A method for the making and utility of gadolinium-labeled albumin microbubbles

Objective

Perfluorocarbon-exposed sonicated dextrose albumin microbubbles (PESDA) binds scavenger receptors and can be noninvasively imaged. To enhance imaging, gadolinium (Gd)-labeled PESDA was developed and tested in a model of vascular inflammation by magnetic resonance imaging (MRI).

Methods and Results

Purified human serum albumin (HSA) (5%) was labeled with Gd via the covalent binding of diethylenetriaminepentacetic acid. Abdominal aortic tissues in Sprague-Dawley rats (n=5 per group) were analyzed by 7-T MRI and scanning electron microscopy to evaluate PESDA binding. Labeling-purified 5% human albumin resulted in an average of 16.1 Gd atoms per albumin molecule as determined by atomic absorption. Forty-eight hours after balloon angioplasty, aortic tissue was enhanced with Gd-PESDA as compared to control tissue. 7-T MRI of explanted tissues was sensitive to the detection of retained PESDA. Enhancement of aortic tissue in vivo was present albeit to a lesser extent than explanted tissue from the same animals.

Conclusions

HSA was successfully labeled, and an albumin-based microbubble with Gd was synthesized. This contrast agent, Gd-PESDA, may serve as an additional agent for the MRI evaluation of innate inflammation and used to noninvasively image early vascular pathophysiologic processes.

Condensed Abstract

In this study, Gd-PESDA microbubbles and were synthesized and shown to detect the binding of these microbubbles using MRI in injured aortic tissue. The method for synthesizing Gd-PESDA is detailed, and the proposed utility of this new contrast agent is discussed.     

Hydration of gas-phase ions formed by electrospray ionization

The hydration of gas-phase ions produced by electrospray ionization was investigated. Evidence that the hydrated ions are formed by two mechanisms is presented. First, solvent condensation during the expansion inside the electrospray source clearly occurs. Second, some solvent evaporation from more extensively solvated ions or droplets is apparent. To the extent that these highly solvated ions have solution-phase structures, then the final isolated gas-phase structure of the ion will be determined by the solvent evaporation process. This process was investigated for hydrated gramicidin S in a Fourier-transform mass spectrometer. Unimolecular dissociation rate constants of isolated gramicidin S ions with between 2 and 14 associated water molecules were measured. These rate constants increased from 16 to 230 s-1 with increasing hydration, with smaller values corresponding to magic numbers.     

KMgF3:Ni2+晶体的占位、局部结构、吸收光谱和顺磁g因子的统一解释

利用完全对角化方法和强场耦合方案,采用半自洽场(semi-SCF)自由Ni2+的d轨道模型和Ni2+-6X-(x=F,Cl,Br,I)络合物的μ-κ-α模型研究,建立了含有过渡族金属离子的晶体的局域结构与吸收光谱和顺磁g因子之间的定量关系,对KMgF3:Ni2+晶体的占位、局域结构、吸收光谱和顺磁g因子作出了统一解释,预测了KMgF3:Ni2+晶体的光谱精细结构.所得理论计算结果与实验值符合得很好.     

