Simian Kidney Cells Research Articles

Massive entry of BK Polyomavirus induces transient cytoplasmic vacuolization of human renal proximal tubule epithelial cells.

BK polyomavirus (BKPyV) is a ubiquitous human virus that establishes a persistent infection in renal tubular epithelial cells and mainly causes disease in kidney transplant recipients. The closely related simian polyomavirus SV40 is known to cause cytoplasmic vacuolization in simian kidney cells, possibly increasing progeny release and cell death. This study aimed to determine whether BKPyV causes cytoplasmic vacuolization in primary human renal proximal tubule epithelial cells (RPTECs) and to investigate its potential role in the replication cycle. Using a large infectious dose (MOI 100-1000), a fraction of RPTECs (10-72%) showed early-wave vacuolization from 3 hours post-infection (hpi), which was mainly reversed by 36 hpi. Independent of the infectious dose, late-wave vacuolization occurred around the timepoint of progeny release. BKPyV receptor binding and internalization were required, as neuraminidase pretreatment and preincubation or treatment with a BKPyV-specific neutralizing antibody prevented early or late-occurring vacuolization. Microscopy revealed that the vacuoles were enlarged acidic endo-/lysosomal structures (dextran, EEA1, Rab5, Rab7, LAMP1, and/or Lysoview positive) that contained membrane-bound BKPyV. Time-lapse microscopy and quantitative PCR revealed that cell death and progeny release preceded late-wave vacuolization, mainly affecting cells directly neighboring the lysed cells. Thus, vacuolization had little impact on cell death or progeny release. Addition of the V-ATPase inhibitor Bafilomycin A1 at 0 hpi blocked vacuolization and BKPyV replication, but addition at 2 hpi only blocked vacuolization, suggesting that continuous endosomal acidification and maturation is needed for vacuole formation, but not for BKPyV replication. Our study shows that a massive uptake of BKPyV in RPTECs induces transient enlargement of endo-/lysosomes and is an early event in the viral replication cycle. Vacuolization gives no clear benefit for BKPyV and is possibly the result of a transiently overloaded endocytic pathway. Focal vacuolization around lysed cells suggests that the spread of BKPyV is preferably local.

First-time detection of porcine reproductive and respiratory syndrome virus (PRRSV) infection in Uruguay.

Within the last two decades, several high-impact viruses have emerged in the global swine population, including porcine reproductive and respiratory syndrome virus (PRRSV). In Uruguay, the more recent serological survey for PRRSV and other notifiable diseases such as Aujeszky's disease virus (ADV) and classical swine fever virus (CSFV) dated from year 2000. The main purpose of this study was to update our information on the infection status of PRRSV, ADV and CSFV in Uruguayan pig herds, in order to keep informed about the epidemiological situation of these notifiable infections in the country. For serological testing, a total of 524 swine serum samples collected during the period 2014-2016 were assayed by commercial ELISAs. Our results revealed the (unexpected) presence of PRRSV antibodies in Uruguayan domestic swine herds and confirmed the absence of ADV and CSFV antibodies in all of the assessed samples. Following such initial finding, PRRSV antibodies were further investigated in 23 retrospective samples collected during 2010-2014. Thirteen of these 23 samples resulted seropositive. Subsequently, a molecular detection approach in frozen serum samples was implemented to confirm PRRSV infection, and viral RNA was identified by reverse transcription-nested polymerase chain reaction (RT-nPCR). Fourteen of 86 evaluated 2014-2016 samples resulted positive for viral RNA, while molecular analysis of four retrospective samples also revealed the presence of PRRSV type 2. Viral isolation of selected samples was carried out in porcine alveolar macrophages (PAM) and MARC 145 simian kidney cells, and the virus identity was confirmed by cytopathic effect (CPE) and immunofluorescence assay (IFA) using specific monoclonal antibodies for PRRSV nucleocapsid. Data reported here evidence for the first time the circulation of PRRSV type 2 in Uruguay, and retrospective serology results suggest that the virus has been infecting pigs in this country at least since 2011.

