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Targeted covalent inhibitors have had a major impact on human health and disease. In the past decade, several drugs with a covalent mechanism of action have been approved for the treatment of various cancers. Unfortunately, due to the lack of chemical diversity found naturally in protein biologics, we have been limited to designing covalent inhibitors that are small molecules or peptides. In recent years, however, through the use of genetic code expansion, we have been able to genetically encode new functionalities into proteins. These new functionalities include incorporating bioreactive unnatural amino acids (Uaas) into proteins, which allow for the formation of covalent bonds between binding proteins and their targets. Developing covalent biologics has great implications for the development of novel therapeutics and diagnostic tools. Chapter 1 introduces the reader to the recent chemical advancement of encoding bioreactive Uaas into proteins as well as successful development of anticancer and antiviral covalent protein drugs. Here, we describe the development of a new electrophilic bioreactive Uaa as well as two applications of protein binders with covalent mechanisms. In chapter 2, we describe the development of genetically encoding a SuFEx reactive meta-fluorosulfate tyrosine (mFSY) into proteins to expand the toolbox of bioreactive Uaas. mFSY has the ability to covalently target different side chain orientations as compared to known fluorosulfate Uaas fluorosulfate-L-tyrosine (FSY) and fluorosulfonyloxy-benzoyl-L-lysine (FSK). Chapter 3 describes the application of covalent biologics for targeted radionuclide therapies. We develop a covalent nanobody that binds to its target irreversibly, achieving exceptional tumor uptake, fast blood clearance, and low background. This radiolabeled covalent nanobody markedly increases radioisotope levels in tumors and extends tumor residence time while maintaining systematic clearance. Finally, in chapter 4 we demonstrate the

Allosteric regulation is classically defined as action at a distance, where a perturbation outside of a protein active site affects function. While this definition has motivated many studies of allosteric mechanisms at the level of protein structure, translating these insights to the allosteric regulation of entire cellular processes - and their crosstalk - has received less attention, despite the broad importance of allostery for cellular regulation foreseen by Jacob and Monod. Here, we revisit an evolutionary model for the widespread emergence of allosteric regulation in colocalized proteins, describe supporting evidence, and discuss emerging advances in mapping allostery in cellular networks that link precise and often allosteric perturbations at the molecular level to functional changes at the pathway and systems levels.

Cells convey information about their extracellular environment to their core functional machineries. Studying the capacity of intracellular signaling pathways to transmit information addresses fundamental questions about living systems. Here, we review how information-theoretic approaches have been used to quantify information transmission by signaling pathways that are functionally pleiotropic and subject to molecular stochasticity. We describe how recent advances in machine learning have been leveraged to address the challenges of complex temporal trajectory datasets and how these have contributed to our understanding of how cells employ temporal coding to appropriately adapt to environmental perturbations.

In this thesis, we studied the role of Bruton's tyrosine kinase (Btk) in B cells. We specifically investigated the role of different receptor signaling pathways in which Btk is involved and the contribution of Btk signaling to the development of leukemia and autoimmune disease.

DNA-encoded library (DEL) technology is a powerful next-generation drug discovery engine. DELs permit the design and synthesis of large, customized molecular libraries, which can be mined for new ligands by affinity selection. More advanced screening technology has emerged that enables direct activity-based DEL screening, but to date, this is limited to biochemical activity assays. Cellular assays, which are vital for discovering physiologically relevant novel chemical matter, are still incompatible with DEL. Here, I describe a prospective workflow that renders DEL compatible with cellular activity assays. Cells are grown directly on DEL beads, which effectively serve as microcarriers. The cells are engineered to secrete a reporter upon stimulation with an agonist, which is liberated from the DEL bead via photocleavage. The microcarrier is functionalized with affinity tag ligands, which capture the affinity-tagged secreted reporter. Toward this goal, I have engineered cell lines that secrete HaloTag-labeled fluorescent protein reporters, shown that reporter cell lines can grow and signal on microcarriers, and explored photocleavage conditions that permit efficient photochemistry while preserving cell viability. Additional studies were conducted to identify appropriate positive control ligands of a model cellular target, STING (stimulator of interferon genes). This thesis lays the groundwork for a DEL-compatible cellular assay paradigm that can be adapted to an array of cell-based assay strategies.

