The isolation, partial purification and preliminary characterization of a growth factor from chick embryo brains

Chicl( brain growth factor (CBGF) is a mitogen isolated from embryonic chick brains thought to have a potential role as a trophic factor involved in nerve dependent amphibian limb regeneration. In addition, CBGF stimulates 3H-thymidine incorporation in chick embryo brain astrocytes in vitro. In this study, cultured chick embryo brain non-neuronal cells were employed in a bioassay to monitor CBGF activity throughout various stages of its pllrification. Cell culture and assay conditions were optimized. Nonneuronal cells grew best on collagen-coated culture dishes in complete medium, were most responsive to a growth stimulus [10% fetal bovine serum (FBS)] at the second and third subcultures, and were healthiest when rendered "quiescent" in medium supplemented with 1% FBS. The most effective bioassay conditions consisted of a minimum 14.5 hour "quiescence" time (24 hours was used), a 6 hour "prestimulation" time, and a 24 hour 3H-thymidine labeling time. Four-day subconfluent primary non-neuronal cells consisted of 6.63% GFAP positive cells; as a result cultures were thought to be mainly composed of astroblasts. CBGF was purified from 18-day chick embryo brains by ultrafiltration through Amicon PM-30 and YM-2 membranes, size exclusion chromatography through a Biogel P6 column, and analytical reverse-phase high-performance liquid chromatography (rp-HPLC). The greatest activity resided in rp-HPLC fraction #7 (10 ng/ml) which was as effective as 10% FBS at stimulating 3H-thymidine incorporation in chick embryo brain nonneuronal cells. Although other researchers report the isolation of a mitogenic fraction consisting of 5'-GMP from the embryonic chick brain, UV absorbance spectra, rp-HPLC elution profiles, and fast atom bombardment (FAB) mass spectra indicated that CBGF is neither 5'-GMP nor 51-AMP. 2 Moreover, commercially available 5t-GMP was inhibitory to 3H-thymidine incorporation in the chick non-neuronal cells, while Sf-AMP had no effect. Upon treatment with pronase, the biological activity of fraction P6-3 increased; this increase was nearly 30% greater than what would be expected from a simple additive effect of any mitogenic activity of pronase alone together with P6-3 alone. This may suggest the presence of an inhibitor protein. The bioactive component may be a protein protected by a nucleoside/nucleotide or simply a nucleoside/nucleotide acting alone. While the FAB mass spectrum of rp-HPLC fraction #7 did not reveal molecular weight or sequence information, the ion of highest molecular weight was observed at m/z 1610; this is consistent with previous estimations of CBGF's size. 3

Isolation and analysis of a corprinus cinereus DNA fragment showing homology to a fimbrial cDNA from ustilago violacea

Surface proteinaceous fibrils, termed fimbriae, were first identified on gram negative bacteria in the 1940s. Fungal fimbriae, discovered some 25 years later, are found on members of all fungal classes. In the present study, polyclonal antiserum raised against the fimbrial proteins of U. vio/acea were used in order to identify antigenically related proteins from Coprinus cinereus and Schizophy//um commune. Two polypeptides with molecular masses of 37 and 39 kDa from C. cinereus were observed and confirm earlier results. A single previously unidentified 50 kDa polypeptide in S. commune crossreacted with the antiserum. The 50 kDa protein was found to consist of 3 isoforms with isoelectric points ranging from 5.6 to 5.8. A fimbrial cDNA derived from U. vio/acea was used to identify DNA restriction fragments from C. cinereus and S. commune showing homology to the fimbrial transcript of U. vio/acea. Heterologous hybridization with this cDNA was used in order to screen a C. cinereus genomic DNA library. A single clone, A2-3A, with a 14 kbp insert showed strong homology to the pfim3-1 cDNA. The region of homology, a 700 bp Xba I fragment, was subcloned into pUG19. This plasmid was refered to as pXX8. DNA sequence determinations of pXX8 and adjacent fragments from A2-3A suggested that the cloned DNA was a portion of the rONA repeat encoding the small subunit rRNA. DNA sequence analysis of pfim3-1 yielded an incomplete open reading frame. The predicted amino acid sequence codes for a 206 amino acid, 22 kDa polypeptide which contains a domain similar to a transmembrane domain from rat leukocyte antigen, GDS3. As well, an untranslated 576 nucleotide domain showed 81 % homology to pXX8 and 830/0 homology to the 188 rRNA sequence of Ustilago maydis. This sequence was found adjacent to a region of adenine-thymine base pairs presumed to represent the polyadenylation sequence of the fimbrial transcript. The size and extent of homology is sufficient to account for the hybridization of pfim3-1 to rDNA. It is suggested that this domain represents a completely novel regulatory domain within eukaryotes that may enable the observed rapid regeneration of fimbriae in U. violacea.

