采用重组侧链二氧酸脱氢酶复合物E2亚单位检测M2抗体及其临床意义

目的 探讨用重组表达的侧链二氧酸脱氢酶复合物E2亚单位（BCOADC—E2）检测M2抗体，以早期诊断原发性胆汁性肝硬化（PBC）。方法 重组表达BCOADC—E2，采用酶联免疫吸附试验（ELISA）和免疫印迹法（IBT）来检测PBC患者的M2抗体，并以健康体检者、自身免疫病患者、其他非胆汁性肝硬化患者为对照组。结果 60例肝病患者血清中检测出抗BCOADC—E2抗体阳性33例，阴性者为27例，阳性率为55％，而健康体检者和疾病对照组血清中M2抗体检测均为阴性。结论 采用重组表达的人侧链二氧酸脱氢酶复合物E2亚单位检测M2抗体，有一定的特异性，对于早期诊断PBC有一定的辅助参考作用。     

人源M2抗原检测M2抗体诊断抗线粒体抗体阴性原发性胆汁性肝硬化

目的 分析抗线粒体抗体（AMA）阴性的原发性胆汁性肝硬化（PBC）患者与AMA阳性PBC患者在临床症状，实验室诊断指标等方面的差异；对比用克隆表达的人源M2抗原建立的酶联免疫吸附试验（ELISA）法测定M2抗体，与经典的间接免疫荧光法测定AMA的敏感性高低。方法 测定63例PBC患者外周血中的AMA与M2抗体。结果 AMA在63例PBC患者中的阳性率为81%。而M2抗体为100%；AMA阴性的PBC患者抗核抗体（ANA），抗平滑肌抗体（ASMA）的阳性率高于AMA阳性的PBC患者，但在临床症状和其他实验室诊断标准上差异并无显著性。结论 酶联免疫吸附试验（ELISA）法检测M2抗体的敏感性高于间接免疫荧光法测定AMA，PBC患者外周血中AMA的阳性与否和疾病并无明显关联。     

原发性胆汁性肝硬化抗线粒体抗体M2亚型的诊断价值

目的探讨抗线粒体亚型（AMA亚型）对原发性胆汁性肝硬化（PBC）的诊断价值。方法应用酶免疫斑点法检测血清中AMA-M2、M4、M9亚型。13例经临床诊断为PBC患者，44例非PBC肝胆病患者。结果13例PBC患者M2均阳性、M4及M9均阴性；44例非PBC肝胆病患者M2、M4、M9均阴性。5例PBC者肝活检4例为Ⅱ期、1例为Ⅳ期。结论血清AMA-M2抗体检测可作为临床诊断PBC的重要血清免疫学指标。     

抗线粒体抗体及其分型对原发性胆汁性肝硬化的诊断价值

目的 研究抗线粒体抗体(AMA)及其分型检测对原发性胆汁性肝硬化(PBC)的诊断价值。 方法 应用间接免疫荧光法测定血清中AMA抗体,用免疫印迹法检测AMA-M2、M4、M9亚型。78例PBC患者、35例其他肝病患者和20名健康体检者检测AMA及M2,其中30例PBC患者检测M4、M9亚型。 结果 78例PBC患者中74例(94.9％)AMA及M2均阳性,1例AMA阳性而M2阴性,3例AMA及M2均阴性。35例其他肝病患者M2均阴性,2例AMA阳性,1例AMA弱阳性。30例M2均阳性PBC患者中,16例M4阳性(53.3％),4例M9阳性(13.3％)。20名健康体检者AMA及M2均阴性。 结论 AMA及其分型,特别是M2抗体检测可作为临床诊断PBC的重要血清免疫学指标。     

人源M2三联体靶抗原检测抗体诊断原发性胆汁性肝硬化

目的 探讨用人源M2三联体靶抗原检测M2抗体在原发性胆汁性肝硬化诊断中的应用价值。方法 间接免疫荧光法检测肝病患者，自身免疫病患者和健康体检者血清中的抗线粒体抗体。以人源M2三联体靶抗原建立的ELISA法与以猪心肌纯化组分作为抗原的免疫印迹法商业试剂盒检测M2抗体。结果 20例原发性胆汁性肝硬化（PBC）均检出M2抗体，28例不明原因肝病6例M2抗体阳性。ELISA法的敏感性和特异性优于经典的间接免疫荧光法和商业试剂盒。结论 以人源M2三联体靶抗原建立的ELISA法具有很高的特异性和敏感性，为原发性胆汁性肝硬化的诊断提供了简便、快速而有效的手段。     

