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PubMed2001

Restrictive dermopathy: case report, subject review with Kaplan-Meier analysis, and differential diagnosis of the lethal congenital contractural syndromes.

We report on a 34-week-old infant with restrictive dermopathy (RD), a rare lethal genodermatosis, characterized by an abnormal skin growth and differentiation with thin, tightly adherent skin that causes a dysmorphic face, generalized flexion joint contractures, and respiratory insufficiency. Kaplan-Meier analysis of 32 previously well-described infants affected with RD showed a median survival of 132 hours. Lethal congenital contractural syndromes, including Pena-Shokeir phenotype, cerebro-oculo facio-skeletal syndrome, and lethal multiple pterygium syndrome, should be considered first in the differential diagnosis. Other lethal contractural syndromes are discussed.

Contracture↗
PubMed2006

Differential proteome analysis of tonsils from children with chronic tonsillitis or with hyperplasia reveals disease-associated protein expression differences.

A proteomic approach has been used to establish a proteome map and differentiate between the protein composition of tonsils from patients with chronic tonsillitis (CT) and that of tonsils with hyperplasia (HPL). Two-dimensional gel analysis was performed with material from four patients with HPL and five patients with CT. An average of approximately 600 spots were detected in each gel. A total of 127 different proteins were identified in 158 spots analyzed by mass spectrometry. Our study revealed disease-associated differences between protein abundance for two protein spots, an HSP27 isoform and UMP-CMP kinase. Both protein spots were more abundant in the CT group. HSP27 ELISA was performed for 32 patients, 12 belonging to the HPL group and 20 to the CT group. ELISA could not be used to differentiate HSP27 isoforms nor to distinguish CT from HPL. HSP27 was found to migrate to two further protein spots in the 2D gels. The differently expressed HSP27 isoform migrated as the most acidic of all the HSP27 isoforms detected, indicating the highest degree of phosphorylation. The sum of all three HSP27 abundances in the gels from the CT group was not different from that of the HPL group, consistent with the ELISA results. Our results suggest that phosphorylation differences caused the observed migration differences of HSP27. Together with the UMP-CMP kinase abundance differences, we conclude that kinase and/or phosphatase activity are different in CT and HPL.

Child↗
PubMed2006

Meta-analysis of differentiating mouse embryonic stem cell gene expression kinetics reveals early change of a small gene set.

Stem cell differentiation involves critical changes in gene expression. Identification of these should provide endpoints useful for optimizing stem cell propagation as well as potential clues about mechanisms governing stem cell maintenance. Here we describe the results of a new meta-analysis methodology applied to multiple gene expression datasets from three mouse embryonic stem cell (ESC) lines obtained at specific time points during the course of their differentiation into various lineages. We developed methods to identify genes with expression changes that correlated with the altered frequency of functionally defined, undifferentiated ESC in culture. In each dataset, we computed a novel statistical confidence measure for every gene which captured the certainty that a particular gene exhibited an expression pattern of interest within that dataset. This permitted a joint analysis of the datasets, despite the different experimental designs. Using a ranking scheme that favored genes exhibiting patterns of interest, we focused on the top 88 genes whose expression was consistently changed when ESC were induced to differentiate. Seven of these (103728_at, 8430410A17Rik, Klf2, Nr0b1, Sox2, Tcl1, and Zfp42) showed a rapid decrease in expression concurrent with a decrease in frequency of undifferentiated cells and remained predictive when evaluated in additional maintenance and differentiating protocols. Through a novel meta-analysis, this study identifies a small set of genes whose expression is useful for identifying changes in stem cell frequencies in cultures of mouse ESC. The methods and findings have broader applicability to understanding the regulation of self-renewal of other stem cell types.

Adaptation, Physiological↗
PubMed1999

Digital image processing for rapid analysis of differentially expressed transcripts on high-density cDNA arrays.

Usage of filter arrays is becoming increasingly attractive for many research laboratories involved in determination of gene-expression profiles. However, analysis of numerous spots, representing genes or partial gene sequences (ESTs), is still tedious work involving the ordered analysis of vast amounts of numerical tabular data. We present a rapid and efficient method for the visual identification of differentially expressed targets on high-density cDNA filter arrays using standard laboratory equipment and standard software, which is available for free. The method we introduce provides an inexpensive alternative, and no changes in the experimental set up are required. Our results were verified by densitometric analyses performed with an established system.

Autoradiography↗
PubMed2002

Global analysis of differentially expressed genes in early gestational decidua and chorionic villi using a 9600 human cDNA microarray.

