Proteomics-based approach for identification and purification of human phosphate binding apolipoprotein from amniotic fluid.

Human amniotic fluid is of both maternal and fetal origin; it protects the fetus and provides the environment for growth and development of the fetus. We used a proteomics-based approach for targeting and purifying human phosphate binding protein, a member of the DING family of proteins from amniotic fluid, using Blue Sepharose CL-6B, DEAE-Sephacel and gel filtration chromatography. The protein had earlier been reported to be serendipitously purified along with PON1 (paraoxonase 1). It was identified using electro-spray-ionization-time-of-flight mass spectrometry and was found to be human phosphate binding protein. Human phosphate binding proteins have been reported to play a role as phosphate scavengers and may have a protective function against phosphate-related disorders, such as atherosclerosis, diabetes and kidney stones.

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.

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% lipids and 9 and 7% 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.

Lipoproteína (a) y otros factores de riesgo en niños chilenos con diabetes mellitus tipo I / Lipoprotein (a) and other risk factors in chilean children with diabetes mellitus type I

Aims- To study serum Lp(a) levels and other metabolic cardiovascular risk factors in children with type I diabetes mellitus (DM) in comparison with sex and age matched nondiabetic children. To determine the influence of diabetes control on serum lipoprotein (a) concentrations. Design- Transversal observational study. Target population: diabetic group: 70 type I DM children with microalbuminuria and no macro-microvascular nor neurological complications, aged from 8 to 15 years; 30 boys, 40 girls. Mean duration of type I DM was 8 ñ 4 years. Non diabetic group: composed by 123 healthy children with no family history of DM, aged from 8 to 15 years, 53 boys, 70 girls. Methods- The lipids profile include: total cholesterol (TC) and triglyceride (TG), cholesterol high-density lipoproteins (C-HDL) cholesterol very-low-density lipoproteins (C-LDL) and cholesterol low-density lipoproteins (C-LDL). ApoAI, APOAII and ApoB, Lp(a) and fructosamine. Results- Fructosamine concentration in diabetic children was 340 ñ 108 uM/1 in 240 ñ 25 uM/I nondiabetic children. Lp(a) serum levels did not significantly differ among both groups 17 ñ 16 mg/dl in diabetics 19 ñ 18 mg/dl in controls. Multivariate analysis showed that in the diabetic children the worsening of metabolic control as reflected by fructosamine, was positively correlated with the increase in total Lp(a) serum concentration. Conclusions- In children aged 8-15 years with uncomplicated IDDM lasting less than 15 years duration (Lp(a) serum levels are positively correlated with the poorest metabolic control

Lipophorin as a lipid carrier in the hemolymph of the horseshoe crab, Limulus polyphemus.

A high-density lipoprotein (HDL) was obtained from the hemolymph of Limulus polyphemus in yields generally less than 30 micrograms/ml by ultracentrifugal flotation. SDS-PAGE revealed two apolipoproteins with masses similar to those of apolipophorins (apoLp-I, 265 +/- 14 kDa; apoLp-II, 89 +/- 6 kDa). Lipid composition was different from both insect lipophorin and crustacean HDL, and showed less diacylglycerols than triacylglycerols (3.8% and 36.2% of total lipids, respectively). Since Limulus polyphemus is closely related to precambrian chelicerates, our results confirm that lipophorin was present early in the evolution of arthropods.

Lipophorin as a lipid carrier in the hemolymph of the horseshoe crab, Limulus polyphemus

A high-density lipoprotein (HDL) was obtained from the hemolymph of Limulus polyphemus in yields generally less than 30 micrograms/ml by ultracentrifugal flotation. SDS-PAGE revealed two apolipoproteins with masses similar to those of apolipophorins (apoLp-I, 265 +/- 14 kDa; apoLp-II, 89 +/- 6 kDa). Lipid composition was different from both insect lipophorin and crustacean HDL, and showed less diacylglycerols than triacylglycerols (3.8 and 36.2 of total lipids, respectively). Since Limulus polyphemus is closely related to precambrian chelicerates, our results confirm that lipophorin was present early in the evolution of arthropods.

