Nucleocytoplasmic distribution of the ovalbumin serpin PI-9 requires a nonconventional nuclear import pathway and the export factor Crm1.

Proteinase inhibitor 9 (PI-9) is a human serpin present in the cytoplasm of cytotoxic lymphocytes and epithelial cells. It inhibits the cytotoxic lymphocyte granule proteinase granzyme B (graB) and is thought to protect cytotoxic lymphocytes and bystander cells from graB-mediated apoptosis. Following uptake into cells, graB promotes DNA degradation, rapidly translocating to the nucleus, where it binds a nuclear component. PI-9 should therefore be found in cytotoxic lymphocyte and bystander cell nuclei to ensure complete protection against graB. Here we demonstrate by microscopy and subcellular fractionation experiments that PI-9 is present in the nuclei of human cytotoxic cells, endothelial cells, and epithelial cells. We also show that the related serpins, PI-6, monocyte neutrophil elastase inhibitor (MNEI), PI-8, plasminogen activator inhibitor 2 (PAI-2), and the viral serpin CrmA exhibit similar nucleocytoplasmic distributions. Because these serpins lack classical nuclear localization signals and are small enough to diffuse through nuclear pores, we investigated whether import occurs actively or passively. Large (approximately 70 kDa) chimeric proteins comprising PI-9, PI-6, PI-8, MNEI, or PAI-2 fused to green fluorescent protein (GFP) show similar nucleocytoplasmic distributions to the parent proteins, indicating that nuclear import is active. By contrast, CrmA-GFP is excluded from nuclei, indicating that CrmA is not actively imported. In vitro nuclear transport assays show that PI-9 accumulates at a rate above that of passive diffusion, that it requires cytosolic factors but not ATP, and that it does not bind an intranuclear component. Furthermore, PI-9 is exported from nuclei via a leptomycin B-sensitive pathway, implying involvement of the export factor Crm1p. We conclude that the nucleocytoplasmic distribution of PI-9 and related serpins involves a nonconventional nuclear import pathway and Crm1p.

Cerebellar cortex lesions prevent acquisition of conditioned eyelid responses.

We have used aspiration and electrolytic lesions to investigate the contributions of cerebellar cortex to the acquisition and expression of conditioned eyelid responses. We show that lesions of the anterior lobe of rabbit cerebellar cortex disrupt the timing of previously learned conditioned eyelid responses. These short-latency responses were used as an indication that the cerebellar cortex was sufficiently damaged and that the underlying pathways necessary for the expression of responses were sufficiently intact to support responses. Rabbits were subsequently trained for 15 daily sessions using a new conditioned stimulus. Whereas rabbits in which lesions had no significant effect on response timing showed rapid acquisition of appropriately timed eyelid responses to the new conditioned stimulus, animals with lesions that disrupt timing showed no significant increases in either amplitude or probability of responses. Histological analysis suggests that damage to the anterior lobe of the cerebellar cortex is necessary and sufficient to abolish timing and prevent acquisition. These data indicate that the cerebellar cortex is necessary for the acquisition of conditioned eyelid responses and are consistent with the hypotheses that (1) eyelid conditioning results in plasticity in both the anterior lobe of the cerebellar cortex and in the anterior interpositus nucleus and (2) induction of plasticity in the interpositus requires intact input from the cerebellar cortex.

Inhibitory control of LTP and LTD: stability of synapse strength.

Although much is known about the induction of synaptic plasticity, the persistence of memories suggests the importance of understanding factors that maintain synaptic strength and prevent unwanted synaptic changes. Here we present evidence that recurrent inhibitory connections in the CA1 region of hippocampus may contribute to this task by modulating the relative ability to induce long-term potentiation and depression (LTP and LTD). Bath application of the gamma-aminobutyric acid (GABA) type A agonist muscimol to hippocampal slices increased the range of frequencies that produce LTD, whereas in the presence of the GABA type A antagonist picrotoxin LTD was induced only at very low stimulation frequencies (0.25-0.5 Hz). Because one source of GABAergic input to CA1 pyramidal cells is via recurrent inhibition, we tested the prediction that elevated postsynaptic spike activity would increase feedback GABA inhibition and favor the induction of LTD. By using an induction stimulation of 8 Hz, which alone produced no net change in synaptic strength, we found that stimulation presented during antidromic activation of pyramidal cell spikes induced LTD. This effect was blocked by picrotoxin. The influence of recurrent inhibition on LTP and LTD displays properties that may decrease the potential for self-reinforcing, runaway changes in synapse strength. A mechanism of this sort may help maintain patterns of synaptic strengths despite the ongoing opportunities for plasticity produced by synapse activation.