The global gene expression profiles of the decidua and chorionic villi of early human pregnancies were analysed by using cDNA microarray technology. Decidual and villous placental tissues were obtained from first trimester abortus and mRNA was extracted for cDNA microarray analysis. The human cDNA microarray [9600 clones, including known regulatory genes and expressed sequence tags (EST)] with colorimetric detection was used to identify differentially expressed genes between early gestational decidua and villi. According to cDNA microarray analysis, we have identified 641 genes with highly expressed mRNA in both decidua and villi, 49 genes with higher expressions in decidua, and 75 genes with higher expression in chorionic villi. These differentially expressed genes were further grouped into categories by their putative functions, including: cell growth-related factors, hormones/cytokines, cell adhesion molecules, signal transduction molecules, apoptosis-related factors, cytoskeleton/extracellular matrix proteins, and EST. Immunohistochemical stainings of cathepsin L, leukaemia inhibitory factor-receptor, and proliferative cell nuclear antigen showed results consistent with the microarray data. Identification of the differentially expressed genes between decidua and villi by microarray provide a global profiling of the gene expression pattern. This work adds to our understanding of placentation by reporting the gene expression profiles during first trimester human pregnancies using cDNA microarray.

Adult↗
PubMed1997

Analysis of differential scanning calorimetry data for proteins. Criteria of validity of one-step mechanism of irreversible protein denaturation.

We consider in this work the analysis of the excess heat capacity C(p)(ex) versus temperature profiles in terms of a model of thermal protein denaturation involving one irreversible step. It is shown that the dependences of ln C(p)(ex) on 1 T (T is the absolute temperature) obtained at various temperature scanning rates have the same form. Several new methods for estimation of parameters of the Arrhenius equation are explored. These new methods are based on the fitting of theoretical equations to the experimental heat capacity data, as well as on the analysis of the dependence d(ln C (p)(ex)) d ( 1 T ) on 1 T . We have applied the proposed methods to calorimetric data corresponding to the irreversible thermal denaturation of Torpedo californica acetylcholinesterase, cellulase from Streptomyces halstedii JM8, and lentil lectin. Criteria of validity for the one-step irreversible denaturation model are discussed.

Journal Article↗
PubMed1996

Numerical aberration and point mutation of p53 gene in human gastric intestinal metaplasia and well-differentiated adenocarcinoma: analysis by fluorescence in situ hybridization (FISH) and PCR-SSCP.

This study examined p53 gene alterations in human gastric mucosa, intestinal metaplasia and well-differentiated (cohesive type) adenocarcinomas to clarify to the role of the p53 gene in gastric tumorigenesis by means of fluorescence in situ hybridization (FISH), polymerase-chain-reaction-single-strand-conformation polymorphism (PCR-SSCP) and immunohistochemistry. Gene alterations were compared with numerical changes of chromosome 17. Samples were obtained from 31 surgically resected stomachs affected with gastric cancer. There was no nuclear p53 protein in cells from normal gastric mucosa. Among 23 specimens of intestinal metaplasia, cells with p53 protein were variably detected in 5 incomplete metaplasias (colonic type). A histological study revealed a mildly dysplastic appearance. PCR-SSCP identified p53-gene mutation in exons 5 and 8 in 2 of the 5 samples. Numerical aberrations of chromosome 17 and the p53 gene were undetectable both in normal mucosa and in intestinal metaplasia. Variable numbers of tumor cells contained p53 protein in 13 (54%) of 24 carcinomas, and PCR-SSCP revealed abnormal bands in 5 of them. Mutations were detected in exons 5, 7, 7, 7 and 8. FISH analysis demonstrated that 6 carcinomas emitted 3 or 4 signals for chromosome 17, and 7 gave one signal for the p53 gene in over 20% of the cells. Ten (77%) of 13 carcinomas examined by FISH appeared to show a p53-gene deletion.

Adenocarcinoma↗
PubMed1988

[Ultrastructural analysis in differential diagnosis of tumors].

Ultrastructures of 25 tumors were analyzed by electron microscopy (EM). Of the 25 cases, there were 9 amine precursor uptake and decarboxylation tumors (APUD) (2 carotid body tumor, 2 medullary carcinoma of thyroid and 5 carcinoid) in which the dense core granules of different sizes were seen in the cytoplasm. 4 cases of malignant melanoma were identified by EM basing on the premelanosome and melanosome in the cells. In 4 carcinomas from different locations, 2 had mucous secretory granules in the cytoplasm and junction complex between the tumor cells. The diagnosis was finally confirmed as adenocarcinoma. The other 2 cases were identified as epidermoid carcinoma or anaplastic carcinoma as desmosome and tonofilaments were found. 4 cases of malignant lymphoma without any cell junction complex were identified. Moreover, there were several cases of mesenchymal cell tumors, such as leiomyoma, rhabdomyosarcoma, chordoma and Schwannoma confirmed by their special organelles. This study shows that the ultrastructural analysis is valuable in the differential diagnosis and classification of tumors.

