Does sharing process differences reduce patient length of stay in the emergency department?

STUDY OBJECTIVE: We assess the ability of the best demonstrated processes (BDP) methodology to decrease emergency department patient length of stay (LOS) in EDs in a large multihospital system. METHODS: Two hundred ninety-one EDs were ranked by LOS, and the fastest and slowest EDs were observed to identify the BDPs. The resulting "meaningful differences" were shared with all EDs throughout the hospital system. LOS studies were repeated after the BDP intervention. Five separate LOS measures were performed during a 19-month period, with 223 to 273 EDs participating in each measure. Three interval times were calculated: arrival to examination room, examination room to physician evaluation, and physician evaluation to discharge. RESULTS: Two hundred ninety-one EDs participated, and 386,837 patient visits were evaluated. Before intervention, the average LOS was 147 minutes for all EDs and 186 minutes in the slowest third. At 19 months after intervention, the average LOS was 139 minutes for all EDs and 157 minutes in the slowest third. Between the initial and final measurement period, there was an 8-minute (5.4%) improvement in LOS on a system-wide basis, and the slowest third of EDs improved LOS by 29 minutes (15.6%). Before intervention, arrival to examination room time was 27 minutes, examination room to physician evaluation was 20 minutes, and evaluation to discharge was 100 minutes. After intervention, these times decreased to 22 (P <.001), 18 (P <.001), and 99 (P =.33) minutes, respectively. The slowest one third of EDs went from 37 to 24 minutes for arrival to examination room time (P <.001), from 25 to 20 minutes for examination room to evaluation time (P <.001), and from 124 to 113 minutes for evaluation to discharge time (P <.001). CONCLUSION: Implementing observed BDP meaningful differences resulted in decreased patient LOS in EDs, particularly in the slowest one third of EDs in the hospital system.

Traffic control: Rab GTPases and the regulation of interorganellar transport.

Membrane proteins, membrane lipids, and luminal contents are exchanged among the intracellular compartments of eukaryotic cells by vesicular transport. This process must be highly ordered to maintain cellular architecture in the face of rapid membrane turnover. The Ras-related Rab GTPases play multiple roles in regulating this traffic.

A novel membrane-anchored Rab5 interacting protein required for homotypic endosome fusion.

The ras-related GTPase rab5 is rate-limiting for homotypic early endosome fusion. We used a yeast two-hybrid screen to identify a rab5 interacting protein, rab5ip. The cDNA sequence encodes a ubiquitous 75-kDa protein with an N-terminal transmembrane domain (TM), a central coiled-coil structure, and a C-terminal region homologous to several centrosome-associated proteins. rab5ip lacking the transmembrane domain (rab5ipTM(-)) had a greater affinity in vitro for rab5-guanosine 5'-O-2-(thio)diphosphate than for rab5-guanosine 5'-3-O-(thio)triphosphate. In transfected HeLa cells, rab5ipTM(-) was partly cytosolic and localized (by immunofluorescence) with a rab5 mutant believed to be in a GDP conformation (GFP-rab5(G78A)) but not with GFP-rab5(Q79L), a GTPase-deficient mutant. rab5ip with the transmembrane domain (rab5ipTM(+)) was completely associated with the particulate fraction and localized extensively with GFP-rab5(wt) in punctate endosome-like structures. Overexpression of rab5ipTM(+) using Sindbis virus stimulated the accumulation of fluid-phase horseradish peroxidase by BHK-21 cells, and homotypic endosome fusion in vitro was inhibited by antibody against rab5ip. rab5ipTM(-) inhibited rab5(wt)-stimulated endosome fusion but did not inhibit fusion stimulated by rab5(Q79L). rab5ip represents a novel rab5 interacting protein that may function on endocytic vesicles as a receptor for rab5-GDP and participate in the activation of rab5.

Conformationally variable Rab protein surface regions mapped by limited proteolysis and homology modelling.

