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Transport and diffusion of molecular oxygen in phosphatidylcholine (PC)-cholesterol membranes and their molecular mechanism were investigated. A special attention was paid to the molecular interaction involving unsaturated alkyl chains and cholesterol. Oxygen transport was evaluated by monitoring the bimolecular collision rate of molecular oxygen and the lipid-type spin labels, tempocholine phosphatidic acid ester, 5-doxylstearic acid, and 16-doxylstearic acid. The collision rate was determined by measuring the spin-lattice relaxation times (T1's) in the presence and absence of molecular oxygen with long-pulse saturation-recovery ESR techniques. In the absence of cholesterol, incorporation of either a cis or trans double bond at the C9-C10 position of the alkyl chain decreases oxygen transport at all locations in the membrane. The activation energy for the translational diffusion of molecular oxygen in the absence of cholesterol is 3.7-6.5 kcal/mol, which is comparable to the activation energy theoretically estimated for kink migration or C-C bond rotation of alkyl chains [Träuble, H. (1971) J. Membr. Biol. 4, 193-208; Pace, R. J., & Chan, S. I. (1982) J. Chem. Phys. 76, 4241-4247]. Intercalation of cholesterol in saturated PC membranes reduces oxygen transport in the headgroup region and the hydrophobic region near the membrane surface but little affects the transport in the central part of the bilayer. In unsaturated PC membranes, intercalation of cholesterol also reduces oxygen transport in and near the headgroup regions. In contrast, it increases oxygen transport in the middle of the bilayer. On the basis of these observations, a model for the mechanism of oxygen transport in the membrane is proposed in which oxygen molecules reside in vacant pockets created by gauche-trans isomerization of alkyl chains and the structural nonconformability of neighboring lipids, unsaturated PC and cholesterol in particular, and oxygen molecules jump from one pocket to the adjacent one or move along with the movement of the pocket itself. The presence of cholesterol decreases oxygen permeability across the membrane in all membranes used in this work in spite of the increase in oxygen transport in the central part of unsaturated PC-cholesterol membranes because cholesterol decreases oxygen transport in and near the headgroup regions, where the major barriers for oxygen permeability are located. Oxygen gradients across the membranes of the cells and the mitochondria are evaluated. Arguments are advanced that oxygen permeation across the protein-rich mitochondrial membranes can be a rate-limiting step for oxygen consumption under hypoxic conditions in vivo.

Phosphatidylinositol (PI) is initially synthesized in mammalian cells with a fatty acid composition similar to that of its precursor, primarily monounsaturated forms of cytidine diphosphodiglyceride (CDP-DAG). However, at the steady state, over 80% of PI exists in the 1-stearoyl, 2-arachidonoyl form. The fatty acid remodelling of PI is due to a number of deacylation/reacylation mechanisms. In the preceding paper we demonstrated that de novo synthesized PI is rapidly deacylated and subsequently reacylated. In this report we present further evidence that cycles of deacylation and reacylation are involved in the remodelling of PI. Incubation of microsomes with CDP-DAG of different fatty acid composition results in quantitative and qualitative differences in lysoPI formation. Additionally, analyses of the resulting lysoPI and PI species reveal that multiple species of fatty acids are incorporated into the 1-position of both PI and lysoPI. Addition of acylation cofactors (fatty acyl CoAs or ATP plus CoA) potentiate reacylation in this system. The addition of stearoyl or myristoyl CoA during de novo synthesis of PI results in the incorporation of these added fatty acids into the I-positive of PI. In addition, some evidence is presented that multiple mechanisms for remodelling of the 1-position of PI may be active in the microsomes, including ATP- and CoA-dependent acylation, ATP-independent, CoA-dependent acylation and CoA-independent mechanisms. Finally, the disappearance of only a subset of lysoPI species upon the addition of acylation cofactors suggests that the reacylation step exhibits some substrate specificity.

Controversy concerning egg cholesterol values exists in recent literature due to varying procedures used for cholesterol determination. The purpose of the present study was to investigate the efficacy of direct sample saponification (Method A) versus saponification of a lipid extract (Method B) for analysis of yolk cholesterol. Method A resulted in a value of 19.1 +/- .4 (SE) mg cholesterol/g of yolk for the National Institute of Standards and Technology (NIST) reference (cholesterol in whole egg powder) as compared with the NIST certified value of 19.0 +/- .2 mg/g. Method B resulted in a significantly lower value of 14.6 +/- .5 mg/g. Egg yolk cholesterol values were determined to be 196 +/- 4.2 mg per egg by Method A and 132 +/- 11 mg per egg by Method B. Various amounts (1, .5, .25 g) of yolk cholesterol assayed by either method proportionately decreased cholesterol values as yolk amount decreased; however, Method B consistently resulted in lower yolk cholesterol. These data suggest that both Methods A and B are valid for determining relative differences between treatments; however, the NIST standard data indicate that for quantification of absolute cholesterol values, direct saponification is more accurate. The NIST standard of cholesterol in whole egg powder should be used as a control for comparing cholesterol data regardless of extraction method used.

Eggs laid by hens experimentally infected with Salmonella enteritidis were assayed for the presence of Serogroup D-specific yolk antibodies. Yolk antibodies were detected with S. enteritidis and Salmonella pullorum antigens in the microantiglobulin test as early as 9 days after inoculation of hens with S. enteritidis. Yolk antibody titers reached peak levels at 3 to 5 wk postinoculation and remained at detectable levels for at least 7 wk postinoculation in eggs from both orally inoculated and horizontally contact-exposed hens. Eggs laid by hens from commercial flocks implicated in epidemiological investigations of human S. enteritidis outbreaks were also tested. Serogroup D-specific yolk antibodies were detected in 5 to 22% of eggs from hens in houses identified as infected by bacteriological culturing of internal organs of hens for S. enteritidis.

