卡介苗作用大鼠膀胱肿瘤细胞／正常移行上皮细胞后TNF—α．、IL-10、IFN-γ的检测及意义

目的：观察卡介苗（BCG）单独作用膀胱肿瘤细胞、正常膀胱移行上皮细胞及其代谢产物作用上述细胞后细胞生长情况及各自细胞培养液上清液中细胞因子（TNF-α．、IL-10、IFN-γ）浓度的变化,探讨其在卡介苗治疗膀胱肿瘤中可能的作用机制。方法：构建大鼠膀胱肿瘤模型,并原代培养大鼠膀胱肿瘤细胞及正常膀胱移行上皮细胞。分别用BCG,普通培养液和细胞培养的代谢产物作用上述细胞。酶联接免疫吸附剂测定法（ELISA法）检测各组细胞上清液中肿瘤坏死因子-α（TNF-α）、白细胞介素-10（IL-10）、干扰素-γ（IFN-γ）的浓度。结果：ELISA法检测各组细胞上清液中TNF-α、IL-10的浓度改变有显著差异,而IFN-γ的浓度无显著差异。结论：BCG可以直接刺激肿瘤细胞自身分泌细胞因子（TNF-α、IL-10）参与调节抑制肿瘤细胞的生长。     

Rapid apoplastic pH measurement in Arabidopsis leaves using a fluorescent dye

In plants, the extracellular space (apoplast) is one of the main places where exchange of molecules occurs between cells. Not only is this compartment involved in the storage of multiple metabolites and ions, including calcium and protons, but it also plays a role in the transmission of signaling molecules for cell-to-cell communication. It has recently been shown multiple times that these two aspects are linked and can influence each other. In particular, apoplast pH was shown as a primary regulator of auxin (IAA) transport in Arabidopsis thaliana. To prove the role of apoplastic pH, we have developed a protocol for apoplastic fluid extraction from Arabidopsis leaves, followed by pH determination using the 8-hydroxypyrene-1,3,6-trisulfonic acid (HPTS) fluorescent dye. This technique successfully allows one to monitor apoplastic pH variations among different plant lines and to link changes in apoplastic pH to cellular responses in the plant.     

Fractionation of Phenolic Compounds Extracted from Propolis and Their Activity in the Yeast Saccharomyces cerevisiae

We have here investigated the activities of Slovenian propolis extracts in the yeast Saccharomyces cerevisiae, and identified the phenolic compounds that appear to contribute to these activities. We correlated changes in intracellular oxidation and cellular metabolic energy in these yeasts with the individual fractions of the propolis extracts obtained following solid-phase extraction. The most effective fraction was further investigated according to its phenolic compounds.     

Ocular Injury by Transient Formaldehyde Exposure in a Rabbit Eye Model

Formaldehyde (FA) is frequently used in sterilizing surgical instruments and materials. Exposure to FA is highly concerned for eye tissues. Rabbit corneal epithelial cells were examined for changes after FA exposure. Our results showed that cell survival decreased 7 days after transient 3 min exposure to more than 100 ppm FA by trypan blue staining while MTT assay detected significant decrease at 20 ppm at 24 hours observation. The decrease of cell survival rate was concentration (up to 600 ppm)- and observation time (1–7 day)- dependent. The cell number decreased after 100 ppm FA exposure for more than 10 min at 7-day observation. The FA treated cells showed increased apoptosis/necrosis and cell cycle accumulation at sub G1 phase as well as mitochondria clustering around nucleus. The in vivo rabbit eye exposure for tear production by Schirmer’s test revealed that the FA-induced overproduction of tear also exhibited observation time (1–10 day)- and FA concentration (20–300 ppm for 5 min exposure)-dependent. Activated extracellular signal-regulated kinase (pERK2) in cornea explants by western blotting was reduced and increased c-Jun amino - terminal kinase (JNK) activation (pJNK) in cornea and conjunctiva was evident at 2 month after exposure to 50–200 ppm FA for 5 min. In conclusion, injury to the eye with transient exposure of up to 100 ppm FA for 3 min decreased corneal cell survival while a more sensitive MTT test detected the cell decrease at 20 ppm FA exposure. Morphology changes can be observed even at 5 ppm FA exposure for 3 min at 7 days after. The FA exposure also increased apoptotic/necrotic cells and sub-G1 phase in cell cycle. Long term effect (2 months after exposure) on the eye tissues even after the removal of FA can be observed with persistent JNK activation in cornea and conjunctiva.     

