Digitonin-Permeabilized Cells Are Exocytosis Competent

Release of norepinephrine from PC12 cells can be stimulated by free Ca2+ in micromolar concentrations after permeabilization with 10 micrograms/ml of digitonin. This release is time and temperature dependent, half-maximal at 0.3 microM Ca2+, and, after washing out of endogenous ATP, half-maximal at about 0.5 mM MgATP when exogenously added. Similar results were obtained with bovine adrenal chromaffin cells using the same protocol. Support for the idea that the mechanism of release from both permeabilized cell types is still exocytosis is demonstrated at the electron microscopic level by immunolabeling chromaffin granule membrane antigens that were introduced into the plasma membrane following stimulation. Electron micrographs furthermore demonstrate that chromaffin granules retain typical dense cores after permeabilization, indicating that leakiness of catecholamines from the granules was not a major factor. Pores, formed by digitonin in the plasma membranes, were utilized to introduce antibodies into such exocytosis-competent cells. Anti-actin and anti-chromaffin granule membrane antibodies show a staining pattern similar to conventionally fixed and stained preparations. Our results demonstrate that pores formed by digitonin do not impair the process of exocytosis although they are big enough to allow macromolecules to pass in both directions. The digitonin-permeabilized cell is therefore an ideal in vitro system with which to study the fusion process between chromaffin granules and the plasma membrane.     

Competent processing techniques for scaffolds in tissue engineering

Engineering a functional tissue ex vivo requires a synchronized effort towards developing technologies for ECM mimicking scaffold and cultivating tissue-specific cells in an integrated and controlled manner. Cell-interactive scaffolds in three dimensions (3D), designed and processed appropriately with an apt biomaterial to yield optimal porosity and mechanical strength is the key in tissue engineering (TE). In order to accomplish these facets in a 3D scaffold, multiple techniques and processes have been explored by researchers all over the world. New techniques offering reasonable flexibility to use blends of different materials for integrated tissue-specific mechanical strength and biocompatibility have an edge over conventional methods. They may allow a combinatorial approach with a mix of materials while incorporating multiple processing techniques for successful creation of tissue-specific ECM mimics. In this review, we analyze the material requirement from different TE perspectives, while discussing pros and cons of advanced fabrication techniques for scale-up manufacturing.     

枯草芽孢杆菌感受态细胞的制备及质粒转化方法研究

为便于枯草芽孢杆菌工业化生产应用,对Spizizen创立的枯草芽孢杆菌DNA转化方法进行改进.用GMI和GMII溶液处理枯草芽孢杆菌野生型菌株BS501a、营养缺陷型突变株DBl342和非营养缺陷型突变株WB800,用改进的方法制备感受态细胞,用7.5kb质粒pSBPTQ进行转化,并研究RNA、酵母粉、水解酪蛋白、培养方法对枯草芽孢杆菌质粒转化的影响.结果表明,该方法适用于不同基因型枯草芽孢杆菌的质粒转化,营养缺陷型突变株DBl342的转化率为750 CFU/μg/DNA,非营养缺陷型突变株WB800转化率为1 070 CFU/xg DNA,野生型菌株BS501a转化率为270 CFU/μg/DNA.根据影响转化效率的因素,推测在该方法中,枯草芽孢杆菌质粒转化原理:一定生物量的枯草芽孢杆菌在外界营养条件和钙、镁离子作用下,细胞壁和细胞膜形成缺陷,使外源DNA转入枯草芽孢杆菌细胞内.     

