RESUMEN

BACKGROUND: Eltrombopag is an oral, non-peptide thrombopoietin receptor agonist that has shown efficacy and safety in chronic immune thrombocytopenia (ITP). However, ethnic differences in eltrombopag exposure have been reported: area under the curve exposure to eltrombopag was 87% greater among ITP patients of East Asian descent than among ITP patients of non-East Asian ITP descent. OBJECTIVES: To evaluate the efficacy and safety of eltrombopag by using, in Japanese ITP patients, lower starting (12.5 mg) and maximum (50 mg) doses of eltrombopag than the standard starting (50 mg) and maximum (75 mg) doses approved in the USA and Europe. PATIENTS: We examined 23 Japanese patients with previously treated chronic ITP with a platelet count of < 30,000 µL(-1) in a multicenter study comprising a randomized, double-blind, placebo-controlled phase for 6-week evaluation (15 eltrombopag, and eight placebo) and an open-label phase for 6-month evaluation (23 eltrombopag). RESULTS AND CONCLUSIONS: The response rate (platelet count of ≥ 50,000 µL(-1) ) at week 6 of the 6-week double-blind phase was 60% in eltrombopag-treated patients and 0% in placebo-treated patients. Ten of 23 patients (43.5%) responded for ≥ 75% of predefined assessment visits during the 6-month open-label phase. Notably, 22% (5/23) of patients responded to 12.5 mg of eltrombopag, which was administered within the first 3 weeks of eltrombopag treatment. Bleeding decreased with eltrombopag treatment as compared with baseline. Eltrombopag was generally well tolerated; one patient experienced a transient ischemic attack on day 9. Eltrombopag (12.5-50 mg) is effective for the management of Japanese patients with chronic ITP (NCT00540423).

Asunto(s)

Pueblo Asiatico , Benzoatos/administración & dosificación , Plaquetas/efectos de los fármacos , Fármacos Hematológicos/administración & dosificación , Hemorragia/prevención & control , Hidrazinas/administración & dosificación , Púrpura Trombocitopénica Idiopática/tratamiento farmacológico , Pirazoles/administración & dosificación , Administración Oral , Adulto , Anciano , Benzoatos/efectos adversos , Benzoatos/farmacocinética , Plaquetas/inmunología , Plaquetas/metabolismo , Enfermedad Crónica , Método Doble Ciego , Femenino , Fármacos Hematológicos/efectos adversos , Fármacos Hematológicos/farmacocinética , Hemorragia/sangre , Hemorragia/etnología , Hemorragia/inmunología , Humanos , Hidrazinas/efectos adversos , Hidrazinas/farmacocinética , Japón/epidemiología , Masculino , Persona de Mediana Edad , Placebos , Recuento de Plaquetas , Púrpura Trombocitopénica Idiopática/sangre , Púrpura Trombocitopénica Idiopática/etnología , Púrpura Trombocitopénica Idiopática/inmunología , Pirazoles/efectos adversos , Pirazoles/farmacocinética , Receptores de Trombopoyetina/agonistas , Receptores de Trombopoyetina/sangre , Factores de Tiempo , Resultado del Tratamiento

RESUMEN

BACKGROUND: CD5-positive (CD5+) diffuse large B-cell lymphoma (DLBCL) shows poor prognosis and frequent central nervous system (CNS) relapses under anthracycline-containing chemotherapy. The aim of this study was to determine the prognosis and CNS relapse incidence of CD5+ DLBCL in the rituximab era. PATIENTS AND METHODS: We analyzed 337 patients with CD5+ DLBCL who received chemotherapy with (R-chemotherapy group; n = 184) or without (chemotherapy group; n = 153) rituximab. RESULTS: No significant difference was found in clinical background comparisons between the two groups. In the R-chemotherapy group, 60% of the patients were older than 65 years at diagnosis. Both the complete response rate and overall survival (OS) were significantly better in the R-chemotherapy group (P = 0.0003 and P = 0.002, respectively). Multivariate analysis confirmed that chemotherapy without rituximab was associated with unfavorable OS. However, the probability of CNS relapse did not differ between the two groups (P = 0.89). The CNS relapse was strongly associated with short OS (P < 0.0001). In the R-chemotherapy group, 83% of patients who experienced CNS relapse had parenchymal disease. CONCLUSIONS: Our results indicate that rituximab improves the OS of patients with CD5+ DLBCL but does not decrease the CNS relapse rate. More effective treatments with CNS prophylaxis are needed for CD5+ DLBCL patients.

