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Studies on the submicrosomal fractions of bovine oligodendroglia: lipid composition and glycolipid biosynthesis.

Oligodendroglia were isolated from bovine brain, and a "crude" microsomal fraction obtained from cell homogenates was subfractionated into myelin (MP), plasma membranes (PM), Golgi (GF), smooth (SER) and rough (RER) endoplasmic membranes using discontinuous-sucrose gradient centrifugation. The submicrosomal fractions were characterized by ultrastructural examination and analysis of the specific organelle markers. The myelin and plasma membrane rich fractions contained characteristically the highest amounts of the lipid with lower mole percentages of total phospholipids and phosphatidylcholine, and higher concentrations of phosphatidylethanolamine (+ plasmalogens), cholesterol and galactolipids. Considerable amounts of the typical myelin galactolipids (galacto-cerebrosides, sulfatides and monogalactosyl diglycerides) were also found in the Golgi fraction (GF). The GF fraction had the greatest enrichment of glycolipid-forming galactosyltransferases, and the distribution of these enzymes correlated well with that of the Golgi marker enzymes. The results give evidence that intracellular Golgi apparatus of oligodendroglia is rich in the myelin-specific lipids, and suggest its involvement in the synthesis and processing of myelin lipids.

Animals↗

Compositional and digestibility changes in sprouted barley and canola seeds.

Barley and canola seeds were sprouted over a 5 day period, in laboratory conditions under room temperature (22 degrees C) and room lighting. Following initial hydration, seeds were kept moist by wetting the germination trays at 9 a.m., 1 p.m. and 6 p.m. daily. A parallel germination experiment using 200 g quantities of seeds in petri dishes was conducted. Starting from the second day of germination, and every day, dishes of germinating seeds were removed, oven-dried, weighed and milled for proximate and chemical analysis. Seeds from the main germination experiment were fed in a digestibility trial to Wistar rats. Results indicated that sprouting was associated with depletion of many nutrients in both barley and canola, the major losses being in respect of dry matter, gross energy and triglycerides. In barley (but not in canola) sprouting was associated with significant increases in crude fiber and diglyceride content. In canola, there were significant losses in lipid content and increases in phytosterol and phospholipid content. Digestibility data showed an enhancement in digestibility of nutrients in barley but not in canola, implying that sprouting improved nutritional quality of barley but not canola.

Animals↗

Kinetics of radioiodinated species in subcellular fractions from rat hearts following administration of iodine-123-labelled 15-(p-iodophenyl)-3-(R,S)-methylpentadecanoic acid (123I-BMIPP).

It is recognized that iodine-123-labelled 15-(p-iodophenyl)-3-(R,S)-methylpentadecanoic acid (123I-BMIPP) slowly washes out of the myocardium. The mechanism for the washout was investigated in normal rat hearts by analyses of the subcellular distribution and lipid classes based on the BMIPP metabolism. Rat hearts were excised at 1-120 min after intravenous injection of 123I-BMIPP. After counting the radioactivity, the hearts were digested with Nagarse and homogenized, and then fractionated into the cytosolic, mitochondrial, microsomal and crude nuclear fractions by centrifugations. The radioactivity of each fraction was counted, and the lipid classes were analysed by radio-thin-layer chromatographic and high-performance liquid chromatographic methods. The heart uptake of 123I-BMIPP was maximal at 5 min (6.81%+/-0.36% ID/g), and 41% of the radioactivity disappeared within 120 min. The myocardial radioactivity was immediately distributed into the cytosolic, mitochondrial, microsomal and crude nuclear fractions. The distribution (%) of each fraction was almost identical from 5 min through 120 min. The cytosolic fraction was always the major site of radioactivity deposition (60%), and the time-activity curve of the cytosolic fraction paralleled that of the whole heart throughout the 120-min study period. In the cytosolic fraction, most of the radioactivity was incorporated into the triglyceride class, and the rest was present in the free fatty acid, phospholipid (phosphatidylcholine) and diglyceride classes. In the mitochondrial fraction, the radioactivity was mostly incorporated into the phospholipid class (phosphatidylethanolamine), followed by free fatty acids. The final metabolite of 123I-BMIPP, 123I-p-iodophenylacetic acid (123I-PIPA), initially appeared in the mitochondrial fraction as early as 1 min, and subsequently in the cytosolic fraction at 5 min. Another intermediary metabolite, 123I-p-iodophenyldodecanoic acid (123I-PIPC12), was found only in the mitochondrial fraction after 5 min. In conclusion, the slow washout kinetics of 123I-BMIPP from the myocardium mainly reflects the turnover rate of the triglyceride pool in the cytosol. The BMIPP metabolism, i.e. initial alpha-oxidation followed by subsequent cycles of beta-oxidation, was confirmed in vivo. The participation of the mitochondria in the metabolism was also proven.