The effect of lactic acid on mononuclear cell secretion of proinflammatory cytokines in response to group B streptococci.

This study found that lactate alone had a stimulatory effect (207.1 +/- 16.3%; P = .001) on tumor necrosis factor (TNF)-alpha production by human mononuclear cells with the most profound secretion being at pathologic concentrations of 4-8 mM lactate. Furthermore, exposure of these mononuclear cells to group B streptococci (GBS, 10(5) cfu) resulted in TNF-alpha production of up to 621.1 +/- 42% of control; the combination of lactic acid and GBS increased TNF-alpha production up to 1019.3 +/- 16.1% (P = .001). The combination of GBS and lactate also enhanced the secretion of interleukin (IL)-1beta and IL-6. Lactate in pathologic concentrations, therefore, likely enhances the secretion of these inflammatory mediators and contributes to septic shock and meningitis caused by GBS.

Sequence of a Leishmania major gene encoding an ubiquitin fusion protein.

A gene encoding an ubiquitin-tail protein fusion was isolated from the parasitic protozoan, Leishmania major, and sequenced. The L. major tail protein shares 97, 96, 67, 62, 62 and 61% sequence identity with the tail proteins of Trypanosoma brucei, Trypanosoma cruzi, yeast, Dictyostelium discoideum, human, and Arabidopsis thaliana, respectively. The putative 'zinc finger' nucleic acid-binding domain found in all ubiquitin 'tail' or 'extension' proteins described is also conserved in the L. major sequence. The upstream sequence indicated that this gene is not located at the end of a polyubiquitin sequence.

Antithrombotic therapy for deep arterial injury by angioplasty. Efficacy of common platelet inhibition compared with thrombin inhibition in pigs.

BACKGROUND: Platelet-thrombus formation is a complication of arterial wall deep injury by balloon angioplasty that may lead to acute arterial occlusion and may contribute to restenosis. METHODS AND RESULTS: Because common platelet-inhibitor drugs with a heparin bolus (100 units/kg) may be effective in inhibiting platelet-thrombus formation after arterial angioplasty, these were compared with a bolus of heparin alone (control), the specific thrombin inhibitor hirudin (1.0 mg/kg), and saline (hirudin control) in normal pigs after angioplasty of the common carotid arteries. In the presence of deep arterial wall injury (injury exposing the media), indium-111-labeled platelet deposition (x 10(6)/cm2) was 68.8 +/- 12.3 and 48.1 +/- 16.9 in the control animals. This was significantly reduced by pretreatment with low-dose aspirin (1 mg/kg/day), by high-dose aspirin (20 mg/kg/day) plus dipyridamole, and especially by thrombin inhibition with hirudin. Treatment regimens with aspirin alone (20 mg/kg/day), dipyridamole alone, or sulfinpyrazone were ineffective. Likewise, the incidence of mural thrombosis was 75% and 80% in deeply injured arteries of controls and was significantly reduced to 46% with aspirin plus dipyridamole, 25% with low-dose aspirin, and 0% with hirudin. The incidence of mural thrombosis was unchanged with high-dose aspirin (69%), dipyridamole (90%), or sulfinpyrazone (92%). This mural thrombosis could not be identified by angiography. In the presence of mild injury (deendothelialization), platelet deposition was low (less than 10 x 10(6)/cm2, a single layer) and was not changed by any therapy, including hirudin. CONCLUSIONS: These therapies do not affect platelet adhesion to deeply or mildly injured artery. These data suggest a greater role for thrombin inhibition than with thromboxane or cyclooxygenase inhibition in the pathogenesis of platelet-rich mural thrombosis after deep injury during angioplasty. Antithrombotic therapy for arterial thrombosis by thrombin inhibition appears promising.

Does the emergency room electrocardiogram identify patients with suspected myocardial infarction who are at low risk of acute complications?

To determine the early morbidity of patients admitted to the coronary care unit (CCU) with inconclusive evidence of acute myocardial infarction, the prognostic value of the emergency room electrocardiogram (ECG) was examined prospectively in a blinded fashion in 410 patients presenting with acute chest pain. One hundred and forty one patients (34.4%) had an ECG that was normal, showed ST segment changes less than 1 mm, or was unchanged from a previous recording (group 1). The remaining patients (65.6%, group 2) had ECGs considered abnormal. Thirty-nine patients in group 1 and 226 in group 2 had confirmed infarction. There was one CCU death in group 1 (0.7%) versus 27 (10.0%) in group 2 (p less than 0.001) and the overall hospital mortality for group 1 was 2.1% versus 13.0% in group 2 (p less than 0.001). Twenty-eight patients (19.9%) from group 1 suffered complications in the CCU versus 155 (57.6%) from group 2 (p less than 0.001). No life-threatening arrhythmias occurred in group 1 versus occurrence in 47 patients (17.5%) in group 2 (p less than 0.001). The need for acute intervention was also less for group 1 versus group 2 patients, 14 (9.9%) and 85 (31.6%) respectively (p less than 0.001) with no patient requiring electrical cardioversion in group 1. It is concluded that the emergency room ECG can reliably identify a group of low risk patients presenting with suspected myocardial infarction and so help in establishing priority for admission to the CCU. Furthermore, the risk-benefit of thrombolytic therapy in these low risk patients appears unacceptable.

