Electrical Events Correlated with Contractile Vacuole Activity in the Suctorian, Heliophrya

SYNOPSIS. The surface membrane potentials of suctorian genus Heliophrya were studied with intracellular electrodes. Resting membrane potentials averaged -32 mV, and spontaneous depolarizing potentials occurring at apparently random intervals and having a variety of waveforms were routinely observed. Such spontaneous potentials were correlated in time with visually monitored contractile vacuole activity. Individual contractile vacuoles had unique, although somewhat variable, electrical signatures. In the presence of an intracellular electrode all vacuoles contracted independently, but at approximately the same frequency. The amplitude of the electrical potentials increased when the membrane was hyperpolarized and decreased when it was depolarized. The sign of such potentials reversed at between -10 mV and the zero membrane potential. A 20% decrease in the membrane resistance was measured at the peak of the spontaneous depolarizing potentials.     

Propidium Iodide Competes with Ca2+ to Label Pectin in Pollen Tubes and Arabidopsis Root Hairs

We have used propidium iodide (PI) to investigate the dynamic properties of the primary cell wall at the apex of Arabidopsis (Arabidopsis thaliana) root hairs and pollen tubes and in lily (Lilium formosanum) pollen tubes. Our results show that in root hairs, as in pollen tubes, oscillatory peaks in PI fluorescence precede growth rate oscillations. Pectin forms the primary component of the cell wall at the tip of both root hairs and pollen tubes. Given the electronic structure of PI, we investigated whether PI binds to pectins in a manner analogous to Ca²⁺ binding. We first show that Ca²⁺ is able to abrogate PI growth inhibition in a dose-dependent manner. PI fluorescence itself also relies directly on the amount of Ca²⁺ in the growth solution. Exogenous pectin methyl esterase treatment of pollen tubes, which demethoxylates pectins, freeing more Ca²⁺-binding sites, leads to a dramatic increase in PI fluorescence. Treatment with pectinase leads to a corresponding decrease in fluorescence. These results are consistent with the hypothesis that PI binds to demethoxylated pectins. Unlike other pectin stains, PI at low yet useful concentration is vital and specifically does not alter the tip-focused Ca²⁺ gradient or growth oscillations. These data suggest that pectin secretion at the apex of tip-growing plant cells plays a critical role in regulating growth, and PI represents an excellent tool for examining the role of pectin and of Ca²⁺ in tip growth.The apical wall of tip-growing cells participates directly in the process of growth regulation (McKenna et al., 2009; Winship et al., 2010), yet few methods permit monitoring the wall properties of living cells. Despite this, several recent studies have enhanced our understanding of the apical cell wall. Chemical analyses of isolated pollen tube wall material have revealed a complex mixture of pectic polysaccharides with regions comprising long sequences of polygalacturonic acid. Important patterns of pectin methoxylation have been detected using immunocytochemical approaches, but these are limited to fixed cells (Dardelle et al., 2010). In a recent study, Parre and Geitmann (2005) used microindentation to show significant correlations between wall strength and growth rate. None of these techniques allow for easy investigation of the cell wall during growth.In an earlier study, we found that propidium iodide (PI) vitally stains pollen tubes of lily (Lilium formosanum) and tobacco (Nicotiana tabacum) and in particular reveals with great clarity the thickened apical cell wall (Fig. 1; McKenna et al., 2009). In addition, the apical PI fluorescence oscillates and in lily pollen tubes correlates tightly with oscillations in wall thickness measured by differential interference contrast (DIC) optics. Finally, these studies indicated that the PI fluorescence predicted cell growth rates with high confidence, suggesting that PI binding may provide useful information about the physical and chemical properties of the cell wall.Open in a separate windowFigure 1.PI fluorescence and growth rate oscillate in lily pollen tubes (A and B), Arabidopsis root hairs (C–E), and Arabidopsis pollen tubes (F and G). A, The top panel shows a DIC image of a lily pollen tube, and the bottom panel shows PI fluorescence of the same tube. The PI fluorescence is pseudocolored, with white representing high signal and blue representing low signal. Bar = 10 μm. B, Growth rate (blue) and PI fluorescence (red) are plotted on a line graph. Both oscillate with the same period but with different phases. C, DIC image (top panel) and PI fluorescence image (bottom panel) of an Arabidopsis root hair. Bar = 10 μm. D, Two PI fluorescence images of the same root hair focused on the apex representing peak (top) and trough (bottom) PI signals. Bar = 5 μm. E, A line graph showing the growth rate (blue) and peak PI fluorescence at the apex (red) for the same root hair shown in C and D. F, The top panel shows a DIC image of an Arabidopsis pollen tube, and the bottom panel shows PI fluorescence of the same tube. The PI fluorescence is pseudocolored, with white representing high signal and blue representing low signal. Bar = 5 μm. G, Growth rate (blue) and PI fluorescence (red) are plotted on a line graph. Both oscillate with the same period but with different phases. The growth rate between individual 3-s frames was smaller than the pixel size for our optics in both Arabidopsis cell types; to remove the noise this generated, a four-image (pollen) or five-image (root hair) running average is shown. A.U., Arbitrary units.PI is commonly used to visualize plant cell walls by wide-field fluorescence and confocal microscopy (Fiers et al., 2005; Tian et al., 2006; Estevez et al., 2008) and to select viable cells during cell sorting (Deitch et al., 1982; Jones and Senft, 1985). A positively charged phenanthridine derivative, the propidium ion stains cell walls but does not pass through the intact cell membranes of living cells. It readily diffuses into dead cells and forms highly fluorescent complexes by intercalation between base pairs of double-stranded nucleic acids, thus acting as an excellent indicator for cell vitality (Hudson et al., 1969). Binding to cell walls presumably occurs by a different mechanism, since it is not accompanied by the dramatic increase in fluorescence and shift in absorption and emission maxima observed when PI binds to nucleic acids. The mechanism of PI binding needs further exploration, as does the potential for broader use in other tip-growing plant cells.In this report, we test two hypotheses: first, that PI stains other tip-growing cells with pectin-containing cell walls; and second, that PI and Ca²⁺ bind to the same sites in these walls. This binding would occur through the interaction of partial positive charges caused by localized deficits in π-orbital electrons associated with three of the four nitrogen atoms of PI (Luedtke et al., 2005) coordinating with negatively charged carboxyl and hydroxyl groups on homogalacturonans (HGs), as has been suggested in Oedogonium bharuchae (Estevez et al., 2008).Our findings indicate that both hypotheses are satisfied. Notably, oscillatory changes in apical PI fluorescence occur and are observed to anticipate oscillations in growth rate in Arabidopsis (Arabidopsis thaliana) root hairs and Arabidopsis pollen tubes. In addition, competition studies indicate that PI and Ca²⁺ bind to the same sites in cell walls. Supporting these studies, we demonstrate that pectin methyl esterase (PME) creates more sites for PI binding, presumably by demethoxylating HGs as they are secreted, and that pectinase reduces PI fluorescence dramatically. However, unlike other pectin-binding dyes, PI does not block Ca²⁺ channels at the concentration used in live cell studies, nor does it alter oscillatory growth characteristics. Our findings provide evidence that PI may be employed as a quantitative measure of Ca²⁺-binding sites and thus may have use as an indicator of the degree of cross-linking of HGs and of cell wall extensibility.     

