Identification of Eggshell Membrane Proteins and Purification of Ovotransferrin and β-NAGase from Hen Egg White

Exposure of selected Gram-positive and Gram-negative bacterial pathogens to egg shell membranes (ESM) significantly reduced their thermal resistance and/or inactivated cells. Although the components responsible for this antibacterial activity have not been conclusively identified, several proteins associated with the ESM activity have been identified including β-N-acetylglucosaminidase, lysozyme and ovotransferrin, with each displaying varying degrees of antibacterial activity. Numerous attempts to purify active fractions of β-N-acetylglucosaminidase, lysozyme and ovotransferrin from the ESM proved somewhat limited; however, hen egg white (HEW) β-N-acetylglucosaminidase was purified using a two-step chromatographic procedure, isoelectric focusing followed by cation exchange chromatography. Pure fractions of ovotransferrin were also obtained in the process. SDS-PAGE electrophoresis and Matrix-Assisted Laser Desorption Time-of-Flight Mass Spectrometry were then used to partially characterize the individual protein components. Purified protein fractions such as these will be required in order to fully elucidate the mechanism responsible for the antimicrobial properties associated with the ESM.     

Emulsifying Properties of a Complex between Apoprotein from Hen’s Egg Yolk Low Density Lipoprotein and Egg Yolk Lecithin

In the present work, an apoprotein solution was prepared from hen’s egg yolk low density lipoprotein (LDL) without using detergents; LDL was delipidated with chloroform-methanol (2:1) and solubilized by sonication with 80% ethylene glycol. An apoLDl-phospholipid complex was prepared by sonicating this apoprotein solution with an egg yolk lecithin suspension. Although high-molecular-weight polypeptides of apoLDL formed an insoluble complex with lecithin, its low- molecular-weight polypeptides formed a soluble complex. The soluble apoLDl-phospholipid complex gave only one peak on gel filtration on Sephacryl S-400.

Emulsifying properties of the soluble apoLDl-phospholipid complex were much better than those of either lecithin vesicles or lecithin suspensions with the same concentration of phospholipid, and almost the same as those of LDL. These results seem to show that the superior emulsifying properties of LDL depend on the characteristic structure of its lipid-protein complexes.     

Embryogenesis of Polyembryonic Rice ApⅢ: Structural and Histochemical Studies of Egg Apparatus Around Fertilization

The structural and histochemical changes of the egg apparatus in the polyembryonic rice (Oryza sativa L.), ApⅢ with the highest frequence of additional embryos among the polyembryonic rice investigated, before and after fertilization were studied and compared with those of normal and other polyembryonic rices in a similar developmental period. A total of 2 932 ovules were observed and each of them contained only asingle embryo sac with a set of egg apparatus. Among 1 655 embryo sacs, there were 1 643 embryo sacs (99.27%) with one normal egg apparatus in each embryo sac, and only 12 embryo sacs (0.73%) from the remainder with 4 celled egg apparatus, i.e. two eggs and two synergids. Neither the numerous poly egg apparatus and egg like cells, nor the double set of embryo sacs each containing one egg apparatus and other abnormal egg apparatus in single ovary, which were reported by earlier investigators to have high frequency of embryo production in SB 1 and ApⅣ, were observed. The egg cell was located at the subterminal site of the micropylar end of embryo sac. The cytoplasm of egg cell was rich in protein materials and poly saccharide grains, which did not disappear until the division of zygote. The prominent nucleus was closely surrounded by protein and polysaccharide grains, which did not disappear until the division of zygote. No cytological difference was found between egg cells from the normal and abnormal egg apparatus. The two synergids were fully developed and situated at the upper most part of the micropylar end of the mature embryo sac. In most embryo sacs, the synergids were flask shaped with longer necks, and a widened cap shaped top, in close contact with the micropyle. The synergids had a well developed filiform apparatus. The characteristic appearance of the filiform apparatus as well as the cap neck region of synergids before and after pollen tube penetration were easily distinguishable from the egg cell. The structure, the stainability with Coomassie Brilliant Blue and PAS reaction, the process of accumulation, distribution and disapperance of the cytoplasmic protein materials and polysaccharide grains of the two synergids, the persistent and rarely the receptive synergids before and after pollen tube penetration, were closely similar to those of egg cell of the same developmental stage. In comparison with normal and other polyembryonic rice reported, the size of nucleus and nucleolus and their stainability also strongly resembled those of egg cell. Based on the results observed, the main conclusions are summarized as follows: (1) the additional embryos very frequently developed in the young and mature seed of polyembryonic rice ApⅢ were produced by one or two synergids of normal egg apparatus, rarely by 4 celled egg apparatus; (2) during fertilization, the synergids, in addition to the natural specific function of introducing pollen tube and transferring sperms to egg cell and central cell, could be closely associated with the potentiality to breed one or two additional embryos; and (3) as compared with that of normal or other polyembryonic rice it is firstly disclosed that in a few embryo sacs of ApⅢ, the cytoplasmic and nuclear structure, the active anabolism and catabolism of protein and polysaccharide materials and the delayed disorganization at the mid basal region of the receptive and persistent synergid still remained unchanged before the division of zygote. Such salient features could be the predisposition for the origin of additional embryos in ApⅢ.     

