RESUMEN
Bemisia tabaci (Genn.) was considered a secondary pest in Brazil until 1990, despite being an efficient geminivirus vector in beans and soybean. In 1991, a new biotype, known as B. tabaci B biotype (=B. argentifolii) was detected attacking weed plants and causing phytotoxic problems in Cucurbitaceae. Nowadays, B. tabaci is considered one of the most damaging whitefly pests in agricultural systems worldwide that transmits more than 60 different plant viruses. Little is known about the genetic variability of these populations in Brazil. Knowledge of the genetic variation within whitefly populations is necessary for their efficient control and management. The objectives of the present study were to use RAPD markers (1) to estimate the genetic diversity of B. tabaci populations, (2) to study the genetic relationships among B. tabaci biotypes and two other whitefly species and (3) to discriminate between B. tabaci biotypes. A sample of 109 B. tabaci female individuals obtained from 12 populations in Brazil were analyzed and compared to the A biotype from Arizona (USA) and B biotype from California (USA) and Paraguay. Trialeurodes vaporariorum and Aleurodicus cocois samples were also included. A total of 72 markers were generated by five RAPD primers and used in the analysis. All primers produced RAPD patterns that clearly distinguished the Bemisia biotypes and the two other whitefly species. Results also showed that populations of the B biotype have considerable genetic variability. An average Jaccard similarity of 0.73 was observed among the B biotype individuals analyzed. Cluster analysis demonstrated that, in general, Brazilian biotype B individuals are scattered independently in the localities where samples were collected. Nevertheless, some clusters were evident, joining individuals according to the host plants. AMOVA showed that most of the total genetic variation is found within populations (56.70 per cent), but a significant portion of the variation is found between crops (22.73 per cent). The present study showed that the B biotype is disseminated throughout the sampled areas, infesting several host plants and predominates over the A biotype
Asunto(s)
Animales , Brasil , Variación Genética , Insectos , Técnica del ADN Polimorfo Amplificado AleatorioRESUMEN
BACKGROUND: Hydroxyethyl starch (HES) has a known dose-dependant effect on coagulopathy. The purpose of this study was to determine the effect of HES on coagulopathy after a period of hemorrhagic shock. METHODS: Anesthetized swine underwent a 15-minute, 40% blood volume hemorrhage (28 mL/kg) and a 1-hour shock period, followed by resuscitation with sham resuscitation (group I); 6% HES, 15 mL/kg (group II); 5% albumin, 15 mL/kg (group III); lactated Ringer's solution, 39 mL/kg, and 6% HES, 15 mL/kg (group IV); and lactated Ringer's solution, 39 mL/kg, and 5% albumin, 15 mL/kg (group V). Coagulation function was measured by bleeding time, prothrombin time, partial thromboplastin time, fibrinogen, platelet count, and thromboelastography. RESULTS: Platelet counts decreased significantly (p < 0.05) in all resuscitation groups except the sham resuscitation group. A significant decrease in platelets, fibrinogen levels, and maximum amplitude on thromboelastography was related to a dilutional effect of the fluid given and not a result of HES at the dose tested. CONCLUSION: The linear dose-related coagulopathic effects of HES when given at moderate doses does not seem to be worsened by prolonged periods of hemorrhagic shock. The coagulopathy seen during resuscitation from hemorrhagic shock seems to be a dilutional effect.
Asunto(s)
Derivados de Hidroxietil Almidón/farmacología , Resucitación/métodos , Choque Hemorrágico/terapia , Análisis de Varianza , Animales , Coagulación Sanguínea , Modelos Animales de Enfermedad , Relación Dosis-Respuesta a Droga , Femenino , Hemodinámica , Choque Hemorrágico/fisiopatología , PorcinosRESUMEN
Cyclooxygenase-2 (COX-2) is a highly inducible gene in macrophages by pro-inflammatory cytokines. A major mechanism for cytokine-induced COX-2 expression is stabilization of COX-2 mRNA. In this study, we examined the induction of COX-2 expression by interleukin-1 beta (IL-1 beta) and tumor necrosis factor-alpha (TNF-alpha) in human primary in vitro differentiated macrophages. IL-1 beta (5 ng/mL) or TNF-alpha (1 ng/mL) induced up to an approximately 40-fold increase of COX-2 mRNA in macrophages during a 2 to 2.5-hr incubation. Run-off experiments demonstrated that cytokine stimulation had only a mild effect on the COX-2 transcription rate (approximately 10-40% increase). The translation blocker cycloheximide (CHM) (10 mg/mL) superinduced COX-2 mRNA during 2 hr of incubation and further stabilized the COX-2 mRNA (T1/2 > 4 hr). The CHM-superinduced COX-2 mRNA was subject to a rapid degradation after removal of CHM (T1/2 < 1 hr). Both IL-1 beta and TNF-alpha stabilized cytokine-induced COX-2 mRNA (T1/2 > or = 2 hr). Maximal stabilization of COX-2 mRNA after a short-term stimulation required the continued presence of IL-1 beta in the medium. Long-term treatment of TNF-alpha destabilized the induced COX-2 mRNA. Cells simultaneously treated with both IL-1 beta and TNF-alpha had a reduced induction of COX-2, IL-1 beta, and IL-6 mRNA. In transcription-arrested cells, the translation blocker puromycin affected the TNF-alpha-induced stabilization and destabilization of COX-2 mRNA, but not the IL-1 beta-induced stabilization. The studies suggest that positive and negative regulation of mRNA stability may play a major role in cytokine-mediated COX-2 induction in human macrophages. TNF-alpha may play both pro-inflammatory and protective roles during inflammation by regulation of pro-inflammatory gene transcripts.
