Imperatorin shows selective antitumor effects in SGC-7901 human gastric adenocarcinoma cells by inducing apoptosis, cell cycle arrest and targeting PI3K/Akt/m-TOR signalling pathway.

PURPOSE: The main purpose of the present study was to determine the anticancer properties of imperatorin - a naturally occurring coumarin compound - against SGC-7901 human gastric adenocarcinoma cells and the mouse fibroblast cell line 3T3 (normal cell line). METHODS: Imperatorin effects on apoptosis induction, cell cycle phase distribution and PI3K/Akt/m-TOR signalling pathways were studied. MTT cell viability assay examined the compound's cytotoxic potential, while inverted phase contrast microscopy and fluorescence microscopy techniques were used to study morphological changes induced in SGC-7901 cells by imperatorin. Flow cytometry examined its effects on cell cycle progression while Western blot assay was used to study changes in protein expressions of PI3K/Akt/m-TOR pathway. RESULTS: Imperatorin induced a dose-dependent growth inhibition of the SGC-7901 gastric cancer cells with an IC50 value 62.6 µM, while in case of normal 3T3 mouse fibroblast cells, the drug did not show significant toxicity (IC50 value 195.8 µM), indicating that the drug selectively induced cytotoxicity in gastric cancer cells. The cells became rounded up, shrunken in size and got detached from the monolayer attached to well surface. Cells treated with 10, 75 and 175 µM imperatorin indicated that they began to emit yellow or red fluorescence which is an indication of early or late apoptosis respectively. Imperatorin also induced significant DNA fragmentation along with increasing the fraction of sub-G1 cells, indicating a sub-G1 cell cycle arrest. CONCLUSION: Imperatorin could prove an important lead molecule for the treatment of gastric cancer and deserves further research in vivo against more cell lines.

Injection of Toll-like receptor 4 siRNA into the ventrolateral periaqueductal gray attenuates withdrawal syndrome in morphine-dependent rats.

We assessed the role of the Toll-like receptor 4 (TLR4) gene in the ventrolateral periaqueductal gray (vlPAG) region of morphine-dependent rats on attenuating withdrawal syndrome, and regulating glutamic acid decarboxylase (GAD67), glutamic acid (Glu), and gamma-aminobutyric acid (GABA). After siRNA-mediated downregulation of TLR4, changes were observed in withdrawal behavior and downstream signaling molecules. Rats were injected into the vlPAG with TLR4 siRNA, followed by intraperitoneal injection of morphine for 5 consecutive days, and then naloxone, and the behavioral indices of morphine withdrawal were observed. 'Wet-dog' shakes, teeth chattering, and the total scores of withdrawal reactions were reduced. TLR4 expression and Glu levels were reduced, whereas GAD67 and GABA levels were increased. Overall, these findings indicate that modifying TLR4 gene expression in the vlPAG stimulates expression of the downstream signaling molecule, GAD67, which decreases Glu levels and increases GABA levels. This mechanism may explain the inhibition of withdrawal syndrome in morphine-dependent rats.

Analysis of variants and mutations in the human winged helix FOXA3 gene and associations with metabolic traits.

BACKGROUND/OBJECTIVES: The forkhead factor Foxa3 is involved in the early transcriptional events controlling adipocyte differentiation and plays a critical function in fat depot expansion in response to high-fat diet regimens and during aging in mice. No studies to date have assessed the potential associations of genetic variants in FOXA3 with human metabolic outcomes. SUBJECTS/METHODS: In this study, we sequenced FOXA3 in 392 children, adolescents and young adults selected from several cohorts of subjects recruited at the National Institute of Child Health and Human Development of the National Institutes of Health based on the availability of dual-energy X-ray absorptiometry data, magnetic resonance imaging scans and DNA samples. We assessed the association between variants present in these subjects and metabolic traits and performed in vitro functional analysis of two novel FOXA3 missense mutations identified. RESULTS: Our analysis identified 14 novel variants and showed that the common single-nucleotide polymorphism (SNP) rs28666870 is significantly associated with greater body mass index, lean body mass and appendicular lean mass (P values 0.009, 0.010 and 0.013 respectively). In vitro functional studies showed increased adipogenic function for the FOXA3 missense mutations c.185C>T (p.Ser62Leu) and c.731C>T (p.Ala244Val) compared with FOXA3-WT. CONCLUSIONS: Our study identified novel FOXA3 variants and mutations, assessed the adipogenic capacity of two novel missense alterations in vitro and demonstrated for the first time the associations between FOXA3 SNP rs28666870 with metabolic phenotypes in humans.

