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Isothermal titration calorimetry (ITC) is used for studying the thermodynamics of binding of 12 biphosphonates to human bone. The thermodynamic group approach and a linear regression method are used for predicting the [DELTA]G of binding of all 12 compounds, opening a way to designing novel chemotherapy, immunotherapy and anti-infectious disease drugs having weak bone binding affinity.

A functional angiotensin II receptor-GFP fusion protein: evidence for agonist-dependent nuclear translocation. Am J Physiol Renal Physiol 279: F440-F448, 2000.--We constructed an expression vector for a fusion protein [ANG II type la receptor-green fluorescent protein ([AT.sub.1a]R-GFP)] consisting of enhanced GFP attached to the COOH terminus of the rat [AT.sub.1a]R. Chinese hamster ovary (CHO) cells transfected with [AT.sub.1a]R-GFP demonstrated specific, high-affinity [sup.125]I-labeled ANG II binding ([IC.sub.50] 21 nM). ANG II exposure stimulated sodium-proton exchange and cytoplasmic calcium release to a similar extent in cells transfected with [AT.sub.1a]R or [AT.sub.1a]R-GFP; these responses were desensitized by prior exposure to ANG II and were sensitive to the AT1R blocker losartan. ANG II-driven internalization of [AT.sub.1a]R-GFP in transfected CHO cells was demonstrated both by radioligand binding and by laser scanning confocal microscopy. Colocalization of GFP fluorescence with that of the nuclear stain TOTO-3 in confocal images was increased more than twofold after 1 h of ANG II exposure. We conclude that [AT.sub.1a]R-GFP exhibits similar pharmacological behavior to that of the native [AT.sub.1a]R. Our observations also support previous evidence for the presence of [AT.sub.1a]R in the nucleus and suggest that the density of [AT.sub.1a]R in the nucleus may be regulated by exposure to its ligand. green fluorescent protein; losartan; confocal microscopy; internalization

Transglutaminase-mediated fibronectin multimerization in lung endothelial matrix in response to TNF-[Alpha]. Am J Physiol Lung Cell Mol Physio1279: L161-L174, 2000.--Exposure of lung endothelial monolayers to tumor necrosis factor (TNF)-[Alpha] causes a rearrangement of the fibrillar fibronectin (FN) extracellular matrix and an increase in protein permeability. Using calf pulmonary artery endothelial cell layers, we determined whether these changes were mediated by FN multimerization due to enhanced transglutaminase activity after TNF-[Alpha] (200 U/ml for 18 h. Western blot analysis indicated that TNF-[Alpha] decreased the amount of monomeric FN detected under reducing conditions. Analysis of [sup.125]I-FN incorporation into the extracellular matrix confirmed a twofold increase in high molecular mass (HMW) FN multimers stable under reducing conditions (P [is less than] 0.05). Ehhanced formation of such HMW FN multimers was associated with increased cell surface transglutaminase activity (P [is less than] 0.05). Calf pulmonary artery endothelial cells pretreated with TNF-[Alpha] also formed nonreducible HMW multimers of FN when layered on surfaces precoated with FN. Inhibitors of transglutaminase blocked the TNF-[Alpha]-induced formation of nonreducible HMW multimers of FN but did not prevent either disruption of the FN matrix or the increase in monolayer permeability. Thus increased cell surface transglutaminase after TNF-[Alpha] exposure initiates the enhanced formation of nonreducible HMW FN multimers but did not cause either the disruption of the FN matrix or the increase in endothelial monolayer permeability. tumor necrosis factor-[Alpha]; endothelial cells

The synthesis is reported of a newseries of oligo(aryleneethynylene)(OAE) derivatives of up to ca. 6 nm in molecular length (OAE9) using iterative Pd-mediated Sonogashira cross-coupling methodology.The oligo-p-phenyleneethynylene cores of the molecularwires are functionalized at both termini with pyridyl units for attachmentto gold leads. The molecular structures determined by single-crystalX-ray analysis are reported for OAE4, OAE5, OAE7, and OAE8a. The charge transportcharacteristics of derivatives OAE3–OAE9in single-molecular junctions have been studied using the mechanicallycontrolled break junction technique. The data demonstrate that thejunction conductance decreases with increasing molecular length. Atransition from coherent transport via tunneling to a hopping mechanismis found for OAE wires longer than ca. 3 nm. [ABSTRACT FROM AUTHOR]

