RESUMEN
This paper describes the discovery of alpha-trifluoroketoacetamides as potent antibacterial agents against Gram-positive organisms. The initial SAR indicates that the aryl ethyl side chain is essential in maintaining antibacterial activity. The SAR observations have been utilized to design a bioisostere for the alpha-trifluoroketoacetamide with good activity against Gram-positive organisms.
Asunto(s)
Acetamidas/farmacología , Antibacterianos/farmacología , Fluoroacetatos , Bacterias Grampositivas/efectos de los fármacos , Acetamidas/síntesis química , Antibacterianos/síntesis química , Bacterias Gramnegativas/efectos de los fármacos , Pruebas de Sensibilidad Microbiana , Estructura Molecular , Relación Estructura-Actividad , Ácido Trifluoroacético/químicaRESUMEN
Important resistance patterns in Gram-negative pathogens include active efflux of antibiotics out of the cell via a cellular pump and decreased membrane permeability. A 3-arylpiperidine derivative (1) has been identified by high-throughput assay as a potentiator with an IC(50) approximately 90 microM. This report details the evaluation of the tether length, aryl substitution and the importance of the fluorine on antibiotic accumulation. Evaluation of various tether lengths demonstrated that the two-carbon tethered analogues are optimal. Removal of the fluorine has a modest effect on antibiotic accumulation and the defluorinated analogue 17 is equally potent to the original lead 1.
Asunto(s)
Antibacterianos/farmacología , Escherichia coli/efectos de los fármacos , Bacterias Gramnegativas/efectos de los fármacos , Piperidinas/farmacología , Staphylococcus aureus/efectos de los fármacos , Antibacterianos/síntesis química , Transporte Biológico Activo/fisiología , Resistencia a Medicamentos , Sinergismo Farmacológico , Flúor/química , Bacterias Gramnegativas/patogenicidad , Concentración 50 Inhibidora , Pruebas de Sensibilidad Microbiana/normas , Permeabilidad , Piperidinas/síntesis químicaRESUMEN
The promoter for the Staphylococcus aureus capsule polysaccharide synthesis gene (cap1A) was cloned in front of the firefly luciferase gene for use in a cell extract S. aureus transcription-translation system. The assay is rapid, reproducible, and sensitive and has a lower background level than the radiolabeled amino acid incorporation translation assays. We present data evaluating a transcription inhibitor and a number of protein translation inhibitors in this system.
Asunto(s)
Antibacterianos/farmacología , Genes Reporteros , Luciferasas/genética , Biosíntesis de Proteínas/efectos de los fármacos , Staphylococcus aureus/efectos de los fármacos , Transcripción Genética/efectos de los fármacos , Clonación Molecular , Evaluación Preclínica de Medicamentos/métodos , Pruebas de Sensibilidad Microbiana , Plásmidos , Staphylococcus aureus/genéticaRESUMEN
BACKGROUND: Previous experiments have demonstrated that acute exposure to a high-cholesterol diet (HCD) increases the severity of myocardial infarction in animals. Recent results suggest that the process is modulated by multiple genes and their interactions with circulating cholesterol. DESIGN: In the present study cholesteryl-ester-transfer-protein (CETP) transgenic mice were generated and fed a normal rodent-chow diet, HCD for 1 week, or a HCD for 6 weeks in order to define the role of CETP in myocardial infarction after acute exposure to a HCD. METHODS: Cholesterol levels in mice of all groups were measured. Separate groups of mice were exposed to 30 min of in-vivo occlusion of coronary artery and 2 h of reperfusion. We assessed the sizes of the ischemic zone and infarct using Evans blue and 2,3,5-triphenyltetrazolium chloride. RESULTS: The extent of infarction (percentage infarct/area at risk) was significantly less (P < 0.05) after 1 week of a HCD (18.7 +/- 7.0%) than those for the normal diet group (51.4 +/- 5.5%) and the group fed a HCD for 6 weeks (44.4 +/- 5.2%). Additionally, there was significantly less infiltration of neutrophils into the ischemic-reperfused mouse hearts for mice fed a HCD for 1 week. Levels of reduced and oxidized glutathione in the hearts of CETP mice were measured for separate groups of animals. The reduced:oxidized-glutathione ratio was significantly (P < 0.01) lower for mice fed a HCD for 1 week (1.5 +/- 0.1) than it was for mice fed a normal diet (3.6 +/- 0.3) and a HCD for 6 weeks (3.3 +/- 0.2). CONCLUSIONS: These data suggest that activity of CETP in hypercholesterolemic mice has an acute effect on size of infarct after 1 week of a HCD. This suggests that CETP induces tolerance of ischemia in the mice fed a HCD via mild oxidative stress.
