Enzymatic recycling-based amperometric immunosensor for the ultrasensitive detection of okadaic acid in shellfish

Electrochemical immunosensors based on a competitive indirect enzyme-linked immunosorbent assay (ciELISA) and an enzymatic recycling system were developed for the detection of okadaic acid (OA). OA-ovalbumin (OA-OVA) conjugate was immobilised on screen-printed electrodes (SPEs) and competition of a newly generated monoclonal antibody (MAb) for free and immobilised OA was subsequently performed. Secondary antibodies labelled with alkaline phosphatase (ALP) or horseradish peroxidase (HRP) were used for signal generation. Experimental parameters were firstly optimised by colorimetric ELISA on microtiter wells and on SPEs. The ELISA system was then tested by amperometry at +300 mV vs. Ag/AgCl (detection of p-aminophenol produced by the reaction of p-aminophenyl phosphate with ALP) or -200 mV vs. Ag/AgCl (detection of 5-methyl-phenazinium methyl sulfate, redox mediator in the HRP bioelectrocatalysis). The limits of detection (LODs) with standard solutions were 1.07 and 1.98 microgL(-1) when using ALP and HRP labels, respectively. An electrochemical signal amplification system based on diaphorase (DI) recycling was integrated into the ALP-based immunosensor, decreasing the LOD to 0.03 microgL(-1) and enlarging the working range by two orders of magnitude. Preliminary results with mussel and oyster extracts were obtained and compared with the colorimetric immunoassay, the colorimetric protein phosphatase inhibition assay (PPIA) and LC-MS/MS.     

Regulation of MAPK phosphatase 1 (AtMKP1) by calmodulin in Arabidopsis

The mitogen-activated protein kinases (MAPKs) are key signal transduction molecules, which respond to various external stimuli. The MAPK phosphatases (MKPs) are known to be negative regulators of MAPKs in eukaryotes. We screened an Arabidopsis cDNA library using horseradish peroxidase-conjugated calmodulin (CaM), and isolated AtMKP1 as a CaM-binding protein. Recently, tobacco NtMKP1 and rice OsMKP1, two orthologs of Arabidopsis AtMKP1, were reported to bind CaM via a single putative CaM binding domain (CaMBD). However, little is known about the regulation of phosphatase activity of plant MKP1s by CaM binding. In this study, we identified two Ca(2+)-dependent CaMBDs within AtMKP1. Specific binding of CaM to two different CaMBDs was verified using a gel mobility shift assay, a competition assay with a Ca(2+)/CaM-dependent enzyme, and a split-ubiquitin assay. The peptides for two CaMBDs, CaMBDI and CaMBDII, bound CaM in a Ca(2+)-dependent manner, and the binding affinity of CaMBDII was found to be higher than that of CaMBDI. CaM overlay assays using mutated CaMBDs showed that four amino acids, Trp(453) and Leu(456) in CaMBDI and Trp(678) and Ile(684) in CaMBDII, play a pivotal role in CaM binding. Moreover, the phosphatase activity of AtMKP1 was increased by CaM in a Ca(2+)-dependent manner. Our results suggest that two important signaling pathways, Ca(2+) signaling and the MAPK signaling cascade, are connected in plants via the regulation of AtMKP1 activity. To our knowledge, this is the first report to show that the biochemical activity of MKP1 in plants is regulated by CaM.     

Ca2+ binding site 2 in calcineurin-B modulates calmodulin-dependent calcineurin phosphatase activity

