Simultaneous determination of catecholamines, uric acid and ascorbic acid at physiological levels using poly(N-methylpyrrole)/Pd-nanoclusters sensor

An interesting electrochemical sensor has been constructed by the electrodeposition of palladium nanoclusters (Pd_nano) on poly(N-methylpyrrole) (PMPy) film-coated platinum (Pt) electrode. Cyclic voltammetry, electrochemical impedance spectroscopy (EIS), and scanning electron microscopy were used to characterize the properties of the modified electrode. It was demonstrated that the electroactivity of the modified electrode depends strongly on the electrosynthesis conditions of the PMPy film and Pd_nano. Moreover, the modified electrode exhibits strong electrocatalytic activity toward the oxidation of a mixture of dopamine (DA), ascorbic acid (AA), and uric acid (UA) with obvious reduction of overpotentials. The simultaneous analysis of this mixture at conventional (Pt, gold [Au], and glassy carbon) electrodes usually struggles. However, three well-resolved oxidation peaks for AA, DA, and UA with large peak separations allow this modified electrode to individually or simultaneously analyze AA, DA, and UA by using differential pulse voltammetry (DPV) with good stability, sensitivity, and selectivity. This sensor is also ideal for the simultaneous analysis of AA, UA and either of epinephrine (E), norepinephrine (NE) or l-DOPA. Additionally, the sensor shows strong electrocatalytic activity towards acetaminophen (ACOP) and other organic compounds. The calibration curves for AA, DA, and UA were obtained in the ranges of 0.05 to 1 mM, 0.1 to 10 μM, and 0.5 to 20 μM, respectively. The detection limits (signal/noise [S/N] = 3) were 7 μM, 12 nM, and 27 nM for AA, DA, and UA, respectively. The practical application of the modified electrode was demonstrated by measuring the concentrations of AA, DA, and UA in injection sample, human serum, and human urine samples, respectively, with satisfactory results. The reliability and stability of the modified electrode gave a good possibility for applying the technique to routine analysis of AA, DA, and UA in clinical tests.     

Green tea epigallocatechin-3-gallate inhibits microsomal prostaglandin E2 synthase-1

Prostaglandin (PG)E₂ is a critical lipid mediator connecting chronic inflammation to cancer. The anti-carcinogenic epigallocatechin-3-gallate (EGCG) from green tea (Camellia sinensis) suppresses cellular PGE₂ biosynthesis, but the underlying molecular mechanisms are unclear. Here, we investigated the interference of EGCG with enzymes involved in PGE₂ biosynthesis, namely cytosolic phospholipase (cPL)A₂, cyclooxygenase (COX)-1 and -2, and microsomal prostaglandin E₂ synthase-1 (mPGES-1). EGCG failed to significantly inhibit isolated COX-2 and cPLA₂ up to 30 μM and moderately blocked isolated COX-1 (IC₅₀ > 30 μM). However, EGCG efficiently inhibited the transformation of PGH₂ to PGE₂ catalyzed by mPGES-1 (IC₅₀ = 1.8 μM). In lipopolysaccharide-stimulated human whole blood, EGCG significantly inhibited PGE₂ generation, whereas the concomitant synthesis of other prostanoids (i.e., 12(S)-hydroxy-5-cis-8,10-trans-heptadecatrienoic acid and 6-keto PGF_1α) was not suppressed. Conclusively, mPGES-1 is a molecular target of EGCG, and inhibition of mPGES-1 is seemingly the predominant mechanism underlying suppression of cellular PGE₂ biosynthesis by EGCG.     

The effect of estradiol on COX-2, EP2, and EP4 mRNA expression and the extracellular matrix in the cervix of the hypogonadotrophic, ovariectomized ewe