Asunto(s)

Protocolos de Quimioterapia Combinada Antineoplásica/uso terapéutico , Neoplasias Encefálicas/tratamiento farmacológico , Antígenos CD5/metabolismo , Linfoma de Células B Grandes Difuso/tratamiento farmacológico , Recurrencia Local de Neoplasia/tratamiento farmacológico , Adulto , Anciano , Anciano de 80 o más Años , Anticuerpos Monoclonales de Origen Murino/administración & dosificación , Neoplasias Encefálicas/metabolismo , Neoplasias Encefálicas/secundario , Ciclofosfamida/administración & dosificación , Doxorrubicina/administración & dosificación , Femenino , Estudios de Seguimiento , Humanos , Linfoma de Células B Grandes Difuso/metabolismo , Linfoma de Células B Grandes Difuso/patología , Masculino , Persona de Mediana Edad , Recurrencia Local de Neoplasia/metabolismo , Recurrencia Local de Neoplasia/patología , Prednisona/administración & dosificación , Inducción de Remisión , Estudios Retrospectivos , Rituximab , Tasa de Supervivencia , Resultado del Tratamiento , Vincristina/administración & dosificación , Adulto Joven

RESUMEN

The complete genomic sequence of an aerobic thermoacidophilic crenarchaeon, Sulfolobus tokodaii strain7 which optimally grows at 80 degrees C, at low pH, and under aerobic conditions, has been determined by the whole genome shotgun method with slight modifications. The genomic size was 2,694,756 bp long and the G + C content was 32.8%. The following RNA-coding genes were identified: a single 16S-23S rRNA cluster, one 5S rRNA gene and 46 tRNA genes (including 24 intron-containing tRNA genes). The repetitive sequences identified were SR-type repetitive sequences, long dispersed-type repetitive sequences and Tn-like repetitive elements. The genome contained 2826 potential protein-coding regions (open reading frames, ORFs). By similarity search against public databases, 911 (32.2%) ORFs were related to functional assigned genes, 921 (32.6%) were related to conserved ORFs of unknown function, 145 (5.1%) contained some motifs, and remaining 849 (30.0%) did not show any significant similarity to the registered sequences. The ORFs with functional assignments included the candidate genes involved in sulfide metabolism, the TCA cycle and the respiratory chain. Sequence comparison provided evidence suggesting the integration of plasmid, rearrangement of genomic structure, and duplication of genomic regions that may be responsible for the larger genomic size of the S. tokodaii strain7 genome. The genome contained eukaryote-type genes which were not identified in other archaea and lacked the CCA sequence in the tRNA genes. The result suggests that this strain is closer to eukaryotes among the archaea strains so far sequenced. The data presented in this paper are also available on the internet homepage (http://www.bio.nite.go.jp/E-home/genome_list-e.html/).

Asunto(s)

Genoma Arqueal , Sulfolobus/genética , Proteínas Arqueales/genética , Secuencia de Bases , Mapeo Cromosómico , Cromosomas de Archaea/genética , Codón/genética , Secuencia Conservada , ADN de Archaea/genética , Transporte de Electrón/genética , Duplicación de Gen , Datos de Secuencia Molecular , Sistemas de Lectura Abierta , Plásmidos/genética , ARN de Archaea/genética , Sulfuros/metabolismo , Sulfolobus/metabolismo

RESUMEN

Histone acetylation and deacetylation are closely linked to transcriptional activation and repression, respectively. In acute promyelocytic leukemia (APL), histone deacetylase inhibitors (HDACIs) have a synergistic effect with all-trans retinoic acid (ATRA) in vitro to induce differentiation. Here we report in vitro and in vivo effects of a HDACI, FK228 (formerly FR901228 or depsipeptide), on the human APL cell line NB4. FK228 had a strong and irreversible cytotoxicity compared with another HDACI, trichostatin A. In vivo administration of ATRA or FK228 alone partly inhibited the growth of established tumors of NB4 subcutaneously transplanted in NOD / Shi-scid / scid mice, and the combination was synergistically effective. Histopathological examination revealed that the combination induced apoptosis and differentiation as well as histone acetylation. Intravenous injection of NB4 in NOD / Shi-scid / scid mice followed by combination treatment significantly prevented leukemia death, whereas single administration did not. These findings suggest that FK228 is a promising agent to enhance ATRA-sensitivity in the treatment of APL.

