P2 pyridine N-oxide thrombin inhibitors: a novel peptidomimetic scaffold

In this study, we have demonstrated that the critical hydrogen bonding motif of the established 3-aminopyrazinone thrombin inhibitors can be effectively mimicked by a 2-aminopyridine N-oxide. As this peptidomimetic core is more resistant toward oxidative metabolism, it also overcomes the metabolic liability associated with the pyrazinones. An optimization study of the P(1) benzylamide delivered the potent thrombin inhibitor 21 (K(i) = 3.2 nM, 2xaPTT = 360 nM), which exhibited good plasma levels and half-life after oral dosing in the dog (C(max) = 2.6 microM, t(1/2) = 4.5 h).     

Comparative proteome analyses of human plasma following in vivo lipopolysaccharide administration using multidimensional separations coupled with tandem mass spectrometry

There is significant interest in characterization of the human plasma proteome due to its potential for providing biomarkers applicable to clinical diagnosis and treatment and for gaining a better understanding of human diseases. We describe here a strategy for comparative proteome analyses of human plasma, which is applicable to biomarker identifications for various disease states. Multidimensional liquid chromatography-mass spectrometry (LC-MS/MS) has been applied to make comparative proteome analyses of plasma samples from an individual prior to and 9 h after lipopolysaccharide (LPS) administration. Peptide peak areas and the number of peptide identifications for each protein were used to evaluate the reproducibility of LC-MS/MS and to compare relative changes in protein concentration between the samples following LPS treatment. A total of 804 distinct plasma proteins (not including immunoglobulins) were confidently identified with 32 proteins observed to be significantly increased in concentration following LPS administration, including several known inflammatory response or acute-phase mediators such as C-reactive protein, serum amyloid A and A2, LPS-binding protein, LPS-responsive and beige-like anchor protein, hepatocyte growth factor activator, and von Willebrand factor, and thus, constituting potential biomarkers for inflammatory response.     

Domain exchange between human toll-like receptors 1 and 6 reveals a region required for lipopeptide discrimination

Among the 10 human Toll-like receptors (TLRs), TLR2 appears to be unique in its requirement for cooperation with other TLRs, namely TLR1 and TLR6, to mediate cell signaling. Through reconstitution experiments, we have defined more precisely the function of these human TLRs. Human colonic epithelial cells cotransfected with TLR1 and -2 preferentially respond to a synthetic tripalmitoylated bacterial lipopeptide analogue (Pam(3)CSK(4)). However, examination of a wide variety of lipopeptide derivatives indicates that recognition by human TLR1 and -2 does not strictly correlate with the number or position of the acyl chains on the modified cysteine residue. Conversely, human TLR2 and -6 exclusively respond to lipopeptides possessing a diacylglycerol group. Most surprisingly, we have found that an R stereoisomer of diacylated macrophage-activating lipopeptide 2 (MALP-2) exclusively activates epithelial cells through TLR6 and -2 but not through TLR1 and -2. These results suggest that the chirality of the central carbon of the diacylglycerol group of these agonists is a structural determinant for human TLR recognition. Examination of chimeric receptors, generated by domain exchange between TLR1 and -6, has revealed that leucine-rich repeats 9-12 of the extracellular domain enable these receptors to discriminate between structurally similar lipopeptides. However, additional chimeric constructs reveal that this region alone is not sufficient to generate receptors that can functionally cooperate with TLR2. Our results support the idea that TLR1 and TLR6 diverged during evolution to differentially recognize natural lipoprotein structures and that this function has been conserved with respect to the human receptors.     

An essential tryptophan of Escherichia coli DnaA protein functions in oligomerization at the E. coli replication origin

In the initiation of bacterial DNA replication, DnaA protein recruits DnaB helicase to the chromosomal origin, oriC, leading to the assemble of the replication fork machinery at this site. Because a region near the N terminus of DnaA is required for self-oligomerization and the loading of DnaB helicase at oriC, we asked if these functions are separable or interdependent by substituting many conserved amino acids in this region with alanine to identify essential residues. We show that alanine substitutions of leucine 3, phenylalanine 46, and leucine 62 do not affect DnaA function in initiation. In contrast, we find on characterization of a mutant DnaA that tryptophan 6 is essential for DnaA function because its substitution by alanine abrogates self-oligomerization, resulting in the failure to load DnaB at oriC. These results indicate that DnaA bound to oriC forms a specific oligomeric structure, which is required to load DnaB helicase.     

