RESUMEN
Seeds are continuously exposed to a wide variety of microorganisms in the soil. In addition, seeds contain large amounts of carbon and nitrogen sources that support initial growth after germination. Thus, seeds in the soil can easily promote microbial growth, and seeds are susceptible to decay. Therefore, seed defense against microorganisms is important for plant survival. Seed-microbe interactions are also important issues from the perspective of food production, in seed quality and shelf life. However, seed-microbe interactions remain largely unexplored. In this study, we established a simple and rapid assay system for the antibacterial activity of rice seed crude extracts by colorimetric quantification methods by the reduction of tetrazolium compound. Using this experimental system, the diversity of effects of rice seed extracts on microbial growth was analyzed using Escherichia coli as a bacterial model. We used collections of cultivated rice, comprising 50 accessions of Japanese landraces, 52 accessions of world rice core collections, and of 30 wild Oryza accessions. Furthermore, we attempted to find genetic factors responsible for the diversity by genome-wide association analysis. Our results demonstrate that this experimental system can easily analyze the effects of seed extracts on bacterial growth. It also suggests that there are various compounds in rice seeds that affect microbial growth. Overall, this experimental system can be used to clarify the chemical entities and genetic control of seed-microbe interactions and will open the door for understanding the diverse seed-microbe interactions through metabolites.
RESUMEN
A stable packaging cell line (Vero/BC-F) constitutively expressing fusion (F) protein of the human parainfluenza virus type 2 (hPIV2) was established for production of the F-defective and single round-infectious hPIV2 vector in a strategy for recombinant vaccine development. The F gene expression has not evoked cytostatic or cytotoxic effects on the Vero/BC-F cells and the F protein was physiologically active to induce syncytial formation with giant polykaryocytes when transfected with a plasmid expressing hPIV2 hemagglutinin-neuraminidase (HN). Transduction of the F-defective replicon RNA into the Vero/BC-F cells led to the release of the infectious particles that packaged the replicon RNA (named as hPIV2ΔF) without detectable mutations, limiting the infectivity to a single round. The maximal titer of the hPIV2ΔF was 6.0 × 10(8) median tissue culture infections dose per ml. The influenza A virus M2 gene was inserted into hPIV2ΔF, and the M2 protein was found to be highly expressed in a human lung cancer cell line after transduction. Furthermore, in vivo airway infection experiments revealed that the hPIV2ΔF was capable of delivering transgenes to hamster tracheal cells. Thus, non-transmissible or single round-infectious hPIV2 vector will be potentially applicable to human gene therapy or recombinant vaccine development.
Asunto(s)
Técnicas de Transferencia de Gen , Vectores Genéticos , Virus de la Parainfluenza 2 Humana/genética , Proteínas Virales de Fusión/genética , Animales , Línea Celular , Células Cultivadas , Chlorocebus aethiops , Cricetinae , Humanos , Virus de la Influenza A/genética , Proteínas Recombinantes/genética , Vacunas Sintéticas/genética , Células Vero , Proteínas Virales de Fusión/metabolismo , Proteínas de la Matriz Viral/genéticaRESUMEN
Ewing's sarcoma (EWS) is a malignant bone tumor that frequently occurs in teenagers. Genetic mutations which cause EWS have been investigated, and the most frequent one proved to be a fusion gene between EWS gene of chromosome 22 and the FLI1 gene of chromosome 11. However, a limited numbers of useful biological markers for diagnosis of EWS are available. In this study, we identified ADAMTS4 (a disintegrin and metalloproteinase with thrombospondin motifs) as a possible tumor marker for EWS using the retrovirus-mediated signal sequence trap method. ADAMTS4 is a secreted protein of 837 amino acids with a predicted molecular mass of 98-100 kDa. It is a member of metalloprotease family, is expressed mainly in cartilage and brain, and regulates the degradation of aggrecans. ADAMTS4 has been suggested to be involved in arthritic diseases and gliomas. Herein, we show that ADAMTS4 mRNA was expressed in all primary EWS samples and all EWS-derived cell lines examined, while its expression was detected only in small subpopulations of other solid tumors. Furthermore, ADAMTS4 expression was found to be regulated by EWS-FLI1 fusion gene-dependent manner. We also demonstrated that ADAMTS4 protein was highly expressed in tumor samples of the patients with EWS by using immunohistochemistry. These results suggest that ADAMTS4 is a novel tumor marker for EWS.
