Molecular mechanisms of <Emphasis Type="Italic">Bombyx batryticatus</Emphasis> ethanol extract inducing gastric cancer SGC-7901 cells apoptosis

Bombyx batryticatus is a traditional Chinese medicine. To understand apoptotic effect of B. batryticatus ethanol extract (BBE), we investigated the role of BBE in inducing apoptosis of human gastric cancer cells SGC-7901. Cells treated with BBE and apoptosis was assessed by methyl thiazolyl tetrazolium (MTT) assay, morphological changes, DNA fragmentation and flow cytometry assays. The expression of Bcl-2, Bax and P21 were evaluated by western blot analysis and real time polymerase chain reaction. MTT assay showed that the cytotoxicity of BBE extract on SGC-7901 cells was correlated with treatment time and concentration. After treatment with 6 mg/mL of BBE the microscopy showed that, the majority of SGC-7901 cells were obviously reduced, distorted and grew slowly. Annexin-V/propidium iodide double-staining assay emerge the early apoptosis and the late apoptosis after treatment with different times by laser confocal fluorescence microscopy and flow cytometer. Cell cycle analysis of SGC 79 cells showed that BBE induced cell cycle arrest in the G1 and G2 phases. DNA fragmentation indicated the trend of BBE inducing apoptosis on SGC-7901 cells. The qRT-PCR and western blot analysis indicated that the mRNA and protein expressions of Bax and P21 were significantly up-regulated whereas that of Bc1-2 was down-regulated after treatment with BBE for 24 h. Our results revealed a correlation between gene regulation and BBE-induced apoptosis, which might indicate the potential of BBE in cancer therapy.     

Effects of ligands of α<Superscript>2</Superscript>-adrenoceptors on mRNA level of apoptotic proteins in developing rat brain

The effects of antagonist of α²-adrenoceptors yohimbine and their agonist clonidine on Bax and Bcl-X _L mRNA levels in neonatal rat brain were studied. Yohimbine decreased Bax mRNA level in the cerebellum, increased the ratio between Bcl-X _L and Bax mRNA levels in the cerebellum, cortex, and hippocampus, and increased Bcl-X _L mRNA level in the cortex and hippocampus of 6-day-old-rat pups 24 h after injection. Administration of clonidine 20 min after yohimbine administration abolished its effect on Bcl-X_L mRNA level in the hippocampus. The data obtained indicate that the blockade of α²-adrenoceptors induces antiapoptotic changes in the developing rat brain, some of which can be abolished after coadministration with the agonist of these receptors.     

Differential alternative splicing in brain regions of rats selected for aggressive behavior

Profiles of alternative mRNA isoforms have been determined in three brain regions of rats from an aggressive and a tame line selected for 74 generations. Among 2319 genes with alternatively spliced exons, approximately 84% were confirmed by analyzing public databases. Based on Gene Ontology-guided clustering of alternatively spliced genes, it has been found that the sample was enriched in synapse-specific genes (FDR < 10^–17). Patterns of gene expression in the brains of animals with genetically determined high or low aggression were more frequently found to differ in the use of alternatively spliced exons than in animals environmentally conditioned for increased or lowered propensity to aggression. For the Adcyap1r1 gene, five alternatively spliced mRNA isoforms have been represented differentially in aggressive animals. A detailed analysis of the gene that encodes glutamate ionotropic receptor NMDA type subunit 1 (Grin1) has confirmed significant differences in the levels of its alternatively spliced isoforms in certain brain regions of tame and aggressive rats. These differences may affect the behavior in rats genetically selected for aggression levels.     

RNA binding protein QKI contributes to WT1 mRNA and suppresses apoptosis in ST cells

The RNA binding protein quaking (QKI), a key member of the STAR family, as an upstream gene could involve in much process including cell proliferation, apoptosis, differentiation and so on. However, the roles of QKI in germ cell, especially in swine testis (ST) cells, was not clear currently. And apoptosis plays important roles in the growth and development. The purpose of the present study was to clarify the relationship between QKI and apoptosis in ST cells. Firstly, our results showed that pEF1α-QKI and shQKI3 have clear effects on expression levels of QKI. Secondly, we established that QKI directly binds to WT1 3′UTR by binding with QRE-1 (2046–2052 bp, ACTAAC) only. Furthermore, QKI overexpression significantly increased the expression levels of WT1 and Bcl-2. QKI also has the effect on delaying the degradation of WT1 mRNA. In addition, we verified that QKI had a significantly suppressed apoptosis in ST cells. Finally, pBI-WT1 could make up for shQKI3-induced decrease in WT1, Bcl-2 mRNA levels and suppress apoptosis in ST cells. The results demonstrated that QKI was an important regulatory factor that affects apoptosis by targeting WT1 gene.     

