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1.
Xiang Y Zhou X Hewitt SL Skok JA Garrard WT 《Journal of immunology (Baltimore, Md. : 1950)》2011,186(9):5356-5366
Nonbiased V gene usage for V(D)J joining is essential for providing an optimal immune system, but no cis-acting sequence with this function has been uncovered. We previously identified a recombination silencer and heterochromatin targeting element in the Vκ-Jκ intervening sequence of germline Igκ transgenes, which we termed Sis. We now have generated Sis knockout mice in the endogenous locus. Intriguingly, Sis(-/-) mice exhibit a skewed Igκ repertoire with markedly decreased distal and enhanced proximal Vκ gene usage for primary rearrangement, which is associated with reduced occupancy of Ikaros and CCCTC-binding factor in the Vκ-Jκ intervening sequence in pre-B cells, proteins believed to be responsible for dampening the recombination of nearby Vκ genes and altering higher-order chromatin looping. Furthermore, monoallelic heterochromatin localization is significantly reduced in Sis(-/-) mice for Igκ in cis and IgH loci in trans in pre-B cells. Because Sis(-/-) mice still allelically excluded Igκ and IgH loci and still exhibited IgL isotype exclusion, we concluded that stable localization at pericentromeric heterochromatin is neither necessary nor sufficient for the establishment or maintenance of allelic exclusion. Hence, Sis is a novel multifunctional element that specifies repertoire and heterochromatin localization to Ig genes. 相似文献
2.
Horynová M Takahashi K Hall S Renfrow MB Novak J Raška M 《Protein expression and purification》2012,81(2):175-180
The human UDP-N-acetyl-α-d-galactosamine:polypeptide N-acetylgalactosaminyl-transferase 2 (GalNAc-T2) is one of the key enzymes that initiate synthesis of hinge-region O-linked glycans of human immunoglobulin A1 (IgA1). We designed secreted soluble form of human GalNAc-T2 as a fusion protein containing mouse immunoglobulin light chain kappa secretory signal and expressed it using baculovirus and mammalian expression vectors. The recombinant protein was secreted by insect cells Sf9 and human HEK 293T cells in the culture medium. The protein was purified from the media using affinity Ni-NTA chromatography followed by stabilization of purified protein in 50mM Tris-HCl buffer at pH 7.4. Although the purity of recombinant GalNAc-T2 was comparable in both expression systems, the yield was higher in Sf9 insect expression system (2.5mg of GalNAc-T2 protein per 1L culture medium). The purified soluble recombinant GalNAc-T2 had an estimated molecular mass of 65.8kDa and its amino-acid sequence was confirmed by mass-spectrometric analysis. The enzymatic activity of Sf9-produced recombinant GalNAc-T2 was determined by the quantification of enzyme-mediated attachment of GalNAc to synthetic IgA1 hinge-region peptide as the acceptor and UDP-GalNAc as the donor. In conclusion, murine immunoglobulin kappa secretory signal was used for production of secreted enzymatically active GalNAc-T2 in insect baculovirus expression system. 相似文献
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6.
