RESUMEN

G protein-coupled receptors (GPCRs) are a family of cell membrane receptors that couple and activate heterotrimeric G proteins and their associated intracellular signalling processes after ligand binding. Although the carboxyl terminal of the receptors is essential for this action, it can also serve as a docking site for regulatory proteins such as the ß-arrestins. Prokineticin receptors (PKR1 and PKR2) are a new class of GPCRs that are able to activate different classes of G proteins and form complexes with ß-arrestins after activation by the endogenous agonists PK2. The aim of this work was to define the molecular determinants within PKR2 that are required for ß-arrestin-2 binding and to investigate the role of ß-arrestin-2 in the signalling pathways induced by PKR2 activation. Our data show that PKR2 binds constitutively to ß-arrestin-2 and that this process occurs through the core region of the receptor without being affected by the carboxy-terminal region. Indeed, a PKR2 mutant lacking the carboxy-terminal amino acids retains the ability to bind constitutively to ß-arrestin-2, whereas a mutant lacking the third intracellular loop does not. Overall, our data suggest that the C-terminus of PKR2 is critical for the stability of the ß-arrestin-2-receptor complex in the presence of PK2 ligand. This leads to the ß-arrestin-2 conformational change required to initiate intracellular signalling that ultimately leads to ERK phosphorylation and activation.

Asunto(s)

Unión Proteica , Receptores Acoplados a Proteínas G , Arrestina beta 2 , Arrestina beta 2/metabolismo , Humanos , Células HEK293 , Receptores Acoplados a Proteínas G/metabolismo , Animales , Receptores de Péptidos/metabolismo , Receptores de Péptidos/genética , Transducción de Señal , Sitios de Unión , Fosforilación , Receptores de la Hormona Gastrointestinal/metabolismo , Receptores de la Hormona Gastrointestinal/genética

RESUMEN

OBJECTIVES: The value of alternative autogenous venous conduits for treating critical limb ischaemia (CLI) with infragenicular bypass surgery is well established. In this study, the results of using arm veins as alternative conduits for treating CLI over a 15-year period have been evaluated. METHODS: This was a retrospective study. Between 1991 and 2005. 120 infragenicular bypasses using arm vein conduits (AVCs) were performed in 120 patients. CLI was the main indication (87.5%) for the procedures. The indications for using arm veins were inadequacy or absence of the ipsilateral greater saphenous vein (GSV). Survival, limb salvage, and patency rates were calculated using the Kaplan-Meier method. RESULTS: There was a predominance of male gender (65%), and the group mean age was 68.1 ± 8.3 years. The mean follow-up period was 29.6 ± 26.3 months. The operative mortality (30 days) rate was 7.5%. The main alternative conduit was non-spliced cephalic vein (37.5%). Composite grafts included GSV + AVC (45.2%), AVC + AVC (43.3%) and small saphenous vein + AVC (11.5%). The 5-year primary and secondary patency (SP) rates were 45.2 ± 5.6% and 56.5 ± 5.0%, respectively. The 5-year SP rate was greatest when using non-spliced cephalic vein (65.8 ± 7.6%), but there was no difference in cumulative patency between spliced and non-spliced veins (49.5 ± 8.0% vs. 61.2 ± 6.4%; p = 0.501). The 5-year limb salvage and survival rates were 70.6 ± 5.9% and 59.6 ± 5.8%, respectively. CONCLUSIONS: The favourable long term results of secondary patency and limb salvage rates encourage the use of arm veins as alternative conduits for infragenicular bypass surgery.

Asunto(s)

Isquemia/cirugía , Extremidad Superior/irrigación sanguínea , Injerto Vascular/métodos , Anciano , Enfermedad Crítica , Femenino , Humanos , Isquemia/diagnóstico , Isquemia/mortalidad , Isquemia/fisiopatología , Estimación de Kaplan-Meier , Recuperación del Miembro , Masculino , Persona de Mediana Edad , Estudios Retrospectivos , Factores de Riesgo , Factores de Tiempo , Resultado del Tratamiento , Injerto Vascular/efectos adversos , Injerto Vascular/mortalidad , Grado de Desobstrucción Vascular , Venas/fisiopatología , Venas/trasplante

RESUMEN

OBJECTIVE: To report our experience of long-term results of inframalleolar bypass. DESIGN: Retrospective analysis. MATERIALS AND METHODS: We analysed 122 inframalleolar bypasses performed between January 1991 and June 2005 in 116 patients. Most patients were treated for critical ischaemia (97%). The indication for the use of podalic arteries was a lack of tibial arteries with run-off to the foot. The dorsalis pedis was predominantly used for distal anastomoses (62.3%) and the greater saphenous vein (84.4%) as the conduit. The follow-up periods ranged from 1 to 60 months. The endpoints analysed were graft patency, limb salvage, preservation of deambulation and survival rate. RESULTS: The cumulative patency was 58.2% at 3 years and 53.4% at 5 years. The best results were achieved with the devalvulated greater saphenous veins. Limb salvage was 70.0% at 3 years and 50.4% at 5 years, with preserved deambulation rates of 57.3% and 47.1%, respectively. There were 36 major and 45 minor amputations. At 3 years, the survival rate was 50.2% and the surgical mortality 13%. Female sex was associated with worse results for cumulative patency and limb salvage (P<0.01). CONCLUSIONS: In the long term, inframalleolar bypass is a satisfactory option for limb salvage.

