Confocal Fluorescence Recovery After Photobleaching of Green Fluorescent Protein in Solution

Fluorescence recovery after photobleaching (FRAP) is one of the most widely used approaches to quantitatively estimate diffusion characteristics of molecules in solution and cellular systems. In general, comparison of the diffusion times (t _1/2) from a FRAP experiment provides qualitative estimates of diffusion rates. However, obtaining consistent and reliable quantitative estimates of mobility of molecules using FRAP is hindered by the lack of appropriate standards for calibrating the FRAP set-up (microscope configuration and data fitting algorithms) used in a given experiment. In comparison with other fluorescent markers, the green fluorescent proteins (GFP) possess characteristics that are ideal for use in such experiments. We have monitored the mobility of pure enhanced green fluorescent protein (EGFP) in a viscous solution by confocal FRAP experiments. Our experimentally determined diffusion coefficient of EGFP in a glycerol–water mixture is in excellent agreement with the value predicted for GFP in a solution of comparable viscosity, calculated using the Stokes–Einstein equation. The agreement in the experimentally determined diffusion coefficient and that predicted from theoretical framework improves significantly when one takes into account the effective size of the bleached spot in such experiments. Our results therefore validate the use of GFP as a convenient standard for FRAP experiments. Importantly, we present a simple method to correct for artifacts in the accurate determination of diffusion coefficient of molecules measured using FRAP arising due to the underestimation in the effective size of the bleached spot.     

Five-coordinate aluminum bromides: synthesis, structure, cation formation, and cleavage of phosphate ester bonds

The alkane elimination reaction between Salen((t)Bu)H(2) ligands and diethylaluminum bromide was used to prepare three Salen aluminum bromide compounds salen((t)Bu)AlBr (1) (salen = N,N'-ethylenebis(3,5-di-tert-butylsalicylideneimine)), salpen((t)Bu)AlBr (2) (salpen = N,N'-propylenebis(3,5-di-tert-butylsalicylideneimine)), and salophen((t)Bu)AlBr (3) (salophen = N,N'-o-phenylenenebis(3,5-di-tert-butylsalicylideneimine)). The compounds contain five-coordinate aluminum either in a distorted square pyramidal or a trigonal bipyramidal environment. The bromide group in these compounds could be displaced by triphenylphosphine oxide or triphenyl phosphate to produce the six-coordinate cationic aluminum compounds [salen((t)Bu)Al(Ph(3)PO)(2)]Br (4), [salpen((t)Bu)Al(Ph(3)PO)(2)]Br (5), [salophen((t)Bu)Al(Ph(3)PO)(2)]Br (6), and [salophen((t)Bu)Al[(PhO)(3)PO)](2)]Br (7). All the compounds were characterized by (1)H, (13)C, (27)Al, and (31)P NMR, IR, mass spectrometry, and melting point. Furthermore, compounds 1-3 and 5-7 were structurally characterized by single-crystal X-ray diffraction. Compounds 1-3 dealkylated a series of organophosphates in stoichiometric reactions by breaking the ester C-O bond. Also, they were catalytic in the dealkylation reaction between trimethyl phosphate and added boron tribromide.     

Aluminum phosphinate and phosphates of salen ligands

A new dealkylation reaction between organophosphate esters and Salen aluminum bromide compounds has been used to prepare three new aluminum salen compounds salen((t)Bu)AlOP(O)Ph2 (1) (salen = N,N'-ethylenebis(3,5-di-tert-butylsalicylideneimine)), [(MeOH)Alsalen((t)Bu)[OMePO2(O)]Alsalen((t)Bu)[OMePO2(O)]Alsalen((t)Bu)]Br (2), and [salpen((t)Bu)AlO]2[(BuO)2PO]2 (3) (salpen = N,N'-propylenebis(3,5-di-tert-butylsalicylideneimine)). Compounds 1.MeOH, 2, and 3 were characterized by single-crystal X-ray diffraction. Compound 1 is the first example of a monomeric aluminum Schiff base phosphinate. Compound 2 is a cationic Salen aluminum phosphate, and compound 3 contains an aluminophosphate ring. This work is the first example of the intentional use of an aluminum-based dealkylation reaction to form new compounds.     

