44 research outputs found
Anti-Obesity and Anti-Inflammatory Effects of Novel Carvacrol Derivatives on 3T3-L1 and WJ-MSCs Cells
Get PDF(1) Background: Obesity, a complex metabolic disease resulting from an imbalance between food consumption and energy expenditure, leads to an increase in adipocytes and chronic inflammatory conditions. The aim of this paper was to synthesize a small series of carvacrol derivatives (CD1-3) that are able to reduce both adipogenesis and the inflammatory status often associated with the progression of the obesity disease. (2) Methods: The synthesis of CD1-3 was performed using classical procedures in a solution phase. Biological studies were performed on three cell lines: 3T3-L1, WJ-MSCs, and THP-1. The anti-adipogenic properties of CD1-3 were evaluated using western blotting and densitometric analysis by assessing the expression of obesity-related proteins, such as ChREBP. The anti-inflammatory effect was estimated by measuring the reduction in TNF-α expression in CD1-3-treated THP-1 cells. (3) Results: CD1-3—obtained through a direct linkage between the carboxylic moiety of anti-inflammatory drugs (Ibuprofen, Flurbiprofen, and Naproxen) and the hydroxyl group of carvacrol—have an inhibitory effect on the accumulation of lipids in both 3T3-L1 and WJ-MSCs cell cultures and an anti-inflammatory effect by reducing TNF- α levels in THP-1 cells. (4) Conclusions: Considering the physicochemical properties, stability, and biological data, the CD3 derivative—obtained by a direct linkage between carvacrol and naproxen—resulted in the best candidate, displaying anti-obesity and anti-inflammatory effects in vitro
Helicobacter Pylori ATCC 43629 Outer Membrane Vesicles (OMVS): a vehicle for extracellular dNA (eDNA) transport
No full textImmunolocalization of glutathione S-transferase (GST) and peptide methionine sulphoxide reductase (MSRA) in Ochrobactrum anthopi.
No full textCyanidin reduces preadipocyte differentiation and relative ChREBP expression
No full textAdipogenesis is a continuous process even in adult adipose tissue for the presence of preadipocytes that, when subjected to appropriate stimuli can proliferate and differentiate. ChREBP, the essential transcription factor for lipogenesis, is expressed in all tissues, but mainly in lipogenic organs. In this study, we focused on ChREBP expression during preadipocytes differentiation. Since it was found that cyanidin-3 reduces body weight in mice even in the presence of a high-fat diet, by decreasing levels of blood glucose and by improving insulin sensitivity, we studied the effect of this substance on adipogenic differentiation. For this purpose we used preadipocytes obtained from subcutaneous and visceral human adipose explant tissue, characterized and stimulated to differentiate in selective media. On cytofluorimetric analysis these cells showed mesenchymal markers (CD29, CD90, CD44), whereas they were negative for hematopoietic markers (CD45, CD10, CD117,CD31). ChREBP expression levels were quantified by immunoelectron-microscopy and western blotting analysis. In this report we show that ChREBP is expressed in preadipocytes (both nuclear and cytoplasmic compartments); the cytoplasmic level of ChREBP increased by 50 percent on day seven of differentiation into mature adipocytes. Cyanidin reduced differentiation by 20 percent (as evaluated by red oil O staining) and the expression of ChREBP. In addition, cyanidin-treated cells showed abnormal morphology, a square shape with irregular size, probably due to the fact that cyanidin may interfere with the extracellular matrix. These findings suggest that dietary cyanidin, may have inhibitory effects on adipogenesi
Carvacrol codrugs: a new approach in the antimicrobial plan.
