TgMAPK1 is a Toxoplasma gondii MAP kinase that hijacks host MKK3 signals to regulate virulence and interferon-γ-mediated nitric oxide production.

The parasite Toxoplasma gondii controls tissue-specific nitric oxide (NO), thereby augmenting virulence and immunopathology through poorly-understood mechanisms. We now identify TgMAPK1, a Toxoplasma mitogen-activated protein kinase (MAPK), as a virulence factor regulating tissue-specific parasite burden by manipulating host interferon (IFN)-γ-mediated inducible nitric oxide synthase (iNOS). Toxoplasma with reduced TgMAPK1 expression (TgMAPK1(lo)) demonstrated that TgMAPK1 facilitates IFN-γ-driven p38 MAPK activation, reducing IFN-γ-generated NO in an MKK3-dependent manner, blunting IFN-γ-mediated parasite control. TgMAPK1(lo) infection in wild type mice produced ≥ten-fold lower parasite burden versus control parasites with normal TgMAPK1 expression (TgMAPK1(con)). Reduced parasite burdens persisted in IFN-γ KO mice, but equalized in normally iNOS-replete organs from iNOS KO mice. Parasite MAPKs are far less studied than other parasite kinases, but deserve additional attention as targets for immunotherapy and drug discovery.

A novel solubility-enhanced curcumin formulation showing stability and maintenance of anticancer activity.

Curcumin (CUR) is an active food compound, but its insolubility and instability in water contributes to low bioavailability. In this study, the solubility of CUR was enhanced by utilizing the solubilizing properties of rubusoside (RUB). The solubility of CUR in water increased linearly from 61 µg/mL to 2.318 mg/mL in the presence of RUB ranging from 1% to 10% (w/v). Dynamic light scattering and transmission electron microscopy studies found that CUR and RUB formed CUR-RUB nanoparticle (∼8 nm) complexes. The RUB-solubilized CUR was stable in physiological conditions and did not precipitate when diluted or degrade when spray-dried to a completely reconstitutable powder. Furthermore, cell viability assays demonstrated the efficacy of RUB-solubilized CUR against human colon, breast, and pancreatic cancer cell lines. The development of this new solubilized, stable, and biologically active CUR formulation lays the foundation for future bioavailability improvement.

Paclitaxel-induced apoptosis is blocked by camptothecin in human breast and pancreatic cancer cells.

The combination of paclitaxel (PTX) and topoisomerase I inhibitors such as camptothecin (CPT) constitutes a therapeutic strategy based on anticipated synergism. However, previous in vitro studies have generated contradictory findings for this strategy. The interaction between these drugs can be synergistic or antagonistic, depending on the cell type examined. To gain additional insight into this promising yet controversial strategy, we investigated the interaction between PTX and CPT in three different cell lines (PANC-1, MDA-MB-231 and HL-60) and explored possible underlying mechanisms of synergy or antagonism. Using a novel solubilizing natural compound, rubusoside, water-insoluble PTX and CPT were solubilized to enable the comparison of the effects of single drugs and their combination on cell viability. Intracellular drug concentrations were quantified to examine the effect of CPT on cellular uptake and accumulation of PTX. Flow cytometry and quantitative real-time PCR gene array analyses were used to explore the mechanisms behind the interaction between PTX and CPT. Our studies confirmed that rubusoside-solubilized PTX or CPT maintained cytotoxicity, causing significant reductions in cell viability. However, the efficacy of the combination of PTX and CPT produced varied results based on the cell line tested. CPT antagonistically reduced the cytotoxic activity of PTX in PANC-1 and MDA-MB-231 cells. The effect of CPT on the cytotoxicity of PTX was less pronounced in HL-60 cells, showing neither synergy nor antagonism. Analysis of apoptosis by flow cytometry revealed that upon co-treatment with CPT, apoptosis induced by PTX was attenuated in PANC-1 and MDA-MB-231 cells. In agreement with our cytotoxicity findings, no synergistic or antagonistic effects on apoptosis were observed in HL-60 cells. The antagonism in PANC-1 and MDA-MB-231 cells was not a result of reduced PTX uptake and accumulation because the amount of intracellular PTX was not altered upon co-treatment with CPT. Moreover, higher expression of anti-apoptosis-related transcripts (BCL2L10, CFLAR, HIP1 and TRADD) in PANC-1 cells was observed upon combination treatment over PTX treatment alone. Although exact underlying mechanisms are unknown, the suspected CPT-dependent reduction of intracellular PTX accumulation was ruled out. The findings of antagonism and increased anti-apoptotic gene transcription serve as a precaution to the design of combination drug strategies where a synergistic interaction may not exist.

A rapid ATP affinity-based purification for the human non-receptor tyrosine kinase c-Src.

The non-receptor tyrosine kinase c-Src plays a central role in a variety of cell signaling pathways that regulate cell growth, differentiation, apoptosis, and other important cellular processes. An 85-amino acid N-terminal deletion construct of c-Src (DeltaN85 c-Src) has been structurally characterized and used extensively in biochemical and biophysical studies. In this report, we have established a relatively rapid, simplified purification of DeltaN85 c-Src from recombinant baculovirus-infected insect cells. Q-Sepharose anion-exchange and aminophenyl-ATP affinity chromatography were used to isolate 5mg of >98% pure DeltaN85 c-Src from 900 mg of total soluble protein. The specific activity of DeltaN85 c-Src (20 U mg(-1)) was found to be >or = 5-fold greater than previously reported values. A lag in the autophosphorylation kinetics of DeltaN85 c-Src was observed, and the reaction occurred with observed first-order rate constants k1=0.20+/-0.01 min(-1) and k2=0.38+/-0.01 min(-1) under the experimental conditions used. Steady-state kinetic analysis of peptide phosphorylation by DeltaN85 c-Src gave Km values of 99+/-23 microM and 190+/-30 microM for the peptide and ATP substrates, respectively, and a value of k(cat)=17+/-2s(-1). Overall, we present a dramatically improved purification strategy that represents a simplified, relatively rapid protocol for the isolation of milligram quantities of DeltaN85 c-Src required for rigorous structure-function and inhibition studies that rely on a pre-steady-state kinetic approach.