RESUMEN
Manipulation of mammalian cells has been achieved by the transfection of expression vectors, microinjection, or diffusion of peptidyl mimetics. While these approaches have been somewhat successful, the classic manipulation methods are not easily regulated and can be laborious. One approach to circumvent these problems is the use of HIV TAT-mediated protein transduction. Although this technology was originally described in 1988, few improvements were reported in the subsequent 10 years. In the last few years, significant steps have been taken to advance this technology into a broadly applicable method that allows for the rapid introduction of full-length proteins into primary and transformed cells. The technology requires the synthesis of a fusion protein, linking the TAT transduction domain to the molecule of interest using a bacterial expression vector, followed by the purification of this fusion protein under either soluble or denaturing conditions. The purified fusion protein can be directly added to mammalian cell culture or injected in vivo into mice. Protein transduction occurs in a concentration-dependent manner, achieving maximum intracellular concentrations in less than 5 min, with nearly equal intracellular concentrations between all cells in the transduced population. Full-length TAT fusion proteins have been used to address a number of biological questions, relating to cell cycle progression, apoptosis, and cellular architecture. Described here are the fundamental requirements for the creation, isolation, and utilization of TAT-fusion proteins to affect mammalian cells. A detailed protocol for production and transduction of TAT-Cdc42 into primary cells is given to illustrate the technique.
Asunto(s)
Productos del Gen tat/genética , Transducción Genética , Animales , Células Cultivadas , Productos del Gen tat/aislamiento & purificación , Productos del Gen tat/metabolismo , VIH-1/genética , Humanos , Activación Transcripcional , Transformación Genética , Proteínas Virales de Fusión/química , Proteínas Virales de Fusión/genética , Proteínas Virales de Fusión/metabolismo , Productos del Gen tat del Virus de la Inmunodeficiencia HumanaRESUMEN
The retinoblastoma tumor suppressor protein (pRB) negatively regulates early-G(1) cell cycle progression, in part, by sequestering E2F transcription factors and repressing E2F-responsive genes. Although pRB is phosphorylated on up to 16 cyclin-dependent kinase (Cdk) sites by multiple G(1) cyclin-Cdk complexes, the active form(s) of pRB in vivo remains unknown. pRB is present as an unphosphorylated protein in G(0) quiescent cells and becomes hypophosphorylated (approximately 2 mol of PO(4) to 1 mol of pRB) in early G(1) and hyperphosphorylated (approximately 10 mol of PO(4) to 1 mol of pRB) in late G(1) phase. Here, we report that hypophosphorylated pRB, present in early G(1), represents the biologically active form of pRB in vivo that is assembled with E2Fs and E1A but that both unphosphorylated pRB in G(0) and hyperphosphorylated pRB in late G(1) fail to become assembled with E2Fs and E1A. Furthermore, using transducible dominant-negative TAT fusion proteins that differentially target cyclin D-Cdk4 or cyclin D-Cdk6 (cyclin D-Cdk4/6) and cyclin E-Cdk2 complexes, namely, TAT-p16 and TAT-dominant-negative Cdk2, respectively, we found that, in vivo, cyclin D-Cdk4/6 complexes hypophosphorylate pRB in early G(1) and that cyclin E-Cdk2 complexes inactivate pRB by hyperphosphorylation in late G(1). Moreover, we found that cycling human tumor cells expressing deregulated cyclin D-Cdk4/6 complexes, due to deletion of the p16(INK4a) gene, contained hypophosphorylated pRB that was bound to E2Fs in early G(1) and that E2F-responsive genes, including those for dihydrofolate reductase and cyclin E, were transcriptionally repressed. Thus, we conclude that, physiologically, pRB is differentially regulated by G(1) cyclin-Cdk complexes.
