Heat shock response in the thermophilic fungus Rhizomucor miehei

Changes in the composition of the membrane lipids and cytosol carbohydrates of the thermophilic fungus Rhizomucor miehei in response to heat shock were studied. Under optimal conditions (41–43°C), high trehalose content (8–11%) was found at all stages of growth of submerged culture. Heat shock (51–53°C) for 1 h did not result in enhanced trehalose synthesis, while increase in shock duration to 3 h resulted in a significant increase in trehalose content. The share of sterols and phosphatidic acids in the membrane lipids increased, while the share of phosphatidylcholines and phosphatidylethanolamines decreased. These processes resulted in increased content of non-bilayer lipids, while the unsaturation degree of the fatty acids of the major phospholipids did not decrease. Comparison of resistance to lethal heat shock in the control and experimental variants of R. miehei revealed that this thermophilic fungus exhibited no acquired heat resistance.     

Acquired thermotolerance,membrane lipids and osmolytes profiles of xerohalophilic fungus Aspergillus penicillioides under heat shock

Xerophilic fungi accumulate a large amount of glycerol in the cytosol to counterbalance the external osmotic pressure. But during heat shock (HS) majority of fungi accumulate a thermoprotective osmolyte trehalose. Since glycerol and trehalose are synthesized in the cell from the same precursor (glucose), we hypothesised that, under heat shock conditions, xerophiles growing in media with high concentrations of glycerol may acquire greater thermotolerance than those grown in media with high concentrations of NaCl. Therefore, the composition of membrane lipids and osmolytes of the fungus Aspergillus penicillioides, growing in 2 different media under HS conditions was studied and the acquired thermotolerance was assessed. It was found that in the salt-containing medium an increase in the proportion of phosphatidic acids against a decrease in the proportion of phosphatidylethanolamines is observed in the composition of membrane lipids, and the level of glycerol in the cytosol decreases 6-fold, while in the medium with glycerol, changes in the composition of membrane lipids are insignificant and the level of glycerol is reduced by no more than 30%. In the mycelium trehalose level have increased in both media, but did not exceed 1% of dry weight. However, after exposure to HS the fungus acquires greater thermotolerance in the medium with glycerol than in the medium with salt. The data obtained indicate the interrelation between changes in the composition of osmolytes and membrane lipids in the adaptive response to HS, as well as the synergistic effect of glycerol and trehalose.     

Membrane lipid and cytosol carbohydrate composition in Aspergillus niger under heat shock

A submerged Aspergillus niger culture exposed to heat shock (40–41°C) for 1, 3, and 6 h acquires resistance to a more severe, lethal heat shock (55°C, 20 min). A general trend characteristic of a heat shock occurring during the trophophase or idiophase (regardless of its duration) is an increase in the trehalose level in the cytosol carbohydrate fraction and in the content of sphingolipids and phosphatidic acid in the membrane lipid fraction. Contrary to generally accepted views, no increase in the content of unsaturated fatty acid in the main phospholipid fraction, sterol level, and share of “bilayer” phospholipids was detected. The results obtained are discussed in terms of the current hypotheses concerning membrane protection under heat shock and our own suggestion on this subject.     

The effect of different heat influences on composition of membrane lipids and cytosol carbohydrates in mycelial fungi

Comparison of the changes in the composition of the membrane lipids and soluble cytosol carbohydrates caused by two types of heat influence (within the tolerance zone and heat shock-level) revealed fundamental differences in the stress response of fungal cells. In three fungal species, Aspergillus niger, Pleurotus ostreatus, and Cunninghamella japonica, increased levels of trehalose and phosphatidic acids were observed under heat shock, while heat influences within the tolerance zone had no such effect. Under heat shock, the ratio of saturated fatty acids did not increase in any of the major phospholipids of all the studied species. This is in contradiction with the existing hypothesis and confirms the previously suggested the hypothesis of membrane stabilization by heat-protecting compounds.     