Asunto(s)
Proteínas Portadoras/fisiología , Glicoproteínas/fisiología , Infarto del Miocardio/fisiopatología , Daño por Reperfusión Miocárdica/fisiopatología , Animales , Proteínas de Transferencia de Ésteres de Colesterol , Colesterol en la Dieta/administración & dosificación , Hemodinámica , Hipercolesterolemia/fisiopatología , Masculino , Ratones , Ratones TransgénicosRESUMEN
Plasma levels of tissue plasminogen activator (tPA) increase rapidly in response to specific vasoactive agents, trauma, and neural stimulation. This response has been attributed to acute release of tPA from stored pools within the vascular endothelium and from catecholamine storage vesicles of chromaffin cells. We have tested directly whether tPA can be sorted into the regulated secretory pathway using the murine pituitary-derived neuroendocrine cell line AtT-20 transfected with tPA cDNA. Clones of AtT-20 cells expressing tPA were isolated, and targeting of tPA into the regulated secretory pathway was demonstrated by (1) stimulation of tPA secretion with 8-bromo-cAMP, the secretagogue which promotes the release of dense granule contents; (2) colocalization with ACTH, an endogenous protein that is stored in dense core granules; and (3) retention of newly synthesized tPA in the cell for prolonged periods of time. Laser scanning confocal microscopy analysis of cells immunostained with antibodies to tPA and ACTH showed colocalization at the tips of the neuritic processes under the cytoplasmic membrane, a region where dense granules are known to migrate after maturation. Treatment of the cells with 5 mM 8-bromo-cAMP for 30 min resulted in a 2.41+/-0.36-fold increase in tPA secretion. Both the magnitude of the stimulatory effect and the fraction of the intracellular tPA released were the same regardless of the tPA expression level in the various clones. Pulse-chase experiments showed that a portion of newly synthesized tPA is retained in the cell for at least 4 h and is released into the culture medium in response to 8-bromo-cAMP. These studies indicate that tPA, under the appropriate conditions, can be targeted into the regulated secretory pathway and can be stored for later release by cellular stimuli.
Asunto(s)
Adenohipófisis/metabolismo , Señales de Clasificación de Proteína/fisiología , Activador de Tejido Plasminógeno/metabolismo , 8-Bromo Monofosfato de Adenosina Cíclica/farmacología , Hormona Adrenocorticotrópica/metabolismo , Animales , Células CHO , Línea Celular , Células Clonales , Cricetinae , Espacio Extracelular/metabolismo , Inmunohistoquímica , Líquido Intracelular/metabolismo , Ratones , Factores de Tiempo , Activador de Tejido Plasminógeno/genética , TransfecciónRESUMEN
When the Ay gene is expressed in KK mice, the yellow offspring (KKAy mice) become obese, insulin resistant, hyperglycemic, and severely hypertriglyceridemic, yet they maintain extraordinarily high plasma high-density lipoprotein (HDL) levels. Mice lack the ability to redistribute neutral lipids among circulating lipoproteins, a process catalyzed in humans by cholesteryl ester transfer protein (CETP). To test the hypothesis that it is the absence of CETP that allows these hypertriglyceridemic mice to maintain high plasma HDL levels, simian CETP was expressed in the KKAy mouse. The KKAy-CETP mice retained the principal characteristics of KKAy mice except that their plasma HDL levels were reduced (from 159 +/- 25 to 25 +/- 6 mg/dl) and their free apolipoprotein A-I concentrations increased (from 7 +/- 3 to 22 +/- 6 mg/dl). These changes appeared to result from a CETP-induced enrichment of the HDL with triglyceride (from 6 +/- 2 to 60 +/- 18 mol of triglyceride/mol of HDL), an alteration that renders HDL susceptible to destruction by lipases. These data support the premise that CETP-mediated remodeling of the HDL is responsible for the low levels of that lipoprotein that accompany hypertriglyceridemic non-insulin-dependent diabetes mellitus.
