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Results for “differential analysis”

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PubMed1987

Analysis of differentiation antigens on normal and carcinogen-altered rat epithelial cells in vivo and in culture.

Two murine monoclonal antibodies, 3BG8 and 9BG8, which were raised against a rat tracheal squamous-cell-carcinoma cell line, recognize cell-surface antigens on normal rat squamous epithelium (skin, esophagus, vagina, and cornea) as well as on carcinogen-exposed, immortalized, rat tracheal epithelial cells. Monoclonal antibody 3BG8 binds to a 115-kilodalton cell-surface protein on undifferentiated basal cells of the epithelium, while the binding of the other antibody, 9BG8, occurs in both differentiated and undifferentiated populations of normal squamous epithelium and squamous cell carcinomas. Undifferentiated tracheal carcinomas bound only the 3BG8 antibody. No binding of either antibody was detected on normal tracheal mucociliary epithelium. Only under conditions that induce squamous differentiation of rat tracheal epithelium was binding of 3BG8 and 9BG8 detected. For reasons which are not clear at present, 9BG8 dramatically inhibits the growth of normal tracheal and esophageal cells in primary culture, whereas only 3BG8 affects the growth of carcinogen-altered tracheal cell lines. Based on antigen characterization and distribution, it is concluded that the 3BG8 and 9BG8 epitopes are localized on differentiation antigens which differ from others that have been previously described.

Animals↗
PubMed2007

Analysis of differential gene expression between chronic hepatitis B patients and asymptomatic hepatitis B carriers.

BACKGROUND AND AIMS: Chronic hepatitis B virus (HBV) infection remains a serious global health problem, inducing a spectrum of diseases, including asymptomatic HBV carriage (ASC) and chronic hepatitis B (CHB). ASC and CHB represent different immunological states and their prognoses are diverse. To clarify molecular mechanisms underlying the two infection states, the differentially expressed genes between the two states were screened and identified. METHODS: Subtracted complementary DNA libraries by suppression subtractive hybridization, dot blot hybridization and quantitative real-time PCR were used to identify the differentially expressed genes between subjects with CHB and those with ASC. RESULTS: RNA from peripheral blood mononuclear cells from CHB and ASC subjects was subjected to suppression subtractive hybridization and resulted in isolation of subtracted complementary DNA clones. Eighty-eight randomly sampled clones were rescreened by dot blot hybridization, from which 29 clones were identified as differentially expressed genes. The differential expression of three genes was confirmed by real-time PCR in 23 subjects with CHB and 21 with ASC. CONCLUSIONS: Differentially expressed genes in peripheral blood mononuclear cells between CHB and ASC have been isolated by suppression subtractive hybridization, including some new genes. Of the up-regulated genes in CHB, most are known to be responsive to inflammatory conditions. These genes might provide clues in elucidating the mechanisms of the two different HBV infection states and designing therapeutic targets for HBV infection.

Adult↗
PubMedDate not supplied

Immunohistochemical analysis of differentiation in static and mixed prostate cancer spheroids.

Neoplastic multicellular spheroids are in vitro models of solid tumors employed in drug testing and basic research. This study compares differentiation in static and mixed prostate cancer spheroids. Staining intensity of prostate specific antigen (PSA) was down-regulated upon mixing, from 0.21 +/- 0.03 to 0.13 +/- 0.03 in LNCaP multicellular spheroids, and from 0.13 +/- 0.04 to 0.03 +/- 0.02 in DU 145 multicellular spheroids. This was accompanied by 65% increase in the expression of cytokeratins 8 and 18 in DU 145 spheroids. PSA expression extended 60 micro m within static spheroids and was disrupted in mixed culture. Diminished PSA expression and spatial organization suggests a more aggressive cancer. Higher cytokeratin expression could result from either differentiation towards a luminal phenotype or activation of the Ras pathway during dedifferentiation. Thus, the existing paradigm of differentiation established for normal tissue does not apply for our neoplastic spheroids. Cell dedifferentiation is attributed to improved interstitial transport and synthesis of extracellular matrix.

Animals↗
PubMed1982

Fatty acid analysis for differentiation or Bordetella and Brucella species.

