bluuebunny/crossref_metadata_2025 huggingface dataset:
Crystal structures of fusion proteins with large‐affinity tags
Authors: Smyth Douglas R. , Mrozkiewicz Marek K. , McGrath William J. , Listwan Pawel , Kobe Bostjan
Published: June 2003

The fusion of a protein of interest to a large‐affinity tag, such as the maltose‐binding protein (MBP), thioredoxin (TRX), or glutathione‐S‐transferase (GST), can be advantageous in terms of increased expression, enhanced solubility, protection from proteolysis, improved folding, and protein purification via affinity chromatography. Unfortunately, crystal growth is hindered by the conformational heterogeneity induced by the fusion tag, requiring that the tag is removed by a potentially problematic cleavage step. The first three crystal structures of fusion proteins with large‐affinity tags have been reported recently. All three structures used a novel strategy to rigidly fuse the protein of interest to MBP via a short three‐ to five‐amino acid spacer. This strategy has the potential to aid structure determination of proteins that present particular experimental challenges and are not conducive to more conventional crystallization strategies (e.g., membrane proteins). Structural genomics initiatives may also benefit from this approach as a way to crystallize problematic proteins of significant interest.
A solution NMR study of the binding kinetics and the internal dynamics of an HIV‐1 protease‐substrate complex
Authors: Katoh Etsuko , Louis John M. , Yamazaki Toshimasa , Gronenborn Angela M. , Torchia Dennis A. , Ishima Rieko
Published: June 2003

NMR studies of the binding of a substrate to an inactive HIV‐1 protease construct, containing an active site mutation PRD25N, are reported. Substrate titration measurements monitored by HSQC spectra and a 15N‐edited NOESY experiment show that the chromogenic substrate analog of the capsid/p2 cleavage site binds to PRD25N with an equilibrium dissociation constant, KD, of 0.27 ± 0.05 mM, and upper limits of the association and dissociation rate constants, 2×104 M−1s−1 and 10 s−1, respectively, at 20°C, pH 5.8. This association rate constant is not in the diffusion limit, suggesting that association is controlled by a rare event, such as opening of the protease flaps. Analysis of 15N relaxation experiments reveals a slight reduction of S2 values in the flap region, indicating a small increase in the amplitude of internal motion on the sub‐nsec timescale. In addition, several residues in the flap region are mobile on the conformational exchange timescale, msec–μsec. Flap dynamics of the protease‐substrate complex are compared with those of protease‐inhibitor complexes, and the implications of these results for substrate‐binding models are discussed.
Recognition of cysteine‐containing peptides through prompt fragmentation of the 4‐dimethylaminophenylazophenyl‐4′‐maleimide derivative during analysis by MALDI‐MS
Authors: Borges Chad R. , Watson J. Throck
Published: June 2003

Under specified UV‐MALDI conditions, the 4‐dimethylaminophenylazophenyl‐4'‐maleimide (DABMI) derivative of cysteine‐containing peptides undergoes prompt fragmentation to produce a characteristic mass spectral pattern. As reported previously by others, derivatization with chromophoric DABMI allows cysteine‐containing peptides and proteins to be tracked during HPLC by absorbance at upper UV and visible wavelengths. Here, we describe methodology by which these same peptide derivatives can be recognized by their distinctive MALDI mass spectral fragmentation pattern, then mass mapped, allowing for easy, rapid identification of cysteine‐containing peptides.
Grafting a new metal ligand in the cocatalytic site of B. cereus metallo‐β‐lactamase: Structural flexibility without loss of activity
Authors: Rasia Rodolfo M. , Ceolín Marcelo , Vila Alejandro J.
Published: June 2003

Metallo‐β‐lactamases are zinc enzymes able to hydrolyze the four‐membered ring of β‐lactam antibiotics, representing one of the latest generations of β‐lactamases. These enzymes belong to the zinc metallo‐hydrolase family of the β‐lactamase fold. Enzymes belonging to this family have a bimetallic active site whose structure varies among different members by point substitutions of the metal ligands. In this work, we have grafted new metal ligands into the metal binding site of BcII from Bacillus cereus that mimic the ligands present in other members of this superfamily. We have characterized spectroscopically and modeled the structure of the redesigned sites, which differ substantially from the wild‐type enzyme. Despite the changes introduced in the active site, the mutant enzymes retain almost full activity. These results shed some light on the possible evolutionary origin of these metalloenzymes.
Three monophyletic superfamilies account for the majority of the known glycosyltransferases
Authors: Liu Jing , Mushegian Arcady
Published: June 2003

Sixty‐five families of glycosyltransferases (EC 2.4.x.y) have been recognized on the basis of high‐sequence similarity to a founding member with experimentally demonstrated enzymatic activity. Although distant sequence relationships between some of these families have been reported, the natural history of glycosyltransferases is poorly understood. We used iterative searches of sequence databases, motif extraction, structural comparison, and analysis of completely sequenced genomes to track the origins of modern‐type glycosyltransferases. We show that >75% of recognized glycosyltransferase families belong to one of only three monophyletic superfamilies of proteins, namely, (1) a recently described GPGTF/GT‐B superfamily; (2) a nucleoside‐diphosphosugar transferase (GT‐A) superfamily, which is characterized by a DxD sequence signature and also includes nucleotidyltransferases; and (3) a GT‐C superfamily of integral membrane glycosyltransferases with a modified DxD signature in the first extracellular loop. Several developmental regulators in Metazoans, including Fringe and Egghead homologs, belong to the second superfamily. Interestingly, Tout‐velu/Exostosin family of developmental proteins found in all multicellular eukaryotes, contains separate domains belonging to the first and the second superfamilies, explaining multiple glycosyltransferase activities in one protein.
Structural studies of the interaction of S‐adenosylmethionine with the [4Fe‐4S] clusters in biotin synthase and pyruvate formate‐lyase activating enzyme
Authors: Cosper Michele M. , Cosper Nathaniel J. , Hong Wei , Shokes Jacob E. , Broderick William E. , Broderick Joan B. , Johnson Michael K. , Scott Robert A.
Published: June 2003

