bluuebunny/crossref_metadata_2025_split huggingface dataset:
Kinetic and crystallographic studies on 2‐(β‐D‐glucopyranosyl)‐5‐methyl‐1, 3, 4‐oxadiazole, ‐benzothiazole, and ‐benzimidazole, inhibitors of muscle glycogen phosphorylase b. Evidence for a new binding site
Authors: Chrysina Evangelia D. , Kosmopoulou Magda N. , Tiraidis Constantinos , Kardakaris Rozina , Bischler Nicolas , Leonidas Demetres D. , Hadady Zsuzsa , Somsak Laszlo , Docsa Tibor , Gergely Pal , Oikonomakos Nikos G. 
Published: March 2005

AbstractIn an attempt to identify leads that would enable the design of inhibitors with enhanced affinity for glycogen phosphorylase (GP), that might control hyperglycaemia in type 2 diabetes, three new analogs of β‐D‐glucopyranose, 2‐(β‐D‐glucopyranosyl)‐5‐methyl‐1, 3, 4‐oxadiazole, ‐benzothiazole, and ‐benzimidazole were assessed for their potency to inhibit GPb activity. The compounds showed competitive inhibition (with respect to substrate Glc‐1‐P) with Ki values of 145.2 (±11.6), 76 (±4.8), and 8.6 (±0.7) μM, respectively. In order to establish the mechanism of this inhibition, crystallographic studies were carried out and the structures of GPb in complex with the three analogs were determined at high resolution (GPb‐methyl‐oxadiazole complex, 1.92 Å; GPb‐benzothiazole, 2.10 Å; GPb‐benzimidazole, 1.93 Å). The complex structures revealed that the inhibitors can be accommodated in the catalytic site of T‐state GPb with very little change of the tertiary structure, and provide a rationalization for understanding variations in potency of the inhibitors. In addition, benzimidazole bound at the new allosteric inhibitor or indole binding site, located at the subunit interface, in the region of the central cavity, and also at a novel binding site, located at the protein surface, far removed (∼ 32 Å) from the other binding sites, that is mostly dominated by the nonpolar groups of Phe202, Tyr203, Val221, and Phe252.
The monomer–dimer equilibrium of stromal cell‐derived factor‐1 (CXCL 12) is altered by pH, phosphate, sulfate, and heparin
Authors: Veldkamp Christopher T. , Peterson Francis C. , Pelzek Adam J. , Volkman Brian F. 
Published: March 2005

AbstractChemokines, like stromal cell‐derived factor‐1 (SDF1/CXCL12), are small secreted proteins that signal cells to migrate. Because SDF1 and its receptor CXCR4 play important roles in embryonic development, cancer metastasis, and HIV/AIDS, this chemokine signaling system is the subject of intense study. However, it is not known whether the monomeric or dimeric structure of SDF1 is responsible for signaling in vivo. Previous structural studies portrayed the SDF1 structure as either strictly monomeric in solution or dimeric when crystallized. Here, we report two‐dimensional NMR, pulsed‐field gradient diffusion and fluorescence polarization measurements at various SDF1 concentrations, solution conditions, and pH. These results demonstrate that SDF1 can form a dimeric structure in solution, but only at nonacidic pH when stabilizing counterions are present. Thus, while the previous NMR structural studies were performed under acidic conditions that strongly promote the monomeric state, crystallographic studies used nonacidic buffer conditions that included divalent anions shown here to promote dimerization. This pH‐sensitive aggregation behavior is explained by a dense cluster of positively charged residues at the SDF1 dimer interface that includes a histidine side chain at its center. A heparin disaccharide shifts the SDF1 monomer–dimer equilibrium in the same manner as other stabilizing anions, suggesting that glycosaminoglycan binding may be coupled to SDF1 dimerization in vivo.
Structural basis of the temperature transition of Pf1 bacteriophage
Authors: Thiriot David S. , Nevzorov Alexander A. , Opella Stanley J. 
Published: March 2005