Asunto(s)

Arteriopatías Oclusivas/cirugía , Isquemia/cirugía , Recuperación del Miembro , Extremidad Inferior/irrigación sanguínea , Vena Safena/trasplante , Injerto Vascular , Adulto , Anciano , Anciano de 80 o más Años , Amputación Quirúrgica , Arteriopatías Oclusivas/complicaciones , Arteriopatías Oclusivas/diagnóstico por imagen , Arteriopatías Oclusivas/mortalidad , Arteriopatías Oclusivas/fisiopatología , Arterias/trasplante , Brasil , Femenino , Humanos , Isquemia/diagnóstico por imagen , Isquemia/etiología , Isquemia/mortalidad , Isquemia/fisiopatología , Estimación de Kaplan-Meier , Masculino , Persona de Mediana Edad , Modelos de Riesgos Proporcionales , Radiografía , Reoperación , Estudios Retrospectivos , Medición de Riesgo , Factores de Riesgo , Factores Sexuales , Factores de Tiempo , Resultado del Tratamiento , Injerto Vascular/efectos adversos , Injerto Vascular/mortalidad , Grado de Desobstrucción Vascular

RESUMEN

An ion-chromatographic method combined with electrochemical detection at a copper-based chemically modified glassy carbon electrode (Cu-GC) has been shown to provide a simple analytical approach for the determination of some common organic acids in alkaline medium. Under the optimized isocratic chromatographic conditions (i.e. 0.1 M NaOH plus 80 mM CH3COONa), organic acids such as gallic, ascorbic, gluconic, lactobionic, galacturonic and glucuronic acid could be separated in less than 20 min. Under constant potential amperometric detection (i.e. 0.55 V vs. Ag-AgCl) the Cu-GC modified electrode allowed detection limits between 2 and 5 pmol for all investigated organic acids while the linear dynamic range spanned generally over three orders of magnitude. Examples of applications included the separation and quantitation of some common organic acids in vinegar, honey and tea samples, are given.

Asunto(s)

Ácidos/análisis , Cromatografía por Intercambio Iónico/instrumentación , Cobre/química , Electrodos , Compuestos Orgánicos/análisis , Análisis de los Alimentos

RESUMEN

The alpha chemokine receptor CXCR4 has been shown to be expressed on human hematopoietic progenitor cells and during the megakaryocytic differentiation pathway. Stromal cell-derived factor 1 (SDF-1) is the ligand for CXCR4. In this study, the role of SDF-1alpha in megakaryocytopoiesis was investigated. CD34(+) progenitors purified from peripheral blood were grown in serum-free liquid suspension culture supplemented with thrombopoietin to obtain a virtually pure megakaryocytic progeny. In this condition, the addition of SDF-1alpha gives rise to megakaryocytes (MKs) showing an increased DNA content and a rise of lobated nuclei, as compared with untreated cells: at day 5, approximately 20% of the cells already showed the presence of more than one nuclear lobe versus fewer than 5% in the control cells; at day 12, approximately 85% of the cells were of large size and markedly polyploid, whereas approximately 60% of the control cells were polyploid, showed fewer lobes, and were a smaller size. This effect was dose-dependent and did not affect the megakaryocytic proliferation. Experiments with the mitogen-activated protein kinase (MAPK) inhibitor PD98059 suggested a role for MAPK pathway on SDF-1alpha-induced endomitosis. Furthermore, SDF-1alpha induced a significant increase in the number of proplatelet-bearing MKs and promoted the migration of megakaryocytic cells. Treatment with SDF-1alpha caused reduction in CXCR4 abundance on the plasma membrane, seemingly owing to receptor internalization. Furthermore, the presence of SDF-1alpha did not affect the expression of megakaryocytic markers, indicating that differentiation and polyploidization are independently regulated events.

