Functional characterization and genetic mapping of alk8

The novel type I TGFβ family member receptor alk8 is expressed both maternally and zygotically. Functional characterization of alk8 was performed using microinjection studies of constitutively active (CA), kinase modified/dominant negative (DN), and truncated alk8 mRNAs. CA Alk8 expression produces ventralized embryos while DN Alk8 expression results in dorsalized phenotypes. Truncated alk8 expressing embryos display a subtle dorsalized phenotype closely resembling that of the identified zebrafish dorsalized mutant, lost-a-fin (laf). Single-strand conformation polymorphism (SSCP) analysis was used to map alk8 to zebrafish LG02 in a region demonstrating significant conserved synteny to Hsa2, and which contains the human alk2 gene, ACVRI. Altogether, these functional, gene mapping and phylogenetic analyses suggest that alk8 may be the zebrafish orthologue to human ACVRI (alk2), and therefore extend previous studies of Alk2 conducted in Xenopus.     

Expression of Chx10 and Chx10-1 in the developing chicken retina

We have isolated full-length cDNAs of chick Chx10 and Chx10-1, two members of the paired type homeobox/CVC gene family. A comparison of sequences suggests that Chx10 is closely related to Alx/Vsx-2 and Vsx-2 of zebrafish and goldfish, respectively; while Chx10-1 is closely related to Vsx-1 of zebrafish and goldfish. Chx10 and Chx10-1 are expressed in the early retinal neuroepithelium, but not in the pigment epithelium and lens. The expression of Chx10 is present in most retinal neuroblasts, while Chx10-1 exhibits a novel pattern along the nasotemporal border. In the differentiating retina, both Chx10 and Chx10-1 are restricted to bipolar cells and are maintained at a low level in bipolar cells of the mature retina.     

金线莲抑制斑马鱼黑色素形成的活性组分筛选及机理研究

为探索金线莲中对黑色素形成具有抑制效果的活性组分,本研究对金线莲进行分离、提取,获得总提组、醇沉组与醇提组,利用斑马鱼筛选金线莲具有美白作用的活性组分。将受精后0.75 h的斑马鱼胚胎分别暴露于不同浓度的金线莲总提组、醇沉组、醇提组,72 h时观察结果表明,金线莲醇提物能有效抑制斑马鱼胚胎黑色素和黄色素沉着,浓度越高抑制效果越明显,且不影响胚胎生长发育。进一步采用半定量PCR和整胚原位杂交技术定量和定性地检测黑色素形成相关基因mRNA表达,结果表明金线莲醇提物可以有效降低silv、tyr和tyrp1a等黑色素合成相关基因的转录水平,且具有浓度依赖关系。通过检测酪氨酸酶活性显示,加入醇提物的实验组其酪氨酸酶活性随着处理浓度升高而逐渐降低。此外,在黑色素已经大量形成的情况下,金线莲醇提物仍可通过下调黑色素合成相关基因的mRNA表达及酪氨酸酶活性来抑制黑色素的形成,并且这种抑制效果可在金线莲醇提物撤除后得到恢复。上述实验结果表明,金线莲醇提物能显著抑制斑马鱼黑色素的形成,本文为金线莲在美白产品领域的开发和应用方面提供了有力支撑。     

The zebrafish Pax3 and Pax7 homologues are highly conserved, encode multiple isoforms and show dynamic segment-like expression in the developing brain

This study describes the isolation and characterization of zebrafish homologues of the mammalian Pax3 and Pax7 genes. The proteins encoded by both zebrafish genes are highly conserved (>83%) relative to the known mammalian sequences. Also the neural expression patterns during embryogenesis are very similar to the murine homologues. However, observed differences in neural crest and mesodermal expression relative to mammals could reflect some functional divergence in the development of these tissues. For the zebrafish Pax7 protein we report the first full-length amino acid sequences in vertebrates and show the existence of three additional isoforms which have truncations in the homeodomain and/or the C-terminal region. These novel variants provide evidence for additional isoform diversity of vertebrate Pax proteins.     

