Molecular, Cellular, and Developmental Biology

7-27-09
Contacts:
Ravindra Singh, Biomedical Sciences, (515) 294-8505,
Dan Kuester, News Service, (515) 294-0704, kuester@iastate.edu

ISU researchers find possible treatment for Spinal Muscular Atrophy

Ravindra Singh

AMES, Iowa - Spinal Muscular Atrophy is the second-leading cause of infant mortality in the world.

Ravindra Singh, associate professor in biomedical sciences at Iowa State University's College of Veterinary Medicine, would like to see Spinal Muscular Atrophy lose its high ranking and even slide off the list altogether.

Most Spinal Muscular Atrophy sufferers -- more than 95 percent -- have a mutated or deleted gene called Survival Motor Neuron 1 (SMN1) that doesn't correctly do its job of creating functional SMN proteins. Singh's solution is to replace that poor-performing gene with another gene.

Humans need a certain level of SMN protein to ward off Spinal Muscular Atrophy.

When SMN1 fails to create functioning proteins, Spinal Muscular Atrophy is the result.

There is a gene already in humans that looks very much like SMN1, so much so that it's called SMN2. The SMN2 gene doesn't seem to serve any function that researchers can identify.

Singh has discovered a way of using SMN2 to produce the working SMN protein. When SMN2 makes enough SMN, it compensates for the mutated or malfunctioning SMN1 gene.

All proteins in human bodies are made by copying genes. This copy is called pre-mRNA.

Pre-mRNA then becomes mRNA by splicing out certain parts of the sequence that are non-coding, meaning they don't help the function of the gene.

These non-coding portions of the pre-mRNA are called intronic sequences, sometimes referred to as junk sequence because it is originally copied from junk DNA.

SMN2 normally doesn't produce normal protein because of the presence of a specific intronic sequence in the gene or DNA.

To make SMN2 behave as SMN1, Singh has introduced a small antisense oligonucleotide that blocks this specific intronic sequence.

When the intronic sequence is blocked, SMN2 produces normal proteins and acts, in effect, like SMN1.

"The significance of our work is that we have this stuff called junk DNA in SMN2," said Singh. "We found that we could get SNM2 to behave as SMN1 by introducing a small oligonucleotide. It is a very simple experiment if you think about it."

The resulting proteins are normal just like a regular cell - free from Spinal Muscular Atrophy.

"Our cells are healthy and survive," he said. "From that point of view, this is a major achievement."

Singh, along with his team Natalia Singh and Maria Shishimorova, both of Iowa State University's biomedical services department; Lu Cheng Cao, University of Massachusetts Medical School, Worcester; and Laxman Gangwani, Medical College of Georgia, Augusta, have their research highlighted as the cover story on this month's issue of the journal RNA Biology. Their research is the most downloaded story on the RNA Biology page of the Web site Landes Bioscience.

Spinal Muscular Atrophy affects 1 in 6,000 to 1 in 10,000 children born every year. One in 40 people are carriers of the disease -- they don't have the symptoms, but could pass the disease to their children.

Most children born with the most severe type of SMA die within two years. Using this junk sequence in SMN2 to restore the high levels of functional SMN protein could eliminate Spinal Muscular Atrophy caused by deletion or mutation in SMN1.

Singh believes this technology could also work treating other diseases.

"We know that Parkinson's disease, Alzheimer's disease, cystic fibrosis, multiple sclerosis and cancer all come from genes that are aberrantly spliced," he said.

"If this is a model disease, meaning we succeed in treating Spinal Muscular Atrophy, we will know how to correct splicing of other genes in other diseases," he said.

A short and smart oligonucleotide: A short antisense oligonucleotide (3UP8) targeting a specific intronic sequence corrects aberrant splicing of Survival Motor Neuron 2 (SMN2) and restores high levels of functional SMN protein in patient cells of spinal muscular atrophy (SMA). Prominent green dots (see in "SMN section") represent well-organized SMN bodies (gems) in the nucleus of the 3UP8-treated cells (bottom two panels). Gem contains another protein ZPR1 (see red dots in "ZPR1 section") that co-localizes with SMN protein in the nucleus of the cell (see white dots in "Merge section").

