The effect of media tonicity on Escherichia coli resistance to heating with different gradient

The influence of NaCl water solutions and glycerine hypertonic concentration on the survival of bacteria Escherichia coli B/r heated with different values of heat drop was investigated. It was shown that the transfer of cell suspensions from isotonic conditions to media with raised osmotic pressure, preliminarily heated up to 60 degrees C, and the following heating at this temperature inhibited differences in cell sensitivity to heating at different heat drop. Unlike, it was found that the transfer of cell suspensions from isotonic conditions to hypertonic media before and after heating at 60 degrees C increased differences in resistance of these microorganisms to heating at different heat drop. It is proposed that different resistance of bacteria to damaging action of hyperthermia at different heat drop, and a modified influence of hypertonic solutions on these differences may be due to heat induced destabilization of cell osmotic homeostasis. The extent of expression of this destabilization may be determined by a quantitative ratio of osmotic pressure values in the cell-suspension medium system in particular temperature and tonic environmental conditions.     

渗透压对痢疾志贺菌水通道蛋白glpF基因表达的影响

目的探讨细菌水-甘油通道蛋白（GlpF）的生理功能及其对生长繁殖的影响。方法将用简并PCR发现表达水通道蛋白glpF基因的痢疾志贺菌接种于不同渗透压的液体培养基和添加了GlpF功能抑制剂的相应培养基中,培养不同时间后取培养液检测其生长繁殖量,RT-PCR分析其GlpF的表达。结果痢疾志贺菌GlpF的表达随培养基渗透压的改变而变化,在低渗培养基中的表达低于在等渗环境中;在高渗透压的培养基中,其表达显著高于在等渗培养基中。在加入Hg2＋抑制剂抑制GlpF的表达后,在低渗培养基中,未明显影响细菌的生长繁殖,但在较高渗透压的培养基中,细菌的繁殖量显著少于在未加Hg2＋抑制剂的同样渗透压培养基中。结论在非等渗环境中,细菌GlpF的表达对细菌细胞内外水分的调节,维持胞内环境稳定起到重要作用,尤其在高渗透压环境中更为明显。     

Evaluation of an innate immune reaction to parasites in earthworms

Encapsulation is an essential process of the invertebrate immune system and includes the prophenoloxidase (proPO) cascade. We present an assay for evaluating this immune response, now newly adapted to earthworms. Coelomic fluid is withdrawn and coelomocytes are stained with l-Dopa. We studied assay repeatability and the correlation between number of PO-active cells and infection level of the parasitic protozoan Monocystis sp. in the earthworm Lumbricus terrestris. Our study showed high assay repeatability although the expected negative relationship between PO-active coelomocytes and parasite load was not observed; yet a suggestion toward a positive relationship was detected. This finding is contrary to previous assumptions that presume coelomocyte concentrations to be the independent variable determining parasite load.     

Analysis of the role of microfilaments and microtubules in acquisition of bipolarity and elongation of fibroblasts in hydrated collagen gels

Fibroblasts in situ reside within a collagenous stroma and are elongate and bipolar in shape. If isolated and grown on glass, they change from elongate to flat shape, lose filopodia, and acquire ruffles. This shape change can be reversed to resemble that in situ by suspending the cells in hydrated collagen gels. In this study of embryonic avian corneal fibroblasts grown in collagen gels, we describe for the first time the steps in the acquisition of the elongate shape and analyze the effect of cytoskeleton-disrupting drugs on filopodial activity, assumption of bipolarity, and cell elongation within extracellular matrix. We have previously shown by immunofluorescence that filopodia contain actin but not myosin and are free of organelles. The cell cortex is rich in actin and the cytosol, in myosin. By using antitubulin, we show in the present study that microtubules are aligned along the long axis of the bipolar cell body. The first step in assumption of the elongate shape is extension of filopodia by the round cells suspended in collagen, and this is not significantly affected by the drugs we used: taxol to stabilize microtubules; nocodazole to disassemble microtubules; and cytochalasin D to disrupt microfilaments. The second step, movement of filopodia to opposite ends of the cell, is disrupted by cytochalasin, but not by taxol or nocodazole. The third step, extension of pseudopodia and acquisition of bipolarity similarly requires intact actin, but not microtubules. If fibroblasts are allowed to become bipolar before drug treatment, moreover, they remain so in the presence of the drugs. To complete the fourth step, extensive elongation of the cell, both intact actin and microtubules are required. Retraction of the already elongated cell occurs on microtubule disruption, but retraction requires an intact actin cytoskeleton. We suggest that the cell interacts with surrounding collagen fibrils via its actin cytoskeleton to become bipolar in shape, and that microtubules interact with the actin cortex to bring about the final elongation of the fibroblast.     

