Rasosomes originate from the Golgi to dispense Ras signals.

Ras proteins undergo an incompletely understood trafficking process in the cell. Rasosomes are protein nanoparticles of 80-100 nm diameter that carry lipidated Ras isoforms (H-Ras and N-Ras) as well as their effectors through the cytoplasm and near the plasma membrane (PM). In this study, we identified the subcellular origin of rasosomes and how they spread Ras proteins through the cell. We found no dependency of rasosome formation on galectins, or on the GDP-/GTP-bound state of Ras. We found that significantly more rasosomes are associated with forms of Ras that are localized to the Golgi, namely N-Ras or the singly palmitoylated H-Ras mutant (C181S). To explore the possibility that rasosome originate from the Golgi, we used photoactivatable (PA)-GFP-H-Ras mutants and showed that rasosomes bud from the Golgi in a two-step mechanism. Newly released rasosomes first move in an energy-dependent directed fashion and then convert to randomly diffusing rasosomes. Dual fluorescence time-lapse imaging revealed the appearance of dually labeled rasosomes, indicating a dynamic exchange of cytoplasmic and PM-associated Ras with rasosome-associated Ras. Finally, higher levels of rasosomes correlate with higher levels of ERK phosphorylation, a key marker of Ras downstream signaling. We suggest that H-Ras and N-Ras proteins exchange with rasosomes that can function as carriers of palmitoylated Ras and its signals.

Peripheral arterial tone as an on-line measure of load in a simulated flight task.

Variations in continuous and discrete flight demands were investigated in a simulated flight mission measuring peripheral arterial tone (PAT) from the tip of the finger. A total of 12 participants performed a computer-simulated agricultural flight task. They were required to fly over a specific lane of a simulated corn field (continuous task) and change lanes in response to flags, which appeared at varying intervals (discrete task). The difficulty of the flight task was manipulated by varying the airplane control (single- vs. dual-axis control), while the difficulty of the discrete task was manipulated by varying the amount of lateral change signalled by the flag. PAT amplitude was lower in the difficult level of the continuous task and was further attenuated following the appearance of the flag only when a change in the flight position was required. These results suggest the potential utility of PAT as an on-line measure of the joint continuous and discrete demands of a flight mission.

Deletion of LTbetaR augments male susceptibility to Plasmodium chabaudi.

SUMMARY Disruption of the lymphotoxin beta receptor (LTbetaR) gene has been shown to result in enhanced resistance of female mice to blood-stage Plasmodium chabaudi malaria. Here, we investigate the effect of LTbetaR deletion on host defence of males. In contrast to females, male LTbetaR(-/-) mice do not exhibit any increase in resistance. Conversely, they are even more susceptible than wild-type C57BL/6 mice, which becomes evident after lowering circulating levels of testosterone by castration, which makes C57BL/6 males resistant, whereas LTbetaR(-/-) remain susceptible. Gene-expression analysis using cDNA arrays revealed no differences in immunological responses in spleen of malaria-resistant female and malaria-susceptible castrated male LTbetaR(-/-) mice. In the liver, however, expression levels of plasminogen activator inhibitor PAI1, chemokine CXCL10, dual specificity phosphatase DUSP1, and hydroxysteroid-specific sulfotransferases Sult2a1/2 were decreased 6- to 85-fold in susceptible castrated male LTbetaR(-/-) mice in comparison to resistant female LTbetaR(-/-) mice at maximal parasitaemia, as evidenced by Northern blot analyses. The present data support our previous view that the liver is involved in the combat against malarial blood stages and that down-regulation of the genes DUSP1 and Sult2a1/2 signals dysregulation of protective liver responses, thus possibly contributing to male susceptibility of LTbetaR(-/-) mice.

Primary human liver cells as source for modular extracorporeal liver support--a preliminary report.

