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- The Effect of Conjugated Linoleic Acids (CLA) Supplementation on the Activity of Enzymes Participating in the Formation of Arachidonic Acid in Liver Microsomes of Rats-Probable Mechanism of CLA Anticancer Activity. [JOURNAL ARTICLE]
- Nutr Cancer 2014 Nov 20.:1-11.
The aim of the present research was to examine the effect of conjugated linoleic acids (CLA) supplementation on the activity of enzymes that take part in the synthesis of arachidonic acid (AA) and also to investigate the relation between their activity and the neoplastic process. The enzyme activities were established indirectly, because their measure was the amount of AA formed in vitro, being developed from linoleic acid as determined in liver microsomes of Spraque-Dawley rats. In addition, the indices of Δ(6)-desaturase (D6D) and Δ(5)-desaturase (D5D) were determined. To this aim, the method of high per-formance liquid chromatography with UV/VIS detection was used. Between the examined groups, statistically significant differences were observed in the activities of enzymes as well as D6D. The carcinogenic agent applied (DMBA) was found to significantly increase the activity of the examined enzymes. Negative correlation was found between the activities of desaturases and CLA supplementation, whereas the activity of those enzymes was a little higher in the group of rats with chemically induced cancer process. The neoplastic process has a stimulating effect on the activity of D6D. The decrease of D6D activity, resulting from the presence of CLA in the animals' diet, may confirm the anticancer properties of these isomers.
- Identification of Ser/Thr kinase and Forkhead Associated Domains in Mycobacterium ulcerans: Characterization of Novel Association between Protein Kinase Q and MupFHA. [JOURNAL ARTICLE]
- PLoS Negl Trop Dis 2014 Nov; 8(11):e3315.
Mycobacterium ulcerans, the causative agent of Buruli ulcer in humans, is unique among the members of Mycobacterium genus due to the presence of the virulence determinant megaplasmid pMUM001. This plasmid encodes multiple virulence-associated genes, including mup011, which is an uncharacterized Ser/Thr protein kinase (STPK) PknQ.In this study, we have characterized PknQ and explored its interaction with MupFHA (Mup018c), a FHA domain containing protein also encoded by pMUM001. MupFHA was found to interact with PknQ and suppress its autophosphorylation. Subsequent protein-protein docking and molecular dynamic simulation analyses showed that this interaction involves the FHA domain of MupFHA and PknQ activation loop residues Ser170 and Thr174. FHA domains are known to recognize phosphothreonine residues, and therefore, MupFHA may be acting as one of the few unusual FHA-domain having overlapping specificity. Additionally, we elucidated the PknQ-dependent regulation of MupDivIVA (Mup012c), which is a DivIVA domain containing protein encoded by pMUM001. MupDivIVA interacts with MupFHA and this interaction may also involve phospho-threonine/serine residues of MupDivIVA.Together, these results describe novel signaling mechanisms in M. ulcerans and show a three-way regulation of PknQ, MupFHA, and MupDivIVA. FHA domains have been considered to be only pThr specific and our results indicate a novel mechanism of pSer as well as pThr interaction exhibited by MupFHA. These results signify the need of further re-evaluating the FHA domain -pThr/pSer interaction model. MupFHA may serve as the ideal candidate for structural studies on this unique class of modular enzymes.
- Thymosin Beta 4 Protects Mice from Monocrotaline-Induced Pulmonary Hypertension and Right Ventricular Hypertrophy. [JOURNAL ARTICLE]
- PLoS One 2014; 9(11):e110598.
Pulmonary hypertension (PH) is a progressive vascular disease of pulmonary arteries that impedes ejection of blood by the right ventricle. As a result there is an increase in pulmonary vascular resistance and pulmonary arterial pressure causing right ventricular hypertrophy (RVH) and RV failure. The pathology of PAH involves vascular cell remodeling including pulmonary arterial endothelial cell (PAEC) dysfunction and pulmonary arterial smooth muscle cell (PASMC) proliferation. Current therapies are limited to reverse the vascular remodeling. Investigating a key molecule is required for development of new therapeutic intervention. Thymosin beta-4 (Tβ4) is a ubiquitous G-actin sequestering protein with diverse biological function and promotes wound healing and modulates inflammatory responses. However, it remains unknown whether Tβ4 has any protective role in PH. The purpose of this study is to evaluate the whether Tβ4 can be used as a vascular-protective agent. In monocrotaline (MCT)-induced PH mouse model, we showed that mice treated with Tβ4 significantly attenuated the systolic pressure and RVH, compared to the MCT treated mice. Our data revealed for the first time that Tβ4 selectively targets Notch3-Col 3A-CTGF gene axis in preventing MCT-induced PH and RVH. Our study may provide pre-clinical evidence for Tβ4 and may consider as vasculo-protective agent for the treatment of PH induced RVH.
