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Results for “RNA, Competitive Endogenous”

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PubMed2000

Alleviation of murine leukemia virus repression in embryonic carcinoma cells by genetically engineered primer binding sites and artificial tRNA primers.

The primer binding site (PBS) plays pivotal roles during reverse transcription of retroviruses and also is the target of a cellular host defense impeding the transcription of murine leukemia virus (MLV) harboring a proline (pro) PBS in embryonic cells. Both the PBS and the tRNA primer are copied during reverse transcription and anneal as complementary DNA sequences creating the PBS of the integrated provirus. The pro PBS of MLV can be exchanged by PBS sequences matching endogenous or engineered tRNAs to allow replication of Akv MLV-derived vectors in fibroblasts. Here we use the PBS escape mutant B2 to demonstrate the capacity of the synthetic tRNA(B2) to function in reverse transcription in competition with endogenous tRNAs in fibroblasts and embryonic carcinoma (EC) cells. We further show symmetry between PBS and the primer by the ability of the synthetic tRNA(B2) to confer escape from EC repression of a PBS-Pro vector. Of a panel of vectors with the repressed pro PBS substituted for other natural or artificial PBS sequences, all except one efficiently expressed the neo marker gene when transferred to NIH/3T3 and EC cells, hence avoiding PBS-mediated silencing in EC cells. A non-natural PBS matching an artificially designed tRNA molecule conferred no further relief from repression than that attained with the B2 escape mutant or the natural alternative PBSs. Interestingly, a vector harboring a PBS matching tRNA(Lys1.2) suffered repression similar to the wild-type PBS-Pro but was partially rescued by a single point mutation of the PBS.

3T3 Cells↗

PubMed2006

Decreased B cell activating factor receptor expression on peripheral lymphocytes associated with increased disease activity in primary Sjögren's syndrome and systemic lupus erythematosus.

OBJECTIVE: To analyse B cell activating factor (BAFF) receptor (BAFF-R) expression on peripheral lymphocytes from patients with primary Sjögren's syndrome (pSS) and systemic lupus erythematosus (SLE). PATIENTS AND METHODS: Peripheral blood mononuclear cells from 20 patients with pSS, 19 patients with SLE and 15 controls were examined by flow cytometry to investigate BAFF-R mean fluorescence intensity (MFI) on lymphocytes. BAFF-R mRNA level from isolated blood B cells of nine patients with pSS and eight controls was assessed by real-time quantitative reverse transcription-PCR. BAFF serum level was determined by ELISA. RESULTS: In all subjects, BAFF-R was expressed on all naïve CD27- and memory CD27+ B-cells and was present on <0.5% of T cells. The expression of BAFF-R on B cells was significantly decreased in patients with pSS as compared with controls (MFI = 7.8 vs 10.6, p = 0.001), and was intermediate in patients with SLE (MFI = 9.5). Serum BAFF level was inversely correlated with BAFF-R MFI (p = 0.007), but not because of competition between endogenous BAFF (at observed concentrations in patients) and the monoclonal antibody (11C1) detecting BAFF-R. BAFF-R mRNA levels did not differ between patients with pSS and controls (p = 0.48). BAFF-R MFI decreased after overnight culture with recombinant human BAFF (from 32.5 to 25.4, p = 0.03). Contrary to the serum BAFF level, BAFF-R expression was correlated with extraglandular involvement in pSS and SLE Disease Activity Index. CONCLUSIONS: BAFF-R expression is reduced on peripheral B cells of patients with pSS and SLE. This down-regulation occurs through a post-transcriptional mechanism and could be the consequence of chronic increase in BAFF. BAFF-R levels on B cells could be a novel activity biomarker in autoimmune diseases.

Adult↗

PubMed2002

First cloning and functional characterization of a melatonin receptor in fish brain: a novel one?

