RESUMEN
Heat shock protein 90 (Hsp90), a molecular chaperone, is ubiquitous and involved in numerous cellular processes. To contribute to the relatively small collection of vertebrate Hsp90 sequences in the gene data bank, we cloned and sequenced horse (Equus caballus) Hsp90 alpha and beta cDNAs. This enabled identification of horse-specific primers for development of a convenient PCR-based method that could monitor horse stress tolerance. We analyzed the sequence data comparatively and phylogenetically with other Hsp90 cDNA sequences, and identified vertebrate-specific and isoform-specific conserved regions to facilitate future molecular investigations of Hsp90 functions. We found 4 highly conserved regions to vertebrate Hsp90 exclusively and 27 amino acids conserved among but differing between Hsp90 alpha and Hsp90 beta sequences. Protein-based phylogenetic trees revealed high conservation between mammal species within Hsp90 alpha and beta clusters. Comparison of nucleotide and amino acid substitution levels suggests that horse Hsp90 beta has undergone strong purifying selection, while rat Hsp90 beta and hamster Hsp90 alpha have been positively selected. Surprisingly, fish Hsp90 alpha genes clearly clustered with Hsp90 beta genes, and no distinct placement of fish Hsp90 alpha protein was found. The Hsp90 alpha isoform is apparently the result of beta gene duplication. Our results highlight the importance of organism- and isoform-specific Hsp90 functional analyses in describing the role of Hsp90 in cells.
Asunto(s)
Proteínas HSP90 de Choque Térmico/genética , Caballos/genética , Secuencia de Aminoácidos , Animales , Secuencia de Bases , Clonación Molecular , Proteínas HSP90 de Choque Térmico/química , Humanos , Datos de Secuencia Molecular , Filogenia , Isoformas de Proteínas/química , Isoformas de Proteínas/genética , ARN/química , ARN/genética , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa/veterinaria , Alineación de Secuencia , Análisis de Secuencia de ADN , Homología de Secuencia de Aminoácido , Homología de Secuencia de Ácido NucleicoRESUMEN
Previous biochemical data identified a host cell fraction, designated RAF-2, which stimulated influenza virus RNA synthesis. A 48-kDa polypeptide (RAF-2p48), a cellular splicing factor belonging to the DEAD-box family of RNA-dependent ATPases previously designated BAT1 (also UAP56), has now been identified as essential for RAF-2 stimulatory activity. Additionally, RAF-2p48 was independently identified as an influenza virus nucleoprotein (NP)-interacting protein, NPI-5, in a yeast two-hybrid screen of a mammalian cDNA library. In vitro, RAF-2p48 interacted with free NP but not with NP bound to RNA, and the RAF-2p48-NP complex was dissociated following addition of free RNA. Furthermore, RAF-2p48 facilitated formation of the NP-RNA complexes that likely serve as templates for the viral RNA polymerase. RAF-2p48 was shown, in both in vitro binding assays and the yeast two-hybrid system, to bind to the amino-terminal region of NP, a domain essential for RNA binding. Together, these observations suggest that RAF-2p48 facilitates NP-RNA interaction, thus leading to enhanced influenza virus RNA synthesis.
Asunto(s)
Adenosina Trifosfatasas/metabolismo , Virus de la Influenza A/metabolismo , Proteínas Nucleares/metabolismo , Nucleoproteínas/metabolismo , ARN Viral/biosíntesis , Adenosina Trifosfatasas/aislamiento & purificación , Regulación Viral de la Expresión Génica , Células HeLa , Humanos , Virus de la Influenza A/genética , Proteínas Nucleares/aislamiento & purificación , ARN Helicasas/aislamiento & purificación , ARN Helicasas/metabolismo , Empalme del ARN , ARN Viral/metabolismo , Empalmosomas/metabolismo , Técnicas del Sistema de Dos HíbridosRESUMEN
UNLABELLED: In case of 5-fluorouracil (5-FU)/leucovorin (LV) treatment, which is one of the most effective forms of chemotherapy for colorectal carcinoma, 5-FU is usually continuously infused from the venous route. However, since this continuous infusion limits the patients' active daily life, oral administration is preferable. In the present study, we evaluated the efficacy and side effects of orally administered 5-FU/LV. MATERIAL AND METHODS: In the continuous intravenous infusion group (civ group), colon 26 bearing mice were cannulated into central vein from external jugular vein. From this route, either 5, 10, or 20 mg/kg of 5-FU was continuously infused for 7 days (n = 6). In another group, either 10, 20, 40 mg/kg of 5-FU was infused orally (po group, n = 6). The other 6 animals were used for the non-treatment group. In the next series, 100 mg/kg of LV was added for each group above. Tumor volume, thymidylate synthase inhibition rate (TSIR) and body weight were measured at the end of infusion. During the experimental period, mice had free access to chow and water. RESULTS: The tumor/control (T/C) volumes ratio showed that approximately twice the orally administered 5-FU dose had an anti-tumor effect equal to that of 5-FU administered intravenously. Synergic antitumor effects by LV were only revealed in the civ group. Significant body weight loss was recognized only in the po group at a 5-FU dose of more than 20 mg/kg. In summary, since the modulation effect of LV was recognized only with continuously intravenous infusion of 5-FU, further improvement of oral administration is required in the LV/5-FU combination therapy.
