Vitamin D (VD) reduces the risk of breast cancer and improves disease prognoses. Potential VD analogs are being developed as therapeutic agents for breast cancer treatments. The large-conductance Ca2+-activated K⁺ channel KCa1.1 regulates intracellular Ca2+ signaling pathways and is associated with high grade tumors and poor prognoses. In the present study, we examined the effects of treatments with VD receptor (VDR) agonists on the expression and activity of KCa1.1 in human breast cancer MDA-MB-453 cells using real-time PCR, Western blotting, flow cytometry, and voltage-sensitive dye imaging. Treatments with VDR agonists for 72 h markedly decreased the expression levels of KCa1.1 transcripts and proteins in MDA-MB-453 cells, resulting in the significant inhibition of depolarization responses induced by paxilline, a specific KCa1.1 blocker. The specific proteasome inhibitor MG132 suppressed VDR agonist-induced decreases in KCa1.1 protein expression. These results suggest that KCa1.1 is a new downstream target of VDR signaling and the down-regulation of KCa1.1 through the transcriptional repression of KCa1.1 and enhancement of KCa1.1 protein degradation contribute, at least partly, to the antiproliferative effects of VDR agonists in breast cancer cells.

TP53 gene mutations occurring in patients with myelodysplastic syndrome (MDS) and acute myeloid leukemia (AML) are associated with high-risk karyotypes including 17p abnormalities, monosomal and complex cytogenetics. TP53 mutations in these disorders portend rapid disease progression and resistance to conventional therapeutics. Notably, the size of the TP53 mutant clone as measured by mutation allele burden is directly linked to overall survival (OS) confirming the importance of p53 as a negative prognostic variable. In nucleolar stress-induced ribosomopathies, such as del(5q) MDS, disassociation of MDM2 and p53 results in p53 accumulation in erythroid precursors manifested as erythroid hypoplasia. P53 antagonism by lenalidomide or other therapeutics such as antisense oligonucleotides, repopulates erythroid precursors and enhances effective erythropoiesis. These findings demonstrate that p53 is an intriguing therapeutic target that is currently under investigation in MDS and AML. This study reviews molecular advances in understanding the role of p53 in MDS and AML, and explores potential therapeutic strategies in this era of personalized medicine.

Most breast cancers express estrogen receptor (ER) α, and the antiestrogen drug tamoxifen has been widely used for their treatment. Unfortunately, up to half of all ERα-positive tumors have intrinsic or acquired endocrine therapy resistance. Our recent studies revealed that the ER coactivator Mediator Subunit 1 (MED1) plays a critical role in tamoxifen resistance through cross-talk with HER2. Herein, we assembled a three-way junction (3-WJ) pRNA-HER2apt-siMED1 nanoparticle to target HER2-overexpressing human breast cancer via an HER2 RNA aptamer to silence MED1 expression. We found that these ultracompact RNA nanoparticles are very stable under RNase A, serum, and 8 M urea conditions. These nanoparticles specifically bound to HER2-overexpressing breast cancer cells, efficiently depleted MED1 expression, and significantly decreased ERα-mediated gene transcription, whereas point mutations of the HER2 RNA aptamer on these nanoparticles abolished such functions. The RNA nanoparticles not only reduced the growth, metastasis, and mammosphere formation of the HER2-overexpressing breast cancer cells but also sensitized them to tamoxifen treatment. These biosafe nanoparticles efficiently targeted and penetrated into HER2-overexpressing tumors after systemic administration in orthotopic xenograft mouse models. In addition to their ability to greatly inhibit tumor growth and metastasis, these nanoparticles also led to a dramatic reduction in the stem cell content of breast tumors when combined with tamoxifen treatment in vivo. Overall, we have generated multifunctional RNA nanoparticles that specifically targeted HER2-overexpressing human breast cancer, silenced MED1, and overcame tamoxifen resistance.

