Research Reports , a feature service by the Office of Research Reports of the National Institute of General Medical Sciences, is designed to let you know what some of our grantees are doing. A list of some recent NIGMS grant awards is also included.
This issue of Research Reports contains the following stories:
New Insights into the Genetic Basis of Hypertension
An NIGMS-supported scientist has been using a technique called gene targeting, which is itself the result of many years of NIGMS-supported research, to study the effects of several important genes on high blood pressure.
Illuminating the Signposts on a Cellular Pathway
This story presents an in-depth look at the work of two NIGMS-supported biochemists who are elucidating the many complex steps involved in the pathway that signals cells to divide. This untargeted basic research is yielding knowledge that is of relevance to many different disease processes.
Despite decades of research and great popular interest, the exact cause of high blood pressure remains elusive. The condition, also called hypertension, affects an estimated one in four American adults and can lead to heart attacks, strokes, and kidney failure. It is one of the nation's most serious and most costly medical problems.
Hypertension is also one of the most complex medical problems. It can result from multiple genetic and behavioral factors. In fact, different genetic defects may be responsible for hypertension in different people, according to NIGMS grantee Oliver Smithies, D.Phil. (Oxon), Excellence Professor of Pathology at the University of North Carolina, Chapel Hill.
"Some individuals may be hypertensive because they have a slight defect in [hypothetical] genes B, Q, and Z. Others might be hypertensive because they have a difference in B, M, and S," Dr. Smithies said.
Each of the defects might be so slight that the genes still function within the normal range, Dr. Smithies said. It may just be an unlucky genetic combination that causes hypertension.
The goal of Dr. Smithies' work is to determine which genes regulate blood pressure and therefore might contribute to hypertension. He conducts his studies in mice, which have normal blood pressure values similar to those of humans.
For the past decade, many researchers have been investigating the genetic basis of diseases by using a technique called gene targeting, which enables them to specifically alter chosen genes. Most commonly, gene targeting is used in mice to completely inactivate, or "knock out," a gene the researchers suspect is involved in causing a disease. If the animals develop the disease, or symptoms of it, scientists have evidence that the inactivated gene probably plays a role in the disease.
But for studies of hypertension, which may result from slightly altered rather than entirely ineffective genes, the "knockout" scheme doesn't work well. So Dr. Smithies and his collaborators devised a new way to use gene targeting to study diseases such as hypertension.
This technique, which was developed with NIGMS support, was first described in April 1994 in the Proceedings of the National Academy of Sciences . It enables researchers to breed mice with anywhere from zero to four copies of a specific gene. Normal mice have two copies of most genes. Animals with extra copies of a gene have higher levels of the protein coded by that gene, and animals with fewer copies of the gene have lower levels of the protein. By breeding mice with one to four copies of a gene, scientists are able to investigate the effects of having low, normal, high, and extra-high amounts of the protein coded by that gene. These moderate genetic alterations are more representative of the type of defects seen in hypertension than those produced in "knockout" experiments.
This use of gene targeting is also advantageous because it places the extra genes in their natural chromosomal location. In the original insertion experiments, pioneered about 20 years ago, an inserted gene--usually from another species--pops into a random place in the chromosome. This can disrupt normal genes, and may necessitate altering many animals before finding one in which the inserted gene functions.
Dr. Smithies' laboratory has used gene targeting to study several genes involved in controlling blood pressure. These include the genes for angiotensinogen, angiotensin II receptor, and atrial natriuretic peptide.
Angiotensinogen (AGT) is a protein produced in the liver that circulates in the bloodstream. Through two enzymatic cleavages, it is converted into the potent hormone angiotensin II, which increases blood pressure.
Scientists have recognized that, in humans, mutations somewhere near the AGT gene raise blood pressure. Dr. Smithies' group set out to determine whether alterations within the AGT gene itself can raise blood pressure.
Using gene targeting, they produced mice with zero, one, two, three, or four copies of the AGT gene at its normal chromosomal location. They found that the blood pressure values of the mice increased almost linearly with the number of AGT genes they carried. The increase was small--about 8 mm Hg (millimeters of mercury) per AGT gene--but was significant and reproducible (see Figure 1).