Asunto(s)

Antibacterianos/farmacología , Depsipéptidos , Inhibidores Enzimáticos/farmacología , Inhibidores de Histona Desacetilasas , Leucemia Promielocítica Aguda/patología , Péptidos Cíclicos , Animales , Antibacterianos/administración & dosificación , Apoptosis/efectos de los fármacos , Diferenciación Celular/efectos de los fármacos , Sinergismo Farmacológico , Histonas/metabolismo , Humanos , Ácidos Hidroxámicos/farmacología , Cinética , Leucemia Promielocítica Aguda/tratamiento farmacológico , Ratones , Ratones Endogámicos NOD , Ratones SCID , Trasplante de Neoplasias , Tretinoina/administración & dosificación , Tretinoina/farmacología , Células Tumorales Cultivadas

RESUMEN

Histone deacetylase inhibitors (HDACIs) have been used to focus on the effects of inducing gene expression through the acetylation of histones which results in chromatin remodeling. The study explored whether HDACIs could induce the expression of costimulatory/adhesion molecules on acute myeloid leukemia (AML) cells, thereby effectively inducing tumor immunity. The expression of CD80, CD86, human leukocyte antigen (HLA)-DR, HLA-ABC, and intracellular adhesion molecule-1 (ICAM-1) was tested in human AML cell lines after the addition of HDACI, sodium butyrate (SB). Generally, increased expression of CD86 was observed by SB treatment in a majority of cell lines, and ICAM-1 was expressed in fewer cell lines. Essentially the same results were obtained using other HDACIs such as FR901228, trichostatin A, and trapoxin A. Quantitation of transcripts of CD86 accompanied with RNA synthesis inhibition assay and nuclear run-on assay revealed that SB up-regulates the CD86 expression transcriptionally. Furthermore, chromatin immunoprecipitation experiments showed that HDACI treatment caused remarkable acetylation on histone H3 and H4 at CD86 promoter chromatin in vivo. In 30 clinical AML samples, CD86 expression was significantly increased (P <.001) by SB treatment, and the expression of HLA-DR and ICAM-1 was moderately increased (P <.05) by SB treatment. Finally, the allogeneic mixed leukocyte reaction (allo-MLR) against HL60 cells pretreated with SB was enhanced 4-fold compared with allo-MLR obtained with non-treated HL60 cells. These results suggest that the immunotherapeutic use of HDACIs may become a novel tool for treatment of AML. (Blood. 2000;96:3847-3856)

Asunto(s)

Antígenos CD/fisiología , Moléculas de Adhesión Celular/fisiología , Inhibidores de Histona Desacetilasas , Leucemia Mieloide/patología , Glicoproteínas de Membrana/fisiología , Acetilación/efectos de los fármacos , Enfermedad Aguda , Antígenos CD/efectos de los fármacos , Antígenos CD/genética , Antígenos CD/metabolismo , Antígeno B7-2 , Ácido Butírico/inmunología , Ácido Butírico/farmacología , Moléculas de Adhesión Celular/efectos de los fármacos , Diferenciación Celular , Cromatina , Inhibidores Enzimáticos/inmunología , Inhibidores Enzimáticos/farmacología , Histonas/metabolismo , Humanos , Inmunofenotipificación , Molécula 1 de Adhesión Intercelular/genética , Molécula 1 de Adhesión Intercelular/metabolismo , Interferón gamma/farmacología , Leucemia Mieloide/inmunología , Leucemia Mieloide/metabolismo , Prueba de Cultivo Mixto de Linfocitos , Glicoproteínas de Membrana/efectos de los fármacos , Glicoproteínas de Membrana/genética , Glicoproteínas de Membrana/metabolismo , Regiones Promotoras Genéticas , ARN Mensajero/metabolismo , Transcripción Genética/efectos de los fármacos , Tretinoina/farmacología , Células Tumorales Cultivadas , Regulación hacia Arriba

RESUMEN

Histone deacetylases are promising targets for cancer treatment. Here we studied the in vitro effects of a potent histone deacetylase inhibitor, FK228 (formerly FR901228), on human leukemia / lymphoma cells and cell lines compared with normal hematopoietic cells. In a lymphoma cell line, Raji, a nanomolar concentration of FK228 induced G1 arrest and / or apoptotic cell death, depending on the concentration and exposure time. Growth of lymphoid cell lines including Raji (N = 13) was inhibited by 50% (IC(50)) after 2-day treatment at concentrations of 0.83 to 1.87 ng / ml. Viability of clinical samples from patients with acute lymphoblastic leukemia was decreased by 50% at 0.78 +/- 0.46 ng / ml, whereas the IC(50) values for normal mononuclear cells from peripheral blood and bone marrow were 2.3 +/- 0.96 and 7.8 +/- 1.0 ng / ml, respectively. The IC(50) values for normal progenitor cells were 3.1, 4.4 and 7.8 ng / ml for BFU-E, CFU-GM and CFU-Mix, respectively. Expression levels of HDAC-1 and HDAC-3 proteins, which varied among cell lines, but were stable during the treatment with FK228, did not correlate with the sensitivity to FK288. This novel agent might be useful in the treatment of lymphoid malignancies, because the above concentrations are clinically achievable in vivo according to a recent clinical study.

