Jack R Lancaster, Jr. PhD

Research, Teaching and Service Statement

 

Professor of Pharmacology and Chemical Biology

 

University of Pittsburgh School of Medicine

 

 

 

My research interests have been broad and interdisciplinary. I have made contributions 

 

in many areas, as outlined below, but overall dealing with fundamental mechanistic studies at 

 

the biochemical and biophysical levels. The general areas in which I have worked are the 

 

multiple roles of nitric oxide as an effector and messenger molecule, biological energetics, 

 

membrane ion translocation, and electron transfer. I have been very selective in the papers I 

 

have published and consequently my work is known in several different fields. I describe briefly 

 

below my major research accomplishments, listed chronologically.

 

-- In 1975, utilizing a cytoplasmic membrane preparation of E. coli of "inside-out" 

 

orientation, I showed that reversing the direction of a transmembrane proton gradient results 

 

in the "backwards" uphill translocation of sugar (lactose) [1-3]. This result provided important 

 

support for the Chemiosmotic Theory, for which Peter Mitchell received the Nobel Prize in 

 

Chemistry in 1978. I subsequently published a detailed kinetic model for this system which as 

 

far as I am aware is the only model that accounts for all published kinetic data for this "classic" 

 

transport protein which is the product of the y gene of the lac operon [4,5].

 

-- In 1979, I reported an extensive enzymological and spectroscopic characterization of 

 

spinach nitrite reductase, a siroheme-containing iron-sulfur protein that catalyzes the six-

 

electron reduction of nitrite to ammonia [6,7]. Using EPR spectroscopy, I showed the formation 

 

of heme-bound nitric oxide as an intermediate in the reaction, which (as far as I am aware) was 

 

the first demonstration of enzymatic NO synthesis.

 

-- Also in 1979, I reconstituted the adrenal cortex mitochondrial membrane cytochrome 

 

 (responsible for the committed step in steroidogenesis) into synthetic phospholipid 

 

vesicles [8-12]. I used this experimental system to demonstrate the involvement of the 

 

membrane bilayer in orienting cholesterol (the substrate for the reaction) for delivery to the 

 

active site. In 1980, in a collaborative project we used EPR spectroscopy to determine the 

 

orientation of this protein within the membrane. This was the first description of a specific role 

 

for the membrane in any cytochrome P450 and suggests that the membrane may serve to 

 

“solubilize” hydrophobic substrates and deliver them to the enzyme active site.

 

-- In 1980, I reported in Science the first direct observation, using EPR spectroscopy, of 

 

the element nickel (as Ni(III)) in a biological system [13-15]. This was a critical observation in 

 

showing that this element plays a specific and catalytic role in a recently recognized group of 

 

enzymes that have since been found in a variety of organisms, and was noted in a News and 

 

Views in Nature [16]. In 1988 I edited the first book devoted to the role of this metal in 

 

biological systems [17].

 

-- In 1982, in collaboration with Dr. Thomas Emery, I published the first study of iron 

 

transport in cells using EPR spectroscopy [18].

 

-- From 1983-1986 I published a series of investigations into the molecular mechanisms 

 

of energy coupling in the methanogenic bacteria, an extremely unusual group of organisms that 

 

have been called the "third form of Life". In these papers [19-27], we described several unique 

 

features of the mechanism of energy coupling, including the first report of a sodium-

 

translocating ATPase which is involved in electron transport-driven phosphorylation. These 

 

results were described in two reviews [28,29].

 

-- Using EPR spectroscopy, in 1983 in collaboration with two nutritionists I published a 

 

paper in Science that showed the destruction of iron-sulfur centers and the simultaneous 

 

formation of iron-nitrosyl complexes in the organism Clostridium botulinum upon the addition 

 

of nitrite [30]. This result provided a significant advance in understanding one of the "unsolved 

 

problems" of nutrition, the mechanism of prevention of botulism by the addition of nitrite to 

 

foods. Nitrite appears to be oxidized to the reactive molecule nitric oxide (NO) which destroys 

 

iron-containing enzyme function. Perhaps most importantly, this result presaged the many 

 

papers later describing an identical effect of nitric oxide produced as an immune effector 

 

molecule, as described in more detail below.

