The role of oocyte transcription, the 5'UTR, and translation repression and derepression in Drosophila gurken mRNA and protein localization.
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The establishment of the major body axes of the Drosophila egg and future embryo requires strict regulation of gurken mRNA and protein localization. Here, we show that grk mRNA and protein localization is dependent on synthesis of grk transcripts in the oocyte nucleus and on RNA localization elements in the 5' portion of the transcript. We also show that gurken mRNA and protein localization is dependent on region-specific translation of gurken transcripts and identify K10 as a probable negative regulator of gurken translation. (+info)
Mechanisms related to [18F]fluorodeoxyglucose uptake of human colon cancers transplanted in nude mice.
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[18F]Fluorodeoxyglucose ([18F]FDG), a glucose analogue, has been widely used for tumor imaging. To investigate the mechanisms related to [18F]FDG uptake by tumors, an experiment involving nude mice was performed. METHODS: Human colon cancer cell lines SNU-C2A, SNU-C4 and SNU-C5 were transplanted to nude mice. Using immunohistochemical staining and Western blot, the expression of glucose transporter (Glut) isoforms (Glut-1 through -5) in xenografted tumors was analyzed. For the analysis of messenger ribonucleic acid (mRNA) expression, reverse-transcription polymerase chain reaction and Northern blot were used and the enzyme activity of hexokinase in cancer tissues was measured by continuous spectrophotometric rate determination. RESULTS: [18F]FDG uptake in SNU-C4 and SNU-C5 cells was higher than in normal colon cells. Among these cells and xenografted tumors, SNU-C5 showed the highest level of [18F]FDG uptake, followed by SNU-C4 and SNU-C2A. An immunostaining experiment showed intense staining of Glut-1 in SNU-C5 tumors but somewhat faint staining in SNU-C4. SNU-C5 tumors also showed positive staining with Glut-3, although this was not the case with SNU-C2A and SNU-C4. Western blot analysis showed the expression of Glut-1 and Glut-3 in all tumors. Experiments involving Northern blot analysis and reverse-transcription polymerase chain reaction confirmed the overexpression of Glut-1 mRNA in all tumors, with the highest level in SNU-C5. The level of Glut-3 mRNA was also elevated in SNU-C5 tumors but not in SNU-C2A and SNU-C4. The enzyme activity of hexokinase did not vary among different tumors. CONCLUSION: Gluts, especially Glut-1, are responsible for [18F]FDG uptake in a nude mouse model of colon cancer rather than hexokinase activity. Increased numbers of glucose transporters at the plasma membrane of cancer cells is attributed to an increased level of transcripts of glucose transporter genes and may be a cause of increased [18F]FDG uptake, at least in colon cancer tumors. (+info)
Expression of B7 costimulatory molecules by salivary gland epithelial cells in patients with Sjogren's syndrome.
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OBJECTIVE: To investigate the expression of B7 costimulatory molecules in the lymphoepithelial lesions of salivary gland (SG) biopsy tissues and in SG epithelial cell lines derived from patients with Sjogren's syndrome (SS). METHODS: B7.1 and B7.2 protein expression was studied by immunohistochemistry in minor SGs obtained from 11 patients with SS and 10 disease control patients with nonspecific sialadenitis and in cultured SG epithelial cell lines obtained from minor SGs from 15 SS patients and 15 control patients. B7.1 and B7.2 messenger RNA (mRNA) expression by SG epithelial cell lines was examined by reverse transcription-polymerase chain reaction (RT-PCR). RESULTS: In biopsy tissues from SS patients, but not control patients, ductal and acinar epithelial cells showed increased expression of both B7.1 and B7.2. Intense spontaneous B7.1 protein expression (as well as HLA-ABC, but not B7.2 or HLA-DR) was also found in 73% of SG epithelial cell lines from SS patients versus 13% of those from control patients (P < 0.01). Interferon-y treatment induced, or up-regulated, B7.1, B7.2, and HLA-DR expression in all SG epithelial cell lines tested. B7.1 and B7.2 expression by SG epithelial cell lines was also verified at the mRNA level by RT-PCR. CONCLUSION: Human SG epithelia are intrinsically capable of expressing B7 proteins upon activation. In SS patients, the expression of B7 molecules by SG epithelial tissues and by SG epithelial cell lines indicates the activated status of SG epithelial cells in this disorder and, possibly, their capacity for presenting antigens to T cells. (+info)
Expression of both P1 and P2 purine receptor genes by human articular chondrocytes and profile of ligand-mediated prostaglandin E2 release.
