Immunohistochemical analysis of 14-3-3 sigma and related proteins in hyperplastic and neoplastic breast lesions, with particular reference to early carcinogenesis

In order to confirm the role of 14-3-3 sigma (sigma) as a tumor suppressor in breast carcinogenesis, we have studied the expression of 14-3-3sigma immunohistochemically in usual ductal hyperplasia (UDH), ductal carcinoma in situ (DCIS) and invasive ductal carcinoma (IDC) breast lesions. Immunostaining for estrogen receptor alpha (ERalpha), p53 and estrogen-responsive RING finger protein (Efp) was also carried out. Immunohistochemically, expression of 14-3-3sigma was seen in 92% UDH lesions and gradually decreased from 65% in DCIS to 23% in IDC. The expression of ERalpha decreased gradually from UDH to DCIS to IDC, while p53 showed an inverse staining pattern to that of ERalpha. The expression of Efp showed no significant difference among the three breast lesions. Hence, the present immunohistochemical study confirmed 14-3-3sigma as a tumor suppressor in breast carcinogenesis. A similar immunohistochemical analysis was then carried out on columnar cell hyperplasia with atypia (CCHA), in which the expression pattern of tumor suppressor 14-3-3sigma, ERalpha and p53 suggested that it might be possible that CCHA is a precancerous lesion.     

氨基葡萄糖胶囊干预膝骨关节炎患者外周血单个核细胞中软骨代谢相关基因的表达

背景:目前关于氨基葡萄糖的研究多集中于对膝骨关节炎的治疗作用,但关于其对膝骨关节炎患者外周血中软骨代谢相关基因影响的研究有限。目的:观察氨基葡萄糖胶囊对膝骨关节炎的治疗效果及对软骨代谢相关基因表达的影响。方法:选取2017年3月至2019年2月郑州大学附属郑州中心医院收治的90例膝骨关节炎患者,另选取同期医院体检中心收入的40例健康受试者,检测并对比健康受试者与膝骨关节炎患者治疗前外周血单个核细胞中软骨寡聚基质蛋白、Ⅱ型胶原纤维α1、聚糖蛋白、特异性组织抑制物3基因表达水平。采用随机数表法将膝骨关节炎患者分为常规组和研究组,分别给予双氯芬酸钠缓释片、双氯芬酸钠缓释片+氨基葡萄糖胶囊治疗12周,对比治疗前后2组外周血单个核细胞中各基因表达水平、Lequesne指数,统计治疗期间2组不良反应发生情况。结果与结论:①与健康受试者比较,膝骨关节炎患者外周血单个核细胞中软骨寡聚基质蛋白、特异性组织抑制物3 mRNA相对表达量升高(P<0.05),Ⅱ型胶原纤维α1、聚糖蛋白mRNA相对表达量降低(P<0.05);②治疗前研究组和常规组外周血单个核细胞中各基因表达水平比较,差异均无显著性意义(P>0.05);治疗后2组外周血单个核细胞中软骨寡聚基质蛋白、特异性组织抑制物3 mRNA相对表达量均低于治疗前,且研究组低于常规组(P<0.05);治疗后2组外周血单个核细胞中Ⅱ型胶原纤维α1、聚糖蛋白mRNA相对表达量均高于治疗前,且研究组高于常规组(P<0.05);③治疗前研究组和常规组Lequesne指数比较,差异均无显著性意义(P>0.05);治疗后2组Lequesne指数均低于治疗前,且研究组低于常规组(P<0.05);④研究组和常规组不良反应发生率比较,差异均无显著性意义(P>0.05);⑤结果表明,氨基葡萄糖胶囊可有效改善膝骨关节炎患者临床症状且安全可靠,可能通过抑制软骨寡聚基质蛋白、特异性组织抑制物3基因表达,促进Ⅱ型胶原纤维α1、聚糖蛋白基因表达实现的。     

一株新的鼠抗人2IgB7-H3功能性单克隆抗体的研制及其生物学特性的鉴定

研制功能性鼠抗人2IgB7-H3单克隆抗体。以人2IgB7-H3基因转染细胞L929/2IgB7-H3为免疫原,常规免疫6~8周龄的雌性BALB/c小鼠;利用B淋巴杂交瘤技术,将免疫小鼠脾脏细胞和骨髓瘤细胞株SP2/0融合,L929/2IgB7-H3细胞为抗原筛选阳性细胞;经间接免疫荧光标记法对杂交瘤细胞进行反复筛选和多次克隆化培养;采用快速定性试纸法鉴定单抗Ig亚类;利用竞争抑制性实验分析该单抗识别的抗原表位;采用MTT法分析该单抗在体外阻断DC对T细胞的促增殖效应。结果:获得了1株持续稳定分泌鼠抗人2IgB7-H3单克隆抗体的杂交瘤细胞株(命名为7D7),该单抗可特异识别L929/2IgB7-H3分子和介导有效的共刺激信号。该单抗的成功获得和其生物学特性的初步鉴定,为进一步研究B7-H3两个异构体在DC细胞及肿瘤细胞上的作用机制奠定了物质基础。     

Stochastic and Coherence Resonance in an In Silico Neural Model

We show that it is possible for chaotic systems to display the main features of stochastic and coherence resonance. In particular, a model of coupled nonlinear oscillators which emulates the transmembrane voltage activities in CA3 neurons, operating in a chaotic regime and in the presence of noise, can exhibit coherence resonance and stochastic resonance. Certain firing frequencies become more "rhythmic" for some optimal values of noise intensity. The effect of noise in different coupling pathways is investigated. We found that the effect of coherence resonance and stochastic resonance are more prominent if noise is presented in either electric field or gap junction coupling pathways. Frequency sensitivity of the model is investigated as a preliminary step in illustrating the principles of possible epileptic seizure control strategies using "chaos control" concepts. Significant effects of stochastic resonance are observed in the 4-8 Hz range. Weaker effects can be found in the 1-4 Hz and 8-10 Hz ranges whereas 0.5 Hz does not exhibit any resonance phenomenon. Our results suggest that: (a) Stochastic resonance could enhance the intrinsic 4-8 Hz rhythms in CA3 neurons more prominently via field coupling pathways. It could also help explain why some reported seizure control strategies using pulse-trains would only be effective at 0.5 Hz. (b) Stochastic resonance-like behavior can occur in the gamma range only if noise is presented via chemical synaptic pathways.     

