RESUMO
Activated hepatic stellate cells differentiate into myofibroblasts, which synthesize and secrete extracellular matrix (ECM) leading to liver fibrosis. It was previously demonstrated that bulleyaconitine A (BLA), an alkaloid from Aconitum bulleyanum, inhibits proliferation and promotes apoptosis of human hepatic Lieming Xu-2 (LX-2) cells. In this study, we analyzed the effect of BLA on the production of ECM and related proteins by LX-2 cells activated with acetaldehyde (AA). The cells were randomized into the control group, AA group (cells activated with 400 µM AA), and BLA+AA group (cells cultured in the presence of 400 µM AA and 18.75 µg/ml BLA). In the BLA+AA group, the contents of collagens I and III and the expression of α-smooth muscle actin and transforming growth factor-ß1 (TGF-ß1) were statistically significantly higher than in the control, but lower than in the AA group. Expression of MMP-1 in the BLA+AA group was also significantly higher than in the AA group, but lower than in the control. Expression of TIMP-1 in the BLA+AA group was significantly higher than in the control, but lower than in the AA group. Thus, BLA suppressed activation and proliferation of LX-2 cells by inhibiting TGF-ß1 signaling pathway and decreasing the content of collagens I and III by reducing the MMP-1/TIMP-1 ratio.
Assuntos
Acetaldeído , Aconitina , Actinas , Colágeno Tipo I , Matriz Extracelular , Células Estreladas do Fígado , Inibidor Tecidual de Metaloproteinase-1 , Fator de Crescimento Transformador beta1 , Células Estreladas do Fígado/efeitos dos fármacos , Células Estreladas do Fígado/metabolismo , Humanos , Acetaldeído/farmacologia , Acetaldeído/análogos & derivados , Aconitina/farmacologia , Aconitina/análogos & derivados , Colágeno Tipo I/metabolismo , Colágeno Tipo I/genética , Matriz Extracelular/metabolismo , Matriz Extracelular/efeitos dos fármacos , Inibidor Tecidual de Metaloproteinase-1/metabolismo , Inibidor Tecidual de Metaloproteinase-1/genética , Fator de Crescimento Transformador beta1/metabolismo , Fator de Crescimento Transformador beta1/genética , Actinas/metabolismo , Actinas/genética , Metaloproteinase 1 da Matriz/metabolismo , Metaloproteinase 1 da Matriz/genética , Linhagem Celular , Colágeno Tipo III/metabolismo , Colágeno Tipo III/genética , Proliferação de Células/efeitos dos fármacos , Aconitum/química , Cirrose Hepática/metabolismo , Cirrose Hepática/patologiaRESUMO
Patients with dementia with Lewy body(DLB), Alzheimer disease (AD), frontotemporal dementia (FTP), progressive supranuclear palsy (PSP) and healthy controls in the Department of Neurology, the First Affiliated Hospital of Zhengzhou University from August 2019 to March 2021 were recruited, with 3 in each group. Phosphorylated α-synuclein from the skin of DLB patients was detected by skin micro-biopsy and compared with patients with AD, FTP, PSP and health controls. Phosphorylated α-synuclein was found in the skin nerves of the DLB patients, while no α-synuclein were detected in the skin samples of others. Skin phosphorylated α-synuclein may potentially become a diagnostic biomarker of DLB, however further studies are warranted to assess its sensitivity and specificity.
