Revealing the dynamic allosteric changes required for formation of the cysteine synthase complex by hydrogen-deuterium exchange MS

CysE and CysK, the last two enzymes of the cysteine biosynthetic pathway, engage in a bienzyme complex, cysteine synthase, with yet incompletely characterized three-dimensional structure and regulatory function. Being absent in mammals, the two enzymes and their complex are attractive targets for antibacterial drugs. We have used hydrogen/deuterium exchange MS to unveil how complex formation affects the conformational dynamics of CysK and CysE. Our results support a model where CysE is present in solution as a dimer of trimers, and each trimer can bind one CysK homodimer. When CysK binds to one CysE monomer, intratrimer allosteric communication ensures conformational and dynamic symmetry within the trimer. Furthermore, a long-range allosteric signal propagates through CysE to induce stabilization of the interface between the two CysE trimers, preparing the second trimer for binding the second CysK with a nonrandom orientation. These results provide new molecular insights into the allosteric formation of the cysteine synthase complex and could help guide antibacterial drug design

Structure and Oxygen Affinity of Crystalline des-His-146β Human Hemoglobin in the T State *

To correlate directly structure with function, the oxygen affinity and the three-dimensional structure of crystals of the T quaternary state of des-His-146beta human hemoglobin have been determined by polarized absorption microspectrophotometry and x-ray diffraction crystallography. In des-His-146beta, the COOH-terminal histidine residues of the beta chains of hemoglobin A have been removed. Oxygen binding to crystalline des-His hemoglobin is non-cooperative and independent of pH. The oxygen affinity is 1.7-fold greater than that of the crystalline state of hemoglobin A. Removal of His-146beta results in a small movement of the truncated COOH-terminal peptide and in a very small change in quaternary structure. Previously, similar studies on T state crystals of des-Arg-141alpha hemoglobin showed that removal of the COOH termini of the alpha chains results in much larger effects on oxygen affinity and on quaternary structure. Kinetic studies in solution reveal that at pH 7.0, the rates of CO combination with deoxygenated des-His-146beta in the absence and presence of inositol hexaphosphate are 2.5- and 1.3-fold, respectively, more rapid than for hemoglobin A. The values for des-Arg are 7.6- and 3.9-fold. The properties of the T state of hemoglobin both in the crystal and in solution are influenced to a greater degree by the interactions associated with Arg-141alpha than those associated with His-146beta

Metric Features of a Dipolar Model

The lattice spin model, with nearest neighbor ferromagnetic exchange and long range dipolar interaction, is studied by the method of time series for observables based on cluster configurations and associated partitions, such as Shannon entropy, Hamming and Rohlin distances. Previous results based on the two peaks shape of the specific heat, suggested the existence of two possible transitions. By the analysis of the Shannon entropy we are able to prove that the first one is a true phase transition corresponding to a particular melting process of oriented domains, where colored noise is present almost independently of true fractality. The second one is not a real transition and it may be ascribed to a smooth balancing between two geometrical effects: a progressive fragmentation of the big clusters (possibly creating fractals), and the slow onset of a small clusters chaotic phase. Comparison with the nearest neighbor Ising ferromagnetic system points out a substantial difference in the cluster geometrical properties of the two models and in their critical behavior.Comment: 20 pages, 15 figures, submitted to JPhys

High- and low-affinity PEGylated hemoglobin-based oxygen carriers: differential oxidative stress in a Guinea pig transfusion model

Hemoglobin (Hb)-based oxygen carriers (HBOCs) are an investigational replacement for blood transfusions and are known to cause oxidative damage to tissues. To investigate the correlation between their oxygen binding properties and these detrimental effects, we investigated two PEGylated HBOCs endowed with different oxygen binding properties - but otherwise chemically identical - in a Guinea pig transfusion model. Plasma samples were analyzed for biochemical markers of inflammation, tissue damage and organ dysfunction; proteins and lipids of heart and kidney extracts were analyzed for markers of oxidative damage. Overall, both HBOCs produced higher oxidative stress in comparison to an auto-transfusion control group. Particularly, tissue 4-hydroxynonenal-adducts, tissue malondialdehyde adducts and plasma 8-oxo-2'-deoxyguanosine exhibited significantly higher levels in comparison with the control group. For malondialdehyde adducts, a higher level in the renal tissue was observed for animals treated with PEG-Hboxy, hinting at a correlation between the HBOCs oxygen binding properties and the oxidative stress they produce. Moreover, we found that the high-affinity HBOC produced greater tissue oxygenation in comparison with the low affinity one, possibly correlating with the higher oxidative stress it induced

