Genomic organization of the rat aspartyl-tRNA synthetase gene family: A single active gene and several retropseudogenes

The genomic organization of the gene encoding rat aspartyl-tRNA synthetase (AspRS), a class II aminoacyl-tRNA synthetase (aaRS), was determined. A single active gene and several pseudogenes were isolated from a rat genomic DNA library and characterized. The active DRS1 gene encoding the rat AspRS spans approximately 60 kb and is divided into 16 exons. Exons 8–16, encoding the nt-binding domain of the synthetase, are clustered in the 3′-region of the gene, whereas exons 3, 4, and 5, encoding the anticodon-binding domain are separated by large introns (up to 15 kb) containing LINE sequences. One of the pseudogenes, ΨDRSI, has a nt sequence 93% identical to that of the complete cDNA sequence of rat AspRS but several stop codons interrupt the coding sequence, thus identifying ΨDRS1 to an inactive processed pseudogene. Two repetitive elements from the LINE family are inserted into ΨDRS1. Calculation of nt substitution rates suggests that ΨDRS1 sequences arose approximately 27 Myr ago. The other pseudogene, ΨDRS2, should be more ancient. Taken together, these results clearly demonstrate that the AspRS gene family is composed of only one active gene. The availability of the gene structure of AspRS could help to clarify molecular evolution of class II aaRS.     

草菇不同菌株RT-qPCR参考基因筛选

适合的参考基因是应用实时荧光定量PCR (RT-qPCR)技术进行基因表达分析的前提。本研究以7个食用菌常用参考基因(β-TUB 1、GPD、ACTB、Ras、α-TUB、β-TUB 2和SPRYp)为候选基因,利用RT-qPCR检测其在草菇常用生产菌株(CPS)V844、V5、V971和V844继代退化菌株(SDS) T8、T12、T16、T20中的表达;用Genorm、NormFinder和BestKeeper 3种软件分析候选基因的表达稳定性,并结合几何平均数法筛选出最佳参考基因。结果表明,SPRYp、α-TUB和β-TUB 2基因适用于草菇常用生产菌株检测,SPRYp、GPD和α-TUB基因适用于草菇继代退化检测,SPRYp基因适用于两种条件的检测。两两差异分析表明,草菇常用生产菌株的最佳参考基因组合为SPRYp、α-TUB和β-TUB 2,继代退化菌株的最佳参考基因组合为SPRYp和GPD,两种条件混合菌株的最佳参考基因组合为SPRYp和α-TUB。     

Structural characterization of the chicken SF-1/Ad4BP gene

SF-1/Ad4BP was identified as a master regulator controlling steroidogenic P-450 genes and belongs to the steroid hormone receptor superfamily. It is expressed in the adrenal cortex, gonads, and pituitary gonadotroph. Targeted disruption of the mouse SF-1/Ad4BP gene showed that it plays a critical role in the development of the steroidogenic tissues and pituitary gonadotroph. We have recently cloned the chicken SF-1/Ad4BP cDNA and have now cloned the chicken SF-1/Ad4BP gene and analyzed its promoter activity. This gene consists of seven exons as well as mammalian counterparts and spans about 15 kb. In mice, the gene encodes another protein, ELP, but we could not find the open reading frame of ELP in the chicken SF-1/Ad4BP gene. The promoter of this gene included five putative cis elements (E, CCAAT, GC and TATA boxes and a GA-rich element), although no TATA box has been found in mammalian counterparts. The E and CCAAT boxes moderately affected promoter activity and the GA-rich element and TATA box were essential for the expression of the chicken SF-1/Ad4BP gene.     

Characterisation of the beta-tubulin gene of Cylicocyclus nassatus?

mRNA and genomic DNA were isolated from adult Cylicocyclus nassatus, and the mRNA was reverse transcribed. The cDNA was PCR amplified using degenerate primers designed according to the alignment of the β-tubulin amino acid sequences of other species. To complete the coding sequence, the 3′ end was amplified with the 3′-RACE, and for amplification of the 5′ end the SL1-primer was used. The cDNA of the β-tubulin gene of C. nassatus spans 1429 bp and encodes a protein of 448 amino acids. Specific primers were developed from the cDNA sequence to amplify the genomic DNA sequence and to analyse the genomic organisation of the β-tubulin gene. The complete sequence of the genomic DNA of the β-tubulin gene of C. nassatus has a size of 2652 bp and is organised into nine exons and eight introns. The identities with the exons of the gru-1 β-tubulin gene of Haemonchus contortus range between 79% and 97%.     

