Expansion of CD34+ cells from human umbilical cord blood by FL and/or TPO gene transfected human marrow stromal cell lines

To elucidate the effect of gene transfected marrow stromal cell on expansion of human cord blood CD34⁺ cells, a culture system was established in which FL and TPO genes were transfected into human stromal cell line HFCL. To establish gene transfected stromal cells co-culture system, cord blood CD34⁺ cells were purified by using a magnetic beads sorting system. The number of all cells and the number of CD34⁺ cells and CFC (CFU-GM and BFU-E) were counted in different culture systems. The results showed that in all 8 culture systems, SCF+IL-3+HFT manifested the most potent combination, with the number of total nucleated cells increasing by (893.3 ±52.1)-fold, total progenitor cells (CFC) by (74.5 ±5.2)-fold and CD34⁺ cells by 15.7-fold. Maximal expansions of CFC and CD34⁺ cells were observed at the end of the second week of culture. Within 14 days of culture, (78.1 ± 5.5)-fold and (57.0 ± 19.7)-fold increases in CFU-GM and BFU-E were obtained. Moreover, generation of LTC-IC from amplified CD34⁺ cells within 28 days was found only in two combinations, i.e. SCF+IL-3+FL+TPO and SCF+IL-3+HFT, and there was no significant difference between these two groups statistically. These results suggest that human umbilical cord blood CD34⁺ cells can be extensively expandedex vivo by using gene transfected stromal cells along with cytokines.     

结核病实验诊断技术

结核病诊断一直是控制结核病疫情的关键,快速准确、敏感特异、简便低廉的诊断方法是目前迫切需要的.从结核分枝杆菌快速诊断噬菌体法、AMPLICOR(R) MTB试验、Gen-Probe分子生物学诊断方法到T-SPOT.TB和QuantiFeron-Gold Test免疫学检测方法,结核病实验诊断方法在不断改进和完善.近年来...     

Differential Response of Floret Opening in Male-Sterile and Male-Fertile Rices to Methy Jasmonate

Effects of methyl jasmonate (MeJA) on floret opening were investigated in male-sterile and male-fertile rice ( Oryza sativa subsp. indica ). Excised or intact panicles with several top florets flowered one day before the experiments were soaked in MeJA, salicylic acid (SA), 24-epibrassinolide (24-epiBL) or control solution for 2 min. MeJA at the concentration of 4 mmol/L significantly promoted rice floret opening; the response of floret opening of male-sterile rice to MeJA was more sensitive than that of male-fertile rice (such as maintenance line and conventional variety). The percentage of finally opened-floret per panicle of Zhenshan 97A (cytoplasmic male sterile line) or Pei'ai 64S (thermo-sensitive genic male sterile line) treated with MeJA was over 40% in the day of experiment, that of Zhenshan 97B (maintenance) and Pei'ai 64 (conventional variety) was (21±1.5)% and (25±2.1)%, respectively; floret opening of intact Pei'ai 64S panicles in the field could also be induced by 2-mmol/L MeJA. The percentage of opened floret in all control panicles was 0% or less than 5% in the day of experiment. The percentage of fertilized-grain of Zhenshan 97A and Longtepu A could be increased over 24% by spraying panicles with 2-mmol/L MeJA solution （circa 6.0 μmol per panicle）. The lag time (time from the finishing of treatment to first floret opening) of male sterile line responsing to MeJA was 10 min shorter than that of male fertile line. MeJA induction on the rice floret opening could markedly be inhibited by SA solution at the concentration of 1 mmol/L, and the inhibition of SA on MeJA induction on floret opening of male-sterile rice could be nullified by MeJA treatment again. MeJA effect on floret opening of male-sterile rice could be clearly synergized by pretreatment with 24-epibrassinolide (24-epiBL).     