- Determination of Absolute Configuration in Chiral Solvents with Nuclear Magnetic Resonance. A Combined Molecular Dynamics/Quantum Chemical Study. [JOURNAL ARTICLE]
- J Phys Chem A 2014 Nov 20.
Nuclear magnetic resonance (NMR) spectroscopy is omnipresent in chemical analysis. However, chirality of a molecule can only be detected indirectly by NMR, e.g., by monitoring its interaction with another chiral object. In the present study, we investigate the spectroscopic behavior of chiral molecules placed into a chiral solvent. In this case, the solvent-solute interaction is much weaker, but the application range of such NMR analysis is wider than for a specific chemical shift agent. Two alcohols and an amine were used as model systems, and differences in NMR chemical shifts dependent on the solute-solvent chirality combination were experimentally detected. Combined quantum mechanic/molecular mechanic (QM/MM) computations were applied to reveal the underlying solute-solvent interactions. NMR shielding was calculated using the density functional theory (DFT). While the experimental observations could not be reproduced quantitatively, the modeling provided a qualitative agreement and detailed insight into the essence of solvent-solute chiral interactions. The potentials of mean force (PMF) obtained using molecular dynamics (MD) and the weighted histogram analysis method (WHAM) indicate that the chiral interaction brings about differences in conformer ratios, which are to a large extent responsible for the NMR shifts. The MD results also predicted slight changes in the solvent structure, including the radial distribution function (RDF), to depend on the solvent/solute chirality combination. Apart from the conformer distribution, an effective average solvent electrostatic field was tested as another major factor contributing to the chiral NMR effect. The possibility to simulate spectral effects of chiral solvents from the first principles opens up the way to NMR spectroscopic determination of the absolute configuration for a larger scale of compounds, including those not forming specific complexes.
- Revealing the macromolecular targets of complex natural products. [JOURNAL ARTICLE]
- Nat Chem 2014 Dec; 6(12):1072-1078.
Natural products have long been a source of useful biological activity for the development of new drugs. Their macromolecular targets are, however, largely unknown, which hampers rational drug design and optimization. Here we present the development and experimental validation of a computational method for the discovery of such targets. The technique does not require three-dimensional target models and may be applied to structurally complex natural products. The algorithm dissects the natural products into fragments and infers potential pharmacological targets by comparing the fragments to synthetic reference drugs with known targets. We demonstrate that this approach results in confident predictions. In a prospective validation, we show that fragments of the potent antitumour agent archazolid A, a macrolide from the myxobacterium Archangium gephyra, contain relevant information regarding its polypharmacology. Biochemical and biophysical evaluation confirmed the predictions. The results obtained corroborate the practical applicability of the computational approach to natural product 'de-orphaning'.
- The Protective Effect of Capparis ovata on 6-Mercaptopurine-induced Hepatotoxicity and Oxidative Stress in Rats. [JOURNAL ARTICLE]
- J Pediatr Hematol Oncol 2014 Nov 19.
Capparis ovata is a member of Capparidacaeae family has been used in phytomedicine with a lot of positive effects such as an antioxidative, antihyperlipidemic, anti-inflammatory, and antihepatotoxic agent. The aim of this study was to research the protective effect of C. ovata on 6-mercaptopurine (6-MP) induced to hepatotoxicity and oxidative stress in rats. The rats were divided into 4 groups: control, 6-MP, C. ovataovate, and 6-MP+C. ovata. A complete blood count was performed, liver function test and antioxidant enzymes levels such as superoxide dismutase, glutathione peroxidase, catalase, and malondialdehyde were measured in blood before and after a 14-day test period. White blood cell and platelet counts were lower in the 6-MP group than other 3 groups (P<0.005). Hepatic transaminase levels were higher in 6-MP group than the 3 groups (P<0.05). Superoxide dismutase, glutathione peroxidase, and CAT levels were lower and malondialdehyde was higher in blood samples in 6-MP group than other 3 groups (P<0.005). In conclusion, our tests were showed that C. ovata may be useful in patients receiving 6-MP therapy to prevent hepatotoxicity and in order to maintain uninterrupted therapy possibly reducing the risk of relapse. Although additional studies ensure that Capparis does not affect 6-MP antileukemic activity. We believe these results are important contribution to the literature.
