Oleosomes in flag leaves of wheat; their distribution,composition and fate during senesence and rust-infection

Oleosomes, up to 14m in diameter, were found in mesophyll and bundle sheath cells of the flag and lower leaves of wheat cv Professeur Marchal. They develop in flag leaves at least 10 d before anthesis, possibly from fatty acids secreted by the plastids, and persist in mature and senescing leaf tissue. Oleosomes are bordered with an osmiophilic layer rather than a unit membrane. The major lipids of oleosomes, isolated 20 d after anthesis, are triacylglycerols (50%) and sterol or wax exter (34%). The dominant fatty acids of both lipid classes are plamitic (16:0) and stearic (18:0) acids which accounts for the low osmiophilia of the oleosomes. The function of the oleosomes is unknown but they may act as short-term energy reserves. Oleosomes persist in leaves infected with brown rust, even in cells penetrated by haustoria. Yellowish-brown oleosomes found in senescing and rust-infected leaves may be formed by the release and coalescence of pigmented plastoglobuli.     

Influence of nitrogen deficiency on biochemical composition of the green alga <Emphasis Type="Italic">Botryococcus</Emphasis>

A study has been carried out to investigate the influence of nitrogen deficiency on intracellular lipid composition, including total fatty acid composition of lipids, polar lipids, and triacylglycerols, of the alga Botryococcus braunii Kütz IPPAS H-252 in batch culture. Under nitrogen limitation, the alga accumulates lipids as triacylglycerols and the total fatty acid (FA) composition changes: trienoic acids decrease (from 52.8–57.2 to 19.5–24.7% of the total FAs) and the oleic acid increases (from 1.1–1.2 to 17.1–24.4%) as does the saturated acids (from 23.7–26 to 32.9–46.1%). A similar rearrangement in the FA spectrum occurs at later times in the control culture, but it is less pronounced. Under nitrogen limitation, considerable changes in the polar lipid FAs are registered at day 13: saturated acids increase (from 28.6–35.5 to 76.8%) and all polyenoic acids markedly decrease (from 56.9–64.1 to 6.8%). Changes in the triacylglycerol fatty acid spectrum are seen on day 7: the oleic acid increases (from 14.7 to 34.2%) and remains at a high level till the end of the culture. In the control, triacylglycerols with large contents of oleic acid are detected at day 13, the total lipids and triacylglycerols still remaining unchanged.     

Formation of oleosomes (storage lipid bodies) during embryogenesis and their breakdown during seedling development in cotyledons of Sinapis alba L.

Electron microscopic and biochemical investigations of developing embryonic mustard cotyledons provided no evidence for the widely accepted hypothesis that oleosomes of fat-storing tissues originate from the endoplasmic reticulum and are surrounded by a unit- or half-unit membrane. In contrast, it was found that the first lipid droplets appear (about 12–14 d after pollination) in the ground cytoplasm near the surface of plastids. Subsequently these nascent lipid droplets, which lack any detectable boundary structure at this stage, become encircled by a cisterna of rough endoplasmic reticulum. At the same time an osmiophilic coat of about 3 nm thickness becomes detectable at the lipid/water interface. In the cotyledon cells of germinating seedlings a centrifugally moving front of fat degradation moves from the central vacuoles(s) towards the cell periphery, leaving behind collapsed coats of oleosomes which are depleted of their lipid contents (saccules). Although saccules appear tripartite in cross section, they are structurally different from endoplasmic reticulum membranes. The oleosome coats can be isolated from oleosome preparations by extracting lipids with organic solvents. The coat material is insoluble in detergents like Triton X-100 or deoxycholate and shows a tripartite, lamellar structure (similar to collapsed saccules) under the electron microscope. Upon dissolution with dodecylsulfate, polyacrylamide gel electrophoresis revealed a polypeptide composition (9 major bands) which is qualitatively different from that of the endoplasmic reticulum membrane. Also the buoyant densities of defatted oleosome coats and defatted endoplasmic reticulum membranes are very different. It is concluded that oleosome lipids accumulate in the ground cytoplasm and are bounded by a lamellar structure originating de novo from proteinaceous elements synthesized by specific regions of the endoplasmic reticulum.Abbreviation ER endoplasmic reticulum     

Triacylglycerol biosynthesis in developing seeds of Tropaeolum majus L. and Limnanthes douglasii R. Br.

