Acid-phosphatase activity in sieve-tube members of Tilia americana

Summary Acid phosphatases are localized on the internal strands of sieve-tube members in the secondary phloem of Tilia americana. Companion cells and certain strand parenchyma cells associated with sieve-tube members show a high activity of acid phosphatases.This research has been supported by NSF Grant GB-615.     

Plastids in sieve elements and companion cells of Tilia americana

Summary Contrary to an earlier report, the sieve elements and companion cells of Tilia americana contain plastids. In young sieve elements and companion cells the plastids contain a moderately electronopaque matrix and internal membranes; the latter are very numerous in the plastids of the sieve elements. Plastids of mature sieve elements contain an electron-transparent matrix, apparently fewer internal membranes than the plastids of young elements, and a single starch grain each. The plastids of companion cells undergo little or no structural modification during cellular differentiation, and apparently contain no starch.This research has been supported by the National Science Foundation, grants GB-5950 and GB-8330.     

Neuroprotective Evaluation of Tilia americana and Annona diversifolia in the Neuronal Damage Induced by Intestinal Ischemia

Tilia americana and Annona diversifolia are plants widely distributed in Mexico and sold in markets for their medicinal properties on the central nervous system (CNS) including possible neuroprotection. Pharmacological studies have corroborated CNS activities due to flavonoid constituents, but evidence of their neuroprotector effects are lacking. This study was conducted to test aqueous and organic extracts of these two plants for neuroprotective effects in a novel experimental model of intestinal ischemia in situ. T. americana and A. diversifolia aqueous and organic extracts were administrated to guinea pigs at an oral dose of 100 and 300 mg/kg for 15 days. Twenty four hours after the last administration, the animals were anesthetized and intestinal ischemia in situ was induced by clamping for 80 min selected branches of the superior mesenteric artery. Ischemic segments placed in an in vitro organ bath were stimulated electrically (0.3 Hz frequency, 3.0 ms duration, 14 V intensity) and chemically (ACh; 1 × 10^?9 to 1×10^?5 M). Neuroprotection was considered present when the depressed contractile response of the ischemic tissue to electrical stimulation was normalized in the treated animals. Results showed that pretreatment with the T. americana hexane and aqueous extracts, but not with those from A. diversifolia, significantly improved responses of the ischemic tissue. These results suggest that T. americana possesses neuroprotective effects against neuronal damage induced by ischemia, and that flavonoids as well as non-polar constituents are involved. Our study supports the use of this plant in folk medicine and suggests its possible effectiveness for stroke prevention.     

Exudation from Aphid Stylets during the Period from Dormancy to Bud break in Tilia americana (L.)

Aphid stylet exudation during the period from dormancy to budbreak was studied in Tilia americana by means of detached branchesbrought from the woods into conditions of warmth and extendedphotoperiod. During physiological dormancy little exudationwas obtained. Thereafter until mid-March exudation was morevariable, but its onset accelerated progressively until mid-March,after which persistent exudation could be obtained from allbranches within 2 days. When the buds were removed from onebranch at this time, exudation persisted for only 5 days comparedwith 14 to 21 days for branches with buds. To account for theseresults it is suggested that a hormonal factor is produced bythe buds which results in sieve-tube activation, that the factoris virtually absent during physiological dormancy, and thereafterrequires a few weeks to become fully active. Determinations of sugar concentration, level of exudation, andbud dry weight indicated that exudation was most intense beforethe bud sinks were active and then rapidly falls off. It issuggested that the stylet acts as a sink competing with thenatural sinks for solutes from a limited region of the stem.Other interpretations are also considered.     

Mitosis

Within the last decade, the study of mitosis has evolved into a multidisciplinary science in which findings from fields as diverse as chromosome biology and cytoskeletal architecture have converged to present a more cohesive understanding of the complex events that occur when cells divide. The largest strides have been made in the identification and characterization of regulatory enzymes (kinases and phosphatases) that modulate mitotic activity, as well as a number of the proteins and structural components (spindle, chromosomes, nuclear envelope) which carry out the mitotic instructions. One emerging theme appears to be that molecular signalling, which can involve modification of components (such as phosphorylation) or even their specific destruction, monitors the state of the mitotic cell at all stages. One of the major challenges for the future will be the identification of addititonal targets of the signalling machinery, as well as new regulatory components and their targets on the chromosomes, on the spindle, and at the cleavage furrow.     

Active compounds and anti-inflammatory activity of the methanolic extracts of the leaves and callus from Tilia americana var. mexicana propagated plants

Plant Cell, Tissue and Organ Culture (PCTOC) - Tilia americana var. mexicana is used in Mexican traditional medicine to treat anxiety and inflammatory processes. Several glycosides derived from...     

