RESUMO
BACKGROUND: Although CD4+ memory T cells are considered the primary latent reservoir for HIV-1, replication competent HIV has been detected in tissue macrophages in both animal and human studies. During in vitro HIV infection, the depleted nucleotide pool and high dUTP levels in monocyte derived macrophages (MDM) leads to proviruses with high levels of dUMP, which has been implicated in viral restriction or reduced transcription depending on the uracil base excision repair (UBER) competence of the macrophage. Incorporated dUMP has also been detected in viral DNA from circulating monocytes (MC) and alveolar macrophages (AM) of HIV infected patients on antiretroviral therapy (ART), establishing the biological relevance of this phenotype but not the replicative capacity of dUMP-containing proviruses. RESULTS: As compared to in vitro differentiated MDM, AM from normal donors had sixfold lower levels of dTTP and a sixfold increased dUTP/dTTP, indicating a highly restrictive dNTP pool for reverse transcription. Expression of uracil DNA glycosylase (UNG) was eightfold lower in AM compared to the already low levels in MDM. Accordingly, ~ 80% of HIV proviruses contained dUMP, which persisted for at least 14-days due to low UNG excision activity. Unlike MDM, AM expression levels of UNG and SAM and HD domain containing deoxynucleoside triphosphate triphosphohydrolase 1 (SAMHD1) increased over 14 days post-HIV infection, while dUTP nucleotidohydrolase (DUT) expression decreased. These AM-specific effects suggest a restriction response centered on excising uracil from viral DNA copies and increasing relative dUTP levels. Despite the restrictive nucleotide pools, we detected rare replication competent HIV in AM, peripheral MC, and CD4+ T cells from ART-treated donors. CONCLUSIONS: These findings indicate that the potential integration block of incorporated dUMP is not realized during in vivo infection of AM and MC due to the near absence of UBER activity. In addition, the increased expression of UNG and SAMHD1 in AM post-infection is too slow to prevent integration. Accordingly, dUMP persists in integrated viruses, which based on in vitro studies, can lead to transcriptional silencing. This possible silencing outcome of persistent dUMP could promote viral latency until the repressive effects of viral dUMP are reversed.
Assuntos
Infecções por HIV , HIV-1 , DNA Viral/genética , HIV-1/fisiologia , Humanos , Macrófagos Alveolares , Monócitos/metabolismo , Nucleotídeos/metabolismo , Proteína 1 com Domínio SAM e Domínio HD/metabolismo , Uracila/metabolismo , Uracila-DNA Glicosidase/metabolismo , Replicação ViralRESUMO
Previous short-hairpin RNA knockdown studies have established that depletion of human uracil DNA glycosylase (hUNG) sensitizes some cell lines to 5-fluorodeoxyuridine (FdU). Here, we selectively inhibit the catalytic activity of hUNG by lentiviral transduction of uracil DNA glycosylase inhibitor protein into a large panel of cancer cell lines under control of a doxycycline-inducible promoter. This induced inhibition strategy better assesses the therapeutic potential of small-molecule targeting of hUNG. In total, 6 of 11 colorectal lines showed 6- to 70-fold increases in FdU potency upon hUNG inhibition ("responsive"). This hUNG-dependent response was not observed with fluorouracil (FU), indicating that FU does not operate through the same DNA repair mechanism as FdU in vitro. Potency of the thymidylate synthase inhibitor raltitrexed (RTX), which elevates deoxyuridine triphosphate levels, was only incrementally enhanced upon hUNG inhibition (<40%), suggesting that responsiveness is associated with incorporation and persistence of FdU in DNA rather than deoxyuridine. The importance of FU/A and FU/G lesions in the toxicity of FdU is supported by the observation that dT supplementation completely rescued the toxic effects of U/A lesions resulting from RTX, but dT only increased the IC50 for FdU, which forms both FU/A and FU/G mismatches. Contrary to previous reports, cellular responsiveness to hUNG inhibition did not correlate with p53 status or thymine DNA glycosylase expression. A model is suggested in which the persistence of FU/A and FU/G base pairs in the absence of hUNG activity elicits an apoptotic DNA damage response in both responsive and nonresponsive colorectal lines. SIGNIFICANCE STATEMENT: The pyrimidine base 5-fluorouracil is a mainstay chemotherapeutic for treatment of advanced colorectal cancer. Here, this study shows that its deoxynucleoside form, 5-fluorodeoxyuridine (FdU), operates by a distinct DNA incorporation mechanism that is strongly potentiated by inhibition of the DNA repair enzyme human uracil DNA glycosylase. The hUNG-dependent mechanism was present in over 50% of colorectal cell lines tested, suggesting that a significant fraction of human cancers may be sensitized to FdU in the presence of a small-molecule hUNG inhibitor.
