抑癌基因p53与细胞自噬

细胞自噬(autophagy)是一种在进化上高度保守的代谢通路,它发生的分子机制和信号调控途径相当复杂,其中mTOR信号通路和Beclin1复合物发挥了最重要的调控作用,p53也是细胞自噬重要的调节因子。研究发现,p53可通过多种途径调节细胞自噬水平,这主要决定于它的亚细胞定位。在细胞核中,p53可通过多种方式上调细胞自噬;而在细胞质中,p53对细胞自噬具有负性调节作用,可抑制细胞自噬的发生。探究清楚p53与细胞自噬之间的调控关系将有助于人类正确认识由于细胞自噬功能异常所诱导的肿瘤的发生发展过程,从而最终攻克各种肿瘤性疾病。     

Intramembrane protease PARL defines a negative regulator of PINK1- and PARK2/Parkin-dependent mitophagy

Mutations in PINK1 and PARK2/Parkin are a main risk factor for familial Parkinson disease. While the physiological mechanism of their activation is unclear, these proteins have been shown in tissue culture cells to serve as a key trigger for autophagy of depolarized mitochondria. Here we show that ablation of the mitochondrial rhomboid protease PARL leads to retrograde translocation of an intermembrane space-bridging PINK1 import intermediate. Subsequently, it is rerouted to the outer membrane in order to recruit PARK2, which phenocopies mitophagy induction by uncoupling agents. Consistent with a role of this retrograde translocation mechanism in neurodegenerative disease, we show that pathogenic PINK1 mutants which are not cleaved by PARL affect PINK1 kinase activity and the ability to induce PARK2-mediated mitophagy. Altogether we suggest that PARL is an important intrinsic player in mitochondrial quality control, a system substantially impaired in Parkinson disease as indicated by reduced removal of damaged mitochondria in affected patients.     

Dual‐color oligo‐FISH can reveal chromosomal variations and evolution in Oryza species

Fluorescence in situ hybridization using probes based on oligonucleotides (oligo‐FISH) is a useful tool for chromosome identification and karyotype analysis. Here we developed two oligo‐FISH probes that allow the identification of each of the 12 pairs of chromosomes in rice (Oryza sativa). These two probes comprised 25 717 (green) and 25 215 (red) oligos (45 nucleotides), respectively, and generated 26 distinct FISH signals that can be used as a barcode to uniquely label each of the 12 pairs of rice chromosomes. Standard karyotypes of rice were established using this system on both mitotic and meiotic chromosomes. Moreover, dual‐color oligo‐FISH was used to characterize diverse chromosomal abnormalities. Oligo‐FISH analyses using these probes in various wild Oryza species revealed that chromosomes from the AA, BB or CC genomes generated specific and intense signals similar to those in rice, while chromosomes with the EE genome generated less specific signals and the FF genome gave no signal. Together, the oligo‐FISH probes we established will be a powerful tool for studying chromosome variations and evolution in the genus Oryza.     

Discriminative detection of mercury (II) and hydrazine using a dual‐function fluorescent probe

A dual‐function fluorescent probe (Probe 1 ) was developed for discriminative detection of Hg²⁺ and N₂H₄. Probe 1 could discriminatively detect Hg²⁺ and N₂H₄ through two different reaction sites, with the mechanism for Probe 1 for Hg²⁺ depending on a desulfurization reaction and for N₂H₄ depending on the Schiff‐base reaction. N₂H₄ had minimal effect on Hg²⁺ detection in dimethyl sulfoxide (DMSO)/H₂O solution, but Hg²⁺ could interfere with N₂H₄ detection in DMSO/buffer solution. Different concentrations of Hg²⁺ and N₂H₄ resulted in different blue shades of Probe 1 test strips, and the shade of blue was different with the same concentration of Hg²⁺ or N₂H₄, as observed under ultraviolet light at 365 nm wavelength.     