The control oligonucleotide (F8) that targets an unrelated sequence had no effect on SMN or ZPR1 levels in patient cells (top two panels). Cell were transfected with the same amount of antisense oligonucleotide (compare the color intensity in "Oligo section").

7-27-09
Contacts:
James Reecy, Animal Science, (515) 294-9269, jreecy@iastate.edu
Dan Kuester, News Service, (515) 294-0704, kuester@iastate.edu

Iowa State University researcher is part of cattle-genome mapping team

AMES, Iowa -- An Iowa State University faculty member is part of an international team that mapped the cattle genome.

James Reecy, director of the Office of Biotechnology and associate professor of animal science, took part in the annotation portion of the mapping project that manually examined the computer-generated genetic sequencing.

The group's findings are being published in the current edition of the journal Science. In the past, animal genome sequencing has focused mostly on small animals, such as rat and dog.

"This is the first time cattle were sequenced for a whole genome assembly," said Reecy. "It is important that we have the entire genome for a large animal."

The research was funded in part by the National Institutes of Health, which was interested in how this sequencing could help understand the previously mapped human genome.

"It was very beneficial for humans," said Reecy. "We found a new gene sequence in cattle that hadn't been seen in humans, which helped us to improve the annotation of the human genome."

The grouped mapped the genome of Taurine cattle, or non-humped cattle, which includes many breeds such as Angus, Shorthorn and most other beef and dairy breeds that are common to colder weather areas such as North America and Western Europe.

Cattle were chosen by researchers to be mapped because the species fills a need in the list of animals that can help understand animal and human genes. Mapping cattle also helps fill in needed information in the evolution of different species.

Cattle fit a unique evolutionary group. That, along with the agricultural benefit is why the species was chosen, said Reecy.

That benefit for farmers includes the possibility of learning more quickly which genes are associated with which traits of importance in cattle.

"We can increase the efficiency with which we can say, 'This gene is associated with this trait.'" said Reecy. "This will help us answer questions like, 'Can we improve milk production? Can we improve the healthfulness of beef?' This will allow us to do things quickly that otherwise would have taken us years."

The lead researchers for the project were from Georgetown University, Washington, D.C.; Baylor College of Medicine, Houston, Texas; and Commonwealth Scientific and Industrial Research Organization, Australia; and was funded partially through the United States Department of Agriculture, CSIRO and National Cattlemen's Beef Association.

May 09

Seeds of change

What genes and molecular mechanisms pilot plant cells along paths to become either starchy endosperm, transfer layer or aleurone is something Philip Becraft, associate professor in the Departments of Genetics, Development and Cell Biology and Agronomy wants to know.

Cell fate determination is an important area of study in all organisms, but understanding these pathways at the molecular level for seed development could help researchers boost the production of beneficial compounds naturally produced in grains, alter phosphorous content in animal manure to reduce negative environmental impacts (aleuronic mineral storage capacity includes phosphorus which finds its way from feed to manure to waterways) or even help the brewmeister brew better beer.

"Every developmental event is regulated by a specific mechanism," says Becraft. "Some dictated by positive regulators and others by negative regulators."

In corn, starch grains and protein bodies accumulate in mature starchy endosperm cells of the growing kernel. The transfer layer pumps maternal solutes into the endosperm. The aleurone, a single layer of cells surrounding the endosperm has a mineral storage function and "serves as a digestive tissue in the germinating seed to feed the growing seedling," says Becraft.

To map out events determining cell fate, Becraft exploits a genetic marker for anthocyanin, a purple pigment localized to the aleurone layer. By looking for mutations that disrupt or change the pigmentation of a corn kernel, Becraft is finding his way to specific genes that are controlling normal aleurone differentiation.

In a related project, Becraft and his colleagues have developed a high-throughput PCR (polymerase chain reaction) based method to rapidly screen mutations in genes essential for grain development.

Focusing on a class of genes called dek (for defective kernel)/emp (for empty pericarp), the team is amplifying insertion site DNA from a mutant corn line highly riddled with natural genetic elements called transposons - recognizable mobile genetic elements that randomly hop around the genome and can cause mutations when they insert into specific genes.

