Diversity, structure, and expression of the gene for p26, a small heat shock protein from Artemia

p26, a small heat shock protein, is thought to protect Artemia embryos from stress during encystment and diapause. Full-length p26 cDNAs were compared and used to determine phylogenetic relationships between several Artemia species. The alpha-crystallin domain of p26 was the most conserved region of the protein and p26 from each Artemia species contained characteristic amino-terminal WD/EPF and carboxy-terminal VPI motifs. Sequence conservation suggested the importance of p26 to oviparously developing Artemia embryos and indicated common functions for the protein during development and stress resistance, although as shown by modeling some species-specific p26 amino acid substitutions may have adaptive significance. The p26 gene obtained from A. franciscana exhibited a unique sHSP intron arrangement with an intron in the 5'-untranslated region. Computer-assisted analysis revealed heat shock elements and other putative cis regulatory sequences but their role in gene regulation is unknown. In contrast to previous results for which Northern blots were analyzed, p26 gene expression was observed in ovoviviparous embryos by use of PCR-based methodology, but the p26 protein was not detected.     

The Potential Role of As-sumo-1 in the Embryonic Diapause Process and Early Embryo Development of Artemia sinica

During embryonic development of Artemia sinica, environmental stresses induce the embryo diapause phenomenon, required to resist apoptosis and regulate cell cycle activity. The small ubiquitin-related modifier-1 (SUMO), a reversible post-translational protein modifier, plays an important role in embryo development. SUMO regulates multiple cellular processes, including development and other biological processes. The molecular mechanism of diapause, diapause termination and the role of As-sumo-1 in this processes and in early embryo development of Artemia sinica still remains unknown. In this study, the complete cDNA sequences of the sumo-1 homolog, sumo ligase homolog, caspase-1 homolog and cyclin B homolog from Artemia sinica were cloned. The mRNA expression patterns of As-sumo-1, sumo ligase, caspase-1, cyclin B and the location of As-sumo-1 were investigated. SUMO-1, p53, Mdm2, Caspase-1, Cyclin B and Cyclin E proteins were analyzed during different developmental stages of the embryo of A. sinica. Small interfering RNA (siRNA) was used to verify the function of sumo-1 in A. sinica. The full-length cDNA of As-sumo-1 was 476 bp, encoding a 92 amino acid protein. The As-caspases-1 cDNA was 966 bp, encoding a 245 amino-acid protein. The As-sumo ligase cDNA was 1556 bp encoding, a 343 amino acid protein, and the cyclin B cDNA was 739 bp, encoding a 133 amino acid protein. The expressions of As-sumo-1, As-caspase-1 and As-cyclin B were highest at the 10 h stage of embryonic development, and As-sumo ligase showed its highest expression at 0 h. The expression of As-SUMO-1 showed no tissue or organ specificity. Western blotting showed high expression of As-SUMO-1, p53, Mdm2, Caspase-1, Cyclin B and Cyclin E at the 10 h stage. The siRNA caused abnormal development of the embryo, with increased malformation and mortality. As-SUMO-1 is a crucial regulation and modification protein resumption of embryonic diapause and early embryo development of A. sinica.     

Oligomerization, chaperone activity, and nuclear localization of p26, a small heat shock protein from Artemia franciscana

Artemia franciscana embryos undergo encystment, developmental arrest and diapause, the last characterized by profound metabolic dormancy and extreme stress resistance. Encysted embryos contain an abundant small heat shock protein termed p26, a molecular chaperone that undoubtedly has an important role in development. To understand better the role of p26 in Artemia embryos, the structural and functional characteristics of full-length and truncated p26 expressed in Escherichia coli and COS-1 cells were determined. p26 chaperone activity declined with increasing truncation of the protein, and those deletions with the greatest adverse effect on protection of citrate synthase during thermal stress had the most influence on oligomerization. When produced in either prokaryotic or eukaryotic cells the p26 alpha-crystallin domain consisting of amino acid residues 61-152 existed predominantly as monomers, and p26 variants lacking the amino-terminal domain but with intact carboxyl-terminal extensions were mainly monomers and dimers. The amino terminus was, therefore, required for efficient dimer formation. Assembly of higher order oligomers was enhanced by the carboxyl-terminal extension, although removing the 10 carboxyl-terminal residues had relatively little effect on oligomerization and chaperoning. Full-length and carboxyl-terminal truncated p26 resided in the cytoplasm of transfected COS-1 cells; however, variants missing the complete amino-terminal domain and existing predominantly as monomers/dimers entered the nuclei. A mechanism whereby oligomer disassembly assisted entry of p26 into nuclei was suggested, this of importance because p26 translocates into Artemia embryo nuclei during development and stress. However, when examined in Artemia, the p26 oligomer size was unchanged under conditions that allowed movement into nuclei, suggesting a process more complex than just oligomer dissociation.     

