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PubMedDate not supplied

Proteome analysis of rhoptry-enriched fractions isolated from Plasmodium merozoites.

The rhoptries of Plasmodium species participate in merozoite invasion and modification of the host erythrocyte. However, only a few rhoptry proteins have been identified using conventional gene identification protocols. To investigate the protein organization of this organelle and to identify new rhoptry proteins, merozoite rhoptries from three different Plasmodium rodent species were enriched by sucrose density gradient fractionation, and subjected to proteome analysis using multidimensional protein identification technology (MudPIT); 148 proteins were identified. To distinguish abundant cellular contaminants from bona fide organellar proteins, a differential analysis comparing the proteins in the rhoptry-enriched fractions to proteins identified from whole cell lysates of P. berghei mixed asexual blood stages was undertaken. In addition, the proteins detected were analyzed for the presence of transmembrane domains, secretory signal peptide, cell adhesion motifs, and/or rhoptry-specific tyrosine-sorting motifs. Combining the differential analysis and bioinformatic approaches, a set of 36 proteins was defined as being potentially located to the Plasmodium rhoptries. Among these potential rhoptry proteins were homologues of known rhoptry proteins, proteases, and enzymes involved in lipid metabolism. Molecular characterization and understanding of the supramolecular organization of these novel potential rhoptry proteins may assist in the identification of new intervention targets for the asexual blood stages of malaria.

Animals↗
PubMed1999

Differential display analysis can reveal patterns of gene expression in immortalised hepatoma cells which are similar to those observed in young adult but not old adult liver cells.

We used the differential display technique to examine whether there were any patterns of gene expression which were characteristic of both young adult rat liver and of immortalised rat hepatoma cell lines, but not of old adult rat liver. No genes were detected which appeared to be clearly expressed in young liver and immortalised cell lines, but not in old liver. However, 14 genes were detected in old liver which were down-regulated in young liver and the hepatoma cell lines. This observation lends support to the idea that immortalisation of malignant cells may involve, at least in some aspects, a reversal of the ageing process in these cells and that the genes involved have a recessive action.

Aging↗
PubMed2006

Differential display analysis of gene expression in two immunologically distinct strains of Eimeria maxima.

Gene expression during sporulation and sporozoite excystation of two strains of Eimeria maxima was analyzed using the mRNA differential display technique. The two strains, the Guelph strain (GS) and a single sporocyst-derived strain (M6) from Florida, have been shown to be immunologically distinct. We isolated and cloned a 453-bp complimentary DNA (cDNA) fragment (GS-453) found only in GS sporozoites. In GS, this mRNA begins to be expressed during the earliest stages of oocyst sporulation and is continuously expressed up to and including in the excysted sporozoite. In all Northern blots, digoxigenin (DIG)-labeled GS-453 probe recognized an mRNA of approximately 1.6 kb from GS but not from RNA of M6. Southern blots using various endonucleases and probed with DIG-labeled GS-453 demonstrated that the genomes of both strains contained sufficiently similar sequences to permit hybridization with the probe, but the pattern of hybridization differed between the two strains. Extensive searches of the GenBank, European Molecular Biology Laboratory, and various apicomplexan expressed sequence tag databases using the DNA or inferred amino acid sequences of GS-453 cDNA clone did not identify similarity to any existing sequences.

Animals↗
PubMed2005

Analysis of differential protein expression during growth states of Ferroplasma strains and insights into electron transport for iron oxidation.

To investigate the metabolic biochemistry of iron-oxidizing extreme acidophiles, a proteomic analysis of chemomixotrophic and chemo-organotrophic growth, as well as protein expression in the absence of organic carbon, was carried out in Ferroplasma species. Electron transport chain inhibitor studies, spectrophotometric analysis and proteomic results suggest that oxidation of ferrous iron may be mediated by the blue copper-haem protein sulfocyanin and the derived electron passes to a cbb3 terminal electron acceptor. Despite previous suggestions of a putative carbon dioxide fixation pathway, no up-regulation of proteins typically associated with carbon dioxide fixation was evident during incubation in the absence of organic carbon. Although a lack of known carbon dioxide fixation proteins does not constitute proof, the results suggest that these strains are not autotrophic. Proteins putatively involved in central metabolic pathways, a probable sugar permease and flavoproteins were up-regulated during chemo-organotrophic growth in comparison to the protein complement during chemomixotrophic growth. These results reflect a higher energy demand to be derived from the organic carbon during chemo-organotrophic growth. Proteins with suggested function as central metabolic enzymes were expressed at higher levels during chemomixotrophic growth by Ferroplasma acidiphilum Y(T) compared to 'Ferroplasma acidarmanus' Fer1. This study addresses some of the biochemical and bioenergetic questions fundamental for survival of these organisms in extreme acid-leaching environments.