Partículas lipoproteicas: concepto, aislamiento e implicancias clínicas / Lipoprotein particles: concept, isolation and clinical significance

La utilización de la cromatografía de afinidad con concanavalina A o de cromatografía de inmunoafinidad con anticuerpos anti apoB permite obtener dos grupos de lipoproteínas: las que contienen apoA y las que contienen apoB. El subfraccionamiento por cromatografía de inmunoafinidad secuencial de las partículas lipoproteicas que contienen apoA permite obtener a su vez tres mayores partículas lipoproteicas: LP-A-I, LP-A-I:A-II y LP-A-II. El subfraccionamiento a través de inmunoprecipitación secuencial o cromatografía de inmunoafinidad secuencial de las partículas lipoproteicas que contienen apoB permite obtener cinco mayores grupos de partículas: LP-B, LP-B:C, LP-B:E, LP-B:C:E y LP-A-II:B. La diferencia entre normo e hiperlipoproteinémicos sería el resultado de cambios cuantitativos (y no cualitativos) de las partículas lipoproteicas. En hipercolesterolémicos se destaca un aumento de LP-B en tanto que en hipertrigliceridémicos aumentan la LP-B-C y LP-B:C:E. Las drogas hipolipemiantes, independientemente de su mecanismo de acción, afectan en diferentes sentidos las concentraciones de las partículas lipoproteicas que contienen apoA y apoB. Bajas concentraciones de LP-A-I y elevadas de LP-B:C y LP-B:C:E se asocian con riesgo aterogénico

Partículas lipoproteicas: concepto, aislamiento e implicancias clínicas / Lipoprotein particles: concept, isolation and clinical significance

La utilización de la cromatografía de afinidad con concanavalina A o de cromatografía de inmunoafinidad con anticuerpos anti apoB permite obtener dos grupos de lipoproteínas: las que contienen apoA y las que contienen apoB. El subfraccionamiento por cromatografía de inmunoafinidad secuencial de las partículas lipoproteicas que contienen apoA permite obtener a su vez tres mayores partículas lipoproteicas: LP-A-I, LP-A-I:A-II y LP-A-II. El subfraccionamiento a través de inmunoprecipitación secuencial o cromatografía de inmunoafinidad secuencial de las partículas lipoproteicas que contienen apoB permite obtener cinco mayores grupos de partículas: LP-B, LP-B:C, LP-B:E, LP-B:C:E y LP-A-II:B. La diferencia entre normo e hiperlipoproteinémicos sería el resultado de cambios cuantitativos (y no cualitativos) de las partículas lipoproteicas. En hipercolesterolémicos se destaca un aumento de LP-B en tanto que en hipertrigliceridémicos aumentan la LP-B-C y LP-B:C:E. Las drogas hipolipemiantes, independientemente de su mecanismo de acción, afectan en diferentes sentidos las concentraciones de las partículas lipoproteicas que contienen apoA y apoB. Bajas concentraciones de LP-A-I y elevadas de LP-B:C y LP-B:C:E se asocian con riesgo aterogénico

[Biochemistry of the development cycle of Triatoma infestans (vinchuca). X. Hemolymph lipoproteins of females]. / Bioquímica del ciclo evolutivo del Triatoma infestans (vinchuca). X. Lipoproteinas hemolinfaticas de hembras.

A lipoprotein of high density (HDL) and three of very high density (VHDL-I, VHDL-f and VHDL-II) were separated by ultracentrifugation from T. infestans female hemolymph. Lipids were mainly transported by HDL, whereas VHDL-II presented the highest protein content. In all the lipoproteins, 1,2 and 1,3-diacylglycerols, triacylglycerols, free fatty acids, cholesterol, cholesterol esters and hydrocarbons, were present. Phosphatidylcholine and phosphatidylethanolamine were also identified. The main lipids were represented by phospholipids, 1,2 and 1,3-diacylglycerols and hydrocarbons. All lipoproteins were delipidated to study the corresponding apolipoproteins. They were examined by SDS-polyacrylamide gel electrophoresis in the presence of 2-mercaptoethanol. Apo-HDL was separated into four intense polypeptide bands of approximate MW 19,000; 45,000; 86,000 and 200,000. Apo-VHDL-I was fractionated into three polypeptide bands--a major one of MW 18,500 and two minor ones of 28,000 and 45,000. The apolipoproteins belonging to VHDL-f that only appeared in females, were separated into 10 bands; the major ones corresponded to MW of 58,000, 86,000 and 160,000. In these apolipoproteins, the specimens of 58,000 and 160,000 showed positive carbohydrate reaction. Yet, in the apo-VHDL-II the most intense protein subunits primarily corresponded to bands of MW 160,000 and 86,000. Apparently, the four lipoproteins would share two polypeptide chains.