Asunto(s)
Quinasas CDC2-CDC28 , Proteínas Portadoras , Proteínas de Ciclo Celular , Ciclina E/metabolismo , Quinasas Ciclina-Dependientes/metabolismo , Ciclinas/metabolismo , Genes Supresores de Tumor , Proteínas Serina-Treonina Quinasas/metabolismo , Proteínas Proto-Oncogénicas , Proteína de Retinoblastoma/metabolismo , Células Cultivadas , Ciclina D , Ciclina E/genética , Quinasa 2 Dependiente de la Ciclina , Quinasa 4 Dependiente de la Ciclina , Quinasa 6 Dependiente de la Ciclina , Inhibidor p16 de la Quinasa Dependiente de Ciclina/metabolismo , Proteínas de Unión al ADN/metabolismo , Factores de Transcripción E2F , Factor de Transcripción E2F4 , Fase G1 , Humanos , Fosforilación , Proteínas Represoras/metabolismo , Proteína 1 de Unión a Retinoblastoma , Tetrahidrofolato Deshidrogenasa/genética , Factores de Tiempo , Factores de Transcripción/metabolismoRESUMEN
Progression of cells through the G1 phase of the cell cycle requires cyclin D:Cdk4/6 and cyclin E:Cdk2 complexes; however, the duration and ordering of these complexes remain unclear. To address this, we synthesized a peptidyl mimetic of the Cdk4/6 inhibitor, p16INK4a that contained an NH2-terminal TAT protein transduction domain. Transduction of TAT-p16 wild-type peptides into cells resulted in the loss of active, hypophosphorylated pRb and elicited an early G1 cell cycle arrest, provided cyclin E:Cdk2 complexes were inactive. We conclude that cyclin D:Cdk4/6 activity is required for early G1 phase cell cycle progression up to, but not beyond, activation of cyclin E:Cdk2 complexes at the restriction point and is thus nonredundant with cyclin E:Cdk2 in late G1.
Asunto(s)
Quinasas CDC2-CDC28 , Inhibidor p16 de la Quinasa Dependiente de Ciclina/fisiología , Quinasas Ciclina-Dependientes/fisiología , Ciclinas/fisiología , Fase G1/fisiología , Proteínas Serina-Treonina Quinasas/fisiología , Proteínas Proto-Oncogénicas , Proteína de Retinoblastoma/metabolismo , Línea Celular , Ciclina D , Ciclina E/fisiología , Quinasa 2 Dependiente de la Ciclina , Quinasa 4 Dependiente de la Ciclina , Quinasa 6 Dependiente de la Ciclina , Humanos , Queratinocitos , Fosforilación , TransfecciónAsunto(s)
Proteínas de Ciclo Celular , Movimiento Celular , Cruzamientos Genéticos , Productos del Gen tat/genética , Proteínas Asociadas a Microtúbulos/genética , Proteínas Supresoras de Tumor , Movimiento Celular/genética , Separación Celular , Inhibidor p27 de las Quinasas Dependientes de la Ciclina , Citometría de Flujo , Humanos , Células Jurkat , Desnaturalización Proteica , Proteínas Recombinantes de Fusión/genética , Células Tumorales CultivadasRESUMEN
rpoS homologues were identified in several Erwinia species using Escherichia coli rpoS sequences as probes. The rpoS gene from Erwinia carotovora was cloned and the deduced amino acid sequence had 91% identity to E. coli RpoS. The latter sigma factor regulates the stationary phase inducible HPII catalase activity of E. coli. In an E. coli rpoS mutant, the E. carotovora rpoS gene was also able to regulate synthesis of this catalase. The presence of a similar catalase in E. carotovora suggests that the structural gene for this may be part of the rpoS 'regulon' in Erwinia also. This study also showed that there are several differences in the gene organization of the rpoS region of the E. coli and E. carotovora chromosomes.
