Characterization of the inhibitory effect of glucocorticoids on the DNA replication of adult rat hepatocytes growing at various cell densities

Dexamethasone inhibited the basal and EGF-stimulated DNA synthesis of adult rat hepatocytes in primary culture. The inhibition was glucocorticoid-specific: It was shown by dexamethasone and hydrocortisone, but not by progesterone, testosterone, or estradiol; and was counteracted by the glucocorticoid antagonist RU-38486 in a concentration-dependent manner. Dexamethasone acted by decreasing the rate of entry into S-phase (kG1/S), while cell cycle parameters were unaffected. The steroid was able to decrease the kG1/S severalfold even when added more than 20 hr after EGF, half-maximal effect occurring 11 hr after the addition of dexamethasone. Densely populated areas were much more sensitive to the inhibition by dexamethasone than sparsely populated areas within the same culture dish: A moderate (10 nM) concentration of dexamethasone nearly abolished the DNA synthesis in densely populated areas of hepatocyte cultures with only marginal effect on sparsely populated cells.     

Detection of lipopolysaccharides blotted to polyvinylidene difluoride membranes

Bacterial lipopolysaccharide (LPS) blotted to polyvinylidene difluoride (PVDF) membranes was detected by a technique adapted from current methodologies used to detect glycoproteins. PVDF-bound LPS was coupled to a hapten and localized on the membrane by Western blotting with an antibody-alkaline phosphatase conjugate specific for the hapten. Immobilon blots could be made reversibly transparent for photography and densitometry.     

Kinetic studies on the inactivation of 5-lipoxygenase by 5(S)-hydroperoxyeicosatetraenoic acid

The oxygenation of arachidonic acid (AA) by guinea-pig neutrophil 5-lipoxygenase terminates prematurely at a substrate utilization of only 50%. In the presence of dithiothreitol (DTT), reaction progress continues longer but still terminates prematurely, at about 70% substrate turnover. The addition of more substrate during the first 60 seconds of the initial reaction resulted in continued product formation. However, at times after 120 seconds, the addition of more AA could not produce additional product formation. Together, these results indicate a time-dependent (t1/2 = 0.5-1.0 min), irreversible loss of enzyme activity. To determine if the product 5-hydroperoxy-6,8,11,14-eicosatetraenoic acid (5-HPETE) mediates the inactivation, it was tested for its ability to irreversibly inhibit the enzyme and found to inactivate 5-lipoxygenase with Ki = 0.05 +/- 0.01 microM and ki = 1.4 +/- 0.4 min-1. DTT changed the apparent affinity of 5-HPETE (Ki = 0.33 +/- 0.09 microM) but had no effect on the rate of inactivation (ki = 1.26 +/- 0.62 min-1). In contrast, the hydroxy derivative of 5-HPETE, 5-hydroxy-6,8,11,14-eicosatetraenoic acid (5-HETE), is a reversible, time-independent inhibitor with Ki = 6.3 +/- 0.9 microM regardless of DTT. The ability of thiols to protect 5-lipoxygenase from production inactivation is due, at least in part, to a non-enzymatic reaction between DTT and 5-HPETE that converts the hydroperoxy acid to a material that can no longer inactivate the enzyme.     

Promotion of Stomatal Opening by Indoleacetic Acid and Ethrel in Epidermal Strips of Vicia faba L

Indole-3-acetic acid (IAA), at concentrations of 0.01 to 1.0 millimolar, and ethephon (0.3% v/v Ethrel) promote stomatal opening when applied to epidermal peels of Vicia faba L. in light or dark. The effect of ethylene is seen by 30 minutes and maximal opening (over two times that of untreated controls) occurs after only 60 to 90 minutes in the light. Stomatal opening by IAA and Ethrel in both light and dark is prevented by 0.14 millimolar AgCl. It is suggested that the effect of added IAA, but not that of light, is linked to ethylene production. The possible role of ethylene in stomatal opening during fungal infection is discussed. The stomates of Vicia faba provide a new system to study the effects of ethylene on certain membrane-regulated processes.     

Requirement for BSF-1 in the induction of antigen-specific B cell proliferation by a thymus-dependent antigen and carrier-reactive T cell line

We report here a role of B cell stimulatory factor 1 (BSF-1) in the induction of antigen-specific proliferation of affinity-purified small B lymphocytes by a thymus-dependent antigen and a carrier-reactive T cell line. By using an ovalbumin-reactive T cell line (designated Hen-1), which does not produce BSF-1 following activation, it was possible to demonstrate that the antigen-specific proliferative response of trinitrophenyl (TNP)-binding B cells to TNP-ovalbumin required exogenous BSF-1 in addition to direct interaction with irradiated Hen-1 T cells. The activation obtained under these conditions was highly efficient, being sensitive to antigen doses as low as 0.001 microgram/ml. The addition of saturating amounts of BSF-1 did not alter the antigen-specificity or the requirements for hapten-carrier linkage or major histocompatibility complex-restricted T-B interaction in this system. The involvement of BSF-1 was confirmed by the ability of 11B11 anti-BSF-1 antibody to specifically suppress the response of TNP-binding B cells to TNP-ovalbumin, BSF-1, and irradiated Hen-1 T cells. Finally, this response was augmented by addition of the monokine interleukin 1. These data indicate that the proliferative response of small B cells to the thymus-dependent antigen and carrier-reactive T cell line used in our experiments can be regulated by the same factors that govern B cell proliferation induced by thymus-independent type 2 antigens or anti-IgM antibodies.     

Active site of human liver aldehyde dehydrogenase

Bromoacetophenone (2-bromo-1-phenylethanone) functions as an affinity reagent for human aldehyde dehydrogenase (EC 1.2.1.3) and has been found specifically to label a unique tryptic peptide in the enzyme. Amino-terminal sequence analysis of the labeled peptide after purification by two different procedures revealed the following sequence: Val-Thr-Leu-Glu-Leu-Gly-Gly-Lys. Radioactivity was found to be associated with the glutamate residue, which was identified as Glu-268 by reference to the known amino acid sequence. This paper constitutes the first identification of an active site of aldehyde dehydrogenase.