The Serpentine Sign: A Reliable Endoscopic and Radiographic Finding in Empty Nose Syndrome.

OBJECTIVE: Empty nose syndrome (ENS) is a relatively uncommon disease that greatly impacts the quality of life and presents diagnostic challenges. We sought to identify objective clinical findings unique to patients with ENS, and in doing so identified compensatory mucosal hypertrophy in an alternating, undulating swelling on endoscopy and coronal computerized tomography (CT) that we have termed the "Serpentine Sign." Here, we investigated whether this radiographic finding is a reliable manifestation in ENS patients. METHODS: Retrospective review was undertaken to identify ENS patients with past turbinoplasty, an ENS6Q score of at least 11/30, and symptomatic improvement with the cotton placement test. Control patients without complaints of ENS symptoms (ENS6Q < 11) were identified for comparison. ENS and control patients had coronal CT imaging available to evaluate for the Serpentine Sign, as well as ENS6Q scores, and histologic analysis of nasal tissue. RESULTS: 34 ENS and 74 control patients were evaluated for the presence of the Serpentine Sign. Of the 34 patients with ENS, 18 exhibited this radiographic finding on CT imaging (52.9%) compared to 0 of the 74 control patients (p < 0.0001). Further analysis demonstrated that ENS patients with the Serpentine Sign had lower median scores on ENS6Q than ENS patients without (17.5 vs. 22, p = 0.033). Histology revealed disorganized subepithelium rich in seromucinous glands similar to the nasal septum swell body. CONCLUSION: The "Serpentine Sign" is a unique presentation of hypertrophic change to the nasal septum soft tissue that is specific to ENS patients and may serve as a reliable radiographic and endoscopic finding in diagnosis. LEVEL OF EVIDENCE: 4 Laryngoscope, 134:1089-1095, 2024.

Targeted replacement of full-length CFTR in human airway stem cells by CRISPR-Cas9 for pan-mutation correction in the endogenous locus.

Cystic fibrosis (CF) is a monogenic disease caused by impaired production and/or function of the CF transmembrane conductance regulator (CFTR) protein. Although we have previously shown correction of the most common pathogenic mutation, there are many other pathogenic mutations throughout the CF gene. An autologous airway stem cell therapy in which the CFTR cDNA is precisely inserted into the CFTR locus may enable the development of a durable cure for almost all CF patients, irrespective of the causal mutation. Here, we use CRISPR-Cas9 and two adeno-associated viruses (AAVs) carrying the two halves of the CFTR cDNA to sequentially insert the full CFTR cDNA along with a truncated CD19 (tCD19) enrichment tag in upper airway basal stem cells (UABCs) and human bronchial epithelial cells (HBECs). The modified cells were enriched to obtain 60%-80% tCD19+ UABCs and HBECs from 11 different CF donors with a variety of mutations. Differentiated epithelial monolayers cultured at air-liquid interface showed restored CFTR function that was >70% of the CFTR function in non-CF controls. Thus, our study enables the development of a therapy for almost all CF patients, including patients who cannot be treated using recently approved modulator therapies.

High-Efficiency, Selection-free Gene Repair in Airway Stem Cells from Cystic Fibrosis Patients Rescues CFTR Function in Differentiated Epithelia.

Cystic fibrosis (CF) is a monogenic disorder caused by mutations in the Cystic Fibrosis Transmembrane Conductance Regulator (CFTR) gene. Mortality in CF patients is mostly due to respiratory sequelae. Challenges with gene delivery have limited attempts to treat CF using in vivo gene therapy, and low correction levels have hindered ex vivo gene therapy efforts. We have used Cas9 and adeno-associated virus 6 to correct the ΔF508 mutation in readily accessible upper-airway basal stem cells (UABCs) obtained from CF patients. On average, we achieved 30%-50% allelic correction in UABCs and bronchial epithelial cells (HBECs) from 10 CF patients and observed 20%-50% CFTR function relative to non-CF controls in differentiated epithelia. Furthermore, we successfully embedded the corrected UABCs on an FDA-approved porcine small intestinal submucosal membrane (pSIS), and they retained differentiation capacity. This study supports further development of genetically corrected autologous airway stem cell transplant as a treatment for CF.

