RESUMEN
We previously reported that upper thoracic exposure to ionizing radiation (IR) accelerates fatty streak formation in C57BL/6 mice and that such effects are inhibited by overexpression of the antioxidant enzyme CuZn-superoxide dismutase (SOD). Notably, IR-accelerated lesion formation is strictly dependent on a high fat diet (i.e., atherogenic lipoproteins) but does not involve alterations in circulating lipid or lipoprotein levels. We thus proposed that IR promotes changes in the artery wall that enhance the deposition of lipoprotein lipids. To address this hypothesis, we examined the effects of IR on aortic accumulation and degradation of low density lipoproteins (LDL). Ten-week-old C57BL/6 mice were exposed to a single (8-Gy) dose of (60)Co radiation to the upper thoracic area or were sham irradiated (controls) and were then placed on the high fat diet. Five days postexposure, the mice received either (125)I-labeled LDL ((125)I-LDL) (which was used to measure intact LDL) or (125)I-labeled tyramine cellobiose ((125)I-TC)-LDL (which was used to measure both intact and cell-degraded LDL) via tail vein injection. On the basis of trichloroacetic acid (TCA)-precipitable counts in retroorbital blood samples, > or =95% of donor LDL was cleared within 24 h and there were no differences in time-averaged plasma concentrations of the two forms of LDL among irradiated and control mice. Aortic values increased markedly within the first hour and thereafter exhibited a slow increase up to 24 h. There were no differences between irradiated and control mice at 1 h, when values primarily reflected LDL entry, but a divergence was observed thereafter. At 24 h, (125)I-TC-associated counts were 1.8-fold higher in irradiated mice (P = 0.10). In contrast, (125)I-LDL-associated counts were 30% lower in irradiated mice (P< 0.05), suggesting that most of the retained (125)I-TC was associated with LDL degradation products. Consistent with the proposed involvement of oxidative or redox-regulated events, IR-induced LDL degradation was lower in SOD-transgenic than wild-type mice (P<0.05). The importance of LDL oxidation was suggested by observations that IR-induced LDL degradation was significantly reduced by preenriching LDL with alpha-tocopherol. On the basis of these results, we propose that IR elicits SOD-inhibitable changes in the artery wall that enhance LDL oxidation and degradation leading to the deposition of LDL-borne lipids. These studies provide additional support for the role of oxidation in lipoprotein lipid deposition and atherogenesis and suggest that IR promotes an arterial environment that stimulates this process in vivo.
Asunto(s)
Aorta/metabolismo , Aorta/efectos de la radiación , Lipoproteínas LDL/metabolismo , Vitamina E/farmacología , Animales , Arteriosclerosis/prevención & control , Celobiosa/metabolismo , Celobiosa/farmacocinética , Modelos Animales de Enfermedad , Depuradores de Radicales Libres/farmacología , Humanos , Radioisótopos de Yodo , Peroxidación de Lípido/efectos de los fármacos , Peroxidación de Lípido/efectos de la radiación , Lipoproteínas LDL/efectos de los fármacos , Lipoproteínas LDL/farmacocinética , Lipoproteínas LDL/farmacología , Ratones , Ratones Endogámicos C57BL , Ratones Transgénicos , Oxidación-Reducción/efectos de los fármacos , Oxidación-Reducción/efectos de la radiación , Radiación Ionizante , Superóxido Dismutasa/farmacología , Factores de Tiempo , Tiramina/metabolismo , Tiramina/farmacocinéticaRESUMEN
Cryo-electron microscopy was used to analyze the structure of lipoprotein particles in density gradient subfractions of human very low density lipoprotein (VLDL), intermediate density lipoprotein (IDL), and low density lipoprotein (LDL). Lipoproteins from a normolipidemic subject with relatively large and buoyant LDL (pattern A) and from a subject with a predominance of small dense LDL (pattern B) were compared. Projections of VLDL in vitreous ice were heterogeneous in size, but all were circular with a relatively even distribution of contrast. Selected projections of LDL, on the other hand, were circular with a high density ring or rectangular with two high density bands. Both circular and rectangular LDL projections decreased in average size with increasing subfraction density, but were found in all of 10 density gradient subfractions, both in pattern A and in pattern B profiles. Preparations of total IDL contained particles with the structural features of VLDL as well as particles resembling LDL. IDL particles resembling LDL were observed in specific density gradient subfractions in the denser region of the VLDL;-IDL density range. Within the group of IDL particles resembling LDL considerable heterogeneity was observed, but no structural features specific for the pattern A or pattern B lipoprotein profile were recognized. The observed structural heterogeneity of the apolipoprotein B-containing serum lipoproteins may reflect differences in the composition of these particles that may also influence their metabolic and pathologic properties.
