Intracellular transport,organelle biogenesis and establishment of golgi identity: Impact of brefeldin a on the activity of lipid synthesizing enzymes

• 1.1. The effect of brefeldin A (BFA) on generation of transport vesicles, synthesis of phospho-glycerides, sphingosine and ceramides, and utilization of the sphingolipid precursors in the formation of sphingomyelin and glycosphingolipids in Golgi was investigated.
• 2.2. In the presence of 5–10 gmg/ml BFA, the incorporation of [³H]palmitate into glycerides, phosphoglycerides and sphingolipids decreased 45–60%, and the production of endoplasmic reticulum transport vesicles was reduced 30–50%.
• 3.3. In Golgi membranes, the presence of 5–10 gmg/ml BFA in the mixture, assembled to generate Golgi vesicles, evoked inhibitory effect on the synthesis of sphingomyelin, glycosphingolipids and phosphatidylcholine. On average, the synthesis of the sphingolipids and phosphatidylcholine and production of Golgi transport vesicles declined to 30–40%.
• 4.4. Addition of 5–10 gmg/ml BFA to the assay mixture prepared to measure the activity of cytidylyltransferase, phosphocholine diacylglyceroltransferase, and serine palmitoyltransferase, caused up to 50% inhibition of the enzymes involved in the synthesis of phosphatidylcholine and up to 70% inhibition of the enzyme generating 3-ketosphinganine.
• 5.5. The results suggest that BFA inhibits the synthesis of phosphoglycerides and sphingolipids. This, at first, is displayed in reduced production of endoplasmic reticulum and Golgi transport vesicles, while the depletion of sphingolipids abrogates the identity of Golgi membranes.

     

Type II alveolar cells in organotypic culture: A model system for the study of surfactant synthesis

• 1.1. Type II cells, maintained in an organotypic system where in vivo morphologic characteristics are retained, were utilized to study surfactant phospholipid and protein synthesis. Cellular components were separated into a surfactant and a residual fraction by sucrose density centrifugation. The major phospholipid of the surfactant fraction is phosphatidylcholine (70%). Phosphatidylglycerol accounts for 4.0% of the total surfactant phospholipids. Phosphatidylcholine is also the major phospholipid of the residual fraction, constituting 50% of this fraction's phospholipids; phosphatidylglycerol is also present (4.2%).
• 2.2. Type II cells in organotypic culture are capable of metabolizing exogenous glucose. The uptake of glucose from the incubation medium is concentration dependent. Glucose can be utilized by the type II cell for surfactant and residual phosphatidylcholine synthesis. The rate of incorporation of glucose into residual phosphatidylcholine is three times the incorporation rate into surfactant phosphatidylcholine.
• 3.3. Palmitate and choline can also be utilized for surfactant and residual phosphatidylcholine synthesis. Palmitate incorporation into residual phosphatidylcholine is five times greater than the incorporation into surfactant phosphatidylcholine. Surfactant phosphatidylcholine is labelled with choline at a rate one-fourth that of residual phosphatidylcholine. In contrast to glucose and palmitate, choline incorporation into surfactant phosphatidylcholine is linear only after a 15 min lag period. The labelling of residual phosphatidylcholine with choline is biphasic.
• 4.4. Type II cells in organotypic culture can also synthesize the protein moiety of surfactant. Leucine was found to incorporate linearly into surfactant proteins for approx. 8 h.

     

Metabolic energy and cytoskeletal requirements for synthesis and secretion by acini from rat mammary gland—ii. Intracellular transport and secretion of protein and lactose

• 1.1. Iodoacetate, 2,4-dinitrophenol, cyanide and cycloheximide inhibited protein secretion as well as synthesis by acini (alveoli) from rat mammary gland. Cytochalasin B and vinblastine inhibited protein secretion and marginally reduced protein synthesis. Colchicine was without effect on protein synthesis but inhibited secretion.
• 2.2. Intracellular protein transport was altered during incubation with metabolic and cytoskeletal inhibitors. Cycloheximide, iodoacetate. 2,4-dinitrophenol and Cytochalasin B appeared to block protein synthesis on polysomes of rough endoplasmic reticulum. Vinblastine inhibited protein transport from rough endoplasmic reticulum to Golgi apparatus and colchicine appeared to cause accumulation of protein in several endomembrane fractions.
• 3.3. Iodoacetate reduced acinar lactose content but was without effect on lactose synthetase activity. Cyanide, cycloheximide and vinblastine reduced lactose synthetase activity but not tissue lactose concentration. Cytochalasin B reduced glucose incorporation but was without effect on lactose content and lactose synthetase activity. Colchicine and 2,4-dinitrophenol did not alter glucose incorporation, lactose content or lactose synthetase activity. Lactose secretion was inhibited by all metabolic and cytoskeletal inhibitors examined.
• 4.4. Results indicated that sustained protein secretion depended on continued protein synthesis and that lactose secretion was coupled to protein secretion.

