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1.
J Transl Med ; 21(1): 730, 2023 10 17.
Article in English | MEDLINE | ID: mdl-37848935

ABSTRACT

BACKGROUND: Lysosomes are closely linked to autophagic activity, which plays a vital role in pancreatic ductal adenocarcinoma (PDAC) biology. The survival of PDAC patients is still poor, and the identification of novel genetic factors for prognosis and treatment is highly required to prevent PDAC-related deaths. This study investigated the germline variants related to lysosomal dysfunction in patients with PDAC and to analyze whether they contribute to the development of PDAC. METHODS: The germline putative pathogenic variants (PPV) in genes involved in lysosomal storage disease (LSD) was compared between patients with PDAC (n = 418) and healthy controls (n = 845) using targeted panel and whole-exome sequencing. Furthermore, pancreatic organoids from wild-type and KrasG12D mice were used to evaluate the effect of lysosomal dysfunction on PDAC development. RNA sequencing (RNA-seq) analysis was performed with established PDAC patient-derived organoids (PDOs) according to the PPV status. RESULTS: The PPV in LSD-related genes was higher in patients with PDAC than in healthy controls (8.13 vs. 4.26%, Log2 OR = 1.65, P = 3.08 × 10-3). The PPV carriers of LSD-related genes with PDAC were significantly younger than the non-carriers (mean age 61.5 vs. 65.3 years, P = 0.031). We further studied a variant of the lysosomal enzyme, galactosylceramidase (GALC), which was the most frequently detected LSD variant in our cohort. Autophagolysosomal activity was hampered when GALC was downregulated, which was accompanied by paradoxically elevated autophagic flux. Furthermore, the number of proliferating Ki-67+ cells increased significantly in pancreatic organoids derived from Galc knockout KrasG12D mice. Moreover, GALC PPV carriers tended to show drug resistance in both PDAC cell line and PDAC PDO, and RNA-seq analysis revealed that various metabolism and gene repair pathways were upregulated in PDAC PDOs harboring a GALC variant. CONCLUSIONS: Genetically defined lysosomal dysfunction is frequently observed in patients with young-onset PDAC. This might contribute to PDAC development by altering metabolism and impairing autophagolysosomal activity, which could be potentially implicated in therapeutic applications for PDAC.


Subject(s)
Carcinoma, Pancreatic Ductal , Pancreatic Neoplasms , Humans , Mice , Animals , Middle Aged , Proto-Oncogene Proteins p21(ras) , Pancreatic Neoplasms/pathology , Carcinoma, Pancreatic Ductal/genetics , Carcinoma, Pancreatic Ductal/pathology , Germ Cells/metabolism , Lysosomes/metabolism , Lysosomes/pathology , Pancreatic Neoplasms
2.
Curr Issues Mol Biol ; 43(3): 2059-2067, 2021 Nov 19.
Article in English | MEDLINE | ID: mdl-34889900

ABSTRACT

A declined salivary gland function is commonly observed in elderly people. Advanced glycation end products (AGEs) are believed to contribute to the pathogenesis of aging. Although physical exercise is shown to increase various organ functions in human and experimental models, it is not known whether it has a similar effect in the salivary glands. In the present study, we evaluated the AGEs burden in the salivary gland in the aging process and the protective effect of physical exercise on age-related salivary hypofunction. To accelerate the aging process, rats were peritoneally injected with D-galactose for 6 weeks. Young control rats and d-galactose-induced aging rats in the old group were not exercised. The rats in the physical exercise group ran on a treadmill (12 m/min, 60 min/day, 3 days/week for 6 weeks). The results showed that the salivary flow rate and total protein levels in the saliva of the d-galactose-induced aging rats were reduced compared to those of the young control rats. Circulating AGEs in serum and secreted AGEs in saliva increased with d-galactose-induced aging. AGEs also accumulated in the salivary glands of these aging rats. The salivary gland of aging rats showed increased reactive oxygen species (ROS) generation, loss of acinar cells, and apoptosis compared to young control mice. However, physical exercise suppressed all of these age-related salivary changes. Overall, physical exercise could provide a beneficial option for age-related salivary hypofunction.


Subject(s)
Aging/metabolism , Galactose/metabolism , Glycation End Products, Advanced/metabolism , Salivary Glands/metabolism , Animals , Biomarkers , Glycation End Products, Advanced/blood , Physical Conditioning, Animal , Rats , Reactive Oxygen Species/metabolism , Salivary Glands/pathology , Salivary Glands/physiopathology , Salivation
3.
Biomed Pharmacother ; 137: 111297, 2021 May.
Article in English | MEDLINE | ID: mdl-33493968

