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Simultaneous Expression (simultaneous + expression)
Selected AbstractsCo-localization of multiple ErbB receptors in stratified epithelium of oral squamous cell carcinomaTHE JOURNAL OF PATHOLOGY, Issue 3 2001Roberto Bei Abstract The expression of all four ErbB receptors was compared by immunohistochemistry, using receptor-specific polyclonal antisera, in 32 invasive, 11 in situ carcinomas, six benign lesions, and 22 samples of histologically normal mucosa adjacent to specimens of carcinoma originating from oral cavity epithelium. Among invasive and in situ carcinoma, EGFR expression was the most prevalent (in 29/32 and 8/11 cases, respectively) followed by ErbB2 (17/32 and 2/11) and ErbB4 (9/32 and 1/10), while ErbB3 was only detected in invasive tumours (12/32). Specific patterns included invasive tumours with expression of EGFR (8/32) or ErbB4 (1/32) alone, as well as different receptor combinations (EGFR+ErbB2, EGFR+ErbB4, EGFR+ErbB2+ErbB3, EGFR+ErbB2+ErbB4, and all four receptors). Simultaneous expression of three or four ErbB receptors correlated with tumour invasion (p=2.2×10,4) and localized in the intermediate epithelial cell layer of well and moderately differentiated tumours. No other significant correlation with clinico-pathological features was noticed. Some benign lesions and histologically normal mucosa adjacent to carcinomas showed weak immunostaining of EGFR (10/28), ErbB2 (4/28) or ErbB4 (3/28). By comparison, overexpression, as indicated by increased staining intensity, was observed in invasive tumours for EGFR (18/32), ErbB2 (8/32), ErbB4 (3/32), and ErbB3 (3/32). Statistical evaluation demonstrated a significant association of EGFR or ErbB2 overexpression with invasive carcinoma when compared with benign lesions and apparently normal epithelium (p=5.2×10,7 and p=5×10,3, respectively). Tumour-specific overexpression of ErbB receptors and their co-expression, most frequently involving EGFR and ErbB2, in the same cell layer of neoplastic epithelium, implicate receptor heterodimers in the pathogenesis of oral squamous carcinoma. Copyright © 2001 John Wiley & Sons, Ltd. [source] Simultaneous expression and maturation of the iron-sulfur protein ferredoxin in a cell-free systemBIOTECHNOLOGY & BIOENGINEERING, Issue 1 2006Marcus E. Boyer Abstract The model iron-sulfur (Fe-S) protein ferredoxin (Fd) from Synechocystis sp. PCC 6803 has been simultaneously produced and matured in a cell-free production system. After 6 h of incubation at 37°C, Fd accumulated to >450 µg/mL. Essentially all was soluble, and 85% was active. Production and maturation of the protein in the cell-free system were found to be dependent in a coupled manner on the concentration of the supplemented iron and sulfur sources, ferrous ammonium sulfate and cysteine, respectively. The recombinant expression of ISC helper proteins during cell extract preparation did not increase cell-free Fd accumulation or activity, although the efficiency of iron and cysteine utilization increased. Fd maturation was independent of protein production rate, and proceeded at a constant rate throughout the period of active translation. In addition, incubation of denatured apo Fd with cell-free reaction components resulted in recovery of Fd activity, supporting the interpretation that maturation mechanisms did not act co-translationally. Incubation at 28°C increased total and active protein accumulation, but decreased the ratio of active to total Fd produced. In summary, the high product yields and folding efficiency make the cell-free system described here an attractive platform for the study of Fe-S protein production and maturation. The system enables both small-volume, high throughput investigations as well as larger scale production. To our knowledge, this is the first demonstration of directed, high-yield production and maturation of an Fe-S protein in a cell-free system. © 2006 Wiley Periodicals, Inc. [source] Biosynthesis of the Vitamin E Compound ,-Tocotrienol in Recombinant Escherichia coli CellsCHEMBIOCHEM, Issue 15 2008Christoph Albermann Dr. Abstract The biosynthesis of natural products in a fast growing and easy to manipulate heterologous host system, such as Escherichia coli, is of increasing interest in biotechnology. This procedure allows the investigation of complex natural product biosynthesis and facilitates the engineering of pathways. Here we describe