Assuntos
Antineoplásicos/farmacologia , Neoplasias Colorretais/patologia , Floxuridina/farmacologia , Fluoruracila/farmacologia , Quinazolinas/farmacologia , Tiofenos/farmacologia , Uracila-DNA Glicosidase/antagonistas & inibidores , Linhagem Celular Tumoral , Dano ao DNA , Ensaios de Seleção de Medicamentos Antitumorais , Humanos , Uracila-DNA Glicosidase/metabolismoRESUMO
dUTP is a close structural congener of dTTP and can be readily incorporated into DNA opposite to adenine during DNA replication leading to non-mutagenic dU/A base pairs ('uracilation'). We find that dU/A pairs located within DNA transcriptional templates optimized for either T7 RNA polymerase (T7 RNAP) or human RNA polymerase II (pol II) have inhibitory and mutagenic effects on transcription. The data for T7 RNAP establishes that even a single dU/A pair can inhibit promoter binding and transcription initiation up to 30-fold, and that inhibitory effects on transcription elongation are also possible. Sequencing of the mRNA transcribed from uniformly uracilated DNA templates by T7 RNAP indicated an increased frequency of transversion and insertion mutations compared to all T/A templates. Strong effects of dU/A pairs on cellular transcription activity and fidelity were also observed with RNA pol II using uracil base excision repair (UBER)-deficient human cells. At the highest levels of template uracilation, transcription by RNA pol II was completely blocked. We propose that these effects arise from the decreased thermodynamic stability and increased dynamics of dU/A pairs in DNA. The potential implications of these findings on gene regulation and disease are discussed.
Assuntos
Reparo do DNA , RNA Polimerases Dirigidas por DNA/genética , DNA/genética , Desoxiuridina/metabolismo , RNA Polimerase II/genética , RNA/genética , Transcrição Gênica , Proteínas Virais/genética , Pareamento de Bases , Sequência de Bases , Linhagem Celular Tumoral , DNA/metabolismo , Replicação do DNA , RNA Polimerases Dirigidas por DNA/metabolismo , Fibroblastos/citologia , Fibroblastos/metabolismo , Humanos , Cinética , Mutação , Regiões Promotoras Genéticas , Ligação Proteica , RNA/metabolismo , RNA Polimerase II/metabolismo , Termodinâmica , Proteínas Virais/metabolismoRESUMO
BACKGROUND: Inflammation plays a major role in the pulmonary artery hypertension (PAH) and the acute lung injury (ALI) diseases. The common feature of these complications is the dysfunction of pulmonary microvascular endothelial cells (PMVECs). Fasudil, the only Rho kinase (ROCK) inhibitor used in clinic, has been proved to be the most promising new drug for the treatment of PAH, with some anti-inflammatory activity. Therefore, in the present study, the effect of fasudil on lipopolysaccharide (LPS)-induced inflammatory injury in rat PMVECs was investigated. METHODS: LPS was used to make inflammatory injury model of rat PMVECs. Thereafter, the mRNA and protein expression of pro-inflammatory factors was evaluated by reverse transcription-polymerase chain reaction (RT-PCR) and enzyme-linked immunosorbent assay (ELISA) assay respectively. Intracellular reactive oxygen species (ROS) levels were measured by the confocal laser scanning system. The activities of superoxide dismutase (SOD), glutathione peroxidase (GSH-Px) and the content of malondialdehyde (MDA) were determined by using commercial kits according to the manufacturer's instructions. Western blot assay was used to detect the protein expression of nuclear factor kappa B (NF-κB) p65. RESULTS: Fasudil effectively prevented inflammatory injury induced by LPS, which is manifested by the decrease of pro-inflammatory cytokines interleukin-6 (IL-6) and monocyte chenotactic protein-1 (MCP-1). Meanwhile, fasudil dramatically reduced the levels of ROS and MDA, and also elevated the activities of SOD and GSH-Px. Furthermore, the nuclear translocation of NF-κB p65 induced by LPS was also suppressed by fasudil. Additionally, the ROS scavengers N-Acetylcysteine (N-Ace) was also found to inhibit the nuclear translocation of NF-κB and the mRNA expression of IL-6 and MCP-1 induced by LPS, which suggested that ROS was essential for the nuclear translocation of NF-κB. CONCLUSIONS: The present study revealed that fasudil reduced the expression of inflammatory factors, alleviated the inflammatory and oxidative damage induced by LPS in rat PMVECs via ROS-NF-κB signaling pathway.
