RESUMEN
Despite novel targeted agents, prognosis of metastatic renal cell cancer (RCC) remains poor, and experimental therapeutic strategies are warranted. Transfection of tumor cells with co-stimulatory molecules and/or cytokines is able to increase immunogenicity. Therefore, in our clinical study, 10 human leukocyte antigen (HLA)-A(*)0201(+) patients with histologically-confirmed progressive metastatic clear cell RCC were immunized repetitively over 22 weeks with 2.5-40 × 10(6) interleukin (IL)-7/CD80 cotransfected allogeneic HLA-A(*)0201(+) tumor cells (RCC26/IL-7/CD80). Endpoints of the study were feasibility, safety, immunological and clinical responses. Vaccination was feasible and safe. In all, 50% of the patients showed stable disease throughout the study; the median time to progression was 18 weeks. However, vaccination with allogeneic RCC26/IL-7/CD80 tumor cells was not able to induce TH1-polarized immune responses. A TH2 cytokine profile with increasing amounts of antigen-specific IL-10 secretion was observed in most of the responding patients. Interferon-γ secretion by patient lymphocytes upon antigen-specific and non-specific stimulation was substantially impaired, both before and during vaccination, as compared with healthy controls. This is possibly due to profound tumor-induced immunosuppression, which may prevent induction of antitumor immune responses by the gene-modified vaccine. Vaccination in minimal residual disease with concurrent depletion of regulatory cells might be one strategy to overcome this limitation.
Asunto(s)
Vacunas contra el Cáncer/uso terapéutico , Carcinoma de Células Renales/terapia , Interleucina-7/inmunología , Neoplasias Renales/terapia , Adulto , Anciano , Antígeno B7-1/metabolismo , Vacunas contra el Cáncer/administración & dosificación , Línea Celular Tumoral , Femenino , Antígenos HLA/análisis , Humanos , Masculino , Persona de Mediana Edad , Linfocitos T/inmunología , TransfecciónRESUMEN
Genes and cDNAs encoding plant protein kinases highly homologous to the animal GSK-3/shaggy subfamily were isolated from Arabidopsis thaliana, Brassica napus, Petunia hybrida and Nicotiana tabacum using the P. hybrida PSK6 GSK-3/shaggy related cDNA as a probe. All the derived protein sequences contained the characteristic catalytic domain of GSK-3/shaggy protein kinases. Sequence comparisons within the catalytic domain with other plant GSK-3/shaggy like kinases clearly indicate that the novel sequences form an isolated group of genes termed the PSK6 group. All the proteins within this group possess an amino-terminal extension which contains short amino acid motifs highly conserved between species and possibly implicated in mitochondrial targeting. Northern hybridisation experiments and reverse transcriptase PCR analysis demonstrated that these novel cDNAs are predominantly expressed in developing pollen. The three genes isolated from P. hybrida and A. thaliana show the same genomic organisation into 12 introns and 13 exons. Although the size of the introns varies, their positions are conserved between genes and species. The comparison of these gene structures and the analysis of deduced protein sequences belonging to different plants hold important information to understand the function of individual members. They suggest that some of the characterised sequences represent most likely true orthologues whereas others must be paralogues. They also allow us to discuss the evolution of the plant GSK-3/shaggy like gene family with regard to plant speciation.
Asunto(s)
Proteínas Quinasas Dependientes de Calcio-Calmodulina/genética , Proteínas de Drosophila , Evolución Molecular , Regulación de la Expresión Génica de las Plantas , Plantas/enzimología , Plantas/genética , Polen/enzimología , Proteínas Quinasas/genética , Proteínas Serina-Treonina Quinasas/genética , Secuencia de Aminoácidos , Animales , Arabidopsis/enzimología , Arabidopsis/genética , Brassica/enzimología , Brassica/genética , Proteínas Quinasas Dependientes de Calcio-Calmodulina/química , Secuencia Conservada , Regulación del Desarrollo de la Expresión Génica , Regulación Enzimológica de la Expresión Génica , Glucógeno Sintasa Quinasa 3 , Datos de Secuencia Molecular , Familia de Multigenes , Filogenia , Proteínas de Plantas/química , Proteínas de Plantas/genética , Plantas Tóxicas , Proteínas Quinasas/química , Proteínas Serina-Treonina Quinasas/química , Alineación de Secuencia , Homología de Secuencia de Aminoácido , Nicotiana/enzimología , Nicotiana/genéticaRESUMEN
In the framework of the complete genome sequencing programme of the crucifer Arabidopsis thaliana, a 23.9-kb fragment from the long arm of chromosome IV has been analysed. This paper presents a methodological approach, integrating computerized predictions, database screening, the sequencing of cognate cDNAs and a PCR-based detection of expression that allows the accumulation of an important amount of information from an anonymous sequence. This work revealed the organization of novel genes and the vestige of a copia-like retrotransposon. The gene AtRH1 encodes the first member of a new subfamily of the plant DEAD box RNA helicases. A recurrent and complete search of dbEST has been used to evaluate the number of different RNA helicases expressed in A. thaliana. On the 18 discriminated members of the family, only a small number seems to be expressed at a relatively high level. The putative gene AtTS1 encodes a novel terpene synthase in A. thaliana, and the genes G14587-5 and G14587-6 encode unknown proteins. This study illustrates most of the situations that could be encountered during the analysis of an anonymous sequence from A. thaliana.
