Inactivation of rabbit muscle glycogen synthase by glycogen synthase kinase-3. Dominant role of the phosphorylation of Ser-640 (site-3a)

Rabbit skeletal muscle glycogen synthase, a rate-limiting enzyme for glycogen biosynthesis, is regulated by multisite phosphorylation. The protein kinase glycogen synthase kinase 3 (GSK-3) phosphorylates 4 Ser residues (Ser-640, Ser-644, Ser-648, and Ser-652; also known as sites 3a, 3b, 3c, and 4, respectively) at the COOH terminus of the subunit. Phosphorylation of these sites by GSK-3 is sequential, from COOH- to NH2-terminal, and is wholly dependent on prior phosphorylation by casein kinase II at Ser-656 (site 5). Expression in Escherichia coli was used to generate mutant forms of glycogen synthase, S640A, S644A, and S648A, in which site 3a, site 3b, or site 3c was changed to Ala, respectively. The purified enzymes had -/+ glucose-6-P activity ratios in the range of 0.8-0.9. Phosphorylation by casein kinase II and GSK-3 gave results consistent with the model of obligate sequential action of GSK-3. Phosphorylation at site 5, sites 4 + 5, or sites 3c + 4 + 5 had no measurable effect on activity. When sites 3b + 3c + 4 + 5 were phosphorylated, modest inactivation resulted. Additional phosphorylation at site 3a, however, was potently inactivating, reducing the -/+ glucose-6-P activity ratio to 0.1 and increasing the glucose-6-P concentration needed for half-maximal activation by an order of magnitude. Introduction of each additional phosphate, in the order site 4, 3c, 3b, and 3a, caused an incremental reduction in the mobility of the subunit when analyzed by polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate. The results of this study demonstrate that GSK-3 phosphorylation of site 3a (Ser-640), and to a lesser extent, site 3b, correlates with inactivation of glycogen synthase by GSK-3. Evidence is also presented for an allosteric mechanism of inactivation whereby modification of one subunit influences the activity state of adjacent subunits.     

Effects of spray drying conditions on the physicochemical and antioxidant properties of the Gac (Momordica cochinchinensis) fruit aril powder

Gac fruit aril has an attractive orange red colour and very high level of carotenoids, giving it exceptional antioxidant properties. However, spray drying of this material has not been successful and malto dextrin is considered as a suitable drying aid to preserve its colour and antioxidant properties. This paper reports the effects of inlet drying air temperature (120, 140, 160, 180 and 200 °C) and maltodextrin addition (10%, 20% and 30%) on the physicochemical and antioxidant properties of the Gac aril powder. Moisture content and bulk density, colour characteristics, total carotenoid content (TCC), encapsulation efficiency and total antioxidant activity (TAA) were significantly affected by maltodextrin concentration and the inlet air temperatures. However, pH, a_w and water solubility index were not significantly influenced by the spray drying conditions. Overall, a good quality Gac powder in terms of colour, TCC and TAA can be produced by spray-drying at inlet temperature of 120 °C and adding maltodextrin concentration at 10% w/v.     

Inorganic ligand-modified, colloid-enhanced ultrafiltration: A novel method for removing uranium from aqueous solution

Inorganic ligand-modified, colloid-enhanced ultrafiltration (ILM-CEUF) is as a novel membrane-based separation method for selectively removing target ions from aqueous solution. Traditional colloid-enhanced ultrafiltration (CEUF) is a well-established membrane-based separation technique that can be used to separate metal ions from other aqueous solution components. Ligand-modified, colloid-enhanced ultrafiltration (LM-CEUF) uses organic ligands that selectively complex target ions and also associate with a water-soluble colloid, such as a surfactant micelle or polyelectrolyte. The colloid, associated -ligand, and target ion are then concentrated using an ultrafilter, producing a filtrate with a low concentration of the target ion. While traditional LM-CEUF techniques are able to provide quantitative separations of a variety of ionic pollutants, the high costs of the chelating agents make such techniques nonviable in most remediation schemes. This study investigated the replacement of organic ligands with carbonate for the selective removal of U(VI) from aqueous solution.In slightly to moderately basic solutions containing carbonate, UO₂(CO₃)₃⁴⁻ can be made to dominate the U(VI) speciation. Using poly(diallyldimethylammonium) chloride, the effectiveness and efficiency of ILM-CEUF for removing U(VI) from other aqueous solution components was investigated as a function of carbonate concentration, pH, and ionic strength. Uranium separations of greater than 99.6% were achieved; even in the presence of large excesses of competing ions. The specific separation of U(VI) from Sr²⁺ was also examined.     

