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PubMed1978

Some observations on the calcium ion binding to the eggshell matrix.

The calcified matrix of the hen eggshell has been demineralized with the EDTA. Aliquots of this material are soluble in water and have been characterized by column chromatography and by chemical analyses. Of particular interest is the high hexosamine and uronic acid content, which confirms the protein-polysaccharide nature of this water-soluble material. The calcium ion binding to the eggshell matrix has been studied by the equilibrium dialysis technique at different pH values, with both free and blocked carboxylic groups. The material with the free carboxylic side chain groups binds more calcium ions with increasing pH value. When the carboxylic groups have been previously blocked with a water-soluble carbodiimide, the calcium ion binding rapidly decreases. The residual capacity to bind calcium ions in the material with the carboxylic functions modified is probably due to the sulfate ions. In agreement with previous observations on other calcified substrates, the calcium ion binding seems to depend on the presence of ionized carboxylic functions of the matrix.

Amino Acids↗
PubMed1990

Multiple proteins are produced from the dec-1 eggshell gene in Drosophila by alternative RNA splicing and proteolytic cleavage events.

The defective chorion-1 gene (dec-1) in Drosophila encodes follicle cell proteins necessary for proper eggshell assembly. A distinctive feature of the gene is the production of multiple products by both alternative RNA splicing and proteolytic processing events. DNA and protein sequencing studies have revealed several dec-1 protein products. The predominant translation product, fc106, has a vitelline membrane-like N-terminal domain followed by a glutamine, methionine-rich central region, largely in the form of 26 amino acid repeats. During late stage 10 the N-terminal portion of fc106 is cleaved, yielding s80, a major eggshell protein. Conceptual translation of the DNA sequence as well as molecular analyses of several dec-1 mutants suggest that the less abundant alternatively spliced RNAs encode primary translation products with different carboxy terminal ends. These results are discussed with respect to previous genetic analyses of dec-1 mutants as well as with respect to potential protein-protein interactions which may underlie stabilization of this complex extracellular structure.

Amino Acid Sequence↗
PubMed1986

Eggshell conductance--Fick's or Stefan's law?

The diffusion of gases through the avian eggshell has usually been interpreted as an example of Fick's law, and this appears to be the best explanation for gas exchange through narrow long pores. Many eggshells, however, contain more elaborate pores, of which the funnel-shaped type is the most common. It is suggested that most of the resistance to diffusion through these types of pores is best explained by Stefan's law of diffusion through apertures. The implications of this theory to the understanding of pore functions are discussed.

Animals↗
PubMed1988

Diffusive resistance of avian eggshell pores.

Resistance to gas diffusion through the avian eggshell resides in the microscopic pores which penetrate the shell. We calculated the resistance to water vapor diffusion of individual pores in the shells of 23 species of avian eggs, based on measurements of pore dimensions taken from drawings of 321 pore casts published by Tyler (1962, 1964, 1965) and Tyler and Simkiss (1959). Diffusive resistances were calculated from Fick's first law, using a 100-segment model of each pore. In addition, we added 2 series resistances, calculated from Stefan's law, to account for boundary layer resistances at the inner and outer pore apertures. Convective resistances for the same 100-segment model were computed from Poiseuille's law. A special, symmetrically branching model is presented for the diffusive resistance of the branched pores of ostrich eggshells, based on the drawings of Tyler and Simkiss (1959). The total aperture resistance was less than 6.2% of total pore resistance, while the outside aperture effect was on average only 1.5%. The calculated average pore conductance for all species was 5.4 micrograms (day Torr)-1, about three times higher than the average value of 1.6 micrograms (day Torr)-1 obtained by dividing measured shell conductance by the number of pores (Ar and Rahn, 1985). A possible explanation for this discrepancy is advanced. However, it is to be noted that in spite of the discrepancy, both calculated and functional values of pore conductance appear to be independent of egg mass.

Animals↗
PubMed1989

The relationship between eggshell porosity and air space gas tensions measured before and during the parafoetal period and their effects on the hatching process in the domestic fowl.

