bluuebunny/crossref_metadata_2025_split huggingface dataset:
Novel and economical purification of recombinant proteins: Intein‐mediated protein purification using in vivo polyhydroxybutyrate (PHB) matrix association
Authors: Banki Mahmoud Reza , Gerngross Tillman U. , Wood David W. 
Published: May 2005

AbstractThis work combines two well‐established technologies to generate a breakthrough in protein production and purification. The first is the production of polyhydroxybutyrate (PHB) granules in engineered strains of Escherichia coli. The second is a recently developed group of self‐cleaving affinity tags based on protein splicing elements known as inteins. By combining these technologies with a PHB‐specific binding protein, a self‐contained protein expression and purification system has been developed. In this system, the PHB‐binding protein effectively acts as an affinity tag for desired product proteins. The tagged product proteins are expressed in E. coli strains that also produce intracellular PHB granules, where they bind to the granules via the PHB‐binding tag. The granules and attached proteins can then be easily recovered following cell lysis by simple mechanical means. Once purified, the product protein is self‐cleaved from the granules and released into solution in a substantially purified form. This system has been successfully used at laboratory scale to purify several active test proteins at reasonable yield. By allowing the bacterial cells to effectively produce both the affinity resin and tagged target protein, the cost associated with the purification of recombinant proteins could be greatly reduced. It is expected that this combination of improved economics and simplicity will constitute a significant breakthrough in both large‐scale production of purified proteins and enzymes and high‐throughput proteomics studies of peptide libraries.
Domain swapping in the low‐similarity isomerase/hydratase superfamily: The crystal structure of rat mitochondrial Δ3, Δ2‐enoyl‐CoA isomerase
Authors: Hubbard Paul A. , Yu Wenfeng , Schulz Horst , Kim Jung‐Ja P. 
Published: May 2005

AbstractTwo monofunctional Δ3, Δ2‐enoyl‐CoA isomerases, one in mitochondria (mECI) and the other in both mitochondria and peroxisomes (pECI), belong to the low‐similarity isomerase/hydratase superfamily. Both enzymes catalyze the movement of a double bond from C3 to C2 of an unsaturated acyl‐CoA substrate for re‐entry into the β‐oxidation pathway. Mutagenesis has shown that Glu165 of rat mECI is involved in catalysis; however, the putative catalytic residue in yeast pECI, Glu158, is not conserved in mECI. To elucidate whether Glu165 of mECI is correctly positioned for catalysis, the crystal structure of rat mECI has been solved. Crystal packing suggests the enzyme is trimeric, in contrast to other members of the superfamily, which appear crystallographically to be dimers of trimers. The polypeptide fold of mECI, like pECI, belongs to a subset of this superfamily in which the C‐terminal domain of a given monomer interacts with its own N‐terminal domain. This differs from that of crotonase and 1,4‐dihydroxy‐2‐naphtoyl‐CoA synthase, whose C‐terminal domains are involved in domain swapping with an adjacent monomer. The structure confirms Glu165 as the putative catalytic acid/base, positioned to abstract the pro‐R proton from C2 and reprotonate at C4 of the acyl chain. The large tunnel‐shaped active site cavity observed in the mECI structure explains the relative substrate promiscuity in acyl‐chain length and stereochemistry. Comparison with the crystal structure of pECI suggests the catalytic residues from both enzymes are spatially conserved but not in their primary structures, providing a powerful reminder of how catalytic residues cannot be determined solely by sequence alignments.
The solution structure of the oxidative stress‐related protein YggX from Escherichia coli
Authors: Osborne Michael J. , Siddiqui Nadeem , Landgraf Dirk , Pomposiello Pablo J. , Gehring Kalle 
Published: May 2005

AbstractYggX is a highly conserved protein found only in eubacteria and is proposed to be involved in the bacterial response to oxidative stress. Here we report the solution structure of YggX from Escherichia coli determined by nuclear magnetic resonance spectroscopy. The structure of YggX displays a fold consisting of two N‐terminal antiparallel β‐sheets and three α‐helices, which shares significant structural similarity to the crystal structure of a hypothetical protein PA5148 from Pseudomonas aeruginosa. Previous studies propose YggX as an iron binding protein that is involved in cellular iron trafficking. Our data indicate that the protein alone does not bind iron in vitro, suggesting other cofactors or different conditions may be necessary for metal binding.
Asymmetric effect of domain interactions on the kinetics of folding in yeast phosphoglycerate kinase
Authors: Osváth Szabolcs , Köhler Gottfried , Závodszky Péter , Fidy Judit 
Published: May 2005