ABSTRACT

Patients with diabetes commonly experience hyposalivation, which induces discomfort in eating, swallowing, dryness, smell, and speaking, as well as increases the incidence of periodontal disease. Dipeptidyl peptidase-4 (DPP4) inhibitors are frequently used as antidiabetic drugs that lower glucose levels by utilizing similar mechanisms; however, additional protective functions of each gliptin have been discovered. In this study, the protective roles of gemigliptin, a DPP4 inhibitor, against salivary dysfunction under diabetic conditions were investigated. Streptozotocin-induced diabetic rats received gemigliptin 10 mg/kg or 100 mg/kg via oral gavage for 3 weeks. The weights of salivary gland tissues, saliva secretion, and antioxidant capacity in salivary glands were reduced after diabetes induction, but were significantly preserved following gemigliptin treatment. In salivary gland analysis, expression of apoptotic proteins, as well as amylase and aquaporin-5 (AQP5) protein expression, were increased following gemigliptin treatment. Furthermore, the number of TUNEL-positive cells decreased after gemigliptin treatment. Therefore, gemigliptin has protective roles against salivary dysfunction observed in diabetes, mediated via antioxidant, anti-apoptotic, and salivary secretion mechanisms. These results may help in selecting a suitable drug for patients with diabetes experiencing salivary dysfunction.


Subject(s)
Antioxidants/pharmacology , Diabetes Mellitus, Experimental/drug therapy , Dipeptidyl-Peptidase IV Inhibitors/pharmacology , Piperidones/pharmacology , Pyrimidines/pharmacology , Salivary Gland Diseases/prevention & control , Salivary Glands/drug effects , Salivation/drug effects , Animals , Apoptosis/drug effects , Diabetes Mellitus, Experimental/chemically induced , Diabetes Mellitus, Experimental/physiopathology , Male , Oxidative Stress/drug effects , Rats, Sprague-Dawley , Reactive Oxygen Species/metabolism , Salivary Gland Diseases/etiology , Salivary Gland Diseases/physiopathology , Salivary Glands/metabolism , Salivary Glands/physiopathology , Streptozocin
4.
Pharmaceutics ; 14(1)2021 Dec 27.
Article in English | MEDLINE | ID: mdl-35056946

ABSTRACT

Polydatin (resveratrol-3-O-ß-mono-D-glucoside) is a polyphenol that can be easily accessed from peanuts, grapes, and red wine, and is known to have antiglycation, antioxidant, and anti-inflammatory effects. Diabetes mellitus is a very common disease, and diabetic complications are very common complications. The dry mouth symptom is one of the most common oral complaints in patients with diabetes mellitus. Diabetes mellitus is thought to promote hyposalivation. In this study, we aimed to investigate the improvement effect of polydatin on diabetes-induced hyposalivation in db/db mouse model of type 2 diabetes. We examined salivary flow rate, TUNEL assay, PAS staining, and immunohistochemical staining for AGEs, RAGE, HMGB1, 8-OHdG, and AQP5 to evaluate the efficacy of polydatin in the submandibular salivary gland. Diabetic db/db mice had a decreased salivary flow rate and salivary gland weight. The salivary gland of the vehicle-treated db/db mice showed an increased apoptotic cell injury. The AGEs were highly accumulated, and its receptor, RAGE expression was also enhanced. Expressions of HMGB1, an oxidative cell damage marker, and 8-OHdG, an oxidative DNA damage marker, increased greatly. However, polydatin ameliorated this hypofunction of the salivary gland and inhibited diabetes-related salivary cell injury. Furthermore, polydatin improved mucin accumulation, which is used as a damage marker for salivary gland acinar cells, and decreased expression of water channel AQP5 was improved by polydatin. In conclusion, polydatin has a potent protective effect on diabetes-related salivary gland hypofunction through its antioxidant and anti-glycation activities, and its AQP5 upregulation. This result suggests the possibility of the use of polydatin as a therapeutic drug to improve hyposalivation caused by diabetes.

5.
Food Sci Nutr ; 8(6): 2895-2903, 2020 Jun.
Article in English | MEDLINE | ID: mdl-32566207

ABSTRACT

Neovascularization in the retina is common pathophysiology of diabetic retinal microvasculopathy and exudative macular degeneration. Our study assessed the inhibitory activity of an ethanol-based extract of Aucuba japonica (AJE) on abnormal angiogenesis in the retina with a hyperoxia-induced neovascular retinopathy model. The inhibitory effects of aucubin, quercetin, and kaempferol, bioactive compounds, from A. japonica, on retinal vascular hyperpermeability were also examined. On the 7th postnatal day (P7), the C57BL/6 pups were exposed to a hyperoxic environment with 75% oxygen to develop the experimental angiogenesis in retinas. On the 12th postnatal day (P12), the pups were then returned to the normal atmospheric pressure of oxygen. From P12 to P16, the administration was intraperitoneal. The dose per day was 250 mg per kg weight. Retinal neovascularization was measured with retinal flat mounts prepared on P17. We also measured the vascular leakage mediated by the vascular endothelial growth factor (VEGF) in retinas. Mice treated with AJE had markedly smaller neovascular lesions, in comparison with vehicle-administered mice. AJE downregulated the expression of both VEGF protein and mRNA. In addition, aucubin, quercetin, and kaempferol ameliorated VEGF-induced retinal vascular leakage. The results of our study suggest that AJE is a potent antiangiogenic substance. AJE could also serve as a therapeutic agent for abnormal growth of vessels in the retina in patients with ischemic retinopathy. The bioactive compounds of AJE may be responsible for its antiangiogenic abilities.

