Detection of DD3 mRNA level in peripheral blood of patients with prostate cancer and its clinical value / 中国基层医药

Objective To study the level and clinical value of differential display code 3 (DD3) in peripheral blood of patients with prostate cancer.Methods 27 patients with prostate cancer from April 2014 to April 2015 in Affiliated Hospital of Hangzhou Normal University were researched.26 patients with non prostate diseases were selected as control group.DD3 mRNA levels were detected in peripheral blood of all patients.DD3 absorbance value of three groups of patients,the relationship between the relative content of DD3 mRNA and the clinical characteristics of prostate cancer,DD3 mRNA in urine and peripheral blood of patients with prostate cancer and benign prostatic hyperplasia were observed.Results In the patients with benign hyperplasia of prostate and non prostate diseases,a total of 4 patients did not appear to be DD3 specific bands.In prostate cancer patients,all patients were found to have DD3 specific bands.The relative content of patients with non prostate diseases was (0.18 ± 0.05) copies/mL.The relative content of benign prostatic hyperplasia patients was (0.30 ± 0.09) copies/mL.The relative content of prostate cancer patients was (0.78 ± 0.23) copies/mL.The positive expression rates of DD3 mRNA in peripheral blood and urine of patients with prostate cancer were 85.18％ (23/27),51.85％ (14/27),respectively,which were significantly higher than those in patients with benign prostatic hyperplasia[21.42％ (6/28),7.14％ (2/27)] (x2 =22.416,13.319,all P ＜ 0.05).Conclusion The specific expression of DD3 mRNA in peripheral blood of patients with prostate cancer can be used as an effective basis for judging the patients' condition,and it has certain value for the treatment and prognosis of patients with prostate cancer.

Screening of Stilbene Glucosides Biosynthesis-related Genes in Polygonum multiflorum Thunb. by mRNA Differential Display Reverse Transcription Polymerase Chain Reaction / 广州中医药大学学报

Objective To screen and clone the genes related to stilbene glucosides biosynthesis in Polygonum multiflorum Thunb. Methods The differentially expressed genes in the root, stem and leaf of Polygonum multiflorum which have different contents of stilbene glucosides were screened by differential display reverse transcription polymerase chain reaction (DDRT-PCR). After pMD19-T carrier was inserted into the obtained differential genes for sequencing and comparison, the gene function was analyzed. Results Fifty-one differentially expressed cDNA fragments were found. Of them, 9 were used for the identification by semi-quantitative PCR. The identification results presented 3 positive fragments, one fragment was specifically expressed in the stem and leaf of Polygon-um multiflorum Thunb., sharing high homology with glycine dehydrogenase, and 2 were specifically expressed in the root of Polygonum multiflorum Thunb., having high homology with enoyl-CoA hydratase and aminopeptidase N, respectively. Conclusion Three homologous gene sequences obtained through DDRT-PCR provide a basis for the further study of biosynthetic pathway of stilbene glucoside from Polygonum multiflorum Thunb..

Expresión diferencial durante la interacción Solanum tuberosum - Phytophthora infestans / Differential expression during Solanum tuberosum- Phytophthora infestans interaction

La papa (Solanum tuberosum L.) es el cuarto cultivo más importante a nivel mundial y es el producto agrícola con mayor demanda de fungicidas, insecticidas y fertilizantes quí­micos. Las pérdidas mundiales ocasionadas por Phytophthora infestans (Mont.) de Bary en este cultivo, ascienden a 6,7 billones de dólares al año y su control quí­mico genera un aumento en los costos, perjudica la salud humana y el ambiente. Todo esto justifica la búsqueda constante de mecanismos alternativos para el control de la enfermedad, entre ellos la obtención de variedades resistentes mediante cisgenesis usando genotipos silvestres. Como un aporte en este sentido, y dada la falta de conocimiento de lo que controla y constituye la diferencia entre una respuesta compatible e incompatible, en el presente estudio se compararon los perfiles de expresión génica obtenidos mediante Despliegue Diferencial de variedades resistentes y susceptibles durante su interacción con P. infestans. Los resultados evidenciaron diferencias en la expresión génica, tanto a distintos tiempos post-inoculación como en el tipo de cambio de expresión, incluyendo la presencia y ausencia de bandas diferenciales y el aumento o disminución en su intensidad. Al analizar las secuencias de fragmentos diferencialmente expresados, se encontró que algunos fragmentos sobre-expresados en las variedades susceptibles, tení­an homología con secuencias que codifican para una serina-acetiltranferasa y para la subunidad Β de la RNA polimerasa. Por su parte, fragmentos sobre-expresados en la variedad resistente, tení­an homología con una secuencia codificante para un dominio transmembranal.

