Changes in lipophorins are related to the activation of phenoloxidase in the haemolymph of Locusta migratoria in response to injection of immunogens

In Locusta migratoria, activation of phenoloxidase in the haemolymph in response to injection of laminarin is age-dependent: being absent in fifth instar nymphs and newly emerged adults, and only becoming evident four days after the final moult. This pattern of change in phenoloxidase activation correlates with the pattern of change in the concentration of apolipophorin-III (apoLp-III) in the haemolymph. Injection of a conspecific adipokinetic hormone (Lom-AKH-I) has no effect on the phenoloxidase response in nymphs or newly emerged adults but, in adults older than four days, co-injection of the hormone with laminarin prolongs the activation of phenoloxidase in the haemolymph: a similar enhancement of the response to laminarin is observed in locusts that have been starved for 48 h but not injected with AKH-I. During most of the fifth stadium, injection of laminarin results in a decrease in the level of prophenoloxidase in the haemolymph; an effect that is not observed in adults of any age. Marked changes in the concentration of apoLp-III, and the formation of LDLp in the haemolymph, are observed after injection of laminarin (or LPS) and these are remarkably similar, at least qualitatively, to those that occur after injection of AKH-I. The involvement of lipophorins in the activation of locust prophenoloxidase in response to immunogens is discussed

The Evolution of Sex Determination and the DMRT1 Gene in the Japanese Gecko (Gekko japonicus)

There are different sex-determining mechanisms in our environment, which are separated into two groups known as genotypic sex determination (GSD) and environmental sex determination. The most well-known mechanism in the ESD group is temperature-dependent sex determination (TSD). In this study, the presence of the Doublesex and mab-3-related Transcription Factor (Dmrt1) gene was observed during embryonic development in geckos with a TSD mechanism. To do this, I observed the rate of transcription of the Dmrt1 gene in the Gecko species Gekko japonicus. Pregnant geckos were caught around Nanjing, China. Once the females laid their eggs, the eggs were then randomly placed in one of three different temperature regimes (24°C, 28°C, 32°C). The embryos, the main target, were dissected at two points during development and total RNA was extracted. A relative rate of transcription of the Dmrt1 gene was assessed using quantitative PCR (qPCR). I was not able to quantify the use of the Dmrt1 gene between males and females. However, my study does not allow me to exclude the possibility that G. japonicus may use a GSD system with a TSD override, instead of the earlier proposed TSD system

Mid-century gothic : the agency and intimacy of uncanny objects in post-war British literature and culture

This thesis reassesses the years 1945-1955 as a hingepoint in British culture, a moment when literature, film and art responded to the wartime hiatus of consumer capitalism by resisting the turn towards conspicuous consumption and selfcommodification. This resistance can be discerned in a gothic impulse in post-war culture, in which uncanny encounters with haunted, recalcitrant or overassertive objects proliferated, and provided a critique of the subject/object relationship on which consumerism was predicated. In the opening chapter, the ubiquity of bombsite rubble is brought into dialogue with mid-century mural painting both in literature and at the Festival of Britain. In the second chapter, Barbara Jones’s Black Eyes and Lemonade exhibition of ephemera is considered alongside the work of the Independent Group. The third chapter examines how the period’s new media and computing hardware further complicated the status of the subject, through an analysis of the work of George Orwell, Alan Turing and William Grey Walter. In the fourth chapter, haunted furniture and domestic ephemera threaten to become rival subjectivities, in works including Elizabeth Bowen’s The Heat of the Day and Marghanita Laski’s The Victorian Chaise Longue. The fifth chapter considers the ways in which mid-century clothes and apparel enabled or restricted the autonomy of their wearers, through a comparative analysis of the Coronation, the British Everest expedition, and Britten’s coronation opera Gloriana. Finally, the onset of atomic anxiety is explored through stories about bombs, prosthetics and bodily penetration including Powell and Pressburger’s The Small Back Room. The thesis concludes that the intimacy and agency of these unruly objects remain as half-submerged cultural signposts offering an alternative understanding of twentieth-century materialism

