Electromagnetic field in human sperm cryopreservation improves fertilizing potential of thawed sperm through physicochemical modification of water molecules in freezing medium.

Physicochemical properties of water molecules as the main compositions of the freezing media can be affected by the electromagnetic fled. The purpose of this study was to apply extremely low repetition rate electromagnetic fields (ELEFs) to change the molecular network of water molecules existing in freezing media used for human sperm cryopreservation. First, different time periods and pulsed electromagnetic fields were used to evaluate the physiochemical properties of water. The lowest rate of cluster size, surface tension, viscosity, and density was observed for water samples exposed to 1000 Hz ELEF for 60 min (P < 0.05) that could be results in small ice crystal formation. Therefore, this treatment was selected for further evaluations in human sperm freezing because there was minimal probability of amorphous ice crystallization in this group. To assess fertilizing potential, human semen samples were subjected to ELEF (1000 Hz) water-made freezing medium and cryopreserved. The highest percentage of total motility, progressive motility, viability, membrane integrity, mitochondrial membrane potential, DNA integrity, and TAC were obtained in frozen ELEF as compared to other groups. The percentage of viable spermatozoa (Annexin V-/PI-) in frozen ELEF was significantly higher than in frozen control. The level of ROS was significantly lower in frozen ELEF when compared to frozen control. It can be concluded that the modification of physicochemical properties of water existing in cryopreservation media by ELEF is a suitable strategy to improve the outcome of cryopreservation.

Proteomic analysis and microtubule dynamicity of human sperm in electromagnetic cryopreservation.

The proteomic changes, microtubule dynamicity, and quality parameters of human sperm were investigated during cryopreservation in an extremely low electromagnetic field (ELEF) condition. Semen samples were obtained from 210 healthy individuals with normospermia and then were divided into three experimental groups: fresh control, frozen control, and frozen ELEF group. Shotgun proteomics was performed to assess the identification of microtubule proteins of the sperm in experimental groups. Microtubule dynamicity, secondary, and tertiary structure modifications of tubulins, characteristics of transmission electron microscopy of sperm as well as sperm quality parameters were evaluated. The expression ratios of α- and ß-tubulins were significantly increased after cryopreservation compared with fresh control while this ratio was not significantly different in frozen ELEF group. The expression ratio of tubulin polymerization-promoting protein was significantly decreased after cryopreservation compared with fresh control. The length, width, and the activity of microtubule, secondary, and tertiary structures of tubulins, motility, and the viability of the sperm were decreased in frozen control as compared with fresh control. The microtubule activity, secondary, and tertiary structures of sperm tubulin in frozen ELEF group were higher than frozen control. Transmission electron microscopy of microtubules showed that the size of the width and length of the microtubules in frozen ELEF group were greater than frozen control. Motility, viability, and reactive oxygen species levels were improved in frozen ELEF group when compared with frozen control. While the microtubule dynamicity of the sperm was affected by the cryopreservation, this trait was improved during the electromagnetic cryopreservation resulted in better motility and viability.

α-Cyperone of Cyperus rotundus is an effective candidate for reduction of inflammation by destabilization of microtubule fibers in brain.

