Arginine-rich intracellular delivery peptides noncovalently transport protein into living cells

Plasma membranes of plant or animal cells are generally impermeable to peptides or proteins. Many basic peptides have previously been investigated and covalently cross-linked with cargoes for cellular internalization. In the current study, we demonstrate that arginine-rich intracellular delivery (AID) peptides are able to deliver fluorescent proteins or beta-galactosidase enzyme into animal and plant cells, as well as animal tissue. Cellular internalization and transdermal delivery of protein could be mediated by effective and nontoxic AID peptides in a neither fusion protein nor conjugation fashion. Therefore, noncovalent AID peptides may provide a useful strategy to have active proteins function in living cells and tissues in vivo.     

Multisurface Glass Roller Bottle for Growth of Animal Cells in Culture

A multisurface glass roller bottle has been constructed for the growth of animal cells in culture. This bottle contains five concentrically placed glass cylinders that provide additional surfaces for the growth of animal cells. The bottle occupies the same space as a standard roller bottle, but it contains nine times the surface area of a standard bottle. L and HeLa cells can be grown in the bottle with cell yields 5- to 10-fold greater than in a standard bottle. L cells can be induced to produce interferon in the multisurface bottle.     

Basement membrane remodeling guides cell migration and cell morphogenesis during development

Basement membranes (BMs) are thin, dense forms of extracellular matrix that underlie or surround most animal tissues. BMs are enormously complex and harbor numerous proteins that provide essential signaling, mechanical, and barrier support for tissues during their development and normal functioning. As BMs are found throughout animal tissues, cells frequently migrate, change shape, and extend processes along BMs. Although sometimes used only as passive surfaces by cells, studies in developmental contexts are finding that BMs are often actively modified to help guide cell motility and cell morphogenesis. Here, I provide an overview of recent work revealing how BMs are remodeled in remarkably diverse ways to direct cell migration, cell orientation, axon guidance, and dendrite branching events during animal development.     

Generation of rat induced pluripotent stem cells: the analysis of reprogramming and culturing media

The rat represents very important, superior in many respects to the mous, animal model for studying pharmacology, physiology, ageing, cardiovascular etc. However, numerous attempts to derive rat ES cells necessary to carry out loss-of-gene-function studies have not been successful thus far. Therefore rat induct pluripotent stem cells (or riPS) should provide a notable alternative to ES cell, allowing to study gene functions in this valuable animal model. Here we report an improved lentivirus-based riPS derivation protocol that makes use of small inhibitors of MEK and GSK3. We show that the excision of proviruses does not affect neither karyotype and pluripotency state of these cells. Also, we propose genetic tool for an improvement of the quality of riPS cells in culture. These data may prompt further iPS-based gene targeting in rat as well as the development iPS-based gene therapies, using this animal model.     

供体细胞与哺乳动物体细胞核移植

哺乳动物体细胞核移植(克隆)技术在转基因动物生产、珍稀动物资源复原与保护、生物学基础研究等方面业已显示出重要的应用价值,而目前该技术还与诱导多能干细胞技术一同被认为是创制患者特异性多能干细胞,为再生医学临床"细胞治疗"提供素材的最佳手段。但是,体细胞克隆的效率仍不理想,关键机制还不清楚,严重制约了该技术的推广。因此,如何提高克隆效率已成为人们普遍关心的首要问题。在体细胞克隆技术所涉及的各环节中,供体细胞是影响克隆效率的最关键因素之一。该文从供体细胞的生物学因素和技术因素两方面进行了回顾,旨在为进一步探寻建立物种或供体细胞个性化准备方案,为提高动物克隆效率提供参考。     

Insulin acts as a myogenic differentiation signal for neural stem cells with multilineage differentiation potential

