福建省慢性HBV感染者HBV基因S区、基本核心启动子区和前C区基因变异特征分析

为研究福建省慢性HBV感染者HBV基因多样性及变异规律,了解该人群HBV-DNA的病毒学特征。收集慢性HBV感染者血清标本,通过巢式PCR法扩增其HBV基因序列,比对NCBI数据库中标准基因型序列,分析HBV基因S区,基本核心启动子区(BCP)及前C区的序列变异情况,并对这些变异可能造成的病毒抗原表达,疫苗逃逸,患者病症改变等情况进行探讨。最终成功扩增82例HBV全长基因序列,其中B基因型56例,C基因型26例。基因组特定功能区序列分析发现慢性HBV感染者HBV基因在S区(23.2%)、BCP区(61.0%)和前C区(29.3%)均出现了不同程度的变异。其中主蛋白(HBsAg)主要抗原决定簇a决定簇45.8%位点出现了变异,这些变异位点中包括与肝炎重症化及免疫逃逸密切相关的位点(aa126、aa129、aa145等)。位点G1896A(19.5%),G1764A(11.0%)和A1762T(9.8%)依然是BCP/前C区的主要突变位点。而位点A1752G(25.6%)高突变率的出现在BCP区应引起关注。此外位点G1764A(χ~2=5.742,P=0.030)、A1896G(χ~2=14.392,P=0.000)以及A1762T/G1764A(χ~2=7.289,P=0.012)的突变更容易发生在HBeAg阴性的样品中;而位点A1846T(χ~2=11.882,P=0.003)、A1762T(χ~2=6.561,P=0.038)和A1896G(χ~2=6.958,P=0.030)的突变与HBV-DNA的病毒载量存在一定相关性。总之,福建省慢性HBV感染者在HBV不同基因功能区域均存在不同程度的变异,一些与HBeAg表达情况、HBV-DNA载量、疫苗免疫逃避及肝细胞癌发生具有相关联的变异位点已经出现,BCP区A1752G位点的高频率出现应值得关注,对于这些变异位点的患者应加强监测。     

山东省潍坊市慢性乙型肝炎患者HBVP区基因耐药突变检测及相关因素分析

探讨慢性乙型肝炎(Chronic hepatitis B,CHB)患者多聚酶基因逆转录保守区(P区)位点突变情况。选择212例行核苷(酸)类似物抗病毒治疗的CHB患者,采用PCR产物直接测序法检测HBV P基因区耐药变异位点,同时检测其HBV基因型。结果表明,HBV的P基因区突变位点有173、180、18l、184、204、236和250,主要的耐药位点为204和180,分别占35.8%和23.5%。180位点在不同年龄组之间比较均有显著差异,204位点在30岁以下组与41~50岁组、51~60岁组间比较有显著差异(P0.05,P0.01);180位点联合204位点突变率为66.6%,l81位点联合236位点突变率为23.3%;HBV C基因型患者年龄明显大于B基因型患者(P0.01)。M204V/I多以联合L180M突变的形式存在,突变率与年龄有关,HBV基因型和HBV P区耐药位点的检测对CHB患者的治疗和病情预后具有重要的临床意义。     

2010年舟山某医院拉米夫定无良好应答乙肝患者耐药监测分析

目的观察2010年舟山某医院拉米夫定单药治疗无良好应答的乙肝患者其基因型和P区耐药位点突变的关系。方法随机选取舟山某医院就诊的124例拉米夫定单药治疗无良好应答的本地乙肝患者,男64例,女60例,平均年龄(46.92±15.16)岁,用实时荧光定量PCR检测其基因型和HBV-DNA,并用毛细管电泳测序技术对P区进行突变位点检测。结果 124例患者35例(28.23%)B型和89例(71.77%)C基因型。C型HBV-DNA为(5.83±0.91)log10 copies/mL,高于B型的(5.07±1.13)log10 copies/mL,差异具有统计学意义(t=3.55,P<0.01)。拉米夫定在B基因型中的耐药率为45.71%(16/35),低于C基因型的65.17%(58/89),差异具有统计学意义(2χ=3.951,P<0.05)。拉米夫定耐药株在B基因型中rtM204 I/V/S位点突变率为68.75%(11/16),高于C基因型的37.93%(22/58),差异具有统计学意义(2χ=4.821,P<0.05)。结论对于本地区拉米夫定治疗无良好应答的C基因乙肝患者,应加强拉米夫定的耐药位点筛查,而拉米夫定治疗无良好应答的B基因乙肝患者应加强rtM204 I/V/S位点检测。     