Asunto(s)
Interleucina-1/farmacología , Isoenzimas/genética , Macrófagos/efectos de los fármacos , Prostaglandina-Endoperóxido Sintasas/genética , Estabilidad del ARN/efectos de los fármacos , Factor de Necrosis Tumoral alfa/farmacología , Diferenciación Celular , Ciclooxigenasa 2 , Sinergismo Farmacológico , Regulación Enzimológica de la Expresión Génica , Humanos , Técnicas In Vitro , Isoenzimas/metabolismo , Lipopolisacáridos/farmacología , Macrófagos/metabolismo , Proteínas de la Membrana , Prostaglandina-Endoperóxido Sintasas/metabolismo , ARN Mensajero/efectos de los fármacos , ARN Mensajero/metabolismo , Activación TranscripcionalRESUMEN
BACKGROUND: Hyperlipidemia inhibits proliferation of endothelial cells (ECs) in culture and angiogenesis in vivo and in arterial explants. Elucidation of the mechanisms may suggest novel therapies against atherosclerosis. METHODS AND RESULTS: Basic fibroblast growth factor (bFGF) expression and mitogenic effects were assessed in bovine aortic ECs incubated with oxidized LDL (ox-LDL). Compared with native LDL and lipoprotein-free controls, ox-LDL reduced bFGF mRNA levels in a time- and concentration-dependent manner, 100 microg/mL producing a maximum reduction of 40% to 50% within 24 to 48 hours. There were commensurate reductions in intracellular and extracellular bFGF concentrations, DNA and total RNA syntheses, and cell replication. FGF receptor 1 and beta-actin mRNA levels were unchanged. Ox-LDL accelerated bFGF mRNA degradation in actinomycin D-treated cells. However, inhibition of bFGF expression by ox-LDL was attenuated by cyclohexamide, indicating a requirement for continuous new protein synthesis for posttranscriptional destabilization. Reduced syntheses of DNA and total RNA were completely restored by bFGF but not by vascular endothelial growth factor. Inhibition of total RNA synthesis achieved by exposing cells to a bFGF-neutralizing antibody was similar in magnitude to that induced by ox-LDL. CONCLUSIONS: Cytotoxic effects of ox-LDL on ECs are attributable in part to suppression of bFGF expression.
Asunto(s)
Endotelio Vascular/citología , Factor 2 de Crecimiento de Fibroblastos/antagonistas & inhibidores , Lipoproteínas LDL/farmacología , Animales , Bovinos , División Celular/efectos de los fármacos , Células Cultivadas , ADN/biosíntesis , Dactinomicina/farmacología , Factores de Crecimiento Endotelial/farmacología , Endotelio Vascular/efectos de los fármacos , Espacio Extracelular/metabolismo , Factor 2 de Crecimiento de Fibroblastos/genética , Factor 2 de Crecimiento de Fibroblastos/metabolismo , Líquido Intracelular/metabolismo , Linfocinas/farmacología , Inhibidores de la Síntesis del Ácido Nucleico/farmacología , Fragmentos de Péptidos/farmacología , ARN/biosíntesis , ARN Mensajero/antagonistas & inhibidores , Proteínas Tirosina Quinasas Receptoras/metabolismo , Receptor Tipo 1 de Factor de Crecimiento de Fibroblastos , Receptores de Factores de Crecimiento de Fibroblastos/metabolismo , Factor A de Crecimiento Endotelial Vascular , Factores de Crecimiento Endotelial VascularRESUMEN
N,N-Dipalmitylglycyl-apolipoprotein E (129-169) peptide (dpGapoE) is an efficient gene delivery system for both plasmids and antisense oligodeoxynucleotides (ODNs). To develop a new and efficient approach to the regulation of cholesteryl ester transfer protein (CETP) expression, we used dpGapoE to transfect phosphorothioate antisense ODNs against nucleotides 329 to 349 of human CETP cDNA into a human CETP-stably transfected Chinese hamster ovary (CHO) cell line (hCETP-CHO). After transfection, translocation to the nuclei and concentration in nuclear structures were observed in >95% of the cells at 6 and 12 hours by fluorescence microscopy. No membrane disruption was observed after transfection of ODNs by dpGapoE. Although the translocation stability of phosphorothioate ODNs in the nuclei continued for >48 hours, it had weakened after 24 hours. Cellular CETP mRNA levels gradually declined, and the maximum reduction in the mRNA level (>50%) was observed at 36 hours, after which the mRNA level started to recover. CETP activity in the culture medium declined over 72 hours. The maximum reduction in CETP activity was observed at 36 hours (53.8% of control). Neither CETP mRNA nor CETP activities changed throughout the experiment after the transfection of sense phosphorothioate ODNs delivered by dpGapoE complex or naked antisense ODNs. We conclude that (1) the novel synthetic dpGapoE was a highly effective and nontoxic vehicle for the nuclear delivery of antisense ODNs into hCETP-CHO cells and (2) antisense ODNs selectively inhibited both CETP expression and activity in an hCETP-CHO cell line. This approach may enable gene regulation in vivo and could possibly be used as an antiatherosclerotic agent to alter high density lipoprotein metabolism.