ETHNOPHARMACOLOGICAL RELEVANCE: Cyperus rotundus L. (Cyperaceae), commonly known as purple nutsedge or nut grass is one of the most invasive and endemic weeds in tropical, subtropical and temperate regions. This plant has been extensively used in traditional medicine for anti-arthritic, antidiarrheal and antiplatelet properties as well as treatment for several CNS disorders such as epilepsy, depression and inflammatory disorders. Inflammation is evidently occurring in pathologically susceptible regions of the Alzheimer's disease (AD) brain as well as other disorders. Many cellular processes are responsible in chronic inflammation. Microtubule-based inflammatory cell chemotaxis is a well-recognized process that influences production of cytokines and phagocytosis. The effect of α-Cyperone, one of main ingredients of Cyperus rotundus on microtubule assembly and dynamics has not been examined and is the purpose of this investigation. MATERIALS AND METHODS: Microtubules and tubulin were extracted in order to explore their interaction with α-Cyperone by utilization of turbidimetric examinations, intrinsic fluorescence and circular dichroism spectroscopy (CD) studies. The molecular docking analysis was executed in order to facilitate a more detail and stronger evidence of this interaction. The BINding ANAlyzer (BINANA) algorithm was used to evaluate and further substantiate the binding site of α-Cyperone. RESULTS: It was demonstrated that α-Cyperone had a pronounced influence on the tubulin structure, decreased polymerization rate and reduced concentration of polymerized tubulin in vitro. The CD deconvolution analysis concluded that significant conformational changes occurred, demonstrated by a drastic increase in content of ß-strands upon binding of α-Cyperone. The fluorescence spectroscopy revealed that a static type of quenching mechanism is responsible for binding of α-Cyperone to tubulin. Upon characterization of various biophysical parameters, it was further deduced that ligand binding was spontaneous and a single site of binding was confirmed. Transmission electron microscopy revealed that upon binding of α-Cyperone to microtubule the number and complexity of fibers were noticeably decreased. The computational analysis of docking suggested that α-Cyperone binds preferably to ß-tubulin at a distinct location with close proximity to the GTP binding and hydrolysis site. The ligand interaction with ß-tubulin is mostly hydrophobic and occurs at amino acid residues that are exclusively on random coil. The BINANA 1.2.0 algorithm which counts and tallies close molecular interaction by performing defined set of simulations revealed that amino acid residues Arg 48 and Val 62 have registered the highest scores and are possibly crucial in ligand-protein interaction. CONCLUSION: α-Cyperone binds and interacts with tubulin and is capable of distinctly destabilizing microtubule polymerization. The effect of this interaction could result in reduction of inflammation which would be highly beneficial for treatment of inflammatory diseases such as AD.

Simultaneous Analysis of Wnt and NF-κB Signaling Pathways in Doxorubicin Sensitive and Methotrexate Resistant PLC/ PRF/5 Cells.

OBJECTIVE: Multi-drug resistance (MDR) is a controversial issue in traditional chemo- therapy of aggressive cancers, including hepatocellular carcinoma. The major cause of MDR is suggested to be the aberrant activation of the main signaling pathways such as Wnt and nuclear factor kappa-light-chain-enhancer of activated B cells (NF- κB) which have key roles in the maintenance of cancer stem cells (CSCs). Therefore, the evaluation of their alterations could be essential in chemo-resistant cancers such as Hepatocellular carcinoma. The main purpose of this study was to investigate the alteration of the mentioned pathways in the chemotherapy resistant cancer cells by assessing their major molecular parameters. MATERIALS AND METHODS: In this experimental study, methylthiazol tetrazolium (MTT) assay, acridine orange/ethidium bromide (AO/EtBr) and Hoechst 33342 staining, DNA fragmentation and colony formation methods were employed to investigate the cytotoxic effects of methotrexate (MTX) and doxorubicin (DOX) on PLC/PRF/5 cells. Moreover, the expression of 11 important genes involved in MDR was performed by semi-quantitative reverse transcriptase-polymerase chain reaction (RT-PCR). RESULTS: PLC/PRF/5 cells (Alexander) were sensitive to DOX and normally resist- ant to MTX. In addition, the results obtained from RT-PCR analysis revealed that ß-catenin expression was significantly reduced and ABCG2 significantly overex- pressed 4.85 and 3.34 times (P value<0.05) in DOX and MTX treated cells, respec- tively. Furthermore, a considerable expression of HIF-1α and p65 were detected only in MTX-resistant cells. CONCLUSION: Anti-cancer drugs may have more than one target in tumor cells. They not only participate in deregulation of Wnt but also alter NF-κB activation. Moreover, HIF-1α was the only anti-apoptotic protein that was significantly induced in the chem- oresistant cells.

Amelioration by chicory seed extract of diabetes- and oleic acid-induced non-alcoholic fatty liver disease (NAFLD)/non-alcoholic steatohepatitis (NASH) via modulation of PPARα and SREBP-1.