RtH238：一个与耐药无关的基因型依赖位点

目的：探讨乙型肝炎病毒（hepatitis B virus,HBV）序列rt238 位点的氨基酸多态性与抗病毒治疗、耐药突变及基因型等的关 系。方法：采用P 区测序法对76 例阿德福韦（adefovir,ADV）基因耐药患者、52 例拉米夫定（Lamivudine,LAM）基因耐药患者和50 例未经抗病毒治疗的慢乙肝患者的血清病毒进行P区测序。结果：在76 例ADV基因耐药患者中,rt238 的三种不同类型氨基酸 在HBV基因型中的分布差异显著（x2=40.196,P=0.000）。RtH238 主要出现在基因型B 患者中,rtN238 主要出现在基因型C 患者 中,而rtT238的HBV 序列全部为基因型C。控制HBV基因型,rt238 氨基酸类型与rt181、rt236位点突变无明显相关。rtT238 只出 现在ADV和LAM基因耐药组。Rt238 位点的氨基酸在不同治疗组中的分布无统计学意义(P=0.127)。结论:Rt238位点的氨基酸多 态性与HBV 基因型显著相关,是一个基因型依赖位点,rtT238可能是基因型C 相关的潜在耐药突变。     

C 蛋白基因多态性对脓毒血症患者血小板功能的影响

目的:探讨蛋白C基因突变及基因型频率分布和脓毒血症患者血小板功能及血清TXB2水平的相关性。方法:纳入112例脓毒血症患者,健康人群50例为对照组。采用PCR-RFLP法测定所有受试者蛋白C基因rs 17999808C/A位点和rs1799809位点基因型及突变率。入院24小时内测定脓毒血症患者血小板计数、最大聚集率及血清TXB2水平,并对其进行SOFA评分。结果:病例组和对照组rs17999808位点和rs1799809位点间基因型分布频率无统计学差异(P0.05)。rs 17999808基因型分布C/C占81.48%、C/A占12.96%、A/A占5.55%。rs1799809位点G/G占76.54%、G/C占15.43%、C/C占8.02%。rs17999808位点和rs1799809位点突变率分贝为12.03%、15.74%。病例死亡34例,死亡率30.35%。rs17999808位点突变纯合子患者(A/A)死亡率及SOFA评分明显增高,和野生纯合子及突变杂合子患者差异有统计学意义(P0.05)。rs17999808位点C/C野生纯合子患者血小板计数和TXB2浓度明显高于C/A和A/A患者,血小板聚集率低于后两者,差异有统计学意义(P0.05)。突变纯合子A/A患者较C/C、C/A患者两两相比,差异有统计学意义(P0.05)。rs17999809位点突变和TXB2浓度有相关性(P0.05)。结论:蛋白C基因rs17999808位点突变增加了脓毒血症患者死亡风险,这可能和其改变血小板功能有关。     

HPV16 变异体在新疆地区宫颈癌及癌前病变中的分布研究

目的:研究新疆地区HPV16 E6、E7、LCR基因突变情况,分析HPV16变异体在宫颈癌及癌前病变发生发展中的作用。方法:选择HPV16阳性的宫颈癌及癌前病变患者,提取基因组DNA,利用PCR扩增HPV16 DNA E6、E7基因及LCR区核苷酸片段,正反向测序。与HPV16基因序列分析比对,分析核苷酸突变位点。结果:E6基因突变率为80.00%(92/115)主要突变位点T350G(59.78%)、T178G(18.47%);E7突变率为54.78%(63/115),主要突变位点A647G(33.33%)、T846C(26.98%);LCR突变率为23.48%(27/115),主要突变位点为C24T(74.07%)、C13T(25.92%)。维吾尔族T350G突变率较汉族妇女显著升高,而汉族A647G、T846C、C24T突变率显著高于维吾尔族,差异具有统计学意义(P0.05)。维吾尔族宫颈癌组T350G突变率显著高于炎症组(P0.05),且随病变严重程度增加突变率上升,汉族T350G、A647G、T846C、C24T突变率炎症组、宫颈病变组显著高于宫颈癌组(P0.05),维吾尔族C24T突变率炎症组显著高于宫颈癌组(P0.05),差异均具有统计学意义(P0.05)。结论:HPV16E6、E7突变可能与宫颈病变进展有关,T350G突变可能是维吾尔族宫颈癌高发的原因之一。     

