Jodie A. Robinson

Wolbachia-Mediated Resistance to Dengue Virus Infection and Death at the Cellular Level

Background Dengue is currently the most important arthropod-borne viral disease of humans. Recent work has shown dengue virus displays limited replication in its primary vector, the mosquito Aedes aegypti, when the insect harbors the endosymbiotic bacterium Wolbachia pipientis. Wolbachia-mediated inhibition of virus replication may lead to novel methods of arboviral control, yet the functional and cellular mechanisms that underpin it are unknown. Methodology/Principal Findings Using paired Wolbachia-infected and uninfected Aedes-derived cell lines and dengue virus, we confirm the phenomenon of viral inhibition at the cellular level. Although Wolbachia imposes a fitness cost to cells via reduced proliferation, it also provides a significant degree of protection from virus-induced mortality. The extent of viral inhibition is related to the density of Wolbachia per cell, with highly infected cell lines showing almost complete protection from dengue infection and dramatically reduced virus titers compared to lines not infected with the bacteria. Conclusions/Significance We have shown that cells infected with Wolbachia display inhibition of dengue virus replication, that the extent of inhibition is related to bacterial density and that Wolbachia infection, although costly, will provide a fitness benefit in some circumstances. Our results parallel findings in mosquitoes and flies, indicating that cell line models will provide useful and experimentally tractable models to study the mechanisms underlying Wolbachia-mediated protection from viruses.

Peroxisome proliferator–activated receptor α in the human breast cancer cell lines MCF‐7 and MDA‐MB‐231

Peroxisome proliferator–activated receptor (PPAR) α is a ligand‐activated transcription factor that has been linked with rodent hepatocarcinogenesis. It has been suggested that PPARα mRNA expression levels are an important determinant of rodent hepatic tumorigenicity. Previous work in rat mammary gland epithelial cells showed significantly increased PPARα mRNA expression in carcinomas, suggesting the possible role of this isoform in rodent mammary gland carcinogenesis. In this study we sought to determine whether PPARα is expressed and dynamically regulated in human breast cancer MCF‐7 and MDA‐MB‐231 cells. Having established the presence of PPARα in both cell types, we then examined the consequence of PPARα activation, by its ligands Wy‐14,643 and clofibrate, on proliferation. With real‐time reverse transcriptase–polymerase chain reaction, we showed that PPARα mRNA was dynamically regulated in MDA‐MB‐231 cells and that PPARα activation significantly increased proliferation of the cell line. In contrast, PPARα expression in MCF‐7 cells did not change with proliferation during culture and was present at significantly lower levels than in MDA‐MB‐231 cells. However, PPARα ligand activation still significantly increased the proliferation of MCF‐7 cells. The promotion of proliferation in breast cancer cell lines following PPARα activation was in stark contrast to the effects of PPARγ‐activating ligands that decrease proliferation in human breast cancer cells. Our results established the presence of PPARα in human breast cancer cell lines and showed for the first time that activation of PPARα in human breast cancer cells promoted proliferation. Hence, this pathway may be significant in mammary gland tumorigenesis.

Peroxisome proliferator-activated receptor alpha in the human breast cancer cell lines MCF-7 and MDA-MB-231.

Peroxisome proliferator–activated receptor (PPAR) α is a ligand‐activated transcription factor that has been linked with rodent hepatocarcinogenesis. It has been suggested that PPARα mRNA expression levels are an important determinant of rodent hepatic tumorigenicity. Previous work in rat mammary gland epithelial cells showed significantly increased PPARα mRNA expression in carcinomas, suggesting the possible role of this isoform in rodent mammary gland carcinogenesis. In this study we sought to determine whether PPARα is expressed and dynamically regulated in human breast cancer MCF‐7 and MDA‐MB‐231 cells. Having established the presence of PPARα in both cell types, we then examined the consequence of PPARα activation, by its ligands Wy‐14,643 and clofibrate, on proliferation. With real‐time reverse transcriptase–polymerase chain reaction, we showed that PPARα mRNA was dynamically regulated in MDA‐MB‐231 cells and that PPARα activation significantly increased proliferation of the cell line. In contrast, PPARα expression in MCF‐7 cells did not change with proliferation during culture and was present at significantly lower levels than in MDA‐MB‐231 cells. However, PPARα ligand activation still significantly increased the proliferation of MCF‐7 cells. The promotion of proliferation in breast cancer cell lines following PPARα activation was in stark contrast to the effects of PPARγ‐activating ligands that decrease proliferation in human breast cancer cells. Our results established the presence of PPARα in human breast cancer cell lines and showed for the first time that activation of PPARα in human breast cancer cells promoted proliferation. Hence, this pathway may be significant in mammary gland tumorigenesis.