Interactions between the endocrine and immune systems in locusts

The prophenoloxidase cascade in the haemolymph of mature adult Locusta migratoria migratorioides (R & F) is activated in response to injection of laminarin, a -1,3 glucan. Co-injection of adipokinetic hormone-I (Lom-AKH-I) and laminarin prolongs the activation of the enzyme in a dose-dependent manner. However, injections of bacterial lipopolysaccharide (LPS) do not activate prophenoloxidase unless AKH is co-injected, when there is a dose-dependent increase in the level of phenoloxidase that persists in the haemolymph for several hours. Even when AKH is co-injected, the highest levels of phenoloxidase activity are always greater after injection of laminarin than after LPS, and these two immunogens must activate the prophenoloxidase cascade by quite distinct pathways. In the present study, interactions between the endocrine and immune systems were examined with respect to activation of prophenoloxidase and the formation of nodules: injection of LPS induces nodule formation in adult locusts. With LPS from Pseudomonas aeruginosa, nodules form exclusively in dense accumulations in the anterior portion of the abdomen on either side of the dorsal blood vessel associated with the dorsal diaphragm. However, with LPS from Escherichia coli, fewer nodules are formed but with a similar distribution, except that occasionally some nodules are aligned additionally on either side of the ventral nerve cord. Co-injection of Lom-AKH-I with LPS from either bacteria stimulates greater numbers of nodules to be formed. This effect of coinjection of AKH on nodule formation is seen at low doses of hormone with only 0.3 or 0.4 pmol of Lom-AKH-1, respectively, increasing the number of nodules by 50%. Injections of octopamine or 5-hydroxytryptamine do not mimic either of the actions of Lom-AKH-I described here. Co-injection of an angiotensin-converting enzyme inhibitor, captopril, reduces nodule formation in response to injections of LPS but has no effect on the activation of phenoloxidase. Co-injection of an inhibitor of eicosanoid synthesis, dexamethasone, with LPS influences nodule formation (with or without AKH) in different ways according to the dose of dexamethasone used, but does not affect activation of prophenoloxidase. Eicosanoid synthesis is important for nodule formation, but not for the activation of the prophenoloxidase cascade in locust haemolymph

Development of 'Redox Arrays' for identifying novel glutathionylated proteins in the secretome

Proteomics techniques for analysing the redox status of individual proteins in complex mixtures tend to identify the same proteins due to their high abundance. We describe here an array-based technique to identify proteins undergoing glutathionylation and apply it to the secretome and the proteome of human monocytic cells. The method is based on incorporation of biotinylated glutathione (GSH) into proteins, which can then be identified following binding to a 1000-protein antibody array. We thus identify 38 secreted and 55 intracellular glutathionylated proteins, most of which are novel candidates for glutathionylation. Two of the proteins identified in these experiments, IL-1 sRII and Lyn, were then confirmed to be susceptible to glutathionylation. Comparison of the redox array with conventional proteomic methods confirmed that the redox array is much more sensitive, and can be performed using more than 100-fold less protein than is required for methods based on mass spectrometry. The identification of novel targets of glutathionylation, particularly in the secretome where the protein concentration is much lower, shows that redox arrays can overcome some of the limitations of established redox proteomics techniques

Bacterial influence on uranium oxidation reduction reactions : implications for environmental remediation and isotopic composition

Thesis (Ph. D.)--Massachusetts Institute of Technology, Dept. of Nuclear Science and Engineering, 2007.Includes bibliographical references (p. 239-247).The bacterial influence on the chemistry and speciation of uranium has some important impacts on the environment, and can be exploited usefully for the purposes of environmental remediation of uranium waste contamination. It is important to understand both from a scientific and environmental perspective how different types of bacteria can affect the chemistry and speciation of uranium. Analysis of the kinetics of uranium reduction, to determine the influence of external governing factors, can help us to understand the mechanisms of uranium reduction in vitro and aid in the design of more effective uranium remediation schemes in the environment. Bacterial reduction kinetics are found to fit well to a first order exponential decay model. Using this model we have determined the dependence of the rate of bacterial uranium reduction on several parameters, including bacterial density and pH. Understanding the reduction kinetics is also an important step in the determination of the extent of isotopic separation that occurs as a result of the bacterial reduction process. Here, we demonstrate that isotopes of uranium, the heaviest naturally occurring element, are subject to fractionation when uranium serves as a terminal electron acceptor during anaerobic bacterial respiration, resulting in an enrichment of 235U in the reaction product, UO2. The manganese oxidizing bacterium Leptothrix discophora produces manganese oxides with can both adsorb uranyl and partially oxidize UO2. Determination of if and how bacteria can influence the oxidation of uranium is important because oxidation will increase the solubility and mobility of uranium in the environment.(cont.) Although oxidation of UO2 by biologically precipitated manganese oxides occurs to some degree, reduced uranium remains associated with the manganese oxides in a surface complex and is not significantly mobilized. Taken together, a more complete knowledge of how bacteria can influence the speciation of uranium in the environment will improve not only our fundamental understanding of bacterial interactions with uranium, but also how we can effectively model uranium transport in the environment and our abilities to clean-up uranium contaminated soils and groundwater both cheaply and safely.by Lisa Maureen Mullen.Ph.D

