Endothelial cell fatty acid unsaturation mediates cold-induced oxidative stress

Ultraprofound hypothermia (< 5 degrees C) induces changes to cell membranes such as liquid-to-gel lipid transitions and oxidative stress that have a negative effect on membrane function and cell survival. We hypothesized that fatty acid substitution of endothelial cell lipids and alterations in their unsaturation would modify cell survival at 0 degrees C, a temperature commonly used during storage and transportation of isolated cells or tissues and organs used in transplantation. Confluent bovine aortic endothelial cells were treated with 18-carbon fatty acids (C18:0, C18:1n-9, C18:2n-6, or C18:3n-3), C20:5n-3 or C22:6n-3 (DHA), and then stored at 0 degrees C without fatty acid supplements. Storage of control cells caused the release of lactate dehydrogenase (LDH) and a threefold increase in lipid peroxidation (LPO) when compared to control cells not exposed to cold. Pre-treating cells with C18:0 decreased the unsaturation of cell lipids and reduced LDH release at 0 degrees C by 50%, but all mono- or poly-unsaturated fatty acids increased injury in a concentration-dependent manner and as the extent of fatty acid unsaturation increased. DHA-treatment increased cell fatty acid unsaturation and caused maximal injury at 0 degrees C, which was prevented by lipophilic antioxidants BHT or vitamin E, the iron chelator deferoxamine, and to a lesser extent by vitamin C. Furthermore, the cold-induced increase in LPO was reduced by C18:0, vitamin E, or DFO but enhanced by DHA. In conclusion, the findings implicate iron catalyzed free radicals and LPO as a predominant mechanism of endothelial cell injury at 0 degrees C, which may be reduced by increasing lipid saturation or treating cells with antioxidants.     

Influence of glutamic acid on the endogenous respiration of Bacillus subtilis

Clifton, C. E. (Stanford University, Stanford, Calif.), and John Cherry. Influence of glutamic acid on the endogenous respiration of Bacillus subtilis. J. Bacteriol. 91:546-550. 1966.-Amino acids serve as the major initial endogenous substrate for Bacillus subtilis. The endogenous activity of freshly harvested washed cells is high and falls off rapidly with time of shaking at 30 C to lower but still significant levels. The rate of O(2) consumption after the addition of glutamic acid also decreases as the cells age, but more slowly than noted for endogenous respiration. When cells were fed glutamate as soon as possible after harvesting, an apparent stimulation of endogenous respiration was noted. However, endogenous activity was inhibited if the cell suspensions were shaken for at least 1 hr before addition of the glutamate. Similar results were obtained with glycerol or glucose as exogenous substrates. Variation in rates of respiration with age of the cells, inherent instability of B. subtilis, and possible utilization of substances initially excreted by the cells appear to account for the variations noted regarding the influence of an exogenous substrate on endogenous respiration.     

Physiological Studies on the Recovery of Salt Tolerance by Staphylococcus aureus After Sublethal Heating

Cultures of S. aureus in 100 mM potassium phosphate buffer heated at 52 C for 15 min lost their tolerance to 7.5% NaCl. After incubation in a complex growth medium or in a diluted dialyzed medium in which unheated cells were unable to grow, salt tolerance was regained. Heat injury caused 30% loss of lipid. During recovery, the concentration of C(15) and C(17) fatty acids returned to normal, and there appeared to be an oversynthesis of C(16) and C(18) unsaturated acids. Penicillin abolished the latter reaction without affecting recovery; chloramphenicol did not affect fatty acid oversynthesis but reduced recovery. The K/Na ratio was 12.6 in control cells and 3.4 in injured cells, where it remained during the recovery of salt tolerance. Aspartate uptake was about 10% of the control level after injury and about 35% at recovery. Control cells grew without a lag on subculture, but injured cells which had regained their salt tolerance needed about 2 more h of incubation. Cells recovering with penicillin needed 6 more h, and cells recovering with chloramphenicol did not grow without a prolonged lag. Cells of S. aureus, therefore, may recover their salt tolerance while various membrane functions are still damaged.     