Ergosterol peroxides as phospholipase A2 inhibitors from the fungus Lactarius hatsudake

Four ergosterol derivatives (1–4) have been isolated for the first time from the fruiting bodies of a basidiomycete fungus, Lactarius hatsudake, through activity-guided fractionation. Their structures were determined, using spectroscopic analysis, as: (22E,24R)-ergosta-5,7,22-dien-3β-ol (ergosterol, 1); 5,8-epidioxy-(22E,24R)-ergosta-6,22-dien-3β-ol (ergosterol peroxide, 2); 5,8-epidioxy-(24S)-ergosta-6-en-3β-ol (3); and (22E,24R)-ergosta-7,22-dien-3β,5,6β-triol (cerevisterol, 4). Compounds 2 and 3 showed selective inhibitory activity against Crotalus adamenteus venom phospholipase A₂ (PLA₂) enzyme, but not against Apis mellifcra bee venom PLA₂. The antiphospholipase A₂ activity of compounds 2 and 3 are reported here for the first time.     

Two non-reactive ternary complexes of estrogenic 17beta-hydroxysteroid dehydrogenase: crystallization and preliminary structural analysis.

Human estrogenic 17β-hydroxysteroid dehydrogenase (17β-HSD1, EC1.1.1.62) is an important enzyme that catalyses the last step of active estrogen formation. 17β-HSD1 plays a key role in the proliferation of breast cancer cells. The three-dimensional structures of this enzyme and of the enzyme-estradiol complex have been solved (Zhu et al., 1993, J. Mol. Biol. 234:242; Ghosh et al., 1995, Structure 3:503; Azzi et al., 1996, Nature Struct. Biol. 3:665). The determination of the non-reactive ternary complex structure, which could mimic the transition state, constitutes a further critical step toward the rational design of inhibitors for this enzyme (Ghosh et al. 1995, Structure 3:503; Penning, 1996, Endocrine-Related Cancer, 3:41).

To further study the transition state, two non-reactive ternary complexes, 17β-HSD1–EM519-NADP⁺ and 17β-HSD1–EM553-NADP⁺ were crystallized using combined methods of soaking and co-crystallization. Although they belong to the same C2 space group, they have different unit cells, with a=155.59 Å, b=42.82 Å, c=121.15 Å, β=128.5° for 17β-HSD1–EM519-NADP⁺, and a=124.01 Å, b=45.16 Å, c=61.40 Å, β=99.2° for 17β-HSD1–EM553-NADP⁺, respectively. Our preliminary results revealed that the inhibitors interact differently with the enzyme than do the natural substrates.     

The contribution of beta1 integrins to neuronal migration and differentiation depends on extracellular matrix molecules

The interaction of β1 integrin receptors and different extracellular matrix molecules during neuronal development was investigated by comparing both migration and morphological differentiation of D3 wild-type embryonic stem (ES) cell line-derived neural precursor cells with those of the β1 integrin knockout ES cell line G201. Analysing neurosphere explants on laminin and fibronectin as major β1 integrin ligands, the maximal spreading of outward migrating neuronal cells was determined. Compared with gelatine as a standard substrate, migration was found to be significantly increased for D3-derived neurospheres on fibronectin and laminin-1. These matrix effects were found to be even enhanced for G201 preparations. In addition, also the differentiation of wild-type and β1 integrin −/− neurones – as determined by MAP-2- and HNK-1-immunoreactive processes – was found to be increased on fibronectin and laminin when compared to gelatine standards. In the respective knockout preparations on these matrices, again perturbation effects were less pronounced than on gelatine. Our observations indicate that laminin and fibronectin are involved both in β1 integrin-dependent and -independent signalling mechanisms during neurogenesis. Upregulation of compensatory mechanisms such as β1 integrin-independent receptors for laminin and fibronectin might be responsible for the much less pronounced perturbations of G201 neural precursor migration and differentiation on these two substrates than on gelatine.     

Charophyte growth in small temperate water bodies: Extreme isotopic disequilibrium and implications for the palaeoecology of shallow marl lakes

A small pond containing the charophyte Chara hispida was monitored over a one-year period for changes in growth, water chemistry, water level and stable isotopic composition. Chara growth was found to be seasonal, with maximum growth occurring from late April to July. During this period, pH rose to > 10 while the dissolved inorganic carbon (DIC) and calcium fell as a result of photosynthesis and calcification. Large gradients in pH, water temperature and irradiance were found within the Chara sward and measurements showed that most growth and photosynthesis occurred within the upper 20 cm of the water column. Chara oospore formation was also found to be seasonal but dependent upon environmental conditions.