Assessment of radioactivity associated with a low ore grade open cast mine at Banduhurang, Jharkhand, India and estimation of occupational exposure to the miners

The study summarizes radiological characteristics of Banduhurang open cast mine which includes qualitative and quantitative behavior of ²²²Rn concentration, external gamma radiation level over the mine pit as well as in its adjoining environment, long-lived alpha (LLα) activity concentration associated with the respirable size of ore dust and assessment of dose to the mine workers in 2006–2008. The investigations reveal that geometric means (χ_g) of measured radon concentration were 36.39, 38.69, 26.64 and 24 Bq m^?3 with respective geometric standard deviations (σ_g) were 1.52, 1.55, 1.36 and 1.68 Bq m^?3 and χ_g of gamma absorbed dose rates were 0.54, 0.64, 0. 45 and 0.15 μGy h^?1 with respective σ_g were 1.63, 1.53, 1.52 and 1.72 μGy h^?1 over the mine pit, ore yard, waste yard and in the surrounding environment within a 10 km radius to the mine, respectively. The χ_g of LLα activity was observed to be 16 mBq m^?3 with σ_g of 1.9 mBq m^?3. The annual mean effective dose equivalent received by the member radiation workers of Banduhurang mine was estimated to 1.41 mSv y^?1, which is about 7% of the prescribed dose limits of 20 mSv y^?1.     

Purification and Characterization of a Novel Heparin Degrading Enzyme from <Emphasis Type="Italic">Aspergillus flavus</Emphasis> (MTCC-8654)

A heparinase-producing fungus was isolated, and the strain was taxonomically characterized as Aspergillus flavus by morphophysiological and 26S rRNA gene homology studies. The culture produced intracellular heparinase enzyme, which was purified 40.5-fold by DEAE-Sephadex A-50, CM-Sephadex C-50, and Sephadex G-100 column chromatography. Specific activity of the purified enzyme was found to be 44.6 IU/μg protein and the molecular weight of native as well as reduced heparinase was 24 kDa, showing a monomeric unit structure. Peptide mass spectrum showed poor homogeneity with the database in the peptide bank. The enzyme activity was maximum at 30 °C in the presence of 300 mM NaCl at pH 7.0. In the presence of Co²⁺, Mn²⁺ ions, and reducing agents (β-mercaptoethanol, dithiothreitol), enzyme activity was enhanced and inhibited by iodoacetic acid. These observations suggested that free sulfohydryl groups of cysteine residues were necessary for catalytic activity of the enzyme. The enzyme was also inhibited by histidine modifier, DEPC, which suggests that along with cysteine, histidine may be present at its active site. The enzyme showed a high affinity for heparin as a substrate with K _m and V _max as 2.2 × 10⁻⁵ M and 30.8 mM min⁻¹, respectively. The affinity of the enzyme for different glycosaminoglycans studied varied, with high substrate specificity toward heparin and heparin-derived polysaccharides. Depolymerization of heparin and fractionation of the oligosaccharides yielded heparin disaccharides as main product.     

A Bis(silaselenone) with Two Donor‐Stabilized SiSe Bonds from an Unexpected Stereoconvergent Hydrolysis of a Diselenadisiletane

SiSe matters : Diselenadisiletane 2 , formed from direct reaction of a racemic silylene 1 with elemental selenium, gives the first bis(silaselenone) upon hydrolysis with water ( 3 ; see picture, C gray, H white, N blue, O red, Se purple, Si green; d(Si?Se)=215 pm). The reaction is stereoconvergent: only racemic forms of 3 are obtained from a mixture of racemic and meso forms of 2 .

     

Preparation and nonlinear characterization of zinc selenide nanoparticles embedded in polymer matrix

Nanocomposites of ZnSe nanoparticles embedded in polyvinyl alcohol (PVA) matrix have been prepared by in-situ synthesis. ZnSe/PVA nanocomposites are characterized by X-ray diffraction (XRD), Transmission electron microscopy (TEM), Fourier transform infrared spectroscopy (FTIR) and UV/Vis spectra. The nanocomposite structure is confirmed by the blue-shift of the absorption edge. The nonlinear refractive index and two-photon absorption (TPA) coefficient are measured by the Z-scan technique using low power CW He–Ne laser light. The results show that the ZnSe nanocomposite films show large optical nonlinearity and the magnitude of the third-order nonlinear susceptibility χ⁽³⁾ is calculated to be 2.62×13^?11 m²/V².