Green Fluorescent Protein Fusion Research Articles

Aminopeptidase N expression in the endometrium could affect endometrial receptivity

Aminopeptidase N (ANPEP) is a membrane-bound zinc-dependent peptidase. Although it is widely believed that ANPEP acts as an important angiogenesis regulatory factor, there are few studies about its function in the female reproductive system. In our previous research, we applied Isobaric Tags for Relative and Absolute Quantitation (iTRAQ) to analyze the influence of different controlled superovulation treatments for Assisted Reproductive Technology, In Vitro Fertilization and Embryo Transfer (IVF-ET)) patients from a global proteomic perspective to search for potential biomarkers associated with endometrium receptivity and embryo implantation. ANPEP is one of the proteins that demonstrated differential expression between different treatment groups and may be closely associated with endometrial receptivity. In this study, we assessed the expression of ANPEP in the endometrium of mice at different ages and found it to be highest in the mature period. We also detected ANPEP expression in the endometrium of IVF-ET patients in the proliferative, preimplantation and implantation stages, and the highest expression level of ANPEP was found in the last group. Human primary endometrial stromal cells were infected with an adenovirus expression vector containing the ANPEP gene and a green fluorescent protein (GFP) fusion protein; the mRNA levels of HOXA-10, LIF, and integrin β3 were found to be increased. Therefore, we conclude that ANPEP could be involved in the regulation of endometrial receptivity.

The Arabidopsis receptor kinase STRUBBELIG undergoes clathrin-dependent endocytosis.

Signaling mediated by cell surface receptor kinases is central to the coordination of growth patterns during organogenesis. Receptor kinase signaling is in part controlled through endocytosis and subcellular distribution of the respective receptor kinase. For the majority of plant cell surface receptors, the underlying trafficking mechanisms are not characterized. In Arabidopsis, tissue morphogenesis requires the atypical receptor kinase STRUBBELIG (SUB). Here, we studied the endocytic mechanism of SUB. Our data revealed that a functional SUB–enhanced green fluorescent protein (EGFP) fusion is ubiquitinated in vivo. We further showed that plasma membrane-bound SUB:EGFP becomes internalized in a clathrin-dependent fashion. We also found that SUB:EGFP associates with the trans-Golgi network and accumulates in multivesicular bodies and the vacuole. Co-immunoprecipitation experiments revealed that SUB:EGFP and clathrin are present within the same protein complex. Our genetic analysis showed that SUB and CLATHRIN HEAVY CHAIN (CHC) 2 regulate root hair patterning. By contrast, genetic reduction of CHC activity ameliorates the floral defects of sub mutants. Taken together, the data indicate that SUB undergoes clathrin-mediated endocytosis, that this process does not rely on stimulation of SUB signaling by an exogenous agent, and that SUB genetically interacts with clathrin-dependent pathways in a tissue-specific manner.

Weisiensin B inhibits primary and lateral root development by interfering with polar auxin transport in Arabidopsis thaliana

Weisiensin B, a new ent-kaurene diterpenoid isolated from Isodon weisiensis (C. Y. Wu) H. Hara, exhibited phytotoxic effects on root growth and lateral root development in Arabidopsis thaliana seedlings. Primary root growth and lateral root formation in A. thaliana seedlings were significantly inhibited by 10–20 μM weisiensin B. Additionally, the role of weisiensin B in response to polar auxin transport in A. thaliana roots was investigated using a PIN promoter (PIN::GUS), a green fluorescent protein (GFP) fusion protein reporter (PINs::PINs:GFP), and DR5::GUS and DR5::GFP reporter genes. The results indicated that weisiensin B reduced the expression of PIN2, PIN3, PIN4, PIN7, and AUX1 genes and significantly decreased the abundance of PIN2-GFP, PIN3-GFP, PIN4-GFP, PIN7-GFP, and AUX1-GFP fusion proteins at their respective cellular locations, simultaneously causing auxin accumulation in the root apex. These results suggest that weisiensin B interferes with polar auxin transport in A. thaliana roots, resulting in auxin accumulation in the root meristematic cells and the inhibition of root growth and lateral root development.

The Cryptococcus neoformans monocarboxylate transporter Jen4 is responsible for increased 3-bromopyruvate sensitivity.

