Molecular Evolution of VEF-Domain-Containing PcG Genes in Plants

Arabidopsis VERNALIZATION2 （VRN2）, EMBRYONIC FLOWER2 （EMF2）, and FERTILIZATION-INDEPENDENT SEED2 （FIS2） are involved in vernalization-mediated flowering, vegetative development, and seed development, respectively. Together with Arabidopsis VEF-L36, they share a VEF domain that is conserved in plants and animals. To investigate the evolution of VEF-domain-containing genes （VEF genes）, we analyzed sequences related to VEF genes across land plants. To date, 24 full-length sequences from 11 angiosperm families and 54 partial sequences from another nine families were identified. The majority of the full-length sequences identified share greatest sequence similarity with and possess the same major domain structure as Arabidopsis EMF2. EMF2-1ike sequences are not only widespread among angiosperms, but are also found in genomic sequences of gymnosperms, lycophyte, and moss. No FIS2- or VEF-L36-1ike sequences were recovered from plants other than Arabidopsis, including from rice and poplar for which whole genomes have been sequenced. Phylogenetic analysis of the full-length sequences showed a high degree of amino acid sequence conservation in EMF2 homologs of closely related taxa. VRN2 homologs are recovered as a clade nested within the larger EMF2 clade. FIS2 and VEF-L36 are recovered in the VRN2 clade. VRN2 clade may have evolved from an EMF2 duplication event that occurred in the rosids prior to the divergence of the eurosid I and eurosid II lineages. We propose that dynamic changes in genome evolution contribute to the generation of the family of VEF-domain-containing genes, Phylogenetic analysis of the VEF domain alone showed that VEF sequences continue to evolve following EM F2NRN2 divergence in accordance with species relationship. Existence of EMF2-1ike sequences in animals and across land plants suggests that a prototype form of EMF2 was present prior to the divergence of the plant and animal lineages. A proposed sequence of events, based on domain organization and occurrence of intermediate seque     

Antioxidant activity of mangostin in cell-free system and its effect on K562 leukemia cell line in photodynamic therapy

Mangostin （MAG）, a kind of xanthone widely used in diet and medicine, has antioxidant, anti-inflammatory, antimicrobial, and anticancer activities. On account of its antioxidant activity, MAG might protect cancer cells from free radical damage in photodynamic therapy （PDT） during which reactive oxygen species production was stimulated leading to irreversible tumor cell injury. In this study, the antioxidant activity of MAG was investigated and the influence of MAG on K562 cells in 5-aminolevulinic acid （ALA）-based PDT is demon- strated. The results showed that MAG could scavenge hydroxyl radical, superoxide anion, and hydrogen per- oxide and inhibit the formation of malondialdehyde （MDA）, but increase the amounts of singlet oxygen in cell-free systems. MAG inhibits cell proliferation and enhances cell apoptosis, lipid peroxidation, and DNA damage in ALA-PDT on K562 cells. NAN3, a singlet oxygen quencher, suppresses the MAG-induced cell apoptosis, lipid peroxidation, and DNA damage. In con- clusion, MAG enhances the PDT-induced cytotoxicity in K562 cells and singlet oxygen was involved in this process. These results implied that the effect of antioxidants on PDT might be determined by its sensitization ability to singlet oxygen.     

Alb4 of Arabidopsis Promotes Assembly and Stabilization of a Non Chlorophyll-Binding Photosynthetic Complex,the CF1CF0-ATP Synthase

All members of the YidC/Oxal/Alb3 protein family are evolutionarily conserved and appear to function in membrane protein integration and protein complex stabilization. Here, we report on a second thylakoidal isoform of Alb3, named Alb4. Analysis of Arabidopsis knockout mutant lines shows that AIb4 is required in assembly and/or stability of the CF1CF0-ATP synthase （ATPase）. alb4 mutant lines not only have reduced steady-state levels of ATPase subunits, but also their assembly into high-molecular-mass complexes is altered, leading to a reduction of ATP synthesis in the mutants. Moreover, we show that Alb4 but not AIb3 physically interacts with the subunits CF1β and CF0ll. Summarizing, the data indicate that AIb4 functions to stabilize or promote assembly of CF1 during its attachment to the membrane-embedded CF0 part.     