Melatonin, a neuroendocrine transducer of photoperiod, influences a number of physiological functions and behaviors through specific seven transmembrane domains receptors. We report here the first full-length cloning and functional characterization of a melatonin receptor (P2.6) in a fish, the pike (Teleost). P2.6 encodes a protein that is approximately 80% identical to melatonin receptors previously isolated partially in non-mammals and classified as members of the Mel(1b) subtype; but, it shares only 61% identity with the full-length human Mel(1b) melatonin receptor (hMT2). Expression of P2.6 results in ligand binding characteristics similar to that described for endogenous melatonin receptors. Selective antagonists of the hMT2 (4-phenyl-2-propionamidotetraline and luzindole) were poor competitors of 2-[125I]iodomelatonin binding to the recombinant receptor. In Chinese hamster ovary cells expressing both the cystic fibrosis transmembrane conductance regulator chloride channel and P2.6 receptor, melatonin counteracted the forskolin induced activation of the channel. The results are best explained by a selective inhibition of the adenylyl cyclase. By reverse transcription-polymerase chain reaction, P2.6 mRNA appeared expressed in the optic tectum and, to lesser extent, in the retina and pituitary. In conclusion, these results, together with those of a phylogenetic analysis, suggest that P2.6 might belong to a distinct subtype group within the vertebrate melatonin receptor family.

Amino Acid Sequence↗

PubMed1995

Use of a marked erythropoietin gene for investigation of its cis-acting elements.

To examine the function of conserved noncoding regions in the erythropoietin (Epo) gene, we have prepared clones and pools of Hep3B cells stably transfected with a marked 4.1-kilobase Epo gene and deletions thereof. The marked transcripts had single base substitutions at three sites in the coding portion of Exon 5, enabling them to be distinguished from endogenous Epo mRNA by ribonuclease protection and competitive polymerase chain reaction. The basal expression and hypoxic induction of the marked Epo gene that had no deletions were indistinguishable from that of the endogenous Epo gene. Likewise, deletion of conserved intervening sequence 1 had minimal effect on hypoxic induction. In contrast, a 3'-deletion that included the conserved 3'-enhancer element resulted in a substantial, but not complete, suppression of hypoxic induction while a 3'-deletion downstream of the enhancer resulted in enhancement. A 188-base pair deletion of a conserved 3'-untranslated region in Exon 5 had minimal effect on hypoxic induction. However, the truncated Epo mRNA had a markedly prolonged half-life (15 h) in comparison to the endogenous Epo mRNA (2.0 h) or the marked full-length Epo mRNA (2.1 h). Further deletions in the 3'-UTR showed that a relatively small region of approximately 50 bases is responsible for the relatively rapid turnover of Epo mRNA. These experiments provide information on cis-acting elements of the Epo gene that cannot be obtained from conventional reporter gene transfection experiments.

Base Sequence↗

PubMedDate not supplied

Poly(A)+ RNA from the mucosa of rat jejunum induces novel Na(+)-dependent and Na(+)-independent leucine transport activities in in oocytes of Xenopus laevis.

