The Intramolecular Loss of Fluorescence by Lysine Derivatized with Naphthalenedialdehyde

This is the publisher's version, also available electronically from http://www.opticsinfobase.org/as/abstract.cfm?URI=as-44-5-858.Derivatization of primary amines such as amino acids and peptides with naphthalenedialdehyde (NDA) in the presence of cyanide ion yields cyanobenzo[f]-isoindole (CBI) adducts that are highly fluorescent. However, the fluorescence is seriously quenched with amines that possess more than one primary amine site, as is the case with lysine. Although it was found that the adsorption of CBI2-lysine on a solid substrate restored the fluorescence, the reason for the solution quenching, with respect to results for mono-derivatized amines, was investigated. The experiments to probe the quenching were based on the assumption that the mechanism responsible for quenching involved a charge-transfer (CT) excited state. Thus, it was found that the solvent properties of viscosity and polarity affected the lifetime and quantum yield of fluorescence in a manner consistent with the proposed mechanism

Broadband Coupling into a Single-Mode, Electroactive Integrated Optical Waveguide for Spectroelectrochemical Analysis of Surface-Confined Redox Couples

Pushing the sensitivity of spectroelectrochemical techniques to routinely monitor changes in spectral properties of thin molecular films (i.e., monolayer or submonolayer) adsorbed on an electrode surface has been a goal of many investigators since the earliest developments in this field. 1 It was initially recognized that exploiting the evanescent field generated by total internal reflection at the interface of an optically transparent electrode (such as a thin film of tin oxide or indium tin oxide (ITO) on glass or quartz) has the inherent advantage of selectively probing only the near-surface region, as opposed to bulk sampling with transmission based techniques. Furthermore, by utilizing the multiple reflections in an attenuated total reflectance (ATR) geometry, an enhancement in sensitivity can be realized, and as the thickness of the ATR element is decreased, the number of reflections increases, yielding a substantial sensitivity enhancement. [2][3][4][5][6] Itoh and Fujishima were the first to show the advantages of reducing the thickness of an ATR element overcoated with a transparent conductive oxide to the integrated optical waveguide (IOW) regime. Using a four-mode, gradient index waveguide coated with a transparent, conductive tin oxide layer, they demonstrated large sensitivity enhancements, relative to a single pass transmission experiment, for spectroelectrochemical measurements of methylene blue. 7,8 Other research groups subsequently described similar gradient index, multilayer, electroactive waveguide structures, but they did not make use of the technology to explore the spectroelectrochemistry of (sub)monolayer coverage films. [9][10][11][12][13] We recently described a single-mode, electroactive planar IOW (the EA-IOW) having a step refractive index profile. It was fabricated by sputtering a Corning 7059 glass layer (400 nm) over soda lime glass or quartz, followed by a 200-nm layer of SiO 2

Bim and Bmf synergize to induce apoptosis in Neisseria gonorrhoeae infection

Abstract: Bcl-2 family proteins including the pro-apoptotic BH3-only proteins are central regulators of apoptotic cell death. Here we show by a focused siRNA miniscreen that the synergistic action of the BH3-only proteins Bim and Bmf is required for apoptosis induced by infection with Neisseria gonorrhoeae (Ngo). While Bim and Bmf were associated with the cytoskeleton of healthy cells, they both were released upon Ngo infection. Loss of Bim and Bmf from the cytoskeleton fraction required the activation of Jun-N-terminal kinase-1 (JNK-1), which in turn depended on Rac-1. Depletion and inhibition of Rac-1, JNK-1, Bim, or Bmf prevented the activation of Bak and Bax and the subsequent activation of caspases. Apoptosis could be reconstituted in Bim-depleted and Bmf-depleted cells by additional silencing of antiapoptotic Mcl-1 and Bcl-XL, respectively. Our data indicate a synergistic role for both cytoskeletal-associated BH3-only proteins, Bim, and Bmf, in an apoptotic pathway leading to the clearance of Ngo-infected cells. Author Summary: A variety of physiological death signals, as well as pathological insults, trigger apoptosis, a genetically programmed form of cell death. Pathogens often induce host cell apoptosis to establish a successful infection. Neisseria gonorrhoeae (Ngo), the etiological agent of the sexually transmitted disease gonorrhoea, is a highly adapted obligate human-specific pathogen and has been shown to induce apoptosis in infected cells. Here we unveil the molecular mechanisms leading to apoptosis of infected cells. We show that Ngo-mediated apoptosis requires a special subset of proapoptotic proteins from the group of BH3-only proteins. BH3-only proteins act as stress sensors to translate toxic environmental signals to the initiation of apoptosis. In a siRNA-based miniscreen, we found Bim and Bmf, BH3-only proteins associated with the cytoskeleton, necessary to induce host cell apoptosis upon infection. Bim and Bmf inactivated different inhibitors of apoptosis and thereby induced cell death in response to infection. Our data unveil a novel pathway of infection-induced apoptosis that enhances our understanding of the mechanism by which BH3-only proteins control apoptotic cell death