Utilization of membranous lipid substrates by membranous enzymes: activation of the latent sphingomyelinase of hen erythrocyte membrane

Previous studies demonstrated that hen erythrocytes have an inoperative, latent sphingomyelinase which is activated when the cells are hemolyzed in a hypotonic medium. Within minutes after hemolysis about 60-80% of the sphingomyelin (SPM) of the RBC "ghost" membrane was hydrolyzed. In this paper, expression of sphingomyelinase activity was further investigated. The percentage of total SPM hydrolyzed depended on the volume of the hypotonic hemolyzing buffer. Thus, suspending the erythrocytes in 4 vol of the buffer resulted in clumping of the hemolyzed "ghosts" and no hydrolysis of SPM. In comparison, suspension in 19 vol of the hypotonic buffer showed no clumping and sphingomyelinase activity was fully expressed. But centrifugation of the latter or, alternatively, addition of concanavalin A induced clumping and elimination of sphingomyelinase activity. Hen RBC could also be hemolyzed in an isotonic medium in the presence of Triton X-100, mellitin, halothane, and phospholipase C. Activation of the latent sphingomyelinase occurred at concentrations of these reagents which caused cell lysis. Hen RBC were dispersed in an isotonic medium containing glutaraldehyde (0.1%) or formaldehyde (10%). This rendered the cells resistant to hemolysis, even when subsequently dispersed in a hypotonic medium or water. But incubation of the "fixed" cells in a hypotonic or isotonic medium activated the enzyme, resulting in hydrolysis of 60% of the cellular SPM. In contrast, when glutaraldehyde was included in the hypotonic buffer, hemolysis occurred but sphingomyelinase activity was eliminated.     

Degradation of Phospholipids and Liquefaction of Pressed Baker’s Yeast by Acetic Acid Vapour

When pressed baker’s yeast (Saccharomyces cerevisiae) was exposed to the vapour of acetic acid, autolysis of yeast cells was induced in 3 or 4 hr. In order to elucidate the mechanism of the autolysis caused by the AcOH-treatment, we investigated variations in the lipid content of yeast cells during the treatment. The degradation of phospholipids and the accumulation of free fatty acids occurred within 3 hr. Formic acid exerted a similar effect on the pressed yeast. The effect of propionic acid was not seen in 3hr but was after 18 hr. When the homogenate of fresh yeast cells was incubated in the acidic region below pH 4.5 for 1 hr, phospholipids were hydrolyzed and free fatty acids were accumulated. Such deacylation of phospholipids was observed even at pH 6 on incubation for 12hr, but not observed at pH 7 or above pH 9. At pH 8, although phospholipids were somewhat degraded, free fatty acids almost never accumulated but diacylglycerol did accumulate.

Therefore, yeast cells have inherently phospholipid-acylhydrolases and, on AcOH-treatment, such enzymes may degrade membrane phospholipids to induce the autolysis of pressed yeast.     

Thermal inactivation kinetics of Bacillus subtilis spores suspended in buffer and in oils

The heat resistance of Bacillus subtilis 5230 and A spores freeze dried and suspended in buffer or oils was investigated. As expected, spores were more resistant to heat when suspended in oils than in buffer. This was ascribed to the low a _w of oils and to their content of free fatty acids. Linear survivor curves were obtained for spores suspended in buffer at 105°C or above and for B. subtilis A spores suspended in a vegetable oil. However, the survivor curves of the spores suspended in mineral oil (strain 5230) or olive oil (both strains) were concave upward with a characteristic tailing. The tailing could not be ascribed to spore clumping or to a specific heat injury that can be circumvented by Ca-dipicolinate. It is possibly due to another mechanism of injury or to the activation at high temperature of a normally dormant germination system.     

Insulin effect on in vivo gluconeogenesis from [3-14C]pyruvate in the starved rat

During a 24 hr fast rats received 4 subcutaneous injections of insulin, and 15 min after the last injection they were given an intravenous pulse of [3-14C]pyruvate. The amount of [14C]glucose in blood 2 min after the tracer did not differ between insulin treated and control animals, whereas at 5 and 10 min values were significantly lower in the former group. At 10 min after the tracer, liver [14C]glycogen specific activity and [14C]fatty acid amount were higher in the insulin treated animals than in controls while plasma concentration of gluconeogenic amino acids was lower in the first group. Similar changes but less pronounced and more retarded were found in 24 hr fasted rats given only one insulin dose 15 min before the [3-14C]pyruvate pulse. Results indicate that gluconeogenesis from pyruvate is not directly modified by insulin treatment. Effects found at 5 and/or 10 min after the tracer and reported effects after prolonged insulin treatments may be caused by one or all of the following possibilities: enhanced utilization of the new-formed glucose, reduced availability of gluconeogenic substrates, and counteracting action on gluconeogenic hormones.     

