Effects of dimethylsulfoxide (DMSO) on the digestive-lysosomal system in Paramecium caudatum

The effects of DMSO (dimethylsulfoxide) on cell growth and on the digestive-lysosomal system of axenically grown Paramecium caudatum were studied. A general protocol of exposing cells to different concentrations of DMSO at the beginning of each of the four processes in the digestive cycle enabled us to analyze the effect of DMSO at each step. Vacuole formation and the beginning of a digestive cycle were initiated by adding latex beads to the cells. Maximum cell densities at stationary phase of growth were found to be inversely proportional to DMSO between 0.5 and 1.75%, and the duration of the generation time was exponentially proportional. At 2% DMSO cellular division was completely blocked, and above 2% it was cytotoxic. P. caudatum survived for 8 h in 4% DMSO and died instantaneously in 10%. This inhibitory effect on growth was reversible, though this reversibility might depend on the duration and level of DMSO exposure. DMSO exerted a dose- and time-dependent inhibitory effect on the rate of DV formation but had little effect on the acidification-condensation and the lysosome fusion-digestion processes. The size of the DV formed was also reduced, and this effect was dose-but not time-dependent; vacuole size reduction occurred immediately with DMSO exposure, and no further reduction was observed during exposures of up to 24 h. DMSO at 3 and 4% inhibited vacuole defecation, but the cells could overcome this inhibition when exposed to DMSO for longer periods.(ABSTRACT TRUNCATED AT 250 WORDS)     

Polar uncoating of tobacco mosaic virus (TMV) with dimethylsulfoxide (DMSO) and subsequent reassembly of partially stripped TMV

Summary Increasing concentrations of dimethylsulfoxide (DMSO) strip tobacco mosaic virus (TMV) stepwise from the 3 end. The RNA tail increases in length up to 2,000 nucleotides (nu) reaching a region of very strong protein-RNA affinity. Thereafter, uncoating occurs from the other end and produces a second RNA tail 500 nu long. Further stripping of TMV proceeds from both ends, the long tail increasing in length up to 4,000 nu and the short one increasing more moderately and remaining below 2,000 nu. The region of strongest protein-RNA affinity is located between 4,000 and 5,000 nu away from the 3 end.Using the same conditions as for in vitro TMV reassembly, it is possible to recoat the RNA tails with viral protein preferentially in the 5 direction.The advantages of DMSO in studies of TMV protein-RNA interactions are discussed.     

Relation between enzyme release and metabolic changes in reversible anoxic injury of myocardial cells

Cultured adult cardiac myocytes were exposed to anoxia under substrate-free conditions. When compared to the metabolic changes in the oxygen deficient organ, those in the anoxic cell culture proceed in a similar, yet prolonged manner. Release of cytosolic enzymes starts with minor energetic disturbances and proceeds in close correlation to the actual ATP decay. Below 2 μmol. ATP/g_WW, an increasing number of cells becomes irreversibly damaged, but above, 30 min reoxygenation leads to extensive recovery of the whole preparation. The results indicate that leakage of cytosolic enzymes during the early stage of anoxia is due to a gradual protein release from the individual cells, related to reversible membrane alterations.     

Functional preservation of the mammalian kidney. III. Ultrastructural effects of perfusion with dimethylsulfoxide (DMSO)

Isolated rabbit kidneys were perfused at 37 ° C with a cell-free solution containing 0.0, 1.4, 2.1, and 2.8 m DMSO for 60 min. Electron microscopic examination of cortical structures revealed relatively high sensitivity of proximal tubular cells to DMSO-induced alterations, which included cytoplasmic clarification and disruption of microvilli. Glomeruli exhibited proliferation of rough endoplasmic reticulum, but were otherwise well preserved after perfusion with and without DMSO. Vascular endothelial cells were also intact after perfusion with DMSO, suggesting that DMSO-induced resistance changes are not necessarily associated with degeneration of capillary cells. Perfusion without DMSO resulted in good preservation of all cortical structures. These studies suggest that ultrastructural effects of DMSO must be considered in assessment of damage done in whole kidneys during freeze-preservation studies.     

