rab3A attachment to the synaptic vesicle membrane mediated by a conserved polyisoprenylated carboxy-terminal sequence.

rab3A is a small neuronal GTP-binding protein specifically localized to synaptic vesicles. Membrane-bound rab3A behaves like an intrinsic membrane protein in vitro, but reversibly dissociates from synaptic vesicles after exocytosis in vivo. Here we demonstrate that rab3A is attached to synaptic vesicle membranes by a carboxy-terminal Cys-X-Cys sequence that is posttranslationally modified. This modification is inhibited by compactin in a mevalonate-dependent manner, suggesting that the Cys-X-Cys sequence represents a novel polyisoprenylation sequence. Isolation of a rab3 homolog from D. melanogaster reveals high evolutionary conservation of rab3A, including its carboxy-terminal Cys-X-Cys sequence. The posttranslational modifications of soluble and membrane-bound rab3A are biochemically different, but both require the carboxy-terminal Cys-X-Cys sequence and are faithfully reproduced in nonneuronal cells. Our results suggest that the carboxy-terminal Cys-X-Cys sequence of rab3A is polyisoprenylated and is used as its regulatable membrane anchor. Furthermore, the hydrophobic modification of rab3A and its correct intracellular targeting to synaptic vesicles are independent, presumably consecutive events.

Absence of exercise-induced MRI enhancement of skeletal muscle in McArdle's disease.

To assess the role of glycogenolysis in mediating exercise-induced increases in muscle water as monitored by changes in muscle proton relaxation times on magnetic resonance imaging (MRI) and cross-sectional area (CSA), five patients with myophosphorylase deficiency (MPD) were compared with seven controls. Absolute and relative work loads were matched during ischemic handgrip and graded cycling, respectively. Relaxation times of active muscle did not increase after handgrip in MPD (T1: 1 +/- 14%, P greater than 0.1; T2: 4 +/- 4%, P greater than 0.1) but did in controls (T1: 59 +/- 30%, P less than 0.005; T2: 26 +/- 9%, P less than 0.005). The volume of exercised muscles, estimated by CSA, increased in both groups after handgrip (controls: 13.8 +/- 3.5%, n = 7, P less than 0.0001; MPD: 7.5 +/- 1.5%, n = 4, P less than 0.005), but the change was greater in controls (P less than 0.02). Ischemic handgrip in controls resulted in a large increase in finger flexor signal intensity (SI) on short tau-inversion recovery images (25 +/- 7%, n = 3; P less than 0.005 compared with preexercise) and a further increase with subsequent reflow (43 +/- 11%, n = 3; P less than 0.001 compared with rest); in MPD, SI did not increase. The ratio of active to inactive muscle SI did not increase from rest to maximal cycle exercise in MPD (0 +/- 20%, n = 2, P greater than 0.1) but did in normals (73 +/- 36%, n = 3; P less than 0.001).(ABSTRACT TRUNCATED AT 250 WORDS)

A MRI gradient waveform model for automated sequence calibration.

In order to set up magnetic resonance imaging (MRI) procedures of arbitrary voxel dimensions, slice orientation, and sequence timing in a reasonable time, some form of automatic gradient pulse calibration is required. One such method, involving simulation of gradient waveforms, is presented. Waveforms are modeled based on measurements of the step response. The model used divides each transition into three time regions: a "start" region in the first 0.3 ms, a "slew" region, and a "tail" region representing decay of the eddy current compensation error. In the "slew" region, the time derivative of the gradient, G' (t), is expressed as a function of G(t). The first two regions are nonlinear with respect to demand. The mean error in the simulated gradient is generally less than 0.04 mT m-1 in spin echo sequences. Image signal/noise ratios resulting from sequences calibrated using the model are within 5% of those of empirically calibrated sequences.

Low resolution spin echo: a simple timesaving technique for MRI liver exams.