Reports of non-neural differentiation of neural stem cells (NSCs) have been challenged by alternative explanations for expanded differentiation potentials. In an attempt to demonstrate the plasticity of NSC, neurospheres were generated from single retrovirally labeled embryonic cortical precursors. In a defined serum-free insulin-containing media, 40% of the neurospheres contained both myogenic and neurogenic differentiated progeny. The number of NSCs displaying multilineage differentiation potential declines through gestation but does exist in the adult animal. In this system, insulin appears to function as a survival and dose-dependent myogenic differentiation signal for multilineage NSCs (MLNSC). MLNSC-derived cardiomyocytes contract synchronously, respond to sympathetic and parasympathetic stimulation, and regenerate injured heart tissues. These studies provide support for the hypothesis that MLNSCs exist throughout the lifetime of the animal, and potentially provide a population of stem cells for cell-based regenerative medicine strategies inside and outside of the nervous system.     

Bovine mature adipocytes readily return to a proliferative state

The dynamics of human and animal adipogenesis has been defined using several traditional cell systems including stromal vascular cells and adipocyte-related cell lines. But a relatively new cell system using progeny cells stemming from the dedifferentiation of purified cultures of mature adipocytes may be used for studying the development and biology of adipocytes. In this research, we show that isolated (and purified) mature adipocytes derived from Wagyu cattle dedifferentiate into progeny cells, and that these spindle-shaped, proliferative-competent daughter cells possess ability to proliferate. We outline the optimum cell culture system and offer precautionary thoughts for effective mature adipocyte culture. Collectively, this represents a novel cell model which may provide new insights into cell development, physiology and use as a model for animal production/composition, tissue engineering and disease treatment.     

Myosin V and the endoplasmic reticulum: the connection grows

In this issue, Estrada et al. (2003) provide new and important insights into how the endoplasmic reticulum (ER) of budding yeast cells is inherited. Together with other studies in plant and animal cells, the results of Estrada et al. (2003) support the idea that myosin V acts as a universal motor for the transport of ER membranes.     

A selection system for unequal crossing-over: Plasmid construction and initial studies in Escherichia coli

Unequal crossing-over is involved in genetic duplication and deletion in such diverse genetic systems as Drosophila, bacteria, and animal viruses. It is proposed to be involved in the form of unequal sister chromatid exchange in gene amplification in cultured animal cells and during carcinogenesis. Studies of the process of unequal crossing-over have been hampered by the lack of genetic systems allowing specific selection for cells that have undergone such unequal crossing-over. We report here on the construction of plasmids designed to provide specific selection of unequal crossing-over. One such plasmid was studied in Escherichia coli. We show that kanamycin resistance is generated, as predicted, by the expected unequal crossover event.     

Continuous Attractor Network Model for Conjunctive Position-by-Velocity Tuning of Grid Cells

The spatial responses of many of the cells recorded in layer II of rodent medial entorhinal cortex (MEC) show a triangular grid pattern, which appears to provide an accurate population code for animal spatial position. In layer III, V and VI of the rat MEC, grid cells are also selective to head-direction and are modulated by the speed of the animal. Several putative mechanisms of grid-like maps were proposed, including attractor network dynamics, interactions with theta oscillations or single-unit mechanisms such as firing rate adaptation. In this paper, we present a new attractor network model that accounts for the conjunctive position-by-velocity selectivity of grid cells. Our network model is able to perform robust path integration even when the recurrent connections are subject to random perturbations.     

Replication properties of human adenovirus in vivo and in cultures of primary cells from different animal species