Asunto(s)

Antibacterianos/toxicidad , Antibióticos Antineoplásicos/toxicidad , Apoptosis/efectos de los fármacos , Depsipéptidos , Inhibidores Enzimáticos/toxicidad , Leucemia/tratamiento farmacológico , Linfoma/tratamiento farmacológico , Péptidos Cíclicos , División Celular/efectos de los fármacos , Células Madre Hematopoyéticas/efectos de los fármacos , Células Madre Hematopoyéticas/enzimología , Histona Desacetilasa 1 , Inhibidores de Histona Desacetilasas , Histona Desacetilasas/biosíntesis , Humanos , Concentración 50 Inhibidora , Leucemia/enzimología , Leucemia/patología , Leucemia de Células B/tratamiento farmacológico , Leucemia de Células B/enzimología , Leucemia de Células B/patología , Leucemia de Células T/tratamiento farmacológico , Leucemia de Células T/enzimología , Leucemia de Células T/patología , Linfoma/enzimología , Linfoma/patología , Leucemia-Linfoma Linfoblástico de Células Precursoras/tratamiento farmacológico , Leucemia-Linfoma Linfoblástico de Células Precursoras/enzimología , Leucemia-Linfoma Linfoblástico de Células Precursoras/patología , Células Tumorales Cultivadas/efectos de los fármacos

RESUMEN

Transcription factor c-Myb plays important roles in cell survival and differentiation in immature hematopoietic cells. Here we demonstrate that c-Myb is acetylated at the carboxyl-terminal conserved domain by histone acetyltransferase p300 both in vitro and in vivo. The acetylation sites in vivo have been located at the lysine residues of the conserved domain (K471, K480, K485) by the use of the mutant Myb (Myb-KAmut), in which all three lysine residues are substituted into alanine. Electrophoretic mobility shift assay reveals that Myb-KAmut shows higher DNA binding activity than wild type c-Myb and that acetylation of c-Myb in vitro by p300 causes dramatic increase in DNA binding activity. Accordingly, transactivation activity of both mim-1 and CD34 promoters by Myb-KAmut is higher than that driven by wild type c-Myb. Furthermore, the bromodomain of p300, in addition to the histone acetyltransferase (HAT) domain, is required for effective acetylation of c-Myb, and hGCN5 is revealed to be a factor acetyl-transferase for c-Myb in vitro. We present a new manner of post-translational modification of the c-Myb protein and the potential significance of the acetylation in c-Myb.

Asunto(s)

Acetiltransferasas/fisiología , Proteínas de Ciclo Celular/fisiología , Proteínas Proto-Oncogénicas c-myb/metabolismo , Proteínas de Saccharomyces cerevisiae , Transactivadores/fisiología , Acetilación , Secuencia de Aminoácidos , Animales , Línea Celular , Secuencia Conservada , ADN/metabolismo , Histona Acetiltransferasas , Humanos , Datos de Secuencia Molecular , Conejos , Factores de Transcripción , Factores de Transcripción p300-CBP

RESUMEN

We previously showed that aminobisphosphonates (aminoBPs), potent inhibitors of bone resorption, increased the number of osteoclasts and granulocytes, and enhanced the cell size of osteoclasts in vivo, indicating that aminoBPs have a profound effect on murine haemopoiesis. The possible effect of an aminoBP (4-amino-1-hydroxybutylidene-1,1-bisphosphonate; AHBuBP) on murine haemopoiesis in vivo was examined in more detail. Macroscopically, AHBuBP induced the whitened bone marrow (BM) and splenomegaly. Flow cytometric analysis indicated that in BM, AHBuBP reduced the number of mature monocyte-macrophage lineage cells and erythroid cells 1 and 2 d after treatment, respectively, whereas it enhanced granulopoiesis on day 4. In the spleen, both erythropoiesis and granulopoiesis were significantly increased. BM haemopoietic progenitors of granulocyte lineage and of monocyte-macrophage lineage (CFU-G, CFU-M and CFU-GM) were well maintained by the injection of AHBuBP, and even a small increment in these progenitors was observed 2-4 d after treatment. Immunohistochemical examination of BM demonstrated that residential macrophages of erythroblastic islands disappeared. Increased numbers of osteoclasts, as well as enlarged cell size, was confirmed up to 7 d after the treatment, implicating that the inhibition of bone resorption was not due to the reduced generation of osteoclasts by AHBuBP. These results suggest (1) that AHBuBP treatment in vivo rapidly deleted mature residential macrophages from BM, (2) that mature macrophages once deleted did not reappear even when CFU-M and CFU-GM increased in number and the number of Mac-1+/Gr-1- cells recovered to normal, (3) that BM erythropoiesis was significantly decreased due to the lack of erythroblastic islands, and (4) that compensatory erythropoiesis was evoked in the spleen to induce splenomegaly.

