Hyperglycemia exacerbates colon cancer malignancy through hexosamine biosynthetic pathway.

Hyperglycemia is a common feature of diabetes mellitus, considered as a risk factor for cancer. However, its direct effects in cancer cell behavior are relatively unexplored. Herein we show that high glucose concentration induces aberrant glycosylation, increased cell proliferation, invasion and tumor progression of colon cancer. By modulating the activity of the rate-limiting enzyme, glutamine-fructose-6-phosphate amidotransferase (GFAT), we demonstrate that hexosamine biosynthetic pathway (HBP) is involved in those processes. Biopsies from patients with colon carcinoma show increased levels of GFAT and consequently aberrant glycans' expression suggesting an increase of HBP flow in human colon cancer. All together, our results open the possibility that HBP links hyperglycemia, aberrant glycosylation and tumor malignancy, and suggest this pathway as a potential therapeutic target for colorectal cancer.

Transport of inorganic phosphate in Leishmania infantum and compensatory regulation at low inorganic phosphate concentration.

BACKGROUND: Proliferation of Leishmania infantum depends on exogenous inorganic phosphate (P(i)) but little is known about energy metabolism and transport of P(i) across the plasma membrane in Leishmania sp. METHODS: We investigated the kinetics of 32P(i) transport, the influence of H+ and K+ ionophores and inhibitors, and expression of the genes for the Na+:P(i) and H+:P(i) cotransporters. RESULTS: The proton ionophore FCCP, bafilomycin A1 (vacuolar ATPase inhibitor), nigericin (K+ ionophore) and SCH28080 (an inhibitor of H+, K(+)-ATPase) all inhibited the transport of P(i). This transport showed Michaelis-Menten kinetics with K0.5 and V(max) values of 0.016 +/- 0.002 mM and 564.9 +/- 18.06 pmol x h(-1) x 10(-7) cells, respectively. These values classify the P(i) transporter of L. infantum among the high-affinity transporters, a group that includes Pho84 of Saccharomyces cerevisiae. Two sequences were identified in the L. infantum genome that code for phosphate transporters. However, transcription of the PHO84 transporter was 10-fold higher than the PHO89 transporter in this parasite. Accordingly, P(i) transport and LiPho84 gene expression were modulated by environmental P(i) variations. CONCLUSIONS: These findings confirm the presence of a P(i) transporter in L. infantum, similar to PHO84 in S. cerevisiae, that contributes to the acquisition of inorganic phosphate and could be involved in growth and survival of the promastigote forms of L. infantum. GENERAL SIGNIFICANCE: This work provides the first description of a PHO84-like P(i) transporter in a Trypanosomatide parasite of the genus Leishmania, responsible for many infections worldwide.

Inorganic phosphate uptake in Trypanosoma cruzi is coupled to K(+) cycling and to active Na(+) extrusion.

BACKGROUND: Orthophosphate (Pi) is a central compound in the metabolism of all organisms, including parasites. There are no reports regarding the mechanisms of Pi acquisition by Trypanosoma cruzi. METHODS: (32)Pi influx was measured in T. cruzi epimastigotes. The expression of Pi transporter genes and the coupling of the uptake to Na(+), H(+) and K(+) fluxes were also investigated. The transport capacities of different evolutive forms were compared. RESULTS: Epimastigotes grew significantly more slowly in 2mM than in 50mM Pi. Influx of Pi into parasites grown under low Pi conditions took place in the absence and presence of Na(+). We found that the parasites express TcPho84, a H(+):Pi-symporter, and TcPho89, a Na(+):Pi-symporter. Both Pi influx mechanisms showed Michaelis-Menten kinetics, with a one-order of magnitude higher affinity for the Na(+)-dependent system. Collapsing the membrane potential with carbonylcyanide-p-trifluoromethoxyphenylhydrazone strongly impaired the influx of Pi. Valinomycin (K(+) ionophore) or SCH28028 (inhibitor of (H(+)+K(+))ATPase) significantly inhibited Pi uptake, indicating that an inwardly-directed H(+) gradient energizes uphill Pi entry and that K(+) recycling plays a key role in Pi influx. Furosemide, an inhibitor of the ouabain-insensitive Na(+)-ATPase, decreased only the Na(+)-dependent Pi uptake, indicating that this Na(+) pump generates the Na(+) gradient utilized by the symporter. Trypomastigote forms take up Pi inefficiently. CONCLUSIONS: Pi starvation stimulates membrane potential-sensitive Pi uptake through different pathways coupled to Na(+) or H(+)/K(+) fluxes. GENERAL SIGNIFICANCE: This study unravels the mechanisms of Pi acquisition by T. cruzi, a key process in epimastigote development and differentiation to trypomastigote forms.

