Comparative molecular modeling analysis of­5‐amidinoindole and benzamidine binding to thrombin and trypsin: specific H‐bond formation contributes to high 5‐amidinoindole potency and selectivity for thrombin and factor Xa

The coagulation cascade enzymes thrombin and factor Xa are known to have specificity pockets very similar to those of trypsin and plasmin. However, comparative molecular modeling analysis of the crystal structures of benzamidine–thrombin and benzamidine–trypsin, in conjunction with a docking analysis of 5‐amidinoindole and related inhibitors in both enzymes reveals subtle differences between the specificity sites of the two types of enzymes. Specifically, thrombin and factor Xa, which have an alanine residue at position 190, exhibit increased activities for the rigid and more bulky bicyclic derivatives of benzamidine (e.g. amidinobenzofuran, amidinothiophene and amidinoindole), because of additional hydrophobic and H‐bond interactions between the inhibitors and the specificity sites, whereas enzymes with a serine residue at position 190, like trypsin and plasmin, exhibit little difference in activity among the same set of compounds because of the orientational restriction imposed on the inhibitors by Ser190, which forms an additional H‐bond with the amidino group of the inhibitors. Enzymes of both groups show similar responses to the flexible aminobenzamidine since the smaller size and the rotatable anilino group of the inhibitor would allow the inhibitor to achieve favorable electrostatic interactions with both groups of enzymes. 5‐Amidinoindole is the most dramatic example of the rigid bicyclic type inhibitor. Based on our docking analysis, we propose that a selective H‐bond with the hydroxyl group of the catalytic Ser195 and the subtle differences in steric fit imposed by Ala/Ser at position 190 explain the high potency and selectivity of 5‐amidinoindole for thrombin and factor Xa. Copyright © 1999 John Wiley & Sons, Ltd.     

Estimation of disease severity in the NHS cervical screening programme. Part I: artificial cut‐off points and semi‐quantitative solutions

R. G. Blanks
Estimation of disease severity in the NHS cervical screening programme. Part I: artificial cut‐off points and semi‐quantitative solutions Objective: Current cytology and histology classifications are based on ordered categories and have a strong emphasis on providing information that decides a woman's management rather than the best estimate of disease severity. This two‐part paper explores the use of a quantitative approach to both cytology and histology disease severity measurements. Methods: In Part I the problem of artificial cut‐off points is discussed and a simple semi‐quantitative solution to the problem is proposed. This closely relates to the revised British Society for Clinical Cytology (BSCC) terminology. The estimates of disease severity are designed as extensions of the existing methods, with an emphasis on probability rather than certainty, as a more natural way of approaching the problem. Borderline changes are treated as categorical variables, but koilocytosis, mild, moderate and severe dyskaryosis, and ?invasive as quasi‐continuous and the disease severity estimated as a grade number (GN) with any value between 0–4 and the margin of error as a calculated grade range (CGR). Results: As an example, if the reader is unsure between moderate dyskaryosis (HSIL favouring CIN2) and mild dyskaryosis (LSIL favouring CIN1) they can register this uncertainty as a probability, such as 60%/40% moderate/mild. With 2 and 1 as the mid‐points of the grade numbers for moderate and mild dyskaryosis the GN value is ((60 × 2) + (40 × 1))/100 = 1.6. The CGR is 1.5 ? 0.4 to 1.5 + 0.6 = 1.1 to 2.1. The GN (CGR) estimate of disease severity is therefore 1.6 (1.1–2.1). In a similar manner the disease severity from all slides showing koilocytosis or dyskaryosis can be estimated as a number between 0 and 4 with an associated error. Histology can be treated in a similar way. Conclusions: This semi‐quantitative approach provides a framework more suitable for research and audit of disease severity estimates. It avoids the paradox inherent in the current systems using artificial cut‐points to produce categories whereby increasing agreement can only be achieved by losing information.     

Glucocorticoids coordinately regulate type I collagen proα1 promoter activity through both the glucocorticoid and transforming growth factor β response elements: A novel mechanism of glucocorticoid regulation of eukaryotic genes

Glucocorticoids have previously been shown to decrease Type 1 collagen synthesis in vivo and in fibroblast cell culture. Several studies have demonstrated that glucocorticoids decrease Type 1 procollagen gene expression. These latter studies have included uridine incorporation into proα1(I) and proα2(1) mRNas and nuclear run-off experiments. Using the ColCat 3.6 plasmid, which contains part of the 5' flanking regionof the proα1 (1) coullagen gene and the reporter gene, chljoramphenicol acetyltransferase, the present studies demonstrate by stable transfection of fetal rat skin fibrolblasts that dexamethasone down regulates the promoter activity of the proα1(I) collagen gene. The glucocorticoid-mediated down-regulastionof procolljagen gene expression was demonstrated using the ColCat 3.6, 2.4, 1.7, or 0.9 plasmid. In addition, competitive oligonucleotide transfection experiments and site specific mutation of the glucocorticoid response element (GRE) in the whoulue ColCat 3.6 plasmid did not elimiinatre the effect. The ipossibility existed that another cis-element inthe 5' flanking region of the proα1(I) collagen gene was also required for the glucocorticoid-mediated down-regulation of procollagen gene expression, since TGF-β has been shown to stimulate collagen proα1(I) and proα2(I) gene activities. Dexamethasone treatment of non-transfected skin fibroblasts did result in a decrease of transforming growth factor-β. The decrease of CVAT activity by dexamethasone was brought back to control value by the addition of exogenous TGF-β to the culture media. Gel mobility studies demonstrated that glucocorticoid treatment of rat skin fibroblasts decreased glucocorticoid recptor binding to the GRE and TGF-β activator protein to the TGF-β element which were brought back to control values by coordinate exogenous TGF-β treatment. Thus the interaction of these TGF-β molecules with cellular membrane receptors and subsequent rtransduction is dramatically decreased resulting in less signals to regulate collagen gene expression. These data indicate that glucocorticoids coordinately regulate procollagen gene expfrssion through both the GRE and TGF-β elements. Depression of procollagen gene expression by glucocorticoids through the TGF-β element is mediated by decreased TGF-β secretion, possibly involving a secondary effect on regulatory protein(s) encoded by noncollagenous protein gene(s). The present studies provide the bassis for a novel mechanism of glucocorticoid-mediated regulation of eukaryotic genes containing the TGF-β element. © 1995 Wiley-Liss, Inc.