Involvement of membrane protein GDE2 in retinoic acid-induced neurite formation in Neuro2A cells

We show that a glycerophosphodiester phosphodiesterase homolog, GDE2, is widely expressed in brain tissues including primary neurons, and that the expression of GDE2 in neuroblastoma Neuro2A cells is significantly upregulated during neuronal differentiation by retinoic acid (RA) treatment. Stable expression of GDE2 resulted in neurite formation in the absence of RA, and GDE2 accumulated at the regions of perinuclear and growth cones in Neuro2A cells. Furthermore, a loss-of-function of GDE2 in Neuro2A cells by RNAi blocked RA-induced neurite formation. These results demonstrate that GDE2 expression during neuronal differentiation plays an important role for growing neurites.     

DNA methylation imprints on the IG-DMR of the Dlk1-Gtl2 domain in mouse male germline

Mouse genomes show a large cluster of imprinted genes at the Dlk1-Gtl2 domain in the distal region of chromosome 12. An intergenic-differentially methylated region (IG-DMR) located between Dlk1 and Gtl2 is specifically methylated in the male germline; IG-DMR regulates the parental allele-specific expression of imprinted genes. Here, we show the resetting of IG-DMR methylation marks during male germ-cell differentiation. For parental allele-specific methylation analysis, polymorphisms were detected in a 2.6-kb IG-DMR in three mouse strains. Bisulfite methylation analysis showed erasure of the marks by E14 and re-establishment before birth. The IG-DMR methylation status was maintained in spermatogonia and spermatocytes of mature testes. The IG-DMR methylation status established before birth is thus maintained throughout the lifetime in the male germline.     

Enhanced biological activity of polymeric osteopontin

Osteopontin is a multifunctional glycoprotein with roles in immunomodulation, inflammatory response, tissue mineralization, and tissue remodeling, which are mediated primarily through integrins. Transglutaminase 2 selectively cross-links proteins by isopeptide bonding. Osteopontin is one of the substrates of this enzyme and undergoes polymerization; however, the biological meaning of this polymerization remains unknown. Using recombinant osteopontin polymerized with purified transglutaminase 2, we examined cell adhesion, spreading, focal contact formation, and migration of SW480 or HUVE cells. All of these cellular behaviors were dramatically enhanced with polymeric osteopontin. These enhancements of cellular functions imply that polymerization might modulate physiological and pathological functions of osteopontin.     

Genetic networks responsive to sodium butyrate in colonic epithelial cells

We performed microarray and computational gene network analyses to identify the detailed mechanisms by which sodium butyrate (SB) induces cell growth arrest and the differentiation of mouse colonic epithelial MCE301 cells. Two thousand six hundred four differentially expressed probe sets were identified in the cells treated with 2mM SB and were classified into four groups. Of these, the gradually increased group and the gradually and remarkably decreased group contained the genetic networks for cellular development and cell cycles or canonical pathways for fatty acid biosynthesis and pyrimidine metabolism, respectively. The present results provide a basis for understanding the detailed molecular mechanisms of action of SB in colonic epithelial cells.     

Hypertrophy and atrophy inversely regulate Caveolin-3 expression in myoblasts

Caveolin-3 (Cav-3) is a muscle-specific membrane protein crucial for myoblast differentiation, as loss of the protein due to mutations within the gene causes an autosomal dominant form of limb girdle muscular dystrophy 1-c. Here we show that along with p38 activity the PI3-kinase/AKT/mTOR pathway is required for proper Cav-3 up-regulation during muscle differentiation and hypertrophy, as confirmed by the marked increase of Cav-3 expression in hypertrophied C2C12 cells transfected with an activated form of AKT. Accordingly, Cav-3 expression was further increased during hypertrophy of L6C5 myoblasts treated with Arg(8)-vasopressin and in hypertrophic muscles of MLC/mIGF-1 transgenic mice. In contrast, Cav-3 expression was down-regulated in C2C12 myotubes exposed to atrophic stimuli such as starvation or treatment with dexamethasone. This study clearly suggests that Cav-3 expression is causally linked to the maturation of muscle phenotype and it is tightly regulated by hypertrophic and atrophic stimuli.     