The partial pressure of gases in the air space were measured before and during the parafoetal period for domestic fowl eggs of the same initial weight but varying in eggshell conductance. Embryos developed and hatched normally from eggs with a wide range of shell conductance and resultant air space gas partial pressures. Air space PO2 levels measured just before pipping ranged from 55 to 65 mm Hg in low and high conductance eggs, respectively, whilst PCO2 levels ranged from 75 to 55 mm Hg. With increasing shell conductance embryos membrane-penetrated and pipped the shell later, but hatch time was unaffected. Membrane penetration and external pipping appeared to occur in response to the partial pressures of gas in the air space but not in response to one particular level of air space PO2 or PCO2. During the parafoetal stage air space PO2 decreased at about 3.8 mm Hg/h and air space PCO2 increased at about 1.8 mm Hg/h and these rates of change were unaffected by eggshell conductance.

Animals↗
PubMed1991

Three-dimensional reconstruction of innermost chorion layer of Drosophila grimshawi and Drosophila melanogaster eggshell mutant fs(1)384.

A low-resolution three-dimensional structure of the crystalline innermost chorionic layer (ICL) of the Hawaiian species Drosophila grimshawi and the Drosophila melanogaster eggshell mutant fs(1)384 has been calculated from electron microscope images of tilted negatively stained specimens. The isolated ICL of Drosophila grimshawi is a three-layer structure, about 36 nm thick, whereas the ICL of Drosophila melanogaster eggshell mutant fs(1)384 is a single layer, about 12 nm thick. Each unit in both crystalline structures includes octamers made up of four heterodimers. Crosslinks between the structural elements, both within and between unit cells form an interconnecting network, apparently important in maintaining the integrity of the layer. A model which may account for the ICL self-assembly formation in vivo and the ICL observed lattice polymorphism is proposed, combining data from the three-dimensional reconstruction work and secondary structure features of the ICL component proteins s36 and s38.

Animals↗
PubMed1989

A preliminary study of the translocation of aldicarb across the duck eggshell.

The duck eggshell has the reputation of being more permeable than that of the domestic hen. If this is true, the developing embryo could be at greater risk from xenobiotic agents, since toxicants picked up on the feathers could be transferred to the embryo during incubation. This study looked for such an effect on the developing embryo after the application of aldicarb to the eggshell. At 72 hr, the eggs were painted with 3, 7, 11, or 15 microM aldicarb in 500 microliters water. The eggs were then incubated to Day 24. The gross morphological measurements were then recorded. A similar study was made using domestic hen eggs; these were treated after 36 hr incubation and incubated to Day 17. Direct injection into the yolk sac of both species was used for further comparison. There was a statistically significant reduction (P less than 0.01) in the middle web toe length with 11 and 15 microM aldicarb and the tarsometatarsus length with 7, 11, and 15 microM. Compared with the duck control group, the group given 15 microM aldicarb had reductions of approximately 8% in the tarsometatarsus and approximately 9% in the middle web toe. No statistically significant changes were produced in the chick embryos.

Aldicarb↗
PubMed1987

Possible eggshell protein gene from Schistosoma mansoni.

We have identified and sequenced a cDNA clone of a mRNA found only in mature female schistosomes. This mRNA is not detectably synthesized by female worms from single sex infections (unisexual females), by males or by the developing miracidia in the eggs. The clone hybridises to a highly abundant polyadenylated mRNA of approximately 1500 nucleotides. The nucleotide sequence of the clone predicts a polypeptide comprising two repetitive regions. A pentapeptide repeat with the consensus sequence Gly-Tyr-Asp-Lys-Tyr, and a region rich in histidine residues. Hybrid selected mRNA translated in vitro with [3H]tyrosine as labelled amino acid yields a polypeptide of 48 kDa (p48) that corresponds to the major [3H]tyrosine labelled translation product of female worm total mRNA. p48 does not label with [35S]methionine and is absent from the translation products of male and unisexual female mRNAs. The amino acid sequence of p48 has significant homologies to silk moth chorion proteins and we suggest that it is one of the major components of the schistosome eggshell probably accounting for the high level of [3H]tyrosine incorporation into the vitellaria of Schistosoma mansoni. The tyrosine content of the polypeptide suggests that it may play a role in phenol oxidase mediated cross-linking of the schistosome eggshell and in support of this we find that mushroom phenol oxidase will cause the specific cross-linking of p48 in in vitro translation products.

