The synthesis of lipids and long chain fatty acids in young and aging cultures of Penicillium chrysogenum was studied by identifying the intracellular location of radioactively labeled intermediates in the vegetative mycelium and identifying the lipids and fatty acids into which the label from 1-14C-palmitic acid was incorporated.Previous work has indicated that the pH of the growth medium of submerged cultures of Penicillium chrysogenum dropped from pH 7.4 in newly inoculated cultures to 3.2-3.8 during the 20-40 hour growth period. Young cultures were defined as those harvested before the pH began to drop and aging cultures were those harvested after the pH stabilized at near 3.4. Changes in the fatty acid composition of various cell fractions of the mycelium harvested as the pH of the medium declined suggested that a change in utilization of lipids employed in the synthesis of structural components in the cell may have occurred as a result of a shift in fatty acid metabolism as the culture aged.Young and aging cultures were incubated with l-14C-palmitic acid and harvested after 2, 10, 60, and 120 minutes. Samples of mycelia from each harvest were examined by light and electron microscopy and were prepared for autoradiography. In addition lipids were extracted from sonified mycelial samples, analyzed for total lipids, lipid classes, and phospholipid components by thin layer chromatography. Fatty acids were identified and quantified by gas liquid chromatography with percent distribution of label in fatty acids determined by liquid scintillation spectrometry. Results of biochemical analysis of some of the major lipid components were compared with electron microscope autoradiographs and related to changes in the location of labeled fatty acid in cell organelles or cell parts as the cells aged.In young cultures 70% of the radioactive label was recovered in phospholipids while about 15% was recovered in the free fatty acid component. By contrast, in aging cultures approximately 20% of the label was recovered in the phospholipids and 80f was recovered from the free fatty acid component. Electron autoradiographic data tend to support these biochemical findings in that numerous grains occur over the membranal components of the young cells and over cytoplasmic areas of lipid depots in aging cells. Phospholipids differed markedly also with large amounts of an unidentified phospholipid type found in aging cells not seen in young cells.Light and electron microscope observations of hyphal cells showed significant alterations in cell morphology over the forty hour growth period. Young cells were long, slender, with dense cytoplasm and thin cell walls. As the culture aged, cells became progressively shorter, thicker and more clubby in appearance with prominent lipid inclusions. The vacuolar lipid depots of aging cells were ,determined to be composed of free fatty acids with 20% of label being incorpoated into C20 and C21 long chain fatty acids. The synthesis of fatty acid chains longerthan C18 has not been previously reported in intact cultures of Penicillium chrysogenum.The lipid metabolism of young cells of Penicillium chrysogenum differed from that of aging cells in many aspects. Young cells incorporated label from precursor palmitic acid into membranes. However, as the cells aged, lipids were diverted to storage. Young and aging cells differed in the amounts and composition of total lipids, lipid classes, phospholipid components and fatty acids. Differences in morphology between young and aging cells could be demonstrated by light and electron microscopy. These structural changes paralleled the biochemical changes indicating a functional dissimilarity existed between the young and aging cells.

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The overall objective of this study was the optimization of a model enzymatic system in organic solvent media for the biosynthesis of selected phenolic lipid compounds. The model enzymatic system consisted of cinnamic acid and oleyl alcohol as substrates using commercial immobilized lipase (Novozym 435) from Candida antarctica. The experimental findings showed that an increase in the hydrophobicity of the solvent mixture and a decrease in the aw values of the reaction medium increased the initial enzymatic activity and bioconversion yield; the use of an iso-octane and butanone solvent mixture (85:15, v/v) and an initial aw of 0.05 resulted in an initial enzymatic activity of 192.7 nmol product/g enzyme/min and a corresponding bioconversion yield of 95.3% after a 16-day reaction period.