     

Lipid biosynthesis in amphibian oocyte and embryonic cell-free preparations

• 1.1. The utilization of [2-³H]glycerol-3-phosphate in the synthesis of lipids during early embryogenesis was studied in cell-free preparations from oocytes or embryos of Bufo arenarum Hensel
• 2.2. The precursor was incorporated in all stages of development up to gill circulation, which indicates that oocytes and embryos have the enzymatic machinery necessary to synthesize at least part of their own lipids.
• 3.3. A significant decrease in the labeling of most lipids took place after fertilization, especially in gastrulas, but at gill circulation lipid synthesis was highly stimulated.
• 4.4. The incorporation pattern is similar in unfertilized oocyte, fertilized oocyte and gastrulas, where phosphatidylglycerol has the highest amount of radioactivity. At gill circulation stage phosphatidylethanolamine and neutral lipid biosynthesis also became significant.
• 5.5. The results suggest a different regulation of the biosynthetic lipid routes through the appearance of new enzymes or modulators of preexisting enzymes during amphibian development.

     

Properties and topology of enzymes methylating phosphatidylethanolamine to phosphatidylcholine in sarcoplasmic reticulum

• 1.1. The synthesis of phosphatidylcholine (PC) by stepwise methylation of phosphatidylethanolamine (PE) is carried out by two enzymes in sarcoplasmic reticulum (SR) membrane of rabbit fast-twitch skeletal muscles.
• 2.2. Two methyltransferases (Met I and Met II) have a different pH optimum and affinity for methyl donor—5-adenosyl-L-methionine (SAM).
• 3.3. Met I is an integral SR membrane protein which active site faces the cytoplasmic surface of the membrane.
• 4.4. Met II is a peripheral, loosely bound protein, localized mainly on the extracytoplasmic (luminal) part of the SR membrane.

     

Association of cytosolic lipids with fatty acid synthase from lactating mammary gland

• 1.1. Membrane-free cytosol contained over 4% of both the total lipids and phospholipids present in homogenates of lactating rat mammary gland, and much of this lipid was associated with a high molecular weight complex isolated from cytosol by gel exclusion chromatography or by density gradient centrifugation.
• 2.2. This complex principally consisted of polypeptides with apparent molecular weights of 220 and 116kDa. Lipids associated with this complex were transferred to endoplasmic reticulum and to intracellular lipid droplet precursors of milk lipid globules upon incubation in a cell-free system.
• 3.3. This lipoprotein complex was abundant in cytosol from lactating mammary gland, but was diminished in amount in cytosol from involuted mammary glands. The 220 kDa constituent of this complex was identified as the monomer of fatty acid synthase.
• 4.4. These results suggest that fatty acid synthase complex in lactating mammary gland may function in transfer of lipids necessary for formation or growth of lipid droplet precursors of milk lipid globules.

     

Rat liver endoplasmic reticulum protein kinases

• 1.1. Rat liver microsomal membranes were studied for the presence of protein kinases. Microsomal proteins solubilized with Triton X-100 were analyzed by means of ion exchange chromatography.
• 2.2. Protein kinase activity was detected in the column fractions using specific assays for cAMP-dependent protein kinase, cGMP-dependent protein kinase, protein kinase C, Ca²⁺/calmodulin-dependent protein kinase and casein kinases.
• 3.3. Fractions with protein kinase activity were further analyzed by SDS-polyacrylamide gel electrophoresis.
• 4.4. The results indicate that cAMP-dependent protein kinase type I and II, casein kinases I and II, protein kinase C proenzymes I and II and Ca²⁺ /calmodulin kinase II are associated with the membranes of endoplasmic reticulum (ER).

     

Effect of hibernation on liver and kidney metabolism in 13-lined ground squirrels

• 1.1. Metabolic rates and adenine nucleotide content of liver and kidney from hibernating ground squirrels were measured and compared to rats to study the biochemical adaptation to hibernation.
• 2.2. High rates of renal and hepatic gluconeogenesis were observed in squirrels, particularly from propionate and glycerol compared to rat.
• 3.3. During hibernation and starvation soluble phosphoenolpyruvate carboxykinase activity was increased in both liver and kidney.
• 4.4. Although metabolic rates are decreased during hibernation the results suggest that the enzymic complement is maintained at high activity even during torpor.