Asunto(s)
Proteínas Portadoras/fisiología , Diabetes Mellitus Tipo 2/sangre , Glicoproteínas , Lipoproteínas HDL/sangre , Animales , Apolipoproteína A-I/metabolismo , Glucemia/metabolismo , Proteínas Portadoras/genética , Fenómenos Químicos , Química Física , Proteínas de Transferencia de Ésteres de Colesterol , Femenino , Expresión Génica , Insulina/sangre , Lipasa/sangre , Lipoproteínas HDL/química , Masculino , Ratones , Ratones Endogámicos C57BL , Ratones Mutantes , Ratones Transgénicos , Fosfolípidos/sangre , Triglicéridos/sangreRESUMEN
Oxazolidinone-resistant mutants of Staphylococcus aureus, isolated with a spiral plating technique, had a 16-fold higher MIC (2 versus 32 microg/ml) of eperezolid when compared to the parental sensitive strain. Eperezolid inhibited in vitro protein translation with 50% inhibitory concentrations of 30 microM for the oxazolidinone-sensitive S30 extract and 75 microM for the resistant extract. Experiments mixing various combinations of S100 and crude ribosome preparations from oxazolidinone-sensitive and -resistant S. aureus strains in a transcription-translation assay demonstrated that the resistant determinant resided within the ribosomal fraction. Ribosomes from the oxazolidinone-resistant strain bound less drug than ribosomes from the sensitive strain, indicating that the ribosome is the site of action for the oxazolidinones. These experiments demonstrate that an alteration of the ribosome is responsible for some or all of the oxazolidinone resistance observed in the S. aureus mutant.
Asunto(s)
Acetamidas/farmacología , Antibacterianos/farmacología , Mutación/fisiología , Oxazoles/farmacología , Ribosomas/metabolismo , Staphylococcus aureus/efectos de los fármacos , Sistema Libre de Células , Oxazolidinonas , Biosíntesis de Proteínas , Ribosomas/efectos de los fármacos , Staphylococcus aureus/genética , Transcripción GenéticaRESUMEN
The oxazolidinones are a novel class of antibiotics that act by inhibiting protein synthesis. It as been reported that the drug exerts its primary activity on the initiation phase of translation. In order to study the possibility of direct interaction between the drug and the ribosome, we have developed a binding assay using 14C-labelled eperezolid (PNU-100592; formerly U-100592). Eperezolid binds specifically to the 50S ribosomal subunit of Escherichia coli. The specific binding of eperezolid is dose dependent and is proportional to the ribosome concentrations. Scatchard analysis of the binding data reveals that the dissociation constant (Kd) is about 20 microM. The binding of eperezolid to the ribosome is competitively inhibited by chloramphenicol and lincomycin. However, unlike chloramphenicol and lincomycin, eperezolid does not inhibit the puromycin reaction, indicating that the oxazolidinones have no effect on peptidyl transferase. In addition, whereas lincomycin and, to some extent, chloramphenicol inhibit translation termination, eperezolid has no effect. Therefore, we conclude that the oxazolidinones inhibit protein synthesis by binding to the 50S ribosomal subunit at a site close to the site(s) to which chloramphenicol and lincomycin bind but that the oxazolidinones are mechanistically distinct from these two antibiotics.