Species of Root-knot Nematodes and Fungal Egg Parasites Recovered from Vegetables in Almería and Barcelona,Spain

Intensive vegetable production areas were surveyed in the provinces of Almería (35 sites) and Barcelona (22 sites), Spain, to determine the incidence and identity of Meloidogyne spp. and of fungal parasites of nematode eggs. Two species of Meloidogyne were found in Almería—M. javanica (63% of the samples) and M. incognita (31%). Three species were found in Barcelona, including M. incognita (50%), M. javanica (36%), and M. arenaria (14%). Solanaceous crops supported larger (P < 0.05) nematode numbers than cucurbit crops in Almería but not in Barcelona. Fungal parasites were found in 37% and 45% of the sites in Almería and Barcelona, respectively, but percent parasitism was never greater than 5%. Nine fungal species were isolated from single eggs of the nematode. The fungi included Verticillium chlamydosporium, V. catenulatum, Fusarium oxysporum, F. solani, Fusarium spp., Acremonium strictum, Gliocladium roseum, Cylindrocarpon spp., Engiodontium album, and Dactylella oviparasitica. Two sterile fungi and five unidentified fungi also were isolated from Meloidogyne spp. eggs.     

Destruction of Enteric Bacteria in Liquid Egg with β-Propiolactone

Liquid whole egg or egg white, inoculated with Escherichia coli 1485, Salmonella senftenberg ATCC 8400, or Salmonella typhimurium 84-I, was treated with concentrations of β-propiolactone ranging from 0.05 to 0.3%. Egg white containing 1 × 10³ to 1 × 10⁶ cells of E. coli 1485 per ml was sterilized in 1 hr at 27 C by lactone concentrations of 0.2 and 0.3%. Egg white containing 1 × 10⁵ cells of S. senftenberg ATCC 8400 per ml was sterilized in 12 hr at 10 C by 0.1% lactone and in 2 to 3 hr by 0.3% lactone at the same temperature.

Liquid whole egg inoculated with 1 × 10⁵ cells of either species of Salmonella was sterilized in 4 to 5 hr at 10 C with 0.2% lactone or in 2 to 3 hr by 0.3% lactone at this temperature. A mild heat treatment of either 15 min at 37 C or 1 min at 55 C markedly shortened the exposure times required for sterilization by β-propiolactone at 10 C.

