31P NMR study on the autophosphorylation of insulin receptors in the plasma membrane

A nonradioactive ³¹P nuclear magnetic resonance (NMR) spectroscopy protocol has been developed and used to investigate in vitro autophosphorylation of insulin receptors. Optimum experimental conditions have been explored, and the effects of Mn²⁺ and phosphocreatine (PCr) on the determination of the phosphorylation reaction have been assayed. The method was used to monitor the time courses of the phosphorylation reaction in solution. The results from this NMR study were in agreement with observations of insulin receptor phosphorylation made by using Western blotting.      

Amplification of noble gas ion lines in the afterglow of a pulsed hollow cathode discharge and possible benefit for analytical glow discharge mass spectrometry

A high-current pulsed hollow cathode discharge was used to study the role of atomic and ionic metastables involved in ionization plasma processes. We observed the enhancement of the spectral emission lines of noble gas ions in the afterglow. A study of the processes that involve atomic and ionic metastables is of great interest since it should lead to a better understanding of and enhanced control over the ionization mechanisms crucial to analytical glow discharge mass spectrometry (GDMS) analysis. Figure Time profile of Ti, Ti⁺, and Ne⁺ spectral lines     

Observation of salt-induced β-lactoglobulin aggregation using sedimentation field-flow fractionation

Sedimentation field-flow fractionation (SdFFF) was applied in order to characterize particle sizes of β-lactoglobulin aggregates induced by Ca²⁺ or Zn²⁺. Aggregation induced by Zn²⁺ was faster than that induced by Ca²⁺. Effects of Zn²⁺ and β-lactoglobulin concentrations, as well as contact time, on the aggregation of β-lactoglobulin were examined. All factors exhibited a combined effect on the size of aggregates, whereby larger aggregates were obtained at increased concentrations of Zn²⁺ and β-lactoglobulin. At fixed concentrations of 2% (w/v) β-lactoglobulin and 10 mM Zn²⁺, the particle size of the aggregates increased from 0.19 μm (at 15 min) to 0.38 μm (at 2880 min). Further, a hyphenated technique of SdFFF and inductively coupled plasma–optical emission spectrometry (ICP–OES) was used to examine whether intermolecular ionic bridges take part in salt-induced β-lactoglobulin aggregation. With SdFFF–ICP–OES, protein–cation–protein cross-linkages were observed for β-lactoglobulin aggregation induced by Zn²⁺, but not for that induced by Ca²⁺.      

Pattern recognition analysis for <Superscript>1</Superscript>H NMR spectra of plasma from hemodialysis patients

¹H NMR spectroscopic and pattern recognition-based methods (NMR-PR) were applied to the metabolic profiling studies on hemodialysis (HD). Plasma samples were collected from 37 patients before and after HD and measured by 600 MHz NMR spectroscopy. Each spectrum was data-processed and subjected to principal component analysis for pattern recognition. Spectral patterns of plasma between pre- and post-dialyses were clearly discriminated, together with significant fluctuations in the levels of creatinine, trimethylamine-N-oxide, glucose, lactate, and acetate, which were quantitated. We have first observed the significant elevation of lactate levels in post-dialysis plasma. The present study has demonstrated the high feasibility of NMR-PR method for monitoring the dialysis condition and comprehensive profiling of the change of low-molecular-weight metabolites in HD. Figure PCA for 1H NMR spectra of plasma from HD patients     

Lead‐sensitive PNIPAM microgels modified with crown ether groups

A series of lead‐sensitive poly(N‐isopropylacrylamide) microgels with pendant crown ether groups were prepared. Their cation‐sensitive behaviors were studied by dynamic light scattering. When ionic strength is not controlled, adding salts causes the microgel particles to deswell. However, when the salt effect is ruled out by keeping a constant ionic strength, adding Pb²⁺ results in much larger swelling. The Pb²⁺‐induced swelling was explained by the formation of host–guest complex between Pb²⁺ and the pendant crown ether groups, which increases the hydrophilicity of the polymer and accordingly the degree of swelling. The lead sensitivity of the microgels increases with increasing crown ether content. For the modified microgel with the highest crown ether content, it swells to ～430% of its original volume at [Pb²⁺] = 10 mM. Other cations also increase the swelling degree of the modified microgels. The extent of the cation‐induced swelling mainly depends on their affinity to the pendant crown ether groups. © 2010 Wiley Periodicals, Inc. J Polym Sci Part A: Polym Chem 48: 4120–4127, 2010     

Effect of interferents present in the internal solution or in the conducting polymer transducer on the responses of ion-selective electrodes

The effect of interferents present on the opposite side of the Pb²⁺-selective membrane has been studied for both internal solution and all-solid-state sensors with a conducting polymer (CP) transducer. For interferents with moderate selectivity coefficients (sodium cations) present in the internal solution or in the CP transducer phase, super-Nernstian responses were obtained. For sensors containing strongly discriminated interferents (lithium ions), however, responses typical of conventional electrodes are observed, despite the low activity of primary ions on the opposite side of the membrane. This effect is attributed to hindered incorporation of interfering ions into the membrane, which also impairs the long term stability of the potential. Because of the relatively small absolute amounts of interferents in the transducer of all-solid-state sensors, their exchange for primary ions occurs quickly. Thus, transformation of the sensor to one with a micromolar detection limit and high potential stability is observed.      