Asunto(s)
Apolipoproteínas E/farmacología , Proteínas Portadoras/antagonistas & inhibidores , Glicoproteínas , Oligonucleótidos Antisentido/administración & dosificación , Fragmentos de Péptidos/farmacología , Animales , Apolipoproteínas E/genética , Transporte Biológico , Células CHO , Proteínas Portadoras/genética , Proteínas Portadoras/metabolismo , Línea Celular Transformada , Núcleo Celular/metabolismo , Supervivencia Celular/efectos de los fármacos , Proteínas de Transferencia de Ésteres de Colesterol , Cricetinae , Medios de Cultivo/metabolismo , Humanos , Oligonucleótidos Antisentido/metabolismo , Oligonucleótidos Antisentido/farmacología , Fragmentos de Péptidos/genética , ARN Mensajero/metabolismo , TransfecciónRESUMEN
-Macrophages secrete matrix metalloproteinases (MMPs) that may weaken the fibrous cap of atherosclerotic plaque, predisposing its fissuration. The 92-kDa gelatinase B (MMP-9) has been identified in abdominal aortic aneurysms and in atherosclerotic tissues. Fluvastatin, through the inhibition of the isoprenoid pathway, inhibits major processes of atherogenesis in experimental models (smooth muscle cell migration and proliferation and cholesterol accumulation in macrophages). We studied the effect of fluvastatin on the activity of MMP-9 in mouse and human macrophages in culture. Conditioned media of cells treated for 24 hours with fluvastatin were analyzed by gelatin zymography. In mouse macrophages, fluvastatin (5 to 100 micromol/L) significantly inhibited in a dose-dependent manner MMP-9 activity from 20% to 40% versus control. The drug, at a concentration as low as 5 micromol/L, inhibited MMP-9 activity ( approximately 30%) in human monocyte-derived macrophages as well. Phorbol esters (TPA, 50 ng/mL) stimulated MMP-9 activity by 50%, and fluvastatin inhibited this enhanced activity up to 50% in both mouse and human macrophages. The above results on the secretion of MMP-9 were confirmed by Western blotting and ELISA. The inhibitory effect of fluvastatin was overcome by the simultaneous addition of exogenous mevalonate (100 micromol/L), a precursor of isoprenoids. Fluvastatin's effect was fully reversible, and the drug did not cause any cellular toxicity. The statin did not block directly the in vitro activation of the secreted protease. Similar data were obtained with simvastatin. Altogether, our data indicate an inhibition of MMP-9 secretion by the drug. This effect is mediated by the inhibition of synthesis of mevalonate, a precursor of numerous derivatives essential for several cellular functions.
Asunto(s)
Colagenasas/metabolismo , Ácidos Grasos Monoinsaturados/farmacología , Inhibidores de Hidroximetilglutaril-CoA Reductasas/farmacología , Indoles/farmacología , Macrófagos Peritoneales/efectos de los fármacos , Animales , Células Cultivadas , Fluvastatina , Humanos , Immunoblotting , Macrófagos Peritoneales/metabolismo , Metaloproteinasa 9 de la Matriz , Ratones , Tasa de Secreción/efectos de los fármacos , Simvastatina/farmacologíaRESUMEN
Hepatic scavenger receptors (SR) may play a protective role by clearing modified lipoproteins before they target the artery wall. To gain insight into this hypothesized function, transgenic mice expressing hepatic bovine SR (TgSR) were created and studied when fed chow, and during diet-induced hyperlipidemia. SR overexpression resulted in extensive hepatic parenchymal cell uptake of fluorescently labeled acetylated human low density lipoprotein (DiI ac-hLDL) and a twofold increase in 125I-acetylated-LDL clearance. Food intake and cholesterol absorption was indistinguishable between control and TgSR mice. In chow-fed mice, lipoprotein cholesterol was similar in control and TgSR mice. However, on a 3-wk high fat/cholesterol (HFHC) diet, the rise in apoB containing lipoproteins was suppressed in TgSR+/- and TgSR+/+ mice. The rise in HDL was similar in control and TgSR+/- mice, but significantly elevated in the TgSR+/+ mice. Overall, on chow, the ratio of apo-B containing lipoprotein cholesterol to HDL cholesterol was similar for all groups (control = 0.33; TgSR+/- = 0.32; TgSR+/+ = 0.38). However, after 3 wk on the HFHC diet, this ratio was markedly higher in control (2.34 +/- 0.21) than in either TgSR+/- (1.00 +/- 0.24) or TgSR+/+ (1.00 +/- 0.19) mice. In TgSR+/- mice, hepatic cholesteryl esters were reduced by 59%, 7 alpha-hydroxylase mRNA levels were elevated twofold, and a significant increase in fecal bile acid flux was observed after the 3-wk HFHC diet. These results suggest SR may play a protective role in liver by preventing diet-induced increases in apoB containing lipoproteins.
