A re-evaluation of the role of host defence peptides in mammalian immunity

Host defence peptides are found in all classes of life and are a fundamental component of the innate immune response. Initially it was believed that their sole role in innate immunity was to kill invading microorganisms, thus providing direct defence against infection. Evidence now suggests that these peptides play diverse and complex roles in the immune response and that, in higher animals, their functions are not restricted to the innate immune response. In in vitro experiments certain host defence peptides have been demonstrated to be potent antimicrobial agents at modest concentrations, although their antimicrobial activity is often strongly reduced or ablated in the presence of physiological concentrations of ions such as Na(+) and Mg(2+). In contrast, in experiments done in standard tissue culture media, the composition of which more accurately represents physiological levels of ions, mammalian host defence peptides have been demonstrated to have a number of immunomodulatory functions including altering host gene expression, acting as chemokines and/or inducing chemokine production, inhibiting lipopolysaccharide induced pro-inflammatory cytokine production, promoting wound healing, and modulating the responses of dendritic cells and cells of the adaptive immune response. Animal models indicate that host defence peptides are crucial for both prevention and clearance of infection. As interest in the in vivo functions of host defence peptides is increasing, it is important to consider whether in mammals the direct antimicrobial and immunomodulatory properties observed in vitro are physiologically relevant, especially since many of these activities are concentration dependent. In this review we summarize the concentrations of host defence peptides and ions reported throughout the body and compare that information with the concentrations of peptides that are known have antimicrobial or immunomodulatory functions in vitro.     

Expanded utility of the native chemical ligation reaction

The post-genomic era heralds a multitude of challenges for chemists and biologists alike, with the study of protein functions at the heart of much research. The elucidation of protein structure, localization, stability, post-translational modifications, and protein interactions will steadily unveil the role of each protein and its associated biological function in the cell. The push to develop new technologies has necessitated the integration of various disciplines in science. Consequently, the role of chemistry has never been so profound in the study of biological processes. By combining the strengths of recombinant DNA technology, protein splicing, organic chemistry, and the chemoselective chemistry of native chemical ligation, various strategies have been successfully developed and applied to chemoselectively label proteins, both in vitro and in live cells, with biotin, fluorescent, and other small molecule probes. The site-specific incorporation of molecular entities with unique chemical functionalities in proteins has many potential applications in chemical and biological studies of proteins. In this article, we highlight recent progress of these strategies in several areas related to proteomics and chemical biology, namely, in vitro and in vivo protein biotinylation, protein microarray technologies for large-scale protein analysis, and live-cell bioimaging.     

HILIC-MS/MS method for the quantitation of nucleotides in infant formula and adult nutritional formula: First Action 2011.21

Official Method 2011.21 is for the quantitation of the following nucleotides: adenosine 5'-monophosphate (AMP), guanosine 5'-monophosphate (GMP), uridine 5'-monophosphate (UMP), cytidine 5'-monophosphate (CMP), and inosine 5'-monophosphate (IMP) in infant formula and adult/pediatric nutritional formula. It uses hydrophilic interaction liquid chromatography with tandem mass spectrometry (HILIC-MS/MS). Preparation of the internal standards was conducted using centrifugal ultrafiltration and the standards are AMP- (13)C10, (15)N5; GMP-(13)C10, (15)N5; UMP-(13)C9, (15)N2; and15 CMP- (13)C9, (15)N3. Data were collected by using multiple reaction monitoring of the product ions of protonated molecules of the five nucleotides generated by positive-electrospray ionization. The HILIC conditions were conducted with ammonium formate (30 mmol/L) in water (pH 2.5, adjusted with formic acid) and methanol. The LOD and LOQ of the standard solution were 0.005-0.01 and 0.01-0.03 microg/mL, respectively. Recovery data were collected for intraday and interday testing and ranged from 98.1 to 108.9% with an RSD of 0.7-5.4%. The analytical range of the method is between 0.04 to 5 microg/mL for standard solution.     

Mechanistic study of the deamination reaction of guanine: a computational study

The mechanism for the deamination of guanine with H(2)O, OH(-), H(2)O/OH(-) and for GuaH(+) with H(2)O has been investigated using ab initio calculations. Optimized geometries of the reactants, transition states, intermediates, and products were determined at RHF/6-31G(d), MP2/6-31G(d), B3LYP/6-31G(d), and B3LYP/6-31+G(d) levels of theory. Energies were also determined at G3MP2, G3MP2B3, G4MP2, and CBS-QB3 levels of theory. Intrinsic reaction coordinate (IRC) calculations were performed to characterize the transition states on the potential energy surface. Thermodynamic properties (ΔE, ΔH, and ΔG), activation energies, enthalpies, and Gibbs free energies of activation were also calculated for each reaction investigated. All pathways yield an initial tetrahedral intermediate and an intermediate in the last step that dissociates to products via a 1,3-proton shift. At the G3MP2 level of theory, deamination with OH(-) was found to have an activation energy barrier of 155 kJ mol(-1) compared to 187 kJ mol(-1) for the reaction with H(2)O and 243 kJ mol(-1) for GuaH(+) with H(2)O. The lowest overall activation energy, 144 kJ mol(-1) at the G3MP2 level, was obtained for the deamination of guanine with H(2)O/OH(-). Due to a lack of experimental results for guanine deamination, a comparison is made with those of cytosine, whose deamination reaction parallels that of guanine.     

Determination of dissolved thiols using solid-phase extraction and liquid chromatographic determination of fluorescently derivatized thiolic compounds

A method employing solid-phase extraction coupled with HPLC separation of thiol-monobromobimane (mBBr) derivatives was developed and optimized to quantify dissolved thiols at concentrations as low as 0.1 nM for glutathione (GSH) and gamma-glutamylcysteine (gammaEC) in natural waters. The reducing reagent, tri-n-butylphosphine (TBP), is needed for complete derivatization. At the optimal addition of TBP ([TBP]/[mBBr] = -0.4-1.6), no interference from copper was observed. The thiol fluorescence signal was totally suppressed if the mole ratio of TBP to mBBr was 2.6 or greater. Consistent recovery of thiols standards in a NaCl solution (0.5 M) was obtained using the Waters HLB reversed-phase resin, and blank levels of GSH and gammaEC were extremely low (less than 0.03 nM). The detection limits for GSH, gammaEC and phytochelatin-2 (PC-2) were 0.03, 0.03, and 0.06 nM, respectively.     

Product applications and technology directions with SiGe BiCMOS

In this paper, we highlight the effectiveness and flexibility of SiGe BiCMOS as a technology platform over a wide range of performance and applications. The bandgap-engineered SiGe heterojunction bipolar transistors (HBTs) continue to be the workhorse of the technology, while the CMOS offering is fully foundry compatible for maximizing IP sharing. Process customization is done to provide high-quality passives, which greatly enables fully integrated single-chip solutions. Product examples include 40-Gb/s (OC768) components using high-speed SiGe HBTs, power amplifiers compatible for cellular applications, integrated voltage-controlled oscillators, and very high-level mixed-signal integration. It is argued that such key enablements along with the lower cost and higher yields attainable by SiGe BiCMOS technologies will provide competitive solutions for the communication marketplace.