Conformational changes in chemically modified Escherichia coli thioredoxin monitored by H/D exchange and electrospray ionization mass spectrometry

Hydrogen/deuterium (H/D) exchange in combination with electrospray ionization mass spectrometry and near-ultraviolet (UV) circular dichroism (CD) was used to study the conformational properties and thermal unfolding of Escherichia coli thioredoxin and its Cys32-alkylated derivatives in 1% acetic acid (pH 2.7). Thermal unfolding of oxidized (Oxi) and reduced (Red) -thioredoxin (TRX) and Cys-32-ethylglutathionyl (GS-ethyl-TRX) and Cys-32-ethylcysteinyl (Cys-ethyl-TRX), which are derivatives of Red-TRX, follow apparent EX1 kinetics as charge-state envelopes, H/D mass spectral exchange profiles, and near-UV CD appear to support a two-state folding/unfolding model. Minor mass peaks in the H/D exchange profiles and nonsuperimposable MS- and CD-derived melting curves, however, suggest the participation of unfolding intermediates leading to the conclusion that the two-state model is an oversimplification of the process. The relative stabilities as measured by melting temperatures by both CD and mass spectral charge states are, Oxi-TRX, GS-ethyl-TRX, Cys-ethyl-TRX, and Red-TRX. The introduction of the Cys-32-ethylglutathionyl group provides extra stabilization that results from additional hydrogen bonding interactions between the ethylglutathionyl group and the protein. Near-UV CD data show that the local environment near the active site is perturbed to almost an identical degree regardless of whether alkylation at Cys-32 is by the ethylglutathionyl group, or the smaller, nonhydrogen-bonding ethylcysteinyl group. Mass spectral data, however, indicate a tighter structure for GS-ethyl-TRX.     

The development of 2-benzimidazole substituted pyrimidine based inhibitors of lymphocyte specific kinase (Lck)

This communication details the synthesis, biological activity, and binding mode of a novel class of 2-benzimidazole substituted pyrimidines. The most potent analogs disclosed showed low nanomolar activity for the inhibition of Lck kinase and a representative analog was co-crystallized with Hck (a structurally related member of the Src family kinases).     

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.     

NKG2D-RAE-1 receptor-ligand variation does not account for the NK cell defect in nonobese diabetic mice

NK cells from NOD mice induced with poly(I:C) in vivo exhibit low cytotoxicity against a range of target cells, but the genetic mechanisms controlling this defect are yet to be elucidated. Defects in the expression of NKG2D and its ligands, the RAE-1 molecules, have been hypothesized to contribute to the reduced NK function present in NOD mice. In this study, we show that segregation of the NK-mediated killing phenotype did not correlate with the NOD Raet1 haplotype and that the large alterations in NKG2D expression previously reported on NK cells expanded in vitro were not observed in primary, poly(I:C)-elicited NK cells in vivo. Additional studies indicate a complex genetic control of defective NOD NK cells including genes linked to the MHC and possibly those that are associated with an altered cytokine response to the TLR3-agonist poly(I:C).     

Dissemination of Fluoroquinolone-Resistant Campylobacter spp. within an Integrated Commercial Poultry Production System

While characterizing the intestinal bacterial community of broiler chickens, we detected -proteobacterial DNA in the ilea of 3-day-old commercial broiler chicks (J. Lu, U. Idris, B. Harmon, C. Hofacre, J. J. Maurer, and M. D. Lee, Appl. Environ. Microbiol. 69:6816-6824, 2003). The sequences exhibited high levels of similarity to Campylobacter jejuni and Campylobacter coli sequences, suggesting that chickens can carry Campylobacter at a very young age. Campylobacter sp. was detected by PCR in all samples collected from the ilea of chicks that were 3 to 49 days old; however, it was detected only in the cecal contents of chickens that were at least 21 days old. In order to determine whether the presence of Campylobacter DNA in young chicks was due to ingestion of the bacteria in food or water, we obtained commercial broiler hatching eggs, which were incubated in a research facility until the chicks hatched. DNA sequencing of the amplicons resulting from Campylobacter-specific 16S PCR performed with the ileal, cecal, and yolk contents of the day-of-hatching chicks revealed that Campylobacter DNA was present before the chicks consumed food or water. The 16S rRNA sequences exhibited 99% similarity to C. jejuni and C. coli sequences and 95 to 98% similarity to sequences of other thermophilic Campylobacter species, such as C. lari and C. upsaliensis. The presence of C. coli DNA was detected by specific PCR in the samples from chicks obtained from a commercial hatchery; however, no Campylobacter was detected by culturing. In order to determine whether the same strains of bacteria were present in multiple levels of the integrator, we cultured Campylobacter sp. from a flock of broiler breeders and their 6-week-old progeny that resided on a commercial broiler farm. The broiler breeders had been given fluoroquinolone antibiotics, and we sought to determine whether the same fluoroquinolone-resistant strain was present in their progeny. The isolates were typed by pulsed-field gel electrophoresis, which confirmed that the parental and progeny flocks contained the same strain of fluoroquinolone-resistant C. coli. These data indicate that resistant C. coli can be present in multiple levels of an integrated poultry system and demonstrated that molecular techniques or more sensitive culture methods may be necessary to detect early colonization by Campylobacter in broiler chicks.     