Characteristic sequence bits from the transposable elements are amplified along with small segments from the corn genes into which they have inserted. "If one of the inserts is responsible for the phenotype then we see it in all the mutant samples but not in the wild type," says Becraft, comparing wild type corn plant DNA with mutant siblings.

Using this method, Becraft expects to identify and determine the function of some 250-400 mutant genes within five years. "These belong to a class of some 350 to 900 essential genes," says Becraft, which are identified as a class of mutants causing lethal abortion events in seed development - another visually identifiable phenotype.

After determining the predicted biochemical function of the genes identified, "we look to see what is modified. Are the activities of these genes or gene products altered in any of the mutants?" These essential genes identify crucial steps in various cellular, developmental and metabolic processes and represent likely targets for manipulating seed properties.

Becraft's high-throughput methodology will appear in an upcoming issue of The Plant Journal.

4-8-09

ISU researcher identifies protein that concentrates carbon dioxide in algae

AMES, Iowa -- Increasing levels of carbon dioxide in the atmosphere are a concern to many environmentalists who research global warming.

The lack of atmospheric carbon dioxide (CO2) concentration, however, actually limits the growth of plants and their aquatic relatives, microalgae.

For plants and microalgae, CO2 is vital to growth. It fuels their photosynthesis process that, along with sunlight, manufactures sugars required for growth.

CO2 is present in such a limiting concentration that microalgae and some plants have evolved mechanisms to capture and concentrate CO2 in their cells to improve photosynthetic efficiency and increase growth.

An Iowa State University researcher has now identified one of the key proteins in the microalgae responsible for concentrating and moving that CO2 into cells.

"This is a real breakthrough," said Martin Spalding, professor and chair of the department of genetics, development and cell biology. "No one had previously identified any of the proteins that are involved in transporting CO2 in microalgae."

The main protein that Spalding and his team have identified that is responsible for transporting CO2 is called HLA3.

The research by Spalding; Deqiang Duanmu, a graduate student in Spalding's department; and Amy Miller, Kempton Horken and Donald Weeks, all from the University of Nebraska, Lincoln; is published in the current issue of the journal Proceedings of the National Academy of Sciences of the United States of America.

Now that the HLA3 protein has been identified, Spalding believes there are several possibilities to use the gene that encodes this protein.

The recent explosion of interest in using microalgae for production of biofuels raises the possibility of increasing photosynthesis and productivity in microalgae by increasing expression of HLA3 or other components of the CO2 concentrating mechanism, according to Spalding.

Since all plants need CO2 to thrive, introducing the HLA3 gene into plants that do not have the ability to concentrate CO2, could help those plants grow more rapidly.

Spalding says several plants would be candidates for the HLA3 protein.

"One of the things we've been working on is the prospect that we may be able to take components of the CO2 concentrating mechanism for microalgae, such as this HLA3, and put it into something like rice and improve photosynthesis for rice," said Spalding.

Rice and other commodity crops such as wheat and soybeans do not have any CO2 concentrating mechanism.

Contacts:
Martin Spalding, Genetics, Development and Cell Biology, (515) 294-1749,

4-1-09

Researchers examine bacterial rice diseases, search for genetic solutions

AMES, Iowa -- As a major food source for much of the world, rice is one of the most important plants on earth.

Keeping it safe from disease has become, in part, the task of a group of three researchers from Iowa State University and one from Kansas State University.

The researchers are looking at two bacterial diseases of rice. The most costly is bacterial blight of rice, which is caused by a bacterium called Xanthomonas oryzae pathovar oryzae, and can diminish yield by up to 50 percent.

"This is the most important bacterial disease in rice, and in some areas, it is the most important rice disease of any kind," said Adam Bogdanove, an associate professor of plant pathology who is part of the ISU research team.

The team is also studying bacterial leaf streak of rice caused by the closely related bacterium Xanthomonas oryzae pathovar oryzicola. Bacterial leaf streak is usually not as damaging as bacterial blight, but it is increasing in importance in many areas of the world, particularly Southeast Asia.