 

Currently, my research involves studies of the biochemical, molecular biological, and 

 

biophysical mechanisms of action of nitric oxide and its interactions with reactive oxygen 

 

species. My work in this area in mammalian systems began with a sabbatical year at Emory 

 

University in 1987-1988, which I took because of the graduation of my three Ph.D. students and 

 

the last year of support from my American Heart Association Established Investigatorship 

 

Award. For this year, I studied the mechanism of programmed cell death by tumor necrosis 

 

factor (TNF). This cytokine is produced by macrophages, and plays a central role in the immune 

 

response to inflammation, infection, and transformation. TNF induces cell “suicide” in 

 

numerous transformed cells in culture as well as virally and microbially-infected cells, and so is 

 

a key mediator of the host response to many disease states. We showed for the first time that a 

 

very early event in the killing of several cell lines by TNF is a specific inhibition of mitochondrial 

 

electron transfer [31], which was the first direct demonstration of mitochondrial involvement in 

 

programmed cell death. 

 

On returning to Utah State University from my sabbatical in 1988, a remarkable series of 

 

events began to unfold in the literature involving nitric oxide, a molecule which I had previously 

 

demonstrated was formed by a nonmicrobial system (plants [6]) and that destruction of iron-

 

sulfur centers can result in a cellular toxic effect [30]. In classic studies published by Hibbs it was 

 

shown that certain immune cells (activated macrophages) can kill tumor cells through the 

 

production of nitric oxide. I contacted Hibbs (who was aware of my previous work) and we 

 

demonstrated unequivocally using EPR spectroscopy the formation of cellular iron-NO 

 

complexes by NO synthesized by activated macrophages from L-arginine, the substrate for the 

 

enzyme that makes NO [32]. In 1987-88 it was shown by Ignarro and Moncada that Furchgott’s 

 

endothelium-derived relaxing factor (EDRF) possesses properties identical to NO, meaning that 

 

it regulates control of vascular tone and blood pressure. NO transmits its message through 

 

stimulation of cyclic GMP production, which was studied extensively by Murad and Ignarro 

 

beginning in the 1970’s. Finally, in 1988 Garthwaite showed that neuronal cells produce NO as a 

 

neurotransmitter. The discovery of mammalian NO has been described as one of the most 

 

important discoveries in biomedicine, as evidence by (among other things) the selection of NO 

 

as “Molecule of the Year” by Science in 1992 and the awarding of the 1998 Nobel Prize in 

 

Physiology or Medicine to Furchgott, Ignarro, and Murad. The spectacular growth of this field is 

 

emphasized by the fact that there have been over 130,000 papers on NO in biology, most of 

 

which have appeared within the past seven years. 

 

Our application of EPR spectroscopy allows the study of NO cellular actions in both in 

 

vitro and in vivo systems. In addition to studying the biochemical reactions of NO in cells in 

 

culture, using this technique we were able to observe for the first time NO (bound to cellular 

 

iron) in blood and tissue of rejecting organs (heart [33] and bowel [34]). We applied this 

 

technique to several other clinically important conditions, including diabetes [35] (including the 

 

first description of the application of an inhibitor of NO synthase selective for the inducible 

 

isoform, aminoguanidine [36]), whooping cough (pertussis) [37], and the biochemistry of NO 

 

cellular actions when induced by inflammatory cytokines and mediators [38,39].

 

In addition to using EPR spectroscopy as a tool, we have pursued six general areas of NO 

 

and reactive oxygen chemistry/biology.

 

Oxidant Stress and NO:

 

One of the central questions in reactive nitrogen and oxygen biology is what determines 

 

whether NO will function as an antioxidant or a prooxidant. We have developed an in vitro

 

model system which mimics the current paradigm for this, namely, that low-level NO is 

 

antioxidative and high NO is prooxidative. Specifically, we have shown that low levels of NO 

 

prevent oxidant killing in cells in culture while higher levels increase oxidant killing [40].