LSCHL4 from Japonica Cultivar,Which Is Allelic to NAL 1, Increases Yield of Indica Super Rice 93-11

The basic premise of high yield in rice is to improve leaf photosynthetic efficiency and coordinate the sourcesink relationship in rice plants. Quantitative trait loci （QTLs） related to morphological traits and chlorophyll content of rice leaves were detected at the stages of heading to maturity, and a major QTL （qLSCHL4） related to flag leaf shape and chlorophyll content was detected at both stages in recombinant inbred lines constructed using the indica rice cultivar 93-11 and the japonica rice cultivar Nipponbare. Map-based cloning and expression analysis showed that LSCHL4 is allelic to NAL1, a gene previously reported in narrow leaf mutant of rice. Overexpression lines transformed with vector carrying LSCHL4 from Nipponbare and a near-isogenic line of 93-11 （NIL-9311） had significantly increased leaf chlorophyll content, enlarged flag leaf size, and improved panicle type. The average yield of NIL-9311 was 18.70% higher than that of 93-11. These results indicate that LSCHL4 had a pleiotropic function. Exploring and pyramiding more high-yield alleles resem- bling LSCHL4 for super rice breeding provides an effective way to achieve new breakthroughs in raising rice yield and generate new ideas for solving the problem of global food safety.     

The anorexic effect of Ex4/Fc through GLP-1 receptor activation in high-fat diet fed mice

Exendin-4 （Ex4）, a peptide initially found in the saliva of the Gila monster, can activate the signaling pathway of the incre- tin hormone glucagon-like peptide-1 （GLP-1） through the GLP-1 receptor （GLP-1R）. We previously reported that a chimera protein consisting of Ex4 and mouse IgG heavy chain constant regions （Ex4/Fc） can exert biological effects of GLP-1, such as improving glycemic control and ameliorating manifestations in diabetic mice. The aim of this study was to determine whether Ex4/Fc is effective in modulating energy homeostasis in mice. Our results showed that in vivo expres- sion of Ex4/Fc by intramuscular injection of the plasmid en- coding Ex4/Fc followed by local electroporation effectively decreased food intake in the mice on high-fat diet （HFD） feeding. In addition, the reduced energy intake was associated with the decreased excrements from the Ex4/Fc-treated HFD mice but not the Fc control mice. Remarkably, the Ex4/Fc- treated HFD mice displayed significantly lower triglyceride （TG） levels when compared with the control mice. Interest- ingly, while the leptin levels were not changed, the circulating ghrelin levels were higher in Ex4/Fc mice than those in the Fc control mice. These results suggested that Ex4/Fc can improve energy metabolism and lipid metabolism through GLP-1R in mice under excessive nutrition conditions.     

The role of MAPK and FAS death receptor pathways in testicular germ cell apoptosis induced by lead

The aim of the present study is to investigate gene expression involved in the signal pathway of MAPK and death signal receptor pathway of FAS in lead- induced apoptosis of testicular germ cells. First, cell viabilities were determined by MTT assay. Second, using single cell gel-electrophoresis test （comet assay） and TUNEL staining technique, apoptotic rate and cell apoptosis localization of testicular germ cells were measured in mice treated with 0.15%, 0.3%, and 0.6% lead, respectively. Third, the immunolocalization of K-ras, c-fos, Fas, and active caspase-3 proteins was determined by immunohistochemistry. Finally, changes in the translational levels of K-ras, c-fos, Fas, and active caspase-3 were further detected by western blot analysis. Our results showed that lead could significantly induce testicular germ cell apoptosis in a dose-dependent manner （P 〈 0.01）. The mechanisms were closely related to the increased expressions of K-ras, c-fos, Fas, and active caspase-3 in apoptotic germ cells. In conclusion, K-ras/c-fos and Fas/caspase-3 death signafing receptor pathways were involved in the lead-induced apoptosis of the testicular germ cells in mice.     