Linking ascorbic acid production in Ribes nigrum with fruit development and changes in sources and sinks

Background and Aims

Understanding the synthesis of ascorbic acid (l-AsA) in green tissues in model species has advanced considerably; here we focus on its production and accumulation in fruit. In particular, our aim is to understand the links between organs which may be sources of l-AsA (leaves) and those which accumulate it (fruits). The work presented here tests the idea that changes in leaf and fruit number influence the accumulation of l-AsA. The aim was to understand the importance of leaf tissue in the production of l-AsA and to determine how this might provide routes for the manipulation of fruit tissue l-AsA.

Methods

The experiments used Ribes nigrum (blackcurrant), predominantly in field experiments, where the source–sink relationship was manipulated to alter potential leaf l-AsA production and fruit growth and accumulation of l-AsA. These manipulations included reductions in reproductive capacity, by raceme removal, and the availability of assimilates by leaf removal and branch phloem girdling. Natural variation in fruit growth and fruit abscission is also described as this influences subsequent experimental design and the interpretation of l-AsA data.

Key Results

Results show that fruit l-AsA concentration is conserved but total yield of l-AsA per plant is dependent on a number of innate factors many of which relate to raceme attributes. Leaf removal and phloem girdling reduced fruit weight, and a combination of both reduced fruit yields further. It appears that around 50 % of assimilates utilized for fruit growth came from apical leaves, while between 20 and 30 % came from raceme leaves, with the remainder from ‘storage’.

Conclusions

Despite being able to manipulate leaf area and therefore assimilate availability and stored carbohydrates, along with fruit yields, rarely were effects on fruit l-AsA concentration seen, indicating fruit l-AsA production in Ribes was not directly coupled to assimilate supply. There was no supporting evidence that l-AsA production occurred predominantly in green leaf tissue followed by its transfer to developing fruits. It is concluded that l-AsA production occurs predominantly in the fruit of Ribes nigrum.     

Bi-specific TCR-anti CD3 redirected T-cell targeting of NY-ESO-1- and LAGE-1-positive tumors

NY-ESO-1 and LAGE-1 are cancer testis antigens with an ideal profile for tumor immunotherapy, combining up-regulation in many cancer types with highly restricted expression in normal tissues and sharing a common HLA-A*0201 epitope, 157–165. Here, we present data to describe the specificity and anti-tumor activity of a bifunctional ImmTAC, comprising a soluble, high-affinity T-cell receptor (TCR) specific for NY-ESO-1_157–165 fused to an anti-CD3 scFv. This reagent, ImmTAC-NYE, is shown to kill HLA-A2, antigen-positive tumor cell lines, and freshly isolated HLA-A2- and LAGE-1-positive NSCLC cells. Employing time-domain optical imaging, we demonstrate in vivo targeting of fluorescently labelled high-affinity NYESO-specific TCRs to HLA-A2-, NY-ESO-1_157–165-positive tumors in xenografted mice. In vivo ImmTAC-NYE efficacy was tested in a tumor model in which human lymphocytes were stably co-engrafted into NSG mice harboring tumor xenografts; efficacy was observed in both tumor prevention and established tumor models using a GFP fluorescence readout. Quantitative RT-PCR was used to analyze the expression of both NY-ESO-1 and LAGE-1 antigens in 15 normal tissues, 5 cancer cell lines, 10 NSCLC, and 10 ovarian cancer samples. Overall, LAGE-1 RNA was expressed at a greater frequency and at higher levels than NY-ESO-1 in the tumor samples. These data support the clinical utility of ImmTAC-NYE as an immunotherapeutic agent for a variety of cancers.     

In Planta Assessment of the Role of Thioredoxin h Proteins in the Regulation of S-Locus Receptor Kinase Signaling in Transgenic Arabidopsis