Asunto(s)
Hiperlipoproteinemia Tipo II/prevención & control , Hígado/metabolismo , Proteínas de la Membrana , Receptores Inmunológicos/biosíntesis , Receptores de Lipoproteína , Animales , Secuencia de Bases , Ácidos y Sales Biliares/análisis , Bovinos , Colesterol/metabolismo , Dieta , Heces/química , Hiperlipoproteinemia Tipo II/etiología , Lípidos/sangre , Ratones , Ratones Transgénicos , Datos de Secuencia Molecular , ARN Mensajero/análisis , Receptores Inmunológicos/genética , Receptores de LDL/fisiología , Receptores Depuradores , Receptores Depuradores de Clase B , Esteroles/sangreRESUMEN
BACKGROUND: Severe combined immunodeficient (SCID) mice develop Epstein-Barr virus (EBV) containing human lymphoproliferative disease (LPD) tumors when reconstituted with human peripheral blood leukocytes (PBLs) from EBV-seropositive donors, but LPD tumors do not develop in the presence of immunosuppressive agents, such as cyclosporine A or corticosteroids. METHODS: Therefore, LPD development in SCID mice was used as a model to explore the relationship among B cells, T cells, and EBV in vivo. SCID mice were engrafted with PBLs isolated by leukapheresis from a single EBV-seropositive donor. Purified populations of CD3+ lymphocytes (T cells) or CD19+ lymphocytes (B cells) were isolated and engrafted into SCID mice. RESULTS: SCID mice engrafted with purified CD3+ lymphocytes (T cells) or CD19+ lymphocytes (B cells) did not develop LPD. In contrast, mice engrafted with purified B cells developed LPD if they were co-engrafted with purified T cells or if they were inoculated with infectious EBV. CONCLUSIONS: This study confirms the requirement of T cells or active EBV infection in the development of LPD in animals engrafted with B cells latently infected with EBV. A greater understanding of the cellular and viral interactions leading to transformation and malignancy may allow the development of specific interventional therapies for malignancies in the immunosuppressed host.
Asunto(s)
Transformación Celular Neoplásica/inmunología , Infecciones por Herpesviridae/inmunología , Infecciones por Herpesviridae/virología , Herpesvirus Humano 4 , Trastornos Linfoproliferativos/inmunología , Trastornos Linfoproliferativos/virología , Infecciones Tumorales por Virus/inmunología , Infecciones Tumorales por Virus/virología , Animales , Antígenos CD/inmunología , Antígenos CD19 , Antígenos de Diferenciación de Linfocitos B/inmunología , Linfocitos B/inmunología , Linfocitos B/patología , Linfocitos B/trasplante , Complejo CD3/inmunología , Transformación Celular Neoplásica/patología , Trasplante de Células , Citometría de Flujo , Infecciones por Herpesviridae/patología , Humanos , Inyecciones , Trastornos Linfoproliferativos/patología , Ratones , Ratones SCID , Linfocitos T/inmunología , Linfocitos T/patología , Infecciones Tumorales por Virus/patologíaRESUMEN
ApoE is a 34-kDa apoprotein that mediates lipoprotein binding to the low density lipoprotein (LDL) receptor and to the LDL receptor-related protein. Receptor binding is mediated by a highly basic, alpha-helical sequence of approximately 15 amino acids that interacts with cysteine-rich repeat regions of the receptor. To determine the relationship between the receptor binding and lipid associating properties of apoE, we have synthesized a series of apoE peptides containing all (residues 129-169) or part (residues 139-169, 144-169, and 148-169) of the receptor-binding domain. The lipophilicity of these peptides was increased by modification of their N termini by acylation with either palmitic acid (C16-apoE peptide) or the N,N-distearyl derivative of glycine (diC18-Gly-apoE peptide). The unmodified peptides demonstrated low affinity for lipid surfaces (Kd > 10(-5) M) and moderate alpha-helicity in the presence of lipid (40%) and had no effect on LDL uptake by fibroblasts. N-Palmitoyl peptides had increased affinity for lipid (Kd approximately 10(-6) M) and increased alpha-helicity (55%) in the presence of lipid. The addition of the C16-apoE-(129-169)-peptide to 125I-LDL enhanced its uptake and degradation by fibroblasts 8-10-fold; however, < 50% of the degradation was mediated by the LDL receptor. By contrast, the diC18-Gly-apoE-(129-169)-peptide was essentially nonexchangeable (Kd < or = 10(-9) M) and highly helical (78%) in the presence of lipid. The addition of the diC18-Gly-apoE-(129-169)-peptide to 125I-LDL enhanced the specific uptake and degradation of LDL by both LDL receptor-mediated and non-LDL receptor-mediated mechanisms. Uptake and degradation of methylated LDL containing diC18-Gly-apoE-(129-169) revealed that the lipoprotein-bound peptide is the active agent. In agreement with this finding, a mutant diC18-Gly-apoE peptide (Arg142-->Gln) was much less effective than the wild-type peptide in potentiating binding, uptake, and degradation of 125I-LDL. Complexes of diC18-Gly-apoE-(129-169), apoA-I, and 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine containing four to six copies of the peptide/particle displayed an affinity for the LDL receptor similar to that of apoE-L-alpha-dimyristoylphosphatidylcholine discs containing four copies of apoE.(ABSTRACT TRUNCATED AT 400 WORDS)