Animals↗
PubMed1988

Identification and localisation of the products of a putative eggshell precursor gene in the vitellarium of Schistosoma mansoni.

An abundant 0.9 kb female-specific mRNA in Schistosoma mansoni is thought to code for an egg-shell precursor protein [Bobek et al. (1986) Proc. Natl. Acad. Sci. USA 83, 5544-5548]. This gene contains two ORFs. A recombinant plasmid was constructed that expresses a fusion protein containing a glycine- and tyrosine-rich polypeptide coded for by one of these ORFs. Antisera raised against homogenates of female, but not of male, S. mansoni recognise this fusion protein, providing direct evidence that this ORF is used by S. mansoni. In comparative Western blots of S. mansoni homogenates from males and females affinity purified antibodies that react with the fusion protein react exclusively with proteins from females, recognising a 28 kDa polypeptide and a smear of immunoreactive material probably caused by oxidative crosslinking. In immunohistology, the affinity purified antibodies react with mature vitelline cells in female schistosomes. The immunoreactive material is localised in the so-called 'vitelline droplets' that are morphologically very similar to 'shell globules', known to contain egg-shell precursors, that are found in Fasciola hepatica. In situ hybridisation shows that the eggshell precursor gene is only transcribed in immature vitelline cells and has a short half-life. Taken together, these observations provide persuasive evidence that the 0.9 kb mRNA codes for an eggshell precursor.

Animals↗
PubMed1989

Identification, expression and in situ hybridization of an eggshell protein gene from Fasciola hepatica.

The molecular basis of egg formation in the parasitic liver fluke, Fasciola hepatica, was investigated by isolating and characterizing an abundant cDNA from a female genital complex cDNA library. It was expressed in Escherichia coli as a beta-galactosidase fusion protein, which was purified and used to produce polyclonal antibodies. Using immunoblots, the antiserum recognized two soluble constituents of isolated egg shells, both significantly larger than predicted from cDNA sequencing. Using in situ hybridization, the message was detected in cells in the adult vitelline follicles. Eggshell protein mRNA expressed in E. coli will provide a source of precursor protein for further studies of parasite eggshell formation.

Animals↗
PubMed1992

Eggshell precursor proteins of Fasciola hepatica, II. Microheterogeneity in vitelline protein B.

At least 3 structural protein precursors of the eggshell are synthesized and stockpiled in the extensive vitelline cells of the liver fluke Fasciola hepatica L. One of these, vitelline protein B, consists of a closely related family of proteins that owes its apparent electrophoretic heterogeneity to variations in the Tyr to DOPA conversion as well as to subtle variations in the primary sequence. The efficiency of the Tyr to DOPA conversion ranges from a maximum of about 90% to a minimum of 55% in the protein. Trypsin digestion in borate buffer at pH 8 was used to produce DOPA-peptides for sequencing. Notably, trypsin does not cleave Arg/Lys-DOPA sequences at borate concentrations greater than 0.15 M. Peptides with DOPA-containing sequences most frequently have flanking amino acids such as Lys, Ser, or Asp on the N-terminal side and Gly or Asp on the C-terminal side. All protein variants fall within a narrow molecular weight range (30-33 kDa), a pI range of 6.9 to 8.3, and the collective majority would appear to share a common N-terminal sequence up to residue 28. The results suggest some combination of the following: variations in post-translational hydroxylation, alternative post-transcriptional splicing and/or the existence of multiple gene copies of eggshell precursors. The latter have been shown to occur in the blood fluke Schistosoma mansoni [15].

Amino Acid Sequence↗
PubMed1984

Effects of suppression of eggshell calcification and of 1,25(OH)2D3 on Mg2+, Ca2+ and Mg2+HCO-3 ATPase, alkaline phosphatase, carbonic anhydrase and CaBP levels--I. The laying hen uterus.