AbstractThe filamentous bacteriophage Pf1 undergoes a reversible temperature‐dependent transition that is also influenced by salt concentrations. This structural responsiveness may be a manifestation of the important biological property of flexibility, which is necessary for long, thin filamentous assemblies as a protection against shear forces. To investigate structural changes in the major coat protein, one‐ and two‐dimensional solid‐state NMR spectra of concentrated solutions of Pf1 bacteriophage were acquired, and the structure of the coat protein determined at 0°C was compared with the structure previously determined at 30°C. Despite dramatic differences in the NMR spectra, the overall change in the coat protein structure is small. Changes in the orientation of the C‐terminal helical segment and the conformation of the first five residues at the N‐terminus are apparent. These results are consistent with prior studies by X‐ray fiber diffraction and other biophysical methods.
Distinguishing multiple chemotaxis Y protein conformations with laser‐polarized 129Xe NMR
Authors: Lowery Thomas J. , Doucleff Michaeleen , Ruiz E. Janette , Rubin Seth M. , Pines Alexander , Wemmer David E. 
Published: March 2005

AbstractThe chemical shift of the 129Xe NMR signal has been shown to be extremely sensitive to the local environment around the atom and has been used to follow processes such as ligand binding by bacterial periplasmic binding proteins. Here we show that the 129Xe shift can sense more subtle changes: magnesium binding, BeF3− activation, and peptide binding by the Escherichia coli chemotaxis Y protein. 1H‐15N correlation spectroscopy and X‐ray crystallography were used to identify two xenon‐binding cavities in CheY that are primarily responsible for the shift changes. One site is near the active site, and the other is near the peptide binding site.
NMR characterization of the Escherichia coli nitrogen regulatory protein IIANtr in solution and interaction with its partner protein, NPr
Authors: Wang Guangshun , Peterkofsky Alan , Keifer Paul A. , Li Xia 
Published: March 2005

AbstractThe solution form of IIANtr from Escherichia coli and its interaction with its partner protein, NPr, were characterized by nuclear magnetic resonance (NMR) spectroscopy. The diffusion coefficient of the protein (1.13 × 10−6 cm/sec) falls between that of HPr (′9 kDa) and the N‐terminal domain of E. coli enzyme I (∼30 kDa), indicating that the functional form of IIANtr is a monomer (∼18 kDa) in solution. Thus, the dimeric structure of the protein found in the crystal is an artifact of crystal packing. The residual dipolar coupling data of IIANtr (covering residues 11–155) measured in the absence and presence of a 4% polyethyleneglycol‐hexanol liquid crystal alignment medium fit well to the coordinates of both molecule A and molecule B of the dimeric crystal structure, indicating that the 3D structures in solution and in the crystal are indeed similar for that protein region. However, only molecule A possesses an N‐terminal helix identical to that derived from chemical shifts of IIANtr in solution. Further, the 15N heteronuclear nuclear Overhauser effect (NOE) data also support molecule A as the representative structure in solution, with the terminal residues 1–8 and 158–163 more mobile. Chemical shift mapping identified the surface on IIANtr for NPr binding. Residues Gly61, Asp115, Ser125, Thr156, and nearby regions of IIANtr are more perturbed and participate in interaction with NPr. The active‐site His73 of IIANtr for phosphoryl transfer was found in the Nδ1‐H tautomeric state. This work lays the foundation for future structure and function studies of the signal transducing proteins from this nitrogen pathway.
Bcl‐XL as a fusion protein for the high‐level expression of membrane‐associated proteins
Authors: Thai Khang , Choi Jungyuen , Franzin Carla M. , Marassi Francesca M. 
Published: March 2005

