Respiratory metabolism of L-929 cells at different water contents and volumes

Oxygen consumption was measured in mouse L-929 cells whose volumes and water contents were reduced by adding sorbitol to the medium. The volume of water lost due to a given sorbitol supplement exceeded the loss in apparent cell volume. An explanation is given for this discrepancy. The rate of oxygen uptake in the absence of exogenous respiratory substrate was essentially the same in cells whose total volume was reduced by 45%, amounting to a loss of about 70% of the total cell water, compared to controls at 'physiological' volume and water content. Cells under these same conditions responded to added substrates (pyruvate, glucose, and glutamine) and inhibitors (iodoacetate and 2-deoxyglucose) in nearly the same way as control cells. These observations are in accord with and add to previous work showing that very large fluctuations in cell volume and water content have only modest effects on the rates and directions of a variety of metabolic processes. The results are interpreted in terms of current views on the composition and organization of the aqueous compartments of eucaryotic cells.     

Some structural,biochemical and biophysical characteristics of L-929 cells growing in the presence of hyperosmotic sorbitol concentrations

Mouse L-929 cells (a fibroblast-like line) were transferred from normal growth medium to one supplemented with 0.3 M sorbitol, doubling the normal external osmotic pressure. After a short lag phase and minimal cell death, the cells began to grow, and the growth rate reached that of controls after about one week. These chronically grown cells (S) have been compared to those of control cultures (C) with regard to general morphology, ability to reverse when returned to normal condition, water content, volume and selected metabolic parameters. S-cell cultures exhibited considerable heterogeneity but most contained vesicle-like cytoplasmic structures, sometimes in abundance. These structures do not appear to be completely bounded by membranes, but that is uncertain. S cells become larger and contain more water than C cells; however, the ratio of total water to total dry mass is indistinguishable from controls suggesting regulation at that level. S and C cells were found to be remarkably similar, on a per cell basis, with regard to their rate of respiration and the incorporation of glucose into metabolites and macromolecules. These results are interpreted in terms of current views on the composition and organization of the aqueous compartments of animal Cells.     

Beta-D-xyloside induced modulations of glycosaminoglycans, proliferation, and cytoskeletal organization of rat liver myofibroblast-like cells (transformed fat storing cells)

Transformed fat storing cells, i.e. myofibroblast-like cells are the major source of proteoglycans in injured liver. In the present study p-nitrophenyl-beta-D-xylopyranoside (PNP-Xyl), a specific metabolic inhibitor of proteoglycan synthesis, was used to analyze some details of altered glycosaminoglycan metabolism, proliferation, morphology and cytoskeletal organization of myofibroblast-like cells (secondary cultures of fat storing cells) under conditions of abrogated proteoglycan synthesis. PNP-Xyl increased dose-dependently the synthesis of [35S] sulfate-labelled medium glycosaminoglycans, among which chondroitin sulfate formation was stimulated predominantly. The distribution and composition of glycosaminoglycans in the cellular and cell surface compartments were affected differently. Production of medium hyaluronan was reduced by more than 40% at 5 mM PNP-Xyl. The compound inhibited dose-dependently the mitotic activity of myofibroblast-like cells without affecting viability. The morphologic appearance was changed at 5 mM PNP-Xyl and the organization and expression of desmin and smooth muscle iso-alpha-actin, both important markers of myofibroblast-like cells, were also modified by PNP-Xyl. Inhibition of proliferation, morphologic changes, and cytoskeletal disorganization were fully and rapidly reversible upon removal of the drug. The results support the notion of a direct or indirect role of proteoglycans in maintaining important functions of myofibroblast-like cells in culture.     