A monopartite nuclear localization sequence regulates nuclear targeting of the actin binding protein myopodin

Myopodin is an actin bundling protein that shuttles between nucleus and cytoplasm in response to cell stress or during differentiation. Here, we show that the myopodin sequence 58KKRRRRARK66, when tagged to either enhanced green fluorescent protein (EGFP) or to enhanced cyan fluorescent protein-CapG (ECFPCapG), is able to target these proteins to the nucleolus in HeLa or HEK293T cells. By contrast, 58KKRR61-ECFP-CapG accumulates in the nucleus. Mutation of 58KKRRRRARK66 into alanine residues blocks myopodin nuclear import and promotes formation of cytoplasmic actin filaments. A second putative nuclear localization sequence, 612KTSKKKGKK620, displays much weaker activity in a heterologous context, and appears not to be functional in the full length protein. Thus myopodin nuclear translocation is dependent on a monopartite nuclear localization sequence.     

A PKC-mediated backup mechanism of the MXXCW motif-linked switch for initiating tyrosine kinase activities

The cysteine in the M/IXXCW motif is conserved in all but one (threonine in place of cysteine) of the human protein tyrosine kinases (PTKs). We showed that all RET-PTC-1 mutants in which the C in this motif (C376) was replaced with glycine, lysine, threonine or serine lost their activity in vitro. However, the C376T/S mutants showed normal tyrosine phosphorylation in vivo (in cells). Further analyses reveled that protein kinase C (PKC) initiated the activities of the C376T/S mutants in cells. We conclude that the M/IXXCW motif-mediated mechanisms which initiate PTK activities are partially replaced by a PKC-mediated mechanism.     

N-terminal glycine-specific protein conjugation catalyzed by microbial transglutaminase

Here, we report the N-terminal glycine (Gly) residue of a target protein can be a candidate primary amine for site-specific protein conjugation catalyzed by microbial transglutaminase (MTG) from Streptomyces mobaraensis. Gly5-enhanced green fluorescent protein (EGFP) (EGFP with five additional Gly residues at its N-terminus) was cross-linked with Myc-dihydrofolate reductase (DHFR) (DHFR with the myc epitope sequence at its N-terminus) to yield DHFR-EGFP heterodimers. The reactivities of additional peptidyl linkers were investigated and the results obtained suggested that at least three additional Gly residues at the N-terminus were required to yield the EGFP-DHFR heterodimeric form. Site-directed mutagenesis analysis revealed marked preference of MTG for amino acids adjacent to the N-terminal Gly residue involved in the protein conjugation. In addition, peptide-protein conjugation was demonstrated by MTG-catalyzed N-terminal Gly-specific modification of a target protein with the myc epitope peptide.     

Covalent labeling of cell-surface proteins for in-vivo FRET studies

Fluorescence resonance energy transfer (FRET) is a powerful technique to reveal interactions between membrane proteins in live cells. Fluorescence labeling for FRET is typically performed by fusion with fluorescent proteins (FP) with the drawbacks of a limited choice of fluorophores, an arduous control of donor-acceptor ratio and high background fluorescence arising from intracellular FPs. Here we show that these shortcomings can be overcome by using the acyl carrier protein labeling technique. FRET revealed interactions between cell-surface neurokinin-1 receptors simultaneously labeled with a controlled ratio of donors and acceptors. Moreover, using FRET the specific binding of fluorescent agonists could be monitored.     

Testis mediated gene transfer: In vitro transfection in goat testis by electroporation

Testis mediated gene transfer (TMGT) is a potential tool for making transgenic mice having more than 90% success rate. However, this method needs further standardization before it can be adapted in other species including livestock. In order to standardize the TMGT in goat, buck testes (n = 20) collected from the slaughter house were injected with a vector driving green fluorescent protein (GFP) expression under a cytomegalovirus (CMV) promoter. Then, the testes were subjected to electroporation with predetermined voltage, pulse length, pulse interval and number of pulses. Seminiferous tubules were isolated from the electroporated testis and cultured in-vitro. The expression was checked at regular intervals. Green fluorescence was observed on different days in different samples. It suggests transient integration of the plasmid into the seminiferous tubules. This in-vitro transfection of seminiferous tubule using electroporation will provide valuable baseline information.     