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Pea root plastids were isolated by differential centrifugation and resulting crude plastid fraction was purified by centrifugation through 10%(v/v) Percoll. Marker enzymes indicated that greater than 50% of the plastids were recovered essentially free from mitochondrial and endoplasmic reticulum contamination. The optimum in vitro conditions for glycerolipid biosynthesis from (U-$ sp{14}$C) glycerol-3-phosphate have been determined. Total glycerolipid biosynthesis was approximately 15 nmole/hr/mg protein in the presence of 200 $ mu$M glycerol-3-phosphate, 0.5 mM each of NADH and NADPH, 15 mM KH$ sb2$CO$ sb3$, 0.05 mM CoA, and 2 mM each of ATP and MgCl$ sb2$, 100 mM Bis Tris Propane (pH 7.5) and incubated at the standard temperature of 25$ sp circ$C. ATP, Coenzyme A and a divalent cation are absolutely required for glycerolipid biosynthesis, whereas reduced nucleotides and bicarbonate improve the synthesis to varying degrees. Dihydroxyacetone phosphate had little effect, while dithiothreitol, detergent and Mn$ sp{2+}$ inhibited activity. Under the optimum conditions, isolated pea root plastids mainly synthesized approximately 15% phosphatidic acid, 16% phosphatidylcholine, 13% phosphatidylglycerol, 32% triacylglycerol. Galactolipid synthesis occurred only when UDP-galactose was supplied. Different concentrations of some cofactors resulted in alterations of glycerolipid distribution. Phospholipase A$ sb2$ and Rhizopus lipase digestions of phospholipids and neutral lipids revealed that radioactive fatty acids were preferentially esterified to position sn 2 of each glycerolipid with generally 2-4 times as much radioactivity as position sn 1. Pea root plastids are composed of approximately 62% phospholipid, 24% neutral lipid and 14% glycolipid. Within these classes PG, TAG, and the galactolipids are the major components representing 24, 12, and 12% of the total plastid lipids.

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Au cours de l'obésité, l'excès de lipides circulants, est stocké dans les organes périphériques, principalement dans le foie. Ce stockage ectopique de lipide peut avoir, à long terme, des conséquences délétères sur le métabolisme glucidique. Au long cours, l'excès de lipides hépatiques peut conduire au développement de stéatopathies métaboliques pouvant évoluer vers la cirrhose et le cancer du foie. Dans l'hépatocyte, le métabolisme et l'homéostasie lipidique sont finement régulés par la balance entre la synthèse (LDN) et l'utilisation (ß-oxydation) des lipides. Ces deux voies métaboliques sont sous le contrôle de plusieurs facteurs de transcriptions comme ChREBP, SREBP1 PPARƴ ou PPARa. La compaction et l'accessibilité de la chromatine sont des éléments cruciaux pour la régulation indirecte de l'activité de ces facteurs de transcription. Dans le noyau, la compaction de l'ADN est régulée par les histones mais aussi par les protéines High Mobility Group (HMG). Parmi la famille des protéines HMG, la protéine High Mobility Group box 1 (HMGB1), principalement localisée dans le noyau, est capable de réguler de façon indirecte la transcription de gènes dans de nombreux tissus. En plus de son rôle nucléaire, HMGB1 peut être activement sécrétée par les cellules innées au cours de phénomènes inflammatoires aigus. Chez la souris, la délétion totale de cette protéine entraine une hypoglycémie périnatale létale. De plus, chez la souris, les concentrations circulantes d'HMGB1 sont augmentées lors d'un stress métabolique induit par un régime hyper-lipidique (HFD). Tous ces résultats suggèrent un rôle d'HMGB1 dans le métabolisme hépatique et énergétique ainsi que dans les processus inflammatoires de bas-bruits associés au stress métabolique. [...] / During obesity, the excess of circulating lipids, are stored in the peripheral organs, and mainly in the liver. This ectopic storage of lipids may have long-term deleterious consequences on carbohydrate metabolism. Over time, excess of intra-hepatic lipids can lead to the development of steatohepatitis that can evolve to cirrhosis and liver cancer. In the hepatocyte, lipid homeostasis is finely regulated by the balance between synthesis (de novo lipogenesis-DNL) and degradation (ß-oxidation) of lipids. These two metabolic pathways are under the control of several transcription factors like ChREBP, SREBP1, PPARƴ or PPARa. The compaction and accessibility of chromatin are crucial parameters, which regulate the activity of these transcription factors. In the nucleus, the compaction of DNA is regulated by histones but also by High Mobility Group (HMG) proteins. Among the HMG protein family, High Mobility Group box 1 protein (HMGB1), mainly located in the nucleus, is able to indirectly regulate gene transcription in many tissues. In addition to its nuclear role, HMGB1 can be actively secreted by innate immunes cells during acute inflammatory reactions. In mice, the global deletion of Hmgb1 gene leads to perinatal lethality due to a severe hypoglycemia. Moreover, preliminary data from our laboratory show that circulating concentrations of HMGB1 are increased in mice subjected to high fat diet (HFD). All these results support a role of HMGB1 in hepatic and energetic metabolism but also in tissue-low grade inflammation related to metabolic stress. [...]