DNA-encoded library (DEL) technology is a powerful next-generation drug discovery engine. DELs permit the design and synthesis of large, customized molecular libraries, which can be mined for new ligands by affinity selection. More advanced screening technology has emerged that enables direct activity-based DEL screening, but to date, this is limited to biochemical activity assays. Cellular assays, which are vital for discovering physiologically relevant novel chemical matter, are still incompatible with DEL. Here, I describe a prospective workflow that renders DEL compatible with cellular activity assays. Cells are grown directly on DEL beads, which effectively serve as microcarriers. The cells are engineered to secrete a reporter upon stimulation with an agonist, which is liberated from the DEL bead via photocleavage. The microcarrier is functionalized with affinity tag ligands, which capture the affinity-tagged secreted reporter. Toward this goal, I have engineered cell lines that secrete HaloTag-labeled fluorescent protein reporters, shown that reporter cell lines can grow and signal on microcarriers, and explored photocleavage conditions that permit efficient photochemistry while preserving cell viability. Additional studies were conducted to identify appropriate positive control ligands of a model cellular target, STING (stimulator of interferon genes). This thesis lays the groundwork for a DEL-compatible cellular assay paradigm that can be adapted to an array of cell-based assay strategies.

The complex molecular machinery of the cell controls its response to various signals and environmental conditions. A natural approach to study these molecular mechanisms and cellular processes is with protein interaction networks. Due to the complexity of these networks, sophisticated computational techniques are required to extract biological insights from them. In this thesis, I develop and apply network-based algorithms for three different challenges. 1. I develop a novel, highly-scalable algorithm for network-based label prediction methods that enables the integration of functional annotations and interaction networks across many species in order to predict the functions of genes in newly-sequenced bacteria. 2. To overcome the limitations of experimental approaches to find human proteins and processes that are hijacked by SARS-CoV-2, I adapt network propagation approaches for predicting human interactors of the virus. 3. Large-scale experimental techniques to screen chemicals for toxicity have tested their effects on many individual proteins. I integrate human protein-protein interactions with this data to gain insights into the molecular networks those chemicals affect. For each of these research problems, I perform comprehensive evaluations and downstream analyses to demonstrate both the accuracy of our approaches and their utility in obtaining a broader understanding of the molecular systems in question.

The complex molecular machinery of the cell controls its response to various signals and environmental conditions. A natural approach to study these molecular mechanisms and cellular processes is with protein interaction networks. Due to the complexity of these networks, sophisticated computational techniques are required to extract biological insights from them. In this thesis, I develop and apply network-based algorithms for three different challenges. 1. I develop a novel, highly-scalable algorithm for network-based label prediction methods that enables the integration of functional annotations and interaction networks across many species in order to predict the functions of genes in newly-sequenced bacteria. 2. To overcome the limitations of experimental approaches to find human proteins and processes that are hijacked by SARS-CoV-2, I adapt network propagation approaches for predicting human interactors of the virus. 3. Large-scale experimental techniques to screen chemicals for toxicity have tested their effects on many individual proteins. I integrate human protein-protein interactions with this data to gain insights into the molecular networks those chemicals affect. For each of these research problems, I perform comprehensive evaluations and downstream analyses to demonstrate both the accuracy of our approaches and their utility in obtaining a broader understanding of the molecular systems in question.

Heparan sulfate proteoglycans (HSPG) are macromolecular glyco-conjugates expressed ubiquitously on the cell surface and in the extracellular matrix where they interact with a wide range of ligands to regulate many aspects of cellular function. The capacity of the side glycosaminoglycan chain heparan sulfate (HS) being able to interact with diverse protein ligands relies on its complex structure that is generated by a controlled biosynthesis process, involving the actions of glycosyl-transferases, sulfotransferases and the glucuronyl C5-epimerase. It is believed that activities of the modification enzymes control the HS structures that are designed to serve the biological functions in a given cell or biological status. In this review, we briefly discuss recent understandings on the roles of HSPG in cytokine stimulated cellular signaling, focusing on FGF, TGF-beta, Wnt, Hh, HGF and VEGF.