Asunto(s)
Apolipoproteínas E/química , Péptidos/metabolismo , Receptores de LDL/metabolismo , Secuencia de Aminoácidos , Apolipoproteínas E/metabolismo , Sitios de Unión , Células Cultivadas , Humanos , Datos de Secuencia MolecularRESUMEN
BACKGROUND: Directed enzyme pro-drug therapy incorporates the delivery of a gene to a cancer cell that will be specifically expressed and will confer sensitivity to a therapeutic agent. Tumor-specific gene expression can be achieved by coupling the promoter for the carcinoembryonic antigen (CEA) to a gene such as herpes simplex virus thymidine kinase (HSV-tk), which phosphorylates ganciclovir to a potent DNA synthesis inhibitor. METHODS: Retroviral vectors were constructed to contain the CEA promoter coupled to HSV-tk (LN-CEA-TK) and were used to transduce the CEA-expressing pancreatic carcinoma cell line BXPC3. Recombinant pancreatic carcinoma cell lines expressing HSV-tk (BXPC3CEA-TK) were then tested for sensitivity to the toxic effects on ganciclovir after engraftment into severe combined immunodeficient mice. Tumors were generated by subcutaneous inoculation of 20 x 10(6) tumor cells consisting of BXPC3 and/or BXPC3CEA-TK cells in ratios of 100:0, 90:10, 50:50, 10:90, and 0:100. After 3 days mice received daily ganciclovir (0.1 mg/kg) or phosphate-buffered saline solution by intraperitoneal injection and were monitored for tumor growth. RESULTS: All severe combined immunodeficient mice inoculated with BXPC3 or BXPC3CEA-TK cells in any proportion developed large pancreatic tumors. As expected, a significant reduction in tumor size was seen in the BXPC3CEA-TK engrafted mice receiving ganciclovir compared with mice receiving phosphate-buffered saline solution or mice engrafted with only BXPC3. In addition, all animals with any fraction of cells expressing HSV-tk exhibited a significant reduction in tumor growth, including animals with only 10% of cells expressing HSV-tk. CONCLUSIONS: These results suggest the potential utility of directed enzyme pro-drug therapy in patients with CEA-expressing pancreatic carcinoma.
Asunto(s)
Ganciclovir/uso terapéutico , Terapia Genética , Neoplasias Pancreáticas/terapia , Profármacos/uso terapéutico , Retroviridae/genética , Timidina Quinasa/genética , Animales , Secuencia de Bases , Antígeno Carcinoembrionario/genética , Línea Celular , Ganciclovir/metabolismo , Humanos , Ratones , Ratones SCID , Datos de Secuencia Molecular , Profármacos/metabolismo , Simplexvirus/enzimologíaAsunto(s)
Arteriosclerosis/sangre , Lipoproteínas LDL/sangre , Receptores de Lipoproteína/metabolismo , Ácidos Siálicos/química , Acetilación , Adulto , Células Cultivadas , Fibroblastos/metabolismo , Humanos , Radioisótopos de Yodo , Lipoproteínas LDL/química , Macrófagos/metabolismo , Masculino , Persona de Mediana Edad , Ácido N-AcetilneuramínicoRESUMEN
We have studied the interaction of LDL and Lp[a] with fibroblasts. Our studies suggest that Lp[a] does not effectively compete with LDL for binding to the LDL receptor, and does not efficiently suppress the activity of the intracellular enzyme HMG-CoA reductase. However, Lp[a-], formed by reduction of the disulfide bond between apo[a] and apoB, behaves much like homologous LDL, whether or not apo[a] is removed from the mixture, and in spite of the fact that one or more apoB disulfides may also have been cleaved. In our studies we also noted that Lp[a] often enhanced binding of 125I-LDL by fibroblasts. Further investigation has suggested that this interaction is time-dependent. Experiments in receptor-negative fibroblasts indicate that the enhancement is not related to the presence of the LDL receptor; however, it is inhibited by the removal of calcium from the medium. The presence of sialic acid at millimolar concentrations in the medium inhibits much of the Lp[a]-enhanced binding of 125I-LDL to the cells. These studies suggest that Lp[] may in some way enhance LDL binding to cells, perhaps via interaction with cell surface glycosaminoglycans or proteoglycans or with collagen.
