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PubMed2006

The potential of differential mobility analysis coupled to MS for the study of very large singly and multiply charged proteins and protein complexes in the gas phase.

As previously demonstrated by the technique of gas-phase electrophoretic mobility molecular analyzer (GEMMA) introduced by Kaufman and colleagues, differential mobility analysis (DMA) of charge-reduced electrospray ions in the gas phase is a useful complement to MS for studying large proteins and their weakly bound complexes. Several limitations of GEMMA, the solutions for which have the potential to greatly improve its performance, are discussed here, including DMA resolution and transmission. A quantitative theory of charge reduction kinetics for dried multiply charged globular proteins at atmospheric pressures is also presented, showing that the charge reduction time must be carefully chosen to maximize a singly charged ion signal, while avoiding survival of contaminating multiply charged species. Because charge reduction limits the range of masses analyzable by MS, we also consider the potential of a parallel-plate DMA coupled in series to an MS for DMA-MS studies without charge reduction.

Biotechnology↗
PubMed2002

Differential display analysis of gene expression in mammals: a p53 story.

Differential display is used worldwide as a method to identify changes in gene expression and to discover novel genes that are involved in important biological pathways. The principle of differential display is the systematic amplification of the 3' termini of messenger RNAs by using anchored oligo-dT primers in combination with upstream arbitrary primers. The separation of the polymerase chain reaction products by gel electrophoresis and their direct comparison allows the identification of differentially regulated genes. Recently, fluorescent differential display was established as the first nonradioactive differential display system with equivalent sensitivity to originally 33P isotopic labeling method. Because of its simplicity, sensitivity, reproducibility and automation, which increase the throughput and accuracy, differential display has become one of the most widely used gene-screening methods in biomedical research involving mammals. This chapter provides a glimpse of the application of differential display in search of target genes of the p53 tumor suppressor gene.

Animals↗
PubMed2001

Protocol for using signal peptide differential display and representational difference analysis to isolate differentially expressed cDNAs from fetal mouse brain.

Comparing female and male brain structures reveals a variety of sex differences in many vertebrates. Some of these differences are thought to be induced during the fetal period by the effect of steroid hormones produced in the gonads. Not much is known about molecular mechanisms involved in gender-specific development of the brain. We have taken a broad approach to isolate sex-specific genes from 18.5 days post coitum brain (A. Eriksson, C. Wahlestedt and K. Nordqvist. 1999. Isolation of sex-specific cDNAs from fetal mouse brain using mRNA differential display and representational difference analysis. Mol. Brain Res., 74, 91-97). Female and male mouse brains were screened with the signal peptide differential display, developed in our laboratory, and with a modified representational difference analysis of cDNA. The resulting sex-specific fragments were verified by semi-quantitative RT-PCR. Here we describe these methods in detail.

Animals↗
PubMed2006

In silico identification and comparative analysis of differentially expressed genes in human and mouse tissues.

BACKGROUND: Screening for differentially expressed genes on the genomic scale and comparative analysis of the expression profiles of orthologous genes between species to study gene function and regulation are becoming increasingly feasible. Expressed sequence tags (ESTs) are an excellent source of data for such studies using bioinformatic approaches because of the rich libraries and tremendous amount of data now available in the public domain. However, any large-scale EST-based bioinformatics analysis must deal with the heterogeneous, and often ambiguous, tissue and organ terms used to describe EST libraries. RESULTS: To deal with the issue of tissue source, in this work, we carefully screened and organized more than 8 million human and mouse ESTs into 157 human and 108 mouse tissue/organ categories, to which we applied an established statistic test using different thresholds of the p value to identify genes differentially expressed in different tissues. Further analysis of the tissue distribution and level of expression of human and mouse orthologous genes showed that tissue-specific orthologs tended to have more similar expression patterns than those lacking significant tissue specificity. On the other hand, a number of orthologs were found to have significant disparity in their expression profiles, hinting at novel functions, divergent regulation, or new ortholog relationships. CONCLUSION: Comprehensive statistics on the tissue-specific expression of human and mouse genes were obtained in this very large-scale, EST-based analysis. These statistical results have been organized into a database, freely accessible at our website http://gln.ibms.sinica.edu.tw/product/HMDEG/EST/index.php, for easy searching of human and mouse tissue-specific genes and for investigating gene expression profiles in the context of comparative genomics. Comparative analysis showed that, although highly tissue-specific genes tend to exhibit similar expression profiles in human and mouse, there are significant exceptions, indicating that orthologous genes, while sharing basic genomic properties, could result in distinct phenotypes.

