Hybrid embryonic stem cell-derived tetraploid mice show apparently normal morphological,physiological, and neurological characteristics

ES cell-tetraploid (ES) mice are completely derived from embryonic stem cells and can be obtained at high efficiency upon injection of hybrid ES cells into tetraploid blastocysts. This method allows the immediate generation of targeted mouse mutants from genetically modified ES cell clones, in contrast to the standard protocol, which involves the production of chimeras and several breeding steps. To provide a baseline for the analysis of ES mouse mutants, we performed a phenotypic characterization of wild-type B6129S6F(1) ES mice in relation to controls of the same age, sex, and genotype raised from normal matings. The comparison of 90 morphological, physiological, and behavioral parameters revealed elevated body weight and hematocrit as the only major difference of ES mice, which exhibited an otherwise normal phenotype. We further demonstrate that ES mouse mutants can be produced from mutant hybrid ES cells and analyzed within a period of only 4 months. Thus, ES mouse technology is a valid research tool for rapidly elucidating gene function in vivo.     

Glucocorticosteroids modify Langerhans cells to produce TGF-β and expand regulatory T cells

Although glucocorticosteroids (GCSs) have been used for many decades in transplantation and (auto)inflammatory diseases, the exact mechanisms responsible for their immunosuppressive properties are not fully understood. The purpose of this study was to characterize the effects of oral GCSs on the cutaneous immune response. We analyzed, by immunofluorescence staining and quantitative RT-PCR, residual skin biopsy material from a clinical study in which we had used oral GCS as positive control for determining the effects of candidate anti-inflammatory compounds on epicutaneous patch tests of Ni-allergic patients. Expectedly, oral GCS treatment led to a reduction of clinical symptoms and infiltrating leukocytes. Notably, we observed increased numbers of dermal FOXP3(+)CD25(+) T cells and epidermal Langerhans cells (LCs) that were associated with upregulated mRNA expression of TGF-β in lesions of GCS-treated Ni-allergic patients. To investigate this phenomenon further, we exposed purified LCs to GCS. They exhibited, in contrast to GCS-nonexposed LCs, 1) a more immature phenotype, 2) higher intracellular amounts of TGF-β, and 3) increased receptor activator for NF-κB expression, conditions that reportedly favor the expansion of regulatory T cells (Tregs). Indeed, we observed an enhancement of functionally suppressive FOXP3(+) T cells when CD3(+) cells were incubated with GCS-pretreated LCs. The expansion of Tregs was inhibited by TGF-β blockage alone, and their suppressive activity was neutralized by a combination of anti-TGF-β and anti-IL-10 Abs. Our data show that systemically applied GCSs endow LCs with Treg-promoting properties and thus shed new light on the mechanisms of GCS-mediated immunosuppression.     

PMR5, an acetylation protein at the intersection of pectin biosynthesis and defense against fungal pathogens

Powdery mildew (Golovinomyces cichoracearum), one of the most prolific obligate biotrophic fungal pathogens worldwide, infects its host by penetrating the plant cell wall without activating the plant's innate immune system. The Arabidopsis mutant powdery mildew resistant 5 (pmr5) carries a mutation in a putative pectin acetyltransferase gene that confers enhanced resistance to powdery mildew. Here, we show that heterologously expressed PMR5 protein transfers acetyl groups from [¹⁴C]‐acetyl‐CoA to oligogalacturonides. Through site‐directed mutagenesis, we show that three amino acids within a highly conserved esterase domain in putative PMR5 orthologs are necessary for PMR5 function. A suppressor screen of mutagenized pmr5 seed selecting for increased powdery mildew susceptibility identified two previously characterized genes affecting the acetylation of plant cell wall polysaccharides, RWA2 and TBR. The rwa2 and tbr mutants also suppress powdery mildew disease resistance in pmr6, a mutant defective in a putative pectate lyase gene. Cell wall analysis of pmr5 and pmr6, and their rwa2 and tbr suppressor mutants, demonstrates minor shifts in cellulose and pectin composition. In direct contrast to their increased powdery mildew resistance, both pmr5 and pmr6 plants are highly susceptibile to multiple strains of the generalist necrotroph Botrytis cinerea, and have decreased camalexin production upon infection with B. cinerea. These results illustrate that cell wall composition is intimately connected to fungal disease resistance and outline a potential route for engineering powdery mildew resistance into susceptible crop species.     

Advanced electron paramagnetic resonance on the catalytic iron–sulfur cluster bound to the CCG domain of heterodisulfide reductase and succinate: quinone reductase