In the last decades, 3-bromopyruvate (3BP) has been intensively studied as a promising anticancer and antimicrobial agent. The transport of this drug inside the cell is a critical step for its toxicity in cancer and microorganisms. The Cryptococcus neoformans is the most sensitive species of microorganisms toward 3BP. Its cells exhibit the highest uptake rate of 3BP among all tested fungal strains. In Saccharomyces cerevisiae cells, the Jen1 transporter was found to be responsible for 3BP sensitivity. The deletion of Jen1 resulted in the abolishment of 3BP mediated transport. We functionally characterized the Jen4 protein, a Jen1 homologue of C. neoformans, and its role in the phenotypic 3BP sensitivity. The deletion of the CNAG_04704 gene, which encodes Jen4, was found to impair the mediated transport of 3BP and decrease 3BP sensitivity. Further heterologous expression of Jen4 in the S. cerevisiae jen1Δ ady2Δ strain restored the mediated transport of 3BP. The application of a green fluorescent protein fusion tag with the CNAG_04704, revealed the Jen4 labeled on the plasma membrane. The identification of 3BP transporters in pathogen cells is of great importance for understanding the mechanisms of 3BP action and to anticipate the application of this compound as an antimicrobial drug.

SlSWEET1a is involved in glucose import to young leaves in tomato plants.

Sugar allocation from source to sink (young) leaves, critical for plant development, relies on activities of plasma membrane sugar transporters. However, the key sugar unloading mechanism to sink leaves remains elusive. SWEET transporters mediate sugar efflux into reproductive sinks; therefore, they are promising candidates for sugar unloading during leaf growth. Transcripts of SlSWEET1a, belonging to clade I of the SWEET family, were markedly more abundant than those of all other 30 SlSWEET genes in young leaves of tomatoes. High expression of SlSWEET1a was also detected in reproductive sinks, such as flowers. SlSWEET1a was dominantly expressed in leaf unloading veins, and the green fluorescent protein (GFP) fusion protein was localized to the plasma membrane using Arabidopsis protoplasts, further implicating this carrier in sugar unloading. In addition, yeast growth assays and radiotracer uptake analyses further demonstrated that SlSWEET1a acted as a low-affinity (Km ~100 mM) glucose-specific carrier with a passive diffusion manner. Finally, virus-induced gene silencing of SlSWEET1a expression reduced hexose accumulation to ~50% in young leaves, with a parallel 2-fold increase in mature leaves. Thus, we propose a novel function for SlSWEET1a in the uptake of glucose into unloading cells as part of the sugar unloading mechanism in sink leaves of tomato.

The Catalytic Activity of the PAH1‐encoded Phosphatidate Phosphatase is Required for Lipid Biosynthesis in the Oleaginous Yarrowia lipolytica

The PAH1 gene encodes phosphatidate phosphatase (PAP) which catalyzes the conversion of phosphatidate (PA) to diacylglycerol used for the synthesis of triacylglycerol (TAG). Previous research in our lab showed that the lack of Pah1 (i.e. pah1Δ) in Y. lipolytica causes a significant decrease in cellular lipid content and TAG levels, while the phospholipid levels are increased. However, it is unclear whether this phenotype is due to the loss of the catalytic activity of Pah1 or due to some other function of the protein (e.g. transcriptional regulation). The objective of this work was to confirm that the loss of the catalytic activity of Pah1 is responsible for the phenotypes presented by pah1Δ cells.Yarrowia lipolytica pah1Δ cells expressing either wild‐type Pah1 fused with Green fluorescent protein (GFP) or a catalytically inactive Pah1 (i.e., D350E) fused with GFP were grown in media that favor lipid biosynthesis. PAP activity was measured following the release of water‐soluble 32Pi from chloroform‐soluble [32P] PA, while lipid profiles were analyzed by thin layer chromatography. The expression of the GFP fusions was confirmed by immunoblot analysis using antibodies directed against GFP. The results showed that the pah1Δ strain expressing an inactive Pah1 presented a decrease in TAG levels as well as overall lipid content and an increase in phospholipids compared to the pah1Δ strain expressing the wild‐type Pah1. These results showed that the catalytic activity of Pah1 is required for TAG biosynthesis in Y. lipolytica.Support or Funding InformationThis work was supported, in whole or in part, by National Institutes of Health Grant R15GM124602‐01 from the United States Public Health Service. The content is solely the responsibility of the authors and does not necessarily represent the official views of the National Institutes of Health.This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Cell-Free Expression and Photo-Crosslinking of the Human Neuropeptide Y2 Receptor.

