Animal Tissue Cells Research Articles

An optimized protocol for isolation of soluble proteins from microalgae for two-dimensional gel electrophoresis analysis

Two-dimensional gel electrophoresis (2-DE)is a core proteomic technique to studyprotein expression and function in livingorganisms. Although it has been extensivelyused for investigation of bacterial, yeast,animal and plant tissue cells, there islimited information about the use of 2-DEin microalgal research. In this study, anumber of key chemical reagents, includingacetone, trichloroacetic acid, urea,thiourea, dithiothreitol, and tributylphosphine, were quantitatively evaluatedfor 2-DE of green microalgae, using Haematococcus pluvialis as a model system.The goal was to maximize the number andstaining intensity of protein spots whileminimizing streaking and smearing on thesecond dimensional SDS gel. Compared tonon-frozen immobilized pH gradients (IPG)strips, freezing of the IPG strips at –20 °C after isoelectric focusing (IEF)enhanced protein resolubilization andtransfer into the SDS gel, and thusimproved resolution while eliminatingvertical point streaking on the SDS gel. Itwas also confirmed that manipulation ofsample loading capacity is a simple,effective purification strategy forselective investigation of the proteins ofinterest and of varying abundances. Theprotocol was also successfully applied toprofiling protein expression in H.pluvialis under external stressconditions, indicating its potentialusefulness in further proteomics studies ofthis organism and related species.

Construction of Bacterial Artificial Chromosome (BAC/PAC) Libraries

Large-insert genomic libraries are necessary for physical mapping of large chromosomal regions, for isolation of complete genes, and for use as intermediates in DNA sequencing of entire genomes. Construction of BAC and PAC libraries is detailed in the unit, including preparation of PAC or BAC vector DNA for cloning by digestion with BamHI or EcoRI, dephosphorylation with alkaline phosphatase, and purification through pulsed-field gel electrophoresis (PFGE). For the preparation of high-molecular weight DNA for cloning, procedures for embedding total genomic DNA from lymphocytes or animal tissue cells are also provided. Other protocols detail partial digestion of genomic DNA with MboI or with a combination of EcoRI endonuclease and EcoRI methylase (including methods for optimizing the extent of digestion), and subsequent size fractionation by preparative PFGE. Finally, the isolation of BAC and PAC plasmid DNA for analyzing clones is also presented.

Endogenous Fluorescence Identifies Dead Cells In Plants

Abstract Various fluorescent stains and vital dyes have been used to identify dead cells in animal tissues and celi lines. In plants, fluorescein diacetate and propidium iodide have been used to label nuclei and to identify necrotic cells in plant protoplasts and 4,6-diamidino-2-phenylindole (DAPI) has been used to mark senescing cells in sections of roots. However, these dyes may be problematic when used with intact plant tissue with well-developed cells walls which may impede dye penetration. Endogenous fluorescence has been used to identify dead cells in intact and sectioned plant tissues. Published procedures typically employ ultraviolet (UV) excitation wavelengths of 340-380 nm and emission wavelengths of 400- 425 nm, thus requiring a UV filter set.

Luminometric Quantitation of Photinus pyralis Firefly Luciferase and Escherichia coli β-Galactosidase in Blood-Contaminated Organ Lysates

Firefly luciferase and Escherichia coli β-galactosidase chemiluminescent reporter gene assays are rapid and sensitive means of detecting reporter enzyme activities in cell lysates of both eukaryotic and prokaryotic systems. In these assays, expression vectors containing the luciferase or β-galactosidase genes are transferred to cells in culture or animal tissues in vivo. Crude cell or organ lysates are then prepared and submitted to enzyme assays. The level of enzyme activity is proportional to the efficiency of gene delivery and expression. When used with modified substrates that emit light when cleaved by the appropriate enzyme, luciferase and β-galactosidase activity can be detected luminometrically. Attempts to apply these assays to cell lysates contaminated with blood, as from any whole organ lysate, have had questionable results thus far because of light absorption by hemoglobin in the ranges of light emission by both of these assays. We have made several adjustments to standard chemiluminescent reporter gene assay protocols to minimize errors in quantitation contributed by hemoglobin. To this end, we have developed a method for quantitating the protein due to blood and due to the organ itself in a blood-contaminated organ lysate. We have also found that the use of a colorimetric protein assay that is unaffected by hemoglobin absorbance is preferred for protein quantitation. In conclusion, luciferase and β-galactosidase assays can be applied to blood-contaminated organ lysates; however, the luciferase assay proved to be superior due to minimal endogenous activity and lower absorption by hemoglobin of light emitted by the enzyme product.

