Recombinant eggshell ovocalyxin-32: expression, purification and biological activity of the glutathione S-transferase fusion protein

The avian eggshell is a highly ordered biomineral composed mainly of calcium carbonate associated with an organic matrix composed of proteins, glycoproteins and proteoglycans. This structure provides the developing embryo with protection from physical damage and microbial invasion. Ovocalyxin-32 (OCX-32) is a 32 kDa eggshell-specific matrix protein which has been cloned and demonstrates 30% identity with the mammalian carboxypeptidase inhibitor, latexin. In order to further study its function, recombinant OCX-32 protein was expressed in E. coli. The protein was extracted from inclusion bodies and purified by sequential DEAE Sepharose and Ni2+ metal ion affinity chromatographies as a 58 kDa GST-fusion protein. The refolded GST-OCX-32 significantly inhibited bovine carboxypeptidase and also inhibited the growth of Bacillus subtilis. The results suggest that OCX-32 may show similar activity to the fusion protein and reinforce the antimicrobial properties of the eggshell by providing protection to the developing avian embryo. OCX-32 is the first example of an eggshell specific protein to be successfully cloned and expressed in a prokaryotic system. The association of an antimicrobial protease inhibitor with the outer eggshell and cuticle of the table egg may enhance the food safety of this product.     

Structure of the cuticle of the common house cricket with reference to the location of lipids

Cuticle segments from the thorax, abdomen, and jumping legs of the house cricket. Acheta domesticus, were examined using histological techniques for light microscopy, scanning and transmission electron microscopy, and direct examination of frozen-fractured cuticle. The surface of untreated cuticle is covered by a lipid film which obscures fine surface detail. Standard EM preparative procedures, as well as washing the cuticle with ethanol before examination, remove this film exposing previously covered openings to dermal gland ducts and wax canals. An epicuticle, exocuticle, mesocuticle, endocuticle, and a deposition layer were present in all transverse sections of cuticle. Light microscopy showed that the exocuticle and mesocuticle are heavily impregnated with lipids, whereas there is little lipid associated with the endocuticle. Frozen-fractured cuticle clearly shows the ‘plywood’ structure of the meso- and endocuticle, while the exocuticle fractures as if it were a solid sheet. The epicuticle is composed of a dense homogeneous layer, cuticulin, outer epicuticle, and the outer membrane. Superficial wax was detected only in cuticle samples prepared using vinylcyclohexane dioxide as a polar dehydrant. The results were used to construct a comprehensive model of the cuticle of A. domesticus.     

Fine structure of the cuticle of the black widow spider with reference to surface lipids

The surface and transverse sections of the cephalothorax, abdomen, and walking leg cuticle of the black widow spider, Latrodectus hesperus, were examined by scanning and transmission electron microscopy. Cuticle that was untreated prior to normal EM preparative procedures was compared with cuticle subjected to lipid solvents and/or concentrated alkali. The surface of untreated dorsal cephalothorax cuticle contained droplets and a lipid film that obscured fine surface detail. Immersing the cuticle in chloroform: methanol removed the droplets and lipid film, exposing previously covered openings to dermal gland ducts. An epicuticle, exocuticle, and endocuticle were present in all transverse sections of cuticle as was a complex system of pore and wax canals that connected the epidermis with the cuticle surface. The epicuticle of the walking leg was composed of three sublayers: outer membrane, outer epicuticle, and the dense homogeneous layer. A cuticulin layer was not observed. Lipid solvents did not significantly alter the morphology of any of these layers or the contents of the wax/pore canals.     