Implication des peptides RALFs dans les communications cellulaires lors du développement du gamétophyte femelle chez Solanum chacoense et Arabidopsis thaliana.

Chez les angiospermes, la reproduction passe par la double fécondation. Le tube pollinique délivre deux cellules spermatiques au sein du gamétophyte femelle. Une cellule féconde la cellule œuf pour produire un zygote; l’autre féconde la cellule centrale pour produire l’endosperme. Pour assurer un succès reproductif, le développement du gamétophyte femelle au sein de l’ovule doit établir un patron cellulaire qui favorise les interactions avec le tube pollinique et les cellules spermatiques. Pour ce faire, un dialogue doit s’établir entre les différentes cellules de l’ovule lors de son développement, de même que lors de la fécondation. D’ailleurs, plusieurs types de communications intercellulaires sont supposées suite à la caractérisation de plusieurs mutants développementaux. De même, ces communications semblent persister au sein du zygote et de l’endosperme pour permettre la formation d’un embryon viable au sein de la graine. Malgré les développements récents qui ont permis de trouver des molécules de signalisation supportant les modèles d’interactions cellulaires avancés par la communauté scientifique, les voies de signalisation sont de loin très incomplètes. Dans le but de caractériser des gènes encodant des protéines de signalisation potentiellement impliqués dans la reproduction chez Solanum chacoense, l’analyse d’expression des gènes de type RALF présents dans une banque d’ESTs (Expressed Sequence Tags) spécifiques à l’ovule après fécondation a été entreprise. RALF, Rapid Alcalinization Factor, est un peptide de 5 kDa qui fait partie de la superfamille des «protéines riches en cystéines (CRPs)», dont les rôles physiologiques au sein de la plante sont multiples. Cette analyse d’expression a conduit à une analyse approfondie de ScRALF3, dont l’expression au sein de la plante se limite essentiellement à l’ovule. L’analyse de plantes transgéniques d’interférence pour le gène ScRALF3 a révélé un rôle particulier lors de la mégagamétogénèse. Les plantes transgéniques présentent des divisions mitotiques anormales qui empêchent le développement complet du sac embryonnaire. Le positionnement des noyaux, de même que la synchronisation des divisions au sein du syncytium, semblent responsables de cette perte de progression lors de la mégagamétogénèse. L’isolement du promoteur de même que l’analyse plus précise d’expression au sein de l’ovule révèle une localisation sporophytique du transcrit. La voie de signalisation de l’auxine régule également la transcription de ScRALF3. De surcroît, ScRALF3 est un peptide empruntant la voie de sécrétion médiée par le réticulum endoplasmique et l’appareil de Golgi. En somme, ScRALF3 est un important facteur facilitant la communication entre le sporophyte et le gamétophyte pour amener à maturité le sac embryonnaire. L’identification d’un orthologue potentiel chez Arabidopsis thaliana a conduit à la caractérisation de AtRALF34. L’absence de phénotype lors du développement du sac embryonnaire suggère, cependant, de la redondance génétique au sein de la grande famille des gènes de type RALF. Néanmoins, les peptides RALFs apparaissent comme d’importants régulateurs lors de la reproduction chez Solanum chacoense et Arabidopsis thaliana.

Molecular cloning and homology modelling of a subtilisin-like serine protease from the marine fungus, Engyodontium album BTMFS10

An alkaline protease gene (Eap) was isolated for the first time from a marine fungus, Engyodontium album. Eap consists of an open reading frame of 1,161 bp encoding a prepropeptide consisting of 387 amino acids with a calculated molecular mass of 40.923 kDa. Homology comparison of the deduced amino acid sequence of Eap with other known proteins indicated that Eap encode an extracellular protease that belongs to the subtilase family of serine protease (Family S8). A comparative homology model of the Engyodontium album protease (EAP) was developed using the crystal structure of proteinase K. The model revealed that EAP has broad substrate specificity similar to Proteinase K with preference for bulky hydrophobic residues at P1 and P4. Also, EAP is suggested to have two disulfide bonds and more than two Ca2? binding sites in its 3D structure; both of which are assumed to contribute to the thermostable nature of the protein.