Triacylglycerols of both Tropaeolum majus L. and Limnanthes douglasii R. Br. are predominantly esterified with very long-chain acyl groups at each position of the glycerol backbone. In order to elucidate whether these acyl groups are directly chanelled into the triacylglycerols via the stepwise acylation of glycerol-3-phosphate, seed oil formation has been investigated in developing embryos of both plant species. [1-¹⁴C]Acetate labelling experiments using embryos at different stages of development, as well as the determination of the properties of the microsomal acyl-CoA:sn-glycerol-3-phosphate acyltransferase (EC 2.3.1.15) and acyl-CoA:sn-1-acylglycerol-3-phosphate acyltransferase (EC 2.3.1.51), revealed differences between the two plant species, especially with respect to the incorporation of very longchain acyl groups into the C2 position of the triacylglycerols. In microsomal fractions of developing embryos of L. douglasii both a glycerol-3-phosphate and a 1-acylglycerol-3-phosphate acyltransferase were detected which utilize very long-chain acyl-CoA thioesters as substrates. Thus, in seeds of L. douglasii very long-chain acyl groups can enter not only the C1, but also the C2 position of the triacylglycerols in the course of de-novo biosynthesis. A comparison of the properties of the acyltransferases of developing embryos with those of the corresponding activities of leaves indicates an embryo specific expression of an erucoyl-CoA-dependent microsomal 1-acylglycerol-3-phosphate acyltransferase in L. douglasii. The microsomal glycerol-3-phosphate acyltransferase of developing embryos of T. majus displayed properties very similar to those of the corresponding activity of L. douglasii. On the other hand, the microsomal 1-acylglycerol-3-phosphate acyltransferases of the two plant species showed strikingly different substrate specificities. Irrespective of the acyl groups of 1-acylglycerol-3-phosphate and regardless of whether acyl-CoA thioesters were offered separately or in mixtures, the enzyme of T. majus, in contrast to that of L. douglasii, was inactive with erucoyl-CoA. These results of the enzyme studies correspond well with those of the [1-¹⁴C]acetate labelling experiments and thus indicate that T. majus has developed mechanisms different from those of L. douglasii for the incorporation of erucic acid into the C2 position of its triacylglycerols.Abbreviations GPAT acyl-CoA:sn-glycerol-3-phosphate acyltransferase (EC 2.3.1.15) - LPAT acyl-CoA:sn-1-acylglycerol-3-phosphate acyltransferase (EC 2.3.1.51) This work was supported by the Bundesministerium für Forschung und Technologie (Förderkennzeichen 0316600A).     

Membranous appendices of spherosomes (oleosomes)

Spherosomes (oleosomes) of cotyledons of rape (Brassica napus L.), sunflower (Helianthus annuus L.), and watermelon (Citrullus vulgaris, Schrad.) seedlings are delimited by a half unit membrane that appears to be continuous with each of the osmiophilic layers of a tripartite unit membrane forming a handlelike appendix of the spherosomes. Prior to any noticeable utilization of the spherosomal storage fat, ribosomes were found to be attached to these handles. At later stages appendices of the spherosomes are smooth, showing a diameter of about 22 nm that greatly exceeds the thickness of any other unit membrane profiles identical in structure and diameter osomes appears to be continuous with the thick lipid layer of the handles. In intermediate stages of fat depletion the spherosomal bodies become invaginated with cytoplasmic material. Finally vesicles with cytoplasmic contents surrounded by a membrane with a typically thick lipid layer are left in the cells. Membrane profiles indentical in structure and diameter to the spherosomal appendices were also present in electron micrographs of the lipolytic membrane fraction recovered from sucrose density gradients after centrifugation of a microsomal cell fraction. The ultrastructural observations are taken for evidence that the spherosomal appendices represent the lipase-carrying membranes isolated previously (Theimer and Rosnitschek, 1978). A novel hypothesis for development and utilization of fat-storing spherosomes is also proposed.     

