Stabilization and sclerotization ofRaja erinacea egg capsule proteins

Synopsis Sclerotization of skate egg capsule occurs after secretion of capsule precursors from the shell gland and involves a form of quinone tanning in which catechols are introduced in utero and subsequently oxidized to quinones by catechol oxidase. A latent form of enzyme is incorporated in the capsular matrix during secretion. Oxidase activity increases concomitantly with increasing catechol and quinone contents. Six major proteins ranging in size from 95kDa to 20kDa comprise the skate egg capsule, all of which contain elevated levels of glycine, serine, proline and tyrosine. Hydroxyproline occurs in all but one protein, however, none has an amino acid composition typical of collagen. Solubilization of two proteins from pre-tanned capsule requires reducing agents indicating that an early event leading to matrix stabilization is mediated by disulfide bonds. Stabilization of the other proteins along with the disulfide bonded proteins directly correlates with increasing catechol content, catechol oxidase activity and quinone formation.     

Levels of connectivity between longnose skate (Dipturus oxyrinchus) in the Mediterranean Sea and the north-eastern Atlantic Ocean

Sequencing of a partial region of the mitochondrial control region has revealed no shared haplotypes between longnose skate (Dipturus oxyrinchus L.) sampled in the north-eastern Atlantic (Norway and Rockall) and those sampled in the Mediterranean (Mallorca). Bayesian estimation of the migration rate suggests little, if any, gene flow occurs between the regions and that the populations separated 20,000?years ago. These conclusions provide a genetic basis for long-standing observations, based on egg capsule and adult size, that longnose skate in the Mediterranean may be genetically isolated from other stocks. This result has important conservation implications for the threatened longnose skate.     

Seamount egg‐laying grounds of the deep‐water skate Bathyraja richardsoni

Highly localized concentrations of elasmobranch egg capsules of the deep‐water skate Bathyraja richardsoni were discovered during the first remotely operated vehicle (ROV) survey of the Hebrides Terrace Seamount in the Rockall Trough, north‐east Atlantic Ocean. Conductivity–temperature–depth profiling indicated that the eggs were bathed in a specific environmental niche of well‐oxygenated waters between 4·20 and 4·55° C, and salinity 34·95–35·06, on a coarse to fine‐grained sandy seabed on the seamount's eastern flank, whereas a second type of egg capsule (possibly belonging to the skate Dipturus sp.) was recorded exclusively amongst the reef‐building stony coral Solenosmilia variabilis. The depths of both egg‐laying habitats (1489–1580 m) provide a de facto refuge from fisheries mortality for younger life stages of these skates.     

Photocontrol of chloroplast and mitochondrial polypeptide levels in euglena

Two dimensional polyacrylamide gel electrophoresis resolved protein from intact chloroplasts of wild type Euglena gracilis Klebs var. bacillaris Cori into 185 polypeptides of which 55 were localized on the whole cell polypeptide map. Of these chloroplast polypeptides, the relative amounts of 49 increased, the relative amounts of two decreased, and the relative amounts of four polypeptides were unaltered by exposure of dark grown resting cells to light for 72 hours. Proteins from intact purified mitochondria obtained from a bleached mutant (W₁₀BSmL) lacking plastids were resolved into 193 polypeptides of which 44 were localized on the whole cell polypeptide map from wild type cells. Of these mitochondrial polypeptides, the relative amount of one increased, the relative amounts of 12 were unaltered, and the relative amounts of 31 decreased after exposure of the dark grown resting cells to light. Since it is known that the development of the chloroplast in Euglena occurs without a net increase in total cellular protein and without a change in the size of the cellular amino acid pools, the degradation of mitochondrial polypeptides represents a major source of amino acids for the synthesis of chloroplast polypeptides.     

cDNA cloning and characterization of Type I procollagen alpha1 chain in the skate Raja kenojei

