Site-specific amide hydrogen/deuterium exchange in E. coli thioredoxins measured by electrospray ionization mass spectrometry

Mass spectrometry as an analytical tool to study protein folding and structure by hydrogen/deuterium exchange is a relatively new approach. In this study, site-specific amide deuterium content was measured in oxidized and reduced E. coli thioredoxins by using the b(n) ions in electrospray ionization CID MS/MS experiments after 20-s incubation in D(2)O phosphate-buffered solution (pH 5.7). The deuterium levels correlated well with reported NMR-determined H/D exchange rate constants. The deuterium measured by y(n) ions, however, showed much less reliable correlation with rate exchange data. In general, residues in alpha helices and beta sheets, when measured by b(n) ions, showed low incorporation of deuterium while loops and turns had high deuterium levels. Most amide sites in the two protein forms showed similar deuterium levels consistent with the expected similarity of their structures, but there were some differences. The turn consisting of residues 18-22 in particular showed more variability in deuterium content consistent with reported structural differences in the two forms. The deuterium uptake by thioredoxins alkylated at Cys-32 by S-(2-chloroethyl)glutathione and S-(2-chloroethyl)cysteine, in peptides 1-24 and 45-58, was similar to that observed for oxidized and reduced thioredoxins, but several residues, particularly Leu-53 and Thr-54, showed slightly elevated deuterium levels, suggesting that structural changes had occurred from alkylation of the protein at Cys-32. It is concluded that b(n) ions are reliable for determining the extent of site-specific amide hydrogen isotope exchange and that mass spectrometry is useful as a complementary technique to NMR and other analytical methods for probing regional structural characteristics of proteins.     

Fractionally colouring total graphs

Bchzad and Vizing have conjectured that given any simple graph of maximum degree , one can colour its edges and vertices with +2 colours so that no two adjacent vertices, or two incident edges, or an edge and either of its ends receive the same colour. We show that for any simple graphG, V(G)E(G) can be fractionally coloured with +2 colours.     

The complexity of central series in nilpotent computable groups

The terms of the upper and lower central series of a nilpotent computable group have computably enumerable Turing degree. We show that the Turing degrees of these terms are independent even when restricted to groups which admit computable orders.     

Prospects of anionic nanolipoplexes in nanotherapy: transmission electron microscopy and light scattering studies

Currently nanosystems composed of polynucleotides and lipid vesicles (nanolipoplexes) are considered to be promising tools for gene therapeutics. Successful in vivo application of these vectors depends on their physicochemical, technological and biological characteristics including morphology, size distribution, molecular interactions and stability. Anionic nanoliposomes (DPPC:DCP:CHOL) were prepared by two different techniques, namely the conventional thin-film hydration method followed by extrusion, and the heating method (HM), in which no volatile solvent or detergent is used. A non-viral and non-cationic gene transfer vector was constructed by incorporating plasmid DNA (pcDNA3.1/His B/lacZ) to the HM-nanoliposomes by the electrostatic mediation of Ca²⁺ ions. Transfection efficiency of the nanolipoplexes was evaluated using a human bronchial epithelial cell line (16HBE14o-) in the presence of serum. Particle characterisation, stability of the formulations and lipid–DNA interaction studies were performed using transmission electron microscopy (TEM) and light scattering. TEM pictures of nanolipoplexes showed presence of two to four closely packed vesicles with signs of fusion. Efficient delivery of plasmid DNA and subsequent β-galactosidase expression was achieved using the anionic nanolipoplexes. Transfection efficiency increased with lipid:DNA ratio up to 7:1 (w/w), where transfection efficiency was 12-fold higher than in untreated cells. Further increase in lipid ratio decreased transfection. These nanolipoplexes appear to be safe, stable and efficient in the protection and delivery of DNA to different cells and tissues.     

Validity and Reliability of the Reflux Symptom Index (RSI)

