Harpagophytum procumbens Extract Ameliorates Allodynia and Modulates Oxidative and Antioxidant Stress Pathways in a Rat Model of Spinal Cord Injury.

Spinal cord injury (SCI) is a deliberating disorder with impairments in locomotor deficits and incapacitating sensory abnormalities. Harpagophytum procumbens (Hp) is a botanical widely used for treating inflammation and pain related to various inflammatory and musculoskeletal conditions. Using a modified rodent contusion model of SCI, we explored the effects of this botanical on locomotor function and responses to mechanical stimuli, and examined possible neurochemical changes associated with SCI-induced allodynia. Following spinal cord contusion at T10 level, Hp (300 mg/kg, p.o.) or vehicle (water) was administered daily starting 24 h post-surgery, and behavioral measurements made every-other day until sacrifice (Day 21). Hp treatment markedly ameliorated the contusion-induced decrease in locomotor function and increased sensitivity to mechanical stimuli. Determination of Iba1 expression in spinal cord tissues indicated microglial infiltration starting 3 days post-injury. SCI results in increased levels of 4-hydroxynonenal, an oxidative stress product and proalgesic, which was diminished at 7 days by treatment with Hp. SCI also enhanced antioxidant heme oxygenase-1 (HO-1) expression. Concurrent studies of cultured murine BV-2 microglial cells revealed that Hp suppressed oxidative/nitrosative stress and inflammatory responses, including production of nitric oxide and reactive oxygen species, phosphorylation of cytosolic phospholipases A2, and upregulation of the antioxidative stress pathway involving the nuclear factor erythroid 2-related factor 2 and HO-1. These results support the use of Hp for management of allodynia by providing resilience against the neuroinflammation and pain associated with SCI and other neuropathological conditions.

An Investigation into the Immunomodulatory Activities of Sutherlandia frutescens in Healthy Mice.

Sutherlandia frutescens is a medicinal plant that has been traditionally used in southern Africa for cancers, infections, and inflammatory conditions. We recently published experiments demonstrating that an aqueous extract of S. frutescens possessed potent immune-stimulatory activity. This work was carried out with murine macrophages, an immune cell type that plays a pivotal role in host defense from infection and in shaping host inflammatory and immune responses. Here, we conducted a series of follow-up experiments to explore the impact of consuming S. frutescens on host response to bacterial challenge using healthy mice. We found that feeding mice a diet containing S. frutescens failed to significantly alter host response to systemic infection by either a gram-positive or gram-negative bacterium (i.e., L. monocytogenes and E. coli, respectively). In contrast to the in vitro observations, we found no evidence that S. frutescens consumption stimulated in vivo inflammatory responses; instead, consumption of S. frutescens tended to diminish in vivo inflammatory responses. Several possible reasons for this are discussed.

Phytochemicals and botanical extracts regulate NF-κB and Nrf2/ARE reporter activities in DI TNC1 astrocytes.

The increase in oxidative stress and inflammatory responses associated with neurodegenerative diseases has drawn considerable attention towards understanding the transcriptional signaling pathways involving NF-κB (nuclear factor kappa-light-chain-enhancer of activated B cells) and Nrf2 (Nuclear Factor Erythroid 2-like 2). Our recent studies with immortalized murine microglial cells (BV-2) demonstrated effects of botanical polyphenols to inhibit lipopolysaccharide (LPS)-induced nitric oxide (NO) and enhance Nrf2-mediated antioxidant responses (Sun et al., 2015). In this study, an immortalized rat astrocyte (DI TNC1) cell line expressing a luciferase reporter driven by the NF-κB or the Nrf2/Antioxidant Response Element (ARE) promoter was used to assess regulation of these two pathways by phytochemicals such as quercetin, rutin, cyanidin, cyanidin-3-O-glucoside, as well as botanical extracts from Withania somnifera (Ashwagandha), Sutherlandia frutescens (Sutherlandia) and Euterpe oleracea (Açaí). Quercetin effectively inhibited LPS-induced NF-κB reporter activity and stimulated Nrf2/ARE reporter activity in DI TNC1 astrocytes. Cyanidin and the glycosides showed similar effects but only at much higher concentrations. All three botanical extracts effectively inhibited LPS-induced NF-κB reporter activity. These extracts were capable of enhancing ARE activity by themselves and further enhanced ARE activity in the presence of LPS. Quercetin and botanical extracts induced Nrf2 and HO-1 protein expression. Interestingly, Ashwagandha extract was more active in inducing Nrf2 and HO-1 expression in DI TNC1 astrocytes as compared to Sutherlandia and Açaí extracts. In summary, this study demonstrated NF-kB and Nrf2/ARE promoter activities in DI TNC1 astrocytes, and further showed differences in ability for specific botanical polyphenols and extracts to down-regulate LPS-induced NF-kB and up-regulate the NRF2/ARE activities in these cells.