Analysis of naturally produced technetium and plutonium in geologic materials

In uncontaminated natural materials, plutonium and technetium exist exclusively as products (daughters) of nuclear reactions in which uranium is the principal reactant (parent). Under conditions of chemical stability over geologic periods of time, the relative abundances of daughter and parent elements are fixed by the rates of nuclear reactions and the decay of the daughter radionuclide. The state of this nuclear secular equilibrium condition is the primary basis of the geochemical study of these elements in nature. Thus, it is critical that nuclear parent and daughter abundances are measured in the same sample. We have developed a quantitative procedure for measuring subpicogram quantities of plutonium and technetium in gram quantities of geologic matrices such as uranium ores. The procedure takes advantage of the aggressive properties of sodium peroxide/hydroxide fusion to ensure complete dissolution and homogenization of complex materials, the precision provided by isotope dilution techniques, and the extreme sensitivity offered by thermal ionization mass spectrometry. Using this technique, a quantitative aliquot can be removed for uranium analysis by isotope dilution thermal ionization mass spectrometry or α spectrometry. Although the application of the procedure is unique, the analytical concepts may find more general application in studies of environmental contamination by nuclear materials. To assess the precision and accuracy of the analytical results, blanks and standards were analyzed routinely for a 1-year period to ensure quality control of our sample analyses. The average technetium blank is 5 ± 4 fg (n = 8), and that for plutonium is 0.17 ± 0.15 pg (n = 7). Thus, the detection limit for technetium (defined as 3 times the standard deviation of the average blank) is 11 fg, and that for plutonium is 0.44 pg. To assess the procedural precision, Canadian Reference Material BL-5 was analyzed routinely with samples. The results of seven replicate analyses for technetium in this standard reference material yield a technetium concentration of 59.0 fg/g, with a remarkably small standard deviation of 0.6 fg, 1.0% of the average value. The results of six replicate analyses for the concentration of plutonium in BL-5 give 1.012 pg/g, with an equally small standard deviation of 0.016, 1.6% of the average value. No direct measure of accuracy can be done on the technetium or plutonium analyses, because no standard reference material exists for these elements. To help constrain the accuracy of our measurements, equilibrium technetium/uranium and plutonium/uranium abundances were calculated using the nuclear reaction code MCNP. For technetium, such calculations are relatively insensitive to variations in model parameters, and measurements fall within a 21% high/low bias. For plutonium, the calculations are very sensitive to model parameters and hence inherently less precise. Indirectly, spike and isotope mix calibrations made from weighted quantities of certified isotopes (both technetium and plutonium) can be used to determine the bias of the measurement system for these elements. These calibrations show that the measurement system is biased by no more than ±1.5%.     

Intraocular laser surgical probe for membrane disruption by laser-induced breakdown

A fiber probe has been designed as a surgical aid to cut intraocular membranes with laser-induced breakdown as the mechanism. The design of the intraocular laser surgical probe is discussed. A preliminary retinal damage distance has been calculated with breakdown threshold, spot size, and shielding measurements. Collateral mechanical-damage effects caused by shock wave and cavitation are discussed.     

Benzodiazepine receptor function in the chick social separation–stress procedure.

The role of benzodiazepine (BZ) receptors in modulating social separation–induced distress vocalizations (DVocs) and stress-induced analgesia (SIA) were examined in 8-day-old cockerels (Gallus gallus). In Experiment 1, the BZ agonist chlordiazepoxide (CDP; 5.0 mg/kg) reversed both DVocs and SIA in isolated chicks. Coadministration of the BZ antagonist flumazenil (0.01, 0.03, or 0.10 mg/kg) reversed CDP anxiolytic effects. In Experiment 2, the BZ agonists alprazolam (ALP; 0.065, 0.125, 0.25, or 0.50 mg/kg) and lorazepam (LOR; 0.125, 0.25, 0.50, or 1.0 mg/kg) dose dependently reversed social separation–induced DVocs and SIA. The ED??s for ALP and LOR in attenuating DVOcs were 0.19 and 0.34 mg/kg, respectively. These data strongly support the theory that CDP anxiolytic effects are mediated by BZ receptor activity in the chick social separation procedure (Exp 1) and that this model is sensitive to BZ agonists of different potencies (Exp 2). (PsycINFO Database Record (c) 2010 APA, all rights reserved)     

Variations in isomer distribution in commercially available conjugated linoleic acid

Conjugated linoleic acid (CLA) has been reported to have anticarcinogenic and antiatherogenic properties, to repartition body fat, to build bone mass, to normalize glucose tolerance, and to reduce hyperglycemia and diabetes. CLA products are now commercially available, and there is considerable interest in studying CLA because of this range of reported beneficial effects. However, little is known about the composition of these preparations. Representative commercial CLA products in capsule or liquid (aqueous or oily) form were analyzed for their CLA content and isomer composition using gas chromatography (GC), silver ion-high performance liquid chromatography (Ag⁺-HPLC) and spectroscopic techniques. The content of CLA in the preparations varied widely. Based on the GC-internal standard technique, total CLA varied from 20 to 89% by total weight and 28 to 94% of total fat. One product contained no CLA. The isomer distributions were generally of two types: those with two major CLA positional isomers, and those with four major CLA positional isomers. All the CLA preparations in capsule form contained the four isomer mixture, while the liquid preparations contained from two to four CLA positional isomers.     

Tissue imaging using nanospray desorption electrospray ionization mass spectrometry

Ambient ionization imaging mass spectrometry is uniquely suited for detailed spatially resolved chemical characterization of biological samples in their native environment. However, the spatial resolution attainable using existing approaches is limited by the ion transfer efficiency from the ionization region into the mass spectrometer. Here, we present a first study of ambient imaging of biological samples using nanospray desorption ionization (nano-DESI). Nano-DESI is a new ambient pressure ionization technique that uses minute amounts of solvent confined between two capillaries comprising the nano-DESI probe and the solid analyte for controlled desorption of molecules present on the substrate followed by ionization through self-aspirating nanospray. We demonstrate highly sensitive spatially resolved analysis of tissue samples without sample preparation. Our first proof-of-principle experiments indicate the potential of nano-DESI for ambient imaging with a spatial resolution of better than 12 μm. The significant improvement of the spatial resolution offered by nano-DESI imaging combined with high detection efficiency will enable new imaging mass spectrometry applications in clinical diagnostics, drug discovery, molecular biology, and biochemistry.