δ¹⁸O_w rose rapidly during summer as evaporation progressed and this was correlated with the δ¹⁸O_DIC, and to some extent with δ¹⁸O_c of the Chara encrusted calcite. However, extreme isotopic disequilibrium was observed between the δ¹⁸O_c and the δ¹⁸O_w and also between the δ¹³C_c and the δ¹³C_DIC. This arose from the high pH allowing atmospheric CO₂ to enter the water and combine directly with OH⁻.

It is concluded that, within shallow eutrophic lakes containing Chara swards, inferences of climate (e.g. air temperature) cannot be made from observations of the isotopic composition of Chara carbonates. However in combination with other geochemical data, disequilibrium events may be identifiable in ancient lake basins and taken as evidence for lake shallowing and/or eutrophication.     

Regulation of sex steroid formation by interleukin-4 and interleukin-6 in breast cancer cells

Sex steroids play a predominant role in the development and differentiation of normal mammary gland as well as in the regulation of hormone-sensitive breast cancer growth. There is evidence suggesting that local intracrine formation of sex steroids from inactive precursors secreted by the adrenals namely, dehydroepiandrosterone (DHEA) and 4-androstenedione (4-dione) play an important role in the regulation of growth and function of peripheral target tissues, including the breast. Moreover, human breast carcinomas are often infiltrated by stromal/immune cells secreting a wide spectra of cytokines. These might in turn regulate the activity of both immune and neoplastic cells. The present study was designed to examine the action of cytokines on 17β-hydroxysteroid dehydrogenase (17β-HSD) and 3β-hydroxysteroid dehydrogenase/isomerase (3β-HSD) activities in human breast cancer cells. The various types of human 17β-HSD (five types) and 3β-HSD (two types), because of their tissue- and cell-specific expression and substrate specificity, provide each cell with necessary mechanisms to control the level of intracellular active androgens and estrogens. We first investigated the effect of exposure to IL-4 and IL-6 on reductive and oxidative 17β-HSD activities in both intact ZR-75-1 and T-47D human breast cancer cells. In ZR-75-1 cells, a 6 d exposure to IL-4 and IL-6 decreased E₂-induced cell proliferation, the half maximal inhibitory effect being exerted at 88 and 26 pM, respectively. In parallel, incubation with IL-4 and IL-6 increased oxidative 17β-HSD activity by 4.4- and 1.9-fold, respectively, this potent activity being observed at ₅₀ values of 22.8 and 11.3 pM, respectively. Simultaneously, reductive 17β-HSD activity leading to E₂ formation was decreased by 70 and 40% by IL-4 and IL-6, respectively. Moreover, IL-4 and IL-6 exerted the same regulatory effects on 17β-HSD activities when testosterone and 4-dione were used as substrates, thus strongly suggesting the expression of the type 2 17β-HSD ZR-75-1 cells. In contrast, in T-47D cells, IL-4 increased the formation of E₂, whereas IL-6 exerts no effect on this parameter. However, we found that T-47D cells failed to convert testosterone efficiently into 4-DIONE, thus suggesting that there is little or no expression of type 2 17β-HSD in this cell line. The present findings demonstrate that the potent regulatory effects of IL-4 and IL-6 on 17β-HSD activities depend on the cell-specific gene expression of various types of 17β-HSD enzymes. We have also studied the effect of cytokines on the regulation of the 3β-HSD expression in both ZR-75-1 and T-47D human breast cancer cells. Under basal culture conditions, there is no 3β-HSD activity detectable in these cells. However, exposure to IL-4 caused a rapid and potent induction of 3β-HSD activity, whereas IL-6 failed to induce 3β-HSD expression. Our data thus demonstrate that cytokines may play a crucial role in sex steroid biosynthesis from inactive adrenal precursors in human breast cancer cells.     

Anti-diabetic properties of the Canadian lowbush blueberry Vaccinium angustifolium Ait.