Inhibition of Gli/hedgehog signaling in prostate cancer cells by "cancer bush" Sutherlandia frutescens extract.

Sutherlandia frutescens is a medicinal plant, traditionally used to treat various types of human diseases, including cancer. Previous studies of several botanicals link suppression of prostate cancer growth with inhibition of the Gli/hedgehog (Gli/Hh) signaling pathway. Here we hypothesized the anti-cancer effect of S. frutescens was linked to its inhibition of the Gli/Hh signaling in prostate cancer. We found a dose- and time-dependent growth inhibition in human prostate cancer cells, PC3 and LNCaP, and mouse prostate cancer cell, TRAMP-C2, treated with S. frutescens methanol extract (SLE). We also observed a dose-dependent inhibition of the Gli-reporter activity in Shh Light II and TRAMP-C2QGli cells treated with SLE. In addition, SLE can inhibit Gli/Hh signaling by blocking Gli1 and Ptched1 gene expression in the presence of a Gli/Hh signaling agonist (SAG). A diet supplemented with S. frutescens suppressed the formation of poorly differentiated carcinoma in prostates of TRAMP mice. Finally, we found Sutherlandioside D was the most potent compound in the crude extract that could suppress Gli-reporter in Shh Light II cells. Together, this suggests that the S. frutescens extract may exert anti-cancer effect by targeting Gli/Hh signaling, and Sutherlandioside D is one of the active compounds.

Inhibition of Hedgehog-Signaling Driven Genes in Prostate Cancer Cells by Sutherlandia frutescens Extract.

Sutherlandia frutescens (L) R. Br. (Sutherlandia) is a South African botanical that is traditionally used to treat a variety of health conditions, infections and diseases, including cancer. We hypothesized Sutherlandia might act through Gli/ Hedgehog (Hh)-signaling in prostate cancer cells and used RNA-Seq transcription profiling to profile gene expression in TRAMPC2 murine prostate cancer cells with or without Sutherlandia extracts. We found 50% of Hh-responsive genes can be repressed by Sutherlandia ethanol extract, including the canonical Hh-responsive genes Gli1 and Ptch1 as well as newly distinguished Hh-responsive genes Hsd11b1 and Penk.

Unveiling the anti-inflammatory activity of Sutherlandia frutescens using murine macrophages.

Sutherlandia frutescens is a botanical widely used in southern Africa for treatment of inflammatory and other conditions. Previously, an ethanolic extract of S. frutescens (SFE) has been shown to inhibit the production of reactive oxygen species (ROS) and nitric oxide (NO) by murine neurons and a microglia cell line (BV-2 cells). In this study we sought to confirm the anti-inflammatory activities of SFE on a widely used murine macrophage cell line (i.e., RAW 264.7 cells) and primary mouse macrophages. Furthermore, experiments were conducted to investigate the anti-inflammatory activity of the flavonol and cycloartanol glycosides found in high quantities in S. frutescens. While the SFE exhibited anti-inflammatory activities upon murine macrophages similar to that reported with the microglia cell line, this effect does not appear to be mediated by sutherlandiosides or sutherlandins. In contrast, chlorophyll in our extracts appeared to be partly responsible for some of the activity observed in our macrophage-dependent screening assay.

