Blastocyst attachment and morphogenesis of ectoplacental cone in mouse

Mouse blastocyst attaches on the antimesometrial side of the uterus through mural trophoblasts. Later the polar trophoblasts begin proliferation, and rapid multiplication towards the mesometrial side of the uterus occurs resulting in the formation of an excrescence designated as ectoplacental cone. The morphogenesis of ectoplacental cone, viewedin utero, initiates on day 6post-coitum when microvilli of the trophoblast and the uterine epithelial cells are lost and as a result of this opposing membranes appear interlocked with each other. Soon following the invasion by surrounding trophoblasts the necrosis of the epithelial cells starts. Mitochondriae of the epithelial cells, at this stage, are shrunken and lack well defined cristae. Several leucocytes are seen at the site and few electron dense structures appear wedged between the trophoblasts and epithelial cells. At places the cell membrane is studded with the basement membrane of the uterine epithelium giving an impression of a bristle coated membrane. By day 7post-coitum the basement membrane has almost disappeared leaving trophoblast cells to develop close contact with stromal cells. Collagen fibres appeared between the trophoblasts and the stromal cells, many large inclusions of high electron density representing engulfed necrotic epithelial cells are discernible. On day 8post-coitum the ectoplacental cone is fully developed. Four types of trophoblast cells can be identified in it: (i) basal cells lying on the base of the cone, are polyhedral and compactly arranged. They have a large nucleus and well developed nucleoli, (ii) central cells forming the middle area of the cone are of two types; one contained several osmiophilic granules enclosing translucent area (eccentric) and a well developed golgi complex around the nucleus, while the other has many heterophagosomes, vacuoles and residual bodies and (iii) peripheral cells contained several pleomorphic structures resembling secondary lysosomes. Minute dense granules and band of microfibrils on the apical region of these cells are seen. Dense granules probably release lytic proteins at the site and microfibrils help in forming cytoplasmic projections.     

<Emphasis Type="Italic">Xenopus laevis</Emphasis> peroxiredoxins: Gene expression during development and characterization of the enzymes

Reactive oxygen species (ROS) are produced via catabolic and anabolic processes during normal embryonic development, and ROS content in the cell is maintained at a certain level. Peroxiredoxins are a family of selenium-independent peroxidases and play a key role in maintaining redox homeostasis of the cell. In addition to regulating the ROS level, peroxiredoxins are involved in intracellular and intercellular signaling, cell differentiation, and tissue development. The time course of peroxiredoxin gene (prx1–6) expression was studied in Xenopus laevis during early ontogeny (Nieuwkoop and Faber stages 10–63). The highest expression level was observed for prx1 at these developmental stages. The prx1, prx3, and prx4 expression level changed most dramatically in response to oxidative stress artificially induced in X. laevis embryos. In X. laevis adults, prx1–6 were all intensely expressed in all organs examined, the prx1 expression level being the highest. The X. laevis prx1–6 genes were cloned and expressed in Escherichia coli, and physico-chemical characteristics were compared for the recombinant enzymes. The highest peroxidase activity and thermal stability were observed for Prx1 and Prx2. It was assumed that Prx1 plays a leading role in X. laevis early development.     

TGFα Reactivates Imprinted <Emphasis Type="Italic">Igf2</Emphasis> in the Parthenogenetic Mice Embryos and Placenta

Imprinted genes play important roles in the mammalian development. In the parthenogenetic embryos (PE), there is only expression of maternally expressed genes. Therefore, PEs are appropriate experimental models to study genomic imprinting controlling mechanisms. The maternally expressed H19 and paternally expressed Igf2 are reciprocally imprinted genes in normal embryos. Here, we studied effect of transforming growth factor alpha (TGFα) treatment in vitro (10 ng/ml at the morula stage) on the expression of Igf2/H19 locus in mice PE (9.5 days of gestation, 25 somites) and their placentas (PP). Using RT-PCR, we showed that TGFα reactivated maternally imprinted Igf2 gene in parthenogenetic embryos and placentas. In spite of similar Tgfα expression in the preimplantation stages, its expression in the 9.5-day parthenogenetic embryos is significantly less than in normal embryos (NE). In our experiments, it was shown that reactivation of Igf2 gene occurred independently of H19 gene. In vitro TGFα treatment of mouse PE reactivated paternally expressed Igf2 gene in the PE and PP. In the PE and PP, both Igf2 and H19 were expressed. It seems that TGFα can play an important role as modulator of the Igf2/H19 locus.     