Brain-derived neurotrophic factor in human subjects with function-altering melanocortin-4 receptor variants.

BACKGROUND: In rodents, hypothalamic brain-derived neurotrophic factor (BDNF) expression appears to be regulated by melanocortin-4 receptor (MC4R) activity. The impact of MC4R genetic variation on circulating BDNF in humans is unknown. OBJECTIVE: The objective of this study is to compare BDNF concentrations of subjects with loss-of-function (LOF) and gain-of-function (GOF) MC4R variants with those of controls with common sequence MC4R. METHODS: Circulating BDNF was measured in two cohorts with known MC4R sequence: 148 subjects of Pima Indian heritage ((mean±s.d.): age, 15.7±6.5 years; body mass index z-scores (BMI-Z), 1.63±1.03) and 69 subjects of Hispanic heritage (10.8±3.6 years; BMI-Z, 1.57±1.07). MC4R variants were characterized in vitro by cell surface expression, receptor binding and cyclic AMP response after agonist administration. BDNF single-nucleotide polymorphisms (SNPs) rs12291186, rs6265 and rs7124442 were also genotyped. RESULTS: In the Pima cohort, no significant differences in serum BDNF was observed for 43 LOF subjects versus 65 LOF-matched controls (age, sex and BMI matched; P=0.29) or 20 GOF subjects versus 20 GOF-matched controls (P=0.40). Serum BDNF was significantly associated with genotype for BDNF rs12291186 (P=0.006) and rs6265 (P=0.009), but not rs7124442 (P=0.99); BDNF SNPs did not interact with MC4R status to predict serum BDNF. In the Hispanic cohort, plasma BDNF was not significantly different among 21 LOF subjects, 20 GOF subjects and 28 controls (P=0.79); plasma BDNF was not predicted by BDNF genotype or BDNF-x-MC4R genotype interaction. CONCLUSIONS: Circulating BDNF concentrations were not significantly associated with MC4R functional status, suggesting that peripheral BDNF does not directly reflect hypothalamic BDNF secretion and/or that MC4R signaling is not a significant regulator of the bulk of BDNF expression in humans.

Association between TIM-1 gene polymorphisms and allergic rhinitis in a Han Chinese population.

BACKGROUND: TIM-1, a member of the T cell immunoglobulin and mucin (TIM) domain family, is involved in T-cell differentiation and has been implicated in allergic diseases. An association between TIM-1 and allergic rhinitis, however, has not been established. OBJECTIVE: To investigate whether TIM-1 gene polymorphisms were associated with allergic rhinitis in a Han Chinese population. METHODS: Two TIM-1 promoter single nucleotide polymorphisms (SNPs), -416G>C and -1454G>A, were examined in 185 allergic rhinitis patients of Han Chinese ethnicity using polymerase chain reaction (PCR) and restriction fragment length polymorphism. Additionally, exon 4 insertion/deletion polymorphisms in the TIM-1 gene were analyzed by PCR, polyacrylamide gel electrophoresis, and silver staining. The relationship between gene polymorphisms and serum specific IgE levels in this Han Chinese population was also evaluated. RESULTS: We found that the -416G>C and -1454G>A SNPs were associated with allergic rhinitis susceptibility in this Han Chinese population. No statistically significant differences in the distribution of genotype or allele frequencies of 5383_5397ins/del and 5509_5511delCAA in exon 4 were observed. The -416G>C and -1454G>A SNPs were associated with the level of serum IgE specific to house dust mites in patients with allergic rhinitis. CONCLUSIONS: These results suggest that TIM-1 gene polymorphisms (-416G>C and -1454G>A) are associated with allergic rhinitis susceptibility in a Han Chinese population.

LINC01096 knockdown inhibits progression of triple-negative breast cancer by increasing miR-3130-3p.