陇中黄土高原半干旱区不同种植年限紫花苜蓿土壤线虫群落分布特征

为明确长期种植紫花苜蓿对土壤线虫群落演变的影响,以不同种植年限紫花苜蓿为研究对象(2 a、9 a、18 a),并以农田为对照,采用 Illumina MiSeq测序技术研究了陇中黄土高原半干旱区紫花苜蓿土壤线虫群落结构及其多样性,探讨影响其群落变化的主控因子。结果表明: 所获土壤样品线虫种群隶属于2纲7目16科21属,其中,色矛纲为黄绵土优势线虫类群(44.6%～81.4%),相对丰度随苜蓿种植年限延长而降低。针线属、螺旋属、剑线属、Pristionchus、茎属、盆咽属、长针属、艾普鲁斯属、Isolaimium和野外垫刃属为苜蓿地特有线虫,其中,针线属(54.1%)、螺旋属(23.9%)、剑线属(21.9%)分别为2 a、9 a、18 a苜蓿地优势线虫属。紫花苜蓿土壤均以植物寄生线虫为优势类群(31.8%～67.1%),其相对丰度随苜蓿年限延长先降后升。冗余分析显示,土壤速效磷和全氮含量是影响紫花苜蓿土壤线虫群落结构的主导环境因子。     

The propensity of alkoxide and aryloxide derivatives of tungsten carbonyls to aggregate in solution. Synthesis and X-ray structures of dinuclear, trinuclear and tetranuclear complexes derived from the MeOW(CO)5 anion

The complex [Et₄N][W(CO)₅OMe] (1) has been prepared from the reaction of the photochemically generated W(CO)₅THF adduct and [Et₄N][OH] in methanol. Complex 1 was shown to undergo rapid CO dissociation in THF to quantitatively provide the dimeric dianion, [W(CO)₄OMe]₂²⁻. The resulting THF insoluble salt [Et₄N]₂[W(CO)₄OMe]₂ (2) has been structurally characterized by X-ray crystallography, with the doubly bridging methoxide ligands being in an anti configuration. Complex 2 was found to subsequently react with excess methoxide ligand in a THF slurry to afford the face-sharing octahedron complex [Et₄N]₃[W₂(CO)₆(OMe)₃] (3) which contains three doubly bridging methoxide groups. In the absence of excess methoxide ligand complex 2 cleanly yields the tetrameric complex [Et₄N]₄[W(CO)₃OMe]₄ (4) which has been structurally characterized as a cubane-like arrangement with triply bridging μ³-methoxide groups and W(CO)₃ units. Although complex 3 was not characterized in the solid state, the closely related glycolate derivative [Et₄N]₃[W₂(CO)₆(OCH₂CH₂OH)₃] (5) was synthesized and its structure determined by X-ray crystallography. The trianions of complex 5 are linked in the crystal lattice by strong intermolecular hydrogen bonds. Crystal data for 2: space group P2₁/n, a = 7.696(2), b = 22.019(4), c = 9.714(2) Å, β = 92.22(3)°, Z = 4, R = 6.43%. Crystal data for 4: space group Fddd, a = 12.433(9), b = 24.01(2), c = 39.29(3) Å, Z = 8, R = 8.13%. Crystal data for 5: space group P2₁2₁2₁, a = 11.43(2), b = 12.91(1), c = 29.85(6) Å, Z = 8, R = 8.29%. Finally, the rate of CO ligand dissociation in the closely related aryloxide derivatives [Et₄N][W(CO)₅OR] (R = C₆H₅ and 3,5-F₂C₆H₃) were measured to be 2.15 × 10⁻² and 1.31 × 10⁻³ s⁻¹, respectively, in THF solution at 5°C. Hence, the value of the rate constant of 2.15 × 10⁻² s⁻¹ establishes a lower limit for the first-order rate constant for CO loss in the W(CO)₅OMe⁻ anion, since the methoxide ligand is a better π-donating group than phenoxide.     

