Somatic hypermutation of an immunoglobulin transgene in kappa transgenic mice

Initial studies of somatically acquired mutations in immunoglobulin V regions from hybridomas and myelomas that are not derived from joining aberrations, suggested a controlled and specific hypermutation process, because spontaneous mutation rates observed for other genes are extremely low. Some evidence for the idea that mutations are introduced during V-gene rearrangement came from the clustering of mutations at the joining sites, from the absence of mutations in unrearranged V genes and from the low level of mutations in only partially (D-J) rearranged nonproductive heavy-chain alleles. Another model in which mutations accumulate with each cell division, rather than being introduced all at once, was supported by the finding that immunoglobulin genes of hybridomas derived from a single mouse frequently had several mutations in common, and so might be derived from the same precursor cell whose daughters then accumulated additional mutations. But the common mutations in some cases could be due to as yet unidentified related germline genes, or could represent the effect of antigen selection for certain amino acids. To try to detect hypermutation in the absence of V-gene rearrangement, we isolated B lymphocytes with endogenous heavy-chain gene mutations from transgenic mice carrying pre-rearranged kappa-transgenes. We found that these kappa-transgenes were also somatically mutated. This and other observations indicated that: ongoing rearrangement is not required for mutation; there are signals for hypermutation in the transgenes; the mutations are found only in the variable region, so the constant region may not be a target; different transgene insertion sites are compatible with hypermutations and more than one transgene is expressed in the same cell.     

Induction of somatic hypermutation in immunoglobulin genes is dependent on DNA polymerase iota

Somatic hypermutation of immunoglobulin genes is a unique, targeted, adaptive process. While B cells are engaged in germinal centres in T-dependent responses, single base substitutions are introduced in the expressed Vh/Vl genes to allow the selection of mutants with a higher affinity for the immunizing antigen. Almost every possible DNA transaction has been proposed to explain this process, but each of these models includes an error-prone DNA synthesis step that introduces the mutations. The Y family of DNA polymerases--pol eta, pol iota, pol kappa and rev1--are specialized for copying DNA lesions and have high rates of error when copying a normal DNA template. By performing gene inactivation in a Burkitt's lymphoma cell line inducible for hypermutation, we show here that somatic hypermutation is dependent on DNA polymerase iota.     

Ablation of XRCC2/3 transforms immunoglobulin V gene conversion into somatic hypermutation

After gene rearrangement, immunoglobulin V genes are further diversified by either somatic hypermutation or gene conversion. Hypermutation (in man and mouse) occurs by the fixation of individual, non-templated nucleotide substitutions. Gene conversion (in chicken) is templated by a set of upstream V pseudogenes. Here we show that if the RAD51 paralogues XRCC2, XRCC3 or RAD51B are ablated the pattern of diversification of the immunoglobulin V gene in the chicken DT40 B-cell lymphoma line exhibits a marked shift from one of gene conversion to one of somatic hypermutation. Non-templated, single-nucleotide substitutions are incorporated at high frequency specifically into the V domain, largely at G/C and with a marked hotspot preference. These mutant DT40 cell lines provide a tractable model for the genetic dissection of immunoglobulin hypermutation and the results support the idea that gene conversion and somatic hypermutation constitute distinct pathways for processing a common lesion in the immunoglobulin V gene. The marked induction of somatic hypermutation that is achieved by ablating the RAD51 paralogues is probably a consequence of modifying the recombination-mediated repair of such initiating lesions.     

用局部刚度修正法处理结构优化的全局约束问题

研究了当前在结构优化设计中处理全局约束问题的方法，进而提出了局部刚度修正法，即只要修改部分构件的刚度就可使全局约束条件得到满足。通过算例和工程实践表明，对规模为1000个节点、50个设计变量的杆系结构，在Pentium Ⅲ计算机上，只要运行40min就可得到满意解。该方法简便易行，不用做灵敏度分析，收敛快、计算量小，是解决大型工程结构优化设计问题的好方法。     

