RESUMO
Protein and peptide drugs hold great promise as therapeutic agents. But there are shortcomings: Many recombinant proteins are quickly degraded by proteolytic enzymes or are rapidly cleared by kidney filtration resulting in a short circulating half-life. Additionally they are prone to be recognized by the immune system resulting in the generation of neutralizing and non-neutralizing antibodies. PEGylation, a process by which polyethylene glycol chains are attached to protein and peptide drugs, can overcome these and other shortcomings. By increasing the molecular mass of proteins and peptides and shielding them from proteolytic enzymes, PEGylation primarily improves pharmacokinetics and helps to prevent adverse drug reactions.
Assuntos
Polietilenoglicóis/química , Proteínas Recombinantes/administração & dosagem , Animais , Química Farmacêutica , Desenho de Fármacos , Enzimas/química , Humanos , Proteínas Recombinantes/farmacocinética , Compostos de Sulfidrila/químicaRESUMO
On 11 November 2010, the German Bundestag approved the law on the Reorganization of the Pharmaceutical Market (AMNOG). This act went into force on 01 January 2011. The aim was to "confine the rapidly rising pharmaceutical expenditure of public health insurance." A better way should be defined for a "fair competition" and a "stronger focus on the well-being of patients" through a new balance between innovation and affordability of medicines. Obviously, advocates and opponents of this new law argue differently. Those in favor of the AMNOG argue that a fair evaluation of the incremental clinical benefits of newly approved drugs should build the basis for fair price negotiations, that prices should be paid, which more realistically reflect the value of the innovation, and that the system will receive urgently needed relief from the immense burden of the rather small group of patent-protected drugs. Those opposing the new law see the danger of delayed launches or even withdrawals of new medicines that can benefit patients, erosion of Germany's strength in pharmaceutical innovation, and the loss of high-qualified and high-paying jobs in research and development.
Assuntos
Aprovação de Drogas/economia , Aprovação de Drogas/legislação & jurisprudência , Custos de Medicamentos/legislação & jurisprudência , Indústria Farmacêutica/economia , Indústria Farmacêutica/legislação & jurisprudência , Necessidades e Demandas de Serviços de Saúde/economia , Necessidades e Demandas de Serviços de Saúde/legislação & jurisprudência , Alemanha , HumanosRESUMO
In prokaryotes and eukaryotes mobile genetic elements frequently disrupt the highly conservative structures of chromosomes, which are responsible for storage of genetic information. The factors determining the site for integration of such elements are still unknown. Transfer RNA (tRNA) genes are associated in a highly significant manner with different putative mobile genetic elements in the cellular slime mold Dictyostelium discoideum. These results suggest that tRNA genes in D. discoideum, and probably tRNA genes generally in lower eukaryotes, may function as genomic landmarks for the integration of different transposable elements in a strictly position-specific manner.
Assuntos
Dictyostelium/genética , RNA de Transferência/genética , Sequência de Bases , Mapeamento Cromossômico , Dados de Sequência Molecular , Hibridização de Ácido Nucleico , Sondas de Oligonucleotídeos , Polimorfismo de Fragmento de Restrição , RNA de Transferência de Ácido Glutâmico/genética , RNA de Transferência de Lisina/genética , RNA de Transferência de Valina/genéticaRESUMO
The reciprocal chromosomal translocation t(4;11) is correlated with infant, childhood, adult and therapy-related high-risk acute leukemia. Here, we investigated the biological effects of MLL.AF4, AF4.MLL or the combination of both reciprocal fusion proteins in a conditional in vitro cell culture model system. Several parameters like cell growth, cell cycling capacity, apoptotic behavior and growth transformation were investigated under physiological and stress conditions. Co-transfected cells displayed the highest resistance against apoptotic triggers, cell cycling capacity and loss-of-contact inhibition. These analyses were complemented by gene expression profiling experiments and specific gene signatures were established for each of the three cell lines. Interestingly, co-transfected cells strongly upregulate the homeobox gene Nanog. In combination with Oct4, the Nanog homeoprotein is steering maintenance of pluripotency and self-renewal in embryonic stem cells. Transcription of Nanog and other stem cell factors, like Oct4 and Bmi1, was verified in biopsy material of t(4;11) patient cells which express both reciprocal t(4;11) fusion genes. In conclusion, the presence of both reciprocal MLL fusion proteins confers biological properties known from t(4;11) leukemia, suggesting that each of the two fusion proteins contribute specific properties and, in combination, also synergistic effects to the leukemic phenotype.