Cell-based extracorporeal liver support is an option to assist or replace the failing organ until regeneration or until transplantation can be performed. The use of porcine cells or tumor cell lines is controversial. Primary human liver cells, obtained from explanted organs found to be unsuitable for transplantation, are a desirable cell source as they perform human metabolism and regulation. The Modular Extracorporeal Liver Support (MELS) concept combines different extracorporeal therapy units, tailored to suit the individual and intra-individual clinical needs of the patient. A multi-compartment bioreactor (CellModule) is loaded with human liver cells obtained by 5-step collagenase liver perfusion. A cell mass of 400 g - 600 g enables the clinical application of a liver lobe equivalent hybrid organ. A detoxification module enables single pass albumin-dialysis via a standard high-flux dialysis filter, and continuous veno-venuous hemodiafiltration may be included if required. Cells from 54 human livers have been isolated (donor age: 56 +/- 13 years, liver weight: 1862 +/- 556 g resulting in a viability of 55.0 +/- 15.9%). These grafts were not suitable for LTx, due to steatosis (54%), cirrhosis (15%), fibrosis (9%), and other reasons (22%). Out of 36 prepared bioreactors, 10 were clinically used to treat 8 patients with liver failure. The overall treatment time was 7-144 hours. No adverse events were observed. Initial clinical applications of the bioreactor evidenced the technical feasibility and safety of the system.

Extracorporeal liver support: porcine or human cell based systems?

Initial results of the clinical use of primary porcine liver cells for extracorporeal liver support are being reviewed as the cell source is controversial. According to Eurotransplant data 20-25% of explanted donor livers are not transplanted, due to factors such as steatosis or cirrhosis. This number corresponds to the number of patients with acute liver failure who require bridging therapy to transplantation. Primary human liver cells from transplant discards can be isolated, purified and maintained in bioreactors and provide an alternative for cell-based extracorporeal liver support therapy. A four-compartment bioreactor enables recovery from preservation and isolation injury in a three-dimensional network of interwoven capillary membranes with integrated oxygenation, rendering the liver cells from these discarded donor organs viable for clinical utilization. Patient contact with additional animal-derived biomatrix and fetal calf serum can be avoided. The initiation of an in vitro cultivation phase allows cell stabilization, quality control, and immediate availability of a characterized system without cryopreservation. The hypothesis of this paper is that with appropriate logistics and four-compartment bioreactor technology, cells from human liver transplant discards can serve the demand for cell-based therapy, including extracorporeal liver support.

The application of ethics to engineering and the engineer's moral responsibility: perspectives for a research agenda.

There are different possibilities for defining the areas for the application of ethics to engineering. They range from descriptive analysis of engineers' relationship to moral criteria and extend to normative issues on how engineers should design more "sustainable" technology. In this paper, a frame of reference is proposed, which makes it possible to elaborate in a transparent manner goals for analysis of the scope of ethics in engineering Its point of departure is marked by two questions. 1) which types of situation in the practice of engineering require ethical reflection? and 2) to what extent are engineers expected to assume moral responsibility in the practice of their profession? The answers to both of these questions presuppose reflection on the societal processes of setting definitions and of making ascriptions. Understanding these processes of societal "construction" of demands for ethical reflection in engineering and of engineers' moral responsibilities should be an important objective of the analysis of ethics in engineering.

Hydroxyurea and periodicity in myeloproliferative disease.

Three patients, one with polycythaemia vera (PV) and two with chronic myeloid leukaemia (CML), are described who had cycling of blood counts which became apparent whilst receiving hydroxyurea therapy. Significant periodicity was confirmed with the use of the Lomb periodogram. This is Fourier power spectral analysis tailored for unevenly sampled data. The patient with PV had marked oscillations of platelet counts with a periodicity of 29 d and an amplitude of (202-588)x10(9)/L. Smaller oscillations of neutrophil, monocyte and lymphocyte numbers and Hb levels occurred with a similar periodicity. Anticipatory changes in hydroxyurea dosage or the maintenance of a constant dose did not abolish periodicity, but a change in therapy to the non-cycle-specific drug anagrelide dampened and abolished the cycling. One of the patients with CML had tremendous and clear oscillations in white cell, platelet and Hb levels, with a mean periodicity of 74 d. The other had erratic counts which were confirmed to be significantly periodic (64 d), on spectral analysis. A change in therapy to busulphan in both these patients again dampened and abolished the cycling. Hydroxyurea, which is a cell-cycle-specific agent, probably exacerbates the periodicity which may be present in some patients with myeloproliferative disease. A change in therapy to non-cycle-acting compounds such as busulphan or anagrelide results in much more stable counts in such patients.

