Transgenic pig expressing the enhanced green fluorescent protein produced by nuclear transfer using colchicine-treated fibroblasts as donor cells

Fetal-derived fibroblast cells were transduced with replication defective vectors containing the enhanced green fluorescent protein (EGFP). The transgenic cells were treated with colchicine, which theoretically would synchronize the cells into G2/M stage, and then used as donor nuclei for nuclear transfer. The donor cells were transferred into the perivitalline space of enucleated in vitro matured porcine oocytes, and fused and activated with electrical pulses. A total of 8.3% and 28.6% of reconstructed oocytes showed nuclear envelope breakdown and premature chromosome condensation 0.5 and 2 hr after activation, respectively. Percentage of pronuclear formation was 62.5, 12 hr after activation. Most (91.4%) of the 1-cell embryos with pronuclei did not extrude a polar body. Most (77.2%) embryos on day 5 were diploid. Within 2 hr after fusion, strong fluorescence was detectable in most reconstructed oocytes (92.3%). The fluorescence in all NT embryos became weak 15 hr after fusion and disappeared when culture to 48 hr. But from day 3, cleaved embryos at the 2- to 4-cell stage started to express EGFP again. On day 7, 85.8% of cleaved embryos expressed EGFP. A total of 9.4% of reconstructed embryos developed to blastocyst stage and 71.5% of the blastoctysts expressed EGFP. After 200 reconstructed 1-cell stage embryos were transferred into four surrogate gilts, three recipients were found to be pregnant. One of them maintained to term and delivered a healthy transgenic piglet expressing EGFP. Our data suggest that the combination of transduction of somatic cells by a replication defective vector with the nuclear transfer of colchicine-treated donors is an alternative to produce transgenic pigs. Furthermore, the tissues expressing EGFP from descendents of this pig may be very useful in future studies using pigs that require genetically marked cells.     

Using Nanotechnology for the Substitution of Hazardous Chemical Substances

It is often assumed that nanotechnology (NT) holds the potential to provide a substantial contribution to the solution of various ecological problems, including high consumption of energy and materials and the generation of waste. However, problems surrounding the use and release of hazardous substances remain largely unexplored. For this reason, the Scientific Technical Option Assessment (STOA) Panel of the European Parliament initiated a study on “The Role of Nanotechnology in Chemical Substitution.” The subject and aim of the study was an investigation into preexisting and potential applications of NT that could lead to a reduction in hazardous substances by providing substitutes for them. In terms of method, it was based on electronic searches of the literature, expert interviews, and an expert workshop. This article discusses the results of the project. It focuses on the methodological challenges and the principal problems resulting from a combination of the broad and ill‐defined concept of NT and the specific concept of hazardous substances. The hazardous substances addressed had to be reduced to a manageable number, and the term substitution was understood according to the characteristics of NT and the way in which the latter could reduce the use of hazardous substances. Although several applications of NT were identified that could lead to a considerable reduction in the use of hazardous substances, ambiguities in both the concept of NT and the concept of substitution in relation to NT prevent a comprehensive assessment of the potential of NT in respect to substitution.     

AN UNRECOGNIZED ANCIENT LINEAGE OF GREEN PLANTS PERSISTS IN DEEP MARINE WATERS1

We provide molecular phylogenetic evidence that the obscure genera Palmophyllum Kütz. and Verdigellas D. L. Ballant. et J. N. Norris form a distinct and early diverging lineage of green algae. These palmelloid seaweeds generally persist in deep waters, where grazing pressure and competition for space are reduced. Their distinctness warrants recognition as a new order, the Palmophyllales. Although phylogenetic analyses of both the 18S rRNA gene and two chloroplast genes (atpB and rbcL) are in agreement with a deep‐branching Palmophyllales, the genes are in conflict about its exact phylogenetic placement. Analysis of the nuclear ribosomal DNA allies the Palmophyllales with the prasinophyte genera Prasinococcus and Prasinoderma (Prasinococcales), while the plastid gene phylogeny placed Palmophyllum and Verdigellas as sister clade to all other Chlorophyta.     

Pluripotency potential of human adipose-derived stem cells marked with exogenous green fluorescent protein

Musculoskeletal tissues regeneration requires rapid expansion of seeding cells both in vitro and in vivo while maintaining their multilineage differentiation ability. Human adipose-derived stem cells (ASCs) are considered to contain multipotent mesenchymal stem cells. Monolayer cultures of human ASCs were isolated from human lipoaspirates and passaged 3 times and then infected with replication-incompetent adenoviral vectors carrying green fluorescent protein (Ad/GFP) genes. Then, Ad/GFP infected human ASCs were transferred to osteogenic, chondrogenic, adipogenic, and myogenic medium. The morphological characterization of induced cells was observed using phase-contrast microscopy and fluorescence microscopy. The expression of marker proteins or genes was measured by immunocytochemical and RT-PCR analysis. Osteopontin (OPN), and osteocalcin (OCN) were positive in osteogenic lineages, aggrecan and SOX9 were positive in chondrogenic ones, peroxisome proliferator-activated receptor (PPAR-γ2) and lipoprotein lipase (LPL) were positive in adipogenic ones, and myogenin and myod1 was positive in myogenic ones. At the same time, the results of fluorescence microscopic imaging proved that the high level of GFP expression during ASCs differentiation maintained stable nearly 2 months. So the exogenous GFP and multilineage potential of human ASCs had no severe influences on each other. Since the human ASCs can be easily obtained and abundant, it is proposed that they may be promising candidate cells for further studies on tissue engineering. Imaging with expression of GFP facilitates the research on ASCs physiological behavior and application in tissue engineering during differentiation both in vitro and in vivo.     

