Saturated fat ingestion stimulates proatherogenic inflammation in polycystic ovary syndrome.

Inflammation and dyslipidemia are often present in polycystic ovary syndrome (PCOS). We determined the effect of saturated fat ingestion on circulating heat shock protein-70 (HSP-70) and mononuclear cell (MNC) toll-like receptor-2 (TLR2) gene expression, activator protein-1 (AP-1) activation, and matrix matalloproteinase-2 (MMP-2) protein in women with PCOS. Twenty reproductive-age women with PCOS (10 lean, 10 with obesity) and 20 ovulatory controls (10 lean, 10 with obesity) participated in the study. HSP-70 was measured in serum and TLR2 mRNA and protein, AP-1 activation, and MMP-2 protein were quantified in MNC from blood drawn while fasting and 2, 3, and 5 h after saturated fat ingestion. Insulin sensitivity was derived from an oral glucose tolerance test (ISOGTT). Androgen secretion was assessed from blood drawn while fasting and 24, 48, and 72 h after human chorionic gonadotropin (HCG) administration. In response to saturated fat ingestion, serum HSP-70, TLR2 gene expression, activated AP-1, and MMP-2 protein were greater in lean women with PCOS compared with lean controls and in women with PCOS and obesity compared with controls with obesity. Both PCOS groups exhibited lower ISOGTT and greater HCG-stimulated androgen secretion compared with control subjects of their respective weight classes. Lipid-stimulated proatherogenic inflammation marker responses were negatively correlated with ISOGTT and positively correlated with abdominal adiposity and HCG-stimulated androgen secretion. In PCOS, saturated fat ingestion stimulates proatherogenic inflammation independent of obesity. This effect is greater when PCOS is combined with obesity compared with obesity alone. Abdominal adiposity and hyperandrogenism may perpetuate proatherogenic inflammation.NEW & NOTEWORTHY This paper demonstrates that in polycystic ovary syndrome (PCOS), ingestion of saturated fat triggers a molecular pathway of inflammation known to drive atherogenesis. This effect is independent of obesity as it occurs in lean women with PCOS and not in lean ovulatory control subjects. Furthermore, the combined effects of PCOS and obesity are greater compared with obesity alone.

Effectiveness of Huachansu injection combined with chemotherapy for treatment of gastric cancer in China: a systematic review and Meta-analysis.

OBJECTIVE: to evaluate the effectiveness and safety of Huachansu (HCS) injection plus chemotherapy in the treatment of gastric cancer. METHODS: A thorough and systematic retrieval of randomized controlled trials (RCTs) concerning HCS injection for treating gastric cancer was conducted in several electronic databases from inception to May 10, 2018. The quality of the RCTs was assessed by the Cochrane risk of bias tool. And the data about objective remission rate, performance status, adverse drug reactions (ADRs) and other outcomes were extracted and analyzed by Review Manager 5.3 and Stata 13.0 software. RESULTS: A total of 14 RCTs with 976 participants were involved in the current Meta-analysis. The results suggested that HCS injection combined with chemotherapy was associated with better effects than receiving conventional chemotherapy alone in respect of improving the objective response rate [RR = 1.18, 95% CI (1.03, 1.37), Z = 2.32, P = 0.02], and performance status [RR = 1.84,95% CI (1.43, 2.36), Z = 4.74, P < 0.000 01]. In addition, HCS injection combined with chemotherapy could relieve pain for patients with gastric cancer. CONCLUSION: This Meta-analysis revealed that HCS injection plus chemotherapy might more effective than chemotherapy in treating gastric cancer. Nevertheless, more large-scale and rigorously designed RCTs should be performed to validate this finding.

Background subtraction via time continuity and texture consistency constraints.

