Inhibition of estradiol-induced early osteoarthritic changes by tamoxifen.

Previous studies on osteoarthritic changes induced by intraarticular injections of estradiol benzoate (EB) suggest estrogen as a risk factor in the development of knee osteoarthritis (OA). The present study observed the anti-arthritic effects of tamoxifen (TMX). Oophorectomized rabbits were subjected to intraarticular injections of vehicle control, EB, TMX, or EB/TMX for 3 weeks. The cartilage changes were assessed by light and scanning electron microscopic examination, enzyme histochemical analysis, and the amount of alcian blue stain binding to glycosaminoglycans. EB injections resulted in cell necrosis, chondrocyte clonings, and pittings, whereas the vehicle control, TMX, and EB/TMX-injected groups showed no histologic abnormalities. Histochemical analysis showed that the numbers of lactate dehydrogenase (LDH)-reactive chondrocytes in the EB-injected group were significantly reduced when compared to other groups (p less than 0.001). The injections of EB/TMX significantly reduced the chondrocyte numbers in the lateral superficial layer (p less than 0.05), compared with the vehicle injection. TMX-injected group revealed slight although insignificant decreases in chondrocyte numbers. The amount of alcian blue stains, representing the relative amount of proteoglycans, significantly decreased only in the superficial layer of the EB- and EB/TMX-injected groups (p less than 0.05). TMX, when concurrently injected with EB, antagonized the chondrodestructive effects of estradiol at the early stage of knee OA in rabbits. The results suggest the potential therapeutic use of TMX at the early stage of OA.     

Mechanism of activation of cholera toxin by ADP-ribosylation factor (ARF): both low- and high-affinity interactions of ARF with guanine nucleotides promote toxin activation

Activation of adenylyl cyclase by cholera toxin A subunit (CT-A) results from the ADP-ribosylation of the stimulatory guanine nucleotide binding protein (GS alpha). This process requires GTP and an endogenous guanine nucleotide binding protein known as ADP-ribosylation factor (ARF). One membrane (mARF) and two soluble forms (sARF I and sARF II) of ARF have been purified from bovine brain. Because the conditions reported to enhance the binding of guanine nucleotides by ARF differ from those observed to promote optimal activity, we sought to characterize the determinants influencing the functional interaction of guanine nucleotides with ARF. High-affinity GTP binding by sARF II (apparent KD of approximately 70 nM) required Mg2+, DMPC, and sodium cholate. sARF II, in DMPC/cholate, also enhanced CT-A ADP-ribosyltransferase activity (apparent EC50 for GTP of approximately 50 nM), although there was a delay before achievement of a maximal rate of sARF II stimulated toxin activity. The delay was abolished by incubation of sARF II with GTP at 30 degrees C before initiation of the assay. In contrast, a maximal rate of activation of toxin by sARF II, in 0.003% SDS, occurred without delay (apparent EC50 for GTP of approximately 5 microM). High-affinity GTP binding by sARF II was not detectable in SDS. Enhancement of CT-A ADP-ribosyltransferase activity by sARF II, therefore, can occur under conditions in which sARF II exhibits either a relatively low affinity or a relatively high affinity for GTP. The interaction of GTP with ARF under these conditions may reflect ways in which intracellular membrane and cytosolic environments modulate GTP-mediated activation of ARF.     

Mechanism of adenylate kinase. Structural and functional demonstration of arginine-138 as a key catalytic residue that cannot be replaced by lysine

Replacement of the arginine-138 of adenylate kinase (AK) by lysine or methionine resulted in a decrease in kcat by a factor of 10(4), increases in Km by a factor of 10-20, and relatively little changes in dissociation constants. Proton nuclear magnetic resonance (NMR) studies were then undertaken to obtain structural information for quantitative interpretation of the kinetic data. Since the lysine mutant (R138K) represents a conservative mutation with surprisingly large effects on kinetics, structural studies were focused on the wild type (WT) and R138K. The results and conclusions are summarized as follows: (i) The aromatic spin systems of WT and R138K were assigned from total correlated spectroscopy (TOCSY). Comparison of the chemical shifts of aromatic protons, one-dimensional spectra, TOCSY, and nuclear Overhauser enhanced spectroscopy (NOESY) indicated that the conformation of R138K was almost unperturbed relative to that of WT. Thus Arg-138 is not important for the tertiary structure. (ii) Proton NMR titrations with AMP and MgATP suggested that substrate binding affinities and substrate-induced conformational changes are nearly identical between WT and R138K. Thus arginine-138 should not be involved in stabilizing the first substrate in the binary complex. (iii) Notable differences were observed between the proton NMR spectra of the WT and R138K complexes with the reaction mixture, which agrees with the perturbation in the Km values of R138K. The differences were analyzed in detail by using a "static reaction mixture'--p1, p5-bis(5'-adenosyl)pentaphosphate (MgAP5A). The aromatic spin systems of WT + MgAP5A and R138K + MgAP5A were partially assigned from various two-dimensional spectra.(ABSTRACT TRUNCATED AT 250 WORDS)     

Mechanism of adenylate kinase. Are the essential lysines essential?

Using site-specific mutagenesis, we have probed the structural and functional roles of lysine-21 and lysine-27 of adenylate kinase (AK) from chicken muscle expressed in Escherichia coli. The two residues were chosen since according to the nuclear magnetic resonance (NMR) model [Mildvan, A. S., & Fry, D. C. (1987) Adv. Enzymol. 58, 241-313], they are located near the alpha- and the gamma-phosphates, respectively, of adenosine 5'-triphosphate (ATP) in the AK-MgATP complex. In addition, a lysine residue (Lys-21 in the case of AK) along with a glycine-rich loop is considered "essential" in the catalysis of kinases and other nucleotide binding proteins. The Lys-27 to methionine (K27M) mutant showed only slight increases in kcat and Km, but a substantial increase (1.8 kcal/mol) in the free energy of unfolding, relative to the WT AK. For proper interpretation of the steady-state kinetic data, viscosity-dependent kinetics was used to show that the chemical step is partially rate-limiting in the catalysis of AK. Computer modeling suggested that the folded form of K27M could gain stability (relative to the wild type) via hydrophobic interactions of Met-27 with Val-179 and Phe-183 and/or formation of a charge-transfer complex between Met-27 and Phe-183. The latter was supported by an upfield shift of the methyl protons of Met-27 in 1H NMR. Other than this, the 1H NMR spectrum of K27M is very similar to that of WT, suggesting little perturbation in the global or even local conformations.(ABSTRACT TRUNCATED AT 250 WORDS)     

Three-dimensional growth and morphogenesis of mouse submandibular epithelial cells in serum-free primary culture

Mouse submandibular epithelial cells can be grown in primary culture using the collagen gel matrix and a chemically defined medium consisting of insulin, transferrin, cholera toxin, and BSA (or FGF). Sustained cell growth leading to a 5–10-fold increase in cell number was observed in less than 2 weeks. In the presence of these additives, clumps of cells proliferate by extending ‘star-like’ projections into the matrix, resulting in three-dimensional outgrowths. The morphology of these outgrowths can be modulated to form a ‘cyst-like’ appearance by deleting BSA and adding cortisol to the basal medium containing insulin, transferrin, cholera toxin and FGF. In brief, a serum-free medium for sustained growth has been devised and a simple manipulation of supplements can modulate the three-dimensional colony morphology in the collagen gel matrix. Finally, the resulting outgrowths can produce epidermal growth factor (EGF) in response to dihydrotestosterone.