Evolution of a Vκ gene family 总被引:2,自引:0,他引:2
To examine the evolution of multigene families we have selected as an example an immunoglobulin light chain variable region subgroup (V24) which has been extensively characterized in inbred mice (Mus musculus domesticus). Homologous genes have been isolated and sequenced from Mus pahari, a genetically and geographically isolated species believed to be the oldest living representative of the genus. Southern blot analysis using probes corresponding to individual genes in this subgroup reveals changes in the overall size of the family occurring at the level of individual genes but not at the level of the entire family. Nucleotide sequence analysis indicates an absence of regulatory sequences such as the CAT and TATA boxes 5 to the coding region, but a decanucleotide sequence involved in light chain expression is highly conserved. Within coding regions highly complex patterns of variation are seen which appear to reflect quite different selective pressures on various subregions of the coding sequence. Complementarity determining regions (CDR) are conserved to different extents, with the first CDR region in all family members being among the most conserved segments of the molecule. Conservation is similarly variable among framework segments, indicating complex and variable evolutionary pressures not only at the level of individual genes or their products but also at subregions within homologous molecules. 相似文献
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目的:探讨PD-L1 Ig(Programmed death ligand-1 immunoglobulin,程序性死亡配体-1免疫球蛋白)在缓解肾移植急性排斥反应中的免疫调节作用。方法:选择健康成年雄性Lewis大鼠24只作为供体,另取24只健康雄性Wistar大鼠作为受体。所选大鼠3个月龄,体重300 g±30 g,创建肾移植大鼠模型,并将其随机分为三组,空白对照组,对照组,和PD-L1 Ig组。空白对照组:灌注液,肾动脉灌注3分钟。PD-L1 Ig组:含1 m L的PD-L1 Ig的灌注液,肾动脉灌注3分钟。对照组不进行额外灌注操作。术后观察三组大鼠食欲、精神状态及生存情况。7天后取血标本观察肌酐、尿素氮、γ-干扰素(Interferon-γ, IFN-γ),白细胞介素-2(Interleukin-2, IL-2),白细胞介素-10 (Interleukin-10, IL-10)水平,同时三组各处死3只大鼠行肾脏病理学检查。结果:1.病理:对照组及空白对照组大鼠较模型大鼠有改善,仍有较多的炎细胞浸润,间质水肿,红细胞管型,肾小管上皮细胞脱落。而PD-L1 Ig组大鼠较模型大鼠、对照组及空白对照组有显著改善。2.生存状态和肾功能:PD-L1 Ig组大鼠生存时间最长50天,平均存活时间和体重均显著高于对照组和空白对照组,差异均有统计学意义(P0.05);血肌酐、尿素氮水平显著低于对照组和空白对照组,差异均有统计学意义(P0.05)。3.细胞因子变化:PD-L1 Ig组IFN-γ、IL-2水平显著低于空白对照组、对照组,差异均有统计学意义(P0.05);PD-L1 Ig组IL-10水平显著高于空白对照组、对照组,差异均有统计学意义(P0.05)。结论:PD-L1 Ig可降低IL-2和IFN-γ,提升IL-10,调节移植肾急性排斥反应的细胞因子平衡,改善肾功能,延长移植肾存活时间。 相似文献
10.
Kagefumi Todo Orie KogaMiwako Nishikawa Masaki Hikida 《Biochemical and biophysical research communications》2014
It has been shown that cytoplasmic tail of the IgG1 B cell receptors (BCRs) are essential for the induction of T-dependent immune responses. Also it has been revealed that unique tyrosine residue in the cytoplasmic tail of IgG2a has the potential of being phosphorylated at tyrosine and that this phosphorylation modulates BCR signaling. However, it still remains unclear whether such phosphorylation of IgG cytoplasmic tail is involved in the regulation of BCR surface expression. In order to approach the issue, we established and analyzed the cell lines which express wild-type or mutated forms of IgG1 BCR. As the result, we found that IgG1 BCR expressed normally on the surface of A20 B cell line independent of the cytoplasmic tail. In contrast, IgG1 BCR whose cytoplasmic tyrosine was replaced with glutamic acid which mimics phosphorylated tyrosine, was expressed most efficiently on the surface of non-B lineage cells and Igβ-down-regulated B cell lines. These results suggest that tyrosine residue in IgG cytoplasmic tail is playing a essential role for the efficient expression of IgG BCR on the cell surface when BCR associated signaling molecules, including Igβ, are down-regulated. 相似文献
11.
