In Vivo Perturbation of Membrane-Associated Calcium by Freeze-Thaw Stress in Onion Bulb Cells : Simulation of This Perturbation in Extracellular KCl and Alleviation by Calcium

Incipient freeze-thaw stress in onion bulb scale tissue is known to cause enhanced efflux of K⁺, along with small but significant loss of cellular Ca²⁺. During the post-thaw period, irreversibly injured cells undergo a cytological aberration, namely, `protoplasmic swelling.' This cellular symptom is thought to be caused by replacement of Ca²⁺ from membrane by extracellular K⁺ and subsequent perturbation of K⁺ transport properties of plasma membrane. In the present study, onion (Allium cepa L. cv Sweet Sandwich) bulbs were slowly frozen to either −8.5°C or −11.5°C and thawed over ice. Inner epidermal peels from bulb scales were treated with fluorescein diacetate for assessing viability. In these cells, membrane-associated calcium was determined using chlorotetracycline fluorescence microscopy combined with image analysis. Increased freezing stress and tissue infiltration (visual water-soaking) were paralleled by increased ion leakage. Freezing injury (−11.5°C; irreversible) caused a specific and substantial loss of membrane-associated Ca²⁺ compared to control. Loss of membrane-associated Ca²⁺ caused by moderate stress (−8.5°C; reversible) was much less relative to −11.5°C treatment. Ion efflux and Ca²⁺-chlorotetracycline fluorescence showed a negative relationship. Extracellular KCl treatment simulated freeze-thaw stress by causing a similar loss of membrane-associated calcium. This loss was dramatically reduced by presence of extracellular CaCl₂. Our results suggest that the loss of membrane-associated Ca²⁺, in part, plays a role in initiation and progression of freezing injury.     

Freezing Injury in Onion Bulb Cells: II. Post-thawing Injury or Recovery

Onion (Allium cepa L.) bulbs were subjected for 12 days to either a moderate freeze (−4 C) or a severe freeze (−11 C). They were then thawed slowly over ice. During 7 to 12 days following the thaw, the injury progressed with time in the severely frozen bulbs, but appeared completely repaired in the moderately frozen bulbs. This was shown by the following post-thawing changes.     

离子胁迫诱导洋葱鳞茎内表皮细胞凋亡

通过不同浓度离子胁迫诱导剂(NaCl、CaCl2)对洋葱鳞茎内表皮细胞进行不同时间的处理,发现0.1M、0.5M的NaCl和CaCl2处理2小时即可诱导出细胞凋亡现象,随处理时间延长直至10小时,细胞核凋亡的形态学变化和凋亡小体更加明显,基因组DNA降解更加梯状条带化。本实验对离子诱导的洋葱鳞茎内表皮细胞凋亡现象做了较系统的描述,为植物细胞凋亡的研究及细胞凋亡实验教学提供了经济、快捷、有效的诱导方法。     

Extracellular Calcium Sensing by Glial Cells: Low Extracellular Calcium Induces Intracellular Calcium Release and Intercellular Signaling

Abstract: Glial cells in primary mixed cultures or purified astrocyte cultures from mouse cortex respond to reduced extracellular calcium concentration ([Ca²⁺]_e) with increases in intracellular calcium concentration ([Ca²⁺]_i) that include single-cell Ca²⁺ oscillations and propagated intercellular Ca²⁺ waves. The rate and pattern of propagation of low [Ca²⁺]_e-induced intercellular Ca²⁺ waves are altered by rapid perfusion of the extracellular medium, suggesting the involvement of an extracellular messenger in Ca²⁺ wave propagation. The low [Ca²⁺]_e-induced Ca²⁺ response is abolished by thapsigargin and by the phospholipase antagonist U73122. The low [Ca²⁺]_e-induced response is also blocked by replacement of extracellular Ca²⁺ with Ba²⁺, Zn²⁺, or Ni²⁺, and by 100 µM La³⁺. Glial cells in lowered [Ca²⁺]_e(0.1–0.5 mM) show an increased [Ca²⁺]_i response to bath application of ATP, whereas glial cells in increased [Ca²⁺]_e (10–15 mM) show a decreased [Ca²⁺]_i response to ATP. These results show that glial cells possess a mechanism for coupling between [Ca²⁺]_e and the release of Ca²⁺ from intracellular stores. This mechanism may be involved in glial responses to the extracellular environment and may be important in pathological conditions associated with low extracellular Ca²⁺ such as seizures or ischemia.     

