Research Interests
Dennis Bobilya
We are using our in vitro model of the blood-brain barrier to investigate various aspects of nutritional and biomedical physiology. We are investigating nutrient transport into the brain, with special emphasis on how the BBB attempts to maintain brain zinc homeostasis during periods of zinc malnutrition. This research includes studies to characterize the mechanism(s) of zinc transport by mammalian cells. We are also evaluating the consequences of heat stress and head trauma on the integrity of the BBB. We are initiating studies on drug transport across the BBB into the brain.
Current Projects
Grant # R15 NS35285-02.
Influence of Zinc Status on the Blood-Brain Barrier.
What is the problem? Zinc is an essential nutrient that has hundreds of critical physiological roles important to human health and well-being; when zinc metabolism is perturbed, human health suffers. The prevailing evidence indicates that even a transient period of mild zinc deficiency can result in subclinical changes in eating behavior (Henkin 1975, Catalanotto 1978, Shatzman 1981, Ruz 1991) and learning behavior and aggression (Sandstead 1985, Golub 1988), as well as impaired gestational development (Keen 1993), growth rate (Golub 1988, Hambidge 1976), and immune function (Ruz 1991, Vruwink 1991). Elevated concentrations of zinc in brain cerebrospinal fluid has been implicated in the induction of Alzheimer's disease, Parkinson disease, and other neurological disorders (Bush 1994, Lee 2000). Many possible neurological roles have been proposed for zinc, yet little is known about the maintenance of cerebral zinc homeostasis.
Our hypothesis is that the blood-brain barrier actively regulates zinc movement between the brain and the circulatory system, and that this zinc transport process is under physiological control. This regulation comprises the mechanism by which the brain maintains zinc homeostasis.
What do we propose to do about it? The blood-brain barrier is the interface between the cerebrospinal fluid of the brain and the plasma of the vascular system. We have developed an in vitro model of the blood-brain barrier (Bobilya 1995) and propose to use it to examine the regulation of zinc transport and metabolism at this important physiological location in the body (Bobilya 1997). We have experience manipulating the zinc status of endothelial cells in vitro (McClung 1999).
We propose to induce a moderate zinc deficiency in cultures of porcine brain capillary endothelial cells (BCEC) and then evaluate their transport of zinc (in and out of BCEC) and their expression of individual proteins to identify candidates for participation in zinc transport and brain-zinc homeostasis. We recently refined procedures for imposing an in vitro zinc deficiency on cultures of artery endothelial cells. The artery endothelial cells increased their uptake of zinc by 66% in response to moderate zinc deficiency while remaining healthy. Kinetic analysis determined this increase in uptake to be due to an increase in transporter number. Meanwhile, we have refined our procedures for isolating brain capillaries and cultivating BCEC. We expect the BCEC to have an even greater response to zinc deficiency than the artery endothelial cells, due to their importance in brain homeostasis.
The BCEC are responsible for maintaining the integrity and homeostasis of the interstitial fluid bathing the neurological cells of the brain. Neurological functions are prioritized in the distribution of scarce essential nutrients by the organism. Therefore, it is reasonable to assume that the delivery of zinc to the brain would be prioritized during periods of low zinc intake. Plasma zinc decreases during zinc deficiency, but zinc uptake into the brain needs to be maintained. This would likely be accomplished by increasing the number of zinc transporters. We will measure differential expression of proteins under conditions of zinc deficiency to identify participants in zinc transport and brain-zinc homeostasis.
Putative zinc uptake transporters have recently been identified in Saccharomyces cerevisiae (ZRT1, ZRT2) and Arabidopsis thaliana (ZIP1, ZIP2, ZIP3) by David Eide's laboratory at the University of Missouri. A related gene (hZIP2) has been identified in humans and transfected into human K562 erythroleukemia cells, which respond by increasing their uptake of zinc. Dr. Eide will share probes and antibodies for these transporters to test whether these candidates contribute to zinc homeostasis in BCEC. We will also separate the cellular proteins by 2-dimensional gel electrophoresis (2-D PAGE) and compare expression among zinc status treatments.
The broad, long-term objectives of this project are twofold: 1) to identify the mechanisms that the brain uses to maintain brain-zinc homeostasis, and 2) to identify specific proteins that facilitate the movement of zinc through biological membranes in brain capillary cells.
Specific Objective #1.
Determine the influence that zinc status has on zinc homeostasis by cultured BCEC. The import and export of zinc will be tested in cells grown under in vitro environments that are deficient, adequate, and excessive in zinc. BCEC grown under these different zinc environments will also be examined by 2-D PAGE and tested for their expression of specific proteins, evaluating putative zinc transporters.
Specific Objective #2.
Determine the influence of zinc status on the expression of specific proteins by capillary blood vessels isolated from swine brains. Pigs will be fed diets that are deficient, adequate, and excessive in their zinc contents. The brain capillaries will be isolated from the pigs and analyzed by 2-D PAGE and tested for their expression of specific proteins, evaluating putative zinc transporters.
BCEC Capillary Fragments on day of isolation at 100X magnification.
Primary culture of BCEC on day 3 at 100X magnification.
.
Primary culture of BCEC at confluency on day 7 at 100X magnification
BCEC were photographed by graduate student Holly Lehmann, fall of 1999.
US Department of Agriculture
Hatch Project # NH00358
Regulation of zinc transport by endothelial cells.