Assuntos
Apoptose , Ciclo Celular , Cromossomos Humanos Par 4/genética , Proteína de Leucina Linfoide-Mieloide/genética , Proteína de Leucina Linfoide-Mieloide/metabolismo , Proteínas de Fusão Oncogênica/genética , Proteínas de Fusão Oncogênica/metabolismo , Animais , Células Cultivadas , Proteínas Inibidoras de Quinase Dependente de Ciclina/genética , Perfilação da Expressão Gênica , Humanos , Camundongos , Mutação/genética , Reação em Cadeia da Polimerase , Fatores de Transcrição/genéticaRESUMO
The MLL gene is frequently involved in chromosomal translocations associated with high-risk acute leukaemia. Infant and therapy-related acute leukaemia patients display chromosomal breakpoints preferentially clustered in the telomeric portion of the MLL breakpoint cluster region (SCII). Here, we demonstrate that SCII colocalizes with a gene-internal promoter element in the mouse and human MLL gene, respectively. The mRNA generated encodes an N-terminally truncated version of MLL that still exhibits many functional regions, including the C-terminal SET-domain. Etoposide-induced DNA double-strand breaks colocalize with the binding site of RNA polymerase II and the transcription initiation region, but not with a nearby Topo II consensus sequence. Thus, the observed genomic instability of the human MLL gene is presumably linked to transcriptional processes. The consequences of this novel finding for the creation of chromosomal translocations, the biology of the MLL protein and for MLL-mediated acute leukaemia are discussed.
Assuntos
Proteína de Leucina Linfoide-Mieloide/genética , Regiões Promotoras Genéticas , Recombinação Genética , Transcrição Gênica , Animais , Cromatina , Etoposídeo , Histona-Lisina N-Metiltransferase , Humanos , Camundongos , RNA Polimerase IIRESUMO
Different Dictyostelium discoideum strains contain between 2 and 200 copies of a retrotransposable element termed DRE (Dictyostelium repetitive element). From the analysis of more than 50 elements, it can be concluded that DRE elements always occur 50 +/- 3 nucleotides upstream of tRNA genes. All analyzed clones contain DRE in a constant orientation relative to the tRNA gene, implying orientation specificity as well as position specificity. DRE contains two open reading frames which are flanked by nonidentical terminal repeats. Long terminal repeats (LTRs) are composed of three distinct modules, called A, B, and C. The tRNA gene-proximal LTR is characterized by one or multiple A modules followed by a single B module (AnB). With respect to the distal LTR, two different subforms of DRE have been isolated. The majority of isolated clones contains a distal LTR composed of a B module followed by a C module (BC), whereas the distal LTR of the other subform contains a consecutive array of a B module, a C module, a slightly altered A module, another B module, and another C module (BC.ABC). Full-length as well as smaller transcripts from DRE elements have been detected, but in comparison with the high copy number in D. discoideum strains derived from the wild-type strain NC4, transcription is rather poor.
