Structural and biochemical analysis of the Leptinotarsa decemlineata (Coleoptera; Chrysomeloidea) crystalline chorionic layer

The developmental aspects of the Leptinotarsa decemlineata crystalline chorionic layer (CCL) morphogenesis, its composition and its supramolecular structure were studied. The mature Leptinotarsa decemlineata eggshell consists of the vitelline membrane and the CCL, while the follicle cell remnants following their degeneration after oogenesis completion constitute the outer chorionic layer. The vitelline membrane and the CCL layers are formed through continuous material deposition from the follicular epithelium, whereas the main morphogenic factor during most insect eggshell formation, namely the follicle cell and oocyte microvilli, are seemingly involved only in vitelline membrane formation. Analysis of the CCL morphogenesis showed that this layer is assembled from a fiber-like pre-crystalline material, which accumulates at the vitelline membrane-follicle cell interface. The mature CCL is about 1 microm thick and exhibits a periodicity of approximately 10 nm, while computer image analysis studies of thin-sectioned CCL revealed the existence of crystalline layers parallel to the CCL surface. Finally, SDS-PAGE-electrophoresis of purified CCLs showed that this crystalline layer is of a proteinaceous nature and is most likely composed of 3-5 polypeptides with a molecular weight ranging in between 28-60 kDa. Overall, these data exemplify for the first time the nature and supramolecular arrangement of a crystalline layer and its constituent molecules in Coleoptera.     

Characterization of the eggshell of Haemonchus contortus--I. Structural components.

1. By transmission electron microscopy, the eggshell of Haemonchus contortus was seen to be similar to previously studied nematodes, with an outer vitelline layer bounded by a trilaminate membrane, a broad medial region, containing chitin, and an electron dense basal region, containing lipid and protein. 2. Exposure of Haemonchus contortus eggs to proteases resulted in disruption of the shell with removal of components of the outer, medial and basal regions. Exposure to chitinase depleted fibrillar components of the medial region of the shell, while collagenase had no effect. 3. Chloroform/methanol extraction of fresh eggshells caused a minor condensation of the outer, vitelline layer and some depletion of the basal layer. 4. After normal hatching, shells appeared similar to those treated with protease and chitinase, but also lacked the basal, lipid layer. 5. Extracts of isolated unhatched eggshells and hatched eggshells, and extracts of biotin-labelled whole fresh eggs showed three major protein bands when run on sodium dodecyl sulphate-polyacrylamide gels indicating that these three proteins are most likely structural in nature and do not participate in the release of the larva from the eggshell. 6. Biotin-labelled protein bands were degraded by proteases and chitinase, but not collagenase or lipase.     

Phosphoproteins of the chicken eggshell calcified layer

The chicken eggshell matrix is a complex mixture of proteins and proteoglycans. It also contains phosphoproteins that are thought to affect mineralization of the matrix. Several of the matrix phosphoproteins, such as the major component osteopontin, have already been identified as phosphoproteins in other tissues, but the phosphorylation status of the eggshell matrix forms was unknown. The phosphopeptides, obtained after cleavage of the matrix proteins with several different cleavage methods, were enriched by anion-exchange chromatography and reversible binding to titanium oxide and identified by LC-MS(n) or pseudo-MS(n) analysis following neutral loss scanning. Altogether we identified 39 phosphorylated matrix proteins, 22 of which were not known to be phosphorylated before. Eight of the proteins were identified as eggshell matrix components for the first time. Together these proteins contained more than 150 different phosphorylation sites, 103 of which were determined with high confidence. Among the major phosphorylated proteins of the chicken eggshell matrix were osteopontin and the eggshell-specific proteins ovocleidin-17, ovocleidin-116, and ovocalyxin-32.     