Vitellogenin has been localized at the electron microscopical level in the liver of the cockerel using a colloidal gold technique. White leghorn cockerels were treated with 17 beta-oestradiol to induce vitellogenesis. Pieces of liver were removed from control and experimental birds on the 4th and 8th days following hormone treatment, and embedded in Lowicryl K4M. Vitellogenin was isolated from the plasma of oestradiol-treated cockerels, and the antibody to it elicited in rabbits and made vitellogenin-specific by affinity chromatography on lipovitellin-Sepharose columns. At the light microscopical level, the intensity of immunohistochemical staining was considerably above background levels in oestradiol-treated cockerels. At the electron microscopical level, gold particles indicating antigenic sites of vitellogenin were largely confined to the rough endoplasmic reticulum, Golgi apparatus, immature and lysosomes and phagosomes within hepatocytes and sinusoidal cells respectively. These observations strongly suggest that the intracellular pathway of vitellogenin secretion in chicken hepatocytes under the experimental conditions studied involves external stimuli and secretory vacuoles. The labelling of lysosomes may reflect catabolic turnover (crinophagy).

Due to the numerous proposed cardiovascular benefits associated with consumption of omega-3 fatty acids, marketing of an egg enriched by omega-3 fatty acid may benefit the egg producer. Effects on yolk composition of a standard laying hen diet enriched with 3% menhaden oil (test diet), versus an isocaloric (control) diet containing no added fat, were evaluated for 18 wk. Dietary menhaden oil did not alter egg production, egg weight, total yolk fat, or yolk cholesterol. However, yolk contents of omega-6 and omega-3 fatty acids were influenced by diet. Arachidonic acid decreased and eicosapentaenoic acid increased in eggs from hens fed the test diet following 1 wk of dietary treatment. Docosahexaenoic acid and linolenate increased in eggs from hens fed the test diet at 2 and 3 wk of the trial, respectively. These alterations in yolk composition resulted in a decrease in the ratio of omega-6 to omega-3 fatty acids from 18 for eggs from hens fed the control diet to 3 for eggs from hens fed the test diet. At Weeks 14 and 18, hens (n = 10 per diet) were killed and necropsied. No change in gross scoring of hepatic lipidosis was observed. Histologically, significantly greater scores for hepatocellular lipid infiltration were recorded for liver sections from hens fed menhaden oil than for control hens. Increased microscopic hepatic lipid infiltration observed with dietary omega-3 administration may have significance for flocks predisposed to fatty liver syndrome and may also provide a unique system in which to study the effects of dietary omega-3 fatty acids on liver lipid metabolism.

Studies were conducted to determine the effect of divergent selection for plasma cholesterol response to adrenocorticotropin (ACTH) on the levels and relationships between plasma and yolk cholesterol in Japanese quail. Cholesterol data were obtained in Generation 25, following seven generations of relaxed selection, from birds maintained under a normal environment with no exposure to exogenous ACTH. Levels of plasma and yolk cholesterol were determined at 22 and 28 wk. Plasma cholesterol levels of quail in the low cholesterol line were significantly (P less than .01) lower than levels in the high line at both ages (224 versus 383 and 209 versus 326 mg/100 mL, respectively). In contrast, yolk cholesterol levels were significantly (P less than .01) higher in the low line than in the high line (24.1 versus 21.5 and 21.1 versus 16.9 mg cholesterol/g yolk at 22 and 28 wk, respectively). A significant line by sex interaction was present at both ages for plasma cholesterol with females having higher cholesterol values than males in the low line and males having higher values than females in the high line. A negative relationship was observed between changes in plasma and yolk cholesterol in the selected lines. Greater deposition of cholesterol in the yolk of the line with lower plasma cholesterol indicates that excretion rate may play a role in explaining genetic differences in plasma cholesterol.

A new method for the preparation of liposomes is described that avoids the use of pharmaceutically unacceptable solvents and energy-expensive procedures such as sonication. The method is based on the initial formation of a proliposome mixture containing lipid, ethanol and water, which is converted to lipsomes by a simple dilution step. Measurements using 6-carboxyfluorescein as a marker indicate that water-soluble drugs can be trapped with extremely high efficiency (65-80% depending on lipid composition). The structural organization of the proliposome mixture and the final liposomes were characterized using electron microscopy and 31P-NMR.

Adult Culex quinquefasciatus, maintained from emergence on sugar, were fed blood and then fed either sugar (control) or water (starving) for 7 days. Analysis of ovaries and egg rafts for protein, lipids and glycogen showed that only glycogen levels were diminished by starvation. Eggs from both control and starving females, however, were equally viable. Nonbloodfed starving females lived longer than bloodfed starving females. These results suggest that the blood meal maximizes fertility, not longevity.

A simple and rapid enzyme immunoassay for specific antibodies in chicken egg yolk is described. As a model system, the levels of anti-Salmonella IgG in the yolk of eggs obtained from various produce retailers were compared. Polyester cloth coated with Salmonella typhimurium lipopolysaccharide was used to capture specific egg yolk antibodies, which were then detected using an anti-chicken IgG-peroxidase conjugate. This assay, requiring less than 30 min to complete, revealed considerable differences in the relative levels of anti-Salmonella IgG in the egg yolks. Anti-Salmonella IgG levels were generally lower in eggs obtained from large produce retailers than in eggs obtained from a small local farm. This assay may provide a rapid and economical means of monitoring the levels of Salmonella contamination in chicken rearing facilities.

