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PubMed2005

[Effects of macrophages on the biological behaviors and VEGF receptor mRNA, Hoxb2 mRNA, and integrin alphavbeta3 expressions of vascular endothelial cells].

OBJECTIVE: To explore the mechanism by which macrophages regulate angiogenesis by co-culturing human umbilical vein endothelial cells (ECV-304) with human macrophage cells (U937) stimulated by concanavalin A (ConA). METHODS: Monolayer ECV-304 cells growing to 60% confluence were co-cultured with 1 x 10(5)/ml U937 cells in the presence or absence of ConA (ConA+U937+ECV-304 and U937+ECV-304 groups, respectively), with non-treated and ConA-treated ECV-304 cells serving as the control groups (ECV-304 and ConA+ECV-304 groups, respectively). Forty-eight h later, U937 cells were removed from the cell co-culture for examining changes in DNA synthesis of ECV-304 cells with (3)H-TdR incorporation assay and for cell cycle analysis with flow cytometry. RT-PCR was employed to assess the influence of macrophages stimulated by ConA on the expression of the target genes. With immunofluorescent method, the changes in the expression of integrin receptor alphavbeta3 of ECV-304 were determined. RESULTS: A significant increase in S-phase ECV-304 cells with enhanced DNA synthesis was observed after co-culture of the cells with ConA-stimulated U937 cells (P<0.01), which also resulted in significant up-regulation of the expressions of KDR mRNA (0.879+/-0.003), Hoxb2 mRNA (0.947+/-0.003) and integrin receptor alphavbeta3 (10.26+/-1.73). CONCLUSION: Macrophages can accelerate the proliferation, migration and adhesion of the vascular endothelial cells to the basilar membrane matrix by affecting their cell cycle, DNA synthesis, expression of KDR mRNA, Hoxb2 mRNA and integrin alphavbeta3, so as to modulate the angiogenetic process of the latter cells.

Cell Proliferation↗
PubMed1992

Inhibition of tumor necrosis factor-alpha (TNF-alpha) and interleukin-1 beta (IL-1 beta) messenger RNA (mRNA) expression in HL-60 leukemia cells by pentoxifylline and dexamethasone: dissociation of acivicin-induced TNF-alpha and IL-1 beta mRNA expression from acivicin-induced monocytoid differentiation.

We have previously noted that the glutamine antagonist acivicin (alpha S,5S-alpha-amino-3-chloro-4,5-dihydro-5-isoxazoleacetic acid) induces monocytoid differentiation of freshly isolated human myeloid leukemia cells and cells of the myeloid leukemia cell line HL-60, and that the differentiation is accompanied by increases in expression of tumor necrosis factor-alpha (TNF-alpha) and interleukin-1 beta (IL-1 beta). Because we also showed that TNF-alpha and IL-1 beta can act synergistically to cause monocytoid differentiation of HL-60 cells, we hypothesized that acivicin-induced TNF-alpha and IL-1 beta, in an autocrine manner, caused the differentiation. The purpose of the present study was to determine the causal roles of TNF-alpha and IL-1 beta in the acivicin-induced differentiation of HL-60 cells by the use of dexamethasone (DEX) and pentoxifylline (PTX), two drugs that effectively inhibit expression of TNF-alpha and IL-1 beta. Acivicin caused a monocytoid differentiation of the cells as manifest by diminished cell growth, morphologic maturation of the cells, increased ability to generate hydrogen peroxide in response to acute treatment with phorbol myristate acetate, and increased expression of nonspecific esterase and the surface antigens CD14 and CD11b. Acivicin treatment also caused the cells to have diminished steady-state expression of messenger RNA (mRNA) for c-myc and c-myb, and increased expression of mRNA for TNF-alpha and IL-1 beta. DEX and PTX did not alter cell growth, and did not block the acivicin-induced block in growth. PTX caused a slight increase in nonspecific esterase expression, but DEX had no effect on this, and neither drug diminished the acivicin-induced increase in nonspecific esterase. Although neither drug alone lessened the acivicin enhancement of hydrogen peroxide production, DEX and PTX together reduced this. DEX did not modify the acivicin-induced morphologic maturation of the cells, but PTX alone or PTX with DEX potentiated the acivicin-induced increase in mature cells. Basal CD14 and CD11b expression were slightly reduced by DEX and PTX, but neither drug modified the acivicin-induced increases. DEX and PTX reduced the acivicin-induced increases in TNF-alpha and IL-1 beta mRNA expression, but they had little or no effect on the acivicin-induced decreases in expression of mRNA for c-myc and c-myb. Thus, DEX and PTX effectively block the acivicin-induced expression of TNF-alpha and IL-1 beta, but they have little influence on the acivicin-induced differentiation process.(ABSTRACT TRUNCATED AT 250 WORDS)

