Sequence variations of Env signal peptide alleles in different clinical stages of HIV infection

The human immunodeficiency virus has been shown to increase its infectivity throughout the course of infection. This virus selection property has been associated with genome mutations and recombinations among virus variants, causing amino acid residue alterations in important viral proteins. In order to explore the contribution of Env signal peptide (Env-sp) to Env glycoprotein expression and its possible relationship to increased virus infectivity observed at late stages of infection, we characterized Env-sp sequences derived from twelve patients at “early” and “late” stages of HIV infection without antiretroviral therapy use. In spite of the remarkable overall similarity between both stages, we observed the deletion of a sequence of neutral and basic residues at the Env-sp amino terminus in virus from early stage specimens and the insertion of basic residues in the hydrophobic region on late-stage viral isolates. The Env-sp sequence alterations may have viral adaptive functions during HIV infection.     

Identification and Characterization of an Ecto-Pyrophosphatase Activity in Intact Epimastigotes of Trypanosoma rangeli

In this study, we performed the molecular and biochemical characterization of an ecto-enzyme present in Trypanosoma rangeli that is involved with the hydrolysis of extracellular inorganic pyrophosphate. PCR analysis identified a putative proton-pyrophosphatase (H⁺-PPase) in the epimastigote forms of T. rangeli. This protein was recognized with Western blot and flow cytometry analysis using an antibody against the H⁺-PPase of Arabidopsis thaliana. Immunofluorescence microscopy confirmed that this protein is located in the plasma membrane of T. rangeli. Biochemical assays revealed that the optimum pH for the ecto-PPase activity was 7.5, as previously demonstrated for other organisms. Sodium fluoride (NaF) and aminomethylenediphosphonate (AMDP) were able to inhibit approximately 75% and 90% of the ecto-PPase activity, respectively. This ecto-PPase activity was stimulated in a dose-dependent manner by MgCl₂. In the presence of MgCl₂, this activity was inhibited by millimolar concentrations of CaCl₂. The ecto-PPase activity of T. rangeli decreased with increasing cell proliferation in vitro, thereby suggesting a role for this enzyme in the acquisition of inorganic phosphate (Pi). Moreover, this activity was modulated by the extracellular concentration of Pi and increased approximately two-fold when the cells were maintained in culture medium depleted of Pi. All of these results confirmed the occurrence of an ecto-PPase located in the plasma membrane of T. rangeli that possibly plays an important role in phosphate metabolism of this protozoan.     

Evidences for Viral Strain Selection in Late Stages of HIV Infection: An Analysis of Vpu Alleles

One of the most studied topics about AIDS disease is the presence of different progression levels in patients infected by HIV. Several studies have shown that this progression is directly associated with host genetics, although viral factors are also known to play a role. Here we explore the contribution of Vpu protein in the evolution of viral population. The sequence variation of Vpu was analyzed during HIV infection in peripheral blood monocyte cells of 12 patients in different clinical stages of HIV-1 infection early and late stages of infections, separated by at least 4 years. The clustering analysis of Vpu sequences showed higher diversity of early alleles, non-random distribution of sequences, and viral evolution strains selection. Forty-two amino acid modifications were found in the multiple alignments of the 57 different alleles found for early stage were 23 modifications were found in the late stage dataset. Interestingly fourteen alteration of early stage were located in conserved site related with Vpu functions alterations while these alterations appear with less frequency in the late stage of infection. Moreover, late stage alleles tend to be similar with the Vpu wild type sequence, suggesting viral selection toward populations harboring more efficient variants during the course of infection. This would contribute to higher infectivity and viral replication actually observed at the aggressive late stages of infection. These data, in conjunction with in vitro experiments, will be important to elucidation of the physiological relevance of Vpu protein in the pathogenic mechanisms of AIDS.     

Dynamics of lipid accumulation by the fat body of Rhodnius prolixus: the involvement of lipophorin binding sites

In insects, lipids are stored in the fat body, mainly as triacylglycerol (TAG). In Rhodnius prolixus, a hematophagous hemipteran, lipids are accumulated after blood meal to be used later on. In adult females, at the second day after feeding, the amount of TAG was 57+/-17 microg/fat body, it increased almost five times and at fourth day it was 244+/-35 microg/fat body. TAG content remained constant until day 13, but it then decreased and, at day 20th it was very low (31+/-4.9 microg/fat body). Radiolabeled free fatty acid was used to follow lipid accumulation by the fat body, as it was previously shown that, in R. prolixus, injected free fatty acids associate with lipophorin, a major hemolymphatic lipoprotein. (3)H-palmitic acid was injected into the hemocoel of R. prolixus females. It disappeared from the hemolymph very rapidly, and radioactivity was incorporated by the fat body. Sixty minutes after injection, radioactivity in the fat body was found mainly in TAGs. The capacity of the fat body to incorporate fatty acids from the hemolymph varied according to the days after blood meal, and it was maximal around the fourth day. Lipophorin binding to specific sites in fat body membrane preparations also showed variation at different days. When membranes obtained from insects at the second, fifth and tenth days were compared, binding was highest at fifth day after feeding.     

