In hip osteoarthritis, Nordic Walking is superior to strength training and home-based exercise for improving function.

This observer-blinded, randomized controlled trial compared the short- and long-term effects of 4 months of supervised strength training (ST) in a local fitness center, supervised Nordic Walking (NW) in a local park, and unsupervised home-based exercise (HBE, control) on functional performance in 60+-year-old persons (n = 152) with hip osteoarthritis (OA) not awaiting hip replacement. Functional performance [i.e., 30-s chair stand test (primary outcome), timed stair climbing, and 6-min walk test] and self-reported outcomes (i.e., physical function, pain, physical activity level, self-efficacy, and health-related quality of life) were measured at baseline and at 2, 4, and 12 months. Based on intention-to-treat-analyses improvements [mean (95% CI)] after intervention in number of chair stands were equal in all three groups at 4 months [ST: 0.9 (0.2-1.6), NW: 1.9 (0.8-3.0), HBE: 1.1 (0.1-2.0)] but greater in the NW group [1.4 (0.02-2.8)] than in the ST group at 12 months. Generally, improvements in functional performance were greater (P < 0.001-P < 0.03) after NW compared with HBE and ST at all follow-up time points. Furthermore, NW was superior (P < 0.01) to HBE for improving vigorous physical activity and to both ST and HBE for improving (P < 0.01) mental health. These data suggest that NW is the recommended exercise modality compared with ST and HBE.

Pain and knee function in relation to degree of bone bruise after acute anterior cruciate ligament rupture.

It is unknown whether the bone bruise that occurs in connection with acute anterior cruciate ligament (ACL) rupture is causing pain and dysfunction. We followed prospectively 17 patients [10 men, seven women, mean age 28 years (range 23-34)] with acute ACL rupture for 2 months. A magnetic resonance imaging (MRI) scan was performed shortly after the injury, and at 2 weeks, 1 month and 2 months. The patients reported the level of pain every day and filled in a Knee injury and Osteoarthritis Outcome Score sheet in connection with MRI. For every MRI of the knee, volume of bone bruise was calculated, and intensity was visually graded. Our study showed a reduction of the pain to 50% approximately 2 weeks after the injury, at which time the bone bruise was at maximum. There was a significant relationship between pain and the volume and intensity of the bone bruise in the medial tibia condyle, as well as pain and the bone bruise volume of the lateral femoral condyle. Patients with bone bruise of the medial tibia and patients with meniscal lesions had more pain. It is suggested that pain and decreased function after acute ACL injury most likely is related to soft tissue and cartilage injury and not to bone bruise.

Profilin plays a role in cell elongation, cell shape maintenance, and flowering in Arabidopsis.

Profilin (PFN) is an ubiquitous, low-M(r), actin-binding protein involved in the organization of the cytoskeleton of eukaryotes including higher plants. PFNs are encoded by a multigene family in Arabidopsis. We have analyzed in vivo functions of Arabidopsis PFN by generating transgenic plants carrying a 35S-PFN-1 or 35S-antisense PFN-1 transgene. Etiolated seedlings underexpressing PFN (PFN-U) displayed an overall dwarf phenotype with short hypocotyls whose lengths were 20% to 25% that of wild type (WT) at low temperatures. Light-grown PFN-U plants were smaller in stature and flowered early. Compared with equivalent cells in WT, most cells in PFN-U hypocotyls and roots were shorter, but more isodiametric, and microscopic observations of etiolated PFN-U hypocotyls revealed a rough epidermal surface. In contrast, light-grown seedlings overexpressing PFN had longer roots and root hair although etiolated seedlings overexpressing PFN were either the same size or slightly longer than WT seedlings. Transgenic seedlings harboring a PFN-1-GUS transgene directed expression in root and root hair and in a ring of cells at the elongating zone of the root tip. As the seedlings matured PFN-1-GUS was mainly expressed in the vascular bundles of cotyledons and leaves. Our results show that Arabidopsis PFNs play a role in cell elongation, cell shape maintenance, polarized growth of root hair, and unexpectedly, in determination of flowering time.

Expression and characterization of recombinant Rhodocyclus tenuis high potential iron-sulfur protein.