Cell Differentiation↗
PubMed1991

Tumor necrosis factor-alpha mRNA accumulation in human myelomonocytic cell lines. Role of transcriptional regulation by DNA sequence motifs and mRNA stabilization.

The cytokine TNF mediates many of the pathologic signs of cachexia, inflammation, and sepsis. The current work describes the regulation of TNF in human myelomonocytic cell lines after PMA stimulation. The cell lines exhibit a low level of constitutive TNF mRNA expression. Within 2 to 4 h of PMA exposure, steady state levels of TNF mRNA are markedly elevated in all myelomonocytic cell lines studied. This rise is due to increased mRNA stability, which increased by almost twofold, and to an overall increase in transcription, which rises by more than sixfold. At the level of the genomic TNF gene, a DNase I hypersensitive site is detected within the TNF promoter between -200 to -100 bp relative to the transcription initiation site. Although absent in nonexpressing erythroleukemia cell lines, the DNase I site is present in uninduced myelomonocytic cell lines and is not changed after PMA induction. The PMA induction of c-fos mRNA correlated well with TNF gene induction; expression of genes encoding other proteins in the AP-1 complex (junB and junD) were also induced by PMA. The nuclear extracts from resting and induced ML-1 cells contain proteins binding specifically to the AP-1, AP-2, and NF kappa B sequence located within the TNF promoter. PMA induction increases the level of a number of specific binding complexes relative to the resting cells. The regulatory mechanisms of the human and murine TNF genes are discussed.

Base Sequence↗
PubMed1987

[Cloning of DNA complementary to mRNA for proopiomelanocortin from the bovine, rat and human hypophysis. Hormonal regulation of proopiomelanocortin mRNA in the rat hypophysis].

Cloning of DNA and complementary mRNA of bovine, rat and human proopiomelanocortin (POMC) was carried out. A structural analysis of the cloned cDNA of POMC was performed. Using restriction fragments of bovine, rat and human POMC cloned cDNA, probes for molecular hybridization based on one-chain bacteriophage M13 were made. Using the dot-hybridization technique with labeled [32P] POMC cDNA, the effect of 17 beta-estradiol and adrenalectomy on the POMC mRNA level in rat hypophysis was studied. The hormone was shown to significantly decrease the POMC mRNA content at a concentration of 4 and 8 micrograms per 100 g of body weight 4 hours after injection. Adrenalectomy caused a 2-3-fold increase in the POMC mRNA level in rat hypophysis after a period of 5-7 days. The possibility of polyhormonal control of POMC genome transcription is discussed.

Adrenalectomy↗
PubMed1985

Molecular cloning of DNA sequences complementary to rat liver glucose-6-phosphate dehydrogenase mRNA. Nutritional regulation of mRNA levels.

