Resistance of SKW6 cell to apoptosis induction with anti-Fas antibody upon transduction of a reverse fragment to a cDNA encoding human 6A8 α-mannosidase

The effect of transduction with a reverse fragment to a cDNA encoding human 6A8 ?-mannosidase on apoptosis induction of human B cell line SKW6 by anti-Fas antibody was tested. Apoptosis-inducer of anti-Fas monoclonal antibody was used to induce apoptosis in SKW6 cells. Giemsa’s staining, Annexin-V-FLUOS staining and DNA ladder test were used to determine the events of apoptosis. Indirect immunofluorescent staining with anti-Fas antibody was performed to detect the surface Fas expression. In a time-course test of 12, 24 and 36 h for apoptosis induction by anti-Fas antibody, DNA ladder was observed in the wild-type SKW6 cells in a time-dependent fashion. Mock transduction had no effect on DNA ladder production. However, no DNA ladder was detected in the rAAV-antisense 6A8 cDNA-transduced SKW6. Results from Annexin-V-FLUOS staining on anti-Fas antibody-treated cells revealed that the staining-positive rate in the rAAV-antisense 6A8 cDNA-transduced SKW6 cells was decreased in comparison to that in the wild-type and the mock-transduced cells. Giemsa’s staining observation showed that the number of dying (with apoptotic bodies) and dead cells was reduced in the rAAV-antisense 6A8 cDNA-transduced SKW6 cells in comparison with that in the wild-type and the mock-transduced cells upon anti-Fas antibody induction. The transduction did not affect the expression of Fas molecular on cell surface. 100% cells in all the groups showed Fas expression. The SKW6 cells became resistant to apoptosis induction by anti-Fas antibody upon transduction with a reverse fragment to a cDNA encoding human 6A8 a-mannosidase. The transduction did not affect the expression of Fas molecule on cells.     

Resistance of SKW6 cell to apoptosis induction with anti-Fas antibody upon transduction of a reverse fragment to a cDNA encoding human 6A8 α-mannosidase

The effect of transduction with a reverse fragment to a cDNA encoding human 6A8 α-mannosidase on apoptosis induction of human B cell line SKW6 by anti-Fas antibody was tested. Apoptosis-inducer of anti-Fas monoclonal antibody was used to induce apoptosis in SKW6 cells. Giemsa's staining, Annexin-V-FLUOS staining and DNA ladder test were used to determine the events of apoptosis. Indirect immunofluorescent staining with anti-Fas antibody was performed to detect the surface Fas expression. In a time-course test of 12, 24 and 36 h for apoptosis induction by anti-Fas antibody, DNA ladder was observed in the wild-type SKW6 cells in a time-dependent fashion. Mock transduction had no effect on DNA ladder production. However, no DNA ladder was detected in the rAAV-antisense 6A8 cDNA-transduced SKW6. Results from Annexin-V-FLUOS staining on anti-Fas antibody-treated cells revealed that the staining-positive rate in the rAAV-antisense 6A8 cDNA-transduced SKW6 cells was decreased in comparison to that in the wild-     

Transglutaminase 3 expression in C57BL／6J mouse embryo epidermis and the correlation with its differentiation

Epidermal-type transglutaminase 3 (TGM3) is involved in the cross-linking of structural proteins to form the cornified envelope in the epidermis. In the present study, we detected the expression of TGM3 in the mouse embryo using RT-PCR.TGM3 mRNA is weakly presented from E11.5 to E14.5 and increases significantly from E15.5 to birth. Then we determined the spatial and temporal expression pattern of TGM3 in the skin and other organs by in situ hybridization. We found a deprivation of TGM3 in skin at E11.5, while a rich supply in periderm cells and a weak expression in basal cells from E12.5 to E14.5. From the period of E15.5 to E16.5, after keratinization in the epidermis, TGM3 was expressed in the granular and cornified layers. The electron microscopic observation of the C57BL/6J mouse limb bud skin development provided several morphological evidences for the epidermal differentiation. The above findings suggest that the expression of TGM3 plays a important role in the epidermis differentiation in embryogenesis.     

Androgen receptor coregulator ARA267-α interacts with death receptor-6 revealed by the yeast two-hybrid

ARA267-α is a newly identified androgen receptor coactivator. In order to further elucidate its precise role in cells, using the ARA267-α fragment containing four PHD and one SET conserved domains as bait we revealed an ARA267-α-PHD-SET-interacting protein, death receptor-6 (DR6), in the yeast two-hybrid screening. DR6 is the member of TNF receptor family and has a death domain in its intracellular cytoplasmic portion (DR6cp) to mediate the cell apoptosis. The interaction between ARA267-α-PHD-SET and DR6cp was confirmed in vitro and in vivo. Our finding implied that androgen signaling pathway might cross talk with apoptosis signaling pathway through the interaction between ARA267-α and DR6.     