人源M2三联体靶抗原BPO-E2融合蛋白的克隆表达与鉴定

目的用基因工程的方法克隆表达抗线粒体抗体二亚型（AMA-M2）的3个靶抗原侧链二氧酸脱氢酶复合体E2（BCOADC-E2）、丙酮酸脱氢酶复合体E2（PDC-E2）、2-氧戊二酸脱氢酶复合体E2（OGDC-E2）及其连接形成的三联体BPO-E2融合蛋白，以用于人原发性胆汁性肝硬化（PBC）的早期发现和临床诊断。方法针对BCOADC-E2、PDC-E2、OGDC-E2的cDNA序列设计引物，从正常人的淋巴细胞中提取RNA，通过反转录PCR方法扩增得到相应的基因片段，经测定序列验证后插入表达载体pET28a（＋），构建重组表达载体pET28a（＋）-BCOADC-E2、pET28a（＋）-PDC-E2、pET28a（＋）-OGDC-E2、pET28a（＋）-BPO-E2，转化大肠杆菌BL21（DE3）后诱导表达蛋白质。表达蛋白经SDS-PAGE、Western-blot等鉴定。结果经核苷酸序列测定和酶切鉴定结果表明，成功地构建了4种重组质粒。IPTG诱导表达后，获得4种融合蛋白。经免疫学鉴定，重组抗原片段具有AMA-M2的免疫原性。结论重组表达的融合蛋白将有利于原发性胆汁性肝硬化（PBC）的实验室诊断。     

抗线粒体抗体M2亚型IgG和IgA在原发性胆汁性肝硬化诊断中的作用

目的 评价以新型天然M2抗原和BPO融合蛋白M2-3E(BPO)为靶抗原的酶联免疫吸附(ELISA)法(抗-M2-3E ELISA)检测抗线粒体抗体M2亚型(AMA-M2)IgG和IgA抗体在原发性胆汁性肝硬化(PBC)诊断中的敏感性、特异性以及其临床意义.方法 分别用间接免疫荧光法(IFL)、以丙酮酸脱氢酶复合体为靶抗原的ELISA法(抗-PDC ELlSA),抗-M2-3E ELISA法检测107例PBC患者,87例疾病对照患者和26名健康体检者的AMA-M2 IgG和(或)IgA抗体.检测ALT,AST,总胆红素、白蛋白、Y-谷氨酰转肽酶、碱性磷酸酶、血清肌酐、IgG、IgM及IgA.对于符合正态分布的计量数据采用t检验,不符合正态分布者采用秩和检验. 结果 107例PBC患者抗-M2-3E ELISA法AMA-M2 IgG的检出率(90.6%)高于IFL法(81.3%)和抗-PDCELISA法(72.9%),t值分别为4.32和6.03,P值均<0.05.抗M2-3E ELISA法检测AMA-M2 IgA的检出率为55.1%,特异性为97.2%,而总体AMA-M2 IgG和(或)IgA的检出率为92.5%,特异性为95.2%.20例IFL检测AMA-M2 IgG为阴性的PBC患者中,抗-M2-3E ELISA法检出率为45%,AMA-M2 IgG和(或)IgA的检出率为55%,抗-M2-3E ELISA可以在超过一半的IFL阴性的患者中检出AMA-M2 IgG和(或)IgA.AMA-M2 IgG呈阳性的患者(97例)比呈阴性的患者(10例)有更加严重的组织学变化和更高的IgG,IgM和碱性磷酸酶水平.结论 抗-M2-3E ELISA法具有比IFL和抗-PDC ELISA法更高的敏感性和特异性,可作为第一轮AMA-M2的筛查.AMA-M2 IgG阳性可能说明该PBC患者有更加严重的疾病,但是AMA-M2 IgG和IgA都不能单独用于诊断PBC.     

