No abstract

Zinc is essential for all bacteria, but excess amounts of the metal can have toxic effects. To address this, bacteria have developed tightly regulated zinc uptake systems, such as the ZnuABC zinc transporter which is regulated by the Fur-like zinc uptake regulator (Zur). In Pseudomonas aeruginosa, a Zur protein has yet to be identified experimentally, however, sequence alignment revealed that the zinc-responsive transcriptional regulator Np20, encoded by np20 (PA5499), shares high sequence identity with Zur found in other bacteria. In this study, we set out to determine whether Np20 was functioning as Zur in P. aeruginosa. Using RT-PCR, we determined that np20 (hereafter known as zur) formed a polycistronic operon with znuC and znuB. Mutant strains, lacking the putative znuA, znuB, or znuC genes were found to grow poorly in zinc deplete conditions as compared to wild-type strain PAO1. Intracellular zinc concentrations in strain PAO-Zur (Δzur) were found to be higher than those for strain PAO1, further implicating the zur as the zinc uptake regulator. Reporter gene fusions and real time RT-PCR revealed that transcription of znuA was repressed in a zinc-dependent manner in strain PAO1, however zinc-dependent transcriptional repression was alleviated in strain PAO-Zur, suggesting that the P. aeruginosa Zur homolog (ZurPA) directly regulates expression of znuA. Electrophoretic mobility shift assays also revealed that recombinant ZurPA specifically binds to the promoter region of znuA and does not bind in the presence of the zinc chelator N,N′,N-tetrakis(2-pyridylmethyl) ethylenediamine (TPEN). Taken together, these data support the notion that Np20 is the P. aeruginosa Zur, which regulates the transcription of the genes encoding the high affinity ZnuABC zinc transport system.

Ultraviolet photoelectron spectroscopy in an ion beam was used to investigate the electronic properties of isolated DNA oligonucleotides [dA(5)-4H](4-) and [dT(5)-4H](4-), carrying four excess negative charges. We find the fourth adiabatic electron affinity to be slightly negative for [dA(5)-4H](4-), while it is positive for [dT(5)-4H](4-). This implies a significant influence of the base composition on energetics, which is in turn relevant for analytic applications and also for charge transport properties.

Non-melanoma skin cancer and other epithelial tumors overexpress cyclooxygenase-2 (COX-2), differentiating them from normal cells. COX-2 metabolizes arachidonic acid to prostaglandins including, the J-series prostaglandins, which induce apoptosis by mechanisms including endoplasmic reticulum (ER) stress. Arachidonoyl-ethanolamide (AEA) is a cannabinoid that causes apoptosis in diverse tumor types. Previous studies from our group demonstrated that AEA was metabolized by COX-2 to J-series prostaglandins. Thus, the current study examines the role of COX-2, J-series prostaglandins, and ER stress in AEA-induced apoptosis. In tumorigenic keratinocytes that overexpress COX-2, AEA activated the PKR-like ER kinase (PERK), inositol requiring kinase-1 (IRE1), and activating transcription factor-6 (ATF6) ER stress pathways and the ER stress apoptosis-associated proteins, C/EBP homologous protein-10 (CHOP10), caspase-12, and caspase-3. Using an ER stress inhibitor, it was determined that ER stress was required for AEA-induced apoptosis. To evaluate the role of COX-2 in ER stress-apoptosis, HaCaT keratinocytes with low endogenous COX-2 expression were transfected with COX-2 cDNA or an empty vector and AEA-induced ER stress-apoptosis occurred only in the presence of COX-2. Moreover, LC-MS analysis showed that the novel prostaglandins, 15-deoxyΔ(12,14) PGJ2 -EA and Δ(12) PGJ2 /PGJ2-EA, were synthesized from AEA. These findings suggest that AEA will be selectively toxic in tumor cells that overexpress COX-2 due to the metabolism of AEA by COX-2 to J-series prostaglandin-ethanolamides (prostamides). Hence, AEA may be an ideal topical agent for the elimination of malignancies that overexpress COX-2.

