Anthony R. Means, PhD - US grants

The overall objective of the proposed research is to establish how Ca 2 ion and cAMP interact to regulated differentiation and function of the cytoskeleton of the testicular Sertoli cell using the Sertoli cell-enriched testis and isolated Sertoli cells as model systems. We will first evaluate changes in the synthesis distribution, and assembly of microtubule (tubulin) and microfilaments (actin) together with proteins involved in the action of Ca 2 ion (calmodulin, myosin light chain kinase) and cAMP (Protein kinase inhibitor, catalytic subunit of cAMP-dependent protein kinase). Studies will include localization of the proteins by immunofluorescence and immunocytochemical methods, quantitation of rates of synthesis and degradation as well as steady state levels of the proteins, and determination of mRNA levels by hybridization to complementary cDNA probes. In all instances we will evaluate how the parameters may be altered by Ca 2 ion and cAMP. Next we will investigate how the assembly/disassembly of microtubules is regulated especially in regards to the roles of Ca 2 ion Calmodulin and cAMP. For this aim permiablized Sertoli cells and sperm tail pieces will be used as mucleation sites for 6S tubulin. Changes in enzymes regulated by Ca 2 ion and cAMP will be determined during Sertoli cell differentiation and in response to hormonal stimulation. The enzymes include those involved in glycogen metabolism (glycogen synthase, phosphorylase, phosphorylase kinase) and contractility (myosin light chain kinase). Finally we will evaluate roles for cAMP, Ca 2 ion, protein phosphorylation and the cytoskeleton in regulation of specific protein secretion from isolated Sertoli cells in culture.

The long term objective of the proposed research is to determine how the structure of Ca++ binding proteins dictate function. The primary system to be utilized is calmodulin dependent regulation of smooth muscle myosin light chain kinase (MLCK) that results in the phosphorylation of myosin light chain (LC) required for myosin movement. Calmodulin, MLCK and LC have been expressed in bacteria. Site specific mutagenesis will be employed together with direct binding and enzyme activity assays to determine the interaction of the three proteins in molecularly precise turns. Portions of calmodulin, troponin C, S100 beta and parvalbumin cDNA will be used to create hybrid proteins in bacteria. The isolated proteins will be evaluated for binding to and activation of MLCK as well as activation of calmodulin kinase II, calcineurin and cGMP phosphodiesterase. Both MLCK and calmodulin kinase 11 contain a substrate inhibitory region contiguous to the calmodulin binding site. Portions of these regulatory areas will be substituted between the two enzymes to examine the functional consequences. We will attempt to define the interaction of the pseudosubstrate region of MLCK with the constitutive enzyme to allow the eventual development of pharmacological agents to specifically inhibit the vascular smooth muscle form of MLCK. Finally, gene substitution by homologous recombination will be utilized to define functions of calmodulin in a genetically fascile fungal system and second site suppressors will be employed to identify calmodulin targets in this organism. Together these studies should contribute to a better understanding of how calmodulin serves as a multipurpose regulator of cellular homeostasis.

The overall objective of the proposed research is to establish the mechanism of action of intracellular second messengers at the molecular level. The primary model will be how Ca++ acts to alter cell function via interaction with its ubiquitous intracellular receptor, calmodulin. We will focus on three areas. First it will be determined whether Ca++ plays a role in maintaining the intracellular levels of calmodulin. This will be accomplished by manipulating intracellular Ca++ levels and measuring calmodulin, calmodulin mRNA and rate of transcription of the calmodulin gene. We will evaluate the portion of the calmodulin gene necessary for Ca++ regulation by gene transfer methods. Finally we will use DNA/protein binding methods to determine if calmodulin interacts specifically with regions of the calmodulin gene. The second aim is to evaluate the consequences of altered calmodulin levels on cell functions such as growth, proliferation, and cell cycle. Eukaryotic expression vectors will be used to introduce calmodulin genes into cells under control of regulatable promoters. The constructions will allow production of both the sense and antisense strands of cDNA. Third we will evaluate the specific interaction of calmodulin with a model target enzyme, myosin light chain kinase (MLCK). The remainder of the MLCK cDNA will be cloned and the calmodulin binding fragment identified by the use of bacterial expression vectors and calmodulin binding assays of the peptides produced. The precise molecular interaction of MLCK and calmodulin will be solved by crystallographic procedures. Finally we will determine whether differences exist in the calmodulin binding domains of a phosphatase (calcineurin) and a multisubstrate kinase, calmodulin kinase II. These studies will provide the first details of interaction of an intracellular receptor with its acceptor proteins in chemically precise terms.

