一种新的以影像为基础的细胞增殖和细胞毒性检测方法

3-(4,5-二甲基-2-噻唑)-2,5-二苯基溴化四唑盐)（MTT）比色法是传统上检测细胞增殖和细胞毒性的常用方法.
CloneSelectTM成像系统是一种以影像为基础的用于分析细胞生长的可视检测系统.本研究采用人结直肠癌HCT116细胞系,运用CloneSelect成像系统和MTT方法分别检测药物阿的平的细胞毒性,并采用Bland Altman作图法比较两种实验方法获得的pEC50值,分析两种研究方法获得的结果的一致性. 结果表明,CloneSelectTM成像系统和MTT法获得的pEC50值具有较好的一致性.与MTT方法相比,基于影像的CloneSelectTM成像分析技术检测快速、无损伤且结果更准确,获取资料不损伤细胞,允许后续其它时间点或动力学检测. 研究提示,这种新的以影像为基础的检测技术可以替代MTT方法,用于分析不同药物的抗细胞增殖活性.     

A series of novel directional cloning and expression vectors for blunt-end ligation of PCR products

Novel directional cloning and expression vectors were developed for blunt-end ligation of PCR products that are suitable for high-throughput cloning and simplifying the screening procedure. The PCR products, without further processing, are cloned into vectors digested with SchI and, following transformation, the desired recombinants give typical blue colonies on selectable plates. The principle of this selection strategy is that the construction also generates a full-length ideal lacO gene. To the best of our knowledge, this is the first time that this lacO reconstruction strategy has been applied in the selection of recombinants.     

Changes in RNA,DNA, protein contents and growth of turbot Scophthalmus maximus larvae and juveniles

The growth potential of turbot Scophthalmus maximus larvae and juveniles was studied using nucleic acid‐based indices and protein variables. The experiment was carried out from 4 to 60 days post hatching (dph). A significant increase in instantaneous growth rate during metamorphosis and retarded growth rate during post‐metamorphic phase were observed. Ontogenetic patterns of DNA, RNA and protein all showed developmental stage‐specific traits. The RNA:DNA ratio decreased up to 12 dph, then increased rapidly till 19 dph and fluctuated until 35 dph followed by a decline to the end. The RNA:DNA ratio was positively correlated with growth rate of juveniles during the post‐metamorphic phase, whereas this ratio was not a sensitive indicator of growth during the pre‐metamorphic phase and metamorphosis. The protein:DNA ratio showed a similar tendency to the RNA:DNA ratio. Changes of DNA content and protein:DNA ratio revealed that growth of S. maximus performed mainly by hyperplasia from 4 to 12 dph and hypertrophy until 21 dph during the pre‐metamorphic larval phase. Growth was dominantly hypertrophical from the early‐ to mid‐metamorphosing phase and hyperplastic thereafter. The results show that the DNA content and protein:DNA ratio can evaluate growth rates of larval and juvenile S. maximus on a cellular level.     

Anti-convulsant Effect and Mechanism of Astragalus mongholicus Extract In Vitro and In Vivo: Protection Against Oxidative Damage and Mitochondrial Dysfunction

Astragalus mongholicus (AM) is a traditional medicinal herb used as a neuroprotective agent for its anxiolytic, antidepressant, antiamnestic, and antiaggresive effects. However, the mechanisms underlying its anti-convulsant properties are not well studied. In the present study, we examined the anticonvulsant effects on pentylenetetrazol (PTZ)-induced seizures in mice and the possible mechanisms of protection against oxidative damage and mitochondrial dysfunction in vitro. The behavioral studies showed that the root extract of AM had powerful anticonvulsant effects against seizures induced by PTZ and the biochemical studies showed that root extract of AM inhibited PTZ-induced increase in lipid peroxidation, protein oxidation and reactive oxygen species, and enhanced mitochondrial function. Electron spin resonance spectroscopy studies demonstrated that the extracts from the root and aerial parts of AM possess potent effects on scavenging hydroxyl and lipid free radicals. We found that AM extract significantly protected malondialdehyde-induced oxidative damage by ameliorating activities of the mitochondrial complexes I, II, malate dehydrogenase and mitochondrial membrane potential. These data suggest that the anti-convulsant effects of AM extract may be mediated by its protective actions against oxidative damage and amelioration of mitochondrial dysfunction.     

pKILLIN: a versatile positive-selection cloning vector based on the toxicity of Killin in Escherichia coli

The invention of DNA cloning over 40 years ago marked the advent of molecular biology. The technique has now become a routine practice in any modern biomedical laboratory. Although positive-selection of recombinants in DNA cloning seems to be superior to blue/white selection based on the disruption of the lacZ gene, it is rarely practiced due to its high background, lack of multiple cloning sites, and inability to express the genes of interest or purify the protein products. Here we report the creation of a new positive-selection cloning vector dubbed pKILLIN, which overcomes all of the above pitfalls. The essence behind its high cloning efficiency is the extreme toxicity and small size of the toxic domain of killin, a recently discovered p53 target gene. Insertion inactivation of killin within the multiple cloning site via either blunt- or sticky-end ligation not only serves as a highly efficient cloning trap, but also may allow any cloned genes to be expressed as His-tagged fusion proteins for subsequent purification. Thus, pKILLIN is a versatile positive-selection vector ideal for cloning PCR products, making DNA libraries, as well as routine cloning and bacterial expression of genes.     

Precise mapping of a quantitative trait locus interval for spike length and grain weight in bread wheat (Triticum aestivum L.)

