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.     

Effects of cytosolic Ca2+ on the Ca2+ content of the sarcoplasmic reticulum in saponin-permeabilized rat ventricular trabeculae

After a brief presentation of the development of free walking interpreted as learning dynamical equilibrium, the problem of sensory integration in the process of walking development is discussed. A critical review of the role of vision in the development of posturo-locomotor task is presented, along with recent test results on the development of the vestibular system. A final section presents the development of head stabilization and coordination as a necessary means to assist sensory integration. It is suggested that if sensory information is necessary to enhance posturo-locomotor skills, a good mastery of walking is in turn necessary to increase the efficiency of sensory integration.     

Survival of cylindrical implants in composite grafted maxillary sinuses

PURPOSE: This retrospective study investigated the survival of dental implants placed in the maxilla after composite grafting of the sinus and an average of 55 months of loading. PATIENTS AND METHODS: Maxillary sinuses of 88 patients were grafted with autogenous cancellous bone combined with dense hydroxyapatite particles. After an average healing period of 3.4 months, hydroxyapatite-coated titanium endosseous implants were placed. A total of 388 implants were placed in grafted sinus floors, and 82 were placed in onlay grafted nonsinus position in the canine region. The implants were loaded with overdentures and fixed bridges 4 months (mean) after implantation, with a follow-up for a mean of 55 months. RESULTS: The cumulative implant survival was calculated according to the Kaplan-Meier method. Implant survival from the time of loading was 89% in full reconstructed cases and 90% in partially edentulous cases. The overall cumulative implant survival rate, including the loss in the surgical stage, was 82%. CONCLUSION: Implant loss in composite grafted maxillae after 70 months of follow-up was similar to loss in nongrafted maxillae.     

Infection by Mycobacterium tuberculosis promotes human alveolar macrophage apoptosis

The effect of Mycobacterium tuberculosis infection on the viability of healthy (control) human alveolar macrophages was evaluated by staining with ethidium homodimer and calcein to discriminate live from dead cells. Infection with M. tuberculosis H37Ra or H37Rv increased macrophage mortality at 6 days from the control level of 3.8% +/- 0.7% to 28.7% +/- 6.9% or 12.6% +/- 3.1%, respectively (P < 0.001 for comparisons of all conditions). A role for tumor necrosis factor alpha (TNF-alpha) in the M. tuberculosis-induced cytolysis of alveolar macrophages was demonstrated by increased cytotoxicity following the addition of exogenous TNF-alpha to the cultures and by enhancement of macrophage survival when M. tuberculosis-infected alveolar macrophages were treated with pentoxifylline or anti-TNF-alpha antibody. The cytolytic mechanism was determined to be apoptosis by the demonstration of a characteristic internucleosomal ladder of genomic DNA by agarose gel electrophoresis, by finding nuclear fragmentation and condensation by electron microscopy, and by in situ terminal transferase-mediated nick end labeling of fragmented DNA in alveolar macrophages infected with M. tuberculosis in vitro. The latter technique was employed to reveal extensive apoptosis within caseating granulomas from lung tissue samples from clinical tuberculosis cases. The induction of apoptosis in alveolar macrophages by M. tuberculosis may play a role in the macrophage-pathogen interaction of tuberculosis in vivo.