mosaic-zebrafish-transgenesis-for-functional-genomic-analysis

Mosaic Zebrafish Transgenesis for Functional Genomic Analysis of Candidate Cooperative Genes in Tumor Pathogenesis

Comprehensive genomic analysis has uncovered surprisingly large numbers of genetic alterations in various types of cancers.  To robustly and efficiently ...

inducing-hairy-roots-agrobacterium-rhizogenes-mediated-transformation

Inducing Hairy Roots by <em>Agrobacterium rhizogenes</em>-Mediated Transformation in Tartary Buckwheat (<em>Fagopyrum tataricum</em>)

Tartary buckwheat (TB) [Fagopyrum tataricum (L.) Gaertn] possesses various biological and pharmacological activities because it contains abundant ...

Mayo Clinic

Huaibei Normal University

Rice (Oryza sativa L.) roots were fed with L-ascorbic acid (AsA) and its putative precursors to observe AsA and oxalate concentrations and the resistance of rice to chilling, water stress, and Al toxicity. AsA concentration was significantly enhanced in both shoots and roots of rice seedlings by feeding with D-glucose or L-galactono-gamma-lactone. AsA or L-galactono-gamma-lactone treatment increased accumulation of oxalate mainly in soluble form, while these treatments decreased electrolyte leakage from root cells, H2O2 and lipid peroxidation level in rice seedlings subjected to chilling, water stress, and Al toxicity. They also alleviated the inhibition on root growth by Al. These results indicated that AsA and its immediate precursor protected plants against the oxidative damages induced by various stresses. However, 0.5 mM AsA and 10 mM L-galactono-gamma-lactone treatment had no significant effect on superoxide dismutase and catalase activity and ascorbate-peroxidase activities. Enhanced Al resistance caused by AsA and L-galactono-gamma-lactone may possibly be resulted from increased level of oxalate, which acts as metal chelator. Thus it is proposed that manipulation of AsA and oxalate biosynthesis through enhancement of L-galactono-gamma-lactone level in plants could be a strategy for improving abiotic stress tolerance.

Effect of intermediates on ascorbic acid and oxalate biosynthesis of rice and in relation to its stress resistance.

GM1 ganglioside was reported to mediate the amyloid beta-protein (Abeta) secretion and accumulation in the pathogenesis of Alzheimer's disease (AD). The objective of this project was to comprehend the underlying molecular changes related to amyloid beta-protein precursor (APP) processing pathway induced by GM1. Using suppression subtractive hybridisation (SSH), we detected one prominent sequence with increased expression in human neuroblastoma cells that stably transfected with human APP695 cDNA treated with GM1. This transcript has high identity to human Ubiquilin 1 gene. Differential expression was initially confirmed by dot blot hybridization. This result was further authenticated with quantitative real-time polymerase chain reaction (RT-PCR) analysis. Furthermore, using Western blots, we discovered that GM1 stimulated the expression of Ubiquilin 1 in human neuroblastoma cells and rat cortical neurons while other gangliosides Asialo-GM1 and GD1b did not. Ubiquilin 1 is one of the candidate genes of AD, which have been shown to modulate the gamma-secretase components in the proteolytic processing of APP, and is therefore a putative candidate for further investigation of GM1 mechanisms in the etiology and pathology of AD.

GM1 up-regulates Ubiquilin 1 expression in human neuroblastoma cells and rat cortical neurons.

Cyclin-dependent protein kinases (CDKs) are required for various cell cycle events both in mitosis and in meiosis. During the meiotic prophase of Saccharomyces cerevisiae, only one CDK, Cdc28, which forms a complex with B-type cyclins, Clb5 or Clb6, promotes not only the onset of premeiotic DNA replication but also the formation of meiotic double-strand breaks (DSBs). In this study, we showed that Cdc28 exhibits punctate staining on chromosomes during meiotic prophase I. Chromosomal localization of Cdc28, dependent on Clb5 and/or Clb6, is frequently observed in zygotene and pachytene, when formation of the synaptonemal complex (SC) occurs. Interestingly, the CDK localization is independent of DSB formation, but rather dependent on meiosis-specific chromosome components such as Red1, Hop1 and a cohesin subunit Rec8. Compromised CDK activity in meiotic prophase leads to defective SC formation without affecting DSB formation. These results suggest that CDK-dependent phosphorylation regulates meiotic chromosome morphogenesis.