Against over-estimating the role of ethics in technology development.

The role of ethics in technology development has been often questioned, especially in the early days of societal reflection of technology. However, the situation has changed dramatically. Ethical consideration now is generally declared to be indispensable in shaping technology in a socially acceptable and sustainable way. The expectations of ethics are large; often even a kind of "New Ethics" is postulated. In the present paper an over-estimation of the role of ethics for technology development is rejected. It is argued that ethical reflection is, indeed, indispensable in certain problem areas and situation types; but there is, on the other hand, space for technology development free from the requirement for ethical reflection. The absence of a requirement for ethical reflection, however, always has to be considered relative to some "morale provisoire" (provisional morality) as an accepted normative framework within which technology development may occur without explicit ethical reflection. If this framework, however, is doubted or is shown to be insufficient the situation changes completely. Ethical reflection in this case becomes necessary, to consider this normative framework in order to offer modifications or supplements.

Effect of intravenous nitroglycerin on heparin dosage requirements in coronary artery disease.

Patients admitted to the coronary care unit who received both intravenous nitroglycerin and heparin were studied to evaluate heparin dosage requirements. Physicians ordered all nitroglycerin and heparin doses as well as coagulation studies without knowledge of this study. Activated partial thromboplastin time (APTT) values obtained during steady-state heparin administration were considered therapeutic if the ratio of APTT/APTT-baseline was > or = 1.5. Sixty patients with myocardial infarction or unstable angina were included in the study. The initial therapeutic heparin dose of 1,014 +/- 151 units/hour produced an APTT ratio of 2.0 +/- 0.5. At the time of the initial therapeutic dose, the nitroglycerin dose was 110 +/- 108 micrograms/min. There was a significant correlation between the initial therapeutic dose and both total (r = 0.56; p = 0.0001) and lean (r = 0.26; p < 0.05) body weight. Comparison of patients with nitroglycerin doses < and > or = 100 micrograms/min revealed a significant difference in the initial therapeutic dose (971 +/- 147 vs 1,077 +/- 136 U/hour, p < 0.01), but not the initial therapeutic dose standardized to total body weight (14.0 +/- 2.5 vs 13.5 +/- 2.7 U/kg/hour). Similarly, analysis of variance revealed a significant difference in the initial therapeutic dose (p < 0.05), but not the initial therapeutic dose standardized to weight among 5 different nitroglycerin dosage ranges (10 to 533 micrograms/min). Neither aspirin use, thrombolytic therapy nor decreasing or discontinuing the nitroglycerin dose significantly affected heparin requirements. Thus, contrary to prior reports, clinically significant heparin resistance induced by nitroglycerin was not found.

Prognostic significance from 10-year follow-up of a qualitatively normal planar exercise thallium test in suspected coronary artery disease.

