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PubMed2004

A gene family of cathepsin L-like proteases of filarial nematodes are associated with larval molting and cuticle and eggshell remodeling.

Cysteine proteinases are involved in a variety of important biological processes and have been implicated in molting and tissue remodeling in free living and parasitic nematodes. We show that in the lymphatic filarial nematode Brugia pahangi molting of third-stage larvae (L3) to fourth-stage larvae is dependent on the activity of a cathepsin L-like cysteine protease (CPL), which can be detected in the excretory/secretory (ES) products of molting L3. Directed cloning of a cysteine protease gene in B. pahangi and analysis of the expressed sequence tag (EST) and genomic sequences of the closely related human lymphatic filarial nematode Brugia malayi have identified a family of CPLs. One group of these enzymes, Bm-cpl-1, -4, -5 and Bp-cpl-4, is highly expressed in the B. malayi and B. pahangi infective L3 larvae. Immunolocalization indicates that the corresponding enzymes are synthesized and stored in granules of the glandular esophagus of L3 and released during the molting process. Functional analysis of these genes in Brugia and closely related CPL genes identified in the filarial nematode Onchocerca volvulus and the free living model nematode Caenorhabditis elegans indicate that these genes are also involved in cuticle and eggshell remodeling.

Animals↗
PubMed2005

Preparation and application of a novel core/shell organic nanoparticle as a fluorescence probe in the selective determination of Cr(VI).

A novel core/shell organic nanoparticles, anthracene/poly-acrylamide (AN/PAM), has been prepared successfully. Based on the fluorescence quenching of AN/PAM nanoparticles by Cr(VI), a method for the selective determination of Cr(VI), without separation of Cr(VI) in water, was developed. Furthermore, the reaction mechanism between nano-AN/PAM and Cr(VI) was also discussed. The synthesis and reaction conditions were investigated in detail. The assay is characterized by short reaction time, very few interference stable fluorescence signals, simple instruments and sensitivity. Under optical experimental conditions, a limit of detection of 0.02 microg/ml was achieved. The calibration curve was linear over the concentration range 0.04-2.00 microg/ml with a correlation coefficient of 0.9924. The proposed method has been applied to the selective quantification of Cr(VI) in synthetic samples and waste-water samples with the satisfactory results.

Animals↗
PubMed2006

Minor proteins and enzymes of the Drosophila eggshell matrix.

The Drosophila eggshell provides an in vivo model system for extracellular matrix assembly, in which programmed gene expression, cell migrations, extracellular protein trafficking, proteolytic processing, and cross-linking are all required to generate a multi-layered and regionally complex architecture. While abundant structural components of the eggshell are known and are being characterized, less is known about non-abundant structural, regulatory, and enzymatic components that are likely to play critical roles in eggshell assembly. We have used sensitive mass spectrometry-based analyses of fractionated eggshell matrices to validate six previously predicted eggshell proteins and to identify eleven novel components, and have characterized the expression patterns of many of their mRNAs. Among these are several putative structural or regulatory (non-enzymatic) proteins, most larger in mass than the major eggshell proteins and often showing preferential expression in follicle cells overlying specific structural features of the eggshell. Of particular note are the putative enzymes, some likely to be involved in matrix cross-linking (two yellow family members previously implicated in eggshell integrity, a heme peroxidase, and a small-molecule oxidoreductase) and others possibly involved in matrix proteolysis or adhesion (proteins related to cathepsins B and D). This work provides a framework for future molecular studies of eggshell assembly.

Animals↗
PubMed2006

Testing phylogenetic implications of eggshell characters in side-necked turtles (Testudines: Pleurodira).

Amniote egg and eggshell morphology is a rich source of characters to link aspects of reproductive biology with systematics. Extensive work concerning both anatomy and phylogenetic assignability has been done on fossil bird and dinosaur eggs, but little is known for extant sauropsids. The utility of eggshell characters for phylogenetic analyses is tested and discussed for extant side-necked turtles (Pleurodira), and the diversity of egg ultrastructure is examined in several species. Egg gross morphology and eggshell ultrastructure of 12 species of extant side-necked turtles was documented using scanning electron microscopy. Thirteen eggshell characters were scored and mapped on a composite phylogeny and ancestral character states were reconstructed. Many of the characters do not show a phylogenetic signal according to a test comparing the number of steps on the chosen phylogeny with that on randomly generated trees. The presence of conservative, clade-supporting features could be demonstrated, and the following clades are supported by several characters: the Elseya-Emydura entity, short-necked Australasian chelids, is backed by two characters, and two additional characters could potentially support this group. Three characters support the monophyly of South American chelids, whereas two characters argue for the exclusion of Hydromedusa, a long-necked form resembling Australian chelids rather than South American forms, from this clade.

Animals↗
PubMed2003

fc177, a minor dec-1 proprotein, is necessary to prevent ectopic aggregation of the endochorion during eggshell assembly in Drosophila.

