A quantitative in silico analysis calculates the angiotensin I converting enzyme (ACE) inhibitory activity in pea and whey protein digests

Angiotensin I converting enzyme (ACE) inhibitory peptides can induce antihypertensive effects after oral administration. By means of an ACE inhibitory peptide database, containing about 500 reported sequences and their IC(50) values, the different proteins in pea and whey were quantitatively evaluated as precursors for ACE inhibitory peptides. This analysis was combined with experimental data from the evolution in ACE inhibitory activity and protein degradation during in vitro gastrointestinal digestion. Pea proteins produced similar in silico scores and were degraded early in the in vitro digestion. High ACE inhibitory activity was observed after the simulated stomach phase and augmented slightly in the simulated small intestine phase. The major whey protein beta-lactoglobulin obtained the highest in silico scores, which corresponded with the fact that degradation of this protein in vitro only occurred from the simulated small intestine phase on and resulted in a 10-fold increase in the ACE inhibitory activity. Whey protein obtained total in silico scores of about 124 ml/mg, compared to 46 ml/mg for pea protein, indicating that whey protein would be a richer source of ACE inhibitory peptides than pea protein. Although beta-lactoglobulin is only partially digested, a higher ACE inhibitory activity was indeed found in the whey (IC(50) = 0.048 mg/ml) compared to the pea digest (IC(50) = 0.076 mg/ml). In silico gastrointestinal digestion of the highest scoring proteins in pea and whey, vicilin and albumin PA2, and beta-lactoglobulin, respectively, directly released a number of potent ACE inhibitory peptides. Several other ACE inhibitory sequences resisted in silico digestion by gastrointestinal proteases. Briefly, the quantitative in silico analysis will facilitate the study of precursor proteins on a large scale and the specific release of bioactive peptides.     

Effects of dietary fat source and subtherapeutic levels of antibiotic on the bacterial community in the ileum of broiler chickens at various ages

The effect of dietary fat source (soy oil or a mixture of lard and tallow) and dietary supplementation with antibiotics (a combination of avilamycin at 10 mg kg of feed(-1) and salinomycin at 40 mg kg of feed(-1)) on the bacterial community in the ileum of broiler chickens at different ages (7, 14, 21, and 35 days) was studied using PCR with denaturing gradient gel electrophoresis (DGGE) analysis and bacteriological culture. The bacterial origin of fragments in DGGE profiles was identified by sequencing. Bacterial enumeration results, together with PCR-DGGE profiles, showed that the composition of the microflora was age dependent and influenced by dietary fat source and antibiotic supplementation. An increased incidence of streptococci, enterobacteria, and Clostridium perfringens with age of the chickens was demonstrated. Lactobacilli and C. perfringens were the bacterial groups most strongly affected by the dietary treatments. Moreover, different strains (clonal variants of the alpha-toxin gene) of C. perfringens type A were detected in response to age, dietary fat source, and dietary supplementation with antibiotics.     

C andH NMR spectroscopy as a tool in the configurational analysis of iridoid glucosides

The ¹³C NMR data of 51 iridoid glucosides or glucoside acetates are tabulated. The collection includes 20 pairs of C-6, C-7 or C-8 epimers. Three parameters in using the data for the configurational assignment of 6-O-substituents are given. The chemical shift for C-9 in a range of substituted compounds is shown to be numerically related to the stereochemistry at C-8. This allows the determination of the configuration at this centre for most types of substitution patterns by calculation of the C-9 shift using increments for each substituent. Such increments are given for 25 substituents in three different solvents. A method for simulation of spectra of unknown iridoid glucosides is presented. By this method, the structures of five novel iridoid glucosides have been elucidated, and that of tecomoside has been revised. The methods used to assign the configurations to C-6 and C-8 epimeric iridoid glucosides by ¹H NMR spectroscopy are discussed and a table with selected data is presented. It is suggested that the structures in the literature for ajugol and myoporoside should be interchanged. Consequently, Horeau's method has failed in these instances. Finally, the differences in the ¹³C NMR spectra of pairs of C-6 and C-8 epimeric iridoid glucosides have been interpreted as originating from cis/trans-interactions.     