Blackbody infrared radiative dissociation of oligonucleotide anions

The dissociation kinetics of a series of doubly deprotonated oligonucleotide 7-mers [d(A) ₇ ^2? , d(AATTAAT)^2?, d(TTAATTA)^2?, and d(CCGGCCG)^2?] were measured using blackbody infrared radiative dissociation in a Fourier-transform mass spectrometer. The oligonucleotides dissociate first by cleavage at the glycosidic bond leading to the loss of a neutral nucleobase, followed by cleavage at the adjacent (5′) phosphodiester bond to produce structurally informative a-base and w type ions. From the temperature dependence of the unimolecular dissociation rate constants, Arrhenius activation parameters in the zero-pressure limit are obtained for the loss of base. The measured Arrhenius parameters are dependent on the identity of the nucleobase. The process involving the loss of an adenine base from the dianions, d(A) ₇ ^2? , d(AATTAAT)^2?, and d(TTAATTA)^2? has an average activation energy (E _a ) of ～1.0 eV and a preexponential factor (A) of 10¹⁰ s^?1. Both guanine and cytosine base loss occurs for d(CCGGCCG)^2?. The average Arrhenius parameters for the loss of cytosine and guanine are E _a =1.32 ± 0.03 eV and A=10^13.3±0.3 s^?1. No loss of thymine was observed for mixed adenine-thymine oligonucleotides. Neither base loss nor any other fragmentation reactions occur for d(T) ₇ ^2? over a 600 s reaction delay at 207 °C, a temperature close to the upper limit accessible with our instrument. The Arrhenius parameters indicate that the preferred cleavage sites for mixed oligonucleotides of similar mass-to-charge ratio will be strongly dependent on the internal energy of the precursor ions. At low internal energies (effective temperatures below 475 K), loss of adenine and subsequent cleavage of the adjacent phosphoester bonds will dominate, whereas at higher energies, preferential cleavage at C and G residues will occur. The magnitude of the A factors ≤10¹³ s^?1 measured for the loss of the three nucleobases (A, G, and C) is indicative of an entropically neutral or disfavored process as the rate limiting step for this reaction.     

Method development for the determination of nickel in petroleum using line-source and high-resolution continuum-source graphite furnace atomic absorption spectrometry

Several sample preparation methods have been investigated for the direct determination of nickel in crude oil using graphite furnace atomic absorption spectrometry (GF AAS). Xylene was found unsuitable as solvent because of the poor long-term stability of the solutions and the resulting contamination of the equipment. Isobutyl methyl ketone (IBMK) solutions exhibited better stability, but the sensitivity of the organic nickel salt used for the standard solutions showed a high day-to-day variability. An oil-in-water emulsion using Triton X-100 as surfactant gave the best results. Using high-resolution continuum-source (HR-CS) GF AAS, it could be observed that up to 50% of the nickel in crude oil, most likely low molecular weight nickel porphyrins, were lost already at pyrolysis temperatures >400 °C, whereas the rest of the nickel as well as the nickel standard were stable up to 1300 °C. The nickel absorption at a secondary line at 232.138 nm was recorded simultaneously with that at the primary line at 232.003 nm, expanding the dynamic working range by an order of magnitude. The best characteristic mass obtained was m₀=27 pg and the limit of detection was around 0.07 μg g⁻¹ Ni in oil, based on an emulsion of 2 g oil in 10 ml. The accuracy of the procedure was verified by analyzing the certified reference material (CRM) NIST SRM 1634c, Trace Metals in Residual Fuel Oil.     

Die K4[Ag4O4] — Strukturfamilie

K₄[Ag₄0₄] Structure Type M₄[Ag₄O₄] (M ? Li? Cs) and M₄[Cu₄O₄] (M ? Li? Rb) have been prepared anew; as an example the crystal structure of K₄[Ag₄O₄] has been revised. Contrary to our first report [2, 3] it crystallizes in the space-group I4 m2 with the “ring” [Ag₄O₄]^4? which is not plane, however. Each two O^2? (trans-arrangement) are rather (0.02 Å) above and below the plane of the “ring”, respectively. The new parameters are given in the text. The distances, for example d(Ag⁺·O^2?) = 2.058 Å and the Madelung Part of Lattice Energy, MAPLE, are both in a very good agreement with the measurements and calculations, respectively, which have been done on other ternary oxides with silver.     

2′-Methyl and 1′-xylosyl derivatives of 2′-hydroxyflexixanthin are major carotenoids of Hymenobacter species

Bacteria related to the genus Hymenobacter are colored intensely red due to their carotenoid pigmentation. Using high-resolution mass spectrometry and comparison to the literature reports, we previously isolated and presumptively identified seven structurally related carotenoids from representative Hymenobacter-like strains. After further analysis using one- and two-dimensional ¹H NMR, UV-vis, and circular dichroism spectroscopies, we determined that the major carotenoid present in these strains is 2′-hydroxyflexixanthin, as proposed previously by others in a related organism. Furthermore, we have identified three novel, related carotenoids abundant in these strains as 1′-xylosyl-2′-hydroxyflexixanthin, 2′-methoxyflexixanthin, and 3-deoxy-2′-methoxyflexixanthin.     