The diverse reactions catalyzed by the radical‐SAM superfamily of enzymes are thought to proceed via a set of common mechanistic steps, key among which is the reductive cleavage of S‐adenosyl‐L‐methionine (SAM) by a reduced [4Fe‐4S] cluster to generate an intermediate deoxyadenosyl radical. A number of spectroscopic studies have provided evidence that SAM interacts directly with the [4Fe‐4S] clusters in several of the radical‐SAM enzymes; however, the molecular mechanism for the reductive cleavage has yet to be elucidated. Selenium X‐ray absorption spectroscopy (Se‐XAS) was used previously to provide evidence for a close interaction between the Se atom of selenomethionine (a cleavage product of Se‐SAM) and an Fe atom of the [4Fe‐4S] cluster of lysine‐2,3‐aminomutase (KAM). Here, we utilize the same approach to investigate the possibility of a similar interaction in pyruvate formate‐lyase activating enzyme (PFL‐AE) and biotin synthase (BioB), two additional members of the radical‐SAM superfamily. The results show that the latter two enzymes do not exhibit the same Fe‐Se interaction as was observed in KAM, indicating that the methionine product of reductive cleavage of SAM does not occupy a well‐defined site close to the cluster in PFL‐AE and BioB. These results are interpreted in terms of the differences among these enzymes in their use of SAM as either a cofactor or a substrate.
Solution structure and thermal stability of ribosomal protein L30e from hyperthermophilic archaeonThermococcus celer
Authors: Wong Kam‐Bo , Lee Chi‐Fung , Chan Siu‐Hong , Leung Tak‐Yuen , Chen Yu Wai , Bycroft Mark
Published: June 2003

To understand the structural basis of thermostability, we have determined the solution structure of a thermophilic ribosomal protein L30e fromThermococcus celerby NMR spectroscopy. The conformational stability ofT. celerL30e was measured by guanidine and thermal‐induced denaturation, and compared with that obtained for yeast L30e, a mesophilic homolog. The melting temperature ofT. celerL30e was 94°C, whereas the yeast protein denatured irreversibly at temperatures >45°C. The two homologous proteins also differ greatly in their stability at 25°C: the free energy of unfolding was 45 kJ/mole forT. celerL30e and 14 kJ/mole for the yeast homolog. The solution structure ofT. celerL30e was compared with that of the yeast homolog. Although the two homologous proteins do not differ significantly in their number of hydrogen bonds and the amount of solvent accessible surface area buried with folding, the thermophilicT. celerL30e was found to have more long‐range ion pairs, more proline residues in loops, and better helix capping residues in helix‐1 and helix‐4. A K9A variant ofT. celerL30e was created by site‐directed mutagenesis to examine the role of electrostatic interactions on protein stability. Although the melting temperatures of the K9A variant is ∼8°C lower than that of the wild‐type L30e, their difference in Tmis narrowed to ∼4.2°C at 0.5 M NaCl. This salt‐dependency of melting temperatures strongly suggests that electrostatic interactions contribute to the thermostability ofT. celerL30e.
Folding specificity induced by loop stiffness
Authors: Spagnolo Laura , Ventura Salvador , Serrano Luis
Published: June 2003

To test the importance of loop stiffness in restricting the heterogeneity of transition state ensemble, we relaxed the distal loop of 10 unstable redesigned hydrophobic core mutants of α‐spectrin SH3 domain. This was achieved by replacing Asp48 by Gly at the tip of the distal hairpin. Although the change was local in nature, the effect on stabilization was not uniform across the core mutants tested. There is an inverse rough correlation between the stabilization and the increase in buried hydrophobic volume, with respect to the wild type. Interestingly enough, proteins that although unstable are properly folded become molten globule‐like after relaxation of the distal loop. These results highlight the importance of stiffness in restricting the conformational heterogeneity of a protein during the folding reaction. An interplay between unspecific hydrophobic interactions and constraint induced by polar interactions, or in this case local stiffness, is essential to achieve a well‐ordered folded structure.
Crystal structure of a phosphatase with a unique substrate binding domain from Thermotoga maritima
Authors: Shin Dong Hae , Roberts Anne , Jancarik Jaru , Yokota Hisao , Kim Rosalind , Wemmer David E. , Kim Sung‐Hou
Published: June 2003

We have determined the crystal structure of a phosphatase with a unique substrate binding domain from Thermotoga maritima, TM0651 (gi 4981173), at 2.2 Å resolution by selenomethionine single‐wavelength anomalous diffraction (SAD) techniques. TM0651 is a member of the haloacid dehalogenase (HAD) superfamily, with sequence homology to trehalose‐6‐phosphate phosphatase and sucrose‐6F‐phosphate phosphohydrolase. Selenomethionine labeled TM0651 crystallized in space group C2 with three monomers per asymmetric unit. Each monomer has approximate dimensions of 65 × 40 × 35 Å3, and contains two domains: a domain of known hydrolase fold characteristic of the HAD family, and a domain with a new tertiary fold consisting of a six‐stranded β‐sheet surrounded by four α‐helices. There is one disulfide bond between residues Cys35 and Cys265 in each monomer. One magnesium ion and one sulfate ion are bound in the active site. The superposition of active site residues with other HAD family members indicates that TM0651 is very likely a phosphatase that acts through the formation of a phosphoaspartate intermediate, which is supported by both NMR titration data and a biochemical assay. Structural and functional database searches and the presence of many aromatic residues in the interface of the two domains suggest the substrate of TM0651 is a carbohydrate molecule. From the crystal structure and NMR data, the protein likely undergoes a conformational change upon substrate binding.
The inactivating factor of glutamine synthetase, IF7, is a “natively unfolded” protein
Authors: Muro‐Pastor M. Isabel , Barrera Francisco N. , Reyes José C. , Florencio Francisco J. , Neira José L.
Published: June 2003

