Measurement of trace elements in murine liver tissue samples: Comparison between ICP-MS/MS and TXRF

BackgroundTrace elements exhibit essential functions in many physiological processes. Thus, for research focusing on trace element homeostasis and metabolism analytical methods allowing for multi-element analyses are fundamental. Small sample amounts may be a big challenge in trace element analyses especially if also other end points want to be addressed in the same sample. Therefore, the aim of the present study was to examine trace elements (iron, copper, zinc, and selenium) in murine liver tissue prepared by a RIPA buffer-based lyses method.Methods and resultsAfter centrifugation, lysates and pellets were obtained and trace elements were analyzed with TXRF in liver lysates. The results were compared to that obtained by a standard microwave-assisted acidic digestion with subsequent ICP-MS/MS analysis of the same liver tissue, liver lysates, and remaining pellets. In addition, trace element concentrations, determined in murine serum with both methods, were compared. For serum samples, both TXRF and ICP-MS/MS provide similar and highly correlating results. Furthermore, in liver lysate samples prepared with RIPA buffer, comparable trace element concentrations were measured by TXRF as with the standard digestion technique and ICP-MS/MS. Only marginal amounts of trace elements were detected in the pellets.ConclusionTaken together, the results obtained by the present study indicate that the RIPA buffer-based method is suitable for sample preparation for trace element analyses via TXRF, at least for the here investigated murine liver samples.     

Bioenergetics of Neurotransmitter Transport

Neurotransmitter transporters are essential components in the recycling of neurotransmitters released during neuronal activity. These transporters are the targets for important drugs affecting mood and behavior. They fall into at least four gene families, two encoding proteins in the plasma membrane and two in the synaptic vesicle membrane, although the known vesicular transporters have not all been cloned. Each of these transporters works by coupling the downhill movement of small ions such as Na⁺, Cl^–, K⁺, and H⁺ to the uphill transport of neurotransmitter. Plasma membrane transporters move the transmitter into the cytoplasm by cotransport with Na⁺. Many transporters also couple Cl^– cotransport to transmitter influx and these all belong to the NaCl-coupled family, although within the family the coupling stoichiometry can vary. Transporters for glutamate couple influx of this excitatory amino acid to Na⁺ and H⁺ influx and K⁺ efflux. Transporters in synaptic vesicles couple H⁺ efflux to neurotransmitter transport from the cytoplasm to the vesicle lumen.     

Synaptic plasma membrane-bound acetylcholinesterase activity is not affected by docosahexaenoic acid-induced decrease in membrane order

We investigated the effect of administration of docosahexaenoic acid (C22:6, n-3; 300 mg/kg.day, for 12 weeks) on the degree of membrane order and membrane-bound acetylcholinesterase activity of the cerebral cortex synaptic plasma membrane in male Wistar rats. Docosahexaenoic acid levels in the synaptic plasma membrane increased significantly by 16% over levels in control rats concomitant with an increase in the molar ratio of docosahexaenoic acid to arachidonic acid. Synaptic plasma membrane order, assessed by 1,6-diphenyl-1,3,5-hexatriene, which measures order of the bulk internal hydrophobic lipid core, decreased significantly in the docosahexaenoic acid-fed rats. Lateral mobility of both global and annular lipids measured by pyrene also increased. Acetylcholinesterase activity of the synaptic plasma membrane was unaffected, and synaptic plasma membrane phospholipid contents increased in the docosahexaenoic acid-fed rats, with a concomitant decrease in the cholesterol/phospholipid molar ratio. Lipid peroxide and reactive oxygen species, indicators of tissue oxidative stress, decreased in both the cerebral cortex synaptosome and homogenate of the docosahexaenoic acid-fed rats. Arrhenius plot showed a break point in acetylcholinesterase activity at 22 degrees C and 24 degrees C in plasma membranes from docosahexaenoic acid-fed and control rats, respectively. The present experiment indicates that chronic administration of docosahexaenoic acid does not affect synaptic acetylcholinesterase activity and evoke oxidative stress, although it increases the disorder of the global and annular lipids of rat synaptic plasma membranes.     

