Lipid composition of cells and its variation during degradation of a culture of Streptococcus diacetilactis

The process of dying of a culture of Str. diacetilactis under conditions of prolonged culturing is accompanied by substantial changes in the composition of the cell lipids. In the lipids of young cells the basic components are mono- and diglucosyldiglycerides, as well as phospholipids--two glucophospholipids, cardiolipin, and phosphatidylglycerin; triglycerides and sterols are present in negligible amounts. As the Str. diacetilactis culture develops and undergoes degradation, an increase is observed in the total amount of extractable lipids. In this case the fraction of phospholipids increases (chiefly on account of cardiolipin), while the fraction of neutral glucolipids decreases. It was shown that in cell homogenates there is a negligible peroxidation of lipids, which is intensified as the culture dies off. There is a parallel acceleration of the uptake of O2 by cell suspensions from cultures of different ages. The detected changes in the lipids are correlated with the morphological and physiological indices of the state of the culture.     

Studies on the origin of milk fat. A further study of bovine serum lipoproteins and an estimation of their contribution to milk fat

1. Tritium-labelled palmitic acid combined in olive-oil triglycerides was introduced into the rumen of a lactating cow and the specific radioactivity of the lipids of milk and of the lipoproteins of both jugular and mammary venous serum was measured. 2. As previously found in a similar experiment with [(3)H]stearic acid, the specific radioactivity of the triglyceride fraction of the dextran sulphate-precipitable lipoproteins reached a maximum earlier and greater than that of the milk fat. 3. This fraction was the only lipid separated that had a significant arteriovenous difference in concentration, and is therefore identified as the main circulating lipid precursor of milk fat. 4. Although the non-esterified fatty acids showed no arteriovenous difference in concentration, they showed a negative difference in specific radioactivity that could have occurred only at the expense of the triglycerides of the precipitable lipoproteins. 5. The mean specific radioactivity of the triglycerides immediately after removal from the blood is calculated and shown to be very close in value to that of the corresponding fraction in mammary venous serum. 6. By comparison of the mean specific radioactivities of milk fat and of this precursor, its contribution is calculated as 36%. 7. This value is discussed with reference to the concentration of C(16) and C(18) fatty acids in milk fat and it is concluded that substantial amounts of these acids must have been derived from a source other than preformed circulating lipids.     

Assessing Mixing Quality of a Copovidone-TPGS Hot Melt Extrusion Process with Atomic Force Microscopy and Differential Scanning Calorimetry

Atomic force microscopy (AFM) and modulated differential scanning calorimetry (mDSC) were used to evaluate the extent of mixing of a hot melt extrusion process for producing solid dispersions of copovidone and D-α-tocopherol polyethylene glycol 1000 succinate (TPGS 1000). In addition to composition, extrusion process parameters of screw speed and thermal quench rate were varied. The data indicated that for 10% TPGS and 300 rpm screw speed, the mixing was insufficient to yield a single-phase amorphous material. AFM images of the extrudate cross section for air-cooled material indicate round domains 200 to 700 nm in diameter without any observed alignment resulting from the extrusion whereas domains in extrudate subjected to chilled rolls were elliptical in shape with uniform orientation. Thermal analysis indicated that the domains were predominantly semi-crystalline TPGS. For 10% TPGS and 600 rpm screw speed, AFM and mDSC data were consistent with that of a single-phase amorphous material for both thermal quench rates examined. When the TPGS concentration was reduced to 5%, a single-phase amorphous material was achieved for all conditions even the slowest screw speed studied (150 rpm).     

Glomerular sclerotic lesions in the rat. Histochemical analysis of their macromolecular and cellular composition

The present study provides a histochemical analysis of the macromolecular and cellular composition of focal and segmental glomerular hyalinosis and sclerosis (FSGHS) in the rat with special reference to the different types of lipids present and to the participation of monocytes. FSGHS was induced in male Wistar rats by unilateral nephrectomy (UN) or puromycin aminonucleoside (PAN) injections. Histochemical analysis of glomeruli with FSGHS in both models after 20 weeks of proteinuria revealed massive deposits of lipids. These lipid accumulations were shown to consist mainly of free and esterified cholesterol; triglycerides and phospholipids were present in small amounts. Monocytes, identified by the alpha-naphthyl acetate method for non-specific esterase activity were scanty in glomeruli affected by FSGHS with an average glomerular count of 0.1 in UN- and 0.2 in PAN-treated rats. When present, no preferential localization of monocytes in the lesions was observed. The progressive glomerular damage occurring once the process of hyalinosis and sclerosis has started may be related to the paucity of "scavenging" monocytes. Cholesterol may be one of the substances involved in the development of these glomerular changes.     