Asunto(s)
Protocolos de Quimioterapia Combinada Antineoplásica/administración & dosificación , Neoplasias del Colon/tratamiento farmacológico , Administración Oral , Animales , Esquema de Medicación , Fluorouracilo/administración & dosificación , Infusiones Intravenosas , Leucovorina/administración & dosificación , RatonesRESUMEN
Transcription and replication of the influenza virus RNA genome take place in the nuclei of infected cells. Ribonucleoprotein (RNP) complexes consisting of viral RNA, RNA polymerase, and nucleocapsid protein (NP) are proven to be the catalytic unit for RNA synthesis, while it has been indicated that the viral RNA polymerase activity is modulated by host-derived nuclear factors. Here we have identified such host factors present in nuclear extracts prepared from uninfected HeLa cells with biochemical complementation assays using the in vitro RNA synthesis system. The stimulatory activity was not absorbed to phosphocellulose but was tightly bound to Q-Sepharose. The eluate recovered from Q-Sepharose was able to stimulate the RNA synthesis catalyzed by both RNP complexes and purified RNA polymerase and NP. The stimulatory activity was further separated into two distinct fractions, designated RAF-1 (RNA polymerase activating factor-1) and RAF-2 fractions, through phenyl-Sepharose column chromatography. When these fractions were fractionated through a gel filtration column, RAF-1 and RAF-2 activities were recovered in fractions corresponding to the molecular mass of 350 kDa and 60 kDa, respectively. Furthermore, the RAF-2 fraction was shown to contain an inhibitory activity, tentatively designated RIF-1 (RNA polymerase inhibitory factor-1). RIF-1 sedimented as fast as bovine serum albumin in glycerol density gradient centrifugation. Roles of these host factors are discussed in the context of viral RNA transcription and replication.
Asunto(s)
Factores Biológicos/metabolismo , ARN Polimerasas Dirigidas por ADN/metabolismo , Virus de la Influenza A/enzimología , Proteínas Nucleares/metabolismo , Factores Biológicos/aislamiento & purificación , Núcleo Celular/metabolismo , Cromatografía en Gel , Cromatografía por Intercambio Iónico , Genoma Viral , Células HeLa , Humanos , Virus de la Influenza A/genética , Peso Molecular , Proteínas Nucleares/aislamiento & purificación , ARN Viral/biosíntesis , Transcripción GenéticaRESUMEN
Autocrine motility factor (AMF) a tumor-secreted 55 kDa cytokine induces tumor cell motility by a signal transduction pathway mediated by interaction with its receptor (AMFR) a cell surface glycoprotein of 78 kDa (gp78). Here, AMF secreted by the metastatic LMF4 human oral squamous-cell carcinoma (SCC) cells, induced dose- and time-dependent morphological changes and chemotaxis of the producing cells. Expression of AMFR mRNA was associated with the metastatic ability of SCC cell variants. The data presented show for the first time that SCC cells produce AMF and express AMFR and the expression is related to their invasiveness and metastatic potentials.
