Differences in the frequency and disposition of plasmodesmata resulting from root cell elongation

Planta - Differences in plasmodesmatal organization and frequency between cells which have and have not undergone wall expansion, were studied in four plant species (Trifolium repens L., Raphanus...     

Using fluorescence for improvement of the quantitative analysis of micronucleus in cell culture

The micronucleus test is of unquestionable importance, being widely used in studies with several purposes. Standardization of the technique has been proposed by several groups mainly when in vivo assays are performed. In cell cultures the staining method that predominates is the Feulgen's reaction, since it is specific for DNA. In this work, we evaluated the use of two fluorescent stains, SYTOX green and propidium iodide, in substitution for the Feulgen's reaction. Based on the results we concluded that the fluorescence microscopy allows the reliable detection of micronuclei, presenting the following advantages: the technique is easy and fast to perform, it avoids acid treatments; the fluorescence is long lasting; micronucleus counting becomes easier and more reliable by the use of RNAase treatment; the technique is more sensitive to detect the smallest micronuclei.     

Using fluorescence for improvement of the quantitative analysis of micronucleus in cell culture

The micronucleus test is of unquestionable importance, being widely used in studies with several purposes. Standardization of the technique has been proposed by several groups mainly when in vivo assays are performed. In cell cultures the staining method that predominates is the Feulgen's reaction, since it is specific for DNA. In this work, we evaluated the use of two fluorescent stains, SYTOX green^® and propidium iodide, in substitution for the Feulgen's reaction. Based on the results we concluded that the fluorescence microscopy allows the reliable detection of micronuclei, presenting the following advantages: the technique is easy and fast to perform, it avoids acid treatments; the fluorescence is long lasting; micronucleus counting becomes easier and more reliable by the use of RNAase treatment; the technique is more sensitive to detect the smallest micronuclei.     

A case of elevated spontaneous micronucleus frequency derived from chromosome 2

This work tested the hypothesis that the content of spontaneous micronuclei in lymphocytes in an apparently healthy normal human subject, who exhibited an unusually high micronucleus frequency, was non-random. Several DNA probes were used in fluorescent in-situ hybridization (FISH), beginning with a probe generated from the subject's micronuclei. Micronuclei obtained from peripheral blood lymphocytes by microdissection were subjected to random amplification of polymorphic DNA (RAPD-PCR), and a unique PCR product was then used to isolate a cosmid clone from a human genomic library. This clone hybridized to chromosome 2. Subsequently, commercial probes were included in FISH analyses of micronuclei from the subject and age- and sex-matched controls. No significant differences were found between subject and controls in the percentages of micronuclei hybridizing with a centromere probe for the X chromosome or a painting probe for chromosome 3. However, the subject had a very highly significant increase (p<0.0001) in chromosome 2 in micronuclei over a level that might be expected to be present by chance. Characterization of micronuclei may be a promising tool in studies of mechanisms of inherited or induced chromosome instability. The strength of the strategy employed in this study is that, by characterizing the chromosomes present in micronuclei, this work has advanced from an observation of chromosomal instability to a foundation for study of the mechanism underlying the observation.     

Changes in the cellular structure of the salivary glands in Chironomus larvae resulting from mechanical cell damage

Rapid morphological changes were observed in some cells of hand-isolated salivary glands of Ch. thummi larvae. The nuclear envelope, routinely closely fitting the tightly packaged polytene chromosomes, was seen to lose its contact with the chromosomes and to attain a smooth round shape. Then unfolding of the chromosomes occurred, their banding patterns becoming clearly evident, probably through widening the interband regions; the chromosome length increased by about 20%. We argue that the changes observed were induced during gland isolation by lesions of the cell basal envelope in the sites of the fat body connections to the salivary gland.     

Degassing‐assisted patterning of cell culture surfaces

We developed an alternative patterning technique which is capable of producing both topographic and biochemical features for cell culture studies. This technique is based on microaspiration induced with a degassed poly (dimethylsiloxane) (PDMS) mold. After degassing in a rough vacuum chamber and placed on a sample surface, liquid solution can be aspired through channels and cavities created in the PDMS mold. Depending on the composition of the solution and the associated drying or incubation processes, a variety of surface patterns can be produced without applying external pressure. For demonstration, we fabricated agarose gel microwells and biomolecule patterns either on a glass plate or in a cell culture Petri dish, both applicable for cell culture studies. Biotechnol. Bioeng. 2010. 105: 854–859. © 2009 Wiley Periodicals, Inc.     

Changes in cell morphology of Listeria monocytogenes and Shewanella putrefaciens resulting from the action of protamine.

Protamine, which is an antibacterial basic peptide, was shown to alter the cell morphology of Listeria monocytogenes and Shewanella putrefaciens. Atomic force microscopy revealed that protamine smoothed the surface of cells, formed holes in the cell envelope, and caused fusion of S. putrefaciens cells. Immunoelectron microscopy of protamine-treated cells of both L. monocytogenes and S. putrefaciens showed great damage to the cell wall and condensation of the cytoplasm. Respiration of the cells was decreased due to treatment with sublethal concentrations of protamine, probably due to leakage or loss of cell envelope potential. It was concluded that protamine disrupted the outer surface structure and condensed the cytoplasm of sensitive cells and, in sublethal concentrations, altered membrane structures, thereby eliminating respiration.     

