Sompop Bencharit

Structural basis of heroin and cocaine metabolism by a promiscuous human drug-processing enzyme.

We present the first crystal structures of a human protein bound to analogs of cocaine and heroin. Human carboxylesterase 1 (hCE1) is a broad-spectrum bioscavenger that catalyzes the hydrolysis of heroin and cocaine, and the detoxification of organophosphate chemical weapons, such as sarin, soman and tabun. Crystal structures of the hCE1 glycoprotein in complex with the cocaine analog homatropine and the heroin analog naloxone provide explicit details about narcotic metabolism in humans. The hCE1 active site contains both specific and promiscuous compartments, which enable the enzyme to act on structurally distinct chemicals. A selective surface ligand-binding site regulates the trimer-hexamer equilibrium of hCE1 and allows each hCE1 monomer to bind two narcotic molecules simultaneously. The bioscavenger properties of hCE1 can likely be used to treat both narcotic overdose and chemical weapon exposure.

Crystal Structure of Human Carboxylesterase 1 Complexed with the Alzheimer's Drug Tacrine: From Binding Promiscuity to Selective Inhibition

Human carboxylesterase 1 (hCE1) is a broad-spectrum bioscavenger that plays important roles in narcotic metabolism, clinical prodrug activation, and the processing of fatty acid and cholesterol derivatives. We determined the 2.4 A crystal structure of hCE1 in complex with tacrine, the first drug approved for treating Alzheimers disease, and compare this structure to the Torpedo californica acetylcholinesterase (AcChE)-tacrine complex. Tacrine binds in multiple orientations within the catalytic gorge of hCE1, while it stacks in the smaller AcChE active site between aromatic side chains. Our results show that hCE1s promiscuous action on distinct substrates is enhanced by its ability to interact with ligands in multiple orientations at once. Further, we use our structure to identify tacrine derivatives that act as low-micromolar inhibitors of hCE1 and may provide new avenues for treating narcotic abuse and cholesterol-related diseases.

Molecular basis for pH-dependent mucosal dehydration in cystic fibrosis airways.

Significance Cystic fibrosis (CF) is caused by mutations in the cystic fibrosis transmembrane conductance regulator (CFTR) gene, which codes for a chloride/bicarbonate channel whose absence leads to dehydration and acidification of CF airways. A contributing factor to CF lung disease is dysregulation of the epithelial Na+ channel (ENaC), which exacerbates mucus dehydration. Here, we show that ENaC hyperactivity in CF airways is direct consequence of acidic airway surface liquid (ASL) and that ASL hydration is restored by raising ASL pH. Additionally, we show that short palate lung and nasal epithelial clone 1, the most abundant gene in airway epithelia, is the extracellular pH-sensitive factor that inhibits ENaC in normal but not CF airways. We suggest that future CF therapy be directed toward raising the pH of CF airways. The ability to maintain proper airway surface liquid (ASL) volume homeostasis is vital for mucus hydration and clearance, which are essential aspects of the mammalian lung’s innate defense system. In cystic fibrosis (CF), one of the most common life-threatening genetic disorders, ASL dehydration leads to mucus accumulation and chronic infection. In normal airways, the secreted protein short palate lung and nasal epithelial clone 1 (SPLUNC1) effectively inhibits epithelial Na+ channel (ENaC)-dependent Na+ absorption and preserves ASL volume. In CF airways, it has been hypothesized that increased ENaC-dependent Na+ absorption contributes to ASL depletion, and hence increased disease. However, this theory is controversial, and the mechanism for abnormal ENaC regulation in CF airways has remained elusive. Here, we show that SPLUNC1 is a pH-sensitive regulator of ENaC and is unable to inhibit ENaC in the acidic CF airway environment. Alkalinization of CF airway cultures prevented CF ASL hyperabsorption, and this effect was abolished when SPLUNC1 was stably knocked down. Accordingly, we resolved the crystal structure of SPLUNC1 to 2.8 Å. Notably, this structure revealed two pH-sensitive salt bridges that, when removed, rendered SPLUNC1 pH-insensitive and able to regulate ASL volume in acidic ASL. Thus, we conclude that ENaC hyperactivity is secondary to reduced CF ASL pH. Together, these data provide molecular insights into the mucosal dehydration associated with a range of pulmonary diseases, including CF, and suggest that future therapy be directed toward alkalinizing the pH of CF airways.

Defining Salivary Biomarkers Using Mass Spectrometry-Based Proteomics: A Systematic Review

Recent advancements in mass spectrometric proteomics provide a promising result in utilizing saliva to explore biomarkers for diagnostic purposes. However, the issues of specificity or redundancy of disease-associated salivary biomarkers have not been described. This systematic review was therefore aimed to define and summarize disease-related salivary biomarkers identified by mass spectrometry proteomics. Peer-reviewed articles published through July 2009 within three databases were reviewed. Out of 243 articles, 21 studies were selected in this systematic review with conditions including Sjögrens syndrome, squamous cell carcinoma, dental caries, diabetes, breast cancer, periodontitis, gastric cancer, systemic sclerosis, oral lichen planus, bleeding oral cavity, and graft-versus-host disease. The sample size ranged from 3-41 in both diseased and control subjects, with no consensus on sample collection protocol. One hundred eighty biomarkers were identified in total; 87 upregulated, 63 downregulated, and 30 varying based on disease. Except for Sjögrens syndrome, the majority of studies with the same disease produce inconsistent biomarkers. Larger sample size and standardization of sample collection/treatment protocol may improve future studies.

