Electrochemical detection of extracellular hydrogen peroxide released from RAW 264.7 murine macrophage cells based on horseradish peroxidase-hydroxyapatite nanohybrids

A new method developed for the reliable determination of extracellular and intracellular H(2)O(2) is very useful for gaining a full understanding of the role that H(2)O(2) plays in pathology and physiology, and the relationship between H(2)O(2) and environmental stresses and lipid peroxidation. This work developed and validated an electrochemical approach for the determination of extracellular H(2)O(2) released from RAW 264.7 murine macrophage cells. This approach is based on the electrocatalytic reduction of the released H(2)O(2) at the biosensor of HRP-HAP/GC, which was fabricated by depositing the horseradish peroxidase-hydroxyapatite (HRP-HAP) nanohybrids on a glassy carbon (GC, 3 mm in diameter) electrode. The biosensor exhibited a rapid response (less than 2 s), a low detection limit (0.1±0.02 μM), a wide linear range (5 μM to 0.82 mM), as well as good stability and repeatability. In addition, the common interfering species, such as uric acid (UA), ascorbic acid (AA), glucose, and 3,4-dihydroxyphenylacetic acid (DOPAC), etc., did not cause any interference due to the use of a low operating potential (-400 mV, versus SCE). Therefore, this work has demonstrated a simple and effective sensing platform for the detection of extracellular H(2)O(2) released from cells such as RAW 264.7 cells, which has potential utility to bioelectroanalytical chemistry, cellular biology, and pathophysiology.     

Conformational changes of hapten-protein conjugates resulting in improved broad-specificity and sensitivity of an ELISA for organophosphorus pesticides

The type of hapten linkage to a carrier protein can play an important role in determining the nature of the resulting antibody response. Generic haptens using three types of linkers were synthesized (a monocarboxylic acid, an unsaturated hydrocarbon and a carboxamido spacer). These haptens were conjugated to bovine serum albumin (BSA) and used as immunogens to produce broad-specificity monoclonal antibodies (MAbs) to organophosphorus pesticides (OPs). Three-dimensional (3D) structures of hapten-lysine conjugates were optimized using molecular modeling (MM) to mimic conformations of hapten-BSA conjugates. The results from MM studies revealed a change of the 3D conformation and electrostatic potential of hapten 1 when the monocarboxylic acid linker was coupled to lysine. This result was consistent with the observed high-cross-reactivity of the corresponding MAb-H1 for the OPs. The competitive indirect enzyme-linked immunosorbent assay based on MAb-H1 is ideally suited to be used as a screening method for OP contaminants.     

Extraction tool and matrix effects on arsenic speciation analysis in cell lines

Arsenic glutathione (As–GSH) complexes have been suggested as possible metabolites in arsenic (As) metabolism. Extensive research has been performed on the toxicological and apoptotic effects of As, while few reports exist on its metabolism at the cellular level due to the analytical challenges. In this study, an efficient extraction method for arsenicals from cell lines was developed. Evaluation of extraction tools; vortex, ultrasonic bath and ultrasonic probe and solvents; water, chemicals (methanol and trifluoroacetic acid), and enzymes (pepsin, trypsin and protease) was performed. GSH effect on the stability of As–GSH complexes was studied. Arsenic metabolites in dimethylarsino glutathione (DMA(GS)) incubated multiple myeloma cell lines were identified following extraction. Intracellular GSH concentrations of myeloma cell lines were imitated in the extraction media and its corresponding effect on the stability and distribution of As metabolites was studied. An enhancement in both extraction recoveries and time efficiency with the use of the ultrasonic probe was observed. Higher stabilities for the As species in water, pepsin and trypsin were obtained. The presence of 0.5 mM GSH in the extraction media (PBS, pH 7.4) could not stabilize the As–GSH complexes compared to the 5 mM GSH, where high stabilization of the complexes was observed over a 5 day storage study. Finally, the speciation analysis of the DMA(GS) culture incubated cell lines in the presence or absence of GSH revealed the important role GSH plays in the preservation of DMA(GS) identity. Hence, caution is required during the extraction of arsenicals especially the As–GSH complexes, since their identification is highly dependent on GSH concentration.     