Oncolytic adenoviruses have emerged as a promising approach for the treatment of tumors resistant to other treatment modalities. However, preclinical safety studies are hampered by the lack of a permissive nonhuman host. Screening of a panel of primary cell cultures from seven different animal species revealed that porcine cells support productive replication of human adenovirus type 5 (Ad5) nearly as efficiently as human A549 cells, while release of infectious virus by cells from other animal species tested was diminished by several orders of magnitude. Restriction of productive Ad5 replication in rodent and rabbit cells seems to act primarily at a postentry step. Replication efficiency of adenoviral vectors harboring different E1 deletions or mutations in porcine cells was similar to that in A549 cells. Side-by-side comparison of the viral load kinetics in blood of swine and mice injected with Ad5 or a replication-deficient adenoviral vector failed to provide clear evidence for virus replication in mice. In contrast, evidence suggests that adenovirus replication occurs in swine, since adenoviral late gene expression produced a 13.5-fold increase in viral load in an individual swine from day 3 to day 7 and 100-fold increase in viral DNA levels in the Ad5-infected swine compared to the animal receiving a replication-deficient adenovirus. Lung histology of Ad5-infected swine revealed a severe interstitial pneumonia. Although the results in swine are based on a small number of animals and need to be confirmed, our data strongly suggest that infection of swine with human adenovirus or oncolytic adenoviral vectors is a more appropriate animal model to study adenoviral pathogenicity or pharmacodynamic and toxicity profiles of adenoviral vectors than infection of mice.     

Changes of the metabolism of the colon cancer cell line SW-480 under serum-free and serum-reduced growth conditions

Serum of animal origin, like foetal calf serum (FCS), is used as a standard supplement for media to cultivate mammalian cells, mostly due to its growth-supporting properties. Unfortunately, animal serum has many disadvantages like the risk of contamination, high costs, fluctuations within the composition of different batches and the high amount of foetuses, which have to be harvested. To avoid all this, it is necessary to provide alternatives, which combine as many positive properties of the animal serum as possible but do not influence the cellular metabolism negatively. Today, several serum-free complete media as well as serum substitutes are commercially available. In the present study, a serum substitute, a serum-reduced medium and a serum-free medium were evaluated concerning their influence on the metabolism on the colon cancer cell line SW-480. The evaluation of morphological changes of the cells was done by microscopic analysis whereas differences in the volatile metabolome were analysed by solid phase micro extraction (SPME) followed by gas chromatography/mass spectrometry (GC/MS).     

Pathophysiology of stroke and stroke-induced retinal ischemia: emerging role of stem cells

The current review focuses on pathophysiology, animal models and molecular analysis of stroke and retinal ischemia, and the role of stem cells in recovery of these disease conditions. Research findings associated with ischemic stroke and retinal ischemia have been discussed, and efforts towards prevention and limiting the recurrence of ischemic diseases, as well as emerging treatment possibilities with endothelial progenitor cells (EPCs) in ischemic diseases, are presented. Although most neurological diseases are still not completely understood and reliable treatment is lacking, animal models provide a major step in validating novel therapies. Stem cell approaches constitute an emerging form of cell-based therapy to treat ischemic diseases since it is an attractive source for regenerative therapy in the ischemic diseases. In this review, we highlight the advantages and limitations of this approach with a focus on key observations from preclinical animal studies and clinical trials. Further research, especially on treatment with EPCs is warranted.     

A unifying new model of cytokinesis for the dividing plant and animal cells

Cytokinesis ensures proper partitioning of the nucleocytoplasmic contents into two daughter cells. It has generally been thought that cytokinesis is accomplished differently in animals and plants because of the differences in the preparatory phases, into the centrosomal or acentrosomal nature of the process, the presence or absence of rigid cell walls, and on the basis of 'outside-in' or 'inside-out' mechanism. However, this long-standing paradigm needs further reevaluation based on new findings. Recent advances reveal that plant cells, similarly to animal cells, possess astral microtubules that regulate the cell division plane. Furthermore, endocytosis has been found to be important for cytokinesis in animal and plant cells: vesicles containing endocytosed cargo provide material for the cell plate formation in plants and for closure of the midbody channel in animals. Thus, although the preparatory phases of the cell division process differ between plant and animal cells, the later phases show similarities. We unify these findings in a model that suggests a conserved mode of cytokinesis.     

Manipulating the host to study bacterial virulence

The ability to manipulate animal hosts as well as bacterial pathogens greatly expands the utility of in vivo models of infection. For example, the construction of mice that harbor human tissues or express specific transgenes can provide ligand-receptor interactions that are essential for pathogenesis. Interactions between virulence factors and specific host defenses can sometimes be resolved by challenging selectively immuno deficient mice with bacteria containing virulence gene mutations. Transgenic animals expressing inducible reporters can be used to conveniently identify cells in which specific response pathways have been activated during infection. These and other approaches promise to improve the quality of information obtainable from in vivo assessments of pathogenesis.     