渗透压突然改变对大肠埃希菌水通道蛋白GlpF基因表达的影响

目的 探讨水-甘油通道蛋白(glycerol protein ficilitator,GlpF)的生理功能及对细菌生长繁殖的影响.方法 将大肠埃希菌接种于等渗透压的液体培养基(1 IM)培养,18h后,突然改变培养液的渗透压,在30、60、120 min时间点检测细菌的A600nm值,RT-PCR分析其GlpF的表达.结果 突然改变渗透压后,大肠埃希菌数量均有不同程度的减少.在1/2 IM组、1/4 IM组细菌的A600nm值与其各自对照组相比,变化并不明显,但其细菌GlpF表达量明显降低.在高渗组,2 IM组的A600nm值与等渗组相比变化不大,而其GlpF表达量明显高于等渗组.结论 在环境渗透压突然改变时,细菌可以通过调控GlpF的表达来实现对细菌细胞内外水份的调节,以维持胞内环境稳定.     

Evidence for fascin cross-links between the actin filaments in coelomocyte filopodia

Coelomocyte filopodia are composed primarily of actin and a 57000 D protein, which is antigenically related to the actin cross-linking protein found in needles produced in vitro from extracts of sea urchin oocytes. In this report we present electron micrographs of the filopodia which show longitudinal striations corresponding to bundled, F-actin filaments and transverse bands corresponding to a cross-linking protein. The periodicities of the spacings are 90 and 130 Å respectively. Interestingly, the filaments in the filopodia are less well ordered than those in needles, probably as a result of the speed with which the filopodia are assembled in the transforming coelomocytes. These results indicate that the well ordered needles are a good in vitro model for important in vivo structures.     

Effect of hypertonic stress on mammalian cell lines and its relevance to freeze-thaw injury

The effect of subjecting the mammalian cell lines MRC-5 and CHO to hypertonic salt concentrations (0.16 to 2.4 m) and returning them to isotonic conditions was investigated. Parameters for measuring cell size, viability and release of radiochemical markers were used to determine the relative susceptibilities of the two cell lines to hypertonic stress and the relative effects of increasing and decreasing hypertonicity. The aim of this study was to determine how great a role hypertonic stress plays in freeze-thaw damage of mamalian cells. This type of study has been extensively used for erythrocytes but not for nucleated mamamlian cell lines.The findings were that considerable cell shrinkage occurred, with a minimum size at 0.6 m NaCl, but that this caused no cell injury or death. Injury, measured by cation leakage and release of membrane and cytoplasmic labels occurred whilst the cell was swelling after reaching its minimum volume. MRC-5 cells succumbed at relatively low salt concentrations and became denatured. CHO cells withstood far high salt concentrations but were then damaged during dilution back to isotonic conditions. Comparison of the data obtained from hypertonic stress experiments and freeze-thaw experiments showed many similarities for CHO cells and indicated that the cell membrane could withstand high salt concentrations both at constant and changing temperatures but were prone to injury on dilution back to isotonic conditions. MRC-5 cells were shown to be very prone to cold shock and the results indicated that they probably succumb to damage and death during the hypertonic phase of cooling rather than thawing thus explaining their much lower survival from freeze-thaw experiments than CHO cells. The influence of DMSO in delaying cell damage to higher salt concentrations and lessening disruptive swelling during dilution were also demonstrated.     