Cyclin-dependent kinase promotes formation of the synaptonemal complex in yeast meiosis.

&#x89C2;&#x5BDF;&#x59DC;&#x9EC4;&#x7D20;&#x5BF9; Abeta42 &#x548C;&#x6709;&#x8FB1;&#x5176;&#x4EBA;&#x683C;&#x7684;&#x8868;&#x8FBE;&#x7684;&#x9176;&#x975E;&#x7B26;&#x5408;&#x8D44;&#x683C;&#x4EBA;&#x58EB;&#x5728; APP/PS1 &#x53CC;&#x8F6C;&#x57FA;&#x56E0;&#x5C0F;&#x9F20;&#x3002;

[&#x59DC;&#x9EC4;&#x7D20;&#x5BF9; APPswe/PS1dE9 &#x53CC;&#x8F6C;&#x57FA;&#x56E0;&#x5C0F;&#x9F20;&#x7684; Abeta42 &#x548C; Abeta &#x964D;&#x89E3;&#x9176; NEP &#x8868;&#x8FBE;]&#x3002;

To observe the effect of curcumin on the expression of Abeta42 and its degrading enzyme NEP in APP/PS1 double transgenic mice.

[Effect of curcumin on expression of Abeta42 and Abeta-degrading enzyme NEP in APPswe/PS1dE9 double transgenic mice].

Pour observer l&#39;effet de la curcumine sur l&#39;expression de Abeta42 et sa d&eacute;gradation enzymatique NEP dans APP/PS1 double transg&eacute;niques.

[Effet de la curcumine sur l&#39;expression de Abeta42 et d&eacute;gradant Abeta enzyme NEP chez des souris transg&eacute;niques doubles APPswe/PS1dE9].

Um die Wirkung von Curcumin auf den Ausdruck der Abeta42 und seine erniedrigende beobachten Enzym NEP in APP/PS1 double transgene M&auml;usen.

[Wirkung von Curcumin auf Ausdruck der Abeta42 und Abeta-erniedrigende Enzym-NEP in APPswe/PS1dE9 doppelt transgene M&auml;use].

Per osservare l&#39;effetto della curcumina sull&#39;espressione di Abeta42 e sua degradazione enzimatica NEP in APP/PS1 doppio topi transgenici.

[Effetto della curcumina sull&#39;espressione di Abeta42 e degradante Abeta enzima NEP in topi transgenici doppi APPswe/PS1dE9].

Abeta42 &#x3068;&#x305D;&#x306E;&#x5206;&#x89E3;&#x306E;&#x5F0F;&#x306B;&#x5BFE;&#x3059;&#x308B;&#x30AF;&#x30EB;&#x30AF;&#x30DF;&#x30F3;&#x306E;&#x5F71;&#x97FF;&#x3092;&#x89B3;&#x5BDF;&#x3059;&#x308B;&#x306B;&#x306F;&#x3001;&#x9175;&#x7D20;&#x30CD;&#x30C3;&#x30D7; &#x30A2;&#x30D7;&#x30EA;/PS1 &#x306E;&#x30C8;&#x30E9;&#x30F3;&#x30B9;&#x30B8;&#x30A7;&#x30CB;&#x30C3;&#x30AF; &#x30DE;&#x30A6;&#x30B9;&#x3092;&#x30C0;&#x30D6;&#x30EB;&#x3057;&#x307E;&#x3059;&#x3002;