Characteristics of the Vegetative Growth of Bacillus popilliae

Growth characteristics of the insect pathogen, Bacillus popilliae Dutky, were studied by propagation in shaken flasks and in 2-liter fermentors. Maximal populations between 5 × 10⁸ and 2 × 10⁹ viable cells per milliliter of culture medium routinely were obtained in incubation periods of 18 to 24 hr at 30 C in a medium composed of 1.5% yeast extract, 0.6% K₂HPO₄, and 0.2% glucose or trehalose. The carbohydrate required for growth in liquid media was fermented with the formation of 2 meq of acid per mmole of carbohydrate utilized; acid products ordinarily were not subsequently metabolized. B. popilliae is an aerobe, and the amount of growth obtained varied with aeration to an optimum at oxygen absorption rates of about 0.5. Maximal populations persist in a culture for periods of only 1 to 4 hr; cessation of growth was followed immediately by rapid death of cultures, so that less than 1% of the cells remained viable after 48 hr, and viability often was lost entirely by the end of 72 hr of incubation. No cytological evidence for spore formation was observed under any growth condition. Death was not associated with lysis of the cells, although extensive granulation ultimately occurred. Continuous neutralizaiton, augmented buffering, various techniques of dialysis, or slow feeding of the carbohydrate did not markedly alleviate the characteristic death of the cultures.     

Induction of Prodigiosin Biosynthesis after Shift-Down in Temperature of Nonproliferating Cells of Serratia marcescens

Nonpigmented bacteria obtained by growth of Serratia marcescens at 38 C synthesized prodigiosin at 25 C if certain individual amino acids were added to cultures of nonproliferating cells. In order of effectiveness, the amino acids were: DL-histidine, L-proline, L-hydroxyproline, DL-alanine, L-alanine, DL-aspartic acid, D-alanine, DL-proline, L-serine, L-ornithine, L-glutamic acid, and D-proline. DL-Histidine at its optimal concentration (20 mg/ml) induced formation of prodigiosin (198 mug of prodigiosin per mg of bacterial protein) after incubation of cultures for 54 hr. Lower concentrations (10 mg/ml) of the other amino acids usually were optimum but less prodigiosin was synthesized, and the maximal amount of pigment occurred between 36 and 48 hr. DL-Methionine was not effective alone but at a low concentration (40 mug/ml) enhanced and accelerated biosynthesis of prodigiosin in the presence of other suitable amino acids. Addition of 2 mg of L-proline per ml at 0 hr induced formation of only 30 mug of prodigiosin after incubation for 42 hr, but addition at 36 hr of 5 mg more of L-proline per ml increased synthesis to 120 mug at 42 hr. Again, DL-methionine markedly augmented prodigiosin biosynthesis in these cultures. Synthesis of prodigiosin ceased if cultures were shifted from 25 to 38 C. Prodigiosin biosynthesis by the nonproliferating cells was maximum when cultures were aerated, the amount of bacterial protein was about 2.0 mg/ml, and amino acids were added at 0 hr. Bacteria synthesized prodigiosin most efficiently when they were harvested from aerated cultures grown at 38 C for 24 hr in a complete medium in a fermentor.     