Incorporation of unnatural amino acids (uAAs) via codon reassignment is a powerful approach for introducing novel chemical and biological properties to synthesized polypeptides. However, the site-selective incorporation of multiple uAAs into polypeptides is hampered by the limited number of reassignable nonsense codons. This challenge is addressed in the current work by developing Escherichia coli in vitro translation system depleted of specific endogenous tRNAs. The translational activity in this system is dependent on the addition of synthetic tRNAs for the chosen sense codon. This allows site-selective uAA incorporation via addition of tRNAs pre- or cotranslationally charged with uAA. We demonstrate the utility of this system by incorporating the BODIPY fluorophore into the unique AGG codon of the calmodulin(CaM) open reading frame using in vitro precharged BODIPY-tRNACysCCU. The deacylated tRNACysCCU is a poor substrate for Cysteinyl-tRNA synthetase, which ensures low background incorporation of Cys into the chosen codon. Simultaneously, p-azidophenylalanine mediated amber-codon suppression and its post-translational conjugation to tetramethylrhodamine dibenzocyclooctyne (TAMRA-DIBO) were performed on the same polypeptide. This simple and robust approach takes advantage of the compatibility of BODIPY fluorophore with the translational machinery and thus requires only one post-translational derivatization step to introduce two fluorescent labels. Using this approach, we obtained CaM nearly homogeneously labeled with two FRET-forming fluorophores. Single molecule FRET analysis revealed dramatic changes in the conformation of the CaM probe upon its exposure to Ca2+ or a chelating agent. The presented approach is applicable to other sense codons and can be directly transferred to eukaryotic cell-free systems.

We assessed the effects of equine chorionic gonadotropin (eCG) on oocyte in vitro maturation (IVM), apoptosis, and follicle-stimulating hormone receptor (FSHR), luteinizing hormone receptor (LHR), and gonadotropin-releasing hormone receptor (GnRHR) expression and mRNA levels. Cumulus-oocyte complexes (COCs) were recovered from sheep ovaries and pooled in groups, before being cultured in IVM media containing varying eCG concentrations. Maturation and apoptosis rates were then calculated. Expression of FSHR, LHR, and GnRHR mRNA in oocytes was measured using quantitative reverse transcription polymerase chain reaction. Protein levels were ascertained by western blotting. Matured oocytes displayed and released an intact first polar body. Sheep oocyte maturation rates gradually increased as eCG concentration was raised from 0 to 20 µg/mL. Apoptosis rates of eCG-treated oocytes were lower than those of the control group, and were lowest using 20 µg/mL eCG. FSHR, LHR, and GnRHR mRNA expression increased (P < 0.01, P < 0.05, and P < 0.05, respectively, compared to 0 µg/mL eCG) with eCG concentration, being highest following exposure to 20 µg/mL. FSHR and GnRHR protein levels were significantly higher in oocytes administered 20 µg/mL eCG compared with those matured in the absence of eCG. eCG dose positively correlated with FSHR, LHR, and GnRHR mRNA and protein expression. In conclusion, eCG enhances maturation and decreases apoptosis of oocytes undergoing IVM, and heightens FSHR, LHR, and GnRHR expression. Such increased expression may facilitate oocyte IVM. These findings contribute to our understanding of the mechanisms of underlying hormonal control of sheep oocyte IVM, advancing ovine reproductive methods.

5-hydroxytryptamine receptor 1A (5-HT1AR) is closely associated with cognitive functions. Selective serotonin reuptake inhibitors (SSRIs) can protect individuals from brain damage following ischemia/hypoxia. To investigate the function of SSRIs in vascular dementia (VD), we established a rat model of VD, and observed the effect of SSRIs on the expression of 5-HT1AR mRNA and neurotransmitters. Male SD rats (6 months) were randomly assigned into sham, model, and SSRI groups (N = 30). VD was achieved by permanent ligation of the bilateral common carotid artery. Escitalopram, a highly selective 5-HT reabsorption inhibitor, was ip injected into the rats for three consecutive weeks. The Morris water-maze was used to test learning and memory. H&E staining for neuronal injury was conducted on cortical and hippocampal tissues. HPLC was used to determine the levels of dopamine (DA), 5-HT, and norepinephrine (NE). RT-PCR was used to determine expression of 5-HT1AR mRNA. As compared to control rats, model animals demonstrated elongated escape latency, lower platform crossing times, and significant injuries to hippocampal CA1 neurons. This was accompanied by reductions in DA, 5-HT, and NE levels in hippocampal tissues, as well as reduced cortical 5-HT and decreased 5-HT1AR mRNA expression (P < 0.05). Escitalopram treatments reduced escape latency, elevated platform crossing times, improved CA1 neuronal damage, increased DA and 5-HT levels in hippocampal and cortical neurons, as well as elevated expression of 5-HT1AR mRNA (P < 0.05). Therefore, SSRIs may improve cognitive dysfunction of VD rats, possibly by stimulating expression of neurotransmitters and protecting neurons.