Polythiophene (Pth) bilayer films each consists of a nanotube layer and a compact layer had been prepared by electrochemical polymerization and using anodic aluminum oxide (AAO) membranes as template. The films were treated by mechanical polishing and freeze drying to improve the degree of orientation of nanotubes and their surface smoothness. A treated Pth bilayer film can adhere strongly on glass with its nanotube surface by applying a dragging preload. The applied dragging preload caused the original vertically aligned Pth nanotube array to tilt opposite to the direction of preload, which induced a superstrong shear adhesion force of up to 144 ± 30 N cm−2 for the film with a tube length of 26 μm as the film was dragged along the direction of preload. However, the shear adhesion force opposite to the direction of dragging preload was relatively low (44 ± 17 N cm−2). The direction-dependent force management of Pth bilayer films is quite similar to that gecko does, indicating the potential for developing advanced adhesive systems. © 2011 Wiley Periodicals, Inc. J Appl Polym Sci, 2012 [ABSTRACT FROM AUTHOR]

We report the results of a series of chemical, EPR, ENDOR, and HYSCORE spectroscopic investigations of the mechanism of action (and inhibition) of GcpE, E-1-hydroxy-2-methyl-but-2-enyl-4-diphosphate (HMBPP) synthase, also known as IspG, an Fe4S4 cluster-containing protein. We find that the epoxide of HMBPP when reduced by GcpE generates the same transient EPR species as observed on addition of the substrate, 2-C-methyl-D-erythritol-2, 4-cyclo-diphosphate. ENDOR and HYSCORE spectra of these transient species (using 2H, 13C and 17O labeled samples) indicate formation of an Fe-C-H containing organometallic intermediate, most likely a ferraoxetane. This is then rapidly reduced to a ferracyclopropane in which the HMBPP product forms an η2-alkenyl π- (or π/σ) complex with the 4th Fe in the Fe4S4 cluster, and a similar "metallacycle" also forms between isopentenyl diphosphate (IPP) and GcpE. Based on this metallacycle concept, we show that an alkyne (propargyl) diphosphate is a good (Ki ~ 300 nM) GcpE inhibitor, and supported again by EPR and ENDOR results (a 13C hyperfine coupling of ~7 MHz), as well as literature precedent, we propose that the alkyne forms another π/σ metallacycle, an η2-alkynyl, or ferracyclopropene. Overall, the results are of broad general interest because they provide new mechanistic insights into GcpE catalysis and inhibition, with organometallic bond formation playing, in both cases, a key role. [ABSTRACT FROM AUTHOR]

Smad7, a protein induced by transforming growth factor-beta1 (TGF-beta1) in many target cells, inhibits TGF-beta1 signal transduction and is thought to mediate an intracellular negative feedback response that limits TGF-beta1 effects. It is possible that overexpression of Smad7 could block specified effects of TGF-beta1 on mesangial cells, a TGF-beta target in glomerular disease. Smad7 mRNA was induced by TGF-beta1 within 1 h in a concentration-dependent manner in a transformed mouse mesangial cell (MMC) line. Uptake of (14)C-spermidine from the medium by MMC and the transcriptional activity of a segment of the human collagen pro-alpha2 type 1 chain (COL1A2) promoter fused to a luciferase reporter gene were used as indices of TGF-beta1. Treatment with TGF-beta1 increased (14)C-spermidine uptake rate in a time-, concentration-, and temperature-dependent manner. For example, exposure to 1 ng/ml TGF-beta1 for 15 h increased uptake approximately twofold, a response that was attenuated by cycloheximide. Transfection of Smad7 expression vector into MMC abrogated both TGF-beta1-dependent stimulation of spermidine uptake and COL1A2 promoter activity. It is concluded that: (1) TGF-beta1 induces Smad7 in MMC; (2) (14)C-spermidine uptake is a convenient quantitative index of TGF-beta1 effect in these cells; and (3) overexpression of Smad7 is a highly effective method of blocking at least some mesangial cell effects of TGF-beta1 that may warrant evaluation in vivo in experimental glomerular disease.