Changes in lipoprotein composition during larval-pupal metamorphosis of an insect, Manduca sexta

During the transition from the last feeding larval stage to the pupal stage of the tobacco hornworm, Manduca sexta, significant changes occur in the properties of lipophorin, the major hemolymph lipoprotein. Within the first 24 h after cessation of feeding, the larval lipophorin (HDLp-L) is first converted to a higher density form (HDLp-W2) and then HDLp-W2 is converted to a lower density form (HDLp-W1). HDLp-W1 remains in the hemolymph until pupation, when another form, HDLp-P, with a density between HDLp-W1 and HDLp-L, is present. Although all the lipophorins contain identical apoproteins, they differ in lipid content and composition; the differences in density being primarily related to diacylglycerol content. The conversion of HDLp-L to HDLp-W1 is accompanied by a loss of hydrocarbon and uptake of carotenes. These latter changes in lipophorin composition reflect alterations in cuticular lipid composition. HDLp-L was radiolabeled in the apoproteins by injecting animals with 3H-amino acids early in the last larval stage. Subsequently HDLp-L was isolated at the end of the larval stage, HDLp-W2 and HDLp-W1 were isolated during the wandering stage, and HDLp-P was isolated after pupation. The specific activity of the apoproteins in the four lipophorins was not significantly different, suggesting that the observed alterations in lipophorin properties do not require synthesis of new apoproteins but result from retailoring the lipid composition of preexisting molecules. Examination of the hemolymph of individual animals during these transitions showed that only one species of lipoprotein was present, never a mixture of two or more species. These observations suggest that the lipoprotein conversions are precisely timed and that lipoprotein metabolism during larval development and pupation cannot be considered a static process. The unique finding of these studies was that synthesis of lipophorin apoproteins proceeds actively during the first part of the fifth instar but then ceases and does not recommence during the wandering or early pupal stages.     

Lipoprotein biosynthesis in the larvae of the tobacco hornworm, Manduca sexta

Lipoprotein biosynthesis in larvae of the tobacco hornworm (Manduca sexta) was investigated. By immunoblotting, it was shown that the apoproteins are present in the fat body, but not in the midgut. Fat body incubated in vitro with [35S]methionine secreted labeled apoproteins. However, when the density of the secreted particle was determined, it was found at 1.24-1.28 g/ml instead of 1.15 g/ml, which is the density of the circulating lipoprotein. Lipid analysis of immunoprecipitated lipoprotein secreted by the fat body showed a phospholipid/diacylglycerol ratio of 8.3 rather than 0.9, the ratio found in the circulating lipoprotein. When labeled oleic acid or triolein was fed to larvae, it was found that greater than 98% of the label in the circulating lipoprotein was in diacylglycerol. In studies using animals raised on a fat-free diet, it was shown that the circulating lipoprotein has properties comparable to those of the material secreted in vitro by the fat body and that this diacylglycerol-poor particle can be converted to the normal lipoprotein by feeding a bolus of triolein. These data support the hypothesis that the fat body makes and secretes a "nascent" lipoprotein which contains apoproteins and phospholipid, but is devoid of diacylglycerol. The diacylglycerol is then picked up from the midgut to complete assembly of the mature circulating lipoprotein.     

Lipoprotein interconversions in an insect, Manduca sexta. Evidence for a lipid transfer factor in the hemolymph

