Absence Of Diisopropylfluorophosphate Research Articles

Allopregnanolone and perampanel as adjuncts to midazolam for treating diisopropylfluorophosphate-induced status epilepticus in rats.

Combinations of midazolam, allopregnanolone, and perampanel were assessed for antiseizure activity in a rat diisopropylfluorophosphate (DFP) status epilepticus model. Animals receiving DFP followed by atropine and pralidoxime exhibited continuous high‐amplitude rhythmical electroencephalography (EEG) spike activity and behavioral seizures for more than 5 hours. Treatments were administered intramuscularly 40 min after DFP. Seizures persisted following midazolam (1.8 mg/kg). The combination of midazolam with either allopregnanolone (6 mg/kg) or perampanel (2 mg/kg) terminated EEG and behavioral status epilepticus, but the onset of the perampanel effect was slow. The combination of midazolam, allopregnanolone, and perampanel caused rapid and complete suppression of EEG and behavioral seizures. In the absence of DFP, animals treated with the three‐drug combination were sedated but not anesthetized. Animals that received midazolam alone exhibited spontaneous recurrent EEG seizures, whereas those that received the three‐drug combination did not, demonstrating antiepileptogenic activity. All combination treatments reduced neurodegeneration as assessed with Fluoro‐Jade C staining to a greater extent than midazolam alone, and most reduced astrogliosis as assessed by GFAP immunoreactivity but had mixed effects on markers of microglial activation. We conclude that allopregnanolone, a positive modulator of the GABAA receptor, and perampanel, an AMPA receptor antagonist, are potential adjuncts to midazolam in the treatment of benzodiazepine‐refractory organophosphate nerve agent–induced status epilepticus.

Infrared Reflection−Absorption Spectroscopy and Polarization-Modulated Infrared Reflection−Absorption Spectroscopy Studies of the Organophosphorus Acid Anhydrolase Langmuir Monolayer

The secondary structure of the organophosphorus acid anhydrolase (OPAA) Langmuir monolayer in the absence and presence of diisopropylfluorophosphate (DFP) in the subphase was studied by infrared reflection-absorption spectroscopy (IRRAS) and polarization-modulated IRRAS (PM-IRRAS). The results of both the IRRAS and the PM-IRRAS indicated that the alpha-helix and the beta-sheet conformations in OPAA were parallel to the air-water interface at a surface pressure of 0 mN.m-1 in the absence of DFP in the subphase. When the surface pressure increased, the alpha-helix and the beta-sheet conformations became tilted. When DFP was added to the subphase at a concentration of 1.1 x 10(-5) M, the alpha-helix conformation of OPAA was still parallel to the air-water interface, whereas the beta-sheet conformation was perpendicular at 0 mN.m-1. The orientations of both the alpha-helix and the beta-sheet conformations did not change with the increase of surface pressure. The shape of OPAA molecules is supposed to be elliptic, and the long axis of OPAA was parallel to the air-water interface in the absence of DFP in the subphase, whereas the long axis became perpendicular in the presence of DFP. This result explains the decrease of the limiting molecular area of the OPAA Langmuir monolayer when DFP was dissolved in the subphase.

Single-chain urokinase-type plasminogen activator does not possess measurable intrinsic amidolytic or plasminogen activator activities