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In the present study, the potential of low-melting point lipids, namely methyl palmitate (MP) and menhaden stearine (MS), were studied for delivery of water-soluble materials to fish larvae. Addition of the solid emulsifier, sorbitan monopalmitate (SMP) resulted in higher RE compared to the liquid emulsifier sorbitan sesquioleate (SSO). Additions of SMP were found to have a positive effect on the stability of lipid spray beads (LSB) at concentrations >10% (w/w) of lipid. LSB were shown to retain riboflavin with the highest RE when the lipid matrix was composed of a mixture of MP+10% SMP+1O% ethyl cellulose (w/w). Use of MP facilitated digestive breakdown of LSB by larvae of zebrafish, Brachydanio rerio, and glowlight tetra, Hemigrammus erythrozonus, as indicated by release of riboflavin from ingested LSB. LSB composed of MS performed better than LSB composed of MP when particulate glycine was incorporated within LSB. Highest retention efficiencies (RE) and delivery efficiencies (DE) were achieved by LSB composed of 100% MS. RE and DE of LSB were further improved when glycine was encapsulated as aqueous droplets rather than in a particulate form. The overall performance of LSB containing tyrosine showed that nutrients with similar water-solubilities to tyrosine can be very effectively delivered to early fish larvae. Visual observation indicated that LSB composed of 100% MS were broken down by larvae of clownfish, Amphiprion percula. Zein-bound particles (ZBP) were not efficient in delivering riboflavin to fish larvae due to high leaching rates. In an attempt to improve ZBP, we developed complex particles (CP) containing LSB incorporated in a dietary mixture bound by zein, to obtain a single particle type. The potential of LSB for delivery of free amino acids (FAA) was evaluated when LSB were incorporated within CP. RE and DE for tyrosine were significantly greater than those for alanine, glycine and serine. There was a significant inverse correlation between RE for FAA and their solubilities where FAA with low solubilites resulted in higher RE. Development of CP with high RE and DE for amino acids is an important step towards formulating a diet to allow early weaning of fish larvae onto artificial diets. / Graduation date: 2003

Germinating pea (Pisum sativum L.) roots were divided into five sequential 0.5 cm segments from the root tip. Pooled segments were analyzed for their protein, starch and lipid content as an indirect indication of plastid function. Fresh weights of root segments were lowest in the tips (4.45mug per segment) and progressively higher up to the fifth segment (11.09mug per segment). Total protein, starch and lipid content, on a per segment basis, were all highest in zone 1 (tip segment) and progressively lower up to zone 5. Plastids were isolated from each of the five root segments and analyzed for their capacity for lipid biosynthesis under several different in vitro conditions. Collectively, the observations presented here suggest that the relative contributions of plastids to the overall physiology of germinating pea roots gradually diminishes as root development proceeds, and that plastids isolated from progressively older root zones have increased capacity for glycolytic and/or pentose phosphate metabolism. (Abstract shortened by UMI.)