A full-length cDNA of the Type I procollagen alpha1 [pro-alpha1(I)] chain (4388 bp), coding for 1463 amino acid residues in the total length, was determined by RACE PCR using a cDNA library constructed from 4-week embryo of the skate Raja kenojei. The helical region of the skate pro-alpha1(I) chain consisted of 1014 amino acid residues - the same as other fibrillar collagen alpha chains from higher vertebrates. Comparison on denaturation temperatures of Type I collagens from the skate, rainbow trout (Oncorhynchus mykiss) and rat (Rattus norvegicus) revealed that the number of Gly-Pro-Pro and Gly-Gly in the alpha1(I) chains could be directly related to the thermal stability of the helix. The expression property of the skate pro-alpha1(I) chain mRNA and phylogenetic analysis with other vertebrate pro-alpha1(I) chains suggested that skate pro-alpha1(I) chain could be a precursor form of the skate Type I collagen alpha1 chain. The present study is the first evidence for the primary structure of full-length pro-alpha1(I) chain in an elasmobranch.     

X-ray diffraction analysis of the 3D organization of collagen fibrils in the wall of the dogfish egg case

The wall of the egg case of the dogfish,Scyliorhinus canicula, contains a network-forming collagen assembled into a regular three-dimensional (3D) structure. It accomplishes supportive, protective and filtering functions for the embryo contained within it. The collagen molecules in the egg case are organized into a body-centred unit cell of dimensions (mean ± s.d.) (11.6 ± 1.0) nm X (11.6 ± 1.0) nm X (81.6 ± 3.2) nm, which belongs to the I422 space group. At a higher hierarchical level, the collagen molecules assemble into parallel arrays of fibrils, ca. 100 nm in diameter, which aggregate to form laminae ca. 0.5 μm thick. These laminae are organized into a plywood-like structure and account for 98% of the thickness of the wall of the egg case. X-ray diffraction patterns of the wall of the egg case were taken along mutually perpendicular directions, one being perpendicular to the surface of the egg case. Three different kinds of diffraction pattern were observed. One of the patterns was characteristic of an X-ray direction perpendicular to the laminae in the egg case (along the x-direction). The two other patterns were obtained with the X-rays directed parallel to the plane of the laminae, either along the capsule long axis (z) or perpendicular to this (y). These two patterns were observed interchangeably in either of the x- or y-directions depending on the specimen. The diffraction patterns were analysed and interpreted taking into consideration the 3D electron microscope data of the egg case. The results confirm and extend previous findings from transmission electron microscopy and low-angle X-ray diffraction and they suggest that there is only one major type of ordered collagen arrangement in the wall of the egg case.     

The effect of PH on fibrillogenesis of collagen in the egg capsule of the dogfish, Scyliorhinus canicula

The collagen of the egg capsule of the dogfish, Scyliorhinus canicula is stored and secreted by the secretory cells of the D-zone of the nidamental gland (Rusaou?n-Innocent, 1990b). The collagen appears to pass through several morphologically distinct textures during storage, secretion and fibril formation which may represent different lyotropic liquid crystalline phases (Knight et al., 1993). In the present communication we report evidence that a fall in hydrogen ion concentration induces fibrillogenesis during the secretion of the dogfish egg capsule. In an attempt to understand the factors involved in collagen assembly, we investigated the effects of subjecting isolated collagen storage granules in vitro to solutions ranging in pH from 2-11 and Na(+), K(+), Ca(++), Mg(++), Zn(++) and Cu(++) ions at concentrations varying from 0.01-0.5 M. From pH 2 to pH 4 most granules appeared completely amorphous; from pH 5 to pH 7 granules showed the following previously reported liquid crystalline textures: isotropic, lamellar, micellar, hexagonal columnar, transversely banded twisted nematic, and unbanded twisted nematic. At pH 8 granules showed both the hexagonal columnar phase (phase IV) and small quantities of the final fibrillar phase together with a previously undescribed texture. The latter texture, which we refer to as phase VII, had a D period (17.5 nm) half that of the lamellar texture (phase II) and the final egg capsule fibrils (phase VI). From pH 9 to pH 11, only the final fibrillar texture (phase VI) together with small quantities of the new texture (phase VII) were present. Na(+), K(+), Ca(++), Mg(++), Zn(++) and Cu(++) ions did not appear to have an observable effect on the phases found in isolated granules at pH 7.0. The role of pH in collagen storage and fibrillogenesis was confirmed by direct estimation of the pH in vivo using vital staining with neutral red, a range of pH indicators applied to unfixed cryostat sections and direct measurements of the pH of the jelly within the egg capsule. The implications of these findings for the mechanism of collagen storage and fibrillogenesis in the dogfish egg capsule and other collagenous systems are discussed.     