The fatty acid composition of Bordetella pertussis (13 strains), B. parapertussis (3 strains), B. bronchiseptica (6 strains), Brucella abortus (6 strains), B. melitensis (4 strains) and B. suis (5 strains) was determined. Both genera contained straight-chain saturated and mono-unsaturated acids as well as cyclopropane substituted isomers, but the overall differences between the two genera were distinct. The Brucella species contained exclusively C16 to C19 acids. Minor amounts of hydroxylated acids (tentatively identified as 3-hydroxy-hexadecanoate and 3-hydroxy-octadecanoate) could only be detected after thin-layer chromatography and substantial concentration. The Bordetella species, on the other hand, contained a number of both non-hydroxylated and hydroxylated fatty acids of chain-lengths varying from C10 to C19. Intra-generically, no clear distinction between the Brucella species could be detected. The three species of Bordetella, on the other hand, were clearly distinguished by their fatty acid patterns. B. pertussis did not contain cyclopropane fatty acids encountered in significant amounts in the two other species, and B. bronchiseptica was characterized by containing 2-hydroxy-dodecanoate in significant amounts.

Bordetella↗
PubMed2006

Proteomic analysis of differential protein expression in early process of pancreatic regeneration in pancreatectomized rats.

AIM: A broad-range proteomic approach was applied to investigate the complexity of the mechanisms involved in pancreatic regeneration for identification of new targets of diabetes treatment and potential markers of pancreatic stem cells. METHODS: A regeneration pancreatic model was induced by 90% partial pancreatectomy (Px) in rats. Changes in the protein expression in regenerating rat pancreas on the third day after Px, as compared with rats that received sham surgery, were analyzed by using 2-D gel electrophoresis (2-DE), mass spectrometry (MS), and mass fingerprinting. RESULTS: 2-DE revealed 91 spots with at least 1.5-fold increases in expression at 3 d after pancreatectomy and 53 differentially expressed proteins that were identified by peptide mass fingerprinting (PMF). These included cell growth-related, lipid and energy metabolism-related, protein and amino acid metabolism-related proteins, and signal transduction proteins. Vimentin, CK8, L-plastin, hnRNP A2/B1, and AGAT are associated with embryogenesis and cell differentiation, and may be new potential pancreatic stem cells markers. CONCLUSION: The proteome profiling technique provided a broad-based and effective approach for the rapid assimilation and identification of adaptive protein changes during pancreas regeneration induced by pancreatectomy. Our data clarify the global proteome during the pancreatic proliferation and differentiation processes, which is important for better understanding of pancreatic regeneration and for discovering of protein biomarkers for pancreatic stem cells.

Amidinotransferases↗
PubMed1998

Sodium dodecyl sulfate polyacrylamide gel electrophoresis of canine urinary proteins for the analysis and differentiation of tubular and glomerular diseases.

A preliminary investigation was performed to evaluate the use of a new, noninvasive technique for the localization of canine renal lesions by electrophoresis of urinary proteins. Urine specimens from six clinically healthy, nonproteinuric dogs and 12 dogs with persistent proteinuria were examined by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS PAGE). Urine electrophoretic patterns of proteinuric dogs were classified as glomerular (n = 4), tubular (n = 2), or mixed (glomerular and tubular) (n = 6), based on the number and molecular weight of the silver-stained protein bands. Renal tissues from biopsies or necropsies were obtained from eight of the dogs with proteinuric disease. Interpretation of seven of eight electrophoretograms agreed with the histologic interpretation of renal lesions. We concluded SDS PAGE is a potentially valuable technique for detection and localization of renal lesions in dogs with proteinuric disease.

Journal Article↗
PubMed2003

Analysis of differential substrate selectivities of CYP2B6 and CYP2E1 by site-directed mutagenesis and molecular modeling.

Human CYP2B6 and CYP2E1 were used to investigate the extent to which differential substrate selectivities between cytochrome P450 subfamilies reflect differences in active-site residues as opposed to distinct arrangement of the backbone of the enzymes. Reciprocal CYP2B6 and CYP2E1 mutants at active-site positions 103, 209, 294, 363, 367, and 477 (numbering according to CYP2B6) were characterized using the CYP2B6-selective substrate 7-ethoxy-4-trifluoromethylcoumarin, the CYP2E1-selective substrate p-nitrophenol, and the common substrates 7-ethoxycoumarin, 7-butoxycoumarin, and arachidonic acid. This report is the first to study the active site of CYP2E1 by systematic site-directed mutagenesis. One of the most intriguing findings was that substitution of CYP2E1 Phe-477 with valine from CYP2B6 resulted in significant 7-ethoxy-4-trifluoromethylcoumarin deethylation. Use of three-dimensional models of CYP2B6 and CYP2E1 based on the crystal structure of CYP2C5 suggested that deethylation of 7-ethoxy-4-trifluoromethylcoumarin by CYP2E1 is impeded by van der Waals overlaps with the side chain of Phe-477. Interestingly, none of the CYP2B6 mutants acquired enhanced ability to hydroxylate p-nitrophenol. Substitution of residue 363 in CYP2E1 and CYP2B6 resulted in significant alterations of the metabolite profile for the side chain hydroxylation of 7-butoxycoumarin. Probing of CYP2E1 mutants with arachidonic acid indicated that residues Leu-209 and Phe-477 are critical for substrate orientation in the active site. Overall, the study revealed that differences in the side chains of active-site residues are partially responsible for differential substrate selectivities across cytochrome P450 subfamilies. However, the relative importance of active-site residues appears to be dependent on the structural similarity of the compound to other substrates of the enzyme.

