noted an increased number of glycans having up to four outer branches.
Serine proteinase inhibition by the active site titrant N α‐(N,N‐dimethylcarbamoyl)‐α‐azaornithine p‐nitrophenyl ester
Authors: Ascenzi Paolo , Balliano Gianni , Gallina Carlo , Polticelli Fabio , Bolognesi Martino
Published: March 2003

Kinetics for the hydrolysis of the chromogenic active‐site titrant N α‐(N,N‐dimethylcarbamoyl)‐α‐azaornithine p‐nitrophenyl ester (Dmc‐azaOrn‐ONp) catalysed by bovine β‐trypsin, bovine α‐thrombin, bovine Factor Xa, human α‐thrombin, human Factor Xa, human Lys77‐plasmin, human urinary kallikrein, Mr 33 000 and Mr 54 000 species of human urokinase, porcine pancreatic β‐kallikrein‐A and ‐B and Ancrod (the coagulating serine proteinase from the Malayan pit viper Agkistrodon rhodostoma venom) have been obtained between pH 6.0 and 8.0, at 21.0 °C, and analysed in parallel with those for the enzymatic cleavage of N α‐(N,N‐dimethylcarbamoyl)‐α‐azalysine p‐nitrophenyl ester (Dmc‐azaLys‐ONp). The enzyme kinetics are consistent with the minimum three‐step catalytic mechanism of serine proteinases, the rate‐limiting step being represented by the deacylation process. Bovine β‐trypsin kinetics are modulated by the acid–base equilibrium of the His57 catalytic residue (pKa ≈ 6.9). Dmc‐azaOrn‐ONp and Dmc‐azaLys‐ONp bind stoichiometrically to the serine proteinase active site, and allow the reliable determination of the active enzyme concentration between 1.0 × 10−6 m and 3.0 × 10−4 m. The affinity and the reactivity for Dmc‐azaOrn‐ONp (expressed by Ks and k+2/Ks, respectively) of the serine proteinases considered are much lower than those for Dmc‐azaLys‐ONp. The very different affinity and reactivity properties for Dmc‐azaOrn‐ONp and Dmc‐azaLys‐ONp have been related to the different size of the ornithine/lysine side chains, and to the ensuing different positioning of the active‐site titrants upon binding to the enzyme catalytic centre (i.e. to P1‐S1 recognition). These data represent the first detailed comparative investigation on the catalytic properties of serine proteinases towards an ornithine derivative (i.e. Dmc‐azaOrn‐ONp).
The gene encoding polyneuridine aldehyde esterase of monoterpenoid indole alkaloid biosynthesis in plants is an ortholog of theα/β hydrolase super family
Authors: Dogru Emine , Warzecha Heribert , Seibel Frank , Haebel Sophie , Lottspeich Friedrich , Stöckigt Joachim
Published: March 2003

The biosynthesis of the anti‐arrhythmic alkaloid ajmaline is catalysed by more than 10 specific enzymes. In this multistep process polyneuridine aldehyde esterase (PNAE) catalyses a central reaction by transforming polyneuridine aldehyde into epi‐vellosimine, which is the immediate precursor for the synthesis of the ajmalane skeleton. PNAE was purified from cell suspension cultures of Rauvolfia serpentina. The N‐terminal sequence and endoproteinase LysC fragments of the purified protein were used for primer design and for the amplification of specific PCR products leading to the isolation of PNAE‐encoding cDNA from a R. serpentina library. The PNAE cDNA was fused with a C‐terminal His‐tag, expressed in Escherichia coli and purified to homogeneity using Ni‐affinity chromatography. The pure enzyme shows extraordinary substrate specificity, completely different to other esterases. Sequence alignments indicate that PNAE is a new member of the α/β hydrolase super family.
Biological activities of lipopolysaccharides are determined by the shape of their lipid A portion
Authors: Schromm Andra B. , Brandenburg Klaus , Loppnow Harald , Moran Anthony P. , Koch Michel H. J. , Rietschel Ernst TH. , Seydel Ulrich
Published: March 2003