Genome-wide identification,systematic analysis and characterization of <Emphasis Type="Italic">SRO</Emphasis> family genes in maize (<Emphasis Type="Italic">Zea mays</Emphasis> L.)

SIMILAR TO RCD ONE (SRO) is a small plant-specific gene family, which play essential roles in plant growth and development as well as in abiotic stresses. However, the function of SROs in maize is still unknown. In our study, six putative SRO genes were isolated from the maize genome. A systematic analysis was performed to characterize the ZmSRO gene family. The ZmSRO gene family was divided into two groups according to the motif and intron/exon analysis. Phylogenetic analysis of them with other plants showed that the clades of SROs along with the divergence of monocot and dicot and ZmSROs were more closely with OsSROs. Many abiotic stress response and hormone-induced cis-regulatory elements were identified from the promoter region of ZmSROs. Furthermore, RNA-seq analysis indicated that SRO genes were widely expressed in different tissues and development stages in maize, and the expression divergence was also obviously observed. Analyses of expression in response to PEG6000 and NaCl treatment, in addition to exogenous application of ABA and GA hormones showed that the majority of the members display stress-induced expression patterns. Taken together, our results provide valuable reference for further functional analysis of the SRO gene family in maize, especially in abiotic stress responses.     

Kelch-motif containing acyl-CoA binding proteins AtACBP4 and AtACBP5 are differentially expressed and function in floral lipid metabolism

Key message

We herein demonstrated two of the Arabidopsis acyl-CoA-binding proteins (ACBPs), AtACBP4 and AtACBP5, both function in floral lipid metabolism and they may possibly play complementary roles in Arabidopsis microspore-to-pollen development. Histological analysis on transgenic Arabidopsis expressing β-glucuronidase driven from the AtACBP4 and AtACBP5 promoters, as well as, qRTPCR analysis revealed that AtACBP4 was expressed at stages 11–14 in the mature pollen, while AtACBP5 was expressed at stages 7–10 in the microspores and tapetal cells. Immunoelectron microscopy using AtACBP4- or AtACBP5-specific antibodies further showed that AtACBP4 and AtACBP5 were localized in the cytoplasm. Chemical analysis of bud wax and cutin using gas chromatographyflame ionization detector and GC-mass spectrometry analyses revealed the accumulation of cuticular waxes and cutin monomers in acbp4, acbp5 and acbp4acbp5 buds in comparison to the wild type (Col-0). Fatty acid profiling demonstrated a decline in stearic acid and an increase in linolenic acid in acbp4 and acbp4acbp5 buds, respectively, over Col-0. Analysis of inflorescences from acbp4 and acbp5 revealed that there was an increase of AtACBP5 expression in acbp4, and an increase of AtACBP4 expression in acbp5. Deletion analysis of the AtACBP4 and AtACBP5 5′-flanking regions indicated the minimal promoter activity for AtACBP4 (?145/+103) and AtACBP5 (?181/+81). Electrophoretic mobility shift assays identified a pollen-specific cis-acting element POLLEN1 (AGAAA) mapped at AtACBP4 (?157/?153) which interacted with nuclear proteins from flower and this was substantiated by DNase I footprinting.

