Role of substrates and products of PI 3-kinase in regulating activation of Rac-related guanosine triphosphatases by Vav.

Mitogen stimulation of cytoskeletal changes and c-jun amino-terminal kinases is mediated by Rac small guanine nucleotide-binding proteins. Vav, a guanosine diphosphate (GDP)-guanosine triphosphate (GTP) exchange factor for Rac that stimulates the exchange of bound GDP for GTP, bound to and was directly controlled by substrates and products of phosphoinositide (PI) 3-kinase. The PI 3-kinase substrate phosphatidylinositol-4,5-bisphosphate inhibited activation of Vav by the tyrosine kinase Lck, whereas the product phosphatidylinositol-3,4,5-trisphosphate enhanced phosphorylation and activation of Vav by Lck. Control of Vav in response to mitogens by the products of PI 3-kinase suggests a mechanism for Ras-dependent activation of Rac.

Amino acid residues in the CDC25 guanine nucleotide exchange factor critical for interaction with Ras.

Previously we found that negatively charged residues at positions 62, 63, and 69 of H-Ras are involved in binding to the CDC25 guanine nucleotide exchange factor (GEF). Using site-directed mutagenesis, we have changed conserved, positively charged residues of CDC25GEF to glutamic acid. We find the nonfunctional CDC25R1374E mutant and the nonfunctional H-RasE63K mutant cooperate in suppression of the loss of CDC25 function in Saccharomyces cerevisiae. Also, peptides corresponding to residues 1364 to 1383 of CDC25GEF inhibit interaction between GEFs and H-Ras. We propose that residues 1374 of CDC25GEF and 63 of H-Ras form an ion pair and that when this ion pair is reversed, functional interaction can still occur.

Identification of residues of the H-ras protein critical for functional interaction with guanine nucleotide exchange factors.

Ras proteins are activated in vivo by guanine nucleotide exchange factors encoded by genes homologous to the CDC25 gene of Saccharomyces cerevisiae. We have taken a combined genetic and biochemical approach to probe the sites on Ras proteins important for interaction with such exchange factors and to further probe the mechanism of CDC25-catalyzed GDP-GTP exchange. Random mutagenesis coupled with genetic selection in S. cerevisiae was used to generate second-site mutations within human H-ras-ala15 which could suppress the ability of the Ala-15 substitution to block CDC25 function. We transferred these second-site suppressor mutations to normal H-ras and oncogenic H-rasVal-12 to test whether they induced a general loss of function or whether they selectively affected CDC25 interaction. Four highly selective mutations were discovered, and they affected the surface-located amino acid residues 62, 63, 67, and 69. Two lines of evidence suggested that these residues may be involved in binding to CDC25: (i) using the yeast two-hybrid system, we demonstrated that these mutants cannot bind CDC25 under conditions where the wild-type H-Ras protein can; (ii) we demonstrated that the binding to H-Ras of monoclonal antibody Y13-259, whose epitope has been mapped to residues 63, 65, 66, 67, 70, and 73, is blocked by the mouse sos1 and yeast CDC25 gene products. We also present evidence that the mechanism by which CDC25 catalyzes exchange is more involved than simply catalyzing the release of bound nucleotide and passively allowing nucleotides to rebind. Most critically, a complex of Ras and CDC25 protein, unlike free Fas protein, possesses significantly greater affinity for GTP than for GDP. Furthermore, the Ras CDC25 complex is more readily dissociated into free subunits by GTP than it is by GDP. Both of these results suggest a function for CDC25 in promoting the selective exchange of GTP for GDP.

Distinct subclasses of small GTPases interact with guanine nucleotide exchange factors in a similar manner.