Hemolymph lipoproteins (lipophorins) of adult Manduca sexta are disinct from larval forms in density, lipid content, composition, and the presence of a third, low molecular weight apoprotein. Generally, only one lipoprotein species exists in M. sexta hemolymph during any given life stage. Progression through the life cycle results in alterations of existing lipoproteins to produce new forms, without new protein synthesis. The observed alterations in lipoprotein density could result from facilitated lipid transfer in insect hemolymph. An in vitro assay of facilitated lipid transfer was developed which employs a high density lipophorin from the wandering larva (density = 1.18 g/ml) as acceptor and adult low density lipophorin (density = 1.03 g/ml) as donor. Adult lipophorin-deficient hemolymph was shown to catalyze a time-dependent equilibration of the starting lipoproteins to produce a new intermediate lipophorin, Lp-I. Hydrodynamic experiments on the donor, acceptor, and product lipoproteins excluded fusion as the mechanism whereby Lp-I is produced. Thus, it is concluded that Lp-I results from facilitated net lipid transfer from low to high density lipoprotein. Furthermore, experiments conducted with radioiodinated donor and radioiodinated acceptor lipoproteins demonstrated that apoprotein exchange does not occur during the lipid transfer reaction. When donor lipoprotein was labeled in the lipid moiety with carbon-14, evidence of diacylglycerol and phospholipid exchange was obtained. Partial characterization of the lipid transfer factor revealed a relationship between incubation time, donor concentration, acceptor concentration, lipophorin-deficient hemolymph concentration, and transfer activity, as measured by Lp-I production. It is concluded that lipophorin-deficient hemolymph contains one or more factor(s) that catalyze net lipid transfer as well as diacylglycerol and phospholipid exchange between lipophorins to produce a single form at equilibrium.     

Solubilization and purification of hepatic microsomal trans-2-enoyl-CoA reductase: evidence for the existence of a second long-chain enoyl-CoA reductase

The present study describes the solubilization and purification of a NADPH-specific trans-2-enoyl-CoA reductase from rat liver microsomes. The final preparation was purified to near homogeneity and had a minimal molecular weight of 51,000 +/- 2,000, as judged by sodium dodecylsulfate (SDS)-polyacrylamide gel electrophoresis. This enzyme specifically used NADPH, as cofactor, and was chromatographically (2',5'-ADP-agarose) separated from another trans-2-enoyl-CoA reductase which utilized either NADH or NADPH as cofactor. The NADPH-specific trans-2-enoyl-CoA reductase catalyzed the reduction of trans-2-enoyl-CoAs from 4 to 16 carbon units. The Km values for crotonyl-CoA, trans-2-hexenoyl-CoA, and trans-2-hexadecenoyl-CoA were 20, 0.5, and 1.0 microM, while the Km value for NADPH was 10 microM. Although N-ethylmaleimide, heat treatment, and limited proteolysis with trypsin affected the reduction of short-chain (C4) and long-chain (C16) substrates equally, and in spite of the fact that a single protein band was observed on SDS-gels, at the present time one cannot state unequivocally that the purified preparation contained only one reductase. trans-2-Hexenoyl-CoA, for example, did not inhibit the reduction of trans-2-hexadecenoyl-CoA to palmitoyl-CoA and trans-2-decenoyl-CoA to decanoyl-CoA whereas it strongly inhibited the conversion of crotonyl-CoA to butyryl-CoA. The potential implications of this finding are discussed. Finally, the reductase preparation was shown not to contain either heme, nonheme iron, or a flavin prosthetic group.     

Differences in amino acid transport and phospholipid contents during the cell cycle ofCandida albicans

Drugs like L-ethionine, 1,10-phenanthroline and 3-(2-thienyl)-DL-alanine which arrest Saccharomyces cerevisiae cells in the G1 phase, were unable to arrest Candida albicans cells. However, C. albicans could be arrested in G1 after a prolonged stationary phase. As compared to normal cells, there was a selective reduction in the level of accumulation of valine and glutamate in G1-arrested cells, while the phospholipid polar head group ratio was not significantly altered. When G1-arrested C. albicans cells were again allowed to grow, the level of different phospholipids started increasing at about the time of bud emergence (2.5 h) whereas reduced levels of accumulated valine and glutamate recovered within 1 h. The recovery of phospholipids and amino acid transport are two distinct events during the progression of C. albicans cells from G1 to S phase.     

Substrate recognition by the 2 micron circle site-specific recombinase: effect of mutations within the symmetry elements of the minimal substrate.