Sensitive methods recently developed to measure laser-induced fluorescence from trapped ions have been applied to study the dynamics of double-and single-stranded oligonucleotides. In this paper, the fraying of duplex terminal base pairs has been identified by measuring the donor fluorescence as a function of temperature from an oligonucleotide duplex labeled with a pair of FRET dyes. Comparison of the degree of dissociation of 14-mer duplexes observed in the mass spectra with the fluorescence intensity of the donor enables intermediate conformations of the unzipping duplex at the weaker binding end of the duplex to be identified. The autodetachment of electrons from double-and single-stranded oligonucleotide anions has been observed in a gas phase environment. To characterize this process, measurements were performed on 7-mers prepared without FRET fluorophores attached. The dependence of the decay rates of trapped anions have been measured as a function of charge state and temperature for various base compositions. An exceptionally strong dependence of the decay rate on base composition has been identified. The physical basis for this process will be discussed. ( T he structures and conformational changes of biomolecules in the gas phase have been studied by a variety of methods [1,2]. One of the advantages of working in the gas phase is that it provides an opportunity to observe dynamical processes which may be obscured or not present in a condensed phase environment. Quadrupole ion traps provide a controlled environment in which ions can be probed for a theoretically unlimited time while in thermal equilibrium with the surrounding buffer gas. Ion trap instrumentation developed in our laboratory [3] has been used to carry out fluorescence measurements of trapped ions, and recently has been used to measure fluorescence resonance energy transfer (FRET) in trapped oligonucleotide ions [4]. The extension of FRET methods, widely used in solution studies [5], to measurements of trapped biomolecule ions provides the opportunity to directly correlate changes in fluorescence intensity with changes in the average conformation of biomolecules.This paper describes the application of ion trapping technology and fluorescence measurements to the study of oligonucleotides in the gas phase. First, the unique capability of correlating in-situ fluorescence with mass spectra allows the investigation of the gasphase dissociation dynamics of double-stranded oligonucleotide anions, and a continuation of previous studies [4] will be presented. The results of FRET and mass spectrometry experiments with trapped 14-mer doublestranded oligonucleotides display characteristics consistent with an intermediate state related to the dissociation dynamics into single strands. Second, the ability to store ions for long periods in controlled thermal environments has led to the observation of electron autodetachment of multiply charged oligonucleotide anions. The time and temperature dependence of the detachment rates and the dependence of these ...

DNA damage from (+/-)-anti-benzo[a]pyrene-7,8-dihydrodiol-9,10-epoxide (BPDE) at a hotspot TP53 gene sequence was electrochemically detected. BPDE was exposed to gold electrode immobilized double-stranded DNA oligomers followed by voltammetric measurements in the presence of redox-active C(12)H(25)V(2+)C(6)H(12)V(2+)C(12)H(25) (V(2+) = 4,4'-bipyridyl or viologen, C12-viologen). Square wave voltammograms from BPDE-exposed DNA-modified electrodes showed the emergence of a C12-viologen-DNA complex at -0.37 V versus Ag/AgCl. The peak current intensity of this redox wave was dependent on both BPDE concentration and exposure time. Controls with alternate xenobiotics and DNA sequences showed this redox wave to be primarily due to BPDE damage at the wild-type DNA sequence. The detection limit was determined to be approximately 170 nM BPDE. Mass spectrometry and UV thermal melting experiments provided insight into the BPDE reaction and mirrored the sensor results. This report demonstrates that an electrochemical hybridization sensor can be used to detect sequence-related xenobiotic DNA damage.