Spatially and temporally-restricted expression of two T-box genes during zebrafish embryogenesis

T-box genes are conserved in all animal species. We have identified two members of the T-box gene family from the zebrafish, Danio rerio. Zf-tbr1 and zf-tbx3 share high amino acid identity with human, murine, chick and Xenopus orthologs and are expressed in specific regions during zebrafish development.     

The zebrafish Hairy/Enhancer-of-split-related gene her6 is segmentally expressed during the early development of hindbrain and somites

Several vertebrate genes of the Hairy/Enhancer-of-split (HES) family are involved in paraxial mesoderm segmentation and intersomitic boundary establishment/maintenance. Here, we show that the zebrafish hairy-related gene, her6, highly homologous to the mammalian and chicken HES-1 genes, is expressed in the posterior part of each segmented somite and in stripes in the anterior presomitic mesoderm (PSM), and also in a dynamic, segmentally restricted pattern during hindbrain segmentation, with all rhombomeres expressing her6 at different time points and at different levels.     

化肥减量配施有机肥对药用菊花产量、品质和药理活性的影响

采用田间小区试验,设置5个有机肥无机肥配施处理(100%化肥和14%、28%、56%、84%有机肥替代化肥处理),测定各处理对药用菊花农艺性状、产量、矿质元素吸收、有效成分含量的影响。并采用酶标仪和MTT试剂盒测定不同处理菊花水提物体外抗氧化活性及对H₂O₂致损的LO2肝细胞的保护作用。结果表明: 与100%化肥处理相比,化肥配施有机肥可以保证药用菊花产量,甚至低比例配施处理(14%有机肥替代化肥)还可以增产达8.3%。随着化肥减量配施有机肥比例的提高,菊花花中N、Mg含量呈上升趋势,而Ca和P含量分别在56%和28%有机肥替代处理有最大值。化肥减量配施有机肥可以显著增加药用菊花中绿原酸、木犀草苷和3,5-O-二咖啡酰基奎宁酸的含量,各成分含量随着有机肥比例的升高呈逐渐上升的趋势,上升幅度分别为3.3%～12.8%、15.7%～30.1%和9.5%～29.7%。各处理菊花水提液均有一定的体外抗氧化活性,且随着有机肥比例的升高呈先上升后下降的趋势;菊花水提液能显著提高H₂O₂致损的LO2肝细胞存活率,28%有机肥替代化肥处理细胞存活率最高,为91.2%,与模型组相比呈现极显著差异。综合产量、养分吸收、有效成分含量、体外抗氧化活性、对H₂O₂致损的LO2肝细胞的保护作用等指标,以及有机肥生态友好的特点,确定药用菊花栽培上以28%有机肥替代化肥的效果最佳。     

Phylogeny, expression and enzyme activity of zebrafish cyp19 (P450 aromatase) genes

The cyp19 encodes P450 aromatase, the enzyme catalyzing the conversion of estrogens from androgens. Estrogens affect the dimorphic, anatomical, functional and behavioral aspects of development of both males and females. In zebrafish, two cyp19 genes, cyp19a and cyp19b were found. They are expressed in ovary and brain, respectively. Expression of cyp19b can be detected by 11 days post-fertilization (dpf) by in situ hybridization in the olfactory bulbs, ventral telencephalic region and the hypothalamus of the brain in both male and female, where it is generally known to be affecting the reproductive function and sexual behavior. COS-1 clones permanently expressing the enzymes have been isolated. Both aromatase enzymes encoded by these two genes are functional in COS-1 cells and they can use androstenedione and testosterone equally efficiently. The presence of two functional cyp19 in zebrafish has its evolutionary and physiological importance.     

