Robert G. Elkin

PROTEOLYSIS OF JAPANESE QUAIL AND CHICKEN PLASMA APOLIPOPROTEIN B AND VITELLOGENIN BY CATHEPSIN D : SIMILARITY OF THE RESULTING PROTEIN FRAGMENTS WITH EGG YOLK POLYPEPTIDES

Plasma very-low density lipoprotein (VLDL) and vitellogenin (VTG) from mature female Japanese quail (Coturnix coturnix japonica) and chickens (Gallus domesticus) were isolated and digested in vitro with cathepsin D (EC3.4.23.5). The incubation mixtures were then reduced and subjected to gradient (4.5-18%) SDS-polyacrylamide gel electrophoresis. Protein fragments were stained with either Coomassie Brilliant Blue R-250 (VLDL digests) or Coomassie Brilliant Blue R-250 containing 20 mM AlCl3 (VTG digests). Fragments resulting from the in vitro enzymatic digestion of quail and chicken plasma VLDL-apolipoprotein B (apo B) and VTG closely resembled those produced in vivo and isolated from egg yolks of each respective species. Phosvitin, a proteolytically derived fragment of VTG, primarily existed as a single band (M(r) approximately 42 kDa) in Japanese quail yolk granules. In contrast, chicken phosvitin mainly consisted of a cluster of phosphoproteins ranging in size from approximately 37 to 45 kDa. In addition to reporting a novel species difference in phosvitin moieties, the present study is the first to examine the role of cathepsin D in the generation of egg yolk proteins from plasma precursors in Japanese quail. Confirmatory evidence also was provided concerning the important role of this aspartic endopeptidase in the proteolytic cleavage of plasma VLDL-apo B and VTG in the chicken.

Comparative assessment of bone among wild-type, restricted ovulator and out-of-production hens

1. The aim of this study was to assess bone characteristics in restricted ovulator (RO) hens. These hens generally are unable to ovulate due to a point mutation in the oocyte VLDL receptor gene whose protein product mediates the uptake of yolk precursors. Because these hens do not have the cyclic calcium (Ca) metabolism associated with egg formation, they could be a useful model for studying bone metabolism. 2. RO hens had greater humerus, femur and tibia ash concentrations than wild-type (WT) and out-of-production (OP) hens. Bone mineral content and density obtained with dual-energy X-ray absorptiometry (DXA) were highly correlated with the results of conventional bone assays. 3. Gross and histological examination of the femurs confirmed the presence of extremely dense medullary bone deposition in the RO hens. However, the composition of non-collagenous protein extracts of medullary bone was similar for the two genotypes. 4. Analysis of medullary bone extracts for glycosaminoglycans (GAG) confirmed the presence of large amounts of keratan sulphate (KS) in the matrix of medullary bone. 5. Plasma Ca, total GAG and KS concentrations of RO hens were markedly higher than WT and OP hens. The changes in plasma calcium and keratan sulphate are probably a reflection of elevated Ca-binding yolk precursor molecules and intensive medullary bone formation in response to increased plasma oestrogen observed by others in RO hens.

A review of duck nutrition research

A review of the literature on the nutrition of ducks revealed that although several areas have been intensively researched (protein requirements, dietary effects on carcass composition, and vitamin...

Biliary bile acid profiles of domestic fowl as determined by high performance liquid chromatography and fast atom bombardment mass spectrometry.

1. The biliary bile acid profiles of domestic chickens (Gallus domesticus), turkeys (Meleagris gallopavo), and ducks (Anas platyrhynchos) were determined by high performance liquid chromatography (HPLC) and fast atom bombardment mass spectrometry (FAB-MS). 2. Chenodeoxycholyltaurine and cholyltaurine were the predominant bile acids in chicken and turkey bile, whereas duck bile contained primarily chenodeoxycholyltaurine and phocaecholyltaurine. 3. Allocholyltaurine was also detected in chicken and turkey bile, but not in duck bile. 4. FAB-MS analyses of individual HPLC peak fractions from chicken and duck bile extracts confirmed the presence of either taurine-conjugated dihydroxy- or trihydroxycholanoates. 5. Direct FAB-MS analyses of avian bile extracts not subjected to HPLC permitted a rapid assessment of the relative proportion of taurine-conjugated dihydroxy- to trihydroxycholanoates.