The γ-gliadin gene content of a derivative from a somatic hybrid between bread wheat and tall wheatgrass

A PCR-based strategy was applied to obtain the DNA sequence of γ-gliadin open reading frames present in line II-12, a derivative from a somatic hybrid between bread wheat (Triticum aestivum L.) cv. Jinan177 and tall wheatgrass (Lophopyrum ponticum, 10×). A total 50 analysable sequences were obtained, 18 from II-12 and 16 each from the parents. Amplicon length ranged from 720 to 936 bp, corresponding to a putative mature protein of 239–309 residues. The primary structure of these putative proteins comprised five domains, of which only two varied in length. Phylogenetic analyses showed that the mature γ-gliadin sequences fell into four major clades. Group 1 contained sequences shared between II-12 and L. ponticum, suggesting that some L. ponticum γ-gliadin genes are present in the introgression line. Group 3 has five Jinan177 and five II-12 sequences, indicating that II-12 also carries wheat versions of Gli-1. Group 2 and 4 comprised four and two II-12, three and one Jinan177 as well as one and four L. ponticum sequences, respectively. Fewer genes encoding coeliac disease epitopes were present in II-12 than in the wheat donor parent. Three II-12 γ-gliadins and one from the wheat parent contained an odd number of cysteine residues, and two of them had an additional cysteine residue at the amino end of domain V. The possible use of II-2 for improving quality of bread wheat is discussed.     

A simulation test of the resource‐averaging hypothesis of ecotone formation

Abstract. The pattern at an ecotone may indicate the processes that created that ecotone. Such patterns may in turn affect the responses of ecotones to environmental change. The resource averaging hypothesis suggests a process for the development of tree lines that should produce patterns that are modifications of patterns in soil resources. A computer simulation model that embodies the resource averaging hypothesis is used to generate tree‐line patterns. Different spatial patterns in the variation of soil resources are represented in the model. The patterns of tree line computed by the simulation closely correspond to the patterns of soil resources that were input. These patterns are compared to patterns recorded in the field and by aerial photography. For the patterns of soil resources observed at some alpine tree lines, the model cannot produce the kinds of patterns of vegetation observed. Resource averaging alone cannot be an explanation of such tree lines.     

Targeted Integration of Single-Copy Transgenes in Drosophila melanogaster Tissue-Culture Cells Using Recombination-Mediated Cassette Exchange

Transfection of transgenes into Drosophila cultured cells is a standard approach for studying gene function. However, the number of transgenes present in the cell following transient transfection or stable random integration varies, and the resulting differences in expression level affect interpretation. Here we developed a system for Drosophila cell lines that allows selection of cells with a single-copy transgene inserted at a specific genomic site using recombination-mediated cassette exchange (RMCE). We used the φC31 integrase and its target sites attP and attB for RMCE. Cell lines with an attP-flanked genomic cassette were transfected with donor plasmids containing a transgene of interest (UAS-x), a dihydrofolate reductase (UAS-DHFR) gene flanked by attB sequences, and a thymidine kinase (UAS-TK) gene in the plasmid backbone outside the attB sequences. In cells undergoing RMCE, UAS-x and UAS-DHFR were exchanged for the attP-flanked genomic cassette, and UAS-TK was excluded. These cells were selected using methotrexate, which requires DHFR expression, and ganciclovir, which causes death in cells expressing TK. Pure populations of cells with one copy of a stably integrated transgene were efficiently selected by cloning or mass culture in ∼6 weeks. Our results show that RMCE avoids the problems associated with current methods, where transgene number is not controlled, and facilitates the rapid generation of Drosophila cell lines in which expression from a single transgene can be studied.     

Paclitaxel administration and its effects on clinically relevant human cancer and non cancer cell lines

Comparisons of the effects of clinically relevant concentrations of the anticancer agent paclitaxel on growth, viability, and apoptosis were determined using in vitro human cell cultures. Growth of the cervical cancer cell line, HeLa-S3, was significantly reduced, and apoptotic index was significantly increased, after 24 h in cultures treated with 12 nM paclitaxel. In contrast, hepatic carcinoma (HEpG2) cells capable of detoxifying paclitaxel were only affected at paclitaxel concentrations ge120 nM. The previously uncharacterized non-cancerous human microvessel endothelial cell line HMEC-1, was more sensitive to paclitaxel treatment than both HeLa-S3 and HEpG2 cells, demonstrating decreased growth and increased apoptosis with 1.2 nM paclitaxel. These results are significant in the design of in vitro cell culture systems to study drug metabolism and toxicity.     