Artifactual Elevation of the Apparent Levels of Phosphatidic Acid and Phosphatidylinositol 4,5-Bisphosphate during Short-Term Labeling of Plant Tissue with Radioactive Precursor

Some pulvini of Samanea saman Mehr. labeled with radioactive phosphate show apparent remarkable elevations of the levels of phosphatidic acid and phosphatidylinositol 4,5-bisphosphate. The elevated levels, however, appear to be illusory and to result from rapid initial incorporation of label into these phospholipids relative to others. These results demonstrate the need for caution in interpreting apparent changes in the levels of phosphatidic acid or inositol phospholipids in cultures or plants labeled with radioactive precursors.     

Characterization of the heat shock response in cultured sugarcane cells : I. Physiology of the heat shock response and heat shock protein synthesis

Effect of heat shock on the growth of cultured sugarcane cells (Saccharum officinarum L.) was measured. Heat shock (HS) treatment at 36 to 38°C (2 hours) induced the development of maximum thermotolerance to otherwise nonpermissive heat stress at 54°C (7 minutes). Optimum thermotolerance was observed 8 hours after heat shock. Development of thermotolerance was initiated by treatments as short as 30 minutes at 36°C. Temperatures below 36°C or above 40°C failed to induce maximum thermotolerance. In vivo labeling revealed that HS at 32 to 34°C induced several high molecular mass heat shock proteins (HSPs). A complex of 18 kilodalton HSPs required at least 36°C treatment for induction. The majority of the HSPs began to accumulate within 10 minutes, whereas the synthesis of low molecular mass peptides in the 18 kilodalton range became evident 30 minutes after initiation of HS. HS above 38°C resulted in progressively decreased HSP synthesis with inhibition first observed for HSPs larger than 50 kilodaltons. Analysis of two-dimensional gels revealed a complex pattern of label incorporation including the synthesis of four major HSPs in the 18 kilodalton range and continued synthesis of constitutive proteins during HS.     

Combinatorial Action of Different Stress Factors on the Composition of Membrane Lipids and Osmolytes of Aspergillus niger

Adaptive response (changes in the composition of osmolytes and membrane lipids) of the mycelial fungus Aspergillus niger to combinatorial action of oxidative and heat (or osmotic) shocks was studied. Oxidative shock was found to cause no significant changes in the composition of osmolytes. A combination of oxidative shock with other stressors was shown to suppress their adaptive responses, such as accumulation of trehalose (during heat shock) and polyols (during osmotic shock). A common pattern of the changes in membrane lipids observed for all the studied stress factors was an increase in the proportion of non-bilayer phosphatidic acids, which was more pronounced in the case of combinatorial stress effects. No significant changes in the degree of unsaturation of membrane phospholipids were observed. Thus, the studied combinatorial shocks did not result in an additive response and caused a decrease in the amount of osmolytes compared with individual stressors, which weakened the adaptive response of the fungus.

     

ASSEMBLY OF LIPIDS INTO MEMBRANES IN ACANTHAMOEBA PALESTINENSIS : I. Observations on the Specificity and Stability of Choline-14C and Glycerol-3H as Labels for Membrane Phospholipids

In order to determine the feasibility of using radioactive precursors as markers for membrane phospholipids in Acanthamoeba palestinensis, the characteristics of phospholipids labeled with choline-¹⁴C and glycerol-³H were examined. Choline-¹⁴C was found to be a specific label for phosphatidyl choline. There was a turnover of the radioactive moiety of phosphatidyl choline at a rate that varied with the concentration of nonradioactive choline added to the growth medium. Radioactivity was lost from labeled phosphatidyl choline into the acid-soluble intracellular pool and from the pool into the extracellular medium. This loss of radioactivity from cells leveled off and an equilibrium was reached between the label in the cells and in the medium. Radioactive choline was incorporated into phosphatidyl choline by cell-free microsomal suspensions. This incorporation leveled off with the attainment of an equilibrium between the choline-¹⁴C in the reaction mixture and the choline-¹⁴C moiety of phosphatidyl choline in the microsomal membranes. Therefore, a choline exchange reaction may occur in cell-free membranes, as well as living A. palestinensis. In contrast to choline-¹⁴C, the apparent turnover of glycerol-³H-labeled phospholipids was not affected by large concentrations of nonradioactive choline or glycerol in the medium. The radioactivity in lipids labeled with glycerol-³H consisted of 33% neutral lipids and 67% phospholipids. Phospholipids labeled with glycerol-³H turned over slowly, with a concomitant increase in the percentage of label in neutral lipids, indicating a conversion of phospholipids to neutral lipids. Because most (~96%) of the glycerol-³H recovered from microsomal membranes was in phospholipids, whereas only a minor component (~2%) of the glycerol-³H was in the phospholipids isolated from nonmembrane lipids, glycerol-³H was judged to be a specific marker for membrane phospholipids.     