Thesis (Ph.D.)--University of Rochester. School of Medicine & Dentistry. Dept. of Pharmacology and Physiology, 2016., Heart failure is one of the leading causes of death both in the United States and worldwide; therefore, discovering new drugs for the treatment of heart failure is a critical area of research. Cyclic nucleotide phosphodiesterases (PDEs) may represent compelling therapeutic targets in the treatment of heart failure. However, the function of phosphodiesterase 1C (PDE1C) remains largely unknown. Herein, we studied the expression, regulation, function, and underlying mechanisms of PDE1C in the pathogenesis of cardiac remodeling and dysfunction in vitro and in vivo. PDE1C expression is upregulated in mouse and human failing hearts, and by conducting comparative studies in cardiomyocytes isolated from wild type and global genetic PDE1C knockout (PDE1C-KO) mice, we determined that PDE1C depletion protects against cell death and hypertrophy induced by Ang II or isoproterenol treatment, indicating a potent cardioprotective role for PDE1C. This protective effect was reliant on cAMP/PKA activation, and involved PDE1C coupling to adenosine A2 receptors, indicating that PDE1C may regulate cAMP produced by these receptors. Furthermore, when subjected to pressure overload-induced heart failure, PDE1C-KO mice developed significantly reduced cardiac hypertrophy, fibrosis, apoptosis, and dysfunction relative to PDE1C-WT mice. Furthermore, treatment of PDE1C-KO TAC mice with an adenosine receptor antagonist reversed many of the protective effects of PDE1C-KO. Finally, as PDE1C inhibition or depletion appears to potentiate a particular cAMP pool in cardiomyocytes, we hypothesized that PDE1C inhibition could potentiate cAMP efflux, activating the extracellular cAMP-adenosine pathway, which could potentially act on either cardiomyocytes or fibroblasts in a protective fashion. Remarkably, we found that this pathway does appear to be active in cardiomyocytes, and that inhibition of any element of this pathway significantly reduced the protective effects of PDE1C depletion or inhibition on