Identical twins discordant for multiple sclerosis have a shift in their T-cell receptor repertoires

CD4 T‐cells have an important role in the autoimmune response in multiple sclerosis (MS). We investigate the possibility that a shift occurs in the T‐cell receptor (TR) repertoire of identical twins discordant for MS. We compare the CDR3 spectratype distributions of 24 different TR V beta (TRBV) segments in naïve CD4 T‐cells from discordant MS twins and from healthy identical twins. We also compare the CDR3 spectratype distributions in unrelated healthy pairs, formed by combining members of different healthy twins, with the CDR3 spectratype distributions in unrelated pairs of MS patients and in unrelated pairs of their apparently healthy cotwins, formed by combining members of different discordant twins. We use the correlation coefficient (r‐value) as a measure of similarity of CDR3 spectratypes in each pair, and we test for the significance of the difference between r‐values from the different pairs. We observe that the r‐value for the CDR3 spectratype distributions among discordant twins differs significantly from the corresponding r‐value for the healthy twins for two TRBV segments. Further, the r‐values, for both the unrelated MS patient pairs and the unrelated pairs of their apparently healthy cotwins, differ significantly from the r‐values for healthy unrelated pairs of individuals. We conclude that both the MS patients and their apparently healthy cotwins have shifts in their CDR3 repertoires. Because we study naïve CD4 T‐cells, we postulate that CDR3 repertoire shifts precede MS and predispose to MS, but are unlikely to be sufficient to cause MS.     

High-throughput single strand conformation polymorphism mutation detection by automated capillary array electrophoresis: validation of the method

Capillary array electrophoresis (CAE) is a novel technique, which allows for high throughput analysis of DNA fragments. When screening for mutations in whole populations or large patient groups it is necessary to have robust and well-characterized setups for high throughput analysis. For large-scale mutation screening, we have developed procedures for single strand conformation polymorphism (SSCP) assays using CAE (CAE-SSCP) whereby we may increase both the sensitivity and the throughput compared to conventional SSCP analysis. In this study we have validated CAE-SSCP by 1) comparing detection by slab-gel based SSCP with CAE-SSCP of mutations in the MYH7, MYL2, and MYL3 genes encoding sarcomere proteins from patients suffering from hypertrophic cardiomyopathy; and 2) by constructing a series of 185 mutants having substitution mutations, as well as insertion/deletion mutations, or some combinations of these, in different sequence contexts in four exons and different positions relative to the end of the amplicon (three from the KCNQ1 gene, encoding a cardiac potassium channel, and one from the TNNI3 gene encoding cardiac troponin I). The method identified 181 out of 185 mutations (98%), and the data suggest that the position of mutation in the fragment had no effect on the sensitivity. Analysis of the specificity of the method showed that only very few mutants could not be distinguished from each other and there were no false positives.     

抗CD3单克隆抗体重、轻链可变区基因的克隆及序列分析

目的：抗CD3单克隆抗体（WuT3)可变区基因克隆及序列分析。方法；采用RT-PCR技术，从WuT3杂交瘤细胞总RNA中扩增VH，VL片段，经酶切后通过链接反应构建重组克隆载体，测序鉴定。结果：通过国际联机检索发现VH，VL基因与Ig同源，分别符合小鼠IgVH,Vк基因特征。VH基因属于鼠重链VH第ⅡB亚类，全长363bp，可编码121个氨基酸；VL基因属于鼠к轻链第Ⅲ亚类，全长330bp，可编码110个氨基酸，结论：成功获得WuT3单抗的重，轻链可变区基因。     

Intermediate-term toxicity of repeated orally administered doses of the anti-malarial β-artemether in dogs

The artemisinin derivative beta-artemether, an anti-malarial, was evaluated for its toxicity and tolerability in a 2-week, multiple-dose study in dogs. Eight beagle dogs (4 females, 4 males) were given beta-artemether by oral gavage 3 times daily at 45 mg/kg/dosing (a total daily dose-level of 135 mg/kg) for 2 weeks. This beta-artemether dose regime was well tolerated. Body weight changes were normal although feed consumption during the treatment period reduced compared to that of the pre-trial period. Clinical signs were transient spells of soft to liquid feces. On completion of the treatment period, the animals were sacrificed and submitted to a full macroscopic post-mortem examination. Designated organs were weighed and a complete light microscopic examination was performed on 43 selected tissues from 1 animal per sex, and on the liver, kidneys, thymus, mandibular lymph nodes and lungs of the three other animals per sex. Major findings were high liver weight and histopathologic findings of slight diffuse hepatocellular hypertrophy and distal tubular dilatation, together with flattened epithelium, in the kidneys. With the dose regime used in this trial beta-artemether produced no clinical or apparent histopathological signs of neurotoxicity in dogs.