Freezing Behavior of Intra- and Extracellular Water of Cultured Green Cells of Lavender as Measured by NMR

Three new proteins which inhibit protein synthesis in rabbit reticulocyte lysates were isolated from an extract of sponge gourd (Luffa cylindricd) seeds by chromatography on a AF-Blue Toyopearl column followed by FPLC with a Mono S column. These three protein-synthesis inhibitory proteins (PSIs) have molecular masses of 19kDa, 15kDa, and 9kDa, and were designated 19K-PSI, 15K-PSI, and 9K-PSI, respectively. Although the 19K-PSI had no effect on protein synthesis in HeLa cells, its inhibitory activity on the cell-free protein synthesis was 340- and 83-fold stronger than those of ricin A-chain and luffin-a, respectively, probably due to hydrolyzing mRNA. The inhibitory activities of 15K-and 9K-PSIs on the cell-free protein synthesis were weaker than those of ricin A-chain and luffin-a. The 19K-PSI was a glycoprotein having an ordinary amino acid composition, three intramolecular disulfide bonds and a blocked N-terminal residue, while the 15K-PSI was extraordinarily rich in glycine and the 9K-PSI in arginine and glutamic acid (and/or glutamine). The amino acid composition of 19K-PSI was: Ser₂₇Glx₃Gly₁₆₄Tyr₇Lys₉His₆, and that of 9K-PSI was: Asx₃Glx₂₅Pro₂Gly₅Lys₂His₂Arg₂₅Trp₃.     

Dehydration of Onion Cells: A Comparison of Freezing vs. Desiccation and Living vs. Dead Cells

Measurements were made of the amount of liquid water present in the epidermal cells of onion at various degrees of dehydration caused by slow extracellular freezing and by desiccation. This was achieved by using a pulsed NMR spectrometer during freezing stress and by weighing the epidermal pieces during desiccation. Measurements were made on the extent of cell survival by direct microscopic observation (plasmolysis and protoplasmic streaming). Onion epidermal cells (Allium cepa L. cv. Downing Yellow Globe) were found to survive freezing temperatures as low as –20°C and an equivalent desiccation stress. This equivalence opposes the reports by others on Hordeum vulgare and on Solanum sp. of greater injury by freezing than by an equivalent dehydration due to desiccation. The discrepancy -has been explained in terms of the limitations of the conductivity method used by those authors to evaluate the injury. The freezing and desiccation curves correspond to the equation: L _t=L₀Δt_m/t+L_u where L_t and L₀ are the amounts of liquid water at temperature t and O°C respectively. Δt_m is the freezing point depression of the cell sap and L_u is the amount of liquid water which does not freeze. These results demonstrate that the dehydration of onion cells during both freezing and desiccation duplicates the dehydration of ordinary aqueous solutions. This was equally true for living and dead cells, and suggests that the negative turgor invoked by others is not significantly involved in the dehydration of living Allium cepa epidermis cells. An explanation is proposed for these contradictory results.     

Studies on Freezing Injury of Plant Cells: I. Relation between Thermotropic Properties of Isolated Plasma Membrane Vesicles and Freezing Injury

The thermotropic transition of plasma membrane of Dactylis glomerata was studied by using fluorescence polarization of embedded fluorophore, 1,6-diphenyl-1,3,5-hexatriene. Under the presence of 35% ethylene glycol, reversible thermotropic transitions were observed in isolated plasma membrane vesicles in nearly the same temperature range as the temperature of freezing injury to cells. In liposomes prepared from isolated plasma membranes, however, the thermotropic transitions occurred at much lower temperatures in comparison with those of intact membrane vesicles. Following treatment with pronase, the thermotropic transition also shifted downward.

Thus, the thermotropic properties of plasma membranes appeared to be dependent on the membrane proteins. In vitro freezing of the isolated plasma membrane vesicles without addition of any cryoprotectant, such as sorbitol, resulted in an irreversible alteration both in the fluorescence anisotropy values and the temperatures for the thermotropic transition, suggesting an irreversible alteration in the membrane structure, presumably changes in lipid-protein interactions and protein conformation.