After disinfection was complete, the lactone-treated liquid whole egg was reinoculated with low cell numbers of either species of Salmonella to determine the presence of residual lactone or toxic products. Liquid whole egg treated with 0.2% lactone would support the growth of salmonellae after 13 to 14 hr at 10 C. A heat treatment of 45 min at 37 C or 10 min at 55 C immediately after addition of 0.2% lactone allowed growth of the salmonellae in the lactone-treated liquid whole egg. No evidence of residual toxicity from the lactone treatment was found.

The amount of lactone needed to prevent the outgrowth of low cell numbers of either strain of Salmonella in liquid whole egg was quantitated. Liquid whole egg containing 0.06 to 0.07% lactone would not support salmonellae growth from inocula of 1 to 10 cells per ml of egg. Lactone concentrations above 0.08% prevented outgrowth of salmonellae inocula of 10 to 200 cells per ml of liquid whole egg.

     

“Egg transformation” induced by irradiated pollen in Nicotiana: critical appraisal of Chyi and Sanford's observations

Summary This note strongly refutes the suggestion by Chyi and Sanford (1985) that egg transformation by the use of lethally irradiated pollen in Nicotiana reported by the present author could be explained on the basis of mutation, contamination or other causes, and suggests that their conclusion is based on faulty experimentation and interpretation. It also shows that, contrary to these authors' assertions, their results, in fact, contain certain significant demonstrations of egg transformation supporting the present author's work.     

Comparison of Four Distinct Idiotypes of Monoclonal Antibodies against Hen’s Egg Ovomucoid

Two monoclonal antibodies (mAb 41B3 and mAb 51B3), directed against hen’s egg ovomucoid (OM) and different from those we previously reported (mAb 23E5 and mAb 32A8), were prepared and purified. A competitive radioimmunoassay using ¹²⁵I-labeled OM showed that both mAb 41B3 and mAb 51B3 could bind all three homologous domains (domains I (DI), II (DII) and III (Dili)) of OM and that they reacted most efficiently with Dill. To analyze the paratope specificities of these four mAbs, a sandwich assay and a competitive radioimmunoassay were done. Only the pair mAb 41B3 and mAb 51B3 could not simultaneously bind the OM or domains in the sandwich assay. Only mAb 41B3 inhibited the binding of mAb 51B3 to antigens and vice versa in the competitive radioimmunoassay. These results suggest that mAb 41B3 and mAb 51B3 recognized a closely related site distinct from the epitopes for mAb 23E5 or mAb 32A8. These mAbs may be useful for general studies on epitopes of protein antigens as well as for analyses of the antigenic determinants of OM.     

Variations in Fecundity and Egg Size of Female Nile Tilapia, Oreochromis niloticus, from Man-made Lakes of Côte D'Ivoire

Fecundity and oocyte size of Oreochromis niloticus females were studied over a period of two annual cycles in six small agropastoral and three large hydroelectric reservoirs of Côte d'Ivoire. Important differences in fecundity and oocyte size were observed among populations and within the same population between successive years. An inverse correlation was found between size and number of oocytes produced by females. This inverse relationship occurred for a constant spawn weight during the first year of study, but not during the second year. Monthly mean residuals of regressions between fecundity and female body weight have shown a seasonal variation in fecundity. The peak of fecundity corresponded with the maximum resource availability and the flooding eminence, which may have a great impact on parents and offspring fitness.     

Association of the FADS2 Gene with ω-6 and ω-3 PUFA Concentration in the Egg Yolk of Japanese Quail

This study focused on the association of polymorphisms of the FADS2 gene with fatty acid profiles in egg yolk of eight Japanese quail lines selected for high and low ω-6:ω-3 PUFA ratio (h2 = 0.36–0.38). For the identification of polymorphisms within the FADS2 gene 1350 bp of cDNA sequence were obtained encoding 404 amino acids. Five synonymous SNPs were found by comparative sequencing of animals of the high and low lines. These SNPs were genotyped by single base extension on 160 Japanese quail. The association analysis, comprising analysis of variance and family based association test (FBAT), revealed significant effects of SNP3 and SNP4 genotypes on the egg yolk fatty acid profiles, especially the ω-6 and ω-3 PUFAs (P < 0.05). No effects of the other SNPs were found—indicating that these are not in linkage disequilibrium with the causal polymorphism. The results of this study promote FADS2 as a functional candidate gene for traits related to ω-6 and ω-3 PUFA concentration in the egg yolk.     

