Phloem unloading follows an extensive apoplasmic pathway in cucumber (Cucumis sativus L.) fruit from anthesis to marketable maturing stage

The phloem unloading pathway remains unclear in fruits of Cucurbitaceae, a classical stachyose-transporting species with bicollateral phloem. Using a combination of electron microscopy, transport of phloem-mobile symplasmic tracer carboxyfluorescein, assays of acid invertase and sucrose transporter, and [(14)C]sugar uptake, the phloem unloading pathway was studied in cucumber (Cucumis sativus) fruit from anthesis to the marketable maturing stage. Structural investigations showed that the sieve element-companion cell (SE-CC) complex of the vascular bundles feeding fruit flesh is apparently symplasmically restricted. Imaging of carboxyfluorescein unloading showed that the dye remained confined to the phloem strands of the vascular bundles in the whole fruit throughout the stages examined. A 37 kDa acid invertase was located predominantly in the cell walls of SE-CC complexes and parenchyma cells. Studies of [(14)C]sugar uptake suggested that energy-driven transporters may be functional in sugar trans-membrane transport within symplasmically restricted SE-CC complex, which was further confirmed by the existence of a functional plasma membrane sucrose transporter (CsSUT4) in cucumber fruit. These data provide a clear evidence for an apoplasmic phloem unloading pathway in cucumber fruit. A presumption that putative raffinose or stachyose transporters may be involved in soluble sugars unloading was discussed.     

蔗糖磷酸化酶的分离纯化及其催化合成α-熊果苷的研究

考察了肠膜明串珠菌(Leuconostoc mesenteroides)G123厌氧发酵产蔗糖磷酸化酶下游的分离纯化工艺.收集的菌体经超声破碎得到粗酶液,通过硫酸铵沉淀、透析、阴离子交换层析分离后获得了电泳纯的蔗糖磷酸化酶,酶活回收率为31.7%,酶的分子量约为55.7 kD,纯化后的蔗糖磷酸化酶比活为115.3 U/mg.该酶在中性及偏酸性(pH5.5-8.0)情况下,酶稳定性较好,较报道的肠膜明串珠菌(Leuconostoc mesenteroides)B-1149的pH稳定范围宽.同时该酶在37℃保存2 h,酶活几乎没有下降.利用获得的纯酶以氢醌和蔗糖为底物催化合成α-熊果苷,在23 U/mL的酶反应体系中,60%蔗糖、5%氢醌、pH7.5,37℃,反应12 h,氢醌转化率达到16.3%,α-熊果苷的产量为20g/L.     

Characterization of the structure and function of Escherichia coli DegQ as a representative of the DegQ-like proteases of bacterial HtrA family proteins

HtrA family proteins play a central role in protein quality control in the bacterial periplasmic space. DegQ-like proteases, a group of bacterial HtrA proteins, are characterized by a short LA loop as compared with DegP-like proteases, and are found in many bacterial species. As a representative of the DegQ-like proteases, we report that Escherichia coli DegQ exists in?vivo primarily as a trimer (substrate-free) or dodecamer (substrate-containing). Biochemical analysis of DegQ dodecamers revealed that the major copurified protein substrate is OmpA. Importantly, wild-type DegQ exhibited a much lower proteolytic activity, and thus higher chaperone-like activity, than DegP. Furthermore, using cryo-electron microscopy we determined high-resolution structures of DegQ 12- and 24-mers in the presence of substrate, thus revealing the structural mechanism by which DegQ moderates its proteolytic activity.     

TNF and IL-1 exhibit distinct ubiquitin requirements for inducing NEMO–IKK supramolecular structures

Nuclear factor κB (NF-κB) essential modulator (NEMO), a regulatory component of the IκB kinase (IKK) complex, controls NF-κB activation through its interaction with ubiquitin chains. We show here that stimulation with interleukin-1 (IL-1) and TNF induces a rapid and transient recruitment of NEMO into punctate structures that are anchored at the cell periphery. These structures are enriched in activated IKK kinases and ubiquitinated NEMO molecules, which suggests that they serve as organizing centers for the activation of NF-κB. These NEMO-containing structures colocalize with activated TNF receptors but not with activated IL-1 receptors. We investigated the involvement of nondegradative ubiquitination in the formation of these structures, using cells deficient in K63 ubiquitin chains or linear ubiquitin chain assembly complex (LUBAC)-mediated linear ubiquitination. Our results indicate that, unlike TNF, IL-1 requires K63-linked and linear ubiquitin chains to recruit NEMO into higher-order complexes. Thus, different mechanisms are involved in the recruitment of NEMO into supramolecular complexes, which appear to be essential for NF-κB activation.     

Differential Actin-regulatory Activities of Tropomodulin1 and Tropomodulin3 with Diverse Tropomyosin and Actin Isoforms