乙型肝炎病毒X蛋白的优势氨基酸序列与热点突变位点的生物信息学分析

乙型肝炎病毒X蛋白(hepatitis B virus X protein,HBx)全长154个氨基酸,与肝癌发生密切相关.为确定HBx的优势氨基酸序列和热点突变位点,在GenBank中下载所有HBx的氨基酸序列13950条,剔除插入突变、缺失突变和起始密码子非甲硫氨酸的序列,最后保留7126条.通过分析这7126条序列,计算出HBx每个位点的氨基酸分布情况,出现频率最高的氨基酸为该位点的优势氨基酸,其他氨基酸为突变氨基酸.154个位点的优势氨基酸组成HBx优势氨基酸序列.突变率>10.0%的热点突变位点有32个.其中第36、42、44、87、88和127位氨基酸有4种(突变率>1.0%)以上突变形式,具有较高的多态性.与肝癌密切相关的K130M/V131I双突变率为34.7%.通过7126条HBx序列与优势序列的同源性比较,随机选出其中50条序列(2条与优势序列同源性<75%,48条同源性为76%~99%),与23条参考序列及优势序列共同构建系统发生树.结果显示,HBx优势氨基酸序列属于基因型C,这与基因型C为全球主要流行型一致.本研究首次系统性分析了GenBank中HBx的优势序列,确定了32个HBx热点突变位点和6个多态性较高的位点,为基于HBx突变的基础和应用研究奠定了基础.     

乙型肝炎病毒Ⅹ蛋白的优势氨基酸序列与热点突变位点的生物信息学分析

乙型肝炎病毒X蛋白(hepatitis B virus X protein,HBx)全长154个氨基酸,与肝癌发生密切相关。为确定HBx的优势氨基酸序列和热点突变位点,在GenBank中下载所有HBx的氨基酸序列13 950条,剔除插入突变、缺失突变和起始密码子非甲硫氨酸的序列,最后保留7 126条。通过分析这7 126条序列,计算出HBx每个位点的氨基酸分布情况,出现频率最高的氨基酸为该位点的优势氨基酸,其他氨基酸为突变氨基酸。154个位点的优势氨基酸组成HBx优势氨基酸序列。突变率10.0%的热点突变位点有32个。其中第36、42、44、87、88和127位氨基酸有4种(突变率1.0%)以上突变形式,具有较高的多态性。与肝癌密切相关的K130M/V131I双突变率为34.7%。通过7 126条HBx序列与优势序列的同源性比较,随机选出其中50条序列(2条与优势序列同源性75%,48条同源性为76%~99%),与23条参考序列及优势序列共同构建系统发生树。结果显示,HBx优势氨基酸序列属于基因型C,这与基因型C为全球主要流行型一致。本研究首次系统性分析了GenBank中HBx的优势序列,确定了32个HBx热点突变位点和6个多态性较高的位点,为基于HBx突变的基础和应用研究奠定了基础。     

内蒙古敖汉旗地区乙肝患者HBV基因型分布研究

目的:了解内蒙古赤峰市敖汉旗地区乙型肝炎病毒(HBV)基因型、血清型的分布特征。方法:收集2014年在赤峰市敖汉旗医院住院治疗的慢性乙型肝炎患者,采集血清标本,巢式PCR方法扩增HBV S区,测序后应用Mega6.0构建系统发育树确定该地区乙型肝炎病毒主要基因型、血清型,并分析HBV S基因a决定簇变异情况。结果:在72例HBV患者中,B基因12例(16.7%),C基因型40例(55.65%),D基因型20例(27.8%)。不同性别患者的基因型分布差异有统计学意义(P0.01)。血清亚型分析结果显示:adw2血清亚型15例(20.8%)、adrq+血清亚型37例(51.4%)、ayw2血清亚型20例(27.8%)。HBV S基因"a"抗原决定簇突变率为34.7%。结论:内蒙古赤峰市敖汉旗地区HBV基因型以C型为主,其次是D型和B型,血清型主要为adrq+,有34.7%患者携带HBV S基因a决定簇变异株。     