Analysis of Siamese Crocodile (<Emphasis Type="Italic">Crocodylus siamensis</Emphasis>) Eggshell Proteome

The proteins and pigment of the eggshell of the Siamese crocodile (Crocodylus siamensis) were analysed. For proteomic analysis, various decalcification methods were used when the two main surface layers were analyzed. These layers are important for antimicrobial defense of egg (particularly the cuticle). We found 58 proteins in both layers, of which 4 were specific for the cuticle and 26 for the palisade (honeycomb) layer. Substantial differences between proteins in the eggshell of crocodile and previously described birds’ eggshells exist (both in terms of quality and quantity), however, the entire proteome of Crocodilians has not been described yet. The most abundant protein was thyroglobulin. The role of determined proteins in the eggshell of the Siamese crocodile is discussed. For the first time, the presence of porphyrin pigment is reported in a crocodilian eggshell, albeit in a small amount (about 2 to 3 orders of magnitude lower than white avian eggs).     

Microstructure of matrix and mineral components of eggshells from White Leghorn chickens (Gallus gallus)

The avian eggshell is a composite structure of organic matrix and mineral (calcium carbonate) that is rapidly and sequentially fabricated in the oviduct in <24 hr. The eggshell is an excellent vehicle for the study of biomineralization processes and the role of the organic matrix in the mineral-matrix composite. The organic matrix components of eggshells from White Leghorn chickens (Gallus gallus) were examined by transmission electron microscopy (TEM) and optical microscopy. The mineral phase was analyzed by TEM, scanning electron microscopy (SEM), X-ray compositional microanalysis, and electron diffraction. Ultrastructural examination of the matrices within the calcified eggshell reveals a complex architecture that differs within each of the major zones of the eggshell: the shell membranes, the mammillary zone, the palisade region, and the cuticle. The mammillary layer consists of the calcium reserve assembly (CRA) and crown region, each with a unique substructure. TEM images show that the matrix of the CRA consists of a dense, flocculent material partially embedded within the outer shell membrane (a mostly noncalcified region of the shell). The mantle of the collagen fibers of the shell membranes is rich in polyanions (cuprolinic blue-positive), as is the CRA matrix. The CRA is capped by a centrally located calcium reserve body sac (CRB sac) that contains numerous 300–400 nm, electron-dense, spherical vesicles. Directly above the CRB sac is a zone of matrix consisting of stacks of interconnected vesicles (similar in morphology to CRA vesicles) that are interspersed with a granular material. The palisade region, the largest of the mineralized zones, contains hollow vesicles ∼450 nm (s.d. = 75 nm) in diameter, with a crescent-shaped, electron-dense fringe. An interconnecting matrix material is also found between the vesicles in the palisades region. The cuticle is composed of two layers, a mineralized inner layer and an outer layer consisting of only organic matrix. The bulk of the mineral within the eggshell is calcite, with small amounts of needlelike hydroxyapatite in the inner cuticle and occasionally, vaterite micro crystals found at the base of the palisade (cone) region. The well-crystallized calcite crystals within the palisade are columnar, typically ∼20 μm wide by 100–200 μm long; aside from numerous entrapped vesicles and occasional dislocations, they are relatively defect-free. The bulk of the matrix found in the palisade and crown regions are thought to be residual components of the rapid mineralization process. The unique matrix structure within the CRB corresponds to the region of preferentially solubilized calcite used by the developing embryo and the hydroxyapatite found in the inner cuticle may play a role in the cessation of mineral growth. © 1996 Wiley-Liss, Inc.     

Enhanced C-type lysozyme content of wood duck (Aix sponsa) egg white: an adaptation to cavity nesting?