RESUMEN
Fractions obtained from pine cone extract (PCE) of Pinus parviflora Sieb. et Zucc. have been shown to suppress the growth of influenza virus. The inhibitory effects of one of the fractions, Fraction VII, on the formation of RNA-viral protein complex and the viral RNA synthesis were investigated. The formation of M1-RNA or NP-RNA complex was inhibited when M1 or NP was preincubated with the PCE fraction. The in vitro viral RNA synthesis was inhibited by the PCE fraction, while this inhibitory effect was titrated out by the increasing concentration of M1 protein. These results suggest that the major target of the PCE fraction was M1 protein.
Asunto(s)
Antineoplásicos/farmacología , Gammainfluenzavirus/fisiología , Hemaglutininas Virales/metabolismo , Extractos Vegetales/farmacología , ARN Viral/metabolismo , Árboles , Proteínas Virales de Fusión , Proteínas Virales/metabolismo , Replicación Viral/efectos de los fármacos , Antineoplásicos/metabolismo , Secuencia de Bases , Cápside/biosíntesis , Cápside/aislamiento & purificación , Cápside/metabolismo , Clonación Molecular , Cartilla de ADN , Glicoproteínas/metabolismo , Hemaglutininas Virales/biosíntesis , Hemaglutininas Virales/efectos de los fármacos , Histidina , Gammainfluenzavirus/efectos de los fármacos , Datos de Secuencia Molecular , Reacción en Cadena de la Polimerasa , ARN Viral/efectos de los fármacos , Proteínas Recombinantes/biosíntesis , Proteínas Recombinantes/aislamiento & purificación , Proteínas Recombinantes/metabolismo , Lugares Marcados de Secuencia , Transcripción Genética/efectos de los fármacos , Proteínas del Núcleo Viral/biosíntesis , Proteínas del Núcleo Viral/aislamiento & purificación , Proteínas del Núcleo Viral/metabolismo , Proteínas Virales/biosíntesis , Proteínas Virales/efectos de los fármacosRESUMEN
Fourteen patients with recurrent breast cancer were treated with a combination of mitoxantrone (10 mg/m2 iv, day 1), vincristine (1 mg/m2 iv, day 1) and prednisolone (30 mg/day day 1-7). Cycles were repeated every 4 weeks and all patients received more than 3 cycles. The objective response rate was 70.0% (7 of 10). In two patients, the treatment has been continued now for more than one year. In these 2 patients, the duration of response is more than 40 weeks. The toxicity was primarily myelosuppression. Five of 14 patients (35.7%) had leukopenia of Grade 3 and 4. These patients received G-CSF therapy and recovered well from leukopenia. Nausea and vomiting were well tolerated, probably by prednisolone. This regimen was especially superior in the maintenance of quality of life because hair loss was much less compared to CAF (cyclophosphamide, ADM, 5-fluorouracil) treatment. In conclusion, MVP treatment is highly effective and safe for the treatment of recurrent breast cancer.
Asunto(s)
Protocolos de Quimioterapia Combinada Antineoplásica/uso terapéutico , Neoplasias de la Mama/tratamiento farmacológico , Recurrencia Local de Neoplasia/tratamiento farmacológico , Adulto , Anciano , Neoplasias de la Mama/patología , Esquema de Medicación , Femenino , Humanos , Persona de Mediana Edad , Mitoxantrona/administración & dosificación , Prednisolona/administración & dosificación , Vincristina/administración & dosificaciónRESUMEN
A human oral squamous-cell-carcinoma cell line, HOC313, was found to produce a factor which stimulates cell motility in an autocrine manner. The motility factor of HOC313 cells also promoted the locomotory activity of B16 murine melanoma cells reported to be sensitive to autocrine motility factor (AMF). HOC313 cells were found to express a large amount of AMF-receptor mRNA. In addition, the cell motility activity of HOC313 cells was completely blocked by pertussis toxin, a known inhibitor of AMF activity, suggesting that the motility factor of HOC313 cells may be AMF or a closely related factor. Immunocytochemical analysis has revealed that the AMF-like factor of HOC313 cells diminishes the cell-surface expression of adhesive molecule E-cadherin. These results suggest that down-regulation of E-cadherin may be involved in the cell-motility activity induced by the AMF-like factor of HOC313 cells.