A comparative study of the replicative properties of antitumor recombinant vaccinia viruses in cultivated U87 human glioblastoma cells and simian CV-1 kidney cells

At present, virotherapy is one of the rapidly developing areas in the treatment of cancer, and its advantage is the selective destruction of cancer cells with the minimal destructive effect on the healthy cells of the body. Orthopoxviruses provide a promising basis for the design of oncolytic drugs. They have a number of advantages over other viral vectors, and one of these advantages is the large capacity of their genomes. It allows them to be furnished with genes encoding proteins with antitumor properties. In this study, we compare the replicative properties of ten variants of the vaccinia virus (LIVP strain of VACV) tested on cultures of human glioblastoma cells and simian CV-1 kidney cells. Some of these viruses have additional genes, such as those encoding the granulocyte-macrophage colony-stimulating factor, apoptosis-inducing protein TRAIL, and green fluorescent protein. We have also studied VACV with five virulence genes deleted, namely, the genes encoding hemagglutinin, the γ-interferon-binding protein, thymidine kinase, the complement-binding protein, and the Bcl2-like inhibitor of apoptosis. The reactogenicity and neurovirulence of this disarmed derivative are much less than in the original LIVP strain. It has been found that derivatives of the vaccinia virus with the deleted thymidine kinase gene most intensely replicate in the glioblastoma cell culture.

Efficient propagation of archetype JC polyomavirus in COS-7 cells: evaluation of rearrangements within the NCCR structural organization after transfection

John Cunningham virus (JCPyV) is an ubiquitous human pathogen that causes disease in immunocompromised patients. The JCPyV genome is composed of an early region and a late region, which are physically separated by the non-coding control region (NCCR). The DNA sequence of the NCCR distinguishes two forms of JCPyV, the designated archetype and the prototype, which resulted from a rearrangement of the archetype sequence. To date, the cell culture systems for propagating JCPyV archetype have been very limited in their availability and robustness. Prior to this study, it was demonstrated that JCPyV archetype DNA replicates in COS-7 simian kidney cells expressing SV40 TAg and COS-7 cells expressing HIV-1 Tat. Based on these observations, the present study was conducted to reproduce an in vitro model in COS-7 cells transfected with the JCPyV archetype strain in order to study JCPyV DNA replication and analyze NCCR rearrangements during the viral life cycle. The efficiency of JCPyV replication was evaluated by quantitative PCR (Q-PCR) and by hemagglutination (HA) assay after transfection. In parallel, sequence analysis of JCPyV NCCR was performed. JCPyV efficiently replicated in kidney-derived COS-7 cells, as demonstrated by a progressive increase in viral load and virion particle production after transfection. The archetypal structure of NCCR was maintained during the viral cycle, but two characteristic point mutations were detected 28 days after transfection. This model is a useful tool for analyzing NCCR rearrangements during in vitro replication in cells that are sites of viral persistence, such as tubular epithelial cells of the kidney.

Patterned two-photon photoactivation illuminates spatial reorganization in live cells.

Photoactivatable fluorescent proteins offer the possibility to optically tag and track the location of molecules in their bright state with high spatial and temporal resolution. Several reports of patterned photoactivation have emerged since the development of a photoactivatable variant of the green fluorescent protein (PaGFP) and the demonstration of two-photon activation of PaGFP. To date, however, there have been few methods developed to quantify the spatial reorganization of the photoactivated population. Here we report on the use of singular value decomposition (SVD) to track the time-dependent distribution of fluorophores after photoactivation. The method was used to describe live-cell actin cytoskeleton behavior in primary murine T-cells, in which a dynamic cytoskeleton is responsible for the reorganization of membrane proteins in response to antigen peptide recognition. The method was also used to observe immortalized simian kidney (Cos-7) cells, in which the cytoskeleton is more stable. Both cell types were transfected with PaGFP fused to the F-actin binding domain of utrophin (UtrCH). Photoactivation patterns were written in the samples with a pair of galvanometric scanning mirrors in circular patterns that were analyzed by transforming the images into a time series of radial distribution profiles. The time-evolution of the profiles was well-described by the first two SVD component states. For T-cells, we find that actin filaments are highly mobile. Inward transport from the photoactivation region was observed and occurred on a 1−2 s time scale, which is consistent with retrograde cycling. For Cos-7 cells, we find that the actin is relatively stationary and does not undergo significant centripetal flow as expected for a resting fibroblast. The combination of patterned photoactivation and SVD analysis offers a unique way to measure spatial redistribution dynamics within live cells.

Comparative study of human and mouse pregnane X receptor agonistic activity in 200 pesticides using in vitro reporter gene assays

