Characterization of 3-Iodothyronamine In Vitro Dynamics by Mathematical Modeling

3-Iodothyronamine (T₁AM) is regarded as a hormone-like substance thanks to its endogenous nature, its interaction with specific receptors trace amine-associated receptor 1 and its biological effects. We characterized T₁AM transport and conversion in an in vitro culture of H9c2 murine cells, after a T₁AM bolus injection. Samples of cell medium culture and cell lysate were assayed by high-performance liquid chromatography coupled to tandem mass spectrometry. We performed comparative experiments by adding to T₁AM bolus amino oxidase inhibitors as iproniazid, pargyline (monoamine oxidase, MAO inhibitors), aminoguanidine, and semicarbazide (semicarbazide-sensitive amino oxidase, SSAO inhibitors). A mathematical model was developed, based on the assumption that T₁AM is transported with a mechanism that is typical of hormone transport (i.e., EGF or insulin). We noticed that surface receptors downregulation could play a major role in T₁AM dynamics. We also estimated that T₁AM catabolism is mainly affected by MAO inhibitors, which produce a dramatic decrease in the kinetic constants related to T₁AM degradation, while no significant changes were observed in experiments with SSAO inhibitors.     

对生玉米离体培养再生体系的建立

以对生玉米的雌、雄幼穗为外植体,研究了不同质量浓度激素及其组合对愈伤组织诱导、再分化苗和试管苗生根的影响,建立了对生玉米离体培养再生体系。结果表明：长度为15-17 mm的雌、雄幼穗能够诱导出质量较好的愈伤组织,但只有来自于雄幼穗的愈伤组织才能再生成苗。适合愈伤组织诱导的培养基为Ms+1．5-2．0 mg·L-12,4-D+0．5 mg·L-16-BA+0．5 mg·L-1 NAA+500 mg·L-1脯氨酸+1000 mg·L-1水解酪蛋白+30 g·L-1蔗糖;适合愈伤组织再分化培养基为MS+1．5-2．0 mg·L-1 6-BA+0．1 mg·L-1 NAA+500 mg·L-1水解酪蛋白+30 g·L-1 蔗糖;适合试管苗生根培养基为1／2 MS+0．25-0．5 mg·L-1 IBA+20 g·L-1蔗糖。     

In Vitro System for Production of Mouse Mammary Tumor Virus

An in vitro system for production, purification, and concentration of mouse mammary tumor virus is described. Monolayer cultures of C(3)H mouse mammary tumor cells propagated at 34 C in roller bottles in the presence of dexamethasone, a glucocorticoid hormone, release B-type particles which possess ribonucleic acid and a ribonucleic acid-dependent deoxyribonucleic acid polymerase. One thousandfold concentration by ultracentrifugation with subsequent gradient fractionation yielded > 7 x 10(10) particles per ml in the 1.16- to 1.18-g/ml region. Mouse mammary tumor virus produced in this system was free of detectable C-type virus.     

仙鹤藓属（Atrichum）藓类植物组织培养再生体系的建立

报道了仙鹤藓（Atrichum undulatum(Hedw.)P.Beauv.)和仙鹤藓小形变种（Atrichum undulatum var.minus(Hedw.)Par.)的组织培养再生体系的建立。为研究仙鹤藓属（Atrichum)藓类愈伤组织的诱导和再分化,将仙鹤藓和仙鹤藓小形变种原丝体接种在含有4%葡萄糖和0.2-2.0mg/L 6-BA的MS培养基上,培养一个月后,成功地诱导出疏松、易碎的绿色愈伤组织。愈伤组织诱导和常规继代培养较适合的培养基为含4%葡萄糖和1-2mg/L 6-BA的MS培养基。当将继代培养5次的脱分化藓类愈伤组织转移到含4%葡萄糖但无任何激素的MS培养基上时,能再分化形成原丝体,而在无任何碳源的Benecke培养基土培养时,能再分化形成经原丝体阶段发育来的直立配子体。     

仙鹤藓属(Atrichum)藓类植物组织培养再生体系的建立

报道了仙鹤藓(Atrichum undulatum(Hedw.)P.Beauv.)和仙鹤藓小形变种(Atrichum undulatum var.minus(Hedw.)Par.)的组织培养再生体系的建立.为研究仙鹤藓属(Atrichum)藓类愈伤组织的诱导和再分化,将仙鹤鲜和仙鹤藓小形变种原丝体接种在含有4%葡萄糖和0.2～2.0 mg/L 6-BA的MS培养基上,培养一个月后,成功地诱导出疏松、易碎的绿色愈伤组织.愈伤组织诱导和常规继代培养较适合的培养基为含4%葡萄糖和1～2 mg/L6-BA的MS培养基.当将继代培养5次的脱分化藓类愈伤组织转移到含4%葡萄糖但无任何激素的MS培养基上时,能再分化形成原丝体,而在无任何碳源的Benecke培养基上培养时,能再分化形成经原丝体阶段发育来的直立配子体.     