Quantitative examination of a perfusion microscope for the study of osmotic response of cells

The perfusion microscope was developed for the study of the osmotic response of cells. In this microscope, the cells are immobilized in a transparent chamber mounted on the stage and exposed to a variety of milieus by perfusing the chamber with solutions of different concentrations. The concentration of the supplied solution is controlled using two variable-speed syringe pumps, which supply an isotonic solution and a hypertonic solution. Before using this system to characterize the osmotic response of cells, the change in the concentration of NaCl solution flowing through the chamber is examined quantitatively using a laser interferometer and an image processing technique. The NaCl concentration is increased from an isotonic condition to a hypertonic condition abruptly or gradually at a given constant rate, and decreased from a hypertonic condition to an isotonic condition. It is confirmed that the concentration is nearly uniform in the cross direction at the middle of the chamber, and the change in the NaCl concentration is reproducible. The average rate of increase or decrease in the measured concentration agrees fairly well with the given rate when the concentration is changed gradually at a constant rate. The rate of the abrupt change is also determined to be the highest limit achieved by the present method. As the first application of using the perfusion microscope for biological studies, the volume change of cells after exposure to a hypertonic solution is measured. Then, the hydraulic conductivity of the cell membrane is determinedfrom the comparison of the volume change between the experiment and the theoretical estimation for the measured change in the NaCl concentration of the perfused solution.     

Osmotic tolerance of in vitro produced porcine blastocysts assessed by their morphological integrity and cellular actin filament organization

This experiment investigated the osmotic tolerance limits of the morphology and the cellular actin filament organization of porcine blastocysts. In vitro produced Day 6 blastocysts were subjected to osmotic treatments with sucrose solutions of different osmolalities (75, 150, 210, 600, 1200, and 2400 mOsm) and one isotonic solution (NCSU-23, 285 mOsm). Blastocysts were then either fixed immediately, or cultured for 18 h and subsequently fixed with formalin. The morphology of the treated blastocysts was examined under a stereomicroscope and the integrity of the cellular actin filaments of the blastocysts was examined by confocal microscopy after staining with Alexa Fluor 488 phalloidin. The results indicated that there was a significant relationship between the osmotic levels and the probability of blastocysts exhibiting disrupted cellular actin filaments. In addition, blastocysts also collapsed in proportion to the levels of osmotic treatments. The osmotic tolerance limits which would maintain 70% of the blastocysts with their original morphology immediately after the treatment were 90 and 170%, respectively, of isotonicity. After 18 h of culture, the osmotic tolerance limits were 61 and 163%, respectively, of isotonicity. Similarly, the osmotic conditions relative to isotonicity which would maintain the integrity of cellular actin filaments in 70% of treated blastocysts had to be within the range of 87 and 147% immediately after the treatment and 87 and 169% after 18 h of culture. Collectively, these data indicate that in vitro produced porcine blastocysts are very sensitive to osmotic stress. This information can be used to optimize cryopreservation procedures for porcine embryos.     

A small diffusion pore in the outer membrane of Pseudomonas aeruginosa

The permeability properties of the outer membrane of Pseudomonas aeruginosa were re-examined, since the reported conclusions are conflicting [Decad, M. G. and Nikaido, H. (1976) J. Bacteriol. 128, 325-336; Caulcott, C. A., Brown, M. R. W. and Gonda, I. (1984) FEMS Microbiol. Lett. 21, 119-123]. On the basis of the experimental evidence to be described below we conclude that the exclusion limit of the outer membrane of P. aeruginosa is smaller than the size of uncharged disaccharides but larger than the size of hexose. This conclusion is based on the following evidence. Penetration of monosaccharides into the expanded periplasm was large and that of disaccharides was small, after the cells were plasmolyzed with 600 mosM NaCl. A significant amount of protein was released after osmotic down-shock of cells treated with the hypertonic monosaccharides but not of cells treated with the hypertonic saccharides larger than disaccharides. Centrifuged pellets of cells treated with hypertonic di, tri and tetrasaccharides weighed about 15-20% less than that of cells treated with the isotonic monosaccharide, suggesting that the osmotic pressure was exerted on the outer membrane causing dehydration and shrinking of the cells. By contrast, cells treated with the hypertonic pentose and hexoses weighed about 0.1% and 6% less, respectively, than cells treated with the isotonic saccharide, suggesting that pentose diffused through the outer membrane freely.     

小檗碱激活人结肠癌细胞容积敏感的氯通道

本文旨在研究小檗碱对人结肠癌细胞(SW480)氯通道的作用.采用膜片钳技术记录小檗碱激活的SW480全细胞氯电流,并用高渗和低渗灌流液、以及氯通道阻断剂研究该电流的生理学和药理学特性.结果显示,当细胞处在等渗液中,可在SW480细胞膜上记录到微弱且稳定的背景电流;小檗碱(10 nmol/L)可诱发SW480细胞迅速产生...     