Information spielt in der Natur eine zentrale Rolle. Als intrinsischer Teil des genetischen Codes ist sie das Grundgerüst jeder Struktur und ihrer Entwicklung. Im Speziellen dient sie auch Organismen, ihre Umgebung wahrzunehmen und sich daran anzupassen. Die Grundvoraussetzung dafür ist, dass sie Information ihrer Umgebung sowohl messen als auch interpretieren können, wozu Zellen komplexe Signaltransduktionswege entwickelt haben. In dieser Arbeit konzentrieren wir uns auf Signalprozesse in S.cerevisiae die von osmotischem Stress (High Osmolarity Glycerol (HOG) Signalweg) und der Stimulation mit α-Faktor (Pheromon Signalweg) angesprochen werden. Wir wenden stochastische Modelle an, die das intrinsische Rauschen biologischer Prozesse darstellen können, um verstehen zu können wie Signalwege die ihnen zur Verfügung stehende Information umsetzen. Informationsübertragung wird dabei mit einem Ansatz aus Shannons Informationstheorie gemessen, indem wir sie als einen Kanal in diesem Sinne auffassen. Wir verwenden das Maß der Kanalkapazität, um die Genauigkeit des Phosphorelays einschränken zu können. In diesem Modell, simuliert mit dem Gillespie Algorithmus, können wir durch die Analyse des Signalverhaltens den Parameterraum zusätzlich stark einschränken. Eine weitere Herangehensweise der Signalverarbeitung beschäftigt sich mit dem “Crosstalk” zwischen HOG und Pheromon Signalweg. Wir zeigen, dass die Kontrolle der Signalspezifizität vor allem bei Scaffold-Proteinen liegt, die Komponenten der Signalkaskade binden. Diese konservierten Motive zellulärer Signaltransduktion besitzen eine geeignete Struktur, um Information getreu übertragen zu können. Im letzten Teil der Arbeit untersuchen wir potentielle Gründe für die evolutionäre Selektion von Scaffolds. Wir zeigen, dass ihnen bereits durch die Struktur des Mechanismus möglich ist, Informationsgenauigkeit zu verbessern und einer verteilten Informationsweiterleitung sowohl dadurch als auch durch ihre Robustheit überlegen sind., Information plays a ubiquitous role in nature. It provides the basis for structure and development, as it is inherent part of the genetic code. It also enables organisms to make sense of their environments and react accordingly. For this, a cellular interpretation of information is needed. Cells have developed sophisticated signaling mechanisms to fulfill this task and integrate many different external cues with their help. Here we focus on signaling that senses osmotic stress (High Osmolarity Glycerol (HOG) pathway) as well as α-factor stimulation (pheromone pathway) in S.cerevisiae. We employ stochastic modeling to simulates the inherent noisy nature of biological processes to assess how systems process the information they receive. This information transmission is evaluated with an information theoretic approach by interpreting signal transduction as a transmission channel in the sense of Shannon. We use channel capacity to both constrain as well as quantify the fidelity in the phosphorelay system of the HOG pathway. In this model, simulated with the Gillespie Algorithm, the analysis of signaling behavior allows us to constrain the possible parameter sets for the system severely. A further approach to signal processing is concerned with the mechanisms that conduct crosstalk between the HOG and the pheromone pathway. We find that the control for signal specificity lies especially with the scaffold proteins that tether signaling components and facilitate signaling by trans-location to the membrane and shielding against miss-activation. As conserved motifs of cellular signal transmission, these scaffold proteins show a particularly well suited structure for accurate information transmission. In the last part of this thesis, we examine the potential reasons for an evolutionary selection of the scaffolding structure. We show that due to its structure, scaffolds are increasing information transmission fidelity and outperform a distributed signal in this regard.

Scope: 8-prenyl quercetin (PQ) is a typical prenylflavonoid distributed in plant foods and shows higher potential bioactivity than its parent quercetin (Q) although the mechanisms are not fully understood. This study aims to clarify the anti-inflammatory effects and molecular mechanisms of PQ in cell and animal models, compared to Q. Methods and results: RAW264.7 cells were treated with PQ or Q to investigate the influence on the production of inducible nitric oxide synthase (iNOS), cyclooxygenase-2 (COX-2) and protein kinases by Western blotting. Nitric oxide (NO) and prostaglandin E2 (PGE2) were measured by the Griess method and ELISA, respectively. Cytokines were assayed by the multiplex technology. Mouse paw edema was induced by lipopolysaccharide (LPS). The results revealed that PQ had stronger inhibition on the productions of iNOS, COX-2, NO, PGE2, and 12 kinds of cytokines, than Q. PQ also showed in vivo anti-inflammatory effect by attenuating mouse paw edema. Molecular data revealed that PQ had no competitive binding to Toll-like receptor 4 (TLR4) with LPS, but directly targeted SEK1-JNK1/2 and MEK1-ERK1/2. Conclusion: PQ as a potential inhibitor revealed anti-inflammatory effect in both cell and animal models at least by targeting SEK1-JNK1/2 and MEK1-ERK1/2.