AKT1 and AKT2, the AKT isoforms that are highly expressed in skeletal muscle, have distinct and overlapping functions, with AKT2 more important for insulin-stimulated glucose metabolism. In adipocytes, AKT2 versus AKT1 has greater susceptibility for insulin-mediated redistribution from cytosolic to membrane localization, and insulin also causes subcellular redistribution of AKT Substrate of 160 kDa (AS160), an AKT2 substrate and crucial mediator of insulin-stimulated glucose transport. Although skeletal muscle is the major tissue for insulin-mediated glucose disposal, little is known about AKT1, AKT2 or AS160 subcellular localization in skeletal muscle. The major aim of this study was to determine insulin's effects on the subcellular localization and phosphorylation of AKT1, AKT2 and AS160 in skeletal muscle. Rat skeletal muscles were incubated ex vivo ± insulin, and differential centrifugation was used to isolate cytosolic and membrane fractions. The results revealed that: 1) insulin increased muscle membrane localization of AKT2, but not AKT1; 2) insulin increased AKT2 phosphorylation in the cytosol and membrane fractions; 3) insulin increased AS160 localization to the cytosol and membranes; and 4) insulin increased AS160 phosphorylation in the cytosol, but not membranes. These results demonstrate distinctive insulin effects on the subcellular redistribution of AKT2 and its substrate AS160 in skeletal muscle.

Degenerate Clostridium beijerinckii strain (DG-8052) can be partially recovered by supplementing CaCO3 to fermentation media. Genome resequencing of DG-8052 showed no general regulator mutated. This study focused on transcriptional analysis of DG-8052 and its response to CaCO3 treatment via microarray. The expressions of 5168 genes capturing 98.6% of C. beijerinckii NCIMB 8052 genome were examed. The results revealed that with addition of CaCO3 565 and 916 genes were significantly up-regulated, and 704 and 1044 genes significantly down-regulated at acidogenic and solventogenic phase of DG-8052, respectively. These genes are primarily responsible for glycolysis to solvent/acid production (poR, pfo), solventogensis (buk, ctf, aldh, adh, bcd) and sporulation (spo0A, sigE, sigma-70, bofA), cell motility and division (ftsA, ftsK, ftsY, ftsH, ftsE, mreB, mreC, mreD, rodA), and molecular chaperones (grpE, dnaK, dnaJ, hsp20, hsp90), etc. The functions of some altered genes in DG-8052, totalling 5.7% at acidogenisis and 8.0% at sovlentogenisis, remain unknown. The response of the degenerate strain to CaCO3 was suggested significantly pleiotropic. This study reveals the multitude of regulatory function that CaCO3 has in clostridia and provides detailed insights into degeneration mechanisms at gene regulation level. It also enables us to develop effective strategies to prevent strain degeneration in future.

The purpose of this study was to investigate the underlying mechanism of metallothionein (MT) protection from depleted uranium (DU) using a proteomics approach to search for a DU toxicity-differential protein. MT-/- and MT+/+ mice were administrated with a single dose of DU (10 mg/kg, i.p.) or equal volume of saline. After 4 days, protein changes in kidney tissues were evaluated using a proteomics approach. A total of 13 differentially expressed proteins were identified using two-dimensional electrophoresis and matrix-assisted laser desorption/ionization time-of-flight mass spectrometry. The validating results showed that the expression of aminoacylase-3 (ACY-3) and the mitochondrial ethylmalonic encephalopathy 1 (ETHE1) decreased significantly after DU exposure; in addition, the reduction in MT-/- mice was more significant than that in MT+/+ mice. The results also showed that exogenous ETHE1 or ACY-3 could increase the survival rate of human embryonic kidney 293 (HEK293) cells after DU exposure. A specific siRNA of ETHE1 significantly increased cell apoptosis rates after DU exposure, whereas exogenous ETHE1 significantly decreased cell apoptosis rates. In summary, ACY-3 and ETHE1 might involve in protection roles of MT. ETHE1 could be a new sensitive molecular target of DU-induced cell apoptosis.