Regulation of Mitosis in Living Cells. I. Mitosis under Controlled Conditions

A quantitative method has been devised to study mitosis in vitro by phase contrast and polarization microscopy. Mitosis in cell-wall-free endosperm cells of Haemanthus kathrinar Baker (the African blood lily) has been divided into 18 arbitrary stages or events. The time course for the various stapes, as well as the percentage of cells that proceed from one stage to another during a four hour observation period, are presented. Cells that were in prophase when selected for study proceeded from nuclear membrane breakdown to melaphase in 60 minutes and remained in melaphase for 30 minutes. Only 13 minutes was required to proceed from onset of anaphase to mid-anaphasc. Mid-anaphase provides a clear and precise baseline for determining the time required for succeeding stages to appear. The cell plate made its appearance 40 minutes after mid-anaphase and was completely formed 20 minutes later. The nuclear membranes also became evident at this latter time and nucleoli were visible 30 minutes later. Thus, the average time for a cell observed initially in prophase to proceed from nuclear membrane breakdown to formation of two daughter cells was just over three hours. A high percentage of cells that were in late prophase or later stages of mitosis at the time of initial observation completed mitosis during the observation period. The effect of the length of time a cell is subjected to experimental conditions upon its subsequent behaviour is assessed. These results form the basis for future studies of the effects of chemicals, particularly herbicides, upon cells in mitosis as observed in vitro by phase contrast and polarization microscopy.     