The objective of this proposal is to obtain partial funding for a FASEB Summer Research Conference on Calcium and Cell Function, to be held July 1014, 1989 at the Vermont academy in Saxtons River, VT. This conference is held under the auspices of the Federation of the American Societies of Experimental Biology (FASEB) and will employ a format similar to that of the Gordon Research Conferences. Nine major sessions are scheduled with a invited speakers participating in each. The chairperson of each session will intro- duce the topic, provide a historical perspective and place the topic in context with the conference theme. Poster sessions will be held each afternoon of the meeting and all attenders will be invited to participate. Because of the breadth of this conference, informal workshops will be arranged to focus on critical issues in the field. This is the only major conference that covers the entire spectrum of calcium research rather than focus on a single subdiscipline. As such the biannual conference is always oversubscribed. Great efforts are made to include physiology, cell biology, biochemistry, pharmacology, molecular biology and genetics methods and approaches as relevant to the field. This meeting is the only one to be held in North America or Europe on Calcium this year and is the only regularly scheduled meeting of its kind in and country. The topics to be covered will be hormonal regulation of calcium homeostasis; calcium regulation in excitable cells; calcium release mechanisms; calcium regulation in smooth muscle; calcium and the protein phosphorylation cycle; calcium in growth and development; novel calcium control mechanisms; genetic analysis of calcium function; and structure/function of calcium binding proteins.

The long term objective is to evaluate how changes in calcium homeostasis in the beta cell of the pancreas results in development of very early onset non-immune diabetes. Transgenic mice have been produced in which the rat insulin II gene promotor has been ligated to a chicken calmodulin minigene. Expression of this minigene is specific to the insulin-producing beta cells and results in an increased calmodulin content. Calmodulin is a calcium binding protein that is present at high concentration in beta cells and serves to regulate several enzymes involved in glucose-mediated insulin secretion. The 5X increase in calmodulin occurs early in embryonic beta cell development and results in diabetes that becomes evident within a few hours after birth. The disease is characterized by increased blood glucose, decreased insulin in both pancreas and serum as well as gradual depletion of beta cells. Except for reduced insulin content, beta cells in the late embryo appear normal whereas those of 2 day neonates are markedly abnormal. Results suggest that excess calmodulin interferes with beta cell function leading to diabetes. The diabetic state and excess calmodulin are involved in beta cell toxicity. We propose to complete analysis of both fetal and neonatal pancreas in the existing transgenic lines. Analysis will include electron microscopic examination and immunogold localization of calmodulin and the islet hormones. Pancreatic islets will be isolated and evaluated for insulin secretion in response to glucose, glucose metabolism, glucose sensitive K+ channels, Ca++ conductance and changes in intracellular Ca++. The second aim is to determine whether diabetes is specifically due to elevated calmodulin or increased Ca++ buffering capacity. We will generate new lines of transgenic mice in which the transgene will be a mutant calmodulin molecule that binds calcium normally but cannot bind to or activate calmodulin-dependent enzymes. The glycemic state of these mice will be evaluated by measurement of blood glucose and insulin. If these are also diabetic, similar experiments as described for the current transgenic lines will be performed. The final aim is to address the importance of normal levels of calmodulin on development of the endocrine pancreas and proliferation and differentiated function of beta cells. This problem will be evaluated utilizing transgenic mice that express either a calmodulin antisense RNA or a calmodulin binding protein in beta cells. The purpose is to decrease calmodulin levels and question the functional significance of this reduction. Completion of these aims will provide insight into molecular events that could produce diabetes with characteristics similar to those present in both IDDM and NIDDM in humans.