Fos-related antigen-1 transgenic mouse as a model for systemic sclerosis: A potential role of M2 polarization

Objectives:To investigate the systemic sclerosis–related phenotype in fos-related antigen-1 transgenic mice and its underlying mechanisms.Methods:Lung and skin sections of constitutive fos-related antigen-1 transgenic mice and wild-type mice were examined by tissue staining and immunohistochemistry. The tricuspid regurgitation pressure gradient was measured by transthoracic echocardiography with a Doppler technique. To assess the impact of fos-related antigen-1 expression on macrophage function, bone marrow–derived mononuclear cells were derived from mice that expressed fos-related antigen-1 under the control of doxycycline and wild-type littermates. These bone marrow–derived mononuclear cells were induced to differentiate into macrophages with or without doxycycline, and analyzed for gene and protein expression. Finally, lung explants obtained from systemic sclerosis patients and control donors were subjected to immunohistochemistry.Results:The lungs of fos-related antigen-1 transgenic mice showed excessive fibrosis of the interstitium and thickening of vessel walls, with narrowing lumen, in an age-dependent manner. The tricuspid regurgitation pressure gradient was significantly elevated in fos-related antigen-1 transgenic versus control mice. Increased dermal thickness and the loss of subdermal adipose tissue were also observed in the fos-related antigen-1 transgenic mice. These changes were preceded by a perivascular infiltration of mononuclear cells, predominantly consisting of alternatively activated or M2 macrophages. Overexpressing fos-related antigen-1 in bone marrow–derived mononuclear cell cultures increased the expression of M2-related genes, such as Il10, Alox15, and Arg1. Finally, fos-related antigen-1-expressing M2 macrophages were increased in the lung tissues of systemic sclerosis patients.Conclusions:The fos-related antigen-1 transgenic mouse serves as a genetic model of systemic sclerosis that recapitulates the major vascular and fibrotic manifestations of the lungs and skin in systemic sclerosis patients. M2 polarization mediated by the up-regulation of fos-related antigen-1 may play a critical role in the development of systemic sclerosis

Overexpression of Mcl-1 exacerbates lymphocyte accumulation and autoimmune kidney disease in lpr mice

Cell death by apoptosis has a critical role during embryonic development and in maintaining tissue homeostasis. In mammals, there are two converging apoptosis pathways: the ‘extrinsic’ pathway, which is triggered by engagement of cell surface ‘death receptors’ such as Fas/APO-1; and the ‘intrinsic’ pathway, which is triggered by diverse cellular stresses, and is regulated by prosurvival and pro-apoptotic members of the Bcl-2 family of proteins. Pro-survival Mcl-1, which can block activation of the proapoptotic proteins, Bax and Bak, appears critical for the survival and maintenance of multiple haemopoietic cell types. To investigate the impact on haemopoiesis of simultaneously inhibiting both apoptosis pathways, we introduced the vavP-Mcl-1 transgene, which causes overexpression of Mcl-1 protein in all haemopoietic lineages, into Faslpr/lpr mice, which lack functional Fas and are prone to autoimmunity. The combined mutations had a modest impact on myelopoiesis, primarily an increase in the macrophage/monocyte population in Mcl-1tg/lpr mice compared with lpr or Mcl-1tg mice. The impact on lymphopoiesis was striking, with a marked elevation in all major lymphoid subsets, including the non-conventional double-negative (DN) T cells (TCRβ+ CD4– CD8– B220+ ) characteristic of Faslpr/lpr mice. Of note, the onset of autoimmunity was markedly accelerated in Mcl-1tg/lpr mice compared with lpr mice, and this was preceded by an increase in immunoglobulin (Ig)-producing cells and circulating autoantibodies. This degree of impact was surprising, given the relatively mild phenotype conferred by the vavP-Mcl-1 transgene by itself: a two- to threefold elevation of peripheral B and T cells, no significant increase in the non-conventional DN T-cell population and no autoimmune disease. Comparison of the phenotype with that of other susceptible mice suggests that the development of autoimmune disease in Mcl-1tg/lpr mice may be influenced not only by Ig-producing cells but also other haemopoietic cell types