Bioquímica del ciclo evolutivo del Triatoma infestans (vinchuca): X. Lipoproteinas hemolinfáticas de hembras / Biochemistry of the evolutive cycle of Triatoma infestans (venchuca): X. Hemolymph lipoproteins of females

En la hemolinfa de Triatoma infestans hembras se separaron por ultracentrifugación una lipoproteína de alta densidad, HDL y tres de muy alta densidad, VHDL-I, VHDL-f y VHDL-II. La proporción de lípidos es mayor en HDL y la de proteínas en VHDL-II. En todas las lipoproteínas se reconoció la presencia de 1,2 y 1,3-diacilgliceroles, triacilgliceroles, ácidos grasos libres, colesterol, ésteres de colesterol e hidrocarburos. Entre los fosfolípidos se reconocieron la fosfatidilcolina y fosfatidiletanolamina. Los lípidos mayoritarios fueron fosfolípidos, 1,2 y 1,3-diacilgliceroles e hidrocarburos. Para estudiar las correspondientes apoliproteínas se deslipidizaron las lipoproteínas. Se examinaron por electroforesis en gel de poliacrilamida con SDS en presencia de 2-mercaptoetanol. Las apo-HDL se separaron en 4 bandas polipeptídicas intensas de PM aproximados 19 000, 45 000, 86 000 y 200 000. Las apo-VHDL-I fraccionaron en 3 bandas polipeptídicas, una mayoritaria de PM 18 500 y dos débiles de 28 000 y 45 000. Las apo-VHDL-f que sólo aparecen en hembras se separaron en 10 bandas. Las mayoritarias correspondieron a los PM 58 000, 86 000 y 160 000. Las bandas de 58 000 y 160 000 presentaron reacción positiva de hidratos de carbono. En las apo-VHDL-II las subunidades proteicas más intensas correspondieron a las bandas de PM 86 000 y 160 000. Aparentemente, estas cuatro lipoproteínas tendrian dos cadenas polipeptídicas comunes

Bioquímica del ciclo evolutivo del Triatoma infestans (vinchuca): X. Lipoproteinas hemolinfáticas de hembras / Biochemistry of the evolutive cycle of Triatoma infestans (venchuca): X. Hemolymph lipoproteins of females

En la hemolinfa de Triatoma infestans hembras se separaron por ultracentrifugación una lipoproteína de alta densidad, HDL y tres de muy alta densidad, VHDL-I, VHDL-f y VHDL-II. La proporción de lípidos es mayor en HDL y la de proteínas en VHDL-II. En todas las lipoproteínas se reconoció la presencia de 1,2 y 1,3-diacilgliceroles, triacilgliceroles, ácidos grasos libres, colesterol, ésteres de colesterol e hidrocarburos. Entre los fosfolípidos se reconocieron la fosfatidilcolina y fosfatidiletanolamina. Los lípidos mayoritarios fueron fosfolípidos, 1,2 y 1,3-diacilgliceroles e hidrocarburos. Para estudiar las correspondientes apoliproteínas se deslipidizaron las lipoproteínas. Se examinaron por electroforesis en gel de poliacrilamida con SDS en presencia de 2-mercaptoetanol. Las apo-HDL se separaron en 4 bandas polipeptídicas intensas de PM aproximados 19 000, 45 000, 86 000 y 200 000. Las apo-VHDL-I fraccionaron en 3 bandas polipeptídicas, una mayoritaria de PM 18 500 y dos débiles de 28 000 y 45 000. Las apo-VHDL-f que sólo aparecen en hembras se separaron en 10 bandas. Las mayoritarias correspondieron a los PM 58 000, 86 000 y 160 000. Las bandas de 58 000 y 160 000 presentaron reacción positiva de hidratos de carbono. En las apo-VHDL-II las subunidades proteicas más intensas correspondieron a las bandas de PM 86 000 y 160 000. Aparentemente, estas cuatro lipoproteínas tendrian dos cadenas polipeptídicas comunes (AU)

