bluuebunny/crossref_metadata_2025 huggingface dataset:
Galactosyl‐mimodye ligands for Pseudomonas fluorescensβ‐galactose dehydrogenase
Authors: Mazitsos C. F. , Rigden D. J. , Tsoungas P. G. , Clonis Y. D.
Published: March 2003

Protein molecular modelling and ligand docking were employed for the design of anthraquinone galactosyl‐biomimetic dye ligands (galactosyl‐mimodyes) for the target enzyme galactose dehydrogenase (GaDH). Using appropriate modelling methodology, a GaDH model was build based on a glucose‐fructose oxidoreductase (GFO) protein template. Subsequent computational analysis predicted chimaeric mimodye‐ligands comprising a NAD‐pseudomimetic moiety (anthraquinone diaminobenzosulfonic acid) and a galactosyl‐mimetic moiety (2‐amino‐2‐deoxygalactose or shikimic acid) bearing an aliphatic ‘linker’ molecule. In addition, the designed mimodye ligands had an appropriate in length and chemical nature ‘spacer’ molecule via which they can be attached onto a chromatographic support without steric clashes upon interaction with GaDH. Following their synthesis, purification and analysis, the ligands were immobilized to agarose. The respective affinity adsorbents, compared to other conventional adsorbents, were shown to be superior affinity chromatography materials for the target enzyme, Pseudomonas fluorescensβ‐galactose dehydrogenase. In addition, these mimodye affinity adsorbents displayed good selectivity, binding low amounts of enzymes other than GaDH. Further immobilized dye‐ligands, comprising different linker and/or spacer molecules, or not having a biomimetic moiety, had inferior chromatographic behavior. Therefore, these new mimodyes suggested by computational analysis, are candidates for application in affinity labeling and structural studies as well as for purification of galactose dehydrogenase.
Functional reconstitution of the HIV receptors CCR5 and CD4 in liposomes
Authors: Devesa François , Chams Vida , Dinadayala Premkumar , Stella Alexandre , Ragas Aude , Auboiroux Henri , Stegmann Toon , Poquet Yannick
Published: March 2003

Reconstitution of membrane proteins allows their study in a membrane environment that can be manipulated at will. Because membrane proteins have diverse biophysical properties, reconstitution methods have so far been developed for individual proteins on an ad hoc basis. We developed a postinsertion reconstitution method for CCR5, a G protein coupled receptor, with seven transmembrane α helices and small ecto‐ and endodomains. A His6‐tagged version of CCR5 was expressed in mammalian cells, purified using the detergent N‐dodecyl‐β‐d‐maltoside (DDM) and reconstituted into preformed liposomal membranes saturated with DDM, removing the detergent with hydrophobic polystyrene beads. We then attempted to incorporate CD4, a protein with a single transmembrane helix and a large hydrophilic ectodomain into liposomal membranes, together with CCR5. Surprisingly, reconstitution of this protein was also achieved by the method. Both proteins were found to be present together in individual liposomes. The reconstituted CCR5 was recognized by several monoclonal antibodies, recognized its natural ligand, and CD4 bound a soluble form of gp120, a subunit of the HIV fusion protein that uses CD4 as a receptor. Moreover, cells expressing the entire fusion protein of HIV bound to the liposomes, indicating that the proteins were intact and that most of them were oriented right side out. Thus, functional coreconstitution of two widely different proteins can be achieved by this method, suggesting that it might be useful for other proteins.
tRNA‐dependent amino acid discrimination by yeast seryl‐tRNA synthetase
Authors: Gruic‐Sovulj Ita , Landeka Irena , Söll Dieter , Weygand‐Durasevic Ivana
Published: March 2003

The ability of aminoacyl‐tRNA synthetases to distinguish between similar amino acids is crucial for accurate translation of the genetic code. Saccharomyces cerevisiae seryl‐tRNA synthetase (SerRS) employs tRNA‐dependent recognition of its cognate amino acid serine [Lenhard, B., Filipic, S., Landeka, I., Skrtic, I., Söll, D. & Weygand‐Durasevic, I. (1997) J. Biol. Chem.272, 1136–1141]. Here we show that dimeric SerRS enzyme complexed with one molecule of tRNASer is more specific and more efficient in catalyzing seryl‐adenylate formation than the apoenzyme alone. Sequence‐specific tRNA–protein interactions enhance discrimination of the amino acid substrate by yeast SerRS and diminish the misactivation of the structurally similar noncognate threonine. This may proceed via a tRNA‐induced conformational change in the enzyme's active site. The 3′‐terminal adenosine of tRNASer is not important in effecting the rearrangement of the serine binding site. Our results do not provide an indication for a readjustment of ATP binding in a tRNA‐assisted manner. The stoichiometric analyses of the complexes between the enzyme and tRNASer revealed that two cognate tRNA molecules can be bound to dimeric SerRS, however, with very different affinities.
Regulated expression and intracellular localization of cystatin F in human U937 cells
Authors: Nathanson Carl‐Michael , Wassélius Johan , Wallin Hanna , Abrahamson Magnus
Published: March 2003