There is a degree of cervical relaxation in the ewe at estrus that is regulated by changes in prostaglandin synthesis, prostaglandin receptor expression, and changes in the cervical extracellular matrix. It is likely that these are regulated by changes in periovulatory hormones, particularly estradiol. This study determined the effect of estradiol benzoate on the mRNA expression of cyclooxygenase-2 (COX-2) and the prostaglandin E receptors EP₂ and EP₄, the concentration of cervical hyaluronan, and the proportion of smooth muscle and collagen in the cervix of the hypogonadotrophic ovariectomized ewe (Ovis aries). Ovariectomized hypogonadotrophic ewes were given 100 μg estradiol benzoate, and their cervices were collected 0, 24, and 48 h thereafter to determine the expression of cervical COX-2, EP₂, and EP₄ mRNA by in situ hybridization, the concentration of hyaluronan by ELISA, and the proportion of smooth muscle and collagen by Masson's trichrome staining. Estradiol benzoate increased the mRNA expression of COX-2 and EP₄ within 24 h after treatment (P < 0.05), whereas EP₂ mRNA, hyaluronan, and the ratio of smooth muscle to collagen did not change within 48 h after treatment. The COX-2, EP₂, and EP₄ mRNA expression were greatest in the smooth muscle layers (P < 0.05) and least in the luminal epithelium (P < 0.05). In conclusion, we inferred that estradiol regulates cervical COX-2 and EP₄ mRNA expression and may regulate cervical relaxation via the synthesis of prostaglandin E₂ and activation of the PGE₂ receptors EP₂ and EP₄.     

Prostaglandin E2 modulates components of the Wnt signaling system in bone and prostate cancer cells

Both Wnt signaling and prostaglandin E₂ (PGE₂) play pivotal roles in bone development, remodeling, osteoporosis and prostate cancer (PCa) bone metastases. We investigated the effects of PGE₂ on Wnt signaling in osteoblast-lineage cells and Wnt-inhibitor expression in PCa cells. We demonstrate that low dose PGE₂ (0.1 μM) promotes Wnt signaling while higher doses of PGE₂ (1.0-10 μM) inhibit these same parameters in osteoblast-lineage cells. The differential effects of low vs high-dose PGE₂ on pre-osteoblasts may be attributed to dose-dependent modulation of prostaglandin receptor (EP) subtype expression; with lower doses increasing the expression the cAMP-stimulatory EP4 receptor subtype and higher doses increasing the expression of the cAMP-inhibitory EP3 receptor subtype. Moreover, we demonstrate that high expression levels of COX-2 and PGE₂ promote the secretion of Wnt inhibitors from prostate cancer cells. These data demonstrate that there are dose-dependent effects of PGE₂ on Wnt activation in osteoblast-lineage cells and Wnt-inhibitor expression in PCa cells which may have clinical implications in the management.     

The role of the central arachidonic acid-thromboxane A2 cascade in cardiovascular regulation during hemorrhagic shock in rats

The aim of the current study was to elucidate the underlying central mechanism(s) of the cardiovascular effects evoked by centrally injected melittin and arachidonic acid (AA) in hemorrhaged hypotensive condition, specifically, from central AA release from the cell membrane under the influence of phospholipase A₂ (PLA₂) to central thromboxane A₂ (TXA₂) signaling via the cyclooxygenase (COX) pathway. As the main control of the study, melittin (3 μg) or AA (150 μg) was injected intracerebroventricularly (i.c.v.) after the hemorrhage procedure, which was performed by withdrawing a total volume of 2.2 ml of blood/100 g body weight over a period of 10 min. Both treatments generated a pressor response and abolished the hypotension-induced hemorrhage. Pretreatment with the PLA₂ inhibitor mepacrine (500 μg; i.c.v.) completely blocked the pressor response to melittin in the hemorrhagic hypotensive state. Pretreatments with the nonselective COX inhibitor indomethacin (200 μg; i.c.v.) or the TXA₂ synthesis inhibitor furegrelate (250 or 500 μg; i.c.v.) were made to test the role of central COX activity and, subsequently, the TXA₂ signaling pathway in the melittin- or AA-mediated reversal of hemorrhagic hypotension. Indomethacin completely prevented the pressor response to melittin and AA in the hemorrhaged, hypotensive state, but furegrelate did so only partially.In conclusion, these findings suggest that central COX activity and, subsequently, the central TXA₂ signaling pathway, are, at least in part, involved in the melittin- or AA-induced reversal effect during hemorrhagic shock.     

