Localized DNA Demethylation at Recombination Intermediates during Immunoglobulin Heavy Chain Gene Assembly

Multiple epigenetic marks have been proposed to contribute to the regulation of antigen receptor gene assembly via V(D)J recombination. Here we provide a comprehensive view of DNA methylation at the immunoglobulin heavy chain (IgH) gene locus prior to and during V(D)J recombination. DNA methylation did not correlate with the histone modification state on unrearranged alleles, indicating that these epigenetic marks were regulated independently. Instead, pockets of tissue-specific demethylation were restricted to DNase I hypersensitive sites within this locus. Though unrearranged diversity (D_H) and joining (J_H) gene segments were methylated, DJ_H junctions created after the first recombination step were largely demethylated in pro-, pre-, and mature B cells. Junctional demethylation was highly localized, B-lineage-specific, and required an intact tissue-specific enhancer, Eμ. We propose that demethylation occurs after the first recombination step and may mark the junction for secondary recombination.     

Kinetic Control in Protein Folding for Light Chain Amyloidosis and the Differential Effects of Somatic Mutations

Light chain amyloidosis is a devastating disease where immunoglobulin light chains form amyloid fibrils, resulting in organ dysfunction and death. Previous studies have shown a direct correlation between the protein thermodynamic stability and the propensity for amyloid formation for some proteins involved in light chain amyloidosis. Here we investigate the effect of somatic mutations on protein stability and in vitro fibril formation of single and double restorative mutants of the protein AL-103 compared to the wild-type germline control protein. A scan rate dependence and hysteresis in the thermal unfolding and refolding was observed for all proteins. This indicates that the unfolding/refolding reaction is kinetically determined with different kinetic constants for unfolding and refolding even though the process remains experimentally reversible. Our structural analysis of AL-103 and AL-103 delP95aIns suggests a kinetic coupling of the unfolding/refolding process with cis–trans prolyl isomerization. Our data reveal that the deletion of proline 95a (AL-103 delP95aIns), which removes the trans–cis di-proline motif present in the patient protein AL-103, results in a dramatic increment in the thermodynamic stability and a significant delay in fibril formation kinetics with respect to AL-103. Fibril formation is pH dependent; all proteins form fibrils at pH 2; reactions become slower and more stochastic as the pH increases up to pH 7. Based on these results, we propose that, in addition to thermodynamic stability, kinetic stability (possibly influenced by the presence of cis proline 95a) plays a major role in the AL-103 amyloid fibril formation process.     

人心肌肌球蛋白轻链1与重链和肌动蛋白的结合

在测得中国人心肌肌球蛋白轻链 1cDNA的核苷酸序列 ,并获得一株单克隆抗体 (HCMLC1 8)的基础上 ,用PCR方法 ,以中国人心肌肌球蛋白轻链 1的cDNA为模板 ,分别获得中国人心肌肌球蛋白轻链 1的各为 98个氨基酸的N端和C端片段cDNA的克隆并进行了表达。同时进行了其表达产物和大鼠心肌肌球蛋白重链和人心肌肌动蛋白以及单克隆抗体结合的研究 ,发现三者均和轻链 1的N端相结合 ,结合位点各不相同。这些结合位点可能均位于轻链 1的分子表面 ,而且如果轻链 1在实验状态下先与肌动蛋白结合 ,则有可能影响轻链与重链间的彼此结合。肌动蛋白在体外能以不同位点结合肌球蛋白重链和轻链 ,可能在肌肉收缩过程中具有重要的生理意义     

Cep63 and Cep152 Cooperate to Ensure Centriole Duplication

Centrosomes consist of two centrioles embedded in pericentriolar material and function as the main microtubule organising centres in dividing animal cells. They ensure proper formation and orientation of the mitotic spindle and are therefore essential for the maintenance of genome stability. Centrosome function is crucial during embryonic development, highlighted by the discovery of mutations in genes encoding centrosome or spindle pole proteins that cause autosomal recessive primary microcephaly, including Cep63 and Cep152. In this study we show that Cep63 functions to ensure that centriole duplication occurs reliably in dividing mammalian cells. We show that the interaction between Cep63 and Cep152 can occur independently of centrosome localisation and that the two proteins are dependent on one another for centrosomal localisation. Further, both mouse and human Cep63 and Cep152 cooperate to ensure efficient centriole duplication by promoting the accumulation of essential centriole duplication factors upstream of SAS-6 recruitment and procentriole formation. These observations describe the requirement for Cep63 in maintaining centriole number in dividing mammalian cells and further establish the order of events in centriole formation.     