Molecular cloning and homology modelling of a subtilisin-like serine protease from the marine fungus, Engyodontium album BTMFS10

An alkaline protease gene (Eap) was isolated for the first time from a marine fungus, Engyodontium album. Eap consists of an open reading frame of 1,161 bp encoding a prepropeptide consisting of 387 amino acids with a calculated molecular mass of 40.923 kDa. Homology comparison of the deduced amino acid sequence of Eap with other known proteins indicated that Eap encode an extracellular protease that belongs to the subtilase family of serine protease (Family S8). A comparative homology model of the Engyodontium album protease (EAP) was developed using the crystal structure of proteinase K. The model revealed that EAP has broad substrate specificity similar to Proteinase K with preference for bulky hydrophobic residues at P1 and P4. Also, EAP is suggested to have two disulfide bonds and more than two Ca2? binding sites in its 3D structure; both of which are assumed to contribute to the thermostable nature of the protein.

Down-regulation of the CSLF6 gene results in decreased (1,3;1,4)-beta-D-glucan in endosperm of wheat

(1,3;1,4)-beta-d-Glucan (beta-glucan) accounts for 20% of the total cell walls in the starchy endosperm of wheat (Triticum aestivum) and is an important source of dietary fiber for human nutrition with potential health benefits. Bioinformatic and array analyses of gene expression profiles in developing caryopses identified the CELLULOSE SYNTHASE-LIKE F6 (CSLF6) gene as encoding a putative beta-glucan synthase. RNA interference constructs were therefore designed to down-regulate CSLF6 gene expression and expressed in transgenic wheat under the control of a starchy endosperm-specific HMW subunit gene promoter. Analysis of wholemeal flours using an enzyme-based kit and by high-performance anion-exchange chromatography after digestion with lichenase showed decreases in total beta-glucan of between 30% and 52% and between 36% and 53%, respectively, in five transgenic lines compared to three control lines. The content of water-extractable beta-glucan was also reduced by about 50% in the transgenic lines, and the M(r) distribution of the fraction was decreased from an average of 79 to 85 x 10(4) g/mol in the controls and 36 to 57 x 10(4) g/mol in the transgenics. Immunolocalization of beta-glucan in semithin sections of mature and developing grains confirmed that the impact of the transgene was confined to the starchy endosperm with little or no effect on the aleurone or outer layers of the grain. The results confirm that the CSLF6 gene of wheat encodes a beta-glucan synthase and indicate that transgenic manipulation can be used to enhance the health benefits of wheat products.

Germin, a protein marker of early plant development, is an oxalate oxidase

Germin is a homopentameric glycoprotein, the synthesis of which coincides with the onset of growth in germinating wheat embryos. There have been detailed studies of germin structure, biosynthesis, homology with other proteins, and of its value as a marker of wheat development. Germin isoforms associated with the apoplast have been speculated to have a role in embryo hydration during maturation and germination. Antigenically related isoforms of germin are present during germination in all of the economically important cereals studied, and the amounts of germin-like proteins and coding elements have been found to undergo conspicuous change when salt-tolerant higher plants are subjected to salt stress. In this report, we describe how circumstantial evidence arising from unrelated studies of barley oxalate oxidase and its coding elements have led to definitive evidence that the germin isoform made during wheat germination is an oxalate oxidase. Establishment of links between oxalate degradation, cereal germination, and salt tolerance has significant implications for a broad range of studies related to development and adaptation in higher plants. Roles for germin in cell wall biochemistry and tissue remodeling are discussed, with special emphasis on the generation of hydrogen peroxide during germin-induced oxidation of oxalate.

The effect of environment on endosperm cell-wall development in Triticum aestivum during grain filling: an infrared spectroscopic imaging study

One of the major factors contributing to the failure of new wheat varieties is seasonal variability in end-use quality. Consequently, it is important to produce varieties which are robust and stable over a range of environmental conditions. Recently developed sample preparation methods have allowed the application of FT-IR spectroscopic imaging methods to the analysis of wheat endosperm cell wall composition, allowing the spatial distribution of structural components to be determined without the limitations of conventional chemical analysis. The advantages of the methods, described in this paper, are that they determine the composition of endosperm cell walls in situ and with minimal modification during preparation. Two bread-making wheat cultivars, Spark and Rialto, were selected to determine the impact of environmental conditions on the cell-wall composition of the starchy endosperm of the developing and mature grain, focusing on the period of grain filling (starting at about 14 days after anthesis). Studies carried out over two successive seasons show that the structure of the arabinoxylans in the endosperm cell walls changes from a highly branched form to a less branched form. Furthermore, during development the rate of restructuring was faster when the plants were grown at higher temperature with restricted water availability from 14 days after anthesis with differences in the rate of restructuring occurring between the two cultivars.

