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Helianthus tuberosus is economically important species. To improve characters of this energetic plant via genetic modification, production of callus tissue and plant regeneration are the first steps. A new, potentially energetic cultivar Albik was used in this study to test callus induction and regeneration. Callus was produced on leaves, petioles, apical meristems and stems from field-harvested plants but was totally non-morphogenic. Its induction started in the cortex and vascular bundles as confirmed by histological analysis. The surface of heterogeneous callus was partially covered with a membranous extracellular matrix surface network visible in scanning and transmission electron microscopies. The results clearly indicate that: (i) the morphogenic capacity of callus in topinambur is genotype dependent, (ii) cv. Albik of H. tuberosus proved recalcitrant in in vitro regeneration, and (iii) extracellular matrix surface network is not a morphogenic marker in this cultivar.
Dermatan sulfate (DS) widespread as a component of extracellular matrix proteoglycans, is characterized by great bio-reactivity and remarkable structural heterogeneity due to distinct degrees of sulfation and glucuronosyl epimerization and different polymerization degrees. However, DS metabolism under various biological conditions is poorly known. Dupuytren’s contracture is a benign fibromatosis leading to complex remodeling of the palmar fascia structure and properties. However, it remains unclear whether the disease affects the structure of DS, which is the major tissue glycosaminoglycan. Thus the aim of the study was to examine the structure of the total DS in Dupuytren’s fascia. DS chains were extracted from 5 samples of normal fascia and 7 specimens of Dupuytren’s tissue by papain digestion followed by fractionation with cetylpyridinium chloride. Then, DS structure analysis was performed comprising the evaluation of its molecular masses and sensitivity to hyaluronidase and chondroitinase B. Dupuytren’s contracture is associated with significant remodeling of DS chain structure revealed by (1) a distinct profile of chain molecular masses characterized by the appearance of long size components as well as the increase in the content of small size chains; (2) a different glucuronosyl epimerization pattern connected with the enhanced content of glucuronate disaccharide blocks; (3) chain oversulfation. These structural alterations in total DS may modify the GAG interactions especially affecting collagen fibrillogenesis and growth factor availability. Thus, Dupuytren’s contracture associated DS remodeling may promote the phenomena typical for advanced disease: apoptosis and reduction in cell number as well as the appearance of dense pseudotendinous collagen matrix
Procollagen C-endopeptidase (BMP-1) and N-endopeptidase (ADAMTS-2) are key enzymes for correct and efficient conversion of fibrillar procollagens to their self assembling monomers. Thus, they have an essential role in building and controlling the quality of extracellular matrices (ECMs). Here, we tested inhibition of activity of the largest variant of BMP-1, a recombinant mammalian tolloid (mTld), in vitro by three synthetic peptides with conservative amino-acid sequences found in chordin using procollagen type I as a substrate. We also verified the specific action of best inhibitory 16 amino-acid peptide in the procollagen type I cleavage assay with the use of ADAMTS-2 (procollagen N-endopeptidase). Subsequently, we determined the critical residues and minimal sequence of six amino acids in the original 16 amino-acid peptide required to maintain the inhibitory potential. Studies on the interactions of 6 and 16 amino acid long peptides with the enzyme revealed their binding to non-catalytic, regulatory domains of mTld; the inhibitory activity was not due to the competition of peptides with the substrate for the enzyme active center, because mTld did not cleave the peptides. However, in the presence of mTld both peptides underwent cyclization by disulfide bond formation. Concluding, we have shown that procollagen C-endopeptidase may be specifically blocked via its non-catalytic domains by synthetic peptide consisting of 6 amino acids in the sequence found in highly conservative region of chordin. Thus, we hypothesize that the 6 amino-acid peptide could be a goodcandidate for anti-fibrotic drug development.
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