Archaeal Proteins↗
PubMed2006

[Differential proteins analysis for human nasal polyposis and normal nasal mucosa].

OBJECTIVE: To establish two-dimensional polyacrylamide gel electrophoresis (2-DE) map with high resolution and reproducibility from human nasal polyposis and normal nasal mucosa, and to identify differential expression proteins of 2-DE map. METHOD: Samples of human nasal polyposis and normal nasal mucosa (each sample group containing seven cases) were obtained. The total proteins were extracted and separated by immobilized pH gradient (IPG)-based 2-DE. The silver-stained 2-DE were scanned with digital Image Scanner and analyzed with ImageMaster 2-DE Elite 4.01 software. Peptide mass fingerprint (PMF) of differential protein spots was obtained with matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF-MS). The peptide mass fingerprints were searched in Swiss-Prot and TreMBL database by PeptIdent software, and then differential expression proteins were identified. RESULT: (1) 2-DE for a randomly selected 1 sample from each of the 2 groups was repeated 3 times respectively to analyze the reproductive of the method. The image analysis showed: For the polyposis tissues, the average proteins spots of three 2-DE maps were 891 +/- 67 and 767 +/- 83, spots were matched with the average matching rate of 86.1%. The average deviations of matched spot position were (1.13 +/- 0.16) mm in IEF direction and 1.45 +/- 0.21) mm in SDS-PAGE direction, respectively. For the nasal mucosa tissues, the average proteins spots of three 2-DE maps were 936 +/- 62 and 821 +/- 78, spots were matched with the average matching rate of 87.7%. Comparing the average electrophoresis profiles of the two tissues, each sample contained 7 cases. The proteins spots of nasal polyposis and nasal mucosa tissues were 1532 and 1617. A total of 1 065 proteins spots were matched between the two tissues average electrophoresis profiles. (2)Twenty dif-ferential expression protein spots were incised from silver staining gel randomly and digested in gel by TPCK Trypsin. Sixteen PMF were obtained by MALDI-TOF-MS, and 11 differential expression proteins were identified. CONCLUSION: In this study, the well-resolved reproducible 2-DE map of human nasal polyposis and nasal mucosa were established. Certain differential proteins were identified.

Adult↗
PubMed2001

Differential expression analysis by gene array of cell cycle modulators in human corneal epithelial cells stimulated with epidermal growth factor (EGF), hepatocyte growth factor (HGF), or keratinocyte growth factor (KGF).

PURPOSE: To identify and differentiate cell cycle and differentiation genes that are up-regulated or down-regulated in human corneal epithelial cells in response to alternative epithelium-modulating cytokines epidermal growth factor (EGF), hepatocyte growth factor (HGF) or keratinocyte growth factor (KGF). METHODS: Primary cultures human corneal epithelial cell (HCE) were treated with 25 ng/ml of EGF, 25 ng/ml HGF, 25 ng/ml KGF, or vehicle for 8 hours. Complementary DNA (cDNA) probes were synthesized from total cellular RNA isolated from the HCE cells. The cDNA probes were hybridized to the Atlas human cell cycle/differentiation array membrane. RNAse protection assay was used to confirm up-regulation of the serine/threonine-protein kinase PITALRE gene by EGF, KGF, and HGF. RESULTS: The expression of one hundred and eleven cell cycle and differentiation genes was monitored with the gene array system. It was found that these epithelial cell-modulating cytokines shared similar effects on some of the cell cycle and differentiation genes that were monitored, but had specific effects on some cytokines. Up-regulation of PITALRE gene expression was confirmed using RNAse protection assay. CONCLUSION: EGF, HGF and KGF had differential effects on cell cycle- and differentiation-related gene expression in corneal epithelial cells. For example, all three mitogenic growth factors up-regulated the expression of cyclin D1 (BCL-1 oncogene) and serine/threonine-protein kinase PITALRE in the primary cultured human corneal epithelial cells. However, EGF and KGF, but not HGF, up-regulated expression of the E2F-1 pRB-binding protein gene. Thus, while these three epithelial mitogens have similar effects on many genes that were analyzed, important differences were noted that may relate to differing effects of these growth factors on corneal epithelial cells. Studies to analyze the significance of the identified differences among these growth factors are in progress.