The question whether single-chain urokinase-type plasminogen activator (Sc-uPA) possesses an enzymatic activity has been a subject of intense investigation for a number of years but still remains unresolved. Recent studies from several laboratories suggest that Sc-uPA or its plasmin-resistant mutants obtained by site-directed mutagenesis possess significant, albeit low, amidolytic and plasminogen activator activities, ranging from 0.1% to 1% of that observed for two-chain urokinase (Tc-uPA). In an effort to characterize these putative intrinsic activities, Sc-uPA was repeatedly treated with dansyl-Glu-Gly-Arg chloromethyl ketone (dansyl-EGRck) or diisopropyl fluorophosphate (DFP) (0.1-0.25 mM added thrice over a period of 24 h at 0 degrees C). This treatment exhaustively inactivated the Tc-uPA contaminant but did not affect Sc-uPA, as evidenced by the lack of significant incorporation of radiolabeled inhibitor in Sc-uPA and full activation of the inhibitor-treated Sc-uPA by plasmin. Assayed in the presence of excess DFP or dansyl-EGRck to ensure trapping of any Tc-uPA generated in the assay mixture, Sc-uPA (84 micrograms/mL, 10,500 latent units/mL) did not elicit any detectable cleavage of the chromogenic substrate S-2444 (detection limit 0.1 unit of Tc-uPA/mL). However, if the Tc-uPA inhibitors were removed prior to assay, a trace amount of amidolytic activity invariably reappeared in the Sc-uPA preparation. Incorporation experiments with [3H]DFP suggested that the appearance of this amidolytic activity was due to formation of Tc-uPA. Plasminogen activator assay of DFP- and dansyl-EGRck-treated Sc-uPA (0.45-2.25 microM), performed in the presence of these inhibitors and Trasylol (10 microM) to ensure entrapment of any Tc-uPA or plasmin generated in the reaction mixture, showed no significant cleavage of 125I-labeled plasminogen (detection limit 0.1 nM). However, if dansyl-EGRck and DFP were removed from the inhibitor-treated Sc-uPA and the assay was performed in the presence of Trasylol alone, there was significant cleavage of 125I-plasminogen due to contamination by Tc-uPA. Fibrin, a positive effector of plasminogen activation by Tc-uPA or Sc-uPA preparations in the absence of DFP and dansyl-EGRck, did not promote cleavage of plasminogen or S-2444 by Sc-uPA in the presence of the Tc-uPA inhibitors.(ABSTRACT TRUNCATED AT 400 WORDS)

Loading of fura-2/AM with an aid of DFP on single smooth muscle cells prepared from guinea pig taenia coli

Procedures for loading fura 2 acetoxymethyl ester (fura 2/AM) into smooth muscle cells isolated from gu inea pig taenia coli have been investigated. It was difficult to load these cells with fura 2 in the absence of diisopropylfluorophosphate (DFP). The presence of DFP, a potent cholinesterase (ChE) inhibitor during the loading, significantly enhanced the incorporation of the fura 2 into the cells. More than 80% of the single cells treated with DFP and fura 2/AM were viable. DFP did not affect the ability of the cell to shorten in response to either Ca 2+ or carbachol (CCh). The single cells contracted transiently with caffeine and the intracellular Ca 2+ concentration increased simultaneously. The results indicate that the amount of fura 2/AM incorporated into the single smooth muscle cells depends on the activity of ChE or various serine proteases located outside the cells and suppression of these enzymes induces more efficient incorporation, which permits shorter incorporation periods. Since the presence of DFP may shorten the incubation time significantly, the viability of these cells is improved. The procedure might be applicable for measuring simultaneously the contraction of cells and the behavior of intracellular Ca 2+ in the same cells.

Simultaneous recording of tension and fluorescence ratio of the fura-2-loaded longitudinal muscle of guinea-pig ileum: responses to muscarinic agents

Procedures for loading fura 2 acetoxymethyl ester (fura 2/AM) into smooth muscle cells isolated from gu inea pig taenia coli have been investigated. It was difficult to load these cells with fura 2 in the absence of diisopropylfluorophosphate (DFP). The presence of DFP, a potent cholinesterase (ChE) inhibitor during the loading, significantly enhanced the incorporation of the fura 2 into the cells. More than 80% of the single cells treated with DFP and fura 2/AM were viable. DFP did not affect the ability of the cell to shorten in response to either Ca2+ or carbachol (CCh). The single cells contracted transiently with caffeine and the intracellular Ca2+ concentration increased simultaneously. The results indicate that the amount of fura 2/AM incorporated into the single smooth muscle cells depends on the activity of ChE or various serine proteases located outside the cells and suppression of these enzymes induces more efficient incorporation, which permits shorter incorporation periods. Since the presence of DFP may shorten the incubation time significantly, the viability of these cells is improved. The procedure might be applicable for measuring simultaneously the contraction of cells and the behavior of intracellular Ca2+ in the same cells.

Identification of a second class of IgG Fc receptors on human neutrophils. A 40 kilodalton molecule also found on eosinophils.

We describe a newly recognized 40 kD FcR for IgG on human neutrophilic granulocytes. An mAb (IV3) developed against the IgG FcR of K562 cells, and specific as well for a 40 kD FcR on human monocytes and platelets, was found to purify by affinity adsorption a 40 kD protein from detergent lysates of surface-radioiodinated neutrophils. This protein, proteolytically degraded to 33 kD when purified in the absence of diisopropylfluorophosphate, is distinct from the 51-73 kD protein precipitated by the anti-neutrophil FcR mAb, 3G8, previously described by others. Complete inhibition of binding of rabbit IgG-coated erythrocytes to neutrophils was achieved only when both antibodies, IV3 and 3G8, were used. Fab fragments of IV3 were as effective inhibitors as the intact molecule. IV3 IgG or Fab fragments completely and selectively inhibited immune complex-mediated generation of superoxide by human neutrophils; superoxide generation by other stimulants was not abrogated by IV3. This antibody (IV3) bound also to human eosinophils and completely inhibited the binding of IgG-coated erythrocytes to eosinophils. IV3 appears to define the human homolog of the murine macrophage FcRII identified initially by mAb 2.4G2 and present in the mouse on both neutrophils and eosinophils.