The nuclear receptor, pregnane X receptor (PXR), is a ligand-dependent transcription factor that regulates genes involved in xenobiotic metabolism. Recent studies have shown that PXR activation may affect energy metabolism as well as the endocrine and immune systems. In this study, we characterized and compared the agonistic activities of a variety of pesticides against human PXR (hPXR) and mouse PXR (mPXR). We tested the hPXR and mPXR agonistic activity of 200 pesticides (29 organochlorines, 11 diphenyl ethers, 56 organophosphorus pesticides, 12 pyrethroids, 22 carbamates, 12 acid amides, 7 triazines, 7 ureas, and 44 others) by reporter gene assays using COS-7 simian kidney cells. Of the 200 pesticides tested, 106 and 93 activated hPXR and mPXR, respectively, and a total of 111 had hPXR and/or mPXR agonistic activity with greater or lesser inter-species differences. Although all of the pyrethroids and most of the organochlorines and acid amides acted as PXR agonists, a wide range of pesticides with diverse structures also showed hPXR and/or mPXR agonistic activity. Among the 200 pesticides, pyributicarb, pretilachlor, piperophos and butamifos for hPXR, and phosalone, prochloraz, pendimethalin, and butamifos for mPXR, acted as particularly potent activators at low concentrations in the order of 10 −8–10 −7 M. In addition, we found that several organophosphorus oxon- and pyributicarb oxon-metabolites decreased PXR activation potency compared to their parent compounds. These results suggest that a large number of structurally diverse pesticides and their metabolites possess PXR-mediated transcriptional activity, and their ability to do so varies in a species-dependent manner in humans and mice.

Proline Isomerase Pin1 Represses Terminal Differentiation and Myocyte Enhancer Factor 2C Function in Skeletal Muscle Cells

Reversible proline-directed phosphorylation at Ser/Thr-Pro motifs has an essential role in myogenesis, a multistep process strictly regulated by several signaling pathways that impinge on two families of myogenic effectors, the basic helix-loop-helix myogenic transcription factors and the MEF2 (myocyte enhancer factor 2) proteins. The question of how these signals are deciphered by the myogenic effectors remains largely unaddressed. In this study, we show that the peptidyl-prolyl isomerase Pin1, which catalyzes the isomerization of phosphorylated Ser/Thr-Pro peptide bonds to induce conformational changes of its target proteins, acts as an inhibitor of muscle differentiation because its knockdown in myoblasts promotes myotube formation. With the aim of clarifying the mechanism of Pin1 function in skeletal myogenesis, we investigated whether MEF2C, a critical regulator of the myogenic program that is the end point of several signaling pathways, might serve as a/the target for the inhibitory effects of Pin1 on muscle differentiation. We show that Pin1 interacts selectively with phosphorylated MEF2C in skeletal muscle cells, both in vitro and in vivo. The interaction with Pin1 requires two novel critical phospho-Ser/Thr-Pro motifs in MEF2C, Ser98 and Ser110, which are phosphorylated in vivo. Overexpression of Pin1 decreases MEF2C stability and activity and its ability to cooperate with MyoD to activate myogenic conversion. Collectively, these findings reveal a novel role for Pin1 as a regulator of muscle terminal differentiation and suggest that Pin1-mediated repression of MEF2C function could contribute to this function.

Differential infectious entry of human influenza A/NWS/33 virus (H1N1) in mammalian kidney cells

In this report we focused our interest on the early events of the replication cycle of NWS/33 human influenza A (NWS) virus in MDCK (canine), LLC-MK2 (simian), and NSK (swine) kidney cells, with different susceptibility upon infection. We have previously demonstrated that actin organization induces restriction to viral replication during the early stages of NWS virus infection in simian kidney cells. To explore how cell endocytic mechanisms are hijacked by NWS virus and may modulate the outcome of viral infection, the effect of drugs affecting selectively the entry via clathrin-coated pits, caveolar/raft-dependent endocytosis and macropinocytosis was analyzed. Results point to critical differences in terms of internalization pathways exploited by NWS virus to enter the examined cell models. Moreover, we show that some ways of entry do not allow an effective virus internalization, depending on the cell type. Understanding how specific cell functions/components may regulate early phases of viral replication allows us to deepen our knowledge on influenza virus infection and provides new insights for anti-viral researches.

The fibrodysplasia ossificans progressiva R206H ACVR1 mutation activates BMP-independent chondrogenesis and zebrafish embryo ventralization

Patients with classic fibrodysplasia ossificans progressiva, a disorder characterized by extensive extraskeletal endochondral bone formation, share a recurrent mutation (R206H) within the glycine/serine-rich domain of ACVR1/ALK2, a bone morphogenetic protein type I receptor. Through a series of in vitro assays using several mammalian cell lines and chick limb bud micromass cultures, we determined that mutant R206H ACVR1 activated BMP signaling in the absence of BMP ligand and mediated BMP-independent chondrogenesis that was enhanced by BMP. We further investigated the interaction of mutant R206H ACVR1 with FKBP1A, a glycine/serine domain-binding protein that prevents leaky BMP type I receptor activation in the absence of ligand. The mutant protein exhibited reduced binding to FKBP1A in COS-7 simian kidney cell line assays, suggesting that increased BMP pathway activity in COS-7 cells with R206H ACVR1 is due, at least in part, to decreased binding of this inhibitory factor. Consistent with these findings, in vivo analyses of zebrafish embryos showed BMP-independent hyperactivation of BMP signaling in response to the R206H mutant, resulting in increased embryonic ventralization. These data support the conclusion that the mutant R206H ACVR1 receptor in FOP patients is an activating mutation that induces BMP signaling in a BMP-independent and BMP-responsive manner to promote chondrogenesis, consistent with the ectopic endochondral bone formation in these patients.