High dynamic range characterization of the trauma patient plasma proteome

Although human plasma represents an attractive sample for disease biomarker discovery, the extreme complexity and large dynamic range in protein concentrations present significant challenges for characterization, candidate biomarker discovery, and validation. Herein we describe a strategy that combines immunoaffinity subtraction and subsequent chemical fractionation based on cysteinyl peptide and N-glycopeptide captures with two-dimensional LC-MS/MS to increase the dynamic range of analysis for plasma. Application of this "divide-and-conquer" strategy to trauma patient plasma significantly improved the overall dynamic range of detection and resulted in confident identification of 22,267 unique peptides from four different peptide populations (cysteinyl peptides, non-cysteinyl peptides, N-glycopeptides, and non-glycopeptides) that covered 3,654 different proteins with 1,494 proteins identified by multiple peptides. Numerous low abundance proteins were identified, exemplified by 78 "classic" cytokines and cytokine receptors and by 136 human cell differentiation molecules. Additionally a total of 2,910 different N-glycopeptides that correspond to 662 N-glycoproteins and 1,553 N-glycosylation sites were identified. A panel of the proteins identified in this study is known to be involved in inflammation and immune responses. This study established an extensive reference protein database for trauma patients that provides a foundation for future high throughput quantitative plasma proteomic studies designed to elucidate the mechanisms that underlie systemic inflammatory responses.     

The influence of tidal cycles and freshwater inflow on the distribution and movement of an estuarine resident fish Acanthopagrus butcheri

Movement patterns and habitat utilization by black bream Acanthopagrus butcheri (Sparidae), an estuarine resident species, were investigated using acoustic telemetry in a small estuary on the east coast of Tasmania, Australia. Thirty‐four adult A. butcheri were tracked for periods of up to 187 days between August 2005 and January 2006. Although able to tolerate a wide range of salinities, the fish spent most of the time within the upper and middle regions of the estuary, where brackish conditions dominated. The species exhibited extensive movements linked to tidal cycles, with small‐scale upstream movements during incoming tides and downstream movements during out going tides. The extent of these movements was positively correlated with the tidal height difference between consecutive tidal peaks and troughs. Freshwater inflows and resultant changes in salinity also significantly influenced distribution and movement patterns. Fish moved downstream during the periods of heavy inflows, returning upstream as salinities increased to c. >10. During the peak of spawning period (November to December) fish moved into the upper region of the estuary, where they aggregated to spawn. Periodic increases in freshwater discharge, however, resulted in fish leaving the spawning grounds and moving downstream. Towards the end of the spawning season (January), the fish became more dispersed throughout the entire estuarine system.     

Mutations in the Bacillus subtilis β Clamp That Separate Its Roles in DNA Replication from Mismatch Repair

