Successful implantation of in vitro cultured rabbit embryos after uterine transfer: A role for mucin

The functional role of the mucin layer for development of rabbit embryos was examined by uterine transfer of embryos with different thicknesses of mucin. Embryos collected at various intervals after human chorionic gonadotropin (hCG) injection were cultured until 90 hr post-coitum (p.c.) and transferred to the uterus of synchronized recipients. When embryos collected at 20 or 25 hr p.c. were used for transfer, no implantation occurred. By contrast, embryos collected at 35 or 40 hr p.c. developed to term at high rates (53 and 80%, respectively). The thickness of the mucin layer on the embryos was different between these two groups. Embryos collected before 25 hr p.c. have less than 11.2 ± 0.2 μm of thickness of mucin and embryos collected after 35 hr p.c. have more than 34.3 ± 5.5 μm. To examine whether mucin deposition is required for in vitro cultured rabbit blastocysts to continue development after uterine transfer, embryos were collected at 20 hr p.c., cultured for 60 or 70 hr in vitro, and then temporarily transferred to the oviducts of recipient does to add mucin. These embryos were recovered from the oviducts at 24 hr after transfer, classified according to the thickness of mucin deposition, and transferred again to the uterus of synchronized recipients. Twenty live young were obtained from 67 embryos with a 20–40 μm thick mucin layer. No live young were obtained from 57 embryos with less than a 20 μm thick mucin. The thickness of the mucin layer appears to be an important factor for successful implantation of rabbit embryos. © 1996 Wiley-Liss, Inc.     

Stereoselective metabolism of two keto-pyrrol analogues of 8-hydroxy-2-dipropyl-aminotetralin by freshly isolated rat hepatocytes

In vitro metabolism models have been used to determine the relative metabolic stability of novel 2-aminotetralin analogues for the treatment of CNS diseases. Few of these new compounds had been produced as stereochemically pure materials and the achiral analytical techniques, used initially, measured the average metabolic clearance of the two enantiomers of the racemic mixtures. A chiral HPLC assay, using a Chiral AGP column, was developed for two of these racemic analogues and was used to measure the clearance of the enantiomers from suspensions of freshly isolated rat hepatocytes. Robust separations were obtained for both compounds and a number of metabolic products. The enantiomers of only one analogue were subject to different rates of metabolism. The extent of the difference was dependent upon the initial starting concentration of the incubation. The identity of certain metabolites was investigated using LC/MS. The enantioselectivity appears to have arisen from the restricted hydroxylation of one analogue compared to that of the other. © 1996 Wiley-Liss, Inc.     

Effect of continuous-wave and amplitude-modulated 2.45 GHz microwave radiation on the liver and brain aminoacyl-transfer RNA synthetases of in utero exposed mice

Investigations have been carried out concerning the effects of microwave (MW) exposure on the aminoacyl-transfer ribonucleic acid (tRNA) synthetase of the progeny of females that were exposed during their entire period of gestation (19 days). The changes caused by continuous-wave (CW) and amplitude-modulated (AM) MW radiation have been compared. CFLP mice were exposed to MW radiation for 100 min each day in an anechoic room. The MW frequency was 2.45 GHz, and the amplitude modulation had a 50 Hz rectangular waveform (on/off ratio, 50/50%). The average power density exposure was 3 mW/cm², and the whole body specific absorption rate (SAR) was 4.23 ± 0.63 W/kg. The weight and mortality of the progeny were followed until postnatal day 24. Aminoacyl-tRNA synthetase enzymes and tRNA from the brains and livers of the offspring (461 exposed, 487 control) were isolated. The aminoacyl-tRNA synthetase activities were determined. The postnatal increase of body weight and organ weight was not influenced by the prenatal MW radiation. The activity of enzyme isolated from the brain showed a significant decrease after CW MW exposure, but the changes were not significant after 50 Hz AM MW exposure. The activity of the enzyme isolated from liver increased under CW and 50 Hz modulated MW. © 1996 Wiley-Liss, Inc.     