纳米卟啉金属有机骨架对斑马鱼神经系统发育的作用*

目的:研究纳米卟啉金属有机骨架(NPMOF)对斑马鱼幼鱼神经系统发育的作用。方法:斑马鱼胚胎在发育6 h(hpf)后随机分为两组:对照组(n=500)和暴露组(n=500),对照组斑马鱼孵化于E3溶液中,暴露组于100 mg/L的NPMOF-E3溶液中,持续暴露至28、48、72、96或120 hpf。以20条斑马鱼为一批,在28、48、72、96和120 hpf分别提取3批实验组和对照组的总RNA用于RT-PCR;在120 hpf,实验组和对照组分别取20条PTU处理过的斑马鱼用于整体胚胎原位杂交,同样是在120 hpf,每组取150条用于免疫荧光染色,30条用于行为学测试。NPMOF的形状和尺寸用透射电镜测定,其光学特性由紫外分光光度计和荧光光谱仪测定;通过免疫荧光、整体胚胎原位杂交和RT-PCR方法检测各类神经细胞的发育;行为学测试用来监测斑马鱼运动状态的改变。结果:与正常组比较,NPMOF暴露组中神经发育相关基因的表达明显升高(P＜0.05),müller细胞和星形胶质细胞的数量明显增加(P＜0.05),神经元和少突胶质细胞的分布与形态未显示出差异;行为学测试中,斑马鱼的总运动距离、快速运动时间和运动速度的值显著增加(P＜0.05),总静止时间明显降低(P＜0.05)。结论:100 mg/L NPMOF溶液的持续暴露对斑马鱼神经系统发育,特别是视网膜中müller细胞和脑中星形胶质细胞的发育有促进作用。     

Influence of DNA repair gene polymorphisms of hOGG1, XRCC1, XRCC3, ERCC2 and the folate metabolism gene MTHFR on chromosomal aberration frequencies

We have studied the effect of genetic polymorphisms in the DNA repair genes hOGG1, XRCC1, XRCC3, ERCC2 and the MTHFR gene in the folate metabolism on the frequencies of cells with chromosomal aberrations (CA), chromosome-type aberrations (CSA), chromatid-type aberrations (CTA), chromatid breaks (CTB) and chromatid gaps (CTG) scored in peripheral blood lymphocytes from 651 Norwegian subjects of Caucasian descendant. DNA was extracted from fixed cell suspensions. The log-linear Poisson regression model was used for the combined data which included age, smoking, occupational exposure and genotype for 449 subjects.

Our results suggest that individuals carrying the hOGG1 326Cys or the XRCC1 399Gln allele have an increased risk of chromosomal damage, while individuals carrying the XRCC1 194Trp or the ERCC2 751Gln allele have a reduced risk regardless of smoking habits and age.

Individuals carrying the XRCC1 280His allele had an increased risk of CSA which was only apparent in non-smokers. This was independent of age.

A protective effect of the XRCC3 241Met allele was only found in the older age group in non-smokers for CA, CSA and CTA, and in smokers for CSA. In the youngest age group, the opposite effect was found, with an increased risk for CA, CTA and CTG in smokers. Carrying the MTHFR 222Val allele gave an increased risk for chromosome and chromatid-type aberrations for both non-smokers and smokers, especially for individuals in the older age group, and with variable results in the youngest age group. The variables included in the different regression models accounted, however, for only 4–10% of the variation. The frequency ratio for CTG was significantly higher than for CTA and CTB for only 7 of the 43 comparisons performed. Some of the gap frequencies diverge from the trend in the CA, CSA, CTA and CTB results.     