Mutational Specificity of γ-Radiation-Induced Guanine−Thymine and Thymine−Guanine Intrastrand Cross-Links in Mammalian Cells and Translesion Synthesis Past the Guanine−Thymine Lesion by Human DNA Polymerase η

Comparative mutagenesis of γ- or X-ray-induced tandem DNA lesions G[8,5-Me]T and T[5-Me,8]G intrastrand cross-links was investigated in simian (COS-7) and human embryonic (293T) kidney cells. For G[8,5-Me]T in 293T cells, 5.8% of progeny contained targeted base substitutions, whereas 10.0% showed semitargeted single-base substitutions. Of the targeted mutations, the G → T mutation occurred with the highest frequency. The semitargeted mutations were detected up to two bases 5′ and three bases 3′ to the cross-link. The most prevalent semitargeted mutation was a C → T transition immediately 5′ to the G[8,5-Me]T cross-link. Frameshifts (4.6%) (mostly small deletions) and multiple-base substitutions (2.7%) also were detected. For the T[5-Me,8]G cross-link, a similar pattern of mutations was noted, but the mutational frequency was significantly higher than that of G[8,5-Me]T. Both targeted and semitargeted mutations occurred with a frequency of ∼16%, and both included a dominant G → T transversion. As in 293T cells, more than twice as many targeted mutations in COS cells occurred in T[5-Me,8]G (11.4%) as in G[8,5-Me]T (4.7%). Also, the level of semitargeted single-base substitutions 5′ to the lesion was increased and 3′ to the lesion decreased in T[5-Me,8]G relative to G[8,5-Me]T in COS cells. It appeared that the majority of the base substitutions at or near the cross-links resulted from incorporation of dAMP opposite the template base, in agreement with the so-called “A-rule”. To determine if human polymerase η (hpol η) might be involved in the mutagenic bypass, an in vitro bypass study of G[8,5-Me]T in the same sequence was carried out, which showed that hpol η can bypass the cross-link incorporating the correct dNMP opposite each cross-linked base. For G[8,5-Me]T, nucleotide incorporation by hpol η was significantly different from that by yeast pol η in that the latter was more error-prone opposite the cross-linked Gua. The incorporation of the correct nucleotide, dAMP, by hpol η opposite cross-linked T was 3−5-fold more efficient than that of a wrong nucleotide, whereas incorporation of dCMP opposite the cross-linked G was 10-fold more efficient than that with dTMP. Therefore, the nucleotide incorporation pattern by hpol η was not consistent with the observed cellular mutations. Nevertheless, at and near the lesion, hpol η was more error-prone compared to a control template. The in vitro data suggest that translesion synthesis by another Y-family DNA polymerase and/or flawed participation of an accessory protein is a more likely scenario in the mutagenesis of these lesions in mammalian cells. However, hpol η may play a role in correct bypass of the cross-links.

Novel Inhibitors for Murine and Human Leukemia Inhibitory Factor Based on Fused Soluble Receptors

Fusion proteins of the extracellular parts of cytokine receptors, also known as cytokine traps, turned out to be promising cytokine inhibitors useful in anti-cytokine therapies. Here we present newly designed cytokine traps for murine and human leukemia inhibitory factor (LIF) as prototypes for inhibitors targeting cytokines that signal through a heterodimer of two signaling receptors of the glycoprotein 130 (gp130) family. LIF signals through a receptor heterodimer of LIF receptor (LIFR) and gp130 and induces the tyrosine phosphorylation of STAT3 leading to target gene expression. The analysis of various receptor fusion and deletion constructs revealed that a truncated form of the murine LIF receptor consisting of the first five extracellular domains was a potent inhibitor for human LIF. For the efficient inhibition of murine LIF, the cytokine-binding module of murine gp130 had to be fused to the first five domains of murine LIFR generating mLIF-RFP (murine LIFR fusion protein). The tyrosine phosphorylation of STAT3 and subsequent gene induction induced by human or murine LIF are completely blocked by the respective inhibitor. Furthermore, both inhibitors are specific and do not alter the bioactivities of the closely related cytokines interleukin (IL)-6 and oncostatin M. The gained knowledge on the construction of LIF inhibitors can be transferred to the design of inhibitors for related cytokines such as IL-31, IL-27, and oncostatin M for the treatment of inflammatory and malignant diseases.

