Improved method of determining muramic acid from environmental samples

Muramic acid is an amino sugar that forms part of the peptidoglycan in prokaryotic cell walls. Since muramic acid is found only in prokaryotes it has been used as a measure of bacterial and cyanophyte biomass. Successful application of sensitive capillary gas‐liquid chromatographic (GLC) analysis required neutralization of the acid‐hydrolysate of a biomass sample followed by centrifugation to remove humic acids. After a further fractionation on a cation exchange column followed by derivatization and GLC analysis, recoveries of 98 ±9.5 (X± S.D.) % of authentic muramic acid from estuarine sediments with sensitivities of 10^‐13 mol were achieved. The structure of the GLC derivative was established by GLC infrared analysis and GLC mass spectrometry. The improvements in reproduci‐bility and sensitivity have allowed detection of ¹³C enrichments in muramic acid from the detrital microbiota incubated with relabeled precursors.     

Determination of the sedimentary microbial biomass by extractible lipid phosphate

Summary The measurement of lipid phosphate is proposed as an indicator of microbial biomass in marine and estuarine sediments. This relatively simple assay can be performed on fresh, frozen or frozen-lyophilized sediment samples with chloroform methanol extraction and subsequent phosphate determination. The sedimentary lipid phosphate recovery correlates with the extractible ATP and the rate of DNA synthesis. Pulse-chase experiments show active metabolism of the sedimentary phospholipids. The recovery of added ¹⁴C-labeled bacterial lipids from sediments is quantitative. Replicate analyses from a single sediment sample gave a standard deviation of 11%. The lipid extract can be fractionated by relatively simple procedures and the plasmalogen, diacyl phospholipid, phosphonolipid and non-hydrolyzable phospholipid content determined. The relative fatty acid composition can be readily determined by gas-liquid chromatography.The lipid composition can be used to define the microbial community structure. For example, the absence of polyenoic fatty acids indicates minimal contamination with benthic micro-eukaryotes. Therefore the high content of plasmalogen phospholipids in these sediments suggests that the anaerobic prokaryotic Clostridia are found in the aerobic sedimentary horizon. This would require anaerobic microhabitats in the aerated zones.     

Effects of Grazing by Estuarine Gammaridean Amphipods on the Microbiota of Allochthonous Detritus

Estuarine gammaridean amphipods grazing at natural population density on detrital microbiota affected the microbial community composition, biomass, and metabolic activity without affecting the physical structure of the leaves. Total microbial biomass estimated by adenosine triphosphate and lipid phosphate or observed by scanning electron microscopy was greater on grazed than on ungrazed detritus. The rates of oxygen consumption, poly-β-hydroxybutyrate synthesis, total lipid biosynthesis, and release of ¹⁴CO₂ from radioactively prelabeled microbiota were higher on grazed than on ungrazed leaves, indicating stimulation of the metabolic activity of grazed detrital microbes. This was true with rates based either on the dry leaf weight or microbial biomass. Alkaline phosphatase activity was lower in the grazed system, consistent with enhanced inorganic phosphate cycling. The loss of ¹⁴C from both total lipid and poly-β-hydroxybutyrate of microorganisms prelabeled with ¹⁴C was greater from grazed than ungrazed microbes. There was a faster decrease in the ¹⁴C-glycolipid than in the ¹⁴C-neutral lipid or ¹⁴C-phospholipid fractions. Analysis of specific phospholipids showed losses of the metabolically stable [¹⁴C]glycerolphosphorylcholine derived from phosphatidylcholine and much more rapid metabolism of the bacterial lipid phosphatidylglycerol measured as [¹⁴C]glycerolphosphorylglycerol with amphipod grazing. The biochemical data supported scanning electron microscopy observations of a shift as the grazing proceeded from a bacterial/fungal community to one dominated by bacteria.     