Asunto(s)
Proteínas ADAM/biosíntesis , Procolágeno N-Endopeptidasa/biosíntesis , Sarcoma de Ewing/enzimología , Proteínas ADAM/genética , Proteína ADAMTS4 , Adolescente , Animales , Línea Celular Tumoral , ADN Complementario/genética , Proteínas de Unión al ADN/genética , Proteínas de Unión al ADN/metabolismo , Regulación Neoplásica de la Expresión Génica , Humanos , Inmunohistoquímica , Ratones , Células 3T3 NIH , Proteínas de Fusión Oncogénica/genética , Proteínas de Fusión Oncogénica/metabolismo , Procolágeno N-Endopeptidasa/genética , Proteína Proto-Oncogénica c-fli-1/genética , Proteína Proto-Oncogénica c-fli-1/metabolismo , ARN Mensajero/biosíntesis , ARN Mensajero/genética , Proteína EWS de Unión a ARN , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Sarcoma de Ewing/genética , Sarcoma de Ewing/patología , Factores de Transcripción/genética , Factores de Transcripción/metabolismo , Transfección , Regulación hacia ArribaRESUMEN
Mixed-lineage-leukemia (MLL) fusion oncogenes are closely involved in infant acute leukemia, which is frequently accompanied by mutations or overexpression of FMS-like receptor tyrosine kinase 3 (FLT3). Earlier studies have shown that MLL fusion proteins induced acute leukemia together with another mutation, such as an FLT3 mutant, in mouse models. However, little has hitherto been elucidated regarding the molecular mechanism of the cooperativity in leukemogenesis. Using murine model systems of the MLL-fusion-mediated leukemogenesis leading to oncogenic transformation in vitro and acute leukemia in vivo, this study characterized the molecular network in the cooperative leukemogenesis. This research revealed that MLL fusion proteins cooperated with activation of Ras in vivo, which was substitutable for Raf in vitro, synergistically, but not with activation of signal transducer and activator of transcription 5 (STAT5), to induce acute leukemia in vivo as well as oncogenic transformation in vitro. Furthermore, Hoxa9, one of the MLL-targeted critical molecules, and activation of Ras in vivo, which was replaceable with Raf in vitro, were identified as fundamental components sufficient for mimicking MLL-fusion-mediated leukemogenesis. These findings suggest that the molecular crosstalk between aberrant expression of Hox molecule(s) and activated Raf may have a key role in the MLL-fusion-mediated-leukemogenesis, and may thus help develop the novel molecularly targeted therapy against MLL-related leukemia.
Asunto(s)
Regulación Neoplásica de la Expresión Génica , Proteínas de Homeodominio/genética , Leucemia/etiología , Proteína de la Leucemia Mieloide-Linfoide/fisiología , Quinasas raf/metabolismo , Proteínas ras/fisiología , Enfermedad Aguda , Animales , Ratones , Proteínas de Fusión Oncogénica , Receptor Cross-TalkRESUMEN
CCAAT/enhancer binding proteins (C/EBPs) have an important function in granulocytic differentiation, and are also involved in the leukemogenesis of acute myeloid leukemia (AML). Their involvement in myelomonocytic leukemia, however, is still unclear. Therefore, the expression and function of C/EBPs in myelomonocytic cells with MLL-fusion genes were investigated. Retinoic acid (RA) induced monocytic differentiation in the myelomonocytic cell lines with MLL-fusion genes, THP-1, MOLM-14 and HF-6 cells, accompanied by monocytic differentiation with the upregulation of C/EBPalpha and C/EBPepsilon. Monocytic differentiation by RA treatment was confirmed in primary AML cells using a clonogenic assay. When the activity of C/EBPalpha or C/EBPepsilon was introduced into HF-6 cells, their cellular growth was arrested through differentiation into monocytes with the concomitant marked downregulation of Myc. Cebpe mRNA was upregulated by the induction of C/EBPalpha-ER, but not vice versa, thus suggesting that C/EBPepsilon may have an important function in the differentiation process. Introduction of Myc isoforms into HF-6 cells partially antagonized the C/EBPs effects. These findings suggest that the ectopic expression of C/EBPepsilon, as well as C/EBPalpha, can induce the monocytic differentiation of myelomonocytic leukemic cells with MLL-fusion gene through the downregulation of Myc, thus providing insight into the development of novel therapeutic approaches.
