Human chromosome 21 dosage: effect on the expression of the interferon induced antiviral state

Human primary skin fibroblasts trisomic for chromosome 13, 18, or 21 and diploid human skin fibroblasts were induced for an antiviral response with human interferon. The cells that were trisomnic for chromosome 21 were three to seven times more sensitive to protection by human interferon than the normal diploid or trisomic 18 or 13 fibroblasts. The differential response in trisomnic 21 cells is consistent with the known assignment of the human antiviral gene to chromosome 21.     

Interferon binding: the first step in establishment of antiviral activity

Chick cells incubated at 1 degrees C with interferon fail to develop antiviral activity, but this activity appears subsequent to a 7-hour incubation at 37 degrees C after removal of interferon by repeated washings. Treatment with actinomycin D blocks the development of the latter activity. Cells incubated with interferon at 1 degrees C for up to 1 hour and then washed and incubated for 2 hours at 37 degrees C develop a degree of antiviral activity proportional to the concentration of interferon at initial incubation; at any concentration, the antiviral activity increased with the duration of initial incubation at 1 degrees C, but a maximal response was reached at 10 or 20 minutes. Treatment with trypsin after incubation with interferon at 1 degrees C inhibited development of antiviral activity. Interferon is rapidly bound to a superficial cell site, and this binding is necessary for development of antiviral activity in chick cells.     

An early event in the interferon-induced transmembrane signaling process

Human interferon stimulates a transient two- to threefold increase in the concentration of diacylglycerol and inositol tris-phosphate within 15 to 30 seconds of cell exposure to interferon. Antibodies to interferon inhibit this effect. The stimulation was measurable in isolated cell membranes exposed to interferon. Human alpha and beta, but not gamma, interferon stimulate this increase in cells containing the appropriate interferon receptor. The effect was proportional to the number of interferon receptors. Both the diacylglycerol increase and antiviral effects induced by interferon could be correlated in terms of dose dependence. Thus, a transient diacylglycerol increase is an early event in the interferon-induced transmembrane signaling process.     

Interferon-resistant cell line lacks fatty acid cyclooxygenase activity

A clone of L1210 mouse leukemia cells selected for resistance to both the antiviral and anticellular properties of mouse interferon were essentially devoid of fatty acid cyclooxygenase activity. Experiments in which broken cell preparations were mixed or the two cell types were cultivated together failed to indicate the presence of a diffusible enzyme inhibitor. Fatty acid lipoxygenase activity of similar magnitude was detectable in both cell types. A selective impairment of fatty acid cyclooxygenase in interferon-resistant cells is consistent with recently described data suggesting that this enzyme may play a crucial role in mediating the antiviral and anticellular effects of interferon.     

鸡重组α干扰素与鸡重组γ干扰素抗新城疫病毒活性研究

将鸡IFN-α和IFN-γ原核表达产物经Ni2＋-NTA亲和层析法纯化,进行SDS-PAGE分析,再将纯化并已测定浓度的鸡重组IFN-α和IFN-γ稀释到1/10倍;用细胞病变抑制法检测重组表达蛋白的抗新城疫病毒活性,并利用感染病毒的鸡胚来测定鸡重组表达蛋白的抗病毒活性。结果显示,SDS-PAGE可检测到相对分子质量为25kU左右的蛋白条带,纯化的鸡IFN-α和IFN-γ抗新城疫病毒复制的活性分别为4.32×103 U/mg和2.65×103 U/mg;当鸡IFN-α和IFN-γ的比例为125∶1时,其联合抗新城疫病毒复制的活性最佳。抗鸡胚感染病毒活性的测定结果表明,与病毒对照组相比,IFN-α和IFN-γ及IFN-α和γ联合组,HA滴度都有明显的降低;并且从鸡胚的平均死亡时间上,联合干扰素对新城疫病毒的抑制作用更强于单个干扰素的应用。这证明了鸡IFN-α、IFN-γ及其联合干扰素对新城疫病毒的复制有明显的抑制作用。     

Systemic lupus erythematosus: presence in human serum of an unusual acid-labile leukocyte interferon

A previously undescribed species of human leukocyte, or alpha, interferon is present in the serum of many patients with systemic lupus erythematosus. It was shown to be alpha-interferon by neutralization with specific antiserums, affinity column chromatography, and antiviral activity on bovine cells. However, 23 of 30 interferon samples tested were inactivated by incubation at pH 2, a characteristic of human "immune," or gamma, interferon. Multiple samples of interferon from the same patient had similar biological properties, but samples from different patients were not all identical, suggesting that several variants of this species of human alpha-interferon may exist.     