Asunto(s)

Difosfonatos/farmacología , Hematopoyesis/efectos de los fármacos , Animales , Médula Ósea/efectos de los fármacos , Resorción Ósea , Macrófagos/metabolismo , Ratones , Bazo/efectos de los fármacos

RESUMEN

We investigated the effect of the histone deacetylase inhibitors (HDIs), trichostatin A and trapoxin A on leukemia cells and cell lines from the viewpoint of differentiation induction. TSA induced differentiation in erythroid cell lines by itself, whereas it synergistically enhanced the differentiation that was directed by all-trans retinoic acid (ATRA) or vitamin D3 in U937, HL60 and NB4 cells. The combined treatment of HDI with ATRA induced differentiation in ATRA-resistant HL60 and NB4 cells. The transcriptional expression during the treatment with HDI was examined in HL60, U937 and MEG-O1. Cell cycle-regulator genes (p21waf1 and p16INK4A) were upregulated or constantly expressed, erythroid-specific genes (GATA-1, beta-globin) were silent or downregulated, and housekeeping genes (beta-actin and GAPDH) were constantly expressed. Twelve of 35 (34%) clinical samples from AML patients ranging from M0 to M7 also displayed both phenotypical and morphological changes by the treatment with TSA alone. HDIs are thus the potent inducer or enhancer of differentiation in acute myeloid leukemia and regulate transcription in an ordered manner.

Asunto(s)

Antineoplásicos/uso terapéutico , Inhibidores Enzimáticos/farmacología , Inhibidores de Histona Desacetilasas , Leucemia Mieloide/tratamiento farmacológico , Péptidos , Células 3T3 , Enfermedad Aguda , Animales , Antibacterianos/uso terapéutico , Diferenciación Celular/efectos de los fármacos , Línea Celular , Células Precursoras Eritroides/efectos de los fármacos , Regulación Neoplásica de la Expresión Génica/efectos de los fármacos , Ácidos Hidroxámicos/uso terapéutico , Megacariocitos/efectos de los fármacos , Ratones , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Células Tumorales Cultivadas

RESUMEN

Analysis of reports about incidental and accidental events in nursing care were made using a reliability engineering method. Unnatural working hours, such as evening duty, night duty falling next to a holiday, two consecutive night-duty shifts, and two consecutive evening-duty shifts were major factors in the occurrence of errors. In a mixed-division ward (a ward containing patients belonging to different divisions), rule-based errors happened more frequently than in a single-division ward. Also, less experienced nursing staffs made errors more frequently than experienced nursing staffs.

Asunto(s)

Errores Médicos/estadística & datos numéricos , Atención de Enfermería , Análisis y Desempeño de Tareas , Servicio de Cardiología en Hospital , Humanos , Japón

Asunto(s)

Diferenciación Celular/efectos de los fármacos , Células Madre Hematopoyéticas/citología , Inhibidores de Histona Desacetilasas , Leucemia/tratamiento farmacológico , Humanos

RESUMEN

Aortic smooth muscle cells (A-SMC) undergo phenotypic transition to a synthetic and proliferative state and become fibroblast-like cells upon serial passage with culture on plastic dishes, especially in the presence of serum. Such fibroblast-like cells (M-SMC) derived from A-SMC may correspond to the cells identified pathologically as myofibroblasts. We examined the effects of type IV collagen gels used as a culture substrate on the morphology and proliferation of M-SMC. The M-SMC underwent extreme elongation in shape when cultured on rigid type IV collagen gels, and eventually formed cell-to-cell junctions with the elongated processes. In contrast, M-SMC showed a spindle-like cell shape on dishes coated with a type IV collagen solution or type I collagen solution, or on type I collagen gels or fragile type IV collagen gels. Cell proliferation was totally repressed by culture on rigid type IV collagen gels for over 10 days, while the highest proliferative activity was seen for cells grown on dishes coated with type IV collagen solution. The expression of smooth muscle myosin heavy chains, specific markers for contractile A-SMC, was acquired by M-SMC cultured on rigid type IV collagen gels for 3 days, while M-SMC cultured on type IV collagen-coated dishes continued to show no expression. These results suggest that the quiescent and contractile phenotype of A-SMC might be restored in M-SMC by culture on rigid type IV collagen gels, even after they have become myofibroblastic.