Asunto(s)
Proteína alfa Potenciadora de Unión a CCAAT/biosíntesis , Proteínas Potenciadoras de Unión a CCAAT/biosíntesis , Diferenciación Celular , Monocitos/metabolismo , Proteína de la Leucemia Mieloide-Linfoide/biosíntesis , Proteínas de Fusión Oncogénica/biosíntesis , Proteína alfa Potenciadora de Unión a CCAAT/genética , Proteínas Potenciadoras de Unión a CCAAT/genética , Línea Celular Tumoral , Regulación hacia Abajo , N-Metiltransferasa de Histona-Lisina , Humanos , Leucemia Mieloide Aguda/genética , Leucemia Mieloide Aguda/metabolismo , Proteína de la Leucemia Mieloide-Linfoide/genética , Proteínas de Fusión Oncogénica/genética , Isoformas de Proteínas/genética , Isoformas de Proteínas/metabolismo , Proteínas Proto-Oncogénicas c-myc/genética , Proteínas Proto-Oncogénicas c-myc/metabolismo , ARN Mensajero/biosíntesis , ARN Mensajero/genéticaAsunto(s)
Leucemia-Linfoma Linfoblástico de Células Precursoras/genética , Leucemia-Linfoma Linfoblástico de Células Precursoras/patología , Receptor alfa de Factor de Crecimiento Derivado de Plaquetas/genética , Translocación Genética , Cromosomas Humanos Par 11 , Cromosomas Humanos Par 4 , Humanos , Lactante , Estructura Terciaria de Proteína , Receptor alfa de Factor de Crecimiento Derivado de Plaquetas/químicaRESUMEN
AIM: Loss of oestrogen synthesis capacity after menopause contributes to increases in arterial stiffness and calcification. Exercise training improves arterial stiffness and calcification. However, the mechanism of exercise training-induced improvement of arterial stiffness and calcification remains unclear. METHOD: We examined the mechanism by using aortas of sham-operated rats (sham control; SC), ovariectomized rats (OVX control; OC), OVX plus treatment with vitamin D(3) plus nicotine (VDN) rats (OV sedentary; OVSe), which is an animal model of endothelial dysfunction and arterial calcification, and voluntary running wheel exercise for 8 weeks plus OVX plus VDN rats (OV exercise; OVEx). RESULTS: The arterial tissue calcium and endothelin-1 (ET-1: a vasoconstrictor peptide and a potent regulator of arterial calcification) levels were significantly higher in OVSe rats compared with the SC and OC rats, whereas these levels in the OVEx rats were significantly lower than in the OVSe rats. Additionally, arterial expression of endothelial nitric oxide synthase (eNOS), which is an enzyme that produces nitric oxide (NO: a vasodilator substance), was reduced in OVSe rats. However, exercise training prevented the decrease in eNOS expression. Moreover, there was a significant positive correlation between arterial calcium level and arterial ET-1 level. CONCLUSION: These findings suggest that exercise training-induced improvement of ET-1 and NO prevents the impairment of endothelial function after menopause in females, and this improvement may result in less arterial calcification.
Asunto(s)
Enfermedades de la Aorta/prevención & control , Calcinosis/prevención & control , Endotelio Vascular/fisiopatología , Actividad Motora/fisiología , Animales , Enfermedades de la Aorta/metabolismo , Enfermedades de la Aorta/fisiopatología , Calcinosis/metabolismo , Calcinosis/fisiopatología , Calcio/metabolismo , Citrato (si)-Sintasa/metabolismo , Modelos Animales de Enfermedad , Endotelina-1/análisis , Endotelio Vascular/metabolismo , Estradiol/sangre , Femenino , Músculo Esquelético/enzimología , Óxido Nítrico Sintasa de Tipo III/metabolismo , Ovariectomía , Posmenopausia/metabolismo , Posmenopausia/fisiología , Ratas , Ratas Sprague-DawleyRESUMEN
Transforming growth factor-beta (TGF-beta)-stimulated clone-22 (TSC-22) was originally isolated as a TGF-beta-inducible gene. In this study, we identified TSC-22 as a potential leukemia suppressor. Two types of FMS-like tyrosine kinase-3 (Flt3) mutations are frequently found in acute myeloid leukemia: Flt3-ITD harboring an internal tandem duplication in the juxtamembrane domain associated with poor prognosis and Flt3-TKD harboring a point mutation in the kinase domain. Comparison of gene expression profiles between Flt3-ITD- and Flt3-TKD-transduced Ba/F3 cells revealed that constitutive activation of Flt3 by Flt3-TKD, but not Flt3-ITD, upregulated the expression of TSC-22. Importantly, treatment with an Flt3 inhibitor PKC412 or an Flt3 small interfering RNA decreased the expression level of TSC-22 in Flt3-TKD-transduced cells. Forced expression of TSC-22 suppressed the growth and accelerated the differentiation of several leukemia cell lines into monocytes, in particular, in combination with differentiation-inducing reagents. On the other hand, a dominant-negative form of TSC-22 accelerated the growth of Flt3-TKD-transduced 32Dcl.3 cells. Collectively, these results suggest that TSC-22 is a possible target of leukemia therapy.
