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A novel photoreactive analog of cholesterol, 3alpha-(4-azido-3-[125I]iodosalicylic)-cholest-5-ene ([125I]azido-cholesterol), was used to label both native acetylcholine receptor (AChR)-rich membranes from Torpedo californica and affinity-purified Torpedo AChRs reconstituted into lipid vesicles. In both cases all four AChR subunits incorporated [125I]azido-cholesterol on an equal molar basis and neither the pattern nor the extent of labeling was affected by the presence of the agonist carbamylcholine. Labeled regions in each of the AChR subunits were initially mapped by Staphylococcus aureus V8 protease digestion to large fragments which contain the AChR transmembrane segments. Sites of [125I]azido-cholesterol incorporation were further mapped by exhaustive tryptic digestion of the V8 protease subunit fragments alphaV8-20 (alphaSer-173-Glu-338), alphaV8-10 (alphaAsn-339-Gly-439), and gammaV8-14 (gammaLeu-373-Pro-489). The digests were separated by reverse-phase high-performance liquid chromatography and labeled peptides identified by amino-terminal sequence analysis. [125I]Azido-cholesterol labeling was localized to peptides that contain almost exclusively the alpha-M4, alpha-M1 and gamma-M4 membrane spanning segments. These results establish that the binding domain for cholesterol is at the lipid-protein interface of the AChR.

Circular dichroism (CD) and attenuated total reflection Fourier transform infrared (ATR-FTIR) spectroscopy are used to establish the secondary structure of peptides containing one or more transmembrane segments (M1-M4) of the Torpedo californica nicotinic acetylcholine receptor (AChR). Peptides containing the M2-M3 and M1-M2-M3 transmembrane segments of the AChR beta-subunit and the M4 segment of the alpha- and gamma-subunits were isolated from proteolytic digests of receptor subunits, purified, and reconstituted into lipid vesicles. For each peptide, an amide I vibrational frequency centered between 1650 and 1656 cm-1 and negative CD absorption bands at 208 and 222 nm indicate that the peptide is largely alpha-helical. In addition, the CD spectrum of a tryptic peptide of the alpha-subunit containing the M1 segment is also consistent with a largely alpha-helical structure. However, secondary structure analysis of the alpha-M1 CD spectrum indicates the presence of other structures, suggesting that the M1 segment may represent either a distorted alpha-helix, likely the consequence of several proline residues, or may not be entirely alpha-helical. Overall, these findings are consistent with studies that indicate that the transmembrane region of the AChR comprises predominantly, if not exclusively, membrane-spanning alpha-helices.

IntroductionDental microbial biofilm initiates gingival inflammation, and its suppression is the current dominant strategy for treating periodontitis. However, the host response to the biofilm is largely responsible for the connective tissue breakdown including alveolar bone loss, which is mediated by proinflammatory cytokines and matrix metalloproteinases (MMPs).MethodsThe current study compared the efficacy of a novel host-modulation compound, a chemically modified curcumin (CMC 2.24), to that of its parent compound (natural curcumin), in both lipopolysaccharide (LPS) (a bacterial endotoxin)-induced cell culture and in vivo models of periodontitis.ResultsIn cell culture, both CMC 2.24 and curcumin appeared similarly effective in suppressing LPS-induced cytokine (IL-1β and TNF-α) secretion by mononuclear inflammatory cells; however, CMC 2.24 significantly reduced MMP-9 secretion by 78% (P<0.05) whereas curcumin was ineffective. In vivo, CMC 2.24 administration was more effective than curcumin in suppressing (a) IL-1β in gingival tissue and (b) MMP-9 in both gingiva and plasma, the latter indicating a reduced severity of systemic inflammation. The difference in primary clinical outcome between the two treatments was that CMC 2.24 reduced the pathologically excessive alveolar bone loss, assessed morphometrically at multiple sites, by 80%–90% (P<0.01), whereas curcumin, surprisingly, either increased (P<0.05) or had no effect on alveolar bone loss at these sites.ConclusionThese data, plus that from previous studies, support the therapeutic potential of CMC 2.24 in the management of inflammatory periodontal disease and its ability to reduce the risk of associated systemic diseases. The current study also indicates that the MMP-9 inhibitor efficacy is associated with the ability of CMC 2.24 (but not curcumin) to inhibit alveolar bone loss in this rat model of periodontitis.

Background: Although retrograde peri‐implantitis (RPI) is not a common sequela of dental implant surgery, its prevalence has been reported in the literature to be 0.26%. Incidence of RPI is reported to increase to 7.8% when teeth adjacent to the implant site have a previous history of root canal therapy, and it is correlated with distance between implant and adjacent tooth and/or with time from endodontic treatment of adjacent tooth to implant placement. Minimum 2 mm space between implant and adjacent tooth is needed to decrease incidence of apical RPI, with minimum 4 weeks between completion of endodontic treatment and actual implant placement. The purpose of this study is to compile all available treatment modalities and to provide a decision tree as a general guide for clinicians to aid in diagnosis and treatment of RPI. Methods: Literature search was performed for articles published in English on the topic of RPI. Articles selected were case reports with study populations ranging from 1 to 32 patients. Any case report or clinical trial that attempted to treat or rescue an implant diagnosed with RPI was included. Results: Predominant diagnostic presentation of a lesion was presence of sinus tract at buccal or facial abscess of apical portion of implant, and subsequent periapical radiographs taken demonstrated a radiolucent lesion. On the basis of case reports analyzed, RPI was diagnosed between 1 week and 4 years after implant placement. Twelve of 20 studies reported that RPI lesions were diagnosed within 6 months after implant placement. A step‐by‐step decision tree is provided to allow clinicians to triage and properly manage cases of RPI on the basis of recommendations and successful treatments provided in analyzed case reports. It is divided between symptomatic and asymptomatic implants and adjacent teeth with vital and necrotic pulps. Conclusions: Most common etiology of apical RPI is endodontic infection from neighboring teeth, which was diagnosed within 6 months after implant placement. Most common findings, radiographically and clinically, are lesions around implant apex and sinus tract. A small number of implants did not improve with treatment. Decision tree provides a path to diagnose and treat lesions to facilitate their management. Further studies are needed to focus on histologic data around periapical microbiota to establish specific etiology and differential diagnoses compared with marginal peri‐implantitis and other implant‐related conditions.