Glutamine synthetase (GS) is the key enzyme responsible for the primary assimilation of ammonium in all living organisms, and it catalyses the synthesis of glutamine from glutamic acid, ATP, and ammonium. One of the recently discovered mechanisms of GS regulation involves protein‐protein interactions with a small 65‐residue‐long protein named IF7. Here, we study the structure and stability of IF7 and its binding properties to GS, by using several biophysical techniques (fluorescence, circular dichroism, Fourier transform infrared and nuclear magnetic resonance spectroscopies, and gel filtration chromatography) which provide complementary structural information. The findings show that IF7 has a small amount of residual secondary structure, but lacks a well defined tertiary structure, and is not compact. Thus, all of the studies indicate that IF7 is a “natively unfolded” protein. The binding of IF7 to GS, its natural binding partner, occurs with an apparent dissociation constant ofKD= 0.3 ± 0.1 μM, as measured by fluorescence. We discuss the implications for the GS regulation mechanisms of IF7 being unfolded.
Interaction of the 18.5‐kD isoform of myelin basic protein with Ca2+‐calmodulin: Effects of deimination assessed by intrinsic Trp fluorescence spectroscopy, dynamic light scattering, and circular dichroism
Authors: Libich David S. , Hill Christopher M.D. , Bates Ian R. , Hallett F. Ross , Armstrong Souzan , Siemiarczuk Aleksander , Harauz George
Published: June 2003

The effects of deimination (conversion of arginyl to citrullinyl residues) of myelin basic protein (MBP) on its binding to calmodulin (CaM) have been examined. Four species of MBP were investigated: unmodified recombinant murine MBP (rmMBP‐Cit0), an engineered protein with six quasi‐citrullinyl (i.e., glutaminyl) residues per molecule (rmMBP‐qCit6), human component C1 (hMBP‐Cit0), and human component C8 (hMBP‐Cit6), both obtained from a patient with multiple sclerosis (MS). Both rmMBP‐Cit0 and hMBP‐Cit0 bound CaM in a Ca2+‐dependent manner and primarily in a 1:1 stoichiometry, which was verified by dynamic light scattering. Circular dichroic spectroscopy was unable to detect any changes in secondary structure in MBP upon CaM‐binding. Inherent Trp fluorescence spectroscopy and a single‐site binding model were used to determine the dissociation constants: Kd = 144 ± 76 nM for rmMBP‐Cit0, and Kd = 42 ± 15 nM for hMBP‐Cit0. For rmMBP‐qCit6 and hMBP‐Cit6, the changes in fluorescence were suggestive of a two‐site interaction, although the dissociation constants could not be accurately determined. These results can be explained by a local conformational change induced in MBP by deimination, exposing a second binding site with a weaker association with CaM, or by the existence of several conformers of deiminated MBP. Titration with the collisional quencher acrylamide, and steady‐state and lifetime measurements of the fluorescence at 340 nm, showed both dynamic and static components to the quenching, and differences between the unmodified and deiminated proteins that were also consistent with a local conformational change due to deimination.
Analysis of accessible surface of residues in proteins
Authors: Lins Laurence , Thomas Annick , Brasseur Robert
Published: June 2003

We analyzed the total, hydrophobic, and hydrophilic accessible surfaces (ASAs) of residues from a nonredundant bank of 587 3D structure proteins. In an extended fold, residues are classified into three families with respect to their hydrophobicity balance. As expected, residues lose part of their solvent‐accessible surface with folding but the three groups remain. The decrease of accessibility is more pronounced for hydrophobic than hydrophilic residues. Amazingly, Lysine is the residue with the largest hydrophobic accessible surface in folded structures. Our analysis points out a clear difference between the mean (other studies) and median (this study) ASA values of hydrophobic residues, which should be taken into consideration for future investigations on a protein‐accessible surface, in order to improve predictions requiring ASA values. The different secondary structures correspond to different accessibility of residues. Random coils, turns, and β‐structures (outside β‐sheets) are the most accessible folds, with an average of 30% accessibility. The helical residues are about 20% accessible, and the difference between the hydrophobic and the hydrophilic residues illustrates the amphipathy of many helices. Residues from β‐sheets are the most inaccessible to solvent (10% accessible). Hence, β‐sheets are the most appropriate structures to shield the hydrophobic parts of residues from water. We also show that there is an equal balance between the hydrophobic and the hydrophilic accessible surfaces of the 3D protein surfaces irrespective of the protein size. This results in a patchwork surface of hydrophobic and hydrophilic areas, which could be important for protein interactions and/or activity.
Predicting the topology of transmembrane helical proteins using mean burial propensity and a hidden‐Markov‐model‐based method
Authors: Zhou Hongyi , Zhou Yaoqi
Published: June 2003

