The biosynthesis and turnover of lipid during the differentiation of Dictyostelium discoideum

Despite the fact that there are only relatively slight changes in lipid composition during the differentiation of Dictyostelium discoideum, the rates of lipid synthesis were found to vary considerably. Polar lipid synthesis declined markedly during aggregation and pseudoplasmodium formation and then increased during the terminal stages of differentiation. Several neutral lipid classes (sterol, the diacylglycerols and triacylglycerol) exhibited similar changes in synthetic rates, although the effects were somewhat less pronounced. In contrast, the rates of synthesis of steryl ester and free fatty acid increased slightly throughout the differentiation period, so that, by the end of the later stages of fruiting body culmination, the rates were essentially doubled. Finally, the synthesis of an unknown component increases at least 10-fold during differentiation. Of the newly synthesized lipid, only triacylglycerol and polar lipid exhibited marked turnover. Accumulation of radioactivity in steryl ester and free fatty acid continued after the removal of radioactive acetate, presumably due to the incorporation of fatty acid produced by polar lipid degradation.     

Effect of inhibition of protein synthesis on lipid metabolism in Lactobacillus plantarum.

In Lactobacillus plantarum 17-5, lipid synthesis appears to be correlated with protein synthesis. Inhibition of protein synthesis by chloramphenicol (50 mug/ml) caused the nearly simultaneous inhibition of incorporation of radioactive oleic acid into polar lipids before the cessation of growth. In addition, de novo fatty acid synthesis, as determined by the incorporation of radioactive acetate into cellular lipids, was also inhibited. Removal of the antibiotic resulted in the resumption of growth, protein synthesis, and polar lipid synthesis. Inhibition of protein synthesis by leucine deprivation also produced a marked reduction in the incorporation of radioactive oleic acid into the total polar lipids at about the same time that growth stopped (30 to 60 min after the removal of leucine). However, the different classes of lipids behaved differently. For example, the incorporation of oleic acid into cardiolipin was inhibited immediately upon removal of leucine from the cultures, whereas incorporation into phosphatidyl-glycerol was maintained at near normal rates for 60 min after the removal of leucine and then ceased. In contrast, the accumulation of radioactive oleic acid in a neutral lipid identified as diglyceride occurred to a much greater extent in leucine-deprived cultures than in control (+ leucine) cultures. Upon addition of leucine to leucine-deprived cultures, the rates of synthesis of phosphatidyl-glycerol and cardiolipin returned to normal; the amount of radioactivity in the diglyceride fraction decreased to normal levels concomitantly with increased phospholipid synthesis.     

Active Hydrocarbon Biosynthesis and Accumulation in a Green Alga,Botryococcus braunii (Race A)

Among oleaginous microalgae, the colonial green alga Botryococcus braunii accumulates especially large quantities of hydrocarbons. This accumulation may be achieved more by storage of lipids in the extracellular space rather than in the cytoplasm, as is the case for all other examined oleaginous microalgae. The stage of hydrocarbon synthesis during the cell cycle was determined by autoradiography. The cell cycle of B. braunii race A was synchronized by aminouracil treatment, and cells were taken at various stages in the cell cycle and cultured in a medium containing [¹⁴C]acetate. Incorporation of ¹⁴C into hydrocarbons was detected. The highest labeling occurred just after septum formation, when it was about 2.6 times the rate during interphase. Fluorescent and electron microscopy revealed that new lipid accumulation on the cell surface occurred during at least two different growth stages and sites of cells. Lipid bodies in the cytoplasm were not prominent in interphase cells. These lipid bodies then increased in number, size, and inclusions, reaching maximum values just before the first lipid accumulation on the cell surface at the cell apex. Most of them disappeared from the cytoplasm concomitant with the second new accumulation at the basolateral region, where extracellular lipids continuously accumulated. The rough endoplasmic reticulum near the plasma membrane is prominent in B. braunii, and the endoplasmic reticulum was often in contact with both a chloroplast and lipid bodies in cells with increasing numbers of lipid bodies. We discuss the transport pathway of precursors of extracellular hydrocarbons in race A.     

