RESUMEN
In the adult, tissue-specific stem cells are thought to be responsible for the replacement of differentiated cells within continuously regenerating tissues, such as the liver, skin, and blood system. In this review, we will consider the factors that influence stem cell fate, taking as a primary example the cell fate determination of hematopoietic stem cells.
Asunto(s)
Linaje de la Célula/fisiología , Células Madre Hematopoyéticas/fisiología , Regeneración/fisiología , Adulto , Animales , Apoptosis/fisiología , Adhesión Celular/fisiología , Diferenciación Celular/fisiología , División Celular/fisiología , Movimiento Celular/fisiología , Humanos , Modelos BiológicosRESUMEN
Clonogenic multipotent mouse hematopoietic stem cells (HSCs) and progenitor cells are contained within the c-kit(+) (K) lineage(-/lo) (L) Sca-1(+) (S) population of hematopoietic cells; long-term (LT) and short-term (ST) HSCs are Thy-1.1(lo). c-kit is a member of the receptor tyrosine kinase family, a class of receptors that are important in the proliferation and differentiation of hematopoietic cells. To establish whether the Flk-2/Flt3 receptor tyrosine kinase was expressed on the most primitive LT-HSCs, we sorted highly purified multipotent stem and progenitor cells on the basis of Flk-2 surface expression and used them in competitive reconstitution assays. Low numbers of Flk-2(-) HSCs gave rise to long-term multilineage reconstitution in the majority of recipients, whereas the transfer of Flk-2(+) multipotent cells resulted in mostly short-term multilineage reconstitution. The KLS subset of adult mouse bone marrow was analyzed for Flk-2 and Thy-1.1 expression. Three phenotypically and functionally distinct populations were isolated: Thy(lo) Flk-2(-) (LT-HSCs), Thy(lo) Flk-2(+) (ST-HSCs), and Thy(-) Flk-2(+) multipotent progenitors. The loss of Thy-1.1 and gain of Flk-2 expression marks the loss of self-renewal in HSC maturation. The addition of Flk-2 antibody to the lineage mix allows direct isolation of LT-HSC from adult bone marrow as c-kit(+) lin(-) Sca-1(+) Flk-2(-) from many strains of mice. Fetal liver HSCs are contained within Flk-2(-) and Flk-2(+) KTLS cells.
Asunto(s)
Separación Celular/métodos , Células Madre Hematopoyéticas/citología , Células Madre Hematopoyéticas/metabolismo , Proteínas Proto-Oncogénicas/metabolismo , Proteínas Tirosina Quinasas Receptoras/metabolismo , Animales , Antígenos Ly/metabolismo , Biomarcadores , Diferenciación Celular , Feto/citología , Feto/inmunología , Feto/metabolismo , Hematopoyesis , Trasplante de Células Madre Hematopoyéticas , Células Madre Hematopoyéticas/inmunología , Hígado/citología , Hígado/inmunología , Hígado/metabolismo , Proteínas de la Membrana/metabolismo , Ratones , Ratones Congénicos , Ratones Endogámicos C57BL , Fenotipo , Proteínas Proto-Oncogénicas/genética , Proteínas Proto-Oncogénicas c-kit/metabolismo , Proteínas Tirosina Quinasas Receptoras/genética , Antígenos Thy-1/metabolismo , Tirosina Quinasa 3 Similar a fmsRESUMEN
Cytokine-mobilized peripheral blood hematopoietic stem cells (MPB HSC) are widely used for transplantation in the treatment of malignancies, but the mechanism of HSC mobilization is unclear. Although many HSC in bone marrow (BM) cycle rapidly and expand their numbers in response to cytoreductive agents, such as cyclophosphamide (CY), and cytokines, such as granulocyte colony-stimulating factor (G-CSF), MPB HSC are almost all in the G(0) or G(1) phase of the cell cycle. This has raised the question of whether a subset of noncycling BM HSC is selectively released, or whether cycling BM HSC are mobilized after M phase, but before the next S phase of the cell cycle. To distinguish between these possibilities, mice were treated with one dose of CY followed by daily doses of G-CSF, and dividing cells were marked by administration of bromodeoxyuridine (BrdU) during the interval that BM HSC are expanding. After CY and 4 days of G-CSF, 98.5% of the 2n DNA content long-term repopulating MPB (LT)-HSC stained positively for BrdU, and therefore derived from cells that divided during the treatment interval. Next, LT-HSC from mice previously treated with a single dose of CY, which kills cycling cells, and 3 daily doses of G-CSF, were nearly all killed by a second dose of CY, suggesting that CY/G-CSF causes virtually all LT-HSC to cycle. Analysis of cyclin D2 messenger RNA (mRNA) expression and total RNA content of MPB HSC suggests that these cells are mostly in G(1) phase. After CY/G-CSF treatment, virtually all BM LT-HSC enter the cell cycle; some of these HSC then migrate into the blood, specifically after M phase, and are rapidly recruited to particular hematopoietic organs.