Effects of prolonged incubation of isolated fat cells on their response to hormones stimulating lipolysis and glucose metabolism

1. The metabolism of isolated fat cells from parametrial adipose tissue of starved normal rats was studied during 8hr. incubation. 2. There was a three- to eight-fold increase in conversion of glucose into carbon dioxide, fatty acids and glycerol during the fourth to eighth hours of incubation in 4% albumin buffer over that seen during the first 4hr. of incubation. 3. The addition of growth hormone and dexamethasone to fat cells at the start of the incubation period accelerated lipolysis during the first 4hr. of incubation but no further effect was seen during the fourth to eighth hours of incubation. Addition of growth hormone and dexamethasone to fat cells that had been incubated for 4hr. did not accelerate lipolysis during the next 4hr. whether fat cells were incubated with or without glucose. 4. Fat cells incubated for prolonged periods also displayed a reduced sensitivity to the lipolytic action of adrenocorticotrophic hormone. 5. During prolonged incubation there was no damage to the cells as judged by the retention of two soluble cytoplasmic enzymes, lactate dehydrogenase and malate dehydrogenase, within the cells.     

Growth temperature affects accumulation of exogenous fatty acids and fatty acid composition in <Emphasis Type="Italic">Schizosaccharomyces pombe</Emphasis>

The incorporation of exogenously supplied fatty acids, palmitic acid, palmitoleic acid, oleic acid and linoleic acid, was examined in the yeast Schizosaccharomyces pombe at two growth temperatures, 20 °C and 30 °C. Fatty acids supplied to S. pombe in the growth medium were found to be preferentially incorporated into the cells, becoming a dominant species. The relative increase in exogenous fatty acids in cells came at the expense of endogenous oleic acid as a proportion of total fatty acids. Lowering the temperature at which the yeast were grown resulted in decreased levels of incorporation of the fatty acids palmitic acid, palmitoleic acid and linoleic acid compared to cells supplemented at 30 °C. In addition, the relative amount of the endogenously produced unsaturated fatty acid oleic acid, while greatly reduced compared to unsupplemented cells, was increased in cells supplemented with fatty acids at 20 °C compared to supplemented cells at 30 °C. The differential production of oleic acid in S. pombe cells indicates that regulation of unsaturated fatty acid levels, possibly by control of the stearoyl-CoA desaturase, is an important control point in membrane composition in response to temperature and diet in this species.     

Growth promotion of transfected hepatoma cells by liver fatty acid binding protein

Former studies have linked hepatocyte growth with liver fatty acid binding protein (L-FABP) of rat liver cytosol. In search for the roles of L-FABP in hepatocytes, we previously stably transfected rat L-FABP sense and antisense cDNAs into rat hepatoma HTC cells that do not contain L-FABP RNA or protein, thereby providing a zero-background, homologous cell model of L-FABP-expression suitable for controlled studies of its intracellular functions in hepatocyte-derived cells. The present study demonstrates the abilities of L-FABP to promote DNA synthesis and cell growth, preserve cell morphology, extend survival, and act cooperatively with unsaturated fatty acids in the transfected hepatoma cells in the absence of serum. Following removal of serum, the three control L-FABP-nonexpressing cell lines increased in cell lines increased in cell number for 24 hr and thereafter declined, whereas the three L-FABP-expressing cell lines exhibited a 39% higher rate of DNA synthesis per cell at 24 hr and grew in cell number for 48 hr. As a result, at 72 hr there were 2.5-fold (avg.) as many L-FABP-expressing cells than L-FABP-nonexpressing cells. In addition, the L-FABP-expressing cells retained their original polygonal morphology at 48 hr, when in contrast most of the control nonexpressing cells were spherical in shape with membrane blebs. In an effort to identify the agonists that collaborate with L-FABP in the growth promotion and preservation of cell morphology, various free fatty acids were examined at 48 hr for their ability to elminate the differences in behavior of the two cell types in the serum-free medium. The unsaturated fatty acids, oleic acid (18:1 ω9), linoleic acid (18:2ω6), α-linolenic acid (18: 3ω3), and arachidonic acid (20:4ω6), at 1 μM markedly elevated the level of DNA synthesis in the more depressed control L-FABP-nonexpressing cells and moderately raised it in the less depressed L-FABP-expressing cells. In accord, the control L-FABP-nonexpressing cells needed 10^?6–10^?5 M linoleic acid to achieve the extent of DNA synthesis attained by the expressing cells in the absence of added fatty acid. At 10 μM linoleic acid, their levels of DNA synthesis were equal. In contrast, five saturated fatty acids had no detectable effect on DNA synthesis. In addition, linoleic acid at 1 μM, but not the saturated fatty acid palmitic acid (16:0), prevented the above morphological alterations in the control L-FABP-nonexpressing cells observed in the absence of serum, thereby retaining their original polygonal morphology and that of the expressing cells. The findings are consistent with the concept that L-FABP improves the efficacy of the utilization of unsaturated fatty acid ligands of L-FABP in the formation, integrity, and fluidity of cell membranes that are involved in cell growth, morphology, and survival. © 1993 Wiley-Liss, Inc.     

