Refinement of the three-dimensional solution structure of barley serine proteinase inhibitor 2 and comparison with the structures in crystals.

The three-dimensional structure of barley serine proteinase inhibitor, CI-2, has been determined using nuclear magnetic resonance spectroscopy. The present structure determination is a refinement of the structure previously determined by us, using in the present case stereo-specific assignments, and a virtually complete set of assignments of the two-dimensional nuclear Overhauser spectrum. The structure determination is based on the identification of more than 1300 nuclear Overhauser effects, of which 961 were used in the structure calculation as distance restraints, and on 94 dihedral angle restraints, of which 31 are for chi 1 angles in defined chiral centers. These have been used to calculate a series of 20 three-dimensional structures using a combination of distance geometry, simulated annealing and restrained molecular dynamics. Each of the 20 structures was in agreement within less than 0.5 A of each of the distance restraints and with all dihedral angle restraints. When compared to the geometric average structure of the 20 refined structures the root-mean-square differences for the backbone atoms were 0.8 (+/- 0.2) A and for all atoms were 1.6 (+/- 0.2) A. By comparison, the values obtained for the structures determined previously were 1.4 (+/- 0.2) A and 2.1 (+/- 0.1) A, respectively. The structures were also compared to the structure determined in the crystalline state by X-ray diffraction showing root-mean-square differences of 1.6 (+/- 0.2) A and 2.8 (+/- 0.2) A for the backbone and all atoms, respectively. Common features of the solution structure and the two crystal structures are the four-stranded beta-structure, composed of a pair of parallel strands, and three pairs of antiparallel beta-strands flanked on one side by a 12-residue alpha-helix and on the other side by a loop containing the serine proteinase binding site. The new analysis of the structure has revealed an additional pair of antiparallel beta-strands, consisting of residues 65 to 67 and 81 to 83, that was not seen in either of the crystal structures or the previous solution structure. Identification of this was based on nuclear magnetic resonance evidence for the hydrogen bond (67HN to 81CO) not reported previously. Also the presence of a bifurcated hydrogen bond involving Phe69 CO and HN atoms of Ala77 and Gln78 was observed in solution but not in crystals. Minor differences between the two structures were observed in the phi-angles of residues Met59 and Glu60 in the inhibitory site.     

The stabilization of locust cuticle

Cuticle from the metathoracic femur of adult locusts (Locusta migratoria) is characterized with respect to changes in water content and in protein extractability during maturation. The swelling behaviour and extractability of fully-sclerotized cuticle are compared to those of chemically-modified, unsclerotized cuticle.It is concluded that although dehydration may contribute to the stabilization of cuticle, it cannot account for the observed differences. The properties of mature cuticle can best be explained by the assumption that covalent cross-links are present between protein molecules.     

Dihydrofurocoumarin glucosides from Angelica archangelica and Angelica silvestris

A water-soluble root extract of Angelica archangelica subsp. litoralis afforded, in addition to adenosine, coniferin and the two known dihydrofurocoumarin glycosides, apterin and 1′-O-β-d-glycopyranosyl-(S)-marmesin (marmesinin), two new dihydrofuranocoumarin glycosides, 1′-O-β-d-glucopyranosyl-(2S, 3R)-3-hydroxymarmesin, and 2′-β-d-glucopyranosyloxymarmesin. For the latter a 2S-configuration was demonstrated, the stereochemistry at position 1′ remaining undefined. Roots of A. silvestris similarly afforded 1′-O-β-d-glucopyranosyl-(2S, 3R)-3-hydroxymarmesin. By correlation with the aglycone 2S,3R)-3-hydroxymarmesin obtained in this work, the absolute configurations (2S,3R) were established for the known dihydrofurocoumarin diesters smirniorin and smirnioridin.     

IgG subclass antibodies to human and bacterial HSP60 are not associated with disease activity and progression over time in axial spondyloarthritis

Introduction

Spondyloarthritis (SpA), an interrelated group of rheumatic diseases, has been suggested to be triggered by bacterial infections prior to the development of an autoimmune response that causes inflammation of the spinal and peripheral joints. Because human heat shock protein 60 (HSP60), recently renamed HSPD1, and bacterial HSP60 are highly homologous, immunological cross-reactivity has been proposed as a mechanism of disease initiation. However, previous investigations of the humoral immune response to HSP60 in SpA patients have lacked determination of immunoglobulin G (IgG) subclasses and patient follow-up. In this study, we have focused on these parameters in a cohort of axial SpA patients with a well-established set of clinical characteristics, including MRI changes and human leukocyte antigen B27.