AbstractThe aim of this work is to shed more light on the effect of domain–domain interactions on the kinetics and the pathway of protein folding. A model protein system consisting of several single‐tryptophan variants of the two‐domain yeast phosphoglycerate kinase (PGK) and its individual domains was studied. Refolding was initiated from the guanidine‐unfolded state by stopped‐flow or manual mixing and monitored by tryptophan fluorescence from 1 msec to 1000 sec. Denaturant titrations of both individual domains showed apparent two‐state unfolding transitions. Refolding kinetics of the individual domains from different denaturant concentrations, however, revealed the presence of intermediate structures during titration for both domains. Refolding of the same domains within the complete protein showed that domain–domain interactions direct the folding of both domains, but in an asymmetric way. Folding of the N domain was already altered within 1 msec, while detectable changes in the folding of the C domain occurred only 60–100 msec after initiating refolding. All mutants showed a hyperfluorescent kinetic intermediate. Both the disappearance of this intermediate and the completion of the folding were significantly faster in the individual N domain than in the complete protein. On the contrary, folding of the individual C domain was slower than in the complete protein. The presence of the C domain directs the refolding of the N domain along a completely different pathway than that of the individual N domain, while folding of the individual C domain follows the same path as within the complete protein.
Strain relief at the active site of phosphoserine aminotransferase induced by radiation damage
Authors: Dubnovitsky Anatoly P. , Ravelli Raimond B.G. , Popov Alexander N. , Papageorgiou Anastassios C. 
Published: May 2005

AbstractThe X‐ray susceptibility of the lysine‐pyridoxal‐5′‐phosphate Schiff base in Bacillus alcalophilus phosphoserine aminotransferase has been investigated using crystallographic data collected at 100 K to 1.3 Å resolution, complemented by on‐line spectroscopic studies. X‐rays induce deprotonation of the internal aldimine, changes in the Schiff base conformation, displacement of the cofactor molecule, and disruption of the Schiff base linkage between pyridoxal‐5′‐phosphate and the Lys residue. Analysis of the “undamaged” structure reveals a significant chemical strain on the internal aldimine bond that leads to a pronounced geometrical distortion of the cofactor. However, upon crystal exposure to the X‐rays, the strain and distortion are relaxed and eventually diminished when the total absorbed dose has exceeded 4.7 × 106 Gγ. Our data provide new insights into the enzymatic activation of pyridoxal‐5′‐phosphate and suggest that special care should be taken while using macromolecular crystallography to study details in strained active sites.
The expression of human leukocyte antigen‐DR and CD25 on circulating T cells in cutaneous lupus erythematosus and correlation with disease activity
Authors: Wenzel Joerg , Henze Stephanie , Brähler Sabine , Bieber Thomas , Tüting Thomas 
Published: May 2005

Background:  Lupus erythematosus (LE) is a chronic autoimmune disease with a broad clinical spectrum reaching from primarily cutaneous manifestations [cutaneous LE (CLE)] up to systemic disease [systemic LE (SLE)]. In patients with SLE, the expression of activation markers on circulating T cells reflects disease activity. Here, we investigated whether this also holds true for patients with CLE.Patients and methods:  The expression of the activation markers human leukocyte antigen (HLA)‐DR and CD25 on circulating T lymphocytes was measured by flow cytometry in 24 patients suffering from different types of active CLE. Simultaneously, the disease activity was assessed clinically using a CLE activity index. Eighteen healthy donors were analyzed for control purposes.Results:  HLA‐DR was expressed on a significantly elevated percentage of both CD4+ and CD8+ circulating T cells in active CLE patients when compared with healthy controls. The percentage of HLA‐DR‐expressing T lymphocytes closely correlated with the disease activity. Interestingly, in disseminated scarring chronic discoid LE, a significantly increased percentage of CD25+ cells was observed only in the subset of skin‐homing cutaneous lymphocyte antigen (CLA)+CD4+ and CD8+ T cells.Conclusion:  Our results provide evidence that activation markers on peripheral blood T cells might help to objectively assess the disease activity in CLE. Furthermore, a significant population of CD25+CLA+CD8+ T cells can only be detected in a subgroup of patients with disseminated scarring CLE and might reflect the systemic expansion of activated cytotoxic T lymphocytes involved in destruction of epidermal tissue.
Fermentation patterns of small‐bale silage and haylage produced as a feed for horses
Authors: Müller C. E. 
Published: May 2005