The nutritional regulation of rat liver glucose-6-phosphate dehydrogenase was studied using a cloned DNA complementary to glucose-6-phosphate dehydrogenase mRNA. The recombinant cDNA clones were isolated from a double-stranded cDNA library constructed from poly(A+) RNA immunoenriched for glucose-6-phosphate dehydrogenase mRNA. Immunoenrichment was accomplished by adsorption of polysomes with antibodies directed against glucose-6-phosphate dehydrogenase in conjunction with protein A-Sepharose and oligo(dT)-cellulose chromatography. Poly(A+) RNA encoding glucose-6-phosphate dehydrogenase was enriched approximately 20,000-fold using these procedures. Double-stranded cDNA was synthesized from the immunoenriched poly(A+) RNA and inserted into pBR322 using poly(dC)-poly(dG) tailing. Escherichia coli MC1061 was transformed, and colonies were screened for glucose-6-phosphate dehydrogenase cDNA sequences by differential colony hybridization. Plasmid DNA was purified from clones which gave positive signals, and the identity of the glucose-6-phosphate dehydrogenase clones was verified by hybrid-selected translation. A collection of glucose-6-phosphate dehydrogenase cDNA plasmids with overlapping restriction maps was obtained. Northern blot analysis of rat liver poly(A+) RNA using nick-translated, 32P-labeled cDNA inserts revealed that the glucose-6-phosphate dehydrogenase mRNA is 2.3 kilobases in length. RNA blot analysis showed that refeeding fasted rats a high carbohydrate diet results in a 13-fold increase in the amount of hybridizable hepatic glucose-6-phosphate dehydrogenase mRNA which parallels the increase in enzyme activity. These results suggest that the nutritional regulation of hepatic glucose-6-phosphate dehydrogenase occurs at a pretranslational level.

Animals↗
PubMed1983

Rat liver mitochondrial aspartate aminotransferase mRNA: isolation of mRNA by polyacrylamide gel electrophoresis.

In order to obtain an enriched fraction of mRNA for mAAT we applied the following method: preparation of cytoplasmic polysomes; isolation of total RNA; isolation of mRNA by chromatography on oligo(dT)-cellulose column; polyacrylamide gel electrophoresis; extraction of mRNA from the gel. The procedure seems to allow isolation of mRNA enriched fraction for mAAT. An enriched fraction of mRNAs can be useful for the preparation of corresponding cDNAs.

Animals↗
PubMed1978

Detection and relative quantitation of mRNA for creatine kinase isoenzymes in mRNA from myogenic cell cultures and embryonic chicken tissues.

The presence of mRNA coding for creatine kinase M (Mck) and creatine kinase B (B-CK) in RNA from myogenic and fibrogenic cell cultures, embryonic muscle, and embryonic brain tissue was demonstrated by "in vitro" translation in a heterologous cell-free protein-synthesizing system from rabbit reticulocytes. The products were isolated by sensitive immunochemical methods and their identity with isolated M-CK and B-CK was shown by the following criteria: (a) the in vitro synthesized creatine kinases react with the specific antibody against these antigens; (b) the labeled peptides co-migrate with purified creatine kinase on sodium dodecyl sulfate gels in single bands; (c) the labeled peptides form homo- and heterodimers with isolated enzymatically active creatine kinase, thus behaving like authentic creatine kinases. The assay was shown to be reproducible and gave a linear response with increasing amounts of RNA, allowing relative quantitation of mRNA in polysomal RNA for the creatine kinases M and B. MRNA for M-CK was detected in polusomal RAN and total cellular RNA from myogenic cells. It is also present in polysomal RNA from enbryonic muscle and the fraction binding to oligo(dT)-cellulose. mRNA for B-CK could be found in RNA extracted from young myogenic cultures and the fraction of polysomal embryonic brain RNA binding to oligo(dT)-cellulose.

Animals↗
PubMed1994

The mRNA (guanine-7-)methyltransferase domain of the vaccinia virus mRNA capping enzyme. Expression in Escherichia coli and structural and kinetic comparison to the intact capping enzyme.

The mRNA (guanine-7-)methyltransferase active site of the heterodimeric vaccinia virus mRNA capping enzyme was previously localized to the carboxyl-terminal third of the large subunit, D1R, associated with the small subunit, D12L (Cong, P., and Shuman, S. (1992) J. Biol. Chem. 267, 16424-16429; Higman, M. A., Bourgeois, N., and Niles, E. G. (1992) J. Biol. Chem. 267, 16430-16437). A plasmid was constructed which directs the coexpression of the carboxyl terminus of the D1R subunit from amino acids 498 to 844 and the D12L subunit in Escherichia coli. The mRNA (guanine-7-)methyltransferase catalytic activity in the isolated domain was found to be kinetically equivalent to that present in the intact enzyme. Through mobility shift and ultraviolet photolinkage analyses, both domains were shown to bind RNA in a saturable fashion. RNA binding was localized predominantly to the large subunit, but a low level of linkage of RNA to D12L was also observed. A low, but reproducible, level of mRNA (guanine-7-)methyltransferase activity was detected in the isolated D1R498-844 subunit demonstrating that the active site resides solely within the large subunit of the capping enzyme. This activity is enhanced 30- to 50-fold by the association of the D12L subunit.

