Freezing the effect of eutectic crystallization on biological membranes

Thylakoids from isolated spinach chloroplasts were frozen in the presence of various concentrations of inorganic and organic salts, amino acids and sugars and the kinetics of inactivation of cyclic photophosphorylation with phenazine methosulfate and of electron transport reactions were measured as a function of temperature.During freezing of membranes in the presence of neutral nontoxic compounds membrane damage did not occur until the eutectic temperature was reached. Then photophosphorylation became rapidly inactivated. With weakly membrane-toxic compounds there was a slow inactivation during freezing followed by rapid inactivation at the eutectic temperature. Freezing in the presence of strongly membrane-toxic compounds led to inactivation of photophosphorylation before the eutectic temperature was reached. The temperature at which eutectic crystallization occurred was dependent on the nature of the solutes present. The ratio between solute and membranes was also important: the lower the initial concentration of solutes added to membrane suspensions the lower the temperature at which eutectic solidification occurred. Some compounds such as mannitol crystallized gradually during the decrease in temperature; in this case inactivation of photophosphorylation took place parallel to the crystallization process.In contrast to photophosphorylation, electron transport reactions were not decreased during eutectic freezing in the presence of neutral membrane-protective compounds. Rather a stimulation of electron transport was observed. However, in the presence of inorganic salts or of sodium succinate, electron transport reactions were also inactivated in addition to photophosphorylation during eutectic solidification. This inactivation seems to be a salt effect and may not directly be related to the crystallization process. Various soluble enzymes and the Ca²⁺-dependent ATPase of thylakoids were not affected by eutectic crystallization.The results demonstrate that eutectic crystallization which may take place during freezing is a factor in membrane damage and has to be considered as a possible cause of membrane alterations in in vitro studies on freezing resistance.     

Sesquiterpene lactones from Vernonia scorpioides and their in vitro cytotoxicity

Fresh leaves of Vernonia scorpioides are widely used in Brazil to treat a variety of skin disorders. Previous in vivo studies with extracts of this species had also demonstrated a high antitumor potential. This paper reports isolation of four sesquiterpene lactones (hirsutinolides and glaucolides), together with diacetylpiptocarphol, 8-acetyl-13-etoxypiptocarphol, luteolin, apigenin, and ethyl caffeate from fresh leaves and flowers of Vernonia scorpioides. The hypothesis that hirsutinolide 3 is formed during extraction was verified theoretically using Density Functional Theory. The effects of isolated compounds on in vitro tumor cells were investigated, as well as their genotoxicity by means of an in vitro comet assay. The results indicate that glaucolide 2 and hirsutinolide 4 are toxic to HeLa cells. These compounds were genotoxic in vitro, a property that appears to be related to the presence of their epoxy groups, which has been a more reliable indication of toxicity than substitution on C-13 or the presence of α,β-unsaturated keto-groups. These results need to be replicated in vivo in order to ascertain their toxicity.     

Leishmania tarentolae: Utility as an in vitro model for screening of antileishmanial agents

Primary screens for antileishmanial compounds use Leishmania species pathogenic to humans that must be handled under biosafety conditions that cannot be adopted or guaranteed everywhere. Leishmania tarentolae, a parasite isolated from the gecko Tarentolae annularis, has not been considered pathogenic to humans. Promastigotes of L. tarentolae have been previously used as a eukaryotic expression system for the production of recombinant proteins and in the amplification of genes involved in resistance to antileishmanial drugs. To validate the use of this Leishmania species in the screening of antileishmanial drugs, the sensitivity of axenic and intracellular amastigotes of L. tarentolae was compared to the sensitivity showed by Leishmania species causative of human leishmaniasis. The ability of L. tarentolae to grow as axenic amastigotes is first described while its ability to infect several mammalian cells has been confirmed. L. tarentolae amastigotes offer a suitable model for the in vitro screening of compounds for antileishmanial activity.     

