Does initiation of an ambulance pre-alert call reduce the door to needle time in acute myocardial infarct?

OBJECTIVES: To assess the effect an ambulance pre-alert call for patients with suspected acute myocardial infarction (AMI) would have on door to needle (DTN) times. METHODS: We carried out back to back audits of DTN times following the initiation of the pre-alert calls. PARTICIPANTS: All patients thrombolysed within the emergency department between July 2003 and April 2004 (inclusive). STATISTICAL ANALYSIS: Mean DTN times and time to ECG pre-change and post-change were compared using the Two sample t test. The Fisher's exact test was used to compare pre-change and post-change proportions of patients seen within guideline times. RESULTS: In total, 73 patients were thrombolysed with 40 of these arriving by ambulance. Eighteen of these 40 were pre-change and 22 were post-change. Four patients were excluded. Fifty per cent of the pre-change group had a DTN time of <30 minutes compared with 91% of the post-change group (p = 0.005, Fisher's exact test). The phase one mean DTN time was found to be significantly greater than that for phase two (Two sample t test, p = 0.016; 95% CI 1.6 to 14.6). CONCLUSIONS: There was a significant reduction in DTN times after the introduction of the pre-alert call.

The retinal rod Na(+)/Ca(2+),K(+) exchanger contains a noncleaved signal sequence required for translocation of the N terminus.

The retinal rod Na(+)/Ca(2+),K(+) exchanger (RodX) is a polytopic membrane protein found in photoreceptor outer segments where it is the principal extruder of Ca(2+) ions during light adaptation. We have examined the role of the N-terminal 65 amino acids in targeting, translocation, and integration of the RodX using an in vitro translation/translocation system. cDNAs encoding human RodX and bovine RodX through the first transmembrane domain were correctly targeted and integrated into microsomal membranes; deletion of the N-terminal 65 amino acids (aa) resulted in a translation product that was not targeted or integrated. Deletion of the first 65 aa had no effect on membrane targeting of full-length RodX, but the N-terminal hydrophilic domain no longer translocated. Chimeric constructs encoding the first 65 aa of bovine RodX fused to globin were translocated across microsomal membranes, demonstrating that the sequence could function heterologously. Studies of fresh bovine retinal extracts demonstrated that the first 65 aa are present in the native protein. These data demonstrate that the first 65 aa of RodX constitute an uncleaved signal sequence required for the efficient membrane targeting and proper membrane integration of RodX.

Role of the Rab3A-binding domain in targeting of rabphilin-3A to vesicle membranes of PC12 cells.

Rab3A is a small GTPase implicated in the docking of secretory vesicles in neuroendocrine cells. A putative downstream target for Rab3A, rabphilin-3A, is located exclusively on secretory vesicle membranes. It contains near its C terminus two C2 domains that bind Ca2+ in a phospholipid-dependent manner and an N-terminal, Rab3A-binding domain that includes a Cys-rich region. We have determined that the Cys-rich domain binds two Zn2+ ions and is necessary but not sufficient for efficient binding of rabphilin to Rab3A. A minimal Rab3A-binding domain consists of residues 45 to 170 of rabphilin. HA1-tagged Rab3A and a green fluorescent protein (GFP)-rabphilin fusion were used to examine the roles of Rab3A and of rabphilin domains in the subcellular localization of these proteins. A Rab3A mutant (T54A) that does not bind rabphifin in vitro colocalized with the GFP-rabphilin fusion, indicating that Rab3A targeting is independent of its interaction with rabphilin. Deletion of the C2 domains of rabphilin reduced membrane association of GFP-rabphilin but did not cause mistargeting of the membrane-associated fraction. However, disruption of the zinc fingers, which drastically reduced Rab3A binding, did not reduce membrane association. These results suggest that the C2 domains are required for efficient membrane attachment of rabphilin in PC12 cells and that Rab3A binding may act to target the protein to the correct membrane.

Interaction cloning of Rabin3, a novel protein that associates with the Ras-like GTPase Rab3A.