6.
Molecules ; 24(24)2019 Dec 04.
Article in English | MEDLINE | ID: mdl-31817154

ABSTRACT

In the present study, we examined the potent retinoprotective effects of an ethanol-based extract of Aucuba japonica (AJE) and its active ingredient, aucubin, on N-methyl-N-nitrosourea (MNU)-induced retinal degeneration in mice. Retinal degeneration was induced by an intraperitoneal injection of MNU (60 mg/kg). AJE (250 mg/kg) and aucubin (15 mg/kg) were orally administered for 1 week after the MNU injection. Electroretinography (ERG) and histological examinations were performed. Retinal apoptosis and oxidative DNA damage were also quantified. The retinoprotective abilities of AJE and aucubin were also assessed in primary cultured retinal cells. Morphologically, MNU induced a remarkable decrease in the outer nuclear layer, which contains photoreceptor cells. However, this layer was well preserved in the AJE- and aucubin-administered mice. The ERG responses significantly decreased in both a- and b-wave amplitudes in the MNU-injected mice. In the AJE and aucubin-treated mice, ERG responses were significantly increased. In addition, a terminal deoxynucleotidyl transferase dUTP nick end-labeling (TUNEL) assay and immunohistochemical staining for 8-hydroxydeoxyguanosine (8-OHdG) revealed that both AJE and aucubin attenuated MNU-induced photoreceptor cell apoptosis and oxidative DNA damage. Furthermore, the in vitro assay also showed that AJE and aucubin have potent anti-oxidative and anti-apoptotic activities in primary cultured retinal cells. These results indicate that AJE and aucubin have potent retinoprotective effects, and that this retinoprotective activity is as a result of the potency of the bioactive compound, aucubin. These pharmacological characteristics suggest the additional application of AJE or aucubin in the treatment of patients with retinal degenerative diseases.


Subject(s)
Iridoid Glucosides/therapeutic use , Magnoliopsida/chemistry , Retinal Degeneration/prevention & control , Animals , Apoptosis/drug effects , Cell Survival/drug effects , Cells, Cultured , DNA Damage , Disease Models, Animal , Iridoid Glucosides/pharmacology , Male , Methylnitrosourea , Mice, Inbred C57BL , Oxidative Stress/drug effects , Photoreceptor Cells, Vertebrate/drug effects , Photoreceptor Cells, Vertebrate/pathology , Plant Extracts/analysis , Retina/drug effects , Retina/pathology , Retina/physiopathology , Retinal Degeneration/chemically induced , Retinal Degeneration/pathology , Retinal Degeneration/physiopathology
7.
Molecules ; 24(20)2019 Oct 10.
Article in English | MEDLINE | ID: mdl-31658696

ABSTRACT

Advanced glycation end products (AGEs) is a causative factor of various chronic diseases, including chronic kidney disease and atherosclerosis. AGE inhibitors, such as aminoguanidine and pyridoxamine, have the therapeutic activities for reversing the increase in AGEs burden. This study evaluated the inhibitory effects of aucubin on the formation of methylglyoxal (MGO)-modified AGEs in vitro. We also determined the potential activity of aucubin in reducing the AGEs burden in the kidney, blood vessel, heart, and retina of exogenously MGO-injected rats. Aucubin inhibited the formation of MGO-modified AGE-bovine serum albumin (IC50 = 0.57 ± 0.04 mmol/L) and its cross-links to collagen (IC50 = 0.55 ± 0.02 mmol/L) in a dose-dependent manner. In addition, aucubin directly trapped MGO (IC50 = 0.22 ± 0.01 mmol/L) in vitro. In exogenous MGO-injected rats, aucubin suppressed the formation of circulating AGEs and its accumulation in various tissues. These activities of aucubin on the MGO-derived AGEs in vitro and in vivo showed its pharmacological potential for inhibiting AGEs-related various chronic diseases.