Asunto(s)

Quimiocinas CXC/farmacología , Células Madre Hematopoyéticas/citología , Megacariocitos/citología , Ploidias , Diferenciación Celular , Células Cultivadas , Quimiocina CXCL12 , Replicación del ADN/efectos de los fármacos , Humanos

RESUMEN

A nickel-based composite electrode obtained by anodic electrodeposition of nickel (III) oxyhydroxide film on the gold electrode substrate was characterized as an amperometric sensor and successfully applied to the determination of underivatised amino acids in flow-through systems. The electrodeposition of nickel oxyhydroxide films was obtained by cycling a gold electrode between 0.0 V and +1.0 V vs. a saturated calomel electrode in a 80 microM Ni2+ solution buffered at pH 10 with NaHCO3/Na2CO3. The resulting Au-Ni composite electrode exhibits good stability in alkaline medium and can be used as an amperometric sensor of underivatised amino acids at a fixed applied potential (+0.55 V vs. Ag/AgCl). The detection limits (S/N=3) for all investigated compounds ranged between 5 and 30 pmol injected, while the linear ranges spanned over two or three orders of magnitude. The contents of several free amino acids in two sample cheeses from different brands were evaluated by calibration graphs.

Asunto(s)

Aminoácidos/análisis , Cromatografía por Intercambio Iónico/métodos , Electroquímica/métodos , Electrodos , Resinas de Intercambio Aniónico , Oro/química , Níquel/química

RESUMEN

Disaccharide alditols (DAs) such as maltitol, isomaltitol, and lactitol are increasingly being employed in food industry by virtue of their low hydroscopicity, high stability, and good bulking properties. Still, these compounds are reduced-calorie sweeteners, so they are successfully employed in many dietetic foods, like candies, chocolates, baked products, ice creams, and beverages. Here we describe the determination of maltitol, isomaltitol, and lactitol, along with other common carbohydrates, in some foodstuffs such as toffees, biscuits, creams, sponge cakes, chocolates, roasted malt, and chicory leaves. Separations were accomplished by high-pH anion-exchange chromatography (HPAEC) with pulsed amperometric detection using 40 mM NaOH + 1 mM Ba(CH(3)COO)(2) as the mobile phase. The optimal detection potential (E(DET) = +0.10 V) was established in voltammetric experiments carried out in batch and flowing stream solutions. Under optimized conditions there was no need for both postcolumn addition of strong bases to the eluent and, even more important, column regeneration between runs. A pellicular column with a relatively low ion-exchange capacity was adopted, which allows a rapid separation of sorbitol, isomaltitol, lactitol, maltitol, glucose, fructose, sucrose, and lactose. The presence in the alkaline mobile phase of barium ions improved selectivity and reproducibility besides shorter analysis times as well. Limits of detection were on the order of 10-20 pmol injected. The contents of DAs and other free sugars in some dietetic foods were evaluated by calibration graphs.

Asunto(s)

Cromatografía por Intercambio Iónico/métodos , Maltosa/análogos & derivados , Alcoholes del Azúcar/análisis , Resinas de Intercambio Aniónico , Electroquímica , Concentración de Iones de Hidrógeno , Maltosa/análisis

RESUMEN

Studies on pluripotent hematopoietic stem cells (HSCs) have been hindered by lack of a positive marker, comparable to the CD34 marker of hematopoietic progenitor cells (HPCs). In human postnatal hematopoietic tissues, 0.1 to 0.5% of CD34(+) cells expressed vascular endothelial growth factor receptor 2 (VEGFR2, also known as KDR). Pluripotent HSCs were restricted to the CD34+KDR+ cell fraction. Conversely, lineage-committed HPCs were in the CD34+KDR- subset. On the basis of limiting dilution analysis, the HSC frequency in the CD34+KDR+ fraction was 20 percent in bone marrow (BM) by mouse xenograft assay and 25 to 42 percent in BM, peripheral blood, and cord blood by 12-week long-term culture (LTC) assay. The latter values rose to 53 to 63 percent in LTC supplemented with VEGF and to greater than 95 percent for the cell subfraction resistant to growth factor starvation. Thus, KDR is a positive functional marker defining stem cells and distinguishing them from progenitors.

Asunto(s)

Antígenos CD34/análisis , Hematopoyesis , Células Madre Hematopoyéticas/citología , Proteínas Tirosina Quinasas Receptoras/análisis , Receptores de Factores de Crecimiento/análisis , Animales , Células de la Médula Ósea/citología , Linaje de la Célula , Separación Celular , Células Cultivadas , Factores de Crecimiento Endotelial/farmacología , Femenino , Sangre Fetal/citología , Feto , Citometría de Flujo , Trasplante de Células Madre Hematopoyéticas , Células Madre Hematopoyéticas/química , Células Madre Hematopoyéticas/efectos de los fármacos , Células Madre Hematopoyéticas/fisiología , Humanos , Linfocinas/farmacología , Ratones , Ratones Endogámicos NOD , Ratones SCID , Fenotipo , Embarazo , Proteínas Tirosina Quinasas Receptoras/fisiología , Receptores de Factores de Crecimiento/fisiología , Receptores de Factores de Crecimiento Endotelial Vascular , Ovinos , Trasplante Heterólogo , Factor A de Crecimiento Endotelial Vascular , Factores de Crecimiento Endotelial Vascular

