Syntaxin clusters assemble reversibly at sites of secretory granules in live cells.

Syntaxin resides in the plasma membrane, where it helps to catalyze membrane fusion during exocytosis. The protein also forms clusters in cell-free and granule-free plasma-membrane sheets. We imaged the interaction between syntaxin and single secretory granules by two-color total internal reflection microscopy in PC12 cells. Syntaxin-GFP assembled in clusters at sites where single granules had docked at the plasma membrane. Clusters were intermittently present at granule sites, as syntaxin molecules assembled and disassembled in a coordinated fashion. Recruitment to granules required the N-terminal domain of syntaxin, but not the entry of syntaxin into SNARE complexes. Clusters facilitated exocytosis and disassembled once exocytosis was complete. Syntaxin cluster formation defines an intermediate step in exocytosis.

Single secretory granules of live cells recruit syntaxin-1 and synaptosomal associated protein 25 (SNAP-25) in large copy numbers.

Before secretory vesicles undergo exocytosis, they must recruit the proteins syntaxin-1 and synaptosomal associated protein 25 (SNAP-25) in the plasma membrane. GFP-labeled versions of both proteins cluster at sites where secretory granules have docked. Single-particle tracking shows that minority populations of both molecules are strongly hindered in their mobility, consistent with their confinement in nanodomains. We measured the fluorescence of granule-associated clusters, the fluorescence of single molecules, and the numbers of unlabeled syntaxin-1 and SNAP-25 molecules per cell. There was a more than 10-fold excess of SNAP-25 over syntaxin-1. Fifty to seventy copies each of syntaxin-1 and SNAP-25 molecules were associated with a single docked granule, many more than have been reported to be required for fusion.

Social inequality of stroke mortality among US black populations, 1968 to 1987.

Though the rate of stroke mortality in the United States has been declining for all race-sex-specific groups, rates for blacks are among the highest in the world. Studies of the geographic and social variation of stroke mortality between populations have focused solely on either trends or social determinants, but not on both. In addition, conclusions have concentrated on the comparison of whites to nonwhites. The purpose of this study was to investigate the variation in stroke mortality trends, from 1968 to 1987, between US black populations categorized by the socioeconomic structure of the state economic area. The educational achievement profile of the population (percentage of the state economic area that had not completed high school) was used as an indicator of socioeconomic development. This aggregate study assessed whether (1) an inverse relationship existed between stroke mortality and educational achievement category, and (2) levels and trends of stroke mortality by educational achievement category varied from 1968 to 1987, for each age-gender-specific group (35 to 64, 65 to 74, and 75 to 84 years). Results showed an inverse relationship between educational achievement level of communities and stroke mortality, as well as a rank-ordering in level of educational achievement for all age-gender-specific groups. The absolute difference in rates among educational achievement levels clearly converged over time, with greater convergence in the period 1968 to 1978 than 1979 to 1987. The percent declines were similar across education categories. Results suggest that geographic inequalities in stroke mortality, in relation to socioeconomic structure, have converged over time.(ABSTRACT TRUNCATED AT 250 WORDS)

Vaccinia recombinant virus expressing the rabies virus glycoprotein: safety and efficacy trials in Canadian wildlife.

Twenty-six meadow voles (Microtus pennsylvanicus), ten woodchucks (Marmota monax), thirteen grey squirrels (Sciurus carolinensis), thirteen ring-billed gulls (Larus delawarensis), six red-tailed hawks (Buteo jamaicensis) and eight great horned owls (Bubo virginianus) received vaccinia virus recombinant expressing the rabies virus glycoprotein (V-RG) by direct instillation into the oral cavity. Each of ten coyotes (Canis latrans) received the virus in two vaccine-laden baits. Several voles and most of the gulls died from diseases unrelated to vaccination during the observation period, but all other animals remained healthy and survived. These deaths from causes other than vaccination and the absence of any lesions suggestive of vaccinia infection indicate that it is unlikely that any animal suffered or died as a result of V-RG administration. In addition several animals showed an unexpected high level of rabies neutralizing antibodies.

