Two polymorphs of a covalent complex between papain and a diazomethylketone inhibitor.

The three-dimensional structure of two polymorphs of a ZLFG-CH2-papain covalent complex has been determined by X-ray crystallography. The structures indicate that: (i) the methylene carbon atom of the inhibitor is covalently bound to the Sgamma atom of Cys25 of papain; (ii) the hydrophobic S2 pocket formed by Pro68, Val133, Val157, and Asp158 is occupied by the inhibitor's phenylalanyl P2 side chain; (iii) extensive hydrogen bonding and hydrophobic interactions are responsible for the interaction of the inhibitor with the enzyme. Comparison with similar structures suggests that in covalent complexes preservation of main chain-main chain interactions between the enzyme and the inhibitor may have higher priority than the P-S interactions.

Sequence determination and analysis of S-adenosyl-L-homocysteine hydrolase from yellow lupine (Lupinus luteus).

The coding sequences of two S-adenosyl-L-homocysteine hydrolases (SAHases) were identified in yellow lupine by screenig of a cDNA library. One of them, corresponding to the complete protein, was sequenced and compared with 52 other SAHase sequences. Phylogenetic analysis of these proteins identified three groups of the enzymes. Group A comprises only bacterial sequences. Group B is subdivided into two subgroups, one of which (B1) is formed by animal sequences. Subgroup B2 consist of two distinct clusters, B2a and B2b. Cluster B2b comprises all known plant sequences, including the yellow lupine enzyme, which are distinguished by a 50-residue insert. Group C is heterogeneous and contains SAHases from Archaea as well as a new class of animal enzymes, distinctly different from those in group B1.

Structural studies of cysteine proteases and their inhibitors.

Cysteine proteases (CPs) are responsible for many biochemical processes occurring in living organisms and they have been implicated in the development and progression of several diseases that involve abnormal protein turnover. The activity of CPs is regulated among others by their specific inhibitors: cystatins. The main aim of this review is to discuss the structure-activity relationships of cysteine proteases and cystatins, as well as of some synthetic inhibitors of cysteine proteases structurally based on the binding fragments of cystatins.

Human cystatin C, an amyloidogenic protein, dimerizes through three-dimensional domain swapping.

The crystal structure of human cystatin C, a protein with amyloidogenic properties and a potent inhibitor of cysteine proteases, reveals how the protein refolds to produce very tight two-fold symmetric dimers while retaining the secondary structure of the monomeric form. The dimerization occurs through three-dimensional domain swapping, a mechanism for forming oligomeric proteins. The reconstituted monomer-like domains are similar to chicken cystatin except for one inhibitory loop that unfolds to form the 'open interface' of the dimer. The structure explains the tendency of human cystatin C to dimerize and suggests a mechanism for its aggregation in the brain arteries of elderly people with amyloid angiopathy. A more severe 'conformational disease' is associated with the L68Q mutant of human cystatin C, which causes massive amyloidosis, cerebral hemorrhage and death in young adults. The structure of the three-dimensional domain-swapped dimers shows how the L68Q mutation destabilizes the monomers and makes the partially unfolded intermediate less unstable. Higher aggregates may arise through the three-dimensional domain-swapping mechanism occurring in an open-ended fashion in which partially unfolded molecules are linked into infinite chains.

Expression of a selenomethionyl derivative and preliminary crystallographic studies of human cystatin C.

Human cystatin C, a protein with amyloidogenic properties and a potent inhibitor of papain-like mammalian proteases, has been produced in its full-length form by recombinant techniques and crystallized in two polymorphic forms: cubic and tetragonal. A selenomethionyl derivative of the protein, obtained by Escherichia coli expression and with complete Met-->Se-Met substitution confirmed by mass spectrometry, amino-acid analysis and X-ray absorption spectra, was crystallized in the cubic form. A truncated variant of the protein, lacking ten N-terminal residues, has also been crystallized. The crystals of this variant are tetragonal and, like the two polymorphs of the full-length protein, contain multiple copies of the molecule in the asymmetric unit, suggesting oligomerization of the protein.

[Pulmonary embolism as a complication of nephrotic syndrome--case report and therapeutic management]. / Zatorowatosc pluc wiklajaca zespól nerczycowy--opis przypadku, postepowanie terapeutyczne.

A case of a 41-year-old man, who was treated due to severe steroid and immunoresistant nephrotic syndrome is described. During the pulsed steroid therapy the patient underwent a pulmonary embolism and developed the post-infarction cavern and abscess of the lung. In this study the case history, complications, diagnostic procedures and therapeutic management were described. The probable mechanism of the described complications was carefully discussed.

Leukocyte compartments in the nasal secretion medium.

Nasal secretions are known to play a role in respiratory tract and host defense. Besides the mucociliary transport and biochemical properties of the mucus, we hypothesize the role of a secretory leukocyte compartment. We designed a time-series study to count leukocytes in baseline, physically- and pharmacologically-induced secretions. Twenty-three healthy volunteers and 29 patients participated in the study. In healthy subjects, secretion weights significantly increased from 24 +/- 5 mg (mean +/- SEM) at baseline to 35 +/- 6 mg and 115 +/- 12 mg, respectively, in physically and methacholine-induced secretions (p < 0.05). The leukocyte count did not change between baseline (14,445 +/- 5,010) and physically induced secretions (13,396 +/- 6,8401), but significantly increased after methacholine (28,140 +/- 11,411; p = 0.02). Leukocyte differential counts showed, moreover, an increase of lymphocytes in the methacholine-induced compartment. In patients, secretion weights significantly increased from 70 +/- 12 mg at baseline to 117 +/- 22 mg and 223 +/- 28 mg, respectively. The leukocyte count significantly increased between baseline (172,109 +/- 95,890) and physically induced secretions (410,503 +/- 318,224; p = 0.02), but decreased after methacholine (112,774 +/- 54,860). These data argue for the existence of a secretory leukocyte compartment, with a subcompartment of surface and an intraglandular subcompartment of reserve, the kinetics of which are different in patients and control subjects.

Sputum eosinophilia negates need to perform sputum Gram's stain.

A total of 1878 sputum specimens were evaluated to assess the potential of encountering a sputum Gram's stain with clinically useful positive data in the presence of sputum eosinophilia. Wet preparations were used to assess the adequacy of the specimen and to quantitate eosinophils. Quantitative sputum Gram's stains were performed. When more than 50% of the cells observed on sputum wet preparation were eosinophils, there were no positive Gram's stains. When more than 20% of the cells were eosinophils, there was a 1% prevalence of potentially clinically useful positive Gram's stains. The data strongly suggest that sputum eosinophilia obviates the need to perform a sputum Gram's stain since it is extremely unlikely that it would be useful in diagnosing a bacterial infection of the lower respiratory tract.