G protein-coupled receptors (GPCRs) represent a large family of different proteins, which are involved in physiological processes throughout the entire body. Furthermore, they represent important drug targets. For rational drug design, it is important to get further insights into the binding mode of endogenous ligands as well as of therapeutic agents at the respective target receptors. However, structural investigations usually require homogenous, solubilized and functional receptors, which is still challenging. Cell-free expression methods have emerged in the last years and many different proteins are successfully expressed, including hydrophobic membrane proteins like GPCRs. In this work, an Escherichia coli based cell-free expression system was used to express the neuropeptide Y2 receptor (Y2R) for structural investigations. This GPCR was expressed in two different variants, a C-terminal enhanced green fluorescent fusion protein and a cysteine deficient variant. In order to obtain soluble receptors, the expression was performed in the presence of mild detergents, either Brij-35 or Brij-58, which led to high amounts of soluble receptor. Furthermore, the influence of temperature, pH value and additives on protein expression and solubilization was tested. For functional and structural investigations, the receptors were expressed at 37°C, pH 7.4 in the presence of 1 mM oxidized and 5 mM reduced glutathione. The expressed receptors were purified by ligand affinity chromatography and functionality of Y2R_cysteine_deficient was verified by a homogenous binding assay. Finally, photo-crosslinking studies were performed between cell-free expressed Y2R_cysteine_deficient and a neuropeptide Y (NPY) analog bearing the photoactive, unnatural amino acid p-benzoyl-phenylalanine at position 27 and biotin at position 22 for purification. After enzymatic digestion, fragments of crosslinked receptor were identified by mass spectrometry. Our findings demonstrate that, in contrast to Y1R, NPY position 27 remains flexible when bound to Y2R. These results are in agreement with the suggested binding mode of NPY at Y2R.

Protein and metabolite composition of Arabidopsis stress granules

Stress granules (SGs) are evolutionary conserved aggregates of proteins and untranslated mRNAs formed in response to stress. Despite their importance for stress adaptation, no complete proteome composition has been reported for plant SGs. In this study, we addressed the existing gap. Importantly, we also provide evidence for metabolite sequestration within the SGs. To isolate SGs we used Arabidopsis seedlings expressing green fluorescent protein (GFP) fusion of the SGs marker protein, Rbp47b, and an experimental protocol combining differential centrifugation with affinity purification (AP). SGs isolates were analysed using mass spectrometry-based proteomics and metabolomics. A quarter of the identified proteins constituted known or predicted SG components. Intriguingly, the remaining proteins were enriched in key enzymes and regulators, such as cyclin-dependent kinase A (CDKA), that mediate plant responses to stress. In addition to proteins, nucleotides, amino acids and phospholipids also accumulated in SGs. Taken together, our results indicated the presence of a preexisting SG protein interaction network; an evolutionary conservation of the proteins involved in SG assembly and dynamics; an important role for SGs in moderation of stress responses by selective storage of proteins and metabolites.

Structural biology workflow for the expression and characterization of functional human sodium glucose transporter type 1 in Pichia pastoris

Heterologous expression of human membrane proteins is a challenge in structural biology towards drug discovery. Here we report a complete expression and purification process of a functional human sodium/D-glucose co-transporter 1 (hSGLT1) in Pichia pastoris as representative example of a useful strategy for any human membrane protein. hSGLT1 gene was cloned in two different plasmids to develop parallel strategies: one which includes green fluorescent protein fusion for screening optimal conditions, and another for large scale protein production for structural biology and biophysics studies. Our strategy yields at least 1 mg of monodisperse purified recombinant hSGLT1 per liter of culture, which can be characterized by circular dichroism and infrared spectroscopy as an alpha-helical fold protein. This purified hSGLT1 transports co-substrates (Na+ and glucose) and it is inhibited by phlorizin in electrophysiological experiments performed in planar lipid membranes.

C-X-C Motif Chemokine Receptor 4 Blockade Promotes Tissue Repair After Myocardial Infarction by Enhancing Regulatory T Cell Mobilization and Immune-Regulatory Function.