MR evaluation of the liver at mid-field strength requires relatively lengthy T2-weighted sequences (T2W) for differentiation of benign cavernous hemangiomas from malignant lesions. A short duration T2W, which maintains standard signal-to-noise ratio and also contrast relationships, can be easily implemented by increasing the pixel size in the phase-encoding direction (thus reducing spatial resolution) and proportionally decreasing the number of phase-encoding steps in the matrix (thus reducing acquisition time). Blinded interpretations of a quick (4 min), low resolution (3.4 mm x 1.7 mm pixel) T2W sequence (matrix 64 x 256, FOV 21.7 cm phase x 43.5 cm frequency) were compared to the 17 min standard resolution (1.7 mm x 1.7 mm) T2W sequence (256 x 256 matrix, FOV 43 x 43 cm) in 25 patients suspected of having liver metastasis. Lesions felt to be cavernous hemangiomas showed a 100% (24/24) agreement for interpreter "A" and 96% (22/23) agreement for interpreter "B" when 4 min low resolution T2W was compared to the standard 17 min sequence. Sensitivity (for all types of lesions) of the low resolution T2W sequence ranged from 100% (31/31) for interpreter "A" to 80% (28/35) for interpreter "B." Missed lesions (interpreter "B") were either partially obscured by excessive fat (wrap around) (N = 4), less than 1 cm in size (N = 2), or degraded by motion artifact (N = 1). Thus in many situations low resolution T2 may provide a substantial timesaving alternative to standard T2W particularly where T2W is used primarily for lesion classification in normal sized patients.

Synaptotagmin II. A novel differentially distributed form of synaptotagmin.

Synaptotagmin is a synaptic vesicle membrane protein with properties suggestive of a role in synaptic vesicle exocytosis (Perin, M. S., Fried, V. A., Mignery, G. A., Jahn, R., and Südhof, T. C. (1990) Nature 345, 260-263). Here, we report the structure of a novel form of synaptotagmin named synaptotagmin II that is highly homologous to the originally described synaptotagmin, now referred to as synaptotagmin I. Synaptotagmins I and II exhibit the same overall structure, containing a small intravesicular sequence that is glycosylated, a single transmembrane region, and a large carboxyl-terminal cytoplasmic sequence that includes two copies of an internal repeat homologous to the regulatory region of protein kinase C. The homology between synaptotagmins I and II is not uniformly distributed across the molecule but is highest in their carboxyl-terminal regulatory repeats (88% sequence identity) and lowest in their amino-terminal intravesicular sequences (46% sequence identity). RNA blots demonstrate complementary patterns of expression for synaptotagmins I and II, with synaptotagmin I preferentially expressed in rostral, phylogenetically younger brain regions, and synaptotagmin II predominantly expressed in caudal, phylogenetically older brain regions. With this description of two forms of synaptotagmin, all major synaptic vesicle proteins implicated in membrane traffic have now been shown to be present in several isoforms with differential distributions, suggesting that this is a general organizational principle of the mammalian brain.

Structure and expression of the rat inositol 1,4,5-trisphosphate receptor.

The complete primary structure of the inositol 1,4,5-trisphosphate receptor from rat brain was elucidated using a series of overlapping cDNA clones. Two different sets of clones that either contain or lack a 45-nucleotide sequence in the amino-terminal third of the protein were isolated, suggesting a differential splicing event that results in the biosynthesis of either a 2734- or 2749-amino acid receptor protein. Hydrophobicity analysis demonstrates the presence of a cluster of hydrophobic sequences in the carboxyl-terminal third of the protein that probably comprise eight transmembrane regions and that may form the calcium channel intrinsic to the receptor. The receptor was universally expressed at low levels in all tissues and cultured cells tested. Transfection of a full-length expression construct of the inositol 1,4,5-trisphosphate receptor into COS cells resulted in the biosynthesis of a 260-kDa protein that bound inositol 1,4,5-trisphosphate and formed high molecular weight complexes similar to the native receptor as analyzed by sucrose gradient centrifugations. On the other hand, the protein product synthesized by a mutant receptor construct in which the amino-terminal 418 amino acids were deleted failed to bind inositol 1,4,5-trisphosphate. The mutant receptor still formed high molecular weight complexes, suggesting that it folded normally and that the amino-terminal sequences of the receptor are part of the ligand binding domain.

Structures and chromosomal localizations of two human genes encoding synaptobrevins 1 and 2.