Coarctation of the aorta. Long-term follow-up and prediction of outcome after surgical correction.

The long-term clinical course was studied in 646 patients, who underwent isolated operative repair of coarctation of the aorta at the Mayo Clinic from 1946 to 1981. There were 17 perioperative deaths, and 58 patients were lost to follow-up. Of the 571 patients with long-term follow-up, 11% required subsequent cardiovascular surgery, and 25% developed hypertension. There were 87 late deaths. The mean age at death was 38 years (range, 0-67 years). Estimated survival analysis revealed 91% of patients alive at 10, 84% at 20, and 72% at 30 years after operative repair. The most common cause of late death was coronary artery disease in 32 patients, followed by sudden death, heart failure, cerebrovascular accidents, and ruptured aortic aneurysm. Age, sex, and postoperative systolic blood pressure were found to be independently predictive of survival. For patients less than 14 years of age at the time of initial coarctectomy, survival to 20 years was 91%, and for patients 14 years or older at the time of operation, survival was 79%. The best survivorship was observed in patients operated on at 9 years of age or less. The higher the postoperative systolic pressure, the higher the probability of death. This study has the largest population undergoing repair of coarctation of the aorta with a median follow-up of as long as 20 years. Four main points emerged. 1) Age at the time of initial repair is the most important predictor of long-term survival. Surgery should be offered to patients after age 1 year or sooner if hypertension is severe. 2) Coronary artery disease is the most common cause of late death.(ABSTRACT TRUNCATED AT 250 WORDS)

Isolated atrial septal defect with pulmonary vascular obstructive disease--long-term follow-up and prediction of outcome after surgical correction.

UNLABELLED: We examined the cases of 702 patients found to have isolated atrial septal defect of the secundum or sinus venosus type at catheterization from 1953 to 1978. Forty patients (6%), 34 women and six men, had pulmonary vascular obstructive disease, with a total pulmonary resistance greater than 7 U/m2; of these patients 26 (mean age 47 years) underwent surgical closure and 14 (mean age 44 years) received medical treatment. All patients were followed for at least 4 years, with a median follow-up of 12 years. At the most recent follow-up, 17 of the 40 patients were dead. Of the 22 surgically treated patients with total pulmonary resistance less than 15 U/m2, 19 were alive with significant regression of symptoms. All four surgically treated patients with total pulmonary resistance greater than or equal to 15 U/m2 were dead. Of the five medically treated patients with total pulmonary resistance less than 15 U/m2, four had died, and one was alive with significant progression of symptoms. Of the nine medically treated patients with total pulmonary resistance greater than or equal to 15 U/m2, six had died and the three survivors had progression of symptoms. In the surgically treated group, the following variables correlated with survival: total pulmonary resistance (p less than .00001), pulmonary arteriolar resistance (p less than .00001), pulmonary-to-systemic resistance ratio (p = .004), systemic arterial oxygen saturation (p = .005), and pulmonary arterial oxygen saturation (p = .007). IN CONCLUSION: (1) Atrial septal defect with high total pulmonary resistance is uncommon and predominates in adult female patients.(ABSTRACT TRUNCATED AT 250 WORDS)

Tc-99m tertiary butyl isonitrile: a preliminary clinical study of a new myocardial imaging agent in patients with chest pain.

Tc-99m is a preferred agent for nuclear imaging. Tc-99m tertiary butyl isonitrile (Tc-99m-TBI) is the first of a family of lipophilic cations which allow effective imaging of the human myocardium. Twenty-one patients scheduled for coronary angiography for the investigation of their chest pain were studied after injections of this tracer given during exercise, and on a separate visit, at rest. Despite high uptake in the liver and transiently in the lungs, high quality gated images were obtained allowing the assessment of both myocardial uptake and wall motion. The findings corresponded closely with angiographic and ventriculographic assessment of coronary artery disease and myocardial infarction. Redistribution of Tc-99m-TBI was shown to occur in ischemic myocardium analogous to that occurring with thallium-201 (TI-201). Similar agents with more favourable properties may well replace TI-201 as the myocardial imaging agent of choice in the near future.