Transport of inorganic phosphate in Leishmania infantum and compensatory regulation at low inorganic phosphate concentration.

BACKGROUND: Proliferation of Leishmania infantum depends on exogenous inorganic phosphate (Pi) but little is known about energy metabolism and transport of Pi across the plasma membrane in Leishmania sp. METHODS: We investigated the kinetics of 32Pi transport, the influence of H+ and K+ ionophores and inhibitors, and expression of the genes for the Na+:Pi and H+:Pi cotransporters. RESULTS: The proton ionophore FCCP, bafilomycin A1 (vacuolar ATPase inhibitor), nigericin (K+ ionophore) and SCH28080 (an inhibitor of H+, K+-ATPase) all inhibited the transport of Pi. This transport showed Michaelis-Menten kinetics with K0.5 and Vmax values of 0.016±0.002mM and 564.9±18.06pmol×h-1×10-7cells, respectively. These values classify the Pi transporter of L. infantum among the high-affinity transporters, a group that includes Pho84 of Saccharomyces cerevisiae. Two sequences were identified in the L. infantum genome that code for phosphate transporters. However, transcription of the PHO84 transporter was 10-fold higher than the PHO89 transporter in this parasite. Accordingly, Pi transport and LiPho84 gene expression were modulated by environmental Pi variations. CONCLUSIONS: These findings confirm the presence of a Pi transporter in L. infantum, similar to PHO84 in S. cerevisiae, that contributes to the acquisition of inorganic phosphate and could be involved in growth and survival of the promastigote forms of L. infantum. GENERAL SIGNIFICANCE: This work provides the first description of a PHO84-like Pi transporter in a Trypanosomatide parasite of the genus Leishmania, responsible for many infections worldwide.

Na+-dependent and Na+-independent mechanisms for inorganic phosphate uptake in Trypanosoma rangeli.

BACKGROUND: Trypanosoma rangeli is dependent on the presence of exogenous orthophosphate (Pi) for maximal growth and ecto-phosphatase activity is responsible for Pi supply under low Pi. Here we investigated the mechanisms of Pi uptake. METHODS: We investigated the kinetics of 32Pi transport, its Na+ and H+ dependence, its correlation with the Na+-ATPase and H+-ATPase, and gene expression of the Na+:Pi cotransporter and Na+-ATPase. RESULTS: T. rangeli grown under limiting Pi transports this anion to the cytosol in the absence and presence of Na+, suggesting that influx is mediated by both Na+-independent and Na+-dependent transporters. Cloning studies demonstrated that this parasite expresses a Pi transporter not previously studied in trypanosomatids. The H+ ionophore, carbonylcyanide-p-trifluoromethoxyphenylhydrazone, decreased both components of 32Pi influx by 80-95%. The H+-ATPase inhibitor, bafilomycin A1, inhibited the Na+-independent mechanism. Furosemide, an inhibitor of ouabain-insensitive Na+-ATPase, decreased both uptake mechanisms of 32Pi to the same extent, whereas ouabain had no effect, indicating that the former is the pump responsible for inwardly directed Na+ and the electric gradients required by the transporters. Parasite growth in high Pi had a lower Pi influx than that found in those grown in low Pi, without alteration in TrPho89 expression, showing that turnover of the transporters is stimulated by Pi starvation. CONCLUSIONS: Two modes of Pi transport, one coupled to Na+-ATPase and other coupled to H+-ATPase seem to be responsible for Pi acquisition during development of T. rangeli. GENERAL SIGNIFICANCE: This study provides the first description of the mechanism of Pi transport across the plasma membrane of trypanosomatids.