[APPswe/PS1dE9 &#x30C0;&#x30D6;&#x30EB; &#x30C8;&#x30E9;&#x30F3;&#x30B9;&#x30B8;&#x30A7;&#x30CB;&#x30C3;&#x30AF; &#x30DE;&#x30A6;&#x30B9;&#x306B;&#x304A;&#x3051;&#x308B; Abeta42 &#x3068;&#x306A;&#x5206;&#x89E3;&#x9175;&#x7D20;&#x30CD;&#x30C3;&#x30D7;&#x306E;&#x767A;&#x73FE;&#x306B;&#x5BFE;&#x3059;&#x308B;&#x30AF;&#x30EB;&#x30AF;&#x30DF;&#x30F3;&#x306E;&#x5F71;&#x97FF;]&#x3002;

Abeta42&#xC640; &#xADF8; &#xD0C0;&#xB77D;&#xC758; &#xD45C;&#xD604;&#xC5D0; curcumin&#xC758; &#xD6A8;&#xACFC; &#xAD00;&#xCC30; &#xD558;&#xAE30; &#xC704;&#xD574; &#xC560;&#xD50C; &#xB9AC; &#xCF00;&#xC774; &#xC158;/ps1&#xC5D0; NEP &#xD6A8;&#xC18C; &#xC720;&#xC804;&#xC790; &#xBCC0;&#xD615; &#xC950;&#xB97C; &#xB450; &#xBC88;.

[&#xD6A8;&#xACFC; APPswe/PS1dE9 &#xC774;&#xC911; &#xC720;&#xC804;&#xC790; &#xBCC0;&#xD615; &#xC950;&#xC5D0; Abeta42 &#xBC0F; Abeta &#xC800;&#xD558; &#xD6A8;&#xC18C; NEP&#xC758; &#xD45C;&#xD604;&#xC5D0; curcumin].

Para observar el efecto de la curcumina en la expresi&oacute;n de Abeta42 y su degradaci&oacute;n enzim&aacute;tica NEP en APP/PS1 doble ratones transg&eacute;nicos.

[Efecto de la curcumina en la expresi&oacute;n de la NEP de enzima Abeta42 y degradadoras de Abeta en ratones transg&eacute;nicos dobles APPswe/PS1dE9].

liver-enriched gene 1 (leg1) is a liver-enriched gene in zebrafish and encodes a novel protein. Our preliminary data suggested that Leg1 is probably involved in early liver development. However, no detailed characterization of Leg1 has been reported thus far. We undertook both bioinformatic and experimental approaches to study leg1 gene structure and its role in early liver development. We found that Leg1 identifies a new conserved protein superfamily featured by the presence of domain of unknown function 781 (DUF781). There are two copies of leg1 in zebrafish, namely leg1a and leg1b. Both leg1a and leg1b are expressed in the larvae and adult liver with leg1a being the predominant form. Knockdown of Leg1a or Leg1b by their respective morpholinos specifically targeting their 5'-UTR each resulted in a small liver phenotype, demonstrating that both Leg1a and Leg1b are important for early liver development. Meanwhile, we found that injection of leg1-ATG(MO), a morpholino which can simultaneously block the translation of Leg1a and Leg1b, caused not only a small liver phenotype but hypoplastic exocrine pancreas and intestinal tube as well. Further examination of leg1-ATG(MO) morphants with early endoderm markers and early hepatic markers revealed that although depletion of total Leg1 does not alter the hepatic and pancreatic fate of the endoderm cells, it leads to cell cycle arrest that results in growth retardation of liver, exocrine pancreas and intestine. Finally, we proved that Leg1 is a secretory protein. This intrigued us to propose that Leg1 might act as a novel secreted regulator that is essential for liver and other digestive organ development in zebrafish.

liver-enriched gene 1a and 1b encode novel secretory proteins essential for normal liver development in zebrafish.