Asunto(s)
Acetamidas/metabolismo , Antibacterianos/metabolismo , Cloranfenicol/metabolismo , Lincomicina/metabolismo , Oxazoles/metabolismo , Ribosomas/metabolismo , Escherichia coli/metabolismo , Cinética , Oxazolidinonas , Puromicina/farmacologíaRESUMEN
The oxazolidinones are a new class of synthetic antibiotics with good activity against gram-positive pathogenic bacteria. Experiments with a susceptible Escherichia coli strain, UC6782, demonstrated that in vivo protein synthesis was inhibited by both eperezolid (formerly U-100592) and linezolid (formerly U-100766). Both linezolid and eperezolid were potent inhibitors of cell-free transcription-translation in E. coli, exhibiting 50% inhibitory concentrations (IC50s) of 1.8 and 2.5 microM, respectively. The ability to demonstrate inhibition of in vitro translation directed by phage MS2 RNA was greatly dependent upon the amount of RNA added to the assay. For eperezolid, 128 microg of RNA per ml produced an IC50 of 50 microM whereas a concentration of 32 microg/ml yielded an IC50 of 20 microM. Investigating lower RNA template concentrations in linezolid inhibition experiments revealed that 32 and 8 microg of MS2 phage RNA per ml produced IC50s of 24 and 15 microM, respectively. This phenomenon was shared by the translation initiation inhibitor kasugamycin but not by streptomycin. Neither oxazolidinone inhibited the formation of N-formylmethionyl-tRNA, elongation, or termination reactions of bacterial translation. The oxazolidinones appear to inhibit bacterial translation at the initiation phase of protein synthesis.
Asunto(s)
Acetamidas/farmacología , Antiinfecciosos/farmacología , Oxazoles/farmacología , Oxazolidinonas , Biosíntesis de Proteínas/efectos de los fármacos , Antibacterianos , Proteínas Bacterianas/biosíntesis , Colifagos , Medios de Cultivo , Escherichia coli/efectos de los fármacos , Escherichia coli/genética , Linezolid , Pruebas de Sensibilidad Microbiana , N-Formilmetionina/metabolismo , Poli U/metabolismo , Polirribosomas/efectos de los fármacos , Polirribosomas/metabolismo , Ribosomas/efectos de los fármacos , Ribosomas/metabolismo , Transcripción Genética/efectos de los fármacosRESUMEN
Leukocyte and endothelial cell adhesion molecules (CAMs) are essential for emigration of leukocytes, with the selectins mediating the initial step of leukocyte "rolling" and the beta 2-(CD18) integrins and intercellular adhesion molecule-1 (ICAM-1) being important for firm adhesion and emigration. On the basis of evidence for an inflammatory component in the pathogenesis of atherosclerosis, including increased expression of CAMs, cytokines, and growth factors, we tested the hypothesis that decreased expression of inflammatory CAMs would reduce susceptibility to atherosclerosis. Using C57BL/6 mice fed a high-fat diet, we observed a 50% to 75% reduction in atherosclerotic fatty streaks in mice with homozygous mutations for ICAM-1, P-selectin, CD18, both ICAM-1 and CD18, or both ICAM-1 and P-selectin. In contrast to previous evidence of increased expression of CAMs in atherosclerotic lesions, which does not prove a cause-and-effect relationship, these data indicate directly that the level of expression of CAMs can determine the susceptibility to the formation of atherosclerotic fatty streaks. The results suggest that genetic variation at these loci could influence susceptibility to atherosclerosis and that pharmacological reduction of the expression or function of these CAMs might protect against atherosclerosis.
Asunto(s)
Arteriosclerosis/prevención & control , Molécula 1 de Adhesión Intercelular/metabolismo , Síndrome de Deficiencia de Adhesión del Leucocito/fisiopatología , Alimentación Animal , Animales , Antígenos CD18/genética , Colesterol en la Dieta/administración & dosificación , Grasas de la Dieta/administración & dosificación , Susceptibilidad a Enfermedades , Femenino , Expresión Génica , Homocigoto , Molécula 1 de Adhesión Intercelular/genética , Molécula 1 de Adhesión Intercelular/farmacología , Lípidos/sangre , Ratones , Ratones Endogámicos C57BL , Mutación , Selectina-P/genéticaRESUMEN
High density lipoproteins (HDLs) play a role in two processes that include the amelioration of atheroma formation and the centripetal flow of cholesterol from the extrahepatic organs to the liver. This study tests the hypothesis that the flow of sterol from the peripheral organs to the liver is dependent upon circulating HDL concentrations. Transgenic C57BL/6 mice were used that expressed variable amounts of simian cholesteryl ester-transfer protein (CETP). The rate of centripetal cholesterol flux was quantitated as the sum of the rates of cholesterol synthesis and low density lipoprotein-cholesterol uptake in the extrahepatic tissues. Steady-state concentrations of cholesterol carried in HDL (HDL-C) varied from 59 to 15 mg/dl and those of apolipoprotein AI from 138 to 65 mg/dl between the control mice (CETPc) and those maximally expressing the transfer protein (CETP+). There was no difference in the size of the extrahepatic cholesterol pools in the CETPc and CETP+ animals. Similarly, the rates of cholesterol synthesis (83 and 80 mg/day per kg, respectively) and cholesterol carried in low density lipoprotein uptake (4 and 3 mg/day per kg, respectively) were virtually identical in the two groups. Thus, under circumstances where the steady-state concentration of HDL-C varied 4-fold, the centripetal flux of cholesterol from the peripheral organs to the liver was essentially constant at approximately 87 mg/day per kg. These studies demonstrate that neither the concentration of HDL-C or apolipoprotein AI nor the level of CETP activity dictates the magnitude of centripetal cholesterol flux from the extrahepatic organs to the liver, at least in the mouse.