Assuntos
Lipopolissacarídeos , NF-kappa B , 1-(5-Isoquinolinasulfonil)-2-Metilpiperazina/análogos & derivados , Animais , Células Endoteliais , Interleucina-6/genética , Interleucina-6/metabolismo , Lipopolissacarídeos/toxicidade , NF-kappa B/metabolismo , Inibidores de Proteínas Quinases/farmacologia , Inibidores de Proteínas Quinases/uso terapêutico , RNA Mensageiro/metabolismo , Ratos , Espécies Reativas de Oxigênio/metabolismo , Superóxido Dismutase/metabolismo , Quinases Associadas a rho/metabolismo , Quinases Associadas a rho/farmacologiaRESUMO
Non-dividing cells of the myeloid lineage such as monocytes and macrophages are target cells of HIV that have low dNTP pool concentrations and elevated levels of dUTP, which leads to frequent incorporation of dUMP opposite to A during reverse transcription ("uracilation"). One factor determining the fate of dUMP in proviral DNA is the host cell uracil base excision repair (UBER) system. Here we explore the relative UBER capacity of monocytes (MC) and monocyte-derived macrophages (MDM) and the fate of integrated uracilated viruses in both cell types to understand the implications of viral dUMP on HIV diversification and infectivity. We find that the kinetics for MC infection is compatible with their lifetime in vivo and their near absence of hUNG2 activity is consistent with the retention of viral dUMP at high levels at least until differentiation into macrophages, where UBER becomes possible. Overexpression of human uracil DNA glycosylase in MDM prior to infection resulted in rapid removal of dUMP from HIV cDNA and near complete depletion of dUMP-containing viral copies. This finding establishes that the low hUNG2 expression level in these cells limits UBER but that hUNG2 is restrictive against uracilated viruses. In contrast, overexpression of hUNG2 after viral integration did not accelerate the excision of uracils, suggesting that they may poorly accessible in the context of chromatin. We found that viral DNA molecules with incorporated dUMP contained unique (+) strand transversion mutations that were not observed when dUMP was absent (GâT, TâA, TâG, AâC). These observations and other considerations suggest that dUMP introduces errors predominantly during (-) strand synthesis when the template is RNA. Overall, the likelihood of producing a functional virus from in vitro infection of MC is about 50-fold and 300-fold reduced as compared to MDM and activated T cells. The results implicate viral dUMP incorporation in MC and MDM as a potential viral diversification and restriction pathway during human HIV infection.
Assuntos
Reparo do DNA , Infecções por HIV/genética , Macrófagos/virologia , Monócitos/virologia , Provírus/genética , Uracila/metabolismo , DNA Viral/genética , Nucleotídeos de Desoxiuracil/deficiência , Nucleotídeos de Desoxiuracil/metabolismo , HIV-1/genética , Humanos , Uracila-DNA Glicosidase/metabolismoRESUMO
BACKGROUND: Porcine reproductive and respiratory syndrome (PRRS) viruses are a major cause of disease and economic loss in pigs worldwide. High genetic diversity among PRRSV strains is problematic for successful disease control by vaccination. Mosaic DNA and vaccinia (VACV) vaccines were developed in order to improve protection against heterologous PRRSV strains. METHODS: Piglets were primed and boosted with GP5-Mosaic DNA vaccine and recombinant GP5-Mosaic VACV (rGP5-Mosaic VACV), respectively. Pigs vaccinated with rGP5-WT (VR2332) DNA and rGP5-WT VACV, or empty vector DNA and empty VACV respectively, served as controls. Virus challenge was given to separate groups of vaccinated pigs with VR2332 or MN184C. Necropsies were performed 14 days after challenge. RESULTS: Vaccination with the GP5-Mosaic-based vaccines resulted in cellular reactivity and higher levels of neutralizing antibodies to both VR2332 and MN184C PRRSV strains. In contrast, vaccination of animals with the GP5-WT vaccines induced responses only to VR2332. Furthermore, vaccination with the GP5-Mosaic based vaccines resulted in protection against challenge with two heterologous virus strains, as demonstrated by the significantly lower viral loads in serum, tissues, porcine alveolar macrophages (PAMs), and bronchoalveolar lavage (BAL) fluids, and less severe lung lesions after challenge with either MN184C or VR2332, which have only 85% identity. In contrast, significant protection by the GP5-WT based vaccines was only achieved against the VR2332 strain. Conclusions: GP5-Mosaic vaccines, using a DNA-prime/VACV boost regimen, conferred protection in pigs against heterologous viruses.