Compartmentalization of self-reproducing machineries: Multiplication of microsystems with self-instructing polymerization of amino acids

A theoretical model is presented for the self-instructing polymerization of free amino acids which proceeds inside microsystems which are phase-separated from the solution of thermal polyamino acids. It is shown theoretically that a compartmentalized microsystem fixes inside itself only the process with a faster macromolecular multiplication as time passes, even if the catalytic polymerization alone could spontaneously decrease the corresponding reaction rate. The compartmentalized machinery of macromolecular multiplication cannot reach its stationary state. The machinery is inevitably multiplied and alternates with those with either faster rates of macromolecular multiplication or slower rates of macromolecular degradation during their time development. These results are based upon the dynamic process that any material system acts by itself so as to remove any flow disequilibrium, that is, to maintain the continuity of material flow.     

Cholesteryl ester and triglyceride hydrolysis by an acid lipase from rabbit aorta.

The properties of the triglyceride- and cholesteryl ester-hydrolyzing activity by an acid lipase from rabbit aortic tissue were compared under different experimental conditions. Radiolabeled cholesteryl oleate or triolein was incorporated into phospholipid vesicles by sonication and the resulting preparations were used for in vitro studies. No distinction was observed between triglyceride lipase and cholesterol esterase activity in the aortic cytosol fraction following either thermal inactivation, inhibition by a mercurial, fractionation by ammonium sulfate or acid precipitation, or DEAE-cellulose chromatography. Addition of rabbit lipoproteins to the assay system resulted in inhibition of both cholesterol esterase and triglyceride lipase activity. Parallel changes in the hydrolysis of both substrates also were observed when exogenously added lipids were added to the incubation system in various physical states. Specificities of the enzyme system towards different cholesteryl esters were examined. No differences in the rate of hydrolysis were observed between cholesteryl oleate, palmitate and linoleate. The data suggest that a single acid lipase, presumably of lysosomal origin, has broad specificity towards triglycerides and cholesteryl esters, and may play a role in the hydrolysis of these lipids during intralysosomal degradation of lipoproteins.     

Epigallocatechin gallate increases the formation of cytosolic lipid droplets and decreases the secretion of apoB-100 VLDL

Epigallocatechin gallate (EGCG) increases the formation of cytosolic lipid droplets by a mechanism that is independent of the rate of triglyceride biosynthesis and involves an enhanced fusion between lipid droplets, a process that is crucial for their growth in size. EGCG treatment reduced the secretion of both triglycerides and apolipoprotein B-100 (apoB-100) VLDLs but not of transferrin, albumin, or total proteins, indicating that EGCG diverts triglycerides from VLDL assembly to storage in the cytosol. This is further supported by the observed increase in both intracellular degradation of apoB-100 and ubiquitination of the protein (indicative of increased proteasomal degradation) in EGCG-treated cells. EGCG did not interfere with the microsomal triglyceride transfer protein, and the effect of EGCG on the secretion of VLDLs was found to be independent of the LDL receptor. Thus, our results indicate that EGCG promotes the accumulation of triglycerides in cytosolic lipid droplets, thereby diverting lipids from the assembly of VLDL to storage in the cytosol. Our results also indicate that the accumulation of lipids in the cytosol is not always associated with increased secretion of VLDL.     