Comparative effects of diverse vertebrate gonadotropins on androgen formation in vitro from testes of roosters and mice

A study was carried out to compare the androgen formation activity of gonadotropins from diverse vertebrate species by rooster and mouse testes in vitro. The dispersed testicular interstitial cells from 6- to 7-wk-old mice or testicular slices from 3- to 4-mo-old roosters were incubated with varying doses of luteinizing hormones (LHs)/gonadotropins (GTHs) in Medium 199 containing isobutyl-methyl-xanthine and N-2-hydroxyethylpiperazine-N-2-ethanesulfonic acid buffer (pH 7.4) at 34 degrees C (mice) or 37 degrees C (roosters) for 4 hr under continuous aeration of 95% O2-5% CO2 in a Dubnoff incubator shaken at 100 cycles/min. Androgen in the medium was measured by radioimmunoassay. The results revealed that dose-related androgen formations were obtained both in rooster and mouse systems in response to stimulations of all LHs/GTHs tested. The mouse system was more responsive to mammalian LHs and placental GTHs, less responsive to LHs from chickens, frogs, and turtles, and extremely unresponsive to piscine GTHs. In contrast, the rooster system was highly responsive to LHs from both mammals and chickens in androgen formation; it was also responsive to LHs from turtles and frogs as well as to piscine GTHs, although with relatively lower sensitivity. The rooster testis system is thus suitable for in vitro bioassay of LHs/GTHs from virtually all vertebrate classes, whereas the mouse testis system is more suitable for bioassay of mammalian LHs and placental GTHs. The differential androgen formation potencies of the diverse vertebrate GTHs in testis systems between roosters and mice indicate that a divergence exists in the testicular receptors between the two animal species.(ABSTRACT TRUNCATED AT 250 WORDS)     

Effect of Theanine,r-Glutamylethylamide,on Brain Monoamines and Striatal Dopamine Release in Conscious Rats

Theanine, r-glutamylethylamide, is one of the major components of amino acids in Japanese green tea. Effect of theanine on brain amino acids and monoamines, and the striatal release of dopamine (DA) was investigated. Determination of amino acids in the brain after the intragastric administration of theanine showed that theanine was incorporated into brain through blood-brain barrier via leucine-preferring transport system. The concentrations of norepinephrine, 3,4-dihydroxyphenylacetic acid (DOPAC) and 5-hydroxyindole acetic acid (5HIAA) in the brain regions were unaffected by the theanine administration except in striatum. Theanine administration caused significant increases in serotonin and/or DA concentrations in the brain, especially in striatum, hypothalamus and hippocampus. Direct administration of theanine into brain striatum by microinjection caused a significant increase of DA release in a dose-dependent manner. Microdialysis of brain with calcium-free Ringer buffer attenuated the theanine-induced DA release. Pretreatment with the Ringer buffer containing an antagonist of non-NMDA (N-methyl-D-aspartate) glutamate receptor, MK-801, for 1 hr did not change the significant increase of DA release induced by theanine. However, in the case of pretreatment with AP-5, (±)-2-amino-5-phosphonopentanoic acid; antagonist of NMDA glutamate receptor, the theanine-induced DA release from striatum was significantly inhibited. These results suggest that theanine might affect the metabolism and/or the release of some neurotransmitters in the brain, such as DA.     

Antigen retrieval trial for post-embedding immunoelectron microscopy by heating with several unmasking solutions.