Helices in membrane spanning regions are more tightly packed than the helices in soluble proteins. Thus, we introduce a method that uses a simple scale of burial propensity and a new algorithm to predict transmembrane helical (TMH) segments and a positive‐inside rule to predict amino‐terminal orientation. The method (the topology predictor of transmembrane helical proteins using mean burial propensity [THUMBUP]) correctly predicted the topology of 55 of 73 proteins (or 75%) with known three‐dimensional structures (the 3D helix database). This level of accuracy can be reached by MEMSAT 1.8 (a 200‐parameter model‐recognition method) and a new HMM‐based method (a 111‐parameter hidden Markov model, UMDHMMTMHP) if they were retrained with the 73‐protein database. Thus, a method based on a physiochemical property can provide topology prediction as accurate as those methods based on more complicated statistical models and learning algorithms for the proteins with accurately known structures. Commonly used HMM‐based methods and MEMSAT 1.8 were trained with a combination of the partial 3D helix database and a 1D helix database of TMH proteins in which topology information were obtained by gene fusion and other experimental techniques. These methods provide a significantly poorer prediction for the topology of TMH proteins in the 3D helix database. This suggests that the 1D helix database, because of its inaccuracy, should be avoided as either a training or testing database. A Web server of THUMBUP and UMDHMMTMHP is established for academic users at http://www.smbs.buffalo.edu/phys_bio/service.htm. The 3D helix database is also available from the same Web site.
Role of a solvent‐exposed tryptophan in the recognition and binding of antibiotic substrates for a metallo‐β‐lactamase
Authors: Huntley James J.A. , Fast Walter , Benkovic Stephen J. , Wright Peter E. , Dyson H. Jane
Published: June 2003

Numerous X‐ray crystal structures of the metallo‐β‐lactamase from Bacteroides fragilis and related organisms show a β‐hairpin loop immediately adjacent to the active‐site zinc atom(s). Both crystallographic and NMR information show that the end of this β‐hairpin loop, which contains a solvent exposed tryptophan residue, Trp49, is highly flexible in the absence of substrates or other ligands, giving rise in some of the X‐ray structures to a lack of observable electron density in this region. We report an investigation of the role of this mobile, solvent‐exposed tryptophan using site‐directed mutagenesis, steady state kinetics measurements and characterization by NMR. Trp49 appears to have a role both in substrate binding and in promotion of catalysis. Substitution of this residue with a number of different side chains indicates that the binding interaction depends on the bulky hydrophobic and aromatic nature of the indole ring, which can provide relatively non‐specific interactions with a variety of antibiotic substrates. In this way, the tryptophan at this position provides a large degree of the breadth of substrate specificity for the metallo‐β‐lactamase. Previous studies established that the antibiotic binding site was sufficiently plastic that the derivatization of existing antibiotics is unlikely to result in the successful treatment of bacterial infections incorporating this resistance element. Rather, a more productive approach may be to design therapeutics directed towards this solvent‐exposed tryptophan residue.
Construction of a sequence motif characteristic of aminergic G protein–coupled receptors
Authors: Huang Enoch S.
Published: June 2003

An approach to discover sequence patterns characteristic of ligand classes is described and applied to aminergic G protein–coupled receptors (GPCRs). Putative ligand‐binding residue positions were inferred from considering three lines of evidence: conservation in the subfamily absent or underrepresented in the superfamily, any available mutation data, and the physicochemical properties of the ligand. For aminergic GPCRs, the motif is composed of a conserved aspartic acid in the third transmembrane (TM) domain (rhodopsin position 117) and a conserved tryptophan in the seventh TM domain (rhodopsin position 293); the roles of each are readily justified by molecular modeling of ligand‐receptor interactions. This minimally defined motif is an appropriate computational tool for identifying additional, potentially novel aminergic GPCRs from a set of experimentally uncharacterized “orphan” GPCRs, complementing existing sequence matching, clustering, and machine‐learning techniques. Motif sensitivity stems from the stepwise addition of residues characteristic of an entire class of ligand (and not tailored for any particular biogenic amine). This sensitivity is balanced by careful consideration of residues (evidence drawn from mutation data, correlation of ligand properties to residue properties, and location with respect to the extracellular face), thereby maintaining specificity for the aminergic class. A number of orphan GPCRs assigned to the aminergic class by this motif were later discovered to be a novel subfamily of trace amine GPCRs, as well as the successful classification of the histamine H4 receptor.
Effects of denaturants and substitutions of hydrophobic residues on backbone dynamics of denatured staphylococcal nuclease
Authors: Ohnishi Satoshi , Shortle David
Published: June 2003

Analysis of residual dipolar couplings (RDCs) in the Δ131Δ fragment of staphylococcal nuclease has demonstrated that its ensemble‐averaged structure is resistant to perturbations such as high concentrations of urea, low pH, and substitution of hydrophobic residues, suggesting that its residual structure is encoded by local side‐chain/backbone interactions. In the present study, the effects of these same perturbations on the backbone dynamics of Δ131Δ were examined through 1H‐15N relaxation methods. Unlike the global structure reported by RDCs, the transverse relaxation rates R2 were quite sensitive to denaturing conditions. At pH 5.2, Δ131Δ exhibits an uneven R2 profile with several characteristic peaks involving hydrophobic chain segments. Protonation of carboxyl side chains by lowering the pH reduces the values of R2 along the entire chain, yet these characteristic peaks remain. In contrast, high concentrations of urea or the substitution of 10 hydrophobic residues eliminates these peaks and reduces the R2 values by a greater amount. The combination of low pH and high urea leads to further decreases in R2. These denaturant‐induced increases in backbone mobility are also reflected in decreases in 15N NOEs and in relaxation interference parameters, with the former reporting an increase in fast motions and the latter a decrease in slow motions. Comparison between the changes in chain dynamics and the corresponding changes in Stokes radius and the patterns of RDCs suggests that regional variations in backbone dynamics in denatured nuclease arise primarily from local contacts between hydrophobic side chains and local interactions involving charged carboxyl groups.
Recombinant production and solution structure of PcTx1, the specific peptide inhibitor of ASIC1a proton‐gated cation channels
Authors: Escoubas Pierre , Bernard Cédric , Lambeau Gérard , Lazdunski Michel , Darbon Hervé
Published: June 2003