Asunto(s)
Regulación Leucémica de la Expresión Génica , Regulación Neoplásica de la Expresión Génica , Genes Supresores de Tumor , Leucemia/terapia , Proteínas Represoras/uso terapéutico , Tirosina Quinasa 3 Similar a fms/química , Animales , Células HL-60 , Humanos , Leucemia/inmunología , Masculino , Ratones , Ratones Endogámicos BALB C , Trasplante de Neoplasias , Células U937 , Tirosina Quinasa 3 Similar a fms/inmunologíaRESUMEN
We recently demonstrated that STAT5 can induce a variety of biological functions in mouse IL-3-dependent Ba/F3 cells; STAT5-induced expression of pim-1, p21(WAF/Cip1), and suppressor of cytokine signaling-1/STAT-induced STAT inhibitor-1/Janus kinase binding protein is responsible for induction of proliferation, differentiation, and apoptosis, respectively. In the present study, using a constitutively active STAT5A (STAT5A1*6), we show that STAT5 induces macrophage differentiation of mouse leukemic M1 cells through a distinct mechanism, autocrine production of IL-6. The supernatant of STAT5A1*6-transduced cells contained sufficient concentrations of IL-6 to induce macrophage differentiation of parental M1 cells, and STAT3 was phosphorylated on their tyrosine residues in these cells. Treatment of the cells with anti-IL-6 blocking Abs profoundly inhibited the differentiation. We also found that the STAT5A1*6 transactivated the IL-6 promoter, which was mediated by the enhanced binding of NF-kappaB p65 (RelA) to the promoter region of IL-6. These findings indicate that STAT5A cooperates with Rel/NF-kappaB to induce production of IL-6, thereby inducing macrophage differentiation of M1 cells in an autocrine manner. In summary, we have shown a novel mechanism by which STAT5 induces its pleiotropic functions. Cytokines
Asunto(s)
Proteínas de Unión al ADN/fisiología , Interleucina-6/genética , Macrófagos/inmunología , Proteínas de la Leche , FN-kappa B/fisiología , Transactivadores/fisiología , Animales , Anticuerpos/inmunología , Apoptosis , Comunicación Autocrina , Células COS , Diferenciación Celular , Línea Celular , Proteínas de Unión al ADN/genética , Proteínas de Unión al ADN/metabolismo , Interleucina-6/biosíntesis , Interleucina-6/inmunología , Leucemia Mieloide Aguda , Macrófagos/citología , Ratones , Mutación , Fosforilación , Regiones Promotoras Genéticas , Factor de Transcripción STAT3 , Factor de Transcripción STAT5 , Transactivadores/genética , Transactivadores/metabolismo , Factor de Transcripción ReIA , Activación Transcripcional , Células Tumorales CultivadasRESUMEN
In a search for a human sequence related to a recently identified type I cytokine receptor delta1, which turned out to be a receptor subunit for a cytokine called TSLP, we have now identified a novel human type I cytokine receptor from a human T lymphocyte cDNA library. The deduced amino acid sequence of 371 residues has a typical signal sequence and a membrane-spanning region. The mature protein is predicted to have a molecular mass of 39,698 Da. The N-terminal extracellular region contains two fibronectin type III-like domains, four conserved cysteine residues, and a WSXWS box-like motif. The C-terminal intracellular region contains box 1 and box 2-like motifs. Thus, it has common characteristics of type I cytokine receptor family members, and we tentatively termed this protein CRLF2, which stands for cytokine receptor-like factor 2. Northern blot analysis revealed CRLF2 mRNA in liver, kidney, heart, and skeletal muscle. The fetal liver also expresses CRLF2 transcripts. The gene for CRLF2 was mapped to the pseudoautosomal region, Xp22.3 and Yp11.3 by FISH analysis, a region where genes encoding the IL-3 receptor alpha and the GM-CSF receptor alpha chains are also located. The biological function of this newly identified receptor is now under investigation.