Zinc has been recognized as an essential nutrient in the diet of humans for many years. In 1974, the US National Research Council established a Recommended Dietary Allowance (RDA) for zinc in the diet of Americans (NRC 1974). Hundreds of biochemical and physiological mechanisms have been identified that depend on zinc's participation (Vallee 1993). To fulfill these essential roles, zinc must be available in the proper concentration in the correct location at the optimal time. Excessive or deficient availability of zinc leads to profound pathologies. Optimal health relies upon the ability of the body (and cells) to redistribute zinc in order to match the current need for zinc with the available supply of zinc. Regulating zinc uptake into cells is likely to be a principal way of controlling the intracellular availability of zinc. This project will attempt to increase our understanding of how zinc gets into cells, and how this process is regulated. Understanding the mechanism(s) and regulation of how zinc is transported in the body will contribute to methods for analyzing a person's zinc status (deficient, adequate, toxic). It will also improve our understanding of why pathologies develop in zinc deficiency and zinc toxicity.
Researchers have been trying to characterize how cells of the body get their zinc for several years with limited success. This important question remains somewhat elusive. Transport mechanisms of other nutrients have been successfully examined by focusing on the plasma membrane proteins which have a high affinity for the nutrient under investigation. Zinc is involved in interactions with a nearly infinite number of other biological molecules; including amino acids, nucleic acids, and proteins. This has made it difficult to unravel the specific interactions that are responsible for transporting zinc into cells. But, some recent discoveries may have enabled this area of research to move forward.
Albumin is another biomolecule whose route of cellular uptake has been difficult to describe. Albumin also binds to a nearly infinite number of other substances, so it was very difficult to identify a plasma membrane protein responsible for mediating albumin uptake. This field was significantly advanced by the discovery of an albumin receptor (albondin) in endothelial cells and synthesis of antibodies for that receptor (Schnitzer 1992, Schnitzer 1994, Tiruppathi 1996). The antibodies for albondin interfere with albumin uptake. Our laboratory is experienced in examining zinc uptake mechanisms. We are skilled in the culture of endothelial cells from several sources. Dr. Schnitzer has agreed to share his antibody for albondin and his expertise in albumin transport with us to test this hypothesis. We are well-suited to examine the role of albumin in zinc uptake by endothelial cells.
Endothelial cells are an appropriate model for these investigations because they form the walls of blood vessels. Consequently, nutrient transport is one of their most important functions. But, not all endothelial cells are alike; endothelial cells vary depending upon the blood vessel they form (e.g., artery, vein, capillary) and the tissue they infiltrate (e.g., brain, lung, muscle). The first objective will be to develop procedures appropriate to testing our hypothesis. Endothelial cells will be isolated from different locations in the body and tested for the ability of the albondin antibody to interfere with albumin uptake, since albumin transport varies among tissues. Once a cell preparation is identified that is responsive to the albondin antibody, then the antibody will be tested for its influence on zinc transport and metabolism.
Specific Aims:
- Identify procedures for the isolation and cultivation of endothelial cells that are highly responsive to the antibody for the albumin transport receptor (albondin).
- Test the hypothesis that cotransport with albumin is a significant route for zinc uptake into endothelial cells.
- Determine the influence of albumin on zinc transport through endothelial cells (blood vessel walls).
These objectives will be pursued using in vitro models constructed of cultured cells. These models will enable us to exquisitely manipulate the environmental conditions of the cells and probe their metabolism of zinc. In addition to the identified objectives, all other promising avenues will be pursued that identify the mechanism(s) and regulation of zinc transport by cells.
Ribbon model of Human Serum Albumin.
No consensus on location of the zinc binding site.
From: Protein Data Bank (http://www.rcsb.org/pdb/index.html)
Original Source: Sugio, S., Kashima, A., Mochizuki, S., Noda, M., Kobayashi, K. (1999) Crystal Structure of Human Serum Albumin at 2.5 Angstrom Resolution. Protein Eng. 12:439.
Non-Lethal Technology Innovation Center (NTIC)
Non-lethal weapons and the blood-brain barrier: The effects of thermal and kinetic energy.
Understanding the interactions between non-lethal weapons and the human body at the cellular level is essential to the safe employment of current non-lethal systems and the development of advanced ones. Research in this area is just beginning to develop. The lack of understanding of how the basic structures of the body react to the thermal changes or physical kinetic impact caused by some classes of non-lethal weapons leads to indecision both about development of these classes of weapons and their employment. There are two types of potential reaction, in particular, that must be understood; the cellular damage caused by different levels of thermal and kinetic energy and the potential changes in the function of multi-cellular systems caused by the different levels of thermal or kinetic energy. Of the multi-cellular systems that make-up the human body few are more critical than the blood-brain barrier (BBB). The BBB is the primary means of preventing contamination of the brain with toxins circulating in the blood stream. While the BBB prevents contamination of the brain, it must also allow for the passage of brain essential materials and the removal of waste products.
The project evaluates the effects of both thermal and kinetic energy on the BBB. The proposed research will evaluate the damage to and changes in function of the BBB caused by the application of thermal and kinetic energy. Cell damage as well as BBB permeability (a measure of function) changes will be evaluated using a variety of recognized analytic tools. This will be a joint research project of the University of New Hampshire (UNH) and the Pennsylvania State University (PSU). The BBB model developed by the UNH will be used to conduct a series of trials of the insult to the BBB caused by both thermal and kinetic energy. Trials will be conducted at both universities. A commercially available Thermal Cycler will be used for the thermal trials component of the project. A laser ablation technique employed at PSU for cell injury studies will be used to apply measured impact forces to the BBB models. In addition, a specially developed precision blunt impact kinetic energy source will be developed for the kinetic energy trials as an efficient alternative to the laser ablation technique. The results of the program support the safe development of new, more effective non-lethal weapons and add to the body of knowledge on the BBB.
