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BEN/DM-GRASP/SC1 expression during mouse facial development: differential expression and regulation in molars and incisors.

The cell adhesion molecule BEN/DM-GRASP/SC1 is expressed in a variety of tissues during embryogenesis. Here, we studied the expression pattern of BEN/DM-GRASP/SC1 in different organs involved in facial mouse development, especially in the developing teeth. BEN/DM-GRASP/SC1 was expressed in nose, whisker, gland, and tongue epithelia, as well as in myogenic mesenchyme. In molars, BEN/DM-GRASP/SC1 was firstly expressed in the condensed mesenchyme and thereafter expression was confined to mesenchymal cells of the dental follicle. In contrast, in incisors, transient BEN/DM-GRASP/SC1 expression was restricted to epithelium. In tissue recombination experiments, BEN/DM-GRASP/SC1 expression in mesenchyme was activated by molar, but not incisor epithelium.

Activated-Leukocyte Cell Adhesion Molecule↗

Differential expression of two cathepsin Es during metamorphosis-associated remodeling of the larval to adult type epithelium in Xenopus stomach.

Cathepsin E (CE) was purified from the foregut of Xenopus laevis tadpoles as a mature dimeric form. The purified enzyme was a typical CE among aspartic proteinases with respect to pH dependence of proteolytic activity, susceptibility to pepstatin, and having N-linked high-mannose type oligosaccharide chains. We isolated two cDNAs for the CE (CE1 and CE2) from adult stomach. The amino acid sequence of the N-terminal region of the purified CE coincided with the corresponding sequence predicted from CE1. Northern blot analysis and in situ hybridization were performed. The CE1 mRNA was highly expressed in surface mucous cells and gland cells constituting the larval epithelium of the foregut of pro-metamorphic tadpoles. As metamorphosis began and progressed, CE1 mRNA drastically decreased in amount, and subsequently both CE1 and CE2 mRNAs gradually increased. The increase in CE2 mRNA was detected shortly after the increase in CE1 mRNA. The decrease in CE1 expression correlated with degeneration of the larval type epithelium, while the increases in both CE1 and CE2 expression correlated with formation of the adult type epithelium. Thus, cathepsin E gene expression was differentially regulated during metamorphosis-associated remodeling of the larval to adult type epithelium in stomach.

Amino Acid Sequence↗

Induced expression of NMDAR2 proteins and differential expression of NMDAR1 splice variants in dysplastic neurons of human epileptic neocortex.

Immunocytochemistry was used to study the expressions of glutamate receptor subunit proteins for NMDAR2A/B, NMDAR1 splice variants, and AMPA Glu-R2/3 in human brain resected for intractable epilepsy associated with cortical dysplasia. NMDAR2A/B intensely labeled dysplastic neurons showing staining in both the cell bodies and dendritic profiles. However, nondysplastic neurons were not immunoreactive to NMDAR2A/B. The antibody selective to NMDAR1 splice variants of NR1-1a. -1b, -2a, and -2b labeled dysplastic neurons, but few nondysplastic neurons. In contrast, the antibody to splice variants of NR1-1a, -1b, 2a, -2b, -3a, -3b, -4a, and -4b labeled both dysplastic and nondysplastic neurons. The different labeling patterns by these two antibodies indicate that variants of NMDAR1-3a, -3b, -4a, and -4b are present in nondysplastic neurons. Both dysplastic neurons and nondysplastic neurons were immunoreactive to AMPA GluR2/3, but denser immunoreactivity was observed in dysplastic neurons. We also found that the locations of dysplastic neurons labeled by NMDAR2A/B were related to focal epileptic EEG seizure onsets or spiking and to focal behavioral seizure types. Our results suggest that there is hyperexcitability of dysplastic cortical regions, at least in part, from the presence of NMDAR2 subunits and selectively expressed NMDAR1 splice variants in dysplastic neurons.

Adolescent↗

Differential expression of Pmp10 in cell culture infected with Chlamydia pneumoniae CWL029.