Origin of the short circuit decay profile and maintenance of the cation transport capacity of the larval lepidopteran midgut in vitro and in vivo

The larval midguts of Hyalophora cecropia and Manduca sexta contain two primary cell types: a columnar cell and a goblet cell. Employing scanning electron microscopy, goblet cells were found to contain within their cavities semi-viscous matrix plugs. Massive release or removal of goblet matrix plugs is noted following a variety of physiological insults to the tissues, including stretching, gaseous carbon dioxide anesthesia, gut evacuation, gut excision in the absence of cold anesthesia, and mounting the tissue in a chamber designed for the study of cation transport. A reduction in the capacity to actively transport cations in vitro or in vivo follows each of these treatments.When a current (short-circuit current = I_SC) is imposed across the isolated larval midgut that is equal but opposite in direction to the natural electromotive force generated by the tissue, a characteristic irreversible I_SC (and potential = P.D.) decay profile is obtained. This decay profile normally consists of three phases : a transient increase in I_SC, a rapid decay in I_SC and a slower but continuous decay in I_SC. The transient increase in I_SC is an artifact associated with anoxia. The duration of this transient increase in I_SC is related to elapsed time between mounting the midgut in a chamber designed for measuring I_SC and the time at which the hemolymph side of the tissue is bathed in oxygenated saline. The rapid decay is associated with massive release of matrix plugs, an increase in membrane K⁺ conductance and a reduced capacity to transport K⁺. The slower decay is associated with further loss of plugs coupled with cell death. Cell death is caused by inadequacies in the saline normally employed to bathe the midgut epithelium in vitro. These inadequacies promote tissue histolysis and disruption of the normal epithelial topology.     

Synthesis and storage of a neurohormone in insect brains in vitro

The build-up of neurosecretory material in the median neurosecretory cells and fibre tracts of cultured cockroach brains was demonstrated by staining and bioassay. Examination of the cultured brains by electron microscopy showed active production of neurosecretory granules after 3 days in vitro. The close correlation of the results obtained by these different methods of assay leaves little doubt that a neurohormone is being synthesized and stored.     

Histochemical lipid studies on Schistosoma mansoni adults maintained in situ and in vitro

Haseeb M. A., Eveland L. K. and Fried B. 1984. Histochemical lipid studies on Schistosoma mansoni adults maintained in situ and in vitro. International Journal for Parasitology14: 83–88. Schistosoma mansoni male and female adults were incubated at 37°C for 0.5 and 1.0 h in Earle's balanced salt solution containing 0.1% glucose and 0.5% lactalbumin hydrolysate, then examined by histochemistry and scanning electron microscopy. Histochemical analysis of cryostat sections stained with Oil Red O showed that males contain neutral lipid mainly in the parenchyma and tubercles, while females contain neutral lipid in the vitellaria. Neutral lipids are released from the tubercles of both paired and unpaired males maintained in vitro. There is evidence of in situ lipid transfer from males to blood vessel walls. Neutral lipid was not seen in females from unisexual infections. Sudan Black B staining fo total lipids is positive in tubercles, parenchyma, and vitellaria. Nile Blue Sulphate stains acidic lipids in male caecal walls. Scanning electron microscopy reveals no tegumental damage.     

The effect of freezing on thylakoid membranes in the presence of organic acids

The effect of salts of organic acids on washed and non-washed chloroplast membranes during freezing was investigated. Thylakoids were isolated from spinach leaves (Spinacia oleracea L.) and, prior to freezing, salts of various organic acids or inorganic salts or both were added. Freezing occurred for 3 to 4 hours at −25 C. After thawing membrane integrity was investigated by measuring the activity of cyclic photophosphorylation.     

Effects of high-temperature stress on various biomembranes of leaf cells in situ and in vitro

The sensitivity of photosynthetic and respiratory functions to supraoptimal temperature stress was compared after heating of leaves, protoplasts and membrane systems of spinach (Spinacia oleracea L. cv. Monatol) and lettuce (Valerianella locusta [L.] Betcke) in situ and in vitro.