Animals↗
PubMed2000

Identification of genes highly expressed in G2-arrested Chinese hamster ovary cells by differential display analysis.

Abnormal cell cycle regulation is believed to be an important step in tumorigenesis. In mammalian cells, DNA damage commonly leads to cell cycle arrest in G2; however, little is known about the detailed biochemical mechanisms underlying the DNA damage-induced G2 arrest. In order to identify genes differentially expressed in association with G2 arrest, differential display analysis was performed between exponentially growing Chinese hamster ovary (CHO) cells and G2-arrested CHO cells induced by etoposide, SN-38, or X-radiation. We identified five cDNA clones whose expression was up-regulated in G2-arrested CHO cells. Sequence analysis revealed that three clones were homologous to known genes: isogene I of translation initiation factor eIF-4A, ribosomal protein L13, and translation repressor NAT1. The remaining two clones showed no homology to known genes. These results indicate that DNA damage can alter the expression of multiple genes, including translational regulators.

Animals↗
PubMed2002

Thyroid hormone gene targets in ROS 17/2.8 osteoblast-like cells identified by differential display analysis.

Thyroid hormone plays an important role in bone development and metabolism. We used a polymerase chain reaction (PCR)-based mRNA differential display (DD) analysis to obtain a profile of thyroid hormone-responsive genes in osteoblast-like cells (ROS 17/2.8). ROS 17/2.8 cells were treated with 10(-8) M triiodothyronine (T(3)) for 2 and 24 hours. Total RNA was isolated, reverse-transcribed, and amplified using a total of 72 combinations (2 hours) and 240 combinations (24 hours) of 5' and 3' primers. At the 2-hour time point, 1 true-positive novel clone was identified and shown to be the mitochondrial gene, subunit 6 of ATP synthase (ATPase-6). At the 24-hour time point, 3 differentially expressed (DE) mRNAs were confirmed as true-positives including; nonmuscle alkali myosin light chain (NM aMLC), ATPase-6, and one novel clone. T(3)-induction of ATPase-6 mRNA in ROS 17/2.8 cells was seen at 2 and 4 hours, but was maximal at 24 hours (2.1-fold). T(3) induction of ATPase-6 mRNA was increased to fourfold in ROS 17/2.8 cells cultured at a low density. NM aMLC mRNA was modestly upregulated by T(3) in ROS 17/2.8 cells by 1.4-fold, and induction was augmented at low cell density to 1.7-fold. T(3) action on NM aMLC and on the mitochondrial gene ATPase 6, represent novel targets and potential mediators of thyroid hormone action on bone. Cell type, and the extent of cell differentiation, influences T(3) regulation of genes in osteoblast-derived cells.

Adenosine Triphosphatases↗
PubMed2001

DNA microarray analysis of differential gene expression of 6-year-old rat neural striatal progenitor cells during early differentiation.

EGF-responsive striatal progenitor cells from rat brain have been maintained in culture in the form of neurospheres for six years without exhausting their renewal capacity. The events surrounding differentiation of stem cells in the brain after a long progenitorship remain a mystery. Using DNA microarray analysis we investigated differential gene expression, comparing progenitor cells in their neurosphere state with the cells 24 hours after induction of differentiation. Eighty-one genes showed increased expression in the differentiated condition. Genes associated with cellular growth, neurite outgrowth, and synaptogenesis were activated, including both anti-apoptotic and pro-apoptotic genes. Two transmitter- related genes, acetylcholine receptor-beta and glutamate receptor-beta-unit were also elevated-, these genes not only fit the profile of early neural development, but also reflect the characteristics of striatal neurons. In addition, there are approximately 30 expressed sequence tags (ES7) increased during neural differentiation. Forty-seven genes showed decreased expression; half of them are known genes related to the cell cycle, cell adhesion, transcription, and signaling. Tbe signaling and cell cycle genes may be responsible for the life-long self-renewal. These data demonstrate for the first time that life-long quiescent stem cells retain the potential to become activated and develop into specific types of brain cells. The six-year long-term neural stem cells are an excellent model for studying developmental neurobiological processes and aging.