L-929 cells under hyperosmotic conditions. Water, Na+, and K+

Changes in cell water content resulting from sorbitol addition to the environment of L-929 cells were evaluated gravimetrically using 14C-labeled polyethylene glycol as a probe of extracellular space. Reductions in cell water were proportional to sorbitol supplements up to 0.6 molal, above which no further measurable decrease occurred. No volume regulation occurred for at least 1 h but the percentage of cell water lost was quickly regained when physiological conditions were restored. The amount of cell water lost because of a given hyperosmotic exposure was found to exceed the loss of cell volume. That discrepancy could be the result of an overestimation of extracellular space and/or an underestimation of cell volume reduction as a result of in-folding of the cell surface. Na+ and K+ were also measured in cells of variable water content and volume: no significant change occurred in the amounts of these ions per cell, but large increases in total cell concentration resulted from hyperosmotic exposure. The sum of Na+ and K+ concentrations exceeds the total osmotic pressure of the medium indicating that an appreciable fraction of Na+ and K+ must be bound to fixed charges within the cells. The results are evaluated in the context of intracellular organization.     

Survival of Saccharomyces cerevisiae Y5 during starvation in the presence of osmotic supports

The viability of starved or dying Saccharomyces cerevisiae Y5 cells was extended for up to 1,500 h by adding osmotic supports (0.6 M sorbitol or mannitol) to the starvation media. Replacement of these polyols with 0.3 M KCl, 0.5 M MgSO4, or 0.2 M MgCl2 postponed the death of cells incubated in N-free medium, but accelerated it in distilled water.     

Survival of Saccharomyces cerevisiae Y5 during starvation in the presence of osmotic supports.

The viability of starved or dying Saccharomyces cerevisiae Y5 cells was extended for up to 1,500 h by adding osmotic supports (0.6 M sorbitol or mannitol) to the starvation media. Replacement of these polyols with 0.3 M KCl, 0.5 M MgSO4, or 0.2 M MgCl2 postponed the death of cells incubated in N-free medium, but accelerated it in distilled water.     

Reactions of yeast cells to glycerol treatment alterations to membrane structure and glycerol uptake

Summary Ultrastructural alterations to the plasmalemma and tonoplast ofSaccharomyces cerevisiae were studied after incubation in hypertonic solutions of glycerol and sorbitol. After 20 to 30 minutes incubation in glycerol, the cells had shrunk to about 40% of their original volume. Large depressions of the plasmalemma were then always found associated with the typical plasmalemma invaginations. The vacuoles of treated cells changed to an irregular form, the tonoplast intramembranous particles were clustered, and large smooth areas appeared. After 6 to 12 hours incubation, cell and vacuole volume, as well as plasmalemma and tonoplast ultrastructure, had reverted to normal. The rate of recovery was strongly temperature dependent.Protoplasts could be similarly shrunk, but no alterations to the plasmalemma ultrastructure were then observed; however, the tonoplast revealed particle clustering as observed in whole cells. Protoplasts also reverted to normal volume and ultrastructure after prolonged incubation. Cells and protoplasts treated with sorbitol showed similar phenomena, but remained shrunken.By the use of radioactive tracers, glycerol was shown to penetrate cells, protoplasts and isolated vacuoles, but no uptake of sorbitol could be demonstrated.During the glycerol permeation period (0.5 to 6 hours), numerous vesicles were found in the cytoplasm and these were possibly engulfed by the vacuole. Associated with the engulfment, patches of tonoplast intramembranous particles were found in a semicrystalline array. Osmotic stress induced alterations to membrane ultrastructure, due to the use of cryoprotective agents, are discussed.A preliminary note of the paper was given at the Sixth European Congress on Electron Microscopy, Jerusalem, 1976.     