Base Sequence↗
PubMed1994

Identification of an activity that interacts with the 3'-untranslated region of c-myc mRNA and the role of its target sequence in mediating rapid mRNA degradation.

Single or multiple copies of the pentameric motif, AUUUA, are found in the 3'-untranslated domain of most immediate early mRNAs. A broad body of data links this 5-base element to the selective degradation of these mRNAs. However, AUUUA motifs are neither always necessary nor always sufficient to tag an mRNA for rapid degradation. Here, we describe a cytoplasmic, protease-sensitive factor in Balb/c3T3 cells that interacts with the 3'-untranslated region of c-myc mRNA. The factor recognizes a 39-base uridine-rich domain adjacent to, but distinct from, the c-myc AUUUA motif. In chimeric mRNA constructs, the 39-base binding element cooperates with adjacent sequence material to destabilize beta-globin mRNA.

3T3 Cells↗
PubMed1992

The vaccinia virus mRNA (guanine-N7-)-methyltransferase requires both subunits of the mRNA capping enzyme for activity.

Plasmid vectors capable of expressing the large and small subunits of the vaccinia virus mRNA capping enzyme were constructed and used to transform Escherichia coli. Conditions for the induction of the dimeric enzyme or the individual subunits in a soluble form were identified, and the capping enzyme was purified to near homogeneity. Proteolysis of the capping enzyme in bacteria yields a 60-kDa product shown previously to possess the mRNA triphosphatase and guanyltransferase activities (Shuman, S. (1990) J. Biol. Chem. 265, 11960-11966) was isolated and shown by amino acid sequence analysis to be derived from the NH2 terminus of D1R. The individual subunits lacked methyltransferase activity when assayed alone. However, mixing the D1R and D12L subunits permitted reconstitution of the methyltransferase activity, and this appearance in activity accompanied the association of the subunits. In contrast, mixing the D12L subunit with the D1R-60K proteolytic fragment failed to yield methyltransferase activity or result in a physical association of the two proteins. These results demonstrate that the methyltransferase active site requires the presence of the D12L subunit with the carboxyl-terminal portion of the D1R subunit. Furthermore, since the mRNA triphosphatase and guanyltransferase active sites reside in the NH2-terminal domain of the D1R subunit, and the methyltransferase activity is found in the carboxyl-terminal portion of this subunit and D12L, there must be at least two separate active sites in this enzyme.

Chromatography↗
PubMed1996

Conditional mutants of the yeast mRNA capping enzyme show that the cap enhances, but is not required for, mRNA splicing.

The 5' end of eukaryotic mRNAs are modified by the addition of a 7-methyl guanosine (m7G) cap. The role of the cap in translation has been well established. Additionally, studies conducted in vitro or in microinjected Xenopus oocytes have implicated the cap in RNA processing and transport. To determine the fate of uncapped mRNA in intact yeast cells, conditional alleles of the gene encoding the capping enzyme guanylyltransferase subunit (CEG1) were generated. RNA analysis of temperature-sensitive ceg1 strains revealed an accumulation of unspliced pre-mRNAs and a corresponding decrease in spliced mRNAs at the restrictive temperature. A substantial proportion of spliced mRNA was also uncapped. Therefore, the cap appears to stimulate, but is not absolutely required for, splicing in vivo. In addition, steady-state levels of several mRNAs were decreased, perhaps due to increased degradation of uncapped mRNAs. In contrast to splicing, mRNA polyadenylation and transport to the cytoplasm were unaffected.

Alleles↗
PubMed1998

Distribution of prepro-nociceptin/orphanin FQ mRNA and its receptor mRNA in developing and adult mouse central nervous systems.