Asunto(s)
Médula Ósea/efectos de los fármacos , Ciclofosfamida/farmacología , Factor Estimulante de Colonias de Granulocitos/farmacología , Movilización de Célula Madre Hematopoyética , Células Madre Hematopoyéticas/efectos de los fármacos , Animales , Células Sanguíneas/citología , Linaje de la Célula , Movimiento Celular , Células Cultivadas , Ciclina D2 , Ciclinas/biosíntesis , Ciclinas/genética , Replicación del ADN/efectos de los fármacos , Células Madre Hematopoyéticas/citología , Metafase , Ratones , Ratones Endogámicos C3H , Ratones Endogámicos C57BL , Ratones Transgénicos , Especificidad de Órganos , ARN Mensajero/biosíntesis , Proteínas Recombinantes de Fusión/análisis , Bazo/citologíaRESUMEN
A series of thiazolothiazepines were prepared and tested against purified human immunodeficiency virus type-1 integrase (HIV-1 IN) and viral replication. Structure-activity studies reveal that the compounds possessing the pentatomic moiety SC(O)CNC(O) with two carbonyl groups are in general more potent against purified IN than those containing only one carbonyl group. Substitution with electron-donating or -withdrawing groups did not enhance nor abolish potency against purified IN. By contrast, compounds with a naphthalene ring system showed enhanced potency, suggesting that a hydrophobic pocket in the IN active site might accommodate an aromatic system rather than a halogen. The position of sulfur in the thiazole ring appears important for potency against IN, as its replacement with an oxygen or carbon abolished activity. Further extension of the thiazole ring diminished potency. Compounds 1, 19, and 20 showed antiviral activity and inhibited IN within similar concentrations. These compounds inhibited IN when Mn(2+) or Mg(2+) was used as cofactor. None of these compounds showed detectable activities against HIV-1 reverse transcriptase, protease, virus attachment, or nucleocapsid protein zinc fingers. Therefore, thiazolothiazepines are potentially important lead compounds for development as inhibitors of IN and HIV replication.
Asunto(s)
Inhibidores de Integrasa VIH/síntesis química , VIH-1 , Tiazepinas/síntesis química , Tiazoles/síntesis química , Tiazolidinedionas , Adhesión Celular/efectos de los fármacos , Línea Celular , Inhibidores de Integrasa VIH/química , Inhibidores de Integrasa VIH/farmacología , Inhibidores de la Proteasa del VIH/síntesis química , Inhibidores de la Proteasa del VIH/química , Inhibidores de la Proteasa del VIH/farmacología , Magnesio/química , Manganeso/química , Proteínas de la Nucleocápside/química , Inhibidores de la Transcriptasa Inversa/síntesis química , Inhibidores de la Transcriptasa Inversa/química , Inhibidores de la Transcriptasa Inversa/farmacología , Relación Estructura-Actividad , Tiazepinas/química , Tiazepinas/farmacología , Tiazoles/química , Tiazoles/farmacología , Replicación Viral/efectos de los fármacos , Dedos de Zinc/efectos de los fármacosRESUMEN
The rapid emergence of human immunodeficiency virus (HIV) strains resistant to available drugs implies that effective treatment modalities will require the use of a combination of drugs targeting different sites of the HIV life cycle. Because the virus cannot replicate without integration into a host chromosome, HIV-1 integrase (IN) is an attractive therapeutic target. Thus, an effective IN inhibitor should provide additional benefit in combination chemotherapy. A four-point pharmacophore has been identified based on the structures of quinalizarin and purpurin, which were found to be potent IN inhibitors using both a preintegration complex assay and a purified enzyme assay in vitro. Searching with this four-point pharmacophore in the 'open' part of the National Cancer Institute three-dimensional structure database produced 234 compounds containing the pharmacophore. Sixty of these compounds were tested for their inhibitory activity against IN using the purified enzyme; 19 were found to be active against IN with IC50 values of less than 100 microM, among which 10 had IC50 values of less than 10 microM. These inhibitors can further serve as leads, and studies are in progress to design novel inhibitors based on the results presented in this study.