Cell Cycle↗
PubMed1992

Review of the use of pattern analysis to differentiate true drug and placebo responses.

Placebo response in patients assigned active drug is a troubling source of variance in antidepressant studies. This paper summarizes a series of studies utilizing pattern analysis to distinguish between placebo and true drug responses. Analysis of the persistence, speed, and timing of onset of patients' improvement during antidepressant therapy reveals distinct patterns of response which are likely to be attributable to placebo and true drug effects. While true drug effects seem to be characterized by a 2-week delay in onset followed by persistent improvement, placebo effects seem to be characterized by abrupt, transient improvement. Gradual responses on placebo may be due to spontaneous remission. The heuristic and clinical implications of pattern analysis are discussed.

Adolescent↗
PubMed2005

Comparative gene expression analysis by differential clustering approach: application to the Candida albicans transcription program.

Differences in gene expression underlie many of the phenotypic variations between related organisms, yet approaches to characterize such differences on a genome-wide scale are not well developed. Here, we introduce the "differential clustering algorithm" for revealing conserved and diverged co-expression patterns. Our approach is applied at different levels of organization, ranging from pair-wise correlations within specific groups of functionally linked genes, to higher-order correlations between such groups. Using the differential clustering algorithm, we systematically compared the transcription program of the fungal pathogen Candida albicans with that of the model organism Saccharomyces cerevisiae. Many of the identified differences are related to the differential requirement for mitochondrial function in the two yeasts. Distinct regulation patterns of cell cycle genes and of amino acid metabolic genes were also revealed and, in some cases, could be linked to the differential appearance of cis-regulatory elements in the gene promoter regions. Our study provides a comprehensive framework for comparative gene expression analysis and a rich source of hypotheses for uncharacterized open reading frames and putative cis-regulatory elements in C. albicans.

Candida albicans↗
PubMed1996

Application of random amplified polymorphic DNA analysis to differentiate strains of Salmonella enteritidis.

A random amplified polymorphic DNA (RAPD) fingerprinting method has been developed to differentiate Salmonella enteritidis isolates. A total of 65 arbitrary primers were screened with S. enteritidis isolates of different phage types. This allowed selection of a panel of primers capable of detecting DNA polymorphisms among S. enteritidis isolates. This panel was used to examine a panel of 29 isolates of S. enteritidis which had been previously characterized by other subtyping methods, including phage typing (PT) (n = 7), ribotyping (RT) (n = 13), and pulsed-field gel electrophoresis (PFGE). Applied collectively, these three methods resolved the collection into 20 different subtypes. However, by the RAPD fingerprinting method alone, 14 RAPD subtypes were revealed. Eight isolates of S. enteritidis phage type 8 that failed to be discriminated by other typing methods (PT, RT, and PFGE) were resolved into three different subtypes by RAPD analysis. In contrast, isolates that were derived from the same sources were not differentiated by any of the subtyping methods employed, including PT, RT, PFGE, and RAPD analysis. This RAPD approach to S. enteritidis subtyping provided more discriminatory power than did any of several other subtyping methods applied individually. Once the challenging step of primer identification was accomplished, determinations of the appropriate concentrations of arbitrary primer, DNA template, and MG2+ ion were also necessary for optimal discriminatory power. The bacterial DNA used in this RAPD protocol was obtained by boiling the bacterial sample. This simple procedure yielded DNA that produced fingerprint patterns as consistent as those obtained from phenol-chloroform-extracted DNA. Clearly, when appropriately constituted primer sets are identified and employed, RAPD analysis provides a simple, rapid, and powerful subtyping method for S. enteritidis.

Bacterial Typing Techniques↗
PubMed2005

Microarray analysis of differentially expressed genes of primary tumors in the canine central nervous system.

