Development of genetic markers in cyanobacteria and stability of genetically marked strains in soil

Reporter marker GUS (-glucuronidase gene from Escherichia coli) and luc operon from the American firefly were introduced into cyanobacteria and the stability of these markers in soil was examined. To transfer the integrational vector into cyanobacteria, the genomic DNA library of Synechocystis sp. or Anabaena cylindrica maintained in pBR 322, pCY 100 and pCY 101 were transformed with HB 101 containing pRL 528 and selected for Cm^r and Amp^r. These clones of HB 101 containing pRL 528 and the vectors carrying different cyanobacterial chromosomal DNA fragments were used for triparental mating with HB 101 [pRK 2013/pRK 2073] and cyanobacteria. The frequency of transconjugants for integrational vectors was between 2.1 × 10^–5 and 4.0 × 10^–4. The transfer frequency of RSF 1010 based vectors (pDSK 519 and pCY 106) was 1.0–4.5 × 10^–4 in Synechocystis sp. whereas A. cylindrica failed to maintain these vectors. Low frequency transfer (2.0–2.3 × 10^–6) of RK 2 based vectors pVK 100 and pCY 104 was observed in A. cylindrica but these were unable to replicate in Synechocystis sp. The vectors in general were stable at least by 74.9% for 60 days of incubation in BG-11 medium. The markers were less stable in A. cylindrica (74.9–84.2%) compared to Synechocystis sp. (80.1–88.8%) at 60 days of incubation. Integrational vectors were almost 85% stable in both the strains. The RK 2 derivative of pCY 104 was less stable in A. cylindrica (74.9–77.3%) than the RSF 1010-based vector pCY 106 in Synechocystis sp. (80.1–81.0%). A maximum of 64.7% of the markers were lost in soil. The chromosomal markers through integrational vectors were found to be highly stable and 68.2–72.7% of these markers were retained in cyanobacteria at 60 days of incubation. Plasmid markers were less stable, with a loss of 64.7–48.7% at the end of the experiment. In A. cylindrica 58–65% of the RK 2 vector was lost whereas in Synechocystis sp. 49–61% of RSF 1010 was lost at 60 days of incubation.     

Ex vivo thickness measurement of cartilage covering the temporomandibular joint

Articular cartilage covers the temporomandibular joint (TMJ) and provides smooth and nearly frictionless articulation while distributing mechanical loads to the subchondral bone. The thickness of the cartilage is considered to be an indicator of the stage of development, maturation, aging, loading history, and disease. The aim of our study was to develop a method for ex vivo assessment of the thickness of the cartilage that covers the TMJ and to compare that with two other existing methods. Eight porcine TMJ condyles were used to measure cartilage thickness. Three different methods were employed: needle penetration, micro-computed tomography (micro-CT), and histology; the latter was considered the gold standard. Histology and micro-CT scanning results showed no significant differences between thicknesses throughout the condyle. Needle penetration produced significantly higher values than histology, in the lateral and anterior regions. All three methods showed the anterior region to be thinner than the other regions. We concluded that overestimated thickness by the needle penetration is caused by the penetration of the needle through the first layer of subchondral bone, in which mineralization is less than in deeper layers. Micro-CT scanning method was found to be a valid method to quantify the thickness of the cartilage, and has the advantage of being non-destructive.     

84K杨PagC3H3基因表达模式分析

木质素作为木材的主要组成成分,通常是由3种单体聚合而成,在其生物合成过程中,共有10个酶家族参与负责将苯丙胺酸转化为单体木质素,其中C3H是在对-香豆酰辅酶A（p-coumaroyl CoA）到咖啡酰辅酶A（caffeoyl CoA）的羟基化过程和G/S单体形成中的关键控制酶类,探究PagC3H3基因表达模式,对于进一步了解该基因功能具有重要意义。该研究通过定量PCR对PagC3H3基因的组织特异性表达进行分析;克隆得到了长度为2 035 bp的PagC3H3的启动子序列,预测含有多个顺式作用元件;同时,将获得的PagC3H3的启动子序列构建植物表达载体pBI121-PagC3H3pro：：GUS,进行拟南芥瞬时转化,结果显示PagC3H3基因在84K杨的根、中部茎节和基部茎节中的表达量较高;瞬时转化拟南芥,GUS染色表明：在下胚轴和根中GUS活性较强,由此推测PagC3H3基因在木质素合成过程中发挥作用。     

Expression of the Tobacco Ext 1.4 Extensin Gene Upon Mechanical Constraint and Localization of Regulatory Regions

Abstract: The regulation of the Ext 1.4 gene encoding a tobacco ( Nicotiana tabacum L.) extensin was studied in response to mechanical constraints. Transgenic plants carrying chimeric Ext 1.4 promoter/GUS (β-glucuronidase)/ nos terminator or Ext 1.4 3'-end constructs were obtained. Expression of gene fusions was found in tissues where mechanical stresses occur, e.g., during germination, as well as in root and stem tissues. Chimeric genes were successively and transiently expressed in different tissues during germination, i.e., at the tip of the root and then in the hypocotyl, during their growth through the seed coat. Moreover, they were expressed in cortical cells surrounding the emergence of adventitious and lateral roots and developmentally-regulated in nodes. The expression of Ext 1.4 could be induced by imposing mechanical constraints due to curving of either the stems or roots. Expression then occurred in cells where it does not normally occur, i.e., in cortical cells of internodes and in the distal piliferous zone of roots. Accumulation of RNAs occurs several days after the start of the constraint. Promoter regions involved in regulation of expression of Ext 1.4 in stems, roots, and in seedlings upon mechanical constraint could be localized. Moreover, the 3' non-coding region was shown to modulate expression in roots. These results suggest that the regulation of Ext 1.4 following mechanical stress is dependent on both tissue-specific and mechanical-responsive elements.     

