Granulomatous mycosis fungoides presenting as poikiloderma.

Granulomatous mycosis fungoides (MF) is a rare subtype of MF, characterized by the histological presence of a granulomatous reaction, but distinct clinical characteristics are not present. A 41-year-old healthy man presented with poikiloderma, ichthyosis and erythematous scaly plaque. Histological examination of a biopsy taken from poikilodermic skin showed a granulomatous reaction to epidermotropic atypical lymphocytes. However, in other areas there were only findings of conventional MF without granuloma. Granulomatous MF may be associated with poikiloderma.

Long-range effect of mutation of calcium binding aspartates [correction of asparates] on the catalytic activity of alkaline protease from Pseudomonas aeruginosa.

Apart from a catalytic domain, the alkaline protease of Pseudomonas aeruginosa has a novel parallel beta-helix domain stabilized through Ca2+ binding. In order to clarify the importance of the beta-helix structure in maturation of the enzyme, aspartic residue D-356 or D-365 in the Ca2+ binding sequence motif was replaced with L-alanine, and the catalytic activity of each mutant was assayed. These mutants did not show any proteolytic activity, although the composition of their polypeptide chains was the same as that of the wild type except for the mutated alanine residue. These results suggest that D-356 and D-365 are important in control of the beta-helix folding induced by Ca2+ binding and that incomplete beta-helix folding due to the lack of their side-chains affects the maturation of the enzyme in the long-range order.

Structural gene and complete amino acid sequence of Vibrio alginolyticus collagenase.

The DNA encoding the collagenase of Vibrio alginolyticus was cloned, and its complete nucleotide sequence was determined. When the cloned gene was ligated to pUC18, the Escherichia coli expression vector, bacteria carrying the gene exhibited both collagenase antigen and collagenase activity. The open reading frame from the ATG initiation codon was 2442 bp in length for the collagenase structural gene. The amino acid sequence, deduced from the nucleotide sequence, revealed that the mature collagenase consists of 739 amino acids with an Mr of 81875. The amino acid sequences of 20 polypeptide fragments were completely identical with the deduced amino acid sequences of the collagenase gene. The amino acid composition predicted from the DNA sequence was similar to the chemically determined composition of purified collagenase reported previously. The analyses of both the DNA and amino acid sequences of the collagenase gene were rigorously performed, but we could not detect any significant sequence similarity to other collagenases.

Thermolysin catalyzed semisynthesis of peptide hormones by introduction of Phe-NH2 or Tyr-NH2 at the carboxyl termini.

The semisynthesis of C-terminal peptides of gastrin, calcitonin gene-related peptides (rat and human), and cholecystokinin, or of human neuropeptide Y was achieved by introduction of Phe-NH2 or Tyr-NH2 at their carboxyl termini, respectively. Thermolysin or the related enzyme was used for the purpose. The coupling was usually performed in the presence of high concentration of organic cosolvent, where the secondary hydrolysis was negligibly small.

Degradation of IgA proteins by Pseudomonas aeruginosa elastase.

Human colostral IgA and myeloma proteins of both IgA1 and IgA2 subclasses were susceptible to cleavage by Pseudomonas aeruginosa elastase. Detailed analysis of the cleavage products of IgA myeloma proteins revealed complete degradation of Fab with no evidence of intact Fab fragments as intermediate cleavage products. In contrast, both IgA1 and IgA2 proteins were resistant to cleavage by alkaline protease from P. aeruginosa. The susceptibility of human IgA proteins to elastase suggests a mechanism by which P. aeruginosa might evade the potentially protective function of IgA by producing this enzyme.

Degradation of soluble laminin and depletion of tissue-associated basement membrane laminin by Pseudomonas aeruginosa elastase and alkaline protease.