The complete genome of Chlamydia pneumoniae contains a total of 21 genes encoding polymorphic membrane proteins (Pmp). From this large Pmp family three genes, pmp8, pmp10 and pmp11, were cloned and antibodies against recombinant full-length Pmp proteins were produced. Indirect immunofluorescence microscopy of HEp-2 cells infected with C. pneumoniae CWL029 was performed with the Pmp antibodies in combination with a Chlamydia-specific anti-lipopolysaccharide (LPS) antibody. This double staining technique clearly showed that expression of Pmp10 was differential. Additional double staining with monoclonal antibodies to the surface of C. pneumoniae elementary bodies and the anti-LPS antibody resulted in identification of seven monoclonal antibodies that reacted identically to the Pmp10 antibody indicating that Pmp10 is an immunodominant protein. Finally, the molecular mechanism responsible for differential expression is suggested to be variation in the guanine residues in the polyG tract of pmp10.

Antibodies, Monoclonal↗

Expression of tumor-associated differentially expressed Gene-14 (TADG-14/KLK8) and its protein hK8 in uterine endometria and endometrial carcinomas.

To clarify the biological behavior of TADG-14/KLK8, we investigated TADG-14/KLK8 mRNA by semiquantitative RT-PCR and hK8 expression by immunohistochemistry using 37 normal endometria and 44 endometrial carcinoma tissues. TADG-14/KLK8 mRNA expression levels were significantly higher in proliferative compared to secretory phase endometria (p = 0.0143). Levels of TADG-14/KLK8 mRNA expression correlated with hK8 protein levels. hK8 was detected in 73.3% (11/15) of endometria with a significantly higher detection rate in the proliferative compared to secretory and atrophic phase endometria (p = 0.0002). High expression of hK8 was found in 61.4% of endometrial carcinomas compared to 35.1% of endometrial tissue samples (p = 0.0187). hK8 expression was significantly higher in stage I (p = 0.0433, 0.0038) and grade 1/2 (G1/2) of the tumors (p = 0.0195, 0.0044). We suggest that expression of TADG-14/KLK8may be regulated by sex steroid hormones in endometria. Our results indicate that elevated TADG-14/KLK8 expression is an early event in endometrial carcinogenesis, and may potentially serve as a useful early biomarker for the detection of endometrial carcinomas in menopausal women.

Biomarkers, Tumor↗

Differential expression of gap junction proteins in the canine sinus node.

Electrical coupling of pacemaker cells at gap junctions appears to play an important role in sinus node function. Although the major cardiac gap junction protein, connexin43 (Cx43), is expressed abundantly in atrial and ventricular muscle, its expression in the sinus node has been a subject of controversy. The objectives of the present study were to determine whether Cx43 is expressed by sinus node myocytes, to characterize the spectrum of connexin expression phenotypes in sinus node pacemaker cells, and to define the spatial distribution of different connexin phenotypes in the intact sinus node. To fulfill these objectives, we performed high-resolution immunohistochemical analysis of disaggregated adult canine sinus node preparations. Using enhanced tissue preservation and antigen retrieval techniques, we also performed immunohistochemical studies on sections of intact canine sinus node tissue. Analysis of disaggregated sinus node preparations revealed three populations of pacemaker cells distinguished on the basis of connexin immunohistochemical phenotype: approximately 55% of cells expressed only connexin40 (Cx40); 30% to 35% of cells expressed Cx43, connexin45 (Cx45), and Cx40; and the remaining cells had no detectable connexin expression. In immunostained sections of intact sinus node, Cx43- and Cx45-positive cells were limited in their distribution and were observed in discrete bundles that appeared to abut atrial myocytes. In contrast, Cx40 immunoreactive signal was widely distributed in the sinus node region. These results indicate that subsets of pacemaker cells express distinct connexin phenotypes. Differential expression of connexins could create regions within the sinus node with different conduction properties, thereby contributing to the nonuniform conduction properties seen in this tissue.

Animals↗

Differential expression and regulation of the vascular endothelial growth factor receptors neuropilin-1 and neuropilin-2 in rat uterus.