The overall goals of the proposed research are to examine regulatory mechanisms that influence post-mitotic development of testicular germ cells. The major calcium binding protein in mammalian sperm is calspermin. We have cloned the CDNA encoding this protein. Analysis of the sequence and of the gene revealed that calspermin is derived from a transcription unit that also encodes a novel calcium-calmodulin dependent protein kinase. This cDNA has also been cloned and sequenced. Whereas the kinase mRNA is first detected in early primary spermatocytes and declines to a low level following the last meiotic division, calspermin mRNA is first expressed in pachytene primary spermatocytes and continues to increase as cell complete meiosis and undergo terminal differentiation. We have isolated genomic DNA that encodes the entire transcription unit and propose to characterize the structural organization of the gene. We will then utilize transfected mammalian cells, a testicular transcription system and transgenic mice to determine the molecular mechanisms that control cell specific mRNA expression. The hypothesis is that alternative transcriptional initiation followed by a requisite unique RNA processing event are involved in calspermin production. Expression vectors have been used to produce calspermin and the kinase and to identify key amino acids required for function and/or regulation of activity. A constitutive kinase has been used to develop mammalian cell lines that express the enzyme in a regulated fashion and to identify a transcriptional DNA element that is trans-activated by the active enzyme. We propose to characterize the mechanism by which the overexpression of the kinase results in a G2 cell cycle block and the transcription factor responsive to the kinase. Transgenic mice will be utilized to evaluate the consequences of altering the concentration of the kinase on spermatogenesis and sperm motility. Different promoters will be employed to change the temporal expression and/or amount of the proteins during germ cell development. We predict that the kinase will play a role in the onset, progression or completion of meiosis whereas calspermin will be involved in terminal differentiation of the spermatid or sperm motility.

The long term objective is to evaluate how changes in calcium homeostasis in the beta cell of the pancreas results in development of very early onset non-immune diabetes. Transgenic mice have been produced in which the rat insulin II gene promotor has been ligated to a chicken calmodulin minigene. Expression of this minigene is specific to the insulin-producing beta cells and results in an increased calmodulin content. Calmodulin is a calcium binding protein that is present at high concentration in beta cells and serves to regulate several enzymes involved in glucose-mediated insulin secretion. The 5X increase in calmodulin occurs early in embryonic beta cell development and results in diabetes that becomes evident within a few hours after birth. The disease is characterized by increased blood glucose, decreased insulin in both pancreas and serum as well as gradual depletion of beta cells. Except for reduced insulin content, beta cells in the late embryo appear normal whereas those of 2 day neonates are markedly abnormal. Results suggest that excess calmodulin interferes with beta cell function leading to diabetes. The diabetic state and excess calmodulin are involved in beta cell toxicity. We propose to complete analysis of both fetal and neonatal pancreas in the existing transgenic lines. Analysis will include electron microscopic examination and immunogold localization of calmodulin and the islet hormones. Pancreatic islets will be isolated and evaluated for insulin secretion in response to glucose, glucose metabolism, glucose sensitive K+ channels, Ca++ conductance and changes in intracellular Ca++. The second aim is to determine whether diabetes is specifically due to elevated calmodulin or increased Ca++ buffering capacity. We will generate new lines of transgenic mice in which the transgene will be a mutant calmodulin molecule that binds calcium normally but cannot bind to or activate calmodulin-dependent enzymes. The glycemic state of these mice will be evaluated by measurement of blood glucose and insulin. If these are also diabetic, similar experiments as described for the current transgenic lines will be performed. The final aim is to address the importance of normal levels of calmodulin on development of the endocrine pancreas and proliferation and differentiated function of beta cells. This problem will be evaluated utilizing transgenic mice that express either a calmodulin antisense RNA or a calmodulin binding protein in beta cells. The purpose is to decrease calmodulin levels and question the functional significance of this reduction. Completion of these aims will provide insight into molecular events that could produce diabetes with characteristics similar to those present in both IDDM and NIDDM in humans.

