bluuebunny/crossref_metadata_2025 huggingface dataset:
Deamidation and disulfide bridge formation in human calbindin D28k with effects on calcium binding
Authors: Vanbelle Christophe , Halgand Frédéric , Cedervall Tommy , Thulin Eva , Åkerfeldt Karin S. , Laprévote Olivier , Linse Sara
Published: March 2005

Calbindin D28k (calbindin) is a cytoplasmic protein expressed in the central nervous system, which is implied in Ca2+ homeostasis and enzyme regulation. A combination of biochemical methods and mass spectrometry has been used to identify post‐translational modifications of human calbindin. The protein was studied at 37°C or 50°C in the presence or absence of Ca2+. One deamidation site was identified at position 203 (Asn) under all conditions. Kinetic experiments show that deamidation of Asn 203 occurs at a rate of 0.023 h−1 at 50°C for Ca2+‐free calbindin. Deamidation is slower for the Ca2+‐saturated protein. The deamidation process leads to two Asp iso‐forms, regular Asp and iso‐Asp. The form with regular Asp 203 binds four Ca2+ ions with high affinity and positive cooperativity, i.e., in a very similar manner to non‐deamidated protein. The form with β‐aspartic acid (or iso‐Asp 203) has reduced affinity for two or three sites leading to sequential Ca2+ binding, i.e., the Ca2+‐binding properties are significantly perturbed. The status of the cysteine residues was also assessed. Under nonreducing conditions, cysteines 94 and 100 were found both in reduced and oxidized form, in the latter case in an intramolecular disulfide bond. In contrast, cysteines 187, 219, and 257 were not involved in any disulfide bonds. Both the reduced and oxidized forms of the protein bind four Ca2+ ions with high affinity in a parallel manner and with positive cooperativity.
Modulation of cooperativity in Mycobacterium tuberculosis NADPH‐ferredoxin reductase: Cation‐and pH‐induced alterations in native conformation and destabilization of the NADP+‐binding domain
Authors: Bhatt Anant Narayan , Shukla Nidhi , Aliverti Alessandro , Zanetti Giuliana , Bhakuni Vinod
Published: March 2005

FprA, a Mycobacterium tuberculosis NADPH‐ferredoxin reductase, consists of two structural domains, a FAD‐binding and a NADP‐binding domain, respectively. For the first time, we demonstrated that native FprA, on thermal treatment underwent partial denaturation with unfolding of only the FAD‐binding domain and release of the protein‐bound flavin. The NADP‐binding domain of this protein is highly resistant to denaturation under these conditions. However, the presence of either 150 mM NaCl or KCl or 10 μM MgCl2 or CaCl2 or slightly acidic pH of 6.0 resulted in a highly cooperative and complete thermal unfolding of the protein. Physicochemical investigations showed that the monovalent cations or low concentrations of divalent cations induced compaction of the protein conformation. However, divalent cations at higher concentrations resulted in FAD release leading to stabilization of an enzymatically inactive apoenzyme. Detailed thermal denaturation studies on the native protein and the isolated NADP‐binding domain showed that cations and pH 6.0 destabilized only the heat‐stable NADP‐binding domain. The experimental studies demonstrate that modulation of intramolecular ionic interactions induce significant conformational changes in the NADP‐binding domain of FprA, resulting in a substantial increase in the structural cooperativity of the whole molecule. The results presented in this paper are of importance as they demonstrate alterations in the native three‐dimensional structure of FprA and cooperativity in protein molecule on slight alteration of pH or modification of ionic interactions in protein.
Crystal structure of a novel polyisoprenoid‐binding protein from Thermus thermophilus HB8
Authors: Handa Noriko , Terada Takaho , Doi‐Katayama Yukiko , Hirota Hiroshi , Tame Jeremy R.H. , Park Sam‐Yong , Kuramitsu Seiki , Shirouzu Mikako , Yokoyama Shigeyuki
Published: March 2005

