Investigation of sequential behavior of carboxyl protease and cysteine protease activities in virus-infected Sf-9 insect cell culture by inhibition assay

Proteases produced during the culture of Spodoptera frugiperda Sf-9 cells infected with Autographa californica nuclear polyhedrosis virus (AcNPV) were assayed with various protease inhibitors. This inhibitory analysis revealed that: (1) carboxyl and cysteine proteases were predominantly produced by the insect cells infected with recombinant AcNPV, the gene of which encoded a variant of green fluorescent protein in a portion of the polyhedrin gene of the baculovirus, and (2) the protease activity was almost completely blocked by pepstatin A (carboxyl protease inhibitor) and E64 (cysteine protease inhibitor) in an additive manner in the presence of EDTA. Utilizing the additive property of the inhibitors, the inhibition-based protease assay discriminated between the two protease activities and elucidated the sequential behavior of the carboxyl and cysteine proteases produced in the virus-infected Sf-9 cell culture. The carboxyl protease(s) existed in the virus-infected cells all the time and their level in the medium continuously increased. Uninfected cells also contained a carboxyl protease activity, the level of which was similar to that of the virus-infected cells. At a certain time after virus infection, the cysteine protease activity was largely increased in the virus-infected cells and a significant amount of the protease(s) was released into the medium, due to the cell membranes losing their integrity. The behavior of intracellular and extracellular cysteine protease activities coincided with that of a recombinant protein whose expression was under the control of the viral polyhedrin promoter. Similar examinations with wt-AcNPV-infected and uninfected insect cells showed that the inhibition-based protease assay was useful for analyzing the carboxyl protease and cysteine protease activities emerging in the insect cell (Sf-9)/baculovirus expression system.     

Microemulsions Containing Medium-Chain Glycerides as Transdermal Delivery Systems for Hydrophilic and Hydrophobic Drugs

We evaluated the ability of microemulsions containing medium-chain glycerides as penetration enhancers to increase the transdermal delivery of lipophilic (progesterone) and hydrophilic (adenosine) model drugs as well as the effects of an increase in surfactant blend concentration on drug transdermal delivery. Microemulsions composed of polysorbate 80, medium-chain glycerides, and propylene glycol (1:1:1, w/w/w) as surfactant blend, myvacet oil as the oily phase, and water were developed. Two microemulsions containing different concentrations of surfactant blend but similar water/oil ratios were chosen; ME-lo contained a smaller concentration of surfactant than ME-hi (47:20:33 and 63:14:23 surfactant/oil/water, w/w/w). Although in vitro progesterone and adenosine release from ME-lo and ME-hi was similar, their transdermal delivery was differently affected. ME-lo significantly increased the flux of progesterone and adenosine delivered across porcine ear skin (4-fold or higher, p < 0.05) compared to progesterone solution in oil (0.05 ± 0.01 μg/cm²/h) or adenosine in water (no drug was detected in the receptor phase). The transdermal flux of adenosine, but not of progesterone, was further increased (2-fold) by ME-hi, suggesting that increases in surfactant concentration represent an interesting strategy to enhance transdermal delivery of hydrophilic, but not of lipophilic, compounds. The relative safety of the microemulsions was assessed in cultured fibroblasts. The cytotoxicity of ME-lo and ME-hi was significantly smaller than sodium lauryl sulfate (considered moderate-to-severe irritant) at same concentrations (up to 50 μg/mL), but similar to propylene glycol (regarded as safe), suggesting the safety of these formulations.     

Comparison of chloroplast DNAs by specific fragmentation with EcoRI endonuclease

Summary Mutants ofEscherichia coli K12, deficient in up to three major outer membrane proteinsb,c andd have been constructed. Mutants that lack the lipopolysaccharide sugar heptose are deficient in proteinb. All heptose-deficient strains are supersensitive to lysozyme, various antibiotics and detergents. They excrete the periplasmic enzyme ribonuclease I. Mutants deficient in proteinsc and/ord have the same sensitivity towards these compounds as the parent strain. Cells of single, double and triple mutants are all rod-shaped. Electrophoretic analysis of cell evelope proteins indicates that in some mutants the protein deficiency is partially compensated for by increased amounts of one or two of the other major outer membrane proteins. Heptose-deficient strains have an increased amount of 2-keto-3-deoxyoctonate.     

Electroporation of cultured adult rat hepatocytes with the c-myc gene potentiates DNA synthesis in response to epidermal growth factor

The human c-myc gene was introduced and transiently expressed in adult rat hepatocyte cultures by the technique of electroporation and its effect on DNA synthesis was examined. Epidermal growth factor (EGF) has been found to stimulate a wave of DNA synthesis in electroporated rat hepatocytes. Hepatocyte cultures electroporated with the c-myc gene showed a potentiation of this EGF effect exhibiting rates of DNA synthesis up to 50% greater than those of control electroporated cultures, as determined by [3H]thymidine labeling of cell nuclei. This potentiation was dependent on the amount of c-myc DNA transfected. The potentiation was due neither to an alteration in the dose-response of the stimulatory effect of EGF nor to a change in the time course of the DNA synthesis wave.