The Ras-related GTPases are small, 20- to 25-kDa proteins which cycle between an inactive GDP-bound form and an active GTP-bound state. The Ras superfamily includes the Ras, Rho, Ran, Arf, and Rab/YPT1 families, each of which controls distinct cellular functions. The crystal structures of Ras, Rac, Arf, and Ran reveal a nearly superimposible structure surrounding the GTP-binding pocket, and it is generally presumed that the Rab/YPT1 family shares this core structure. The Ras, Rac, Ran, Arf, and Rab/YPT1 families are activated by interaction with family-specific guanine nucleotide exchange factors (GEFs). The structural determinants of GTPases required for interaction with family-specific GEFs have begun to emerge. We sought to determine the sites on YPT1 which interact with GEFs. We found that mutations of YPT1 at position 42, 43, or 49 (effector loop; switch I), position 69, 71, 73, or 75 (switch II), and position 107, 109, or 115 (alpha-helix 3-loop 7 [alpha3-L7]) are intragenic suppressors of dominant interfering YPT1 mutant N22 (YPT1-N22), suggesting these mutations prevent YPT1-N22 from binding to and sequestering an endogenous GEF. Mutations at these positions prevent interaction with the DSS4 GEF in vitro. Mutations in the switch II and alpha3-L7 regions do not prevent downstream signaling in yeast when combined with a GTPase-defective (activating) mutation. Together, these results show that the YPT1 GTPase interacts with GEFs in a manner reminiscent of that for Ras and Arf in that these GTPases use divergent sequences corresponding to the switch I and II regions and alpha3-L7 of Ras to interact with family-specific GEFs. This finding suggests that GTPases of the Ras superfamily each may share common features of GEF-mediated guanine nucleotide exchange even though the GEFs for each of the Ras subfamilies appear evolutionarily unrelated.

Lck regulates Vav activation of members of the Rho family of GTPases.

Vav is a member of a family of oncogene proteins that share an approximately 250-amino-acid motif called a Dbl homology domain. Paradoxically, Dbl itself and other proteins containing a Dbl domain catalyze GTP-GDP exchange for Rho family proteins, whereas Vav has been reported to catalyze GTP-GDP exchange for Ras proteins. We present Saccharomyces cerevisiae genetic data, in vitro biochemical data, and animal cell biological data indicating that Vav is a guanine nucleotide exchange factor for Rho-related proteins, but in similar genetic and biochemical experiments we fail to find evidence that Vav is a guanine nucleotide exchange factor for Ras. Further, we present data indicating that the Lck kinase activates the guanine nucleotide exchange factor and transforming activity of Vav.

Identification of a dominant-negative mutation in the yeast CDC25 guanine nucleotide exchange factor for Ras.

In previous studies we changed five conserved amino acid residues in the catalytic domain of the yeast Ras-specific guanine nucleotide exchange factor CDC25GEF (Park et al., 1994). One of the substitutions (R1489E) resulted in a molecule which could bind Ras but was catalytically inactive. These observations suggested that CDC25R1489E might be a dominant-negative mutant. Here we report further experiments which confirm the dominant-negative phenotype of CDC25R1489E. Two lines of evidence indicate that the CDC25R1489E mutant exhibits Ras-specific binding in vivo. First, expression of CDC25R1489E in a wild-type yeast strain caused a partial inhibition of growth which was reversed by overexpression of the wild-type yeast RAS2 protein. Second, expression of CDC25R1489E in a yeast strain containing a temperature-sensitive, dominant-negative RAS2 mutation (RAS2val19ala22) suppressed the temperature-sensitive phenotype. The latter findings suggest that the CDC25R1489E protein bound the mutant RAS2 protein thereby releasing the wild-type CDC25 protein for activation of the wild-type RAS1 protein. Further, using a protein-protein binding assay and guanine nucleotide exchange assay (release of [3H]-GDP) in vitro, we demonstrate that the CDC25R1489E protein can bind wild-type Ras protein but is unable to catalyze GDP-GTP exchange. Thus, the results of genetic and biochemical experiments demonstrate that CDC25R1489E encodes a dominant-negative GEF which blocks the Ras signaling pathway by binding wild-type Ras in a catalytically inactive complex.

Ventricular fibrillation in patients without significant structural heart disease: a multicenter experience with implantable cardioverter-defibrillator therapy.