Assuntos
Elementos de DNA Transponíveis , Dictyostelium/genética , RNA de Transferência/genética , Sequências Repetitivas de Ácido Nucleico , Sequência de Aminoácidos , Animais , Sequência de Bases , DNA , Genoma , Dados de Sequência Molecular , Fases de Leitura Aberta , Mapeamento por Restrição , Alinhamento de SequênciaRESUMO
The Dictyostelium discoideum NC4 genome harbors approximately 150 individual copies of a retrotransposable element called the Dictyostelium repetitive element (DRE). This element contains nonidentical terminal repeats (TRs) consisting of conserved building blocks A and B in the left TR and B and C in the right TR. Seven different-sized classes of RNA transcripts from these elements were resolved by Northern (RNA) blot analysis, but their combined abundance was very low. When D. discoideum cells were grown in the presence of the respiratory chain blocker antimycin A, steady-state concentrations of these RNA species increased 10- to 20-fold. The D. discoideum genome contains two DRE subtypes, the full-length 5.7-kb DREa and the internally deleted 2.4-kb DREb. Both subtypes are transcribed, as confirmed by analysis of cloned cDNA. Primary transcripts from the sense strand originate at nucleotide +1 and terminate at two dominant sites, located 21 or 28 nucleotides upstream from the 3' end of the elements. The activity of a reasonably strong polymerase II promoter in the 5'-terminal A module is slightly upregulated by the tRNA gene located 50 +/- 4 nucleotides upstream and drastically reduced by the adjacent B module of the DRE. Transcripts from the opposite DNA strand (complementary-sense transcripts) were also detected, directed by an internally located polymerase II promoter residing within the C module. This latter transcription was initiated at multiple sites within the oligo(dA12) stretch which terminates DREs.
Assuntos
Elementos de DNA Transponíveis , DNA Fúngico/genética , Dictyostelium/enzimologia , Dictyostelium/genética , Regulação Fúngica da Expressão Gênica , Regiões Promotoras Genéticas , RNA Polimerase II/biossíntese , RNA Polimerase II/genética , Transcrição Gênica , Animais , Sequência de Bases , Núcleo Celular/metabolismo , Sequência Conservada , Primers do DNA , DNA Fúngico/metabolismo , Genes Fúngicos , Dados de Sequência Molecular , RNA Polimerase II/metabolismo , Proteínas Recombinantes de Fusão/biossíntese , Proteínas Recombinantes de Fusão/metabolismo , Sequências Repetitivas de Ácido Nucleico , Mapeamento por Restrição , Retroviridae/genética , beta-Galactosidase/biossíntese , beta-Galactosidase/metabolismoRESUMO
We investigated the use of the prokaryotic tetracycline operator-repressor system as a regulatory device to control the expression of Dictyostelium discoideum tRNA genes. The tetO1 operator fragment was inserted at three different positions in front of a tRNA(Glu) (Am) suppressor gene from D. discoideum, and the tetracycline repressor gene was expressed under the control of a constitutive actin 6 promoter. The effectiveness of this approach was determined by monitoring the expression of a beta-galactosidase gene engineered to contain a stop codon that could be suppressed by the tRNA. When these constructs were introduced into Dictyostelium cells, the repressor bound to the operator in front of the tRNA gene and prevented expression of the suppressor tRNA. Addition of tetracycline (30 micrograms/ml) to the growth medium prevented repressor binding, allowed expression of the suppressor tRNA, and resulted in beta-galactosidase synthesis. The operator-repressor complex interfered with tRNA gene transcription when the operator was inserted immediately upstream (position +1 or -7) of the mature tRNA coding region. Expression of a tRNA gene carrying the operator at position -46 did not respond to repressor binding. This system could be used to control the synthesis of any protein, provided the gene contained a translational stop signal.