Incidence of type II diabetes is rapidly increasing worldwide. In order to identify complementary or alternative approaches to existing medications, we studied anti-diabetic properties of Vaccinium angustifolium Ait., a natural health product recommended for diabetes treatment in Canada. Ethanol extracts of root, stem, leaf, and fruit were tested at 12.5 μg/ml for anti-diabetic activity in peripheral tissues and pancreatic β cells using a variety of cell-based bioassays. Specifically, we assessed: (1) deoxyglucose uptake in differentiated C2C12 muscle cells and 3T3-L1 adipocytes; (2) glucose-stimulated insulin secretion (GSIS) in β TC-tet pancreatic β cells; (3) β cell proliferation in β TC-tet cells; (4) lipid accumulation in differentiating 3T3-L1 cells; (5) protection against glucose toxicity in PC12 cells. Root, stem, and leaf extracts significantly enhanced glucose transport in C2C12 cells by 15–25% in presence and absence of insulin after 20 h of incubation; no enhancement resulted from a 1 h exposure. In 3T3 cells, only the root and stem extracts enhanced uptake, and this effect was greater after 1 h than after 20 h; uptake was increased by up to 75% in absence of insulin. GSIS was potentiated by a small amount in growth-arrested β TC-tet cells incubated overnight with leaf or stem extract. However, fruit extracts were found to increase ³H-thymidine incorporation in replicating β TC-tet cells by 2.8-fold. Lipid accumulation in differentiating 3T3-L1 cells was accelerated by root, stem, and leaf extracts by as much as 6.5-fold by the end of a 6-day period. Stem, leaf, and fruit extracts reduced apoptosis by 20–33% in PC12 cells exposed to elevated glucose for 96 h. These results demonstrate that V. angustifolium contains active principles with insulin-like and glitazone-like properties, while conferring protection against glucose toxicity. Enhancement of proliferation in β cells may represent another potential anti-diabetic property. Extracts of the Canadian blueberry thus show promise for use as a complementary anti-diabetic therapy.     

Pustulan and branched β-galactofuranan from the phytopathogenic fungus Guignardia citricarpa, excreted from media containing glucose and sucrose

Guignardia citricarpa is a phytopathogenic fungus and the causal agent of citrus black spot. Incubation in a semi-defined media resulted in formation of exopolysaccharides [EPS(s)]. A medium containing glucose gave rise to a (1→6)-linked β-glucan (200 kD), pustulan, which was characterized by NMR and methylation analysis. A sucrose-containing medium provided a homogalactan (376 kD) and methylation analysis showed nonreducing end- (20%), 6-O- (53%) and 5,6-di-O-substituted Galf units (27%). An HMQC spectrum of the homogalactan showed C-1/H-1 signals at δ 108.2/4.820, 108.3/4.820 and 107.1/5.079, corresponding to three types of β- -Galf units. A DEPT analysis showed inverted signals (CH₂) at δ 67.8 and 67.2, corresponding to 6-O-substituted β- -Galf units, whereas a C-5 signal at δ 77.0 suggests 5-O-substitution, confirming a novel structure for a β-galactofuranan.     

Effect of temperature and pH on 31P nuclear magnetic resonances of phospholipids in cholate micelles

Accurate and precise determination of phospholipid composition by ³¹P NMR spectroscopy requires correct assignments and adequate spectral resolution. Because temperature and pH may affect chemical shifts (δ), our first aim was to establish the temperature coefficient (Δδ/ΔT) of common phospholipid classes when using sodium cholate as detergent. This parameter can then be used to aid in resonance assignments. The second goal was to investigate the pH dependence of δ so that, in addition to temperature, pH control can be used to minimize spectral overlap. For phosphatidylcholine, sphingomyelin, dihydrosphingomyelin and phosphatidylglycerol, δ values were invariant with pH and temperature. Whereas the Δδ/ΔT for phosphatidylinositol was 4 × 10⁻³ ppm/°C, regardless of pH, these coefficients were highly pH-dependent for phosphatidic acid, phosphatidylethanolamine and phosphatidylserine, exhibiting maximal variations with the deprotonation of the headgroup, particularly for phosphatidic acid. These trends indicate the importance of H-bonding on δ and Δδ/ΔT for phospholipid resonances.     

Cannulation in situ of equine umbilicus. Identification by gas chromatography-mass spectrometry (GC-MS) of differences in steroid content between arterial and venous supplies to and from the placental surface

Equine umbilicus was cannulated in utero and a series of cord plasma samples removed for analysis. After steroid extraction and derivatisation, gas chromatographic-mass spectrometric (GC-MS) analysis demonstrated large differences in steroid content between the plasma samples obtained from the umbilical artery and vein, the blood supplies leading to and from the placental surface, respectively. 3β-Hydroxy-5,7-androstadien-17-one, dehydroepiandrosterone, pregnenolone, 3β-hydroxy-5-pregnan-20-one, 5-pregnene-3β,20β-diol and 5β-pregnane-3β,20β-diol were identified as major constituents in extracts from umbilical arterial plasma samples, mostly as unconjugated steroids. Together with 5-pregnane-3,20-dione, these steroids were identified in extracts from umbilical venous plasma samples but at significantly reduced levels to those determined in arterial plasma samples. Oestradiol-17, dihydroequilin-17 and dihydroequilenin-17 were identified in extracts (mostly sulphate-conjugated) from both umbilical arterial and venous plasma samples, much larger amounts being detected in the plasma sampled from, rather than to, the placental surface. Equilin, equilenin, oestrone, oestradiol-17β, dihydroequilin-17β and dihydroequilenin-17β were not detected in the present studies. Isomers of 5(10)-oestrene-3,17β-diol together with 5(10),7-oestradiene-3,17β-diol and its possible oxidative artifact, 5(10),7,9-oestratriene-3,17β-diol, were tentatively identified only in sulphate-conjugated extracts from umbilical venous plasma samples. No glucuronic acid-conjugated steroids could be detected. The implications of this work in the elucidation of the biosynthetic pathways leading to both the formation of oestrogens and C₁₈ neutral steroids at the placental surface are discussed.     