OBJECTIVE: Triple-negative breast cancer (TNBC) is an aggressive type of breast cancer. Long noncoding RNAs (lncRNAs) have been reported to be involved in the development of TNBC. However, the role and mechanism of LINC01096 in TNBC are largely unclear. This work aims to investigate the effect of LINC01096 on cell viability, apoptosis, migration, and invasion of TNBC cells, as well as explore the interaction between LINC01096 and microRNA (miR)-3130-3p. PATIENTS AND METHODS: Sixty TNBC patients were recruited. T47-D and BT-549 cells were cultured for experiments in vitro. The expression levels of LINC01096 and miR-3130-3p were detected by quantitative Real Time-Polymerase Chain Reaction (qRT-PCR). Cell viability, apoptosis, migration, and invasion were determined by MTT, flow cytometry, and trans-well assays. The target association between LINC01096 and miR-3130-3p was confirmed by the luciferase reporter assay. RESULTS: The expression of LINC01096 was enhanced in TNBC tissues and cells. High expression of LINC01096 predicted poor outcomes of patients with TNBC. Silence of LINC01096 led to the suppression of cell viability, migration, and invasion, as well as the promotion of apoptosis in TNBC cells. MiR-3130-3p was targeted by LINC01096 and lowly expressed in TNBC. Overexpression of miR-3130-3p repressed cell viability, migration, and invasion, while it induced apoptosis. However, the knockdown of miR-3130-3p induced the opposite effect. This was weakened by inhibiting LINC01096. CONCLUSIONS: Knockdown of LINC01096 inhibited cell viability, migration, and invasion; however, it promoted apoptosis in TNBC by up-regulating miR-3130-3p, indicating a novel target for the treatment of TNBC.

ARHGAP15 regulates lung cancer cell proliferation and metastasis via the STAT3 pathway.

OBJECTIVE: Lung cancer, which is typically diagnosed at later stages, is a leading cause of cancer death among both males and females given its highly invasive and rapidly metastasizing nature. Rho GTPase activating protein 15 (ARHGAP15) is a member of the RhoGAP family and functions in multiple biological processes, such as cell proliferation and migration. However, the effect of ARHGAP15 in lung cancer and its underlying molecular mechanisms remain unclear. PATIENTS AND METHODS: In this study, immunohistochemistry and Real Time PCR were performed to detect ARHGAP15 expression in lung cancer tissues and cells. Proliferation, transwell, and Western blot assays were further performed to explore the role and underlying mechanism of ARHGAP15 in lung cancer. RESULTS: Reduced ARHGAP15 expression was observed in lung cancer tissues and cells. In vitro upregulation of ARHGAP15 in lung cancer cells strongly suppressed cell proliferation, migration, and invasion and was accompanied by reduced matrix metalloproteinase-2 (MMP2), MMP9, vascular endothelial growth factor (VEGF) expression, and the phosphorylation of the signal transducer and activator of transcription-3 (p-STAT3). In contrast, interleukin-6 (IL-6) had the opposite effect and the induction of IL-6 was counteracted by ARHGAP15 upregulation. In addition, the proliferation, migration, and cell invasion induced by ARHGAP15 silencing were potentially inhibited by the STAT3 inhibitor AG490 (100 µM), MMP2, MMP9, VEGF, and p-STAT3 levels decreased. CONCLUSIONS: These results suggest that ARGFAP15 suppressed the proliferation and metastasis of lung cancer cells, which may occur through the inhibition of MMP2, MMP9, and VEGF expression via the STAT3 pathway inactivation.

Oxidative stress, lysosomal damage and dysfunctional autophagy in molluscan hepatopancreas (digestive gland) induced by chemical contaminants.

