In animals, sporadic injections of the mitochondrial toxin 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) selectively damage dopaminergic neurons but do not fully reproduce the features of human Parkinson's disease. We have now developed a mouse Parkinson's disease model that is based on continuous MPTP administration with an osmotic minipump and mimics many features of the human disease. Although both sporadic and continuous MPTP administration led to severe striatal dopamine depletion and nigral cell loss, we find that only continuous administration of MPTP produced progressive behavioral changes and triggered formation of nigral inclusions immunoreactive for ubiquitin and ␣-synuclein. Moreover, only continuous MPTP infusions caused long-lasting activation of glucose uptake and inhibition of the ubiquitin-proteasome system. In mice lacking ␣-synuclein, continuous MPTP delivery still induced metabolic activation, but induction of behavioral symptoms and neuronal cell death were almost completely alleviated. Furthermore, the inhibition of the ubiquitinproteasome system and the production of inclusion bodies were reduced. These data suggest that continuous low-level exposure of mice to MPTP causes a Parkinson-like syndrome in an ␣-synucleindependent manner.neurodegeneration ͉ mitochondria ͉ neuronal inclusions ͉ Lewy bodies

Recently, we have found that partially unfolded lysozyme exerts broad spectrum antimicrobial action in vitro against Gram-negative and Gram-positive bacteria independent of its catalytic activity. In parallel, an internal peptide (residues 98 -112) of hen egg white lysozyme, obtained after digestion with clostripain, possessed broad spectrum antimicrobial action in vitro. This internal peptide is part of a helix-loop-helix domain (87-114 sequence of hen lysozyme) located at the upper lip of the active site cleft of lysozyme. The helix-loophelix (HLH) structures are known motifs commonly found in membrane-active and DNA-binding proteins. To evaluate the contribution of the HLH peptide to the antimicrobial properties of lysozyme, the HLH sequence and its secondary structure derivatives of chicken and human lysozyme were synthesized and tested for antimicrobial activity against several bacterial strains. We found that the full HLH peptide of both chicken and human lysozymes was potently microbicidal against both Gram-positive and Gram-negative bacteria and the fungus Candida albicans. The N-terminal helix of HLH was specifically bactericidal to Gram-positive bacteria, whereas the C-terminal helix was bactericidal to all tested strains. Outer and inner membrane permeabilization studies, as well as measurements of transmembrane electrochemical potentials, provided evidence that HLH peptide and its C-terminal helix domain kill Gram-negative bacteria by crossing the outer membrane via self-promoted uptake and causing damage to the inner membrane through channel formation. The results are discussed in terms of proposed mechanisms for the catalytically independent antimicrobial activity of lysozyme that offer a new strategy for the design of potential antimicrobial drugs in the treatment of infectious diseases.

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Chicken egg white lysozyme exhibits antimicrobial activity against both Gram‐positive and Gram‐negative bacteria. Fractionation of clostripain‐digested lysozyme yielded a pentadecapeptide with antimicrobial activity but without muramidase activity. The peptide was isolated and its sequence found to be I‐V‐S‐D‐G‐N‐G‐M‐N‐A‐W‐V‐A‐W‐R (amino acids 98–112 of chicken egg white lysozyme). A synthesized peptide of identical sequence had the same bactericidal activity as the natural peptide. Replacement of Trp 108 with tyrosine significantly reduced the antibacterial capacity of the peptide. By replacement of Trp 111 with tyrosine the antibacterial activity was lost. Replacement of Asn 106 with the positively charged arginine strongly increased the antibacterial capacity of I‐V‐S‐D‐G‐N‐G‐M‐N‐A‐W‐V‐A‐W‐R. The peptide I‐V‐S‐D‐G‐N‐G‐M consisting of the eight amino acids of the N‐terminal side had no bactericidal properties, whereas the peptide N‐A‐W‐V‐A‐W‐R of the C‐terminal side retained some bactericidal activity. Replacement of asparagine 106 by arginine (R‐A‐W‐V‐A‐W‐R) increased the bactericidal activity considerably. The D enantiomer of R‐A‐W‐V‐A‐W‐R was as active as the L form against five of the tested bacteria, but substantially less active against Serratia marcescens, Micrococcus luteus,Staphylococcus aureus, Staphylococcus epidermidis and Staphylococcus lentus. For these bacterial species some stereospecific complementarity between receptor structures of the bacteria and the peptide can be assumed.