RESUMO
[This corrects the article DOI: 10.1371/journal.pone.0208801.].
RESUMO
Porcine reproductive and respiratory syndrome virus (PRRSV), is a highly mutable RNA virus that affects swine worldwide and its control is very challenging due to its formidable heterogeneity in the field. In the present study, DNA vaccines constructed with PRRSV GP5-Mosaic sequences were complexed to cationic liposomes and administered to experimental pigs by intradermal and intramuscular injection, followed by three boosters 14, 28 and 42 days later. The GP5-Mosaic vaccine thus formulated was immunogenic and induced protection from challenge in vaccinated pigs comparable to that induced by a wild type (VR2332) GP5 DNA vaccine (GP5-WT). Periodic sampling of blood and testing of vaccine-induced responses followed. Interferon-γ (IFN-γ) mRNA expression by virus-stimulated peripheral blood mononuclear cells (PBMCs) of GP5-Mosaic-vaccinated pigs was significantly higher compared to pigs vaccinated with either GP5-WT or empty vector at 21, 35 and 48 days after vaccination. Cross-reactive cellular responses were also demonstrated in GP5-Mosaic vaccinated pigs after stimulation of PBMCs with divergent strains of PRRSV. Thus, significantly higher levels of IFN-γ mRNA were detected when PBMCs from GP5-Mosaic-vaccinated pigs were stimulated by four Genotype 2 strains (VR2332, NADC9, NADC30 and SDSU73), which have at least 10% difference in GP5 amino acid sequences, while such responses were recorded only upon VR2332 stimulation in GP5-WT-vaccinated pigs. In addition, the levels of virus-specific neutralizing antibodies were higher in GP5-Mosaic or GP5-WT vaccinated pigs than those in vector-control pigs. The experimental pigs vaccinated with either the GP5-Mosaic vaccine or the GP5-WT vaccine were partially protected from challenge with VR2332, as measured by significantly lower viral loads in sera and tissues and lower lung lesion scores than the vector control group. These data demonstrate that the GP5-Mosaic vaccine can induce cross-reactive cellular responses to diverse strains, neutralizing antibodies, and protection in pigs.
Assuntos
Vírus da Síndrome Respiratória e Reprodutiva Suína/imunologia , Vacinas Virais/imunologia , Animais , Anticorpos Neutralizantes/imunologia , Interferon gama/metabolismo , Leucócitos Mononucleares/metabolismo , Síndrome Respiratória e Reprodutiva Suína , Vírus da Síndrome Respiratória e Reprodutiva Suína/patogenicidade , RNA Mensageiro/metabolismo , Suínos , Proteínas Virais/imunologia , Vacinas Virais/uso terapêuticoRESUMO
BACKGROUND: It has been recognized that the expression of type I interferon (IFNα/ß) may be suppressed during infection with porcine reproductive, respiratory syndrome virus (PRRSV). This causes profound negative effects on both the innate and adaptive immunity of the host resulting in persistence of infection. OBJECTIVE: Test the effects of PRRSV infection of porcine alveolar macrophages (PAMs), the main target cell, on the expression of interferon beta (IFNß) and downstream signaling events. METHODS: In order to examine those effects, PAMs harvested from lungs of healthy PRRSV-free animals were infected with virulent, attenuated, infectious clone-derived chimeric viruses, or field PRRS virus strains. Culture supernatants from the infected PAMs were tested for IFNß protein expression by means of indirect ELISA and for bioactivity by a vesicular stomatitis virus plaque reduction assay. The expression of the Mx protein was assayed to ascertain signaling events. RESULTS: These experiments demonstrated that PRRSV does induce variably, the expression of bioactive IFNß protein in the natural host cell. To further elucidate the effects of PRRSV infection on IFNß signaling, Mx-1 an interferon stimulated gene (ISG), was also tested for expression. Interestingly, Mx-1 expression by infected PAMs generally correlated with IFNß production. CONCLUSION: The results of this study demonstrate that the induction of IFNß and signaling in PAMs after PRRSV infection is variable.