The spike characteristics length, spikelet density and fertile floret number are related yield components and are important in cereal improvement. QSpl.nau-2D is a major quantitative trait locus controlling spike length (SPL) detected in the recombinant inbred line population developed by crossing wheat (Triticum aestivum) cultivars Nanda2419 with Wangshuibai. In this study, to validate its genetic effect and determine its precise location, QSpl.nau-2D’s near-isogenic line (NIL) was developed using Mianyang99-323 as the recurrent parent through marker-assisted selection. Field trials showed that the NIL not only had significantly longer spikes on average than the recurrent parent but also had significantly higher grain weight, but did not differ in spikelet number and kernel number per spike. In the F2 population derived from a cross of the NIL with Mianyang99-323, QSpl.nau-2D functioned like a single gene and conditioned the SPL in a partially dominant manner, and was thus designated as HL1 (for head length). To precisely map HL1, 89 recombinants, consisting of 11 genotypes, were identified in the NIL-derived F2 population of 674 plants by using markers in the Xwmc25–Xgpw4080 interval. Phenotyping these lines showed that the introduction of a 0.9-cM interval flanked by Xcfd53 and DG371 in Nanda2419 resulted in longer spikes and a higher grain weight in the NIL. The availability of markers closely linked to HL1 could facilitate its use in breeding programs.     

Primordial follicle assembly was regulated by notch signaling pathway in the mice

Notch signaling pathway, a highly conserved cell signaling system, exists in most multicellular organisms. The objective of this study was to examine Notch signaling pathway in germ cell cyst breakdown and primordial follicle formation. The receptor and ligand genes of Notch pathway (Notch1, Notch2, Jagged1, Jagged2 and Hes1) were extremely down-regulated after newborn mouse ovaries were cultured then exposed to DAPT or L-685,458 in vitro (P < 0.01). Since DAPT or L-685,548 inhibits Notch signaling pathway, the expression of protein LHX8 and NOBOX was significantly reduced during the formation of the primordial follicles. Down-regulated mRNA expression of specific genes including Lhx8, Figla, Sohlh2 and Nobox, were also observed. The percentages of female germ cells in germ cell cysts and primordial follicles were counted after culture of newborn ovaries for 3 days in vitro. The result showed female germ cells in cysts was remarkably up-regulated while as the oocytes in primordial follicles was significantly down-regulated (P < 0.05). In conclusion, Notch signaling pathway may regulate the formation of primordial follicle in mice.     

Expression of ghrelin receptor (GHSR-1a) in rat epididymal spermatozoa and the effects of its activation

In this study we demonstrated the expression of the ghrelin receptor GHSR-1a in rat spermatids and epididymal spermatozoa, as well as some effects of ghrelin on the spermatozoa in vitro. For the demonstration of GHSR-1a the immunocytochemical, immunofluorescence and Western blotting techniques were applied using three different types of antibodies. The response of spermatozoa to ghrelin was tested in a series of in vitro experiments and their effects were evaluated using confocal microscopy and flow cytometry. GHSR-1a protein was found as expressed in the Golgi and acrosomes of spermatids and acrosome regions or the head cell membrane of epididymal spermatozoa. The GHSR-1a expression in spermatozoa was also confirmed by Western blot. No differences were found in percentage of spermatozoa showing annexin-V binding and expression of active form caspase-3 between control and ghrelin-treated spermatozoa. This result may indicate no pro-apoptotic effects of ghrelin neither at 10^?9 nor 10^?6 mol/L concentration. Ghrelin (10^?6 mol/L) increased free intracellular calcium ion concentration in the rat spermatozoa. Moreover, stimulation with 10^?6 mol/L ghrelin increased, while 10^?4 mol/L ghrelin decreased the number of spermatozoa showing progressive motility. In conclusion, the expression of the GHSR-1a receptor in spermatozoa, as well as ghrelin influences on sperm motility and intracellular calcium ion concentration suggest that such biological effects of ghrelin may be produced under in vivo conditions.     

鹅掌楸贵州烂木山居群的微卫星遗传多样性及空间遗传结构

濒危植物鹅掌楸(Liriodendron chinense)目前仅零散分布于我国亚热带及越南北部地区, 残存居群生境片断化较为严重。研究濒危植物片断化居群的遗传多样性及小尺度空间遗传结构(spatial genetic structure)有助于了解物种的生态进化过程以及制定相关的保育策略。本研究采用13对微卫星引物, 对鹅掌楸的1个片断化居群进行了遗传多样性及空间遗传结构的研究, 旨在揭示生境片断化条件下鹅掌楸的遗传多样性及基因流状况。研究结果表明: 鹅掌楸烂木山居群内不同生境斑块及不同年龄阶段植株的遗传多样性水平差异不显著(P>0.05), 居群内存在寨内和山林2个遗传分化明显的亚居群。烂木山居群个体在200 m以内呈现显著的空间遗传结构, 而2个亚居群内的个体仅在20 m的距离范围内存在微弱或不显著的空间遗传结构。鹅掌楸的空间遗传结构强度较低(Sp = 0.0090), 且寨内亚居群的空间遗传结构强度(Sp = 0.0067)要高于山林亚居群(Sp = 0.0053)。鹅掌楸以异交为主, 种子较轻且具翅, 借助风力传播, 在一定程度上降低了空间遗传结构的强度。此外, 居群内个体密度及生境特征也对鹅掌楸的空间遗传结构产生了一定影响。该居群出现显著的杂合子缺失, 近交系数(F_IS)为0.099 (P < 0.01), 表明生境片断化的遗传效应正逐渐显现。因此, 对鹅掌楸的就地保护应注意维护与强化生境的连续性, 促进基因交流。迁地保护时, 取样距离应不小于20 m, 以涵盖足够多的遗传变异。