Animals↗

Congenital esophageal atresia: lipase activity is present in the esophageal pouch and stomach.

Lipolytic activity was studied in aspirates from the esophageal pouch and from the stomach of eight infants with congenital esophageal atresia. Lipolytic activity, tested with doubly labeled ([3H]glyceryl, [14C]fatty acid) long-chain triglyceride was present in esophageal and gastric aspirates. The activity in esophageal aspirates was in the range of 2.7-130 nmol/min/ml aspirate and that in gastric aspirates was in the range of 2.9-40.4 nmol/min/ml aspirate. The reaction products of lipolytic activity in esophageal and gastric aspirates were a mixture of mono- and diglycerides, glycerol, and free fatty acids. The lipolytic activity at the two sites--esophagus and stomach--varied with respect to pH optimum (5.0-7.6 and 6.0-6.5, respectively) and reaction products (glycerol 41.6 +/- 20% and 7.3 +/- 4.6%, respectively). These findings confirm the earlier observations that digestion of dietary fat is initiated in the stomach and suggest that the lipolytic activity present in gastric contents originates concomitantly from the oral-esophageal area as well as from the stomach. These studies do not exclude the possibility that the lipolytic activity in the stomach of infants with esophageal atresia could originate in regurgitated intestinal contents.

Esophageal Atresia↗

Biochemical and physiological characterization of the efrotomycin fermentation.

An efrotomycin fermentation was characterized through physical, chemical and biochemical studies. Growth of the actinomycete, Nocardia lactamdurans occurred during the first 50 h of the fermentation cycle at the expense of glucose, protein, and triglycerides. The initiation of efrotomycin biosynthesis was observed when glucose dropped to a low concentration. Upon glucose depletion, cell growth ceased and a switch in the respiratory quotient occurred. Efrotomycin biosynthesis was supported by the utilization of soybean oil and starch. Analysis of triglyceride metabolism showed that no diglycerides or monoglycerides accumulated during the fermentation. The activity of extracellular enzymes (lipase, protease, and amylase) increased during the cell growth phase and decreased significantly after 150 h. The concentrations of DNA, tetrahydro-vitamin K2 (a membrane component), and free amino acids in the supernatant increased dramatically late in the fermentation cycle (225 h), indicating massive cell lysis. During this same time period, a reduction in cellular respiratory activity and efrotomycin biosynthesis were observed.

Amino Acids↗

Relationship between chemical and physical indexes and column and HPSE chromatography methods for evaluating frying oil.

Standard chemical and physical indexes, such as color index, acid value, and K270 were compared with the polar content and the specific thermoxidative and hydrolytic compounds originated during 75 potato fryings with sunflower oil. The color index, acid value, K270 and total polar content showed a significant increase (p < 0.05) after the first 30 fryings, followed by a tendency of the last three indexes to reach a near-steady state. Triglyceride polymers and triglyceride dimers showed a significant increase (p < 0.05) after 30 fryings, while the oxidized triglycerides were not increased significantly after 30 fryings. The amount of diglycerides and free fatty acids related to hydrolytic alteration did not change significantly throughout the frying operations. The significant correlations between acid value and total polar content, triglyceride dimers, triglycerides polymers and oxidized triglycerides, or between color index and polar content and triglyceride polymers or between K270 and triglyceride polymers indicate that these simple and standard methods can be applied and they are as useful as more specific methods of evaluation in the monitoring of frying operations, provided that initial values of all of these indexes are available.

Color↗

Role of protein kinase C in the regulation of cytosolic Ca2+ in A431 cells: separation of growth factor and bradykinin pathways.