Complementary DNA clones have been isolated recently from rat (D2) and rabbit kidney (rBAT) which induce increased Na(+)-independent Leu and Lys transport activities (System b0, +) when expressed in oocytes of Xenopus laevis. These cDNAs encode type II membrane glycoproteins which show significant homology to the heavy chain of the human and mouse 4F2 surface antigen (4F2hc). Injection of human 4F2hc cRNA into oocytes also results in induction of Leu/Lys transport activity, but with differing cation requirements for the two amino acids (Na(+)-dependent for Leu, Na(+)-independent for Lys: system y+L). System y+L is a newly discovered zwitterionic/cationic amino acid transporter first described in human erythrocytes. Here we have examined the characteristics of Leu transport in Xenopus oocytes microinjected with mRNA from the mucosa of rat jejunum. L-Leu uptake during 10 min (0.2 mM, 20 degrees C) reached 20 pmol/oocyte compared with endogenous uptake by water-injected oocytes of typically 3-4 pmol/oocyte. The expressed transport activity was 80% Na(+)-dependent. The Na(+)-dependent component of the expressed flux was saturable (Km app 0.20 mM) and inhibited by Lys, but not by Ala or Phe. The minor Na(+)-independent component of expressed Leu transport activity was also saturable (Km app 0.10 mM). Amino acid inhibition studies resolved this flux into two main components, one of which was inhibited by Lys, Ala and Phe and another which was only inhibited by Lys. There was a small residual component of Na(+)-independent Leu transport which was insensitive to inhibition by Lys. Experiments utilizing polymerase chain reaction (PCR) demonstrated the presence of both D2 and 4F2hc message in rat jejunum. Hybrid-depletion of jejunal mRNA with an antisense oligonucleotide complementary to D2 had no effect on the expression of Na(+)-linked Leu transport activity, but reduced the smaller Na(+)-independent component of Leu transport by 40%, suggesting only a minor role of D2 in the expression of rat intestinal Leu transport activity. Although the properties of Na(+)-dependent Leu transport were, with the exception of a lack of inhibition by Ala and Phe, consistent with erythrocyte y+L, hybrid-depletion of jejunal mRNA with an antisense oligonucleotide complementary to 4F2hc had no detectable effect on the expressed transport activity. We conclude, therefore, that mRNA from rat jejunum encodes novel Na(+)-dependent and Na(+)-independent transport activities unrelated to the D2/4F2hc glycoproteins.(ABSTRACT TRUNCATED AT 400 WORDS)

Amino Acid Sequence↗

PubMed2000

Extracellular collagen regulates expression of transforming growth factor-beta1 gene.

A complex interrelationship exists between the extracellular matrix and cytokine signaling in articular chondrocytes. We sought to determine whether the extracellular matrix serves as a regulatory component of transforming growth factor-beta1 expression. Bovine articular chondrocytes were isolated and resuspended in alginate, yielding final extracellular protein concentrations of 0 to 1.5% (wt/vol) for type-II or type-I collagen. Cultures were maintained for 7 days in the presence or absence of transforming growth factor-beta1-supplemented medium (10 ng/ml). The amount of transforming growth factor-beta1 mRNA was examined with quantitative competitive reverse transcription-polymerase chain reaction analysis. The results indicate that exogenous transforming growth factor-beta1 stimulates endogenous transforming growth factor-beta1 mRNA expression approximately 8-fold. This effect depended on the concentration of extracellular type-II collagen. As the concentration of extracellular type-II collagen is increased, the expression of transforming growth factor-beta1 mRNA decreases in both basal and transforming growth factor-beta1-stimulated cultures. Exogenous extracellular type-I collagen also served to negatively modulate transforming growth factor-beta1 gene expression but with a different concentration profile. The results demonstrate that transforming growth factor-beta1 mRNA expression was upregulated by exogenous transforming growth factor-beta1 and was downregulated by extracellular type-I and type-II collagens. The profoundly different effects on transforming growth factor-beta1 expression by the two collagens are consistent with those reported for mammary epithelial cells and likely serve as a negative feedback mechanism to preserve tissue homeostasis.

Animals↗

PubMed2000

Testicular recrudescence in the male black bear (Ursus americanus): changes in testicular luteinizing hormone-, follicle-stimulating hormone-, and prolactin-receptor ribonucleic acid abundance and dependency on prolactin.