We evaluated the effect of chicory (Cichorium intybus L.) seed extract (CI) on hepatic steatosis caused by early and late stage diabetes in rats (in vivo), and induced in HepG2 cells (in vitro) by BSA-oleic acid complex (OA). Different dosages of CI (1.25, 2.5 and 5 mg/ml) were applied along with OA (1 mM) to HepG2 cells, simultaneously and non-simultaneously; and without OA to ordinary non-steatotic cells. Cellular lipid accumulation and glycerol release, and hepatic triglyceride (TG) content were measured. The expression levels of sterol regulatory element-binding protein-1c (SREBP-1c) and peroxisome proliferator-activated receptor alpha (PPARα) were determined. Liver samples were stained with hematoxylin and eosin (H&E). Significant histological damage (steatosis-inflammation-fibrosis) to the cells and tissues and down-regulation of SREBP-1c and PPARα genes that followed steatosis induction were prevented by CI in simultaneous treatment. In non-simultaneous treatment, CI up-regulated the expression of both genes and restored the normal levels of the corresponding proteins; with a greater stimulating effect on PPARα, CI acted as a PPARα agonist. CI released glycerol from HepG2 cells, and targeted the first and the second hit phases of hepatic steatosis. A preliminary attempt to characterize CI showed caffeic acid, chlorogenic acid, and chicoric acid, among the constituents.

Induction of G1 cell cycle arrest and cyclin D1 down-regulation in response to pericarp extract of Baneh in human breast cancer T47D cells.

BACKGROUND AND THE PURPOSE OF THE STUDY: Natural products from plants have an important role in the development and production of new drugs mainly for cancer therapy. More recently, we have shown that the pericarp methanolic extract of Pistacia atlantica sub kurdica (with local name of Baneh) as a rich source of active biological components with high antioxidant and radical scavenging activities, has ability to cease proliferation and induce apoptosis in T47D human breast cancer cells. The present study aimed to clarify whether Baneh extract able to alter cell cycle progression of T47D cells or not. METHODS: In order to study the possible effect of Baneh extract on cell cycle of T47D cells, we evaluated cell cycle distribution and its regulatory proteins by flow cytometry and western blot analysis respectively. RESULTS: Baneh extract induced G0/G1 cell cycle arrest in conjunction with a marked decrease in expression of cyclin D1 and cdk4 that was strongly dependent on time of exposure. In parallel, Dox-treated T47D cells in early time points were accumulated on S phase, but after 48 h cell cycle progression was inhibited on G2/M. Dox promoted striking accumulation of cyclin B1 rapidly and enhanced cyclin A abundance. CONCLUSION: Taken together, our results establish that the antitumor activity of the pericarp extract of Baneh partly is mediated via cell cycle arrest and downregulation of cyclin D1 and cdk4 expression. These findings warrant further evaluation regarding the mechanism(s) of action of this promising anticancer agent.

Induction of apoptosis and cell cycle arrest by pericarp polyphenol-rich extract of Baneh in human colon carcinoma HT29 cells.

Plants as important source of natural active components with anticancer effects commonly are different in structure and biological properties. The pericarp of Pistacia atlantica sub kurdica with local name of Baneh, a rich source of active phytochemicals, contains noticeable amounts of polyphenolic compounds, flavonoids and anthocyanins. Therefore, the antiproliferative, apoptosis induction and cell cycle alterations of Baneh were evaluated in human colon carcinoma HT29 cells. The Baneh extract (0.7 mg/ml) resulted in 50% growth inhibition similar to 500 nM of Doxorubicin (Dox) in HT29 cells after 72 h. The down-regulation of cyclin A protein by Baneh extract induced S phase delay in cell cycle progression of HT29 cells. Unlike the Baneh extract, Dox showed G2/M accumulation of HT29 cells which was associated with an increase in cyclin A and cyclin B1 protein expression. Furthermore, the induction of apoptosis following Baneh extract and Dox treatment in HT29 cells was confirmed by DNA fragmentation and translocation of phosphatidylserine. The morphological characteristics of apoptosis were also observed in HT29 cells exposed to the Baneh extract and Dox. These results suggest that due to the existence of bioactive components, methanolic extract of the Baneh has significant cytotoxic effects against human colon carcinoma HT29 cells.