Immuno-stimulatory activity of a polysaccharide-enriched fraction of Sutherlandia frutescens occurs by the toll-like receptor-4 signaling pathway.

ETHNOPHARMACOLOGICAL RELEVANCE: Sutherlandia frutescens (L.) R. Br. is an indigenous plant of southern Africa that has been traditionally used for various cancers, infections, and inflammatory conditions. AIM OF THE STUDY: Our aim was to investigate the potential immuno-stimulatory activity of a polysaccharide-enriched fraction (SFPS) from a decoction of S. frutescens. MATERIALS AND METHODS: RAW 264.7 cells (a murine macrophage cell line) were used to determine the activities of SFPS on macrophage function. The production of reactive oxygen species (ROS), nitric oxide (NO), and inflammatory cytokines were evaluated in the cells treated with or without SFPS. CLI-095, a toll-like receptor (TLR) 4-specific inhibitor, was used to identify whether or not SFPS exerts its effects through TLR4. An antagonist of endotoxin, polymyxin B, was used to evaluate whether endotoxin present in SFPS contributed to its immune-stimulatory activity. RESULTS: SFPS exhibited potent immune-stimulatory activity by macrophages. The production of ROS, NO, and tumor necrosis factor (TNF-α) were increased upon exposure to SFPS in a dose-dependent manner. All of these activities were completely blocked by co-treatment with CLI-095, but only partially diminished by polymyxin B. CONCLUSION: We demonstrate for the first time potent immune-stimulatory activity in a decoction prepared from S. frutescens. We believe that this immune stimulatory activity is due, in part, to the action of polysaccharides present in the decoction that acts by way of TLR4 receptors and the nuclear factor kappa-light-chain-enhancer of activated B cells (NF-κB) signaling pathway. These findings provide a plausible mechanism through which we can understand some of the medicinal properties of S. frutescens.

Antioxidant potential of Sutherlandia frutescens and its protective effects against oxidative stress in various cell cultures.

BACKGROUND: Sutherlandia frutescens (L.) R.Br. (SF) is a South African plant that is widely used to treat stress, infections, cancer, and chronic diseases, many of which involve oxidative stress. The aim of the study was to quantitatively assess the antioxidant potential of SF extracts in cell-free system as well as in cell lines. METHODS: Dried SF vegetative parts were extracted using six different solvents, and the extracts were assessed for total phenolic and flavonoid contents, total reducing power, iron chelating capacity, and free radical scavenging power, including, scavenging of hydroxyl radicals, superoxide anions, nitric oxide, and hydrogen peroxide. We further investigated the freeze-dried hot water extract of SF (SFE) to assess its effect against oxidative stress induced by tert-butyl hydroperoxide (t-BHP), an organic peroxide. Three different cell lines: Chinese hamster ovary (CHO), human hepatoma (HepaRG), and human pulmonary alveolar carcinoma (A549) cells, were employed to determine cell viability, intracellular reactive oxygen species (ROS) levels, and reduced to oxidized glutathione levels (GSH/GSSG). RESULTS: The results indicated that: (1) SF extracts have significant antioxidant potential that is dependent upon the nature of the extraction solvent and (2) SFE protects against tBHP-induced oxidative stress in cells by scavenging ROS and preserving intracellular GSH/GSSG. CONCLUSION: Oxidative stress is implicated in a number of disorders, and due to the public's concerns about synthetic antioxidants, various natural antioxidants are being explored for their therapeutic potential. Our findings support claims for S. frutescens being a promising adjunctive therapeutic for oxidative stress-related health problems.

Sutherlandia frutescens ethanol extracts inhibit oxidative stress and inflammatory responses in neurons and microglial cells.