Examination of Thermophilic Methane-Producing Digesters by Analysis of Bacterial Lipids

Thermophilic methane-producing digesters were examined by the analysis of lipids to determine the microbial biomass, community structure, and nutritional status of the microbes within the digesters. The digesters received a daily feedstock of cattle feed and Bermuda grass, with some digesters receiving additional supplements of propionate, butyrate, or nitrate. Microbial biomass, measured as total extractable lipid phosphate, was decreased in slurries from digesters receiving continuous addition of the fermentation intermediates propionate or butyrate as compared with slurries from control digesters receiving the feedstock alone. In slurries from digesters that received continuous addition of nitrate, the microbial biomass was higher than in the slurries from control digesters. The control digesters had ca. 2.5 × 10¹¹ bacteria per g (dry weight) as determined from total extractable lipid phosphate. Shifts in microbial community structure were observed by analysis of ester-linked phospholipid fatty acids. Statistical analysis of the patterns of phospholipid fatty acids indicated that the digesters receiving different supplements could be distinguished from the control digester and from each other. Poly-β-hydroxybutyric acid, an indicator of metabolic stress, was detected in slurries from all the digesters. Slurries from the nitrate-amended digester had the highest concentration of poly-β-hydroxybutyric acid, whereas slurries from the propionate-amended digester had the lowest concentration. These chemical analyses offer a quantitative means to correlate shifts in microbial biomass, community structure, and nutritional status in complex fermentation systems to the production of a specific end product.     

Towards valorization of pectin-rich agro-industrial residues: Engineering of Saccharomyces cerevisiae for co-fermentation of d-galacturonic acid and glycerol

Pectin-rich plant biomass residues represent underutilized feedstocks for industrial biotechnology. The conversion of the oxidized monomer d-galacturonic acid (d-GalUA) to highly reduced fermentation products such as alcohols is impossible due to the lack of electrons. The reduced compound glycerol has therefore been considered an optimal co-substrate, and a cell factory able to efficiently co-ferment these two carbon sources is in demand. Here, we inserted the fungal d-GalUA pathway in a strain of the yeast S. cerevisiae previously equipped with an NAD-dependent glycerol catabolic pathway. The constructed strain was able to consume d-GalUA with the highest reported maximum specific rate of 0.23 g g_CDW⁻¹ h⁻¹ in synthetic minimal medium when glycerol was added. By means of a ¹³C isotope-labelling analysis, carbon from both substrates was shown to end up in pyruvate. The study delivers the proof of concept for a co-fermentation of the two ‘respiratory’ carbon sources to ethanol and demonstrates a fast and complete consumption of d-GalUA in crude sugar beet pulp hydrolysate under aerobic conditions. The future challenge will be to achieve co-fermentation under industrial, quasi-anaerobic conditions.     

Synthesis and properties of 35S, 14C and 3H labeled S-alkyl glycerol ethers and derivatives

Radioactive S-alkyl glycerol ethers have been synthesized with ³⁵S, ¹⁴C and ³H labels as well as ³H/³⁵S double labels.The synthesized compounds were converted to various derivatives which can serve to characterize the S-alkyl glycerol ethers. These included the isopropChemical analysis, IR, NMR, zonal TLC profile scans and GLC showed all the products to be > 99% pure.The GLC behaviour of the aldehyde and acetate derivatives of both S-alkyl glycerol ethers and O-alkyl glycerol ethers on EGSS-X was compared.     

峡谷型喀斯特不同生态系统的土壤微生物数量及生物量特征

采用样地调查与室内分析相结合的方法,研究了峡谷型喀斯特水田、旱地、草地、灌丛、人工林、次生林6种生态系统不同深度土壤微生物数量、微生物生物量特征及其分形关系。结果表明:峡谷型喀斯特不同生态系统的土壤微生物数量及组成不同,微生物数量均以次生林最高,旱地最低,其组成数量均为细菌放线菌真菌,细菌是土壤微生物的主要类群,数量多达26.66×105—71.64×105cfu/g,占全部微生物比例为87.00%—95.50%,其次为放线菌数量,为1.45×105—3.78×105cfu/g,所占比例为4.21%—12.39%,真菌数量最小,为0.07×105—0.23×105cfu/g,所占比例仅为0.24%—0.61%,不足1%。不同生态系统土壤微生物生物量碳(MBC)、氮(MBN)、磷(MBP)的含量不同,次生林MBC与MBN最高,人工林MBP最高,旱地MBC最低,草地MBN与MBP最低;各生态系统均为MBCMBNMBP。不同生态系统的MBC/SOC、MBN/TN、MBP/TP分别为0.44%—0.97%、2.13%—3.13%、1.46%—2.13%,差异不显著;MBC/MBN在3.06—6.54之间,其中次生林极显著高于其他生态系统,其他生态系统差异不显著。不同生态系统土壤微生物数量及生物量均随土层加深而减少,且具有良好分形关系,均达到了极显著水平(P0.01)。探讨土壤微生物活性为提高石灰土土壤肥力、促进喀斯特植被迅速恢复提供依据。     