Asunto(s)

Músculo Liso Vascular/citología , Animales , Bovinos , División Celular , Tamaño de la Célula , Células Cultivadas , Colágeno , Medios de Cultivo , Fibroblastos/citología , Geles , Humanos , Fenotipo

RESUMEN

The complete sequence of the genome of an aerobic hyper-thermophilic crenarchaeon, Aeropyrum pernix K1, which optimally grows at 95 degrees C, has been determined by the whole genome shotgun method with some modifications. The entire length of the genome was 1,669,695 bp. The authenticity of the entire sequence was supported by restriction analysis of long PCR products, which were directly amplified from the genomic DNA. As the potential protein-coding regions, a total of 2,694 open reading frames (ORFs) were assigned. By similarity search against public databases, 633 (23.5%) of the ORFs were related to genes with putative function and 523 (19.4%) to the sequences registered but with unknown function. All the genes in the TCA cycle except for that of alpha-ketoglutarate dehydrogenase were included, and instead of the alpha-ketoglutarate dehydrogenase gene, the genes coding for the two subunits of 2-oxoacid:ferredoxin oxidoreductase were identified. The remaining 1,538 ORFs (57.1%) did not show any significant similarity to the sequences in the databases. Sequence comparison among the assigned ORFs suggested that a considerable member of ORFs were generated by sequence duplication. The RNA genes identified were a single 16S-23S rRNA operon, two 5S rRNA genes and 47 tRNA genes including 14 genes with intron structures. All the assigned ORFs and RNA coding regions occupied 89.12% of the whole genome. The data presented in this paper are available on the internet homepage (http://www.mild.nite.go.jp).

Asunto(s)

Archaea/genética , ADN de Archaea/genética , Genoma , Archaea/metabolismo , Secuencia de Bases , Datos de Secuencia Molecular , Sistemas de Lectura Abierta , Oxígeno/metabolismo , Reacción en Cadena de la Polimerasa , ARN de Archaea/genética , Secuencias Repetitivas de Ácidos Nucleicos , Mapeo Restrictivo

RESUMEN

1. When injected intraperitoneally into mice in doses larger than those used clinically, all the amino derivatives of bisphosphonates (aminoBPs) tested induce a variety of inflammatory reactions such as induction of histidine decarboxylase (HDC, the histamine-forming enzyme), hypertrophy of the spleen, atrophy of the thymus, hypoglycaemia, ascites and accumulation of exudate in the thorax, and an increase in the number of macrophages and/or granulocytes in the peritoneal cavity of blood. On the other hand, dichloromethylene bisphosphonate (Cl2MBP) a typical non-aminoBP, has no such inflammatory actions. In the present study, we found that this agent can suppress the inflammatory actions of aminoBPs. 2. Cl2MBP, when injected into mice before or after injection of 4-amino-1-hydroxybutylidene-1,1-bisphosphonic acid (AHBuBP; a typical aminoBP), inhibited the induction of HDC activity by AHBuBP in a dose- and time-dependent manner. The increase in HDC activity induced by AHBuBP was largely suppressed by the injection of an equimolar dose of Cl2MBP. Cl2MBP also inhibited other AHBuBP-induced inflammatory reactions, as well as the inflammatory actions of two other aminoBPs. However, Cl2MBP did not inhibit the increase in HDC activity induced by lipopolysaccharide (LPS). 3. We have previously reported that AHBuBP augments the elevation of HDC activity and the production of interleukin-1beta (IL-1beta) that are induced by LPS. These actions of AHBuBP were also inhibited by Cl2MBP. 4. Based on these results and reported actions of bisphosphonates, the mechanisms underlying the contrasting effects of aminoBPs and Cl2MBP, a non-aminoBP are discussed. The results suggest that combined administration of Cl2MBP and an aminoBP in patients might be a useful way of suppressing the inflammatory side effects of aminoBPs.

Asunto(s)

Antiinflamatorios/farmacología , Difosfonatos/antagonistas & inhibidores , Difosfonatos/farmacología , Animales , Resorción Ósea/prevención & control , Relación Dosis-Respuesta a Droga , Hematopoyesis/efectos de los fármacos , Histidina Descarboxilasa/metabolismo , Interleucina-1/biosíntesis , Lipopolisacáridos/farmacología , Masculino , Ratones , Ratones Endogámicos BALB C