噬菌体展示 protein A 及 protein L Ig 结合单结构域随机组合文库及 Ig 亲和筛选 总被引:8,自引:1,他引:7
Protein A 和 protein L 是细菌产生的两种结构和功能均不同的免疫球蛋白 (immunoglobulin , Ig) 结合分子,在细菌的致病中起重要作用 . 用含 SacⅠ位点的特定引物 PCR 分别扩增制备 protein A 的 A 、 B 、 C 、 D 抗体结合结构域和 protein L 的 B3 抗体结合结构域,各结构域 DNA 片段经 SacⅠ酶切后,再随机连接形成各种不同长度的分子组合文库,将该文库呈现在噬菌体表面构建了噬菌体展示 Ig 结合分子单结构域随机组合文库,所建组合文库容量为 2.3×106个菌落形成单位,滴度为 4.1×1011TU/ml , 包含各种单结构域片段,并以随机方式连接 . 用人 Ig 对该文库进行 4 轮亲和筛选,随机挑选 36 个代表性的阳性克隆进行序列测定分析表明,亲和筛选获得了多种非天然形式存在的新的 Ig 结合分子结构,其中 32 个克隆具有由 protein L 的单结构域和 protein A 的单结构域间隔重复排列而成的特征性 (MDPL-MDPA)n 结构 . 对噬菌体展示 Ig 结合分子单结构域随机组合文库的体外分子进化研究的尝试,为 Ig 结合分子的结构和功能研究提供了一新的途径,也为 Ig 结合分子的定向改造打下基础 . 相似文献
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目的:初步探索NF-κB和HIF-1在宫颈上皮内瘤变和宫颈鳞癌中的表达及临床病理学意义。方法:以宫颈上皮内瘤变和宫颈鳞癌病例为研究对象,应用免疫组织化学方法检测NF-κB和HIF-1的表达情况并分析其表达在宫颈癌变中的意义。结果:NF-κB在正常宫颈上皮、CINⅠ、CINⅡ、CINⅢ、鳞癌的阳性表达率分别为16%,36%,44%,68%,72%。总体比较存在显著性差异x2=21.636,p〈0.01。组间两两比较显示正常上皮与CINⅢ和鳞癌有显著性差异。HIF-1α在正常宫颈上皮、CINⅠ、CINⅡ、CINⅢ、鳞癌的阳性表达率分别为12%,20%,40%,68%,76%。总体比较存在显著性差异x2=32.733,p〈0.01。组间两两比较显示正常上皮与CINⅢ(x2=16.333 p〈0.001)和鳞癌(x2=20.779 p〈0.001)有显著性差异。结论:NF-κB和HIF-1与宫颈鳞癌的发生有关,可能作为早期诊断的标志物。 相似文献
13.
EB病毒LMP1在鼻咽癌细胞中通过NFκB促进Igκ表达 总被引:5,自引:1,他引:4
利用已建立的受四环素调控LMP1表达的鼻咽癌细胞系,用受CMV启动子调控的NFκB报道基因质粒pNFκB-luc的荧光素酶表达分析NFκB的活性,并以核蛋白的Western印迹方法观察LMP1表达前后核内NFκB组分p65量的改变,用全蛋白Western印迹分析Igκ蛋白质的表达等方法,探讨在鼻咽癌中,EB病毒LMP1蛋白是否通过核转录因子NFκB促进免疫球蛋白κ轻链(Igκ)基因的表达。结果显示 相似文献
14.
Yong Chen Chongguang Chen Evangelia Kotsikorou Diane L. Lynch Patricia H. Reggio Lee-Yuan Liu-Chen 《The Journal of biological chemistry》2009,284(3):1673-1685
We demonstrated previously that the protein GEC1 (glandular epithelial cell
1) bound to the human κ opioid receptor (hKOPR) and promoted cell
surface expression of the receptor by facilitating its trafficking along the
secretory pathway. Here we showed that three hKOPR residues
(Phe345, Pro346, and Met350) and seven GEC1
residues (Tyr49, Val51, Leu55,
Thr56, Val57, Phe60, and Ile64)
are indispensable for the interaction. Modeling studies revealed that the
interaction was mediated via direct contacts between the kinked hydrophobic
fragment in hKOPR C-tail and the curved hydrophobic surface in GEC1 around the
S2 β-strand. Intramolecular Leu44-Tyr109
interaction in GEC1 was important, likely by maintaining its structural
integrity. Microtubule binding mediated by the GEC1 N-terminal domain was
essential for the GEC1 effect. Expression of GEC1 also increased cell surface
levels of the GluR1 subunit and the prostaglandin EP3.f receptor, which have
FPXXM and FPXM sequences, respectively. With its widespread
distribution in the nervous system and its predominantly hydrophobic
interactions, GEC1 may have chaperone-like effects for many cell surface
proteins along the biosynthesis pathway.κ opioid receptor
(KOPR)2 is one of the
three major types of opioid receptors mediating effects of opioid drugs and
endogenous opioid peptides. Stimulation of KOPR generates many effects in
vivo, for example antinociception (especially for visceral chemical pain,
antipruritis, and water diuresis
(1). The KOPR agonist
nalfurafine (TRK-820) is used clinically in Sweden for the treatment of uremic
pruritus in kidney dialysis patients
(2). Because KOPR agonists
produce profound sedative effects, it has been proposed that KOPR agonists may
be useful in treating mania, antagonists as anti-depressants, and partial
agonists for the management of mania depression
(3). KOPR antagonists may also
be useful for curbing cocaine craving and as anti-anxiety drugs
(4,
5).KOPR, a member of the rhodopsin subfamily of the seven-transmembrane
receptor superfamily, is coupled preferentially to pertussis toxin-sensitive G
proteins, namely Gi/o proteins
(6). KOPR has been found to
interact with several non-G protein-binding partners, such as
Na+,H+-exchanger regulatory
factor-1/ezrin-radixin-moesin-binding phosphoprotein-50 and the δ opioid
receptor. These interactions have influence on signal transduction and
trafficking of the receptor
(7–9).
