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.     

Pulmonary vascular response to leukotriene D4 in unanesthetized sheep: role of thromboxane

We examined the pulmonary vascular response to an intravenous leukotriene D4 (LTD4) injection of (1 microgram X kg-1 X min-1 for 2 min) immediately followed by infusion of 0.133 microgram X kg-1 X min-1 for 15 min in awake sheep prepared with lung lymph fistulas. LTD4 resulted in rapid generation of thromboxane A2 as measured by an increase in plasma thromboxane B2 concentration. The thromboxane B2 generation was associated with increases in pulmonary arterial and pulmonary arterial wedge pressures while left atrial pressure did not change significantly. Pulmonary lymph flow (Qlym) increased (P less than 0.05) transiently from base line 6.87 +/- 1.88 (SE) ml/h to maximum value of 9.77 +/- 1.27 at 15 min following the LTD4 infusion. The maximum increase in Qlym was associated with an increase in the estimated pulmonary capillary pressure. The increase in Qlym was not associated with a change in the lymph-to-plasma protein concentration (L/P) ratio. Thromboxane synthetase inhibition with dazoxiben (an imidazole derivative) prevented thromboxane B2 generation after LTD4 and also prevented the increases in pulmonary vascular pressures and Qlym. We conclude that LTD4 in awake sheep increases resistance of large pulmonary veins. The small transient increase in Qlym can be explained by the increase in pulmonary capillary pressure. Thromboxane appears to mediate both the pulmonary hemodynamic and lymph responses to LTD4 in sheep.     

Effect of prostacyclin on pulmonary vascular response to thrombin in awake sheep

We determined the effects of infusion of prostacyclin (PGI2) and 6-alpha-carba-PGI2 (6-cPGI2), a stable PGI2 analogue, on pulmonary transvascular fluid and protein fluxes after intravascular coagulation induced by thrombin. Studies were made in control awake sheep prepared with lung lymph fistulas (n = 6) and in similarly prepared awake sheep pretreated with either 6-cPGI2 (n = 5) or PGI2 (n = 5). Both prostacyclin compounds (500 ng X kg-1 X min-1) were infused intravenously. All groups were challenged with 80 U/kg thrombin. Pulmonary arterial pressure (Ppa), pulmonary vascular resistance (PVR), pulmonary lymph flow (Qlym), lymph protein clearance (Qlym X lymph/plasma protein concentration ratio), and neutrophil and platelet counts were determined. In vitro tests assessed sheep neutrophil chemotaxis and chemiluminescence and platelet aggregation. In both 6-cPGI2 and PGI2 groups, the increases in Qlym after thrombin were less than those in the control group. The increase in lymph protein clearance in the 6-cPGI2 group was the same as that in control, whereas the increase in clearance in the PGI2 group was reduced. PVR and Ppa increased to a greater extent in the 6-cPGI2 group than in the control group, whereas the increases in PVR and Ppa were inhibited in the PGI2 group. Neutrophil and platelet counts decreased after thrombin in PGI2 and 6-cPGI2 groups, as they did in the control group. Neither 6-cPGI2 altered neutrophil chemotaxis induced by thrombin and chemiluminescence induced by opsonized zymosan. Both prostacyclin compounds inhibited platelet aggregation induced by ADP or thrombin.(ABSTRACT TRUNCATED AT 250 WORDS)     

Mechanism of peptidoleukotriene-induced increases in pulmonary transvascular fluid filtration