Metabolically optimised BHK cell fed-batch cultures

The aim of this work was the optimisation of a fed-batch culture by metabolic confinement of BHK21 cells producing an antibody/cytokine fusion protein with potential application in tumour-targeted therapy. Previous results showed that at very low nutrient concentrations, a metabolic shift towards more efficient metabolic pathways occurs. The application of those results in the optimisation of a fed-batch culture resulted in higher cell growth (0.020 vs. 0.016 h(-1)) and cell viability, higher maximum cell concentration (2.5 vs. 1.1x10(6) cell ml(-1)), longer culture span (17 versus nine days) and higher product titre (60% increase), in relation to batch culture. This was achieved by maintaining glucose at 0.3 mM and glutamine at 0.2 mM through the addition of a concentrated solution based on the estimations of future nutrient consumption and growth rates through off line measurements. The production of toxic metabolites such as lactate and ammonia was reduced, especially the lactate production, which was markedly decreased due to the metabolic confinement of the cells. In conclusion, it was possible to increase the final titre of the recombinant antibody/cytokine fusion protein by confining the metabolism of the cells to an energetically more efficient state.     

Changes in the Ultrastructure of Prasiola crispa ssp. antarctica under Salinity Stress

The supralittoral green alga Prasiola crispa, collected in Antarctica, was grown at salinities ranging from 0.35 %o to 175 %o. Its ultrastructure was examined by electron microscopy and compared with the ultrastructure of plants grown under standard conditions (35 %o). The thalli had very thick cell walls, making up approximately 50% of the thallus volume. Most of the protoplast was occupied by a single chloroplast. Numerous starch granules in the chloroplast were enlarged under hypo-osmotic conditions and reduced in volume when subjected to hyperosmotic stress indicating a metabolic shift towards low molecular weight organic osmolytes under the latter conditions. Whilst vacuoles were lacking in plants cultured at salinities between 0.35 %o and 35 %o, they developed under hyperosmotic conditions and possibly may serve as compartments accumulating inorganic ions. Growth, photosynthesis and dark respiration were strongly inhibited under higher salinities, whilst they remained almost unaffected under lower salinities (Jacob et al., 1991).     

L-929 cells under hyperosmotic conditions

Changes in cell water content resulting from sorbitol addition to the environment of L-929 cells were evaluated gravimetrically using¹⁴C-labeled polyethylene glycol as a probe of extracellular space. Reductions in cell water were proportional to sorbitol supplements up to 0.6 molal, above which no further measurable decrease occurred. No volume regulation occurred for at least 1 h but the percentage of cell water lost was quickly regained when physiological conditions were restored. The amount of cell water lost because of a given hyperosmotic exposure was found to exceed the loss of cell volume. That discrepancy could be the result of an overestimation of extracellular space and/or an underestimation of cell volume reduction as a result of infolding of the cell surface. Na⁺ and K⁺ were also measured in cells of variable water content and volume: no significant change occurred in the amounts of these ions per cell, but large increases in total cell concentration resulted from hyperosmotic exposure. The sum of Na⁺ and K⁺ concentrations exceeds the total osmotic pressure of the medium indicating that an appreciable fraction of Na⁺ and K⁺ must be bound to fixed charges within the cells. The results are evaluated in the context of intracellular organization.     

大豆和豆粕水提物致草鱼原代肝细胞损伤作用的研究

为了探讨大豆和豆粕对肝细胞是否具有损伤作用及其作用机制,以大豆水提物(Soybean aqueous extract,SAE)和豆粕水提物(Soybean meal aqueous extract, SMAE)为实验材料,以草鱼(Ctenopharyngodon idella)离体培养的原代肝细胞为试验对象, SAE和SMAE分别以终浓度为0、0.5、5.0和10.0 mg/mL加入到细胞培养液中。采用CCK-8法检测细胞活力,电子显微镜观察细胞超微结构, Hoechst 33285染色观察细胞核形态,检测细胞培养液中乳酸脱氢酶(LDH)和丙二醛(MDA)的含量,检测细胞内超氧化物歧化酶(SOD)和还原性谷胱甘肽(GSH)的活性,流式细胞仪检测细胞凋亡率、线粒体膜电位(MMP)及活性氧(ROS)。利用转录组技术分析对照组、SAE组(5.0 mg/mL)和SMAE组(5.0 mg/mL)肝细胞差异表达基因(Differentially expressed genes, DEGs)及其富集的相关通路。结果表明,随着水提物浓度的升高,肝细胞活力逐渐下降,有明显的剂量效应关系(P<0.0...     