Genetic deletion of mPGES-1 accelerates intestinal tumorigenesis in APC(Min/+) mice

The induced synthesis of bioactive prostanoids downstream of cyclooxygenase-2 (COX-2) and prostaglandin H₂ (PGH₂) exerts a critical event in colorectal carcinogenesis. Here we demonstrate that APC^Min/+ mice with genetic deletion of microsomal prostaglandin E synthase-1 (mPGES-1), which catalyses the terminal conversion of PGH₂ into PGE₂, surprisingly develop more and generally larger intestinal tumors than do mPGES-1 wild type littermates (mean number of tumors/intestine 80 vs. 38, p < 0.0005, mean tumor diameter 1.64 vs. 1.12 mm, p < 0.0005). No deviation regarding the expression of other PGE₂ related enzymes (COX-1, COX-2, mPGES-2, cPGES, and 15-PGDH) or receptors (EP1-4) was obvious among the mPGES-1 deficient mice. PGE₂ levels were suppressed in tumors of mPGES-1 deficient animals, but the concentrations of other PGH₂ derived prostanoids were generally enhanced, being most prominent for TxA₂ and PGD₂. Thus, we hypothesise that a redirected synthesis towards other lipid mediators might (over)compensate for loss of mPGES-1/PGE₂ during intestinal tumorigenesis. Nevertheless, our results question the suitability for mPGES-1 targeting therapy in the treatment or prevention of colorectal cancer.     

miR-143 decreases COX-2 mRNA stability and expression in pancreatic cancer cells

Small non-coding RNAs, microRNAs (miRNA), inhibit the translation or accelerate the degradation of message RNA (mRNA) by targeting the 3′-untranslated region (3′-UTR) in regulating growth and survival through gene suppression. Deregulated miRNA expression contributes to disease progression in several cancers types, including pancreatic cancers (PaCa). PaCa tissues and cells exhibit decreased miRNA, elevated cyclooxygenase (COX)-2 and increased prostaglandin E₂ (PGE₂) resulting in increased cancer growth and metastases. Human PaCa cell lines were used to demonstrate that restoration of miRNA-143 (miR-143) regulates COX-2 and inhibits cell proliferation. miR-143 were detected at fold levels of 0.41 ± 0.06 in AsPC-1, 0.20 ± 0.05 in Capan-2 and 0.10 ± 0.02 in MIA PaCa-2. miR-143 was not detected in BxPC-3, HPAF-II and Panc-1 which correlated with elevated mitogen-activated kinase (MAPK) and MAPK kinase (MEK) activation. Treatment with 10 μM of MEK inhibitor U0126 or PD98059 increased miR-143, respectively, by 187 ± 18 and 152 ± 26-fold in BxPC-3 and 182 ± 7 and 136 ± 9-fold in HPAF-II. miR-143 transfection diminished COX-2 mRNA stability at 60 min by 2.6 ± 0.3-fold in BxPC-3 and 2.5 ± 0.2-fold in HPAF-II. COX-2 expression and cellular proliferation in BxPC-3 and HPAF-II inversely correlated with increasing miR-143. PGE₂ levels decreased by 39.3 ± 5.0% in BxPC-3 and 48.0 ± 3.0% in HPAF-II transfected with miR-143. Restoration of miR-143 in PaCa cells suppressed of COX-2, PGE₂, cellular proliferation and MEK/MAPK activation, implicating this pathway in regulating miR-143 expression.     

A nanocomposite/crude extract enzyme-based xanthine biosensor

A novel amperometric biosensor for xanthine was developed based on covalent immobilization of crude xanthine oxidase (XOD) extracted from bovine milk onto a hybrid nanocomposite film via glutaraldehyde. Toward the preparation of the film, a stable colloids solution of core–shell Fe₃O₄/polyaniline nanoparticles (PANI/Fe₃O₄ NPs) was dispersed in solution containing chitosan (CHT) and H₂PtCl₆ and electrodeposited over the surface of a carbon paste electrode (CPE) in one step. Scanning electron microscopy (SEM), Fourier transform infrared (FTIR) spectrophotometry, cyclic voltammetry (CV), and electrochemical impedance spectroscopy (EIS) were used for characterization of the electrode surface. The developed biosensor (XOD/CHT/Pt NPs/PANI/Fe₃O₄/CPE) was employed for determination of xanthine based on amperometric detection of hydrogen peroxide (H₂O₂) reduction at –0.35 V (vs. Ag/AgCl). The biosensor exhibited a fast response time to xanthine within 8 s and a linear working concentration range from 0.2 to 36.0 μM (R² = 0.997) with a detection limit of 0.1 μM (signal/noise [S/N] = 3). The sensitivity of the biosensor was 13.58 μA μM⁻¹ cm⁻². The apparent Michaelis–Menten (K_m) value for xanthine was found to be 4.7 μM. The fabricated biosensor was successfully applied for measurement of fish and chicken meat freshness, which was in agreement with the standard method at the 95% confidence level.     