We previously reported that αA-crystallin and protease-activated receptor are involved in protection of astrocytes against C2-ceramide- and staurosporine-induced cell death (Li et al., 2009). Here, we investigated the molecular mechanism of αA-crystallin-mediated cytoprotection. We found that the expression of mutants mimicking specific phosphorylation of αA-crystallin increases the protection of astrocytes. However, the expression of mutants mimicking unphosphorylation of αA-crystallin results in loss of protection. These data revealed that the phosphorylation of αA-crystallin at Ser122 and Ser148 is required for protection. Furthermore, we explored the mechanism of cytoprotection of astrocytes by αA-crystallin. Application of specific inhibitors of p38 and ERK abrogates the protection of astrocytes by over-expression of αA-crystallin. Thus, p38 and ERK contribute to protective processes by αA-crystallin. This is comparable to our previous results which demonstrated that p38 and ERK regulated protease-activated receptor-2 (PAR-2)/αB-crystallin-mediated cytoprotection. Furthermore, we found that PAR-2 activation increases the expression of αA-crystallin. Thus, endogenous αA-crystallin protects astrocytes via mechanisms, which regulate the expression and/or phosphorylation status of αA-crystallin.

Specific phosphorylation of αA-crystallin is required for the αA-crystallin-induced protection of astrocytes against staurosporine and C2-ceramide toxicity.

Ultraviolet (UV) radiation can cause oxidative stress in insects. To gain insight into the roles of different antioxidant enzymes of Helicoverpa armigera adults in response to oxidative stress caused by UV-A at a molecular level, three antioxidant enzyme genes [encoding copper zinc superoxide dismutase (Cu/ZnSOD), catalase (CAT) and glutathione peroxidase (GPX)] were cloned. The deduced amino acid sequences of these genes are similar to that of other insects. In addition, the expression profiles of genes from a classic antioxidant defense system, comprising Cu/ZnSOD, manganese superoxide dismutase (MnSOD), CAT, thioredoxin peroxidase, GPX and glutathione-S-transferase, were also determined. The results indicated that all antioxidant enzyme genes (except MnSOD in females) were significantly upregulated at certain time points (from 30 to 90min) in both male and female adults of H. armigera following UV-A radiation. We also found that longer periods of radiation exposure did not yield higher levels of mRNA expression. Furthermore, changes of determined physiological parameters (glutathione, Cu/ZnSOD and MnSOD activity) were basically consistent with the gene expression profiles. We therefore infer that the different antioxidant enzymes of H. armigera act in a coordinated manner at the transcriptional level against oxidative stress caused by UV-A radiation.

The molecular characterization of antioxidant enzyme genes in Helicoverpa armigera adults and their involvement in response to ultraviolet-A stress.

Protease-activated receptors (PARs) are seven transmembrane-domains G-protein-coupled receptors with four members: PAR-1, PAR-2, PAR-3, and PAR-4. The scaffold proteins β-arrestin 1 and β-arrestin 2 have been shown mediating responses to various receptor agonists, including PAR-1 and PAR-2. The aim of this study was to investigate whether the signaling adaptor β-arrestin 1 is (i) associated with apoptosis of astrocytes and (ii) involved in thrombin-induced PAR-1-mediated cytoprotection. Here, we found firstly that staurosporine-induced apoptosis, detected as cleavage of caspase 3 is more than 3-times higher in β-arrestin 1-lacking astrocytes than in control cells. This indicates that β-arrestin 1 is important to protect astrocytes from apoptosis. Secondly, PAR-1 activation by thrombin protects non-silenced and β-arrestin 1-deficient astrocytes from staurosporine-induced chemical toxicity. Furthermore, application of thrombin rescues β-arrestin 1-lacking astrocytes from apoptosis by enhanced Akt (Ser 473) phosphorylation. Rescue from cell death was measured by quantification of the cleavage of caspase 3. Thus, we conclude that the thrombin-activated PI3K/Akt signaling cascades play pivotal roles in survival of β-arrestin 1-deficient astrocytes. Our most striking novel finding is that β-arrestin 1 inhibits long-term thrombin-stimulated phosphorylation of Akt (Ser 473). This has been demonstrated by enhanced Akt (Ser 473) phosphorylation in astrocytes with knockdown of β-arrestin 1. Blockade of the PI3K/Akt signaling pathway by LY294002 abrogates the protection caused by thrombin treatment. In addition, we also found that thrombin-induced phosphorylation of Akt (Ser 473) is increased by transactivation of the EGF and PDGF receptors in β-arrestin 1-silenced astrocytes.