Doxycycline (DOTC) and oxytetracycline (OTC) were dissolved in drinking water (0.5 g/l) and supplied to laying hens for 7 consecutive days. Eggs laid were collected daily during and after medication, and the antibiotic concentrations in the yolk and albumin were determined by the cup-plate method with Bacillus cereus var. mycoides ATCC 11778. The concentrations of both antibiotics were increased in yolk day by day with the advance in medication, reached peaks 2 days after withdrawal and then declined gradually. Mean peak concentrations in the yolk were 6.70 micrograms/g for DOTC and 1.42 micrograms/g for OTC. Peak concentrations in the albumin occurred in the middle stage of medication, where the mean values were 12.24 micrograms/g for DOTC and 1.03 micrograms/g for OTC. DOTC was detected in albumin until 24 days after withdrawal and for 2 days more in yolk than in albumin. OTC was detected in yolk until 9 days after withdrawal. The depletion period of OTC was shorter for the albumin, where the residue disappeared in all eggs 6 days after withdrawal. In spite of similarities between DOTC and OTC in structure, DOTC was deposited in higher concentrations and lasted for a longer period in eggs. This characteristic was considered due to its greater lipophilicity, closely correlated with oral absorption and tissue penetration.

1. The possibility that biotin in stored eggs migrates from the yolk into the albumen was re-investigated. 2. Sets of eggs collected from 12 Single Comb White Leghorn hens were stored at 5, 8, 12, 15, or 18 degrees C for 5, 10, 15, 20, or 25 days and then frozen. 3. For each set, yolk and albumen samples adjacent to the yolk membrane were each pooled and assayed for protein-bound biotin by radioligand exchange with non-linear regression analysis of the data. 4. In contrast to previous work reported by Bush and White (Comp. Biochem. Physiol. 93B, 543-547, 1989), no evidence of biotin diffusion was found. 5. Non-linear regression was used to re-evaluate the data from the earlier study, confirming the results, although less strikingly. 6. The discrepancy is partly due to the method of data analysis, although differences in breed or number of hens may also be contributing factors.

1. Exposure of laying hens to elevated ambient temperatures (30-40 degrees C) resulted in a significant increase in the biotin concentrations in plasma and egg yolk. 2. Exogenous oestrogen administration to immature pullets increased plasma biotin three-fold. The increase was six-fold when the birds were simultaneously exposed to an ambient temperature of 35 degrees C. 3. It is proposed that ambient temperature affects the balance between thyroid and ovarian hormones resulting in increased circulating levels of biotin and deposition of the vitamin in egg yolk.

Mixed micelles prepared from egg lecithin and the sodium salt of glycocholic acid markedly inhibited amphotericin B toxicity to mammalian cells without significantly affecting the antifungal effects of the drug.

To dissect the regulatory and structural requirements for Listeria monocytogenes intracellular growth and cell-to-cell spread, we designed a protocol based on transposon mutagenesis and the isolation of mutants which form small plaques in monolayers of mouse L2 cell fibroblasts. Two different transposable elements were used to generate libraries of insertion mutants: Tn916 and a derivative of Tn917-lac, Tn917-LTV3. Ten classes of mutants were isolated and evaluated for growth and cell-to-cell spread in J774 mouse macrophagelike cells, Henle 407 human epithelial cells, and mouse bone marrow-derived macrophages. Mutants were also evaluated for secretion of hemolysin and phospholipase (assayed by egg yolk opacity) and association with F-actin in the cytoplasm of cells, using NBD-phallacidin staining. The ten classes of mutants included (i) mutants showing abortive intracellular and extracellular growth; (ii) mutants showing abortive intracellular growth; (iii) rough mutants; (iv) mutants showing greatly reduced hemolysin and phospholipase secretion but showing normal growth in cells and little or no association with F-actin; (v) mutants with mutations mapping to an open reading frame (ORF) adjacent to hlyA and referred to as ORF U, lacking phospholipase activity, and with 50% normal hemolysin activity; (vi) mutants with reduced secretion of both hemolysin and phospholipase; (vii) nonhemolytic mutants with mutations mapping to the structural gene, hlyA; (viii) mutants with 25% normal hemolysin secretion and absolutely no association with F-actin; (ix) mutants with mutations mapping to ORF U, lacking phospholipase activity, and with normal hemolysin activity; and (x) mutants showing a mixed-plaque morphology but normal for all other parameters.

Chickens were vaccinated subcutaneously twice, at 13 and 17 weeks of age. The vaccines used were the whole organisms of Mycoplasma gallisepticum (MG) adjuvanted with multilamellar positively charged (MPC) liposomes or oil-emulsion. Other chickens received the same bacterins but supplemented with Salmonella typhimurium cell wall protein mitogen (STP) (50 micrograms/dose). At 21 weeks of age, each bird was challenged in the right and left caudal thoracic air sacs. The challenge dose/chicken was 1.3 x 10(5) CFU of MG (R-strain). A significant immunoglobulin (Ig) response specific to MG was observed in sera of chickens collected 3 weeks after the first and second vaccination with MG adjuvanted with MPC liposomes or oil-emulsion. The same two treatments had highly significant MG-titers in eggs collected during the first and second month post challenge. Both groups had highly significant protection (P less than 0.05) against MG transmission in eggs layed during the first month post challenge. Vaccination with MG organisms adjuvanted to MPC liposomes or oil-emulsion resulted in higher egg production, during the first month following challenge, in comparison to the unvaccinated-challenged birds; the same two groups had higher egg production in the second month following challenge compared to unvaccinated-challenged birds, but not significantly different (P greater than 0.05). The addition of STP to bacterins containing MG organisms adjuvanted to MPC liposomes or oil-emulsion, resulted in a significant reduction (P less than 0.05) of the Ig-specific to MG in sera and in a significant drop in egg production (P less than 0.05) during the first month following challenge.