The minimal substrate for the 2 microns circle site-specific recombinase FLP consists of a nearly perfect 13-base-pair dyad symmetry with an 8-base-pair core. By using a series of chemically synthesized FLP substrates in in vitro FLP recombination and FLP-binding assays, we have identified four positions within each of the symmetry elements that are important contact points for the FLP protein. Furthermore, the binding and recombination data provide evidence for cooperativity between the two symmetry elements of a substrate and between the symmetry elements of two partner substrates during FLP recombination.     

Glycolipid and phospholipid composition of wheat and barley chloroplasts

Composition of complex lipid of chloroplasts of two cultivars of wheat and barley was determined at tillering, ear emergence and grain filling stages. The chloroplast lipids, MGDG, DGDG and PG were maximum at grain filling stage in both wheat and barley. PC content showed variations at different stages in both the crops while no significant changes were observed in PI concentrations at grain filling stage.     

Chemical modification of Escherichia coli succinyl-CoA synthetase with the adenine nucleotide analogue 5''-p-fluorosulphonylbenzoyladenosine.

Escherichia coli succinyl-CoA synthetase (EC 6.2.1.5) was irreversibly inactivated on incubation with the adenine nucleotide analogue 5'-p-fluorosulphonylbenzoyladenosine (5'-FSBA). Optimal inactivation by 5'-FSBA took place in 40% (v/v) dimethylformamide. ATP and ADP protected the enzyme against inactivation by 5'-FSBA, whereas desulpho-CoA, an analogue of CoA, did not. Inactivation of succinyl-CoA synthetase by 5'-FSBA resulted in total loss of almost four thiol groups per alpha beta-dimer, of which two groups appeared to be essential for catalytic activity. 5'-FSBA at the first instance appeared to interact non-specifically with non-essential thiol groups, followed by a more specific reaction with essential thiol groups in the ATP(ADP)-binding region. Plots of the data according to the method of Tsou [(1962) Sci. Sin. 11, 1535-1558] revealed that, of the two slower-reacting thiol groups, only one was essential for catalytic activity. When succinyl-CoA synthetase that had been totally inactivated by 5'-FSBA was unfolded in acidic urea and then refolded in the presence of 100 mM-dithiothreitol, 85% of the activity, in comparison with the appropriate control, was restored. These data are interpreted to indicate that inactivation of succinyl-CoA synthetase by 5'-FSBA involves the formation of a disulphide bond between two cysteine residues. Disulphide bond formation likely proceeds via a thiosulphonate intermediate between 5'-p-sulphonylbenzoyladenosine and one of the reactive thiol groups of the enzyme.     

Inducing sporulation in Alternaria solani II. Effect of light

Among the three different light sources viz. incandescent electric light, infra-red light and sunlight, only sunlight was effective in inducing sporulation in petri dish cultures ofA. solani. The intensity of sporulation depended upon the age and growth stage of the cultures, duration and number of exposures to light and the presence or absence of a sporulating zone in the culture. Maximum sporulation was obtained in the case of 6 days old partially grown cultures by inducing the formation of sporulating zone which appeared in 24 hours after every exposure of 60 minutes to sunlight.     

Effect of zinc on increasing oxygen affinity of sickle and normal red blood cells

We have hypothesized a state of zinc deficiency in sickle cell disease (SCD). This could at least partially explain the growth problems, hypogonadism, and slow healing leg ulcers associated with SCD. Preliminary findings revealed abnormally low red blood cell zinc levels in 10 of 16 patients studied. Before suggesting zinc supplementation in SCD we thought it important to look at the effect of zinc on red cell metabolism and function. It was found that zinc chloride added to normal and SCD blood to a final concentration of 1.5 × 10^?3 M caused a left-shift of the blood oxygen affinity curve (increased oxygen affinity) varying from 1.5 to 3.5 mm Hg change in half saturation (p50). This curve shifting property has important implications for SCD since recent work with cyanate suggests that such shifts are very beneficial in treatment of SCD. Thus zinc supplementation in SCD, in addition to its potential role in correcting wound healing and growth problems, may have a beneficial effect on the basic pathological process. Data are given which suggest that zinc and 2, 3-diphosphoglycerate may not be competing for the same site on the hemoglobin molecule.