Subject(s)
Glycation End Products, Advanced/blood , Iridoid Glucosides/pharmacology , Pyruvaldehyde/pharmacology , Animals , Rats , Rats, Sprague-Dawley
8.
Nucl Med Commun ; 39(7): 691-698, 2018 Jul.
Article in English | MEDLINE | ID: mdl-29893751

ABSTRACT

PURPOSE: To evaluate the diagnostic accuracy of fluorine-18-fluorodeoxyglucose PET/computed tomography (F-FDG PET/CT) for lymph node (LN) metastasis and the prognostic significance of F-FDG PET/CT LN parameters in patients with resectable pancreatic cancer. PATIENTS AND METHODS: Patients with resectable pancreatic cancer who underwent staging F-FDG PET/CT between May 2007 and September 2016 were retrospectively enrolled and analyzed through medical record and image re-evaluation. The diagnostic accuracy of F-FDG PET/CT in predicting LN metastasis was evaluated and compared with that of contrast-enhanced abdominal computed tomography (CECT). Prognostic variables, including LN parameters assessed by F-FDG PET/CT [standardized uptake value (SUV)LN and LN/tumor SUV ratio], that affect disease-free survival (DFS) and overall survival (OS) were evaluated by regression analysis. RESULTS: When predicting LN metastasis, F-FDG PET/CT showed greater sensitivity, positive predictive value, negative predictive value, and accuracy than CECT. Among prognostic factors affecting DFS, PET-positive LN (P=0.008), and LN/tumor SUV ratio (P=0.003) were found to be significant by regression analysis. Among the variables affecting OS, lymphovascular invasion (P=0.018) and the LN/tumor SUV ratio (P=0.046) were found to be significant. CONCLUSION: F-FDG PET/CT showed higher diagnostic accuracy in predicting LN metastasis than CECT in patients with resectable pancreatic cancer. Only the LN/tumor SUV ratio of F-FDG PET/CT was an independent prognostic variable in both DFS and OS.


Subject(s)
Fluorodeoxyglucose F18 , Pancreatic Neoplasms/diagnostic imaging , Pancreatic Neoplasms/pathology , Positron Emission Tomography Computed Tomography , Adult , Aged , Aged, 80 and over , Disease-Free Survival , Female , Humans , Lymphatic Metastasis , Male , Middle Aged , Pancreatic Neoplasms/surgery
9.
Dent Mater J ; 37(6): 950-956, 2018 Nov 30.
Article in English | MEDLINE | ID: mdl-29515045

ABSTRACT

This study evaluated the flexural mechanical properties of various thermoplastic denture base polymers (six polyamides, four acrylic resins, polyester, polypropylene, and polycarbonate) by three different testing conditions; specimens were tested in water bath at 37°C (Wet/Water, by ISO 20795-1), or in ambient air (Wet/Air) after being immersed in distilled water for 50 h, or after desiccation for 7 days (Dry/Air). The mean ultimate flexural strength (UFS) and flexural modulus (FM) for most products ranged from 27 to 61 MPa and from 611 to 1,783 MPa respectively, which failed to meet the minimum requirements of the international standard, except for polycarbonate (89 and 2,245 MPa). The mean UFS and FM values were ranked Dry/Air>Wet/Air>Wet/Water (p<0.05). In conclusion, the flexural mechanical properties of denture base polymers varied with the products and were significantly affected by the testing medium (air or water) and specimen conditions (wet or dry).


Subject(s)
Dental Materials/chemistry , Denture Bases/standards , Polymers/chemistry , Acrylic Resins/chemistry , Air , Desiccation , Elastic Modulus , Flexural Strength , Materials Testing , Nylons/chemistry , Polycarboxylate Cement/chemistry , Polyesters/chemistry , Polypropylenes/chemistry , Water
10.
Clin Mol Hepatol ; 23(3): 230-238, 2017 Sep.
Article in English | MEDLINE | ID: mdl-28669175

ABSTRACT

BACKGROUND/AIMS: Tenofovir disoproxil fumarate (TDF) monotherapy for 48 weeks provided a virological response comparable to that of TDF and entecavir (ETV) combination therapy in patients infected with ETV-resistant hepatitis B virus (HBV). Little long-term data in routine clinical practice are available regarding the optimal treatment of patients with ETV-resistant HBV. METHODS: We investigated the long-term antiviral efficacy of combination therapy of TDF+lamivudine (LAM) or TDF+ETV compared to that of TDF monotherapy in 73 patients with resistance to both LAM and ETV. RESULTS: Patients were treated with TDF monotherapy (n=12), TDF+LAM (n=19), or TDF+ETV (n=42) for more than 6 months. The median duration of TDF-based rescue therapy was 37 months. Virologic response (VR) was found in 63 patients (86.3%). The rates of VR among the three groups (TDF monotherapy, TDF+LAM, and TDF+ETV) were not statistically different (log-rank P=0.200) at 12 months (59.3%, 78.9%, and 51.8%, respectively) or at 24 months (88.4%, 94.7%, and 84.2%). In addition, treatment efficacy of TDF-based combination or TDF monotherapy was not statistically different with ETV-resistant strains or exposure to other antiviral agents. In multivariate analysis, only lower baseline HBV DNA level was an independent predictor for VR (hazard ratio, 0.723; 95% confidence interval, 0.627-0.834; P<0.001). CONCLUSIONS: TDF monotherapy was as effective as combination therapy of TDF+LAM or TDF+ETV in maintaining long-term viral suppression in chronic hepatitis B patients with resistance to both LAM and ETV. HBV DNA level at the start of TDF rescue therapy was the only independent predictor of subsequent VR.