Expression and localization of GhH6L,a putative classical arabinogalactan protein in cotton （Gossypium hirsutum）

Arabinogalactan proteins （AGPs） are a large family of highly glycosylated of hydroxyproline-rich glycoproteins that play important roles in plant growth, development, and signal transduction. A cDNA encoding a putative classical AGP named GhH6L was isolated from cotton fiber cDNA libraries, and the deduced protein contains 17 copies of repetitive motif of X-Y-proline-proline-proline （where X is serine or alanine and Y is threonine or serine）. Northern blotting analysis and quantitative RT-PCR results showed that it was preferentially expressed in 10 days post-anthesis （dpa） fibers and was also developmentally regulated. A promoter fragment was isolated from cotton （Gossypium hirsutum） by genome walking PCR. Expression of β-glucuronidase （GUS） gene under the GhH6L promoter was examined in the transgenic Arabidopsis plants; only petiole and pedicel were stained, no staining was detected in other tissues. Subcellular localization indicated that GhH6L was localized to the plasma membrane and in the cytoplasm. These data further our understanding of GhH6L as well as shed light on functional insight to GhH6L in cotton.     

The effects of detergents DDM and β-OG on the singlet excited state lifetime of the chlorophyll a in cytochrome b_6f complex from spinach chloroplasts

The singlet excited state lifetime of the chlorophyll a (Chl a) in cytochrome b6f (Cyt b6f) complex was reported to be shorter than that of free Chl a in methanol, but the value was different for Cyt b6f com-plexes from different sources (~200 and ~600 ps are the two measured results). The present study demonstrated that the singlet excited state lifetime is associated with the detergents n-dodecyl-β-D- maltoside (DDM) and n-octyl-β-D-glucopyranoside (β-OG), but has nothing to do with the different sources of Cyt b6f complexes. Compared with the Cyt b6f dissolved in β-OG, the Cyt b6f in DDM had a lower fluorescence yield, a lower photodegradation rate of Chl a, and a shorter lifetime of Chl a excited state. In short, the singlet excited state lifetime, ~200 ps, of the Chl a in Cyt b6f complex in DDM is closer to the true in vivo.     

The effects of detergents DDM and β-OG on the singlet excited state lifetime of the chlorophyll a in cytochrome b6f complex from spinach chloroplasts

The singlet excited state lifetime of the chlorophyll a (Chi a) in cytochrome b6f (Cyt b6f) complex was reported to be shorter than that of free Chl a in methanol, but the value was different for Cyt b6f complexes from different sources (～200 and ～600 ps are the two measured results). The present study demonstrated that the singiet excited state lifetime is associated with the detergents n-dodecyl-β-D-maltoside (DDM) and n-octyl-β-D-glucopyranoside (β-OG), but has nothing to do with the different sources of Cyt b6f complexes. Compared with the Cyt b6f dissolved in β-OG, the Cyt b6f in DDM had a lower fluorescence yield, a lower photodegradation rate of Chl a, and a shorter lifetime of Chl a excited state. In short, the singlet excited state lifetime, ～200 ps, of the Chl a in Cyt b6f complex in DDM is closer to the true in vivo.     

RNA interference of a trehalose-6-phosphate synthase gene reveals its roles in the biosynthesis of chitin and lipids in Heortia vitessoides(Lepidoptera:Crambidae)

Trehalose 6-phosphate synthase(TPS),an enzyme that hydrolyzes two glucose molecules to yield trchalose,plays a pivotal role in various physiological processes.In this study,we cloned the trehalose-6-phosphate synthase gene(HvTPS)and investigated its expression patterns in various tssues and d:velopmental stages in Heortia vitessoides Moore(Lepidoptera:Crambidac).HvTPS was highly expressed in the fat body and after pupation or before molting.We knocked down TPS in H.vitessoides by RNA interference and found that 3.0μg of dsHvTPS resulted in optimal interference at 24 h and 36 h post-injection and caused a sharp decline in the survival rate during the 5th instar larval-pupal stage and obviously abnormal or lethal phenotypes.Additionally.compared to the controls,TPS activity and trehalose contents were significantly lower and the glucose content was significantly higher 24 h or 36 h after injection with 3.0μg of dsHIvTPS.Furthermore,the silencing of HvTPS suppressed the cxpression of six key genecs in the chitin biosynthesis pathway and one key gene related to lipid catabolism.The expression levels of two genes associated with lipid biosynthesis were upregulated.These results strongly suggest that HvTPS is essential for the normal growth and development of H.vitessoides and provide a reference for further studies of the utility of key genes involved in chitin and lipid biosynthesis for controlling insect development.     