Bactericidal properties of aprotinin, a proteinase inhibitor and possibly a defence molecule in bovine species, and of chicken egg white lysozyme, known as muramidase, were investigated. Incubation of various bacteria in the presence of either aprotinin or lysozyme showed that both proteins killed Gram-positive as well as Gram-negative bacteria without addition of complement or EDTA. Denaturation of the two proteins by dithiothreitol did not lead to loss of their bactericidal potency. Electron microscopic examination of Escherichia coli incubated either with lysozyme or aprotinin revealed that the bacterial cytoplasms gradually disintegrated. Both aprotinin and lysozyme were demonstrated within the affected cytoplasm by immunogold labelling. The results suggest that the bactericidal potency of lysozyme is not only due to muramidase activity but also to its cationic and hydrophobic properties. The bactericidal activity of aprotinin is probably also related to both these properties rather than to its activity as proteinase inhibitor.

Antimicrobial peptides are present in men, animals and plants and represent an important component of the innate immunity. Nevertheless they can also be generated through proteolytical digestion of food proteins. Thus, food proteins can be regarded not only for their nutritive value but also as a possible resource to increase the natural defence of the organism against invading pathogens. Consequently food proteins can be considered as component of nutritional immunity. Antimicrobial peptides generated from food proteins present the great advantage to be derived from harmless substances, therefore one can expect their safety for use in medicine and in food industry. Many biologically active peptides have been produced from food proteins, in particularly from milk proteins. The possibility that proteins can be tailored and their fragments modelled to achieve a particular function is recently giving rise to increased interest. This strategy has had particular success with food proteins like lactoferrin and lysozyme. Both bactericidal domains of these proteins have been extensively investigated. A number of short peptides with high bactericidal activity have been developed from the bactericidal domain of lysozyme through the strategy "tailoring and modelling". Ovotransferrin, alpha--lactalbumin and beta-lactoglobulin are further examples of food proteins which are a source of antimicrobial peptides. The observation that antimicrobial peptides can be generated through proteolytical digestion of parent proteins, which usually have another physiological function in the organism, led us to consider these latter as multifunctional molecules. This raises the question, whether multifunctionality is an intrinsic property of many proteins or limited to a few.

The increasing development of bacterial resistance to traditional antibiotics has reached alarming levels, thus necessitating the strong need to develop new antimicrobial agents. These new antimicrobials should possess both novel modes of action as well as different cellular targets compared with the existing antibiotics. Lysozyme, muramidase, and aprotinin, a protease inhibitor, both exhibit antimicrobial activities against different microorganisms, were chosen as model proteins to develop more potent bactericidal agents with broader antimicrobial specificity. The antibacterial specificity of lysozyme is basically directed against certain Gram-positive bacteria and to a lesser extent against Gram-negative ones, thus its potential use as antimicrobial agent in food and drug systems is hampered. Several strategies were attempted to convert lysozyme to be active in killing Gram-negative bacteria which would be an important contribution for modern biotechnology and medicine. Three strategies were adopted in which membrane-binding hydrophobic domains were introduced to the catalytic function of lysozyme, to enable it to damage the bacterial membrane functions. These successful strategies were based on either equipping the enzyme with a hydrophobic carrier to enable it to penetrate and disrupt the bacterial membrane, or coupling lysozyme with a safe phenolic aldehyde having lethal activity toward bacterial membrane. In a different approach, proteolytically tailored lysozyme and aprotinin have been designed on the basis of modifying the derived peptides to confer the most favorable bactericidal potency and cellular specificity. The results obtained from these strategies show that proteins can be tailored and modelled to achieve particular functions. These approaches introduced, for the first time, a new conceptual utilization of lysozyme and aprotinin, and thus heralded a great opportunity for potential use in drug systems as new antimicrobial agent.