Electrochemical measurement of hydrogen peroxide in plasma: evaluation of freshwater prawn (Macrobrachium rosenbergii) and crucian carp (Cyprinus carpio) phagocytes under natural conditions

An electrochemical technique for the real-time detection of hydrogen peroxide (H2O2) was employed to describe respiratory burst activity (RBA) of phagocytes in plasma which can be used to evaluate the ability of immune system and disease resistance. The method is based upon the electric current changes, by redox reaction on platinum electrode of extracellular hydrogen peroxide (H2O2) released from phagocytes stimulated by the zymosan at 680 mV direct current (d.c.). Compared with the control, activation of respiratory burst by zymosan particles results in a high amperometric response, and a current peak was obtained during the whole monitoring process. The peak current was proved by addition of Cu2+ and other controls, to be the result of intense release of H2O2 from phagocytes. The peak area was calculated and used to evaluate the quantity of effective H2O2, which represents the quantity of H2O2 beyond the clearance of related enzymes in plasma. According to Faraday's law, the phagocytes' ability of prawns to generate effective H2O2 was evaluated from 1.253 x 10(-14) mol/cell to 6.146 x 10(-14) mol/cell, and carp from 1.689 x 10(-15) mol/cell to 7.873 x 10(-15) mol/cell. This method is an acute and quick detection of extracellular effective H2O2 in plasma and reflects the capacity of phagocytes under natural conditions, which could be applied for selecting species and parents with high immunity for breeding in aquaculture.     

New aspects of biosynthesis and metabolism of <Emphasis Type="Italic">N</Emphasis>-acyldopamines in rat tissues

Possible biosynthetic pathways of N-acyldopamines in rat tissues were compared. It was shown that an insignificant amount of the conjugation products was formed during the incubation of arachidonic acid and dopamine, whereas the substitution of tyrosine for dopamine resulted in the productive biosynthesis of N-arachidonoyldopamine. The biosynthesis presumably involves several closely conjugated enzymatic stages, and free fatty acids rather than their CoA esters served as the starting substrates. The decarboxylation stage probably precedes the stage of catechol system formation, because N-acetyltyramine (a probable intermediate) was easily oxidized by monophenol monooxygenase to N-acyldopamine, whereas N-acyltyrosine is hydrolyzed under these conditions. Biosynthesis of N-acyldopamines in a cell-free medium was accompanied by their methylation. The possibility of oxidative metabolism of N-acyldopamines, which could serve as co-substrates or inhibitors of different oxidoreductases, was shown for the first time.     

Elevated CO2 increases nitrogen fixation and decreases soil nitrogen mineralization in Florida scrub oak

We report changes in nitrogen cycling in Florida scrub oak in response to elevated atmospheric CO₂ during the first 14 months of experimental treatment. Elevated CO₂ stimulated above-ground growth, nitrogen mass, and root nodule production of the nitrogen-fixing vine, Galactia elliottii Nuttall. During this period, elevated CO₂ reduced rates of gross nitrogen mineralization in soil, and resulted in lower recovery of nitrate on resin lysimeters. Elevated CO₂ did not alter nitrogen in the soil microbial biomass, but increased the specific rate of ammonium immobilization (NH₄⁺ immobilized per unit microbial N) measured over a 24-h period. Increased carbon input to soil through greater root growth combined with a decrease in the quality of that carbon in elevated CO₂ best explains these changes. These results demonstrate that atmospheric CO₂ concentration influences both the internal cycling of nitrogen (mineralization, immobilization, and nitrification) as well as the processes that regulate total ecosystem nitrogen mass (nitrogen fixation and nitrate leaching) in Florida coastal scrub oak. If these changes in nitrogen cycling are sustained, they could cause long-term feedbacks to the growth responses of plants to elevated CO₂. Greater nitrogen fixation and reduced leaching could stimulate nitrogen-limited plant growth by increasing the mass of labile nitrogen in the ecosystem. By contrast, reduced nitrogen mineralization and increased immobilization will restrict the supply rate of plant-available nitrogen, potentially reducing plant growth. Thus, the net feedback to plant growth will depend on the balance of these effects through time.     