Nociceptin/orphanin FQ (N/OFQ) and its receptor share similarities to opioids and their receptors in terms of the molecular structure and signaling pathway, but the two systems exhibit different actions in vivo. To understand the mechanism of N/OFQ-system actions, we examined, by in situ hybridization analysis, the distribution of preproN/OFQ and N/OFQ receptor mRNAs in the developing and adult mouse central nervous systems (CNS). In most neural regions, preproN/OFQ mRNA was mainly expressed in a small population of middle-sized neurons. These neurons were scattered between large projection-type neurons or within the neuropil, suggestive of interneurons. In some other nuclei (lateral septum, bed nucleus of the stria terminalis, reticular thalamic nucleus, inferior colliculus, and rostral periolivery nucleus), preproN/OFQ mRNA was expressed in a number of large projection-type neurons. By contrast, N/OFQ receptor mRNA was evenly expressed in most neurons of the adult CNS. Considering the inhibitory actions of N/OFQ, the distinct cellular expression pattern of the N/OFQ system suggests that the release of N/OFQ from interneurons may lower neuronal and synaptic activities of neighboring neurons, leading to integration or modulation of local circuits. Furthermore, the cellular expression pattern, distinct from that of the opioid system, may provide a possible molecular/cellular basis for the different in vivo actions of N/OFQ and opioids. In embryonic stages, both preproN/OFQ and N/OFQ receptor mRNAs were highly and widely expressed in the mantle zone, suggesting the possible importance of N/OFQ signaling in CNS development.

Age Factors↗
PubMed1995

Subsets of midbrain dopaminergic neurons in monkeys are distinguished by different levels of mRNA for the dopamine transporter: comparison with the mRNA for the D2 receptor, tyrosine hydroxylase and calbindin immunoreactivity.

The midbrain dopamine system can be divided into two groups of cells based on chemical characteristics and connectivity. The dorsal tier neurons, which include the dorsal pars compacta and the ventral tegmental area, are calbindin-positive, and project to the shell of the nucleus accumbens. The ventral tier neurons are calbindin-negative and project to the sensorimotor striatum. This study examined the distribution of the mRNAs for the dopamine transporter molecule (DAT) and the D2 receptor in the midbrain of monkeys by using in situ hybridization. The distribution patterns were compared to that of tyrosine hydroxylase and calbindin immunohistochemistry. The results show that high levels of hybridization for DAT and the D2 receptor mRNA are found in the ventral tier, calbindin-negative neurons and relatively low levels are found in the dorsal, calbindin-positive tier. Within the dorsal tier, the dorsal substantia nigra pars compacta has the least amount of both messages. These results show that in monkeys, the ventral tegmental area and the dorsal pars compacta form a dorsal continuum of dopamine neurons which express lower levels of mRNA for DAT and D2 receptor than the ventral tier. DAT has been shown to be involved in the selective neurotoxicity of N-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP). Different levels of DAT mRNA and calbindin may explain the differential effects of MPTP neurotoxicity.

Animals↗
PubMed1990

Regulation of Ig gene expression in normal lymphocytes. I. The half-life of secreted mu chain mRNA differs from that of membrane mu chain mRNA in resting and activated B cells.

Resting and activated B lymphocytes were used to study the stability of mu-specific precursor and mature mRNA. Resting cells which predominantly process the mu precursor towards micron do so rather slowly as reflected in a precursor half-life (T1/2) of 1-2 h. The small amount of secreted mu chain mRNA is fairly stable (T1/2 approximately 8 h) compared to membrane mu chain (T1/2 approximately 4 h). After activation the precursor processing is very fast (T1/2 approximately 10 min) and the stability of mu2, which now predominates, increases (T1/2 approximately 16 h) while the half-life of micron remains at about 4 h. The data indicate that normal B cells regulate mu-specific mRNA stability differently from tumor cells of the B cell lineage.

Animals↗
PubMed1986

Generation of histone mRNA 3' ends by endonucleolytic cleavage of the pre-mRNA in a snRNP-dependent in vitro reaction.

Incubation of SP6 generated mouse histone H4 mRNA precursors in nuclear extracts of HeLa cells yields processed mRNA species which end on the 3' adenosine of the conserved terminal ACCA sequence not unlike ten different histone mRNAs isolated from sea urchin embryos which end either on the 3' C or A. In the presence of 20 mM EDTA, the cleaved off 3' spacer portions of the RNA transcripts are readily detected, hence the 3' ends of histone mRNAs arise as a consequence of endonucleolytic cleavage(s) of the precursor RNA. The in vitro cleavage reaction is specifically inhibited by human antisera of the Sm-serotype, but not by control sera and can be rescued by the addition of a preparation of partially purified small nuclear RNPs to the antibody-depleted extract. Interestingly, the snRNP preparation is sufficient to elicit 3' processing of pre-mRNA in the absence of added nuclear extract.