Single Comb White Leghorn laying hens that were 60 wk of age were fed wheat and soybean meal diets containing either 0 or 1% cholesterol. Birds were artificially inseminated, and fertilized eggs were collected for incubation after a plateau of egg cholesterol content was reached. Posthatch chicks were raised with starter diets containing either 0 or .5% cholesterol. Samples of developing embryos and posthatch chicks at various stages were prepared for cholesterol analysis. As compared with controls, cholesterol content of eggs from hens fed 1.0% cholesterol diet was increased by approximately 70%. Embryos from the cholesterol-loaded eggs had significantly higher (P less than .05) cholesterol content. The plasma total cholesterol (TC) level in chicks from cholesterol-loaded eggs, when compared with TC in control eggs, was significantly higher at hatching but decreased to the same level by 2 wk after hatching. Cholesterol feeding to newly hatched chicks elevated plasma TC and high-density lipoprotein cholesterol levels. The TC contents of liver and heart, but not skeletal muscle, were significantly higher in chicks fed the .5% cholesterol starter diet than those fed the cholesterol-free diet. These results show that cholesterol metabolism in developing embryos and posthatch chicks is influenced by cholesterol in both maternal and chick diets.

An experiment was conducted to evaluate the effects of dietary corn oil (CO) and commercial free fatty acids on the fatty acid composition of blood plasma and egg yolk lipids and on the reproductive performance of Japanese quail. When quail were fed a semi-purified low-fat basal mix, the substitution of 3% of CO for corn starch (CS) improved egg weight (11.2 versus 10.8 g), reduced late embryonic mortality (16.7 versus 28.4%), and improved hatchability (75.7 versus 64.1%). Diets in which palmitic acid or oleic acid were substituted on a weight basis for 3% of CS performed as well as those in which CO was used. When linoleic acid replaced 3% of CS, late embryonic mortality was reduced (13.7 versus 28.4%) and hatchability was improved (80.1 versus 64.1%), but fertility was depressed (85.8 versus 93.6%). The substitution of linolenic acid for 3% of CS depressed feed consumption, body weight, and egg weight relative to the low-fat CS diet and did not improve the low 18-day embryonic livability and low hatchability. The fatty acid compositions of plasma and yolk were influenced by the lipid composition of the diets. Quail fed 3% of CO deposited more linoleic acid in the egg than those fed 3% of linoleic acid (15.2 versus 11.7%). Thus, quail fed a simplified basal breeder diet containing .6% of linoleic acid showed decreased embryonic livability and hatchability and these parameters achieved normal levels in response to the substitution of 3% of either CO, palmitic acid, oleic acid, or linoleic acid for CS.

Extraction of specific IgG antibodies from egg yolks of immunized hens is a convenient and economical approach to the production of diagnostic antibody reagents. In this study, two methods for the extraction of yolk IgG were constructed and compared. That either method could extract at least 15 mg of yolk IgG preparation from 1 ml of egg yolk of hyperimmunized hens clearly indicates that both methods are reliable and efficient. Yolk IgG preparations obtained by method II were purer and possessed higher antibody activity than the yolk IgG preparations produced by method I, so method II is superior to method I. Nevertheless, the yolk IgG preparations produced by method I and method II contained 22% and 16% unrelated proteins respectively. Therefore, these two methods should primarily be used for the rough extraction of hens yolk IgG antibodies. If yolk antibody reagents with higher purity are required, the yolk IgG preparations produced by both methods should be further purified.

1. The elimination kinetics of four macrolide antibiotics (tylosin, erythromycin, spiramycin and josamycin) in eggs were determined separately for albumen and yolk after oral administration through either drinking water or diet or after intramuscular injection. 2. Residues were assayed by a plate diffusion technique in cylinders with Micrococcus luteus as the test-organism. 3. Drug excretion was usually over a longer time in the yolk. Spiramycin was the most highly excreted in the egg whereas seven to eight times less tylosin and erythromycin was transferred. The conditions for the use of macrolide antibiotics in laying hens are discussed.

The fat content and the concentrations of glucose and triglyceride in day-old chicks hatched from 27 and 60 wk-old broiler-breeder hens were determined from pooled samples of residual yolk and blood serum, respectively. Serum glucose and triglyceride levels were unaffected (P greater than .05) by breeder age, although there was a linear (P less than .001; r = .67) relationship between these characteristics and chick weight. Yolk fat, adjusted for chick weight, was on the average 13% greater (P less than .05) in chicks from old breeders than chicks from young breeder hens. Yolk wet weight was not affected (P greater than .05) by breeder age. Results indicate that breeder age may affect chick performance through alterations in the fat content of residual yolk.