Animals↗
PubMed1998

Analysis of differential gene expression in human colorectal tumor tissues by RNA arbitrarily primed-PCR: a technical assessment.

RNA arbitrarily primed (RAP)-PCR is a powerful tool for studying differential gene expression in cancer cells. Systematic analysis of human tumor samples may provide a list of markers with potential application to the diagnosis, prognostic assessment, and treatment of the disease. Nevertheless, because of characteristics inherent to the samples and technique, artifactual results are likely. We have assessed the effects of several factors on RAP-PCR performance to determine the sensitivity and reproducibility of the technique, as well as the accuracy of its results, under different conditions in human cell lines and in a series of 129 paired human normal colonic mucosa-colorectal carcinoma samples. Our results show that RAP-PCR provides reliable fingerprints in a relatively wide spectrum of circumstances, including variations in RNA concentration and contamination by DNA. Densitometric analysis indicated that relative band-intensity variations more than 20% were reproducible in 95% of the cases. Serial analysis of paired normal-tumor cases yielded a number of bands that were recurrently either underexpressed or overexpressed in tumor relative to normal mucosa. These differentially expressed bands are prime targets of research because they represent candidate tumor-specific up- or down-regulated genes with a relevant role in carcinogenesis.

Colorectal Neoplasms↗
PubMed2005

Differential proteomic analysis of bronchoalveolar lavage fluid in asthmatics following segmental antigen challenge.

Allergic asthma is characterized by persistent airway inflammation and remodeling. Bronchoalveolar lavage conducted with fiberoptic bronchoscopy has been widely used for investigating the pathogenesis of asthma and other lung disorders. Identification of proteins in the bronchoalveolar lavage fluid (BALF) and their expression changes at different stages of asthma could provide further insights into the complex molecular mechanisms involved in this disease. In this report, we describe the first comprehensive differential proteomic analysis of BALF from both asthmatic patients and healthy subjects before and 24 h after segmental allergen challenge. Our proteomic analysis involves affinity depletion of six abundant BALF proteins, SDS-PAGE fractionation, protein in-gel digestion, and subsequent nano-LC-MS/MS analysis in conjunction with database searching for protein identification and semiquantitation. More than 1,500 distinct proteins were identified of which about 10% displayed significant up-regulation specific to the asthmatic patients after segmental allergen challenge. The differentially expressed proteins represent a wide spectrum of functional classes such as chemokines, cytokines, proteases, complement factors, acute phase proteins, monocyte-specific granule proteins, and local matrix proteins, etc. The majority of these protein expression changes are closely associated with many aspects of the pathophysiology of asthma, including inflammation, eosinophilia, airway remodeling, tissue damage and repair, mucus production, and plasma infiltration. Importantly a large portion of these proteins and their expression changes were identified for the first time from BALF, thus providing new insights for finding novel pathological mediators and biomarkers of asthma.

Adult↗
PubMed2004

Two-step differential expression analysis reveals a new set of genes involved in thyroid oncocytic tumors.

Thyroid oncocytic adenomas are a class of tumors characterized by the presence of abundant mitochondria. We performed a differential display RT-PCR analysis on two oncocytic adenomas and their paired controls. We then carried out a microarray analysis using the 460 selected, differentially expressed clones on four other oncocytomas and their paired controls. Thirty genes, 12 encoded by mitochondrial DNA and 18 nuclear-encoded, were overexpressed by a factor of at least 2 in the tumors compared with the controls. Seven of the 18 nuclear-encoded genes are involved in protein metabolism: DKFZP434I116, B3GTL, SNX19, RP42, SENP1, UBE2D3, and the CTSB gene, which is known to be particularly deregulated in most thyroid tumors. Other genes are implicated in signal transduction (ITGAV) or tumorigenesis (AF1q). Immunohistochemistry allowed us to confirm overexpression of the ITGAV and CTSB genes at the protein level and showed a marked relocation of the CTSB protein. We confirmed the overexpression of the AF1q oncogene in 56% of 18 oncocytic tumors by quantitative RT-PCR analysis, which attested to the heterogeneity of these tumors. Our results show an increased expression of genes involved in protein metabolism in oncocytoma, the significance of which requires investigation.