Selective inhibitory effect of organophosphates on UDP-glucuronyl transferase activities in rat liver microsomes

The effects of acute and subacute administration of diisopropylfluorophosphate (DFP) and acute administration of Soman, Sarin and Tabun on UDP-glucuronyltransferase (GT) activity towards 4-nitrophenol, 4-methylumbelliferone, phenolphthalein and testosterone in rat liver microsomes were investigated. Twenty-four hours after a single injection of DFP, the activity of GT towards 4-nitrophenol and 4-methylumbelliferone was inhibited, and the inhibitory effect continued for 3 days. The activity had recovered by 7 days after injection. The activity of GT towards phenolphthalein and testosterone was not affected at any time after injection. Soman, Sarin and Tabun showed the same effect as DFP after a single injection. After daily DFP injections, the activity of GT towards 4-nitrophenol and 4-methylumbelliferone was decreased to the same level as found following acute treatment with DFP. The in vitro addition of DFP to liver microsomes did not affect GT activity towards 4-nitrophenol. It is suggested that these changes are not due to a direct effect of DFP. Furthermore, the effects of two enzyme inducers on GT activity in the presence and absence of DFP were investigated. In the 3-methylcholanthrene (MC) pretreatment group, DFP inhibited only the GT activity towards 4-nitrophenol and 4-methylumbelliferone. On the other hand, in the phenobarbital (PB) pretreatment group, DFP did not inhibit the GT activity towards 4-nitrophenol and 4-methylumbelliferone. It was also demonstrated that MC pretreatment increased the mortality in the DFP-treated rats but that PB pretreatment suppressed it. These results suggest that DFP and other organophosphorus agents may be useful agents for studies on the heterogeneity of GT.

Autocatalytic activation of C1r subcomponent of the first component of human complement.

Autoactivation of the proenzyme form of a subunit of the first component (C1r) was performed in the presence and absence of diisopropyl fluorophosphate (DFP). The time-course of autoactivation of zymogen C1r followed a sigmoidal curve and was accelerated by addition of the enzyme C1r and by increasing the concentration of C1r, suggesting that autoactivation of C1r consists of two intermolecular reactions, i.e. zymogen(C1r)- and enzyme(C1r)-catalyzed reactions. In the presence of 10 mM DFP, the enzyme-catalyzed autoactivation of C1r was completely inhibited, while the zymogen-catalyzed autoactivation still proceeded depending upon C1r concentration. These results suggested that the zymogen-catalyzed autoactivation of C1r is a DFP-insensitive second-order reaction and is mediated by an active site generated in a single chain C1r through a conformational change (Kassahara et al. (1982) FEBS lett. 141, 128-131). Based on these results, a possible reaction process of autoactivation of C1r was proposed, as follows: (formula; see text) where C1r represents a conformational isomer which catalyzes the autoactivation of C1r, and the rate constants, k2 and k3, are of second-order. Utilizing a computer, we simulated the autoactivation of C1r and found the above scheme to be a reasonable model of C1r autoactivation. Evidence which supports the formation of a conformational isomer of C1r, C1r, as an intermediate in its autoactivation was also obtained by a surface radiolabeling method.

Salt-induced transformation of the human myometrial estrogen receptor

The effect of KC1 on the binding characteristics and sedimentation properties of the human myometrial estrogen receptors was studied. The dissociation constant for the hormone-receptor complex increased with increasing ionic strength and the increase was greater at 0°C than at 20°C. At 20°C the association rate constants were 19.3 (no salt added), 29.44 (0.15 M KCl) and 24.23 (0.4 M KCl) × 10 5 M −1s −1 for receptors in cytosol prepared in the presence of 2mM diisopropylfluorophosphate (DFP), a serine protease inhibitor. The corresponding values for receptors prepared in the absence of DFP were 11.42, 14.23 and 12.72 × 10 5M −1s −1. The change in entropy for the formation of the estrogen receptor complex was positive under all conditions studied which implies conformational changes in the receptor protein. The 9.0 S receptor form obtained when the proteolytical activity in the cytosol is inhibited with DFP was irreversibly transformed to a 5.3 S form by 0.4 M KCl. When 0.15 M KCl was used, a broad peak sedimenting at 6–7 S was obtained. The sedimentation of the 4.3 S proteolytically transformed receptor form was not affected by increased ionic strength and/or preincubation at 30°C. The salt-induced transformation of the 9.0 S form was very fast and was obtained at 0, 20 and 30°C. The results indicate that the hormone binding site of the receptor molecule is not greatly affected by salt. The conformational changes occur likely quite distant from the active binding site.

