Selective increase in cytokeratin synthesis in cultured rat hepatocytes in response to hormonal stimulation

Addition of a combination of insulin, dexamethasone and EGF at seeding time to cultured rat hepatocytes in serum-free medium caused a selective increase in the biosynthesis of particular cytokeratin components. This increase was prominent during the first day in culture. No significant increases were detected in the absence of hormones or in the presence of either hormones added alone or in pairs, except in the case of insulin plus dexamethasone, which yielded an effect close to that obtained with the three factors. Interestingly, the latter condition also maintained a high level of albumin production over a 6-day period in culture.     

Methods for study of mutations and mutagenesis in human lymphocytes

Detailed methods are presented for measurement and study of in vivo mutations and in vitro mutagenesis in human lymphocytes. The methods described include preparation of conditioned medium containing interleukin-2, enumeration of mutant clones, in vitro mutagenesis, and expansion of mutant clones for further study.     

Enhancement of eosinophil effector function by soluble factors released by S. mansoni: role of proteases

Schistosomulum-released products (SRP) have been shown to enhance both expression of rat and human eosinophil Fc receptors and IgG-dependent cytotoxicity. The present work provides additional evidence of the secretion of eosinophil-enhancing factors by schistosomula and other developmental stages of schistosomes, including adult worms. The heat lability, as well as the strong inhibition of the stimulating activity of SRP by the protease inhibitor Trasylol, suggest that thermolabile proteases secreted by the parasite are involved in this mechanism. The purification of the schistosome proteases by preparative isoelectric focusing and gel filtration demonstrated that neutral proteases able to hydrolyze the collagenase substrates Azocoll and Z-Gly-Pro-Leu-Gly-Pro are able to significantly enhance eosinophil effector functions. Purified Clostridium histolyticum collagenase was also able to mimic the enhancing effect of schistosome proteases, suggesting involvement of a collagenase-like activity of the enzymes in the eosinophil stimulation.     

Studies on functional domains of the regulatory subunit of bovine heart adenosine 3':5'-monophosphate-dependent protein kinase

The functional domains of the regulatory subunit of isozyme II of cAMP-dependent protein kinase were studied. It was shown using Edman degradation that the regulatory subunit contained a phosphorylated residue which was very close in primary sequence to the site most sensitive to hydrolysis by low trypsin concentrations as postulated previously (Corbin, J.D., Sugden, P.H., West, L., Flockhart, D.A., Lincoln, T.M., and McCarthy, D. (1978) J. Biol. Chem. 253, 3997-4003). Catalytic subunit incorporated 0.9 mol of 32P from [gamma-32P]ATP into a preparation of regulatory subunit that contained 1.1 mol of endogenous phosphate. After phosphorylation by the catalytic subunit, the regulatory subunit contained 2.2 mol of chemical phosphate. The effects of heat denaturation upon the rate and extent of phosphorylation of the regulatory subunit were compared with the effects of these treatments upon the cAMP binding and inhibitory domains. These data suggested that the regulatory subunit required factors in addition to an intact phosphorylatable primary sequence in order for inhibitory activity to be expressed. Such factors might be part of the secondary or tertiary structure of the protein. These studies are discussed with respect to the mechanism of inhibition of catalytic activity, and a model of the regulatory subunit structure is proposed.     

Effects of detergents on catalytic activity of human endometase/matrilysin 2, a putative cancer biomarker

Matrix metalloproteinases (MMPs) are a family of hydrolytic enzymes that play significant roles in development, morphogenesis, inflammation, and cancer invasion. Endometase (matrilysin 2 or MMP-26) is a putative early biomarker for human carcinomas. The effects of the ionic and nonionic detergents on catalytic activity of endometase were investigated. The hydrolytic activity of endometase was detergent concentration dependent, exhibiting a bell-shaped curve with its maximum activity near the critical micelle concentration (CMC) of nonionic detergents tested. The effect of Brij-35 on human gelatinase B (MMP-9), matrilysin (MMP-7), and membrane-type 1 MMP (MT1-MMP) was further explored. Their maximum catalysis was observed near the CMC of Brij-35 (∼ 90 μM). Their IC₅₀ values were above the CMC. The inhibition mechanism of MMP-7, MMP-9, and MT1-MMP by Brij-35 was a mixed type as determined by Dixon’s plot; however, the inhibition mechanism of endometase was noncompetitive with a K_i value of 240 μM. The catalytic activities of MMPs are influenced by detergents. Monomer of detergents may activate and stabilize MMPs to enhance catalysis, but micelle of detergents may sequester enzyme and block the substrate binding site to impede catalysis. Under physiological conditions, a lipid or membrane microenvironment may regulate enzymatic activity.