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PubMed2005

[Pro-apoptotic efficiencies of three reconstructed human caspase-8 on cervical cancer cell line HeLa].

BACKGROUND & OBJECTIVE: Apoptosis is a kind of evolutional high conservative cell death. Transferring high active pro-apoptotic molecules into cancer cells to induce apoptosis is a potential strategy for cancer gene therapy. Based on our previous generation of reconstructed human caspase-8, which can continuously induce apoptosis of cervical cancer cell line HeLa, by reversing its large and small subunits, this study was designed to investigate the pro-apoptotic efficiencies of 3 reconstructed human caspase-8 (Casp8CD, Rev8, and Rev8L) on HeLa cells, and to explore the feasibility of reconstructed human caspase-8 as potential apoptosis-inducing candidates. METHODS: The eukaryotic expression vectors pIRES2-EGFP carrying Casp8CD, Rev8, and Rev8L genes were transfected into HeLa cells, and breast cancer MCF-7 cells. Expressions and pro-apoptotic effects of Casp8CD, Rev8, and Rev8L genes were observed under fluorescent microscope, and their pro-apoptotic efficiencies were assessed by MTT assay and cells counting. The flexibilities of linking-peptides between subunits of Rev8 and Rev8L were analyzed by bioinformatics. RESULTS: Expressions of the 3 reconstructed caspase-8 genes were observed under fluorescent microscope, and the HeLa and MCF-7 cells expressing Rev8 or Rev8L genes displayed typical apoptotic volume decrease (AVD). MTT assay showed that compared with control cells, A(570) values of Rev8- and Rev8L-transfected cells began to decrease 20 h after transfection. Cell counting results indicated that cell death ratio of Casp8CD-, Rev8-, and Rev8L-transfected cells were 16.9%, 52.3%, and 47.7%, respectively, 24 h after transfection; and 12.9%, 51.6%,and 61.2%,respectively,48 h after transfection. Bioinformatics analysis showed that the linking-peptides between subunits of Rev8 and Rev8L were flexible. CONCLUSION: Rev8 and Rev8L molecules have similar pro-apoptotic effects and efficiencies, but over-expressed Casp8CD had no significant pro-apoptotic effects.

Apoptosis↗

PubMed2005

Plasma protein profiling for diagnosis of pancreatic cancer reveals the presence of host response proteins.

Plasma protein profiling using separations coupled to matrix-assisted laser desorption ionization mass spectrometry (MALDI MS) has great potential in translational research; it can be used for biomarker discovery and contribute to disease diagnosis and therapy. Previously reported biomarker searches have been done solely by MS protein profiling followed by bioinformatics analysis of the data. To add to current methods, we tested an alternative strategy for plasma protein profiling using pancreatic cancer as the model. First, offline solid-phase extraction is done with 96-well plates to fractionate and partially purify the proteins. Then, multiple profiling and identification experiments can be conducted on the same protein fractions because only 5% of the fractions are used for MALDI MS profiling. After MALDI MS analysis, the mass spectra are normalized and subjected to a peak detection algorithm. Over three sets of mass spectra acquired using different instrument variables, approximately 400 unique ion signals were detected. Classification schemes employing as many as eight individual peaks were developed using a training set with 123 members (82 cancer patients) and a blinded validation set with 125 members (57 cancer patients). The sensitivity of the study was 88%, but the specificity was significantly lower, 75%. The reason for the low specificity becomes apparent upon protein identification of the ion signals used for the classification. The identifications reveal only common serum proteins and components of the acute phase response, including serum amyloid A, alpha-1-antitrypsin, alpha-1-antichymotrypsin, and inter-alpha-trypsin inhibitor.

Acute-Phase Proteins↗

PubMedDate not supplied

[An intron-free methyl jasmonate inducible geranylgeranyl diphosphate synthase gene from Taxus media and its functional identification in yeast].

