Hemoglobin regulates the migration of glioma cells along poly(ε‐caprolactone)‐aligned nanofibers

Aligned fibers have been shown to facilitate cell migration in the direction of fiber alignment while oxygen (O₂)‐carrying solutions improve the metabolism of cells in hypoxic culture. Therefore, U251 aggregate migration on poly(ε‐caprolactone) (PCL)‐aligned fibers was studied in cell culture media supplemented with the O₂ storage and transport protein hemoglobin (Hb) obtained from bovine, earthworm and human sources at concentrations ranging from 0 to 5 g/L within a cell culture incubator exposed to O₂ tensions ranging from 1 to 19% O₂. Individual cell migration was quantified using a wound healing assay. In addition, U251 cell aggregates were developed and aggregate dispersion/cell migration quantified on PCL‐aligned fibers. The results of this work show that the presence of bovine or earthworm Hb improved individual cell viability at 1% O₂, while human Hb adversely affected cell viability at increasing Hb concentrations and decreasing O₂ levels. The control data suggests that decreasing the O₂ tension in the incubator from 5 to 1% O₂ decreased aggregate dispersion on the PCL‐aligned fibers. However, the addition of bovine Hb at 5% O₂ significantly improved aggregate dispersion. At 19% O₂, Hb did not impact aggregate dispersion. Also at 1% O₂, aggregate dispersion appeared to increase in the presence of earthworm Hb, but only at the latter time points. Taken together, these results show that Hb‐based O₂ carriers can be utilized to improve O₂ availability and the migration of glioma spheroids on nanofibers. © 2014 American Institute of Chemical Engineers Biotechnol. Prog., 30:1214–1220, 2014     

Analysis of the neutral polysaccharide fraction of MCP and its inhibitory activity on galectin-3

The pH-modified citrus pectin (MCP) has been demonstrated to inhibit galectin-3 in cancer progression. The components and structures of MCP related to this inhibition remained unknown. In this paper, we fractionated MCP on DEAE-cellulose column into a homogenous neutral fraction MCP-N (about 20?kDa) and a pectin mixture fraction MCP-A (wide molecular distribution on Sepharose CL-6B chromatography). Both MCP-N and MCP-A inhibited hemagglutination mediated by galectin-3 with minimum inhibition concentration (MIC) 625 and 0.5?μg/ml, respectively. MCP-N was identified to be a type I arabinogalactan (AG-I) with a main chain of β-1→4-galactan. MCP-N was digested by α-L-arabinofuranosidase to give its main chain structure fraction (M-galactan, around 18?kDa), which was more active than the original molecule, MIC 50?μg/ml. The acidic degradation of M-galactan increased the inhibitory activity, MIC about 5 times lower than M-galactan. These results above showed that the functional motif of the β-1→4-galactan fragment might lie in the terminal residues rather than in the internal region of the chain. Therefore, MCP-N and its degraded products might be developed to new potential galectin-3 inhibitors. This is the first report concerning the fractionation of MCP and its components on galectin-3 inhibition. The information provided in this paper is valuable for screening more active galectin-3 inhibitors from natural polysaccharides.     

Development of a Loop-Mediated Isothermal Amplification Assay for Porcine Circovirus Type 2

In this study, the loop-mediated isothermal amplification (LAMP) method was used to develop a rapid and simple detection system for porcine circovirus type 2 (PCV2). According to the PCV2 sequences published in GenBank, multiple LAMP primers were designed targeting conserved sequences of PCV2. Using the DNA extracted from PCV2 isolates HUN-09 and SD-09 as the template, LAMP reactions in a PCV2 LAMP system was performed, the amplification products were detected by adding SYBR Green I and could be observed di...     

The Combination of RAD001 and MK-2206 Exerts Synergistic Cytotoxic Effects against PTEN Mutant Gastric Cancer Cells: Involvement of MAPK-Dependent Autophagic,but Not Apoptotic Cell Death Pathway

In the current study, we showed that the combination of mammalian target of rapamycin (mTOR) inhibitor RAD001 (everolimus) and Akt inhibitor MK-2206 exerted synergistic cytotoxic effects against low-phosphatase and tensin homolog (PTEN) gastric cancer cells (HGC-27 and SNU-601 lines). In HGC-27 cells, RAD001 and MK-2206 synergistically induced G1/S cell cycle arrest, growth inhibition, cell death but not apoptosis. RAD001 and MK-2206 synergistically induced light chain 3B (LC3B) and beclin-1 expression, two important autophagy indicators. Meanwhile, the autophagy inhibitor 3-methyladenine (3-MA) and chloroquine inhibited the cytotoxic effects by RAD001 and MK-2206, suggesting that autophagic, but not apoptotic cell death was important for the cytotoxic effects by the co-administration. We observed that the combination of RAD001 and MK-2206 exerted enhanced effects on Akt/mTOR inhibition, cyclin D1 down-regulation and ERK/MAPK(extracellular signal-regulated kinase/mitogen-activated protein kinases) activation. Intriguingly, MEK/ERK inhibitors PD98059 and U0126 suppressed RAD001 plus MK-2206-induced beclin-1 expression, autophagy induction and cytotoxicity in HGC-27 cells. In conclusion, these results suggested that the synergistic anti-gastric cancer cells ability by RAD001 and MK-2206 involves ERK-dependent autophagic cell death pathway.     