RESUMEN

Human immunodeficiency virus (HIV) entry is mediated not only by the CD4 receptor, but also by interaction with closely related molecules that act as membrane coreceptors. We have analyzed mRNA expression and/or cell membrane exposition of the coreceptors most widely used by diverse HIV-1 strains (CXCR4, CCR5, and CCR3) on purified hematopoietic progenitor cells (HPCs) induced in liquid suspension culture to unilineage differentiation/maturation through the erythroid (E), granulocytic (G), megakaryocytic (Mk), and monocytic (Mo) lineages. Reverse transcriptase-polymerase chain reaction (RT-PCR) and cytofluorimetric analysis showed the presence of both CXCR4 and CCR5 in quiescent HPCs, but failed to detect CCR3-specific transcripts. Chemokine expression in HPC progenies showed that CXCR4 receptor is detected on the majority of MKs from early to late stages of maturation, whereas it is moderately decreased in the Mo lineage. In the G pathway, two distinct cell populations, CXCR4(+) and CXCR4(-), were observed: morphological analysis of the sorted populations showed that the CXCR4(+) cells were largely eosinophils and the CXCR4(-) were granulocytes of the neutrophilic series. Furthermore, in the E pathway, CXCR4 was almost completely absent. CCR5 expression is restricted to Mo cultures, ie, approximately 30% to 80% cells throughout all monocytopoietic differentiation/maturation stages. Finally, CCR3 mRNA is always absent in all the unilineage cultures. Evaluation of CD4 expression by flow cytometry on both quiescent HPCs and differentiating unilineage precursors showed that the CD4 receptor is present on approximately 15% of the starting CD34(+) HPC population, highly expressed in the Mo lineage up to 80% at terminal maturation, present on 20% to 30% of maturing Mks, and not detectable in either the E or G lineage. Expression of CD4 receptor together with CXCR4 and/or CCR5 coreceptor in the four lineages correlates with hematopoietic precursor susceptibility to T-lymphotropic and macrophage (M)-tropic HIV strains infection: (1) CD4(-) G and E cells were resistant to both M-tropic and T-lymphotropic strains; (2) HPC-derived Mks were susceptible to T-tropic, but resistant to M-tropic, infection; (3) Mo differentiating cells efficiently replicate both HIV strains. Furthermore, we showed that the CXCR4 and CCR5 ligands (stromal-derived factor 1 and macrophage-inflammatory protein-1alpha [MIP-1alpha], MIP-1beta and RANTES, respectively) inhibit HIV replication in both maturing Mo and Mk cells. Taken together, our data show a lineage-specific modulation of chemokine receptor/coreceptor during hematopoietic cell differentiation and extend previous observations on the relationship between the expression of HIV receptor/coreceptors, susceptibility, and chemokine-mediated resistance to HIV infection.

Asunto(s)

Quimiocinas/fisiología , Infecciones por VIH/virología , VIH-1/fisiología , Células Madre Hematopoyéticas/patología , Células Madre Hematopoyéticas/virología , Linfocitos T/virología , Adulto , Linaje de la Célula , Infecciones por VIH/inmunología , Infecciones por VIH/patología , Células Madre Hematopoyéticas/fisiología , Humanos , Masculino , Receptores CCR3 , Receptores CCR5/fisiología , Receptores CXCR4/fisiología , Receptores de Quimiocina/fisiología , Linfocitos T/inmunología , Replicación Viral

RESUMEN

B-MYB is an ubiquitous protein required for mammalian cell growth. In this report we show that B-MYB transactivates its own promoter through a 120 bp segment proximal to the transcription start site. The B-MYB-responsive element does not contain myb-binding sites and gel-shift analysis shows that SP1, but not B-MYB, protein contained in SAOS2 cell extracts binds to the 120 bp B-myb promoter fragment. B-MYB-dependent transactivation is cooperatively increased in the presence of SP1, but not SP3 overexpression. When the SP1 elements of the B-myb promoter are transferred in front of a heterologous promoter, an increased response to B-MYB results. In contrast, c-MYB, the prototype member of the Myb family, is not able to activate the luciferase construct containing the SP1 elements. With the use of an SP1-GAL4 fusion protein, we have determined that the cooperative activation occurs through the domain A of SP1. These observations suggest that B-MYB functions as a coactivator of SP1, and that diverse combinations of myb and SP1 sites may dictate the responsiveness of myb-target genes to the various members of the myb family.