Acute myocardial infarction (MI) elicits an inflammatory response that drives tissue repair and adverse cardiac remodeling. Inflammatory cell trafficking after MI is controlled by C-X-C motif chemokine ligand 12 (CXCL12) and its receptor, C-X-C motif chemokine receptor 4 (CXCR4). CXCR4 antagonists mobilize inflammatory cells and promote infarct repair, but the cellular mechanisms are unclear. We investigated the therapeutic potential and mode of action of the peptidic macrocycle CXCR4 antagonist POL5551 in mice with reperfused MI. We applied cell depletion and adoptive transfer strategies using lymphocyte-deficient Rag1 knockout mice; DEREG mice, which express a diphtheria toxin receptor-enhanced green fluorescent protein fusion protein under the control of the promoter/enhancer region of the regulatory T (Treg) cell-restricted Foxp3 transcription factor; and dendritic cell-depleted CD11c-Cre iDTR mice. Translational potential was explored in a porcine model of reperfused MI using serial contrast-enhanced magnetic resonance imaging. Intraperitoneal POL5551 injections in wild-type mice (8 mg/kg at 2, 4, 6, and 8 days) enhanced angiogenesis in the infarct border zone, reduced scar size, and attenuated left ventricular remodeling and contractile dysfunction at 28 days. Treatment effects were absent in splenectomized wild-type mice, Rag1 knockout mice, and Treg cell-depleted DEREG mice. Conversely, treatment effects could be transferred into infarcted splenectomized wild-type mice by transplanting splenic Treg cells from POL5551-treated infarcted DEREG mice. Instructive cues provided by infarct-primed dendritic cells were required for POL5551 treatment effects. POL5551 injections mobilized Treg cells into the peripheral blood, followed by enhanced Treg cell accumulation in the infarcted region. Neutrophils, monocytes, and lymphocytes displayed similar mobilization kinetics, but their cardiac recruitment was not affected. POL5551, however, attenuated inflammatory gene expression in monocytes and macrophages in the infarcted region via Treg cells. Intravenous infusion of the clinical-stage POL5551 analogue POL6326 (3 mg/kg at 4, 6, 8, and 10 days) decreased infarct volume and improved left ventricular ejection fraction in pigs. These data confirm CXCR4 blockade as a promising treatment strategy after MI. We identify dendritic cell-primed splenic Treg cells as the central arbiters of these therapeutic effects and thereby delineate a pharmacological strategy to promote infarct repair by augmenting Treg cell function in vivo.

Sequential trafficking of Env and Gag to HIV-1 T cell virological synapses revealed by live imaging

BackgroundHIV infection is enhanced by cell adhesions that form between infected and uninfected T cells called virological synapses (VS). VS are initiated by an interaction between Env and CD4 on cell surfaces and result in the recruitment of virus assembly to the site of cell–cell contact. However, the recruitment of Env to the VS and its relationship to Gag recruitment is not well defined.ResultsTo study the trafficking of HIV-1 Env through the VS, we constructed a molecular clone of HIV carrying a green fluorescent protein-Env fusion protein called, HIV Env-isfGFP-∆V1V2. The Env-isfGFP-∆V1V2 fusion protein does not produce virus particles on its own, but can be rescued by cotransfection with full-length HIV constructs and produce virus particles that package the fluorescent Env. These rescued fluorescent Env can participate in VS formation and can be used to directly image CD4-dependent Env transfer across VS from donor to target cells. The movements of fluorescently tagged Gag and Env to the VS and transfer into target cells can be also tracked through live imaging. Time lapse live imaging reveals evidence of limited Env accumulation at the site of cell–cell contact shortly after cell adhesion, followed by Gag re-distribution to contact area. Both Gag and Env can be recruited to form button-like spots characteristic of VS.ConclusionsEnv and Gag are recruited to the VS in a coordinated temporal sequence and subsequently transfer together across the synapse into the target cell. Env accumulations, when observed, are earlier than Gag re-distribution to the contact area during formation of VS.

Bioluminescence calcium imaging of network dynamics and their cholinergic modulation in slices of cerebral cortex from male rats.