Interaction of collagen with hydrophobic protein granules in the egg capsule of the dogfish scyliorhinus canicula

The egg capsule of the dogfish is a composite material containing collagenous fibrils and 2 mum spherical hydrophobic protein granules. The latter appear to owe much of their hydrophobicity to an exceptionally high tyrosine content (approximately 20% of total amino acid residues). The hydrophobic component appears to form as an emulsion in the secretory granules of the D and E zone gland cells of the nidamental gland. Droplets of the hydrophobic material appear to become coated with remarkably regular layers of radially-arranged collagen molecules which form a series of concentric, evenly spaced layers around each hydrophobic granule. Numerous disclinations were seen where the layers around adjacent granules interfered with one another. The layers are thought to represent a lamellar liquid crystalline phase previously described for this collagen (Knight et al., 1993). The fine structural appearance of the concentric layers and evidence for radial arrangement of collagen molecules within them is compatible with the suggestion that the layers are built from a dumbbell-shaped unit approximately 35 nm long with hydrophobic groups concentrated at the ends. This unit may represent a dumbbell-shaped molecule or an oligomer of two or more molecules lying parallel with one another in a head-to-tail arrangement. Such a unit can be readily incorporated into models for the micellar, hexagonal columnar and final fibrillar phases previously described for this collagen (Knight et al., 1993). Evidence from the TEM study of stretched egg capsule wall suggests that there is a mechanical interaction between the hydrophobic granules and the collagen fibrils in the fully formed material. We suggest that the radial, concentric layered arrangement of collagen molecules is established by hydrophobic interactions within the liquid crystalline material and locked into place by oxidative covalent cross-linking to give a 3-dimensional cross-linked meshwork of collagen fibrils and hydrophobic granules. The latter arrangement helps to account for the high tensilestrength and toughness of this material.     

The chemical composition of the egg shells of the potato cyst-nematode, Heterodera rostochiensis Woll

1. Eggs of the potato cyst-nematode (Heterodera rostochiensis Woll.) were isolated by sieving a suspension of crushed cysts. Eggs were broken open by ultrasonic vibration and the egg shells separated from the released larvae by centrifuging in a potassium tartrate density gradient. About 1 mg. of dried egg shells was obtained from 1000 cysts. 2. The major constituent of the egg shells was protein (59%, calculated from nitrogen content). About 80% of the egg shells went into solution on acid hydrolysis. Of the 18 amino acids determined with the Technicon Auto-Analyser, proline was most abundant and, with aspartic acid, glycine and serine, made up about 64% by weight of the total amino acids. The small amounts of aromatic and sulphur-containing amino acids, and the presence of hydroxy-proline, indicate a collagen-like protein. 3. The egg shells gave a positive van Wisselingh colour test for chitin, and glucosamine was detected in their acid hydrolysate by chromatography. The glucosamine content of the egg shells, determined by the Elson-Morgan colorimetric method, was 7%, corresponding to about 9% chitin. 4. Dried egg shells contained about 7% of lipid, 6% of carbohydrate and 3% of ash. Polyphenols (3% by weight of the egg shells) were detected in the acid hydrolysates. 5. Neither the collagen nor the chitin showed evidence of crystallinity when examined by X-ray diffraction.     

Short-chain basement membrane collagen. Further characterization and its biosynthesis by F-9 embryonal carcinoma cells

The paper describes further characterization of the 55-kDa short-chain collagen from lens capsule. Lens capsules were extracted with 5.5 M guanidine.HCl and the extracted material was fractionated on agarose A-5M followed by high-pressure liquid chromatography (HPLC). By amino acid composition, the major fraction obtained from HPLC was found to be different than type-IV collagen fragments. The 55-kDa short-chain collagen on pepsin digestion produced a 45-kDa pepsin-resistant fragment. The undifferentiated embryonal carcinoma (F-9) cells were found to synthesize increased amounts of 55-kDa short-chain collagen. The identity of this biosynthesized molecule with 55-kDa short-chain collagen from lens capsules was established by immunoprecipitation experiments. The results indicated a close similarity or identical nature of the short-chain collagens from these two sources.     