Methods

IgG subclass antibodies (IgG1, IgG2, IgG3 and IgG4) against recombinant HSP60 of three reactive arthritis-related bacteria; human HSP60; and the microorganisms Chlamydia trachomatis and C. pneumoniae were determined by ELISA. Serum samples collected from 2004 to 2006 and in 2010 and 2011 from 39 axial SpA patients were analyzed and compared with samples from 39 healthy controls. The Mann-Whitney U test and Wilcoxon matched pairs test were used to compare the antibody levels in different and paired groups, respectively. P < 0.01 was considered significant. The Spearman nonparametric correlation was used to determine correlation between antibody levels and between antibody levels and the disease parameters.

Results

Elevated levels of IgG1 and IgG3 to human HSP60 and IgG1 to HSP60 of Salmonella enterica Enteritidis were observed in SpA patients compared with healthy controls at both time points. The antibody levels were almost constant over time for IgG1, whereas high levels of IgG3 to human HSP60 tended to decrease over time. The antibody response to human HSP60 was predominantly of the IgG3 subclass, and patients with high levels of IgG3 to this antigen had low levels of IgG1, indicating an inverse association. Different IgG subclasses were produced against bacterial and human HSP60 in the same serum sample, IgG1 and IgG3, respectively, indicating that there was no cross-reaction.

Conclusions

A significant association was observed between axial SpA and the presence of IgG1/IgG3 antibodies to human HSP60 and of IgG1 to S. enterica Enteritidis and C. trachomatis. Generation of antibodies to human HSP60 was independent of the presence of antibodies to bacterial HSP60. No association was observed between clinical and MRI changes with antibodies over time. Altogether, such antibodies do not reflect the disease activity in these patients.This study has been approved by the Regional Research Ethics Committee of Central Jutland, Denmark. Trial registration numbers: 20050046 and 20100083     

Characterization of a novel missense mutation in the prodomain of GDF5, which underlies brachydactyly type C and mild Grebe type chondrodysplasia in a large Pakistani family

All TGF-beta family members have a prodomain that is important for secretion. Lack of secretion of a TGF-beta family member GDF5 is known to underlie some skeletal abnormalities, such as brachydactyly type C that is characterized by a huge and unexplained phenotypic variability. To search for potential phenotypic modifiers regulating secretion of GDF5, we compared cells overexpressing wild type (Wt) GDF5 and GDF5 with a novel mutation in the prodomain identified in a large Pakistani family with Brachydactyly type C and mild Grebe type chondrodyslplasia (c527T>C; p.Leu176Pro). Initial in vitro expression studies revealed that the p.Leu176Pro mutant (Mut) GDF5 was not secreted outside the cells. We subsequently showed that GDF5 was capable of forming a complex with latent transforming growth factor binding proteins, LTBP1 and LTBP2. Furthermore, secretion of LTBP1 and LTBP2 was severely impaired in cells expressing the Mut-GDF5 compared to Wt-GDF5. Finally, we demonstrated that secretion of Wt-GDF5 was inhibited by the Mut-GDF5, but only when LTBP (LTBP1 or LTBP2) was co-expressed. Based on these findings, we suggest a novel model, where the dosage of secretory co-factors or stabilizing proteins like LTBP1 and LTBP2 in the microenvironment may affect the extent of GDF5 secretion and thereby function as modifiers in phenotypes caused by GDF5 mutations.     

Smaller stomata require less severe leaf drying to close: A case study in Rosa hydrida