Asunto(s)
Proteínas Portadoras/metabolismo , HDL-Colesterol/sangre , Colesterol/metabolismo , Glicoproteínas , Hígado/metabolismo , Animales , Apolipoproteína A-I/sangre , Proteínas Portadoras/genética , Proteínas de Transferencia de Ésteres de Colesterol , LDL-Colesterol/sangre , VLDL-Colesterol/sangre , Cinética , Lipoproteínas LDL/metabolismo , Macaca fascicularis , Masculino , Ratones , Ratones Endogámicos C57BL , Ratones Transgénicos , Especificidad de Órganos , Valores de ReferenciaRESUMEN
Two inhibitors of cynomolgus monkey cholesteryl ester transfer protein were evaluated. One, a monoclonal antibody made against purified cynomolgus monkey cholesteryl ester transfer protein, was capable of severely inhibiting triglyceride transfer, but had a variable effect on cholesteryl ester transfer. At low antibody to antigen ratios, there was what appeared to be a stoichiometric inhibition of cholesteryl ester transfer, but at high antibody to antigen ratios the inhibition of cholesteryl ester transfer was completely relieved, even though triglyceride transfer remained blocked. Fab fragments of the antibody had no effect whatsoever on cholesteryl ester transfer, but were capable of completely blocking triglyceride transfer. The other inhibitor, 6-chloromecuric cholesterol, severely inhibited cholesteryl ester transfer with minimal inhibition of triglyceride transfer. When both inhibitors were added to the assay, both cholesteryl ester and triglyceride transfer were inhibited; an indication that the inhibitors did not compete for the same binding site on cholesteryl ester transfer protein. When the antibody was given subcutaneously to cynomolgus monkeys at a dose which inhibited triglyceride transfer in the plasma by more than 90%, there was no detectable effect on the high density lipoprotein (HDL) cholesterol level, but the HDL triglyceride levels decreased from 13 +/- 2 to 1 +/- 0 mol/mol of HDL (mean +/- S.D.); an indication that the antibody uncoupled cholesteryl ester and triglyceride transfer in vivo. The 6-chloromecuric cholesterol could not be evaluated in vivo because it is a potent lecithin:cholesterol acyltransferase inhibitor. The fact that cholesteryl ester transfer can be inhibited without effect on triglyceride transfer and, conversely, that triglyceride transfer can be inhibited without effect on cholesteryl ester transfer indicates that these two lipids are not transferred by a single, non-discriminatory process.
Asunto(s)
Proteínas Portadoras/metabolismo , Glicoproteínas , Metabolismo de los Lípidos , Animales , Sitios de Unión , Transporte Biológico , Células CHO , Proteínas Portadoras/genética , Proteínas de Transferencia de Ésteres de Colesterol , HDL-Colesterol/metabolismo , Cricetinae , Fragmentos Fab de Inmunoglobulinas/metabolismo , Macaca fascicularis , Masculino , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Triglicéridos/sangreRESUMEN
The effect of cage population density on plasma lipids and the development of atherosclerosis was examined in female C57BL/6 mice. Mice were housed at a density of one, two or five animals per cage and fed an atherogenic diet for 28 weeks. Subsequently, the animals were bled, sacrificed, the hearts removed and the extent of fatty lesion development in the aorta examined and quantified. As the population density increased, there was a statistically significant increase in total cholesterol levels, VLDL+LDL cholesterol levels, the VLDL+LDL/HDL ratio and lesion severity. These differences are due to the psychosocial stress associated with living within a confined space with high population density over an extended period of time.