Acid‐sensing ion channels (ASICs) are thought to be important ion channels, particularly for the perception of pain. Some of them may also contribute to synaptic plasticity, learning, and memory. Psalmotoxin 1 (PcTx1), the first potent and specific blocker of the ASIC1a proton‐sensing channel, has been successfully expressed in the Drosophila melanogaster S2 cell recombinant expression system used here for the first time to produce a spider toxin. The recombinant toxin was identical in all respects to the native peptide, and its three‐dimensional structure in solution was determined by means of 1H 2D NMR spectroscopy. Surface characteristics of PcTx1 provide insights on key structural elements involved in the binding of PcTx1 to ASIC1a channels. They appear to be localized in the β‐sheet and the β‐turn linking the strands, as indicated by electrostatic anisotropy calculations, surface charge distribution, and the presence of residues known to be implicated in channel recognition by other inhibitor cystine knot (ICK) toxins.
Solution structure of Vibrio cholerae protein VC0424: A variation of the ferredoxin‐like fold
Authors: Ramelot Theresa A. , Ni Shuisong , Goldsmith‐Fischman Sharon , Cort John R. , Honig Barry , Kennedy Michael A.
Published: June 2003

The structure of Vibrio cholerae protein VC0424 was determined by NMR spectroscopy. VC0424 belongs to a conserved family of bacterial proteins of unknown function (COG 3076). The structure has an α‐β sandwich architecture consisting of two layers: a four‐stranded antiparallel β‐sheet and three side‐by‐side α‐helices. The secondary structure elements have the order αβαββαβ along the sequence. This fold is the same as the ferredoxin‐like fold, except with an additional long N‐terminal helix, making it a variation on this common motif. A cluster of conserved surface residues on the β‐sheet side of the protein forms a pocket that may be important for the biological function of this conserved family of proteins.
Structure and properties of recombinant human pyridoxine 5′‐phosphate oxidase
Authors: Musayev Faik N. , Di Salvo Martino L. , Ko Tzu‐Ping , Schirch Verne , Safo Martin K.
Published: June 2003

Pyridoxine 5′‐phosphate oxidase catalyzes the terminal step in the synthesis of pyridoxal 5′‐phosphate. The cDNA for the human enzyme has been cloned and expressed in Escherichia coli. The purified human enzyme is a homodimer that exhibits a low catalytic rate constant of ∼0.2 sec−1 and Km values in the low micromolar range for both pyridoxine 5′phosphate and pyridoxamine 5′‐phosphate. Pyridoxal 5′‐phosphate is an effective product inhibitor. The three‐dimensional fold of the human enzyme is very similar to those of the E. coli and yeast enzymes. The human and E. coli enzymes share 39% sequence identity, but the binding sites for the tightly bound FMN and substrate are highly conserved. As observed with the E. coli enzyme, the human enzyme binds one molecule of pyridoxal 5′‐phosphate tightly on each subunit.
The role of side chain conformational flexibility in surface recognition by Tenebrio molitor antifreeze protein
Authors: Daley Margaret E. , Sykes Brian D.
Published: June 2003

Two‐dimensional nuclear magnetic resonance spectroscopy was used to investigate the flexibility of the threonine side chains in the β‐helical Tenebrio molitor antifreeze protein (TmAFP) at low temperatures. From measurement of the 3Jαβ 1H‐1H scalar coupling constants, the χ1 angles and preferred rotamer populations can be calculated. It was determined that the threonines on the ice‐binding face of the protein adopt a preferred rotameric conformation at near freezing temperatures, whereas the threonines not on the ice‐binding face sample many rotameric states. This suggests that TmAFP maintains a preformed ice‐binding conformation in solution, wherein the rigid array of threonines that form the AFP‐ice interface matches the ice crystal lattice. A key factor in binding to the ice surface and inhibition of ice crystal growth appears to be the close surface‐to‐surface complementarity between the AFP and crystalline ice, and the lack of an entropic penalty associated with freezing out motions in a flexible ligand.
Structural analysis of UBL5, a novel ubiquitin‐like modifier
Authors: McNally Teresa , Huang Qiulong , Janis Richard S. , Liu Zhihong , Olejniczak Edward T. , Reilly Regina M.
Published: June 2003

UBL5 is a widely expressed human protein that is strongly conserved across phylogeny. Orthologs of UBL5 occur in every eukaryotic genome characterized to date. The yeast ortholog of UBL5, HUB1, was reported to be a ubiquitin‐like protein modifier important for modulation of protein function. However, unlike ubiquitin and all other ubiquitin‐like modifiers, UBL5 and its yeast ortholog HUB1 both contain a C‐terminal di‐tyrosine motif followed by a single variable residue instead of the characteristic di‐glycine found in all other ubiquitin‐like modifiers. Here we describe the three‐dimensional structure of UBL5 determined by NMR. The overall structure of the protein was found to be very similar to ubiquitin despite the low ∼25% residue similarity. The signature C‐terminal di‐tyrosine residues in UBL5 are involved in the final β sheet of the protein. This is very different to the di‐glycine motif found in ubiquitin, which extends beyond the final β sheet. In addition, we have confirmed an earlier report of an interaction between UBL5 and the cyclin‐like kinase, CLK4, which we have determined is specific and does not extend to other cyclin‐like kinase family members.
Characterization of Hypoxia‐Induced Ventilatory Depression in Newborn Piglets
Authors: St‐Jacques Rene , Filiano James J. , Darnall Robert A. , St‐John Walter M.
Published: June 2003