The high potential iron-sulfur protein (HiPIP) from Rhodocyclus tenuis strain 2761 has been overproduced in Escherichia coli from its structural gene, purified to apparent homogeneity, and then characterized by an array of methods. UV-visible spectra of the reduced and oxidized recombinant protein were similar to those of the native protein. EPR of the oxidized protein shows g values of 2. 11, 2.03, and 2.03. ESI-MS gave a mass difference of 350 Da between the holoprotein and acid-treated protein, consistent with incorporation of a [Fe(4)S(4)] cluster in the holoprotein. The observed mass of the apoprotein was 6296.6 Da compared to the expected average molecular mass of 6297.2 Da of the apoprotein. The reduction potential was determined using cyclic and square-wave voltammetry to be 321 and 314 mV versus NHE, respectively. All the observed properties of the recombinant protein parallel those of the native protein or those of native HiPIPs in general, indicating correct folding and incorporation of the iron-sulfur cluster.

The crystal structure of a major allergen from plants.

BACKGROUND: Profilins are small eukaryotic proteins involved in modulating the assembly of actin microfilaments in the cytoplasm. They are able to bind both phosphatidylinositol-4,5-bisphosphate and poly-L-proline (PLP) and thus play a critical role in signaling pathways. Plant profilins are of interest because immunological cross-reactivity between pollen and human profilin may be the cause of hay fever and broad allergies to pollens. RESULTS: The determination of the Arabidopsis thaliana profilin isoform I structure, using multiwavelength anomalous diffraction (MAD) to obtain structure-factor phases, is reported here. The structure of Arabidopsis profilin is similar to that of previously determined profilin structures. Conserved amino acid residues in profilins from plants, mammals, and lower eukaryotes are critically important in dictating the geometry of the PLP-binding site and the overall polypeptide fold. The main feature distinguishing plant profilins from other profilins is a solvent-filled pocket located in the most variable region of the fold. CONCLUSIONS: Comparison of the structures of SH3 domains with those of profilins from three distinct sources suggests that the mode of PLP binding may be similar. A comparison of three profilin structures from different families reveals only partial conservation of the actin-binding surface. The proximity of the semi-conserved actin-binding site and the binding pocket characteristic of plant profilins suggests that epitopes encompassing both features are responsible for the cross-reactivity of antibodies between human and plant profilins thought to be responsible for type I allergies.

Arabidopsis profilins are functionally similar to yeast profilins: identification of a vascular bundle-specific profilin and a pollen-specific profilin.

Four members of the Arabidopsis profilin (pfn) multigene family have been cloned, sequenced and analyzed. By RNA gel blot analysis it has been shown that these four genes fall into two groups: one group (pfn1 and pfn2) is expressed in all organs of the plant and the other group (pfn3 and pfn4) in floral tissues only. Based on amino acid sequence alignment Arabidopsis profilins can be divided into the same two groups: PFN1 and PFN2 are 89% identical and PFN3 and PFN4 are 91% identical. Between these two groups they are 71-75% identical. The Arabidopsis profilins bind poly-L-proline and can complement both the Saccharomyces cerevisiae profilin deletion mutant and the Schizosaccharomyces pombe cdc3-124/profilin mutation, showing that the plant profilins are functionally similar to yeast profilins despite the low amino acid sequence homology. Analysis of pfn promoter-GUS fusion genes in transgenic Arabidopsis shows that pfn2 is specifically expressed in the vascular bundles of roots, hypocotyls, cotyledons, leaves, sepals, petals, stamen filaments and stalks of developing seeds, whereas expression of pfn4 is restricted to mature and germinating pollen grains.

Total synthesis of a miniferredoxin.

A miniferredoxin has been designed based on the Desulfovibrio gigas ferredoxin II structure and has been successfully synthesized. The 31 amino acid apoprotein was synthesized via standard Fmoc solid phase peptide synthesis and in vitro cluster insertion carried out. The UV-visible spectrum of the miniferredoxin (peak at 300 nm and a shoulder at 405 nm) shows the same features as that of the D. gigas ferredoxin. Cyclic voltammetry indicated a quasireversible electrode process with a midpoint potential of -370mV vs NHE, which demonstrates that the miniferredoxin is redox active. From these and EPR studies, we propose the incorporation of a Fe4S4 cluster.