会议报道：北京生物入侵国际研讨会: 2004年6月6日－11日

 Scientists and policy-makers are now well aware of the ecological and societal problems associated with the introduction of species by humans across natural barriers to dispersal. Some introduced species spread abundantly in their new habitats and have negative effects on existing species, both agricultural and natural. Such introduced, invasive species can alter fire, nutrient, and hydrological regimes and have large economic costs. In many parts of the world, this awareness has come rather late: commerce and travel have already carried large numbers of plants, animals, and microbes between many regions. In contrast, China's relative self-remove from global trade during much of the second half of the 20th century may have earned it a partial reprieve from invasions from abroad and reduced the number of invasions from China to elsewhere. If so, there could be a new wave of species invasions between China and North America impending. China's trade with the U.S. increased by 460% from 1992 to 2002, according to the U.S. Department of Commerce. This may be the critical moment to apply the lessons learned from invasions across other oceans and forestall new invasions across the Pacific. We need to match growth in trade with growth in the science and policy needed to prevent and control introductions. To this end, some 100 scientists and representatives of governmental and non-governmental organisations from eight countries gathered on June 6-11 for the Beijing International Symposium on Biological Invasions, subtitled "Species Exchanges Between Eastern Asia and North America: Threats to Environment and Economy". The Institute of Botany of the Chinese Academy of Sciences and the Sino-Ecologists Club Overseas organized the meeting, under the leadership of Xinguo Han and Ming Dong for the Institute and Shili Miao for the Club. The National Science Foundation of China, the U.S. Geological Survey, and the Asian Ecology Section of the Ecological Society of America provided additional support. Shili Miao and Jianhui Huang coordinated the program and logistics, and the Beijing Botanical Garden provided the venue, appropriately stocked with introduced plants from around the world, presumably non-invasive ones. There were two keynote addresses. Harold Mooney (Stanford, USA) highlighted the need for scientists to develop quantitative tools that managers can use to analyze the extent and the impacts of invasions. Richard Mack (Washington State, USA) noted that the phylogenetic, latitudinal, and climatic similarities between China and the U.S. may predispose introductions from one to the other to bloom into invasions. The 37 other talks and the posters focused on current patterns of invasion in eastern Asia, the theory of invasions, techniques to manage invasions, and policies to prevent them.     

Dual-Targeting of Arabidopsis 6-Phosphogluconolactonase 3 （PGL3）to Chloroplasts and Peroxisomes Involves Interaction with Trx m2 in the Cytosol

The oxidative pentose-phosphate pathway （OPPP） represents a central branch of cellular metabolism emanating from glucose-6-phosphate （G6P） to provide reductive power （NADPH） and sugar phosphates for anabolic biosyntheses. In plant cells, the oxidative OPPP branch is found in the cytosol and in plastids, consisting of glucose-6-phosphate dehydrogenase （G6PD）, 6-phosphogluconolactonase （PGL）, and 6-phosphogluconate dehydrogenase （6PGD）. These enzymes are encoded by small gene families in the nuclear genome, which, in Arabidopsis, comprise six G6PD, five 6-PGL, and three 6-PGD isoforms （Kruger and von Schaewen, 2003）. Specific targeting motifs at the C-terminus of 6-PGL and 6-PGD isoforms suggested that the OPPP may also occur in peroxisomes （Reumann et al., 2004）.     

Structural basis of the ultrasensitive calcium indicator GCaMP6

GCaMP is one of the most widely used calcium indicators in neuronal imaging and calcium cell biology.The newly developed GCaMP6 shows superior brightness and ultrasensitivity to calcium concentration change.In this study,we determined crystal structures of Ca2+-bound GCaMP6 monomer and dimer and presented detailed structural analyses in comparison with its parent version GCaMP5G.Our analyses reveal the structural basis for the outperformance of this newly developed Ca2+indicator.Three substitution mutations and the resulting changes of local structure and interaction explain the ultrasensitivity and increased fluorescence intensity common to all three versions of GCaMP6.Each particular substitution in the three GCaMP6 is also structurally consistent with their differential sensitivity and intensity,maximizing the potential of using GCaMP6 in solving diverse problems in neuronal research and calcium signaling.Our studies shall also be beneficial to further structure-guided optimization of GCaMP and facilitate the design of novel calcium indicators.     