Synthesis and evaluation of 1,5-diaryl-substituted tetrazoles as novel selective cyclooxygenase-2 (COX-2) inhibitors

A series of 1,5-diaryl-substituted tetrazole derivatives was synthesized via conversion of readily available diaryl amides into corresponding imidoylchlorides followed by reaction with sodium azide. All compounds were evaluated by cyclooxygenase (COX) assays in vitro to determine COX-1 and COX-2 inhibitory potency and selectivity. Tetrazoles 3a-e showed IC₅₀ values ranging from 0.42 to 8.1 mM for COX-1 and 2.0 to 200 μM for COX-2. Most potent compound 3c (IC₅₀ (COX-2) = 2.0 μM) was further used in molecular modeling docking studies.     

Large-scale expression, purification, and characterization of an engineered prostacyclin-synthesizing enzyme with therapeutic potential

Recently, we reported that a novel hybrid enzyme (TriCat enzyme), engineered by linking human cyclooxygenase-2 (COX-2) with prostacyclin (PGI₂) synthase (PGIS) together through a transmembrane domain, was able to directly integrate the triple catalytic (TripCat) functions of COX-2 and PGIS and effectively convert arachidonic acid (AA) into the vascular protector, PGI₂ [K.H. Ruan, H. Deng, S.P. So, Biochemistry 45 (2006) 14003-14011]. In order to confirm the important biological activity and evaluate its therapeutic potential, it is critical to characterize the properties of the enzyme using the purified protein. The TriCat enzyme cDNA was subcloned into a baculovirus vector and its protein was expressed in Sf-9 cells in large-scale with a high-yield (∼4% of the total membrane protein), as confirmed by Western blot and protein staining. The Sf-9 cells’ membrane fraction, rich in TriCat enzyme, exhibited strong TriCat functions (K_m = 3 μM and K_cat = 100 molecules/min) for the TriCat enzyme and was 3-folds faster in converting AA to PGI₂ than the combination of the individual COX-2 and PGIS. Another superiority of the TriCat enzyme is its dual effect on platelet aggregation: it completely inhibited platelet aggregation at the low concentration of 2 μg/ml and then displayed the ability to reverse the initially aggregated platelets to their non-aggregated state. Furthermore, multiple substrate-binding sites were confirmed in the single protein by high-resolution NMR spectroscopy, using partially purified TriCat enzyme. These studies have clearly demonstrated that the isolated TriCat enzyme protein functions in the selective biosynthesis of the vascular protector, PGI₂, and revealed its potential for anti-thrombosis therapeutics.     

Differential role of cyclooxygenase-1 and -2 on renal vasoconstriction to α1-adrenoceptor stimulation in normotensive and hypertensive rats

Aims

Hypertension is associated with the impairment of renal cyclooxygenase (COX) activity, which regulates vascular tone, salt and water balance and renin release. We aimed to evaluate the functional role of COX isoforms in kidneys isolated from spontaneously hypertensive rats (SHR) after α₁-adrenoceptor (α₁-AR) stimulation.

Main methods

Male six-month-old SHR and normotensive Wistar-Kyoto rats (WKY) were used. The kidneys were isolated to measure perfusion pressure and COX-1- or COX-2-derived prostanoids in response to α₁-AR activation.