Heterogeneity of serum lipoproteins during the fetal and neonatal development of the pig

Serum lipoproteins from fetal, neonatal and adult pigs were characterized with the use of lipid analysis, polyacrylamide gradient gel electrophoresis, two-dimensional immunoelectrophoresis and zonal ultracentrifugation. Almost all serum cholesterol was found in LDL during the early stages of fetal development, while low but increasing levels appeared in the fetal pig HDL by the end of the gestation period. In the fetal pig, most of the serum triglycerides could be found in the HDL fraction. After the start of suckling, the levels of serum triglycerides and cholesterol increased. Most of these exogenous lipids were found in the chylomicrons + VLDL + LDL fraction of the newborn pig serum. The molecular weights of the native serum lipoproteins were calculated as being 2.0-2.4 X 10(5) daltons for newborn pig HDL and 1.4-1.7 X 10(6) daltons for newborn pig LDL. Minor changes in the molecular weight distributions were detected within these ranges for both HDL and LDL during fetal and neonatal development of the pig. Zonal ultracentrifugation of neonatal pig serum partly separated the LDL into three subfractions, whereas neonatal HDL appeared as one broad fraction.     

Glycerolipid metabolism during early amphibian embryogenesis. Neutral lipid involvement in fertilization triggered events

In order to study glycerolipid metabolism during early embryonic development, Bufo arenarum Hensel female toads were administered [1-3-(14)C]glycerol together with hormonal stimulus. Triglycerides comprised 60% of the total label in all developmental stages analyzed. The highest specific activity was observed in phosphatidic acid which underwent a net decrease as a function of development. Phosphatidylcholine was the most actively synthesized phosphoglyceride. A significant decrease in the total lipid labeling, mainly accounted for by triglycerides, was detected at fertilization time concomitant with a net diminution in the content of these neutral lipids. Subcellular studies showed that yolk platelets contain about 99% of the total oocyte triglycerides suggesting that massive breakdown would be confined to this fraction. Present findings evidence that the de novo lipid biosynthesis is operating during the developmental analyzed period. In addition, they would suggest the active role of triglycerides in the energetic events taking place around fertilization.     

Organic matter evolution during co-composting of the organic fraction of municipal waste and poultry manure

The study concerned the evolution of organic matter, and the humification process, during the co-composting of the organic fraction of municipal solid waste (OMSW) and poultry manure (PM); the study was made with two different mixtures (OMSW:PM ratios of 3:2 and 2:3, wet weight:wet weight) and two different particle sizes (1 and 0.2cm). The results suggested that the composting process proceeded unhindered throughout the degradation of easily degradable materials like hemicellulose, and that of the rather less degradable cellulose and lipids, and the concentration of recalcitrant material, i.e. a ligno-humic (LU) fraction. These processes were more evident for mixtures with lower particle size. Throughout the composting, in all mixtures studied, humification proceeded by the formation of a new HA fraction, which was probably the result of the partial degradation and solubilization of more complex insoluble organic molecules, i.e. humin fraction.     

Influence of diet on the modeling of adipose tissue triglycerides during growth

We have studied the accretion of lipids in growing mice. We measured the rates of synthesis and degradation of triglycerides in epididymal fat pads of mice maintained for 44 days on a low-fat, high-carbohydrate diet (I) or a high-fat, low-carbohydrate diet (II). 2H2O was added to the drinking water for 14 days. Rates of incorporation/washout of 2H to/from C1 of triglyceride-glycerol showed that triglyceride synthesis was greater than triglyceride degradation (net triglyceride balance was approximately 2.5 times greater in II than in I). The data also show that the contribution of de novo lipogenesis to triglyceride-bound palmitate was approximately 3 times greater in I than in II. This was consistent with a greater relative intake of carbohydrate in I vs. II. The rates of incorporation and washout of newly synthesized (2H-labeled) palmitate into and from triglycerides were also measured. Those data suggested a remodeling of triglyceride-bound fatty acids. On measuring the profile of triglyceride-bound fatty acids, we observed a decrease in the relative abundance of triglyceride-bound palmitate and stearate and an increase in triglyceride-bound oleate and linoleate. This was observed in I and II. In summary, diet substantially affects the deposition and modeling of triglycerides in adipose tissue during growth. 2H2O can be used to examine the mechanisms responsible for the accumulation of triglycerides, e.g., factors that affect 1) triglyceride synthesis and degradation and 2) the source of fatty acids that are used in esterification.     

