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Turning farm pests into living factories Scientists at MU are turning caterpillars into living
factories of a human heart protein. They hope their research will not
only help design drugs that strengthen the heart but will also open a
new era in scientific study. In a laboratory at the MU Dalton Cardiovascular Research
Center, associate professor Calvin Hale injects cabbage loopers, which
are common farm pests, with a virus that makes them carriers of a protein
stitched into the surfaces of human heart cells. "I think their work is extremely exciting, and I'm
actually trying to incorporate it into my own research," said Craig
Gatto, a cell biologist at Illinois State University. "I'm trying
to get in on the ground floor before it becomes routine in other laboratories."
Hale is working with a team of MU researchers on the cardiac
sodium-calcium exchanger, a protein found in the cell membranes of heart
muscle. Calcium ions in a muscle cell cause it to contract. What
the exchanger does is pump calcium ions out of heart cells and sodium
ions into them, thus allowing heart cells to relax. "This protein may play a more important role in heart
disease than previously believed," said David Kass, a professor at
the Johns Hopkins University School of Medicine. The MU research "should
speed up our ability to understand how this protein works." MU scientists inserted the gene for the exchanger into
a virus that targets only insects. "The virus infects a caterpillar and hijacks its
DNA so that its cells start producing more viruses," Hale explained.
In the process, the caterpillars also produce the exchanger
in unprecedented amounts, making the experiment unique. "We're the only people doing what we're doing,"
Hale said. Previously, one time-consuming method researchers employed
grew the heart protein using insect cells kept in petri dishes. One caterpillar,
however, can make as much exchanger as can be grown much more slowly in
10 petri dishes. "The possibility of getting so much more protein
using the caterpillars is an order of magnitude better than using the
cells," Gatto said. "Here, you can actually get enough protein
to look at structurally." Keeping a colony of 1,000 caterpillars in 50 Styrofoam
cups fed on a vitamin-enriched pinto bean mash is far cheaper and easier
for the MU research team than maintaining 10,000 petri dishes. And the colony of cabbage loopers maintains itself as
well. "We hold about 5 percent of the caterpillars aside
to become moths," Hale said. "They breed and provide us with
our next generation." Hale said he got the idea to use caterpillars from one
of his partners, MU molecular biologist Elmer Price, who tried the method
four years ago to produce a protein. Hale started work on the process
in January 1998 after almost a year of planning, and is hoping to have
the three-dimensional structure of the exchanger in a year. "These proteins are really important," Hale
said. "Research on this type of protein has lagged far behind in
terms of understanding how they work. We hope to change that." The exchanger is made of about 970 amino acids arranged
like beads on a string, and it is unique to vertebrates. "There is no other known protein like it," Hale
explained. "The human, the cow and the dog variant differ only by
a few amino acids, suggesting that these subtle differences are not really
all that important. It's amazing how close they are to one another across
vertebrate species." Although the amino acid sequence for the exchanger was
discovered 10 years ago, no one really knows yet how the heart protein
twists and folds three-dimensionally. "It's like having a piece of paper for origami,"
Hale said. "Is the shape of the paper going to be a cone or a swan?
We know the size of the piece of paper, but we don't know how it's folded."
Knowing the shape of the exchanger gives researchers the
chance to figure out how drugs might inhibit it. Keeping the exchanger
from working would in turn keep more calcium in heart cells, making heart
contractions all the stronger. "We don't want to know the shape just to know the
shape," Hale said. "We want to develop drugs that might possibly
increase the heart's muscle tone." MU researchers use the caterpillars of the farm pest Trichoplusia
ni, a small brown moth known commonly as the cabbage looper. Hale said
they live only about six to eight weeks, "from egg to dead moth,
cradle to grave." Each caterpillar is injected individually after its fourth
molting, when it is 10 to 12 days old and about the length of a dime.
The stainless-steel needle used to inject them is so fine that a caterpillar's
size in relation to the needle is roughly equivalent to that of a human
to the needles used in blood drives. One person can inject about 100 caterpillars an hour.
The virus used, though harmless to humans, would prove lethal to the caterpillars
by the fifth day if they were left alone. "They shrivel and turn black," Hale said. "It's
not a pretty sight." Instead, the infected caterpillars are frozen 3 1/2 days
after injection. Twenty to 60 caterpillars are then ground up, or "homogenized,"
with a foot-long device called a polytron, which resembles a butter churn
with a knife at the end of its stick. "We make a little milkshake out of them," Hale
said. "The first grinding is pretty gross. We separate out feet and
antennas and that sort of junk." By January, researchers had homogenized 1,500 caterpillars
that, lined up end to end, would be almost 100 feet long. Once their extract
had been purified, 100 microliters of the exchanger was obtained, which
amounts to about two drops. "It's only a few drops," Hale explained. "But
it's highly concentrated." The concentration of the exchanger in these few drops
is 21.5 milligrams per milliliter, which is roughly equal to the amount
of sugar found in dry champagne. Dry champagne may not be all that sweet, but for scientists
at MU, the taste of success is. "We had more protein than anyone else had ever made,"
Hale said proudly. The purified extract is shipped to the Center for Advanced
Research in Biotechnology in Maryland. After scientists crystallize the
extract, they bombard the crystal with an intense beam of X-rays. Researchers then study the way the deadly beams reflect
off the atoms inside the crystal. By examining the angles at which the
X-rays were scattered, researchers can figure out how atoms are arranged
in the protein. "We're really hoping that other people working with membrane proteins try our technique," Hale said. "We're hoping that this technique will really start a whole new era in studying membrane proteins." |