Assuntos
Dictyostelium/genética , Regulação Fúngica da Expressão Gênica , Genes Supressores , RNA de Transferência/genética , Animais , Sequência de Bases , Clonagem Molecular , DNA Polimerase II/metabolismo , DNA Recombinante , Cinética , Dados de Sequência Molecular , Regiões Operadoras Genéticas , Resistência a Tetraciclina/genética , Transcrição GênicaRESUMO
Chromosomal rearrangements of the human MLL gene are a hallmark for aggressive (high-risk) pediatric, adult and therapy-associated acute leukemias. These patients need to be identified in order to subject these patients to appropriate therapy regimen. A recently developed long-distance inverse PCR method was applied to genomic DNA isolated from individual acute leukemia patients in order to identify chromosomal rearrangements of the human MLL gene. We present data of the molecular characterization of 414 samples obtained from 272 pediatric and 142 adult leukemia patients. The precise localization of genomic breakpoints within the MLL gene and the involved translocation partner genes (TPGs) was determined and several new TPGs were identified. The combined data of our study and published data revealed a total of 87 different MLL rearrangements of which 51 TPGs are now characterized at the molecular level. Interestingly, the four most frequently found TPGs (AF4, AF9, ENL and AF10) encode nuclear proteins that are part of a protein network involved in histone H3K79 methylation. Thus, translocations of the MLL gene, by itself coding for a histone H3K4 methyltransferase, are presumably not randomly chosen, rather functionally selected.
Assuntos
Leucemia/genética , Proteína de Leucina Linfoide-Mieloide/genética , Proteínas de Fusão Oncogênica/genética , Translocação Genética/genética , Doença Aguda , Adulto , Criança , Aberrações Cromossômicas , Mapeamento Cromossômico , DNA/genética , DNA/isolamento & purificação , Histona-Lisina N-Metiltransferase , Histonas/metabolismo , Humanos , MetilaçãoAssuntos
Fitoterapia/tendências , Extratos Vegetais/efeitos adversos , Extratos Vegetais/uso terapêutico , Plantas Medicinais , Serviços de Informação sobre Medicamentos , Alemanha , Humanos , Naturologia , Educação de Pacientes como Assunto , Fitoterapia/efeitos adversos , Extratos Vegetais/provisão & distribuição , Plantas Medicinais/efeitos adversosRESUMO
The one-hybrid method for genetic screening in yeast was used to search a Dictyostelium discoideum cDNA library for DNA-binding proteins that interact with the C-module of the Dictyostelium repetitive element. The C-module was formerly shown to contain two high affinity, sequence-specific binding sites for a nuclear protein factor of unknown function (CMBF). The bait DNA sequence was bound in vivo by a cDNA-encoded protein whose derived amino acid sequence showed high homology to nucleosome core histone H3, but not to partially available CMBF sequences. The D. discoideum histone H3 homolog is encoded by a single gene and shows significant sequence variation at the amino terminus of the protein, including a triple-serine insertion not found in any other histone H3.
Assuntos
DNA Complementar/genética , Dictyostelium/genética , Histonas/genética , Sequência de Aminoácidos , Animais , Sequência de Bases , Clonagem Molecular , DNA Complementar/química , DNA Complementar/isolamento & purificação , Escherichia coli/genética , Histonas/química , Dados de Sequência Molecular , Nucleossomos/metabolismo , Alinhamento de Sequência , Transformação Genética , Leveduras/genéticaRESUMO
A total of 68 different tRNA genes from the cellular slime mold Dictyostelium discoideum have been isolated and characterized. Although these tRNA genes show features common to typical nuclear tRNA genes from other organisms, several unique characteristics are apparent: (1) the 5'-proximal flanking region is very similar for most of the tRNA genes; (2) more than 80% of the tRNA genes contain an "ex-B motif" within their 3'-flanking region, which strongly resembles characteristics of the consensus sequence of a T-stem/T-loop region (B-box) of a tRNA gene; (3) probably more than 50% of the tRNA genes in certain D. discoideum strains are associated with a retrotransposon, termed DRE (Dictyostelium repetitive element), or with a transposon, termed Tdd-3 (Transposon Dictyostelium discoideum). DRE always occurs 50 (+/- 3) nucleotides upstream and Tdd-3 always occurs 100 (+/- 20) nucleotides downstream from the tRNA gene. D. discoideum tRNA genes are organized in multicopy gene families consisting of 5 to 20 individual genes. Members of a particular gene family are identical within the mature tRNA coding region while flanking sequences are idiosyncratic.