Asunto(s)
Proteínas Bacterianas/genética , Escherichia coli/genética , Genes Bacterianos , Pectobacterium carotovorum/genética , Factor sigma/genética , Secuencia de Aminoácidos , Secuencia de Bases , Catalasa/metabolismo , Clonación Molecular , Datos de Secuencia MolecularRESUMEN
Deletion of antigen-activated T cells after an immune response and during peripheral negative selection after strong T cell receptor (TCR) engagement of cycling T cells occurs by an apoptotic process termed TCR antigen-induced cell death (AID). By analyzing the timing of death, cell cycle markers, BrdU-labeled S phase cells, and phase-specific centrifugally elutriated cultures from stimulated Jurkat T cells and peripheral blood lymphocytes, we found that AID occurs from a late G1 check point prior to activation of cyclin E:Cdk2 complexes. T cells stimulated to undergo AID can be rescued by effecting an early G1 block by direct transduction of p16INK4a tumor suppressor protein or by inactivation of the retinoblastoma tumor suppressor protein (pRb) by transduced HPV E7 protein. These results suggest that AID occurs from a late G1 death check point in a pRb-dependent fashion.
Asunto(s)
Apoptosis/fisiología , Fase G1/fisiología , Linfocitos/citología , Linfocitos/ultraestructura , Receptores de Antígenos de Linfocitos T/fisiología , Ciclo Celular/fisiología , Quinasas Ciclina-Dependientes/metabolismo , Activación Enzimática , Humanos , Células Jurkat/citología , Células Jurkat/metabolismo , Células Jurkat/ultraestructura , Activación de Linfocitos/fisiología , Linfocitos/metabolismo , Fosforilación , Proteína de Retinoblastoma/metabolismo , Fase S/fisiología , Sensibilidad y EspecificidadRESUMEN
Carotenoid synthesis in Escherichia coli, when transformed with plasmid containing a carotenoid gene cluster from Erwinia herbicola (pPL376), is regulated by RpoS. When the plasmid was transformed into E. coli mutants that were oxyR minus, the intracellular carotenoid concentration dramatically increased from that observed in an oxyR plus allele. The higher carotenoid concentration in these mutants correlated with an increase in rpoS transcription as indicated by beta-galactosidase activity from a rpoS::lacZ promoter fusion. This indication of a higher concentration of carotenoids correlated with an increased resistance to hydrogen peroxide and near-ultraviolet radiation (310-400 nm; near-UV).
Asunto(s)
Proteínas Bacterianas/metabolismo , Proteínas Bacterianas/fisiología , Carotenoides/biosíntesis , Proteínas de Unión al ADN , Erwinia/genética , Escherichia coli/genética , Proteínas Represoras/fisiología , Factor sigma/metabolismo , Factores de Transcripción/fisiología , Proteínas Bacterianas/genética , Carotenoides/genética , Erwinia/metabolismo , Escherichia coli/efectos de la radiación , Proteínas de Escherichia coli , Estrés Oxidativo , Plásmidos/metabolismo , Regiones Promotoras Genéticas , Factor sigma/genética , Espectrofotometría Atómica , Transcripción Genética , Rayos UltravioletaRESUMEN
A hybridization assay for the detection of Pneumocystis carinii was developed using a repetitive DNA fragment of P.c. hominis. The assay was specific as different micro-organisms typically found in the respiratory tract, normal human lung DNA (A 549 cell line) and normal rat lung DNA did not react with the repetitive probe. In a slot blot (SB) hybridization assay, the repetitive probe was able to detect as few as 100 P.c. hominis organisms with no false-positives. The results of the SB hybridization assay were compared with an immunofluorescence (IFA) assay for the detection of P.c. hominis in 84 induced sputum (IS) samples obtained from 52 human immunodeficiency virus (HIV)-seropositive patients, 22 HIV-seronegative patients and 10 healthy individuals. Samples from 24 patients clinically diagnosed with P. carinii pneumonia (PCP) were positive for P.c. hominis by both assays. In addition, the SB assay detected P.c. hominis in 14 patients (10 HIV-positive and four HIV-negative) who were negative by IFA. All 14 samples showed a positive PCR signal for the P.c. hominis dihydrofolate reductase gene, further confirming the presence of P.c. hominis in these specimens. Twelve of these patients had a clinical course highly suggestive of PCP and were either on P. carinii prophylaxis or P. carinii chemotherapy. The other two samples were from HIV-positive patients who had respiratory illness due to causes other than P.c. hominis (disseminated histoplasmosis and fatal Bordetella pneumonia). Detection of P.c. hominis in these samples suggests that these patients may have subclinical colonization by P.c. hominis. Furthermore, P.c. hominis was detected in all 12 sequential IS samples from six AIDS patients who had primary episodes of PCR using the SB assay, while P.c. hominis was detected only in eight samples by IFA (66.6%). All six patients developed recurrent PCP within 6 months from the time the assays were performed, further illustrating the potential of the SB hybridization assay in monitoring PCP recurrence. Thus, the ability of the SB hybridization assay to detect a low parasite load suggests that this assay may become an important supplemental tool, along with current cytological methods, for detecting P.c. hominis in patient populations with lower burdens of the organism and in identifying asymptomatic carriers of the parasite in healthy and immunosuppressed individuals.
