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PubMed2002

Differential display: a critical analysis.

Differential display (DD) is a well-established analytical tool for measuring gene expression that is still popular due to its documented success and ability to identify novel genes not yet available for analysis by more powerful microarray hybridization. For a comprehensive analysis of all mRNAs in a given cell, it is statistically predicted that at least 240 different DD primer combinations are required. This prediction, however, has never been empirically tested. Using far more primer combinations than that predicted to evaluate 90% of the mRNAs in a cell, plus other modifications, we identified and confirmed the induction of five mRNAs by hydrogen peroxide in HA-1 hamster cells. However, five other known oxidant-inducible mRNAs were not identified by DD. Filter microarray hybridization did not result in the identification of any additional species modulated twofold or greater but previous two-dimensional protein gel electrophoresis identified 15 induced protein species. We conclude that the current statistical prediction for comprehensive analysis of all the mRNAs in a given cell is inaccurate, at least in our hands, and further conclude that DD is a useful but less than comprehensive method for assessing changes in mRNA levels.

Animals↗
PubMed2002

Porcine mesenchymal stem cells. Induction of distinct mesenchymal cell lineages.

The potential of mesenchymal stem and progenitor cells (MSC) to replicate undifferentiated and to mature into distinct mesenchymal tissues suggests these cells as an attractive source for tissue engineering. The objective was to establish a protocol for the isolation of porcine MSC from bone marrow and to demonstrate their ex vivo differentiation into various mesenchymal tissue cells. MSC from passage 2 were selected for differentiation analysis. Differentiation along the osteogenic lineage was documented by deposition of calcium, visualization of alkaline phosphatase activity, and by analysis of osteogenic marker genes. Adipocytes were identified morphologically and by gene-expression analysis. Deposition of type II collagen and histological staining of proteoglycan indicated chondrogenic differentiation. Therefore, porcine MSC may be introduced as a valuable model system with which to study the mesenchymal lineages for basic research and tissue engineering.

Adipocytes↗
PubMed1999

Expression of an IL-1 receptor antagonist during mouse hepatocarcinogenesis demonstrated by differential display analysis.

The differential display technique was applied for identification of genes that have altered expression in mouse hepatocellular carcinomas relative to normal liver. Three genes were identified. The IL-1 receptor antagonist (IL-1ra) was expressed in hepatocellular carcinomas, whereas the major urinary protein (MUP) and cytochrome P-450 naphthalene hydroxylase (cyp2F2) genes were down-regulated. Because IL-1ra is a natural antagonist of IL-1, and because the latter has been reported to suppress the growth of hepatic cells, we also studied the expression of IL-1ra in hepatocarcinogenesis. IL-1ra was immunohistochemically detected in tumor cells in approximately 70% of hepatocellular adenomas and carcinomas, whereas early preneoplastic hepatocytic foci, as well as normal hepatocytes surrounding the lesions, were negative. In addition, 20% of human hepatocellular carcinomas were also partly positive for IL-1ra. RT-PCR analysis demonstrated that mouse hepatic tumors contain both secreted and intracellular forms of IL-1ra. On the other hand, there were no differences in levels of IL-1alpha and IL-1beta between hepatic tumors and normal liver in mice, suggesting that the majority of tumors create a microenvironment that inhibits the actions of IL-1. Furthermore, IL-1ra-positive adenomas contained more proliferating cell nuclear antigen-positive cells than IL-1ra-negative adenomas, indicating a link with high proliferation activity, although this was no longer evident in carcinomas. The observed altered gene expression may be related to biological phenotypes of hepatic tumors, and IL-1ra in particular may positively influence tumor cell growth through its antagonism of IL-1.

Animals↗
PubMedDate not supplied

Expression of testicular germ cell genes identified by differential display analysis.