AbstractAn Escherichia coli plasmid vector for the high‐level expression of hydrophobic membrane proteins is described. The plasmid, pBCL, directs the expression of a target polypeptide fused to the C terminus of a mutant form of the anti‐apoptotic Bcl‐2 family protein, Bcl‐XL, where the hydrophobic C terminus has been deleted, and Met residues have been mutated to Leu to facilitate CNBr cleavage after a single Met inserted at the beginning of the target sequence. Fusion protein expression is in inclusion bodies, simplifying the protein purification steps. Here we report the high‐level production of PLM, a membrane protein that is a member of the FXYD family of tissue‐specific and physiological‐state–specific auxiliary subunits of the Na,K‐ATPase, expressed abundantly in heart and skeletal muscle. We demonstrate that milligram quantities of pure, isotopically labeled protein can be obtained easily and in little time with this system.
Solution structure of At3g04780.1‐des15, an Arabidopsis thaliana ortholog of the C‐terminal domain of human thioredoxin‐like protein
Authors: Song Jikui , Tyler Robert C. , Wrobel Russell L. , Frederick Ronnie O. , Vojtek Frank C. , Jeon Won Bae , Lee Min S. , Markley John L. 
Published: March 2005

AbstractThe structure of At3g04780.1‐des15, an Arabidopsis thaliana ortholog of the C‐terminal domain of human thioredoxin‐like protein, was determined by NMR spectroscopy. The structure is dominated by a β‐barrel sandwich. A two‐stranded anti‐parallel β‐sheet, which seals off one end of the β‐barrel, is flanked by two flexible loops rich in acidic amino acids. Although this fold often provides a ligand binding site, the structure did not reveal an appreciable cavity inside the β‐barrel. The three‐dimensional structure of At3g04780.1‐des15 provides an entry point for understanding its functional role and those of its mammalian homologs.
Helix periodicity, topology, and dynamics of membrane‐associated α‐Synuclein
Authors: Bussell Robert , Ramlall Trudy Fiona , Eliezer David 
Published: March 2005

AbstractThe protein α‐Synuclein (aS) is a synaptic vesicle‐associated regulator of synaptic strength and dopamine homeostasis with a pathological role in Parkinson's disease. The normal function of aS depends on a membrane‐associated conformation that is adopted upon binding to negatively charged lipid surfaces. Previously we found that the membrane‐binding domain of aS is helical and suggested that it may exhibit an unusual structural periodicity. Here we present a study of the periodicity, topology, and dynamics of detergent micelle‐bound aS using paramagnetic spin labels embedded in the micelle or attached to the protein. We show that the helical region of aS completes three full turns every 11 residues, demonstrating the proposed 11/3 periodicity. We also find that the membrane‐binding domain is partially buried in the micelle surface and bends toward the hydrophobic interior, but does not traverse the micelle. Deeper submersion of certain regions within the micelle, including the unique lysine‐free sixth 11‐residue repeat, is observed and may be functionally important. There are no long‐range tertiary contacts within this domain, indicating a highly extended configuration. The backbone dynamics of the micelle‐bound region are relatively uniform with a slight decrease in flexibility observed toward the C‐terminal end. These results clarify the topological features of aS bound to membrane‐mimicking detergent micelles, with implications for aS function and pathology.
Dioxane contributes to the altered conformation and oligomerization state of a designed engrailed homeodomain variant
Authors: Hom Geoffrey K. , Lassila J. Kyle , Thomas Leonard M. , Mayo Stephen L. 
Published: March 2005

AbstractOur goal was to compute a stable, full‐sequence design of the Drosophila melanogaster engrailed homeodomain. Thermal and chemical denaturation data indicated the design was significantly more stable than was the wild‐type protein. The data were also nearly identical to those for a similar, later full‐sequence design, which was shown by NMR to adopt the homeodomain fold: a three‐helix, globular monomer. However, a 1.65 Å crystal structure of the design described here turned out to be of a completely different fold: a four‐helix, rodlike tetramer. The crystallization conditions included ∼25% dioxane, and subsequent experiments by circular dichroism and sedimentation velocity analytical ultracentrifugation indicated that dioxane increases the helicity and oligomerization state of the designed protein. We attribute at least part of the discrepancy between the target fold and the crystal structure to the presence of a high concentration of dioxane.
Effect of 2 weeks of endurance training on uncoupling protein 3 content in untrained human subjects
Authors: Schrauwen P. , Russell A. P. , Moonen‐Kornips E. , Boon N. , Hesselink M. K. C. 
Published: March 2005