Background subtraction has attracted enormous interest in the field of moving object detection. However, when there are complex scenarios such as illumination changes, dynamic background, and noise, the moving object area obtained by background subtraction often has holes, noise, and shadows. This paper proposes a novel background update model based on matrix factorization, which uses the temporal continuity of video content to solve the problems of holes, noise, and shadows. Moreover, in some cases, the texture consistency of the object is also a factor worth considering. The neighborhood weighed local binary pattern (NWLBP) is introduced to optimize the background update model, which is very effective for suppressing background or foreground shadow. The effectiveness of our method is confirmed by extensive experiments on public data sets and real shot video. Compared with the existing state-of-the-art moving object detection methods, the proposed methods can accurately establish the background model and locate the moving object region robustly.

Mechanisms of Cancer Induction by Tobacco-Specific NNK and NNN.

Tobacco use is a major public health problem worldwide. Tobacco-related cancers cause millions of deaths annually. Although several tobacco agents play a role in the development of tumors, the potent effects of 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK) and N'-nitrosonornicotine (NNN) are unique. Metabolically activated NNK and NNN induce deleterious mutations in oncogenes and tumor suppression genes by forming DNA adducts, which could be considered as tumor initiation. Meanwhile, the binding of NNK and NNN to the nicotinic acetylcholine receptor promotes tumor growth by enhancing and deregulating cell proliferation, survival, migration, and invasion, thereby creating a microenvironment for tumor growth. These two unique aspects of NNK and NNN synergistically induce cancers in tobacco-exposed individuals. This review will discuss various types of tobacco products and tobacco-related cancers, as well as the molecular mechanisms by which nitrosamines, such as NNK and NNN, induce cancer.

The transcription factor DREAM represses the deubiquitinase A20 and mediates inflammation.

Here we found that the transcription repressor DREAM bound to the promoter of the gene encoding A20 to repress expression of this deubiquitinase that suppresses inflammatory NF-κB signaling. DREAM-deficient mice displayed persistent and unchecked A20 expression in response to endotoxin. DREAM functioned by transcriptionally repressing A20 through binding to downstream regulatory elements (DREs). In contrast, binding of the transcription factor USF1 to the DRE-associated E-box domain in the gene encoding A20 activated its expression in response to inflammatory stimuli. Our studies define the critical opposing functions of DREAM and USF1 in inhibiting and inducing A20 expression, respectively, and thereby the strength of NF-κB signaling. Targeting of DREAM to induce USF1-mediated A20 expression is therefore a potential anti-inflammatory strategy for the treatment of diseases associated with unconstrained NF-κB activity, such as acute lung injury.

Part I-mechanism of adaptation: high nitric oxide adapted A549 cells show enhanced DNA damage response and activation of antiapoptotic pathways.

Our previous studies demonstrate that A549, a human lung adenocarcinoma line, could be adapted to the free radical nitric oxide (NO([Symbol: see text])). NO([Symbol: see text]) has been shown to be overexpressed in human tumors. The original cell line, A549 (parent), and the newly adapted A549-HNO (which has a more aggressive phenotype) serves as a useful model system to study the role of NO([Symbol: see text]) in tumor biology. It is well known that DNA damage response (DDR) is altered in cancer cells and NO([Symbol: see text]) is known to cause DNA damage. Modulations in molecular mechanisms involved in DNA damage response in A549-HNO cells can provide better insights into the enhanced growth behavior of these cells. Thus, here, we carried out a series of time course experiments by treating A549 and A549-HNO cells with NO([Symbol: see text]) donor and examining levels of proteins involved in the DDR pathway. We observed induced expression of key components of DDR pathway in A549-HNO cells. The HNO cells showed sustained expression of key proteins involved in both nonhomologous end joining (NHEJ) and homologous recombination pathways, whereas parent cells only expressed low levels of NHEJ pathway proteins. Further with prolonged NO([Symbol: see text]) exposure, ATR, Chk1, and p53 were activated and upregulated in HNO cells. Activation of p53 results in inhibition of apoptosis through induced Mcl1 expression. It also leads to cell cycle modulation. Interestingly, several reports show that cancer stem cells have enhanced expression of proteins involved in DNA damage response and also activated an antiapoptotic response. Our results here suggest that our HNO adapted A549 cells have increased activation of DNA damage response pathway proteins which can lead to better DNA repair function. Enhanced DDR leads to activation of antiapoptosis response and modulation in the cell cycle which may lead to better survival of these cells under harsh conditions. Thus, our present investigation further supports the hypothesis that HNO exposure leads to survival of these cells.