Proteomic analysis of the acid-soluble organic matrix of the chicken calcified eggshell layer

The major difference between inorganic minerals and biominerals is the presence of an organic matrix consisting of proteins, glycoproteins, proteoglycans, and polysaccharides, which is synthesized by specialized cells under genetic control before or during mineralization. The organic matrix is thought to play a major role in the assembly of the biomineral and determination of its mechanical properties. The recent elucidation of the chicken genome provided an opportunity to explore the matrix proteome of a biomineral using up-to-date MS-based technology. We identified 520 proteins in this matrix including the ten matrix proteins already known before. The identified proteins were divided into three abundance groups using the exponentially modified protein abundance index described recently which was roughly calibrated with the few known data on protein yield derived from Edman sequence analysis. A small group of 32 highly abundant proteins contained the presently known eggshell-specific proteins and all of the other known eggshell matrix constituents identified before with much less sensitive conventional methods. The present study, which is the first comprehensive proteomic study of a vertebrate biomineral, is intended as a starting point for the detailed molecular characterization of eggshell matrix proteins, their interactions in the matrix network and functional studies.     

Crystallization and preliminary x-ray analysis of ovocleidin-17 a major protein of the gallus gallus eggshell calcified layer

In this work, we report the crystallization of ovocleidin-17, the major protein of the avian eggshell calcified layer and the preliminary X-ray characterization of this soluble protein which is implied into the CaCO(3) formation of the eggshell in avians. Crystals belong to one of the trigonal space group P3 with cell dimensions a= b= 59.53 A and c = 83.33 A, and alpha=beta= 90 degrees and gamma=120 degrees. Crystals diffract up to 3.0 A.     

Structural details of crystalline cellulose from higher plants

It is commonly assumed that cellulose from higher plants contains the Ialpha and Ibeta crystalline allomorphs together with surface and disordered chains. For cellulose Ialpha, the evidence for its presence in higher plants is restricted to the C-4 signals in the solid-state (13)C NMR spectrum, which match those of crystalline cellulose Ialpha from algal sources. Algal cellulose Ialpha can be converted to the Ibeta form by high-temperature annealing. We used this approach to generate cellulose samples differing in Ibeta content from flax fibers and celery collenchyma, which respectively are representative of textile (secondary-wall) and primary-wall cellulose. It was then possible to isolate the detailed spectral contributions of the surface, Ibeta and Ialpha-like phases from linear combinations of the observed (13)C NMR and FTIR spectra. The (13)C NMR spectra resembled those of highly crystalline tunicate or algal cellulose Ibeta and Ialpha, with slight differences implying increased disorder and minor conformational discrepancies. The FTIR spectrum of the Ibeta form was closely similar to its more crystalline counterparts, but the FTIR spectrum of the Ialpha form was not. In addition to increased bandwith indicative of lower order, it showed substantial differences in the profile of hydroxyl stretching bands. These results confirm that higher plants synthesize cellulose Ibeta but show that the Ialpha-like chains, although conformationally quite similar to crystalline algal cellulose Ialpha, sit in a different hydrogen-bonding environment in higher plants. The differences are presumably occasioned by the small diameter of the crystallites and the influence of the crystallite surface on chain packing.     

Identification and localization of lysozyme as a component of eggshell membranes and eggshell matrix.

The avian eggshell is a composite biomaterial composed of non-calcifying eggshell membranes and the overlying calcified shell matrix. The calcified shell forms in a uterine fluid where the concentration of different protein species varies between the initial, rapid calcification and terminal phases of eggshell deposition. The role of these avian eggshell matrix proteins during shell formation is poorly understood. The properties of the individual components must be determined in order to gain insight into their function during eggshell mineralization. In this study, we have identified lysozyme as a component of the uterine fluid by microsequencing, and used western blotting, immunofluorescence and colloidal-gold immunocytochemistry to document its localization in the eggshell membranes and the shell matrix. Furthermore, Northern blotting and RT-PCR indicates that there is a gradient to the expression of lysozyme message by different regions of the oviduct, with significant albeit low levels expressed in the isthmus and uterus. Lysozyme protein is abundant in the limiting membrane that circumscribes the egg white and forms the innermost layer of the shell membranes. It is also present in the shell membranes, and in the matrix of the calcified shell. Calcite crystals grown in the presence of purified hen lysozyme exhibited altered crystal morphology. Therefore, in addition to its well-known anti-microbial properties that could add to the protective function of the eggshell during embryonic development, shell matrix lysozyme may also be a structural protein which in soluble form influences calcium carbonate deposition during calcification.     