Tryptic proteolysis of the small GTPases Rab4 and Rab5 is a multi-step, nucleotide-dependent process. Using N-terminal peptide sequencing, matrix-assisted laser desorption ionization-time-of-flight MS and molecular modelling, we identified the three initial sites of proteolysis in Rab5 as Arg-4, Arg-81 and Arg-197. Arg-4 and Arg-81 lie within regions previously implicated in Rab5 endocytic function, and Arg-197 lies in a region involved in membrane targeting. Topologically, Arg-81 lies within the conformationally variable Switch II region shown to be important for protein-protein interactions of other GTPases. Homology modelling studies on Rab5 indicate that the Arg-81 side chain is buried in the Rab5 GTP conformation, but is solvent-accessible in the GDP conformation, explaining the dependence of proteolysis on nucleotides. Peptide mapping of Rab4 was performed to take advantage of additional scissile bonds within Switch II to determine more precisely the limits of the nucleotide-dependent protease-accessible region. The Rab4 cleavage sites corresponded to Arg-81 and Pro-87 of Rab5, and taken together with the finding that Rab5 was not cleaved at Arg-91 this analysis defines an eight-residue surface-exposed conformationally variable region lying in the centre of Switch II. A sequence comparison of Rab proteins shows these eight residues to have a loosely conserved motif that we term Switch II(v) for its relative variability. C-terminal to Switch II(v) is a highly conserved Rab-specific YYRGA motif that we term Switch II(c) for its constant sequence. N-terminal to Switch II(v) is a sequence-invariant G-domain involved in nucleotide binding and hydrolysis. We propose that the Rab Switch II(v) region imparts specificity to nucleotide-dependent protein-protein interactions.

Evidence for a symmetrical requirement for Rab5-GTP in in vitro endosome-endosome fusion.

Early endosome fusion, which has been extensively characterized using an in vitro reconstitution assay, is Rab5-dependent. To examine the requirement for Rab5 on both fusion partners, we prepared cytosol and endosomes depleted of Rab5. Unlike control cytosol, Rab5-depleted cytosol was only marginally active in the in vitro endosome fusion. However, fusion could be restored by the addition of wild-type Rab5 or Rab5 D136N, a mutant whose nucleotide specificity favors xanthine over guanine. The addition of Rab5 D136N restored fusion only in the presence of XTP. In the absence of XTP or in the presence of XDP, Rab5 D136N failed to restore fusion. When fusion was carried out with endosomal vesicles depleted of Rab GTPases (by preincubation of vesicles with GDP dissociation inhibitor), together with cytosol immunodepleted of Rab5, fusion was virtually absent. We then used immunodepleted cytosol and GDP dissociation inhibitor-treated vesicles to determine whether Rab5 is required by both fusion partners. Using separate sets of endosomal vesicles, we found that priming both sets of Rab5-depleted vesicles with Rab5 Q79L, a GTPase-defective mutant, substantially stimulated endosome fusion. Priming one set of vesicles with Rab5 Q79L and a second set of vesicles with Rab5 S34N failed to activate fusion. When both sets of Rab5-depleted vesicles were primed with Rab5 D136N supplemented with XTP, endosome fusion was stimulated, similar to that observed with Rab5 Q79L. However, when one set of vesicles was preincubated with Rab5 D136N plus XTP and the second set with Rab5 D136N and XDP, no stimulation of fusion was observed. We conclude that Rab5-GTP is required on both fusion partners for docking and fusion of early endosomes. To confirm the fusion of Rab5-GTP-positive vesicles in vivo, we expressed GFP-Rab5 Q79L in fibroblasts and observed fusion of Rab5-positive vesicles. We failed to record fusion of Rab5-positive vesicles with Rab5-negative vesicles. We conclude that Rab5-GTP is required on both sets of endosomes for fusion in vitro and in living cells.

Emergency department ultrasound services by emergency physicians: model for gaining hospital approval.

We anticipate that over the next few years, emergency physician use of emergency department ultrasound will become the standard of care. However, many EDs are hampered in their efforts to gain hospital approval for emergency physician use of ultrasound because of the lack of publicized information regarding the goals of such use, the scope of emergency physician ultrasound privileges, emergency physician ultrasound credentialing criteria, and ED ultrasound quality-improvement plans. In this article we address these issues and provide an example of a proposal used successfully to gain hospital approval for ED use of ultrasound by emergency physicians.

Specific and effective interaction of a guanine nucleotide analogue with small G proteins.