Looking for reference genes for real-time quantitative PCR experiments in Rhodnius prolixus (Hemiptera: Reduviidae).

Quantitative real-time PCR (qPCR) has become one of the most used techniques to measure gene expression. However, normalization of gene expression data against reference genes is essential, although these are usually used without any kind of validation. The expression of seven genes was compared in organs of Rhodnius prolixus under diverse conditions, using published software to test gene expression stability. Rp18S and elongation factor 1 (RpEF -1) were the most reliable genes for normalization in qPCR when gene expression in different organs was compared. Moreover, both genes were found to be the best references when transcript levels were compared in the posterior midgut of insects infected with Trypanosoma cruzi. Rp18S was also the best reference gene in the fat bodies of unfed and fed insects. By contrast, RpEF-1 was found to be the best reference gene for comparison between posterior midguts, and RpMIP or RpActin should be used to compare gene expression in the ovaries. Although Rp18S is indicated here as the best reference in most cases, reports from the literature show that it is difficult to find an optimum reference gene. Nevertheless, validation of candidate genes to be taken as references is important when new experimental conditions are tested to avoid incorrect data interpretation.

Lipophorin interaction with the midgut of Rhodnius prolixus: characterization and changes in binding capacity.

Several classes of lipids are transported in insect hemolymph by lipophorin, a major hemolymphatic lipoprotein. The binding of lipophorin to the midgut of the hematophagous insect Rhodnius prolixus was characterized in a midgut membrane preparation, using purified lipophorin radiolabelled in protein moiety ((125)I-HDLp). Lipophorin specific binding to membranes achieved equilibrium after 30-40 min, was sensitive to pH, and was maximal at pH 7.0. In the presence of increasing concentrations of membrane protein, corresponding increases in lipophorin binding were observed. The specific binding of lipophorin to the membrane preparation was a saturable process, with K(d)=0.9+/-0.06 x 10(-7) M and a maximal binding capacity of 70+/-11 ng lipophorin/microg of membrane protein. Lipophorin binding did not depend on calcium, but it was affected by ionic strength and was inhibited in the presence of increasing salt concentrations. Suramin interfered with lipophorin binding to the midgut receptor, and it was abolished in the presence of 2 mM suramin, but at concentrations between 0.05 and 0.2 mM it was slightly increased. Condroitin 4-sulfate also affected lipophorin binding, which was reduced to 56% of control. Pre-incubation of the midgut membrane preparation with trypsin or at high temperature inhibited binding. Midgut capacity to bind lipophorin varied at different days after blood meal. It was highest at second day after feeding, and then gradually decreased.

Purification and characterization of a lipid transfer particle in Rhodnius prolixus: phospholipid transfer.

In this study we report the purification and characterization of a lipid transfer particle (LTP) from Rhodnius prolixus hemolymph, and its participation in phospholipid and diacylglycerol transfer processes. (3)H-diacylglycerol labeled low density lipophorin from Manduca sexta ((3)H-LDLp) was incubated with R. prolixus lipophorin (Lp) in the presence of Rhodnius hemolymph. Following incubation and isolation, both lipoproteins showed equivalent amounts of (3)H-labeled lipids. Hemolymph was subjected to KBr gradient ultracentrifugation. SDS-PAGE analysis of gradient fractions showed the enrichment of bands with molecular masses similar to the M. sexta LTP standard. LTP containing fractions were assayed and lipid transfer activity was observed. Purification of LTP was accomplished by (i) KBr density gradient ultracentrifugation, (ii) size exclusion, (iii) Cu(++) affinity and (iv) ion exchange chromatographies. LTP molecular mass was estimated approximately 770 kDa, comprising three apoproteins, apoLTP-I (315 kDa), apoLTP-II (85 kDa) and apoLTP-III (58 kDa). Phospolipid content of (32)P-LTP was determined after two-dimensional TLC. (32)P-phospholipid-labeled and unlabeled lipophorins, purified from R. prolixus were incubated in the presence of LTP resulting in the time-dependent transfer of phospholipids. LTP-mediated phospholipid transfer was not a selective process.