Synaptobrevins 1 and 2 are small integral membrane proteins specific for synaptic vesicles in neurons. Two cosmid clones containing the human genes encoding synaptobrevins 1 and 2 (gene symbols SYB1 and SYB2, respectively) were isolated and characterized. The coding regions of the synaptobrevin genes are highly homologous to each other and are interrupted at identical positions by introns of different size and sequence. Each gene is organized into five exons whose boundaries correspond to those of the protein domains. Exon I contains part of the initiator methionine codon whereas exon II encodes the variable and immunogenic amino-terminal domain of the synaptobrevins. The third exon comprises the highly conserved central domain of the synaptobrevins, exon IV encodes most of the transmembrane region, and exon V contains the last residues of the transmembrane region and the small intravesicular carboxyl terminus. Comparisons of the synaptobrevin sequences in five species from Drosophila with man indicate a selective conservation of sequences adjacent to the synaptic vesicle surface, suggesting a function at the membrane-cystosol interface. The chromosomal localizations of the human and mouse SYB1 and SYB2 genes were determined using hybrid cell lines. SYB1 was localized to the short arm of human chromosome 12 and to mouse chromosome 6 whereas SYB2 was found on the distal portion of the short arm of human chromosome 17 and on mouse chromosome 11. A PstI restriction fragment length polymorphism was identified at the SYB2 locus.

Structure of a novel InsP3 receptor.

Inositol 1,4,5-trisphosphate (InsP3) constitutes a major intracellular second messenger that transduces many growth factor and neurotransmitter signals. InsP3 causes the release of Ca2+ from intracellular stores by binding to specific receptors that are coupled to Ca2+ channels. One such receptor from cerebellum has previously been extensively characterized. We have now determined the full structure of a second, novel InsP3 receptor which we refer to as type 2 InsP3 receptor as opposed to the cerebellar type 1 InsP3 receptor. The type 2 InsP3 receptor has the same general structural design as the cerebellar type 1 InsP3 receptor with which it shares 69% sequence identity. Expression of the amino-terminal 1078 amino acids of the type 2 receptor demonstrates high affinity binding of InsP3 to the type 2 receptor with a similar specificity but higher affinity than observed for the type 1 receptor. These results demonstrate the presence of several types of InsP3 receptor in brain and raise the possibility that intracellular Ca2+ signaling may involve multiple pathways with different regulatory properties dependent on different InsP3 receptors.

Fast short-tau inversion-recovery MR imaging.

To enhance the versatility of the short-tau inversion-recovery (STIR) sequences, the authors determined a range of repetition time (TR) and inversion time (TI) combinations that suppress signal intensity from fat by study of both patient and phantom images. To make fast STIR images, variations in the following pulsing conditions were studied with use of an interactive computer program: decreasing the TR, limiting the number of excitations, and limiting the number of phase-encoding steps. The authors found that (a) STIR imaging need not be time consuming, (b) fat suppression can be accomplished at shorter TR by using shorter TI, and (c) short-TR fast STIR imaging is sensitive to enhancement with gadopentetate dimeglumine.

Synaptophysin: structure of the human gene and assignment to the X chromosome in man and mouse.

Synaptophysin is an integral membrane protein of small synaptic vesicles in brain and endocrine cells. We have determined the structure and organization of the human synaptophysin gene and have established the chromosome localizations in man and mouse. Analysis of a cosmid clone containing the human synaptophysin gene (SYP) revealed seven exons distributed over approximately 20 kb, when compared with the previously published cDNA sequence. The exon-intron boundaries have been identified and do not correlate with functional domains. One intron interrupts the 3' untranslated region. Chromosomal localization of the human and murine genes for synaptophysin established the human SYP locus on the X chromosome in subbands Xp11.22-p11.23 and the mouse synaptophysin gene locus (Syp) on the X chromosome in region A-D. In addition, an Eco0109 RFLP has been identified and used in genetic mapping of the human SYP locus and supports the order TIMP-SYP-DXS14 within a span of approximately 4-7 centimorgans.

Effect of perfusion on exercised muscle: MR imaging evaluation.

An ischemic clamp model of exercise was used to evaluate the potential role of blood flow in mediating changes in the magnetic resonance imaging appearance of skeletal muscle. Proton relaxation times of muscle were serially estimated in 10 healthy subjects (a) before exercise, (b) after exercise in the presence of vascular occlusion (VO1), (c) during vascular reocclusion after 1 minute of reperfusion (VO2), and (d) after reinstitution of continuous flow. T1 and T2 of active muscles were increased during VO1. During VO2, there were additional increases in relaxation times of active muscles. Reinstitution of continuous flow was associated with a continuous decrease in the T2 of exercised muscle. Hence, blood flow was not required for increases in T1 and T2 with exercise. Additional relaxation time increases occurred after a brief period of reperfusion; however, continuous flow was associated with a decrease in T2.