Role of Ca2+ in sorbitol release from rat inner medullary collecting duct (IMCD) cells under hypoosmotic stress

The role of Ca2+ was studied in the release of the organic osmolyte sorbitol from rat IMCD cells in response to hypoosmotic stress. When cells were exposed to hypoosmotic media, sorbitol release was greatly reduced in Ca-free media which, on readmission of Ca2+, returned to control values. Under isoosmotic conditions, the ionophore A23187 stimulated sorbitol release without any effect on cell volume. Addition of trifluoperazine, a calmodulin inhibitor, but not the protein kinase C inhibitor H-7, inhibited the osmotically-activated sorbitol release. These results suggest that sorbitol release is a calmodulin-dependent event, possibly activated by a rise in intracellular calcium as a result of cell swelling.     

myo-Inositol Metabolism in 41 A3 Neuroblastoma Cells: Effects of High Glucose and Sorbitol Levels

Neuroblastoma cells were used to determine the effect of high carbohydrate and polyol levels on myo-inositol metabolism. The presence of elevated concentrations of glucose or sorbitol caused a significant decrease in both inositol accumulation and incorporation into phospholipid. These conditions, however, did not alter the accumulation of the other phospholipid head groups or the growth rate and water content of the cells. Two weeks of growth in either of the modified conditions was necessary to obtain a maximal effect on inositol incorporation. In contrast, growth in elevated concentrations of fructose, mannitol, or dulcitol had no effect on inositol metabolism. The reduced inositol accumulation and incorporation into lipids seen with glucose or sorbitol supplementation resulted in a decrease in the total phosphatidylinositol content of the cell without changing the levels of the other phospholipids. Kinetic analysis of cells grown in the presence of elevated glucose indicated that V'max for inositol uptake was significantly decreased with little change in the K'm. These data suggest that glucose decreases myo-inositol uptake in this system by noncompetitive inhibition. Cells grown in the presence of increased glucose also had elevated levels of intracellular sorbitol and decreased levels of myo-inositol. These results suggest that the high levels of glucose and sorbitol which exist in poorly regulated diabetes may be at least partially responsible for diabetic neuropathy via a reduction in the cellular content of myo-inositol and phosphatidylinositol. This system may be a useful model to determine the effect of reduced inositol phospholipid levels on neural cell function.     

Temperature dependence of the preferential interactions of ribonuclease A in aqueous co-solvent systems: thermodynamic analysis.

The temperature dependence of preferential solvent interactions with ribonuclease A in aqueous solutions of 30% sorbitol, 0.6 M MgCl2, and 0.6 M MgSO4 at low pH (1.5 and 2.0) and high pH (5.5) has been investigated. This protein was stabilized by all three co-solvents, more so at low pH than high pH (expect 0.6 M MgCl2 at pH 5.5). The preferential hydration of protein in all three co-solvents was high at temperatures below 30 degrees C and decreased with a further increase in temperature (for 0.6 M MgCl2 at pH 5.5, this was not significant), indicating a greater thermodynamic instability at low temperature than at high temperature. The preferential hydration of denatured protein (low pH, high temperature) was always greater than that of native protein (high pH, high temperature). In 30% sorbitol, the interaction passed to preferential binding at 45% for native ribonuclease A and at 55 degrees C for the denatured protein. Availability of the temperature dependence of the variation with sorbitol concentration of the chemical potential of the protein, (delta mu(2)/delta m3)T,p,m2, permitted calculation of the corresponding enthalpy and entropy parameters. Combination with available data on sorbitol concentration dependence of this interaction parameter gave (approximate) values of the transfer enthalpy, delta H2,tr, and transfer entropy delta S2,tr. Transfer of ribonuclease A from water into 30% sorbitol is characterized by positive values of the transfer free energy, transfer enthalpy, transfer entropy, and transfer heat capacity. On denaturation, the transfer enthalpy becomes more positive. This increment, however, is small relative to both the enthalpy of unfolding in water and to the transfer enthalpy of the native protein from water a 30% sorbitol solution.     