Mobilization and Acquisition of Sparingly Soluble P-Sources by Brassica Cultivars under P-Starved Environment I. Differential Growth Response,P-Efficiency Characteristics and P-Remobilization

Phosphorus （P） starvation is highly notorious for limiting plant growth around the globe. To combat P-starvation, plants constantly sense the changes in their environment, and elicit an elegant myriad of plastic responses and rescue strategies to enhance P-solublization and acquisition from bound soil P-forms. Relative growth responses, P-solublization and P- acquisition ability of 14 diverse Brassica cultivars grown with sparingly soluble P-sources （Rock-P （RP） and Ca3（PO4）2 （TCP）） were evaluated in a solution culture experiment. Cultivars showed considerable genetic diversity in terms of biomass accumulation, concentration and contents of P and Ca in shoots and roots, P-stress factor （PSF） and P use efficiency. Cultivars showed variable P-stress tolerance, and cultivars depicting low PSF and high P-efficiency values were better adaptable to P-starvation. In experiment 2, after initial feeding on optimum nutrition for 12 d after transplanting （DAT）, class-I （low P-tolerant （Oscar and Con-II）） and class-II （low P-sensitive （Gold Rush and RL-18）） cultivars were exposed to P-free environment for 25 d. All of the cultivars remobilized P from above ground parts to their roots during growth in P-free environment, the magnitude of which was variable in tested cultivars. P-concentrations （[P]s） at 37 DAT were higher in developing compared with developed leaves. Translocation of absorbed P from metabolically inactive to active sites in P- stressed plants may have helped class-I cultivars to establish a better rooting system, which provided a basis for enhanced P-utilization efficiency （PUE） and tolerance against P-stress. By supplying TCP and RP spatially separated from other nutrients in split root study, class-I cultivars were still able to mobilize RP and TCP more efficiently compared with class-II cultivars. To compare the growth behavior under P-stress, cultivars were grown in pots for 41 d after sowing, using a soil low in P （NaHCO3-extractable P = 3.97 mg/kg, M     

Hydrogen peroxide induces the activation of the phospholipase C-γ1 survival pathway in PC12 cells： protective role in apoptosis

It has been reported that phospholipase C-γ1 （PLC-γ1） plays an important protective role in hydrogen peroxide （H2O2）-induced pheochromocytoma （PC） 12 cells death. However, most studies have used high doses of H2O2 and the downstream targets of PLC-γ1 activation remain to be identified. The present study was designed to examine the roles of PLC-γ1 signaling pathway in the apoptosis of PC12 cells induced by low dose of H2O2, as well as the downstream factors involved in this pathway. Low-dose treatment of H2O2 resulted in PLC-γ1 tyrosine phosphorylation in a time-dependent manner and H2O2 killed the PC12 cells by inducing necrosis. In contrast, pretreatment of PCI2 cells with U73122, a specific inhibitor of PLC, markedly increased the percentage of dead cells. The mode of cell death was converted to apoptosis as determined by Hoechst/PI nuclear staining and fluorescence microscopy. Western blot analysis demonstrated that the expression of Bcl-2 protein and the activation of pro-caspase-3 were not significantly affected by low dose of H2O2 alone. However, after pretreatment with U73122, Bcl-2 protein expression was dramatically decreased and the activation of pro-caspase-3 was significantly increased. We concluded that PLC-γ1 plays an important protective role in H2O2-induced PC12 cells death. Bcl-2 and caspase-3 probably participate in the signaling pathway as downstream factors.     

