Rapid identification of substrates for novel proteases using a combinatorial peptide library.

Fluorogenic substrates for assaying novel proteolytic enzymes could be rapidly identified using an easy, solid-phase combinatorial assay technology. The methodology was validated with leader peptidase of Escherichia coli using a subset of an intramolecularly quenched fluorogenic peptide library. The technique was extended toward the discovery of substrates for a new aspartic protease of pharmaceutical relevance (human napsin A). We demonstrated for the first time known to us that potent fluorogenic substrates can be discovered using extracts of cells expressing recombinant enzyme to screen the peptide library. The straightforward and rapid optimization of protease substrates greatly facilitates the drug discovery process by speeding up the development of high throughput screening assays and thus helps more effective exploitation of the enormous body of information and chemical structures emerging from genomics and combinatorial chemistry technologies.

Hydroxamic acid derivatives as potent peptide deformylase inhibitors and antibacterial agents.

Low-molecular-weight beta-sulfonyl- and beta-sulfinylhydroxamic acid derivatives have been synthesized and found to be potent inhibitors of Escherichia coli peptide deformylase (PDF). Most of the compounds synthesized and tested displayed antibacterial activities that cover several pathogens found in respiratory tract infections, including Chlamydia pneumoniae, Mycoplasma pneumoniae, Haemophilus influenzae, and Moraxella catarrhalis. The potential of these compounds as antibacterial agents is discussed with respect to selectivity, intracellular concentrations in bacteria, and potential for resistance development.

Purification and characterization of active recombinant human napsin A.

Recombinant human napsin A expressed in human embryonic kidney 293 cells was purified to homogeneity by a single-step procedure using part of napsin A propeptide as affinity ligand. N-Terminal amino-acid sequencing of the purified enzyme identified the mature form of napsin A. Treatment of purified napsin A with endoglycosidases F and H resulted in a decrease in its molecular mass from 39 kDa to approximately 37 kDa, confirming that napsin A is glycosylated. The kinetic properties were analyzed by using two fluorogenic synthetic substrates K(Dabsyl)-TSLLMAAPQ-Lucifer yellow (DS1) and K(Dabsyl)-TSVLMAAPQ-Lucifer yellow (DS3). The Km values obtained were 1.7 microM and 6.2 microM, respectively. A substrate-specificity study using a napsin A-targeted peptide library confirmed the preference of napsin A for hydrophobic residues at positions P1 and P1'. Adjacent positions, P2-P4 and P2'-P4', appeared less restricted in distribution of amino acids. A pH optimum between 4.0 and 5.5 at room temperature was determined. The purified enzyme was fully active for more than 10 h at pH 5.0 and 6.0, while a half-life of 4 h was determined at pH 7.0 and 37 degrees C.

Laparoscopic versus open adrenalectomy: outcome in 35 consecutive patients.

BACKGROUND: The use of minimally invasive techniques in the surgical treatment of adrenal masses has been used to remove a wide variety of adrenal tumors. AIMS: We have reviewed our experience with laparoscopic adrenalectomy and compared laparoscopic vs. open surgical approach. METHODS: The outcome of 35 consecutive patients who underwent adrenalectomy over a 3-year period has been analyzed retrospectively. Differences in operating time, blood loss, period of hospitalization, use of parenteral analgesia, resumption of oral feeding, complications, and time to return to normal activity after 18 coelioscopic vs. 17 open consecutive adrenalectomies have been considered. RESULTS: The average operative time was longer (mean 160 vs. 148 min, p = 0.48) and postoperative complications lower (4 vs. 5 cases, p =0.73), although not statistically significant, for the laparoscopic compared to the open surgical approach, whereas blood loss (30 vs. 165 ml; p = 0.01), postoperative analgesia (3.4 vs. 5.0 days, p = 0.02), time to restart oral feeding (3.0 vs. 4.7 days, p = 0.001), average time of hospitalization (4.5 vs. 9.6 days, p = 0.001), time to return to normal activity (21 vs. 37 days, p = 0.001) were all statistically significant. CONCLUSIONS: Laparoscopic adrenalectomy can be considered the method of choice for managing almost all adrenal masses, because of its lower morbidity and shorter postoperative recovery.