Part II-mechanism of adaptation: A549 cells adapt to high concentration of nitric oxide through bypass of cell cycle checkpoints.

Previous work has shown enhanced survival capacity in high nitric oxide (HNO)-adapted tumor cells. In Part I of this series of manuscripts, we have shown that A549-HNO cells demonstrate an improved growth profile under UV and X-ray radiation treatment. These cells exhibit increased expression of proteins involved in DNA damage recognition and repair pathway, both the non-homologous end joining pathway and homologous recombination. These include Ku80, DNA-PK, XLF ligase and MRN complex proteins. Further, the A549-HNO cells show high levels of ATM, ATR, Chk1 and Chk2, and phospho-p53. Activation of these molecules may lead to cell cycle arrest and apoptosis due to DNA damage. This is observed in parent A549 cells in response to NO donor treatment; however, the A549-HNO cells proliferate and inhibit apoptosis. Cell cycle analysis showed slowed progression through S phase which will allow time for DNA repair. Thus, to better understand the increased growth rate in A549-HNO when compared to the parent cell line A549, we studied molecular mechanisms involved in cell cycle regulation in A549-HNO cells. During the initial time period of NO donor treatment, we observe high levels of cyclin/Cdk complexes involved in regulating various stages of the cell cycle. This would lead to bypass of G1-S and G2-M checkpoints. The HNO cells also show much higher expression of Cdc25A. Cdc25A activates Cdk molecules involved in different phases of the cell cycle. In addition, there is enhanced phosphorylation of the Rb protein in HNO cells. This leads to inactivation of Rb/E2F checkpoint regulating G1-S transition. This may lead to faster progression in S phase. Thus, all of these perturbations in HNO cells lead to accelerated cell cycle progression and a higher growth rate. We also assessed expression of cell cycle inhibitors in HNO cells. Interestingly, the HNO cells show a significant decline in p21CIP1 at initial time points, but with prolonged exposure, the levels were much higher than those of the parent cells. This suggests an initial bypass of cell cycle checkpoints as p21CIP1 can inhibit the activity of all cyclin/Cdk complexes. p21CIP1 is also known to inhibit p53-induced apoptosis. This could be important during later phases of the cell cycle to allow time for repair of damaged DNA and thus better survival of HNO cells.

Part II. Initial molecular and cellular characterization of high nitric oxide-adapted human tongue squamous cell carcinoma cell lines.

It is not understood why some head and neck squamous cell carcinomas, despite having identical morphology, demonstrate different tumor aggressiveness, including radioresistance. High levels of the free radical nitric oxide (NO) and increased expression of the NO-producing enzyme nitric oxide synthase (NOS) have been implicated in tumor progression. We previously adapted three human tongue cancer cell lines to high NO (HNO) levels by gradually exposing them to increasing concentrations of an NO donor; the HNO cells grew faster than their corresponding untreated ("parent") cells, despite being morphologically identical. Herein we initially characterize the HNO cells and compare the biological properties of the HNO and parent cells. HNO/parent cell line pairs were analyzed for cell cycle distribution, DNA damage, X-ray and ultraviolet radiation response, and expression of key cellular enzymes, including NOS, p53, glutathione S-transferase-pi (GST-pi), apurinic/apyrimidinic endonuclease-1 (APE1), and checkpoint kinases (Chk1, Chk2). While some of these properties were cell line-specific, the HNO cells typically exhibited properties associated with a more aggressive behavior profile than the parent cells (greater S-phase percentage, radioresistance, and elevated expression of GST-pi/APE1/Chk1/Chk2). To correlate these findings with conditions in primary tumors, we examined the NOS, GST-pi, and APE1 expression in human tongue squamous cell carcinomas. A majority of the clinical samples exhibited elevated expression levels of these enzymes. Together, the results herein suggest cancer cells exposed to HNO levels can develop resistance to free radicals by upregulating protective mechanisms, such as GST-pi and APE1. These upregulated defense mechanisms may contribute to their aggressive expression profile.

