Synergistic amelioration between Ligusticum striatum DC and borneol against cerebral ischemia by promoting astrocytes-mediated neurogenesis.

ETHNOPHARMACOLOGICAL RELEVANCE: Ligusticum chuanxiong Hort (LCH), with the accepted name of Ligusticum striatum DC in "The Plant List" database, is a widely used ethnomedicine in treating ischemic stroke, and borneol (BO) is usually prescribed with LCH for better therapy. Our previous study confirmed their synergistic effect on neurogenesis against cerebral ischemia. However, the underlying mechanism is still unclear. AIM OF THE STUDY: More and more evidence indicated that astrocytes (ACs) might be involved in the modulation of neurogenesis via polarization reaction. The study was designed to explore the synergic mechanism between LCH and BO in promoting astrocyte-mediated neurogenesis. MATERIALS AND METHODS: After primary cultures and identifications of ACs and neural stem cells (NSCs), the oxygen-glucose deprivation (OGD) model and the concentrations of LCH and BO were optimized. After the OGD-injured ACs were treated by LCH, BO, and their combination, the conditioned mediums were used to culture the OGD-injured NSCs. The proliferation, migration, and differentiation of NSCs were assessed, and the secretions of BDNF, CNTF, and VEGF from ACs were measured. Then the expressions of C3 and PTX3 were detected. Moreover, the mice were performed a global cerebral ischemia/reperfusion model and treated with LCH and (or) BO. After the assessments of Nissl staining, the expressions of Nestin, DCX, GFAP, C3, PTX3, p65 and p-p65 were probed. RESULTS: The most appropriate duration of OGD for the injury of both NSCs and ACs was 6 h, and the optimized concentrations of LCH and BO were 1.30 µg/mL and 0.03 µg/mL, respectively. The moderate OGD environment induced NSCs proliferation, migration, astrogenesis, and neurogenesis, increased the secretions of CNTF and VEGF from ACs, and upregulated the expressions of C3 and PTX3. For the ACs, LCH further increased the secretions of BDNF and CNTF, enhanced PTX3 expression, and reduced C3 expression. Additionally, the conditioned medium from LCH-treated ACs further enhanced NSC proliferation, migration, and neurogenesis. The in vivo study showed that LCH markedly enhanced the Nissl score and neurogenesis, and decreased astrogenesis which was accompanied by downregulations of C3, p-p65, and p-p65/p65 and upregulation of PTX3. BO not only decreased the expression of C3 in ACs both in vitro and in vivo but also downregulated p-p65 and p-p65/p65 in vivo. Additionally, BO promoted the therapeutic effect of LCH for most indices. CONCLUSION: A certain degree of OGD might induce ACs to stimulate the proliferation, astrogenesis, and neurogenesis of NSCs. LCH and BO exhibited a marked synergy in promoting ACs-mediated neurogenesis and reducing astrogenesis, in which LCH played a dominant role and BO boosted the effect of LCH. The mechanism of LCH might be involved in switching the polarization of ACs from A1 to A2, while BO preferred to inhibit the formation of A1 phenotype via downregulating NF-κB pathway.

Nanog suppresses the expression of vasa by directly regulating nlk1 in the early zebrafish embryo.

Nanog is a homeodomain transcription factor that is essential for maintenance of pluripotency and self-renewal of embryonic stem cells (ESCs). In the present study, we demonstrate that zebrafish Nanog (zNanog) directly binds to the promoter region of zebrafish nlk1 (znlk1) by ChIP-Seq analysis and that it up-regulates the expression of znlk1 in fibroblast-like embryonic cells of Danio rerio (ZEM-2S cells) and in zebrafish embryos at 30% epiboly both at the mRNA and protein levels. In addition, compared with control (MO-C) embryos at 30% epiboly, the mRNA and protein expression of vasa and the numbers of vasa-positive cells were increased in embryos injected with zNanog morpholino (MO-zNanog). Further, injection of znlk1 mRNA into zNanog-depleted embryos restored the expression of vasa and the number of vasa-positive cells. These data indicated that zNanog up-regulates the expression of znlk1 through directly binding to the znlk1 promoter, thereby suppressing the expression of vasa. Vasa is a marker gene for PGCs. Our results suggest that zNanog plays a role in restraint of PGC cell number through regulating the expression of znlk1 in the early embryonic development. The current results provide fundamental information to support further investigation regarding the regulatory mechanism of zNanog during the development of PGCs.

Knockdown of zebrafish Nanog increases primordial germ cells during early embryonic development.