Calcium signaling systems in nonexcitable cells involve activation of Ca2+ entry across the plasma membrane and release from intracellular stores as well as activation of Ca2+ pumps and inhibition of passive Ca2+ pathways to ensure exact regulation of free cytosolic Ca2+ concentration [( Ca2+]i). A431 cells loaded with fura-2 cells were used as a model system to examine regulation of Ca2+ entry and intracellular release. Epidermal growth factor (EGF) and transforming growth factor alpha (TGF-alpha) both stimulated Ca2+ entry and release while bradykinin appeared only to release Ca2+ from intracellular stores. The possible role of protein kinase C (PKC) in modulating the [Ca2+]i response to these agonists was examined by four methods. Low concentrations of TPA (2 x 10(-10) M) had no effect on Ca2+ release due to EGF, TGR-alpha or bradykinin but resulted in a rapid return of [Ca2+]i to baseline levels for EGF or TGF-alpha. Addition of the PKC inhibitor staurosporine (1 and 10 nM) completely inhibited the action of TPA on EGF-induced [Ca2+]i changes. An inhibitor of diglyceride kinase (R59022) mimicked the action of TPA. Down-regulation of PKC by overnight incubation with 0.1 or 1 microM TPA produced the converse effect, namely prolonged Ca2+ entry following stimulation with EGF or TGF-alpha. To show that one effect of TPA was on Ca2+ entry, fura-2 loaded cells were suspended in Mn2+ rather than Ca2+ buffers. Addition of EGF or TGF-alpha resulted in Ca2+ release and Mn2+ entry. TPA but not the inactive phorbol ester, 4-alpha-phorbol-12,13-didecanoate, inhibited the Mn2+ influx. Thus, PKC is able to regulate Ca2+ entry due to EGF or TGF-alpha in this cell type. A431 cells treated with higher concentrations of TPA (5 x 10(-8) M) inhibited not only Ca2+ entry but also Ca2+ release due to EGF/TGF-alpha but had no effect on bradykinin-mediated Ca2+ release, suggesting differences in the regulation of the intracellular stores responsive to these two classes of agonists. Furthermore, sequential addition of EGF or TGF-alpha gave a single transient of [Ca2+]i, showing a common pool of Ca2+ for these agonists. In contrast, sequential addition of EGF (or TGF-alpha) and bradykinin resulted in two [Ca2+]i transients equal in size to those obtained with a single agonist.(ABSTRACT TRUNCATED AT 400 WORDS)

Alkaloids↗

Involvement of lysosome-like particles in the metabolism of endogenous myocardial triglycerides during ischemia/reperfusion. Uptake and degradation of triglycerides by lysosomes isolated from rat heart.

The hormonal regulation and enzymatic basis of endogenous lipolysis in heart are not yet completely elucidated. The lysosomal fraction from rat heart appeared to be markedly enriched in triglycerides and a significant reduction in triglycerides in this fraction was found after prolonged perfusion or stimulation of lipolysis with glucagon. The enhanced rate of lipolysis, measured as glycerol release from the isolated perfused rat heart, was abolished 10-15 min after continuous glucagon administration. Omission of glucagon for another 60 min restored the ability of glucagon to stimulate lipolysis, indicating the limited availability of endogenous triglycerides and the presence of a transfer-system for triglycerides from a non-metabolically active pool to a metabolically active pool. The enhanced lipolysis induced by low-flow ischemia was found to be inhibited by the lysosomotropic agent methylamine (5 mM). Methylamine-perfusion during low-flow ischemia was accompanied by an increased recovery of myocardial triglycerides in the lysosomal fraction. The possible role of lysosome-like particles in myocardial triglyceride homeostasis was further investigated by studying the kinetics of uptake and degradation of labeled triglycerides by membrane-particles recovered in the subcellular fraction enriched with lysosomal marker enzymes. It appeared that isolated lysosomal membranes take up added triglycerides at an average rate of 30 nmoles/min/g protein. The bulk of these triglycerides taken up is stored whereas 20% is degraded to diglycerides and free fatty acids. More than 90% of the free fatty acids formed were released from the lysosomes into the supernatant. The uptake and degradation of triglyceride-filled liposomes by isolated myocardial lysosomes was inhibited during incubation with methylamine (5 mM). On the other hand, a lowering of pH during in vitro incubation increased the rate of uptake and degradation of added triglycerides by isolated lysosomes. These results indicate that lysosomes or lysosome-like particles are involved in the enhanced lipolysis during myocardial ischemia.

Acid-Base Equilibrium↗

Lipid composition of Microsporum gypseum.

The lipid composition of Microsporum gypseum has been studied. The lipids amounted to 10.1% and phospholipids to 1.1% of the mycelial dry weight. Phosphatidyl choline, phosphatidyl serine and phosphatidyl ethanolamine were the major components, while lysophosphatidyl choline, and phosphatidyl inositol were present in smaller quantities. Neutral lipids consisted of monoglycerides, diglycerides, triglycerides, free and esterified cholesterol.

