Magnetic relaxation switches capable of sensing molecular interactions

Highly sensitive, efficient, and high-throughput biosensors are required for genomic and proteomic data acquisition in complex biological samples and potentially for in vivo applications. To facilitate these studies, we have developed biocompatible magnetic nanosensors that act as magnetic relaxation switches (MRS) to detect molecular interactions in the reversible self-assembly of disperse magnetic particles into stable nanoassemblies. Using four different types of molecular interactions (DNA-DNA, protein-protein, protein-small molecule, and enzyme reactions) as model systems, we show that the MRS technology can be used to detect these interactions with high efficiency and sensitivity using magnetic relaxation measurements including magnetic resonance imaging (MRI). Furthermore, the magnetic changes are detectable in turbid media and in whole-cell lysates without protein purification. The developed magnetic nanosensors can be used in a variety of biological applications such as in homogeneous assays, as reagents in miniaturized microfluidic systems, as affinity ligands for rapid and high-throughput magnetic readouts of arrays, as probes for magnetic force microscopy, and potentially for in vivo imaging.     

Copolymers of ethylene imine and N-(2-hydroxyethyl)-ethylene imine as tools to study effects of polymer structure on physicochemical and biological properties of DNA complexes

A series of five poly[(ethylene imine)-co-N-(2-hydroxyethyl-ethylene imine)] copolymers with similar molecular weights and different degrees of branching was established to study structure-function relationship with regard to physicochemical and biological properties as gene delivery systems. Copolymers were synthesized by acid-catalyzed ring-opening copolymerization of aziridine and N-(2-hydroxyethyl)-aziridine in aqueous solution and characterized by GPC-MALLS, (1)H- and (13)C NMR, IR, potentiometric titration, and ion exchange chromatography. Complexation of DNA was determined by agarose gel electrophoresis, and complex sizes were quantitated by PCS. Cytotoxicity of the copolymers in fibroblasts was assessed by MTT-assay, LDH-assay, and hemolysis. The transfection efficiency was determined using the reporter plasmid pGL3 in 3T3 mouse fibroblasts. The copolymers obtained by solution polymerization had relatively low molecular weights of about 2000 Da, and the degree of branching increased with increasing ethylene imine ratio. The pK(a) as well as the buffer capacity increased proportional to the number of primary and secondary amines. Higher branched polymers showed stronger complexation and condensation of DNA, formed smaller polymer/DNA complexes, and induced the expression of plasmids to a higher extent than less branched polymers. In vitro cytotoxic effects and the hemolysis of erythrocytes decreased with decreased branching. Our results indicate that the basicity and degree of protonation of the polymers depending on their amount of primary and secondary amines seem to be important factors both for their transfection efficiency and for their cytotoxicity in gene transfer.     

Solution structures of staphylococcal nuclease from multidimensional, multinuclear NMR: Nuclease-H124L and its ternary complex with Ca2+ and thymidine-3′,5′-bisphosphate

The solution structures of staphylococcal nuclease (nuclease) H124L and itsternary complex, (nuclease-H124L)pdTpCa²⁺, were determinedby ab initio dynamic simulated annealing using 1925 NOE, 119 , 20¹ and 112 hydrogen bond constraints for the free protein,and 2003 NOE, 118 , 20 ¹ and 114 hydrogen bondconstraints for the ternary complex. In both cases, the final structuresdisplay only small deviations from idealized covalent geometry. In structuredregions, the overall root-mean-square deviations from mean atomic coordinatesare 0.46 (±0.05) Å and 0.41 (±0.05) Å for thebackbone heavy atoms of nuclease and its ternary complex, respectively. Thebackbone conformations of residues in the loop formed byArg⁸¹–Gly⁸⁶, which is adjacent to the activesite, are more precisely defined in the ternary complex than in unligatednuclease. Also, the protein side chains that show NOEs and evidence forhydrogen bonds to pdTp (Arg³⁵, Lys⁸⁴,Tyr⁸⁵, Arg⁸⁷, Tyr¹¹³, andTyr¹¹⁵) are better defined in the ternary complex. As has beenobserved previously in the X-ray structures of nuclease-WT, the binding ofpdTp causes the backbone of Tyr¹¹³ to change from an extendedto a left-handed -helical conformation. The NMR structures reportedhere were compared with available X-ray structures: nuclease-H124L [Truckseset al. (1996) Protein Sci., 5, 1907–1916] and the ternary complex ofwild-type staphylococcal nuclease [Loll and Lattman (1989) Proteins Struct.Funct. Genet., 5, 183–201]. Overall, the solution structures ofnuclease-H124L are consistent with these crystal structures, but smalldifferences were observed between the structures in the solution and crystalenvironments. These included differences in the conformations of certain sidechains, a reduction in the extent of helix 1 in solution, and many fewerhydrogen bonds involving side chains in solution.     