Abstract Wild waterfowl species often nest in conditions where high humidity and microbial contamination may influence egg survival and quality. Albumen is traditionally regarded as the major impediment to microbial contamination of eggs, and its composition and activity may be selected by environmental pressures. Egg white protein from the eggs of wood duck (Aix sponsa), hooded merganser (Lophodytes cucullatus), Canada goose (Branta canadensis), and mute swan (Cygnus olor) was evaluated in order to compare the antimicrobial defenses of these species. Ovotransferrin and ovalbumin were identified in all species, but c-type lysozyme was present only in wood duck and hooded merganser egg white samples. Wood duck egg white showed the greatest bacterial activity as well as the highest lysozyme content. Egg white from wood duck and hooded merganser possessed greater lysozyme activity under acidic conditions, suggesting a c-type lysozyme with a pH optimum lower than that of Gallus gallus c-type lysozyme or the presence of g-type lysozyme. Ovotransferrin bacteriostatic activity appeared to be similar across the species investigated. The results suggest that lysozyme and ovotransferrin play a role in the antimicrobial defense of the avian egg. High levels of the broad-acting c-type lysozyme appear to have evolved in the albumen of the wood duck in order to ensure proper development of the embryo in the humid conditions of the cavity nest.     

藏马鸡卵壳的扫描电镜观察

利用扫描电镜对我国特有珍禽──藏马鸡的卵壳进行了超微结构观察。电镜下显示：藏马鸡卵壳从内向外由壳膜层、锥体层、海绵层和表层等组成。壳膜层内层致密、含少量纤维,外层为纵横交错成网状的纤维结构,锥体层由许多乳头状突起密集排列组成,海绵层为似沉积岩层的层状结构,表层在卵壳最外面,上由具保护性的透明蛋白质薄膜覆盖。与同属的褐马鸡的卵壳进行比较,其超微结构存在差异。     

Structure of the shell and tertiary membranes of eggs of softshell turtles (Trionyx spiniferus)

Eggs of the turtle Trionyx spiniferus are rigid, calcareous spheres averaging 2.5 cm in diameter. The eggshell is morphologically very similar to avian eggshells. The outer crystalline layer is composed of roughly columnar aggregates, or shell units, of calcium carbonate in the aragonite form. Each shell unit tapers to a somewhat conical tip at its base. Interior to the crystalline layer are two tertiary egg membranes: the outer shell membrane and the inner shell membrane. The outer shell membrane is firmly attached to the inner surface of the shell, and the two membranes are in contact except at the air cell, where the inner shell membrane separates from the outer shell membrane. Both membranes are multi-layered, with the inner shell membrane exhibiting a more fibrous structure than the outer shell membrane. Numerous pores are found in the eggshell, and these generally occur at the intersection of four or more shell units.     

Chemical composition and antimicrobial activity of cuticular and internal lipids of the insect Rhynchophorus palmarum

Insect cuticle lipids are involved in various types of chemical communication between species, and reduce the penetration of insecticides, chemicals, and toxins, as well as provide protection against the attack of microorganisms, parasitic insects, and predators. Ecological studies related to the insect Rhynchophorus palmarum are well-known; however, very little is known about its resistance mechanisms, which includes its lipid composition and its importance, specifically the cuticle layer. This study aimed to characterize the cuticle and internal lipid compounds of the male and female R. palmarum adult insects and to evaluate the presence of antimicrobial activity. We performed by gas chromatography coupled to mass spectrometry (GC–MS) analyzes of lipid extracts fractions and we identified 10 methyl esters of fatty acids esters of C14 to C23, with variation between the sexes of C22:0, C21:0, present only in male cuticle, and C20:2 in female. The lipid content of this insect showed relevant amount of C16:1, C18:1, and C18:2. The antimicrobial activity of the cuticular and internal fractions obtained was tested, which resulted in minimum inhibitory concentrations between 12.5 and 20 μg/ml against Gram-positive bacteria (Staphylococcus epidermidis, Enterococcus faecalis), Gram-negative (Pseudomonas aeruginosa, Escherichia coli, Klebsiella pneumonia), and fungal species (Candida albicans e Candida tropicalis). The antimicrobial effect of the R. palmarum cuticle open perspectives for a new source to bioinsecticidal strategies, in addition to elucidating a bioactive mixture against bacteria and fungi.     