Alpha helical structures in the leader sequence of human GLUD2 glutamate dehydrogenase responsible for mitochondrial import

Human glutamate dehydrogenase (hGDH) exists in two highly homologous isoforms with a distinct regulatory and tissue expression profile: a housekeeping hGDH1 isoprotein encoded by the GLUD1 gene and an hGDH2 isoenzyme encoded by the GLUD2 gene. There is evidence that both isoenzymes are synthesized as pro-enzymes containing a 53 amino acid long N-terminal leader peptide that is cleaved upon translocation into the mitochondria. However, this GDH signal peptide is substantially larger than that of most nuclear DNA-encoded mitochondrial proteins, the leader sequence of which typically contains 17-35 amino acids and they often form a single amphipathic α-helix. To decode the structural elements that are essential for the mitochondrial targeting of human GDHs, we performed secondary structure analyses of their leader sequence. These analyses predicted, with 82% accuracy, that both leader peptides are positively charged and that they form two to three α-helices, separated by intermediate loops. The first α-helix of hGDH2 is strongly amphipathic, displaying both a positively charged surface and a hydrophobic plane. We then constructed GLUD2-EGFP deletion mutants and used them to transfect three mammalian cell lines (HEK293, COS 7 and SHSY-5Y). Confocal laser scanning microscopy, following co-transfection with pDsRed2-Mito mitochondrial targeting vector, revealed that deletion of the entire leader sequence prevented the enzyme from entering the mitochondria, resulting in its retention in the cytoplasm. Deletion of the first strongly amphipathic α-helix only was also sufficient to prevent the mitochondrial localization of the truncated protein. Moreover, truncated leader sequences, retaining the second and/or the third putative α-helix, failed to restore the mitochondrial import of hGDH2. As such, the first N-terminal alpha helical structure is crucial for the mitochondrial import of hGDH2 and these findings may have implications in understanding the evolutionary mechanisms that led to the large mitochondrial targeting signals of human GDHs.     

Phenotypic behavior of C2C12 myoblasts upon expression of the dystrophy-related caveolin-3 P104L and TFT mutants

Caveolin-3 (Cav-3) is the main scaffolding protein present in myofiber caveolae. We transfected C2C12 myoblasts with dominant negative forms of Cav-3, P104L or DeltaTFT, respectively, which cause the limb-girdle muscular dystrophy 1-C. Both these forms triggered Cav-3 loss during C2C12 cell differentiation. The P104L mutation reduced myofiber formation by impaired AKT signalling, accompanied by dramatic expression of the E3 ubiquitin ligase Atrogin. On the other hand, the DeltaTFT mutation triggered hypertrophic myotubes sustained by prolonged AKT activation, but independent of increased levels of follistatin and interleukin 4 expression. These data suggest that separated mutations within the same dystrophy-related gene may cause muscle degeneration through different mechanisms.     

Application of Cell-Surface Engineering for Visualization of Yeast in Bread Dough: Development of a Fluorescent Bio-Imaging Technique in the Mixing Process of Dough

Cell-surface engineering (Ueda et al., 2000) has been applied to develop a novel technique to visualize yeast in bread dough. Enhanced green fluorescent protein (EGFP) was bonded to the surface of yeast cells, and 0.5% EGFP yeasts were mixed into the dough samples at four different mixing stages. The samples were placed on a cryostat at ?30 °C and sliced at 10 μm. The sliced samples were observed at an excitation wavelength of 480 nm and a fluorescent wavelength of 520 nm. The results indicated that the combination of the EGFP-displayed yeasts, rapid freezing, and cryo-sectioning made it possible to visualize 2-D distribution of yeast in bread dough to the extent that the EGFP yeasts could be clearly distinguished from the auto-fluorescent background of bread dough.     

低分子肝素的绿色荧光蛋白荧光标记及其活性检测

增强型绿色荧光蛋白（EGFP）是生物领域常用的标记物. 本实验首先利用高碘酸法活化低分子肝素（LMWH）,与EGFP连接得到LMWH EGFP.然后用Sephacryl S-200 HR对其进行初步分离,再用Sephadex G-10 HR进行脱盐纯化. 采用Sephacryl S 200 HR检测LMWH EGFP的纯度,为单一对称峰. 经检测,LMWH-EGFP具有良好的热稳定性和耐碱性. 通过荧光分光检测器检测LMWH-EGFP的λEx为488 nm,λEm为509 nm. 通过抗凝实验发现LMWH EGFP仍具有抗凝活性. 本实验建立了LWMH荧光标记的方法,为多糖的荧光标记提供了新的选择.