Hip joint replacement surgery for idiopathic osteoarthritis aggregates in families

In order to determine whether there is a genetic component to hip or knee joint failure due to idiopathic osteoarthritis (OA), we invited patients (probands) undergoing hip or knee arthroplasty for management of idiopathic OA to provide detailed family histories regarding the prevalence of idiopathic OA requiring joint replacement in their siblings. We also invited their spouses to provide detailed family histories about their siblings to serve as a control group. In the probands, we confirmed the diagnosis of idiopathic OA using American College of Rheumatology criteria. The cohorts included the siblings of 635 probands undergoing total hip replacement, the siblings of 486 probands undergoing total knee replacement, and the siblings of 787 spouses. We compared the prevalence of arthroplasty for idiopathic OA among the siblings of the probands with that among the siblings of the spouses, and we used logistic regression to identify independent risk factors for hip and knee arthroplasty in the siblings. Familial aggregation for hip arthroplasty, but not for knee arthroplasty, was observed after controlling for age and sex, suggesting a genetic contribution to end-stage hip OA but not to end-stage knee OA. We conclude that attempts to identify genes that predispose to idiopathic OA resulting in joint failure are more likely to be successful in patients with hip OA than in those with knee OA.     

Sulphate respiration from hydrogen in Desulfovibrio bacteria: a structural biology overview

Sulphate-reducing organisms are widespread in anaerobic enviroments, including the gastrointestinal tract of man and other animals. The study of these bacteria has attracted much attention over the years, due also to the fact that they can have important implications in industry (in biocorrosion and souring of oil and gas deposits), health (in inflamatory bowel diseases) and the environment (bioremediation). The characterization of the various components of the electron transport chain associated with the hydrogen metabolism in Desulfovibrio has generated a large and comprehensive list of studies. This review summarizes the more relevant aspects of the current information available on the structural data of various molecules associated with hydrogen metabolism, namely hydrogenases and cytochromes. The transmembrane redox complexes known to date are also described and discussed. Redox-Bohr and cooperativity effects, observed in a few cytochromes, and believed to be important for their functional role, are discussed. Kinetic studies performed with these redox proteins, showing clues to their functional inter-relationship, are also addressed. These provide the groundwork for the application of a variety of molecular modelling approaches to understanding electron transfer and protein interactions among redox partners, leading to the characterization of several transient periplasmic complexes. In contrast to the detailed understanding of the periplasmic hydrogen oxidation process, very little is known about the cytoplasmic side of the respiratory electron transfer chain, in terms of molecular components (with exception of the terminal reductases), their structure and the protein–protein interactions involved in sulphate reduction. Therefore, a thorough understanding of the sulphate respiratory chain in Desulfovibrio remains a challenging task.     

Structural evidence of a fourth Gla residue in fish osteocalcin: biological implications

Osteocalcin is a small (45 amino acids) secreted protein found to accumulate in bone and dentin of many organisms by interacting with calcium and hydroxyapatite, through the presence of three gamma-carboxylated residues. In this work, we describe the first X-ray crystal structure for a nonmammalian osteocalcin, obtained at 1.4 A resolution, purified from the marine teleost fish Argyrosomus regius. The three-dimensional fit between the A. regius structure and that of the only other known X-ray structure, the porcine osteocalcin, revealed a superposition of the Calpha atoms of their metal chelating residues, Gla and Asp, showing that their spatial distribution is consistent with the interatomic distances of calcium cations in the hydroxyapatite crystals. In both structures, the protein forms a tight globular arrangement of their three alpha-helices while the remaining residues, at N- and C-terminal regions, have essentially no secondary structure characteristics. This study revealed the presence of a fourth gamma-carboxylation at Glu(25), not previously detected in the structure of the porcine osteocalcin or in any other of the sequentially characterized mammalian osteocalcins (human, cow, and rat). A confirmation of the fourth Gla residue in A. regius osteocalcin was achieved via LC-MS analysis. These four doubly charged residues are, together with Asp(24), concentrated in a common surface region located on the same side of the molecule. This further suggests that the known high affinity of osteocalcin for bone mineral may be derived from the clustering of calcium binding sites on this surface of the molecules.     