Nanog is a homeodomain transcription factor that plays a prominent role in maintaining the pluripotency and self-renewal capacity of embryonic stem cells (ESCs) in mammals. Medaka Nanog is necessary for S-phase transition and proliferation during embryonic development. However, whether Nanog regulates the proliferation of primordial germ cells (PGCs) during embryonic development has not yet been investigated. In this study, we identified the homologue of the mammalian Nanog gene in zebrafish (zNanog). The expression of both zNanog mRNA and protein was demonstrated in the spermatogonia (male germ stem cells) of the testis and the early oocytes of the ovary. During the embryonic development, zNanog mRNA is expressed in the cytoplasm of PGCs, and its protein is localized to the PGC nuclei. We also found that zNanog depletion using morpholinos resulted in the increases and aberrant localization of PGCs in the zebrafish embryos from the sphere stage to the 50% epiboly stage. These data indicated that zNanog inhibits the PGCs proliferation in early embryonic development of zebrafish.

Cloning and Characterization of ifitm1 and ifitm3 Expression During Early Zebrafish Development - CORRIGENDUM.

The authors apologise for errors in the corresponding authors details given on page 1 of the article. Below is the correct information of the corresponding author and email address : 1) Wei-Wei Xue, Huan-Nan Wang, Zhi-Meng Wang, Meng-Xi Qiu, Jing Che, Feng-Jiao Deng* and Jiang-Dong Liu* 2) *All correspondence to: Feng-Jiao Deng and Jiang-Dong Liu. e-mail: fish4@whu.edu.cn 3) All authors are from the same one laboratory. The second laboratory was superfluous and should be deleted.

Cloning and characterization of ifitm1 and ifitm3 expression during early zebrafish development.

The family of interferon-inducible transmembrane proteins (IFITMs) plays a crucial role in inhibiting proliferation, promoting homotypic cell adhesion and mediating germ cell development. In the present study, the full-length cDNAs of zebrafish ifitm1 (744 bp) and ifitm3 (702 bp) were obtained by rapid amplification of cDNA ends (RACE). Reverse transcription polymerase chain reaction (RT-PCR) analysis showed that ifitm1 mRNA was expressed in the ovary, testis, brain, muscle, liver and kidney, while ifitm3 mRNA was only detected in the ovary. Based on in situ hybridization, ifitm1 mRNA was found to be strongly expressed in the ooplasm from stage I to stage II and ifitm3 mRNA was also strongly expressed in the ooplasm from stage I to stage II, furthermore ifitm3 expression ultimately localized to the cortex region beneath the plasma membrane of stage IV oocytes. During development, ifitm1 expression was initially detected in the enveloping layer cells and deep layer cells of shield stage embryos. Then, throughout the segmentation phase (10.25-24 hours post-fertilization (hpf)), ifitm1 expression was mainly detected in the head, trunk and tail regions. Unlike ifitm1, ifitm3 expression was initially detected in sphere stage embryos and was then broadly expressed throughout the embryo from the 70% epiboly stage to 24 hpf. Interestingly, ifitm3 was also expressed in primordial germ cells (PGCs) from the bud stage to 24 hpf. This expression analysis indicates that zebrafish ifitm1 may play a critical role in early organogenesis and may perform immune or hematopoietic functions and ifitm3 might be necessary for PGC migration and the formation of female germ cells.

Compressed matrix thin film (CMTF)-assisted laser desorption ionization mass spectrometric analysis.

The inhomogeneous re-crystallization process of matrix materials is the major concerns associated with matrix assisted laser desorption/ionization (MALDI) analysis. We describe here the approach termed compressed matrix thin film (CMTF) in order to make a uniform matrix deposition. In this approach, solid matrix particles are compressed under 10 MPa of pressure by a compressor that is regularly used in infrared spectroscopic analysis. Then aqueous samples can be deposited on the surface of the matrix film. Major advantages of the CMTF approach are summarized as follows. (1) Reproducible sample preparation procedure. Size and thickness of matrix thin films can be controlled by using a fixed mold.force and known amount of matrix materials. (2) Significantly decreased shot-to-shot variations and enhanced reproducibility. (3) Tolerance for in situ salt washing. Because matrix materials are hydrophobic, salts can be washed away while proteins or peptides are retained on the surface of matrix thin films through hydrophobic interactions. (4) Improved sensitivity. The hydrophobic coating of MALDI sample plate by matrix thin films prevents the spreading of samples across the plate and confines analytes to a small area, leading to increased local concentration. (5) A new means for tissue analysis. Tissue sections can be directly transferred to the uniform surface of matrix materials for reproducible and quantitative comparison of different molecules in different localization. The proposed CMTF should be an enabling technique for mass spectrometric analysis with improved correlations between signal intensities and sample quantities.

Zebrafish Nanos interacts with and regulates the phosphorylation of Mylz2.