Immunogenicity and efficacy of two rabies vaccines in wild-caught, captive raccoons.

The immunogenicity and efficacy of two rabies vaccines in wild-caught, captive raccoons (Procyon lotor) were investigated. Raccoons were fed Ontario Slim (OS) baits containing a recombinant vaccinia virus-rabies glycoprotein (VRG) oral rabies vaccine, or they were given an intramuscular (IM) injection of IMRAB(®) 3 rabies vaccine. Blood samples collected before treatment and from weeks 1 to 16 posttreatment were assessed for the presence of rabies virus antibody (RVA). There were significantly more positive responders in the group that received an IM injection of IMRAB 3 (18/27) than in the group that consumed VRG in OS baits (VRG-OS; 4/ 26). There were no significant associations among age, sex, and seroconversion. Of those animals that mounted a humoral immune response to vaccination, RVA was first detected between weeks 1 and 5, with the majority of initial seroconversions detectable at week 2. A subsample of 50 raccoons (19 VRG-OS, 18 IMRAB 3, and 13 controls) from the longitudinal serology study was challenged with live raccoon variant rabies virus 442 days after initial treatment. There were significantly more survivors in the group that received IMRAB 3 (13/18) than in the VRG-OS (5/19) or control (2/13) groups. All 15 raccoons that demonstrated a serologic response survived challenge regardless of treatment. Of the 35 raccoons with no detectable serologic response, 30 (86%) succumbed to rabies virus infection (14/15 VRG-OS, 5/7 IMRAB 3, and 11/13 controls).

Immunization of foxes against rabies with a vaccinia recombinant virus expressing the rabies glycoprotein.

Foxes were vaccinated orally (by bait), gastrically (by stomach tube) and by scarification with a vaccinia recombinant virus expressing the rabies glycoprotein. Neutralizing antibodies against rabies virus were detected at two weeks postvaccination in 8/8 foxes in the bait-fed group, in 3/6 foxes inoculated by stomach tube and in 2/2 of the scarified foxes. After challenge at three months postvaccination with street rabies virus, all foxes that had developed antibodies were protected. The high rate of seroconversion, high levels of antibodies, and resistance to challenge suggest that this recombinant virus might be a suitable vaccine for oral immunization of foxes against rabies.

[S]-AR-R 15896AR-A novel anticonvulsant: acute safety, pharmacokinetic and pharmacodynamic properties.

A rational, chemical, synthetic effort to identify promising low-affinity uncompetitive N-methyl-D-aspartic acid receptor antagonists for use as antiepileptic drugs led to the discovery of AR-R 15035AR, or [RS]-alpha-phenyl-2-pyridine-ethanamine.2HCl. Chiral separation followed by intensive in vivo screening resulted in the selection of the [S] enantiomer, AR-R 15896AR, as the best compound for further preclinical development. AR-R 15896AR prevented tonic seizures in rodents for up to 6 to 8 h in response to maximal electroshock (MES), 4-aminopyridine, bicuculline, or strychnine, as well as characteristic seizures following injections of N-methyl-DL-aspartic or kainic acids. AR-R 15896AR was ineffective in two kindling models of epilepsy, did not produce tolerance to MES, and was devoid of proconvulsant and phencyclidine-like properties in mice and rats, respectively. Therapeutic indices for AR-R 15896AR were comparable to or exceeded those for standard anticonvulsants. Orally administered AR-R 15896AR rapidly entered the rat brain and was eliminated in parallel from the plasma and plasma-free compartment. A dose-response relationship between plasma and brain levels after p.o. or i.v. administration of AR-R 15896AR and protection against MES was highly correlative. The time course for loss of protection against MES mirrored the elimination of the compound from brain and plasma. The total brain concentration (25 microM) of drug at the ED50 value (approximately 3 mg/kg) for protection against MES seizures was consistent with the reported affinity of AR-R 15896AR at the N-methyl-D- aspartic acid binding site (IC50 value = 1.3 microM). The present findings demonstrated the attractiveness of AR-R 15896AR as a candidate for further development to treat epilepsy.