Clinical & Research Interests

Dr. Denlinger is board-certified in General, Pulmonary and Critical Care Medicine. He attends an ambulatory Pulmonary Clinic and works in a multi-disciplinary intensive care unit. Areas of interest include asthma, bronchiectasis, Mycobacterial diseases, lung infections, and sepsis.

He has studied the roles of extracellular nucleotides as paracrine stress hormones. A specific focus has been on a nucleotide receptor, P2X7, and its role in amplifying innate immune responses to infectious and allergic stimuli recognized by Toll Like Receptors. The Denlinger lab is interested in the human P2RX7 gene with respect to the regulation of its expression and function as well as its contribution to airway disorders. This gene encodes a nonselective cation channel expressed by leukocytes and epithelial cells that is involved in amplification of innate immune cytokine responses and microbial killing. An active project focuses on the post-transcriptional control mechanisms of P2RX7 expression in human bronchial epithelial cells. We also have a functional screening assay that has proven reliable in identifying subjects with variant alleles. Using this assay as an epidemiological tool in collaboration with several investigators at the UW Asthma Center, we are testing the hypothesis that attenuated P2X7 function contributes to increased asthma symptoms and/or biomarkers driven by respiratory viruses and atypical bacteria.

The diabetes epidemic. We are in the midst of a worldwide diabetes epidemic. About 171 million people have diabetes and this figure is expected to double in the next 20 years. In the United States, 21 million people (7% of the population) have diabetes and this number is also rapidly growing. Diabetes is the leading cause of blindness, kidney failure, limb amputations, and a major risk factor for premature cardiovascular disease.

Diabetes results from an absolute or a relative insulin deficiency. Pancreatic ß-cells sense blood glucose and respond by secreting insulin. Insulin lowers blood glucose by promoting its clearance from the circulation and by inhibiting gluconeogenesis. In type 1 diabetes, there is an absolute insulin deficiency due to autoimmune destruction of the cells that produce insulin, the pancreatic ß-cells. However, in type 2 diabetes, there is an increased requirement for insulin, caused by a dampened response to the hormone, coupled with a failure to meet this increased requirement. Obesity and diabetes. Virtually everyone who is obese is insulin resistant. But, although >80% of people with type 2 diabetes are obese, most obese people do not develop diabetes. In order to avoid developing diabetes, an insulin resistant person must compensate for insulin resistance by producing more insulin. This can occur through an expansion in ß-cell mass or through increased ß-cell insulin secretion.

Research Interests
Molecular mechanisms of ion channel function

The electrical signals responsible for neuronal communication and cardiac rhythmicity depend on potassium channels, proteins that regulate the movement of potassium ions across cell membranes. The disruption of these channels by inherited diseases or drugs can lead to neurological defects or cardiac arrhythmias.

Work in our lab focuses on voltage-gated potassium channels encoded by the human Ether-à-go-go-Related Gene, or hERG1. In 1995, we showed that hERG1 expressed in Xenopus oocytes produces currents with the unique biophysical and pharmacological properties of the cardiac repolarizing current known as I_Kr. This work established hERG1 channels as a potential target for acquired long QT syndrome (LQTS), in which block of I_Kr by drugs intended for a wide range of therapeutic targets can trigger life-threatening ventricular arrhythmias known as torsades de pointes. In addition, these studies explained the underlying cause of inherited type 2 LQTS (LQT2), which had been linked to the hERG1 gene, as a loss or reduction of cardiac I_Kr

Research Description:

Work in my laboratory focuses on Prokaryotic and eukaryotic RNA polymerases (RNAPs) and transcription factors and their roles in RNA synthesis and its regulation. We study the structure and function of the initiation factor, E. coli RNAP sigma70 subunit, by a concerted use of protein and physical chemistry, monoclonal antibodies (MAbs), molecular genetics, computer-based sequence and structure analysis, and biochemistry. We have overproduced and purified all seven known E. coli sigma factors and have made MAbs to them that can be used in measuring their levels in the cell under various growth conditions, inhibiting them, and immunoaffinity purifying them. We are carrying out systematic site-directed mutagenesis of sigma70 to determine more precisely the region involved in the binding of sigma to core RNAP (composed of alpha, beta and beta prime subunits). We have developed a powerful new method, using histidine-tagged RNAP subunit fragments, to map epitopes of our various MAbs easily and quickly. We have recently utilized a variation of this method, employing "far-Western blotting", to map interaction domains and have identified a major binding site for sigma70 within the region of amino acids 260-309 of the beta prime subunit of core RNAP.