Autophagy is a highly conserved evolutionary survival or defence process that enables cells and organisms to survive periods of environmental stress by breaking down cellular organelles and macromolecules in autolysosomes to provide a supply of nutrients for cell maintenance. However, autophagy is also a part of normal cellular physiology that facilitates the turnover of cellular constituents under normal conditions: it can be readily augmented by mild environmental stress; but becomes dysfunctional with severe oxidative stress leading to cellular pathology. The molluscan hepatopancreas or digestive gland provides a versatile and environmentally relevant model to investigate lysosomal autophagy and stress-induced dysfunctional autophagy. This latter process has been implicated in many animal and human disease conditions, including degenerative and neurodegenerative diseases, as well as obesity related conditions. Many environmental pollutants have also been found to induce dysfunctional autophagy in molluscan hepatopancreatic digestive cells, and in this study, the marine blue mussel Mytilus galloprovincialis was exposed for 7 days to: 0.1 µM, 1 µM and 10 µM concentrations of fluoranthene and phenanthrene (PAHs); chlorpyrifos and malathion (organophosphorus compounds); atrazine (triazine herbicide); copper (transition metal) and dodecylbenzene sulphonic acid (LAS, surfactant). The marine snail or periwinkle, Littorina littorea, was also exposed to phenanthrene, chlorpyrifos and copper. Indices of oxidative stress, cell injury and dysfunctional autophagy were measured (i.e., lysosomal membrane stability, protein carbonyls, lipofuscin, and lysosomal accumulation of lipid or lipidosis). Evidence of oxidative stress, based on the elevation of lipofuscin and protein carbonyls, was found for all compounds tested; with chlorpyrifos being the most toxic to both species. Dysfunctional autophagy was induced by all of the compounds tested in both species, except for atrazine in mussels. This failure of normal autophagy was consistently associated with oxidative stress. Autophagic dysfunction is an important emerging feature in the aetiology of many disease conditions in animals and humans; and an explanatory conceptual mechanistic model has been developed for dysregulation of autophagy in response to oxidative stress.

The IVBF dual-blade plate and its applications.

A recently developed simple device, the intervertebral body fixation dual-blade plate, was used in 88 cases of different spinal disorders. This patients in the first series were operated on from 1984 to 1986. The device is biomechanically simple and its application safe and easy. Using a proper bone grafting technique, it is a reliable device to establish spinal stability in spine surgery through an anterior approach. Its application in cases of fractures and fracture-dislocations of the thoracolumbar spine with paraplegia, tuberculous spondylitis, and primary tumors of vertebral bodies is presented. The midterm results, covering a follow-up period of 24-49 months (mean, 32 months) were satisfactory; there were no serious complications directly related to the device. There were four cases of pseudarthrosis due to insufficient bone graft technique. Of these, there were three cases of failure of the dual-blade plate.

Studies on gastrin in duodenal ulcer.

By immunocytochemical method and radioimmunoassay, the gastrin secreting cells (G cells) and gastrin concentration in antral mucosa, gastric juice and serum in 20 patients with duodenal ulcer (DU) were studied. The number of G cells and gastrin concentration in antral mucosa showed no significant difference as compared with normal control. The number of G cells in patients with DU and antral atrophy was much higher than those with antral atrophy but with DU. It indicated that G cells were increased in number in DU, and the gastrin concentration in gastric juice (271.11 +/- 255.25 pg/ml) was much higher than in sera (74.71 +/- 43.07 pg/ml). G cells were distributed in different parts of pyloric glands, showing that gastrin in gastric juice should come directly from G cells. The disturbance of feedback mechanism in regulating gastric acidity might be an important role in hypersecretion of gastric acid in DU. The increase of gastrin concentration in gastric juice might be closely related to hyperplasia of parietal cells.

Fine-mapping of an Arabidopsis cell death mutation locus.

An Arabidopsis cell death mutation locus was mapped to chromosome 2 between /GS1 and mi421. The YAC clone ends, CIC9A3R, CIC11C7L, CIC2G5R and RFLP marker CDs3 within this interval, were used to probe TAMU BAC library and 31 BAC clones were obtained. A BAC contig encompassing the mutation locus, which consists of T6P5, T7M23, T12A21, T8L6 and T18A18, was identified by Southern hybridization with the BAC ends as probes. 11 CAPS and 12 STS markers were developed in this region. These results will facilitate map-based cloning of the genes and sequencing of the genomic DNA in this region.

[Biodegradation and biocompatibility of a chitosan film].

The biodegradability and biocompatibility of a chitosan film were investigated in mice. The results showed that chitosan films had a mild inflammatory reaction in the early days of grafting, and after 16 weeks the inflammation basically subsided. Chitosan films were easily biodegraded. Chitosan is a novel natural absorbable medical film material and has a good developmental prospect.