Geranylgeranyl diphosphate synthase (GGPPS, EC: 2.5.1.29) catalyzes the biosynthesis of geranylgeranyl diphosphate (GGPP), which is a key precursor for diterpenes including Taxol, one of the most potent antitumor drugs. In order to investigate the role of GGPP synthase in taxol biosynthesis, we cloned, characterized and functionally expressed the GGPP synthase gene from Taxus media. A 3743-bp genomic sequence of T. media was isolated by genome walking strategy which contained an 1182-bp open reading frame (ORF) encoding a 393-amino acid polypeptide that showed high similarity to other plant GGPPSs. Subsequently the full-length cDNA of the GGPPS gene of T. media (designated TmGGPPS) was amplified by RACE. Bioinformatic analysis showed that TmGGPPS was an intron-free gene and its deduced polypeptide contained all the five conserved domains and functional aspartate-rich motifs of the prenyltransferases. By constructing the phylogenetic tree of plant GGPPSs, it was found that plant-derived GGPPSs could be divided into two classes, angiosperm and gymnosperm classes, which might have evolved in parallel from the same ancestor. To our knowledge this was the first report that the geranylgeranyl diphosphate synthase genes were free of intron and evolved in parallel between angiosperms and gymnosperms. The coding sequence of TmGGPPS was expressed in yeast mutant (SFNY368) lacking of GGPP synthase activity through functional complementation, and the transgenic yeast showed to have activity of GGPP synthase. This was also the first time to use SFNY368 to identify the function of plant-derived GGPPSs. Furthermore, investigation of the impact of methyl jasmonate (MeJA) on the expression of TmGGPPS revealed that MeJA-treated T. media cultured cells had much higher expression of TmGGPPS than untreated cells.

Acetates↗

PubMedDate not supplied

[Molecular cloning and characterization of a novel ice gene from Capsella bursapastoris].

A new ice gene (designated as Cbice53, an inducer of CBF expression) was cloned from Capsella bursa-pastoris by rapid amplification of cDNA ends (RACE). The full-length cDNA of Cbice53 was 1811 bp long with a 1476 bp open reading frame (ORF) encoding a Myc-like protein of 492 amino acids. The predicted CbICE53 protein contained a potential basic helix-loop-helix, a nuclear localization signal (NLS), a RNA-binding regions RGG box, N-glycosylation and kinase phosphorylation sites. Bioinformatic analysis revealed that CbICE53 was highly homologous to ICE1 from Arabidopsis thaliana. Transcription of Cbice53 gene was induced transiently during salt and cold treatments, suggesting that it was involved in someway in cold-acclimation process. Our study implies that the Cbice53 gene is a new member of the ice gene family and may exert functions in cold- and salt-responsiveness in C. bursa-pastoris.

Adaptation, Physiological↗

PubMed2005

Application of restriction fragment differential display-polymerase chain reaction in study on differential expression profiles of human diseases.

OBJECTIVE: To establish the restriction fragment differential display-polymerase chain reaction (RFDD-PCR) as an efficient technique for constructing and studying the gene expression profile of human tissues. METHODS: The tissues of mamma adenocarcinoma (T), cancerometastasis lymph node (L) and normal mammary (N) from one mammary infiltrating ductal carcinoma case were collected, and the gene expression profile of each kind of tissue was constructed using RFDD-PCR technique at equal pace according to the operating manual of Qbio-gene Company. Then all fragments of the three gene expression profiles were separated and displayed by electrophoresis. With the use of gene database at the website http://www.Qbio-gene.com/display, the authors identified the names of the probable fragments by bioinformatics analysis. Through comparison of the three profiles, the numbers and types of most differentially expressed gene fragments were displayed. RESULTS: The expression profiles of the three kinds of tissue have been constructed covering 1716 fragments of mammary adenocarcinoma, 1769 of cancerometastasis lymph nodes and 1922 of normal mammary tissue. Among these 5407 fragments, 39.39% were exactly the same. While 33.9% sequences of T and L showed differences in abundance or presence, 40.9% of T and N and 39.6% fragments of L and N were observed differentially expressed. These differentially expressed gene fragments were found to relate with metastasis, differentiation, inflammation and so on. CONCLUSION: RFDD-PCR is an efficient technique for research in human diseases genomics as a mass screening for complete gene expression profile with high-flux. Through comparison among three or more profiles, the screening for candidate genes of a certain disease can be accomplished, and there is probably a chance to identify novel gene or expressed sequence tag.