Arabidopsis STAY-GREEN2 Is a Negative Regulator of Chlorophyll Degradation during Leaf Senescence

Chlorophyll （Chl） degradation causes leaf yellowing during senescence or under stress conditions. For Chl breakdown, STAY-GREEN1 （SGR1） interacts with Chl catabolic enzymes （CCEs） and light-harvesting complex II （LHCII） at the thylakoid membrane, possibly to allow metabolic channeling of potentially phototoxic Chl breakdown intermediates. Among these Chl catabolic components, SGR1 acts as a key regulator of leaf yellowing. In addition to SGR1 （At4g22920）, the Arabidopsis thaliana genome contains an additional homolog, SGR2 （At4g11910）, whose biological function remains elusive. Under senescence-inducing conditions, SGR2 expression is highly up-regulated, similarly to SGR1 expression. Here we show that SGR2 function counteracts SGR1 activity in leaf Chl degradation; SGR2-overexpressing plants stayed green and the sgr2-1 knockout mutant exhibited early leaf yellowing under age-, dark-, and stress-induced senescence conditions. Like SGR1, SGR2 interacted with LHCII but, in contrast to SGR1, SGR2 interactions with CCEs were very limited. Furthermore, SGR1 and SGR2 formed homo- or heterodimers, strongly suggesting a role for SGR2 in negatively regulat- ing Chl degradation by possibly interfering with the proposed CCE-recruiting function of SGR1. Our data indicate an antagonistic evolution of the functions of SGR1 and SGR2 in Arabidopsis to balance Chl catabolism in chloroplasts with the dismantling and remobilizing of other cellular components in senescing leaf cells.     

Mammalian cell display for rapid screening scFv antibody therapy

Human antibodies are beginning to draw attention for use in immune gene therapy. The efficient generation of effective therapeutic monoclonal antibodies suitable for the treatment of cancers and infectious diseases would be enormously valuable. Antibody display methods are increasingly used to screen human monoclonal antibodies. Here we report the construction of a mammalian cell display method derived from a naive antibody repertoire, for which human single- chain variable fragments （scFv） have been transiently dis- played on 293T cell surfaces based on a pDisplay vector. The sizes of the current pDisplay-scFv antibody repertoires have been estimated to be 0.74 × 10^7. An immunoblot assay confirmed the expression of the scFv antibody library. The subceilular distribution of ErbB3-scFv expression plasmid facilitated the display of ErbB3 scFv on the cell membrane surface and the efficiency of the display was evaluated by fluorescence-activated cell sorting. This method of mamma- lian cell display was verified by successfully screening ErbB3 scFv candidates. A published scFv control was used to confirm the feasibility of the ErbB3 scFv screening process. Three ErbB3 scFv candidates were produced and they were found to have affinity similar to the published scFv candidate. Thus, the present screening system provided an optimal alternative for rapid acquisition of a novel candi- date scFv sequence to target genes with high affinity in vitro.     

Baculovirus Host-Range

Baculoviruses are used as microbial insecticides, protein expression vectors, epitope display platforms, and most recently as vectors for gene therapy. Understanding the mechanisms that control baculovirus host-range and tissue tropisms are important for assessing their safety and for improving their properties for these biotechnology applications. In the past two decades some progress has been made and several baculovirus genes that influence host-range have been identified. Despite this progress, our understanding of the underlying mechanisms that restrict baculovirus host-range is still limited. Here we review what is currently known about baculovirus genes that influence virus host-range     

Conserved Functions of Arabidopsis and Rice CC-Type Glutaredoxins in Flower Development and Pathogen Response

Glutaredoxins （GRXs） are ubiquitous oxidoreductases that play a crucial role in response to oxidative stress by reducing disulfides in various organisms. In planta, three different GRX classes have been identified according to their active site motifs. CPYC and CGFS classes are found in all organisms, whereas the CC-type class is specific for higher land plants. Recently, two Arabidopsis CC-type GRXs, ROXY1 and ROXY2, were shown to exert crucial functions in petal and anther initiation and differentiation. To analyze the function of CC-type GRXs in the distantly related monocots, we isolated and characterized OsROXY1 and OsROXY2-two rice homologs of ROXY1. Both genes are expressed in vegetative and reproductive stages. Although rice flower morphology is distinct from eudicots, OsROXY1/2 floral expression patterns are similar to their Arabidopsis counterparts ROXY1/2. Complementation experiments demonstrate that OsROXY1 and OsROXY2 can fully rescue the roxyl floral mutant phenotype. Overexpression of OsROXY1, OsROXY2, and ROXY1 in Arabidopsis causes similar vegetative and reproductive plant developmental defects. ROXY1 and its rice homologs thus exert a conserved function during eudicot and monocot flower development. Strikingly, overexpression of these CC-type GRXs also leads to an increased accumulation of hydrogen peroxide levels and hyper-susceptibility to infection from the necrotrophic pathogen Botrytis cinerea, revealing the importance of balanced redox processes in flower organ develop- ment and pathogen defence.     