Patients may fail to absorb cobalamin (vitamin B12) bound to food even when they have adequate intrinsic factor to absorb free cobalamin normally. We studied cobalamin transfer from egg yolk cobalamin-binding protein to human saliva and gastric juice as a model of this important first step in cobalamin assimilation. The cobalamin-binding protein of egg yolk eluted with human R binder on Sephadex gel chromatography and bound cobalamin with a comparable affinity, but it did not cross-react with R binder immunologically. Transfer of cobalamin from egg yolk to saliva or gastric juice R binder did not occur at neutral pH. Slight transfer (8%-12% of the 57Co-cobalamin bound to egg yolk) occurred when the saliva was acidified to pH 1.5. This minor transfer by acid was not inhibited by pepstatin A, a pepsin inhibitor. Acidification caused variable transfer to gastric juice R binder (12%-40%) that appeared to be partially due to residual gastric pepsin activity. Adding 1200 U of pepsin per milliliter enhanced cobalamin transfer to saliva or gastric juice R binders (39%-58% transfer). At no time was cobalamin transferred directly to intrinsic factor; R binder-deficient gastric juice failed to accept cobalamin from egg yolk. The transfer of cobalamin from egg yolk to human R binder requires both an acid pH and pepsin activity. While as little as 30 U of pepsin added per milliliter of saliva promoted transfer of cobalamin, the requirement for an acid pH was very strict. Virtually no transfer occurred when pH exceeded 2.0, regardless of the amount of pepsin present. Acid provided an optimal pH for pepsin activity and, to a lesser extent, affected transfer by a mechanism unrelated to pepsin. Our data suggest that compromised pepsin secretion and, probably even more importantly, compromised acid secretion interfere with transfer of food cobalamin to R binder.

An experiment was conducted to determine if adding supplemental cholesterol to the feed or first adding it to the supplemental fat source of laying hen diets would result in differences in egg yolk and liver cholesterol levels. Five levels of cholesterol (0, .5, 1, 2, and 4%) and three levels of animal tallow (0, 4, and 8%) were used. The diets were randomly assigned and fed for 35 days to individually caged hens within each of six replicates. Eggs laid on or near Days 0, 7, 14, 21, 28, and 35 were used for cholesterol analysis. Liver cholesterol, egg production, and feed intake were also assessed. Mixing cholesterol with the fat source before feed incorporation did not promote higher yolk or liver cholesterol levels and were essentially the same as the method in which powdered cholesterol was added directly to the feed. A linear increase in yolk and liver cholesterol was observed with 0, .5, and 1% dietary cholesterol. Yolk cholesterol also increased linearly during the first 14 days of cholesterol administration. Further increases in yolk cholesterol, however, were not obtained with either the higher levels of dietary cholesterol or the extended feeding times.

The titers of hemagglutinins to human erythrocytes were investigated using sera and eggs from hens immunized by a single intraperitoneal injection of crude blood group substance from OSe saliva mixed with Freund's complete adjuvant. Elevation of the hemagglutinin titer in egg yolk followed the increase of serum hemagglutinin with 1- to 2-week time-lag. The maximum hemagglutinin titer of egg yolk was 1/2 to 1/8 of the serum hemagglutinin titer. The total hemagglutinin quantity in egg yolk exceeded the serum quantities of the antibody in sacrificed hens. Egg-white transiently contained a very low titer of hemagglutinin. The yolk hemagglutinin scarcely migrated at all to the white after storage of the egg for 3 months at 5 degrees C. These results indicate that egg yolk from hens immunized with blood group substances is an appropriate source of blood group hemagglutinins.

Hens were immunized intraperitoneally with OSe saliva mixed with Freund's complete adjuvant, and the properties of hemagglutinins in sera and egg yolk were compared. Both serum and egg yolk hemagglutinins agglutinated O, A1, B and A1B erythrocytes to a similar degree, and both showed somewhat stronger agglutinating potencies at 5 degrees C than at 25 degrees C or 37 degrees C. The degrees of agglutination were little improved by the addition of 20% BSA or PVP medium solution for potentiating incomplete agglutinin. The antiglobulin-augmentation reaction with specific anti-chicken-Igs revealed that the principal immunoglobulins of the serum hemagglutinins produced during the early and hyperimmune phases of immune response were IgM and IgG, respectively; whereas the presence in egg yolk of both IgM and IgG hemagglutinins was demonstrated throughout the antibody producing periods. The yolk probably contained IgM incomplete hemagglutinins, because addition of an antiserum to chicken-IgM strongly enhanced the yolk hemagglutinin. The secretor status of six saliva specimens, OSe, Ose, ASe, Ase, BSe and Bse, was tested by the agglutinin-inhibition reaction using one of the following reagents: Ulex lectin, chicken antiserum, and egg yolk hemagglutinin. High inhibition titers of OSe saliva were obtained with the serum hemagglutinin (of the early and hyperimmune phases) and the yolk hemagglutinin of the eggs laid in the hyperimmune phase. The test with Ulex lectin showed a moderate grade of inhibition, and the use of yolk hemagglutinin produced in the early stage of immune response resulted in very low inhibition titers. Yolk hemagglutinins produced in the early and hyperimmune stages were more strongly inhibited by ASe and BSe salivas than by other hemagglutinin reagents. These results indicate the availability of blood group agglutinins in the yolks of eggs laid during the hyperimmune stage.

Mutagenicity of oil of charred egg yolk (called ranyu in Japanese), which is commercially available and consumed as a health food in Japan, was tested on Salmonella typhimurium strains TA98 and TA100 with and without metabolic activation. Both strains showed a high response to the oil, and the number of His+ revertant colonies with strain TA98 was 15,000-20,000 for 1 g equivalent amount of oil. The mutagens were purified by acid extraction, chloroform extraction after alkalization, dialysis, adsorption to blue cotton, passing through a Sephadex LH-20 column and several stages of high-pressure liquid chromatography with reverse-phase columns. At least 7 heterocyclic amine mutagens were detected. Two of them were suggested to be 2-amino-3-methylimidazo[4,5-f]quinoline (IQ) and 2-amino-6-methyldipyrido [1,2-a:3',2'-d]imidazole (Glu-P-1). One was suggested to be 2-amino-3,4-dimethylimidazo[4,5-f]quinoline (MeIQ). The others were not identified but distinguishable from 12 known heterocyclic amine mutagens. The estimated minimum contents of IQ and Glu-P-1 were 1.1 ng/g and 4.8 ng/g, respectively.