Asunto(s)
Apolipoproteínas B/sangre , Apolipoproteínas B/ultraestructura , Lipoproteínas/sangre , Lipoproteínas/ultraestructura , Apolipoproteínas B/aislamiento & purificación , Centrifugación por Gradiente de Densidad , Microscopía por Crioelectrón/métodos , Electroforesis en Gel de Poliacrilamida , Humanos , Lipoproteínas/aislamiento & purificación , Lipoproteínas IDL , Lipoproteínas LDL/sangre , Lipoproteínas LDL/aislamiento & purificación , Lipoproteínas LDL/ultraestructura , Lipoproteínas VLDL/sangre , Lipoproteínas VLDL/aislamiento & purificación , Lipoproteínas VLDL/ultraestructura , Valores de Referencia , SacarosaRESUMEN
Platelet-activating factor acetylhydrolase (PAF-AH) is transported by lipoproteins in plasma and is thought to possess both anti-inflammatory and anti-oxidative activity. It has been reported that PAF-AH is recovered primarily in small, dense LDL and HDL following ultracentrifugal separation of lipoproteins. In the present studies, we aimed to further define the distribution of PAF-AH among lipoprotein fractions and subfractions, and to determine whether these distributions are affected by the lipoprotein isolation strategy (FPLC versus sequential ultracentrifugation) and LDL particle distribution profile. When lipoproteins were isolated by FPLC, the bulk (approximately 85%) of plasma PAF-AH activity was recovered within LDL-containing fractions, whereas with ultracentrifugation, there was a redistribution to HDL (which contained approximately 18% of the activity) and the d>1.21 g/ml fraction (which contained approximately 32%). Notably, re-ultracentrifugation of isolated LDL did not result in any further movement of PAF-AH to higher densities, suggesting the presence of dissociable and nondissociable forms of the enzyme on LDL. Differences were noted in the distribution of PAF-AH activity among LDL subfractions from subjects exhibiting the pattern A (primarily large, buoyant LDL) versus pattern B (primarily small, dense LDL) phenotype. In the latter group, there was a relative depletion of PAF-AH activity in subfractions in the intermediate to dense range (d=1.039-1.047 g/ml) with a corresponding increase in enzyme activity recovered within the d>1.21 g/ml ultracentrifugal fraction. Thus, there appears to be a greater proportion of the dissociable form of PAF-AH in pattern B subjects. In both populations, most of the nondissociable activity was recovered in a minor small, dense LDL subfraction. Based on conjugated dienes as a measure of lipid peroxidation, variations in PAF-AH activity appeared to contribute to variations in oxidative behavior among ultracentrifugally isolated LDL subfractions. The physiologic relevance of PAF-AH dissociability and the minor PAF-AH-enriched oxidation-resistant LDL subpopulation remains to be determined.
