Double mutant cycle analysis of aspartate 69, 97, and 103 to asparagine mutants in the m2 muscarinic acetylcholine receptor

Double mutant cycles provide a method for analyzing the effects of a mutation at a defined position in the protein structure on the properties of an amino acid at a second site. This approach was used to map potential interactions between aspartates 69, 97, and 103 in the m2 muscarinic acetylcholine receptor transmembrane helices 2 and 3. Receptors containing single and double aspartate to asparagine mutants were expressed in Chinese hamster ovary cells and their effects on ligand binding, signal transduction, and thermal stability determined. Analysis of the double mutant cycles showed that the mutations had approximately additive effects on ligand binding, signal transduction, and thermal stability. Ligand binding and thermal inactivation results support the conclusion that aspartate-103 is the ligand amine counterion. Effector coupling properties of the mutant receptors showed that aspartate-103 was also required for signal transduction activity. The mutation of aspartate-69 to asparagine completely eliminated signal transduction by the agonists acetylcholine, carbachol, and pilocarpine but not oxotremorine M, which caused reduced but significant inhibition of adenylyl cyclase and stimulation of phospholipase C. In contrast, adenylyl cyclase stimulation by the asparagine-69 mutant was elicited only by acetylcholine and carbachol but not by oxotremorine M. The variation in agonist-dependent effector coupling properties provides evidence that the asparagine-69 mutant can exist in activated receptor states that are different from the wild-type m2 muscarinic receptor.     

The ongoing evolution of behavioral couples therapy: retrospect and prospect

Behavioral couples therapy (BCT) was first described thirty years ago, and the conceptual basis of the approach has been evolving ever since. Whilst BCT in its current form is a well established treatment for relationship problems, there are significant limitations to its efficacy, and there are a number of new developments intended to enhance the efficacy of the approach. Currently BCT primarily is a skills training therapy, targeting change in couple's interactions to promote greater relationship satisfaction. Current developments focus on broadening this traditional emphasis on changing couple interactions to greater attention to reducing negative emotional responses to partner behaviors (increasing acceptance), and increasing self-directed personal change efforts. Future developments of BCT are likely to pay greater attention to different types of couple interaction which are associated with sustained relationship satisfaction, the individual vulnerabilities partners bring to relationships, and the environmental stresses impinging upon the couple.     

Impaired innervation of cultured human muscle overexpressing betaAPP experimentally and genetically: relevance to inclusion-body myopathies

To investigate whether abnormally accumulated betaAPP may be responsible for denervation of muscle fibers that are present in hereditary inclusion-body myopathy (h-IBM) and sporadic inclusion-body myositis (s-IBM), we cultured five h-IBM and eight normal muscle biopsies. In eight other experiments, a 3 kb human 751-betaAPP-cDNA was transferred, using adenovirus vector, into cultured normal myotubes immediately after myoblast fusion. In all experiments, cultured muscle fibers were co-cultured with fetal rat spinal cord. Controls had no detectable betaAPP epitopes, whereas betaAPP epitopes were greatly increased in cultured h-IBM muscle and in cultured normal muscle after betaAPP-gene transfer. Innervated normal cultured muscle fibers were continuously contracting and fully cross-striated, and they had acetylcholine receptors (AChRs) and acetylcholinesterase (AChE) accumulated only at the neuromuscular junctions (NMJs). By contrast, both groups of betaAPP-overexpressing cultured muscle fibers were not contracting and not cross-striated; and did not have NJMs containing AChRs and AChE. Our results suggest that over-expression of betaAPP in cultured muscle fibers inhibits their innervation, and that the accumulation of betaAPP in muscle fibers of both h- and s-IBM patients may be responsible for their not becoming or remaining properly innervated or reinnervated, i.e. a 'myogenous-dysinnervation' mechanism.     

Analysis of T cell receptor Vbeta gene usage during the course of disease in patients with chronic hepatitis B

Polyvinyl siloxane impression materials have applications in a variety of indirect procedures in prosthodontics and restorative dentistry. Favourable handling properties, good patient acceptance and excellent physical properties have resulted in their popularity in today's practice. In this review, the chemistry and important physical properties of polyvinyl siloxanes are summarized, and recent clinical questions of improved hydrophilics, tray adhesives, disinfection, and glove-induced polymerization inhibition are addressed.     

A prospective randomized trial of Teflon versus polyethylene stents for distal malignant biliary obstruction

BACKGROUND AND STUDY AIMS: Clogging of biliary stents continues to be a major clinical problem. Different polymer materials may have different effects on clogging. In vitro studies have shown a direct relation between the frictional coefficient of a polymer and the amount of encrusted material. Teflon appeared to be the best polymer for biliary stents. Two different types of stents made of Teflon have been tested in clinical practice and showed favourable patency rates. However, a randomized trial has never been performed. We compared the patency of an Amsterdam-type polyethylene stent with a Teflon stent in a prospective randomized trial. PATIENTS AND METHODS: Between September 1995 and November 1996, 42 patients received a Teflon stent and 42 patients a polyethylene stent. All patients had a distal malignant biliary stricture without a previous drainage procedure. Diagnoses included carcinoma of the pancreas (n = 76), papilla (n = 1), bile duct (n = 5) and metastases (n = 2). The internal and external diameter (10 Fr), length (9 cm) and stent design (a straight stent with two side flaps and one side hole at each end) were similar for both stents. RESULTS: A reduction in bilirubin of more than 20% within one week was seen in 91% of the patients. Early complication rates were similar in both groups (10%). The median follow-up was 142 days. Stent dysfunction occurred in 28 Teflon and 29 polyethylene stents. The thirty-day mortality was 14% in both groups. Patient survival did not differ significantly between the groups (median survival: Teflon 165 days, polyethylene 140 days). The median stent patency was 83 days for Teflon and 80 days for polyethylene stents, and was not significantly different either. CONCLUSION: Teflon material did not improve patency in biliary stents with an Amsterdam-type design.     

The present system of quantities and units for radiation protection

The International Commission on Radiation Units and Measurements has over the last decade developed operational quantities, the ambient, directional and personal dose equivalent, suitable for the measurement of radiation fields in a variety of circumstances. Experience with the use of these quantities to represent the dose limitation quantities defined by the International Commission on Radiological Protection in 1977 has been an important part of recent radiation protection metrology. The definition by International Commission on Radiological Protection in 1991 of new limitation quantities, the equivalent dose and the effective dose has necessitated a redirection of this work. The metrology field has made good progress, however. It has found that for photons, at least above 50 keV, the effective dose can be measured by the ambient dose equivalent about as well as the former effective dose equivalent. Unfortunately, for neutrons the existing and already quite severe complications have been made somewhat worse by the new quantities although not any worse in the important region between 0.1 and 1 MeV. Neutron measurements over a broad energy range are the subject of extensive evaluation and some new suggestions as the metrology field wrestles with these problems. Values of wR constitute an important part of the International Commission on Radiological Protection recommendations. A brief history of the development of higher relative biological effectiveness values for fission neutrons and alpha particles leading to the selection of 20 for wR in each case, is provided.