Effect of hyperglycaemic conditions on the response of human periodontal ligament fibroblasts to mechanical stretching

Objectives: The aim of the present study was to investigate the impact of high glucose concentration on the response of human periodontal ligament fibroblasts (PDLFs) to cyclic tensile strain. Materials and methods: Human PDLFs were incubated under normal or high glucose conditions, and then were subjected to cyclic tensile stretching (8 per cent extension, 1 Hz). Gene expression was determined by quantitative real-time polymerase chain reaction. Intracellular reactive oxygen species (ROS) were determined by the 2',7'-dichlorofluorescein-diacetate assay, activation of mitogen-activated protein kinase (MAPK) was monitored by western analysis and osteoblastic differentiation was estimated with Alizarin Red-S staining. Results: Cyclic tensile stretching of PDLF leads to an immediate activation of extracellular signal-regulated kinase (ERK) and c-Jun N-terminal kinase (JNK), as well as to the increased expression of the transcription factor c-fos, known to regulate many osteogenesis-related genes. At later time points, the alkaline phosphatase and osteopontin genes were also upregulated. Hyperglycaemic conditions inhibited these effects. High glucose conditions were unable to increase ROS levels, but they increased the medium's osmolality. Finally, increase of osmolality mimics the inhibitory effect of hyperglycaemia on MAPK activation, c-fos and osteoblast-specific gene markers' upregulation, as well as osteogenic differentiation capacity. Conclusion: Our findings indicate that under high glucose conditions, human PDLFs fail to adequately respond to mechanical deformation, while their strain-elicited osteoblast differentiation ability is deteriorated. The aforementioned effects are most probably mediated by the increased osmolality under hyperglycaemic conditions

Early responses of human periodontal ligament fibroblasts to cyclic and static mechanical stretching

OBJECTIVE To compare the mechanotransduction caused by cyclic and static mechanical strains in human periodontal ligament fibroblasts (hPDLFs) cultured under identical conditions. MATERIALS AND METHODS hPDLFs, originating from the same donors, were exposed either to cyclic or to static tensile strain using specially designed devices and under identical culture conditions. Activation of all members of mitogen-activated protein kinases (MAPKs) was monitored by western immunoblot analysis. Expression levels of immediate/early genes c-fos and c-jun were assessed with quantitative real-time polymerase chain reaction. RESULTS Time course experiments revealed that both types of stresses activate the three members of MAPK, that is ERK, p38, and JNK, with cyclic stress exhibiting a slightly more extended activation. Further downstream, both stresses upregulate the immediate/early genes c-fos and c-jun, encoding components of the activator protein-1 (AP-1), a key transcription factor in osteoblastic differentiation; again cyclic strain provokes a more intense upregulation. Six hours after the application of both strains, MAPK activation and gene expression return to basal levels. Finally, cells exposed to cyclic stress for longer periods are distributed approximately perpendicular to the axis of the applied strain, whereas cells exposed to static loading remain in a random orientation in culture. CONCLUSION The findings of the present study indicate similar, although not identical, immediate/early responses of hPDLs to cyclic and static stretching, with cyclic strain provoking a more intense adaptive response of these cells to mechanical deformation

Senescent human periodontal ligament fibroblasts after replicative exhaustion or ionizing radiation have a decreased capacity towards osteoblastic differentiation

Loss of teeth increases with age or after genotoxic treatments, like head and neck radiotherapy, due to periodontium breakdown. Periodontal ligament fibroblasts represent the main cell type in this tissue and are crucial for the maintenance of homeodynamics and for its regeneration. Here, we have studied the characteristics of human periodontal ligament fibroblasts (hPDLF) that became senescent after replicative exhaustion or after exposure to ionizing radiation, as well as their ability for osteoblastic differentiation. We found that senescent hPDLF express classical markers of senescence, as well as a catabolic phenotype, as shown by the decrease in collagen type I and the increase of MMP-2 expression. In addition, we observed a considerably decreased expression of the major transcription factor for osteoblastic differentiation, i.e. Runx2, a down-regulation which was found to be p53-dependent. In accordance to the above, senescent cells have a significantly decreased alkaline phosphatase gene expression and activity, as well as a reduced ability for osteoblastic differentiation, as found by Alizarin Red staining. Interestingly, cells from both type of senescence express similar characteristics, implying analogous functions in vivo. In conclusion, senescent hPDLF express a catabolic phenotype and express a significantly decreased ability towards an osteoblastic differentiation, thus probably affecting tissue development and integrity

Bioactive Metabolites of the Stem Bark of <i>Strychnos aff. darienensis</i> and Evaluation of Their Antioxidant and UV Protection Activity in Human Skin Cell Cultures

