Modeling endophilin-mediated Aβ disposal in glioma cells

Autophagy dysregulation has emerged in age-related neurological diseases (Ulland et al.; Matheoud et al.; Ashkenazi et al.). Alzheimer Disease (AD), the most common progressive neurodegenerative disorder, is characterized by the accumulation of amyloid-β (Aβ) plaques caused by aberrant Aβ metabolism (Qiang et al.; Sevigny et al.; Ittner et al.). Glia constitute the brain immune system and ingest extracellular Aβ for degradation via the autophagy-lysosome machinery (Ries and Sastre; Cho et al.). Here, we model the molecular rationale for this clearance process in glioma cells by showing that miR34a inhibits autophagy-mediated disposal of Aβ fibrils and identifying two novel direct targets of miR34a, endophilin-3 and cathepsin B (CTSB, a previously reported enzyme for Aβ degrading (Sun et al.)). Bioinformatics analyses revealed that endophilin-3 expresses at a significantly lower level in neurodegenerative diseases. Its gain-of-function substantially promotes both uptake and degradation of Aβ while small interfering RNA (siRNA)-mediated endophilin-3 knockdown slowed down Aβ clearance and blocked autolysosome formation. Mechanistically, gene ontology (GO) analysis of the endophilin-3 interactome identified by mass spectrometry uncovered enriched components involved in actin binding (with the highest score). Importantly, we validated that the actin-binding protein phostensin interacted with endophilin-3. Phostensin knockdown restored endophilin-3-mediated up-regulation of Aβ clearance. Thus, our findings indicate that miR34a inhibits Aβ clearance by targeting endophilin-3 and CTSB at multiple steps including uptake and autophagy-mediated degradation.     

沙棘H3K9乙酰化修饰全基因组分析

为了绘制沙棘H3K9乙酰化修饰图谱,确定H3K9乙酰化修饰所调控的基因,该实验通过Western blot验证抗体与组蛋白的结合能力和ChIP-seq验证抗体富集效率,获得全基因组范围内沙棘H3K9乙酰化修饰图谱和调控基因。实验结果表明,H3K9ac抗体与复合物具有较强的结合能力。对富集到的DNA片段进行高通量测序,分别获得2.2×10~7和3.6×10~7条原始序列;唯一比对序列广泛分布于沙棘基因组中,并且在结构基因中的两端具有明显的富集。对富集区进行峰的预测结果显示,共预测出1 011个峰;对峰所处部位基因进行功能预测结果发现,H3K9ac对于沙棘细胞代谢和信号转导基因的表达具有重要调控作用。沙棘片段化DNA的富集以及高通量测序结果证明,抗体能够用于研究沙棘的组蛋白修饰类型,并且绘制了沙棘第一张H3K9乙酰化修饰遗传图谱草图,鉴定出沙棘H3K9乙酰化修饰所调控的基因,为今后研究组蛋白修饰对沙棘基因表达的调控方式奠定了基础。     

Antipeptide antibodies directed against the carboxy-terminal region of SV40 structural proteins VP2 and VP3

Rabbits were immunized with a synthetic heptapeptide of the sequence Arg-Asn-Arg-Ser-Ser-Arg-Ser corresponding to the carboxy-terminal region of the SV40 viral proteins VP2 and VP3. The raised antibodies recognize the viral proteins in enzyme-linked immunosorbent (ELISA) and Western blot assay. Specificity of the antibodies were confirmed by competition experiments. The antibodies recognize VP2 and VP3 in infected cells by immunofluorescence and in subcellular fractions by ELISA. No interaction with virions was observed.     

Ciliary polypeptides and glycoconjugates of wild-type and mutant Tetrahymena thermophila: Starved versus nonstarved

To investigate the role of cilia in mating interactions of Tetrahymena thermophila, ciliary membrane-rich fractions were isolated from two wild-type strains, a non-discharge mucocyst mutant which possesses mating behavior similar to wild-type, and a mating mutant which is able to costimulate cells of complementary mating type but cannot enter into pair formation. In each case, proteins from the ciliary membrane-rich fractions of starved, mating-competent (“initiated”) cells were compared with those from non-starved, mating-incompetent (“non-initiated”) cells, by gel electro-phoresis and lectin blotting. In stained gels, a 43 kDa polypeptide was reduced or absent in initiated cells but present in non-initiated cells, in all strains. In silver-stained gels, a 25 kDa polypeptide was present in all strains, both initiated and non-initiated. In blots probed with Con A-peroxidase, a 25 kDa glycoprotein was present in ciliary membrane fractions from non-initiated cells and absent in membranes of initiated cells of the two wild-type strains and the mucocyst mutant, but is present in initiated and non-initiated cells of the mating mutant (several hypotheses are presented to explain these findings). In addition, ciliary proteins of the mating mutant included at least two unique Con A-binding polypeptides. Our results support the idea that development of mating competence during starvation involves an extensive remodeling of ciliary membranes, and identify a 25 kDa glyco-conjugate as having a potential role in control of pair formation during mating. © 1992 Wiley-Liss, Inc.     

