Membrane-enveloped vesicles travel among the compartments of the cytoplasm of eukaryotic cells, delivering their specific cargo to programmed locations by membrane fusion. The pairing of vesicle v-SNAREs (soluble N-ethylmaleimide-sensitive factor attachment protein receptors) with target membrane t-SNAREs has a central role in intracellular membrane fusion. We have tested all of the potential v-SNAREs encoded in the yeast genome for their capacity to trigger fusion by partnering with t-SNAREs that mark the Golgi, the vacuole and the plasma membrane. Here we find that, to a marked degree, the pattern of membrane flow in the cell is encoded and recapitulated by its isolated SNARE proteins, as predicted by the SNARE hypothesis.

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To fuse transport vesicles with target membranes, proteins of the SNARE (soluble N-ethylmaleimide-sensitive factor attachment protein receptors) complex must be located on both the vesicle (v-SNARE) and the target membrane (t-SNARE). In yeast, four integral membrane proteins, Sed5, Bos1, Sec22 and Bet1 (refs 2-6), each probably contribute a single helix to form the SNARE complex that is needed for transport from endoplasmic reticulum to Golgi. This generates a four-helix bundle, which ultimately mediates the actual fusion event. Here we explore how the anchoring arrangement of the four helices affects their ability to mediate fusion. We reconstituted two populations of phospholipid bilayer vesicles, with the individual SNARE proteins distributed in all possible combinations between them. Of the eight non-redundant permutations of four subunits distributed over two vesicle populations, only one results in membrane fusion. Fusion only occurs when the v-SNARE Bet1 is on one membrane and the syntaxin heavy chain Sed5 and its two light chains, Bos1 and Sec22, are on the other membrane where they form a functional t-SNARE. Thus, each SNARE protein is topologically restricted by design to function either as a v-SNARE or as part of a t-SNARE complex.

MRI studies using the manual tracing method have shown a smaller-than-normal hippocampal volume in patients with posttraumatic stress disorder (PTSD). However, these studies have yielded inconsistent results, and brain structures other than the hippocampus have not been well investigated. A recently developed, fully automated method called voxel-based morphometry enables an exploration of structural changes throughout the brain by applying statistical parametric mapping to high-resolution MRI. Here we first used this technology in patients with PTSD. Participants were 9 victims of the Tokyo subway sarin attack with PTSD and 16 matched victims of the same traumatic event without PTSD. The voxel-based morphometry showed a significant gray-matter volume reduction in the left anterior cingulate cortex (ACC) in trauma survivors with PTSD compared with those without PTSD. The severity of the disorder was negatively correlated with the gray-matter volume of the left ACC in PTSD subjects. There were no significant differences in other gray-matter regions or any of the white-matter regions between two groups. The present study demonstrates evidence for structural abnormalities of ACC in patients with PTSD. Together with previous functional neuroimaging studies showing a dysfunction of this region, the present findings provide further support for the important role of ACC, which is pivotally involved in attention, emotional regulation, and conditioned fear, in the pathology of PTSD.F indings from neuroimaging studies of patients with posttraumatic stress disorder (PTSD) have suggested that some brain pathology plays an important role in the disorder (1, 2). Six structural MRI studies have shown that combat-related or childhood physically and͞or sexually abused subjects with PTSD have a smaller-than-normal hippocampal volume (3-8). However, almost the same number of studies failed to show reduced hippocampal volume in chronically maltreated children (9-11), survivors of acute traumatic events (12), nonalcoholic combat veterans (13), and alcoholic patients (14) with PTSD. In contrast, brain structures other than the hippocampus have received less attention, although a few studies have reported whole-brain volume reduction (9), reduced total white-matter volume (7), smaller corpus callosum (9), larger superior temporal gyrus gray-matter volume (15), and attenuation of frontal lobe asymmetry (11). Therefore, it is unclear whether structural abnormality in brain structures other than the hippocampus exists in patients with PTSD.In contrast to the emphasis on the hippocampus in previous structural MRI studies, symptom-provocation and cognitiveactivation studies using functional neuroimaging have revealed greater activation of the amygdala, anterior paralimbic structures, Broca's region, and other neocortical regions and a failure of activation of anterior cingulate cortex (ACC) in response to trauma-related stimuli in individuals with PTSD (1, 2, 16). Furthermore, one study (17) showed a reduced N-acetylaspartate͞creatine ratio...

