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The interplay of albumin (BSA) and lysozyme (LYZ) adsorbed simultaneously on titanium was analyzed by gel electrophoresis and BCA assay. It was found that BSA and lysozyme adsorb cooperatively. Additionally, the isoelectric point of the respective protein influences the adsorption. Also, the enzymatic activity of lysozyme and amylase (AMY) in mixtures with BSA was considered with respect to a possible influence of protein-protein interaction on enzyme activity. Indeed, an increase of lysozyme activity in the presence of BSA could be observed. In contrast, BSA does not influence the activity of amylase., (Copyright © 2016 Elsevier B.V. All rights reserved.)

Background: The efficacy and safety profile of ciclesonide (CIC) in the treatment of asthma was evaluated in a large patient population in a real-life setting in Germany., Methods: 24,037 patients with persistent mild/moderate bronchial asthma were enrolled into three observational studies with identical design. Data were pooled and analyzed. Patients received ciclesonide (160 μg/day) and were observed for 3 months. FEV(1), PEF, NO, asthma episodes, use of rescue medication and adverse drug reactions (ADR) were recorded., Results: Mean (95% CI) FEV(1) significantly increased from 80.7 [80.5; 80.9]% of predicted at baseline to 90.1 [89.9; 90.2]% after 3 months (n = 20,297), mean PEF significantly increased from 338 [335; 340] l/min to 392 [390; 395] l/min (n = 8100). NO was significantly reduced from 53.6 [51.8; 55.4] ppb to 26.2 [25.2; 27.1] ppb (n = 971). The percentage of patients with daily symptoms declined from 24.3% to 1.9%, night-time symptoms from 13.3% to 1.3%, and β(2)-agonists use from 26.9% to 8.8%. ADRs were reported by 51 patients (0.2%). Most frequent ADRs were: dysphonia (n = 11), cough (n = 10), dyspnoea, throat irritation, and oral candidiasis (n = 5 each). 46 patients terminated the study prematurely, 41 due to ADR and 5 due to unknown/missing reason. One patient died due to cardiac failure (no causal relation)., Conclusion: These observational studies under real-life conditions support findings from controlled clinical studies regarding efficacy and tolerability of ciclesonide in patients with mild to moderate bronchial asthma. No unexpected ADRs were detected., (Copyright © 2010 Elsevier Ltd. All rights reserved.)

We have cloned and ligated a full-length bovine link protein (LP) in the pMAL-c2 vector and overexpressed it in fusion with maltose-binding protein (MBP) in Escherichia coli. We have demonstrated dose-dependent binding of MBP/LP to biotinylated hyaluronan in a dot blot assay. A greater percentage of the expressed fusion protein was soluble, monomeric, and undegraded when the growth temperature was lowered, the growth medium was supplemented with zinc, and metal chelators were omitted from the lysis buffers. Similar effects were observed when we tested the effects of lower growth temperature and zinc supplementation on another construct consisting of MBP in fusion with the first proteoglycan tandem repeat of LP. Our results suggest zinc may be necessary for the folding and disulfide bond formation of recombinant LP. In addition, a greater amount of monomeric MBP/LP produced at 27 degrees C with zinc supplementation bound to biotinylated hyaluronic acid-binding region of aggrecan than MBP/LP produced at 27 or 37 degrees C without zinc. This suggests that recombinant LP may have a conformational requirement for zinc necessary for binding to aggrecan. Factor Xa cleavage of MBP/LP expressed in the presence of zinc yielded much more intact LP product than cleavage of MBP/LP expressed without zinc. These data indicate a structural role of zinc that allows MBP/LP to fold in a manner such that it is resistant to proteolytic degradation., (Copyright 1997 Academic Press.)

The previously available sequence for bovine aggrecan included only the KS domain, the C-terminal portion of the CS-2 domain, and the entire CS-3 and G3 domains. We have isolated cDNA clones for previously uncharacterized portions of the bovine aggrecan sequence, and, when we combined them with previously published incomplete sequences, have obtained a complete sequence for the entire core protein. The bovine aggrecan sequence, which is a composite of new sequence data and previously published incomplete sequences, is 2327 residues in length. Although there is significant conservation of G1, G2, and G3 globular domains between species, there are differences in the length of the interglobular domain, in the number of KS domain hexapeptide repeats and CS domain repeats, and in alternative splicing within the G3 domain. The bovine aggrecan KS domain contains 24 repeats of a hexapeptide motif. The largely uncharacterized CS-1 domain of bovine aggrecan was found to contain 27 variable repeats of a 21-residue consensus sequence. A notable feature of the bovine CS-1 domain is in the distribution of single Ser-Gly dipeptides, the majority of which are separated by 7 or 8 amino acids, compared to the human, where discrete pairs of Ser-Gly dipeptides are separated by 13 amino acids. The CS-2 domain contains a total of six "homology domains" with 4 complete and 2 partial approximately 100-residue repeats. Each "homology domain" contains a "nodal" region with few sites for CS chain addition that is highly conserved between species, suggesting a possible role in aggrecan biosynthesis or catabolism.

