Very High Density Lipoproteins Research Articles

Green Tea Powder Decreased Egg Weight Through Increased Liver Lipoprotein Lipase and Decreased Plasma Total Cholesterol in an Indigenous Chicken Breed.

Simple SummaryTons of green tea powder (GTP) are produced and cast off during green tea processing. It is suggested that GTP could increase immunity and health, and so improve animal production performance. We demonstrated that one percent of GTP supplemented in the diet did not affect egg production. However, long time GTP inclusion resulted in decreased egg weight and increased feed-to-egg ratio. Combined with plasma lipid concentration, the decreased egg weight might be because of lower plasma lipid concentration, increased plasma orexin A, and liver lipoprotein lipase expression in chickens fed a diet containing GTP.Whether or not green tea promotes egg production is unclear. Huainan partridge chickens at 20 weeks of age were divided into two groups, with one group fed a basal diet (control) and one fed a basal diet plus 10 g/kg green tea powder (GTP) for 12 weeks. Egg production (EP) and feed intake (FI) were recorded daily. Plasma lipid parameters, and apolipoprotein-B (Apo-B), 3-hydroxy-3-methylglutaryl coenzyme A reductase (HMGR), and lipoprotein lipase (LPL) expression were determined every four weeks. Egg production and FI showed no significant difference between the two groups (p > 0.05). Egg weight was 47.58 g in the control group, which was higher than that of the GTP group, and the feed-to-egg ratio (FCR) was 4.62 in the control group, which was lower than that of the GTP group after 12 weeks feeding. Compared with the control group, plasma orexin A (p < 0.05), high-density lipoprotein (HDL), apolipoprotein A (Apo A), and very high-density lipoprotein (VHDL) (p < 0.01, respectively) were increased. Plasma glucose (Glu), free fatty acid (FFA), apolipoprotein B (Apo B), triglyceride (TG), total cholesterol (TC) (p < 0.01, respectively), and low density lipoprotein (LDL) (p < 0.05) were decreased in the GTP group after 8 weeks feeding. The LPL expression in the liver was increased in the GTP group after 8 to 12 weeks feeding when compared to the control group (p < 0.05). Chickens fed GTP did not affect EP, but decreased egg weight, which might be because of lower plasma lipid concentration, increased plasma Orexin A, and liver LPL expression.

Very high-density lipoprotein and vitellin as carriers of novel biliverdins IXα with a farnesyl side-chain presumably derived from heme A in Spodoptera littoralis

Bilins in complex with specific proteins play key roles in many forms of life. Biliproteins have also been isolated from insects; however, structural details are rare and possible functions largely unknown. Recently, we identified a high-molecular weight biliprotein from a moth, Cerura vinula, as an arylphorin-type hexameric storage protein linked to a novel farnesyl biliverdin IXα; its unusual structure suggests formation by cleavage of mitochondrial heme A. In the present study of another moth, Spodoptera littoralis, we isolated two different biliproteins. These proteins were identified as a very high-density lipoprotein (VHDL) and as vitellin, respectively, by mass spectrometric sequencing. Both proteins are associated with three different farnesyl biliverdins IXα: the one bilin isolated from C. vinula and two new structurally closely related bilins, supposed to be intermediates of heme A degradation. The different bilin composition of the two biliproteins suggests that the presumed oxidations at the farnesyl side-chain take place mainly during egg development. The egg bilins are supposedly transferred from hemolymph VHDL to vitellin in the female. Both biliproteins show strong induced circular dichroism activity compatible with a predominance of the M-conformation of the bilins. This conformation is opposite to that of the arylphorin-type biliprotein from C. vinula. Electron microscopy of the VHDL-type biliprotein from S. littoralis provided a preliminary view of its structure as a homodimer and confirmed the biochemically determined molecular mass of ∼350 kDa. Further, images of S. littoralis hexamerins revealed a 2 × 3 construction identical to that known from the hexamerin from C. vinula.