Asunto(s)

Proteínas de Ciclo Celular , Proteínas de Unión al ADN/genética , Regiones Promotoras Genéticas , Proteínas de Saccharomyces cerevisiae , Factor de Transcripción Sp1/metabolismo , Transactivadores/genética , Activación Transcripcional , Proteínas E1A de Adenovirus/metabolismo , Sitios de Unión , Línea Celular , Proteínas de Unión al ADN/metabolismo , Proteínas Fúngicas/genética , Proteínas Fúngicas/metabolismo , Proteína Vmw65 de Virus del Herpes Simple/metabolismo , Humanos , FN-kappa B/metabolismo , Oligonucleótidos/farmacología , Unión Proteica , Proteínas Proto-Oncogénicas/metabolismo , Proteínas Proto-Oncogénicas c-myb , Proteínas Recombinantes de Fusión , Factor de Transcripción Sp1/genética , Factor de Transcripción Sp3 , Transactivadores/metabolismo , Factores de Transcripción/genética , Factores de Transcripción/metabolismo , Transcripción Genética , Activación Transcripcional/efectos de los fármacos , Transfección

RESUMEN

Cyclic voltammetry was used to investigate the electrochemical behavior of an Au/Cu electrode towards the electrooxidation of thiocyanate ion in alkaline medium. The effects of pH, copper loading, scan rate and applied potential on the electrocatalytic oxidation of thiocyanate have been investigated. Flow injection experiments and ion-chromatography (IC) were performed to characterise the electrode as an amperometric sensor for the thiocyanate determination. The effects of carbonate concentration and common interferents on the retention time were also estimated. The electrode stability, precision, limit of detection and linear range were evaluated at a constant applied potential of 0.7 V vs. Ag/AgCl. Calibration plots, obtained in IC, were linear from 1.0 to 195 microM (correlation coefficient of 0.9984). The detection limit (LOD) was 0.5 microM (29 ppb) in a 50 microlitres injection. An example of analytical application, which includes the IC separation and detection of thiocyanate ion present in human urine, is given.

Asunto(s)

Tiocianatos/análisis , Cromatografía por Intercambio Iónico , Cobre , Electroquímica , Electrodos , Oro , Humanos , Tiocianatos/orina

RESUMEN

We have evaluated the susceptibility to human immunodeficiency virus (HIV)-1 infection of in vitro grown megakaryopoietic progenitors/precursors and maturing megakaryocytes (MKs), based on the following approach: (1) human hematopoietic progenitor cells (HPCs), stringently purified from peripheral blood and grown in serum-free liquid suspension culture supplemented with thrombopoietin (Tpo), generated a relatively large number of >/= 98% to 99% pure megakaryocytic precursors and then mature-terminal MKs; (2) at different days of culture (ie, 0, 5, 8, 10) the cells were inoculated with 0.1 to 1.0 multiplicity of infection (m.o.i.) of the lymphotropic NL4-3 or 0.1 m.o.i. of the monocytotropic BaL-1 HIV-1 strain; (3) finally, the presence of viral mRNA and proteins was analyzed by reverse transcriptase-polymerase chain reaction (RT-PCR)/in situ hybridization and antigen capture assays, respectively, on day 2 to 12 of culture. MKs derived from day 0 and day 5 BaL-1-challenged cells do not support viral replication as assessed by p24 enzyme-linked immunosorbent assay (ELISA) and RT-PCR. On the contrary, HIV transcripts and proteins were clearly detected in all NL4-3 infection experiments by RT-PCR and p24 assay, respectively, with the highest viral expression in day 5 to 8 challenged MKs. In situ hibridization studies indicate that the percentage of HIV+ MKs varies from at least 1% and 5% for day 0 and day 5 infected cells, respectively. Production of an infectious viral progeny, evaluated by the capability of culture supernatants from day 5 NL4-3-challenged MKs to infect C8166 T-lymphoblastoid cell line, was consistently observed (viral titer, approximately 5 x 10(3) tissue culture infectious dose50/mL/10(6) cells). Exposure of MKs to saturating concentration of anti-CD4 OKT4A monoclonal antibody (MoAb), which recognizes the CD4 region binding with the gp120 envelope glycoprotein, markedly inhibited HIV infection, as indicated by a reduction of p24 content in the supernatants: because the inhibitory effect was incomplete, it is apparent that the infection is only partially CD4-dependent, suggesting that an alternative mechanism of viral entry may exist. Morphologic analysis of day 12 MKs derived from HPCs infected at day 0 showed an impaired megakaryocytic differentiation/maturation: the percentage of mature MKs was markedly reduced, in that approximately 80% of cells showed only one nuclear lobe and a pale cytoplasm with few granules. Conversely, megakaryocytic precursors challenged at day 5 to 8 generated fully mature day 10 to 12 MKs showing multiple nuclear segmentation. Thus, the inhibitory effect of HIV on the megakaryopoietic gene program relates to the differentiation stage of cells subjected to the viral challenge. Finally, HPCs treated with 20 or 200 ng/mL of recombinant Tat protein, analyzed at different days of culture, showed an impaired megakaryocytopoiesis comparable to that observed in HIV-infected cells, thus suggesting that Tat is a major mediator in the above described phenomena. These results shed light on the pathogenesis of HIV-related thrombocytopenia; furthermore, they provide a model to investigate the effects of HIV on megakaryocytic differentiation and function.

