Native polyacrylamide electrophoresis in the presence of Ponceau Red to study oligomeric states of protein complexes

Native polyacrylamide electrophoresis in the presence of two reversible protein anionic stains (Ponceau S and Ponceau 2R) was used to study the oligomeric states of soluble proteins. A mild binding of the used protein stains to nondissociated protein oligomers imposed a charge shift on the proteins resulting into separation of protein species according to their size under physiological conditions. Adsorbed stains could be easily removed after electrophoresis by washing of polyacrylamide gel with buffer and protein complexes could be visualized either by the detection of their enzyme activity or by using a nonspecific protein stain. The specific detection of enzyme activity of glycosidases, lactate dehydrogenase, or phosphatases was shown as an example.     

The UHPLC-DAD fingerprinting method for analysis of extracellular metabolites of fungi of the genus <Emphasis Type="Italic">Geosmithia</Emphasis> (Acomycota: Hypocreales)

A new simple ultra-high-performance liquid chromatography method with diode array detection (UHPLC-DAD) was developed for chemical fingerprinting analysis of extracellular metabolites in fermentation broth of Geosmithia spp. The SPE method employing Oasis MCX strong cation-exchange mixed-mode polymeric sorbent was chosen for extraction of the metabolites. The analyses were performed on an Acquity UPLC BEH C18 column (100 × 2.1 mm i.d.; particle size, 1.7 μm; Waters) using a gradient elution program with an aqueous solution of trifluoroacetic acid and acetonitrile as the mobile phase. The applicability of the method was proved by analysis of 38 strains produced by different species and isolated from different sources (hosts). The results revealed the correlation of obtained UHPLC-DAD fingerprints with taxonomical identity.     

Finite-sample density and its small sample asymptotic approximation

To derive the exact density of a statistic, which can be intractable, is sometimes a difficult problem. The exact densities of estimates of the shift or regression parameters can be derived with the aid of score functions. Moreover, extremely accurate approximations can be obtained by the small sample asymptotics, based on the saddlepoint method. It is of interest to compare these two approaches, at least for small samples. We numerically compare the exact densities of estimates of the shift parameter with their small sample approximations for various parent distributions of the data. For some distributions both methods are in surprising concordance even under very small samples.     

A palladium-catalyzed three-component cross-coupling of conjugated dienes or terminal alkenes with vinyl triflates and boronic acids

A three-component coupling of vinyl triflates and boronic acids to alkenes catalyzed by palladium is reported. Using 1,3-dienes, selective 1,2-alkene difunction-alization is observed, whereas the use of terminal alkenes results in 1,1-alkene difunctionalization. The reaction outcome is attributed to the formation of stabilized, cationic Pd-π-allyl intermediates to regulate β-hydride elimination.     

Synthesis of monomeric Fe(II) and Ru(II) complexes of tetradentate phosphines

rac-Bis[{(diphenylphosphino)ethyl}-phenylphosphino]methane (DPPEPM) reacts with iron(II) and ruthenium(II) halides to generate complexes with folded DPPEPM coordination. The paramagnetic, five-coordinate Fe(DPPEPM)Cl(2) (1) in CD(2)Cl(2) features a tridentate binding mode as established by (31)P{(1)H} NMR spectroscopy. Crystal structure analysis of the analogous bromo complex, Fe(DPPEPM)Br(2) (2) revealed a pseudo-octahedral, cis-α geometry at iron with DPPEPM coordinated in a tetradentate fashion. However, in CD(2)Cl(2) solution, the coordination of DPPEPM in 2 is similar to that of 1 in that one of the external phosphorus atoms is dissociated resulting in a mixture of three tridentate complexes. The chloro ruthenium complex cis-Ru(κ(4)-DPPEPM)Cl(2) (3) is obtained from rac-DPPEPM and either [RuCl(2)(COD)](2) [COD = 1,5-cyclooctadiene] or RuCl(2)(PPh(3))(4). The structure of 3 in both the solid state and in CD(2)Cl(2) solution features a folded κ(4)-DPPEPM. This binding mode was also observed in cis-[Fe(κ(4)-DPPEPM)(CH(3)CN)(2)](CF(3)SO(3))(2) (4). Addition of an excess of CO to a methanolic solution of 1 results in the replacement of one of the chloride ions by CO to yield cis-[Fe(κ(4)-DPPEPM)Cl(CO)](Cl) (5). The same reaction in CH(2)Cl(2) produces a mixture of 5 and [Fe(κ(3)-DPPEPM)Cl(2)(CO)] (6) in which one of the internal phosphines has been substituted by CO. Complexes 2, 3, 4, and 5 appear to be the first structurally characterized monometallic complexes of κ(4)-DPPEPM.     