Get PDFOBJECTIVE:The increasing prevalence of antibiotic-resistant bacterial infections led to identify alternative strategies for a novel therapeutic approach. In this study, we synthesized ten carvacrol codrugs - obtained linking the carvacrol hydroxyl group to the carboxyl moiety of sulphur-containing amino acids via an ester bond - to develop novel compounds with improved antimicrobial and antibiofilm activities and reduced toxicity respect to carvacrol alone. METHOD:All carvacrol codrugs were screened against a representative panel of Gram positive (S. aureus and S. epidermidis), Gram negative (E. coli and P. aeruginosa) bacterial strains and C. albicans, using broth microdilution assays. FINDINGS:Results showed that carvacrol codrug 4 possesses the most notable enhancement in the anti-bacterial activity displaying MIC and MBC values equal to 2.5 mg/mL for all bacterial strains, except for P. aeruginosa ATCC 9027 (MIC and MBC values equal to 5 mg/mL and 10 mg/mL, respectively). All carvacrol codrugs 1-10 revealed good antifungal activity against C. albicans ATCC 10231. The cytotoxicity assay showed that the novel carvacrol codrugs did not produce human blood hemolysis at their MIC values except for codrugs 8 and 9. In particular, deepened experiments performed on carvacrol codrug 4 showed an interesting antimicrobial effect on the mature biofilm produced by E. coli ATCC 8739, respect to the carvacrol alone. The antimicrobial effects of carvacrol codrug 4 were also analyzed by TEM evidencing morphological modifications in S. aureus, E. coli, and C. albicans. CONCLUSION:The current study presents an insight into the use of codrug strategy for developing carvacrol derivatives with antibacterial and antibiofilm potentials, and reduced cytotoxicity
A method to study the C924T polymorphism of the thromboxane A2 receptor gene
No full textThe thromboxane A2 receptor (TBXA2R) gene is a member of the G-protein coupled superfamily with seven-transmembrane regions. It is involved in atherogenesis progression, ischemia, and myocardial infarction. Here we present a methodology of patient genotyping to investigate the post-transcriptional role of the C924T polymorphism (rs4523) situated at the 3' region of the TBXA2 receptor gene. This method relies on DNA extraction from whole blood, polymerase chain reaction (PCR) amplification of the TBXA2 gene portion containing the C924T mutation, and identification of wild type and/or mutant genotypes using a restriction digest analysis, specifically a restriction fragment length polymorphism (RFLP) on agarose gel. In addition, the results were confirmed by sequencing the TBXA2R gene. This method features several potential advantages, such as high efficiency and the rapid identification of the C924T polymorphism by PCR and restriction enzyme analysis. This approach allows a predictive study for plaque formation and atherosclerosis progression by analyzing patient genotypes for the TBXA2R C924T polymorphism. Application of this method has the potential to identify subjects who are more susceptible to atherothrombotic processes, in particular subjects in a high-risk, aspirin-treated group
Helicobacter pylori ATCC 43629/NCTC 11639 Outer Membrane Vesicles (OMVs) from biofilm and planktonic phase associated with extracellular DNA (eDNA)
No full textHelicobacter pylori persistence is associated with its capacity to develop biofilms as a response to changing environmental conditions and stress. Extracellular DNA (eDNA) is a component of H. pylori biofilm matrix but the lack of DNase I activity supports the hypothesis that eDNA might be protected by other extracellular polymeric substances (EPS) and/or Outer Membrane Vesicles (OMVs), which bleb from the bacteria surface during growth. The aim of the present study was to both identify the eDNA presence on OMVs segregated from H. pylori ATCC 43629/NCTC 11639 biofilm (bOMVs) and its planktonic phase (pOMVs) and to characterize the physical-chemical properties of the OMVs. The presence of eDNA in bOMVs and pOMVs was initially carried out using DNase I-gold complex labeling and Transmission Electron Microscope analysis (TEM). bOMVs and pOMVs were further isolated and physical-chemical characterization carried out using dynamic light scattering (DLS) analysis. eDNA associated with OMVs was detected and quantified using a PicoGreen spectrophotometer assay, while its extraction was performed with a DNA Kit. TEM images showed that eDNA was mainly associated with the OMV membrane surfaces; while PicoGreen staining showed a four-fold increase of dsDNA in bOMVs compared with pOMVs. The eDNA extracted from OMVs was visualized using gel electrophoresis. DLS analysis indicated that both planktonic and biofilm H. pylori phenotypes generated vesicles, with a broad distribution of sizes on the nanometer scale. The DLS aggregation assay suggested that eDNA may play a role in the aggregation of OMVs, in the biofilm phenotype. Moreover, the eDNA associated with vesicle membrane may impede DNase I activity on H. pylori biofilms. These results suggest that OMVs derived from the H. pylori biofilm phenotype may play a structural role by preventing eDNA degradation by nucleases, providing a bridging function between eDNA strands on OMV surfaces and promoting aggregation
Carvacrol reduces adipogenic differentiation by modulating autophagy and ChREBP expression.