By yeast two-hybrid (Y2H) assay using the hKOPR C-tail to screen a human brain
cDNA library, we identified GEC1, also named GABAA
receptor-associated protein like 1 (GABARAPL1), to be a binding partner of
hKOPR (10).GEC1 cDNA was first cloned as an early estrogen-regulated mRNA from guinea
pig endometrial glandular epithelial cells by Pellerin et al.
(11). Subsequently, it was
cloned from other species, including human and house mouse
(12). Interestingly, the amino
acid sequences of GEC1 are completely conserved among all these species except
orangutan, in which Arg99 substitutes for His99.
Northern blot and immunoblotting analyses revealed that it has widespread
tissue distribution
(12–14).
In particular, GEC1 was found to be abundant in the central nervous system and
expressed throughout the rat brain
(14,
15). This wide tissue
distribution and the high sequence identity across species strongly suggest
that GEC1 has important biological functions in mammalian cells.Based on sequence similarity, GEC1 is classified as a member of
microtubule-associated proteins (MAPs), which also include GABAA
receptor-associated protein (GABARAP), Golgi-associated ATPase enhancer of 16
kDa (GATE16), GABARAP-like 3 (GABARAPL3), light chain 3 (LC3) of MAP 1A/1B,
and the yeast autophagy protein 8 (Atg8)
(12,
13). Among these homologues,
GEC1 share the highest identity with GABARAPL3 (93%), followed by GABARAP
(86%), GATE16 (61%), Atg8 (55%), and LC3 (∼30%).A growing body of evidence shows that this protein family is closely
related to two distinct biological functions. Studies mainly on GABARAP,
GATE16, and GEC1 indicate that they promote intracellular protein trafficking
by enhancing vesicle fusion
(10,
16–21).
In addition, they facilitate degradation of proteins and intracellular
organelles via autophagy-related pathways, which is bolstered largely by
research on Atg8 and LC3 (22,
23).We previously reported that GEC1 interacted with the hKOPR C-tail and
enhanced cell surface levels of hKOPR stably expressed in CHO cells. GEC1
expression enhances hKOPR expression through facilitating its anterograde
trafficking along the protein biosynthesis pathway without affecting
degradation of the receptor
(10). This represented the
first biological function reported for GEC1. Mansuy et al.
(24) demonstrated that GEC1
interacted with tubulin and promoted microtubule bundling in vitro,
and that green fluorescence protein-tagged GEC1 was localized in the
perinuclear vesicles with a scattered pattern. Our electron microscopic
studies in the rat brain showed that GEC1 was associated with ER, Golgi
apparatus, endosome-like vesicles, and plasma membranes and scattered in
cytoplasm in neurons (14). In
addition, N-ethylmaleimide-sensitive factor, a protein critical for
intracellular membrane-trafficking events, binds directly to GEC1
(10).In this study, we employed Y2H techniques to determine the amino acid
residues in both GEC1 and hKOPR C-tail involved in the interaction. Further
studies were then carried out in mammalian cells to examine if elimination of
the interaction affected the effect of GEC1 on hKOPR expression. In addition,
we generated a molecular model of GEC1 based on the x-ray crystal structure of
GABARAP and found that the residues involved in hKOPR binding formed
hydrophobic patches on the exterior surface of GEC1. Moreover, we found that
the cytosolic tail of AMPA receptor subunit GluR1 has the same FPXXM
motif as that found in the hKOPR C-tail to be involved in GEC1 binding and
that GEC1 expression up-regulated GluR1. 相似文献
15.