Using differential display reverse transcriptase-polymerase chain reaction (DDRT-PCR) we identified transcripts encoding for the RNA helicase mDEAH9, Ran binding protein 5 (RanBP5), and 3 novel complementary DNAs designated GC3, GC12, and GC14 in developing testicular germ cells. Sources of RNA for the initial DDRT-PCR screen were purified mouse type A spermatogonia, adult mouse wild-type testis, and W/W(v) mutant mouse testis. We identified cDNA fragments for mDEAH9, RanBP5, GC3, GC12, and GC14 in testis and type A spermatogonia samples from wild-type mice, but not in samples from the W/W(v) mouse testis. These same transcripts were absent in Northern blots of testis RNA from mice treated with busulfan 30 days prior, but were present in testis RNA from wild-type mice at 5, 15, 25, and 40 days of age. The mDEAH9 gene was expressed in many tissues, whereas RanBP5 and GC12 genes were expressed predominantly in the testis with much lower expression in other tissues. The expression of GC3 and GC14 were limited to the testis as evidenced by Northern blot and RT-PCR analyses. The mDEAH9 transcript was not detected in cultured interstitial cells but was found at low levels in cultured immature Sertoli cells, whereas the RanBP5, GC3, GC12, and GC14 transcripts were not detected in either cultured testicular interstitial cells or cultured Sertoli cells. RT-PCR analyses of isolated spermatogonia, pachytene spermatocytes, and round spermatids revealed that mDEAH9, RanBP5, GC3, GC12, and GC14 genes were expressed in all 3 cellular populations. In situ hybridization analyses of testis samples from 40-day-old mice localized expression of mDEAH9, RanBP5, GC3, GC12, and GC14 to the seminiferous tubules. RanBP5 expression appeared to be regulated during the cycle of the seminiferous epithelium, with the highest expression in stages III through VII. Expression of GC14 was greatest in the meiotic germ cell populations.

Animals↗
PubMed1994

Improvements to the differential display method for gene analysis.

Differential display requires two primers to identify discordantly expressed mRNAs. Anchored primers with at least one G residue were superior to those which had one C residue and those ending in A or T were the least efficient. Arbitrary primers with GC pairs at the 5'end were superior to primers with GC pairs at the 3'end. We found that screening of amplified DNA-fixed membranes by a slot blot manifold and hybridization with DNA probes made from sample RNA rapidly re-screened with DNA fragments for differential expression, requires less RNA and is faster than Northern analysis. Similarly, hybridization of DNA fragments to plasmid DNA fixed to membranes and direct PCR sequencing rapidly determine homology to common laboratory plasmids prior to any further manipulation. These modifications permit rapid isolation and characterization of DNA fragments identified by differential display.

Animals↗
PubMed2026

A Comprehensive Analysis of Differential Protein Expression in the Plasma of Rheumatoid Arthritis Patients Utilizing Data-Independent Acquisition (DIA) Proteomics Technology.

BACKGROUND: Rheumatoid Arthritis (RA) is a Prevalent Autoimmune Disorder Affecting Millions of People Worldwide. A Thorough Understanding of Its Clinical and Pathological Features Is Essential to Improve Patient Outcomes. METHODS: This Study Combined Data-Independent Acquisition Proteomics and Enzyme-Linked Immunosorbent Assay (ELISA) to Identify and Validate Potential Plasma Protein Biomarkers for the Early Diagnosis of RA. RESULTS: Differential Proteomic Analysis Identified Differentially Expressed Proteins Between Patients With RA and Healthy Controls and Characterized Their Functions. Gene Ontology and Kyoto Encyclopedia of Genes and Genomes Enrichment Analyses Were Performed to Explore Protein Functions and Associated Biological Pathways. The STRING Database and the Metascape Platform Were Used to Conduct an in-Depth Analysis of the Protein-Protein Interaction Network, Highlighting the Functional Attributes and Interconnections of Upregulated Proteins and Identifying Key Protein Complexes Involved in RA. ELISA Analysis of Plasma Samples Revealed Significantly Elevated SERPINA3 Levels in Patients With RA, Which Were Positively Correlated With Disease Activity Indicators-Including Erythrocyte Sedimentation Rate, C-Reactive Protein, and Disease Activity Score 28-But Were Not Correlated With Rheumatoid Factor or Its Subtypes. CONCLUSIONS: This Study Provides New Insights and Identifies Potential Biomarkers for the Early Diagnosis of RA.