RESUMEN

1. Aminobisphosphonates (aminoBPs), potent inhibitors of bone resorption, have been reported to induce inflammatory reactions such as fever and an increase in acute phase proteins in human patients, and to induce the histamine-forming enzyme, histidine decarboxylase, in mice. In the present study, we examined the effect of aminoBP, 4-amino-1-hydroxybutylidene-1,1-bisphosphonic acid (AHBuBP), on the production of the pro-inflammatory cytokines, IL-1 and TNFalpha, in mice. 2. Intraperitoneal injection of AHBuBP did not itself produce detectable levels of IL-1 (alpha and beta) and TNFalpha in the serum. However, the elevation of serum IL-1 induced by lipopolysaccharide (LPS) was greatly augmented in mice injected with AHBuBP 3 days before the LPS injection, whereas the LPS-induced elevation of serum TNFalpha was almost completely abolished. 3. Spleen and bone marrow cells taken from mice injected with AHBuBP produced IL-1beta in vitro spontaneously, and the production was augmented following the addition of LPS. Cells that accumulated in the peritoneal cavity in response to AHBuBP produced a particularly large amount of IL-1beta. However, AHBuBP treatment of mice did not lead to an impairment of the in vitro production of TNFalpha by these three types of cells. 4. Liposomes encapsulating dichloromethylene bisphosphonate (a non-amino BP) selectively deplete phagocytic macrophages. When an intraperitoneal injection of these liposomes was given 2 days after an injection of AHBuBP, there was a marked decrease in the LPS-induced elevation of serum IL-1 (alpha and beta) (LPS being injected 3 days after the injection of AHBuBP). 5. These results indicate that AHBuBP has contrasting effects on the in vivo LPS-induced production of IL-1 and TNFalpha in mice, enhancing the production of IL-1 by phagocytic macrophages and suppressing the production of TNFalpha, although underling mechanisms remain to be clarified.

Asunto(s)

Alendronato/farmacología , Interleucina-1/metabolismo , Factor de Necrosis Tumoral alfa/metabolismo , Animales , Médula Ósea/metabolismo , Células Cultivadas , Lipopolisacáridos/farmacología , Liposomas/farmacología , Macrófagos/efectos de los fármacos , Masculino , Ratones , Ratones Endogámicos BALB C , Fagocitosis , Bazo/metabolismo , Factores de Tiempo

RESUMEN

The c-MYB proto-oncogene encodes a transcription factor which plays an important role in hematopoiesis. We detected truncated c-MYB mRNA (2.0 kb) and c-Myb protein (55 kDa) in the TK-6 cell line, which was established from a patient with chronic myelogenous leukemia in T cell blast crisis. Mutated c-MYB cDNA clone (WTK-1) was isolated from a TK-6 cell cDNA library and sequenced in its entirety. Compared with the wild-type human c-MYB sequence, the WTK-1 sequence diverged at the 3' ends of exons 9. A termination codon was present as the second codon downstream from the point of divergence and was followed by a previously unknown rearranged sequence. The conceptual protein encoded by WTK-1 (Myb(TK-6)) comprises 402 amino acids and lacks the negative regulatory domain of the normal c-Myb, reminiscent of the activated form of Myb protein. Luciferase reporter assay in NIH3T3 cells showed that the expression vector encoding Myb(TK-6) stimulated Myb-regulated mim-1 promoter more effectively than that encoding wild-type human c-Myb, suggesting that Myb(TK-6) is functional as a transcription factor, and thus as a potential transforming protein. Southern blot and mutant allele-specific polymerase chain reaction analyses showed that the same rearrangement of the c-MYB gene in TK-6 was present in late, but not in early, specimens obtained from the patient, indicating that this mutation had been acquired during disease progression.

Asunto(s)

Cromosomas Humanos Par 6/genética , Leucemia Mielógena Crónica BCR-ABL Positiva/metabolismo , Proteínas Proto-Oncogénicas/genética , Transactivadores/genética , Adulto , Secuencia de Bases , Crisis Blástica/metabolismo , ADN Complementario/genética , ADN de Neoplasias/genética , Células HL-60 , Células HeLa , Humanos , Masculino , Datos de Secuencia Molecular , Proto-Oncogenes Mas , Proteínas Proto-Oncogénicas/metabolismo , Proteínas Proto-Oncogénicas c-myb , Análisis de Secuencia de ADN , Transactivadores/metabolismo , Activación Transcripcional , Células Tumorales Cultivadas