The assimilation of glycerol into lipid acyl chains and associated carbon backbones of Nannochloropsis salina varies under nitrogen replete and deplete conditions

Mixotrophic cultivation can increase microalgae productivity, yet the associated lipid metabolism remains mostly unknown. Stable isotope labeling was used to track assimilation of glycerol into the triacylglyceride (TAG) and membrane lipids of Nannochloropsis salina. In N-replete media, glycerol uptake and ¹³C incorporation into acyl chains were, respectively, 6-fold and 12-fold higher than in N-deplete conditions. In N-replete cultures, 42% of the carbon in the consumed glycerol was assimilated into lipid acyl chains, mostly in membrane lipids rather than TAG. In N-deplete cultures, only 11% of the limited amount of consumed glycerol was fixed into lipid acyl chains. Labeled lipid-associated glycerol backbones were predominantly ¹³C₃ labeled, suggesting that intact glycerol molecules were directly esterified with fatty acids/polar head groups. However, the presence of singly and doubly labeled lipid-bound glycerol species suggested that some glycerol also went through the central carbon metabolism before forming glycerol-3-phosphate destined for lipid esterification. ¹³C incorporation was higher in the saturated and monounsaturated than the polyunsaturated acyl chains of TAG, indicating the flux of carbon from glycerol went first to de novo fatty acid synthesis before acyl editing reactions. The results demonstrate that nitrogen availability influences both glycerol consumption and utilization for lipid synthesis in Nannochloropsis, providing novel insights for developing mixotrophic cultivation strategies.     

Thylakoid membrane biogenesis in Chlamydomonas reinhardtii 137+: cell cycle variations in the synthesis and assembly of polar glycerolipid

The synthesis and assembly of thylakoid membrane polar glycerolipid (glycolipid, phospholipid, and ether lipid) have been monitored in synchronous cultures of the green alga Chlamydomonas reinhardtii 137+. A "pulse" protocol using radioactive acetate as the lipogenic precursor was devised to allow assessment of both processes during the 24-h (12-h light/12-h dark) vegetative cell cycle. Under these conditions, acetate incorporation into each chromatographically resolved lipid at the cellular level reliably reflects lipid synthesis, and the appearance of radiolabeled lipid in purified photosynthetic membrane is indicative of the lipid assembly attendant to thylakoid biogenesis. Our results demonstrate that polar glycerolipid is synthesized by the alga and is assembled into its thylakoid membrane continuously, but differentially, with respect to cell cycle time. Synthesis and assembly are most rapid during the photoperiod (mid-to-late G1), reach maximum rates at mid- photoperiod, and are comparatively negligible in the dark (S, M, and early-to-mid G1). The extent to which synthesis and assembly vary within this general kinetic pattern, though, is characteristic of each thylakoid lipid, suggesting that the processes take place in a multistep manner with some temporal coordination among the different lipid types. Parallelism between the cyclic patterns of polar lipid synthesis at the cellular level and of polar lipid assembly into photosynthetic membrane at the subcellular level indicates that lipid production is not only essential to continuing thylakoid biogenesis but is also the critical determinant of the kinetics of thylakoid lipid assembly.     

Aflatoxin formation,lipid synthesis,and glucose metabolism by Aspergillus parasiticus during incubation with and without agitation

Glucose utilization, growth of mold, and synthesis of aflatoxin and total lipid by Aspergillus parasiticus were studied with cultures that were incubated statically and with agitation. With both cultural conditions, maximal toxin formation occurred at 5 days which coincided with the end of rapid mold growth and rapid uptake of glucose. The toxin concentration decreased as incubation continued. The pattern for formation and depletion of total lipid was similar to that for aflatoxin. Maximal yields of toxin and of total lipid did not coincide with maximal production of mold mycelium. Incubation with agitation enhanced mold growth, consumption of glucose, and production of aflatoxin and total lipid during the first 3 days. Generally, more growht occured in agitated cultures, but maximal yields of aflatoxin and total lipid were lower than in quiescent cultures. The need for limited, but not excessive, O₂ for synthesis of aflatoxin and lipid also was demonstrated by varying the volume of medium in flasks that were incubated quiescently. Incorporation of [1-¹⁴C] glucose into aflatoxin indicated that limiting the O₂ supply and thereby favoring glucose catabolism via the Embden-Meyerhof pathway enhanced toxin formation. Aflatoxin formation also was greater when oxidative respiration of the mold was restricted by a metabolic inhibitor. Results suggest that the degree of aeration of the culture is important in controlling biosynthesis of aflatoxin.     