Residues 10-18 within the C5a receptor N terminus compose a binding domain for chemotaxis inhibitory protein of Staphylococcus aureus

Chemotaxis inhibitory protein of Staphylococcus aureus (CHIPS) is excreted by the majority of S. aureus strains and is a potent inhibitor of C5a- and formylated peptide-mediated chemotaxis of neutrophils and monocytes. Recently, we reported that CHIPS binds to the C5a receptor (C5aR) and the formylated peptide receptor, thereby blocking activation by C5a and formylated peptides, respectively. The anaphylatoxin C5a plays an important role in host immunity and pathological inflammatory processes. For C5a a two-site binding model is proposed in which C5a initially binds the C5aR N terminus, followed by interaction of the C5a C-terminal tail with an effector domain on the receptor. We have shown here that CHIPS does not affect activation of the C5aR by a peptide mimic of the C5a C terminus. Moreover, CHIPS was found to bind human embryonic kidney 293 cells expressing only the C5aR N terminus. Deletion and mutation experiments within this C5aR N-terminal expression system revealed that the binding site of CHIPS is contained in a short stretch of 9 amino acids (amino acids 10-18), of which the aspartic acid residues at positions 10, 15, and 18 plus the glycine at position 12 are crucial. Binding studies with C5aR/C3aR and C5aR/IL8RA chimeras confirmed that CHIPS binds only to the C5aR N terminus without involvement of its extracellular loops. CHIPS may provide new strategies to block the C5aR, which may lead to the development of new C5aR antagonists.     

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     

FLS2-BAK1 Extracellular Domain Interaction Sites Required for Defense Signaling Activation

Signaling initiation by receptor-like kinases (RLKs) at the plasma membrane of plant cells often requires regulatory leucine-rich repeat (LRR) RLK proteins such as SERK or BIR proteins. The present work examined how the microbe-associated molecular pattern (MAMP) receptor FLS2 builds signaling complexes with BAK1 (SERK3). We first, using in vivo methods that validate separate findings by others, demonstrated that flg22 (flagellin epitope) ligand-initiated FLS2-BAK1 extracellular domain interactions can proceed independent of intracellular domain interactions. We then explored a candidate SERK protein interaction site in the extracellular domains (ectodomains; ECDs) of the significantly different receptors FLS2, EFR (MAMP receptors), PEPR1 (damage-associated molecular pattern (DAMP) receptor), and BRI1 (hormone receptor). Repeat conservation mapping revealed a cluster of conserved solvent-exposed residues near the C-terminus of models of the folded LRR domains. However, site-directed mutagenesis of this conserved site in FLS2 did not impair FLS2-BAK1 ECD interactions, and mutations in the analogous site of EFR caused receptor maturation defects. Hence this conserved LRR C-terminal region apparently has functions other than mediating interactions with BAK1. In vivo tests of the subsequently published FLS2-flg22-BAK1 ECD co-crystal structure were then performed to functionally evaluate some of the unexpected configurations predicted by that crystal structure. In support of the crystal structure data, FLS2-BAK1 ECD interactions were no longer detected in in vivo co-immunoprecipitation experiments after site-directed mutagenesis of the FLS2 BAK1-interaction residues S554, Q530, Q627 or N674. In contrast, in vivo FLS2-mediated signaling persisted and was only minimally reduced, suggesting residual FLS2-BAK1 interaction and the limited sensitivity of co-immunoprecipitation data relative to in vivo assays for signaling outputs. However, Arabidopsis plants expressing FLS2 with the Q530A+Q627A double mutation were impaired both in detectable interaction with BAK1 and in FLS2-mediated responses, lending overall support to current models of FLS2 structure and function.     

Similarity between contemporary vegetation and plant remains in the surface sediment in Mediterranean lakes

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High-resolution mapping by YAC fragmentation of a 2.5-Mb Xp22 region containing the human RS, KFSD and CLS disease genes

The disease loci for X-linked Retinoschisis (RS), Keratosis follicularis spinulosa decalvans (KFSD), and Coffin-Lowry syndrome (CLS) have been localized to the same, small region in Xp22 on the human X Chromosome (Chr). To generate a high-resolution map of the available contig in this area, we have used the YAC fragmentation vectors pBP108/ADE2 and pBP109/ADE2 and generated fragmented YACs from a 2.5-Mb YAC (y939H7) spanning the mentioned disease gene candidate regions. Forty-seven fragmented YACs were generated and analyzed, ranging in size from 170 kb to over 2400 kb. The resulting YAC fragmentation panel was used to construct a detailed restriction map of the region and has been used to bin clones and markers. As a deletion panel, it will present a valuable resource for further mapping. Received: 31 December 1996 / Accepted: 22 February 1997