Regression of childhood Barrett's esophageal mucosa by antireflux surgery and bipolar electrocoagulation

The authors report a case of a 13-year-old girl with Barrett's esophagus who underwent antireflux surgery and was subsequently treated with endoscopic thermal coagulation using bipolar electrocoagulation. Follow-up endoscopy 15 months after completion of the endoscopic therapy showed normal esophageal mucosa without intestinal metaplasia. Longer follow-up is needed to assess the long-term effects of endoscopic treatment of the Barrett's mucosa with thermal coagulation, and this procedure should still be considered under investigation.     

Casein kinase 1 is tightly associated with paired-helical filaments isolated from Alzheimer's disease brain

The protein kinase activity tightly associated with paired helical filaments (PHFs) purified from the brain tissue of individuals with Alzheimer's disease has been characterized in vitro. The activity is shown to phosphorylate casein, an exogenous substrate, with a maximal velocity of approximately 2 nmol/min/mg, suggesting it comprises a significant component of the total protein in the PHF preparation. On the basis of substrate selectivity, isoquinoline sulfonamide inhibitor selectivity, in-gel renaturation assays, and western analysis, the activity consists of closely related members of the alpha branch of the casein kinase 1 family of protein kinases. Because of its tight association with PHFs and its phosphate-directed substrate selectivity, casein kinase 1 is positioned to participate in the pathological hyperphosphorylation of tau protein that is observed in neurodegenerative diseases such as Alzheimer's disease.     

Evaluation of bovine-derived bone protein with a natural coral carrier as a bone-graft substitute in a canine segmental defect model

The efficacy of a bone-graft substitute (bovine-derived bone protein in a carrier of natural coral) in the healing of a segmental defect of a weight-bearing long bone was evaluated. Twenty dogs, divided into two groups, underwent bilateral radial osteotomies with creation of a 2.5 cm defect. On one side of each dog, the defect was filled with autogenous cancellous bone graft. Contralateral defects received, in a blinded randomized fashion, cylindrical implants consisting of natural coral (calcium carbonate) or calcium carbonate enhanced with a standard dose of bovine-derived bone protein (3.0 mg/implant; 0.68 mg bone protein/cm3). The limbs were stabilized with external fixators, and all animals underwent monthly radiographs. They were killed at 12 (group 1) or 24 (group 2) weeks, and regenerated bone was studied by biomechanical testing and histology. Radiographic union developed in all 20 radii with autogenous cancellous bone grafts and in all 10 of the radii with the composite implants. None of the radii with implants of calcium carbonate alone showed radiographic evidence of union. This represented a statistically significant difference between implant types. In addition, calcium carbonate implants both with and without bone protein demonstrated radiographic evidence of near total resorption of the radiodense carrier by 12 weeks. This resorption facilitated radiographic evaluation of healing. Mean values for biomechanical parameters of radii with the composite implants exceeded those for the contralateral controls at 12 and 24 weeks; the difference was statistically significant at 12 weeks. Histology revealed scant residual calcium carbonate carrier at either time in the defects with calcium carbonate implants; however, a moderate amount was present in defects with the composite implants. In these specimens, the residual carrier was completely surrounded by newly formed bone that may have insulated the calcium carbonate from further degradation. The present study used a carrier of granular calcium carbonate reconstituted with bovine type-I collagen to deliver an osteoinductive protein to the defect site. This carrier is of nonhuman origin (eliminating the risk of disease transmission or antigenicity) and resorbs rapidly. In this model, bovine-derived bone protein in a natural coral carrier performed consistently better than the gold standard autogenous cancellous bone graft in terms of the amount of bone formation and strength of the healed defect. This may have implications for removal of hardware or resumption of weight-bearing in certain clinical situations. These data also indicate that coralline calcium carbonate alone represents a poor option as a bone-graft substitute in this critical-sized segmental defect model.     

Partial activation of muscle phosphorylase by replacement of serine 14 with acidic residues at the site of regulatory phosphorylation