Structural basis for recruitment of tandem hotdog domains in acyl-CoA thioesterase 7 and its role in inflammation

Acyl-CoA thioesterases (Acots) catalyze the hydrolysis of fatty acyl-CoA to free fatty acid and CoA and thereby regulate lipid metabolism and cellular signaling. We present a comprehensive structural and functional characterization of mouse acyl-CoA thioesterase 7 (Acot7). Whereas prokaryotic homologues possess a single thioesterase domain, mammalian Acot7 contains a pair of domains in tandem. We determined the crystal structures of both the N- and C-terminal domains of the mouse enzyme, and inferred the structure of the full-length enzyme using a combination of chemical cross-linking, mass spectrometry, and molecular modeling. The quaternary arrangement in Acot7 features a trimer of hotdog fold dimers. Both domains of Acot7 are required for activity, but only one of two possible active sites in the dimer is functional. Asn-24 and Asp-213 (from N- and C-domains, respectively) were identified as the catalytic residues through site-directed mutagenesis. An enzyme with higher activity than wild-type Acot7 was obtained by mutating the residues in the nonfunctional active site. Recombinant Acot7 was shown to have the highest activity toward arachidonoyl-CoA, suggesting a function in eicosanoid metabolism. In line with the proposal, Acot7 was shown to be highly expressed in macrophages and up-regulated by lipopolysaccharide. Overexpression of Acot7 in a macrophage cell line modified the production of prostaglandins D2 and E2. Together, the results link the molecular and cellular functions of Acot7 and identify the enzyme as a candidate drug target in inflammatory disease.

Effect of the peroxisome proliferator-activated receptor beta activator GW0742 in rat cultured cerebellar granule neurons

The ligand‐activated transcription factor peroxisome proliferator‐activated receptor β (PPARβ) is present in the brain and is implicated in the regulation of genes with potential roles in neurotoxicity. We sought to examine the role of PPARβ in neuronal cell death by using the PPARβ ligand GW0742. Primary cultures of rat cerebellar granule neurons were prepared from 7‐day‐old pups. Reverse transcriptase‐polymerase chain reaction and in situ hybridization were used to verify that PPARβ mRNA was present in neurons. After 10–12 days in culture, the neuronal cells were incubated in the presence of GW0742, and cell death was measured with a lactate dehydrogenase release (LDH) assay. After 24 hr of exposure, PPARβ activation by GW0742 was not inherently toxic to cerebellar granule neurons. However, toxicity was observed after 48 hr, with cell death mediated via an apoptotic mechanism. In an effect opposite to that observed with PPARα‐activating ligands, PPARβ activation exhibited neuroprotective properties. Treatment with GW0742 significantly reduced cell death during a 12‐hr exposure to low‐KCl media. These results clearly reinforce very specific roles for the PPAR isoforms in neurons and suggest that PPARβ is worthy of further investigation regarding its potential role as a therapeutic target in neurodegenerative states.

Effects of peroxisome proliferator-activated receptor γ ligands ciglitazone and 15-deoxy-Δ12,14-prostaglandin J2 on rat cultured cerebellar granule neuronal viability

Peroxisome proliferator‐activated receptor γ (PPARγ) has been the focus of studies assessing its potential neuroprotective role. These studies have shown either neuroprotection or neurotoxicity by PPARγ ligands. Comparison of these studies is complicated by the use of different PPARγ ligands, mechanisms of neurotoxicity induction, and neuronal cell type. In this study, we compared the effects of the synthetic PPARγ ligand ciglitazone with an endogenous PPARγ ligand, 15‐deoxy‐Δ12,14‐prostaglandin J2 (15‐deoxy PGJ2), on inherent neurotoxicity and neuroprotection using a reduction in extracellular KCl in rat cultured cerebellar granule neurons (CGN). We also assessed the effects of these ligands on c‐Jun protein expression, which is up‐regulated on induction of low‐KCl‐mediated neuronal apoptosis as well as being associated with PPAR in other cell types. We showed that PPARγ mRNA is expressed in CGN cultures and observed ciglitazone‐ and 15‐deoxy PGJ2‐mediated inherent neurotoxicity that was concentration and time dependent. c‐Jun was only modestly increased in the presence of ciglitazone but was markedly up‐regulated by 15‐deoxy PGJ2 after 12 hr. Treatment of CGN cultures with ciglitazone simultaneous with KCl withdrawal resulted in a modest, time‐dependent neuroprotection. Such neuroprotection after KCl withdrawal was not observed with 15‐deoxy PGJ2. Despite the absence of neuroprotection, 15‐deoxy PGJ2 markedly inhibited the early up‐regulation of c‐Jun during KCl withdrawal. These studies suggest that ciglitazone and 15‐deoxy PGJ2 have markedly different effects on inherent and low‐KCl‐induced toxicity and c‐Jun expression in CGN, indicating potential non‐PPARγ mechanisms.

Identification of a non-purple tartrate-resistant acid phosphatase: an evolutionary link to Ser/Thr protein phosphatases?