Steroid hormone 20-hydroxyecdysone regulation of the very-high-density lipoprotein (VHDL) receptor phosphorylation for VHDL uptake

During the metamorphic stage of holometabolous insects, the biosynthetic precursors needed for the synthesis of a large number of adult proteins are acquired from the selective absorption of storage proteins. The very-high-density lipoprotein (VHDL), a non-hexameric storage protein, is consumed by the fat body from the hemolymph through VHDL receptor (VHDL-R)-mediated endocytosis. However, the mechanism of the uptake of VHDL by a VHDL-R remains unclear. In this study, a VHDL-R from Helicoverpa armigera was found to be involved in 20E-regulated VHDL uptake through the regulation of steroid hormone 20-hydroxyecdysone (20E). The transcripts of VHDL-R were detected mainly in the fat body and integument during the wandering stage. The transcription of VHDL-R was upregulated by 20E through the ecdysteroid receptor (EcRB1) and Ultraspiracle (USP1). In addition, 20E stimulates the phosphorylation of VHDL-R through protein kinase C for ligand binding. VHDL-R knockdown in larvae results the inhibition of development to adulthood. These data imply that 20E regulates VHDL-R on both transcriptional and posttranslational levels for VHDL absorption.

The role of phospholipid transfer protein in lipoprotein-mediated neutralization of the procoagulant effect of anionic liposomes.

Serum has the ability to neutralize the procoagulant properties of anionic liposomes, with transfer of phospholipids (PLs) to both high-density lipoprotein (HDL) and low-density lipoprotein (LDL) particles. Phospholipid transfer protein (PLTP) mediates transfer of PLs between HDL and other lipoproteins and conversion of HDL into larger and smaller particles. To examine the role of PLTP in the neutralization of procoagulant liposomes. Procoagulant liposomes were incubated with different lipoproteins in the presence or absence of PLTP, and then tested for their ability to stimulate thrombin formation. In the absence of added PLTP, the lipoprotein-enriched fraction, total HDL, HDL(3) and very high-density lipoprotein (VHDL) were all able to neutralize the procoagulant properties of the liposomes. In these samples, endogenous PLTP was present, as judged by Western blotting. In contrast, no PLTP was present in LDL, HDL(2) and lipoprotein-deficient serum, all of which displayed no ability to neutralize the procoagulant liposomes. The phospholipid (PL) transfer activity was dependent on both enzyme (PLTP) and PL acceptor (lipoproteins). After treatment of the VHDL fraction with antiserum against PLTP, the neutralization of procoagulant activity was reduced, but could be regained by the addition of active PLTP. The neutralizing activity was dependent on a catalytically active form of PLTP, and addition of a low activity form of PLTP had no effect. In conclusion, PLTP was found to mediate transfer of anionic PLs to HDL and LDL, thereby neutralizing the effect of procoagulant liposomes, resulting in a reduction of procoagulant activity.

VHDL, a larval storage protein from the corn earworm, Helicoverpa zea, is a member of the vitellogenin gene family

The hemolymph of last instar larvae of the corn earworm, Helicoverpa zea contains a blue very high-density lipoprotein (VHDL) that is selectively taken up into fat body prior to pupation. Its amino-terminal sequence was determined by Edman degradation, and used to design a degenerate primer for PCR amplification. With 5′ and 3′ RACE techniques, the entire cDNA coding for VHDL was amplified and sequenced. Conceptual translation reveals a 173 kDa protein that contains a 15 amino acid signal sequence immediately before the experimentally determined N-terminus of the mature protein. The protein contains a typical lipoprotein N-terminal domain, and shows high sequence similarity to vitellogenins from Lepidoptera and other insect species. VHDL mRNA was not detectable in adult H. zea, and antibodies raised against VHDL did not react with adult hemolymph or yolk proteins. Therefore VHDL, although a member of the vitellogenin gene family, seems to be distinct from the vitellogenin expressed in adult females.