Tunicamycin enhances the antiviral and anticellular activity of interferon

The inhibitory effects of interferon on virus multiplication and cell growth are significantly enhanced by treatment with tunicamycin. Potentiation of antiviral activity was found only with enveloped viruses and not with nonbudding viruses. Changes in the plasma membrane of treated cells may account for this effect, since enveloped viruses bud from the cell surface as a terminal step.     

重组猪α干扰素理化性状及其体外抗病毒活性研究(摘要)(英文)

[目的]研究重组猪α干扰素(rPoIFN-α)半成品理化性状并对其体外抗病毒活性进行测试与鉴定。[方法]用HEp-2/VSV体系对3批rPoIFN-α蛋白进行抗病毒活性检测,以重组人IFN-α为参比品,测定干扰素效价;用0.25%胰蛋白酶HCl以及鼠抗猪α干扰素单克隆抗体作用已知效价的rPOIFN-α半成品,并检测各批次抗病毒活性,对rPoIFN-α理化性状进行评价;在猪肾细胞株(PK-15)上检测rPoIFN-α对猪细小病毒(PPV)和猪伪狂犬病毒(PRV)的致细胞病变抑制效应,观察rPoIFN-α的体外抗病毒活性。[结果]HEp-2/VSV体系滴定rPoIFN-α半成品效价可达1.5×105IU/ml,比活性达1.1×106IU/mg;rPoIFN-α经0.25%胰蛋白酶37℃作用1h,效价残留率低于1%,经HCl(pH=2.0)处理72h效价残留率高达95%以上,经56℃处理30min效价残留率高于47%,经鼠抗猪α干扰素单克隆抗体37℃作用1h后效价残留率约为1%;体外抗病毒试验表明,用50和500IU/mlr PoIFN-α可分别抑制PRV和PPV对PK-15细胞株致细胞病变效应。[结论]rPoIFN-α具有IFN-α的基本理化性状,其在体外可分别抑制PRV、PPV对PK-15细胞株致细胞病变效应,但剂量有差别。     

重组猪α干扰素理化性状及其体外抗病毒活性研究

[目的]研究重组猪α干扰素（rPoIFN-α）半成品理化性状并对其体外抗病毒活性进行测试与鉴定。[方法]用HEp-2/VSV体系对3批rPoIFN-α蛋白进行抗病毒活性检测,以重组人IFN-α为参比品,测定干扰素效价;用0.25%胰蛋白酶HCl以及鼠抗猪α干扰素单克隆抗体作用已知效价的rPoIFN-α半成品,并检测各批次抗病毒活性,对rPoIFN-α理化性状进行评价;在猪肾细胞株（PK-15）上检测rPoIFN-α对猪细小病毒（PPV）和猪伪狂犬病毒（PRV）的致细胞病变抑制效应,观察rPoIFN-α的体外抗病毒活性。[结果]HEp-2/VSV体系滴定rPoIFN-α半成品效价可达1.5×105IU/ml,比活性达1.1×106IU/mg;rPoIFN-α经0.25%胰蛋白酶37℃作用1h,效价残留率低于1%,经HCl（pH=2.0）处理72h效价残留率高达95%以上,经56℃处理30min效价残留率高于47%,经鼠抗猪α干扰素单克隆抗体37℃作用1h后效价残留率约为1%;体外抗病毒试验表明,用50和500IU/mlrPoIFN-α可分别抑制PRV和PPV对PK-15细胞株致细胞病变效应。[结论]rPoIFN-α具有IFN-α的基本理化性状,其在体外可分别抑制PRV、PPV对PK-15细胞株致细胞病变效应,但剂量有差别。     

Suppression of gamma interferon production by inactivated feline leukemia virus

Supernatants from cultures of normal feline lymphocytes stimulated with Staphylococcus enterotoxin A showed antiviral activity, characterized as a gamma-like interferon. With the addition of inactivated feline leukemia virus, markedly less interferon was produced. The reduction in interferon production was not attributable to lowered lymphocyte viability or reduced mitogenic properties of Staphylococcus enterotoxin A and appears to be a direct retroviral effect. This finding may reflect clinically relevant events that may contribute to the development of the feline or human states of acquired immunodeficiency.     