Biochemical changes accompanying the long-term starvation of Micrococcus luteus cells in spent growth medium

Changes in the biochemical properties of Micrococcus luteus cells were studied during the transition to a dormant state after incubation in an extended stationary phase. The overall DNA content after 150 days of starvation was similar to its initial level, while the RNA content decreased by 50%. Total lipids and protein, phospholipids and membrane proteins declined rapidly within the first 1–10 days of starvation. After 180 days of starvation, cells contained 43% of the protein and 35% of the lipid initially present. Starvation for 120 days resulted in the loss of phosphatidylglycerol and, to some extent, of phosphatidylinositol, giving a membrane whose phospholipids consisted mainly of cardiolipin. The membrane fluidity declined during starvation, as judged by diphenyl hexatriene fluorescence anisotropy measurements. Oxidase activities declined to zero within the first 20–30 days of starvation, while the dehydrogenases and cytochromes were more stable. The activities of some cytoplasmic enzymes were lost very rapidly, while NADPH-linked isocitrate dehydrogenase had 30% of its initial activity after 120 days of starvation. For all parameters tested there were significant fluctuations during the first 10–20 days of starvation, which may reflect cryptic growth in the culture.Abbreviations MPN Most probable number - DPH Diphenyl hexatriene     

Effects of sodium chloride on the plasma membrane of halotolerant Dunaliella primolecta: an electron spin resonance study

Electron spin resonance spectroscopy was used to monitor the in vivo microviscosity of the plasma membrane and lipid extracts of the salt tolerant alga, Dunaliella primolecta. The fluidity of the plasma membrane decreased as the algae were adapted to and suspended in higher sodium chloride concentrations [2–24% (w/v)]. Both biochemical modification and a physical interaction between Na⁺ and lipids were implicated.When the microviscosity of the plasma membrane and that of lipid extracts were determined as a function of temperature, two or three lipid phase transformations were observed. There were always transformations at 9–14° C and 39–43° C. These were interpreted as the onset and completion of the lipid phase transition of at least a major lipid component of the membrane, possibly the entire membrane. These transformation temperatures were independent of the salt concentration to which the algae were adapted or suspended. This suggests that D. primolecta exists with some of its membrane in the solid-fluid mixed lipid state. With a NaCl concentration of 8% (w/v) or greater in the growth medium, a third transformation occurred around 20–22° C. It was the result of a lipid-lipid interaction and was not related to adaptation.Abbreviations ESR electron spin resonance spectroscopy - 2 T hyperfine splitting - S order parameter - 5-DS or 5-doxyl-stearate 2-(3-carboxylpropyl)-4,4-dimethyl-2-tridecyl-3-oxazolidinyloxyl     

Chromoplasts of Tropaeolum majus L.: Lipid synthesis in whole organelles and subfractions

Isolation of tubulous chromoplasts from Tropaeolum majus L. petals was achieved in pure form. Their main substructures-lipid bodies, tubules, and envelope membranes-have been enriched. Whole chromoplasts as well as substructures have been tested for their activities in lipid synthesis. The following activities were found: fatty acid synthesis from acetate, glycosyl transfer reactions from UDP-galactose and UDP-glucose to galactolipids and sterols, acyltransferase reactions from palmitoyl-CoA, and a very active acyl-CoA hydrolase (EC 3.1.2.2.). Fatty acid synthesis was restricted to whole chromoplasts. Glycosyl- and acyltransferases were essentially confined to envelope membranes, whereas acyl-CoA hydrolase was found in all fractions. The chemical composition of chromoplast subfractions was determined. The lipid bodies consisted mainly of galactolipids and carotenoid esters in a 1:1 ratio, together with small amounts of protein.     

Half unit membranes surrounding osmiophilic granules (lipid droplets) of the so-called lipotubuloid inOrnithogalum

Summary Osmiophilic granules ofOrnithogalum umbellatum lipotubuloids are surrounded by half unit membranes 2–3 nm thick and are filled with nearly homogenous osmiophilic material. It is the presence of this single line membrane that distinguishes the osmiophilic granules from bodies surrounded by tripartite unit membranes (lysosomes, peroxisomes, glyoxysomes) and allows to recognize them as lipid droplets.     