Topical Corticosteroid Pretreatment Mitigates Cellular Damage After Caustic Injury to the Nasal Upper Airway Epithelium.

BACKGROUND: Topical corticosteroids are currently employed to reduce established airway inflammation; their prophylactic use might help limit cellular damage against harmful stimuli. OBJECTIVES: To determine the effects of a prophylactic topical application of budesonide (BD) on an in vivo nasal epithelium injury model induced by trichloroacetic acid (TCA). METHODS: C57Bl/6 mice were exposed to intranasal TCA topical application. Three groups received topical intranasal BD, saline solution, or no intervention prior to a single topical exposure to TCA. Controls were not exposed to TCA. Whole nasal cavity coronal sections were analyzed at 1, 3, and 6 days postinjury at tissue and cellular levels using histopathological analysis, immunofluorescent staining, and fresh tissue RNA microarray analysis. RESULTS: Prophylactic topical corticosteroid exposure protected the nasal epithelium from acute damage, maintaining epithelial thickness and cell survival. Six days following TCA exposure, epithelial and cellular changes were less pronounced on the BD-treated group compared to all exposure groups. The microarray analysis was used to evaluate the gene transcripts in all treatment groups. Ciliary tip protein, Sentan, and submucosal protein S100b were identified as potential factors in epithelial airway protection; immunofluorescent staining corroborated their presence and location within the respiratory epithelium. CONCLUSION: Topical corticosteroid treatment to the nasal epithelium can mitigate several of the early deleterious effects of acute epithelial damage in experimental airway injuries caused by TCA. These findings suggest a novel, direct cytoprotective effect of corticosteroids on the nasal epithelium, and the potential of expanding the use of prophylactic periprocedural topical corticosteroids for respiratory epithelial tissues.

Porcine small intestine submucosal grafts improve remucosalization and progenitor cell recruitment to sites of upper airway tissue remodeling.

BACKGROUND: To better understand upper airway tissue regeneration, the exposed cartilage and bone at donor sites of tissue flaps may serve as in vivo "Petri dishes" for active wound healing. The pedicled nasoseptal flap (NSF) for skull-base reconstruction creates an exposed donor site within the nasal airway. The objective of this study is to evaluate whether grafting the donor site with a sinonasal repair cover graft is effective in promoting wound healing. METHODS: In this multicenter, prospective trial, subjects were randomized to intervention (graft) or control (no graft) intraoperatively after NSF elevation. Individuals were evaluated at 2, 6, and 12 weeks postintervention with endoscopic recordings. Videos were graded (Likert scale) by 3 otolaryngologists blinded to intervention on remucosalization, crusting, and edema. Scores were analyzed for interrater reliability and cohorts compared. Biopsy and immunohistochemistry at the leading edge of wound healing was performed in select cases. RESULTS: Twenty-one patients were randomized to intervention and 26 to control. Subjects receiving the graft had significantly greater overall remucosalization (p = 0.01) than controls over 12 weeks. Although crusting was less in the small intestine submucosa (SIS) group, this was not statistically significant (p = 0.08). There was no overall effect on nasal edema (p = 0.2). Immunohistochemistry demonstrated abundant upper airway basal cell progenitors in 2 intervention samples, suggesting that covering grafts may facilitate tissue proliferation via progenitor cell expansion. CONCLUSION: This prospective, randomized, controlled trial indicates that a porcine SIS graft placed on exposed cartilage and bone within the upper airway confers improved remucosalization compared to current practice standards.

Selective expansion of human regulatory T cells in nasal polyps, and not adjacent tissue microenvironments, in individual patients exposed to steroids.