     

Interrelationships between gluconeogenesis and uricogenesis in chicken liver

• 1.1. Experiments performed on isolated hepatocytes and perfused liver of starved chickens showed that gluconeogenesis from lactate, glycerol and fructose was inhibited by 22–100% on addition of urate precursors.
• 2.2. The inhibition was associated with an increased rate of urate formation.
• 3.3. 2,4-Dinitrophenol (40 μM), 2-bromooctanoate (2 mM) and 3-mercaptopicolinate (3MPA) (0.5 mM) were inhibitory with respect to gluconeogenesis but did not significantly affect the rate of urate formation.
• 4.4. The possible interrelationships between gluconeogenesis and uricogenesis are considered in terms of a competition for ATP and for other metabolites between the two pathways.
• 5.5. An interplay of both pathways at the level of anion transfer across the inner mitochondrial membrane is also discussed.

     

Effects of phosphatidylserine on the oxidation of low density lipoprotein

• 1.1. LDL was incubated in the presence of 1 μ M CuSO₄ for 18 hr at 37°C. The content of lipoperoxides was found to be approx. 40 nmol MDA equivalents/mg LDL protein. The addition of 50 μM phosphatidylserine (PS) reduced the content of lipoperoxides to 15% of control values.
• 2.2. The electrophoretic mobility observed for LDL oxidized in the presence of PS approximated the mobility observed for native LDL.
• 3.3. The formation of conjugated dienes was strongly inhibited when LDL was oxidized in the presence of PS.
• 4.4. The addition of 50 μM phosphatidylcholine, phosphatidylglycerol and cardiolipin did not alter the extent of LDL oxidation.
• 5.5. PS did not inhibit the oxidation of LDL mediated by J774 macrophages in the presence of Ham's F-10 culture medium. Under these conditions, PS was found to be an excellent substrate for oxidation.

     

Activation of 3-methyl-branched fatty acids in rat liver

• 1.1. Subcellular fractionation of rat liver revealed that 3-methylmargaric acid, a monobranched phytanic acid analogue, can be activated by mitochondria, endoplasmic reticulum and peroxisomes.
• 2.2. Indirect data (effects of pyrophosphate and Triton X-100) suggested that the peroxisomal activation of 3-methylmargaric, 2-methylpalmitic and palmitic acid is catalyzed by different enzymes.
• 3.3. Despite many attempts, column chromatography of solubilized peroxisomal membrane proteins so far did not provide more conclusive data. On various matrices, lignoceroyl-CoA synthetase clearly eluted differently from the synthetases acting on 3-methylmargaric, 2-methylpalmitic and palmitic acid. The latter three however, tended to coelute together, although often not in an identical manner.

     

How the atmosphere and the presence of substrate affect the photo and non-photoinactivation of heme enzymes by uroporphyrin I

• 1.1. The effect of URO I on the activity of ALA-D, PBGase, deaminase and URO-D, both in aerobiosis and anaerobiosis, was studied.
• 2.2. Photoinactivation of the enzymes was much lower in an anaerobic than in an aerobic atmosphere.
• 3.3. Dark inactivation in the absence of oxygen was lower than its presence.
• 4.4. Preincubation in the presence of ALA or PBG protected the enzymic activity of ALA-D, PBGase and deaminase against URO I-inactivation both under u.v. light and in the dark.
• 5.5. Photoinactivating action of URO I would be mediated by reactive oxygen species generated by the excited porphyrin after its absorption of light. Dark inactivation, in aerobiosis, can also be partly mediated by amino acid oxidation, although to a lesser extent than that observed under u.v. light.

     

Length of hibernation and the brain's influence on temperature-induced spermatogenic DNA synthesis in Helix aspersa

• 1.1. In ovotestis of hibernating snails, the male cell line is very sensitive to temperature. An elevated temperature from 5 to 25°C induces spermatogenic DNA synthesis and formation of spermatids and spermatozoa in 4 weeks.
• 2.2. The DNA synthesis gradually increases in male cell line with the lengthening of hibernation.
• 3.3. Brain removal produces a marked increase of DNA synthesis after 1 and 6 months of hibernation (× 3.5 and 3.3) but not after 12 months.