Testicular recrudescence in male black bears (Ursus americanus) is initiated in January and completed in May. The goals of this study in the black bear were to determine 1) if testicular abundance of LH-receptor (LHr), FSH-receptor (FSHr), and prolactin-receptor (PRLr) mRNA changes during recrudescence; 2) if these changes in mRNA abundance are associated with changes in serum LH, PRL, and testosterone (T) concentrations; and 3) if the spring increase in serum PRL concentrations is required for testicular recrudescence. Serum was obtained monthly from nine male bears for 2 yr, except in July and August. To suppress endogenous PRL, four bears were treated with Parlodel LAR, 50 mg per 70 kg body weight, monthly from January through May, whereas five bears served as controls. Testicular biopsies were obtained in January, March, and May and analyzed for LHr, FSHr, and PRLr mRNA abundance using reverse transcriptase-competitive polymerase chain reaction. The LHr and PRLr mRNA abundance was low in January, increased in March, and remained high in May, whereas the FSHr mRNA abundance remained constant. Serum concentrations of PRL and T increased in March, coincident with the increase in testicular LHr and PRLr mRNA abundance. Suppression of serum PRL concentrations during testicular recrudescence 1) prevented the increase in testicular LHr and PRLr mRNA abundance observed among control bears in March, 2) lowered serum T concentrations in March and April, and 3) resulted in reduced testis size in May. We conclude that testicular LHr and PRLr mRNA are seasonally regulated, and that PRL has a role in testicular recrudescence in the black bear.

Animals↗

PubMed1976

Radiation leukemia in C57BL/6 mice. II. Lack of ecotropic virus expression in the majority of lymphomas.

The expression of endogenous ecotropic viruses in radiation-induced thymomas of C57BL/6 mice was examined. Competition radioimmunoassays for AKR MuLV gp71, p30, and p12 were used for viral antigen expression. 3 of 40 lymphomas had readily detectable ecotropic gp71 at levels of 95-689 ng/mg protein; the remainder of the tumors had no detectable gp71 (less than 1.0 ng/mg protein). 30 thymomas were characterized by the presence of MuLV p30 at levels of 1-10 ng/mg protein, levels that were comparable to those found in thymus extracts from age-matched, nonirradiated control. 10 tumors were characterized by having p30 levels of 10-30 ng/mg protein. In one tumor significant levels of AKR MuLV p12 were detectable. Since B-tropic and N-tropic viruses from C57BL/6 mice have glycoproteins (gp71) indistinguishable from AKR MuLV gp71 and the N-tropic virus had a p12 serologically identical to AKR MuLV p12, these results demonstrate that overt endogenous B-tropic virus was detectable in 2 of 40 thymomas and endogenous N-tropic virus was detectable in 1 of 40 thymomas. The lack of overt expression of gp71 or p12 was also confirmed by cytotoxicity assays using monospecific antisera to these viral proteins. Radiation-induced lymphomas were also examined for the presence of reverse transcriptase after chromatography of tissue extracts on poly G-Sepharose. One tumor, which was characterized by the lack of gp71, also had no detectable reverse transcriptase; whereas one tumor with gp71 was characterized by readily detectable levels of reverse transcriptase in cellular extracts. The presence of viral RNA was examined using AKR cDNA. Low levels of RNA capable of hybridizing with AKR cDNA were found in age-matched, nonirradiated mice; these hybrids had Tm's of 72 degrees C, while hybrids with AKR MuLV 70S RNA had Tm's of 80 degrees C. In 1 of 12 thymomas the concentration of hybridizable RNA and the Tm of the hybrids were identical to control values. In 9 of 12 thymomas the concentration of hybridizable sequences increased approximately three-to fivefold and the Tm of these hybrids varied from 73 to 75 degrees C. In 1 of 12 thymomas the concentration of hybridizable sequences increased over 100-fold, hybridized completely with AKR MuLV cDNA, and the hybrids had Tm's of 79 degrees C. This thymoma was also characterized by the presence of the AKR MuLV type of gp71 and p12. One tumor was characterized by a 10-to 100-fold increase in hybridizable sequences, which only partially hybridized with AKR MuLV cDNA, and hybrids had a Tm of 73 degrees C. This tumor was characterized by the presence of AKR MuLV gp71 but not AKR MuLV p12. The results taken together demonstrate that overt endogenous ecotropic virus expression is only rarely detectable in radiation-induced thymomas of C57BL/6 mice.

AKR murine leukemia virus↗

PubMed2000

Enhancement of the basal-level activity of HIV-1 long terminal repeat by HIV-1 nucleocapsid protein.