Assuntos
Doença de Alzheimer , Doença por Corpos de Lewy , Doença de Alzheimer/diagnóstico , Biópsia , Humanos , Doença por Corpos de Lewy/diagnóstico , PeleRESUMO
On-chip photonics devices relying on the weak, volatile thermo-optic or electro-optic effects of silicon usually suffer from high insertion loss (IL) and a low refractive index coefficient. In this paper, we designed two novel 1 × 1 and 1 × 2 phase-change optical switches based on a signal-mode Si waveguide integrated with a Ge2Sb2Te5 (GST) top clad layer, respectively. The three-state switch including amorphous GST (a-GST), face centered cubic crystalline phase (FCC-GST) and hexagonal crystalline phase (HCP-GST) operated by utilizing the dramatic difference in the optical constants between the amorphous and two crystalline phases of GST. In the case of the 1 × 1 optical switch, an extinction ratio (ER) of 8.9 dB and an extremely low IL of 0.8 dB were achieved using an optimum GST length of only 2 µm. While for the 1 × 2 optical switch, low ILs in the range of 0.15 â¼ 0.35 dB for both 'cross' (a-GST) and 'bar' (FCC-GST and HCP-GST) states were also obtained. Additionally, we found that both ILs and mode losses of the switch with HCP-GST were about half lower than those with FCC-GST, which means FCC-GST could be instituted by HCP-GST in the traditional ovonic switch with the consideration of low loss. This research provides the fundamental understanding for the realization of low loss and non-volatile Si-GST hybrid optical switches, with potential for future communication networks.
RESUMO
Studies have shown that the crystallization phase state of Ge2Sb2Te5 (GST) can be reversibly modulated by femtosecond (fs) laser multiple pulses, which have excellent applications in reconfigurable multi-level operation fields. In this study, the temporal-spatial crystalline evolution dynamics of amorphous GST film is investigated during two fs laser pulses excitation through a pump-probe shadowgraph imaging technique. A quasi-amorphous phase state, which is different from that in the initial as-deposited amorphous GST, is emerged through the first fs laser pulse excitation with a pulse energy lower than crystallization threshold. The experimental results reveal that a crystallization enhancement effect can be induced through the second pulse excitation based on this quasi-amorphous surface structure. The stimulative cluster generated in the quasi-amorphous reduces the amorphous-to-crystalline phase transition threshold for the second fs laser pulse irradiation. The spatially-resolved phase-transition threshold extension effect in a horizontal direction is proposed with the increasing pulse number to summarize the mechanism of the crystallization enhancement effect. The specific-grain-appearance (coarse grains and fine grains representing different phase transition approach) distributed area induced by single and double fs laser pulses irradiation are experimentally demonstrated corresponding to threshold extension theory.
RESUMO
Heterosis has greatly contributed to conventional plant breeding and is widely used to increase crop plant productivity. However, although some studies have explored the mechanisms of heterosis at the genomic and transcriptome level, these mechanisms still remain unclear. The growth and development of maize seedlings and immature embryos have an important impact on subsequent production. This study investigated differentially expressed genes (DEGs) between parents and reciprocal hybrids in the seedling leaves, roots, and immature embryo 15 days after pollination using amplified fragment length polymorphism (AFLP)-based transcript profiling (cDNA-AFLP). We isolated 180, 170, and 108 genes from the leaves, roots, and immature embryos, respectively, that were differentially expressed between hybrids and parents. Sequencing and functional analysis revealed that 107 transcript-derived fragments in the roots and leaves and 90 in the immature embryos were involved in known functions, whereas many DEGs had roles in plant growth and development, photosynthesis, signal transduction, and seed germination. Quantitative reverse-transcription polymerase chain reaction analysis of relative expression levels between reciprocal hybrids and both parental genotypes of selected genes produced results that were consistent with cDNA-AFLP. We validated the expression patterns of 15 selected genes related to heterosis formation and revealed that most showed non-additive expression in one or both hybrids, including dominant, underdominant, and overdominant expression. This indicates that gene-regulatory interactions among parental alleles play an important role in heterosis during the early developmental stages of maize.
Assuntos
Quimera , Perfilação da Expressão Gênica , Hibridização Genética , Transcriptoma , Zea mays/genética , Regulação da Expressão Gênica de Plantas , Genes de Plantas , Germinação/genética , Vigor Híbrido/genética , Endogamia , Reprodutibilidade dos TestesRESUMO
Chronic obstructive pulmonary disease (COPD) is a growing cause of morbidity and mortality throughout the world. The purpose of our study was to uncover biomarkers and explore its pathogenic mechanisms at the molecular level. The gene expression profiles of COPD samples and normal controls were downloaded from Gene Expression Omnibus. Matlab was used for data preprocessing and SAM4.0 was applied to determine the differentially expressed genes (DEGs). Furthermore, a protein-protein interaction (PPI) network was constructed by mapping the DEGs into PPI data, and functional analysis of the network was conducted with BiNGO. A total of 348 DEGs and 765 interactive genes were identified. The hub genes were mainly involved in metabolic processes and ribosome biogenesis. Several genes related to COPD in the PPI network were found, including CAMK1D, ALB, KIT, and DDX3Y. In conclusion, CAMK1D, ALB, KIT, and DDX3Y were chosen as candidate genes, which have the potential to be biomarkers or candidate target molecules to apply in clinical diagnosis and treatment of COPD.