A century of tree line changes in sub‐Arctic Sweden shows local and regional variability and only a minor influence of 20th century climate warming

Aim Models project that climate warming will cause the tree line to move to higher elevations in alpine areas and more northerly latitudes in Arctic environments. We aimed to document changes or stability of the tree line in a sub‐Arctic model area at different temporal and spatial scales, and particularly to clarify the ambiguity that currently exists about tree line dynamics and their causes. Location The study was conducted in the Torneträsk area in northern Sweden where climate warmed by 2.5 °C between 1913 and 2006. Mountain birch (Betula pubescens ssp. czerepanovii) sets the alpine tree line. Methods We used repeat photography, dendrochronological analysis, field observations along elevational transects and historical documents to study tree line dynamics. Results Since 1912, only four out of eight tree line sites had advanced: on average the tree line had shifted 24 m upslope (+0.2 m year^?1 assuming linear shifts). Maximum tree line advance was +145 m (+1.5 m year^?1 in elevation and +2.7 m year^?1 in actual distance), whereas maximum retreat was 120 m downslope. Counter‐intuitively, tree line advance was most pronounced during the cooler late 1960s and 1970s. Tree establishment and tree line advance were significantly correlated with periods of low reindeer (Rangifer tarandus) population numbers. A decreased anthropozoogenic impact since the early 20th century was found to be the main factor shaping the current tree line ecotone and its dynamics. In addition, episodic disturbances by moth outbreaks and geomorphological processes resulted in descent and long‐term stability of the tree line position, respectively. Main conclusions In contrast to what is generally stated in the literature, this study shows that in a period of climate warming, disturbance may not only determine when tree line advance will occur but if tree line advance will occur at all. In the case of non‐climatic climax tree lines, such as those in our study area, both climate‐driven model projections of future tree line positions and the use of the tree line position for bioclimatic monitoring should be used with caution.     

Transversal dose distribution optimization for laser-accelerated proton beam medical applications by means of Geant4

The main purpose of this paper is to quantitatively study the possibility of delivering dose distributions of clinical relevance with laser-driven proton beams. A Monte Carlo application has been developed with the Geant4 toolkit, simulating the ELIMED (MEDical and multidisciplinary application at ELI-Beamlines) transport and dosimetry beam line which is being currently installed at the ELI-Beamlines in Prague (CZ). The beam line will be used to perform irradiations for multidisciplinary studies, with the purpose of demonstrating the possible use of optically accelerated ion beams for therapeutic purposes. The ELIMED Geant4-based application, already validated against reference transport codes, accurately simulates each single element of the beam line, necessary to collect the accelerated beams and to select them in energy. Transversal dose distributions at the irradiation point have been studied and optimized to try to quantitatively answer the question if such kind of beam lines, and specifically the systems developed for ELIMED in Prague, will be actually able to transport ion beams not only for multidisciplinary applications, such as pitcher-catcher nuclear reactions (e.g. neutrons), PIXE analysis for cultural heritage and space radiation, but also for delivering dose patterns of clinical relevance in a future perspective of possible medical applications.     

Model of ribosomal translocation coupled with intra- and inter-subunit rotations

The ribosomal translocation involves both intersubunit rotations between the small 30S and large 50S subunits and the intrasubunit rotations of the 30S head relative to the 30S body. However, the detailed molecular mechanism on how the intersubunit and intrasubunit rotations are related to the translocation remains unclear. Here, based on available structural data a model is proposed for the ribosomal translocation, into which both the intersubunit and intrasubunit rotations are incorporated. With the model, we provide quantitative explanations of in vitro experimental data showing the biphasic character in the fluorescence change associated with the mRNA translocation and the character of a rapid increase that is followed by a slow single-exponential decrease in the fluorescence change associated with the 30S head rotation. The calculated translation rate is also consistent with the in vitro single-molecule experimental data.