Synchrony during aggression in adult male Atlantic spotted dolphins <Emphasis Type="Italic">(Stenella frontalis)</Emphasis>

Synchrony among Atlantic spotted dolphins (Stenella frontalis) is crucial for successfully overcoming bottlenose dolphins (Tursiops truncatus) during interspecies aggression (Cusick and Herzing 2014). The present study examined synchrony in adult Atlantic spotted dolphins during aggressive encounters with bottlenose dolphins. Across group size, aggressive behaviors increased preceding synchrony, peaked during synchrony, and decreased dramatically after synchrony. Although smaller groups (< 10 dolphins) became synchronous more frequently than larger groups (> 10 dolphins), larger groups remained synchronous longer; however, smaller groups exhibited greater aggressive behaviors during synchrony, suggesting that additional aggressive behaviors may be necessary to compensate for the smaller group size, whereas larger groups may be able to rely on synchrony with less aggression. Disorganized squawk bouts synchronized as physical synchrony began, but only if coupled with escalating aggression.     

Expression of iron-regulators in the bone tissue of rats with and without iron overload

Recently, more and more studies indicate that iron overload would cause osteopenia or osteoporosis. However, the molecular mechanism of it remains unclear. Moreover, very little is known about the iron metabolism in bone tissue at present. Therefore, the mRNA expression of iron-regulators, transferrin receptor1 (Tfr1), divalent metal transporter1 (Dmt1?+?IRE and Dmt1???IRE), ferritin (FtH and FtL), and ferroportin1 (Ireg1), and the localization of ferroportin1 protein were examined in the bone tissue of rats. In addition, the mRNA expression of each gene was compared between groups of rats with and without iron overload. The results showed that ferroportin1 protein was localized in the cytoplasm of osteoblast, osteocyte, chondrocyte and osteoclast of rats’ femur. The six iron-regulatory genes, Tfr1, ferritin (FtH and FtL), (Dmt1?+?IRE and Dmt1???IRE) and ferroportin1 (Ireg1), were found in femurs of rats. In addition, significantly up-regulated expression of FtH and FtL mRNA, and markedly down-regulated expression of Tfr1, Dmt1?+?IRE and Ireg1 mRNA, were observed in the iron overload group compared with the control group. The result indicates that ferroportin1 protein is localized in the cytoplasm of bone cells of rats. Tfr1, Dmt1, ferritin and ferroportin1 exist in bone tissue of rats, and they may be involved in the pathological process of iron overload-induced bone lesion.     

Cytotoxic and toxicogenomic effects of silibinin in bladder cancer cells with different <Emphasis Type="Italic">TP53</Emphasis> status

Silibinin is a natural phenol found in the seeds of the milk thistle plant. Recent data have shown its effectiveness for preventing/treating bladder tumours. Therefore, in this study we investigated the cytotoxic and toxicogenetic activity of silibinin in bladder cancer cells with different TP53 statuses. Two bladder urothelial carcinoma cell lines were used: RT4 (wild-type TP53 gene) and T24 (mutated TP53 gene). Cell proliferation, clonogenic survival, apoptosis rates, genotoxicity and relative expression profile of FRAP/mTOR, FGFR3, AKT2 and DNMT1 genes and of miR100 and miR203 were evaluated. Silibinin promoted decreased proliferation and increased late apoptosis in TP53 mutated cells. Increased early apoptosis rates, primary DNA damage, and decrease of cell colonies in the clonogenic survival assay were detected in both RT4 and T24 cell lines. Down-regulation of FRAP/mTOR, AKT2, FGFR3, DNMT1 and miR100 expression occurred in RT4 cells. Modulation of miR203 was observed in both cell lines. In conclusion, despite the reduction of clone formation in both cell lines, the toxicogenomic effect of silibinin on FRAP/mTOR, AKT2, FGFR3, DNMT1 and miR100 was dependent on the TP53 status. Taken together, the data confirmed the role of silibinin as an antiproliferative compound, whose mechanism of action was related to the TP53 status.     