Adenine Nucleotide Translocator 2↗
PubMed2005

A search for genes modulated by interleukin-6 alone or with interleukin-1beta in HepG2 cells using differential display analysis.

Interleukin-1 and interleukin-6 are principal cytokines involved in regulation of expression of acute-phase proteins. In the joint action of both cytokines IL-1 can suppress or enhance the IL-6-dependent induction of gene expression. Here, we report changes in the transcriptome profile of HepG2 cells exposed to IL-6 alone, or IL-1 and IL-6. Cytokine-responsive genes were identified by differential display analysis. Validation of observed changes in the transcript level was carried out using the slot blot method. Out of 88 cDNA species modulated by IL-6, only 38 represent different known genes whereas 18 clones match genomic clones in NCBI data with hypothetical cDNA sequences (the remaining 32 clones showed no homology with the database or represented several clones of the same gene). In the experiments with HepG2 cells prestimulated for 3 h with IL-1 and then stimulated with IL-6, 43 cDNA fragments were amplified. Twenty-three of them represent known genes while 10 clones have inserts matching hypothetical cDNA sequences in NCBI data. The identified transcripts modulated by IL-6 or both cytokines in HepG2 cells code for intracellular proteins of various function. The largest groups represent genes engaged in metabolism, protein synthesis and signaling pathways. Among all genes identified as differentially regulated under stimulation by IL-6, or IL-1/IL-6, six were detected in both types of stimulation. None of the typical genes coding for plasma acute phase proteins was identified in our experiments. This indicates that differential display cannot be used to characterize the profile of a given transcriptome. On the other hand, it is a useful technique for detection of new genes responding to IL-6 alone or IL-6 in combination with IL-1.

Acute-Phase Proteins↗
PubMed2004

Metabolomic differential display analysis of the white-rot basidiomycete Phanerochaete chrysosporium grown under air and 100% oxygen.

When the cultural atmosphere of the white-rot basidiomycete, Phanerochaete chrysosporium, was changed from air to 100% oxygen, the lyophilized mycelial weight increased and thickening of extracellular glucan layer was observed in 2-3 days. To better understand the oxygen-stress responsive mechanism of P. chrysosporium, the metabolomic differential display analysis was performed using metabolites isolated from fungal cells grown under either air or 100% O(2) atmosphere. In the GC-MS total ion chromatogram of methanol-extracts from fungal cells, at least 183 peaks were detected and 53 compounds were identified. Among them, veratryl alcohol (VA), threonate, and erythronate were identified as oxygen-stress responsive metabolites. The intracellular concentration of VA increased dramatically within 1 h after an oxygen purge, indicating that VA production is sensitive to the oxygen stress in P. chrysosporium.

Air↗
PubMed2005

Differential proteome analysis of conditioned media to detect Smad4 regulated secreted biomarkers in colon cancer.

Smad4 is a tumor suppressor gene primarily involved in carcinogenesis of the pancreas and colon. The functional inactivation of Smad4 is a late step genetically. In pancreatic carcinogenesis, loss of Smad4 marks the transition to invasive growth. In colorectal cancers, the frequency of Smad4 inactivation is markedly increased in metastatic cancers. We have established cell biological models, re-expressing Smad4 in deficient human cancer cells, in which we could show that Smad4 is adequate to suppress tumor growth through suppression of angiogenic and invasive properties. Thus, pairs of Smad4-re-expressing and Smad4-deficient cells are prone to model the progression from premalignant stages to carcinomas in the carcinogenic process and may provide access to Smad4 targets of high clinical relevance. We present here a "differential secretome analysis", comparing all the proteins released in vitro from the Smad4-deficient and Smad4-re-expressing SW480 human colon carcinoma cells. The differential secretome catalog comprises more than 25 proteins including proteases and protease inhibitors, as well as established tumor biomarkers. In conclusion, this approach proved to be a sensitive tool to specifically detect Smad4 targets relevant for tumor-stroma interactions. It is also able to reflect complex alterations of cellular physiology. Moreover, the results support our hypothesis that human tumor markers detectable in serum may be identified through differential secretome analyses.