Potato (Solanum tuberosum L.) is the fourth most important crop worldwide; also, is the agriculture product with most fungicides, insecticides and chemical fertilizers requirement. Worldwide losses caused by Phytophthora infestans (Mont.) de Bary in this crop, amount to 6,7 billion dollars per year and its chemical control increase production costs, harming human health and environment. For these reasons, is necessary constant research for alternative mechanisms to control disease, including development of resistant varieties using cis-genesis from wild genotypes. As a contribution in this way, and the lack of knowledge of what controls and is the difference between compatible and incompatible interaction, in this study we compared gene expression profiles obtained by Differential Display from resistant and susceptible varieties, during their interaction with P. infestans. The results showed differences in gene expression between resistant and susceptible varieties, at different times post-inoculation as well as exchange expression rate, including the presence and absence of differential bands and increase or decrease in their intensity. After analyzing the sequences of differential expressed fragments, we found that some overexpressed fragments from susceptible varieties had homology with an encoded sequence for a serine-acetyltransferase and for a RNA Polymerase Β subunit. On the other hand, overexpressed fragments from resistant variety, had homology with an encoded sequence for a transmembrane domain.

A phage display combined with DNA affinity magnetic system can be applied to a screening of DNA binding proteins, such as transcription factors / Un fago pantalla combinado con un sistema de afinidad magnético al ADN se puede aplicar al exámen de las proteínas de unión al ADN, tales como los factores de transcripción

Here we introduce a new approach for the screening of DNA binding proteins, using a phage library based on a phage display technique. In principal, a complementary DNA (cDNA) library based on the recombinant bacteriophage T7 expressing target proteins on its capsid (phage display) is constructed. These phage particles are hybridized with a biotinylated target DNA fragment which is immobilized on the surface of streptavidin paramagnetic particle (SA-PMP). The phage particles are released from the target DNA fragment by a nuclease treatment and the recovered phages are used to the next round of hybridization. These processes are repeated three times to amplify the target phages in the population. This simple method is faster, and more systemic than other current methods (e.g. yeast one hybrid system). As a proof of this principle, we tried to isolate transcription factors which specifically bind to the promoter region of the Arabidopsis thaliana AtGST11 gene. Two obtained candidates, RING zinc finger protein and AtHB6, showed DNA binding activity to the AtGST11 promoter region. We could validate that our new application of phage display is a superior method for isolation of DNA binding proteins with a broad range of potential applications.

A Winged-Helix Transcription Factor Foxg1 Induces Expression of Mss4 Gene in Rat Hippocampal Progenitor Cells

Foxg1 (previously named BF1) is a winged-helix transcription factor with restricted expression pattern in the telencephalic neuroepithelium of the neural tube and in the anterior half of the developing optic vesicle. Previous studies have shown that the targeted disruption of the Foxg1 gene leads to hypoplasia of the cerebral hemispheres with severe defect in the structures of the ventral telencephalon. To further investigate the molecular mechanisms by which Foxg1 plays essential roles during brain development, we have adopted a strategy to isolate genes whose expression changes immediately after introduction of Foxg1 in cultured neural precursor cell line, HiB5. Here, we report that seventeen genes were isolated by ordered differential displays that are up-regulated by over-expression of Foxg1, in cultured neuronal precursor cells. By nucleotide sequence comparison to known genes in the GeneBank database, we find that nine of these clones represent novel genes whose DNA sequences have not been reported. The results suggest that these genes are closely related to developmental regulation of Foxg1.