Mechanical characterization of an unusual elastic biomaterial from the egg capsules of marine snails (Busycon spp.)

Egg capsule material serves as a putative protection mechanism for developing snail embryos facing the perils of the marine environment. We conducted the first quantitative study of this acellular structural protein with the goals of characterizing its chemical and mechanical properties and the relationship of these properties to its biological protective function. We have found that this protein polymer exhibits long-range elasticity with an interesting recoverable yield evidenced by an order of magnitude decrease in elastic modulus (apparent failure) that begins at 3%-5% strain. This material differs significantly from other common structural proteins such as collagen and elastin in mechanical response to strain. Qualitative similarities in stress/strain behavior to keratin, another common structural protein, are more than coincidental when composition and detailed mechanical quantification are considered. This suggests the possibility of alpha-helical structure and matrix organization that might be similar in these two proteins. Indeed, the egg capsule protein may be closely related to vertebrate keratins such as intermediate filaments. We conclude that while this material's bimodal tensile properties may serve as useful protection against the impact loading egg capsules encounter in the intertidal zone, the full biological importance of these capsules is not known.     

Skate Bathyraja spp. egg predation in the eastern Bering Sea

Predation on skate eggs by snails was examined for three skate species at seven nursery sites in three regions (north, middle and south) of the eastern Bering Sea. Mean predation levels were 6·46% for the Alaska skate Bathyraja parmifera , 2·65% for the Aleutian skate Bathyraja aleutica and 22·25% for the Bering skate Bathyraja interrupta . Predation levels were significantly higher at the middle and north sites than the south sites for all species combined. Predation levels decreased with increasing egg-case densities at all nursery sites, and the highest predation levels occurred where egg-case densities were very low. Predated egg-case density increased with increasing snail densities across all nursery sites examined. The Oregon triton Fusitriton oregonensis was the most abundant snail species at all nursery sites and displayed ability to drill holes in the egg case of B. parmifera. Holes left by predatory snails in egg cases of B. parmifera were significantly larger, and of different shape at the middle site compared to the south site. Holes in B. parmifera were also significantly larger than those in egg cases of B. interrupta across all sites examined. Egg cases of B. aleutica possess surface spines that cover the egg case and may inhibit predation by snails at a greater rate than that of the B. parmifera and B. interrupta , which have a smoother egg-case surface.     

The localization and synthesis of some collagen types in developing mouse embryos.

The location of type IV (basement membrane)collagen in early post-implantation mouse embryos was examined by immunoperoxidase reactions using a specific immunoglobulin raised against mouse lens capsule collagen. Reaction was positive in the earliest embryos studied--on the fifth day of gestation (the day of detection of the copulation plug is the first day). It was found only in the primitive endoderm adjacent to the blastocoelic cavity. Subsequently in development, strong staining reactions were found in the parietal endoderm, Reichert's membrane and an acellular layer which separates the visceral endoderm of the egg cylinder from the ectoderm. In tenth to eighteenth day visceral yolk sacs, the mesodermal portion was stained, which is consistent with the presence of basement membranes around blood vessels. The endodermal portion of the visceral yolk sac did not react, while small amounts were found in the amnion. By incubation of various embryonic tissues with tritiated amino acids, purification of the biosynthesized secreted collagens and their partial characterization, the differential expression of several collagen genes was detected. Identification of collagen types was made by: reaction with specific antibodies to type I and IV collagens; electrophoretic mobility; sensitivity to reduction and to collagenase; analysis of the proportions of 3-hydroxyproline, 4-hydroxyproline and hydroxylysine; and CNBr peptides. In agreement with the data of Minor et al. (1976a) for the rat, mouse parietal endoderm synthesizes large amounts of type IV collagen. In contrast to their findings, however, the 165,000 molecular weight polypeptide is not converted to one of 100,000 after reduction, alkylation and repepsinization (Dehm and Kefalides, 1978). The endoderm of the visceral yolk sac was shown to be synthesizing primarily type I collagen, while the mesoderm layer of this membrane synthesized both type I and IV collagens. Little or no type IV collagen synthesis was detected in the endoderm of the visceral yolk sac. If it is correct that the visceral endoderm of the early embryo makes a major contribution to the formation of the endoderm portion of the visceral yolk sac, then it is clear that a switch in collagen gene expression must occur as it does so.     