Sutherlandia frutescens (L.) R.Br. (SF) is a medicinal plant indigenous to southern Africa and used in folk and contemporary remedies for stress, chronic diseases, cancer, and HIV/AIDS. While previous studies have focused on physiological effects of SF on cellular and systemic abnormalities associated with these diseases, little is known about its effects in the brain and immune cells in the central nervous system. Results of this study indicate that ethanol extracts of SF (SF-E) suppress NMDA-induced reactive oxygen species (ROS) production in neurons, and LPS- and IFNγ-induced ROS and nitric oxide (NO) production in microglial cells. SF-E's action on microglial cells appears to be mediated through inhibition of the IFNγ-induced p-ERK1/2 signaling pathway which is central to regulating a number of intracellular metabolic processes including enhancing STAT1α phosphorylation and filopodia formation. The involvement of SF in these pathways suggests the potential for novel therapeutics for stress and prevention, and/or treatment of HIV/AIDS as well as other inflammatory diseases in the brain.

Magnolia polyphenols attenuate oxidative and inflammatory responses in neurons and microglial cells.

BACKGROUND: The bark of magnolia has been used in Oriental medicine to treat a variety of remedies, including some neurological disorders. Magnolol (Mag) and honokiol (Hon) are isomers of polyphenolic compounds from the bark of Magnolia officinalis, and have been identified as major active components exhibiting anti-oxidative, anti-inflammatory, and neuroprotective effects. In this study, we investigate the ability of these isomers to suppress oxidative stress in neurons stimulated by the ionotropic glutamate receptor agonist N-methyl-D-aspartate (NMDA) and oxidative and inflammatory responses in microglial cells activated by interferon-γ (IFNγ) and lipopolysaccharide (LPS). We also attempt to elucidate the mechanism and signaling pathways involved in cytokine-induced production of reactive oxygen species (ROS) in microglial cells. METHODS: Dihydroethidium (DHE) was used to assay superoxide production in neurons, while CM-H2DCF-DA was used to test for ROS production in murine (BV-2) and rat (HAPI) immortalized microglial cells. NADPH oxidase inhibitors (for example, diphenyleneiodonium (DPI), AEBSF, and apocynin) and immunocytochemistry targeting p47phox and gp91phox were used to assess the involvement of NADPH oxidase. Western blotting was used to assess iNOS and ERK1/2 expression, and the Griess reaction protocol was employed to determine nitric oxide (NO) concentration. RESULTS: Exposure of Hon and Mag (1-10 µM) to neurons for 24 h did not alter neuronal viability, but both compounds (10 µM) inhibited NMDA-stimulated superoxide production, a pathway known to involve NADPH oxidase. In microglial cells, Hon and Mag inhibited IFNγ±LPS-induced iNOS expression, NO, and ROS production. Studies with inhibitors and immunocytochemical assay further demonstrated the important role of IFNγ activating the NADPH oxidase through the p-ERK-dependent pathway. Hon and, to a lesser extent, Mag inhibited IFNγ-induced p-ERK1/2 and its downstream pathway for ROS and NO production. CONCLUSION: This study highlights the important role of NADPH oxidase in mediating oxidative stress in neurons and microglial cells and has unveiled the role of IFNγ in stimulating the MAPK/ERK1/2 signaling pathway for activation of NADPH oxidase in microglial cells. Hon and Mag offer anti-oxidative or anti-inflammatory effects, at least in part, through suppressing IFNγ-induced p-ERK1/2 and its downstream pathway.

Stimulation of BK virus DNA replication by NFI family transcription factors.