Fat body fructose-2,6-bisphosphate content and phosphorylase activity correlate with changes in hemolymph glucose concentration during fasting and re-feeding in larval Manduca sexta.

Fasting of second-day fifth instar larval Manduca sexta leads to a rapid decrease in hemolymph glucose concentration from 3.39+/-0.29 to 0.33+/-0.06 mM in 1 h, along with a decrease in the fructose-2,6-bisphosphate content in the fat body (from 5.92+/-0.31 to 2.80+/-0.47 nmol fructose-2,6-bisphosphate/g fat body in 3 h) and activation of fat body glycogen phosphorylase (from 16% to 55-65% phosphorylase a). During re-feeding an increase in the glucose level in the hemolymph was observed (from 0.36+/-0.05 to 3.91+/-0.36 mM in 3 h), along with an increase in the fructose-2,6-bisphosphate level in the fat body (from 2.88+/-0.47 to 6.66+/-0.42 nmol fructose-2,6-bisphosphate/g fat body in 3 h) and inactivation of fat body glycogen phosphorylase (from 56% to 16% phosphorylase a). These data are consistent with the hypothesis that a decrease in hemolymph glucose both activates fat body glycogen phosphorylase and causes a decrease in fat body fructose-2,6-bisphosphate content. Both of these changes would favor conversion of stored glucose to trehalose in the fat body. When second-day larvae were decapitated, the changes in hemolymph glucose and fat body fructose-2,6-bisphosphate were very similar to those observed in fasting whole insects. These data are consistent with a direct role for glucose in controlling carbohydrate metabolism in Manduca sexta.

Ectonucleotide diphosphohydrolase activities in hemocytes of larval Manduca sexta.

In this work, we describe the ability of living hemocytes from an insect (Manduca sexta, Lepidoptera) to hydrolyze extracellular ATP. In these intact cells, there was a low level of ATP hydrolysis in the absence of any divalent metal (8.24 +/- 0.94 nmol of Pi/h x 10(6) cells). The ATP hydrolysis was stimulated by MgCl2 and the Mg2+-dependent ecto-ATPase activity was 15.93 +/- 1.74 nmol of Pi/h x 10(6) cells. Both activities were linear with cell density and with time for at least 90 min. The addition of MgCl2 to extracellular medium increased the ecto-ATPase activity in a dose-dependent manner. At 5 mM ATP, half-maximal stimulation of ATP hydrolysis was obtained with 0.33 mM MgCl2. This stimulatory activity was not observed when Ca2+ replaced Mg2+. The apparent Km values for ATP-4 and Mg-ATP2- were 0.059 and 0.097 mM, respectively. The Mg2+-independent ATPase activity was unaffected by pH in the range between 6.6 and 7.4, in which the cells were viable. However, the Mg2+-dependent ATPase activity was enhanced by an increase of pH. These ecto-ATPase activities were insensitive to inhibitors of other ATPase and phosphatase activities, such as oligomycin, sodium azide, bafilomycin A1, ouabain, furosemide, vanadate, sodium fluoride, tartrate, and levamizole. To confirm the observed hydrolytic activities as those of an ecto-ATPase, we used an impermeant inhibitor, DIDS (4,4'-diisothiocyanostilbene-2,2'-disulfonic acid), as well as suramin, an antagonist of P2-purinoreceptors and inhibitor of some ecto-ATPases. These two reagents inhibited the Mg2+-independent and the Mg2+-dependent ATPase activities to different extents. Interestingly, lipopolysaccharide, a component of cell walls of gram-negative bacteria that increase hemocyte aggregation and phagocytosis, increased the Mg2+-dependent ecto-ATPase activity in a dose-dependent manner but did not modify the Mg2+-independent ecto-ATPase activity.