Subject(s)
Antiviral Agents/therapeutic use , Guanine/analogs & derivatives , Hepatitis B, Chronic/drug therapy , Lamivudine/therapeutic use , Tenofovir/therapeutic use , Adult , Aged , Area Under Curve , DNA, Viral/analysis , Drug Resistance, Viral/drug effects , Drug Therapy, Combination , Female , Guanine/therapeutic use , Hepatitis B e Antigens/blood , Humans , Male , Middle Aged , Proportional Hazards Models , ROC Curve , Tenofovir/pharmacology , Treatment Outcome
11.
PLoS One ; 12(6): e0179088, 2017.
Article in English | MEDLINE | ID: mdl-28654679

ABSTRACT

We investigated the association between autoimmune thyroid disease and systemic lupus erythematosus (SLE) using nationwide insurance claims data for the entire Korean population. Claims data for the period 2009-2013 were retrieved from the National Health Insurance System database. SLE and thyroid disease were identified using the International Classification of Diseases codes and medication information. Logistic regression analyses were used to evaluate the association between SLE and thyroid disease. The study used records from 17,495 patients with SLE and 52,485 age- and sex-matched control subjects. A greater prevalence of Graves' disease (0.94% vs. 0.46%, P < 0.001), Hashimoto's thyroiditis (2.68% vs. 0.80%, P < 0.001), and thyroid cancer (1.81% vs. 1.30%, P < 0.001) was observed in SLE patients than in control subjects. Multivariate regression analyses demonstrated that SLE was significantly associated with an increased risk of both autoimmune thyroid disease and thyroid cancer (Graves' disease: odds ratio [OR] 2.07, 95% confidence interval [CI] 1.70-2.53; Hashimoto's thyroiditis: OR 3.42, 95% CI 3.00-3.91; thyroid cancer: OR 1.40, 95% CI 1.22-1.60). Age- and sex- stratified analyses revealed that the risk of autoimmune thyroid disease in SLE patients was increased for all age groups and the female group. An association between thyroid cancer and SLE was identified only in the 20- to 59-year-old age group and in the female group. Using a large population-based study, we demonstrated that patients with SLE are at a greater risk of developing thyroid disease than matched control individuals.


Subject(s)
Lupus Erythematosus, Systemic/epidemiology , Thyroid Diseases/epidemiology , Adolescent , Adult , Age Factors , Aged , Aged, 80 and over , Databases, Factual , Female , Humans , Male , Middle Aged , Prevalence , Republic of Korea , Risk , Sex Factors , Young Adult
12.
BMC Cancer ; 16: 35, 2016 Jan 22.
Article in English | MEDLINE | ID: mdl-26801760

ABSTRACT

BACKGROUND: c-Met signaling has been implicated in oncogenesis especially in cells with c-met gene amplification. Since 20 % of gastric cancer patients show high level of c-Met expression, c-Met has been identified as a good candidate for targeted therapy in gastric cancer. Herein, we report our newly synthesized c-Met inhibitor by showing its efficacy both in vitro and in vivo. METHODS: Compounds with both triazolopyrazine and pyridoxazine scaffolds were synthesized and tested using HTRF c-Met kinase assay. We performed cytotoxic assay, cellular phosphorylation assay, and cell cycle assay to investigate the cellular inhibitory mechanism of our compounds. We also conducted mouse xenograft assay to see efficacy in vivo. RESULTS: KRC-00509 and KRC-00715 were selected as excellent c-Met inhibitors through biochemical assay, and exhibited to be exclusively selective to c-Met by kinase panel assay. Cytotoxic assays using 18 gastric cancer cell lines showed our c-Met inhibitors suppressed specifically the growth of c-Met overexpressed cell lines, not that of c-Met low expressed cell lines, by inducing G1/S arrest. In c-met amplified cell lines, c-Met inhibitors reduced the downstream signals including Akt and Erk as well as c-Met activity. In vivo Hs746T xenograft assay showed KRC-00715 reduced the tumor size significantly. CONCLUSIONS: Our in vitro and in vivo data suggest KRC-00715 is a potent and highly selective c-Met inhibitor which may have therapeutic potential in gastric tumor with c-Met overexpression.