A COMPARATIVE STUDY OF THE METABOLISM OF DE NOVO SYNTHESIZED FATTY ACIDS FROM ACETATE AND GLUCOSE, AND EXOGENOUS FATTY ACIDS, IN SLICES OF RABBIT CEREBRAL CORTEX DURING DEVELOPMENT

Slices of rabbit cerebral cortex, from the foetal stage to the adult have been used to compare lipid synthesis from fatty acids synthesized de novo from [U-¹⁴C]glucose and [1-¹⁴C]acetate, with lipid synthesis from exogenous albumin-bound [1-¹⁴C]palmitate. Incorporation into cellular lipid has been determined in terms of DNA, protein, wet wt. of tissue and wet weight of whole brain. On a wet wt. basis, maximum incorporation of glucose carbon into lipid occurred in the foetal brain while lipid synthesis from acetate and palmitate was maximum at 4–14 days after birth. Glucose and acetate were incorporated into a diversity of lipids (with increasing amounts of phosphatidylcholine synthesized during maturation), while palmitate was incorporated into the free fatty acid and triglyceride fractions. A greater proportion of acetate was incorporated into fatty acids of chain-length longer than C16 compared with the incorporation of palmitate. However, on a molar basis de novo synthesized and exogenous palmitate were elongated, desaturated and incorporated into phospholipids at a similar rate, while exogenous palmitate was incorporated to a greater extent than de nova synthesized fatty acid into the triglyceride fraction. This difference in metabolism may be due to the different size of the non-esterified fatty acid pool in the two situations. At the period of their most active formation, the very long-chain fatty acids may be synthesized from a pool of the C18 series of fatty acids (saturated and monoenoic) not in equilibrium with the bulk of C₁₈ acids in cerebral lipids. This could be a pool of acyl groups derived from ethanolamine phospholipids.     

Accumulation of incomplete metabolic side products of lipid A in Salmonella typhimurium during inhibition of 3-deoxy-D-manno-octulosonate incorporation by a new class of antibacterial agents

A new class of antibacterial agents for Gram-negative bacteria, rationally designed to inhibit the incorporation of 3-deoxy-D-manno-octulosonate into lipopolysaccharide (LPS), was recently reported. In Salmonella typhimurium, where the lipid A species are well characterised, it was previously demonstrated that the addition of a compound which inhibits the enzyme 3-deoxy-manno-octulosonate cytidylytransferase (CMP-KDO synthetase; EC 2.7.7.38) leads to rapid accumulation of lipid A derivatives. The major lipid A species, IVA (O-(2-amino-2-deoxy-beta-D-glucopyranosyl)-(1-6)-2-amino-2-deoxy-alpha-D - glucose, acylated at positions 2, 3, 2', 3' with beta-hydroxymyristoyl groups and bearing phosphates at positions 1 and 4'), was shown to be converted mainly to LPS by pulse-chase experiments in the absence of inhibitor. Labelled precursor (IVA) was also chased to other more polar lipid A derivatives. During chase in the presence of inhibitor, there was no conversion to LPS, while the major lipid A species was converted to the same polar lipid A derivatives as in chase without inhibitor. Our data indicate that despite the accumulation of several species of lipid A derivatives during inhibition of LPS synthesis, only IVA is destined for synthesis of mature LPS when LPS synthesis resumes. The more polar lipid A derivatives would thus represent aberrant side reaction products which occur when the pathway is inhibited.     