1. Eighteen Warren SSL hens of 71 weeks of age were forced-moulted by ad libitum feeding of a high-zinc diet (10,000 ppm zinc for 2 days followed by 5,000 ppm zinc-supplement diet for 4 days). From the start of the treatment, eggs were collected and 3 hens were slaughtered on days 0, 2, 3, 4, 5 and 6 of the study. 2. Zinc analyses were carried out on the different components of the eggs and on liver, pancreas, kidney, different yolky follicles of the ovary and various segments of the oviduct. 3. Seven-, six- and threefold increases in zinc concentration were found in pancreas, liver and kidney, respectively. 4. The shell gland and isthmus, but not the magnum, also showed slight but significant increases in Zn content. 5. Zinc accumulation was also high and almost identical in ovarian follicles F1 to F4 but slightly less in F5 and F6 follicles. 6. In the egg, a significant increase in zinc concentration was only observed in the yolk.

Two studies were conducted to determine the effects of anticoccidial agents on the production and reproduction of White Leghorns. In Experiment 1, nicarbazin (NCZ) was fed at 0, 20, 50, and 100 ppm. Hen-day egg production, egg weight, the egg-yolk DNC (4-4'-dinitrocarbanilide) level, and egg-yolk mottling were affected by the treatments. When response was evidenced, the relationship between those variables and the level of NCZ was basically linear. Decreased egg production occurred from Days 5 and 6 of the treatment through Days 1 and 2 of withdrawal. On Days 9 and 10 of treatment, the control hens peaked at 92% hen-day production, while hens fed 20, 50, and 100 ppm of NCZ peaked late--at 90, 82, and 80%, respectively. Compared to the controls, egg weight was reduced linearly as the level of dietary NCZ increased. The egg-yolk DNC level increased from Days 3 and 4 of treatment through Days 9 and 10 of withdrawal. Egg yolk mottling generally increased along with the level and duration of feeding NCZ. If the NCZ was mistakenly fed to White Leghorn layers, ill effects would be alleviated within 10 days after drug withdrawal. In Experiment 2, halofuginone (3 ppm), maduramicin (5 ppm), monensin (100 ppm), narasin (70 ppm), nicarbazin (125 ppm), robenidine (33 ppm), and salinomycin (60 ppm) were fed to White Leghorn hens at the levels specified in parentheses. Nicarbazin reduced egg production, depressed egg weight, reduced shell thickness, and caused egg-yolk mottling; but internal egg quality, as measured by Haugh Units, was unaffected. Halofuginone, maduramicin, monensin, narasin, robenidine, and salinomycin did not have a meaningful effect on the variables measured when fed to White Leghorn layers.

The mechanism of poly(ethylene glycol)-induced fusion of phospholipid vesicles was studied based on the "osmophobic association" theory which was recently proposed both theoretically [Ito, T., Yamazaki, M., & Ohnishi, S. (1989) Biochemistry 28, 5626-5630] and also experimentally [Yamazaki, M., Ohnishi, S., & Ito, T. (1989) Biochemistry 28, 3710-3715]. Osmophobic association and fusion were detected by measuring the light scattering of the vesicle suspension; the former was detected from the increase in light scattering induced by the addition of PEG, and the latter was from the irreversibility of the increase in light scattering. Threshold concentrations of PEG were required not only for osmophobic association but also for fusion. The threshold concentration for fusion depended on the molecular weight of PEG and also on the electrostatic repulsive interaction between phospholipid vesicles, which was manipulated by the use of vesicles with negative surface charge; increasing the molecular weight of PEG lowered the threshold concentration, and increasing the electrostatic repulsive interaction raised it. In addition, a transient leakage of internal contents from the vesicles was observed at the concentration that caused fusion. When the surface charge of the vesicle was varied, the threshold for fusion coincided with that for osmophobic association, provided that the latter exceeded 22 wt % of PEG 6000. However, when the threshold for osmophobic association was less than 22 wt %, the threshold for fusion remained approximately 22 wt %, irrespective of the difference in the threshold for osmophobic association.(ABSTRACT TRUNCATED AT 250 WORDS)

The solubilization of multilamellar egg yolk lecithin liposomes by sodiumtaurodeoxycholate in aqueous phase was studied by ultrafiltration as a function of time, bile salt and cholesterol concentration. The corresponding equilibrium states were analysed. Complete solubilization was achieved at total bile salt/lecithin molar mixing ratios of approximately 5. The minimum ratio to start solubilization was 0.1, corresponding to a free bile salt concentration of only 5% of the critical micelle concentration (CMC). Mean equilibrium constants for the partition of bile salts between non-filterable aggregates and filterable mixed micelles and also the free bile salt concentration were determined. Sodiumtaurodeoxycholate had a higher affinity for small mixed micelles than for lamellar mixed aggregates especially in the presence of cholesterol, which reduces the degree and rate of the solubilization process. A non-homogeneous distribution of bile salts in the lipid phase was detected at low bile salt concentrations.