Stomata formed at high relative air humidity (RH) close less as leaf dries; an effect that varies depending on the genotype. We here quantified the contribution of each stomatal response characteristic to the higher water loss of high RH-grown plants, and assessed the relationship between response characteristics and intraspecific variation in stomatal size. Stomatal size (length multiplied by width), density and responsiveness to desiccation, as well as pore dimensions were analyzed in ten rose cultivars grown at moderate (60%) or high (85%) RH. Leaf morphological components and transpiration at growth conditions were also assessed. High growth RH resulted in thinner (11%) leaves with larger area. A strong positive genetic correlation of daytime and nighttime transpiration at either RH was observed. Stomatal size determined pore area (r = 0.7) and varied by a factor of two, as a result of proportional changes in length and width. Size and density of stomata were not related. Following desiccation, high RH resulted in a significantly lower (6–19%) decline of transpiration in three cultivars, whereas the relative water content (RWC) of high RH-expanded leaflets was lower (29–297%) in seven cultivars. The lower RWC of these leaflets was caused by (a) higher (33–72%) stable transpiration and/or (b) lower (12–143%) RWC at which this stable transpiration occurred, depending on the cultivar. Stomatal size was significantly correlated with both characteristics (r = 0.5 and -0.7, respectively). These results indicate that stomatal size explains much of the intraspecific variation in the regulation of transpiration upon water deprivation on rose.     

Proteolytic cleavage of the Chlamydia pneumoniae major outer membrane protein in the absence of Pmp10

The genome of the obligate intracellular bacteria Chlamydia pneumoniae contains 21 genes encoding polymorphic membrane proteins (Pmp). While no function has yet been attributed to the Pmps, they may be involved in an antigenic variation of the Chlamydia surface. It has previously been demonstrated that Pmp10 is differentially expressed in the C. pneumoniae CWL029 isolate. To evaluate whether the absence of Pmp10 in the outer membrane causes further changes to the C. pneumoniae protein profile, we subcloned the CWL029 isolate and selected a clone with minimal Pmp10 expression. Subsequently, we compared the proteome of the CWL029 isolate with the proteome of the subcloned strain and identified a specific cleavage of the C-terminal part of the major outer membrane protein (MOMP), which occurred only in the absence of Pmp10. In contrast, when Pmp10 was expressed we predominantly observed full-length MOMP. No other proteins appeared to be regulated according to the presence or absence of Pmp10. These results suggest a close association between MOMP and Pmp10, where Pmp10 may protect the C-terminal part of MOMP from proteolytic cleavage.     

Regional differences in degree of resilin cross-linking in the desert locust, Schistocerca gregaria

Various cuticular regions from the desert locust, Schistocerca gregaria, were quantitatively analyzed for two cross-linking amino acids, dityrosine and trityrosine, characteristic constituents of the rubberlike cuticular protein, resilin. These amino acids were found in all regions of cuticle investigated, but in widely varying amounts. In fully mature adult locusts the largest amounts of di- and trityrosine were obtained from the prealar arms and wing-hinges, structures possessing long-range elasticity and being involved in energy storage in the flight system. In structures where deformations tend to occur more slowly, such as the clypeo-labral springs and tracheae, di- and trityrosine are less abundant. In sclerotized cuticle from femur and tibia, as well as in cornea and in the highly stretchable intersegmental membranes of mature females, they are only found in trace amounts and are probably unrelated to elasticity. The trityrosine-to-dityrosine ratio in the various cuticular regions vary from nearly equal amounts of the two amino acids to about ten times more dityrosine than trityrosine, indicating that the regions differ in degree of cross-linking; the tracheal wall is the material with the highest trityrosine-to-dityrosine ratio. In some cuticular regions the ratio increases during maturation from newly moulted (teneral) adults to reproductively active locusts; the most pronounced increase was observed for the wing-hinges, and only a small increase was observed for the abdominal tergal plates. In most cuticular regions in fifth instar locust nymphs the contents of di- and trityrosine corresponded to the contents measured for the adult cuticular regions, but only trace amounts of the two amino acids were obtained from the region of the nymphal wing base which corresponds to the wing-hinge containing cuticular region in adult locusts.     

Identification and characterization of a novel Chlamydia trachomatis reticulate body protein

The genome of the obligate intracellular bacterium Chlamydia trachomatis comprises 894 genes predicted by computer-based analysis. As part of a large-scale proteome analysis of C. trachomatis, a small abundant protein encoded by a previously unrecognized novel 204-bp open reading frame was identified by tandem mass spectrometry. No homology of this protein was observed to proteins from other organisms. The protein was conserved in C. trachomatis but not found in Chlamydia pneumoniae. Using proteomics, we show that the expression of the protein is initiated at the middle of the developmental cycle. The protein is rapidly degraded and is only present in reticulate or intermediate bodies, suggesting a possible function in the intracellular stage of C. trachomatis development. We have termed the protein '7-kDa reticulate body protein'.