Intestinal lactase: maturational excess expression of mRNA over enzyme protein.

The mechanism of decline of intestinal lactase during mammalian development remains uncertain. Despite a major loss of catalytic activity, lactase mRNA appears to persist at detectable concentrations in adult rats. We quantified lactase activity, total lactase protein, and lactase mRNA in rats aged 7, 11, 15, 18, 22, 30, and 60 days using the 7S ribosomal RNA as the developmental control. The active lactase fraction was 0.81 of total lactase for all age groups except 60-day-old animals, in which it declined to 0.60 (P = 0.004), indicating that conversion of active lactase to inactive species contributed to the lower activity in the adult. Northern blots revealed a single discrete 6.8-kb message at all ages. Although lactase activity and immunoprotein decreased coordinately to a minimum by day 30 (20% of the 7-day value), lactase mRNA doubled to a maximum at day 22 and was maintained at 7-day concentrations even in 60-day adults. The lactase mRNA-to-protein ratio was low at 7 days (0.19) but more than doubled (0.50) by 22 days, achieved a fivefold increase (1.0) by 30 days, and persisted at 0.77 in adults. The relative excess of lactase message during maturation suggests that translational or post-translational events may be paramount in the developmental regulation of lactase gene expression.

Effects of filipin on the structure and biological activity of enveloped viruses.

The interaction of the polyene antibiotic filipin with membrane-bound cholesterol in vesicular stomatitis (VS), influenza, and Rauscher leukemia virions was studied. Exposure of virions to filipin resulted in a series of depressions and ridges in the envelope of VS virions, with a periodicity of 15 to 20 nm perpendicular to the long axis of the particle; similar morphological alterations were observed in negatively stained preparations, in thin-sectioned virions, and in protease-treated virions that lack surface glycoproteins. This morphological effect was specific for filipin, since the envelopes of VS virions that had been treated with another polyene antibiotic, amphotericin B, exhibited markedly different morphology. Morphological alterations induced by filipin in influenza and Rauscher leukemia virions differed from those seen in VS virions. The infectivity of filipin-treated VS virions was reduced up to 500-fold, whereas influenza virions were resistant to filipin treatment. Incorporation of filipin into the virions was demonstrated, and no release of either lipids or proteins from virions was detected after filipin treatment. A stoichiometry of approximately 1 mol of bound filipin per mol of cholesterol was found in both intact and protease-treated VS virions. The equilibrium dissociation constant for filipin-cholesterol interaction was approximately 74-fold larger in intact than in protease-treated VS virions. The initial rate of association of filipin with cholesterol in intact virions was slower than that in protease-treated particles. The fluidity of lipids in VS viral membranes, as probed by a stearic acid derivative spin label, was markedly reduced when either intact or protease-treated virions were treated with filipin.

Permeability properties of the membrane of vesicular stomatitis virions.

Observations of the light-scattering properties of several enveloped viruses indicate that virions (vesicular stomatitis, SV5 and influenza), in common with other membrane systems, are osmotically active, responding to NaCl gradients by swelling in hypo-osmolar solutions and shrinking in hyperosmolar solutions. The permeability barrier responsible for this osmotic response in vesicular stomatitis virions was modified both by protease treatment to remove the viral glycoprotein and by treatment with the polyene antibiotic filipin, an agent known to interact with cholesterol in liposomes and membranes. Filipin altered the kinetic and equilibrium permeability behavior of virions but the extent of leakage of osmotic shocking agent was less than that in lecithin/cholesterol and lecithin/ergosterol liposomes and in ergosterol-containing ciliary membranes. Negative-staining electron microscopy revealed that filipin treatment caused structural changes in the viral membrane. Intact virions exhibited appreciably larger responses to osmotic change than did protease-treated virus particles. Thus, the osmotic barrier in intact vesicular stomatitis virions may not be exclusively lipid in nature.