The performance of quadrupole ion traps using argon or air as the buffer gas was evaluated and compared to the standard helium only operation. In all cases a pure buffer gas, not mixtures of gases, was investigated. Experiments were performed on a Bruker Esquire ion trap, a Finnigan LCQ, and a Finnigan ITMS for comparison. The heavier gases were found to have some advantages, particularly in the areas of sensitivity and collision-induced dissociation efficiency; however, there is a significant resolution loss due to dissociation and/or scattering of ions. Additionally, the heavier gases were found to affect ion activation and deactivation during MS/MS, influencing the product ion intensities observed. Finally, the specific quadrupole ion trap design and the ion ejection parameters were found to be crucial in the quality of the spectra obtained in the presence of heavy gases. Operation with static pressures of heavy gases can be beneficial under certain design and operating conditions of the quadrupole ion trap. T he quadrupole ion trap mass spectrometer (QITMS) comprises a large and continually growing segment of mass spectrometry. The QITMS possesses many advantages over other mass analyzers, including ease of use, size, cost, and ability to perform MS n analysis. With this greatly increased usage comes the motivation to enhance and extend its abilities. One area that has seen sporadic investigation is the buffer gas used within QITMS. The use and function of the buffer gas has been of interest to researchers for many years prior to commercialization of the QITMS [1][2][3].The buffer gas is an important component of the QITMS and can affect many aspects of its operation. When injecting ions into the QITMS, one function of the buffer gas is to reduce ion kinetic energy. As the ions enter the trapping volume, they will encounter neutral buffer gas atoms or molecules and collide with them. One possible outcome of these collisions is the transfer of kinetic energy from the ions to the neutral target (the buffer gas). This reduction of ion kinetic energy is termed collisional cooling, and the process aids in ion trapping and therefore can improve sensitivity. Many collisions can occur once the ion is nominally trapped, yielding a condensed cloud of ions close to the center of the trapping volume. With all of the ions condensed to the center of the QITMS and possessing similarly low kinetic energies, mass analysis can be performed with higher resolution and sensitivity [3].A collision also can result in ion kinetic energy being converted into ion internal energy. The maximum amount of internal energy that can be deposited into the ion in a single collision is the center-of-mass kinetic energy, E com , and can be calculated using the following eq:where M n is the mass of the neutral buffer gas atom or molecule, M p is the mass of the ion, E lab is the ion's laboratory frame kinetic energy and E com is the centerof-mass kinetic energy. Increasing the internal energy of an ion is the desired outcome when performing coll...

A unique collision-induced dissociation pattern was observed for protonated polyproline peptides of length n in which y nϪ2 and/or y nϪ4 ions were formed in much higher abundance than any other product ions. Cleavage occurs only at every other amide bond, such that product ions are formed only from the losses of even numbers of proline residues. Exclusive losses of even numbers of proline residues were not observed from sodiated peptides. Further study of the tandem mass spectrometry (MS/MS) patterns of protonated proline-rich peptides showed that the substitution of alanine in the second position of polyproline peptides did not prevent the dominant formation of y nϪ2 and y nϪ4 ions. The loss of ProAla to form the y 8 ion from (ProAlaPro 8 NH 2 ϩH) ϩ was as abundant as the loss of ProPro from (Pro 10 NH 2 ϩH) ϩ . However, modification of the peptides that presumably affected the location of the proton on the peptide did alter the MS/MS spectra. Pro 10 and Pro 5 with blocked N-termini or with arginine substituted for the first proline residue did not form abundant y nϪ2 or y nϪ4 ions. MS 3 and double resonance experiments showed that dissociation of intermediate y n product ions can produce y nϪ2 ions, but are not necessary dissociation pathway intermediates. This analysis suggests that the ionizing proton must be located at the N-terminus for the peptide ion to dissociate in this manner. (J Am Soc Mass Spectrom 2007, 18, 2198 -2203) © 2007 American Society for Mass Spectrometry P roline (Pro) is the only naturally occurring amino acid that is bonded to the nitrogen atom of the peptide backbone. The absence of the amide hydrogen prevents polyproline peptides from forming alpha helices. Instead, two possible helical structures of polyproline can be found in solution, named ProI and ProII. ProI exists in solution as a right-handed helix with 3.3 residues per turn, and all peptide bonds are cis bonds. ProII is a left-handed helix with 3.0 residues per turn, and all peptide bonds are trans bonds. The type of helix formed varies with the type of solvent in which the polyproline is dissolved. ProI is found in less protic solvents such as butanol or propanol, whereas ProII exists in more protic solvents such as water. Counterman and Clemmer [1] have shown with ion mobility spectrometry and supporting computational studies that when polyproline peptides are introduced into the gas phase from propanol solutions, features of the ProI helix persist in the gas phase. This was observed for many different lengths of polyproline chains. However, electrospray ionization (ESI) of aqueous solutions did not produce ion mobility data consistent with ProII helices. It was reasoned that the elongated structure of the ProII helix could not be stabilized in the absence of solvent [1,2]. It already has been established that the solvent choice has no effect on the dissociation energetics of polyproline peptides [3]. Interestingly, a unifying feature was established for all polyproline peptides examined, regardless of the original solvent....