How does a protein with a given amino acid sequence manage to achieve its bioactive and amazingly organized three-dimensional structure? This process, known as protein folding, is one of the most fundamental yet poorly understood events in chemistry and biology. Most studies performed in the past have focused on the in vitro folding of full-length biopolymers starting from unfolded states generated by high concentrations of denaturants or high temperature. However, these types of unfolded states rarely exist in living cells! Moreover, polypeptide chains start sampling conformational space (and possibly even fold) way before the protein amino acid sequence has been fully synthesized, during a process known as translation. In order to fully understand protein folding, it is therefore important to take the cellular context into account. This is even more important in the case of protein misfolding, i.e., folding gone wrong, which leads to protein aggregation and several deadly neurodegenerative and brain disorders such as Alzheimer's disease, spinocerebellar ataxia and Huntington's chorea. Thus, understanding protein folding/misfolding will lead to both fundamental knowledge and long-term benefits to human health. 

Understanding how symbiotic associations between plants and microbes develop is an important biological question that is particularly relevant in modern agriculture and economy.

We seek to understand the molecular mechanisms controlling the establishment of plant - microbe symbioses and the stimulation of plant growth by microbial signals. Our goal is to maintain soil quality and sustainability while protecting the environment over the long term and reducing costs for food and Biofuel production.

We transfer information gained from model plants such as Medicago truncatula or Brachypodium distachyon to crops such as soybean, alfalfa and maize in order to take full advantage of the fantastic  opportunities offered by these symbiotic associations to our agriculture.

Research Focus: Cell Separation and Abscission in Arabidopsis thaliana

The research efforts of the Patterson lab are directed towards understanding the mechanisms that regulate cell separation and cell-cell adhesion. Of particular interest is the process of abscission in which the organs of a plant are detached from the main body of the plant in a developmentally regulated program. Our focus has been to identify the genes which control this process using floral organ abscission in Arabidopsis as a model system. We are using two basic approaches: 1) a broad base phenotypic screen for delayed abscission mutants from a collection of T-DNA tagged Arabidopsis plants and 2) a reverse genetic approach using PCR in which we have targeted specific cell wall hydrolytic enzymes including endo-beta-1,4- glucanases (cellulases), polygalacturonases, pectin lyases, and pectin methylesterases. These approaches allow us to identify both regulatory and structural genes controlling abscission. Abscission and cell separation are important agricultural traits regulating processes including fruit drop, foliage loss, and cut flower longevity, and our ability to understand these processes will ultimately lead to many aspects of crop improvement.

Iron is crucial to cell viability because it is a component of proteins that function in a large number of physiological processes including respiration and cell division. However, excess iron can be toxic because it participates in the production of potentially lethal oxidizing agents. All organisms possess specific iron-binding and other proteins that function in concert to allow cells to make use of the essential properties of iron while minimizing its potentially toxic attributes. Genetic or nutritional perturbations of iron metabolism impair the health of nearly one-third of the world’s population.

Mammalian iron metabolism is modulated through changes in the synthesis of proteins required for the uptake, storage, and use of iron. Synthesis of these proteins is controlled post-transcriptionally by regulatory RNA binding proteins, the iron regulatory proteins (IRPs). Two IRPs, IRP1 and IRP2, exist and each can independently regulate the use of IRE-containing mRNA. Under appropriate conditions IRPs bind stem-loop structures (IREs) in the mRNAs encoding proteins of iron metabolism thereby regulating the translation or stability of the affected mRNA. In this manner iron, and other factors that regulate IRP RNA binding activity, alter the uptake and metabolic fate of iron.

Molecular genetics; RNA and RNA-protein interactions; developmental biology

Understanding how mRNAs are regulated. It is not enough to make an mRNA: you have to know how and when to use it. mRNAs are controlled at many levels — they can be turned on or off, be destroyed or stabilized, and can be moved within the cell. These controls are critical in biology — in development, viral replication and human pathologies, for example. Our work aims at identifying the molecular mechanisms that execute these controls. The powerful molecular genetics of the yeast, S. cerevisiae, is invaluable for this purpose, and has led to the identification of key players in the process.

Focusing on a regulatory hot spot. The region of the mRNA between the termination codon and the poly(A) tail — the 3’ untranslated region (3’UTR) — often governs when, where and how much protein an mRNA produces. A key first step in figuring out how 3’UTRs work is identifying the regulators they bind to. Using methods we developed, we identified and cloned 3’UTR regulators in both C. elegans and yeast. At the same time, we have identified mRNA targets for regulators we already knew. The challenges now are to understand how these mRNA-protein complexes, located in the 3’UTR, regulate an mRNA’s expression, how they are integrated, and how they evolve.

Studying the regulation of gene expression at the interface of chemistry, biology and genomics