 

Cellular Responses to Nitric Oxide:

 

We have reasoned that, like reactive oxygen species, cells must have evolved protective 

 

mechanisms against injury from NO overproduction. We provided the first evidence for this in 

 

1995 where we found that, surprisingly, cells become resistant to killing by NO when they are 

 

pretreated for 8-12 hours with a very small dose of NO, and this resistance involves new protein 

 

synthesis [41]. We also have shown the protective actions of two other protective proteins 

 

(heme oxygenase [42, 43] and metallothionein [44]) against NO damage, and also reported a 

 

unique cellular mechanism for detoxification of nitrite [45]. This work was the first to identify 

 

a preconditioning response to NO and to identify heme oxygenase as a protective protein that 

 

Dynamics of NO in the Vasculature and Tissue:

 

NO is electrically neutral and is one of the eleven smallest molecules in Nature (of the more 

 

than 500 million known) [46]. We have studied several physical properties of this unique 

 

biomolecule which are important to its biological actions. Using a combination of Fick’s Laws 

 

of Diffusion and mathematical modeling, I have presented the first quantitative descriptions 

 

of the diffusion of NO [47-50]. Application of these results reveals several important (and not 

 

generally appreciated) concepts: in tissue, the spatial extent of NO diffusion away from a single 

 

cell producing it steadily is exceedingly large, encompassing a tissue volume which contains 

 

500,000 to 1,000,000 cells. In addition, in 1994 utilization of these modeling techniques 

 

allowed the first demonstration that the hemoglobin in the blood will be a potent sink for 

 

NO, and raised a serious concern with regard to the postulate that EDRF is free NO. We 

 

subsequently demonstrated that enclosing hemoglobin within erythrocytes slows the reaction 

 

with NO by nearly 1000-fold, thus helping solve this conundrum [51]. This work provided the 

 

theoretical background for the demonstration of the importance of scavenging of NO by plasma 

 

hemoglobin in patients with sickle cell disease, and gave rise to the basis for clinical trials 

 

testing the efficacy of NO inhalation as a potential therapy [52]. In other work [53], we were 

 

able to estimate the in vivo lifetime of NO (ie, in the tissue around the blood vessel); utilizing 

 

these measurements and further modeling, we also proposed a second function for NO in 

 

facilitating tissue oxygenation (in addition to vasodilation), by extending the zone of adequate 

 

oxygenation away from the surface of a vessel. We also have been involved in studies that 

 

seriously challenge the proposal that hemoglobin acts physiologically to carry NO [54, 55]. 

 

Membrane Reactivity of NO:

 

In 1998 we demonstrated that the hydrophobic solubility of NO results in an acceleration of 

 

NO reaction with O2 within hydrophobic tissue compartments such as membranes [56]. We 

 

also were able to demonstrate that this increase in reactions also results in increased rate of 

 

formation of reactive nitrogen oxides (•NO2 and perhaps N2O3), and that the origin of this “lens” 

 

effect is due purely to a partitioning of NO and O2 into the hydrophobic phase [57]. Thus, the 

 

membrane is an important location for both NO disappearance and the appearance of reactive 

 

nitrogen intermediates which have been implicated in many aspects of NO effects, including 

 

DNA damage and nitrosothiol formation. This also raises the possibility that scavenging of these 

 

intermediates is a major physiological action of the lipid soluble vitamin E.

 

Cellular Mechanisms of Formation of Iron-Nitrosyl Complexes:

 

We have recently found that the iron in the ubiquitous EPR-observable iron-NO complexes 

 

originates from the so-called “chelatable iron pool” (CIP) in cells [58]. This finding has important 

 

implications in the potent effects of NO on cellular iron homeostasis as well as the multiple 

 

effects of NO on the pro-oxidative actions of this pool of iron. 

 

Predicting Reactive Nitrogen Species Chemistry in the Biological Milieu:

 

I have recently presented a comprehensive computer modeling approach to predicting the 

 

most important reactions of NO and its descendents under simulated biological conditions 

 

[59]. The application of this approach has provided many specific (and several surprising) 

 

experimentally testable predictions and also suggest new insights into the actions of NO as 

 

a reactive species and also as a messenger, perhaps the most important being that under 

 

essentially all biologically relevant conditions the overwhelmingly preponderant chemistry that 

 

occurs when cells are exposed to NO is oxidation (as opposed to for example nitrosation or 

 

Cellular Mechanisms of Protein Nitrosothiol Formation from NO:

 

Cysteine nitrosation (producing nitrosothiol) has been proposed as an important protein 

 

posttranslational modification, although the mechanism of formation is essentially unknown. 

 

We have recently proposed that cellular protein nitrosothiols are formed from transfer of 

 

nitroso groups from DNIC to protein cysteine thiols. This finding has critically important 

 

implications for multiple biological actions of NO [60].