抗人小细胞肺癌单克隆抗体的重链变区基因的体外扩增、克隆和序列分析

本文从抗人小细胞肺癌单克隆抗体杂交瘤细胞中抽提总RNA,合成第一链cDNA,直接用PCR技术(polymerase chain reaction)扩增出351bp的重链变区基因(V_H),克隆至pUCV_(NP)-PCR载体上,经筛选得一批插入片段为351bp的阳性克隆,经核苷酸序列分析研究,证实已获得了该单克隆抗体的重链可变区基因。     

Role of Glycosaminoglycan Sulfation in the Formation of Immunoglobulin Light Chain Amyloid Oligomers and Fibrils

Primary amyloidosis (AL) results from overproduction of unstable monoclonal immunoglobulin light chains (LCs) and the deposition of insoluble fibrils in tissues, leading to fatal organ disease. Glycosaminoglycans (GAGs) are associated with AL fibrils and have been successfully targeted in the treatment of other forms of amyloidosis. We investigated the role of GAGs in LC fibrillogenesis. Ex vivo tissue amyloid fibrils were extracted and examined for structure and associated GAGs. The GAGs were detected along the length of the fibril strand, and the periodicity of heparan sulfate (HS) along the LC fibrils generated in vitro was similar to that of the ex vivo fibrils. To examine the role of sulfated GAGs on AL oligomer and fibril formation in vitro, a κ1 LC purified from urine of a patient with AL amyloidosis was incubated in the presence or absence of GAGs. The fibrils generated in vitro at physiologic concentration, temperature, and pH shared morphologic characteristics with the ex vivo κ1 amyloid fibrils. The presence of HS and over-O-sulfated-heparin enhanced the formation of oligomers and fibrils with HS promoting the most rapid transition. In contrast, GAGs did not enhance fibril formation of a non-amyloidogenic κ1 LC purified from urine of a patient with multiple myeloma. The data indicate that the characteristics of the full-length κ1 amyloidogenic LC, containing post-translational modifications, possess key elements that influence interactions of the LC with HS. These findings highlight the importance of the variable and constant LC regions in GAG interaction and suggest potential therapeutic targets for treatment.     

Cerebrospinal Fluid Immunoglobulin Kappa Light Chain in Clinically Isolated Syndrome and Multiple Sclerosis

Background

Oligoclonal bands (OCB) are the most widely used CSF test to support the diagnosis of MS and to predict conversion of clinically isolated syndrome (CIS) to multiple sclerosis (MS). Since OCB tests are based on non-quantitative and difficult to standardise techniques, measurement of immunoglobulin kappa free light chains (KFLC) may represent an easier to use quantitative test.

Methods

KFLC were measured in CSF and serum of 211 patients using ELISA. These include patients without any inflammatory central nervous system reaction (NIND, n = 77), MS (n = 20), viral CNS infections (V-CNS-I, n = 10), neuroborreliosis (NB, n = 17) and other bacterial CNS infections (B-CNS-I, n = 10). Furthermore a cohort of 77 patients with CIS, including 39 patients that remained CIS over follow-up of two years (CIS-CIS) and 38 patients that developed MS over the same follow-up time (CIS-MS).

Results

CSF-serum ratio of KFLC (Q KFLC) was elevated in all patients with MS, 86.8% of patients with CIS-MS and 61.5% of patients with CIS-CIS. It was significantly elevated in CIS with presence of OCB (p<0.001). Q KFLC significantly correlated with other CSF variables such as CSF leukocyte count (p<0.001, R = 0.46), CSF CXCL13 levels (p<0.001, R = 0.64) and also intrathecal IgG synthesis (p<0.001, R = 0.74) as determined by nephelometry and quotient diagram. OCB were detected in 66.7% of CIS-CIS and in 92.1% of CIS-MS.

Conclusions

Although the measurement of CSF KFLC is a rapid and quantitative easy to standardize tool, it is almost equal but not superior to OCB with regard to diagnostic sensitivity and specificity in patients with early MS.     

Genetic Control of Variable and Constant Regions of Immunoglobulin Heavy Chains

IMMUNOGLOBULIN polypeptide chains consist of two well defined regions designated the “variable region” and the “constant region”. Whereas great diversity exists in amino-acid sequences of variable regions, the constant regions of a given subclass of heavy chains (C_H)^* are essentially invariant in sequence^{1, 2}. Exceptions are the allelic forms, such as the rabbit allotypes A14 and A15^{3, 4}, where a threonine-alanine interchange occurs in the constant region of γ chains (Appella, Chersi, R. G. M. and Dubiski, in preparation). The markers unique to a chains (for example, A14-A15) are closely linked to allotypic markers at the a locus (a1, a2, a3)^{3, 4} which seem to be present on four different Ig heavy chain classes (α, γ, ε, µ)^5–7. These puzzling observations can be explained if the a locus determinants are variable region markers which reflect genetically controlled differences in some relatively constant residues within the V_H region sequences⁷.     

