A cDNA for the estradiol-regulated 24K protein: Control of mRNA levels in MCF-7 cells

We have previously demonstrated an estradiol-regulated 24 kDa (24K) protein in human breast cancer tissue culture cells and human tumor biopsies. The presence of 24K correlates well with the presence of steroid hormone receptors. In order to further study the hormonal regulation of the 24K protein and gene, we have isolated cDNA clones corresponding to the 24K mRNA.Poly(A)⁺ RNA isolated from the MCF-7 human breast cancer cell line was translated in a cell-free translation system containing [³⁵S]-methionine. The translation products were immunoprecipitated with a 24K monoclonal antibody, and thein vitro synthesis of 24K protein was confirmed by sodium dodecylsulfate (SDS) polyacrylamide gel electrophoresis. The same poly(A)⁺ RNA was used to construct an oligo(dT)-primed cDNA library in thegt11 expression vector system. The library was screened with a highly specific polyclonal antibody raised against 24K protein purified by immunoaffinity chromatography. Four recombinant clones reacting with the antibody by virtue of antigen expression were isolated and three were used in hybridization-selected translation. Three clones were able to hybridize specifically to a messenger RNA (mRNA) that yielded a Mr 24,000 protein when translatedin vitro and analyzed by SDS/polyacrylamide gel electrophoresis. This protein was also immunoprecipitable by the 24K monoclonal antibody. MCF-7 mRNA size fractionated by formaldehyde-agarose gel electrophoresis was transferred to nitrocellulose paper and hybridized to a nick-translated 24K cDNA clone. A single band of hybridization corresponding to a mRNA size of approximately 0.9–1.0 kilobase (kb) was observed. Using this same technique, 24K cDNA was hybridized to mRNA extracted from MCF-7 cells that had been treated for varying periods with either estradiol, nafoxidine, or tamoxifen. The 24K mRNA was elevated by the addition of estradiol, and clearly diminished by nafoxidine and tamoxifen.These results demonstrate that we have isolated cDNA clones for the study of the hormonal regulation of the 24K gene in breast cancer cells, and have shown that the mRNA is regulated by estradiol.     

长链非编码RNA lnc-CCDC33-1:1在甲状腺乳头状癌中的表达及临床意义

目的：检测lnc-CCDC33-1:1在甲状腺乳头状癌（PTC）中的表达,并分析其临床意义。方法：选取2021年11月至2022年3月杭州市萧山区第一人民医院收治的120例PTC患者,收集120例PTC组织及30例癌旁正常甲状腺组织。采用qRT-PCR检测lnc-CCDC33-1:1在PTC组织及癌旁组织中的表达。分析本地队列和TCGA队列中lnc-CCDC33-1:1表达水平与PTC患者临床病理因素的相关性。采用受试者工作特征（ROC）曲线评价lnc-CCDC33-1:1对PTC的诊断价值。结果：与癌旁正常甲状腺组织比,lnc-CCDC33-1:1在PTC组织中表达明显升高（P ＜0.001）。ROC曲线显示曲线下面积为0.803（95%CI =0.736～0.869,P ＜0.001）。本地队列显示lnc-CCDC33-1:1 表达与PTC肿瘤大小（P =0.048）、腺外侵犯（P =0.019）、T分期（P =0.011）和淋巴结转移（P =0.009）相关,TCGA组数据显示lnc-CCDC33-1:1表达水平与PTC腺外侵犯（P =0.036）和淋巴结转移（P ＜0.001）相关。结论：lnc-CCDC33-1:1在PTC中表达异常升高,与PTC高危特征相关,可能是潜在的诊断标志物和治疗靶点。     

RNA甲基化修饰在肺动脉高压中的研究进展

肺动脉高压（pulmonary hypertension, PH）是以肺血管显著重构及血管负荷进行性增大为特征的一类综合征,疾病进展常伴随右心室重构及肥厚,最终导致右心衰而致死亡。近年来,表观遗传机制特别是RNA甲基化的研究进展,揭示了表观遗传修饰与PH之间的密切关系,并为诊断和治疗PH提供了新的潜在靶点。N6-甲基腺苷(m6A)、N7-甲基鸟苷(m7G)是真核细胞中最普遍和最丰富的内部转录后RNA修饰类型。本文主要对m6A甲基化修饰,m7G甲基化修饰在PH发生发展过程中的作用及其机制进行综述。     

247例丙型肝炎病毒(HCV)RNA FQ-PCR定量结果分析

目的为了了解丙型肝炎病毒载量在两性间的流行状况.方法荧光定量PCR用于HCV RNA定量检测.结果男性患者阳性率为20.27%(30/148),病毒拷贝对数值为4.18±1.04;而女性分别为29.29%(29/99),4.28±1.07.结论男女HCV阳性病人的阳性率(P=0.103,χ2=2.656)和病毒量(P=0.487 ,t=0.699)在男女两性间无统计学差异.     

Are microproerythroblasts in human bone marrow real or artefacts? A cytochemical note

Early erythroid precursors were studied in human bone marrow smears to provide more information on small proerythroblasts--"microproerythroblasts"--using a silver reaction to demonstrate silver stained nucleolar organizer regions (AgNORs) and light microscopic densitometry of large irregularly shaped nucleoli and cytoplasm stained for RNA. No significant differences were found for the density of such nucleoli and basophilic cytoplasm between characteristic large proerythroblasts with a nuclear diameter larger that 9 microm (K2 and K1 erythroblasts) and small proerythroblasts--"microproerythroblasts" representing a subpopulation of K1/2 erythroblasts (early basophilic erythroblasts), which are characterized by a smaller nuclear diameter. In addition, large irregularly shaped nucleoli of "microproerythroblasts" possessed numerous silver stained particles representing AgNORs similar to those of large proerythroblasts. The number of AgNORs in "microproerythroblasts" was slightly, but significantly, smaller than that in large characteristic proerythroblasts.     