Amalaki Rasayana (AR) is a common Ayurvedic herbal formulation of Phyllanthus emblica fruits and some other ingredients, and is used for general good health and healthy aging. We reported it to improve life history traits and to suppress neurodegeneration as well as induced apoptosis in Drosophila. The present study examines responses of Drosophila reared on AR-supplemented food to crowding, thermal or oxidative stresses. Wild-type larvae/flies reared on AR-supplemented food survived the various cell stresses much better than those reared on control food. AR-fed mutant park13 or DJ-1 beta Delta93 (Parkinson's disease model) larvae/flies, however, showed only partial or no protection, respectively, against paraquat-induced oxidative stress, indicating essentiality of DJ-1 beta for AR-mediated oxidative stress tolerance. AR feeding reduced the accumulation of reactive oxygen species (ROS) and lipid peroxidation even in aged (35-day-old) wild-type flies while enhancing superoxide dismutase (SOD) activity. We show that while Hsp70 or Hsp83 expression under normal or stress conditions was not affected by AR feeding, Hsp27 levels were elevated in AR-fed wild-type control as well as heat-shocked larvae. Therefore, besides the known anti-oxidant activity of Phyllanthus emblica fruits, dietary AR also enhances cellular levels of Hsp27. Our in vivo study on a model organism shows that AR feeding significantly improves tolerance to a variety of cell stresses through reduced ROS and lipid peroxidation on the one hand, and enhanced SOD activity and Hsp27 on the other. The resulting better homeostasis improves life span and quality of organism's life.

We investigated the influence of the quaternary ammonium salt (QAS) called IM (N-(dodecyloxycarboxymethyl)- N,N,N-trimethyl ammonium chloride) on yeast cells of the parental strain and the IM-resistant mutant (EO25 IMR) growth. The phenotype of this mutant was pleiotropic. The IMR mutant exhibited resistance to ethanol, osmotic shock and oxidative stress, as well as increased sensitivity to UV. Moreover, it was noted that mutant EO25 appears to have an increased resistance to clotrimazole, ketoconazole, fluconazole, nystatin and cycloheximide. It also tolerated growth in the presence of crystal violet, DTT and metals (selenium, tin, arsenic). It was shown that the presence of IM decreased ergosterol level in mutant plasma membrane and increased its unsaturation. These results indicate changes in the cell lipid composition. Western blot analysis showed the induction of Pma1 level by IM. RT-PCR revealed an increased PMA1 expression after IM treatment.

The controlled synthesis of silver nanoparticles (AgNPs) using cell-free filtrate of Fusarium oxysporum fungus was investigated. The effect of fungal incubation period on nanoparticle formation and nitrate reductase enzyme activity was studied using UV-visible spectroscopy and Harley assay, respectively. The highest AgNP formation was observed in the cell-free filtrate of biomass harvested at the early stationary phase where the NR enzyme activity is the maximum. Mixing of the cell-free filtrates of fungal cultures obtained at 23, 28, and 33 °C with silver nitrate solution confirms the higher productivity of AgNP biosynthesis using the cell-free filtrate of fungus incubated at 28 °C. The effect of some factors such as carbon and nitrate sources and light in fungal incubation period on nitrate reductase induction and AgNP formation was also evaluated. In conclusion, increasing nitrate and carbon sources and presence of light induced NR enzyme and produced AgNPs with smaller size, higher monodispersity, and productivity. Results revealed that the presence of ammonium prevents the NR enzyme secretion and causes to the lower productivity of AgNPs.

Matrix metalloproteinase enzymes (MMPs) play important role in inflammation, malignant cell proliferation, invasion and angiogenesis by mediating extracellular matrix degradation. Doxycycline, a synthetic tetracycline, behaves as a MMP inhibitor at a subantimicrobial dose and inhibits tumor cell proliferation, invasion and angiogenesis. The aberrant activity of nuclear factor kappa B (NF-κB) causes activation of MMPs and thereby proliferation and invasion of cancer cells. The aim of this study was to investigate the effects of doxycycline on the expression of MMPs in lipopolysaccharide (LPS)-induced PC3 human prostate cancer cells and the possible role of NF-κB signaling.