PAR-1 activation rescues astrocytes through the PI3K/Akt signaling pathway from chemically induced apoptosis that is exacerbated by gene silencing of β-arrestin 1.

The metazoan liver exhibits a remarkable capacity to regenerate lost liver mass without leaving a scar following partial hepatectomy (PH). Whilst previous studies have identified components of several different signaling pathways that are essential for activation of hepatocyte proliferation during liver regeneration, the mechanisms that enable such regeneration to occur without accompanying scar formation remain poorly understood. Here we use the adult zebrafish liver, which can regenerate within two weeks following PH, as a new genetic model to address this important question. We focus on the role of Digestive-organ-expansion-factor (Def), a nucleolar protein which has recently been shown to complex with calpain3 (Capn3) to mediate p53 degradation specifically in the nucleolus, in liver regeneration. Firstly, we show that Def expression is up-regulated in the wild-type liver following amputation, and that the defhi429/+ heteroozygous mutant (def+/-) suffers from haploinsufficiency of Def in the liver. We then show that the expression of pro-inflammatory cytokines is up-regulated in the def+/- liver, which leads to distortion of the migration and the clearance of leukocytes after PH. Transforming growth factor β (TGFβ) signalling is thus activated in the wound epidermis in def+/- due to a prolonged inflammatory response, which leads to fibrosis at the amputation site. Fibrotic scar formation in def+/- is blocked by the over-expression of Def, by the loss-of-function of p53, and by treatment with anti-inflammation drug dexamethasone or TGFβ-signalling inhibitor SB431542. We finally show that the Def- p53 pathway suppresses fibrotic scar formation, at least in part, through the regulation of the expression of the pro-inflammatory factor, high-mobility group box 1. We conclude that the novel Def- p53 nucleolar pathway functions specifically to prevent a scar formation at the amputation site in a normal amputated liver.

Haploinsufficiency of Def activates p53-dependent TGFβ signalling and causes scar formation after partial hepatectomy.

Thrombin was found to stimulate astrocytes proliferation. In this study, we want to clarify whether thrombin-activated protease-activated receptor will affect the glucose metabolism signaling pathways to accelerate the proliferation of astrocytes. In addition, we study if thrombin has effects on cell cycle transition to promote astrocytes proliferation. We firstly observed that thrombin activated protease-activated receptor 1 (PAR-1) inducing the increases of intracellular Ca(2+) and ROS production, which contribute to the astrocytes' proliferation. We further confirmed that ROS stabilized HIF-1α, the latter subsequently accelerated glucose uptake in astrocytes. On the other hand, we demonstrated that thrombin triggered PI3K/Akt/cyclin D1 signal transduction, which may promote the cell cycle transition to enhance astrocytes proliferation. As a result, we discovered three signaling pathways mainly accounting for cell proliferation induced by thrombin: (1) thrombin-stimulated ERK, JNK/ROS/HIF-1α and (2) PI3K/Akt/ROS/HIF-1α pathways to increase expression of hexokinase 2 which mediated glucose metabolism in astrocytes, and (3) thrombin stimulates PAR-1/PI3K/Akt/cyclin D1 to promote the cell cycle transition and finally to increase cell proliferation.