Rapid degradation of polar lipids in frost damaged winter wheat crown and root tissue

The ettect of a lethal frost on the lipid composition of crown and root tissue of winter wheat after thawing was investigated. The data show rapid degradation of polar lipids with an increase first in diglycerides and later in unesterified fatty acids, while active synthesis of triglycerides was maintained during the first 6 hr after thawing. Polyunsaturated species of polar lipids were preferentially degraded. Extensive losses of total linoleic and linolenic acids suggest lipoxygenase action. The observed lipid degradation after frost is probably the result of decompartmentation following damage to membranes.     

Comparative analysis of the chemical composition of mixed and pure cultures of green algae and their decomposed residues by C nuclear magnetic resonance spectroscopy

It is known that macromolecular organic matter in aquatic environments, i.e., humic substances, is highly aliphatic. These aliphatic macromolecules, predominantly paraffinic in structure, are prevalent in marine and lacustrine sediments and are believed to originate from algae or bacteria. A comparative study of mixed and pure cultures of green algae and their decomposed residues was performed by using solid-state C nuclear magnetic resonance spectroscopy as the primary analytical method. Results obtained in this study confirm the presence of components that are chemically refractory and that are defined as alghumin and hydrolyzed alghumin. These were detected in heterogeneous, homogeneous, and axenic biomasses composed of several genera of Chlorophyta. Although the chemical composition of algal biomass varied with culture conditions, the chemical structure of the alghumin and hydrolyzed alghumin, demonstrated by C nuclear magnetic resonance spectroscopy appeared to be constant for members of the Chlorophyta examined in this study. The alghumin was dominated by carbohydrate-carbon, with minor amounts of amide or carboxyl carbon and paraffinic carbon, the latter surviving strong hydrolysis by 6 N HCI (hydrolyzed alghumin). Bacterial decomposition of heterogeneous algal biomass labeled with C was conducted under both aerobic and anaerobic conditions to determine chemical structure and stability of the refractory material. The refractory fraction ranged from 33% in aerobic to 44% in anaerobic cultures. The refractory fraction recovered from either aerobic or anaerobic degradation comprised 40% alghumin, which represented an enrichment by 10% relative to the proportion of alghumin derived from whole cells of algae. The paraffinic component in the hydrolyzed alghumin of whole algal cells was found to be 1.8% and increased to 5.1 and 6.9% after aerobic and anaerobic bacterial degradation, respectively. It is concluded that members of the Chlorophyta contain a common insoluble structure composed of paraffinic carbon that is resistant to chemical and bacterial degradation under conditions used in this study. The paraffinic structure is identical to those constituting humin of aquatic origin. Thus, alga-derived macromolecular compounds deposited in aquatic environments (alghumin) probably contribute to sedimentary humic substances.     

Are allogenic or xenogenic screws and plates a reasonable alternative to alloplastic material for osteosynthesis—A histomorphological analysis in a dynamic system

Despite invention of titanium and resorbable screws and plates, still, one of the main challenges in bone fixation is the search for an ideal osteosynthetic material. Biomechanical properties, biocompatibility, and also cost effectiveness and clinical practicability are factors for the selection of a particular material. A promising alternative seems to be screws and plates made of bone. Recently, xenogenic bone pins and screws have been invented for use in joint surgery.In this study, screws made of allogenic sheep and xenogenic human bone were analyzed in a vital and dynamic sheep-model and compared to conventional titanium screws over a standard period of bone healing of 56 days with a constant applied extrusion force. Biomechanical analysis and histomorphological evaluation were performed.After 56 days of insertion xenogenic screws made of human bone showed significantly larger distance of extrusion of on average 173.8 μm compared to allogenic screws made of sheep bone of on average 27.8 and 29.95 μm of the titanium control group. Severe resorption processes with connective tissue interposition were found in the histomorphological analysis of the xenogenic screws in contrast to new bone formation and centripetal vascularization of the allogenic bone screw, as well as in processes of incorporation of the titanium control group.The study showed allogenic cortical bone screws as a substantial alternative to titanium screws with good biomechanical properties. In contrast to other reports a different result was shown for the xenogenic bone screws. They showed insufficient holding strength with confirmative histomorphological signs of degradation and insufficient osseointegration. Before common clinical use of xenogenic osteosynthetic material, further evaluation should be performed.     