Key findings

The basal perfusion pressure was higher in SHR kidneys compared with WKY kidneys (95 ± 11 vs. 68 ± 6 mm Hg, P < 0.05). Phenylephrine induced a greater vasopressor response in SHR kidneys (EC₅₀ of 1.89 ± 0.58 nmol) than WKY kidneys (EC₅₀ of 3.30 ± 0.54 nmol, P < 0.05 vs. SHR). COX-1 inhibition decreased the α₁-AR-induced vasoconstrictor response in WKY but did not affect SHR response, while COX-2 inhibition diminished the response in SHR. Both basal prostacyclin (PGI₂) and thromboxane A₂ (TxA₂) values were higher in SHR kidney perfusates (P < 0.05) and were reduced by COX-1 and COX-2 inhibitors in both strains. Furthermore, phenylephrine increased PGI₂ through COX-2 in WKY and through COX-1 in SHR, but the agonist did not significantly modify TxA₂ in both strains.

Significance

The data suggest that COX-1contributes to vasoconstrictor effects in WKY kidneys and that COX-2 has the same effect in SHR kidneys. The results also suggest that basal release of COX-2-derived vasoconstrictor prostanoids is involved in renal vascular hypersensitivity in SHR.     

Photo-crosslinking of proteins in intact cells reveals a dimeric structure of cyclooxygenase-2 and an inhibitor-sensitive oligomeric structure of microsomal prostaglandin E2 synthase-1

We have characterized the structures of cyclooxygenase-2 (COX-2) and microsomal prostaglandin E₂ synthase-1 (mPGES-1) in intact cells using bifunctional and photo-activatable crosslinking agents. A dimeric complex was detected for COX-2 by both crosslinking approaches, consistent with the crystal structure of the enzyme. For mPGES-1, treatment of A549 cells with disuccinimidyl suberate yielded immunoreactive protein bands corresponding to a dimer (33 kDa) and a trimer (45 kDa), as observed for the isolated enzyme. Photo-crosslinking with photoactivatable methionine in intact cells generated complexes with molecular weights corresponding to the dimer (33 kDa) and two putative trimer forms (50 and 55 kDa). Treatment with the selective mPGES-1 inhibitor MF63 prevented the formation of the 50 and 55 kDa crosslinked complexes, while an inactive structural analogue had no effect. Our data indicate that COX-2 forms a dimer in intact cells and that mPGES-1 has an oligomeric structure that can be disrupted by a selective inhibitor.     

The development of an electrochemical immunosensor using a thiol aromatic aldehyde and PAMAM-functionalized Fe3O4@Au nanoparticles

In this work, a novel thiol aromatic aldehyde was synthesized. It can be used as a substrate to directly immobilize antibodies on a gold electrode, for which no additional chemical cross-linker is required. It was also applied as a linker to prepare Fe₃O₄@Au/PAMAM/Ab₂–horseradish peroxidase bioconjugates, which introduced multiple enzymes onto a sensing interface owing to the high surface-to-volume ratio of Fe₃O₄@Au nanoparticles and many functional groups of the poly(amidoamine) dendrimer (PAMAM). The introduced multiple enzymes greatly improved the detection signal. Under optimal conditions, the proposed electrochemical immunosensor exhibited desirable performance for detection of IgG in the range 0.005–50 ng ml⁻¹ with a detection limit of 3 pg ml⁻¹ based on a signal-to-noise ratio of 3. It has great potential application in the area of clinical analysis.     

A novel hypothesis regarding the possible involvement of cytosolic phospholipase 2 in insulin-stimulated proliferation of vascular smooth muscle cells