Assuntos
Mapeamento de Interação de Proteínas/métodos , Mapas de Interação de Proteínas/genética , Doença Pulmonar Obstrutiva Crônica/genética , Transdução de Sinais/genética , Biomarcadores/metabolismo , Proteína Quinase Tipo 1 Dependente de Cálcio-Calmodulina/genética , Proteína Quinase Tipo 1 Dependente de Cálcio-Calmodulina/metabolismo , RNA Helicases DEAD-box/genética , RNA Helicases DEAD-box/metabolismo , Perfilação da Expressão Gênica , Redes Reguladoras de Genes , Predisposição Genética para Doença/genética , Humanos , Antígenos de Histocompatibilidade Menor , Modelos Genéticos , Análise de Sequência com Séries de Oligonucleotídeos , Proteínas Proto-Oncogênicas c-kit/genética , Proteínas Proto-Oncogênicas c-kit/metabolismo , Doença Pulmonar Obstrutiva Crônica/metabolismo , Albumina Sérica/genética , Albumina Sérica/metabolismo , Albumina Sérica HumanaRESUMO
Heterosis is the superior performance of heterozygous individuals and has been widely exploited in plant breeding, although the underlying regulatory mechanisms still remain largely elusive. To understand the molecular basis of heterosis in maize, in this study, roots and leaves at the seedling stage and embryos and endosperm tissues 15 days after fertilization of 2 elite hybrids and their parental lines were used to estimate the levels and patterns of cytosine methylation by the methylation-sensitive amplification polymorphism method. The relative total methylation levels were lower in all the tissues of all hybrids than their corresponding mid-parent values, and the number of demethylation events was higher in the hybrids. These results implied that the decreasing trend and demethylation in hybrids relative to their parents may enable the derepression and possibly expression of many genes that were associated with the phenotypic variation in hybrids. To further analyze the observed methylation pattern changes, a total of 63 differentially displayed DNA fragments were successfully sequenced. Basic Local Alignment Search Tool analysis showed that 11 fragments shared similarity with known functional proteins in maize or other plant species, including metabolism, transposon/retrotransposon, development, stress response, and signal transduction, which indicated that these genes might play a significant role in maize hybrid vigor.
Assuntos
Metilação de DNA , Regulação da Expressão Gênica de Plantas , Vigor Híbrido , Hibridização Genética , Zea mays/genética , EndogamiaRESUMO
We developed EST-PCR markers specific to barley chromosome 2H, for the purpose of effectively tracing alien chromosomes or chromosome parts in the wheat genetic background. The target alien chromosome 2H confers high resistance to pre-harvest sprouting, which is a worldwide natural disaster in wheat. A total of 120 primer pairs were selected by combining the wheat group 2 chromosomes of the EST database and the genome sequences of the new model plant Brachypodium distachyon. Seventy-seven of 120 primer pairs were polymorphic and 31 of 120 primer pairs were monomorphic between a set of wheat-barley chromosome 2H disomic addition/substitution lines and their parents by agarose gel electrophoresis and polyacrylamide gel electrophoresis. Thirty of 77 polymorphic primer pairs including primer pair P120 derived from the basi gene were chromosome 2H-specific. These markers are expected to be valuable in screening of wheat-barley chromosome 2H recombination lines and pre-harvest sprouting resistant varieties.
