Suppressive effect of topoisomerase inhibitors on JC polyomavirus propagation in human neuroblastoma cells

JC polyomavirus (JCPyV) causes progressive multifocal leukoencephalopathy (PML), a fatal demyelinating disease of the central nervous system, in immunocompromised patients. Because no drugs have been approved for treating PML, many antiviral agents are currently being investigated for this purpose. The inhibitory effects of the topoisomerase I inhibitors topotecan and β‐lapachone were assessed by investigating viral replication, propagation and viral protein 1 (VP1) production in cultured cells. JCPyV replication was assayed using the human neuroblastoma cell line IMR‐32 transfected with the JCPyV plasmid and RT‐ PCR combined with Dpn I treatment. Dpn I digests the input plasmid DNA containing methylated adenosine, but not newly replicated JCPyV DNA, in IMR‐32 cells. It was found that JCPyV replicates less in IMR‐32 cells treated with topotecan or β‐lapachone than in untreated cells. Moreover, drug treatment of JCI cells, which are IMR‐32 cells persistently infected with JCPyV, led to a reduction in the amount of JCPyV DNA and population of VP1‐positive cells. These results demonstrate that topotecan and β‐lapachone affects JCPyV propagation in human neuroblastoma cell lines, suggesting that topotecan and β‐lapachone could potentially be used to treat PML.     

JC virus as a marker of human migration to the Americas

JC virus is a ubiquitous human polyomavirus present in populations worldwide. Seven genotypes differing in DNA sequence by approximately 1-3% characterize three Old World population groups (African, European and Asian) as well as Oceania. It is possible to follow Old World populations into the New World by the JC virus genotypes they carried. The first population to settle in the Americas, the Native Americans, brought with them type 2A from northeast Asia. European settlers arriving after Columbus carried primarily type 1 and type 4. Africans brought by the slave trade carried type 3 and type 6.     

Role of JC virus agnoprotein in virion formation

JC virus (JCV) belongs to the polyomavirus family of double-stranded DNA viruses and causes progressive multifocal leukoencephalopathy in humans. JCV encodes early proteins (large T antigen, small T antigen, and T' antigen) and four late proteins (agnoprotein, and three viral capsid proteins, VP1, VP2, and VP3). In the current study, a novel function for JCV agnoprotein in the morphogenesis of JC virion particles was identified. It was found that mature virions of agnoprotein-negative JCV are irregularly shaped. Sucrose gradient sedimentation and cesium chloride gradient ultracentrifugation analyses revealed that the particles of virus lacking agnoprotein assemble into irregularly sized virions, and that agnoprotein alters the efficiency of formation of VP1 virus-like particles. An in vitro binding assay and immunocytochemistry revealed that agnoprotein binds to glutathione S-transferase fusion proteins of VP1 and that some fractions of agnoprotein colocalize with VP1 in the nucleus. In addition, gel filtration analysis of formation of VP1-pentamers revealed that agnoprotein enhances formation of these pentamers by interacting with VP1. The present findings suggest that JCV agnoprotein plays a role, similar to that of SV40 agnoprotein, in facilitating virion assembly.     

Efficient propagation of progressive multifocal leukoencephalopathy-type JC virus in COS-7-derived cell lines stably expressing Tat protein of human immunodeficiency virus type 1

The high incidence of progressive multifocal leukoencephalopathy (PML) in AIDS patients compared with many other immunosuppressive diseases suggests that HIV-1 infection is strictly related to the activation of JC virus (JCV) propagation. In this report, propagation of PML-type JCV in COS-7-derived cell lines stably expressing HIV-1 Tat (COS-tat cells) has been examined. In COS-tat cells, production of viral particles and replication of genomic DNA were markedly increased compared to COS-7 cells, as judged by HA and real-time PCR analyses. These results demonstrate that COS-tat cells provide a useful model system for studying HIV-1 Tat-mediated propagation of PML-type JCV.     

CPT11 prevents virus replication in JCI cells persistently infected with JC polyomavirus