Vascular endothelial growth factor (VEGF) is a potent modulator of vascular remodeling and angiogenesis in the uterus. Recently, neuropilins (Npn), semaphorin receptors associated with neuronal guidance, were demonstrated to bind VEGF isoforms with high affinity, facilitating VEGF(165) binding to the tyrosine kinase receptor VEGFR2. The current studies examined rat uterus neuropilin expression and regulation. Npn-1 and Npn-2 transcripts and 135-kDa proteins were observed in uterine extracts. Both uterine vascular endothelial cells and glandular epithelium expressed Npn-1 immunoreactivity, whereas Npn-2 was restricted to the glandular epithelium. In hormone-replaced ovariectomized animals, progesterone increased uterine 6.5-kb Npn-1 messenger RNA (mRNA) expression approximately 2-fold compared with that in tissues from ovariectomized controls. 17ss-Estradiol alone had no effect, but blunted the progesterone response; by contrast, Npn-2 mRNA expression was decreased by estrogen. VEGFR2 mRNA was coregulated with Npn-1. Consistent with these results, Npn-1 mRNA expression was augmented nearly 7- and 4-fold at metestrus and diestrus, respectively, during periods of high progesterone; Npn-2 mRNA expression was not significantly altered during the estrous cycle. The regulated expression and differential localization of neuropilins in the rat uterus suggest that these receptors may participate in hormonally regulated changes occurring throughout the female reproductive cycle.

Animals↗

Differential expression of somatostatin receptor subtypes in human peripheral blood mononuclear cell subsets.

BACKGROUND: Somatostatin (SS)-binding sites have been demonstrated in human lymphoid tissues and peripheral blood cells. However, not much is known with respect to the SS receptor subtype (sst) expression pattern and the expression of SS itself in the immune system. OBJECTIVE: The aim of this study was to evaluate the mRNA expression of the five known sst (sst(1-5)) in peripheral blood mononuclear cell (sub)populations. Moreover, the expression of the mRNAs encoding SS and the SS-like peptide cortistatin (CST) in immune cell subsets was studied. METHODS: RT-PCR and quantitative PCR were performed to evaluate sst, SS and CST mRNA expression in cells in the basal or activated state. Fluorescence-activated cell sorter (FACS) analysis using fluorescent SS was performed to visualize sst protein on cell membranes. RESULTS: B- and T-lymphocytes selectively expressed sst(3) mRNA. sst(3) expression in B-lymphocytes was significantly lower compared with T-lymphocytes. Unstimulated, freshly isolated monocytes did not express any sst mRNA. Upon activation, monocytes selectively expressed sst(2) mRNA, whereas T-lymphocyte activation upregulated sst(3) expression. sst(2) mRNA expression on monocytes was confirmed by FACS analysis. B- and T-lymphocytes did not express SS mRNA, while both cell types expressed CST mRNA. CST mRNA expression was downregulated following T-lymphocyte activation. CONCLUSION: We demonstrate for the first time unequivocally that human peripheral blood B- and T-lymphocytes selectively express sst(3), whereas monocytes do not express sst. However, upon activation, monocytes are induced to express sst(2A). No expression of SS mRNA was detected in any cell type, whereas all cell types expressed CST mRNA. The differential expression of sst and CST mRNA in lymphocytes and monocytes suggests a functional significance for the CST-sst interaction in immune cells, but further studies should be performed to evaluate the significance of sst and CST in these cells.

B-Lymphocytes↗

Identification of differentially expressed mRNA transcripts in drug-resistant versus parental human melanoma cell lines.

BACKGROUND: Malignant melanoma resistance to chemotherapy remains a major limitation to treatment. Our aim was to identify genes associated with drug resistance, in order to better understand the molecular events underlying the drug-resistant phenotype. MATERIALS AND METHODS: A human melanoma cell line and its drug-resistant variants obtained by selection with MNNG or 6-thioguanine were used. Alterations in gene expression were characterized by differential display reverse transcription-polymerase chain reaction (DDRT-PCR). Prominent mRNA fragments present in selected variants and not in the parental cells were identified and characterized by cloning and sequencing. Differential expression was confirmed by real-time RT-PCR. RESULTS: Three functionally distinct transcriptional products were demonstrated: the chaperonin subunit TCP 1-zeta-6A (CCT6A), the hyaluronan receptor CD44 and LPPR-2, the lipid phosphate phosphatase-related protein type-2. CONCLUSION: Genes with altered expression were identified in drug-resistant variants. The identified molecules may provide new insights into the molecular basis for melanoma resistance to chemotherapy.

Base Sequence↗

Differential expression of Mr 70,000 heat shock protein in normal, premalignant, and malignant human uterine cervix.