Decorin is a small leucine-rich proteoglycan which is a component of the extracellular matrix of many connective tissues. We have developed a method for expression and purification of decorin as a fusion protein in Escherichia coli. A PCR product coding for the 330-amino-acid-residue secreted form of bovine decorin core protein was cloned into the vector pMal-c for expression in E. coli as a maltose-binding protein (MBP) fusion. Expressed MBP-decorin tended to form insoluble aggregates resistant to degradation by E. coli intracellular proteases. Fusion protein was therefore solubilized from bacterial inclusion bodies using guanidine HCl and refolded by dilution of the chaotrope to minimally denaturing conditions and disulfide shuffling. Final purification included amylose resin affinity chromatography and size exclusion chromatography. The 79-kDa MBP-decorin fusion protein could be completely cleaved by factor Xa protease in 24 h to yield 43-kDa MBP and 36-kDa decorin core protein. The decorin core protein was separated from MBP and factor Xa protease by DEAE-Sephacel chromatography. Using a solid-phase assay, we have characterized its binding affinity for extracellular matrix components known to interact with tissue-derived decorin including collagen type VI, collagen type I, and fibronectin. The purification and refolding protocol described here may be generally applicable to bacterial expression of other members of this growing family of related small leucine-rich proteoglycan core proteins.

The first proteoglycan tandem repeat (PTR) of bovine link protein has been cloned in the pMAL-c vector and overexpressed in fusion with maltose-binding protein (MBP) in Escherichia coli. The fusion protein can be isolated from the soluble phase of the bacterial lysate by amylose affinity chromatography. The PTR domain can be cleaved from the MBP domain with factor Xa protease. Evidence using zinc affinity chromatography is presented which indicates that at least one of the zinc-binding sites of bovine link protein is contained within the first PTR domain. Zinc affinity chromatography was then incorporated as the final purification step of the MBP/PTR protein. Evidence for the binding of MBP/PTR to hyaluronic acid (HA) is demonstrated by coprecipitation with HA using cetylpyridinium chloride. Binding is specific since MBP/PTR does not coprecipitate with chondroitin sulfate. Binding is also demonstrated in an ELISA assay on HA-coated plates. In this assay, binding could be inhibited by the addition of HA or HA oligosaccharides.

To define the influence of time in culture on gene expression for extracellular matrix proteins we have examined the progression of changes in gene expression for chondrocyte extracellular matrix proteins from the time the chondrocytes are initially isolated from their native cartilaginous matrix through 13 days of high-density culture. We have also determined the effect of matrix depletion and shape change by enzymatically resuspending cells after 6 days in culture and sampling the replated cells at intervals up to 13 days. Northern blots of chondrocyte RNA were hybridized with probes for collagen alpha 1(II), alpha 1(I), aggrecan, link protein, and decorin mRNA. The steady-state level of alpha 1(II) collagen mRNA dropped to 45% of the initial value within 24 h, with a further decrease to 21% by Day 3. A similar decline occurred, but less rapidly in ascorbate supplemented cultures with values of 78%, at 24 h and 62% at Day 3. Very low levels of alpha 1(I) collagen mRNA were detectible in cells maintained for 2 weeks without ascorbate supplementation. Type I collagen alpha 1(I) mRNA was not detected in freshly isolated chondrocytes or at the earliest times in culture but was increasingly abundant from Days 5-13 in the presence of ascorbate. Ascorbic acid supplementation altered the pattern of aggrecan expression over time. Without ascorbate there was an increase in steady-state aggrecan mRNA with time in culture, but in the presence of ascorbate, aggrecan mRNA levels peaked at early culture times and progressively diminished. Decorin steady-state mRNA levels in cultures not supplemented with ascorbic acid steadily increased over time in culture following a lag of several days. In cultures treated with ascorbate, however, there was a progressive increase in decorin steady-state mRNA levels from the first day in culture. Resuspending chondrocytes by digestion of the cell layer with pronase and collagenase at Day 6, which resulted in a transient shape change as well as matrix depletion, resulted in a greater than 2-fold increase in alpha 1(II) mRNA at Day 7 in ascorbate supplemented cultures. Only with ascorbate was there a small increase in decorin mRNA at Day 7, after resuspension. Aggrecan mRNA, however, showed a 3-fold increase without ascorbate and a 10-fold increase with ascorbate within 24 h of resuspension. Similarly, link protein steady-state mRNA showed an 8-fold increase without ascorbate and a 9-fold increase with ascorbate within 24 h after resuspension.

The collagen content, as determined by hydroxyproline assay, of experimental granulation tissue in rats was observed to increase rapidly 21 days, and less rapidly to 90 days of tissue development. Resistance of the collagen to pepsin digestion reached a maximum at 21 days, suggesting more extensive or more stable crosslinking at that time. Type V collagen and the expected collagen types I and III were present in pepsin extracts of the granulation tissue as determined by SDS-polyacrylamide gel electrophoresis. Over 3 months of tissue development the relative quantity of type V collagen, as evidenced by changes in the alpha B chain, varied in parallel with the changing vascularity of the tissue, suggesting an association with capillary endothelial cells and angiogenesis.