Asunto(s)
Receptores de Citocinas/química , Receptores de Citocinas/genética , Secuencias de Aminoácidos , Secuencia de Aminoácidos , Animales , Secuencia de Bases , Clonación Molecular , Secuencia Conservada , Perfilación de la Expresión Génica , Humanos , Inmunoglobulinas , Hibridación Fluorescente in Situ , Ratones , Datos de Secuencia Molecular , Mapeo Físico de Cromosoma , Estructura Terciaria de Proteína , Subunidades de Proteína , ARN Mensajero/análisis , ARN Mensajero/genética , Receptores de Citocinas/clasificación , Alineación de Secuencia , Cromosoma X/genéticaRESUMEN
In the search for stromal-derived growth factors, we have identified a novel secreted short form of immune suppressor factor (ISF) using a combination of a genetic approach and retrovirus-mediated functional screening. This protein, which we termed ShIF, was isolated based on its ability to support proliferation of a mutant clone S21, which was established from Ba/F3 cells that are usually interleukin-3-dependent but became dependent on a stroma cell line ST2 after chemical mutagenesis. ISF, a membrane protein harboring six transmembrane domains, was reported to have immunosuppressive functions. The coding region of ShIF started from the third transmembrane domain of ISF. Biochemical analysis demonstrated that ShIF was expressed in both the secreted and membrane-bound forms of 27-kDa protein, which was supposed to have an internal ATG present in the third transmembrane domain of ISF as a start codon. In addition to the full-length form of ISF, a major protein with a molecular size of 27 kDa was also expressed through the proteolytic process of ISF. ShIF resembles this naturally occurring short form of ISF (sISF). Deletion analysis of the major domains of ISF cDNA revealed that ShIF is an active functional domain of ISF with a capability to support proliferation of S21 cells. Enforced expression of ShIF in MS10 cells, bone marrow stroma cells that do not express endogenous ShIF or ISF, conferred on the cells an ability to support the growth of S21 cells as well as bone marrow cells. Interestingly, ShIF shows a high sequence homology to the C-terminal part of a 95-kDa yeast vacuolar H (+)-ATPase subunit, Vph1p (39%), and a 116-kDa proton pump (VPP1) (54%) of the rat and bovine synaptic vesicle. Therefore, it is possible that ShIF also acts as a proton pump and somehow prevents the cells from undergoing apoptosis.
Asunto(s)
División Celular , Sustancias de Crecimiento/química , Bombas de Protones , ATPasas de Translocación de Protón/química , ATPasas de Translocación de Protón Vacuolares , Secuencia de Aminoácidos , Animales , Secuencia de Bases , Línea Celular , Técnicas de Cocultivo , Cartilla de ADN , ADN Complementario , Sustancias de Crecimiento/genética , Sustancias de Crecimiento/metabolismo , Hidrólisis , Ratones , Microscopía Fluorescente , Datos de Secuencia Molecular , Mutagénesis , Procesamiento Proteico-Postraduccional , Retroviridae/genéticaRESUMEN
We have recently cloned a cDNA for a full-length form of MgcRacGAP. Here we show using anti-MgcRacGAP antibodies that, unlike other known GAPs for Rho family, MgcRacGAP localized to the nucleus in interphase, accumulated to the mitotic spindle in metaphase, and was condensed in the midbody during cytokinesis. Overexpression of an N-terminal deletion mutant resulted in the production of multinucleated cells in HeLa cells. This mutant lost the ability to localize in the mitotic spindle and midbody. MgcRacGAP was also found to bind alpha-, beta-, and gamma-tubulins through its N-terminal myosin-like domain. These results indicate that MgcRacGAP dynamically moves during cell cycle progression probably through binding to tubulins and plays critical roles in cytokinesis. Furthermore, using a GAP-inactive mutant, we have shown that the GAP activity of MgcRacGAP is required for cytokinesis, suggesting that inactivation of the Rho family of GTPases may be required for normal progression of cytokinesis.
Asunto(s)
División Celular/fisiología , Activadores de GTP Fosfohidrolasa/metabolismo , Proteínas Activadoras de GTPasa/metabolismo , Huso Acromático/metabolismo , Sitios de Unión , Proteínas Activadoras de GTPasa/genética , Células Gigantes , Células HeLa , Humanos , Microtúbulos/metabolismo , Mutación , Unión Proteica , Estructura Terciaria de Proteína/genética , ARN Mensajero/biosíntesisRESUMEN
To identify the key molecules that regulate differentiation of hematopoietic cells, we carried out retrovirus-mediated functional screening for cDNAs whose expression suppresses IL-6-induced differentiation of mouse myeloid leukemic M1 cells. From this screening, we obtained a full length cDNA encoding a mouse homologue of human MgcRacGAP. Overexpression of the anti-sense MgcRacGAP profoundly inhibited IL-6-induced macrophage-differentiation of M1 cells. On the other hand, overexpression of the full-length form of MgcRacGAP alone enhanced macrophage differentiation of M1 cells in response to IL-6, and induced macrophage differentiation of HL-60 leukemic cells. To determine how this protein regulates differentiation and proliferation, an antibody against MgcRacGAP was prepared. Immunohistochemical studies revealed that MgcRacGAP mainly localizes in the nucleus in interphase, accumulates on the mitotic spindle in metaphase, and is condensed in the midbody during cytokinesis. Overexpression of an N-terminal domain deletion mutant, which lacks the ability to localize to the midbody through association with tubulins, or a GAP-inactive mutant resulted in the formation of multinucleated cells in HeLa cells as well as in hemopoietic cells. Interestingly, MgcRacGAP in the midbody was phosphorylated probably on serine and threonine residues. These results indicate that MgcRacGAP regulates cytokinesis and cellular differentiation as a regulator of Rho family of GTPase and suggest that this process is controlled by some serine/threonine kinases.
