Ectopic Pax2 expression in chick ventral optic cup phenocopies loss of Pax2 expression

Pax2 is essential for the development of the urogenital system, neural tube, otic vesicle, optic cup and optic tract [Dressler, G.R., Deutsch, U., et al., 1990. PAX2, a new murine paired-box-containing gene and its expression in the developing excretory system. Development 109 (4), 787-795; Nornes, H.O., Dressler, G.R., et al., 1990. Spatially and temporally restricted expression of Pax2 during murine neurogenesis. Development 109 (4), 797-809; Eccles, M.R., Wallis, L.J., et al., 1992. Expression of the PAX2 gene in human fetal kidney and Wilms’ tumor. Cell Growth Differ 3 (5), 279-289]. Within the visual system, a loss-of-function leads to lack of choroid fissure closure (known as a coloboma), a loss of optic nerve astrocytes, and anomalous axonal pathfinding at the optic chiasm [Favor, J., Sandulache, R., et al., 1996. The mouse Pax2(1Neu) mutation is identical to a human PAX2 mutation in a family with renal-coloboma syndrome and results in developmental defects of the brain, ear, eye, and kidney. Proc. Natl. Acad. Sci. U. S. A. 93 (24), 13870-13875; Torres, M., Gomez-Pardo, E., et al., 1996. Pax2 contributes to inner ear patterning and optic nerve trajectory. Development 122 (11), 3381-3391]. This study is directed at determining the effects of ectopic Pax2 expression in the chick ventral optic cup past the normal developmental period when Pax2 is found. In ovo electroporation of Pax2 into the chick ventral optic cup results in the formation of colobomas, a condition typically associated with a loss of Pax2 expression. While the overexpression of Pax2 appears to phenocopy a loss of Pax2, the mechanism of the failure of choroid fissure closure is associated with a cell fate switch from ventral retina and retinal pigmented epithelium (RPE) to an astrocyte fate. Further, ectopic expression of Pax2 in RPE appears to have non-cell autonomous effects on adjacent RPE, creating an ectopic neural retina in place of the RPE.     

Heterologous expression of lipoprotein-associated phospholipase A2 in different expression systems

Lipoprotein-associated phospholipase A(2) (Lp-PLA(2)) is a key enzyme involved in atherosclerosis, and has been considered as a new target for drug discovery. The major difficulty for high-throughput screening of Lp-PLA(2) inhibitors and for functional studies was their fast and efficient production. Purification of native Lp-PLA(2) from human plasma was complicated and produced a very low yield. We herein examined the feasibility of expressing and purifying recombinant Lp-PLA(2) in different heterologous expression systems. The fusion Lp-PLA(2) was expressed at high levels and exhibited strong enzyme activity in insect cell-baculovirus expression system. The functional enzyme could also be produced in Pichia pastoris. The inclusion of a Kozak sequence increased greatly the expression level of recombinant Lp-PLA(2) in insect cells, but had little effect on the expression of recombinant Lp-PLA(2) in P. pastoris and Escherichia coli. P. pastoris-produced Lp-PLA(2) could be purified rapidly and conveniently through a one-step procedure, while baculovirus-produced Lp-PLA(2) could be efficiently purified through a two-step procedure. This ability to readily produce recombinant Lp-PLA(2) could provide a screening model for Lp-PLA(2) inhibitors and will facilitate further studies on this enzyme.     

TRAF2 expression in differentiated muscle

Recent data involving traf2 knockout mice have suggested a necessity of the protein in viability of skeletal muscle tissue. traf2−/− mice are born with decreased muscle mass that is hypothesized to be due to the increased circulating tumor necrosis factor in these mice. We show that TRAF2 protein is present at high levels in terminally differentiated skeletal muscle in the developing mouse. In vitro differentiation of mouse myoblasts displays a dramatic increase in TRAF2 protein levels. Although basal NF-κB activity decreases during myogenesis, TNF-induced NF-κB activity is 10 times greater in myotubes compared with myoblasts, presumably because of the stockpiling of TRAF2 protein in these cells. This may represent a strong anti-apoptotic TRAF2-mediated response specifically tailored to myotubes. These data help explain why muscle integrity is at risk in traf2−/− mice. J. Cell. Biochem. 71:461–466, 1998. © 1998 Wiley-Liss, Inc.     

棉铃虫核多角体病毒进化保守性基因iap2基因表达载体的构建及表达

分析棉铃虫核多角体病毒基因组 ,结合GenBank中已知的序列 ,发现iap2基因位于其基因组的BamHⅠ F片段上 ,回收此片段作为模板 ,设计引物 ,通过PCR扩增得到了抗细胞凋亡基因iap2的DNA片段。将扩增产物克隆到pGEM T载体上 ,再进一步将插入片段酶切并连接到表达载体pET 2 8a上 ,构建了重组质粒pET iap2。DNA序列分析结果表明 ,克隆得到的DNA序列与所发表序列完全相同。含重组质粒pET iap2的大肠杆菌BL2 1 (DE3)表达了抗细胞凋亡蛋白IAP2。     

pEGFP-ORF2质粒在Vero细胞中的有效表达

目的：确立pEGFP—ORF2质粒在不同细胞密度,不同转染时间,及血清存在与否时在细胞中的有效表达,为进一步研究HSV-2LATORF2对细胞的作用,及阐明HSV-2潜伏复发机制打下基础。方法：采用脂质体法将pEGFP—ORF2与pEGFP—C2两个真核表达载体转染Vero细胞,观察荧光蛋白表达。结果：有无血清对转染效果无明显差异。在细胞密度为4．0×10^4cells／ml,转染后84hEGFP表达量较其它条件下高,且在相同条件下pEGFP—ORF2与pEGFP—C2的表达无明显差异。结论：血清对表达效果的影响不大,细胞的接种密度及转染时间对转染有明显作用,以荧光蛋白作为标签鉴定蛋白表达的方法切实可行。     