Reductions in I kappa B epsilon and changes in NF-kappa B activity during B lymphocyte differentiation

The levels and stability of IkappaBepsilon have been examined in unstimulated and stimulated splenic B cells and compared with that of IkappaBalpha and IkappaBbeta. Primary murine splenic B cells but not T cells were found to contain high levels of IkappaBepsilon protein, equivalent to levels of the abundant IkappaBalpha. Most agents that activate IkappaBalpha and IkappaBbeta degradation do not induce rapid degradation of IkappaBepsilon. Interestingly, however, the levels of IkappaBepsilon, but not of IkappaBalpha or IkappaBbeta, are dramatically reduced upon the stimulation of B cells both in vivo and in vitro. Since IkappaBepsilon exhibits substrate specificity for NF-kappaB Rel homodimers, this suggested the possibility that changes in NF-kappaB-responsive genes might also occur during this transition. Consistent with this hypothesis, we found that a NF-kappaB reporter construct sensitive to p65/RelA homodimers is activated at the time that IkappaBepsilon levels decline following B cell stimulation. In IgG(+) B cell lines, which contain low levels of IkappaBepsilon, this same reporter construct was inactive, suggesting that the increases in Rel homodimer activity that accompany B cell stimulation are transient. However, there are differences in the level of expression of NF-kappaB-responsive genes in these IgG(+) B cell lines compared with their IgM(+) counterparts. From these data, we conclude that there are transient changes in NF-kappaB activity due to reductions in IkappaBepsilon, which might contribute to long-term, persistent changes that accompany B cell differentiation. We propose an important role for IkappaBepsilon in the differential regulation of nuclear NF-kappaB activity in stimulated B cells.     

B lymphocyte development in rabbit: progenitor B cells and waning of B lymphopoiesis

In mammals that use gut-associated lymphoid tissues for expansion and somatic diversification of the B cell repertoire, B lymphopoiesis occurs early in ontogeny and does not appear to continue throughout life. In these species, including sheep, rabbit, and cattle, little is known about the pathway of B cell development and the time at which B lymphopoiesis wanes. We examined rabbit bone marrow by immunofluorescence with anti-CD79a and anti-mu and identified both proB and preB cells. The proB cells represent the vast majority of B-lineage cells in the bone marrow at birth and by incorporation of 5-bromo-2'-deoxyuridine, they appear to be a dynamic population. PreB cells reach maximum levels in the bone marrow at 3 wk of age, and B cells begin to accumulate at 7 wk of age. We cloned two VpreB and one lambda5 gene and demonstrated that they are expressed within B-lineage cells in bone marrow. VpreB and lambda5 coimmunoprecipitated with the mu-chain in lysates of 293T cells transfected with VpreB, lambda5, and mu, indicating that VpreB, lambda5, and mu-chains associate in a preB cell receptor-like complex. By 16 wk of age, essentially no proB or preB cells are found in bone marrow and by PCR amplification, B cell recombination excision circles were reduced 200-fold. By 18 mo of age, B cell recombination excision circles were reduced 500- to 1000-fold. We suggest that B cell development in the rabbit occurs primarily through the classical, or ordered, pathway and show that B lymphopoiesis is reduced over 99% by 16 wk of age.     

Enhanced IgA class switching in marginal zone and B1 B cells relative to follicular/B2 B cells

Mouse splenic marginal zone (MZ) B cells and B1 B cells enriched in the peritoneal cavity respond preferentially to T cell-independent Ags compared with follicular (FO)/B2 B cells. Despite the differential responses of B cell subsets to various stimuli, and despite the need for multiple stimuli to induce IgA class switching, the relative contribution of B cell subpopulations to IgA production is unknown. By culturing purified B cell populations, we find that MZ and peritoneal B1 cells switch more readily to IgA than do splenic FO or peritoneal B2 cells in BLyS/LPS/TGF-beta. Addition of IL-4, IL-5, and anti-IgD dextran to the cultures enhances IgA switching in FO/B2 and MZ B cells to a similar frequency, but this treatment suppresses IgA class switching in B1 cells. Thus, IgA switching differs among purified B cell subsets, suggesting that individual B cell populations could contribute differentially to IgA expression in vivo, depending on available stimuli.     