Location of the dipteran specificity region in a lepidopteran-dipteran crystal protein from Bacillus thuringiensis.

Two highly related crystal protein genes from Bacillus thuringiensis subsp. kurstaki HD-1, designated cryIIA and cryIIB (previously named cryB1 and cryB2, respectively), were used to study host range specificity. Their respective gene products are 87% identical but exhibit different toxicity spectra; CryIIA is toxic to both mosquito and tobacco hornworm larvae, whereas CryIIB is toxic only to the latter. Hybrids of the cryIIA and cryIIB genes were generated, and their resultant gene products were assayed for toxicity. A short segment of CryIIA corresponding to residues 307 through 382 was shown to be sufficient for altering host range specificity-i.e., when this region replaced the corresponding segment of CryIIB, the resulting hybrid protein acquired toxicity against mosquitoes. The CryIIA and CryIIB polypeptides differ by only 18 amino acids in this region, indicating that very few amino acid changes can have a substantial effect on the toxicity spectra of these proteins.     

Structural and chemical characterization of the S layer of a Pseudomonas-like bacterium.

Sections and freeze-fractured preparations showed an S layer on the surface of Pseudomonas-like strain EU2. Polyacrylamide gel electrophoresis of cell envelopes extracted with 1% sodium dodecyl sulfate (SDS) at room temperature showed three proteins (45K, 55K, and 110K). The 55K protein was identified as the S-layer protein. Incubation in 1.5 M guanidine hydrochloride removed the S layer from cell envelopes and dissociated the structure into subunits. The soluble 55K protein reassembled into planar sheets upon removal of the guanidine hydrochloride by dialysis. Electron microscopy and image processing indicated that these sheets had p4 symmetry in projection with a lattice constant of 13.2 +/- 0.1 nm (corresponding to 9.3 nm between adjacent fourfold axes). In some instances these reassemblies appeared to form small three-dimensional crystals which gave particularly clear views of the structure in projection because of the superimposition of information from a number of layers. A model is proposed with molecules having rounded lobes connected by a narrower linker region and joining at the lobes to form the fourfold axes of the array. The pattern superficially resembles those of other bacterial S layers, such as those of Aeromonas salmonicida, Aeromonas hydrophila, and Azotobacter vinelandii. Extraction of cell envelopes with 1% SDS at 50 degrees C released the 110K protein from the envelopes and removed an amorphous backing layer from the S layer. The 45K protein displayed heat-modifiable migration in SDS-polyacrylamide gel electrophoresis and was insoluble in SDS at 50 degrees C or in high concentrations of guanidine hydrochloride, suggesting that it was associated with the peptidoglycan.     

The eggshell of Drosophila melanogaster. VIII. Morphogenesis of the wax layer during oogenesis

Utilizing freeze-fracturing and conventional electron microscopy methods, we have studied the details of morphogenesis and construction of the wax layer envelope from Oregon R and mutants of Drosophila melanogaster egg' s during oogenesis. The wax layer is synthesized and secreted by the follicular cells in the 10b of lipid vesicles during static 10b. During secretion (stages 10b, 11 and 12) the lipid vcsicles are accumulated on the vitelline membrane surface and become flat. At the late stage of choriogenisis (stages 13, 14) the lipid vesicles are compressed tightly between the vitelline membrane and the other already constructed eggshell layers, so the wax layer becomes very thin and is hardly seen in crossfractured views.     

Salmonella enterica isolates from layer farm environments are able to form biofilm on eggshell surfaces

This study examined the eggshell biofilm forming ability of Salmonella enterica isolates recovered from egg farms. Multicellular behaviour and biofilm production were examined at 22 and 37°C by Congo red morphology and the crystal violet staining assay. The results indicated that the biofilm forming behaviour of Salmonella isolates was dependent on temperature and associated with serovars. Significantly greater biofilm production was observed at 22°C compared with 37°C. The number of viable biofilm cells attached to eggshells after incubation for 48 h at 22°C was significantly influenced by serovar. Scanning electron microscopic examination revealed firm attachment of bacterial cells to the eggshell surface. The relative expression of csgD and adrA gene was significantly higher in eggshell biofilm cells of S. Mbandaka and S. Oranienburg. These findings demonstrate that Salmonella isolates are capable of forming biofilm on the eggshell surface and that this behaviour is influenced by temperature and serovar.     