We evaluated the hypothesis that a ‘central oxygen detector’ in the brainstem is necessary for depressions of ventilatory activity to be manifested in the newborn. Decerebrate piglets, ventilated with 100% O2, were studied following neuromuscular blockade. The vagi and carotid sinus nerves were sectioned bilaterally in order to remove the influence of the peripheral chemoreceptors. Activity of the phrenic nerve was recorded as the index of the central respiratory rhythm. This activity declined and, in some preparations, ceased upon ventilation with air or a hypoxic gas, at either normocapnia or hypercapnia. The degree of depression in hypercapnic hypoxia was greatest in the youngest piglets. Following a medial section of the brainstem, extending to the caudal pons, the depression was reduced. In some preparations, a similar reduction followed the placement of radiofrequency lesions in the caudal ventromedial pons. We conclude that a region of the caudal mesencephalon or pons is necessary for the manifestation of depressions of ventilatory activity in the newborn pig.
Acute Hypervolaemia Improves Arterial Oxygen Pressure in Athletes with Exercise‐Induced Hypoxaemia
Authors: Zavorsky Gerald S. , Walley Keith R. , Hunte Garth S. , McKenzie Donald C. , Sexsmith George P. , Russell James A.
Published: June 2003

The aim of this study was to determine the effect of acute plasma volume expansion on arterial blood‐gas status during 6.5 min strenuous cycling exercise comparing six athletes with and six athletes without exercise‐induced arterial hypoxaemia (EIAH). We hypothesized that plasma volume expansion could improve arterial oxygen pressure in a homogeneous sample of athletes ‐ those with EIAH. In this paper we have extended the analysis and results of our recently published surprising findings that lengthening cardiopulmonary transit time did not improve arterial blood‐gas status in a heterogeneous sample of endurance cyclists. One 500 ml bag of 10% Pentastarch (infusion condition) or 60 ml 0.9% saline (placebo) was infused prior to exercise in a randomized, double‐blind fashion on two different days. Power output, cardiac output, oxygen consumption and arterial blood gases were measured during strenuous exercise. Cardiac output and oxygen consumption were not affected by acute hypervolaemia. There were group × condition interaction effects for arterial oxygen pressure and alveolar‐arterial oxygen pressure difference, suggesting that those with hypoxaemia experienced improved arterial oxygen pressure (+4 mmHg) and lower alveolar‐arterial oxygen pressure difference (‐2 mmHg) with infusion. In conclusion, acute hypervolaemia improves blood‐gas status in athletes with EIAH. The impairment of gas exchange occurs within the first minute of exercise, and is not impaired further throughout the remaining duration of exercise. This suggests that arterial oxygen pressure is only minimally mediated by cardiac output.
Intravenous Magnesium Reduces the Rate of Glucose Disposal in Lactating Sheep
Authors: Gow I. F. , Mitchell E. , Wait M.
Published: June 2003

Magnesium deficiency is generally associated with an impaired ability to dispose of glucose. In order to test whether or not increasing the peripheral free Mg concentration ([Mg2+]) would enhance glucose disposal, we have carried out glucose infusions in sheep with and without simultaneous infusion of Mg. Basal plasma glucose levels were higher in lactating sheep (‘lactators’) than in non‐lactating sheep (controls) (P < 0.05). The glucose disposal rate (KG) with no added Mg was greater in lactators than controls (P < 0.05). When Mg was added to the infusate, KG in lactators was reduced (P < 0.005). Infusion of Mg depressed basal insulin levels in controls and lactators (P < 0.0001 for both). The insulin response to the intravenous glucose tolerance test (IVGTT) was lower in lactators compared with controls (P < 0.0001); however, after correcting for the reduced basal insulin level when Mg was included during the IVGTT, there was no difference between the two groups. We conclude that intravenous Mg at the doses used in this study leads to a decrease in basal insulin secretion, and that increasing serum [Mg2+] reduces glucose disposal in lactating sheep.
Dynamic Exercise Attenuates Spontaneous Baroreceptor Reflex Sensitivity in Conscious Rats
Authors: Waki Hidefumi , Kasparov Sergey , Katahira Kiyoaki , Shimizu Tsuyoshi , Murphy David , Paton Julian F. R.
Published: June 2003

In humans, it has been reported that dynamic exercise attenuates spontaneous baroreceptor reflex sensitivity (sBRS), which is an index of the gain of the baroreceptor‐cardiac reflex. We demonstrated previously that endogenously produced NO from endothelial nitric oxide synthase (eNOS) within the nucleus tractus solitarii (NTS), the central terminal site of baroreceptor afferents, depressed sBRS. In this study, we investigated whether eNOS activity within the NTS plays any role in down‐modulating the sBRS during dynamic exercise. In conscious Wistar rats arterial pressure and heart rate (HR) were monitored continuously and chronically using radiotelemetry before and during wheel cage running at 6 m min−1 for 10 min. sBRS was determined by a time‐series method. During dynamic exercise systolic blood pressure (SBP) and HR were significantly increased (SBP: 138 ± 2 vs. 125 ± 2 mmHg, P < 0.001; HR: 447 ± 6 vs. 362 ± 8 beats min−1, P < 0.001) while sBRS was significantly decreased (0.53 ± 0.03 vs. 1.08 ± 0.08 ms mmHg−1, P < 0.001). In sino‐aortic denervated rats the change in SBP in response to dynamic exercise was significantly larger than that in baroreceptor‐intact rats (denervated: 21.6 ± 2.5 mmHg; intact: 12.0 ± 2.8 mmHg, P < 0.05). In contrast, denervation made no difference to the change in HR. Although disabling eNOS activity in the NTS by adenoviral‐directed expression of a dominant negative mutant form of eNOS increased resting sBRS (1.48 ± 0.20 vs. 1.09 ± 0.15 ms mmHg−1, P < 0.05), the absolute level reached during dynamic exercise was identical to control. These results demonstrate that during dynamic exercise (i) the sBRS decreases around the operating point of the baroreceptor‐cardiac reflex function curve in normotensive rats, (ii) the baroreceptor reflex operates to limit the rise in arterial pressure, and (iii) the attenuation of sBRS is not mediated by changes in eNOS activity within the NTS.
Dynamics of a Transgene Expression in Acute Rat Brain Slices Transfected with Adenoviral Vectors
Authors: Stokes C. E. L. , Murphy D. , Paton J. F. R. , Kasparov S.
Published: June 2003