Assuntos
Regulação da Expressão Gênica , Interações Hospedeiro-Patógeno , Interferon beta/genética , Macrófagos Alveolares/imunologia , Síndrome Respiratória e Reprodutiva Suína/imunologia , Vírus da Síndrome Respiratória e Reprodutiva Suína/imunologia , Transdução de Sinais , Animais , Ensaio de Imunoadsorção Enzimática , Regulação da Expressão Gênica/imunologia , Interferon beta/metabolismo , Pulmão/virologia , Macrófagos Alveolares/virologia , Proteínas de Resistência a Myxovirus/genética , SuínosRESUMO
OBJECTIVE: To study the effect of drought stress on the growth of Glycyrrhiza uralensis and its drought resistance characteristic. METHOD: The growth in dicators of G. uralensis including leaf, stem, root and biomass were measured when two-years-old G. uralensis had lived in drought stress soil for 60 days. RESULT: The growth of all organs of G. uralensis was restrained because of drought stress, especially to up ground organs. CONCLUSION: It is obvious that the restraining effect of drought stress on G. uralensis possesses organ speciality. Both yield and quality of G. uralensis will be satisfied when it grows in the soil with relative water content of 50%.
Assuntos
Desastres , Glycyrrhiza uralensis/crescimento & desenvolvimento , Plantas Medicinais/crescimento & desenvolvimento , Glycyrrhiza uralensis/anatomia & histologia , Componentes Aéreos da Planta/anatomia & histologia , Componentes Aéreos da Planta/crescimento & desenvolvimento , Raízes de Plantas/anatomia & histologia , Raízes de Plantas/crescimento & desenvolvimento , Plantas Medicinais/anatomia & histologiaRESUMO
Cell migration is central to a number of normal and disease processes. Small organic molecules that inhibit cell migration have potential as both research probes and therapeutic agents. We have identified two tetrahydroisoquinoline natural product analogs with antimigratory activities on Madin-Darby canine kidney epithelial cells: a semisynthetic derivative of quinocarmycin (also known as quinocarcin), DX-52-1, and a more complex synthetic molecule, HUK-921, related to the naphthyridinomycin family. It has been assumed that the cellular effects of reactive tetrahydroisoquinolines result from the alkylation of DNA. We have reported previously that the primary target of DX-52-1 relevant to cell migration appears to be the membrane-cytoskeleton linker protein radixin. Here we extend the analysis of the protein targets of DX-52-1, reporting that the multifunctional carbohydrate-binding protein galectin-3 is a secondary target of DX-52-1 that may also be relevant to the antimigratory effects of both DX-52-1 and HUK-921. All known inhibitors of galectin-3 target its beta-galactoside-binding site in the carbohydrate recognition domain. However, we found that DX-52-1 and HUK-921 bind galectin-3 outside of its beta-galactoside-binding site. Intriguingly HUK-921, although a less potent inhibitor of cell migration than DX-52-1, had far greater selectivity for galectin-3 over radixin, exhibiting little binding to radixin, both in vitro and in cells. Overexpression of galectin-3 in cells led to a dramatic increase in cell adhesion on different extracellular matrix substrata as well as changes in cell-cell adhesion and cell motility. Galectin-3-overexpressing cells had greatly reduced sensitivity to DX-52-1 and HUK-921, and these compounds caused a change in localization of the overexpressed galectin-3 and reversion of the cells to a more normal morphology. The converse manipulation, RNA interference-based silencing of galectin-3 expression, resulted in reduced cell-matrix adhesion and cell migration. In aggregate, the data suggest that DX-52-1 and HUK-921 inhibit a carbohydrate binding-independent function of galectin-3 that is involved in cell migration.