Lipopolysaccharide (LPS) represents a major virulence factor of Gram‐negative bacteria (‘endotoxin’) that can cause septic shock in mammals including man. The lipid anchor of LPS to the outer membrane, lipid A, has a peculiar chemical structure, harbours the ‘endotoxic principle’ of LPS and is responsible for the expression of pathophysiological effects. Chemically modified lipid A can be endotoxically inactive, but may express strong antagonistic activity against LPS, a property that can be utilized in antisepsis treatment. We show here that these different biological activities are directly correlated with the molecular shape of lipid A. Only (hexaacyl) lipid A with a conical/concave shape, the cross‐section of the hydrophobic region being larger than that of the hydrophilic region, exhibited strong interleukin‐6 (IL‐6)‐inducing capacity. Most strikingly, a correlation between a cylindrical molecular shape of lipid A and antagonistic activity was established: IL‐6 induction by enterobacterial LPS was inhibited by cylindrically shaped lipid A except for compounds with reduced headgroup charge. The antagonistic action is interpreted by assuming that lipid A molecules intercalate into the cytoplasmic membrane of mononuclear cells, and subsequently blocking of the putative signaling protein by the lipid A with cylindrical shape.
Analysis of a binding difference between the two dsRNA‐binding domains in TRBP reveals the modular function of a KR‐helix motif
Authors: Daviet Laurent , Erard Monique , Dorin Dominique , Duarte Mariela , Vaquero Catherine , Gatignol Anne
Published: March 2003

Double‐stranded RNA‐binding proteins constitute a large family with conserved domains called dsRBDs. One of these, TRBP, a protein that binds HIV‐1 TAR RNA, has two dsRBDs (dsRBD1 and dsRBD2), as indicated by computer sequence homology. However, a 24‐amino‐acid deletion in dsRBD2 completely abolishes RNA binding, suggesting that only one domain is functional. To analyse further the similarities and differences between these domains, we expressed them independently and measured their RNA‐binding affinities. We found that dsRBD2 has a dissociation constant of 5.9 × 10−8 m, whereas dsRBD1 binds RNA minimally. Binding analysis of 25‐amino‐acid peptides in TRBP and other related proteins showed that only one peptide in TRBP and one in Drosophila Staufen bind TAR and a GC‐rich TAR‐mimic RNA. Whereas a 25‐mer peptide derived from dsRBD2 (TR5) bound TAR RNA, the equivalent peptide in dsRBD1 (TR6) did not. Molecular modelling indicates that this difference can mainly be ascribed to the replacement of Arg by His residues. Mutational analyses in homologous peptides also show the importance of residues K2 and L3. Analysis of 15‐amino‐acid peptides revealed that, in addition to TR13 (from TRBP dsRBD2), one peptide in S6 kinase has RNA‐binding properties. On the basis of previous and the present results, we can define, in a broader context than that of TRBP, the main outlines of a modular KR‐helix motif required for binding TAR. This structural motif exists independently from the dsRBD context and therefore has a modular function.
Monoclonal antibodies to vascular endothelial growth factor‐D block its interactions with both VEGF receptor‐2 and VEGF receptor‐3
Authors: Achen Marc G. , Roufail Sally , Domagala Teresa , Catimel Bruno , Nice Edouard C. , Geleick Detlef M. , Murphy Roger , Scott Andrew M. , Caesar Carol , Makinen Taija , Alitalo Kari , Stacker Steven A.
Published: March 2003

Vascular endothelial growth factor‐D (VEGF‐D), the most recently discovered mammalian member of the VEGF family, is an angiogenic protein that activates VEGF receptor‐2 (VEGFR‐2/Flk1/KDR) and VEGFR‐3 (Flt4). These receptor tyrosine kinases, localized on vascular and lymphatic endothelial cells, signal for angiogenesis and lymphangiogenesis. VEGF‐D consists of a central receptor‐binding VEGF homology domain (VHD) and N‐terminal and C‐terminal propeptides that are cleaved from the VHD to generate a mature, bioactive form consisting of dimers of the VHD. Here we report characterization of mAbs raised to the VHD of human VEGF‐D in order to generate VEGF‐D antagonists. The mAbs bind the fully processed VHD with high affinity and also bind unprocessed VEGF‐D. We demonstrate, using bioassays for the binding and cross‐linking of VEGFR‐2 and VEGFR‐3 and biosensor analysis with immobilized receptors, that one of the mAbs, designated VD1, is able to compete potently with mature VEGF‐D for binding to both VEGFR‐2 and VEGFR‐3 for binding to mature VEGF‐D. This indicates that the binding epitopes on VEGF‐D for these two receptors may be in close proximity. Furthermore, VD1 blocks the mitogenic response of human microvascular endothelial cells to VEGF‐D. The anti‐(VEGF‐D) mAbs raised to the bioactive region of this growth factor will be powerful tools for analysis of the biological functions of VEGF‐D.
The heme‐independent manganese‐peroxidase activity depends on the presence of the C‐terminal domain within the Streptomyces reticuli catalase‐peroxidase CpeB
Authors: Zou Peijian , Schrempf Hildgund
Published: March 2003

Streptomyces reticuli produces a heme‐containing homodimeric enzyme (160 kDa), the catalase‐peroxidase CpeB, which is processed to the enzyme CpeC during prolonged growth. CpeC contains four subunits of 60 kDa each that do not include the C‐terminal portion of the progenitor subunits. A genetically engineered cpeB gene encodes a truncated subunit lacking 195 of the C‐terminal amino acids; four of these subunits assemble to form the enzyme CpeD. Heme binds most strongly in CpeB, least in CpeD. The catalase‐peroxidase CpeB and its apo‐form (obtained after extraction of heme) catalyze the peroxidation of Mn(II) to Mn(III), independent of the presence or absence of the heme inhibitor KCN. CpeC and CpeD, in contrast, do not exhibit manganese‐peroxidase activity.