目的:探讨大鼠肝脏缺血再灌注损伤NF-κB和ICAM-1表达情况及NAC的保护作用机制。方法:45只雄性SD大鼠随机分成三组:假手术组(Sham组,n=5);缺血再灌注损伤组(I/R组,n=20)缺血60min后分别再灌注1、3、6、12h;N-乙酰半胱氨酸组(NAC组,n=20):先自阴茎背静脉给大鼠注射溶于生理盐水的NAC,20min后再按I/R组处理。在各规定的再灌注时间点,分别采用western-blot和免疫组化方法测定肝组织中NF-κB和ICAM-1的表达。结果:I/R组和NAC组再灌注1、3、6、12h后,NF-κB的表达均明显高于Sham组(p〈0.01),于再灌注3h达到高峰;ICAM-1的表达均明显高于Sham组(p〈0.01),于再灌注6h达到高峰。NAC组再灌注1、3、6h与I/R组相同时间点比较:NF-κB和ICAM-1的表达均低于I/R组(p〈0.05)。NAC组再灌注12h与I/R组相同时间点比较:NF-κB和ICAM-1的表达虽然在数值上有所减少,但统计学上无差异(p〉0.05)。结论:大鼠肝脏缺血再灌注后NF-κB和ICAM-1表达增加,NAC可抑制NF-κB激活,减少ICAM-1表达减轻大鼠肝脏缺血再灌注损伤。 相似文献
16.
Antibodies recognize antigens through six hypervariable loops, five of which have a limited set of conformations known as canonical structures. For κ light chains, the majority of CDR-L3 [the third hypervariable loop of the light chain variable domain (VL)] adopts the type 1 canonical structure (CS1), with a cis-proline at position 95. Here, we present the design and structural studies of the monoclonal antibody mAb15 and related mutants that contained a series of progressively germline mutations only in the heavy chain variable domain (VH) that ultimately led to an increase of more than 11 °C in the melting temperature (Tm) of the antigen-binding fragment (Fab). The all-trans CDR-L3 structure in the wild type is significantly different from any known CDR-L3 canonical structures. In the thermally stable mutants, the L94L-S95L peptide bond adopts an energetically unfavorable non-X-proline cis conformation, but the overall CDR-L3 loop converted to CS1. The stabilized VH appears to function as a specific molecular chaperone that facilitated the trans-cis isomerization of S95L. Thus, it is plausible that proline is the evolutionary choice to maintain overall structure and stability for VL. These results provide new insights into the evolution of CS1 and suggest a potential molecular switch mechanism at position 95 that links CDR-L3 structural diversity and antibody stability and will have implications for antibody engineering. 相似文献
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Wei Bai Jing Zhou Na Zhou Qin Liu Jian Cui Wei Zou Wei Zhang 《Biochemical and biophysical research communications》2018,495(3):2282-2288
The potential role of hypoxia in mediating the receptor for advanced glycation end products (RAGE) expression deserves to be confirmed. And the role of RAGE in hypoxia-induced chemotaxis and inflammation is still unclear. In present study, THP-1?cells were pretreated with siRNA to block HIF1α, NF-κ B, or RAGE, followed by exposed to hypoxia (combined with H2O2 or SNP), and then RAGE expression, nuclear translocation of HIF1α and NF-κ B, release of TNF-α and IL-1β, as well as expression of MCP-1 and CCR2 were measured. The results revealed that RAGE mRNA and protein in THP-1?cells were significantly increased after exposed into hypoxia atmosphere, especially into the solution containing SNP or H2O2. Moreover, SNP or H2O2 exposure could further amplify hypoxia-induced nuclear translocation of HIF-1α and NF-κ B. Knockdown HIF-1α or NF-κ B by siRNAs could reduce hypoxia- and oxidative stress-induced RAGE hyper-expression. And pretreatment THP-1?cells with RAGE siRNA or NF-κ B siRNA could reduce hypoxia- and oxidative stress-induced expression of MCP-1 and CCR2, and release of TNF-α and IL-1β. Thus, hypoxia not only increases RAGE expression in THP-1?cells by promoting nuclear translocation of NF-κ B and HIF1α, but also regulates chemotaxis and pro-inflammatory cytokines release, which may be partially mediated through upregulation of RAGE expression. 相似文献
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