Asunto(s)
Diferenciación Celular , División Celular , Proteínas Activadoras de GTPasa/metabolismo , Proteínas de la Leche , Proteínas de Unión al GTP rho/metabolismo , Animales , Proteínas de Unión al ADN/metabolismo , Activadores de GTP Fosfohidrolasa/metabolismo , Proteínas Activadoras de GTPasa/genética , Células HeLa , Humanos , Inmunohistoquímica , Interleucina-6/farmacología , Macrófagos/efectos de los fármacos , Macrófagos/metabolismo , Ratones , Modelos Biológicos , FN-kappa B/metabolismo , Factor de Transcripción STAT5 , Transactivadores/metabolismoRESUMEN
We report here the identification of a novel member of the low-density lipoprotein receptor (the LDL receptor) family through signal sequence trap screening of a mouse lymphocyte cDNA library. The protein was termed LDL receptor-related protein 9 (LRP9). LRP9 is a type I membrane protein predicted to contain 696 amino acids with a calculated molecular mass of 74 764 Da. The NH(2)-terminal half of LRP9 contains two CUB domains separated by a single ligand-binding repeat. The second CUB domain is followed by a cluster of three additional ligand-binding repeats and a transmembrane domain. The COOH-terminal intracellular region contains a proline-rich region. LRP9 mRNA was expressed in the liver, kidney, lung, and heart at high levels, and in the spleen and brain at low levels. In situ hybridization analysis of mouse liver, kidney, and brain detected LRP9 transcripts in hepatocytes, sinusoidal lining cells, peritubular capillaries, choroid plexus, ependyma of the third ventricle, pia matter, and hippocampus. In particular, high levels of expression were observed in the vascular walls. Apolipoprotein E (apoE)-enriched beta-VLDL stimulated cellular cholesteryl ester formation in ldl-A7/LRP9. These results raise the possibility that this newly identified receptor, which is expressed in the liver, may play a physiological role in the uptake of apoE-containing lipoproteins.
Asunto(s)
Apolipoproteínas E/metabolismo , Lipoproteínas LDL/fisiología , Proteínas de Transporte de Membrana , Receptores de LDL/metabolismo , Receptores de LDL/fisiología , Adulto , Secuencia de Aminoácidos , Animales , Secuencia de Bases , Western Blotting , Línea Celular , Mapeo Cromosómico , Clonación Molecular , ADN Complementario/aislamiento & purificación , Humanos , Hibridación in Situ , Proteínas Relacionadas con Receptor de LDL , Lipoproteínas VLDL/metabolismo , Ratones , Datos de Secuencia Molecular , Especificidad de Órganos/genética , Biosíntesis de Proteínas , Estructura Terciaria de Proteína/genética , ARN Mensajero/biosíntesis , Ratas , Receptores de LDL/genética , Receptores de LDL/aislamiento & purificación , Análisis de Secuencia de Proteína , Homología de Secuencia de AminoácidoRESUMEN
OBJECTIVE: To determine the accuracy and repeatability of pulse dye densitometry (PDD) in measuring blood volume (BV) by comparing it with the conventional method using 51Cr-labeled red blood cells (RI method) and by assessing sequential measurements. DESIGN: Prospective clinical study. SETTING: University hospital. PATIENTS AND PARTICIPANTS: Eleven adult ICU patients who received cardiac surgery (1st ICU day). INTERVENTIONS: None. MEASUREMENTS AND RESULTS: After injecting indocyanine green (10 or 20 mg) into the right atrium, its arterial concentration was continuously monitored at the nose and finger by PDD, and BV was calculated by back extrapolating the logarithmic dye concentration on the dye elimination curve between 2.5 and 5.5 min after mean transit time to each mean transit time with the least squares method. These measurements were repeated in eight patients and performed only once in the other three, and the BV was measured concurrently by the RI method one time. The Bland-Altman method was used for evaluating differences between methods and within methods. The (percentage) biases and standard deviations between the PDD and RI methods and between the successive measurements by PDD at the finger and nose were 0.26 +/- 0.491 (8.8 +/- 15.3%) and 0.004 +/- 0.251 (0.06 +/- 5.9%) with the probe on a nostril, and 0.16 +/- 0.561 (2.5 +/- 14.4%) and 0.19 +/- 0.311 (4.7 +/- 7.3%) using the finger probe. The bias between methods was less than 10%, and the repeatability of PDD was better. CONCLUSIONS: As PDD can measure BV with good repeatability and with a small bias compared to the RI method, serial changes in BV can be evaluated at the bedside of critically ill patients noninvasively and repeatedly.
