Adjuvant activity of non-ionic block copolymers. IV. Effect of molecular weight and formulation on titre and isotype of antibody.

The adjuvant activity of block copolymers varies with the lengths of the chains of polyoxypropylene (POP) and polyoxyethylene (POE). This project evaluated the adjuvant activity of new copolymers with long polyoxypropylene chains in mice immunized with TNP-hen egg albumin in 2% squalone-in-water emulsions. Two of the new copolymers, L141 and L180.5, not only stimulated higher antibody titres to TNP than older preparations, but also induced a higher percentage of the IgG2 isotypes. The smaller older copolymers, L101 and L121, induced higher absolute levels of IgG3 antibody and relative increases in IgG1. Incorporation of immunomodulators (cell wall skeletons, monophosphoryl lipid A or threonyl muramyl dipeptide) increased mean IgG titres but also increased variability of responsiveness among individuals.

Insulin metabolism in rat hepatocytes. Evidence for generation of an insulin fragment missing a portion of the B chain involved in receptor binding.

Insulin metabolism by isolated rat hepatocytes was studied, utilizing A14 [125I]monoiodoinsulin, [3H]PheB1 semisynthetic insulin, and [3H]insulins synthesized by rat islets. Degradation was assessed by gel filtration, polyacrylamide gel electrophoresis, precipitation by anti-insulin antibody, and binding to specific insulin receptors on IM-9 human lymphocytes. When incubations were performed at 15 degrees C or less, insulin bound to hepatocytes remained intact for up to 2 h. At 37 degrees C we detected the generation of an insulin fragment with an apparent molecular weight of approximately 5000 whose electrophoretic mobility was greater than that of insulin. The fragment bound well to anti-insulin antibodies but poorly to insulin receptors. Information about the structure of the fragment was obtained by comparing the metabolism of [3H]PheB1 semisynthetic insulin with that of [3H]PheB1,24,25 insulin. The data suggest that the fragment contains PheB1 but is missing the PheB24 and PheB25. Treatment of the fragment with trypsin and carboxypeptidase B did not affect its electrophoretic mobility indicating that the fragment is also missing ArgB22. Incubation in the presence of 0.1 mM chloroquine led to accumulation of both intact insulin and the insulin fragment, suggesting that both are degraded by lysosomes. The results of this study suggest the presence of two pathways for insulin degradation in liver: a chloroquine-insensitive pathway by which a portion of the B chain consisting of at least 10 amino acids is removed and a chloroquine-sensitive pathway by which both insulin and the fragment are degraded.

Uptake of vasoactive intestinal peptide by rat liver.

We have investigated the uptake and degradation of vasoactive intestinal peptide (VIP) by rat livers. When liver was perfused with 125I-VIP, less than 20% of the radioactivity was recovered as intact peptide. 125I-VIp bound to specified high-affinity sites on isolated hepatocytes. Half-maximal inhibition of binding occurred at about 1 nM unlabeled VIP. Cell-bound 125I-VIP was degraded to low-molecular-weight products. The percent of 125I-VIP that was bound and degraded was approximately the same at both extremes of the range of VIP concentrations (25-250 pg/ml) reported in portal vein plasma. The lysosomotropic agent chloroquine inhibited 125I-VIP degradation and led to the accumulation of cell-bound 125I-VIP. We conclude that a) most of the VIP secreted from the gastrointestinal tract into portal blood is removed during its passage through the liver, b) VIP binds to specific high-affinity sites on hepatocytes and is probably internalized and degraded by lysosomes, and c) uptake of VIP by liver may serve to prevent the peptide from exerting deleterious systemic effects.

125I-insulin metabolism by rat liver cells: preferential binding and degradation of insulin labeled at TyrA14.

The binding and degradation of highly purified A14-[125I]monoiodoinsulin and A19-[125I]monoiodoinsulin were compared in isolated rat hepatocytes. A14-125I-insulin was bound more rapidly and was degraded more rapidly than was A19-125I-insulin. The difference in the rate of degradation between the two preparations was apparent when either tricholoroacetic acid precipitation or binding to specific insulin receptors on cultured human lymphocytes was used for assay. These results provide evidence that binding to its receptor is the first step in insulin degradation by liver cells.

Extraction and quantification of solutes in solidified agar culture media.

A method is described for determining the concentration of certain solutes in solidified culture media. The method is based upon the finding that under specified conditions the concentration of solute in an agar gel (C(g)) is related to the concentration of solute in a centrifugally extracted gel supernatant (C(s)) by the ratio, C(g)/C(s), which is characteristic for each solute. The method avoids direct assays of the gels and instead involves assaying the supernatants from inoculated and uninoculated (control) gels with conventional liquid assay techniques and then calculating solute concentrations in the inoculated gels by use of the C(g)/C(s) ratios determined from the controls. Uninoculated agar gels containing known concentrations of various solutes and similar gels inoculated with Neurospora crassa or Escherichia coli were centrifuged at various times, and the supernatants were assayed for solute concentrations. The solute concentrations in the supernatants from the inoculated gels multiplied by the C(g)/C(s) ratios for those solutes determined at the same times for the uninoculated controls gave calculated solute concentrations in the inoculated gels. The differences between these calculated solute concentrations and those initially present in the inoculated gels indicated the amounts of solutes utilized from the gels by the microorganisms at various incubation times.

Hepatic mono-oxygenase activity and hepatocellular morphology in chickens treated with 3-methylcholanthrene.

Hepatic drug metabolism in the chicken was investigated. White leghorn chickens were administered 20 mg of 3-methylcholanthrene (3MC) per kg 72 and 48 hr before killing. Levels of hepatic cytochrome P-450 were increased approximately 4-fold. In vitro ethylmorphine N-demethylase (ND) activity was enhanced approximately 1.7-fold, aniline hydroxylase (AH) was increased 2.5-fold, aryl hydrocarbon hydroxylase was increased 20-fold, and NADPH-cytochrome c reductase was unchanged. The Vmax was increased for both ND and AH activities, but the KM for demethylation was depressed whereas that for hydroxylation of aniline was increased. The metabolism of hexobarbital in vivo was not enhanced by 3MC treatment. In brief, the distinctive features of the hepatic mono-oxygenase system of the 3MC-treated chicken were: (a) enhanced ethylmorphine N-demethylase activity, (b) a shift in the Soret peak in the CO-difference spectrum of reduced cytochrome P-450 from the control value of 452 nm to 449 nm, and (c) proliferation and pronounced vesiculation of the hepatic endoplasmic reticulum as revealed by electron-microscopic examination.

Correlation of induced drug metabolism with titer of duck hepatitis virus in chickens.

While chickens infected with duck hepatitis virus showed no signs of clinical illness, their levels of hepatic cytochrome P-450 in response to phenobarbital induction and their microsomal aryl hydrocarbon hydroxylase activities in response to 3-methylcholanthrene induction were each found to correlate with the titer of virus recovered from the livers. These clear correlations indicate that avian hepatic drug metabolism is significantly modified during viral infection.