Cytokine-dependent and cytokine-independent roles for Mcl-1: genetic evidence for multiple mechanisms by which Mcl-1 promotes survival in primary T lymphocytes

Myeloid cell leukemia sequence-1 (Mcl-1) is a critical anti-apoptotic factor in T lymphocytes. However, in spite of the many pro-apoptotic proteins with proposed binding to Mcl-1, the specific interactions by which Mcl-1 regulates primary T-cell survival under different conditions have not been fully explored. Further, how different trophic cytokines modulate the specific role(s) of Mcl-1 is unknown. Here, we use genetic mouse models to dissect the roles of Mcl-1 in primary T lymphocytes. Using the inducible Mcl-1-floxed estrogen receptor-Cre fusion protein (Mcl-1f/fERCre) deletion system in combination with genetic modification of other B-cell lymphoma 2 (Bcl-2) family members, we show that loss of pro-apoptotic Bcl-2 homologous antagonist/killer (Bak) rescues the survival of Mcl-1-deficient T cells in the presence of IL-7. Without IL-7, the survival of Mcl-1-deficient cells cannot be rescued by loss of Bak, but is partially rescued by overexpression of Bcl-2 or loss of Bcl-2-interacting mediator of cell death (Bim). Thus, Mcl-1 and Bcl-2 have a shared role, the inhibition of Bim, in promoting T-cell survival during cytokine withdrawal. Finally, we show that other common gamma-chain (γc) cytokines differentially modulate the roles of Mcl-1. IL-15 has effects similar to those of IL-7 in memory T cells and naïve CD8+ cells, but not naïve CD4+ cells. However, IL-4 maintains Mcl-1 and Bcl-2 but also upregulates Bim and Bcl-2-associated X protein (Bax), thus altering the cell's dependence on Mcl-1

A patient with Graves' disease who survived despite developing thyroid storm and lactic acidosis

A 56-year-old woman with Graves' disease presented with the complaints of diarrhea and palpitations. Physical examination and laboratory data revealed hypothermia and signs of mild hyperthyroidism, heart failure, hepatic dysfunction with jaundice, hypoglycemia, and lactic acidosis. The patient was diagnosed as having developed the complication of thyroid storm in the absence of marked elevation of the thyroid hormone levels, because of the potential hepatic and cardiac dysfunctions caused by heavy alcohol drinking. A year later, after successful treatment, the patient remains well without any clinical evidence of heart failure or hepatic dysfunction. Thyroid storm associated with lactic acidosis and hypothermia is a serious condition and has rarely been reported. Prompt treatment is essential even if the serum thyroid hormone levels are not markedly elevated. We present a report about this patient, as her life could eventually be saved