Assuntos
Dictyostelium/genética , RNA de Transferência/genética , Sequências Reguladoras de Ácido Nucleico/genética , Sequências Repetitivas de Ácido Nucleico/genética , Animais , Sequência de Bases , Códon/genética , Elementos de DNA Transponíveis , DNA de Protozoário/genética , DNA de Cadeia Simples , Éxons , Íntrons , Dados de Sequência Molecular , Família Multigênica , Homologia de Sequência do Ácido NucleicoRESUMO
The C-module-binding factor, CMBF, is a nuclear DNA-binding protein which was originally identified through its specific binding to a promoter element within the retrotransposable element TRE5-A of Dictyostelium discoideum AX2 cells. In order to analyse putative physiological functions of CMBF for the TRE5-A-hosting D. discoideum cells, we used a novel strategy to create mutant cell lines which stably underexpressed functional CMBF. An amber (UAG) translation stop codon was introduced into the chromosomal copy of the CMBF-encoding gene (cbfA), and an amber suppressor tRNA gene was expressed in the same mutant cells. Due to the low efficiency of translation stop codon suppression in this system all recovered cell lines expressed <20 % of wild-type CMBF levels. The mutant cell lines displayed strong growth phenotypes when plated on their natural food source, bacteria. We show evidence that growth reduction was due to impaired phagocytosis of bacteria in the mutants. All obtained mutants showed a strong developmental defect which was defined by the formation of very small fruiting bodies. The strength of the developmental phenotype appeared to depend upon the residual CMBF levels maintained in the mutants. We propose that CMBF is a general transcription regulator which supports the normal expression of several genes required for the maintenance of high proliferation rates of D. discoideum amoebae as well as proper aggregation and development. Our results demonstrate that amber stop codon suppression may be a useful strategy to stably underexpress proteins whose coding genes cannot be successfully disrupted by homologous recombination.
Assuntos
Códon de Terminação/genética , Proteínas de Ligação a DNA/genética , Proteínas de Ligação a DNA/metabolismo , Dictyostelium/crescimento & desenvolvimento , Proteínas Nucleares/metabolismo , Proteínas de Protozoários , Supressão Genética/genética , Transporte Ativo do Núcleo Celular , Sequência de Aminoácidos , Animais , Divisão Celular , Proteínas de Ligação a DNA/química , Dictyostelium/citologia , Dictyostelium/genética , Dictyostelium/microbiologia , Expressão Gênica/genética , Regulação da Expressão Gênica no Desenvolvimento , Genes Supressores/genética , Dados de Sequência Molecular , Sinais de Localização Nuclear , Proteínas Nucleares/química , Proteínas Nucleares/genética , Fagocitose , Fenótipo , Pinocitose , Biossíntese de ProteínasAssuntos
Anticorpos Monoclonais/farmacologia , Diabetes Mellitus Tipo 1/imunologia , Diabetes Mellitus Tipo 1/terapia , Hipoglicemiantes/uso terapêutico , Imunoterapia , Insulina/uso terapêutico , Anticorpos Monoclonais/uso terapêutico , Anticorpos Monoclonais Humanizados , Complexo CD3/imunologia , Linfócitos T CD8-Positivos/efeitos dos fármacos , Diabetes Mellitus Tipo 1/patologia , Humanos , Células Secretoras de Insulina/imunologia , Células Secretoras de Insulina/patologiaRESUMO
An inducible expression system that indirectly regulates gene expression through the use of an inducible suppressor tRNA has been used to express both endogenous and exogenous genes in Dictyostelium. The tetracycline repressor and tRNA suppressor (Glu) are expressed from a single G418 selectable vector, while a gene engineered to contain a stop codon is expressed from a separate hygromycin selectable vector. beta-Galactosidase could be induced over 300 fold with this system, and the extent of induction could be varied depending upon the amount of tetracycline added. It took 3 days to fully induce expression, and about 3 days for expression to decrease to baseline after removal of the tetracycline. Dictyostelium myosin II heavy chain could also be expressed in an inducible manner, although the induction ratio was not as high as beta-galactosidase and the maximum expression level was not as high as wild-type levels. A significant accumulation of the truncated peptide indicates that complete suppression of the stop codon was not achieved. Partial phenotypic reversion was observed in null mutants inducibly expressing myosin II. RacB could also be inducibly expressed, whereas the protein could not be expressed from a constitutive promoter, presumably because expression at high levels is lethal. Therefore, the inducible tRNA system can be used to control expression of endogenous Dictyostelium genes.