Severe forms of chronic rhinosinusitis (CRS), a common upper airway inflammatory disorder, are associated with nasal polyps (NPs). NP disease is ameliorated by glucocorticoid (GC) treatment, whose cellular effects are poorly understood. We therefore assessed the influence of GC therapy on NPs in CRS patients, focusing on regulatory T (Treg) cells. Treg cell populations were analyzed by flow cytometry in NPs and control tissues from GC-treated CRS patients and controls. After GC exposure, selective expansion of Treg cells was seen within NPs, and not blood or adjacent ethmoid tissues. To confirm direct GC effects, NPs from the same patients were biopsied prior to, and following, 1week of oral GC exposure. Direct expansion of Tregs into the same NP bed was detected in 4/4 CRS patients following GC exposure. Treg cell spikes into NPs were secondary to cellular recruitment given limited Ki67 expression within these regulatory cells. Chemokine gene expression profiling identified several chemokines, notably CCL4, induced within NPs upon GC treatment. Neutralization of chemokine receptor/ligand interactions using CCR4 small molecule antagonists reduced Treg migration towards GC-treated NPs in an ex vivo migration assay. Our findings suggest that the common use of GCs in the treatment of NP disease leads to recruitment of Treg cells from peripheral sites into NP tissues, which may be critical to the anti-inflammatory effect of GCs. Mechanistically Treg expansion appears to be conferred, in part, by chemokine receptor/ligand interactions induced following corticosteroid therapy.

Air pollutants cause release of hydrogen peroxide and interleukin-8 in a human primary nasal tissue culture model.

BACKGROUND: A component of primary innate defense of the nasal mucosa against inhaled pathogens includes continuous, low-level release of hydrogen peroxide (H2 O2 ) into luminal secretions. Epidemiologically, an association exists between poor air quality and increased prevalence of sinonasal disease. To understand the effects of particulate matter (PM) in nasal mucosa, we studied the release of H2 O2 and interleukin 8 (IL-8) after PM exposure. METHODS: Human nasal specimens were collected from surgery and cultured in serum-free growth medium. Cell integrity and recovery during culture was monitored by lactate dehydrogenase (LDH) release into the medium. Cultures were exposed to PM for 24 hours in the presence/absence of diphenyleneiodonium sulfate (DPI; a nicotinamide adenine dinucleotide phosphate [NADPH] oxidase inhibitor). Luminex cytokine and Amplex-Red H2 O2 assays were performed. RESULTS: LDH levels dropped rapidly within 2 days, indicative of stabilization and cell recovery after harvest. All cultures released H2 O2 into the medium. Exposure to PM (20 µg/cm(2) ) increased H2 O2 levels significantly (94.6 ± 7.7 nM) compared to untreated controls (55.8 ± 4.0 nM; p = 0.001). PM-induced H2 O2 production was partially inhibited by DPI (80.1 ± 3.8nM), indicating that cellular NADPH oxidase may be a primary source of H2 O2 production. Exposure to PM increased IL-8 levels in a dose-dependent fashion (control = 2301 ± 412 MFI; 20 µg/cm(2) = 5002 ± 1327 MFI; 40 µg/cm(2) = 8219 ± 1090 MFI; p = 0.022). CONCLUSION: PM increases the quantity of H2 O2 released by nasal epithelial cells, indicating that PM can contribute to oxidative stress in part by activating a normal cellular defense mechanism. Exposure to PM resulted in elevated IL-8 levels and mucin production in explants. Efforts to reduce airborne PM may lead to reduced H2 O2 and mucin production in sinonasal epithelium.

Human ethmoid sinus mucosa: a promising novel tissue source of mesenchymal progenitor cells.

INTRODUCTION: The identification of new progenitor cell sources is important for cell-based tissue engineering strategies, understanding regional tissue regeneration, and modulating local microenvironments and immune response. However, there are no reports that describe the identification and isolation of mesenchymal progenitor cells (MPCs) from paranasal sinus mucosa, and compare the properties of MPCs between tissue sources within the sinonasal cavity. We report here the identification of MPCs in the maxillary sinus (MS) and ethmoid sinus (ES). Furthermore, we contrast these MPCs in the same individuals with MPCs from two additional head and neck tissue sources of the inferior turbinate (IT) and tonsil (T). METHODS: These four MPC sources were exhaustively compared for morphology, colony-forming potential, proliferation capability, immunophenotype, multilineage differentiation potential, and ability to produce soluble factors. RESULTS: MS-, ES, IT-, and T-MPCs showed similar morphologies and surface phenotypes, as well as adipogenic, osteogenic, and chondrogenic differentiation capacity by immunohistochemistry and qRT-PCR for defined lineage-specific genes. However, we noted that the colony-forming potential and proliferation capability of ES-MPCs were distinctly higher than other MPCs. All MPCs constitutively, or upon stimulation, secrete large amounts of IL-6, IL-8, IL-10, IFN-γ, and TGF-ß. After stimulation with TNF-α and IFN-γ, ES-MPCs notably demonstrated significantly higher secretion of IL-6 and IL-10 than other MPCs. CONCLUSIONS: ES-MPCs may be a uniquely promising source of MPCs due to their high proliferation ability and superior capacity toward secretion of immunomodulatory cytokines.