Prevalence of hepatitis B virus in chronic hepatitis B patients

The aim of the current study was to detect HBV by Real time - PCR in chronic hepatitis B patients. Fifty-eight sera of chronic hepatitis B patients were subjected during the period March 2009 to April 2010 in Ilam cities in West of Iran. Sera assayed by real-time PCR and ELISA methods. Twenty serum samples from healthy volunteers and non-hepatitis B patients and negative for hepatitis B seromarkers served as negative controls for the study. Among fifty-eight sera, ELISA showed fifty-five (94.8%) of the samples were positive for HBsAg and three (5.2%) negative results obtained while real-time PCR specified fifty-eight (100%) positive results in chronic hepatitis B patients. HBsAg status did not necessarily reflect HBV DNA level in the serum, as 5.2% of chronic Hepatitis B patients were positive for HBV DNA but negative for HBsAg. HBV DNA was not found to be positive amongst any of the negative controls. Real time - PCR is a sensitive and reproducible assay for HBV DNA quantization.     

Multiplication of hepatitis B virus in fulminant hepatitis B.

The presence in serum of hepatitis B e antigen (HBeAg) and hepatitis B virus DNA, which are each regarded as reflecting multiplication of hepatitis B virus, were looked for one to five days after the onset of hepatic encephalopathy in 64 patients with fulminant hepatitis B. HBeAg and hepatitis B virus DNA were found in the serum of only 24 (37%) and six (9%) patients, respectively. Hepatitis B virus DNA was absent from the serum in all 13 patients positive for anti-HBs. These findings indicate that replication of hepatitis B virus stopped after the onset of hepatic encephalopathy in most of the patients and support the view that an enhanced immune response stops the replication. Agents that inhibit viral multiplication would probably not have any effect at this stage of the disease.     

Cathepsin B in osteoblasts

Active cathepsin B has been found in cell extract and medium of human osteoblast-like cells and MG-63 cells. The released form is stable at neutral and alkaline pH and, in both cell types, intracellular and extracellular cathepsin B activities are increased by interleukin-1 beta (IL-1beta) and parathyroid hormone (PTH). To evaluate the physiological role of cathepsin B in osteoblasts, we investigated the production and secretion of this enzyme in normal human synovial fibroblasts and modulation by IL-1beta and PTH. Lactate secretion concurrent with release of cathepsin B and comparable responses in osteoblasts were also examined. Our data show that synovial fibroblasts respond differently to treatment with the two agents, suggesting a cell-specific regulation of cathepsin B and possible involvement in osteoblast physiology. Cathepsin B involvement was then evaluated in the activation of plasminogen activator (PA) in MG-63 cells using two specific inhibitors of cathepsin B, CA074 and CA074-Me, in constitutive conditions and after treatment with IL-1beta. As results of PA activity obtained in the presence of IL-beta were in contrast with previous reports, we examined the activities of PA, pro-PA activated with trypsin, and plasmin in cell extract and media of MG-63 cells after 24-h treatment with IL-1beta. Results show that in normal conditions and in the presence of IL-1beta, cathepsin B is involved in the activation of PA. Moreover, IL-1beta stimulates PA, pro-PA activated by trypsin, and plasmin activity in medium, whereas in cell extract it stimulates pro-PA activated by trypsin and plasmin activity. IL-1beta has no effect on cell extract-associated PA.     

B chromosomes in plants

Abstract

B chromosomes (Bs) can be described as “selfish chromosomes”, a term that has been used for the repetitive DNA which comprises the bulk of the genome in large genome species, except that Bs have a life of their own as independent chromosomes. They can accumulate in number by various processes of mitotic or meiotic drive, especially in the gametophyte phase of the life cycle of flowering plants. This parasitic property of drive ensures their survival and spread in natural populations, even against a gradient of harmful effects on the host plant phenotype. B chromosomes are inhabitants of the nucleus and they are subject to control by “genes” in the A chromosome (As) complement. This interaction with the As, together with the balance between drive and harmful effects makes a dynamic system in the life of a Bs. In this review, we concentrate mainly on recent developments in the Bs of rye and maize, two of the species currently receiving most attention. We focus on their population dynamics and on the molecular basis of their structural organisation and mechanisms of drive, as well as on their mode of origin and potential applications in plant biotechnology.