Heterodisulfide reductase (Hdr) is a key enzyme in the energy metabolism of methanogenic archaea. The enzyme catalyzes the reversible reduction of the heterodisulfide (CoM-S-S-CoB) to the thiol coenzymes M (CoM-SH) and B (CoB-SH). Cleavage of CoM-S-S-CoB at an unusual FeS cluster reveals unique substrate chemistry. The cluster is fixed by cysteines of two cysteine-rich CCG domain sequence motifs (CX_31–39CCX_35–36CXXC) of subunit HdrB of the Methanothermobacter marburgensis HdrABC complex. We report on Q-band (34 GHz) ⁵⁷Fe electron-nuclear double resonance (ENDOR) spectroscopic measurements on the oxidized form of the cluster found in HdrABC and in two other CCG-domain-containing proteins, recombinant HdrB of Hdr from M. marburgensis and recombinant SdhE of succinate: quinone reductase from Sulfolobus solfataricus P2. The spectra at 34 GHz show clearly improved resolution arising from the absence of proton resonances and polarization effects. Systematic spectral simulations of 34 GHz data combined with previous 9 GHz data allowed the unambiguous assignment of four ⁵⁷Fe hyperfine couplings to the cluster in all three proteins. ¹³C Mims ENDOR spectra of labelled CoM-SH were consistent with the attachment of the substrate to the cluster in HdrABC, whereas in the other two proteins no substrate is present. ⁵⁷Fe resonances in all three systems revealed unusually large ⁵⁷Fe ENDOR hyperfine splitting as compared to known systems. The results infer that the cluster’s unique magnetic properties arise from the CCG binding motif.     

Split-Cre Complementation Indicates Coincident Activity of Different Genes In Vivo

Cre/LoxP recombination is the gold standard for conditional gene regulation in mice in vivo. However, promoters driving the expression of Cre recombinase are often active in a wide range of cell types and therefore unsuited to target more specific subsets of cells. To overcome this limitation, we designed inactive “split-Cre” fragments that regain Cre activity when overlapping co-expression is controlled by two different promoters. Using transgenic mice and virus-mediated expression of split-Cre, we show that efficient reporter gene activation is achieved in vivo. In the brain of transgenic mice, we genetically defined a subgroup of glial progenitor cells in which the Plp1- and the Gfap-promoter are simultaneously active, giving rise to both astrocytes and NG2-positive glia. Similarly, a subset of interneurons was labelled after viral transfection using Gad67- and Cck1 promoters to express split-Cre. Thus, split-Cre mediated genomic recombination constitutes a powerful spatial and temporal coincidence detector for in vivo targeting.     

<Emphasis Type="Italic">Saccharomyces cerevisiae</Emphasis>: a versatile eukaryotic system in virology

The yeast Saccharomyces cerevisiae is a well-established model system for understanding fundamental cellular processes relevant to higher eukaryotic organisms. Less known is its value for virus research, an area in which Saccharomyces cerevisiae has proven to be very fruitful as well. The present review will discuss the main achievements of yeast-based studies in basic and applied virus research. These include the analysis of the function of individual proteins from important pathogenic viruses, the elucidation of key processes in viral replication through the development of systems that allow the replication of higher eukayotic viruses in yeast, and the use of yeast in antiviral drug development and vaccine production.     

Dynamics of trigger factor interaction with translating ribosomes

In all organisms ribosome-associated chaperones assist early steps of protein folding. To elucidate the mechanism of their action, we determined the kinetics of individual steps of the ribosome binding/release cycle of bacterial trigger factor (TF), using fluorescently labeled chaperone and ribosome-nascent chain complexes. Both the association and dissociation rates of TF-ribosome complexes are modulated by nascent chains, whereby their length, sequence, and folding status are influencing parameters. However, the effect of the folding status is modest, indicating that TF can bind small globular domains and accommodate them within its substrate binding cavity. In general, the presence of a nascent chain causes an up to 9-fold increase in the rate of TF association, which provides a kinetic explanation for the observed ability of TF to efficiently compete with other cytosolic chaperones for binding to nascent chains. Furthermore, a subset of longer nascent polypeptides promotes the stabilization of TF-ribosome complexes, which increases the half-life of these complexes from 15 to 50 s. Nascent chains thus regulate their folding environment generated by ribosome-associated chaperones.     

环境因子对木棉种子萌发的影响

木棉(Bombax malabaricum DC.)是一种可以生长在干热河谷的落叶大乔木.河谷内,木棉成年树木生长良好,有大量结实,而其周围却很少有种苗出现.通过室内条件研究木棉种子的物理特性和萌发的生物学特性,可以了解该物种种子萌发对环境因子的需求,为探讨木棉在干热河谷内自然种群更新难和萌发阶段对这一特殊生境的适应性提供理论依据.经测定,木棉种子千粒重为39.08 g,比同科的其它物种要轻.四唑(TTC,1.O％)染色测定种子生活力的结果表明有生活力种子占64.0％.种皮对吸胀无障碍,种子在25℃吸胀2d时就开始萌发.检测了环境因子对种子萌发和幼苗生长的影响,结果表明,种子萌发对光照不敏感而且对温度有较广的适应性,15-35℃都可以萌发,25-35℃萌发率无显著差异,但随温度升高,萌发速率加快,幼苗长势增加;萌发过程对渗透胁迫敏感,聚乙二醇(PEG)浓度为0.10 g/mL时萌发率较对照显著下降,0.15 g/mL时种子就不能萌发;室温吸胀24h的种子对热激敏感,42℃热激2h后萌发率就已经显著下降;室湿下水杨酸(SA,1,10,100 mg/L)浸种24h,没有提高种子在萌发期间对干旱和热激的抗性.可见,高温和干旱是限制木棉种子成功萌发的关键因子,然而发现,温度较高时水分过大也会造成萌发后幼苗的死亡.