Metabolism of triacylglycerol in developing rat brain

Metabolism of triacylglycerol (TAG) in developing brain has been examined. TAG is a relatively minor fraction of brain lipid in both suckling and adult rats and cannot be accounted for as entrapped blood. When glycerol tri[1-¹⁴C]oleate and [2-³H]glycerol trioleate were simultaneously injected intracerebrally into suckling rats, both labels appeared in diacylglycerol and the major phospholipids; acyl chain label was incorporated more extensively at early time points, with choline phosphoglycerides being most actively labeled. With [1-¹⁴C]fatty acids and [2-³H] glycerol administration, the specific activity of TAG was much greater than that of the more abundant phospholipids. Although direct acyl exchange between TAG and phospholipids was not demonstrated, relationships of TAG to selective mechanisms of phosphoglyceride synthesis were indicated.Abbreviations used TAG triacylglycerol - DAG diacylcerol - HPLC high performance liquid chromatography - CoA coenzyme A - BSA bovine serum albumin - TLC thin layer chromatography - DPM disintegrations per minute - ATP adenosine triphosphate - GLC gas liquid chromatography - PC choline, phosphoglyceride - PE ethanolamine phosphoglyceride - PS serine phosphoglyceride - PI inositol phosphoglyceride     

川西亚高山森林土壤呼吸和微生物生物量碳氮对施氮的响应

随着全球大气氮沉降的明显增加,将有可能显著影响我国西部地区受氮限制的亚高山森林生态系统。土壤微生物是生态系统的重要组成部分,是土壤物质循环和能量流动的重要参与者。由于生态系统类型、土壤养分、氮沉降背景值等的差异,土壤呼吸和土壤生物量碳氮对施氮的响应存在许多不确定性。而施氮会不会促进亚高山森林生态系统中土壤呼吸和微生物对土壤碳氮的固定?基于此假设,选择了川西60年生的四川红杉(Larix mastersiana)亚高山针叶林为研究对象,通过4个水平的土壤施氮控制试验(CK:0 g m~(-2) a~(-1)、N1:2 g m~(-2)a~(-1)、N2:5 g m~(-2) a~(-1)、N3:10 g m~(-2)a~(-1)),监测了土壤呼吸及土壤微生物生物量碳氮在一个生长季的动态情况。结果表明:施氮对土壤呼吸各指标和土壤微生物碳氮都有极显著的影响,施氮能促进土壤全呼吸、自养呼吸、异养呼吸通量和土壤微生物生物量碳氮的增长,施氮使土壤呼吸通量提高了11%—15%,土壤微生物量碳提高了5%—9%,土壤微生物量氮提高了23%—34%。在中氮水平下(5 g m~(-2) a~(-1))对土壤呼吸的促进最显著。相关分析发现,土壤呼吸与微生物生物量碳氮和微生物代谢商极呈显著正相关,微生物量碳氮与土壤温度呈极显著的正相关,与土壤湿度呈极显著负相关。通过一般线性回归拟合土壤呼吸速率与土壤10 cm温湿度的关系,发现土壤呼吸速率与土壤温度呈极显著的正相关,与土壤湿度极显著负相关(P0.001),中氮水平下土壤温度敏感性系数Q_(10)值(7.10)明显高于对照(4.26)。     

Biodegradation kinetics of 2,4,6-Trichlorophenol by an acclimated mixed microbial culture under aerobic conditions