Ghrelin Interacts with Human Plasma Lipoproteins

Ghrelin, a peptide hormone produced predominantly by the stomach, stimulates food intake and GH secretion. The Ser(3) residue of ghrelin is mainly modified by a n-octanoic acid. In the human bloodstream, ghrelin circulates in two forms: octanoylated and desacylated. We previously demonstrated that ghrelin is desoctanoylated in human serum by butyrylcholinesterase (EC 3.1.1.8) and other esterase(s), whereas in rat serum, only carboxylesterase (EC 3.1.1.1) is involved. The aims of this study were to determine the role of lipoprotein-associated enzymes in ghrelin desoctanoylation and the role of lipoproteins in the transport of circulating ghrelin. Our results show that ghrelin desoctanoylation mostly occurred in contact with low-density lipoproteins (LDLs) and lipoprotein-poor plasma subfractions. Butyrylcholinesterase and platelet-activating factor acetylhydrolase (EC 3.1.1.47) were responsible for the ghrelin hydrolytic activity of the lipoprotein-poor plasma and LDL subfractions, respectively. Moreover, we observed that ghrelin is associated with triglyceride-rich lipoproteins (TRLs), high-density lipoproteins (HDLs), very high-density lipoproteins (VHDLs), and to some extent LDLs. In conclusion, we report that the presence of the acyl group is necessary for ghrelin interaction with TRLs and LDLs but not HDLs and VHDLs. Ghrelin interacts via its N- and C-terminal parts with HDLs and VHDLs. This suggests that, whereas TRLs mostly transport acylated ghrelin, HDLs and VHDLs transport both ghrelin and des-acyl ghrelin.

Hemocyanin lipid uptake in Polybetes pythagoricus is altered by fenitrothion

The spider very high density lipoprotein (VHDL), which contains hemocyanin as the major apoprotein, transports most of the circulating lipids. In this work, the effect of the pesticide fenitrothion (FS) on the ability of VHDL-apoproteins to uptake different lipids was investigated. For this, VHDL was delipidated using Triton X-100 and recombined with different radiolabeled lipids in the presence or the absence of FS. The oligomeric structural integrity was maintained after delipidation as shown by non-denaturating PAGE. In the presence of the insecticide, palmitic acid uptake decreased by 28.2 and 62.4% after treating the apolipoprotein with 10 and 20 ppm FS, respectively. Decreases in the uptake of cholesterol, triolein, and phosphatidylcholine caused by FS were 29, 23, and 31% using 10 ppm, and 40, 44, and 29% using 20 ppm FS, respectively. Fluorescence measurements with the hydrophobic probes diphenylhexatriene (DPH) and diphenylhexatrienyl-propionic acid (DPH-PA) indicate that FS induces a red shift, decreases the intensity and increases the anisotropy of the emission of these probes in the VHDL. These results indicate that insecticide binding to the lipoprotein enhances the environment polarity and restricts the mobility of these probes at their binding site. These changes at the hydrophobic VHDL binding sites could lead to the decreased affinity for lipids and hydrophobic ligands. It is inferred that FS could alter the normal lipid exchange between this lipoprotein and tissues.

Cloning and expression of the VHDL receptor from fat body of the corn ear worm, Helicoverpa zea

In Noctuids, storage proteins are taken up into fat body by receptor-mediated endocytosis. These include arylphorin and a second, structurally unrelated very high-density lipoprotein (VHDL). Previously, we have isolated a single storage protein receptor from the corn earworm, Helicoverpa zea, which binds both VHDL and arylphorin. The receptor protein is a basic, N-terminally blocked, ∼80 kDa protein that is associated with fat body membranes. Microsequencing of proteolytic fragments of the isolated receptor protein revealed internal sequences that were used to clone the complete cDNA of the VHDL receptor by 3′ and 5′ RACE techniques. The receptor protein, when expressed in vitro via a suitable insect expression vector, reacted with antibodies against the native VHDL receptor and bound strongly to its ligand VHDL, thus confirming that the cloned cDNA represents indeed the previously purified VHDL receptor. The receptor protein and a second, similar protein also found associated with the fat body membrane show considerable homology to putative basic juvenile hormone suppressible proteins cloned previously from other Noctuid species. Sequence analysis revealed that the receptor is likely a peripheral membrane protein that may mediate the selective uptake of VHDL.BJHSP basic juvenile hormone suppressible protein FITC fluorescein isothiocyanate HRP horseraddish peroxidase RACE rapid amplification of cDNA ends VHDL very high density lipoprotein

Cloning and expression of the VHDL Receptor from fat body of the corn ear worm, Helicoverpa zea

In Noctuids, storage proteins are taken up into fat body by receptor-mediated endocytosis. These include arylphorin and a second, structurally unrelated very high-density lipoprotein (VHDL). Previously, we have isolated a single storage protein receptor from the corn earworm, Helicoverpa zea, which binds both VHDL and arylphorin. The receptor protein is a basic, N-terminally blocked, approximately 80 kDa protein that is associated with fat body membranes. Microsequencing of proteolytic fragments of the isolated receptor protein revealed internal sequences that were used to clone the complete cDNA of the VHDL receptor by 3' and 5' RACE techniques. The receptor protein, when expressed in vitro via a suitable insect expression vector, reacted with antibodies against the native VHDL receptor and bound strongly to its ligand VHDL, thus confirming that the cloned cDNA represents indeed the previously purified VHDL receptor. The receptor protein and a second, similar protein also found associated with the fat body membrane show considerable homology to putative basic juvenile hormone suppressible proteins cloned previously from other Noctuid species. Sequence analysis revealed that the receptor is likely a peripheral membrane protein that may mediate the selective uptake of VHDL.