We present a quantitative account of the expression dynamics of a transgene (enhanced green fluorescent protein, EGFP) in acute brain slices transfected with an adenoviral vector (AVV) under control of the human cytomegalovirus (HCMV) promoter. Micromolar concentrations of EGFP could be detected in brainstem and hippocampal slices as early as 7 h after in vitro transfection with a viral titre of 4.4 × 109 plaque‐forming units (pfu) ml−1. Although initially EGFP appeared mainly in glia, it could be detected in neurones with longer incubation times of 10‐12 h. However, fluorescence was never detected within some populations of neurones, such as hippocampal pyramidal cells, or within the hypoglossal motor nucleus. The density of cells expressing EGFP peaked at 10 h and then decreased, possibly suggesting that high concentrations of EGFP are toxic. The age of the animal significantly affected the speed of EGFP accumulation: after 10 h of incubation in 30‐day‐old rats only 4.88 ± 0.51 cells/10 000 μm2 were fluorescent compared to 7.28 ± 0.39 cells/10 000 μm2 in 12‐day‐old rats (P < 0.05). HCMV promoter‐driven transgene expression depended on the activity of protein kinase A, and was depressed with a cAMP/protein kinase A antagonist (20 μM Rp‐cAMPS; P < 0.0005). This indicates that expression of HCMV‐driven constructs is likely to be skewed towards cellular populations where cAMP‐dependent signalling pathways are active. We conclude that acute transfection of brain slices with AVVs within hours causes EGFP expression in micromolar concentrations and that such transfected cells may remain viable for use in physiological experiments.
The Use of Fluorescent Nuclear Dyes and Laser Scanning Confocal Microscopy to Study the Cellular Aspects of Arterial Remodelling in Human Subjects with Critical Limb Ischaemia
Authors: Coats P. , Jarajapu Y. P. R. , Hillier C. , McGrath J. C. , Daly C.
Published: June 2003

Resistance arteries isolated from patients with critical limb ischaemia (CLI), a hypotensive/hypoperfusion state of the lower leg, have been shown to undergo morphological changes opposite to those observed in hypertension, that is, decreased wall thickness, reduced cross‐sectional area and a decreased wall : lumen ratio. The aim of this present study was to use laser scanning confocal microscopy (LSCM) to study intact resistance arteries isolated from patients with CLI, specifically to identify the cellular aspects of the morphological changes identified in ischaemic subcutaneous and skeletal muscle resistance vessels. Using LSCM, a significant reduction in adventitial and medial thickness, cross‐sectional area and wall : lumen ratio was confirmed in resistance arteries from both distal ischaemic subcutaneous and skeletal muscle vascular beds when compared with corresponding arteries from the proximal non‐ischaemic sites. The cellular composition of the adventitial, medial and intimal layers of these distal ischaemic arteries was significantly different compared with proximal non‐ischaemic arteries. These differences in the distal arteries were characterised by hypoplasia in the adventitial and medial layers of the arterial wall and hypertrophy in the intimal layer. The differences observed in both distal ischaemic vascular beds (subcutaneous and skeletal muscle) were similar.
Effect of Polyphenolic Compounds on the Renal Na+,K+‐ATPase during the Restoration of Normotension after Experimentally Induced Hypertension in Rats
Authors: Javorková Veronika , Pechánová Olga , Andriantsitohaina Ramaroson , Vrbjar Norbert
Published: June 2003

It is commonly known that consumption of foods and beverages rich in polyphenols is associated with a lower incidence of cardiovascular disease. The purpose of this study was to assess whether the application of red wine polyphenols influences the kinetic properties of renal Na+,K+‐ATPase in rats in which hypertension has been experimentally induced by the nitric oxide synthase inhibitor L‐NAME. Treatment with polyphenols during the recovery from hypertension to normotension resulted in the complete revival of the functional properties of the Na+,K+‐ATPase, as indicated by the total restoration of Km, KNa (concentration of Na+ necessary to achieve half‐maximal reaction velocity) and Vmax for enzyme activation by ATP and/or Na+ to pre‐hypertension values. Two positive effects of polyphenols during the recovery period are indicated: a restoration of the affinity of the ATP and Na+ binding sites to control values and a probable increase in the number of Na+,K+‐ATPase molecules to a level comparable to that in control conditions, as suggested by the complete renewal of Vmax.
Effects of Chronic Systemic Hypoxia on Contraction Evoked by Noradrenaline in the Rat Iliac Artery
Authors: Bartlett I. S. , Marshall Janice M.
Published: June 2003