Two HIV-1 proteins, Tat and NCp7 (NC), have zinc finger-like structures. NC is a virion protein and has been shown to accumulate in the nucleus 8 h postinfection. Since transcription factors with zinc fingers assist the transcriptional activity of both RNA polymerases II and III, we examined the effect of NC on HIV-1 LTR-directed gene expression. The HIV-1 NC binds to the HIV-1 LTR and results in a mobility shift in polyacrylamide gel electrophoresis. Competition assays with cold probes revealed that the binding of NC and formation of a DNA-protein complex could be prevented by the addition of excess unlabeled LTR self-probe, but not the HIV-1 V3 envelope gene. The DNase I footprint analysis showed that NC binds to six regions within HIV-1 LTR, four of which are near the transcription start site. The NC alone enhances LTR basal-level activity in RNA runoff experiments. When the general transcription factors (GTFs) were added in the assay, NC enhances NF-kappaB, Sp1, and TFIIB-induced HIV-1 LTR-directed RNA transcription. RNA transcription directed by the adenovirus major late promoter, however, is not significantly affected by NC in the cell-free system. Transient transfection of human T lymphocytes with the plasmids containing HIV-1 nc or gag showed enhancement of LTR-CAT activity. Moreover, transfection of HIV-1 provirus containing mutations in NC zinc-finger domains dramatically decreases the enhancement activity in human T cells, in which HIV-1 LTR is stably integrated into the cellular genome. These observations show that NC binds to HIV-1 LTR and cooperatively enhances GTFs and NF-kappaB induced HIV-1 LTR basal-level activity. NC may play the role of a nucleation protein, which binds to LTR and enhances basal-level transcription by recruiting cellular transcription factors to the HIV-1 promoter in competition with cellular promoters.

Base Sequence↗

PubMed2002

Differential activation of the IkappaBalpha and mouse mammary tumor virus promoters by progesterone and glucocorticoid receptors.

The glucocorticoid and progesterone receptors (GR and PR) are structurally homologous and bind a common hormone response element (HRE). The mechanisms by which receptors activate specific promoters when multiple steroids are present in a cell is a critical question in endocrinology. To investigate how co-existing steroid receptors regulate gene transcription, we have compared two hormone-responsive promoters in T47D/A1-2 human breast cancer cells expressing both the GR and PR. The promoters chosen were those for the mouse mammary tumor virus (MMTV) and the gene for IkappaBalpha, the inhibitor of the ubiquitous transcription factor, nuclear factor kappa B (NFkappaB). Several differences between glucocorticoid and progestin activation of the IkappaBalpha and MMTV promoters were revealed. Both steroids activated the endogenous IkappaBalpha promoter, while only glucocorticoids activated a stably integrated MMTV promoter. In combination, progestins enhanced glucocorticoid activation of IkappaBalpha, but antagonized glucocorticoid activation of MMTV. These differences in steroid receptor competition were further demonstrated when levels of the PR were reduced by prolonged treatment with progestin. Under these conditions, the PR no longer competes effectively with the GR for activation of the MMTV promoter. However, on the IkappaBalpha promoter, the GR and PR still activate the promoter in a cooperative fashion. Another difference between the two promoters is their chromatin structure. In this cell line, the MMTV promoter chromatin is "closed" and insensitive to restriction enzyme cleavage, while the IkappaBalpha promoter is "open." Using PR antagonists, we demonstrate that at least one cofactor complex, the BRG-1 chromatin remodeling complex, differentially contributes to activation of both promoters.

Animals↗

PubMed2000

Interleukin-2 expression in human carcinoma cell lines and its role in cell cycle progression.