Get PDFOBJECTIVE:Obesity is the result of white adipose tissue accumulation where excess of food energy is stored to form triglycerides. De novo lipogenesis (DNL) is the continuous process of new fat production and is driven by the transcription factor ChREBP. During adipogenesis, white adipocytes change their morphology and the entire cell volume is occupied by one large lipid droplet. Recent studies have implicated an essential role of autophagy in adipogenic differentiation, cytoplasmic remodelling and mitochondria reorganization. The phenolic monoterpenoid carvacrol (2-methyl-5-[1-methylethyl]phenol), produced by numerous aromatic plants, has been shown to reduce lipid accumulation in murine 3T3-L1 cells during adipogenic differentiation by modulating genes associated with adipogenesis and inflammation. Therefore, the aim of this study was to evaluate whether carvacrol could affect autophagy and ChREBP expression during adipogenic differentiation. METHODS:The study was carried on by using the murine 3T3-L1 and the human WJ-MSCs (Wharton's jelly-derived mesenchymal stem cells) cell lines. Cells undergoing adipogenic differentiation were untreated or treated with carvacrol. Adipogenic differentiation was assessed by analyzing cellular lipid accumulation with Oil-Red O staining and by ultrastructural examination with TEM. Autophagy was evaluated by western immunoblotting of autophagy markers LC3B and p62/SQSTM and by ultrastructural examination of autophagic bodies. Autophagic flux was evaluated by using autophagy inhibitor cloroquine (CQ). ChREBP expression levels was assessed by both western blotting and immunoelectron microscopy and ChREBP activity by analysis of adipogenic target genes expression. RESULTS:We found that carvacrol reduced adipogenic differentiation of about 40% and 30% in, respectively, 3T3-L1 and in WJ-MSCs cells. The effect of carvacrol on adipogenic differentiation correlated with both reduction of autophagy and reduction of ChREBP expression. CONCLUSION:The results support the notion that carvacrol, through its effect on autophagy (essential for adipocyte maturation) and on ChREBP activity, could be used as a valuable adjuvant to reduce adipogenic differentiation
Preparation, Characterization, and Biological Evaluation of a Hydrophilic Peptide Loaded on PEG-PLGA Nanoparticles
Get PDFThe encapsulation of peptides and proteins in nanosystems has been extensively investigated for masking unfavorable biopharmaceutical properties, including short half-life and poor permeation through biological membranes. Therefore, the aim of this work was to encapsulate a small antimicrobial hydrophilic peptide (H-Ser-Pro-Trp-Thr-NH2, FS10) in PEG-PLGA (polyethylene glycol-poly lactic acid-co-glycolic acid) nanoparticles (Nps) and thereby overcome the common limitations of hydrophilic drugs, which because they facilitate water absorption suffer from rapid degradation. FS10 is structurally related to the well-known RNAIII inhibiting peptide (RIP) and inhibits S. aureus biofilm formation. Various parameters, including different method (double emulsion and nanoprecipitation), pH of the aqueous phase and polymeric composition, were investigated to load FS10 into PEG-PLGA nanoparticles. The combination of different strategies resulted in an encapsulation efficiency of around 25% for both the double emulsion and the nanoprecipitation method. It was found that the most influential parameters were the pH—which tailors the peptides charge—and the polymeric composition. FS10-PEG-PLGA nanoparticles, obtained under optimized parameters, showed size lower than 180 nm with zeta potential values ranging from −11 to −21 mV. In vitro release studies showed that the Nps had an initial burst release of 48–63%, followed by a continuous drug release up to 21 h, probably caused by the porous character of the Nps. Furthermore, transmission electron microscopy (TEM) analysis revealed particles with a spherical morphology and size of around 100 nm. Antimicrobial assay showed that the minimum inhibitory concentration (MIC) of the FS10-loaded Nps, against S. aureus strains, was lower (>128 µg/mL) than that of the free FS10 (>256 µg/mL). The main goal of this work was to develop polymeric drug delivery systems aiming at protecting the peptide from a fast degradation, thus improving its accumulation in the target site and increasing the drug-bacterial membrane interactions
Exogenous human immunodeficiency virus type‐1 Tat protein selectively stimulates a phosphatidylinositol‐specific phospholipase C nuclear pathway in the Jurkat T cell line
No full textWe investigated the effect of extracellular Tat protein of human immunodeficiency virus-type 1 (HIV-1) on the phosphatidylinositol (PI) cycle, which represents a major signal transduction pathway in lymphoid cells. Recombinant Tat, recombinant HIV-1 p24 and cross-linked anti-CD3 monoclonal antibody (mAb) were added in culture for 1-60 min to Jurkat lymphoblastoid CD4+ T cells. The stimulation of T cell receptor by cross-linked anti-CD3 mAb resulted in a rapid increase of the phosphatidylinositol-specific phospholipase C (PI-PLC) activity in whole cell lysates. On the other hand, Tat protein, either alone or in combination with anti-CD3 mAb, showed little effect on the PI turnover of whole cell extracts. Tat, however, selectively stimulated a nuclear-specific PI-PLC with a peak of activity after 30 min from the addition in culture to Jurkat cells. Interestingly, this time corresponded to that required for the uptake and nuclear localization of recombinant Tat protein, as demonstrated by electron microscope immunocytochemistry experiments with anti-Tat mAb. Moreover, exogenous Tat reached the nucleus of Jurkat cells in a bioactive form, as shown in a HIV-1 long terminal repeat-chloramphenicol acetyl transferase transactivation assay. The specific increase of a nuclear PI-PLC activity was further demonstrated by the ability of Tat to stimulate PI turnover also when added directly to isolated nuclei. As a whole, these data demonstrate that Tat selectively stimulates a nuclear polyphosphoinositide hydrolysis, which appears to be independent of the cellular PI turnover. The relevance of these findings for a better understanding of the biological functions of extracellular Tat is discussed