Methanol induction optimization for scFv antibody fragment production in Pichia pastoris

Fibronectin splice variant ED B (extracellular domain B) is a promising marker for angiogenesis in growing solid tumors. Currently, recombinant antibodies against ED B are being investigated concerning their potential use, for either therapeutic or diagnostic purposes. Single-chain antibody fragments directed against the ED B can be efficiently expressed in Pichia pastoris; thus, a recombinant strain of the methylotropic yeast P. pastoris was used for this work. Three different forms of scFv antibody fragment are found in the supernatant from this fermentation: covalent homodimer, associative homodimer, and monomer. Both homodimeric forms can be converted to the monomeric form (under reducing conditions) and be efficiently radiolabeled, whereas the monomeric form of scFv already present in the supernatant cannot. It was also found that the fraction of protein in the monomeric form is highly dependent on the mode of induction rather than scFv concentration. This suggests that the monomeric form of the scFv present in the supernatant might be a result of events occurring at the expression, secretion, or folding level. A high cell density fermentation protocol was developed by optimizing methanol induction, yielding the highest scFv antibody fragment production rate and product quality; cell concentration at the induction point and specific methanol uptake rate were found to be the most important control variables. A decrease in specific methanol uptake rate led to a higher specific production rate for the scFv antibody fragment (5.4 microg g(cell) h(-1)). Product quality, i.e., percentage of product in a homodimeric form, also increased with the decrease in methanol uptake rate. Furthermore, the volumetric productivity depended on cell concentration at the induction point, increasing with the increase of cell concentration up to 320 g L(-1) wet cell weight (WCW). The reduction of the methanol feeding rate for induction, and consequently of the oxygen uptake rate, have important consequences for optimizing product titers and quality and thus on the scale-up of this production process; hence one of the major limitations upon high cell density cultivation in bioreactors is keeping the high oxygen transfer rate required. From the results obtained, a scale-up strategy was developed based on the available oxygen transfer rates at larger scales, allowing the definition of the optimum biomass concentration for induction and methanol feeding strategy for maximization of product titer and quality.     

饲喂黄曲霉毒素B_1大鼠肝中AFB_1-DNA加合物的免疫组织化学研究

应用免疫组织化学ＳＰ法对４例饲喂黄曲霉毒素Ｂ１的Ｗｉｓｔａｒ大鼠肝组织中ＡＦＢ１－ＤＮＡ加合物的染色及常规ＨＥ染色发现,４例肝细胞核均出现异型性,但未见肝细胞坏死、增生灶及肝细胞癌;免疫组化揭示部分肝细胞核出现棕黄色、不均质状的ＡＦＢ１－ＤＮＡ加合物,阳性细胞数占１０～８５％。结果提示免疫组织化学方法可作为一种ＡＦＢ１的定位方法,ＡＦＢ１在动物致癌过程中ＡＦＢ１－ＤＮＡ加合物可能具有启动作用     

A preliminary analysis of the three-dimensional structure of dimeric di-haem split-Soret cytochrome c from Desulfovibrio desulfuricans ATCC 27774 at 2.5-Å resolution using the MAD phasing method: a novel cytochrome fold with a stacked-haem arrangement

Haem-containing proteins are directly involved in electron transfer as well as in enzymatic functions. The "split-Soret" cytochrome (SSC) was isolated from the sulfate- and nitrate-reducing bacterium Desulfovibrio desulfuricans ATCC 27774 and has no significant nitrate or nitrite reductase activity. The protein received its name due its unusual spectral properties. It is a dimer containing two identical subunits of 26.3 kDa, each with two haem-c groups. A preliminary model for the three-dimensional structure of this cytochrome was derived using the Multiple Wavelength Anomalous Dispersion (MAD) phasing method. This model shows that SSC is indeed a dimer containing four haems at one end of the molecule. In each monomer the two haems have their edges overlapped within van der Waals contacts with an iron-to-iron distance of 9?Å. The polypeptide chain of each monomer supplies the sixth axial ligand to the haems of the other monomer. This work shows that SSC constitutes a new class of cytochrome. The stacking of the two haems in the monomer within van der Waals distances of each other, and also the short (van der Waals) distances between the two monomers in the dimeric molecule are unprecedented in hemoproteins. This particular haem arrangement is an excellent model for the spectral study (undertaken several years ago) of haem-haem interaction using the aggregated haem undecapeptide derived from mammalian cytochrome c.     

