Medication‐related osteonecrosis of the jaw (MRONJ) is a rare but serious drug‐related adverse event. To identify pharmacogenomic markers of MRONJ associated with bisphosphonate therapy, we conducted ...a genomewide association study (GWAS) meta‐analysis followed by functional analysis of 5,008 individuals of European ancestry treated with bisphosphonates, which includes the largest number of MRONJ cases to date (444 cases and 4,564 controls). Discovery GWAS was performed in randomly selected 70% of the patients with cancer and replication GWAS was performed in the remaining 30% of the patients with cancer treated with intravenous bisphosphonates followed by meta‐analysis of all 3,639 patients with cancer. GWAS was also performed in 1,369 patients with osteoporosis treated with oral bisphosphonates. The lead single‐nucleotide polymorphism (SNP), rs2736308 on chromosome 8, was associated with an increased risk of MRONJ with an odds ratio (OR) of 2.71 and 95% confidence interval (CI) of 1.90–3.86 (P = 3.57*10−8) in the meta‐analysis of patients with cancer. This SNP was validated in the MRONJ GWAS in patients with osteoporosis (OR: 2.82, 95% CI: 1.55–4.09, P = 6.84*10−4). The meta‐analysis combining patients with cancer and patients with osteoporosis yielded the same lead SNP rs2736308 on chromosome 8 as the top SNP (OR: 2.74, 95% CI: 2.09–3.39, P = 9.65*10−11). This locus is associated with regulation of the BLK, CTSB, and FDFT1 genes, which had been associated with bone mineral density. FDFT1 encodes a membrane‐associated enzyme, which is implicated in the bisphosphonate pathway. This study provides insights into the potential mechanism of MRONJ.
To carry out an immunoassay analysis of biomarkers expressed in gingival crevicular fluid (GCF) with the main goal of finding a useful diagnostic pattern to distinguish between resorbing deciduous ...teeth and nonresorbing controls.
A split-mouth design was used in this study with a total of 22 GCF samples collected from 11 patients in the mixed dentition. For each child, one deciduous molar with radiographic evidence of root resorption was used as the test tooth whereas the contralateral first permanent molar with formed roots was used as the control tooth. Samples were processed with immunoassays using a panel of selected biomarkers including interleukin-1 beta (IL-1b), interleukin-1 receptor antagonist (IL-1RA), nuclear factor kappa B ligand (RANKL), osteoprotegerin (OPG), matrix metalloproteinase-9 (MMP-9), and dentin sialoprotein (DSP).
There were no statistically significant differences in levels of IL-1b, OPG, and MMP-9 between test and control sites (P > .05). IL-1RA was the only biomarker to show a significant down-regulation (P = .04) in GCF samples collected from resorbing teeth. RANKL data showed a heavily skewed distribution and was deemed unreliable. Only one deciduous GCF sample had detectable levels of DSP; therefore, no further statistical calculation was applicable because of the limited amount of data for this biomarker.
This study indicated that IL1-RA is down-regulated in GCF from resorbing primary molars, thus suggesting this cytokine as a potential analyte to be included in a panel that can discriminate between resorbing and nonresorbing teeth.
Vacuolar H+-ATPases (V-ATPases) are a family of highly conserved proton pumps that couple hydrolysis of cytosolic ATP to proton transport out of the cytosol. How ATP is supplied for V-ATPase-mediated ...hydrolysis and for coupling of proton transport is poorly understood. We have reported that the glycolytic enzyme aldolase physically associates with V-ATPase (Lu, M., Holliday, L. S., Zhang, L., Dunn, W. A., and Gluck, S. L. (2001) J. Biol. Chem. 276, 30407–30413). Here we show that aldolase interacts with three different subunits of V-ATPase (subunits a, B, and E). The binding sites for the V-ATPase subunits on aldolase appear to be on distinct interfaces of the glycolytic enzyme. Aldolase deletion mutant cells were able to grow in medium buffered at pH 5.5 but not at pH 7.5, displaying a growth phenotype similar to that observed in V-ATPase subunit deletion mutants. Abnormalities in V-ATPase assembly and protein expression observed in aldolase deletion mutant cells could be fully rescued by aldolase complementation. The interaction between aldolase and V-ATPase increased dramatically in the presence of glucose, suggesting that aldolase may act as a glucose sensor for V-ATPase regulation. Taken together, these findings provide functional evidence that the ATP-generating glycolytic pathway is directly coupled to the ATP-hydrolyzing proton pump through physical interaction between aldolase and V-ATPase.