Experimental Sepsis Impairs Humoral Memory in Mice

Patients with sepsis are often immune suppressed, and experimental mouse models of sepsis also display this feature. However, acute sepsis in mice is also characterized by a generalized B cell activation and plasma cell differentiation, resulting in a marked increase in serum antibody concentration. Its effects on humoral memory are not clearly defined. We measured the effects of experimental sepsis on long-term immunological memory for a defined antigen: we induced colon ascendens stent peritonitis (CASP) 8 weeks after 2 rounds of immunization with ovalbumin. Four weeks later, the antigen-specific bone marrow plasma cell count had doubled in immunized non-septic animals, but remained unchanged in immunized septic animals. Sepsis also caused a decrease in antigen-specific serum antibody concentration. We conclude that sepsis weakens humoral memory by impeding the antigen-specific plasma cell pool’s development, which is not complete 8 weeks after secondary immunization.     

Oxidative stress and lipid mediators induced in alveolar macrophages by ultrafine particles

In ambient aerosols, ultrafine particles (UFP) and their agglomerates are considered to be major factors contributing to adverse health effects. Reactivity of agglomerated UFP of elemental carbon (EC), Printex 90, Printex G, and diesel exhaust particles (DEP) was evaluated by the capacity of particles to oxidize methionine in a cell-free in vitro system for determination of their innate oxidative potential and by alveolar macrophages (AMs) to determine production of arachidonic acid (AA), including formation of prostaglandin E2 (PGE2), leukotriene B4 (LTB4), reactive oxygen species (ROS), and oxidative stress marker 8-isoprostane. EC exhibiting high oxidative potential induced generation of AA, PGE2, LTB4, and 8-isoprostane in canine and human AMs. Printex 90, Printex G, and DEP, showing low oxidative capacity, still induced formation of AA and PGE2, but not that of LTB4 or 8-isoprostane. Aging of EC lowered oxidative potential while still inducing production of AA and PGE2 but not that of LTB4 and 8-isoprostane. Cellular ROS production was stimulated by all particles independent of oxidative potential. Particle-induced formation of AA metabolites and ROS was dependent on mitogen-activated protein kinase kinase 1 activation of cytosolic phospholipase A2 (cPLA2) as shown by inhibitor studies. In conclusion, cPLA2, PGE2, and ROS formation was activated by all particle types, whereas LTB4 production and 8-isoprostane were strongly dependent on particles' oxidative potential. Physical and chemical parameters of particle surface correlated with oxidative potential and stimulation of AM PGE2 and 8-isoprostane production.     

Use-dependent plasticity in barrel cortex: intrinsic signal imaging reveals functional expansion of spared whisker representation into adjacent deprived columns

We used optical imaging of intrinsic cortical signals, elicited by whisker stimulation, to define areas of activation in primary sensory cortex of normal hamsters and hamsters subjected to neonatal follicle ablation at postnatal day seven (P7). Follicle ablations were unilateral, and spared either C-row whiskers or the second whisker arc. This study was done to determine if the intrinsic cortical connectivity pattern of the barrel cortex, established during the critical period, affects the process of representational plasticity that follows whisker follicle ablation. Additionally, we tested the ability to monitor such changes in individual cortical whisker representations using intrinsic signal imaging. Stimulation of a single whisker yielded peak activation of a barrel-sized patch in the somatotopically appropriate location in normal cortex. In both row and arc-spared animals, functional representations corresponding to spared follicles were significantly stronger and more oblong than normal. The pattern of activation differed in the row-sparing and arc-sparing groups, in that the expansion was preferentially into deprived, not spared areas. Single whisker stimulation in row-spared cases preferentially activated the corresponding barrel arc, while stimulation of one whisker in arc-spared cases produced elongated activation down the barrel row. Since whisker deflection normally has a net inhibitory effect on neighboring barrels, our data suggest that intracortical inhibition fails to develop normally in deprived cortical columns. Because thalamocortical projections are not affected by follicle ablation after P7, we suggest that the effects we observed are largely cortical, not thalamocortical.     

Report from the second cytomegalovirus and immunosenescence workshop

The Second International Workshop on CMV & Immunosenescence was held in Cambridge, UK, 2-4th December, 2010. The presentations covered four separate sessions: cytomegalovirus and T cell phenotypes; T cell memory frequency, inflation and immunosenescence; cytomegalovirus in aging, mortality and disease states; and the immunobiology of cytomegalovirus-specific T cells and effects of the virus on vaccination. This commentary summarizes the major findings of these presentations and references subsequently published work from the presenter laboratory where appropriate and draws together major themes that were subsequently discussed along with new areas of interest that were highlighted by this discussion.