Two New Cyclic Pentapeptides from the Marine‐Derived Fungus Aspergillus versicolor

Two new cyclic pentapeptides, named versicotides A ( 1 ) and B ( 2 ), were obtained from a marine‐derived fungus strain ZLN‐60, identified as Aspergillus versicolor. Their structures were established on the basis of chemical and spectroscopic evidence. Versicotides are new cyclic pentapeptides which contain an L ‐alanine residue, two anthranilic acid (=2‐aminobenzoic acid) residues, and two N‐methyl‐L ‐alanine residues. Antitumor activities were evaluated by the SRB and MTT methods.     

Bruceines K and L from the Ripe Fruits of Brucea javanica

Bruceine K ( 1 ), a pentacyclic C₂₀‐quassinoid bearing a unique 12,20‐epoxy moiety, and bruceine L ( 2 ), along with the ten known compounds (6S,7E)‐6,9,10‐trihydroxy‐ and (6S,7E)‐6,9‐dihydroxymegastigma‐4,7‐dien‐3‐one ( 3 and 4 , resp.), cleomiscosins A–C, luteoline, quercetine, bruceantinol, pinoresinol, and thevetiaflavone, were isolated from the ripe fruits of Brucea javanica. Bruceines K ( 1 ) and L ( 2 ) were determined to be (1β,2α,11β,12β,14ξ,15β)‐12,20‐epoxy‐1,2,11,13,14,15‐hexahydroxypicras‐3‐en‐16‐one and (1β,2α,11β,12β,15β)‐13,20‐epoxy‐1,2,11,12‐tetrahydroxy‐16‐oxo‐15‐(senecioyloxy)picras‐3‐en‐21‐oic acid methyl ester (senecioic acid=3‐methylbut‐2‐enoic acid), respectively, on the basis of NMR (¹H‐ and ¹³C‐NMR, DEPT, ¹H,¹H‐COSY, NOESY, HMQC, and HMBC) and ESI‐MS data. Among the known compounds, (6S,7E)‐6,9,10‐trihydroxy‐ and (6S,7E)‐6,9‐dihydroxymegastigma‐4,7‐dien‐3‐one ( 3 and 4 , resp.), cleomiscosin C, luteoline, quercetine, and thevetiaflavone were isolated for the first time from the Brucea plants.     

New Phenylphenalene Derivatives from Water Hyacinth (Eichhornia crassipes)

Water hyacinth (Eichhornia crassipes) is a cause of great concern in terms of environmental and agricultural impacts in many parts of the world. Phytochemical investigation of water hyacinth led to the isolation of six new phenylphenalenes, 2,3‐dihydro‐3,9‐dihydroxy‐5‐methoxy‐4‐phenyl‐1H‐phenalen‐1‐one ( 1 ), 2,3‐dihydro‐8‐methoxy‐9‐phenyl‐1H‐phenalene‐1,4‐diol ( 2 ), 2,3‐dihydro‐4,8‐dimethoxy‐9‐phenyl‐1H‐phenalen‐1‐ol ( 3 ), 2,3‐dihydro‐9‐(4‐hydroxyphenyl)‐8‐methoxy‐1H‐phenalene‐1,4‐diol ( 4 ), 2,6‐dimethoxy‐9‐phenyl‐1H‐phenalen‐1‐one ( 5 ), and 7‐(4‐hydroxyphenyl)‐5,6‐dimethoxy‐1H‐phenalen‐1‐one ( 6 ), together with the four known compounds 7 – 10 . Their structures were elucidated by spectrometric methods including 1D‐ and 2D‐NMR, and MS analysis. These compounds may be involved in allelopathic interactions of water hyacinth with neighboring plants.     