Aggression and empathy as genetic differentiation factors of urban population

Permanent residents of Kharkiv (637 men and 856 women at the age of 45–65 years) are tested on the level of aggression and empathy. The average aggression level (41.7 points) is higher in migrants (born outside Kharkiv) than in indigenous people (36.3 points); the average empathy level is lower in migrants (3.2 points) than in indigenous people (5.5 points). The average values of the aggression and empathy indices are not associated with ethnicity and degree of miscegenation. The correlation between spouses (r) by these personal features is within 0.20–0.31; the marriage conjugation index (K) is 0.13–0.18. Genotyping of the married couples for the rs2235186 SNP of X-linked monoaminooxidase (MAO-A) gene detected a positive marriage assortativeness: the C × CC and T × TT pairs are developed more frequently than during panmixia; the C × TT and T × CC pairs, less frequently. The T allele is coupled with increased aggression level and decreased empathy level. The phenotypes of heterozygous women indicate the intermediate inheritance of these traits.     

The poplar ARGOS-LIKE gene promotes leaf initiation and cell expansion,and controls organ size

We identified a Populus nigra auxin-regulated gene involved in organ size (PnARGOS)-LIKE, encoding one organ size related protein in black poplar. It is homologous to AtARGOS and AtARGOS-LIKE genes of Arabidopsis thaliana. ABRE-like, G-box, GATA and I-box motifs were discovered in the promoter region of the poplar ARGOS-LIKE gene. In wild type aspen (Populus tremula) plants, an ortholog of the PnARGOS-LIKE gene (PtrARGOS-LIKE) was noticeably expressed in actively dividing and expanding young leaves and calli, whereas its mRNA content increased in response to exogenous 6-benzylaminopurine, 1-naphthaleneacetic acid, and 24-epibrassinolide. Expression of the PtrARGOS-LIKE gene was reduced under a salinity treatment. In addition, we generated transgenic tobacco and aspen plants with an up-regulated expression of the PnARGOS-LIKE gene. A constitutive expression of the gene contributed to an increase in size of stems and leaves of the transgenic tobacco plants. In the transgenic aspen, a constitutive expression of the PnARGOS-LIKE gene promoted an increase in the frequency of leaf initiations and in leaf length and area. The size of transgenic tobacco and aspen leaves increased due to the enlargement of individual cells. The results show the significance of the PnARGOS-LIKE gene for control of leaf initiation and organ growth by cell expansion in poplar.     

Connection between proliferation rate and temozolomide sensitivity of primary glioblastoma cell culture and expression of <Emphasis Type="Italic">YB-1</Emphasis> and <Emphasis Type="Italic">LRP/MVP</Emphasis>

Glioblastomas (GBL) are the most common and aggressive brain tumors. They are distinguished by high resistance to radiation and chemotherapy. To find novel approaches for GBL classification, we obtained 16 primary GBL cell cultures and tested them with real-time PCR for mRNA expression of several genes (YB-1, MGMT, MELK, MVP, MDR1, BCRP) involved in controlling cell proliferation and drug resistance. The primary GBL cultures differed in terms of proliferation rate, wherein a group of GBL cell cultures with low proliferation rate demonstrated higher resistance to temozolomide. We found that GBL primary cell cultures characterized by high proliferation rate and lower resistance to temozolomide expressed higher mRNA level of the YB-1 and MDR1 genes, whereas upregulated expression of MVP/LRP mRNA was a marker in the group of GBL with low proliferation rate and high resistance. A moderate correlation between expression of YB-1 and MELK as well as YB-1 and MDR1 was found. In the case of YB-1 and MGMT expression, no correlation was found. A significant negative correlation was revealed between mRNA expression of MVP/LRP and MELK, MDR1, and BCRP. No correlation in expression of YB-1 and MVP/LRP genes was observed. It seems that mRNA expression of YB-1 and MVP/LRP may serve as a marker for GBL cell cultures belonging to distinct groups, each of which is characterized by a unique pattern of gene activity.     