The objective of this study was to achieve a better quantitative understanding of the kinetics of 2,4,6-trichlorophenol (TCP) biodegradation by an acclimated mixed microbial culture. An aerobic mixed microbial culture, obtained from the aeration basin of the wastewater treatment plant, was acclimated in shake flasks utilizing various combinations of 2,4,6-TCP (25–100 mg l⁻¹), phenol (300 mg l⁻¹) and glycerol (2.5 mg l⁻¹) as substrates. Complete primary TCP degradation and a corresponding stoichiometric release of chloride ion were observed by HPLC and IEC analytical techniques, respectively. The acclimated cultures were then used as an inoculum for bench scale experiments in a 4 l stirred-tank reactor (STR) with 2,4,6-TCP as the sole carbon/energy (C/E) source. The phenol acclimated mixed microbial culture consisted of primarily Gram positive and negative rods and was capable of degrading 2,4,6-TCP completely. None of the predicted intermediate compounds were detected by gas chromatography in the cell cytoplasm or supernatant. Based on the disappearance of 2,4,6-TCP, degradation was well modelled by zero-order kinetics which was also consistent with the observed oxygen consumption. Biodegradation rates were compared for four operating conditions including two different initial 2,4,6-TCP concentrations and two different initial biomass concentrations. While the specific rate constant was not dependent on the initial 2,4,6-TCP concentration, it did depend on the initial biomass concentration (X _init). A lower biomass concentration gave a much higher zero-order specific degradation rate. This behaviour was attributed to a lower average biomass age or cell retention time (θ_x) for these cultures. The implications of this investigation are important for determining and predicting the potential risks associated with TCP, its degradation in the natural environment or the engineering implications for ex situ treatment of contaminated ground water or soil.     

Evidence for Transaldolase Activity in the Isolated Heart Supplied with [U-13C3]Glycerol

Studies of glycerol metabolism in the heart have largely emphasized its role in triglyceride synthesis. However, glycerol may also be oxidized in the citric acid cycle, and glycogen synthesis from glycerol has been reported in the nonmammalian myocardium. The intent of this study was to test the hypothesis that glycerol may be metabolized to glycogen in mammalian heart. Isolated rat hearts were supplied with a mixture of substrates including glucose, lactate, pyruvate, octanoate, [U-¹³C₃]glycerol, and ²H₂O to probe various metabolic pathways including glycerol oxidation, glycolysis, the pentose phosphate pathway, and carbon sources of stored glycogen. NMR analysis confirmed that glycogen production from the level of the citric acid cycle did not occur and that the glycerol contribution to oxidation in the citric acid cycle was negligible in the presence of alternative substrates. Quite unexpectedly, ¹³C from [U-¹³C₃]glycerol appeared in glycogen in carbon positions 4–6 of glucosyl units but none in positions 1–3. The extent of [4,5,6-¹³C₃]glucosyl unit enrichment in glycogen was enhanced by insulin but decreased by H₂O₂. Given that triose phosphate isomerase is generally assumed to fully equilibrate carbon tracers in the triose pool, the marked ¹³C asymmetry in glycogen can only be attributed to conversion of [U-¹³C₃]glycerol to [U-¹³C₃]dihydroxyacetone phosphate and [U-¹³C₃]glyceraldehyde 3-phosphate followed by rearrangements in the nonoxidative branch of the pentose phosphate pathway involving transaldolase that places this ¹³C-enriched 3-carbon unit only in the bottom half of hexose phosphate molecules contributing to glycogen.     

Bacterial abundance and diversity in the Barbados Trench determined by phospholipid analysis

Abstract: The Barbados trench is characterized by large fields of volcanoes and mounds located over a distance of 30 km above the northern slope of a basement ridge corresponding to an inactive transform fault. Sediments from various locations were collected and analyzed for their lipid contents. Bacterial input to the overall biomass was estimated through the analysis of phospholipid ester-linked fatty acid (PLFA) profiles and glycerol ether lipids. Results indicated a eubacterial biomass estimated to be 10⁹cells (g dry wt)⁻¹. Individual fatty acid profiles revealed the presence of sulfur-oxidizing bacteria common to many deep-sea sites but also a large contribution of type I and type II methanotrophs to the eubacterial biomass. The presence of methanotrophs was further supported by the analysis of specific biomarkers of these microorganisms as well as some unusual trans fatty acid isomers. Anaerobic bacteria and presumbly sulfate reducing bacteria were also present, as well as archaebacteria and primarily methanogens, as indicated by glycerol ether lipid analysis.     