论伪造、变造国家有价证券罪

文章就伪造、变造国家有价证券罪的沿革、概念、构成、适用、处罚等问题进行了探讨。     

Two levels of protection for the B cell genome during somatic hypermutation

Somatic hypermutation introduces point mutations into immunoglobulin genes in germinal centre B cells during an immune response. The reaction is initiated by cytosine deamination by the activation-induced deaminase (AID) and completed by error-prone processing of the resulting uracils by mismatch and base excision repair factors. Somatic hypermutation represents a threat to genome integrity and it is not known how the B cell genome is protected from the mutagenic effects of somatic hypermutation nor how often these protective mechanisms fail. Here we show, by extensive sequencing of murine B cell genes, that the genome is protected by two distinct mechanisms: selective targeting of AID and gene-specific, high-fidelity repair of AID-generated uracils. Numerous genes linked to B cell tumorigenesis, including Myc, Pim1, Pax5, Ocab (also called Pou2af1), H2afx, Rhoh and Ebf1, are deaminated by AID but escape acquisition of most mutations through the combined action of mismatch and base excision repair. However, approximately 25% of expressed genes analysed were not fully protected by either mechanism and accumulated mutations in germinal centre B cells. Our results demonstrate that AID acts broadly on the genome, with the ultimate distribution of mutations determined by a balance between high-fidelity and error-prone DNA repair.     

细菌脱硫抑制硫化矿自燃实验研究

通过室内细菌脱硫柱浸实验,将硫化矿石颗粒表面的硫降低到临界值以下,使脱硫后矿石自燃倾向性等级下降.结果表明,44d最大脱硫率可达61.82%,脱硫率随着时间延长而逐渐增大.对矿石自燃倾向性分析显示,硫化矿石的5d氧化增重率从2.044%降低到0.902%,自燃点从209.6℃升高到319.8℃,自燃倾向性等级由Ⅰ级降为Ⅲ级,降低了硫化矿石的自燃风险.     

Structural basis for removal of adenine mispaired with 8-oxoguanine by MutY adenine DNA glycosylase

The genomes of aerobic organisms suffer chronic oxidation of guanine to the genotoxic product 8-oxoguanine (oxoG). Replicative DNA polymerases misread oxoG residues and insert adenine instead of cytosine opposite the oxidized base. Both bases in the resulting A*oxoG mispair are mutagenic lesions, and both must undergo base-specific replacement to restore the original C*G pair. Doing so represents a formidable challenge to the DNA repair machinery, because adenine makes up roughly 25% of the bases in most genomes. The evolutionarily conserved enzyme adenine DNA glycosylase (called MutY in bacteria and hMYH in humans) initiates repair of A*oxoG to C*G by removing the inappropriately paired adenine base from the DNA backbone. A central issue concerning MutY function is the mechanism by which A*oxoG mispairs are targeted among the vast excess of A*T pairs. Here we report the use of disulphide crosslinking to obtain high-resolution crystal structures of MutY-DNA lesion-recognition complexes. These structures reveal the basis for recognizing both lesions in the A*oxoG pair and for catalysing removal of the adenine base.     

Treatment of diabetes and atherosclerosis by inhibiting fatty-acid-binding protein aP2

Adipocyte fatty-acid-binding protein, aP2 (FABP4) is expressed in adipocytes and macrophages, and integrates inflammatory and metabolic responses. Studies in aP2-deficient mice have shown that this lipid chaperone has a significant role in several aspects of metabolic syndrome, including type 2 diabetes and atherosclerosis. Here we demonstrate that an orally active small-molecule inhibitor of aP2 is an effective therapeutic agent against severe atherosclerosis and type 2 diabetes in mouse models. In macrophage and adipocyte cell lines with or without aP2, we also show the target specificity of this chemical intervention and its mechanisms of action on metabolic and inflammatory pathways. Our findings demonstrate that targeting aP2 with small-molecule inhibitors is possible and can lead to a new class of powerful therapeutic agents to prevent and treat metabolic diseases such as type 2 diabetes and atherosclerosis.     

Cyclin is degraded by the ubiquitin pathway.

Cyclin degradation is the key step governing exit from mitosis and progress into the next cell cycle. When a region in the N terminus of cyclin is fused to a foreign protein, it produces a hybrid protein susceptible to proteolysis at mitosis. During the course of degradation, both cyclin and the hybrid form conjugates with ubiquitin. The kinetic properties of the conjugates indicate that cyclin is degraded by ubiquitin-dependent proteolysis. Thus anaphase may be triggered by the recognition of cyclin by the ubiquitin-conjugating system.     