Properties of the estrogen receptor in the human oviduct and its interaction with ethinylestradiol and mestranol in vitro.

Biochemical properties of estrogen binding are investigated in the cytosol and nuclear fractions of the human Fallopian tube. Sucrose density ultracentrifugation and dextrancoated charcoal adsorption techniques are used for characterization of [3H]estradiol uptake in the human oviduct. There is indirect evidence for the presence of protease which can be inhibited by diisopropylfluorophosphate (DFP). When DFP is present in buffer of low ionic strength, the cytosol receptor sediments at 8S and 4S peaks. In the absence of DFP, the 4S peak alone is demonstrated. The proteolytic inhibitor does not alter the estrogen-binding capacity in the human oviduct. The dissociation constant (Kd) for [3H]estradiol in cytosol is 2 X 10(-10) M without DFP and 1.5 X 10(-10) M with DFP. The presence of protease in cytosol of human oviducts is confirmed by hydrolysis of benzoyl-arginine nitroanalide. The enzymatic activity is inhibited by DFP. Nuclear estrogen receptor sediments at 4S after extraction with 0.4 M KCL buffer. A rapid nuclear accumulation of [-3H]estradiol is seen at 25 C, with reciprocal depletion of cytoplasmic receptor in human oviduct tissue minces. The synthetic estrogens ethinylestradiol (17 alpha-ethynyl-1,3,5-estratriene-3,17 beta-diol) and mestranol [3-methoxy-17 alpha-ethinyl-1,3-5(10)-estratiene-17 beta-OL] are competitors for the estrogen receptor in the human Fallopian tube. Inhibition of oviductal estrogen binding is 500 times greater with ethinylestradiol than with mestranol (ki = 0.75 X 10(-9) M for ethinylestradiol; Ki = 3.74 X 10(-7) M for mestranol). The estrogen receptor in the human Fallopian tube shows properties similar to those of the estrogen receptor of the human uterus. However, the determination of the number of binding sites in the oviduct is not influenced by proteolytic enzyme activity.

Nuclear binding of estradiol in human myometrium

The nuclear uptake of human myometrial estrogen receptors in cytosol prepared in a low salt medium in the presence and absence of diisopropylfluorophosphate (DFP) has been studied. The [ 3H]-estradiol receptor complex in the cytosol prepared in the presence of DTP showed a much greater nuclear binding activity than the receptor complex in the cytosol prepared in the absence of DFP. This difference in the nuclear translocation was obtained in spite of the greater binding capacity of the receptors in cytosol prepared in the absence of DFP. Receptors prepared in the presence of DFP seclimented at 9.0 S, 5.3 S and 4.2 S. Sucrose density gradient centrifugation of the cytoplasmic receptors after incubation with nuclei showed that radioactivity seclimenting at 9.0 S disappeared and no significant changes in the 5.3 S and 4.2 S areas was observed. Nuclear uptake of [ 3H]-estradiol receptor complex from cytosol prepared in the absence of DFP was also dependent on the presence of the 9.0 S receptor complex while the 4.2 S proteolytic fragment did not show any nuclear binding activity. A correlation coefficient of 0.99 was obtained when the decrease in the amount of 9.0 S receptor form was plotted against the amount of [ 3H]-estradiol extracted from the nuclear pellet.

Effects of proteases and protease inhibitors on the 4.5 S and 8 S androgen receptor.