The β clamp is an essential replication sliding clamp required for processive DNA synthesis. The β clamp is also critical for several additional aspects of DNA metabolism, including DNA mismatch repair (MMR). The dnaN5 allele of Bacillus subtilis encodes a mutant form of β clamp containing the G73R substitution. Cells with the dnaN5 allele are temperature sensitive for growth due to a defect in DNA replication at 49°C, and they show an increase in mutation frequency caused by a partial defect in MMR at permissive temperatures. We selected for intragenic suppressors of dnaN5 that rescued viability at 49°C to determine if the DNA replication defect could be separated from the MMR defect. We isolated three intragenic suppressors of dnaN5 that restored growth at the nonpermissive temperature while maintaining an increase in mutation frequency. All three dnaN alleles encoded the G73R substitution along with one of three novel missense mutations. The missense mutations isolated were S22P, S181G, and E346K. Of these, S181G and E346K are located near the hydrophobic cleft of the β clamp, a common site occupied by proteins that bind the β clamp. Using several methods, we show that the increase in mutation frequency resulting from each dnaN allele is linked to a defect in MMR. Moreover, we found that S181G and E346K allowed growth at elevated temperatures and did not have an appreciable effect on mutation frequency when separated from G73R. Thus, we found that specific residue changes in the B. subtilis β clamp separate the role of the β clamp in DNA replication from its role in MMR.Replication sliding clamps are essential cellular proteins imparting a spectacular degree of processivity to DNA polymerases during genome replication (24, 39-41). Encoded by the dnaN gene, the β clamp is a highly conserved bacterial sliding clamp found in virtually all eubacterial species (reviewed in reference 7). The β clamp is a head-to-tail, ring-shaped homodimer that encircles double-stranded DNA (1, 39). In eukaryotes and archaea, the analog of the β clamp is proliferating cell nuclear antigen (PCNA) (15, 28, 40, 41). Eukaryotic PCNA is a ring-shaped homotrimer that also acts to encircle DNA, increasing the processivity of the replicative DNA polymerases (40, 41). Although the primary structures of the β clamp and PCNA are not conserved, the tertiary structures of these proteins are very similar, demonstrating structural conservation among bacterial, archaeal, and eukaryotic replication sliding clamps (28, 39-41; reviewed in reference 6).The function of the β clamp is not limited to its well-defined role in genome replication. The Escherichia coli β clamp binds Hda, which also binds the replication initiation protein DnaA, regulating the active form of DnaA complexed with ATP (19, 37, 43). This allows the β clamp to regulate replication initiation through the amount of available DnaA-ATP. In Bacillus subtilis, the β clamp binds YabA, a negative regulator of DNA replication initiation (12, 29, 52). It has also been suggested that the B. subtilis β clamp sequesters DnaA from the replication origin during the cell cycle through the binding of DnaA to YabA and the binding of YabA to the β clamp (70). Thus, it is hypothesized that in E. coli and B. subtilis, the β clamp influences the frequency of replication initiation through interactions with Hda and YabA, respectively.The E. coli and B. subtilis β clamp has an important role in translesion DNA synthesis during the replicative bypass of noncoding bases by specialized DNA polymerases belonging to the Y family (20, 33). The roles of the E. coli β clamp in translesion synthesis are well established (5, 8, 30, 31). Binding sites on the E. coli β clamp that accommodate translesion polymerases pol IV (DinB) and pol V (UmuD₂′C) have been identified, and the consequence of disrupting their association with the β clamp has illustrated the critical importance of the β clamp to the activity of both of these polymerases (4, 5, 8, 26, 30, 31, 48, 49).In addition to the involvement of the β clamp in replication initiation, DNA replication, and translesion synthesis, the E. coli and B. subtilis β clamp also functions in DNA mismatch repair (MMR) (45, 46, 64). The MMR pathway recognizes and repairs DNA polymerase errors, contributing to the overall fidelity of the DNA replication pathway (reviewed in references 42 and 60). In both E. coli and B. subtilis, deletion of the genes mutS and mutL increases the spontaneous mutation frequency several hundredfold (13, 25, 63). In E. coli, MutS recognizes and binds mismatches, while MutL functions as a “matchmaker,” coordinating the actions of other proteins in the MMR pathway, allowing the removal of the mismatch and resynthesis of the resulting gap (reviewed in references 42 and 60). MutS and MutL of E. coli and B. subtilis physically interact with the β clamp (45, 46, 51, 64). Interaction between the B. subtilis β clamp and MutS is important for efficient MMR and organization of MutS-green fluorescent protein (GFP) into foci in response to replication errors, while the function of MutL binding to the β clamp is unknown (64).These studies show that the β clamp is critical for several aspects of DNA metabolism in E. coli and B. subtilis. In E. coli, many dnaN alleles have been examined and used to define the mechanistic roles of the β clamp in vivo (5, 18, 24, 30, 31, 48, 49, 73). A limitation in studying the mechanistic roles of the B. subtilis β clamp is that only two dnaN alleles (β clamp) are available, dnaN5 and dnaN34 (36) (www.bgsc.org/), and both of these alleles do not support growth at temperatures above 49°C, suggesting that they may cause similar defects (36) (www.bgsc.org/). Of these two dnaN alleles, only dnaN5 has been investigated in any detail (36, 53, 64). The mutant β clamp encoded by dnaN5 contains a G73R substitution [dnaN5(G73R)] in a surface-exposed residue located on the outside rim of the β clamp (53, 64). Our previous studies with this allele showed that dnaN5(G73R) confers an increase in mutation frequency at 30°C and 37°C (64). Further characterization of dnaN5(G73R) showed that the increased mutation frequency is caused by a partial defect in MMR (64). Additionally, dnaN5(G73R)-containing cells have a reduced ability to support MutS-GFP focus formation in response to mismatches (64). These results support the hypothesis that G73R in the β clamp causes a defect in DNA replication at 49°C (36) and impaired MMR manifested by a defect in establishing the assembly of MutS-GFP foci in response to replication errors (64).To understand the roles of the B. subtilis β clamp in MMR and DNA replication, we examined the dnaN5 and dnaN34 alleles. We found that the nucleotide sequences of dnaN5 and dnaN34 and the phenotypes they produce were identical, both producing the G73R missense mutation. We analyzed in vivo β clamp^G73R protein levels and found that the β clamp^G73R protein accumulated to wild-type levels at elevated temperatures. To identify amino acid residues that would restore DNA replication at elevated temperatures, we isolated three intragenic suppressors of dnaN5(G73R) that conferred growth of B. subtilis cells at 49°C. Epistasis analysis and determination of the mutation spectrum showed that each dnaN allele isolated in this study caused an MMR-dependent increase in mutation frequency. Additionally, we found that the β clamp binding protein YabA can reduce the efficiency of MMR in vivo when yabA expression is induced. Thus, we have identified residues in the β clamp that are critical for DNA replication and MMR in B. subtilis. We also found that a β clamp binding protein, YabA, can reduce the efficiency of MMR in vivo.     

Patterns of sequence conservation in presynaptic neural genes

Background  

The neuronal synapse is a fundamental functional unit in the central nervous system of animals. Because synaptic function is evolutionarily conserved, we reasoned that functional sequences of genes and related genomic elements known to play important roles in neurotransmitter release would also be conserved.