Characterization of human upper airway epithelial progenitors.

BACKGROUND: New epithelial cells are generated through the proliferation and differentiation of resident progenitor cells in the nasal cavity. In several upper airway diseases, such as cystic fibrosis and chronic rhinosinusitis, self-renewing progenitor cells may be functionally defective, or compromised in their ability, to regenerate cells that maintain normal mucociliary clearance. Herein, we describe our early work to define and characterize a rare population of human nasal epithelial putative progenitors. METHODS: Single-cell suspensions of human ethmoid sinus tissues were prepared following endoscopic sinus surgery. Cell surface antibodies were analyzed as candidate markers for detecting progenitor cells. A panel of antibodies, including epithelial cell adhesion molecule (EpCAM, epithelial cells), CD45 (hematopoietic cells), nerve growth factor receptor (NGFR/CD271), intercellular adhesion molecule-1 (ICAM1/CD54), and integrin-α6 (ITGA6/CD49f) were used to resolve epithelial progenitor candidates by high-dimensional flow cytometry and the gating technique of fluorescence minus one (FMO) controls. RESULTS: A rare population of approximately 0.06% of total ethmoid cells was discriminated as EpCAM(-) CD45(-) NGFR(+) ICAM1(+) by surface markers. Use of ITGA6 was excluded based on FMO control analysis. This lineage-negative population was purified to 99% homogeneity by cell sorting and analyzed by immunofluorescence microscopy. Sorted cells were subsequently confirmed to uniformly express the transcription factor p63. Upon in vitro culture, lineage-negative clonal cells were confirmed to spontaneously differentiate into epithelial lineage-positive cells. CONCLUSION: Using the NGFR and ICAM1 cellular coordinates, we have identified a promising population of native human nasal epithelial progenitor cells that require more formal investigation for their role in upper airway regeneration.

Frizzled-8 receptor is activated by the Wnt-2 ligand in non-small cell lung cancer.

BACKGROUND: Wnt-2 plays an oncogenic role in cancer, but which Frizzled receptor(s) mediates the Wnt-2 signaling pathway in lung cancer remains unclear. We sought to (1) identify and evaluate the activation of Wnt-2 signaling through Frizzled-8 in non-small cell lung cancer, and (2) test whether a novel expression construct dominant negative Wnt-2 (dnhWnt-2) reduces tumor growth in a colony formation assay and in a xenograft mouse model. METHODS: Semi-quantitative RT-PCR was used to identify the expression of Wnt-2 and Frizzled-8 in 50 lung cancer tissues from patients. The TCF reporter assay (TOP/FOP) was used to detect the activation of the Wnt canonical pathway in vitro. A novel dnhWnt-2 construct was designed and used to inhibit activation of Wnt-2 signaling through Frizzled-8 in 293T, 293, A549 and A427 cells and in a xenograft mouse model. Statistical comparisons were made using Student's t-test. RESULTS: Among the 50 lung cancer samples, we identified a 91% correlation between the transcriptional increase of Wnt-2 and Frizzled-8 (p<0.05). The Wnt canonical pathway was activated when both Wnt-2 and Frizzled-8 were co-expressed in 293T, 293, A549 and A427 cells. The dnhWnt-2 construct we used inhibited the activation of Wnt-2 signaling in 293T, 293, A549 and A427 cells, and reduced the colony formation of NSCLC cells when ß-catenin was present (p<0.05). Inhibition of Wnt-2 activation by the dnhWnt-2 construct further reduced the size and mass of tumors in the xenograft mouse model (p<0.05). The inhibition also decreased the expression of target genes of Wnt signaling in these tumors. CONCLUSIONS: We demonstrated an activation of Wnt-2 signaling via the Frizzled-8 receptor in NSCLC cells. A novel dnhWnt-2 construct significantly inhibits Wnt-2 signaling, reduces colony formation of NSCLC cells in vitro and tumor growth in a xenograft mouse model. The dnhWnt-2 construct may provide a new therapeutic avenue for targeting the Wnt pathway in lung cancer.