Axonal transport of phospholipids in rat visual system.

Abstract— Seventeen day old rats were injected intraocularly with a phospholipid precursor, [³²P]phosphate, and a glycoprotein precursor, [³H]fucose. Animals were killed between 1 h and 21 days later, and structures of the visual pathway (retina, optic nerve, optic tract, lateral geniculate body, and superior colliculus) were dissected. Radioactivity in phospholipids ([³²P] in solvent-extracted material) and in glycoproteins ([³H] in solvent-extracted residue) was determined. Incorporation of [³H]fucose into retinal glycoproteins peaked at 6–8 h. Labelled glycoproteins were present in superior colliculus by 2h after injection, indicating a rapid rate of transport; maximal labelling was at 8–10 h after injection. Incorporation of [³²P]phosphate into retinal phospholipids peaked at 1 day after injection. Phospholipids were also rapidly transported since label was present in the superior colliculus by 3 h after injection: however, maximal labelling did not occur until 5–6 days. These results indicate that newly synthesized phospholipids enter a preexisting pool, part of which is later committed to transport at a rapid rate. Transported phospholipids were catabolized at the nerve endings with a maximum half-life of several days; there was minimal recycling of precursor label. Lipids were fractionated by thin-layer chromatography, and radioactivity in individual phospholipid classes determined. Choline and ethanolamine phosphoglycerides were the major transported phospholipids, together accounting for approx 85% of the total transported lipid radioactivity. At early time points, the ratio of radioactivity in choline phosphoglycerides to that in ethanolamine phosphoglycerides increased in structures progressively removed from the site of synthesis (retina) but by 2 days approached a constant value. In each structure, choline phosphoglyceride-ethanolamine phosphoglyceride radioactivity ratios decreased with time, rapidly at first, but plateaued by 2 days. These results indicate that choline phosphoglycerides are committed to transport sooner than ethanolamine phosphoglycerides. Some experiments were also conducted using [2-³H]glycerol as a phospholipid precursor. Results concerning incorporation of this precursor into individual phospholipid classes and their subsequent axonal transport were comparable to those obtained using [³²P]phosphate, with the following exceptions: (a) incorporation of [2-³H]glycerol into retinal phospholipids was relatively rapid (near-maximal levels at 1 h after injection) although transport to the superior colliculus showed an extended time course very similar to [³²P]-labelled lipids; (b) [2-³H]glycerol was somewhat less efficient than [³²P]phosphate in labelling lipids committed to transport relative to labelling those which remained in the retina; and (c) [2-³H]glycerol did not label plasmalogens.     

Soil microbial biomass influence on strontium availability in mine soil

Abstract

The main aim of our work was to assess whether strontium (Sr) affects soil microbial biomass size and activity, and the involvement of said biomass in the availability process of the metal. In addition, information concerning the distribution and mobility of the stable element within ecosystems may allow the prediction of the behaviour of its radioisotope counterpart, ⁹⁰Sr. Samples were collected in the surroundings of a strontium mine and characterised for total and diethylene triamine pentaacetic acid (DTPA)-extractable Sr, total organic C (TOC), microbial biomass C (MBC), MBC/TOC ratio and metabolic quotient (qCO₂). Moreover, MBC and DTPA-extractable Sr were measured during a 45-day incubation experiment of samples soils amended with maize. Overall, increased levels of total Sr had a negative effect on both TOC and MBC. DTPA-extractable Sr was significantly correlated to MBC/TOC suggesting a possible role of soil microbial biomass in the mobilisation of the element. The synthesis of new microbial biomass after soil amendment was negatively affected by the initial content of DTPA-extractable Sr. Conversely, there was a linear positive relationship between newly formed MBC and DTPA-extractable Sr during the incubation, indicating that soil microbial biomass may promote the mobilisation of Sr. These findings indicate that soil amendment with easily degradable organic substrate significantly increases Sr mobility and availability.     