After heating of whole leaves or protoplasts, endogenous respiration was not or only slightly affected at temperatures which caused a marked decrease of photosynthesis. This was manifested when mitochondria and thylakoids were isolated from heat-treated leaves. In the presence of exogenous substrates, mitochondrial electron transport and phosphorylation were even somewhat stimulated compared to the controls.

Inactivation of net CO₂ uptake of whole leaves following heat stress and of the photochemical activities of chloroplast membranes isolated from heat-treated leaves of the same origin occurred nearly simultaneously. In protoplasts, photosynthesis was inactivated at temperatures far below those which caused drastic changes in the integrity of the tonoplast and the plasmalemma. This indicates that damage occurring within the chloroplasts rather than alterations in the compartmentation of the cell is responsible for the high sensitivity of photosynthesis to supraoptimal temperature stress.

Mitochondria and thykaloids isolated from the same preparation of intact leaves under comparable conditions and subjected to heat treatment in vitro, however, were inactivated nearly in the same temperature range. Thus, mitochondria are much more stable within their cytoplasmic environment.

     

Effects of prolonged freezing stress on the photosynthetic apparatus of moderately hardy leaves as assayed by chlorophyll fluorescence kinetics

Abstract Moderately frost-hardy leaves of the wintergreen broadleaf woody shrubs Pyracantha coccinea and Ligustrum ovalifolium and the winter annual herb Spinacia oleracea were subjected to extended freezing stress up to 15 d at temperatures 2–8°C above the mean lethal temperature (LT₅₀). After thawing, the fast kinetics of in vivo chlorophyll fluorescence of photosystem II (PSII) and the potential of linear photosynthetic electron transport of isolated thylakoid membranes was measured at room temperature. The lower the minimum freezing temperature and the longer the time of exposure, the greater was the suppression of the fluorescence signals of the leaves and decrease of the electron transport capacity of the thylakoid membranes. The pattern of inactivation of PSII -mediated electron flow, i.e. inhibition of photoreaction to photochemistry and/or electron donation to the photochemical reaction, during long-term freezing at temperatures somewhat above the LT₅₀ of the leaves was similar to that observed earlier after relatively brief exposure of leaves and isolated thylakoid membranes to more severe freezing stress. As injury occurred during freezing in complete darkness, it is likely that prolonged winter stress under natural environmental conditions causes changes in the photosynthetic apparatus of moderately hardy leaves which are not due to photoinhibition.     

Ligh-dependent activity of glutamate synthase in vitro

Glutamate synthase from rice (Oryza sativa) green leaves was assayed in a chloroplast reconstituted system. The enzyme activity was totally dependent on externally supplied thylakoid membranes and ferredoxin in the light. Glutamate synthase activity was also detected from etiolated leaves with photoreduced ferredoxin as an electron donor.     

Low immunogenicity of allogeneic human umbilical cord blood-derived mesenchymal stem cells in vitro and in vivo

Evaluation of the immunogenicity of human mesenchymal stem cells (MSCs) in an allogeneic setting during therapy has been hampered by lack of suitable models due to technical and ethical limitations. Here, we show that allogeneic human umbilical cord blood derived-MSCs (hUCB-MSCs) maintained low immunogenicity even after immune challenge in vitro. To confirm these properties in vivo, a humanized mouse model was established by injecting isolated hUCB-derived CD34+ cells intravenously into immunocompromised NOD/SCID IL2γnull (NSG) mice. After repeated intravenous injection of human peripheral blood mononuclear cells (hPBMCs) or MRC5 cells into these mice, immunological alterations including T cell proliferation and increased IFN-γ, TNF-α, and human IgG levels, were observed. In contrast, hUCB-MSC injection did not elicit these responses. While lymphocyte infiltration in the lung and small intestine and reduced survival rates were observed after hPBMC or MRC5 transplantation, no adverse events were observed following hUCB-MSC introduction. In conclusion, our data suggest that allogeneic hUCB-MSCs have low immunogenicity in vitro and in vivo, and are therefore “immunologically safe” for use in allogeneic clinical applications.     