In silico identification of cancer genes by high throughput analyses / 西安交通大学学报(医学版)

Objective To identify potential candidate genes related to the cancerous phenotype by analyzing databases publicly available. Methods Using a data-mining tool called Digital Differential Display (DDD) from the Cancer Gene Anatomy Project database, ESTs from 17 different tumor types were analyzed for differential expression. Results We obtained 130 up-regulated and 159 down-regulated genes, most of which are related to cytoskeleton, ribosomal subunit, substance metabolism, cell cycle, signal conduction, transcription and translation. These genes appear most frequently on chromosome 12 but rarely on chromosome 21 and Y. Conclusion In silico identification is a high-throughput screening strategy. Our study may lay a foundation for identification of future caner markers and provide a new thought for screening strategy of cancer markers.

A porcine gene, PBK, differentially expressed in the longissimus muscle from Meishan and Large White pig

An investigation of differences in gene expression in the longissimus muscle of Meishan and Large White pigs was undertaken, using the mRNA display technique. A fragment of one differentially expressed gene was isolated and sequenced, whereupon the complete cDNA sequence was then obtained by using the rapid amplification of cDNA ends (RACE). The nucleotide sequence of the gene is not related to any known porcine gene. Sequence analysis revealed that the open reading frame of this gene encodes a protein with 322 amino acids, thus displaying high sequence identity with the PDZ binding kinase (PBK) of eleven other animal species - dog, horse, cattle, human, chimpanzee, crab-eating macaque, rhesus monkey, rat, mouse, gray short-tailed opossum and platypus, so it can be defined as the porcine PBK gene. This gene was finally assigned GeneID: 100141310. Phylogenetic tree analysis revealed that the swine PBK gene has a closer genetic relationship with the PBK gene of platypus. Gene expression analysis of eight tissues of a Meishan x Large White cross showed that the porcine PBK gene is differentially expressed in various tissues. Our experiment established the primary foundation for further research on this gene.

Genes e epilepsia II: expressão gênica diferencial: [revisão] / Genes and epilepsy II: differential gene expression in epilepsy: [review]

Nesta revisão, introduzimos abordagens investigativas, assim como discutimos os principais achados de expressão gênica diferencial em tecido epiléptico humano e em modelos experimentais. As alterações observadas no cérebro de indivíduos epilépticos sugerem que eventos moleculares específicos refletem diferentes expressões do quadro fisiopatológico. É possível que diferentes combinações da expressão de genes associados à morte celular, metabolismo de radicais livres, transmissão sináptica, resposta imune e de neurotrofinas reflitam propriedades características de diferentes populações neuronais e gliais, que determinam as distintas respostas de cada área cerebral. A compreensão dessas particularidades moleculares será muito importante para o desenvolvimento de uma estratégia de intervenção visando reduzir neurotoxicidade e disfunções sinápticas que ocorrem durante a epileptogênese e a fase crônica em pacientes epilépticos.

We introduce some investigative appnacher and findings on differential gene expression in human epileptic time as well as in animal models of epilepsy. Molecular alterations observed in the epileptic brain suggest that they may disclose different psychopathological stages. It is possible that different gene expression combinations involved in cell death, reactive oxygen metabolism, synaptic transmission and immune response and of neurotrophins reflect distinct functional properties of different neuronal and glial populations, which determine specific brain region responses. Understanding the molecular patterns of gene expression following epileptogenic insults will be of great importance for the development of treatments aiming to reduce neurotoxicity and subtle synaptic dyfunctions present in the early stages as well as during the chronic phase of epilepsy.

Differential gene expression and mitotic cell analysis of the drought tolerant soybean (Glycine max L. Merrill Fabales, Fabaceae) cultivar MG/BR46 (Conquista) under two water deficit induction systems