The pathophysiologic similarities of many human and canine cancers support the role of the domestic dog as a model for brain tumor research. Here we report the construction of a custom canine brain-specific cDNA microarray and the analysis of gene expression patterns of several different types of canine brain tumor. The microarray contained 4000 clones from a canine brain specific cDNA library including 2161 clones that matched known genes or expressed sequence tags (ESTs) and 25 cancer-related genes. Our study included 16 brain tumors (seven meningiomas, five glial tumors, two ependymomas, and two choroid plexus papillomas) from a variety of different dog breeds. We identified several genes previously found to be differentially expressed in human brain tumors. This suggests that human and canine brain tumors share a common pathogenesis. In addition, we also found differentially expressed genes unique to either meningiomas or the glial tumors. This report represents the first global gene expression analysis of different types of canine brain tumors by cDNA microarrays and might aid in the identification of potential candidate genes involved in tumor formation and progression.

Animals↗
PubMed2003

Controlling false-negative errors in microarray differential expression analysis: a PRIM approach.

MOTIVATION: Theoretical considerations suggest that current microarray screening algorithms may fail to detect many true differences in gene expression (Type II analytic errors). We assessed 'false negative' error rates in differential expression analyses by conventional linear statistical models (e.g. t-test), microarray-adapted variants (e.g. SAM, Cyber-T), and a novel strategy based on hold-out cross-validation. The latter approach employs the machine-learning algorithm Patient Rule Induction Method (PRIM) to infer minimum thresholds for reliable change in gene expression from Boolean conjunctions of fold-induction and raw fluorescence measurements. RESULTS: Monte Carlo analyses based on four empirical data sets show that conventional statistical models and their microarray-adapted variants overlook more than 50% of genes showing significant up-regulation. Conjoint PRIM prediction rules recover approximately twice as many differentially expressed transcripts while maintaining strong control over false-positive (Type I) errors. As a result, experimental replication rates increase and total analytic error rates decline. RT-PCR studies confirm that gene inductions detected by PRIM but overlooked by other methods represent true changes in mRNA levels. PRIM-based conjoint inference rules thus represent an improved strategy for high-sensitivity screening of DNA microarrays. AVAILABILITY: Freestanding JAVA application at http://microarray.crump.ucla.edu/focus

Algorithms↗
PubMed2002

How to identify patients in need of psychological intervention.

The identification of skin cancer patients in need of psychosocial intervention is a necessary prerequisite to relieving their distress and specifically supporting their coping strategies. The influence of early support even on prognosis has been shown in melanoma patients. Only a few patients ask for support themselves. The workload of doctors and nurses limits their ability to identify patients in need of support. Conducting interviews during daily hospital routine is time-consuming and cannot be implemented adequately. The screening of psychosocial risk patients needs to be based on standardized instruments. They should be easy to implement during the every-day routine. The Hornheide questionnaire with 27 items, the short form of the questionnaire with 9 items, and the Hornheide Screening Instrument (HSI) with 7 interview questions have been developed to achieve this aim in skin cancer patients. The Hornheide questionnaire offers a differentiated analysis of distress, and facilitates an accurate intervention according to the individual distress of the patient. The short form does not provide a differentiated analysis of distress. Its advantage is the speedy identification of patients at psychosocial risk. The HSI is for use during the anamnestic interview. In correlation with reduction of hospitalization time the HSI is most practicable for routine clinical implementation.

Female↗
PubMed2003

Global analysis of differentially expressed genes in oxidized LDL-treated human aortic smooth muscle cells.

Oxidized low density lipoproteins (OxLDL) play a key role in atherogenesis and induce a wide range of biological effects on smooth muscle cells. We used two commercially available cDNA microarray systems with a total of 35,932 human genes to determine differentially expressed genes in OxLDL-treated human aortic smooth muscle cells (HASMC) and to identify novel genes responsive to this agonist. We found a significant increase in expression of 180 and a significant decrease in expression of 192 named genes after treatment by OxLDL, compared with native LDL. Real time-PCR analysis confirmed microarray data for seven of eight tested genes. The differentially regulated genes were grouped into 16 classes based on the functions of the corresponding protein products. Our data demonstrate that OxLDL predominantly elevates expression of genes involved in cell-cell interactions, membrane transport, oncogenesis, apoptosis, and transcription and decreases expression of genes responsible for protein and nucleic acid biosynthesis, lipid metabolism, and humoral responses. Interestingly, we identify for the first time expression of metastasis-related protein (MB2) and novel scavenger receptor SREC-II in HASMC and these were upregulated 12- and 3-fold by OxLDL treatment, respectively. These findings have major implications for understanding atherogenic effect of OxLDL.