Abstract

In Arabidopsis thaliana, six acyl-CoA-binding proteins (ACBPs), designated as AtACBP1 to AtACBP6, have been identified to function in plant stress and development. AtACBP4 and AtACBP5 represent the two largest proteins in the AtACBP family. Despite having kelch-motifs and sharing a common cytosolic subcellular localization, AtACBP4 and AtACBP5 differ in spatial and temporal expression. Histological analysis on transgenic Arabidopsis expressing β-glucuronidase driven from the respective AtACBP4 and AtACBP5 promoters, as well as, qRT-PCR analysis revealed that AtACBP4 was expressed at stages 11–14 in mature pollen, while AtACBP5 was expressed at stages 7–10 in the microspores and tapetal cells. Immunoelectron microscopy using AtACBP4- or AtACBP5-specific antibodies further showed that AtACBP4 and AtACBP5 were localized in the cytoplasm. Chemical analysis of bud wax and cutin using gas chromatography-flame ionization detector and GC-mass spectrometry analyses revealed the accumulation of cuticular waxes and cutin monomers in acbp4, acbp5 and acbp4acbp5 buds, in comparison to the wild type. Analysis of inflorescences from acbp4 and acbp5 revealed that there was an increase of AtACBP5 expression in acbp4, and an increase of AtACBP4 expression in acbp5. Deletion analysis of the AtACBP4 and AtACBP5 5′-flanking regions indicated the minimal promoter region for AtACBP4 (?145/+103) and AtACBP5 (?181/+81). Electrophoretic mobility shift assays identified a pollen-specific cis-acting element POLLEN1 (AGAAA) within AtACBP4 (?157/?153) which interacted with nuclear proteins from flower and this was substantiated by DNase I footprinting. These results suggest that AtACBP4 and AtACBP5 both function in floral lipidic metabolism and they may play complementary roles in Arabidopsis microspore-to-pollen development.

     

Construction of a recombinant <Emphasis Type="Italic">Lactococcus lactis</Emphasis> strain expressing a fusion protein of Omp22 and HpaA from <Emphasis Type="Italic">Helicobacter pylori</Emphasis> for oral vaccine development

Objectives

To develop orally administrated anti-Helicobacter pylori vaccination, a Lactococcus lactis strain was genetically constructed for fusion expression of H. pylori protective antigens HpaA and Omp22.

Results

The fusion gene of omp22 and hpaA with an adapter encoding three glycines was cloned from a plasmid pMAL-c2x-omp22-hpaA into Escherichia coli MC1061 and L. lactis NZ3900 successively using a shutter vector pNZ8110. Expression of the fusion gene in L. lactis was induced with nisin resulting in production of proteins with molecular weights of 50 and 28 kDa. Both of them were immunoreactive with mouse anti-H. pylori sera as determined via western blotting. Oral vaccination of BALB/c mice using the L. lactis strain carrying pNZ8110-omp22-hpaA elicited significant systematic humoral immune response (P < 0.05).

Conclusions

This is the first report showing that a fusion protein of two H. pylori antigens was efficiently expressed in L. lactis with immunogenicity. This is a considerable step towards H. pylori vaccines.

     

Genome-wide identification and analysis of the U-box family of E3 ligases in grapevine

E3 ubiquitin ligases play essential roles in determining the specificity of ubiquitination and subsequent protein degradation. The plant U-box (PUB) family of E3 ligases has been implicated in biotic and abiotic stress signaling and developmental events in various species. A comprehensive bioinformatics analysis identified 56 PUB genes in the grapevine (Vitis vinifera L.) genome. Based on conserved motifs, the VvPUB family was classified into seven subclasses. Expansion of this family was driven by chromosomal, segmental, and tandem duplications. Microarray expression profiling revealed that three PUB genes were preferentially expressed in pollen, four in leaf, and five in root. Moreover, a large number of PUB genes were differentially expressed under abiotic stresses and eight PUB genes likely participated in defense against powdery mildew. The microarray expression data were verified by RT-qPCR. Genome-wide identification of VvPUB genes and examination of their expression will give insights into the functions of U-box genes in grapevine.     