AbstractThe fermentation quality of small‐bale silage and haylage for feeding to horses in Sweden, and using a conventional high‐density hay baler, was investigated in two experiments. Treatments studied were use of additives (inoculants containing lactic acid bacteria and a chemical additive consisting of hexamethylenetetramine, sodium nitrate, sodium benzoate and sodium propionate), the influence of dry‐matter (DM) content of wilted herbage and the effect of number of stretch film layers on fermentation pattern and aerobic stability. All silages and haylages were made from predominantly Timothy swards and were well fermented as indicated by low levels of ammonia and butyric acid. Values of pH were higher and concentrations of organic acids were lower in haylages than in the silages. This was not considered to be indicative of a poor fermentation in the haylage but of a restricted fermentation due to the high DM content of the herbage. The additives enhanced aerobic storage stability because of inhibition of mould growth. The only statistically significant effect of varying the number of stretch film layers was a higher content of CO2inside the bales when ten layers of stretch film were applied compared with six layers.
Competition between Artemisia frigida and Cleistogenes squarrosa under different clipping intensities in replacement series mixtures at different nitrogen levels
Authors: Gao Y. Z. , Wang S. P. , Han X. G. , Patton B. D. , Nyren P. E. 
Published: May 2005

AbstractThe relative competitive ability of Artemisia frigida Willd. (C3, semi‐shrub) and Cleistogenes squarrosa (Trin.) Keng (C4, perennial bunchgrass), grown in a greenhouse, was studied under three nitrogen fertilizer levels, three defoliation intensities and five competitive replacement series in a factorial arrangement. Relative yield total and the respective crowding coefficients for each species were calculated with reference to aboveground, below‐ground and total biomass. The index of aggressivity was also determined relative to total biomass. Cleistogenes squarrosa was a consistently weak competitor at all nitrogen fertilizer and clipping levels compared with A. frigida. Artemesia frigida had a higher relative yield when grown in mixtures with C. squarrosa than in monocultures, whereas the yield of C. squarrosa was lower in mixtures with A. frigida than in monocultures. The relative competitive ability of A. frigida over C. squarrosa declined significantly at different clipping intensities with increasing nitrogen fertilizer. The interaction of nitrogen fertilizer with clipping was significant, although the clipping effect on aggressivity was not significant.
Sward characteristics important for intake in six Lolium perenne varieties
Authors: Smit H. J. , Tas B. M. , Taweel H. Z. , Elgersma A. 
Published: May 2005

AbstractAn experiment was conducted to determine the genetic variation among diploid perennial ryegrass (Lolium perenne L.) varieties for sward structural characteristics considered to be important for intake by cattle. Assessments were made between June and September in 2000 and 2001. Six varieties (Abergold, Respect, Agri, Herbie, Barezane and Barnhem) were subjected to a cutting experiment where swards were cut after 3 to 4 weeks of regrowth during the growing season. The variables, measured in three 2‐week periods, were herbage mass of dry matter (DM), sward surface height (SSH), bulk density, proportion of green leaf, tiller density, tiller weight, extended tiller height, length of sheath and length of leaf blade. Significant differences among varieties were found in both years for herbage mass of DM, SSH, bulk density, proportion of green leaf, tiller density, tiller weight and length of sheath. The results show that there is significant genetic variation among diploid perennial ryegrass varieties for sward characteristics important for intake during grazing.
Effects of winter grazing on spring production of lucerne under Mediterranean conditions
Authors: Chocarro C. , Lloveras J. , Fanlo R. 
Published: May 2005