Salt treatment induces frost hardiness in leaves and isolated thylakoids from spinach

Frost hardiness of spinach (Spinacia oleracea L.) leaves was increased by high concentrations of NaCl in the hydroponic culture medium. Freezing damage was determined by measurement of slow chlorophyll fluorescence quenching after freezing of leaves. Both the osmolality of the leaf sap and forst hardiness of the leaves were linearly correlated with the salt concentration in the hydroponic culture medium. Freezing damage occurred, irrespective of the extent of frost hardening, when dehydration of cells during extracellular ice formation decreased cellular volume to approximately 14% of the volume of unfrozen cells. The resistance of isolated, washed thylakoids against mechanical and chemical damage by freezing was investigated. Chemical damage by freezing caused by salt accumulation was measured as release of chloroplast coupling factor (CF1; EC 3.6.1.3), and mechanical damage was measured as release of the lumenal protein plastocyanin from the membranes during an in-vitro freeze-thaw cycle. Isolated thylakoids from salt-treated frost-hardy spinach and those from plants hardened under natural conditions did not exhibit improved tolerance against chemical freezing stress exerted by high salt concentrations. They were, however, more hardy than thylakoids from unhardened control leaves against mechanical damage by freezing.Abbreviation CF1 peripheral part of chloroplast coupling factor ATPase     

Schistosoma mansoni: Ultrastructural damages due to complement on schistosomula in vitro

The ultrastructural damage induced by complement in vitro on the schistosomula of Schistosoma mansoni was studied using transmission and scanning electron microscopy. The sequence of events which leads to the killing of the schistosomula is as follows: (a) the lytic activity starts at the anterior end of the schistosomula; (b) lesions progress simultaneously in two distinct directions: from the anterior to the posterior end, and from the outer membrane to the muscle layer; (c) “bubbles” appear around parasite which might correspond to increased membrane permeability; (d) the lytic activity of the late complement components occur in the syncytium; (e) the schistosomula lose their tegument completely, exposing the muscle layer. These findings and our previous work suggest that the activation of the alternate pathway and late complement components is sufficient, without antibodies or cells, to kill schistosomula in vitro.     

Changes in macrophage status in vivo during infection with and immunity to Leishmania enriettii

The changing status of peritoneal macrophages in guinea pigs infected with Leishmania enriettii has been examined. It was possible to demonstrate that, at certain times during a primary infection and following attempted reinfection of immune animals, the response of peritoneal macrophages to lymphokine contact in vitro was altered. At these times the harvested cells appeared to behave in vitro as if they had been at least partially activated in vivo before removal. They were unresponsive to lymphokine in the migration inhibition assay, and contact with lymphokine in culture caused a rapid increase in the level of glucose oxidation in these cells. It is suggested that changes in the response of macrophages to lymphokine in vitro may be one way of monitoring activation in vivo.     

Crucial role of vinexin for keratinocyte migration in vitro and epidermal wound healing in vivo

In the process of tissue injury and repair, epithelial cells rapidly migrate and form epithelial sheets. Vinexin is a cytoplasmic molecule of the integrin-containing cell adhesion complex localized at focal contacts in vitro. Here, we investigated the roles of vinexin in keratinocyte migration in vitro and wound healing in vivo. Vinexin knockdown using siRNA delayed migration of both HaCaT human keratinocytes and A431 epidermoid carcinoma cells in scratch assay but did not affect cell proliferation. Induction of cell migration by scratching the confluent monolayer culture of these cells activated both EGFR and ERK, and their inhibitors AG1478 and U0126 substantially suppressed scratch-induced keratinocyte migration. Vinexin knockdown in these cells inhibited the scratch-induced activation of EGFR, but not that of ERK, suggesting that vinexin promotes cell migration via activation of EGFR. We further generated vinexin (−/−) mice and isolated their keratinocytes. They similarly showed slow migration in scratch assay. Furthermore, vinexin (−/−) mice exhibited a delay in cutaneous wound healing in both the back skin and tail without affecting the proliferation of keratinocytes. Together, these results strongly suggest a crucial role of vinexin in keratinocyte migration in vitro and cutaneous wound healing in vivo.     