Activation of gibberellin 2-oxidase 6 decreases active gibberellin levels and creates a dominant semi-dwarf phenotype in rice （Oryza sativa L.）

Gibberellin (GA) 2-oxidase plays a key role in the GA catabolic pathway through 2β-hydroxylation.In the present study,we isolated a CaMV 35S-enhancer activation tagged mutant,H032.This mutant exhibited a dominant dwarf and GA-deficient phenotype,with a final stature that was less than half of its wild-type counterpart.The endogenous bioactive GAs are markedly decreased in the H032 mutant,and application of bioactive GAs (GA3 or GA4) can reverse the dwarf phenotype.The integrated T-DNA was detected 12.8 kb upstream of the OsGA2ox6 in the H032 genome by TAIL-PCR.An increased level of OsGA2ox6 mRNA was detected at a high level in the H032 mutant,which might be due to the enhancer role of the CaMV 35S promoter.RNAi and ectopic expression analysis of OsGA2ox6 indicated that the dwarf trait and the decreased levels of bioactive GAs in the H032 mutant were a result of the up-regulation of the OsGA2ox6 gene.BLASTP analysis revealed that OsGA2ox6 belongs to the class III of GA 2-oxidases,which is a novel type of GA2ox that uses C20-GAs (GA12 and/or GA53) as the substrates.Interestingly,we found that a GA biosynthesis inhibitor,paclobutrazol,positively regulated the OsGA2ox6 gene.Unlike the over-expression of OsGA2ox1,which led to a high rate of seed abortion,the H032 mutant retained normal flowering and seed production.These results indicate that OsGA2ox6 mainly affects plant stature,and the dominant dwarf trait of the H032 mutant can be used as an efficient dwarf resource in rice breeding.     

Heat shock induction of a 65 kDa ATP—binding proteinase in rat C6 glioma cells

The 45,55,65 and 100kDa ATP-binding proteinases(ATP-BPases) of the heat-shocked (44℃ for 30 min,recovery for 12h) rat C6 glioma cells were purified by DEAE-ionexchange and ATP-affinity chromatography.Their molecular masses,isoelectric points (pI),pH-optima and other properties were analyzed by native proteinase gels.It was shown that the 65 kDa ATP-BPase is specifically induced by heat shock and not detectable in control cells.Its N-terminal 1-9amino acid sequence was determined by Edman degradation,but no homologies to other proteins in the protein data bases were found.30 and 31kDa proteinases can be cleaved from the 45,55 and 65 kDa proteinases to which they are linked.A possible relationship of the heat-induced 65 kDa ATP-BPase with the ATP-dependent proteinases (ATP-DPases) in prokaryotes and eukaryotes is discussed.     

Vocal individuality and rhythm in male and female duet contributions of a nonhuman primate

Duetting,or the stereotypical,repeated and often coordinated vocalizations between 2 individuals arose independently multiple times in the Order Primates.Across primate species,there exists substantial variation in terms of timing,degree of overlap,and sex-specificity of duet contributions.There is increasing evidence that primates can modify the timing of their duet contributions relative to their partner,and this vocal flexibility may have been an important precursor to the evolution of human language.Here,we present the results of a fine-scale analysis of Gursky’s spectral tarsier Tarsius spectrumgurskyae duet phrases recorded in North Sulawesi,Indonesia.Specifically,we aimed to investigate individual-level variation in the female and male contributions to the duet,quantify individual-and pair-level differences in duet timing,and measure temporal precision of duetting individuals relative to their partner.We were able to classify female duet phrases to the correct individual with an 80%accuracy using support vector machines,whereas our classification accuracy for males was lower at 64%.Females were more variable than males in terms of timing between notes.All tarsier phrases exhibited some degree of overlap between callers,and tarsiers exhibited high temporal precision in their note output relative to their partners.We provide evidence that duetting tarsier individuals can modify their note output relative to their duetting partner,and these results support the idea that flexibility in vocal exchanges—a precursor to human language—evolved early in the primate lineage and long before the emergence of modern humans.     

New challenge for bionics—brain-inspired computing

By definition,bionics is the application of biological mechanisms found in nature to artificial systems in order to achieve specific functional goals.Successful examples range from Velcro,the touch fastener inspired by the hooks of burrs,to self-cleaning material,inspired by the surface of the lotus leaf.Recently,a new trend in bionics—Brain-Inspired Computing (BIC)-has captured increasing attention.Instead of learning from burrs and leaves,BIC aims to understand the brain and then utilize its operating principles to achieve powerful and efficient information processing.     