A normal exercise thallium-201 scintigram has been shown to confer an excellent prognosis over a 1- to 4-year follow-up period. However, progression of coronary disease could result in cardiovascular mortality with increasing time. Therefore, the vital status of 309 patients with normal stress thallium myocardial imaging was determined after an average of 10.3 years. Deaths were classified as cardiac or noncardiac. Statistical analysis was performed using Kaplan-Meier survival curves. Standardized mortality ratios were calculated and compared with those of an age- and sex-matched general population. Follow-up was complete in 288 patients (93%). Of 18 deaths, only 3 were cardiac; the remaining 15 were mainly secondary to cancer. Thus, cardiac mortality was 1% and total mortality 6.3% at 10 years. In addition, both all-cause and cardiac mortality rates were significantly less than would be expected in an age- and sex-adjusted segment of the general population. Thus, normal exercise thallium scintigraphy retains its high negative predictive value for death < or = 10 years after initial testing. This supports the use of stress thallium imaging to predict which patients with suspected coronary artery disease are at low risk for cardiac death and thus do not need invasive testing.

Early and rapid prediction of patency of the infarct-related coronary artery by using left ventricular wall thickness as measured by two-dimensional echocardiography.

OBJECTIVES: The aim of this study was to determine whether echocardiography can distinguish between persistent coronary occlusion and reperfusion. BACKGROUND: There are no adequate clinical or noninvasive laboratory markers to accurately predict successful reperfusion in an acute myocardial infarction. METHODS: In a closed chest swine model, the effect of reperfusion on myocardial wall thickness was studied by comparing a 150-min total coronary artery occlusion (group 1) with 120 min of occlusion followed by 30 min of reperfusion (group 2) in the area of risk as measured by echocardiography. Wall thickness was measured at baseline and at 90 and 150 min. RESULTS: In group 1 (n = 4), there was no appreciable change in mean wall thickness from 90 min to 150 min of occlusion at either end-diastole or end-systole (0.54 +/- 0.02 to 0.52 +/- 0.03 cm, 0.55 +/- 0.03 to 0.54 +/- 0.03 cm, respectively; p = NS). In contrast, in group 2 (n = 6), an increase in mean wall thickness from 0.53 +/- 0.02 to 0.97 +/- 0.05 cm at end-diastole and from 0.56 +/- 0.04 to 1.04 +/- 0.07 cm at end-systole was found from 90 min of occlusion to 30 min of reperfusion (p < 0.001). Reperfusion resulted in an increase in wall thickness of 83 +/- 11% at end-diastole and 92 +/- 17% at end-systole. In contrast, persistent coronary occlusion showed minimal changes of -3.0 +/- 5% at end-diastole and -2.0 +/- 6% at end-systole. CONCLUSIONS: This study confirms the hypothesis that an increase in wall thickness can accurately distinguish between reperfusion and permanent coronary occlusion.

Adjunctive thrombolytic therapy for angioplasty in ischemic rest angina: results of a double-blind randomized pilot study.

OBJECTIVES: A multicenter pilot study was instituted to assess the role of intracoronary thrombolytic therapy during angioplasty for ischemic rest angina. BACKGROUND: Acute thrombotic coronary occlusion is increased during angioplasty for unstable angina, and intracoronary thrombolytic agents have been used to maintain patency. Prophylactic use of intracoronary thrombolytic agents has been advocated in certain high risk subgroups, although no studies have randomized therapy. METHODS: Ninety-three patients with either unstable angina and pain at rest (trial A, 66 patients) or postinfarction pain at rest (trial B, 27 patients) were randomized in double-blind fashion to administration of either intracoronary urokinase, 150,000 U, or saline solution placebo given immediately before angioplasty. Cineangiograms of the culprit lesion were recorded and analyzed in blinded fashion by a core laboratory for definite or possible (haziness) filling defects 15 min after angioplasty or after acute closure. RESULTS: Urokinase decreased filling defects at 15 min after angioplasty in comparison with placebo (14% vs. 29%, respectively, p = 0.08). Four patients in each treatment group developed acute vessel closure. However, although urokinase significantly reduced the incidence of filling defects in trial A (3% vs. 23%, p = 0.03), the drug had no effect at the selected dose in trial B (42% vs. 43%, respectively). Acute vessel closure occurred significantly more frequently in trial B than in trial A, and urokinase at the selected dose also had no effect. Ischemic events after angioplasty appeared to be related more to dissection than to thrombosis, although redilation, which was more frequent after placebo administration, may have reduced their incidence as well as that of acute closure. CONCLUSIONS: These data suggest a possible role for intracoronary urokinase during angioplasty for unstable angina. The lack of effect after infarction may represent a greater thrombus burden or degree of plaque disruption. A trial utilizing higher doses of urokinase in a larger patient group is in progress.