Modeling and Optimization of Direct Chill Casting to Reduce Ingot Cracking

Approximately 68% of the aluminum produced in the United States is first cast into ingots prior to further processing into sheet, plate, extrusions, or foil. The direct chill (DC) semi-continuous casting process has been the mainstay of the aluminum industry for the production of ingots due largely to its robust nature and relative simplicity. Though the basic process of DC casting is in principle straightforward, the interaction of process parameters with heat extraction, microstructural evolution, and development of solidification stresses is too complex to analyze by intuition or practical experience. One issue in DC casting is the formation of stress cracks [1-15]. In particular, the move toward larger ingot cross-sections, the use of higher casting speeds, and an ever-increasing array of mold technologies have increased industry efficiencies but have made it more difficult to predict the occurrence of stress crack defects. The Aluminum Industry Technology Roadmap [16] has recognized the challenges inherent in the DC casting process and the control of stress cracks and selected the development of 'fundamental information on solidification of alloys to predict microstructure, surface properties, and stresses and strains' as a high-priority research need, and the 'lack of understanding of mechanisms of cracking as a function of alloy' and 'insufficient understanding of the aluminum solidification process', which is 'difficult to model', as technology barriers in aluminum casting processes. The goal of this Aluminum Industry of the Future (IOF) project was to assist the aluminum industry in reducing the incidence of stress cracks from the current level of 5% to 2%. Decreasing stress crack incidence is important for improving product quality and consistency as well as for saving resources and energy, since considerable amounts of cast metal could be saved by eliminating ingot cracking, by reducing the scalping thickness of the ingot before rolling, and by eliminating butt sawing. Full-scale industrial implementation of the results of the proposed research would lead to energy savings in excess of 6 trillion Btu by the year 2020. The research undertaken in this project aimed to achieve this objective by a collaboration of industry, university, and national laboratory personnel through Secat, Inc., a consortium of aluminum companies. During the four-year project, the industrial partners and the research team met in 16 quarterly meetings to discuss research results and research direction. The industrial partners provided guidance, facilities, and experience to the research team. The research team went to two industrial plants to measure temperature distributions in commercial 60,000-lb DC casting ingot production. The project focused on the development of a fundamental understanding of ingot cracking and detailed models of thermal conditions, solidification, microstructural evolution, and stress development during the initial transient in DC castings of the aluminum alloys 3004 and 5182. The microstructure of the DC casting ingots was systematically characterized. Carefully designed experiments were carried out at the national laboratory and university facilities as well as at the industrial locations using the industrial production facilities. The advanced computational capabilities of the national laboratories were used for thermodynamic and kinetic simulations of phase transformation, heat transfer and fluid flow, solidification, and stress-strain evolution during DC casting. The achievements of the project are the following: (1) Identified the nature of crack formation during DC casting; (2) Developed a novel method for determining the mechanical properties of an alloy at the nonequilibrium mushy zone of the alloy; (3) Measured heat transfer coefficients (HTCs) between the solidifying ingot and the cooling water jet; (4) Determined the material constitutive model at high temperatures; and (5) Developed computational capabilities for the simulation of cracking formation in DC casting ingot. The models and the database developed in this project have been used to predict crack formation and to determine the optimal conditions for DC casting. The results demonstrated that cracking formation is strongly affected by casting conditions and the composition of the alloy. Scrap rate due to crack formation can be significantly reduced by controlling the cast speed and the concentrations of the alloy

ERK2 phosphorylation of serine 77 regulates Bmf pro-apoptotic activity

B-cell lymphoma 2 (Bcl-2) homology 3 (BH3)-only proteins represent a class of pro-apoptotic factors that neutralize pro-survival Bcl-2 proteins, and, in some cases, directly activate Bax. The mechanisms of control and the role of BH3-only proteins, such as Bcl-2 like protein 11 extra large and Bad are well studied. By contrast, relatively little is known about the regulation and role of Bcl-2 modifying factor (Bmf). The B-RAF oncogene is mutated in ∼8% of human tumors. We have previously shown that Bmf is upregulated at the transcript level and is required for apoptosis induced by targeting B-RAF signaling in tumor cells harboring mutant B-RAF. In this study, we show that Bmf is regulated at the post-translational level by mutant B-RAF-MEK-ERK2 signaling. Extracellular signal-regulated kinase (ERK2) directly phosphorylates Bmf on serine 74 and serine 77 residues with serine 77 being the predominant site. In addition, serine 77 phosphorylation reduces Bmf pro-apoptotic activity likely through a mechanism independent of altering Bmf localization to the mitochondria and/or interactions with dynein light chain 2 and the pro-survival proteins, B-cell lymphoma extra large, Bcl-2 and Mcl-1. These data identify a novel mode of regulation in Bmf that modulates its pro-apoptotic activity in mutant B-RAF tumor cells