Inhibition of apoptosis by p26: implications for small heat shock protein function during Artemia development

p26, an abundantly expressed small heat shock protein, is thought to establish stress resistance in oviparously developing embryos of the crustacean Artemia franciscana by preventing irreversible protein denaturation, but it might also promote survival by inhibiting apoptosis. To test this possibility, stably transfected mammalian cells producing p26 were generated and their ability to resist apoptosis induction determined. Examination of immunofluorescently stained transfected 293H cells by confocal microscopy demonstrated p26 is diffusely distributed in the cytoplasm with a minor amount of the protein in nuclei. As shown by immunoprobing of Western blots, p26 constituted approximately 0.6% of soluble cell protein. p26 localization and quantity were unchanged during prolonged culture, and the protein had no apparent ill effects on transfected cells. Molecular sieve chromatography in Sepharose 6B revealed p26 oligomers of about 20 monomers, with a second fraction occurring as larger aggregates. A similar pattern was observed in sucrose gradients, but overall oligomer size was smaller. Mammalian cells containing p26 were more thermotolerant than cells transfected with the expression vector only, and as measured by annexin V labeling, Hoescht 33342 nuclear staining and procaspase-3 activation, transfected cells effectively resisted apoptosis induction by heat and staurosporine. The ability to confer thermotolerance and limit heat-induced apoptosis is important because Artemia embryos are frequently exposed to high temperature in their natural habitat. p26 also blocked apoptosis in transfected cells during drying and rehydration, findings with direct relevance to Artemia life history characteristics because desiccation terminates cyst diapause. Thus, in addition to functioning as a molecular chaperone, p26 inhibits apoptosis, an activity shared by other small heat shock proteins and with the potential to play an important role during Artemia embryo development.     

Cloning and expression patterns of the brine shrimp (Artemia sinica) glycogen phosphorylase (GPase) gene during development and in response to temperature stress

Glycogen serves as a metabolic reserve and is involved in macromolecular synthesis. Glycogen phosphorylase (GPase) is a key enzyme involved in intracellular glycogen catabolism, catalyzing the first step in glycogen degradation. In the diapause, GPase catalyzes glycogen into the closely related molecule, sorbitol. In this study, the full-length cDNA of the GPase gene (2,790 bp) was isolated from Artemia sinica for the first time by rapid amplification of cDNA ends technology. The GPase gene encoded a protein of 853 amino acids belonging to the Glycosyltransferase GTB type superfamily. The expression pattern and location of GPase were investigated at various stages during the embryonic development of A. sinica using real-time PCR and in situ hybridization. High GPase expression was detected at the 0 and 5 h stages. Subsequently, expression declined and was maintained at a low level during the stages from 10 to 40 h following by a small increase at day 3. Expression was downregulated at temperatures ranging from 25 to 20 °C and was subsequently upregulated in the range 15–5 °C. In situ hybridization assays showed wide distribution of the GPase gene during different developmental stages. From the results of this study, we conclude that the GPase gene expression is stress-related and might play an important role in Artemia development and metabolism.     

Identification of a novel DNA methyltransferase 2 from the brine shrimp, Artemia franciscana

DNA methyltransferase 2 (Dnmt2) is a dual-specificity DNA methyltransferase, which contains a weak DNA methyltransferase and novel tRNA methyltransferase activity. However, its biological function is still enigmatic. To elucidate the expression profiles of Dnmt2 in Artemia franciscana, we isolated the gene encoding a Dnmt2 from A. franciscana and named it as AfDnmt2. The cDNA of AfDnmt2 contained a 1140-bp open reading frame that encoded a putative Dnmt2 protein of 379 amino acids exhibiting 32% approximately 39% identities with other known Dnmt2 homologs. This is the first report of a DNA methyltransferase gene in Crustacean. By using semi-quantitative RT-PCR, AfDnmt2 was found to be expressed through all developmental stages and its expression increased during resumption of diapause cysts development. Southern blot analysis indicated the presence of multiple copies of AfDnmt2 genes in A. franciscana.     