两种细胞株滴定重组猪α干扰素效价的比较研究

采用水泡性口炎病毒（VSV）作为攻击病毒,以微量细胞病变抑制法对同批次不同浓度的8组重组猪α干扰素（rPoIFN-α）样品进行了效价检测,以中国药品生物制品检定所指定使用的标化重组人α干扰素为效价滴定的参比标准,对在牛肾细胞（MDBK）株和人喉癌上皮细胞（HEp-2）株所得结果进行比较,并对细胞与待测样品按＂一步法＂和先加细胞,待细胞长满单层再加待测样品的＂两步法＂所得的rPoIFN-α抗病毒效价进行比较。结果表明：MDBK细胞株与HEp-2细胞株对于rPoIFN-α抗病毒活性检测结果无显著性差异,均可用于rPoIFN-α抗病毒活性的检测,重组人α干扰素滴定系统可用于rPoIFN-α效价检测。两种加样方法所得结果基本相同,推荐使用＂一步法＂。     

NMRAL1对流感病毒复制的调控机制

【目的】流感病毒是一种人兽共患病原,常引起大流行,给人类健康造成巨大威胁,且流感病毒易发生变异,能不断逃逸宿主细胞的免疫反应,对现有抗流感药物产生耐药性,因此寻找抵抗流感的新方法迫在眉睫。研究通过探索NMRAL1(NmrA-like family domain-containing protein 1)对流感病毒复制的影响,并揭示其发挥作用的分子机制,为抗流感药物研发提供潜在靶点。【方法】采用siRNA干扰技术在A549细胞中下调表达NMRAL1,并通过Western Blot检测siRNA干扰后NMRAL1的表达水平;在下调表达NMRAL1的细胞中,分别感染A/Anhui/2/2005 (AH05) (H5N1)和A/WSN/33 (H1N1) 两株不同亚型流感病毒,利用蚀斑试验检测感染病毒后24和48 h细胞上清中的病毒滴度。为确定NMRAL1影响流感病毒复制的具体阶段,在HEK293T细胞中瞬时转染NMRAL1-Myc-pCAGGS质粒过表达NMRAL1,通过双荧光素酶报告系统检测过表达NMRAL1对流感病毒聚合酶活性的影响;使用免疫荧光技术对流感病毒NP蛋白进行染色,通过激光共聚焦试验观察下调表达NMRAL1对感染病毒后3、4、5、6和8 h NP蛋白在被感染细胞中的定位情况的影响,判断下调表达NMRAL1是否影响流感病毒的入核和出核过程;利用Western Blot检测下调表达NMRAL1对流感病毒各病毒蛋白表达的影响和对流感病毒激活I型干扰素通路下游IFN刺激基因（ISGs）表达的影响,利用间接免疫荧光试验进一步研究NMRAL1对流感病毒复制的影响。【结果】Western Blot检测发现NMRAL1 siRNA能显著下调NMRAL1表达,在下调表达NMRAL1的A549细胞中分别感染H5N1和H1N1病毒,并通过蚀斑试验检测感染病毒后细胞上清中的病毒滴度,结果显示在下调表达NMRAL1的细胞中,感染流感病毒后24和48 h收取的细胞上清中病毒滴度显著下降,表明NMRAL1能促进不同亚型流感病毒的复制;为进一步探索NMRAL1调控流感病毒复制的具体机制,利用双荧光素酶报告系统检测流感病毒聚合酶活性,发现过表达NMRAL1对流感病毒聚合酶活性无明显影响;激光共聚焦试验结果显示下调NMRAL1表达不影响NP蛋白的入核和出核过程,同时Western Blot检测表明下调NMRAL1表达不影响各病毒蛋白的表达;但荧光定量PCR试验结果显示下调NMRAL1表达能够促进流感病毒感染诱导的IFN-β mRNA水平上升,且Western Blot检测发现下调表达NMRAL1促进I型干扰素通路下游的MxA和IFITM3抗病毒蛋白的表达,与此同时,间接免疫荧光试验结果显示下调NMRAL1表达可显著抑制流感病毒复制。【结论】在流感病毒感染过程中,NMRAL1不影响流感病毒的入侵以及转录翻译过程,而是通过抑制I型干扰素通路激活从而抑制MxA、IFITM3等抗病毒因子的表达,最终促进流感病毒复制。研究证实宿主因子NMRAL1正调控流感病毒的复制,丰富了参与流感病毒复制的宿主因子网络。     

Interferon: production by chick erythrocytes activated by cell fusion

In the presence of Sendai virus inactivated with ultraviolet light, nucleated chick erythrocytes can be fused with several types of human cells to form heterokaryons. Although chick erythrocytes alone cannot be stimulated by Sendai virus to produce interferon, fusion with a human cell (AH-1) which itself may produce human interferon results in heterokaryons in which the erythrocyte genome is activated and chick interferon is produced. When nucleated chick erythrocytes are fused with another type of human cell (HeLa clone S-3) which does not produce human interferon when stimulated, no chick interferon is detectable, despite morphologic changes suggestive of activation of the erythrocyte nuclei.     