Transformed cells differ from normal cells in their pattern of heat shock protein expression. Here, we report differential expression of a Mr 70,000 heat shock protein (HSP70) in squamous cell carcinomas of the uterine cervix compared to normal or premalignant uterine cervix. Expression of HSP70 was measured using a mouse mAb against HSP70 by an ELISA. A significant increase in the level of expression of HSP70 was observed in fresh tumor cells as compared to normal or dysplastic ones (P < 0.001). The induction of HSP70 in tumor cells subjected to hyperthermia was less than that in normal or premalignant cells. Strong cytoplasmic and nuclear immunostaining was observed in cancerous tissues using anti-HSP70 mAb, biotinylated rabbit antimouse immunoglobulins, avidin combined in vitro with biotinylated horseradish peroxidase, and diaminobenzidine tetrachloride. HSP70 immunostaining was not detected in normal or dysplastic tissue sections. The results were confirmed by immunoprecipitation using anti-HSP70 mAb. These results are the first demonstration of overexpression of HSP70 in squamous cell carcinomas of the uterine cervix as compared to that in dysplastic or normal uterine cervix cells. We conclude that tumor cells differ from normal cells in their pattern of HSP70 expression, and increased levels of HSP70 correlate with an increase in tumor size.

Adult↗

Differential expression of members of the E2F family of transcription factors in rodent testes.

BACKGROUND: The E2F family of transcription factors is required for the activation or repression of differentially expressed gene programs during the cell cycle in normal and abnormal development of tissues. We previously determined that members of the retinoblastoma protein family that interacts with the E2F family are differentially expressed and localized in almost all the different cell types and tissues of the testis and in response to known endocrine disruptors. In this study, the cell-specific and stage-specific expression of members of the E2F proteins has been elucidated. METHODS: We used immunohistochemical (IHC) analysis of tissue sections and Western blot analysis of proteins, from whole testis and microdissected stages of seminiferous tubules to study the differential expression of the E2F proteins. RESULTS: For most of the five E2F family members studied, the localizations appear conserved in the two most commonly studied rodent models, mice and rats, with some notable differences. Comparisons between wild type and E2F-1 knockout mice revealed that the level of E2F-1 protein is stage-specific and most abundant in leptotene to early pachytene spermatocytes of stages IX to XI of mouse while strong staining of E2F-1 in some cells close to the basal lamina of rat tubules suggest that it may also be expressed in undifferentiated spermatogonia. The age-dependent development of a Sertoli-cell-only phenotype in seminiferous tubules of E2F-1 knockout males corroborates this, and indicates that E2F-1 is required for spermatogonial stem cell renewal. Interestingly, E2F-3 appears in both terminally differentiated Sertoli cells, as well as spermatogonial cells in the differentiative pathway, while the remaining member of the activating E2Fs, E2F-2 is most concentrated in spermatocytes of mid to late prophase of meiosis. Comparisons between wildtype and E2F-4 knockout mice demonstrated that the level of E2F-4 protein displays a distinct profile of stage-specificity compared to E2F-1, which is probably related to its prevalence and role in Sertoli cells. IHC of rat testis indicates that localization of E2F-5 is distinct from that of E2F-4 and overlaps those of E2F-1 and E2F-2. CONCLUSION: The E2F-1 represents the subfamily of transcription factors required during stages of DNA replication and gene expression for development of germ cells and the E2F-4 represents the subfamily of transcription factors that help maintain gene expression for a terminally differentiated state within the testis.

Animals↗

cDNA microarray in isolation of novel differentially expressed genes related to human glioma and clone of a novel full-length gene.

BACKGROUND: This investigation was undertaken to obtain differentially expressed genes related to human glioma using cDNA microarray and the characterization of one novel full-length gene. METHODS: Total RNA was extracted from human glioma tissues and normal brain tissues, and mRNA was used to make probes. After hybridization and washing, the results were scanned using a computer system. The gene named 681F05 clone was an expressed gene to human glioma through four-time hybridization and scanning. Subsequently northern blot analysis was performed by northern blot, 5'RACE and bioinformatics. RESULTS: Fifteen differentially expressed genes to human glioma were obtained through four-time hybridization and scanning. Northern blot analysis confirmed that 681F05 clone was low-expressed in human brain tissues and over-expressed in human glioma tissues. The analysis of BLASTn and BLASTx showed that 681F05 clone is two cDNA clones encoding two novel proteins that are highly identified to the cyclophilin isoform 10 of C. Elgans, respectively. Sequence analysis revealed the two cDNA clones are two different splicing variants of a novel cycophilin-like gene (PPIL3a and PPIL3b). CONCLUSIONS: cDNA microarray technology can be successfully used to identify differentially expressed genes. The novel full-length gene of human PPIL3 may be correlated with the formation of human glioma.