Bioquímica del ciclo evolutivo del Triatoma infestans (Vinchuca): VIII. Estudio preliminar de las apolipoproteinas hemolinfáticas de machos adutos / Biochemistry of the evoluation of Triatoma infestans (Vinchuca): VIII. Preliminary study of hemolymph apolipoproteins of adult males

Las tres lipoproteínas de alta (HDL) y muy alta densidad (VHDL-I y VHDL-II) fueron separadas de la hemolinfa de machos adultos de T. infestans. Ellas fueron deslipidizadas y las correspondientes apolipoproteínas se examinaron por electroforesis en gel de dodecil sulfato sodio en poliacrilamida en presencia de 2-mercapto-etanol. Luego, fueron separadas por enfoque isoelétrico en un gradiente de pH de 5 a 8. Los tres grupos de apolipoproteínas fueron separados en varias cadenas polipeptídicas, las que tenían en común una unidad glicoproteica de peso molecular 86 000. Las VHDL-II presentaron la composición más simple, con predominio de una banda de 86 000. La apo-HDL fue la más complicada y mostró bandas intensas con peso molecular tan alto como 210 000 y tan bajo como 17 000, conjuntamente con bandas de 44 000 y 86 000 y otras de menor intensidad. La apo-VHDL-I se separó en cadenas polipeptídicas en el rango de 86 000 a 17 000. Aparentemente, las distintas apoproteínas están formadas por la asociación, al menos en parte, de polipéptidos comunes

Bioquímica del ciclo evolutivo del Triatoma infestans (Vinchuca): VIII. Estudio preliminar de las apolipoproteinas hemolinfáticas de machos adutos / Biochemistry of the evoluation of Triatoma infestans (Vinchuca): VIII. Preliminary study of hemolymph apolipoproteins of adult males

Las tres lipoproteínas de alta (HDL) y muy alta densidad (VHDL-I y VHDL-II) fueron separadas de la hemolinfa de machos adultos de T. infestans. Ellas fueron deslipidizadas y las correspondientes apolipoproteínas se examinaron por electroforesis en gel de dodecil sulfato sodio en poliacrilamida en presencia de 2-mercapto-etanol. Luego, fueron separadas por enfoque isoelétrico en un gradiente de pH de 5 a 8. Los tres grupos de apolipoproteínas fueron separados en varias cadenas polipeptídicas, las que tenían en común una unidad glicoproteica de peso molecular 86 000. Las VHDL-II presentaron la composición más simple, con predominio de una banda de 86 000. La apo-HDL fue la más complicada y mostró bandas intensas con peso molecular tan alto como 210 000 y tan bajo como 17 000, conjuntamente con bandas de 44 000 y 86 000 y otras de menor intensidad. La apo-VHDL-I se separó en cadenas polipeptídicas en el rango de 86 000 a 17 000. Aparentemente, las distintas apoproteínas están formadas por la asociación, al menos en parte, de polipéptidos comunes (AU)

[Biochemistry of the growth cycle of Triatoma infestans (Vinchuca). VIII. Preliminary study of hemo-lymphatic apolipoproteins in the adult male]. / Bioquímica del ciclo evolutivo del Triatoma infestans (Vinchuca). VIII. Estudio preliminar de las apolipoproteinas hemolinfaticas de machos adultos.

The three lipoproteins of high (HDL) and very high density (VHDL-I and VHDL-II) were separated from the hemolymph of adult males of T. infestans. They were delipidated and the corresponding apolipoproteins examined by sodium dodecyl sulphate (SDS) gel electrophoresis in polyacrylamide in the presence of 2-mercaptoethanol. They were also iso-electro focused in a pH gradient of 5 to 8. All three groups of apolipoproteins were separated in several polypeptide chains, but all of them had in common a glycoproteic unit of PM 86 000. The VHDL-II presented the simplest composition with predominance of the 86 000 band. Apo-HDL was the most complicated and showed intense bands of PM as high as 210 000 and as low as 17 000 together with 44 000 and 86 000 and less intense bands. The apo-VHDL-I was separated in polypeptides chains in the range of 86 000 to 17 000. The different apoproteins are apparently formed by the association at least in part of common polypeptides.