Calcineurin is the Ca(2+)- and calmodulin-dependent Ser/Thr phosphatase. Human calcineurin-Aalpha and wild-type or mutated calcineurin-Bs were coexpressed in Escherichia coli and purified by calmodulin-Sepharose affinity chromatography. Four calcineurin-B mutants were studied. Each had a single conserved Glu in the 12th position of one EF-hand Ca(2+) binding site replaced by a Lys, resulting in the loss of Ca(2+) binding to that site. Phosphatase activities of the enzymes toward a (32)P-labeled phosphopeptide substrate were measured. Inactivating Ca(2+) binding sites 1, 2, or 3 in calcineurin-B reduced Ca(2+)-dependent phosphatase activity of the enzymes in the absence of calmodulin with the site 2 mutation being most effective. Inactivating Ca(2+) binding site 4 did not change enzyme activity or sensitivity to Ca(2+) in either the absence or presence of calmodulin. The calmodulin-dependent phosphatase activity of the enzymes containing site 1, 2, or 3 mutations in calcineurin-B was also decreased compared to enzyme with wild-type calcineurin-B. Of these enzymes, the one with the site 2 mutation was most profoundly affected as determined by the magnitude of the shift in Ca(2+) concentration dependence. Binding of a fluorescein-labeled calmodulin to the wild-type and the site 2 mutant enzymes was examined using fluorescence polarization measurements. The decrease in Ca(2+) sensitivity for the enzyme with calcineurin-B site 2 inactivated is apparently due to a decrease in the affinity of that enzyme for calmodulin at low Ca(2+) concentrations. These data support a role for Ca(2+) binding site 3 in the carboxyl half of calcineurin-B in transmitting the Ca(2+) signal to calcineurin-A and indicate that site 2 in the amino half of calcineurin-B is critical for enzyme activation.     

Assessment of markers of bone formation under controlled environmental factors and their correlation with serum minerals in adult sheep as a model for orthopaedic research

Eighteen healthy skeletally mature (3 years old) ewes, with an average weight of 45 kg, of the Portuguese Churra da Terra Quente breed were used to evaluate the normal values of total and bone-specific isoform of alkaline phosphatase serum activities (ALP and BALP, respectively) and serum osteocalcin (OC) and their correlation with the serum minerals - calcium (Ca), phosphorus (P), magnesium (Mg) and ionized calcium (Ca(2+)). The sheep were maintained under controlled environmental conditions (constant diurnal photoperiod cycle and identical husbandry and feeding) for six weeks before the collection of the blood samples. The measurement of the total ALP and serum minerals was performed with automated biochemistry analysers using the BioMérieux kits, the serum electrolyte Ca(2+) Diametrics Medical, Inc specific cassettes and the BALP and OC METRATM kits from QUIDEL Corporation. The mean +/- standard deviation values obtained were: total ALP 90.17 +/- 85.72 U/L, BALP 15.0 +/- 5.44 U/L, ratio BALP/ total ALP 29.28 +/- 24.22, OC 13.02 +/- 1.87 ng/mL, Ca 2.57 +/- 0.37 mmol/L, P 2.13 +/- 0.42 mmol/L, Mg 1.04 +/- 0.13 mmol/L, Ca(2+) 1.29 +/- 0.04 mmol/L. Significant correlations were observed between the total ALP and Ca (r = 0.5939; P = 0.05) and OC and Ca (r = 0.5706; P = 0.05). Reference to the serum values of bone turnover parameters in sheep could be of great value in research and could provide complementary non-invasive information on the bone healing process, particularly with regard to obtaining an early prognosis of fracture healing.     

Using total fluorescence increase (signal mass) to determine the Ca2+ current underlying localized Ca2+ events

The feasibility of determining localized Ca(2+) influx using only wide-field fluorescence images was explored by imaging (using fluo-3) single channel Ca(2+) fluorescence transients (SCCaFTs), due to Ca(2+) entry through single openings of Ca(2+)-permeable ion channels, while recording unitary channel currents. Since the image obtained with wide-field optics is an integration of both in-focus and out-of-focus light, the total fluorescence increase (DeltaF(total) or "signal mass") associated with a SCCaFT can be measured directly from the image by adding together the fluorescence increase due to Ca(2+) influx in all of the pixels. The assumptions necessary for obtaining the signal mass from confocal linescan images are not required. Two- and three-dimensional imaging was used to show that DeltaF(total) is essentially independent of the position of the channel with respect to the focal plane of the microscope. The relationship between Ca(2+) influx and DeltaF(total) was obtained using SCCaFTs from plasma membrane caffeine-activated cation channels when Ca(2+) was the only charge carrier of the inward current. This relationship was found to be linear, with the value of the slope (or converting factor) affected by the particular imaging system set-up, the experimental conditions, and the properties of the fluorescent indicator, including its binding capacity with respect to other cellular buffers. The converting factor was used to estimate the Ca(2+) current passing through caffeine-activated channels in near physiological saline and to estimate the endogenous buffer binding capacity. In addition, it allowed a more accurate estimate of the Ca(2+) current underlying Ca(2+) sparks resulting from Ca(2+) release from intracellular stores via ryanodine receptors in the same preparation.     

