Characterization of microsatellite DNA markers in a critically endangered species,Mekong giant catfish,Pangasianodon gigas

Microsatellite DNA markers for a critically endangered Mekong giant catfish (Pangasianodon gigas Roberts and Vidthayanon, 1991) were developed from fin clips collected from captive fish using (GT)₁₅ probe. The number of alleles per locus ranged from two to four. The expected heterozygosities ranged from 0.13 to 0.68. Also, these primers were successfully amplified in four closely related species, Pangasius bocourti, Pangasius conchophilus, Pangasius larnaudii and Pangasius sanitwongsei with the number of alleles per locus ranged from 1 to 13, 1 to 16, 1 to 12 and 1 to 4, respectively. These markers should prove to be very useful for the evaluation of genetic diversity for this species and other related Pangasius species.     

Participation of the DNA-binding cytokine amphoterine in triggering the processes of tissue reparation

Author's studies and literature references indicate that the DNA-binding cytokine: amphoterine, being a non-histone component of chromatin, acts in extracellular milieu as cytokine regulating gene expression in target cells. The amphoterine effects are mediated by its interaction with the cell surface receptors including those of the final glucosiding product (RAGE), which leads to activation of the ERK and p38 MAP-kinases as well as NF-kappaB. Amphoterine prompts inflammatory response by means of activating synthesis of interleukins-1, -6 and -8 as well as tumour necrosis factors (TNF-alpha) and activates tissue regeneration by means of involvement of the precursor cells into the damage foci and induction of the cells' differentiation. These properties of amphoterine suggest that appearance of this protein in extracellular space signals of a tissue damaging.     

Changes in the free-radical state and the level of free iron during the development of drug resistance in tumor cells

The free-radical state of K562 human erythroleukemia cells changes during the development of resistance to doxorubicin, an antitumor antibiotic with prooxidant action widely used in oncology. It was found that the level of superoxide anion in the resistant cells decreased. The addition of doxorubicin to the culture medium induced a much smaller increase in O ₂ ^? generation in the resistant cells than in the sensitive cells. Again, the semiquinone-type EPR signal with a g-factor of 2.006 considerably decreased in the resistant cells grown without doxorubicin as compared with the sensitive cells under the same conditions. The EPR study has shown that the level of paramagnetic nitrosyl complexes of nonheme iron in the resistant cells decreased, which indicates that the content of free nonheme iron declined in development of drug resistance. In addition, we have found with the use of RT-PCR that the level of mRNA of the transferrin receptor decreased in the resistant cells. The data suggest that the suppression of free-radical processes during the development of resistance of K562 cells to doxorubicin is a coordinated redox-dependent adaptive response.     

Zinc ions stimulate the cooperative RNA binding of hordeiviral gammab protein

A small regulatory gammab protein of the Poa semilatent hordeivirus (PSLV) contains two zinc finger-like motifs separated by a basic motif in the N-terminal part and a C-terminal coiled-coil motif. Interactions of the recombinant PSLV gammab protein and its mutants with various RNAs (ssRNA, dsRNA, ssRNA oligonucleotides) and ssDNA were studied in gel-shift assays. The results demonstrated that zinc ions are essential for effective nucleic-acid-binding activity of the gammab protein, suggesting the important role of zinc finger motifs in these interactions. Deletion of the C-proximal coiled-coil region did not affect highly cooperative RNA-protein binding, indicating that the N-terminal part of the protein contributes to the protein-protein interactions needed for the protein-RNA cooperativity.     

The extreme N-terminal domain of a hordeivirus TGB1 movement protein mediates its localization to the nucleolus and interaction with fibrillarin

The hordeiviral movement protein encoded by the first gene of the triple gene block (TGBp1) of Poa semilatent virus (PSLV), interacts with viral genomic RNAs to form RNP particles which are considered to be a form of viral genome capable of cell-to-cell and long-distance transport in infected plants. The PSLV TGBp1 contains a C-terminal NTPase/helicase domain (HELD) and an N-terminal extension region consisting of two structurally and functionally distinct domains: an extreme N-terminal domain (NTD) and an internal domain (ID). This study demonstrates that transient expression of TGBp1 fused to GFP in Nicotiana benthamiana leaves results in faint but obvious fluorescence in the nucleolus in addition to cytosolic distribution. Mutagenesis of the basic amino acids inside the NTD clusters A ¹¹⁶KSKRKKKNKK¹²⁵ and B ¹⁷⁵KKATKKESKKQTK¹⁸⁷ reveals that these clusters are indispensable for nuclear and nucleolar targeting of PSLV TGBp1 and may contain nuclear and nucleolar localization signals or their elements. The PSLV TGBp1 is able to bind to fibrillarin, the major nucleolar protein (AtFib2 from Arabidopsis thaliana) in vitro. This protein–protein interaction occurs between the glycine-arginine-rich (GAR) domain of fibrillarin and the first 82 amino acid residues of TGBp1. The interaction of TGBp1 with fibrillarin is also visualized in vivo by bimolecular fluorescence complementation (BiFC) during co-expression of TGBp1 or its deletion mutants, and fibrillarin as fusions to different halves of YFP in N. benthamiana plants. The sites responsible for nuclear/nucleolar localization and fibrillarin binding, have been located within the intrinsically disordered TGBp1 NTD. These data could suggest that specific functions of hordeivirus TGBp1 may depend on its interaction with nucleolar components.     