The isoprenoid quinones exist widely among prokaryotes and eukaryotes. They play essential roles in respiratory electron transport and in controlling oxidative stress and gene regulation. In the isoprenoid quinone biosynthetic pathway, polyprenyl pyrophosphates are used as isoprenoid side‐chain precursors. Here we report the crystal structure of a novel polyprenyl pyrophosphate binding protein, TT1927b, from Thermus thermophilus HB8, complexed with its ligand. This protein belongs to the YceI‐like family in the Pfam database, and its sequence homologs are present in a broad range of bacteria and archaea. The structure consists of an extended, eight‐stranded, antiparallel β‐barrel. In the hydrophobic pore of the barrel, the protein binds the polyisoprenoid chain by hydrophobic interactions. Its overall structure resembles the lipocalin fold, but there is no sequence homology between TT1927b and the lipocalin family of proteins.
The conformation of the pore region of the M2 proton channel depends on lipid bilayer environment
Authors: Duong‐Ly Krisna C. , Nanda Vikas , DeGrado William F. , Howard Kathleen P.
Published: March 2005

The M2 protein from influenza A virus is a 97‐amino‐acid protein with a single transmembrane helix that forms proton‐selective channels essential to virus function. The hydrophobic transmembrane domain of the M2 protein (M2TM) contains a sequence motif that mediates the formation of functional tetramers in membrane environments. A variety of structural models have previously been proposed which differ in the degree of helix tilt, with proposed tilts ranging from ∼15° to 38°. An important issue for understanding the structure of M2TM is the role of peptide–lipid interactions in the stabilization of the lipid bilayer bound tetramer. Here, we labeled the N terminus of M2TM with a nitroxide and studied the tetramer reconstituted into lipid bilayers of different thicknesses using EPR spectroscopy. Analyses of spectral changes provide evidence that the lipid bilayer does influence the conformation. The structural plasticity displayed by M2TM in response to membrane composition may be indicative of functional requirements for conformational change. The various structural models for M2TM proposed to date—each defined by a different set of criteria and in a different environment—might provide snapshots of the distinct conformational states sampled by the protein.
Misfolded forms of glyceraldehyde‐3‐phosphate dehydrogenase interact with GroEL and inhibit chaperonin‐assisted folding of the wild‐type enzyme
Authors: Polyakova Oxana V. , Roitel Olivier , Asryants Regina A. , Poliakov Alexei A. , Branlant Guy , Muronetz Vladimir I.
Published: March 2005

We studied the interaction of chaperonin GroEL with different misfolded forms of tetrameric phosphorylating glyceraldehyde‐3‐phosphate dehydrogenase (GAPDH): (1) GAPDH from rabbit muscles with all SH‐groups modified by 5,5′‐dithiobis(2‐nitrobenzoate); (2) O‐R‐type dimers of mutant GAPDH from Bacillus stearothermophilus with amino acid substitutions Y283V, D282G, and Y283V/W84F, and (3) O‐P‐type dimers of mutant GAPDH from B. stearothermophilus with amino acid substitutions Y46G/S48G and Y46G/R52G. It was shown that chemically modified GAPDH and the O‐R‐type mutant dimers bound to GroEL with 1:1 stoichiometry and dissociation constants Kd of 0.4 and 0.9 μM, respectively. A striking feature of the resulting complexes with GroEL was their stability in the presence of Mg‐ATP. Chemically modified GAPDH and the O‐R‐type mutant dimers inhibited GroEL‐assisted refolding of urea‐denatured wild‐type GAPDH from B. stearothermophilus but did not affect its spontaneous reactivation. In contrast to the O‐R‐dimers, the O‐P‐type mutant dimers neither bound nor affected GroEL‐assisted refolding of the wild‐type GAPDH. Thus, we suggest that interaction of GroEL with certain types of misfolded proteins can result in the formation of stable complexes and the impairment of chaperonin activity.
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

In 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

Chemokines, 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

The 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

The 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

The 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

An 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

The 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

The 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

Our 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

Aim:  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

Background 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

Aims:  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

Aim:  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

Aim:  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

Aims 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.
Changes in triceps surae muscle architecture with sarcopenia
Authors: Morse C. I. , Thom J. M. , Birch K. M. , Narici M. V.
Published: March 2005

Aim:  To investigate whether sarcopenia was evenly distributed among the three components of the triceps surae (TS) muscle group.