Figure 1. Effect of the number of AGT genes on blood pressure in mice.
"This shows that a change in the angiotensinogen gene makes a change in blood pressure that the animal can't compensate for," Dr. Smithies said.
This is significant, he said, because mice, as well as humans, have a battery of regulatory mechanisms to maintain their blood pressure. Somehow, an alteration in the AGT gene overcomes those mechanisms and results in mice with increased blood pressure. It is very possible that a similar effect would occur in humans who had defects in the AGT gene, he said.
Angiotensin II Receptor Gene
Although the end-product of angiotensinogen, angiotensin II, can powerfully affect blood pressure, it cannot do so without binding to a specific cell-surface receptor. Dr. Smithies wanted to determine whether the blood pressure of mice would be affected if he genetically crippled this receptor.
Angiotensin II binds to two known receptor types, types 1 and 2. Type 1 receptors are further divided into A and B subtypes. Dr. Smithies and his collaborators chose to study the type 1A receptor, which predominates in most tissues and is thought to mediate the known functions of angiotensin II.
Using gene targeting, the investigators bred mice with zero, one, or two copies of the type 1A receptor gene. Again, the results were significant. Mice with only one copy of the gene had blood pressure values that were reduced by 12 mm Hg to 17 mm Hg, depending on the method of measurement. Mice that completely lacked the receptor gene had blood pressure values that were even more reduced--by 24 mm Hg to 43 mm Hg.
These results show that with only one copy of, or without, the gene for type 1A angiotensin II receptor, mice have lower blood pressure, indicating that the gene is responsible for keeping blood pressure up at a normal level. The study also suggests that mutations in this gene may affect blood pressure in humans as well as mice.
Atrial Natriuretic Peptide Gene
In some human hypertensive patients, a diet high in salt increases blood pressure. To better understand why this happens, Dr. Smithies and his coworkers investigated the genetic basis of hypertension in combination with high salt intake. In this study they investigated the role of atrial natriuretic peptide (ANP), a 28-amino-acid polypeptide produced in the heart. ANP lowers blood pressure when given as a drug, but scientists still do not know whether it is involved in hypertension in humans.
Atrial natriuretic peptide is produced by the enzymatic cleavage of a larger precursor, proANP. Dr. Smithies' laboratory used gene targeting to produce mice with zero, one, and two copies of the proANP gene. They then put the mice on diets with different amounts of salt for at least 2 weeks to see how their blood pressure was affected.
When fed diets with standard levels of salt, the blood pressure of mice that lacked the proANP gene was elevated by 8 mm Hg (see Figure 2). These results show that without the gene for ANP, mice have higher blood pressure, indicating that the proANP gene is responsible for keeping blood pressure down to within a normal range.
Figure 2. Effect of the number of proANP genes on blood pressure in mice.
Because human hypertensive patients are more likely to have slightly defective genes than to have entirely dysfunctional ones, the researchers next measured the blood pressure of mice with only one functional copy of the proANP gene. These mice had normal blood pressure when fed diets with a standard amount of salt. But when fed high-salt diets, the mice were hypertensive, with blood pressure elevated by 27 mm Hg. On the same high-salt diet, control mice with two copies of the proANP gene maintained normal blood pressure by drinking more water and excreting salt in their urine (see Figure 3).
Figure 3. Effect of the number of proANP genes and salt intake on blood pressure in mice.
Clearly, alterations in the proANP gene cause salt-sensitive hypertension in mice. It is very possible that a similar effect occurs in humans. These data may cause clinical researchers to re-evaluate some of their earlier studies of ANP, Dr. Smithies said.
About 10 years ago, researchers recognized that ANP might play a role in hypertension in humans, he said. Initially, scientists thought it would play an important role, but their enthusiasm was dampened after a series of family and genetic studies yielded conflicting results. These studies didn't examine the critical variable--the effect of high-salt diets on blood pressure, Dr. Smithies said.