Feeding laying hens a bioavailable soy sterol mixture fails to enrich their eggs with phytosterols or elicit egg yolk compositional changes

Coronary heart disease (CHD) is the leading cause of death in the United States. Elevated levels of plasma total cholesterol (TC), and particularly plasma low density lipoprotein-cholesterol (LDLC), are primary contributing factors to CHD. Dietary plant sterols (phytosterols) have been shown to significantly reduce plasma TC and LDLC in humans, primarily through inhibition of intestinal cholesterol absorption, and are potentially effective agents for reduction of CHD risk. Although a variety of phytosterol-containing foods are currently available, phytosterol-enriched eggs, which represent a potential value-added product, are conspicuously absent from the marketplace. Therefore, the objectives of this study were 1) to enrich shell eggs with phytosterols; and 2) to determine if feeding phytosterols to hens elicits egg compositional changes, particularly that of yolk cholesterol content. Sixteen 32-wk-old White Leghorn hens were fed a corn-soy-based layer diet without (n = 8) or with (n = 8) 1 g of supplemental soy sterols/100 g of diet for 28 d.. Hen performance was determined on an individual basis, and 1 egg/hen per week was collected, processed, and analyzed for yolk cholesterol, CP, crude fat (CF), and phytosterol content. There was no effect (P > 0.05) of supplemental dietary phytosterols on 28-d weight gain, feed consumption, feed efficiency, plasma TC, hen-day egg production, egg weights, egg component weights, and yolk cholesterol, CP, and CF contents. Small amounts of campesterol were present in most of the eggs (average of 0.29 and 1.02 mg/yolk for control vs. soy sterol-fed hens, respectively; P < or = 0.05), whereas only 3 of the 80 analyzed eggs contained trace amounts of beta-sitosterol and none contained any detectable stigmasterol. It was concluded that phytosterols are either poorly absorbed from the chicken intestine or, if they are absorbed, they are efficiently secreted back into the intestinal lumen, most likely via as yet uncharacterized adenosine triphosphate-binding cassette transporters.

Dietary self-selection and the regulation of protein and energy intake in chicks

Studies were conducted to determine whether chicks could regulate their protein intake independent of total energy intake in self-selection feeding trials. Day-old White Mountain cockerels were reared in electrically-heated battery brooders and given access to either a 23% protein control ration (no choice) or two diets containing 10% or 60% protein with or without supplemental amino acids. The latter were added to either improve the dietary amino acid balance or to alter plasma and brain levels of free large neutral amino acids (tryptophan, isoleucine, leucine, valine, phenylalanine, and tyrosine) which have been implicated in the neuroregulation of feed intake. Both feed and water were provided ad lib, and the location of the feed troughs within each pen were changed daily. Body weights and feed intakes were measured daily, and total calorie and protein intakes were calculated. Chicks offered 10% and 60% protein diets with no supplemental amino acids exhibited reduced weight gains and markedly higher protein intakes as compared to birds fed either the control ration or those given a choice between 10% and 60% protein diets supplemented with methionine. The higher protein consumption by chicks fed the unsupplemented diets most likely was a result of an attempt to compensate for a dietary methionine deficiency. Chicks fed the 10% and 60% protein diets supplemented with amino acids grew at a slower rate than those fed the 23% protein control diet. In general, plasma and brain data did not support a proposed relationship between certain large neutral amino acid ratios and protein or energy intake.

Characterization of Ascites-Derived Ovarian Tumor Cells from Spontaneously Occurring Ovarian Tumors of the Chicken: Evidence for E-Cadherin Upregulation

Ovarian cancer, a highly metastatic disease, is the fifth leading cause of cancer-related deaths in women. Chickens are widely used as a model for human ovarian cancer as they spontaneously develop epithelial ovarian tumors similar to humans. The cellular and molecular biology of chicken ovarian cancer (COVCAR) cells, however, have not been studied. Our objectives were to culture COVCAR cells and to characterize their invasiveness and expression of genes and proteins associated with ovarian cancer. COVCAR cell lines (n = 13) were successfully maintained in culture for up to19 passages, cryopreserved and found to be viable upon thawing and replating. E-cadherin, cytokeratin and α-smooth muscle actin were localized in COVCAR cells by immunostaining. COVCAR cells were found to be invasive in extracellular matrix and exhibited anchorage-independent growth forming colonies, acini and tube-like structures in soft agar. Using RT-PCR, COVCAR cells were found to express E-cadherin, N-cadherin, cytokeratin, vimentin, mesothelin, EpCAM, steroidogenic enzymes/proteins, inhibin subunits-α, βA, βB, anti-müllerian hormone, estrogen receptor [ER]-α, ER-β, progesterone receptor, androgen receptor, and activin receptors. Quantitative PCR analysis revealed greater N-cadherin, vimentin, and VEGF mRNA levels and lesser cytokeratin mRNA levels in COVCAR cells as compared with normal ovarian surface epithelial (NOSE) cells, which was suggestive of epithelial-mesenchymal transformation. Western blotting analyses revealed significantly greater E-cadherin levels in COVCAR cell lines compared with NOSE cells. Furthermore, cancerous ovaries and COVCAR cell lines expressed higher levels of an E-cadherin cleavage product when compared to normal ovaries and NOSE cells, respectively. Cancerous ovaries were found to express significantly higher ovalbumin levels whereas COVCAR cell lines did not express ovalbumin thus suggesting that the latter did not originate from oviduct. Taken together, COVCAR cell lines are likely to improve our understanding of the cellular and molecular biology of ovarian tumors and its metastasis.

