Rapid detection of anti-hepatitis A virus neutralizing antibodies in a microplate enzyme immunoassay

The slow growth of hepatitis A virus (HAV) in cell culture is one of the primary pitfalls in the development of sensitive and rapid methods for the detection and quantification of HAV and associated neutralizing antibodies. Currently, in vitro assays frequently require 8 days or more to detect and quantify the presence of HAV neutralizing antibodies. This study describes a rapid immunoassay that allowed the detection of anti-HAV neutralizing antibodies in only 3 days. This microplate-based enzymic assay may be applicable in virological diagnostics, in evaluating the immunogenicity of HAV vaccines and in quantifying neutralizing antibodies during the course of HAV infection.

Fatty acyl-CoA esters inhibit glucose-6-phosphatase in rat liver microsomes

In native rat liver microsomes glucose 6-phosphatase activity is dependent not only on the activity of the glucose-6-phosphatase enzyme (which is lumenal) but also on the transport of glucose-6-phosphate, phosphate and glucose through the respective translocases T1, T2 and T3. By using enzymic assay techniques, palmitoyl-CoA or CoA was found to inhibit glucose-6-phosphatase activity in intact microsomes. The effect of CoA required ATP and fatty acids to form fatty acyl esters. Increasing concentrations (2-50 microM) of CoA (plus ATP and 20 microM added palmitic acid) or of palmitoyl-CoA progressively decreased glucose-6-phosphatase activity to 50% of the control value. The inhibition lowered the Vmax without significantly changing the Km. A non-hydrolysable analogue of palmitoyl-CoA also inhibited, demonstrating that binding of palmitoyl-CoA rather than hydrolysis produces the inhibition. Light-scattering measurements of osmotically induced changes in the size of rat liver microsomal vesicles pre-equilibrated in a low-osmolality buffer demonstrated that palmitoyl-CoA alone or CoA plus ATP and palmitic acid altered the microsomal permeability to glucose 6-phosphate, but not to glucose or phosphate, indicating that T1 is the site of palmitoyl-CoA binding and inhibition of glucose-6-phosphatase activity in native microsomes. The type of inhibition found suggests that liver microsomes may comprise vesicles heterogeneous with respect to glucose-6-phosphate translocase(s), i.e. sensitive or insensitive to fatty acid ester inhibition.

Thermal inactivation of γ-glutamyltranspeptidase and Enterococcus faecium in milk-based systems

SummaryUntreated whole milk, skim milk, sweetened milks, sweetened and unsweetened creams and ice-cream mixes were preincubated with a culture of Enterococcus faecium, then subjected to heat treatment in a pilot-scale plate heat exchanger using a hold of 15 s at 76°C. The liquids were examined for γ-glutamyltranspeptidase (GGTP) activity and total streptococcal count before and after heat treatment and the results plotted against water activity (aw). There was a good correlation between reduction in GGTP activity, destruction of streptococci and aw, demonstrating the potential of the enzymic assay for assessing the severity of HTST heat treatments above the minimum for pasteurization.

Inhibition of steroid 5α-reductase by specific aliphatic unsaturated fatty acids

Human or rat microsomal 5 alpha-reductase activity, as measured by enzymic conversion of testosterone into 5 alpha-dihydrotestosterone or by binding of a competitive inhibitor, [3H]17 beta-NN-diethulcarbamoyl-4-methyl-4-aza-5 alpha-androstan-3-one ([3H]4-MA) to the reductase, is inhibited by low concentrations (less than 10 microM) of certain polyunsaturated fatty acids. The relative inhibitory potencies of unsaturated fatty acids are, in decreasing order: gamma-linolenic acid greater than cis-4,7,10,13,16,19-docosahexaenoic acid = cis-6,9,12,15-octatetraenoic acid = arachidonic acid = alpha-linolenic acid greater than linoleic acid greater than palmitoleic acid greater than oleic acid greater than myristoleic acid. Other unsaturated fatty acids such as undecylenic acid, erucic acid and nervonic acid, are inactive. The methyl esters and alcohol analogues of these compounds, glycerols, phospholipids, saturated fatty acids, retinoids and carotenes were inactive even at 0.2 mM. The results of the binding assay and the enzymic assay correlated well except for elaidic acid and linolelaidic acid, the trans isomers of oleic acid and linoleic acid respectively, which were much less active than their cis isomers in the binding assay but were as potent in the enzymic assay. gamma-Linolenic acid had no effect on the activities of two other rat liver microsomal enzymes: NADH:menadione reductase and glucuronosyl transferase. gamma-Linolenic acid, the most potent inhibitor tested, decreased the Vmax. and increased Km values of substrates, NADPH and testosterone, and promoted dissociation of [3H]4-MA from the microsomal reductase. gamma-Linolenic acid, but not the corresponding saturated fatty acid (stearic acid), inhibited the 5 alpha-reductase activity, but not the 17 beta-dehydrogenase activity, of human prostate cancer cells in culture. These results suggest that unsaturated fatty acids may play an important role in regulating androgen action in target cells.