Despite decades of research, obesity and diabetes remain major health problems in the USA and worldwide. Among the many complications associated with diabetes is an increased risk of cardiovascular diseases, including myocardial infarction and heart failure. Recently, microRNAs have emerged as important players in heart disease and energy regulation. However, little work has investigated the role of microRNAs in cardiac energy regulation. Both human and animal studies have reported a significant increase in circulating free fatty acids and triacylglycerol, increased cardiac reliance on fatty acid oxidation, and subsequent decrease in glucose oxidation which all contributes to insulin resistance and lipotoxicity seen in obesity and diabetes. Importantly, MED13 was initially identified as a negative regulator of lipid accumulation in Drosophilia. Various metabolic genes were downregulated in MED13 transgenic heart, including sterol regulatory element-binding protein. Moreover, miR-33 and miR-122 have recently revealed as key regulators of lipid metabolism. In this review, we will focus on the role of microRNAs in regulation of cardiac and total body energy metabolism. We will also discuss the pharmacological and non-pharmacological interventions that target microRNAs for the treatment of obesity and diabetes.

The aim of the present study is to explore the effects of exogenous insulin-like growth factor-1 (IGF1) on hyperglycemia-induced apoptosis of Schwann cells via neuritin-mediated pathway. Neuritin was identified with immunohistochemistry. Exogenous IGF1 was used to prevent possible changes in neuritin expression and apoptosis of Schwann cells isolated from rat sciatic nerves and cultured in high-glucose media. Neuritin silencing or overexpressing lentivirus transfection of Schwann cells was conducted. Expressions of neuritin at levels of transcription or translation were measured using quantitative PCR or Western blot. Caspase-3 and caspase-9 fluorometric assays were performed. Bcl-2 and Bax were assayed using Western blotting. Apoptosis of Schwann cells was measured using FACS analysis and TUNEL assay. A pathway of IGF1 action in relation to neuritin was explored. Neuritin and Bcl-2 protein were localized in Schwann cells of rats' sciatic nerves. In vitro, apoptosis increased with downregulated neuritin expression, which was prevented by exogenous IGF1 treatment in contrast to without, in Schwann cells isolated from rat sciatic nerve and cultured in high-glucose and serum-free media. A phosphatidylinositol-3-kinase (PI3K) inhibitor treatment blocked the action of IGF1. The inhibitor did not affect the apoptosis rate that decreased obviously after neuritin was overexpressed in Schwann cells. The apoptosis rate increased drastically after neuritin was silenced, and the resultant apoptosis was suppressed by a caspase inhibitor treatment but not affected by exogenous IGF1. The activities of caspase-3 and caspase-9 changed positively with apoptosis. An anti-apoptotic protein (Bcl-2) not Bax increased or decreased in neuritin-overexpressed or neuritin-silenced Schwann cells, respectively. Bcl-2-selective inhibitor blocked the anti-apoptotic effect of neuritin. IGF1 or neuritin was not found to affect glucose levels in media during the experiment. The anti-apoptotic effect of IGF1 on Schwann cells inflicted by hyperglycemia is mediated at least by neuritin, a novel neurotrophic factor, through PI3K and Bcl-2.

