Efficient preparation of surface imprinted magnetic nanoparticles using poly (2-anilinoethanol) as imprinting coating for the selective recognition of glycoprotein.

In view of the significance of glycoprotein biomarkers for early clinical diagnostics and treatments of diseases, it is essential to develop efficient and selective enrichment approaches for glycoproteins. Molecularly imprinted polymers (MIPs) have found important applications for separation and enrichment of glycoproteins. In this study, we use boronate affinity-based controllable oriented surface imprinting to prepare glycoprotein-imprinted magnetic nanoparticles. A glycoprotein was first immobilized onto the surface of boronic acid functionalized magnetic nanoparticles by boronate affinity. Subsequently, self-polymerization of 2-anilinoethanol was carried out to form thin imprinting coating on the magnetic nanoparticles surface with appropriate thickness. After removing the template with an acidic solution containing sodium dodecyl sulfate, 3D cavities complementary to the template were efficiently formed in the imprinting layer. The imprinting coating was highly hydrophilic and presented limited residual boronic acid, thus non-specific binding was avoided. Using horseradish peroxidase as a model target, the effects of imprinting conditions on the properties and performance of the prepared MIPs were investigated. The obtained MIPs exhibited several highly favorable features, including excellent specificity, high binding strength and low binding pH. The MIPs were successfully applied to the analysis of transferrin (TRF) in human serum.

[Segmental and regional innervation of acupoint "Taixi" (KI 3) area in the rat: a horseradish peroxidase method study].

OBJECTIVE: To study the peripheral and central neural involvements of acupoint "Taixi" (KI 3) by using Horseradish Peroxidase (HRP). METHODS: Ten male SD rats were used in the present study. 5 pL of 1% HRP was injected into the excavate site between the apex of medial malleolus and the Achilles tendon of the left hind foot, a corresponding site of acupoint "Taixi" (KI 3) in the human body. After 36 surviving hours, the rat's brain, spinal cord and dorsal root ganglia (DRG) of the lumbar segments (L) were dissected from the perfused experimental animals, and their tissue sections were examined with 3, 3', 5, 5' -tetramethyl-benzidine histochemistry for revealing HRP-labeling. RESULTS: All the HRP-labeling appeared ipsilaterally to the injection side. Numerous HRP-labeled neurons were detected in the DRGs of L4 - L6 segments with their number being L5> L4 >L6. A longitudinal column of HRP-labeled motoneurons was found in the dorsolateral and mediolateral portions of the spinal cord, distributing in the lamina IX from the caudal L4 to the rostral L6. Additionally, the transganglionic HRP-labeled central prolection axonal terminals were found to be dense in the central part of laminae I-II from L4 to the rostral L6, and to scatter in the central part of gracile nucleus. CONCLUSION: HRP-labeled primary afferent and efferent innervating acupoint "Taixi" (KI 3) are DRGs of L4-L6, the dorsolateral and mediolateral motoneuron columns of L4-L6, and the centrally projecting axonal terminals of laminae I-II of the spinal cord and the gracile nucleus.

Effects of oxidizing adulterants on detection of 11-nor-delta9-THC-9-carboxylic acid in urine.

Bleach, nitrite, chromate, and hydrogen peroxide-peroxidase are effective urine adulterants used by the illicit drug users to conceal marijuana-positive results. Methods for detecting nitrite and chromate are available. Effects of other oxidizing agents that could possibly be used as adulterants and are difficult to detect or measure are presented in this report. Urine samples containing 40 ng/mL of 11-nor-delta9-THC-9-carboxylic acid (THC-acid) were treated with 10 mmol/L of commonly available oxidizing agents. Effects of horseradish peroxidase of activity 10 unit/mL and extracts from 2.5 g of red radish (Raphanus sativus, Radicula group), horseradish (Armoracia rusticana), Japanese radish (Raphanus sativus, Daikon group), and black mustard seeds (Brassica nigra), all with 10 mmol/L of hydrogen peroxide, were also examined. After 5 min, 16 h and 48 h of exposure at room temperature (23 degrees C) the specimens were tested by a gas chromatographic-mass spectrometric method for THC-acid. A control group treated with sodium hydrosulfite to reduce the oxidants, was also tested to investigate the effect of oxidizing agents on THC-acid in the extraction method. THC-acid was lost completely in the extraction method when treated with chromate, nitrite, oxone, and hydrogen peroxide/ferrous ammonium sulfate (Fenton's reagent). Some losses were also observed with persulfate and periodate (up to 25%). These oxidants, and other oxidizing agents like permanganate, periodate, peroxidase, and extracts from red radish, horseradish, Japanese radish and black mustard seeds destroyed most of the THC-acid (> 94%) within 48 h of exposure. Chlorate, perchlorate, iodate, and oxychloride under these conditions showed little or no effect. Complete loss was observed when THC-acid was exposed to 50 mmol/L of oxychloride for 48 h. Several oxidizing adulterants that are difficult to test by the present urine adulterant testing methods showed considerable effects on the destruction of THC-acid. The time and temperature for these effects were similar to those used by most laboratories to collect and test specimens. In several cases, the loss of THC-acid was > 94%.