The restricted ovulator chicken strain: An oviparous vertebrate model of reproductive dysfunction caused by a gene defect affecting an oocyte-specific receptor

A unique non-laying strain of chickens with heritable hyperlipidemia and aortic atherosclerosis was first described in 1974. Subsequent work established that the phenotype results from a naturally occurring point mutation in the gene specifying the very low density lipoprotein (VLDL) receptor, a 95-kDa membrane protein which normally mediates the massive uptake of the main circulating hepatically-synthesized yolk precursors, VLDL and vitellogenin. As a result, hens of the mutant strain termed “restricted ovulator” (R/O) have approximately 5-fold elevations in circulating cholesterol and triglyceride concentrations compared with normal layers, and hepatic lipogenesis and cholesterogenesis are markedly attenuated due to feedback inhibition. R/O hens also exhibit hyperestrogenemia, hypoprogesteronemia, elevated circulating gonadotropins, and up-regulated pituitary progesterone receptor mRNA and isoforms. The ovaries of R/O hens are abnormal in that they lack a follicular hierarchy and contain many small preovulatory follicles of various colors, shapes, and sizes. However, since R/O hens occasionally lay eggs, it is possible that endocytic receptors other than the VLDL receptor may be able to facilitate oocyte growth and/or that yolk precursor uptake can occur via a nonspecific bulk process. A mammalian model of impaired fecundity with abnormal lipoprotein metabolism also has been described, but different mechanisms are likely responsible for its reproductive dysfunction. Nevertheless, as our understanding of the molecular physiology and biochemistry of avian oocyte growth continues to expand, in part due to studies of the R/O model, new analogies may emerge between avian and mammalian systems, which ultimately could help to answer important questions in reproductive biology.

Uptake of Yolk Very Low Density Lipoprotein by Chicken and Quail Embryos Is Not Mediated by a Homologue of the Oocyte Vitellogenesis Receptor

Abstract In chicken (Gallus domesticus) embryos, a limited amount of yolk engulfment occurs via coated invaginations at the yolk sac membrane apical surface. Because the presence of these so-called “coated pits” is associated with receptor-mediated endocytosis, the purpose of the present study was to demonstrate the existence on the yolk sac membrane of receptor sites for the interaction with very low density lipoprotein (VLDL), the major component of egg yolk. Ligand blotting experiments revealed the presence of a VLDL-binding protein (Mr ∼95 kDa) in yolk sac membranes of both chicken and Japanese quail (Coturnix coturnix japonica) embryos 8 days of age and older. However, these VLDL-binding proteins were present in very low abundance relative to that of another apolipoprotein B receptor that is found in the plasma membrane of chicken and quail oocytes (the so-called oocyte vitellogenesis receptor [OVR]; Mr 95 kDa). Furthermore, no signals were detected when chicken and quail yolk sac membrane proteins were probed with a rabbit polyclonal antibody raised against the 14 C-terminal amino acids of the chicken OVR. It was concluded that chicken and quail yolk sac membrane VLDL-binding proteins were structurally different from the chicken OVR and that receptor-mediated endocytosis plays a minor role in the uptake of yolk VLDL by developing avian embryos.

Comparative utilization of d- and l-methionine by the white pekin duckling (Anas platyrhynchos)

1. 1. White Pekin ducklings (Anas platyrhynchos) were fed a sulfur amino acid-deficient corn-peanut meal basal diet supplemented with either d- or l-methionine (MET) at levels of 0,0.04, 0.08, or 0.16%. 2. 2. Although liver and kidney d-amino acid oxidase activities were induced in birds fed d-MET, their weight gains were only approximately 78% of those fed l-MET at each level of supplementation. 3. 3. Ducklings fed the basal diet supplemented with 0.08 or 0.16% d-MET had significantly (P<0.05) higher plasma MET levels than birds fed an equimolar amount of l-MET. 4. 4. It was concluded that d-MET was a less efficacious source of dietary MET activity than l-MET for White Pekin ducklings.