The effect of alpha-tocopherol and its acetate on the membrane structure of egg yolk phosphatidylcholine (egg PC) dispersions was investigated using phosphate-31 nuclear magnetic resonance (31P-NMR) and small-angle X-ray diffraction. The incorporation of alpha-tocopherol into egg PC dispersions induced a change in the 31P-NMR spectrum from a multilamellar bilayer line shape to a hexagonal HII one. The phase transition by alpha-tocopherol was also confirmed by small-angle X-ray diffraction analysis. The amount of hexagonal HII phase increased with increase in concentration of alpha-tocopherol. Egg PC dispersions containing a molar ratio of 0.8 of alpha-tocopherol gave a 31P-NMR spectrum of an approximately hexagonal HII type at 37 degrees C. The amount of hexagonal HII phase increased with increasing temperature, indicating that the alpha-tocopherol-induced phase transition is thermotropic and that the transition temperature of egg PC membranes from the lamellar to the hexagonal HII phase is lowered by alpha-tocopherol. The incorporation of alpha-tocopherol acetate did not induce any phase transition. This fact indicates that the hydroxyl group of alpha-tocopherol may play an important role in the hexagonal HII phase formation of egg PC dispersions.

Biochemical studies in lipid composition of yolk granules from the crayfish Rhynchocinetes typus is shown to be composed mainly of cholesterol and phospholipids. Thin layer chromatography analysis demonstrates phosphatidyl-choline, -ethanolamine and -serine as its major phospholipidic components. In vivo labeling experiments using 32P-orthophosphoric acid suggest that two of the major yolk phospholipid components, phosphatidyl -choline and -serine could be synthesized in the hepatopancreas and subsequently transported via hemolymph to the growing oocyte.

A fully automated HPLC determination of the coccidiostat meticlorpindol in whole egg, egg white and yolk is described. The sample homogenate is dialysed on-line against water. The dialysate is concentrated on-line on a short reversed-phase (RP) column. The contents of this column are transferred to the reversed-phase analytical column by means of the mobile phase. Meticlorpindol is detected using an absorbance detector at 270 nm. Linear calibration graphs are obtained in the range 40-900 ng/g in whole egg and egg white (detection limit 10 ng/g) and 80-1800 ng/g in yolk (detection limit 20 ng/g). Out of 111 commercially obtained egg samples 12 contained meticlorpindol with levels varying from 10 to 433 ng/g. A group of laying hens, kept in cages, received 10 mg/kg of Lerbek (meticlorpindol and methylbenzoquate; Dow Chemical) in the feed for 10 days. Meticlorpindol residues in the eggs rose to a level of 622 ng/g. Meticlorpindol was found in the eggs until 6 days after withdrawal of the medicated feed. Another group received 110 mg/kg in the feed. Meticlorpindol residues rose to levels of 4480 ng/g in the eggs, 5880 ng/g in the egg white and 2660 ng/g in the yolk. Meticlorpindol was found in the eggs and the egg white until 14 days and in the yolk until 8 days after withdrawal of the medicated feed.

Adenosine deaminase (adenosine aminohydrolase; E.C. 3.5.4.4) has been purified 4686-fold from egg yolk. The procedure developed was used to isolate the enzyme from eight chicken eggs. An easily prepared affinity column employing purine riboside was used as the final step in the purification. The method developed permits the rapid isolation and a high recovery of the protein. The specific activity of the enzyme preparation obtained is 81.4 mU/mg.

Egg yolk was spiked with p,p'-dicofol (p,p'-DCF) (0.1-2.0 micrograms/gm), p,p'-dichlorobenzophenone (p,p'-DCBP) (0.1-2.0 micrograms/gm), and 1,1-bis(4-chlorophenyl)-2,2-dichloroethylene (p,p'-DDE) (0.05-1.0 micrograms/gm). The fortified egg yolk (2-5 g) was mixed with acetonitrile to extract non-fat organic materials. After removal of acetonitrile, the spiked chemicals were separated with a column chromatograph packed with acid alumina. Recovery efficiencies for p,p'-DCBP and p,p'-DDE were determined by gas chromatography, and for p,p'-dicofol by high performance liquid chromatography. The recovery efficiencies for p,p'-dicofol, p,p'-DCBP and p,p'-DDE were 77.2-93.8%, 84.1-101.1%, and 88.5-96.0%, respectively.

1. Gas-liquid chromatography studies were done on sterols in plasma and the digestive gland-gonad (DGG) complex of Biomphalaria glabrata snails fed lettuce vs hen's egg yolk. 2. The major sterols present in the DGG of both populations were cholesterol, stigmasterol, beta-sitosterol, campesterol, and desmosterol. 3. The percentage composition of cholesterol in the DGG of yolk vs lettuce fed snails was 82 and 51, respectively. 4. The elution profiles of sterols in the plasma of yolk vs lettuce fed snails were similar; both contained desmosterol, campesterol, and stigmasterol, negligible amounts of cholesterol, and unidentified sterols. 5. The high lipid diet increased the level of cholesterol in the DGG but not in the plasma.

1. Developing oocytes of the Japanese quail accumulated 0-44 microgram of each 1 microgram of 125I-labelled iodide after intra-muscular injection of doses up to 500 microgram iodide as NaI but only 0-007 microgram after injection of more than this: the abrupt change in the rate of accumulation was attributed to saturation of the iodide transport mechanism. 2. The proportion of available iodide transferred into the oocytes appeared to be more dependent on the amount of iodide injected and the total weight of growing oocytes than on a requirement for either a store of iodide for the embryo or an iodide excretory pathway for the hen. 3. The thyroid was about four times more active in accumulating iodide than the oocyte. 4. The percentage of iodide accumulated by the plasma was the same at all dose rates.

Egg production type chickens were given the drug Probucol in their diets to determine its effect on egg production characteristics, liver lipid metabolism, egg yolk lipid synthesis, and egg yolk cholesterol concentration. In none of the three trials conducted did Probucol feeding affect egg production or body weight. The drug reduced total liver lipogenesis as measured by incorporation of 14C-acetate by surviving liver slices. Relative incorporation of 14C-acetate into cholesterol by liver was increased by the drug. However, in ovo incorporation of 14C-acetate in total yolk lipids remains unchanged and relative incorporation into yolk cholesterol is reduced. As a result, egg yolk cholesterol content is reduced by 5% with the .10% dietary level of drug administration.