Debundling, selection and release of SWNTs using fluorene-based photocleavable polymers

Photocleavable polymers based on 9,9-dialkylfluorene backbone and o-nitrobenzylether were designed and synthesized to obtain stable (n,m) enriched suspensions of semiconducting SWNTs in toluene. Photoirradiation of the suspensions triggered the precipitation of the SWNTs and TEM images indicate close packing of SWNTs pointing at partial removal of the coating polymer.     

Pentafluoropyridine as a fluorinating reagent for preparing a hydrocarbon soluble β-diketiminatolead(II) monofluoride

A well-designed method for the preparation of a β-diketiminatolead(II) monofluoride has been developed using LPbNMe(2) (L = [CH{C(Me)(2,6-iPr(2)C(6)H(3)N)}(2)]) and pentafluoropyridine (C(5)F(5)N). The resulting LPbF was used for the synthesis of amidinatosilicon(II) monofluoride. Moreover the activation of a ketone was observed when the LPbF was treated with PhCOCF(3).     

Stigmasterol-based novel low molecular weight/mass organic gelators

Conjugates consisting of stigmasterol and L-phenylalanine, interconnected through short-chained dicarboxylic acyls by ester and amide bonds, respectively, were synthesized as potential low molecular weight/mass organic gelators (LMWGs/LMMGs). Their physico-chemical properties were subjected to investigation, especially their ability to form gels reversibly based on changes of the environmental conditions. Other self-assembly properties detectable by UV-VIS traces were measured in systems consisting of two miscible solvents (water/acetonitrile) with varying solvent ratios and using constant concentrations of the studied compounds. Partition and diffusion coefficients and solubility in water were calculated for the target conjugates. The conjugate 3a was the only compound from this series capable of forming a gel in 1-octanol. All three conjugates 3a-3c displayed supramolecular characteristics in the UV-VIS spectra.     

Application of colloidal palladium nanoparticles for labeling in electron microscopy

The application of palladium nanoparticles as electron-dense markers for labeling in both transmission and scanning electron microscopy requires their conjugation to a specific protein. The conjugation protocol described here includes the dihydrolipoic acid (DHLA) capping of Pd nanoparticles (8 nm equivalent diameter) and their subsequent covalent attachment to functional protein molecules such as streptavidin, protein A, or avidin. The single-step reaction was mediated using the cross-linking agent ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDC). The final Pd conjugates were fully functional, as demonstrated by labeling of ultrathin resin sections of either bovine serum albumin or secretory granules of the salivary gland isolated from the partially fed female Ixodes ricinus tick. The results of bovine serum labeling were quantified, statistically evaluated, and compared with results obtained using commercially available gold particle conjugates (10 nm diameter). The highest values of labeling density were achieved using both streptavidin-Pd (106 ± 7 particles/μm2) and protein A-Au conjugates (130 ± 18 particles/μm2) compared to a commercial streptavidin-Au (66 ± 16 particles/μm2) and protein A-Pd conjugates (70 ± 11 particles/μm2). The concentrations of both DHLA and EDC, pH during conjugation, and finally thorough washing away of unbound proteins crucially influenced conjugation.