Redox regulation of inflammatory processes is enzymatically controlled

Redox regulation depends on the enzymatically controlled production and decay of redox active molecules. NADPH oxidases, superoxide dismutases, nitric oxide synthases, and others produce the redox active molecules superoxide, hydrogen peroxide, nitric oxide, and hydrogen sulfide. These react with target proteins inducing spatiotemporal modifications of cysteine residues within different signaling cascades. Thioredoxin family proteins are key regulators of the redox state of proteins. They regulate the formation and removal of oxidative modifications by specific thiol reduction and oxidation. All of these redox enzymes affect inflammatory processes and the innate and adaptive immune response. Interestingly, this regulation involves different mechanisms in different biological compartments and specialized cell types. The localization and activity of distinct proteins including, for instance, the transcription factor NFκB and the immune mediator HMGB1 are redox-regulated. The transmembrane protein ADAM17 releases proinflammatory mediators, such as TNFα, and is itself regulated by a thiol switch. Moreover, extracellular redox enzymes were shown to modulate the activity and migration behavior of various types of immune cells by acting as cytokines and/or chemokines. Within this review article, we will address the concept of redox signaling and the functions of both redox enzymes and redox active molecules in innate and adaptive immune responses

DIGE analysis of rat skeletal muscle proteins using nonionic detergent phase extraction of young adult versus aged gastrocnemius tissue

Contractile weakness and loss of muscle mass are critical features of the aging process in mammalians. Age-related fibre wasting has a profound effect on muscle metabolism, fibre type distribution and the overall physiological integrity of the neuromuscular system. This study has used mass spectrometry-based proteomics to investigate the fate of the aging rat muscle proteome. Using nonionic detergent phase extraction, this report shows that the aged gastrocnemius muscle exhibits a generally perturbed protein expression pattern in both the detergent-extracted fraction and the aqueous protein complement from senescent muscle tissue. In the detergent-extracted fraction, the expression of ATP synthase, isocitrate dehydrogenase, enolase, tropomyosin and beta-actin was increased. Different isoforms of creatine kinase and prohibitin showed differential changes. In the aqueous fraction, malate dehydrogenase, sulfotransferase, triosephosphate isomerase, aldolase, cofilin-2 and lactate dehydrogenase showed increased levels. Interestingly, differential effects on dissimilar 2-D spots of the same protein species were shown for Cu/Zn superoxide dismutase, albumin, annexin A4 and phosphoglycolate phosphatase. Mitochondrial Hsp60, Hsp71 and nucleoside diphosphate kinase B exhibited a reduced abundance in aged muscle. The majority of altered proteins were found to be involved in mitochondrial metabolism, glycolysis, metabolic transportation, regulatory processes, the cellular stress response, detoxification mechanisms and muscle contraction

Engineering of TIMP-3 as a LAP-fusion protein for targeting to sites of inflammation

Tissue inhibitor of metalloproteinase (TIMP)‐3 is a natural inhibitor of a range of enzymes that degrade connective tissue and are involved in the pathogenesis of conditions such as arthritis and cancer. We describe here the engineering of TIMP‐3 using a novel drug‐delivery system known as the ‘LAP technology’. This involves creating therapeutic proteins in fusion with the latency‐associated peptide (LAP) from the cytokine TGF‐? to generate proteins that are biologically inactive until cleavage of the LAP to release the therapy. LAP‐TIMP‐3 was successfully expressed in mammalian cells and the presence of the LAP resulted in a 14‐fold increase in the quantity of recombinant TIMP‐3 produced. LAP‐TIMP‐3 was latent until release from the LAP by treatment with matrix metalloproteinase when it could inhibit proteases of the adamalysins and adamalysins with thrombospondin motifs families, but not matrix metalloproteinases, indicating that this version of TIMP‐3 is a more specific inhibitor than the native protein. There was sufficient protease activity in synovial fluid from human joints with osteoarthritis to release TIMP‐3 from the LAP fusion. These results demonstrate the potential for development of TIMP‐3 as a novel therapy for conditions where upregulation of catabolic enzymes are part of the pathology