Carbon and nitrogen cycling during old-field succession: Constraints on plant and microbial biomass

Soil C and N dynamics were studied in a sequence of old fields of increasing age to determine how these biogeochemical cycles change during secondary succession. In addition, three different late-successional forests were studied to represent possible "steady state" conditions. Surface soil samples collected from the fields and forests were analyzed for total C, H₂O-soluble C, total N, potential net N mineralization, potential net nitrification, and microbial biomass. Above-and belowground plant biomass was estimated within each of the old field sites.Temporal changes in soil organic C, total N and total plant biomass were best described by a gamma function [y =at ^b e ^ctd +f] whereas a simple exponential model [y =a(l – e^–bt ) + c] provided the best fit to changes in H₂O-soluble C, C:N ratio, microbial C, and microbial N. Potential N mineralization and nitrification linearly increased with field age; however, rates were variable among the fields. Microbial biomass was highly correlated to soil C and N pools and well correlated to the standing crop of plant biomass. In turn, plant biomass was highly correlated to pools and rates of N cycling.Patterns of C and N cycling within the old field sites were different from those in a northern hardwood forest and a xeric oak forest; however, nutrient dynamics within an oak savanna were similar to those found in a 60-yr old field. Results suggest that patterns in C and N cycling within the old-field chronosequence were predictable and highly correlated to the accrual of plant and microbial biomass.     

Chemical structure of wall teichoic acid isolated from Enterococcus faecium strain U0317

Wall teichoic acid (WTA) was isolated from Enterococcus faecium strain U0317 and structurally characterized using ¹H, ¹³C, and ³¹P NMR spectroscopy, including two-dimensional COSY, TOCSY, ROESY, HMQC, and HMBC experiments. Further compositional determination was undertaken using classical chemical methods and HF treatment followed by GLC and GLC–MS analyses. The repeating unit of WTA consisted of two residues of 2-acetamido-2-deoxy-d-galactose, glycerol (Gro), and phosphate, and has the structure shown below:→6)-α-d-GalpNAc-(1→3)-β-d-GalpNAc-(1→2)-Gro-(3→P→     

Lipid composition of the electron transport membrane of Haemophilus parainfluenzae

The principal lipids associated with the electron transport membrane of Haemophilus parainfluenzae are phosphatidylethanolamine (78%), phosphatidylmonomethylethanolamine (0.4%), phosphatidylglycerol (18%), phosphatidylcholine (0.4%), phosphatidylserine (0.4%), phosphatidic acid (0.2%), and cardiolipin (3.0%). Phospholipids account for 98.4% of the extractible fatty acids. There are no glycolipids, plasmalogens, alkyl ethers, or lipo amino acid esters in the membrane lipids. Glycerol phosphate esters derived from the phospholipids by mild alkaline methanolysis were identified by their staining reactions, mobility on paper and ion-exchange column chromatography, and by the molar glycerol to phosphate ratios. Eleven diacyl phospholipids can be separated by two-dimensional thin-layer chromatography. Each lipid served as a substrate for phospholipase D, and had a fatty acid to phosphate ratio of 2:1. Each separated diacyl phospholipid was deacylated and the glycerol phosphate ester was identified by paper chromatography in four solvent systems. Of the 11 separated phospholipids, 3 were phosphatidylethanolamines, 2 were phosphatidylserines, and 2 were phosphatidylglycerols. Phosphatidylcholine, cardiolipin, and phosphatidic acid were found at a single location. Phosphatidylmonomethylethanolamine was found with the major phosphatidylethanolamine. Three distinct classes of phospholipids are separable according to their relative fatty acid compositions. (i) The trace lipids consist of two phosphatidylethanolamines, two phosphatidylserines, phosphatidylcholine, phosphatidic acid, and a phosphatidylglycerol. Each lipid represents less than 0.3% of the total lipid phosphate. These lipids are characterized by high proportions of the short (C(10) to C(14)) and long (C(19) to C(22)) fatty acids with practically no palmitoleic acid. (ii) The major phospholipids (93% of the lipid phosphate) are phosphatidylethanolamine, phosphatidylmonomethylethanolamine, and phosphatidylglycerol. These lipids contain a low proportion of the short (C(19)) fatty acids. Palmitic and palmitoleic acids represent over 80% of the total fatty acids. (iii) The fatty acid composition of the cardiolipin is intermediate between the other two classes. Both palmitoleic and the longer fatty acids represent a significant proportion of the total fatty acid.     