Asunto(s)

Infecciones por VIH , VIH-1 , Megacariocitos/virología , Adulto , Diferenciación Celular , Células Cultivadas , Susceptibilidad a Enfermedades , Humanos , Masculino , Megacariocitos/citología

RESUMEN

B-MYB expression is associated with cell proliferation and recent studies have suggested that it promotes the S phase of mammalian cells. Based on its homology to the transcription factors c-MYB and A-MYB, B-MYB is thought to be involved in transcriptional regulation; however, its activity is not detectable in several cell lines. It was postulated that B-MYB function may depend on the presence of a cofactor, and recent studies suggested that B-MYB is phosphorylated specifically during S phase in murine fibroblasts. In this report we provide evidence that the product of the human B-myb gene can be activated in vivo by coexpression with cyclin A or cyclin E. Transfection studies showed that B-MYB was a weak transcriptional activator in SAOS-2 cells and was unable to promote their proliferation. In contrast, overexpression of both B-MYB and cyclin A or cyclin E caused a drastic increase in the number of SAOS-2 cells in S phase. Also, overexpression of cyclin A and cyclin E in SAOS-2 cells enhanced the ability of B-MYB, but not c-MYB, to transactivate various promoters, including the cdc2 promoter, the HIV-1-LTR, and the simian virus 40 minimal promoter. A direct role for cyclin-dependent activation of B-MYB was demonstrated using an in vitro transcription assay. These observations suggest that one mechanism by which cyclin A and E may promote the S phase is through modification and activation of B-MYB.

Asunto(s)

Proteínas de Ciclo Celular , Ciclina B , Ciclinas/fisiología , Proteínas de Unión al ADN/metabolismo , Transactivadores , Factores de Transcripción/metabolismo , Ciclo Celular , División Celular , Línea Celular , Ciclina B1 , Ciclinas/metabolismo , Regulación de la Expresión Génica , Humanos , Fosforilación , Activación Transcripcional

RESUMEN

The effects of some divalent nonelectroactive cations (DNCs), such as Sr(II), Ba(II), and Ca(II) on the electrochemical oxidation of alditols and carbohydrates at gold electrodes in pulsed amperometric detection have been investigated. It seems that in the presence of DNCs in alkaline solutions, two competitive processes are involved: polyhydroxy compound complexation in the following order of metal ion binding affinity, Ca(II) > Sr(II) > Ba(II), and inhibition on the onset of gold oxide formation in the same order. This last effect leads to an increased activity of the electrode surface; at the optimized value of detection potential of d-sorbitol, E(DET) = +50 mV vs Ag|AgCl, which is ∼100 mV lower in comparison to the maximum value observed with blank carrier electrolytes (i.e., 0.58 M NaOH), there is an increase in sensitivity of ∼50%, and 30% in the presence of 1.0 mM Sr(II), and 1.0 mM Ba(II), respectively. However, the current response of sample molecules results increased only when Ba(II) or Sr(II) ions were already contained in the alkaline media, that is the experimental condition normally occurring in flow injection and liquid chromatography systems. The voltammetric response, observed upon additions of Ba(II) or Sr(II) to an alkaline electrolyte containing d-sorbitol, showed progressive decrease of the anodic current. Irrespective of the experimental condition adopted, i.e., cation addition to the solution before or after the carbohydrate, the presence of Ca(II) has an adverse effect on the anodic currents. These findings have been explained by a rapid formation of adducts between polyhydroxy compounds and DNCs in sodium hydroxide solutions.

RESUMEN

c-myb, a protooncogene prevalently expressed in the hematopoietic tissue, is a transcription factor that contains a DNA-binding domain and an acidic domain and is able to transactivate specific viral and cellular genes. In this report, we show that c-myb can stimulate apoptosis in both the murine promyelocytic 32D and the human osteosarcoma SAOS2 cell lines when coexpressed with p53. Apoptosis is accompanied by increased transactivation of the cell death-associated BAX gene. This effect is c-myb specific, because B-myb is not able to cooperate with p53 in the induction of BAX transcription and apoptosis. Immunoprecipitation studies and gel shift analysis indicate that c-myb does not directly interact with the BAX promoter or the p53 protein but, rather, cooperates through an indirect mechanism. Consistent with the existence of a functional link between c-myb and p53, we also observed that c-myb represses p53-induced activation of the WAF-1 promoter and induces proliferation of SAOS2 cells growth arrested by p53. These results might contribute to the elucidation of the mechanisms underlying p53-dependent pathways of oncogene-induced apoptosis and provide a further example of DNA-binding independent myb activity.

