Burden and outcome of prevalent ischemic brain disease in a national acute stroke registry.

BACKGROUND AND PURPOSE: Previous overt stroke and subclinical stroke are frequent in patients with stroke; yet, their clinical significance and effects on stroke outcome are not clear. We studied the burden and outcome after acute ischemic stroke by prevalent ischemic brain disease in a national registry of hospitalized patients with acute stroke. METHODS: Patients with ischemic stroke in the National Acute Stroke Israeli prospective hospital-based registry (February to March 2004, March to April 2007, and April to May 2010) with information on previous overt stroke and subclinical stroke per computed tomography/MRI (n=3757) were included. Of them, a subsample (n=787) was followed up at 3 months. Logistic regression models were computed for outcomes in patients with prior overt stroke or subclinical stroke, compared with patients with first stroke, adjusting for age, sex, vascular risk factors, stroke severity, and clinical classification. RESULTS: Two-thirds of patients had a prior overt stroke or subclinical stroke. Death rates were similar for patients with and without prior stroke. Adjusted odds ratios (OR; 95% confidence interval [CI]) for disability were increased for patients with prior overt stroke (OR, 1.31; 95% CI, 1.03-1.66) and subclinical stroke (OR, 1.45; 95% CI, 1.16-1.82). Relative odds of Barthel Index≤60 for patients with prior overt stroke (OR, 2.04; 95% CI, 1.14-3.68) and with prior subclinical stroke (OR, 2.04; 95% CI, 1.15-3.64) were twice higher than for patients with a first stroke. ORs for dependency were significantly increased for patients with prior overt stroke (OR, 1.95; 95% CI, 1.19-3.20) but not for those with subclinical stroke (OR, 1.36; 95% CI, 0.84-2.19). CONCLUSIONS: In our national cohort of patients with acute ischemic stroke, nearly two thirds had a prior overt stroke or subclinical stroke. Risk of poor functional outcomes was increased for patients with prior stroke, both overt and subclinical.

Asymmetric fluorogenic organophosphates for the development of active organophosphate hydrolases with reversed stereoselectivity.

In order to enhance the enzymatic detoxification rate of organophosphorus (OP) nerve agents we have searched for more active variants of recombinant mammalian paraoxonase (PON1). We have previously identified three key positions in PON1 that affect OP hydrolysis: Leu69, Val346 and His115, that significantly enhance the hydrolysis of cyclosarin (GF), soman, chlorpyrifos-oxon (ChPo), O-isopropyl-O-(p-nitrophenyl)methylphosphonate (IMP-pNP) and diisopropyl fluorophosphate (DFP). GC/FPD analysis compared to residual AChE inhibition assay displayed stereoselective hydrolysis of GF, soman and IMP-pNP, indicating that wild type PON1 and its variant V346A are more active toward the less toxic P(+) optical isomer. In order to obtain new PON1 variants with reversed stereoselectivity, displaying augmented activity toward the more toxic isomer P(-) of nerve agents, we synthesized new asymmetric fluorogenic OPs (Flu-OPs). Six Flu-OPs were prepared containing either ethyl (E), cyclohexyl (C) or pinacolyl (P) alkyl radicals attached to methyl-phosphonyl (MP) moiety analogous to the structure of VX, GF and soman, respectively. The fluorescent moieties are either 3-cyano-4-methyl-7-hydroxy coumarin (MeCyC) or 1,3-dichloro-7-hydroxy-9,9-dimethyl-9H-acridin-2-one (DDAO). The kinetics of AChE and BChE inhibition by these new Flu-OPs display k(i) values 8.5x10(4) to 8.5x10(7) and 5x10(4) to 2x10(6)M(-1)min(-1), respectively. EMP-MeCyC and EMP-DDAO are the most active inhibitors of AChE whereas CMP-MeCyC and CMP-DDAO are better inhibitors of BChE than AChE, indicating accommodation of bulky cyclohexyl group inside the active site of BChE. PMP-MeCyC and PMP-DDAO are the least active inhibitors of both AChE and BChE. CMP-MeCyC and CMP-DDAO were significantly detoxified only by the five-site mutations PON1 variant L69V/S138L/S193P/N287D/V346A. Degradation kinetics of Flu-OPs measured by increase in absorbance of the released fluorogenic group was fit by a two exponential function, indicating faster hydrolysis of the less toxic optical isomer. Interestingly, wt PON1 caused only 50% degradation of racemic EMP-MeCyC, CMP-MeCyC and CMP-DDAO indicating complete hydrolysis of P(+) isomer. This remarkable stereoselectivity was used for the enzymatic separation of the P(-) isomer of CMP-MeCyC. The bimolecular rate constant k(i) for human AChE inhibition by the isolated P(-) isomer of CMP-MeCyC is five-fold larger than that of its P(+) isomer. The marked preference of wt PON1 toward P(+) stereo-isomer of CMP-MeCyC and CMP-DDAO renders their P(-) stereo-isomers suitable for the selection of new OP hydrolase variants with reversed stereoselectivity.

Enhanced stereoselective hydrolysis of toxic organophosphates by directly evolved variants of mammalian serum paraoxonase.