The isolation of plasma membrane from frog cardiac muscle cells

A vesicular preparation consisting largely of the plasma membrane of frog cardiac cells was isolated and its enzymatic activities and lipid content were investigated.The enriched plasma membrane preparation was obtained by (1) mildly homogenizing washed ventricles, (2) separating away the cellular debris by low speed differential centrifugation, (3) separating the plasma membrane fraction from other membranous components by centrifugation to equilibrium in various sucrose gradients. The frog cardiac plasma membranes were found to be concentrated between specific gravities of 1.07 and 1.11. In the membrane fraction the specific activities of membrane marker enzymes: 5′-nucleotides (EC 3.1.3.5), alklaline phosphatase (EC 3.1.3.1) and Na⁺---:K⁺)-activated ATPase were, respectively, 19, 15, and 14 times greater than in the homogenate. Activities of mitochondrial marker enzymes were either very low or absent. No unusual lipid types were found. Cardiolipin was less than 0.1% (by wt) in the membrane fraction. The molar ratio of phospholipid to cholesterol was approximatley 3 : 2.     

Lipid composition of positively buoyant eggs of reef building corals

Lipid composition of the eggs of three reef building corals, Acropora millepora, A. tenuis and Montipora digitata, were determined. Sixty to 70% of the egg dry weight was lipid, which consisted of wax esters (69.5–81.8%), triacylglycerols (1.1–8.4%) and polar lipids c/mainly phospholipids (11.9–13.2%). Montipora digitata also contained some polar lipids typical of the thylakoid membrane in chloroplasts, probably due to the presence of symbiotic zooxanthellae in the eggs. The wax esters appeared to be the major contributor to positive buoyancy of the eggs, and specific gravity of wax esters in A. millepora was estimated to be 0.92. Among the fatty acids of the wax esters, 34.9–51.3% was hexadecanoic acid (16:0) while the major fatty acids in polar lipids were octadecenoic acid (18:1), hexadecanoic acid (16:0), eicosapentaenoic acid (20:5) and eicosatetraenoic acid (20:4). The wax ester appears to be the main component of the 4.5 6.0 m diameter lipid droplets which fill most of the central mass of the coral eggs.     

Intercellular compartmentation of basic carbon pathways in motor organs (pulvini) of leaves of Phaseolus coccineus L.

The activities of enzymes which catalyze one step in each of the five major carbon pathways in green plants were measured in secondary pulvini and other tissues of Phaseolus coccineus L. leaves. We were able to detect activities of fumarase (EC 4.2.1.2; tricarboxylic-acid pathway), NAD-glyceraldehyde-phosphate dehydrogenase (NAD-GAPDH, EC 1.2.1.12; glycolysis), 6-phosphogluconate dehydrogenase (6-PGDH, EC 1.1.1.44; oxidative pentose-phosphate pathway), ribulose-1, 5-bisphosphate carboxylase (Rubisco, EC 4.1.1.39; photosynthetic carbon-reduction pathway), and of hydroxypyruvate reductase (HP-R, EC 1.1.1.81; photosynthetic carbon-oxidation pathway). On a protein basis the activities of Rubisco and HP-R in pulvinar regions were very low (below 1 and 2 mol · (kg protein) ^–-1 · h^–-1, respectively), but the activities of fumarase and NAD-GAPDH were between 10- and 5-fold higher compared with the laminar tissue (up to 7 and 50 mol · (kg protein)^–-1 · h^–-1, respectively). Similarly, the protein specific activities of 6-PGDH were increased in the pulvinus (3–4 compared with approx. 1 mol · (kg protein)^–-1 · h^–-1 in the leaf blade). No differences in specific activities were detected between day and night positions of the leaves. By applying quantitative histochemical techniques we determined the longitudinal and transversal compartmentation of the activities of fumarase, NAD-GAPDH, and 6-PGDH in pulvinar tissues. Levels of activity of all three enzymes increased towards the middle part of the pulvinus. Here, expressed on a dry-weight (DW) basis, the analysis of cross sections showed highest activities in the outer parts of the extensor in the order given, approx. 0.6, 5, and 0.25 mol · (kg DW)^–-1 · h^–-1 for fumarase, NAD-GAPDH and 6-PGDH. When related to protein, levels of activity were comparably high within the inner parts of extensor and flexor, and partly also in the abaxial part of the bundle (fumarase, 6-PGDH). The tissue-specific compartmentation of the respective activities is discussed in relation to leaf movement and shows parallels with guard-cell function.Abbreviations Chl chlorophyll - DW dry weight - GAPDH glyceraldehyde-phosphate dehydrogenase - HP-R hydroxypyruvate reductase - Rubisco ribulose-1,5-bisphosphate carboxylase - 6-PGDH 6-phosphogluconate dehydrogenase This investigation was supported by a grant from the Deutsche Forschungsgemeinschaft.     

