Effects of transferrin conjugates of artemisinin and artemisinin dimer on breast cancer cell lines.

Transferrin (Tf) conjugates of monomeric artemisinin (ART) and artemisinin dimer were synthesized. The two conjugates, ART-Tf and dimer-Tf, retained the original protein structure, and formed stable aggregates in aqueous buffer. ART-Tf induced declines in proteins involved in apoptosis (survivin), cell cycling (cyclin D1), oncogenesis (c-myelocytomatosis oncogene product (c-MYC)), and dysregulated WNT signaling (beta-catenin) in both the human prostate (DU145) and breast (MCF7) cancer cell lines. Both ART-Tf and dimer-Tf induced down-regulation of survivin, c-MYC and mutated human epidermal growth factor receptor-2 (ERBB2 or HER2) in the BT474 breast cancer cell line. To our knowledge, this is the first demonstration that an ART derivative can cause a decline of ERBB2 in a human cancer cell line. Potential mechanisms for the observed effects are presented. Both transferrin conjugates strongly inhibited the growth of BT474 cells in the same concentration range that the conjugates caused declines in the levels of ERBB2, survivin, and c-MYC, while showing essentially no toxicity towards MCF10A normal breast cells.

Identification, cloning, expression, and purification of Francisella lpp3: an immunogenic lipoprotein.

The severe and fatal human disease, tularemia, results from infection with the Gram-negative pathogen Francisella tularensis. Identification of surface outer membrane proteins, specifically lipoproteins, has been of interest for vaccine development and understanding the initiation of disease. We sought to identify Francisella live vaccine strain lipoproteins that could be a component of a subunit vaccine and have adjuvant properties as TLR2 agonists. We have identified a membrane lipoprotein of Francisella LVS isolated by sarkosyl extraction and gel filtration chromatography that is recognized by sera from LVS-vaccinated individuals and tularemia patients, indicating its potential diagnostic value. Sequencing of the protein by mass spectrometry indicated that it encodes the FTL_0645 open reading frame of F. holarctica LVS, which is 100% identical/homologous to FTT1416c of F. tularensis Schu S4. The predicted 137 amino acid lipoprotein encoded by FTL_0645 ORF, was expressed in Escherichia coli, purified, and demonstrated to be a lipoprotein. This recombinant lipoprotein, named Flpp3, was able to activate TLR2 and induce an immunogenic response in mice, suggesting that the E. coli-expressed Flpp3 is palmitoylated and closely resembles the native protein in structure and immunogenicity. Taken together, these data suggest that Flpp3 could be a candidate for inclusion in a F. tularensis vaccine.

Identification and type III-dependent secretion of the Yersinia pestis insecticidal-like proteins.

Plague, or the Black Death, is a zoonotic disease that is spread from mammal to mammal by fleas. This mode of transmission demands that the causative agent of this disease, Yersinia pestis, is able to survive and multiply in both mammals and insects. In recent years the complete genome sequence of a number of Y. pestis strains have been determined. This sequence information indicates that Y. pestis contains a cluster of genes with homology to insecticidal toxin encoding genes of the insect pathogen Photorhabdus luminescens. Here we demonstrate that Y. pestis KIM strains produced the encoded proteins. Production of the locus-encoded proteins was dependent on a gene (yitR) encoding a member of the LysR family of transcriptional activators. Evidence suggests the proteins are type III secretion substrates. N terminal amino acids (100 to 367) of each protein fused to an epitope tag were secreted by the virulence plasmid type III secretion type. A fusion protein comprised of the N-terminus of YipB and the enzymatic active component of Bordetella pertussis adenylate cyclase (Cya) was translocated into both mammalian and insect cells. In conclusion, a new class of Y. pestis type III secreted and translocated proteins has been identified. We hypothesize that these proteins function to promote transmission of and infection by Y. pestis.

Free fatty acid impairment of nitric oxide production in endothelial cells is mediated by IKKbeta.

OBJECTIVE: Free fatty acids (FFA) are commonly elevated in diabetes and obesity and have been shown to impair nitric oxide (NO) production by endothelial cells. However, the signaling pathways responsible for FFA impairment of NO production in endothelial cells have not been characterized. Insulin receptor substrate-1 (IRS-1) regulation is critical for activation of endothelial nitric oxide synthase (eNOS) in response to stimulation by insulin or fluid shear stress. METHODS AND RESULTS: We demonstrate that insulin-mediated tyrosine phosphorylation of IRS-1 and serine phosphorylation of Akt, eNOS, and NO production are significantly inhibited by treatment of bovine aortic endothelial cells with 100 micromol/L FFA composed of palmitic acid for 3 hours before stimulation with 100 nM insulin. This FFA preparation also increases, in a dose-dependent manner, IKKbeta activity, which regulates activation of NF- kappaB, a transcriptional factor associated with inflammation. Similarly, elevation of other common FFA such as oleic and linoleic acid also induce IKKbeta activation and inhibit insulin-mediated eNOS activation. Overexpression of a kinase inactive form of IKKbeta blocks the ability of FFA to inhibit insulin-dependent NO production, whereas overexpression of wild-type IKKbeta recapitulates the effect of FFA on insulin-dependent NO production. CONCLUSIONS: Elevated levels of common FFA found in human serum activate IKKbeta in endothelial cells leading to reduced NO production, and thus may serve to link pathways involved in inflammation and endothelial dysfunction.