三种兽药添加剂对土壤赤子爱胜蚓的毒理学研究

喹乙醇、阿散酸和土霉素是应用极广的兽药添加剂,在畜牧业的发展中起着特殊的作用,但其使用不当或过量使用会给环境带来很大危害.采用土壤中的重要生物赤子爱胜蚓作为生物材料,分别研究了喹乙醇、阿散酸和土霉素对赤子爱胜蚓的急性毒性和对赤子爱胜蚓体腔细胞DNA的损伤.结果表明,喹乙醇溶液法LC₅₀>2000mg·L^-1,滤纸法LC₅₀>5.71×10^-2mg·cm^-2,而阿散酸和土霉素在所设最大受试剂量内没有毒性.单细胞凝胶电泳试验结果表明,阿散酸没有引起蚯蚓体腔细胞DNA明显损伤;喹乙醇和土霉素均能引起一定程度的DNA损伤,中、高剂量组与阴性对照相比有极显著差异(P<0.01).因此,有必要加强对以上兽药添加剂,特别是喹乙醇和土霉素的管理,控制用药时间和剂量,以确保人体健康和环境安全.     

Hemolysis of human red blood cells by freezing and thawing in solutions containing polyvinylpyrrolidone: relationship with postthypertonic hemolysis and solute movements

An investigation was made into the effects of the presence of polyvinylpyrrolidone (PVP) on changes in human red blood cells suspended in hypertonic solutions, on posthypertonic hemolysis, and on freezing at temperatures down to ?12 °C.PVP is very effective at reducing hemolysis when the red blood cells are frozen at temperatures down to ?12 °C. However, the membranes of the cells recovered on thawing have become very permeable to sodium and potassium ions and there is a much increased hemolysis if the cells are resuspended in an isotonic solution of sodium chloride.The presence of PVP does not affect the dehydration of the cells or the development of a change in membrane permeability when the cells are shrunken in hypertonic solutions at 0 °C. Neither does its presence in the hypertonic solution reduce the extent of posthypertonic hemolysis at 4 °C (as measured by the hemolysis on resuspension in an isotonic solution of sodium chloride), but it is more effective than sucrose at reducing hemolysis when present in the resuspension solution. It is concluded that the PVP is able to prevent swelling and hemolysis of cells which are very permeable to cations by opposing the colloid osmotic pressure due to the hemoglobin. However, this does not explain how PVP is able to protect cells against freezing damage at high cooling rates, and a mechanism by which it might do this is discussed.     

Are osmotic forces involved in influenza virus-cell fusion?

The kinetics of the fusion process of unsealed and resealed erthyrocyte ghosts with influenza virus (A/PR8/34, A/Chile 1/83), were measured under hypotonic, isotonic and hypertonic conditions using a recently developed fluorescence assay (Hoekstraet al. (1984)Biochemistry 23:5675–5681]. No correlation between the external osmotic pressure and kinetics and extent of fusion was observed. Influenza viruses fuse as effectively with unsealed ghosts as with resealed ghosts. It is concluded that osmotic forces as well as osmotic swelling of cells are not necessary for virus-cell membrane fusion.     

The influenza virus-induced fusion of erythrocyte ghosts does not depend on osmotic forces

The role of osmotic forces and cell swelling in the influenza virus-induced fusion of unsealed or resealed ghosts of human erythrocytes was investigated under isotonic and hypotonic conditions using a recently developed fluorescence assay (Hoekstra, D., De Boer, T., Klappe, K., Wilschut, J. (1984) Biochemistry 23, 5675-5681). The method is based on the relief of fluorescence selfquenching of the fluorescent amphiphile octadecyl rhodamine B chloride (R18) incorporated into the ghost membrane as occurs when labeled membranes fuse with unlabeled membranes. No effect neither of the external osmotic pressure nor of cell swelling on virally mediated ghost fusion was established. Influenza virus fused unsealed ghosts as effectively as resealed ghosts. It is concluded that neither osmotic forces nor osmotic swelling of cells is necessary for virus-induced cell fusion. This is supported by microscopic observations of virus-induced fusion of intact erythrocytes in hypotonic and hypertonic media. A disruption of the spectrin-actin network did not cause an enhanced cell fusion at acidic pH of about 5 or any fusion at pH 7.4.     