Biomarkers, Tumor↗
PubMed1998

Fluorescent differential display analysis of gene expression in apoptotic neuroblastoma cells.

Identification of differentially expressed genes will provide leads in the elucidation of the molecular mechanisms underlying neuronal cell death associated with neurodegenerative disorders. Using a high-throughput fluorescent differential display (FDD) system based on an automated DNA sequencer, we analyzed global patterns of gene expression during the apoptosis of neuroblastoma SH-SY5Y cells induced by a neurotoxin, colchicine. Initial screening of approximately 24000 cDNA bands displayed with 320 primer combinations has revealed 263 fragments showing differential expression patterns, suggesting that approximately 1% of transcripts are modulated in their expression level. Of these differentially displayed bands, we cloned 18 fragments composed of 17 distinct species and confirmed differential expression of each species by reverse transcription-PCR or Northern blot hybridization, thereby proving the reliability of the approach. These include eight derived from seven known genes, five homologous to expressed sequence tags (ESTs), and five totally lacking any homology to those deposited in the database. Among these, a novel transcript SAI1 induced prominently was characterized further and revealed to encode a putative RNA-binding protein NAPOR (neuroblastoma apoptosis-related RNA-binding protein), containing three copies of evolutionarily conserved RNA recognition motif. Since several RNA-binding proteins have been known to play crucial roles in other apoptosis systems, it is conceivable that NAPOR is also involved in the process of neuronal cell death.

Amino Acid Sequence↗
PubMed2005

Differential display analysis of gene expression in brains from hepatitis C-infected patients.

OBJECTIVES: Hepatitis C virus (HCV) infection is often associated with cognitive dysfunction, fatigue and depression. The current study was undertaken to determine whether HCV infection affects gene expression in brain tissue. DESIGN: We analysed the gene expression pattern in brain tissue in a group of HCV-infected patients compared with HCV-negative controls. METHODS: Brain tissue samples were obtained at autopsy from three HCV-positive patients and three HCV-negative control patients. The analysis of gene expression was conducted using differential display and reverse Northern hybridization. Only those genes that were up or downregulated more than 1.8 times were considered to be differentially expressed. RESULTS: Altogether, 29 differentially expressed genes were identified by differential display and subsequently confirmed by reverse Northern hybridization. A prominent finding was the downregulation of mitochondrial oxidative phosphorylation genes in HCV-infected patients. The impairment of brain oxidative/energy metabolism has previously been suggested to be the proximate cause of many disorders that impair mentation. Another finding was the downregulation of some ribosomal protein genes and several genes involved in transcription regulation, perhaps reflecting reduced metabolic activities. CONCLUSION: Our findings suggest for the first time that there may be a biological basis for the neuropsychiatric symptoms and cognitive impairment associated with HCV infection.

Aged↗
PubMed2004

Analysis of differentially expressed parasite genes and proteins using transcriptomics and proteomics.

At any particular point in time, the full complement of transcribed RNAs and relevant proteins of a cell are known as the transcriptome and proteome, respectively. The composition of these two populations changes throughout the life cycle of a parasite or in response to environmental factors, such as drug treatments. Comparing the changes in the composition of the transcriptome and proteome between different life-cycle stages or in the same stage but under different conditions can be of particular interest, as it can allow the identification of potentially important differentially expressed genes and proteins. Combining the analysis of both the transcriptome and proteome in tandem allows changes in RNA transcripts to be followed right through to changes in the level of protein expression. The protocols in this chapter describe methods for analyzing the transcriptome, by using suppression subtraction hybridization to construct subtracted complementary DNA libraries, and the proteome, by using two-dimensional sodium dodecyl sulfate-polyacrylamide gel electrophoresis. These two methods are then integrated to allow the global changes in RNA and protein expression to be examined. The protocols have been adapted for working on parasites and contain extensive notes.