Mutagenicity of the 1-Nitropyrene-DNA Adduct N-(Deoxyguanosin-8-yl)-1-aminopyrene in Mammalian Cells

The mutagenesis of the major DNA adduct N-(deoxyguanosin-8-yl)-1-aminopyrene (C8-AP-dG) formed by 1-nitropyrene was compared with the analogous C8-dG adducts of 2-aminofluorene (AF) and N-acetyl-2-aminofluorene (AAF) in simian kidney (COS-7) cells. The DNA sequence chosen for this comparison contained 5'-CCATC GCTACC-3' that has been used for solution NMR investigations. The structural and conformational differences among these lesions are well-established [Patel, D. J., Mao, B., Gu, Z., Hingerty, B. E., Gorin, A., Basu, A. K., and Broyde,S. (1998) NMR solution structures of covalent aromatic amine-DNA adducts and their mutagenic relevance. Chem. Res. Toxicol. 11, 391- 407.]. Accordingly, we found a notable difference in the viability of the progeny, which showed that the AAF adduct was most toxic and that the AF adduct was least toxic, with the AP adduct exhibiting intermediate toxicity. However, analysis of the progeny showed that translesion synthesis was predominantly error-free. Only low-level mutations (<3%) were detected with G-->T as the dominant type of mutation by all three DNA adducts. When C8-AP-dG was evaluated in a repetitive 5'-CGC GCG-3' sequence, higher mutational frequency ( approximately 8%) was observed. Again, G-->T was the major type of mutations in simian kidney cells, even though in bacteria CpG deletions predominate in this sequence [Hilario, P., Yan, S., Hingerty, B. E., Broyde, S., and Basu, A. K. (2002) Comparative mutagenesis of the C8-guanine adducts of 1-nitropyrene,and 1,6- and 1,8-dinitropyrene in a CpG repeat sequence: A slipped frameshift intermediate model for dinucleotide deletion. J. Biol. Chem. 277, 45068- 45074.]. Mutagenesis of C8-AP-dG in a 12-mer containing the local DNA sequence around codon 273 of the p53 tumor suppressor gene, where the adduct was located at the second base of this codon, was also investigated. In this 5'-GTGC GTGTTTGT-3' site, the mutations were slightly lower but not very different from the progeny derived from the 5'-CGC GCG-3' sequence. However, the mutational frequency increased by more than 50% when the 5'-C to the adduct was replaced with a 5-methylcytosine (5-MeC). With a 5-MeC, the most notable change in mutation was the enhancement of G-->A, which occurred 2.5 times relative to a 5'-C. The C8-AP-dG adduct in codon 273 dodecamer sequence with a 5'-C or 5-MeC was also evaluated in human embryonic kidney (293T) cells. Similar to COS cells, targeted mutations doubled with a 5-MeC 5' to the adduct. Except for an increase in G-->C transversions, the results in 293T were similar to that in COS cells. We conclude that C8-AP-dG mutagenesis depends on the type of cell in which it is replicated, the neighboring DNA sequence, and the methylation status of the 5'-C.

Mutagenic potential of DNA–peptide crosslinks mediated by acrolein-derived DNA adducts

Current data suggest that DNA–peptide crosslinks are formed in cellular DNA as likely intermediates in the repair of DNA–protein crosslinks. In addition, a number of naturally occurring peptides are known to efficiently conjugate with DNA, particularly through the formation of Schiff-base complexes at aldehydic DNA adducts and abasic DNA sites. Since the potential role of DNA–peptide crosslinks in promoting mutagenesis is not well elucidated, here we report on the mutagenic properties of Schiff-base-mediated DNA–peptide crosslinks in mammalian cells. Site-specific DNA–peptide crosslinks were generated by covalently trapping a lysine-tryptophan-lysine-lysine peptide to the N 6 position of deoxyadenosine (dA) or the N 2 position of deoxyguanosine (dG) via the aldehydic forms of acrolein-derived DNA adducts (γ-hydroxypropano-dA or γ-hydroxypropano-dG, respectively). In order to evaluate the potential of DNA–peptide crosslinks to promote mutagenesis, we inserted the modified oligodeoxynucleotides into a single-stranded pMS2 shuttle vector, replicated these vectors in simian kidney (COS-7) cells and tested the progeny DNAs for mutations. Mutagenic analyses revealed that at the site of modification, the γ-hydroxypropano-dA-mediated crosslink induced mutations at only ∼0.4%. In contrast, replication bypass of the γ-hydroxypropano-dG-mediated crosslink resulted in mutations at the site of modification at an overall frequency of ∼8.4%. Among the types of mutations observed, single base substitutions were most common, with a prevalence of G to T transversions. Interestingly, while covalent attachment of lysine-tryptophan-lysine-lysine at γ-hydroxypropano-dG caused an increase in mutation frequencies relative to γ-hydroxypropano-dG, similar modification of γ-hydroxypropano-dA resulted in decreased levels of mutations. Thus, certain DNA–peptide crosslinks can be mutagenic, and their potential to cause mutations depends on the site of peptide attachment. We propose that in order to avoid error-prone replication, proteolytic degradation of proteins covalently attached to DNA and subsequent steps of DNA repair should be tightly coordinated.