α1-Antitrypsin deficiency (AATD) is an inherited disease characterized by emphysema and liver disease. AATD is most often caused by a single amino acid substitution at position 342 in the mature ...protein, resulting in the Z mutation of the AAT gene (ZAAT). This substitution is associated with misfolding and accumulation of ZAAT in the endoplasmic reticulum (ER) of hepatocytes, causing a toxic gain of function. ERdj3 is an ER luminal DnaJ homologue, which, along with calreticulin, directly interacts with misfolded ZAAT. We hypothesize that depletion of each of these chaperones will change the fate of ZAAT polymers. Our study demonstrates that calreticulin modulation reveals a novel ZAAT degradation mechanism mediated by exosomes. Using human PiZZ hepatocytes and K42, a mouse calreticulin-deficient fibroblast cell line, our results show ERdj3 and calreticulin directly interact with ZAAT in PiZZ hepatocytes. Silencing calreticulin induces calcium independent ZAAT–ERdj3 secretion through the exosome pathway. This co-secretion decreases ZAAT aggregates within the ER of hepatocytes. We demonstrate that calreticulin has an inhibitory effect on exosome-mediated ZAAT–ERdj3 secretion. This is a novel ZAAT degradation process that involves a DnaJ homologue chaperone bound to ZAAT. In this context, calreticulin modulation may eliminate the toxic gain of function associated with aggregation of ZAAT in lung and liver, thus providing a potential new therapeutic approach to the treatment of AATD-related liver disease.
Structured
Objectives
Orthodontic treatment consists of numerous appliance activations that rely on stimulation of osteoclasts at alveolar bone sites. However, the action of osteoclast‐like cells on ...dentin (“odontoclasts”) is a pathological side effect of orthodontic treatment. The aim of this article is twofold: (a) To report preliminary results from ongoing cell culture experiments to identify unique markers of dentin resorption, and (b) To discuss our work using nanoparticle tracking analysis (NTA) and exosomes for developing biological fluid‐based biopsies to monitor clastic cell activity.
Setting and sample population
Twelve healthy volunteers in permanent dentition.
Material and methods
For the in vitro experiments, murine clastic cell precursors were cultured on dentin or bone slices for 7 days and phage‐display biopanning was used to identify molecular surface differences between osteoclasts and odontoclasts. In the human study, gingival crevicular fluid (GCF) samples were collected using different tools and analysed for protein and exosome recovery.
Results
Biopanning generated antibody fragments that were uniquely reactive to odontoclasts. Numerous nanoparticles in the size range of exosomes were detected in all of the human GCF samples.
Conclusions
Our results support that there are molecular differences between osteoclasts and odontoclasts. Emerging technologies may allow the use of exosomes in GCF as a clinical tool to detect markers of root resorption.
Several members of the SLC26A family of transporters, including SLC26A3 (DRA), SLC26A5 (prestin), SLC26A6 (PAT-1; CFEX) and SLC26A9, form multi-protein complexes with a number of molecules (e.g., ...cytoskeletal proteins, anchoring or adaptor proteins, cystic fibrosis transmembrane conductance regulator, and protein kinases). These interactions provide regulatory signals for these molecules. However, the identity of proteins that interact with the Cl
/HCO
exchanger, SLC26A4 (pendrin), have yet to be determined. The purpose of this study is to identify the protein(s) that interact with pendrin.
A yeast two hybrid (Y2H) system was employed to screen a mouse kidney cDNA library using the C-terminal fragment of SLC26A4 as bait. Immunofluorescence microscopic examination of kidney sections, as well as co-immunoprecipitation assays, were performed using affinity purified antibodies and kidney protein extracts to confirm the co-localization and interaction of pendrin and the identified binding partners. Co-expression studies were carried out in cultured cells to examine the effect of binding partners on pendrin trafficking and activity.