Characterization of lipophorin binding to the midgut of larval Manduca sexta.

Lipophorin binding to the midgut of Manduca sexta larvae was characterized in a midgut membrane preparation, using iodinated larval high-density lipophorin ((125)I-HDLp-L). The iodination procedure did not change the affinity of the preparation for lipophorin. In the presence of increasing concentrations of membrane protein, corresponding increases in lipophorin binding were observed. The time-course of lipophorin binding to the membranes was affected by the lipophorin concentration in the medium, and at a low lipoprotein concentration, a longer time was required for equilibrium to be reached. The specific binding of lipophorin to the midgut membrane was a saturable process with a K(d) = 1.5+/-0.2x10(-7) M and a maximal binding capacity = 127+/-17 ng lipophorin/microg of membrane protein. Binding did not depend on calcium, was maximal around pH 5.5, was strongly inhibited by an increase in the ionic strength, and abolished by suramin. However, suramin did not completely displace lipophorin that was previously bound to the membrane preparation. The lipid content of the lipophorin did not significantly affect the affinity of the membrane preparation for lipoprotein.

Developmental changes in the response of larval Manduca sexta fat body glycogen phosphorylase to starvation, stress and octopamine.

Fasting or starvation of 1(st)- and 2(nd)-day fifth instar Manduca sexta larvae leads to rapid activation of fat body glycogen phosphorylase. Under feeding conditions, 21-29% of the phosphorylase was found in the active form. However, after only one hour of starvation, the active form increased to 55-65%. In larvae on the 3(rd)-day there was a slower increase in the activation, requiring three hours of starvation to reach a maximum of 60-65%. No activation was observed in 4(th)-day larvae after three hours of starvation. When 1(st)- or 2(nd)-day larvae were decapitated, the time-course of activation of glycogen phosphorylase was very similar to that observed in intact insects. However, activation of glycogen phosphorylase following decapitation was only observed in 1(st)- and 2(nd)-day larvae. In 2(nd)-day larvae, octopamine promoted activation of glycogen phosphorylase and 100-pmol of octopamine promoted maximum activation. Higher amounts of injected octopamine caused a decrease in activation. The injection of 100 pmol of octopamine caused a 50-55% activation of phosphorylase within 30 minutes. The simultaneous injection of the alpha-adrenergic receptor antagonist phentolamine with octopamine blocked the octopamine effect in 1(st)- and 2(nd)-day feeding larvae. However, the activation of glycogen phosphorylase observed in ligated/decapitated larvae on the 1(st)- and 2(nd)-day was not abolished by injection of phentolamine. All of these data suggest that factors other than adipokinetic hormone and octopamine may be involved in the activation of glycogen phosphorylase during fasting or starvation in the early part of the fifth larval stage of M. sexta.

Fatty acid incorporation by Rhodnius prolixus midgut.

[(14)C]Oleic acid injected into the hemocoel of Rhodnius prolixus females was shown to rapidly associate with lipophorin particles. Half of the lipophorin-associated [(14)C]oleic acid was transferred in about 5 min to different organs, but the midgut was the main organ to take it up on day 10 after a blood meal. The rate of [(14)C]oleic acid incorporation by the midgut was high up to 15 min after injection and then declined. The [(14)C]oleic acid incorporated by the midgut was found in phospholipids (58.6%) and neutral lipids (37.4%). The midgut capacity to incorporate [(14)C]oleic acid varied on different days after a meal: it increased up to day 10 and then decreased. The fate of the [(14)C]lipids synthesized by the midgut was followed and it was observed that 10 days after feeding diacylglycerol was the main lipid released to hemolymph and that most of phospholipids and triacylglycerols remained associated with the midgut. The metabolism of free fatty acids in Rhodnius prolixus females is discussed in the context of major biological events that follow a blood meal such as digestion and oogenesis.