Enhanced release of rosmarinic acid from Coleus blumei permeabilized by dimethyl sulfoxide (DMSO) while preserving cell viability and growth

Continuous permeabilization of preconditioned Coleus blumei cells with dimethyl sulfoxide (DMSO) is shown to be an effective strategy for the enhanced release of rosmarinic acid (RA) while preserving cell viability. When nonpreconditioned cells were permeabilized with DMSO, they lost their viability at DMSO concentrations higher than a critical value located between 0.1% and 0.5% DMSO. Product release was low [0.49 g RA/100 g dry cell weight (DCW)] at 0.1% DMSO. Preconditioning cells at 0.1% DMSO ensured high viability at DMSO concentrations of 0.5%, 1.0%, and 1.5%. Product release reached a maximum of 2.85 g RA/100 g DCW at 0.5% DMSO, which was 66.4% of the total rosmarinic acid produced. (c) 1992 John Wiley & Sons, Inc.     

On the lability of dimethylsulfoxide (DMSO) coordinated to the [Ru(II)-NO(+)] species: X-ray structures of mer-[RuCl(3)(DMSO)(2)(NO)] and mer-[RuCl(3)(CD(3)CN)(DMSO)(NO)

The syntheses of nitrosyl–dimethylsulfoxide–ruthenium(II) complexes with general formula mer-[RuCl₃(L)(DMSO)(NO)] (L=DMSO or CD₃CN) is reported. The mer-[RuCl₃(DMSO)₂(NO)] (1) complex was obtained from the reaction of [RuCl₃(NO)] with the sulfoxide ligand in acetone. The mer-[RuCl₃(CD₃CN)(DMSO)(NO)] (2) compound was obtained from mer-[RuCl₃(DMSO)₂(NO)] maintained in deuterated acetonitrile. These data suggest a slow kinetic reaction due the low lability of the DMSO ligand coordinated to the {Ru^II–NO⁺} species. The crystal and molecular structures of (1) and (2) have been determined from X-ray studies. Crystal data: for (1), monoclinic, P2₁/c, a=8.8340(2) Å, b=12.0230(3) Å, c=13.7064(4) Å, β=114.546(2)°, Z=4, R1=0.0429; for (2), monoclinic, P21/n, a=10.0180(7) Å, b=9.5070(7) Å, c=13.3340(9) Å, β=102.264(4)°, Z=4, R1=0.0472. The spectroscopic characterization of (1), in solid state (infrared spectrum) and in solution (nuclear magnetic resonance and cyclic voltammetry) is also described.     

Evaluation of freezing injury and freeze-thaw injury to membranes of Saskatoon serviceberry twigs by measuring HCN release

The influence of thawing on freeze-injured Saskatoon serviceberry ( Amelanchier alnifolia Nutt.) twigs was evaluated by refreezing freeze-thawed twigs and comparing the HCN release at -5°C fro these twigs to the HCN release at -5°C from twigs that had not been thawed. An effect of thawing depended on the physiological state of the twigs, on the degree of freezing stress, or on both. Manifestation of membrane injury does not have an absolute dependence on thawing. Post-thaw temperature influences manifestation of injury, since twigs warmed to 30°C released more HCN than twigs warmed to 1°C when refrozen to -5°C. Although thawing and post-thaw conditions can influence the magnitude of membrane injury, the critical event leading to injury occurs while plants are frozen.     

Determination of high-energy phosphate compounds and inorganic phosphate by reversed-phase high-performance liquid chromatography: evaluation of myocardial metabolic status in aerobically perfused and hypoxic mouse heart

The present paper describes a simple HPLC method designed for measuring high-energy phosphate (HEP) compounds in a single run and inorganic phosphate (P_i) in an other short run under the same HPLC conditions. Inorganic phosphate was estimated by using thymidine phosphorylase (EC 2.4.2.4) which catalyzes a reaction involving inorganic phosphate to produce 2-deoxyribose 1-phosphate and thymine. The thymine/P_i stoichiometry was 1. The method provides a reproducible instrument for evaluating myocardial high-energy metabolism under physiological and pathological conditions.     

Regulation of arachidonic acid release by enzyme(s) of rat brain cortex

Brain cortex membranes labeled with [14C]arachidonic acid were used as the source of substrate and enzyme for the assay of arachidonic acid (AA) liberation. A significant amount of AA was released Ca2(+)-independently, mainly from phosphatidic acid, polyphosphoinositides and phosphatidylserine. Quinacrine, inhibitor of phospholipase A2 (PLA2), suppressed AA release by 60% and neomycin, inhibitor of phospholipase C (PLC) by about 30%. Both inhibitors applied together have an additive effect. Physiological calcium level elevated AA liberation by 50%, whereas 2 mM calcium enhanced this process by a further 30%. Carbachol, exclusively in the presence of calcium, activated AA release selectively from phosphatidylinositol and diglycerides. We suggest that Ca2(+)-independent PLA2 and PLC play an important role in AA liberation, and that physiological increments of calcium may have serious implications.     