Cystatin F is a cysteine peptidase inhibitor recently discovered in haematopoietic cells by cDNA cloning. To further investigate the expression, distribution and properties of the native human inhibitor the promyeloid cell line U937 has been studied. The cells expressed relatively large quantities of cystatin F, which was found both secreted and intracellularly. The intracellular levels were unusually high for a secreted cystatin (≈ 25% of the cystatin F in 2‐ or 4‐day culture medium). By contrast, U937 cells contained only 3–4% of the related inhibitor, cystatin C. Cystatin F purified from lysates of U937 cells showed three major forms carrying two, one or no carbohydrate chains. Immunocytochemistry demonstrated a marked cytoplasmic cystatin F staining in a granular pattern. Double staining with a marker for endoplasmic reticulum revealed no colocalization for cystatin F. Analysis of the promoter region of the cystatin F gene (CST7) showed that it, like that of the cystatin C gene (CST3), is devoid of typical TATA‐ and CAAT‐box elements. In contrast to the cystatin C promoter, it does not contain multiple Sp1 binding sites, but has a unique site for C/EBPα, possibly explaining the restricted expression of the cystatin F gene. Cells stimulated with all‐trans retinoic acid to differentiate them towards a granulocytic pathway, showed a strong (≈ 18‐fold) down‐regulation of intracellular cystatin F and almost abolished secreted levels of the inhibitor. Stimulation with tetradecanoyl phorbol acetate, causing monocytic differentiation, also resulted in down‐regulation (two fold to threefold) of cystatin F expression, whereas the cystatin C expression was essentially unaltered in both experiments. The results suggest that cystatin F as an intracellular cysteine peptidase inhibitor with readily regulated expression, may be a candidate to control the cysteine peptidase activity known to be essential for antigen presentation in different blood cell lineages.
Dinucleoside polyphosphates stimulate the primer independent synthesis of poly(A) catalyzed by yeast poly(A) polymerase
Authors: Sillero María A. Günther , de Diego Anabel , Osorio Hugo , Sillero Antonio
Published: March 2003

Novel properties of the primer independent synthesis of poly(A), catalyzed by the yeast poly(A) polymerase are presented. The commercial enzyme from yeast, in contrast to the enzyme from Escherichia coli, is unable to adenylate the 3′‐OH end of nucleosides, nucleotides or dinucleoside polyphosphates (NpnN). In the presence of 0.05 mm ATP, dinucleotides (at 0.01 mm) activated the enzyme velocity in the following decreasing order: Gp4G, 100; Gp3G, 82; Ap6A, 61; Gp2G, 52; Ap4A, 51; Ap2A, 41; Gp5G, 36; Ap5A, 27; Ap3A, 20, where 100 represents a 10‐fold activation in relation to a control without effector. The velocity of the enzyme towards its substrate ATP displayed sigmoidal kinetics with a Hill coefficient (nH) of 1.6 and a Km(S0.5) value of 0.308 ± 0.120 mm. Dinucleoside polyphosphates did not affect the maximum velocity (Vmax) of the reaction, but did alter its nH and Km(S0.5) values. In the presence of 0.01 mm Gp4G or Ap4A the nH and Km(S0.5) values were (1.0 and 0.063 ± 0.012 mm) and (0.8 and 0.170 ± 0.025 mm), respectively. With these kinetic properties, a dinucleoside polyphosphate concentration as low as 1 µm may have a noticeable activating effect on the synthesis of poly(A) by the enzyme. These findings together with previous publications from this laboratory point to a potential relationship between dinucleoside polyphosphates and enzymes catalyzing the synthesis and/or modification of DNA or RNA.
Expression and purification of the recombinant subunits of toluene/o‐xylene monooxygenase and reconstitution of the active complex
Authors: Cafaro Valeria , Scognamiglio Roberta , Viggiani Ambra , Izzo Viviana , Passaro Irene , Notomista Eugenio , Piaz Fabrizio Dal , Amoresano Angela , Casbarra Annarita , Pucci Piero , Di Donato Alberto
Published: March 2003

This paper describes the cloning of the genes coding for each component of the complex of toluene/o‐xylene monooxygenase from Pseudomonas stutzeri OX1, their expression, purification and characterization. Moreover, the reconstitution of the active complex from the recombinant subunits has been obtained, and the functional role of each component in the electron transfer from the electron donor to molecular oxygen has been determined.

The coexpression of subunits B, E and A leads to the formation of a subcomplex, named H, with a quaternary structure (BEA)2, endowed with hydroxylase activity.

Tomo F component is an NADH oxidoreductase. The purified enzyme contains about 1 mol of FAD, 2 mol of iron, and 2 mol of acid labile sulfide per mol of protein, as expected for the presence of one [2Fe−2S] cluster, and exhibits a typical flavodoxin absorption spectrum.

Interestingly, the sequence of the protein does not correspond to that previously predicted on the basis of DNA sequence. We have shown that this depends on minor errors in the gene sequence that we have corrected.

C component is a Rieske‐type ferredoxin, whose iron and acid labile sulfide content is in agreement with the presence of one [2Fe−2S] cluster. The cluster is very sensitive to oxygen damage.

Mixtures of the subcomplex H and of the subunits F, C and D are able to oxidize p‐cresol into 4‐methylcathecol, thus demonstrating the full functionality of the recombinant subunits as purified.

Finally, experimental evidence is reported which strongly support a model for the electron transfer. Subunit F is the first member of an electron transport chain which transfers electrons from NADH to C, which tunnels them to H subcomplex, and eventually to molecular oxygen.
Evidence for general stabilization of mRNAs in response to UV light
Authors: Bollig Frank , Winzen Reinhard , Kracht Michael , Ghebremedhin Beniam , Ritter Birgit , Wilhelm Arno , Resch Klaus , Holtmann Helmut
Published: March 2003