Assuntos
Etiquetas de Sequências Expressas/metabolismo , Hordeum/genética , Reação em Cadeia da Polimerase/métodos , Triticum/genética , Sequência de Bases , Cromossomos de Plantas , Primers do DNA/metabolismo , Eletroforese em Gel de Ágar , Genes de Plantas/genética , Marcadores Genéticos , Dados de Sequência Molecular , Mapeamento Físico do Cromossomo , Alinhamento de SequênciaRESUMO
We analyzed genetic diversity and population genetic structure of four artificial populations of wild barley (Hordeum brevisubulatum); 96 plants collected from the Songnen Prairie in northeastern China were analyzed using amplified fragment length polymorphism (AFLP), specific-sequence amplified polymorphism (SSAP) and methylation-sensitive amplified polymorphism (MSAP) markers. Indices of (epi-)genetic diversity, (epi-)genetic distance, gene flow, genotype frequency, cluster analysis, PCA analysis and AMOVA analysis generated from MSAP, AFLP and SSAP markers had the same trend. We found a high level of correlation in the artificial populations between MSAP, SSAP and AFLP markers by the Mantel test (r > 0.8). This is incongruent with previous findings showing that there is virtually no correlation between DNA methylation polymorphism and classical genetic variation; the high level of genetic polymorphism could be a result of epigenetic regulation. We compared our results with data from natural populations. The population diversity of the artificial populations was lower. However, different from what was found using AFLP and SSAP, based on MSAP results the methylation polymorphism of the artificial populations was not significantly reduced. This leads us to suggest that the DNA methylation pattern change in H. brevisubulatum populations is not only related to DNA sequence variation, but is also regulated by other controlling systems.
Assuntos
Metilação de DNA/genética , Epigênese Genética , Hordeum/genética , Polimorfismo Genético , Análise do Polimorfismo de Comprimento de Fragmentos Amplificados , Sequência de Bases , Marcadores Genéticos , Genética Populacional , Filogenia , Especificidade da EspécieRESUMO
Aralia elata is an important medicinal plant in China; it produces large amounts of oleanane type triterpene saponins. A full-length cDNA encoding ß-amyrin synthase (designated as AeAS) was isolated from young leaves of A. elata by reverse transcription-PCR. The full-length cDNA of AeAS was found to have a 2292-bp open reading frame, encoding a protein with 763 amino acid residues. The deduced amino acid sequence of AeAS showed the highest identity (97%) to Panax ginseng ß-amyrin synthase. When AeAS cDNA was expressed in Escherichia coli, an 87.8-kDa recombinant protein was detected by SDS-PAGE and Western blotting. The sequence was also heterologously expressed in the yeast Pichia pastoris, and production of ß-amyrin was detected by HPLC. Tissue expression pattern analysis by real-time reverse transcription-PCR revealed that AeAS is strongly expressed in leaves and stems, and weakly expressed in roots and flowers.
Assuntos
Aralia/enzimologia , Aralia/genética , Genes de Plantas/genética , Transferases Intramoleculares/genética , Plantas Medicinais/enzimologia , Plantas Medicinais/genética , Árvores/enzimologia , Sequência de Aminoácidos , Sequência de Bases , Western Blotting , Cromatografia Líquida de Alta Pressão , Clonagem Molecular , DNA Complementar/genética , Eletroforese em Gel de Poliacrilamida , Regulação da Expressão Gênica de Plantas , Transferases Intramoleculares/química , Dados de Sequência Molecular , Filogenia , Saponinas/biossíntese , Alinhamento de Sequência , Análise de Sequência de DNA , Árvores/genética , Triterpenos/metabolismoRESUMO
A series of expressed sequence tags-derived polymerase chain reaction (EST-PCR) markers specific to chromosome 2Ai#2 from Thinopyrum intermedium were developed in this study using a new integrative approach. The target alien chromosome confers high resistance to barley yellow dwarf virus (BYDV), which is a severe virus disease in wheat. To generate markers evenly distributed on 2Ai#2, a total of 105 primer pairs were designed based on mapped ESTs from 8 bins of wheat chromosome 2B with intron-prediction by aligning ESTs with genomic sequences of the new model plant Brachypodium distachyon. Eight and seven polymorphic markers on the short arm and the long arm of chromosome 2Ai#2, respectively, were obtained with a polymorphism rate of 14.3%. These chromosome 2Ai#2-specific EST-PCR markers were then used in tracing and exploring the structural variation of the alien chromosome in the population derived from the immature embryo culture of the cross between N452, a 2Ai#2(2D) substitution line, and common wheat CB037. Two centric fusion of translocations involving 2Ai#2 short or long arm with wheat chromosome 2D and some new genetic stocks including telosomes with the alien chromosome short or long arm were identified in the SC(3) generations, which provided basic materials to further study the mechanism of the BYDV resistance. BYDV tests in two field seasons suggest that the BYDV resistance was mainly conferred by the short arm, gene interaction on both arms of the alien chromosome was discussed.