Humans↗
PubMed1976

Differentiation of channel models by noise analysis.

Differentiation of membrane channel models based on fluctuation (or noise) analysis is discussed. The theory is particularly useful in distinguishing a single-conductance model (Hodgkin-Huxley formalism) from a multiconductance model. When applied to the frog node of Ranvier, it seems likely that the potassium channels of the membrane may have multiconductance states.

Animals↗
PubMed1988

Correlation of aromatase activity with histological differentiation of breast cancer--a morphometric analysis.

Differentiation is a term that indicates the degree to which a tumor resembles histologically the tissue or cell of origin. A system to quantitate the proportion of breast cancer cells participating in glandular differentiation or remaining within ducts was employed. The degree of tumor differentiation of 58 primary breast cancers was correlated with estrogen (ER) and progesterone receptors (PR) and tumor aromatase activity. There was a significant association between tumor differentiation (greater than or equal to 2% cancer cells exhibiting glandular differentiation) and the presence of ER or PR in tumors. Conversely, there was no correlation between tumor differentiation and measurable tumor aromatase activity.

Aromatase↗
PubMed2006

[Proteomics of breast cancer: From differential to functional analysis].

From differential analysis to identify biomarkers, to functional analysis for finding new therapeutic targets, proteomics bring new comprehensive information for a better understanding of the molecular basis of oncology and new perspectives for the clinic. However the major limitation of proteomic investigations, more generally of post-genomic approaches, remains the molecular and cellular complexity of the mammary gland that is still a major challenge.

Biomarkers, Tumor↗
PubMed2005

DNA microarray data imputation and significance analysis of differential expression.

MOTIVATION: Significance analysis of differential expression in DNA microarray data is an important task. Much of the current research is focused on developing improved tests and software tools. The task is difficult not only owing to the high dimensionality of the data (number of genes), but also because of the often non-negligible presence of missing values. There is thus a great need to reliably impute these missing values prior to the statistical analyses. Many imputation methods have been developed for DNA microarray data, but their impact on statistical analyses has not been well studied. In this work we examine how missing values and their imputation affect significance analysis of differential expression. RESULTS: We develop a new imputation method (LinCmb) that is superior to the widely used methods in terms of normalized root mean squared error. Its estimates are the convex combinations of the estimates of existing methods. We find that LinCmb adapts to the structure of the data: If the data are heterogeneous or if there are few missing values, LinCmb puts more weight on local imputation methods; if the data are homogeneous or if there are many missing values, LinCmb puts more weight on global imputation methods. Thus, LinCmb is a useful tool to understand the merits of different imputation methods. We also demonstrate that missing values affect significance analysis. Two datasets, different amounts of missing values, different imputation methods, the standard t-test and the regularized t-test and ANOVA are employed in the simulations. We conclude that good imputation alleviates the impact of missing values and should be an integral part of microarray data analysis. The most competitive methods are LinCmb, GMC and BPCA. Popular imputation schemes such as SVD, row mean, and KNN all exhibit high variance and poor performance. The regularized t-test is less affected by missing values than the standard t-test. AVAILABILITY: Matlab code is available on request from the authors.

Algorithms↗
PubMed1994

Molecular polymorphism as a tool for differentiating ground beetles (Carabus species): application of ubiquitin PCR/SSCP analysis.