Characterization of nuclear membranes and endoplasmic reticulum isolated from plant tissue

Nuclei, nuclear membranes and rough endoplasmic reticulum (rER) were isolated from onion root tips and stems. Structural preservation and purity of the fractions was determined by electron microscopic and biochemical methods. Gross compositional data (protein, phospholipid, nonpolar lipids, sterols, RNA, DNA), phospholipid and fatty acid patterns, enzyme activities (ATPases, ADPase, IDPase, glucose-6-phosphatase, 5'-nucleotidase, acid phosphatase, and NADH- and NADPH-cytochrome C reductases), and cytochrome contents were determined. A stable, high salt-resistant attachment of some DNA with the nuclear membrane was observed as well as the association of some RNA with high salt-treated nuclear and rER membranes. The phospholipid pattern was identical for both nuclear and rER membranes and showed a predominance of lecithin (about 60%) and phosphatidyl ethanolamine (20-24%). Special care was necessary to minimize lipid degradation by phospholipases during isolations. Nonpolar lipids, mostly sterols and triglycerides, accounted for 35-45% of the membrane lipids. Sterol contents were relatively high in both membrane fractions (molar ratios of sterols to phospholipids ranged from 0.12 to 0.43). Sitosterol accounted for about 80% of the total sterols. Palmitic, oleic, and linoleic acids were the most prevalent acids in membrane-bound lipids as well as in storage lipids and occurred in similar proportions in phospholipids, triglycerides and free fatty acids of the membrane. About 80% of the fatty acids in membrane phospholipids and triglycerides were unsaturated. A cytochrome of the b5 type was characterized in these membranes, but P-450-like cytochromes could not be detected. Both NADH and NADPH-cytochrome c reductases were found in nuclear and rER membranes and appeared to be enriched in rER membranes. Among the phosphatases, Mg2+-ATPase and, to lesser extents, ADPase, IDPase and acid phosphatase activities occurred in the fractions, but significant amounts of monovalent ion-stimulated ATPase, 5'-nucleotidase and glucose-6-phosphatase activities did not. The results obtained emphasize that the close biochemical similarities noted between rER and nuclear membranes of animal cells extend to these fractions from plant cells.     

Evidence for the tight binding of human mucus proteinase inhibitor to highly glycosylated macromolecules in sputum

Two extraction procedures of non-purulent sputum for the isolation of human mucus proteinase inhibitor (MPI) in its free and bound forms have been assayed. The dissociating procedure involved sputum homogenization in 1M NaCl and 4% (w/v) trichloroacetic treatment. When the soluble material was applied to a CM-Trisacryl column, a non-negligible, MPI-related inhibitory activity was recovered with the highly glycosylated constituents not retained on the column; the amount of MPI released in a free form was retained and eluted from the column according to the basic character of this inhibitor. The non-dissociating procedure consisted in a high water dilution (1:12) of sputum, known to bring into solution the macromolecular, fibrillar constituents, which was followed by ultrafiltration on selected Mr cut-off membranes. All the inhibitory activity was recovered with the high Mr (greater than 100,000) fraction which was shown on SDS-PAGE to be essentially composed of strongly glycosylated material; on electrophoretic analysis under non-reducing conditions, the MPI activity was visualized as three bands which corresponded to the inhibitor released from this high Mr fraction in the presence of SDS. As mucin-type molecules are the major, highly glycosylated constituents of bronchial secretions, it is suggested that they are responsible for the entrapping of MPI within their macromolecular network; it would appear that, as well as for lysozyme, electrostatic interactions occur between the acid charges of mucins and the basic charges of MPI. The possible in vivo consequences of these interactions on MPI activity are discussed.     

Patterns of fatty acid release from endogenous substrates by human platelet homogenates and membranes.