G proteins are molecular switches that use a cycle of GTP binding and hydrolysis to regulate a wide variety of cellular biochemical processes. Because the functional state of these proteins is allosterically determined by bound guanine nucleotides, a nucleotide analogue with protein specificity might have pharmacological or biochemical value. The binding of [alpha-32P]GTP to four small G proteins immobilized on nitrocellulose was competed by a series of analogues with modifications at multiple sites. One analogue, N2-(p-n-butylphenyl)guanosine 5'-(beta,gamma-difluoromethylene)triphosphate, had a approximately 40-fold higher affinity for one small G protein than for two of the others. Systematic analysis of each modification in the synthetic nucleotide revealed that specificity was conferred by the carbon substitution in the beta,gamma-phosphoanhydride bond. These observations were then extended to purified proteins of known sequence in solution by filtration binding studies with H-ras and rab5. Ras was 9-fold more discriminant between guanosine-5'-(beta,gamma-difluoromethylene)triphosphate and guanosine-5'-O-(3-thiotriphosphate) than was rab5, and the Q79L GTPase-defective mutant of rab5 was 6-fold more discriminant than wild-type rab5. Guanosine-5'-(beta,gamma-difluoromethylene)triphosphate protected a 20-kDa fragment of rab5 from tryptic proteolysis with greater efficacy than guanosine-5'-O-(3-thiotriphosphate) or guanosine-5'-(beta,gamma-imido)triphosphate despite its lower affinity, and GMP stabilized a conformation indistinguishable from apo-rab5. These results identify a synthetic guanine nucleotide analogue with differential affinity for closely related G proteins, determine the atomic substitution in the analogue that confers specificity, demonstrate discrimination by the analogue between wild-type and a point-mutant G protein, and establish efficacy of the analogue in inducing conformational change of a target protein disproportionate to the affinity of the interaction.

Functional and structural interactions of the Rab5 D136N mutant with xanthine nucleotides.

Rab5 is a Ras-related GTPase which regulates endosomal fusion. The D136N mutant of Rab5, which was predicted to switch specificity from guanine to xanthine nucleotides, was expressed in E. coli, extracted with urea, purified by column chromatography, and refolded by stepwise dialysis against buffer containing XDP. The purified protein bound xanthine nucleotides with considerably higher affinity than guanine nucleotides. In vitro prenylation of the mutant protein was highly dependent on xanthosine diphosphate. In contrast, both the wild type and mutant proteins were protected from proteolysis equally well by non-cognate and cognate triphosphate nucleosides at high concentration. The D136N Rab5 mutant appears to be a valuable reagent in conjunction with xanthine nucleotides for the study of protein-nucleotide interactions in systems in which multiple GTPases are active, although interactions with non-cognate nucleotides should be evaluated if they are present at high concentration.

Ligand-stimulated beta 2-adrenergic receptor internalization via the constitutive endocytic pathway into rab5-containing endosomes.

The small GTPase rab5 appears to be rate-limiting for the constitutive internalization of transferrin receptor and for fluid-phase endocytosis. However, it is unknown whether rab5 regulates receptors whose internalization is stimulated by the binding of ligand, and whether such receptors change the underlying rate of the endocytic pathways they utilize. As a model for ligand-stimulated endocytosis, we used transfected HEK293 cells expressing high levels of an epitope-tagged human beta 2-adrenergic receptor. Nearly all receptors were on the cell surface in the absence of agonist, but within ten minutes of agonist addition > 50% of receptors internalized and colocalized extensively with rab5. Hypertonic sucrose blocked beta 2-adrenergic receptor internalization, as well as that of transferrin receptor, suggesting a clathrin-mediated process. In contrast, an inhibitor of potocytosis had little effect upon beta 2-adrenergic receptor internalization, suggesting that this process did not require active caveolae. Consistent with this finding, caveolin was not detectable in the 12 beta 6 line, as assessed by western blotting with a polyclonal anti-caveolin antibody. Stimulated receptor internalization did not affect the rate or capacity of the constitutive endocytic pathway since there was no detectable increase in fluid-phase endocytosis after addition of beta-agonist, nor was there a significant change in the amount of surface transferrin receptor. Altogether, these data suggest that beta 2-adrenergic receptors internalize by a clathrin-mediated and rab5-regulated constitutive endocytic pathway. Further, agonist-stimulated receptor internalization has no detectable effect upon the function of this pathway.

Pilot study of intravenous magnesium sulfate in refractory cardiac arrest: safety data and recommendations for future studies.