Phosphorylation of glycogen phosphorylase at residue Ser14 triggers a conformational transition that activates the enzyme. The N-terminus of the protein, in response to phosphorylation, folds into a 310 helix and moves from its location near a cluster of acidic residues on the protein surface to a site at the dimer interface where a pair of arginine residues form charged hydrogen bonds with the phosphoserine. Site-directed mutagenesis was used to replace Ser14 with Asp and Glu residues, analogs of the phosphoserine, that might be expected to participate in ionic interactions with the arginine side chains at the dimer interface. Kinetic analysis of the mutants indicates that substitution of an acidic residue in place of Ser14 at the site of regulatory phosphorylation partially activates the enzyme. The S14D mutant shows a 1.6-fold increase in Vmax, a 10-fold decrease in the apparent dissociation constant for AMP, and a 3-fold decrease in the S0.5 for glucose 1-phosphate. The S14E mutant behaves similarly, showing a 2.2-fold increase in Vmax, a 6-fold decrease in the apparent dissociation constant for AMP, and a 2-fold decrease in the S0.5 for glucose 1-phosphate. The ability of the mutations to enhance binding of AMP and glucose 1-phosphate and to raise catalytic activity suggests that the introduction of a carboxylate side chain at position 14 promotes docking of the N-terminus at the subunit interface and concomitant stabilization of the activated conformation of the enzyme. Like the native enzyme, both mutants show significant activity only in the presence of the activator, AMP. Full activation, analogous to that provided by covalent phosphorylation of the enzyme, likely is not achieved because of differences in the charge and the geometry of ionic interactions at the phosphorylation site.     

Choline acetyltransferase (ChAT) immunoreactivity in paraffin sections of normal and diseased intestines

There is increasing interest in localizing nerves in the intestine, especially specific populations of nerves. At present, the usual histochemical marker for cholinergic nerves in tissue sections is acetylcholinesterase activity. However, such techniques are applicable only to frozen sections and have uncertain specificity. Choline acetyltransferase (ChAT) is also present in cholinergic nerves, and we therefore aimed to establish a paraffin section immunocytochemical technique using an anti-ChAT antibody. Monoclonal anti-choline acetyltransferase (1.B3.9B3) and a biotin-streptavidin detection system were used to study the distribution of ChAT immunoreactivity (ChAT IR) in paraffin-embedded normal and diseased gastrointestinal tracts from both rats and humans. Optimal staining was seen after 6-24 hr of fixation in neutral buffered formalin and overnight incubation in 1 microgram/ml of 1.B3.9B3, with a similar distribution to that seen in frozen sections. In the rat diaphragm (used as a positive control), axons and motor endplates were ChAT IR. Proportions of ganglion cells and nerve fibers in the intramural plexi of both human and rat gastrointestinal tracts were also ChAT IR, as well as extrinsic nerve bundles in aganglionic segments of Hirschsprung's disease. Mucosal cholinergic nerves, however, were not visualized. In addition, non-neuronal cells such as endothelium, epithelium, and inflammatory cells were ChAT IR. We were able to localize ChAT to nerves in formalin-fixed, paraffin-embedded sections. The presence of ChAT IR in non-neuronal cells indicates that this method should be used in conjunction with other antibodies. Nevertheless, it proves to be a useful technique for studying cholinergic neuronal distinction in normal tissues and pathological disorders.     

Regulation of cytoplasmic pH in osteoclasts. Contribution of proton pumps and a proton-selective conductance

Osteoclasts resorb bone by secreting protons into an extracellular resorption zone through vacuolar-type proton pumps located in the ruffled border. The present study was undertaken to evaluate whether proton pumps also contribute to intracellular pH (pHi) regulation. Fluorescence imaging and photometry, and electrophysiological methods were used to characterize the mechanisms of pH regulation in isolated rabbit osteoclasts. The fluorescence of single osteoclasts cultured on glass coverslips and loaded with a pH-sensitive indicator was measured in nominally HCO(3-)-free solutions. When suspended in Na(+)-rich medium, the cells recovered from an acute acid load primarily by means of an amiloride-sensitive Na+/H+ antiporter. However, rapid recovery was also observed in Na(+)-free medium when K+ was used as the substitute. Bafilomycin-sensitive, vacuolar-type pumps were found to contribute marginally to pH regulation and no evidence was found for K+/H+ exchange. In contrast, pHi recovery in high K+ medium was largely attributed to a Zn(2+)-sensitive proton conductive pathway. The properties of this conductance were analyzed by patch-clamping osteoclasts in the whole-cell configuration. Depolarizing pulses induced a slowly developing outward current and a concomitant cytosolic alkalinization. Determination of the reversal potential during ion substitution experiments indicated that the current was due to H+ (equivalent) translocation across the membrane. The H+ current was greatly stimulated by reducing pHi, consistent with a homeostatic role of the conductive pathway during intracellular acidosis. These results suggest that vacuolar-type proton pumps contribute minimally to the recovery of cytoplasmic pH from intracellular acid loads. Instead, the data indicate the presence of a pH- and membrane potential-sensitive H+ conductance in the plasma membrane of osteoclasts. This conductance may contribute to translocation of charges and acid equivalents during bone resorption and/or generation of reactive oxygen intermediates by osteoclasts.