Quantitation of interaction of lipids with polymer surfaces in cell culture

As cell culture medium development efforts have progressed towards leaner, serum-free, and chemically defined formulations, it has become increasingly important to ensure that the appropriate concentrations of all nutrients are maintained and delivered at point of use. In light of concurrent efforts to progress to disposable polymeric storage and culture platforms, the characterization and control of medium component interactions with container surfaces can be a key issue in ensuring consistent delivery of these medium formulations. These studies characterize the interactions of lipids with culture surfaces typically encountered in the bioprocess industry using model systems. The extent and kinetics of lipid association with polymeric surfaces were determined using radio-labeled linoleic acid and cholesterol. The effect of methyl-beta-cyclodextrin, a component commonly used to solubilize lipids in culture media, on association kinetics was also examined. In addition, loss of lipids across a sterilizing membrane filter was quantified. We find that there is potential for significant loss of hydrophobic components due to non-specific binding to surfaces at timescales relevant to a typical cell culture process. The extent of loss is dependent on the nature of the hydrophobic component as well as the type of surface. These studies highlight the potential of the extracellular environment to modify medium composition and also emphasize the importance of medium formulation strategies, including those used in the delivery of hydrophobic components. It is noted, however, that the level of loss is very dependent on the specific system including the composition of the culture medium used.     

A novel culture morphology resulting from applied mechanical strain

Summary To demonstrate that cells both perceive and respond to external force, a strain/relaxation regimen was applied to normal human fetal and aged dermal fibroblasts cultured as monolayers on flexible membranes. The precisely controlled protocol of stretch (20% elongation of the culture membrane) at 6.67 cycles/min caused a progressive change in the monolayers, such that the original randomly distributed pattern of cells became a symmetric, radial distribution as the cell bodies aligned parallel to the applied force. High cell density interfered with the success of re-alignment in the fetal cell cultures observed, which may reflect a preference in this cell strain for cell-cell over cell-matrix contacts. The chronologically aged cells observed did not demonstrate this feature, aligning efficiently at all seeding densities examined. The role of microfilaments in force perception and transmission was investigated through the addition of cytochalasin D in graded doses. Both intercellular interactions and cytoskeletal integrity mediate the morphological response to mechanical strain.     

Increased micronucleus frequency after oral administration of cadmium in dogs

Cadmium (Cd) is a toxic heavy metal that has been classified as a human carcinogen by the International Agency for Research on Cancer. The genotoxic effects of cadmium oxide (CdO) were investigated in cultured dog lymphocytes after a short-term oral CdO administration by the micronucleus (MN) test. The dogs were given 10 mg CdO/kg body weight per day for 3 and 28 d, respectively group I (n=7) and group II (n=6). Blood samples were collected at the beginning of feeding and at 4 and 29 d after Cd administration and cultured for 72 h. Whereas no significant increase in the MN frequency in group I was observed (p=0.398), a significant MN induction with CdO was found in group II (p=0.028) when compared with initial MN frequencies of dogs in both groups. Our results suggest that CdO might be directly and/or indirectly genotoxic after a monthly oral administration of CdO in dogs.     

Changes in citrus leaf flavonoid concentrations resulting from blight-induced zinc-deficiency

Increased flavonoid concentrations were found to correlate with the elevated levels of leaf phenolic compounds occurring in blight-induced zinc-deficient citrus. In orange (Citrus sinensis L.) leaves, the increases occurred primarily in hesperidin and diosmin, whereas in grapefruit (C. paradisi Macf.) the largest increases occurred in naringin and rhoifolin. Zinc-deficiency occurring in the blighted citrus leaves appeared to be the important contributing factor to the increased flavonoid content. Although the leaves from trees with blight were typically smaller than leaves from unaffected trees, the increased flavonoid content was not significantly due to a concentration effect. Large differences occurred in the percent increases in concentrations of certain citrus leaf flavonoids. While large increases occurred for a number of flavanone and flavone glycosides, much smaller percent increases occurred for other minor flavone glycosides, and the polymethoxyflavone aglycones. The parallel increases occurring in the concentrations of certain flavone glycosides and their flavanone analogs provide a further indication that flavanone glycosides are precursors in the biosynthesis of flavone glycosides in citrus.     