Osteoblastic γ-aminobutyric acid, type B receptors negatively regulate osteoblastogenesis toward disturbance of osteoclastogenesis mediated by receptor activator of nuclear factor κB ligand in mouse bone

The prevailing view is that signaling machineries for the neurotransmitter GABA are also expressed by cells outside the CNS. In cultured murine calvarial osteoblasts, mRNA was constitutively expressed for both subunits 1 and 2 of metabotropic GABA(B) receptor (GABA(B)R), along with inhibition by the GABA(B)R agonist baclofen of cAMP formation, alkaline phosphatase (ALP) activity, and Ca(2+) accumulation. Moreover, baclofen significantly inhibited the transactivation of receptor activator of nuclear factor-κB ligand (RANKL) gene in a manner sensitive to a GABA(B)R antagonist, in addition to decreasing mRNA expression of bone morphogenetic protein-2 (BMP2), osteocalcin, and osterix. In osteoblastic MC3T3-E1 cells stably transfected with GABA(B)R1 subunit, significant reductions were seen in ALP activity and Ca(2+) accumulation, as well as mRNA expression of osteocalcin, osteopontin, and osterix. In cultured calvarial osteoblasts from GABA(B)R1-null mice exhibiting low bone mineral density in tibia and femur, by contrast, both ALP activity and Ca(2+) accumulation were significantly increased together with promoted expression of both mRNA and proteins for BMP2 and osterix. No significant change was seen in the number of multinucleated cells stained for tartrate-resistant acid phosphatase during the culture of osteoclasts prepared from GABA(B)R1-null mice, whereas a significant increase was seen in the number of tartrate-resistant acid phosphatase-positive multinucleated cells in co-culture of osteoclasts with osteoblasts isolated from GABA(B)R1-null mice. These results suggest that GABA(B)R is predominantly expressed by osteoblasts to negatively regulate osteoblastogenesis through down-regulation of BMP2 expression toward disturbance of osteoclastogenesis after down-regulation of RANKL expression in mouse bone.     

A rapid micro method for counting cells “in situ” using a fluorogenic alkaline phosphatase enzyme assay

Summary A new method has been developed to count cells “in situ”, based on a fluorogenic enzyme assay that measures the activity of alkaline phosphatase. Increasing cell number was shown to correlate closely with alkaline phosphatase activity and this relationship did not change with time in culture. The alkaline phosphatase assay (ALP assay) was able to estimate relative cell numbers over a range from about 10⁴ to 5×10⁵ for many cell types, including Hep-2, a derivative of HeLa, several human colorectal cell lines SW1222, SW837, LS174T and HT29, a normal human diploid cell strain MRC5 and a rodent line NIH-3T3. The ALP assay is rapid and efficient, making it a useful method for studying growth assays. Editor's Statement This paper describes a quick method for quantitation of cell number in microcultures. Such procedures are valuable for the many situations in which minimizing cells and medium volume is desirable, although somewhat specialized equipment is required for the procedure. An alternative procedure for quantitation of cells in microtiter culture appeared previously in this journal (McCaffrey, et al., 24∶247–252).     