Genetic effects of oxidative DNA damages: comparative mutagenesis of the imidazole ring-opened formamidopyrimidines (Fapy lesions) and 8-oxo-purines in simian kidney cells

Genetic effects of oxidative DNA damages: comparative mutagenesis of the imidazole ring-opened formamidopyrimidines (Fapy lesions) and 8-oxo-purines in simian kidney cells

Genetic effects of oxidative DNA damages: comparative mutagenesis of the imidazole ring-opened formamidopyrimidines (Fapy lesions) and 8-oxo-purines in simian kidney cells

Fapy·dG and 8-oxo-7,8-dihydro-2′-deoxyguanosine (8-oxo-dG) are formed in DNA by hydroxyl radical damage. In order to study replication past these lesions in cells, we constructed a single-stranded shuttle vector containing the lesion in 5′-TGT and 5′-TGA sequence contexts. Replication of the modified vector in simian kidney (COS-7) cells showed that Fapy·dG is mutagenic inducing primarily targeted Fapy·G→T transversions. In the 5′-TGT sequence mutational frequency of Fapy·dG was ∼30%, whereas in the 5′-TGA sequence it was ∼8%. In parallel studies 8-oxo-dG was found to be slightly less mutagenic than Fapy·dG, though it also exhibited a similar context effect: 4-fold G→T transversions (24% versus 6%) occurred in the 5′-TGT sequence relative to 5′-TGA. To investigate a possible structural basis for the higher G→T mutations induced by both lesions when their 3′ neighbor was T, we carried out a molecular modeling investigation in the active site of DNA polymerase β, which is known to incorporate both dCTP (no mutation) and dATP (G→T substitution) opposite 8-oxo-G. In pol β, the syn-8-oxo-G:dATP pair showed greater stacking with the 3′-T:A base pair in the 5′-TGT sequence compared with the 3′-A:T in the 5′-TGA sequence, whereas stacking for the anti-8-oxo-G:dCTP pair was similar in both 5′-TGT and 5′-TGA sequences. Similarly, syn-Fapy·G:dATP pairing showed greater stacking in the 5′-TGT sequence compared with the 5′-TGA sequence, while stacking for anti-Fapy·G:dCTP pairs was similar in the two sequences. Thus, for both lesions less efficient base stacking between the lesion:dATP pair and the 3′-A:T base pair in the 5′-TGA sequence might cause lower G→T mutational frequencies in the 5′-TGA sequence compared to 5′-TGT. The corresponding lesions derived from 2′-deoxyadenosine, Fapy·dA and 8-oxo-dA, were not detectably mutagenic in the 5′-TAT sequence, and were only weakly mutagenic (<1%) in the 5′-TAA sequence context, where both lesions induced targeted A→C transversions. To our knowledge this is the first investigation using extrachromosomal probes containing a Fapy·dG or Fapy·dA site-specifically incorporated, which showed unequivocally that in simian kidney cells Fapy·G→T substitutions occur at a higher frequency than 8-oxo-G→T and that Fapy·dA is very weakly mutagenic, as is 8-oxo-dA.