Colloidal-gold immunocytochemical localization of osteopontin in avian eggshell gland and eggshell.

During mineralization of the avian eggshell, there is a sequential and orderly deposition of both matrix and mineral phases. Therefore, the eggshell is an excellent model for studying matrix-mineral relationships and the regulation of mineralization. Osteopontin, as an inhibitor of crystal growth, potently influences the formation of calcium phosphate and calcium carbonate biominerals. The purpose of this study was to characterize matrix-mineral relationships, specifically for osteopontin, in the avian eggshell using high-resolution transmission (TEM) and scanning (SEM) electron microscopy to gain insight into how calcite crystal growth is structured and compartmentalized during eggshell mineralization. Osteopontin was localized at the ultrastructural level by colloidal-gold immunocytochemistry. In EDTA-decalcified eggshell, an extensive matrix network was observed by TEM and SEM throughout all regions and included interconnected fibrous sheets, irregularly shaped aggregates, vesicular structures, protein films, and isolated protein fibers. Osteopontin was associated with protein sheets in the highly mineralized palisades region; some of these features defined boundaries that compartmentalized different eggshell structural units. In fractured and undecalcified eggshell, osteopontin was immunolocalized on the {104} crystallographic faces of calcite-its natural cleavage plane. The specific occlusion of osteopontin into calcite during mineralization may influence eggshell structure to modify its fracture resistance.     

Bacteriological larvicides of dipteran disease vectors.

The apparent success in vector control observed between 1950 and 1970 was followed by worldwide resistance to organosynthetic insecticides wherever they were used intensively. Insect resistance to one or more categories of insecticides has limited the effectiveness of these compounds, and their non-selective mode of action adversely affects non-target organisms. This scenario highlights the need for selective agents in integrated vector control programs. This article gives an overview of the main fundamental and applied research topics on entomopathogenic bacteria in relation to their role in vector control.     

The isolation of mitochondria from dipteran flight muscle

Procedures for the isolation of mitochondria from dipteran flight muscle have been investigated in an attempt to determine the extent and to identify the causes of deterioration associated with isolation. In the light of the results obtained isolation procedures have been improved by minimising mechanical damage, avoiding the development of anoxic conditions, and by the use of an isolation medium of a more physiological nature, containing the potassium salt of an organic anion as the principal osmoeffector, phosphate as the principal buffer, and low concentrations of free Mg²⁺. The oxidative capacity of mitochondria isolated by the improved method is adequate to support the in vivo requirements of the flight system.     

Chromatic aberration of a dipteran corneal lens

Summary The longitudinal chromatic aberration (variation in the position of focus with wavelength) of corneal facet lenses of the houseflyMusca domestica is measured directly. The result is shown to agree with that calculated using the thick-lens formulas, the measured lens parameters and the dispersion of the refractive index of the lenses, measured with an interference microscope. The longitudinal chromatic aberration between the two wavelengths of peak absorption of fly rhabdomeres (360 nm and 495 nm) is about 2.5 m and comparable to the depth of focus of the lens, assuming the lens to be diffraction limited. Chromatic aberration is therefore expected to have little effect on optical image quality in the fly; in particular the effect on the modulation transfer function at the receptor level and on the angular sensitivity of the rhabdomeres is insignificant.Abbreviations LCA longitudinal chromatic aberration - MTF modulation transfer function     

Radiation sensitivity of Bacillus cereus with and without a crystalline surface protein layer

The radiation sensitivity of four strains of Bacillus cereus was investigated with attention to bacterial surface structure. All four strains were sensitive to radiation with gamma rays (D(10)=0.4 kGy). No crystalline surface protein layer could be detected on the cell surface. When cultured on solid media, an S-layer covered the cells of the two strains, and they were 2.6 times as resistant to radiation as the two reference strains without an S-layer. In SDS-PAGE, a major 97-kDa band from the resistant strains from plate cultures was replaced by a ca. 85-kDa protein band in samples from broth cultures. Electron microscopy, SDS-PAGE, Western blot and fluorescent antibody staining indicated that the higher resistance to radiation of the clinical strains from plate cultures was associated with the presence of the S-layer on the cell surface.     