     

Endogenous Diurnal and Nocturnal Activity of Guard Cells as Indicated by Stomatal Aperture and Protoplasmic Streaming

The hourly stomatal apertures on epidermal strips of Antirrhinum majus, Rheum rhaponticum, Vicia faba, and Zebrina pendula fixed by Lloyd's technique were compared with the number of stomata found to contain streaming at the same time. With all four species there was an indication of an endogenously controlled rhythm in stomatal opening with both increases and decreases in aperture and streaming during daytime hours in either the light or dark. A similar endogenously activated rhythm was also found at night in Rheum and Vicia. Some relationships exist between changes in stomatal aperture and streaming. Streaming in guard cells may only be a byproduct of metabolism, but as such, it serves the useful purpose as a mixer of chemical activity.     

Studies on Freezing Injury in Plant Cells : II. Protein and Lipid Changes in the Plasma Membranes of Jerusalem Artichoke Tubers during a Lethal Freezing in Vivo

Plasma membranes were isolated from both unfrozen and frozen tissues of Jerusalem artichoke tubers (Helianthus tuberosus L.) in high purity utilizing an aqueous two-polymer phase partition system. Although the recovery of the plasma membranes was decreased significantly by freezing of tissues even at the nonlethal temperature (−5°C), the isolated plasma membrane samples were considered to be representative of the plasma membranes in situ. Freezing of the tissues at sublethal temperatures resulted in marked changes in the chemical composition of the plasma membrane. Those are losses of sterols and phosphatidylethanolamine from the plasma membranes, and a change of specific proteins with relatively high molecular weights into low molecular weight peptides. These specific proteins were designated as frost susceptible proteins. The properties of the plasma membrane ATPase seem to be not affected so much by the in vivo freezing of cells. However, inhibition of the plasma membrane ATPase by N,N′-dicyclohexylcarbodiimide (DCCD) was relatively low before and after freezing in vivo at the nonlethal temperature at −5°C, but was markedly enhanced by freezing in vivo at sublethal temperatures below −10°C. From the results, it is assumed either that the enzyme molecule was partially modified, especially at the presumed DCCD binding sites or that the DCCD had become more accessible to the enzyme as a result of increased permeability of the plasma membranes. These observed changes are discussed in connection with the mechanism of cell injury.     

Effects of Root Excision on Swelling of Leaf Sheath Cells and on the Arrangement of Cortical Microtubules in Onion Seedlings

Unlike abundantly-leafed mature plants, 2- to 3-leafed youngonion seedlings (Allium cepa L. cv. Senshu-Chuko) showed noswelling of leaf sheaths even when they were kept under long-dayconditions. When their roots were excised, however, the seedlingsshowed sign of swelling. Before swelling became evident, changesin the arrangement of cortical microtubules occurred in leafsheath cells. The microtubules, which were oriented transverselyto the cell axis in unexcised seedlings, were oriented longitudinallyor obliquely in excised seedlings. Such effects of root excisionwere observed only in seedlings grown under long-day conditions,but not under short-day conditions. (Received July 14, 1984; Accepted September 26, 1984)     

Freezing Injury and Phospholipid Degradation in Vivo in Woody Plant Cells: III. Effects of Freezing on Activity of Membrane-bound Phospholipase D in Microsome-enriched Membranes

Freeze-thawing of microsome-enriched membranes from living bark tissues of black locust trees, especially those from less hardy tissues, caused a drastic increase in sensitivity to Ca²⁺ and a complete loss of the regulatory action of Mg²⁺ in membrane-bound phospholipase D activity with endogenous (membrane-bound) substrates. Also, the freeze-thaw cycle made phospholipase D in these membranes more resistant to digestion by proteases. Thus, the regulatory properties of the membrane-bound phospholipase D seem to be dependent on the nature of the membranes and on the interaction between the enzyme and membranes as well. The alteration of regulatory properties by freezing was protected by sucrose, at lower concentrations, and more effectively for membranes from hardy tissues than for membranes from less hardy tissue. Addition of partially purified soluble phospholipase D to the reaction system containing membranes caused only a slight stimulation of the degradation of endogenous phospholipids. Phospholipid degradation in vivo during freezing of less hardy tissue may be catalyzed mainly by the bound enzyme. Disintegration of the tonoplast, however, besides releasing soluble phospholipase D into the cytosol, would release organic acids (lowering the pH) and free Ca²⁺. Both factors would stimulate drastically the membrane-bound phospholipase D, causing degradation of membrane phospholipids.     