The activity of neuronal ensembles was monitored in neocortical slices from male rats using wide-field bioluminescence imaging of a calcium sensor formed with the fusion of green fluorescent protein and aequorin (GA) and expressed through viral transfer. GA expression was restricted to pyramidal neurons and did not conspicuously alter neuronal morphology or neocortical cytoarchitecture. Removal of extracellular magnesium or addition of GABA receptor antagonists triggered epileptiform flashes of variable amplitude and spatial extent, indicating that the excitatory and inhibitory networks were functionally preserved in GA-expressing slices. We found that agonists of muscarinic acetylcholine receptors largely increased the peak bioluminescence response to local electrical stimulation in layer I or white matter, and gave rise to a slowly decaying response persisting for tens of seconds. The peak increase involved layers II/III and V and did not result in marked alteration of response spatial properties. The persistent response involved essentially layer V and followed the time course of the muscarinic afterdischarge depolarizing plateau in layer V pyramidal cells. This plateau potential triggered spike firing in layer V, but not layer II/III pyramidal cells, and was accompanied by recurrent synaptic excitation in layer V. Our results indicate that wide-field imaging of GA bioluminescence is well suited to monitor local and global network activity patterns, involving different mechanisms of intracellular calcium increase, and occurring on various timescales.

A Flexible and Scalable High-Throughput Platform for Recombinant Membrane Protein Production.

Integral membrane proteins (MP) are implicated in many disease processes and are the primary targets of numerous marketed drugs. Despite recent advances in the areas of MP solubilization, stabilization, and reconstitution, it remains a time-consuming task to identify the combination of constructs and purification conditions that will enable MP structure-function studies outside of the lipid bilayer. In this chapter, we describe a strategy for rapidly identifying and optimizing the solubilization and purification conditions for nearly any recombinant MP, based on the use of a noninvasive fluorescent probe (His-Glow) that specifically binds to the common hexahistidine affinity tag of expressed targets. This His-Glow approach permits fluorescent size-exclusion chromatography (FSEC) without the need for green fluorescent protein (GFP) fusion. A two-stage detergent screening strategy is employed at the solubilization stage, whereby appropriate detergent families are identified first, followed by optimization within these families. Screening up to 96 unique combinations of solubilization conditions and constructs can be achieved in less than 24h. At the outset of each new project, we screen six different detergents for each construct and the subsequent implementation of a simple thermostability challenge further aids in the identification of constructs and conditions suitable for large-scale production. Our strategy streamlines the parallel optimization of appropriate production conditions for multiple MP targets to rapidly enable downstream biochemical, immunization, or structural studies.

Functional Analysis of Three Rice Chloroplast Transit Peptides

Chloroplast transit peptides (CTPs) can be used to transport non-chloroplastic proteins into the chloroplasts. Here, we studied the CTPs of three rice (Oryza sativa L.) chloroplast-localized proteins and found that their CTPs could be used to transport non-chloroplast-localized proteins into the chloroplasts. Fusion proteins lacking the CTP remained located in the cytoplasm. Furthermore, we constructed green fluorescent protein fusion vectors with the three CTPs and three non-chloroplast-localized proteins, Ghd10, MULTI-FLORET SPIKELET1 (MFS1), and SHORTENED UPPERMOST INTERNODE 1 (SUI1). After transforming these constructs into rice protoplasts, the fusion proteins all localized in the chloroplasts. Collectively, our results showed that these CTPs can transport non-chloroplast-localized proteins into the chloroplasts, and more importantly, these CTPs can be applied to engineer chloroplast metabolism.

Genome-Wide Analysis of Watermelon HSP20s and Their Expression Profiles and Subcellular Locations under Stresses.

Watermelon (Citrullus lanatus L.), which is an economically important cucurbit crop that is cultivated worldwide, is vulnerable to various adverse environmental conditions. Small heat shock protein 20s (HSP20s) are the most abundant plant HSPs and they play important roles in various biotic and abiotic stress responses. However, they have not been systematically investigated in watermelon. In this study, we identified 44 watermelon HSP20 genes and analyzed their gene structures, conserved domains, phylogenetic relationships, chromosomal distributions, and expression profiles. All of the watermelon HSP20 proteins have a conserved the α-crystallin (ACD) domain. Half of the ClHSP20s arose through gene duplication events. Plant HSP20s were grouped into 18 subfamiles and a new subfamily, nucleo-cytoplasmic XIII (CXIII), was identified in this study. Numerous stress- and hormone-responsive cis-elements were detected in the putative promoter regions of the watermelon HSP20 genes. Different from that in other species, half of the watermelon HSP20s were repressed by heat stress. Plant HSP20s displayed diverse responses to different virus infections and most of the ClHSP20s were generally repressed by Cucumber green mottle mosaic virus (CGMMV). Some ClHSP20s exhibited similar transcriptional responses to abscisic acid, melatonin, and CGMMV. Subcellular localization analyses of six selected HSP20- green fluorescence protein fusion proteins revealed diverse subcellular targeting. Some ClHSP20 proteins were affected by CGMMV, as reflected by changes in the size, number, and distribution of fluorescent granules. These systematic analyses provide a foundation for elucidating the physiological functions and biological roles of the watermelon HSP20 gene family.

