Alisa Tietz

Relationship Between Vitellogenin and Vitellin in a Marine Shrimp (Penaeus semisulcatus) and Molecular Characterization of Vitellogenin Complementary DNAs

Abstract The source of yolk proteins in crustacean ovaries has been the subject of controversy for several decades, and both extraovarian and intraovarian synthesized proteins have been implicated. To offer a new insight into the relationship of vitellogenin (VTG) and vitellin (VT), a comparison of extraovarian VTG and ovarian VT of the marine shrimp Penaeus semisulcatus was performed at the protein and cDNA levels. Two cDNAs (7920 and 2068 nucleotides [nt]) were sequenced for VTG from the ovary and one cDNA (7920 nt) was sequenced from the hepatopancreas. VTG cDNA from hepatopancreas was similar to VTG cDNA from ovary. Although a VTG gene was also found in the males, ∼7.8-kilobase transcripts were only detected in the ovary and hepatopancreas of females. The mRNA expression pattern was related to the stage of ovarian development and to the molt cycle, as determined by real-time polymerase chain reaction assay. VTG and VT apoproteins were composed of two and three major subunits, respectively, as shown by SDS-PAGE. N-terminal sequences of these subunits revealed the presence of a cleavage site at a consensus motif for a subtilisin-like endoprotease in VTG and VT and an additional cleavage site in VT revealed by an unidentified endoprotease. These results indicate that penaeid shrimps constitute a unique model for vitellogenesis, showing intraovarian gene expression and synthesis of yolk protein.

Triglyceride digestion and absorption in the locust, locust a migratoria

Abstract Locusts were fed glyceryl tri[I−14C]oleate. The triglyceride was rapidly hydrolyzed and 14C-labeled fatty acids were found in the lumen. Large amounts of 14C-labeled acids were found in the phospholipids of the intestinal wall, hemolymph diglycerides and fat body glycerides. Only trace amounts of free acids were detected in these tissues. When double labeled triolein, [2−3H]glyceryl tri[I−14C]oleate was fed, the 3H/14C ratio of the lipids isolated from the above tissues was very markedly lower than that of the fed triolein. Free [3H]glycerol was found. To test whether fatty acids or diglycerides were released from the intestine into the hemolymph, the following observations were made: (I) when bovine serum albumin was injected into the hemocoel soon after feeding, 14C-labeled fatty acids could be isolated from the hemolymph. (2) [14C]Oleic acid was incorporated into hemolymph lipoproteins. The acid was rapidly utilized in vitro by the fat body and subsequently 14C-labeled diglycerides were found in the lipoproteins. (3) Diglyceride uptake in vitro by the fat body was demonstrated.

Lipid composition of the halotolerant alga, Dunaliella bardawil

Abstract Dunaliella bardawil , a halotolerant unicellular alga capable of accumulating large amounts of β-carotene, contains 30% of its total carbon content as lipids. These lipids are equally divided between neutral and polar lipids. The major neutral lipid component is β-carotene. The polar lipid fraction is composed of (in descending order) monogalactosyl- and digalactosyldiacylglycerol, trimethylhomoserine diacylglycerol, sulfoquinovosyldiacylglycerol, phosphatidylglycerol, phosphatidylcholine and phosphatidylethanolamine. The following fatty acids were identified: palmitic, 3- trans -hexadecenoic, hexadecatetraenoic, stearic, oleic, linoleic and linolenic. Globules isolated from D. bardawil contained most of the β-carotene of the cells, plus small amounts of other neutral lipids.

Isolation and properties of a lipoprotein from the haemolymph of the locust, Locusta migratoria

Abstract A yellow lipoprotein was isolated from the haemolymph of the migratory locust by ethanol precipitation and sucrose gradient centrifugation. The lipoprotein contained 30% lipids, 80% of which were diglycerides and phospholipids. The yellow colour was due to the presence of carotenoids. A mol w of 340,000 ± 50,000 was calculated from sedimentation equilibrium measurements. By electrophoresis in sodium dodecyl sulphate-gels the lipoproteins separated into several polypeptides. The capacity of the purified lipoprotein to promote diglyceride release was lower than that of the haemolymph. Full activity was restored by the addition of a protein from the haemolymph, which by itself showed little activity.