AbstractThe effects of one severe winter‐grazing of lucerne were studied over 3 years in an experiment in the Ebro Valley, Spain. In this region the crop is harvested six to seven times per season and winter grazing is a traditional practice. On average, winter‐grazing reduced the yield at the first harvest in spring by 200 kg dry matter (DM) ha−1. This limited yield reduction of 0·06 was accompanied by an increase in the proportion of lucerne in the herbage DM from 0·54 to 0·62, a reduction in the proportion of weeds from 0·39 to 0·36, and a reduction in the proportion of dead material from 0·06 to 0·02. The crude protein concentration and the in vitro DM digestibility increased by 20 g kg−1 DM and 0·03, respectively. The traditional practice, i.e. of grazing lucerne with sheep once in the winter season, results in only a limited reduction in yield in the spring. In addition, the spring crop has a higher nutritive value.
Soil organic carbon stocks in verges and urban areas of Flanders, Belgium
Authors: Mestdagh I. , Sleutel S. , Lootens P. , Van Cleemput O. , Carlier L. 
Published: May 2005

AbstractSoil organic carbon (SOC) stocks in different terrestrial ecosystems have already been calculated. However, SOC stocks of grassy verges and grass‐covered urban areas have never been reported. In this study the total grass‐covered area of verges and urban areas in different agro‐pedological regions of Flanders, Belgium was calculated and SOC stocks estimated. The total grassy area of verges along roads, waterways and railways was calculated to be 18 027 ha, 9530 ha for gardens and parks and 2360 ha for recreations areas. Total SOC stock for grass‐covered verges, extrapolated to a depth of 1 m, was estimated to be 3520 kt SOC. Total SOC stock for grassy vegetation in urban areas was 1738 kt SOC. These total SOC stocks equate, respectively, to 0·10 and 0·05 of the total SOC stock in Flemish grassland.
Intake characteristics of perennial ryegrass varieties when grazed by yearling beef cattle under rotational grazing management
Authors: Orr R. J. , Cook J. E. , Young K. L. , Champion R. A. , Rutter S. M. 
Published: May 2005

AbstractFour intermediate‐heading perennial ryegrass (Lolium perenne L.) varieties, which in previous studies had been associated with high‐ or low‐intake characteristics when swards containing them had been continuously stocked with sheep, were sown as monocultures. They were rotationally grazed, using 1‐d paddocks, with core groups of four yearling Simmental × Holstein beef heifers in 2002 and 2003 and ingestive and ruminative behaviour, and sward factors, were measured. There were two diploid (Belramo and Glen) and one tetraploid (Rosalin) perennial ryegrass varieties and one tetraploid hybrid (Lolium × boucheanum Kunth) (AberExcel) variety. Intake rate (IR) was significantly higher in August 2003 for heifers grazing Glen than those grazing Belramo [27·5 vs. 20·6 g dry matter (DM) min−1; P = 0·019], but there were no significant differences between varieties in two other measurement periods. This is in contrast to previous results with sheep when IR were significantly higher for Glen than Belramo and for AberExcel than Rosalin. Total jaw movement rates during grazing were significantly higher for heifers on the tetraploid swards than those on the diploid swards (87·7 vs. 83·6 jaw movements min−1; P = 0·023) in September 2002. Ruminating time was significantly lower for heifers on the tetraploid swards than those on the diploid swards (453 vs. 519 min 24 h−1; P = 0·012) in July 2002. Digestibility of grass snips was significantly higher on the tetraploid than the diploid swards [697 vs. 680 g digestible organic matter (DOM) kg−1 DM; P = 0·042] in September 2003 and, within diploids, was significantly higher for Glen than Belramo (696 vs. 663 g DOM kg−1 DM; P = 0·014). There were significant differences in sheath tube and leaf lengths and in the population density of tillers between and within ploidies, which might have been expected to have influenced intake characteristics, but this was not generally found under rotational grazing with cattle. In order to separate the effects of defoliation interval from those of grazing style of the different ruminant species, it is suggested that grass variety evaluations using continuously stocked cattle swards are required.
The relationship between tiller appearance in spring and contribution to dry‐matter yield in perennial ryegrass (Lolium perenne L.) cultivars differing in heading date
Authors: Laidlaw A. S. 
Published: May 2005

