Search

Organophosphorus (OP) compounds inhibit central and peripheral acetylcholinesterase (AChE) activity, overstimulating cholinergic receptors and causing autonomic dysfunction (e.g., bronchoconstriction, excess secretions), respiratory impairment, seizure and death at high doses. Current treatment for OP poisoning in the United States includes reactivation of OP-inhibited AChE by the pyridinium oxime 2-pyridine aldoxime (2-PAM). However, 2-PAM has a narrow therapeutic index and its efficacy is confined to a limited number of OP agents. The bis-pyridinium oxime MMB4, which is a more potent reactivator than 2-PAM with improved pharmaceutical properties and therapeutic range, is under consideration as a potential replacement for 2-PAM. Similar to other pyridinium oximes, high doses of MMB4 lead to off-target effects culminating in respiratory depression and death. To understand the toxic mechanisms contributing to respiratory depression, we evaluated the effects of MMB4 (0.25–16 mM) on functional and neurophysiological parameters of diaphragm and limb muscle function in rabbits and rats. In both species, MMB4 depressed nerve-elicited muscle contraction by blocking muscle endplate nicotinic receptor currents while simultaneously prolonging endplate potentials by inhibiting AChE. MMB4 increased quantal content, endplate potential rundown and tetanic fade during high frequency stimulation in rat but not rabbit muscles, suggesting species-specific effects on feedback mechanisms involved in sustaining neurotransmission. These data reveal multifactorial effects of MMB4 on cholinergic neurotransmission, with the primary toxic modality being reduced muscle nicotinic endplate currents. Evidence of species-specific effects on neuromuscular function illustrates the importance of comparative toxicology when studying pyridinium oximes and, by inference, other quaternary ammonium compounds.

The current study was performed to assess the precision of the principal subvisible particle measurement methods available today. Special attention was given to identifying the sources of error and the factors governing analytical performance.The performance of individual techniques was evaluated using a commercial biologic drug product in a prefilled syringe container. In control experiments, latex spheres were used as standards and instrument calibration suspensions.The results reported in this manuscript clearly demonstrated that the particle measurement techniques operating in the submicrometer range have much lower precision than the micrometer size-range methods. It was established that the main factor governing the relatively poor precision of submicrometer methods in general and inherently, is their low sampling volume and the corresponding large extrapolation factors for calculating final results.The variety of new methods for submicrometer particle analysis may in the future support product characterization; however, the performance of the existing methods does not yet allow for their use in routine practice and quality control.

Electrophysiological and ultrastructural studies were performed on phrenic nerve-hemidiaphragm preparations isolated from wild-type and acetylcholinesterase (AChE) knockout (KO) mice to determine the compensatory mechanisms manifested by the neuromuscular junction to excess acetylcholine (ACh). The diaphragm was selected since it is the primary muscle of respiration, and it must adapt to allow for survival of the organism in the absence of AChE. Nerve-elicited muscle contractions, miniature endplate potentials (MEPPs) and evoked endplate potentials (EPPs) were recorded by conventional electrophysiological techniques from phrenic nerve-hemidiaphragm preparations isolated from 1.5- to 2-month-old wild-type (AChE(+/+)) or AChE KO (AChE(-/-)) mice. These recordings were chosen to provide a comprehensive assessment of functional alterations of the diaphragm muscle resulting from the absence of AChE. Tension measurements from AChE(-/-) mice revealed that the amplitude of twitch tensions was potentiated, but tetanic tensions underwent a use-dependent decline at frequencies below 70 Hz and above 100 Hz. MEPPs recorded from hemidiaphragms of AChE(-/-) mice showed a reduction in frequency and a prolongation in decay (37%) but no change in amplitude compared to values observed in age-matched wild-type littermates. In contrast, MEPPs recorded from hemidiaphragms of wild-type mice that were exposed for 30 min to the selective AChE inhibitor 5-bis(4-allyldimethyl-ammoniumphenyl)pentane-3-one (BW284C51) exhibited a pronounced increase in amplitude (42%) and a more marked prolongation in decay (76%). The difference between MEPP amplitudes and decays in AChE(-/-) hemidiaphragms and in wild-type hemidiaphragms treated with BW284C51 represents effective adaptation by the former to a high ACh environment. Electron microscopic examination revealed that diaphragm muscles of AChE(-/-) mice had smaller nerve terminals and diminished pre- and post-synaptic surface contacts relative to neuromuscular junctions of AChE(+/+) mice. The morphological changes are suggested to account, in part, for the ability of muscle from AChE(-/-) mice to function in the complete absence of AChE.