"If we look at the data with hindsight, we see it would be better to do these experiments again now and test individuals specifically for their sensitivity to salt," he said. "Then we might find that ANP is important in human hypertension."
Dr. Smithies and his collaborators are also using gene targeting to examine other genes potentially involved in blood pressure regulation. These include renin and angiotensin-converting enzyme, the two enzymes that convert AGT into angiotensin II. Soon he hopes to study mice with defects in multiple genes, a condition that mimics hypertension in humans.
Each of these studies teaches us more about the genes involved in blood pressure regulation and provides scientists with another piece to fit into the complex puzzle of hypertension. In the future, these pieces may even help achieve a cure for the condition that --directly or indirectly--causes about 700,000 U.S. deaths a year.
Dr. Smithies' coworkers on these studies included Hsung-Suk Kim, Ph.D., John H. Krege, M.D., Masaki Ito, Ph.D., Thomas M. Coffman, M.D., and Simon W.M. John, Ph.D.
Because of its direct implications in human hypertension, Dr. Smithies' work is funded not only by NIGMS, but also by the National Heart, Lung, and Blood Institute and the National Institute of Diabetes and Digestive and Kidney Diseases.
Alisa Zapp Machalek
John SWM, Krege JH, Oliver PM, Hagaman JR, Hodgin JB, Pang SC, Flynn TG, Smithies O. Genetic decreases in atrial natriuretic peptide and salt-sensitive hypertension. Science 1995; 267:679-81.
Kim H-S, Krege JH, Kluckman KD, Hagaman JR, Hodgin JB, Best CF, Jennette JC, Coffman TM, Maeda N, Smithies O. Genetic control of blood pressure and the angiotensinogen locus. Proc Natl Acad Sci USA 1995; 92:2735-9.
Ito M, Oliverio MI, Mannon PJ, Best CF, Maeda N, Smithies O, Coffman TM. Regulation of blood pressure by the type 1A angiotensin II receptor gene. Proc Natl Acad Sci USA 1995; 92:3521-5.
Smithies O, Kim H-S. Targeted gene duplication and disruption for analyzing quantitative genetic traits in mice. Proc Natl Acad Sci USA 1994; 91:3612-5.
Smithies O, Maeda N. Gene targeting approaches to complex genetic diseases: atherosclerosis and essential hypertension. Proc Natl Acad Sci USA 1995; 92:5266-72.
Until recently, scientists studying the biochemical steps leading to cell division entered unknown territory when they looked at the middle part of the pathway. They knew how growth factors (or "mitogens") attach themselves to the cell's outer membrane and they had an idea of how the cell eventually reacts to the incoming signal by dividing, but the steps between the beginning and the end of the path remained in shadow. Now, thanks to a flood of findings from many laboratories, the sliver of space between a cell's membrane and its nucleus has begun to look like a Cecil B. De Mille production, featuring a biochemical "cast of thousands."
Remarkably, the biochemical players involved in cell signaling are substantially the same in all living cells, whether yeast or human. The high degree of similarity in the signaling pathways of such divergent organisms indicates just how crucial these pathways are. All cells must react quickly and appropriately to incoming signals, and a breakdown anywhere in the system can have dire consequences. For example, if the message to divide is not inactivated after division occurs, then unregulated cell division--even cancer--may result.
A detailed picture of a cell signaling pathway--with every step clearly marked--will not only give scientists a better idea of how healthy cells work, it will also show clinicians precisely where to aim their therapies when they want to fix a signaling system that has become snarled.
Among the researchers who have provided a more detailed picture of the steps leading to cell division are NIGMS grantees Alan Wolfman, Ph.D., of the Cleveland Clinic Foundation and Michael Weber, Ph.D., of the University of Virginia. Drs. Wolfman and Weber and their colleagues have focused their attention on the ras signaling pathway. The ras gene is one of a family of "oncogenes" that was discovered almost two decades ago. Oncogenes were named for their first-identified ability to cause tumors ("onkos" is Greek for "mass"), but they only cause cancer if something goes wrong.