Evidence that water can reduce the kinetic stability of protein-hydrophobic ligand interactions

The first quantitative comparison of the thermal dissociation rate constants measured for protein-ligand complexes in their hydrated and dehydrated states is described. Rate constants, measured using surface plasmon resonance spectroscopy, are reported for the dissociation of the 1:1 complexes of bovine β-lactoglobulin (Lg) with the fatty acids (FA), palmitic acid (PA), and stearic acid (SA), in aqueous solution at pH 8 and at temperatures ranging from 5 to 45 °C. The rate constants are compared to values determined from time-resolved blackbody infrared radiative dissociation measurements for the gaseous deprotonated (Lg+FA)(n-) ions, where n = 6 and 7, at temperatures ranging from 25 to 66 °C. Notably, the hydrated (Lg+PA) complex is kinetically less stable than the corresponding gas phase (Lg+PA)(n-) ions at all temperatures investigated; the hydrated (Lg+SA) complex is kinetically less stable than the gaseous (Lg+SA)(n-) ions at temperatures <45 °C. The greater kinetic stability of the gaseous (Lg+FA)(n-) ions originates from significantly larger, by 11-12 kcal mol(-1), E(a) values. It is proposed that the differences in the dissociation E(a) values measured in solution and the gas phase reflect the differential hydration of the reactant and the dissociative transition state.     

Reliable Determinations of Protein–Ligand Interactions by Direct ESI-MS Measurements. Are We There Yet?

The association-dissociation of noncovalent interactions between protein and ligands, such as other proteins, carbohydrates, lipids, DNA, or small molecules, are critical events in many biological processes. The discovery and characterization of these interactions is essential to a complete understanding of biochemical reactions and pathways and to the design of novel therapeutic agents that may be used to treat a variety of diseases and infections. Over the last 20 y, electrospray ionization mass spectrometry (ESI-MS) has emerged as a versatile tool for the identification and quantification of protein–ligand interactions in vitro. Here, we describe the implementation of the direct ESI-MS assay for the determination of protein–ligand binding stoichiometry and affinity. Additionally, we outline common sources of error encountered with these measurements and various strategies to overcome them. Finally, we comment on some of the outstanding challenges associated with the implementation of the assay and highlight new areas where direct ESI-MS measurements are expected to make significant contributions in the future.     

Thermal decomposition of multiply charged T-rich oligonucleotide anions in the gas phase. Influence of internal solvation on the arrhenius parameters for neutral base loss

Arrhenius activation parameters (E(a), A) for the loss of neutral nucleobases from a series of T-rich, doubly and triply deprotonated 15- and 20-mer oligodeoxynucleotides (ODN) containing a single reactive base (X = A or C) with the sequence, XT14, XT19 and T19X, have been determined using the blackbody infrared radiative dissociation technique. The A-containing anions are significantly more reactive (> or =3000 times) than the C-containing ions over the temperature range investigated. Importantly, the Arrhenius parameters for the loss of AH exhibit a strong dependence on size of the ODN and, to some extent, the charge state; the Arrhenius parameters increase with size and charge (Ea = 29-39 kcal mol(-1), A = 10(15)-10(20) s(-1)). In contrast, the parameters for the loss of CH are much less sensitive to size (Ea = 35-39 kcal mol(-1), A = 10(14)-10(17) s(-1)). The results are consistent with a greater contribution from the internal solvation of the reactive base to the Arrhenius parameters for the loss of A, compared with C, from the 15- and 20-mers. To further probe differences in internal solvation of A and C, hydrogen/deuterium exchange was carried out on AT19(-3), T19A(-3), CT19(-3) and T19C(-3) using D2O as the exchange reagent. However, the H/D exchange results did not reveal any differences in internal solvation within the ODN anions. Arrhenius parameters for the dissociation of noncovalent complexes of T20(-3) and the neutral nucleobase AH or CH have also been determined. Differences in the parameters indicate differences in the nature of the intermolecular interactions. It is proposed that neutral A-T interactions (i.e., base-base), which originate in solution, dominate in the case of (T20 + AH)(-3), while charge solvation, involving CH and a deprotonated phosphate group, is present for (T20 + CH)(-3).