Amino Acid Sequence↗
PubMed1999

Characterization, molecular cloning, and differential expression analysis of laccase genes from the edible mushroom Lentinula edodes.

The effect of different substrates and various developmental stages (mycelium growth, primordium appearance, and fruiting-body formation) on laccase production in the edible mushroom Lentinula edodes was studied. The cap of the mature mushroom showed the highest laccase activity, and laccase activity was not stimulated by some well-known laccase inducers or sawdust. For our molecular studies, two genomic DNA sequences, representing allelic variants of the L. edodes lac1 gene, were isolated, and DNA sequence analysis demonstrated that lac1 encodes a putative polypeptide of 526 amino acids which is interrupted by 13 introns. The two allelic genes differ at 95 nucleotides, which results in seven amino acid differences in the encoded protein. The copper-binding domains found in other laccase enzymes are conserved in the L. edodes Lac1 proteins. A fragment of a second laccase gene (lac2) was also isolated, and competitive PCR showed that expression of lac1 and lac2 genes was different under various conditions. Our results suggest that laccases may play a role in the morphogenesis of the mushroom. To our knowledge, this is the first report on the cloning of genes involved in lignocellulose degradation in this economically important edible fungus.

Amino Acid Sequence↗
PubMed1999

Reverse transcription-PCR differential display analysis of Escherichia coli global gene regulation in response to heat shock.

A reverse transcription (RT)-PCR technique was developed to analyze global gene regulation in Escherichia coli. A novel combination of primers designed specifically for the start and stop regions of E. coli genes (based on the findings of Fislage et al. [R. Fislage, M. Berceanu, Y. Humboldt, M. Wendt, and H. Oberender, Nucleic Acids Res. 25:1830-1835, 1997]) was used as an alternative to the poly(T) primers often used in eukaryotic RT-PCR. The validity of the technique was demonstrated by applying it to heat shock analysis. Specifically, RT-PCR-amplified total RNA from heat-shocked and non-heat-shocked cells were hybridized with slot blots of the Kohara set (U. Kohara, K. Akiyama, and K. Isono, Cell 50:495-508, 1987; S. Chuang, D. Daniels, and F. Blattner, J. Bacteriol. 175:2026-2036, 1993). The signals obtained for heat-shocked and control cultures of each clone were compared, and differences in intensity were evaluated by calculating induction ratios. Clones that were considered significantly induced were subsequently mapped by the Southern blot technique in order to determine specific gene upregulation. Also, for several genes, Northern blotting and total RNA dot blotting were performed to confirm that the transcript levels in the original RNA samples were different. This technique extended previously described methods for studying global gene regulation in E. coli by incorporating a PCR amplification step in which global, mRNA-specific primers were used. In addition, the method employed here can be easily extended to study E. coli global gene regulation in response to additional environmental stimuli.

Base Sequence↗
PubMed2002

Proteomic analysis of differentially expressed Chlamydia pneumoniae genes during persistent infection of HEp-2 cells.

Recent data have shown that the respiratory pathogen Chlamydia pneumoniae expresses an altered gene transcription profile during gamma interferon (IFN-gamma)-induced persistent infection in vitro. In the present study, we examined, by proteomics, expression of C. pneumoniae proteins labeled intracellularly with [(35)S]methionine/cysteine under normal conditions or IFN-gamma-mediated persistence. The identity of differentially expressed proteins during persistent infection was determined by matching spots to those of proteins identified in C. pneumoniae elementary bodies by matrix-assisted laser desorption ionization mass spectrometry. Upon treatment with 50 U of IFN-gamma per ml, a marked upregulation of major outer membrane protein (MOMP), heat shock protein 60 (Hsp-60/GroEL), and proteins with functions in DNA replication (GyrA), transcription (RpoA, PnP), translation (Rrf), glycolysis (PgK, GlgP), and type III secretion (SctN) was observed at 24 h of infection. In contrast, no significant decreases in bacterial protein expression were found in C. pneumoniae-infected cells due to IFN-gamma treatment. Upregulation of C. pneumoniae proteins involved in diverse functions during persistent infection may allow the organism to resist the inhibitory effects of IFN-gamma while retaining basic functions. Future studies should examine the differential expression of chlamydial proteins during the developmental cycle under IFN-gamma pressure to obtain a finer representation of the gene products involved in establishing persistence.