Amino Acid Sequence↗

Genes differentially expressed in human lung fibroblast cells transformed by glycidyl methacrylate.

OBJECTIVE: To define the differences in gene expression patterns between glycidyl methacrylate (GMA)-transformed human lung fibroblast cells (2BS cells) and controls. METHODS: The mRNA differential display polymerase chain reaction (DD-PCR) technique was used. cDNAs were synthesized by reverse transcription and amplified by PCR using 30 primer combinations. After being screened by dot blot analysis, differentially expressed cDNAs were cloned, sequenced and confirmed by Northern blot analysis. RESULTS: Eighteen differentially expressed cDNAs were cloned and sequenced, of which 17 were highly homologous to known genes (homology = 89%-100%) and one was an unknown gene. Northern blot analysis confirmed that eight genes encoding human zinc finger protein 217 (ZNF217), mixed-lineage kinase 3 (MLK-3), ribosomal protein (RP) L15, RPL41, RPS 16, TBX3, stanniocalcin 2 (STC2) and mouse ubiquitin conjugating enzyme (UBC), respectively, were up-regulated, and three genes including human transforming growth factor beta inducible gene (Betaig-h3), alpha-1,2-mannosidase 1A2 (MAN 1A2) gene and an unknown gene were down-regulated in the GMA-transformed cells. CONCLUSION: Analysis of the potential function of these genes suggest that they may be possibly linked to a variety of cellular processes such as transcription, signal transduction, protein synthesis and growth, and that their differential expression could contribute to the GMA-induced neoplastic transformation.

Air Pollutants, Occupational↗

Differential expression of individual suppressor tRNA(Trp) gene gene family members in vitro and in vivo in the nematode Caenorhabditis elegans.

Eight different amber suppressor tRNA (suptRNA) mutations in the nematode Caenorhabditis elegans have been isolated; all are derived from members of the tRNA(Trp) gene family (K. Kondo, B. Makovec, R. H. Waterston, and J. Hodgkin, J. Mol. Biol. 215:7-19, 1990). Genetic assays of suppressor activity suggested that individual tRNA genes were differentially expressed, probably in a tissue- or developmental stage-specific manner. We have now examined the expression of representative members of this gene family both in vitro, using transcription in embryonic cell extracts, and in vivo, by assaying suppression of an amber-mutated lacZ reporter gene in animals carrying different suptRNA mutations. Individual wild-type tRNA(Trp) genes and their amber-suppressing counterparts appear to be transcribed and processed identically in vitro, suggesting that the behavior of suptRNAs should reflect wild-type tRNA expression. The levels of transcription of different suptRNA genes closely parallel the extent of genetic suppression in vivo. The results suggest that differential expression of tRNA genes is most likely at the transcriptional rather than the posttranscriptional level and that 5' flanking sequences play a role in vitro, and probably in vivo as well. Using suppression of a lacZ(Am) reporter gene as a more direct assay of suptRNA activity in individual cell types, we have again observed differential expression which correlates with genetic and in vitro transcription results. This provides a model system to more extensively study the basis for differential expression of this tRNA gene family.

Alleles↗

[A study of differential expression of HLA class II antigens in human lymphocytes].

Flow cytometric data of human lymphocyte subset in routine samples of patients have often shown differential expression of HLA Class II (Class II) antigens on T and B lymphocytes in our laboratory. I2 and I3 (both Coulter) were used as monoclonal antibodies for detection of Class II antigens. I2 was able to recognize HLA-DR except for haplotype of HLA-DR7, whose frequency was 1.8% in Japanese. Differential expression of Class II antigens of B lymphocytes in the present study was noticed in approximately 10% of our samples in frequency. In all of the 7 follow-up cases with renal transplantation, decrease of I2 expression on B lymphocytes was consistently noticed. On the other hand, increase of Class II expression on T lymphocytes was often recognized in autoimmune diseases, compared with those of diseases. Two of seven cases of hematologic neoplastic disorders were B cell type of malignant lymphoma and failed to express I2 despite the presence of I3 in terms of differential expression of Class II. In conclusion, the present study indicates that acquired decrease of HLA-DR expression on B lymphocytes was recognized in all of the studied patients with renal transplantation and that lack or decrease of HLA-DR expression on neoplastic B lymphocytes might be one of the characteristics of monoclonal growth.