Freezing injury in cold-acclimated and unhardened spinach leaves

Spinach plants (Spinacia oleracea L.) were frost-hardened by cold-acclimation to 1° C or kept in an unhardy state at 20°/14° C in phytotrons. Detached leaves were exposed to temperatures below 0°C. Rates of photosynthetic CO₂ uptake by the leaves, recorded after frost treatment, served as a measure of freezing injury. Thylakoid membranes were isolated from frost-injured leaves and their photosynthetic activities tested. Ice formation occurred at about-4° to-5° C, both in unhardened and cold-acclimated leaves. After thawing, unhardened leaves appeared severely damaged when they had been exposed to-5° to-8° C. Acclimated leaves were damaged by freezing at temperatures between-10° to-14° C. The pattern of freezing damage was complex and appeared to be identical in hardened and unhardened leaves: 1. Inactivation of photosynthesis and respiration of the leaves occurred almost simultaneously. 2. When the leaves were partly damaged, the rates of photosynthetic electron transport and noncyclic photophosphorylation and the extent of light-induced H⁺ uptake by the isolated thylakoids were lowered at about the same degree. The dark decay of the proton gradient was, however, not stimulated, indicating that the permeability of the membrane to-ward protons and metal cations had not increased. 3. As shown by partial reactions of the electron transport system, freezing of leaves predominantly inhibited the oxygen evolution, but photosystem II and photosystem I-dependent electron transport were also impaired. 4. Damage of the chloroplast envelope was indicated by a decline in the percentage of intact chloroplasts found in preparations from injured leaves. The results are discussed in relation to earlier studies on freezing damage of thylakoid membranes occurring in vitro.Abbreviations Chl chlorophyll - DCPIP 2,6-dichlorophenol indophenol - HEPES N-2-hydroxyethylpiperazine-N-2-ethane sulfonic acid - MES 2(N-morpholino) ethane sulfonic acid     

Simulation of in situ freezing damage of the photosynthetic apparatus by freezing in vitro of thylakoids suspended in complex media

Chloroplast thylakoid membranes were isolated from leaves of unhardened and cold-acclimated spinach (Spinacia oleracea L.). For freezethaw treatment, the membranes were suspended in complex media composed to simulate the solute concentrations in the chloroplast stroma in the unhardened and hardened states of the leaves. In particular, high concentrations of amino acids were applied for simulating the hardened state. After frost treatment, photosynthetic activities and chlorophyll fluorescence parameters of the thylakoids were tested to determine the degree of freezing damage. The results revealed a pattern of freezing injury similar to that observed upon frost treatment of thylakoids in situ. A major manifestation of damage was the inhibition of photosynthetic electron transport. Uncoupling of photophosphorylation, which is the dominating effect of freezing of thylakoids suspended in binary solutions (e.g., containing one sugar and one inorganic salt), was also visible but less pronounced in the complex media. Thylakoids obtained from cold-acclimated leaves did not exhibit an increased frost tolerance in vitro, as compared with thylakoids from unhardened plants. The results, furthermore, indicated a strong protective effect of free amino acids at the concentrations and composition found in chloroplasts of hardened leaves. The presence of inorganic salts in the complex media slightly stabilized rather than damaged the membranes during freezing. It is concluded that inactivation of thylakoids in situ may be understood as the destabilizing action of the combined solutes surrounding the thylakoids, occurring when solute concentration is raised due to freezing of water.Abbreviations Chl chlorophyll - DCMU 3-(3,4-dichlorophenyl)-1,1-dimethylurea - Hepes 4-(2-hydroxyethyl)-1-piper-azineethanesulfonic acid - PSI photosystem I - PSII photosystem II     