Construction of Bacterial Artificial Chromosome (BAC/PAC) Libraries

This unit describes the construction of BAC and PAC libraries. Two vectors, pCYPAC2 and pPAC4 have been used for preparing PAC libraries, and a new BAC vector pBACe3.6 has been developed for construction of BAC libraries. A support protocol describes preparation of PAC or BAC vector DNA for cloning by digestion with BamHI or EcoRI, simultaneous dephosphorylation with alkaline phosphatase, and subsequent purification through pulsed-field gel electrophoresis (PFGE). For the preparation of high-molecular weight DNA for cloning, support protocols provide procedures for embedding total genomic DNA from lymphocytes or animal tissue cells, respectively, in InCert agarose. Another support protocol details the next steps for the genomic DNA: partial digestion with MboI or with a combination of EcoRI endonuclease and EcoRI methylase, and subsequent size fractionation by preparative PFGE. The final support protocol covers the isolation of BAC and PAC plasmid DNA for analyzing clones.

ヒアルロン酸の生物学的活性を考える

Hyaluronan (HA) is an unbranched glycosaminoglycan (GAG) which is the only nonsulfated disaccharide units of D-glucuronic acid and N-acetyl-D-glucosamine. HA is synthesized by most cells in various animal tissues and has by far the largest molecular size, with an average molecular weight of several million. HA plays important roles in tissue development, tissue hydration, cell surface-matrix interactions, cell migration, inflammation, and wound healing. The biosynthesis of HA is unique among the GAG. Rather than being made in the Golgi apparatus, HA is synthesized via HA synthase (HAS) which is located at the plasma membrane. Recently, it has been reported that 3 subtypes of HAS, named HAS 1, HAS 2 and HAS 3, have been cloned and that each of those plays a different role in HA production in vivo. Several lines of evidence demonstrated that expression of HAS and synthesis of HA are regulated by a variety of mechanisms other than cytokines and that the inducible HA production generates HA with relatively high molecular weight. Furthermore, recent observation demonstrated that HA with low molecular weight showed various biological activities such as induction of chemokines and iNOS. These data may suggest that HA plays crucial roles both in continuation of inflammatory responses and in restoration and maintenance of tissue homeostasis.

Comparative Quantitative Chromatographic Determination of Formaldehyde in Different Groups of Physiological and Pathological Hard Tissues of Teeth

Taking into consideration that HCHO level in cells of plant, animal and human tissues as well as in body fluids depends from physiological state of an organism in the current study it was decided to find out if there are changes of HCHO level in different physiological and pathological hard tissues of teeth. The obtained results showed in all 4 groups of teeth separately analysed that there were some regularities in the level of HCHO as far as similar physiological or pathological states are concerned. This was best seen when comparing the obtained results with mean HCHO level of the studied groups of teeth.

Dielectric phenomenon of living matter

Surges of current in the polarization and depolarization curves for living tissue refer to trapped charges on domain boundaries within the cytoplasm or on cell membranes. An electric field is shown to provoke an elastic deformation of living cells with a relaxation nature. The diagram of a cell with domains of neutral-normal and polarized-excited states is described. An electromechanical model with an inertial element is used to describe the cooperative correlation between domains. A new technique of voltage-stimulated polarization in response to a ramp voltage enables the assessment of the visco-elastic characteristics of animal tissue cells. It reveals the nature of fine liquid crystalline domains of hierarchical structures in cytoplasm and cell membranes with respective threshold voltages that initiate their discrete polarizations. The positive increment of the permittivity spectra in the range of 20 to 10/sup 6/ Hz of intact leaves differs from those exhibiting relaxation behavior, and is shown to be anomalous. Voltages above membrane thresholds on fruit tissue samples cause membrane disruption and this is studied as an electrical breakdown process. The extreme accuracy of utilized methods elucidate otherwise intangible intracellular morphological changes, including cell transformations caused by chemical and thermal treatments. Dielectric methods are shown as the least invasive, compared with traditional biomedical examinations employing mostly histochemical treatments which alter the structure, at least, by artefacts of space charges of the treatment medium.