Searching for process information in the aroma of cell cultures

Aroma emissions from living cells can provide valuable information about the metabolic and physiological condition of those cells. Electronic noses are chemical gas-sensor arrays that use artificial neural network models to evaluate aromas. They can interpret the complex aroma information emitted from cultures of bacteria, yeast cells and animal cells. Potential applications for electronic noses range from medical diagnosis to industrial bioprocessing.     

Mass production of animal cells in nude mice with retention of cell specific markers.

Biochemical analysis of macromolecular constituents of somatic cells in culture frequently requires the production of large quantities of cells from small initial populations, often a laborious and expensive procedure. Here we show that nude mice, because of their genetic immune deficiency, provide a relatively simple and reliable means of growing large quantities of cells irrespective of the species or tissue of origin. Most established animal cell lines are capable of growing as large tumors in these athymic mice. The passage of mammalian cells in nude mice does not cause either the loss or modification of specific biochemical, chromosomal, antigenic or other cellular markers, nor the induction of malignant transformation of the host cells. Tumors formed by the injected cells grow as localized, encapsulated masses, and host cell admixture due to elements of the vascular system in the tumor is variable but not extensive. Pure cultures of the original cell type can easily be recovered from the tumors if they are derived from previously established cell lines. The use of nude mice for large scale growth of animal cells is particularly advantageous for cell lines which are not adapted to mass suspension culture for highly differentiated functional tumor cells which cannot be grown in vitro.     

Mitochondrial involvement in tracheary element programmed cell death

The mitochondria pathway is regarded as a central component of some types of programmed cell death (PCD) in animal cells where specific signals cause the release of cytochrome c from mitochondria to trigger a proteolytic cascade involving caspases. However, plant cells lack canonical caspases, therefore a role for the mitochondria in programmed cell death in plant cells is not obvious. Using plant cells which terminally differentiate, we provide evidence supporting the involvement of mitochondria in PCD, however the release of cytochrome c is insufficient to trigger the PCD. Prior to execution of cellular autolysis initiated by the rupture of the large central vacuole to release sequestered hydrolases, mitochondria adopt a definable morphology, the inner membrane depolarizes prior to death, and cytochrome c is released from mitochondria. However, PCD can be blocked despite translocation of cytochrome c. These results suggest a role for the mitochondria in this PCD but do not support the current animal model for a causative role of cytochrome c in triggering PCD.     

植物细胞中间纤维角蛋白性质的分析

角蛋白是植物细胞中间纤维的主要成分。应用选择性抽提和生物化学技术,分离纯化了豌豆根尖细胞58-、52 kD、白菜子叶52kD和胡萝卜悬浮细胞64kD角蛋白,测定了它们的氨基酸组成,结果表明上述角蛋白与动物细胞中间纤维角蛋白的氨基酸组成有较大的相似性。比较了动、植物细胞角蛋白的肽谱,结果显示它们之间存在较大的差异,但是植物细胞间角蛋白的肽谱比较一致,这提示它们属于同一蛋白家族,为植物中间纤维及其角蛋白的存在提供了新的论据。     

Transgenic mice expressing the diphtheria toxin receptor are sensitive to the toxin

Whereas diphtheria and the mechanism of action of diphtheria toxin, the bacterial molecule that induces the disease, have been studied and understood for some time, the receptor that allows animal cells to bind the toxin escaped identification until recently. The receptor was identified by its ability to confer toxin-sensitivity to mouse cells, which are normally toxin-resistant. Although mice are also naturally resistant, we now demonstrate that transgenic mice expressing the diphtheria toxin receptor are as sensitive to the toxin as are humans and other toxin-sensitive animals. These transgenic mice provide a suitable model for studying modern antidotes for diphtheria.