AbstractAim:  The mitochondrial uncoupling protein‐3 (UCP3) is able to lower the proton gradient across the inner mitochondrial membrane, thereby uncoupling substrate oxidation from ATP production and dissipating energy as heat. What the effect of endurance training on UCP3 is, is still controversial. Endurance‐trained athletes are characterized by lower levels of UCP3, but longitudinal studies in rodents reported no effect of endurance training on muscular UCP3 levels. Here, we examined the effect of a 2‐week training programme on skeletal muscle UCP3 protein content in untrained human subjects, and hypothesized that UCP3 will be reduced after the training programme.Methods:  Nine untrained men [age: 23.3 ± 3.2 years; BMI: 22.6 ± 2.6 kg m−2; maximal power output (Wmax): 3.8 ± 0.6 W kg−1 body weight] trained for 2 weeks. Before and at least 72 h after the training period, muscle biopsies were taken for determination of UCP3 protein content.Results:  UCP3 protein content tended to be lower after the training programme [95 ± 10 vs. 109 ± 12 arbitrary units (AU), P = 0.08]. Cytochrome c content tended to increase with 33% in response to endurance training (52 ± 6 vs. 39 ± 6 AU, P = 0.08). The ratio UCP3 relative to cytochrome c tended to decrease significantly upon endurance training (2.0 ± 0.4 vs. 3.2 ± 0.6 AU, P = 0.01).Conclusion:  A short‐term (2‐week) endurance training programme decreased UCP3 protein levels and significantly reduced the ratio of UCP3 to cytochrome c.
Role of Na+,K+ pumps in restoring contractility following loss of cell membrane integrity in rat skeletal muscle
Authors: Clausen T. , Gissel H. 
Published: March 2005

AbstractBackground and aim:  In skeletal muscles, electrical shocks may elicit acute loss of force, possibly related to increased plasma membrane permeability, induced by electroporation (EP). We explore the role of the Na+,K+ pumps in force recovery after EP.Methods:  Isolated rat soleus or extensor digitorum longus (EDL) muscles were exposed to EP paradigms in the range 100–800 V cm−1, and changes in tetanic force, Na+,K+ contents, membrane potential, 14C‐sucrose space and the release of the intracellular enzyme lactic acid dehydrogenase (LDH) were characterized. The effects of Na+,K+ pump stimulation or inhibition were followed.Results:  Electroporation caused voltage‐dependent loss of force, followed by varying rates and degrees of recovery. EP induced a reversible loss of K+ and gain of Na+, which was not suppressed by tetrodotoxin, but associated with increased 14C‐sucrose space and release of LDH. In soleus, EP at 500 V cm−1 induced complete loss of force, followed by a spontaneous, partial recovery. Stimulation of active Na+,K+ transport by adrenaline, the β2‐agonist salbutamol, calcitonin gene‐related peptide (CGRP) and dibutyryl cyclic AMP increased initial rate of force recovery by 183–433% and steady‐state force level by 104–143%. These effects were blocked by ouabain (10−3 m), which also completely suppressed spontaneous force recovery. EP caused rapid and marked depolarization, followed by a repolarization, which was accelerated by salbutamol. Also in EDL, EP caused complete loss of force, followed by a spontaneous partial recovery, which was markedly stimulated by salbutamol.Conclusion:  Electroporation induces reversible depolarization, partial rundown of Na+,K+ gradients, cell membrane leakage and loss of force. This may explain the paralysis elicited by electrical shocks. Na+,K+ pump stimulation promotes restoration of contractility, possibly via its electrogenic action. The major new information is that the Na+,K+ pumps are sufficient to compensate a simple mechanical leakage. This may be important for force recovery in leaky muscle fibres.
Neuronal nitric oxide synthase and splanchnic blood flow in anaesthetized rats
Authors: Jansson L. , Carlsson P.‐O. , Bodin B. , Andersson A. , Källskog Ö. 
Published: March 2005