Evidence for water-organized structure in the cuticular water barrier of the American cockroach,Periplaneta americana

Summary Cuticle discs cut from the wings ofPeriplaneta and cuticular lipid extracts were analysed by differential scanning calorimetry. Two major endothermic events, one beginning at 7.1±0.3°C, and the other increasing with temperature above 24.7±0.7°C, were associated with extractable lipids in intact cuticle discs. Heating progressively destroyed the molecular structures responsible for the high temperature event but had no effect on the lower one. These results complement changes in cuticle permeability observed in a recent study and thought to be associated with structural change in the cuticular water barrier. The molecular structures responsible for both events depended on the presence of water in the cuticle. Cuticular lipid extracts lack the molecular organization found in intact cuticle, even when water is present.     

Fine structure and formation of eggshells in marine Gastrotricha

Summary The fine structure of the gastrotrich eggshell in the hermaphroditic species Turbanella ocellata (Hummon 1974) and the parthenogenetic species Aspidiophorus sp. is described using transmission electron microscopy. The presented evidence strongly suggests that the shell is produced by the egg itself prior to oviposition in both species. The layed egg in Aspidiophorus sp. is provided with a special attachment stalk that is also preformed in the mother animal. Freshly layed eggs of T. ocellata are adhesive all around their surface and lack any specialized structures for attachment. Formation of the spiny eggshell of Aspidiophorus sp. appears to begin with a sudden release of special vesicles containing the preformed spines of the outer eggshell covering. Additional material appears to be secreted by the egg in a more gradual process after the initial vesicle release. The formation of the two fibrous layers in the eggshell of T. ocellata is less well understood and deposition of eggshell material could be seen either as a continuous process or as two separate steps, similar to the events observed for Aspidiophorus sp. For T. ocellata, Tetranchyroderma sp. and Aspidiophorus sp. it is demonstrated that formation of the cuticle occurs as an independent process from that of eggshell formation. This is significantly different from the basic mode of cuticle formation in the annelid line of evolution. The paper argues further that the data support earlier claims of a pronounced difference between the Gastrotricha-Macrodasyida and the Gastrotricha-Paucitubulatina and agree well with the postulated ties of the Gastrotricha and Nematoda. The phylogenetic importance of the eggshell fine-structure is discussed in the framework of present theories on aschelminth phylogeny.Abbreviations cus cuticular spines - cut cuticle - cov coated vesicles - cv cup-shaped vesicles - dp dense particles - ep epidermis - emb embryo - erl lacunae of smooth ER - fgb fibrous and granular bodies - fl fibrous layer - ga Golgi apparatus - gc gut cell - gv Golgi vesicles - im intercellular matrix - isp intercellular space - isl inner shell layer - ld lipid droplet - mdb medium-dense bodies - mvb multivesicular bodies - oc oocyte - od oviduct - osl outer shell layer - o egg - sv spiny vesicles - sh eggshell - st egg-stalk - sl spiny layer - sub substrate - trm trilaminate membrane - yb yolk bodies - yg yolk granule - yoc young oocyte This work was supported by NSF Grant # GB-42211 to R.M. Rieger     

Proteomic analysis provides new insight into the chicken eggshell cuticle

The cuticle is the outermost layer of the avian eggshell, whose protein constituents remain virtually unknown. We hypothesize that cuticle components play a major role in microbial resistance, since eggs with incomplete or absent cuticle are more susceptible to bacterial contamination. In this study we extracted proteins from the outermost non-calcified layer of the cuticle of chicken eggs and subjected them to LC/MS/MS proteomic analysis. We identified 47 cuticle proteins with high confidence and reproducibility. Two proteins, similar to Kunitz-like protease inhibitor and ovocalyxin-32 (a carboxypeptidase A inhibitor), were the most abundant of the cuticle proteins. A number of proteins known to have antimicrobial activity in the egg were detected (lysozyme C, ovotransferrin, ovocalyxin-32, cystatin, ovoinhibitor) as well as possible new candidates (myeloperoxidase, ovocalyxin-36 and members of the SERPIN family). This is the first comprehensive report of cuticle proteome, a starting point to determine cuticle function and the molecular basis of its antimicrobial properties.     