Maintenance of pluripotency in human embryonic stem cells cultured on a synthetic substrate in conditioned medium

Realizing the potential clinical and industrial applications of human embryonic stem cells (hESCs) is limited by the need for costly, labile, or undefined growth substrates. Here we demonstrate that trypsin passaging of the hESC lines, HUES7 and NOTT1, on oxygen plasma etched tissue culture polystyrene (PE‐TCPS) in conditioned medium is compatible with pluripotency. This synthetic culture surface is stable at room temperature for at least a year and is readily prepared by placing polystyrene substrates in a radio frequency oxygen plasma generator for 5 min. Modification of the polystyrene surface chemistry by plasma etching was confirmed by X‐ray photoelectron spectroscopy (XPS) and time‐of‐flight secondary ion mass spectrometry (ToF‐SIMS), which identified elemental and molecular changes as a result of the treatment. Pluripotency of hESCs cultured on PE‐TCPS was gauged by consistent proliferation during serial passage, expression of stem cell markers (OCT4, TRA1‐60, and SSEA‐4), stable karyotype and multi‐germlayer differentiation in vitro, including to pharmacologically responsive cardiomyocytes. Generation of cost‐effective, easy‐to‐handle synthetic, defined, stable surfaces for hESC culture will expedite stem cell use in biomedical applications. Biotechnol. Bioeng. 2010;105: 130–140. © 2009 Wiley Periodicals, Inc.     

Membrane trafficking in plants: new discoveries and approaches

The general organization and function of the endomembrane system is highly conserved in eukaryotic cells. In addition, increasing numbers of studies demonstrate that normal plant growth and development are dependent on specialized tissue and subcellular-specific components of the plant membrane trafficking machinery. New approaches, including chemical genomics and proteomics, will likely accelerate our understanding of the diverse functions of the plant endomembrane system.     

Relation between insulin resistance and fast-migrating LDL subfraction as characterized by capillary isotachophoresis

The proportion of the electronegative low density lipoprotein [LDL(-)] subfraction, which is atherogenic, is increased in type 2 diabetes but is not reduced by glycemic control. Therefore, we evaluated the ability of a new technique, capillary isotachophoresis (cITP), to quantify charge-based LDL subfractions and examined the relation between insulin resistance and the cITP fast-migrating (f) LDL levels. Seventy-five 10-year-old boys were included. The two cITP LDL subfractions, fLDL and major LDL subfractions, were proportional to the LDL protein content within the range of 0.1-0.8 mg/ml LDL protein. Levels of cITP fLDL were positively correlated with triglyceride (TG) levels and negatively correlated with LDL size. Insulin resistance as assessed by the homeostasis model assessment (HOMA-IR) was positively correlated (P < 0.01) with cITP fLDL levels (r = 0.41). The relation between HOMA-IR and cITP fLDL levels depended on TG levels but was independent of body mass index and LDL size. cITP lipoprotein analysis is an accurate and sensitive method for quantifying charge-based LDL subfractions in human plasma, and insulin resistance is related to cITP fLDL independent of LDL size.     

Discovery of C-(1-aryl-cyclohexyl)-methylamines as selective,orally available inhibitors of dipeptidyl peptidase IV

The successful launches of dipeptidyl peptidase IV (DPP IV) inhibitors as oral anti-diabetics warrant and spur the further quest for additional chemical entities in this promising class of therapeutics. Numerous pharmaceutical companies have pursued their proprietary candidates towards the clinic, resulting in a large body of published chemical structures associated with DPP IV. Herein, we report the discovery of a novel chemotype for DPP IV inhibition based on the C-(1-aryl-cyclohexyl)-methylamine scaffold and its optimization to compounds which selectively inhibit DPP IV at low-nM potency and exhibit an excellent oral pharmacokinetic profile in the rat.