These bacteria damage rice by entering the plant and taking control of certain rice cell processes, eventually killing the rice cells. Pathovar oryzae does this in the vascular system of the plant, which typically allows the bacterium to spread faster and cause more damage than is its cousin, oryzicola, which is limited to growth in the tissue between the veins.

Some types of rice are naturally resistant to the Xanthomonas bacteria. Bogdanove and other researchers -- Bing Yang, Iowa State assistant professor of genetics development and cell biology; Dan Nettleton, Iowa State professor of statistics; and Frank White, principal investigator and professor of plant pathology at Kansas State University, Manhattan -- are researching why some types of rice are naturally resistant to the bacteria.

In rice varieties that are resistant to the diseases, the team is exposing the plants to the two bacteria. They then check to see which plant genes are activated, and to what extent.

By identifying which genes are turned on, Bogdanove believes the team can identify the genes that are making the plants resistant.

"We are looking at genes of successful plants," he said. "What genes are active and when and how much they are being turned on."

Bogdanove hopes that this effort will aid in breeding the resistance into cultivated varieties that are currently susceptible to the diseases.

Another aspect of the research is aimed at discovering how the bacteria change gene expression in susceptible rice plants.

"If we understand which genes are being manipulated by the pathogens in disease, we can look into different varieties and wild relatives of rice for variants of these genes that are immune to manipulation and bring these genes into cultivated varieties," said Bogdanove. "The idea is to reduce or eliminate susceptibility altogether."

Rice is the major food staple for more than half the world's population. In the United States, rice is planted on almost 3 million acres with yields of around 7,000 pounds per acre in 2007, according the U.S. Department of Agriculture.

American producers grow 95 percent of the rice eaten in this country and the United States is a major exporter as well, according to Bogdanove.

In addition to the benefits to rice, the research should be helpful in understanding and controlling diseases in other cereal crops.

Funding for the project comes from the National Science Foundation through Kansas State University, the lead institution on the project. Of the $3 million award for the project, $2 million is going to Iowa State.

Contacts:
Adam Bogdanove, Plant Pathology, (515) 294- 43421,

3-2-09

Iowa State researchers receive awards for Parkinson's Disease study

AMES, Iowa -- Two researchers in the Iowa Center for Advanced Neurotoxicology (ICAN) at Iowa State University have received awards totaling more than $4 million from the National Institute of Neurological Disorders and Stroke (NINDS), a component of the National Institutes of Health (NIH). The awards represent innovative approaches to funding biomedical research in Parkinson's Disease by NINDS.

Dr. Anumantha Kanthasamy (picture top left), a faculty member in the Department of Biomedical Sciences at ISU's College of Veterinary Medicine and director of ICAN, is the first ISU funding recipient from a new NIH Multi-Principal Investigators Award program. This award is intended to foster interdisciplinary biomedical research among multiple institutions.

Under this award, Kanthasamy will collaborate with Dr. Balaraman Kalayanaraman, chair and professor of the Department of Biophysics at the Medical College of Wisconsin, in developing a novel class of antioxidant-based therapeutic agents for the treatment of Parkinson's Disease. A total of $2.77 million in NIH funding will be provided to their project. Iowa State University will receive approximately $1.4 million from the award over the next five years.

Dr. Arthi Kanthasamy (picture bottom left), also an ICAN researcher in neurotoxicology and a faculty member in the Department of Biomedical Sciences at ISU, has received an award from the NINDS' New Investigator Award program. She will receive a total of $1.28 million for her work in studying the brain inflammatory mechanisms in Parkinson's Disease models. The New Investigator program is designed to support new researchers in transitioning to independence in their research careers. Arthi Kanthasamy currently researches degenerative processes in stroke models and also teaches pharmacology and histology courses to graduate students and veterinary medicine students at ISU.

"We are especially proud to have both Drs. Kanthasamy here in the College of Veterinary Medicine," said Dr. John Thomson, dean of the college. "They are each outstanding neuroscience researchers and their awards are all the more distinguished by the fact that the National Institute of Health funding is strictly based on scientific merit. To receive not only one but two awards for work in Parkinson's Disease reflects positively on the quality of research being conducted at ISU."

