Subtractive phage display technology identifies zebrafish marcksb that is required for gastrulation

In the present study, we used a phage display technique to screen differentially expressed proteins from zebrafish post-gastrula embryos. With a subtractive screening approach, 6 types of single-chain Fv fragments (scFvs) were screened out from an scFv antibody phage display library by biopanning against zebrafish embryonic homogenate. Four scFv fragments (scFv1, scFv3, scFv4 and scFv6) showed significantly stronger binding to the tailbud embryos than to the 30%-epiboly embryos. A T7 phage display cDNA library was constructed from zebrafish tailbud embryos and used to identify the antigens potentially recognized by scFv1, which showed the highest frequency and strongest binding against the tailbud embryos. We acquired 4 candidate epitopes using scFv1 and the corresponding genes showed significantly higher expression levels at tailbud stage than at 30%-epiboly. The most potent epitope of scFv1 was the clone scFv1-2, which showed strong homology to zebrafish myristoylated alanine-rich C-kinase substrate b (Marcksb). Western blot analysis confirmed the high expression of marcksb in the post-gastrula embryos, and the endogenous expression of Marcksb was interfered by injection of scFv1. Zebrafish marcksb showed dynamic expression patterns during embryonic development. Knockdown of marcksb strongly affected gastrulation movements. Moreover, we revealed that zebrafish marcksb is required for cell membrane protrusion and F-actin alignment. Thus, our study uncovered 4 types of scFvs binding to zebrafish post-gastrula embryos, and the epitope of scFv1 was found to be required for normal gastrulation of zebrafish. To our knowledge, this was the first attempt to combine phage display technique with the embryonic and developmental study of vertebrates, and we were able to identify zebrafish marcksb that was required for gastrulation.     

Expression of Microtubule-Associated Proteins During the Early Stages of Neurite Extension by Brain Neurons Cultured in a Defined Medium

Immunoblotting analysis was used to identify the microtubule-associated proteins (MAPs) present in cultures of mouse brain neurons. Polyclonal antibodies were raised against the two main adult brain MAPs, i.e., MAP2 (300 kDa) and tau (60-70 kDa). Whatever the stage of the culture, which was performed in a defined medium (3 or 6 days), the anti-MAP2 serum detected several high-molecular-weight components (including MAP2) and an entity with 62-65 kDa. Anti-tau revealed essentially a major peak of 48 kDa (young tau) but also slightly cross-reacted with the 62-65 kDa entity. During the culture period (0-6 days) the cells developed progressively a dense neuritic network; the concentration of the different MAPs increased in parallel but at different rates depending on the different species. The increase in concentration of the high-molecular-weight components occurred before that of 48-kDa tau. This suggests that high-molecular-weight MAPs and 48-kDa tau might be involved respectively in the initiation and elongation of neurites. In contrast, and since the main developmental changes in tau composition seen in vivo did not occur during the time course of the culture, this transition might be related to later events of neuronal differentiation.     

The History and Advances of Reversible Terminators Used in New Generations of Sequencing Technology

DNA sequencing using reversible terminators, as one sequencing by synthesis strategy, has garnered a great deal of interest due to its popular application in the second-generation high-throughput DNA sequencing technology. In this review, we provided its history of development, classification, and working mechanism of this technology. We also outlined the screening strategies for DNA polymerases to accommodate the reversible terminators as substrates during polymerization; particularly, we introduced the "REAP" method developed by us. At the end of this review, we discussed current limitations of this approach and provided potential solutions to extend its application.     

海南岛东寨港几种红树植物种间生态位研究

采用3种常见的生态位宽度和生态位重叠计测公式,以外来种无瓣海桑扩散区的秋茄+桐花树群落演替系列作为资源轴,定量计测了几种红树植物的生态位宽度和重叠值.结果表明,各树种生态位宽度值排序为桐花树(3.8357)＞秋茄(3.3421)＞木榄(3.3180)＞白骨壤(3.0975)＞无瓣海桑(2.9137)＞海桑(2.5724)＞角果木(1.8523)＞红海榄(1.6897)＞海莲(1.0000),很好地表征了其生态适应性和分布幅度.各树种重叠值中,以秋茄、桐花树、木榄、白骨壤之间的生态位重叠较大,表明其间存在较强的资源利用性竞争.无瓣海桑生态位宽度处于中等程度,与中低潮滩红树植物海桑、桐花树、秋茄和白骨壤的重叠值相对较高,与红海榄、木榄有中度重叠,与角果木有少量重叠,与海莲完全没有重叠.     