Synthesis of medium-chain fatty acids and their incorporation into triacylglycerols by cell-free fractions fromCuphea embryos

During their rapid maturation period, seeds of Cuphea wrightii A. Gray mainly accumulate medium-chain fatty acids (C₈ to C₁₄) in their storage lipids. The rate of lipid deposition (40–50 mg·d^–1·(g fresh weight)^–1) is fourfold higher than in seeds of Cuphea racemosa (L. f.) Spreng, which accumulate long-chain fatty acids (C₁₆ to C₁₈). Measurements of the key enzymes of fatty-acid synthesis in cell-free extracts of seeds of different maturities from Cuphea wrightii show that malonyl-CoA synthesis may be a triggering factor for the observed high capacity for fatty-acid synthesis. Experiments on the incorporation of [1-¹⁴C]acetate into fatty acids by purified plastid preparations from embryos of Cuphea wrightii have demonstrated that the biosynthesis of medium-chain fatty acids (C₈ to C₁₄) is localized in the plastid. Thus, in the presence of cofactors for lipid synthesis (ATP, NADPH, NADH, acyl carrier protein, and sn-glycerol-3-phosphate), purified plastid fractions predominantly synthesized free fatty acids, 30% of which were of medium chain length. Transesterification of the freshly synthesized fatty acids to coenzyme A and recombination with the microsomal fraction of the embryo homogenate induced triacylglycerol synthesis. It also stimulated fatty-acid synthesis by a factor 2–3 and increased the relative amount of medium-chain fatty acids bound to triacylglycerols, which corresponded to about 60–80% in this lipid fraction.Abbreviations ACP acyl carrier protein - FW fresh weight This work was supported by the Bundesminister für Forschung und Technologie. The authors thank S. Borchert for her suggestions for plastid preparation.     

Lipid synthesis in germinating Tradescantia pollen

Synthesis of neutral and polar lipids in pollen of Tradescantia paludosa during germination and tube growth was studied by the incorporation of acetate-[1-¹⁴C] into lipids in the presence and absence of inhibitors of RNA and protein synthesis. The proteins required for the synthesis of both neutral lipids and phospholipids are not made de novo during germination but are already present in the mature ungerminated pollen grain and they are functionally stable during the first 2 hr of pollen growth.     

Patterns of DNA synthesis during pollen embryogenesis in henbane

Continued DNA synthesis in the generative cell nucleus, followed by mitosis and cytokinesis, results in the formation of pollen embryoids in cultured anthers of H. niger. In contrast, the nucleus of the vegetative cell undergoes no DNA synthesis after it is cut off, or synthesizes DNA only during a limited number of cell cycles. DNA synthetic patterns in the generative and vegetative cell nuclei confirm the ontogeny of embryoids described in this plant.     

RADIOAUTOGRAPHIC LOCALIZATION OF THE INCREASED SYNTHESIS OF PHOSPHATIDYLINOSITOL IN RESPONSE TO PANCREOZYMIN OR ACETYLCHOLINE IN GUINEA PIG PANCREAS SLICES

A technique is described for measuring the incorporation of myo-inositol-2-³H into the lipid of various regions of the guinea pig pancreatic acinar cell by radioautography. Stimulation of enzyme secretion with either pancreozymin or acetylcholine was associated with increased graining in both the basophilic cytoplasm and the nonbasophilic cytoplasm. Kinetic studies suggested that the incorporation of myo-inositol-2-³H was stimulated independently in the two regions. Most of the increment in graining due to stimulation with pancreozymin or acetylcholine plus eserine was abolished if the tissue was extracted with 2:1 chloroform-methanol before radioautography. On chromatography of lipid extracts of pancreas, the only lipid showing a detectable increment in radioactivity on stimulation with pancreozymin was phosphatidylinositol. Thus, essentially all of the increment in graining is likely to be due to increased incorporation of tritium into phosphatidylinositol. These studies, coupled with earlier studies employing differential centrifugation, indicate that on stimulation of enzyme secretion there is increased synthesis of phosphatidylinositol in the rough-surfaced endoplasmic reticulum and in the smooth-surfaced Golgi membranes. The significance of these observations is discussed in connection with membrane circulation presumed to occur in the pancreatic acinar cell on stimulation of protein secretion. It is suggested that the increased synthesis of phosphatidylinositol may be concerned with the formation of new endoplasmic reticulum and possibly Golgi membrane to replace that which is presumably converted to membrane of the zymogen granules during intracellular protein transport.     