Lipid composition during sexual development of the noble crayfish Astacus astacus and effect of a fungal infection

Summary Previous published results (Hall et al., 1995) showed that two plasma proteins involved in the immune response in the crayfish (Pacifastacus leniusculus) are lipoproteins, suggesting an apparent functional association between lipid transport and the immune response system in crustaceans. The crayfish Astacus astacus is sensitive to a disease caused by the fungus Aphanomyces astaci, which is responsible for high mortalities in the natural environment. Since vitellogenesis is accompanied by a massive accumulation and transport of lipids to ovaries, the present study examines the effect of the crayfish plague fungus A. astaci on lipid transport during sexual development in the crayfish A. astacus. Comparison of the plasma lipid composition between animals sampled before and at the end of the ovarian development cycle revealed that mature females contained more lipids than males or immature females. The high-density lipo-protein (HDL) hemolymph fractions contained four to seven times more lipids than the very high-density lipoprotein (VHDL) fractions; triacylglycerols (TAG) were detected only in the HDL of females. There were no significant changes in hemolymph lipid levels in vitellogenic females subjected to an experimental fungal infection. However, in males exposed to the same experimental conditions, there was a significant decrease in phospholipids (PL) concomitant with an increase in diacylglycerols (DAG) and cholesterol (CH). No differences were observed in the lipid content of either the hepatopancreas (of males and females) or the ovaries due to infection, except a slight decrease in the ovarian PL level. Clotting protein was found relatively more abundant in plasma samples obtained from infected animals after subjecting them to polyacrylamide gel electrophoresis under native conditions. The results of the present study suggest that long-term exposure to a sub lethal dose of the fungus may allow in future studies, a better evaluation of the effect of an infection on lipid composition in shrimp plasma and tissue samples.

Molecular characterization of the bifunctional VHDL-CP from the hemolymph of white shrimp Penaeus vannamei

A very high-density lipoprotein (VHDL) purified from the hemolymph of the white shrimp Penaeus vannamei is shown to be identical to the clotting protein (CP) previously reported from the same organism based on size, subunits and N-terminal amino acid sequence. The ∼440-kDa protein, a homodimer of ∼200-kDa subunits, was present in KBr gradient fractions ranging in density from 1.155 to 1.212 g/ml. Samples of VHDL after purification by strong cation exchange chromatography were subjected to electrophoresis on native polyacrylamide gels. Lipids associated with the VHDL were detected by Sudan Black and Oil Red O staining and comprise 9–15% of the purified protein. Circular dichroism of VHDL-CP indicates that the α-helix content of the VHDL-CP is 32%, while β-sheets correspond to 33%, closely resembling the secondary structure of CP from the shrimp Penaeus monodon and, remarkably, the secondary structure of very high-density lipophorin E (VHDLpE) from the tobacco hornworm, Manduca sexta.

Seasonal variation in plasma lipids, lipoproteins, apolipoprotein A-I and vitellogenin in the freshwater turtle, Chrysemys picta

An analysis of plasma lipids and lipoprotein fractions was performed over the course of the annual ovarian cycle of the female turtle, Chrysemys picta. Determinations of total plasma triglycerides, cholesterol, vitellogenin and apolipoprotein A-I (apoA-I) were made. The lipid and protein composition of the lipoprotein fractions [very low density lipoprotein (VLDL), low density lipoprotein (LDL), high density lipoprotein (HDL) and very high density lipoprotein (VHDL)] were also observed over the same period. Plasma triglyceride and vitellogenin levels were significantly increased in the spring preovulatory period and fall recrudescent phase. Total plasma cholesterol levels were significantly elevated only at the onset of the fall recrudescent phase and apoA-I levels were highest during the postoviposition/ovarian arrest phase. The triglyceride content of VLDL was highest in preovulatory animals and there were apparent seasonal changes in the expression of apoA-I and apoE of HDL/VHDL. We conclude that the coordinate regulation of lipids and protein contributes to seasonal ovarian growth and clearance of lipids from plasma, both of which are most likely under hormonal control.