Tropomodulins (Tmods) are F-actin pointed end capping proteins that interact with tropomyosins (TMs) and cap TM-coated filaments with higher affinity than TM-free filaments. Here, we tested whether differences in recognition of TM or actin isoforms by Tmod1 and Tmod3 contribute to the distinct cellular functions of these Tmods. We found that Tmod3 bound ∼5-fold more weakly than Tmod1 to α/βTM, TM5b, and TM5NM1. However, surprisingly, Tmod3 was as effective as Tmod1 at capping pointed ends of skeletal muscle α-actin (α_sk-actin) filaments coated with α/βTM, TM5b, or TM5NM1. Tmod3 only capped TM-coated α_sk-actin filaments more weakly than Tmod1 in the presence of recombinant αTM2, which is unacetylated at its NH₂ terminus, binds F-actin weakly, and has a disabled Tmod-binding site. Moreover, both Tmod1 and Tmod3 were similarly effective at capping pointed ends of platelet β/cytoplasmic γ (γ_cyto)-actin filaments coated with TM5NM1. In the absence of TMs, both Tmod1 and Tmod3 had similarly weak abilities to nucleate β/γ_cyto-actin filament assembly, but only Tmod3 could sequester cytoplasmic β- and γ_cyto-actin (but not α_sk-actin) monomers and prevent polymerization under physiological conditions. Thus, differences in TM binding by Tmod1 and Tmod3 do not appear to regulate the abilities of these Tmods to cap TM-α_sk-actin or TM-β/γ_cyto-actin pointed ends and, thus, are unlikely to determine selective co-assembly of Tmod, TM, and actin isoforms in different cell types and cytoskeletal structures. The ability of Tmod3 to sequester β- and γ_cyto-actin (but not α_sk-actin) monomers in the absence of TMs suggests a novel function for Tmod3 in regulating actin remodeling or turnover in cells.     

Does Campylobacter jejuni Form Biofilms in Food-Related Environments?

Campylobacter jejuni is one of the most frequent causes of bacterial gastrointestinal food-borne infection worldwide. This species is part of the normal flora of the gastrointestinal tracts of animals used for food production, including poultry, which is regarded as the primary source of human Campylobacter infections. The survival and persistence of C. jejuni in food processing environments, especially in poultry processing plants, represent significant risk factors that contribute to the spread of this pathogen through the food chain. Compared to other food-borne pathogens, C. jejuni is more fastidious in its growth requirements and is very susceptible to various environmental stressors. Biofilm formation is suggested to play a significant role in the survival of C. jejuni in the food production and processing environment. The aims of this minireview were (i) to examine the evidence that C. jejuni forms biofilms and (ii) to establish the extent to which reported and largely laboratory-based studies of C. jejuni biofilms provide evidence for biofilm formation by this pathogen in food processing environments. Overall existing studies do not provide strong evidence for biofilm formation (as usually defined) by most C. jejuni strains in food-related environments under the combined conditions of atmosphere, temperature, and shear that they are likely to encounter. Simple attachment to and survival on surfaces and in existing biofilms of other species are far more likely to contribute to C. jejuni survival in food-related environments based on our current understanding of this species.     

向日葵单染色体显微分离及特异文库的构建

采用玻璃针分离法,通过显微操作系统成功地分离到内葵杂3号三交种和单交种的随体染色体,经两轮LA．PCR扩增得到250～1500bp的DNA片段。用各自的基因组DNA标记成探针,与随体染色体扩增产物进行Southern杂交,显示杂交信号,证明内葵杂3号三交种和单交种随体染色体DNA已被成功扩增。将第2轮PCR产物构建质粒文库,得到三交种和单交种克隆数分别约为2．26×10^5和2．57×10^5。各随机挑取30个重组子进行分析,发现插入片段大小分别为200-700bp和200～500bp,平均插入片段大小分别为535bp和480bp。这是染色体微分离与微克隆技术首次在向日葵上的应用。     

Effects of NaCl stress on the growth and photosynthetic characteristics of Ulmus pumila L. seedlings in sand culture

The effects of NaCl stress on the growth and photosynthetic characters of Ulmus pumila L. seedlings were investigated under sand culture condition. With increasing NaCl concentration, main stem height, branch number, leaf number, and leaf area declined, while Na⁺ content and the Na⁺/K⁺ ratio in both expanded and expanding leaves increased. Na⁺ content was significantly higher in expanded leaves than in those just expanding. Chlorophyll (Chl) a and Chl b contents declined as NaCl concentration increased. The net photosynthetic rate, intercellular CO₂ concentration, stomatal conductance, and transpiration rate also declined, but stomatal limitation value increased as NaCl concentration increased. Both the maximal quantum yield of PSII photochemistry and the effective quantum yield of PSII photochemistry declined as NaCl concentration rose. These results suggest that the accumulation of Na⁺ in already expanded leaves might reduce damage to the expanding leaves and help U. pumila endure high salinity. The reduced photosynthesis in response to salt stress was mainly caused by stomatal limitation.     

UV-irradiation mutation of tetraspores of Gracilariopsis lemaneiformis and screening of thermotolerant strains

The rhodophyte Gracilariopsis lemaneiformis is one of the most economically important marine algae in China. In order to extend the cultivation period and enlarge the cultivation area, mutagenic breeding was used in this study for screening strains with thermal tolerance. The tetraspores were mutated by UV irradiation, and then thermotolerant strains were screened by exposing developing tetraspores to a range of higher temperatures. Two heat-tolerant mutants, MT-17 and MT-18, were obtained and physiologically compared with cultivar 981 and the wild strains. The results demonstrated that the two mutants grew at significantly higher rates than wild strains and similar to that of cultivar 981 both in the laboratory and the sea. The malondialdehyde contents in MT-17 and MT-18 were all significantly lower than the wild-type after 3-day heat stress, and that in MT-18 was lower than cultivar 981. The superoxide dismutase activities of MT-17 and MT-18 were significantly higher than the wild-type, and those of MT-18 and cultivar 981 were at the same level all through the treatment. The heat shock protein 70 gene expressions of two mutants was higher than the wild-type and that of MT-18 remained at the same level as that of cultivar 981 after 4 h heat shock. All these indicate that the two mutants were more tolerant to high temperature than the wild-type. RSAP (restriction site amplified polymorphism) analysis indicated the genetic background of the two mutants may be changed. The mutagenesis and selection process may help to develop heat-tolerant G. lemaneiformis cultivars in the future.