Superovulation and embryo collection in nulliparous Boer goat does immunized against a recombinant ovine alpha-subunit inhibin

With the purpose of eliciting a superovulatory response, 12 adult nulliparous Boer goat does were actively immunized against a recombinant a-subunit of ovine inhibin (roIHN-alpha; two injections of 100 mg 4 weeks apart). Another 12 control Boer goat does were treated with physiological saline and acted as controls. One year later the immunized animals were boostered by the administration of another dose (100 mg) of the immunogen. Following treatment, blood samples were collected twice weekly for the periods of 16 and 12 weeks, respectively, to monitor the inhibin binding ability with the aid of a radio-tracer binding assay. Throughout the experiment, estrus detection was conducted twice daily with the aid of an aproned intact buck. From the first day after treatment to 48 h after standing estrus, ovarian activity was monitored daily by transrectal ultrasonography. On alternate estrous cycles, does were mated and 6 days later flushed transcervically to recover embryos. All goats treated with the roIHN-alpha produced antibodies reactive to the native bovine inhibin tracer-the titre increasing from 2.9 +/- 0.4 to a maximum of 21.9 +/- 2.9% binding after the second injection. The antibody titre gradually subsided over the next 16 weeks. The booster injection restored an elevated antibody titre (11.7 +/- 0.4%), which was maintained until the end of the sampling period 12 weeks later. In the control goats only trace amounts of antibody were recorded throughout the trial. In the roIHN-alpha-immunized goats the number of follicles reaching a diameter of > 4 mm was 14.6 +/- 1.2 per doe. A positive correlation was recorded between the follicle number and antibody titre (r=0.61; P < 0.01). The number of follicles ovulating per doe (6.9 +/- 0.7) followed the same tendency-however, the proportion decreased with increasing follicle numbers. A relatively weak correlation was recorded between the inhibin binding ability and number of ovulations (r=0.27; P < 0.05). In the control goats the majority (92%) of follicles exceeding 4 mm in diameter ovulated (2.5 +/- 0.1 follicles/doe). Embryo collection proved unsatisfactory (42% versus 39% recovery for immunized and control animals, respectively)-presumably because the uterine lumen of the nulliparous does was too narrow to permit effective flushing. In the group of immunized goats the occurrence of short estrous cycles (< 15 days) recorded was 34% versus only 6% in the controls. Overall, immunization of goats against roIHN-alpha led to an almost six-fold increase in number of ovarian follicles, a three-fold increase in ovulations and, despite the low recovery rate, a more than three-fold increase in ova or embryos recovered. It may be concluded that treatment of female goats with roIHN-alpha leads to an inhibin antibody response, accompanied by enhanced ovarian activity. The response was, however, accompanied by a large proportion of retained follicles and a high incidence of short estrous cycles. These problems need to be further investigated before rendering the method fit for application in embryo transfer programs in goats. (C) 2007 Elsevier B.V. All rights reserved.

Expression and regulation of Nkd-1, an intracellular component of Wnt signalling pathway in the chick embryo

The Wnt family of secreted signalling molecules control a wide range of developmental processes in all metazoans. The intracellular response to Wnt signalling depends on the choice of signalling cascade activated in the responding cell. Cells can activate either the canonical pathway that modulates gene expression to control cellular differentiation and proliferation, or the non-canonical pathway that controls cell polarity and movement. Recent work has identified the protein Naked Cuticle to act as an intracellular switch to promote the non-canonical pathway at the expense of the canonical pathway. We have cloned chick Naked Cuticle-1 (cNkd-1) and show that it is expressed in a dynamic manner during early embryogenesis. We show that it is expressed in the somites and in particular regions where cells are undergoing movement. Lastly, we show that the expression of cNkd-1 is regulated by Wnt expression originating from the neural tube. This study provides evidence that non-canonical Wnt signalling plays a part in somite development.

Investigation of the fracture mode for hard and soft wheat endosperm using the loading-unloading bending test

Investigation of the fracture mode for hard and soft wheat endosperm was aimed at gaining a better understanding of the fragmentation process. Fracture mechanical characterization was based on the three-point bending test which enables stable crack propagation to take place in small rectangular pieces of wheat endosperm. The crack length can be measured in situ by using an optical microscope with light illumination from the side of the specimen or from the back of the specimen. Two new techniques were developed and used to estimate the fracture toughness of wheat endosperm, a geometric approach and a compliance method. The geometric approach gave average fracture toughness values of 53.10 and 27.0 J m(-2) for hard and soft endosperm, respectively. Fracture toughness estimated using the compliance method gave values of 49.9 and 29.7 J m(-2) for hard and soft endosperm, respectively. Compressive properties of the endosperm in three mutually perpendicular axes revealed that the hard and soft endosperms are isotropic composites. Scanning electron microscopy (SEM) observation of the fracture surfaces and the energy-time curves of loading-unloading cycles revealed that there was a plastic flow during crack propagation for both the hard and soft endosperms, and confirmed that the fracture mode is significantly related to the adhesion level between starch granules and the protein matrix.