Self-assembly of Mutant Huntingtin Exon-1 Fragments into Large Complex Fibrillar Structures Involves Nucleated Branching

Huntingtin (HTT) fragments with extended polyglutamine tracts self-assemble into amyloid-like fibrillar aggregates. Elucidating the fibril formation mechanism is critical for understanding Huntington's disease pathology and for developing novel therapeutic strategies. Here, we performed systematic experimental and theoretical studies to examine the self-assembly of an aggregation-prone N-terminal HTT exon-1 fragment with 49 glutamines (Ex1Q49). Using high-resolution imaging techniques such as electron microscopy and atomic force microscopy, we show that Ex1Q49 fragments in cell-free assays spontaneously convert into large, highly complex bundles of amyloid fibrils with multiple ends and fibril branching points. Furthermore, we present experimental evidence that two nucleation mechanisms control spontaneous Ex1Q49 fibrillogenesis: (1) a relatively slow primary fibril-independent nucleation process, which involves the spontaneous formation of aggregation-competent fibrillary structures, and (2) a fast secondary fibril-dependent nucleation process, which involves nucleated branching and promotes the rapid assembly of highly complex fibril bundles with multiple ends. The proposed aggregation mechanism is supported by studies with the small molecule O4, which perturbs early events in the aggregation cascade and delays Ex1Q49 fibril assembly, comprehensive mathematical and computational modeling studies, and seeding experiments with small, preformed fibrillar Ex1Q49 aggregates that promote the assembly of amyloid fibrils. Together, our results suggest that nucleated branching in vitro plays a critical role in the formation of complex fibrillar HTT exon-1 aggregates with multiple ends.     

肾素－Ⅱ转基因的初步研究

将注射２４ｋｂ小鼠肾素－２（Ｒｅｎ－２）基因的４２５枚受精卵再植于２２只假孕大鼠输卵管内（每只约２０枚）。怀孕的１６只母鼠共生得子代鼠９７只,用ＰＣＲ及Ｓｏｕｔｈｅｒｎ印迹杂交技术对存活的６０只子代鼠进行鉴定,发现有两只大鼠携带Ｒｅｎ－２基因。此两只转基因大鼠整合外源基因的拷贝数约为１～２个,血压均未见升高。     

Newly evolved highly repeated DNA sequences ofTupaia glis (Tupaiidae,Scandentia)

We have studied highly repeated DNA sequences ofTupaia glis (Tupaiidae, Scandentia) with restriction endonucleases and Southern blotting techniques. Five highly repeated DNA fragments have been isolated fromT. glis and hybridized with genomic DNAs (cleaved by different restriction enzymes) of several non-human primate species and one insectivore (E. europaeus), in order to highlight eventual differences or similarities of their highly repeated DNA sequences. Our first preliminary findings suggest that the newly isolated highly repeated DNA fragments ofT. glis are distinct from both non-human primates and insectivore, the two taxonomic groups considered most similar to the Tupaiidae.     

背部烫伤大鼠脑及肝脏热休克蛋白70（hsp70）mRNA的诱导

本文首先用α－＾３２Ｐ标记的热休克蛋白７０（ｈｓｐ７０）ｃＤＮＡ为探针,研究了背部烫伤对大鼠脑肝ｈｓｐ７０ｍＲＮＡ的诱导。结果表明,烫伤大鼠脑肝ｈｓｐ７０ｍＲＮＡ的表达明显增加,烫伤后５ｍｉｎ内开始出现,２４ｈ后恢复正常。由于烫伤后脑肝温度的增加不超过１℃。因此温度升高不大可能是该基因表达增加的原因。在此基础上用ｈｓｐ７０的抗体,采用Ｗｅｓｔｅｒｎ ｂｌｏｔ的方法,分析了烫伤大鼠脑肝ｈｓｐ７０的变     

Southern blotting analyses of strains ofCampylobacter fetus using the conserved region ofsapA

Chromosomal DNA of 27 strains of Campylobacter fetus was analyzed by Southern blotting with a probe of the conserved region of sapA. The probe hybridized with 23 strains that produced type A lipopolysaccharide. These strains had more than six sapA homologs. In Southern blots of SalI-digested chromosomal DNA separated by pulsed-field gel electrophoresis, one fragment from 19 strains and two fragments from 4 strains hybridized. These data indicate that multiple sapA homologs are localized to a limited region on the chromosomal DNA of C. fetus and thus suggest the possibility of developing a typing system using this method. Received: 28 June 1995 / Accepted: 19 September 1995     

MICA, a new polymorphic HLA-related antigen, is expressed mainly by keratinocytes, endothelial cells, and monocytes

 MICA is a new polymorphic gene in the HLA region expressed in epithelial cell lines and gastrointestinal epithelium. Little is yet known about the MICA protein, and the pattern of its expression by freshly isolated cells has not been established. In the present experiments, we used antibodies raised in rabbits against α1 and α2 domain-peptides to study the expression of MICA. By western blot and immunoprecipitation, we detected a band of 62 000 M _r in various cell lines (THP-1, U937, HeLa, A431, Raji, MOLT-4, and HUV-EC-C) and in freshly isolated keratinocytes, endothelial cells, and monocytes but not in CD4⁺ and CD8⁺ T cells, and CD19⁺ cells (B lymphocytes). It was not possible to up-regulate the expression of MICA in different cells by stimulation with γ-interferon, but the expression of MICA was induced in phytohemagglutinin-stimulated T cells. We confirmed that MICA is expressed at the cell surface by flow cytometry. Results of immunoprecipitation studies of β₂-microglobulin (β₂m)- or MICA-depleted, metabolically labeled HeLa cells indicated that MICA was not associated with β₂m. Although the function of MICA is still unknown, its restricted pattern of tissue expression, the fact that it is expressed on the cell surface, and its polymorphic nature suggest that this new molecule, encoded close to HLA class I, may play a role in the interaction between epithelial cells and cells of the immune system. Received: 21 May 1997 / Revised: 15 July 1997