- Effect of cyclin-dependent kinase 7 silencing on cisplatin sensitivity in endometrial carcinoma cells. [JOURNAL ARTICLE]
- Mol Med Rep 2014 Nov 19.
The aim of the present study was to determine the effect of cyclin‑dependent kinase 7 (CDK7) silencing on the sensitivity of the HEC‑1‑A endometrial carcinoma cell line to cisplatin [cis‑dichlorodiammineplatinum (II), or DDP]. Four CDK7 siRNA fragments were designed and synthesized based on the gene sequence of CDK7 and transfected into HEC‑1‑A cells. The RNA interference of the fragments was confirmed by semi‑quantitative polymerase chain reaction (PCR) and western blot analyses. The CDK7‑423 siRNA fragment exhibited the most marked silencing of CDK‑7 (>70%), and was chosen for the subsequent experiments in HEC‑1‑A endometrial carcinoma cells. The sensitivity of the cells to a chemotherapeutic agent (cisplatin) was determined before and after transfection of the siRNA, using a MTT cytotoxicity assay, flow cytometry and Hoechst/propidium iodide (PI) double‑staining immunofluorescence microscopy. The results of the MTT cytotoxicity assay showed that the half maximal inhibitory concentration of cisplatin was reduced from 45.12 µg/ml to 3.200 µg/ml following the inhibition of CDK7 expression levels, indicating a significantly increased cytotoxicity in the treated cells (P<0.05). The flow cytometry analysis showed that the mean rate of apoptosis in the CDK7 low‑expression group was 37.57%, which was significantly higher than the rate in the parental cells (11.66%) (P<0.05). Hoechst/PI co‑immunofluorescence microscopy revealed that the number of apoptotic bodies in the CDK7 low‑expression HEC‑1‑A cells was significantly increased as compared with the parental cells. Downregulation of CDK7 expression levels in HEC‑1‑A endometrial carcinoma cells via the transfection of CDK7 siRNA may significantly enhance cancer cell sensitivity to cisplatin chemotherapy and increasing apoptosis. CDK7 is a novel promising treatment for endometrial carcinoma that requires further in‑depth study.
- Evaluation of the masseter muscle elasticity with the use of acoustic coupling agents as references in strain sonoelastography. [JOURNAL ARTICLE]
- Dentomaxillofac Radiol 2014 Nov 20.:20140302.
Objective: To verify the use of a single coupling agent as a reference to obtain the elasticity index (EI) ratios and to investigate the EI ratios of the masseter muscles of healthy volunteers. Methods: Muscle phantoms with known elasticity (20, 40, and 60 kPa in the Young's modulus) were examined by a strain type sonoelastography using the coupling agent as a reference. Eight examiners tested the soft (with 7 kPa) and hard (with 40 kpa) reference coupling agents separately. The correlation coefficients were determined between the EI ratio and the Young's modulus of muscle phantoms. The interclass correlation coefficients were calculated for the inter- and intra-examiner agreement. Results: Strong correlations were found between the EI ratios and the Young's modulus for both the soft and hard references. The variations of EI ratios were larger with the soft coupling agent than with the hard coupling agent, and they increased in the phantoms with 60 kPa elasticity. There were no differences in the EI ratios of the masseter muscle at rest between male and female, and between the right and left sides. The ratio increased during clenching. Conclusions: The hard reference coupling agent was suitable for obtaining the EI ratio of the masseter muscle. No differences were found in the masseter muscle EI ratios neither between sexes nor between the right and left sides at rest, and the rations increased with widening of their variations during clenching.
- Myocardial clearance of technetium-99m-teboroxime in reperfused injured canine myocardium. [JOURNAL ARTICLE]
- EJNMMI Res 2014.:42.