mRNA stabilization plays an important role in the changes in protein expression initiated by inducers of inflammation or direct cell stress such as UV light. This study provides evidence that stabilization in response to UV light differs from that induced by proinflammatory stimuli such as bacterial lipopolysaccharide or interleukin (IL)‐1. Firstly, UV‐induced stabilization is independent of the p38 MAP kinase pathway, which has previously been shown to mediate stabilization induced by IL‐1 or lipopolysaccharide. UV‐induced mRNA stabilization was insensitive to the dominant negative forms of p38 MAP kinase and its substrate MAP kinase‐activated protein kinase 2 (MK2), or to the p38 MAP kinase inhibitor SB 203580, demonstrating that it occurs through a different signaling mechanism. Secondly, UV‐induced stabilization exhibits a different transcript selectivity. Activation of the p38 MAP kinase pathway, by expressing active MAP kinase kinase 6, induced stabilization only of transcripts containing AU‐rich elements. UV light also induced stabilization of transcripts lacking AU‐rich elements. This effect could not be mimicked by expressing MEKK1, an upstream activator of the p38, JNK, ERK and NF‐κB pathways. UV light also stabilized endogenous histone mRNA, which lacks AU‐rich elements and a poly(A) tail. This effect was not mimicked by active MAP kinase kinase 6 and not sensitive to a p38 MAP kinase inhibitor. This suggests that UV light induces stabilization through a mechanism that is independent of p38 MAP kinase and affects a broad spectrum of mRNAs.
Stimulated biosynthesis of flavins in Photobacterium phosphoreum IFO 13896 and the presence of complete rib operons in two species of luminous bacteria
Authors: Kasai Sabu , Sumimoto Takumi
Published: March 2003

Photobacterium phosphoreum IFO 13896 emits light strongly when cultured in medium containing 3% NaCl, but only weakly in medium containing 1% NaCl. It is known that dim or dark mutants appear frequently and spontaneously from this parent strain. To confirm that riboflavin biosynthesis is stimulated when the lux operon is active, the amount of light emitted and flavins synthesized under strongly or weakly light emitting conditions was determined. In comparison with the parent strain cultured in 3% NaCl, the same strain cultured in 1% NaCl emitted 1/36 the light and produced 1/4 the flavins, while three dim or dark mutants, M1, M2 and M3 cultured in 3% NaCl, emitted almost no light, 1/58 the light and 1/10 the light and produced 1/8, 1/5 and 1/3 the amount of flavins, respectively. From these results, we deduced that the genes for riboflavin synthesis, rib genes, are organized in an operon in this strain. In P. phosphoreum NCMB 844, it has been reported that a rib gene cluster is present just downstream of the lux operon. However, among rib genes, the gene for pyrimidine deaminase/pyrimidine reductase, ribD, was not found in this cluster. Because a complete rib operon seems to be necessary for efficient regulation at the transcriptional level, we expected ribD to be present downstream of this cluster and sequenced this region, using SUGDAT, Sequencing Using Genomic DNA As a Template. We could not find this gene but found a gene for hybrid‐cluster protein (prismane protein). To find ribD in a different region, a partial ribD sequence was amplified and sequenced using a PCR‐based method, and subsequently the genomic DNA was sequenced in both directions from this partial sequence using SUGDAT. Because ribC was found just downstream of ribD, we sequenced further downstream of ribC and confirmed that another complete set of rib genes, ribD, ribC, ribBA, and ribE, is present in P. phosphoreum. The presence of a complete rib operon in P. phosphoreum explains why this species can synthesize flavins at enhanced levels to sustain a strong light emission. Furthermore, we sequenced the rib operon in Vibrio fischeri, another representative luminous bacterium, in a manner similar to that described above, and confirmed that a complete operon is present also in this species. The organization of rib genes in an operon in the Proteobacteria γ‐subdivision is discussed.
Activation of transcription of the human presenilin 1 gene by 12‐O‐tetradecanoylphorbol 13‐acetate
Authors: Pastorcic Martine , Das Hriday K.
Published: March 2003

We have recently identified an Ets element controlling over 90% of the basal expression of the human presenilin 1 (PS1) gene. We have also shown that Ets1 and Ets2 act as transactivators of the PS1 gene by cotransfection experiments in SK‐N‐SH neuronal cells. The PS1 gene is widely but differentially expressed across tissues and the expression in brain appears to be restricted to neurons. To gain further insight into the regulation of the gene we have examined the regulation of PS1 by 12‐O‐tetradecanoylphorbol 13‐acetate (TPA). SK‐N‐SH neuronal cells were treated with 0.2 µm TPA for 30 min to 24 h and the level of expression of the endogenous PS1 gene was measured by Northern blot analysis. A two‐ to threefold increase in the level of PS1 mRNA appeared 4–8 h after the addition of TPA. A similar increase in transcription activity was observed in nuclear run off experiments, indicating that the increased mRNA level results from an activation in the initiation of transcription of PS1. Consistently, TPA also increased the level of PS1 protein. No activation of the PS1 endogenous gene by TPA was observed in hepatoma HepG2 cells. Next we tested the effect of TPA on the expression of the PS1 promoter by transfecting fusion genes including various fragments of the PS1 promoter linked to a CAT reporter into SK‐N‐SH cells. TPA also stimulated the expression of the PS1CAT constructs. Generally wild type constructs −687/+178, −118/+178, −22/+178 including the short −35/+6 fragment showed a minor two‐ to threefold activation by TPA. Point mutations eliminating the −10 Ets motif or the −6 CREB/AP1 motif did not decrease the stimulation by TPA. Thus TPA appears to activate the transcription of the PS1 gene by a mechanism which does not require these elements.
Reconstructing the replication complex of AcMNPV
Authors: Hefferon Kathleen L. , Miller Lois K.
Published: March 2003

Baculoviruses are well known for their large, circular, double‐stranded DNA genomes. The type member, AcMNPV, is the best characterized and undergoes a succession of early, late and very late gene expression during its infection cycle. The viral genes involved in DNA replication have previously been identified and their products are required for the activation of late gene expression. In this study, we FLAG‐ and HA‐tagged the replication late expression factors of AcMNPV, examined their expression and functional activities by CAT assay and Western blot analysis, and determined their subcellular localization in transfected cells by subcellular fractionation and immunofluorescent microscopy. We found that all replication LEFs with the exception of P143 and P35 resided in the nucleus of transfected cells. We further investigated the interactions among various replication LEFs using both yeast two‐hybrid and coprecipitation strategies. A summary of the interactive properties of the replication LEFs is presented and a model for a putative AcMNPV replication complex is offered.
Identification and characterization of a novel activated RhoB binding protein containing a PDZ domain whose expression is specifically modulated in thyroid cells by cAMP
Authors: Mircescu Hortensia , Steuve Séverine , Savonet Valérie , Degraef Chantal , Mellor Harry , Dumont Jacques E. , Maenhaut Carine , Pirson Isabelle
Published: March 2003