Membrane lipids and cytosol carbohydrates in <Emphasis Type="Italic">Aspergillus niger</Emphasis> under osmotic,oxidative, and cold impact

The composition of the membrane lipids and cytosol soluble carbohydrates under three kinds of unfavorable impacts (osmotic, oxidative, and cold) was studied. Changes in the composition of the membrane lipids, specifically, increasing content of phosphatidic acids and decreasing levels of phosphatidylcholines and phosphatidylethanolamines, were the general response to the impacts. The degree of fatty acid unsaturation increased in all dominant phospholipids under osmotic shock, only in cardiolipins and phosphatidic acids under oxidative stress, and only in phosphatidylcholines under cold shock. Increased sterol content was observed only under cold and osmotic treatments. No general pattern was revealed in the composition of cytosol carbohydrates in response to stresses. Oxidative stress had almost no effect on the carbohydrate composition, while osmotic and cold treatments resulted in increased glycerol content and decreased total carbohydrate content. The mechanisms of fungal response to various stress impacts are discussed.     

Dynamics of the cytosol soluble carbohydrates and membrane lipids in response to ambient pH in alkaliphilic and alkalitolerant fungi

Comparative composition of lipids and cytosol soluble carbohydrates at different ambient pH values was studied for two obligately alkaliphilic fungi (Sodiomyces magadii and S. alkalinus) and for two alkalitolerant ones (Acrostalagmus luteoalbus and Chordomyces antarcticus). The differences and common patterns were revealed in responses to pH stress for the fungi with different types of adaptation to ambient pH. While trehalose was one of the major cytosol carbohydrates in alkaliphilic fungi under optimal growth conditions (pH 10.2), pH decrease to 7.0 resulted in doubling its content. In alkalitolerant fungi trehalose was a minor component and its level did not change significantly at different pH. In alkalitolerant fungi, arabitol and mannitol were the major carbohydrate components, with their highest ratio observed under alkaline conditions and the lowest one, under neutral and acidic conditions. In alkaliphiles, significant levels of arabitol were revealed only under alkaline conditions, which indicated importance of trehalose and arabitol for alkaliphily. Decreased pH resulted in the doubling of the proportion of phosphatidic acids among the membrane lipids, which was accompanied by a decrease in the fractions of phosphatidylcholines and sterols. Alkalitolerant fungi also exhibited a decrease in sterol level at decreased pH, but against the background of increased proportion of one of phospholipids. Decreased unsaturation degree in the fatty acids of the major phospholipids was a common response to decreased ambient pH.     

Effect of retinoic acid on the acylation of phospholipids in granulocytes in vitro

The influence of retinoic acid on the incorporation of [1-14C]palmitic acid and [1-14C]arachidonic acid into phospholipids was examined in guinea pig peritoneal granulocytes. All-trans-retinoic acid inhibited the incorporation of both fatty acids into phosphatidic acid and phosphatidylinositol. However, it stimulated the incorporation of both fatty acids into phosphatidylcholine but not other phospholipids. All-trans-retinoic acid was more effective than 13-cis-retinoic acid. The influence of all-trans-retinoic acid on the acylation of phospholipids was concentration-dependent with significant effect occurring at 2.1 microM. The loss of labeled fatty acids from prelabeled phospholipids and the transport of labeled fatty acids into granulocytes were not responsive to the presence of retinoic acid in the incubation media. These results suggest that retinoic acid may affect the activities of acyltransferases involved in the synthesis of phosphatidic acid, phosphatidylinositol and phosphatidylcholine.     

