Probing fibronectin-surface interactions: a multitechnique approach

The development of adhesive as well as antiadhesive surfaces is essential in various biomaterial applications. In this study, we have used a multidisciplinary approach that combines biological and physicochemical methods to progress in our understanding of cell-surface interactions. Four model surfaces have been used to investigate fibronectin (Fn) adsorption and the subsequent morphology and adhesion of preosteoblasts. Such experimental conditions lead us to distinguish between anti- and proadhesive substrata. Our results indicate that Fn is not able to induce cell adhesion on antiadhesive materials. On adhesive substrata, Fn did not increase the number of adherent cells but favored their spreading. This work also examined Fn-surface interactions using ELISA immunoassays, fluorescent labeling of Fn, and force spectroscopy with Fn-modified tips. The results provided clear evidence of the advantages and limitations of each technique. All of the techniques confirmed the important adsorption of Fn on proadhesive surfaces for cells. By contrast, antiadhesive substrata for cells avoided Fn adsorption. Furthermore, ELISA experiments enabled us to verify the accessibility of cell binding sites to adsorbed Fn molecules.     

An Improved Enzyme Assay for Molybdenum-Reducing Activity in Bacteria

Molybdenum-reducing activity in the heterotrophic bacteria is a phenomenon that has been reported for more than 100 years. In the presence of molybdenum in the growth media, bacterial colonies turn to blue. The enzyme(s) responsible for the reduction of molybdenum to molybdenum blue in these bacteria has never been purified. In our quest to purify the molybdenum-reducing enzyme, we have devised a better substrate for the enzyme activity using laboratory-prepared phosphomolybdate instead of the commercial 12-phosphomolybdate we developed previously. Using laboratory-prepared phosphomolybdate, the highest activity is given by 10:4-phosphomolybdate. The apparent Michaelis constant, K _m for the laboratory-prepared 10:4-phosphomolybdate is 2.56 ± 0.25 mM (arbitrary concentration), whereas the apparent V _max is 99.4 ± 2.85 nmol Mo-blue min⁻¹ mg⁻¹ protein. The apparent Michaelis constant or K _m for NADH as the electron donor is 1.38 ± 0.09 mM, whereas the apparent V _max is 102.6 ± 1.73 nmol Mo-blue min⁻¹ mg⁻¹ protein. The apparent K _m and V _max for another electron donor, NADPH, is 1.43 ± 0.10 mM and 57.16 ± 1.01 nmol Mo-blue min⁻¹ mg⁻¹ protein, respectively, using the same batch of molybdenum-reducing enzyme. The apparent V _max obtained for NADH and 10:4-phosphomolybdate is approximately 13 times better than 12-phoshomolybdate using the same batch of enzyme, and hence, the laboratory-prepared phosphomolybdate is a much better substrate than 12-phoshomolybdate. In addition, 10:4-phosphomolybdate can be routinely prepared from phosphate and molybdate, two common chemicals in the laboratory.     

Purification of pediocin PA-1 by immunoaffinity chromatography

A one-step purification procedure for purifying pediocin PA-1, a class IIa bacteriocin produced by Pediococcus acidilactici UL5, was developed based on column immunoaffinity chromatography with specific antipediocin PA-1 polyclonal antibodies coupled to cyanogen bromide-activated SepharoseTM. About 13.3 microg/mL purified pediocin PA-1 was obtained from 15 mL P. acidilactici UL5 culture supernatant, as measured by enzyme-linked immunosorbent assay. The specific activity and average recovery of the eluted pediocin PA-1 were about 6602 AU/mg and 53.3%, respectively. This is the first report of successful purification of pediocin PA-1 by immunoaffinity using pediocin PA-1-specific polyclonal antibodies.     

Determination of etofibrate, fenofibrate, and atorvastatin in pharmaceutical preparations and plasma using differential pulse polarographic and square wave voltammetric techniques

Etofibrate, fenofibrate, and atorvastatin were determined in their pharmaceutical preparations and human plasma using differential pulse polarographic and square wave voltammetric techniques by reduction at a dropping-mercury working electrode versus Ag/AgCl reference electrode. The reversibility of the electrode reactions was tested using cyclic voltammetry, and they were found to be irreversible reduction reactions. Optimum conditions such as pH, scan rate, and pulse amplitude were studied, and validation of the proposed methods was performed. The proposed methods proved to be accurate, precise, robust, and specific for determination of the 3 drugs. The relative standard deviation values were <2%, indicating that these methods are precise. Limits of detection and quantitation were in the ranges of 0.037-0.21 and 0.12-0.71 microg/mL, respectively, indicating high sensitivity.     