Separation and Characterization of Inositol Phospholipids from the Pulvini of Samanea saman

To supplement current thin-layer chromatographic methods for separation and quantitation of plant phospholipids, an alternative method, high-performance liquid chromatography was developed. The major inositol-containing lipids from the pulvini of Samanea saman Merr. were identified as phosphatidylinositol, phosphatidylinositol phosphate, and phosphatidylinositol bisphosphate based on comigration with authentic standards on high-performance liquid chromatography and on thin-layer chromatography. The patterns of incorporation of radioactivity into the putative phosphatidylinositol and phosphatidylinositol phosphate were consistent with these identifications when pulvini were labeled with [³H]glycerol, [³H]inositol, or [³²P]orthophosphate. Analysis of the products of enzymic hydrolysis, of chemical deacylation, and of `fingerprint' methanolysis of these phospholipids confirmed the identifications.     

Glyceride metabolism in cultured cells dissociated from rat cerebral cortex

Abstract— [1-¹⁴C]stearic acid and [2-³H]glycerol were rapidly taken up and esterified into triacylglycerol and phospholipids by rat brain cells cultivated in monolayers. Expressed in terms of pool size, the incorporation of glycerol and stearate into triacylglycerol was 6- and 8-fold, respectively, higher than the incorporation into the choline phosphoglycerides. Tritium-labelled glycerol in both triacylglycerol and glycerophosphatides was diluted more rapidly than the [¹⁴C] labelled fatty acids. Chase experiments indicated a transfer of fatty acid from one lipid class to another, mainly from triacylglycerol to phospholipids, with no apparent loss of radioactivity. The accumulation of triacylglycerol in the brain cells was a function of both the presence of exogenous fatty acids in the culture medium and the metabolic needs of the cells; as long as the cells were involved in active formation of membranes the proportion of triacylglycerol was relatively small; its concentration increased while cell division slowed down in older, fully monolayered cultures.     

THE METABOLIC TURNOVER OF MOLECULAR SPECIES OF PHOSPHATIDYLINOSITOL and ITS PRECURSOR PHOSPHATIDIC ACID IN GUINEA-PIG CEREBRAL HEMISPHERES

Abstract– The molecular species composition of phosphatidylinositol from guinea-pig cerebral hemispheres was studied and found similar to that of phosphatidylinositol from ox cerebral hemispheres. In both cases the tetraenoic species was predominant. Phosphatidic acid from guinea-pig cerebral hemispheres contained two major molecular species; the monoenoic and hexaenoic (33.4 and 24 mol/100 mol respectively). In order to study the metabolism of molecular species of phosphatidic acid and phosphatidylinositol in the cerebral hemispheres, guinea-pigs were injected intracisternally with ³²P_i and [U-¹⁴C]glucose. After 5 min of isotopic exchange, the specific radioactivity of ³²P in phosphatidylinositol was nearly equal to that in phosphatidic acid, whereas specific radioactivity of ¹⁴C in the glycerol was 1.4 times and in the fatty acids nearly 0.5 times that in the phosphatidic acid respectively, indicating metabolic heterogeneity of both phospholipids. The glycerol specific radioactivity was different in all the molecular species of phosphatidic acid being greatest in the monoenoic and least in the tetranenoic species. When the molecular species were arranged in this way, the order was representative of their relative rates of synthesis by acylation of glycerol-3-phosphate. An almost opposite order was obtained when the molecular species were arranged according to their phosphate/glycerol radioactivity ratios, indicating the relative contribution of the diacylglycerol kinase pathway to their formation. When the specific radioactivity values and ratios of phosphatidylinositol were similarly considered, the orders of the molecular species were, on the whole, similar to that of phosphatidic acid. This indicated that synthesis de novo (Paulus & Kennedy , 1960) was operative in the formation of most of its molecular species, but due to other considerations it was concluded that part of the tetraenoic, and probably the whole of saturated phosphatidylinositol may be formed by transacylation reactions. The results are discussed in terms of the experimental limitations of previous and present techniques for the analysis of phospholipid molecular species.