Lipid bilayer fusion is mediated by SNAREs (soluble N-ethylmaleimide-sensitive factor attachment protein receptors) located on the vesicle membrane (v-SNAREs) and the target membrane (t-SNAREs). The assembled v-SNARE/t-SNARE complex consists of a bundle of four helices, of which one is supplied by the v-SNARE and the other three by the t-SNARE. For t-SNAREs on the plasma membrane, the protein syntaxin supplies one helix and a SNAP-25 protein contributes the other two. Although there are numerous homologues of syntaxin on intracellular membranes, there are only two SNAP-25-related proteins in yeast, Sec9 and Spo20, both of which are localized to the plasma membrane and function in secretion and sporulation, respectively. What replaces SNAP-25 in t-SNAREs of intracellular membranes? Here we show that an intracellular t-SNARE is built from a 'heavy chain' homologous to syntaxin and two separate non-syntaxin 'light chains'. SNAP-25 may thus be the exception rather than the rule, having been derived from genes that encoded separate light chains that fused during evolution to produce a single gene encoding one protein with two helices.

The purpose of this study is to investigate whether the diffusional anisotropy of water molecules is disrupted in the pyramidal and extra-pyramidal regions in patients with amyotrophic lateral sclerosis (ALS). We studied seven patients with probable ALS (four women, mean age +/- SD, 57.3 +/- 6.2 years old) according to the criteria of the World Federation of Neurology. A control group consisted of 11 age- and sex-matched volunteers (six women, 57.1 +/- 4.5) without disorders affecting the central nervous system. Voxel-based diffusion tensor analysis was made with statistical parametric mapping (SPM99). We also created the normalized corticospinal tractography from the diffusion tensor data. The significant fractional anisotropy (FA) decrease in the ALS group was found in the right frontal subgyral white matter and left frontal precentral white matter. These clusters with significant FA decrease corresponded well to the average group map of the corticospinal tract in a standard reference frame. These results suggested that the combination of voxel-based diffusion tensor analysis and diffusion tensor tractography might help determine the location of the affected neuronal tissues among ALS patients in a non-invasive manner.

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In vitro reconstitution of functions of membrane proteins is often hampered by aggregation, misfolding, or lack of post-translational modifications of the proteins attributable to overexpression. To overcome this technical obstacle, we have developed a method to express multimeric integral membrane proteins in extracellular (budded) baculovirus particles that are released from Sf9 cells co-infected with multiple transmembrane proteins. We applied this method to the reconstitution of ␥-secretase, a membrane protease complex that catalyzes the intramembrane cleavage of ␤-amyloid precursor protein to release A␤ peptides, the major component of amyloid deposits in Alzheimer brains as well as of Notch. When we co-infected Sf9 cells with human presenilin 1 (PS1), nicastrin, APH-1a, and PEN-2, a high-molecular-weight membrane protein complex that contained PS1 exclusively in its fragment form associated with three other cofactor proteins was reconstituted and recovered in a highly ␥-secretase-active state in budded virus particles, whereas nonfunctional PS1 holoproteins massively contaminated the parental Sf9 cell membranes. The relative ␥-secretase activity (per molar PS1 fragments) was concentrated by ϳ2.5 fold in budded virus particles compared with that in Sf9 membranes. The budded baculovirus system will facilitate structural and functional analyses of ␥-secretase, as well as screening of its binding molecules or inhibitors, and will also provide a versatile methodology for the characterization of a variety of membrane protein complexes.