Knockdown of miR-210 decreases hypoxic glioma stem cells stemness and radioresistance

Glioma contains abundant hypoxic regions which provide niches to promote the maintenance and expansion of glioma stem cells (GSCs), which are resistant to conventional therapies and responsible for recurrence. Given the fact that miR-210 plays a vital role in cellular adaption to hypoxia and in stem cell survival and stemness maintenance, strategies correcting the aberrantly expressed miR-210 might open up a new therapeutic avenue to hypoxia GSCs. In the present study, to explore the possibility of miR-210 as an effective therapeutic target to hypoxic GSCs, we employed a lentiviral-mediated anti-sense miR-210 gene transfer technique to knockdown miR-210 expression and analyze phenotypic changes in hypoxic U87s and SHG44s cells. We found that hypoxia led to an increased HIF-2α mRNA expression and miR-210 expression in GSCs. Knockdown of miR-210 decreased neurosphere formation capacity, stem cell marker expression and cell viability, and induced differentiation and G0/G1 arrest in hypoxic GSCs by partially rescued Myc antagonist (MNT) protein expression. Knockdown of MNT could reverse the gene expression changes and the growth inhibition resulting from knockdown of miR-210 in hypoxic GSCs. Moreover, knockdown of miR-210 led to increased apoptotic rate and Caspase-3/7 activity and decreased invasive capacity, reactive oxygen species (ROS) and lactate production and radioresistance in hypoxic GSCs. These findings suggest that miR-210 might be a potential therapeutic target to eliminate GSCs located in hypoxic niches.     

In Situ pH Measurement in Partly Frozen Aqueous Solution Using the Fluorescent Probe 8-Hydroxypyrene-1,3,6-Trisulfonic Acid

A method based on the fluorescence probe 8-hydroxypyrene-1,3,6-trisulfonic acid for in situ measurement of pH in partly frozen aqueous solutions was developed using multifrequency, phase-modulated fluorescence spectroscopy inherently correcting for light scattering. The probe was determined to have pK _a = 7.72 ± 0.03 at 25.0 °C extrapolated to zero ionic strength with as derived from temperature dependence (5 to 25 °C investigated). Ionic strength dependence of pK _a determined experimentally was described using Debye–Hückel formalism for ionic strength up to 3 M. Temperature and ionic strength dependence were combined to yield for determination of pH at subzero temperatures with α experimentally determined from the ratio between fluorescence intensity after excitation at 454 and 415 nm, α = FI(454 nm)/2.5·FI(415 nm). Fluorescence could be described as a decay of a single excited state with a fluorescence life time of 5.40 ± 0.05 ns at 25 °C, and excited state acid–base equilibration was shown not to interfere with the pH measurement. Using the method, pH of a 0.25 M phosphate buffer with pH = 6.8 at 25 °C was shown to decrease gradually to pH = 4.2 in the ice slurry at −13 °C.     

The complete mitochondrial genome of Taeniogonalos taihorina (Bischoff) (Hymenoptera: Trigonalyidae) reveals a novel gene rearrangement pattern in the Hymenoptera

The family Trigonalyidae is considered to be one of the most basal lineages in the suborder Apocrita of Hymenoptera. Here, we determine the first complete mitochondrial genome of the Trigonalyidae, from the species Taeniogonalos taihorina (Bischoff, 1914). This mitochondrial genome is 15,927 bp long, with a high A + T-content of 84.60%. It contains all of the 37 typical animal mitochondrial genes and an A + T-rich region. The orders and directions of all genes are different from those of previously reported hymenopteran mitochondrial genomes. Eight tRNA genes, three protein-coding genes and the A + T-rich region were rearranged, with the dominant gene rearrangement events being translocation and local inversion. The arrangements of three tRNA clusters, trnY–trnM–trnI–trnQ, trnW–trnL2–trnC, and trnH–trnA–trnR–trnN–trnS–trnE–trnF, and the position of the cox1 gene, are novel to the Hymenoptera, even the insects. Six long intergenic spacers are present in the genome. The secondary structures of the RNA genes are normal, except for trnS2, in which the D-stem pairing is absent.     