Ca2+ influx via TRPC channels induces NF-kappaB-dependent A20 expression to prevent thrombin-induced apoptosis in endothelial cells.

NF-kappaB signaling is known to induce the expression of antiapoptotic and proinflammatory genes in endothelial cells (ECs). We have shown recently that Ca(2+) influx through canonical transient receptor potential (TRPC) channels activates NF-kappaB in ECs. Here we show that Ca(2+) influx signal prevents thrombin-induced apoptosis by inducing NF-kappaB-dependent A20 expression in ECs. Knockdown of TRPC1 expressed in human umbilical vein ECs with small interfering RNA (siRNA) suppressed thrombin-induced Ca(2+) influx and NF-kappaB activation in ECs. Interestingly, we observed that thrombin induced >25% of cell death (apoptosis) in TRPC1-knockdown ECs whereas thrombin had no effect on control or control siRNA-transfected ECs. To understand the basis of EC survival, we performed gene microarray analysis using ECs. Thrombin stimulation increased only a set of NF-kappaB-regulated genes 3- to 14-fold over basal levels in ECs. Expression of the antiapoptotic gene A20 was the highest among these upregulated genes. Like TRPC1 knockdown, thrombin induced apoptosis in A20-knockdown ECs. To address the importance of Ca(2+) influx signal, we measured thrombin-induced A20 expression in control and TRPC1-knockdown ECs. Thrombin-induced p65/RelA binding to A20 promoter-specific NF-kappaB sequence and A20 protein expression were suppressed in TRPC1-knockdown ECs compared with control ECs. Furthermore, in TRPC1-knockdown ECs, thrombin induced the expression of proapoptotic proteins caspase-3 and BAX. Importantly, thrombin-induced apoptosis in TRPC1-knockdown ECs was prevented by adenovirus-mediated expression of A20. These results suggest that Ca(2+) influx via TRPC channels plays a critical role in the mechanism of cell survival signaling through A20 expression in ECs.

NF-kappaB regulates thrombin-induced ICAM-1 gene expression in cooperation with NFAT by binding to the intronic NF-kappaB site in the ICAM-1 gene.

Activation of NF-kappaB is essential for protease-activated receptor-1 (PAR-1)-mediated ICAM-1 expression in endothelial cells. Here we show that PAR-1 activation induces binding of both p65/RelA and NFATc1 to the NF-kappaB binding site localized in intron-1 of the ICAM-1 gene to initiate transcription in endothelial cells. We discovered the presence of two NF-kappaB binding sites in intron-1 (+70, NF-kappaB site 1; +611, NF-kappaB site 2) of the human ICAM-1 gene. Chromatin immunoprecipitation results showed that thrombin induced binding of p65/RelA and of NFATc1 specifically to intronic NF-kappaB site 1 of the ICAM-1 gene. Electrophoretic mobility shift and supershift assays confirmed the binding of p65/RelA and NFATc1 to the intronic NF-kappaB site 1 in thrombin-stimulated cells. Thrombin increased the expression of ICAM-1-promoter-intron 1-reporter (-1,385 to +234) construct approximately 25-fold and mutation of intronic NF-kappaB site 1 markedly reduced thrombin-induced reporter expression. Moreover, inhibition of calcineurin, knockdown of either NFATc1 or p65/RelA with siRNA significantly reduced thrombin-induced ICAM-1 expression and polymorphonuclear leukocyte adhesion to endothelial cells. In contrast, NFATc1 knockdown had no effect on TNF-alpha-induced ICAM-1 expression. Thus these results suggest that p65/RelA and NFATc1 bind to the intronic NF-kappaB site 1 sequence to induce optimal transcription of the ICAM-1 gene in response to thrombin in endothelial cells.