The application of propidium iodide staining to the study of the macronucleus and micronuclei in the suctorian, Heliophrya sp

Morphological changes in the macronucleus and micronuclei of the ciliated protozoon Heliophrya chapmani were investigated using the nucleic acid-specific stain propidium iodide. The fluorescence patterns of nuclei observed in propidium iodide preparations correspond well with those observed using more conventional DNA-specific methods, such as the Feulgen stain. The advantages of propidium iodide staining (minimal cell loss during staining, rapidity of the staining process, and the avoidance of cell damage during hydrolysis) make this method a quick and efficient alternative in the cytochemical study of the protozoan nucleus.     

The Stalk of the Suctorian Tokophrya infusionum: Histochemistry, Biochemistry, and Physiology

Tokophrya infusionum, a sessile suctorian with an external stalk and adhesive disc, has in its life cycle a ciliated, swimming embryo which metamorphoses into the adult form. The addition of NiCl₂ to the medium induced metamorphosis immediately; however, other salts had no effect. Incomplete metamorphosis, without stalk formation, occurred if the organism began metamorphosis before its anterior (stalk-forming) end touched a substrate. The stalk was studied by histochemical and biochemical technics to determine its composition. The stalk stained with mercury-bromphenol blue, and Alcian blue under a variety of conditions, but not with PAS. These results suggest that the stalk contains protein and sulfate groups, possibly in the form of a sulfated protein-polysaccharide. The stalk was insoluble in several common laboratory reagents, but did dissolve in hot 6 N HCl, 2 N NaOH, and papain. It was evident from amino acid analysis of the stalk and of the whole organism that 15% of the total protein is located in the stalk.     