The Y2H studies identified IQ motif-containing GTPase-activating protein 1 (IQGAP1) as a protein that binds to SLC26A4's C-terminus. Co-immunoprecipitation experiments using affinity purified anti-IQGAP1 antibodies followed by western blot analysis of kidney protein eluates using pendrin-specific antibodies confirmed the interaction of pendrin and IQGAP1. Immunofluorescence microscopy studies demonstrated that IQGAP1 co-localizes with pendrin on the apical membrane of B-intercalated cells, whereas it shows basolateral expression in A-intercalated cells in the cortical collecting duct (CCD). Functional and confocal studies in HEK-293 cells, as well as confocal studies in MDCK cells, demonstrated that the co-transfection of pendrin and IQGAP1 shows strong co-localization of the two molecules on the plasma membrane along with enhanced Cl
/HCO
exchanger activity.
IQGAP1 was identified as a protein that binds to the C-terminus of pendrin in B-intercalated cells. IQGAP1 co-localized with pendrin on the apical membrane of B-intercalated cells. Co-expression of IQGAP1 with pendrin resulted in strong co-localization of the two molecules and increased the activity of pendrin in the plasma membrane in cultured cells. We propose that pendrin's interaction with IQGAP1 may play a critical role in the regulation of CCD function and physiology, and that disruption of this interaction could contribute to altered pendrin trafficking and/or activity in pathophysiologic states.
Periodontal diseases are multifactorial, caused by polymicrobial subgingival pathogens, including Porphyromonas gingivalis, Treponema denticola, and Tannerella forsythia. Chronic periodontal ...infection results in inflammation, destruction of connective tissues, periodontal ligament, and alveolar bone resorption, and ultimately tooth loss. Enoxacin and a bisphosphonate derivative of enoxacin (bis-enoxacin) inhibit osteoclast formation and bone resorption and also contain antibiotic properties. Our study proposes that enoxacin and/or bis-enoxacin may be useful in reducing alveolar bone resorption and possibly bacterial colonization. Rats were infected with 10(9) cells of polymicrobial inoculum consisting of P. gingivalis, T. denticola, and T. forsythia, as an oral lavage every other week for twelve weeks. Daily subcutaneous injections of enoxacin (5 mg/kg/day), bis-enoxacin (5, 25 mg/kg/day), alendronate (1, 10 mg/kg/day), or doxycycline (5 mg/day) were administered after 6 weeks of polymicrobial infection. Periodontal disease parameters, including bacterial colonization/infection, immune response, inflammation, alveolar bone resorption, and systemic spread, were assessed post-euthanasia. All three periodontal pathogens colonized the rat oral cavity during polymicrobial infection. Polymicrobial infection induced an increase in total alveolar bone resorption, intrabony defects, and gingival inflammation. Treatment with bis-enoxacin significantly decreased alveolar bone resorption more effectively than either alendronate or doxycycline. Histologic examination revealed that treatment with bis-enoxacin and enoxacin reduced gingival inflammation and decreased apical migration of junctional epithelium. These data support the hypothesis that bis-enoxacin and enoxacin may be useful for the treatment of periodontal disease.
Celotno besedilo
Dostopno za:
DOBA, IZUM, KILJ, NUK, PILJ, PNG, SAZU, SIK, UILJ, UKNU, UL, UM, UPUK
Binding between vacuolar H+-ATPases (V-ATPases) and microfilaments is mediated by an actin binding domain in the B-subunit. Both isoforms of mammalian B-subunit bind microfilaments with high ...affinity. A similar actinbinding activity has been demonstrated in the B-subunit of yeast. A conserved "profilin-like" domain in the B-subunit mediates this actin-binding activity, named due to its sequence and structural similarity to an actin-binding surface of the canonical actin binding protein profilin. Subtle mutations in the "profilin-like" domain eliminate actin binding activity without disrupting the ability of the altered protein to associate with the other subunits of V-ATPase to form a functional proton pump. Analysis of these mutated B-subunits suggests that the actin-binding activity is not required for the "housekeeping" functions of V-ATPases, but is important for certain specialized roles. In osteoclasts, the actin-binding activity is required for transport of V-ATPases to the plasma membrane, a prerequisite for bone resorption. A virtual screen led to the identification of enoxacin as a small molecule that bound to the actin-binding surface of the B2-subunit and competitively inhibited B2-subunit and actin interaction. Enoxacin disrupted osteoclastic bone resorption in vitro, but did not affect osteoblast formation or mineralization. Recently, enoxacin was identified as an inhibitor of the virulence of Candida albicans and more importantly of cancer growth and metastasis. Efforts are underway to determine the mechanisms by which enoxacin and other small molecule inhibitors of B2 and microfilament binding interaction selectively block bone resorption, the virulence of Candida, cancer growth, and metastasis.
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