Publications

The factors operative during normal and disease states which govern the degradation of the glomerular extracellular matrix (ECM, glomerular basement membrane and mesangial matrix) remain poorly understood. These structures consist primarily of types IV and V collagen, which are highly resistant to the action of classical interstitial collagenases. Here we report that an intrinsic glomerular cell type, the human mesangial cell, secretes: 1) a neutral proteinase with specific activity against type IV basement membrane collagen and, 2) an inhibitory protein with the characteristics of the tissue inhibitor of metalloproteinases (TIMP). The purified enzyme has a neutral pH optimum, consists of a basic (pI 8.4) protein doublet with molecular weight of 66 and 68 kilodaltons, and is inhibited by calcium and zinc chelators. The enzyme degrades both soluble and basement membrane type IV collagen, but does not specifically degrade types I or V collagen or casein. Immunohistochemistry of cultured cells demonstrated homogeneous staining for the neutral proteinase antigen, while the TIMP antigen was detected in fewer than one-third of cultured cells. These findings suggest that the synthesis of these proteins may be independently regulated. The secretion of these factors may play an important role in the turnover of the glomerular ECM under basal or pathologic conditions.

We have reported [Correia et al. (1987) Arch. Biochem. Biophys. 258, 436-443] that administration of 3,5-dicarbethoxy-4-ethyl-2,6-dimethyl-1,4-dihydropyridine (DDEP) to untreated, phenobarbital (PB) pretreated, or dexamethasone (DEX) pretreated rats results in relatively selective inactivation of cytochrome P-450 (P-450) isozymes h (CYP2C11), k (CYP2C6), and p (CYP3A). Such inactivation involves destruction of P-450 prosthetic heme predominantly by N-ethylation in untreated and PB-pretreated rats, whereas in DEX-pretreated rats, it also appears to be associated with prosthetic heme alkylation of the apocytochrome presumably at the active site. The cause for this differential course of DDEP-mediated P-450 heme destruction is unclear. Since this process is absolutely dependent on NADPH-mediated DDEP metabolism and can be reproduced in vitro, in search of mechanistic clues, we have examined DDEP metabolism by liver microsomes from the three rat sources as well as by isolated purified rat liver P-450h and P-450k. HPLC analyses of microsomal incubations of DDEP with NADPH, in the presence of an esterase inhibitor, revealed the presence of two major products: deethylated pyridine (DP) and 4-ethylpyridine (4-EDP) with product ratios (DP/4-EDP) of 1.4, 1.4, and 0.7 for reactions catalyzed by liver microsomes from untreated, PB-pretreated, and DEX-pretreated rats, respectively. The corresponding mean product ratios for P-450h- and P-450k-catalyzed reactions were 4.2 and 5.5, respectively. On the other hand, partition ratios (DP formed/P-450 destroyed) ranged from 12.0, 10.5, and 4.8, respectively, for incubations of liver microsomes from untreated, PB-pretreated, and DEX-pretreated rats to 9.5 and 28.9 for purified P-450h- and P-450k-catalyzed reactions, respectively. However, DP formation in all these microsomal systems was comparable, and although 4-EDP formation was greatly stimulated by DEX pretreatment, it does not appear to be a destructive pathway. In view of this, our findings reported herein suggest that the active site environment of P-450's h, k, and p apparently determines not only the pattern of DDEP metabolism but also the differential course of prosthetic heme destruction.

The rex gene of the type I human T-cell leukaemia virus (HTLV-I) encodes a phosphorylated nuclear protein of relative molecular mass 27,000 which is required for viral replication. The Rex protein acts by promoting the cytoplasmic expression of the incompletely spliced viral messenger RNAs that encode the virion structural proteins. To identify the biologically important peptide domains within Rex, we introduced a series of mutations throughout its sequence. Two distinct classes of mutations lacking Rex biological activity were identified. One class corresponds to trans-dominant repressors as they inhibit the function of the wild-type Rex protein. The second class of mutants, in contrast, are recessive negative, rather than dominant negative, as they are not appropriately targeted to the cell nucleus. These results indicate the presence of at least two functionally distinct domains within the Rex protein, one involved in protein localization and a second involved in effector function. The trans-dominant Rex mutants may represent a promising new class of anti-viral agents.