Changes in the cell cycle during culture of mouse-Chinese hamster cell hybrids

Cell cycle studies, using PLM analysis, were carried out on a mouse-Chinese hamster cell hybrid and its derivatives which stably retained all parental chromosomes during the year of study. Parameter estimates were obtained from the PLM curves, using conjugate gradient curve fitting procedures. The hybrid initially grew very slowly, and all phases (especially G₁) were longer than those of either parent. During propagation, mean generation time decreased progressively, and the phase times approached those of the mouse parent (which had the longer G₁ and S). DNA replication could be scored separately in mouse and hamster chromosome sets; initially termination was highly asynchronous, but during growth asynchrony was progressively reduced as DNA synthesis in the hamster set was prolonged. We conclude that cell hybrids may undergo progressive modifications of the cell cycle, even in the absence of significant chromosome segregation, and suggest that such changes may at least partly account for the great variety of relationships between the growth rates and phase times of parent and hybrid cells which have been reported. Because of the complexity of these changes in the cycles of interspecific cell hybrids, we believe that somatic cell genetic analysis of the regulation of the cell cycle would be more usefully applied to intraspecific hybrids whose parents differ in only one specific cycle characteristic.     

Effect of thiols on micronucleus frequency in gamma-irradiated mammalian cells

The effects of the thiols cysteamine, WR-1065, and WR-255591 on radiation-induced micronucleus (MN) frequency and cell killing were compared in cultured Chinese hamster ovary cells. MN were measured using the cytochalasin B assay of Fenech and Morley (1985), which minimizes the effect of cytokinetic perturbations on MN expression. The dose-response curves for MN induction were curvilinear both for control cells at doses between 1.5 and 4.5 Gy and for thiol-treated cells at doses between 3 and 9 Gy. Protection against MN induction by each thiol was independent of radiation dose. Furthermore, there was a close correlation between the degree of modification of MN induction and cell survival by each thiol, i.e., the MN frequency closely predicted the survival level regardless of the presence of absence of the thiols. A similar predictive relationship has also been reported by us for cell survival and DNA double-strand break (DSB) induction in this cell line following treatment with these same thiols. Collectively, these data support the hypothesis that, for DNA-repair-proficient mammalian cells treated with radiomodifying agents that do not alter DNA-repair processes, MN and DSB induction are predictive of the level of radiation lethality and of each other.     

Changes in the radioresistance of bacteria after the inhibition of proteosynthesis in the preirradiation phase

The strain ofEscherichia coli WP₂ (try_v) was irradiated with UV light, at a dosage of 240 erg/mm. Proteosynthesis was inhibited by the elimination of the essential amino acid from the cultivation medium. Changes in radioresistance were followed during 45 minutes of starvation and during the subsequent 45 minutes of restitution after the addition of the essential amino acid. The radioresistance of the cells showed a linear increase immediately after the removal of the essential amino acids, proportional to the duration of the inhibition of proteosynthesis. The increase in radioresistance was shown to be reversible. After the addition of the essential amino acid there was an immediate decrease in radioresistance which was most marked in the first 15 minutes.     

Changes in the jaw-opening reflex during anesthesia resulting from auricular electrostimulation

The effect of auricular electrostimulation on the jaw opening reflex and affective behaviour of adult cats was studied in chronic experiments during stimulation of the lip. Auricular electrostimulation was shown to facilitate the jaw opening reflex and to inhibit the affective component of the pain.     

Changes of cell membrane fluidity for mesenchymal stem cell spheroids on biomaterial surfaces

BACKGROUNDThe therapeutic potential of mesenchymal stem cells (MSCs) in the form of three-dimensional spheroids has been extensively demonstrated. The underlying mechanisms for the altered cellular behavior of spheroids have also been investigated. Cell membrane fluidity is a critically important physical property for the regulation of cell behavior, but it has not been studied for the spheroid-forming cells to date.AIMTo explore the association between cell membrane fluidity and the morphological changes of MSC spheroids on the surface of biomaterials to elucidate the role of membrane fluidity during the spheroid-forming process of MSCs.METHODSWe generated three-dimensional (3D) MSC spheroids on the surface of various culture substrates including chitosan (CS), CS-hyaluronan (CS-HA), and polyvinyl alcohol (PVA) substrates. The cell membrane fluidity and cell morphological change were examined by a time-lapse recording system as well as a high-resolution 3D cellular image explorer. MSCs and normal/cancer cells were pre-stained with fluorescent dyes and co-cultured on the biomaterials to investigate the exchange of cell membrane during the formation of heterogeneous cellular spheroids.RESULTSWe discovered that vesicle-like bubbles randomly appeared on the outer layer of MSC spheroids cultured on different biomaterial surfaces. The average diameter of the vesicle-like bubbles of MSC spheroids on CS-HA at 37 °C was approximately 10 μm, smaller than that on PVA substrates (approximately 27 μm). Based on time-lapse images, these unique bubbles originated from the dynamic movement of the cell membrane during spheroid formation, which indicated an increment of membrane fluidity for MSCs cultured on these substrates. Moreover, the membrane interaction in two different types of cells with similar membrane fluidity may further induce a higher level of membrane translocation during the formation of heterogeneous spheroids. CONCLUSIONChanges in cell membrane fluidity may be a novel path to elucidate the complicated physiological alterations in 3D spheroid-forming cells.