S1P(2) receptors mediate inhibition of glioma cell migration through Rho signaling pathways independent of PTEN

Sphingosine 1-phosphate (S1P) induced the inhibition of glioma cell migration. Here, we characterized the signaling mechanisms involved in the inhibitory action by S1P. In human GNS-3314 glioblastoma cells, the S1P-induced inhibition of cell migration was associated with activation of RhoA and suppression of Rac1. The inhibitory action of S1P was recovered by a small interference RNA specific to S1P₂ receptor, a carboxyl-terminal region of Gα12 or Gα13, an RGS domain of p115RhoGEF, and a dominant-negative mutant of RhoA. The inhibitory action of S1P through S1P₂ receptors was also observed in both U87MG glioblastoma and 1321N1 astrocytoma cells, which have no protein expression of a phosphatase and tensin homolog deleted on chromosome 10 (PTEN). These results suggest that S1P₂ receptors/G_12/13-proteins/Rho signaling pathways mediate S1P-induced inhibition of glioma cell migration. However, PTEN, recently postulated as an indispensable molecule for the inhibition of cell migration, may not be critical for the S1P₂ receptor-mediated action in glioma cells.     

Polyhydroxyalkanoates – what are the uses? Current challenges and perspectives

Over the past few decades, a considerable attention has been focused on the microbial polyhydroxyalkanoates (PHAs) owing to its multifaceted properties, i.e. biodegradability, biocompatibility, non-toxicity and thermo-plasticity. This article presents a critical review of the foregoing research, current trends and future perspectives on the value added applications of PHAs in the biomedical, environmental and industrial domains of life.     

Improving phosphorus availability in two upland soils of Vietnam using Tithonia diversifolia H.

Phosphorus was added to two acidic upland soils (a Cambisol and a Ferralsol) at two rates (9 mg P kg⁻¹ and 145 mg P kg⁻¹) either in an inorganic P form (KH₂PO₄) or as a green manure (Tithonia diversifolia H. at 2.5 g kg⁻¹ and 40 g kg⁻¹). The effect of P source on the chemical availability of P was assessed in an incubation experiment by measuring resin extractable P, soluble molybdate reactive (DMR-P) and unreactive P (DMU-P). Soil pH and extractable Al were monitored during the incubation period of 49 days. Green manure addition caused an immediate and sustained increase in soil pH and an immediate and sustained decrease in extractable Al. Labile P (resin P + DMR-P + DMU-P) was increased more by P added as a green manure than when added in inorganic form in one soil (Ferralsol), while it decreased or did not differ in the other one (Cambisol). In both soils, the concentrations of soluble DMU-P were frequently higher where Tithonia had been added. The effects of green manure amendment on physical factors governing the phosphorus supply through diffusive transport were also investigated. Aggregate size distribution was substantially changed by green manure amendment due to a shift in the percentage of microaggregates (<250 μm in diameter) to larger sizes. Changes in soil aggregation as a consequence of green manure amendment led to a reduction in specific surface area (SSA) of the whole soil. Coupled with the large increase in effective cation exchange capacity caused by green manure amendment in both soils, and the decrease in SSA, there was an increase in the net negative surface charge density in both soils. In summary, at a large addition rate – and in addition to the well-known effect derived from the extra supply in P, green manure amendment may improve the chemical availability and diffusive supply of P through the following mechanisms: (i) an increase in soil pH increasing the solubility of phosphate sources; (ii) a decrease in extractable Al reducing the fixation of added P; (iii) increased macro-aggregation and reduced specific surface area and porosity leading to fewer sorption sites for P and hence enhanced diffusion rates; and (iv) increased negative charges and reduced positive charges at the soil surface resulting in a net increase in repulsive force for P. The induced changes in most measured soil properties were smaller in the Ferralsol than in the Cambisol. This revised version was published online in June 2005 with a corrected article title.     

从绿色荧光蛋白的发现谈如何提出新颖的科研问题

为了使研究生在获得专业知识的同时,还可以依据文献提出新的科研问题,并设计实验方案来解决提出的问题,我们以绿色荧光蛋白为案例教学,谈谈在其发现过程中是如何提出问题以及提出问题所用的思维方式。启发研究生运用求异和置换的思维方式来提出新颖的科研问题,并设计实验来回答提出的问题,从而为研究生完成学术论文奠定良好的基础。     

Effect of Intranigral Administration of 6-Hydroxydopamine and 5, 7-Dihydroxytryptamine on Rat Brain Tryptamine

Earlier experiments have shown that unilateral electrolytic lesions of the substantia nigra result in significant reductions in the rate of accumulation of rat striatal tryptamine. For elucidation of the type of neuronal degeneration that is associated with tryptamine depletion, the effects of intranigral injections of 6-hydroxydopamine or 5,7-dihydroxytryptamine, which would affect, respectively, dopamine- or indoleamine-containing neurons, have been assessed. Nigral 6-hydroxydopamine lesions resulted in an ipsilateral reduction in the rate of accumulation of striatal tryptamine, but no changes were observed after nigral injections of 5,7-dihydroxytryptamine. The present results suggest that decreases in the pargyline-induced accumulation of striatal tryptamine may be associated with lesions of the nigral dopamine-containing cell bodies. Alternatively, there may exist specific tryptamine-containing neurons that are damaged by 6-hydroxytryptamine and unaffected by 5,7-dihydroxytryptamine.