Asunto(s)
Fibroblastos/metabolismo , Lipoproteína(a)/metabolismo , Lipoproteínas LDL/metabolismo , Apolipoproteínas/metabolismo , Apolipoproteínas B/metabolismo , Apoproteína(a) , Unión Competitiva , Transporte Biológico Activo , Calcio/metabolismo , Células Cultivadas , Humanos , Hidroximetilglutaril-CoA Reductasas/metabolismo , Ácido N-Acetilneuramínico , Unión Proteica , Receptores de LDL/metabolismo , Ácidos Siálicos/metabolismo , Piel/metabolismoRESUMEN
BACKGROUND: Immunocompromised organ transplant recipients have a high incidence of B cell lymphomas (BCL). Severe combined immunodeficient (SCID) mice develop human BCL when engrafted with Epstein-Barr virus (EBV) transformed and immortalized B lymphoblastoid cell lines (BLCL). Because a lack of effective EBV-specific cytotoxic T lymphocytes (EBV-CTL) is thought to lead to lymphoma development, the SCID mouse model was used to determine the relationship between EBV-infected B cells and EBV-specific CTL in BCL development in vivo. METHODS: EBV-CTL were generated by in vitro stimulation of peripheral blood leukocytes with autologous BLCL. CD8+ CTL were isolated from CTL populations by depletion of CD4+ cells. SCID mice were engrafted with BLCL, EBV-CTL were adoptively transferred into engrafted SCID mice either immediately or 7 days after engraftment, and the animals were monitored for the development of BCL. Statistical significance was determined by the log rank test. RESULTS: SCID mice engrafted with BLCL rapidly developed BCL (mean, 20 days). SCID mice engrafted with BLCL and human leukocyte antigen-identical EBV-CTL or CD8+ EBV-CTL had a significant delay in BCL development (p < 0.05), whereas some mice did not develop BCL. In contrast, human leukocyte antigen-nonidentical EBV-CTL did not significantly delay BCL development. CONCLUSIONS: This study showed the role of EBV-CTL in inhibiting the development of BCL. A greater understanding of the cellular and viral interactions leading to B-cell transformation and malignancy may allow the development of specific interventional therapies in patients who have received immunosuppressants.
Asunto(s)
Inmunoterapia Adoptiva , Linfoma de Células B/terapia , Trasplante de Órganos/efectos adversos , Linfocitos T Citotóxicos/inmunología , Adulto , Animales , Linfocitos B/inmunología , Células Madre Hematopoyéticas/inmunología , Herpesvirus Humano 4/genética , Herpesvirus Humano 4/inmunología , Humanos , Activación de Linfocitos , Ratones , Ratones SCIDRESUMEN
25R)-26-Hydroxycholesterol, 25-hydroxycholesterol, and 3 beta-hydroxy-5 alpha-cholest-8(14)-en-15-one, all extraordinarily potent suppressors of sterol synthesis and 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) reductase activity in mammalian cells, have been studied with respect to their effects on the metabolism of low density lipoproteins (LDL) by human hepatocarcinoma (HepG2) cells. The three oxysterols differed markedly in their effects on LDL metabolism, as measured by the combination of cell-associated plus degraded 125I-LDL. The 26-hydroxysterol, at concentrations from 0.1 microM to 75 microM, lowered LDL metabolism. In contrast, the 25-hydroxysterol and the 15-ketosterol, at concentrations from 0.05 microM to 2.5 microM, caused an increase in LDL metabolism. At higher concentrations of these oxysterols, LDL metabolism was suppressed. However, upon increasing the concentration of the 15-ketosterol further to 75 microM, an extraordinary 9-fold increase in LDL metabolism was observed. In contrast to their effects on LDL metabolism, the 25-hydroxysterol and the 15-ketosterol caused simple concentration-dependent decreases in the levels of HMG-CoA reductase activity under the same conditions.
Asunto(s)
Colestenonas/farmacología , Hidroxicolesteroles/farmacología , Hidroximetilglutaril-CoA Reductasas/metabolismo , Lipoproteínas LDL/metabolismo , Carcinoma Hepatocelular , Humanos , Inhibidores de Hidroximetilglutaril-CoA Reductasas , Cinética , Neoplasias Hepáticas , Estructura Molecular , Células Tumorales CultivadasRESUMEN
Human plasma apolipoprotein (apo) D is a glycoprotein with an apparent molecular weight of 29,000 M(r). It is present, mainly, in high density lipoproteins (HDL) and very high density lipoproteins (VHDL). Western blot analysis of HDL and VHDL using rabbit antibodies to human apoD revealed major immunoreactive bands at 29,000 and 38,000 M(r), with minor bands ranging from 50,000 to and 80,000 M(r). Only the 29,000 M(r) band corresponding to apoD remained when the electrophoresis was conducted under reducing conditions, demonstrating that apoD is cross-linked to other proteins via disulfide bonds. The broad pattern of immunoreactivity was also observed under nonreducing conditions when the blood was collected into a solution of sulfhydryl-trapping reagents, or when these reagents were added to the isolated lipoproteins. These results indicated that the disulfide bonds were not the result of disulfide exchange during the experimental procedures. On the basis of amino acid sequencing and reactions to antibodies, the 38,000 M(r) band was identified as an apoD-apoA-II heterodimer. The apoD-apoA-II was also demonstrated in plasma. In both HDL and plasma, the apoD-apoA-II heterodimer constituted the major form of apoD. Disulfide-linked heterodimers of apoD and apoB-100 were also found in low and very low density lipoproteins, and in whole plasma. It is concluded that a fraction of human apoD, like other cysteine-containing apolipoproteins, exists as a disulfide-linked heterodimer with other apolipoproteins in all major human lipoprotein fractions.