α1β1-Integrin is an essential signal for neurite outgrowth induced by thrombospondin type 1 repeats of SCO-spondin

In the central and peripheral nervous systems a heterogeneous group of proteins constituting the thrombospondin superfamily provides a cue for axonal pathfinding. They either contain or are devoid of the tripeptide RGD, and the sequence(s) and mechanism(s) which trigger in vitro their neurite-promoting activity have remained unclear. In this study, we reconsider the problem of whether sequences present in the thrombospondin type 1 repeats (TSRs), and independent of the well-known RGD-binding site, may activate integrins and account for their neurite-promoting activity. SCO-spondin is a newly identified member of the thrombospondin superfamily, which shows a multidomain organization with a great number of TSR motifs but no RGD sequence. Previous research has implicated oligopeptides derived from SCO-spondin TSRs in in-vitro development of various neuronal cell types. In this study, we investigate whether function-blocking antibodies directed against integrin subunits can block these effects in cell line B104, cloned from a neuroblastoma of the rat central nervous system. By two different approaches: flow cytometry revealing short-term effects and cell cultures revealing long-term effects, we show that: (a) activation of cell metabolism, (b) changes in cell size and structure, and (c) neurite-promoting activity induced by TSR oligopeptides are inhibited by function-blocking antibodies to 1-subunit. Using a panel of function-blocking antibodies directed against various integrin -subunits we show that the 1-subunit might be the partner of the 1-subunit in B104 cells. Thus, we demonstrate that an original sequence within a TSR motif from SCO-spondin promotes neurite outgrowth through an intracellular signal driven by integrins, independently of an RGD-binding site.     

Identification of an amino acid sequence motif in the cytoplasmic domain of the NCAM-140 kDa isoform essential for its neuritogenic activity

The functions of the extracellular domains of neural cell adhesion molecule (NCAM) have been studied extensively, whereas the roles of the cytoplasmic domains of the transmembrane forms of NCAM are less elucidated. We investigated the importance of the cytoplasmic domain of the 140-kDa NCAM isoform (cytNCAM-140) and of the 180-kDa NCAM isoform (cytNCAM-180) in NCAM-induced neurite extension by estimating NCAM-dependent neurite outgrowth from PC12-E2 cells grown in coculture with NCAM-negative or NCAM-positive fibroblasts. PC12-E2 cells were transiently transfected with expression plasmids encoding cytNCAM-140, cytNCAM-180, the constitutively active form of the mitogen-activated protein (MAP) kinase/extracellular signal-regulated kinase kinase (MEK2), and the enhanced variant of the green fluorescent protein (EGFP). EGFP expression was used for identification of transfected cells. We found that expression of cytNCAM-180 had no effect on NCAM-stimulated neuritogenesis, whereas expression of cytNCAM-140 strongly inhibited this process. However, if MEK2 was expressed concomitantly with cytNCAM-140, neurite outgrowth was rescued, indicating that cytNCAM-140 is involved in signaling via the Ras-MAP kinase pathway. PC12-E2 cells were subsequently transiently transfected with constructs encoding a series of fragments of cytNCAM-140 and various full-length cytNCAM-140 mutants, and the residues Thr-Glu-Val-Lys-Thr (839-843) were identified as essential in NCAM-stimulated neuritogenesis. The combined substitution of Glu(840) and Lys(842) with Ala abrogated the effect of the construct, assigning a critical role to these two residues.     

Optimisation of overlap extension PCR-based mutagenesis of a GC-rich DNA template: application to the human α2C-adrenoceptor cDNA

PCR-based overlap extension mutagenesis was applied to introduce a Thr³⁸¹ to Lys mutation in the _2C-adrenoceptor (_2C AR) coding sequence. This cDNA contains 71% G+C nucleotides and conventional PCR procedures were inefficient in generating a corresponding amplification product. Only the combined use of a pre-PCR denaturation step at 100 °C followed by quick chilling on ice and the addition of 1 M of a commercial GC Melt reagent and 5% (v/v) dimethylsulphoxide with the Advantage GC cDNA PCR kit yielded efficient amplification during the three successive PCR steps of the overlap extension procedure. Transient expression of the mutant Thr³⁸¹Lys _2C AR in Cos-7 cells was performed to determine the binding profile for a series of ₂ AR ligands using [³H]RX 821002.     

Genetic evidence for posterior specification by convergent extension in the Xenopus embryo

Genetic studies substantiate that mesodermal convergent extension expressed behind the anteroposterior borderline, in the form of a gradient with the posterior apex after gastrulation, regulates morphogenesis of the posterior zone at the dorsal and dorso-lateral levels which is in full agreement with the model of dorsalization–caudalization. In contrast, how anteroposterior specification of mesodermal tissues occurs at the ventral and latero-ventral levels is not yet understood.