Role of phospholipids in the protein stability of an insect lipoprotein, lipophorin from Rhodnius prolixus.

Lipophorin (Lp) is the major lipoprotein in insect hemolymph. The structural organization proposed for Lp is basically the same as that suggested for vertebrate lipoproteins, consisting of a hydrophobic core containing neutral lipids, stabilized in the aqueous environment by surrounding polar moieties of protein and phospholipids at the particle surface. After complete removal of phospholipids from Lp by phospholipase A2, the particle remains soluble [Gondim, K. C., Atella, G. C., Kawooya, J. K., & Masuda, H. (1992) Arch. Insect Biochem. Physiol. 20, 303-314]. However, studies on the roles of phospholipid on the structural stability of Lp are still lacking. In the present work, we have studied the structure and stability of dephospholipidated lipophorin (d-Lp). Trypsinolysis of d-Lp indicated no exposure of new cleavage sites on the protein when compared to Lp. However, an enhanced rate of proteolysis of the apoproteins (especially apolipophorin II) was observed in d-Lp. Circular dichroism analysis indicated that the secondary structure of Lp was not significantly affected by phospholipid removal. Furthermore, the exposure of tryptophan residues to the aqueous solvent in d-Lp was the same as in Lp, as indicated by intrinsic fluorescence emission spectra and fluorescence quenching experiments. Interestingly, d-Lp was more resistant to denaturation by guanidine hydrochloride than Lp. d-Lp was also found to be less sensitive than Lp to structural changes induced by hydrostatic pressure. Taken together, these results indicate that, although changes in its structural organization were subtle, dephospholipidated lipophorin may have additional protein-protein and/or protein-neutral lipid interactions that are responsible for the observed increase in stability. Therefore, phospholipids are not only not essential for Lp stability, but their presence in the particle seems to result in a less stable structure in the aqueous environment.

Lipophorin density variation during oogenesis on Rhodnius prolixus.

The density of lipophorin was determined in adult females of Rhodnius prolixus on different days after a meal. Several populations od lipoproteins, differing in density but always in the range of HDL, were found in the hemolymph. The density of the major population was analyzed and a complex profile of density variation was found associated with the principal metabolic events in these insects digestion and oogenesis. During the initial three days after the blood meal, with the onset of the digestive process, the density of lipophorin decreased from 1.1185 g/l to 1.1095 g/l, associated with the transfer of lipids from midgut to the lipophorin particles. During the period of intense vitellogenesis and lipid uptake by the ovary, the lipophorin density started to increase and reached the value, 1.1322 g/l, and remained stable up to the end of oogenesis. As soon as the requirement of lipids to build up the oocytes ceased, the density of lipophorin decreased to its initial value associated with the transfer of lipids from fat body to lipophorin. Soon after the blood meal the midgut was the main source of lipids capable of replenishing the lipophorin particles, while the fat body assumed this function during the succeeding days and reached its maximum capacity around day 10, as estimated by the rate of lipid transfer. The principal lipids transferred were phospholipids and diacylglycerols. Except in the protein/lipid ratio no major changes were observed among different lipids isolated from lipophorin of different densities.

Characterization and immunocytochemical localization of lipophorin binding sites in the oocytes of Rhodnius prolixus.

Purified lipophorin, metabolically labelled with 32P exclusively in the phospholipid moiety, was used to study the process of phospholipid delivery to the oocyte. The kinetics of phospholipid transfer "in vitro," from lipophorin to the oocytes, was linear at least up to 4 h and was impaired by low temperature. A net transfer of phospholipids from lipophorin particles to the oocytes was observed. The rate of phospholipid uptake was dependent on the concentration of lipophorin in the medium and was shown to be a saturable process. The addition of a molar excess of purified unlabelled lipophorin to the culture medium resulted in a substantial decrease in the transfer of [32P]phospholipids, but no reduction occurred in the presence of a molar excess of albumin. The lipophorin binding sites were localized in the oocytes by immunogold techniques using two different protocols for oocyte fixation. Strong labelling was observed especially at the microvilli. No labelling was detected in the yolk granules.