Human carcinomas were shown to express mRNA and protein for IL-2R alpha, beta and gamma chains. Recently, human carcinomas were also shown to constitutively express protein and mRNA for IL-2 in vivo and in vitro. Here we report that the expression levels of cytoplasmic IL-2 as well as IL-2Rbeta- and gamma-chain in human carcinoma cells change during the cell cycle progression. Carcinoma cells synchronized in the G2/M phase of the cell cycle expressed significantly more intracytoplasmic IL-2 as well as IL-2Rbeta and gamma proteins than tumor cells in the G0/G1 phase. The level of mRNA for IL-2 was 5-10-fold higher in the M phase than in the G0/G1-phase, as shown by quantitative competitive RT-PCR. Expression of the cyclin-dependent kinase (CDK) inhibitor p27kip1 in these carcinoma cells was found to be high in the G0/G1 phase, nearly absent in the S phase, and it increased again in the G2/M phase of the cell cycle. In synchronized cells, the decrease in p27 expression coincided with high levels of expression of IL-2. Using the IL-2 specific antisense oligonucleotide to block synthesis of endogenous IL-2 in tumor cells, we observed increased levels of p27 as well as p21. The antisense oligonucleotides specific for p27 or p21 blocked expression of these proteins but not of IL-2. Thus, endogenous IL-2 is important in regulating expression of p27 as well as p21 and, therefore, in controlling cell cycle progression of tumor cells, while its own expression remains independent of the CDK inhibitors.

Blotting, Western↗

PubMed1995

Developmental expression of endothelin receptors in cerebellar neurons differentiating in culture.

We describe the identification and expression of endothelin (ET) receptor subtypes in differentiating cultured cerebellar neurons. Using [125I]ET-1 and the subtype-selective ligands BQ-123 and sarafotoxin 6c as selective ligands for the ETA and ETB receptors, respectively, we found that cerebellum from 8-day-old rats displayed only the ETB receptor subtype. We next cultivated cerebellar granule cell neurons to study ET receptor differentiation between 2 and 22 days in vitro. Using the above reagents, we found that while unlabeled ET-1 displayed monophasic competition curves, BQ-123 and sarafotoxin 6c displayed partial displacement curves, indicating the presence of both ETA and ETB receptors on these neurons. The proportion of ETB receptors gradually decreased from day 2 onwards the proportion of ETA receptors gradually increased. On days 2, 3, 4, and 5 of culture, the ETB:ETA receptor ratios were 90:10, 70:30, 60:40, and 40:60, respectively. There was no further change in receptor subtype ratio beyond day 5 and up to day 22. Northern blot analysis showed that ETB receptor message expression was 6.9-fold higher than that of ETA receptor expression on day 2, but steadily decreased with time, whereas ETA receptor message expression was minimal on day 2 and maximal by day 3 and 4. By day 7, receptor message was of equal abundance, which was in good agreement with the binding studies. This novel, developmentally regulated process predicts the existence of endogenous mediators of neuronal ET receptor expression.

Animals↗

PubMed1998

Lipopolysaccharide (LPS)- and muramyl dipeptide (MDP)-induced anorexia during refeeding following acute fasting: characterization of brain cytokine and neuropeptide systems mRNAs.

We investigated the effectiveness of lipopolysaccharide (LPS) and muramyl dipeptide (MDP) administered into the brain to induce anorexia in acutely fasted Wistar rats allowed to refeed. We also assayed for changes in mRNA levels of IL-1 system components, TNF-alpha, TGF-beta1, glycoprotein 130 (gp 130), leptin receptor (OB-R), pro-opiomelanocortin (POMC), neuropeptide Y (NPY), glucocorticoid receptor (GR), and CRF receptor (CRF-R) in selected brain regions. The data show that LPS and MDP induced anorexia differentially during refeeding. LPS-induced anorexia was of a stronger magnitude and duration than that of MDP. RNase protection assays showed that LPS and MDP significantly increased the expression of IL-1beta, IL-1 receptor type I, and TNF-alpha mRNAs in the cerebellum, hippocampus, and hypothalamus; LPS was more potent in all cases. MDP treatment, on the other hand, induced a stronger increase in hypothalamic levels of IL-1 receptor antagonist (IL-1Ra) and TGF-beta1 mRNAs relative to LPS. In addition, competitive RT-PCR analysis showed that LPS induced an eleven-fold increase in IL-1alpha mRNA in the hypothalamus relative to vehicle. These findings suggest that LPS and MDP mediate anorexia through different cytokine mechanisms. A stronger up-regulation of anti-inflammatory cytokines (IL-1Ra and TGF-beta1) mRNA expression by MDP may be involved in the weaker MDP-induced anorexia relative to LPS. No significant changes were observed in the peptide components examined except for an up-regulation in cerebellar gp 130 mRNA and down-regulation of hypothalamic GR mRNA expression in response to LPS or MDP. This study shows that LPS and MDP induce anorexia in fasted rats allowed to refeed, and suggests an important role for endogenous cytokine-cytokine interactions.