The data show for the first time that a bacterial catalase‐peroxidase has a heme‐independent manganese‐peroxidase activity, which depends on the presence of the C‐terminal domain.
Fidelity analysis of HIV‐1 reverse transcriptase mutants with an altered amino‐acid sequence at residues Leu74, Glu89, Tyr115, Tyr183 and Met184
Authors: Jonckheere Heidi , De Clercq Erik , Anné Jozef
Published: March 2003

Substitution of particular residues postulated to have a role in active site architecture can alter the overall fidelity of DNA polymerization by HIV‐1. The effects of this kind of substitution were determined in a lacZ‐based assay using HIV‐1 reverse transcriptase with specifically mutated residues. We found that the reported higher fidelity of nucleotide incorporation by the Met184→Val and Glu89→Gly mutant reverse transcriptases (RTs) was not reflected in a substantial increase in the overall fidelity for these RT mutants. For the 3TC‐resistant Met184→Val RT mutant an almost wild‐type level of overall mutation frequency was observed, while the foscarnet‐resistant RTs harbouring the Glu89→Gly mutation showed only a twofold decrease in mutation frequency. The Tyr183→Phe mutant RT displayed a slightly lower fidelity than wild‐type RT. Conversely, the ddI‐resistant RT mutant containing the Leu74→Val mutation showed a 3.5‐fold higher fidelity compared to the wild‐type enzyme. Finally, the Tyr115→Ala substitution rendered the enzyme substantially more error‐prone for DNA polymerization. These results correlate with three‐dimensional structural studies of the polymerase active site and confirm the postulated impact of the Leu74, Tyr183 and Tyr115 RT residues on the overall fidelity of DNA polymerization by HIV‐1 RT.
Solution structure of the antimicrobial peptide gaegurin 4 by 1H and 15N nuclear magnetic resonance spectroscopy
Authors: Park Sang‐Ho , Kim Yun‐Kyong , Park Jung‐Won , Lee ByeongJae , Lee Bong‐Jin
Published: March 2003

Gaegurin 4 (GGN4) is a 37‐residue antimicrobial peptide isolated from the skin of a Korean frog, Rana rugosa. This peptide shows a broad range of activity against prokaryotic cells but shows very little hemolytic activity against human red blood cells. The solution structure of GGN4 was studied by using circular dichroism (CD) and NMR spectroscopy. CD investigations revealed that GGN4 adopts mainly an α‐helical conformation in trifluoroethanol/water solution, in dodecylphosphocholine and in SDS micelles, but adopts random structure in aqueous solution. By using both homonuclear and heteronuclear NMR experiments, complete 1H and 15N resonance assignments were obtained for GGN4 in 50% trifluoroethanol/water solution. The calculated structures of GGN4 consist of two amphipathic α‐helices extending from residues 2–10 and from residues 16–32. These two helices are connected by a flexible loop spanning between the residues 11 and 15. By using enzyme digestion and matrix‐assisted laser desorption/ionization mass spectroscopy, we confirmed that GGN4 contains a disulfide bridge formed between the residues Cys31 and Cys37 in its C‐terminus. The effect of disulfide bridge on the structure and the activity of GGN4 was investigated. The reduced form of GGN4 revealed a similar activity and conformation to native GGN4, suggesting that the disulfide bridge does not strongly affect the conformation and the antimicrobial activity of GGN4.
Functional properties of recombinant Azospirillum brasilense glutamate synthase, a complex iron–sulfur flavoprotein
Authors: Stabile Helena , Curti Bruno , Vanoni Maria Antonietta
Published: March 2003