原发性胆汁性肝硬化52例患者自身抗体的检测

目的 研究血清学指标M2亚型抗线粒体抗体（AMA-M2），核包膜蛋白（GP210），及核多点抗体（SP100）对原发性胆汁性肝硬化（primary biliary cirrhosis，PBC）的诊断意义。方法 研究对比52例患者的临床表现、实验室检查、自身抗体及并发病种。结果 52例患者中AMA-M2阳性率为61％，GP210阳性率30％，SP100阳性率为29％，其中GP210在AMA-M2阴性的PBC患者中阳性率为62％。SPl00在AMA-M，阴性的PBC患者中阳性率为87％，9．6％PBC患者M2与GP210同时出现，5．8％PBC患者M2与SP100同时出现。结论 SPl00、GP210与传统AMA-M2同时检测可增加对PBC诊断的特异性并提高PBC的检出率。     

原发性胆汁性肝硬化抗线粒体抗体M2亚型检测方法和临床评估

目的　研究抗线粒体抗体 (AMA)M2 亚型检测对原发性胆汁性肝硬化 (PBC)的诊断价值。方法　应用酶联免疫吸附试验 (ELISA) ,检测人血清中M2 抗体含量。共检测正常人 2 0名 ,PBC患者 3 7例 ,其他肝病患者 5 0例。结果　ELISA法检测M2 抗体重复性好 ,灵敏度高 ,特异性强。 3 7例PBC患者中有 3 6例阳性 (>5U/ml) ,只有 1例阴性。而其它肝胆系患者 5 0例 ,3例为假阳性。灵敏度为 97.3 % ,特异性为 94.0 %。正常人均为阴性。PBC患者M2 抗体滴度为 (73 .66± 5 5 .93 )U/ml,较正常人 (1.60± 1.13 )U /ml显著升高 (P <0 .0 1) ,其他肝胆系患者M2 抗体滴度 (2 .90±3 .16)U/ml与正常人相比无显著差别 (P >0 .0 5 )。结论　M2 抗体检查可作为临床诊断PBC的重要血清免疫学指标     

检测M2抗体早期发现原发性胆汁性肝硬化

原发性胆汁性肝硬化 (primarybiliarycirrhosis,PBC)是一种慢性肝内胆汁淤积性疾病 ,其病因和发病机制尚不完全清楚。高滴度的抗线粒体抗体 (AMA)是PBC的重要血清学指标 ,AMA可被分为M1～M 9共 9个亚型 ,其中只有M2为PBC特异性抗体[1] 。我们建立了M2抗体检测ELISA法[2 5] 。在本研究中我们用该方法在体检者中筛查PBC特异性M2抗体。一、对象与方法1.研究对象 :2 0 0 1年 1月至 2 0 0 1年 6月上海长征医院成批体检者 185 2例 ,均是由单位安排工作人员集体作健康检查 ,其中男 84 8例 ,女 10 0 4例 ,年龄为 2 6～ 82周岁 ,平均(4 …     

Classification and characterization of hereditary types 2A, 2B, 2C, 2D, 2E, 2M, 2N, and 2U (unclassifiable) von Willebrand disease.

All variants of type 2 von Willebrand disease (VWD) patients, except 2N, show a defective von Willebrand factor (VWF) protein (on cross immunoelectrophoresis or multimeric analysis), decreased ratios for VWF:RCo/Ag and VWF:CB/Ag and prolonged bleeding time. The bleeding time is normal and FVIII:C levels are clearly lower than VWF:Ag in type 2N VWD. High resolution multimeric analysis of VWF in plasma demonstrates that proteolysis of VWF is increased in type 2A and 2B VWD with increased triplet structure of each visuable band (not present in types 2M and 2U), and that proteolysis of VWF is minimal in type 2C, 2D, and 2E variants that show aberrant multimeric structure of individual oligomers. VWD 2B differs from 2A by normal VWF in platelets, and increased ristocetine-induced platelet aggregation (RIPA). RIPA, which very likely reflects the VWF content of platelets, is normal in mild, decreased in moderate, and absent in severe type 2A VWD. RIPA is decreased or absent in 2M, 2U, 2C, and 2D, variable in 2E, and normal in 2N. VWD 2M is usually mild and characterized by decreased VWF:RCo and RIPA, a normal or near normal VWF multimeric pattern in a low resolution agarose gel. VWD 2A-like or unclassifiable (2U) is distinct from 2A and 2B and typically featured by low VWF:RCo and RIPA with the relative lack of high large VWF multimers. VWD type 2C is recessive and shows a characteristic multimeric pattern with a lack of high molecular weight multimers, the presence of one single-banded multimers instead of triplets caused by homozygosity or double hereozygosity for a mutation in the multimerization part of VWF gene. Autosomal dominant type 2D is rare and characterized by the lack of high molecular weight multimers and the presence of a characteristic intervening subband between individual oligimers due to mutation in the dimerization part of the VWF gene. In VWD type 2E, the large VWF multimers are missing and the pattern of the individual multimers shows only one clearly identifiable band, and there is no intervening band and no marked increase in the smallest oligomer. 2E appears to be less well defined, is usually autosomal dominant, and accounts for about one third of patients with 2A in a large cohort of VWD patients.     