Acetylmuramyl-Alanyl-Isoglutamine↗

PubMed1975

Interspecies antigenic determinants of the reverse transcriptases and p30 proteins of mammalian type C viruses.

The major internal structural proteins (p30) of type C viruses isolated from several mammalian species were studied by radioimmunoprecipitation and competitive radioimmunoassays. Three antigenically distinguishable sets of interspecies determinants could be demonstrated by both methods. One set of determinants shared by viruses of rodent origin (mouse and rat) can be detected readily in feline leukemia viruses but not in other type C viral groups. The p30 proteins of murine viruses also contain a second discrete set of antigenic determinants related to those in infectious primate viruses and endogenous porcine viruses, but not detected in the feline leukemia virus group. The p30 proteins of endogenous viruses of baboons and domestic cats share yet a third set of cross-reactive determinants not detected in type C viruses isolated from other species of animals. Enzyme inhibition studies performed with antisera raised toward the reverse transcriptases of these same groups of type C viruses showed the same patterns of immunological cross-reactions as observed with p30 proteins. The antigenic cross-reactions between the homologous proteins of type C virus isolated from genetically distant animals may reflect transmission of type C viruses across species barriers.

Animals↗

PubMed1979

Primate retroviruses: intracistronic mapping of type D viral gag gene by use of nonconditional replication mutants.

Nonconditional replication mutants of squirrel monkey retrovirus (SMRV), an endogenous type D virus of primates, are shown to be defective in post-translational processing of nonglycosylated virus-coded structural proteins. Utilizing such mutants, in combination with sensitive radioimmunological assays, we demonstrate the existence of a 72,000-molecular-weight precursor polyprotein (Pr72gag) encoded by a region of the SMRV genome designated gag. Post-translational cleavage of this precursor polyprotein gives rise to virion structural proteins of 35,000 (p35), 16,000 (p16), 12,000 (p12), and 9,000 (p9) molecular weight. Three of these viral proteins, p35, p16, and p9, are shown to be phosphorylated. Analysis of viral antigen expression in cell lines nonproductively infected with either of two replication-defective SMRV mutants or mink cells productively infected with wild-type SMRV resulted in the detection of several SMRV Pr72gag intermediate cleavage products. Adjacent proteins within such intermediates are identified by use of specific competition immunoassays, and the intracistropic order of individual structural proteins with SMRV Pr72gag was tentatively deduced as NH2-p16-p12-p35-p9-COOH.

Animals↗

PubMed1992

The amyloid beta-protein precursor promoter. A region essential for transcriptional activity contains a nuclear factor binding domain.