Pyridine N-oxide derivatives are inhibitory to the human SARS and feline infectious peritonitis coronavirus in cell culture

Objectives: Evaluation of a wide variety of pyridine N-oxide derivatives on their inhibitory activity against feline coronavirus (FIPV strain) and human SARS-CoV (Frankfurt strain-1) in cell culture. Methods: FIPV and SARS-CoV were exposed to confluent Crandel feline kidney (CRFK) and simian kidney (Vero) cell cultures in the presence of serial concentrations of the test compounds. The anti-cytopathic activity of the pyridine N-oxide derivatives was monitored by spectrophotometric analysis. Results and conclusions: A wide variety of pyridine N-oxide derivatives have been found to be inhibitory against feline coronavirus (FIPV strain) and human SARS-CoV (Frankfurt strain-1) in CRFK and simian kidney (Vero) cell cultures, respectively. The oxide part on the pyridine moiety proved indispensable for anti-coronavirus activity. The potency and virus specificity of the pyridine N-oxide derivatives varied depending the nature and specific location of substituents (i.e. alkyl, halogeno, nitro, etc.) on the different parts of the molecule. The most selective compounds were active in the higher microgram per litre range, being non-toxic at 50–100 mg/L. There was a poor structure-antiviral activity relationship (SAR) for the pyridine N-oxide derivatives against Fe-CoV and SARS-CoV. One of the most active and selective compounds was shown to inhibit Fe-CoV replication at the transcriptional level.

Mutagenesis of 8-Oxoguanine Adjacent to an Abasic Site in Simian Kidney Cells: Tandem Mutations and Enhancement of G→T Transversions

Clustered DNA damages are well-established characteristics of ionizing radiation. As a model clustered lesion in the same strand of DNA, we have evaluated the mutagenic potential of 8-oxoguanine (8-oxoG) adjacent to a uracil in simian kidney cells using a phagemid vector. The uracil residue would be excised by the enzyme uracil DNA glycosylase in vivo generating an abasic site (AP site). A solitary uracil in either GUGTC or GTGUC sequence context provided >60% progeny containing GTGTC indicating that dAMP incorporation opposite the AP site or uracil occurred, but a >30% population showed replacement of U by A, C, or G, which suggests that dTMP, dGMP, or dCMP incorporation also occurred, respectively, opposite the AP site. While the preference for targeted base substitutions at the GUG site was T >> C > A > G, the same at the GUC site was T >> A > C > G. We conclude that base incorporation opposite an AP site is sequence-dependent. For 8-oxoG, as compared to 23-24% G-->T mutants from a single 8-oxoG in a TG(8-oxo)T sequence context, the tandem lesions UG(8-oxo)T and TG(8-oxo)U generated approximately 60 and >85% progeny, respectively, that did not contain the TGT sequence. A significant fraction of tandem mutations were detected when uracil was adjacent to 8-oxoG. What we found most interesting is that the total targeted G(8-oxo)-->T transversions that included both single and tandem mutations at the TG(8-oxo)U site was nearly 60% relative to about 30% at the UG(8-oxo)T site. A higher mutational frequency at the TG(8-oxo)U sequence may arise from a change in DNA polymerase that is more error prone. Thermal melting experiments showed that the Tm for the 8-oxoG:C pair in the TG(8-oxo)(AP*) sequence in a 12-mer was lower than the same in a (AP*)G(8-oxo)T 12-mer with deltadeltaG 0.8 kcal/mol (where AP* represents tetrahydrofuran, the model abasic site). When the 8-oxoG:C pair in each sequence was compared with a 8-oxoG:A pair, the former was found to be more stable than the latter. The preference for C over A opposite 8-oxoG for the (AP*)G(8-oxo)T 12-mer duplex with a deltadeltaG of 1.6 kcal/mol dropped to 0.4 kcal/mol in the TG(8-oxo)(AP*) 12-mer duplex. This suggests that the polymerase discrimination to incorporate dCMP over dAMP would be less efficient in the TG(8-oxo)(AP*) sequence relative to (AP*)G(8-oxo)T. Additionally, the efficiency of recognition and excision of A opposite 8-oxoG by a mismatch repair protein may be impaired in the TG(8-oxo)(AP*) sequence context.

Mutagenic specificity of 2-acetylaminonaphthalene-derived DNA adduct in mammalian cells

2-Acetylaminonaphthalene (2-AAN) has been recognized as a urinary bladder carcinogen in humans. The deacetylated form, 2-aminonaphthalene (2-AN), is metabolized in vivo and reacts primarily with guanine residues in DNA, resulting in the formation of dG- N 2-aminonaphthalene (dG- N 2-AN) adduct. Phosphoramidite chemical procedure has recently been established in our laboratory to prepare oligodeoxynucleotides containing a single dG- N 2-acetylaminonaphthalene (dG- N 2-AAN) adduct. Oligodeoxynucleotides ( 5′TCCTCCTNXCCTCTC, where X is dG or dG- N 2-AAN and N is C, A, T or G) with different bases 5′ flanking to the lesion were prepared and were inserted into a single-strand shuttle vectors and used to establish the mutational frequency and specificity of dG- N 2-AAN adduct in simian kidney cells. dG- N 2-AAN adduct promoted preferential incorporation of dCMP, the correct base, opposite the lesion. When the 5′ flanking base to the lesion was C, A or T, the mutational frequency was under 2.1%. When G flanked to the lesion, the mutational frequency was slightly increased to 4.2%. Misincorporation of dAMP, dTMP, and/or dGMP varied depending on the 5′ flanking base. When dG- N 2-AAN was positioned at codon 61 of noncoding strand of human c-Ha-ras1 gene ( 5′TCCTCCTXGCCTCTC, where X is dG- N 2-AAN), the mutational frequency was 6.7%; G → T transversions (4.7%), followed by G → A transition (2.0%), were observed. These results demonstrated that dG- N 2-AAN is a weak mutagenic lesion in mammalian cells. The influence of 5′ flanking sequence context was observed on the mutational frequency and specificity of this adduct.