Bio-inspired colorimetric detection of Hg<Superscript>2+</Superscript> and Pb<Superscript>2+</Superscript> heavy metal ions using Au nanoparticles

Heavy metal ions are highly toxic species which can cause long-term damage to biological systems. These species are known to disrupt biological events at the cellular level, cause significant oxidative damage, and are carcinogens. The production of simple, in-field detection methods that are highly sensitive for these cations is highly desirable in response to global pollution. In that regard, bio-inspired colorimetric sensing systems have been developed to detect Hg²⁺ and Pb²⁺, and other cations, down to nmol L⁻¹ concentrations. The benefits of these systems, which are reviewed herein, include cost-effective production, facile usage, and a visual color change for the detection method. Such advantages are significant positive steps for heavy metal ion detection, especially in regions where sophisticated laboratory studies are prohibited. Figure Biological-based colorimetric detection of heavy metal cations. The materials on the left are independent Au nanoparticles in solution, functionalized with heavy metal binding biomolecules, which, upon metal addition, aggregate to evolve a detectable solution color change.

Size prediction of recombinant human growth hormone nanoparticles produced by supercritical fluid precipitation

Solution-enhanced dispersion by supercritical fluids (SEDS) was applied to produce nano-sized recombinant human growth hormone (hGH) particles. Ethanol was used to help the supercritical carbon dioxide to extract water from the aqueous protein solution. Various sizes of hGH nanoparticles were successfully prepared with a narrow particle size distribution from aqueous ethanol solution without using any additive. The theoretical particle sizes were deduced from the calculated droplet sizes based on a modified Jasuja’s equation. The calculated mean particle sizes and the experimentally obtained ones were compared and the results showed an excellent correlation coefficient (R ²) of 0.995. Figure Distribution of hGH Nano-particles     

DNA cleavage promoted by Cu2+ complex of cyclen containing pyridine subunit

The tetraazamacrocycle crown ether (cyclen) containing two pyridine subunits was prepared by a modified procedure and the interaction of its metal complexes with DNA was studied by agarose gel electrophoresis analysis. The results indicate that the Cu²⁺ complex as nuclease model can promote the hydrolysis of phosphodiester bond of supercoiled DNA. The rate of degradation of the supercoiled DNA (form I) to nicked DNA (form II) obtained at physiological condition in the presence of 2.14 mM Cu²⁺ complex is 2.31 × 10^–3 min⁻¹. The dependence of the rate of supercoiled DNA cleavage from the complex concentration shows an unusual profile and a hydrolytic cleaving mechanism of two monometallic complexes through cooperation from two-point binding to DNA is proposed.

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Graphical abstract DNA cleavage promoted by metal complex of cyclen containing pyridine subunit Ying Li, Xiao-Min Lu, Xin Sheng, Guo-Yuan Lu*, Ying Shao and Qiang Xu* The copper complex of tetraazamacrocycle crown ether (cyclen) containing two pyridine subunits can promote the hydrolysis of phosphodiester bond of supercoiled DNA and a hydrolytic mechanism of two monometallic complexes through cooperation from two-point binding to DNA is proposed.

     

Recent advances in capillary electrophoretic analysis of individual cells

Because variability exists within populations of cells, single-cell analysis has become increasingly important for probing complex cellular environments. Capillary electrophoresis (CE) is an excellent technique for identifying and quantifying the contents of single cells owing to its small volume requirements and fast, efficient separations with highly sensitive detection. Recent progress in both whole-cell and subcellular sampling has allowed researchers to study cellular function in the areas of neuroscience, oncology, enzymology, immunology, and gene expression.      