Lutein protects against β-amyloid peptide-induced oxidative stress in cerebrovascular endothelial cells through modulation of Nrf-2 and NF-κb

In the present study, we determined the protective role of lutein against Aβ _25–35 peptide-induced oxidative stress and apoptosis in bEND.3 cells. Cell viability was determined through MTT assay. Reactive oxygen species, lipid peroxides, and antioxidant enzyme activities were evaluated to analyze the oxidative stress status. NF-κB and Nrf-2 downstream target protein expressions were determined through western blot. Apoptosis was analyzed through caspase activities and subG1 accumulation. The results showed that Aβ _25–35 significantly increased (p < 0.001) oxidative stress biomarkers. Aβ _25–35 significantly up-regulated NF-κB nuclear expression and down-regulated Nrf-2 levels and HO-1 and, NQO-1 expressions. Aβ _25–35 induced apoptosis through decreasing mitochondrial membrane potential and increasing caspase 9 and 3 activities. Lutein pre-treatment significantly (p < 0.001) improved cell viability and decreased ROS levels (p < 0.001) and lipid peroxidation (p < 0.01). Lutein prevented Aβ _25–35-induced NF-κB nuclear expressions and up-regulated Nrf-2 expressions. Further, lutein also improved mitochondrial membrane potential and down-regulated caspase activities and subG1 accumulation. The present study shows the protective role of lutein against Aβ _25–35-induced toxicity by modulating Nrf-2 and NF-κB expressions in cerebrovascular endothelial cells.     

Behavioral interactions under multiple stressors: temperature and salinity mediate aggression between an invasive and a native fish

Interspecific aggression is a critical determinant of the success and competitive superiority of many invasive over native species. While single abiotic stressors can alter aggression levels, the manner in which multiple stressors may alter the strength and outcome of interspecific interactions and hence the invasion potential of a species is still poorly understood, even though multiple stressors are prevalent in many ecosystems. Furthermore, multiple stressors may interact to produce synergistic or antagonistic effects on individual level behaviors, thereby modulating invasive-native species interactions in unexpected ways. Here we examined the effect of two key abiotic stressors in freshwater ecosystems-temperature and salinity-on interspecific aggression between the invasive eastern mosquito fish (Gambusia holbrooki) and juveniles of the native Australian bass (Macquaria novemaculeata). Under controlled laboratory conditions, individuals were exposed to low or high salinity levels (15 and 35‰), and low or high temperatures (21 and 28 °C), and the frequency of interspecific aggressive behaviors was scored. The effect of temperature and salinity on interspecific aggression was antagonistic for both M. novemaculeata and G. holbrooki. While elevated temperature promoted aggression, elevated salinity partially or entirely negated this effect. Moreover, regardless of temperature, M. novemaculeata was more aggressive than G. holbrooki under elevated salinity. In addition to this, the native displayed more aggression to smaller than larger heterospecifics when exposed to elevated salinity alone, while G. holbrooki showed no such preference. These results highlight the importance of considering the interplay between multiple abiotic stressors and behavioral interactions between invasive and native species, combined with the modulating effect of species-specific and size based responses to those stressors.     

Effect of influenza A virus and bacterial lipopolysaccharide on proliferation and expression of cytokines and other cellular factors in the endothelial cell line ECV-304

Viral infection and bacterial lipopolysaccharide (LPS) cause endothelial-cell dysfunction. The aim of the current study was to investigate the effect of influenza A virus and LPS from Escherichia coli on the proliferative activity and gene expression of cytokines and cellular factors (TNFα, TGFβ, IFN-γ, MMP-9, NF-κB, Rho A, eNOS, and iNOS) in human endothelial cells ECV-304. It was found that ECV-304 cells infected with very low infectious doses of influenza virus acquired the capacity for the long-term active proliferation (over eight passages). Addition of LPS from E. coli reduced the virus-stimulated cell proliferation. It was shown that influenza virus and LPS affected the gene expression of cytokine and other cellular factors. When endothelial cells were infected with influenza A virus in the presence of LPS, there was a significant increase in the expression of several genes and the expression pattern of certain genes was modified. Expression of MMP-9 gene inhibited by the virus and LPS separate exposure significantly increased during the first day after addition of the virus and LPS simultaneously. The same was true for the IFN-γ gene expression. TNFα gene was active only for 1–3 days whereas the expression of TGFβ, eNOS, iNOS, NF-κB and Rho A genes increased significantly on the fifth day, as it was observed with the cells treated with LPS only. Thus, the influenza A virus and LPS change the physiological state of endothelial cells. This occurred during various time periods (as well as at various degrees of viral infection) produced by different cellular factors and, possibly, involved different signaling pathways.