Animals↗
PubMed2002

[Differential proteomic analysis of human lung adenocarcinoma cell line A-549 and of normal cell line HBE].

To explore the differential proteomic expressions between human lung adenocarcinoma cell line A-549 and normal cell line HBE, a series of methods, including immobilized pH gradient-two dimensional polyacrylamide gel electrophoresis, silver staining, PDQuest 2-DE software analysis, peptide mass fingerprinting based on matrix-assisted laser desorption/ionization time of flight mass spectrometry (MALDI-TOF-MS) and SWISS-PROT database searching, were used to separate and identify the differential proteomic expressions between A-549 and HBE. The results showed that the good 2-DE pattern including high resolution and reproducibility was obtained. After silver staining, the 2-DE image analysis by PDQuest 2-DE software detected average (890 +/- 38) spots in A-549, and (757 +/- 27) spots in HBE. The average positional deviation of the matched spots between A-549 and HBE 2-DE maps was (2.85 +/- 0.48) mm in IEF direction, and (2.69 +/- 0.37) mm in SDS-PAGE direction. The differential proteomic expression analysis found that there were 535 matched spots between A-549 and HBE 2-DE maps, 355 spots that were not matched in A-549, 222 spots that were not matched in HBE. 18 differential spots (8 spots in A-549 and 10 spots in HBE) were cut off from silver staining gel at random, digested in gel with TPCK-trypsin, measured with MALDI-TOF-MS and searched in the SWISS-PROT database with PeptIdent software. 18 protein were preliminarily identified. These proteins were related to cell signal transduction, cell metabolism, proliferation and differentiation etc. There was a significant difference at protein level between human lung adenocarcinoma cell line A-549 and normal cell line HBE. It suggests that the differential expression analysis of proteomes may be useful to further study of the related proteins and the molecular markers of lung adenocarcinoma.

Adenocarcinoma↗
PubMed2005

Differential proteomic analysis of proteins induced by glucocorticoids in cultured murine podocytes.

BACKGROUND: The glomerular podocyte is the kidney cell most affected during the development of nephrotic syndrome, and mutations in podocyte proteins are responsible for a variety of inherited forms of nephrotic syndrome. Although glucocorticoids are a primary treatment for nephrotic syndrome, neither their target cell nor mechanism of action are known. In order to describe the proteome of the podocyte, and to identify podocyte proteins whose expression is altered by glucocorticoids, we performed a differential proteomic analysis of control and dexamethasone-treated cultured murine podocytes. METHODS: Podocyte proteins were separated by two-dimensional-polyacrylamide gel electrophoresis (PAGE) and identified by matrix-assisted laser desorption time-of-flight (MALDI-TOF) mass spectrometry and peptide fingerprinting. Comparisons of stained two-dimensional-PAGE separations were used to identify proteins whose expression was altered by treatment with the glucocorticoid dexamethasone, and these results were confirmed by quantitative Western blotting. RESULTS: A total of 106 protein spots yielded MALDI-TOF results, and 92 were identified by protein fingerprinting. Of the 88 unique proteins and four protein isoforms identified, six proteins were found whose expression was altered by dexamethasone. The proteome of cultured murine podocytes is particularly rich in actin cytoskeletal proteins and proteins involved in responses to cellular stress. The change in expression of three proteins [ciliary neurotrophic factor (CNTF), alphaB-crystallin, and heat shock protein 27 (hsp27)] was confirmed by quantitative Western blotting. CONCLUSION: Three proteins with known roles in protecting cells from injury were up-regulated by dexamethasone, demonstrating that glucocorticoids exert a direct effect on cultured podocytes resulting in changes in the expression of proteins with potential relevance to the therapeutic action of glucocorticoids in diseases such as nephrotic syndrome.

Animals↗