Colorimetric detection of immunoglobulin G by use of functionalized gold nanoparticles on polyethylenimine film

A method based on use of functionalized gold nanoparticles on polyethylenimine film has been developed for colorimetric detection of immunoglobulin G (IgG). The immunogold nanoparticles were immobilized on quartz slides by recognition between antibody and antigen, with the antigen chemically adsorbed on the polyethylenimine film. By measurement of the UV–visible spectra of the immobilized immunogold, detection of h-IgG was achieved. The detection limit for h-IgG by use of this method can be as low as 0.01 μg mL⁻¹. This method is quite promising for numerous applications in immunoassay. Figure     

Evaluation of two proteomics technologies used to screen the membrane proteomes of wild-type <Emphasis Type="Italic">Corynebacterium glutamicum</Emphasis> and an <Emphasis Type="SmallCaps">L</Emphasis>-lysine-producing strain

The membrane proteomes of a wild-type Corynebacterium glutamicum and an L-lysine-producing strain were quantitatively analyzed by two complementary proteomics techniques—anion exchange chromatography AIEC/SDS-PAGE and 16BAC-PAGE/SDS-PAGE—and the results were compared. Although both techniques allow for the fast screening of differences in protein abundance, AIEC/SDS-PAGE was superior to 16BAC-PAGE/SDS-PAGE with respect to protein separation, it was more suitable for relative protein quantification, and allowed more differentially regulated proteins to be detected (the succinate dehydrogenase complex, an ABC-type cobalamin/Fe³⁺ siderophore transport system, the maltose binding protein, and a subunit of the cytochrome bc-aa₃ supercomplex were upregulated, while a periplasmic component of an ABC-type transporter and an iron-regulated ABC-type transporter were downregulated in the producer). The results indicate the important role of tricarboxylic acid cycle enzymes as well as the adaptation of transport processes in L-lysine-producing cells. Since the only genetic differences between the wild type and the L-lysine producer occur between four central metabolic enzymes in the cytoplasm, our study illustrates the complex effects of metabolic engineering on cell physiology and the power of the new AIEC/SDS-PAGE proteomics approach to detect these effects.      

Highly sensitive spectrofluorimetric determination of trace amounts of lecithin

A new spectrofluorimetric method was developed for the determination of trace amounts of lecithin using the ciprofloxacin (CIP)–terbium (Tb³⁺) ion complex as a fluorescent probe. In a buffer solution at pH=5.60, lecithin can remarkably reduce the fluorescence intensity of the CIP–Tb³⁺ complex at λ=545 nm. The reduced fluorescence intensity of the Tb³⁺ ion is proportional to the concentration of lecithin. Optimum conditions for the determination of lecithin were also investigated. The linear range and detection limit for the determination of lecithin were 1.0×10⁻⁶–3.0×10⁻⁵ mol L⁻¹ and 3.44×10⁻⁷ mol L⁻¹, respectively. This method is simple, practical, and relatively free of interference from coexisting substances. Furthermore, it has been successfully applied to assess lecithin in serum samples.      

Studies on toxic oil syndrome: development of an enzyme-linked immunosorbent assay for 3-(<Emphasis Type="Italic">N</Emphasis>-phenylamino)propane-1,2-diol in human urine

The fatty acid esters of 3-(N-phenylamino)propane-1,2-diol (PAP) are biomarkers of toxic oil batches that caused toxic oil syndrome (TOS), an intoxication that caused over 400 deaths and affected 20,000 people in Spain in 1981. PAP esters are converted into PAP by human pancreatic lipase. The in vivo biotransformation of PAP in two mouse strains generated potentially toxic metabolites. Here we report an enzyme-linked immunosorbent assay (ELISA) for PAP detection incorporating antibodies generated using PAP-hapten derivatives 1 and 2. The immunizing haptens were designed to recognize the phenylamino and hydroxymethylene moieties of the PAP structure. The antisera raised against 1-HCH showed greater affinity for free PAP, as demonstrated in competitive experiments using either 1-BSA or 2-BSA as coating antigens. The developed ELISA detects PAP at a threshold of 130 μg L⁻¹ and can be used over a wide range of pH and ionic strength values. The assay can be applied to human urine samples, after a simple treatment method, with good recovery according to the correlation obtained when analyzing blind spiked urine samples. Figure Development of an ELISA for PAP in human urine     

Determination of norfloxacin in human urine by capillary electrophoresis with electrochemiluminescence detection

A fast and sensitive approach that can be used to detect norfloxacin in human urine using capillary electrophoresis with end-column electrochemiluminescence (ECL) detection of is described. The separation column was a 75-μm i.d. capillary. The running buffer was 15 mmol L⁻¹ sodium phosphate (pH 8.2). The solution in the detection cell was 50 mmol L⁻¹ sodium phosphate (pH 8.0) and 5 mmol L⁻¹ The ECL intensity varied linearly with norfloxacin concentration from 0.05 to 10 μmol L⁻¹. The detection limit (S/N=3) was 0.0048 μmol L⁻¹, and the relative standard deviations of the ECL intensity and the migration time for eleven consecutive injections of 1.0 μmol L⁻¹ norfloxacin (n=11) were 2.6% and 0.8%, respectively. The method was successfully applied to the determination of norfloxacin spiked in human urine without sample pretreatment. The recoveries were 92.7–97.9%.      