The self-incompatibility (SI) response of the Brassicaceae is mediated by allele-specific interaction between the stigma-localized S-locus receptor kinase (SRK) and its ligand, the pollen coat-localized S-locus cysteine-rich protein (SCR). Based on work in Brassica spp., the thioredoxin h-like proteins THL1 and THL2, which interact with SRK, have been proposed to function as oxidoreductases that negatively regulate SRK catalytic activity. By preventing the spontaneous activation of SRK in the absence of SCR ligand, these thioredoxins are thought to be essential for the success of cross pollinations in self-incompatible plants. However, the in planta role of thioredoxins in the regulation of SI signaling has not been conclusively demonstrated. Here, we addressed this issue using Arabidopsis thaliana plants transformed with the SRKb-SCRb gene pair isolated from self-incompatible Arabidopsis lyrata. These plants express an intense SI response, allowing us to exploit the extensive tools and resources available in A. thaliana for analysis of SI signaling. To test the hypothesis that SRK is redox regulated by thioredoxin h, we expressed a mutant form of SRKb lacking a transmembrane-localized cysteine residue thought to be essential for the SRK-thioredoxin h interaction. We also analyzed transfer DNA insertion mutants in the A. thaliana orthologs of THL1 and THL2. In neither case did we observe an effect on the pollination responses of SRKb-expressing stigmas toward incompatible or compatible pollen. Our results are consistent with the conclusion that, contrary to their proposed role, thioredoxin h proteins are not required to prevent the spontaneous activation of SRK in the A. thaliana stigma.Many flowering plants possess self-incompatibility (SI), a genetic system that promotes outcrossing by preventing self-fertilization. In the Brassicaceae family, the SI response is controlled by haplotypes of the S locus, each of which contains two genes that encode highly polymorphic proteins, the S-locus receptor kinase (SRK), which is a plasma membrane resident single-pass transmembrane Ser/Thr receptor kinase displayed at the surface of stigma epidermal cells (Stein et al., 1991; Takasaki et al., 2000), and the S-locus Cys-rich protein (SCR), which is the pollen coat-localized ligand for SRK (Schopfer et al., 1999; Kachroo et al., 2001; Takayama et al., 2001). SRK and SCR exhibit allele-specific interactions, whereby only SRK and SCR encoded by the same S-locus haplotype interact. In a self-pollination, the binding of this “self” pollen-borne SCR to the extracellular domain of SRK activates the SRK kinase, thereby triggering a cellular response in stigma epidermal cells that causes inhibition of pollen germination and tube penetration into the stigma epidermal cell wall (for review, see Tantikanjana et al., 2010).Tight regulation of SRK kinase activity and its signaling cascade is critical for productive pollen-stigma interactions because constitutive (i.e. SCR-independent) activity of the receptor is expected to result in sterile stigmas that reject both compatible and incompatible pollen. In the classical view of ligand-activated receptor kinases, the receptor occurs as catalytically inactive monomers in the absence of ligand and only becomes activated upon ligand-induced dimerization (for review, see Lemmon and Schlessinger, 2010). However, some receptor kinases in both animals (Chan et al., 2000; Ehrlich et al., 2011) and plants (Giranton et al., 2000; Wang et al., 2005, 2008; Shimizu et al., 2010; Bücherl et al., 2013) form catalytically inactive dimers or oligomers in the absence of ligand, with receptor activation presumably resulting from ligand-induced higher order oligomerization or conformational changes (Lemmon and Schlessinger, 2010). Similar to the latter receptors, SRK forms oligomers in unpollinated stigmas, i.e. in the absence of SCR (Giranton et al., 2000), at least partly via ligand-independent dimerization domains located within the SRK extracellular domain (Naithani et al., 2007). It has been proposed that these ligand-independent SRK oligomers are maintained in an inactive state by thioredoxins, the ubiquitous small oxidoreductases that reduce disulfide bridges in proteins (Buchanan and Balmer, 2005). This hypothesis is supported by the following observations: (1) two Brassica napus thioredoxins, the Thioredoxin H-Like proteins THL1 and THL2, were identified as SRK interactors in a yeast (Saccharomyces cerevisiae) two-hybrid screen that used the B. napus SRK₉₁₀ kinase domain as bait (Bower et al., 1996); (2) when purified from pistils or insect cells, the Brassica oleracea SRK₃ variant was found to exhibit constitutive autophosphorylation activity in vitro, and this activity was inhibited by Escherichia coli-expressed THL1 proteins and was restored by addition of pollen coat proteins containing self but not of pollen coat proteins containing nonself SCR (Cabrillac et al., 2001); (3) the catalytic activity of THL1 was required for its inhibition of SRK₃ autophosphorylation activity in vitro (Cabrillac et al., 2001); and (4) antisense suppression of THL1/THL2 gene expression in the stigmas of a self-compatible B. napus strain reportedly produced a low-level constitutive incompatibility (Haffani et al., 2004), as might be expected if the THL1/THL2 proteins prevent the spontaneous activation of SRK-mediated signaling in stigmas.These observations notwithstanding, the in planta role of thioredoxin h proteins as negative regulators of SRK activity has not been conclusively demonstrated. To date, this proposed function has only been evaluated in a self-compatible strain of B. napus (Haffani et al., 2004). Consequently, it is not known if the proposed inhibitory effect of these thioredoxins on SRK catalytic activity is manifested in self-incompatible stigmas and if it applies to all SRK variants, be they derived from Brassica spp. or other self-incompatible species of the Brassicaceae such as Arabidopsis lyrata.In this study, we tested the in planta role of thioredoxin h proteins in the regulation of SI signaling using a transgenic self-incompatible Arabidopsis thaliana model that we generated by transforming A. thaliana with the SRKb-SCRb gene pair isolated from the Sb haplotype of self-incompatible A. lyrata (Kusaba et al., 2001; Nasrallah et al., 2002, 2004). We had previously shown that the stigmas of A. thaliana SRKb-SCRb transformants can exhibit an SI response that is as robust as the SI response observed in naturally self-incompatible A. lyrata, demonstrating that A. thaliana, which harbors nonfunctional S-locus haplotypes (Kusaba et al., 2001; Sherman-Broyles et al., 2007; Shimizu et al., 2008; Boggs et al., 2009c), has nevertheless retained all other factors required for SI. In view of the availability in A. thaliana of a highly efficient transformation method and numerous genetic resources, the A. thaliana SRK-SCR transgenic model has enabled the use of experimental approaches that are difficult or impossible to implement in Brassica species and has thus proven to be an invaluable platform for in planta analysis of SRK and SI signaling (Liu et al., 2007; Boggs et al., 2009a, 2009b; Tantikanjana et al., 2009; Tantikanjana and Nasrallah, 2012).We therefore used this transgenic A. thaliana self-incompatible model to determine if abolishing the proposed SRK-thioredoxin h interaction or eliminating expression of the major thioredoxin h proteins expressed in stigmas would affect the outcome of self- or cross pollination. To this end, we expressed a mutant form of SRKb that lacked the Cys residue previously shown to be required for the interaction of SRK with THLs (Mazzurco et al., 2001), and we analyzed plants carrying knockout insertional mutations in thioredoxin h genes. Our results are inconsistent with the proposed role of thioredoxin h proteins as negative regulators of SRK catalytic activity and SI signaling.     