Stimulation of Mg2+, Ca2+ and Mg2+HCO-3 dependent ATPase activity in mitochondrial and microsomal fractions from the uteri of laying hens is demonstrated. ATPase activity was greatest with 5 mM concentrations of Mg2+ at pH 8.5, and at pH 7.4-7.8 following the addition of bicarbonate. Suppression of eggshell calcification, induced by insertion of a thread into the uterus, did not alter Mg2+, Ca2+ and Mg2+HCO-3 ATPase activities. Alkaline phosphatase activity was generally low, and was unaffected by suppression of eggshell calcification. Levels of carbonic anhydrase and calcium binding protein were lower in the uteri of hens laying shell-less eggs. Injections of 1,25(OH)2D3 in hens laying shell-less eggs did not alter CaBP levels or enzyme activities. It is concluded that factors other than 1,25(OH)2D3 and gonadal hormones are involved in the regulation of uterine CaBP levels.

Adenosine Triphosphatases↗
PubMed1985

The effects of eggshell porosity on blood-gas and acid-base status of domestic fowl embryos within eggs of the same weight.

Eggshell porosity affects the metabolism and growth of the developing embryo and is likely, therefore, to influence blood-gas and acid-base status. PO2 in the airspace and in blood from the allantoic vein, PCO2 in the airspace and in blood from the allantoic artery and allantoic vein, and pH of blood from the allantoic artery and vein are all affected by shell porosity. Low porosity eggshells result in an increased retention of carbon dioxide within the egg, in partial compensation for which an increase in the level of bicarbonate in the blood is predicted. It is concluded that some of the wide variation in blood physiology data between eggs at the same stage of incubation, which has been recorded in the scientific literature, is the result of variation in shell porosity between eggs.

Acid-Base Equilibrium↗
PubMed1992

Relationship between shell structure and movement of Salmonella enteritidis across the eggshell wall.

1. Ultrastructural analyses of the eggshells of one strain of commercial layers indicated that the cuticular layer is rarely present as an even covering over the shell surface and the paired shell membranes are invariably pitted with holes larger than bacterial dimensions. 2. The above conditions pertain irrespective of the age of the laying bird. 3. Bacterial penetration of the eggshell is independent of pore numbers. 4. In the absence of a functional cuticle and with the shell membranes removed, bacteria are checked in their movement by structural modifications in the mammillary layer.

Age Factors↗
PubMed1992

Pleistocene geochronology and palaeothermometry from protein diagenesis in ostrich eggshells: implications for the evolution of modern humans.

Proteinaceous residues incorporated within the crystal structure of ostrich eggshells (OES) are retained without loss over geological time exceeding 10 million years. Degradation of the polypeptides, including hydrolysis to smaller peptide fragments and eventual release of free amino acids, decomposition, and racemization and epimerization occur at regular, predictable rates dependent on ambient temperature. The extent of isoleucine epimerization (aIle/Ile ratio) in OES follows linear first-order reversible kinetics in controlled-temperature laboratory simulations of time up to an aIle/Ile ratio in excess of 1.0. The hydrolysis of leucine also follows a predictable pattern, but deviates from first-order kinetics. A nonlinear mathematical model has been developed that adequately describes the pattern of leucine hydrolysis through a wide temperature range. Arrhenius parameters were derived from laboratory experiments combined with rate constant values found for 14C-dated OES from stratified caves in southern Africa. These parameters for isoleucine epimerization and leucine hydrolysis differ by ca. 10%, allowing the simultaneous solution of the two equations for temperature, independent of sample age. Although the uncertainty of the simultaneous temperature is relatively high (+/- 10 degrees C), it provides an effective means of identifying burned samples. If sample age is known, palaeotemperatures (the integrated thermal history experienced by an eggshell as opposed to an 'instantaneous' temperature) can be calculated with a precision of better than +/- 1 degrees C. The ages of levels at Border Cave, South Africa, from which anatomically modern human skeletal remains have been recovered, are dated by the extent of isoleucine epimerization in associated OES.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1988

Cloning and analysis of the dec-1 female-sterile locus, a gene required for proper assembly of the Drosophila eggshell.

