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Since 1981, the ***/AIDS pandemic has killed an estimated 25 million people worldwide.
Although mortality rates have declined since the 1980s, especially in developed countries,
the disease still claims millions of lives every year. In 2005 alone, it is estimated
that between 2.4 and 3.3 million people died of AIDS, the advanced stage of ***.
Sha Jin - Approximately 33 million people are living with *** virus and then over one
million people in the United States are infected with this virus.
Although incurable - meaning it cannot be eradicated from the human body - *** can be
controlled by drugs.
Sha Jin - Currently the treatment of the *** infection heavily relies on what we call HAART
therapy, which refers to highly active anti-retroviral therapy.
But there are problems with these drugs. Most of them have strong side effects. Roughly
a quarter of all patients treated with *** drugs stop therapy within the first year due
to the effects of high toxicity.
This obstacle motivates scientists all over the world to discover better treatments for
*** and AIDS. One of these researchers is Sha Jin, assistant professor of biomedical
engineering at the University of Arkansas. In her lab at the Engineering Research Center
in south Fayetteville, Jin works with HeLa cells to develop a molecular "probe" that
can simultaneously detect ***-related enzyme produced by the people infected with ***,
as well as toxicity levels of compounds used in *** drugs.
Sha Jin - We tested a tube developed in my lab and we found that if we add *** inhibitor
to the cells we receive higher signaling because cells are showing us red or yellow color that
is indicating the *** protease activity was inhibited by its inhibitor.
Ralph Henry - So what Sha Jin in biomedical engineering has done is something I think
is really cleaver. She knows the protein sequence that the *** protease will recognize and clip.
What she's done is to take two proteins that will actually respond to light, but when they
are in close proximity to each other they'll behave in one way and when they are not in
close proximity to each other they'll behave in a very different way, and you can see that.
What she's done is to take the protein sequence that this *** 1 protease attacks and essentially
join these two light responsive proteins, and she can express in HeLa cells this light
responsive protein combo along with expression of the *** 1 protease. The protease is active
when it's expressed, and you know this because it goes and clips the target sequence that
she put in between the two light responsive proteins.
Sha Jin - Basically we are using a DNA cloning technique to create a new probe, which is
able to tell us which chemical compound has anti-*** activity.
Jin genetically engineered a biological screening system, by applying fluorescent proteins,
one green and one red, to generate what is known as Fšrster resonance energy transfer
signals, or FRET. FRET is a mechanism of energy transfer between a donor and receptor chromophore,
which is the part of a molecule that is responsible for its color. This process - the energy transfer
-prompts reactions from cells in the form of color.
Sha Jin - Using the cell, we will see the cell showing yellow or green color, indicating
the lower signal because of the cleavage by the protease. So the two proteins separate
and the distance is definitely longer than 10 nanometers. Then there is not that strong
signal.
In Jin's project, FRET signals responded to the presence of *** protease, which is a family
of enzymes, or proteins, that are essential for the life-cycle of ***. The signals also
responded to various compounds that are designed to block, or inhibit, *** protease. When applying
the fluorescent proteins to create FRET signals, Jin observed green and yellow cells in the
absence of protease inhibitors. She saw red when the protease inhibitors were added to
the cells. The red cells indicated *** protease loses its activity due to the presence of
protease inhibitors in the cell-culture medium. Meanwhile, healthy cells growing on the culture
plate allowed her to measure toxicity levels, that is, the extent to which toxic inhibitors
affected healthy cells.
Ralph Henry - One of the clever things that Dr. Jin has done is she's created a platform,
a cell line that gives you a visible signal when this *** protease is working and when
it's not. So not only is she able to use these cells in screening drugs that are currently
being developed, but even in developing new drugs that we haven't even looked at. So this
same cell line, it's a specialized cell line of HeLa. It's not just a normal HeLa cell.
It's not a HeLa cell that's expressing an *** protease -- it's expressing a construct
that's sensitive to that protease. The point is that cell line may be valuable for the
next ten years in coming up with the number of new drugs that could be useful in attacking
***.
Jin's novel detection system confirmed previous studies showing that the compounds nelfinavir
and lopinavir are toxic to cells, since many healthy cells died after only two days in
incubation. Indinavir and ritonavir showed less toxicity.
With HeLa, Jin is establishing a cell line that will allow her to consistently express
the detection system. HeLa cells are ideal for this for several reasons. They grow rapidly
and can be handled in a straightforward manner. They grow in large quantity and can be grown
in cell culture at much less expense than many other culture cells. And finally, targeted
genes can be easily delivered to HeLa cells.
Ralph Henry -Sha Jin's approach of having a viral protein attacking something that's
visible and changing its visibility -- that whole concept -- I think she will continue
to expand on, to impact drug development research that goes beyond just ***. That whole platform
has tremendous potential.