Signaling mechanism of protease activated receptor 1-induced proliferation of astrocytes: stabilization of hypoxia inducible factor-1α triggers glucose metabolism and accumulation of cyclin D1.

The protease-activated receptors are a group of unique G protein-coupled receptors, including PAR-1, PAR-2, PAR-3 and PAR-4. PAR-2 is activated by multiple trypsin-like serine proteases, including trypsin, tryptase and coagulation proteases. The clusters of phosphorylation sites in the PAR-2 carboxyl tail are suggested to be important for the binding of adaptor proteins to initiate intracellular signaling to Ca(2+) and mitogen-activated protein kinases. To explore the functional role of PAR-2 carboxyl tail in controlling intracellular Ca(2+), ERK and AKT signaling, a series of truncated mutants containing different clusters of serines/threonines were generated and expressed in HEK293 cells. Firstly, we observed that lack of the complete C-terminus of PAR-2 in a mutated receptor gave a relatively low level of localization on the cell plasma membrane. Secondly, the shortened carboxyl tail containing 13 amino acids was sufficient for receptor internalization. Thirdly, the cells expressing truncation mutants showed deficits in their capacity to couple to intracellular Ca(2+) and ERK and AKT signaling upon trypsin challenge. In addition, HEK293 cells carrying different PAR-2 truncation mutants displayed decreased levels of cell survival after long-lasting trypsin stimulation. In summary, the PAR-2 carboxyl tail was found to control the receptor localization, internalization, intracellular Ca(2+) responses and signaling to ERK and AKT. The latter can be considered to be important for cell death control.

ArticleTotal : 2	Year
Publication title Cited by 7	2015
Publication title Cited by 10	2020

Article	Year
Effect of intermediates on ascorbic acid and oxalate biosynthesis of rice and in relation to its stress resistance. Plant physiology and biochemistry : PPB / Société française de physiologie végétale\| PubMed ID: 16310370	2005
GM1 up-regulates Ubiquilin 1 expression in human neuroblastoma cells and rat cortical neurons. Neuroscience letters\| PubMed ID: 16949204	2006
Cyclin-dependent kinase promotes formation of the synaptonemal complex in yeast meiosis. Genes to cells : devoted to molecular & cellular mechanisms\| PubMed ID: 20825495	2010
[Effect of curcumin on expression of Abeta42 and Abeta-degrading enzyme NEP in APPswe/PS1dE9 double transgenic mice]. Zhongguo Zhong yao za zhi = Zhongguo zhongyao zazhi = China journal of Chinese materia medica\| PubMed ID: 21809590	2011
liver-enriched gene 1a and 1b encode novel secretory proteins essential for normal liver development in zebrafish. PloS one\| PubMed ID: 21857963	2011
Specific phosphorylation of αA-crystallin is required for the αA-crystallin-induced protection of astrocytes against staurosporine and C2-ceramide toxicity. Neurochemistry international\| PubMed ID: 22414529	2012
The molecular characterization of antioxidant enzyme genes in Helicoverpa armigera adults and their involvement in response to ultraviolet-A stress. Journal of insect physiology\| PubMed ID: 22750691	2012
PAR-1 activation rescues astrocytes through the PI3K/Akt signaling pathway from chemically induced apoptosis that is exacerbated by gene silencing of β-arrestin 1. Neurochemistry international\| PubMed ID: 24378649	2014
Haploinsufficiency of Def activates p53-dependent TGFβ signalling and causes scar formation after partial hepatectomy. PloS one\| PubMed ID: 24801718	2014
Signaling mechanism of protease activated receptor 1-induced proliferation of astrocytes: stabilization of hypoxia inducible factor-1α triggers glucose metabolism and accumulation of cyclin ... Neurochemistry international\| PubMed ID: 25280834	2014
The intracellular carboxyl tail of the PAR-2 receptor controls intracellular signaling and cell death. Cell and tissue research\| PubMed ID: 25519044	2015

Zhihui Zhu

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