A novel antigen retrieval procedure was carried out in the post-embedding immunogold electron microscopy method to improve the stainability of the samples. This was done by weakly fixing cultured Helicobacter pylori (ATCC43504) and embedding in Lowicryl K4M. Before staining with the anti-H. pylori antibody, the ultrathin sections were mounted on a nickel grid and heated at 121C for 15 min, 99C for 40 min, and 65C for 24 hr in distilled water, 0.1 M phosphate buffer (pH 7.4), 0.01 M EDTA (pH 7.2), 0.05 M Tris buffer (pH 10.0), 0.8 M urea (pH 7.2), 0.01 M citric acid (pH 6.0), or a commercially available target unmasking fluid (S1699; pH 6.0). Antigen retrieval in the Tris buffer solution generally showed better stainability than the classical post-embedding method without any antigen retrieval. At 65C for 24 hr, better stainability of the ultrasections was observed for each of the solutions used except for the phosphate buffer compared to the control. We suggest that the antigen retrieval method should be applied for routine use even by in post-embedding immunogold electron microscopy.     

Effect of fatty acids on the proliferation of concanavalin A-stimulated rat lymph node lymphocytes

1. The effect of a range of fatty acids upon concanavalin A-stimulated [3H]thymidine incorporation into rat lymphocytes was investigated. 2. All fatty acids tested inhibited the response to mitogen but the extent of the inhibition was dependent upon the fatty acid concentration used, the time of addition of fatty acid and the duration of exposure of the cells to fatty acid. 3. All fatty acids were inhibitory at concentrations of 50 microM or above; at lower concentrations some were inhibitory and some were stimulatory. Above 50 microM the inhibitory effect was concentration dependent; the greater the fatty acid concentration, the greater the inhibition. 4. The longer the lymphocytes were exposed to the fatty acid the greater was the inhibitory effect. This was true if the fatty acids were added at the same time as the mitogenic stimulus or if they were added before or after the stimulus. Some fatty acids maintained their inhibitory effect when added 24 or 48 hr after the mitogenic stimulus. 5. Generally unsaturated fatty acids were more inhibitory than saturated fatty acids; the greatest inhibition of proliferation was caused by eicosapentaenoate and arachidonate and the least inhibition by myristate and palmitate. 6. Inhibition was greater in the absence of serum. 7. Inhibition by unsaturated fatty acids could be partially or totally relieved by addition in combination with myristate or palmitate, suggesting that the inhibitory effect of fatty acids may be due to alteration of membrane fluidity caused by an imbalance of fatty acids presented to the cells. 8. PGE2 levels were similar in the medium of cells grown in the presence of fatty acids with varying inhibitory effects, indicating that PGE2 production is not the sole mechanism of suppression of the proliferative response. 9. Although the mechanism by which fatty acids exert their effect remains to be determined, these results indicate that lymphocyte proliferation and so an immune response could be influenced by dietary lipid manipulation.     

Effect of hypothermia on cell kinetics and response to hyperthermia and X rays

Hyperthermia is a potent radio enhancer. Studies using hypothermia in combination with irradiation have given confusing results due to lack of uniformity in experimental design. This report shows that hypothermia might have potential significance in the treatment of malignant cells with both thermo- and radiotherapy. Reuber H35 hepatoma cells, clone KRC-7 were used to study the effect of hypothermia on cell kinetics and subsequent response to hyperthermia and/or X rays. Cells were incubated at 8.5 degrees C or between 25 and 37 degrees C for 24 hr prior to hyperthermia or irradiation. Hypothermia caused sensitization to both hyperthermia and X rays. Maximum sensitization was observed between 25 and 30 degrees C and no sensitization was found at 8.5 degrees C. At 25 degrees C maximum sensitization was achieved in approximately 24 hr, cell proliferation was almost completely blocked, and cells gradually accumulated in the G2 phase of the cell cycle. In contrast to the effect of hypothermia on either hyperthermia or X rays alone, thermal radiosensitization was decreased in hypothermically pretreated cells (24 hr at 25 degrees C) compared to control cells (37 degrees C). The expression of thermotolerance and the rate of development at 37 degrees C after an initial heating at 42.5 degrees C were not influenced after preincubation at 25 degrees C for 24 hr. The expression of thermotolerance for heat or heat plus X rays during incubation at 41 degrees C occurred in a significantly smaller number of cells after 24 hr preincubation at 25 degrees C. The enhanced thermo- and radiosensitivity in hypothermically treated cells disappeared in approximately 6 hr after return to 37 degrees C.     