Ordinarily, ras provides the blueprint for a protein (called Ras) that is produced when a cell is preparing to divide. Like a light switch, Ras exists in one of two states, "on" or "off." Most of the time, Ras is turned off. But when a growth factor arrives at the cell's outer membrane, Ras gets turned on and relays the "divide" message to the next player in the signal pathway. (Ras and the growth factors do not interact directly. Instead, a recently discovered complex of two proteins attached to the inner face of a cell's membrane serves as an intermediary to pass the growth factor signal from the outside world to Ras.)
But what is the next player after Ras? The scientists did not know. They realized that somewhere down the line an enzyme named mitogen-activated protein kinase (MAP kinase) would spring into action as a final regulator of cell division. (Recall that "mitogen" is another term for growth factor. Kinase is a term for a specific group of enzymes involved in many cellular processes, including cell division.) But, due to its structural peculiarities, Ras cannot "turn on" MAP kinase directly. There has to be at least one intermediary. Drs. Weber and Wolfman and their coworkers set out to find it. The researchers used a straightforward biochemical approach to trap their molecular quarry. They coated silicon beads with purified Ras protein, then splashed various candidate proteins over the beads to see whether any of them formed a complex with Ras. It appears that an important player in the process is a protein called Raf-1 kinase, which itself is made by an oncogene called raf .
Once it is activated, the Raf-1 kinase kicks off a "cascade" of protein kinases, which results ultimately in a signal entering the cell's nucleus. Each kinase in the chain activates many molecules of the next kinase in line. Unfortunately, when speaking of these kinases, scientists sound as if they are in an echo chamber. Raf-1 kinase is, strictly speaking, a kinase kinase kinase, meaning that it turns on a kinase (namely, MAP kinase kinase), which (at long last!) activates MAP kinase. Dr. Weber points out that protein cascades, while aggravating in nomenclature, are a very efficient means of amplifying an incoming signal. A single molecule of Raf-1 kinase can activate many molecules of MAP kinase kinase, every one of which may activate even larger numbers of MAP kinase molecules.
The discovery of how the MAP kinase is activated was something of a breakthrough in the scientists' understanding of the ras signaling pathway, but it was only the beginning. The researchers have also been trying to learn more about certain MAP kinase kinases, called MEK-1 and MEK-2. Using the same method they had employed earlier, the scientists determined that the cell's "preferred" pathway involves a three-part complex of the Ras, Raf-1 kinase, and MEK-1 proteins. MEK-2, although structurally very similar to MEK-1, differs from it in the very region where, the scientists believe, MEK-1 binds to Raf-1 kinase. The presence of both MEK-1 and MEK-2 in normal cells may be an instance of biological redundancy, the researchers say. Such redundancy permits the cell to regulate its response to incoming growth signals. So, for example, the cell can respond with two waves of MAP kinase production, the first--mediated by MEK-1--peaking a few minutes after the arrival of a mitogen, and another--mediated by MEK-2--yielding increased levels of MAP kinase a few hours after the signal's arrival.
Dr. Weber has also been searching for how the Raf-1 kinase is activated and then turned off. He and his colleagues have now been able to inactivate the protein by a process called dephosphorylation, and also to block this inactivation by adding additional proteins. This test tube work has led them to postulate the existence of proteins that may regulate Raf-1 kinase activation in living organisms.
This very basic research in biochemistry could have many clinical implications. Elucidation of the steps in the ras signaling system will aid understanding of all processes in which the growth or differentiation of cells and tissues plays a part, including the development of embryos, wound healing, limb regeneration, the formation of blood cells, and the growth of nerve cells.