A Simplified System for Evaluating Cell Mechanosensing and Durotaxis In Vitro

The composition and mechanical properties of the extracellular matrix are highly variable between tissue types. This connective tissue stroma diversity greatly impacts cell behavior to regulate normal and pathologic processes including cell proliferation, differentiation, adhesion signaling and directional migration. In this regard, the innate ability of certain cell types to migrate towards a stiffer, or less compliant matrix substrate is referred to as durotaxis. This phenomenon plays an important role during embryonic development, wound repair and cancer cell invasion. Here, we describe a straightforward assay to study durotaxis, in vitro, using polydimethylsiloxane (PDMS) substrates. Preparation of the described durotaxis chambers creates a rigidity interface between the relatively soft PDMS gel and a rigid glass coverslip. In the example provided, we have used these durotaxis chambers to demonstrate a role for the cdc42/Rac1 GTPase activating protein, cdGAP, in mechanosensing and durotaxis regulation in human U2OS osteosarcoma cells. This assay is readily adaptable to other cell types and/or knockdown of other proteins of interests to explore their respective roles in mechanosignaling and durotaxis.     

A Case Study of the Dynamics of In Vitro DNA Evolution Under Constant Selection Pressure

Although thousands of in vitro selection and evolution experiments have been performed to seek different types of targets, most of them have only inspected the terminal evolutionary pool for patterns. In addition, to rapidly obtain the most favorable target, many experiments have been carried out under increasing selection pressure. However, increasing selection pressure seldom occurs in natural evolution. We studied the dynamic features of DNA in vitro evolution in the presence of the Mnt repressor under sequential constant selection pressure. When evolving under a constant pressure from an initial random pool of DNA, our system showed a clear, sharp, and reproducible crossover from a random population to an advantageous population (higher binding affinities of DNA sequences to the Mnt repressor). This crossover occurs after a long latent period during which there are no obvious changes in the population phenotype. We demonstrated that the existence of the crossover is caused by a significant sequence-nonspecific binding in the repressor–DNA system. After the crossover, the population settled in a stationary distribution of genotypes, which responded immediately to a subsequent sudden increase in selection pressure. We also experimentally tested the linear correlation between the evolution speed and sequence diversity (Fisher’s theorem) in our system. X. Yang and X. Liu contributed equally to this work.     

Synaptic Potentiation Facilitates Memory-like Attractor Dynamics in Cultured In Vitro Hippocampal Networks

Collective rhythmic dynamics from neurons is vital for cognitive functions such as memory formation but how neurons self-organize to produce such activity is not well understood. Attractor-based computational models have been successfully implemented as a theoretical framework for memory storage in networks of neurons. Additionally, activity-dependent modification of synaptic transmission is thought to be the physiological basis of learning and memory. The goal of this study is to demonstrate that using a pharmacological treatment that has been shown to increase synaptic strength within in vitro networks of hippocampal neurons follows the dynamical postulates theorized by attractor models. We use a grid of extracellular electrodes to study changes in network activity after this perturbation and show that there is a persistent increase in overall spiking and bursting activity after treatment. This increase in activity appears to recruit more “errant” spikes into bursts. Phase plots indicate a conserved activity pattern suggesting that a synaptic potentiation perturbation to the attractor leaves it unchanged. Lastly, we construct a computational model to demonstrate that these synaptic perturbations can account for the dynamical changes seen within the network.     

芳香堆心菊离体再生体系的建立

芳香堆心菊(Helenium aromaticum)全株具芳香气味, 且头状花序仅含管状花, 是研究菊科植物花香和花型的良好材料, 但目前尚缺乏对其转基因技术体系的研究。为建立高效的芳香堆心菊离体再生体系, 以叶片、茎段和下胚轴为外植体, 进行25组不同激素及不同浓度配比的不定芽诱导研究。结果表明, 以芳香堆心菊叶片为外植体, 培养基为MS+ 0.2 mg·L^-1 NAA+1 mg·L^-1 6-BA+0.2 mg·L^-1 TDZ, 培养20天后愈伤组织诱导率高达100%, 丛生芽的诱导率为62.10%; 将不定芽接种于1/2MS培养基中进行生根培养, 16天即可生根, 且生根率为63.33%; 生根后继续培养14天现蕾, 开花率达93.33%。此外, 研究表明芳香堆心菊的再生受外植体来源、激素种类和浓度的影响。2,4-D不利于芳香堆心菊不定芽的诱导, 适宜浓度的6-BA和TDZ组合能有效促进芳香堆心菊不定芽的形成。研究初步建立了芳香堆心菊组织培养条件下的离体再生体系, 为建立其遗传转化体系奠定了坚实的基础。研究结果还可用于后续有关菊科植物花香和花型的研究。     

Improved Luciferase Tagging System for Listeria monocytogenes Allows Real-Time Monitoring In Vivo and In Vitro

An improved system for luciferase tagging Listeria monocytogenes was developed by constructing a highly active, constitutive promoter. This construct gave 100-fold-higher activity in broth than any native promoter tested and allowed for imaging of lux-tagged L. monocytogenes in food products, during murine infections, and in tumor targeting studies.     