Asunto(s)
Apolipoproteínas/sangre , Lipoproteínas/sangre , Secuencia de Aminoácidos , Apolipoproteína A-II/química , Apolipoproteínas/química , Apolipoproteínas/inmunología , Apolipoproteínas D , Reacciones Cruzadas , Disulfuros/química , Humanos , Lipoproteínas/química , Datos de Secuencia Molecular , Peso Molecular , Conformación ProteicaRESUMEN
The functional molecular mass of the macrophage receptor for acetylated low density lipoprotein (Ac-LDL) was determined in membranes by radiation inactivation analysis. Membranes from tumors induced by the mouse macrophage cell line P388D1 were frozen and irradiated with high-energy electrons. Residual binding activity indicated a minimum functional molecular mass of 35,000 Da, considerably smaller than the active 260,000 M(r) protein seen on ligand blots under nonreducing conditions. Scatchard analysis of receptor binding gave no evidence of partially inactivated molecules. The receptor protein, purified by affinity chromatography and preparative gel electrophoresis, was incubated with dithiothreitol (0.1-100 mM) and retested for binding activity. Active subunits of 158,000 and 80,000 M(r) could be demonstrated by ligand blotting, with quantitative conversion of binding activity to the 80,000 M(r) species at 10 mM dithiothreitol. At 100 mM dithiothreitol, all binding activity was lost. Further size reduction was not detected by silver staining. These data suggest that the isolated mouse macrophage Ac-LDL receptor is a trimer with one class of SH groups involved in trimerization and another in the actual binding site. The monomeric species is fully active in vitro under mild reducing conditions. The radiation inactivation data also suggest that each monomeric unit is fully active and capable of functioning independently in the binding of ligands in the membrane.
Asunto(s)
Moléculas de Adhesión Celular , Lipoproteínas LDL/metabolismo , Macrófagos/metabolismo , Receptores de LDL/metabolismo , Animales , Membrana Celular/metabolismo , Cromatografía , Cromatografía de Afinidad , Cromatografía por Intercambio Iónico , Durapatita , Electroforesis en Gel de Poliacrilamida , Humanos , Hidroxiapatitas , Cinética , Lipoproteínas LDL/sangre , Lipoproteínas LDL/aislamiento & purificación , Ratones , Peso Molecular , Receptores de LDL/aislamiento & purificación , Receptores DepuradoresRESUMEN
Low density lipoproteins (LDL) isolated from the plasma of patients with angiographically demonstrable coronary heart disease (CHD) induced accumulation of triglycerides, free cholesterol, and cholesteryl esters in cultured macrophages, smooth muscle cells, and endothelial cells derived from uninvolved intima of human aorta, but not in skin fibroblasts or hepatoma cells. The sialic acid content of LDL from CHD patients was 40-75% lower than that from healthy donors. There was a negative correlation between LDL sialic acid content and the LDL-induced accumulation of total intracellular cholesterol. Neuraminidase treatment of LDL from normal healthy donors produced sialic acid-depleted LDL (Ds-LDL) which was able to stimulate intracellular lipid accumulation. Neuraminidase treatment of LDL from CHD patients further increased its capacity to induce intracellular lipid accumulation. Sialic acid-poor LDL isolated by affinity chromatography of LDL from CHD patients induced a 2- to 4-fold increase of free and esterified cholesterol in human intimal smooth muscle cells. Binding, uptake, and degradation of 125I-labeled Ds-LDL by macrophages and endothelial cells were 1.5- to 2-fold higher than for native LDL. Binding and uptake of Ds-LDL was inhibited 64-93% by the addition of 20-fold excess acetylated LDL (Ac-LDL); in the inverse experiment, the level of inhibition was 35-54%. These data indicate that a sialic acid-poor form of LDL isolated from CHD patients can interact with both native and scavenger LDL receptors. A sialic acid-poor form of LDL may be a naturally occurring ligand that interacts with the scavenger receptor(s) on macrophages and endothelial cells.