Purified Pseudomonas aeruginosa elastase and alkaline protease rapidly cleaved soluble laminin, with each enzyme yielding different cleavage products. These cleavage fragments were separated from the intact laminin A and B polypeptide chains by sodium dodecyl sulfate-polyacrylamide gradient gel electrophoresis and detected by their characteristic Coomassie blue staining patterns. Pseudomonas elastase produced rapid and extensive degradation of both A and B chains, including the disulfide-rich regions. Apparently complete degradation to limit digests was obtained after 30 min with a substrate/enzyme ratio of 30:0.5. Under similar conditions, alkaline protease rapidly degraded the A chain while slowly degrading the B chain. In addition, immunoreactive laminin was released from authentic basement membranes after incubation with either enzyme as detected by an enzyme-linked immunoabsorption assay and by immunofluorescence. The results from these studies suggest a direct role for elastase and alkaline protease in both tissue invasion and hemorrhagic tissue necrosis in P. aeruginosa infections.

Comparison of the subsite specificity of the mammalian neutral endopeptidase 24.11 (enkephalinase) to the bacterial neutral endopeptidase thermolysin.

A comparison has been made of the specificity of the mammalian neutral metalloendopeptidase, endopeptidase 24.11, with that of the bacterial neutral metalloendopeptidase thermolysin. A series of synthetic oligopeptides which have previously been studied as substrates for thermolysin and used in computer modeling were examined as substrates for the mammalian enzyme. It was found that P1, P2, and P'3 subsite interactions in the mammalian enzyme, although similar to those found in thermolysin, are less restrictive spatially and are considerably less dependent on hydrophobic interactions. This difference was maximally expressed with the synthetic substrate dansyl-D-alanylglycylnitrophenylalanylglycine which is a substrate for the mammalian enzyme, but not for the bacterial enzyme. A comparison of substrates in the free acid form with their corresponding amides showed that binding to the mammalian enzyme is dependent in part on an ionic interaction between the substrate carboxylate group and the enzyme. Such an ionic interaction was not observed with the bacterial enzyme.

Effects of proteases on the structure and activity of Pseudomonas aeruginosa exotoxin A.

The effects of various proteases on the enzymatic or biological activity and structure of exotoxin A from Pseudomonas aeruginosa were systematically studied. The toxin was extremely resistant to treatment with various enzymes. The lethality of the toxin disappeared upon treatment with P. aeruginosa protease and elastase, thermolysin, and trypsin with a long incubation time (5h) in the presence of a high enzyme concentration (molar concentration of enzyme to toxin, 1:10 or 1:20), but was little altered by either alpha-chymotrypsin or subtilisin. The decrease of adenosine diphosphate ribosylation activity was moderate when the same treatment was used, regardless to the protease source, except in the case of papain, which was tested in the presence of reducing agents. The increase in activation of the treated toxin determined in the presence of a denaturant and a reducing agent was less than that of the intact toxin, except in the case of trypsin. The differences in disc and sodium dodecyl sulfate gel electropherograms of the toxins treated with these proteases, except for those treated with papain, suggested that the toxins had been nicked by the protease, which resulted in their degradation by sodium dodecyl sulfate treatment. Papain degraded the toxin into fragments and caused the disappearance of lethality or a marked decrease of adenosine diphosphate ribosylation activity.

alpha-Chymotrypsin as the catalyst for peptide synthesis.

alpha-Chymotrypsin (EC 3.4.21.1)-catalysed syntheses of peptides were performed with various N-acylated amino acid or peptide esters as donors, and amino acid derivatives, peptides or their derivatives as acceptors. Under optimal conditions the synthesis was almost quantitative. As acceptor nucleophiles, free amino acids or the ester derivatives were inadequate, but amino acid amides or hydrazides, di- or tri-peptides, or the amides, hydrazides and esters of the peptides were useful. The nucleophile specificity for synthesis was markedly similar to the leaving-group specificity in hydrolysis; hydrophobic or bulky amino acid residues were most effecient at both P1' and P2' positions [notation of Schechter & Berger (1967) Biochem. Biophys. Res. Commun. 27, 157-162], but L-proline as well as D-amino acid residues were the worst choices. The synthesis was further dependent on the solubility of the products synthesized; a higher yield of products was expected with lower solubility. As donor esters, good substrates were all useful. Accordingly, fragment condensation was possible by using N-acylated peptide esters and various peptides. The present study suggested that alpha-chymotrypsin may become a useful tool for peptide synthesis.