Bacterial Proteins↗
PubMed2006

Proteome and differential expression analysis of membrane and cytosolic proteins from Mycobacterium avium subsp. paratuberculosis strains K-10 and 187.

Little is known of protein expression in Mycobacterium avium subsp. paratuberculosis and how this contributes to pathogenesis. In the present study, proteins from both membranes and cytosol were prepared from two strains of M. avium subsp. paratuberculosis, i.e., laboratory-adapted strain K-10 and a recent isolate, strain 187, obtained from a cow exhibiting clinical signs of Johne's disease. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis of cytosol and membrane proteins from K-10 and 187 showed marked differences in protein expression. Relative levels of protein expression from both M. avium subsp. paratuberculosis strains were measured by using amine-reactive isobaric tagging reagents (iTRAQ) and tandem mass spectroscopy. Protein identification and relative expression data were obtained for 874 membrane and cytosolic proteins from the M. avium subsp. paratuberculosis proteome. These data showed a number of significant differences in protein expression between strain K-10 and clinical isolate 187. Examples of proteins expressed at higher levels in clinical isolate 187 compared to strain K-10 are AtpC, RpoA, and several proteins involved in fatty acid biosynthesis. In contrast, proteins such as AhpC and several proteins involved in nitrogen metabolism were expressed at higher levels in strain K-10 compared to strain 187. These data may provide insights into the proteins whose expression is important in natural infection but are modified once M. avium subsp. paratuberculosis is adapted to laboratory cultivation. Results from these studies will provide tools for developing a better understanding of M. avium subsp. paratuberculosis infection in the host and offer potential as diagnostic reagents and vaccine candidates.

Bacterial Proteins↗
PubMed1975

Synchronous germination and outgrowth of fractionated Bacillus subtilis spores: tool for the analysis of differentiation and division of bacterial cells.

A fraction of Bacillus subtilis 168 spores, purified by Urografin isopycnic density centrifugation, heat activated, and inoculated in nutrient broth plus glucose, germinated and outgrew very synchronously. Synchrony was documented by nuclear staining and fluorescence microscopy, and by determining the variation of the buoyant density of the cells during outgrowth. Cell mass increased at constant rates and the passage from one rate to the next was dependent upon deoxyribonucleic acid (DNA) synthesis. DNA synthesis inhibitors induced the formation of anucleated sister cells in a vast majority of the population, indicating that septation was programmed and became independent from DNA synthesis very early in the cell cycle.

Azo Compounds↗
PubMed1988

Microculture assay for human macrophage maturation in vitro. Cell-ELISA analysis of differentiation antigen expression.

The terminal differentiation of monocytes (mo) to macrophages (MO) is essential for the complex functioning of the mononuclear phagocyte system. Similar to in vivo MO maturation, blood mo transform to MO when cultured in vitro. This offers a model system for experimental studies of functional defects within this cell lineage. In order to develop a microassay for the evaluation of MO maturation in vitro we monitored the antigenic phenotype of MO during differentiation in vitro. The cells were grown either in suspension on hydrophobic Teflon foils, or as plastic adherent monolayers. Surface antigens were visualized on single cells with the immunoperoxidase slide technique or quantitated with the enzyme-linked immunosorbent assay (ELISA). As MO mature in vitro they increase in cell size, in the amount of beta-2-microglobulin, the myeloid antigen CD14 and HLA-DR antigen expression as measured with cell-ELISA, both in microwell cultures and on single cells. Upon terminal differentiation mo-MO express differentiation-specific antigens (transferrin receptor, surface transferrin and antigens of the Max series) which are indicative of a successful MO maturation. Monocyte cultures in microplates and the subsequent evaluation of differentiation antigen expression could be used as an experimental system to study the modulation of MO differentiation in vitro and may serve as an in vitro parameter of MO function in vivo.

Antigens, Differentiation↗