Controlled assembly of SNAP-PNA-fluorophore systems on DNA templates to produce fluorescence resonance energy transfer

The SNAP protein is a widely used self-labeling tag that can be used for tracking protein localization and trafficking in living systems. A model system providing controlled alignment of SNAP-tag units can provide a new way to study clustering of fusion proteins. In this work, fluorescent SNAP-PNA conjugates were controllably assembled on DNA frameworks forming dimers, trimers, and tetramers. Modification of peptide nucleic acid (PNA) with the O6-benzyl guanine (BG) group allowed the generation of site-selective covalent links between PNA and the SNAP protein. The modified BG-PNAs were labeled with fluorescent Atto dyes and subsequently chemo-selectively conjugated to SNAP protein. Efficient assembly into dimer and oligomer forms was verified via size exclusion chromatography (SEC), electrophoresis (SDS-PAGE), and fluorescence spectroscopy. DNA directed assembly of homo- and hetero-dimers of SNAP-PNA constructs induced homo- and hetero-FRET, respectively. Longer DNA scaffolds controllably aligned similar fluorescent SNAP-PNA constructs into higher oligomers exhibiting homo-FRET. The combined SEC and homo-FRET studies indicated the 1:1 and saturated assemblies of SNAP-PNA-fluorophore:DNA formed preferentially in this system. This suggested a kinetic/stoichiometric model of assembly rather than binomially distributed products. These BG-PNA-fluorophore building blocks allow facile introduction of fluorophores and/or assembly directing moieties onto any protein containing SNAP. Template directed assembly of PNA modified SNAP proteins may be used to investigate clustering behavior both with and without fluorescent labels which may find use in the study of assembly processes in cells

Effect Of Combined Germination, Dehulling And Boiling On Mineral, Sucrose, Stachyose, Fibrulose, And Phytic Acid Content Of Different Chickpea Cultivars

Chickpea is a good source of high quality protein, carbohydrates, vitamins (thiamine and niacin), and minerals. However, its use in industry has been limited by variation in composition with cultivar and also the presence of oligosaccharides, trypsin inhibitors, phytic acids, tannin, and haemagglutinin. Different technologies have been studied to eliminate or minimise the undesirable factors in chickpeas. None of the studied traditional technologies has been found to effectively eliminate or minimise all the undesirable factors in chickpeas. It is not clear whether a combination of these traditional technologies, more especially cooking of germinated and dehulled chickpeas, will significantly reduce all the antinutritional factors. The physical characteristics, stachyose, sucrose, phytic acid, fibrulose, and mineral content of different chickpeas cultivar were determined and compared with reference to infant and child nutrition. The selected cultivars were (1) dehulled and boiled before drying; (2) dehulled followed by soaking and boiling before drying; (3) boiled without dehulling before drying; and germinated, boiled followed by drying and dehulling . The effects of the processing on mineral, sugar, dietary fibre content were evaluated. Desiwere found to have lower seed weight, hydration capacity and swelling capacity compared to kabuli. Seed density, hydration index and swelling index did not vary with cultivar. The mineral density, stachyose, fibrulose, and hull content increased significantly (p<0.05) with the decrease of seed weight whereas phytic acid content did not vary. All processes resulted in an increase in calcium, phosphorous, zinc, and phytic acid and a decrease in potassium, iron, magnesium, sucrose, stachyose and fibrulose content regardless of cultivar type. Germination for 72 hrs followed by boiling, drying and dehulling resulted in highest reduction in antinutritional factors with minimal nutrient loss.It is feasible to use chickpeas as an excellent source of infant follow-on formula/weaning food with minimal mineral fortification and use of low phytic acid cultivars

Control of pancreatic β cell regeneration by glucose metabolism.

Recent studies revealed a surprising regenerative capacity of insulin-producing β cells in mice, suggesting that regenerative therapy for human diabetes could in principle be achieved. Physiologic β cell regeneration under stressed conditions relies on accelerated proliferation of surviving β cells, but the factors that trigger and control this response remain unclear. Using islet transplantation experiments, we show that β cell mass is controlled systemically rather than by local factors such as tissue damage. Chronic changes in β cell glucose metabolism, rather than blood glucose levels per se, are the main positive regulator of basal and compensatory β cell proliferation in vivo. Intracellularly, genetic and pharmacologic manipulations reveal that glucose induces β cell replication via metabolism by glucokinase, the first step of glycolysis, followed by closure of K(ATP) channels and membrane depolarization. Our data provide a molecular mechanism for homeostatic control of β cell mass by metabolic demand