Assuntos
Brachypodium/genética , Etiquetas de Sequências Expressas , Luteovirus/patogenicidade , Triticum/genética , Mapeamento Cromossômico , Cromossomos de Plantas , Marcadores Genéticos , Doenças das Plantas/genética , Doenças das Plantas/virologia , Imunidade Vegetal/genética , Polimorfismo Genético/genética , Recombinação Genética , Alinhamento de Sequência , Análise de Sequência de DNA , Translocação Genética , Triticum/virologiaRESUMO
HGVbase (Human Genome Variation database; http://hgvbase.cgb.ki.se, formerly known as HGBASE) is an academic effort to provide a high quality and non-redundant database of available genomic variation data of all types, mostly comprising single nucleotide polymorphisms (SNPs). Records include neutral polymorphisms as well as disease-related mutations. Online search tools facilitate data interrogation by sequence similarity and keyword queries, and searching by genome coordinates is now being implemented. Downloads are freely available in XML, Fasta, SRS, SQL and tagged-text file formats. Each entry is presented in the context of its surrounding sequence and many records are related to neighboring human genes and affected features therein. Population allele frequencies are included wherever available. Thorough semi-automated data checking ensures internal consistency and addresses common errors in the source information. To keep pace with recent growth in the field, we have developed tools for fully automated annotation. All variants have been uniquely mapped to the draft genome sequence and are referenced to positions in EMBL/GenBank files. Data utility is enhanced by provision of genotyping assays and functional predictions. Recent data structure extensions allow the capture of haplotype and genotype information, and a new initiative (along with BiSC and HUGO-MDI) aims to create a central repository for the broad collection of clinical mutations and associated disease phenotypes of interest.
Assuntos
Bases de Dados de Ácidos Nucleicos , Variação Genética , Genoma Humano , Polimorfismo de Nucleotídeo Único , Sequência de Bases , Mapeamento Cromossômico , Sistemas de Gerenciamento de Base de Dados , Frequência do Gene , Doenças Genéticas Inatas/genética , Humanos , Armazenamento e Recuperação da Informação , Internet , Controle de Qualidade , Integração de SistemasRESUMO
Four years after the original sequence submission, we have re-annotated the genome of Mycoplasma pneumoniae to incorporate novel data. The total number of ORFss has been increased from 677 to 688 (10 new proteins were predicted in intergenic regions, two further were newly identified by mass spectrometry and one protein ORF was dismissed) and the number of RNAs from 39 to 42 genes. For 19 of the now 35 tRNAs and for six other functional RNAs the exact genome positions were re-annotated and two new tRNA(Leu) and a small 200 nt RNA were identified. Sixteen protein reading frames were extended and eight shortened. For each ORF a consistent annotation vocabulary has been introduced. Annotation reasoning, annotation categories and comparisons to other published data on M.pneumoniae functional assignments are given. Experimental evidence includes 2-dimensional gel electrophoresis in combination with mass spectrometry as well as gene expression data from this study. Compared to the original annotation, we increased the number of proteins with predicted functional features from 349 to 458. The increase includes 36 new predictions and 73 protein assignments confirmed by the published literature. Furthermore, there are 23 reductions and 30 additions with respect to the previous annotation. mRNA expression data support transcription of 184 of the functionally unassigned reading frames.