Application of Coriphosphine Staining to the Study of the Macronuclear Anlage of Stylonychia mytilus

SYNOPSIS. Coriphosphine staining of Stylonychia mytilus exconjugants at different times after separation reveals some details of the developing macronucleus. Green fluorescence is seen in both bands and heterochromatic blocks of the polytene chromosomes. No red fluorescence was observed along these chromosomes. Fragments of the old macronucleus and the pycnotic micronuclei have red or orange fluorescence. Red fluorescence is characteristic also of nucleoli in the new macronucleus.     

The Unique Position of the Degenerating Macronucleus in Tetrahymena tropicalis1

In contrast to the situation in 13 other species of the Tetrahymena pyriformis complex, in which the condensed degenerating old macronucleus lies in the posterior end of the cell during the late stages of conjugation, in Tetrahymena tropicalis that nucleus is found in the anterior portion. This developmental characteristic may be useful for taxonomic purposes as well as being of value in investigations on nucleocytoplasmic interaction.     

An Ultrastructural and Cytochemical Study of the Cell Surface of a Suctorian Ciliate1

The surfaces of the main cell body, tentacle shaft, and knob of Discophrya collini, a freshwater suctorian ciliate, were characterized using various cytochemical techniques. Cells prepared for conventional transmission electron microscopy exhibited a 50–60 nm thick fuzzy layer over the cell body surface; this layer was absent from the tentacle knob. A thick (240 nm), two-layered surface coat surrounding the main cell body was stained with ruthenium red. This heavy coat was absent from the surface of the knob where a thin, dense, ruthenium red-positive layer and projecting filaments were present. Freeze-etched material revealed a “particle region” (150–250 nm in thickness) closely associated with the outer cell surface of the suctorian. Fixed specimens were treated with four different lectins and analyzed with electron microscopy in order to obtain information about the carbohydrate composition of the outer surface of D. collini. Concanavalin A bound to the surface of the cell body and tentacle shaft as a dense, particulate layer (80 nm thick) but thinned to 13–16 nm over the surface of the knob. Wheat germ agglutinin-treated cells also displayed a heavy, electron-dense layer (128 nm thick) that surrounded the main cell body and tentacle shaft, but only scattered patches of bound wheat germ agglutinin were observed on the surface of the knob. Discophrya treated with Helix agglutinin or peanut agglutinin appeared similar to control cells. Suctorians were treated with lectins in vivo in an attempt to inhibit capture and ingestion of their prey, Tetrahymena pyriformis, by masking prey receptor sites on the knob. Concanavalin A and, to a lesser degree, wheat germ agglutinin, successfully inhibited attachment of the prey organism. Helix agglutinin and peanut agglutinin had little effect on prey capture.     

The History of Staining the Development of Cytological Staining

Perhaps no other period has contributed more to our knowledge of the cell than the period 1875-1895. During these years most fundamental cytological phenomena were seen and described. Mitosis, maturation and fertilization, the great cornerstones of cytology, were firmly laid by the remarkable researches of Flemming, Strasburger, Van Beneden, Oscar and Richard Hertwig, Boveri and many others. Upon these researches experimental cytology developed and the significance of the morphological phenomena to inheritance and development was pointed out by such masters as W. Roux, Weismann, O. Hertwig, Boveri and E. B. Wilson.     

Recombination and Assortment in the Macronucleus of TETRAHYMENA THERMOPHILA: A Theoretical Study by Computer Simulation

The compound nature of the macronucleus of Tetrahymena thermophila presents multiple opportunities for recombination between genes on the same macronuclear chromosome. Such recombinants should be detectable through their assortment at subsequent amitotic macronuclear divisions. Thus, a macronucleus that is initially AB/ab should produce recombinant assortees of the genotypes Ab/aB. Computer simulation shows that, when the recombination frequency is two or fewer times per cell cycle, recombinant assortees are produced at experimentally measurable frequencies of less than 40%. At higher recombination frequencies, linked genes appear to assort independently. The simulations also show that recombination during macronuclear development can be distinguished from recombination in subsequent cell cycles only if the first appearance of recombinant assortees is 100 or more fissions after conjugation. The use of macronuclear recombination and assortment as a means of mapping macronuclear genes is severely constrained by the large variances in assortment outcomes; with experimentally small sample sizes, such mapping is impossible.     