In a search for genes regulated in response to cAMP we have identified a new protein, p76RBE, whose mRNA and protein expression is enhanced in thyrocytes following thyrotropin stimulation of the cAMP transduction cascade. This protein presents important similarities with Rhophilin and contains different protein–protein interaction motifs. The presence of HR1 and PDZ motifs as well as a potential PDZ binding domain motif suggests that p76RBE could be implicated in targeting or scaffolding processes. By yeast two‐hybrid screenings and coimmunoprecipitation, we show here that p76RBE is a specific binding protein of RhoB and binds selectively to the GTP‐bound form of this small GTPase. p76RBE also binds in vitro to components of the cytoskeleton, including cytokeratin 18. p76RBE is essentially cytoplasmic in transfected COS‐7 mammalian cells and seems to be recruited to an endosomal compartment when coexpressed with the activated form of RhoB. p76RBE was shown to be mainly expressed in tissues with high secretion activity. Our data suggest that p76RBE could play a key role between RhoB and potential downstream elements needed under stimulation of the thyrotropin/cAMP pathway in thyrocytes and responsible for intracellular motile phenomena such as the endocytosis involved in the thyroid secretory process.
Characterization of γ‐tocopherol methyltransferases from Capsicum annuum L and Arabidopsis thaliana
Authors: Koch Maria , Lemke Rainer , Heise Klaus‐Peter , Mock Hans‐Peter
Published: March 2003

Tocopherols are essential micronutrients in human and animal nutrition due to their function as lipophilic antioxidants. They are exclusively synthesized by photosynthetic organisms including higher plants. Despite the attributed beneficial health effects and many industrial applications, research on the tocopherol biosynthetic pathway and its regulation in plants is still limited. In the work presented here we performed a detailed biochemical characterization of a γ‐tocopherol methyltransferase (γ‐TMT) from Arabidopsis thaliana and of a γ‐TMT purified from Capsicum annuum fruits, a tissue with high accumulation of tocopherols. The biochemical characteristics of both enzyme preparations were remarkably similar including substrate specificities. Both enzymes converted δ‐ and γ‐ into β‐ and α‐tocopherol, respectively, but β‐tocopherol was not accepted as a substrate, pointing to a specific methylation at the C(5)‐position of the tocopherol aromatic head group. A kinetic analysis performed with the Arabidopsis enzyme was consistent with an iso‐ordered bi‐bi type reaction mechanism. Our results emphasize the role of γ‐TMT in regulating the spectrum of accumulated tocopherols in plants.
The influence of temperature and osmolyte on the catalytic cycle of cytochrome c oxidase
Authors: Kornblatt Jack A. , Hill Bruce C. , Marden Michael C.
Published: March 2003

The influence of temperature on cytochrome c oxidase (CCO) catalytic activity was studied in the temperature range 240–308 K. Temperatures below 273 K required the inclusion of the osmolyte ethylene glycol. For steady‐state activity between 278 and 308 K the activation energy was 12 kcal·mol−1; the molecular activity or turnover number was 12 s−1 at 280 K in the absence of ethylene glycol. CCO activity was studied between 240 and 277 K in the presence of ethylene glycol. The activation energy was 30 kcal·mol−1; the molecular activity was 1 s−1 at 280 K. Ethylene glycol inhibits CCO by lowering the activity of water. The rate limitation in electron transfer (ET) was not associated with ET into the CCO as cytochrome a was predominantly reduced in the aerobic steady state. The activity of CCO in flash‐induced oxidation experiments was studied in the low temperature range in the presence of ethylene glycol. Flash photolysis of the reduced CO complex in the presence of oxygen resulted in three discernable processes. At 273 K the rate constants were 1500 s−1, 150 s−1 and 30 s−1 and these dropped to 220 s−1, 27 s−1 and 3 s−1 at 240 K. The activation energies were 5 kcal·mol−1, 7 kcal·mol−1, and 8 kcal·mol−1, respectively. The fastest rate we ascribe to the oxidation of cytochrome a3, the intermediate rate to cytochrome a oxidation and the slowest rate to the re‐reduction of cytochrome a followed by its oxidation. There are two comparisons that are important: (a) with vs. without ethylene glycol and (b) steady state vs. flash‐induced oxidation. When one makes these two comparisons it is clear that the CCO only senses the presence of osmolyte during the reductive portion of the catalytic cycle. In the present work that would mean after a flash‐induced oxidation and the start of the next reduction/oxidation cycle.
A single amino acid substitution of Leu130Ile in snake DNases I contributes to the acquisition of thermal stability
Authors: Takeshita Haruo , Yasuda Toshihiro , Nakajima Tamiko , Mogi Kouichi , Kaneko Yasushi , Iida Reiko , Kishi Koichiro
Published: March 2003