Nanos genes were required for differentiation of the anterior-posterior body axis in the Drosophila embryo. In addition to this somatic function, the nanos protein plays critical roles in the migration and survival of PGCs in vertebrates and invertebrates, but the regulating mechanisms of Nanos is largely undefined during the embyro development. In this study, we report a novel interaction between zebrafish Nanos and myosin light chain II (Mylz2). This stable complex, which is formed through the RNA-binding zinc finger domain and fourteen amino acids at the C-terminus of Nanos, was confirmed by GST pull-down and co-immunoprecipitation. Both of these two parts are required for the interaction with Mylz2, but ninety-one amio acids at N-terminus of Nanos are not required. Using a phospho-myosin light chain 2 (Ser19) antibody, we demonstrate that Nanos downregulates the phosphorylation of the Mylz2 by cotransfecting PRK-FLAG-Na and PRK-HA-My Mylz2 in 93T cell. We discuss the biological roles of the interaction between Nanos and Mylz2 and the Nanos regulating phosphorylation of the Mylz2 in PGCs.

Discovery, characterization and expression of a novel zebrafish gene, znfr, important for notochord formation.

Genes specifically expressed in the notochord may be crucial for proper notochord development. Using the digital differential display program offered by the National Center for Biotechnology Information, we identified a novel EST sequence from a zebrafish ovary library (No. XM_701450). The full-length cDNA of this transcript was cloned by performing 3' and 5'-RACE and was further confirmed by PCR and sequencing. The resulting 614 bp gene was found to encode a novel 94 amino acid protein that did not share significant homology with any other known protein. Characterization of the genomic sequence revealed that the gene spanned 4.9 kb and was composed of four exons and three introns. RT-PCR gene expression analysis revealed that our gene of interest was expressed in ovary, kidney, brain, mature oocytes and during the early stages of embryogenesis. During embryonic development, znfr mRNA was found to be expressed in the embryonic shield, chordamesoderm and the vacuolated notochord cells by in situ hybridization. Based on this information, we hypothesize that this novel gene is an important maternal factor required for zebrafish notochord formation during early embryonic development. We have thus named this gene znfr (zebrafish notochord formation related).

Znrg, a novel gene expressed mainly in the developing notochord of zebrafish.

The notochord, a defining characteristic of the chordate embryo is a critical midline structure required for axial skeletal formation in vertebrates, and acts as a signaling center throughout embryonic development. We utilized the digital differential display program of the National Center for Biotechnology Information, and identified a contig of expressed sequence tags (no. Dr. 83747) from the zebrafish ovary library in Genbank. Full-length cDNA of the identified gene was cloned by 5'- and 3'- RACE, and the resulting sequence was confirmed by polymerase chain reaction and sequencing. The cDNA clone contains 2,505 base pairs and encodes a novel protein of 707 amino acids that shares no significant homology with any known proteins. This gene was expressed in mature oocytes and at the one-cell stage, and persisted until the 5th day of development, as determined by RT-PCR. Transcripts were detected by whole-mount RNA in situ hybridization from the two-cell stage to 72 h of embryonic development. This gene was uniformly distributed from the cleavage stage up to the blastula stage. During early gastrulation, it was present in the dorsal region, and became restricted to the notochord and pectoral fin at 48 and 72 h of embryonic development. Based on its abundance in the notochord, we hypothesized that the novel gene may play an important role in notochord development in zebrafish; we named this gene, zebrafish notochord-related gene, or znrg.

Cloning and characterization of the SSB-1 and SSB-4 genes expressed in zebrafish gonads.

The protein of the gustavus (gus) gene has a typical SOCS box domain and repeats in the splA and RyR (SPRY) domains. GUS can interact with Vasa and is necessary for the specification of germ cells. We cloned two zebrafish genes, SSB-1 and SSB-4 (SPRY domain SOCS box proteins). Phylogenetic analysis shows that zebrafish SSB-1 and SSB-4 are clustered into clades of SSB-1-like and SSB-4-like genes from other species. RT-PCR analysis of tissues revealed that zebrafish SSB-1 and -4 are expressed in the ovary and testis. We investigated the spatial expression patterns of zebrafish SSB-1 and -4 in embryos from the two-cell stage to 72 h postfertilization (hpf) using whole-mount in situ hybridization. SSB-1 and -4 transcripts were present in all blastomeres during the early embryonic stages, but the genes differ in their expression pattern. SSB-4 mRNA was located in the region of the primordial germ cells in 24 and 72 hpf embryos, but SSB-1 mRNA was not detected at these stages. We hypothesize that SSB-4 plays a role in the early development of germ cells.

Cloning, characterization and expression of zvep, a novel vitelline envelope-specific gene in the zebrafish ovary.