Bypass surgery for chronic stable angina: predictors of survival benefit and strategy for patient selection.

The variable mortality risk associated with chronic stable angina calls for careful selection of patients for coronary artery bypass grafting (CABG) if the aim of management is to prolong life. The randomized and observational studies done in the last 20 years have identified the variables relevant to patient selection and thus have provided a rational basis for such clinical decisions. These studies showed that the sicker the patient, as gauged by relevant measures of coronary disease and cardiovascular morbidity, the more likely it is that CABG will prolong life. A CABG-related improvement in survival is therefore more likely to occur the worse the left ventricular function; the greater the number of diseased vessels; the more proximal the location of coronary lesions (more muscle is threatened by such lesions); the greater the severity of the lesions as determined by angiography; the more severe the angina; the more easily provocable the ischemia or the more extreme the measures of ischemia; and, within limits, the older the patient. Greater survival gain after CABG also occurs in patients with peripheral vascular disease, in patients with baseline electrocardiographic ST-segment and T-wave changes, and probably in women. Thus, patients are likely to live longer after CABG if they have left main disease; three-vessel disease with left ventricular dysfunction (ejection fraction less than 50%), class III or IV angina, provocable ischemia, or disease in the proximal left anterior descending coronary artery; two-vessel disease with proximal left anterior descending artery involvement; and two-vessel disease with class III or IV angina as well as either severe left ventricular dysfunction alone or moderate left ventricular dysfunction together with at least one proximal lesion. When the decision of whether to do CABG is less clear-cut, the presence of peripheral vascular disease, female sex, baseline electrocardiographic ST-segment and T-wave changes, or older age (over 60 but under 80 years) should weigh in favor of doing CABG. In general, patients with single-vessel disease do not seem to derive survival benefit from CABG.

Effect of coronary occlusion and myocardial viability on myocardial activity of technetium-99m-sestamibi.

The timing effect of sestamibi administration with respect to the onset of myocardial ischemia and reperfusion was studied in swine. In different groups of animals sestamibi was administered prior to coronary artery occlusion, during occlusion, or 1/2 hour following reperfusion. Sestamibi administered prior to coronary occlusion resulted in an insignificant decrease in 99mTc activity in the ischemic zone. However, infarct zone activity was reduced to 62 +/- 14% of the nonischemic zone. In contrast, administration during coronary occlusion resulted in similar significant reductions of both ischemic and infarct zone activity. Administration of sestamibi during reperfusion resulted in normal ischemic zone activity and markedly reduced activity in the infarct zone. Significantly reduced activity in the infarct zone was found to be independent of the timing of sestamibi administration with respect to the onset of myocardial ischemia and/or reperfusion. Thus, cell viability appears required for uptake and retention of isotope activity.

Effect of early reperfusion on use of triphenyltetrazolium chloride to differentiate viable from non-viable myocardium in area of risk.