Freezing of isolated thylakoid membranes in complex media. VI. The effect of pH

Thylakoid membranes isolated from spinach leaves (Spinacia oleracea L. cv. Monatol) were used as a model biomembrane system for evaluating the significance of the hydrogen ion activity for cryoprotection. After freeze-thaw treatment in a buffered complex medium adjusted to various pH, light-induced photosynthetic membrane reactions were determined at optimum proton concentration. When thylakoids were suspended at hydrogen ion activities above and below the physiologically important pH range, irreversible inhibition of membrane functions was significantly less distinct after freezing at -15 degrees C than after storage for the same time at 0 degree C. It is suggested that thylakoid preservation at subfreezing temperatures could be due to temperature- and concentration-induced changes of the proton activity in the unfrozen part of the system and retardation of the temperature-dependent aging processes of the isolated membranes. In addition, the increase in the concentration of cryoprotective compounds during freezing could stabilize chloroplast membranes against the deleterious effect of unfavorable high and low proton concentrations. Thylakoid injury brought about by lowering the pH was primarily due to dissociation of the chloroplast coupling factor (CF1), which increased the proton permeability of the membranes and caused inhibition of photophosphorylation. In media adjusted to more alkaline pH, inactivation of the water oxidation system was an initial result of membrane damage. Then, noncyclic photophosphorylation was limited by photosystem II-mediated electron flow. Photosystem I-driven electron transport was substantially more stable over a wide pH range.     

Salt-induced redox-independent phosphorylation of light harvesting chlorophyll a/b proteins in Dunaliella salina thylakoid membranes

This study investigated the regulation of the major light harvesting chlorophyll a/b protein (LHCII) phosphorylation in Dunaliella salina thylakoid membranes. We found that both light and NaCl could induce LHCII phosphorylation in D. salina thylakoid membranes. Treatments with oxidants (ferredoxin and NADP) or photosynthetic electron flow inhibitors (DCMU, DBMIB, and stigmatellin) inhibited LHCII phosphorylation induced by light but not that induced by NaCl. Furthermore, neither addition of CuCl₂, an inhibitor of cytochrome b₆f complex reduction, nor oxidizing treatment with ferricyanide inhibited light- or NaCl-induced LHCII phosphorylation, and both salts even induced LHCII phosphorylation in dark-adapted D. salina thylakoid membranes as other salts did. Together, these results indicate that the redox state of the cytochrome b₆f complex is likely involved in light- but not salt-induced LHCII phosphorylation in D. salina thylakoid membranes.     

KPNA7, a nuclear transport receptor,promotes malignant properties of pancreatic cancer cells in vitro

Pancreatic cancer is an aggressive malignancy and one of the leading causes of cancer deaths. The high mortality rate is mostly due to the lack of appropriate tools for early detection of the disease and a shortage of effective therapies. We have previously shown that karyopherin alpha 7 (KPNA7), the newest member of the alpha karyopherin family of nuclear import receptors, is frequently amplified and overexpressed in pancreatic cancer. Here, we report that KPNA7 expression is absent in practically all normal human adult tissues but elevated in several pancreatic cancer cell lines. Inhibition of KPNA7 expression in AsPC-1 and Hs700T pancreatic cancer cells led to a reduction in cell growth and decreased anchorage independent growth, as well as increased autophagy. The cell growth effects were accompanied by an induction of the cell cycle regulator p21 and a G1 arrest of the cell cycle. Interestingly, the p21 induction was caused by increased mRNA synthesis and not defective nuclear transport. These data strongly demonstrate that KPNA7 silencing inhibits the malignant properties of pancreatic cancer cells in vitro and thereby provide the first evidence on the functional role for KPNA7 in human cancer.