Some observations on the collagen fibrils of the egg capsule of the dogfish, Scyliorhinus canicula

The egg capsule of the dogfish Scyliorhinus canicula is a collagenous material with a laminated, plywood (orthogonal) construction. The collagen fibrils which constitute the bulk of the egg capsule wall have a unique, highly ordered structure (Knight and Hunt, 1974; 1976, 1986; Gathercole et al., 1993) which is thought to represent a smectic A liquid crystalline phase (Knight et al., 1993). The egg capsule is extremely strong and chemically inert (Hunt, 1985). It is stored, secreted and formed by the nidamental gland (Rusaou?n 1976, 1990 a, b; Knight and Feng, 1992). During intracellular storage, secretion and fibrillogenesis, the dogfish egg capsule collagen appears to pass through a remarkable series of textures within a lyotropic liquid crystalline phase diagram (Knight et al., 1993). In the present communication, further observations on the ultrastructure of the collagen fibrils and their arrangement within the laminae of the fully-formed egg capsule are reported. The effect of tilting ultrathin sections of fibrils in the goniometer stage of a transmission electron microscope are described, demonstrating that the crystalline lattice within the fibril appeared twisted more or less regularly into a long pitch helix. Other observations indicated that some of the fibrils were in turn twisted round one another to form fibres which therefore had a coiled-coil structure. The fibres are arranged parallel to one another in the laminae which are stacked to give an orthogonal plywood construction. The effects of staining fibrils with cuprolinic blue and with tannic acid are reported. Reduction in the water content of the fibrils before fixation appeared to move some of the fibrils through the part of the lyotropic phase transition diagram converting them from smectic A to smectic C. Finally, evidence is presented that the fibrils shrank, but remarkably, still retained a longitudinally-ordered but modified, molecular arrangement even after boiling in water for periods of up to 10 min. These observations are discussed in relation to other collagens.     

Nitrogen partitioning and blood meal utilization by Aedes aegypti (Diptera Culicidae)

The utilization of the blood meal by mosquitoes was investigated by first feeding females quantities of blood ranging from 1 to 5 mg, and then analyzing the faeces for the various by-products of protein catabolism that were subsequently eliminated. The nitrogeneous waste products in order of importance were uric acid, histidine, ammonia and arginine. Only traces of the other amino acids were excreted.The total amount of each faecal substance varied linearly with the quantity of blood ingested, however their relative proportions did not change. Regardless of blood meal size the quantily of uric acid and ammonia produced indicates that about 80% of the non-histidine and arginine amino acids are deaminated and utilized for metabolic purposes other than egg protein synthesis.Most of the histidine and about one half of the arginine content of the blood were excreted as free amino acids, but the other amino acids were lost in trace amounts.Nineteen per cent of the total ingested amino acids was incorporated into soluble yolk proteins and this proportion was constant even for small blood meals that result in a reduction in the numbers of eggs produced.The comparative aspects of nitrogen partitioning and blood meal utilization by haematophagous insects, as well as the factors that affect blood meal utilization and fecundity in A. aegypti are discussed.     

Secretion and stabilization of the layers of the egg capsule of the dogfish Scyuorhinus canicula

The egg capsule of the dogfish is a unique, collagcnous, layered structure secreted by the nidamental gland which has nine, remarkably discrete, transverse zones of tubular glands. The present paper traces the origin of the four layers of the capsule to particular zones within the gland. Evidence is presented for the existence of DOPA, DOPA oxidase. protein(s) rich in tyrosyl residues and a peroxidase within the same storage granules within the secretory cells of the C and E zones. It is suggested that these interact when secreted to cross-link the inner and outer surfaces of the egg capsule. Evidence is presented that the middle layer which forms the bulk of the thickness of the egg capsule and has the highest collagen content may be partly stabilized by the peroxidation of tyrosyl residues. The mechanical significance of crosslinking in this system and the possible cytological mechanisms involved in the secretion of the tanning agents and enzymes are discussed.     