Asunto(s)

Apoptosis/genética , Genes p53 , Oncogenes , Proteínas Proto-Oncogénicas c-bcl-2 , Animales , Northern Blotting , Cloranfenicol O-Acetiltransferasa/genética , Humanos , Ratones , Plásmidos , Regiones Promotoras Genéticas , Proteínas Proto-Oncogénicas/genética , Proteínas Proto-Oncogénicas/metabolismo , Proteínas Proto-Oncogénicas c-myc/genética , Proteínas Proto-Oncogénicas c-myc/metabolismo , ARN Mensajero/biosíntesis , Fase S , Transfección , Células Tumorales Cultivadas , Proteína p53 Supresora de Tumor/genética , Proteína p53 Supresora de Tumor/metabolismo , Proteína X Asociada a bcl-2

RESUMEN

The retinoblastoma protein family has been implicated in growth control and modulation of the activity of genes involved in cell proliferation, such as B-myb. Recent evidence indicates that the product of the B-myb gene is necessary for the growth and survival of several human and murine cell lines. Upon overexpression, B-myb induces deregulated cell growth of certain cell lines. Here we show that B-myb overexpression is able to induce DNA synthesis in p107 growth-arrested human osteosarcoma cells (SAOS2). p107 might exert its growth-suppressive activity by regulating B-myb gene transcription. Indeed, p107 down-modulated B-myb promoter activity and drastically decreased E2F-mediated transactivation. Finally, B-myb was able to stimulate DNA synthesis of both stably and transiently transfected human glioblastoma cells (T98G). Altogether, these data provide definitive evidence that the human B-myb protein is involved in growth control of human cells, and that p107 has a significant role in regulating B-myb gene activity.

Asunto(s)

Proteínas de Ciclo Celular , Ciclo Celular , Proteínas de Unión al ADN/metabolismo , Proteínas Nucleares/metabolismo , Transactivadores , Factores de Transcripción/metabolismo , Proteínas E2 de Adenovirus/metabolismo , Animales , Neoplasias Óseas , División Celular , Línea Celular , Supervivencia Celular , Replicación del ADN , Proteínas de Unión al ADN/biosíntesis , Regulación Neoplásica de la Expresión Génica , Glioblastoma , Homeostasis , Humanos , Cinética , Luciferasas/biosíntesis , Luciferasas/metabolismo , Ratones , Oncogenes , Osteosarcoma , Plásmidos , Proteínas Recombinantes/metabolismo , Proteína de Retinoblastoma/biosíntesis , Proteína p107 Similar a la del Retinoblastoma , Fase S , Factores de Transcripción/biosíntesis , Transcripción Genética , Activación Transcripcional , Transfección , Células Tumorales Cultivadas

RESUMEN

Zinc finger genes encode proteins that act as transcription factors. The myeloid zinc finger 1 (MZF1) gene encodes a zinc finger protein with two DNA-binding domains that recognize two distinct consensus sequences, is preferentially expressed in hematopoietic cells, and may be involved in the transcriptional regulation of hematopoiesis-specific genes. Reverse transcription-PCR analysis of human peripheral blood CD34+ cells cultured under lineage-restricted conditions demonstrated MZF1 expression during both myeloid and erythroid differentiation. Sequence analysis of the 5'-flanking region of the CD34 and c-myb genes, which are a marker of and a transcriptional factor required for hematopoietic proliferation and differentiation, respectively, revealed closely spaced MZF1 consensus binding sites found by electrophoretic mobility shift assays to interact with recombinant MZF1 protein. Transient or constitutive MZF1 expression in different cell types resulted in specific inhibition of chloramphenicol acetyltransferase activity driven by the CD34 or c-myb 5'-flanking region. To determine whether transcriptional modulation by MZF1 activity plays a role in hematopoietic differentiation, constructs containing the MZF1 cDNA under the control of different promoters were transfected into murine embryonic stem cells which, under defined in vitro culture conditions, generate colonies of multiple hematopoietic lineages. Constitutive MZF1 expression interfered with the ability of embryonic stem cells to undergo hematopoietic commitment and erythromyeloid colony formation and prevented the induced expression of CD34 and c-myb mRNAs during differentiation of these cells. These data indicate that MZF1 plays a critical role in hematopoiesis by modulating the expression of genes involved in this process.