Substrate utilization for energy production and lipid synthesis in oligodendrocyte-enriched cultures prepared from rat brain

The metabolism of oligodendrocytes has been studied using cultures of oligodendrocyte-enriched glial cells isolated from cerebra of 5–8-day old rats. Cultures containing 60–80% oligodendrocytes were incubated for 16h with [3-¹⁴C]acetoacetate, d-[3-¹⁴C]3-hydroxybutyrate, [U-¹⁴C]glucose, l-[U-¹⁴C]glutamine and [1-¹⁴C]pyruvate or [2-¹⁴C]pyruvate in the presence or absence of other oxidizable substrates. Labelled CO₂ was collected as an index of oxidative metabolism and the incorporation of label into total lipids, fatty acids and cholesterol was used as an index of the de novo synthesis of lipids. Glucose, acetoacetate, D-3-hydroxybutyrate, pyruvate and l-lactate were measured to determine substrate utilization and product formation under various conditions. Our results indicate that glucose is rapidly converted to lactate and is a relatively poor substrate for oxidative metabolism and lipid synthesis. Ketone bodies were used as an energy source and as precursors for the synthesis of fatty acids and cholesterol. Preferential incorporation of acetoacetate into cholesterol was not observed. Exogenous pyruvate was incorporated into both the glycerol skeleton of complex lipids and into cholesterol and fatty acids. l-Glutamine appeared to be an important substrate for the energy metabolism of these cells.     

Phospholipid Composition and Metabolism of Micrococcus denitrificans

The phospholipid composition of Micrococcus denitrificans was unusual in that phosphatidyl choline (PC) was a major phospholipid (30.9%). Other phospholipids were phosphatidyl glycerol (PG, 52.4%), phosphatidyl ethanolamine (PE, 5.8%), an unknown phospholipid (5.3%), cardiolipin (CL, 3.2%), phosphatidyl dimethylethanolamine (PDME, 0.9%), phosphatidyl monomethylethanolamine (PMME, 0.6%), phosphatidyl serine (PS, 0.5%), and phosphatidic acid (0.4%). Kinetics of ³²P incorporation suggested that PC was formed by the successive methylations of PE. Pulse-chase experiments with pulses of ³²P or acetate-1-¹⁴C to exponentially growing cells showed loss of isotopes from PMME, PDME, PS, and CL with biphasic kinetics suggesting the same type of multiple pools of these lipids as proposed in other bacteria. The major phospholipids, PC, PG, and PE, were metabolically stable under these conditions. The fatty acids isolated from the complex lipids were also unusual in being a simple mixture of seven fatty acids with oleic acid representing 86% of the total. Few free fatty acids and no non-extractable fatty acids associated with the cell wall or membrane were found.     

In vivo incorporation of acetate into the acyl moieties of polar lipids from etiolated leek seedlings

Seven-day-old leek seedlings actively synthesize lipids in vivo from [1-¹⁴C]acetate, both in the light and in the dark. In the dark, phospholipid synthesis is more effective than galactolipid synthesis. Whatever the time of acetate incorporation by the etiolated seedlings, very long chain fatty acids having from 20 to 26 carbon atoms are found in all the polar lipids, including the acyl-CoAs. All of the labelled very long chain fatty acids incorporated into the polar lipids are saturated. On the other hand, the labelled C₁₈-fatty acids are unsaturated in phospholipids and galactolipids and almost no label is found in the saturated or unsaturated C₁₈-fatty acids of the acyl-CoAs.     