Relationship between copper- and zinc-induced oxidative stress and proline accumulation in<Emphasis Type="Italic"> Scenedesmus</Emphasis> sp.

A 4-h exposure of Scenedesmus sp. to Cu or Zn enhanced intracellular levels of both test metals and proline. The level of intracellular proline increased markedly up to 10 µM Cu, but higher concentrations were inhibitory. However, intracellular proline consistently increased with increasing concentration of Zn in the medium. Cu and Zn induced oxidative stress in the test alga by increasing lipid peroxidation and membrane permeability, and by reducing SH content. Pretreatment of the test alga with 1 mM proline for 30 min completely alleviated Cu-induced lipid peroxidation, minimized K⁺ efflux and also reduced depletion of the SH pool. But proline pretreatment could only slightly reduce Zn-induced oxidative stress. Interestingly, proline pretreatment increased the level of Cu (25–54%) and Zn (19–49%) inside the cells. It did not affect the activities of superoxide dismutase, ascorbate peroxidase or catalase, but improved glutathione reductase activity under Cu and Zn stress. A comparison of the effects of proline pretreatment on lipid peroxidation by Cu, Zn, methyl viologen and ultraviolet-B radiation suggests that proline protects cells from metal-induced oxidative stress by scavenging reactive oxygen species rather than by chelating metal ions. Pretreatment of cells with a known antioxidant (ascorbate) and a hydroxyl radical scavenger (sodium benzoate) considerably reduced metal-induced lipid peroxidation and proline accumulation. However, sodium benzoate had a very mild effect on Zn-induced lipid peroxidation and proline accumulation. The present study demonstrates that proline possibly acts by detoxifying reactive oxygen species, mainly hydroxyl radicals, rather than by improving the antioxidant defense system under metal stress.Abbreviations APOX Ascorbate peroxidase - CAT Catalase - GR Glutathione reductase - MDA Malondialdehyde - MV Methyl viologen - ROS Reactive oxygen species - SH Sulphydryl - SOD Superoxide dismutase - UV-B Ultraviolet-B radiation     

Environmentally-induced changes in the neutral lipids and intracellular vesicles of Saccharomyces cerevisiae and Kluyveromyces fragilis

Saccharomyces cerevisiae, grown aerobically or anaerobically under conditions which induce a requirement for a sterol and an unsaturated fatty acid, synthesized approximately the same amounts of neutral lipid and intracellular low-density vesicles, although the neutral lipids in aerobically-grown cells contained more esterified sterol and less triacylglycerol than those in anaerobically-grown cells. Kluyveromyces fragilis synthesized much less neutral lipid and a smaller quantity of low-density vesicles than S. cerevisiae whether grown at 30°C (generation time 1.1 h) or 20°C (generation time 2.1 h). Both yeasts synthesized highly saturated triacylglycerols, relatively unsaturated phospholipids, and esterified sterols with an intermediate degree of unsaturation irrespective of the conditions under which they were grown. Free sterols in the yeasts were rich in ergosterol and 22(24)-dehydroergosterol, while the esterified sterol fractions were richer in zymosterol.     