Assuntos
Genes Bacterianos/genética , Genoma Bacteriano , Mycoplasma pneumoniae/genética , Fases de Leitura Aberta/genética , Sequência de Aminoácidos , Proteínas de Bactérias/química , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Biologia Computacional , Espectrometria de Massas , Dados de Sequência Molecular , Mycoplasma pneumoniae/química , Análise de Sequência com Séries de Oligonucleotídeos , Filogenia , RNA Bacteriano/análise , RNA Bacteriano/genética , RNA Mensageiro/análise , RNA Mensageiro/genética , Alinhamento de SequênciaRESUMO
The barley chromosome in wheat was identified by genomic in situ hybridization (GISH) in which biotin labelled total genomic DNA of barley Betzes was used as probe and the unlabelled total DNA of common wheat Chinese Spring (CS) as blocking DNA. A series of wheat materials were tested as follows: two disomic alien substitution and monosomic alien addition lines, 2n = 43; two monosomic alien substitution lines, 2n = 42; seven disomic alien substitution lines, 2n = 42. RFLP probe psr131 on the short arm of the homologous group 2 was used to analyze the barley chromosome in wheat. The result indicated that there was a same band in barley Betzes and substitution line A5. The chromosome 2A of A5 was substituted by the chromosome 2H of barley. These materials will be useful in transferring the valuable genes in the chromosome 2H to wheat.
Assuntos
Cromossomos , Hordeum/genética , Hibridização In Situ , Polimorfismo de Fragmento de Restrição , Triticum/genéticaRESUMO
The aim of the present study was to compare the activity levels within the two bellies of the lateral pterygoid muscle between different jaw positions to test the hypothesis that the upper head is primarily a stabilizer. Electromyographic (EMG) recordings, using monopolar concentric needle electrodes, were made from 14 healthy subjects during mandibular rest position (RP), clenching in intercuspal position and jaw opening, first about 10 mm and then about 25 mm. Both bellies had very little activity during RP. The activity level of the superior belly was high during clenching and large opening (LO) with a dip during low opening degree. This pattern differed from that of the inferior belly where the activity was relatively low during clenching and then gradually increased to its highest level during LO. The results support that the lower belly is primarily a jaw opener while the superior belly acts as a stabilizer keeping the disc and condyle in a functionally stable position during clenching and jaw movements.
Assuntos
Eletromiografia , Músculos Pterigoides/fisiologia , Potenciais de Ação/fisiologia , Adulto , Análise de Variância , Oclusão Dentária , Eletrodos Implantados , Humanos , Masculino , Mandíbula/fisiologia , Côndilo Mandibular/anatomia & histologia , Côndilo Mandibular/fisiologia , Movimento , Contração Muscular/fisiologia , Agulhas , Músculos Pterigoides/anatomia & histologia , Disco da Articulação Temporomandibular/anatomia & histologia , Disco da Articulação Temporomandibular/fisiologia , Dimensão VerticalRESUMO
MOTIVATION: Numerous homologous sequences from diverse species can be retrieved from databases using programs such as BLAST. However, due to multigene families, evolutionary relationship often cannot be easily determined and proper functional assignment becomes difficult. Thus, discrimination between orthologues and paralogues within BLAST output lists of homologous sequences becomes more and more important. RESULT: We therefore developed a method that attempts to construct a reconciled tree from a gene tree of selected sequences and its corresponding phylogenetic tree of the species involved (species tree). An interface on the Web is developed to enable users to analyse the BLAST result. BLAST outputs are parsed and, for the selected sequences, multiple alignments are constructed either globally or for local regions. Bootstrapped trees are returned and compared with the expected species tree. In cases of discrepancies, gene duplications are assumed and a reconciled tree is computed. The reconciled tree shows probable orthologues and paralogues as predicted.
Assuntos
Bases de Dados Factuais , Homologia de Sequência do Ácido Nucleico , Biologia Computacional/métodos , Evolução Molecular , Internet , Filogenia , Alinhamento de Sequência/métodos , Análise de Sequência de DNA/métodos , Validação de Programas de ComputadorRESUMO
SUMMARY: Network Analysis Interface for Linking HMMER results (NAIL) is a web-based tool for the analysis of results from a HMMER protein database-search. NAIL facilitates the selection of protein hits and the creation of an alignment, which can be used for a new sequence similarity search.