Azospirillum brasilense glutamate synthase is a complex iron–sulfur flavoprotein that catalyses the NADPH‐dependent reductive transfer of glutamine amide group to the C(2) carbon of 2‐oxoglutarate to yield l‐glutamate. Its catalytically active αβ protomer is composed of two dissimilar subunits (α subunit, 164.2 kDa; β subunit, 52.3 kDa) and contains one FAD (at Site 1, the pyridine nucleotide site within the β subunit), one FMN (at Site 2, the 2‐oxoglutarate/l‐glutamate site in the α subunit) and three different iron–sulfur clusters (one 3Fe−4S center on the α subunit and two 4Fe−4S clusters of unknown location). A plasmid harboring the gltD and gltB genes, the genes encoding the glutamate synthase β and α subunits, respectively, each one under the control of the T7/lac promoter of pET11a was found to be suitable for the overproduction of glutamate synthase holoenzyme in Escherichia coli BL21(DE3) cells. Recombinant A. brasilense glutamate synthase could be purified to homogeneity from overproducing E. coli cells by ion exchange chromatography, gel filtration and affinity chromatography on a 2′,5′ ADP–Sepharose 4B column. The purified enzyme was indistinguishable from that prepared from Azospirillum cells with respect to cofactor content, N‐terminal sequence of the subunits, aggregation state, kinetic and spectroscopic properties. The study of the recombinant holoenzyme allowed us to establish that the tendency of glutamate synthase to form a stable (αβ)4 tetramer at high protein concentrations is a property unique to the holoenzyme, as the isolated β subunit does not oligomerize, while the isolated glutamate synthase α subunit only forms dimers at high protein concentrations. Furthermore, the steady‐state kinetic analysis of the glutamate synthase reaction was extended to the study of the effect of adenosine‐containing nucleotides. Compounds such as cAMP, AMP, ADP and ATP have no effect on the enzyme activity, while the 2′‐phosphorylated analogs of AMP and NADP(H) analogs act as inhibitors of the reaction, competitive with NADPH. Thus, it can be ruled out that glutamate synthase reaction is subjected to allosteric modulation by adenosine containing (di)nucleotides, which may bind to the putative ADP‐binding site at the C‐terminus of the α subunit. At the same time, the strict requirement of a 2′‐phosphate group in the pyridine nucleotide for binding to glutamate synthase (GltS) was established. Finally, by comparing the inhibition constants exhibited by a series of NADP+ analogs, the contribution to the binding energy of the various parts of the pyridine nucleotide has been determined along with the effect of substituents on the 3 position of the pyridine ring. With the exception of thio‐NADP+, which binds the tightest to GltS, it appears that the size of the substituent is the factor that affects the most the interaction between the NADP(H) analog and the enzyme.
Recursive use of evolutionary conservation data in molecular modeling of membrane proteins
Authors: Torres Jaume , Arkin Isaiah T.
Published: March 2003

Membrane proteins are currently the most biomedically important family of proteins, serving as targets for the majority of pharmaceutical agents. It is also clear that they are invariably abundant in all of the genomes sequence so far, representing up to a third of all open reading frames. Finally, and regrettably, it is clear that they are highly resistant to structural elucidation, representing less than 0.2% of the Protein Data Bank. Recent accomplishments in genome sequencing efforts, however, may help offset this imbalance through the availability of evolutionary conservation data.

Herein, we develop a novel approach, utilizing a combination of evolutionary conservation data and global searching molecular dynamics simulations to model membrane proteins, deriving a model for the multidrug H+ antiporter EmrE, a transmembrane four‐helix bundle. Structures resulting from an extensive, rotational molecular dynamics search, were evaluated by comparing the residue specific interaction energy and the evolutionary conservation data. Subsequent rounds of molecular dynamics, in which confinement of the search space was undertaken in order to achieve a self consistent result, point to a structure that best satisfies the evolutionary conservation data. As the conservation patterns calculated for each of the helices suggested that the different conservation pattern for helix 3 (as well as being the most conserved) might be due to the oligomeric nature of EmrE, a dodecamer of helices was constructed based on the result of a search of helix 3 as a trimer. The resulting interaction energy per residue in the final model is in reasonable agreement with the evolutionary data and consistent with recent site directed mutagenesis experiments, pointing to the strength of this method as a general tool.
Identification of hypoxia‐responsive messengers expressed in human microvascular endothelial cells using differential display RT‐PCR
Authors: Roland Isabelle , Minet Emmanuel , Ernest Isabelle , Pascal Thierry , Michel Gaëtan , Remacle José , Michiels Carine
Published: March 2003