Asunto(s)
Arteriosclerosis/psicología , Dieta Aterogénica , Análisis de Varianza , Animales , Arteriosclerosis/sangre , Colesterol/sangre , HDL-Colesterol/sangre , LDL-Colesterol/sangre , VLDL-Colesterol/sangre , Femenino , Ratones , Ratones Endogámicos C57BL , Densidad de PoblaciónRESUMEN
Tissue-type plasminogen activator (t-PA) is composed of structurally autonomous domains. From the N-terminus of t-PA, a finger-like domain (F), an epidermal growth factor-like domain (G), two kringle domains (K1 and K2) and a serine protease domain (P) can be discerned. The K2 domain of t-PA is known to be involved in lysine binding, fibrin binding and fibrin-dependent plasminogen activation. To study the functional autonomy of the K2 domain in t-PA we constructed, with the aid of a cassette t-PA gene [Rehberg et al. (1989) Protein Engng, 2, 371-377], mutant t-PA genes coding for four molecules (FGK1K2P, FGK2K1P, GK1K2P and GK2K1P) in which the K2 domain was placed in two different positions in t-PA. The DNAs of wild-type t-PA and the t-PA variants were expressed in Chinese hamster ovary cells and the recombinant proteins were purified by affinity chromatography. All molecules were expressed in their single-chain form and could be converted to their two-chain form. With these molecules, lysine binding, fibrin binding and fibrin-dependent plasminogen activation were studied. All variants showed affinity for lysyl-Sepharose and aminohexyl-Sepharose. Reversal of the K domains (FGK2K1P versus FGK2K1P and GK1K2P versus GK2K1P) resulted in a 23-47% weaker interaction to both lysyl-Sepharose and aminohexyl-Sepharose. Deleting the F domain (FGK1K2P versus GK1K2P and FGK2K1P versus GK2K1P) resulted in a 20-70% improvement of the interactions lysyl-Sepharose and aminohexyl-Sepharose.(ABSTRACT TRUNCATED AT 250 WORDS)
Asunto(s)
Kringles/genética , Plasminógeno/metabolismo , Activador de Tejido Plasminógeno/metabolismo , Animales , Células CHO , Cricetinae , Electroforesis en Gel de Poliacrilamida , Activación Enzimática , Fibrina/metabolismo , Fibrinógeno/metabolismo , Mutagénesis Sitio-Dirigida , Unión Proteica , Conformación Proteica , Proteínas Recombinantes/metabolismo , Sefarosa/análogos & derivados , Sefarosa/metabolismo , Relación Estructura-Actividad , Activador de Tejido Plasminógeno/genética , TransfecciónRESUMEN
A substantial percentage of people who develop coronary artery atherosclerosis have plasma cholesterol levels in the "desirable" range. The principal lipid abnormality in most of these individuals is a low plasma high-density lipoprotein (HDL) level (HDL cholesterol levels of 35 mg/dL or less). As a result, low HDL levels are not only recognized as a risk factor for the disease, but are considered the single best predictor of an individual's likelihood of developing coronary heart disease. Yet we are only now beginning to understand what regulates plasma HDL levels and why they are low in some individuals. Cholesteryl ester transfer protein (CETP), a plasma protein that shuttles neutral lipids (cholesteryl esters and triglycerides) back and forth between lipoproteins in the circulation, appears to play a key role in HDL metabolism, and recent studies using transgenic mice expressing that protein have broadened our understanding of the metabolic pathways that control plasma HDL levels. In this article, we review some of the key observations regarding CETP's role in HDL metabolism, with special emphasis on the discoveries made using transgenic mice, and we discuss these observations in the context of a model linking plasma triglyceride metabolism with low HDL levels.