Synthesis and characterization of Desulfovibrio gigas rubredoxin and rubredoxin fragments.

The 52-residue Desulfovibrio gigas rubredoxin peptide chain has been synthesized and a procedure for chain folding around iron(II) developed. The folded, stable synthetic rubredoxin can be subjected to purification, and reversibly oxidized and reduced. Ultraviolet/visible absorption and CD spectra of both forms show all the same features as native D. gigas rubredoxin, and the symmetric and asymmetric Fe-S stretching bands in the resonance Raman spectrum can be identified. In addition, the matrix-assisted laser desorption mass spectrum of a peptide sample exposed to trace amounts of iron is dominated by a peak at 5735Da very close to the value for the calculated molecular mass. Details in the ultraviolet/visible bandshape and mass spectrum, however, indicate remaining impurities. In comparison, a previously synthesized 25-residue rubredoxin fragment with the non-conserved positions 13-35 and 51-52 omitted and Val5-Glu50 anchored via glycine folds gives the correct molecular mass and ultraviolet/visible spectrum, but is much more labile than the 52-residue protein. This shows that non-conserved residues are crucial in protein folding and that chemical metalloprotein synthesis offers alternative prospects to microbiological protein engineering.

Cloning and characterisation of the gene encoding cytochrome c4 from Pseudomonas stutzeri.

By use of a degenerate oligodeoxyribonucleotide probe corresponding to the N-terminal amino acid (aa) sequence and Southern blot analysis, the gene encoding the di-heme cytochrome c4 (CC4) from Pseudomonas stutzeri has been cloned. The aa sequence deduced from the nucleotide sequence shows a 20-aa signal peptide and a 190-aa mature protein with 79% identity to the Azotobacter vinelandii CC4 sequence, which had earlier been determined by aa sequencing.

Redox potential and electrostatic effects in competitive inhibition of dual-path electron transfer reactions of spinach plastocyanin.

Redox inactive ions with high positive charges lower the rate constant for oxidation of several plant plastocyanins (PC) by small positively charged inorganic reaction partners. The rate constant decrease is commonly attributed to competitive inhibition where the redox inactive ions are bound to the negatively charged remote electron transfer (ET) site of PC and block this site sterically. We have investigated the effects of the inhibitor [NH3)5Co(NH2)Co(NH3)5]5+ on the ET reactions of spinach PC with [Co(phen)3]3+ (phen = 1,10-phenanthroline) and the electrically neutral analogue [Co(phen-SO3)3] (phen-SO3 = 5-sulfonato-1,10-phenanthroline) at the ionic strengths mu = 0.1 M and 0.03 M. Inhibition of the [Co(phen)3]3+ reactions is notably smaller for PC(II) reduction than for PC(I) oxidation. This is indicative of a redox potential increase of PC(II)/PC(I) on inhibitor attachment. The effect amounts to 16 mV at mu = 0.1 M and 31 mV at mu = 0.03 M. These data, and analysis in terms of ET theory show that inhibition cannot be caused solely by steric blocking. Driving force and interreactant electrostatic work terms are equally important. The PC(I)/[Co(phen-SO3)3] reaction exhibits a more entangled pattern. The rate constant first increases slightly with increasing inhibitor concentration, then drops, and approaches a constant value not far from the original value. This pattern is in line with association between the negatively charged -SO3- groups of the Co(III) complex and the inhibitor, and ET of the associate at both ET sites of PC.

Synthesis and characterization of a 25-residue rubredoxin(II)-like metalloprotein and its valine-leucine mutant.

An iron-sulfur metalloprotein containing the 5-12 and 35-50 residues of Desulfovibrio gigas rubredoxin has been synthesized by Fmoc solid phase peptide synthesis and subsequent peptide folding. A Gly links the two residue chains between Val-5 and Glu-50. Sybyl Tripos structure optimization indicates only minor structural changes of the folded synthetic protein compared to the similar residue positions in the native protein. The UV-VIS spectrum of the reduced synthetic protein is very similar to that of native D. gigas rubredoxin and the molecular mass determined by laser mass spectrometry has the expected value (+/- 2D). No metal is transferred to the gas phase by the laser beam merely by mixing the peptide and iron(II), substantiating that the folding procedure is a necessary pre-requisite for protein formation. The Val-->Leu41 chemical mutant has also been synthesized and behaves in a closely similar fashion.