大肠杆菌感受态细胞转化能力的影响因素

探讨了大肠杆菌菌株、细菌生长状态、转化溶液、抗冻剂及保存时间、质粒长度和纯度对感受态细胞转化能力的影响。结果表明,以100 mmol/L CaCl2为缓冲液,采用经活化培养的A600为0.55的TG1制备的感受态细胞,在冰上放置6h后转化,所得转化率最高,可达2×106-4×107cfu/μg DNA(pUC19)。随着质粒长度增加和纯度降低,转化率有所下降。若感受态细胞要保存备用,以15％甘油为抗冻剂优于7％DMSO,但添加抗冻剂对转化率有抑制作用。贮于甘油的感受态细胞在-70℃冻存两个月后仍有较理想的转化率。     

Plantlet Production from Morphogenetically Competent Cell Suspensions of Daylily

Cell suspensions of the diploid daylily cultivar Autumn Blazewere produced from larger masses of tissue by culture in thebasal medium of Murashige and Skoog supplemented with 10 percent v/v coconut water and 2 mg 1^–12,4–D. By drasticallylowering the level of 2,4–D, followed by transferral toa modified White's or Schenk and Hildebrandt medium, clustersgrow and ultimately give rise to embryonic structures. A finalperiod in a semi-solid medium stimulates shoot and root growthto the point where successful transplanting of plantlets tosoil is assured provided safeguards to prevent ‘dampingoff’ and desiccation are taken. Normal plantlet formationmay be arrested in the formation of neomorphs which do not seemper se to be capable of further development but they can giverise to morphologically normal plantlets after they are stimulatedto form callus which, in turn, is given an appropriate sequenceof stimuli. Hemerocallis, daylily, totipotent cells, micropropagation, tissue culture.     

Competent Diplococcus pneumoniae Accept Both Single- and Double-Stranded Deoxyribonucleic Acid

The transforming activity of fractionated complementary strands of Diplococcus pneumoniae deoxyribonucleic acid (DNA) bands at the position of fully denatured DNA in CsCl at pH 11.0, and is completely (> 99.8%) destroyed by digestion with exonuclease-I. These results prove that pure single strands transform the normally prepared competent cells of this species. Their efficiency is about 0.5% that of native DNA of comparable size.     

Teneur en eau et en azote chez l'abeille en fonction de l'isolement

Résumé La diffèrence de mortalité notée entre les abeilles isolées et groupées n'est pas due à une perturbation du métabolisme de l'eau et de l'azote. Ceci étant également vrai pour les surces, il est possible de généraliser et de dire que le métabolisme généraliser et de dire que le métabolisme général ne semble jouer aucun rôle déterminant dans cet effet de groupe, observé tant chez les abeilles d'hiver que chez les abeilles d'été.

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Zusammenfassung Die Sterblichkeitsdifferenz zwischen isolierten und gruppierten Winter- oder Sommerbienen ist nicht auf eine Perturbation des Metabolismus von Wasser und Stickstoff zurückzuführen, wie es ja auch bereits vom Zuckergehalt festgestellt wurde.Der Metabolismus spielt also keine entscheidende Rolle in diesem Gruppeneffekt.

     

感受态细胞制备与保存方法的比较研究

目的 :确立一个能制备高转化率感受态细胞并长期维持其感受性的实验方案。方法 :比较CaCl2 法、TSS法、超高效法制备感受态细胞的效果 ,选用三者中较好的方法进一步探讨不同生长期 (OD值 0 .2～ 1.1)的细菌对制备感受态细胞的影响 ,并分别比较了不同冷冻保护剂 (7%DMSO ,10 %甘油 )于 - 2 0℃、- 80℃冰箱保存感受态细胞的效果。结果 :三种方法获得的感受态细胞转化率差异极显著 (P <0 .0 1)。采用超高效法 ,OD值为 0 .36 (或 0 .5 8)时收集菌体可获得 1.1× 10 8的高转化率的感受态细胞 ,以 7%的DMSO为冷冻保护剂保存感受态细胞可维持 10 7以上的转化率 4 0d以上     

Structure and Replication of Chromosomes in Competent Cells of Bacillus subtilis

Competent cultures of Bacillus subtilis 168 were fractionated on gradients of Renografin-76 to obtain a population enriched for competent cells. The cells in this fraction contained two nuclear bodies. The competent cell fraction synthesized deoxyribonucleic acid and ribonucleic acid at reduced rates compared to the noncompetent cell fraction and appeared to divide synchronously upon incubation. The state of the chromosome in competent cells was determined by density transfer experiments and marker frequency analyses. The results are consistent with a competent cell possessing two, or a multiple of two, chromosomes, one complete and the other partially duplicated. During subsequent growth the partially completed chromosome replicates preferentially.     