Insulin (INS) via INS receptor acts as a mitogen in vascular smooth muscle cells (VSMCs) through stimulation of multiple signaling mechanisms, including p42/44 mitogen-activated protein kinase (ERK1/2) and phosphatidyl inositol-3 kinase (PI3K). In addition, cytosolic phospholipase 2 (cPLA₂) is linked to VSMCs proliferation. However, the upstream mechanisms responsible for activation of cPLA₂ are not well defined. Therefore, this investigation used primary cultured rat VSMCs to examine the role of PI3K and ERK1/2 in the INS-dependent phosphorylation of cPLA₂ and proliferation induced by INS. Exposure of VSMCs to INS (100 nM) for 10 min increased the phosphorylation of cPLA₂ by 1.5-fold (p < 0.01), which was blocked by the cPLA₂ inhibitor MAFP (10 μM; 15 min). Similarly, the PI3K inhibitor LY294002 (10 μM; 15 min) and ERK1/2 inhibitor PD98059 (20 μM; 15 min) abolished the INS-mediated increase in cPLA₂ phosphorylation by 59% (p < 0.001), and by 75% (p < 0.001), respectively. Further, inhibition of cPLA₂ with cPLA₂ inhibitor MAFP abolished the INS-stimulated ERK1/2 phosphorylation by 65% (p < 0.01). Incubation of rat VSMCs with INS resulted in an increase of VSMCs proliferation by 85% (p < 0.001). The effect of INS on VSMCs proliferation was significantly (p < 0.01) reduced by pretreatment with MAFP. Thus, we hypothesized that INS stimulates VSMCs proliferation via a mechanism involving the PI3K-dependent activation of cPLA₂ and release of arachidonic acid (AA), which activates ERK1/2 and further amplifies cPLA₂ activity.     

Flurbiprofen,A Cyclooxygenase Inhibitor,Reduces the Brain Arachidonic Acid Signal in Response to the Cholinergic Muscarinic Agonist,Arecoline, in Awake Rats

Cholinergic muscarinic receptors, when stimulated by arecoline, can activate cytosolic phospholipase A₂ (cPLA₂) to release arachidonic acid (AA) from membrane phospholipid. This signal can be imaged in the brain in vivo using quantitative autoradiography following the intravenous injection of radiolabeled AA, as an increment in a regional brain AA incorporation coefficient k*. Arecoline increases k* significantly in brain regions having muscarinic M_1,3,5 receptors in wild-type but not in cyclooxygenase (COX)-2 knockout mice. To further clarify the roles of COX enzymes in the AA signal, in this paper we imaged k* following arecoline (5 mg/kg i.p.) or saline in each of 81 brain regions of unanesthetized rats pretreated 6 h earlier with the non-selective COX inhibitor flurbiprofen (FB, 60 mg/kg s.c.) or with vehicle. Baseline values of k* were unaffected by FB treatment, which however reduced by 80% baseline brain concentrations of prostaglandin E₂ (PGE₂) and thromboxane B₂ (TXB₂), eicosanoids preferentially derived from AA via COX-2 and COX-1, respectively. In vehicle-pretreated rats, arecoline increased the brain PGE₂ but not TXB₂ concentration, as well as values for k* in 77 of the 81 brain regions. FB-pretreatment prevented these arecoline-provoked changes. These results and those reported in COX-2 knockout mice suggest that the AA released in brain following muscarinic receptor-mediated activation is lost via COX-2 to PGE₂ but not via COX-1 to TXB₂, and that increments in k* following arecoline largely represent replacement by unesterified plasma AA of this loss.     

Nanoparticles facilitate gene delivery to microorganisms via an electrospray process

In this study, we developed a technique for delivering genes to microorganisms via electrospray of gold nanoparticles. During the electrospray process, charged monodisperse nano-droplets (a mixture of pET30a-GFP plasmid and nano-sized gold particles) were accelerated and deposited on a thin layer of non-competent Escherichia coli cells. Via antibiotic selection, transformed cells containing green fluorescent protein appeared on the agar plates. PCR amplification and restriction enzyme analysis further confirmed that pET30a-GFP plasmid had successfully been delivered into the non-competent E. coli cells. The transformation efficiencies were optimized under different electrospray conditions. Among several electrospray buffer solutions, CaCl₂ (0.01 M) was found to be the best for gene delivery. Furthermore, gold nanoparticles (NPs, 50 nm diameter) significantly improved plasmid transformation efficiency by 5- 7 fold (up to 2 × 10⁶ CFU/μg plasmid) compared with that obtained using naked plasmid. Electronic microscopy images and gel electrophoresis showed that the morphology of plasmids remained unchanged during the electrospray process, but cellular membrane integrity was reduced after being electrosprayed with gold NPs and CaCl₂ buffer solutions. This gene delivery method has the potential to work for many other microorganisms.     