the cloning and the heterologous expression of tocochromanol (vitamin E) biosynthesis genes in E. coli. Tocochromanols are synthesized solely in photosynthetic organisms (cyanobacteria, algae, and higher green plants). For recombinant tocochromanol biosynthesis, the genes encoding hydroxyphenylpyruvate dioxygenase (hpd), geranylgeranylpyrophosphate synthase (crtE), geranylgeranylpyrophosphate reductase (ggh), homogentisate phytyltransferase (hpt), and tocopherol-cyclase (cyc) were cloned in a stepwise fashion and expressed in E. coli. Recombinant E. coli cells were cultivated and analyzed for tocochromanol compounds and their biosynthesis precursors. The expression of only hpd from Pseudomonas putida or crtE from Pantoea ananatis resulted in the accumulation of 336 mgL,1 homogentisate and 84 ,gL,1 geranylgeranylpyrophosphate in E. coli cultures. Simultaneous expression of hpd, crtE, and hpt from Synechocystis sp. under the control of single tac-promoter resulted in the production of methyl-6-geranylgeranyl-benzoquinol (67.9 ,g,g,1). Additional expression of the tocopherol cyclase gene vte1 from Arabidopsis thaliana resulted in the novel formation of a vitamin E compound,,-tocotrienol (15 ,g,g,1),in E. coli. [source] Optimized green fluorescent protein variants provide improved single cell resolution of transgene expression in ascidian embryosDEVELOPMENTAL DYNAMICS, Issue 2 2006Robert W. Zeller Abstract The green fluorescent protein (GFP) is used extensively to monitor gene expression and protein localization in living cells, particularly in developing embryos from a variety of species. Several GFP mutations have been characterized that improve protein expression and alter the emission spectra to produce proteins that emit green, blue, cyan, and yellow wavelengths. DsRed and its variants encode proteins that emit in the orange to red wavelengths. Many of these commercially available fluorescent proteins have been "codon optimized" for maximal levels of expression in mammalian cells. We have generated several fluorescent protein color variants that have been codon optimized for maximal expression in the ascidian Ciona intestinalis. By analyzing quantitative time-lapse recordings of transgenic embryos, we demonstrate that, in general, our Ciona optimized variants are detected and expressed at higher levels than commercially available fluorescent proteins. We show that three of these proteins, expressed simultaneously in different spatial domains within the same transgenic embryo are easily detectable using optimized fluorescent filter sets for epifluorescent microscopy. Coupled with recently developed quantitative imaging techniques, our GFP variants should provide useful reagents for monitoring the simultaneous expression of multiple genes in transgenic ascidian embryos. Developmental Dynamics 235:456,467, 2006. © 2005 Wiley-Liss, Inc. [source] Comparison of the specificity, stability and individual rate constants with respective activation parameters for the peptidase activity of cruzipain and its recombinant form, cruzain, from Trypanosoma cruziFEBS JOURNAL, Issue 24 2001Wagner A. S. Judice The Trypanosoma cruzi cysteine protease cruzipain contains a 130-amino-acid C-terminal extension, in addition to the catalytic domain. Natural cruzipain is a complex of isoforms, because of the simultaneous expression of several genes, and the presence of either high mannose-type, hybrid monoantennary-type or complex biantenary-type oligosacharide chains at Asn255 of the C-terminal extension. Cruzipain and its recombinant form without this extension (cruzain) were studied comparatively in this work. S2 to S2, subsite specificities of these enzymes were examined using four series of substrates derived from the internally quenched fluorescent peptide Abz-KLRFSKQ-EDDnp (Abz, ortho -aminobenzoic acid; EDDnp, N -(2,4-dinitrophenyl)-ethylenediamine). Large differences in the kinetic parameters were not observed between the enzymes; however, Km values were consistently lower for the hydrolysis of most of the substrates by cruzain. No difference in the pH,activity profile between the two enzymes was found, but in 1 m NaCl cruzipain presented a kcat value significantly higher than that of cruzain. The activation energy of denaturation for the enzymes did not differ significantly; however, a negative entropy value was observed for cruzipain denaturation whereas the value for cruzain was positive. We