Major sclerotial polypeptides of psychrophilic pathogenic fungi: Intracellular localization and antigenic relatedness

Summary Major sclerotial polypeptides from the psychrophiles,Myriosclerotinia borealis (W 51),Coprinus psychromorbidus (LRS 131),Typhula idahoensis (W 21), andTyphula incarnata (W 21) were purified by using polyacrylamide gel electrophoresis and electroelution. Polyclonal antibodies were raised against these major sclerotial polypeptides. Immunofluorescence microscopy showed that the major sclerotial polypeptides from all four psychrophilic species were sequestered in discrete protein bodies of cultured and field-grown sclerotia. Western blot analysis indicated that all antisera reacted positively with their respective antigens, the major sclerotial polypeptides. Reciprocal immunological cross-reactions were observed between the major sclerotial polypeptides ofM. borealis (W 51) andT. idahoensis (W 21). Antiserum to the major sclerotial polypeptides of bothM. borealis andT. idahoensis also recognized the major sclerotial polypeptides ofC. psychromorbidus (LRS 131). It is suggested that the major sclerotial polypeptides of these psychrophilic plant pathogens may act as storage proteins.Abbreviations W 51 Myriosclerotinia borealis (W 51) - LRS 131 Coprinus psychromorbidus (LRS 131) - W 21 Typhula idahoensis (W 21) - W 29 Typhula incarnata (W 29) - anti W 51 antiserum to the major sclerotial polypeptide ofM. borealis W 51 - anti LRS 131 antiserum to the major sclerotial polypeptides ofC. psychromorbidus (LRS 131) - anti W 21 antiserum to the major sclerotial polypeptides ofT. idahoensis (W 21) - anti W 29 antiserum to the major sclerotial polypeptides ofT. incarnata (W 29) - SDS sodium dodecylsulfate - kDa kilodalton - PAGE polyacrylamide gel electrophoresis - HRP horseradish peroxidase - PBS phosphate buffered saline - TBS Tris buffered saline - FITC fluorescein isothiocyanate     

Quantitative Estimation of Ipomeamarone by Chromatostrip Technique

Method of analysing a microquantity of ipomcamarone was established by silica gel chromatostrip technique for its separation and the subsequent colorimetric determination of the 2.4-dinitrophenylhydrazone in alkaline condition. Preliminary analysis of ipomeamarone based on this method was carried out for sweet potato roots infected by the black rot during ninety six hours period.     

Serological Detection of Grapevine leafroll virus 2 Using an Antiserum Developed against the Recombinant Coat Protein†

Grapevine leafroll associated virus 2 (GLRaV 2) is one of the important components in the leafroll disease complex. The coat protein gene of GLRaV 2 was cloned into a protein expression vector pMAL‐c2x and the recombinant protein, consisting of the maltose binding protein (MBP) and GLRaV 2 coat protein (CP), was expressed in Escherichia coli. The recombinant MBP‐CP was used to raise a high quality antiserum. When used in Western blot analysis, the anti‐MBP‐CP antiserum produced specific reaction to the recombinant protein as well as to the viral coat protein of GLRaV 2. In Immunosorbent electron microscopy study, the anti‐MBP‐CP antibodies strongly decorated the GLRaV 2 virions. Using the newly developed antiserum, an indirect plate‐trapped antigen enzyme‐linked immunosorbent assay method was developed and successfully implemented for virus detection. A field survey was conducted to evaluate the virus infection status by GLRaV 2 and GLRaV 3 using antibodies developed against their respective recombinant coat proteins.     

Cloning and sequence analysis of genome from the Inner Mongolia strain of the endogenous betaretroviruses (enJSRV)

In order to amplify the complete genome of enJSRV from the strain of Inner Mongolia (enJSRV-NM), we used enJSRV-specific and JSRV-specific DNA probes in dot blot hybridization. Seven pairs of primers were designed based on Genbank sequences. Seven fragments were obtained by PCR and were cloned into the PMD19- T vectors. The recombinant plasmids were sequenced and analyzed. The results showed that the genome was 7 942 bp in length and contained four overlapping open reading frames corresponding to the gag, pro, pol and env genes as well as an additional open reading frame (orf-x) that overlaps the 3' end of the pol gene. The nucleotide acid sequences of the enJSRV-NM loci were compared with the sequences of South Africa enJS56A1 strain (Accession No. AF153615) and USA JSRV21 strain (Accession No. AF105220). The nucleotide acid identities were 99.2% and 92.3% respectively. Two zinc fingers were found in the NC region in the predicted amino acid sequence. However, the YXXM motif, which is a reliable molecular marker for the infectious exogenous virus, was not found in the TM region. It was found that the enJSRV-NM region was 90%-98% identical at the amino acid level to its exogenous infectious counterparts in most of the retroviral genome. This is the first nucleotide sequence of enJSRV reported in P.R China. The resource work has provided a wide range of information useful not only for expression genomics and annotation of genomic DNA sequence, but also for further research on the clinical diagnosis of OPA.