A mutant allele of BARA/LIN-9 rescues the cdk4-/- phenotype by releasing the repression on E2F-regulated genes.

It has been proposed that C. elegans LIN-9 functions downstream of CDK4 in a pathway that regulates cell proliferation. Here, we report that mammalian BARA/LIN-9 is a predominantly nuclear protein that inhibits cell proliferation. More importantly, we demonstrate that BARA/LIN-9 also acts downstream of cyclin D/CDK4 in mammalian cells since (i) its antiproliferative effect is partially blocked by coexpression of cyclin D1, and (ii) a mutant form that lacks the first 84 amino acids rescues several phenotypic alterations observed in mice null for cdk4. Interestingly, mutation of BARA/LIN-9 restores the expression of E2F target genes in CDK4 null MEFs, indicating that the wild-type protein plays a role in the expression of genes required for the G1/S transition.

Ca2+ influx induced by protease-activated receptor-1 activates a feed-forward mechanism of TRPC1 expression via nuclear factor-kappaB activation in endothelial cells.

Thrombin activation of protease-activated receptor-1 induces Ca(2+) influx through store-operated cation channel TRPC1 in endothelial cells. We examined the role of Ca(2+) influx induced by the depletion of Ca(2+) stores in signaling TRPC1 expression in endothelial cells. Both thrombin and a protease-activated receptor-1-specific agonist peptide induced TRPC1 expression in human umbilical vein endothelial cells, which was coupled to an augmented store-operated Ca(2+) influx and increase in endothelial permeability. To delineate the mechanisms of thrombin-induced TRPC1 expression, we transfected in endothelial cells TRPC1-promoter-luciferase (TRPC1-Pro-Luc) construct containing multiple nuclear factor-kappaB (NF-kappaB) binding sites. Co-expression of dominant negative IkappaBalpha mutant prevented the thrombin-induced increase in TRPC1 expression, indicating the key role of NF-kappaB activation in mediating the response. Using TRPC1 promoter-deletion mutant constructs, we showed that NF-kappaB binding sites located between -1623 and -871 in the TRPC1 5'-regulatory region were required for thrombin-induced TRPC1 expression. Electrophoretic mobility shift assay utilizing TRPC1 promoter-specific oligonucleotides identified that the DNA binding activities of NF-kappaB to NF-kappaB consensus sites were located in this domain. Supershift assays using NF-kappaB protein-specific antibodies demonstrated the binding of p65 homodimer to the TRPC1 promoter. Inhibition of store Ca(2+) depletion, buffering of intracellular Ca(2+), or down-regulation of protein kinase Calpha downstream of Ca(2+) influx all blocked thrombin-induced NF-kappaB activation and the resultant TRPC1 expression in endothelial cells. Thus, Ca(2+) influx via TRPC1 is a critical feed-forward pathway responsible for TRPC1 expression. The NF-kappaB-regulated TRPC1 expression may be an essential mechanism of vascular inflammation and, hence, a novel therapeutic target.

Different requirements for the cytostatic and apoptotic effects of type I interferons. Induction of apoptosis requires ARF but not p53 in osteosarcoma cell lines.

The regulation of cell growth is one of the most important effects of type I interferons (IFNs). This response may involve a cytostatic effect or the induction of apoptosis depending on the cell context. Often the growth-inhibitory response of type I IFNs is studied in tumor cell lines carrying mutations of tumor suppressor genes, and therefore, the growth-inhibitory effect can be influenced by inactivation of these important regulators of cell proliferation. In this report, we explored the role of the ARF-p53 pathway in the growth-inhibitory effect of type I IFNs. We found that p53 is only induced in cells that express p14(ARF) (p19(ARF) in mouse cells). Surprisingly, mouse embryonal fibroblasts that are null for p19(ARF) or P53, even after transformation with oncogenic RAS, respond as well as wild type to the growth-inhibitory effect of type I IFNs. Similarly, human ARF(-/-) U2OS and P53(-/-) SAOS-2 cells show a significant decrease in cell proliferation. However, only SAOS-2 or U2OS reconstituted with inducible p14(ARF) undergo apoptosis in response to IFN beta treatment, and this effect was not inhibited by expression of dominant negative p53. These data suggest that (i) at least in specific cell types, the induction of apoptosis by type I IFNs requires an ARF pathway that is p53-independent and (ii) the cytostatic and pro-apoptotic effects of type I IFNs employ different pathways.