Integumental organs of unknown function on chelipeds of deep-sea crabs,genus Hypsophrys

Homolid crabs (Hypsophrys) from water deeper than 700 m in the Straits of Florida and Arabian Sea have smooth darkened oval spots contrasting with the surrounding roughened integument on inner and outer surfaces of each pincer at the base of the fixed finger. Cuticle is thinner over these spots than over surrounaing tissues. Beneath each spot is an organ composed of two markedly contrasting layers of tissue: (1) an outer, densely staining layer of tightly packed tubules, relatively straight and perpendicular to the overlying surface proximally but progressively convoluted and narrowed distally, finally ending blindly in association with the overlying thinned cuticle; (2) an inner layer of relatively large, eosinophilic, irregular cells with dark nuclei also trending at a right angle to the integument and bulging into the hemal sinus of the hand but separated from it by an epidermal lining. Droplets secreted from the inner layer apparently move into and along the tubules. Similar organs are known in no other crabs. The function is unknown but the structure suggests that they may be photophores.     

Antimicrobial properties of avian eggshell-specific C-type lectin-like proteins

C-type lectin-like proteins are major components of the calcified eggshell of multiple avian species. In this study, two representative avian C-type lectin-like proteins, ovocleidin-17 and ansocalcin, were purified from decalcified chicken and goose eggshell protein extracts and investigated for carbohydrate binding activity as well as antimicrobial activity. Purified ovocleidin-17 and ansocalcin were found to bind bacterial polysaccharides, and were bactericidal against Bacillus subtilis, Staphylococcus aureus and Pseudomona aeruginosa. Bactericidal activity was found to be enhanced in the presence of calcium but was not dependent on its presence. The results suggest that avian C-type lectin-like proteins may play an important antimicrobial role in defence of the avian embryo.     

白额鹱卵壳的扫描电镜观察

本文报道白额鹱卵壳的壳膜、孔锥层、海绵层、表层等的超微结构,并对卵壳元素进行ＴＮ－５５００能谱分析。     

Structure of shells from eggs of kinosternid turtles

Shells from eggs of five species of kinosternid turtle (Sternotherus minor, Kinosternon flavescens, K. baurii, K. Hirtipes, and K. alamosae) were examined with light and scanning electron microscopy. Except for possible differences among species in thickness of eggshells, structure of shells from all eggs was similiar. In general, kinosternid turtles lay eggs having a rigid calcareous layer composed of calcium carbonate in the form of aragonite. The calcareous layer is organized into individual shell units with needlelike crystallites radiating from a common center. Most of the thickness of the eggshell is attributable to the calcareous layer, with the fibrous shell membrane comprising only a small fraction of shell thickness. Pores are found in the calcareous layer, but they are not numereous. The outer surface of the eggshells is sculptured and may have a thick, organic layer in places. The outer surface of the shell membrane of decalcified eggshells is studded with spherical cores which presumably nucleate growth of shell units during shell formation. The shell membrane detaches from eggs incubated to hatching, carrying with it remnants of the calcareous layer. Such changes in shell structure presumably reflect withdrawal of calcium from the eggshell by developing embryos.     

Zur substruktur der cuticula der egel (Hirudinea)

Zusammenfassung Die Cuticula der Hirudineen besteht aus zwei Anteilen, einer Innenund einer Außenschicht. Die etwa 1 dicke Innenschicht wird von Lamellen aufgebaut, zwischen denen luftgefüllte Kammern eingeschlos sen Bind. Durch these Bauweise vereint die Cuticula in sick zugleich Festigkeit und Elastizität, wodurch she die bei Kontraktionen des Egelkörpers entstehenden Spannungen aufzunehmen vermag. Die Außenschicht wird von einem dichten Borstenbesatz gebildet, der ein gutes Haften des Schleimbelages ermöglicht. Das epidermale Plasma ist mit der Oberfläche der Cuticula durch Kanälchen verbunden. Sie deenen wahrscheinlich der Abscheidung von Sekreten.

