Mutations in the dynamin 2 gene have been identified in patients with autosomal dominant forms of centronuclear myopathy (CNM). Dynamin 2 is a ubiquitously expressed ϳ100-kDa GTPase that assembles around the necks of vesiculating membranes and promotes their constriction and scission. It has also been implicated in regulation of the actin and microtubule cytoskeletons. At present, the cellular functions of dynamin 2 that are affected by CNM-linked mutations are not well defined, and the effects of these mutations on the physical and enzymatic properties of dynamin have been not examined. Here, we report the expression, purification, and characterization of four CNMassociated dynamin mutants. All four mutants display higher than wild-type GTPase activities, and more importantly, the mutants form high order oligomers that are significantly more resistant than wild-type dynamin 2 to disassembly by guanine nucleotides or high ionic strength. These observations suggest that the corresponding wild-type residues serve to prevent excessive or prolonged dynamin assembly on cellular membranes or inappropriate self-assembly in the cytoplasm. To our knowledge, this report contains the first identification of point mutations that enhance the stability of dynamin polymers without impairing their ability to bind and/or hydrolyze GTP. We envision that the formation of abnormally large and stable complexes of these dynamin mutants in vivo contributes to their role in CNM pathogenesis. Autosomal dominant CNM2 is a congenital disorder that commonly results in muscle weakness and wasting, ptosis, and ophthalmoplegia (reviewed in Ref. 1). As the name implies, the most evident histopathological feature is the presence of a large number of muscle fibers of centrally (rather than peripherally) located nuclei. Other characteristics include a relative increase in the number of type I fibers, hypertrophy of these fibers, and the presence of sarcoplasmic strands distributed radially around the central nuclei. In 2005, Bitoun et al. (2) reported the identification of four mutations in the DNM2 (dynamin 2) gene in patients with autosomal dominant CNM, and an additional seven mutations have since been identified (3, 4). DNM2 is a ubiquitously expressed ϳ100-kDa GTPase that assembles into helical polymers around the necks of vesiculating membranes, thereby providing force for their constriction and scission (reviewed in Refs. 5-8). In addition to its well characterized roles in endocytosis and Golgi budding, DNM2 is also implicated in regulation of the actin (9, 10) and microtubule (11, 12) cytoskeletons. The DNM2 molecule consists of five functional domains: an N-terminal catalytic domain; a so-called "middle domain" implicated in dynamin-dynamin interactions; a PH domain involved in phosphoinositide binding; a GTPase effector domain, which interacts with the catalytic domain and stimulates its GTPase activity; and a C-terminal proline/arginine-rich domain, which mediates interactions of dynamin with other proteins. Most of the currently kn...

BACKGROUND The Activity-regulated cytoskeleton-associated protein, Arc, is an immediate-early gene product implicated in various forms of synaptic plasticity. Arc promotes endocytosis of AMPA type glutamate receptors and regulates cytoskeletal assembly in neuronal dendrites. Its role in endocytosis may be mediated by its reported interaction with dynamin 2 (Dyn2), a 100 kDa GTPase that polymerizes around the necks of budding vesicles and catalyzes membrane scission. METHODS Enzymatic and turbidity assays are used in this study to monitor effects of Arc on dynamin activity and polymerization. Arc oligomerization is measured using a combination of approaches, including size exclusion chromatography, sedimentation analysis, dynamic light scattering, fluorescence correlation spectroscopy, and electron microscopy. RESULTS We present evidence that bacterially-expressed His6-Arc facilitates the polymerization of Dyn2 and stimulates its GTPase activity under physiologic conditions (37°C and 100 mM NaCl). At lower ionic strength Arc also stabilizes pre-formed Dyn2 polymers against GTP-dependent disassembly, thereby prolonging assembly-dependent GTP hydrolysis catalyzed by Dyn2. Arc also increases the GTPase activity of Dyn3, an isoform of implicated in dendrite remodeling, but does not affect the activity of Dyn1, a neuron-specific isoform involved in synaptic vesicle recycling. We further show in this study that Arc (either His6-tagged or untagged) has a tendency to form large soluble oligomers, which may function as a scaffold for dynamin assembly and activation. CONCLUSIONS and GENERAL SIGNIFICANCE The ability of Arc to enhance dynamin polymerization and GTPase activation may provide a mechanism to explain Arc-mediated endocytosis of AMPA receptors and the accompanying effects on synaptic plasticity. This study represents the first detailed characterization of the physical properties of Arc.