Expression of dual oxidases and secreted cytokines in chronic rhinosinusitis.

BACKGROUND: The airway epithelium generates reactive oxygen species (ROS) as a first line of defense. Dual oxidases (DUOX1 and DUOX2) are the H2 O2 -producing isoforms of the nicotinamide adenine dinucleotide phosphate (NADPH) oxidase family in the airway epithelium. The purpose of this study was to explore the molecular expression, function, and regulation of DUOXs in chronic rhinosinusitis (CRS). METHODS: Human nasal tissue samples and nasal secretions were collected from 3 groups of patients undergoing sinus surgery (normal, n = 7; CRS with polyposis [CRSwP], n = 6; CRS without polyposis [CRSsP], n = 6). Nasal secretions were studied for cytokine and H2 O2 content. Tissue samples were used to determine DUOX mRNA and protein expression. RESULTS: DUOX1 mRNA level (80.7 ± 60.5) was significantly increased in CRSwP compared to normal (2.7 ± 1.2) and CRSsP (2.3 ± 0.5, p = 0.042). DUOX2 mRNA levels were increased in both CRSwP (18.6 ± 9.9) and CRSsP (4.0 ± 1.3) compared to normal (1.1 ± 0.3; p = 0.008). DUOX protein was found in the apical portion of the nasal epithelium and protein expression was increased in CRSwP and CRSsP. H2 O2 production was significantly higher in CRSwP (160.9 ± 59.4 nM) and CRSsP (81.7 ± 5.6 nM) compared to normal (53.5 ± 11.5 nM, p = 0.032). H2 O2 content of nasal secretions correlated tightly with DUOX expression (p < 0.001). Cytokines (eotaxin, monokine-induced by interferon γ [MIG], tumor necrosis factor [TNF]-α, interleukin [IL]-8) showed significantly higher levels in nasal secretions from CRSwP compared to normal (p < 0.05). Levels of eotaxin, MIG, and TNF-α correlated closely with DUOX expression. CONCLUSION: DUOX1 and DUOX2 were identified as factors upregulated in CRS. Close correlations between DUOX expression and H2 O2 release, and correlation between key inflammatory cytokines and DUOX expression, indicate DUOX in the inflammatory response in CRS.

Low-frequency pulsed ultrasound in the nasal cavity and paranasal sinuses: a feasibility and distribution study.

BACKGROUND: Bacterial biofilms have been implicated in refractory rhinosinusitis. Biofilms have been shown to respond to treatment with low-frequency ultrasound (LFU) therapy in vitro, and exposure to LFU has shown efficacy in wound repair and topical drug delivery in other fields. This preliminary study was designed to evaluate the safety and feasibility of LFU for use in the nasal cavity and paranasal sinuses. METHODS: This was an experimental observational study. Six cadaver heads were used to deliver a mixture of Renografin and methylene blue solvent to the paranasal sinuses via LFU both before and after resident endoscopic sinus dissection. Sinus computed tomography (CT) scans of the cadaver heads were performed before and after mixture delivery, and blinded assessments were made for distribution to individual sinuses. Mucosa was harvested from 2 subsites to evaluate LFU-treated cadaver tissue. RESULTS: Predissection, LFU delivered solution to 12 of 12 inferior and middle turbinates, 6 of 12 of the superior turbinates and ethmoid sinuses, and 1 of 12 maxillary sinuses as shown by contrast radiography. Postdissection, all heads showed delivery to the maxillary and sphenoid sinuses, with 8 of 12 sinus cavities showing delivery to the ethmoid region, and 4 of 11 to the frontal recess. Using hematoxylin and eosin (H&E) staining of tissue frozen sections, harvested tissue demonstrated no architectural damage to the mucosal layer from LFU exposure. CONCLUSION: LFU appears to be capable of reliably delivering topical solution to the turbinates and ethmoid region preoperatively and to all sinuses, except the frontal, postoperatively. The nasal epithelium does not appear to be disrupted histologically from LFU at this time and distance. This data provides a foundation for a prospective human protocol studying the efficacy of this modality in the treatment of patients with chronic rhinosinusitis and biofilm formation.