We purified pancreatic deoxyribonucleases I (DNases I) from three snakes, Elaphe quadrivirgata, Elaphe climacophora and Agkistrodon blomhoffii, and cloned their cDNAs. Each mature snake DNase I protein comprised 262 amino acids. Wild‐type snake DNases I with Leu130 were more thermally unstable than wild‐type mammalian and avian DNases I with Ile130. After substitution of Leu130Ile, the thermal stabilities of the snake enzymes were higher than those of their wild‐type counterparts and similar to mammalian wild‐type enzyme levels. Conversely, substituting Ile130Leu of mammalian DNases I made them more thermally unstable than their wild‐type counterparts. Therefore, a single amino acid substitution, Leu130Ile, might be involved in an evolutionally critical change in the thermal stabilities of vertebrate DNases I. Amphibian DNases I have a Ser205 insertion in a Ca2+‐binding site of mammalian and avian enzymes that reduces their thermal stabilities [Takeshita, H., Yasuda, T., Iida, R., Nakajima, T., Mori, S., Mogi, K., Kaneko, Y. & Kishi, K. (2001) Biochem. J.357, 473–480]. Thus, it is plausible that the thermally stable wild‐type DNases I of the higher vertebrates, such as mammals and birds, have been generated by a single Leu130Ile substitution of reptilian enzymes through molecular evolution following Ser205 deletion from amphibian enzymes. This mechanism may reflect one of the evolutionary changes from cold‐blooded to warm‐blooded vertebrates.
Trophoblast‐like human choriocarcinoma cells serve as a suitable in vitro model for selective cholesteryl ester uptake from high density lipoproteins
Authors: Wadsack Christian , Hrzenjak Andelko , Hammer Astrid , Hirschmugl Birgit , Levak‐Frank Sanja , Desoye Gernot , Sattler Wolfgang , Malle Ernst
Published: March 2003

As human choriocarcinoma cells display many of the biochemical and morphological characteristics reported for in utero invasive trophoblast cells we have studied cholesterol supply from high density lipoproteins (HDL) to these cells. Binding properties of 125I‐labeled HDL subclass 3 (HDL3) at 4 °C were similar for BeWo, JAr, and Jeg3 choriocarcinoma cell lines while degradation rates at 37 °C were highest for BeWo. Calculating the selective cholesteryl ester (CE)‐uptake as the difference between specific cell association of [3H]CE‐labeled HDL3 and holoparticle association of 125I‐labeled HDL3 revealed that in BeWo cells, the selective CE‐uptake was slightly lower than holoparticle association. However, the pronounced capacity for specific cell association of [3H]CE‐HDL3 and selective [3H]CE‐uptake in excess of HDL3–holoparticle association, and cAMP–mediated enhanced cell association of [3H]CE‐HDL3 in JAr and Jeg3 suggested the scavenger receptor class B, type I (SR‐BI) to be responsible for this pathway. Abundant expression of SR‐BI (but not SR‐BII, a splice variant of SR‐BI) could be observed in JAr and Jeg3 but not in BeWo cells using RT‐PCR, Northern and Western blot analysis, and immunocytochemical technique. Adenovirus‐mediated overexpression of SR‐BI in all three choriocarcinoma cell lines resulted in an enhanced capacity for cell association of [3H]CE‐HDL3 (20‐fold in BeWo; fivefold in JAr and Jeg3). The fact that exogenous HDL3 remarkably increases proliferation in JAr and Jeg3 supports the notion that selective CE‐uptake and subsequent intracellular generation of cholesterol is coupled to cellular growth. From our findings we propose that JAr and Jeg3 cells serve as a suitable in vitro model to study selective CE‐supply to human placental cells.
Structure, mRNA expression and linkage mapping of the brain‐type fatty acid‐binding protein gene (fabp7) from zebrafish (Danio rerio)
Authors: Liu Rong‐Zong , Denovan‐Wright Eileen M. , Wright Jonathan M.
Published: March 2003

The brain fatty acid‐binding protein (B‐FABP) is involved in brain development and adult neurogenesis. We have determined the sequence of the gene encoding the B‐FABP in zebrafish. The zebrafish B‐FABP gene spans 2370 bp and contains four exons interrupted by three introns. The coding sequence of zebrafish B‐FABP gene is identical to its cDNA sequence and the coding capacity of each exon is the same as that for the human and mouse B‐FABP genes. A 1249 bp sequence 5′ upstream of exon 1 of the zebrafish B‐FABP gene was cloned and sequenced. Several brain development/growth‐associated transcription factor binding elements, including POU‐domain binding elements and the proposed lipogenic‐associated transcription factor NF‐Y elements, were found within the 5′ region of the B‐FABP gene. RT‐PCR analysis using mRNA extracted from different tissues of adult zebrafish demonstrated that the zebrafish B‐FABP mRNA was predominant in brain with lower levels in liver, testis and intestine, but not in ovary, skin, heart, kidney and muscle. Quantitative RT‐PCR revealed a similar tissue‐specific distribution for zebrafish B‐FABP mRNA except that very low levels of B‐FABP mRNA, normalized to β‐actin mRNA, were detected in the heart and muscle RNA, but not in liver RNA. Zebrafish B‐FABP mRNA was detected by RT‐PCR in embryos beyond 12 h postfertilization, suggesting a correlation of zebrafish B‐FABP mRNA expression with early brain development. Radiation hybrid mapping assigned the zebrafish B‐FABP gene to linkage group 17. Conserved syntenies of the zebrafish B‐FABP gene and the human and mouse orthologous B‐FABP genes were observed by comparative genomic analysis.
Purification and cDNA cloning of a cellulase from abalone Haliotis discus hannai
Authors: Suzuki Ken‐ichi , Ojima Takao , Nishita Kiyoyoshi
Published: March 2003