The Staining of the Synaptonemal Complex for Light Microscopic Study in the Mouse

Demonstration of the synaptonemal complex for light microscopy has until now been based on staining with silver. After fixation at pH 9-10 it is also possible to visualize synaptonemal complexes with several nonspecific protein stains such as Coomassie brilliant blue, Giemsa, fast green, light green and Stains All. Although staining with silver gives the best contrast between synaptonemal complexes and the background, the other dyes have a number of advantages, such as more even staining, easy extractability, and lower cost than silver.     

Contribution to the Staining of Neuroglia

The chemistry of Weigert's glia staining method is critically discussed. An investigation of the Heidelberger Victoria blue staining method has shown that Victoria blue may be replaced by other phenylmethane dyes as methyl violet, ethyl violet, and crystal violet. It was found that the exposure of the stained section to sunlight is an oxidation process. Artificial ultra violet rays or chemical oxidation agents give the same effect. Frozen sections fixed in formalin or alcohol may be stained in a concentrated aqueous solution of any of the above mentioned phenylmethane dyes, dried, and exposed to ultra violet rays for 30 minutes, then treated with 1/10 N. iodine solution, differentiated in xylol anilin and cleared in xylol. The glia cell body as well as the fibrils are clearly differentiated from the nervous elements and connective tissue.     

PAP染色法在微生态学中应用的研究——Ⅰ.细菌染色方法的建立

在微生态学中应用过氧化物酶—抗过氧化物酶(PAP)染色法鉴定细菌的研究还未见报道。本文报告了用埃希氏大肠杆菌(O_(111)B_4)腹腔感染小鼠,取其多种脏器制石蜡切片,建立PAP染色程序。确定了第一抗体(兔抗埃希氏大肠杆菌O_(111)B_4型血清)最佳染色滴度为1:800～3200。观察到埃希氏大肠杆菌(O_(111)B_4)定位于组织器官上的状态。在细菌鉴定上PAP染色法较其它方法更具有优点。     

History of Staining the Staining of Blood and Parasitic Protozoa

By the term “blood stain” one ordinarily means a compound dye formed from the chemical union of an acid and a basic dye, and usually a compound of the eosin-methylene-blue group. It is well known today that the sodium salt of a color acid (e. g. eosin) and the chloride of a dye base (e. g. methylene blue) may be converted by simple metathesis into sodium chloride plus the compound dye (e. g. methylene blue eosinate), the latter being insoluble in water unless an excess is present of either the acid or the basic dye. In modern blood stains a compound dye of this type is dissolved in methyl alcohol and mixed with water on the slide at the moment of staining.     

Micronuclei, DNA single-strand breaks and DNA-repair activity in mice exposed to 1,3-butadiene by inhalation

We investigated single-strand breaks and endonuclease III-sensitive sites in DNA along with gamma-irradiation-specific DNA-repair activity in hepatocytes and frequencies of micronuclei in polychromatic bone-marrow erythrocytes of male NMRI mice (2 months old, weight 30-35 g) during sub-acute inhalation exposure to 1,3-butadiene (28 days, 500 mg/m3) and up to 28 days after the exposure. Concentrations of 1,3-butadiene in blood, an indicator of internal exposure, moderately increased during the exposure period. The most interesting finding was that gamma-irradiation-specific DNA-repair activity gradually increased during exposure, being significantly higher compared with control levels on days 7 and 28 of exposure (P = 0.005 and 0.035, respectively), reaching a maximum on day 1 after the termination of exposure (P = 0.003) and then returning to control levels. A significant correlation between gamma-irradiation-specific DNA-repair activity and the concentration of 1,3-butadiene in blood (R = 0.866, P = 0.050) supports a possible induction of DNA-repair activity by the exposure to 1,3-butadiene and formation of its metabolites. The initial increase in micronucleus frequency (micronuclei per 1000 cells) in the exposed mice continuously decreased from 20.4 +/- 5.1 (day 3) to 15.1 +/- 3.2 (day 28) within the exposure period, and subsequently from 12.4 +/- 5.1 to 4.6 +/- 1.6 in the period following termination of the 1,3-butadiene exposure, while micronucleus frequencies in control animals were significantly lower (from 1.7 +/- 1.5 to 4.2 +/- 0.8).