Aorta↗
PubMed1992

Analysis of differential accumulation of winged bean Kunitz chymotrypsin inhibitor mRNA species by a sequence-specific termination method.

Winged bean Kunitz chymotrypsin inhibitor (WCI) is encoded by a multigene family and accumulation of its mRNA is restricted in mid-maturation stage seeds and tuberous roots. In this paper, we analyzed the accumulation of mRNA derived from each WCI gene using a novel method: sequence-specific termination analysis. The results demonstrated that the accumulation of each WCI mRNA was differentially regulated in winged bean plants.

Base Sequence↗
PubMed2002

DNA microarray analysis of differentially expressed genes responsive to bisphenol A, an alkylphenol derivative, in an in vitro mouse Sertoli cell model.

To identify genes elicited by bisphenol A (BPA) in Sertoli cells, we carried out a microarray analysis of TTE3 cells (a mouse Sertoli cell line) treated with BPA. BPA (100, 200 and 400 microM) induced cell death concentration-dependently, with levels being 25%, 33% and 96%, respectively. Of the 1,081 genes analyzed, 3 genes showed decreased levels of expression while the remaining 10 genes showed increased levels in the cells treated with a subtoxic dose of BPA (200 microM). The expressions of six genes were confirmed by the TaqMan assay. These findings suggest that DNA microarray analysis is a useful tool for investigating the molecular mechanisms of the toxic effects of BPA in testicular cells.

Animals↗
PubMed2005

Analysis of differential protein expression in Acidithiobacillus ferrooxidans grown under different energy resources respectively using SELDI-ProteinChip technologies.

Surface-enhanced laser desorption/ionization (SELDI)-time of flight is an affinity-based mass spectrometric method in which proteins of interest are selectively absorbed to a chemically modified surface on a chip, which allows proteomic analysis with limited material requirements. This characteristic makes it a valuable technique for microbiologists handling problematic samples, such as low cell number cultures. In this study, we explored differential-expressed proteome of Acidithiobacillus ferrooxidans cultivated with Fe(2+) and elemental sulfur separately by adopting the protein biochip SELDI approach. The cell lysates of A. ferrooxidans were applied onto Ciphergen ProteinChip WCX2, SAX2 and IMAC-Cu arrays. Proteins bound to the chips were analyzed on a ProteinChip Reader Model PBS II. A summary of the molecular masses of the differentially regulated proteins found on WCX2, IMAC-Cu and SAX2 was obtained and 28 differentially expressed proteins were found on the molecular weight range of 5.0 to 25 kDa.

Acidithiobacillus↗
PubMed1987

Analysis of differential survival of syngeneic islets transplanted into hyperglycemic C57BL/6J versus C57BL/KsJ mice.

C57BL/KsJ (BKs) male mice were more sensitive to diabetes induction by administration of multiple low-doses of streptozotocin (Sz) than were C57BL/6J (B6) male mice. Analysis of islet size and insulin content of the two parental strains did not indicate that differences in drug sensitivity could be attributed to an effect of genetic background on islet size or insulin content. 50 BKs islets implanted into the spleens of BKs male mice made diabetic by Sz were eliminated within 12 days posttransplantation, whereas an equal number of B6 islets implanted into the spleens of diabetic B6 recipients were retained, even though the numbers of islets implanted were insufficient to effect remission from hyperglycemia. In contrast to the rapid loss of islets implanted into spleens of hyperglycemic BKs recipients, BKs islets implanted into spleens of normoglycemic recipients were not eliminated, thus suggesting that the basis for the differential survival between the B6 and BKs strains reflected their ability to survive hyperglycemic stress rather than a differential ability to replicate. Since BKs beta cells have been shown to respond to hyperglycemia by expression of an endogenous retroviral gene that cannot be expressed by B6 beta cells, the possibility that this differential survival represents a strain difference in autoreactivity against islet cells is raised.

Animals↗