OBJECTIVES: This study was undertaken to characterize the outcome of survivors of ventricular fibrillation with no or minimal structural heart disease who received an implantable cardioverter-defibrillator. BACKGROUND: The prognosis among survivors of ventricular fibrillation with minimal or no structural cardiac abnormalities remains unclear. Since the advent of implantable cardioverter-defibrillators, this question takes on added importance. METHODS: This 10-center retrospective study provided information on 28 survivors of ventricular fibrillation (mean age 42 years) with minimal or no structural abnormalities who were treated with an implantable cardioverter-defibrillator. RESULTS: Ventricular tachyarrhythmias (polymorphic in all but one patient) were induced during baseline programmed stimulation in 39% of patients. During a median 30.6-month follow-up period after implantable cardioverter-defibrillator implantation, there were no cardiac deaths and two noncardiac deaths. Sixteen patients experienced 36 shock episodes (total 88 shocks). The majority of shocks were classified as "indeterminate"; one patient received 47 "spurious" shocks during one shock episode and each of four patients received one "appropriate" shock. Ventricular arrhythmias were not inducible in any of these latter four patients. CONCLUSIONS: Survivors of ventricular fibrillation with minimal or no structural cardiac abnormalities receiving an implantable cardioverter-defibrillator have an excellent 3-year survival rate. The occurrence, albeit infrequent, of appropriate implantable cardioverter-defibrillator shocks in this group suggests that these patients have a potential risk of recurrent cardiac arrest whose fatal outcome may be avoided by implantable cardioverter-defibrillator therapy.

Improved method for measuring the catalytic activity of tryptophan synthase alpha subunit in cell extracts.

An improved method has been developed for measuring the catalytic activity of tryptophan synthase alpha-subunit in cell extracts using the indole-3-glycerol phosphate (InGP)----tryptophan reaction. The method involves the chemical and enzymatic synthesis of the substrate InGP immediately before use and avoids the preparation of salt-free hydroxylamine. The method is more convenient, safer and more reliable than the traditional method employing the InGP----indole reaction.

Analysis of interaction between Ras and CDC25 guanine nucleotide exchange factor using yeast GAL4 two-hybrid system.

Our results demonstrate that the GAL4 two-hybrid system can be useful for studying interactions of the wild-type and mutant forms of Ras proteins with the CDC25 guanine nucleotide exchange factor (CDC25-GEF). In addition, our findings show that a negative result in the GAL4 two-hybrid system does not indicate that the two proteins tested do not interact under all conditions but only that they do not interact under the specific conditions examined. We recommend that the two-hybrid system be employed in combination with other approaches, including molecular genetic analyses and in vitro binding experiments, for the study of Ras and CDC25-GEF interactions.

Operative mortality with implantation of the automatic cardioverter-defibrillator.

Operative mortality was studied in 939 consecutive patients undergoing initial implantation of an automatic implantable cardioverter-defibrillator (AICD) at 15 hospitals. Twenty-nine (3.1%) patients died during the first 30 days after surgery. Among patients who survived beyond the first 30 postoperative days, ejection fraction data were available in 219; compared with the mortality group, these survivors had a significantly higher ejection fraction (34 +/- 15 vs 26 +/- 10%, respectively, p less than 0.001), despite similar age, sex, underlying heart disease, type of presenting arrhythmia and prevalence of concomitant surgery. The causes of perioperative death were sudden in 7 (24%), tachyarrhythmic/nonsudden in 5 (17%), cardiac nonarrhythmic in 9 (31%), and noncardiac in 8 (28%). Twenty-four (83%) of the deaths occurred before hospital discharge, and in all 9 instances of in-hospital sudden and tachyarrhythmic/nonsudden death, the initial recorded rhythm was sustained ventricular tachycardia or fibrillation; in 5 (56%) of these 9 patients the AICD had been in a deactivated state since implantation. Other possible contributory factors in the 12 sudden or tachyarrhythmic/nonsudden deaths included acute myocardial ischemia or infarction in 2 (17%), and "device proarrhythmia" in 3 (25%) that were AICD-related in 2 and secondary to an antitachycardia pacemaker in another; defibrillation threshold testing was not performed in 3 patients (1 of whom had terminal ventricular fibrillation). Thus, in this multicenter experience with thoracotomy requiring AICD implantation, operative (30-day) mortality was 3.1% and correlated inversely with left ventricular ejection fraction.(ABSTRACT TRUNCATED AT 250 WORDS)

Arterial occlusive disease: a function of vessel bifurcation angle.