Asunto(s)

Antígenos CD34/genética , Proteínas de Unión al ADN/fisiología , Regulación del Desarrollo de la Expresión Génica , Hematopoyesis , Células Madre Hematopoyéticas/citología , Oncogenes , Regiones Promotoras Genéticas , Proteínas Proto-Oncogénicas/genética , Proteínas Represoras/genética , Factores de Transcripción/fisiología , Secuencia de Aminoácidos , Secuencia de Bases , Diferenciación Celular , Células Cultivadas , Secuencia de Consenso , Proteínas de Unión al ADN/metabolismo , Humanos , Factores de Transcripción de Tipo Kruppel , Datos de Secuencia Molecular , Proteínas Proto-Oncogénicas c-myb , ARN Mensajero/genética , Dedos de Zinc

RESUMEN

Chronic myelogenous leukemia evolves in two clinically distinct stages: a chronic and a blast crisis phase. The molecular changes associated with chronic phase to blast crisis transition are largely unknown. We have identified a cDNA clone, DR-nm23, differentially expressed in a blast-crisis cDNA library, which has approximately 70% sequence similarity to the putative metastatic suppressor genes, nm23-H1 and nm23-H2. The deduced amino acid sequence similarity to the proteins encoded by these two latter genes is approximately 65% and includes domains and amino acid residues (the leucine zipper-like and the RGD domain, a serine and a histidine residue in the NH2- and in the COOH-terminal portion of the protein, respectively) postulated to be important for nm23 function. DR-nm23 mRNA is preferentially expressed at early stages of myeloid differentiation of highly purified CD34+ cells. Its constitutive expression in the myeloid precursor 32Dc13 cell line, which is growth-factor dependent for both proliferation and differentiation, results in inhibition of granulocytic differentiation induced by granulocyte colony-stimulating factor and causes apoptotic cell death. These results are consistent with a role for DR-nm23 in normal hematopoiesis and raise the possibility that its overexpression contributes to differentiation arrest, a feature of blastic transformation in chronic myelogenous leukemia.

Asunto(s)

Granulocitos/citología , Granulocitos/metabolismo , Proteínas de Unión al GTP Monoméricas , Nucleósido-Difosfato Quinasa , Factores de Transcripción/genética , Secuencia de Aminoácidos , Animales , Apoptosis , Secuencia de Bases , Diferenciación Celular , Línea Celular , Clonación Molecular , Cartilla de ADN/genética , ADN Complementario/genética , Expresión Génica , Células Madre Hematopoyéticas/citología , Células Madre Hematopoyéticas/metabolismo , Humanos , Ratones , Datos de Secuencia Molecular , Nucleósido Difosfato Quinasas NM23 , Fenotipo , Reacción en Cadena de la Polimerasa , Homología de Secuencia de Aminoácido

RESUMEN

We have utilized highly purified hematopoietic progenitor and stem cells (HPCs, HSCs) from normal peripheral blood to develop methodology for: (a) efficient transfer into HPCs of a non-hematopoietic membrane reporter, i.e., the nerve growth factor receptor complementary DNA; and (b) effective gene transduction of putative HSCs, i.e., cells initiating Dexter-type long-term culture (LTC-ICs). Purified HPCs induced into cycling by growth factors (interleukin 3, interleukin 6, c-kit ligand) were transduced with the N2 retroviral vector containing the neomycin resistance (neor) gene. More than 80% of transduced HPCs were resistant to the toxic G418 level. Thereafter, the HPCs were effectively transduced with the LNSN retroviral vector containing a nerve growth factor receptor complementary DNA; the nerve growth factor receptor was detected on > or = 18% of the transduced HPCs. These experiments provide a new tool from which (a) to monitor expression of a transduced membrane report on hematopoietic cells, particularly at the level of HPCs/HSCs, and (b) to characterize the transduced cells by double- and triple-labeling membrane antigen analysis. Purified HPCs/HSCs grown in Dexter-type LTC were transduced at 1 week by exposure to supernatant N2 retroviral particles in the absence of exogenous hematopoietic growth factors. The procedure, devoid of toxic effects, allowed an efficient neor transduction into LTC-ICs. Thus, we consistently detected neomycin-resistant mRNA in the clonal progeny of HPCs produced in LTC at 5-8 weeks in both the nonadherent and adherent fractions; this timing of expression coincides with that of HPC production by LTC-ICs, thereby indicating the effective transduction of the LTC-ICs. These experiments represent a first step toward development of preclinical models for gene transfer into human peripheral blood HSCs by complex retroviral vectors.

Asunto(s)

Genes Reporteros/genética , Células Madre Hematopoyéticas , Transfección/métodos , Adulto , Resistencia a Medicamentos , Vectores Genéticos , Humanos , Masculino , Neomicina , Receptores de Factor de Crecimiento Nervioso/genética