Biotinylated probes of artemisinin with labeling affinity toward Trypanosoma brucei brucei target proteins

We studied the target proteins of artemisinin in Trypanosoma brucei brucei using the affinity-labeling method. We designed and synthesized four biotinylated probes of artemisinin for use as molecular tools. Their in vitro trypanocidal activities (data not shown) proved that they mimicked the biological action of artemisinin. We assessed the chemical stability for all of the probes in the parasite culture medium and lysate using reversed-phase high-performance liquid chromatography (HPLC). After 3-h incubations, the probes remained undecomposed in a range of 40 to 65% in the parasite culture medium, whereas approximately 80% of the probes remained stable in the parasite lysate. Using liquid chromatography mass spectrometry (LC–MS), we demonstrated that, with respect to all of the probes, uptakes into the parasite ranging from 81 to 96% occurred after 30-min incubations. In a competitive binding assay between artemisinin and the four biotinylated probes, we searched for the trypanosomal target protein of artemisinin. Consequently, we observed that only the diazirine-free probe 5 could provide the desired result with high affinity-labeling efficiency. Using the horseradish peroxidase-tagged streptavidin–biotin method, we showed that artemisinin could specifically bind to candidate target proteins of approximately 60, 40, and 39 kDa.     

Deep Sequencing-guided Design of a High Affinity Dual Specificity Antibody to Target Two Angiogenic Factors in Neovascular Age-related Macular Degeneration

The development of dual targeting antibodies promises therapies with improved efficacy over mono-specific antibodies. Here, we engineered a Two-in-One VEGF/angiopoietin 2 antibody with dual action Fab (DAF) as a potential therapeutic for neovascular age-related macular degeneration. Crystal structures of the VEGF/angiopoietin 2 DAF in complex with its two antigens showed highly overlapping binding sites. To achieve sufficient affinity of the DAF to block both angiogenic factors, we turned to deep mutational scanning in the complementarity determining regions (CDRs). By mutating all three CDRs of each antibody chain simultaneously, we were able not only to identify affinity improving single mutations but also mutation pairs from different CDRs that synergistically improve both binding functions. Furthermore, insights into the cooperativity between mutations allowed us to identify fold-stabilizing mutations in the CDRs. The data obtained from deep mutational scanning reveal that the majority of the 52 CDR residues are utilized differently for the two antigen binding function and permit, for the first time, the engineering of several DAF variants with sub-nanomolar affinity against two structurally unrelated antigens. The improved variants show similar blocking activity of receptor binding as the high affinity mono-specific antibodies against these two proteins, demonstrating the feasibility of generating a dual specificity binding surface with comparable properties to individual high affinity mono-specific antibodies.     

Phenotypic variation in nurse traits and community feedbacks define an alpine community

Much is known about facilitation, but virtually nothing about the underlying genetic and evolutionary consequences of this important interaction. We assessed the potential of phenotypic differences in facilitative effects of a foundation species to determine the composition of an Alpine community in Arizona. Two phenotypes of Geum rossii occur along a gradient of disturbance, with 'tight' competitive cushions in stable conditions and 'loose' facilitative cushions in disturbed conditions. A common-garden study suggested that field-based traits may have a genetic basis. Field experiments showed that the reproductive fitness of G. rossii cushions decreased with increasing facilitation. Finally, using a dual-lattice model we showed that including the cost and benefit of facilitation may contribute to the co-occurrence of genotypes with contrasting facilitative effects. Our results indicate that changes in community composition due to phenotypic differences in facilitative effects of a foundation species may in turn affect selective pressures on the foundation species.     

Arthropod food webs in organic and conventional wheat farming systems of an agricultural long‐term experiment: a stable isotope approach

• 1 Agricultural intensification not only alters the structure of arthropod communities, but also may affect biotic interactions by altering the availability of basal resources. We analyzed variations in stable isotope ratios (¹⁵N/¹⁴N and ¹³C/¹²C) of fertilizers, plants, prey and generalist predators in organic and conventional farming systems in a long‐term agricultural experiment [DOK trial (bioDynamic, bioOrganic, Konventionell)]. Two basal resources with pronounced differences in carbon isotope signatures, wheat litter (C₃ plant) and maize litter (C₄ plant), were used to uncover differences in food web properties between the two farming systems (conventional versus organic).
• 2 Predators incorporated significantly higher proportions of carbon from wheat sources in organically managed fields, suggesting that they were more closely linked to wheat‐consuming prey in this system. The δ¹⁵N values of three predaceous species were more than 2‰ greater in summer than in spring.
• 3 The results obtained suggest that generalist predators consumed higher proportions of herbivore prey in the organic system and that starvation and intraguild predation rates increased in some predator species with time.
• 4 Because the effects of farming system and sampling date on predators were species‐specific, conserving a diverse natural enemy community including species with different phenologies and sensitivities to management practices may, in the long term, be a good strategy for maintaining high pest suppression throughout the growing season.