Asunto(s)
Lipoproteínas LDL/aislamiento & purificación , Fosfolipasas A/metabolismo , Factor de Activación Plaquetaria/metabolismo , 1-Alquil-2-acetilglicerofosfocolina Esterasa , Adulto , Centrifugación por Gradiente de Densidad , Cromatografía Líquida de Alta Presión/métodos , Femenino , Humanos , Concentración de Iones de Hidrógeno , Lipoproteínas HDL/aislamiento & purificación , Lipoproteínas LDL/sangre , Lipoproteínas LDL/química , Lipoproteínas VLDL/aislamiento & purificación , Masculino , Oxidación-Reducción , Tamaño de la Partícula , Fenotipo , Fosfolipasas A/análisis , Factor de Activación Plaquetaria/análisisRESUMEN
Measurements of electrophoretic mobility and particle size of low density lipoproteins (LDL) allowed use of standard electrokinetic theory to quantitate LDL charge characteristics from subjects with predominance of large LDL (pattern A, n = 9) or small LDL (pattern B, n = 8). Pattern A LDL was found to have significantly lower (P < or = 0.001) mobility (-0.22 +/- 0.01 micron s-1 cm V-1), surface potential (-4.2 +/- 0.3 mV) and charge density (-500 +/- 34 esu/cm2) than pattern B LDL (-0.25 +/- 0.01 micron s-1 cm V-1, -4.9 +/- 0.3 mV, and -580 +/- 30 esu/cm2), but no significant difference in particle valence (-22.0 +/- 1.4 for pattern A vs. -21.8 +/- 1.9 for pattern B). Thus, the greater mobility of pattern B LDL is due to similar net charge residing on a smaller particle. Comparison of subfractions in pattern B relative to pattern A LDL revealed greater surface potential in all pattern B subfractions and greater charge density in fractions of d > or = 1.032 g/ml. In a subset of subjects incubation with neuraminidase produced significant reductions in all LDL charge parameters for all subfractions, but did not abolish the differences between pattern A and B. Thus increased surface potential and charge density of unfractionated pattern B LDL is due both to charge properties of particles across the size and density spectrum as well as enrichment of pattern B LDL with smaller, denser particles that have higher surface charge density.
Asunto(s)
Lipoproteínas LDL/química , Adulto , Anciano , Centrifugación por Gradiente de Densidad , Estudios de Cohortes , Conductividad Eléctrica , Electroforesis en Gel de Agar , Femenino , Humanos , Lipoproteínas LDL/sangre , Lipoproteínas LDL/clasificación , Lipoproteínas LDL/metabolismo , Masculino , Persona de Mediana Edad , Neuraminidasa/metabolismo , Tamaño de la Partícula , Propiedades de SuperficieRESUMEN
The genetic polymorphism of apoB EcoRI and XbaI restriction sites and the 3' VNTR hypervariable region was examined in nine human hepatoma derived liver cell lines and related to the cells' ability to secrete lipids and apoB. EcoRI and XbaI genotypes appeared to be unrelated to triglyceride, cholesterol and apoB accumulating in the medium. The VNTR consisted of alleles with 47 to 67 repeats; however, these repeats were not associated with elevated concentrations of lipid or apoB. Data suggest that in the hepatoma cell lines, apoB polymorphisms in EcoRI, XbaI and the VNTR hypervariable region are not sufficient in themselves to account for triglyceride, cholesterol and apoB in the medium. It is possible that intracellular apoB synthesis and/or degradation as well as postsecretory apoB binding and uptake are responsible for the variability of apoB and lipid accumulation in the culture medium.