Time course for labeling of brain membrane phosphoinositides and other phospholipids after intracerebral injection of [32P]-ATP. Evaluation by an improved HPTLC procedure

An improved two-dimensional HPTLC procedure was developed for separating phospholipids including individual phosphoinositides, phosphatidic acids and plasmalogens. This procedure was used to examine the time course for uptake of label by phospholipids in brain subcellular membranes after intracerebral injection of [gamma-32P]-ATP. There were considerable differences in the phospholipid labeling pattern among different subcellular fractions. In particular, a high proportion of labeled phosphatidylinositol 4,5-bisphosphates and phosphatidic acids was found in the myelin fraction during the initial 4 hr after injection. In other subcellular fractions, labeling of phosphoinositides was maximum at 2 hr, but with prolonged time, poly-phosphoinositides started to show a decline in radioactivity whereas labeling of other phospholipids continued to show a steady increase instead. Results indicate at least two different modes for the uptake of label by brain membrane phospholipids after intracerebral injection of [32P]-ATP.     

Alterations in Glial Cell Metabolism During Recovery from Chronic Osmotic Stress

NMR spectroscopy of F98 glioma cell extracts showed that chronic hypertonic conditions largely increased the intracellular content of small, osmotically active molecules. Moreover, hypertonic stress decreased the incorporation of ¹³C-labeled amino acids into the cellular proteins albeit their cytosolic concentrations were increased, which reflects an inhibition of protein synthesis under these conditions. Reincubation with isotonic medium restored almost completely the control values for the cytosolic metabolites but not for amino acid incorporation into the protein. An increased amount of ¹³C label was found in the phospholipids, which indicates stimulation of membrane synthesis processes due to the recovery-induced cell swelling. On the other hand, chronic hypotonic conditions largely decreased the steady state concentration and synthesis of small, cytosolic molecules, whereas the effect on the incorporation of ¹³C-labeled amino acids into the cellular proteins was variable. Reincubation with isotonic medium partially restored the depressed cytosolic metabolite content and also the incorporation of labeled amino acids into cellular protein, but induced an inhibition of phospholipid synthesis. The results verify that readaptation of glial cell metabolism during recovery from chronic osmotic stress is impaired or at least seriously retarded.     

Formation of phospholipids from sn-glycerol-3-phosphate and free fatty acids or their derivatives by homogenates of soybean cotyledons

Cell-free homogenates of soybean cotyledons contain a sn-glycerol-3-phosphate acyltransferase system which incorporated [U-¹⁴C]-sn-glycerol-3-phosphate into 5 labelled lipids when incubated with palmitic acid in the presence of ATP and CoA. In decreasing order of incorporation of label, the lipids were: lysophosphatidic acid, monoacylglycerol, phosphatidic acid, diacylglycerol and triacylglycerol. The substrate specificity of the acyltransferase system was investigated with the fatty acids shown in order of decreasing rates of reaction; palmitate > stearate > oleate > linoleate > linolenate > laurate. Making these acids more soluble as triethanolamine salts or as polyoxyethylene sorbitan esters did not greatly enhance these rates of reaction. Activity was found in a 10000 g pellet containing plastids, mitochondria and glyoxysomes and also in the lipid layer; the activity in these particulate fractions was enhanced by the addition of cytosol which itself had little activity when gentle methods of cell disruption were used. During cotyledon development the total acyltransferase activity increased, although its specific activity slowly declined due to more rapid synthesis of other proteins. During germination total activity decreased but there was a transient increase in specific activity due to more rapid degradation of other proteins.     

Fat Metabolism in Higher Plants: XL. Synthesis of Fatty Acids in the Initial Stage of Seed Germination

To understand more fully organelle membrane assemblage, the synthesis of the first fatty acids by the germinating pea, Pisum sativum, was studied by the incorporation of either tritiated water or acetate-1-¹⁴C into lipids by the intact, initially dry seed. After a lag phase, labeling proceeded linearly. This lag phase ended when uptake of water had increased the seed weight to 185% of its original weight. The first fatty acids synthesized were palmitic and stearic followed shortly after by long chain saturated fatty acids (C₂₀-C₂₆). The synthesis of very long chain acids was consistently characteristic of several other seeds in early stages of germination. The majority of the radioactive acids were present in phospholipids and were localized in particulate fractions. The acyl components of phosphatidyl glycerol were highly labeled. The very long chain acids were found predominantly in the waxes. Pulse labeling indicated little turnover of the labeled fatty acids. Evidence is presented indicating that the enzymes for fatty acid synthesis are already present in the dry seed and participate in the synthesis of fatty acids once a critical water content of the seed is achieved.     