Structural analysis of a new glycosphingolipid from the lipopolysaccharide-lacking bacterium Sphingomonas adhaesiva.

A new glycosphingolipid, GSL-4B, was isolated from Sphingomonas adhaesiva and found to share the ceramide moiety with GSL-1 and GSL-3 from Sphingomonas capsulata studied earlier [Kawahara, K.; Moll, H.; Knirel, Y. A.; Seydel, U.; Z?hringer, U. Eur. J. Biochem. 2000, 267, 1837-1846]. It is heterogeneous with respect to the long-chain bases erythro-2-amino-1,3-octadecanediol (sphinganine), (13Z)-erythro-2-amino-13-eicosene-1,3-diol, and (13Z)-erythro-2-amino-13,14-methylene-1,3-eicosanediol which in GSL-4B are present in the ratios of 1.1:1.0:1.1, and all bearing amide-linked (S)-2-hydroxymyristic acid. Methylation analysis and MALDI-TOF-MS along with 1H and 13C NMR spectroscopy showed that the carbohydrate part of GSL-4B has the structure of alpha-D-Glcp-(1-->4)-alpha-D-Galp-(1-->6)-alpha-D-Glcp-(1-->4)-alpha-D-GlcpA-(1-->1)-Cer     

A re-evaluation of aff. Megaloolithidae eggshell fragments from the uppermost Cretaceous of the Pyrenees and implications for crocodylomorph eggshell structure

The Upper Cretaceous outcrops of the Pyrenees yield one of the most extensive and continuous records of paleoological remains anywhere in the world. Most of eggs and eggshells have been referred to the oofamily Megaloolithidae. In this study, we present a revision of eggshell fragments from the Blasi 2 locality, lattermost Maastrichtian in age, previously assigned to aff. Megaloolithidae. The presence of a blocky extinction pattern and basal knobs supports a crocodilian affinity of these materials. We classify them as Krokolithidae indet. Three structural layers can be recognised in the Blasi 2 eggshells, a feature that is shared with other recent eggshells (e.g. Crocodylus porosus and Crocodylus niloticus) and fossil crocodylomorph eggshells (Krokolitheswilsoni), which were previously described as single layered. The new proposed affinity of the Blasi 2 eggshells reduces the Megaloolithidae oodiversity of the last few million years of the Cretaceous in the Pyrenees to only two valid ootaxa, Megaloolithusmamillare and Megaloolithusbaghensis. The lack of more complete material precludes the erection of new ootaxa based on the Blasi 2 material.     

Structural and functional analysis of pyruvate kinase from Corynebacterium glutamicum.

Pyruvate kinase activity is an important element in the flux control of the intermediate metabolism. The purified enzyme from Corynebacterium glutamicum demonstrated a marked sigmoidal dependence of the initial rate on the phosphoenolpyruvate concentration. In the presence of the negative allosteric effector ATP, the phosphoenolpyruvate concentration at the half-maximum rate (S0.5) increased from 1.2 to 2.8 mM, and cooperation, as expressed by the Hill coefficient, increased from 2.0 to 3.2. AMP promoted opposite effects: the S0.5 was decreased to 0.4 mM, and the enzyme exhibited almost no cooperation. The maximum reaction rate was 702 U/mg, which corresponded to an apparent kcat of 2,540 s-1. The enzyme was not influenced by fructose-1,6-diphosphate and used Mn2+ or Co2+ as cations. Sequence determination of the C. glutamicum pyk gene revealed an open reading frame coding for a polypeptide of 475 amino acids. From this information and the molecular mass of the native protein, it follows that the pyruvate kinase is a tetramer of 236 kDa. Comparison of the deduced polypeptide sequence with the sequences of other bacterial pyruvate kinases showed 39 to 44% homology, with some regions being very strongly conserved.