Amplification of rainbow trout microsatellites in Brachymystax lenok.

Brachymystax lenok is a cold freshwater fish accustomed to inhabit relatively high concentration of dissolved oxygen and clean upper streams. Here we present 13 polymorphic microsatellite primer pairs from rainbow trout to amplify in 32 B. lenok individuals from Ussuri River of China. The number of alleles ranged from two to seven with an average of 3.9 per locus. Observed heterozygosity ranged from 0.0625 to 0.9677. One locus showed significant deviation from Hardy-Weinberg equilibrium. These 13 loci will provide a good basis for investigation of B. lenok population structure and genetic diversity in different distribution region.

Fast resistive reconnection regime in the nonlinear evolution of double tearing modes.

Phases of nonlinear double tearing modes are studied numerically. The first two phases lead to the formation and growth of magnetic islands and are followed by a fast reconnection phase to complete the process, driven by a process of neighboring magnetic separatrices merging and magnetic islands coupling. The fast growth can be understood as a result of the island interaction equivalent to a steadily inward flux boundary driven. Resistivity dependences for various phases are studied and shown by scaling analysis for the first time. It is found that after an early Sweet-Parker phase with a eta(1/2)-scale, a slow nonlinear phase in a Rutherford regime with a eta(1)-scale is followed by the fast reconnection phase with a eta(1/5)-scale.

[mRNA expression of hyaluronan synthase in the endolymphatic sac cells of guinea pigs].

OBJECTIVE: To detect the mRNA expression of hyaluronan synthase in the endolymphatic sac cells of guinea pigs. METHODS: After consulting gene bank, we analyzed conservative sequence of hyaluronan synthases in different species, detected the mRNA expression of hyaluronan synthase in the endolymphatic sac cells of guinea pigs with oligonucleotide probe by hybridization in situ. RESULTS: mRNA of hyaluronan synthase was strongly expressed in some epithelial cells of endolymphatic sac, coupled with negative expression in negative control groups. CONCLUSION: It confirms that endolymphatic sac cells can synthesize hyaluronan.

[Expression of alpha and beta subunit isoforms of Na, K-ATPase in the guinea pig endolymphatic sac].

OBJECTIVE: To investigate the expression and significance of alpha and beta subunit isoforms of Na, K-ATPase in the guinea pig endolymphatic sac. METHODS: The distribution of alpha and beta subunit isoforms of Na, K-ATPase in endolymphatic sac of guinea pig was identified by immunocytochemistry and in situ hybridization. RESULTS: The expression of Na, K-ATPase alpha and beta subunit isoforms varied among different cell regions of the endolymphatic sac. Epithelial cells of the endolymphatic sac were observed to contain the alpha 1, beta 1 and beta 2 subunit isoforms, and the expression of beta 2 was stronger than that of beta 1. Subepithelial cells contained alpha 1 and alpha 2 subunit isoforms and the expression of alpha 1 was stronger than that of alpha 2. No expression of alpha 3 subunit isoform was observed in endolymphatic sac. CONCLUSION: Na, K-ATPase of endolymphatic sac consists of different alpha and beta subunit isoforms which, working in concert, serve to maintain homeostasis in inner ear.

Deficiency in fatty acid synthase leads to premature cell death and dramatic alterations in plant morphology.

An Arabidopsis mosaic death1 (mod1) mutant, which has premature cell death in multiple organs, was isolated. mod1 plants display multiple morphological phenotypes, including chlorotic and curly leaves, distorted siliques, premature senescence of primary inflorescences, reduced fertility, and semidwarfism. The phenotype of the mod1 mutant results from a single nuclear recessive mutation, and the MOD1 gene was isolated by using a map-based cloning approach. The MOD1 gene encodes an enoyl-acyl carrier protein (ACP) reductase, which is a subunit of the fatty acid synthase complex that catalyzes de novo synthesis of fatty acids. An amino acid substitution in the enoyl-ACP reductase of the mod1 mutant causes a marked decrease in its enzymatic activity, impairing fatty acid biosynthesis and decreasing the amount of total lipids in mod1 plants. These results demonstrate that a deficiency in fatty acid biosynthesis has pleiotropic effects on plant growth and development and causes premature cell death.