Public reporting burden for this collection of information is estimated to average 1 hour per response, including the time for reviewing instructions, searching existing data sources, gathering and maintaining the data needed, and completing and reviewing this collection of information. Send comments regarding this burden estimate or any other aspect of this collection of information, including suggestions for reducing this burden to Department of Defense, Washington Headquarters Services, Directorate for Information Studies have demonstrated that benzodiazepine compounds are effective at antagonizing seizure activity produced by the organophosphate (OP) cholinesterase inhibitor soman. In this present study we have investigated the pharmacokinetics of midazolam and its associated effects on electroencephalographic (EEG) activity following intramuscular (im) injection to soman-exposed guinea pigs (Crl:(HA)BR).Prior to experiments, the animals were surgically implanted with EEG leads to monitor seizure activity. For the study, animals were administered the following pretreatment/OP/treatment regimen. Pyridostigmine bromide (0.026 mg/kg, im) was given 30 min prior to soman (56 gtg/kg, 2 x LDso; subcutaneously, sc), followed in one minute by atropine sulfate (2 mg/kg, im) and pralidoxime chloride (25 mg/kg, im). All animals receiving this regimen developed seizure activity. Midazolam 0.8 mg/kg, im, was administered 5 min after onset of seizure activity. Based on EEG data, animals were categorized as either seizure-terminated or seizure not-terminated at 30 min following anticonvulsant administration. Serial blood samples were collected *In conducting the research described in this report, the investigators complied with the regulations and standards of the Animal Welfare Act and adhered to the principles of the Guide for the Care and Use of Laboratory Animals (NRC 1996 for the plasma midazolam analysis; the assay was accomplished with a gas chromatograph/mass spectrometer. The mean time to seizure termination was 8.8 ± 1.6 min. The mean time-plasma concentration data were fit to standard pharmacokinetic models. The following parameter estimates were determined from the model-fit for seizure terminated and not-terminated animals respectively: apparent volumes of distribution (Vd) were 1.4 and 1.7 1/kg; area under the time-concentration curves (AUC), 15,990 and 15,120 ng . minrml; times to maximal plasma concentration (Tma,), 1.66 and 2.91 min and maximal plasma concentrations (Cmnx) 535.1 and 436.6 ng/ml. These data indicate that im injection of midazolam is effective at terminating ongoing soman-induced seizure activity. Additionally, the relatively short Tmax and latency to seizure termination demonstrate the rapidity of drug absorption and action respectively.

Intramuscular (i.m.) diazepam is included by the US military as an anticonvulsant in the standard therapeutic regimen for organophosphorus nerve agent intoxication. In this study we investigated the pharmacokinetics of diazepam after i.m. administration while monitoring pharmacodynamic (electroencephalogram, EEG) data in soman-exposed guinea pigs. Prior to experiments the animals were surgically implanted with EEG leads to monitor seizure activity. For the study, animals were administered pyridostigmine (0.026 mg x kg(-1) i.m.) 30 min prior to soman (56 microg x kg(-1), 2 x LD50; subcutaneously, s.c.), which was followed in 1 min by atropine sulfate (2 mg x kg(-1) i.m.) and pralidoxime chloride (25 mg x kg(-1) i.m.). All animals receiving this regimen developed seizure activity. Diazepam (10 mg x kg(-1) i.m.) was administered 5 min after onset of seizure activity. Based on EEG data, animals were categorized as either seizure terminated or not terminated at 30 min after diazepam. Serial blood samples were obtained from each animal. Diazepam (10 mg x kg(-1) i.m.) terminated seizure activity in 52% of the animals within 30 min. The pharmacokinetics were characterized by a one-compartment model with first-order absorption and elimination. The maximum plasma concentrations (Cmax) were 991 and 839 ng x ml(-1) for seizure terminated and not terminated, respectively. Mean plasma concentrations of diazepam were significantly different (P < 0.05) for seizure terminated vs not terminated groups at 30 min. The plasma Cmax in seizure-terminated animals in this study is similar to the minimum range of plasma diazepam (200-800 ng x ml(-1)) reported to suppress seizure activity in humans. It has been reported in an earlier study that the minimum effective i.m. dose (0.1 mg x kg(-1)) required to prevent soman-induced convulsions in Rhesus monkeys produces a mean Cmax of 50 ng x ml(-1) for diazepam. The data from our current study suggest that a higher dose (and corresponding Cmax) is necessary to terminate ongoing seizure activity.