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OBJECTIVE: To assess the expression and function of purine receptors in articular chondrocytes. METHODS: Reverse transcriptase-polymerase chain reaction (RT-PCR) was used to screen human chondrocyte RNA for expression of P1 and P2 purine receptor subtypes. Purine-stimulated prostaglandin E2 (PGE2) release from chondrocytes, untreated or treated with recombinant human interleukin-1alpha (rHuIL-1alpha), was assessed by radioimmunoassay. RESULTS: RT-PCR demonstrated that human articular chondrocytes transcribe messenger RNA for the P1 receptor subtypes A2a and A2b and the P2 receptor subtype P2Y2, but not for the P1 receptor subtypes A1 and A3. The P1 receptor agonists adenosine and 5'-N-ethylcarboxamidoadenosine did not change PGE2 release from chondrocytes. The P2Y2 agonists ATP and UTP stimulated a small release of PGE2 that was potentiated after pretreatment with rHuIL-1alpha. PGE2 release in response to ATP and UTP cotreatment was not additive, but release in response to coaddition of ATP and bradykinin (BK) or UTP and BK was additive, consistent with ATP and UTP competition for the same receptor site. The potentiation of PGE2 release in response to ATP and UTP after rHuIL-1alpha pretreatment was mimicked by phorbol myristate acetate. CONCLUSION: Human chondrocytes express both P1 and P2 purine receptor subtypes. The function of the P1 receptor subtype is not yet known, but stimulation of the P2Y2 receptor increases IL-1-mediated PGE2 release. (+info)
Overexpression of the multidrug resistance-associated protein (MRP1) in human heavy metal-selected tumor cells.
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Cellular and molecular mechanisms involved in the resistance to cytotoxic heavy metals remain largely to be characterized in mammalian cells. To this end, we have analyzed a metal-resistant variant of the human lung cancer GLC4 cell line that we have selected by a step-wise procedure in potassium antimony tartrate. Antimony-selected cells, termed GLC4/Sb30 cells, poorly accumulated antimony through an enhanced cellular efflux of metal, thus suggesting up-regulation of a membrane export system in these cells. Indeed, GLC4/Sb30 cells were found to display a functional overexpression of the multidrug resistance-associated protein MRP1, a drug export pump, as demonstrated by Western blotting, reverse transcriptase-polymerase chain reaction and calcein accumulation assays. Moreover, MK571, a potent inhibitor of MRP1 activity, was found to markedly down-modulate resistance of GLC4/Sb30 cells to antimony and to decrease cellular export of the metal. Taken together, our data support the conclusion that overexpression of functional MRP1 likely represents one major mechanism by which human cells can escape the cytotoxic effects of heavy metals. (+info)
UCP4, a novel brain-specific mitochondrial protein that reduces membrane potential in mammalian cells.
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Uncoupling proteins (UCPs) are a family of mitochondrial transporter proteins that have been implicated in thermoregulatory heat production and maintenance of the basal metabolic rate. We have identified and partially characterized a novel member of the human uncoupling protein family, termed uncoupling protein-4 (UCP4). Protein sequence analyses showed that UCP4 is most related to UCP3 and possesses features characteristic of mitochondrial transporter proteins. Unlike other known UCPs, UCP4 transcripts are exclusively expressed in both fetal and adult brain tissues. UCP4 maps to human chromosome 6p11.2-q12. Consistent with its potential role as an uncoupling protein, UCP4 is localized to the mitochondria and its ectopic expression in mammalian cells reduces mitochondrial membrane potential. These findings suggest that UCP4 may be involved in thermoregulatory heat production and metabolism in the brain. (+info)
Differential regulation of Bcl-2, AP-1 and NF-kappaB on cardiomyocyte apoptosis during myocardial ischemic stress adaptation.
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Acute ischemia followed by prolonged reperfusion has been shown to induce cardiomyocyte apoptosis. In this report, we demonstrate that myocardial adaptation to ischemia induced by repeated cyclic episodes of short-term ischemia each followed by another short duration of reperfusion reduced cardiomyocyte apoptosis and DNA fragmentation. This was associated with the induction of the expression of Bcl-2 mRNA and translocation and activation of NF-kappaB. Another transcription factor, AP-1, remained unaffected by repeated ischemia and reperfusion, but exhibited significant upregulation by a single episode of 30 min ischemia followed by 2 h of reperfusion. This activation of AP-1 was inhibited by a scavenger of oxygen free radicals, DMTU. Thirty minutes ischemia and 120 min reperfusion downregulated the induction of the expression of Bcl-2 mRNA, but moderately activated NF-kappaB binding activity. This was associated with an increased number of apoptotic cells and DNA fragmentation in cardiomyocytes which were attenuated by DMTU. The results of this study indicate that Bcl-2, AP-1 and NF-kappaB differentially regulate cardiomyocyte apoptosis mediated by acute ischemia and prolonged reperfusion. (+info)
Molecular cloning and characterization of a novel angiopoietin family protein, angiopoietin-3.
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Using homology-based PCR, we have isolated cDNA encoding a novel member (491 amino acids) of the angiopoietin (Ang) family from human adult heart cDNA and have designated it angiopoietin-3 (Ang3). The NH2-terminal and COOH-terminal portions of Ang-3 contain the characteristic coiled-coil domain and fibrinogen-like domain that are conserved in other known Angs. Ang3 has a highly hydrophobic region at the N-terminus (approximately 21 amino acids) that is typical of a signal sequence for protein secretion. Ang3 mRNA is most abundant in adrenal gland, placenta, thyroid gland, heart and small intestine in human adult tissues. Additionally, Ang3 is a secretory protein, but is not a mitogen in endothelial cells. (+info)