BackgroundTartrate-resistant acid phosphatases (TRAcPs), also known as purple acid phosphatases (PAPs), are a family of binuclear metallohydrolases that have been identified in plants, animals and fungi. The human enzyme is a major histochemical marker for the diagnosis of bone-related diseases. TRAcPs can occur as a small form possessing only the ~35 kDa catalytic domain, or a larger ~55 kDa form possessing both a catalytic domain and an additional N-terminal domain of unknown function. Due to its role in bone resorption the 35 kDa TRAcP has become a promising target for the development of anti-osteoporotic chemotherapeutics.FindingsA new human gene product encoding a metallohydrolase distantly related to the ~55 kDa plant TRAcP was identified and characterised. The gene product is found in a number of animal species, and is present in all tissues sampled by the RIKEN mouse transcriptome project. Construction of a homology model illustrated that six of the seven metal-coordinating ligands in the active site are identical to that observed in the TRAcP family. However, the tyrosine ligand associated with the charge transfer transition and purple color of TRAcPs is replaced by a histidine.ConlusionThe gene product identified here may represent an evolutionary link between TRAcPs and Ser/Thr protein phosphatases. Its biological function is currently unknown but is unlikely to be associated with bone metabolism.

A generic screening platform for inhibitors of virus induced cell fusion using cellular electrical impedance

Fusion of the viral envelope with host cell membranes is an essential step in the life cycle of all enveloped viruses. Despite such a clear target for antiviral drug development, few anti-fusion drugs have progressed to market. One significant hurdle is the absence of a generic, high-throughput, reproducible fusion assay. Here we report that real time, label-free measurement of cellular electrical impedance can quantify cell-cell fusion mediated by either individually expressed recombinant viral fusion proteins, or native virus infection. We validated this approach for all three classes of viral fusion and demonstrated utility in quantifying fusion inhibition using antibodies and small molecule inhibitors specific for dengue virus and respiratory syncytial virus.

Anti-proliferative effects of novel Glyco-lipid-arsenicals (III) on MCF-7 human breast cancer cells

Arsenic trioxide appears to be effective in the treatment of pro-myelocytic leukaemia. The substituted phenylarsen(III)oxides are highly polar, they have a high tendency to undergo oxidation to As (V) and to form oligomers, to prevent this we protected the As-(OH)(2) group as cyclic dithiaarsanes. To increase the compounds biological stability and passive diffusion we conjugated the compound of interest with lipoamino acids (Laas). Alternatively, we further conjugated the dithiaarsane derivative with a carbohydrate to utilize active transport systems and to target compound. We investigated two novel glyco-lipid arsenicals (III) (compounds 9 and 11) for their ability to initiate MCF-7 breast cancer cell death and characterized the mechanism by which death was initiated. A significant decrease in MCF-7 cell proliferation was observed using 1 microM and 10 microM compound (11) and 10 microM of compound (9). Treatment with compound (11) triggered apoptosis of MFC-7 cells while compound (9) induced inhibition of cellular proliferation was not via rapid induction of apoptosis and more likely reflected necrosis and/or alterations in the cell cycle. Differences in the anti-proliferative potency of the two compounds indicate that structural modifications influence effectiveness.

Peroxisome proliferator-activated receptor ? in the human breast cancer cell lines MCF-7 and MDA-MB-231

Peroxisome proliferator–activated receptor (PPAR) α is a ligand‐activated transcription factor that has been linked with rodent hepatocarcinogenesis. It has been suggested that PPARα mRNA expression levels are an important determinant of rodent hepatic tumorigenicity. Previous work in rat mammary gland epithelial cells showed significantly increased PPARα mRNA expression in carcinomas, suggesting the possible role of this isoform in rodent mammary gland carcinogenesis. In this study we sought to determine whether PPARα is expressed and dynamically regulated in human breast cancer MCF‐7 and MDA‐MB‐231 cells. Having established the presence of PPARα in both cell types, we then examined the consequence of PPARα activation, by its ligands Wy‐14,643 and clofibrate, on proliferation. With real‐time reverse transcriptase–polymerase chain reaction, we showed that PPARα mRNA was dynamically regulated in MDA‐MB‐231 cells and that PPARα activation significantly increased proliferation of the cell line. In contrast, PPARα expression in MCF‐7 cells did not change with proliferation during culture and was present at significantly lower levels than in MDA‐MB‐231 cells. However, PPARα ligand activation still significantly increased the proliferation of MCF‐7 cells. The promotion of proliferation in breast cancer cell lines following PPARα activation was in stark contrast to the effects of PPARγ‐activating ligands that decrease proliferation in human breast cancer cells. Our results established the presence of PPARα in human breast cancer cell lines and showed for the first time that activation of PPARα in human breast cancer cells promoted proliferation. Hence, this pathway may be significant in mammary gland tumorigenesis.