UNLABELLED: Recent case reports have evidenced a temporal association between administration of i.v. magnesium sulfate (M) and resuscitation from prolonged cardiac arrest refractory to standard (S) ACLS attempts. However, speculation has arisen that M as a vasodilator, may decrease aortic diastolic and coronary perfusion pressure (CPP), aortic systolic and cerebral perfusion pressures (CePP), which may decrease resuscitation rates and neurologic recovery, as compared to standard ACLS alone (SA). OBJECTIVE: To resolve positive beginning evidence vs. negative theory, we conducted a pilot study of M+S vs. SA in refractory cardiac arrest on resuscitation rates (% R, return of stable pulses > 30 min without CPR, first in-hospital cardiac arrest > 5-min duration) and neurologic recovery/survival to hospital discharge (SHD). METHODS: All patients from 1 January 1990-31 December 1991 at Rose Hospital, in cardiac arrest refractory to S through the first epinephrine dose (including 3 defibrillation attempts with pulseless VT/VF) were included in the data analysis, except: (1) patients with trauma, known poisoning, < 18 years, pregnancy excluded; (2) Standard ACLS alone patients with cardiac arrest < 5-min duration were not included in the SA comparison group, because the shortest cardiac arrest time before i.v. MgSO4 administration in the M+S group was 5 min. M+S (N = 29) and SA (N = 33) groups were also comparable on mean age (72-73 years) in this open-label prospective case-matched control group study. RESULTS: SHD rates were nearly equivalent between M+S (5.2%) and SA (4.5%). Complete or partial neurologic recovery, as best neurologic status post-R, occurred in 21% (6/29) M+S patients vs. 9% (3/33) SA (P = 0.17), even though cardiac arrest time on the study code call for resuscitated patients averaged shorter with SA (14.2 min) than M+S (19.8 min). M was frequently administered late in the resuscitation attempt--code call to M administration averaged 16.5 min (< 10 min in only 4/28 patients). A trend toward increased % R with M was evidenced: 21% (7/33) SA vs. 35% (10/29) M+S (P = 0.21). A temporal association between M administration and first return of spontaneous circulation (ROSC) was also documented in 4 of 10 M+S patients (pulseless electrical activity (3)/pulseless VT (1)), who had first ROSC/R occur within 0.5-2.25 min following first i.v. M bolus delivery, after 11-30 min (mean = 20 min) of continuous pulseless rhythm refractory to standard ACLS. All M+S resuscitations occurred within the dose range 2.5-5 g (i.v. push): 3/6 (50%) and 7/13 (54%) R occurred with 1-3 g and 4-5 g MgSO4, respectively (at least 11/13 patients had peripheral i.v. delivery with 4-5 g M). Analyzing post-ROSC hypotension proved important, as 50% of pts with first recorded systolic BP post-ROSC < 90 mmHg were resuscitated vs. 83% with > 90 mmHg (P = 0.10). A trend toward increased post-ROSC hypotension was evidenced with i.v. MgSO4: Recorded first or second systolic BP < 90 mmHg post-ROSC occurred in 66% of M+S vs. 42% of SA patients. All 3 M+S patients having a wide open i.v. levophed infusion as vasopressor support, started immediately post-ROSC/i.v. MgSO4 with systolic BP < 90 mmHg and continued at least 15 min (titrating to a systolic BP approximately 110 mmHg), had a temporal association between M delivery and R after 14-30 min of continuous pulselessness refractory to S. CONCLUSION: Human research determining whether i.v. MgSO4 increases long-term survival from refractory cardiac arrest should be vigorously pursued, as it is safe to proceed given the above described considerations.

Biochemical and functional characterization of a recombinant GTPase, Rab5, and two of its mutants.

Biochemical, structural, and functional properties of Rab5 wild-type (WT) protein were compared with those of Q79L and N133I mutants. The detergent 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonate increased guanine nucleotide binding to Rab5 WT approximately 10-fold. The single-step catalytic rate of Rab5 WT exceeded that of Q79L 12.2-fold, but the steady-state GTPase rate was only 2.8-fold greater because GDP dissociation was rate-limiting and GDP dissociation was 3.6-fold slower than for Q79L. In contrast, dissociation rates of GTP were indistinguishable. Binding to Rab5 N133I was not detectable. GTP protected Rab5 WT and Q79L from any apparent proteolysis by trypsin. A 20-kDa fragment was the major product of digestion in the presence of GDP, and 12- and 8-kDa fragments were the major products in the absence of added guanine nucleotides. Rab5 N133I underwent no apparent proteolysis with 10 mM GTP or GDP, suggesting a "triphosphate" conformation may be induced in Rab5 N133I by either GTP or GDP. Partially geranylgeranylated Rab5 WT stimulated endosome fusion in vitro, whereas unmodified Rab5 WT did not. Processed Rab5 Q79L failed to inhibit endosome fusion, and Rab5 N133I could not be geranylgeranylated. These findings identify biochemical and structural features of Rab5 proteins, providing data for the interpretation of functional assays.