Cytochrome c3 from Desulfovibrio gigas: crystal structure at 1.8 A resolution and evidence for a specific calcium-binding site.

Crystals of the tetraheme cytochrome c3 from sulfate-reducing bacteria Desulfovibrio gigas (Dg) (MW 13 kDa, 111 residues, four heme groups) were obtained and X-ray diffraction data collected to 1.8 A resolution. The structure was solved by the method of molecular replacement and the resulting model refined to a conventional R-factor of 14.9%. The three-dimensional structure shows many similarities to other known crystal structures of tetraheme c3 cytochromes, but it also shows some remarkable differences. In particular, the location of the aromatic residues around the heme groups, which may play a fundamental role in the electron transfer processes of the molecule, are well conserved in the cases of hemes I, III, and IV. However, heme II has an aromatic environment that is completely different to that found in other related cytochromes c3. Another unusual feature is the presence of a Ca2+ ion coordinated by oxygen atoms supplied by the protein within a loop near the N-terminus. It is speculated that this loop may be stabilized by the presence of this Ca2+ ion, may contribute to heme-redox perturbation, and might even be involved in the specificity of recognition with its redox partner.     

Elucidation of the mechanism of lactic acid growth inhibition and production in batch cultures of Lactobacillus rhamnosus

Batch cultures of Lactobacillus rhamnosus were carried out at different pH values in order to study the limitation of growth and lactic acid production by the hydrogen ion, non-dissociated lactic acid and internal lactate concentrations. The effect of pH between 5 and 6.8 was studied at non-limiting concentrations of glucose; this is more significant for the lactic acid fermentation rate than for the maximum specific growth rate, as shown by the incomplete substrate consumption at lower values of medium pH and by the constant maximum cell mass obtained within the range of pH values studied. To check whether these results were a direct consequence of the different concentrations of the non-dissociated form of lactic acid at different external pH values, specific growth rates and lactic acid productions rates were calculated for each external pH value. The same specific growth rates were observed at the same non-dissociated lactic acid concentrations only at pH values of 5 and 5.5. For higher values of pH (pH > 6) the specific growth rate falls to zero as the non-dissociated lactic acid concentration decreases. This shows that generalisations made from studies performed within very narrow ranges of pH are not valid and that the non-dissociated form of lactic acid is not the only inhibiting species. The internal pH was measured experimentally for each external pH value in order to calculate the internal lactate ion concentration. This form is described to be the inhibitory one. The results obtained confirmed that the specific growth rate reached zero at approximately the same lactate concentration for all the pH values studied. Received: 31 January 1997 / Received revision: 15 May 1997 / Accepted: 19 May 1997     

Encounter with New Resources Causes Polarized Growth of the Cord-Forming Basidiomycete Phanerochaete velutina on Soil

Abstract The development and physiology of cord-forming saprotrophic basidiomycetes, which form extensive and persistent mycelial networks in woodland ecosystems, can be conveniently studied on non-sterile soil in laboratory microcosms mimicking field conditions. Morphological responses of Phanerochaete velutina mycelial systems to resource encounters, and decay partitioning following encounters, varied according to whether simulated woody litter was unsterile or autoclaved and on whether encounter took place at the mycelial foraging front or behind the margin (simulating litter fall onto established systems in the field). Results show that encounter of discrete resources by P. velutina is rapidly communicated to the entire mycelial system; that resource capture takes high priority at the expense of continued system extension and decay-derived carbon reallocation; and that polarized growth toward newly encountered resources, previously considered to occur infrequently with this species, may be readily detected using image analysis techniques. Potential advantages of polarized development of P. velutina are discussed.