Exploitation of structural and regulatory diversity in glutamate racemases

Glutamate racemase is an enzyme essential to the bacterial cell wall biosynthesis pathway, and has therefore been considered as a target for antibacterial drug discovery. We characterized the glutamate racemases of several pathogenic bacteria using structural and biochemical approaches. Here we describe three distinct mechanisms of regulation for the family of glutamate racemases: allosteric activation by metabolic precursors, kinetic regulation through substrate inhibition, and D-glutamate recycling using a d-amino acid transaminase. In a search for selective inhibitors, we identified a series of uncompetitive inhibitors specifically targeting Helicobacter pylori glutamate racemase that bind to a cryptic allosteric site, and used these inhibitors to probe the mechanistic and dynamic features of the enzyme. These structural, kinetic and mutational studies provide insight into the physiological regulation of these essential enzymes and provide a basis for designing narrow-spectrum antimicrobial agents.     

Morphogenetic and cytogenetic effects of 7,12-dimethylbenz(a)anthracene onHaworthia callus in vitro

Zusammenfassung 7,12-Dimethylbenz(a)anthracen (DMBA) in verschiedenen Konzentrationen anstelle von Auxin, Kinetin und Kokosmilch im Nährmedium induziert die Bildung von Wurzeln und beblätterten Sprossen auf den Kalli vonHaworthia variegata. Dieses Ergebnis gleicht demjenigen im vollständigen Kontrollmedium ohne DMBA, mit dem Unterschied, dass unter dem Einfluss von DMBA weniger Sprosse und eine grössere Anzahl von Wurzeln gebildet werden. Cytologische Untersuchungen an Kallus- und Wurzelzellen aus dem DMBA-Medium zeigten eine normale Konstitution des Chromosomensatzes ohne chromosomale Aberrationen.

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This study was supported in part by a summer research grant from Lafayette College Faculty Research Funds.     

Differential activation of the inflammasome by caspase-1 adaptors ASC and Ipaf

Specific adaptors regulate the activation of initiator caspases; for example, FADD and Apaf-1 engage caspases 8 and 9, respectively. The adaptors ASC, Ipaf and RIP2 have each been proposed to regulate caspase-1 (also called interleukin (IL)-1 converting enzyme), which is activated within the 'inflammasome', a complex comprising several adaptors. Here we show the impact of ASC-, Ipaf- or RIP2-deficiency on inflammasome function. ASC was essential for extracellular ATP-driven activation of caspase-1 in toll-like receptor (TLR)-stimulated macrophages. Accordingly, ASC-deficient macrophages exhibited defective maturation of IL-1beta and IL-18, and ASC-null mice were resistant to lipopolysaccharide-induced endotoxic shock. Furthermore, activation of caspase-1 in response to an intracellular pathogen (Salmonella typhimurium) was abrogated severely in ASC-null macrophages. Unexpectedly, Ipaf-deficient macrophages activated caspase-1 in response to TLR plus ATP stimulation but not S. typhimurium. Caspase-1 activation was not compromised by loss of RIP2. These data show that whereas ASC is key to caspase-1 activation within the inflammasome, Ipaf provides a special conduit to the inflammasome for signals triggered by intracellular pathogens. Notably, cell death triggered by stimuli that engage caspase-1 was ablated in macrophages lacking either ASC or Ipaf, suggesting a coupling between the inflammatory and cell death pathways.     

Vascular growth factors in neuropsychiatry

Recent advances in understanding the cellular and molecular basis of psychiatric illnesses have shed light on the important role played by trophic factors in modulating functional parameters associated with disease causality and drug action. Disease mechanisms are now thought to involve multiple cell types, including neurons and endothelial cells. These functionally distinct but interactively coupled cell types engage in cellular cross talk via shared and common signaling molecules. Dysregulation in their cellular signaling pathways influences brain function and alters behavioral performance. Multifunctional trophic factors such as VEGF and EPO that possess both neurotrophic and angiogenic actions are of particular interest due to their ability to rescue structural and plasticity deficits in neurons and vasculature. Obtaining insight into the behavioral, cellular and molecular actions of multi-functional trophic factors has the potential to open new and transformative therapeutic approaches.     

Primary structure of Torpedo californica acetylcholinesterase deduced from its cDNA sequence

Acetylcholinesterase, an essential enzyme of the nervous system, rapidly terminates the action of acetylcholine released into the synapse. Acetylcholinesterase is also found (in lower abundance) in extrajunctional areas of muscle and nerve and on erythrocyte membranes. Hydrodynamic analyses of the native enzyme and characterization of its dissociated subunits have revealed multiple enzyme forms which can be divided into two classes: dimensionally asymmetric forms which are usually found within the synapse and contain a collagen-like structural subunit disulphide-linked to the catalytic subunits; and globular forms which appear to be widely distributed on the outer surface of cell membranes. Both forms have been characterized in the ray Torpedo californica and, although their catalytic behaviours seem to be identical, they differ slightly in amino-acid composition, peptide maps and reactivity with certain monoclonal antibodies. Here, we report the complete amino-acid sequence of an acetylcholinesterase inferred from the sequence of a complementary DNA clone. The 575-residue protein shows significant homology with the C-terminal portion of thyroglobulin.     

The accuracy of extrapolation (time series) methods: Results of a forecasting competition

In the last few decades many methods have become available for forecasting. As always, when alternatives exist, choices need to be made so that an appropriate forecasting method can be selected and used for the specific situation being considered. This paper reports the results of a forecasting competition that provides information to facilitate such choice. Seven experts in each of the 24 methods forecasted up to 1001 series for six up to eighteen time horizons. The results of the competition are presented in this paper whose purpose is to provide empirical evidence about differences found to exist among the various extrapolative (time series) methods used in the competition.     

Common sequence variants on 20q11.22 confer melanoma susceptibility

We conducted a genome-wide association pooling study for cutaneous melanoma and performed validation in samples totaling 2,019 cases and 2,105 controls. Using pooling, we identified a new melanoma risk locus on chromosome 20 (rs910873 and rs1885120), with replication in two further samples (combined P < 1 x 10(-15)). The per allele odds ratio was 1.75 (1.53, 2.01), with evidence for stronger association in early-onset cases.