Asunto(s)
Determinación del Volumen Sanguíneo/métodos , Anciano , Procedimientos Quirúrgicos Cardíacos , Radioisótopos de Cromo , Colorantes , Densitometría/métodos , Femenino , Humanos , Técnicas de Dilución del Indicador , Verde de Indocianina , Unidades de Cuidados Intensivos , Masculino , Persona de Mediana Edad , Estudios Prospectivos , Reproducibilidad de los ResultadosRESUMEN
In a search for key molecules that prevent murine M1 leukemia cells from undergoing interleukin (IL)-6-induced differentiation into macrophages, we isolated an antisense complementary DNA (cDNA) that encodes full-length mouse MgcRac-GTPase-activating protein (GAP) through functional cloning. Forced expression of this antisense cDNA profoundly inhibited IL-6-induced differentiation of M1 cells into macrophage lineages. We also isolated a full-length human MgcRacGAP cDNA, which encodes an additional N-terminal polypeptide of 105 amino acid residues compared with the previously published human MgcRacGAP. In human HL-60 leukemic cells, overexpression of the full-length form of human MgcRacGAP alone induced growth suppression and macrophage differentiation associated with hypervacuolization and de novo expression of the myelomonocytic marker CD14. Analyses using a GAP-inactive mutant and 2 deletion mutants of MgcRacGAP indicated that the GAP activity was dispensable, but the myosin-like domain and the cysteine-rich domain were indispensable for growth suppression and macrophage differentiation. The present results indicated that MgcRacGAP plays key roles in controlling growth and differentiation of hematopoietic cells through mechanisms other than regulating Rac GTPase activity.
Asunto(s)
Proteínas Activadoras de GTPasa/genética , Regulación de la Expresión Génica/fisiología , Hematopoyesis/fisiología , Células Madre Hematopoyéticas/citología , Células Madre Hematopoyéticas/fisiología , Secuencia de Aminoácidos , Animales , Diferenciación Celular/efectos de los fármacos , Diferenciación Celular/fisiología , División Celular/efectos de los fármacos , División Celular/fisiología , ADN Complementario/genética , ADN Complementario/aislamiento & purificación , Regulación de la Expresión Génica/efectos de los fármacos , Células HL-60 , Hematopoyesis/efectos de los fármacos , Humanos , Interleucina-6/farmacología , Leucemia Experimental/patología , Leucemia Experimental/fisiopatología , Ratones , Datos de Secuencia Molecular , Oligonucleótidos Antisentido , Alineación de SecuenciaRESUMEN
A member of the tumor necrosis factor (TNF) superfamily, human TNFSF14 (hTNFSF14)/HVEM-L (herpes virus entry mediator ligand) was isolated as a cellular ligand for HVEM/TR2 and human lymphotoxin beta receptor (LTbetaR). TNFSF14 induces apoptosis and suppresses tumor formation. We have isolated a cDNA clone for a mouse homologue of hTNFSF14 by signal sequence trap (SST) screening which we recently developed. The deduced amino acid sequence of the mouse TNFSF14 (mTNFSF14) cDNA comprised 239 amino acid residues and was 77% identical to the hTNFSF14 protein. In Northern blot analysis, 2.1 kb and 4.2kb mTNFSF14 transcripts were detected in spleen and lung, and in heart, respectively. Fluorescence in situ hybridization analysis localized the mTNFSF14 gene Tnfsf14 to chromosome 17 which is tightly linked with Tnf, Lta, and Ltb.
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Proteínas de la Membrana/genética , Familia de Multigenes/genética , Factor de Necrosis Tumoral alfa/genética , Secuencia de Aminoácidos , Animales , Secuencia de Bases , Clonación Molecular , Perfilación de la Expresión Génica , Ligamiento Genético/genética , Humanos , Hibridación Fluorescente in Situ , Proteínas de la Membrana/química , Ratones , Datos de Secuencia Molecular , Mapeo Físico de Cromosoma , ARN Mensajero/análisis , ARN Mensajero/genética , Alineación de Secuencia , Homología de Secuencia de Aminoácido , Miembro 14 de la Superfamilia de Ligandos de Factores de Necrosis Tumoral , Factor de Necrosis Tumoral alfa/químicaRESUMEN
Hematopoietic cell growth and differentiation are controlled by a number of cytokines. Ligand stimulation induces rapid phosphorylation of the tyrosine residues of the cytokine receptor and a variety of cellular molecules. Among them, Janus kinases (JAKs) and signal transducers and activators of transcription (STATs) have recently been found to play a unique role in cytokine receptor-mediated intracellular signaling and hematopoietic cell development. Abnormal signaling of the JAK-STAT pathway results in hematopoietic disorders, including severe combined immunodeficiency and leukemia.
