Comparison of the effects of interleukin-1 beta on proteoglycan synthesis by human skin and post-burn normal scar explant cultures.

The effect of interleukin (IL)-1 beta on proteoglycan (PG) synthesis and secretion, into culture medium by normal human skin and post-burn human normal scar using tissue explants in culture, was investigated. Following exposure of different tissues to labeling with Na2[35SO4] in the presence and absence of IL-1 beta, the extractable [35SO4]PG (isolated from 0.15 M NaCl and 4 M Gdm. Cl extracts), non-extractable [35SO4]PG (isolated after papain treatment of residual tissue), and [35SO4]PG secreted into culture medium were analyzed for contents and distribution. The contents of [35SO4]PG as measured by [35SO4] incorporation indicate differences in [35SO4]PG production of extractable and non-extractable PGs and also in the PGs released into the culture medium. Examination of the sizes of [35SO4]PGs on Sepharose CL-6 beta columns with and without treatment of IL-1 beta shows that the size of non-extractable [35SO4]PG decreases after IL-1 beta treatment. Cellulose acetate plate electrophoresis of these [35SO4]PG fractions shows that the distribution of PGs alters after treatment with IL-1 beta. These results indicate that burn wound healing abnormalities (scarring) is related to a change in the level of PGs, and may be modified by IL-1 beta treatment.

Mutation of the heme-binding crevice of flavocytochrome b2 from Saccharomyces cerevisiae: altered heme potential and absence of redox cooperativity between heme and FMN centers.

Kinetic and thermodynamic properties of yeast flavocytochrome b2 (EC 1.1.2.3) are modified by the product pyruvate, which binds to the flavosemiquinone (FSQ) form of the prosthetic flavin and decreases the thermodynamic driving force for electron transfer from FSQ to heme. Pyruvate inhibits flavocytochrome b2, but the catalytic competence of pyruvate-ligated FSQ in intramolecular electron transfer to heme is unclear; one kinetic study suggested pyruvate prevented this reaction [Tegoni, M, Janot J.-M., & Labeyrie, F. (1990) Eur. J. Biochem. 190, 329-342], while laser flash photolysis indicated pyruvate was essential [Walker, M. C., & Tollin, G. (1991) Biochemistry 30, 5546-5555]. To address this problem, wild-type (WT) and mutant (L36I) flavocytochromes b2 have been expressed in Escherichia coli. Both forms incorporated heme and FMN prosthetic groups and were catalytically active. The mutation L36I was a conservative substitution within the heme-binding crevice and was designed to alter the midpoint potential (Em) of the heme to alter the pyruvate-FSQ/heme equilibrium. Potentiometric titrations yielded Em values (pH 7.0, 25 degrees C) of +8 and -28 mV for WT and L36I forms, respectively. The FMN midpoint potentials in the absence of pyruvate (-58 mV, n = 2) were identical within experimental error in WT and L36I species and were also identical (+5 mV, n = 1) in the presence of pyruvate. These results indicated the absence of redox cooperativity between FMN and heme.(ABSTRACT TRUNCATED AT 250 WORDS)

Proteoglycans in human burn hypertrophic scar from a patient with Ehlers-Danlos syndrome.

Proteoglycans (PGs) from human burn hypertrophic scar of a patient with Ehlers-Danlos syndrome were extracted with 4M guanidinium chloride and purified by DEAE-cellulose chromatography. Differential ethanol precipitation of the PG fraction obtained after ion-exchange chromatography yielded two low mol.-wt. PGs, on rich in glucuronic acid (PGGLCA; Mr 66 kDa) and the other rich in iduronic acid (PGIDOA; Mr 48 kDa). In PGGLCA, 84% of the glycosaminoglycan chains are composed of GlcA----GalNAc(SO4) units, whereas in PGIDOA, the chains contain 95% IdoA----GalNAc(SO4) disaccharide units. Upon treatment with testicular hyaluronidase, the PGs gave different-sized oligosaccharides. Chondroitinase ABC digestion of PGGLCA or PGIDOA gave a single protein core (Mr approximately 20 kDa). The presence of glucosamine and sialic acid in PGGLCA and PGIDOA suggests that both contain N-linked oligosaccharides.

Proteoglycan synthesis in human skin and burn scar explant cultures.

The synthesis of proteoglycans (PG) by normal human skin, and normal and hypertrophic scars were compared using tissue explants in culture. Newly synthesized PG were labelled with [35S]Na2SO4. Significant differences were found in the proportion of [35S]-radio-labelled incorporation of PG in the tissue and accumulation of [35S]PG in culture medium in the different tissues. The rate of PG biosynthesis in all three tissue types occurred in two phases. There was an initial phase of PG synthesis occurring at 0-3 h and a later phase that occurred at 3-18 h [35S]-labelled PG were isolated and characterized by Sepharose CL-6B chromatography and cellulose acetate electrophoresis. The results showed that the hypertrophic scar tissue and its culture medium contained higher proportions of dermatan sulphate (DS), chondroitin sulphate (CS) and DS' PG than the normal skin fractions. These results suggest that abnormal scarring is related to a change in the level of PG synthesis during the burn injury repair process.

Purification and characterization of iduronic acid-rich and glucuronic acid-rich proteoglycans implicated in human post-burn keloid scar.

Small proteoglycans (PGs), extracted from human keloid scar tissue with 4M guanidinium chloride and fractionated by DEAE-cellulose chromatography, were separated by ethanol precipitation into one L-iduronic acid-rich and one D-glucuronic acid-rich fraction. The size of the L-iduronic acid-rich PG was 102 kDa with a 27 kDa glycosaminoglycan chain, that of the D-glucuronic acid-rich PG was 90 kDa with a 26 kDa glycosaminoglycan chain, and the protein core of both PGs was 14.5 kDa. The two PGs carried sulfate groups mostly attached at C-4 of the 2-amino-2-deoxy-D-galactose units. The N-terminal amino acid sequence of both was similar to human bone PGII (decorin), normal and hypertrophic scar, and human dermal tissue PG.