Role of estrogen and androgen in maintaining the preovulatory follicle

Summary The effects of Nitromifene citrate (CI 628), an antiestrogen, and Flutamide, an antiandrogen, on the ultrastructure and viability of the preovulatory follicle and granulosa cells were examined both in vivo and in vitro. In vivo administration of either antihormone induced degeneration within the granulosa cells. In some of the affected granulosa cells, the nuclear material was condensed while the cytoplasm and associated organelles were unaltered. In others, the density of the cytoplasm was reduced, the smooth endoplasmic reticulum was dilated but the nucleus remained unaltered. In vitro, either antihormone reduced granulosa-cell viability but the granulosa cells were twenty times more sensitive to CI 628 than to Flutamide. In addition, exposure to CI 628 induced nuclear condensation without affecting the cytoplasm, while Flutamide induced the deterioration of the cytoplasm without altering the nucleus. These observations suggest that: (1) both estrogen and androgens control the viability of the granulosa cells and thereby the follicle, (2) the action of estrogen and androgen is mediated through receptors within the granulosa cells since these antihormones prevent the nuclear uptake of their respective hormone, (3) the granulosa cells of preovulatory follicles appear to be more dependent on estrogen than on androgen, and (4) each steroid appears to have a specific role in maintaining the granulosa cell; estrogens control the integrity of the nucleus while androgens preserve the cytoplasmic organization of the granulosa cell.The authors are indebted to Dr. Neri of Schering AG for donating the Flutamide and to Dr. Westland of Warner-Lambert/Parke-Davis for providing CI-628     

Cellulase production in continuous culture by Trichoderma reesei on xylose-based media

Trichoderma reesei (QM 9414) produced cellulase in continuous culture, on media containing xylose (1%) supplemented with sorbose (0.3%) to induce cellulase production. Maximum cell mass of 4.54 kg/m(3) occurred at pH 4.0 and a dilution rate of 0.0391 h(-1) where residual substrate was 0.43 kg/m(3), but no cellulase was produced. Maximum cellulase production of 0.69 FPU occurred at pH 3.5 and a dilution rate of 0.0110 h(-1), where cell mass production was 2.56 kg/m(3) and residual substrate was 0.15 kg/m(3). Monod kinetic constants, corrected for endogenous metabolism, were 0.091 h(-1), 0.469 kg/m(3), 0.00923 h(-1), and 0.470 kg cells/kg xylose at pH 3.5, for the maximum specific growth rate, Michaelis-Menten coefficient, endogenous metabolism coefficient, and yield coefficient, respectively. Specific growth rate fitted a maturation time model, which predicted decreasing maturation time with increasing pH.     

Mass Isolation and Preservation of Viable Sperm Cells in Brassica campestris var.purpurea