JC polyomavirus (JCPyV) is the causative agent of the demyelinating disease of the central nervous system known as progressive multifocal leukoencephalopathy (PML), which occurs in immunocompromised patients. Moreover, patients treated with natalizumab for multiple sclerosis or Crohn disease can develop PML, which is then termed natalizumab‐related PML. Because few drugs are currently available for treating PML, many antiviral agents are being investigated. It has been demonstrated that the topoisomerase I inhibitors topotecan and β‐lapachone have inhibitory effects on JCPyV replication in IMR‐32 cells. However, both of these drugs have marginal inhibitory effects on virus propagation in JC1 cells according to RT‐PCR analysis. In the present study, the inhibitory effect of another topoisomerase I inhibitor, 7‐ethy‐10‐[4‐(1‐piperidino)‐1‐piperidino] carbonyloxy camptothecin (CPT11), was assessed by investigating viral replication, propagation, and viral protein 1 (VP1) production in cultured cells. JCPyV replication was assayed using real‐time PCR combined with Dpn I treatment in IMR‐32 cells transfected with JCPyV DNA. It was found that JCPyV replicates less in IMR‐32 cells treated with CPT11 than in untreated cells. Moreover, CPT11 treatment of JCI cells persistently infected with JCPyV led to a dose‐dependent reduction in JCPyV DNA and VP1 production. Additionally, the inhibitory effect of CPT11 was found to be stronger than those of topotecan and β‐lapachone. These findings suggest that CPT11 may be a potential anti‐JCPyV agent that could be used to treat PML.

     

Irisolidone, an isoflavone metabolite, represses JC virus gene expression via inhibition of Sp1 binding in human glial cells

Progressive multifocal leukoencephalopathy (PML) is a fatal demyelinating disease that results from an oligodendrocyte infection caused by the JC virus. Therefore, inhibiting the expression of JC virus is important for preventing and/or treating PML. This study found that irisolidone, an isoflavone metabolite, significantly inhibited the JC virus expression in primary cultured human astrocytes and glial cell lines. Studies examining the underlying mechanism revealed that a mutation of the Sp1 binding site downstream of the TATA box (Sp1-II) dramatically diminished the inhibitory activity of irisolidone. In addition, an irisolidone treatment repressed Sp1 binding to Sp1-II site, which is important for the basal JC virus promoter activity. The results suggest that the inhibitory effect of irisolidone against the JC virus may be attributed at least in part to the suppression of Sp1 binding to the JC virus promoter region. Therefore, the inhibition of the JC virus expression by irisolidone might provide therapeutic potential for PML caused by the JC virus.     

Genetic diversity of JC virus in the Saami and the Finns: implications for their population history

The JC virus (JCV) genotyping method was used to gain insights into the population history of the Saami and the Finns, both speaking Finno-Ugric languages and living in close geographic proximity. Urine samples from Saami and Finns, collected in northern and southern Finland, respectively, were used to amplify a 610-bp JCV-DNA region containing abundant type-specific mutations. Based on restriction site polymorphisms in the amplified fragments, we classified JCV isolates into one of the three superclusters of JCV, type A, B, or C. All 15 Saami isolates analyzed and 41 of 43 Finnish isolates analyzed were classified as type A, the European type, and two samples from Finns were classified as type B, the African/Asian type. We then amplified and sequenced a 583-bp JCV-DNA region from the type A isolates of Saami and Finns. According to type-determining nucleotides within the region, we classified type A isolates into EU-a1, -a2, or -b. Most type A isolates from Saami were classified as EU-a1, while type A isolates from Finns were distributed among EU-a1, EU-a2, and EU-b. This trend in the JCV-genotype distribution was statistically significant. On a phylogenetic tree based on complete sequences, most of the type A isolates from Saami were clustered in a single clade within EU-a1, while those from Finns were distributed throughout EU-a1, EU-a2, and EU-b. These findings are discussed in the context of the population history of the Saami and the Finns. This study provides new complete JCV DNA sequences derived from populations of anthropological interest.     

Genetic diversity of JC virus in the modern Filipino population: implications for the peopling of the Philippines

The Philippines is generally believed to have been established by various peoples who migrated from neighboring areas. To gain new insights into the peopling of the Philippines, we used the JC virus (JCV) genotyping approach. We collected about 50 urine samples on each of two representative islands of the Philippines, Luzon and Cebu. DNA was extracted from the urine samples and used to amplify the 610-bp region (IG region) of the viral genome. For each island, we determined about 20 IG sequences, from which a neighbor-joining phylogenetic tree was constructed to classify the JCV isolates detected into distinct genotypes. The predominant genotype detected was SC, the Southeast Asian genotype. Minor JCV genotypes were SC/Phi, B1-a, and B3. SC/Phi was a subcluster of SC and has not been detected in areas other than the Philippines. B1-a was detected previously in mainland China, Pamalican Island (Palawan, Philippines), and Taiwan (an aboriginal tribe). B3 was classified in this study into two subgroups, one (B3-a) containing three Luzon isolates and several Chinese, Thai, and Uzbek isolates, the other (B3-b) containing two Luzon, one Cebu, and one Indonesian isolate. These findings suggest that the modern Filipino population was formed not only by Southeast Asians carrying SC but also by a few distinct ethnic groups carrying SC/Phi, B1-a, and B3-a or -b.     