Single-Cell RNA Sequencing of Tumor-Infiltrating NK Cells Reveals that Inhibition of Transcription Factor HIF-1α Unleashes NK Cell Activity

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Molecular epidemiology of norovirus variants detected in children under five years of age in Hyderabad,India

PurposeNoroviruses are common viral agents in acute diarrhea in all age groups worldwide. Norovirus has been classified into 10 genogroups, GI to GX with over 48 genotypes among them the GII.4 genotype has evolved over time with a clear pattern of periodic variant replacement. Immunity is strain or genotype specific with little or no protection conferred across genogroups. The present study was aimed to determine the epidemiology, prevalent genotypes of norovirus in children below five years of age in the Hyderabad region, India.MethodsThe stool samples and clinical data were collected from 458 children below 5 years of age comprising of cases with acute gastroenteritis (n ?= ?366) and a control group (n ?= ?92) admitted to the pediatric ward. All the samples were tested for Norovirus by ELISA and RT-PCR. Sequencing was done for predominant strains.Results10.3% (n ?= ?38) of cases and 3.2% (n ?= ?3) of the control group were found to be Norovirus positive. Predominant genotypes were GII-82.5% followed by GI-12.5%.ConclusionSequencing and Phylogenetic analyses of 20 GII.4 strains was done. All of the isolates are clustered away from published the GII.4 variants thus suggesting the appearance of a new variant.     

Sequence analysis of L RNA of Lassa virus

The L RNA of three Lassa virus strains originating from Nigeria, Ghana/Ivory Coast, and Sierra Leone was sequenced and the data subjected to structure predictions and phylogenetic analyses. The L gene products had 2218-2221 residues, diverged by 18% at the amino acid level, and contained several conserved regions. Only one region of 504 residues (positions 1043-1546) could be assigned a function, namely that of an RNA polymerase. Secondary structure predictions suggest that this domain is very similar to RNA-dependent RNA polymerases of known structure encoded by plus-strand RNA viruses, permitting a model to be built. Outside the polymerase region, there is little structural data, except for regions of strong alpha-helical content and probably a coiled-coil domain at the N terminus. No evidence for reassortment or recombination during Lassa virus evolution was found. The secondary structure-assisted alignment of the RNA polymerase region permitted a reliable reconstruction of the phylogeny of all negative-strand RNA viruses, indicating that Arenaviridae are most closely related to Nairoviruses. In conclusion, the data provide a basis for structural and functional characterization of the Lassa virus L protein and reveal new insights into the phylogeny of negative-strand RNA viruses.     

Turnip crinkle virus coat protein mediates suppression of RNA silencing in Nicotiana benthamiana

All of the protein products of Turnip crinkle virus (TCV; Tombusviridae, Carmovirus) were tested for their ability to suppress RNA silencing of a reporter gene after transient expression in Agrobacterium-infiltrated Nicotiana benthamiana leaves. Only the capsid protein, P38, showed suppression activity, although this was not obvious when P38 was expressed as part of a TCV infection of the same tissues. When P38 was expressed from a PVX vector, symptoms with enhanced severity that correlated with increased PVX RNA accumulation were observed. This contradiction between ectopic expression of P38 and TCV infection could be accounted for if the active determinant of suppressor activity within P38 was sequestered within the capsid protein structure. The N-terminal 25 amino acids were shown to be important for this activity. This region forms part of the unexposed R-domain that interacts with the RNA within the virus particle. This observation throws light on some of the complex biology exhibited by TCV.     

人乳腺癌MDR1基因稳定沉默细胞的筛选及其生物学特性

目的筛选MDR1沉默的人乳腺癌细胞并比较其生物学特性。方法采用BLOCK-iT Lentiviral RNAi Expres-sion System生产表达MDR1基因shRNA的慢病毒载体,转导敏感人乳腺癌细胞MCF-7和耐阿霉素人乳腺癌细胞MCF-7/ADM,杀稻瘟菌素(blasticidin)筛选获得MDR1稳定沉默细胞MCF-7/RNAi和MCF-7/ADM/RNAi。定量RT-PCR检测MDR1mRNA表达,流式细胞术检测P-GP蛋白表达和功能,MTT法检测药物敏感性。结果经10 mg/L blasticidin筛选12 d获得MCF-7/RNAi和MCF-7/ADM/RNAi细胞。MCF-7、MCF-7/RNAi、MCF-7/ADM和MCF-7/ADM/RNAi细胞的MDR1mRNA相对表达水平分别为1、0.13、17.14和2.01;P-GP表达分别为(1.5±0.3)%、(1.2±0.2)%、(89.4±3.6)%和(16.3±1.9)%;细胞内Rh123的潴留分别为(92.4±3.1)%、(90.6±4.0)%、(13.6±1.6)%、(72.4±2.8)%;IC50值分别为0.90、0.92、19.61和4.04 mg/L。结论应用慢病毒载体稳定沉默MDR1基因可有效逆转人乳腺癌细胞耐药性。