We describe a method for measuring the release of fatty acids from endogenous substrates of human platelet homogenates and membranes. The method depends on the availability of lipids whose fatty acids are odd-chained and therefore suitable as internal reference compounds that, at the time of lipid extraction, can be added to an incubation to permit subsequent quantification of the content of free fatty acids or fatty acids esterified to specific lipids. We found four types of lipolytic activities in human platelets. In homogenates at pH 4.0 a triglyceride lipase operated as shown by the synchrony of triglyceride degradation and release of glycerol and those fatty acids that are the predominant constituents of triglycerides. However, enough arachidonic acid was released at this pH level to suggest some phospholipid breakdown, since triglycerides hold relatively small amounts of this acid. With membranous preparations, in the alkaline pH range there were two peaks of fatty acid release with accompanying degradation of phospholipids. At pH 8.5, where release of the saturated acids, palmitic and stearic, predominated, their sum was 3.5 times that of arachidonic acid. At pH 9.5 the release of palmitic and stearic acids was only slightly below their peak values; however, the release of arachidonic acid nearly equaled the sum of the saturated acids. Linoleic acid was not released in representative amounts by those reactions that released arachidonic acid, despite the overwhelming propensity of both to be esterified at the 2-position of phospholipids. Pertinently, the choline phospholipids are linoleic-rich and the non-choline phospholipids linoleic-poor, while both have a generous endowment of arachidonic acid. With this in mind, we raise the possibility that the phospholipase A2 of human platelets is an endoenzyme because of its tendency to act on those phospholipids that are thought to comprise the inner layer of the cell membrane.     

Lipids of Rhizopus arrhizus Fischer

The lipids of Rhizopus arrhizus Fischer mycelia and sporangiospores were extracted, isolated, and separated by thin-layer, liquid, and gas chromatography. Structural confirmations of the compounds were made by a gas chromatographmass spectrometer combination. The n-heptane fraction contained squalene (1%) as a major hydrocarbon constituent. Other major lipid classes detected were free fatty acids, naturally occurring methyl esters of fatty acids, triglycerides, sterols, and polar lipids. The polar lipids (44.4%) were found in the highest concentrations, and the triglycerides (22.1%), sterols (16.7%), and free fatty acids (11.7%) were present in lesser concentrations. This is the first report of naturally occuring methyl esters of long-chain fatty acids being present in fungal mycelium. There appears to be a preference for incorporation of unsaturated acids into the complex lipids, with the exception of the triglycerides. The major saturated fatty acids in the mycelium were palmitic (C(16)) and arachidic (C(20)), whereas the major unsaturated acids were oleic (C(18:1)) and linoleic (C(18:2)), respectively.     

Association of a lipoprotein-like particle with bovine fetuin

Fetuin is a major protein of fetal bovine serum that exhibits heterogeneity and has been found to be associated with some biological active growth factors. Preliminary studies indicated that commercial fetuin preparations contain lipids. We investigated in detail the nature of lipids associated with fetuin by using ultracentrifugation and agarose gel chromatography followed by lipid analysis. Fetuin was associated with a variety of lipids, predominantly cholesterol, cholesteryl ester with smaller amounts of phospholipids, triglycerides, and free fatty acids. Adjustment of fetuin preparation for various densities followed by ultracentrifugation resulted in a fraction with a density 1.063-1.21 g/ml (1-2% of total protein) that contained the bulk of the lipids. This fraction eluted as a single peak upon high pressure liquid chromatography and agarose gel chromatography. Delipidation of the lipoprotein-like particle followed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis indicated a major band in the range of fetuin itself. These studies suggest that a fraction of fetuin (or isoform) binds lipids forming a particle with floating characteristics similar to high density lipoproteins.     

Lipids in developing and mature rice grain

The neutral fraction of nonstarch lipids in developing brown rice (Oryza sativa L., cv IR42) was accumulated up to 16 days after flowering (DAF), but phospholipids and glycolipids increased only up to 8 DAF. Fatty acids accumulated in nonstarch lipids until 12 DAF. However, the proportion of linolenic acid in the lipid fraction decreased and that of oleic acid increased during this period. Accumulation of fat-by-hydrolysis in the brown rice occurred until 20 DAF and followed closely that of starch. The proportion of linolenic acid decreased and that of linoleic acid increased until 16 DAF. The fatty acid composition of fat-by-hydrolysis and starch lipids were identical and fat-by-hydrolysis accounted for 48% by weight of starch lipids. Nonstarch lipids were mainly composed of triglycerides and were located in the bran and embryo of mature brown rice. Starch lipids were mainly composed of lysophosphatidyl choline, free fatty acids and lysophosphatidyl ethanolamine, and were located in the endosperm.