Methods:  Muscle volume (VOL), fibre fascicle length (Lf), pennation angle (θ) and physiological cross‐sectional area (PCSA = VOL/Lf) were measured in vivo for the lateral (GL) and medial (GM) heads of the gastrocnemius muscles and for the soleus muscle (SOL), in 15 young males (YM, aged 25.3 ± 4.5 years) and 12 elderly males (EM aged 73.8 ± 4.4 years).

Results:  In the EM, VOL of all three muscles was significantly smaller than in the YM; differences were: 27% for the GL (P < 0.01), 29% for the GM (P < 0.01) and 17% for the SOL (P < 0.05). In total, TS VOL was 22% smaller in EM than in YM (P < 0.01). In the EM, values of θ were significantly smaller than in the YM; by 15–18% for the GL, GM and SOL (P < 0.05). In the EM, Lf of the GM was 16% smaller than in the YM (P < 0.01); no significant differences were found in the other muscles. PCSA of the GL and GM were both found to be smaller in EM by 19% (P < 0.01) and 14.5% (P < 0.05), respectively. No difference was observed in the SOL PCSA between YM and EM. Interestingly, probably because of the prevalent contribution of the SOL to PCSA distribution of each muscle to the TS PCSA, the relative TS PCSA was not different between YM and EM. Furthermore, the Lf/muscle length ratio did not differ between YM and EM.

Conclusion:  The present study shows that the relative PCSA composition of the TS is maintained with ageing and that the PCSA is scaled down harmonically with the decrease in muscle volume and fascicle length. Such observation suggests that the relative contribution of the components of the TS muscle to the total force developed by this muscle group is maintained with ageing.
Acute hyperoxaemia‐induced effects on regional blood flow, oxygen consumption and central circulation in man
Authors: Rousseau A. , Bak Z. , Janerot‐Sjöberg B. , Sjöberg F.
Published: March 2005

Aim:  Despite numerous in vitro and animal studies, circulatory effects and mechanisms responsible for the vasoconstriction seen during hyperoxaemia are yet to be ascertained. The present study set out to: (i) set up a non‐invasive human model for the study of hyperoxia‐induced cardiovascular effects, (ii) describe the dynamics of this effect and (iii) determine whether hyperoxaemia also, by vasoconstriction alters oxygen consumption (O2).

Methods:  The study comprised four experiments (A, B, C and D) on healthy volunteers examined before, during and after 100% oxygen breathing. A: Blood flow (mL min−1·100 mL−1 tissue), venous occlusion plethysmography was assessed (n = 12). B: Blood flow was recorded with increasing transcutaneous oxygen tension (PtcO2) levels (dose–response) (n = 8). C: Heart rate (HR), stroke volume, cardiac output (CO) and systemic vascular resistance (SVR) was assessed using echocardiography (n = 8). D: O2 was measured using an open circuit technique when breathing an air‐O2 mix (fraction of inhaled oxygen: FiO2 = 0.58) (n = 8).

Results:  Calf blood flow decreased 30% during O2 breathing. The decrease in calf blood flow was found to be oxygen dose dependent. A similar magnitude, as for the peripheral circulation, of the effect on central parameters (HR/CO and SVR) and in the time relationship was noted. Hyperoxia did not change O2. An average of 207 (93) mL O2 per subject was washed in during the experiments.