Adenocarcinoma↗

PubMed2005

[Cloning and expression in yeasts of the class V chitinase cDNA gene from Trichoderma harzianum].

To explore the integrated bio-control mechanism of Trichoderma harzianum and acquire some bio-control associated novel genes, a Trichoderma harzianum mycelium cDNA library has been constructed and thereby randomly selected clones were sequenced and analyzed by bioinformatics analysis. Full-length cDNA, encoding class v chitinase (ChiV), successfully cloned. The results indicated that the ORF of ChiV was 1194 bp, encoding 397 aa, deduced molecular weight 44 kD. The gene was ligated to the vector of pYES2 and thereby transformed to the yeast of H158 species. The enzyme activity reached the peak expression after cultured for 60 h induced by beta-galactose. The optimal temperature of ChiV is 37 degrees C and the optimal pH is 6 and 8.

Chitinases↗

PubMed2005

Molecular cloning of a novel LOS2 gene from Capsella bursa-pastoris.

A new LOS2 gene was cloned from C. bursa-pastoris by rapid amplification of cDNA ends (RACE). The full-length cDNA of C. bursa-pastoris LOS2 gene (designated as Cblos2) was 1694 bp containing a 1332 bp open reading frame (ORF) encoding a 444 amino acid protein. The predicted CbLOS2 protein contained enolase-N domain, enolase domain, conserved putative DNA-binding and repression domains like LOS2 from A. thaliana. Bioinformatic analysis indicated that CbLOS2 had similarity with other enolase proteins. Cold acclimation assay revealed that Cblos2 expressed constitutively in C. bursa-pastoris and was involved in the cold acclimation process, implying CbLOS2 was a bi-functional enolase.

Acclimatization↗

PubMed2005

SMYD3-NY, a novel SMYD3 mRNA transcript variant, may have a role in human spermatogenesis.

Identification of genes specifically expressed in adult and fetal testis is important to further our understanding of testis development and function. In this study, a novel SMYD3 transcript variant, termed SMYD3-NY (GenBank Accession No. AY186742), was identified by hybridization of adult and fetal human testis cDNA probes with a human cDNA microarray. SMYD3-NY transcript was expressed at 2.3-fold higher levels in adult human testis than in fetal testis, with a low expression level in human spermatozoa. Bioinformatical analysis showed that SMYD3-NY protein has the SET domain that is involved in histone methyltransferase (HTMase) activity. Southern blotting showed that SMYD3-NY is distributed in several tissues, including testis. In summary, SMYD3-NY is a novel transcript variant of the SMYD3 gene, and SMYD3-NY protein may influence transcriptional regulation during spermatogenesis via HTMase activity.

Adult↗

PubMed2005

[Subcellular localization and tissues expression profile of hGPCRc: an orphan G protein-coupled receptor].