Asunto(s)
Lípidos/biosíntesis , Lipoproteínas LDL/metabolismo , Receptores de LDL/metabolismo , Ácidos Siálicos/metabolismo , Adulto , Células Cultivadas , Enfermedad Coronaria/metabolismo , Humanos , Líquido Intracelular/metabolismo , Lipoproteínas LDL/química , Lipoproteínas LDL/efectos de los fármacos , Masculino , Persona de Mediana Edad , Ácido N-AcetilneuramínicoRESUMEN
Reduced hepatic membrane receptors for acetylated low-density lipoprotein (acetyl-LDL) and maleylated BSA (Mal-BSA) with apparent molecular masses of 35 kDa, 85 kDa and 15 kDa have been extracted from rat liver and separated by affinity chromatography as described by us previously [Ottnad, Via, Sinn, Freidrich, Ziegler & Dresel (1990) Biochem. J. 265, 689-698]. Binding of these three reduced scavenger receptors to oxidatively modified LDL has been now examined. Competition studies with receptor-phosphatidylcholine complexes and 131I-acetyl-LDL and 131I-Mal-BSA as ligands were conducted. Mal-BSA, acetyl-LDL and fully oxidized LDL were used as competitors, and differentiated in the three receptors three types of binding site: a class I binding site for acetyl-LDL, Mal-BSA and fully oxidized LDL; a class II binding site recognizing only 131I-Mal-BSA and class III binding sites recognizing 131I-Mal-BSA and fully oxidized LDL. The results of competition studies with mildly oxidized LDL and polyadenylic acid demonstrated that the binding sites might be even more heterogenous. Thus there is evidence that the reconstituted receptors either have several binding sites for each of the various ligands or are functionally different, despite the fact that they do not differ in their apparent molecular masses.
Asunto(s)
Lipoproteínas LDL/metabolismo , Hígado/metabolismo , Receptores de LDL/metabolismo , Albúmina Sérica Bovina , Albúminas/metabolismo , Animales , Sitios de Unión , Unión Competitiva , Membrana Celular/metabolismo , Humanos , Masculino , Peso Molecular , Oxidación-Reducción , Fosfatidilcolinas/metabolismo , Ratas , Receptores de LDL/química , Receptores de LDL/aislamiento & purificaciónRESUMEN
Oxidation of low density lipoprotein (LDL) causes changes in the biological properties of LDL that may be important in atherogenesis. That LDL oxidation is accompanied by lipid peroxidation has been demonstrated, but previous analyses of the products of LDL oxidation have not included measurement of specific lipid hydroperoxy and hydroxy derivatives. In this study, LDL was isolated from plasma of normal volunteers and exposed to oxygenated buffer and 5 microM CuSO4 for 24 h. Oxidized LDL showed decreased linoleate (18:2) and arachidonate (20:4) content with increased concentrations of thiobarbituric acid reactive substances (TBARS) and hydroxy and hydroperoxy 18:2 and 20:4. The electrophoretic mobility of the LDL protein also was increased by oxidation. After reduction, the hydroxy fatty acids were characterized by gas chromatography-mass spectrometric analysis of the trimethylsilyl ether methyl ester derivatives. The hydroperoxy and hydroxy derivatives accounted for approximately 70% of the linoleate consumed during LDL oxidation and represented 45-fold more product than was measured by TBARS analysis. Numerous biological properties, including cytotoxic and chemoattractant activities of hydroperoxy and hydroxy fatty acids, have been reported, but the manner in which they may contribute to atherogenesis requires further study.
Asunto(s)
Ácidos Araquidónicos/metabolismo , Ácidos Linoleicos/metabolismo , Peroxidación de Lípido , Lipoproteínas LDL/metabolismo , Catálisis , Cobre , HumanosRESUMEN
The binding characteristics of reduced hepatic membrane proteins for acetylated low-density lipoprotein (acetyl-LDL) and maleylated bovine serum albumin (Mal-BSA) have been examined. Two receptor activities were extracted from hepatic membranes in the presence of octyl beta-D-glucoside and beta-mercaptoethanol, and were separated by chromatography on Mal-BSA-Sepharose 4B. The receptors were revealed by ligand blotting. The active binding proteins had apparent molecular masses of 35 and 15 kDa in SDS/polyacrylamide gels. Equilibrium studies with protein-phosphatidylcholine complexes indicated that the reduced 35 kDa protein expresses two binding sites for Mal-BSA and one for acetyl-LDL, whereas the 15 kDa protein-phosphatidylcholine complex binds 131I-Mal-BSA and 131I-acetyl-LDL with a 4:1 stoichiometry. 131I-Mal-BSA binding was linear with both proteins, with a Kd of 4.8 nM at the 35 kDa protein and a Kd of 5.6 nM at the 15 kDa protein. The 35 kDa protein displayed saturable binding of 131I-acetyl-LDL with a Kd of 5 nM; the 15 kDa binding protein bound 131I-acetyl-LDL with a Kd of 2.3 nM. A 85 kDa protein was obtained by Mal-BSA-Sepharose chromatography when the hepatic membranes had been solubilized with Triton X-100 in presence of GSH/GSSG. This protein displayed saturable 131I-Mal-BSA binding with a Kd of 30 nM and 131I-acetyl-LDL binding with a Kd of 6.5 nM. The 131I-Mal-BSA binding capacity was four times higher than that of 131I-acetyl-LDL. Competition studies with the 35 kDa, 15 kDa and 85 kDa proteins binding Mal-BSA, acetyl-LDL, formylated albumin and polyanionic competitors provide evidence for the existence of more than one class of binding sites at the reduced binding proteins.