A database of naturally occurring human urinary peptides and proteins for use in clinical applications

Owing to its availability, ease of collection and correlation with (patho-) physiology, urine is an attractive source for clinical proteomics. However, the lack of comparable datasets from large cohorts has greatly hindered development in this field. Here we report the establishment of a high resolution proteome database of naturally occurring human urinary peptides and proteins - ranging from 800-17,000 Da - from over 3,600 individual samples using capillary electrophoresis coupled to mass spectrometry, yielding an average of 1,500 peptides per sample. All processed data were deposited in an SQL database, currently containing 5,010 relevant unique urinary peptides that serve as classifiers for diagnosis and monitoring of diseases, including kidney and vascular diseases. Of these, 352 have been sequenced to date. To demonstrate the applicability of this database, two examples of disease diagnosis were provided: For renal damage diagnosis, patients with a specific renal disease were identified with high specificity and sensitivity in a blinded cohort of 131 individuals. We further show definition of biomarkers specific for immunosuppression and complications after transplantation (Kaposi's sarcoma). Due to its high information content, this database will be a powerful tool for the validation of biomarkers for both renal and non-renal diseases

Interaction of organophosphorus compounds with muscarinic receptors in sh-sy5y human neuroblastoma cells

Human neuroblastoma cells (line SH-SY5Y) were used to examine the Interaction of single exposure to organophosphorus compounds (OPs) with muscarinic receptors. In this study, SH-SY5Y cells were exposed for 30 min to concentrations of paraoxon, diisopropyl phosphorofluoridate (DFP), phenyl saligenln cyclic phosphate (PSP), and mipafox (N,Nâ\u80\u99-diisopropyl phosphorodiamide fluoridate) that ranged between 10-9M and 10-3M (10-2M for mipafox). Ability to interfere with muscarinic receptor binding was determined by change in the binding of the nonspecific antagonist [3H]-N-mcthy I scopolamine (3H-NMS). Concentrations of paraoxon >0.5 x 10-3M and PSP 1 x 10-3M significantly inhibited the binding of a saturating concentration of 3H-NMS. Concentrations of >10-5M paraoxon or PSP could significantly inhibit the binding of a half-saturating concentration of 3H-NMS. Studies using specific antagonists for muscarinic subtypes (pirenzepine for M1, AFDX-116 for M2, and 4-DAMP for M3) indicated that SH-SY5Y cells have muscarinic receptors most sensitive to the specific antagonist for the M3 subtype (IC50 of 10-8M for 4-DAMP compared to 2.5 x 10-6M and 2.7 x 10-5M for pirenzepine and AFDX-116, respectively). As M3 receptor stimulation results in formation of inositol phosphates from membrane phospholnositides, the capability of OPs to alter levels of inositol phosphates and agonist-stimulated increases in inositol phosphate formation was examined. Intact cells were prelabeled with [3H]myo-inositol and then incubated for 15 min with the OPs before addition of 10-5M to 10-3M carbachol. Levels of inositol phosphates were determined as the amount of aqueous soluble radiolabeled product extracted from the reaction mixture. Paraoxon and PSP, but not mipafox or DFP, decreased basal levels of inositol phosphates in a concentration-related manner. This could be overcome in cells stimulated with carbachol, a muscarinic agonist, and with sodium fluoride, which does not act at muscarinic receptors. These results indicate that certain OPs, upon acute exposure, interact with muscarinic receptors, but that they also have effects on levels of Inositol phosphates that may be associated with another site of action in SH-SY5Y cells. © 1994 Taylor and Francis

Comparative in vitro study of the inhibition of human and hen esterases by methamidophos enantiomers

