Gene transfer by adenovirus in smooth muscle cells.

We report adenovirus-mediated gene transfer into airway smooth muscle cells in cultured cells and organ-cultured tracheal segments. Incubation of cultured rat tracheal myocytes with virus (5 x 10(8) pfu/ml) for 6 h resulted in beta-galactosidase expression in 94.8 +/- 2.5% of cells (n = 4). Following incubation of thin (less than 200 microns diameter) equine trachealis muscle segments with virus in organ culture (5 x 10(8)-5 x 10(10) pfu/ml) the average expression of the Lac Z gene was approximately 19 +/- 10% (n = 9). Expression was markedly improved, however, in segments from neonatal rats (13-21 days). In two experiments in which the mucosa and serosa were removed, nearly all cells expressed beta-galactosidase, whereas in a third experiment in which the tissue was not dissected, about 40% of cells were stained. Viral infection had no effect on tension development of strips following organ culture. In vitro gene transfer may provide a useful method to alter protein expression and examine the effect of this alteration on excitation/contraction coupling in smooth muscle.

Threonine synthase of Escherichia coli: inhibition by classical and slow-binding analogues of homoserine phosphate.

L-threo-3-Hydroxyhomoserine phosphate, derived from the antimetabolites L-threo-3-hydroxyaspartate and L-threo-3-hydroxyhomoserine [Shames, S. L., Ash, D. E., Wedler, F. C., and Villafranca, J. J. (1984) J. Biol. Chem. 258, 15331-15339], is a classical competitive inhibitor of threonine synthase (Ki = 6 microM) with structural elements of both substrate and product. L-2-Amino-5-phosphonovaleric acid also inhibits the enzyme competitively with a Ki (31 microM), comparable to Km for L-homoserine phosphate. In contrast, a structural analogue of Hse-P, L-2-amino-3-[(phosphonomethyl)thio]propionic acid exhibits a Ki = 0.11 microM (ca. 100-fold less than Km for L-Hse-P), along with "slow, tight" inhibition kinetics. Nuclear magnetic resonance was used with these inhibitors to probe for pyridoxal phosphate-catalyzed hydrogen-deuterium exchange reactions characteristic of substrates. With L-threo-3-hydroxy-homoserine phosphate, H-D exchange occurs only at the C-alpha position, but for homoserine in the presence of phosphate and for L-2-amino-5-phosphonovaleric acid and L-amino-3[(phosphonomethyl)thio]propionic acid (APMTP), H-D exchange occurs at C-alpha and stereospecifically at C-beta. For L-homoserine plus phosphate and L-2-amino-5-phosphonovaleric acid, the rate of H-D exchange at C-alpha is 8-45 times faster than at C-beta. For L-2-amino-3-[(phosphonomethyl)thio]propionic acid, the C-alpha to C-beta exchange rate ratio is near unity, due to a 700-fold decrease in the C-alpha rate for the analogue. Taken with information from molecular modeling, these data can be interpreted in terms of the current working hypothesis for the catalytic mechanism. Specifically, the slow, tight inhibition by APMTP results from its being carried further into the catalytic cycle than other analogues prior to forming an intermediate that is blocked from further catalysis.

Nuclear translocation of aflatoxin B1 - protein complex.

The in vitro binding of [3H]-AFB1 to various proteins was studied by equilibrium dialysis. At 23 +/- 1 degree C, [3H]-AFB1 binding activity (mmol/mol) decreased as follows: pyruvate kinase > albumin-NLS > albumin > carbonic anhydrase > RNase > histones. The nuclear translocation and activation of AFB1 and AFB-protein complexes was investigated using isolated rat liver nuclei in the presence of ATP and a NADPH regenerating system. Proteins containing NLS such as histones and albumin-NLS facilitated AFB1 translocation into the nucleus where activation and adduct formation took place.

The in vivo disposition of aflatoxin B1 in rat liver.

The disposition of a non-toxic i.p. dose of [3H]-aflatoxin B1 (0.70 micrograms/kg) in the blood, plasma, and liver was studied in male Wistar rats. Uptake into the blood, plasma, and liver was biphasic; there was an initial rapid rise (0-2 hr) followed by a second phase (2-12 hr) of a gradual increase. Most of the radioactivity in the blood was bound noncovalently to albumin. Distribution of radioactivity in the subcellular fractions of liver showed that the microsomes exhibited the highest labeling which increased over the time course; labeling of the cytosol reached a maximum at 2 hr then decreased to a new steady state, whereas the mitochondria and nuclei reached a plateau. When the content of aflatoxin B1 in the nuclear subfractions was examined, greater than 92% of the total radioactivity was found in the deoxyribonucleoprotein fraction, and 84% of this was bound noncovalently. These results suggest that aflatoxin B1 is transported from the site of injection through the blood to the liver and its subcellular and subnuclear fractions primarily in a noncovalent form.