The quantitative immuno-PCR (qIPCR) technology combines the advantages of flexible and robust immunoassays with the exponential signal amplification power of PCR. The qIPCR allows one to detect antigens using specific antibodies labeled with double-stranded DNA. The label is used for signal generation by quantitative PCR. Because of the efficiency of nucleic acid amplification, qIPCR typically leads to a 10- to 1,000-fold increase in sensitivity compared to an analogous enzyme-amplified immunoassay. A standard protocol of a qIPCR assay to detect human interleukin 6 (IL-6) using a sandwich immunoassay combined with real-time PCR readout is described here. The protocol includes initial immobilization of the antigen, and coupling of this antigen with antibody-DNA conjugates is then carried out by (a) the stepwise assembly of biotinylated antibody, streptavidin and biotinylated DNA, (b) the use of a biotinylated antibody and an anti-biotin-DNA conjugate or (c) the employment of an anti-IL-6 antibody-DNA conjugate. Following the assembly of signal-generating immunocomplexes, real-time PCR is used to amplify and record the signal. Depending on the coupling strategy, the qIPCR assays require 4-7 h with only about 3 h hands-on-time. The use of qIPCR assays enables the detection of rare biomarkers in complex biological samples that are poorly accessible by conventional immunoassays. Therefore, qIPCR offers novel opportunities for the biomedical analysis of, for instance, neurodegenerative diseases and viral infections as well as new tools for the development of novel pharmaceuticals.

Self-assembled oligomeric nanostructures consisting of bisbiotinylated DNA fragments connected by the protein streptavidin (STV) are studied by dynamic scanning force microscopy (SFM) operating in air. A comparison of the images taken in repulsive and attractive regimes is systematically made on DNA and STV structures. Stable and reproducible SFM images are obtained in the attractive regime by using a special feedback circuit, called Q-control. On the other hand, when SFM is operating in the repulsive regime, deformation of the structures that reduce the resolution and the image quality are clearly observable. The heights of both DNA and STV have been measured as a function of the tip/molecule interaction forces. This study offers the possibility to suggest a different mechanical behavior of DNA with respect to STV.

Purpose. To characterize via electron spectroscopy for chemical analysis(ESCA) the surface of spray-dried particles of trehalose plus aprotein (bovine serum albumin). Additionally, to show how and whythe addition of a surfactant reduces protein adsorption, and by thismechanism could reduce protein instability during spray-drying.

Chimera Biotec, founded in 2000 as a spin-off from academic research in Germany, has implemented its proprietary DNA-antibody conjugates within the highly innovative Imperacer product line, enabling the ultra-sensitive detection of proteins and other antigens. The ready-to-use, commercially available Imperacer kits offer a performance-enhancing tool to improve the limit of detection of conventional ELISA protocols by more than 1,000-fold. Hence, the detection and quantification of trace amounts of basically any antigen, ranging from large proteins to small molecules such as hormones or biogenic amines, can be accomplished with an Imperacer-boosted immunoassay.

Gastrointestinal angiodysplasias are the most common cause of obscure chronic digestive blood loss. Push-enteroscopy is likely to detect and to treat vascular lesions. Push-enteroscopy was performed in 83 patients (mean age 62 years) presenting with iron deficiency anemia of obscure origin. A nonrevealing preliminary evaluation included esophagogastroduodenoscopy, colonoscopy and, in 50% of the patients, small bowel barium studies. We employed a 240-cm Olympus push-enteroscope (XSIF-100), 11.3 mm in diameter. A potential bleeding lesion was observed in 49 patients (59%). Gastrointestinal angiodysplasias were the most common lesion (33 patients). Electrocoagulation (bicap) of angiodysplasias was performed when accessible and not diffuse (

Humoral vasoactive substances coming from portal blood have been considered as a possible cause of renal dysfunction in cirrhotic patients. We have thus investigated the effect of perfusion of portal blood from anesthetized dogs on the isolated kidney functions. Both kidneys of a dog were simultaneously perfused on 2 Nizet's pump oxygenators, one kidney serving as control for the other. Renal blood flow was decreased in kidneys perfused with portal blood, as compared to the paired control kidneys perfused with sus-hepatic blood (group A experiments). Addition of polymyxin B to the portal blood restored the renal blood flow to the control level (group B experiments). No significant changes appeared between experimental and control kidneys for glomerular filtration rate, urine output, sodium and water excretion, renin activity, angiotensin II levels, plasmatic PGE2 levels, in group A as well as in group B. We conclude that portal blood of dogs contains vasoactive substances reducing renal blood flow; their action is mediated neither by the renin-angiotensin system nor by changes in renal PGE2 production. The complete abolition of this effect by Polymyxin suggests that these substances may be endotoxins.

The effects of extracellular and intracellular tetraethylammonium (TEA) ions on single Ca2+-activated K+ channels were studied in excised membrane patches from the anterior pituitary clone AtT-20/D16-16 with the patch-clamp technique. The presence of TEA on either surface of the membrane resulted in a decrease in the single-channel current. Dissociation constants at zero voltage for the TEA-receptor complex were calculated to be 52.2 mM and 0.08 mM for external and internal TEA, respectively. The high sensitivity of AtT-20/D16-16 cells to internal TEA is of considerable interest, since in other preparations, the greater TEA sensitivity for Ca2+-activated K+ channels has thus far been found to occur on the external membrane surface. Hill plot analysis of the dose-response data yielded a slope of 0.92, indicating a one-to-one stoichiometry for TEA-receptor binding. The blockade by TEA showed little voltage or current sensitivity over the membrane potential range studied, and could be fully reversed by washout in drug-free solution. The results suggest the presence of TEA receptors on both the external and internal membrane surfaces but with different binding affinities. Occupancy of either site by TEA leads to an apparent decrease in the single-channel conductance of Ca2+-activated K+ channels.