ABSTRACT
We modified the protocol of obtaining of biological scaffolds of rat lungs based on dynamic recording of specific resistivity of working detergent solution (conductometry) during perfusion decellularization. Termination of sodium deoxycholate exposure after attaining ionic equilibrium plateau did not impair the quality of decellularization and preserved structural matrix components, which was confirmed by morphological analysis and quantitative assay of residual DNA.
Subject(s)
Lung , Tissue Scaffolds , Animals , Conductometry , Deoxycholic Acid/chemistry , Detergents/chemistry , Electric Conductivity , Lung/cytology , Male , Rats, Wistar , Solutions , Tissue Engineering , Tissue FixationABSTRACT
Preparation and purification of a recombinant protein are described along with characteristics of its specific (for ε-(γ-Glu)-Lys and D-dimer substrates) and nonspecific (for L-γ-Glu-pNA) isopeptidase activities; the absence of peptidase function for α-(α-Glu)-Lys substrate is noted. It is shown that the protein exhibits muramidase (cell walls of Micrococcus lysodeikticus) and specific glycosidase activities. The latter was determined towards the fluorogenic substrate 4-methylumbelliferyl-tetra-N-acetyl-ß-chitotetraoxide. Antimicrobial activity of recombinant destabilase-lysozyme protein (recDest-Lys) and its 11-membered amphipathic peptide was revealed towards cells of the strict anaerobic Archaean Methanosarcina barkeri, whose cell walls contain no murein. Possible mechanisms of the effect of recDest-Lys on these cells are discussed.
Subject(s)
Endopeptidases/metabolism , Leeches/enzymology , Muramidase/metabolism , Recombinant Proteins/metabolism , Animals , Anti-Infective Agents/chemistry , Anti-Infective Agents/pharmacology , Endopeptidases/chemistry , Endopeptidases/genetics , Fluorescent Dyes/chemistry , Microscopy, Electron, Transmission , Muramidase/chemistry , Muramidase/genetics , Recombinant Proteins/chemistry , Recombinant Proteins/pharmacology , Substrate SpecificityABSTRACT
The relative location of proteins and lipids in particles of medicinal leech salivary gland secretion (SGS) is revealed for the first time. Their sizes and morphology are described. Using scanning electron microscopy and transmission electron microscopy, it was determined that SGS consists of particles of different sizes and form. This picture is supported by confocal laser scanning microscopy of SGS preparations treated with fluorescein isothiocyanate. After incubation with nonionic detergents (Brij 35 and Tween 20), transmission electron microscopy revealed the dissociation of fragments composing protein-lipid particles (PLP), and in this case an increase in free protein concentration determined by a modification of the Lowry method was observed. Perylene probing of lipids in SGS preparations showed that they are concentrated mainly inside PLP and are almost absent on the surface. Cholesterol was detected during SGS probing using the cholesteryl-Bodipy (hydrophobic fluorescent analog of cholesterol) on surface sections during confocal analysis of electron microphotographs of SGS. This analysis detected PLP structures in SGS resembling caveoles full of cholesterol. SGS, preliminary frozen at -70 degrees C, transformed into a multitude of similar small particles visualized by transmission electron microscopy, whose fixed distribution resembled water crystal structure.
Subject(s)
Lipid Metabolism , Salivary Glands/metabolism , Salivary Proteins and Peptides/metabolism , Animals , Cholesterol/metabolism , Hirudo medicinalis/metabolism , Hirudo medicinalis/ultrastructure , Microscopy, Confocal , Particle Size , Polyethylene Glycols/chemistry , Polysorbates/chemistry , Surface-Active Agents/chemistryABSTRACT
BACKGROUND: Since bactericidal properties of some lysozymes are independent of their glycosidase activity, we have investigated this phenomenon for destabilase-lysozyme (DL) from medicinal leech (Hirudo medicinalis). METHODS: Glycosidase activity was determined on Micrococcus luteus, non-enzymatic antibacterial activity of heat-treated DL and of synthetic peptides alpha1, alpha2 and alpha3 (fragments of its primary structure) on M. luteus, Escherichia coli, Bacillus brevis and Streptomyces chrysomallus. RESULTS: Glycosidase activity disappeared after the heating of native DL at 100 degrees C for 40 min. Antibacterial activity of heat-treated DL for M. luteus MDMSU128 and MDMSU140 expressed as minimal inhibitory concentration was 9.8.10(-8) and 12.10(-8) M, respectively, and to E. coli MDMSU52 11.10(-8) M. Antibacterial activity of synthetic peptide alpha1 for M. luteus MDMSU128 and for E. coli MDMSU52 was 8.3.10(-5) and 4.9.10(-5) M, respectively. CONCLUSION: DL is the first invertebrate lysozyme with combined enzymatic and non-enzymatic antibacterial action.