     

A comparative study of porphyrin synthesis by whole blood and haemolysates from different mammalian species

• 1.1. There is no uniform pattern of porphyrin synthesis by whole blood and haemolysates, between the different mammalian species studied.
• 2.2. Coproporphyrin, is the dominant porphyrin synthesised by intact red cells.
• 3.3. Uroporphyrin synthesis increases significantly in the majority of species when the cells are haemolysed.
• 4.4. In the mouse large amounts of protoporphyrin are synthesised by intact red cells which increases further on haemolysis.
• 5.5. The low porphyrin synthesising capacity of cow and sheep red cells is not due to any rate-limiting activity of the enzyme ALA-dehydratase.
• 6.6. The dog has a pattern of porphyrin synthesis and excretion similar to that found in the rabbit, and the possibility exists, that a similar energy-dependent carrier mechanism for movement of uroporphyrin and coproporphyrin across red cell membranes found in the rabbit may be present in this species.
• 7.7. The findings may be of significance in the interpretation of porphyrin excretion patterns in experimental porphyria.

     

Interaction of ligands in phosphorylase A as monitored by crosslinking and enzymatic modifications: Synergism of glucose and caffeine manifested in the exposure of N-terminal segment

• 1.1. Glycogen, caffeine and glucose dissociate phosphorylase a tetramer to dimers with half-maximum effect at 0.16%, 1.1 and 71 mM concentration, respectively, as monitored by crosslinking with dimethyl suberimidate at 18°C.
• 2.2. The above ligands increase the rate of dephosphorylation and tryptic digestion of phosphorylase a at 18°C in the same way with half-maximum effect at 0.04%, 0.1 and 9 mM concentration, respectively.
• 3.3. Caffeine and glucose acted synergistically in tetramer dissociation as well as in the enzymic modifications.
• 4.4. The α-anomer of d-glucose was twice as effective as its mutarotational equilibrium solution.

     

Calcium binding by sarcoplasmic reticulum and mitochondria isolated from an annelid visceral muscle in relation to whole muscle calcium influx and efflux

• 1.1. Nereis pharangeal visceral muscle is composed of obliquely striated fibres with low mitochondrial density and moderately developed sarcoplasmic reticulum.
• 2.2. Isolated mitochondria and sarcoplasmic reticulum showed moderate passive calcium binding but only low ATP-promoted calcium binding which was inhibited by caffeine.
• 3.3. Whole fibres preloaded with Ca⁴⁵ showed a two compartment efflux. The slow, presumably intracellular, compartment accounted for only 10% of total Ca⁴⁵ activity.
• 4.4. Both acetylcholine and high KCl treatments stimulated calcium influx, causing contractures while calcium-free and EGTA treatments inhibited both these contractures and normal spontaneous contractions.
• 5.5. Lanthanum inhibited normal contractility and KCl contractures. Lanthanum also inhibited Ca⁴⁵ influx but was without effect on Ca⁴⁵ efflux.
• 6.6. It is concluded that there is little calcium storage capacity in these visceral muscle fibres and that normal contractions are strongly dependent upon extracellular calcium influx.

     

Role of glutamate dehydrogenase reaction in the control of citrate pool in yeast

• 1.1. Role of NADP-glutamate dehydrogenase in the depletion of citrate was analyzed using permeabilized yeast cells.
• 2.2. Citrate was converted to 2-oxoglutarate, which was then metabolized to glutamate by NADP-glutamate dehydrogenase in the presence of ammonium ion.
• 3.3. Formation of 2-oxoglutarate plus glutamate was in good agreement with the concentration of citrate decreased. Glutamate formation can be a good indicator of the depletion of citrate, because 70% of the citrate decreased was converted to glutamate.
• 4.4. Glycolytic activity was closely correlated with the decrease in citrate under the in situ conditions.
• 5.5. NADP-glutamate dehydrogenase increased in anaerobically grown yeast cells.
• 6.6. An effective depletion of citrate by increased synthesis of NADP-glutamate dehydrogenase can explain the lowered mechanism of citrate causing glycolytic stimulation under the anaerobic growth conditions of yeast.

     

A further contribution to the knowledge of the blood lipid fractions from the african elephant Loxodonta africana

• 1.1. Plasma lipids from 5 African elephants were extracted and fractionated into cholesterol esters, free fatty acids, triglycerides, phosphatidylcholine, phosphatidylethanolamine, phosphatidylinositol, sphingomyelin and glycosphingolipids. The fatty acids of various individual fractions were investigated by gas-chromatography.
• 2.2. All animals, except one, had a high linoleic acid content in cholesterol esters indicating an adequate supply of linoleic acid in the diet.
• 3.3. Phosphatidyl choline had a strong saturated character originating from the presence of unusually high quantities of stearic acid.
• 4.4. Phosphatidylethanolamine was present in small quantities and was characterized by a low content of arachidonic acid.
• 5.5. Sphingomyelin did not contain any long chain saturated acids. Instead it contained 10.2–47.0% of a long chain acid which was most likely monounsaturated.
• 6.6. The presence of significant quantities of glycosphingolipids was established.

     

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