Identification of sulfhydryl-modified cysteine residues in the ligand binding pocket of retinoic acid receptor beta

The diverse biological functions of retinoic acid (RA) are mediated through retinoic acid receptors (RARs) and retinoid X receptors. RARs contain a high affinity binding site for RA which is sensitive to treatment with sulfhydryl modification reagents. In an attempt to identify which Cys residues are important for this loss of binding, we created three site-specific RARbeta mutants: C228A, C258A, and C267A. The affinity for RA of all three mutant receptors was in the range of that of the wild type protein, suggesting that none of these Cys residues are critical for RA binding. Rather, these modified Cys residue(s) function to sterically hinder RA binding; however, the modified Cys residues critical for the inhibition of binding differ depending on the reagent employed. Only modification of Cys228 is necessary to inhibit RA binding when RARbeta is modified by reagents which transfer large bulky groups while both Cys228 and Cys267 must be modified when a small functional group is transferred. These data suggest that both Cys228 and Cys267 but not Cys258 lie in the ligand binding pocket of RARbeta. However, Cys228 lies closer to the opening of the RARbeta ligand binding pocket whereas Cys267 lies more deeply buried.     

Gap junctional intercellular communication contributes to hormonal responsiveness in osteoblastic networks

To evaluate whether intercellular coupling via connexin43 gap junction channels modulates hormonal responsiveness of cells in contact, we have created osteoblastic cell lines deficient in connexin43. Osteoblastic ROS 17/2.8 cells were transfected with a plasmid containing an antisense cDNA construct to rat connexin43. Control transfection did not alter cell-to-cell coupling nor connexin43 mRNA or protein expression relative to nontransfected ROS 17/2.8 cells. In contrast, stable transfection with an antisense connexin43 cDNA resulted in two clones, RCx4 and RCx16, which displayed significant decreases in connexin43 mRNA and protein expression and were dramatically deficient in cell-to-cell coupling. Phenotypically, all transfectants retained osteoblastic characteristics. However, cells rendered connexin43-deficient through antisense transfection displayed a dramatic attenuation in the cAMP response to parathyroid hormone. Alterations in hormonal responses were not due to changes in parathyroid hormone receptor number or binding kinetics nor to alterations in adenylyl cyclase activity. These results indicate that gap junctions may be required for mediating hormonal signals. Furthermore, these experiments support a regulatory role for connexin43-mediated intercellular communication in the modulation of hormonal responses within elaborately networked bone cells.     

Bilateral bronchial balloon dilatation and Strecker stent implantation in a ventilated child with malignant carinal stenosis

Tracheobronchial endoluminal reconstruction and stenting has become a valuable palliative tool in adults with intrathoracic tumors compromising the airways. Tracheobronchial balloon dilatation has been recently used in children and even neonates. We report a case of severe airway obstruction requiring emergency intubation and artificial ventilation in a 5-year-old child with intrathoracic recurrence of a rhabdomyosarcoma. Endoscopic balloon dilatation through the endotracheal tube with subsequent implantation of a non self-expanding metal mesh stent was used successfully, allowing extubation and discharge of the child from ICU.     

Viscosity and fermentability as attributes of dietary fiber responsible for the hypocholesterolemic effect in hamsters

The attribute(s) of soluble dietary fibers responsible for cholesterol lowering is currently uncertain. A series of experiments were conducted in which viscosity and fermentability was assessed independently for their effect on plasma and liver cholesterol concentration. Hamsters were divided into four dietary groups and fed diets containing 0.12% cholesterol and 5% fiber as high viscosity hydroxypropyl methylcellulose (HV-HPMC group), low viscosity hydroxypropyl methylcellulose (LV-HPMC group), high viscosity guar gum (HV-GG group) or low viscosity guar gum (LV-GG group). Hydroxypropyl methylcellulose is essentially nonfermentable, whereas guar gum is highly fermentable. Plasma cholesterol concentrations at 3, 6 and 11 wk and liver cholesterol concentrations at 6 and 11 wk were significantly lower in the HV-HPMC group relative to the LV-HPMC group (P < 0.05). Intestinal content viscosities of the LV-HPMC and HV-GG groups were similar; consequently, these two groups were compared to examine the independent effect of fermentation. Plasma and liver cholesterol were significantly lower in the HV-GG group compared with the LV-HPMC group at 6 wk (P < 0.05), but not at 3 or 11 wk. Hepatic sterol synthesis rates were not affected by any of the diets. This study shows that greater viscosity of intestinal contents is strongly associated with cholesterol reduction, but that the contribution of fiber fermentation remains uncertain.