A telomerase enzymatic assay that does not use polymerase chain reaction, radioactivity, or electrophoresis

A telomerase assay has been developed for high-throughput screening in 96-well microtiter plates. A crude cell lysate which adds telomere repeats to a biotinylated DNA primer is the source of telomerase. The telomerase-extended primer is hybridized to a digoxigenin-labeled telomere antisense DNA probe. The hybrid is further processed by enzyme-linked immunosorbent assay (ELISA) as follows. The biotinylated hybrid is captured on streptavidin-coated microtiter plates. The immobilized hybrid is probed with alkaline phosphatase-antidigoxigenin and detected via chemiluminescent readout. The limit of detection of a chemically synthesized tetra-telomere repeat was about 10 attomoles. Apparent telomerase activity was detected in lysates of 293T cells. The signal to background for the assay (ratio of signal for the complete assay mixture divided by the signal for the assay mixture without primer) was around 10. An automated system that performed unattended runs of up to 17 96-well microtiter plates in 8h was constructed.     

Mlo, a modulator of plant defense and cell death, is a novel calmodulin-binding protein. Isolation and characterization of a rice Mlo homologue

Transient influx of Ca(2+) constitutes an early event in the signaling cascades that trigger plant defense responses. However, the downstream components of defense-associated Ca(2+) signaling are largely unknown. Because Ca(2+) signals are mediated by Ca(2+)-binding proteins, including calmodulin (CaM), identification and characterization of CaM-binding proteins elicited by pathogens should provide insights into the mechanism by which Ca(2+) regulates defense responses. In this study, we isolated a gene encoding rice Mlo (Oryza sativa Mlo; OsMlo) using a protein-protein interaction-based screening of a cDNA expression library constructed from pathogen-elicited rice suspension cells. OsMlo has a molecular mass of 62 kDa and shares 65% sequence identity and scaffold topology with barley Mlo, a heptahelical transmembrane protein known to function as a negative regulator of broad spectrum disease resistance and leaf cell death. By using gel overlay assays, we showed that OsMlo produced in Escherichia coli binds to soybean CaM isoform-1 (SCaM-1) in a Ca(2+)-dependent manner. We located a 20-amino acid CaM-binding domain (CaMBD) in the OsMlo C-terminal cytoplasmic tail that is necessary and sufficient for Ca(2+)-dependent CaM complex formation. Specific binding of the conserved CaMBD to CaM was corroborated by site-directed mutagenesis, a gel mobility shift assay, and a competition assay with a Ca(2+)/CaM-dependent enzyme. Expression of OsMlo was strongly induced by a fungal pathogen and by plant defense signaling molecules. We propose that binding of Ca(2+)-loaded CaM to the C-terminal tail may be a common feature of Mlo proteins.     

In situ enzymatic silver enhancement based on functionalized graphene oxide and layer-by-layer assembled gold nanoparticles for ultrasensitive detection of thrombin

A highly specific in situ amplification strategy was designed for ultrasensitive detection of thrombin by combining the layer-by-layer (LBL) assembled amplification with alkaline phosphatase (ALP) and gold nanoparticles (Au) mediated silver deposition. High-density carboxyl functionalized graphene oxide (FGO) was introduced as a nanocarrier for LBL assembling of alkaline phosphatase decorated gold nanoparticles (ALP-Au), which was further adopted to label thrombin aptamer II. After sandwich-type reaction, numerous ALP were captured onto the aptasensor surface and catalyzed the hydrolysis of ascorbic acid 2-phosphate (AAP), which in situ generated ascorbic acid (AA), reducing Ag(+) to Ag nanoparticles (AgNPs) for electrochemical readout. Inspiringly, the in situ amplification strategy with ethanolamine as an effective blocking agent showed remarkable amplification efficiency, very little nonspecific adsorption, and low background signal, which was favorable to enhance the sensitivity of aptasensor. Our novel dramatic signal amplification strategy, with a detection limit of 2.7fM, showed about 2-3 orders of magnitude improvement in the sensitivity for thrombin detection compared to other universal enzyme-based electrochemical assay.     