TRPV2 expression in rat oral mucosa

The oral mucosa is a highly specialised, stratified epithelium that confers protection from infection and physical, chemical and thermal stimuli. The non-keratinised junctional epithelium surrounds each tooth like a collar and is easily attacked by foreign substances from the oral sulcus. We found that TRPV2, a temperature-gated channel, is highly expressed in junctional epithelial cells, but not in oral sulcular epithelial cells or oral epithelial cells. Dual or triple immunolabelling with immunocompetent cell markers also revealed TRPV2 expression in Langerhans cells and in dendritic cells and macrophages. Electron microscopy disclosed TRPV2 immunoreactivity in the unmyelinated and thinly myelinated axons within the connective tissue underlying the epithelium. TRPV2 labelling was also observed in venule endothelial cells. The electron-dense immunoreaction in junctional epithelial cells, macrophages and neural axons occurred on the plasma membrane, on invaginations of the plasma membrane and in vesicular structures. Because TRPV2 has been shown to respond to temperature, hypotonicity and mechanical stimuli, gingival cells expressing TRPV2 may act as sensor cells, detecting changes in the physical and chemical environment, and may play a role in subsequent defence mechanisms.     

Stam2 expression pattern during embryo development

STAM2 is a tyrosine-phosphorylated protein suggested to be involved in cargo selection during endocytic pathway, regulation of exocytosis and intracellular signaling. Gene trap method was used to create via insertional mutagenesis a mutant mouse line with integration of promoterless βgeo (lacZ-neomycin phosphotransferase fusion) gene in the second intron of Stam2 gene, enabling analysis of its in vivo expression and function. The inserted β-galactosidase (lacZ) reporter gene was used to reveal Stam2 expression during development. Stam2 in situ RNA hybridization and immunostaining confirmed the observed β-galactosidase activity reflecting high Stam2 expression. The homozygous mutant mice showed no overt phenotypic alterations. Stam2 expression was detected after E9.5 in the gut, notochord, neural tube and heart. In the nervous system it was located in the floor, roof and basal plates of the developing neural tube, and in the developing cortex, hippocampus and olfactory bulbs. Toward the end of gestation, Stam2 expression appeared in the testis and ovary, lungs, nasal cavity epithelium, kidneys, urogenital sinus, intestine, pancreas, pituitary and adrenal glands, muscles, brown adipose tissue, skin and epithelium of the tongue and oral cavity.     

Cat-2 gene expression

Summary Poly(A)⁴ RNA was isolated from maize scutella of different stages of post-germinative development and translated in vitro in a rabbit reticulocyte translation system. Immunoprecipitation of the translation products with CAT-2-specific antibody was used to quantitate the relative levels of translatable CAT-2 mRNA at each stage. The results show a close correlation between the developmental profile of Cat2 gene expression and the profile of CAT-2 mRNA levels. Evidence that the levels of CAT-2 mRNA are regulated by a temporal regulatory gene (Car1) is presented and the possible mechanism(s) of this regulation discussed.This work was supported by Research Grants No. GM22733 and No. GM33817 from the U.S. National Institutes of Health, Public Health Service to J.G.S. This is paper No. 9933 of the Journal Series of the North Carolina Agricultural Research Service, Raleigh, NC 27695, USA     

Quantitative variation in H-2-antigen expression

Minor differences in the expression of individual H-2K and H-2D antigens were detected on mouse spleen cells. The method involved the use of an¹²⁵I-protein A radioimmunoassay using highly specific anti-H-2 sera to make estimates of the number of cell-bound antibody molecules. The maximum number of antibody binding sites varied for each H-2 antigen reflecting differences of between 10 and 70 percent in the expression of any two antigens. The order of magnitude of expression was D^b>(K^d)=K^k=K^b=D^q>D^d>K^q>D^k. Minor background differences were detectable, but antigen expression was allele-specific and independent of the expression of other K, D or I antigens expressed on the same cell.     

Ceramide inhibits CCN2 expression in fibroblasts

Connective tissue growth factor (CTGF, CCN2) is induced in response to TGFbeta in fibroblasts. In this report, we show that C2 ceramide reduced the ability of TGFbeta to induce CCN2 protein, mRNA and promoter activity in fibroblasts. C2 ceramide reduced the ability of TGFbeta to induce the generic Smad responsive promoter/reporter construct SBE-luciferase. These results suggest that C2 ceramide reduces the action of TGFbeta in fibroblasts via Smad antagonism.     

MOB2基因在真核细胞中的表达研究

通过RT-PCR从培养的HUVECs中扩增MOB2基因片段,将该片段克隆在真核表达载体pEGFP-C1中,转染NIH3T3细胞,经G418筛选,构建稳定表达细胞系,并用荧光显微镜和Western blot对其进行鉴定。经RT-PCR扩增出734 bp基因片段,经测序分析并与GenBank的DNA序列比对分析后,在NIH3T3细胞中表达。G418筛选后,细胞荧光信号较强,Western blot检测,细胞中的融合蛋白能够与抗MOB2的多抗结合。成功地扩增了HUVECs中的MOB2基因全长cDNA并进行真核表达与鉴定。