RESUMEN
Expression of cynomolgus monkey cholesteryl ester transfer protein (CETP) in C57BL/6 mice has been shown to have a profound effect on the lipoprotein profile in those animals. The objective of this study was to examine the effect of CETP expression on the hepatic lipids of the CETP transgenic mice. The triglyceride, cholesterol and phospholipid composition of livers from 6- and 12-month-old transgenic mice were evaluated and compared with those of age-matched C57BL/6 mice. Statistical analysis indicated that fatty liver was more severe in CETP transgenic mice than C57BL/6 controls (p < 0.01); progressed with age (p < 0.01); and developed more rapidly in males than females (p < 0.01). The lipid that accumulated was triglyceride. These data indicate that CETP expression accelerates the development of fatty liver in the C57BL/6 mouse and raise the possibility that CETP may also contribute to the process of hepatic steatosis in man.
Asunto(s)
Proteínas Portadoras/biosíntesis , Hígado Graso/fisiopatología , Glicoproteínas , Hígado/metabolismo , Envejecimiento/metabolismo , Análisis de Varianza , Animales , Colesterol/metabolismo , Proteínas de Transferencia de Ésteres de Colesterol , Ésteres del Colesterol/metabolismo , Hígado Graso/patología , Femenino , Hígado/patología , Macaca fascicularis , Masculino , Ratones , Ratones Endogámicos C57BL , Ratones Transgénicos , Fosfolípidos/metabolismo , Factores Sexuales , Triglicéridos/metabolismoRESUMEN
Despite the definite etiologic link between apolipoprotein (apo) E mutations and type III hyperlipoproteinemia (HLP), it is not clear what additional factors are involved in the development of florid hyperlipidemia and how to explain the wide variability in the expression of the hyperlipidemic phenotype in carriers of receptor binding-defective apoE variants. The present study was designed to determine whether the overexpression of cholesteryl ester transfer protein (CETP), a plasma protein that transfers cholesteryl esters from the high density lipoproteins (HDL) to the very low density lipoproteins (VLDL) and whose activity is increased in hyperlipidemic states, plays a role in the development of hyperlipidemia and beta-VLDL accumulation in type III HLP. We produced double-transgenic mice that co-expressed high levels of simian CETP and either high or low levels of a human receptor binding-defective apoE variant, apoE(Cys-142). We previously reported that apoE(Cys-142) high-expresser mice showed spontaneous hyperlipidemia and accumulation of beta-VLDL, whereas the low-expresser mice showed only a modest increase in VLDL cholesterol. Co-expression of CETP induced a massive transfer of cholesteryl esters from the HDL to the VLDL in both lines of double-transgenic mice. As a result, HDL cholesterol and apoA-I levels were reduced to about 50% of normal, VLDL cholesterol increased 2.5-fold, and the cholesteryl ester content of VLDL reached values similar to those observed in human beta-VLDL. The ratio of defective to normal apoE in VLDL was unaffected by CETP co-expression and was higher in animals expressing high apoE levels. Finally, in spite of an increased accumulation of beta-VLDL in the high-expresser mice, the VLDL of the low-expresser mice maintained pre-beta mobility upon co-expression of CETP. The results of this study demonstrate that the ratio of defective to normal apoE on the VLDL, rather than the cholesteryl ester content of VLDL, is the major factor determining the development of severe hyperlipidemia and the formation and accumulation of beta-VLDL in type III HLP.
Asunto(s)
Apolipoproteínas E/genética , Proteínas Portadoras/genética , HDL-Colesterol/sangre , VLDL-Colesterol/sangre , Glicoproteínas , Hiperlipoproteinemias/etiología , Animales , Apolipoproteínas E/metabolismo , Proteínas Portadoras/metabolismo , Proteínas de Transferencia de Ésteres de Colesterol , Humanos , Hiperlipoproteinemias/sangre , Ratones , Ratones Endogámicos C57BL , Ratones Transgénicos , Triglicéridos/sangreRESUMEN
Human cholesteryl ester transfer protein was purified from lipoprotein-depleted serum or plasma in a three-step procedure utilizing commercially available triazine dyes immobilized on agarose. The method used consisted of successive chromatography steps on Reactive Red 120 agarose (Procion Red H-E3B, Cibachron Brilliant Red 4G-E), CM Sepharose, and Reactive Yellow 86 agarose (Procion Yellow M-8G). Upon analysis by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, the resultant protein preparation displayed two bands of variable intensity. The two components had apparent molecular weights of approximately 64,000 and approximately 65,000, respectively. Both bands reacted strongly to a monoclonal antibody directed against an epitope consisting of the last eight amino acids at the carboxy terminus of human CETP. Yields of cholesteryl ester transfer activity are 10-40% of the activity present in lipoprotein-depleted serum. The activity is approximately 50,000- to 100,000-fold purified relative to the starting material.