Resonance effects in strongly exothermic long-range electron transfer and their possible implications for the behaviour of site-directed mutant proteins.

Long-range electron transfer investigations of hemoproteins, blue copper and iron-sulphur proteins frequently rest on electronically excited metal centres. When the excitation energy approaches the oxidation or reduction potentials of intermediate residues the superexchange view normally used, however, fails and a variety of new dynamic features arise. These all involve population of the intermediate cation or anion residue states which can be partially or wholly vibrationally relaxed. We discuss suitable views and a new theoretical formalism for these phenomena. We also note some important implications for site-directed mutagenesis in long-range, strongly exothermic electron transfer processes.

Effects of NO2-modification of Tyr83 on the reactivity of spinach plastocyanin with cytochrome f.

We have investigated the electron transfer (ET) reactions between turnip cytochrome f, and the native and NO2-Tyr83-modified forms of spinach plastocyanin (PCu) at 10.0 degrees C and ionic strength 0.200 M(NaCl), in both directions as a function of pH. The PCu(II)/cytochrome f(II) rate constants in the pH-range 4-6.8 reflect active and remote binding site protonation. At higher pH, NO2-Tyr83 and positively charged residues on cytochrome f are deprotonated, and both native and NO2-modified PCu exhibit a composite rate constant variation in this pH range. When framed by ET theory this pattern is fully understandable in terms of variations in reduction potentials and electrostatic interactions, caused by the protonation equilibria. The rate constant ratio knitro/knative is, however, only 1.04 for the PCu(II)/cytochrome f(II) reactions in spite of a 18 mV higher reduction potential for NO2-Tyr83-modified PCu. This is much lower than the value of 1.42 expected from ET theory solely on the basis of such a reduction potential effect. A similar effect is seen for PCu(I)/cytochrome f(III) for which the low-pH knitro/knative ratio is 0.51. Notable but smaller effects are also observed for the small reaction partners [Fe(CN)6]3-/4- and [Co(phen)3]3+/2+. The effect of NO2-modification in addition to the reduction potential effect can be resolved into a small reorganization energy increase around the copper atom and a smaller electronic transmission coefficient for ET through the Cu/Cys84/Tyr83 sequence. The former effect dominates in the reactions with the small reaction partners, while the electronic effects contribute significantly for PCu/cytochrome f, supporting the concept that the PCu/cytochrome f ET is at the remote PCu binding site.

Isolation and purification of plastocyanin from spinach stored frozen using hydrophobic interaction and ion-exchange chromatography.

A new simple three-day procedure for preparative isolation and purification of plastocyanin from spinach stored in the frozen state is described. This procedure is based on batch adsorption on ion-exchange resin, ammonium sulphate precipitation, and purification on a Phenyl-Sepharose hydrophobic interaction column and a single Q Sepharose High Performance ion-exchange column. Approximately 100 mg of plastocyanin with an absorbance ratio A278/A597 of 1.10±0.02 in the oxidized state was typically obtained from 12 kg of spinach leaves. The purified spinach plastocyanin is shown to be homogeneous to the resolution of free solution capillary electrophoresis.

Effects of NO2-modification of Tyr83 on the reactivity of spinach plastocyanin with inorganic redox partners [Fe(CN)6]3-/4- and [Co(phen)3]3+/2+.

