Endoscopic biopsies in Ussing chambers evaluated for studies of macromolecular permeability in the human colon

OBJECTIVE: Studies of mucosal permeability to protein antigens in humans are limited to in vitro techniques. The use of surgical specimens for such studies has major shortcomings. Endoscopic biopsies in Ussing chambers have been introduced as a means of studying secretion and transepithelial permeability, but have not been evaluated for studies of protein antigen uptake in human intestine. MATERIAL AND METHODS: Standard forceps biopsies from the sigmoid colon of 24 healthy volunteers were mounted in Ussing chambers with an exposed tissue area of 1.76 mm2. 51Cr-EDTA (paracellular probe) and horseradish peroxidase (HRP; 45 kDa protein antigen) were used as permeability markers. Mucosal permeability, electrophysiology, histology and energy contents of the biopsies were studied over time. To evaluate the ability of the technique to detect permeability changes, the mucosa was modulated with capric acid, a medium-chain fatty acid, known to affect tight junctions. RESULTS: In the Ussing chamber the mucosal biopsies were viable for 160 min with stable levels of ATP and lactate, and only minor changes in morphology. Steady-state permeability with low variability was seen for both markers during the 30-90 min period. Exposure to capric acid induced a rapid decrease in short-circuit current (Isc) and a slower reversible decrease in transepithelial resistance (TER), as well as an increased permeability to 51Cr-EDTA and HRP. CONCLUSIONS: Endoscopic biopsies of human colon are viable in Ussing chambers and are reliable tools for studies of mucosal permeability to protein antigens. The technique offers a broad potential for studies of mucosal function in the pathophysiology of human gastrointestinal diseases.     

Interface connections of a transmembrane voltage sensor

Voltage-sensitive ion channels open and close in response to changes in transmembrane (TM) potential caused by the motion of the S4 voltage sensors. These sensors are alpha-helices that include four or more positively charged amino acids, most commonly arginine. The so-called paddle model, based on the high-resolution structure of the KvAP K+ channel [Jiang, et al. (2003) Nature 423, 33-41], posits that the S4 sensors move within the membrane bilayer in response to TM voltage changes. Direct exposure of S4 sensors to lipid is contrary to the classical expectation that the dielectric contrast between the membrane hydrocarbon core and water presents an insurmountable energetic penalty to burial of electric charges. Nevertheless, recent experiments have shown that a helix with the sequence of KvAP S4 can be inserted across the endoplasmic reticulum membrane. To reconcile this result with the classical energetics argument, we have carried out a molecular dynamics simulation of an isolated TM S4 helix in a lipid bilayer. The simulation reveals a stabilizing hydrogen-bonded network of water and lipid phosphates around the arginines that reduces the effective thickness of the bilayer hydrocarbon core to approximately 10 A in the vicinity of the helix. It suggests that bilayer phospholipids can adapt locally to strongly perturbing protein elements, causing the phospholipids to become a structural extension of the protein.     

Double-blind crossover randomized trial of intravenously administered verapamil. Its use for atrial fibrillation and flutter following open heart surgery

Fourteen patients with onset of atrial fibrillation (11) or flutter (three) and ventricular rate over 120 beats per minute following cardiac surgery were treated with intravenous (IV) doses of verapamil hydrochloride or placebo in a double-blind crossover protocol. Patients with poor left ventricular function, hypotension, atrioventricular block, and taking beta-blockers and disopyramide were excluded. The dosages were 0.075 mg/kg and 0.15 mg/kg given 15 minutes apart, with termination of study on achieving an end point (conversion to sinus rhythm or slowing of ventricular rate to below 100 beats per minute). None reached the end point with placebo but all with verapamil. Baseline ventricular rate was 144 +/- 20 beats per minute, after placebo 143 +/- 16 beats per minute, and after verapamil 89 +/- 7 beats per minute (mean +/- SD). Thus, IV verapamil briefly slows the ventricular rate of atrial tachyarrhythmias following cardiac surgery.     

Differential coupling of m-cholinoceptors to Gi/Go-proteins in failing human myocardium

Muscarinic acetylcholine receptors (mAChRs) mediate their main cardiac effects via pertussis toxin-sensitive G-proteins. Physiological effects differ considerably between atrium and ventricle, and it is unknown to which extent these differences derive from selective receptor-G-protein coupling or further downstream events. We have characterized specific coupling between mAChRs and Gi/Go-protein isoforms in atrial and ventricular myocardium by agonist-dependent photoaffinity labeling with [(32)P]azidoanilido GTP (aaGTP) and immunoprecipitation in sarcolemmal membranes from terminally failing human hearts. The total amount of mAChRs, as determined by specific binding of [(3)H]QNB, was significantly higher in right-atrial (RA +/- SEM, 959 +/- 68 fmol/mg, n = 4) than in left-ventricular membranes (LV, 582 +/- 53 fmol/mg, n = 6). Standardized immunoblots revealed that Gialpha-2 was the predominant subtype in both regions. A 40-kDa splice variant of Goalpha (Goalpha-1 and/or Goalpha-3) was almost exclusively detectable in RA. Levels of Gialpha-3 and a 39-kDa splice variant of Goalpha (Goalpha-2) were also higher in RA. Basal aaGTP binding was higher in RA than in LV for all Gialpha/Goalpha subtypes. The carbachol (10 micromol/l)-induced increase in aaGTP binding was significantly higher in RA than in LV for Goalpha-1/3 (336 +/- 95% of LV, n = 4) and for Gialpha-3 (211 +/- 83%), lower for Gialpha-2 (42 +/- 5%), and was similar in both regions for Goalpha-2 (130 +/- 62%). The differential coupling of mAChRs in human RA and LV suggests that the initiation of different physiological responses to mAChR stimulation starts with signal sorting at the receptor-G-protein level.     

3-Dimensional Cervical Movement Characteristics and the Influence of Thoracic Treatment on a Subgroup of Acute Neck Pain Patients

Objective

The purpose of this study was to investigate the influence of thoracic high-velocity low-amplitude thrust (HVLAT) manipulation on quantitative and qualitative 3-dimensional cervical spine kinematic patterns in a subgroup of patients with acute neck pain.

Variant angina pectoris: a clinical and coronary arteriographic spectrum

The clinical course and coronary arteriographic findings in eight patients with Prinzmetal's variant angina pectoris are reviewed and contrasted to previously reported cases. In six patients with S-T-elevation inferiorly, three had normal coronary arteriograms, one had complete right coronary artery occlusion, one had diffuse triple-vessel disease, and one did not undergo coronary arteriography. In two patients with S-T-elevation anteriorly, severe stenosis of the anterior descending coronary artery was present. Medical treatment in four patients and surgical revascularization of the anterior descending coronary artery in two patients were both accompanied by marked symptomatic improvement. Spontaneous loss of angina occurred in two patients. During 17 months' mean follow-up, seven patients have remained free of angina and one died suddenly. Variant angina pectoris may be accompanied by a variety of coronary arteriographic findings and the prognosis appears more favorable than previously reported.