9318101 Nadler This is a request for shared instrumentation to image and quantitate ion levels and pH in physiological studies on living cells. The requested Noran Odyssey laser scanning con focal microscope will enhance the research of five members of the Department of Pharmacol ogy by allowing the use of novel and innovative experimental approaches. The Noran instru ment has been optimized for the imaging of living tissue in real time. It will be used to investigate the following issues: (1) effects of regulatory substances on stimulus evoked sodium and calcium accumulation in glutamate nerve terminals, (2) NMDA receptor plastici ty in kindling and ontogenesis, (3) role of axon sprouting in facilitating or inhibiting seizure discharge and propagation, (4) role of calcium transients in regulating the cell cycle, (5) role of calmodulin in regulating insulin secretion from pancreatic SYMBOL 98 \f "Symbol" cells, (6) mechanism by which excess calmodulin leads to cardiac hypertrophy, (7) effects of glutamate and GABAA recep tor activation on intracellular calcium and chloride levels in hippocampal neurons, (8) effects of growth stimuli on intracellular pH, (9) role of protein phosphatase activity in the regula tion of cellular calcium levels and DNA synthesis, (10) induction of the protein phosphatase inhibitor 1 gene by calcium and (11) initiation of seizures by spontaneous action potential firing in axon terminals. These studies are aimed at understanding a variety of signaling mechanisms fundamental to cellular physiology. They will also help explain changes in cellular physiology and cell cell communication that occur in response to excessive or inju rious stimuli. u ~ -H- ~CAL3118TMP U. ~CRD0F04TMP #U. ~CAL3527TMP b5 ~CRD142ATMP . ~CRD0F38TMP b5 ~MF0E37 TMP E: ~DOC085BTMP s9 ~CAL2701TMP T~; ~CRD216FTMP X~; ~CAL3639TMP y< 9318101 Nadler This is a request for shared instrumentation to image and quantitate ion levels and pH in physiological studi ( 0 & ( !` ! F ( 0 ( 3 Times New Roman Symbol & Arial 1 Courier 9 " h % % = abstract Deseree King, BIR Deseree King, BIR

The overall goal of the proposed research is to investigate how calmodulin overexpression causes cardiac myocyte hypertrophy and hyperplasia and perhaps heart failure. Calmodulin (CaM) is a ubiquitous intracellular Ca2+ receptor known to regulate cell proliferation in a number of cell types; we have shown that increased CaM levels targeted to cardiac myocytes by the atrial natriuretic hormone (ANF) promoter in transgenic mice lead to cardiac enlargement characterized by exaggerated myocyte hypertrophy and hyperplasia. Furthermore, there is excess mortality in neonates from the line of transgenic mice expressing the highest levels of CaM. The first aim is to evaluate transgenic mice that die prematurely compared with transgenic mice with normal lifespans. The approach is to examine CaM levels, expression of genes related to growth (i.e. proto-oncogenes) or are markers of hypertrophy, and relative levels of activated multifunctional Ca2+ /CaM protein kinase II (CaMK II), a potential mediator or the CaM effect. Aim 2 is to determine the effect of dither reinduced or persistent expression of CaM in the ventricles of transgenic mice. Two approaches will be used: (1) to reinduce the CaM transgene in adult animals of the existing lines using pharmacologic agents known to induce the ANF promoter, again examining changes, over time, in heart size, mortality, and expression of the genes listed above; and (2) to develop a new line of transgenic mice with CaM expression targeted to the heart by the iso=-ANF promoter; use of this promoter should result in continued expression of CaM in the ventricles into adulthood. The animals will be examined as described above. These studies may further define the relationship of CaM levels in cardiac myocytes to the degree of growth response and development of heart failure and possibly identify change in expression of genes, that accompany the progression from cardiac hypertrophy to heart failure in this model. The final aim is to test the hypothesis that CaMK II is central to the cardiomyocyte growth response induced by CaM. The approach is to develop transgenic mice bearing a constitutively active CaMK II gene under control of a cardiomyocyte specific promoter and characterize the cardiac growth response of these animals.