Rab3A is a small, Ras-like GTPase expressed in neuroendocrine cells, in which it is associated with secretory vesicle membranes and regulates exocytosis. Using the yeast two-hybrid system, we have identified a rat brain cDNA encoding a novel 50-kDa protein, which we have named Rabin3, that interacts with Rab3A and Rab3D but not with other small GTPases (Rab3C, Rab2, Ran, or Ras). Several independent point mutations in the effector domain of Rab3A (F51L, V55E, and G56D) which do not alter nucleotide binding by the GTPase abolish the interaction with Rabin3, while another mutation (V52A) appears to increase the interaction. These results demonstrate that the interaction is highly specific. However, a glutathione S-transferase-Rabin3 fusion protein associates only weakly in vitro with recombinant Rab3A and possesses no detectable GTPase-activating protein or nucleotide exchange activity, and Rabin3 overexpressed in adrenal chromaffin cells has no observable effect on secretion. The protein possess a sequence characteristic of coiled-coil domains and a second small region with sequence similarity to a Saccharomyces cerevisiae protein, Sec2p, Sec2p is essential for constitutive secretion in yeast cells and interacts with Sec4p, a close relative of the Rab3A GTPase. Rabin3 mRNA and protein are widely expressed but are particularly abundant in testes.

The Rab3A GTPase interacts with multiple factors through the same effector domain. Mutational analysis of cross-linking of Rab3A to a putative target protein.

Rab3A/smg25A is a small Ras-like guanine nucleotide binding protein implicated in the control of regulated secretion from cells. Rab3A is approximately 30% cytosolic and 70% associated with the membranes of secretory vesicles. It cross-links specifically to a rat brain membrane protein of about 85 kilodaltons (p85). To identify epitopes on Rab3A that are important for its interaction with this putative target protein, we have determined the effects of point mutations on the cross-linking efficiency of Rab3A to p85. Rab3A, which was preincubated with a non-hydrolyzable analog of GTP, cross-linked more efficiently to p85 than did Rab3A-GDP. Rab3A mutants that had decreased nucleotide binding also exhibited poor cross-linking to p85. Mutations in the effector domain, a site important for the interaction of Rab3A with its guanine nucleotide releasing factor, guanine nucleotide dissociation inhibitor, and GTPase-activating protein, eliminated the ability of Rab3A to cross-link to p85. However, short peptides corresponding to the effector domain did not reduce cross-linking efficiency when present at a concentration of 50 microM.

Mutants of Rab3A analogous to oncogenic Ras mutants. Sensitivity to Rab3A-GTPase activating protein and Rab3A-guanine nucleotide releasing factor.

Rab3A is a Ras-like GTPase that is believed to function in regulated secretion. A GTPase activating protein (GAP) and a guanine nucleotide releasing factor (GRF) specific for Rab3A have recently been described. In this study we have described the biochemical activities of Rab3A mutants in codons 31, 81, 135, and 166, which correspond to codons 12, 61, 116, and 146 in Ras. The results demonstrate that simple extrapolations from the properties of Ras mutants are not valid for all small GTPases. S31V-Rab3A and Q81L-Rab3A had a reduced basal GTPase activity, but surprisingly were sensitive to the effects of Rab3A-GAP and insensitive to Rab3A-GRF. Q81L-Rab3A and wild-type Rab3A that were bound to a nonhydrolyzable GTP analog had similar affinities for Rab3A-GAP. In vivo, the percent of Q81L-Rab3A (46%) and wild-type Rab3A (43%) complexed to GTP was similar. These findings indicate that Rab3A-GRF and Rab3A-GAP interact with residues different from those of previously described GAPs and GRFs. Both A166V-Rab3A and N135I-Rab3A had increased intrinsic dissociation rates for GDP. However, the dissociation rate for N135I-Rab3A was > 100-fold higher than that for A166V-Rab3A suggesting that, in vivo, of the two, N135I-Rab3A would more likely be preferentially in the GTP-bound state.