Comparisons were made between responses evoked by noradrenaline (NA) in iliac artery rings from normoxic (N) rats and chronically hypoxic (CH) rats kept in 12% O2 for 3‐4 weeks. At PO2 of 100 mmHg, cumulative concentration‐response curves (CCRC) to NA were greatly depressed in endothelium‐intact (E+) rings, but not endothelium‐denuded (E‐) rings, of CH rats relative to N rats. However, CCRCs evoked by NA in E+ and E‐ rings during nitric oxide (NO) synthase inhibition were similar in N and CH rats. Reducing PO2 to 55 mmHg depressed CCRCs to NA in E+ and E‐ rings of CH and N rats in the absence and presence of NO synthase inhibition. At PO2 of 100 mmHg, CCRCs evoked by phenylephrine were comparable in E+ and E‐ rings of N and CH rats as were CCRCs for the relaxation evoked by isoprenaline, which were similarly rightward shifted by NO synthase inhibition. However, CCRCs evoked by the NO donor sodium nitroprusside were leftward shifted in E‐ rings of CH rats relative to N rats. Further, in the presence of the α2 adrenoceptor inhibitor rauwolscine, CCRCs to NA were comparable in E+ rings of CH and N rats. Thus, the depressive effects of chronic hypoxia on NA‐evoked contractions of iliac artery are additional to those of acute hypoxia. We propose that they reflect a facilitation of the contribution of NO to α2 adrenoceptor‐evoked relaxation that includes an increased sensitivity of the vascular smooth muscle of arteries from CH rats to NO.
Leukocyte Coping Capacity: A Novel Technique for Measuring the Stress Response in Vertebrates
Authors: McLaren G. W. , Macdonald D. W. , Georgiou C. , Mathews F. , Newman C. , Mian R.
Published: June 2003

Methods used to quantify the stress response in animals are vital tools in many areas of biology. Here we describe a new method of measuring the stress response, which provides rapid results and can be used in the field or laboratory. After a stressful event, we measure the capacity of circulating leukocytes to produce a respiratory burst in vitro in response to challenge by phorbol myristate acetate (PMA). During the respiratory burst leukocytes produce oxygen free radicals, and the level of production can be measured directly as chemiluminescence. When in vitro PMA‐stimulated whole blood chemiluminescence is measured directly after a stressful event, we define the response as the leukocyte coping capacity (LCC). In an experiment badgers (Meles meles), which were caught as part of an on‐going population study, were either transported to a central site prior to blood sampling or blood was collected at their site of capture. Transported animals had a significantly lower LCC and showed changes in leukocyte composition that were indicative of stress. We conclude that the stress of transport reduced LCC in badgers and that LCC serves as a quantitative measure of stress. Potential applications of this method are discussed.
Effect of Endothelin Antagonists on the Renal Haemodynamic and Tubular Responses to Ischaemia‐Reperfusion Injury in Anaesthetised Rats
Authors: Ajis Adam , Bagnall Nigel M. , Collis Michael G. , Johns Edward J.
Published: June 2003

In this investigation we have evaluated whether blockade of endothelin receptors influenced the renal haemodynamic and excretory responses to a period of ischaemia and reperfusion in the anaesthetised rat. The renal artery was occluded for 30 min and renal haemodynamic and excretory function followed for 90 min of reperfusion while either saline, the non‐selective endothelin 1 receptor (ETA/ETB) antagonist SB209670 or the selective ETA receptor antagonist UK‐350,926 was infused. In the post‐ischaemic period, renal cortical and medullary perfusions were reduced by 40‐50%. When SB209670 was administered (30 μg kg−1 min−1 I.V.) for 30 min before, during and for 90 min after renal artery occlusion, cortical and medullary perfusions returned to baseline levels, responses different from those obtained during saline infusion (both P < 0.05). In the presence of UK‐350,926 (30 μg kg−1 min−1 I.V.), perfusion in the medulla returned to baseline on clamp removal whereas that in the cortex remained depressed (P < 0.05). Renal ischaemia for 30 min decreased glomerular filtration rate during reperfusion and increased urine flow and sodium excretion 5‐ to 15‐fold. UK‐350,926 (30 μg kg−1 min−1 I.V.) reduced (P < 0.05) fluid excretion prior to ischaemia but during reperfusion, glomerular filtration rate returned to basal levels and there were progressive increases in fluid excretion which were smaller compared to the saline‐treated group (all P < 0.05). The ischaemic challenge may cause release of endothelin, which acts on ETB receptors in the cortex and ETA receptors in the medulla to decrease perfusion. The blunted natriuresis and diuresis during blockade of ETA receptors may result from either a vascular or tubular action of endothelin.
Hindquarters Vasoconstriction Through Central GABAB Receptors in Conscious Rats
Authors: Takemoto Yumi
Published: June 2003

The exogenous application of GABA into the cisterna magna of the freely moving rat decreases hindquarters vascular tone as well as arterial pressure. GABA could influence GABA receptor subtypes A, B or C. However, the hindquarters vascular response to the stimulation of each receptor subtype has not yet been investigated. The present study therefore characterized the response to the GABAB receptor agonist baclofen injected into the cisterna magna of the conscious rat. Intracisternally injected baclofen induced long‐lasting increases in hindquarters vascular resistance and arterial pressure in a dose‐dependent manner. Both actions induced by baclofen were completely blocked by a prior intracisternal injection with the GABAB receptor antagonist CGP 35348 (p‐[3‐aminopropyl]‐p‐diethoxymethylphosphinic acid), and systemically by ganglionic blockade. These actions of baclofen were also abolished centrally by sodium pentobarbital anaesthesia. The results suggest that GABAB receptor stimulation via the cisterna magna induced hindquarters vasoconstriction, probably due to central disinhibition of sympathetic activity.