ABSTRACT
BACKGROUND AND OBJECTIVE: Urokinase-plasminogen activator (uPA) is a serine protease expressed at high basal level in normal gingival cervical fluid. Despite its known pathologic role in tissue proteolysis in periodontitis, little is known concerning uPA physiological function in oral tissue. Recent evidence in cancer cells has implicated the uPA system in DNA repair and anti-apoptotic pathways. This study is aimed to evaluate the protective function of urokinase against oxidative DNA damage in periodontal ligament (PDL) fibroblast, and to propose a new biological role for uPA in oral cavity. MATERIAL AND METHODS: PDL cells were isolated from human wisdom teeth obtained from healthy donors. An oxidative stress model was created in which PDL cells were incubated with 20, 30, 40 and 60 µmol/L hydrogen peroxide. Twenty-four hours before and after peroxide treatment, cells were treated with uPA and amiloride. Cell viability was assessed by 3-(4,5-dimethylthiazol-2-yl)-2,5diphenyltetrazolium bromide assay, apoptosis by DAPI-staining and annexin V/propidium iodide assay, and DNA breaks by alkaline comet assay. For estimating DNA damage level, γ-H2AX expression was studied using flow cytometry and immunostaining. RESULTS: The incubation of the peroxide-treated cells with uPA significantly increased cell viability and decreased apoptosis. A significant decrease in the number of γ-H2AX foci was seen at 30 µmol/L hydrogen peroxide in uPA-treated cells. uPA inhibition as a result of amiloride treatment, in turn, induced a reduction in cell viability. In addition, there was a significant decrease in the levels of DNA damage in uPA-treated groups as measured by the comet assay. CONCLUSION: The present study brings support to the theory that uPA may have a protective role for periodontal tissue and could protect PDL fibroblasts from oxidative DNA damage and apoptosis.
Subject(s)
Apoptosis/drug effects , DNA Damage/drug effects , Fibroblasts/drug effects , Oxidative Stress/drug effects , Periodontal Ligament/cytology , Urokinase-Type Plasminogen Activator/pharmacology , Amiloride/pharmacology , Cell Survival , Cells, Cultured , Flow Cytometry , HumansABSTRACT
Autism spectrum disorders (ASD) are classified as neurological developmental disorders. Several studies have been carried out to find a candidate biomarker linked to the development of these disorders, but up to date no reliable biomarker is available. Mass spectrometry techniques have been used for protein profiling of blood plasma of children with such disorders in order to identify proteins/peptides that may be used as biomarkers for detection of the disorders. Three differentially expressed peptides with mass-charge (m/z) values of 2020 ± 1, 1864 ± 1 and 1978 ± 1 Da in the heparin plasma of children with ASD that were significantly changed as compared with the peptide pattern of the non-ASD control group are reported here. This novel set of biomarkers allows for a reliable blood-based diagnostic tool that may be used in diagnosis and potentially, in prognosis of ASD.
Subject(s)
Autistic Disorder/blood , Autistic Disorder/diagnosis , Biomarkers/blood , Proteomics , 1-Acylglycerophosphocholine O-Acyltransferase/blood , Autistic Disorder/psychology , Child , Child, Preschool , Female , Humans , Intellectual Disability/blood , Intellectual Disability/diagnosis , Intellectual Disability/psychology , Male , Mass Spectrometry , Peptide Fragments/blood , Prognosis , Protein Array Analysis , Reference Values , Sequence Analysis, ProteinABSTRACT
Prolyl endopeptidase (EC 3.4.21.26) (PEP) is present in nearly all investigated mammalian cells and biological fluids and might be involved in the degradation of physiologically important neuropeptides. To be able to investigate the variation of PEP in blood and cerebrospinal fluid (CSF) in human disease, the factors influencing analysis of PEP in these body fluids must be determined. The purpose of the present work was to study the influence of storage conditions, anticoagulation additives, freezing and thawing and substrate solvent on determination of PEP in blood plasma/serum and CSF. It was found that the PEP activity was about 10% higher in plasma (with EDTA and heparinate for anticoagulation) than in serum. Storage at room temperature (20 degrees C) caused a rapid decline in enzyme activity, which was smaller but still considerable at 4 degrees C. Storage at -20 degrees C and -70 degrees C did not decrease the PEP activity. Freezing and thawing of plasma/serum samples showed that the first freeze-thawing cycle produced a 20% reduction in enzyme activity but little further decrease was observed during subsequent cycles of freeze-thawing. In conclusion, PEP activity should preferably be measured within one hour after sampling using EDTA- or heparinate plasma. For long-term storage, samples should be immediately frozen and stored at -20 degrees C or colder. The selection and amount of the organic solvent used to dissolve the fluorogenic substrate strongly influenced the sensitivity of the assay. By developing an optimal solvent system an increase in assay sensitivity of about 400% could be obtained, which for the first time allowed measurement of the PEP activity in CSF.
