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Hartselab Research-Past and Present
The longest ongoing project in our lab is to tease apart which specific properties of Amphotericin B pharmaceutical preparations make them less toxic (and more effective) in hopes of finding a simpler way to increase the drug's therapeutic index. We are specifically interested in how Liposomal and other supramolecular formulations of Amphotericin B alter toxicity and channel formation properties of this drug. We use stopped-flow fluorescence, absorbance and CD methods to probe the structure and activity of Amphotericin preparations. In addition we are beginning studies aimed at characterizing the biological response of monocytes to stimulation by different formulae. We propose that there are essentially three factors which influence the toxicity and efficacy of AmB preparations: 1) direct membrane toxicity via ion channel formation, 2) differences in distribution and delivery to tissues due to differences in serum lipoprotein/protein binding, and 3) initiation of an inflammatory cytokine response. Another interetsing feature of Amphotericin B (AmB) is that it
is one
of the few agents shown to slow the course of prion diseases
(like chronic
wasting disease and mad cow disease) in animals. Prions and amyloid
diseases
like Alzheimer's have many features in common.
More recently, Dr.Turtinen and I have collaborated on the molecular
mechanism of AmB's ability to induce a cytokine response and how
drug delivery
systems can modify this (9).
We have found that there are essentially two categories of AmB drug
delivery
preparations; those that stimulate TNF-a and
IL-6 in monocytes and those that do not. We use new chemiluminescent
antibody-array
detection (see illustration).
Why would anyone care? Well, TNF and IL-6 both stimulate
HIV replication. So if you are prescribing antifungal medication to an
AIDS patient with serious fungal disease, which would you choose, the
one
that may increase viral load or the one that does not? Another possibly
practical benefit of our research! How is it that diferent
liposomal
forms produce different responses? It is likely some combination of
AmB-induced
membrane potential changes, specific ion currents, calcium fluxes or
TLR receptor activation.
That
is a subject of ongoing research. Images of InstantLyso and other dyes.
An epifluorescence image of InstantLyso with live fibroblasts
at 75 nM and excited with blue filter light (~460-490 nm WIB set).
Notice the clearly visible Golgi apparatus which is particularly
targeted in this cell line. We have also developed fluorescent stains for cholesterol rich domains (InstantLipo) which may also effecitvel stain so-called lipid "rafts" and pathological cholesterol inclusions. This probe may be useful for diagnosing cholesterol and other lipid disorders. Most interesting are the totally novel mitochondrial stains developed here at UW-EC. A photo of the filamentous mtiochondria in fibroblasts is shown below. All photos were imaged in the fluorescence microscope facility at UW-Eau Claire. A recent very exciting area of research has come from collaboration with Alan Dispirito of Iowa State University. We are studying a peptide, Methanobactin, that strongly binds and reduces metals in the environment, especially copper (see these papers, 10 and 11 for more detail). It can also reduce Au(III) to gold nanoparticles. This molecule could have major impacts on weathering of minerals and mobilization of toxic metals in the environment. We are beginning 1 and 2D NMR studies on the structure of this peptide-like molecule.
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