Cholesterol↗

Arachidonic acid mobilization in platelets: the possible role of protein kinase C and G-proteins.

A major route for the release of arachidonic acid from platelet phospholipids appears to be catalyzed by a phospholipase A2 that can be stimulated by a rise of cytosolic Ca2+. This paper discusses certain other mechanisms for regulation of this process. Release of arachidonic acid by calcium ionophores is potentiated by pretreatment with stimulators of protein kinase C; e.g. diglyceride, phorbol esters and the terpene diester mezerein. This effect appears to be coincident with phosphorylation of a certain group of proteins (not 47 KDa protein), and is sensitive to depletion of ATP, activation of Ca2+ dependent phosphatase, and the kinase C inhibitor H-7, but is unaffected by Na+/H+ exchange inhibitors. Recent results in other cell types strongly indicate that phospholipase A2 is also directly under control of certain GTP-binding proteins.

Annexins↗

Identification and quantitation of PAF from psoriatic scales.

Platelet activating factor was isolated from scales of psoriatic patients by the procedure of Bligh and Dyer and purified by silica gel thin layer chromatography. The purified PAF was digested with phospholipase C and the resulting diglyceride was derivatized into PFB ethers. The PAF-PFB ethers were analyzed using fused silica capillary chromatography-negative ion chemical ionization mass spectrometry. Different molecular species of PAF were identified by their negative ion mass spectra and by their elution time from the capillary column. All the molecular species had high abdundance (greater than 90%) of the molecular anion. 1-0-Hexadecyl-2-acetyl-GPC (16:0) was the major PAF species representing 51% of the total PAF. 17:0 and 18:1 were the next abundant species representing 15 and 16%, respectively. Several minor PAF molecular species were also present. The amount of each PAF molecular species was quantitated from 1-0-hexadecyl-2-2H3 acetyl-GPC used as the internal standard. Nanogram quantities of PAF were recovered from 100 mg of psoriatic scales. Significant amounts of lysoPAF were also present in these scales. The alkyl chain of the lysoPAF was compared with that of PAF.

Gas Chromatography-Mass Spectrometry↗

The phospholipase A2 from human platelets.

Studies on a purified phospholipase A2 (PLA2) from human platelets show that the enzyme, which is copurified with the plasma membrane fraction, has a MW of approximately 50 K Dalton, requires Ca++, and has a pH optimum of 9.4. Under optimal conditions, PLA2 activity corresponds to at least 13 nmol/min/10(9) platelets. Unsaturated PL are preferred substrates and the enzyme is considerably more active on the aggregated form of the substrate than on the monomers. The specific activity is markedly affected by the quality of the interface, showing variations of more than 10-fold between different substrate forms. In the absence of detergents, a 4-fold increase in rate is observed when both products are present. Maximal rates are obtained at 20 mole percent of products to substrate. 1,2-Diglyceride and phosphatidic acid stimulate the hydrolysis of PC by the purified enzyme, however, in these forms of the substrate, neither of them are hydrolyzed. Activation of this enzyme by some intermediate of the phospholipase C pathway might play a role in the stimulus-linked release of platelet arachidonic acid.

Arachidonic Acid↗

Characterization studies of glucose dehydrogenase.

Porcine liver beta-D-glucose dehydrogenase has been isolated using Triton X-114 to release it from the endoplasmic reticulum. The purified enzyme contains a limited amount (1.7%) of lipid material, including cholesterol, fatty acids, mono and diglycerides, phosphatidylcholine, phosphatidylethanolamine, and cholesterol esters. This enzyme is a tetrameric protein containing an extensive number of hydrophobic residues. This form of glucose dehydrogenase is capable of turning over both beta-D-glucose and alpha-D-glucose-6-phosphate in vivo as indicated from a steady state kinetic analysis at 37 degrees C.

Amino Acids↗

[Thermal inactivation and storage behavior of technologically important enzymes. IV. Spinach lipid-acyl-hydrolase].

The thermal reaction of a lipid-acyl-hydrolase which seems to be important for the quality preservation of vegetable foods, was investigated in spinach. The authors applied a simple in-situ method using thin-layer chromatography which had been developed for the enzyme determination, to follow the thermal inactivation of the lipid-acyl-hydrolase by measuring the decomposition of lecithin, mono- und digalactosyl diglycerides. According to the inactivation curves, the enzyme is relatively little resistant to heat. Since the D- and z-values resulting from the inactivation curves for phospholipase, mono- and digalacto-lipase activities are almost the same, it can be assumed that the lipid-acyl-hydrolase is a multi-function enzyme in spinach.