We examined the effects of leukotrienes C4 (LTC4) and D4 (LTD4) (1 microgram) on the pulmonary vascular filtration coefficient, a measure of vessel wall conductivity to water, and the alterations in pulmonary vascular resistance (PVR) in isolated-perfused guinea pig lungs. We also assessed whether LTC4 and LTD4 increased the permeability to albumin in cultured monolayers of pulmonary artery endothelial cells. In Ringer-perfused and blood-perfused lungs, LTC4 resulted in increases in pulmonary arterial pressure (Ppa) and the pulmonary capillary pressure (Pcap) measured as the equilibration pressure after simultaneous pulmonary arterial and venous occlusions. Pulmonary venous resistance (Rv) increased to a greater extent than arterial resistance (Ra) in both Ringer-perfused and blood-perused lungs challenged with LTC4. The greater increase in PVR in blood-perfused lungs corresponded with a greater elevation of lung effluent thromboxane B2 (TxB2) concentration. The LTC4-stimulated increase in PVR was prevented by pretreatment with meclofenamate (10(-4) M). LTD4 also induced rapid increases in Ppa and Pcap in both Ringer-perfused and blood-perfused lungs; however, Ppa decreased before stabilizing at a pressure higher than base line. The increases in Rv with LTD4 were greater than Ra. The LTD4-stimulated increases in Ra and Rv also paralleled the elevation in TxB2 concentration. As with LTC4, the increases in Ppa, Pcap, PVR, and TxB2 concentration were greater in blood-perfused than in Ringer-perfused lungs. Pretreatment with meclofenamate reduced the magnitude of the initial increase in Ppa, but did not prevent the response.(ABSTRACT TRUNCATED AT 250 WORDS)     

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.     

Pulmonary microcirculatory responses to leukotrienes B4, C4 and D4 in sheep

The pulmonary microvascular responses to leukotrienes B₄, C₄ and D₄ (total dosage of 4 μg/kg i.v.) were examined in acutely-prepared halothane anesthetized and awake sheep prepared with lung lymp fistulas. In anesthetized as well as unanesthetized sheep, LTB₄ caused a marked and transient decrease in the circulating leukocyte count. Pulmonary transvascular protein clearance (pulmonary lymph flow x lymph-to-plasma protein concentration ratio) increased transiently in awake sheep, suggesting a small increase in pulmonary vascular permeability. The mean pulmonary artery pressure (P ) also increased. In the acutely-prepared sheep, the LTB₄-induced pulmonary hemodynamic and lymph flow responses were damped. Leukotriene C₄ increased P to a greater extent in awake sheep than in anesthetized sheep, but did not significantly affect the pulmonary lymph flow rate (Q̇_lym) and lmph-to-plasma protein concentration (L/P) ration in either group. LTD₄ increased P and Q̇_lymp in both acute and awake sheep; Q̇_lym increased without a significant change in the L/P ratio. The LTD₄-induced rise in P occurred in association with an increase in plasma thromboxane B₂ (Txb₂) cocentration. The relativity small increase in Q̇_lym with LTD₄ suggests that the increase in the transvascular fluid filtration rate is the result of a rise in the pulmonary capillary hydrostatic pressure. In conclusion, LTB₄ induces a marked neutropenia, pulmonary hypertension, and may transiently increase lung vascular permeability. Both LTC₄ and LTD₄ cause a similar degree of pulmonary hypertension in awake sheep, but had different lymph flow responses which may be due to pulmonary vasoconstriction at different sites, i.e. greather pre-capillary constriction with LTC₄ because Q̇_lym did not change and greater post-capillary constriction with LTD₄ because Q̇ increased with the same rise in P .     

Microvascular albumin permeability in isolated perfused lung: effects of EDTA

We examined the effects of decreases in perfusate concentrations of calcium and magnesium on the pulmonary vascular permeability in the isolated perfused rabbit lung. The albumin permeability-surface area product (PS) and the albumin reflection coefficient (sigma) were determined in the same lung using 125I- and 131I-labeled albumin tracers. Decreases in vascular Ca2+ and Mg2+ concentrations were induced by adding ethylenediaminetetraacetic acid (EDTA) to the perfusate. Decreases in the concentration of these cations resulted in an increase in the PS from a control value of 1.18 +/- 0.13 X 10(-3) to 7.69 +/- 0.75 X 10(-3) cm3 X min-1 X g wet lung wt-1 and a decrease in the sigma from 0.96 +/- 0.01 to 0.74 +/- 0.02. The decrease in sigma suggests an increase in the calculated equivalent pore radius from 44 to 63 A. The results indicate that Ca2+ and Mg2+ play a role in the maintenance of normal pulmonary vascular permeability to proteins.