Laryngopharyngeal reflux (LPR) is present in up to 50% of patients with voice disorders. Currently, there is no validated instrument that documents symptom severity in LPR. We developed the reflux symptom index (RSI), a self-administered nine-item outcomes instrument for LPR. The purpose of this investigation was to evaluate the psychometric properties of the RSI. For validity assessment, 25 patients with LPR were evaluated prospectively before and six months after b.i.d. treatment with proton pump inhibitors (PPI). Each patient completed the RSI as well as the 30-item voice handicap index (VHI). For reliability assessment, the study patients were given the RSI on two separate occasions before the initiation of treatment. Normative RSI data were derived from 25 age-matched and gender-matched controls taken from an existing database of asymptomatic individuals without any evidence of LPR. The mean RSI (+/- standard deviation) of patients with LPR improved from 21.2 (+/- 10.7) to 12.8 (+/- 10.0), and the mean VHI improved from 52.2 (+/- 24.7) to 41.5 (+/- 25.0) after 6 months of therapy (p = 0.001 and 0.065, respectively). Of the three VHI subscales (emotional, physical, functional), only the functional subscale improved significantly (p = 0.037). Patients who experienced a five point or better improvement in RSI were 11 times more likely to experience a five-point improvement in VHI (95% confidence interval = 1.7, 76.8). For reliability assessment, the first and second pretreatment RSIs were 19.9 (+/- 11.1) and 20.9 (+/- 9.6), respectively (correlation coefficient = 0.81, p < 0.001). The single-item correlation coefficients ranged from 0.41 to 0.91 (p < 0.05 for all items). The mean pretreatment RSI in patients with LPR was significantly higher than controls (21.2 versus 11.6; p < 0.001). The mean RSI of patients with LPR after 6 months of PPI therapy approached that of asymptomatic controls (p > 0.05). The RSI is easily administered, highly reproducible, and exhibits excellent construct and criterion-based validity.     

Cold acclimation improves recovery of cryopreserved grass (Zoysia and Lolium sp.)

Cold acclimation of Lolium L. and Zoysia Willd. Grass cultivars significantly increased regrowth of cryopreserved meristems. One wk of cold acclimation improved recovery following cryopreservation but extended acclimation (4-8 wk) resulted in the best regrowth. Cold acclimation also significantly increased the dehydration tolerance of both Zoysia and Lolium meristems. Lolium apices cold acclimated for 4 wk produced 60-100% regrowth following cryopreservation by slow freezing or encapsulation-dehydration. Cold-acclimated Zoysia had greater than 60% regrowth following encapsulation-dehydration when beads were dehydrated to less than 22% water content. Non-acclimated meristems of both genera had little or no regrowth. Thawed meristems grew quickly without callus formation and the plantlets produced were transplanted to pots in the greenhouse after 4 to 6 wk. Samples of each cultivar were stored in liquid nitrogen as part of the U.S. National Plant Germplasm System.     

Directly detected Mn MRI: Application to phantoms for human hyperpolarized C MRI development

In this work we demonstrate for the first time directly detected manganese-55 (⁵⁵Mn) magnetic resonance imaging (MRI) using a clinical 3 T MRI scanner designed for human hyperpolarized ¹³C clinical studies with no additional hardware modifications. Due to the similar frequency of the ⁵⁵Mn and ¹³C resonances, the use of aqueous permanganate for large, signal-dense, and cost-effective “¹³C” MRI phantoms was investigated, addressing the clear need for new phantoms for these studies. Due to 100% natural abundance, higher intrinsic sensitivity, and favorable relaxation properties, ⁵⁵Mn MRI of aqueous permanganate demonstrates dramatically increased sensitivity over typical ¹³C phantom MRI, at greatly reduced cost as compared with large ¹³C-enriched phantoms. A large sensitivity advantage (22-fold) was demonstrated. A cylindrical phantom (d = 8 cm) containing concentrated aqueous sodium permanganate (2.7 M) was scanned rapidly by ⁵⁵Mn MRI in a human head coil tuned for ¹³C, using a balanced steady state free precession acquisition. The requisite penetration of radiofrequency magnetic fields into concentrated permanganate was investigated by experiments and high frequency electromagnetic simulations, and found to be sufficient for ⁵⁵Mn MRI with reasonably sized phantoms. A sub-second slice-selective acquisition yielded mean image signal-to-noise ratio of ~ 60 at 0.5 cm³ spatial resolution, distributed with minimum central signal ~ 40% of the maximum edge signal. We anticipate that permanganate phantoms will be very useful for testing HP ¹³C coils and methods designed for human studies.     

A modeling concept of a mechanical system having a piecewise linear spring property for its diagnosis

Machine condition monitoring and fault diagnosis of rotating machinery are very important because of the wide use of rotating machinery in industry. Couplings and gears are used in many mechanical systems to connect elements and transmit power. The system is usually modeled as a single-degree-of-freedom system with a piecewise linear spring property, where the mass of main machine is only considered. In the present study, the dynamic behavior of a system with an unsymmetrical nonlinearity and a significant mass of the connecting part was investigated both experimentally and by numerical simulation. In the experiment, a 1/3 sub-harmonic oscillation was observed, but this oscillation was not found in the simulation using a single-degree-of-freedom system, in which the mass of the connecting part was ignored. However, when a two-degrees-of-freedom system was used that considered both the mass of the connecting part and the impact property, the 1/3 sub-harmonic oscillation was observed. Thus it is recognized that an adequate mathematical model for diagnosis in the early stage of abnormality must be selected on the basis of the mass ratio between the connecting part and the main body.