In order to identify new genes overexpressed in endothelial cells exposed to hypoxia, differential display RT‐PCR was performed on total RNA extracted from human microvascular endothelial cells incubated under hypoxia or under normoxic conditions. Northern blot and reverse Northern blot analyses were used to confirm the results. Sequences corresponding to tissue inhibitor of metalloproteinase‐1, prostate tumor inducing factor‐1, enolase‐α and prothymosin‐α were evidenced as overexpressed in hypoxia. These results were confirmed by Western blot and immunofluorescence experiments. Moreover, several elements homologous to partial sequences of cDNA (expressed sequence tag) were also identified, as well as unknown cDNA sequences. The present study suggests that hypoxia can change the expression of numerous genes in endothelial cells, and that mRNA differential display is useful for cloning known and unknown hypoxia‐responsive genes.
Sulfate assimilation in higher plants
Authors: Weber Markus , Suter Marianne , Brunold Christian , Kopriva Stanislav
Published: March 2003

The enzyme catalysing the reduction of adenosine 5′‐phosphosulfate (AdoPS) to sulfite in higher plants, AdoPS reductase, is considered to be the key enzyme of assimilatory sulfate reduction. In order to address its reaction mechanism, the APR2 isoform of this enzyme from Arabidopsis thaliana was overexpressed in Escherichia coli and purified to homogeneity. Incubation of the enzyme with [35S]AdoPS at 4 °C resulted in radioactive labelling of the protein. Analysis of APR2 tryptic peptides revealed 35SO2–3 bound to Cys248, the only Cys conserved between AdoPS and prokaryotic phosphoadenosine 5′‐phosphosulfate reductases. Consistent with this result, radioactivity could be released from the protein by incubation with thiols, inorganic sulfide and sulfite. The intermediate remained stable, however, after incubation with sulfate, oxidized glutathione or AdoPS. Because truncated APR2, missing the thioredoxin‐like C‐terminal part, could be labelled even at 37 °C, and because this intermediate was more stable than the complete protein, we conclude that the thioredoxin‐like domain was required to release the bound SO2–3 from the intermediate. Taken together, these results demonstrate for the first time the binding of 35SO2–3 from [35S]AdoPS to AdoPS reductase and its subsequent release, and thus contribute to our understanding of the molecular mechanism of AdoPS reduction in plants.
Conformational studies of plasminogen activator inhibitor type 1 by fluorescence spectroscopy
Authors: Fa Ming , Bergström Fredrik , Karolin Jan , Johansson Lennart B.‐Å. , Ny Tor
Published: March 2003

The inhibitors that belong to the serpin family are suicide inhibitors that control the major proteolytic cascades in eucaryotes. Recent data suggest that serpin inhibition involves reactive centre cleavage followed by loop insertion, whereby the covalently linked protease is translocated away from the initial docking site. However under certain circumstances, serpins can also be cleaved like a substrate by target proteases. In this report we have studied the conformation of the reactive centre of plasminogen activator inhibitor type 1 (PAI‐1) mutants with inhibitory and substrate properties. The polarized steady‐state and time‐resolved fluorescence anisotropies were determined for BODIPY® probes attached to the P1′ and P3 positions of the substrate and active forms of PAI‐1. The fluorescence data suggest an extended orientational freedom of the probe in the reactive centre of the substrate form as compared to the active form, revealing that the conformation of the reactive centres differ. The intramolecular distance between the P1′ and P3 residues in reactive centre cleaved inhibitory and substrate mutants of PAI‐1, were determined by using the donor‐donor energy migration (DDEM) method. The distances found were 57 ± 4 Å and 63 ± 3 Å, respectively, which is comparable to the distance obtained between the same residues when PAI‐1 is in complex with urokinase‐type plasminogen activator (uPA). Following reactive centre cleavage, our data suggest that the core of the inhibitory and substrate forms possesses an inherited ability of fully inserting the reactive centre loop into β‐sheet A. In the inhibitory forms of PAI‐1 forming serpin‐protease complexes, this ability leads to a translocation of the cognate protease from one pole of the inhibitor to the opposite one.
Deletion of the 6‐kDa subunit affects the activity and yield of the bc1 complex from Rhodovulum sulfidophilum
Authors: Rodgers Simon , Moser Claudio , Martinez‐Julvez Marta , Sinning Irmgard
Published: March 2003

The cytochrome bc1 complex from Rhodovulum sulfidophilum purifies as a four‐subunit complex: the cytochrome b, cytochrome c1 and Rieske iron‐sulphur proteins, which are encoded together in the fbc operon, as well as a 6‐kDa protein. The gene encoding the 6‐kDa protein, named fbcS, has been identified. It is located within the sox operon, which encodes the subunits of sarcosine oxidase. The encoded 6‐kDa protein is very hydrophobic and is predicted to form a single transmembrane helix. It shows no sequence homology to any known protein. The gene has been knocked‐out of the genome and a three‐subunit complex can be purified. This deletion leads to a large reduction in the yield of the isolated complex and in its activity compared to wild‐type. The high quinone content found in the wild‐type complex is, however, maintained after removal of the 6‐kDa protein. Surprisingly, a fourth subunit of approximately 6 kDa is again found to copurify with the Rhv. sulfidophilumbc1 complex when only the fbc operon is expressed heterologously in a near‐relative, Rhodobacter capsulatus, which lacks this small subunit in its own bc1 complex.
Further analysis of the involvement of the envelope anion channel PIRAC in chloroplast protein import
Authors: van den Wijngaard Paul W. J. , Demmers Jeroen A. A. , Thompson Simon J. , Wienk Hans L. J. , de Kruijff Ben , Vredenberg Wim J.
Published: March 2003