The two-step osmotic shock and grinding methods reported by Yang and Zhou (1989) were modified for isolation of viable sperm cells in large quantities from pollen grains of Brassica campestris var. purpurea. Factors affecting the yield and survival of isolated sperm cells have been investigated. These included physiological status of donor flowers, sucrose concentration used for pollen hydration, basic media, protectants and osmotica supplemented in the medium etc. As a result, two procedures have been developed. For osmotic shock method, pollen grains at the day of anthesis were hydrated in 25% sucrose solution for 30 min and, after centrifugation and removal of the supernatant, the pellet was shocked by a medium containing 12.5% sucrose, 0.1 g/L KNO3, 0.36 g/L CaCl2, 2H2O, 0.3% potassium dextran sulphate (PDS), 0.6% bovine serum albumin (BSA), and 0.3% polyvinylpyrrolidone (PVP). The viable sperm yield was 34%. After removal of pollen wall debris by filtration and centrifugation, the sperm cell-rich pellets were resuspended in a medium containing 20% sucrose, 5% sorbitol, 0.1 g/L KNOs, 0.36g/L CaCl2·2H2O, 0.6% BSA and 0.3% PDS, and preserved at 4℃ for two days. For grinding method, the pollen grains hydrated in 30% sucrose solution for 30 min. were resuspended in a medium containing 20% sucrose, 5% sorbitol, 0.1g/L QNO3, 0.36g/L CaCl2·2H2O, 0.3% PDS, 0.6% BSA, 0.3% PVP and 20 μg/ml fluorescein diacetate, then ground with a glass homogenizer to release the sperm cells. The viable sperm yield was up to 86%. Following filtration and centrifugation for removal of pollen wall debris, the sperm cells were stored at 4℃ in the same medium but without supplementation of PVP. Tested by fluorochromatic reaction, the sperm cells could survive up to one week with a gradual decline of viability. Cytological observations revealed that pairs of ellipsoidal sperm cells just released were linked together; one of the pair had a long tail-like extension which also show fluorochromasia. Soon after, the sperm cells separated and turned to be spherical. The present results open a prospect to use isolated viable sperm cells for further experimental manipulations.     

Cytotoxicity of free versus multilayered polyelectrolytes

The cytotoxicity of polyelectrolytes commonly employed for layer-by-layer deposition of polyelectrolyte multilayers (PEMUs) was assessed using rat smooth muscle A7r5 and human osteosarcoma U-2 OS cells. Cell growth, viability, and metabolic assays were used to compare the responses of both cell lines to poly(acrylic acid), PAA, and poly(allylamine hydrochloride), PAH, in solution at concentrations up to 10 mM and to varying thicknesses of (PAA/PAH) PEMUs. Cytotoxicity correlated with increasing concentration of solution polyelectrolytes for both cell types and was greater for the positively charged PAH than for the negatively charged PAA. While metabolism and proliferation of both cell types was slower on PEMUs than on tissue culture plastic, little evidence for direct toxicity on cells was observed. In fact, evidence for more extensive adhesion and cytoskeletal organization was observed with PAH-terminated PEMUs. Differences in cell activity and viability on different thickness PEMU surfaces resulted primarily from differences in attachment for these adhesion-dependent cell lines.     

Systemic multicompartmental effects of the gut microbiome on mouse metabolic phenotypes

To characterize the impact of gut microbiota on host metabolism, we investigated the multicompartmental metabolic profiles of a conventional mouse strain (C3H/HeJ) (n=5) and its germ‐free (GF) equivalent (n=5). We confirm that the microbiome strongly impacts on the metabolism of bile acids through the enterohepatic cycle and gut metabolism (higher levels of phosphocholine and glycine in GF liver and marked higher levels of bile acids in three gut compartments). Furthermore we demonstrate that (1) well‐defined metabolic differences exist in all examined compartments between the metabotypes of GF and conventional mice: bacterial co‐metabolic products such as hippurate (urine) and 5‐aminovalerate (colon epithelium) were found at reduced concentrations, whereas raffinose was only detected in GF colonic profiles. (2) The microbiome also influences kidney homeostasis with elevated levels of key cell volume regulators (betaine, choline, myo‐inositol and so on) observed in GF kidneys. (3) Gut microbiota modulate metabotype expression at both local (gut) and global (biofluids, kidney, liver) system levels and hence influence the responses to a variety of dietary modulation and drug exposures relevant to personalized health‐care investigations.     

Flux control at the ecosystem level

The analysis of metabolic control has reached a high level of understanding of the regulation in cellular metabolic pathways. However, as soon as we leave the realm of cell compartments and enter into the demise of coordination at the organism or ecosystem level, control theory enters unstable ground. Organisms act as individuals. Here, I compare control features at different levels of organization (cell to ecosystem), to indicate how we may approach understanding of control in complex and multiple-species systems.