Plasma membrane phospholipid and sterol synthesis in soybean hypocotyl segments undergoing auxin-induced elongation

Summary Auxin-induced cell elongation necessitates plasma membrane enlargement. The effect of auxin (10 M 2,4-dichlorophenoxyacetic acid) treatment on amount, composition, and rate of synthesis of plasma membrane lipids was examined. Auxin-treated and control soybean (Glycine max L.) hypocotyl segments were incubated with [¹⁴C]acetate for times ranging from 0.5 to 18 h, prior to isolation of plasma membrane by aqueous two-phase partitioning. The composition of individual plasma membrane lipids in elongating segments did not differ from the composition in treatment time-matched control segments, except that after longer auxin treatments, phospholipids had more unsaturated fatty acids. Plasma membrane phospholipid and free sterol content both increased in elongating segments. The relative proportion of sterols and phospholipids in the plasma membrane primarily depended on time after segment excision, for both auxin-treated and control segments. Auxin enhanced the rate of lipid incorporation into the plasma membrane by 6 h, and stimulated the synthesis of some phospholipids and sterols.Abbreviations 2,4-D 2,4-dichlorophenoxyacetic acid - ER endoplasmic reticulum - GC gas chromatography - IAA indole-3-acetic acid - PA phosphatidic acid - PC phosphatidylcholine - PE phosphatidylethanolamine - PG phosphatidylglycerol - PI phosphatidylinositol - PM plasma membrane - PS phosphatidylserine     

Incorporation of polyunsaturated fatty acids into bis(monoacylglycero)phosphate and other lipids of macrophages and of fibroblasts from control and Niemann-Pick patients

On the basis of earlier studies of rabbit pulmonary alveolar macrophages, the incorporation of 14C-labelled polyunsaturated fatty acids into the lipids of human fibroblasts from patients with various phenotypes of Niemann-Pick disease was examined in order to define further the disturbance in metabolism of bis(monoacylglycero)phosphate occurring in these disorders. Docosahexaenoic acid, which had not been studied previously, was found to be incorporated by macrophages into bis(monoacylglycero)phosphate in a highly selective fashion and was therefore used along with arachidonic acid for studies of fibroblasts. Following incubation of fibroblasts in serum-free medium for 60 min, the distribution of arachidonic acid label in lipids was: phosphatidylcholine, 51%; phosphatidylethanolamine, 12%; phosphatidylinositol, 9.5%; and bis(monoacylglycero)phosphate, 2.3%; and of docosahexaenoic acid label was 36, 20, 2.6 and 10.3% respectively. Phosphatidylinositol had the highest specific activity of arachidonic acid label and bis(monoacylglycero)phosphate of docosahexaenoic acid label. Prolongation of incubation to 21 h, with or without removal of label remaining in the medium at 1 h, resulted in proportional redistributions with phosphatidylcholine decreasing and phosphatidylethanolamine increasing. In bis(monoacylglycero)phosphate and phosphatidylinositol, the proportions of arachidonic acid label decreased and increased respectively, whereas the proportions of docosahexaenoic acid label in these lipids were unchanged. As virtually all label taken up by cells was esterified, these redistributions are taken to reflect transacylations. In Niemann-Pick cells, the expected redistribution of arachidonic acid label in bis(monoacylglycero)phosphate failed to occur with cell types A and B which are deficient in sphingomyelinase-phospholipase C, and excess label accumulated after a 21-h incubation. Excess docosahexaenoic acid label also accumulated in the bis(monoacylglycero)phosphate of these cells. The highly selective incorporation of docosahexaenoic acid in two cell types suggests a special role for bis(monoacylglycero)phosphate in the metabolism of n-3 polyunsaturated fatty acids. A high specific activity found early in incubations of macrophages suggests that polyunsaturated fatty acids may be incorporated into phospholipids during de novo synthesis of phosphatidic acid.