The ability of preferredoxin to inactivate a 50‐pS anion channel of the chloroplast inner membrane in the presence of an energy source was investigated using single‐channel recordings. It was found that preferredoxin cannot inactivate the channel when GTP is the only energy source present. From this it is concluded that the precursor has to interact with the , translocon of the inner membrane of chloroplasts (Tic) complex to be able to inactivate the 50‐pS anion channel. The ability of two mutants of preferredoxin with deletions in their transit sequence to inactivate the channel was also tested. Both mutants have been shown to have a similar binding affinity for the chloroplast envelope, but only one is able to fully translocate. The mutants were both able to inactivate the channel in a similar manner. From this it is concluded that full translocation is not necessary for the inactivation of the channel. It is also shown that preferredoxin is capable of inactivating the 50‐pS anion channel in the chloroplast‐attached configuration as was previously found in the inside‐out configuration. From this it is concluded that stromal factors do not influence the protein‐import‐induced inactivation of the 50‐pS anion channel of the chloroplast inner membrane. Finally the effect of the anion channel blocker 4,4′‐diisothiocyanostilbene‐2,2′‐disulfonate (DIDS) on the channel activity and on protein import was investigated. It was found that DIDS blocked the channel. Furthermore the addition of the channel blocker reduces the efficiency of import to 52%. This leads to the conclusion that correct functioning of the channel is important for protein import.
Proteome analysis reveals ubiquitin‐conjugating enzymes to be a new family of interferon‐α‐regulated genes
Authors: Nyman Tuula A. , Matikainen Sampsa , Sareneva Timo , Julkunen Ilkka , Kalkkinen Nisse
Published: March 2003

Interferon (IFN)‐α is a cytokine with antiviral, antiproliferative, and immunomodulatory properties, the functions of which are mediated via IFN‐induced protein products. We used metabolic labeling and two‐dimensional gel electrophoresis followed by MS and database searches to identify potentially new IFN‐α‐induced proteins in human T cells. By this analysis, we showed that IFN‐α induces the expression of ubiquitin cross‐reactive protein (ISG15) and two ubiquitin‐conjugating enzymes, UbcH5 and UbcH8. Northern‐blot analysis showed that IFN‐α rapidly enhances mRNA expression of UbcH5, UbcH6 and UbcH8 in T cells. In addition, these genes were induced in macrophages in response to IFN‐α or IFN‐γ stimulation or influenza A or Sendai virus infections. Similarly, IFNs enhanced UbcH8 mRNA expression in A549 lung epithelial cells, HepG2 hepatoma cells, and NK‐92 cells. Cycloheximide, a protein synthesis inhibitor, did not block IFN‐induced upregulation of UbcH8 mRNA expression, suggesting that UbcH8 is the primary target gene for IFN‐α and IFN‐γ. Ubiquitin conjugation is a rate‐limiting step in antigen presentation and therefore the upregulation of UbcHs by IFNs may contribute to the enhanced antigen presentation by macrophages. Our results show that proteome analysis of cells is a suitable method for identifying previously unrecognized cytokine‐inducible genes.
In vitro inhibition and intracellular enhancement of lysosomal α‐galactosidase A activity in Fabry lymphoblasts by 1‐deoxygalactonojirimycin and its derivatives
Authors: Asano Naoki , Ishii Satoshi , Kizu Haruhisa , Ikeda Kyoko , Yasuda Kayo , Kato Atsushi , Martin Olivier R. , Fan Jian‐Qiang
Published: March 2003