AbstractThe relative contribution of tillers present in April and those appearing in consecutive periods in spring was assessed for perennial ryegrass cultivars in the three maturity groups (early, intermediate‐ and late‐heading). Each group was represented by two diploid and one tetraploid cultivar each in plots in their third (2000) and fourth (2001) harvest years in three replicated blocks receiving an average of 325 kg N ha−1 and cut seven (in 2001) or eight (in 2000) times annually. ‘Main’ tillers and their daughters were marked with colour‐coded PVC‐covered wire loops in early April as were daughters which appeared in consecutive periods between harvests, the loop colour identifying the period of origin of the tiller. Tillers were harvested at cutting height (5 cm) before the plots were harvested and the herbage from tillers with the same colour code bulked per plot. Tillers were identified retrospectively as ‘reproductive’ if they had been decapitated at the previous harvest.Dry‐matter yield was higher in the early than late‐heading cultivars in April and early May but this was reversed in harvests in late May and June. The early heading group had a lower lamina content than the late‐heading group during reproduction growth, both due to the reproductive tillers (mainly those which overwintered) having a lower leaf content and to their being fewer and smaller vegetative tillers during the reproductive phase than for the late‐heading group.Turnover of tillers was high in spring due to decapitation of reproductive tillers and rapid post‐flowering tillering. This was particularly pronounced in the early heading group which also had slightly more tillers marked in April which were subsequently decapitated than in the other maturity groups, i.e. 0·56 compared with 0·44 for the late‐maturing group. Mean ratios of rate of death: rate of tillering for 3 years (1999–2001) for the early and late‐heading groups were 0·8 and 0·4, respectively, for April–May and 1·1 and 2·4, respectively, for June indicating the different patterns in tiller turnover for the two extreme maturity groups. Information on tiller origin and contribution to yield can be used to refine tiller‐based grass growth models.
Interleukin‐10 production by lung macrophages in CBA xid mutant mice infected with Mycobacterium tuberculosis
Authors: Junqueira‐Kipnis Ana Paula , Kipnis Andre , Henao Tamayo Marcela , Harton Marisa , Gonzalez Juarrero Mercedes , Basaraba Randall J. , Orme Ian M. 
Published: May 2005

SummaryMice on the CBA inbred strain background expressing the well characterized mutation designated xid in the cytoplasmic signalling enzyme Bruton's protein kinase have been previously noted to illustrate shifts in T helper type 1 (Th1)/Th2 immunity which is underlined by an apparent failure to produce the regulatory cytokine interleukin‐10. In the current study we examined if this extended to infection with Mycobacterium tuberculosis, which also depends on Th1 immunity. Contrary to expectations, xid mice showed evidence of a transient early susceptibility to pulmonary infection, changes in macrophage morphology, and decreased activation of lung natural killer cells, while showing evidence of substantial IL‐10 production and accumulation in lung lesions macrophages, but paradoxically this did not influence the course of the chronic disease. In addition, macrophages from the lungs of xid mice also expressed high levels of CD14. These observations suggest that the xid mutation in cellular signalling has much wider effects on the immune system than previously thought.
Monoclonal antibodies that identify the CD3 molecules expressed specifically at the surface of porcine γδ‐T cells
Authors: Yang Huaizhi , Parkhouse R. Michael E. , Wileman Thomas 
Published: May 2005

SummaryThe CD3 antigen is a surface structure associated with the T‐cell receptor (TCR) to form a complex involved in antigen recognition and signal transduction. Reports on the structures of the CD3 molecules associated with αβ‐ and γδ‐TCR have been contradictory. To investigate this issue, we raised a panel of monoclonal antibodies (mAb) against purified porcine CD3 molecules. Unlike the conventional anti‐CD3, these mAb reacted specifically with peripheral γδ‐T cells, but not with αβ‐T cells. Immunoprecipitation showed that the antibody recognized a subset of CD3 molecules that were associated with γδ‐TCR. Also unlike the conventional anti‐CD3, these mAb, though directed at two different epitope groups, failed to induce antigenic modulation, T‐cell proliferation and CD3‐redirected cytotoxicity. Taken together, these results suggest that there are differences in the antigenicity, signal transduction potentials and probably structural differences between the CD3 molecules expressed at the surface of αβ‐ and γδ‐T cells.