The Drosophila eggshell is a highly specialized extracellular matrix that forms between the oocyte and the surrounding epithelial follicle cells during late oogenesis. The dec-1 gene, which is required for proper eggshell assembly, produces three proproteins that are cleaved within the vitelline membrane layer to multiple derivatives. The different spatial distributions of the cleaved derivatives suggest that they play distinct roles in eggshell assembly. Using extant dec-1 mutations in conjunction with genetically engineered dec-1 transgenes, we show that, although all three dec-1 proproteins, fc106, fc125, and fc177, are required for female fertility, gross morphological abnormalities in the eggshell are observed only in the absence of fc177. The coalescence of the roof, pillar, and floor substructures of the tripartite endochorion suggested that quantitatively minor fc177 derivatives are necessary to prevent ectopic aggregation of endochorion proteins during the assembly process. Expression of a fc177 cDNA in dec-1 null mutants was sufficient to restore spaces within the endochorion layer. Fc177 may function as a scaffolding protein akin to those utilized in viral morphogenesis.

Animals↗
PubMed1997

A novel cDNA clone of Schistosoma japonicum encoding the 34,000 Dalton eggshell precursor protein.

A cDNA clone encoding the 34 kDa eggshell protein of Schistosoma japonicum was isolated from an adult female cDNA library with a rabbit antiserum raised against the 34 kDa female worm fraction. A 230 bp-insert of this clone (Sj23A) was introduced in frame into the expression plasmid vector, pMAL-c2, and the recombinant fusion protein of the Sj23A transiation product was induced in Escherichia coli. The antiserum raised against the recombinant protein reacted only with the native 34 kDa protein of mature female worms, which localized in the vitelline cells of the vitelline glands. By reverse transcription-polymerase chain reaction (RT-PCR) analysis, it was found that the gene corresponding to the Sj23A was expressed exclusively in mature female worms. The clone Sj23A showed a high degree of homology to the genes for the eggshell precursor proteins of Fasciola hepatica. At the deduced polypeptide level, the Sj23A also had similarities with the F. hepatica-protein sequence, the amino acid composition [high glycine (16%), lysine (12%) and tyrosine (11%)] and the presence of tyrosine residues flanked by glycine. The clone Sj23A also shared an extensive sequence homology with 3 S. mansoni expression sequence tags (ESTs). The present results suggest that the protein encoded by the female-specific Sj23A gene of S. japonicum is widely conserved in trematodes and plays a significant role as a precursor involved in eggshell formation.

Amino Acid Sequence↗
PubMed1982

Chemical characterization of the hen eggshell matrix: isolation of an alkali-resistant peptide.

The eggshell matrix was obtained from hen eggshells using EDTA solutions. The water-soluble organic material was separated on a DEAE-cellulose column equilibrated with 8 M urea using Tris-hydrochloric acid buffer as eluent with a linear gradient of sodium chloride. Five main fractions were obtained which differ in amino acid composition and sugar contents. As is shown from the uronic acid content, the first two fractions eluted from the column are glycoproteins, while the other three contain proteins and glycosaminoglycans From the alkaline hydrolysate of the eggshell matrix, a peptide was isolated which is composed of aspartic acid, threonine, serine, glutamic acid, proline, glycine and alanine in a molar ratio of 2:1:3:7:1:3:1 with a minimum molecular weight of 2158 daltons. The calcium ion binding to this peptide was studied, at different pH values, with both free and blocked carboxyl groups, using murexide as an indicator of free Ca2+. The importance of this acidic peptide in the calcification process of the eggshell matrix is discussed.

Amino Acids↗
PubMed1996

The eggshell of Drosophila melanogaster: IX. Synthesis and morphogenesis of the innermost chorionic layer.

Synthesis and morphogenesis of the innermost chorionic layer (ICL) was investigated by conventional EM methods, freeze-fracturing, tissue culture in Robb's medium, and EM autoradiography. Both autoradiography and fine structure results have shown that ICL-components are secreted prior to other chorion proteins. Their secretion starts on stage 12a but the first layer of ICL molecules is visible at stage 12b. Its thickness is gradually increased during the next stages, taking first, a bilaminar form along with the inner endochorion. Later, at the end of choriogenesis, ICL is detached from the endochorion and takes its final thickness and configuration, consisting of a 3-dimensional crystal, about 40 nm thick. The isolated ICL in conditions of air water interface is a monolayer crystal 10 nm thick. Studies on chorion mutants showed that the amount of protein secreted by the follicle cells is independent to the process of crystallisation. These data show how a proteinaceous extracellular substance is gradually assembled to form a 3-D crystal and how it can be organised to perform functions such as the physiological resistance of the insect eggs against water loss or water uptake, whenever they are laid on substrates with extreme environmental conditions. These functions are performed by ICL in conjunction with the underlying wax layer.

Animals↗
PubMed1998

An autoregulatory cascade of EGF receptor signaling patterns the Drosophila egg.