The genus Strychnos (Loganiaceae) is well-known as a rich source of various bioactive metabolites. In continuation of our phytochemical studies on plants from Amazonia, we examined Strychnos aff. darienensis, collected in Peru. This species has been traditionally used in South America and is still presently used as a drug by the Yanesha tribe in Peru. Phytochemical investigation of this plant led to the isolation and structure elucidation by &#925;uclear&#924;agnetic Resonance and High Resolution Mass Spectroscopy of 14 compounds that belong to the categories of phenolic acids [p-hydroxybenzoic acid (1) and vanillic acid (2)], flavonoids [luteolin, (3),3-O-methyl quercetin (4), strychnobiflavone (5), minaxin (6) and 3&#8217;,4&#8217;,7-trihydroxy-flavone (7)], lignans [syringaresinol-&#946;-D-glucoside (8), balanophonin (9) and ficusal (10)] and alkaloids [venoterpine (11), 11-methoxyhenningsamine (12), diaboline (13) and 11-methoxy diaboline (14)]. The isolated flavonoids&#8212;a class known for its anti-aging activities&#8212;were further evaluated for their biological activities on normal human skin fibroblasts. Among them, only (6), and to a lesser extent (7), exhibited cytotoxicity at 100 &#181;g/ml. All five flavonoids suppressed intracellularreactive oxygen species (ROS) levels, either basal or following stimulation with hydrogen peroxide or both. Moreover, luteolin and strychnobiflavone protected skin fibroblasts against ultraviolet (UV)-irradiation-induced cell death. The isolated flavonoids could prove useful bioactive ingredients in the cosmetic industry

Optimized synthesis of novel prenyl ferulate performed by feruloyl esterases from Myceliophthora thermophila in microemulsions (Applied Microbiology and Biotechnology, (2017), 101, 8, (3213-3226), 10.1007/s00253-017-8089-8)

Five feruloyl esterases (FAEs; EC 3.1.1.73), FaeA1, FaeA2, FaeB1, and FaeB2 from Myceliophthora thermophila C1 and MtFae1a from M. thermophila ATCC 42464, were tested for their ability to catalyze the transesterification of vinyl ferulate (VFA) with prenol in detergentless microemulsions. Reaction conditions were optimized investigating parameters such as the medium composition, the substrate concentration, the enzyme load, the pH, the temperature, and agitation. FaeB2 offered the highest transesterification yield (71.5 ± 0.2%) after 24 h of incubation at 30 °C using 60 mM VFA, 1 M prenol, and 0.02 mg FAE/mL in a mixture comprising of 53.4:43.4:3.2 v/v/v n-hexane:t-butanol:100 mM MOPS-NaOH, pH 6.0. At these conditions, the competitive side hydrolysis of VFA was 4.7-fold minimized. The ability of prenyl ferulate (PFA) and its corresponding ferulic acid (FA) to scavenge 1,1-diphenyl-2-picrylhydrazyl (DPPH) radicals was significant and similar (IC50 423.39 μM for PFA, 329.9 μM for FA). PFA was not cytotoxic at 0.8-100 μM (IC50 220.23 μM) and reduced intracellular reactive oxygen species (ROS) in human skin fibroblasts at concentrations ranging between 4 and 20 μM as determined with the dichloro-dihydro-fluorescein diacetate (DCFH-DA) assay

Mobile-phone radiation-induced perturbation of gene-expression profiling, redox equilibrium and sporadic-apoptosis control in the ovary of <i>Drosophila melanogaster</i>

<p>The daily use by people of wireless communication devices has increased exponentially in the last decade, begetting concerns regarding its potential health hazards. <i>Drosophila melanogaster</i> four days-old adult female flies were exposed for 30 min to radiation emitted by a commercial mobile phone at a SAR of 0.15 W/kg and a SAE of 270 J/kg. ROS levels and apoptotic follicles were assayed in parallel with a genome-wide microarrays analysis. ROS cellular contents were found to increase by 1.6-fold (x), immediately after the end of exposure, in follicles of pre-choriogenic stages (germarium - stage 10), while sporadically generated apoptotic follicles (germarium 2b and stages 7–9) presented with an averaged 2x upregulation in their sub-population mass, 4 h after fly's irradiation with mobile device. Microarray analysis revealed 168 genes being differentially expressed, 2 h post-exposure, in response to radiofrequency (RF) electromagnetic field-radiation exposure (≥1.25x, <i>P < 0.05)</i> and associated with multiple and critical biological processes, such as basic metabolism and cellular subroutines related to stress response and apoptotic death. Exposure of adult flies to mobile-phone radiation for 30 min has an immediate impact on ROS production in animal's ovary, which seems to cause a global, systemic and non-targeted transcriptional reprogramming of gene expression, 2 h post-exposure, being finally followed by induction of apoptosis 4 h after the end of exposure. Conclusively, this unique type of pulsed radiation, mainly being derived from daily used mobile phones, seems capable of mobilizing critical cytopathic mechanisms, and altering fundamental genetic programs and networks in <i>D. melanogaster</i>.</p