Expression and roles of As-NUPR1 protein from Artemia sinica during embryo development and in response to salinity stress

As-NUPR1, a stress-related protein, plays an important role in post-diapause during embryonic development in the brine shrimp Artemia sinica. In the present study, successful expression of As-NUPR1 from the cDNA sequence isolated from A. sinica was demonstrated using a prokaryotic expression system. The recombinant protein consisted of 132 amino acids with a molecular weight of 15 kDa, and a predicted isoelectric point of 7.17. As-NUPR1 polyclonal antibodies were prepared by immunization of Balb/c mice with purified recombinant As-NUPR1 protein as an antigen, and immunological studies were carried out. Expression of As-NUPR1 during different developmental stages of the embryo and in response to salinity stress was analyzed in A. sinica using Western blots. The experimental results showed that the expression of As-NUPR1 is widely distributed at different developmental stages in A. sinica, and there was no tissue or organ specificity. Expression of As-NUPR1 decreased gradually during the diapause termination stage of embryo development, after which there was a general increase in expression after breaking the shell. In addition, As-NUPR1 expression was highly upregulated under conditions of high salinity. These results suggest that the As-NUPR1 protein is a stress-related protein that plays a role in protecting embryos from high salt damage in different embryonic developmental stages, especially during the post-diapause period.     

卤虫中编码DM结构域的DNA序列的克隆和初步研究（简报）

性别决定的分子机制复杂多样,但是处于动物性别决定的基因调控网络底部的一些调控基因具有相当高的保守性。doublesex(dsx)基因和male abnomal-3(mab-3)基因分别是果蝇(Drosophila melanogaster)和线虫(Caenorhabditis elegans)性别决定调控途径末端的重要基因,对这两个基因序列的比较导致了DM结构域的发现,它是已知在性别发育过程中最为保守的DNA结合结构域。目前,已     

Comparative studies on sorting cells from Artemia sinica at different developmental stages for in vitro cell culture

Cell growth in primary cell culture of the brine shrimp (Artemia sinica) embryo at 12 and 20 h after rehydration at 25°C was examined comparatively in modified Leibovitz-15 medium. The cells from A. sinica embryo at 12 h after rehydration were dispersed, and the cells disseminated but did not attach to the surface of wells and multiply at 2 d of culture, and 12 d later, the cells were degenerated and dead. The best growth of the brine shrimp cells was obtained from the prenauplii of A. sinica at 20 h after dormant embryo rehydration. The fibroblast-like cells attached to the well surface and multiplied at 15 d after the primary culture was set up. Confluent monolayer was formed at 50 d. The prenauplii cells have been subcultured up to passage 3 and maintained for approximately 200 d. The reasons for cell growth potential at the different developmental stages of Artemia embryo were discussed.     

The Small Heat Shock Protein p26 Aids Development of Encysting Artemia Embryos, Prevents Spontaneous Diapause Termination and Protects against Stress

Artemia franciscana embryos enter diapause as encysted gastrulae, a physiological state of metabolic dormancy and enhanced stress resistance. The objective of this study was to use RNAi to investigate the function of p26, an abundant, diapause-specific small heat shock protein, in the development and behavior of encysted Artemia embryos (cysts). RNAi methodology was developed where injection of Artemia females with dsRNA specifically eliminated p26 from cysts. p26 mRNA and protein knock down were, respectively, confirmed by RT-PCR and immuno-probing of western blots. ArHsp21 and ArHsp22, diapause-related small heat shock proteins in Artemia cysts sharing a conserved α-crystallin domain with p26, were unaffected by injection of females with dsRNA for p26, demonstrating the specificity of protein knock down. Elimination of p26 delayed cyst release from females demonstrating that this molecular chaperone influences the development of diapause-destined embryos. Although development was slowed the metabolic activities of cysts either containing or lacking p26 were similar. p26 inhibited diapause termination after prolonged incubation of cysts in sea water perhaps by a direct effect on termination or indirectly because p26 is necessary for the preservation of diapause maintenance. Cyst diapause was however, terminated by desiccation and freezing, a procedure leading to high mortality within cyst populations lacking p26 and indicating the protein is required for stress tolerance. Cysts lacking p26 were also less resistant to heat shock. This is the first in vivo study to show that knock down of a small heat shock protein slows the development of diapause-destined embryos, suggesting a role for p26 in the developmental process. The same small heat shock protein prevents spontaneous termination of diapause and provides stress protection to encysted embryos.     

