Central Role of Glutamate Metabolism in the Maintenance of Nitrogen Homeostasis in Normal and Hyperammonemic Brain

Glutamate is present in the brain at an average concentration-typically 10-12 mM-far in excess of those of other amino acids. In glutamate-containing vesicles in the brain, the concentration of glutamate may even exceed 100 mM. Yet because glutamate is a major excitatory neurotransmitter, the concentration of this amino acid in the cerebral extracellular fluid must be kept low-typically µM. The remarkable gradient of glutamate in the different cerebral compartments: vesicles \u3e cytosol/mitochondria \u3e extracellular fluid attests to the extraordinary effectiveness of glutamate transporters and the strict control of enzymes of glutamate catabolism and synthesis in well-defined cellular and subcellular compartments in the brain. A major route for glutamate and ammonia removal is via the glutamine synthetase (glutamate ammonia ligase) reaction. Glutamate is also removed by conversion to the inhibitory neurotransmitter γ-aminobutyrate (GABA) via the action of glutamate decarboxylase. On the other hand, cerebral glutamate levels are maintained by the action of glutaminase and by various α-ketoglutarate-linked aminotransferases (especially aspartate aminotransferase and the mitochondrial and cytosolic forms of the branched-chain aminotransferases). Although the glutamate dehydrogenase reaction is freely reversible, owing to rapid removal of ammonia as glutamine amide, the direction of the glutamate dehydrogenase reaction in the brain in vivo is mainly toward glutamate catabolism rather than toward the net synthesis of glutamate, even under hyperammonemia conditions. During hyperammonemia, there is a large increase in cerebral glutamine content, but only small changes in the levels of glutamate and α-ketoglutarate. Thus, the channeling of glutamate toward glutamine during hyperammonemia results in the net synthesis of 5-carbon units. This increase in 5-carbon units is accomplished in part by the ammonia-induced stimulation of the anaplerotic enzyme pyruvate carboxylase. Here, we suggest that glutamate may constitute a buffer or bulwark against changes in cerebral amine and ammonia nitrogen. Although the glutamate transporters are briefly discussed, the major emphasis of the present review is on the enzymology contributing to the maintenance of glutamate levels under normal and hyperammonemic conditions. Emphasis will also be placed on the central role of glutamate in the glutamine-glutamate and glutamine-GABA neurotransmitter cycles between neurons and astrocytes. Finally, we provide a brief and selective discussion of neuropathology associated with altered cerebral glutamate levels

Security Implications of Insecure DNS Usage in the Internet

The Domain Name System (DNS) provides domain-to-address lookup-services used by almost all internet applications. Because of this ubiquitous use of the DNS, attacks against the DNS have become more and more critical. However, in the past, studies of DNS security have been mostly conducted against individual protocols and applications. In this thesis, we perform the first comprehensive evaluation of DNS-based attacks against a wide range of internet applications, ranging from time-synchronisation via NTP over internet resource management to security mechanisms. We show how to attack those applications by exploiting various weaknesses in the DNS. These attacks are based on both, already known weaknesses which are adapted to new attacks, as well as previously unknown attack vectors which have been found during the course of this thesis. We evaluate our attacks and provide the first taxonomy of DNS applications, to show how adversaries can systematically develop attacks exploiting the DNS. We analyze the attack surface created by our attacks in the internet and find that a significant number of applications and systems can be attacked. We work together with the developers of the vulnerable applications to develop patches and general countermeasures which can be applied by various parties to block our attacks. We also provide conceptual insights into the root causes allowing our attacks to help with the development of new applications and standards. The findings of this thesis are published in in 4 full-paper publications and 2 posters at international academic conferences. Additionally, we disclose our finding to developers which has lead to the registration of 8 Common Vulnerabilities and Exposures identifiers (CVE IDs) and patches in 10 software implementations. To raise awareness, we also presented our findings at several community meetings and via invited articles