大肠杆菌JM109感受态形成因素分析

目的:分析大肠杆菌JM109感受态形成因素,提高转化效率。方法:采用不同生长状态、不同转化溶液、不同保存时间及热激处理时间的细菌制备感受态,分析转化效率。结果:以20mmol/L MgCl2 80mmol/L CaCl2为处理液,经活化培养OD600为0.82的菌液制备感受态细胞,4℃放置12~24h之内,42℃热激处理60s,转化效率最高,可达9.8×106~1.2×107cfu/μg DNA(pUC19)。随着质粒长度增加,转化效率下降。结论:感受态细胞形成与生长状态关系密切,金属离子、有机溶剂对感受态的形成影响显著。感受态形成过程中,细胞可能发生了一系列的生理变化。     

大肠杆菌最佳感受态细胞制备的探讨

本文对４种大肠杆菌菌株在不同生长时期的转化效率分别进行了测定．结果表明,在细菌的生长繁殖过程中,其转化效率是有很大变化的．对于不同的菌株,它们获得高转化率时的活菌数不同．同时,得到了这４种大肠杆菌菌株制备最佳感受态细胞的条件．     

Macromolecular Synthesis in Bacillus subtilis During Development of the Competent State

Rates of deoxyribonucleic acid, ribonucleic acid, and protein synthesis were examined in purified competent cells of Bacillus subtilis during the development of the transformable state. To become competent, a cell must depart from the normal course of vegetative growth and pass through a precompetent phase beginning as early as 90 to 180 min before the appearance of transformability. While in the precompetent state, the cell decreases its rate of deoxyribonucleic acid synthesis and lowers its ratio of ribonucleic acid synthesis to protein synthesis. This altered pattern of synthesis eventually leads to a decreased buoyant density of precompetent cells. Once a cell has become both precompetent and low in density, it can be converted to a competent (transformable) cell. The early alterations in macromolecular synthesis were found in two competence regimens, one utilizing a nutritional step-down and one free of such a shift. The data imply that the precompetent state is a generalized characteristic of the B. subtilis transformation system and is not specific to the procedure used to allow competence development. Since precompetence-specific events occur very early in a competence regimen, we conclude that the induction of precompetence is unrelated to sporulation or a nutritional shift.     

Derivation of Germline Competent Rat Embryonic Stem Cells from DA Rats

The laboratory rat was one of the earliest mammalian species for scientific research and used as animal disease models in physiology,toxicology,behavior,immunology,and tumor-biology for over 150 years (Jacob,1999).However,rat lags far behind mouse in generating human disease models and functional genomic studies because of the lack of authentic rat embryonic stem (ES) cells (Voigt and Serikawa,2009),whereas the first mouse ES cell line was established in 1981 (Evans and Kaufman,1981).By combining two or three kinase inhibitors which target GSK3,MEK and FGF signaling pathways in serum-free N2B27 medium,germline competent rat ES cells were first derived in 2008 (Buehr et al.,2008;Li et al.,2008).     

少量制备大肠杆菌感受态细胞条件探索

目的:为了获得重复性好、转化率高的少量制备感受态细胞的方法,利用不同生长时期的大肠杆菌感受态细胞,进行转化比较。方法:根据普通实验室的实验条件,常规方法提取质粒,氯化钙法转化不同生长时期的大肠杆菌感受态细胞,比较转化率。结果:大肠杆菌感受态细胞的转化率与OD值显著相关,在OD600nm为0.39和0.55时转化率最高,在OD600nm为0.28~0.55之间均可得到理想的转化效果。结论:少量制备感受态细胞方法操作中无需添加任何保护试剂和细胞复苏培养,操作简便、重复性好,实验成本低廉。