Asunto(s)
Apolipoproteínas B/genética , Carcinoma Hepatocelular/genética , Neoplasias Hepáticas/genética , Polimorfismo de Longitud del Fragmento de Restricción , Animales , Secuencia de Bases , Línea Celular , Desoxirribonucleasa EcoRI/metabolismo , Desoxirribonucleasas de Localización Especificada Tipo II/metabolismo , Humanos , Datos de Secuencia Molecular , Secuencias Repetitivas de Ácidos Nucleicos , Células Tumorales CultivadasAsunto(s)
Apolipoproteínas B/metabolismo , Epítopos/metabolismo , Lipoproteínas LDL/metabolismo , Anticuerpos Monoclonales/inmunología , Apolipoproteínas B/inmunología , Western Blotting , Electroforesis en Gel de Poliacrilamida , Ensayo de Inmunoadsorción Enzimática , Epítopos/inmunología , Glicósido Hidrolasas , Humanos , Hidrólisis , Oxidación-Reducción , Especificidad por SustratoRESUMEN
Previous work has shown that low-density lipoproteins (LDL) secreted by hepatoma-derived cell lines have an unusual composition compared to plasma LDL; rather than cholesteryl ester, the hepatoma cell-secreted LDL have a triacylglycerol core. We have found that they also have an increased negative charge, as judged by agarose electrophoresis. Since apolipoprotein B is a glycoprotein containing carbohydrate chains terminated with negatively charged sialic acid residues, we examined whether increased glycosylation of the apolipoprotein B from three hepatoma cell lines (Hep G2, Hep 3B and Huh 7) might account for the differences in LDL charge. The weight percent carbohydrate for Hep G2, Hep 3B and Huh 7 LDL-protein (1.1 +/- 0.2; 1.7 +/- 0.8; 0.4 +/- 0.1) was found to be extremely low compared with the 2.8-9% range we found for plasma LDL-protein, while the amount of LDL-lipid associated carbohydrate from hepatoma LDL was similar to that we found in plasma LDL. Furthermore, desialation of hepatoma cell-secreted LDL with neuraminidase did not normalize the negative charge to that of neuraminidase-treated plasma LDL. Western blots of thrombin proteolytic fragments indicated that, in addition to the T1-T4 fragments seen in plasma apolipoprotein B, apolipoprotein B of hepatoma-derived LDL produced four to five new fragments (T5-T9), suggesting increased exposure of proteolytic sites. Western blotting of the new fragments with antibodies specific for known apolipoprotein B sequences suggests that many of the new cleavage sites cluster in or near the putative LDL receptor recognition site.
Asunto(s)
Apolipoproteínas B/química , Carcinoma Hepatocelular/metabolismo , Lipoproteínas LDL/sangre , Apolipoproteínas B/sangre , Carbohidratos/sangre , Electroforesis en Gel de Poliacrilamida , Endopeptidasas , Glicosilación , Humanos , Hidrólisis , Mapeo Peptídico , Receptores de LDL/metabolismo , Células Tumorales CultivadasRESUMEN
The neutral carbohydrate content of both the protein (apoB) and lipid fractions of low density lipoproteins (LDL) from subjects with a predominance of small, dense LDL (subclass pattern B) was found to be lower than in subjects with larger LDL (subclass pattern A): 45 +/- 12 versus 64 +/- 13 mg/g apoLDL, and 58 +/- 8 versus 71 +/- 8 mg/g apoLDL (P less than 0.0005 for both). Sialic acid content of LDL lipids, but not apoB, was also reduced in subclass pattern B. ApoB and glycolipid carbohydrate content of total LDL and LDL density subfractions declined with increasing LDL density and decreasing particle diameter. Moreover, in LDL subfractions from pattern B subjects, carbohydrate content of LDL apoB, but not LDL glycolipid, was significantly lower in comparison with particles of similar size from pattern A subjects. Thus, in LDL subclass pattern B, reductions in LDL carbohydrate content are associated both with reduced concentrations of larger carbohydrate-enriched LDL subclasses, and with reduced glycosylation of apoB in all LDL particles. LDL glycolipids may vary with overall lipid content of LDL particles, but variation in apoB glycosylation may indicate differences in pathways for LDL production, and reduced apoB glycosylation may reflect the altered metabolic state responsible for LDL subclass pattern B.
Asunto(s)
Carbohidratos/análisis , Lipoproteínas LDL/química , Adulto , Anciano , Colesterol/sangre , Femenino , Humanos , Lipoproteínas LDL/clasificación , Masculino , Persona de Mediana Edad , Ácido N-Acetilneuramínico , Ácidos Siálicos/sangre , Triglicéridos/sangreRESUMEN
Computer programs are presented for automating the collection, processing, and analysis of enzyme-linked immunosorbent assay (ELISA) data. The programs are written to run on an Apple Macintosh computer (equipped with at least 512 kilobytes of random access memory, a single disk drive, and an Apple Image-writer dot matrix printer) interfaced with a Bio-Rad EIA reader. The primary program is written in Pascal with associated data analysis performed by a programmable electronic spreadsheet.