BK polyomavirus (BKV) establishes persistent, low-level, and asymptomatic infections in most humans and causes polyomavirus-associated nephropathy (PVAN) and other pathologies in some individuals. The activation of BKV replication following kidney transplantation, leading to viruria, viremia, and, ultimately, PVAN, is associated with immune suppression as well as inflammation and stress from ischemia-reperfusion injury of the allograft, but the stimuli and molecular mechanisms leading to these pathologies are not well defined. The replication of BKV DNA in cell cultures is regulated by the viral noncoding control region (NCCR) comprising the core origin and flanking sequences, to which BKV T antigen (Tag), cellular proteins, and small regulatory RNAs bind. Six nuclear factor I (NFI) binding sites occur in sequences flanking the late side of the core origin (the enhancer) of the archetype virus, and their mutation, either individually or in toto, reduces BKV DNA replication when placed in competition with templates containing intact BKV NCCRs. NFI family members interacted with the helicase domain of BKV Tag in pulldown assays, suggesting that NFI helps recruit Tag to the viral core origin and may modulate its function. However, Tag may not be the sole target of the replication-modulatory activities of NFI: the NFIC/CTF1 isotype stimulates BKV template replication in vitro at low concentrations of DNA polymerase-α primase (Pol-primase), and the p58 subunit of Pol-primase associates with NFIC/CTF1, suggesting that NFI also recruits Pol-primase to the NCCR. These results suggest that NFI proteins (and the signaling pathways that target them) activate BKV replication and contribute to the consequent pathologies caused by acute infection.

Effect of short term and chronic administration of Sutherlandia frutescens on pharmacokinetics of nevirapine in rats.

Sutherlandia frutescens (sutherlandia), an African herbal supplement was recommended by the South African Ministry of Health for the treatment of AIDS patients. However, no reports yet exist delineating the effect of sutherlandia on pharmacokinetics of antiretroviral agents. Therefore, this investigation aimed at screening the effects of short term and chronic exposure of sutherlandia on oral bioavailability and pharmacokinetics of nevirapine (NVP), a non-nucleoside reverse transcriptase inhibitor, in Sprague Dawley rats. NVP (6 mg/kg) was administered orally alone (control) and with co-administration of sutherlandia; short term (12 mg/kg single dose) and long term (12 mg/kg, once a day for 5 days). No significant difference in the pharmacokinetic parameters of NVP was found upon short-term co-administration of Sutherlandia. However, there was a 50% decrease (p<0.05) in the AUC and C(max) values of NVP after 5 days of chronic exposure with Sutherlandia. In addition, quantitative RT-PCR studies demonstrated a 2-3-fold increase in the hepatic and intestinal mRNA expression of CYP3A2, relative to vehicle control. To further confirm, if this could translate into a clinically relevant pharmacokinetic interaction in patients, we tested this hypothesis employing LS-180 cells as an in vitro induction model for human CYP3A4. Ninety-six hours post treatment, similar to positive control rifampicin (25 µM), sutherlandia extract (300 µg/mL) resulted in elevated m-RNA expression levels and functional activity of CYP3A4 (human homologue of rodent CYP3A2) in LS-180 cells. Taken together, these results suggest that a potential drug-herb interaction is possible when NVP is co-administered with S. frutescens, although this hypothesis still remains to be investigated in a clinical setting.

Inhibition of human BK polyomavirus replication by small noncoding RNAs.

Small noncoding RNAs regulate a variety of cellular processes, including genomic imprinting, chromatin remodeling, replication, transcription, and translation. Here, we report small replication-regulating RNAs (srRNAs) that specifically inhibit DNA replication of the human BK polyomavirus (BKV) in vitro and in vivo. srRNAs from FM3A murine mammary tumor cells were enriched by DNA replication assay-guided fractionation and hybridization to the BKV noncoding control region (NCCR) and synthesized as cDNAs. Selective mutagenesis of the cDNA sequences and their putative targets suggests that the inhibition of BKV DNA replication is mediated by srRNAs binding to the viral NCCR, hindering early steps in the initiation of DNA replication. Ectopic expression of srRNAs in human cells inhibited BKV DNA replication in vivo. Additional srRNAs were designed and synthesized that specifically inhibit simian virus 40 (SV40) DNA replication in vitro. These observations point to novel mechanisms for regulating DNA replication and suggest the design of synthetic agents for inhibiting replication of polyomaviruses and possibly other viruses.

Hepatitis B Virus genotypic differences map structurally close to NRTI resistance hot spots.

