Hydroxyl radical modification of polyguanylic acid: role of modified guanine in circulating SLE anti-DNA autoantibodies

The hydroxyl radical generated by UV irradiation of hydrogen peroxide cause an extensive damage to guanine residues of ribohomopolymer, polyguanylic acid, poly (G) as investigated by spectrophotometric measurements, agarose gel electrophoresis, Sephadex G-200 gel filtration and DEAE Sephadex A-25 column chromatography. Native and ROS-poly (G) were highly immunogenic inducing high titre antibodies in rabbits. The antibodies showed wide range of cross reactivity with various synthetic polynucleotides exhibiting B-, A-, and allied conformations. The diverse antigen binding characteristics of the induced antibodies resembles to those of naturally occurring lupus anti-DNA autoantibodies. Sera from various SLE patients showed preferential binding to ROS-poly (G) than native poly (G), indicating that oxidatively modified guanine residues are better recognised. The significance of these findings in the induction of SLE anti-DNA autoantibodies by oxygen free radicals modified guanine residues in DNA has been discussed.     

蓝氏贾第鞭毛虫中国株组蛋白H2A基因的克隆与重组表达

目的获取蓝氏贾第鞭毛虫中国株GlH2A基因及重组蛋白。方法 PCR扩增获取GlH2A基因,连接至pMD-19T并进行测序分析。连接至pET28a构建表达载体,并转化宿主菌E.coli BL21（DE3）,然后进行异丙基硫代半乳糖苷酶（IPTG）诱导表达。表达产物进行亲和层析纯化,再用Western blotting进行免疫学鉴定。结果序列测定获得蓝氏贾第鞭毛虫中国株GlH2A基因的编码序列,与美国WB株H2A（GL50803_14256,Accession：NW_001844132）序列完全一致。该基因编码124个氨基酸,预测分子量13900Mr,保留了与核小体形成相关的重要氨基酸位点,在大肠埃希菌中获得表达,纯化后纯度达90%以上。Western blotting检测表明重组蛋白能够被抗His6标签抗体识别。结论克隆得到GlH2A基因编码序列;得到了重组GlH2A蛋白,并进行了纯化,为进一步研究组蛋白及其修饰酶在基因转录调控中的作用奠定了基础。     

The N‐terminal and C‐terminal halves of histone H2A.Z independently function in nucleosome positioning and stability

Nucleosome positioning and stability affect gene regulation in eukaryotic chromatin. Histone H2A.Z is an evolutionally conserved histone variant that forms mobile and unstable nucleosomes in vivo and in vitro. In the present study, we reconstituted nucleosomes containing human H2A.Z.1 mutants, in which the N‐terminal or C‐terminal half of H2A.Z.1 was replaced by the corresponding canonical H2A region. We found that the N‐terminal portion of H2A.Z.1 is involved in flexible nucleosome positioning, whereas the C‐terminal portion leads to weak H2A.Z.1‐H2B association in the nucleosome. These results indicate that the N‐terminal and C‐terminal portions are independently responsible for the H2A.Z.1 nucleosome characteristics.     

Global histone modification of histone H3 in colorectal cancer and its precursor lesions

Chromatin remodeling through histone modification is an important mechanism of epigenetic gene dysregulation in human cancers. However, little is known about global alteration of histone status during tumorigenesis and cancer progression. Histone H3 status was examined in benign and malignant colorectal tumors by immunohistochemistry and Western blotting. For immunohistochemical evaluation, 4 anti-histone H3 antibodies, specific to dimethylation at lysine 4 (H3K4me2), acetylation at lysine 9 (H3K9ac), dimethylation at lysine 9 (H3K9me2), and trimethylation at lysine 27 (H3K27me3), were used. On immunohistochemistry, H3K4me2, H3K9ac, and H3K27me3 showed no significant changes between normal and colorectal tumors. On the other hand, the global level of H3K9me2 was distinctly higher in neoplastic cells (adenoma and adenocarcinoma) than in normal glandular cells. In addition, it was significantly higher in adenocarcinoma than in adenoma. Correspondingly, Western blotting confirmed that H3K9me2 expression was significantly higher in adenocarcinomas than in normal colorectal mucosa. No alteration of H3K9me2 was observed with tumor differentiation and with the histological subtypes of colorectal cancers. These results suggest that aberration of the global H3K9me2 level is an important epigenetic event in colorectal tumorigenesis and carcinogenesis involved with gene regulation in neoplastic cells through chromatin remodeling.     