As a member of orphan G protein-coupled receptors (oGPCRs), hGPCRc was cloned from human colon tissue and analyzed by bioinformatic softwares. It was showed that the corresponding amino acids of hGPCRc formed seven-transmembrane domains as the key characteristic of GPCRs. Then, the recombinant GFP-hGPCRc was constructed by fussing hGPCRc into pEGFP-N1 carrying green fluorescent protein (GFP) gene, and CHO-K1 cells were subsequently transfected with the GFP-hGPCRc or pEGFP-N1. The green fluorescence protein expression in the two different transfected cells was observed under the laser scanning confocal microscopy (LSCM). It was showed that green fluorescence protein was distributed in the whole bodies of the cells transfected with pEGFP-N1, but mainly distributed on the plasma membrane and cytoplasm membrane transfected with GFP-hGPCRc. Thus, the localization on the membrane of hGPCRc was accorded with the predication by bioinformatic analysis. The expression analysis of hGPCRc by RT-PCR indicated that hGPCRc was abundantly expressed in heart, kidney, cerebel and colon etc., but absent in liver, cerebra, small intestine and muscle etc. The expressing profile of hGPCRc could provide some useful clues to understanding its effects on embryonic development and physiological functions.

Amino Acid Sequence↗

PubMed2004

[Interaction of connexin 26 with the C-terminal of neuroendocrine specific protein].

OBJECTIVE: To screen and identify the interactive proteins with connexin 26 (Cx26) by the yeast two hybrid technique. METHODS: The whole coding region of Cx26 (GJB2) gene was amplified from normal human genomic DNA by polymerase chain reaction (PCR). The "bait" Cx26 was then subcloned into the vector pGBKT7 plasmid of the MatchMaker Gal4 Two-Hybrid System 3 as a target to screen its interactive proteins ("prey") from the human fetal brain eDNA library by the yeast two hybrid technique. The false positive clones were discarded from the preys by one to one yeast two hybrid method between Cx26 and the preys. The DNAs of the preys were sequenced and BLAST analyzed against the GenBank, and also underwent other bioinformatics analysis. RESULTS: The insert of one positive clone contained 145 amino acids residues that was identical to the C-terminal of the neuroendocrine specific protein (NSP) and the open reading frame of the insert was correct. CONCLUSION: Cx26 is interacted with the C-terminal of NSP. NSP may participate in the process of Cx26 transportation, assembling the connexon, or influencing the functions of its connexons.

Alcohol Oxidoreductases↗

PubMed2005

[Identification and characterization of a novel gene EOLA1 stimulating ECV304 cell proliferation].

OBJECTIVE: To amplify the full-length cDNA and characterize the structure and biological function of a novel expression sequence tag ST55 (GenBank Accession No. BM121646). METHODS: Rapid amplification of cDNA ends was used to clone the full-length of cDNA of ST55 in this study. Then, its tissue distribution was checked by Northern blots, and the associated protein was screened by GAL 4-based yeast two-hybrid. The effect of stable transfection of the cDNA on cell proliferation was evaluated in ECV304 cells. RESULTS: A full-length 1404 bp cDNA was cloned, and it was accepted as a novel human mRNA by GenBank (No. AY074889), named endothelial-overexpressed lipopolysaccharide-associated factor 1 (EOLA1). Bioinformatic analysis found that the EOLA1 encoded 158 amino acids, 17.89 kDa protein, and mapped to chromosome Xq27.4 with 5 exons. EOLA1 expressed in different human normal tissues and cancer cell lines. Using the EOLA1 cDNA as bait, we performed a yeast two-hybrid screening of a human liver cDNA library and identified metallothionein 2A (MT2A) as associated protein. The interaction between EOLA1 and MT2A was confirmed by co-immunoprecipitation experiments. Stable transfection of EOLA1 was noted to stimulate ECV304 cell proliferation (P < 0.05). CONCLUSION: The findings suggest that EOLA1 is a novel gene and the interaction of EOLA1 and MT2A may play an important role in cell protection in inflammation reaction.

Amino Acid Sequence↗

PubMed2005

[Screening of the genes of hepatitis B virus e antigen interacting proteins].