We addressed the ability of various organophosphorus (OP) hydrolases to catalytically scavenge toxic OP nerve agents. Mammalian paraoxonase (PON1) was found to be more active than Pseudomonas diminuta OP hydrolase (OPH) and squid O,O-di-isopropyl fluorophosphatase (DFPase) in detoxifying cyclosarin (O-cyclohexyl methylphosphonofluoridate) and soman (O-pinacolyl methylphosphonofluoridate). Subsequently, nine directly evolved PON1 variants, selected for increased hydrolytic rates with a fluorogenic diethylphosphate ester, were tested for detoxification of cyclosarin, soman, O-isopropyl-O-(p-nitrophenyl) methyl phosphonate (IMP-pNP), DFP, and chlorpyrifos-oxon (ChPo). Detoxification rates were determined by temporal acetylcholinesterase inhibition by residual nonhydrolyzed OP. As stereoisomers of cyclosarin and soman differ significantly in their acetylcholinesterase-inhibiting potency, we actually measured the hydrolysis of the more toxic stereoisomers. Cyclosarin detoxification was approximately 10-fold faster with PON1 mutants V346A and L69V. V346A also exhibited fourfold and sevenfold faster hydrolysis of DFP and ChPo, respectively, compared with wild-type, and ninefold higher activity towards soman. L69V exhibited 100-fold faster hydrolysis of DFP than the wild-type. The active-site mutant H115W exhibited 270-380-fold enhancement toward hydrolysis of the P-S bond in parathiol, a phosphorothiolate analog of parathion. This study identifies three key positions in PON1 that affect OP hydrolysis, Leu69, Val346 and His115, and several amino-acid replacements that significantly enhance the hydrolysis of toxic OPs. GC/pulsed flame photometer detector analysis, compared with assay of residual acetylcholinesterase inhibition, displayed stereoselective hydrolysis of cyclosarin, soman, and IMP-pNP, indicating that PON1 is less active toward the more toxic optical isomers.

General anesthetics attenuate gap junction coupling in P19 cell line.

Gap junction communication is widespread throughout the mammalian nervous system among neurons as well as glia. We addressed the hypothesis that general anesthetics attenuate gap junction mediated coupling in P19 cell line that can differentiate into neuronal-like cells and astrocytes and oligodendrocytes. We characterized the extent of dye coupling over time in the P19 cell line using colocalization of chlormethylbenzamido-1,1 dioctadecyl-3,3,3',3'-tetramethylindocarbocyamine (CM-DiI) and calcein-AM in donor and recipient cells in cocultures. After seeding, the gap junction permeant dye calcein spreads from donor to recipient cells. CM-DiI and calcein fluorescence identified donor and recipient cells, respectively. The extent of intercellular connections was evaluated using cell counting and flow cytometry up to 2 hr after treatment. Clinically relevant concentrations of the intravenous anesthetics propofol (15 microM) and thiopental (10 microM) attenuated gap junction permeability in P19 cell cultures. In contrast, halothane, a volatile anesthetic in a concentration (0.64 mM) relevant to its free aqueous EC50 had no effect on gap junction coupling; however, very high halothane concentrations (2.8 mM) blocked dye transfer by approximately 90%. The results indicate that halothane concentrations pertinent to clinical anesthesia were unable to attenuate gap junction communication in a cell line that can express neuronal and glial gap junction proteins; however, clinically relevant concentrations of propofol and thiopental depressed gap junction coupling.

Ethanol inhibits gap-junctional coupling between P19 cells.

BACKGROUND: Gap junctions are plaques of multiple intercellular channels that connect the cytoplasm of adjacent cells. They provide both electrical and metabolic coupling and are an essential element in normal growth, development, and physiology. Little research exists on the relationship between alcohol administration and gap-junctional function or expression. This study looks at the function and expression of gap junctions after incubation and withdrawal of ethanol with P19 cell cultures. METHODS: Gap-junctional communication was assessed after 24 and 48 hr of exposure to 20 and 40 mM ethanol and after a 24-hr withdrawal period. The seeding technique was used, and diacyl-3,3'-indocarbocyanine iodide/calcein-stained donor cells were seeded on an unstained monolayer and then reviewed by confocal microscope and counted by flow cytometry. Analysis of connexin (Cx) proteins was performed by Western blot, gel electrophoresis, and immunoblots with antibodies for Cx26 and Cx43. RESULTS: All treatment regimens produced similar results, reducing dye coupling by more than 50% without recovery after a 24-hr withdrawal period. Exposing the cells to 20 mM ethanol for 48 hr did not significantly change the levels of Cx26 protein, but ethanol significantly decreased the levels of Cx43 in cultured P19 cells. CONCLUSIONS: This study illustrates that ethanol can inhibit gap-junction function in the P19 cell line. Chronic exposure to 20 mM ethanol selectively decreased the levels of Cx43 protein in the membrane fraction of the cell cultures.

Characterization of early plasma concentrations of midazolam in pigs after administration by an autoinjector.

The treatment of organophosphate-induced poisoning is based mainly on atropine and an oxime. Prompt anticonvulsive intervention is usually also required to terminate the ensuing seizure activity and to prevent delayed permanent brain damage. Midazolam, a water-soluble benzodiazepine agonist, has the advantage of rapid absorption following intramuscular administration. In mass casualty situations, the availability of an autoinjector, filled with midazolam, might be a further advantage. In the present study, the plasma pharmacokinetics of midazolam after administration by an autoinjector was compared with conventional intramuscular (i.m.) administration in two groups of four pigs each. During the first 15 min after injection, significantly higher plasma concentrations of midazolam were detected following autoinjector administration, compared with the i.m. injection. The physiological reflection of the accelerated midazolam absorption was a marked reduction in the time interval required for muscle relaxation, induced by midazolam. It is concluded that a midazolam autoinjector might be helpful in the mass casualty scenario following organophosphate poisoning.