Regulation of Cytokinesis by Exocyst Subunit SEC6 and KEULE in Arabidopsis thaliana

Proper vesicle tethering and membrane fusion at the cell plate are essential for cytokinesis. Both the vesicle tethering complex exocyst and membrane fusion regulator KEULE were shown to function in cell plate formation, but the exact mechanisms still remain to be explored. In this study, using yeast two-hybrid （Y-2-H） assay, we found that SEC6 interacted with KEULE, and that a small portion of C-terminal region of KEULE was required for the interaction. The direct SEC6-KEULE interaction was supported by further studies using in vitro pull-down assay, immunoprecipitation, and in vivo bimolecular florescence complementation （BIFC） microscopy, sec6 mutants were male gametophytic lethal as reported; however, pollen-rescued sec6 mutants （PRsec6） displayed cytokinesis defects in the embryonic cells and later in the leaf pavement cells and the guard cells. SEC6 and KEULE proteins were co-localized to the cell plate during cytokine- sis in transgenic Arabidopsis. Furthermore, only SEC6 but not other exocyst subunits located in the cell plate interacted with KEULE in vitro. These results demonstrated that, like KEULE, SEC6 plays a physiological role in cytokinesis, and the SEC6-KEULE interaction may serve as a novel molecular linkage between arriving vesicles and membrane fusion machin- ery or directly regulate membrane fusion during cell plate formation in plants.     

Excited States and Free Radicals in Biology and Medicine——Contributions from Flash Photolysis and Pulse Radiolysis

Excited states and free radicals are involved in the normal physiology of living systems; in pathological processes including lipid peroxidation, inflammation, Parkinson's disease, cancer, and ageing; in the mechanism of action of drugs, such as quinone antitumour agents, and in photochemotherapies such as the PUVA therapy of skin diseases and PDT of cancer. The chief aim of this text is to introduce the reader to this rapidly expanding field, which lies at the borderlines of physics, chemistry, biology, pharmacology, and medicine, and, in particular, to explore how time—resolved spectroscopic methods have found solutions at the molecular level to biological and medical problems.     

Salicylic Acid Signaling Controls the Maturation and Localization of the Arabiclopsis Defense Protein ACCELERATED CELL DEATH6

ACCELERATED CELL DEATH6 （ACD6） is a multipass membrane protein with an ankyrin domain that acts in a positive feedback loop with the defense signal salicylic acid （SA）. This study implemented biochemical approaches to infer changes in ACD6 complexes and localization. In addition to forming endoplasmic reticulum （ER）- and plasma membrane （PM）-Iocalized complexes, ACD6 forms soluble complexes, where it is bound to cytosolic HSP70, ubiquitinated, and degraded via the proteasome. Thus, ACD6 constitutively undergoes ER-associated degradation. During SA signaling, the soluble ACD6 pool decreases, whereas the PM pool increases. Similarly, ACD6-1, an activated version of ACD6 that induces SA, is present at low levels in the soluble fraction and high levels in the PM. However, ACD6 variants with amino acid substitutions in the ankyrin domain form aberrant, inactive complexes, are induced by a SA agonist, but show no PM localization. SA signaling also increases the PM pools of FLAGELLIN SENSING2 （FLS2） and BRI1-ASSOClATED RECEPTOR KINASE 1 （BAK1）. FLS2 forms complexes ACD6; both FLS2 and BAK1 require ACD6 for maximal accumulation at the PM in response to SA signaling. A plausible scenario is that SA increases the efficiency of productive folding and/or complex formation in the ER, such that ACD6, together with FLS2 and BAK1, reaches the cell surface to more effectively promote immune responses.     

Diversity and ecological distribution of endophytic fungi associated with medicinal plants

A total of 973 isolates of endophytic fungi were recovered from 1144 tissue fragments of the six me-dicinal plant species belonging to 4 families collected in the Beijing Botanical Garden. Of these isolates 778 sporulated and were identified into 21 taxa by morphological characteristics. Among the taxa 11 belonged to Coelomycetes, 6 to Ascomycetes, and 4 to Hyphomycetes. Various numbers of endophytic fungi (5―8 taxa) were obtained from each plant. Alternaria alternata was the dominant species in the 6 plants, and Microsphaeropsis conielloides was also dominant in Eucommia ulmoides. There were high colonization rates (47.9%―63.1%) and isolation rates (0.7―0.93) of endophytic fungi, and they were conspicuously higher in twigs than those in leaves in the 6 plants examined. The colonization and isolation rates of endophytic fungi increased with the twig age. The results based on the analyses of cluster and Sorenson's similarity coefficients indicated that some endophytic fungi showed a certain degree of host and tissue preference.