A manifestation of Alzheimer's disease is the presence of amyloid depositions in brains of afflicted individuals. A major component of these depositions is the amyloid beta-protein, which is a truncated form of the larger amyloid beta-protein precursor (APP). To investigate the regulation of APP gene expression, the APP promoter and selected deletions were placed 5' to the reporter gene chloramphenicol acetyltransferase. The promoter deletions were transfected into different cell lines that showed variant levels of endogenous APP transcripts. Transient transfection assays showed that 96 base pairs 5' to the transcriptional start site are sufficient for cell type-specific promoter activity. A nuclear factor that binds to this region in a sequence-specific manner was identified by mobility shift electrophoresis, DNase footprinting, and methylation interference. The DNase-protected region covers about 25 base pairs on both strands (position -31 to -55). Mutations within this domain revealed a sequence of 12 base pairs that is crucial for factor binding. This sequence overlaps with the consensus sequences for transcription factors AP-1 and AP-4. However, competition experiments suggest that the nuclear factor that binds to the APP promoter is distinct from both AP-1 and AP-4. Factor binding to the characterized recognition sequence is observed in nuclear extracts originating from human, mouse, and rat cells, suggesting a high degree of conservation.

Actins↗

PubMed2001

Selectivity of recombinant human leukotriene D(4), leukotriene B(4), and lipoxin A(4) receptors with aspirin-triggered 15-epi-LXA(4) and regulation of vascular and inflammatory responses.

Aspirin-triggered lipoxin A(4) (ATL, 15-epi-LXA(4)) and leukotriene D(4) (LTD(4)) possess opposing vascular actions mediated via receptors distinct from the LXA(4) receptor (ALX) that is involved in leukocyte trafficking. Here, we identified these receptors by nucleotide sequencing and demonstrate that LTD(4) receptor (CysLT(1)) is induced in human vascular endothelia by interleukin-1beta. Recombinant CysLT(1) receptor gave stereospecific binding with both [(3)H]-LTD(4) and a novel labeled mimetic of ATL ([(3)H]-ATLa) that was displaced with LTD(4) and ATLa ( approximately IC(50) 0.2 to 0.9 nmol/L), but not with a bioinactive ATL isomer. The clinically used CysLT(1) receptor antagonist, Singulair, showed a lower rank order for competition with [(3)H]-ATLa (IC(50) approximately 8.3 nmol/L). In contrast, LTD(4) was an ineffective competitive ligand for recombinant ALX receptor with [(3)H]-ATLa, and ATLa did not compete for [(3)H]-LTB(4) binding with recombinant LTB(4) receptor. Endogenous murine CysLT(1) receptors also gave specific [(3)H]-ATLa binding that was displaced with essentially equal affinity by LTD(4) or ATLa. Systemic ATLa proved to be a potent inhibitor (>50%) of CysLT(1)-mediated vascular leakage in murine skin (200 microg/kg) in addition to its ability to block polymorphonuclear leukocyte recruitment to dorsal air pouch (4 microg/kg). These results indicate that ATL and LTD(4) bind and compete with equal affinity at CysLT(1), providing a molecular basis for aspirin-triggered LXs serving as a local damper of both vascular CysLT(1) signals as well as ALX receptor-regulated polymorphonuclear leukocyte traffic.

Animals↗

PubMed2002

Endogenous receptor for melanin-concentrating hormone in human neuroblastoma Kelly cells.

Melanin-concentrating hormone (MCH), a cyclic nonadecapeptide, is predominantly expressed in mammalian neurons located in the zona incerta and lateral hypothalamus. Current interest in MCH relates to its role in the control of feeding behaviour. Two receptors for MCH were recently found: MCH-R(1) and MCH-R(2). We show here by RT-PCR analysis and immunofluorescence studies that the human neuroblastoma cell line Kelly expresses MCH and MCH-R(1) but not MCH-R(2). In competition assays using 125I-labelled MCH an inhibitory concentration 50% (IC(50)) of 76nM was determined for MCH, indicating a high affinity of Kelly cells for MCH. MCH induces mitogen-activated protein kinase (MAPK) phosphorylation in Kelly cells but no increase in the intracellular free Ca(2+) concentration. This suggests that MCH signals via Galpha(i)/Galpha(0) in these cells. The presence and functionality of MCH-R(1) renders this neuronal cell a very useful model for future structure-activity studies in a physiological environment mimicking the human brain for the evaluation of potential appetite-regulating drugs.

Calcium Signaling↗