DNA Adduct of the Mitomycin C Metabolite 2,7-Diaminomitosene Is a Nontoxic and Nonmutagenic DNA Lesion in Vitro and in Vivo

Mitomycin C (MC) is a cytotoxic and mutagenic antitumor agent that alkylates and cross-links DNA. These effects are dependent on reductive bioactivation of MC. 2,7-Diaminomitosene (2,7-DAM) is the major metabolite of MC in tumor cells, generated by the reduction of MC. 2,7-DAM alkylates DNA in the cell in situ, forming an adduct at the N7 position of 2'-deoxyguanosine (2,7-DAM-dG-N7). To determine the biological effects of this adduct, we have synthesized an oligonucleotide containing a single 2,7-DAM-dG-N7 adduct and inserted it into an M13 bacteriophage genome. Replication of this construct in repair-competent Escherichia coli showed that the adduct was only weakly toxic and generated approximately 50% progeny as compared to control. No mutant was isolated after analysis of more than 4000 progeny phages from SOS-induced or uninduced host cells; therefore, we estimate that the mutation frequency of 2,7-DAM-dG-N7 was less than 2 x 10(-4) in E. coli. Subsequently, to determine if this adduct might be mutagenic in mammalian cells, it was incorporated into a single-stranded shuttle phagemid vector, pMS2, and replicated in simian kidney (COS-7) cells. Analysis of the progeny showed that mutational frequency of a site specific 2,7-DAM-dG-N7 was not higher than the spontaneous mutation frequency in simian kidney cells. In parallel experiments in cell free systems, template oligonucleotides containing a single 2,7-DAM-dG-N7 adduct directed selective incorporation of cytosine in the 5'-32P-labeled primer strands opposite the adducted guanine, catalyzed by Klenow (exo-) DNA polymerase. The adducted templates also supported full extension of primer strands by Klenow (exo-) and T7 (exo-) DNA polymerases and partial extension by DNA polymerase eta. The innocuous behavior of the 2,7-DAM-dG-N7 monoadduct in vivo and in vitro is in sharp contrast to that of the toxic MC-dG-N2 monoadduct reported earlier.

Mutagenic Potential of Benzo[a]pyrene-Derived DNA Adducts Positioned in Codon 273 of the Human P53 Gene

Codon 273 ((5)(')CGT) of the human P53 gene is a mutational hot spot for the environmental carcinogen benzo[a]pyrene. We incorporated a single (+)- or (-)-trans-anti-benzo[a]pyrene diol epoxide (BPDE) DNA adduct at the second position of codon 273 of the human P53 gene and explored the mutagenic potential of this lesion in mammalian cells. Oligodeoxyribonucleotides ((5)(')GAGGTGCG(BPDE)TGTTTGT) modified with (+)- or (-)-trans-dG-N(2)-BPDE were incorporated into single-stranded shuttle vectors and transfected into simian kidney cells. Progeny plasmids were then used to transform Escherichia coli DH10B. Transformants were analyzed by oligodeoxynucleotide hybridization and DNA sequence analysis to establish the mutation frequency and spectrum produced by the adducted base. We determined the mutational frequencies associated with (+)-trans-dG-N(2)-BPDE and (-)-trans-dG-N(2)-BPDE adduction to be 26.5% and 17.5%, respectively. The predominant mutations generated by both stereoisomers were G --> T transversions, with some G --> A transitions. When the cytosine 5' to dG-N(2)-BPDE was replaced by 5-methylcytosine, the mutational frequencies of (+)-trans-dG-N(2)-BPDE and (-)-trans-dG-N(2)-BPDE were reduced to 11.1% and 10.6%, respectively, while the mutational specificity remained unchanged. Thus, the mutational "hot spot" at codon 273 in P53 may reflect either sequence-specific reactivity of BPDE and/or inefficient repair of BPDE-DNA adducts positioned at this site.