Asunto(s)
Infecciones Oportunistas Relacionadas con el SIDA/diagnóstico , Sondas de ADN , Técnicas de Sonda Molecular , Pneumocystis/aislamiento & purificación , Neumonía por Pneumocystis/diagnóstico , Esputo/microbiología , Southern Blotting , Fluoroinmunoensayo , Seronegatividad para VIH , Seropositividad para VIH , Humanos , Hibridación de Ácido Nucleico , Pneumocystis/genética , Secuencias Repetitivas de Ácidos Nucleicos , Sensibilidad y EspecificidadRESUMEN
The first phenotype described for mutations in the Escherichia coli rpoS gene was hypersensitivity to near-ultraviolet radiation and to its oxidative photoproduct, hydrogen peroxide. Initially named nur, this gene is now known to code for a sigma factor, and has acquired new names such as katF and rpoS. The role of its protein product (sigma-38) is to regulate a battery of genes as cells enter and rest in stationary phase. Some of the gene products are involved in protection against oxidants (e.g., catalases) and repair of oxidative damage (e.g., exonuclease III). Sigma-38 may also modulate transcription of certain growth phase genes, including hydroperoxidase I and glutathione reductase. Sigma-38 activity is regulated at transcriptional, translational, and protein stabilization levels. This review describes the complex mechanisms whereby sigma-38 controls various genes, the interaction of sigma-38 with other regulators, and a possible role of sigma-38 in bacterial virulence.
Asunto(s)
Proteínas Bacterianas/genética , Escherichia coli/genética , Genes Bacterianos/genética , Factor sigma/genética , Proteínas Bacterianas/metabolismo , Proteínas de Unión al ADN/metabolismo , Regulación Bacteriana de la Expresión Génica/genética , Proteínas Nucleares/metabolismo , Estrés Oxidativo/fisiología , Fenotipo , Especies Reactivas de Oxígeno/metabolismo , Factor sigma/metabolismo , Transcripción Genética/genéticaRESUMEN
RpoS (sigma-38) is the major regulator of genes for survival of Escherichia coli in the stationary phase. OxyR is a transcriptional regulator that responds to H2O2 induced stress in exponential phase. Once considered to act independently of each other, they are now known to be integrally involved in the expression of several oxidative stress genes. While it is known that in the exponential phase, OxyR is the transcriptional regulator of gor, this study has shown that RpoS regulates gor in the stationary phase. beta-Galactosidase activity of a gor::lacZ promoter fusion showed no induction in a oxyR rpoS double mutant. Challenge of a gor mutant to several oxidants showed that the gene product was not functioning as a classic antioxidant.