Adolescent↗

Nonradioactive detection of differentially expressed genes using complex RNA or DNA hybridization probes.

The analysis of differential gene expression has become increasingly important in recent years. Typically, differentially expressed genes are identified in a primary screening procedure, yielding candidate genes whose differential expression has to be verified. We provide a highly sensitive, efficient and nonradioactive differential screening procedure to analyze numerous candidate genes in a single step. This comprises labeling of poly(A)+ RNA of the cell types analyzed with DIG Chem-Link and differential hybridization to the candidate genes fixed on dot blots. DIG Chem-Link allows, to our knowledge, for the first time efficient and direct nonradioactive labeling of RNA in vitro. Advantages of this method include extremely short exposure times and the feasibility to re-use the probes after prolonged storage. Using this procedure, we isolated several genes that are differentially expressed in maturing Langerhans cells.

Animals↗

Differential expression of egr-1 in osteoarthritic compared to normal adult human articular cartilage.

OBJECTIVE: The purpose of this study was to identify genes that are differentially expressed in normal versus osteoarthritic human articular cartilage as either potential novel therapeutic targets or diagnostic markers of this disease. DESIGN: mRNA was isolated from histologically normal and osteoarthritic adult human articular cartilage. The Differential Display technique was employed which identified differentially expressed genes in the normal and diseased tissue. Northern and reverse Northern hybridization were used to confirm the gene expression pattern. Immunohistochemistry and in-situ hybridization were used to localize expression of Egr-1 protein and mRNA respectively in cartilage. RESULTS: A transcription factor, early growth response protein-1 (Egr-1) was found to be down-regulated more than six-fold in multiple human OA cartilage samples when compared to normal tissue. Immunohistochemistry indicated that Egr-1 was expressed throughout normal adult cartilage, in deep-, mid- and superficial-zones. In contrast, in OA cartilage there was expression of Egr-1 mRNA and protein only in the chondrocytes undergoing cloning. CONCLUSIONS: Egr-1 is differentially expressed in OA versus normal cartilage and because of its role in transcriptional activation and repression and regulation of proliferation, differentiation and apoptosis, Egr-1 may play an important role in the pathogenesis of OA. Up-regulation of Egr-1 may therefore provide a novel therapeutic approach for either the prevention or treatment of OA.

Adult↗

Identification of six differentially expressed genes in response to copper exposure in the aquatic plant Lemna gibba (Duckweed).

Aquatic plants are susceptible to metal pollution and provide an entry point for metals, such as copper, into the aquatic biosphere. Exposure of the aquatic plant Lemna gibba to copper has been associated with the production of reactive oxygen species (ROS) and oxidative damage, caused in large part by the ability of this metal to redox cycle. In particular, copper-mediated production of ROS, a known group of signaling molecules, triggers numerous defense responses in L. gibba. Therefore, the objective of the present study was to examine to what extent acute copper exposure alters gene expression. First, the kinetics of copper uptake was assessed to determine if assimilation occurred within the short exposures needed to induce gene expression. Subsequently, using differential display polymerase chain reaction, we identified six genes with expressions that were putatively altered in response to copper. Differential expression was confirmed by northern hybridization analysis and showed that copper causes an accumulation of transcripts that encode for callose synthase, heat shock protein 90, serine decarboxylase, and the biotin carboxylase subunit of acetyl-coenzyme A carboxylase. Conversely, copper caused a decline in transcript levels for genes encoding the HAP5 subunit of the heme-activated protein (HAP) transcription factor in addition to the chloroplast nucleoid DNA-binding protein CND41. Interestingly, the expressions of these genes are sensitive to cellular ROS levels. We believe that these gene products provide valuable information regarding the molecular mechanisms of copper toxicity.

Amino Acid Sequence↗