Interactions of calcineurin A, calcineurin B, and Ca2+.

Calcineurin B (CN-B) is the Ca(2+)-binding, regulatory subunit of the phosphatase calcineurin. Point mutations to Ca(2+)-binding sites in CN-B were generated to disable individual Ca(2+)-binding sites and evaluate contributions from each site to calcineurin heterodimer formation. Ca(2+)-binding properties of four CN-B mutants and wild-type CN-B were analyzed by flow dialysis confirming that each CN-B mutant binds three Ca2+ and that wild-type CN-B binds four Ca2+. Macroscopic dissociation constants indicate that N-terminal Ca(2+)-binding sites have lower affinity for Ca2+ than the C-terminal sites. Each CN-B mutant was coexpressed with the catalytic subunit of calcineurin, CN-A, to produce heterodimers with specific disruption of one Ca(2+)-binding site. Enzymes containing CN-B with a mutation in Ca(2+)-binding sites 1 or 2 have a lower ratio of CN-B to CN-A and a lower phosphatase activity than those containing wild-type CN-B or mutants in sites 3 or 4. Effects of heterodimer formation on Ca2+ binding were assessed by monitoring (45)Ca2+ exchange by flow dialysis. Enzymes containing wild-type CN-B and mutants in sites 1 and 2 exchange (45)Ca2+ slowly from two sites whereas mutants in sites 3 and 4 exchange (45)Ca2+ slowly from a single site. These data indicate that the Ca2+ bound to sites 1 and 2 is likely to vary with Ca2+ concentration and may act in dynamic modulation of enzyme function, whereas Ca(2+)-binding sites 3 and 4 are saturated at all times and that Ca2+ bound to these sites is structural.     

Enzyme-linked immunosorbent assay for the determination of p21-activated kinase activity

An enzyme-linked immunosorbent assay (ELISA) for the measurement of p21-activated kinase (PAK) activity is described. The development of this method takes advantage of the fact that a phospho-epitope-specific antibody against the regulatory autophosphorylation site sequence of PAK was successfully produced, and after being phosphorylated by PAK, a cross-linked peptide containing the autophosphorylation site of PAK could be recognized on immunoblot by this antibody. This procedure involves coating the cross-linked peptide on microtiter plates, phosphorylating the cross-linked peptide by adding active PAK plus ATP.Mg(2+), and detecting peptide phosphorylation using the phospho-epitope-specific antibody and secondary antibody conjugated with alkaline phosphatase followed by reaction with p-nitrophenyl phosphate (for colorimetric detection) or fluorescein diphosphate (for fluorimetric detection). The PAK activity detected by this method was linearly proportional to the amount of kinase used in the reaction and to the duration of the kinase reaction. Furthermore, fluorimetric detection proved more sensitive than colorimetric detection in terms of both detection limit and signal magnitude. Kinase inhibitor assay revealed that the IC(50) value of staurosporine obtained by this ELISA was very close to that obtained in radioassay. Besides staurosporine, the inhibitory activity of several kinase inhibitors was also tested by the PAK ELISA. The results taken together demonstrate the feasibility and efficacy of this solid phase method for the measurement of PAK activity in a non-radioactive way. Development of this method can be helpful in further high-throughput screening of potential inhibitors of this kinase.     

Ca(2+) function in photosynthetic oxygen evolution studied by alkali metal cations substitution