Compatibility of Neoseiulus paspalivorus and Proctolaelaps bickleyi, candidate biocontrol agents of the coconut mite Aceria guerreronis: spatial niche use and intraguild predation

The eriophyid mite Aceria guerreronis occurs in most coconut growing regions of the world and causes enormous damage to coconut fruits. The concealed environment of the fruit perianth under which the mite resides renders its control extremely difficult. Recent studies suggest that biological control could mitigate the problems caused by this pest. Neoseiulus paspalivorus and Proctolaelaps bickleyi are two of the most frequently found predatory mites associated with A. guerreronis on coconut fruits. Regarding biological control, the former has an advantage in invading the tight areas under the coconut fruit perianth while the latter is more voracious on the pest mites and has a higher reproductive capacity. Based on the idea of the combined use/release of both predators on coconut fruits, we studied their compatibility in spatial niche use and intraguild predation (IGP). Spatial niche use on coconut fruits was examined on artificial arenas mimicking the area under the coconut fruit perianth and the open fruit surface. Both N. paspalivorus and P. bickleyi preferentially resided and oviposited inside the tight artificial chamber. Oviposition rate of P. bickleyi and residence time of N. paspalivorus inside the chamber were reduced in the presence of a conspecific female. Residence of N. paspalivorus inside the chamber was also influenced by the presence of P. bickleyi. Both N. paspalivorus and P. bickleyi preyed upon each other with relatively moderate IGP rates of adult females on larvae but neither species yielded nutritional benefits from IGP in terms of adult survival and oviposition. We discuss the relevance of our findings for a hypothetic combined use of both predators in biological control of A. guerreronis.     

Population dynamics of Aceria guerreronis Keifer (Acari: Eriophyidae) and associated predators on coconut fruits in Northeastern Brazil

Aceria guerreronis Keifer can cause severe damage to coconuts in several countries around the world. Rare studies have been conducted to determine the predatory mites associated with A. guerreronis in Brazil. The study evaluated the prevalence of A. guerreronis and associated predators on the bracts and on the surface of the fruits underneath the bracts, for 12 months, on coconut palms grown along the coast of the States of Alagoas, Paraíba and Pernambuco, Northeastern Brazil. Mites of 10 families were found, but by far the most abundant species was A. guerreronis, corresponding to 99.5% of the mites collected. The prevailing species amongst the predators were the Phytoseiidae mites Neoseiulus baraki (Athias-Henriot) and Neoseiulus paspalivorus (De Leon). These are flat mites that have short limbs, characteristics that allow them to invade the main habitat occupied by A. guerreronis. Other predators were found, but in low numbers, due mainly to their difficulty in reaching the fruit areas most inhabited by A. guerreronis. However, these mites could prey on that pest when it leaves its preferred habitat to disperse. No significant correlations were observed between the levels of abiotic factors and the population levels of A. guerreronis or of the phytoseiids associated with it. This probably occurred due to the simultaneous and conflicting effects of those factors on the mites in the field, or to significant differences between the climatic factors measured in the environment and those prevailing in the habitat occupied by A. guerreronis.     

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.     

Loading of lipophorin particles with phospholipids at the midgut of Rhodnius prolixus

³²P-Labelled midguts (³²P-midguts) of Rhodnius prolixus females were incubated in the presence of nonradioactive purified lipophorin and the release of radioactivity to the medium was analysed. The radioactivity found in the medium was associated with lipophorin phospholipids. When the ³²P-midguts were incubated in the absence of lipophorin, no ³²P-phospholipids were found in the medium. Comparative analysis by thin-layer chromatography of ³²P-phospholipids derived from metabolically labelled ³²P-midgut or lipophorin particles after incubation with ³²P-midgut showed some differences, revealing a possible selectivity in the process of phospholipids transfer. The transfer of phospholipids to lipophorin was linear with time up to 45 min, was saturable with respect to the concentration of lipophorin, and was half-maximal at about 5 mg/ml. The binding of ³²P-lipophorin to the midgut at O°C reached the equilibrium at about 1 h of incubation. The binding of ³²P-lipophorin was inhibited by an excess of nonradioactive lipophorin, which suggests a specific receptor for lipophorin. The capacity of midguts and fat bodies to transfer phospholipids to lipophorin varied during the days following the meal. When lipophorin enzymatically depleted of phospholipids by treatment with phospholipase A² was incubated with ³²P-midguts, the same amount of phospholipids was transferred, indicating a net gain of phospholipids by the particle. © 1995 Wiley-Liss, Inc.