Sphingomyelin Functions as a Novel Receptor for Helicobacter pylori VacA

The vacuolating cytotoxin (VacA) of the gastric pathogen Helicobacter pylori binds and enters epithelial cells, ultimately resulting in cellular vacuolation. Several host factors have been reported to be important for VacA function, but none of these have been demonstrated to be essential for toxin binding to the plasma membrane. Thus, the identity of cell surface receptors critical for both toxin binding and function has remained elusive. Here, we identify VacA as the first bacterial virulence factor that exploits the important plasma membrane sphingolipid, sphingomyelin (SM), as a cellular receptor. Depletion of plasma membrane SM with sphingomyelinase inhibited VacA-mediated vacuolation and significantly reduced the sensitivity of HeLa cells, as well as several other cell lines, to VacA. Further analysis revealed that SM is critical for VacA interactions with the plasma membrane. Restoring plasma membrane SM in cells previously depleted of SM was sufficient to rescue both toxin vacuolation activity and plasma membrane binding. VacA association with detergent-resistant membranes was inhibited in cells pretreated with SMase C, indicating the importance of SM for VacA association with lipid raft microdomains. Finally, VacA bound to SM in an in vitro ELISA assay in a manner competitively inhibited by lysenin, a known SM-binding protein. Our results suggest a model where VacA may exploit the capacity of SM to preferentially partition into lipid rafts in order to access the raft-associated cellular machinery previously shown to be required for toxin entry into host cells

Molecular biology, part 1 : specialty session

Meeting: International Conference on AIDS, 5th, 4-9 June, 1989, Montreal, QC, CAPresenters: David. J. Looney, Joseph G. Sodroski, Manuel A. Navia, Gerald Myers, Masanori Hayami, Jorg W. Eichber

Prophylactic vertebral cement augmentation at the uppermost instrumented vertebra and rostral adjacent vertebra for the prevention of proximal junctional kyphosis and failure following long-segment fusion for adult spinal deformity

Proximal junctional kyphosis (PJK) and proximal junctional failure (PJF) are common problems after long-segment (>5 levels) thoracolumbar instrumented fusions in the treatment of adult spinal deformity (ASD). No specific surgical strategy has definitively been shown to lower the risk of PJK as the result of a multifactorial etiology. The study aimed to assess the incidence of PJK and PJF in patients treated with prophylactic polymethylmethacrylate (PMMA) cement augmentation at the uppermost instrumented vertebrae (UIV) and rostral adjacent vertebrae (UIV+1). This is a retrospective cohort-matched surgical case series at an academic institutional setting. Eighty-five adult patients over a 16-year enrollment period were identified with long-segment (>5 levels) posterior thoracolumbar instrumented fusions for ASD. Primary outcomes measures were PJK magnitude and PJF formation. Secondary outcomes measures were spinopelvic parameters, as well as global and regional sagittal alignment. The impact of adjunctive PMMA use in long-segment (≥5 levels) fusion for ASD was assessed in adult patients aged 18 and older. Patients were included with at least one of the following: lumbar scoliosis >20°, pelvic tilt >25°, sagittal vertical axis >5 cm, central sacral vertical line >2 cm, and thoracic kyphosis >60°. The frequency of PJF and the magnitude of PJK were measured radiographically preoperatively, postoperatively, and at maximum follow-up in controls (Group A) and PMMA at the UIV and UIV+1 (Group B). Eighty-five patients (64±11.1 years) with ASD were identified: 47 control patients (58±10.6) and 38 patients (71±6.8) treated with PMMA at the UIV and UIV+1. The mean follow-up was 27.9 and 24.2 months in Groups A and B, respectively (p=.10). Preoperative radiographic parameters were not significantly different, except the pelvic tilt which was greater in Group A (26.6° vs. 31.4°, p=.03). Postoperatively, the lumbopelvic mismatch was greater in Group B (14.6° vs. 7.9°, p=.037), whereas the magnitude of PJK was greater in controls (9.36° vs. 5.65°, p=.023). The incidence of PJK was 36% (n=17) and 23.7% (n=9) in Groups A and B, respectively (p=.020). The odds ratio of PJK with vertebroplasty was 0.548 (95% confidence interval=0.211 to 1.424). Proximal junctional kyphosis was observed in 6 (12.8%) controls only (p=.031). The UIV+1 angle, a measure of PJK, was significantly greater in controls (10.0° vs. 6.8°, p=.02). No difference in blood loss was observed. No complications were attributed to PMMA use. The use of prophylactic vertebral cement augmentation at the UIV and rostral adjacent vertebral segment at the time of deformity correction appears to be preventative in the development of proximal junctional kyphosis and failure

Treatment with inverse agonists enhances baseline atrial contractility in transgenic mice with chronic beta2-adrenoceptor activation

1. In this study, we investigate whether chronic treatment with β-adrenoceptor (βAR) ligands with inverse agonist activity enhances myocardial β(2)AR-mediated atrial tension more than neutral antagonists in transgenic mice (TG35). These mice exhibit chronic adrenoceptor activation because they possess a greater number of constitutively active receptors than wild type mice due to cardiac-specific overexpression of human β(2)ARs. TG35 and wild type mice were chronically treated for 90 h with three inverse agonists, ICI-118,551, propranolol, and carvedilol, and one neutral antagonist, alprenolol. After 96 h, we compared the basal and isoprenaline-stimulated (10 μM) increase in atrial tension in treated or untreated TG35 mice and wild type mice. In parallel, to determine the effect of chronic βAR ligand treatment on the amounts of G protein receptor kinase-2 (GRK-2) and G proteins, we performed Western blotting on myocardial cytosolic and membrane proteins. 2. Atria from the TG35 mice treated with inverse agonists showed increases in the baseline tension compared to those from alprenolol/vehicle-treated mice. ICI-118,551 and propranolol treatment restored the elevated myocardial G-inhibitory protein (G(i)α) levels to that of wild type. Also, treatment with inverse agonists upregulated G-stimulatory protein (G(s)α) levels and GRK2 above those levels in vehicle-treated TG35 or wild type mice. The increased baseline atrial tension was reversed by the addition of ICI-118,551. 3. Overall, our data suggests that inverse agonists enhance baseline atrial tension more than neutral antagonists. Based on this, we propose that upregulation of the active conformation of the β(2)ARs, G(s)α protein and restoration of G(i)α as three possible mechanisms to explain this enhanced receptor activity. 4. Therefore, the favourable effects of some ligands used in pathological conditions involving chronic adrenoceptor activation may be due to the inverse agonist activity of the ligand