AbstractAims:  To evaluate to what extent the neuronal form of constitutive nitric oxide synthase (nNOS) contributes to the blood perfusion of splanchnic organs, including the islets of Langerhans.Methods:  The nNOS inhibitor 7‐nitroindazole (300 mg kg−1 i.p.) was administered to anaesthetized Sprague–Dawley rats, some of which were pre‐treated with the ganglionic blocker hexamethonium (20 mg kg−1 i.v.) The blood perfusion of the splanchnic organs, including the pancreatic islets was then measured with a microsphere technique.Results:  Nitroindazole decreased total pancreatic, duodenal and renal blood flow, whereas pancreatic islet, colonic and adrenal blood flows were unchanged. A slight increase in mean arterial blood pressure was seen after nitroindazole treatment. Nitroindazole did not affect blood glucose or serum insulin concentrations. In separate experiments, hexamethonium affected none of the studied blood flow values, suggesting that the effects of nNOS‐inhibition were not mediated from the nervous system.Conclusion:  Nitric oxide derived from the activity of nNOS contributes to the blood perfusion in the upper portions of the gastrointestinal tract, viz. the parts supplied by the cranial mesenteric artery, and the kidneys, whilst no effects are seen on colonic or adrenal blood flow. Pancreatic islet blood flow was unaffected by nNOS inhibition, thereby suggesting that NO derived from the other isoforms of NOS maintains the high basal islet blood perfusion.
The effects of different air velocities on heat storage and body temperature in humans cycling in a hot, humid environment
Authors: Saunders A. G. , Dugas J. P. , Tucker R. , Lambert M. I. , Noakes T. D. 
Published: March 2005

AbstractAim:  The purposes of this study were to determine (i) the effects of different facing air velocities on body temperature and heat storage during exercise in hot environmental conditions and (ii) the effects of ingesting fluids at two different rates on thermoregulation during exercise in hot conditions with higher air velocities.Methods:  On five occasions nine subjects cycled for 2 h at 33.0 ± 0.4 °C with a relative humidity of 59 ± 3%. Air velocity was maintained at 0.2 km h−1 (0 WS), 9.9 ± 0.3 km h−1 (10 WS), 33.3 ±2.2 km h−1 (100 WS) and 50.1 ± 3.2 km h−1 (150 WS) while subjects replaced 58.8 ± 6.8% of sweat losses. In the fifth condition, air velocity was maintained at 33.7 ± 2.2 km h−1 and subjects replaced 80.0 ± 6.8% of sweat losses (100.80 WS).Results:  Heat storage, body temperature and rating of perceived exertion were higher in 0 and 10 WS compared with all other conditions. There were no differences in any measured variable between 100 and 150 WS, or between 100 and 100.80 WS. Thus, the evaporative capacity of the environment is increased with higher air velocities, reducing heat storage and body temperature. At higher air velocities, a higher rate of fluid ingestion did not influence heat storage, body temperature or sweat rate.Conclusion:  The finding of previous laboratory studies showing a beneficial effect of high rates of fluid ingestion on thermoregulation during exercise in hot, humid, windstill conditions cannot be extrapolated to out‐of‐doors exercise in which facing air velocities are seldom lower than the athlete's rate of forward progression.
Importance of the renin–angiotensin system in the regulation of arterial blood pressure in conscious mice and rats
Authors: Cholewa B. C. , Meister C. J. , Mattson D. L. 
Published: March 2005

