The functional role of CrkII in actin cytoskeleton organization and mitogenesis

Crk is a member of a family of adapter proteins predominantly composed of Src homology 2 and 3 domains, whose role in signaling pathways is presently unclear. Using an in situ electroporation system which permits the introduction of glutathione S-transferase (GST) fusion proteins into cells, we found that c-CrkII bound to p130(cas), but not to paxillin in serum-starved rat-1 fibroblasts overexpressing the human insulin receptor (HIRc cells) in vivo. 17 nM insulin stimulation dissociated the binding of c-CrkII to p130(cas), whereas 13 nM insulin-like growth factor-I, 16 nM epidermal growth factor (EGF), and 10% serum each showed little or no effect. We found that stress fiber formation is consistent with a change in the p130(cas).c-CrkII interactions before and after growth factor stimulation. Microinjection of either GST-Crk-SH2 or -Crk-(N)SH3 domains, or anti-Crk antibody each inhibited stress fiber formation before and after insulin-like growth factor-I, EGF, and serum stimulation. Insulin stimulation by itself caused stress fiber breakdown and there was no additive effect of microinjection. Microinjection of anti-p130(cas) antibody also blocked stress fiber formation in quiescent cells. Microinjection of the Crk-inhibitory reagents also inhibited DNA synthesis after insulin-like growth factor-I, EGF, and serum stimulation, but not after insulin. These data suggest that the complex containing p130(cas).c-CrkII may play a crucial role in actin cytoskeleton organization and in anchorage-dependent DNA synthesis.     

Unfolding of Plasmodium falciparum triosephosphate isomerase in urea and guanidinium chloride: evidence for a novel disulfide exchange reaction in a covalently cross-linked mutant

The conformational stability of Plasmodium falciparum triosephosphate isomerase (TIMWT) enzyme has been investigated in urea and guanidinium chloride (GdmCl) solutions using circular dichroism, fluorescence, and size-exclusion chromatography. The dimeric enzyme is remarkably stable in urea solutions. It retains considerable secondary, tertiary, and quaternary structure even in 8 M urea. In contrast, the unfolding transition is complete by 2.4 M GdmCl. Although the secondary as well as the tertiary interactions melt before the perturbation of the quaternary structure, these studies imply that the dissociation of the dimer into monomers ultimately leads to the collapse of the structure, suggesting that the interfacial interactions play a major role in determining multimeric protein stability. The Cm(urea)/Cm(GdmCl) ratio (where Cm is the concentration of the denaturant required at the transition midpoint) is unusually high for triosephosphate isomerase as compared to other monomeric and dimeric proteins. A disulfide cross-linked mutant protein (Y74C) engineered to form two disulfide cross-links across the interface (13-74') and (13'-74) is dramatically destablized in urea. The unfolding transition is complete by 6 M urea and involves a novel mechanism of dimer dissociation through intramolecular thiol-disulfide exchange.     

Outcome of moderate carotid artery stenosis in patients who are asymptomatic

PURPOSE: The incidence rate of disease progression and stroke after the diagnosis of a moderate (50% to 79%) carotid stenosis was determined by means of color-flow duplex scanning. METHODS: During a 4-year period, 344 male veterans with moderate internal carotid artery stenoses, on one or both sides, were examined at regular intervals for a mean period of 25 months. Carotid color-flow scans were obtained semiannually. Clinical follow-up was performed to determine the incidence rate of amaurosis fugax, transient ischemic attacks, nonhemispheric symptoms, and strokes. RESULTS: New neurologic symptoms developed in 75 patients (21.8%). Fifty-one (14.8%) had ipsilateral symptoms during follow-up: 18 amaurosis fugax (5.2%), 14 transient ischemic attacks (4%), 5 nonhemispheric symptoms (1.4%), and 14 strokes (4%). Twenty-four patients (6.9%) had contralateral symptoms: 20 strokes (5.8%) and 4 transient ischemic attacks (1.2%). Life-table analysis showed that the annual rate of ipsilateral neurologic events was 8.1%, and the annual rate of stroke was 2.1%. Seventy-five patients (22%) died in the follow-up period. Disease progression to 80% to 99% stenosis or occlusion occurred in 71 of 458 vessels (15.5%). The internal carotid arteries that showed evidence of disease progression had a significantly higher initial peak systolic velocity (251 vs 190 cm/s; P <.0001) and end diastolic velocity (74 vs 52 cm/s; P < 0.0001). Black patients and patients with ischemic heart disease were at a higher risk for disease progression. We could not identify any atherosclerotic risk factors that reliably predicted patients in whom future ipsilateral neurologic symptoms were more likely to develop. However, there was an increased risk of stroke associated with progression of disease. CONCLUSION: Patients who are asymptomatic and who have moderate carotid stenoses are at significant risk for neurologic symptoms and death, but have a relatively low incidence rate of ipsilateral events. The initial flow characteristics in the stenotic vessel are predictive of future disease progression, but they are not helpful in identifying patients in whom symptoms will develop.     

Activation of particulate guanylyl cyclase by Vibrio vulnificus hemolysin

Recently we reported that Vibrio vulnificus hemolysin, an exotoxin produced by V. vulnificus, dilates rat thoracic aorta via elevated cGMP levels without affecting nitric oxide synthase. We investigated the mechanism further by observing the guanylyl cyclase activities in cytosolic, membrane, unfractionated, or reconstituted preparations. Hemolysin did not activate guanylyl cyclase in the membrane or cytosolic fraction, while it activated guanylyl cyclase in unfractionated or reconstituted preparation. The increased activity was not inhibited by the HS-142-1, a microbial polysaccharide which antagonizes atrial natriuretic peptide receptor, or 1H-[1,2,4]oxadiazolo[4,3-a]quinoxalin-1-one (ODQ), a soluble guanylyl cyclase inhibitor. However, it was attenuated by 6-(phenylamino)-5,8-quinolinedione (LY 83.583), which inhibits the catalytic domain of both guanylyl cyclases, and by cholesterol, which blocks hemolysin-incorporation into the membrane. Removing ATP, a cofactor of particulate guanylyl cyclase, attenuated the activation and ATPgammaS, a non-phosphorylating analog, restored it. These results suggest that V. vulnificus hemolysin activates particulate guanylyl cyclase via hemolysin incorporation into the vascular smooth muscle cell membrane in cooperation with certain unidentified cytosolic component(s).     

Endovascular interventions training and credentialing for vascular surgeons

This article reviews issues concerning the training and credentialing of vascular surgeons in the use of endovascular techniques in the peripheral vascular system. These guidelines update a prior document that was published in 1993. They have been rewritten to accommodate the rapid evolution that has occurred in the field and to provide the appropriate requirements that a vascular surgeon should fulfill to be competent in the basic skills needed to safely and effectively perform all presently accepted diagnostic and therapeutic endovascular procedures.