Water soluble fraction of solar-simulated light-exposed crude oil generates phosphorylation of histone H2AX in human skin cells under UVA exposure

Crude oil contains compounds, which have toxic and cancer-causing properties to humans. The oil spilled in environments is usually exposed to sunlight; however, the toxicity of sunlight-exposed oil is poorly understood. In this study, we found that the water soluble fraction (WSF) of crude oil irradiated with solar-simulated light (SSL) generated phosphorylation of histone H2AX (gamma-H2AX) in human skin cells under UVA irradiation, which was due to the formation of DNA double strand breaks (DSBs). Crude oil was exposed to SSL for approximately 7 days. The WSF obtained from unexposed crude oil showed no toxicity, whereas the WSF obtained from crude oil pre-exposed to SSL induced acute cell death on exposure to UVA irradiation (induction of phototoxicity), which was more remarkable in human skin fibroblasts than human skin keratinocytes. gamma-H2AX was detected in both cell lines immediately after treatment with the WSF plus UVA. Interestingly, gamma-H2AX was detectable even at low SSL- and UVA-doses, which induced no cytotoxicity. The WSF of crude oil irradiated with SSL, generated DSBs under UVA irradiation, which were detected by biased sinusoidal field gel electrophoresis. This was confirmed using xrs-5 cells isolated from CHO-K1 cells, which are deficient in a repair enzyme for DSBs; the WSF plus UVA induced a more dramatic decrease in survival in xrs-5 cells than CHO-K1 cells. These findings demonstrate that exposure of crude oil to sunlight makes the WSF phototoxic, generating DSBs accompanying the appearance of gamma-H2AX in human skin cells.     

Prognostic significance of the therapeutic targets histone deacetylase 1, 2, 6 and acetylated histone H4 in cutaneous T-cell lymphoma

Aims:  Aberrant histone acetylation has been associated with malignancy and histone deacetylase (HDAC) inhibitors are currently being investigated in numerous clinical trials. So far, the malignancy most sensitive to HDAC inhibitors has been cutaneous T-cell lymphoma (CTCL). The reason for this sensitivity is unclear and studies on HDAC expression and histone acetylation in CTCL are lacking. The aim of this study was to address this issue.
Methods and results:  The immunohistochemical expression of HDAC1, HDAC2, HDAC6, and acetylated H4 was examined in 73 CTCLs and the results related to histological subtypes and overall survival. HDAC1 was most abundantly expressed ( P  < 0.0001), followed by HDAC2; HDAC6 and H4 acetylation were equally expressed. HDAC2 ( P  = 0.001) and H4 acetylation ( P  = 0.03) were significantly more common in aggressive than indolent CTCL subtypes. In contrast, no differences were observed for HDAC1 and HDAC6. In a Cox analysis, elevated HDAC6 was the only parameter showing significant influence on survival ( P  = 0.04).
Conclusions:  High expression of HDAC2 and acetylated H4 is more common in aggressive than indolent CTCL. HDAC6 expression is associated with a favorable outcome independent of the subtype.     

猪在禽H9N2亚型流感病毒感染人中的作用

目的　了解猪在禽H9N2亚型流感病毒感染人中的作用。方法　用RT PCR扩增目的基因,用PGEM T Vector(美国Promega公司) 4℃过夜连接,重组质粒转入dH5α细菌,筛选阳性菌落,酶切鉴定,送六合通公司自动测序,然后进行进化树分析。结果　两株山东猪H9N2毒株基因组与人及禽分离出的H9N2病毒均有差异,中国内地从人分离出的H9N2毒株的基因组接近鸡的毒株,而香港特区从人分离出的接近鹌鹑的毒株;禽H9N2毒株不仅宿主范围广,同时其基因组具有多样性。结论　禽H9N2亚型毒株是直接感染人,而不是通过所谓的中间宿主猪,然后再感染人。     

Immunocytochemical assessment of mitotic activity with an antibody to phosphorylated histone H3 in the macaque and human endometrium

BACKGROUND: Determination of the mitotic index in sections of endometrium stained with haematoxylin and eosin (H&E) is difficult and time-consuming. We assessed the value of two mitotic markers as immunocytochemical reagents for measuring mitotic rates in endometrium. METHODS: Mitotic protein monoclonal antibody 2 (MPM-2) and anti-phosphorylated histone H3 (Phospho H3) were applied to paraffin sections of rhesus macaque and human endometrium. RESULTS: In estrogen-treated macaque endometrium the mean +/- SEM mitotic indices were: H&E 1.5 +/- 0.25%, Phospho H3 antibody 1.02 +/- 0.23% and MPM-2 antibody 0.69 +/- 0.17%; these were not statistically significantly different, but the Phospho H3 antibody gave a stronger and cleaner signal than the MPM-2 antibody. Comparisons were made between a computer-determined Phospho H3 index, the H&E-determined mitotic index and the Ki-67 index in samples of human endometrium across the cycle. All revealed that the highest proliferative rate occurred during the follicular phase, but the Phospho H3 and the mitotic indices were more highly correlated (R(2) = 0.89, P < 0.001) than the Ki-67 and mitotic indices (R(2) = 0.74, P < 0.05). CONCLUSIONS: The exceptionally high contrast staining and the excellent correlation between the Phospho H3 and mitotic indices validates the specificity of the Phospho H3 antibody as a new tool for the assessment of endometrial mitotic activity.     