The 65-kDa protein from pig heart. A new substrate for Clostridium botulinum ADP-ribosyltransferase (exoenzyme C3).

In the pig heart sarcolemma, a 65 kDa protein is found to be ADP-ribosylated by Clostridium botulinum ADP-ribosyltransferase (exoenzyme C3). ADP-ribosylation of this protein is regulated by guanyl nucleotides and cytosol factor in a fashion similar to that for other C3 substrates. The new exoenzyme C3 substrate was partially purified. This protein is supposed to be a GTP-binding one.

Signal-transducing GTP-binding proteins of mammalian heart and lungs.

Signal-transducing GTP-binding Proteins of Mammalian Heart and Lungs. Journal of Molecular and Cellular Cardiology (1989) 21 (Suppl I) 91-95. Three distinct G-proteins have been found in mammalian heart sarcolemma: Gi (alpha i = 40 kDa, beta = 36 kDa, and lambda less than 14 kDa), Gp (alpha p = 23 kDa, beta = 36 kDa, and lambda less than 14 kDa), and Gs (alpha s = 42 kDa). ADP-ribosylation of sarcolemmal alpha i by pertussis toxin (PT) or preincubation of sarcolemma with protein kinase C and PMA resulted in increased adenylate cyclase activity and blockade of GTP-dependent inhibition by carbachol whereas the GTP-dependent activating effect of isoproterenol on the adenylate cyclase was preserved. ADP-ribosylation of alpha i in sarcolemma by endogenous NADP-sensitive ADP-ribosyltransferase abolished the PT-induced ADP-ribosylation of alpha i. Gpp (NH)p attenuated the PT-induced ADP-ribosylation of alpha i and promoted the cholera toxin (CT)-induced ADP-ribosylation of alpha s. The CT-induced alpha s ADP-ribosylation was enhanced in the presence of heart cytosol. Soluble Gi- and Gs-proteins were identified in lung cytosol. The 40 kDa alpha i in membrane and soluble fractions was ADP-ribosylated by PT, while the soluble 42 kDa alpha s was ADP-ribosylated by CT in lung tissue. The ADP-ribosylation of soluble alpha i by PT-suppressed guanyl nucleotide binding to Gi. The apparent molecular mass of partially purified soluble Gi was 75 kDa.

Purification and some properties of GTP-binding proteins from pig heart plasma membranes.

The major GTP-binding proteins (N-proteins) have been purified from cholate extract of pig heart plasma membranes using chromatography through DEAE-Trisacryl, Sephacryl S-300, Octyl-Sepharose, and DEAE-Sepharose. The hydrophobic chromatography resulted in elution of two GTP-binding activity peaks. The specific GTP gamma S binding of both N-protein preparations was 2.5 nmol/mg of protein with KD value 2.10(-7) M. The first preparation contained three polypeptides with molecular masses 40 kDa, 36 kDa, and 23 kDa. 40 kDa polypeptide was ADP-ribosylated by pertussis toxin. The same protein appeared to be the only substrate of pertussis toxin in crude detergent extract of membranes. The major polypeptides of the second preparation were represented by 37 kDa and 23 kDa polypeptides.

Dicyclohexylcarbodiimide influences the regulatory properties of the adenylate cyclase system in rat reticulocyte membranes.

Treatment of rat reticulocyte plasma membranes with dicyclohexylcarbodiimide (DCCD) decreased the GTP-stimulated adenylate cyclase activity and reduced the number of receptors that bind the agonist with high affinity in the absence of GTP. Besides, the agonist's competition curve was shifted to the right, irrespective of the presence of guanyl nucleotides. The dissociation constant for the antagonist and the number of binding sites did not change. Preincubation of the DCCD-treated membranes with GMP in the presence of isoproterenol restored the regulation of the agonist's affinity by guanyl nucleotides; however, in this case the GTP-independent change in the agonist's affinity was retained. This suggests that one of the DCCD-modified components of the adenylate cyclase system is a regulatory protein.