"We also commend Dr. Anumantha Kanthasamy for his innovative approach in developing an inter-institutional partnership to leverage the strengths of both Iowa State University and the Medical College of Wisconsin," Dean Thomson emphasized. "The effect of this collaboration will yield multiple benefits and advance existing programs for both institutions."

The NIH funding brings additional benefits identified by Dr. Anumantha Kanthasamy.

"I am especially pleased to receive this funding, not only because it supports what I believe is extremely important work in Parkinson's Disease but also because it allows us to recruit and train new graduate students in neurotoxicology and keeps Iowa State at the forefront of biomedical research."

"Both Arti and Anumantha Kanthasamy are outstanding scientists who have already made very significant contributions to their field," says Chitra Rajan, associate vice president for research at Iowa State. "We are extremely proud of their accomplishments and wish them every success."

Anumantha Kanthasamy is a Distinguished Professor and the Lloyd Chair in the Department of Biomedical Sciences in the College of Veterinary Medicine. He is also the chair of the Interdepartmental Toxicology Graduate Program and founding director of ICAN. His research in neurodegenerative disorders has established him as a leading neuroscientist and brought international distinction to Iowa State University. Anumantha Kanthasamy has made several fundamental advances as well as applied contributions that have resulted in two patents and the establishment of a new business in the ISU Research Park.

"Their winning these extremely competitive awards from NIH will allow them to take their research to the next level and bring visibility and prominence to ISU in research on neurotoxicology and neurodegenerative diseases," said Ted Okiishi, interim vice president for research and economic development at Iowa State.

ICAN was created to promote interdisciplinary research related to neurotoxicological problems in both animals and humans. Neurotoxicology bridges the scientific fields of toxicology and neuroscience and plays a key role in the health of humans and animals, as well as related health industries, the economy, and the environment.

Contacts:
Tom Ligouri, College of Veterinary Medicine, (515) 294-4257,

David Hannapel

3-3-09
Contacts:
David Hannapel, Horticulture, (515) 294-9130,

ISU research examines how plants produce high-energy storage organs

AMES, Iowa -- Understanding how plants produce storage organs that humans use as food would be a valuable tool for science and for a hungry world. Iowa State University researcher David Hannapel, professor in horticulture, thinks he has found a key to figuring out the process.

Hannapel studies potatoes and the process that leads to tuber formation. Potatoes are the fourth most important food crop in the world. Like other plants, potatoes collect sunlight in the leaves and turn that energy into sugars using carbon dioxide. In potatoes, late in the growing season, the sugars in the leaves are delivered to underground stems during the process of making starch in the edible tubers.

"We've always known that there was a signal activated in the leaf that was sent down the plant to activate tuber formation," Hannapel said. "But the identity of that signal has never been confirmed."

Recent discoveries have demonstrated the role of a full-length mobile ribonucleic acid (RNA) molecule in a signaling system that activates tuber formation, Hannapel said. RNA is a molecule present in all living organisms that plays a part in protein production and transmission of genetic information.

Hannapel is part of Iowa State University's Plant Sciences Institute and is working with Guru Rao, professor and chair of the Department of Biochemistry, Biophysics and Molecular Biology.

Grants from the National Science Foundation (NSF) and the U.S. Department of Agriculture totaling $3.15 million are funding a study on how plants use RNAs as long-distance signalers. Other members of the research team include RNA biologist Jeff Coller of Case Western Reserve University, Cleveland, Ohio; and William J. Lucas of the University of California-Davis, who is an expert on RNA transport in plants.

According to Hannapel's theory, the signal molecule RNA moves from the leaves to the tubers and communicates to the plant when to activate the pathway that leads to tuber formation. This strand of RNA makes a protein called BEL5. BEL5 protein acts as a master switch that activates other genes that signal to the plant to manufacture its tubers underground.

The BEL5 gene is activated by sunlight in the leaf. The RNA (and other proteins that escort and protect BEL5 on its trip) recognizes when the days are getting shorter and this induces the RNA to start moving.

Hannapel has studied tuberization for more than 20 years and knows that understanding the process takes time.