Multixenobiotic resistance (MXR) efflux transporters constitute a broad-spectrum physiological defense system allowing marine bivalves to cope with environmental challenges. There is, however, scarce information on the type and role that different MXR transporters may have in embryos, which represent the most sensitive stages of bivalves to environmental stress. In this study regulation of MXR-related transporters was investigated in early developmental stages of the Mediterranean mussel (Mytilus galloprovincialis). In vitro fertilization experiments using gametes from naturally-spawning broodstocks were performed to follow embryo development from fertilized eggs (30min post fertilization, pf) to fully developed D-shape veligers (48hpf). Quantitative PCR analyses indicated that ABCB and ABCC transcripts encoding the MXR-related transporters P-glycoproteins (P-gp) and Multidrug resistance proteins (Mrp), respectively, were expressed soon after 30minpf, with ABCC being more expressed than ABCB. Copy numbers of both transcripts were increased in trochophorae and D-veligers. MXR efflux activity assessed using the fluorescent substrate rhodamine 123 and selective P-gp or Mrp inhibitors showed that the P-gp mediated efflux was detected only in D-veligers, while a significant Mrp mediated efflux was detected soon after 30minpf and remained almost unchanged in trochophorae and D-veligers. MXR modulation by propranolol and carbamazepine showed that the pharmaceuticals may act as transcriptional regulators and substrates. Results reported lead to hypothesize that while P-gp aids in xenobiotic efflux performing a prominent protective role, Mrp could be a dual-functioning transporter performing both protective and physiological functions in mussel development.

Hyperhydricity occurs frequently in plant tissue culture and can severely affect commercial micropropagation and genetic improvement of the cultured plantlets. Hyperhydric shoots are charaterzized by high water content, but how this occurs is still a subject of investigation. Silver ion (Ag+) can reduce the extent of hyperhydricity in plants, but its effect on the reversion of hyperhydric plantlets and the underlying mechanism of reversion has not been clarified. In this study, about 67% of the hyperhydric Dianthus chinensis L. plantlets were found to revert to normal condition when the plantlets were cultured in medium supplemented with 29.4μmolL-1AgNO3. Water content and hydrogen peroxide (H2O2) content in the guard cells of these plantlets were reduced, while stomatal aperture and water loss rate were increased. AgNO3 also reduced the content of endogenous ethylene and expression of ethylene synthesis and ethylene signal transduction-associated genes. Reduced accumulation of ethylene consequently led to an increase in stomatal aperture mediated by decreased H2O2 content in the guard cells. These results adequately verified the role of AgNO3 in the reversion of hyperhydricity in D. chinensis L. and also provided clues for exploring the cause of excessive water accumulation in hyperhydric plants.

The pro- or anti-inflammatory bioactivity of dietary essential linoleic acid (LA) and alpha-linolenic acid (ALA) is mainly attributed to rate-limiting delta-6 desaturase (D6D) activity. The aim of this study was to analyze mechanisms of D6D-substrates ALA, LA and D6D-product gamma-linolenic acid (GLA) under D6D-deficient conditions. Fatty acid profiles (GC-MS), D6D gene expression (real-time RT-PCR) and NFκB activity (luciferase assay) were assessed in HEK293 cells. FADS2 gene expression was approved being marginal. Incubation with ALA or LA did not increase D6D products but their elongase products C20:3n-3 and C20:2n-6. Bypassing the D6D, GLA elevated C20:3n-6 and C20:4n-6. LA significantly increased (+18% at 60 μM; p < .001), ALA reduced (-32% at 100 μM; p < .001) and GLA did not specifically change NFκB activity. Our data indicate that D6D might not be essential for the distinct effects of LA and ALA on NFκB activity.

Date fruits have protective effects against liver fibrosis; however their anti-apoptotic effects have not been investigated.

The increased proliferation of activated hepatic stellate cells (HSCs) is associated with hepatic fibrosis and excessive extracellular matrix (ECM)-protein production. We examined the inhibitory effects of the Monascus purpureus-fermented metabolites, ankaflavin and monascin (15 and 30 μM), on the Akt/nuclear factor (NF)-κB and p38 mitogen-activated protein kinase (MAPK) signaling pathways in HSC-T6 (activated hepatic stellate cell line). Ankaflavin and monascin (30 μM) induced apoptosis and significantly inhibited cell growth (cell viabilities: 80.2 ± 5.43% and 62.8 ± 8.20%, respectively, versus control cells; P < 0.05). Apoptosis and G1 phase arrest (G1 phase percentages: 76.1 ± 2.85% and 79.9 ± 1.80%, respectively, versus control cells 65.9 ± 4.94%; P < 0.05) correlated with increased p53 and p21 levels and caspase 3 activity and decreased cyclin D1 and Bcl-2-family protein levels (P < 0.05, all cases). The apoptotic effects of ankaflavin and monascin were HSC-T6-specific, suggesting their potential in treating liver fibrosis.