Metabolism of carbohydrate and lipid reserves in germinated cotton seeds

Utilization of reserve lipid and carbohydrates during germination (0–12 h) and postgerminative growth (12–48 h) was studied in cotton (Gossypium hirsutum L.) seedlings. Raffinose and stachyose were utilized during the germination period and early growth; mobilization was associated with -galactosidase (EC 3.2.1.22) activity. Results from pulse-chase experiments with [³H]raffinose supplied exogenously to 4-h soaked seeds indicated that raffinose-derived catabolites contributed to the coincident increase in cotyledon sucrose and starch, and to the small increase in axis dry weight. Starch appears to be an alternative sink for end products of hydrolysis of reserve carbohydrates prior to the onset of rapid axis growth and cotyledon expansion. Mobilization of neutral lipid commenced at about 16 h after soaking, concomitant with development of key glyoxylate-cycle and other gluconeogenesis-related enzyme activities. Axis dry weight increased three-fold between 24 and 48 h. Results from pulse-chase (3 h, 16 h) experiments in which [2-¹⁴C]acetate was supplied to cotyledons of intact 22-h-old seedlings showed that acetate-derived metabolites were not transported exclusively to the axes, but were partitioned between axes and cotyledons. Only 27% of total incorporated radioactivity was recovered in axes following the chase, 18% was evolved as CO₂, and the rest was recovered in water-soluble substances (20%) and polymers (31%) within the cotyledons. Of the polymers, 55% of the activity was in polysaccharides (Starch, pectic substances, hemicellulose, cellulose), 25% in protein, and 20% in unidentified neutral and acidic compounds. Considering these data, the amount of lipid mobilized, and various routes by which supplied [2-¹⁴C]acetate could be metabolized, it appears that lipidderived compounds contribute only 25–40% of axis dry-weight gain. Lipid-derived substances retained in the cotyledons likely are utilized for expansion and differentiation of the cotyledons into photosynthetic organs.     

Effect of age on the membrane lipid composition of Streptococcus sanguis

The cell membrane of Streptococcus sanguis contains three classes of lipid: neutral lipid, glycolipid and phospholipid. A striking difference in membrane lipid composition between cells in the exponential and in the stationary phases of growth was observed. During the exponential phase, approx. 37–45%, 14–19% and 37–45% of the lipids synthesized were found to be neutral lipid, glycolipid and phospholipid, respectively. The amount of lipid synthesized reached a maximum at the early stationary phase. The amount of phospholipid drastically declined thereafter and that of neutral lipid slightly declined. In contrast, the amount of glycolipid markedly increased and exceeded the amount of phospholipid. The phospholipid present during the exponential phase was found to be mainly phosphatidylglycerol (82–88%) and a small amount of cardiolipin (12–18%). At the stationary phase, the amount of phosphatidylglycerol greatly decreased and reached approx. 16% of that in the early stationary phase, while cardiolipin steadily increased and became the major phospholipid in the late stationary phase. The glycolipid was found to be composed of mainly mono- and diglucosyldiglycerides. At the end of the experiment (after 8 h incubation), the distribution of lipids was found to be: neutral lipid, 46%; glycolipid (monoglucosyldiglyceride, 28%; diglucosyldiglyceride, 13%) 41%; and phospholipid (phosphatidylglycerol, 3%, cardiolipin, 8%) 13%.     

Effect of amphotericin B on the lipids of yeast cells of Sporothrix schenckii

Yeast cells of five strains of Sporothrix schenckii were obtained for partial analysis of lipid composition. Quantitative analysis of lipids and sterols were completed, as well as qualitative analysis of sterols by thin layer chromatography and by ultraviolet spectra. These determinations were made on cells cultured in the absence and presence of amphotericin B at sub-MIC (minimum inhibitory concentration) levels. Marked alterations in lipid content were observed in the amphotericin B-treated cells. The major alterations were the reduction of total lipid (18.7–57.6%) and sterols (48.5–96.7%) after exposure to the polyenic antibiotic. It is concluded that amphotericin B altered the lipid profiles, especially sterols of S. schenckii.     

Expression of recombinant rabbit UDP-GlcNAc:α3-d-mannoside β-1,2-N-acetylglucosaminyltransferase I catalytic domain in Sf9 insect cells