Asunto(s)
Proteínas Portadoras/aislamiento & purificación , Glicoproteínas , Secuencia de Aminoácidos , Proteínas Portadoras/sangre , Proteínas Portadoras/química , Proteínas de Transferencia de Ésteres de Colesterol , Ésteres del Colesterol/sangre , Cromatografía de Afinidad/métodos , Cromatografía por Intercambio Iónico/métodos , Colorantes , ADN Complementario/metabolismo , Electroforesis en Gel de Poliacrilamida/métodos , Humanos , Immunoblotting , Lipoproteínas/sangre , Lipoproteínas/aislamiento & purificación , Datos de Secuencia Molecular , TriazinasRESUMEN
Expression of simian cholesteryl ester transfer protein (CETP) in C57BL/6 mice causes the animals' high density lipoprotein (HDL) levels to decrease. The purpose of these studies was to determine how CETP expression caused that reduction. Chemical analysis showed that the HDL of the CETP transgenic mice had about twice as much triglyceride and only about 60% as much cholesteryl ester as the HDL from the C57BL/6 mice. Both strains of mouse had high levels of a circulating lipase. When plasma from the mice was incubated at 37 degrees C for 5 h, the triglycerides in the HDL were hydrolyzed, and apoA-I was shed from the particle. However, apoA-I was shed from the CETP HDL more rapidly than it was shed from the C57BL/6 HDL. Because "free" apoA-I is rapidly cleared by the kidney, increased production of free apoA-I would be expected to shorten the average life span of apoA-I in the mouse. Kinetic analyses indicated that the life span of apoA-I was significantly reduced in the CETP transgenic mice. It was concluded that CETP expression enriched the core of the HDL with triglyceride, which rendered it vulnerable to lipolysis, causing apoA-I to be shed from the particle. That shortened the life span of apoA-I in the CETP mice, which led to lower plasma levels of the protein.
Asunto(s)
Apolipoproteína A-I/metabolismo , Apolipoproteínas/biosíntesis , Proteínas Portadoras/biosíntesis , Glicoproteínas , Lipoproteínas HDL/sangre , Animales , Apolipoproteína A-I/biosíntesis , Proteínas Portadoras/genética , Colesterol/sangre , Proteínas de Transferencia de Ésteres de Colesterol , Ésteres del Colesterol/sangre , Humanos , Cinética , Macaca fascicularis , Masculino , Ratones , Ratones Endogámicos C57BL , Ratones Endogámicos ICR , Ratones Transgénicos , Fosfolípidos/sangre , Especificidad de la Especie , Factores de Tiempo , Triglicéridos/sangreRESUMEN
Despite the high overall similarity in primary structure between kringle one (K1) and kringle two (K2) of tissue-type plasminogen activator (t-PA) there exists an enormous functional difference. It is thought that, in contrast to K1, K2 mediates lysine binding and fibrin binding and is involved in stimulation of plasminogen activation by fibrin or derivatives as CNBr fragments of fibrinogen. Hypothesizing that sequence differences are responsible for differences in function, we compared the amino acid sequences of K1 and K2 with a consensus kringle sequence. Six consecutive amino acids unique to K2 of t-PA were found, i.e. from Asn248 to Trp253. To test whether these residues are involved in lysine binding, fibrin binding, and fibrin-dependent plasminogen activation, we constructed a set of t-PA mutant proteins containing only a kringle and the protease (P) domain: K2P, K1P, and k1P. In the latter molecule the original amino acid residues Ala160-Ser165 from K1 were substituted by Asn248-Trp253 from K2. As expected, K2P showed enhanced plasminogen activation in the presence of CNBr fragments of fibrinogen, bound to lysine-Sepharose and to a forming fibrin clot. K1P did not show any of these features. In contrast, k1P could be stimulated by CNBr fragments of fibrinogen and bound to lysine-Sepharose and a forming fibrin clot. These results indicate that at least a part of the functional differences between K1 and K2 of t-PA can be localized to a stretch of 6 amino acid residues from Asn248 to Trp253 present in K2.