Female-sterile mutations at the dec-1 (defective chorion-1) locus of Drosophila severely disrupt the organization of the eggshell late in oogenesis. Previous characterization of dec-1 mutations has correlated the defects with failure to accumulate an early eggshell protein that undergoes proteolytic cleavage during choriogenesis. To enable further study of the regulation and processing of dec-1 products, we have molecularly cloned the locus and characterized its transcripts. Chromosome jumping was used to isolate a deficiency breakpoint within the locus. Overlapping genomic clones from a wild-type library were then obtained, and a region including the dec-1 locus was identified by hybridization to cDNA probes complementary to RNA from stage 9-10 egg chambers. Analysis of genomic rearrangements associated with the locus verified its identity. Two transcripts from the locus have been identified and characterized using cDNA clones, RNase protection, and primer extension analyses. A 4.0-kb transcript accumulates maximally in stages 9-10, when the primary follicle cell protein associated with dec-1 mutations is synthesized. A second transcript of 5.8 kb, generated by alternative splicing, accumulates during stages 11-12. These results are discussed in light of previous analysis of dec-1 mutations.

Animals↗
PubMed1978

Isolation of messenger RNA coding for eggshell protein of the DNA-eliminating nematode Ascaris lumbricoides.

Poly(A)-containing RNA from polyploid uterine epithelial cells of Ascaris lumbricoides has been isolated by poly(U)-Sepharose chromatography. The bulk of poly(A)-containing RNA migrates as 18-S RNA in formamide/polyacrylamide gels. In a cell-free wheat germ system, this RNA directs the synthesis of a polypeptide with identical migration behavior in dodecylsulphate/urea/polyacrylamide gels as the polypeptide isolated from the proteinaceous eggshell. The two proteins reveal almost identical peptide patterns in fingerprint analysis. The authentic eggshell protein has been identified as a glycoprotein with a molecular weight of about 10000, as determined by dodecylsulphate/polyacrylamide gel electrophoresis. The apparent discrepancy between mRNA length and the required coding length for the protein is discussed.

Animals↗
PubMed1983

The inhibitory effect of some chlorinated hydrocarbon pesticides on the ATP-dependent Ca2+ binding of the particulate fraction of the eggshell gland mucosa cells.

The pesticide p-p'-DDT and its persistent metabolite p-p'-DDE cause thinning of the eggshells in several species of birds. In earlier investigations on ducks this thinning was found to be associated with a reduction of the ATP-dependent Ca2+ binding to a homogenate of the shell gland mucosal cells by DDE. The activity of a Ca2+-Mg2+-activated ATPase in the homogenate was also decreased on administration of DDE in vivo. We have therefore investigated the in vitro effects of some other chlorinated hydrocarbon pesticides of ecotoxicological interest on the ATP-dependent Ca2+ binding and the Ca2+-Mg2+-activated ATPase activity in a homogenate of the eggshell gland mucosa of the hen and determined the molar concentrations that produced 50% inhibition (=IC50). Several of the investigated compounds, namely toxaphene, chlordane, p-p'-DDD, o-p'-DDE, p-p'-DDT, methoxychlor and PCB (Arochlor 1242), had a similar IC50 to inhibit the Ca2+ binding as p-p'-DDE. Lindane, p-p'-DDA and biphenyl had an IC50 3.3-4 times higher and that of 2.4 D was 13.5 times higher than that of p-p'-DDE. When the IC50 of some of the compounds (p-p'-DDE, PCB, toxaphene, Lindane) was determined that decreased the Ca2+-Mg2+-activated ATPase of the homogenate it was found to be only 18 to 29 per cent of that needed to inhibit the Ca2+ binding by the homogenate. It is therefore probable that some other effect than inhibition of this enzyme is also involved in the Ca2+-binding process and affected by the compounds.

Adenosine Triphosphatases↗