 

Modulation by NO of Roles of the Cellular Chelatable Iron Pool in Models of Ischemia/

 

Reperfusion Injury and Protein Nitrosothiol Formation:

 

Using an experimental method of induction of severe hypoxia in cells we find evidence that 

 

hypoxia-induced cellular iron mobilization into the CIP in this model effects increased formation 

 

of nitrosothiol from NO and greatly increased sensitivity to hydrogen peroxide; this sensitivity 

 

is ablated by NO. The modulation of these actions by NO appears to involve DNIC formation, 

 

which suggests important new roles for these iron-nitrosyl complexes species as an effector of 

 

multiple cellular phenomena of biomedical relevance [61].

 

Other Activities in the NO Field:

 

In 1996, along with five others, Louis Ignarro and I founded The Nitric Oxide Society. 

 

Ignarro was the first President and I Vice president, and in 1999 I succeeded him as President. 

 

In addition to sponsoring biannual international conferences, we publish Nitric Oxide Biology 

 

and Chemistry (Academic Press), which is the only journal exclusively devoted to NO. I served as 

 

Editor-In-Chief 2003-2012. In 1992 I wrote a popular review on the biological actions of NO for 

 

American Scientist [46] which subsequently received the Bronze Prize for Excellence awarded 

 

by the Society of National Association Publications for general science writing, and in 1995 I 

 

founded the Nitric Oxide Home Page on the World Wide Web which was recognized in 1996 by 

 

The Scientist as one of the first web pages devoted to a specific scientific topic is now the 

 

official home page of The Nitric Oxide Society . I have also co-edited along with Lou Ignarro a 

 

Forum on NO and oxidative injury in Free Radical Biology and Medicine [62], authored or 

 

coauthored several reviews [63-73], and edited two books on NO [74, 75]. Finally, I was co-

 

organizer and founder of the First Gordon Research Conference on the Biochemistry and 

 

Molecular Biology of Nitric Oxide (1995) and have been co-organizer of four other international 

 

conferences on NO.

 

I have strived to be an outstanding teacher. Although my AHA Award at Utah State kept 

 

my teaching and administrative duties small, as a result of my success in the classroom in 1988 I 

 

volunteered to take command of the freshman General Chemistry course in an effort to 

 

increase the number of undergraduates majoring in Chemistry. My teaching evaluations from 

 

this class (the first beginning course I have taught) were outstanding; my "GPA" rating by the 

 

students was 3.3, out of 4.0. In addition, in 1988-1990 I wrote and published a national 

 

Newsletter for Saunders College Publishing, which was intended for professors who adopt their 

 

General Chemistry texts. This represented approximately 60% of all such introductory college 

 

courses, nationwide. With this newsletter the professor was able to incorporate "hot" and 

 

exciting new breakthroughs in Chemistry directly into the classroom, since it was keyed to each 

 

of the six texts produced by Saunders and contains references to the original literature. This 

 

was thus a nationwide project designed to make Chemistry more relevant, exciting, and 

 

interesting to perhaps our most important audience, students taking their first, introductory 

 

course in the molecular basis of natural phenomena.

 

Also, as described in my CV, I have taught a variety of courses, mostly during my years in 

 

the Chemistry department at USU. These courses have ranged from graduate core, laboratory, 

 

special topics and seminars in Biochemistry to undergraduate courses in Instrumental Analysis, 

 

Bio-organic, and Biophysical Chemistry. My teaching duties at LSU involved sections on 

 

Pulmonary, Renal, and Musculoskeletal Pathophysiology in a graduate nursing course, a Special 

 

Topics course on nitrogen and oxygen radicals (which I also taught at UAB), and lectures on 

 

nitric oxide to medical and graduate students. In addition, for 20 years I taught the 

 

Bioenergetics Section of the graduate Biochemistry course at the five universities where I have 

 

been (Duke, Utah State, University of Pittsburgh, LSU, and UAB). In addition, at UAB I taught the 

 

Thermodynamics lectures in the Graduate Biomedical Sciences Core Curriculum. These lectures 

 

were always very well received by the students.

 

Finally, I am very proud of the fact that I was selected one of 16 National Speakers for 

 

Sigma Xi, the Scientific Research Society of North America, from 1994-1996. One of my 

 

presentations was to the small undergraduate institution where I received my B.S. in Chemistry, 

 

University of Tennessee at Martin.