Adolescent↗
PubMed1996

Selection of probes for fluorescence in situ hybridization analysis by differential display polymerase chain reaction of mRNA from rhabdomyosarcoma.

Rhabdomyosarcoma (RMS) is a malignancy of skeletal muscle derivation encompassing two major subtypes, embryonal and alveolar, which differ in clinical behavior and genetic markers. Because RMS is a relatively circumscribed tumor system for which the beginnings of a molecular genetic framework are in place, it becomes an ideal model for the application of improved methods of molecular genetic analysis. We have applied the technique known as differential display polymerase chain reaction (DD-PCR) to characterize expression of RNA in rhabdomyosarcoma subtypes. Our studies have shown that DD-PCR generates a characteristic electrophoretic profile that can be used to isolate subtype specific probes for fluorescence in situ hybridization (FISH) analysis. We have isolated two cDNA fragments and obtained clones suitable for FISH mapping to metaphase chromosomes. One probe was mapped to the centromeric region of human chromosome 22 and the other probe to the human chromosome band 6q25-26. This approach demonstrates the utility of DD-PCR as a technique for isolating novel cDNA expressed in tumors and their subsequent use as probes for FISH analysis. As more genes are identified by DD-PCR and their roles in tumorigenesis become defined, they are likely to provide novel targets for future molecular cytogenetic analysis.

Base Sequence↗
PubMed2006

[Practical application of proliferation markers' (MIB-1, PCNA, AgNOR) expression analysis for differential diagnostics of nodular thyroid lesions].

In the study authors tried to estimate the diagnostic value ofAgNOR number and MIB-1 and PCNA expression in thyreocytes from various pathological lesions, with special attention to differential diagnosis of border-line lesions (nodule in adenomatus goiter--adenoma--follicular carcinoma). Fifty four thyroid glands were examined (10 adenomatous goiters, 20 follicular adenomas, 10 follicular carcinomas, 14 papillary carcinomas). Statistical analysis of obtained results allows in most cases to differentiate carcinomas from benign nodular lesions. The best differentiating factor for border-line lesions (especially differentiation between benign nodular lesions, neoplasms, nodules in adenomatous goiter adenomas and adenomas carcinomas) has turned out to be MIB-1. PCNA expression has differed significant between adenomatous goiter and follicular carcinoma and between adenoma and both carcinomas. AgNOR number has only helped in differentiation between nodules in adenomatous goiter and follicular carcinoma - correlations for other lesions have not been statistically significant.

Adolescent↗
PubMed2005

Proteomic analysis of differential protein expression in human atherosclerotic plaque progression.

In this study, differential protein expression was assessed during human atherosclerotic plaque progression. A multifaceted approach was used in which differential protein expression was studied by two-dimensional (2D) gel electrophoresis and validated in individual patients using western blotting and immunohistochemistry. 2D profiles of whole-mount advanced stable lesions were compared to those of plaques containing a thrombus. Mass spectrometry analysis identified vinexin-beta and alpha1-antitrypsin (AAT) in the same spot that was differentially expressed in plaques with a thrombus. Immunohistochemistry and western blotting showed limited expression of both vinexin-beta and AAT in early lesions, whereas high expression of both proteins was found in advanced lesions. Differential expression of vinexin-beta in lesions with a thrombus compared to stable plaques could not be confirmed, indicating the importance of validation of proteomic analysis. For AAT, western blotting of 2D gels revealed expression of six isoforms in advanced plaques, one of which was confirmed to be solely expressed in thrombus-containing plaques. In conclusion, vinexin-beta is expressed in advanced human atherosclerotic plaques, but differential expression of this protein in lesions with a thrombus versus stable plaques could not be confirmed. However, this analysis revealed expression of six isoforms of AAT in advanced plaques, one of which was uniquely expressed in thrombus-containing plaques.

Adaptor Proteins, Signal Transducing↗
PubMed1977

Hypercalcaemia in elderly hospital in-patients: value of discriminant analysis in differential diagnosis.