Production of Pectic Enzymes by Fusarium oxysporum f. sp. cepae and its Involvement in Onion Bulb Rot

Fusarium oxysporum f. sp. cepae produced an exo-polygalacturonase (exo-PG) and endopectin-trans-eliminase (endo-PTE) in a mineral medium supplemented with a restricted supply of either D-galacturonic acid or onion cell walls. These enzymes were also extracted from infected onion tissue, but only endo-PTE caused tissue maceration and cell death. The patterns of host tissue colonization and pectic enzyme production were followed during bulb rot development. Stem plates were invaded within two weeks of inoculation. The pathogen then remained confined to the stem plates for several weeks or months, before spreading to the outer fleshy scales to initiate a basal rot. In most cases the inner leaf sheaths containing the lateral bud remained healthy. Exo-PG activity m stem plate tissue was greatest at two weeks after inoculation, then it declined. Endo-PTE was not detected in newly invaded stem plate tissue, but was recovered from infected stem plates before decay and from the bases of bulb scales and leaf sheaths at the onset of bulb rot. There was no pectic enzyme activity in uninvaded onion tissue. Spread of the fungus and pectic enzyme production in two Caledon Globe genotypes susceptible or tolerant to F. oxysporum f. sp. cepae were similar, but the onset of bulb rot in tolerant genotypes was considerably delayed.     

Use of Potassium and Sucrose by Onion Guard Cells during a Daily Cycle of Osmoregulation

Solute content of stomata from intact onion cotyledons grownunder either greenhouse or growth chamber conditions was followedover the course of a daily light cycle to determine patternsof osmoregulation. Initial opening of stomata was well correlatedwith guard cell potassium accumulation under both growth conditions.Subsequently, however, there was a consistent decrease in guardcell potassium content despite constant or increasing aperture.Although a secondary increase in potassium was sometimes observedduring the second half of the light cycle, guard cell potassiumcontent was poorly correlated with aperture. Sucrose levelsin guard cells increased 60% during the period of decliningpotassium content, suggesting its use as an alternate osmoticum.Guard cells are postulated to use multiple pathways for theproduction of osmotica over the course of a complete daily cycleof stomatal movements. (Received December 5, 1995; Accepted April 9, 1996)     

Prevention of Liver Fibrosis by Intrasplenic Injection of High-Density Cultured Bone Marrow Cells in a Rat Chronic Liver Injury Model

Endothelial progenitor cells (EPCs) from bone marrow have proven to be functional for the prevention of liver fibrosis in chronic liver injury. However, expansion of EPCs in culture is complicated and expansive. Previously, we have established a simple method that could enrich and expand EPCs by simple seeding bone marrow cells in high density dots. The purpose of this study is to evaluate whether cells derived from high-density (HD) culture of rat bone marrow cells could prevent the liver fibrosis in a chronic liver injury rat model, induced by carbon tetrachloride (CCl₄). Flow cytometric analysis showed that cells from HD culture were enriched for EPCs, expressing high levels of EPC markers. Intrasplenic injection of HD cultured bone marrow cells in the CCl₄-induced liver injury rat showed an enhanced antifibrogenic effect compared with animals treated with cells from regular-density culture. The antifibrogenic effect was demonstrated by biochemical and histological analysis 4 weeks post-transplantation. Furthermore, cells from HD culture likely worked through increasing neovascularization, stimulating liver cell proliferation, and suppressing pro-fibrogenic factor expression. HD culture, which is a simple and cost-effective procedure, could potentially be used to expand bone marrow cells for the treatment of liver fibrosis.     