The role of Coenzyme A in lipid synthesis by a particulate fraction from germinating peas

The effect of CoA on fatty acid synthesis by the microsomal fraction from germinating pea (Pisum sativum) was examined. Increasing concentrations of CoA progressively decreased total fatty acid synthesis from [¹⁴C]malonyl-CoA. However, the synthesis of very long chain fatty acids was relatively unaffected so that their proportion in the reaction products increased. Other CoA-esters also decreased total fatty acid synthesis while increasing the relative accumulation of radioactivity in very long chain fatty acids. The addition of CoA also altered the distribution of newly synthesized fatty acids in different lipid fractions. Complex lipid labelling was relatively increased while that of acyl-acyl carrier proteins was decreased. Very long chain fatty acids accumulated in lipids rather than thioesters. The role of CoA in controlling fatty acid synthesis in the pea microsomal fraction is discussed.     

Cytologic and Autoradiographic Studies of the Micronucleus at Meiotic Prophase in Tetrahymena pyriformis

Micronuclear changes of variety 1 of Tetrahymena pyriformis during meiotic prophase have been observed by the light microscope. Morphologic changes in the micronucleus are divided into 6 stages. In stage I, chromatin begins to polarize; in stage II, the micronucleus becomes spindle shaped; and in stage III, one end of the micronucleus protrudes to form a “neck.” In stage IV, where the micronucleus elongates to maximal length, the whole micronucleus consists of 2 chromatin threads pairing longitudinally. One thread probably contains one genome. In stage V, the elongated thread becomes shorter and thicker. Finally, in stage VI, separate chromosomes appear and enter into metaphase. To discover the role of the elongation of the micronucleus, called crescent formation, autoradiographic analysis of RNA and DNA synthesis were undertaken using [³H]uridine and [³H]thymidine. Pulse label and chase experiments show that the crescent in stages II and III is actively synthesizing RNA. Though no remarkable DNA synthesis was observed during meiosis, a small amount of DNA synthesis occurred during the 1st and 2nd prezygotic divisions.     

Morphohistological and flow cytometric analyses of somatic embryogenesis in <Emphasis Type="Italic">Trifolium nigrescens</Emphasis> Viv.

Microscopy and flow cytometry (FCM) were used to study somatic embryogenesis (SE) from zygotic embryos of Trifolium nigrescens Viv. to determine if there were any relationships between characteristics of somatic embryos (morphology, anatomy, genome size stability) and their regenerability. Embryoids were induced on Murashige and Skoog (MS) medium containing 4 mg l⁻¹ 2,4-dichlorophenoxyacetic acid (2,4-D) and 2 mg l⁻¹ N⁶-[2-isopentenyl]-adenine (2iP) either directly from hypocotyls or via an intervening callus, depending on the duration of culture. The morphology of somatic embryos varied from zygotic-like structures to abnormal structures including horn-shaped, polycotyledonary, and fused embryoids. The incidence of abnormalities was higher in callus cultures than in direct regeneration. Horn-shaped embryoids were the most frequent type of abnormal embryos. Only embryoids having zygotic-like morphology regenerated into plantlets. Histological observations revealed that the absence of shoot and root apical meristems along with parenchymatization of embryos were major obstacles to conversion of horn-shaped embryoids. The estimated 2C value for T. nigrescens was 0.9 pg. FCM analysis revealed differences in DNA content between embryoids induced via an intervening callus and those produced directly from explants. Individuals with species-specific as well as increased DNA content were detected among those zygotic-like embryos derived from callus, but all horn-shaped embryoids had increased genome sizes. The observed lack of differences in DNA content between zygotic-like and horn-shaped embryoids, from direct SE, indicated that these phenotypic abnormalities were of physiological origin. The mean DNA content of regenerants was species-specific, suggesting that only diploid embryoids were capable for regeneration into plantlets.     