Metric characterization of cluster dynamics on the Sierpinski gasket

We develop and implement an algorithm for the quantitative characterization of cluster dynamics occurring on cellular automata defined on an arbitrary structure. As a prototype for such systems we focus on the Ising model on a finite Sierpsinski Gasket, which is known to possess a complex thermodynamic behavior. Our algorithm requires the projection of evolving configurations into an appropriate partition space, where an information-based metrics (Rohlin distance) can be naturally defined and worked out in order to detect the changing and the stable components of clusters. The analysis highlights the existence of different temperature regimes according to the size and the rate of change of clusters. Such regimes are, in turn, related to the correlation length and the emerging "critical" fluctuations, in agreement with previous thermodynamic analysis, hence providing a non-trivial geometric description of the peculiar critical-like behavior exhibited by the system. Moreover, at high temperatures, we highlight the existence of different time scales controlling the evolution towards chaos.Comment: 20 pages, 8 figure

Isozyme-Specific Ligands for O-acetylserine sulfhydrylase, a Novel Antibiotic Target

Conceived and designed the experiments: FS PC BC ES AM. Performed the experiments: FS RS ES PF SR. Analyzed the data: FS BC ES PF GEK PFC AM. Contributed reagents/materials/analysis tools: PC PB GC. Wrote the paper: FS GEK BC AM.The last step of cysteine biosynthesis in bacteria and plants is catalyzed by O-acetylserine sulfhydrylase. In bacteria, two isozymes, O-acetylserine sulfhydrylase-A and O-acetylserine sulfhydrylase-B, have been identified that share similar binding sites, although the respective specific functions are still debated. O-acetylserine sulfhydrylase plays a key role in the adaptation of bacteria to the host environment, in the defense mechanisms to oxidative stress and in antibiotic resistance. Because mammals synthesize cysteine from methionine and lack O-acetylserine sulfhydrylase, the enzyme is a potential target for antimicrobials. With this aim, we first identified potential inhibitors of the two isozymes via a ligand- and structure-based in silico screening of a subset of the ZINC library using FLAP. The binding affinities of the most promising candidates were measured in vitro on purified O-acetylserine sulfhydrylase-A and O-acetylserine sulfhydrylase-B from Salmonella typhimurium by a direct method that exploits the change in the cofactor fluorescence. Two molecules were identified with dissociation constants of 3.7 and 33 µM for O-acetylserine sulfhydrylase-A and O-acetylserine sulfhydrylase-B, respectively. Because GRID analysis of the two isoenzymes indicates the presence of a few common pharmacophoric features, cross binding titrations were carried out. It was found that the best binder for O-acetylserine sulfhydrylase-B exhibits a dissociation constant of 29 µM for O-acetylserine sulfhydrylase-A, thus displaying a limited selectivity, whereas the best binder for O-acetylserine sulfhydrylase-A exhibits a dissociation constant of 50 µM for O-acetylserine sulfhydrylase-B and is thus 8-fold selective towards the former isozyme. Therefore, isoform-specific and isoform-independent ligands allow to either selectively target the isozyme that predominantly supports bacteria during infection and long-term survival or to completely block bacterial cysteine biosynthesis.Yeshttp://www.plosone.org/static/editorial#pee

End-to-End Scheduling to Meet Deadlines in Distributed Systems

urce access protocols and thus increase the utilization of resources. iii To my parents, Aldo and Maria-Rosa Bettati iv Acknowledgements My deepest gratitude goes to Prof. Jane Liu, who five years ago took upon her to teach an intimidated student from overseas the tools of the trade of a researcher. She did this as a great teacher, a fabulous advisor, and an invaluable role model. Her continuous encouragement was crucial in helping over the periods of doubt and the occasionally arid portions of the work, and her enthusiasm and energy are always contagious. I would like to thank the other members of my committee, Professors Andrew Chien, KweiJay Lin, Dave Liu, Tony Ng, and Ben Wah, for having agreed to scrutinize and comment on the contents of my thesis. A big "Thank you" goes to all the members of the Real-Time Systems Laboratory. This is a very special group, because joining it not only means acquiring new colleagues, but tying very dear friendships. First I woul

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Abstract Real-Time Intrusion Detection and Suppression in ATM Networks

Distributed mission critical systems require support for ultra-secure communication, in which intrusions must be detected and suppressed in real time, possibly before the affected messages reach the receiver. When the distributed application has real-time requirements, the effects of intrusion are particularly severe. In addition to covered channels and potentially tampered data at the receiver, such systems may experience violations of timing requirements and timing instabilities in components not directly related to the intrusion. Systems with real-time requirements have admission and access control mechanisms in place to ensure that timing requirements can be met during normal operation. Such admission control mechanisms require load profiles of traffic (for example in form of leaky bucket descriptors) so that resources can be appropriately allocated to meet application requirements during system operation. In this paper, we report on our project aiming at real-time detection of intrusions in ATM networks. We take advantage of the specification of the traffic profile during connection setup, and use a traffic modeling technique to determine the profile of the traffic on the connection in an arbitrary point in the network, thus providing a base line for detection of load deviations. We designed and analyzed a security device that uses the profile information, detects violations. The traffic is modeled in an accurate but efficient manner. As a result, our device is able to detect an intrusion within 25 �s, yet is simple enough to be economically realized in existing VLSI technology