Antiproliferative activity of chloroformic extract of Persian Shallot, Allium hirtifolium, on tumor cell lines.

Allium hirtifolim (Persian Shallot) belongs to Allium genus (Alliaceae family). We investigated the in vitro effects of chloroformic extract of A. hirtifolium and its Allicin on the proliferation of HeLa (cervical cancer), MCF7 (human, caucasion, breast, adenocarcinoma) and L929 (mouse, C3H/An, connective) cell lines. Our results showed that components of A. hirtifolium might inhibit proliferation of tumor cell lines. This inhibition in HeLa and MCF-7 cells was dose-dependent. The presence of Allicin was evaluated by TLC method in bulbs and the extract of A. hirtifolium was analyzed by HPLC. MTT test was performed 24, 48 and 72 h after cell culture. A significant decrease in cell lines was observed in HeLa and MCF-7 as compared to L929 cell lines. DNA fragmentation analysis revealed a large number of apoptotic cells in treated HeLa and MCF-7 cell groups, but no effects in L929 cells. Therefore A. hirtifolium might be a candidate for tumor suppression.

Paraoxonase phenotype distribution in a healthy Iranian population.

Human serum paraoxonase (PON1) is a high-density lipoprotein-associated esterase that protects against organophosphate neurotoxicity, and is proposed to play a role in lipid metabolism and the onset of cardiovascular disease. In the present study, paraoxonase activities and phenotype distribution in serum of 132 healthy Iranian individuals aged 17-68 years were assessed using dual substrate method. In the study population, a wide interindividual variability (up to 15-fold) of paraoxonase activity was found. The mean of basal, salt-stimulated paraoxonase and arylesterase activities were 81.8 +/- 57 U/ml, 153.1 +/- 117.5 U/ml and 80.7 +/- 12.8 kU/l, respectively. The ratio of salt-stimulated paraoxonase activity to arylesterase activity was used for definition of phenotypes. Based on the observed ratios, three distinct phenotypes AA (low activity), AB (intermediate activity) and BB (high activity) were determined. The PON1 ratio varied from 0.5 to 6.8. The paraoxonase phenotype frequencies were approximately 48% (AA), 41% (AB) and 11% (BB). In this work, serum triglycerides had significant positive correlation (r = 0.334, P < 0.05) with paraoxonase activity, whereas high-density lipoprotein did not. No significant decrease in paraoxonase activity by smoking was observed. Age and sex had no influences on PON1 activities. In conclusion, the distribution of paraoxonase phenotypes in this Iranian population was trimodal and comparable to that of Caucasians from North America; however, overall enzyme activity was lower than that reported for Caucasians.

Frequency of paraoxonase 192/55 polymorphism in an Iranian population.

Paraoxonase (PON1) is a serum enzyme that plays an important role in prevention of atherosclerosis and also protects against organophosphate-induced neurotoxicity. PON1 displays a high variability in human populations. In this study, PON1-192 and -55 polymorphisms and correlation to serum PON1 activity were investigated in 132 healthy Iranian individuals from Isfahan province. The genotype frequencies for PON1-192 were approximately 48% (QQ), 42.% (QR), and 10% (RR) and for PON1-55 17% (MM), 48% (ML), and 35% (LL). Thus, the frequencies of alleles R and L were 0.31 and 0.59, respectively. PON1 activity toward paraoxon was markedly affected in both polymorphic populations in the following order QQ < QR < RR genotype for PON1-192 and MM < ML < LL genotype for PON1-55. Neither polymorphism significantly affected PON1 activity toward phenylacetate. The RR/LL individuals had the highest PON1 activity and QQ/MM individuals the least. The QR/ML haplotype was the most frequent seen in Iranians, and the RR/MM and QR/MM haplotypes were absent in this population. In conclusion, the frequencies of PON1-192 and -55 polymorphisms in this Iranian population were different from those seen in other Asian populations from Japan and China but similar to those for European Caucasians.