Interferon: lack of detectable uptake by cells

No interferon, or at most a small fraction of that applied, is taken up by cells during the period of induction of antiviral activity. The lack of detectable adsorption was not influenced either by the concentration of interferon or the volume. The results were similar in both chicken and mouse systems.     

Assignment of the murine interferon sensitivity and cytoplasmic superoxide dismutase genes to chromosome 16

Both hybrids of mouse and human microcells and whole cell hybrids generated by the fusion of primary mouse cells and SV40-transformed human fibroblasts were used to establish the syntenic association of the murine cytoplasmic superoxide dismutase and the interferon sensitivity genes on mouse chromosome 16. This assignment adds two new markers to chromosome 16 and provides another example of an evolutionarily conserved linkage. This finding also provides an animal model both for cellular responsiveness to interferon and for Down's syndrome.     

甘草酸二钾体外诱导感染MDV鸡胚成纤维细胞 凋亡发挥抗病毒作用

【目的】探讨甘草酸二钾体外抗MDV的活性及作用机制。【方法】在体外以鸡胚成纤维细胞为MDV增殖模型,利用MTT比色法、噬斑减少法通过抗病毒试验、病毒复制抑制试验、直接灭活试验来评价甘草酸二钾的抗病毒效果;并通过细胞凋亡试验解释其抗病毒机制。【结果】抗病毒试验结果表明,在安全浓度范围内,甘草酸二钾在体外具有较强的抗MDV的活性,其EC50为（893.5±36.99）µg•mL-1,SI>3.36,最大抑制率达94.4%;在病毒复制抑制试验中,甘草酸二钾可以抑制病毒复制的整个过程;病毒直接灭活试验表明甘草酸二钾可直接杀灭病毒粒子且不呈时间依赖性;细胞凋亡试验结果显示甘草酸二钾能够诱导感染MDV的鸡胚成纤维细胞凋亡。【结论】甘草酸二钾在体外具有抗MDV的活性,可以通过诱导感染病毒的鸡胚成纤维细胞凋亡发挥抗病毒作用。     

Positive control of transformed phenotype in hybrids between SV40-transformed and normal human cells

Somatic cell hybrids have been obtained between SV40-transformed Lesch-Nyhan fibroblasts, which are deficient in hypoxanthine-guanine phosphoribosyltransferase (HGPRT) and display glucose-6-phosphate dehydrogenase A (G6PD-A) activity, and late-passage HGPRT-positive W138 human embryo fibroblasts, which display G6PD-B activity. The human-human hybrid clones, which display G6PD-A and G6PD-B and heteropolymers of the two enzyme forms, have the same growth characteristic as the SV40-transformed parental cells and behave as continuous cell lines. The SV40 tumor antigen, the gene for which has been assigned to human chromosome 7, is present in all clones examined.     

Voltage-sensitive calcium channels in normal and transformed 3T3 fibroblasts

Patch clamp recordings of whole-cell and single channel currents revealed the presence of two voltage-sensitive calcium channel types in the membrane of 3T3 fibroblasts. The two calcium channel types were identified by their unitary properties and pharmacological sensitivities. Both calcium channel types were present in all control 3T3 cells, but one type was selectively suppressed in 3T3 cells that had been transformed by activated c-H-ras, EJ-ras, v-fms, or polyoma middle T oncogenes. The presence of voltage-sensitive calcium channels in these nonexcitable cells and the control of their functional expression by transforming oncogenes raises questions about their role in the control of calcium-sensitive processes such as cell motility, cytoskeletal organization, and cell growth.     

Growth inhibitor from BSC-1 cells closely related to platelet type beta transforming growth factor

Purified growth inhibitor from BSC-1 cells and type beta transforming growth factor from human platelets are shown to have nearly identical biological activity and to compete for binding to the same cell membrane receptor. These findings suggest that the growth inhibitor and the type beta transforming growth factor are similar molecules. The data also show that the same purified polypeptide can either stimulate or inhibit cell proliferation depending on the experimental conditions.