Protective effects of DIDS against ethanol-induced gastric mucosal injury in rats

The compound 4,4＇-diisothiocyanostilbene-2,2＇-disulfonic acid （DIDS） is an efficient anion exchanger inhibitor that can block the activities of anion exchanger 2 （AE2）, which plays an indispensable role in gastric acid secretion. DIDS also has potent anti-oxidative and antiapoptosis activities. This study aimed to investigate the effect of DIDS on ethanol-induced mucosal damage in rats and to evaluate the underlying mechanisms that mediate the action of the compound. The rats received 1 ml of absolute ethanol or saline orally. DIDS [50 mg/kg intravenous （i.v.）] was given 5 min before ethanol administration. Gastric lesions were evaluated macroscopically, microscopically, and electron microscopically at 60 min after ethanol challenge. Gastric myeloperoxidase （MPO） activity, malonyldialdehyde （MDA） level, prostaglandin E2 （PGE2） synthesis, and cyclooxygenase-2 （COX-2） expression were assessed. For the evaluation of the effect of DIDS on gastric acid secretion, histamine-stimulatory gastric acid secretion was examined with or without pretreatment of DIDS （50 mg/kg; i.v.）. Ethanol-induced gastric lesions were characterized by increasing gastric MDA level, MPO activity, and COX-2 expression, and decreasing PGE2 synthesis. It was found that DIDS significantly reduced the extent of gastric mucosal damage and reversed tissue MDA level and MPO activity. DIDS further enhanced the expression of COX-2 and reversed the decrease of PGE2. Our results suggested that DIDS is beneficial in rat model of gastric injury through mechanisms that involve inhibiting inflammatory cell infiltration and lipid peroxidation and up-regulating the COX-2/PGE2 pathway.     

Novel benzenediamine derivative FC99 ameliorates zymosan-induced arthritis by inhibiting RORγt expression and Thl7 cell differentiation

Increased IL-17-producing helper T （Thl7） cells have been observed in patients with rheumatoid arthritis （RA）. The retinoic-acid-related orphan nuclear receptor （RORγt） is the master regulator of Thl7 cells. Our previous research showed that FC99 possesses anti-inflammation activity. However, to date the effects of FC99 on RORγt expression in Thl7 cell differentiation have not been investigated yet. In the present study, we found that FC99 significantly attenu- ated arthritis-like symptoms, i.e., suppressing the develop- ment of paw edema in zymosan-induced arthritis （ZIA） mice. H＆E staining showed that the infdtration of inflamma- tory cells in ankle synovial tissues was significantly suppressed. FC99 also reduced the mRNA levels of pro-in- flammatory cytoklnes in ankle synovial tissues as shown by Q-PCR analysis. The protein levels of the pro-inflammatory cytoklnes in sera were also suppressed after FC99 treatment. Moreover, FC99 decreased the RORγt mRNA level in spleen tissues. Thl7 cell percentage was significantly decreased in spleens and draining lymph nodes （dLNs）. The mRNA and protein levels of IL-17A and IL-23 were reduced after FC99 treatment in ZIA mice. Furthermore, in vitro experiments showed that FC99 inhibited the expression of IL-6 in LPS- induced RAW264.7 cells and BMDCs. Moreover, FC99 sig- nificantly inhibited the RORγt expression in PMA-induced CD4＋ T cells and LPS-induced RAW264.7 cells. These data indicate that FC99 improves arthritis-like pathological symp- toms in vivo and in vitro, which might be related to the inhib- ition of RORγt expression in Thl7 cells. Our findings suggest that FC99 may be a potential therapeutic candidate for the treatment of RA and other inflammatory disorders.