Our overall goal is to determine the manner by which the peptidyl-prolyl isomerase, Pin1, acts as a negative regulator of mitosis and evaluate whether this protein may be an attractive target for development of an anti-cancer therapeutic agent. Pin1 was discovered as a protein that interacts with NIMA, a protein Ser/Thr Kinase that is essential for progression from G2 to mitosis in Aspergillus nidulans. Pin1 has been conserved from yeast to man and may be critical for mitotic progression. We cloned Pin1 from Asperigillus and Xenopus, and made antibodies to the protein. We showed that Pin1 will regulate entry into mitosis in Xenopus extracts where it interacts with key components of the p34cdc2 regulatory pathway such as the active forms of the cdc25 protein phosphatase and the plxl protein kinase as well as a number of other mitotic phosphoproteins recognized by the MPM2 antibody. We propose to use the Xenopus Egg/oocyte system to address key issues regarding the prolyl isomerase and protein binding of Pin1 and elucidate critical details of its role(s) as a cell cycle regulator. We will evaluate the tole of Pin1 in the timing of mitotic entry and in the operation of checkpoints governing the G2/M transition as well as examine if Pin1 plays a role in exit from mitosis. In parallel, we will determine if Pin1 is essential for mouse development and focus on its role in development, activation and/or proliferation of T lymphocytes by using the cre/lox system to disrupt the Pin1 gene in mice either globally or specifically in T lymphocytes. We will determine if phenotypic consequences of Pin1 deletion in either vertebrate system requires the prolyl isomerase activity. Completion of these studies will define the role(s) of Pin1 in growth and development, determine its critical cell cycle functions and clarify whether the Pin1 prolyl isomerase is a good candidate for anti-cancer drug development.

[unreadable] DESCRIPTION (provided by applicant): Calcium is a pervasive second messenger and transduces endocrine signaling pathways initiated by binding of many hormones and growth factors to membrane receptors. The primary Ca2+ receptor is calmodulin (CaM) and Ca2+/CaM activates several CaM kinases in endocrine cells. Our long-term objective is to elucidate how a recently discovered CaMKIV cascade participates in Ca2+/CaM-mediated gene expression and regulates cell function. Analysis of Camk4~'~ mice identified a CaMKIV cascade in hematopoeitic stem cells (HSC) and cerebellar granule cells that regulates CREB phosphorylation and CBP levels leading to increased Bcl-2 and BDNF mRNA, respectively. We will determine how hormonal activation of the CaMKIV cascade regulates hematopoiesis by influencing maintenance of HSCs. HSCs isolated from CaMKIV- deficient mice cannot reconstitute bone marrow when transplanted into irradiated recipient host mice in a manner suggesting that CaMKIV regulates stem cell homeostatis by suppressing inappropriate proliferation and exhaustion of HSCs. To evaluate this hypothesis we will: 1) elucidate the importance of the CaMKIV kinase cascade in HSC maintenance in vivo. HSCs will be isolated from mice null for one or more of the CaMKIV cascade proteins and evaluated in bone marrow reconstitution assays; 2) address the idea that the CaMKIV cascade in HSCs is regulated by specific members of the Wnt family of growth factors. The Wnt proteins are required for embryonic development and Wnt 3A and Wnt 5A, have been implicated in HSC maintenance. Wnt 5A has been shown to initiate a Ca2+ signal and we will use isolated HSCs to investigate if Ca2+ activates a CaMKIV cascade that includes CREB and CBP and leads to upregulation of pro-survival genes such as Bcl-2; 3) evaluate the mechanism by which the CaMKIV cascade regulates CBP and identify key target genes by which the cascade influences HSC maintenance. We will use cultured cells to determine the molecular mechanism by which CaMKIV regulates CBP levels. We will use cultured cells and bone marrow transplantation to determine if Bcl-2 is a primary target of the CaMKIV cascade in HSCs and, if not, evaluate the importance of other target genes identified by previous RNA microarray analysis. Identification of such downstream genes may suggest efficacious drug targets for achieving better ex vivo expansion of HSCs for bone marrow transplantation patients. [unreadable] [unreadable]