The limits of post-national citizenship: European Muslims,human rights and the hijab

Innovative approaches to citizenship emerged in the 1990s. Post-national theory suggested that European minorities no longer needed national citizenship because supra-national political structures such as the European Court of Human Rights (ECtHR) offered them protections. Denationalized citizenship held that universal human rights were now available at the national level too as the Council of Europe's member countries had to incorporate human rights principles within their own jurisdictions. New forms of claims-making among European Muslims were cited as evidence of this trend as religious claims, especially relating to the hijab, began to be made through human rights litigation. This paper demonstrates the limits of post-nationalism through a discussion of the outcomes of such claims. While European Muslims are indeed mobilizing around human rights, there is no evidence – at the level of litigation – that this has helped them to win recognition of their religious or cultural rights. This paper explores the reasons for this.     

Molecular Characterization and Evolution of Self-Incompatibility Genes in Arabidopsis thaliana: The Case of the Sc Haplotype

The switch from an outcrossing mode of mating enforced by self-incompatibility to self-fertility in the Arabidopsis thaliana lineage was associated with mutations that inactivated one or both of the two genes that comprise the self-incompatibility (SI) specificity-determining S-locus haplotype, the S-locus receptor kinase (SRK) and the S-locus cysteine-rich (SCR) genes, as well as unlinked modifier loci required for SI. All analyzed A. thaliana S-locus haplotypes belong to the SA, SB, or SC haplotypic groups. Of these three, the SC haplotype is the least well characterized. Its SRKC gene can encode a complete open-reading frame, although no functional data are available, while its SCRC sequences have not been isolated. As a result, it is not known what mutations were associated with inactivation of this haplotype. Here, we report on our analysis of the Lz-0 accession and the characterization of its highly rearranged SC haplotype. We describe the isolation of its SCRC gene as well as the subsequent isolation of SCRC sequences from other SC-containing accessions and from the A. lyrata S36 haplotype, which is the functional equivalent of the A. thaliana SC haplotype. By performing transformation experiments using chimeric SRK and SCR genes constructed with SC- and S36-derived sequences, we show that the SRKC and SCRC genes of Lz-0 and at least a few other SC-containing accessions are nonfunctional, despite SCRC encoding a functional full-length protein. We identify the probable mutations that caused the inactivation of these genes and discuss our results in the context of mechanisms of S-locus inactivation in A. thaliana.