Stimulation of the DNA unwinding activity of human DNA helicase II/Ku by phosphorylation

The Ku autoantigen is a heterodimeric protein of 70- and 83-kDa subunits, endowed with duplex DNA end-binding capacity and DNA helicase activity (Human DNA Helicase II, HDH II). HDH II/Ku is well established as the DNA binding component, the regulatory subunit as well as a substrate for the DNA-dependent protein kinase DNA-PK, a complex involved in the repair of DNA double-strand breaks and in V(D)J recombination in eukaryotes. The effects of phosphorylation by this kinase on the helicase activity of Escherichia coli-produced HDH II/Ku were studied. The rate of DNA unwinding by recombinant HDH II/Ku heterodimer is stimulated at least fivefold upon phosphorylation by DNA-PK_cs. This stimulation is due to the effective transfer of phosphate residues to the helicase rather than the mere presence of the complex. In vitro dephosphorylation of HeLa cellular HDH II/Ku caused a significant decrease in the DNA helicase activity of this enzyme.     

Gill microsomal (Na+,K+)-ATPase from the blue crab Callinectes danae: Interactions at cationic sites

Euryhaline crustaceans tolerate exposure to a wide range of dilute media, using compensatory, ion regulatory mechanisms. However, data on molecular interactions occurring at cationic sites on the crustacean gill (Na⁺,K⁺)-ATPase, a key enzyme in this hyperosmoregulatory process, are unavailable. We report that Na⁺ binding at the activating site leads to cooperative, heterotropic interactions that are insensitive to K⁺. The binding of K⁺ ions to their high affinity sites displaces Na⁺ ions from their sites. The increase in Na⁺ ion concentrations increases heterotropic interactions with the K⁺ ions, with no changes in K_0.5 for K⁺ ion activation at the extracellular sites. Differently from mammalian (Na⁺,K⁺)-ATPases, that from C. danae exhibits additional NH₄⁺ ion binding sites that synergistically activate the enzyme at saturating concentrations of Na⁺ and K⁺ ions. NH₄⁺ binding is cooperative, and heterotropic NH₄⁺ ion interactions are insensitive to Na⁺ ions, but Na⁺ ions displace NH₄⁺ ions from their sites. NH₄⁺ ions also displace Na⁺ ions from their sites. Mg²⁺ ions modulate enzyme stimulation by NH₄⁺ ions, displacing NH₄⁺ ion from its sites. These interactions may modulate NH₄⁺ ion excretion and Na⁺ ion uptake by the gill epithelium in euryhaline crustaceans that confront hyposmotic media.     

Condensed tannins: co-occurrence of procyanidins,prodelphinidins and profisetinidins in the heartwood of Acacia baileyana

The flavonoids and condensed tannins of the heartwood of Acacia baileyana var. purpurea are described. In conformity with other Acacia species, the hydroxylation pattern of the flavonoids is of the resorcinol type but, in sharp contrast, the tannins are heterogeneous consisting of a mixture of the resorcinol and phtoroglucinol series. Dimeric proanthocyanidins of the phloroglucinol type were absent and this exception to the general observation that they invariably co-occur with the polymers may be explained by the relative nucleophilicity of the aromatic A-rings.     

大豆蚜细胞色素氧化酶Ⅱ基因的克隆及其在捕食性天敌昆虫鉴定中的应用

利用通用引物,对大豆蚜Aphis glycines Matsumura的细胞色素氧化酶Ⅱ（COⅡ）基因序列进行克隆和测序。测得的序列长度为810 bp,并与已在GenBank上登录的其他蚜虫COⅡ基因序列进行比较分析,验证其同源性,同时将该序列在GenBank进行了登记 (登录号为DQ265743)。根据此片段的碱基序列设计了1对大豆蚜特异引物,其扩增片段约为270 bp;种特异性检验结果表明,该引物只对大豆蚜具有扩增能力,对其他相关蚜虫种类不具有扩增效果;并用此引物对大豆蚜捕食性天敌进行定性检测,结果表明,在取食过大豆蚜的异色瓢虫Harmonia axyridis、龟纹瓢虫Propylaea japonica、大草蛉Chrysopa septempunctata幼虫以及小花蝽Orius similes 成虫和幼虫等捕食性昆虫的中肠中均能检测到大豆蚜的DNA片段; 而在未取食蚜虫的上述天敌中却未能扩增出来。     