Analysis by enzyme-linked immunosorbent assay and 2-dimensional electrophoresis of haptoglobin in the high-density lipoprotein fraction in cows.

Haptoglobin (Hp) is a hemoglobin (Hb)-binding acute-phase protein. Besides its relevance in inflammation, Hp is involved in the regulation of lipid metabolism. In cattle, in addition to the lipoprotein-deficient fraction, Hp is distributed in high-density lipoprotein (HDL) and very high-density lipoprotein (VHDL) fractions. The purpose of this study was to determine Hp concentrations in the lipoprotein fractions using an enzyme-linked immunosorbent assay (ELISA) based on the affinity with Hb, and also to detect structural differences of HDL Hp from that in the lipoprotein-deficient fraction using 2-dimensional electrophoresis. When purified Hp was used as the antigen for the ELISA, the detection limit was 7.4 ng/ml and linearity was obtained from 14.8 to 475 ng/ml. The correlation coefficient between the ELISA and single radial immunodiffusion was 0.884. The ELISA was shown to be applicable to evaluate Hp concentrations in the lipoprotein fractions. Hp concentrations in the lipoprotein fractions were in the range of 0.94 to 8.77 microg of Hp/ml (n = 4), and concentration ratios were 0.2 to 0.3% of whole serum Hp. Of the lipoprotein fractions, Hp was most abundant in HDL, moderate in VHDL and faint in chylomicrons, the very low-density lipoprotein fraction and low-density lipoprotein fraction. By 2-dimensional electrophoresis, alpha- and beta-chains of serum Hp were each separated into 5 spots, and their isoelectric point (pI) values were from 5.05 to 6.28 in the alpha-chain and from 5.92 to 6.95 in the beta-chain. The pI values of HDL Hp were indistinguishable from those of serum Hp. These results indicate that the ELISA based on the affinity with Hb is useful for evaluating Hp concentrations in lipoprotein fractions, and also suggest that HDL Hp is structurally similar to that in the lipoprotein-deficient fraction.

Dietary Vitamin A Modulates the Concentrations of RRR-α-tocopherol in Plasma Lipoproteins from Calves Fed Milk Replacer

The practice of supplementing milk replacers fed to neonatal calves with high concentrations of vitamin A has raised concerns regarding the effect of excess vitamin A on the bioavailability of vitamin E. A 4 x 2 factorial experiment evaluated the effects of four dietary amounts of vitamin A [0, 1.78 [National Research Council (NRC)(6) requirement, control], 35.6 and 71.2 micromol daily as retinyl acetate] and two forms of vitamin E (RRR-alpha-tocopherol and RRR-alpha-tocopheryl acetate, 155 micromol daily) on plasma RRR-alpha-tocopherol and RRR-gamma-tocopherol and RRR-alpha-tocopherol associated with plasma lipoproteins (Lp) from milk replacer-fed Holstein calves from birth to 28 d of age. The VLDL, LDL, HDL and very high-density lipoprotein (VHDL) fractions were separated by ultracentrifugal flotation, and the amount of vitamin E associated with each fraction was determined by normal-phase HPLC. The amount and distribution of RRR-alpha-tocopherol in Lp fractions were unaffected by the form of dietary vitamin E. Plasma and Lp RRR-alpha-tocopherol concentrations increased with age (P < 0.0001) and were maximal at 28 d of age. Concentrations of RRR-alpha-tocopherol associated with Lp were 25% (P < 0.01) to 39% (P < 0.0001) lower in calves fed 35.6 and 71.2 micromol of vitamin A daily than in control calves at 28 d of age. The RRR-gamma-tocopherol concentrations were unaffected by dietary vitamin A (P >/= 0.05). In conclusion, dietary vitamin A modulated the amount and distribution of RRR-alpha-tocopherol in the circulation of milk replacer-fed neonatal calves. Because of the essential antioxidant role of vitamin E, the health-related consequences associated with the depression of the LP RRR-alpha-tocopherol concentrations in calves fed vitamin A at 35.6 and 71.2 micromol need to be investigated.