"Right now, we have more questions than answers," he said. "But most likely RNA-binding proteins are involved that protect the RNA and deliver it to its site of function." Hannapel's research has already verified that the BEL5 RNA is responsible for signaling the plant to make tubers. "We've taken the RNA of BEL5 and over-expressed it in potato plants, and that causes the plant to produce more potatoes in a shorter period of time," said Hannapel. Having figured out the function of BEL5, Hannapel now wants to understand how the signal RNA works.

And scientific knowledge is moving fast in this area. "It was just three years ago that we discovered these RNAs were moving," he said. Since then, advances have been common and widespread. Scientists now know that there are hundreds of RNAs that traffic through many different plants.

"Full-length, mobile RNAs that travel long distances in plants and act as signals for development and defense are a novel idea in plant biology. The value of our work is that it provides a model for understanding how such signal RNAs are moving and what determines their final destination," Hannapel said.

Rao will work to identify and characterize the proteins that recognize mobile RNAs and facilitate their movement.

"We will use both targeted and random approaches to do this," says Rao. The work also has the possibility of boosting potato production.

"We are fortunate in that the RNAs we study in this process activate tuber formation and in this way, regulate tuber yields," he said. "So this system can potentially be used to enhance crop productivity. When considering calories generated for human consumption per acre, potato is the most productive food crop on the planet and is a critical staple in many developing countries."

02-23-09
Contacts:
Yeon-Kyun Shin, Biochemistry, Biophysics and Molecular Biology, (515) 294-2530,

Cholesterol-reducing drugs may lessen brain function, says ISU researcher

AMES, Iowa -- Research by an Iowa State University scientist suggests that cholesterol-reducing drugs known as statins may lessen brain function.

Yeon-Kyun Shin, a biophysics professor in the department of biochemistry, biophysics and molecular biology, says the results of his study show that drugs that inhibit the liver from making cholesterol may also keep the brain from making cholesterol, which is vital to efficient brain function.

"If you deprive cholesterol from the brain, then you directly affect the machinery that triggers the release of neurotransmitters," said Shin. "Neurotransmitters affect the data-processing and memory functions. In other words -- how smart you are and how well you remember things."

Shin's findings will be published in this month's edition of the journal Proceedings of the National Academy of Sciences of the United States of America.

Cholesterol is one of the building blocks of cells and is made in the liver. Low-density lipoprotein (LDL) -- often referred to as bad cholesterol -- is cholesterol in the bloodstream from the liver on the way to cells in the body. High-density lipoprotein (HDL) -- so-called good cholesterol -- is cholesterol being removed from cells. Too much LDL going to cells and not enough being removed can lead to cholesterol deposits and hardening of the cells.

"If you have too much cholesterol, your internal machinery is not going to be able to take away enough cholesterol from the cells," said Shin. "Then cells harden and you can get these deposits."

Cholesterol-reducing statin drugs are helpful because they keep the liver from synthesizing cholesterol so less of the substance is carried to the cells. This lowers LDL cholesterol.

It is the function of reducing the synthesis of cholesterol that Shin's study shows may also harm brain function.

"If you try to lower the cholesterol by taking medicine that is attacking the machinery of cholesterol synthesis in the liver, that medicine goes to the brain too. And then it reduces the synthesis of cholesterol which is necessary in the brain," said Shin.

In his experiments, Shin tested the activity of the neurotransmitter-release machinery from brain cells without cholesterol present and measured how well the machinery functioned. He then included cholesterol in the system and again measured the protein function. Cholesterol increased protein function by five times.

"Our study shows there is a direct link between cholesterol and the neurotransmitter release," said Shin. "And we know exactly the molecular mechanics of what happens in the cells. Cholesterol changes the shape of the protein to stimulate thinking and memory."

While reducing the cholesterol in the brain may make you have less memory and cognitive skills, more cholesterol in the blood does not make people smarter. Because cholesterol in the blood cannot get across the blood brain barrier, there is no connection to the amount of cholesterol a person eats and brain function.

Shin says that for many people taking cholesterol-lower statins can be very healthful and they should listen to their doctor when taking medication.

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