SNAP-25 is required for a late postdocking step in Ca2+-dependent exocytosis

The Ca2+-activated fusion of large dense core vesicles (LDCVs) with the plasma membrane is reconstituted in mechanically permeabilized PC12 cells by provision of millimolar MgATP and cytosolic proteins. Ca2+-activated LDCV exocytosis was inhibited completely by the type E but not the type A botulinum neurotoxin (BoNT) even though both BoNTs were equally effective in proteolytically cleaving the synaptosome-associated protein of 25 kDa (SNAP-25). The greater inhibition of exocytosis by BoNT E correlated with a greater destabilization of detergent-extracted complexes consisting of SNAP-25, synaptobrevin, and syntaxin. LDCVs in permeable PC12 cells can be poised at a late postdocking, prefusion state by MgATP-dependent priming processes catalyzed by N-ethylmaleimide sensitive factor and priming in exocytosis proteins. BoNT E completely blocked Ca2+-activated LDCV exocytosis in ATP-primed cells, whereas BoNT A was only slightly inhibitory, implying that the C-terminal region of SNAP-25 (Ile181-Gln197) between the cleavage sites for BoNT E and BoNT A is essential for late postdocking steps. A required role for SNAP-25 at this stage was also indicated by inhibition of Ca2+-activated LDCV fusion in ATP-primed cells by a C-terminal peptide antibody. We conclude that plasma membrane SNAP-25, particularly residues 181-197, is required for Ca2+-regulated membrane fusion at a step beyond LDCV docking and ATP utilization.     

Antigen-specific regression of established tumors induced by active immunization with irradiated IL-12- but not B7-1-transfected tumor cells

Transfection of modestly immunogenic tumors to express B7 family co-stimulator molecules results in their rejection by syngeneic mice, suggesting a possible clinical application in cancer patients. Immunization of naive mice with irradiated B7-1-transfected P1.HTR cells is sufficient to induce specific cytolytic T lymphocytes (CTL) and to protect against tumor challenge. However, patients to be treated will have an existing tumor burden; thus, preclinical models should examine therapeutic efficacy in an established tumor setting. Contrary to expectations, immunization of mice with irradiated B7-1-transfected P1.HTR cells had no impact on the growth of pre-established control-transfected tumors. Mice bearing control-transfected P1.HTR tumors successfully rejected living B7-1 transfectants on the contralateral flank, demonstrating the ability of tumor-bearing mice to respond to B7 co-stimulation. Inasmuch as IL-12 is another important factor for CTL maturation, P1.HTR transfectants expressing B7-1 and/or IL-12 were then constructed. Remarkably, regression of pre-established tumors was achieved following immunization with irradiated IL-12 transfectants, even without co-expression of B7-1. Rejection required a shared antigen with the tumor used for immunization, could not be reproduced with rIL-12 alone, depended on host T lymphocytes and correlated with a high IFN-gamma-producing T cell phenotype. In addition, IL-12-facilitated tumor rejection required co-operation with a CTLA-4 ligand provided by the host, and correlated with up-regulation of B7-1 and B7-2 on host antigen-presenting cells. Thus, active immunization in the established tumor setting is benefitted greatly by the provision of IL-12, which may recruit participation of sufficient B7 co-stimulation from the host that it need not be provided exogenously.     

Blood flow velocity changes in the middle cerebral artery induced by processing of hierarchical visual stimuli

Transcranial Doppler ultrasound was used to measure mean flow velocities in both middle cerebral arteries while 16 young subjects performed a visual task involving the processing of hierarchically structured stimuli. Specifically, large (global) letters composed of smaller (local) letters were presented, with the subjects' task being to attend either to the local or to the global level and press a button whenever a target on the designated level occurred. Each run was comprised of a 35-sec period of passive stimulation, followed by 65 sec of active task. A highly significant increase of blood flow was detected upon initiation of the active task, which was clearly present after ca. 4 sec. The flow velocity reached a maximum after 20 sec and remained stable for the remainder of the active condition. No hemispheric differences with respect to global or local conditions were observed.     