Purification of Clathrin Heavy and Light Chain fromDictyostelium discoideum

Clathrin, a protein important for endocytosis, is a hexamer composed of three heavy chains and three light chains. We report here the purification scheme used to isolate the clathrin protein from the simple eukaryote,Dictyostelium discoideum.Using a combination of differential centrifugation and column chromatography, we isolated ∼2 mg of clathrin triskelions from 150–200 g ofDictyosteliumcells. One additional step purified the 30-kDa clathrin light chain to homogeneity. Glycerol gradient centrifugation was used to determine anSvalue of 7.9 for purified clathrin. Rotary shadowed images ofDictyosteliumclathrin revealed trimeric molecules with extended legs measuring 48 ± 5 nm, similar in length to the legs of mammalian and yeast clathrin triskelions. The single clathrin light chain proved resistant to heat treatment, a property also similar to light chains from other species. The conservation of these physical properties inDictyosteliumclathrin demonstrates the potential of this model organism for the study of clathrin structure and function.     

人免疫球蛋白重链可变区基因引物设计方法的改良

针对抗体胚系基因数据库的数据不断更新和完善,为获得人全部免疫球蛋白(Ig)重链可变区基因,改进引物设计方法,自主设计针对可变区基因高度保守的框架区1(FR1)和框架区4(FR4)的引物,提取未经免疫的健康人外周血单个核细胞,通过RT-PCR扩增重链可变区基因.其DNA序列与GenBank数据库和IMGT/V-QUEST软件比对,序列分析符合人免疫球蛋白重链基本框架结构,为胚系基因重排产生的序列.多个克隆的测序结果对比分析显示了良好的多样性.获得的重链序列为研制基因工程抗体及构建噬菌体抗体库奠定了物质基础,也为扩增其他物种Ig可变区基因的引物提供新的设计思路.     

Synthesis of a Mouse Immunoglobulin Light Chain in a Rabbit Reticulocyte Cell-free System

An RNA fraction isolated from a mouse myeloma functions as template for the synthesis of a mouse Ig light chain in a cell free system from rabbit reticulocytes.     

Calnexin and the Immunoglobulin Binding Protein (BiP) Coimmunoprecipitate with AMPA Receptors

To identify proteins that interact with alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionate (AMPA) receptors, we carried out coimmunoprecipitation analyses on detergent-solubilized rat forebrain membranes. Membranes were solubilized with Triton X-100, and immunoprecipitation was done using subunit-specific antibodies to GluR1, GluR2/3, and GluR4 attached to protein Aagarose. Proteins bound to the antibodies were analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis followed by silver staining and western blotting. With solubilization in low ionic strength buffer, several coimmunoprecipitating proteins, with Mr = 17,000-100,000, were identified in silver-stained gels. Western blots were then probed with antibodies to a series of candidate proteins that were chosen based on the molecular masses of the copurifying proteins. Two of these were identified as the molecular chaperones calnexin (90 kDa) and the immunoglobulin binding protein (BiP; 78 kDa). Immunoprecipitation with antibodies to calnexin and BiP demonstrated that glycosylated AMPA receptor subunits were associated. The relationship between AMPA receptors and calnexin and BiP was further studied with immunocytochemistry of the hippocampus. Both calnexin and BiP labeling was present not only in the cell body but also in dendrites of hippocampal pyramidal neurons, where double-label immunofluorescence also showed the presence of AMPA receptor subunits.     

免疫球蛋白重链可变段和T细胞受体可变段的分子进化研究

为阐明免疫球蛋白(Ig)和T细胞受体(TCR)在抗体多样性产生机理上的异同,作者比较了Ig重链可变段(Ig V_H)和TCR可变段(TCR V)的密码子替代率和协同进化,并分析异同的原因。共搜集8种鼠和3种人的TCR α链可变段(V_α),11种鼠和1种人的TCRβ链可变段(V_β),以及2种鼠和4种人的T细胞γ链可变段;同时搜集11种鼠、3种人、3种南美鳄鱼和1种鲨鱼的Ig V_(H_(o))研究结果揭示:(1)对编码蛋白质的密码子来说,TCR V(包括V_α和V_β)的核苷酸替代率为Ig V_H的2.4倍,说明前者有更高的替代率。(2)以协同进化而言,TCR V和Ig V_H的基因重复率分别为1.7×10~(-6)和1.6×10~(-6)/基因年。两者几乎相同,均系低速保持者。TCR V的数目(V_α为100,V_β为30)远少于Ig V_H(数目为300),原因是前者受到主要组织相容性复合体的制约,即受到负选择,这与中性学说观点相一致。文章还讨论了体细胞突变和DNA重排对两类抗体多样性产生上的作用,并探讨了IgV_H和TCR V的假基因问题。     