Differentiation of methylenedioxybenzylpiperazines and ethoxybenzylpiperazines by GC-IRD and GC-MS

The substituted benzylpiperazines, 3,4-methylenedioxybenzylpiperazine, its regioisomer 2,3-methylenedioxybenzylpiperazine and three isobaric ring substituted ethoxybenzylpiperazines have equal mass and many common mass spectral fragment ions. The mass spectra of the three ethoxybenzylpiperazines yield a unique fragment at m/z 107 that allows the discrimination of the three ring substituted ethoxybenzylpiperazines from the two methylenedioxy isomers. Perfluoroacylation of the secondary amine nitrogen of these isomeric piperazines gave mass spectra with differences in relative abundance of some fragment ions, but acylation does not alter the fragmentation pathway and did not provide additional MS fragments of discrimination among these isomers. Gas chromatography coupled with infrared detection provides direct confirmatory data for the structural differentiation between the five isomers. The mass spectra in combination with the vapor phase infrared spectra provide for specific confirmation of each of the isomeric piperazines. The perfluoroacyl derivatives of the ring substituted benzylpiperazines were resolved on a stationary phase of 50% phenyl and 50% methylpolysiloxane. Gas chromatography coupled with time-of-flight mass spectrometric detection provides an additional means of differentiating between the isobaric compounds 3,4-methylenedioxybenzylpiperazine and 4-ethoxybenzylpiperazine, which have similar nominal masses but are different in their calculated exact masses.     

Transient absorption induced by a picosecond electron pulse in the fused silica windows of an optical cell

The transient absorption induced by picosecond pulse radiolysis in the windows of a fused silica optical cell is investigated with pump probe techniques in the UV and the visible range. After excitation with an electron pulse of 7 MeV and an effective duration of around 10 ps the absorbance changes during relaxation are recorded up to nanoseconds with a supercontinuum and a single wavelength probe at 263 nm. The complex spectral signatures and kinetics of the empty cell are set into relation with the transient absorption of water radiolysis. Special care is taken to assure equal irradiation conditions for the comparative measurements over the large spectral range. The results reveal clearly that the transient absorption induced in the fused silica cell is not negligible. The transient signals due to the cell should be considered in picosecond pulse radiolysis of solutions in order to avoid important errors on the time dependent yield of transient species, particularly of those absorbing in the UV.     

Synthesis and anti‐HIV activity of new 2‐thiolumazine and 2‐thiouracil metal complexes

A series of new Cu(II), Pt(II), VO(II), Fe(II), and Co(II) complexes ( 1‐‐5 ) with 3‐methyl‐6,7‐diphenyllumazine are described. Similarly, complexes from 2‐thiouracil with Cu(II) ( 6,7 ) and Pt(II) ( 8 ) have been prepared and characterized by spectroscopic methods. All the complexes were assayed for their anti‐HIV‐1 and HIV‐2 activity by examination of their inhibition of HIV‐induced cytopathogenicity in MT‐4 cells. Compound 3 was found to be the most active inhibitor against HIV‐2 in cell culture (EC₅₀ = >18.9 μ g/mL, selectivity index (SI) = 3), which provided a good lead for further optimization. Compounds 6 and 7 exhibited some activity (EC₅₀ = >7.12 μ g/mL and >2.23 μ g/mL) against HIV‐1 and HIV‐2, but no selectivity was observed (SI <1). © 2010 Wiley Periodicals, Inc. Heteroatom Chem 22:44–50, 2011; View this article online at wileyonlinelibrary.com . DOI 10.1002/hc.20654     

Synthesis of Cyclic Polyamines by Enzymatic Generation of an Amino Aldehyde In Situ

Multifunctional polycationic polyamines, for example, used in drug and gene delivery, have product range limitations in their synthesis methods. Here, we synthesize a polyamine by forming a self‐assembling amino aldehyde from the corresponding amino alcohol with horse liver alcohol dehydrogenase (HLADH), followed by reduction. Circular polyamines were synthesized from 3‐amino‐propan‐1‐ol as starting material, analogous to cyclic polyamines formed from azetidin. The product had an isolated yield of 89.7% or 15.3 g L⁻¹. The predicted range of possible polyamine products by this method is broad since many amino alcohols are putative substrates for HLADH. The enzyme also had activity for 2‐amino‐propan‐1‐ol and 2‐amino‐2‐phenyl‐ethanol, for which the enantioselectivity was 330 (S) and 32 (R), respectively.