Hypertonic induction of aquaporin-5 expression through an ERK-dependent pathway

Aquaporin-5 (AQP5) is a water channel protein expressed in lung, salivary gland, and lacrimal gland epithelia. Each of these sites may experience fluctuations in surface liquid osmolarity; however, osmotic regulation of AQP5 expression has not been reported. This study demonstrates that AQP5 is induced by hypertonic stress and that induction requires activation of extracellular signal-regulated kinase (ERK). Incubation of mouse lung epithelial cells (MLE-15) in hypertonic medium produced a dose-dependent increase in AQP5 expression; AQP5 protein peaked by 24 h and returned to baseline levels within hours of returning cells to isotonic medium. AQP5 induction was observed only with relatively impermeable solutes, suggesting an osmotic pressure gradient is required for induction. ERK was selectively activated in MLE-15 cells by hypertonic stress, and inhibition of ERK activation with two distinct mitogen-activated extracellular regulated kinase kinase (MEK) inhibitors, U0126 and PD98059, blocked AQP5 induction. AQP5 induction was also observed in the lung, salivary, and lacrimal glands of hyperosmolar rats, suggesting potential physiologic relevance for osmotic regulation of AQP5 expression. This report provides the first example of hypertonic induction of an extrarenal aquaporin, as well as the first association between mitogen-activated protein kinase signaling and aquaporin expression.     

Role of the F-actin cytoskeleton in the RVD and RVI processes in Ehrlich ascites tumor cells.

The role of the F-actin cytoskeleton in cell volume regulation was studied in Ehrlich ascites tumor cells, using a quantitative rhodamine-phalloidin assay, confocal laser scanning microscopy, and electronic cell sizing. A hypotonic challenge (160 mOsm) was associated with a decrease in cellular F-actin content at 1 and 3 min and a hypertonic challenge (600 mOsm) with an increase in cellular F-actin content at 1, 3, and 5 min, respectively, compared to isotonic (310 mOsm) control cells. Confocal visualization of F-actin in fixed, intact Ehrlich cells demonstrated that osmotic challenges mainly affect the F-actin in the cortical region of the cells, with no visible changes in F-actin in other cell regions. The possible role of the F-actin cytoskeleton in RVD was studied using 0. 5 microM cytochalasin B (CB), cytochalasin D (CD), or chaetoglobosin C (ChtC), a cytochalasin analog with little or no affinity for F-actin. Recovery of cell volume after hypotonic swelling was slower in cells pretreated for 3 min with 0.5 microM CB, but not in CD- and ChtC-treated cells, compared to osmotically swollen control cells. Moreover, the maximal cell volume after swelling was decreased in CB-treated, but not in CD- or Chtc-treated cells. Following a hypertonic challenge imposed using the RVD/RVI protocol, recovery from cell shrinkage was slower in CB-treated, but not in CD- or Chtc-treated cells, whereas the minimal cell volume after shrinkage was unaltered by either of these treatments. It is concluded that osmotic cell swelling and shrinkage elicit a decrease and an increase in the F-actin content in Ehrlich cells, respectively. The RVD and RVI processes are inhibited by 0.5 microM CB, but not by 0.5 microM CD, which is more specific for actin.     

Dynamic aspects of filopodial formation by reorganization of microfilaments

The coelomocytes of the sea urchin, Strongylocentrotus droebachiensis, may be prevented from clotting with 50 mM ethylene glycol-bis(beta-aminoethyl)-N,N,N',N'-tetraacetate, 50 mM Tris-HCl, pH 7.8 and subsequently separated into various cell types on sucrose gradients. One cell type, the petaloid coelomocyte, spontaneously undergoes a striking morphological transformation to a form exhibiting numerous, t-in cytoplasmic projections (filopodia). Moreover, the transformation is reversible. Ultrastructurally, the formation of the filopodia results from a progressive reorganization of actin-containing filaments into bundles that are radially oriented. The formation of the filament bundles is initiated at the cell's periphery and proceeds inward. Simultaneously, the cytoplasm in between the bundles is withdrawn, exposing finger-like filopodia. Ultimately, the filopodia can be extended by up to four times their original length. Biochemically, actin is the most abundant protein in while cell homogenates and is extractable in milligram quantities via acetone powders. An actomyosin complex may also be isolated from these cells and is presumed to be active in producing the various forms of motility observed.