Structural Studies of Truncated Forms of the Prion Protein PrP

Prions are proteins that adopt self-propagating aberrant folds. The self-propagating properties of prions are a direct consequence of their distinct structures, making the understanding of these structures and their biophysical interactions fundamental to understanding prions and their related diseases. The insolubility and inherent disorder of prions have made their structures difficult to study, particularly in the case of the infectious form of the mammalian prion protein PrP. Many investigators have therefore preferred to work with peptide fragments of PrP, suggesting that these peptides might serve as structural and functional models for biologically active prions. We have used x-ray fiber diffraction to compare a series of different-sized fragments of PrP, to determine the structural commonalities among the fragments and the biologically active, self-propagating prions. Although all of the peptides studied adopted amyloid conformations, only the larger fragments demonstrated a degree of structural complexity approaching that of PrP. Even these larger fragments did not adopt the prion structure itself with detailed fidelity, and in some cases their structures were radically different from that of pathogenic PrP^Sc.     

Acetylation of TUG Protein Promotes the Accumulation of GLUT4 Glucose Transporters in an Insulin-responsive Intracellular Compartment

Insulin causes the exocytic translocation of GLUT4 glucose transporters to stimulate glucose uptake in fat and muscle. Previous results support a model in which TUG traps GLUT4 in intracellular, insulin-responsive vesicles termed GLUT4 storage vesicles (GSVs). Insulin triggers TUG cleavage to release the GSVs; GLUT4 then recycles through endosomes during ongoing insulin exposure. The TUG C terminus binds a GSV anchoring site comprising Golgin-160 and possibly other proteins. Here, we report that the TUG C terminus is acetylated. The TUG C-terminal peptide bound the Golgin-160-associated protein, ACBD3 (acyl-CoA-binding domain-containing 3), and acetylation reduced binding of TUG to ACBD3 but not to Golgin-160. Mutation of the acetylated residues impaired insulin-responsive GLUT4 trafficking in 3T3-L1 adipocytes. ACBD3 overexpression enhanced the translocation of GSV cargos, GLUT4 and insulin-regulated aminopeptidase (IRAP), and ACBD3 was required for intracellular retention of these cargos in unstimulated cells. Sirtuin 2 (SIRT2), a NAD⁺-dependent deacetylase, bound TUG and deacetylated the TUG peptide. SIRT2 overexpression reduced TUG acetylation and redistributed GLUT4 and IRAP to the plasma membrane in 3T3-L1 adipocytes. Mutation of the acetylated residues in TUG abrogated these effects. In mice, SIRT2 deletion increased TUG acetylation and proteolytic processing. During glucose tolerance tests, glucose disposal was enhanced in SIRT2 knock-out mice, compared with wild type controls, without any effect on insulin concentrations. Together, these data support a model in which TUG acetylation modulates its interaction with Golgi matrix proteins and is regulated by SIRT2. Moreover, acetylation of TUG enhances its function to trap GSVs within unstimulated cells and enhances insulin-stimulated glucose uptake.     

转人工miRNA抗玉米粗缩病毒载体玉米的培育及其抗病能力

玉米是重要的粮食作物,水稻黑条矮缩病毒(RBSDV)是玉米粗缩病的病原,由其引起的玉米粗缩病给玉米生产造成重大损失。利用人工mi RNA构建抗病毒植物的技术已经在多种植物中被证明有效,但是在玉米中的尝试未见报道。实验根据玉米zea-mi R159a的前体序列和RBSDV基因组中编码功能蛋白的基因和基因沉默抑制子的序列信息设计引物,构建了用于沉默RBSDV编码基因和基因沉默抑制子的ami RNA(Artificial mi RNA)基因。构建p CAMBIA3301-121-ami RNA植物表达载体,利用农杆菌介导法转化玉米自交系综31(Z31)。对转基因玉米进行分子检测,选择mi RNA表达量高的纯合体株系进行自然发病实验,按0-4的分级标准调查玉米粗缩病的严重度。结果表明,转抗粗缩病毒人工mi RNA载体玉米纯合体株系的抗病表现好于野生型玉米,其中针对基因组6的S6-mi R159转基因玉米抗病情况较好。研究表明利用人工mi RNA技术构建抗病毒病玉米新品种是可行的。     

Development of IRAP-SCAR marker for strain identification in Lentinula edodes

Rapid and correct authentication of commercial strains is still important in today’s mushroom industry. Here for the first time we reported the using of sequence characterized amplified region (SCAR) marker developed from inter-retrotransposon amplified polymorphism (IRAP) marker to identify Lentinula edodes strain. Genomic polymorphism among 44 shiitake cultivars in China were surveyed by 24 IRAP primer combinations, from which primer combination LTR1L/MarY1R could generate a unique and reproducible 1712 bp fragment to distinguish strain No. 4 from other 43 strains. Based on this strain-specific fragment, a SCAR primer pair was designed and its validity was verified by correctly amplifying a single strain-specific fragment from DNA samples of 100 L. edodes strains. Our study lays the foundation for developing strain-specific SCAR marker by retrotransposon-based marker technique in fungi.     

Antimicrobial activity of Lactobacillus reuteri DPC16 supernatants against selected food borne pathogens

Lactobacillus reuteri DPC16 is a human-isolated strain recently patented in New Zealand. The antimicrobial activity of cell-free supernatants from different fermentation processes, with or without glycerol supplementation was studied. When grown in just MRS broth, the cultural supernatant significantly inhibited the growth of selected food-borne pathogens, possibly due to acidic effect as this activity was pH-dependent. The cell-free supernatants from secondary fermentation of DPC16 resting cells in glycerol-supplemented media have shown very different antimicrobial activities. A very potent antimicrobial activity gradually developed during the fermentation process which was observed only when growing in MRS-glycerol broth (such supernatant is denoted MRSg). This strong antimicrobial activity was pH-independent, dose-dependant and affected both Gram-negative and Gram-positive pathogens. Reuterin detected in MRSg is believed to be responsible for these activities. The susceptibility of the selected pathogens (grown to stationary phase) to MRSg was tested and found that exposure to MRSg for 180 min led to a significant reduction in cell viability in all pathogens. These results suggest that this is a reuterin-producing strain, which has potent antimicrobial activity against both Gram-negative and Gram-positive pathogens. These findings have indicated a clear potential of this novel strain in industrial applications.     

Comparative analysis on the diversity of <Emphasis Type="Italic">Auricularia auricula</Emphasis>-<Emphasis Type="Italic">judae</Emphasis> by physiological characteristics,somatic incompatibility and TRAP fingerprinting

Phenotypic traits (physiological characteristics and somatic incompatibility) and genotypic traits (target region amplification polymorphism TRAP) were used to study the diversity of 32 main cultivars of Auricularia auricula-judae in China. Twenty seven important and stable physiological indexes were evaluated; somatic incompatibility test (SIT) reaction was described from three aspects: type, pigment, and intensity; 16 pairs of TRAP primer combinations produced 535 unambiguous and reproducible DNA fragments, of these 524 (97.9%) were polymorphic. Dendrograms were constructed by Unweighted Pair-group Method with Arithmetic Averages (UPGMA) method, and the principal coordinate analysis (PCO) of the three methods (physiological characteristics, SIT intensity and TRAP) exhibited similar clustered patterns, revealing that all the tested strains could be divided into three distinct groups, each of which was correlated with different geographical regions. Most strains originated from the same area were with a narrow genetic basis and could possibly be domesticated from the local wild-type strains, some strains were suspected to be synonymous. The grouping information obtained in the present work provides significant information for further genetic improvement of A. auricula-judae.