Is vasospasm related to platelet deposition? Relationship in a porcine preparation of arterial injury in vivo.

Although aggregating platelets can release potent vasoactive substances in vitro, the importance of platelets in mediating naturally occurring or provoked spasm in vivo is not clear. To investigate the possible role of platelets in arterial spasm following arterial injury induced by angioplasty, quantitative platelet deposition of the dilated arterial segment and the degree of vasoconstriction (average percent diameter narrowing just proximal and distal to the dilated segment) produced during angioplasty of the common carotid arteries were studied in 42 heparinized normal pigs that were killed immediately after the angioplasty procedure. Angiographic films of the carotid arteries were taken before and after the dilatation to assess the vasoconstriction. Vasoconstriction was greater (40% vs 19%, p less than .002) when platelet deposition (X 10(6)/cm2) was in excess of 10, and the severity of vasoconstriction in vivo had a close positive exponential correlation (r = .77, p less than .001) with extent of platelet deposition in 24 untreated pigs. Platelet deposition and vasoconstriction were greater with severe arterial wall injury than with mild injury (58.8 versus 6.9, p less than .0001; 37% vs 21%, p less than .001, respectively). After severe injury in 18 pigs pretreated with 1 mg/kg/day aspirin, platelet deposition decreased (from 58.8 to 19.6, p less than .02) and vasoconstriction decreased (from 37% to 21%, p less than .003) relative to control. After mild injury, platelet deposition and vasoconstriction were mild and unchanged by aspirin. Thus, local vasoconstriction is influenced by the degree of platelet deposition.(ABSTRACT TRUNCATED AT 250 WORDS)

Deep arterial injury during experimental angioplasty: relation to a positive indium-111-labeled platelet scintigram, quantitative platelet deposition and mural thrombosis.

Although it is not clear why coronary occlusion and restenosis occur after successful coronary angioplasty, factors related to the procedure may influence early and late results. The possible adverse effects of a medial tear documented histologically and produced during balloon angioplasty of the common carotid arteries were studied in 30 fully heparinized (100 U/kg body weight) normal pigs. Scanning electron microscopy showed endothelial denudation and extensive platelet deposition in all dilated arterial segments. Visible macroscopic mural thrombus was present within an hour of the procedure in 29 (91%) of the 32 arteries that had a medial tear documented by histologic study; the tear produced an indium-111-labeled platelet deposition of 116.4 +/- 26.5 X 10(6)/cm2 (mean +/- SE) and total thrombotic occlusion in 2 arteries (4%). None of the 24 arteries without a medial tear had a thrombus, and the mean platelet deposition in that group was 7.0 +/- 0.5 X 10(6)/cm2 (p less than 0.0008). In 12 pigs scanned with a gamma camera, visible thrombus was associated with platelet deposition in excess of 20 X 10(6)/cm2 in 12 arteries, 9 of which had a positive indium-111-labeled platelet scintigram. Thus, arterial angioplasty causes deep arterial injury, which appears to be a major cause of mural thrombosis, heavy platelet deposition, a positive indium-111-labeled platelet scintigram and acute arterial occlusion. A positive indium-111-labeled platelet scintigram was always associated with macroscopic thrombus of at least 20 > 10(6) platelets/cm2 and underlying deep arterial injury.

Systemic embolism in chronic left ventricular aneurysm: incidence and the role of anticoagulation.

The incidence and prevention of systemic embolism in patients with chronic left ventricular aneurysm have been controversial. This retrospective study investigated the incidence of clinically evident embolic events and the effect of oral anticoagulation in patients with unequivocal angiographically defined left ventricular aneurysm. Between 1971 and 1979, 76 patients met the ventriculographic criteria and received initial medical management. The median interval from myocardial infarction to ventriculography was 11 months (range 1 month to 16 years) and subsequent median follow-up time was 5 years. Twenty patients receiving anticoagulant therapy were followed up for a total of 40 patient-years and 69 patients not on anticoagulant therapy were followed up for a total of 288 patient-years; 13 patients were included in both subsets. Twenty-eight patients died during follow-up and the 3 and 5 year survival rates were 75 and 61%, respectively. Only one patient not receiving anticoagulant therapy had a clinical embolic event, resulting in an incidence of 0.35 per 100 patient-years. Therefore, in the absence of other predisposing conditions, the extremely low incidence of systemic emboli in these patients with chronic (first documented at least 1 month after myocardial infarction) left ventricular aneurysm does not justify the use of long-term oral anticoagulant therapy.