谷子茎尖体外遗传转化体系的建立与优化

以谷子(Seteria italica)豫谷一号为实验材料,建立了一套简便、稳定的体外茎尖遗传转化体系。通过根癌农杆菌(Agrobacterium tumefaciens)介导的茎尖转化法,对转化受体采取不同的处理方式,待拟转化株长到三叶期后进行PCR鉴定。探明了草丁膦(Basta)喷施处理用于谷子转基因幼苗筛选的最适...     

植物小RNA荧光原位杂交实验方法

小RNA是对植物生长发育十分重要的一类小分子核苷酸,在多种生命过程以及胁迫响应中发挥重要调控作用。对小RNA的定位研究有助于揭示它们的功能,而小RNA荧光原位杂交(sRNA-FISH)是一种通过荧光检测技术对生物体内小RNA进行定性或半定量分析的技术,目前该技术已经在动物体内被广泛应用,而在植物体内的应用还比较少。该文...     

Chemogenomic Approaches for Revealing Drug Target Interactions in Drug Discovery

The drug discovery process has been a crucial and cost-intensive process. This cost is not only monetary but also involves risks, time, and labour that are incurred while introducing a drug in the market. In order to reduce this cost and the risks associated with the drugs that may result in severe side effects, the in silico methods have gained popularity in recent years. These methods have had a significant impact on not only drug discovery but also the related areas such as drug repositioning, drug-target interaction prediction, drug side effect prediction, personalised medicine, etc. Amongst these research areas predicting interactions between drugs and targets forms the basis for drug discovery. The availability of big data in the form of bioinformatics, genetic databases, along with computational methods, have further supported data-driven decision-making. The results obtained through these methods may be further validated using in vitro or in vivo experiments. This validation step can further justify the predictions resulting from in silico approaches, further increasing the accuracy of the overall result in subsequent stages. A variety of approaches are used in predicting drug-target interactions, including ligand-based, molecular docking based and chemogenomic-based approaches. This paper discusses the chemogenomic methods, considering drug target interaction as a classification problem on whether or not an interaction between a particular drug and target would serve as a basis for understanding drug discovery/drug repositioning. We present the advantages and disadvantages associated with their application.     

Tick tock,tick tock: Mouse culture and tissue aging captured by an epigenetic clock

Aging is associated with dramatic changes to DNA methylation (DNAm), although the causes and consequences of such alterations are unknown. Our ability to experimentally uncover mechanisms of epigenetic aging will be greatly enhanced by our ability to study and manipulate these changes using in vitro models. However, it remains unclear whether the changes elicited by cells in culture can serve as a model of what is observed in aging tissues in vivo. To test this, we serially passaged mouse embryonic fibroblasts (MEFs) and assessed changes in DNAm at each time point via reduced representation bisulfite sequencing. By developing a measure that tracked cellular aging in vitro, we tested whether it tracked physiological aging in various mouse tissues and whether anti‐aging interventions modulate this measure. Our measure, termed CultureAGE, was shown to strongly increase with age when examined in multiple tissues (liver, lung, kidney, blood, and adipose). As a control, we confirmed that the measure was not a marker of cellular senescence, suggesting that it reflects a distinct yet progressive cellular aging phenomena that can be induced in vitro. Furthermore, we demonstrated slower epigenetic aging in animals undergoing caloric restriction and a resetting of our measure in lung and kidney fibroblasts when re‐programmed to iPSCs. Enrichment and clustering analysis implicated EED and Polycomb group (PcG) factors as potentially important chromatin regulators in translational culture aging phenotypes. Overall, this study supports the concept that physiologically relevant aging changes can be induced in vitro and used to uncover mechanistic insights into epigenetic aging.     

Tissue engineering strategies to bioengineer the ageing skin phenotype in vitro

Human skin ageing is a complex and heterogeneous process, which is influenced by genetically determined intrinsic factors and accelerated by cumulative exposure to extrinsic stressors. In the current world ageing demographic, there is a requirement for a bioengineered ageing skin model, to further the understanding of the intricate molecular mechanisms of skin ageing, and provide a distinct and biologically relevant platform for testing actives and formulations. There have been many recent advances in the development of skin models that recapitulate aspects of the ageing phenotype in vitro. This review encompasses the features of skin ageing, the molecular mechanisms that drive the ageing phenotype, and tissue engineering strategies that have been utilised to bioengineer ageing skin in vitro.