The egg envelope is a specialized extracellular matrix that surrounds and protects the oocyte and plays significant roles in animal reproductive and developmental processes. Using the NCBI digital differential display program we identified an EST sequence (XM_001340234.1) acquired from zebrafish ovary cDNA libraries in GeneBank. The full-length cDNA of this transcript was obtained by 3'and 5' RACE and further confirmed by PCR and sequencing. The full-length cDNA of the novel gene is 2,720 bp and encodes a protein of 761 amino acids. RT-PCR and Western blot analysis showed its expression in ovary and brain but not in other tissues. In situ hybridization demonstrates that the mRNA is transcribed in ooplasm of stage I, II, and III oocytes. Interestingly, immunohistochemistry on zebrafish ovarian sections showed that protein expression in the vitelline envelope was located to two thin positive lines in the stage III oocytes. These ovarian expression patterns show that this is a new component of the vitelline envelope that is synthesized during early developing oocytes. This protein was named ZVEP (zebrafish vitelline envelope protein) and it did not have any homology with other known vitelline envelope genes. Thus, we found that zvep is a novel gene related to the vitelline envelope in zebrafish.

Germ cell-less expression in zebrafish embryos.

The gene of germ cell-less (gcl) has been shown to be important in early differentiation of germ cells in Drosophila. Although the gcl homologue genes have been identified in some organisms, there is little data on the expression pattern and functional analysis of the gcl gene in zebrafish. In this research, real-time quantitative reverse transcription-polymerase chain reaction showed that the level of gcl mRNA expression rapidly decreases from the 4-cell stage to the sphere stage at which it reaches a minimum, gradually increases from the 50%-epiboly stage, and then remains stable during the posterior stages. Results of in situ hybridization indicated that the transcripts of zebrafish gcl are evenly distributed in all blastomeres from the 2-cell stage to the blastula period, different from that of vasa, nonas1 and dead end mRNA, and condense into some clusters of cells located along the blastoderm margin from the gastrulation period. During subsequent development, the transcripts are segregated as subcellular clumps to a small number of cells that would migrate to the position of the gonad in the dorsal side. In the adult, gcl mRNA was widely expressed in developing germ cells of both ovary and testis. These data suggest that zebrafish gcl have potentially important roles in the formation of primordial germ cells.

[18S-ITS1 sequence of rRNA in bivalves and its application in phylogenetic analysis].

Bivalves constitute a dominant and diverse group of marine animals in China, most of them are of major commercial important species, and studies of their genetic diversity are necessary for the sustainable exploitation and conservation of these bioresources. The objective of the present work is to explore the feasibility of using the ribosomal RNA as a molecular marker for studying the interspecific and intraspecific genetic variations among bivalves. The 18S-ITS1 sequences of 11 individuals at differing taxonomic levels were determined. The sequence were found to exhibit a high degree of length polymorphism among different species, ranging from 558 bp in C.farreri to 784 bp in O. rivularis, mainly resulting from the variation of ITS1, and the percent divergence ranging from 10.7% to 61.7%. The NJ (neighbor-joining) tree inferred from 18S fragment agrees with the previous study based on morphologies and chemical analysis well. The sequence variation was found to vary among 4 individuals of P. martensi (0.6% approximately 1.9%), collected from 4 geographical sites, which involved substitutions,transversion as well as insertions and deletions. All these results show that ITS1 is highly divergent among different species of bivalves and could be used in classification and distinguishing closely related species, and also could be used for molecular systematic studies at relative species, subspecies and population levels.

[Genetic diversity analysis of mitochondrial D-loop region of Chinese sucker (Myxocyprinus asiaticus)].

Chinese sucker, Myxocyprinus asiaticus, is an endemic species of China and also the only representatives of family Catostomidae in Asia. The fish was naturally distributed in Yangtze River and Mingjiang River and now few could be captured because of pollution and overexploitation. The fish has been listed in the second class of preserved animal in China. Studying and assessing its population structure is an imperative and fundamental work for making effective protection strategies. We amplified and sequenced the D-loop region of mtDNA of 8 samples. The size of the D-loop region is about 958 bp. A total of 32 variation loci were detected and the mutation rate was 0.033. All the mutation came from nucleotide substitution except one nucleotide deletion. Most of the nucleotide variations were found between the 55-365 bp region. The individual mutation rate varied from 0-1.36%, which exhibited nucleotide polymorphism to some extent among 8 samples. Compared with RAPD and other PCR-based methods, the directily sequencing of mtDNA D-loop region revealed much more genetic diversity. Meanwhile, the D-loop region of Moxostoma robustum derived from GenBank was aligned with that of Chinese sucker through CLUSTAL software. By comparison, we found that the mutation rate (0.033) of D-loop of Chinese sucker is higher than that of Moxostoma robustum (0.016).