Establishment of a Convenient System for the Culture and Study of Perineurium Barrier In Vitro

The perineurium serves as a selective, metabolically active diffusion barrier in the peripheral nervous system, which is composed of perineurial cells joined together by tight junctions (TJs). Not only are these junctions known to play an essential role in maintaining cellular polarity and tissue integrity, but also limit the paracellular diffusion of certain molecules and ions, whereas loss of TJs barrier function is imperative for tumour growth, invasion and metastasis. Hence, a detailed study on the barrier function of perineurial cells may provide insights into the molecular mechanism of perineural invasion (PNI). In this study, we aimed to develop an efficient procedure for the establishment of perineurial cell lines as a tool for investigating the physiology and pathophysiology of the peripheral nerve barriers. Herein, the isolation, expansion, characterization and maintenance of perineurial cell lines under favourable conditions are presented. Furthermore, the analysis of the phenotypic features of these perineurial cells as well as the barrier function for the study of PNI are described. Such techniques may provide a valuable means for the functional and molecular investigation of perineurial cells, and in particular may elucidate the pathogenesis and progression of PNI, and other peripheral nerve disorders.

     

Dynamics of the Bacterial Intermediate Filament Crescentin In Vitro and In Vivo

Background

Crescentin, the recently discovered bacterial intermediate filament protein, organizes into an extended filamentous structure that spans the length of the bacterium Caulobacter crescentus and plays a critical role in defining its curvature. The mechanism by which crescentin mediates cell curvature and whether crescentin filamentous structures are dynamic and/or polar are not fully understood.

Methodology/Principal Findings

Using light microscopy, electron microscopy and quantitative rheology, we investigated the mechanics and dynamics of crescentin structures. Live-cell microscopy reveals that crescentin forms structures in vivo that undergo slow remodeling. The exchange of subunits between these structures and a pool of unassembled subunits is slow during the life cycle of the cell however; in vitro assembly and gelation of C. crescentus crescentin structures are rapid. Moreover, crescentin forms filamentous structures that are elastic, solid-like, and, like other intermediate filaments, can recover a significant portion of their network elasticity after shear. The assembly efficiency of crescentin is largely unaffected by monovalent cations (K⁺, Na⁺), but is enhanced by divalent cations (Mg²⁺, Ca²⁺), suggesting that the assembly kinetics and micromechanics of crescentin depend on the valence of the ions present in solution.

Conclusions/Significance

These results indicate that crescentin forms filamentous structures that are elastic, labile, and stiff, and that their low dissociation rate from established structures controls the slow remodeling of crescentin in C. crescentus.     

丙型肝炎病毒体外培养系统研究进展

由于缺少丙型肝炎病毒（HCV）的细胞培养系统和小动物模型，因此对其生活周期、作用机制至今仍不是很清楚，从而严重阻碍了丙型肝炎疫苗及相关治疗药物的开发与研制。一直以来，人们研究的HCV体外培养细胞模型包括感染模型和转染模型两种，感染模型由于原代肝细胞培养问题未能解决而难以成功，而转染模型的发展可喜。但是HCV复制子只能在极少数细胞中短暂复制，且产生的病毒量很低。1999年建立了亚基因组复制子，使人们有机会对其进行深入研究，但须人为引入碱基突变。最近建立的全基因复制子不需要引入突变即形成病毒粒子，是一项重大突破。概述了HCV体外培养系统的研究进展。     

A Cremophor-Free Self-Microemulsified Delivery System for Intravenous Injection of Teniposide: Evaluation In Vitro and In Vivo

In order to tackle the problems on low water solubility of teniposide, involvement of toxic surfactant in its injection, and the poor stability during infusion, a Cremophor-free teniposide self-microemulsified drug delivery system (TEN-SMEDDS) was prepared for the first time, characterized, and evaluated in comparison with teniposide injection (VUMON) in vitro and in vivo. The optimized formulation contained N, N-dimethylacetamide, medium-chain triglyceride, lecithin, and dehydrated alcohol besides teniposide. The TEN-SMEDDS could form fine droplets with mean diameter of 282 ± 21 nm and zeta potential of −7.5 ± 1.7 mV after dilution with 5% glucose, which were stable within 4 h. The release of teniposide from TEN-SMEDDS and VUMON was similar. However, the pharmacokinetic behavior of TEN-SMEDDS in rats was different from that of VUMON, evidenced by the lower area under the concentration–time curve and larger volume of distribution in emulsion group. Finally, TEN-SMEDDS was found to distribute more teniposide in most tissues, especially in reticuloendothelial system, after intravenous administration to rats. Importantly, brain drug level in TEN-SMEDDS group was higher than or similar to that in control group, although the emulsion system had a lower plasma drug concentration. In conclusion, the novel SMEDDS prepared here, without toxic surfactant and as an oil solution before use, may be potential for clinical use due to its low toxicity and high store stability. It may be favorable for the treatment of some tumors like cerebroma, since it may achieve the relatively higher drug level in brain but lower blood concentration.KEY WORDS: characterization, pharmacokinetics, self-microemulsified drug delivery system, teniposide, tissue distribution