Role of arginine-tRNA in protein degradation by the ubiquitin pathway

Degradation of intracellular proteins through the ubiquitin and ATP-dependent proteolysis pathway involves several steps. Initially, ubiquitin is covalently linked to the proteolytic substrate in an ATP-requiring reaction. Proteins marked by ubiquitin may then be selectively lysed in a reaction that also requires ATP (for reviews see refs 1-3). A major question concerns the structural features of a protein that make it a specific substrate for ubiquitin-mediated degradation. It was shown that a free alpha-NH2 group is one important feature of the protein structure recognized by the ubiquitin ligation system, and that the half-life in vivo of a protein with an exposed amino terminus depends on its amino terminal residue. We have previously demonstrated that transfer RNA (tRNA) is essential for conjugation of ubiquitin and for the subsequent degradation of proteins with acidic amino termini (aspartate or glutamate). We now show that tRNA is required for post-translational conjugation of arginine to acidic amino termini of proteins, a modification that is essential for their degradation by the ubiquitin pathway.     

Stem cell division is regulated by the microRNA pathway

One of the key characteristics of stem cells is their capacity to divide for long periods of time in an environment where most of the cells are quiescent. Therefore, a critical question in stem cell biology is how stem cells escape cell division stop signals. Here, we report the necessity of the microRNA (miRNA) pathway for proper control of germline stem cell (GSC) division in Drosophila melanogaster. Analysis of GSCs mutant for dicer-1 (dcr-1), the double-stranded RNaseIII essential for miRNA biogenesis, revealed a marked reduction in the rate of germline cyst production. These dcr-1 mutant GSCs exhibit normal identity but are defective in cell cycle control. On the basis of cell cycle markers and genetic interactions, we conclude that dcr-1 mutant GSCs are delayed in the G1 to S transition, which is dependent on the cyclin-dependent kinase inhibitor Dacapo, suggesting that miRNAs are required for stem cells to bypass the normal G1/S checkpoint. Hence, the miRNA pathway might be part of a mechanism that makes stem cells insensitive to environmental signals that normally stop the cell cycle at the G1/S transition.     

A B cell-deficient mouse by targeted disruption of the membrane exon of the immunoglobulin mu chain gene

Of the various classes of antibodies that B lymphocytes can produce, class M (IgM) is the first to be expressed on the membrane of the developing cells. Pre-B cells, the precursors of B-lymphocytes, produce the heavy chain of IgM (mu chain), but not light chains. Recent data suggest that pre-B cells express mu chains on the membrane together with the 'surrogate' light chains lambda 5 and V pre B (refs 2-7). This complex could control pre-B-cell differentiation, in particular the rearrangement of the light-chain genes. We have now assessed the importance of the membrane form of the mu chain in B-cell development by generating mice lacking this chain. We disrupted one of the membrane exons of the gene encoding the mu-chain constant region by gene targeting in mouse embryonic stem cells. From these cells we derived mice heterozygous or homozygous for the mutation. B-cell development in the heterozygous mice seemed to be normal, but in homozygous animals B cells were absent, their development already being arrested at the stage of pre-B-cell maturation.     

盐酸小檗碱通过P13K/AKT信号通路抑制MMP2和MMP9的表达影响膀胱癌T24细胞侵袭性

目的:探讨盐酸小檗碱(Berberine hydrochloride)对膀胱癌T24细胞侵袭能力的影响及机制.方法:采用利用CCK8法检测膀胱癌T24细胞的生存率;transwell侵袭试验检测盐酸小檗碱对T24细胞侵袭能力的影响;Western-blotting分析盐酸小檗碱对P13K/AKT通路相关因子(PI3K、p-PI3K、AKT、p-AKT),细胞基质金属蛋白酶(MMP2、MMP9)蛋白表达的改变.结果:CCK8显示盐酸小檗碱对T24细胞的增殖抑制呈浓度和时间依赖性(P0.05);与对照组比较,经盐酸小檗碱处理后膀胱癌T24细胞侵袭能力明显降低.P13K抑制剂LY294002可通过抑制P13K/AKT通路活化,抑制MMP2和MMP9的表达,相对应的盐酸小檗碱处理后膀胱癌T24细胞p-PI3K、pAKT、MMP2、MMP9蛋白表达下降(P0.05).结论:盐酸小檗碱可有效抑制膀胱癌T24细胞侵袭能力,其机制可能是通过调控P13K/AKT通路抑制MMP2和MMP9的表达.