OBJECTIVE: To screen and clone the genes in hepatocytes which encode protein that can interact with hepatitis B e antigen(HBeAg) by yeast-two hybridization. METHODS: Recombined HBeAg bait plasmid (pGBKT7-eAg) was transformed into yeast AH l09, followed by mating with yeast Yl87 containing liver cDNA library plasmid in 2 x YPDA medium. Diploid yeast was plated on synthetic dropout nutrient medium (SD/-Trp-Leu-Ade-His) which contains X-a-gal for selecting positive blue clones. Then positive clones were selected and plasmids were prepared and sequenced. Finally, bioinformatics analysis was performed. RESULTS: Totally 245 positive colonies were selected and 101 colonies were sequenced. Through sequences alignment, 6 novel genes and 35 recorded genes were screened. CONCLUSION: Genes of HBeAg interacting proteins have been cloned successfully, which brings some new clues for further studies on the biological functions of HBeAg and the related proteins.

Gene Library↗

PubMed2005

[Characteristics and expression of Mip5, a novel gene associated with myocardial ischemia/reperfusion in rats].

OBJECTIVE: To determine the characteristics of a novel gene Mip5 (GenBank accession number AY553870) and its expression under physiological and pathological conditions. METHODS: The characteristics of Mip5 were analyzed by bioinformatic programs including BLAST, spidey, psort, ClustalW and so on. RT-PCR was performed to detect Mip5 expression. RESULTS: Bioinformatic analysis showed that Mip5 gene lied in the 13th chromosome and contained 8 exons and 7 introns, its open reading frame contained 909 bp and its protein production was 302 amino acid residues including 6 kelth domains. Under normal conditions, MIP5 expressed abundantly in the heart, brain and kidney, but its expression could not be detected in the liver and muscle. Expression of Mip5 gene was increased significantly after ischemia-reperfusion compared with the sham groups, and reached its peak at 3 h and recovered at 12 h after the reperfusion. Conclusion Mip5 gene is a novel gene containing a putative open reading frame of 302 amino acids residues and may play an important role in rat cardiomyocytes suffering ischemia processing.

Amino Acid Sequence↗

PubMed2005

[Detection of differentially expressed genes in human autosomal dominant polycystic kidney tissue].

OBJECTIVE: To detect the differentially expressed genes in human polycystic kidney by cDNA microarray. METHODS: The PCR products of 8398 genes were spotted onto a chip in array. Both mRNAs isolated from polycystic kidney tissue and normal kidney tissue were reversely transcribed to cDNAs with the incorporation of fluorescent dUTP (Cy5-dUTP and Cy3-dUTP) for preparing the hybridization probes. The mixed probes were hybridized to the cDNA microarray. Then the cDNA microarray was scanned for the fluorescent signals and the display of differences between the 2 tissues. IGF1 mRNA, one of the up regulated genes was detected by in situ hybridization technique in the two tissues to validate the result from cDNA microarray. RESULTS: The result indicated that the expressions of 263 genes were up regulated while the expressions of 94 genes were down regulated in the polycystic kidney tissue among the 8398 target genes. Bioinformatical analysis of those genes had been performed. The up-regulated genes were mainly the ones of oncogene, cellular skeleton and movement, apoptosis related protein, cell signal transduction protein, and cytokine. The down regulated genes were mainly the ones of anti-oncogene, DNA binding and transcription factors, cell signal transduction protein, and metabolism protein. The IGF1 mRNA expression detected by in situ hybridization was consequently consistent with the cDNA microarray. CONCLUSION: cDNA microarray is an effective and quick method for studying differential expressed genes. Three hundred and fifty-seven differentially expressed genes with different functions were revealed in the polycystic kidney tissue, which may play some roles in the progression of polycystic kidney.

Carbocyanines↗

PubMed2006

Gene clusters for beta-lactam antibiotics and control of their expression: why have clusters evolved, and from where did they originate?