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On the Ultrastructure of the Cuticle of Leeches (Hirudinea)

Electron microscopy of the cuticle of leeches has revealed two layers, an inner layer with air chambers and an outer layer composed of digitiform filaments. The possible functions of both the outer layer and inner layer are discussed.

     

Spatial distribution of calcite and amorphous calcium carbonate in the cuticle of the terrestrial crustaceans Porcellio scaber and Armadillidium vulgare

The crustacean cuticle is an interesting model to study the properties of mineralized bio-composites. The cuticle consists of an organic matrix composed of chitin–protein fibres associated with various amounts of crystalline and amorphous calcium carbonate. It is thought that in isopods the relative amounts of these mineral polymorphs depend on its function and the habitat of the animal. In addition to the composition, the distribution of the various components should affect the properties of the cuticle. However, the spatial distribution of calcium carbonate polymorphs within the crustacean cuticle is unknown. Therefore, we analyzed the mineralized cuticles of the terrestrial isopods Armadillidium vulgare and Porcellio scaber using scanning electron-microscopy, electron probe microanalysis and confocal μ-Raman spectroscopic imaging. We show for the first time that the mineral phases are arranged in distinct layers. Calcite is restricted to the outer layer of the cuticle that corresponds to the exocuticle. Amorphous calcium carbonate is located within the endocuticle that lies below the exocuticle. Within both layers mineral is arranged in rows of granules with diameters of about 20 nm. The results suggest functional implications of mineral distribution that accord to the moulting and escape behaviour of the animals.     

Enhancing the egg's natural defence against bacterial penetration by increasing cuticle deposition

The cuticle is a proteinaceous layer covering the avian egg and is believed to form a defence to microorganism ingress. In birds that lay eggs in challenging environments, the cuticle is thicker, suggesting evolutionary pressure; however, in poultry, selection pressure for this trait has been removed because of artificial incubation. This study aimed to quantify cuticle deposition and to estimate its genetic parameters and its role on trans‐shell penetration of bacteria. Additionally, cuticle proteins were characterised to establish whether alleles for these genes explained variation in deposition. A novel and reliable quantification was achieved using the difference in reflectance of the egg at 650 nm before and after staining with a specific dye. The heritability of this novel measurement was moderate (0.27), and bacteria penetration was dependent on the natural variation in cuticle deposition. Eggs with the best cuticle were never penetrated by bacteria (P < 0.001). The cuticle proteome consisted of six major proteins. A significant association was found between alleles of one of these protein genes, ovocleidin‐116 (MEPE), and cuticle deposition (P = 0.015) and also between alleles of estrogen receptor 1 (ESR1) gene and cuticle deposition (P = 0.008). With the heritability observed, genetic selection should be possible to increase cuticle deposition in commercial poultry, so reducing trans‐generational transmission of microorganisms and reversing the lack of selection pressure for this trait during recent domestication.     

Vaterite Deposition During Eggshell Formation in the Cormorant,Gannet and Shag,and in ‘Shell-less’ Eggs of the Domestic Fowl.

The eggshells of the cormorant (Phalacrocorax carbo), domestic fowl (Gallus domesticus), gannet (Sula bassana), guinea fowl (Numida meleagris), greater flamingo (Phoenicopterus ruber), and shag (Phalacrocorax aristotelis) have been separated into two groups on the basis of the composition of their outer stratum. In the domestic fowl, guinea fowl and greater flamingo the outer stratum is an organic cuticle while in the sea-birds it is an inorganic cover rich in vaterite. The calcareous deposits on the membranes of eggs of the domestic fowl which are shell-less at oviposition have been shown to consist essentially of the vaterite form of calcium carbonate. Reasons for the occurrence of this polymorph of calcium carbonate are discussed with relation to the physiology of the birds.