Lipid composition of the plasma-membrane of the halotolerant alga, Dunaliella salina

Abstract The major lipids of the isolated plasma-membrane of the halotolerant alga Dunaliella salina are diacylglyceroltrimethylhomoserine (DGTS, 23.5%), sterol peroxides (7-dehydroporiferasterol peroxide and ergosterol peroxide, 22%), phosphatidylcholine (13%) and phosphatidylethanolamine (11%). Free sterols comprised 5% of the lipids and contained predominantly 7-dehydroporiferasterol and ergosterol. The major fatty acids of the plasma-membrane were palmitic (31%), oleic (13%), linoleic (20%) and γ-linolenic (17%) acids. In constrast to the whole cells, the plasma-membrane contained less (11%) α-linolenic acid and no 16-carbon unsaturated fatty acids. Sterol peroxides were identified by 1 H-NMR and 13 C-NMR spectroscopy, mass spectrometry, and by comparison on thin-layer chromatography to the product of ergosterol photooxygenation. We believe that this is the first report on the occurrence of sterol peroxides as major constituents of a biological membrane. It is suggested that they may play a role in the unusual membrane-permeability properties of the plasma-membrane of Dunaliella .

Utilization of 2-acyl-sn-glycerol by locust fat body microsomes: Specificity of the acyltransferase system

Abstract Locust fat-body monoacylglycerol acyltransferase specifically acylates 2-acyl- sn -glycerol; 1-acyl and 2-alkyl- sn -glycerol are poor substrates. The system was activated by Tween 20. Maximal activity was obtained at pH 7.0. Release of free CoA could not be shown.

Hydrolysis of glycerides by lipases of the fat body of the locust, Locusta migratoria

Abstract Lipolysis of mono- di- and triacylglycerol by locust fat body preparation was studied. Microsomes and the soluble supernatant contained an acid lipase (pH optimum 5.0–6.0) which hydrolized micellar dispersions of the above lipids. On short incubations, diacylglycerol yielded equimolar amounts of monoacylglycerol and fatty acids. Trioleylglycerol was degraded very slowly; mainly fatty acids were formed. The enzyme was inhibited by detergents, and various salts. Monoacylglycerol was hydrolyzed by the supernatant over a pH of 5.5 to 7.5. In the microsomes the activity continued to increase up to pH 9.0. These activities were not inhibited by Triton X-100. Moreover, at pH 7.8 in the presence of Triton, di- and triacylglycerol were also degraded.

Fat transport in the locust, Locusta migratoria: The role of protein synthesis

Abstract 1. 1. When prelabeled fat body was incubated in the presence of hemolymph, diglycerides were released and incorporated into lipoproteins of the hemolymph. 2. 2. When fat body was incubated with 14 C-labeled amino acids, the acids were incorporated into tissue proteins which were subsequently released into the medium. Protein release was not dependent on the presence of hemolymph. 3. 3. Gel electrophoresis of the released protein showed that the distribution of radioactivity along the gel coincided with the protein bands of the hemolymph. 4. 4. Addition of cycloheximide inhibited the incorporation of amino acids into proteins; the release of preformed proteins was not affected. 5. 5. Cycloheximide did not inhibit the incorporation of 14 C-labeled palmitate into glycerides, neither was the release of glycerides from the “inhibited” tissue affected.

Hormonal activation of protein kinase and lipid mobilization in the locust fat body in vitro

Cyclic nucleotide-activated protein kinase in locust fat body is elevated by extracts containing adipokinetic hormone (AKH) from locust corpora cardiaca or by synthetic redpigment-concentrating hormone (RPCH) which also exhibits lipid-mobilizing activity. Maximal activation is first attained in vitro 90 sec after RPCH is added to the incubation medium and 3.5 min after addition of AKH extract. This activation reflects the increase in endogenous cyclic nucleotides. Incubation of fat body in the presence of exogenous cyclic nucleotides activates lipase, and RPCH stimulation ultimately elevates the level of transported diacylglycerol 2.3-fold. RPCH stimulates fat body lipase in vivo.

The catabolism of lung surfactant by alveolar macrophages

Surfactant was isolated from lung tissue of normal and chlorocyclizine-fed rats. Chlorocyclizine surfactant contained 2.5-3.4 times more phospholipids per mg protein than normal surfactant. Alveolar macrophages, incubated in vitro with normal and chlorocyclizine surfactants hydrolyzed the surfactant phospholipids and incorporated the fatty acids into cellular triacylglycerol. Employing [3H]palmitate-labeled surfactant, it was shown that cells incubated with chlorocyclizine surfactant incorporated 46.2-73.0 nmol of fatty acids per mg protein and were transformed into foam cells. Employing fluorescein or 125I-labeled surfactant, the uptake of surfactant protein by macrophages was shown. No significant differences between protein uptake from normal and chlorocyclizine surfactants were observed. These results suggest that the surfactant phospholipids and protein were catabolized independently.