Fluorescein-labeled “arch-like” DNA probes for electrochemical detection of DNA on gold nanoparticle-modified gold electrodes

In the present study, a gold nanoparticle-modified gold electrode (nanogold electrode) was used to develop a novel fluorescein electrochemical DNA biosensor based on a target-induced conformational change. The nanogold electrode was obtained by electrodepositing gold nanoparticles onto a bare gold electrode. This modification not only immobilized probe oligonucleotides, but also adsorbed fluorescein onto the surface of the gold nanoparticles to form an “arch-like” structure. This article compares the electrochemical signal changes caused by the hybridization of “arch-like” DNA on nanogold electrode and linear DNA on bare gold electrode. The results showed that the adsorption effect of nanogold can enhance the sensitivity of the sensor. The linear range of target ssDNA is from 2.0 × 10⁻⁹ M to 2.0 × 10⁻⁸ M with a correlation coefficient of 0.9956 and detection limit (3σ) of 7.10 × 10⁻¹⁰ M. Additionally, the specificity and hybridization response of this simple sensor were investigated.     

Electrochemical assay of plasmin activity and its kinetic analysis

A sensitive and convenient electrochemical assay of plasmin activity and its kinetic analysis are described. Thus, a ferrocenyl peptide substrate (FcPS) having a plasmin-specific substrate sequence, Lys-Thr-Phe-Lys, and a Cys residue was prepared and immobilized on a gold electrode through the sulfur-gold linkage. The obtained electrode showed a redox signal based on the ferrocene moiety, suggesting the immobilization of FcPS on the electrode. After treatment of this electrode with plasmin, its electrochemical signal was decreased in proportion to an increase of the amount of plasmin. The detection limit for plasmin in this assay system was as low as 50 ng/ml or 0.15 mU/ml. Real-time monitoring of plasmin reaction on the electrode could also be achieved, and the kinetic parameters of this enzymatic reaction could be determined; for example, the k_cat/K_m value was 0.063 μM⁻¹ s⁻¹. Furthermore, a quantitative assay for streptokinase as a plasminogen activator was also demonstrated by using this system.     

Design of novel N-phenylnicotinamides as selective cyclooxygenase-1 inhibitors

A series of N-phenylnicotinamides (1-40) were designed and evaluated in vitro for their COX inhibitory activities. Most of the synthesized compounds were proved to be potent and selective inhibitors of COX-1. Compound 28 showed the most potent COX-1 inhibitory activity (COX-1 IC₅₀ = 0.68 ± 0.07 μM) and good selectivity (COX-2 IC₅₀ >100 μM). This compound may be useful as a lead compound for superior COX-1 inhibitors. On the basis of the biological results, structure-activity relationships for the COX-1-inhibitory activities of the synthesized N-phenylnicotinamides were discussed concisely.     

Electrochemical assay for the determination of nitric oxide metabolites using copper(II) chlorophyllin modified screen printed electrodes

This work presents a novel electrochemical assay for the collective measurement of nitric oxide (NO) and its metabolites nitrite (NO₂⁻) and nitrate (NO₃⁻) in volume miniaturized sample at low cost using copper(II) chlorophyllin (CuCP) modified sensor electrode. Zinc oxide (ZnO) incorporated screen printed carbon electrode (SPCE) was used as a host matrix for the immobilization of CuCP. The morphological changes of the ZnO and CuCP modified electrodes were investigated using scanning electron microscopy. The electrochemical characterization of CuCP–ZnO–SPCE exhibited the characteristic quasi-reversible redox peaks at the potential +0.06 V versus Ag/AgCl. This biosensor electrode showed a wide linear range of response over NO concentrations from 200 nM to 500 μM with a detection limit of 100 nM and sensitivity of 85.4 nA μM⁻¹. Furthermore, NO₂⁻ measurement showed linearity of 100 nM to 1 mM with a detection limit of 100 nM for NO₂⁻ and sensitivity of 96.4 nA μM⁻¹. Then, the concentration of NO₃⁻ was measured after its enzymatic conversion into NO₂⁻. Using this assay, the concentrations of NO, NO₂⁻, and NO₃⁻ present in human plasma samples before and after beetroot supplement were estimated using suitable membrane coated CuCP–ZnO–SPCE and validated with the standard Griess method.