pH stability of the stromelysin-1 catalytic domain and its mechanism of interaction with a glyoxal inhibitor.

The stromelysin-1 catalytic domain(83-247) (SCD) is stable for at least 16 h at pHs 6.0-8.4. At pHs 5.0 and 9.0 there is exponential irreversible denaturation with half lives of 38 and 68 min respectively. At pHs 4.5 and 10.0 irreversible denaturation is biphasic. At 25°C, C-terminal truncation of stromelysin-1 decreases the stability of the stromelysin-1 catalytic domain at pH values >8.4 and <6.0. We describe the conversion of the carboxylate group of (ßR)-ß-[[[(1S)-1-[[[(1S)-2-Methoxy-1-phenylethyl]amino]carbonyl]-2,2-dimethylpropyl]amino]carbonyl]-2-methyl-[1,1'-biphenyl]-4-hexanoic acid (UK-370106-COOH) a potent inhibitor of the metalloprotease stromelysin-1 to a glyoxal group (UK-370106-CO(13)CHO). At pH 5.5-6.5 the glyoxal inhibitor is a potent inhibitor of stromelysin-1 (K(i)=~1µM). The aldehyde carbon of the glyoxal inhibitor was enriched with carbon-13 and using carbon-13 NMR we show that the glyoxal aldehyde carbon is fully hydrated when it is in aqueous solutions (90.4ppm) and also when it is bound to SCD (~92.0ppm). We conclude that the hemiacetal hydroxyl groups of the glyoxal inhibitor are not ionised when the glyoxal inhibitor is bound to SCD. The free enzyme pK(a) values associated with inhibitor binding were 5.9 and 6.2. The formation and breakdown of the signal at ~92ppm due to the bound UK-370106-CO(13)CHO inhibitor depends on pK(a) values of 5.8 and 7.8 respectively. No strong hydrogen bonds are present in free SCD or in SCD-inhibitor complexes. We conclude that the inhibitor glyoxal group is not directly coordinated to the catalytic zinc atom of SCD.

Methylglyoxal, glyoxal, and their detoxification in Alzheimer's disease.

The accumulation of advanced glycation end products (AGEs) in brains with Alzheimer's disease (AD) has been implicated in the formation of insoluble deposits such as amyloid plaques and neurofibrillary tangles. AGEs are also known to activate glia, resulting in inflammation and neuronal dysfunction. As reactive intermediates of AGE formation, neurotoxic reactive dicarbonyl compounds such as glyoxal and methylglyoxal have been identified. One of the most effective detoxification systems for methylglyoxal and glyoxal is the glutathione-dependent glyoxalase system, consisting of glyoxalase I and glyoxalase II. In this study, we have determined the methylglyoxal and glyoxal levels in the cerebrospinal fluid of AD patients compared to healthy controls. Methylglyoxal levels in AD patients were twofold higher than in controls, but this difference was not significant due to the large intergroup variations and the small sample size. However, the concentrations of both compounds were five to seven times higher in CSF than in plasma. We also investigated the glyoxalase I level in AD and healthy control brains. The number of glyoxalase I- positive neurons were increased in AD brains compared to controls. Our findings suggest that glyoxalase I is upregulated in AD in a compensatory manner to maintain physiological methylglyoxal and glyoxal levels.

Experimental gluing of lung parenchyma in rats.

Gelatin-resorcinol-dialdehyde adhesive has been developed from a gelatin-resorcinol-formaldehyde adhesive by replacing the formaldehyde with two less histotoxic dialdehydes, ethandial and pentandial. The aim of the present study was to evaluate the usefulness of this modified composition in gluing defects in lung parenchyma. In 40 male Wistar rats a standardized lung incision 1.0 cm in length and 0.8 cm in depth were closed by application of gelatin-resorcinol-dialdehyde adhesive. For macroscopic and microscopic examination 4 animals were sacrificed on each of postoperative days 2, 7, and 14 and 14 animals on each of postoperative days 28 and 120. Macroscopic examination revealed a tight closure of the parenchymal defects in all postoperative stages. Initially by an adhesive layer and later on by granulation tissue and scar tissue respectively. On microscopic examination an inflammatory tissue response with polymorphonuclear neutrophils and macrophages predominating was found 2 days postoperatively. After 7 days multinucleated giant cells appeared. On postoperative day 14 the tissue response presented a distinct granulomatous character with multinucleated giant cells persisting. After 28 days remnants of adhesive surrounded by granulation tissue were detectable. On postoperative day 120 the adhesive had been completely resorbed and the parenchymal defect was replaced by fibrous scar tissue. The gelatin-resorcinol-adhesive proved effective in tight closure of lung parenchyma in rats. The adhesive is resorbed completely and does not interfere with parenchymal healing.

Mutagenicity on chlorination of products formed by ozonation of naphthoresorcinol in water.

The mutagenicity of products formed by chlorination after ozonation of naphthoresorcinol in aqueous solution was assayed with Salmonella typhimurium strains TA98 and TA100 in the presence and absence of S9 mix from phenobarbital- and 5,6-benzoflavone-induced rat liver. Ozonated and subsequently chlorinated naphthoresorcinol was directly mutagenic, as was ozonated naphthoresorcinol, in both strains tested. The mutagenic activity at chlorination with 8 equivalents of chlorine per mole of naphthoresorcinol after ozonation was markedly higher than that at only ozonation. Of the identified ozonation products of naphthoresorcinol, muconic acid, after chlorination with 2 or 4 equivalents of chlorine per mole of the compound, induced direct mutagenicity against TA98 and TA100. The chlorination of glyoxal with 0.5 and 1 chlorine equivalents per mole of the compound was shown to produce direct mutagenicity toward TA98. The identification of the chlorination products of these compounds is also discussed.