Role of phospholipids in the lipophorin particles of Rhodnius prolixus.

The lipophorin of Rhodnius prolixus metabolically labelled with 32P exclusively in the phospholipid moiety was purified on a potassium bromide gradient and treated with phospholipase A2 in the presence of an excess of fatty acid-free albumin. The treatment completely removed the phospholipids from the particles and generated [32P]-lysophosphatidylcholine, [32P]-lysophosphatidylethanolamine, and free fatty acids that remained bound to albumin. The phospholipid-depleted lipophorin particles remained soluble, indicating that phospholipids are not essential in maintaining the stability of the particles in aqueous solution. Complete removal of phospholipids did not affect the association of apolipophorin III with lipophorin particles. Lipophorin density increased slightly from 1.120 to 1.134 g/ml after treatment. The phospholipid-depleted particles also retained their ability to be recognized and loaded in vitro with phospholipids delivered by the fat body, thus supporting the concept of lipophorin's role as a reusable lipid shuttle for phospholipids.

Transfer of phospholipids from fat body to lipophorin in Rhodnius prolixus.

32P-Labeled fat bodies (32P-fat bodies) of Rhodnius prolixus females were incubated in the presence of non radioactive purified lipophorin and the release of radioactivity to the medium was analysed to answer the question of whether lipophorin is a reusable shuttle for phospholipids. The radioactivity found in the medium was associated with lipophorin phospholipids. When the 32P-fat bodies were incubated in the absence of lipophorin, only a small amount of radioactivity was released and it was not associated with lipophorin, indicating that there was no release of pre-labeled 32P-lipophorin by the tissue. Analysis of 32P-phospholipids transferred from fat bodies to the lipophorin particles by thin-layer chromatography revealed a predominance of phosphatidylethanolamine and phosphatidylcholine, with minor amounts of phosphatidylserine, phosphatidylinositol, and sphingomyelin. The transfer of phospholipids to lipophorin was linear with time up to 45 min and the process was inhibited at low temperature and by the metabolic inhibitors azide and fluoride. The transfer of phospholipids from the fat bodies to lipophorin was saturable with respect to the concentration of lipophorin, which was half-maximal at about 8 mg/ml. A directional movement of phospholipids from the fat body to lipophorin was observed. The net gain of phospholipids in 2 h of incubation with fat body was 8.54 nmol per insect, which corresponds to 6.69% of increase in the lipophorin phospholipid content. The rate of 32P-phospholipid transfer from fat body to lipophorin particles varied during the days after a blood meal increasing up to day 10 and then decreasing in parallel with the process of oogenesis.

The roles of haemolymphatic lipoproteins in the oogenesis of Rhodnius prolixus.

The fates of purified 32P-vitellin and 32P-lipophorin were followed in vitellogenic females of Rhodnius prolixus. While the radioactivity from 32P-vitellin 6 hours after injection was found almost exclusively in the ovary, the radioactivity from injected 32P-lipophorin was found distributed among several organs. In the ovary, the radioactivity from 32P-vitellin was associated with the contents of the yolk granules. 32P-lipophorin delivered a great amount of radioactive phospholipids to the ovary with no accumulation of its protein moiety, as observed after its iodination with 131I. The delivery of phospholipids was inhibited at 0 degrees C and by the metabolic inhibitors, sodium azide and sodium fluoride. Comparison of the radioactivity incorporation from 32P-lipophorin with that of 14C-inulin suggests that the 32P-phospholipids from lipophorin are not taken up by fluid phase endocytosis. The data presented here are compatible with the concept of lipophorin as a carrier of lipids in insects and provide evidence that lipophorin transports phospholipids as shown previously for other classes of lipids. The utilization by the oocytes of the phospholipids transported by lipophorin is discussed.