Conclusion:  This model appears suitable for the investigation of O2‐related effects on the central and peripheral circulation in man. Our findings, based on a more comprehensive (central/peripheral circulation examination) evaluation than earlier made, suggest significant circulatory effects of hyperoxia. Further studies are warranted to elucidate the underlying mechanisms.
Short‐term endurance training results in a muscle‐specific decrease of myostatin mRNA content in the rat
Authors: Matsakas A. , Friedel A. , Hertrampf T. , Diel P.
Published: March 2005

Aim:  Myostatin has been characterized as a negative regulator of skeletal muscle growth. To examine a probable function of myostatin during the adaptation of skeletal muscle in response to training, we analysed the effect of short‐term endurance training on myostatin and insulin‐like growth factor‐I (IGF‐I) mRNA contents in rat skeletal muscles. To assess the impact of the training stimulus, mRNA levels of metabolic genes were analysed simultaneously.

Methods:  Male Wistar rats were trained for 5 days by swimming, while another group remained untrained. Myostatin, IGF‐I, glucose transporter 4 (GLUT4), hexokinase II (HK II) and hydroxyacyl‐CoA dehydrogenase (HAD) mRNA levels were determined by real‐time reverse transcriptase‐polymerase chain reaction (RT‐PCR) in gastrocnemius, vastus lateralis and soleus muscles. A time course experiment was conducted, in order to examine transient changes of myostatin mRNA contents in gastrocnemius 7 and 24 h after one‐swimming session as well as 24 h after a 3‐day swimming training.

Results:  No significant changes in IGF‐I and GLUT4 mRNA levels were found in any of the muscles analysed. mRNA contents of myostatin were significantly reduced in gastrocnemius and vastus lateralis but not in soleus. In agreement to this pattern, we found significantly higher mRNA levels of HK II and HAD in the trained group. The time course experiment revealed significantly reduced myostatin mRNA contents in gastrocnemius 7 but not 24 h post‐exercise. The 3‐day swimming training resulted also in significantly lower myostatin mRNA levels in the trained group.

Conclusion:  This study demonstrated that short‐term endurance training may modulate myostatin mRNA levels, implying a probable role of myostatin in remodelling of skeletal muscle in response to training.
Solute recycling by crops and leaching in a drained arable soil
Authors: Kohler A. , Abbaspour K.C. , Fritsch M. , Schulin R.
Published: March 2005

Preferential flow, as it bypasses the soil matrix, can greatly enhance the leaching of chemicals. When a soil is drained there is the risk that such short‐circuiting results in more or less direct passage of polluting chemicals from the soil to the groundwater. If the groundwater table is shallow the chemicals could be transferred back into the surface soil by hydraulic lift through roots and subsequent release by exudation or from decaying plant residues and again become exposed to leaching by preferential flow, thus strongly enhancing the chance of export via the drains.

We investigated the leaching of bromide in a tile‐drained arable field over 2 years of crop rotation. The site was a former wetland, artificially drained a century ago for agriculture. Bromide was applied over 1.6 ha at a dosage of 10 g Br per m2 in August 1995 after the harvest of wheat. During the 2 years 18% of the applied bromide was exported via the drainage system, most of it in preferential flow events and more than half of it in a single winter storm 5 months after the application. Within 7 months 56% of the applied tracer was leached out of the main root zone into the groundwater. Subsequently the tracer re‐emerged in water taken up by sugar beet in the following season. The beet accumulated 50% of the initially applied bromide in their leaves and released it again after harvest when the leaves were left as green manure on the field. Our results show that this recycling of solutes to the topsoil can have an important influence on their leaching as the solutes are thus again exposed to preferential transport into drains in the course of preferential flow events.
Preferential phosphorus leaching from an irrigated grassland soil
Authors: Toor G. S. , Condron L. M. , Cade‐Menun B. J. , Di H. J. , Cameron K. C.
Published: March 2005