Asunto(s)
Fármacos Anti-VIH/aislamiento & purificación , Inhibidores de Integrasa VIH/aislamiento & purificación , Integrasa de VIH/efectos de los fármacos , Antraquinonas/química , Antraquinonas/farmacología , Fármacos Anti-VIH/química , Fármacos Anti-VIH/farmacología , Línea Celular , Bases de Datos Factuales , Diseño de Fármacos , Evaluación Preclínica de Medicamentos , Inhibidores de Integrasa VIH/química , Inhibidores de Integrasa VIH/farmacología , VIH-1/efectos de los fármacos , VIH-1/fisiología , Humanos , Linfocitos/virología , Estructura Molecular , Relación Estructura-Actividad , Integración Viral/efectos de los fármacosRESUMEN
In previous studies we identified N,N'-bis(salicylhydrazine) (1) as a lead compound against purified recombinant HIV-1 integrase. We have now expanded upon these earlier observations and tested 45 novel hydrazides. Among the compounds tested, 11 derivatives exhibited 50% inhibitory concentrations (IC50) of less than 3 microM. A common feature for activity among these inhibitors is the hydroxyl group of the salicyl moiety. Although the active inhibitors must contain this hydroxyl group, other structural modifications can also influence potency. Removal of this hydroxyl group or replacement with an amino, bromo, fluoro, carboxylic acid, or ethyl ether totally abolished potency against integrase. Several asymmetric structures exhibited similar potency to the symmetric lead inhibitor 1. The superimposition of the lowest-energy conformations upon one another revealed three sites whose properties appear important for ligand binding. Site A is composed of the 2-hydroxyphenyl, the alpha-keto, and the hydrazine moieties in a planar conformation. We propose that this site could interact with HIV-1 integrase by chelation of the metal in the integrase active site as inhibition of HIV-1 integrase catalytic activity and DNA binding were strictly Mn2+-dependent. The hydrophobic sites B and C are probably responsible for complementarity of molecular shape between ligand and receptor. Our data indicate that only those compounds which possessed sites A, B, and C in a linear orientation were potent inhibitors of HIV-1 integrase. Although all the active inhibitors possessed considerable cytotoxicity and no apparent antiviral activity in CEM cells, the study presents useful information regarding ligand interaction with HIV-1 integrase protein.
Asunto(s)
Inhibidores de Integrasa VIH/síntesis química , Integrasa de VIH/química , Integrasa de VIH/metabolismo , VIH-1/fisiología , Hidrazinas/síntesis química , Salicilamidas/síntesis química , Replicación Viral/efectos de los fármacos , Sitios de Unión , Gráficos por Computador , Diseño de Fármacos , Inhibidores de Integrasa VIH/química , Inhibidores de Integrasa VIH/farmacología , VIH-1/efectos de los fármacos , Humanos , Hidrazinas/química , Hidrazinas/farmacología , Cinética , Modelos Moleculares , Estructura Molecular , Oligodesoxirribonucleótidos/química , Oligodesoxirribonucleótidos/metabolismo , Salicilamidas/química , Salicilamidas/farmacología , Relación Estructura-ActividadRESUMEN
In 1901, when optometry first achieved legal status, optometric education in the United States was inadequate. Two-week courses in refraction, correspondence courses, and 2 year apprenticeships were common. Over the next 2 decades, progress was made through the closing of a number of schools and the development of some creditable ones, but across the country optometric education remained unacceptably uneven. There were various calls for improvement, and in 1921 the American Optometric Association (AOA) formed a Council on Optometric Education. In 1921, funds were appropriated by the AOA to fund a nationwide conference on optometric education, which was held in January, 1992. Among other things, this conference resulted in the classification of all schools, closing some in the process; the adoption of minimum entrance requirements; the adoption of subject matter syllabi; and the recommendation of the end of apprenticeship and correspondence courses. Optometric education was forever changed.