Plasma calcium was measured routinely as a part of profile screening of patients admitted to a geriatric department. Pathological hypercalcaemia was found in 1.33% of those screened, the cause being bone metastases (29%), hyperparathyroidism (21%), bronchial carcinoma without bone metastasis (18.5%), lymphosarcoma without bone metastasis (8%) and multiple myeloma (2.5%). There remained a further group of patients with hypercalcaemia and renal failure (21%) in whom diagnosis was often obscure. Where renal function was normal, discriminant analysis showed that the four main diagnostic groups were biochemically distinguishable. Discriminant analysis thus seems likely to be of practical value in the differential diagnosis of hypercalcaemia in elderly patients with normal renal function, but requires prospective validation.

Aged↗
PubMed2002

Proteome analysis of differentially displayed proteins as a tool for investigating ozone stress in rice (Oryza sativa L.) seedlings.

Employing classical two-dimensional electrophoresis (2-DE), amino acid sequencing and immunoblot analysis, we examine for the first time the effect of ozone, a highly notorious environmental pollutant, on rice seedling proteins. Drastic visible necrotic damage to leaf by ozone and consequent increase in ascorbate peroxidase protein(s) was accompanied by rapid changes in the 2-DE protein profiles, over controls. Out of a total of 56 proteins investigated, which were reproducible in repeated experiments, 52 protein spots were visually identified as differentially expressed over controls. Six proteins were N-terminally blocked, and the sequence of 14 proteins could not be determined, whereas 36 proteins were N-terminally and one was internally sequenced. Ozone caused drastic reductions in the major leaf photosynthetic proteins, including the abundantly present ribulose-1, 5-bisphosphate carboxylase/oxygenase, and induction of various defense/stress related proteins. Most prominent change in leaves, within 24 h post-treatment with ozone, was the induced accumulation of a pathogenesis related (PR) class 5 protein, three PR 10 class proteins, ascorbate peroxidase(s), superoxide dismutase, calcium-binding protein, calreticulin, a novel ATP-dependent CLP protease, and an unknown protein. Present results demonstrate the highly damaging effect of ozone on rice seedlings at the level of the proteome.

Air Pollutants↗
PubMed2006

Identification of pathogenesis-related ESTs in the crucifer downy mildew oomycete Hyaloperonospora parasitica by high-throughput differential display analysis of distinct phenotypic interactions with Brassica oleracea.

Crucifer downy mildew is caused by the obligatory biotrophic oomycete Hyaloperonospora parasitica (formerly Peronospora parasitica). So far, isolates infecting Arabidopsis thaliana have proven to be non-pathogenic on other crucifers and, despite its unequivocal merit as a research model, the pathosystem A. thaliana-H. parasitica by itself will not provide all the answers onto crucifer downy mildew genetics and biology. In this report, we present the development of a differential display (DD)-based strategy, suitable for high-throughput analysis of expressed sequence tags (ESTs) in plant-pathogen interactions, in this work applied to the analysis of the pathosystem Brassica oleracea-H. parasitica interaction transcriptome. Our purpose was the mining for pathogen-specific ESTs that can be used in future research for virulence factors and Avr genes. A total of 743 specific cDNAs showing differential expression in B. oleracea seedlings infected with H. parasitica, as opposed to healthy seedlings, were isolated by DD-PCR. We found 21 exclusively H. parasitica cDNAs from 433 sequenced DD clones, 18 encoding for potential new genes. Our results reinforce the abilities of DD-PCR for differential screening of pathosystems transcriptomes, leading to the finding of more new potential genes than the previously used techniques. Both the improved DD-based methodology and the graphical representations based on Venn diagrams from polyominoes are appropriate for large-scale analysis of multiple interaction transcriptomes. The obtained data are also innovative since this is the first approach to study the interaction of H. parasitica with its natural host.

Base Sequence↗
PubMed2006

Oligonucleotide-based analysis of differentially expressed genes in hippocampus of transgenic mice expressing NSE-controlled APPsw.

The complexity of Alzheimer's disease (AD) has made it difficult to examine its underlying mechanisms. A gene microarray offers a solution to the complexity through parallel analysis of most of the genes expressed in the hippocampal tissues from AD-transgenic and age-matched control littermates. This study examined the potential effect of APPsw over-expression on the modulation of genes for AD. To accomplish this, an oligonucleotide array was used with the large-scale screening of the hippocampus mRNA from 12-month-old APPsw-transgenic and control mice. There was a total of 116 differentially expressed genes, 59 up-regulated and 57 down-regulated, in the hippocampal region of the transgenic mice compared with the control mice. Initially, two of each of the down-regulated (Xlr3b and Mup3) and up-regulated genes (Serpina9 and Ccr6) were chosen for further investigation if the magnitude of change in these genes on the oligonucleotide array would correspond to those in the RT-PCR analysis from APPsw-transgenic mice. We also found that the changes in the differentially expressed genes are reliable. Thus, these genes might associate with AD neuropathology in neurodegenerative process of AD, although relevance of long lists altered genes should be evaluated in a future study.