Early life stage salinity tolerance of wild and hatchery-reared juvenile pink salmon Oncorhynchus gorbuscha

Salinity tolerance in wild (Glendale) and hatchery (Quinsam) pink salmon Oncorhynchus gorbuscha (average mass 0·2 g) was assessed by measuring whole body [Na⁺] and [Cl^?] after 24 or 72 h exposures to fresh water (FW) and 33, 66 or 100% sea water (SW). Gill Na⁺, K⁺‐ATPase activity was measured following exposure to FW and 100% SW and increased significantly in both populations after a 24 h exposure to 100% SW. Whole body [Na⁺] and whole body [Cl^?] increased significantly in both populations after 24 h in 33, 66 and 100% SW, where whole body [Cl^?] differed significantly between Quinsam and Glendale populations. Extending the seawater exposure to 72 h resulted in no further increases in whole body [Na⁺] and whole body [Cl^?] at any salinity, but there was more variability among the responses of the two populations. Per cent whole body water (c. 81%) was maintained in all groups of fish regardless of salinity exposure or population, indicating that the increase in whole body ion levels may have been related to maintaining water balance as no mortality was observed in this study. Thus, both wild and hatchery juvenile O. gorbuscha tolerated abrupt salinity changes, which triggered an increase in gill Na⁺, K⁺‐ATPase within 24 h. These results are discussed in terms of the preparedness of emerging O. gorbuscha for the marine phase of their life cycle.     

Leishmania amazonensis: Heme stimulates (Na + K)ATPase activity via phosphatidylinositol-specific phospholipase C/protein kinase C-like (PI-PLC/PKC) signaling pathways

In the present paper we studied the involvement of the phosphatidylinositol-specific PLC (PI-PLC)/protein kinase C (PKC) pathway in (Na⁺ + K⁺)ATPase stimulation by heme in Leishmania amazonensis promastigotes. Heme stimulated the PKC-like activity with a concentration of 50 nM. Interestingly, the maximal stimulation of the PKC-like activity promoted by phorbol ester was of the same magnitude promoted by heme. However, the stimulatory effect of heme is completely abolished by ET-18-OCH₃ and U73122, specific inhibitors of PI-PLC. (Na⁺ + K⁺)ATPase activity is increased in the presence of increased concentrations of heme, being maximally affected at 50 nM. This effect was completely reversed by 10 nM calphostin C, an inhibitor of PKC. Thus, the effect of 50 nM heme on (Na⁺ + K⁺)ATPase activity is completely abolished by ET-18-OCH₃ and U73122. Taken together, these results demonstrate that the heme receptor mediates the stimulatory effect of heme on the (Na⁺ + K⁺)ATPase activity through a PI-PLC/PKC signaling pathway.     

Variation between mouse major urinary protein genes isolated from a single inbred line

We describe ten Charon 4A genomic DNA clones from BALB/c mice which include at least seven different major urinary protein (MUP) genes. We have established the orientation of all seven sequences, and have placed six of them in precise register by means of restriction site maps and Southern blot hybridization with cloned cDNA sequences. Four of the seven genomic sequences (family I sequences) form hybrids with six independent cDNA clones that have a high thermal stability and hybridize more strongly with mRNA from three inbred mouse lines. Hybrids between the remaining three genomic sequences and the cDNA clones have a lower thermal stability and hybridize less strongly with mRNA from the three inbred lines. Homologies between different cloned sequences extend over as much as 15 kb. No clone contains parts of two MUP genes, and no homology has been detected between the 3' flanking region of one MUP gene and the 5' flanking region of another.