The role of Bombyx mori Bmtutl-519 protein in the infection of BmN cells by Nosema bombycis

Bmtutl-519 is an isoform of the Bombyx Turtle protein and a member of the immunoglobulin superfamily (IgSF). The relative expression level of Bmtutl-519 was significantly upregulated when BmN cells were infected by Nosema bombycis. The subcellular localization of Bmtutl-519 was studied using an indirect immunoinfluscent assay (IFA), Co-localization assay, Western blotting, and enhanced green fluorescent protein (EGFP) fusion constructs expressed in BmN cells transfected with a Bmtutl-519 expression plasmid. The results indicate that Bmtutl-519 is distributed in both the cytoplasm and the cell membrane of BmN cells. Bmtutl-519 may be involved in the infection process of N. bombycis as a cell surface receptor or regulatory factor. Interaction analysis of Bmtutl-519 with NbSWP26, a spore wall protein of N. bombycis involved in host cell adherence and infection, showed that the C-terminal heparin-binding motif (HBM) of NbSWP26 mediates the interaction between these two proteins. Mutation of the NbSWP26 HBM at K208G, K209G, K210G, and K213G led to a loss of the ability to bind the Bmtutl-519 protein. Spore adherence and infection assays showed that Bmtutl-519 enhances the binding ability of N. bombycis to the host cell surface, but this did not enhance host cell infection by N. bombycis. In contrast, the sustained high expression of Bmtutl-519 in BmN cells inhibited the proliferation of N. bombycis spores.

44 Misregulation of ten-eleven translocation 3 CXXC domain leads to abnormal formation of 5-hydroxymethylcytosine and expression of pluripotency genes in pig embryos

Ten-eleven translocation (TET) methylcytosine dioxygenases are considered to play an important role in regulation of DNA methylation patterns by converting 5-methylcytosine (5mC) to 5-hydroxymethylcytosine (5hmC). TET3 protein, a member of TET family, is enriched in mature oocytes and early stage embryos and contributes to DNA demethylation of the paternal genome in zygotes. N-terminal CXXC domain of TET3 is thought to be important in catalysing 5mC oxidation through its DNA binding potential. However, it is not clear whether specific DNA binding of CXXC domain is required for 5hmC conversion in mammalian embryos. Here, we investigated the role of TET3 CXXC domain in controlling 5hmC formation in fertilized pig embryos by injecting TET3 CXXC domain into mature pig oocytes as a dominant negative to inhibit the direct binding of TET3 to the genome through the CXXC domain. The CXXC domain of pig TET3 was identified through bioinformatics comparison of TET3 sequences among different species and cloned from mature pig oocyte-derived cDNA. To construct the green fluorescent protein (GFP)-CXXC fusion protein, CXXC sequence was subcloned into N-terminal GFP fusion vector, and then mRNA was synthesised by in vitro transcription. The GFP-CXXC mRNA (100 ng/µL) was injected into oocytes matured in vitro for 36 to 37h. Then, the oocytes were fertilized at 42h post-maturation. Water-injected oocytes served as a control. At 17h post-fertilization, zygotes were collected to analyse 5hmC level by immunocytochemistry. The level of 5hmC was analysed using ImageJ (https://imagej.nih.gov/ij/). Expression of pluripotency-related genes at Day 7 blastocysts was examined through quantitative RT-PCR; ΔΔCt method was used to analyse the quantitative RT-PCR data and Student’s t-test was used for statistical analysis. All experiments were conducted at least three times and P-values of less than 0.05 were considered significant. The GFP-CXXC was exclusively localised in pronuclei, indicating that the CXXC domain may lead to nuclear localization of TET3. The level of 5hmC in zygotes was not altered by the overexpression of GFP-CXXC. Interestingly, in 2-cell stage embryos, the 5hmC level was reduced in GFP-CXXC injected embryos compared with the control group, suggesting that CXXC domain is important for 5hmC formation post-DNA replication. There was an increase in transcript abundance of NANOG and ESRRB in blastocysts developed from GFP-CXXC injected oocytes compared with control blastocysts (P<0.05). There was no difference in the expression of POU5F1 and SOX2. In this study, we found that CXXC domain of TET3 is critical in maintaining the level of 5hmC formation at 2-cell stage and proper level of pluripotency-related genes (NANOG and ESRRB) in blastocysts. Future studies will focus on elucidating mechanisms behind the changes after overexpression of GFP-CXXC in pig embryos.

