Jennifer Schmidt

Molecular Analysis of the Interaction of Anthrax Adenylyl Cyclase Toxin, Edema Factor, with 2(3)-O-(N-(methyl)anthraniloyl)- Substituted Purine and Pyrimidine Nucleotides

Bacillus anthracis causes anthrax disease and exerts its deleterious effects by the release of three exotoxins: lethal factor, protective antigen, and edema factor (EF), a highly active calmodulin-dependent adenylyl cyclase (AC). However, conventional antibiotic treatment is ineffective against either toxemia or antibiotic-resistant strains. Thus, more effective drugs for anthrax treatment are needed. Previous studies from our laboratory showed that mammalian membranous AC (mAC) exhibits broad specificity for purine and pyrimidine nucleotides ( Mol Pharmacol70:878-886, 2006 ). Here, we investigated structural requirements for EF inhibition by natural purine and pyrimidine nucleotides and nucleotides modified with N-methylanthraniloyl (MANT)- or anthraniloyl groups at the 2′(3′)-O-ribosyl position. MANT-CTP was the most potent EF inhibitor (Ki, 100 nM) among 16 compounds studied. MANT-nucleotides inhibited EF competitively. Activation of EF by calmodulin resulted in effective fluorescence resonance energy transfer (FRET) from tryptophan and tyrosine residues located in the vicinity of the catalytic site to MANT-ATP, but FRET to MANT-CTP was only small. Mutagenesis studies revealed that Phe586 is crucial for FRET to MANT-ATP and MANT-CTP and that the mutations N583Q, K353A, and K353R differentially alter the inhibitory potencies of MANT-ATP and MANT-CTP. Docking approaches relying on crystal structures of EF indicate similar binding modes of the MANT nucleotides with subtle differences in the region of the nucleobases. In conclusion, like mAC, EF accommodates both purine and pyrimidine nucleotides. The unique preference of EF for the base cytosine offers an excellent starting point for the development of potent and selective EF inhibitors.

Migration-associated secretion of melanoma inhibitory activity at the cell rear is supported by KCa3.1 potassium channels

Malignant melanoma, characterized by invasive local growth and early formation of metastases, is the most aggressive type of skin cancer. Melanoma inhibitory activity (MIA), secreted by malignant melanoma cells, interacts with the cell adhesion receptors, integrins α4β1 and α5β1, facilitating cell detachment and promoting formation of metastases. In the present study, we demonstrate that MIA secretion is confined to the rear end of migrating cells, while in non-migrating cells MIA accumulates in the actin cortex. MIA protein takes a conventional secretory pathway including coat protein complex I (COPI)- and coat protein complex II (COPII)-dependent protein transport to the cell periphery, where its final release depends on intracellular Ca2+ ions. Interestingly, the Ca2+-activated K+-channel, subfamily N, member 4 (KCa3.1), known to be active at the rear end of migrating cells, was found to support MIA secretion. Secretion was diminished by the specific KCa3.1 channel inhibitor TRAM-34 and by expression of dominant-negative mutants of the channel. In summary, we have elucidated the migration-associated transport of MIA protein to the cell rear and also disclosed a new mechanism by which KCa3.1 potassium channels promote cell migration.

Processing of MIA protein during melanoma cell migration

MIA (melanoma inhibitory activity) protein, identified as a small 11 kDa protein highly expressed and secreted by malignant melanoma cells, plays an important functional role in melanoma development, progression and tumor cell invasion. Recent data describe a direct interaction of MIA protein with cell adhesion receptors integrin α4β1 and integrin α5β1 and extracellular matrix molecules. By modulating integrin activity MIA protein mediates detachment of melanoma cells from surrounding structures resulting in enhanced invasive and migratory potential. However, until today a detailed understanding of the processes of MIA function is missing. In this study, we show that after binding of MIA protein to integrin α5β1, MIA protein is internalized together with this cell adhesion receptor at the cell rear. This mechanism enables tumor cells to migrate in a defined direction as appropriate for invasion processes. Treatment of melanoma cells with PKC‐inhibitors strongly reduced internalization of MIA protein. Endocytosis is followed by dissociation of MIA–integrin complexes. In acidic vesicles MIA protein is degraded while integrins are recycled. Treatment of melanoma cells with MIA inhibitory peptides almost completely blocked the MIA protein uptake into cells. As MIA protein has a major contribution to the aggressive characteristics of malignant melanoma in particular to formation of metastasis, it is important to elucidate the MIA functional mechanism in tumor cells to find novel therapeutic strategies in the fight against skin cancer.

Chromosomal localization of the human AHR locus encoding the structural gene for the Ah receptor to 7p21→p15

The AHR locus encodes the structural gene for the Ah receptor, a ligand activated transcription factor that regulates the expression of a number of enzymes involved in the metabolism of chemical carcinogens and that appears to mediate the tumor promoting properties of compounds such as 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD). Using polymerase chain reaction (PCR), we amplified exon-10 and intron-D of the AHR gene from human genomic DNA. By using PCR analysis of somatic cell hybrids and fluorescence in situ hybridization of metaphase cells, we localized AHR to human chromosome 7, bands p21-->p15. This mapping data should prove useful in determining the role that the AHR locus plays in human cancer incidence and in the identification of human populations with altered susceptibilities to the toxic/carcinogenic effects of planar aromatic hydrocarbons.