Different RBC aggregating properties of the Aalpha2Bbeta2gammaA2 and Aalpha2Bbeta2gammaAgamma' subpopulations of human fibrinogen

Human fibrinogen (TF) has been separated into two fractions: F1 - homodimers with respect to the gamma chain, and F2 - heterodimers composed of gammaA and gamma' polypeptides. Their rouleaux-inducing properties were as follows: (1) both, at the same concentration of 0.8%, were less effective than TF; (2) F1 produced larger rouleaux even under static conditions of a hemocytometer where F2 was silent; (3) F2 induced the process when a suspension was gently sheared between microscopic slides. Since the synthetic peptide gamma'(414-427) inhibited the rouleau formation in a mixture with F2, the C-terminal amino acids of the gamma' polypeptide probably bind the molecule to the cell. The inhibition was feebly visible in the native ratio of F1/F2, implicating a compensatory effect of F1.     

Structure-function of Hb Marseille-Long Island [alpha 2 beta 2 N-methionyl-2(NA2) His----Pro

Suspensions of red cells containing Hb Marseille-Long Island showed decreased oxygen affinity and low interaction with 2,3-diphosphoglycerate. Oxygen equilibrium studies of the purified component confirmed these abnormalities. Oxidation rate measurements of carbonmonoxy-Hb Marseille and carbonmonoxy-Hb A by ferricyanide showed an increased rate for the former, suggesting an increased dissociation constant for carbon monoxide. Nuclear Magnetic Resonance spectra in the high field region revealed small changes in the proximal region of the heme pocket. These results indicated that the mutation causes a perturbation at a distance from the mutation site.     

Task2 potassium channels set central respiratory CO2 and O2 sensitivity

Task2 K⁺ channel expression in the central nervous system is surprisingly restricted to a few brainstem nuclei, including the retrotrapezoid (RTN) region. All Task2-positive RTN neurons were lost in mice bearing a Phox2b mutation that causes the human congenital central hypoventilation syndrome. In plethysmography, Task2^−/− mice showed disturbed chemosensory function with hypersensitivity to low CO₂ concentrations, leading to hyperventilation. Task2 probably is needed to stabilize the membrane potential of chemoreceptive cells. In addition, Task2^−/− mice lost the long-term hypoxia-induced respiratory decrease whereas the acute carotid-body-mediated increase was maintained. The lack of anoxia-induced respiratory depression in the isolated brainstem–spinal cord preparation suggested a central origin of the phenotype. Task2 activation by reactive oxygen species generated during hypoxia could silence RTN neurons, thus contributing to respiratory depression. These data identify Task2 as a determinant of central O₂ chemoreception and demonstrate that this phenomenon is due to the activity of a small number of neurons located at the ventral medullary surface.     

First-order antiferromagnetic transitions of SrMn2P2 and CaMn2P2 single crystals containing corrugated-honeycomb Mn sublattices

SrMn₂P₂ and CaMn₂P₂ are insulators that adopt the trigonal CaAl₂Si₂-type structure containing corrugated Mn honeycomb layers. Magnetic susceptibility χ and heat capacity versus temperature T data reveal a weak first-order antiferromagnetic (AFM) transition at the Néel temperature TN=53(1) K for SrMn₂P₂ and a strong first-order AFM transition at TN=69.8(3) K for CaMn₂P₂. Both compounds exhibit isotropic and nearly T-independent χ(T≤TN), suggesting magnetic structures in which nearest-neighbor moments are aligned at ≈120° to each other. The ³¹P NMR measurements confirm the strong first-order transition in CaMn₂P₂ but show critical slowing down above TN for SrMn₂P₂, thus also evidencing second-order character. The ³¹P NMR measurements indicate that the AFM structure of CaMn₂P₂ is commensurate with the lattice whereas that of SrMn₂P₂ is incommensurate. These first-order AFM transitions are unique among the class of (Ca, Sr, Ba)Mn₂ (P, As, Sb, Bi)₂ compounds that otherwise exhibit second-order AFM transitions. This result challenges our understanding of the circumstances under which first-order AFM transitions occur.