UDP-GlcNAc:3-d-mannoside -1,2-N-acetylglucosaminyltransferase I (GnT I; EC 2.4.1.101) catalyses a key reaction in the conversion of oligomannose to complex and hybridN-glycans. The cytoplasmic tail and transmembrane segment of rabbit GnT I cDNA were replaced with an in-frame cleavable signal sequence and the hybrid construct was inserted into the genome ofAutographa californica nuclear polyhedrosis virus (AcMNPV) under the control of the polyhedrin promoter. Sf9 insect cells were infected with the recombinant baculovirus and the enzymatically active and soluble catalytic domain of GnT I was purified from the medium (1–5 mg 1^–1) in two steps to a specific activity of abut 2 µmol min^–1 mg^–1 protein. Recombinant GnT I has been used for the chemical-enzymatic synthesis of analogues of Man1-6[GlcNAc1-2Man1-3]Man-O-octyl.Abbreviations AcMNPV Autographa californica nuclear polyhedrosis virus - FCS fetal calf serum - 1 µmol min^–1 international enzyme unit - MAG myelin associated glycoprotein - MOI multiplicity of infection - pfu plaque forming units - SDS-PAGE sodium dodecyl sulfate/polyacrylamide gel electrophoresis - Sf9 cells Spodoptera frugiperda insect cells - GnT I UDP-GlcNAc:3-d-mannoside 1,2-N-acetylglucosaminyltransferase I (EC 2.4.1.101)     

The reactivation of nitrate reductase from spinach (Spinacea oleracea L.) inactivated by NADH and cyanide: effects of peroxidase and associated systems

Nitrate reductase of spinach (Spinacea oleracea L.) leaves which had been inactivated in vitro by treatment with NADH and cyanide, was reactivated by incubation with oxidant systems and measured as FMNH₂-dependent activity. Ferricyanide, a purely chemical oxidant, produced rapid maximal reactivation (100%) which was 90% complete in less than 3 min. Reactivation occurred slowly and less completely (30–75% in 30 or 60 min) when the enzyme was incubated with pure horseradish peroxidase alone, depending on using one or 20 units and time. Addition of glucose and glucose oxidase to generate hydrogen peroxide increased reactivation slightly (10–15%) with 20 units of peroxidase but more (30–50%) with one unit and to 75–90% of ferricyanide values. Adding catalase decreased reactivation by more than half either with or without glucose oxidase. Glucose and glucose oxidase alone did not cause reactivation. Addition of superoxide dismutase increased reactivation from 50–75% of ferricyanide values with one unit of peroxidase alone but had no effect on greater reactivation obtained in the presence of glucose oxidase. The addition of p-cresol and manganese together increased reactivation with one unit of peroxidase and in the presence of glucose oxidase by about double, but omission of manganese had no effect. However, as shown previously, although trivalent manganese was formed, the residual presence of manganous ions inhibited reactivation. Nevertheless, peroxidase systems either alone or with additionally generated hydrogen peroxide can induce substantial reactivation of nitrate reductase in physiologically relevant conditions.Abbreviations EDTA ethylenediaminetetraacetic acid - FMN flavine mononucleotide     

First insight into the lipid uptake, storage and mobilization in arachnids: Role of midgut diverticula and lipoproteins

The importance of midgut diverticula (M-diverticula) and hemolymph lipoproteins in the lipid homeostasis of Polybetes phythagoricus was studied. Radioactivity distribution in tissues and hemolymph was analyzed either after feeding or injecting [1-¹⁴C]-palmitate. In both experiments, radioactivity was mostly taken up by M-diverticula that synthesized diacylglycerols, triacylglycerols and phospholipids in a ratio close to its lipid class composition. M-diverticula total lipids represent 8.08% (by wt), mostly triacylglycerols (74%) and phosphatidylcholine (13%). Major fatty acids were (in decreasing order of abundance) 18:1n − 9, 18:2n − 6, 16:0, 16:1n − 7, 18:0, 18:3n − 3. Spider hemocyanin-containing lipoprotein (VHDL) transported 83% of the circulating label at short incubation times. After 24 h, VHDL and HDL-1 (comparable to insect lipophorin) were found to be involved in the lipid uptake and release from M-diverticula, HDL-2 playing a negligible role. Lipoprotein's labelled lipid changed with time, phospholipids becoming the main circulating lipid after 24 h. These results indicate that arachnid M-diverticula play a central role in lipid synthesis, storage and movilization, analogous to insect fat body or crustacean midgut gland. The relative contribution of HDL-1 and VHDL to lipid dynamics indicated that, unlike insects, spider VHDL significantly contributes to the lipid exchange between M-diverticula and hemolymph.