Purification, partial characterization and role in lipid transport to developing oocytes of a novel lipophorin from the cowpea weevil, Callosobruchus maculatus

Lipid transport in arthropods is achieved by highly specialized lipoproteins, which resemble those described in vertebrate blood. Here we describe purification and characterization of the lipid-apolipoprotein complex, lipophorin (Lp), from adults and larvae of the cowpea weevil Callosobruchus maculatus. We also describe the Lp-mediated lipid transfer to developing oocytes. Lps were isolated from homogenates of C. maculatus larvae and adults by potassio bromide gradient and characterized with respect to physicochemical properties and lipid content. The weevil Lp (465 kDa) and larval Lp (585 kDa), with hydrated densities of 1.22 and 1.14 g/mL, contained 34 and 56 percent lipids and 9 and 7 percent carbohydrates, respectively. In both Lps, mannose was the predominant monosaccharide detected by paper chromatography. SDS-PAGE revealed two apolipoproteins in each Lp with molecular masses of 225 kDa (apolipoprotein-I) and 79 kDa (apolipoprotein-II). The lipids were extracted and analyzed by thin-layer chromatography. The major phospholipids found were phosphatidylserine, phosphatidylcholine and phosphatidylethanolamine in adult Lp, and phosphatidylcholine, phosphatidylethanolamine and sphingomyelin in larval Lp. Hydrocarbons, fatty acids and triacylglycerol were the major neutral lipids found in both Lps. Lps labeled in the protein moiety with radioactive iodine (125I-iodine) or in the lipid moiety with fluorescent lipids revealed direct evidence of endocytic uptake of Lps in live oocytes of C. maculatus.

Feulgen staining in malpighian tubules of meliponid bees: a methodological contribution

Whole-mounted Malpighian tubules of larvae from two meliponid bee species fixed in acetic acid-ethanol showed apositive cytoplasmic staining with Schiff reagent when submitted to the Feulgen reaction in which acid hydrolysiswas done in 4 M HCl at room temperature. The ability of various treatments applied before the Feulgen acid hydrolysisto abolish this cytoplasmic staining was examined. The aldehyde groups of phospholipids present in the cytoplasm ofthe Malpighian tubules were blocked or removed by sequential treatment with 5% sodium borohydride and acetonechloroform(1:1, v/v) for 15 min each prior to HCl hydrolysis. This treatment is recommended in order to abolish thecytoplasmic (plasmal) reaction and to allow the reliable quantification of DNA by the Feulgen reaction and thediscrimination of nuclear phenotypes in the Malpighian tubules of meliponid bees.

Obtenção de hemoglobina purificada. Uso de resinas de troca aniônica para a eliminação de fosfolipídeos residuais / Stroma-free hemoglobin: Using anion-exchange chromatography to eliminate residual phospholipids

A hemoglobina humana (Hb)vem sendo usada como ponto de partida para o desenvolvimento de substitutos para o sangue. Antes de ser submetida a reações de modificação química, a Hb precisa ser obtida com alto grau de pureza. Neste trabalho, concentrados de hemácias foram tratados por métodos convencionais para a obtenção de amostras de Hb, sob a forma de carbonil-hemoglobina (HbCO). A técnica de cromatografia de troca iônica foi usada com o objetivo de verificar-se a eficiência de resinas aniônicas na eliminação de fosfolipídeos da parede celular, ainda residuais nas soluções de HbCO. Experimentos foram realizados com duas resinas de troca aniônica, uma fraca e outra forte. Os extratos orgânicos da solução de purificada adicionalmente por meio de cromatografia em resina de troca aniônica forte, AG MP-1, foram analisados por cromatografia líquida de alta pressão (HPLC). Os resultados mostraram que a resina AG MP-1, sob as condições utilizadas, foi capaz de reter grande parte do total de fosfolipídeos pesquisados

Aislamiento de sustancia tensioactiva de pulmones de cerdo: análisis químico y evaluación in vitro e in vivo de la actividad de superficie / Isolation of a pulmonary surfactant in pig lungs. Chemical analysis and in vitro and in vivo evaluation of surface activity

Introducción. Se aisló una sustancia tensioactiva pulmonar (STP) de porcino para su eventual uso en humanos. Se presenta el análisis químico, estudio in vitro de la actividad de superficie y la efectividad in vivo en un modelo animal. Material y métodos. Se obtuvo STP de lavados bronquiales de pulmón porcino mediante centrifugación y solubilidad diferencial. La actividad de superficie se midió con un surfactometro de burbuja pulsátil. Se evaluó in vivo con cobayos adultos deficientes de STP. Resultados. La concentración de proteínas fue menor a 1 mg/100 mg de fosfolípidos. Este extracto mostró: histéresis amplia, Ymin cercana a 0 mN/m, Ymax de 38 mN/m, índice de estabilidad de 1.98. En el cobayo aumentó la PaO2 a más del doble con disminución significativa de la hipercarbia. Conclusiones. El tensioactivo aislado mostró una magnífica actividad de superficie in vitro y mejoró significativamente la hipoxia e hipercarbia en animales sometidos a lavados pulmonares

Free and protein-linked glycoinositolphospholipids in Trypanosoma cruzi

Two glycoinositol phospholipids (GIPL A and GIPL B) have been purified from epimastigotes of Trypanosoma cruzi at the logarithmic phase of growth (2 days). The GIPLs differ mainly in the lipid moiety and are similar to the lipopeptidophosphoglycan (LPPG) previously isolated from epimastigotes at the stationary phase (4-5 days). [3H]-palmitic acid was incorporated into 1-O-hexadecyl-2-O-palmitoylglycerol in GIPL A and into a sphinganine ceramide with palmitic acid and lignoceric acid as the fatty acids in GIPL B. The lipids could be released by incubation with phosphatidylinositol-specific phospholipase C (PI-PLC) or glycosylphosphatidylinositol phospholipase D (GPI-PLD) from rat serum. The oligosaccharides share the common core structure of the glycosylphosphatidilinositol (GPI) membrane anchors. Microheterogeneity was demonstrated, as well as substitution by galactose, which is mainly in the furanose configuration as was previously described for the LPPG. However, methylation analysis indicated that 20 percent of the galactose is present as terminal pyranose units. In infective trypomastigotes, [3H]-palmitic acid was incorporated into the anchor of the Tc-85 glycoprotein. The lipid cleaved by phospholipase C digestion was identified as 1-O-hexadecylglycerol and the main oligosaccharide has the structure of the conserved core of all GPI anchors. [3H]-palmitic acid-labelled Tc-85 released into the culture medium as membrane vesicles showed 80 percent resistance to the action of PI-PLC. However, after mild alkaline hydrolysis, part of the radioactivity was released by the enzyme

Evidence for the presence of ß-D-galactofuranosyl residues (1->3)-linked to a-D-mannopyranose on the cell membrane in all developmental stages of Trypanosoma cruzi

The lipopeptidophosphoglycan (LPPG) component isolated from epimastigote forms of Trypanosoma cruzi has antigenic properties. Using a rabbit antiserum aginst LPPG, specific for ß-D-galactofuranosyl residues (1-3)-linked to alfa-Dmannopyranose, we identified, by indirect immunofluorescence, the presence of epitopes containing ß-D-galactofuranosyl structures on the cell membrane of epimastigote, amastigote and trypomastigote forms of the parasite. The results demonstrade that all developmental stages of T. cruzi contain similar antigenic determinants on their cell body and flagellar membrane