AbstractAim:  The present experiments were designed to determine the mechanism(s) for increased sensitivity to blockade of the renin–angiotensin system in mice in comparison with rats.Methods:  Mice and rats, with indwelling femoral arterial and venous catheters, were chronically administered angiotensin II or pharmacological inhibitors of the renin–angiotensin system as sodium intake was altered.Results:  Increasing sodium intake led to suppression of circulating renin, angiotensin II, and aldosterone in rats and mice in the absence of alterations in arterial blood pressure. Additional experiments demonstrated that continuous intravenous infusion of angiotensin II (20 ng kg−1 min−1) significantly increased arterial blood pressure by approximately 35 mmHg in conscious rats at all levels of sodium intake (n = 6). In contrast, arterial pressure was unaffected by angiotensin II infusion in conscious mice under conditions of low sodium intake, although arterial pressure was increased by 16 mmHg when mice were administered a high sodium intake while infused with angiotensin II (n = 6). In comparison, blockade of the endogenous renin–angiotensin system led to significantly greater effects on arterial pressure in mice than rats. Continuous infusion of captopril (30 μg kg−1 min−1) or losartan (100 μg kg−1 min−1) resulted in a 55–90% greater fall in blood pressure in conscious mice in comparison with conscious rats.Conclusion:  The present studies indicate that arterial pressure in mice is more dependent upon the endogenous renin–angiotensin system than it is in rats, but mice are more resistant to the hypertensive effects of exogenous angiotensin II.
Relationship between protein O‐linked glycosylation and insulin‐stimulated glucose transport in rat skeletal muscle following calorie restriction or exposure to O‐(2‐acetamido‐2‐deoxy‐d‐glucopyranosylidene)amino‐N‐phenylcarbamate
Authors: Arias E. B. , Cartee G. D. 
Published: March 2005

AbstractAims and Background:  Protein O‐linked glycosylation is regulated in vivo by the concentration of hexosamine substrates. Calorie restriction (60% of ad libitum intake) for 20 days causes decreased UDP‐N‐acetylhexosamine levels and increased insulin‐mediated glucose transport in rat skeletal muscle. Conversely, prolonged incubation (19 h) of muscle with O‐(2‐acetamido‐2‐deoxy‐d‐glucopyranosylidene)amino‐N‐phenyl‐carbamate (PUGNAc; an inhibitor of N‐acetyl‐β‐d‐glucosaminidase) is characterized by increased O‐linked glycosylation and insulin resistance. We aimed to determine the calorie restriction effect on O‐linked glycosylation and characterize the temporal relationship between PUGNAc‐induced O‐linked glycosylation and insulin resistance.Hypothesis:  A calorie restriction protocol characterized by decreased muscle hexosamine levels will result in a global reduction in O‐linked glycosylated proteins in muscle, and PUGNAc‐induced insulin resistance will coincide with increased O‐linked glycosylation.Methods:  Plantaris muscle and liver from rats (ad libitum or calorie restricted) were analysed for O‐linked glycosylation using two antibodies against different O‐linked N‐acetylglucosamine epitopes. Also, rat epitrochlearis muscles were incubated for 8.5 h ± 100 μm PUGNAc prior to measurement of [3H]‐3‐O‐methylglucose transport and O‐linked glycosylation.Results:  Calorie restriction did not alter protein O‐linked glycosylated levels in muscle or liver. Incubation with PUGNAc for 8.5 h resulted in increased in O‐linked glycosylation but unaltered basal or insulin‐stimulated glucose transport.Conclusions:  The delay between O‐linked glycosylation and insulin resistance in muscle incubated with PUGNAc suggests an indirect, relatively slow mechanism for insulin resistance. The effect of calorie restriction on insulin action in muscle is unlikely to be the direct result of a global change in protein O‐linked glycosylation.