The Mn-based 122-type pnictides AMn2Pn2 (A= Ca, Sr, Ba; Pn = P, As, Sb, Bi) have received attention owing to their close stoichiometric 122-type relationship to high-Tc iron pnictides. The undoped Mn pnictides are local-moment antiferromagnetic (AFM) insulators like the high-Tc cuprate parent compounds (1–3). The BaMn2Pn2 compounds crystallize in the body-centered tetragonal ThCr2Si2 structure as in AFe2As2 (A = Ca, Sr, Ba, Eu), whereas the (Ca,Sr)Mn2Pn2 compounds crystallize in the trigonal CaAl2Si2-type structure (4). Recently, density-functional theory (DFT) calculations for the 122 pnictide family have suggested that the trigonal 122 transition-metal pnictides that have the CaAl2Si2 structure might compose a new family of magnetically frustrated materials in which to study the potential superconducting mechanism (5, 6). It had previously been suggested on theoretical grounds that CaMn₂Sb₂ is a fully frustrated classical magnetic system arising from proximity to a tricritical point (7–9).The electrical resistivity ρ and heat capacity Cp versus temperature T of single-crystal CaMn₂P₂ were reported in ref. 10. The compound is an insulator at T = 0 and undergoes a first-order transition of some type at 69.5 K. The Raman spectrum of CaMn₂P₂ at T = 10 K showed new peaks compared to the spectrum at 300 K, whereas the authors’ single-crystal X-ray diffraction measurements showed no difference in the crystal structure at 293 and 40 K. They suggested that the results of the two types of measurements could be reconciled if a superstructure formed below 69.5 K (10). The authors’ magnetic susceptibility χ(T) measurements below 400 K revealed no evidence for a magnetic transition.Here we report the detailed properties of trigonal CaMn₂P₂ and SrMn₂P₂ (11) single crystals. We present the results of single-crystal X-ray diffraction (XRD), electrical resistivity ρ in the ab plane (hexagonal unit cell) versus temperature T, isothermal magnetization versus applied magnetic field M(H), magnetic susceptibility χ(T), heat capacity Cp(H,T), and ³¹P NMR measurements. We find from Cp(T), χ(T), and NMR that CaMn₂P₂ exhibits a strong first-order AFM transition at TN=69.8(3) K whereas SrMn₂P₂ shows a weak first-order transition at TN=53(1) K but with critical slowing down on approaching TN from above as revealed from NMR, a characteristic feature of second-order transitions. Thus, remarkably, the AFM transition in SrMn₂P₂ has characteristics of both first- and second-order transitions. The χ(T) data also reveal the presence of strong isotropic AFM spin fluctuations in the paramagnetic (PM) state above TN up to our maximum measurement temperatures of 900 and 350 K for SrMn₂P₂ and CaMn₂P₂, respectively. This behavior likely arises from spin fluctuations associated with the quasi–two-dimensional nature of the Mn spin layers (12) together with possible contributions from magnetic frustration. Our single-crystal XRD data at room temperature and high-resolution synchrotron XRD data at T = 20 K for SrMn₂P₂ and CaMn₂P₂ demonstrate conclusively that there is no structure change of either compound on cooling below their respective TN.Our studies of SrMn₂P₂ and CaMn₂P₂ thus identify the only known members of the class of materials with general formula AMn2Pn2 containing Mn2+ spins S = 5/2 that exhibit first-order AFM transitions, where A = Ca, Sr, or Ba and the pnictogen Pn= P, As, Sb, or Bi. In particular, only second-order AFM transitions are found in CaMn2As2 (13), SrMn2As2 (13–15), CaMn2Sb2 (8, 9, 16–19), SrMn2Sb2 (16, 19), and CaMn2Bi2 (20).     