Cloning and characterization of β-catenin gene in early embryonic developmental stage of Artemia sinica

β-Catenin plays a crucial role in embryonic development and responds to the activation of several signal transduction pathways. In this paper, in order to understand the functions of β-catenin gene in early embryonic development of Artemia sinica, the complete cDNA sequence was cloned for the first time using RACE technology, then the sequence was analyzed by some bioinformatic methods. The expression of the β-catenin gene was investigated at various stages during the embryonic development using quantitative real-time PCR and immunohistochemistry assay. Through the investigation, the result of real-time PCR illustrated that β-catenin gene might relate to the response of A. sinica’s immune system and osmotic pressure system in early embryonic developmental stage. Meanwhile, Immunohistochemistry assay demonstrated that during embryonic development, β-catenin was mainly expressed in the cephalothorax. Besides, we discovered that β-catenin might not be a maternal gene in A. sinica, and this new phenomenon may explain a constitutive and regional expression during the early embryonic development of A. sinica.     

Matrix metalloproteinase-3 induction in rat brain astrocytes: focus on the role of two AP-1 elements

Embryos of the crustacean, Artemia franciscana, undergo alternative developmental pathways, producing either larvae or encysted embryos (cysts). The cysts enter diapause, characterized by exceptionally high resistance to environmental stress, a condition thought to involve the sHSP (small heat-shock protein), p26. Subtractive hybridization has revealed another sHSP, termed ArHsp21, in diapause-destined Artemia embryos. ArHsp21 shares sequence similarity with p26 and sHSPs from other organisms, especially in the alpha-crystallin domain. ArHsp21 is the product of a single gene and its synthesis occurred exclusively in diapause-destined embryos. Specifically, ArHsp21 mRNA appeared 2 days post-fertilization, followed 1 day later by the protein, and then increased until embryo release at day 5. No ArHsp21 protein was detected in embryos developing directly into larvae, although there was a small amount of mRNA at 3 days post-fertilization. The protein was degraded during post-diapause development and had disappeared completely from second instar larvae. ArHsp21 formed large oligomers in encysted embryos and transformed bacteria. When purified from bacteria, ArHsp21 functioned as a molecular chaperone in vitro, preventing heat-induced aggregation of citrate synthase and reduction-driven denaturation of insulin. Sequence characteristics, synthesis patterns and functional properties demonstrate clearly that ArHsp21 is an sHSP able to chaperone other proteins and contribute to stress tolerance during diapause. As such, ArHsp21 would augment p26 chaperone activity and it may also possess novel activities that benefit Artemia embryos exposed to stress.     

Quantitative analysis of retromer complex-related genes during embryo development in the mouse

The retromer complex is a heteropentameric protein unit associated with retrograde transport of cargo proteins from endosomes to the trans-Golgi network. Functional silencing study of the Vps26a gene indicated the important role of the retromer complex during early developmental stages in the mouse. However, individual expression patterns and quantitative analysis of individual members of the retromer complex during the early developmental stages has not been investigated. In this study, we conducted quantitative expression analysis of six retromer complex genes (Vps26a, Vps26b, Vps29, Vps35, Snx1, and Snx2) and one related receptor gene (Ci-mpr) during the eleven embryonic stages with normal MEF (mouse embryonic fibroblast) and Vps26a(-/-) MEF cells. Remarkably, except for Vps26a (maternal expression pattern), all tested genes showed maternal-zygotic expression patterns. And five genes (Vps26b, Vps29, Vps35, Snx2, and Ci-mpr) showed a pattern of decreased expression in Vps26a(-/-) MEF cells by comparative analysis between normal MEF and Vps26a(-/-) MEF cells. However, the Snx1 gene showed a pattern of increased expression in Vps26a(-/-) MEF cells. From our results, we could assume that retromer complex-related genes have important roles during oocyte development. However, in the preimplantation stage, they did not have significant roles.