Drought cause serious yield losses in soybean (Glycine max), roots being the first plant organ to detect the water-stress signals triggering defense mechanisms. We used two drought induction systems to identify genes differentially expressed in the roots of the drought-tolerant soybean cultivar MG/BR46 (Conquista) and characterize their expression levels during water deficit. Soybean plants grown in nutrient solution hydroponically and in sand-pots were submitted to water stress and gene expression analysis was conducted using the differential display (DD) and real time polymerase chain reaction (PCR) techniques. Three differentially expressed mRNA transcripts showed homology to the Antirrhinum majus basic helix-loop-helix transcription factor bHLH, the Arabidopsis thaliana phosphatidylinositol transfer protein PITP and the auxin-independent growth regulator 1 (axi 1). The hydroponic experiments showed that after 100 min outside the nutrient solution photosynthesis completely stopped, stomata closed and leaf temperature rose. Both stress induction treatments produced significant decrease in the mitotic indices of root cells. Axi 1, PITP and bHLH were not only differentially expressed during dehydration in the hydroponics experiments but also during induced drought in the pot experiments. Although, there were differences between the two sets of experiments in the time at which up or down regulation occurred, the expression pattern of all three transcripts was related. Similar gene expression and cytological analysis results occurred in both systems, suggesting that hydroponics could be used to simulate drought detection by roots growing in soil and thus facilitate rapid and easy root sampling.

Identification of Differentially Expressed Genes in Murine Hippocampus by Modulation of Nitric Oxide in Kainic Acid-induced Neurotoxic Animal Model

Kainic acid (KA) causes neurodegeneration, but no consensus has been reached concerning its mechanism. Nitric oxide may be a regulator of the mechanism. We identified differentially expressed genes in the hippocampus of mice treated with kainic acid, together with or without L-NAME, a nonselective nitric oxide synthase inhibitor, using a new differential display PCR method based on annealing control primers. Eight genes were identified, including clathrin light polypeptide, TATA element modulatory factor 1, neurexin III, ND4, ATPase, H+ transporting, V1 subunit E isoform 1, and N-myc downstream regulated gene 2. Although the functions of these genes and their products remain to be determined, their identification provides insight into the molecular mechanism(s) involved in KA-induced neuronal cell death in the hippocampal CA3 area.

Cloning of novel gene GP1 associated with gastric cancer and its premalignant lesions / 基础医学与临床

Objective To explore the differentially expressed genes of gastric cancer,matched normal gastric tissue and premalignant lesions. Methods The differentially expressed cDNA bands were isolated and identified by fluorescent differential display and then reamplified by PCR.After being cloned,all cDNA fragments were sequenced. Through BLAST,the results were compared with GenBank database for homology analysis. The expression of the cDNA fragment in different tissues was confirmed by Northern blot. SMART-RACE(rapid amplication of cDNA ends) was used to amplify the full length cDNA sequence. Bioinformatics analysis was performed to analysis its function. Results A differentially expressed cDNA fragment expressed lower in gastric cancers and premalignant lesions,no homology was found in GenBank. The novel cDNA fragment was given an accession number of EST(CD454989)in GenBank. A full length cDNA sequence of 1362 bp was acquired,who coded 267 amino acids,and was homologous with BAA91562.1,whose physiology function was unknown. Conclusion A differentially expressed gene was found and it may be involved in process of the gastric cancer.

The application of mRNA differential display in screening differential expressedgenes of peripheral blood leucocyte from Uigur and Kazak patients with type 2 diabetes mellitus / 基础医学与临床

Objective To identify the differentially expressed genes in Uigur and Kazak patients with and without type 2 diabetes mellitus.Methods The differentially expressed cDNA bands were isolated by fluorescent mRNA differential display from peripheral blood leucocyte of the Uigur and Kazak patients with type 2 diabetes mellitus and the normal controls. After being cloned, all cDNA fragments were sequenced, then underwent sequence analysis, homogenous comparison,and Northern blot analysis. Results Z5、Z8、Z15 differentially expressed cDNA fragments were found.They were over-expressed in the normal controls and were lower or scarced in the Uigur and Kazak patients with type 2 diabetes mellitus. They were selected for sequencing and hybridization. Z5、Z8 showed highly homologous to cellular repressor of E1A-stimulated genes,Z15 are unknown. Conclusion Three differentially expressed genes may have a potential relation with type 2 diabetes mellitus.