Fabry disease is a lysosomal storage disorder caused by deficient lysosomal α‐galactosidase A (α‐Gal A) activity. Deficiency of the enzyme activity results in progressive deposition of neutral glycosphingolipids with terminal α‐galactosyl residue in vascular endothelial cells. We recently proposed a chemical chaperone therapy for this disease by administration of 1‐deoxygalactonojirimycin, a potent inhibitor of the enzyme, at subinhibitory intracellular concentrations [Fan, J.‐Q., Ishii, S., Asano, N. and Suzuki, Y. (1999) Nat. Med.5, 112–115]. 1‐Deoxygalactonojirimycin served as a specific chaperone for those mutant enzymes that failed to maintain their proper conformation to avoid excessive degradation. In order to establish a correlation between in vitro inhibitory activity and intracellular enhancement activity of the specific chemical chaperone, a series of 1‐deoxygalactonojirimycin derivatives were tested for activity with both α‐Gal A and Fabry lymphoblasts. 1‐Deoxygalactonojirimycin was the most potent inhibitor of α‐Gal A with an IC50 value of 0.04 µm. α‐Galacto‐homonojirimycin, α‐allo‐homonojirimycin and β‐1‐C‐butyl‐deoxygalactonojirimycin were effective inhibitors with IC50 values of 0.21, 4.3 and 16 µm, respectively. N‐Alkylation, deoxygenation at C‐2 and epimerization at C‐3 of 1‐deoxygalactonojirimycin markedly lowered or abolished its inhibition toward α‐Gal A. Inclusion of 1‐deoxygalactonojirimycin, α‐galacto‐homonojirimycin, α‐allo‐homonojirimycin and β‐1‐C‐butyl‐deoxygalactonojirimycin at 100 µm in culture medium of Fabry lymphoblasts increased the intracellular α‐Gal A activity by 14‐fold, 5.2‐fold, 2.4‐fold and 2.3‐fold, respectively. Weaker inhibitors showed only a minimum enhancement effect. These results suggest that more potent inhibitors act as more effective specific chemical chaperones for the mutant enzyme, and the potent competitive inhibitors of α‐Gal A are effective specific chemical chaperones for Fabry disease.
Mechanisms of induction of human tissue inhibitor of metalloproteinases‐1 (TIMP‐1) gene expression by all‐trans retinoic acid in combination with basic fibroblast growth factor
Authors: Bigg Heather F. , McLeod Ronnie , Waters Jasmine G. , Cawston Tim E. , Clark Ian M.
Published: March 2003

The addition of all‐trans retinoic acid (ATRA) in combination with basic fibroblast growth factor (bFGF) to human fibroblasts results in a synergistic induction of tissue inhibitor of metalloproteinases‐1 (TIMP‐1) protein production. The synergistic stimulation of TIMP‐1 protein by ATRA and bFGF increased across 72 h. An incubation of 10 min to 12 h with bFGF alone followed by ATRA gave a similar synergistic induction of TIMP‐1 protein to that seen with both agents together. Treatment of cells with ATRA first followed by bFGF was ineffective. Expression of RARβ mRNA was induced by ATRA alone, but not further induced by ATRA and bFGF; expression of RARγ mRNA was induced by both ATRA or bFGF alone, and further induced by both reagents together; expression of RXRγ was repressed by ATRA alone, but not by ATRA in combination with bFGF. Steady‐state levels of TIMP‐1 mRNA were induced 14 to 40‐fold above control by ATRA and bFGF. Treatment with ATRA and bFGF did not alter the stability of TIMP‐1 mRNA. The induction of TIMP‐1 mRNA by ATRA and bFGF was greatly diminished by cycloheximide and therefore required new protein synthesis. The tyrosine kinase inhibitor genistein caused a dose‐dependent inhibition of TIMP‐1 protein induction by ATRA and bFGF. A MEK1 inhibitor (PD98059) inhibited both basal and induced levels of TIMP‐1. At high concentrations, p38 MAP kinase inhibitors further enhanced the synergistic stimulation of TIMP‐1 protein by ATRA and bFGF, but at these concentrations, p42/44 MAP kinase was strongly activated. These data begin to elucidate the mechanisms by which TIMP‐1 gene expression can be upregulated.
Distribution, cloning, and characterization of porcine nucleoside triphosphate diphosphohydrolase‐1
Authors: Lemmens Raf , Vanduffel Luc , Kittel Agnes , Beaudoin Adrien R. , Benrezzak Ouhida , Sévigny Jean
Published: March 2003

In this study, we have investigated the distribution of the enzyme nucleoside triphosphate diphosphohydrolase‐1 (NTPDase1; EC 3.6.1.5) in a subset of pig tissues by biochemical activity and Western blotting with antibodies against porcine NTPDase1. The highest expression of this enzyme was found in vascular endothelium, smooth muscle, spleen and lung.

The complete cDNA of NTPDase1 from aorta endothelial cells was sequenced using primer walking. The protein consists of 510 amino acids, with a calculated molecular mass of 57 756 Da. The amino‐acid sequence indicated seven putative N‐glycosylation sites and one potential intracellular cGMP‐ and cAMP‐dependent protein kinase phosphorylation site. As expected, the protein has a very high homology to other known mammalian ATPDases and CD39 molecules, and includes all five apyrase conserved regions.