斑马鱼hoxa1a基因调控颅面骨骼发育的功能研究*

hox基因编码一类高度保守的转录因子家族,人类HOXA1的突变会导致阿萨巴斯卡发育不良综合征 (Athabascan brainstem dysgenesis syndrome, ABDS),使人出现因颅骨异常导致的面部畸形和面部麻痹等症状。利用模式生物斑马鱼研究其同源基因hoxa1a的功能机制。首先利用CRISPR/Cas9技术对斑马鱼hoxa1a进行基因编辑,获得了hoxa1a基因突变,T7E1酶切结果显示F₀酶切效率平均为70%。F₁进一步筛选到两种突变类型,分别是插入了8 bp和删除了7 bp的杂合突变体。杂合子自交得到hoxa1a F₂纯合突变体,并且测序验证hoxa1a基因突变成功。5 dpf时,hoxa1a纯合突变体出现颅面发育异常。阿尔新蓝软骨染色和茜素红硬骨染色结果表明,hoxa1a突变体中颅骨发育异常、筛骨板断裂,鳃弓发育出现缺损。成功在斑马鱼中构建ABDS疾病模型,表明hoxa1a突变会造成斑马鱼颅面骨骼发育异常,为其功能机制研究奠定了基础,为人类ABDS疾病的致病机制研究提供了新的思路。     

低温对离体大鼠心室肌复极时程的影响及其机制

目的: 观察低温对离体大鼠心室肌复极时程及Kir2.1蛋白表达的影响,探讨Kir2.1蛋白在其中的作用。 方法:18只健康雄性SD大鼠, 随机分为3组(n=6),即正常对照组(C组)、35℃低温组(H₁组)和32℃低温组(H₂组)。制备Langendorff离体心脏灌注模型,37℃ K-H液平衡灌注15 min后,C组继续灌注37℃ K-H液30 min;H₁组继续灌注35℃的K-H液30 min,H₂组继续灌注32℃的K-H液30 min。记录各组平衡灌注15 min(T₁)和继续灌注30 min(T₂)时HR、左心室前壁三层心肌单相动作电位,计算单相动作电位复极50％、90％的时程(MAPD₅₀、MAPD₉₀)和跨室壁复极离散度(TDR),同时记录心律失常发生情况。取测量电生理的心室肌部位组织以Western blot测定Kir2. 1蛋白表达,以免疫组织化学法测量Kir2. 1蛋白的平均光密度值(AOD)及分布情况。结果: 与T₀比较,T₁时H₁组和H₂组HR显著减慢(P＜0.05), MAPD₅₀、MAPD₉₀显著延长(P＜0.05),TDR显著增大(P＜ 0.05);与C组比较,T₁时H₁组和H₂组HR显著减慢(P＜0.05),MAPD₉₀显著延长(P＜0.05),TDR显著增大(P＜ 0.05),Kir2.1蛋白表达显著减少(P＜0.05), AOD值显著减少(P＜0.05)。与H₁组比较,T₁时H₂组心率显著减慢(P＜0.05),MAPD₅₀、MAPD₉₀显著延长(P＜0.05),TDR显著增大(P＜0.05)。C组Kir2.1蛋白分布正常,H₁、H₂组蛋白分布紊乱。结论: 低温会延长心室复极时程,增加复极离散度,其机制与低温下调Kir2.1蛋白表达、改变Kir2.1蛋白分布有关。     

斑马鱼tbx2b调控心脏房室间隔发育的功能研究*

tbx2是早期心脏发育的关键基因。为进一步探究其对房室间隔(AVC)发育的影响,研究利用CRISPR/Cas9介导的基因敲除技术,成功构建了斑马鱼tbx2b突变体。通过T7E1酶切对其F₀进行敲除效率检测,结果显示平均敲除效率约为57.5%。F₁进一步筛选获得tbx2b杂合突变体,测序结果显示突变类型为11 bp碱基缺失的移码突变。tbx2b杂合子内交获得纯合子,tbx2b纯合突变体在5 dpf死亡并出现早期心脏环化异常表型。斑马鱼整胚原位杂交实验显示在3 dpf tbx2b纯合突变体中, 心脏腔室分化特异性标志基因nppa、nppb表达上调并异位表达在AVC,而AVC发育关键基因has2的表达消失。高效构建tbx2b突变体并初探其对下游基因的影响,为后续深入研究tbx2b对心脏AVC发育的作用奠定了基础,同时加深了人们对早期心脏调控网络的认识。