Novel regulation of Na, K-ATPase by Src tyrosine kinases in cortical neurons

The Na+, K+-ATPase or Na+, K+-pump plays a critical role in ion homeostasis and many cellular events. The Na+, K+-pump activity is regulated by serine/threonine phosphorylation, the role of tyrosine kinases in the regulation, however, is obscure. We now present novel evidence showing that tyrosine phosphorylation activates the Na+, K+-pump in cortical neurons. The electrogenic activity of the Na+, K+-pump was measured using whole-cell voltage clamp. A tonic activity was revealed by an inward current induced by the specific inhibitor ouabain or strophanthidin; an outward current due to activation of the pump was triggered by raising extracellular K+. The inward and outward currents were attenuated by the tyrosine kinase inhibitor genistein, herbimycin A, or lavendustin A, while blocking tyrosine phosphatases increased the pump current. Down-regulation of the pump current was also seen with the Src inhibitor PP1 and intracellularly applied anti-Lyn or anti-Yes antibody. Consistently, intracellular application of Lyn kinase up-regulated the pump current. Immunoprecipitation and western blotting showed tyrosine phosphorylation and a direct interaction between Lyn and the alpha3 subunit of the Na+, K+-pump. The tyrosine phosphorylation of the alpha3 subunit was reduced by serum deprivation. These data suggest that the Na+, K+-ATPase activity in central neurons is regulated by specific Src tyrosine kinases via a protein-protein mechanism and may play a role in apoptosis.     

Diversity of centromeric repeats in two closely related wild rice species, Oryza officinalis and Oryza rhizomatis

Oryza officinalis (CC, 2n=24) and Oryza rhizomatis (CC, 2n=24) belong to the Oryza genus, which contains more than 20 identified wild rice species. Although much has been known about the molecular composition and organization of centromeres in Oryza sativa, relatively little is known of its wild relatives. In the present study, we isolated and characterized a 126-bp centromeric satellite (CentO-C) from three bacterial artificial chromosomes of O. officinalis. In addition to CentO-C, low abundance of CentO satellites is also present in O. officinalis. In order to determine the chromosomal locations and distributions of CentO-C (126-bp), CentO (155 bp) and TrsC (366 bp) satellite within O. officinalis, fluorescence in situ hybridization examination was done on pachytene or metaphase I chromosomes. We found that only ten centromeres (excluding centromere 7 and 2) contain CentO-C arrays in O. officinalis, while centromere 7 comprises CentO satellites, and centromere 2 is devoid of any detectable satellites. For TrsC satellites, it was detected at multiple subtelomeric regions in O. officinalis, however, in O. rhizomatis, TrsC sequences were detected both in the four centromeric regions (CEN 3, 4, 10, 11) and the multiple subtelomeric regions. Therefore, these data reveal the evolutionary diversification pattern of centromere DNA within/or between close related species, and could provide an insight into the dynamic evolutionary processes of rice centromere.     

KNOCKDOWN OF THE IONOTROPIC γ‐AMINOBUTYRIC ACID RECEPTOR (GABAR) RDL GENE DECREASES FIPRONIL SUSCEPTIBILITY OF THE SMALL BROWN PLANTHOPPER,Laodelphax striatellus (HEMIPTERA: DELPHACIDAE)

Insect γ‐aminobutyric acid receptors (GABARs) are important molecular targets of cyclodiene and phenylpyrazole insecticides. Previously GABARs encoding rdl (resistant to dieldrin) genes responsible for dieldrin and fipronil resistance were identified in various economically important insect pests. In this study, we cloned the open reading frame cDNA sequence of rdl gene from fipronil‐susceptible and fipronil‐resistant strains of Laodelphax striatellus (Lsrdl). Sequence analysis confirmed the presence of a previously identified resistance‐conferring mutation. Different alternative splicing variants of Lsrdl were noted. Injection of dsLsrdl reduced the mRNA abundance of Lsrdl by 27–82%, and greatly decreased fipronil‐induced mortality of individuals from both susceptible and resistant strains. These data indicate that Lsrdl encodes a functional RDL subunit that mediates susceptibility to fipronil. Additionally, temporal and spatial expression analysis showed that Lsrdl was expressed at higher levels in eggs, fifth‐instar nymphs, and female adults than in third‐instar and fourth‐instar nymphs. Lsrdl was predominantly expressed in the heads of 2‐day‐old female adults. All these results provide useful background knowledge for better understanding of fipronil resistance related ionotropic GABA receptor rdl gene expressed variants and potential functional differences in insects.