Inhibition of Hsp90 leads to cell cycle arrest and apoptosis in human malignant pleural mesothelioma.

INTRODUCTION: Heat shock protein 90 (Hsp90) is an abundant molecular chaperone that mediates the maturation and stability of a variety of proteins associated with the promotion of cell growth and survival. Inhibition of Hsp90 function leads to proteasomal degradation of its mis-folded client proteins. Recently, Hsp90 has emerged as being of prime importance to the growth and survival of cancer cells and its inhibitors have already been used in phase I and II clinical trials. METHODS: We investigated how 17-allylamino-17-demethoxygeldanamycin (17-AAG), a small molecule inhibitor of Hsp90, is implicated in human malignant pleural mesothelioma (MM). RESULTS: We found that 17-AAG led to significant G1 or G2/M cell cycle arrest, inhibition of cell proliferation, and decrease of AKT, AKT1, and survivin expression in all human malignant pleural mesothelioma cell lines examined. We also observed significant apoptosis induction in all MM cell lines treated with 17-AAG. Furthermore, 17-AAG induced apoptosis in freshly cultured primary MM cells and caused signaling changes identical to those in 17-AAG treated MM cell lines. CONCLUSION: These results suggest that Hsp90 is strongly associated with the growth and survival of MM and that inhibition of Hsp90 may have therapeutic potential in the treatment of MM.

Inhibition of Wnt-2 and galectin-3 synergistically destabilizes beta-catenin and induces apoptosis in human colorectal cancer cells.

Constitutive activation of the Wnt pathway as a result of APC, AXIN1 or CTNNB1 mutations has been found in most colorectal cancers. For a long time, this aberrant Wnt activation has been thought to be independent of upstream signals. However, recent studies indicate that upstream signals retain their ability to regulate the Wnt pathway even in the presence of downstream mutations. Wnt-2 is well known for its overexpression in colorectal cancer. Galectin-3 (Gal-3), a multifunctional carbohydrate binding protein implicated in a variety of biological functions, has recently been reported to interact with beta-catenin. In this study, we investigated roles of Wnt-2 and Gal-3 in the regulation of canonical Wnt/beta-catenin signaling. We found that siRNA silencing of either Wnt-2 or Gal-3 expression inhibited TCF-reporter activity, decreased cytosolic beta-catenin level and induced apoptosis in human colorectal cancer cells containing downstream mutations. More interestingly, we showed that inhibition of both Wnt-2 and Gal-3 had synergistic effects on suppressing canonical Wnt signaling and inducing apoptosis, suggesting that aberrant canonical Wnt/beta-catenin signaling in colorectal cancer can be regulated at multiple levels. The combined inhibition of Wnt-2 and Gal-3 may be of superior therapeutic advantage to inhibition by either one of them, giving rise to a potential development of novel drugs for the targeted treatment of colorectal cancer.

Mutational and pH analysis of ionic residues in transmembrane domains of vesicular acetylcholine transporter.

This research investigated the roles of 7 conserved ionic residues in the 12 putative transmembrane domains (TMDs) of vesicular acetylcholine transporter (VAChT). Rat VAChT in wild-type and mutant forms was expressed in PC12(A123.7) cells. Transport and ligand binding were characterized at different pH values using filter assays. The ACh binding site is shown to exhibit high or low affinity (K(d) values are approximately 10 and 200 mM, respectively). Mutation of the lysine and aspartate residues in TMDs II and IV, respectively, can decrease the fraction of sites having high affinity. In three-dimensional structures of related transporters, these TMDs lie next to each other and distantly from TMDs VIII and X, which probably contain the binding sites for ACh and the allosteric inhibitor vesamicol. Importantly, mutation of the aspartate in TMD XI can create extra-high affinities for ACh (K(d) approximately 4 mM) and vesamicol (K(d) approximately 2 nM compared to approximately 20 nM). Effects of different external pH values on transport indicate a site that must be protonated (apparent pK(a) approximately 7.6) likely is the aspartate in TMD XI. The observations suggest a model in which the known ion pair between lysine in TMD II and aspartate in TMD XI controls the conformation or relative position of TMD XI, which in turn controls additional TMDs in the C-terminal half of VAChT. The pH effects also indicate that sites that must be unprotonated for transport (apparent pK(a) approximately 6.4) and vesamicol binding (apparent pK(a) approximately 6.3) remain unidentified.