Rho Kinase Inhibition Rescues the Endothelial Cell Cerebral Cavernous Malformation Phenotype

Cerebral cavernous malformations (CCM) are vascular lesions causing seizures and stroke. Mutations causing inactivation of one of three genes, ccm1, -2, or -3, are sufficient to induce vascular endothelial cell defects resulting in CCM. Herein, we show that loss of expression of the CCM1, -2, or -3 proteins causes a marked increase in expression of the GTPase RhoA. Live cell imaging with a RhoA-specific biosensor demonstrates increased RhoA activity with loss of CCM1, -2, or -3, with an especially pronounced RhoA activation in both the cytosol and the nucleus with loss of CCM1 expression. Increased RhoA activation was associated with Rho kinase-dependent phosphorylation of myosin light chain 2. Functionally, loss of CCM1, -2, or -3 inhibited endothelial cell vessel-like tube formation and extracellular matrix invasion, each of which is rescued by chemical inhibition or short hairpin RNA knockdown of Rho kinase. The findings, for the first time, define a signaling network for CCM1, -2, and -3 in CCM pathology, whereby loss of CCM1, -2, or -3 protein expression results in increased RhoA activity, with the activation of Rho kinase responsible for endothelial cell dysregulation. The results define Rho kinase as a therapeutic target to rescue endothelial cells from loss of CCM protein function.

Putative stem cells in human dental pulp with irreversible pulpitis: an exploratory study.

INTRODUCTION Although human dental pulp stem cells isolated from healthy teeth have been extensively characterized, it is unknown whether stem cells also exist in clinically compromised teeth with irreversible pulpitis. Here we explored whether cells retrieved from clinically compromised dental pulp have stem cell-like properties. METHODS Pulp cells were isolated from healthy teeth (control group) and from teeth with clinically diagnosed irreversible pulpitis (diseased group). Cell proliferation, stem cell marker STRO-1 expression, and cell odonto-osteogenic differentiation competence were compared. RESULTS Cells from the diseased group demonstrated decreased colony formation capacity and a slightly decreased cell proliferation rate, but they had similar STRO-1 expression and exhibited a similar percentage of positive ex vivo osteogenic induction and dentin sialophosphoprotein expression from STRO-1-enriched pulp cells. CONCLUSIONS Our study provides preliminary evidence that clinically compromised dental pulp might contain putative cells with certain stem cell properties. Further characterization of these cells will provide insight regarding whether they could serve as a source of endogenous multipotent cells in tissue regeneration-based dental pulp therapy.

Identification of the SPLUNC1 ENaC-inhibitory domain yields novel strategies to treat sodium hyperabsorption in cystic fibrosis airway epithelial cultures

The epithelial sodium channel (ENaC) is responsible for Na(+) and fluid absorption across colon, kidney, and airway epithelia. Short palate lung and nasal epithelial clone 1 (SPLUNC1) is a secreted, innate defense protein and an autocrine inhibitor of ENaC that is highly expressed in airway epithelia. While SPLUNC1 has a bactericidal permeability-increasing protein (BPI)-type structure, its NH2-terminal region lacks structure. Here we found that an 18 amino acid peptide, S18, which corresponded to residues G22-A39 of the SPLUNC1 NH2 terminus inhibited ENaC activity to a similar degree as full-length SPLUNC1 (∼2.5 fold), while SPLUNC1 protein lacking this region was without effect. S18 did not inhibit the structurally related acid-sensing ion channels, indicating specificity for ENaC. However, S18 preferentially bound to the βENaC subunit in a glycosylation-dependent manner. ENaC hyperactivity is contributory to cystic fibrosis (CF) lung disease. Unlike control, CF human bronchial epithelial cultures (HBECs) where airway surface liquid (ASL) height was abnormally low (4.2 ± 0.6 μm), addition of S18 prevented ENaC-led ASL hyperabsorption and maintained CF ASL height at 7.9 ± 0.6 μm, even in the presence of neutrophil elastase, which is comparable to heights seen in normal HBECs. Our data also indicate that the ENaC inhibitory domain of SPLUNC1 may be cleaved away from the main molecule by neutrophil elastase, suggesting that it may still be active during inflammation or neutrophilia. Furthermore, the robust inhibition of ENaC by the S18 peptide suggests that this peptide may be suitable for treating CF lung disease.