Targeting Melanoma Metastasis and Immunosuppression with a New Mode of Melanoma Inhibitory Activity (MIA) Protein Inhibition

Melanoma is the most aggressive form of skin cancer, with fast progression and early dissemination mediated by the melanoma inhibitory activity (MIA) protein. Here, we discovered that dimerization of MIA is required for functional activity through mutagenesis of MIA which showed the correlation between dimerization and functional activity. We subsequently identified the dodecapeptide AR71, which prevents MIA dimerization and thereby acts as a MIA inhibitor. Two-dimensional nuclear magnetic resonance (NMR) spectroscopy demonstrated the binding of AR71 to the MIA dimerization domain, in agreement with in vitro and in vivo data revealing reduced cell migration, reduced formation of metastases and increased immune response after AR71 treatment. We believe AR71 is a lead structure for MIA inhibitors. More generally, inhibiting MIA dimerization is a novel therapeutic concept in melanoma therapy.

Heterogeneous transition metal-based fluorescence polarization (HTFP) assay for probing protein interactions

Analyses of protein interactions are fundamental for the investigation of molecular mechanisms responsible for cellular processes and diseases, as well as for drug discovery in the pharmaceutical industry. The present study details the development of a fluorescence polarization assay using melanoma inhibitory activity (MIA) protein-binding compounds and studies of the binding properties of this protein. Since they are dependent on the the lifetime of the fluorescent label, currently available fluorescence polarization assays can only determine interactions with either high- or low-molecular weight interaction partners. Our new approach eliminates this limitation by immobilizing a known binding partner of MIA protein to a well plate and by labeling the target protein using luminescent transition metal labels such as Ru(bpy)3 for binding studies with both high- and low-molecular weight interaction partners. Due to the use of a functionalized surface, we termed our concept heterogeneous transition metal-based fluorescence polarization (HTFP) assay. The assays independence from the molecular weight of potential binding partners should make the technique amenable to investigations on subjects as diverse as multimerization, interactions with pharmacophores, or binding affinity determination.

DNA Staining in Agarose Gels with ZN2+-Cyclen-Pyrene

A pyrene-labeled Zn2+-cyclen complex for the staining of DNA in agarose gels is reported. The metal chelate coordinates reversibly to the DNA phosphate backbone, which induces the formation of pyrene excimers. The typical pyrene excimer emission is used for the detection of the DNA. Staining is limited to agarose gels and is less sensitive than ethidium bromide, but DNA amounts as low as 10 ng and short DNA strands (∼300 b.p.) are detectable. Gel extraction as a standard technique in molecular biology was successfully performed after staining with Zn2+-cyclen-pyrene. Cytotoxicity tests on HeLa and V-79 cells reveal that the zinc-cyclen pyrene probe is significant less toxic compared to ethidium bromide.

Inducing anti-tumor cytokines and an immune response in melanoma by inhibition of MIA using the peptide AR71

BACKGROUND Malignant melanoma is known for its aggressive metastatic spread and suppression of the host immune system. Immunosuppression in melanoma is mediated in part by the protein melanoma inhibitory activity (MIA). OBJECTIVES In this study, we assessed the in vitro and in vivo efficacy of the MIA-inhibitory peptide AR71 in the inhibition of MIA-induced immunosuppression. This follows a previous study that revealed an increase in CD3-positive cells and cleaved caspase-3 in an in vivo model of hepatic metastasis after MIA inhibition. MATERIALS AND METHODS We used Multiplex-ELISAs and qRT-PCR for determining changes in cytokine expression in vitro and in vivo and calcein release assays for determining immune cell response in vitro. RESULTS By evaluating the serum levels of tumor-associated cytokines of the melanoma-bearing mice, we found beneficial decreases of several cytokines, including TNF-alpha, after AR71 treatment. Additionally, we demonstrated an increase of anti-tumor lymphokine-activated killer (LAK) cell cytotoxicity in the presence of the MIA inhibitor AR71. Stimulation of anti-tumor immune responses by AR71 could be observed via increased numbers of NK cells in the metastases-bearing murine livers in vivo. CONCLUSION In summary, inhibition of MIA activity results in reduced immunosuppression in vitro and in vivo.

The fitting of a unilateral shoulder disarticulation prosthesis after electrical burn injury: A case report.

Acquired amputation of an upper extremity is less common than lower extremity amputation. In the pediatric age group the most common causes of amputation are trauma and disease. Traumatic injuries include those from automobile accidents, farm accidents, lawn mower accidents, and high tension electrical wires. In 90% of acquired amputations, a single limb is involved [2]. Surgical intervention may differ from the adult guidelines to conserve all possible length of the residual extremity. Children may be given a disarticulation rather than a transdiaphyseal amputation, which preserves epiphyseal growth plates and also avoids development of terminal overgrowth [2]. Amputations related to electrical burns present additional rehabilitation challenges beyond those typically encountered due to tumors, trauma, or infection. Electrical burn injuries are complicated because of damage to structures including blood vessels, muscles, nerves, tendons, bone and skin. Tissue injury is the result of the conversion of electrical energy into thermal energy or heat. The voltage determines the pathway of the electricity, with high voltage following the most direct