Intact lysimeters (50 cm diameter, 70 cm deep) of silt loam soil under permanent grassland were used to investigate preferential transport of phosphorus (P) by leaching immediately after application of dairy effluent. Four treatments that received mineral P fertilizer alone (superphosphate at 45 kg P ha−1 year−1) or in combination with effluent (at ∼ 40–80 kg P ha−1 year−1) over 2 years were monitored. Losses of total P from the combined P fertilizer and effluent treatments were 1.6–2.3 kg ha−1 (60% of overall loss) during eight drainage events following effluent application. The rest of the P lost (40% of overall loss) occurred during 43 drainage events following a significant rainfall or irrigation compared with 0.30 kg ha−1 from mineral P fertilizer alone. Reactive forms of P (mainly dissolved reactive P: 38–76%) were the dominant fractions in effluent compared with unreactive P forms (mainly particulate unreactive P: 15–56%). In contrast, in leachate following effluent application, particulate unreactive P was the major fraction (71–79%) compared with dissolved reactive P (1–7%). The results were corroborated by 31P nuclear magnetic resonance analysis, which showed that inorganic orthophosphate was the predominant P fraction present in the effluent (86%), while orthophosphate monoesters and diesters together comprised up to 88% of P in leachate. This shows that unreactive P forms were selectively transported through soil because of their greater mobility as monoesters (labile monoester P and inositol hexakisphosphate) and diesters. The short‐term strategies for reducing loss of P after application of dairy effluent application should involve increasing the residence time of applied effluent in the soil profile. This can be achieved by applying effluent frequently in small amounts.
Soil chemistry versus environmental controls on production of CH4 and CO2 in northern peatlands
Authors: Yavitt J. B. , Williams C. J. , Wieder R. K.
Published: March 2005

Rates of organic carbon mineralization (to CO2 and CH4) vary widely in peat soil. We transplanted four peat soils with different chemical composition into six sites with different environmental conditions to help resolve the debate about control of organic carbon mineralization by resource availability (e.g. carbon and nutrient chemistry) versus environmental conditions (e.g. temperature, moisture, pH). The four peat soils were derived from Sphagnum (bog moss). Two transplant sites were in mid‐boreal Alberta, Canada, two were in low‐boreal Ontario, Canada, and two were in the temperate United States. After 3 years in the field, CH4 production varied significantly as a function of peat type, transplant site, and the type–site interaction. All four peat soils had very small rates of CH4 production (< 20 nmol g−1 day−1) after transplant into two sites, presumably caused by acid site conditions (pH < 4.0). One peat soil had small CH4 production rates regardless of transplant site. A canonical discriminant analysis revealed that large rates of CH4 production (4000 nmol g−1 day−1) correlated with large holocellulose content, a large concentration of p‐hydroxyl phenolic compounds in the Klason lignin, and small concentrations of N, Ca and Mn in peat. Significant variation in rates of CO2 production correlated positively with holocellulose content and negatively with N concentrations, regardless of transplant site. The temperature response for CO2 production varied as a function of climate, being greater for peat formed in a cold climate, but did not apply to transplanted peat. Although we succeeded in elucidating some aspects of peat chemistry controlling production of CH4 and CO2 in Sphagnum‐derived peat soils, we also revealed idiosyncratic combinations of peat chemistry and site conditions that will complicate forecasting rates of peat carbon mineralization into the future.
Testing the Rothamsted Carbon Model against data from long‐term experiments on upland soils in Thailand
Authors: Shirato Y. , Paisancharoen K. , Sangtong P. , Nakviro C. , Yokozawa M. , Matsumoto N.
Published: March 2005