Despite the availability of a Hepatitis B Virus (HBV) vaccine, there are approximately 350 million people that are chronically infected with this virus that can cause liver cirrhosis and hepatocellular carcinoma. Currently, most approved anti-HBV drugs are nucleoside RT inhibitors (NRTIs) that target the viral enzyme reverse transcriptase (RT or P gene product). They suppress viral replication very efficiently but require long-term therapies, which invariably lead to the development of drug resistant viral strains with drug resistance mutations at the P gene. Because the reading frames of the P and S (surface antigen) genes partially overlap, selection of NRTI-resistance mutations may impart changes on the surface structural landscape of the virus. Conversely, genotypic differences on viral surface residues may also change the amino acid composition of the P gene and in terms affect HBV RT properties such as susceptibility to NRTIs. Interestingly, several studies have shown that patients infected with HBV from various genotypes respond differently to NRTI therapies. Here, we built a three-dimensional homology model of the catalytic core of HBV RT using HIV-1 RT as a template. We then mapped on the molecular model the residues that vary among various HBV genotypes. Surprisingly, the genotypic variability residues are generally in the vicinity of residues that are involved in NRTI resistance. Our results suggest that emergence of NRTI resistance mutations in HBV RT may be constrained by structural interactions with residues that vary among different genotypes.

Restriction of human polyomavirus BK virus DNA replication in murine cells and extracts.

BK virus (BKV) causes persistent and asymptomatic infections in most humans and is the etiologic agent of polyomavirus-associated nephropathy (PVAN) and other pathologies. Unfortunately, there are no animal models with which to study activation of BKV replication in the human kidney and the accompanying PVAN. Here we report studies of the restriction of BKV replication in murine cells and extracts and the cause(s) of this restriction. Upon infection of murine cells, BKV expressed large T antigen (TAg), but viral DNA replication and progeny were not detected. Transfection of murine cells with BKV TAg expression vectors also caused TAg expression without accompanying DNA replication. Analysis of the replication of DNAs containing chimeric BKV and murine polyomavirus origins revealed the importance of BKV core origin sequences and TAg for DNA replication. A sensitive assay was developed with purified BKV TAg that supported TAg-dependent BKV DNA replication with human but not with murine cell extracts. Addition of human replication proteins, DNA polymerase alpha-primase, replication protein A, or topoisomerase I to the murine extracts with BKV TAg did not rescue viral DNA replication. Notably, addition of murine extracts to human extracts inhibited BKV TAg-dependent DNA replication at a step prior to or during unwinding of the viral origin. These findings and differences in replication specificity between BKV TAg and the TAgs of simian virus 40 (SV40) and JC virus (JCV) and their respective origins implicate features of the BKV TAg and origin distinct from SV40 and JCV in restriction of BKV replication in murine cells.

Identification of a beta-casein-like peptide in breast nipple aspirate fluid that is associated with breast cancer.

AIMS: Nipple aspirate fluid was collected prospectively from women scheduled for diagnostic breast surgery in order to determine protein masses associated with breast cancer, subsets of women with a unique proteomic profile and a breast cancer predictive model. MATERIALS & METHODS: Breast nipple aspirate fluid was collected preoperatively in 163 breasts from 125 women and analyzed for changes in cell morphology and by SELDI-TOF mass spectrometry over approximately a 44 kDa range (1.5-45 kDa) using IMAC30, CM10 and Q10 ProteinChips. RESULTS: Considering all samples, 16 protein masses were associated with the presence of cancer, the most discriminating being 3592, 6570/6580 and 15870 Da. Excluding women with pathologic nipple discharge or those with a papilloma identified an additional protein of 6383 Da. The best cancer detection models included Breast Imaging Reporting and Data System, age, and either the 4262 (best sensitivity: >87%) or 3592 (best specificity: >94%) peak. MALDI-TOF mass spectrometry demonstrated the 3592 peak, which was most discriminating in many of our cancer prediction models, to be a beta-casein-like peptide. CONCLUSION: Differential nipple aspirate fluid proteomic expression exists between women with/without breast cancer. The most discriminating protein identified is a beta-casein-like peptide not previously described. Combining proteomic and clinical information, which are available before surgery, optimizes the prediction of which women have breast cancer.

