Drosophila Me31B is a Dual eIF4E-Interacting Protein

Eukaryotic translation initiation factor 4E (eIF4E) is a key factor involved in different aspects of mRNA metabolism. Drosophila melanogaster genome encodes eight eIF4E isoforms, and the canonical isoform eIF4E-1 is a ubiquitous protein that plays a key role in mRNA translation. eIF4E-3 is specifically expressed in testis and controls translation during spermatogenesis. In eukaryotic cells, translational control and mRNA decay is highly regulated in different cytoplasmic ribonucleoprotein foci, which include the processing bodies (PBs). In this study, we show that Drosophila eIF4E-1 and eIF4E-3 occur in PBs along the DEAD-box RNA helicase Me31B. We show that Me31B interacts with eIF4E-1 and eIF4E-3 by means of yeast two-hybrid system, FRET in D. melanogaster S2 cells and coimmunoprecipitation in testis. Truncation and point mutations of Me31B proteins show two eIF4E-binding sites located in different protein domains. Residues Y401-L407 (at the carboxy-terminus) are essential for interaction with eIF4E-1, whereas residues F63-L70 (at the amino-terminus) are critical for interaction with eIF4E-3. The residue W117 in eIF4E-1 and the homolog position F103 in eIF4E-3 are necessary for Me31B-eIF4E interaction suggesting that the change of tryptophan to phenylalanine provides specificity. Me31B represents a novel type of eIF4E-interacting protein with dual and specific interaction domains that might be recognized by different eIF4E isoforms in different tissues, adding complexity to the control of gene expression in eukaryotes.     

Barnase-barstar high affinity interaction phenomenon as the base for the heterogenous bioluminescence pseudorabies virus' immunoassay

The effective new variant of "sandwich" bioluminescent enzyme immunoassay (BEIA) for the sensitive detection of glycoprotein B (gB) of pseudorabies virus (PrV) was presently developed. The high affinity interaction of barnase-barstar protein pair and photoprotein obelin as bioluminescent marker were for the first time successfully applied to BEIA development. Preliminary the two monoclonal antibodies, 11/5 and 34/2, were raised against gB for ELISA PrV detection. Presently we used the same immuno-"sandwich" principle for BEIA. To do this the two different bioconjugates were elaborated. Recombinant barnase was chemically conjugated with monoclonal anti-PrV's gB IgG, and also barstar was fused in frame to obelin. The characteristics of BEIA method have been compared to ELISA PrV detection. We have shown the proposed here gB-BEIA was 40-fold more sensitive as opposed to gB-ELISA test. The construction might have a broad promise in multiple potential immunological applications.     

Normal development of the tomato clownfish Amphiprion frenatus: live imaging and in situ hybridization analyses of mesodermal and neurectodermal development

The normal embryonic development of the tomato clownfish Amphiprion frenatus was analysed using live imaging and by in situ hybridization for detection of mesodermal and neurectodermal development. Both morphology of live embryos and tissue‐specific staining revealed significant differences in the gross developmental programme of A. frenatus compared with better‐known teleost fish models, in particular, initiation of somitogenesis before complete epiboly, initiation of narrowing of the neurectoderm (neurulation) before somitogenesis, relatively early pigmentation of melanophores at the 10–15 somite stage and a distinctive pattern of melanophore distribution. These results suggest evolutionary adaptability of the teleost developmental programme. The ease of obtaining eggs, in vitro culture of the embryo, in situ staining analyses and these reported characteristics make A. frenatus a potentially important model marine fish species for studying embryonic development, physiology, ecology and evolution.     

Inferring developmental constraint and constraint release: primordial germ cell determination mechanisms as examples

Developmental constraint and its converse constraint release are significant concepts in understanding pattern and process in macroevolution. The purpose of this paper is to propose a two-step method for identifying constraints and constraint release. The first step is a phylogenetic optimization procedure to identify which trait/process is primitive and which is derived. The primitive trait is inferred to be the constraint and the convergently derived trait the release. The second criterion uses sister-clade asymmetry. Clades diagnosed by the constraint will have fewer taxa than clades diagnosed by the release. As an example, we use the process of germ cell specification, in which there are three modes of specification. Our results corroborate previous conclusions that the induced mode is the constraint and the predetermined mode is the release and we speculate on the importance of these two processes in terms of robustness and evolvability.     