Functional properties of wasabi and horseradish.

Wasabi (Wasabi japonica) and horseradish (Cholearia arnoracia) are used as spices of daily foodstuffs. Allylisothiocyanate (AIT) is a potent component in both plants and occurs by grating them. It is well known that AIT shows inhibitory effect on the growth of food poisoning bacteria and fungi. In this work, several functional properties of roots and leaves from wasabi and horseradish were examined in vitro. Each sample showed peroxidase activity. They also exhibited antioxidative and superoxide scavenging potency. Antimutagenic activity was observed toward 2-amino-3,8-dimethylimidazo[4,5-f]quinoxaline [MeIQx], a well-known mutagen/carcinogen in broiled fish and meat. They also decreased His+ revertant colonies of 3-chloro-4-dichloromethyl-5-hydroxy-2(5H)-furanone (MX) in the Ames test, a strong mutagen and carcinogen in chlorine disinfected tap water. Isolation of antimutagenic components in wasabi root was done. Three components including (-)-(R)-7-methylsulfinylheptyl isothiocyanate were identified. These data show that wasabi and horseradish might be potent functional foods for keeping human health.

Impairment of sterol biosynthesis leads to phosphorus and calcium accumulation in Leishmania acidocalcisomes.

The induction of the formation of inclusion vesicles in Leishmania amazonensis by the sterol biosynthesis inhibitors (SBI) ketoconazole and terbinafine has been reported previously. These compartments were recently identified as acidocalcisomes. By the use of electron spectroscopic imaging and energy loss spectroscopy, the presence of calcium, phosphorus and oxygen in the electron-dense inclusions located within the acidocalcisomes has been demonstrated. Endoplasmic reticulum cisternae formed membrane whorls which enclosed large portions of the cytoplasm and sometimes circumscribed acidocalcisomes. In addition, acid phosphatase activity, as well as the endocytic tracers horseradish peroxidase and gold-labelled transferrin and cystatin C were detected within these organelles in both SBI-treated and untreated parasites. These data suggest that impairment of sterol biosynthesis induces the biogenesis of acidocalcisomes and triggers an autophagic process that leads to intersection of the endosomal/lysosomal system with the acidocalcisomes.

Ultrastructural changes in the blood-brain barrier in rats after treatment with nimodipine and flunarizine. A comparison.

The idea of using induced hypertension to treat the symptomatic ischaemia resulting from vasospasm after subarachnoidal hemorrhage, and the effect of this therapy on the blood-brain barrier, is checked in animal experiments. This therapy is combined with the application of nimodipine, which is recognised as the standard medication for prophylaxis of vasospasm. The effects of the induced hypertension combination with Nimodipine and in combination with another calcium antagonist, Flunarizine are compared. Seventy-four narcotised rats, one group with 22 animals treated with Nimodipine and 22 with placebo, and a second group 20 animals treated with Flunarizine and 10 with placebo, are evaluated. The blood pressure is raised to 150-180 mmHg by i.v. application of norfenephrine and measured continuously. The standard tracer, horseradish peroxidase, is applied as indicator for the blood-brain barrier function. 15 minutes later the experimental animals are exsanguinated by perfusion with saline, then perfused with Karnovsky's solution. After removal, the brains are stained for peroxidase to visualise extravasation of the horseradish peroxidase, and after evaluation of the results each brain is assigned to its experimental group. In the Nimodipine group, a significant accumulation (p < 0.001) of perivascular deposits of peroxidase reaction product were found, these were not found in the placebo group. The Flunarizine group does not differ from its placebo group in the number of extravasates, and thus, with respect to protein extravasation, appears better than the Nimodipine group. In electron micrographs of the extravasates one sees intact tight junctions and a neuroendothelial transport, and also vesicles, filled with horseradish peroxidase in the endothelium, the muscle cells, and the brain parenchyma, which arise from pinocytosis. The vesicles, which transport the high-molecular-weight protein, horseradish peroxidase, also transport other proteins and can, therefore, cause a brain edema. It follows from these morphological results that Nimodipine can disrupt the blood brain barrier function and can, therefore, also interfere with cerebral autoregulation, which depends on the resistance of vessels.

Location of single neurons containing HRP for electron microscopy.

A technique is described for finding single neurons containing transported horseradish peroxidase (HRP) for study in the electron microscope. Tissue chopper sections which have been reacted to demonstrate HRP are cleared in glycerol and examined with darkfield light microscopy. The granular reaction product is clearly seen, enabling accurate drawings to be made for later dissection for ultramicrotomy. The neuron sought can then be found within a few microns of the cutting face of the block. While some distortion does occur, most of the cellular morphology remains intact, enabling recognition of specific organelles such as neurosecretory granules in cells which have transported HRP from the neurohypophysis.