Mitosis is swell

Cell volume and dry mass are typically correlated. However, in this issue, Zlotek-Zlotkiewicz et al. (2015. J. Cell Biol. http://dx.doi.org/10.1083/jcb.201505056) and Son et al. (2015. J. Cell Biol. http://dx.doi.org/10.1083/jcb.201505058) use new live-cell techniques to show that entry to mitosis coincides with rapid cell swelling, which is reversed before division.How growth is linked to division by the cell cycle regulatory network is an important open question in cell biology (Turner et al., 2012; Ginzberg et al., 2015). Yet, what is meant by cell growth? Different methods have been used to estimate either the total dry mass of the cell, total protein content, or cell volume. Although these parameters are often highly correlated, they are not the same. In budding yeast, growth parameters are nearly interchangeable as cell density changes only about 1% through the division cycle (Bryan et al., 2010). In contrast, cell density can drop by over 50% during a rapid growth phase in hypertrophic chondrocytes, which are responsible for determining bone length (Cooper et al., 2013). However, because of the current lack of similarly dramatic examples, it is assumed that chondrocytes are a special case and that most animal cells also exhibit little variation in cell density, as recently measured (Bryan et al., 2014). Yet this assumption has not been thoroughly tested because of the difficulty of measuring cell volume in animal cells, which are often irregularly shaped.Measuring cell volume is even more challenging in live cells. Whereas there is an accurate live-cell method for measuring dry mass in quantitative phase microscopy (Sung et al., 2013), live-cell volume measurements of adherent cells have been difficult because of their irregular geometry. Current methods are mostly based on 3D geometric reconstructions from confocal sections. However, confocal microscopy has poor resolution in the z-dimension, and increasing the number of z-sections to better estimate the cell membrane location and improve accuracy can be phototoxic.In this issue, Son et al. and Zlotek-Zlotkiewicz et al. applied two different methods to accurately measure cell volume changes in live cells. Son et al. (2015) used a variation of the suspended microchannel resonator pioneered by the Manalis laboratory (Fig. 1 A; Burg et al., 2007). In this method, the resonance frequency of the device shifts when a cell enters a part of a microchannel because the cell is of a different density than the surrounding media. The change in resonance frequency can therefore be used to calculate the buoyant mass of the cell. Changing the media of the microchannel to one of different density and then performing the same measurement for the same cell allows the accurate calculation of both cell dry mass and volume. One limitation of the microchannel resonator method is that the cells are required to be nonadherent so that they can be moved into and out of the resonator. To measure cell volume of adherent cells, Zlotek-Zlotkiewicz et al. (2015) used a microchamber culture device with a low 15–25-µm adjustable ceiling (Fig. 1 B). Cells were grown in a media containing fluorescent dye–labeled dextran. Cell volume could then be measured from epifluorescence images because the cells displaced the fluorescent dextran in proportion to their volume. This method was combined with quantitative phase microscopy to measure dry mass.Open in a separate windowFigure 1.Two new live-cell measurements of cell volume and mass reveal that cells swell in mitosis. (A) Schematic of microchannel resonator whose frequency is determined by the cells’ buoyant mass. Live-cell measurements in two media of different density allow calculation of cell volume and density (modified from Son et al., 2015). (B) Using epifluorescence microscopy, cell volume can be measured as the amount of dye-labeled dextran displaced in a low-ceiling culture chamber. (C) Cell density is constant through the cell cycle except in mitosis, when cells swell (modified from Son et al., 2015). (D) In the context of an animal tissue, mitotic swelling may generate a larger, rounder space to promote accurate and rapid chromosome segregation.Both Son et al. (2015) and Zlotek-Zlotkiewicz et al. (2015) applied their methods to precisely and noninvasively measure the volume and density dynamics in growing and dividing mammalian cells (Fig. 1 C). During most of the cell cycle, density is constant and dry mass is correlated with volume. However, the researchers found that cell volume, but not dry mass, increases rapidly as cells enter mitosis. This osmotic swelling occurs during prophase and prometaphase before being reversed in anaphase and telophase. Collectively, the work of both teams also determined that mitotic swelling is driven by osmotic water exchange and requires the activity of the Na/H ion exchanger but is not dependent on the actomyosin cortex, endocytosis, or cytokinesis. Whereas previous studies gave contradictory results, the two papers in this issue show that there is a reversible 10–30% volume increase during mitosis depending on the type of cell.The establishment of cell swelling during mitosis raises the question of its function. In laboratory conditions, mitotic animal cells lose surface adhesion and are spherical. This spherical geometry is accompanied by an increase in intracellular hydrostatic pressure (Stewart et al., 2011). In the in vivo context of an animal tissue, an increase in intracellular pressure accompanied by cell swelling would allow cells to push against their neighbors and open up additional space for mitosis (Fig. 1 D; Son et al., 2015; Zlotek-Zlotkiewicz et al., 2015). The mitotic acquisition of a larger, more spherical geometry may be important because physically preventing cells from rounding up retards mitosis and promotes inaccurate chromosome segregation (Lancaster et al., 2013). Alternatively, the dilution of the cytoplasm by swelling might change the physicochemical properties of the intracellular environment to facilitate chromosomal movement and segregation or change the kinetics of biochemical reactions (Son et al., 2015).Live-cell methods that accurately measure volume will most obviously be useful for studies of how cell growth is linked to cell cycle progression but are unlikely to be limited to this application. For example, it would be interesting to follow the dynamics of cell volume and density in other processes in which the surface area to volume ratio can change rapidly, such as cell migration (Traynor and Kay, 2007). Depending on the environment, cells can switch from actin-driven motility to hydrostatic pressure–driven bleb-based motility (Sahai and Marshall, 2003; Zatulovskiy et al., 2014). Because this motility switch strongly depends on the osmolarity of the environment (Fedier and Keller, 1997; Yoshida and Soldati, 2006), it is likely to be accompanied by and perhaps even require cell swelling.Although the swelling of animal cells has been mostly neglected, cell swelling is not unusual in other eukaryotic lineages. Unlike animal cells, which have a flexible cell geometry that can rapidly be remodeled, plant and fungal cells have a stiff cell wall and cannot easily change their geometry. Nevertheless, plants and fungi can use regulated swelling to move on time scales faster than that of growth (Skotheim and Mahadevan, 2005). For example, a stem bending to track the sun is caused by cells on one side swelling, whereas those on the other side shrink. This differential swelling allows the stem to bend because the plant tissue is connected by elastic cell walls. Using such differential swelling, plants and fungi can perform impressive coordinated movements to track the sun, compete for territory, disperse seeds or spores, and catch prey (Attenborough, 1995). Although swelling-based movements have long been appreciated in the context of plants, there is no a priori reason animal cells might not also harness such mechanisms to perform important functions. The further development and dissemination of technologies to accurately measure cell volume, density, and dry mass, such as those described in this issue, will be essential to determine the extent to which animal cells harness swelling.     

Mitosis under control

The mitotic checkpoint is essential to ensure accurate chromosome segregation by allowing a mitotic delay in response to a spindle defect. This checkpoint postpones the onset of anaphase until all the chromosomes are attached and correctly aligned onto the mitotic spindle. The checkpoint functions by preventing an ubiquitin ligase called the anaphase-promoting complex (APC) from ubiquitinylating proteins whose degradation is required for anaphase onset. Loss of this checkpoint results in chromosome missegregation in higher eukaryotes and may contribute to the genomic instability observed in most of the tumour cells.     

Mitosis in mature lungfish retina

The retina of a 32-7 cm Protopterus aethiopicus was fixed in glutaraldehyde and osmium tetroxide. Araldite-embedded sections were examined by optical and electron microscopy. A cell of the inner nuclear layer was found undergoing mitosis, but it is not possible to say to which class this cell belongs. The normal pattern of retinal cell division in developing and postlarval fish and amphibians is discussed; in neither is mitosis in the inner nuclear layer usual. Retinal regeneration after injury occurs in urodele amphibians, with which lungfish have close affinities.