Analysis of small GTPase signaling pathways using p21-activated kinase mutants that selectively couple to Cdc42.

p21-activated kinase 1 (Pak1) is an effector for the small GTPases Cdc42 and Rac. Because Pak1 binds to and is activated by both these GTPases, it has been difficult to precisely delineate the signaling pathways that link extracellular stimuli to Pak1 activation. To separate activation of Pak1 by Cdc42 versus activation by Rac, we devised a genetic screen in yeast that enabled us to create and identify Pak1 mutants that selectively couple to Cdc42 but not Rac1. We recovered several such Pak1 mutants and found that the residues most often affected lie within the p21 binding domain, a region previously known to mediate Pak1 binding to GTPases, but that several mutations also map outside the borders of the p21 binding domain. Pak1 mutants that associate with Cdc42 but not Rac1 were also activated by Cdc42 but not Rac1. In rat 3Y1 cells expressing oncogenic Ha-Ras, the Pak1 mutants defective in Rac1 binding are not activated, suggesting that Ras signals through a GTPase other than Cdc42 to activate Pakl. Similar results were obtained when epidermal growth factor was used to activate Pak1. However, Pak1 mutants that are unable to bind Rac are nonetheless well activated by calf serum, implying that this stimulus may induce Pak activation independent of Rac.

Effect of transforming growth factor beta on rat hepatocyte cell lines depends upon the state of tumorigenic progression.

The effect of transforming growth factor beta (TGF-beta) on morphology, actin cytoskeleton organization, anchorage-dependent proliferation, anchorage-independent proliferation, and alpha-fetoprotein secretion in a series of related hepatocyte-derived cell lines was measured. We reported previously that TGF-beta 1 partially suppressed the transformed phenotype of a ras- transformed SV40-immortalized hepatocyte cell line designated NR4. In the present study, the CWSV14 cell line, which was derived by transfecting rat hepatocytes with SV40 DNA, was analyzed at low (14LP), mid (14MP), and high (14HP) passage. CWSV14 cells are weakly tumorigenic at low passage and spontaneously transform with in vitro passaging. Tumor cell lines (14T1 and 14T2) derived from hepatocellular carcinomas produced by CWSV14 cells were also analyzed. TGF-beta 1 partially suppressed the transformed phenotype of 14HP, 14T1 and 14T2 cells but had no effect on cell proliferation in these cells. In contrast, TGF-beta 1 induced apoptosis in 14LP and 14MP cells. Studies using both the NR4 and 14T1 cells showed that suppression of the transformed phenotype also could be induced by TGF-beta 2 or TGF-beta 3. We conclude that TGF-beta-induced suppression of the transformed phenotype can be observed in ras-and spontaneously transformed hepatocyte cell lines. It is also apparent that the effect of TGF-beta 1 on hepatocyte cell lines differs depending upon the state of progression toward malignancy; that is, while TGF-beta 1 suppressed the transformed phenotype in highly transformed and tumor cell lines representing late stages in progression, it induced apoptosis in weakly or moderately transformed cell lines representing early stages in progression.

Haemodynamic effects of transurethral prostatectomy.

Thoracic bio-impedance cardiography was used to study the haemodynamic changes in 28 patients undergoing transurethral prostatectomy (TURP) under either general or spinal anaesthesia. Cardiac output and mean arterial pressure fell with induction of general anaesthesia, whilst mean arterial pressure and systemic vascular resistance fell with induction of spinal anaesthesia. The transthoracic fluid index fell during resection under general anaesthesia, but no significant haemodynamic changes were seen during resection under either anaesthetic. This study suggests that resection has no specific adverse haemodynamic consequences. Spinal anaesthesia may produce less haemodynamic disturbance than general anaesthesia in patients undergoing TURP and formal comparison of the 2 techniques seems necessary.

Repetitive nocturnal arterial oxygen desaturation and silent myocardial ischemia in patients presenting for vascular surgery.

OBJECTIVE: To determine whether nocturnal respiratory abnormality (cyclic oxygen desaturation and tachycardia) is associated with nocturnal myocardial ischemia in older individuals with ischemic heart disease. DESIGN: Non-invasive monitoring on a single occasion. SETTING: Tertiary care referral hospital. PATIENTS: Thirty four consecutive older (68.5 +/- 6 yrs) patients referred for elective abdominal or carotid reconstructive vascular surgery. RESULTS: Seven patients (21%) had moderately severe nocturnal respiratory abnormality, defined by more than 50 dips in arterial oxygen saturation and increases in heart rate during the night. Two of these seven had clinical risk factors for ischemic heart disease and had nocturnal myocardial ischemia. Ten patients (29%) developed ischemia at some time during the study, of whom seven hand known ischemic heart disease, hypertension, and/or angina. Those with increased nocturnal ischemia showed very low frequency (1-2 cycles per minute) cyclic heart rate oscillations and repetitive nocturnal episodes of arterial oxygen desaturation, similar to patients with sleep apnea. CONCLUSION: Repetitive nocturnal cyclic arterial desaturation and cyclic increases in heart rate are associated with nocturnal myocardial ischemia in individuals with clinical risk factors for ischemic heart disease. Further investigation in a large patient sample utilizing non-invasive monitoring of saturation, heart rate, and blood pressure may provide definitive evidence regarding causation of some of the nocturnal myocardial ischemia occurring in older individuals with vascular disease.