This sampling of titles from the more than 3,000 grant awards NIGMS makes each year is designed to give you an idea of the basic research the Institute supports. A computer printout of all current (new and continuing) NIGMS grants at your institution is available from us, if you request it. Among the awards that were made in the first half of 1995 are the following:
Arizona State University Joann C. Williams: "Structure/Function Relations in Photosynthetic Systems"
Beckman Research Institute Maria T. Mas: "Folding and Dynamics of a Bi-Domain Enzyme"
Brigham and Women's Hospital Igor Kissin: "Acute Tolerance in Opioid/Benzodiazepine Interactions"
Carnegie-Mellon University Eckard Munck: "0 2 Activation by Mossbauer Spectroscopy"
City College of New York Marilyn R. Gunner: "Electron and Proton Transfer in Reaction Centers"
Cleveland Clinic Foundation Alan S. Wolfman: "Association of a MAP Kinase Activator with p21Ras-GTP"
Dana-Farber Cancer Institute Richard D. Kolodner: "Enzymatic Mechanisms of Genetic Recombination in Yeast"
Fred Hutchinson Cancer Research Center Robert W. Levis: "Chromosomal Telomeres in Drosophila"
Georgetown University Mark Danielsen: "Multiple Forms of Dopamine Beta Hydroxylase"
Health Science Center at Syracuse Christopher E. Turner: "Structure and Function of Paxillin"
Indiana University, Bloomington Milos V. Novotny: "Ultrasensitive Methods for Glycoconjugate Analysis"
Jackson Laboratory Eva M. Eicher: "Cytogenetics of the Mouse"
Kansas State University A.S. Borovik: "Helical and Cavity Motifs About Metal Ions"
Marine Biological Laboratory Shinya Inoue: "Mitosis and Related Motility Directly in Living Cells"
Maryland Biotechnology Institute John Moult: "Computer Algorithm for Modeling Protein Structure"
Medical College of Wisconsin Jung-Ja P. Kim: "Structure and Mechanism of a Flavoprotein Dehydrogenase"
Michigan State University Shelagh M. Ferguson-Miller: "Organization and Control of Electron Transfer Chains"
Mount Sinai School of Medicine Saul Maayani: "5-HT Receptors and Their Effectors"
New York University Pamela Cowin: "Desmosomal Proteins and the Cell"
Northwestern University Robert L. Letsinger: "New Methods for Synthesis of Polynucleotides"
Oklahoma Medical Research Foundation Carol F. Webb: "B Cell Regulation by Interleukin 5 Plus Antigen"
Rice University Kathleen S. Matthews: "Physical and Genetic Studies of Regulatory Proteins"
San Diego State University Constantine D. Tsoukas: "CR2 Receptors on Thymocytes"
Scripps Research Institute James A. Hoch: "Genetic Control of Development"
Sloan-Kettering Institute for Cancer Research Kenneth J. Marians: "Topoisomerases and DNA Replication"
State University of New York, Binghamton Eugene S. Stevens: "Determining Carbohydrate Conformation from CD"
University of Alabama at Birmingham Vytas A. Bankaitis: "Phospholipids in Golgi Secretory Function"
University of California, Irvine David A. Brant: "Conformation and Dynamics of Polysaccharides"
University of California-Lawrence Berkeley Laboratory Stephen R. Holbrook: "Structural Characterization of Internal Loops in RNA"
University of California, Santa Barbara Alison Butler: "Biochemistry of Vanadium and Iron"
University of Delaware Douglass F. Taber: "Synthesis of Taxol"
University of Houston-University Park Harold L. Kohn: "Mode of Action of Bicyclomycin"
University of Kentucky Davy Jones: "Molecular Determinants of a Parasite Regulatory Mediator"
University of Massachusetts, Amherst Barbara A. Osborne: "Molecular Analysis of Apoptosis in T-Cells"
University of Mississippi Medical Center Mark O.J. Olson: "Structure and Function of Nucleolar Nonhistone Proteins"
University of Southern California Myron F. Goodman: "Error Correction in DNA Synthesis--A Biochemical Study"
University of Tennessee, Knoxville Donald E. Olins: "Development of Electron Microscope Tomography"
University of Texas Medical Branch, Galveston Louise Prakash: "Repair of DNA Damaged by UV Irradiation in Yeast"
University of Washington Leland H. Hartwell: "Genetic Analysis of the Eukaryotic Cell Cycle"
University of Wyoming Peter E. Thorsness: "Genetic Analysis of Mitochondrial Integrity"
Wake Forest University Leslie B. Poole: "Mechanistic Studies of Alkyl Hydroperoxide Reductase"
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