Differentiation between Carabus species (ground beetle) and subspecies is difficult, although there have been extensive studies. To address this problem we have applied PCR in combination with SSCP analysis focussing on the evolutionally conservative ubiquitin gene to elaborate a new approach to molecular differentiation between species. We report that Carabidae possess an ubiquitin gene and that its gene has a multimeric structure. Differential SSCP analysis was performed with the monomeric form of the gene to generate a clear SSCP pattern. Such PCR/SSCP resulted in reproducible patterns throughout our experiments. Comparing different Carabus species (Carabus granulatus, C. irregularis, C. violaceus and C. auronitens) we could observe clear interspecies differences but no differences between genders. Some species showed some remarkable differences between the individuals. We suggest that the ubiquitin PCR-SSCP technique might be an additional tool for the differentiation of ground beetles.

Animals↗
PubMed1997

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

Identification of differentially-expressed genes provides an important step toward the elucidation of molecular mechanisms underlying a variety of biological processes. A novel PCR-based approach to detect and clone such transcripts is the so-called differential display (DD). We established an improved DD protocol that can be performed on an automated fluorescent DNA sequencer to ensure high throughput as well as operational safety. Using this fluorescent DD (FDD) technique, we analyzed the gene expression profile in the retinoic acid-induced differentiation of a human neuroblastoma cell line SH-SY5Y. Screening with 102 primer combinations at eight different time points revealed 66 cDNA bands with variously different behaviors out of approximately 6000 bands displayed. Subsequent analyses with 15 cloned species confirmed the differential expression of corresponding transcripts in all the cases, thereby demonstrating the high reliability of FDD analysis. These clones were composed of seven novel and eight known genes, the latter of which included those that had never been described in the context of neuronal differentiation. These results indicate that FDD is an effective approach to obtain not only novel genes but also clues to possible novel functions of known genes involved in various biological phenomena.

Amino Acid Sequence↗
PubMed1986

Is the EORTC prognostic index of thyroid cancer valid in differentiated thyroid carcinoma? Retrospective multivariate analysis of differentiated thyroid carcinoma with long follow-up.

The European Organization for Research on Treatment of Cancer (EORTC) Thyroid Cancer Cooperative Group presented a prognostic index in 1979 that included all histologic groups of thyroid carcinomas, and was based on a multivariate analysis of 507 patients with a median follow-up of 40 months. The current report not only uses a multivariate analysis to study the clinical validity and reproducibility of this index on case material consisting of 226 differentiated thyroid carcinomas with a considerably long follow-up (11 years), but also it investigates possible prognostic factors, other than those proposed by the EORTC. Three EORTC variables could be reproduced as important: age at diagnosis, locally advanced disease, and distant metastases. Of the additional histopathologic variables tested, microscopic tumor invasion beyond thyroid capsule (pT4) and marked cellular atypia (MCA) proved to be significant. When the effect of the age-correlated tumor factors pT4 and MCA of differentiated thyroid carcinomas were taken into consideration, age alone lost its prognostic importance as a guide for treatment.

Adult↗
PubMed2002

Differential display: analysis of gene expression during plant cell separation processes.

An essential component in the study of cell growth and development in any organism is the analysis of differential gene expression. There are numerous techniques available for comparison of two or more systems at the molecular level, including subtractive hybridization, reverse transcriptase (RT), polymerase chain reaction (PCR), differential screening of cDNA libraries, and, more recently, cDNA microarrays. Differential display has advantages in that it is relatively less time-consuming and can result in the identification of rare cDNA, which may be missed by conventional cDNA library screening. In addition, cDNA microarrays are a valuable asset to the analysis of regulated gene expression but the technique is expensive to employ. Although we successfully applied differential display to isolate novel mRNAs that are up- and downregulated during cell separation processes in plants, the technique can be applied to any system where two or more mRNA sets are to be compared.

Brassica napus↗
PubMed1995

Isolation of a novel macrophage-specific gene by differential cDNA analysis.