Consequences of Cis-amide Bond Simulation in Opioid Peptides

Summary Six analogs of leucine-enkephalin were synthesized in which a 1,5-disubstituted tetrazole ring was incorporated in order to lock selected peptide bonds in cis geometry. The obtained compounds were examined based on their biological effects in vivo and in vitro. Only one analog was completely inactive in binding assays being very weakly active in the antinociceptive test. The remaining five compounds displayed at least weak receptor affinity and in vivo activity.     

In vitro studies on the expansion of endothelial cell monolayers on components of the basement membrane

The purpose of the present study was to observe the expansion of a monolayer of endothelial cells over specific components of the basement membrane. This was performed in vitro in a monolayer expansion assay over 5 days. The control surface was uncoated glass in the form of coverslips. Test substances were coated at a concentration of 10 μg/ml. The highest expansion was obtained with a high molecular weight fragment mixture of collagen type IV (IV-F, consisting of 75, 120 and 140 KD fragments), followed by fibronectin. Collagens type I, III and IV tetramer gave similar results, less than fibronectin or collagen type IV-F, although all of the above basement membrane coatings promoted expansion significantly above that of the control (P<0.01). The poorest expansion was obtained with laminin, which was significantly less than the control. The pentapeptide GRGDS, related to the fibronectin cell binding region, gave expansion significantly below that of the intact fibronectin molecule, as did the intact collagen type IV molecule compared with type IV-F (P<0.025). This indicates that sequences of the fibronectin molecule other than the cell binding sequence may be involved in promoting endothelial cell expansion. In addition, the integrity of the collagen type IV molecule does not appear necessary for this effect. On the contrary, the higher movement on IV-F may represent an inherent repair mechanism in damaged endothelium. Autoradiographic studies show that endothelial cell proliferation at the expanding front is involved in the migration assay.     

Differences between ecological niches in juvenileSalix cinerea andS. pentandra

Comparison of growth rates ofSalix cinerea andS. pentandra seedlings in terrestric, limosal, and littoral ecophases showed different life strategies of juvenile stages of these willows. Several characters determining ecological niches of both species are summarized.     

A Piece of the Puzzle: The Bone Health Index of the BoneXpert Software Reflects Cortical Bone Mineral Density in Pediatric and Adolescent Patients

Introduction

Suspected osteopathology in chronically ill children often necessitates the assessment of bone mineral density. The most frequently used methods are dual-energy X-ray-absorption (DXA) and peripheral quantitative computed tomography (pQCT). The BoneXpert software provides an automated radiogrammatic method to assess skeletal age from digitalized X-rays of the left hand. Furthermore, the program calculates the Bone Health Index (BHI), a measure of cortical thickness and mineralization, which is obtained from indices of three metacarpal bones. In our study, we analyzed the manner in which BHI information provided by BoneXpert compares with DXA or pQCT measurements in youths.

Study Design

The BHI was retrospectively obtained using digitalized X-rays of the left hand and compared with the results of 203 corresponding DXA readings (Lunar Prodigy, GE Healthcare) of the lumbar vertebrae and femur as well as 117 pQCT readings (XCT 900, Stratec) of the distal radius.

Results

The BHI values showed a strong positive correlation with the DXA readings at each and all lumbar vertebrae (L1 –L4: r = 0.73; P < 0.0001). The age-adjusted Z-score of L1 –L4 and the height-adjusted score showed a positive correlation with the BHI-SDS (standard deviation score, r = 0.23; P < 0.002 and r = 0.27; P < 0.001, respectively). Total bone mineral density, as assessed via pQCT, also positively correlated with the BHI (r = 0.39; P < 0.0001), but the trabecular values displayed only a weak correlation.

Conclusions

The BHI obtained using BoneXpert can be a useful parameter in the assessment of bone health in children in most cases. This technique provides observer-independent information on cortical thickness and mineralization based on X-ray imaging of the hands.     

Amoxicillin-clavulanic acid and ciprofloxacin-treated SPF mice as gnotobiotic model

Applied Microbiology and Biotechnology - The experiment was carried out on 24 SPF BALB/c female mice and lasted for 15&nbsp;days with a 5-day antibiotic (ATB) treatment and then...