George H. Wirtz

CULTURED TROUT LIVER CELLS: UTILIZATION OF SUBSTRATES AND RESPONSE TO HORMONES

SummaryThe characterization of a recently established system for the short-term culture of rainbow trout (Oncorhynchus mykiss) liver cells in chemically defined medium has been extended to studies on the metabolic competence of the cells and the characterization of their response to hormones. Three areas of metabolism have been addressed: a) the utilization of the exogenously added substrates fructose, lactate, glucose, dihydroxyacetone, and glycerol for glucose and lactate formation; b) the effects of the pancreatic hormones insulin and glucagon on cellular glucose formation, lactate formation, and fatty acid synthesis; and c) the effects of insulin and dexamethasone on the estradiol-dependent production of vitellogenin. Incubation of trout liver cells with fructose, lactate, glucose, dihydroxyacetone, or glycerol resulted in enhanced rates of cellular glucose and lactate production. Substrate-induced effects usually were more clearly expressed after extended (20 h) than after acute (5 h) culture periods. Addition of the hormones insulin or glucagon caused dose-dependent alterations in the flux of substrates to glucose and lactate. Rates of de novo synthesis of fatty acids from [14C]acetate were stimulated by insulin and inhibited by glucagon during acute and extended incubation periods. Treatment of liver cells isolated from male trout for 72 h with estradiol induced vitellogenin production and secretion into the medium. However, the addition of insulin or dexamethasone drastically reduced this estrogen-induced vitellogenesis. These results indicate that trout liver cells cultured in defined medium maintain central metabolic pathways, including glycolysis, gluconeogenesis, lipogenesis, and vitellogenesis as well as their responsiveness to various hormones, for at least 72 h. This cell culture system should provide an excellent model to further characterize metabolic processes in fish liver.

The influence of retinal on complement-dependent immune damage to liposomes

Abstract Retinal was incorporated into liposomes containing dipalmitoyllecithin, cholesterol, dicetyl phosphate and galactocerebroside; the latter substance served as antigen. They were compared to control liposomes, lacking retinal, with regard to glucose release due to complement-dependent immune damage in the presence of anticerebroside serum. The liposomes were indistinguishable from each other in the amount of total glucose trapped, light scattering characteristics and phosphate content. The rate and extent of glucose release in 30 min was inhibited by the incorporation of retinal. In addition, inhibition was directyl related to retinal concentration and was also observed in the presence of a wide range of concentrations of antigen and complement. Damage to liposomes in the presence of either guinea pig or human complement was inhibited by retinal; this was in contrast to the erythrocyte system in which the hemolytic activity of guinea pig complement was inhibited while that of human complement was enhanced by retinal. Addition of retinal to performed liposomes did not influence complement-dependent damage. Inhibition occurred only when retinal was present during the initial formation of the model membranes. Inhibition persisted even after washing the liposomes free of any unincorporated retinal. The data indicate that liposomes may be an excellent model for studying the influences of retinal on complement mechanism in membranes.

Characterization of antibodies to vitamin A.

Abstract Vitamin A acid (retinoic acid) was conjugated to human serum albumin by the mixed anhydride reaction. u.v. absorbance and dinitrophenylation indicated that between 25 and 40 retinoic acid residues were conjugated per molecule of protein. Rabbits were immunized with this conjugate and a 1:200 dilution of the resulting antisera bound approximately 70 per cent of the added 3 H-retinol. Various derivatives of vitamin A were tested for their ability to displace 3 H-retinol from the antibody.

Vitamin A in liposomes. Inhibition of complement binding and alteration of membrane structure.

Incorporation of vitamin A aldehyde (retinal) into liposomes had an inhibitory effect on the amount of human complement protein bound in the presence of specific antiserum. The total membrane-bound protein was directly measured on liposomes which were washed after incubation in antiserum and fresh human serum (complement). At every concentration of complement, decreased protein binding was found with liposomes which contained retinal. Binding of the third component of complement (C3) was also measured directly on washed liposomes and was found to be decreased in the presence of retinal. The diminution in protein binding due to retinal was not caused by differences in the amount of antibody bound and this was shown by two experiments. First, specific antibody protein binding to liposomes was directly measured and was essentially unaffected by retinal. Second, liposomes were prepared from lipid extracts of sheep erythrocytes. These liposomes were used as as immunoadsorbants to remove antisheep erythrocyte antibodies. The immunoadsorbant capacity was the same in both the presence and the absence of retinal. A further conclusion from these experiments was that retinal did not change the number of liposomal glycolipid antigen molecules available for antibody binding and thus presumably did not change the total number of lipid molecules present on the outer surface of the liposomes. Retinal did have an effect on the geometric structure of the liposomes. Size distribution measurements were performed in the diameter range of 1-6.35 mum by using an electronic particle size analyzer (Coulter Counter). Liposomes containing retinal were shifted toward smaller sizes and had less total surface area and volume. It was suggested that retinal-containing liposomes may have had a tighter packing of the molecules in the phospholipid bilayer. This effect of retinal on liposomal structure may have been responsible for the observed decreased binding of C3 and total complement protein.