determined the individual rate constants (k1, substrate diffusion; k,1, substrate dissociation; k2, acylation; k3, deacylation) and the respective activation energies and entropies for hydrolysis of Abz-KLRFSKQ-EDDnp determining the temperature dependence of the Michaelis,Menten parameters kcat/Km and kcat as previously described [Ayala, Y.M. & Di Cera, E. (2000) Protein Sci.9, 1589,1593]. Differences between the two enzymes were clearly detected in the activation energies E1 and E,1, which are significantly higher for cruzipain. The corresponding ,S1 and ,S,1 were positive and significantly higher for cruzipain than for cruzain. These results indicate the presence of a larger energy barrier for cruzipain relating to substrate diffusion and dissociation, which could be related to the C-terminal extension and/or glycosylation state of cruzipain. [source] Reprogramming Hansenula polymorpha for penicillin production: expression of the Penicillium chrysogenum pcl geneFEMS YEAST RESEARCH, Issue 7 2007Loknath Gidijala Abstract We aim to introduce the penicillin biosynthetic pathway into the methylotrophic yeast Hansenula polymorpha. To allow simultaneous expression of the multiple genes of the penicillin biosynthetic pathway, additional markers were required. To this end, we constructed a novel host,vector system based on methionine auxotrophy and the H. polymorpha MET6 gene, which encodes a putative cystathionine ,-lyase. With this new host,vector system, the Penicillium chrysogenum pcl gene, encoding peroxisomal phenylacetyl-CoA ligase (PCL), was expressed in H. polymorpha. PCL has a potential C-terminal peroxisomal targeting signal type 1 (PTS1). Our data demonstrate that a green fluorescent protein,PCL fusion protein has a dual location in the heterologous host in the cytosol and in peroxisomes. Mutation of the PTS1 of PCL (SKI-COOH) to SKL-COOH restored sorting of the fusion protein to peroxisomes only. Additionally, we demonstrate that peroxisomal PCL,SKL produced in H. polymorpha displays normal enzymatic activities. [source] c-FLIP expression in colorectal carcinomas: association with Fas/FasL expression and prognostic implicationsHISTOPATHOLOGY, Issue 2 2007P Korkolopoulou Aims:, Disruption of apoptotic cell death has been implicated in tumour aggressiveness in colonic carcinogenesis. The Fas,Fas ligand (FasL) system is involved in the execution of apoptosis induced by the immune system. c-FLIP protein constitutes an inhibitor of Fas and other (TRAIL) death receptor-mediated apoptosis. The aim of this study was to investigate the simultaneous expression of Fas, FasL and c-FLIP in relation to standard clinicopathological parameters and patients' outcome in colorectal cancer. Methods and results:, Levels of Fas, FasL and c-FLIP protein expression were quantified immunohistochemically in paraffin-embedded tissues from 90 patients. Immunopositivity was detected for Fas, FasL and c-FLIP in 71%, 35.5% and 68.8% of cases, respectively. Concurrent expression of Fas/FasL was seen in 28 samples (31%), of which 24 (85.7%) also displayed c-FLIP positivity (P = 0.04). c-FLIP overexpression (> 10%) tended to prevail marginally in higher stage tumours (P = 0.09). Additionally, FasL and c-FLIP adversely affected survival on both univariate (P = 0.001 and P = 0.0024, respectively) and multivariate analysis [hazard ratio (HR) 3.491, P = 0.005 and HR 2.960, P = 0.036, respectively]. Conclusions:, The frequent expression and coexpression of Fas, FasL and c-FLIP in colorectal carcinoma implicates c-FLIP as an inhibitor of the Fas,FasL-induced death pathway in these tumours. Moreover, c-FLIP conveys independent prognostic information in the presence of classical prognosticators. [source] Defining ETS transcription regulatory networks and their contribution to breast cancer progressionJOURNAL OF CELLULAR BIOCHEMISTRY, Issue 3 2007David P. Turner Abstract ETS factors are members of one of the largest families of evolutionarily conserved transcription factors, regulating critical functions in normal cell homeostasis, that when perturbed contribute to tumor progression. The well documented alterations in ETS factor expression and function during breast cancer progression result in pleiotropic effects manifested by the downstream effect on their target genes. Multiple ETS factors bind to the same regulatory sites present on target genes, suggesting redundant or competitive functions. Furthermore, additional