Gap junctions revealed by freeze-fracture electron microscopy.

Gap junctions provide the basis for the formation of elaborate networks of communication between cells in animal tissues. Electron microscopic examination of thin sections of plastic embedded gap junctions has provided valuable information on the anatomy and function of these remarkable structures. Freeze-fracture electron microscopy, however, has made available unique vistas of gap junction-bearing intramembrane surface--surface previously inaccessible to the researcher's eyes. Data on population density, distribution, size, geometry of intramembrane particle packing, and structural responses of gap junction components to experimental manipulation are simply and easily obtained with freeze fracture. Recent developments of sophisticated protocols of immunocytochemistry as applied to freeze-fracture replicas further serve to reinforce the notion that freeze-fracture is a powerful tool for study of gap junctions. Molecular techniques of gap junction gene transfection promise to add a truly unique dimension to investigations of the broad spectrum of functional roles of gap junctions.

Methods forin VivoTissue Electroporation Using Surface Electrodes

AbstractElectroporation of tissues has many potential biomedical applications, including transdermal and targeted drug delivery. Although there are established protocols for electroporation of single cells, electroporation of tissues remains largely unexplored. Here we describe methods for in vivo tissue electroporation with surface electrodes, where either (a) molecules are provided at the outer surface of skin and electroporation is caused in order to transport molecules across the skin or (b) molecules are injected into an animal and internal tissue cells are electroporated to enhance up-take of the injected molecules. Factors considered in our experimental design include selection of electrode material and electrode/tissue geometry. Electrode materials were used which could accommodate a high instantaneous current density and were expected not to form harmful electrochemical products. Due to the complex heterogeneous nature of tissues, the choice of electrical pulse parameters was guided by, but not b...

A cell surface-associated centrosomal layer of microtubule-organizing material in the inner pillar cell of the mouse cochlea.

This investigation provides evidence that pericentriolar material is divorced from the immediate vicinities of centrioles and becomes functionally associated with the plasmalemma during the differentiation of a mammalian cell type. Such events occur prior to the assembly of large transcellular microtubule bundles in columnar epithelial cells called inner pillar cells in the mouse organ of Corti. The microtubules do not radiate from a typical centrosome and its centrioles. They elongate from a microtubule-organizing centre (MTOC), which is deployed as a subapical cell surface-associated layer in each cell. Most of the dense material of this layer, and the tops of most of the microtubules, are initially concentrated around the sides of a cell about 1 microns below its apical surface. In addition, a pair of centrioles is located above the layer, which acts as if it is a pericellular concentration of the pericentriolar material of a modified centrosome. Although microtubule nucleation takes place in a centrosome-like region, 13 protofilament fidelity is not exercised. Most of the microtubules have 15 protofilaments. Microtubule assembly progresses in these cells after the organ of Corti has been isolated for in vitro culture. However, large numbers of microtubules elongate from pericentriolar material juxtaposed against the centrioles. Hence, there is some reversion by the centrosomes of cultured cells to the operational configuration regarded as typical for animal tissue cells in general.

Analysis of inducers of theE.coli lacrepressor system mammalian cells and whole animals

Although the inducible prokaryotic lac repressor system has been successfully adapted for control of gene expression in mammalian cells, little information is available on the pharmacokinetics of beta-galactoside inducers in mammalian cells for optimizing this system. These studies directly measure the cell uptake and clearance in cultured cells and animal tissue cells of lac inducers. In these cells, the beta-galactosides, isopropyl beta-D-thiogalactoside (IPTG) and methyl beta-D-thiogalactoside (MTG), are rapidly taken up, exceeding extracellular levels in less than 2 hours. Greater than 5% of this inducer is found in the nuclear fraction, slightly exceeding the cytoplasmic concentration. Although similar in uptake, IPTG is cleared from the cultured cells significantly faster than MTG. In the mouse, the half-life of both inducers in the blood ranges from 15-30 minutes. HPLC analysis of tissue extracts from inducer-injected mice indicates that the inducer is metabolically stable and functionally able to bind to lac repressor. These results should permit improvement in the adaptation of the lac repressor system to mammalian cells and aid in the development of an adaptable system for gene control in transgenic animals.