Detection of haptoglobin in the high-density lipoprotein and the very high-density lipoprotein fractions from sera of calves with experimental pneumonia and cows with naturally occurring fatty liver.

In addition to the lipoprotein-deficient d > 1.25 fraction, haptoglobin was detected in the high-density lipoprotein (HDL) and the very high-density lipoprotein (VHDL) fractions from sera of calves with experimental pneumonia and cows with naturally occurring fatty liver. It was not found in the chylomicrons, very low-density lipoprotein and low-density lipoprotein fractions. Washing of the HDL fraction did not decrease the haptoglobin concentration. Transferrin and immunoglobulin G were immunoblotted to examine the possibility of contamination of the lipoprotein fractions by the d > 1.25 fraction. The two serum proteins were detected only in the d > 1.25 fraction, not in any lipoprotein fractions. The distribution pattern of haptoglobin in the lipoprotein fractions was distinct from that of serum albumin. Concentrations of haptoglobin in the HDL fractions from pneumonic sera were largely proportional to those in whole sera. Cholesteryl ester concentrations were decreased in sera from calves with pneumonia, as in cows with fatty liver. A protein immunologically related to hemoglobin was also detected in particular in the VHDL fractions from sera of both groups. These results suggest that haptoglobin or a complex with the hemoglobin-like protein may have a role or roles related to the lipid metabolism.

A very-high-density lipoprotein with clotting ability from hemolymph of sand crayfish, Ibacus ciliatus.

A very-high-density lipoprotein (VHDL) with a density of 1.27-1.29 g/ml was the most abundant lipoprotein in the hemolymph of the sand crayfish Ibacus ciliatus. The VHDL isolated by a density gradient ultracentrifugation consisted of 94% protein and 6% lipid reflecting its high density, and phospholipid was a predominant lipid component. The VHDL had an apolipoprotein of molecular mass 195 kDa and its N-terminal amino acid sequence was identified as follows: LQPGLEYQYRYNGRVAA. This sequence was similar to those of clotting proteins from the spiny lobster Panulirus interruptus and the freshwater crayfish Pacifastacus leniusculus. Transglutaminase and Ca2+ also induced the VHDL to clot. Considering large amounts of VHDL in the hemolymph of sand crayfish, the VHDL not only functions as lipid carrier but plays an important role in the defense process of crustacea.

Lipoproteins of the egg perivitelline fluid of Pomacea canaliculata snails (Mollusca: Gastropoda).

The lipid and protein composition of the perivitelline fluid of the eggs of Pomacea canaliculata was investigated. Two lipoproteins (PV 1 and PV 2) and one lipoprotein fraction (PV 3) were detected for the first time in gastropods. They represent 57.0, 7.5, and 35.5% of the egg total proteins, respectively. PV 1 is a glyco-carotene-protein complex with characteristics of a very high-density lipoprotein (VHDL). It has 0.33% lipids, mainly free sterols and phospholipids. The particle has a MW of 300 Kd and is composed of three subunits of 35, 32, and 28 Kd, respectively. PV 2 particle is a VHDL of 400 Kd and 3.75% lipids. The major lipid classes are free sterols and phospholipids and also have significant quantities of energy-providing triacylglycerides and free fatty acids. It is composed of two apoproteins of 67 and 31 Kd. PV 3 density corresponds to a high-density lipoprotein (HDL). It was fractionated into two subfractions "h" and "p". Fraction "h" contains 5.16% lipids, mainly free sterols, phospholipids, and free fatty acids, and two particles of 100 and 64 Kd. Dissociating electrophoresis showed two subunits of 34 and 29 Kd. Fraction "p" is composed of a single particle of 26 Kd that contains 9.5% lipids, which represents 30% of the total egg lipids. It has high levels of a carotenoid pigment. Besides it contains free fatty acids, hydrocarbons, sterified sterols, and triacylglycerides. These three fractions are probably the major supply of lipids and amino acids for the developing embryo.