High-resolution human papillomavirus genotyping by MALDI-TOF mass spectrometry.

We describe a matrix-assisted laser desorption/ionization-time of flight (MALDI-TOF) mass spectrometry (MS)-based assay for human papillomavirus (HPV) genotyping--the restriction fragment mass polymorphism (RFMP) assay, which is based on mass measurement of genotype-specific oligonucleotide fragments generated by TypeIIS restriction endonuclease cleavage after recognition sites have been introduced by PCR amplification. The use of a TypeIIS restriction enzyme makes the RFMP assay independent of sequence and applicable to a wide variety of HPV genotypes, because these enzymes have cleavage sites at a fixed distance from their recognition sites. After PCR amplification, samples are subjected to restriction enzyme digestion with FokI and BtsCI and desalting using Oasis purification plates, followed by analysis by MALDI-TOF MS. Overall, the protocol is simple, takes approximately 4-4.5 h and can accurately detect and identify at least 74 different HPV genotypes.

Formation of polyomavirus-like particles with different VP1 molecules that bind the urokinase plasminogen activator receptor.

Icosahedral virus-like particles formed by the self-assembly of polyomavirus capsid proteins (Py-VLPs) can serve as useful nanostructures for delivering nucleic acids, proteins, and pharmaceuticals into animal cells and tissues. Four predominant surface-exposed loops in the VP1 structure offer potential sites to display sequences that might contribute new targeting specificities. Introduction into each of these loops of sequences derived from the amino-terminal fragment of urokinase plasminogen activator (uPA) or a related phage display peptide reduced the solubility of VP1 molecules when expressed in insect cells, and insertions into the EF loop reduced VP1 solubility least. Coexpression in insect cells of the uPA-VP1 molecules and VP1 containing a FLAG epitope in the HI loop permitted the formation of heterotypic Py-VLPs containing uPA-VP1 and FLAG-VP1. These heterotypic VLPs bound to uPAR on the surfaces of animal cells. Heterotypic Py-VLPs containing ligands for multiple cell surface receptors should be useful for targeting specific cells and tissues.

Inhibition of polyomavirus ori-dependent DNA replication by mSin3B.

When tethered in cis to DNA, the transcriptional corepressor mSin3B inhibits polyomavirus (Py) ori-dependent DNA replication in vivo. Histone deacetylases (HDACs) appear not to be involved, since tethering class I and class II HDACs in cis does not inhibit replication and treating the cells with trichostatin A does not specifically relieve inhibition by mSin3B. However, the mSin3B L59P mutation that impairs mSin3B interaction with N-CoR/SMRT abrogates inhibition of replication, suggesting the involvement of N-CoR/SMRT. Py large T antigen interacts with mSin3B, suggesting an HDAC-independent mechanism by which mSin3B inhibits DNA replication.

Stimulation of DNA replication from the polyomavirus origin by PCAF and GCN5 acetyltransferases: acetylation of large T antigen.

The PCAF and GCN5 acetyltransferases, but not p300 or CBP, stimulate DNA replication when tethered near the polyomavirus origin. Replication stimulation by PCAF and GCN5 is blocked by mutational inactivation of their acetyltransferase domains but not by deletion of sequences that bind p300 or CBP. Acetylation of histones near the polyomavirus origin assembled into chromatin in vivo is not detectably altered by expression of these acetyltransferases. PCAF and GCN5 interact with polyomavirus large T antigen in vivo, PCAF acetylates large T antigen in vitro, and large T-antigen acetylation in vivo is dependent upon the integrity of the PCAF acetyltransferase domain. These data suggest replication stimulation occurs through recruitment of large T antigen to the origin and acetylation by PCAF or GCN5.