Recovery of intact proteins from silver stained gels

Silver stained proteins of a wide molecular weight (MW) range (20-116 kDa) were successfully recovered by both electroblot and electroelution. The recovery was demonstrated for nanogram loads of proteins separated by SDS-PAGE and visualized by silver staining methods compatible and incompatible with mass spectrometry (MS). It was shown that the alcohol/acid and glutaraldehyde fixation steps present in a number of staining procedures did not prevent recovery of intact proteins from gels. It was found that the recovery of intact proteins from silver stained gels was substantially increased upon pre-equilibration in a buffer containing the reducing agent, dithiothreitol (DTT). The effect of destaining on the recovery of silver stained proteins was also investigated. Comparable recovery of intact proteins within a wide MW range from silver stained gels with and without destaining step was demonstrated. Recovery of model proteins from gels visualized using silver staining method compatible with MS showed 52 to 76% yield of that from the unstained gel, depending upon method of the transfer. Comparison of the recovery of intact proteins from gels visualized using other staining procedures was also made. The above findings have implications as to the supposed irreversible nature of protein "fixation" inside polyacrylamide matrix, and confirm lack of binding of proteins in the gel to metal silver deposited on its surface. This method has the potential to be suitable for direct characterization of proteins by matrix-assisted laser desorption ionization-mass spectrometry (MALDI-MS) without additional purification steps.     

The electrical conductivity and magnetic susceptibility of germanium(II) monoselenide in the solid and liquid states

The specific conductivity of germanium(II) monoselenide has been measured, under equilibrium pressure, from room temperature to 802°C. Interpretation of the results leads to the conclusion that the intrinsic conductivity is observed in the solid in the upper temperature range: the calculated energy gap is E_0g = 1.222 ± 0.027 eV which coincides with the value of the optical gap. In liquid GeSe the calculated energy gap is 1.504 ± 0.016 eV. This increase of the energy gap in liquid GeSe is due, presumably, to the strengthening of the intralayer bonds of the structure. Electrical conductivity and magnetic susceptibility measurements confirm the existence of a polymorphic transformation in GeSe at 656°C.     

Dinuclear zinc(II) double-helicates of homochirally substituted bis(dipyrromethene)s

[structure: see text] A series of bis(dipyrromethene)s substituted with aromatic amide and aliphatic ester homochiral auxiliaries have been prepared and complexed with zinc(II) ions to form double-helical dinuclear complexes. CD analysis of the crude complexes revealed that the helicates formed in a diastereoselective manner. The helicates have been resolved into their constituent M and P helices by HPLC, indicating that the helical sense of the complexes is stable to racemization.     

P840-Reaction Centers from Chlorobium tepidum–Quinone Analysis and Functional Reconstitution into Lipid Vesicles

Membranes of Chlorobium tepidum contain about 35, 45 and2–10 molecules of menaquinone-7, chlorobium quinone (1′-oxo-menaquinone-7) and of the polar menaquinone (probably 1′-OH-menaquinone-7) per reaction center, respectively. None of these quinones was retained during the isolation of P840-reaction centers beyond the detection limit of about 0.2 quinones per reaction center, neither in the core complex nor in functionally intact reaction center preparations. The latter is shown to catalyze the formation of an electrochemical proton gradient in the presence of ascorbate and phenazinium methosulfate, when it is incorporated into lipid vesicles.     

Mechanism of palladium-catalyzed diene cyclization/hydrosilylation: direct observation of intramolecular carbometalation

The results of kinetic, deuterium-labeling, and low-temperature NMR studies have established a mechanism for the palladium-catalyzed cyclization/hydrosilylation of dimethyl diallylmalonate (1) with triethylsilane involving rapid, irreversible conversion of the palladium silyl complex [(phen)Pd(SiEt(3))(NCAr)](+) [BAr(4)](-) [Ar = 3,5-C(6)H(3)(CF(3))(2)] (4b) and 1 to the palladium 5-hexenyl chelate complex [(phen)Pd[eta(1),eta(2)-CH(CH(2)SiEt(3))CH(2)C(CO(2)Me)(2)CH(2)CH=CH(2)]](+) [BAr(4)](-) (5), followed by intramolecular carbometalation of 5 to form the palladium cyclopentylmethyl complex trans-[(phen)Pd[CH2CHCH2C(CO2Me)2CH2CHCH2SiEt3](NCAr)]+ [BAr4]- (6), and associative silylation of 6 to release 3 and regenerate 4b.     