Lipoproteins of the egg perivitelline fluid of Pomacea canaliculata snails (Mollusca: Gastropoda)

The lipid and protein composition of the perivitelline fluid of the eggs of Pomacea canaliculata was investigated. Two lipoproteins (PV 1 and PV 2) and one lipoprotein fraction (PV 3) were detected for the first time in gastropods. They represent 57.0, 7.5, and 35.5% of the egg total proteins, respectively. PV 1 is a glyco-carotene-protein complex with characteristics of a very high-density lipoprotein (VHDL). It has 0.33% lipids, mainly free sterols and phospholipids. The particle has a MW of 300 Kd and is composed of three subunits of 35, 32, and 28 Kd, respectively. PV 2 particle is a VHDL of 400 Kd and 3.75% lipids. The major lipid classes are free sterols and phospholipids and also have significant quantities of energy-providing triacylglycerides and free fatty acids. It is composed of two apoproteins of 67 and 31 Kd. PV 3 density corresponds to a high-density lipoprotein (HDL). It was fractionated into two subfractions “h” and “p”. Fraction “h” contains 5.16% lipids, mainly free sterols, phospholipids, and free fatty acids, and two particles of 100 and 64 Kd. Dissociating electrophoresis showed two subunits of 34 and 29 Kd. Fraction “p” is composed of a single particle of 26 Kd that contains 9.5% lipids, which represents 30% of the total egg lipids. It has high levels of a carotenoid pigment. Besides it contains free fatty acids, hydrocarbons, sterified sterols, and triacylglycerides. These three fractions are probably the major supply of lipids and amino acids for the developing embryo. © 1996 Wiley-Liss, Inc.

Detection and quantification of a very high density lipoprotein in different tissues of Triatoma infestans during the last nymphal and adult stages

The presence of a very high density lipoprotein (VHDL), an hexameric protein, was explored in different tissues of Triatoma infestans throughout the last nymphal and adult stages, and in egg extracts by Western blot assays. The VHDL was always detected in both, hemolymph and fat body, during the above mentioned stages and it was also observed in the buffer soluble fraction of testis and egg homogenates. An enzyme-linked immunosorbent assay (ELISA) was used to measure the VHDL titer in these tissues. Hemolymph VHDL reaches a maximum value before the last molt, then it abruptly declines in males and females just after emergence, but during adult life it increases again. Fat body VHDL decreases slowly and continuously during the nymph growth reaching a minimum value prior to molting, and in the first week of adult life the values were even two-fold lower; then, it shows a different cycle of accumulation and depletion in males and females. In adult testis the VHDL undergoes a cycle similar to the one observed in male fat body. This protein increases progressively during embryonic development and, at the time of larval hatching it reaches its maximum value. The hexameric protein presents homologies in its N-terminal sequence with storage hexamerins of Diptera, Lepidoptera and Hymenoptera.

Characterization of lipoprotein fractions with high content of hemocyanin in the hemolymphatic plasma ofPolybetes pythagoricus

In order to evaluate the role of hemocyanin in the hemolymphatic transport of lipids, two hemocyanin-containing lipoprotein fractions, a very-high-density lipoprotein (VHDL) and a high-density lipoprotein (HDL) isolated from plasma of the spider Polybetes pythagoricus, were characterized. VHDL concentration was found to be twofold when compared to that of HDL, and both of them transport 70% of total circulating lipids. Their lipid moieties were mainly composed of phospholipids (65% and 55% in VHDL and HDL, respectively), triacylglycerols, and free fatty acids in a minor degree, and diacylglycerols, cholesterol, and hydrocarbons even in lesser amounts. Although hemocyanin was the predominant apoprotein, two other proteins were also present in minor amounts, especially in VHDL. The two lipoproteins were analyzed by gel filtration chromatography and separated into three subfractions. Two of them were composed of the heptamer (500 kDa) and the monomer (70 kDa) of hemocyanin, respectively, and different proportions of binding lipids. Free fatty acids (37% of total subfraction lipids) were the main lipid classes linked to the heptamer, whereas triacylglycerols (24%) and free fatty acids (22%) predominated among lipids associated with the hemocyanin monomer. The third subfraction contained the hexamer of hemocyanin (420 kDa), two minor polypeptides, and 65% of the VHDL total lipids. This subfraction is the principal means of transport of phosphatidylcholine and triacylglycerols in VHDL. It is here suggested that hemocyanin, as a single apoprotein or associated with other polypeptides, is responsible for the transport of the majority of plasma lipids in this invertebrate. © 1996 Wiley-Liss, Inc.