Hanna Miecznikowska

The antifungal properties of chicken egg cystatin against Candida yeast isolates showing different levels of azole resistance

The increasing incidence of fungal infections together with the emergence of strains resistant to currently available antifungal drugs calls for the development of new classes of antimycotics. Naturally occurring antifungal proteins and peptides are of interest because of low toxicity, immunomodulatory potential and mechanisms of action distinct from those of currently available drugs. In this study, the potent antifungal activity of cystatin, affinity‐purified from chicken egg white (CEWC), against the most frequent human fungal pathogens of the genus Candida was identified and characterised. CEWC inhibited the growth of azole‐sensitive Candida albicans isolates with minimal inhibitory concentration (MIC) values ranging from 0.8 to 3.3 μmol l−1, a potency comparable with those of fluconazole and histatin 5, the antimicrobial peptide of the human saliva. Similarly to histatin 5, CEWC activity was not compromised in azole‐resistant isolates overproducing the multidrug efflux transporters Cdr1p and Cdr2p and did not antagonise fluconazole or amphotericin B. CEWC had candidacidal activity, as revealed by the time‐kill assay, and, similarly to histatin 5, completely inhibited the growth at supra‐MIC concentrations. This was in contrast to the fungistatic effect and trailing growth observed with fluconazole. CEWC inhibited the growth of Candida parapsilosis and Candida tropicalis at similar concentrations, whereas Candida glabrata was more resistant to CEWC.

The influence of substrate peptide length on human β‐tryptase specificity

Combinatorial chemistry approach was applied to design chromogenic substrates of human β‐tryptase. The most active substrate, Ala‐Ala‐Pro‐Ile‐Arg‐Asn‐Lys‐ANB‐NH2, was selected from among over 9 million heptapeptides. The amide of 5‐amino‐2‐nitrobenzoic acid (ANB‐NH2) attached at the C‐terminus served as a chromophore. In order to determine the optimal length of the tryptase substrate, a series of N‐terminally truncated fragments of this substrate was synthesized. Pro‐Ile‐Arg‐Asn‐Lys‐ANB‐NH2, with the determined value of the specificity constant (kcat/KM) above 9 × 106 M−1 s−1, appeared to be the most specific substrate of tryptase. This substrate was twice as active as the parent heptapeptide substrate. We postulate that the optimal size of the pentapeptide substrate for the interaction with human β‐tryptase is associated with the unique structure of this proteinase, comprising four almost identical monomer subunits arranged in a square flat ring with its substrate pockets faced inside, forming a tetramer with a central pore that can be penetrated by this short peptide. Copyright

Designing of Substrates and Inhibitors of Bovine α-Chymotrypsin with Synthetic Phenylalanine Analogues in Position P1

The primary specificity residue of a substrate or an inhibitor, called the P(1) residue, is responsible for the proper recognition by the cognate enzyme. This residue enters the S(1) pocket of the enzyme and establishes contacts (up to 50%) inside the proteinase substrate cavity, strongly affecting its specificity. To analyze the influence on bovine alpha-chymotrypsin substrate activity, aromatic non-proteinogenic amino acid residues in position P(1) with the sequence Ac-Phe-Ala-Thr-X-Anb(5,2)-NH(2) were introduced: L-pyridyl alanine (Pal), 4-nitrophenylalanine - Phe(p-NO(2)), 4-aminophenylalanine - Phe(p-NH(2)), 4-carboxyphenylalanine Phe(p-COOH), 4-guanidine phenylalanine - Phe(p-guanidine), 4-methyloxycarbonyl-phenylalanine - Phe(p-COOMe), 4-cyanophenylalanine - Phe(p-CN), Phe, Tyr. The effect of the additional substituent at the phenyl ring of the Phe residue was investigated. All peptides contained an amide of 5-amino-2-nitrobenzoic acid, which served as a chromophore. Kinetic parameters (k(cat), K(M) and k(cat)/K(M)) of the peptides synthesized with bovine alpha-chymotrypsin were determined. The highest value of the specificity constant k(cat)/K(M), reaching 6.0 x 10(5) [M(-1)xs(-1)], was obtained for Ac-Phe-Ala-Thr-Phe(p-NO(2))-Anb(5,2)-NH(2). The replacement of the acetyl group with benzyloxycarbonyl moiety yielded a substrate with the value of k(cat) more than three times higher. Peptide aldehydes were synthesized with selected residues (Phe, Pal, Tyr, Phe(p-NO(2)) in position P(1) and potent chymotrypsin inhibitors were obtained. The dissociation constant (K(i)) with the experimental enzyme determined for the most active peptide, Tos-Phe-Ala-Thr-Phe(p-NO(2))-CHO, amounted to 1.12 x 10(-8) M.

S13.9 A proline-rich polypeptide (PRP) and its active nonapeptide (NP) fragment affect the expression of a receptor for peanut agglutinin (PNA) on murine thymocytes

Inhibition was dose dependent between 10 -7 and 10 -4 M dex. The dex effect disappeared after 6d of continuous exposure. In addition, total amounts of intracellular 3H-label, HMG, and mucin were reduced after 4 h of dex treatment. However, dex did not alter the stimulatory effect of methacholine on the secretory rate for total H M G and mucin. We suggest that dex may decrease glycoconjugate secretion by inhibiting glucosamine uptake and consequently the biosynthesis and release of glycoconjugates. Failure of dex to inhibit glycoconjugate secretion after 6d suggests that if similar effects occur in vivo, they may be transient. The mechanism by which inhibition is overcome requires further study. (Supported by NIH grants HL19171 and HL42384). 1. Marom et al.: Am. Rev. Respir. Dis., 1984, 129:62-65. 2. Leigh, M. W., Cheng, P. W., Boat, T. F. Biochem., 1989, 28:9440