Producción de anticuerpos antiprogesterona apartir de la yema de huevo de gallinas y del suero sanguíneo de conejos, para ser utilizados en radioinmunoanálisis / Production of antibodies against progesterone from the eggyolk of hens and from rabbit blood serum to be used in radioimmunoassay

Se indujo la producción de anticuerpos contra progesterona en gallinas Leghorn y conejos Nueva Zelanda, con el propósito de utilizarlos para establecer un Radioinmunoanálisis (RIA) para determinar progesterona. La extracción de anticuerpos de la yema de huevo de las gallinas inmunizadas se realizó con 2-propanolyacetona. Los anticuerpos del suero sanguíneo de conejo se extrajeron con solución de sulfato de amomio. Los anticuerpos de la yema del huevo mostraron elevada afinidad y especificidad hacia la progesterona. La sensibilidad del RIA establecido con estos anticuerpos fue de 0.27 ng/ml; los coeficientes de variabilidad intraensayo e intraensayo fueron de 19.6 y 22.2 por ciento, respectivamente. Hubo una correlación de 0.94 entre los valores de progesterona determinados con un estuche comercial y los establecidos con los anticuerpos de la yema. Los anticuerpos obtenidos del suero de conejo también tuvieron elevada afinidad y especificidad hacia la progesterona. La sensibilidad de RIA que utilizó este anticuerpo fue de 0.23 ng/ml, el coeficiente de variabilidad intraensayo de 14.7 por ciento y el interensayo de 22.7 por ciento. Los valores progesterona medidos con un estuche comercial y con el RIA establecido con los anticuerpos del conejo tuvieron una correlación de 0.96. Se concluye que la yema de huevo de gallinas inmunizadas es una fuente práctica de anticuerpos antiprogesterona de calidad comparable a la de los anticuerpos obtenidos a partir de suero sanguíneo de conejos. La producción de anticuerpos antiprogesterona extraídos de la yema del huevo de gallinas inmunizadas, fue 2.5 vecesa mayor que la obtenida del suero sanguíneo de conejos inmunizados contra el mismo antígeno

Cholesterol and phospholipids content of yolk from fertilized and unfertilized hen eggs

Three lipid-containing (granules, low-density lipoproteins (LDL) and infranatant) of fertilized and unfertilized yolks were obtained from hen eggs, either from commercial sources or from Arbor acres henskept by the Pena Branca Aviario Pernambuco and utilized fresh (laid during the previous 7 days). Total cholesterol (TC) and total phospholipid (TP) levels (mg/g yolk, reported as means ñ SD) were determined. In the yolk granules (insoluble fraction) the levels of TC (2.05 ñ 0.36) and TP(0.90 ñ 0.43) of fertilized egg yolks were similar to the levels of TC (2.20 ñ 0.4) and TP (0.90 ñ 0.14) of unfertilized eggs. The levels TC in the LDL from fertilized egg yolks (8.29 ñ 1.63) were not statistically different from those in unfertilized eggs (7.31 ñ 1.50). In contrast, TC was not detected in the infranatant fraction of unfertilized egg yolks, but was present in the infranatant fraction (1.39 ñ 0.69) of fertilized eggs. The TP levels of LDL (0.73 ñ 0.23) and infranatant (0.32 ñ 0.09) fractions of fertilized egg yolks were significantly lower than the levels of TP in the LDL (1.73 ñ 0.51) and infranatant (0.79 ñ 0.59) fractions of unfertilized eggs. Consequently, the TC/TP ratio (mol/mol) increased in the LDL and infranatant fractions of fertilized egg yolks when compared to unfertilized egg yolks. TC levels were similar in the total yolks TC levels were similar in the total yolk of fertilized (10.76 ñ 1.32) and unfertilized (10.33 ñ 1.77) eggs, while the TP levels were significantly lower in the fertilized (1.92 ñ 0.17) than in unfertilized (3.43 ñ 0.97) eggs. These results suggest a transfer of TC from the LDL to the infranatant fraction and a large consumption of TP during the fertilization process

Effect of alkyl chain unsaturation and cholesterol intercalation on oxygen transport in membranes: a pulse ESR spin labeling study.

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.

Fatty acid remodelling of phosphatidylinositol under conditions of de novo synthesis in rat liver microsomes.

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.

Immunoelectron microscopical demonstration of egg-yolk plasma precursor vitellogenin in the hepatocyte of oestradiol-treated cockerels.

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).

Evaluation of two extraction methods for the determination of egg yolk cholesterol.

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.

Detection of Salmonella serogroup D-specific antibodies in the yolks of eggs laid by hens infected with Salmonella enteritidis.

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.

Dietary modification of yolk lipid with menhaden oil.

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.

Sugar-deprivation following a blood meal does not reduce yolk formation and fertility in Culex quinquefasciatus.

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 method for the preparation of liposomes for pharmaceutical applications: characterization of the liposomes.

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.

Plasma and yolk cholesterol levels in Japanese quail divergently selected for plasma cholesterol response to adrenocorticotropin.

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.

Rapid enzyme immunoassay of chicken egg yolk IgG.

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.

Residues of doxycycline and oxytetracycline in eggs after medication via drinking water to laying hens.

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.

Biotin diffusion in stored chicken eggs: a re-assessment.

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.

Modulation of plasma biotin in the fowl (Gallus domesticus) by oestrogen and ambient temperature.

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.

Inhibition of amphotericin B (Fungizone) toxicity to cells by egg lecithin-glycocholic acid mixed micelles.

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.

Isolation of Listeria monocytogenes small-plaque mutants defective for intracellular growth and cell-to-cell spread.

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.

Effect of feeding cholesterol to laying hens and chicks on cholesterol metabolism in pre- and posthatch chicks.

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.

Preliminary data on efficacy of Mycoplasma gallisepticum vaccines containing different adjuvants in laying hens.

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.

Residues of macrolide antibiotics in eggs following medication of laying hens.

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.