Asunto(s)
Procesamiento Automatizado de Datos/instrumentación , Ensayo de Inmunoadsorción Enzimática/métodos , Programas Informáticos , MicrocomputadoresRESUMEN
Two DNA repair enzyme activities, uracil DNA glycosylase and AP endonuclease, were measured in extracts of T- and B-lymphocytes isolated from mice ranging in age from 3 to 24 months. T- and B-lymphocytes had roughly equal levels of AP endonuclease which did not change appreciably with age. T-lymphocytes had roughly twice as high a level of uracil DNA glycosylase as B-lymphocytes; these levels were not affected by age either. This constancy with age contrasts dramatically with increases in both enzymes--roughly 3-fold on a protein basis or 50-fold on a per cell basis--in a transformed line (MPC-11) derived from a carcinogen-induced lymphocytoma. These results are similar to those obtained with cultured murine fibroblasts, wherein a relative constancy was noted with passage of non-transformed cells, followed by dramatic changes upon transformation (La Belle, M & Linn, S, Mutat res 132 (1984) 51). Hence these enzyme assays do not support the notion of a drop in base excision DNA repair capacity as being a causative factor in aging, but suggest instead that DNA repair properties might differ dramatically in transformed vs non-transformed cells.
Asunto(s)
Linfocitos B/enzimología , ADN Glicosilasas , Reparación del ADN , Endodesoxirribonucleasas/metabolismo , N-Glicosil Hidrolasas/metabolismo , Linfocitos T/enzimología , Envejecimiento , Animales , Linfocitos B/citología , Células Cultivadas , ADN-(Sitio Apurínico o Apirimidínico) Liasa , Desoxirribonucleasa IV (Fago T4-Inducido) , Técnicas In Vitro , Cinética , Ratones , Ratones Endogámicos BALB C , Bazo/citología , Bazo/crecimiento & desarrollo , Linfocitos T/citología , Uracil-ADN GlicosidasaRESUMEN
A study of the apurinic/apyrimidinic (AP) endonuclease activities of a mutant line of CHO cells, EM9, and its parental cell line, AA8, was undertaken to determine if the defective DNA repair exhibited by the mutant cell line after exposure to ethyl methanesulfonate was due to a defective AP endonuclease activity. Phosphocellulose chromatography of cell extracts resolved the AP endonuclease activities of both cell lines into two peaks as seen previously in mouse and human cells. No difference was found between the mutant and parental cell lines in the relative amount of AP endonuclease activity present in the two peaks.
Asunto(s)
Endodesoxirribonucleasas/metabolismo , Metanosulfonato de Etilo/toxicidad , Mutación , Animales , Línea Celular , Cricetinae , Cricetulus , ADN Superhelicoidal , ADN-(Sitio Apurínico o Apirimidínico) Liasa , Desoxirribonucleasa IV (Fago T4-Inducido) , Endodesoxirribonucleasas/aislamiento & purificación , Femenino , Cinética , OvarioRESUMEN
Cultures of mouse cells of various population doubling levels (PDL) were examined for DNA-repair capabilities as estimated by (i) the excision of pyrimidine dimers; (ii) unscheduled DNA synthesis (UDS) in response to UV-irradiation or N-methyl-N'-nitrosoguanidine (MNNG) treatment; (iii) the levels of two DNA-repair enzyme activities, uracil DNA glycosylase and AP endonuclease. The responses to ultraviolet light and MNNG decreased rapidly within the first two PDL and more slowly thereafter until essentially no repair was detected by PDL 12. A continuous cell line which emerged from the cultured cells after a crises period had some restoration of repair capability. The amount of uracil DNA glycosylase activity decreased by approximately 40% before the crises period then decreased by 90% in the continuous cell line. In contrast, the amount of AP endonuclease activity present in the precrises cells showed no significant change until PDL 12, then increased 6-7-fold in the continuous cell line.