Isolation and Purification of Organophosphorus Hydrolases Secreted from Acetone-acclimated Phosphorus Accumulating Organisms and Study of Their Properties for Hydrophobic Organophosphorus Sensor.

The present work studied an acclimation method for phosphorus accumulating organisms (PAOs) with a high content of acetone in culture solutions to develop microbial-based enzyme sensors for highly hydrophobic organophosphorus (OP) pesticides. Through three steps of cultivation and acclimation, only rod-shaped bacteria survived among the various PAOs. The extracellular enzymes released from the acclimated PAOs were salted out by using ammonium sulfate, then purified by a dialysis membrane and a DEAE-Sepharose FF anion exchange column. Two enzyme components were successfully separated-both of which showed hydrolase activity on disodium p-nitrophenyl phosphate (enzyme I, 1.57 µmol/(min·µg); enzyme II, 0.88 µmol/(min·µg) at 45°C). Further, SDS-PAGE gel electrophoresis results showed that the molecular weights of enzymes I and II were about 15.11 and 11.98 kDa, respectively. On this basis, the applicability of the enzyme in hydrophobic OP biosensors was demonstrated.

A hydrophobic ionic liquid compartmentalized sampling/labeling and its separation techniques in polydimethylsiloxane microchip capillary electrophoresis.

This paper demonstrates a novel compartmentalized sampling/labeling method and its separation techniques using a hydrophobic ionic liquid (IL)-1-butyl-3-methylimidazolium bis(trifluoromethylsulfonyl)-imidate (BmimNTf2 )-as the immiscible phase, which is capable of minimizing signal losses during microchip capillary electrophoresis (MCE). The MCE device consists of a silica tube connected to a straight polydimethylsiloxane (PDMS) separation channel. Poly(diallyldimethylammonium chloride) (PDDAC) was coated on the inner surface of channel to ease the introduction of IL plugs and enhance the IL wetting on the PDMS surface for sample releasing. Electroosmotic flow (EOF)-based sample compartmentalization was carried out through a sequenced injection into sampling tubes with the following order: leading IL plug/sample segment/terminal IL plug. The movement of the sample segment was easily controlled by applying an electrical voltage across both ends of the chip without a sample volume change. This approach effectively prevented analyte diffusion before injection into MCE channels. When the sample segment was manipulated to the PDDAC-modified PDMS channel, the sample plug then was released from isolation under EOF while IL plugs adsorbed onto channel surfaces owing to strong adhesion. A mixture of flavin adenine nucleotides (FAD) and flavin mononucleotides (FMN) was successfully separated on a 2.5 cm long separation channel, for which the theoretical numbers of plates were 15 000 and 17 000, respectively. The obtained peak intensity was increased 6.3-fold over the corresponding value from conventional electrokinetic injection with the same sampling time. Furthermore, based on the compartmented sample segment serving as an interim reactor, an on-chip fluorescence labeling is demonstrated.

Tempol reduces injury area in rat model of spinal cord contusion injury through suppression of iNOS and COX-2 expression.

The present study focused on the biologic effects of tempol on anti-inflammatory and nitric oxide generation in contusion spinal cord injury (SCI). The animal model of SCI was induced by dropping a 10-g rod (2.0 mm in diameter) at a height of 25 mm. Tempol was injected intraperitoneally a dose of 100 mg/kg at 15 min before SCI. Controls was injected with saline. The contused spinal segments were removed according to time courses, and the expression level of cyclooxygenase-2 (COX-2) and inducible nitric oxide synthase (iNOS) was analyzed along with the size of irreversibly damaged region. After SCI, the relative amounts of COX-2 and iNOS mRNA were peaked at 8 h after post-injury, and then decreased up to 7 days post-injury, and normal level at 14 days. Expression of COX-2 protein was peaked at 8 h post-injury. With the tempol pre-treatment, the immunoreactivity of COX-2 and nitrotyrosine in paraffin-embedded tissue slices was profoundly decreased. The irreversibly damaged area of the spinal cord was peaked at 3 days after SCI. With tempol pre-treatment, the irreversibly damaged area shows a statistically significant decrease at 3 days after SCI. These evidences indicate that tempol pre-treatment reduces irreversibly damaged area on the contusion SCI in rat. The mechanisms of biologic reactions of tempol might be related to the decreased expressions of COX-2 and iNOS in spinal cord cells, neurons and glia. It is expected that the tempol effect on the SCI is not only antioxidant activity but also anti-inflammatory reaction.

The Role of Inducible Nitric Oxide Synthase Following Spinal Cord Injury in Rat

Acute spinal cord injury (SCI) is two-step process that first involves the primary mechanical injury and then the secondary injury is induced by various biochemical reactions. Apoptosis is one of secondary SCI mechanisms and it is thought to play an important role for the delayed neuronal injury. The enhanced formation of nitric oxide (NO) via inducible nitric oxide synthase (iNOS) has been implicated in the pathogenesis of apoptosis in SCI. The level of .iNOS mRNA peaked at 6 hr after SCI and it declined until 72 hr after SCI in a rat model. Double-immunofluorescence staining revealed that iNOS positive cells were stained for ED-1, synaptophysin, GFAP, and oligodendrocyte marker. The terminal deoxynucleotidyl-transferase-mediated dUDP-biotin nick end-labeling (TUNEL) positive cell count was higher for the 72 hr post-SCI group than for the 24 hr post-SCI group. This cell count was also higher going in the caudal direction than in the rostral direction from the epicenter, and especially for the 72 hr group. Treatment with a selective iNOS inhibitor resulted in the reduction of TUNEL-positive cells at the lesion site. These findings suggest that nitric oxide generated by the iNOS of macrophages, neurons, oligodentrocytes, and astrocytes plays an important role for the acute secondary SCI that results from apoptotic cell death.