The Plasma Membrane of Myxosporidian Valve Cells: Freeze Fracture Data

Freeze fracturing of Myxosporidian spores reveals the occurrence of a continuous layer of transmembrane particles all over the surface area of the valve cells which form the spore envelope. These particles are densely packed all over the P face membrane. Due to their polygonal outline, their diameter (6-7 nm) and their central core, they resemble the particles forming the connections of gap junctions which metabolically couple the neighboring cells in animal tissues. In the present report, the role of the transmembrane particles is still hypothetical. However, they might represent a membrane structural specialization of the spores which are submitted to osmotic variations of the fluid external medium. Furthermore similar transmembrane particles are observed at the level of the septate junction which seals the valve cells. In this occurrence, they are arranged in a series of 40 double rows parallel to the suture of the spore envelope. These findings support the view that Myxosporidia are Metazoa and raise the problem of their origin.

Белковый состав ядерного матрикса в клетках различных тканей животных

Белковый состав ядерного матрикса в клетках различных тканей животных

Early and late damages induced by heavy charged particle irradiation in embryonic tissue of Arabidopsis seeds

Early and late effects of accelerated heavy ions (HZE) on the embryonic tissue of Arabidopsis thaliana seeds were investigated seeing that initial cells of the plant eumeristems resemble the original cells of animal and human tissues with continuous cell proliferation. The endpoints measured were lethality and tumorization in the M 1-generation for early effects and embryonic lethality in the M 2-generation for late effects. The biological endpoints are plotted as functions of the physical parameters of the irradiation i.e. ion fluence (p/cm 2), dose (Gray), charge Z and l inear e nergy t ransfer (LET). The results presented contribute to the estimation of the principles of biological HZE effects and thus may help to develop a unified theory which could explain the whole sequence from physical and chemical reactions to biological responses connected with heavy ion radiation. Additionally, the data of this paper may be used for the discussion of the quality factor for heavy ion irradiation needed for space missions and for HZE-application in radio-therapy by use of accelerators (UNILAC, (SIS/ESR), BEVALAC).

Potential and limitations of cultivated fibroblasts in the study of senescence in animals. A review on the murine skin fibroblasts system

Senescence is the last period of the life span, leading to death. It happens in all animals, with the exception of a few didermic species (Hydras) having a stock of embryonic cells and being immortal. The causes of animal senescence are badly known. They depend both on genetic characters (maximum life span of a species) and on medium factors (mean expectation of life of the animals of a species). Animal senescence could depend on cell aging: (1) by senescence and death of the differentiated cells. (2) by modified proliferation of the stem cells of differentiated tissues, (3) by alterations in the extracellular matrices, (4) by interactions between factors (1) (2) and (3) in each tissue, and (5) by interactions between the several tissues of an organism. This complexity badly impedes the experimental study of animal senescence. Normal mammal cells are aging when they are cultivated (in vitro aging). Present literature upon in vitro aging of cultivated human fibroblasts consists essentially of papers devoted to proliferation and differentiation characteristics and not to cell senescence. Murine skin fibroblasts have been studied in our laboratory, using different systems: (1) primary cultures isolated from peeled skins of mouse embryos, (2) mouse derms analysed in the animals, (3) cultivated explants of skins, (4) serial sub-cultures of fibroblasts isolated from these explants, (5) cells cultivated comparably on plane substrates (glass, plastic, collagen films) and on three-dimensional matrices (collagen fibres). In primary cultures (system 1) all the cell generations have been analysed, including the last one until death of the culture. We have shown that many characters are varying with cell generation. All the observed variations were: progressive, non-linear and correlated (intracellular feedbacks). We come to the conclusion that the main effects of cell mitotic age are (1) to depress the plasticity of the chromatin, (2) to change the organization of the cytoplasmic filaments, (3) to change the organization of the extracellular matrix. The collagen fibres are also acting upon nucleus and filaments either in the animals or in the cultures. The phenotype of a fibroblastic cell is thus both age- and environment-dependent. Overall data on in vitro cell aging point to the hypothesis that senescent cells are phenotypic variants and not mutant cells. Aging cell cultures are remarkably useful to the studies on cell proliferation decrease and cell cycle lengthening shown by the stem cells in animal tissues. We propose the hypothesis that the fibroblasts of the vertebrates would be homologous to the pluripotent mesenchyme cells of their embryos.