Late postoperative nocturnal dips in oxygen saturation in patients undergoing major abdominal vascular surgery. Predictive value of pre-operative overnight pulse oximetry.

Twenty-four patients (23 male) who presented for aortic reconstructive surgery were studied with pulse oximetry on a pre-operative night and during the first five postoperative nights. Patients with five or more dips in oxygen saturation of greater than 4% (with a prompt recovery back toward baseline of 3% or more) per hour of monitoring were classified as having a significant abnormality of respiration. Pre-operatively, four of 24 patients (17%) demonstrated such an abnormality. Postoperatively, 12 patients (50%) met these criteria on at least one of the first five postoperative nights and six of these had two or more nights with severe episodic hypoxaemia. Frequent severe episodic dips in arterial oxygen saturation (to less than 85% saturation) occurred in the late postoperative period at a time when oxygen therapy would usually have been discontinued. Pre-operative overnight pulse oximetry studies fail to predict the development of abnormal respiratory patterns in the postoperative period in the majority of patients.

Postoperative hypoxaemia after major abdominal vascular surgery.

We have studied the severity and duration of nocturnal hypoxaemia before operation and for the first five nights after operation after elective major abdominal vascular surgery. Oxygen supplementation was almost 100% effective in keeping oxygen saturation greater than 90% during the early postoperative period; however, 50% of patients spent prolonged periods with an SpO2 less than 85% during at least one night after operation. The risk of severe hypoxaemia persists well beyond the current prescription of supplementary oxygen in these high risk patients. A significant association exists between the mean preoperative overnight saturation value and the nocturnal saturation observed subsequently in the later postoperative period.

Postoperative myocardial ischaemia: temporal association with nocturnal hypoxaemia.

Three patients presenting for elective aortic surgery were studied before operation and for up to 3 days after operation with ambulatory electrocardiographic monitoring and nocturnal pulse oximetry. Supplementary oxygen was administered for the first 2 days after operation and withdrawn on the 3rd morning after operation. Heart rate remained increased for the first 3 days after operation. Two patients developed episodes of myocardial ischaemia during the first 48 h after operation while their arterial oxygen saturation remained greater than 90%. On the third day, and during the third night after operation, both the severity and duration of myocardial ischaemia increased markedly, associated with arterial hypoxaemia. A temporal relationship between decreases in oxygen saturation and fluctuations in ST segment level were observed in the three patients.

Preoperative silent myocardial ischaemia: incidence and predictors in a general surgical population.

We have studied before operation 156 patients aged more than 40 yr presenting for elective vascular or non-vascular surgery, using ambulatory ECG monitoring to detect silent myocardial ischaemia (SMI). The prevalence of SMI was 18.2% in the vascular group (n = 102) and 7.6% in the non-vascular group (n = 54). A history of ischaemic heart disease, or an abnormal ECG suggestive of a previous myocardial infarction, predicted a high risk of SMI (28% compared with 9% in the absence of these variables). However, a significant amount of SMI (36% of the total) occurred in patients without one of the defined risk factors. In addition, 24 of the patients with abdominal aortic disease underwent cardiac gated blood pool (MUGA) scans. Abnormal ventricular wall function was observed in 62.5% of the patients. Twenty-nine percent of the patients studied with MUGA scans had SMI and 21% had ejection fractions less than 40%. A significant association (P less than 0.05) existed between the presence of SMI and a ventricular ejection fraction of less than 40%.

Postoperative obstructive sleep apnoea. Haemodynamic effects of treatment with nasal CPAP.

A 74-year-old man presenting for aortic reconstructive surgery showed severe, previously undiagnosed obstructive sleep apnoea during overnight oximetry monitoring before operation. Postoperatively, in the first 4 hours following extubation, he suffered 238 episodes of respiratory obstruction. These events were associated with frequent arousals, large fluctuations in systolic and diastolic blood pressure. Administration of nasal continuous positive airways pressure abolished the obstructions and allowed an uninterrupted night's sleep, with a significantly reduced blood pressure. Subsequent dips in oxygen saturation as a result of respiratory obstruction recurred on the fifth postoperative night. We conclude that pre-operative overnight oximetry may be useful in identifying those patients at risk of postoperative upper airway obstruction. Use of nasal continuous positive airway pressure may prevent the occurrence of early postoperative obstruction and the associated haemodynamic changes.

Haemodynamic effects of periodic ventilation: abolition with supplementary oxygen.

A 63-yr-old man was noted to breathe with a periodic pattern of ventilation during sleep, both before and after operation for coronary artery grafting, and 6 weeks later after aortic aneurysm repair. Periodic ventilation was associated with cyclic oxygen desaturations and increases in heart rate and arterial pressure. Administration of oxygen was found to abolish consistently the periodic pattern and the associated haemodynamic changes.