The third myrosinase gene TGG3 in Arabidopsis thaliana is a pseudogene specifically expressed in stamen and petal.

Genomic clones and full-length cDNA for the myrosinase gene TGG3 from Arabidopsis thaliana ecotype Columbia were sequenced. The TGG3 gene was similar with the earlier described myrosinase genes and shared the conserved intron/exon splice sites but had an insertion of one nucleotide in exon 5, a deletion of two nucleotides in exon 6 and a deletion of approximately 210 nucleotides in exon 12. These mutations shifted the open reading frame in exon 5 and resulted in a truncated protein. Analysis of the TGG3 DNA sequence from five other Arabidopsis ecotypes showed polymorphisms, but in no case did a functional TGG3 gene appear to be present. Although TGG3 apparently is a pseudogene, it was expressed specifically in stamen and petal according to RT-PCR analysis, while TGG1 and TGG2 transcripts were present in most of the tested tissues. Western blot analysis showed only one myrosinase band of 68 kDa corresponding to TGG1 and TGG2 in flower samples, while no band corresponding to TGG3 was encountered. Apparently only two functional myrosinases are present in this gene family in Arabidopsis.

Targeted disruption of the testicular SPAG5/deepest protein does not affect spermatogenesis or fertility.

In an effort to define the molecular basis for morphogenesis of major sperm tail structures, including outer dense fibers, we recently cloned the Spag5 gene by virtue of its strong and specific leucine-zipper-mediated interaction with Odf1, the 27-kDa major outer dense fiber protein. Spag5 is expressed during meiosis and in round spermatids and is similar, if not identical, to Deepest, a putative spindle pole protein. Here we report the disruption of the Spag5 gene by homologous recombination. Spag5-null mice lack Spag5 mRNA and protein. However, male mice are viable and fertile. Analysis of the process of spermatogenesis and sperm produced in Spag5-null mice did not reveal a major phenotype as a consequence of the knockout event. This result suggests that if Spag5 plays a role in spermatogenesis it is likely compensated for by unknown proteins.

Identification and characterization of soluble and insoluble myrosinase isoenzymes in different organs of Sinapis alba.

Extraction of Sinapis alba seeds under native conditions solubilized 3 myrosinase isoforms, pool I, II and III, which could be separated by ion exchange chromatography. Sequencing of numerous peptides of the I and III isoforms showed that they belonged to the Myrosinase A (MA) family of myrosinases and that they were encoded by different genes. Western blot analysis of S. alba seed proteins, extracted with a sodium dodecyl sulphate-containing buffer, using an anti-myrosinase monoclonal antibody, showed the presence of two additional myrosinase isoforms with approximate molecular sizes of 62 and 59 kDa. These myrosinases, which only could be solubilized from seeds by inclusion of denaturing agents in the extraction buffer, were by sequence analysis identified as MB myrosinases. These isoenzymes or very similar forms were also present in seedling cotyledons. However, from this tissue, they could be extracted with non-denaturing buffers. In addition, cotyledons contained a 65-kDa MB myrosinase not found in seeds. In contrast, seedling cotyledons contained only minute amounts of pool I and no pool III MA myrosinases, emphasizing the tissue-specific expression of the corresponding gene families. Sequence analysis of myrosinase cDNAs generated cDNA by reversed transcription-polymerase chain reaction using degenerate primers with mRNA isolated from seeds, cotyledons and leaves confirmed the result that the MA isoforms were expressed only in seed tissue, while MB myrosinases were found in all tissues investigated. Furthermore, seed and leaf contained unique MB myrosinase transcripts, suggesting organ-specific expression of individual MB genes.