Anion radical [2 + 2] cycloaddition as a mechanistic probe: stoichiometry- and concentration-dependent partitioning of electron-transfer and alkylation pathways in the reaction of the Gilman reagent Me2CuLi.LiI with bis(enones)

Exposure of easily reduced aromatic bis(enones) 1a-1e to the methyl Gilman reagent Me(2)CuLi.LiI at 0 degrees C in tetrahydrofuran solvent provides the products of tandem conjugate addition-Michael cyclization, 2a-2e, along with the products of [2 + 2] cycloaddition, 3a-3e. Complete partitioning of the Gilman alkylation and [2 + 2] cycloaddition pathways may be achieved by adjusting the loading of the Gilman reagent, the rate of addition of the Gilman reagent, and the concentration of the reaction mixture. The Gilman alkylation manifold is favored by the rapid addition of excess Gilman reagent at higher substrate concentrations, while the [2 + 2] cycloaddition manifold is favored by slow addition of the same Gilman reagent at lower concentrations and loadings. Notably, [2 + 2] cycloaddition to form 3a-3e is catalytic in Gilman reagent. Kinetic data reveal that the ratio of 2a and 3a changes such that the cycloaddition pathway becomes dominant upon increased consumption of Gilman reagent. These data suggest a concentration-dependent speciation of the Gilman reagent and differential reactivity of the aggregates present at higher and lower concentrations. While the species present at higher concentration induce Gilman alkylation en route to products 2a-2e, the species present at lower concentration provide products of catalytic [2 + 2] cycloaddition, 3a-3e. Moreover, upon electrochemical reduction of the bis(enones) 1a-1e, or chemically induced single-electron transfer from arene anion radicals, the very same [2 + 2] cycloadducts 3a-3e are formed. The collective data suggest that [2 + 2] cycloadducts 3a-3e arising under Gilman conditions may be products of anion radical chain cyclobutanation that derive via electron transfer (ET) from the Me(2)CuLi.LiI aggregate(s) present at low concentration. These observations provide a link between the Gilman alkylation reaction and related ET chemistry and suggest these reaction paths are mechanistically distinct. This analysis is made possible by the recent observation that easily reduced bis(enones) are subject to intramolecular [2 + 2] cycloaddition upon cathodic reduction or chemically induced ET from arene anion radicals, and is herewith showcased as a novel method of testing for the intermediacy of enone anion radicals.     

Ruthenium(II) dendrimers containing carbazole-based chromophores as branches

Three new luminescent and redox-active Ru(II) complexes containing novel dendritic polypyridine ligands have been synthesized, and their absorption spectra, luminescence properties (both at room temperature in fluid solution and at 77 K in rigid matrix), and redox behavior have been investigated. The dendritic ligands are made of 1,10-phenanthroline coordinating subunits and of carbazole groups as branching sites. The first and second generation species of this novel class of dendritic ligands (L1 and L2, respectively; see Figure 1 for their structural formulas) have been prepared and employed. The metal dendrimers investigated are [Ru(bpy)(2)(L1)](2+) (1; bpy = 2,2'-bipyridine), [Ru(bpy)(2)(L2)](2+) (2), and [Ru(L1)(3)](2+) (3; see Figure 2). For the sake of completeness and comparison purposes, also the absorption spectra, redox behavior, and luminescence properties of L1 and L2 have been studied, together with the properties of 3,6-di(tert-butyl)carbazole (L0) and [Ru(bpy)(2)(phen)](2+) (4, phen = 1,10-phenanthroline). The absorption spectra of the free dendritic ligands show features which can be assigned to the various subunits (i.e., carbazole and phenanthroline groups) and additional bands at lower energies (at lambda > 300 nm) which are assigned to carbazole-to-phenanthroline charge-transfer (CT) transitions. These latter bands are significantly red-shifted upon acid and/or zinc acetate addition. Both L1 and L2 exhibit relatively intense luminescence at room temperature in fluid solution (lifetimes in the nanosecond time scale, quantum yields of the order of 10(-2)-10(-1)) and at 77 K in rigid matrix (lifetimes in the millisecond time scale). Such a luminescence is assigned to CT states at room temperature and to phenanthroline-centered pi-pi triplet levels at 77 K. The room-temperature luminescence of L1 and L2 is totally quenched by acid or zinc acetate. The metal dendrimers exhibit the typical absorption and luminescence properties of Ru(II) polypyridine complexes. In particular, metal-to-ligand charge-transfer (MLCT) bands dominate the visible absorption spectra, and formally triplet MLCT levels govern the excited-state properties. Excitation spectroscopy evidences that all the light absorbed by the dendritic branches is transferred with unitary efficiency to the luminescent MLCT states in 1-3, showing that the new metal dendrimers can be regarded as efficient light-harvesting antenna systems. All the free ligands and metal dendrimers exhibit a rich redox behavior (except L2 and 3, whose redox behavior was not investigated because of solubility reasons), with clearly attributable reversible carbazole- and metal-centered oxidation and polypyridine-centered reduction processes. The electronic interaction between the carbazole redox-active sites of the dendritic ligands is affected by Ru(II) coordination.     