Hermes RNA-binding protein targets RNAs-encoding proteins involved in meiotic maturation, early cleavage, and germline development

The early development of metazoans is mainly regulated by differential translation and localization of maternal mRNAs in the embryo. In general, these processes are orchestrated by RNA-binding proteins interacting with specific sequence motifs in the 3'-untranslated region (UTR) of their target RNAs. Hermes is an RNA-binding protein, which contains a single RNA recognition motif (RRM) and is found in various vertebrate species from fish to human. In Xenopus laevis, Hermes mRNA and protein are localized in the vegetal region of oocytes. A subpopulation of Hermes protein is concentrated in a specific structure in the vegetal cortex, called the germ plasm (believed to contain determinants of the germ cell fate) where Hermes protein co-localizes with Xcat2 and RINGO/Spy mRNAs. The level of total Hermes protein decreases during maturation. The precocious depletion of Hermes protein by injection of Hermes antisense morpholino oligonucleotide (HE-MO) accelerates the process of maturation and results in cleavage defects in vegetal blastomeres of the embryo. It is known that several maternal mRNAs including RINGO/Spy and Mos are regulated at the translational level during meiotic maturation and early cleavage in Xenopus. The ectopic expression of RINGO/Spy or Mos causes resumption of meiotic maturation and cleavage arrests, which resemble the loss of Hermes phenotypes. We found that the injection of HE-MO enhances the acceleration of maturation caused by the injection of RINGO/Spy mRNA, and that Hermes protein is present as mRNP complex containing RINGO/Spy, Mos, and Xcat2 mRNAs in vivo. We propose that as an RNA-binding protein, Hermes may be involved in maturation, cleavage events at the vegetal pole and germ cell development by negatively regulating the expression of RINGO/Spy, Mos, and Xcat2 mRNAs.     

Ginsenosides promote proliferation of chicken primordial germ cells via PKC-involved activation of NF-kappaB

The effect of ginsenosides on proliferation of chicken primordial germ cells (PGCs) was evaluated and involvement of nuclear factor (NF)-kappaB in the signaling pathway was investigated. PGCs were isolated from the genital ridge of 3.5-4 day embryos and cultured in Medium 199 supplemented with 5% FCS and 10 ng/ml LIF. PGCs subcultured on chicken embryonic fibroblast feeder were challenged with ginsenosides alone or in combination with PKC inhibitor H(7) or activator phorbol 12-myristate 13-acetate (PMA) for 24h. Moreover, the translocation of NF-kappaB and degradation level of IkappaBalpha were investigated by Western blot analysis. Results show that PGCs were identified by periodic acid-Schiff, alkaline phosphatase histochemistry as well as c-kit, SSEA-1 and Oct-4 immunocytochemistry. Treatment with ginsenosides at 1-100 microg/ml significantly increased the number and area of PGC colonies in a dose-dependent manner. However, this proliferating effect was obviously attenuated by combined treatment of H(7) (10(-7)-10(-5)M). Similarly, PKC staining of PGC colonies was more intensive after ginsenosides treatment compared with the control group. In addition, treatment with ginsenosides at 1-10 microg/ml stimulated the translocation of NF-kappaB (p65). However, the NF-kappaB translocation and the degradation of IkappaBalpha were significantly blocked by combined treatment with 10(-6)M H(7). These results indicated that ginsenosides promote proliferation of chicken PGCs through activation of PKC-involved NF-kappaB signaling pathway.     

The implications of mitochondrial DNA copy number regulation during embryogenesis

Mutations of mitochondrial DNA (mtDNA) cause a wide array of multisystem disorders, particularly affecting organs with high energy demands. Typically only a proportion of the total mtDNA content is mutated (heteroplasmy), and high percentage levels of mutant mtDNA are associated with a more severe clinical phenotype. MtDNA is inherited maternally and the heteroplasmy level in each one of the offspring is often very different to that found in the mother. The mitochondrial genetic bottleneck hypothesis was first proposed as the explanation for these observations over 20 years ago. Although the precise bottleneck mechanism is still hotly debated, the regulation of cellular mtDNA content is a key issue. Here we review current understanding of the factors regulating the amount of mtDNA within cells and discuss the relevance of these findings to our understanding of the inheritance of mtDNA heteroplasmy.