Egg production by metacercariae of Microphallus papillorobustus: a reproductive insurance?

This study aimed to determine whether metacercariae of Microphallus papillorobustus (Trematoda: Microphallidae) produce eggs by self-fertilization when their intermediate host, the gammarid Gammarus insensibilis, dies before being eaten by predatory definitive hosts. Such a mechanism would allow the parasite to reproduce even if transmission fails. The percentage of metacercariae that produced eggs and the mean number of eggs were significantly higher in metacercariae placed in a Ringer's solution compared with those in salt water or metacercariae remaining inside the dead gammarid. The lack of egg production in metacercariae removed from dead gammarids in the field supported the idea that self-fertilization is not, for this parasite, a mechanism of reproductive insurance when establishment in the definitive hosts fails.     

Agglutinating Activity and Structural Characterization of Scalarin,the Major Egg Protein of the Snail Pomacea scalaris (d’Orbigny, 1832)

Apple snail perivitellins are emerging as ecologically important reproductive proteins. To elucidate if the protective functions of the egg proteins of Pomacea canaliculata (Caenogastropoda, Ampullariidae), involved in embryo defenses, are present in other Pomacea species we studied scalarin (PsSC), the major perivitellin of Pomacea scalaris. Using small angle X-ray scattering, fluorescence and absorption spectroscopy and biochemical methods, we analyzed PsSC structural stability, agglutinating activity, sugar specificity and protease resistance. PsSC aggluttinated rabbit, and, to a lesser extent, human B and A erythrocytes independently of divalent metals Ca²⁺ and Mg²⁺ were strongly inhibited by galactosamine and glucosamine. The protein was structurally stable between pH 2.0 to 10.0, though agglutination occurred only between pH 4.0 to 8.0 (maximum activity at pH 7.0). The agglutinating activity was conserved up to 60°C and completely lost above 80°C, in agreement with the structural thermal stability of the protein (up to 60°C). PsSC was able to withstand in vitro gastrointestinal digestion, and showed no trypsin inhibition activity. The presence of lectin activity has been reported in eggs of other Pomacea snails, but here we link for the first time, this activity to an apple snail multifunctional perivitellin. This novel role for a snail egg storage protein is different from closely related P.canaliculata defensive proteins.     

Isolation of Hen’s Egg Yolk Low Density Lipoprotein by Affinity Chromatography

Curdlan gel shrinks even in water caused syneresis. This syneresis is repressed almost completely by the addition of starch to curdlan before heating, but not repressed by the addition of different sugar compounds even of 10% concentration. The role of starch in repressing syneresis is discussed.     

Culinary Biophysics: on the Nature of the 6X??C Egg

Shell-on eggs cooked by immersion in water at low and constant temperatures (∼60–70 °C) yield yolks with very particular textures. Structure development in such unique cooking conditions is far from understood. The present study shows that egg yolk, despite its compositional complexity, follows typical gelation kinetics found in many globular proteins and that it can develop structure at temperatures as low as 56 °C. It follows that yolk texture is dictated by time/temperature combinations. Under isothermal, low temperature cooking conditions, the thickening and gelation kinetics of egg yolk follow Arrhenius-type kinetic relationships. The energy of activation of these processes was ∼470 kJ mol⁻¹, which agrees well with the values reported for the denaturation and gelation of the thermally labile chicken serum albumin and immunoglobulin Y. Results are related to common foodstuffs in order to allow chefs and home cooks to achieve a priori conceived textures in egg yolks.     