Effects of adding monovalent alkali metal cations to Ca(2+)-depleted photosystem (PS)II membranes on the biochemical and spectroscopic properties of the oxygen-evolving complex were studied. The Ca(2+)-dependent oxygen evolution was competitively inhibited by K(+), Rb(+), and Cs(+), the ionic radii of which are larger than the radius of Ca(2+) but not inhibited significantly by Li(+) and Na(+), the ionic radii of which are smaller than that of Ca(2+). Ca(2+)-depleted membranes without metal cation supplementation showed normal S(2) multiline electron paramagnetic resonance (EPR) signal and an S(2)Q(A)(-) thermoluminescence (TL) band with a normal peak temperature after illumination under conditions for single turnover of PSII. Membranes supplemented with Li(+) or Na(+) showed properties similar to those of the Ca(2+)-depleted membranes, except for a small difference in the TL peak temperatures. The peak temperature of the TL band of membranes supplemented with K(+), Rb(+), or Cs(+) was elevated to approximately 38 degrees C which coincided with that of Y(D)(+)Q(A)(-) TL band, and no S(2) EPR signals were detected. The K(+)-induced high-temperature TL band and the S(2)Q(A)(-) TL band were interconvertible by the addition of K(+) or Ca(2+) in the dark. Both the Ca(2+)-depleted and the K(+)-substituted membranes showed the narrow EPR signal corresponding to the S(2)Y(Z)(+) state at g = 2 by illuminating the membranes under multiple turnover conditions. These results indicate that the ionic radii of the cations occupying Ca(2+)-binding site crucially affect the properties of the manganese cluster.     

Sandwich hybridisation assay for quantitative detection of yeast RNAs in crude cell lysates

Background  

A rapid microtiter plate based sandwich hybridization assay was developed for detection and quantification of single RNA species using magnetic beads. Following solution hybridization target RNA molecules were collected by biotin-streptavidin affinity binding and detected by fluorescence signal generated by alkaline phosphatase. The 18S rRNA and SUC2 mRNA of Saccharomyces cerevisiae were used as model RNA target molecules.     

Effects of dimethyl sulfoxide and polycationic neomycin on stimulation of purified plasma membrane Ca(2+)-pump by negatively charged phospholipids.

At least two reaction steps are involved in the activation of purified plasma membrane Ca(2+)-transport ATPase by negatively charged phospholipids depending on the type of phospholipids (Lehotsky et al. 1992). The effect of negatively charged phospholipids on Ca(2+)-stimulated ATPase (cycling activity) was compared with that of p-nitrophenylphosphatase (E2-form activity) catalyzed by Ca(2+)-pump. PIP like PS, activated Ca(2+)-ATPase activity by modifying ATP activation curve with increasing Vmax of the high affinity site. Ca(2+)-ATPase activity reconstituted in PC was stimulated by DMSO(10%) by a factor of 1.36. The activity stimulation by DMSO was only weak in PS and activity was inhibited in PIP. Also, phosphatase activity catalyzed by Ca(2+)-pump was strongly stimulated by DMSO and was differentially affected by phospholipid head group. Positively charged neomycin (5 mmol/l) had no effect on Ca(2+)-ATPase activity reactivated in PC or PS, but the stimulatory action of PIP was suppressed. Relative stimulation of phosphatase activity by PS was not influenced. Both hydrolytic activities catalyzed by Ca(2+)-transport ATPase were differentially affected by organic solvents and polycations with respect to the kind of the phospholipid.     

Calcineurin,a Type 2B Protein Phosphatase,Modulates the Ca2+-Permeable Slow Vacuolar Ion Channel of Stomatal Guard Cells

The slowly activating vacuolar (SV) channel of plant vacuoles is gated open by cytosolic free Ca2+ and by cytosol-positive potentials. Using vacuoles isolated from broad bean guard cell protoplasts, SV-mediated currents could be measured in the whole-vacuole configuration of a patch clamp as the time-dependent increase in current at cytosol-positive voltages. Time-dependent deactivation of the SV currents when changing from activating to nonactivating voltages (tail currents) was used to calculate the selectivity of the channel to Ca2+ and Cl- with respect to K+. Changing the equilibrium potential for each permeant ion (Ca2+, Cl-, and K+) at least once for individual vacuoles allowed the relative permeabilities (P) of each of these ions to be calculated in a single experiment. The resulting Pca:Pcl:Pk ratio was close to 3:0.1:1. In accord with its characterization as a weakly selective Ca2+ channel, the SV-mediated current density decreased with increasing Ca2+ activity in the vacuole lumen. SV currents were potently modulated by the Ca2+-dependent, calmodulin-stimulated protein phosphatase 2B (calcineurin). At low concentrations ([less than or equal to]0.4 units per mL), calcineurin stimulated SV currents by ~60%, whereas at higher concentrations the phosphatase was inhibitory, reaching ~90% inhibition at 3 units per mL. Bovine calmodulin had no direct effect on SV-mediated currents, although calcineurin stimulated by exogenous calmodulin inhibited SV currents at all concentrations tested with half-maximal inhibition for calcineurin at 0.16 units per mL. The inhibitory effect of calcineurin could be blocked by the pyrethroid deltamethrin, indicating inhibition of SV channels by calcineurin via dephosphorylation. A model is discussed in which vacuolar Ca2+ release through SV channels is subject to both positive feedforward and negative feedback control through cytosolic Ca2+ and dephosphorylation, respectively.     