Assuntos
Bases de Dados Factuais , Internet , Análise de Sequência de Proteína/métodos , SoftwareRESUMO
Microsatellite instability (MSI) caused by defective DNA mismatch repair (MMR) is a hallmark of hereditary nonpolyposis colorectal cancers (HNPCC) but also occurs in about 15% of sporadic tumors. If instability affects microsatellites in coding regions, translational frameshifts lead to truncated proteins often marked by unique frameshift peptide sequences at their C-terminus. Since MSI tumors show enhanced lymphocytic infiltration and our previous analysis identified numerous coding mono- and dinucleotide repeat-bearing candidate genes as targets of genetic instability, we examined the role of frameshift peptides in triggering cellular immune responses. Using peptide pulsed autologous CD40-activated B cells, we have generated cytotoxic T lymphocytes (CTL) that specifically recognize HLA-A2.1-restricted peptides derived from frameshift sequences. Among 16 frameshift peptides predicted from mutations in 8 different genes, 3 peptides conferred specific lysis of target cells exogenously loaded with cognate peptide. One peptide derived from a (-1) frameshift mutation in the TGFbetaIIR gene gave rise to a CTL bulk culture capable of lysing the MSI colorectal cancer cell line HCT116 carrying this frameshift mutation. Given the huge number of human coding microsatellites and assuming only a fraction being mutated and encoding immunologically relevant peptides in MSI tumors, frameshift protein sequences represent a novel subclass of tumor-specific antigens. It is tempting to speculate that a frameshift peptide-directed vaccination approach not only could offer new treatment modalities for existing MSI tumors but also might benefit asymptomatic at-risk individuals in HNPCC families by a prophylactic vaccination strategy.
Assuntos
Linfócitos B/imunologia , Antígenos CD40/imunologia , Neoplasias do Colo/imunologia , Antígeno HLA-A2/genética , Fragmentos de Peptídeos/imunologia , Linfócitos T Citotóxicos/imunologia , Sequência de Aminoácidos , Antígenos CD/imunologia , Pareamento Incorreto de Bases , Ligante de CD40/imunologia , Neoplasias Colorretais Hereditárias sem Polipose/genética , Reparo do DNA , Mutação da Fase de Leitura , Antígeno HLA-A2/química , Humanos , Repetições de Microssatélites/genética , Fragmentos de Peptídeos/química , Proteínas Serina-Treonina Quinases , Receptor do Fator de Crescimento Transformador beta Tipo II , Receptores de Fatores de Crescimento Transformadores beta/genética , Receptores de Fatores de Crescimento Transformadores beta/imunologia , Células Tumorais CultivadasRESUMO
Microsatellite instability (MSI) caused by deficient DNA mismatch-repair functions is a hallmark of cancers associated with the hereditary nonpolyposis colorectal cancer (HNPCC) syndrome but is also found in about 15% of all sporadic tumors. Most affected microsatellites reside in untranslated intergenic or intronic sequences. However, recently few genes with coding microsatellites were also shown to be mutational targets in MSI-positive cancers and might represent important mutation targets in their pathogenesis. The systematic identification of such genes and the analysis of their mutation frequency in MSI-positive cancers might thus reveal major clues to their functional role in MSI-associated carcinogenesis. We therefore initiated a systematic database search in 33,595 distinctly annotated human genes and identified 17,654 potentially coding mononucleotide repeats (cMNRs) and 2,028 coding dinucleotide repeats (cDNRs), which consist of n > or = 6 and n > or = 4 repeat units, respectively. Expression pattern and mutation frequency of 19 of these genes with the longest repeats were compared between DNA mismatch repair-deficient (MSI(+)) and proficient (MSS) cancer cells. Instability frequencies in these coding microsatellite genes ranged from 10% to 100% in MSI-H tumor cells, whereas MSS cancer cells did not show mutations. RT-PCR analysis further showed that most of the affected genes (10/15) were highly expressed in tumor cells. The approach outlined here identified a new set of genes frequently affected by mutations in MSI-positive tumor cells. It will lead to novel and highly specific diagnostic and therapeutic targets for microsatellite unstable cancers.