Asunto(s)
Transferasas Alquil y Aril/genética , Arabidopsis/genética , Genes de Plantas/genética , ARN Nucleotidiltransferasas/genética , Secuencia de Aminoácidos , Mapeo Cromosómico , ADN Complementario/genética , Expresión Génica , Datos de Secuencia Molecular , ARN Helicasas , Retroelementos/genética , Análisis de Secuencia de ADN , Homología de Secuencia de Aminoácido , Homología de Secuencia de Ácido NucleicoRESUMEN
We have isolated and characterized two Arabidopsis thaliana cDNAs and their cognate genes, At beta fruct3 and At beta fruct4, encoding vacuolar forms of invertase. Our sequencing results showed that the gene At beta fruct3 is located downstream of the 3-ketoacyl-acyl carrier protein synthase III gene (AtKasIII). At beta fruct3 and 4 are functional and organized into seven exons and six introns with an identical organization. The At beta fruct3 and At beta fruct4 genes encode, respectively, polypeptides of 648 and 664 residues that contain all the characteristic hallmarks of vacuolar invertases. A. thaliana is the first plant of which both cell-wall (At beta fruct1 and At beta fruct2) and vacuolar (At beta fruct3 and At beta fruct4) genes are characterized. The same number of exons and introns is seen in the genes At beta fruct1, At beta fruct3 and At beta fruct4 as well as in all other invertase genes described to date. However, the position of the third intron is different in At beta fruct3 and At beta fruct4. At beta fruct2 shows a different organization. A neighbour-joining distance tree shows that the A. thaliana vacuolar invertases described here are, as expected, more closely related to vacuolar invertases from other plant species (e.g., carrot) than to the A. thaliana cell-wall invertases. The evolution of plant invertase genes from a common ancestral gene is discussed. Our results demonstrate that in A. thaliana, at least two genes encoding vacuolar invertases are expressed during the development of the plant. Southern blot hybridization experiments suggest the presence of one copy of, respectively, At beta fruct3 and At beta fruct4 per haploid genome, and Northern blot analysis demonstrates that vacuolar invertase genes are highly expressed in stems, roots, flowers and at very low levels in mature leaves.
Asunto(s)
Arabidopsis/genética , Genes de Plantas/genética , Glicósido Hidrolasas/genética , Vacuolas/enzimología , Secuencia de Aminoácidos , Secuencia de Bases , Pared Celular/enzimología , Clonación Molecular , ADN Complementario/genética , ADN de Plantas/genética , Dosificación de Gen , Regulación de la Expresión Génica de las Plantas/fisiología , Intrones/genética , Datos de Secuencia Molecular , Familia de Multigenes/genética , Filogenia , ARN Mensajero/análisis , ARN de Planta/análisis , Mapeo Restrictivo , Alineación de Secuencia , Análisis de Secuencia de ADN , beta-FructofuranosidasaRESUMEN
The molecular nature of a mutant of the C4 plant Amaranthus edulis that has been shown to contain only 5% of the normal activity and protein of phosphoenolpyruvate carboxylase (PEPC) (Dever et al., 1995) has been investigated. Using Northern blot analysis, it has been shown here that the PEPC transcripts are produced in the mutant. In-vitro translation of these transcripts generated two products immunoprecipitable by a PEPC N-terminus-specific antibody. One of these products has the size of the complete PEPC polypeptide, the other is 9kDa smaller and was not revealed when using a PEPC C-terminus-specific antibody. In the mutant plant, using the same N- and C-terminus-specific antibodies, only the larger polypeptide was immunodetected, whilst at a very low level. A sequence analysis of the suspected faulty region of the mRNA revealed incorrect splicing of the last intron of the PEPC pre-mRNA. Two mis-splicings have been identified, both occurring after an AG site, one leading to a protein lacking five amino acids, the other to a truncated protein due to a stop codon generated by a frame shift in the translation. Finally, the sequencing of the boundary between the last intron and exon showed that these inaccurate splicings result from a mutation in the genuine canonical 3'AG splicing site.