Drug Stability↗

Effect of taurine administration on liver lipids in guinea pig.

The oral administration of 0.4% taurine in drinking water for 14 consecutive days showed the following hepatic effects in male guinea pig. The percentage of tauro-conjugated biliary bile acids was increased from 17.2-54.2%; the ratio liver weight/body weight was increased, and fatty change was induced. Liver triglyceride concentration was accordingly increased; diglyceride and phosphatidylcholine concentrations were reduced by the treatment, while phosphatidylethanolamine level was not affected. These changes suggest an adverse effect of taurine administration on phosphatidylcholine hepatic synthesis.

Animals↗

Effects of homogenization pressure and stabilizers on some physical characteristics of a beverage prepared from partially defatted, roasted peanuts.

A low-fat beverage with roasted peanut flavor was developed from peanuts. The potential milk substitute contained 11.8% total solids, 2.0% fat and 3.7% protein, and was whitish orange-yellow in color. The effects of stabilizing additives and homogenization pressure (13.8 x 10(6) Pa or 41.4 x 10(6) Pa) on selected physical characteristics were determined. Suspension stability was improved by addition of carrageenan [Benlacta CM61-B (CM), 0.02-0.04%] or a hydrogenated mono- and diglyceride [Emuldan HV52K (HV, 0.2-0.4%)]. Homogenizing at 41.4 x 10(6) Pa increased viscosity of all samples except that of the controls but did not improve the suspension stability (top:bottom solids) of the beverage. The most viscous formulations (17.5 +/- 0.95 cps) were those containing 0.04% CM. Treatments yielding the best combination, compared to cow's milk values, of high suspension stability (0.5 +/- 0.03, where 1.0 = maximum stability) and low viscosity (3.7 +/- 0.89 cps) were those containing 0.2% HV which were homogenized at 13.8 x 10(6) Pa.

Animals↗

Fatty acid synthesis from 2-14C-acetate in rat testis mitochondrial and cytosol fractions in vitro.

An in vitro system for acetate incorporation into fatty acids by the mitochondrial and the cytosol fractions of rat testis is described. The rate of incorporation of acetate into fatty acids was twice as fast with the mitochondrial as with the cytosol fraction; both systems were stimulated in the presence of adenosine triphosphate, reduced nicotinamide adenine dinucleotide phosphate, coenzyme A, and MgC1(2). The optimum pH was between 7.0-7.5 for the mitochondrial fraction and between 6.5-8.0 for the cytosol fraction. Radio gas chromatography showed that palmitic acid was the most highly labeled acid, followed by stearic acid, in the mitochondrial fraction in accord with the pathway of de novo fatty acid synthesis. Some of the labeled acetate was also incorporated into the 16:1 and 18:1 fatty acids of this fraction. Distribution of radioactivity among the mitochondrial lipid classes was highest in the phospholipids and monoglycerides, followed by diglycerides and cholesterol; little radioactivity was present in the triglyceride fraction. These observations are in accord with studies of the incorporation of labeled metabolites into testicular lipids following intratesticular injection and indicate the validity of the in vitro system for studies of specific reactions occurring in vivo.

Acetates↗

Lipid metabolism of the yellow clam, Mesodesma mactroides: 2-polyunsaturated fatty acid metabolism.

The fate of labeled linoleic, alpha-linolenic, and higher homologs of alpha-linolenic acid administered to the yellow clam, Mesodesma mactroides, was investigated. It was found that the clam incorporated the acids dissolved in sea water and converted 18:2 (n-6) into 20:2 (n-6) and 18:3 (n-3) into 18:4 (n-3) and 20:3 (n-3). The addition of casein hydrolysate to the sea water increased the desaturation capacity of the calm and allowed the conversion of 18:2 (n-6) into 18:3 (n-6) to be demonstrated. An enhanced desaturation of 18:3 (n-3) into 18:4 (n-3) was also demonstrated. After 12 hr admininistration of the acid, no radioactivity was found in arachidonic, 20:5 (n-3), or 22:6 (n-3). Feeding the clams a culture of Phaeodactylun tricornutum previously incubated with 1-14C-alph-linolenic acid demonstrated that all the homologs of the alpha-linolenic series were found in the clam without any important changes. Six hour administration of labled linolenic acid resulted in the incorporation of the acid into diglycerides and phospholipids.

Animals↗