Two nonradioactive assays for phosphotyrosine phosphatases with activity toward the insulin receptor.

Two highly sensitive, nonradiolabeled assays for protein phosphotyrosine phosphatase (PTPase) have been developed. The first assay is based on the use of chemically synthesised phosphotyrosine-containing peptides that can be separated from the dephosphorylated peptide products by HPLC. In this assay, partially purified placental PTPase 1B dephosphorylated three dodecaphosphopeptides (corresponding to insulin receptor autophosphorylation sites at positions PY1146, PY1150, and PY1151) with approximately equal affinity (Km 1.3-2.5 microM), indicating that PTPase 1B shows no distinct preference for the site of dephosphorylation in these peptides. The second assay employs either a phosphopeptide or an autophosphorylated tyrosine kinase domain immobolized on microtiter plate wells. After reaction with PTPase, the remaining unconverted phosphosubstrate is detected in an ELISA using anti-phosphotyrosine antibodies. The latter assay was used to monitor PTPase activity during purification procedures and for characterizing PTPases. Modulation of PTPase activity by orthovanadate, heparin, Zn2+, and EDTA gave similar results in both assays. The immobilized autophosphorylated IR tyrosine kinase domain was a poor substrate for bovine liver alkaline phosphatase and seminal fluid acid phosphatase. The second assay also offers the potential for comparing PTPase activity toward several autophosphorylated tyrosine kinase domains, including those of the insulin, epidermal growth factor, and platelet-derived growth factor receptors.     

Mucin secretion induced by titanium dioxide nanoparticles

Nanoparticle (NP) exposure has been closely associated with the exacerbation and pathophysiology of many respiratory diseases such as Chronic Obstructive Pulmonary Disease (COPD) and asthma. Mucus hypersecretion and accumulation in the airway are major clinical manifestations commonly found in these diseases. Among a broad spectrum of NPs, titanium dioxide (TiO(2)), one of the PM10 components, is widely utilized in the nanoindustry for manufacturing and processing of various commercial products. Although TiO(2) NPs have been shown to induce cellular nanotoxicity and emphysema-like symptoms, whether TiO(2) NPs can directly induce mucus secretion from airway cells is currently unknown. Herein, we showed that TiO(2) NPs (<75 nm) can directly stimulate mucin secretion from human bronchial ChaGo-K1 epithelial cells via a Ca(2+) signaling mediated pathway. The amount of mucin secreted was quantified with enzyme-linked lectin assay (ELLA). The corresponding changes in cytosolic Ca(2+) concentration were monitored with Rhod-2, a fluorescent Ca(2+) dye. We found that TiO(2) NP-evoked mucin secretion was a function of increasing intracellular Ca(2+) concentration resulting from an extracellular Ca(2+) influx via membrane Ca(2+) channels and cytosolic ER Ca(2+) release. The calcium-induced calcium release (CICR) mechanism played a major role in further amplifying the intracellular Ca(2+) signal and in sustaining a cytosolic Ca(2+) increase. This study provides a potential mechanistic link between airborne NPs and the pathoetiology of pulmonary diseases involving mucus hypersecretion.