Synthesis of acyl-CoAs by isolated spinach chloroplasts in relation to added CoA and ATP

The kinetics of incorporation of [2-¹⁴C] acetate into lipids and acyl-CoAs in relation to added CoA and ATP by isolated spinach chloroplasts have been examined. The effect of the concentration of these cofactors on lipid and acyl-CoA synthesis was also studied. In the absence of cofactors, or when only one was present, the incorporation was very low and went mainly into lipids. When both cofactors were present a strong stimulation of both activities occurred. After 25 min, acyl-CoAs were more strongly labeled than lipids and both activities continued linearly for at least 60 min.Abbreviations ACP acyl carrier protein - FFA free fatty acids     

Effects of biogenic amines on fat cells of Glossina morsitans in vitro

Lipid synthesis from leucine by fat cells of Glossina morsitans in vitro was inhibited by dopamine, adrenaline, noradrenaline and octopamine. Noradrenaline and octopamine were most active with maximal response occurring at 10^?4 and 10^?5 M respectively. The release of free fatty acids and the synthesis of proline from alanine by fat cells were stimulated by octopamine but not by the other amines. Maximal release of material from fat cells occurred at an octopamine concentration of 10^?2 M but at higher concentrations the response was diminished.The inhibition of lipid synthesis by octopamine was blocked by the α-adrenergic antagonist phentolamine but not by the β-adrenergic antagonist propranolol. Neither receptor-blocking-agent affected the action of corpora cardiaca extracts upon fat cells indicating that separate receptors are present for amines and the peptide hormones.     

The induction of reversible and irreversible chromosome decondensation by protein synthesis inhibition during meiotic maturation of mouse oocytes

We investigated the effects of puromycin on mouse oocyte chromosomes during meiotic maturation in vitro. Puromycin treatment for 6 hr at 100 μg/ml almost completely, but reversibly, suppressed [³⁵S]methionine incorporation into oocyte protein at all stages of maturation tested. Nevertheless, oocytes treated at the germinal vesicle stage underwent germinal vesicle breakdown (GVBD) and chromosome condensation. These oocytes completed nuclear maturation to metaphase II (MII) if the inhibitor was withdrawn. Prolonged (24-hr) treatment, however, caused the chromsomes to degenerate. The chromosomes of oocytes treated shortly after GVBD for 6 hr remained condensed, but the oocytes failed to form a polar body. However, 24-hr treatment caused the chromosomes to decondense to form an interphase nucleus. Oocytes treated near MI for 6 hr gave off a polar body during the treatment, and their chromosomes decondensed to form a nucleus, which remained as long as the treatment was continued. However, if the puromycin was withdrawn, the chromosomes recondensed to a state morphologically similar to that at MII. Thus, the chromosome decondensation induced by protein synthesis inhibition at MI was reversible. Oocytes treated at MII, several hours after first polar body formation, also underwent chromosome decondensation to form a nucleus. In the continuous presence of puromycin, the chromosomes remained decondensed, but neither DNA synthesis nor mitosis occurred. However, following puromycin withdrawal, these occytes synthesised DNA and underwent mitosis. Thus, protein synthesis inhibition at MII, by parthenogenetically activating the oocytes, caused irreversible chromosome decondensation. Based on these observations, we discussed the roles of protein synthesis in the regulation of oocyte chromosome behaviour during meiotic maturation.     

Evidence for synthesis in vitro of neutral lipids by isolated oocytes of Perinereis cultrifera (Annelida,Polychaeta). A biochemical and autoradiographic study

Summary

Isolated oocytes of Perinereis cultrifera have been incubated in culture media with added [³H]glycerol, [¹⁴C]butyric acid or [¹⁴C]oleic acid. The principal neutral lipid synthesized was triacylglycerol, although incorporation of radioactivity into other lipid categories (sterol, fatty acid, wax ester) was also observed. A more significant percentage of triacylglycerol was labelled after incubation with [³H]glycerol and [¹⁴C]oleic acid than with [¹⁴C]butyric acid. With this precursor, monoacylglycerol appears to be the class of lipid compartment which initially show the most radioactivity. Electron microscopic autoradiography has revealed that labelling after incorporation of glycerol was mainly localized on the lipid droplets but not on the yolk granules. A second metabolic pathway is represented by phospholipid membrane synthesis.