Fast visible dye staining of proteins in one- and two-dimensional sodium dodecyl sulfate-polyacrylamide gels compatible with matrix assisted laser desorption/ionization-mass spectrometry

A fast and matrix assisted laser desorption/ionization-mass spectrometry (MALDI-MS) compatible protein staining method in one- and two-dimensional sodium dodecyl sulfate-polyacrylamide gel electrophoresis (1- and 2-D SDS-PAGE) is described. It is based on the counterion dye staining method that employs oppositely charged two dyes, zincon (ZC) and ethyl violet (EV) to form an ion-pair complex. The protocol, including fixing, staining and quick washing steps, can be completed in 1-1.5 h depending upon gel thickness. It has a sensitivity of 4-8 ng, comparable to that of colloidal Coomassie Brilliant Blue G (CBBG) staining with phosphoric acid in the staining solution. The counterion dye stain does not induce protein modifications that complicate interpretation of peptide mapping data from MS. Considering the speed, sensitivity and compatibility with MS, the counterion dye stain may be more practical than any other dye-based protein stains for routine proteomic researches.     

Liquid chromatography-diode array detection-electrospray ionisation mass spectrometry/nuclear magnetic resonance analyses of the anti-hyperglycemic flavonoid extract of Genista tenera. Structure elucidation of a flavonoid-C-glycoside

The anti-hyperglycemic flavonoid extract obtained from Genista tenera was first studied by liquid chromatography (LC)-diode array detection (DAD) which showed the presence of two major compounds. One of them was identified as genistein-7-O-glucoside. Luteolin-7-O-glucoside was detected as a minor constituent, while luteolin-7,3'-di-O-glucoside and rutin were found in trace amounts. LC-DAD-ESI-MS and NMR were used to confirm the structure of these compounds and allowed the elucidation of the structure of the unknown major compound, which is the flavonoid 5,7,4'-trihydroxyisoflavone-8-C-glucoside.     

Angular hydroxymethyl directed intramolecular Diels-Alder approach for the stereo- and regioselective synthesis of pimaraditerpenes

The stereo- and regioselective synthetic route to pimaraditerpenes, employing an angular hydroxymethyl directed intramolecular Diels-Alder reaction of the decaline intermediate, has been developed. This synthetic approach allows prompt access to both natural pimaraditerpenes and the unnatural regioisomers, which would be potentially new anti-inflammatory pimaraditerpenes.     

Background-free,fast protein staining in sodium dodecyl sulfate polyacrylamide gel using counterion dyes,zincon and ethyl violet

A background-free, fast protein staining method in polyacrylamide gel electrophoresis using an acidic dye, zincon (ZC) and a basic dye, ethyl violet (EV) is described. It is based on the counterion dye staining technique that employs two oppositely charged dyes to form an ion-pair complex in staining solution. The selective binding of free dye molecules to proteins in acidic solution produces bluish violet-colored bands. It is a rapid and end-point staining procedure, involving only fixing and staining steps that are completed in 1-1.5 h. The detection limit of this method is 8-15 ng of protein that is comparable to the sensitivity of the colloidal Coomassie Brilliant Blue G (CBBG) stain. Due to its sensitivity and speed, this stain may be more practical than any other dye-based stains for routine laboratory purposes.     

Transformation of highly ordered large pore silica mesophases (Fm3m, Im3m and p6mm) in a ternary triblock copolymer-butanol-water system

Exceptional control of the phase behavior of highly ordered large pore mesostructured silica (with the choice of Fm3m, Im3m or p6mm symmetry) is achieved using a triblock copolymer (EO(106)PO(70)EO(106)) and butanol at low acid concentrations.     

Cysteine carboxyl O-methylation of human placental 23 kDa protein

C-Terminal carboxyl methylation of a human placental 23 kDa protein catalyzed by membrane-associated methyltransferase has been investigated. The 23 kDa protein substrate methylated was partially purified by DEAE-Sephacel, hydroxyapatite and Sephadex G-100 gel filtration chromatographies. The substrate protein was eluted on Sephadex G-100 gel filtration chromatography as a protein of about 29 kDa. In the absence of Mg2+, the methylation was stimulated by guanine nucleotides (GTP, GDP and GTPgammaS), but in the presence of Mg2+, only GTPgammaS stimulated the methylation which was similar to the effect on the G25K/rhoGDI complex. AFC, an inhibitor of C-terminal carboxyl methylation, inhibited the methylation of human placental 23 kDa protein. These results suggests that the substrate is a small G protein different from the G25K and is methylated on C-terminal isoprenylated cysteine residue. This was also confirmed by vapor phase analysis. The methylated substrate protein was redistributed to membrane after in vitro methylation, suggesting that the methylation of this protein is important for the redistribution of the 23 kDa small G protein for its putative role in intracellular signaling.     

A novel and efficient approach to highly substituted indolizines via 5-endo-trig iodocyclization

A new approach to indolizines has been developed using a 5-endo-trig iodocyclization of allylic esters followed by isomerization and dehydroiodination facilitated by triethylamine at rt. This mild procedure enabled us to synthesize a number of highly substituted indolizines in good yields.     

Three-dimensional effects on the linear adiabatic molecular wavefunctions in the H + H2 system

The adiabatic molecular wavefunctions in the H + H₂ system are obtained in one dimension by solving the double-well potential problem. In three dimensions, the corresponding linear adiabatic molecular wavefunctions are obtained. A comparison between these wavefunctions clearly suggests that the probability of reaction is smaller in three dimensions.     

Bystander CD4+ T cells: crossroads between innate and adaptive immunity

T cells are the central mediators of both humoral and cellular adaptive immune responses. Highly specific receptor-mediated clonal selection and expansion of T cells assure antigen-specific immunity. In addition, encounters with cognate antigens generate immunological memory, the capacity for long-term, antigen-specific immunity against previously encountered pathogens. However, T-cell receptor (TCR)-independent activation, termed “bystander activation”, has also been found. Bystander-activated T cells can respond rapidly and secrete effector cytokines even in the absence of antigen stimulation. Recent studies have rehighlighted the importance of antigen-independent bystander activation of CD4⁺ T cells in infection clearance and autoimmune pathogenesis, suggesting the existence of a distinct innate-like immunological function performed by conventional T cells. In this review, we discuss the inflammatory mediators that activate bystander CD4⁺ T cells and the potential physiological roles of these cells during infection, autoimmunity, and cancer.Subject terms: T cells, CD4-positive T cells     

Determination of estrone and 17 beta-estradiol in human hair by gas chromatography-mass spectrometry

An efficient procedure is described for the determination of estrone and 17 beta-estradiol in hair by gas chromatography-mass spectrometry (GC-MS). The method involves alkyloxycarbonylation with isobutyl chloroformate (isoBCF) of phenolic hydroxy groups after alkaline digestion of hair samples. The resulting isobutyloxycarbonyl derivatives of estrone and 17 beta-estradiol are extracted with hexane and subjected to chlorodifluoroacetyl derivatization in order to protect the remaining alcoholic hydroxy groups. When GC-MS with selected ion monitoring (SIM) was used, the quantitative ions were at m/z 270 and 384 in the electron ionization mass spectra for estrone and 17 beta-estradiol, respectively. The detection limits for SIM of the steroids were 1 and 2 pg, respectively, and the SIM responses were linear with correlation coefficients varying from 0.991 to 0.994 in the concentration range 0.2-4.0 ng g-1 for the estrogens studied. The detection of estrone and 17 beta-estradiol in hair samples was possible in the concentration range of 0.24-1.30 ng g-1. The concentrations of the two estrogens detected were different in male and female hair samples.