Differentiation of embryonic stem cell to astrocytes visualized by green fluorescent protein

Green fluorescent protein (GFP) gene was transfected and expressed in murine embryonic stem (ES) cells under the control of the astrocyte-specific glial fibrillary acidic protein (GFAP) promoter. Stably transfected cells were characterized by immunohistochemistry and by fluorescence microscopy. Cells containing GFP were differentiated to Type I and Type II astrocytes after induction by all-trans retinoic acid. Differentiated cells were expressed GFP and visualized by fluorescence microscopy. Differentiated cells expressed GFP were correlated with the expression of GFAP and morphological change. It demonstrates that the cell line expressed GFP can be used to trace the morphological changes of astrocytes during differentiation, and further for the isolation of astrocytes from the mixed cells differentiated from ES cell.     

The Association between Four Genetic Variants in MicroRNAs (rs11614913, rs2910164, rs3746444, rs2292832) and Cancer Risk: Evidence from Published Studies

MicroRNAs (miRNAs) participate in diverse biological pathways and may act as either tumor suppressor genes or oncogenes. Single nucleotide polymorphisms (SNPs) in miRNA may contribute to cancer development with changes in the microRNA''s properties and/or maturation. Polymorphisms in miRNAs have been suggested in predisposition to cancer risk; however, accumulated studies have shown inconsistent conslusionss. To further validate determine whether there is any potential association between the four common SNPs (miR-196a2C>T, rs11614913; miR-146aG>C, rs2910164; miR-499A>G, rs3746444; miR-149C>T, rs2292832) and the risk for developing risk, a meta-analysis was performed according to the 40 published case-control studies. Odds ratios (ORs) with 95% confidence intervals (CIs) were calculated to assess the extent of the association. The results demonstrated that the rs11614913TT genotype was significantly associated with a decreased cancer risk, in particular with a decreased risk for colorectal cancer and lung cancer, or for Asian population subgroup. In addition, the rs2910164C allele was associated with decreased risk for esophageal cancer, cervical cancer, prostate cancer, and hepatocellular carcinoma (HCC), in particular in Asian population subgroup. Similarly, the rs3746444G allele was observed as a risk factor for cancers in the Asian population. It is concluded that two SNPs prsent in miRNAs(rs11614913TT, and rs2910164C) may protect against the pathogenesis of some cancers, and that the rs3746444 may increase risk for cancer.     

Regulation of the Na+/K+ ATPase by Klotho

Klotho-hypomorphic (Klotho(hm)) mice suffer from renal salt wasting and hypovolemia despite hyperaldosteronism. The present study explored the effect of Klotho on renal Na(+)/K(+) ATPase activity. According to immunohistochemistry and confocal microscopy Na(+)/K(+) ATPase protein abundance in isolated collecting ducts was lower in Klotho(hm) mice than in their wild type littermates (Klotho(+/+)). Analysis with dual electrode voltage clamp recording showed that expression of Klotho in Xenopus oocytes increased the Na(+)/K(+) ATPase pump current. Treatment of Xenopus oocytes with Klotho protein similarly increased the pump current. In conclusion, Klotho increases the membrane abundance and activity of the Na(+)/K(+) ATPase. Decreased Na(+)/K(+) ATPase activity could thus contribute to the volume-depletion of klotho(hm) mice.     

Characterization of base-pair opening in deoxynucleotide duplexes using catalyzed exchange of the imino proton

Using nuclear magnetic resonance line broadening, longitudinal relaxation and magnetization transfer from water, we have measured the imino proton exchange times in the duplex form of the 10-mer d-CGCGATCGCG and in seven other deoxy-duplexes, as a function of the concentration of exchange catalysts, principally ammonia. All exchange times are catalyst dependent. Base-pair lifetimes are obtained by extrapolation to infinite concentration of ammonia. Lifetimes of internal base-pairs are in the range of milliseconds at 35 degrees C and ten times more at 0 degrees C. Lifetimes of neighboring pairs are different, hence base-pairs open one at a time. Lifetimes of d(G.C) are about three times longer than those of d(A.T). The nature of neighbors usually has little effect, but lifetime anomalies that may be related to sequence and/or structure have been observed. In contrast, there is no anomaly in the A.T base-pair lifetimes of d-CGCGA[TA]5TCGCG, a model duplex of poly[d(A-T)].poly[d(A-T)]. The d(A.T) lifetimes are comparable to those of r(A.U) that we reported previously. End effects on base-pair lifetimes are limited to two base-pairs. The low efficiency of exchange catalysts is ascribed to the small dissociation constant of the deoxy base-pairs, and helps to explain why exchange catalysis had been overlooked in the past. This resulted in a hundredfold overestimation of base-pair lifetimes. Cytosine amino proteins have been studied in the duplex of d-CGm5CGCG. Exchange from the closed base-pair is indicated. Hence, the use of an amino exchange rate to evaluate the base-pair dissociation constant would result in erroneous, overestimated values. Catalyzed imino proton exchange is at this time the safest and most powerful, if not the only probe of base-pair kinetics. We propose that the single base-pair opening event characterized here may be the only mode of base-pair disruption, at temperatures well below the melting transition.     