Asunto(s)
Carcinoma de Células Escamosas/química , Glucosa-6-Fosfato Isomerasa/análisis , Neoplasias de la Boca/química , Animales , Cadherinas/análisis , Carcinoma de Células Escamosas/patología , Movimiento Celular/efectos de los fármacos , Cromatografía en Gel , Medios de Cultivo Condicionados , Glucosa-6-Fosfato Isomerasa/antagonistas & inhibidores , Glucosa-6-Fosfato Isomerasa/farmacología , Humanos , Melanoma Experimental/patología , Ratones , Neoplasias de la Boca/patología , Toxina del Pertussis , ARN Mensajero/análisis , ARN Neoplásico/análisis , Células Tumorales Cultivadas , Factores de Virulencia de Bordetella/farmacologíaRESUMEN
The effects of transforming growth factor-beta (TGF-beta) on three human oral squamous cell carcinoma cell lines, HSC-2, HSC-3, and HSC-4, were investigated. Although these cell lines were equally sensitive to epidermal growth factor, responses to TGF-beta were variable. Dose-dependent inhibition of cell growth and [3H]thymidine incorporation of HSC-4 were observed by the addition of TGF-beta, whereas growth inhibitory effects on HSC-2 and HSC-3 were marginal. Moreover, treatment of HSC-4 with TGF-beta led to a more than 300-fold increase in fibronectin secretion into the medium. In contrast, TGF-beta did not increase the secretion of fibronectin on HSC-2 and HSC-3. Scatchard analysis of the binding of TGF-beta suggested that all squamous cell carcinoma cell lines have similar binding properties, with two classes of binding sites for TGF-beta. Affinity labeling of 125I-TGF-beta to cell surface receptors revealed the two major affinity crosslinked bands with Mr values of 65 kDa (type I) and 280 kDa (type III). A concomitant loss of 85 kDa band (type II) was observed in all squamous carcinoma cell lines examined. Although the proportions of type I and type III receptors were variable, the type I receptor, which is reported to be the main functional receptor in mediating the TGF-beta action, was commonly observed in these squamous cell carcinoma cell lines. These results indicate that the heterogeneity in response to TGF-beta between cell lines may be due to the difference in the signal transduction pathway of TGF-beta.
Asunto(s)
Receptores de Superficie Celular/metabolismo , Factores de Crecimiento Transformadores/farmacología , Células Tumorales Cultivadas/citología , Carcinoma de Células Escamosas , División Celular/efectos de los fármacos , Línea Celular , Reactivos de Enlaces Cruzados/metabolismo , Replicación del ADN/efectos de los fármacos , Fibronectinas/análisis , Fibronectinas/biosíntesis , Humanos , Técnicas para Inmunoenzimas , Cinética , Neoplasias de la Boca , Receptores de Factores de Crecimiento Transformadores beta , Succinimidas/metabolismo , Factores de Crecimiento Transformadores/metabolismo , Células Tumorales Cultivadas/efectos de los fármacos , Células Tumorales Cultivadas/metabolismoRESUMEN
A case of a 7-month-old boy with oropharyngeal heterotropic brain tissue is presented. The lesion, which protruded from the right side of the oropharynx, occupied the oropharyngeal space and pressed the tongue forward and downward. A cranial computed tomograph showed that the mass had some radiolucent areas. Surgical treatment was performed to remove the respiratory and feeding distress. Histologically, the lesions consisted of matured glial cells, ependymal clefts with choroid plexus, and scattered pigmented cells in a part of lining cells of cyst wall.
Asunto(s)
Encéfalo , Coristoma/patología , Neoplasias Orofaríngeas/patología , Neoplasias Faríngeas/patología , Humanos , Lactante , MasculinoRESUMEN
Variant sublines LMF3, LMF4 and LMF5 with high metastatic potential were established from a human oral squamous carcinoma cell line HSC-3. These sublines metastasized to the draining lymph nodes after subcutaneous inoculation into nude mice. They were obtained by sequential selection in vivo from the parent HSC-3. At each step, the cells which metastasized to lymph nodes were cultured and reinoculated into nude mice. Two other cell lines HSC-2 and HSC-4 were also established from other patients, that had neither invasive nor metastatic potential. Biologic properties were compared among high metastatic, low metastatic and non-metastatic cells. Metastatic cells grew rapidly and invaded into surrounding tissues at the inoculated site. The incidence of pulmonary colonization after intravenous injection of tumor cells was high in selected variants. Metastatic cells formed diffuse colonies in type I collagen matrix and had a higher tendency to adhere to type IV collagen network.