Asunto(s)
Optometría/historia , Educación/historia , Educación/normas , Historia del Siglo XIX , Historia del Siglo XX , Humanos , Optometría/educación , Optometría/normas , Estados UnidosRESUMEN
Mice lacking the perforin gene were generated by using targeted gene disruption in embryonal stem cells. When infected with lymphocytic choriomeningitis virus (LCMV), perforin-less (-/-) mice showed clear signs of having mounted an immune response based on activation of CD8 T cells but were unable to clear the LCMV infection. This failure to eliminate virus was accompanied by a failure to generate spleen cells capable of lysing LCMV-infected fibroblasts in vitro. Spleen cells from LCMV-infected -/- mice were able to lyse hematopoietic target cells after exposure to phorbol 12-myristate 13-acetate and ionomycin, provided the target cells expressed the Fas antigen. Spleen cells from -/- mice also responded to alloantigen in mixed leukocyte culture by blastogenesis and proliferation. The resulting cells were able to lyse hematopoietic target cells, although not as well as spleen cells from +/+ littermates sensitized in the same manner. However, lysis by -/- cells was again seen only if the target cells expressed Fas antigen. We conclude that perforin-less -/- mice retain and express the Fas lytic pathway as expressed in vitro but that this pathway is insufficient to clear an LCMV infection in vivo.
Asunto(s)
Coriomeningitis Linfocítica/inmunología , Glicoproteínas de Membrana/fisiología , Linfocitos T Citotóxicos/inmunología , Animales , Antígenos de Superficie/inmunología , Linfocitos T CD8-positivos/inmunología , Citotoxicidad Inmunológica , Femenino , Inmunidad Celular , Recuento de Linfocitos , Virus de la Coriomeningitis Linfocítica , Masculino , Ratones , Ratones Endogámicos C57BL , Ratones Endogámicos CBA , Ratones Noqueados , Perforina , Proteínas Citotóxicas Formadoras de Poros , Receptor fasRESUMEN
A major component of amyloid deposits found in Alzheimer disease and Down syndrome is the beta/A4 peptide, which is derived from the Alzheimer amyloid protein precursor (APP). Recent evidence indicates that increases in APP expression and/or beta/A4 peptide accumulation may underlie the amyloidosis characteristic of these diseases. In the present study, transgenic mice carrying the entire human APP gene were studied for expression of human APP. Significant expression of human APP protein was observed in these animals, and this expression paralleled the expression of endogenous APP. These results, which represent a first demonstration of significant human APP expression in transgenic animals, support the use of such animals to study human APP expression and processing in vivo and possibly as models for the amyloidosis associated with Alzheimer disease.
Asunto(s)
Precursor de Proteína beta-Amiloide/biosíntesis , Precursor de Proteína beta-Amiloide/genética , Encéfalo/metabolismo , Precursor de Proteína beta-Amiloide/aislamiento & purificación , Animales , Corteza Cerebral/metabolismo , Cromosomas Artificiales de Levadura , Electroforesis en Gel de Poliacrilamida , Embrión de Mamíferos , Femenino , Expresión Génica , Humanos , Immunoblotting , Masculino , Ratones , Ratones Transgénicos , Células Madre/metabolismoRESUMEN
Change has affected physiological optics graduate programs' fulfillment of their original purpose of providing faculty for the schools and colleges of optometry. Relevant change has been divided into two categories: 1) change in optometry and optometric education, and 2) change in the characteristics of students opting for graduate study. The answer to the question posed is that these programs are still preparing faculty for academic careers, but not in the numbers they once did. A few consequences and options are considered.