Identification of multiple nursery habitats of skates in the eastern Bering Sea

The use of more than a single nursery habitat type is examined for oviparous elasmobranchs using data summarized from studies conducted on the Alaska skate Bathyraja parmifera and the Aleutian skate Bathyraja aleutica in the eastern Bering Sea. The eastern Bering Sea skate species use two discrete areas as nurseries, one for egg deposition and a second for newly emergent juveniles. Egg deposition sites were located along the outer shelf and upper slope near canyons in the eastern Bering Sea. Newly emergent juveniles were found along the outer and middle shelf for B. parmifera and deep‐slope for B. aleutica, suggesting that habitat used by newly emergent juvenile skates is distinct from habitat used for egg deposition and embryo development. In reviewing many studies on oviparous elasmobranchs, similar patterns emerge of habitat use during their early life history. To distinguish these distinct habitats, appropriate terminology is proposed. Egg case nursery is suggested for areas of egg deposition and juvenile nursery is suggested for areas where juveniles aggregate after emergence. Criteria to describe each habitat type are outlined.     

Macromolecular organization of basement membranes. Characterization and comparison of glomerular basement membrane and lens capsule components by immunochemical and lectin affinity procedures

The macromolecular components of bovine glomerular basement membrane (GBM) and lens capsules (anterior and posterior) solubilized by sequential extractions with denaturing agents were quantitated and characterized by polyacrylamide gel electrophoresis, CL-6B filtration, and DEAE-cellulose chromatography with the help of immunochemical techniques. Laminin, entactin, fibronectin, and heparan sulfate proteoglycan were primarily recovered (over 80%) from both basement membranes in a guanidine HCl extract which contained only a limited amount of the total protein (10-14%); most of the remainder of these noncollagenous components could be solubilized by the guanidine in the presence of reducing agent. Although a portion of the Type IV collagen could be obtained by these treatments, effective extraction of this protein depended on exposure to sodium dodecyl sulfate under reducing conditions. Immunoblot analysis revealed a remarkably similar pattern for GBM and lens capsule Type IV collagens with prominent bands of Mr = 390,000, 210,000, and 190,000 being evident. Fibronectin was present in much greater amounts in GBM than lens capsule while the reverse was true for entactin. In both GBM and lens capsules, the entactin (Mr = 150,000) exceeded laminin; the latter protein on immunoblotting was found to contain primarily the alpha-subunit (Mr = 200,000). The size of the heparan sulfate proteoglycan from anterior (Mr = 400,000) and posterior lens capsule (Mr greater than 500,000) was substantially larger than that from GBM (Mr = 200,000). During DEAE-cellulose chromatography under nonreducing conditions in a denaturing solvent, a portion of the Type IV collagen coeluted with the proteoglycan from these membranes. Considerable Bandeiraea simplicifolia I binding activity (alpha-D-galactose specific) was observed in GBM and lens capsule extracts and column fractions which could not be accounted for by laminin alone. Several components which reacted with this lectin were seen on transblots and among these Type IV collagen was identified. In contrast to the basement membranes from bovine tissues, the constituents from human GBM did not react with the B. simplicifolia I lectin.     

Purification, characterization and immunochemical properties of a novel 60-kDa protein of Vibrio anguillarum strains

Vibrio anguillarum strains expressed increased amounts of a novel 60-kDa protein when cells were grown at physiologically elevated temperatures. The relative amounts of the 60-kDa protein were unaltered by changes in osmolarity or ionic concentration of the growth medium in cells grown at optimal growth temperatures. The N-terminal amino acid sequence analysis of the V. anguillarum 60-kDa protein showed extensive (94–89%) sequence identity with the 60-kDa heat shock protein of Yersinia enterocolitica and with Serratia rubidaea GroEL protein. Monoclonal antibodies against the Y. enterocolitica chaperonin reacted with the 60-kDa protein from V. anguillarum strains, and with a temperature-induced protein of similar molecular mass in other Gram-negative pathogens of fish.