Alzheimer Disease↗
PubMed2005

Differential expression analysis of MIM (MTSS1) splice variants and a functional role of MIM in prostate cancer cell biology.

We previously identified MIM-A (missing in metastasis, MTSS1) by differential display techniques as missing in invasive, metastatic bladder cancer cell lines and suggested that MIM-A is a novel putative metastasis suppressor gene. Characterization of the MIM gene revealed a WH2 (Wiskott-Aldrich syndrome protein homology 2) domain in the C-terminus that is known to bind actin monomers and regulate organization of the actin cytoskeleton. Here, we further describe two alternatively splice variants of MIM-A, called MIM(12del) and MIM-B, which share > 50% amino acid sequence homology with MIM-A in the C-terminal domain. We show that expression of all three transcripts is down-regulated in prostate cancer cell lines and tumor samples from patients. In addition, we generated stably-transfected PC-3 cells overexpressing MIM-A to evaluate the importance of MIM-A in prostate cancer biology. The initial experiments show that expression of MIM decreased the number of actin filaments and was associated with a decrease in the G:F actin ratio. Overexpression of MIM-A had no effect on PC-3 cell adhesion to extracellular matrices, as well as no effect on PC-3 motility. Further, overexpression of MIM-A reduced the rate of PC-3 cell proliferation. These results support the hypothesis that MIM-A is an actin-binding protein and implicate a role of MIM-A in the regulation of cellular proliferation. These data suggest that the reduction of MIM-A gene expression in prostate cancer and other cancers may contribute to tumor growth and development, as well as metastasis.

Actins↗
PubMed2005

Microarray analysis detects differentially expressed genes in the pharyngeal region of mice lacking Tbx1.

22q11-deletion (DiGeorge/velocardiofacial) syndrome (22q11DS) is modeled by mutation of murine transcription factor Tbx1. As part of efforts to identify transcriptional targets of Tbx1, we analyzed the transcriptome of the pharyngeal region of Df1/+;Tbx1+/- embryos at 9.5 days of embryonic development using two independent microarray platforms. In this model, embryos are null for Tbx1, with hemizygosity of genes in cis with Tbx1 on one chromosome providing a positive control for array sensitivity. Reduced mRNA levels of genes deleted from Df1 were detected on both platforms. Expression level filtering and statistical analysis identified several genes that were consistently differentially expressed between mutant and wild type embryos. Real-time quantitative PCR and in situ hybridization validated diminished expression of Pax9 and Gcm2, genes known to be required for normal thymus and parathyroid gland morphogenesis, whereas Pax1, Hoxa3, Eya1, and Foxn1, which are similarly required, were not down-regulated. Gbx2, a gene required for normal arch artery development, was down-regulated specifically in the pharyngeal endoderm and the posterior part of pharyngeal arch 1, and is a potential point of cross talk between the Tbx1 and Fgf8 controlled pathways. These experiments highlight which genes and pathways potentially affected by lack of Tbx1, and whose role may be explored further by testing for epistasis using mouse mutants.

Animals↗
PubMed2005

Analysis of differentially expressed transcripts from planthopper-infested wild rice (Oryza minuta).

A subtracted library was constructed from planthopper-infested wild rice (Oryza minuta) by suppression subtractive hybridization in combination with mirror orientation selection. To screen the differentially expressed transcripts in the library, we applied a cDNA microarray containing 960 random clones in a reverse Northern blot analysis using cDNA probes prepared from the mRNAs of control and planthopper-infested samples. On the basis of the signal intensities and expression ratios obtained from experiments performed in triplicate, we selected 383 clones. The elevated expression levels and overall profiles over time were verified by Northern blot analysis. Although Southern blot analysis showed similar copy numbers of the screened genes in O. minuta and O. sativa, it also revealed that the expression profiles had a different pattern. Functional categorization placed the identified transcripts in the categories of subcellular localization, metabolism, and protein fate. The presence of these expressed sequence tags implies that resistance of O. minuta to insect infestation can be achieved not only by an elevated expression of defense-related genes but also by enhanced metabolic activities.

Animals↗