Cardioprotection of electroacupuncture against myocardial ischemia-reperfusion injury by modulation of cardiac norepinephrine release

Augmentation of cardiac sympathetic tone during myocardial ischemia has been shown to increase myocardial O(2) demand and infarct size as well as induce arrhythmias. We have previously demonstrated that electroacupuncture (EA) inhibits the visceral sympathoexcitatory cardiovascular reflex. The purpose of this study was to determine the effects of EA on left ventricular (LV) function, O(2) demand, infarct size, arrhythmogenesis, and in vivo cardiac norepinephrine (NE) release in a myocardial ischemia-reperfusion model. Anesthetized rabbits (n = 36) underwent 30 min of left anterior descending coronary artery occlusion followed by 90 min of reperfusion. We evaluated myocardial O(2) demand, infarct size, ventricular arrhythmias, and myocardial NE release using microdialysis under the following experimental conditions: 1) untreated, 2) EA at P5-6 acupoints, 3) sham acupuncture, 4) EA with pretreatment with naloxone (a nonselective opioid receptor antagonist), 5) EA with pretreatment with chelerythrine (a nonselective PKC inhibitor), and 6) EA with pretreatment with both naloxone and chelerythrine. Compared with the untreated and sham acupuncture groups, EA resulted in decreased O(2) demand, myocardial NE concentration, and infarct size. Furthermore, the degree of ST segment elevation and severity of LV dysfunction and ventricular arrhythmias were all significantly decreased (P < 0.05). The cardioprotective effects of EA were partially blocked by pretreatment with naloxone or chelerythrine alone and completely blocked by pretreatment with both naloxone and chelerythrine. These results suggest that the cardioprotective effects of EA against myocardial ischemia-reperfusion are mediated through inhibition of the cardiac sympathetic nervous system as well as opioid and PKC-dependent pathways.     

A new study of cell disruption to release recombinant thermostable enzyme from Escherichia coli by thermolysis

Extraction of intracellular protein from Escherichia coli is traditionally achieved by mechanical, chemical or enzymatic disruption technology. In this study, a novel thermolysis method was used to disrupt E. coli cells to release a recombinant thermostable esterase. We found that heat treatment of E. coli was highly effective to destroy the integrity of bacterial cell walls and release the recombinant hyperthermophilic esterase at temperatures above 60 degrees C. The effects of temperature, pH and cell concentration on the efficiency of cell disruption were examined. The most effective temperature for cell disruption was at 80 degrees C. The pH and cell concentration had only minor effect on the release of the hyperthermophilic esterase. In addition, we found that the hyperthermophilic esterase could be purified at the early stage of the thermolysis, which is a major advantage of the thermolysis method. Finally, a comparison between thermolysis and traditional methods for the disruption of cells and the release of the thermostable enzyme was made.     

Autophagy decreases alveolar epithelial cell injury by regulating the release of inflammatory mediators

To research the impact of autophagy on alveolar epithelial cell inflammation and its possible mechanism in the early stages of hypoxia, we established a cell hypoxia–reoxygenation model and orthotopic left lung ischemia–reperfusion model. Rat alveolar epithelial cells stably expressing GFP-LC3 were treated with an autophagy inhibitor (3-MA) or an autophagy promoter (rapamycin), followed by hypoxia–reoxygenation treatment for 2, 4, and 6 hr in vitro. In vivo, 20 male Sprague Dawley rats were randomly divided into four groups (model group: No blocking of the hilum in the left lung; control group: Blocking of the hilum in the left lung for 1 hr with dimethyl sulfoxide lavage; 3-MA group: Blocking of the hilum in the left lung for 1 hr with 100 ml/kg of 3-MA (5 μmol/L) solution lavage; and rapamycin group: Blocking of the hilum in the left lung for 1 hr with 100 ml/kg of rapamycin (250 nmol/L) solution lavage) to establish an orthotopic left lung ischemia model. This study demonstrated that rapamycin significantly suppressed the nuclear factor kappa B signaling pathway and limited the expression of proinflammatory factors. A contrary result was found after the 3-MA pretreatment. These findings indicate that autophagy reduces ischemia–reperfusion injury by repressing inflammatory signaling pathways in the early stages of hypoxia in vitro and in vivo. Autophagy could be a new protective method for application in lung ischemia–reperfusion injury.