Adult neurogenesis, the process by which new neurons develop in the adult mammalian central nervous system, was thought to be nonexistent by accepted scientific dogma until the discovery of adult neural stem cells (NSCs) in the 1990s. NSCs have been found in two regions of the adult brain: the subventricular zone of the lateral ventricles and the subgranular zone of the hippocampal dentate gyrus, and have the capacity to differentiate into neurons, astrocytes, and oligodendrocytes. Importantly, hippocampal NSCs play key roles in learning and memory, and have been implicated in a number of pathologies, including Alzheimer’s disease. NSCs reside in complex niches that provide the physical, chemical, and biological signals regulating stem cell maintenance and differentiation. A thorough understanding of NSC biology and niche signals can provide both insight into the mechanisms of adult neurogenesis, and inform stem cell based therapeutics to treat neurological injury and disease. Since the discovery of NSCs, a body of work has emerged characterizing the wide array of signals and intracellular pathways that mediate NSC behavior. Some of these findings, however, point to complex signaling mechanisms, the further study of which requires techniques outside the standard biological “toolbox”. The goal of this dissertation, therefore, was to engineer novel tools to enable the study and control of complex signaling systems in NSCs, and their application towards novel biological discoveries.The work presented here investigates two aspects of NSC biology: heterogeneity and cell-cell signaling. Stem cells are inherently heterogeneous; NSCs give rise to diverse progeny, including neurons and astrocytes, and in vitro, NSCs can differentially respond to the same set of cues. To probe this heterogeneity, a single cell Western blotting (scWestern) platform was developed. scWesterns enabled the interrogation of the proteome of thousands of single cells in about four hours, and multiple

Adult neurogenesis, the process by which new neurons develop in the adult mammalian central nervous system, was thought to be nonexistent by accepted scientific dogma until the discovery of adult neural stem cells (NSCs) in the 1990s. NSCs have been found in two regions of the adult brain: the subventricular zone of the lateral ventricles and the subgranular zone of the hippocampal dentate gyrus, and have the capacity to differentiate into neurons, astrocytes, and oligodendrocytes. Importantly, hippocampal NSCs play key roles in learning and memory, and have been implicated in a number of pathologies, including Alzheimer’s disease. NSCs reside in complex niches that provide the physical, chemical, and biological signals regulating stem cell maintenance and differentiation. A thorough understanding of NSC biology and niche signals can provide both insight into the mechanisms of adult neurogenesis, and inform stem cell based therapeutics to treat neurological injury and disease. Since the discovery of NSCs, a body of work has emerged characterizing the wide array of signals and intracellular pathways that mediate NSC behavior. Some of these findings, however, point to complex signaling mechanisms, the further study of which requires techniques outside the standard biological “toolbox”. The goal of this dissertation, therefore, was to engineer novel tools to enable the study and control of complex signaling systems in NSCs, and their application towards novel biological discoveries.The work presented here investigates two aspects of NSC biology: heterogeneity and cell-cell signaling. Stem cells are inherently heterogeneous; NSCs give rise to diverse progeny, including neurons and astrocytes, and in vitro, NSCs can differentially respond to the same set of cues. To probe this heterogeneity, a single cell Western blotting (scWestern) platform was developed. scWesterns enabled the interrogation of the proteome of thousands of single cells in about four hours, and multiple

The ketogenic diet (KD) is a broad-spectrum therapy for medically intractable epilepsy and is receiving growing attention as a potential treatment for neurological disorders arising in part from bioenergetic dysregulation. The high-fat/low-carbohydrate "classic KD", as well as dietary variations such as the medium-chain triglyceride diet, the modified Atkins diet, the low-glycemic index treatment, and caloric restriction, enhance cellular metabolic and mitochondrial function. Hence, the broad neuroprotective properties of such therapies may stem from improved cellular metabolism. Data from clinical and preclinical studies indicate that these diets restrict glycolysis and increase fatty acid oxidation, actions which result in ketosis, replenishment of the TCA cycle (i.e., anaplerosis), restoration of neurotransmitter and ion channel function, and enhanced mitochondrial respiration. Further, there is mounting evidence that the KD and its variants can impact key signaling pathways that evolved to sense the energetic state of the cell, and that help maintain cellular homeostasis. These pathways, which include PPARs, AMP-activated kinase, mammalian target of rapamycin, and the sirtuins, have all been recently implicated in the neuroprotective effects of the KD. Further research in this area may lead to future therapeutic strategies aimed at mimicking the pleiotropic neuroprotective effects of the KD.