The distribution and excretion of hexachlorobenzene (HCB) after administration to quails of a single oral dose of 4.5 microCi [14C] HCB per 100 g body weight was studied by whole-body autoradiography and liquid scintillation counting. Radiolabeled HCB was distributed throughout the body in 2 h. Peak levels were found 4-8 d after administration, with the highest concentrations in the subcutaneous and abdominal fat, uropygial gland, bone marrow, adrenals, ovarian stroma, and liver, in that order. After 3 wk, substantial amounts of radiolabeled substance were present only in the adipose tissue and the uropygial gland. The main routes of excretion were the egg yolk, uropygial gland, and bile. About 75% of the administered dose was excreted through the egg yolk and about 25% in the feces.

Each of three colostomized laying hens received per os 0.2% L-Lysine with 48 atom-% 15N-excess (15N') labelled in alpha-position in addition to a pelleted laying hen ration of 120 g over a period of 4 days. On the following 4 days they received equal amounts of unlabelled lysine. The eggs laid during the 8 days of the experiment were separated into the white of egg, the yolk and the eggshell, and the to and heavy nitrogen in the individual fractions were determined. Above that, 17 amino acids and their atom-%15N' were determined in the 19 samples of the white and yolk of egg. Of the total 15N' from the lysine fed in the 4 days, 10.1% were found in the yolk, 10.5% in the white of egg and 1.1% in the eggshells of the eggs laid during the 8 days of the experiment. 85% of the total amino acid-15N' of the yolk and 86% of the white of egg detected to be lysine-15N'. The 15N'-amount of the other 16 amino acids was mainly concentrated in the two acid and basic amino acids. Approximately 50% of the non-lysine 15N' in the egg are contained in aspartic acid, glutamic acid, histidine and arginine. A very low incorporation of the labelled lysine only could be detected in the aromatic and sulphur-containing amino acids from both the yolk and the white of egg. 43% of the 15N' was detected in the 10 essential and semi-essential (except lysine) and 57% in the 6 non-essential amino acids of the yolk and 52% and 48% resp. of the white of egg. One can summarize that the incorporation of 15N' into the egg shows the same development as that of the labelled amino acids of the wheat protein and that 15% of the lysine-15N' could be detected in the 16 other amino acids.

Our previous immunofluorescence experiments (1) on actin localization in Xenopus development showed a fluorescent halo around Xenopus yolk platelets. This fact suggests the presence of a sort of actin covering of the yolk platelet; we have called this structure the 'actin-shell'. In this work, by the use of a DNase I-fluorochrome complex, we were able clearly to demonstrate the presence of the actin-shell around Xenopus yolk platelets. A proposal about the function of the actin-shell is made; its presence could mark the difference between autosynthetic and heterosynthetic eggs.

As part of a study of the transfer of proteins and lipids from avian blood to egg yolk, some properties of lipoproteins from the blood of laying hens were compared with those of the low-density lipoprotein (YLP) from egg yolk of the same hens. Although it is known from previous work that particles of the very-low-density lipoprotein (VLDL) of blood are the most likely precursors of YLP, their apoprotein patterns are different, according to electrophoresis and chromatography, with only one protein in common. YLP has the more complicated pattern which does not, however, include apoprotein B (ApoB) the main apoprotein of VLDL. It is suggested that during the transfer of VLDL to yolk, ApoB is cleaved to give smaller yolk apoproteins, especially apovitellenins IV and VI. Some evidence for this suggestion from the similarity of protein digests is presented.

1. Sera from different embryonic stages of development and adult chickens were examined for embryonic-adult differences. 2. An antisera prepared in rabbits to 14 day embryonic chick sera revealed an antigen present in the early stages of embryonic development. The antigen reached a peak on day 14 and was not detectable 5 days post hatching. 3. Gel filtration studies in conjunction with immunodiffusion studies revealed the serum antigen to be in the 160,000 mol wt range. 4. Immunoelectrophoretic analyses of embryonic and adult sera revealed the sera components to increase in quantity and in numbers with development. The serum immunoelectrophoretic profile of newly hatch chick serum closely resembled sera from adult chickens. 5. Polyacrylamide gel electrophoretic comparisons of the sera of the developing chicks and the adult chickens revealed proteins specific to certain developmental stages as well as proteins specific to the adult serum. Qualitative and quantitative serum protein differences were found.

Chicken tissues were intrinsically labeled with a stable isotope of selenium (76Se) and were evaluated for use in a human feeding study. Laying hens were fed a low Se (0.06 ppm) basal diet for 39 days and then fed the basal diet supplemented with 0.3 ppm enriched 76Se (as selenite) for 35 days. Incorporation of 76Se into samples was determined by use of a double isotope dilution technique and a combined gas chromatography/mass spectrometry analysis. The 76Se content of the basal diet was increased by a factor of 9.7 with the addition of the enriched stable isotope. This maximal level of enrichment was approached in egg yolk (9.5-fold) and liver (9.0-fold). Enrichment was lower in egg white (7.2-fold) and breast meat (5.0-fold). Level of enrichment in a given tissue reflected both the turnover rate of the tissue and its natural selenium content. Selenium-depleted laying hens continuously fed 76Se at the 0.3 ppm level produced egg yolks and livers that were enriched sufficiently with the stable isotope for use in a human metabolic study.