Animals↗
PubMed2004

Antipsychotics increase microtubule-associated protein 2 mRNA but not spinophilin mRNA in rat hippocampus and cortex.

Antipsychotic (neuroleptic) drugs induce structural alterations in synaptic terminals and changes in the expression of presynaptic protein genes. Whether there are also changes in corresponding postsynaptic (dendritic) markers has not been determined. We describe the effect of 14-day treatment with typical (haloperidol, chlorpromazine) or atypical (clozapine, olanzapine, risperidone) antipsychotics on the expression of two dendritic protein genes, microtubule-associated protein 2 (MAP2) and spinophilin, using in situ hybridization, in the rat hippocampus, retrosplenial, and occipitoparietal cortices. MAP2 mRNA was increased modestly in the dentate gyrus and retrosplenial cortex by chlorpromazine, risperidone, and olanzapine and in the occipitoparietal cortex by chlorpromazine, haloperidol, and risperidone. None of the antipsychotics affected spinophilin mRNA in any area. Overall, these results show a modulation of MAP2 gene expression, likely reflecting functional or structural changes in the dendritic tree in response to some typical and atypical antipsychotics. The lack of change in spinophilin mRNA suggests that dendritic spines are not affected selectively by the drugs. The data provide further evidence that antipsychotics regulate genes involved in synaptic structure and function. Such actions may underlie their long-term effects on neural plasticity in areas of the brain implicated in the pathology of schizophrenia.

Animals↗
PubMed1994

An improved method for absolute quantification of mRNA using multiplex polymerase chain reaction: determination of renin and angiotensinogen mRNA levels in various tissues.

We have developed a multiplex, competitive, reverse-transcriptase polymerase chain reaction (RT-PCR) method which measures absolute levels of renin, angiotensinogen, and the housekeeping transcript elongation factor-1 alpha (EF-1 alpha) mRNA. Sample RNA was simultaneously titrated with serial dilutions of renin, angiotensinogen, and EF-1 alpha competitor RNAs which flanked the endogenous concentrations of target transcripts. The samples were coreverse transcribed in the presence of random primers and resulting first-strand cDNA was coamplified for 10-15 cycles with [32P]-dCTP and primers for renin angiotensinogen, after which EF-1 alpha primers were added. Amplified DNA was separated by electrophoresis on polyacrylamide gel and radioactivity in the bands was quantified by direct radioanalytical scanning. Three conditions were necessary to obtain absolute quantification of renin and angiotensinogen mRNA levels: (a) exogenous competitor RNA was used to control for tube-to-tube variability in the efficiencies of reverse transcription and amplification; (b) Sample RNA was titrated with flanking concentrations of competitor RNA to correct for intraassay differences in the efficiency of amplification due to concentration differences between competitor and target templates; and (c) a housekeeping transcript EF-1 alpha was used to control for tube-to-tube differences in RNA loading and/or degradation. We show that the multiplex RT-PCR method is precise and accurate over approximately three logs of transcript concentration and sensitive to less than 5 and 0.5 fg for renin and angiotensinogen mRNA, respectively. This method will be useful for absolute quantification of target mRNAs, especially when the amount of sample RNA is limited or unknown and/or the gene expression is low.

Angiotensinogen↗
PubMed1995

Identification in mouse macrophages of a new 4Kb mRNA present in hematopoietic tissues, which shares a short nucleotide sequence with erythropoietin mRNA.

A mRNA of larger size (4 Kb) than that of erythropoietin is found in hematopoietic tissues and macrophages after hybridization of Northern blots with an EPO cRNA probe. The cross-hybridizing fragment is located in the 3' untranslated region of the EPO mRNA. The level of this new mRNA is increased in anemic mice, which suggests that it might correspond to a molecule involved in erythropoietic regulation.

Anemia↗