Expression analysis of genes encoding double B-box zinc finger proteins in maize

The B-box proteins play key roles in plant development. The double B-box (DBB) family is one of the subfamily of the B-box family, with two B-box domains and without a CCT domain. In this study, 12 maize double B-box genes (ZmDBBs) were identified through a genome-wide survey. Phylogenetic analysis of DBB proteins from maize, rice, Sorghum bicolor, Arabidopsis, and poplar classified them into five major clades. Gene duplication analysis indicated that segmental duplications made a large contribution to the expansion of ZmDBBs. Furthermore, a large number of cis-acting regulatory elements related to plant development, response to light and phytohormone were identified in the promoter regions of the ZmDBB genes. The expression patterns of the ZmDBB genes in various tissues and different developmental stages demonstrated that ZmDBBs might play essential roles in plant development, and some ZmDBB genes might have unique function in specific developmental stages. In addition, several ZmDBB genes showed diurnal expression pattern. The expression levels of some ZmDBB genes changed significantly under light/dark treatment conditions and phytohormone treatments, implying that they might participate in light signaling pathway and hormone signaling. Our results will provide new information to better understand the complexity of the DBB gene family in maize.     

Expression analysis of Dickkopf-related protein 3 (Dkk3) suggests its pleiotropic roles for a secretory glycoprotein in adult mouse

Dickkopf-related protein 3 (Dkk3) is the third member of the Dkk gene family and identical to the gene, whose expression was reduced in immortalized cells. Therefore, its another name is reduced expression in immortalized cells. Since the intratumoral introduction of Dkk3 inhibits tumor growth in mouse models of cancers, Dkk3 is likely a tumor suppressor gene. However, the functions of Dkk3 in vivo remain unclear. As the first step to decipher the physiological roles of this gene, we examined the expression pattern of Dkk3 in various tissues from adult mice. In situ hybridization showed that Dkk3 mRNA was detected in the brain, retina, heart, gastrointestinal tract, adrenal glands, thymus, prostate glands, seminal vesicles, testes, and ovaries in a regionally specific manner. Furthermore, we raised anti-mouse Dkk3 antibody and performed immunohistochemistry. Cytoplasmic localization of Dkk3 protein was observed in the cells of the adrenal medulla, while Dkk3 immunoreactivity was observed in the lumen of the stomach and intestine, implying that the Dkk3 protein may be secreted into the lumen of the gastrointestinal tract. These results suggest that Dkk3 has pleiotropic roles for a secretory glycoprotein that acts primarily in the gastrointestinal tract, thymus, endocrine and reproductive organs of the mouse.     

Comparative genomics analysis reveals gene family expansion and changes of expression patterns associated with natural adaptations of flowering time and secondary metabolism in yellow <Emphasis Type="Italic">Camellia</Emphasis>

Yellow-flowering species are unique in the genus Camellia not only for their bright yellow pigments but also the health-improving substances in petals. However, little is known regarding the biosynthesis pathways of pigments and secondary metabolites. Here, we performed comparative genomics studies in two yellow-flowered species of the genus Camellia with distinctive flowering periods. We obtained 112,190 and 89,609 unigenes from Camellia nitidissima and Camellia chuongtsoensis, respectively, and identified 9547 gene family clusters shared with various plant species and 3414 single-copy gene families. Global gene expression analysis revealed six comparisons of differentially expressed gene sets in different developmental stages of floral bud. Through the identification of orthologous pairs, conserved and specific differentially expressed genes (DEGs) between species were compared. Functional enrichment analysis suggested that the gibberellin (GA) biosynthesis pathway might be related to the alteration of flowering responses. Furthermore, the expression patterns of secondary metabolism pathway genes were analyzed between yellow- and red-flowered Camellias. We showed that the key enzymes involved in glycosylation of flavonoids displayed differential expression patterns, indicating that the direct glycosylation of flavonols rather than anthocyanins was pivotal to coloration and health-improving metabolites in the yellow Camellia petals. Finally, the gene family analysis of UDP-glycosyltransferases revealed an expansion of group C members in C. nitidissima. Through comparative genomics analysis, we demonstrate that changes of gene expression and gene family members are critical to the variation of natural traits. This work provides valuable insights into the molecular regulation of trait adaptations of floral pigmentation and flowering timing.