New transport assay demonstrates vesicular acetylcholine transporter has many alternative substrates.

The acetylcholine-binding site in vesicular acetylcholine transporter faces predominantly toward the outside of the vesicle when resting but predominantly toward the inside when transporting. Transport-related reorientation is detected by an ATP-induced decrease in the ability of saturating substrate to displace allosterically bound [(3)H]vesamicol. The assay was used here to determine whether structurally diverse compounds are transported by rat VAChT expressed in PC12(A123.7) cells. Competition by ethidium, tetraphenylphosphonium and other monovalent organic cations with [(3)H]vesamicol is decreased when ATP is added, and the effect depends on proton-motive force. The results indicate that many organic molecules carrying +1 charge are transported, even though the compounds do not resemble acetylcholine in structural details.

Choline is transported by vesicular acetylcholine transporter.

Previously published results appeared to show that vesicular acetylcholine transporter (VAChT) does not transport choline (Ch). Because it is uniquely suited to detect transport of weakly bound substrates, a recently developed assay that detects transmembrane reorientation of the substrate binding site was used to re-examine transport selectivity. Rat VAChT was expressed in PC12(A1237) cells, postnuclear supernatant-containing microvesicles was prepared, and the reorientation assay was conducted with unlabeled Ch and tetramethylammonium (TMA). Also, [(14)C]Ch and [(3)H]acetylcholine (ACh) were used in an optimized accumulation assay. The results demonstrate that Ch is transported at least as well as ACh is, but with sevenfold lower affinity. Even TMA is transported, but with 26-fold lower affinity. Ch transport by VAChT is of interest in view of the possibilities that Ch (i) occurs at higher concentration than ACh does in terminal cytoplasm under some conditions, and (ii) is an agonist for alpha 7 nicotinic receptors.

Transmembrane reorientation of the substrate-binding site in vesicular acetylcholine transporter.

Active transport of acetylcholine (ACh) by vesicular ACh transporter (VAChT) is driven by a proton-motive force established by V-ATPase. A published microscopic kinetics model predicts the ACh-binding site is primarily oriented toward the outside for nontransporting VAChT and toward the inside for transporting VAChT. The allosteric ligand [(3)H]vesamicol cannot bind when the ACh-binding site is outwardly oriented and occupied by ACh, but it can bind when the ACh site is inwardly oriented. The kinetics model was tested in the paper reported here using rat VAChT expressed in PC12(A1237) cells. Equilibrium titrations of [(3)H]vesamicol binding and ACh competition show that ATP blocks competition between vesamicol and ACh in over one-half of the VAChT. NaCl did not mimic ACh chloride, and bafilomycin A(1) and FCCP completely blocked the ATP effect, which shows that it is mediated by a proton-motive force. The data are consistent with reorientation of over one-half of the ACh-binding sites from the outside to the inside of vesicles upon activation of transport. The observations support the proposed microscopic kinetics model, and they should be useful in characterizing effects of mutations on the VAChT transport cycle.

Reliable, sensitive, rapid and quantitative enzyme-based assay for gamma-hydroxybutyric acid (GHB).

Several assays for gamma-hydroxybutyrate (4-hydroxybutyrate, GHB) have been developed based on the enzyme gamma-hydroxybutyrate dehydrogenase (GHB-DH). Enzymatic oxidation of GHB by NAD+ is coupled to diaphorase-mediated reduction of pro-dye to yield colored product. GHB-DH from Ralstonia eutropha was cloned and expressed as a stable fusion protein easily purified by affinity chromatography. Quantitative initial velocity and endpoint versions of the assay in solution are described. Michaelis-Menten parameters for oxidation of GHB and ethanol were estimated. A semi-quantitative "dipstick" version of the assay on paper also is described. Both solution endpoint and "dipstick" assays are sensitive to about 0.05 mg GHB/mL using 10 microL of sample. Ethanol at concentrations possible in urine and agents used to stabilize physiological fluids for forensics analysis do not interfere significantly. The "dipstick" assay also allows detection of GHB in alcoholic beverages after evaporation of about one-fourth drop of beverage before testing. The enzymatic assay for GHB is reliable, sensitive, inexpensive and rapid.