RESUMEN

The expression of mRNA encoding the inflammatory cytokines interleukin-1alpha (IL-1alpha), interleukin-1beta (IL-1beta), interleukin-6 (IL-6), interleukin-8 (IL-8) and tumor necrosis factor alpha(TNF-alpha) have been examined in radicular cysts by in situ hybridization. Furthermore, the biological activity of the contents of radicular cysts (RCC) has been assayed by adding extracts of RCC to cultured human gingival fibroblasts (HGFs) and analyzing the culture medium for the release of inflammatory cytokines. In the epithelial layer, keratinocytes expressed all cytokine mRNAs examined at various levels. Basal layer cells expressed mRNA for each cytokine. In the subepithelial granulation tissue of the cysts, fibroblasts and macrophages expressed mRNA for IL-6, IL-8, IL-1beta and TNF-alpha mRNA at varying levels; especially clear expression of TNF-alpha and IL-1beta mRNA was detected on macrophages. The infiltrating lymphoid cells, largely composed of T cells and plasma cells, expressed these cytokine mRNAs, especially those encoding IL-6 and IL-8, at various levels. In vitro analysis indicated dose-dependent release of both IL-6 and IL-8 by HGFs in response to RCC. After heating to 100 degrees C for 10 min, RCC almost completely failed to stimulate IL-6 release from HGFs. Furthermore, anti-IL-1beta antibody (neutralization test) did not prevent the stimulation of IL-6 release by RCC. Significant amounts of IL-6 and IL-8 were detected in RCC in two cases, and a trace amount of IL-1beta was detected in one case. This study demonstrated the wide expression of mRNA encoding inflammatory cytokines in radicular cyst tissues, and RCC itself was capable of stimulating IL-6 and IL-8 production from HGFs.

Asunto(s)

Citocinas/genética , Quiste Radicular/inmunología , Anticuerpos/farmacología , Células Cultivadas , Líquido Quístico/inmunología , Citocinas/metabolismo , Fibroblastos/inmunología , Humanos , Inmunohistoquímica , Hibridación in Situ , Interleucina-1/genética , Interleucina-1/inmunología , Interleucina-1/metabolismo , Interleucina-6/genética , Interleucina-6/metabolismo , Interleucina-8/genética , Interleucina-8/metabolismo , ARN Mensajero/metabolismo , Quiste Radicular/genética , Factor de Necrosis Tumoral alfa/genética

RESUMEN

The complete sequence of the genome of a hyper-thermophilic archaebacterium, Pyrococcus horikoshii OT3, has been determined by assembling the sequences of the physical map-based contigs of fosmid clones and of long polymerase chain reaction (PCR) products which were used for gap-filling. The entire length of the genome was 1,738,505 bp. The authenticity of the entire genome sequence was supported by restriction analysis of long PCR products, which were directly amplified from the genomic DNA. As the potential protein-coding regions, a total of 2061 open reading frames (ORFs) were assigned, and by similarity search against public databases, 406 (19.7%) were related to genes with putative function and 453 (22.0%) to the sequences registered but with unknown function. The remaining 1202 ORFs (58.3%) did not show any significant similarity to the sequences in the databases. Sequence comparison among the assigned ORFs in the genome provided evidence that a considerable number of ORFs were generated by sequence duplication. By similarity search, 11 ORFs were assumed to contain the intein elements. The RNA genes identified were a single 16S-23S rRNA operon, two 5S rRNA genes and 46 tRNA genes including two with the intron structure. All the assigned ORFs and RNA coding regions occupied 91.25% of the whole genome. The data presented in this paper are available on the internet at http:@www.nite.go.jp.

Asunto(s)

Genes Arqueales , Genoma , Pyrococcus/genética , Cromosomas de Archaea , Codón , ADN de Archaea/genética , ADN de Archaea/aislamiento & purificación , Vectores Genéticos , Biblioteca Genómica , Datos de Secuencia Molecular , Sistemas de Lectura Abierta , Reacción en Cadena de la Polimerasa , ARN de Archaea/genética , ARN de Transferencia/genética , Mapeo Restrictivo , Análisis de Secuencia de ADN , Operón de ARNr/genética

Asunto(s)

Genes Arqueales , Genoma , Pyrococcus/genética , Mapeo Cromosómico , Cromosomas de Archaea , Sistemas de Lectura Abierta , ARN de Archaea/genética , ARN Ribosómico/genética , ARN de Transferencia/genética

RESUMEN

The telomerase activity of various hematologic disorders, including malignant and non-malignant ones is discussed in this paper. In total of 137 cases, each positivity of telomerase activity was MDS = 17/51, overt leukemia from MDS = 6/15, AML = 17/21, ALL = 4/6, CML-CP (chronic phase) = 0/10, CML-BC (blastic crisis) = 4/4, MPD (myeloproliferative disease)-BC = 3/3, CLL = 1/10, MM (multiple myeloma) = 0/6, aplastic anemia = 3/5, essential thrombocytosis = 0/3, and polycythemia vera = 1/3. The MPD-BC showed very high level of telomerase activity as well as CML-BC cases. From the analysis for 18 cases of AML and/or malignant lymphoma patients, significant results showed that the expression of cyclin D/E was not related to telomerase activity in these hematologic disease, as was not the case with breast cancer which was reported formerly.

Asunto(s)

Enfermedades Hematológicas/enzimología , Telomerasa/análisis , Animales , Humanos , Leucemia/enzimología , Linfoma/enzimología