Antibacterial proteins are components of the innate immune system found in many organisms and produced by a variety of cell types. Human blood platelets contain a number of antibacterial proteins in their ␣-granules that are released upon thrombin activation. The present study was designed to purify these proteins obtained from human platelets and to characterize them chemically and biologically. Two antibacterial proteins were purified from platelet granules in a two-step protocol using cation exchange chromatography and continuous acid urea polyacrylamide gel electrophoresis and were designated thrombocidin (TC)-1 and TC-2. Characterization of these proteins using mass spectrometry and Nterminal sequencing revealed that TC-1 and TC-2 are variants of the CXC chemokines neutrophil-activating peptide-2 and connective tissue-activating peptide-III, respectively. TC-1 and TC-2 differ from these chemokines by a C-terminal truncation of 2 amino acids. Both TCs, but not neutrophil-activating peptide-2 and connective tissue-activating peptide-III, were bactericidal for Bacillus subtilis, Escherichia coli, Staphylococcus aureus, and Lactococcus lactis and fungicidal for Cryptococcus neoformans. Killing of B. subtilis by either TC appeared to be very rapid. Because TCs were unable to dissipate the membrane potential of L. lactis, the mechanism of TC-mediated killing most probably does not involve pore formation.

No abstract

The network structure of native and carbodiimide cross-linked gelatin A and B gels was studied based on their rheological behavior. Gelatin A and B contain different numbers of carboxylic acid groups caused by different preparation conditions and had previously shown different characteristics in controlled release applications. It was evaluated to which extent chemical cross-linking densified the network structure of physical gelatin gels. After normalization of the equilibrium shear modulus (G e) with respect to swelling (Q), it was observed that the normalized G e values largely depend on the way gelatin is prepared from collagen. At an equal number of chemical junctions, chemically cross-linked gelatin B gels had a lower elasticity modulus than chemically cross-linked gelatin A gels. This seemed contradictory as gelatin B contains more carboxylic acid groups, available for cross-linking, but is related to a higher probability for intramolecular cross-linking, as was validated quantitatively by chemical and rheological analysis of the number of cross-links. Assuming an ideal network, the average molecular weight of the elastic network chains (M c) was calculated for physical and chemical gelatin A and B networks, and on the basis of M c the mesh sizes of the gels were estimated. The calculated mesh sizes were experimentally confirmed by lysozyme and albumin diffusion. Chemical cross-linking increased the resistance of the gels toward thermal degradation, resulting in a more gradual disintegration of physical cross-links upon heating. Moreover, chemical cross-linking prevented recombination of these cross-links upon cooling.

Cross-linking of gelatin A and B with N,N-(3-dimethylaminopropyl)-N'-ethyl-carbodiimide hydrochloride (EDC) and N-hydroxysuccinimide (NHS) was optimised by varying the NHS/EDC molar ratio at constant EDC concentration. Native and cross-linked gelatin gels were characterised using the degree of swelling, the number of free amine groups, the phase transition temperature, and titration of the carboxylic acid residues. The cross-linking reaction was most efficient at a NHS to EDC molar ratio of 0.2. At higher NHS/EDC molar ratios, the reaction of EDC with NHS becomes more pronounced, thereby reducing the effective amount of EDC for cross-linking. Swelling measurements of cross-linked gelatin gels gave deviating results when no NHS was used, which was explained by heterogeneous localisation of cross-links in the gelatin gel. The incorporation of undesired compounds into the gelatin gels during the cross-linking reaction was not observed. At optimal NHS to EDC molar ratio, gelatin A and B were cross-linked using increasing EDC/COOHgelatin molar ratios. A range of samples varying from very low cross-link density to very high cross-link density (at high EDC/COOHgelatin) was obtained. Stability of the gels is enhanced with increasing cross-link density, but a minimal cross-link density is required to obtain gelatin gels which are stable at 40 degrees C.

No abstract