Asunto(s)
Proteínas Bacterianas/fisiología , Proteínas de Unión al ADN , Escherichia coli/genética , Regulación Bacteriana de la Expresión Génica/fisiología , Glutatión Reductasa/genética , Factor sigma/fisiología , Inducción Enzimática , Escherichia coli/efectos de los fármacos , Escherichia coli/fisiología , Proteínas de Escherichia coli , Etilmaleimida/farmacología , Genes Bacterianos/genética , Glutatión Reductasa/biosíntesis , Glutatión Reductasa/metabolismo , Peróxido de Hidrógeno/farmacología , Oxidantes/farmacología , Estrés Oxidativo , Proteínas Recombinantes de Fusión/biosíntesis , Proteínas Represoras/fisiología , Factores de Transcripción/fisiología , Transcripción Genética , beta-Galactosidasa/genética , beta-Galactosidasa/metabolismoRESUMEN
A repetitive genomic DNA clone (B12-2) that specifically hybridizes to Pneumocystis carinii DNA has been identified. No cross-hybridization to genomic DNA prepared from bacteria, other fungi, protozoa, or mammals was observed. Clone B12-2 is multiply represented in the P. carinii genome. By direct hybridization to DNA prepared from the lungs of immunosuppressed rats, the probe can detect the equivalent of fewer than 1,000 P. carinii organisms. A hybridization assay employing clone B12-2 has been developed to quantitate organism load in the rat model for P. carinii. Application of the assay to track the accumulation of organisms during the immunosuppression regimen as well as to monitor the efficacy of two drug therapies used clinically for the treatment of P. carinii pneumonia is described here. The clone B12-2 hybridization assay for the determination of P. carinii organism load possesses several advantageous features and thus should serve to complement conventional staining and immunohistochemical methods.
Asunto(s)
Sondas de ADN , Pneumocystis/genética , Animales , Antifúngicos/uso terapéutico , Clonación Molecular , ADN de Hongos/genética , Modelos Animales de Enfermedad , Masculino , Hibridación de Ácido Nucleico , Pneumocystis/aislamiento & purificación , Neumonía por Pneumocystis/tratamiento farmacológico , Neumonía por Pneumocystis/microbiología , Ratas , Ratas Sprague-DawleyRESUMEN
Oligonucleotide primers were prepared from a clone (B12) which has been shown to be a repetitive sequence in the rat P. carinii genome. Polymerase chain reaction was employed to amplify both rat and human P. carinii DNA. The detection limit of the assay was approximately 600 ng of total nucleic acid. Amplification products from both the rat and human isolates (ca. 780 bp) were characterized by denaturing gradient gel electrophoresis after digestion with Sau3A. No amplification products were obtained when DNA from the following potential pulmonary pathogens were used in identical reactions: Aspergillus fumigatus, Cryptococcus neoformans, Candida albicans, Mycobacterium avium-intracellulare and cytomegalovirus. In a blind study using the B12 primers, P. carinii DNA was successfully amplified in clinical samples which were positive by direct immunofluorescence assay (IFA) as well as in some specimens not identified by direct IFA.
Asunto(s)
Pneumocystis/aislamiento & purificación , Animales , Secuencia de Bases , ADN de Hongos/aislamiento & purificación , Humanos , Pulmón/microbiología , Datos de Secuencia Molecular , Reacción en Cadena de la Polimerasa , Valor Predictivo de las Pruebas , Ratas , Ratas EndogámicasRESUMEN
Although calcium ions are crucial in a variety of bacterial processes, including spore development, reports of calmodulin in procaryotes have been few. We have purified to homogeneity a calmodulinlike protein (CaLP) from sporulating cells of Bacillus subtilis grown in a chemically defined sporulation medium; purification involved heat treatment, fractionation with ammonium sulfate, affinity chromatography, and gel filtration on high-performance columns. The protein was eluted from a phenothiazine affinity column in a calcium ion-dependent manner, stained poorly with Coomassie blue and silver stain dyes, bound poorly to nitrocellulose filters, and was not an inhibitor of the major intracellular serine proteinase. It stimulated bovine brain phosphodiesterase in a dose- and Ca2(+)-dependent manner and stimulated NAD kinase from peas in a dose-dependent manner. The B. subtilis calmodulin reacted with anti-bovine brain calmodulin antibodies in enzyme-linked immunoabsorbance assays. The amino acid composition data showed it to be distinctly different from eucaryotic calmodulins, having particularly high levels of serine and glycine. The pI of the protein was estimated to be 4.9 to 5.0. The molecular weight was estimated to be 23,000 or 25,000, based on amino acid composition and detergent gel electrophoresis, respectively. The protein reacted with rhodamine isothiocyanate, which blocked its enzyme-activating capacity and greatly increased its electrophoretic mobility and Coomassie dye-binding ability.