Recent technical developments using solid-state technology have enabled rapid image acquisition with single photon emission computed tomography (SPECT) and have led to a renewed interest in technetium-99m-teboroxime (Tc-99m-teboroxime) as a myocardial imaging agent. Tc-99m-teboroxime has demonstrated high myocardial extraction, linear myocardial uptake relative to flow even at high flow rates, rapid uptake and clearance kinetics, and differential clearance in the setting of ischemia. However, the myocardial clearance kinetics of Tc-99m-teboroxime in a model of myocardial injury has not been previously reported. Thus, the purposes of this study were to use a canine model of ischemia-reperfusion to (1) compare Tc-99m-teboroxime clearance kinetics in normal and ischemic-reperfused myocardium and (2) assess the utility of Tc-99m-teboroxime clearance kinetics in determining the severity of injury following ischemia-reperfusion.Thirteen dogs underwent left circumflex coronary artery (LCx) occlusion for either 30 min (IR30, n = 6) or 120 min (IR120, n = 7), followed by reperfusion, and finally Tc-99m-teboroxime administration 120 min after reperfusion. Microsphere blood flows were determined at baseline, during occlusion, after reperfusion, and before euthanasia. Post-mortem, area at risk was determined using Evans blue dye, and viability was determined using triphenytetrazolium chloride (TTC) staining. The hearts were then subdivided into 24 pieces and Tc-99m activity was measured in a well counter.TTC-determined infarct area as a percentage of total left ventricular myocardium was 1.1% ± 0.3% for the IR30 group and 7.5% ± 2.9% for the IR120 group (p < 0.05). During coronary occlusion, both the IR30 and IR120 groups demonstrated decreases in percent wall thickening in the ischemia-reperfusion zone (IRZ) as compared with the normal zone (NZ). In the IR30 group, percent wall thickening in the IRZ recovered during the reperfusion phase as compared with the NZ. In the IR120 group, percent wall thickening in the IRZ remained depressed during the reperfusion phase and through the end of the experiment as compared with the NZ. Final Tc-99m-teboroxime myocardial IRZ/NZ activity ratio was 0.94 ± 0.01 for the IR30 group, compared to 0.80 ± 0.01 for the IR120 group (p < 0.05).Tc-99m-teboroxime demonstrates moderate differential clearance in a model of severe injury with 120 min of ischemia-reperfusion, but only minimal differential clearance in a model of mild injury with 30 min of ischemia-reperfusion. Thus, Tc-99m-teboroxime clearance kinetics may be helpful in differentiating normal and minimally injured from severely injured myocardium.
- Macrophage stimulating agent soluble yeast β-1,3/1,6-glucan as a topical treatment of diabetic foot and leg ulcers: A randomized, double blind, placebo-controlled phase II study. [JOURNAL ARTICLE]
- J Diabetes Investig 2014 Jul; 5(4):392-399.
Dysregulated inflammatory response is believed to be an important factor in the pathogenesis of several late complications of diabetes mellitus. β-Glucans are potent inducers of immune function. The present randomized, double blind, two-center, placebo-controlled study was undertaken to explore safety, tolerability and efficacy of soluble β-1,3/1,6-glucan (SBG) as a local treatment of diabetic foot ulcers.A total of 60 patients with type 1 or 2 diabetes and lower extremity ulcers (Wagner grade 1-2, Ankle/Brachial Index ≥0.7) received SBG or a comparator product (methylcellulose) locally three times weekly up to 12 weeks in addition to conventional management scheme. A total of 54 patients completed the study.A tendency for shorter median time to complete healing in the SBG group was observed (36 vs 63 days, P = 0.130). Weekly percentage reduction in ulcer size was significantly higher in the SBG group than in the methylcellulose group between weeks 1-2, 3-4 and 5-6 (P < 0.05). The proportion of ulcers healed by week 12 was also in favor of SBG (59% vs 37%, P = 0.09), with a significantly higher healing incidence in the SBG group at week 8 (44% vs 17%, P = 0.03). SBG was safe and well tolerated. There was a clinically significant difference regarding the incidence of serious adverse events in favor of the SBG treatment.Local treatment of diabetic lower extremity ulcers with β-1,3/1,6-polyglucose shows good safety results. This β-glucan preparation shows promising potential as a treatment accelerating cutaneous healing. Further studies are required to confirm this effect. This trial was registered with ClinicalTrials.gov (no. NCT00288392).