青海湖自然保护区人类数字足迹及草地生物量的人类活动暴露度的时空模式分析

开展保护区人类活动压力定量评估对保护区内生态系统安全、降低人类活动影响具有重要意义。许多学者从人类活动对生物多样性、生物的生境或生态系统服务及其价值的影响等角度已开展了大量研究, 但由于反映人类活动的统计数据在时空尺度上较粗, 难以精细刻画保护区内短期动态的人类活动干扰。本研究尝试通过记录人的位置到访信息的高时空分辨率数字足迹数据, 以青海湖国家级自然保护区为研究区域, 利用0.01°逐日的定位请求数据和草地生物量数据, 从人类数字足迹覆盖率、数字足迹强度和草地生物量的人类活动暴露度3个指标上对青海湖自然保护区内人类数字足迹入侵强度及其对生态环境的影响开展了研究。研究结果显示, 青海湖保护区人类数字足迹具有“多尖峰、南高北低、景区节律”的时空模式; 每日人类数字足迹覆盖率和足迹强度呈现按月聚集模式, 最大值分别为7.42%和5.24; 草地生物量的人类活动暴露度显示人类数字足迹对青海湖二郎剑-黑马河沿线的草地生物量影响最大, 此时草地生物量的人类活动暴露度水平在热门旅游景点较高, 最高达到2.24。通过位置大数据挖掘青海湖保护区内人类数字足迹的时空变化及其对于生态环境的影响, 不仅证明了数字足迹用于人类活动对于生态环境影响研究的有效性, 也为保护区生态环境精细化的管理提供支撑。     

Foliar stable carbon isotope ratios of Phragmites australis and the relevant environmental factors in marsh wetlands in Tianjin

Aims Phragmites australis marshes in Tianjin play an important role in ecosystem functioning. Wetlands of Tianjin municipality have been suffering from serious nitrogen loading, salinization and water shortage. The foliar stable carbon isotope ratio (δ¹³C) is a good parameter which records environmental change information associated with the plant growth process, and reflects physiological and ecological responses of plants to environment changes. The objective of this study is to investigate the effects of environment stress on the leaf δ¹³C of P. australis in marsh wetlands in Tianjin municipality.Methods This study was conducted in Qilihai, Beidagang, and Dahuangpu marsh wetlands. We investigated the foliar δ¹³C of P. australis and sediment properties, and evaluated the relationships between the foliar δ¹³C and sediment environmental factors. Important findings 1) Foliar δ¹³C ranged from -26.3‰ to -23.6‰, with an average value of -25.8‰. 2) Sediment water and nitrogen status were the important factors affecting reed foliar δ¹³C. Foliar δ¹³C was negatively correlated to sediment relative water content, and positively correlated to sediment total nitrogen and available nitrogen content. In contrast, foliar δ¹³C was not significantly correlated to sediment salinity and phosphorus content. 3) Leaf δ¹³C were significantly positively correlated with leaf nitrogen content, and negatively correlated with leaf carbon and nitrogen ratio across all site. However, these relationships were not detected due to the wetland drainage at Qilihai site in August. Wetland drainage changed the plant water and nitrogen balance, and further affected water and nitrogen utilization strategies of P. australis. Moreover, wetland drainage had stronger effects on these processes than nitrogen loading and salinization.     

Xenopus axin interacts with glycogen synthase kinase-3 beta and is expressed in the anterior midbrain

Axin is encoded by the fused locus in mice and is required for normal vertebrate axis formation. It has recently been shown that axin associates with the adenomatous polyposis coli gene product (APC), β-catenin and glycogen synthase kinase-3 (GSK-3) in a complex that appears to regulate the level of cytoplasmic β-catenin. We have identified the Xenopus homologue of axin through its interaction with GSK-3β. Xenopus axin (Xaxin) is expressed maternally and throughout early development with a low level of ubiquitous expression. Xaxin also shows remarkably high expression in the anterior mesencephalon adjacent to the forebrain–midbrain boundary.