p47phox participates in activation of RelA in endothelial cells

Activation of endothelial cell NF-kappaB by interleukin (IL)-1 constitutes an event critical to the progression of the innate immune response. In this context, oxidants have been associated with NF-kappaB activation, although the molecular source and mechanism of targeting have remained obscure. We found that RelA, essential for NF-kappaB activation by IL-1, was associated with the NADPH oxidase adapter protein p47(phox) in yeast two-hybrid, coprecipitation, and in vitro binding studies. RelA and p47-GFP also colocalized in endothelial cells in focal submembranous dorsoventral protrusions. Overexpression of p47(phox) synergized with IL-1beta in the activation of an artificial kappaB-luciferase reporter and specifically augmented IL-1beta-induced RelA transactivation activity. p47(phox) overexpression also greatly increased IL-1beta-stimulated RelA phosphorylation, whereas it had no effect on I-kappaB degradation or on RelA nuclear translocation or kappaB binding. The tandem SH3 domains of p47(phox) were found to associate with a proline-rich mid-region of RelA (RelA-PR) located between the Rel homology and transactivation domains. The RelA-PR peptide blocked interaction of p47(phox) and RelA, and ectopic expression of RelA-PR abrogated IL-1beta-induced transactivation of the NF-kappaB-dependent E-selectin promoter. Further, suppression of NADPH oxidase function through the inhibitor diphenylene iodonium, the superoxide dismutase mimetic Mn(III) tetrakis(4-benzoic acid)porphyrin (MnTBAP), or expression of a dominant interfering mutant of a separate NADPH oxidase subunit (p67(V204A)) decreased IL-1beta-induced E-selectin promoter activation, suggesting that p47(phox) facilitates NF-kappaB activation through linkage with the NADPH oxidase. IL-1beta rapidly increased tyrosine phosphorylation of IL-1 type I receptor-associated proteins, suggesting that oxidants may operate through inactivation of local protein-tyrosine phosphatases in the proximal IL-1beta signaling pathway leading to RelA activation.     

Free intermingling of mammalian beta-tubulin isotypes among functionally distinct microtubules

Mammalian cells express a spectrum of tubulin isotypes whose relationship to the diversity of microtubule function is unknown. To examine whether different isotypes are segregated into functionally distinct microtubules, we generated immune sera capable of discriminating among the various naturally occurring beta-tubulin isotypes. Cloned fusion proteins encoding each isotype were used first to tolerogenize animals against shared epitopes, and then as immunogens to elicit a specific response. In experiments using these sera, we show that there is neither complete nor partial segregation of beta-tubulin isotypes: both interphase cytoskeletal and mitotic spindle microtubules are mixed copolymers of all expressed beta-tubulin isotypes. Indeed, a highly divergent isotype normally expressed only in certain hematopoietic cells is also indiscriminately assembled into all microtubules both in their normal context and when transfected into HeLa cells.     

Effects of 5-aminolevulinic acid on the inflammatory responses and antioxidative capacity in broiler chickens challenged with lipopolysaccharide

5-Aminolevulinic acid (5-ALA) is an intermediate in haem biosynthesis and has anti-apoptotic, anti-inflammatory, antioxidant, and other pharmacological effects. This study aimed to investigate the effect of dietary supplementation with 5-ALA on growth performance, antioxidant capacity, and inflammatory response of the lipopolysaccharide (LPS)-challenged broiler chickens. The experiment was designed as a 2 × 2 factorial arrangement with dietary 5-ALA (0 or 60 mg/kg) and LPS (injection of saline or 0.5 mg/kg BW) levels as treatments. A total of 240 one-day-old Arbor Acres broilers were distributed into four treatments consisting of six replicates of 10 birds. All the experimental broilers were intraperitoneally injected with LPS or sterile saline at 16, 18, and 20 days of age. Our results showed that dietary 5-ALA supplementation reduced (P < 0.05) the feed to gain before broilers were stimulated with LPS (days 1–15). LPS challenge decreased (P < 0.05) the catalase (CAT), total superoxide dismutase activities and increased the content of malondialdehyde (MDA) in the serum of broiler chickens. However, 5-ALA supplementation had a tendency to increase (P = 0.08) the activity of CAT and decreased (P < 0.05) the content of MDA. LPS challenge showed higher (P < 0.05) interleukin (IL)-1β, IL-6, and IL-10 concentrations in the serum, whereas dietary 5-ALA supplementation decreased (P < 0.05) the levels of IL-1β and IL-6. Additionally, dietary 5-ALA supplementation significantly attenuated (P < 0.05) the upregulation of mRNA expression levels of hepatic toll-like receptor 4 (TLR4), IL-1β, and IL-2 induced by LPS challenge. Moreover, dietary 5-ALA supplementation also enhanced the mRNA expression of 5-aminolevulinate dehydratase, ferrochelatase, and haem oxygenase-1 (HO-1) as compared to the unsupplemented groups. In conclusion, our results suggested that supplementation of 60 mg/kg 5-ALA exhibited LPS-induced anti-inflammatory and antioxidant properties by enhancing the HO-1 expression and inhibiting the TLR4/NF-κB signalling pathway.     

Characterization of bacterial composition and diversity in a long-term petroleum contaminated soil and isolation of high-efficiency alkane-degrading strains using an improved medium

Bacterial community and diversity in a long-term petroleum-contaminated soil of an oilfield were characterized using 16S rRNA gene-based Illumina MiSeq high-throughput sequencing. Results indicated that Proteobacteria (49.11%) and Actinobacteria (24.24%) were the most dominant phyla, and the most abundant genera were Pseudoxanthomonas (8.47%), Luteimonas (3.64%), Alkanindiges (9.76%), Acinetobacter (5.26%) and Agromyces (8.56%) in the soil. Meanwhile a series of cultivations were carried out for isolation of alkane degraders from petroleum-contaminated soil with gellan gum and agar as gelling agents. And the isolates were classified by their 16S rRNA genes. Nine of the isolates including Enterobacter, Pseudomonas,Acinetobacter, Rhizobium, Bacillus, Sphingomonas, Paenibacillus, Variovorax and Rhodococcus showed strong biodegradability of alkane mixture (C9–C30) in a wide range of chain-length, which could be potentially applied in enhancement of bioremediation.