Use of cholinesterase activity as an indicator for the effects of combinations of organophosphorus pesticides in water from environmental sources

Organophosphorus pesticides (OPs) are commonly detected in agricultural products, animal-derived foodstuffs, and environmental samples. Until now, the focus of research has been to evaluate the adverse effect of a single OP. While each OP may be present at concentrations under recognized as “no observed adverse effect level (NOAEL)”, the combined effects of multiple OPs present at these low concentrations have not been sufficiently studied. Therefore, we developed an in vitro testing method to evaluate the toxicity of multiple OPs based on the degree of inhibition of cholinesterase (ChE) activity. This method requires only 10 min to complete and no specialized technology. We examined 15 OPs by this method and categorized them into three groups according to the degree of ChE inhibition. A relationship between the OPs’ chemical structures and the degree of ChE inhibition emerged with the moiety –P–O–CN– showing the strongest action. The degree of ChE inhibition increased with multiple OPs, and the degree of inhibition seemed to be additive. These results demonstrate that the combined toxicity of multiple OPs present in food or environmental samples is an easily determined and toxicologically relevant measure of overall toxicity of complex OPs mixtures. It is possible to apply this testing method as a monitoring technique in water quality management in order to control OPs. As a result, this method can play the role for the potential risk reduction to the ecosystem and may contribute to the preservation of the environment.     

Late asthmatic response causes peripheral airway hyperresponsiveness in dogs treated with metopirone

To determine if late asthmatic response (LAR) is associated with hyperresponsiveness of airway smooth muscle itself, we performed antigen challenge in dogs treated with Metopirone. We studied the contractile response to acetylcholine (ACh) in isolated bronchial and bronchiolar segments 8 h after either saline inhalation (the control group) or antigen challenge in dogs demonstrating immediate asthmatic response (IAR) alone and in dogs demonstrating both IAR and LAR. Airway responses to Ascaris suum antigen were assessed by changes in respiratory resistance measured with the forced oscillation technique at 3 Hz. Concentration-response curves of bronchial preparations to ACh did not differ significantly among three groups consisting of the control, IAR and LAR. However, the contractile response of bronchiolar preparations to ACh was significantly greater in the LAR group when compared to the control and IAR groups at the concentrations of ACh ranging from 10(-6) to 3 x 10(-4) M (p < 0.01). SQ 29548, a receptor antagonist of thromboxane A2 and prostaglandin D2 (PGD2), inhibited LAR-induced hyperresponsiveness to ACh in a concentration-dependent fashion. The bronchiolar preparations obtained from dogs showing LAR contained a significantly higher amount of PGD2 than those obtained from dogs showing IAR alone (p < 0.01, n = 6). These results suggest that LAR is associated with hyperresponsiveness of peripheral airway smooth muscle to ACh, and this augmented response to ACh mediates via PGD2 released during LAR.     

Alkoxyresorufin O-dealkylase assay using a rat hepatocyte spheroid microarray

Hepatocyte multicellular aggregates (spheroids), which maintain high expression of liver functions, have been advocated as a useful culture technique for various cell-based assays. In this study, we investigated the drug metabolic function of a hepatocyte spheroid microarray (HSM) chip, which contained an array of 672 spheroids of primary rat hepatocytes within a 100-mm² region in the center of a poly(methylmethacrylate) plate (24 × 24 mm) and used an alkoxyresorufin (ethoxy-, methoxy-, pentoxy- and benzyloxyresorufin) O-dealkylase assay system. Ethoxyresorufin O-dealkylase (EROD) activity of the HSM chip initiated by 3-methylcholanthrene (3-MC), an inducer of cytochrome P450 enzymes, was 5- to 10-fold higher than that of monolayer hepatocytes, with activity being maintained for at least 2 weeks. We also demonstrated that 3-MC induced EROD, methoxyresorufin O-dealkylase (MROD) and benzyloxyresorufin O-dealkylase (BROD) activities in the HSM chip, while sodium phenobarbital (P450 inducer) induced pentoxyresorufin O-dealkylase (PROD), BROD, EROD and MROD activities. Induction of these activities was confirmed by increased gene expression of the related P450 enzymes. These results showed that the HSM chip had a good response to P450 inducers and that function was maintained for long periods of time. The HSM chip therefore may be a promising cellular platform for drug metabolic assays using hepatocytes.     

A novel l-amino acid ligase from bacillus licheniformis

l-Amino acid alpha-ligase (EC 6.3.2.28) catalyzed formation of alpha-peptide bond in unprotected l-amino acids in an ATP-dependent manner. BL00235 gene in Bacillus licheniformis NBRC12200 coded as a new l-amino acid ligase. BL00235 substrate specificity was strict; only methionine or leucine was acceptable as dipeptide N-terminal residues.     

Micropatterned organoid culture of rat hepatocytes and HepG2 cells

The culture of liver cell organoids (multicellular aggregates) such as spheroids or cylindroids, which can strongly express liver functions, has been advocated as a useful technique that has advantages over monolayer culture. This paper describes a micropatterning technique for obtaining spheroids and cylindroids by using rat hepatocytes or HepG2 cells. We developed culture chips that comprised multiple, circular or rectangular microwells; the bottom surface of each microwell was modified with collagen to create a cell adhesion area, and the entire microwell, excluding the collagen-coated spots, was modified with polyethylene glycol (PEG) to create a nonadhesive area. Rat hepatocytes and HepG2 cells formed uniform spheroids and cylindroids on the circular and rectangular chips, respectively. Consequently, two-dimensional micropatterned chips containing homogeneous spheroids or cylindroids were generated. The expression of liver functions (protein secretion and ammonia removal) was greater in the spheroids and cylindroids than in the monolayer culture, and this expression was maintained for at least 2 weeks of culture. Thus, this chip technology has potential for use in various applications that involve organoid culture.     

Full scale investigation of GCL damage mechanisms in small earth dam retrofit applications under earthquake loading

This paper reports results of full scale testing to further explore potential GCL damage mechanisms in earth dam retrofit applications in seismically active areas; in particular, to a) investigate whether shear displacements could reduce the magnitude of GCL panel overlap during earthquake shaking; b) explore the influence of gravel particles on GCL thickness at localised point of contact; and c) observe the consequences of an accidental exposure of an uncovered GCL to short duration rainfall in terms of moisture content and effects during subsequent compaction. The results of these experiments indicate that even under severe shaking no movements were detected at the GCL panel overlap. Whereas gravel particles were observed to locally reduce the thickness of the GCL to 2.2 mm, no plowing of the particle into the GCL occurred due to a lack of shear displacement at the interface, resulting in no localised internal erosion through the barrier. Furthermore, hydration of GCL panels during construction due to surface wetting was observed to result in a state of hydration less than its post-construction state. These results indicate that although each of the three GCL damage mechanisms cannot be ruled out to ever be relevant in practice, the performance of the GCL retrofitted earth dam tested was satisfactory under even severe Level 2 earthquake shaking, and suggests that the retrofitting of small earth dams with GCLs is a promising strategy to improve their static and seismic resistance.     

A Restart Control Method for Position Sensorless PMSM Drive Systems Without Potential Transformer for Railway Vehicle Traction

This paper proposes a restart control method for position sensorless PMSM drive systems without a potential transformer for railway vehicle traction. This method can estimate the initial rotor speed and position under coasting conditions over the entire speed range. The method can also be used when the back‐EMF voltage is higher than the inverter DC link voltage. The proposed method is verified by experiments using a 200‐kW PMSM.