Expression of the complete cDNA in COS‐7 cells confirmed that NTPDase1 codes for a transmembrane glycoprotein with ecto‐ATPase and ecto‐ADPase activities. Two proteolytic products of NTPDase1, with molecular mass of 54 and 27 kDa, respectively, were consistently present in proteins from transfected COS‐7 cells and in particulate fractions from different tissues. A trypsin cleavage site, giving rise to these two cleavage products, was identified. In order to remain enzymatically active, the two cleavage products have to interact by non–covalent interactions.
Gelsolin as a calcium‐regulated actin filament‐capping protein
Authors: Gremm Dagmar , Wegner Albrecht
Published: March 2003

Various concentrations of gelsolin (25–100 nm) were added to 2 µm polymerized actin. The concentrations of free calcium were adjusted to 0.05–1.5 µm by EGTA/Ca2+ buffer. Following addition of gelsolin actin depolymerization was observed that was caused by dissociation of actin subunits from the pointed ends of treadmilling actin filaments and inhibition by gelsolin of polymerization at barbed ends. The time course of depolymerization revealed an initial lag phase that was followed by slow decrease of the concentration of polymeric actin to reach the final steady state polymer and monomer concentration. The initial lag phase was pronounced at low free calcium and low gelsolin concentrations. On the basis of quantitative analysis the kinetics of depolymerization could be interpreted as capping, i.e. binding of gelsolin to the barbed ends of actin filaments and subsequent inhibition of polymerization, rather than severing. The main argument for this conclusion was that even gelsolin concentrations (100 nm) that exceed the concentration of filament ends (≈ 2 nm), cause the filaments to depolymerize at a rate that is similar to the rate of depolymerization of the concentration of pointed ends existing before addition of gelsolin. The rate of capping is directly proportional to the free calcium concentration. These experiments demonstrate that at micromolar and submicromolar free calcium concentrations gelsolin acts as a calcium‐regulated capping protein but not as an actin filament severing protein, and that the calcium binding sites of gelsolin which regulate the various functions of gelsolin (capping, severing and monomer binding), differ in their calcium affinity
Factors that determine the plasma‐membrane potential in bloodstream forms of Trypanosoma brucei
Authors: Nolan Derek P. , Voorheis H. Paul
Published: March 2003

The plasma‐membrane potential (Δψp) in bloodstream forms of Trypanosoma brucei was studied using several different radiolabelled probes: 86Rb+ and [14C]SCN− were used to report Δψp directly because they distribute in easily measured quantities across the plasma membrane only, and [3H]methyltriphenylphosphonium (MePh3P+) was used to report Δψp only when Δψm had been abolished with FCCP because it reports the algebraic sum of the two potentials when used alone. The unperturbed Δψp had a value of −82 mV and was found to be essentially identical with, and determined almost completely by, the potassium diffusion potential, as evidenced by: (a) the lack of effect of valinomycin on the value obtained under appropriate conditions when any of these probes were used; (b) the close agreement of this measured value with that predicted from the measured distribution of K+ across the plasma membrane (−76 mV); (c) the large effect of changes in the extracellular K+ concentration by substitution with Na+ on Δψp together with the complete lack of effect of substitution of extracellular Na+ by the choline cation or substitution of extracellular Cl− by the gluconate anion on Δψp. The contribution to Δψp by electrogenic pumping of Na+/K+‐ATPase was found to be small (of the order of 6 mV). H+ was not found to be pumped across the plasma membrane or to contribute to Δψp.
Effects of hydrostatic pressure on the structure and biological activity of infectious bursal disease virus
Authors: Tian Shao‐Min , Ruan Kang‐Cheng , Qian Jian‐Fei , Shao Guo‐Qing , Balny Claude
Published: March 2003

The effects of high hydrostatic pressure on the structure and biological activity of infectious bursal disease virus (IBDV), a commercially important pathogen of chickens, were investigated. IBDV was completely dissociated into subunits at a pressure of 240 MPa and 0 °C revealed by the change in intrinsic fluorescence spectrum and light scattering. The dissociation of IBDV showed abnormal concentration dependence as observed for some other viruses. Electron microscopy study showed that morphology of IBDV had an obvious change after pressure treatment at 0 °C. It was found that elevating pressure destroyed the infectivity of IBDV, and a completely pressure‐inactivated IBDV could be obtained under proper conditions. The pressure‐inactivated IBDV retained the original immunogenic properties and could elicit high titers of virus neutralizing antibodies. These results indicate that hydrostatic pressure provides a potential physical means to prepare antiviral vaccine.