Correlation of actinomycin X2 to the lipid profile in static and shaken cultures of Streptomyces nasri strain YG62

Streptomyces nasri strain YG62 produces a broad-spectrum antibiotic designated actinomycin X2. The influence of static and shaken incubation on the production of actinomycin X2 and lipid profiles of S. nasri strain YG62 was investigated. It was found that shaken incubation was superior to the static process for both actinomycin X2 (2-fold) and total lipids (1.6-fold). Triglyceride and phospholipid levels paralleled the actinomycin X2 production with an increase in the triglyceride (2.8-fold) and phospholipid (1.2-fold) concentrations in the shaken culture over the static incubation. Analysis of fatty acid patterns revealed the occurrence of a wide range of fatty acids (C10-C22). The mean percentage of total saturated fatty acids in shaken culture was higher than those of the static culture. The mean percentage of mono-unsaturated fatty acids was almost the same in both cultures. The mean percentage of the total polyunsaturated fatty acids in the static culture was slightly higher than that of the shaken culture. The polyunsaturated/saturated fatty acid ratio (P/S) was higher in the static culture compared with the shaken culture. A positive correlation was recorded between triglycerides, phospholipids and actinomycin X2. A negative correlation on the other hand, was found between fatty acids and actinomycin X2.     

Staining Mitochondria With Hematoxylin After Formalin-Sublimate Fixation; A Rapid Method

Mitochondria were stained in liver, kidney, pancreas, adrenal and intestinal mucosa of rat and mouse. Tissues 1 mm thick, were fixed in a mixture of saturated aqueous HgCl₂, 90 ml; formalin (37-38% HCHO), 10 ml, at room temperature (25°C) for 1 hr. Deparaffinized sections 3-4μ thick were treated with Lugol's iodine (U.S.P.) followed by Na₂S₂O₃ (5%), rinsed in water and the ribonucleic acid removed by any of the following procedures: 0.2 M McIlavaine's buffer, pH 7.0, 2 hr, or 0.2 M phosphate buffer, pH 7.0, 2 hr at 37°C; 0.1% aqueous ribonuclease, 2 hr at 37°C; 5% aqueous trichloracetic acid overnight at 37°C; or 1% KOH at room temperature for 1 hr. After washing in water, sections were treated with a saturated solution of ferric ammonium alum at 37°C for 8-12 hr and colored by Regaud's ripened hematoxylin for 18 hr. They were then differentiated in 1% ferric ammonium alum solution while under microscopic observation.     

Ceramide inhibits development and cytokinesis and induces apoptosis in preimplantation bovine embryos

Ceramide is a second messenger induced by various cellular insults that plays a regulatory role in apoptosis. The objective of the present study was to determine whether ceramide signaling can occur in the preimplantation embryo by testing (1) effects of ceramide on development, cytokinesis, and apoptosis and (2) whether heat shock, which can induce apoptosis in embryos, causes activation of neutral or acidic sphingomyelinases responsible for generation of ceramide. Treatment of embryos > or =16 cells collected at Day 5 after insemination with 50 microM C(2)-ceramide increased caspase-9 activity and the proportion of blastomeres undergoing apoptosis but did not increase caspase-8 activity. Induction of apoptosis was more extensive when culture with ceramide was for 24 hr than for 9 hr. Ceramide also reduced the proportion of embryos that developed to the blastocyst stage when exposure was for 24 hr. At the two-cell stage, a period in development when apoptosis responses are blocked, culture of embryos with ceramide did not increase caspase-9 activity or the proportion of blastomeres that were apoptotic. However, culture with ceramide for 24 hr reduced cell proliferation and caused an increase in multinucleated cells because of inhibition of cytokinesis. Exposure of Day 5 embryos to a heat shock of 41 degrees C for 15 hr increased neutral sphingomyelinase activity but did not change acid sphingomyelinase activity. In conclusion, ceramide can regulate embryo development and apoptosis in a time and stage-of-development dependent manner and ceramide generation can be activated by cellular insult. Thus, the ceramide signaling pathway is present in the preimplantation embryo.