Intercellular signaling through the EGF receptor (EGFR) patterns the Drosophila egg. The TGF alpha-like ligand Gurken signals from the oocyte to the receptor in the overlying somatic follicle cells. We show that in the dorsal follicle cells this initial paracrine signaling event triggers an autocrine amplification by two other EGFR ligands, Spitz and Vein. Spitz only becomes an effective ligand in the presence of the multitransmembrane domain protein Rhomboid. Consequent high-level EGFR activation leads to localized expression of the diffusible inhibitor Argos, which alters the profile of signaling. This sequential activation, amplification, and local inhibition of the EGFR forms an autoregulatory cascade that leads to the splitting of an initial single peak of signaling into two, thereby patterning the egg.

Animals↗
PubMed2001

Restriction of prenatal gas exchange impairs memory consolidation in the chick.

Our aim was to assess the effects of restricting gas exchange during incubation on postnatal memory formation and growth in the chick. Gas exchange across the eggshell was restricted by covering 50% of the eggshell with an impermeable membrane for 4 or 8 days, commencing at days 14 and 10, respectively, of a 21-day incubation. Memory formation was examined postnatally at 1-2 days using a one-trial discriminated bead task, and at 5-6 days using a discriminated wheat task. For both tasks, chicks from eggs wrapped from days 14 to 18 had impaired memory retention at 60 min after training, although learning and labile memory were not impaired. Chicks from eggs wrapped from days 10-18 appeared to be poorer in their ability to form memories, and did not discriminate as well as controls in any of the tasks. Body weights of chicks from wrapped eggs were reduced from 2 days after hatching; chicks from eggs wrapped from day 10 had lower body weights at hatching. We conclude that a period of altered prenatal gas exchange can impair memory consolidation in the chick soon after hatching. The ability to form memories may be permanently altered, as this impairment is still apparent at 5-6 days after hatching. Pre- and postnatal growth was also impaired in the chicks from wrapped eggs. Our results suggest that the extent to which postnatal neurological function and growth is impaired depends on the timing and possibly the duration of the prenatal insult.

Animals↗
PubMed2003

A subunit vaccine against the adenovirus egg-drop syndrome using part of its fiber protein.

In this study, the effectiveness of antibodies against the hexon, fiber or a fiber fragment of an avian adenovirus egg-drop syndrome (EDS), in neutralizing the virus was tested. The fiber protein is responsible for binding the virus to the target cell. The fiber fragment knob-s comprises the carboxy-terminal knob domain and 34 amino acids of the immediately adjacent shaft domain of the adenovirus fiber protein. The hexon, fiber capsid protein and knob-s were produced in E. coli and injected into chickens. Antibodies that were produced against the whole fiber protein showed some hemagglutination inhibition (HI) activity. Antibodies produced against the knob-s protein showed HI activity and serum neutralization (SN) activity similar to the positive control-whole virus vaccine. We assume that production of only part of the fiber enables the protein produced in E. coli to fold correctly. Antibodies produced against the hexon protein showed no SN activity. In summary, knob-s induced SN and HI antibodies against EDS virus at a rate similar to the whole virus and were significantly more efficient than the full-length fiber. The recombinant knob-s protein may be used as a vaccine against pathogenic adenovirus infections.

Adenoviridae Infections↗
PubMed2003

Reproductive success and chlorinated hydrocarbon contamination of resident great blue herons (Ardea herodias) from coastal British Columbia, Canada, 1977 to 2000.

Over the period 1977-2000, eggs of Pacific great blue heron (Ardea herodias fannini) were collected from 23 colonies along the southern coast of British Columbia, Canada, and analyzed for persistent organochlorine (OC) pesticides and polychlorinated biphenyls (PCBs). Concentrations of OC pesticides in eggs declined sharply in the late 1970s, after which there were minimal changes. The sums of PCB congeners were not reduced appreciably during the 1980s and 1990s, but Aroclor 1260 concentrations suggested a sharp decline in PCB contamination of eggs in the late 1970s, similar to that shown for OC pesticides. Eggs collected along or near the Fraser River delta showed higher levels of most pesticides compared to other monitored colonies. Although the delta lands support a long-standing agricultural economy, the primary factors influencing OC levels in the delta colonies were thought to be driven by estuarine processes. We suggest two possible influencing factors were: 1) a greater rate of bioaccumulation in the estuary due to the deposition of particulates collected over a vast area encompassed by the Fraser River watershed; or 2) a higher rate of biomagnification in the estuary due to species differences at lower trophic levels of the heron food chain. Eggs from urban colonies contained higher levels of PCBs. The congener pattern was not clearly different from that observed in less contaminated eggs from rural and pulp mill-influenced colonies, except that colonies in Vancouver had greater proportions of PCB-66, suggesting a local source of Aroclor 1242. Productivity in the coastal heron colonies was highly variable over the period of study, with 71% of recorded colony-wide reproductive failures occurring in colonies near pulp mills. However, the predominant factors influencing reproductive success were probably disturbance by humans and bald eagles, combined with loss and degradation of nesting habitat, and not sublethal toxicity.

Animals↗