A cellulase [endo‐β‐1,4‐d‐glucanase (EC 3.2.1.4)] was isolated from the hepatopancreas of abalone Haliotis discus hannai by successive chromatographies on TOYOPEARL CM‐650M, hydroxyapatite and Sephacryl S‐200 HR. The molecular mass of the cellulase was estimated to be 66 000 Da by SDS/PAGE, thus the enzyme was named HdEG66. The hydrolytic activity of HdEG66 toward carboxymethylcellulose showed optimal temperature and pH at 38 °C and 6.3, respectively. cDNAs encoding HdEG66 were amplified by the polymerase chain reaction from an abalone hepatopancreas cDNA library with primers synthesized on the basis of partial amino‐acid sequences of HdEG66. By overlapping the nucleotide sequences of the cDNAs, a sequence of 1898 bp in total was determined. The coding region of 1785 bp located at nucleotide position 56–1840 gave an amino‐acid sequence of 594 residues including the initiation methionine. The N‐terminal region of 14 residues in the deduced sequence was regarded as the signal peptide as it was absent in HdEG66 protein and showed high similarity to the consensus sequence for signal peptides of eukaryote secretory proteins. Thus, matured HdEG66 was thought to consist of 579 residues. The C‐terminal region of 453 residues in HdEG66, i.e. approximately the C–terminal three quarters of the protein, showed 42–44% identity to the catalytic domains of glycoside hydrolase family 9 (GHF9)‐cellulases from arthropods and Thermomonospora fusca. While the N‐terminal first quarter of HdEG66 showed 27% identity to the carbohydrate‐binding module (CBM) of a Cellulomonas fimi cellulase, CenA. Thus, the HdEG66 was regarded as the GHF9‐cellulase possessing a family II CBM in the N‐terminal region. By genomic PCR using specific primers to the 3′‐terminal coding sequences of HdEG66‐cDNA, a DNA of 2186 bp including three introns was amplified. This strongly suggests that the origin of HdEG66 is not from symbiotic bacteria but abalone itself.
The subtle side to hypoxia inducible factor (HIFα) regulation
Authors: Bilton Rebecca L. , Booker Grant W.
Published: March 2003

The transcription factor hypoxia inducible factor α‐subunit (HIFα) is pivotal in the cellular response to the stress of hypoxia. Post‐translational modification of HIFα by hydroxylase enzymes has recently been identified as a key ‘oxygen sensing’ mechanism within the cell. The absence of the substrate oxygen prevents the hydroxylases from modifying HIFα during hypoxia and allows dramatic up‐regulation of both HIFα protein stability and transcriptional activation capability. In addition to this oxygen‐dependent response, increased HIFα protein levels and/or enhanced transcriptional activity during normoxic conditions can be stimulated by various receptor‐mediated factors such as growth‐factors and cytokines (insulin, insulin‐like growth factor 1 or 2, endothelial growth factor, tumour necrosis factor α, angiotensin‐2). Oncogenes are also capable of HIFα activation. This induction is generally less intense than that stimulated by hypoxia and although not fully elucidated, appears to occur via hypoxia‐independent, receptor‐mediated signal pathways involving either phosphatidyl‐inositol‐3‐kinase/Akt or mitogen activated protein kinase (MAPK) pathways, depending on the cell‐type. Activation of Akt increases HIFα protein synthesis in the cell and results in increased HIFα protein and transcriptional activity. MAPK also activates HIFα protein synthesis and additionally may potentiate HIF1α transcriptional activity via a separate mechanism that does not necessarily require protein stabilization. Here we review the mechanisms and function of receptor‐mediated signals in the multifaceted regulation of HIFα.
Studies on the regulatory properties of the pterin cofactor and dopamine bound at the active site of human phenylalanine hydroxylase
Authors: Solstad Therese , Stokka Anne J. , Andersen Ole A. , Flatmark Torgeir
Published: March 2003

The catalytic activity of phenylalanine hydroxylase (PAH, phenylalanine 4‐monooxygenase EC 1.14.16.1) is regulated by three main mechanisms, i.e. substrate (l‐phenylalanine, L‐Phe) activation, pterin cofactor inhibition and phosphorylation of a single serine (Ser16) residue. To address the molecular basis for the inhibition by the natural cofactor (6R)‐l‐erythro‐5,6,7,8‐tetrahydrobiopterin, its effects on the recombinant tetrameric human enzyme (wt‐hPAH) was studied using three different conformational probes, i.e. the limited proteolysis by trypsin, the reversible global conformational transition (hysteresis) triggered by L‐Phe binding, as measured in real time by surface plasmon resonance analysis, and the rate of phosphorylation of Ser16 by cAMP‐dependent protein kinase. Comparison of the inhibitory properties of the natural cofactor with the available three‐dimensional crystal structure information on the ligand‐free, the binary and the ternary complexes, have provided important clues concerning the molecular mechanism for the negative modulatory effects. In the binary complex, the binding of the cofactor at the active site results in the formation of stabilizing hydrogen bonds between the dihydroxypropyl side‐chain and the carbonyl oxygen of Ser23 in the autoregulatory sequence. L‐Phe binding triggers local as well as global conformational changes of the protomer resulting in a displacement of the cofactor bound at the active site by 2.6 Å (mean distance) in the direction of the iron and Glu286 which causes a loss of the stabilizing hydrogen bonds present in the binary complex and thereby a complete reversal of the pterin cofactor as a negative effector. The negative modulatory properties of the inhibitor dopamine, bound by bidentate coordination to the active site iron, is explained by a similar molecular mechanism including its reversal by substrate binding. Although the pterin cofactor and the substrate bind at distinctly different sites, the local conformational changes imposed by their binding at the active site have a mutual effect on their respective binding affinities.
Structural characterization of three genetic variants of human serum albumin modified in subdomains IIB and IIIA
Authors: Galliano Monica , Watkins Scott , Madison Jeanne , Putnam Frank W. , Kragh‐Hansen Ulrich , Cesati Roberto , Minchiotti Lorenzo
Published: March 2003