Are Cuckoos Maximizing Egg Mimicry by Selecting Host Individuals with Better Matching Egg Phenotypes?

Background

Avian brood parasites and their hosts are involved in complex offence-defense coevolutionary arms races. The most common pair of reciprocal adaptations in these systems is egg discrimination by hosts and egg mimicry by parasites. As mimicry improves, more advanced host adaptations evolve such as decreased intra- and increased interclutch variation in egg appearance to facilitate detection of parasitic eggs. As interclutch variation increases, parasites able to choose hosts matching best their own egg phenotype should be selected, but this requires that parasites know their own egg phenotype and select host nests correspondingly.

Methodology/Principal Findings

We compared egg mimicry of common cuckoo Cuculus canorus eggs in naturally parasitized marsh warbler Acrocephalus palustris nests and their nearest unparasitized conspecific neighbors having similar laying dates and nest-site characteristics. Modeling of avian vision and image analyses revealed no evidence that cuckoos parasitize nests where their eggs better match the host eggs. Cuckoo eggs were as good mimics, in terms of background and spot color, background luminance, spotting pattern and egg size, of host eggs in the nests actually exploited as those in the neighboring unparasitized nests.

Conclusions/Significance

We reviewed the evidence for brood parasites selecting better-matching host egg phenotypes from several relevant studies and argue that such selection probably cannot exist in host-parasite systems where host interclutch variation is continuous and overall low or moderate. To date there is also no evidence that parasites prefer certain egg phenotypes in systems where it should be most advantageous, i.e., when both hosts and parasites lay polymorphic eggs. Hence, the existence of an ability to select host nests to maximize mimicry by brood parasites appears unlikely, but this possibility should be further explored in cuckoo-host systems where the host has evolved discrete egg phenotypes.     

Could a Factor That Does Not Affect Egg Recognition Influence the Decision of Rejection?

Rejection of the parasitic egg is the most important defence of hosts against brood parasites. However, this response is variable among and within species, and egg discrimination is not always followed by egg rejection. Low risk of parasitism and high risk of rejection costs may lead to the acceptance of the parasitic egg even if it has been previously recognized. The main aim of this paper is to answer a relevant question: can a single egg trait provoke the acceptance of an experimental egg previously recognized as foreign? Increased egg mass should hamper the ejection of an egg that has been discriminated because ejection of a heavy egg may imply higher rejection costs for hosts. We have tested this prediction by experimentally parasitizing natural nests of Common Blackbirds (Turdus merula) with non-mimetic model eggs of different mass (heavy, normal-weight, and light) while controlling for potential confounding factors such as egg size and colour. Our results showed that blackbirds more frequently accepted heavy eggs, even when previously recognized. This differential acceptance may be related to insufficient motivation to assume the higher costs that the ejection of a heavy egg could impose.     

Egg size in relation to body size in rotifers: an indication of reproductive strategy?

Egg sizes and body sizes of 43 egg-bearing rotifer species of numerous, mostly tropical, general have been recorded. Larger absolute egg volumes have been found for larger rotifer species, but the increase was lower than expected in proportion to body size, i.e. the relative egg volume decreased with increasing body size. Obviously the relative investment per offspring is smaller in larger rotifer species.     

Biologically Active Human Interferon α-2b Produced in the Egg White of Transgenic Hens

We have previously described the expression of a bacterial protein in the egg white of transgenic chickens using a replication-deficient retroviral vector. Here we report the expression of a glycosylated human protein, interferon -2b (hIFN), in the egg white of transgenic hens. The hIFN secreted into the egg white was biologically active as determined by a viral inhibition assay. Purification and carbohydrate analysis of the hIFN expressed in egg white revealed that two of the six major glycosylated hIFN species match the naturally occurring human hIFN glycovariants. These results support the potential of the hen as a bioreactor for the production of commercially valuable, biologically active, and glycosylated proteins in egg white.