Free to read Cancer is one of the most life-threatening diseases with many forms still regarded as incurable. The conventional cancer treatments have unwanted side effects such as the death of normal cells. A therapy that can accurately target and effectively kill tumor cells could address the inadequacies of the available therapies. Atmospheric gas plasmas (AGP) that are able to specifically kill cancerous cells offer a promising alternative approach compared to conventional therapies. AGP have been shown to exploit tumor-specific genetic defects and a recent trial in mice has confirmed its antitumor effects. The mechanism by which the AGP act on tumor cells but not normal cells is not fully understood. A review of the current literature suggests that reactive oxygen species (ROS) generated by AGP induce death of cancer cells by impairing the function of intracellular regulatory factors. The majority of cancer cells are defective in tumor suppressors that interfere normal cell growth pathways. It appears that pro-oncogene or tumor suppressor-dependent regulation of antioxidant/or ROS signaling pathways may be involved in AGP-induced cancer cell death. The toxic effects of ROS are mitigated by normal cells by adjustment of their metabolic pathways. On the other hand, tumor cells are mostly defective in several regulatory signaling pathways which lead to the loss of metabolic balance within the cells and consequently, the regulation of cell growth. This review article evaluates the impact of AGP on the activation of cellular signaling and its importance for exploring mechanisms for safe and efficient anticancer therapies.

The ketogenic diet (KD) is a broad-spectrum therapy for medically intractable epilepsy and is receiving growing attention as a potential treatment for neurological disorders arising in part from bioenergetic dysregulation. The high-fat/low-carbohydrate "classic KD", as well as dietary variations such as the medium-chain triglyceride diet, the modified Atkins diet, the low-glycemic index treatment, and caloric restriction, enhance cellular metabolic and mitochondrial function. Hence, the broad neuroprotective properties of such therapies may stem from improved cellular metabolism. Data from clinical and preclinical studies indicate that these diets restrict glycolysis and increase fatty acid oxidation, actions which result in ketosis, replenishment of the TCA cycle (i.e., anaplerosis), restoration of neurotransmitter and ion channel function, and enhanced mitochondrial respiration. Further, there is mounting evidence that the KD and its variants can impact key signaling pathways that evolved to sense the energetic state of the cell, and that help maintain cellular homeostasis. These pathways, which include PPARs, AMP-activated kinase, mammalian target of rapamycin, and the sirtuins, have all been recently implicated in the neuroprotective effects of the KD. Further research in this area may lead to future therapeutic strategies aimed at mimicking the pleiotropic neuroprotective effects of the KD.

The National Cancer Act, signed in 1971, aimed to eliminate cancer deaths through a massive increase in research funding. The American Association for the Advancement of Science, the publisher of Science , Science Signaling , and Science Translational Medicine , observed the 40th anniversary of the Cancer Act in 2011, with special research articles and features, found in all three journals, on the state of cancer research 40 years later. This collection of articles explores both breakthroughs and the challenges in cancer research over the last four decades, and lets us know what we might expect in the future., Der US-amerikanische National Cancer Act, welcher im Jahr 1971 in Kraft trat, beabsichtigte durch eine massive Erhöhung der Krebsforschungsgelder, die Gefahr des Krebstodes zu beseitigen. Die American Association for the Advancement of Science, Herausgeber der Fachzeitschriften Science , Science Signaling , und Science Translational Medicine , gedenkt im Jahr 2011 dem 40. Jahrestag des Cancer Act durch die Veröffentlichung von speziellen Forschungsaufsätzen und Sonderbeiträgen aus allen drei Fachzeitschriften, die zum Stand der Krebsforschung 40 Jahre danach Stellung nehmen. Die Sondersammlung untersucht sowohl Durchbrüche als auch Herausforderungen der Krebsforschung über die letzten vier Jahrzehnte und liefert Prognosen über zukünftige Entwicklungen in diesem Forschungsgebiet.