To analyze myelomonocytic differentiation we have used the approach of differential cDNA analysis to isolate novel genes that are preferentially expressed in mature macrophages. Differential screening of a macrophage cDNA library led to the identification of a novel cDNA that showed macrophage lineage- and differentiation stage-specific expression. Transcripts from the gene, which we have termed Mpg-1, are found at a high level in mature human and murine macrophages and at a moderate level in certain myelomonocytic cell lines. The expression of Mpg-1 was found to increase when murine fetal liver hematopoietic progenitor cells were induced to differentiate into macrophages. An Mpg-1-specific transcript was not detected in a wide variety of other tissues and cell lines. The DNA sequence of Mpg-1 (4,214 bp) was obtained from a series of overlapping cDNA, 3' rapid amplification of cDNA ends (RACE), and genomic clones. Primer extension analysis predicted the existence of multiple transcription start sites, ranging from 26 to 117 bp upstream of the 5' proximal ATG of the open reading frame. The predicted 669-amino acid, Mpg-1-encoded protein has potential glycosylation and phosphorylation sites in addition to a signal sequence. The core protein is predicted to have a molecular weight of 71 to 74 kD. Computer-assisted local similarity searches indicate that Mpg-1 is a novel gene that may share a distant ancestry to perforin, a lytic protein found in cytotoxic T lymphocytes and natural killer cells.

Amino Acid Sequence↗
PubMedDate not supplied

Glucocorticoid effects on the skeletal muscle differentiation program: analysis of clonal proliferation, morphological differentiation and the expression of muscle-specific and regulatory genes.

We examined the effect of glucocorticoids on the proliferation and differentiation of skeletal muscle cells using the C2C12 cell line. We found that treatment with glucocorticoids enhanced muscle cell differentiation but had only minor effects on the clonal growth rate of C2C12 cells. The stimulatory effect of glucocorticoids on myogenic differentiation was reflected in the increased expression of muscle-specific genes, creatine kinase (CK) and acetylcholine receptor gamma subunit (AChR). Dexamethasone had no effect on CK and AChR mRNA stability and enhanced transcription from a CAT reporter genes containing the 3.3kb 5' flanking region of the murine CK gene (-3300MCK-CAT). Since dexamethasone did not affect the expression levels of the myogenic regulatory genes such as myoD and myogenin, the enhancement of muscle-specific transcription might reflect an increase in the functional activity of the regulatory proteins. Other possible mechanisms involved in the differentiation-enhancing effect of glucocorticoids are discussed.

Cell Differentiation↗
PubMed1995

High-dose dexamethasone suppression testing versus inferior petrosal sinus sampling in the differential diagnosis of adrenocorticotropin-dependent Cushing's syndrome: a decision analysis.

Differentiation of adrenocorticotropin (ACTH)-dependent Cushing's syndrome between Cushing's disease and the occult ectopic ACTH syndrome is difficult. Simultaneous bilateral inferior petrosal sinus sampling (IPSS) for ACTH levels in response to corticotropin-releasing hormone has high diagnostic accuracy, but its cost-effectiveness has not been analyzed. In this study, decision analysis was used to compare two diagnostic strategies: IPSS versus high-dose dexamethasone suppression (HDD) followed by IPSS in those with a negative HDD test. Sensitivity analyses were performed for all variables. The authors found that at 100% accuracy, IPSS has an incremental cost-effectiveness ratio of $1,000,000 per life saved. Incremental cost, incremental effectiveness, and incremental cost-effectiveness are sensitive to the pretest probability of Cushing's disease, test characteristics, and test costs. As the pretest probability of Cushing's disease decreases, cost per life saved also decreases to less than 85%, the HDD strategy saves more lives and costs less. When the HDD test has a 83% sensitivity rate and a 100% specificity rate, the two strategy remains less expensive. The IPSS strategy saves lives whenever HDD specificity is less than 100%.(ABSTRACT TRUNCATED AT 250 WORDS)

ACTH Syndrome, Ectopic↗