Assuntos
Movimento Celular/efeitos dos fármacos , Galectina 3/antagonistas & inibidores , Isoquinolinas/farmacologia , Alquilação/efeitos dos fármacos , Animais , Sítios de Ligação/fisiologia , Adesão Celular/efeitos dos fármacos , Linhagem Celular , Proteínas do Citoesqueleto/antagonistas & inibidores , Proteínas do Citoesqueleto/genética , Proteínas do Citoesqueleto/metabolismo , DNA/metabolismo , Cães , Galectina 3/biossíntese , Galectina 3/genética , Expressão Gênica , Humanos , Isoquinolinas/química , Proteínas de Membrana/antagonistas & inibidores , Proteínas de Membrana/genética , Proteínas de Membrana/metabolismo , Interferência de RNA , RNA Interferente Pequeno/genéticaRESUMO
Using a fluidized bed as immobilization system, mixed culture methanotrophic attached-films were developed on diatomite particles. The Methane Monooxygenase (MMO) activity was found to increase obviously as soon as the lag phase ended. Greater than 90% of the MMO activity in the bed was attached. Biofilm concentration of 3.3-3.7 mg dry weight cell/g DS was observed. Batch experiments were performed to explore the possibility of producing epoxypropane by a cooxidation process. The effect of methane on the oxidation of propene to epoxypropane and the effect of propene on the growth of methanotroph were also studied. In continuous experiments, optimum mixed gaseous substrates (methane: 35%; propene: 20%; oxygen: 45%) were continuously circulated through the fluidized bed reactor to remove product. Initial epoxypropane productivity was 110-150 mumol/d. The bioreactor operated continuously for 25 d without obvious loss of epoxypropane productivity.
Assuntos
Biofilmes/crescimento & desenvolvimento , Compostos de Epóxi/metabolismo , Methylococcaceae/enzimologia , Methylosinus/enzimologia , Oxigenases/metabolismo , Adesinas Bacterianas/fisiologia , Reatores Biológicos/microbiologia , Células Imobilizadas/efeitos dos fármacos , Células Imobilizadas/enzimologia , Células Imobilizadas/microbiologia , Metano/metabolismo , Metano/farmacologia , Methylococcaceae/efeitos dos fármacos , Methylococcaceae/crescimento & desenvolvimento , Methylosinus/efeitos dos fármacos , Methylosinus/crescimento & desenvolvimento , Oxirredução , Propano/metabolismo , Propano/farmacologiaRESUMO
Commercial Candida rugosa lipase has been separated into two distinct fractions (CRLA and CRLB) by anion-exchange chromatography. As analyzed on SDS-polyacrylamide gel electrophoresis, CRLA and CRLB are homogenous. At high ionic strength, CRLA and CRLB have similar hydrophobicity and UV spectra, suggesting that the open extent of the large hydrophobic pockets of CRLA and CRLB may be similar. At low ionic strength, using "hydrophobic interfacial affinity chromatography", both CRLA and CRLB have been separated into four isofractions. They have different hydrophobicity and UV spectra, suggesting that the open extent of the large hydrophobic pocket of the four forms may be different. Further, the conversion of CRL isoenzymes in the process of organic solvent treatment and ester hydrolysis were examined. The results clearly showed not only that CRLB had been converted to CRLA, but also that CRLA sub-fractions with different open extent of large hydrophobic pocket had been converted
Assuntos
Candida/enzimologia , Lipase/química , Lipase/isolamento & purificação , 2-Propanol/farmacologia , Álcoois/farmacologia , Fracionamento Químico/métodos , Hidrólise , Focalização Isoelétrica/métodos , Isoenzimas/análise , Isoenzimas/química , Isoenzimas/isolamento & purificação , Lipase/análiseRESUMO
Particulate methane monooxygenase (pMMO) has been exfoliated and isolated from membranes of the Methylosinus trichosporium IMV 3011. It appears that the stability of pMMO in the exfoliation process is increased with increasing copper concentration in the growth medium, but extensive intracytoplasmic membrane formed under higher copper concentration may inhibit the exfoliation of active pMMO from membrane. The highest total activity of purified pMMO is obtained with an initial concentration of 6 microM Cu in the growth medium. The purified MMO contains only copper and does not utilize NADH as electron donor. Treatment of purified pMMO with EDTA resulted in little change in copper level, suggesting that the copper in the pMMO is tightly bound with pMMO.