Metamorphosys

Viele Anwendungen im Bereich der eingebetteten Systeme erfordern hohe Rechenleistung und eine zeitnahe Anpassung an sich ändernde Umgebungsbedingungen. Hierfür eignen sich adaptive Systeme, die neben der Flexibilität durch Software eine Anpassung der System-Hardware erlauben. In dieser Arbeit wurde ein hardware-basierter Adaptionsansatz untersucht, der eine zeitnahe dynamische Anpassung verfügbarer rekonfigurierbarer Systemressource anhand des überwachten Anwendungsverhaltens ermöglicht. In diesem Zusammenhang ist ein fünfschichtiges Konzept entstanden, das sämtliche Systemschichten mit in den Adaptionsprozess einbindet. Die notwendigen Mechanismen wurden in einen adaptiven Prozessor integriert. Zu vergleichbaren adaptiven Ansätzen werden äquivalente Leistungsergebnisse erzielt und mindestens 20% weniger Ressourcen benötigt. Des Weiteren schaffen diese Untersuchungen die Grundlagen für den Entwurf von Hochgeschwindigkeitsprozessoren mit dynamisch umdefinierbaren Acceleratoren.Many high-performance, embedded applications work in rapidly changing environments. Adaptive systems combine flexibility of software with the possibility of adapting hardware to varying requirements. Reconfigurable computing devices enable hardware architecture changes in response to the changing environment. In this work we develop a new dynamic method, which allow application-specific hardware changes during application runtime. In order to achieve this goal a new method of hardware-based monitoring and controlling is utilized. Because the system hardware layer has a very restricted view in matters of application goals, it is necessary to involve all system layers into the adaptation process. Therefore we develop the Metamorphosys concept. To proof the quality of the concept, the adaptive processor–as an example system component–was developed. Different benchmark programs have shown that the developed adaptive processor equals other approaches in terms of computing performance but show a reduced utilization of hardware-resources by at least 20%. The results of this work are applicable in the domain of the design of high-performance computers, which are based on fast general-purpose processor-cores and additional configurable accelerator devices

Cystamine and Cysteamine As Inhibitors of Transglutaminases In Vivo

Cystamine is commonly used as a transglutaminase inhibitor. This disulfide undergoes reduction in vivo to the aminothiol compound, cysteamine. Thus, the mechanism by which cystamine inhibits transglutaminase activity in vivo could be due to either cystamine or cysteamine, which depends on the local redox environment. Cystamine inactivates transglutaminases by promoting the oxidation of two vicinal cysteine residues on the enzyme to an allosteric disulfide, whereas cysteamine acts as a competitive inhibitor for transamidation reaction catalyzed by this enzyme. The latter mechanism is likely to result in the formation of a unique biomarker, N -(gamma-glutamyl)cysteamine that could serve to indicate how cyst(e)amine acts to inhibit transglutaminases inside cells and the body

Transglutaminase 6: a protein associated with central nervous system development and motor function.

Transglutaminases (TG) form a family of enzymes that catalyse various post-translational modifications of glutamine residues in proteins and peptides including intra- and intermolecular isopeptide bond formation, esterification and deamidation. We have characterized a novel member of the mammalian TG family, TG6, which is expressed in a human carcinoma cell line with neuronal characteristics and in mouse brain. Besides full-length protein, alternative splicing results in a short variant lacking the second β-barrel domain in man and a variant with truncated β-sandwich domain in mouse. Biochemical data show that TG6 is allosterically regulated by Ca(2+) and guanine nucleotides. Molecular modelling indicates that TG6 could have Ca(2+) and GDP-binding sites related to those of TG3 and TG2, respectively. Localization of mRNA and protein in the mouse identified abundant expression of TG6 in the central nervous system. Analysis of its temporal and spatial pattern of induction in mouse development indicates an association with neurogenesis. Neuronal expression of TG6 was confirmed by double-labelling of mouse forebrain cells with cell type-specific markers. Induction of differentiation in mouse Neuro 2a cells with NGF or dibutyryl cAMP is associated with an upregulation of TG6 expression. Familial ataxia has recently been linked to mutations in the TGM6 gene. Autoantibodies to TG6 were identified in immune-mediated ataxia in patients with gluten sensitivity. These findings suggest a critical role for TG6 in cortical and cerebellar neurons

The Enzymology of 2-hydroxyglutarate, 2-hydroxyglutaramate and 2-hydroxysuccinamate and Their Relationship to Oncometabolites