PON2 and ATP2B2 gene polymorphisms with noise-induced hearing loss

Background

Noise-induced hearing loss (NIHL) is a complex disease induced by a combination of genetic and environmental factors. Paraoxonase2 (PON2) gene involved in the regulation of reactive oxygen species, and affecting the vulnerability of cochlea to NIHL, and ATPase, calcium-transporting, plasma membrane 2 (ATP2B2) gene which encodes plasma membrane calcium-transporting ATPase isoform 2 (PMCA2) are the candidate genes relating to the attack of NIHL. In this study, we investigated whether ATP2B2 and PON2 polymorphisms were associated with NIHL in Chinese of Han nationality population.

Methods

We performed a case-control study between six single nucleotide polymorphisms (SNPs) (rs1719571, rs3209637 and rs4327369 within ATP2B2, rs12026, rs7785846 and rs12704796 within PON2) and NIHL in 454 subjects. All the SNPs were genotypes, using the TaqMan MGB probe assay. Odds ratios (ORs) were calculated with 95% confidence intervals (95% CIs) with logistic regression analysis to test the level of association for SNPs.

Results

In our study, 221 subjects with hearing loss and 233 subjects without hearing loss were recruited. The frequencies of the CG and CG + GG genotype of rs12026 (PON2) conferred risk factors for NIHL with adjusted OR values of 2.62 (95% CI, 1.69–4.06) and 2.48 (95% CI, 1.63–3.78), respectively. This kind of significance was also found at locus rs7785846, where genotypes CT and CT + TT were the risk types, with adjusted ORs of 2.52 (95% CI, 1.62–3.93) and 2.35 (95% CI, 1.54–3.58), respectively. We performed stratified analysis per noise exposure level, when it came to rs7785846 and rs12026 in the >92 dB(A) noise exposure group, the subjects who carried heterozygote were of significantly (P<0.01) higher susceptibility to NIHL than homozygote carriers. By contrast, no significantly higher risk was found for any rs12704796 genotypes or any genotypes in ATP2B2 (P>0.05), which may suggest that these SNPs did not have significant effects on noise susceptibility across noise exposure.

Conclusions

Our research suggested that PON2 might play a role in the etiology of NIHL in Chinese of Han nationality population.     

Identification of SH2B2beta as an inhibitor for SH2B1- and SH2B2alpha-promoted Janus kinase-2 activation and insulin signaling

The SH2B family has three members (SH2B1, SH2B2, and SH2B3) that contain conserved dimerization (DD), pleckstrin homology, and SH2 domains. The DD domain mediates the formation of homo- and heterodimers between members of the SH2B family. The SH2 domain of SH2B1 (previously named SH2-B) or SH2B2 (previously named APS) binds to phosphorylated tyrosines in a variety of tyrosine kinases, including Janus kinase-2 (JAK2) and the insulin receptor, thereby promoting the activation of JAK2 or the insulin receptor, respectively. JAK2 binds to various members of the cytokine receptor family, including receptors for GH and leptin, to mediate cytokine responses. In mice, SH2B1 regulates energy and glucose homeostasis by enhancing leptin and insulin sensitivity. In this work, we identify SH2B2beta as a new isoform of SH2B2 (designated as SH2B2alpha) derived from the SH2B2 gene by alternative mRNA splicing. SH2B2beta has a DD and pleckstrin homology domain but lacks a SH2 domain. SH2B2beta bound to both SH2B1 and SH2B2alpha, as demonstrated by both the interaction of glutathione S-transferase-SH2B2beta fusion protein with SH2B1 or SH2B2alpha in vitro and coimmunoprecipitation of SH2B2beta with SH2B1 or SH2B2alpha in intact cells. SH2B2beta markedly attenuated the ability of SH2B1 to promote JAK2 activation and subsequent tyrosine phosphorylation of insulin receptor substrate-1 by JAK2. SH2B2beta also significantly inhibited SH2B1- or SH2B2alpha-promoted insulin signaling, including insulin-stimulated tyrosine phosphorylation of insulin receptor substrate-1. These data suggest that SH2B2beta is an endogenous inhibitor of SH2B1 and/or SH2B2alpha, negatively regulating insulin signaling and/or JAK2-mediated cellular responses.