The current Organisation for Economic Co-operation and Development (OECD) guidelines for evaluating organophosphorus-induced delayed neuropathy (OPIDN) require the observation of dosed animals over several days and the sacrifice of 48 hens. Adhering to these protocols in tests with enantiomers is difficult because large quantities of the compound are needed and many animals must be utilized. Thus, developing an in vitro screening protocol to evaluate chiral organophosphorus pesticides (OPs) that can induce delayed neuropathy is important. This work aimed to evaluate, in blood and brain samples from hens, human blood, and human cell culture samples, the potential of the enantiomeric forms of methamidophos to induce acetylcholinesterase (AChE) inhibition and/or delayed neurotoxicity. Calpain activation was also evaluated in the hen brain and SH-SY5Y human neuroblastoma cells. The ratio between the inhibition of neuropathy target esterase (NTE) and AChE activities by the methamidophos enantiomers was evaluated as a possible indicator of the enantiomers' abilities to induce OPIDN. The (-)-methamidophos exhibited an IC50 value approximately 6 times greater than that of the (+)-methamidophos for the lymphocyte NTE (LNTE) of hens, and (+)-methamidophos exhibited an IC50 value approximately 7 times larger than that of the (-)-methamidophos for the hen brain AChE. The IC50 values were 7 times higher for the human erythrocyte AChE and 5 times higher for AChE in the SH-SY5Y human neuroblastoma cells. Considering the esterases inhibition and calpain results, (+)-methamidophos would be expected to have a greater ability to induce OPIDN than the (-)-methamidophos in humans and in hens. (C) 2011 Elsevier B.V. All rights reserved.Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP

Mechanisms for consideration for intervention in the development of organophosphorus-induced delayed neuropathy

Organophosphorus-induced delayed neuropathy (OPIDN) is a neurodegenerative disorder characterised by ataxia progressing to paralysis with concomitant central and peripheral distal axonopathy. Symptoms of OPIDN in people include tingling of the hands and feet. This tingling is followed by sensory loss, progressive muscle weakness and flaccidity of the distal skeletal muscles of the lower and upper extremities and ataxia, which appear about 8-14 days after exposure. Some organophosphorus compounds (OPs) that are still used in worldwide agriculture have potential to induce OPIDN, including methamidophos, trichlorfon, dichlorvos and chorpyrifos. This review summarizes experimental attempts to prevent and/or treat OPIDN and the different mechanisms involved in each approach. The initial mechanism associated with development of OPIDN is phosphorylation and inhibition of neuropathy target esterase (NTE). The phosphorylated enzyme undergoes a second reaction known as aging that results in the loss of one of the R groups bound to the phosphorus of the OP. A second mechanism involved in OPIDN is an imbalance in calcium homeostasis. This can lead to the activation of calcium-activated neutral protease and increases in calcium/calmodulin-dependent protein kinases. These events contribute to aberrant phosphorylation of cytoskeletal proteins and protein digestion in the terminal axon that can proceed similarly to Wallerian-type degeneration. Several experimental studies demonstrated alleviation of the signs and symptoms of OPIDN by restoring calcium balance. Other studies have used preadministration of NTE inhibitors, such as carbamates, thiocarbamates, sulfonyl fluorides and phosphinate to prevent OPIDN. Progress is being made, but there is yet no single specific treatment available for use in clinical practice to prevent or alleviate the severe effects of OPIDN. (C) 2012 Elsevier B.V. All rights reserved.Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP

Biochemical, histopathological and clinical evaluation of delayed effects caused by methamidophos isoforms and TOCP in hens: Ameliorative effects using control of calcium homeostasis

This work evaluated the potential of the isoforms of methamidophos to cause organophosphorus-induced delayed neuropathy (OPIDN) in hens. In addition to inhibition of neuropathy target esterase (NTE) and acetylcholinesterase (AChE), calpain activation, spinal cord lesions and clinical signs were assessed. The isoforms (+)-, (+/-)- and (-)-methamidophos were administered at 50 mg/kg orally; tri-ortho-cresyl phosphate (TOCP) was administered (500 mg/kg, po) as positive control for delayed neuropathy. The TOCP hens showed greater than 80% and approximately 20% inhibition of NTE and AChE in hen brain, respectively. Among the isoforms of methamidophos, only the (+)-methamidophos was capable of inhibiting NTE activity (approximately 60%) with statistically significant difference compared to the control group. Calpain activity in brain increased by 40% in TOCP hens compared to the control group when measured 24h after dosing and remained high (18% over control) 21 days after dosing. Hens that received (+)-methamidophos had calpain activity 12% greater than controls. The histopathological findings and clinical signs corroborated the biochemical results that indicated the potential of the (+)-methamidophos to be the isoform responsible for OPIDN induction. Protection against OPIDN was examined using a treatment of 2 doses of nimodipine (1 mg/kg, i.m.) and one dose of calcium gluconate (5 mg/kg, iv.). The treatment decreased the effect of OPIDN-inducing TOCP and (+)-methamidophos on calpain activity, spinal cord lesions and clinical signs. (C) 2012 Elsevier B.V. All rights reserved