The size of androgen receptors from rat ventral and dorsal prostate, dorsal prostate (Dunning) tumor, testis, epididymis, and seminal vesicle was determined using Sephadex G-200 chromatogrpahy and sucrose gradient centrifugation. The protease inhibitor diisopropyl fluorophosphate (DFP) was used to minimize receptor breakdown. An 8-9 S, 85 to 106 A receptor (Mr = 280,000 to 365,000; f/fo = 1.9 to 2.4) observed in unfractionated cytosol prepared in low ionic strength buffer with or without DFP is in equilibrium with a 4.5-5 S, 58 A form (Mr = 117,000; f/fo = 1.8) observed at salt concentrations greater than 0.1 M KCl. Receptor partially purified using (NH4)2SO4 or phosphocellulose chromatography in the absence of DFP was present as smaller fragments of 3.6 S, 37 A and 3.0 S, 23 A. Similar fragments could be generated from the 4.5 S or 8 S receptor by mild trypsin treatment. In addition, ventral prostate contains a DFP-insensitive enzyme which specifically converts the 4.5 S, 58 A receptor to the 3.6 S 37 A fragment. The DFP-insensitive enzyme is partially inhibited by rabbit bile and appears similar to the enzyme seminin, a secretory protein of human prostate. Androgen receptor isolated in the presence of DFP from nuclei labeled in vivo is predominantly 4.5 S, 58 A, with smaller forms (37 and 23 A) appearing in the absence of DFP. The 4.5 S, 58 A nuclear receptors were also in equilibrium with a large 8 S form. Receptor breakdown by DFP-insensitive and sensitive proteases appears to be an in vitro phenomenon. Furthermore, the size of the androgen receptor is not significantly changed during receptor migration from cytoplasm to nucleus.

The Diisopropylfluorophosphate Inhibitable Step in Antigen-Induced Histamine Release from Human Leukocytes

DFP inhibits early events in antigen-induced histamine release from human leukocytes. If added to cells 5 min or more after antigen it is ineffective. If added with antigen it can be removed at 5 min but release will still be inhibited. In contrast, ethylenediaminetetraacetate (EDTA) and 2 deoxyglucose (2DG) still inhibit the reactions when added 5 min after antigen. During incubation of leukocytes for 90 to 120 min at 0 degrees C they react with specific antigen since they subsequently release significant quantities of histamine after washing and reincubation at 37 degrees C without addition of antigen. Such priming at 0 degrees C is at least equivalent to priming for 2 to 4 min at 37 degrees C. During antigen priming at 0 degrees C the cells are not activated beyond the step in the release sequence which is inhibited by diisopropylfluorophosphate (DFP). This is apparent from the undiminished inhibitory activity of DFP on these cells. Furthermore, cells primed with antigen at 0 degrees C in the presence of DFP release as much histamine after washing and incubation at 37 degrees D as control cells primed in the absence of DFP. Incubation of leukocytes with specific antigen at 37 degrees C for 3 min resulted in significant but not quite complete priming for subsequent histamine release in the absence of antigen. Most of these primed cells were not activated beyond the step inhibitable by DFP. However, some had completed the entire sequence including the release of histamine while others had not released their histamine but were not inhibited by DFP from subsequent release. After 5 min incubation with antigen at 37 degrees C almost all leukocytes had progressed beyond the stage which is inhibited by DFP. Incubation of leukocytes at 37 degrees C with DFP but without antigen for up to 15 min followed by washing did not impair subsequent antigen-induced histamine release by these cells. Thus, DFP was inhibitory under these conditions only after antigen activation of leukocytes.

The Conversion of Prothrombin to Thrombin: II. DIFFERENTIATION BETWEEN THROMBIN- AND FACTOR Xa-CATALYZED PROTEOLYSES

Abstract Prothrombin is activated by activated Factor X ± (Factor V and phospholipid) in the presence of diisopropylfluorophosphate which preferentially inhibits thrombin. Under these conditions a single activation fragment, designated Fragment 1·2, is released which represents the complete nonthrombin portion of the prothrombin molecule. The same fragment is seen as a transient species in the absence of diisopropylfluorophosphate when the activation reaction is monitored by disc electrophoresis in 8 m urea. The NH2terminal residue of both prothrombin and Fragment 1·2 is alanine, suggesting that Fragment 1·2 is derived from the NH2-terminal end of the prothrombin molecule. Fragment 1·2 is not cleaved by activated Factor X ± (Factor V and phospholipid), but is cleaved by thrombin to form two fragments which are identical by electrophoresis, ion exchange chromatography, and amino acid composition to Fragment 1, the fragment released from prothrombin on incubation with thrombin, and Fragment 2, the fragment released by activated Factor X when Intermediate 1 is converted to Intermediate 2 (Paper I; Owen, W. G., Esmon, C. T., and Jackson, C. M. (1974) J. Biol. Chem. 249, 594–605). It is proposed that the formation of Intermediate 1 during prothrombin activation is solely the result of thrombin-catalyzed proteolysis of prothrombin.