Xavier University (XU) and the Tulane Cancer Center (TCC) will build a core of human talent that will address scientific problems such as drug resistance and the effect of environmental agents on breast cancer (BC) in the African-American community. A multi-part research and training program will generate data, develop new research programs and train new faculty and African-American students in BC research. The first component will fund two research projects. The Wang and Burow project will elucidate a previously unexplored cellular signaling mechanism that leads to drug resistance in breast carcinoma cells derived from African American women and women of other ethnicities. The Wiese and Hill project will identify and characterize the genes and gene products associated with BC cell proliferation induced by exposure to pesticide mixtures and is relevant to the African American community in Southern States where pesticide exposure is relatively high. The second part of the program aims to increase the number of faculty at XU involved in BC research by supporting two junior faculty members to develop BC research projects with a TCC mentor. The third objective will support research training of XU undergraduates and pharmacy students. The fourth objective will provide workshops, seminars and research opportunities in BC research for the XU community. This program will enhance the understanding of unique aspects of BC development and progression among African American women and will contribute to the elimination of the "mortality gap" between African- American BC patients and women of other ethnicities.

Cyclooxygenase-2 (COX-2) and its product PGE2 are known to promote tumor growth and to enhance the penetration of cancer cells into adjacent tissues. Thus, knowing how the activity of COX2 is regulated at the cellular level has implications for breast cancer therapeutic strategies. The goal is to unravel a new molecular mechanism for regulating the activity of COX-2. In plasma membranes, arachidonic acid (AA) is released by phospholipase A2 (PLA2). COX then converts AA to prostaglandins. The activity of PLA2 is known to vary with cholesterol content in an alternating manner, showing a local minimum at critical sterol mole fractions for maximal superlattice formation. In this study, the cholesterol content in MCF-7 human breast cancer cells was depleted by using methyl-beta-cyclodextrin. A biphasic change in COX-2 activity was observed at certain cell cholesterol content Cr., The original document contains color images.

Cyclooxygenase-2 (COX-2) and its product PGE2 are known to promote tumor growth and to enhance the penetration of cancer cells into adjacent tissues. Thus, knowing how the activity of COX2 is regulated at the cellular level has implications for breast cancer therapeutic strategies. The goal is to unravel a new molecular mechanism for regulating the activity of COX-2. In plasma membranes, arachidonic acid (AA) is released by phospholipase A2 (PLA2). COX then converts AA to prostaglandins. The activity of PLA2 is known to vary with cholesterol content in an alternating manner, showing a local minimum at critical sterol mole fractions for maximal superlattice formation. In this study, the cholesterol content in MCF-7 human breast cancer cells was depleted by using methyl-beta-cyclodextrin. A biphasic change in COX-2 activity was observed at certain cell cholesterol content Cr. The cholesterol content near Cr could serve as a fine-tuning mechanism to regulate COX-2 activity and PGE2 production, and consequently, cancer cell growth and metastasis., The original document contains color images.

Smooth muscle cells in the urinary bladder are involved in the control of urine filling and emptying. We have used mice to study the contractile regulation of urinary bladder smooth muscle during the fetal period. We found that nerve-induced contractions are mainly dependent on muscarinic receptor activation in the newborn whereas adult bladders are equally dependent on muscarinic and purinergic receptor activation. Muscarinic receptor-induced contractions are due to activation of the M3 subtype in both adults and newborns. Activator calcium is mainly recruited from the extracellular space via L-type calcium channels in both adult and newborn tissue. The contractile machinery of the newborn’s bladder tissue is more sensitive to calcium which enables contractions at lower intracellular and extracellular calcium concentrations. Non-muscle myosin is contributing to the force development in the newborn but is not the cause for the increased calcium sensitivity. The increased calcium sensitivity is due to a lower phosphatase activity and a lower expression of the myosin light chain phosphatase subunit MYPT1. The contractions induced by muscarinic receptor agonists are characterized by increased phasic activity in the newborn. This activity is not dependent on K+ channels, calcium release from the sarcoplasmic reticulum or gap junctional transmission. Muscarinic receptor stimulation activates T-type calcium channels in the newborn bladder but not in the adult. The main sensitizing pathways, involving protein kinase C (PKC) and Rho-kinase, are established in the newborn tissue. The effects of PKC activation on muscarinic receptor-induced responses differ in newborn and adult tissue. The muscarinic receptor-induced response of the newborn is abolished in the presence of high PKC activation (using PDBu) whereas the response is potentiated in the adult. The phasic component of the response in the newborn bladder is particularly inhibited due to activation of PKC.