events contribute to, or may be necessary for, target gene regulation. In order to advance our understanding of the ETS-dependent regulation of breast cancer progression and metastasis, this prospect article puts forward a model for examining the effects of simultaneous expression of multiple transcription factors on the transcriptome of non-metastatic and metastatic breast cancer. Compared to existing RNA profiles defined following expression of individual transcription factors, the anti- and pro-metastatic signatures obtained by examining specific ETS regulatory networks will significantly improve our ability to accurately predict tumor progression and advance our understanding of gene regulation in cancer. Coordination of multiple ETS gene functions also mediates interactions between tumor and stromal cells and thus contributes to the cancer phenotype. As such, these new insights may provide a novel view of the ETS gene family as well as a focal point for studying the complex biological control involved in tumor progression. J. Cell. Biochem. 102: 549,559, 2007. © 2007 Wiley-Liss, Inc. [source] Production of l -2,3-butanediol by a new pathway constructed in Escherichia coliLETTERS IN APPLIED MICROBIOLOGY, Issue 6 2004S. Ui Abstract Aims:, A metabolic pathway for l -2,3-butanediol (BD) as the main product has not yet been found. To rectify this situation, we attempted to produce l -BD from diacetyl (DA) by producing simultaneous expression of diacetyl reductase (DAR) and l -2,3-butanediol dehydrogenase (BDH) using transgenic bacteria, Escherichia coli JM109/pBUD-comb. Methods and Results:, The meso -BDH of Klebsiella pneumoniae was used for its DAR activity to convert DA to l -acetoin (AC) and the l -BDH of Brevibacterium saccharolyticum was used to reduce l -AC to l -BD. The respective gene coding each enzyme was connected in tandem to the MCS of pFLAG-CTC (pBUD-comb). The divided addition of DA as a source, addition of 2% glucose, and the combination of static and shaking culture was effective for the production. Conclusions:,l -BD (2200 mg l,1) was generated from 3000 mg l,1 added of DA, which corresponded to a 73% conversion rate. Meso -BD as a by-product was mixed by 2% at most. Significance and Impact of the Study:, An enzyme system for converting DA to l -BD was constructed with a view to using DA-producing bacteria in the future. [source] A microarray's view of life in the desert: adding a powerful evolutionary genomics tool to the packrat's middenMOLECULAR ECOLOGY, Issue 11 2009MARJORIE D MATOCQ Identifying the genetic architecture of adaptive traits is fundamental to understanding how organisms respond to their environment, over both ecological and evolutionary timeframes. Microarray technology that allows us to capture the simultaneous expression of thousands of genes provides unparalleled insight into how organisms cope with their environment at the transcriptional level. Recent studies in Molecular Ecology demonstrate how microarrays can rapidly identify which genes and pathways allow organisms to face some of the most fundamental physiological challenges posed by the environment, including compensation for the hypoxic and thermal stress of high-altitudes (Cheviron et al. 2008) and, in this issue, the biotransformation of toxic plant secondary compounds by mammals (Magnanou et al. 2009). Microarrays (Ekins et al. 1989; Fodor et al. 1991) are glass slides affixed with hundreds to thousands of oligonucleotide or cDNA sequences (probes). Messenger RNA transcripts (typically reverse transcribed to cDNA) are isolated from a tissue/sample of interest and hybridized to the array. Binding to specific probes indicates that a particular gene was transcriptionally active at or near the time of sampling and thus provides a potentially comprehensive measure of gene expression. Although a tremendously powerful tool, commercially produced oligonucleotide arrays are only available for a handful of model organisms. Nonetheless, evolutionary ecologists have exploited this resource by using a cross-species hybridization approach (e.g. Saetre et al. 2004), that is, hybridizing a model organism array with a nonmodel sample (Bar-Or et al. 2007). Magnanou et al. (2009) present a novel example of using a model muroid microarray (Agilent Technologies, Rattus) to study physiological response in a wild, nonmodel muroid, Neotoma. [source] IgA nephropathy and mesangial cell proliferation: shared global gene expression profilesNEPHROLOGY, Issue 2002Hideto SAKAI SUMMARY: It is well established that mesangial cell proliferation plays a major role in glomerular injury and progressive renal injury. the expression of a number of different genes has been reported in proliferative mesangial cells in culture. However, the relevance of these genes to renal injury in general and IgA nephropathy (IgAN) remains to be established. Assessment of gene activity on a global genome-wide scale is a fundamental and newly developed molecular strategy to expand the scope of clinical investigation from a single gene to studying all genes at once in a systematic pattern. Capitalizing on the recently developed methodology of high cDNA array hybridization, the simultaneous expression of thousands of genes in primary human proliferating mesangial cells was monitored and compared with renal tissue of IgAN. Complex [,- 33P]-labelled cDNA targets were prepared from cultured mesangial cells, remnant tissue from five IgAN renal biopsies and four nephrectomies (controls). Each target was hybridized to a high-density array of 18 326 paired target genes. the radioactive hybridization signals were analysed by phosphorimager. Approximately 8212±530 different gene transcripts were detected per target. Close to 5% (386±90 genes) were full-length mRNA human transcripts (HT) and the remainder were expressed sequence tags (EST). Using a relational database, electronic subtraction was performed and matching was carried out to allow identification of 203 HT with shared expression in proliferative mesangial cells and IgAN renal biopsies. In addition hierarchical clustering analysis was performed on the HT of IgAN and controls to establish differential expression profiles of mesangial HT in IgAN and controls. Collectively the presented data constitutes a preliminary renal bioinformatics database of the transcriptional profiles in IgAN. More importantly, the information may help to speed up the discovery of genes underlying human IgAN. [source] Genomic repertoire of human mesangial cells: comprehensive analysis of gene expression by cDNA array hybridizationNEPHROLOGY, Issue 4 2000Naohiro Yano SUMMARY: Knowing when and where a gene is expressed in a cell often provides a strong clue as to its physiological role. It is estimated the human genome contains 80 000,100 000 genes. Assessment of gene activity on a global genome-wide scale is a fundamental and newly developed experimental strategy to expand the scope of biological investigation from a single gene to studying all genes at once in a systematic way. Capitalizing on the recently developed methodology of cDNA array hybridization, we monitored the simultaneous expression of thousands of genes in primary human mesangial cells. Complex ,- 33P-labelled cDNA probes were prepared from cultured mesangial cells. The probe was hybridized to a high-density array of 18 326 paired target genes. The radioactive hybridization signals were analysed by phosphorimager. Bioinformatics from public genomic databases was utilized to assign a chromosomal location of each expressed transcript. Approximately 7460 different gene transcripts were detected in mesangial cells. Close to 13% (957 genes) were full-length mRNA human transcripts (HTs), the remainder 6503 being expressed sequence tags (ESTs). Using special imaging computer software, the transcriptional level of the 957 HTs was compared with the expression of the ribosomal protein S28 (housekeeping gene). The HTs were also classified by function of the gene product and listed with information on their chromosomal loci. To allow comparison between clinical and experimental studies of gene expression, the detected human gene transcripts were cross-referenced to orthologous mouse genes. Thus, the presented data constitute a quantitative preliminary blueprint of the transcriptional map of the human mesangial cell. The information may serve as a resource for speeding up the discovery of genes underlying human glomerular diseases. The complete listing of the full-length expressed genes is available upon request via E-mail: (Abdalla_Rifai@Brown.edu). [source] A huge ,prostate-mimicking' vaginal leiomyoma with simultaneous expression of oestrogen and progesterone receptorsAUSTRALIAN AND NEW ZEALAND JOURNAL OF OBSTETRICS AND GYNAECOLOGY, Issue 5 2006Dah-Shyong YU No abstract is available for this article. [source] | ||||||||||||||||