Establishment of an immunoscreening system using recombinant VP1 protein for the isolation of a monoclonal antibody that blocks JC virus infection

Polyomavirus JC (JCV) infection causes the fatal human demyelinating disease, progressive multifocal leukoencephalopathy. Although the initial interaction of JCV with host cells occurs through direct binding of the major viral capsid protein (VP1) with cell-surface molecules possessing sialic acid, these molecules have not yet been identified. In order to isolate monoclonal antibodies which inhibit attachment of JCV, we established an immunoscreening system using virus-like particles consisting of the VP1. Using this system, among monoclonal antibodies against the cell membrane fraction from JCV-permissive human neuroblastoma IMR-32 cells, we isolated a monoclonal antibody designated as 24D2 that specifically inhibited attachment and infection of JCV to IMR-32 cells. The antibody 24D2 recognized a single molecule of around 60 kDa in molecular weight in the IMR-32 membrane fraction. Immunohistochemical staining with 24D2 demonstrated immunoreactivity in the cell membrane of JCV-permissive cell lines and glial cells of the human brain. These results suggested that the molecule recognized by 24D2 plays a role in JCV infection, and that it might participate as a receptor or a co-receptor in JCV attachment and entry into the cells.     

鸡胚细胞的传代培养和麻疹病毒的增殖

为了建立原代鸡胚细胞的传代培养工艺,探究传代鸡胚细胞对麻疹病毒的敏感性和适应性,本研究将原代鸡胚细胞进行传代培养,分别采用原代鸡胚细胞和传代鸡胚细胞培养麻疹病毒沪-191(Shanghai-191,S-191)株毒种,并对病毒收获液进行滴度检测和基因序列测定。结果显示,原代鸡胚细胞可稳定传代培养至第10代,各代次细胞生长趋势相似;第5代鸡胚细胞染色体检查为正常染色体核型;第8代鸡胚细胞成瘤性检查未见成瘤;采用第3、5代鸡胚细胞制备的麻疹病毒滴度水平高于原代鸡胚细胞,但无显著性差异(n=3,P>0.05),编码病毒核蛋白(nucleoprotein,N)和血凝素蛋白(hemagglutinin,H)的基因序列与S-191株完全一致,未发生变异。本研究证实,原代鸡胚细胞可进行传代培养,各代次鸡胚细胞对麻疹病毒的敏感性不变,产毒水平无显著差异,可用于培养麻疹病毒。     

Inhibitory effect of the carnosine–gallic acid synthetic peptide on MMP‐2 and MMP‐9 in human fibrosarcoma HT1080 cells

Matrix metalloproteinases (MMPs) are a family of zinc‐dependent endopeptidases that degrade extracellular matrix components and play important roles in a variety of biological and pathological processes such as malignant tumor metastasis and invasion. In this study, we constructed carnosine–gallic acid peptide (CGP) to identify a better MMP inhibitor than carnosine. The inhibitory effects of CGP on MMP‐2 and MMP‐9 were investigated in the human fibrosarcoma (HT1080) cell line. As a result, CGP significantly decreased MMP‐2 and MMP‐9 expression levels without a cytotoxic effect. Moreover, CGP may inhibit migration and invasion in HT1080 cells through the urokinase plasminogen activator (uPA)–uPA receptor signaling pathways to inhibit MMP‐2 and MMP‐9. Based on these results, it appears that CGP may play an important role in preventing and treating several MMP‐2 and MMP‐9‐mediated health problems such as metastasis. Copyright © 2014 European Peptide Society and John Wiley & Sons, Ltd.     

Antiproliferative effect of nitric oxide on epidermal growth factor-responsive human neuroblastoma cells

Addition of nitric oxide (NO) donors to NB69 neuroblastoma cells produced a cGMP-independent decrease in cell proliferation, without affecting cell viability or apoptosis. The potency of short half-life NO donors was higher when cell proliferation was stimulated by epidermal growth factor (EGF), as compared with cultures exposed to fetal calf serum (FCS). Immunoprecipitation and western blot analysis of the EGF receptor (EGFR) revealed a significant reduction of its EGF-induced tyrosine phosphorylation in cells treated with the NO donor 2-(N,N-diethylamino)-diazenolate-2-oxide (DEA-NO). When total cell lysates were subjected to western blotting, we observed that DEA-NO also reduced tyrosine phosphorylation in EGF-activated phosphoproteins, but not in those proteins whose tyrosine phosphorylation was evident in the absence of EGF. The effect of NO on EGFR transphosphorylation was concentration-dependent and transient, with a total recovery observed between 1.5 and 3 h after addition of DEA-NO to the cells. When cells were incubated for 15 min with DEA-NO and then washed, the EGFR transphosphorylation returned to control levels immediately, indicating that the interaction of NO with the receptor molecule was fully reversible. NB69 cells expressed both the neuronal and the inducible isoforms of NO synthase (NOS) when cultured in the presence of FCS; under this condition, the NOS inhibitor, N(omega)-nitro-L-arginine methyl ester, produced a small but significant increase in cell proliferation. The results suggest that NO is an endogenous antimitotic agent and that its interaction with EGFR contributes to cytostasis in NB69 cells.     