Assuntos
Dictyostelium/genética , Cadeias Pesadas de Miosina/genética , Proteínas de Protozoários/genética , RNA de Transferência de Ácido Glutâmico/genética , Animais , Western Blotting , Divisão Celular/efeitos dos fármacos , Divisão Celular/genética , Códon de Terminação/genética , Dictyostelium/efeitos dos fármacos , Dictyostelium/crescimento & desenvolvimento , Relação Dose-Resposta a Droga , Doxiciclina/farmacologia , Regulação da Expressão Gênica/efeitos dos fármacos , Genes Supressores , Cinética , Óperon Lac/genética , Mutagênese Sítio-Dirigida , Cadeias Pesadas de Miosina/metabolismo , Plasmídeos/genética , Proteínas de Protozoários/metabolismo , Tetraciclina/farmacologia , Resistência a Tetraciclina/genética , Fatores de Tempo , beta-Galactosidase/efeitos dos fármacos , beta-Galactosidase/genética , beta-Galactosidase/metabolismoRESUMO
A haploid genome of the cellular slime mold Dictyostelium discoideum contains at least 14 non-allelic gene copies coding for a tRNA(ValGUU). The structure, genomic organization, and expression of these genes have been analyzed in relation to stages of the developmental cycle. So far, 13 tRNA(ValGUU) genes have been isolated and characterized. All genes contain identical mature tRNA-coding regions, and consequently identical gene internal promoter elements. However, different genes differ with respect to their 5'- and 3'-flanking regions, although a certain degree of sequence conservation seems apparent. Different members of this tRNA gene family appear to be randomly dispersed along the seven D. discoideum chromosomes, and not clustered at any one genomic location. In vivo expression of individual genes was studied in yeast. All but one tRNA(ValGUU) gene are actively transcribed, though with different efficiencies. There is also evidence that not all of these tRNA genes are constitutively transcribed in Dictyostelium throughout the developmental cycle. One characteristic primary transcript can only be detected in cells of the late preaggregation phase, whereas growing cells, cells in the stationary phase or cells harvested 4 h after the onset of development do not seem to carry this transcript. This product seems to be transcribed from a gene of an unusual structure. Although this particular gene has not yet been isolated, it can be predicted from the sequence of the cDNA synthesized from primary transcription products of this putative gene, that it is composed of nt 1-54 of a 3'-truncated tRNA(ValGUU) gene linked to a bona fide tRNA(ValGUU) gene.
Assuntos
Dictyostelium/genética , Genes Fúngicos , Família Multigênica , RNA de Transferência Aminoácido-Específico/genética , RNA de Transferência de Valina/genética , Alelos , Sequência de Bases , Códon , Ligação Genética , Dados de Sequência Molecular , Conformação de Ácido Nucleico , Saccharomyces cerevisiae/genéticaRESUMO
The expression of an ochre suppressor mutant of the GluII(UUA) tRNA appears to be lethal to Dictyostelium, and offers a novel 'positive negative' strategy to select for targeted gene disruption by homologous recombination. Inclusion of the suppressor tRNA gene decreases the overall transformation frequency by approximately 20-fold. This increases the proportion of targeted gene disruptions to over 90%.