STUDY OBJECTIVE - The aim of the study was to assess the value of triphenyltetrazolium chloride (TTC) staining as an indicator of non-viable myocardium after early reperfusion of ischaemic myocardium. DESIGN - Left anterior descending artery occlusion was performed in pigs for various lengths of time and at two different sites (proximal and distal). After 120 min reperfusion, TTC was injected distal to the occlusion while the remainder of the myocardium was stained with Evans Blue. Myocardial enzymes were measured in non-ischaemic zone, regions of risk and in necrotic zones and related to staining characteristics. SUBJECTS - 31 male Hampshire pigs, weight 34-39 kg, were studied. Twelve were excluded because of resistant ventricular fibrillation or poorly defined areas of infarction. In the remaining 19 pigs, proximal occlusion was carried out in 15 and distal in four. Occlusion lasted for 15 min in six animals, for 30 min in eight (four of which were the animals with distal occlusions), and for 45 min in five. MEASUREMENTS and RESULTS - Biopsies from non-ischaemic zones, regions of risk and necrotic zones were analysed for creatine kinase and lactate dehydrogenase. In the 15 min group, myocardial creatine kinase in the region of risk (red stained) was similar to the non-ischaemic (blue) zone, but in the 30 min distal occlusion group it was reduced. After 30 and 45 min of proximal occlusion, creatine kinase activity in the necrotic (white) zone was reduced compared to the red zone in the same group, and in the red zone of both groups it was reduced compared to the non-ischaemic area. CONCLUSIONS - The red zone, as defined by TTC staining, may be associated with significant creatine kinase depletion after relatively brief periods of occlusion and subsequent reperfusion. This suggests that the red region may be a heterogeneous area of dead and viable cells.

Role of exercise thallium-201 myocardial perfusion scintigraphy in predicting prognosis in suspected coronary artery disease.

While exercise thallium imaging has improved sensitivity and specificity for detection of coronary artery disease (CAD), its predictive value for morbid cardiac events is unclear. Of 532 consecutive patients who underwent exercise thallium imaging, follow-up was complete in 515 (97%) after an average of 36 months (range 31 to 48). Two hundred six patients had an abnormal exercise thallium response and 309 had a normal response. Twenty morbid cardiac events occurred (13 deaths and 7 acute myocardial infarctions [AMI]). Of the 13 patients who died, 12 had abnormal thallium results. Overall, 5.8% of the patients with abnormal thallium results died, in contrast to 0.3% of patients with normal results. Of the 7 patients who had a nonfatal AMI, 3 had abnormal exercise thallium results. Moreover, similar proportions of patients (1.4% and 1.3%) with normal and abnormal exercise thallium results had nonfatal AMI. Presence or absence of pathologic Q waves and inclusion of exercise electrocardiographic results did not significantly alter the results. Thus, although a normal exercise thallium response significantly reduces the likelihood of cardiovascular death, its predictive value for nonfatal AMI is limited. Moreover, the relatively low event rate for patients with a positive exercise thallium response further limits its prognostic value.

Quantitation of absolute area of a coronary arterial stenosis: experimental validation with a preparation in vivo.

The absolute cross-sectional area of a coronary stenosis measured by quantitative coronary angiography correlates well with its hemodynamic significance. We evaluated a combined approach using edge detection applied to the normal segment and videodensitometry applied to the stenosis to determine the absolute cross-sectional area of the stenosis (videodensity method). The results were then compared with those with the edge detection method applied directly to the stenosis. The area of the stenosis by the edge detection method was calculated by analyzing two orthogonal projections for irregular stenoses and with use of the formula for the area of an ellipse (ellipse method). The accuracy of both these techniques was assessed by analyzing digital angiograms acquired from closed-chest dogs in which 10 plastic cylinders with precisely machined circular and irregular lumina were inserted into the coronary arteries. Angiograms of irregular stenoses were acquired in two orthogonal views. The ellipse method applied to circular stenoses was very accurate, with r = .97, average absolute difference (AAD) = 0.21 mm2, and SEE = 0.30. For the videodensity method r = .97, AAD = 0.84 mm2, and SEE = 0.40. Irregular stenoses were better quantitated by the videodensity method applied in one view (AAD = 0.50 mm2, SEE = 0.47) than by the ellipse method applied in two orthogonal projections (AAD = 1.03 mm2, SEE = 0.87). Overall, the two methods were comparable in accuracy (for videodensity, AAD = 0.65 mm2, SEE = 0.71 vs AAD = 0.54 mm2, SEE = 0.79 for ellipse).(ABSTRACT TRUNCATED AT 250 WORDS)