Evaluation of Electrospun PCL-PLGA for Sustained Delivery of Kartogenin

In this study, kartogenin was incorporated into an electrospun blend of polycaprolactone and poly(lactic-co-glycolic acid) (1:1) to determine the feasibility of this system for sustained drug delivery. Kartogenin is a small-molecule drug that could enhance the outcome of microfracture, a cartilage restoration procedure, by selectively stimulating chondrogenic differentiation of endogenous bone marrow mesenchymal stem cells. Experimental results showed that kartogenin did not affect the electrospinnability of the polymer blend, and it had negligible effects on fiber morphology and scaffold mechanical properties. The loading efficiency of kartogenin into electrospun membranes was nearly 100%, and no evidence of chemical reaction between kartogenin and the polymers was detected by Fourier transform infrared spectroscopy. Analysis of the released drug using high-performance liquid chromatography–photodiode array detection indicated an abundance of kartogenin and only a small amount of its major hydrolysis product. Kartogenin displayed a typical biphasic release profile, with approximately 30% being released within 24 h followed by a much slower, constant rate of release up to 28 days. Although additional development is needed to tune the release kinetics and address issues common to electrospun scaffolds (e.g., high fiber density), the results of this study demonstrated that a scaffold electrospun from biodegradable synthetic polymers is a suitable kartogenin delivery vehicle.     

Automated data processing of [1H-decoupled] 13C MR spectra acquired from human brain in vivo

In clinical 13C infusion studies, broadband excitation of 200 ppm of the human brain yields 13C MR spectra with a time resolution of 2-5 min and generates up to 2000 metabolite peaks over 2h. We describe a fast, automated, observer-independent technique for processing [1H-decoupled] 13C spectra. Quantified 13C spectroscopic signals, before and after the administration of [1-13C]glucose and/or [1-13C]acetate in human subjects are determined. Stepwise improvements of data processing are illustrated by examples of normal and pathological results. Variation in analysis of individual 13C resonances ranged between 2 and 14%. Using this method it is possible to reliably identify subtle metabolic effects of brain disease including Alzheimer's disease and epilepsy.     

Saturation of Berge hypergraphs

Given a graph $F$, a hypergraph is a Berge- $F$ if it can be obtained by expanding each edge in $F$ to a hyperedge containing it. A hypergraph $H$ is Berge-$F$-saturated if $H$ does not contain a subhypergraph that is a Berge-$F$, but for any edge $e\in E\left(\overline{H}\right)$, $H+e$ does. The $k$-uniform saturation number of Berge-$F$ is the minimum number of edges in a $k$-uniform Berge-$F$-saturated hypergraph on $n$ vertices. For $k=2$ this definition coincides with the classical definition of saturation for graphs. In this paper we study the saturation numbers for Berge triangles, paths, cycles, stars and matchings in $k$-uniform hypergraphs.