Ultra-high capacity liquid chromatography chip/quadrupole time-of-flight mass spectrometry for pharmaceutical analysis

Nanoflow liquid chromatography/mass spectrometry (nano-LC/MS) has attracted increasing interest in virtue of high sensitivity, low sample consumption, and minimal matrix effect. In this work a HPLC-Chip/quadrupole time-of-flight (Q-TOF) MS device with a new ultra-high capacity small molecule chip (UHC-Chip) which features a 500 nL enrichment column and a 150 mm × 75 μm analytical column, was evaluated with a drug mixture covering a wide range of polarities. Excellent chromatographic precision with 0.1-0.5% RSD for retention time and 1.7-9.0% RSD for peak area, low limit of detection, good chip-to-chip reproducibility and linearity were obtained by using this UHC-Chip. Compared with the standard HPLC-Chip with 40 nL trapping column, the UHC-Chip showed higher enrichment capability and hence gave a higher response in signal detection. Additionally, 4-30 times increase in sensitivity was obtained compared with conventional LC/MS, which indicated that UHC-Chip/MS was a valuable tool for the quantitative analysis of low level impurities and degradation products in pharmaceuticals. Moreover, satisfactory results obtained from trace drug analysis of serum samples further proved its practicality and potential for use in drug testing and development.     

Efficient removal of boron acid by N-methyl-D-glucamine functionalized silica-polyallylamine composites and its adsorption mechanism

A novel boron adsorbent was fabricated by grafting a boric acid chelating group, i.e., N-methyl-D-glucamine, onto the hydrophilic silica-polyallylamine composites (SPC). The boron adsorbent was characterized by scanning electron microscopy (SEM) and TGA method. The adsorption experiment indicated a maximum boron load capacity of ca. 1.55 mmol g(-1). The high load capacity was attributed to specific chemical affinity and physical adsorption. Highly effective removal of boric acid from aqueous solution was observed for the adsorbent even in the synthetic seawater containing high concentration of foreign ions. Analysis of adsorption thermodynamic and kinetics revealed a spontaneous sorption process that is driven by enthalpy change and limited by chemical reaction. The exhausted adsorbent was regenerated for repeated use by treating with 3% HCl solution, followed by neutralizing with 3% NH(3)·H(2)O at ambient temperature. Only 7% capacity loss was observed after five continuous adsorption-regeneration cycles.     

Separation of exenatide analogue mono-PEGylated with 40 kDA polyethylene glycol by cation exchange chromatography

Random PEGylation usually resulted in product mixtures composed of mono-PEGylated isomers and multi-PEGylated attachments. Generally in PEGylation research, separation of the mono-PEGylated isomers was the prerequisite for finding the optimal PEGylation site. However, when peptides or proteins were PEGylated with polyethylene glycol as large as 40 kDA, the physicochemical properties like hydrophobicity and molecular size of the isomers would become too similar to make the routine separation methods, like RP-HPLC, size-exclusion chromatography, SDS-PAGE and capillary isoelectric focusing invalid. This article presented a useful method of successfully separating exenatide analogue (an incretin for diabetic therapy) isomers mono-PEGylated with 40 kDA polyethylene glycol by cation exchange chromatography, which would be a powerful tool for the PEGylation research.     

Star-shaped polymers for DNA sequencing by capillary electrophoresis

The separation and sequencing of DNA are the main objectives of the Human Genome Project, and this project has also been very useful for gene analysis and disease diagnosis. Capillary electrophoresis (CE) is one of the most common techniques for the separation and analysis of DNA. DNA separations are usually achieved using capillary gel electrophoresis (CGE) mode, in which polymer gel is packed into the capillary. Compared with a traditional CGE matrix, a hydrophilic polymer matrix, which can be adsorb by the capillary wall has numerous advantages, including stability, reproducibility and ease of automation. Various water-soluble additives, such as linear poly(acrylamide) (PAA) and poly(N,N-dimethylacrylamide) (PDMA), have been employed as media. In this study, different star-shaped PDMA polymers were designed and synthesized to achieve lower polymer solution viscosity. DNA separations with these polymers avoid the disadvantages of high viscosity and long separation time while maintaining high resolution (10 bp between 271 bp and 281 bp). The influences of the polymer concentration and structure on DNA separation were also determined in this study; higher polymer concentration yielded better separation performance, and star-like polymers were superior to linear polymers. This work indicates that modification of the polymer structure is a potential strategy for optimizing DNA separation.