Screening and identification of differentially expressed genes in colorectal carcinoma / 临床检验杂志

Objective To screen and identify differentially expressed genes in colorectal carcinoma and explore the possible molecular pathogenesis of colorectal carcinoma.Methods The differentially expressed cDNA bands in colorectal carcinoma specimens and matched adjacent normal tissues were isolated by fluorescent mRNA differential display.Following differential display PCR (DD-PCR),all cDNA fragments were sequenced.By using BLAST software,the sequencing results were compared with Genebank database for homologue analysis.RT-PCR was used to detect the expression of one of the differentially expressed genes in colorectal carcinoma samples were identified by semi-RT-PCR.Results BLAST analysis revealed that the cDNA band was homologous to DDX32 (99%) and its up-regulated expression in colorectal carcinoma tissues was confirmed by RT-PCR (P

Analysis of C3orf1 gene over-expressed in human lung cancer cell line 95D with high metastatic potential / 临床检验杂志

Objective To further define the molecular mechanism involved in the metastasis process in lung cancer and screen out the genes expressed differentially in the lung cancer.Methods mRNA differential display (DD-PCR) was employed to search the specific genes related to metastasis.The highly expressed fragments were cloned and sequenced.Compared with the data in GenBank,the homologous genes were found.The anchor primers were designed to validate the candidates from DD-PCR by real-time PCR.The structure of the gene was prognosticated by software.Results Nine differentially expressed genes were found.One of the nine genes,which named C3orf1,showed high different expression within the six tested cancer cells.The gene was 858bp long and encoded 285 amino acids.The molecular weight was about 32.2 kD.Analyzed by the bio-software,it was found that the gene was consisted with seven EGF-like domains (EGF_1),three 2Fe-2S ferredoxin/iron-sulfur binding regions (2FE2S_FER_1),two VWFC domains (VWFC_1),two thiolase active sites (thiolase_3),and so on.Conclusions The gene C3orf1 is over-expressed in the lung cancer 95D cells.It may encode a familiar secretary growth factor protein and play important role in stimulating growth of the cells.

Gene Expression Analysis of CD34~+ Hematopoietic Stem and Progenitor Cells Grown in Different Culture Environments Using Differential Display / 中国生物工程杂志

Objective: To investigate the changes of gene expression in CD34+ hematopoietic stem and progenitor cells (HSPCs) under different growth environments. Methods: Umbilical cord blood mononuclear cells (UCB MNCs) were cultured in static and stirred systems. After 7 days of culture, CD34+ cells were isolated and total RNA was extracted. Gene expression patterns of CD34+ cells from fresh, static and stirred cultures were compared using differential display (DD). Results: 30 gene fragments displayed differential expression levels based on the conditions of DD. One of differentially expressed genes was identified as RAN, which is a member of oncogene RAS family. This gene may be associated with proliferation of hematopoietic cells. Conclusion: Different growth environments induced differential gene expression patterns of CD34+ HSPCs. These differentially expressed genes would give new insights into optimizing in vitro environments for expanding hematopoietic cells.

Selección de fragmentos diferenciales de ADNc relacionados con estrés hídrico en Ullucus tuberosus Loz: (Bassellaceae) «olluco»

A partir de la expresión diferencial de ARNm de plántulas in vitro de dos accesiones de Ullucus tuberosus Loz. «olluco» altamente tolerantes a estrés osmótico, fueron seleccionados 31 fragmentos diferenciales de ADNc relacionados con tolerancia a sequía.

Thirty-one differential fragments of cDNA related to drought tolerance have been selected from the mRNA differential expression of in vitro plantelets belonging to two accessions of Ullucus tuberosus Loz. «olluco» highly tolerant to osmotic stress.

Development and optimization of a fluorescent differential display PCR system for studying bovine embryo development in vitro

Differential display is a widely used methodology to identify genes that are differentially expressed in biological samples. We developed a new protocol for the amplification and recovery of differentially expressed genes from extremely small initial amounts of RNA (10 to 25 pg mRNA) from preimplantation bovine embryos. The cDNAs generated with an anchor primer, associated with a universal sequence, were amplified with an arbitrary primer and a single fluorescently labeled primer. Amplification products were easily visualized with a fluorescence scanner, without the need for radioisotopes. Nineteen isolated fragments were cloned and sequenced, confirming the expected primer sequences and allowing the recognition and identification of gene transcripts involved in bovine embryonic physiology.