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Proteínas de la Leche , Proteínas Tirosina Quinasas/metabolismo , Proteínas Proto-Oncogénicas , Transactivadores/metabolismo , Animales , Diferenciación Celular , Proteínas de Unión al ADN/fisiología , Células Madre Hematopoyéticas/química , Células Madre Hematopoyéticas/enzimología , Humanos , Janus Quinasa 1 , Janus Quinasa 2 , Janus Quinasa 3 , Leucemia/metabolismo , Ratones , Ratones SCID , Factor de Transcripción STAT5 , Transducción de Señal , Transactivadores/fisiologíaRESUMEN
Transfer of the alphabeta TCR genes into T lymphocytes will provide a means to enhance Ag-specific immunity by increasing the frequency of tumor- or pathogen-specific T lymphocytes. We generated an efficient alphabeta TCR gene transfer system using two independent monocistronic retrovirus vectors harboring either of the class II MHC-restricted alpha or beta TCR genes specific for chicken OVA. The system enabled us to express the clonotypic TCR in 44% of the CD4+ T cells. The transduced cells showed a remarkable response to OVA323-339 peptide in the in vitro culture system, and the response to the Ag was comparable with those of the T lymphocytes derived from transgenic mice harboring OVA-specific TCR. Adoptive transfer of the TCR-transduced cells in mice induced the Ag-specific delayed-type hypersensitivity in response to OVA323-339 challenge. These results indicate that alphabeta TCR gene transfer into peripheral T lymphocytes can reconstitute Ag-specific immunity. We here propose that this method provides a basis for a new approach to manipulation of immune reactions and immunotherapy.
Asunto(s)
Técnicas de Transferencia de Gen , Antígenos de Histocompatibilidad Clase II/inmunología , Receptores de Antígenos de Linfocitos T alfa-beta/genética , Retroviridae/genética , Linfocitos T/inmunología , Traslado Adoptivo , Animales , Linfocitos T CD4-Positivos/inmunología , Linfocitos T CD4-Positivos/metabolismo , Línea Celular , Pollos , Células Clonales , Femenino , Genes Codificadores de la Cadena alfa de los Receptores de Linfocito T , Genes Codificadores de la Cadena beta de los Receptores de Linfocito T , Vectores Genéticos/inmunología , Antígenos de Histocompatibilidad Clase II/genética , Hibridomas/metabolismo , Inmunidad Celular/genética , Activación de Linfocitos/genética , Ratones , Ratones Endogámicos BALB C , Ratones Transgénicos , Ovalbúmina/inmunología , Fragmentos de Péptidos/inmunología , Receptores de Antígenos de Linfocitos T alfa-beta/biosíntesis , Retroviridae/inmunología , Linfocitos T/metabolismoRESUMEN
We previously identified a constitutively active form of STAT (signal transducer and activator of transcription) 5A by polymerase chain reaction-driven random mutagenesis followed by retrovirus-mediated expression screening, which had two point mutations in the DNA-binding and transcriptional activation domains, and was designated STAT5A1*6. STAT5A1*6 showed markedly elevated DNA binding and transactivation activities with stable tyrosine phosphorylation and nuclear accumulation, and conferred autonomous cell growth on interleukin 3-dependent Ba/F3 cells. We now report another constitutively active mutant, STAT5A-N642H which has a single point mutation (N642H) in its SH2 domain, identified using the same strategy as that used to identify STAT5A1*6. STAT5A-N642H showed identical properties to those of STAT5A1*6 both biochemically and biologically. Interestingly the mutation in STAT5A-N642H resulted in restoration of the conserved critical histidine which is involved in the binding of phosphotyrosine in the majority of SH2-containing proteins. Introduction of an additional mutation (Y694F) to STAT5A-N642H, which disrupted critical tyrosine 694 required for dimerization of STAT5, abolished all the activities manifested by the mutant STAT5A-N642H, which indicates that dimerization is required for the activity of STAT5A-N642H as was the case for the wild-type STAT5A. The present findings also show that different mutations rendered STAT5A constitutively active, through a common mechanism, which is similar to that of physiological activation.