Determining sulfamonomethoxine and its acetyl/hydroxyl metabolites in chicken plasma under organic solvent-free conditions

A quantitative technique is described for a sample preparation followed by high performance liquid chromatography method for the simultaneous determination of sulfamonomethoxine and its metabolites, N ⁴-acetyl SMM and 2,6-dihydroxy SMM, in chicken plasma. The average recoveries, analytical total time, and limits of quantitation were ≥80% (relative standard deviations (SD) ≤6%), <30 min sample-1 (12 samples in 2 h), and ≤0.09 μg ml⁻¹, respectively. The procedure, performed under 100% aqueous conditions, uses no organic solvents and toxic reagents at all and is, therefore, harmless to the environment and humans.      

In vitro translation with [<Superscript>34</Superscript>S]-labeled methionine,selenomethionine, and telluromethionine

Heteroisotope and heteroatom tagging with [³⁴S]-enriched methionine (Met), selenomethionine (SeMet), and telluromethionine (TeMet) was applied to in vitro translation. Green fluorescent protein (GFP) and JNK stimulatory phosphatase-1 (JSP-1) genes were translated with wheat germ extract (WGE) in the presence of Met derivatives. GFPs containing Met derivatives were subjected to HPLC coupled with treble detection, i.e., a photodiode array detector, a fluorescence detector, and an inductively coupled plasma mass spectrometer (ICP-MS). The activities of JSP-1-containing Met derivatives were also measured. GFP and JSP-1 containing [³⁴S]-Met and SeMet showed comparable fluorescence intensities and enzyme activities to those containing naturally occurring Met. TeMet was unstable and decomposed in WGE, whereas SeMet was stable throughout the experimental period. Thus, although Te was the most sensitive to ICP-MS detection among S, Se, and Te, TeMet was less incorporated into the proteins than Met and SeMet. Finally, the potential of heteroisotope and heteroatom tagging of desired proteins in in vitro translation followed by ICP-MS detection was discussed. Figure TeMet was less incorporated into GFP than Met and SeMet due to its instability in WGE     

Determination of selenium in sediment by isotope-dilution inductively coupled plasma mass spectrometry with an octapole reaction cell

A method is described for determination of selenium in sediment by isotope-dilution inductively coupled plasma mass spectrometry with an octapole reaction cell (ID–ICP–ORCMS). Sediment samples were digested with HNO₃, HClO₄, and HF, and the digestion included an elaborate evaporation process to remove bromine from the digested solution. Simple strong cation-exchange disk filtration was used to remove rare earth elements (REE) from the digested solution, because REE²⁺ seriously interfere with Se isotopes (i.e. ¹⁵⁶Gd²⁺ with ⁷⁸Se⁺, ¹⁶⁰Gd²⁺ with ⁸⁰Se⁺). Addition of acetic acid to the filtrate was examined to improve the sensitivity of ICP–ORCMS measurement of Se⁺ by means of a carbon-enhancement effect. The interfering for selenium isotopes were almost eliminated by use of H₂ as reaction gas. Interference from BrH⁺ formed in the reaction cell was negligible because the Br was removed in the evaporation process. Approximately 99.5% of REE were removed by cation-exchange disk filtration yet more than 99% of Se remained in the filtrate solution. The intensity for Se⁺ was enhanced approximately fourfold by addition of 5% (v/v) of acetic acid whereas that for was barely enhanced. Measured ⁸⁰Se/⁷⁸Se ratios in unspiked digested solutions of the sample were in good agreement with that for an Se standard solution. The analytical results for Se in the certified reference materials MESS-3 and PACS-1 were in good agreement with their certified values, with small uncertainties.      

Novel coumarin-based fluorescent pH indicators,probes and membranes covering a broad pH range

A new family of coumarin-based pH indicators was synthesized. They are sensitive to pH in either weakly acidic or weakly basic solution. The indicators possess moderate to high brightness, excellent photostability and compatibility with light-emitting diodes. The indicators were covalently immobilized on the surface of amino-modified polymer microbeads which in turn were incorporated into a hydrogel matrix to obtain novel pH-sensitive materials. When a mixture of two different microbeads is used, the membranes are capable of optical pH sensing over a very wide range comparable to the dynamic range of the glass electrode (pH 1–11). A new family of coumarin-based pH indicators is synthesized for the use in either weakly acidic or weakly basic solution. The indicators possess moderate to high brightness, excellent photostability and compatibility with light-emitting diodes. Novel pH-sensitive materials are obtained by covalent immobilization of the indicators on the surface of amino-modified polymer microbeads which in turn are incorporated into a hydrogel matrix. Sensing of pH over a very wide range also becomes possible.