Generation of a transgenic mouse model with chondrocyte-specific and tamoxifen-inducible expression of Cre recombinase

Postnatal cartilage development and growth are regulated by key growth factors and signaling molecules. To fully understand the function of these regulators, an inducible and chondrocyte-specific gene deletion system needs to be established to circumvent the perinatal lethality. In this report, we have generated a transgenic mouse model (Col2a1-CreER(T2)) in which expression of the Cre recombinase is driven by the chondrocyte-specific col2a1 promoter in a tamoxifen-inducible manner. To determine the specificity and efficiency of the Cre recombination, we have bred Col2a1-CreER(T2) mice with Rosa26R reporter mice. The X-Gal staining showed that the Cre recombination is specifically achieved in cartilage tissues with tamoxifen-induction. In vitro experiments of chondrocyte cell culture also demonstrate the 4-hydroxy tamoxifen-induced Cre recombination. These results demonstrate that Col2a1-CreER(T2) transgenic mice can be used as a valuable tool for an inducible and chondrocyte-specific gene deletion approach.     

一种研究和鉴别悬浮培养红豆杉细胞凋亡与坏死的新方法

将在动物细胞凋亡研究中应用的Hoechst-PI双重荧光染色法与琥珀酸脱氢酶(SDH)染色法相结合,建立了一种更加完善的、能同时鉴别和研究悬浮培养的植物细胞凋亡及坏死的新方法——Hoechst-PI-SDH三重染色法(H-P-S法)。该方法可直接用于红豆杉悬浮培养细胞,无需对细胞进行去壁、固定及切片等其它方法所必需的步骤,在荧光显微镜下可鉴别活细胞、死细胞及凋亡细胞,并可同时观测细胞凋亡的全部过程。该方法简单、快速、准确,而且克服了因细胞膜通透性差异引起的对死、活细胞判断的困难,可在植物细胞凋亡的研究中广泛应用。     

Assessment of antifungal effects of a novel compound from <Emphasis Type="Italic">Burkholderia cepacia</Emphasis> against <Emphasis Type="Italic">Fusarium solani</Emphasis> by fluorescent staining

A novel compound CF66I produced by Burkholeria cepacia was investigated for its antifungal effects against Fusarium solani by three different fluorescent dyes. Dual staining with propidium iodide (PI) and fluorescein diacetate (FDA) demonstrated high doses of CF66I (120.0 μg ml⁻¹) killed the fungi by acting primarily on the cell membrane. However, at fungistatic concentration (20.0 μg ml⁻¹) of this compound, microscopic observations revealed swelling hyphae with abnormal chitin deposition, as determined by Calcofluor white (CFW) staining, which was indicative of the alterations in cell wall structure. In addition, inhibition of intracellular esterases activity was observed. These results led us to conclude that low doses of CF66I probably inhibited the fungal growth by interfering with the cell metabolic pathways.     

人胎盘滋养层细胞原代的体外培养与改进

目的:建立与改进纯度较高的适于实验研究的人绒毛膜滋养层细胞。方法:采用胰蛋白酶消化法消化人正常妊娠6～8周胎盘组织,以35%、45%2个Percoll密度梯度进行分离纯化,并用免疫组化及透射电镜等技术对其生物学特性、细胞内部结构进行观察。结果:胎盘组织中滋养层细胞角蛋白染色阳性,血管内皮细胞及基质成分波形蛋白染色阳性,经该法分离纯化的细胞角蛋白染色阳性者(滋养层细胞)占90%以上,透射电镜观察示所获细胞有典型滋养层细胞结构。结论:该法简便易行,可获得合乎实验要求的人滋养层细胞,可供后续实验研究。     

Cytoplasm-enriched fragments ofChara: structure and electrophysiology

Summary Cytoplasm-enriched fragments prepared from internodal cells ofChara corallina by centrifugation contain membrane bound vesicles ranging in size from a few m to hundreds of m. If the fragments are incubated in artificial pond water (APW) of pH₀ above 6.5, neutral red stains the inside of many vesicles bright crimson, suggesting the presence of inward proton-pumping. In APW of pH₀ below 6 crimson vesicles are found less frequently. Under such conditions most vesicles remain unstained inside and some develop indistinct pink halos. After a few days most fragments form a central vacuole, which stains red, regardless of the pH₀. The cytoplasmic layer still contains vesicles after vacuole formation.In order to identify the membrane bounding the vesicles various fluorescent probes were applied either by injection into the fragment or directly onto the vesicles released into artificial cytoplasm. Lucifer yellow or 6 COOH-F move readily across the tonoplast in intact cells, but did not enter any vesicles. On the other hand, the fluorescent cationic stain DIOC, which is used to highlight mitochondria and especially endoplasmic reticulum, stained the vesicle membrane. Numerous elliptical or kidney shaped nuclei in the flowing cytoplasm were highlighted with DAPI. In some fragments the nuclei formed large agreggates sometimes filling the width of the fragment.Patch-clamping the vesicles in artificial cytoplasm showed the presence of several kinds of channels, some displaying similar behaviour to the K⁺ channels observed in cytoplasmic droplets.Analogous to the plasmalemma of intact cells, the fragments without vacuoles displayed electrophysiological states dominated by either K⁺ conduction, H⁺ (or OH^–) conduction or the proton pump. On the other hand, excitation transients in fragments were of low amplitude or absent altogether. Detailed comparisons of data from fragments and intact cells are shown. The effect of vacuole formation on fragment electrophysiology was also explored.