Subject(s)
Cell Proliferation/drug effects , Protein Kinase Inhibitors/administration & dosage , Proto-Oncogene Proteins c-met/biosynthesis , Pyrazines/administration & dosage , Stomach Neoplasms/drug therapy , Triazoles/administration & dosage , Animals , Antineoplastic Agents/administration & dosage , Antineoplastic Agents/chemical synthesis , Cell Line, Tumor , Gene Expression Regulation, Neoplastic/drug effects , Humans , Mice , Protein Kinase Inhibitors/chemical synthesis , Proto-Oncogene Proteins c-met/genetics , Pyrazines/chemical synthesis , Signal Transduction/drug effects , Stomach Neoplasms/genetics , Stomach Neoplasms/pathology , Triazoles/chemical synthesis , Xenograft Model Antitumor Assays
13.
J Cardiovasc Ultrasound ; 23(2): 113-7, 2015 Jun.
Article in English | MEDLINE | ID: mdl-26140155

ABSTRACT

Sinus of Valsalva aneurysms are rare. Sinus of Valsalva aneurysms are frequently associated with ventricular septal defect (VSD) and aortic regurgitation. They often remain asymptomatic until abruptly presenting with acute chest pain and heart failure secondary to rupture. Here, we describe a case of 20-year-old man who presented with chest pain with a history of VSD. Initial work-up concluded that the patient had VSD associated membranous septal aneurysm. Four years later, the patient presented with symptoms of heart failure. Work-up showed that the ruptured sinus of Valsalva aneurysm was the cause of symptoms. Due to its close proximity to the aortic annulus, sinus of Valsalva aneurysm should be differentiated from membranous septal aneurysm.

14.
J Biol Chem ; 290(38): 23320-35, 2015 Sep 18.
Article in English | MEDLINE | ID: mdl-26183897

ABSTRACT

Pectin methylesterases (PMEs) catalyze the demethylesterification of homogalacturonan domains of pectin in plant cell walls and are regulated by endogenous pectin methylesterase inhibitors (PMEIs). In Arabidopsis dark-grown hypocotyls, one PME (AtPME3) and one PMEI (AtPMEI7) were identified as potential interacting proteins. Using RT-quantitative PCR analysis and gene promoter::GUS fusions, we first showed that AtPME3 and AtPMEI7 genes had overlapping patterns of expression in etiolated hypocotyls. The two proteins were identified in hypocotyl cell wall extracts by proteomics. To investigate the potential interaction between AtPME3 and AtPMEI7, both proteins were expressed in a heterologous system and purified by affinity chromatography. The activity of recombinant AtPME3 was characterized on homogalacturonans (HGs) with distinct degrees/patterns of methylesterification. AtPME3 showed the highest activity at pH 7.5 on HG substrates with a degree of methylesterification between 60 and 80% and a random distribution of methyl esters. On the best HG substrate, AtPME3 generates long non-methylesterified stretches and leaves short highly methylesterified zones, indicating that it acts as a processive enzyme. The recombinant AtPMEI7 and AtPME3 interaction reduces the level of demethylesterification of the HG substrate but does not inhibit the processivity of the enzyme. These data suggest that the AtPME3·AtPMEI7 complex is not covalently linked and could, depending on the pH, be alternately formed and dissociated. Docking analysis indicated that the inhibition of AtPME3 could occur via the interaction of AtPMEI7 with a PME ligand-binding cleft structure. All of these data indicate that AtPME3 and AtPMEI7 could be partners involved in the fine tuning of HG methylesterification during plant development.


Subject(s)
Arabidopsis Proteins/chemistry , Arabidopsis/chemistry , Carboxylic Ester Hydrolases/chemistry , Enzyme Inhibitors/chemistry , Hypocotyl/chemistry , Multiprotein Complexes/chemistry , Pectins/chemistry , Arabidopsis/genetics , Arabidopsis/metabolism , Arabidopsis Proteins/genetics , Arabidopsis Proteins/metabolism , Binding Sites , Carboxylic Ester Hydrolases/genetics , Carboxylic Ester Hydrolases/metabolism , Enzyme Inhibitors/metabolism , Hydrogen-Ion Concentration , Hypocotyl/genetics , Hypocotyl/metabolism , Molecular Docking Simulation , Multiprotein Complexes/genetics , Multiprotein Complexes/metabolism , Pectins/genetics , Pectins/metabolism , Recombinant Proteins/chemistry , Recombinant Proteins/genetics , Recombinant Proteins/metabolism , Substrate Specificity
15.
Plant Cell ; 26(7): 2996-3009, 2014 Jul.
Article in English | MEDLINE | ID: mdl-25012190

ABSTRACT

Cellulose microfibrils are para-crystalline arrays of several dozen linear (1→4)-ß-d-glucan chains synthesized at the surface of the cell membrane by large, multimeric complexes of synthase proteins. Recombinant catalytic domains of rice (Oryza sativa) CesA8 cellulose synthase form dimers reversibly as the fundamental scaffold units of architecture in the synthase complex. Specificity of binding to UDP and UDP-Glc indicates a properly folded protein, and binding kinetics indicate that each monomer independently synthesizes single glucan chains of cellulose, i.e., two chains per dimer pair. In contrast to structure modeling predictions, solution x-ray scattering studies demonstrate that the monomer is a two-domain, elongated structure, with the smaller domain coupling two monomers into a dimer. The catalytic core of the monomer is accommodated only near its center, with the plant-specific sequences occupying the small domain and an extension distal to the catalytic domain. This configuration is in stark contrast to the domain organization obtained in predicted structures of plant CesA. The arrangement of the catalytic domain within the CesA monomer and dimer provides a foundation for constructing structural models of the synthase complex and defining the relationship between the rosette structure and the cellulose microfibrils they synthesize.