Three new genetic variants of human serum albumin have been detected in Italy by routine clinical electrophoresis. Albumin Milano Slow is common in Northern Italy, while albumins Liprizzi and Trieste, which are fast migrating, are rare and local variants. Isoelectric focusing analysis of the CNBr fragments obtained from the carboxymethylated alloalbumins in all cases localized the mutation to fragment CB5 (residues 330−446). The modified CNBr fragments were isolated on a preparative scale and subjected to tryptic digestion. Sequence determination of the abnormal tryptic peptides revealed that all the variants are caused by single point mutations : Trieste, Lys359←Asn, Milano Slow, Asp375←His, and Liprizzi, Arg410←Cys. These results were confirmed by sequence determination of a variant V8 peptide in the case of Trieste, and by DNA sequence analysis for the other two variants. The DNA analysis showed a G←C transversion at nucleotide position 11 969 for albumin Milano Slow, and a C←T transition at position 13 251 for Liprizzi. The latter represents a mutation at a hypermutable CpG dinucleotide site. Albumins Trieste and Milano Slow, as most of the variants thus far described, have mutations involving residues on the surface of the molecule. In contrast, albumin Liprizzi represents the first example of a mutation in the most active binding pocket of the molecule, placed in subdomain IIIA.
Biogenesis of acetylcholinesterase is impaired, although its mRNA level remains normal, in the glucocorticoid‐treated rat skeletal muscle
Authors: Brank Martina , Zajc‐Kreft Katarina , Kreft Samo , Komel Radovan , Grubič Zoran
Published: March 2003

Acetylcholinesterase (AChE) is responsible for the hydrolysis of acetylcholine in the neuromuscular junction and other cholinergic synapses. Insight into the mechanisms controlling AChE expression in skeletal muscle is important for understanding formation, plasticity, and various dysfunctions of the neuromuscular junction. We have investigated the mechanisms responsible for the decreased AChE activity in the fast rat sternomastoideus muscle after chronic glucocorticoid treatment. Under such conditions fast skeletal muscles become atrophic and loose 30−40 % of their AChE activity. In order to establish at which level synthesis of AChE is affected by glucocorticoids, we studied the effects of chronic dexamethasone treatment at both AChE mRNA and mature enzyme levels. Reduced rate of AChE recovery after subtotal irreversible AChE inhibition was observed during the first week of dexamethasone treatment, but not later. Statistical analyses of four independent northern blots revealed unchanged AChE mRNA levels. At the same time, we observed more than 60 % decrease in the (G1+G2)/A12 ratio of molecular forms at the expense of G forms. It has been generally accepted that globular G1 and G2 molecular forms are synthesized in the rough endoplasmic reticulum as precursors of asymmetric (A) AChE forms, assembled in the Golgi apparatus. Reduced levels of G1 and G2 AChE forms, in combination with unchanged AChE mRNA, are therefore consistent with the reports demonstrating that glucocorticoids downregulate muscle protein synthesis at the translational level. Our findings support but not entirely prove the concept that impaired translation and/or posttranslational control are the primary cause of decreased AChE activity in the glucocorticoid‐treated muscle.
The octapeptide angiotensin II adopts a well‐defined structure in a phospholipid environment
Authors: Carpenter Katharine A. , Wilkes Brian C. , Schiller Peter W.
Published: March 2003

The conformational properties adopted by angiotensin II in a phospholipid micelle solution were studied by NMR spectroscopy and molecular modelling. The octapeptide was found to assume a well‐defined hairpin structure with its C‐ and N‐termini approaching to within 0.76 nm of each other. Three of the residues had fixed side chain configurations; Tyr4 (g+), His6 (g−) and Val3 (g−). Consequently, the His6 and Tyr4 aromatic rings were consistently close together. Conformers containing a cis His6‐Pro7 peptide bond were observed for the peptide in a purely aqueous sample but completely disappeared when lipid vesicles were added to the sample. This result is explained by the existence of a very stable hydrogen bond between the Phe8 NH and the His6 carbonyl group of the lipid‐solvated trans isomer, resulting in the formation of an inverse γ turn centered on Pro7. 1H‐NMR selective line broadening was apparent for several of the angiotensin II protons upon titration of an aqueous sample with less than stoichiometric amounts of 1,2‐dimyristoyl‐sn‐glycero‐3‐phosphocholine bilayer vesicles. The data obtained were consistent with the structure derived for micelle‐bound angiotensin II, indicating that conformations adopted by the peptide hormone in the presence of micelles and lipid‐bilayer vesicles are similar.
Amphiphilic α‐helices are important structural motifs in the α and β peptides that constitute the bacteriocin lactococcin G
Authors: Hauge Håvard Hildeng , Nissen‐Meyer Jon , Nes Ingolf F. , Eijsink Vincent G. H.
Published: March 2003

Lactococcin G (LcnG) is an antimicrobial substance (bacteriocin) consisting of two peptides, LcnG‐α and LcnG‐β. The structures of intact LcnG‐α and LcnG‐β as well as various fragments of these peptides were studied by circular dichroism (CD) under several conditions. All peptides had a non‐structured conformation in aqueous solutions. In the presence of trifluoroethanol, dodecylphosphocholine micelles and (negatively charged) dioleoylglycerophosphoglycerol (Ole2GroPGro) liposomes, varying amounts of α‐helical structure were induced. Comparisons of the various fragments showed that helicity was concentrated in those parts of LcnG‐α and LcnG‐β that would become amphiphilic if an α‐helical structure was adopted. In the presence of zwitterionic dioleoylglycerophosphocholine (Ole2GroPCho) liposomes, the peptides were much less (if at all) structured, suggesting that the excess of positive charge on the antimicrobial peptides needs to be compensated by an excess of negative charge on the membrane. The structuring of LcnG‐α and LcnG‐β in the presence of Ole2GroPGro liposomes was considerably enhanced when both peptides were presented simultaneously to the membranes. Consecutive addition of the two peptides to Ole2GroPGro liposomes did not give this additional structuring, indicating that the individual LcnG‐α and LcnG‐β peptides associate with the membrane in a virtually irreversible manner that makes them inaccessible for interaction with the complementary peptide. The results suggest that upon arrival at and interaction with the target membrane, LcnG‐α and LcnG‐β form a complex that consists of approximately 50 % amphiphilic α‐helices.
4‐Hydroxybenzoyl‐CoA reductase (dehydroxylating) from the denitrifying bacterium Thauera aromatica
Authors: Breese Klaus , Fuchs Georg
Published: March 2003