While beta-lactam compounds were discovered in filamentous fungi, actinomycetes and gram-negative bacteria are also known to produce different types of beta-lactams. All beta-lactam compounds contain a four-membered beta-lactam ring. The structure of their second ring allows these compounds to be classified into penicillins, cephalosporins, clavams, carbapenens or monobactams. Most beta-lactams inhibits bacterial cell wall biosynthesis but others behave as beta-lactamase inhibitors (e.g., clavulanic acid) and even as antifungal agents (e.g., some clavams). Due to the nature of the second ring in beta-lactam molecules, the precursors and biosynthetic pathways of clavams, carbapenems and monobactams differ from those of penicillins and cephalosporins. These last two groups, including cephamycins and cephabacins, are formed from three precursor amino acids that are linked into the alpha-aminoadipyl-L-cysteinyl-D-valine tripeptide. The first two steps of their biosynthetic pathways are common. The intermediates of these pathways, the characteristics of the enzymes involved, the lack of introns in the genes and bioinformatic analysis suggest that all of them should have evolved from an ancestral gene cluster of bacterial origin, which was surely transferred horizontally in the soil from producer to non-producer microorganisms. The receptor strains acquired fragments of the original bacterial cluster and occasionally inserted new genes into the clusters, which once modified, acquired new functions and gave rise to the final compounds that we know. When the order of genes in the Streptomyces genome is analyzed, the antibiotic gene clusters are highlighted as gene islands in the genome. Nonetheless, the assemblage of the ancestral beta-lactam gene cluster remains a matter of speculation.

Anti-Bacterial Agents↗

PubMed2006

[Cloning, expression and subcellular localization of a novel human gene-RNF122].

OBJECTIVE: To clone a functionally unknown gene RNF122 and analyze its expression pattern and subcellular localization. METHODS: PCR was used to clone the novel gene-RNF122 from the mixed human tissue cDNA library. Bioinformatics analysis was used to identify structure characteristics of the gene. Northern blot was used to analyze its expression in normal tissues; RT-PCR was employed for its expression in cell lines and tumor tissues. By co-focal microscope, we identified its subcellular localization with organelle markers. RESULTS: A novel human gene-RNF122 was cloned, which was proved to have been expressed in several normal and tumor tissues and many cell lines, and localized in ER and Golgi apparatus. CONCLUSION: RNF122 is a novel gene which encodes a protein that has a classic RING domain. It is widely expressed in several tissues and cell lines. The encoded protein is localized in ER and Golgi apparatus, which indicates that it may play a role in the process of protein degradation.

Amino Acid Sequence↗

PubMed2006

[Preliminary study of gene expression profiling in human type I and II endometrial carcinoma].

OBJECTIVE: To study gene expression profiling in human type I and II endometrial carcinoma. METHODS: Six Affymetrix human genome genechips were utilized to investigate the differences in gene expression profiles between type I and II endometrial carcinoma with bioinformatic analysis. RESULTS: Many genes were highly expressed in estrogen-dependent endometrial carcinoma, and some of them were involved in the metabolism and conversion of estrogen, while some others in estrogen regulation. CYP2C9, for instance, was involved in the conversion of estrogen sulfate to 16-hydroxy sulfate metabolite, DDC in estrogen-dependent pathogenesis of endometrial carcinoma possibly by DDC interaction with AR to enhance steroid receptor transcription. CONCLUSION: High expression of these genes in estrogen-dependent endometrial carcinoma may provide insights into their roles in the pathogenesis and prognosis of this malignancy.

Adenocarcinoma↗

PubMed2006

GPS@ bioinformatics portal: from network to EGEE grid.

Bioinformatics analysis of data produced by complete genome sequencing projects is one of the major challenges of the current years. Integrating up-to-date databanks and relevant algorithms is a clear requirement of such analysis. Grid computing would be a viable solution to distribute data, algorithms, computing and storage resources for Genomics. Providing bioinformaticians with a good interface to grid infrastructure, such as the one provided by the EGEE European project, is also a challenge to take up. The GPS@ web portal, "Grid Protein Sequence Analysis", aims to provide such a user-friendly interface for these grid genomic resources on the EGEE grid.

Computational Biology↗