Plastocyanin (PCu) from spinach leaves has been singly NO2-modified, purified by FPLC, and the position of modification at Tyr83 confirmed by trypsin digestion and amino-acid sequencing. Electron-transfer reactions of native and NO2-modified PCu with the inorganic redox partners [Fe(CN)6]3- and [Co(phen)3]3+, as oxidants for PCu(I), and [Fe(CN)6]4- and [Co(phen)3]2+ as reductants for PCu(II), have been studied as a function of pH. The acid dissociation constant for the phenolic group on NO2-Tyr83 PCu is 8.78 (average) for reduced, and 8.10 for oxidised protein, as compared to values greater than 10 for native protein. At I = 0.10 M (NaCl) NO2-modification brings about a 20 mV increase in reduction potential at pH less than 7 and deprotonation of the phenolic group a 20-25 mV decrease, both transmitted to and effective at the active site. Deprotonation brings about a 48% increase in rate for [Fe(CN)6]3- and a 47% decrease for [Fe(CN)6]4- in accordance with these changes. In the case of [Co(phen)3]3+, which reacts substantially at the remote site in the vicinity of Tyr83, the influence of deprotonation on the active site is supplemented by the negative charge of the phenolate, and a total increase of 131% is observed. These results can be understood on the basis of the electron-transfer theory, and add support to the belief that electron transfer kinetics of negatively and positively charged reactants are dominated by different sites on PCu for electron transfer, namely adjacent (close to His87) and remote (close to Tyr83), respectively.

A new procedure for fast isolation and purification of plastocyanin from the cyanobacterium Anabaena variabilis.

Methods are described for growing the cyanobacterium A. variabilis and for the isolation and purification of plastocyanin from the grown culture. Cell paste which had been stored at -35°C was suspended in 1 mM MES buffer, pH 6.5 and centrifuged. The supernatant was diluted to a conductivity of 0.12 mS, [Fe(CN)6](3-) added to a concentration of 0.5 mM and the solution loaded on a S Sepharose Fast Flow column. After elution and ultrafiltration, the plastocyanin containing fractions were reloaded on a S Sepharose Fast Flow column for final purification. A typical yield in three days from cells harvested from 3×20 l of medium was 32 mg plastocyanin with a minimum absorbance ratio A278/A597=1.14. This procedure is faster and the yield higher than for previous procedures.

Amyloid in osteoarthritic hip joints. Depositions in cartilage and capsule. Semiquantitative aspects.

Amyloid depositions in tissue from 116 osteoarthritic hip joints were examined. There was no significant correlation between amyloid and age although there was a tendency for joints from older patients to have more marked amyloid degeneration. We found significantly more amyloid in the joint capsules from male patients than from females. No difference in amyloid deposition was found between the right and left side, and pressure-loaded/less pressure-loaded parts of the femoral head contained equal amounts of amyloid. With the exception of two cases amyloid depositions in the joint capsule were always accompanied by amyloid in the joint cartilage (P less than 0.001). Conversely the cartilage was often positive when the capsule was negative for amyloid.

Amyloid deposits in calcified aortic valves.

Thirty-nine severely calcified aortic valves surgically removed were studied for amyloid deposits using the alkaline Congo red stain. Amyloid deposits were found in all the valves, in such quantities that they could be demonstrated using screening magnification (x40). A close topographic relationship was found between amyloid and calcium deposits. In addition, histological evidence was found of prolonged fibroblast proliferation. In the light of more recent studies, which identify Congophilic cytofilaments in fibroblasts, as well as demonstrating fibroblast degeneration and decomposition in senile aortic valves, the following pathogenesis is suggested for "calcific aortic stenosis": Mechanical injury of malformed aortic valves leading to fibroblast proliferation; fibroblast degeneration and decomposition with extracellular accumulation of cellular degradation products (among others Congophilic cytofilaments); calcium deposits in the cellular degradation products resulting in "calcific aortic stenosis".

Pyrophosphate arthritis with local amyloid deposition.

Pyrophosphate and capsular chondromatosis were demonstrated histologically in 15 cases, 14 of which also exhibited local deposition of amyloid. A systematic examination of the joint capsule in 57 consecutive hip replacement operations for osteoarthritis revealed these changes in 6 of the cases (10.5 per cent). Microscopic examination which was done if intraarticular calcifications were grossly visible at operation, showed the same changes in 8 knees, 4 with osteoarthritis and 4 with meniscal disease. The changes were also seen in the menisci. Only one patient was suffering from chronic, polyarticular pyrophosphate arthritis. In the other cases neither change had been expected a priori, and the patients had no signs of systemic articular disease or amyloidosis. Most of them were elderly, but in good health.