Selection of patients for cardiac evaluation before peripheral vascular operations

PURPOSE: This study evaluated the value of preoperative cardiac screening with dipyridamole thallium scintigraphy and radionuclide ventriculography in vascular surgery patients. METHODS: From July 1, 1989, to Dec. 31, 1991, we routinely (irrespective of the patient's cardiac history or symptomatology) performed dipyridamole thallium scintigraphy (DTS) and radionuclide ventriculography (RVG) in 394 patients being considered for an elective vascular operation. Patients with reversible defects on DTS underwent coronary arteriography. RESULTS: DTS results were normal in 146 patients (37%), showed a fixed defect in 75 (19%), and showed a reversible defect in 173 (44%). Patients with and without a history of angina or myocardial infarction had identical rates of reversible defects. Normal left ventricular function (> 50%) was noted in 76% of the patients; 17% had moderate dysfunction (35% to 50%) and 7% had a low ejection fraction (< 35%). The finding of severe coronary artery disease led to cardiac revascularization in 17 patients who had no prior history of cardiac disease and in 13 patients with a history of angina or myocardial infarction. Two deaths and nine major complications were associated with coronary arteriography and cardiac revascularization. Vascular procedures (144 aortic, 53 carotid, 146 infrainguinal) were ultimately performed in 343 patients, with a mortality rate of 1.7% (3.5% aortic, 0% carotid, and 0.7% infrainguinal bypass). The nonfatal perioperative myocardial infarction rate was 2.0%. We monitored all 394 patients for cardiovascular events, with a mean follow-up of 40 months. Patients who underwent cardiac revascularization had a 4-year survival rate of 75%, which was similar to those with a normal DTS. Late cardiac events were significantly more frequent in patients who had either a reversible DTS or RVG < 35%. CONCLUSIONS: Routine cardiac screening of vascular surgery patients had similar impact on patients irrespective of their prior history or current symptoms suggesting coronary artery disease. Routine screening did not result in substantial benefit. Screening studies such as DTS or RVG may be most useful as part of an overall risk versus benefit assessment in patients without active symptoms of coronary artery disease who have less compelling indications for vascular intervention (claudication, moderate-sized aortic aneurysms, or asymptomatic carotid disease).     

Pharmacokinetic monitoring and dose modification of etanidazole in the RTOG 85-27 phase III head and neck trial

PURPOSE: To prospectively evaluate the pharmacokinetic monitoring and drug dose adjustment of Etanidazole (Eta) in patients treated on the RTOG randomized trial for Stage III and IV head and neck cancer. METHODS AND MATERIALS: From June, 1986 to October, 1991, 521 patients were randomized to conventional RT alone or RT plus Eta. The primary goal was to determine whether the addition of Eta to conventional radiation therapy improves local-regional control and tumor-free survival. Of the 264 patients who received Eta, 233 had their drug exposure calculated and the Eta dose and schedule adjusted accordingly to prevent the occurrence of serious peripheral neuropathy. Drug exposure was assessed using the area under the curve (AUC) for a single treatment that was calculated by the integral over time of the serum concentration of Eta. The total drug exposure (total-AUC) was estimated by multiplying the AUC by the number of drug administrations. RESULTS: Eighteen percent of patients developed Grade I and 6% developed Grade II peripheral neuropathy. There was no Grade 3 or 4 peripheral neuropathy. There is a trend for an increased risk of neuropathy by single dose AUC. The minimal difference in incidence of neuropathy by single-dose AUC was due to the use of dose and schedule modification for patients with the higher values. CONCLUSIONS: The pharmacokinetics investigated in this study confirm previous work that monitoring Eta levels, with dose adjustment, allows it to be used safely in the clinic. In a subset analysis there was a statistically significant improvement in local-regional control and survival rates for patients with N0 and N1 disease, that will require confirmation (14). However, the clinical efficacy of Eta in this trial proved to be of little overall benefit.     

Pathophysiology of erectile dysfunction

During the past decade, our knowledge of the hemodynamics, functional anatomy, neurophysiology, and neuropharmacology of erectile function has evolved substantially. The change of smooth muscle tone has emerged as a key factor in erection and detumescence. However, future studies are needed to elucidate the cellular and molecular basis of erectile physiology. With insight into normal physiology we will understand the pathologic process and be able to treat it.