Isolation and Characterization of the Salicylic Acid Induced Gene in Rehmannia glutinosa by Differential Display

Rehmannia glutinosa is a perennial medicinal plant belonging to the family Scrophulariaceae with more than 300 species known in the world, especially in temperate regions. Its roots have been used widely in Korea for medicinal purposes. However, it is commonly infected by various pathogens during storage, causing great damage to the roots, and impedes the intensive farming of the crop. Therefore, an attempt has been made to isolate and screen a resistance gene against the pathogen Fusarium oxysporum using differential display. We treated salicylic acid (SA), and isolated a resistance gene that responds to SA. As a result, we found that SA was involved in plant defense mechanism in pathogenicity tests with SA treated and non-treted plants, and we isolated a partial PR-la gene through differential display polymerase chain reaction (DD-PCR) method.

Cloning and studying on the expression and function of thyroid hormone-response protein-1 gene, a novel thyroid hormone-response gene from neonatal rat brain / 中华内分泌代谢杂志

Objective To is ol ate novel thyroid hormone-response genes, to study the characterizations of the ir expressions and to predict their possible functions in neonatal rats. Methods A neonatal rat model with congenital hypothyr oidism was established and cDNA fragments of novel thyroid hormone-response gen es from cerebral cortex of neonatal rats were obtained by fluorescence-labeled DD-PCR analysis, subcloning and sequencing. Complete cDNAs of novel thyroid hor mone-response genes were cloned by the techniques of electronic clone, RT-PCR and sequencing, their expressions regulated by thyroid hormone were confirmed b y Northern blot analysis, their distributions, transcription levels in different tissues and different brain areas were further observed by semiquantitative RT -PCR analysis, and their possible functions were postulated through bioinformat ic techniques. Results A novel complete cDNA of thyroid hormone-response protein-1 (TRP-1) gene is cloned. It is 973 bp in f ull-length (Gene Bank accession no. AF348365), and its transcription was enhanc ed in cerebral cortex in neonatal hypothyroidism rats. The expression of its mRN A was very extensive, but more abundant in brain. Its transcriptional level in d ifferent brain areas was not uniform, much higher in olfactory bulb. Its encodin g protein had some significant domains and motifs. Conclusion TRP-1 gene is a new thyroid hormone-response gene and may play an important role during normal brain development. Its abnormal expression may b e partially responsible for neurological defects in brain arising from thyroid h ormone deficiency during critical period for perinatal rats.

APPLICATION OF SIMPLIFIED DIFFERENTIAL DISPLAY POLYMERASE CHAIN REACTIONTO ISOLATE CHLOROQUINE-RESISTANT RELATED GENES OFPLASMODIUM BERGHEI / 中国人兽共患病学报

To insight into genetic differences between chloroquine-resistant line (RC) and chloroquine-sensitive strain (N) of Plasmodium berghei. MethodsAfter continous cbloroquine-pressure upon RC line at higher dosage (50mg/kg. d) ,total RNAs from the RC line and the N strain of P. berghei were isolated for simplified differential display reverse transcript polymerase chain reaction (sDDRT-PCR ). The generated differential fragments had been repetitively rescued and identified by PCRs before one pair of suspected differential fragments (N25 and R25 )were cloned and sequenced. Then, their sequences were analyzed through PC-gene program and BLAST search. ResultsThough the identity of two nucleotide sequences of N252 and R251 ,cloned separately from the N25 and R25 fragments, were up to 99.8％ ,their predicted amino acid secondary structures were quite different due to multiple mutations. Compared with the published sequences in GeneBank,EMBL,DDBJ and PDB database ,no similar gene was found ,using BLAST search. However their partial nucleotide sequences (62nt from query 128nt to189nt bore highly homology to part sequence(from 1053nt to1114nt)of rattus norvegicus mRNA for phospholipase B,up to 93.5％ in N251 and 91.9％ in N252 respectively. ConclusionIt is feasible to isolate chloroquine-resistant related genes by using simplified DDRT-PCR combined repetitively rescuing and PCR identifying the interest differential DNAs together with sequence analyses.