Vitamin A: Probe of immune complement reactions☆

Abstract We have confirmed the observation that vitamin A will inhibit the lysis of sensitized erythrocytes by guinea pig complement. We have shown further that vitamin A aldehyde (retinal) will inhibit the lysis of several EA-complement intermediates. in the case of the intermediate EAC′-7, the existence of inhibition was shown to depend on the conditions of assay. It was also found that retinal decreased the decay of C′2 on EAC′1,4,2 cells.

Vitamin A: Prove of immune complement reactions—II enhancement of human complement hemolysis

Abstract The hemolysis of EA by human complement is enhanced by vitamin A aldehyde (retinal); this is opposite to the influence which retinal has on guinea pig complement lysis. The stage or intermediate in the human complement reaction which is susceptible to retinal action is transient, because delay in adding retinal to the reaction mixtures reduces or eliminates the enhancement of hemolysis. This enhancement is markedly pH dependent. EAC142 prepared with human complement displays a reduced C2 decay rate in the presence of retinal.

Thin layer chromatographic analysis of possible aflatoxins within grain dusts

The National Institute for Occupational Safety and Health is interested in assessing the hazards to grain workers associated with respirable grain dusts of all types. One of these hazards could involve the occurrence of mycotoxin producing fungi either upon or within grains. Aflatoxins, types of mycotoxins produced by bothAspergillus flavus andA. parasiticus, are hepatocarcinogens, mutagens, teratogens and toxins. Here, we report an attempt to determine whether settled and/or airborne dusts from barley, corn, flax, oats and Durum as well as spring wheats contain aflatoxins. These dusts were collected at port grain terminals in the Superior-Duluth regions of the United States. The dusts were extracted with and chromatographed upon thin layer plates in a variety of solvents which have been approved for the separation of aflatoxins. Two acceptable aflatoxin B1 confirmatory tests were employed to verify suspected aflatoxin B1 within the extracts. Each dust contained a chloroform-soluble, blue fluorescent compound(s) which possessed an Rf similar to that of aflatoxin B1 upon chromatography of chloroform extracts in chloroform/95% methanol. Methylene chloride/H2O) extracted a blue fluorescent compound(s) from each dust, and the compound(s) possessed Rf intermediate between those of aflatoxin B1 and B2 upon chromatography in acetone/methylene chloride. The methylene chloride/H2O extracted compounds failed to turn yellow upon spraying with 25% sulphuric acid in methanol and subsequent viewing with an ultraviolet source. Our results confirm those of Sorenson et al., who reported that aflatoxins were absent from airborne grain dusts collected from the Superior-Duluth areas of the United States in the fall of 1977. In conclusion, we stress the need for extracting, detecting, and identifying aflatoxins by a variety of analytical procedures including thin layer and high performance liquid chromatography and “approved” confirmatory tests.

Influence of aliphatic amines on the interaction of human C′1 with EAC′4☆

Abstract The activity of human C′1 fixed to EAC′4 cells (EAC′1hu4) was decreased by treatment with alkylamines. The ability of butanediamine to reduce the C′1 was substantially greater than the corresponding monoamine. The ammonium ion by itself, i.e. lacking an alkyl radical, was without effect on EAC′1hu4). In comparing several alkyldiamines with the amino groups present on the first and last carbons, it was found that those compounds with three to eight carbons were essentially equally effective, but that the two carbon compound had only about one-half the C′1-reducing ability. This lesser activity of ethylenediamine compared to the longer compounds was due to a difference in pKs. The pH at which the experiments were normally run (7·3) allowed the longer compounds such as butanediamine, but not ethylenediamine, to be fully protonated. When the experiments were carried out at a pH sufficiently low (6·0) to allow full protonation of ethylenediamine as well as butanediamine, the two compounds were equally active.

Elemental analysis of airborne grain dusts.

The elemental composition of a group of airborne and settled grain dusts is reported. This survey was undertaken as part of a study to systematically describe the chemistry and morphology of these representative dusts. Our data show that airborne or settled grain dusts differ from each other with respect to elemental composition. Such fundamental differences may be related to previously observed differences in the biological activities of the dusts.

Vitamin A: Probe of immune complement reactions—III inhibition of guinea pig complement hemolysis☆

Abstract The vitamin A inhibition of guinea pig complement-mediated hemolysis has been further examined. The results of a dose—response curve with retinal suggest that a single molecule of retinal per hemolytic site is sufficient to inhibit the reaction. It also appears that under our conditions a fraction of the lytic sites is not susceptible to inhibition. The inhibition is markedly pH dependent. The consumption of Cl by EAC4 is unaffected by retinal, while the consumption of C2 by EAC14 is diminished.