4‐Hydroxybenzoyl‐CoA reductase catalyzes an important reaction in the anaerobic metabolism of phenolic compounds, i.e. the reductive removal of an aromatic hydroxyl group. The prosthetic groups and the natural electron donor of the enzyme were investigated and the genes were cloned and sequenced. The enzyme is a molybdenum‐flavin‐iron‐sulfur protein of subunit composition of α2β2γ2. It contains approximately 1.3 flavin nucleotide, probably FAD, 1.9 Mo, 15 Fe, and 12.5 acid‐labile sulfur. Sequence interpretation suggests that the native enzyme contains two [4Fe‐4S] and four [2Fe‐2S] clusters. A 9.8‐kDa ferredoxin with two [4Fe‐4S] clusters functions as the natural electron donor. The genes coding for the three subunits, hcrABC, show high similarities to other molybdenum‐flavin‐iron‐sulfur proteins of the xanthine oxidase family, notably to the three putative 4‐hydroxybenzoyl‐CoA reductase genes in Rhodopseudomonas palustris. In addition, there are close similarities to three open reading frames (orf) in E. coli. A major difference is the presence of an additional domain in the β‐subunit (HcrB, 35 kDa) probably carrying an additional iron‐sulfur cluster. The 82‐kDa α‐subunit (HcrA) contains a Mo‐cofactor‐binding site. The 17‐kDa γ‐subunit (HcrC) harbors two [2Fe‐2S] clusters. Upstream of the hcrCAB region, an ORF was found coding for a regulatory protein of the MarR family. Downstream of the hcrCAB region lies an ORF presumably coding for a hydrophobic permease.
Purification and characterisation of NADH oxidase from Thermus aquaticus YT1 and evidence that it functions in a peroxide‐reduction system
Authors: Toomey David , Mayhew Stephen G.
Published: March 2003

A thermostable enzyme previously identified as an NADH oxidase has been purified from Thermus aquaticus YT‐1 by chromatography on DEAE‐cellulose and AMP‐Sepharose. The enzyme is dimeric with subunits of 54 kDa and one molecule FAD/subunit. The FAD is tightly bound, but it can be removed reversibly by hydrophobic chromatography at low pH. The blue flavin semiquinone is stabilised during photo‐chemical reduction of the enzyme. Chemical reduction by static titration with dithionite ion showed that the enzyme requires about 5 mol dithionite/mol FAD for full reduction, and that reduction occurs in four phases. Reduction by the substrate NADH is incomplete, with the formation of a new long‐wavelength absorption underlying the semiquinone absorption. Amino acid sequencing showed that the T. aquaticus enzyme resembles other microbial flavoenzymes that function in two‐enzyme systems for the reduction of peroxides, and which contain two redox‐active disulphide groups in addition to the flavin. The enzyme catalyses the reduction of O2, ferricyanide ion, 2,6‐dichloroindophenol, and 5,5′dithiobis(2,2′‐dinitrobenzoate), and of cumene hydroperoxide in the presence of the small protein component (AhpC) of the peroxide‐reducing system of Salmonella typhimurium. The reduction of O2 is slow in the absence of exogenous flavin while dye reduction is fast, suggesting that the free flavin that is added to the usual assay for T. aquaticus NADH oxidase functions by mediating electron transfer from enzyme‐bound reduced flavin to O2. The physiological function of the enzyme is probably in peroxide reduction with a small protein analogous to AhpC as the natural electron acceptor.
Immunochemical studies of the lipopolysaccharide O‐specific polysaccharide of Hafnia alvei PCM 1199 related to H. alvei PCM 1205
Authors: Katzenellenbogen Ewa , Kocharova Nina A. , Zatonsky Georgy V. , Mieszala Malgorzata , Gamian Andrzej , Bogulska Maria , Shashkov Aleksander S. , Romanowska Elzbieta , Knirel Yuriy A.
Published: March 2003

On the basis of chemical analyses and NMR spectroscopic studies, it was found that the O‐specific polysaccharide (O‐PS) isolated from the Hafnia alvei PCM 1199 lipopolysaccharide (LPS) is a glycerol teichoic‐acid‐like polymer having a repeating unit of the following structure :

3)‐β‐ D‐Quip4NAc‐(1←1)‐Gro3P‐(O←3)‐β‐
D‐Galp‐(1←3)‐α‐
D‐GlcpNAc‐(1←
2
6
↑∣
1
 OAc
β‐
D‐GlcpNAc
6
∣
OAc
where Qui4NAc is 4‐acetamido‐4,6‐dideoxyglucose and O‐acetylation at both positions is non‐stoichiometric. The glycosidic linkage of the lateral β‐
D‐GlcpNAc residue is acid‐labile and cleaved from the OPS during mild acid hydrolysis or dephosphorylation with 48 % hydrofluoric acid. Comparative analysis revealed that the structure of the H. alvei PCM 1199 O‐PS is similar to that of H. alvei PCM 1205, which differs in the presence of an additional lateral α‐
D‐Glcp residue and the position of one of the O‐acetyl groups only. Accordingly, serological tests revealed a high degree of serological similarity between LPSs and O‐PSs of the two H. alvei strains.