A Comparison of Freezing Injury in Oat and Rye: Two Cereals at the Extremes of Freezing Tolerance

A detailed analysis of cold acclimation of a winter rye (Secale cereale L. cv Puma), a winter oat (Avena sativa L. cv Kanota), and a spring oat cultivar (Ogle) revealed that freezing injury of leaves of nonacclimated seedlings occurred at -2[deg]C in both the winter and spring cultivars of oat but did not occur in winter rye leaves until after freezing at -4[deg]C. The maximum freezing tolerance was attained in all cultivars after 4 weeks of cold acclimation, and the temperature at which 50% electrolyte leakage occurred decreased to -8[deg]C for spring oat, -10[deg]C for winter oat, and -21[deg]C for winter rye. In protoplasts isolated from leaves of nonacclimated spring oat, expansion-induced lysis was the predominant form of injury over the range of -2 to -4[deg]C. At temperatures lower than -4[deg]C, loss of osmotic responsiveness, which was associated with the formation of the hexagonal II phase in the plasma membrane and subtending lamellae, was the predominant form of injury. In protoplasts isolated from leaves of cold-acclimated oat, loss of osmotic responsiveness was the predominant form of injury at all injurious temperatures; however, the hexagonal II phase was not observed. Rather, injury was associated with the occurrence of localized deviations of the plasma membrane fracture plane to closely appressed lamellae, which we refer to as the "fracture-jump lesion." Although the freeze-induced lesions in the plasma membrane of protoplasts of spring oat were identical with those reported previously for protoplasts of winter rye, they occurred at significantly higher temperatures that correspond to the lethal freezing temperature.     

Studies on the Endoplasmic Reticulum : IV. Its Form and Distribution during Mitosis in Cells of Onion Root Tip

Cells of onion and garlic root tips were examined under the electron and phase contrast microscopes after fixation in KMnO₄. Special attention was focused on the distribution and behavior of the endoplasmic reticulum (ER) during the several phases of mitosis. Slender profiles, recognized as sections through thin lamellar units of the ER (most prominent in KMnO₄-fixed material), are distributed more or less uniformly in the cytoplasm of interphase cells and show occasional continuity with the nuclear envelope. In late prophase the nuclear envelope breaks down and its remnants plus cytoplasmic elements of the ER, which are morphologically identical, surround the spindle in a zone from which mitochondria, etc., are excluded. During metaphase these ER elements persist and concentrate as two separate systems in the polar caps or zones of the spindle. At about this same time they begin to proliferate and to invade the ends of the spindle. The invading lamellar units form drape-like partitions between the anaphase chromosomes. In late anaphase, their advancing margins reach the middle zone of the spindle and begin to fray out. Finally, in telophase, while elements of the ER in the poles of the spindle coalesce around the chromosomes to form the new envelope, the advancing edges of those in the middle zone reticulate at the level of the equator to form a close lattice of tubular elements. Within this, which is identified as the phragmoplast, the earliest signs of the cell plate appear in the form of small vesicles. These subsequently grow and fuse to complete the separation of the two protoplasts. Other morphological units apparently participating in mitosis are described. Speculation is provided on the equal division or not of the nuclear envelope and the contribution the envelope fragments make to the ER of the new cell.     

Understanding Odor Information Segregation in the Olfactory Bulb by Means of Mitral and Tufted Cells

Odor identification is one of the main tasks of the olfactory system. It is performed almost independently from the concentration of the odor providing a robust recognition. This capacity to ignore concentration information does not preclude the olfactory system from estimating concentration itself. Significant experimental evidence has indicated that the olfactory system is able to infer simultaneously odor identity and intensity. However, it is still unclear at what level or levels of the olfactory pathway this segregation of information occurs. In this work, we study whether this odor information segregation is performed at the input stage of the olfactory bulb: the glomerular layer. To this end, we built a detailed neural model of the glomerular layer based on its known anatomical connections and conducted two simulated odor experiments. In the first experiment, the model was exposed to an odor stimulus dataset composed of six different odorants, each one dosed at six different concentrations. In the second experiment, we conducted an odor morphing experiment where a sequence of binary mixtures going from one odor to another through intermediate mixtures was presented to the model. The results of the experiments were visualized using principal components analysis and analyzed with hierarchical clustering to unveil the structure of the high-dimensional output space. Additionally, Fisher''s discriminant ratio and Pearson''s correlation coefficient were used to quantify odor identity and odor concentration information respectively. Our results showed that the architecture of the glomerular layer was able to mediate the segregation of odor information obtaining output spiking sequences of the principal neurons, namely the mitral and external tufted cells, strongly correlated with odor identity and concentration, respectively. An important conclusion is also that the morphological difference between the principal neurons is not key to achieve odor information segregation.