Uric acid and HMGB1 are involved in the induction of autoantibodies elicited in mice infected with mouse hepatitis virus A59

We have shown that mice infected with mouse hepatitis virus A59 develop autoantibodies (autoAb) to liver and kidney fumarylacetoacetate hydrolase (FAH). Because it has been proposed that the immune system is stimulated by alarm signals called damage-associated molecular patterns or alarmins, we investigated the participation of uric acid and high-mobility group box protein 1 (HMGB1) in the autoimmune response elicited by mouse hepatitis virus (MHV). Mice subjected to MHV infection had increased plasmatic uric acid concentration that significantly decreased after 20 days of daily treatment with allopurinol and, simultaneously, autoAb to FAH were undetected. Furthermore, this autoAb disappeared after 30 days of treatment with ethyl pyruvate, along with a substantial reduction in serum HMGB1 concentration. Both results indicated a remarkable relationship between the autoimmune process induced by the virus and uric acid and HMGB1 liberation. Unexpectedly, it was found that allopurinol and ethyl pyruvate inhibited the release of both uric acid and HMGB1. Because HMGB1 is activated through binding to interleukin 1β, and that this cytokine is produced by the NLRP3 inflammasome that could be stimulated by uric acid, we propose that both alarmins could be acting in concert with the induction of the autoAb to FAH in MHV-infected mice.     

组蛋白变异体macroH2A1真核表达载体构建及其细胞内定位的研究

目的构建带有血凝素（HA）标签的小鼠组蛋白变异体macroH2A1（mH2A1）的真核表达载体,并观察其在人胚肾293T细胞中的表达定位情况。方法提取内毒素休克的BALB/c小鼠肝脏组织的总RNA,通过逆转录-聚合酶链反应获得内毒素休克小鼠肝脏组织的cDNA,以cDNA为模板使用PCR方法扩增得到mH2A1编码序列,并将其酶切后连接至带有HA标记的载体pcDNA3-HA上;对阳性克隆进行酶切、PCR和测序鉴定。随后将重组质粒瞬时转染293T细胞,利用荧光显微镜观察。结果 PCR、双酶切和测序鉴定表明pcDNA3-mH2A1-HA真核表达质粒构建正确;经转染实验发现,该质粒能够在293T细胞中表达,表达产物主要定位在细胞核中。结论 mH2A1真核表达载体pcDNA3-mH2A1-HA的成功构建及明确其在哺乳动物细胞中的具体核定位,为进一步研究mH2A1作用细胞的信号通路提供了一个重要的工具。     

从我国人群中再次分离到H9N2亚型流感病毒

目的 了解分离流感病毒毒株表面抗原亚型和特性及其来源。方法 病毒通过ＭＤＣＫ细胞分离,用红细胞凝集抑制（ＨＩ）和神经氨酸酶抑制（ＮＩ）测定对病毒株表面抗原进行鉴定和特性分析,人血清中抗体测定采用ＨＩ和中和实验。对患者进行个」案调查。结果 分离物为甲型流感病毒Ｈ９Ｎ２亚型,属Ｇ９类似毒株,它的ＨＡ抗原特性与已经从人、鸡和鸽分离到的Ｈ９Ｎ２亚型毒株均有差异。患者恢复期血清对分离物的ＨＩ抗体滴度为４００     

T lymphocyte expression of complement receptor 2 (CR2/CD21): a role in adhesive cell-cell interactions and dysregulation in a patient with systemic lupus erythematosus (SLE).

Complement receptor 2 (CR2, CD21), the receptor for both the C3d,g portion of human complement component C3 and the Epstein-Barr virus, has been recently described on peripheral T cells. By using dual stain flow cytometric analysis, we have also observed that a peripheral T lymphocyte subpopulation of normal healthy donors bears CR2 in a range varying from 1.1 to 23.2% (mean 12.6%) of total CD3+ cells. T lymphocytes from nine patients with inactive SLE expressed CR2 in a similar range. Three patients with active SLE were also studied. One of them, having neuropathy and glomerulonephritis, displayed an expansion of the CR2 T cell subpopulation which reached as much as 89% of total CD3+ cells. To examine potential functional roles of T cell CR2, cells from a Jurkat-derived CR2 expressing T cell line were found to bind in vitro to human CR2-, complement-coated K562 cell targets in a CR2- and complement-dependent fashion. Based on these studies, we hypothesize that CR2 might act to increase adherence of T cells to nucleated target cells bearing C3d,g, a function which may be relevant to cytotoxicity or other T cell activities requiring cell-cell interaction.