梭曼对大鼠应激性体温过高的作用及中枢和外周胆碱能受体阻断剂对其效应的影响

目的：观察梭曼对大鼠应激性体温过高的抑制作用以及中枢和外周胆碱能受体阻断剂对其效应的影响。方法：用无线遥测技术测量大鼠的体温,观察皮下注射梭曼、东莨菪碱、甲基东莨菪碱和吡啶斯的明对开放环境中大鼠应激性体温过高的影响。用分光光度技术测定血浆中胆碱酯酶活性。结果：①对照组大鼠在开放实验箱中体温升高达0.96℃,而注射梭曼动物体温只升高了0.55℃。中枢性胆碱能受体阻断剂东莨菪碱几乎完全阻断梭曼对应激体温过高的抑制作用,而外周胆碱能受体阻断剂甲基东莨菪碱则能明显增强梭曼对应激性体温过高的抑制作用。②外周抗胆碱酯酶剂吡啶斯的明能使血浆胆碱酯酶的活性降低至52%,同时明显提高开放环境中大鼠应激性体温过高的反应。甲基东莨菪碱几乎可以阻断吡啶斯的明对应激体温过高反应的影响。结论：神经毒剂梭曼可改变大鼠在开放环境中应激性体温过高的反应能力,其作用主要是通过中枢毒蕈碱型胆碱能通路所致。此外,外周胆碱能神经参与大鼠开放应激性体温过高的形成过程。     

The effect of explosive blast loading on human neuroblastoma cells

Diagnosis of mild to moderate traumatic brain injury is challenging because brain tissue damage progresses slowly and is not readily detectable by conventional imaging techniques. We have developed a novel in vitro model to study primary blast loading on dissociated neurons using nitroamine explosives such as those used on the battlefield. Human neuroblastoma cells were exposed to single and triple 50-psi explosive blasts and single 100-psi blasts. Changes in membrane permeability and oxidative stress showed a significant increase for the single and triple 100-psi blast conditions compared with single 50-psi blast and controls.     

Inhibitory effect of acetylsalicylic acid on matrix metalloproteinase - 2 activity in human endothelial cells exposed to high glucose

Matrix metalloproteinases play a major role in the process of angiogenesis, an important feature of diabetes complications, cancer or rheumatoid arthritis. High glucose concentrations were reported to augment metalloproteinase-2 secretion in some cell types. In the present study we investigated the influence of acetylsalicylic acid on metalloproteinase- 2 secretion and expression in endothelial cells cultured for one week in high glucose conditions (25 mM and 33 mM). Metalloproteinase-2 activity was evidenced by gel zymography, the protein was identified by Western blotting, and the gene expression was quantitated by RT-PCR. The results indicated a marked inhibitory effect of acetylsalicylic acid at gene expression level (approximately 43%) and also at secretion level in samples of conditioned media (approximately 30%) and cellular homogenates (approximately 70%). This may suggest that acetylsalicylic acid could have a beneficial effect in preventing the angiogenic process that appears in diabetes complications.     

不同狂犬病毒株在地鼠肾细胞上培养的病毒滴度与免疫原性比较

三个狂犬病毒株,分别经地鼠肾细胞培养,将其抗原含量,病毒滴度和免疫原性进行比较。结果显示,三个病毒株的抗原含量有差异。但差异显著性,CTN-V10M3的毒力最高,CTN-BHK3的毒力最低,aG株的毒力虽然居中,但免疫原性最好,它仍是最适于地鼠肾细胞上培养的毒株。     

dl-alpha-Tocopheryl succinate enhances the effect of gamma-irradiation on neuroblastoma cells in culture

The effect of dl-alpha-tocopheryl (vitamin E) succinate in modifying the radiation response of mouse neuroblastoma (NBP2) and mouse fibroblast (L-cells) cells in culture was studied on the criterion of growth inhibition (due to cell death and inhibition of cell division). Results show that vitamin E succinate markedly enhanced the effect of 60CO-gamma-irradiation on NB cells, but it did not significantly modify the effect of irradiation on mouse fibroblasts. Sodium succinate plus ethanol (0.25% final concentration) did not modify the radiation response of NB cells or fibroblasts. Butylated hydroxyanisole, a lipid soluble antioxidant, also enhanced the effect of irradiation on NB cells, indicating that the effect of vitamin E in modifying the radiation response may be mediated, in part, by antioxidation mechanisms.