Subject(s)
Catalytic Domain , Glucosyltransferases/chemistry , Oryza/enzymology , Cell Membrane/metabolism , Cell Wall/metabolism , Cellulose/metabolism , Glucosyltransferases/genetics , Glucosyltransferases/metabolism , Models, Molecular , Molecular Conformation , Oryza/genetics , Plant Proteins/chemistry , Plant Proteins/genetics , Plant Proteins/metabolism , Protein Binding , Protein Multimerization , Recombinant Proteins , Substrate Specificity
16.
J Mol Biol ; 426(5): 1019-38, 2014 Mar 06.
Article in English | MEDLINE | ID: mdl-24126213

ABSTRACT

Tailed bacteriophages and herpesviruses consist of a structurally well conserved dodecameric portal at a special 5-fold vertex of the capsid. The portal plays critical roles in head assembly, genome packaging, neck/tail attachment, and genome ejection. Although the structures of portals from phages φ29, SPP1, and P22 have been determined, their mechanistic roles have not been well understood. Structural analysis of phage T4 portal (gp20) has been hampered because of its unusual interaction with the Escherichia coli inner membrane. Here, we predict atomic models for the T4 portal monomer and dodecamer, and we fit the dodecamer into the cryo-electron microscopy density of the phage portal vertex. The core structure, like that from other phages, is cone shaped with the wider end containing the "wing" and "crown" domains inside the phage head. A long "stem" encloses a central channel, and a narrow "stalk" protrudes outside the capsid. A biochemical approach was developed to analyze portal function by incorporating plasmid-expressed portal protein into phage heads and determining the effect of mutations on head assembly, DNA translocation, and virion production. We found that the protruding loops of the stalk domain are involved in assembling the DNA packaging motor. A loop that connects the stalk to the channel might be required for communication between the motor and the portal. The "tunnel" loops that project into the channel are essential for sealing the packaged head. These studies established that the portal is required throughout the DNA packaging process, with different domains participating at different stages of genome packaging.


Subject(s)
Bacteriophage T4/chemistry , Bacteriophage T4/metabolism , Capsid Proteins/chemistry , DNA Packaging/physiology , DNA, Viral/chemistry , Virus Assembly/physiology , Bacteriophage T4/genetics , Capsid Proteins/genetics , Capsid Proteins/metabolism , DNA, Viral/genetics , Escherichia coli/virology , Models, Molecular , Protein Conformation , Protein Folding , Virion/genetics
17.
Proteins ; 78(10): 2338-48, 2010 Aug 01.
Article in English | MEDLINE | ID: mdl-20544969

ABSTRACT

Designing a protein sequence that will fold into a predefined structure is of both practical and fundamental interest. Many successful, computational designs in the last decade resulted from improved understanding of hydrophobic and polar interactions between side chains of amino acid residues in stabilizing protein tertiary structures. However, the coupling between main-chain backbone structure and local sequence has yet to be fully addressed. Here, we attempt to account for such coupling by using a sequence profile derived from the sequences of five residue fragments in a fragment library that are structurally matched to the five-residue segments contained in a target structure. We further introduced a term to reduce low complexity regions of designed sequences. These two terms together with optimized reference states for amino-acid residues were implemented in the RosettaDesign program. The new method, called RosettaDesign-SR, makes a 12% increase (from 34 to 46%) in fraction of proteins whose designed sequences are more than 35% identical to wild-type sequences. Meanwhile, it reduces 8% (from 22% to 14%) to the number of designed sequences that are not homologous to any known protein sequences according to psi-blast. More importantly, the sequences designed by RosettaDesign-SR have 2-3% more polar residues at the surface and core regions of proteins and these surface and core polar residues have about 4% higher sequence identity to wild-type sequences than by RosettaDesign. Thus, the proteins designed by RosettaDesign-SR should be less likely to aggregate and more likely to have unique structures due to more specific polar interactions.