A sensitive method for determining exposure to the chemical warfare agent VX is described in which the biomarker ethyl methylphosphonofluoridate (VX-G) is measured in red blood cells (RBCs) following treatment with fluoride ion using isotope-dilution gas chromatography-tandem mass spectrometry. The analyte was isolated via solid-phase extraction and detected using ammonia chemical ionization in the multiple reaction monitoring mode. A good linear relationship was obtained in the quantitative concentration range of 4 ng/mL to 1000 ng/mL with an absolute detection limit of < 1 pg on column. The method has been applied to the analysis of RBCs from a laboratory worker accidentally exposed to VX vapor. Detection and quantitation of VX-G were possible in samples taken as late as 27 days following exposure.

A rapid and sensitive method for the determination of the chemical warfare agent VX in plasma taken from Göttingen minipigs has been developed using isotope-dilution gas chromatography-tandem mass spectrometry (GC-MS-MS). Chromatographic separation was achieved on a 5% diphenyl/95% dimethyl polysiloxane capillary column with a total run time of about 11 min. The analyte was detected using ammonia chemical ionization in the multiple reaction monitoring mode, following a simple extraction with 10% 2-propanol in hexane. A good linear relationship was obtained in the quantitative concentration range of 10 ng/mL to 1000 ng/mL (r(2) = 0.9998) with an average slope of 1.275 +/- 0.037 (n = 7), and an absolute detection limit of 0.4 pg on column. The average recovery for VX was 95% in saline in the concentration range of 50-100 ng/mL. The method was successfully applied to the analysis of VX in minipig plasma in a preliminary toxicokinetic study.

The selective blockade of potassium channels on excitable membranes by 4-aminopyridine (4-AP) leads to facilitation of neurotransmitter release at a wide variety of synapses. This compound has been shown to be efficacious against lethality induced by saxitoxin (STX) and tetrodotoxin (TTX) in guinea pigs. To characterize the actions of 4-AP in guinea pigs we have investigated its pharmacokinetics (PK) and pharmacodynamics following a 2 mg/kg, intramuscular (im) dose in awake chronically instrumented (IN) animals. Animals were chronically instrumented for electrophysiologic recordings of diaphragmatic electromyogram (DEMG), lead II electrocardiogram (ECGII) and electrocorticogram (ECoG). Also, PK studies were carried out in uninstrumented (UN) guinea pigs. Blood and electrophysiologic data were collected at predetermined time intervals up to 4 hours post 4-AP administration. High performance liquid chromatography was used to determine plasma 4-AP concentrations. For IN and UN animals, plasma concentration-time data best fit a one-compartment model, and PK parameter estimates were similar for both groups. Peak plasma levels were found to occur between 16 and 17 min, and the half-lives of elimination were 65 and 71 min for IN and UN animals respectively. Heart and respiratory rates were elevated as early as 5 and 15 min respectively in response to 4-AP administration. The duration of action was approximately 1-1.5 half-lives of elimination beyond peak plasma levels. Maximum ECoG responses were observed between 12-15 min after 4-AP injection; some residual drug effects were still apparent at 240 min. The difference between the heart and respiratory rates and ECoG profiles suggests that these different physiological systems respond with varying degrees of sensitivity to plasma 4-AP concentrations. The stimulation of these systems is consistent with the action of 4-AP in reversing STX- and TTX-induced cardiorespiratory depression and decreased ECoG power in guinea pigs.

A high-performance liquid chromatographic (HPLC) method for the determination of granisetron (GRN) in guinea pig plasma has been developed. Guinea pig plasma spiked with GRN was microfiltered, and the recovered filtrate was directly injected onto the column without any further cleanup procedures. Separation was achieved on a spherical silica column and GRN was detected at 305 nm. Approximately 800-900 injections were made without any evidence of column deterioration. For the standard curves, correlation coefficients ranged from 0.9978-0.9999, and the percent standard deviation (%SD) from the mean area under the curve (AUC) was calculated to be less than 10% for all concentrations, except for the lowest concentration (0.325 ng/microL, 11.3%). Between-day and within-day coefficients of variation (%CV) ranged from 4.9 to 9.5% and 3.6 to 7.6%, respectively. Percent errors for within-day test plasma samples were not greater than 8.2% of the expected concentration for all samples except for 1.125 ng/microL (-14.6%). The limit of sensitivity was found to be 0.019 ng/microL. Estimated recovery of GRN in the microfiltrate was calculated to be 58-59% and 78-81% in plasma and water, respectively. Stability studies indicated that repeated refrigeration and warming (for six days) of microfiltered GRN plasma samples produced no changes in GRN concentrations from day to day. However, microfiltered GRN plasma samples that were repeatedly frozen and thawed demonstrated erratic concentration changes from day to day. The precision, accuracy, and small sample requirements of this method indicate its utility for pharmacokinetic studies in small animals where sample volume may be restrictive.