[unreadable] DESCRIPTION (provided by applicant): The overall objective is to define the Ca2+-dependent signaling pathway in hypothalamic neurons that regulates appetite and body weight to provide novel insights to human obesity. AMPK, originally discovered to protect cells against stresses that deplete ATP, is a key enzyme in the pathways by which ghrelin and leptin act on arcuate nucleus (ARM) hypothalamic neurons to control production of NPY and, thus, appetite, energy homeostasis and body weight. Ca2+ is a ubiquitous 2nd messenger that mediates signaling cascades initiated by hormones and growth factors, in which calmodulin (CaM) is the Ca2+ receptor and Ca2+/CaM- dependent protein kinases (CaMK) transduce the Ca2+/CaM signal resulting in many cell responses. A "CaMK cascade" has been identified composed of Ca2+/CaM, a CaMKK (a or P) and a CaMK (I or IV). Recently the CaMKKs have also been shown to function as AMPKKs and are required to activate AMPK in some human cells. CaMKKp, a brain-specific enzyme, is expressed in the ARM and CaMKKp"'" mice show decreased hypothalamic AMPK phosphorylation/activity as well as NPY/AgRP mRNAs. Indeed, CaMKKp"'" mice share many phenotypes with mice null for NPY or an NPY receptor. Infusion of the only selective CaMKK antagonist, STO-609, into the 3rd ventricle of adult wild-type mice results in acute decreases in food intake and body weight as well as hypothalamic content of NPY/AgRP mRNAs. We hypothesize that CaMKKp serves as a primary AMPKK in NPY/AgRP neurons and that acute inhibition of this enzyme leads to decreased production of NPY which causes decreased food intake, altered energy homeostasis and weight loss. To evaluate this hypothesis we will: 1) utilize genetically altered mice to investigate the physiologically relevant role(s) of the CaMKKs, p and a as AMPKKs in mouse hypothalamus and cultured cells to clarify pathways that regulate NPY gene expression and malonyl CoA level; and 2) define the physical interaction between the CaMKKs and AMPK by employing molecular and biochemical technology to understand the nature of the CaMKK/AMPK signaling complex and how this complex can be inhibited. Our results should validate CaMKKp as a target for drug discovery, which could eventually lead to identification of new therapeutics to treat obesity. [unreadable] [unreadable] [unreadable]

The recently reorganized Duke Comprehensive Cancer Institute (DCCI) is charged to serve as the center of all Duke activities in cancer, with an imperative to oversee all of the Duke cancer operations in the future. As a matrix center in one of the largest biomedical research institutes, the DCCI has been continuously supported by the National Cancer Institute (NCI) for over 36 years, by leveraging Duke's basic research strengths with significant clinical oncology research excellence which is enabled by treating 5,581 new cancer patients annually, enrolling over 1,000 patients annually onto therapeutic clinical trials, and enhanced by the presence of national oncology research resources, such as the data and statistical centers of two NCI-funded cooperative groups. Total membership in the DCCI is 285 members, with members having an increasingly prominent focus in cancer research and increased amount of NCI funding since 2004. Total funding for program members is $331,617,013, of which $269,525,804 is from peer-reviewed sources. A cancer focus is illustrated by $77,785,582 or 23.5% of funding from the NCI, the American Cancer Society or the Department of Defense. From 2004-2008, program members published 6,941 papers in peer-reviewed journals cited in PubMed. Of these publications, 10% are the result of intra-programmatic collaborations and 19% due to inter-programmatic collaborations. In this competitive renewal application, we request support our membership in 11 Research Programs supported by 17 Shared Resources. DCCI members made significant scientific advances including: the development of innovative genomic tests to predict which cancer patients would respond to specific chemotherapy, and basic insight into critical signaling pathways in cancer and developmental biology. The DCCI oversees all cancer related philanthropy at Duke, and these funds have been used to support recruitment of new members, innovative pilot projects, support of multidisciplinary research, and new laboratory and administrative space. This application for ongoing support of the DCC articulates the principles, goals and strategies for propelling the DCCI to fully leverage the enormous advances in biomedical research at Duke and beyond, to improve and extend the lives of cancer patients with cancer and at risk for cancer, and delivering these to society.