Increased expression of the anti-apoptotic protein Bcl-xL in the brain is associated with resilience to stress-induced depression-like behavior

Clinical observations and the results of animal studies have implicated changes in neuronal survival and plasticity in both the etiology of mood disorders, especially stress-induced depression, and anti-depressant drug action. Stress may predispose individuals toward depression through down-regulation of neurogenesis and an increase in apoptosis in the brain. Substantial individual differences in vulnerability to stress are evident in humans and were found in experimental animals. Recent studies revealed an association between the brain anti-apoptotic protein B cell lymphoma like X, long variant (Bcl-xL) expression and individual differences in behavioral vulnerability to stress. The ability to increase Bcl-xL gene expression in the hippocampus in response to stress may be an important factor for determining the resistance to the development of stress-induced depression. Treatment with anti-depressant drugs may change Bcl-xL response properties. In the rat brainstem, expression of this anti-apoptotic gene becomes sensitive to swim stress during the long-term fluoxetine treatment, an effect that appeared concomitantly with the anti-depressant-like action of the drug in the forced swim test, suggesting that Bcl-xL may be a new target for depression therapy. The processes and pathways linking stress stimuli to behavior via intracellular anti-apoptotic protein are discussed here in the context of Bcl-xL functions in the mechanisms of individual differences in behavioral resilience to stress and anti-depressant-induced effects on the behavioral despair.     

RNA binding is more critical to the suppression of silencing function of Cucumber mosaic virus 2b protein than nuclear localization

Previously, we found that silencing suppression by the 2b protein and six mutants correlated both with their ability to bind to double-stranded (ds) small RNAs (sRNAs) in vitro and with their nuclear/nucleolar localization. To further discern the contribution to suppression activity of sRNA binding and of nuclear localization, we have characterized the kinetics of in vitro binding to a ds sRNA, a single-stranded (ss) sRNA, and a micro RNA (miRNA) of the native 2b protein and eight mutant variants. We have also added a nuclear export signal (NES) to the 2b protein and assessed how it affected subcellular distribution and suppressor activity. We found that in solution native protein bound ds siRNA, miRNA, and ss sRNA with high affinity, at protein:RNA molar ratios ~2:1. Of the four mutants that retained suppressor activity, three showed sRNA binding profiles similar to those of the native protein, whereas the remaining one bound ss sRNA at a 2:1 molar ratio, but both ds sRNAs with 1.5-2 times slightly lower affinity. Three of the four mutants lacking suppressor activity failed to bind to any sRNA, whereas the remaining one bound them at far higher ratios. NES-tagged 2b protein became cytoplasmic, but suppression activity in patch assays remained unaffected. These results support binding to sRNAs at molar ratios at or near 2:1 as critical to the suppressor activity of the 2b protein. They also show that cytoplasmically localized 2b protein retained suppressor activity, and that a sustained nuclear localization was not required for this function.     

茎花葱臭木化学成分的研究

从茎花葱臭木种子中分离得到5个化合物,经理化与波谱分析鉴定为β-谷甾醇(1)、没食子酸乙酯(2)、胡萝卜苷(3)、1-O-β-D-吡喃葡萄糖基-(2S,3S,4R,8Z)-2-N-(2 ′-羟基二十四烷酰氨基)十八二氧鞘氨-8-烯(4)和2,3,2″,3″-四氢穗花杉双黄酮(5).这5个化合物均首次从该植物中分离得到.其中化合物5进行细胞毒活性测试,没有显示抑制活性.     

大苞藤黄化学成分研究及其互变异构体超高效液相色谱质谱联用分析

从大苞藤黄枝叶的混合粉碎物中分离到11个化合物,运用光谱手段分别鉴定为neobractatin(1),brasixanthone B (2),5-O-methylxanthone V1 (3),10-O-methylmacluraxanthone (4),isobractatin (5),xanthone V1(6),xerophenone A (7),xerophenone B (8),bractatin (9),macluraxanthone (10)和3-O-methylneobractatin (11).本文首次应用超高效液相色谱-质谱联用技术分离了异构体7和8并测定了其精确分子量.其中化合物6～8为首次从该植物中发现.