Calcium is an essential nutrient for plants; it is involved in developmental processes and in responses to biotic and abiotic factors. Several signals that modify the calcium concentration in the cytoplasm, endoplasmic reticulum, nucleus and/or plastids have been observed. These changes in the calcium concentration in the cell interior are rapidly returned to basal levels, in the meantime, innumerable and complex signaling cascades. This note exposes the mechanisms of calcium transport through the cell membranes of the entrance of calcium in the plant cells., El calcio es un nutriente esencial para las plantas, se encuentra involucrado en procesos de desarrollo y de respuesta a factores bióticos y abióticos. Numerosas señales modifican la concentración de calcio en el citoplasma, núcleo, retículo endoplásmico o plastídios. El incremento del calcio en el citosol es rápidamente disminuido, pero en el lapso de incremento, se forman innumerables y complejas cascadas de señalización que conllevan a la respuesta celular. Este nota expone los mecanismos implicaciones de la entrada del calcio en las células vegetales.

The most significant finding occurred after exposure of human 244B lymphoblastoid cells to a high-peak power pulsed ultrawideband transmitted electric field (UWB TEMF) signal, which had given a wide array of largely negative results. The UWB TEMF exposure (average peak power, 100 kV/m; average pulse width, 780 ps) duration was 90 min (intermittent). The UWB TEMF did not cause an alteration in cell cycle distribution, stabilization of the p53 target genes. There was no loss of mitochondrial membrane potential or release of cytochrome C into the cytosol at 6 hr post-exposure (i.e., no apoptosis). However, at 2 hr a number of gene increase and decrease were detected (12,000 gene microarray system). This suggests that the cells were capable of "sensing" the pulsed UWB TEMF. While the induction of the gene transcription factor NF-kB was observed, no evidence was obtained for downstream activity. The UWB TEMF signal may be necessary, but not sufficient to cause coordinated downstream events. FDTD analysis was performed to determine the doses for exposure cells in suspension or surface attached. An unexpected distribution of energy in the medium is described.

Atrial natriuretic peptide (ANP) and B-type natriuretic peptide (BNP) are cardiac hormones, which are involved in the regulation of blood pressure and fluid homeostasis. The major determinant for ANP and BNP release are atrial and ventricular wall stretch, but also some vasoactive factors such as endothelin-1 (ET-1) can enhance cardiac hormone secretion. The mechanical stretch rapidly activates multiple signal transduction pathways in cardiac cells, but the cellular mechanisms mediating stretch-induced ANP secretion are still unknown. The aim of the present study was to examine the cellular mechanisms of autocrine/paracrine factors and stretch-induced ANP secretion. Genistein, a potent protein tyrosine kinase (PTK) inhibitor, rapidly increased cardiac contractile force and ANP secretion in perfused rat heart. This effect of genistein may be unrelated to the inhibition of PTKs since this stimulation was blocked by a L-type calcium channel antagonist and Ca²⁺/calmodulin-dependent protein kinase II inhibitor. Pregnancy hormone relaxin increased heart rate and ANP secretion in perfused spontaneously beating heart, suggesting that relaxin may have a role in modulating cardiac function. Cellular mechanisms of atrial wall stretch-induced ANP secretion were also studied. This enhanced secretion was blocked by sarcoplasmic reticulum Ca²⁺-ATPase inhibitor thapsigargin and PTK inhibitor lavendustin A, indicating that thapsigargin sensitive Ca²⁺ pools and activation of PTK orPTK cascade have an important role in the regulation of stretch-secretion coupling. In addition, protein phosphatase inhibitor okadaic acid accelerated stretch-induced ANP secretion, suggesting that precise balance of protein kinase and phosphatase activity plays a role in mechanical stretch-induced ANP secretion. Finally interactions of endothelial factors regulating ANP exocytosis were studied. The potent nitric oxide synthase inhibitor L-NAME increased basal and atrial wall stretch-induced ANP s