Subject(s)
Amino Acid Sequence , Models, Molecular , Protein Conformation , Proteins/chemistry , Amino Acids/chemistry , Computational Biology/methods , Databases, Protein , Hydrophobic and Hydrophilic Interactions , Molecular Weight , Peptide Fragments , Protein Folding , Protein Stability , Protein Structure, Secondary , Sequence Homology, Amino Acid , Software , Surface Properties
18.
Bioorg Med Chem Lett ; 20(3): 922-6, 2010 Feb 01.
Article in English | MEDLINE | ID: mdl-20053559

ABSTRACT

Described herein is design, synthesis, and biological evaluation of novel series of 2-aryl-7-(3',4'-dialkoxyphenyl)-pyrazolo[1,5-a]pyrimidines acting as inhibitors of type 4 phosphodiesterase (PDE4) which is known as a good target for the treatment of asthma and COPD. For this purpose, structure optimization was conducted with the aid of structure-based drug design using the known X-ray crystallography. Also, biological effects of these compounds on the target enzyme were evaluated by using in vitro assays, leading to the potent and selective PDE-4 inhibitor (IC(50)<10nM).


Subject(s)
Drug Design , Phosphodiesterase 4 Inhibitors , Phosphodiesterase Inhibitors/chemical synthesis , Pyrazoles/chemical synthesis , Pyrimidines/chemical synthesis , Animals , Crystallography, X-Ray , Cyclic Nucleotide Phosphodiesterases, Type 4/metabolism , Drug Evaluation, Preclinical/methods , Humans , Mice , Phosphodiesterase Inhibitors/pharmacology , Pyrazoles/pharmacology , Pyrimidines/pharmacology
19.
Diabetes Res Clin Pract ; 81(3): 377-80, 2008 Sep.
Article in English | MEDLINE | ID: mdl-18617286

ABSTRACT

AIM: To compare the prevalence of metabolic syndrome (MetS) by combinations of MetS components derived from the National Cholesterol Education Program Adult Treatment Panel III (ATPIII) and International Diabetes Federation (IDF) definitions. METHODS: Four studies with ethnically distinct populations from the Asia-Pacific region were selected from the DETECT-2 study database. The prevalences of combinations of MetS components using the modified ATPIII (modATPIII) and IDF MetS definitions were compared between sexes and across populations. RESULTS: A total of 22,952 participants from Australia, Japan, Korea and Samoa were included. The age-adjusted prevalence of modATPIII MetS varied from 9.4 to 35.8% in men and 10.3 to 57.2% in women; results for IDF were generally higher. Prevalences of the 16 possible MetS component combinations from the modATPIII definition that result in a diagnosis of MetS ranged from 0 to 12.7%. Of those with IDF-defined abdominal obesity, the prevalences of the 11 IDF-defined MetS component combinations ranged from 0.2 to 18.3%. CONCLUSIONS: The large variation in the prevalence of possible MetS component combinations to diagnose MetS may explain the different risk of cardiovascular outcomes associated with MetS in different populations, especially since particular combinations of MetS components are associated with different risk of cardiovascular disease.


Subject(s)
Metabolic Syndrome/classification , Metabolic Syndrome/epidemiology , Australia/epidemiology , Blood Glucose/analysis , Blood Pressure , Body Size , Female , Humans , Japan/epidemiology , Korea/epidemiology , Lipids/blood , Male , Metabolic Syndrome/physiopathology , Prevalence , Risk Factors , Samoa/epidemiology
20.
Metab Syndr Relat Disord ; 6(1): 37-46, 2008 Mar.
Article in English | MEDLINE | ID: mdl-18370835

ABSTRACT

BACKGROUND: To compare the prevalence of metabolic syndrome (MetS) by four MetS definitions in four Asia-Pacific populations, and to compare the prevalence of individual metabolic components. METHODS: Population-based cross-sectional studies from Australia, Japan, Korea, and Samoa were used to assess the World Health Organization (WHO), European Group for the Study of Insulin Resistance (EGIR), modified National Cholesterol Education Program Adult Treatment Panel III (modATPIII), and International Diabetes Federation (IDF) MetS definitions. Age-adjusted MetS prevalences were compared within and between countries and kappa statistics were used to determine the agreement between IDF and the other three definitions. RESULTS: Japanese people had the lowest prevalence of MetS regardless of definition, and Samoans generally the highest prevalence. Age-adjusted prevalences for the four definitions ranged from 16% to 42% in Australia, 3% to 11% in Japan, 7% to 29% in Korea and 17% to 60% in Samoa. With the exceptions of Korean and Japanese males, the highest prevalence of MetS was obtained with the IDF definition. The best overall agreement with IDF MetS definition was for modATPIII, and the worst for EGIR. There were marked differences in the prevalence of MetS between the sexes, with no systematic pattern, and between the prevalences of individual metabolic components. CONCLUSIONS: Differences in the prevalence of MetS and its components, using the various definitions, both within and between populations, indicate that caution is required when comparing studies from different countries. Determining the clinical significance of these differences will require prospective outcome studies.


Subject(s)
Metabolic Syndrome/classification , Metabolic Syndrome/epidemiology , Population , Adult , Aged , Asia/epidemiology , Australia/epidemiology , Cross-Sectional Studies , Female , Health Status Indicators , Humans , Male , Middle Aged , Research Design , Sex Characteristics
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