Expression of seven carbonic anhydrases in red alga Gracilariopsis chorda and their subcellular localization in a heterologous system, Arabidopsis thaliana.

Red alga, Gracilariopsis chorda, contains seven carbonic anhydrases that can be grouped into α-, β- and γ-classes. Carbonic anhydrases (CAHs) are metalloenzymes that catalyze the reversible hydration of CO2. These enzymes are present in all living organisms and play roles in various cellular processes, including photosynthesis. In this study, we identified seven CAH genes (GcCAHs) from the genome sequence of the red alga Gracilariopsis chorda and characterized them at the molecular, cellular and biochemical levels. Based on sequence analysis, these seven isoforms were categorized into four α-class, one β-class, and two γ-class isoforms. RNA sequencing revealed that of the seven CAHs isoforms, six genes were expressed in G. chorda in light at room temperature. In silico analysis revealed that these seven isoforms localized to multiple subcellular locations such as the ER, mitochondria and cytosol. When expressed as green fluorescent protein fusions in protoplasts of Arabidopsis thaliana leaf cells, these seven isoforms showed multiple localization patterns. The four α-class GcCAHs with an N-terminal hydrophobic leader sequence localized to the ER and two of them were further targeted to the vacuole. GcCAHβ1 with no noticeable signal sequence localized to the cytosol. The two γ-class GcCAHs also localized to the cytosol, despite the presence of a predicted presequence. Based on these results, we propose that the red alga G. chorda also employs multiple CAH isoforms for various cellular processes such as photosynthesis.

A novel FAM83H mutation in one Chinese family with autosomal-dominant hypocalcification amelogenesis imperfecta.

Autosomal-dominant hypocalcification amelogenesis imperfecta (ADHCAI) is characterized by soft enamel that easily disintegrates and exposed dark dentin. ADHCAI is caused by mutations in a gene called family with sequence similarity 83 member H (FAM83H). To investigate the molecular genetics of ADHCAI, a Chinese family in which three generations exhibited ADHCAI was recruited. The enamel ultrastructure was analysed by environmental scanning electron microscopy (ESEM), which showed altered enamel rod (prism) structures in ADHCAI patients compared to the structures in healthy controls. Mutational analysis of the FAM83H gene identified a novel nonsense mutation (c.1222A>T) in the affected family members that encodes a stop codon at amino acid position 408, causing premature protein truncation (p. K408X). Green fluorescent protein (GFP) and FAM83H fusion protein analyses in vitro showed that normal cytoplasmic accumulation of the FAM83H protein was prevented by the K408X mutation in both rat dental epithelial SF2 cells and human embryonic kidney 293T cells. The mutant fusion protein localized primarily to the nucleus, in contrast to the cytoplasmic subcellular localization of the wild-type FAM83H protein. Our results provide new genetic evidence that mutations in FAM83H contribute to ADHCAI.

Isolation of a novel protein phosphatase2C in rice and its response to gibberellin

Protein phosphatase 2Cs (PP2Cs) have been referred to act as negative modulators of the protein kinase pathways involved in different environmental stress responses and developmental processes. In Arabidopsis, PP2Cs have been extensively studied and some are known to negatively regulate abscisic acid signaling. In rice, PP2Cs are scarcely characterized functionally. Here, we identified a novel PP2C from rice (OsPP2C34), which is highly inducible by gibberellin (GA) and expressed in various tissues. Subcellular localization analysis in maize protoplasts using a green fluorescence protein fusion vector localized OsPP2C34 to the cytosol. Genetic analysis of T-DNA insertional mutants revealed that plant height and internode length were significantly shorter in mutants than in corresponding wild types under GA treatment. The induction of the GA-inducibleα-amylase genes RAmy3E and OsAmy was delayed in mutant plants. The substrate of OsPP2C34 was identified by immunoblotting using anti serine/threonine antibodies. A 65 kDa protein was phosphorylated in Ospp2c34-1 but dephosphorylated in the wild type during early germination stage. Overall, the present results indicated that OsPP2C34 is involved inα-amylase expression of GA signal transduction pathway.