Metal Ion-dependent Heavy Chain Transfer Activity of TSG-6 Mediates Assembly of the Cumulus-Oocyte Matrix

The matrix polysaccharide hyaluronan (HA) has a critical role in the expansion of the cumulus cell-oocyte complex (COC), a process that is necessary for ovulation and fertilization in most mammals. Hyaluronan is organized into a cross-linked network by the cooperative action of three proteins, inter-α-inhibitor (IαI), pentraxin-3, and TNF-stimulated gene-6 (TSG-6), driving the expansion of the COC and providing the cumulus matrix with its required viscoelastic properties. Although it is known that matrix stabilization involves the TSG-6-mediated transfer of IαI heavy chains (HCs) onto hyaluronan (to form covalent HC·HA complexes that are cross-linked by pentraxin-3) and that this occurs via the formation of covalent HC·TSG-6 intermediates, the underlying molecular mechanisms are not well understood. Here, we have determined the tertiary structure of the CUB module from human TSG-6, identifying a calcium ion-binding site and chelating glutamic acid residue that mediate the formation of HC·TSG-6. This occurs via an initial metal ion-dependent, non-covalent, interaction between TSG-6 and HCs that also requires the presence of an HC-associated magnesium ion. In addition, we have found that the well characterized hyaluronan-binding site in the TSG-6 Link module is not used for recognition during transfer of HCs onto HA. Analysis of TSG-6 mutants (with impaired transferase and/or hyaluronan-binding functions) revealed that although the TSG-6-mediated formation of HC·HA complexes is essential for the expansion of mouse COCs in vitro, the hyaluronan-binding function of TSG-6 does not play a major role in the stabilization of the murine cumulus matrix.     

石蜡包埋组织的DNA提取及其在免疫球蛋白基因重排分析中的应用

基因重排分析在淋巴瘤诊断中具有重要意义.文章应用改良DNA提取方法,从30例淋巴增生性病变石蜡包埋组织获得的DNA虽有不同程度的降解,但适于PCR扩增Ig重链基因重排分析;约1/3病例提出高分子量DNA,可用于DNA印迹杂交.因此,石蜡包埋组织同样可为某些疾患,如淋巴瘤疑难和罕见病例的回顾性分子病理学研究提供基因诊断的DNA来源.     

抗乙肝病毒表面抗原嵌合抗体基因在昆虫细胞中的表达

应用杆状病毒表达系统在昆虫细胞中表达了抗乙肝病毒表面抗原（ＨＢｓＡｇ）人－鼠嵌合抗体重,轻链基因,鼠源单克隆抗体ＯＨ３重,轻链可变区（ＶＨ,ＶＬ）ｃＤＮＡ分别与人免疫球蛋白恒区γ３,ｋ,ｃＤＮＡ拼接成人－鼠嵌合抗体基因,含嵌合抗体的转移载体与线性化病毒ＤＮＡ共转染Ｓｆ９细胞,并通过点杂交ＰＣＲ扩增和Ｓｏｕｔｈｅｒｎｂｌｏｔ分析获得重组病毒。Ｗｅｓｔｅｒｎｂｌｏｔ和竞争ＥＬＩＳＡ表明以重组病毒感染的     

The Immunoglobulin G Heavy Chain (IGHG) genes of the Atlantic bottlenose dolphin, Tursiops truncatus

Dolphin Immunoglobulin G Heavy Chain (IGHG) sequences were obtained by PCR amplification of cDNA from peripheral blood leukocytes using degenerate primers. Analysis of full-length sequences indicated the presence of two expressed isotypes, IGHG1 and IGHG2 that differ mainly in the hinge region of the molecule. Genomic Southern blot analysis indicated that the IGHG1 and IGHG2 genes are most likely present in single copies. The inferred amino acid sequences show greatest similarity between the dolphin and other closely related artiodactyl species. The genetic structure of the IGHG genes were deduced through genomic PCR and revealed that the hinge regions of both IGHG1 and IGHG2 are encoded by a single exon. The transmembrane region of the dolphin IGHG chain shows similarity to the transmembrane region of other mammalian IGHG chains with a canonical CART motif. This is in contrast to the unusual Ser to Gly substitution previously found in the dolphin IGHM transmembrane region, and the functional significance of this variation for B cell antigen-receptor dimer activation remains unknown.