Asunto(s)
Magnoliopsida/genética , Fosfoenolpiruvato Carboxilasa/genética , Proteínas de Plantas/genética , Empalme del ARN , Secuencia de Bases , ADN Complementario/genética , ADN de Plantas/genética , Intrones/genética , Magnoliopsida/enzimología , Datos de Secuencia Molecular , Fosfoenolpiruvato Carboxilasa/deficiencia , Hojas de la Planta/enzimología , Reacción en Cadena de la Polimerasa , Biosíntesis de Proteínas , ARN Mensajero/metabolismo , ARN de Planta/metabolismo , Alineación de Secuencia , Homología de Secuencia de Ácido NucleicoRESUMEN
The exon d of nad1 is located 993 bp upstream of the nad6 gene in the wheat mitochondrial genome. Transcription analyses of both sequences (nad1 exon d and the nad6 gene) were done by Northern hybridization using RNA from wheat seedlings and tissue cultures derived from immature embryos. A complicated pattern was generated with a probe including exon d of nad1 and the whole nad6 gene. An 0.71-kb transcript is specific to nad1 exon d whereas a 1.2-kb transcript is specific to the nad6 gene. Three larger transcripts hybridize to both probes suggesting that nad1 exon d and nad6 are co-transcribed. This co-transcription has been directly demonstrated by cDNA synthesis on mtRNAs and sequencing of the PCR amplification product.
Asunto(s)
Exones , Genes de Plantas , Mitocondrias/enzimología , NAD(P)H Deshidrogenasa (Quinona)/genética , Triticum/enzimología , Triticum/genética , Secuencia de Bases , Células Cultivadas , Cartilla de ADN , Expresión Génica , Intrones , Datos de Secuencia Molecular , NAD(P)H Deshidrogenasa (Quinona)/biosíntesis , Conformación de Ácido Nucleico , Reacción en Cadena de la Polimerasa , ARN/biosíntesis , ARN/química , ARN/metabolismo , Empalme del ARN , ARN Mitocondrial , Transcripción GenéticaRESUMEN
Northern analyses and reverse transcription-polymerase chain-reaction (RT-PCR) experiments, followed by PCR amplification product sequencing, were performed on total mitochondrial (mt) RNAs from wheat seedlings and tissue cultures. It was shown that the rps13 gene, which encodes ribosomal protein S13, and the atp6 gene, which encodes subunit 6 of the ATP synthase complex, were co-transcribed. However, rps13 transcripts were virtually undetectable in seedlings under conditions where atp6 transcripts appeared abundant. In addition, markedly higher steady state transcript levels were observed in tissue culture. Expression of the mitochondrial rps13 gene was confirmed by showing that its transcripts were edited. Slight differences between editing patterns of tissue-culture and whole-plant transcripts were found. Taken together, these results suggest that in vitro culture could disturb the post-transcriptional regulation of gene expression.
Asunto(s)
Proteínas de Plantas/genética , Edición de ARN , Proteínas Ribosómicas/genética , Transcripción Genética , Triticum/genética , Secuencia de Bases , Northern Blotting , Células Cultivadas , ADN de Plantas/genética , Datos de Secuencia Molecular , Reacción en Cadena de la Polimerasa , ATPasas de Translocación de Protón , Semillas/genética , Triticum/citologíaRESUMEN
The mitochondrial nad6 gene of maize was identified and mapped 1 kb downstream from the atp6 gene. It encodes a 220 amino-acid polypeptide. Using Northern hybridization experiments and RT-PCR analysis, we showed that both nad6 and atp6 are co-transcribed in maize mitochondria. RNA editing of the mitochondrial nad6 transcript was studied by cDNA sequencing. Twelve edited sites were identified at the same positions as those already identified in the wheat mitochondria nad6 transcript. Alignments of nucleotide and amino-acid sequences of the mitochondrial nad6 genes of maize, wheat, and Brassica campestris, show that the wheat gene encodes a shorter polypeptide (229 amino acids) than was previously thought.