A clinical observation that patients with aortoiliac occlusive disease have a high aortic bifurcation serves as the stimulus for this study. A review of 100 consecutive abdominal aortograms revealed that patients with occlusive disease had an average bifurcation angle of 38 degrees and those with normal studies or aneurysmal disease had an average angle of 52 degrees. A flow visualization system was constructed to study shear stress using eight Pyrex models with varying bifurcation angles (20 to 90 degrees). The perfusate consisted of a 35% sucrose solution with anion-exchange beads to serve as flow particles. The bifurcations were photographed at three different flow rates. The length of each tracer particle was measured to determine its velocity. Velocity profile curves were constructed and shear stress calculated by the formula shear stress = mu x dv/dr, where mu is viscosity, v is velocity, and r is radius. Results of the study suggest that more acute angles cause a greater shear stress on the inner wall of the bifurcation and decreased shear stress on the outer wall. Two theories of atheroma predilection, high shear stress and low shear stress, are applied to patients with high acute aortic bifurcations.

Clinical management of patients with atrioventricular nodal reentrant tachycardia.

A broad array of therapeutic options is currently available for the management of patients with AV nodal reentrant tachycardia. While acute termination of tachycardias is readily achieved, either by vagal maneuvers or intravenous medication, the decision to embark on a long-term therapeutic plan to prevent recurrences must be clinically individualized. When a chronic pharmacologic approach is desired, electrophysiologic testing is invaluable for confirming the diagnosis and selecting appropriate medication. However, the growing awareness of potential proarrhythmic effects and the inconvenience and expense of lifelong drug therapy, coupled with other advances in the field, have made nonpharmacologic approaches more attractive. This is especially so for symptomatic younger patients. The definitive cure rates achievable with surgery are now being approached by transcatheter AV nodal modification procedures that ablate AV nodal reentrant tachycardia while preserving anterograde AV nodal conduction. Over the next decade, it is likely that the latter technique will become widely used for the long-term management of symptomatic AV nodal reentrant tachycardia.

Evidence that glucose starvation-sensitive mutants are altered in the relB locus.

Genetic mapping studies indicate that the relaxed-control mutants isolated on the basis of sensitivity to glucose starvation contain lesions in the relB locus. These mutants, which are sensitive to protein synthesis inhibitors such as sulfacetamide, exhibit relaxed control of both RNA and phospholipid syntheses.

Post-translational modification of a tryptophan biosynthetic enzyme. Precursor-product relationships in vivo and carbon-energy source dependence.

Purified preparations of the bifunctional, tryptophan biosynthetic enzyme N-(5'-phosphoribosyl)anthranilate isomerase/indole-3-glycerol phosphate synthase of Escherichia coli K12 contain five forms of the protein (molecular mass = 49 kDa) which can be separated by isoelectric focusing or two-dimensional gel electrophoresis. The stability of the enzyme and its different forms was studied in exponentially growing, ammonium-starved and energy-depleted cultures using a dual-labeling, pulse-chase method. Labeled enzyme was isolated by standard purification techniques and by immunoprecipitation from crude extracts. The results demonstrated the following: 1) post-translational modification occurred in vivo, 2) modification occurred to the same extent in growing and ammonium-starved cells, 3) modification was dependent on a carbon-energy source for the cells, and 4) the enzyme was not degraded in growing or nongrowing cells. The pulse-chase data also indicated that one form of the protein (band 1) was the precursor of three other forms (bands 3-5) and that the relative amount of one form (band 2) remained constant.

Unusual sensitivity of Escherichia coli to adenine or adenine plus histidine.

The W3110 strain of Escherichia coli K-12 is unusually sensitive to adenine. Inhibition of growth is relieved by a combination of thiamine and uridine (or cytidine). In the presence of histidine, inhibition is more severe and is relieved by a combination of thiamine, glycine, uridine (or cytidine), and inosine (or guanosine).