We tested the Rothamsted Carbon Model (RothC) against three long‐term (27–28 years) experimental sites on Thai upland soils in order to see how this widely used ‘temperate’ soil carbon turnover model performed in a typical farming region in the tropics. We were able to verify – over a much longer period than had been examined in previous studies – that RothC performs well in a tropical region in plots used for continuous cropping experiments of maize and cassava without organic matter application. However, the model overestimated soil organic carbon (SOC) in some plots to which large amounts of organic matter (rice straw or cassava stalks) were applied. This overestimate could not be attributed to errors in estimating either the amount of C input to the soil or the ratio of decomposable plant materials to resistant plant materials entering the soil. Among many factors affecting SOC dynamics (e.g. weather conditions, soil characteristics, etc.), which are different in tropical regions from temperate regions, we conclude that the activity of soil fauna might be a major factor which makes the performance of RothC worse where much organic matter was applied. We suggest that care should be taken when applying RothC to tropical soils with large amounts of added organic matter.
Dissolved organic carbon and decreasing pH mobilize cadmium and copper in soil
Authors: Strobel B. W. , Borggaard O. K. , Hansen H. C. B. , Andersen M. K. , Raulund‐Rasmussen K.
Published: March 2005

When limed farmland is converted to forestry cadmium (Cd), copper (Cu) and other heavy metals can become mobile because of acidification and increased concentration of dissolved humic substances. The influence of pH and dissolved organic C on amounts and rates of Cd and Cu release was investigated in a cultivated soil by extraction with ∼ 1 mmhydrochloric acid at pH 3 with and without dissolved organic C in the batch mode with weekly replacement of the extraction solution. After 88 weeks, 35–50% of aqua regia‐extractable Cd was extracted; addition of 10 mmdissolved organic C had no effect on the amount dissolved, but it increased the initial rate of release because the organic matter buffered the suspension at a lower pH. The solubility and release rate of Cd decreased as the geochemically active fraction was depleted. This suggests that Cd occurs in the soil in a continuum of binding strengths ranging from readily available to strongly bound forms. The repeated extractions resulted in distribution coefficients (Kd dis) that have log‐linear relationships with pH. This allows prediction of Cd solubility during acidification of soil. Dissolved organic C enhanced the release of Cu from less than 8% (without) to more than 20% (with) of aqua regia‐extractable Cu. Total contents of Cd and Cu cannot be used as measures of the metals' availability during acidification of former limed farmland. Predictions of availability should be based on the solubility as a function of pH and the degree of Cd or Cu depletion from the geochemically active fraction in soil.
Spatial and temporal variation in podzol organic matter studied by pyrolysis‐gas chromatography/mass spectrometry and micromorphology
Authors: Buurman P. , Van Bergen P. F. , Jongmans A. G. , Meijer E. L. , Duran B. , Van Lagen B.
Published: March 2005

A well‐developed podzol hydrosequence that has been partially covered with drift sand, and partially subjected to improved drainage, provides new insights into the causes of variation in soil organic matter chemistry in such soils. While E horizons invariably move towards a dominance of aliphatic components reflecting residual accumulation, the chemistry of organic matter in well‐drained B horizons is determined mainly by decaying roots, which are transformed by microorganisms to humus aggregates. In poorly drained, stratified B horizons, humus coatings dominate and the chemistry is very close to that of dissolved organic carbon. When a sand cover inhibits the supply of fresh litter, microbial decomposition in the A horizon causes a shift in chemistry towards that of the E horizon. Similarly, upon improved drainage and removal of complexed metals from the top of the B horizon, microbial decomposition of all palatable organic matter in the top of the B horizon causes a shift towards E‐horizon chemistry. This is probably the mechanism by which most E horizons in podzols are formed, and not by re‐solution. Marked chemical changes upon improved drainage may take only decades. During microbial decay, small polysaccharide‐derived pyrolysis products (mainly furans, furaldehydes and acetic acid) remain abundant due to the contribution of microbial sugars. Both micromorphology and factor analysis on quantified results of pyrolysis‐gas chromatography/mass spectrometry contribute significantly to the interpretation of the humus chemistry of these profiles and thus to our understanding of soil genesis. Organic chemistry of the investigated podzols can be understood only in the context of their genesis.