Exploring salivary proteomes in edentulous patients with type 2 diabetes

Type 2 diabetes and tooth loss are linked both epidemiologically and pathophysiologically. We applied label-free differential protein expression analysis using multidimensional liquid chromatography/tandem mass spectrometry (2D-LC-MS/MS) to explore the proteomic profile of saliva samples collected from selected type 2 diabetic edentulous patients and non-diabetic controls. Ninety-six peptides corresponding to 52 proteins were differentially expressed between the diabetic edentulous patients and controls (p < 0.05). Some diabetes-related inflammatory biomarkers including glyceraldehyde-3-phosphate dehydrogenase and serum amyloid A were detected with levels increased in diabetic samples. Other biomarkers including amylase, palate, lung and nasal epithelium associated protein (PLUNC), and serotransferrin levels were decreased in diabetic samples. In contrast with previous findings, salivary carbonic anhydrase 6 and alpha-2 macroglobulin levels, however, were decreased in this diabetic patient population. Cluster analysis and principle component analysis demonstrated a differential pattern of protein biomarker expression between diabetic and control subjects. Western blot analysis was completed to confirm the relatively lower expression level of two biomarkers, including PLUNC and amylase in the diabetic group compared to control subjects. The presence of salivary biomarkers specific for diabetes in edentulous subjects mimics those in serum, especially those related to inflammatory/lipid metabolism. While this exploratory study requires further validation with a larger population, it provides proof-of-principle for salivary proteomics for edentulous subjects with diabetes.

Clinical and histological findings of denture stomatitis as related to intraoral colonization patterns of Candida albicans, salivary flow, and dry mouth.

PURPOSE Multifactorial etiological factors contribute to denture stomatitis (DS), a type of oral candidiasis; however, unlike other oral candidiasis, DS can occur in a healthy person wearing a denture. In this study, we therefore attempt to explore the association between candida, denture, and mucosal tissue using (1) exfoliative cytology, (2) the candidal levels present in saliva, on mucosal tissues and on denture surfaces, and (3) the salivary flow rate and xerostomic symptoms. MATERIALS AND METHODS A cross-sectional study enrolled 32 edentulous participants, 17 without DS as controls and 15 with DS (Newtons classification type II and III). Participants with systemic or other known oral conditions were excluded. Participants completed a xerostomia questionnaire, and salivary flow rates were measured. Samples of unstimulated whole saliva (UWS) and stimulated whole saliva (SWS) were collected. UWS was used for fungal culturing. Periodic acid-Schiff (PAS) stain and quantitative exfoliative cytology were performed on samples from affected and unaffected mucosa from each participant. Levels of Candida species (albicans and non-albicans) were determined in salivary samples (expressed as colony-forming units, CFU), as well as from swab samples obtained from denture fitting surfaces, in addition to affected and unaffected mucosa. RESULTS There were no significant differences in salivary flow rates, mucosal wetness, or frequency of reported dry mouth comparing participants with and without DS. Exfoliative cytology of mucosal smears demonstrated significantly higher (p= 0.02) inflammatory cell counts in DS patients, as compared with smears of healthy denture-wearers. Candida albicans was significantly more prevalent in saliva (p= 0.03) and on denture surfaces (p= 0.002) of DS participants, whereas mucosal candidal counts and the presence of cytological hyphae did not show significant difference comparing DS to healthy participants. CONCLUSIONS In this investigation, we presented a unique group of healthy edentulous patients. This population may reflect the general DS population without systemic or other oral diseases. The prominent etiological factor for DS in this population is the presence of candida in denture and saliva. We found that other factors such as saliva flow/xerostomia, fitting of the denture, and the presence of candida in the mucosa, are less important in this population. Therefore, DS treatments in healthy patients should first focus on sanitization of an existing denture and/or fabrication of a new denture.

Development and Applications of Porous Tantalum Trabecular Metal-Enhanced Titanium Dental Implants

BACKGROUND Porous tantalum trabecular metal has recently been incorporated in titanium dental implants as a new form of implant surface enhancement. However, there is little information on the applications of this material in implant dentistry. PURPOSE The purpose of this article is to summarize the contemporary concept on the applications of porous tantalum trabecular metal in implant dentistry. MATERIALS AND METHODS We therefore review the current literature on the basic science and clinical uses of this material. RESULTS Porous tantalum metal is used to improve the contact between osseous structure and dental implants and therefore presumably facilitate osseointegration. Success of porous tantalum metal in orthopedic implants led to the incorporation of porous tantalum metal in the design of root-form endosseous titanium implants. The porous tantalum three-dimensional enhancement of titanium dental implant surface allows for combining bone ongrowth together with bone ingrowth, or osseoincorporation. While little is known about the biological aspect of the porous tantalum in the oral cavity, there seems to be several possible advantages of this implant design. This article reviews the biological aspects of porous tantalum-enhanced titanium dental implants, in particular the effects of anatomical consideration and oral environment to implant designs. CONCLUSIONS We propose here possible clinical situations and applications for this type of dental implant. Advantages and disadvantages of the implants as well as needed future clinical studies are discussed.