Ultrastructural localization of glucoside residues on tissue sections by applying the enzyme-gold approach.

The enzyme-gold approach was applied for ultrastructural localization of glucoside residues in animal and plant tissues. A beta-glucosidase-gold complex was prepared and used on thin tissue sections to reveal the corresponding substrate molecules by electron microscopy. Conditions for preparation of the complex, as well as for its application, were determined. Once applied on thin tissue sections, the glucosidase-gold complex yielded labeling over the rough endoplasmic reticulum, mainly on the ribosomal side of the membranes, and over the dense chromatin in the nucleus. Mitochondria, Golgi apparatus, and secretory granules in liver and pancreatic cells were free of gold particles. In plant cells, the labeling pattern was similar. In addition, the stroma regions of chloroplasts were densely labeled. In the extracellular space, labeling was found over the basal laminae of cells in animal tissues and over the fibrillar wall material bordering the intercellular space in plant tissues. Fungal cell cytoplasm was also labeled, as well as the membrane delineating mycoplasma-like organisms. Control conditions confirmed these labelings, demonstrating the possibility of revealing glucoside residues on tissue sections with high resolution and specificity.

Topographical control of cell behaviour. I. Simple step cues.

The photolithographic techniques of the microelectronics industry have allowed us to fabricate patterned plastic substrata to investigate contact guidance of animal tissue cells. The reactions of cells to single steps on a substratum were examined using time-lapse videorecording and scanning electron microscopy. BHK cells and chick embryonic neural cell processes exhibited gradual inhibition of crossing steps with a concomitant increase in alignment at steps dependent on increasing step height. Comparison of these cells' reactions, with those of chick heart fibroblasts and rabbit neutrophils, at a 5 micron step revealed that the influence of topography is also dependent on cell type, the neutrophils being relatively unaffected. The presence of an adhesive difference at a series of steps altered BHK cells' reactions such that the frequency of crossing was dependent on the direction of approach to a step. Although our data are consistent with Dunn & Heath's proposal (1976) that the inflexibility of the cytoskeleton of a moving cell's protrusion is the cellular property determining such reactions to topography, we have found that, on encountering a topographical feature, the response of a cell may be predictable on a probabilistic basis, i.e. the topographical feature reduces the probability of a cell making a successful protrusion and contact in a given direction, that even the largest features tested did not act as absolute barriers to cell locomotion since a small proportion of a population of cells were able to overcome them, and that other guidance cues could significantly alter a cell's response. Even in situations where it is not the primary cue in directing cell locomotion, topographical control may be an important factor during morphogenesis since it must, at the very least, influence the efficiency of other cues.

Functional reconstitution of lens junction proteins into proteoliposomes

Many cells in animal tissues are connected together through “communicative junctions”, such as gap junctions, and protein oligomers (sometimes called connexons) with an apparent central channel have been isolated from the regions of liver plasma membrane enriched in such junctions. Although ingenious approaches such as dye injections have been used to characterize the properties of the channel, to our knowledge there has been no report of direct reconstitution studies of the type widely used with bacterial channelforming protein, porin, or with its analog from the mitochondrial outer membrane, mitochondrial porin.More recently, membranes from lens fiber have been studied intensively, because they contain a large abundance of one protein, which appears to be the subunit of intercellular junctions. However, the subunits of the lens junction is different from those of liver gap junctions in its molecular weight, peptide map pattern, and immunological specificity.

The Functional Integration of Cells in Animal Tissues.John D. Pitts , Malcolm E. Finbow

Previous articleNext article No AccessNew Biological BooksThe Functional Integration of Cells in Animal Tissues. John D. Pitts , Malcolm E. Finbow William J. LarsenWilliam J. Larsen Search for more articles by this author PDFPDF PLUS Add to favoritesDownload CitationTrack CitationsPermissionsReprints Share onFacebookTwitterLinkedInRedditEmail SectionsMoreDetailsFiguresReferencesCited by The Quarterly Review of Biology Volume 58, Number 3Sep., 1983 Published in association with Stony Brook University Article DOIhttps://doi.org/10.1086/413417 Views: 1Total views on this site Copyright 1983 Stony Brook Foundation, Inc.PDF download Crossref reports no articles citing this article.