Many enzymes make mistakes . Consequently, repair enzymes have evolved to correct these mistakes. For example, lactate dehydrogenase (LDH) and mitochondrial malate dehydrogenase (mMDH) slowly catalyze the reduction of 2-oxoglutarate (2-OG) to the oncometabolite l-2-hydroxyglutarate (l-2-HG). l-2-HG dehydrogenase corrects this error by converting l-2-HG to 2-OG. LDH also catalyzes the reduction of the oxo group of 2-oxoglutaramate (2-OGM; transamination product of l-glutamine). We show here that human glutamine synthetase (GS) catalyzes the amidation of the terminal carboxyl of both the l- and d- isomers of 2-HG. The reaction of 2-OGM with LDH and the reaction of l-2-HG with GS generate l-2-hydroxyglutaramate (l-2-HGM). We also show that l-2-HGM is a substrate of human omega-amidase. The product (l-2-HG) can then be converted to 2-OG by l-2-HG dehydrogenase. Previous work showed that 2-oxosuccinamate (2-OSM; transamination product of l-asparagine) is an excellent substrate of LDH. Finally, we also show that human omega-amidase converts the product of this reaction (i.e., l-2-hydroxysuccinamate; l-2-HSM) to l-malate. Thus, omega-amidase may act together with hydroxyglutarate dehydrogenases to repair certain mistakes of GS and LDH. The present findings suggest that non-productive pathways for nitrogen metabolism occur in mammalian tissues in vivo. Perturbations of these pathways may contribute to symptoms associated with hydroxyglutaric acidurias and to tumor progression. Finally, methods for the synthesis of l-2-HGM and l-2-HSM are described that should be useful in determining the roles of omega-amidase/4- and 5-C compounds in photorespiration in plants

DNA barcodes reveal species-specific mercury levels in tuna sushi that pose a health risk to consumers

Excessive ingestion of mercury--a health hazard associated with consuming predatory fishes--damages neurological, sensory-motor and cardiovascular functioning. The mercury levels found in Bigeye Tuna (Thunnus obesus) and bluefin tuna species (Thunnus maccoyii, Thunnus orientalis, and Thunnus thynnus), exceed or approach levels permissible by Canada, the European Union, Japan, the US, and the World Health Organization. We used DNA barcodes to identify tuna sushi samples analysed for mercury and demonstrate that the ability to identify cryptic samples in the market place allows regulatory agencies to more accurately measure the risk faced by fish consumers and enact policies that better safeguard their health.VoRSUNY DownstateEpidemiology and BiostatisticsN/

Synthesis and Evaluation of 11C-Labeled Triazolones as Probes for Imaging Fatty Acid Synthase Expression by Positron Emission Tomography

Cancer cells require lipids to fulfill energetic, proliferative, and signaling requirements. Even though these cells can take up exogenous fatty acids, the majority exhibit a dependency on de novo fatty acid synthesis. Fatty acid synthase (FASN) is the rate-limiting enzyme in this process. Expression and activity of FASN is elevated in multiple cancers, where it correlates with disease progression and poor prognosis. These observations have sparked interest in developing methods of detecting FASN expression in vivo. One promising approach is the imaging of radiolabeled molecular probes targeting FASN by positron emission tomography (PET). However, although [11C]acetate uptake by prostate cancer cells correlates with FASN expression, no FASN-specific PET probes currently exist. Our aim was to synthesize and evaluate a series of small molecule triazolones based on GSK2194069, an FASN inhibitor with IC50 = 7.7 ± 4.1 nM, for PET imaging of FASN expression. These triazolones were labeled with carbon-11 in good yield and excellent radiochemical purity, and binding to FASN-positive LNCaP cells was significantly higher than FASN-negative PC3 cells. Despite these promising characteristics, however, these molecules exhibited poor in vivo pharmacokinetics and were predominantly retained in lymph nodes and the hepatobiliary system. Future studies will seek to identify structural modifications that improve tumor targeting while maintaining the excretion profile of these first-generation 11C-methyltriazolones

DNA barcodes reveal species-specific mercury levels in tuna sushi that pose a health risk to consumers

Excessive ingestion of mercury—a health hazard associated with consuming predatory fishes—damages neurological, sensory-motor and cardiovascular functioning. The mercury levels found in Bigeye Tuna (Thunnus obesus) and bluefin tuna species (Thunnus maccoyii, Thunnus orientalis, and Thunnus thynnus), exceed or approach levels permissible by Canada, the European Union, Japan, the US, and the World Health Organization. We used DNA barcodes to identify tuna sushi samples analysed for mercury and demonstrate that the ability to identify cryptic samples in the market place allows regulatory agencies to more accurately measure the risk faced by fish consumers and enact policies that better safeguard their health

Transglutaminase activation in neurodegenerative diseases

The following review examines the role of calcium in promoting the in vitro and in vivo activation of transglutaminases in neurodegenerative disorders. Diseases such as Alzheimer's disease, Parkinson's disease and Huntington's disease exhibit increased transglutaminase activity and rises in intracellular calcium concentrations, which may be related. The aberrant activation of transglutaminase by calcium is thought to give rise to a variety of pathological moieties in these diseases, and the inhibition has been shown to have therapeutic benefit in animal and cellular models of neurodegeneration. Given the potential clinical relevance of transglutaminase inhibitors, we have also reviewed the recent development of such compounds