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Results for “RNA, Competitive Endogenous”

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PubMed2002

The cyclin-dependent kinase inhibitor butyrolactone is a potent inhibitor of p21 (WAF1/CIP1 expression).

Butyrolactone I (BL) is a competitive inhibitor of ATP for binding and activation of cyclin-dependent kinases and is a potent inhibitor of cell cycle progression. Treatment of H460 human lung and SW480 human colon cancer cells with doses of BL that exceed the Ki for CDK inhibition but which are much lower than doses required to inhibit MAPK, PKA, PKC, or EGFR lead to a rapid significant reduction of endogenous p21 protein expression. BL-dependent inhibition of p21 expression appears to be p53-independent. BL-dependent p21 degradation was blocked by lactacystin, consistent with the hypothesis that there is accelerated p21 proteasomal degradation in the presence of BL. BL also inhibited the p53-dependent increase of p21 protein expression in cells exposed to the DNA damag-ing agent etoposide, and favored a greater G2/M arrest as compared to the non-BL exposed cells. BL accelerated the degradation of exogenously expressed p21 that was not observed with a C-terminal truncated form of p21. Degradation of exogenous p21 led to a shift to G2 accumulation in the cells exposed to BL. We conclude that BL has effects on the cell cycle beyond its role as a CDK inhibitor and can be used as a novel tool to study the mechanism of p21 degradation and the consequences towards p21- dependent checkpoints.

Acetylcysteine↗

PubMed2006

Nitric oxide synthase and heme oxygenase expressions in human liver cirrhosis.

AIM: Portal hypertension is a common complication of liver cirrhosis. Intrahepatic pressure can be elevated in several ways. Abnormal architecture affecting the vasculature, an increase in vasoconstrictors and increased circulation from the splanchnic viscera into the portal system may all contribute. It follows that endogenous vasodilators may be able to alleviate the hypertension. We therefore aimed to investigate the levels of endogenous vasodilators, nitric oxide (NO) and carbon monoxide (CO) through the expression of nitric oxide synthase (NOS) and heme oxygenase (HO). METHOD: Cirrhotic (n=20) and non-cirrhotic (n=20) livers were obtained from patients who had undergone surgery. The mRNA and protein expressions of the various isoforms of NOS and HO were examined using competitive PCR, Western Blot and immunohistochemistry. RESULTS: There was no significant change in either inducible NOS (iNOS) or neuronal NOS (nNOS) expressions while endothelial NOS (eNOS) was up-regulated in cirrhotic livers. Concomitantly, caveolin-1, an established down-regulator of eNOS, was up-regulated. Inducible HO-1 and constitutive HO-2 were found to show increased expression in cirrhotic livers albeit in different localizations. CONCLUSION: The differences of NOS expression might be due to their differing roles in maintaining liver homeostasis and/or involvement in the pathology of cirrhosis. Sheer stress within the hypertensive liver may induce increased expression of eNOS. In turn, caveolin-1 is also increased. Whether this serves as a defense mechanism against further cirrhosis or is a consequence of cirrhosis, is yet unknown. The elevated expression of HO-1 and HO-2 suggest that CO may compensate in its role as a vasodilator albeit weakly. It is possible that CO and NO have parallel or coordinated functions within the liver and may work antagonistically in the pathophysiology of portal hypertension.

Adult↗

PubMed1997

Expression and induction of cytochrome P-450 1A1 and P-450 2D subfamily in the rat glioma C6 cell line.

The cytochrome P-450 (P-450) monooxygenase system can catalyze the oxidation of a wide variety of endogenous and exogenous compounds, including steroid hormones, fatty acids, drugs and pollutants. The functions of this system are as diverse as the substrates. Though this enzyme system has the highest level of activity in the liver, it is present in other tissues, including brain. In this study, we have established the rat glioma C6 cell line as an in vitro model system to examine the expression and induction of P-450 1A1 and the P-450 2D subfamily. Rat glioma C6 cells were treated with P-450 inducers phenobarbital (PB) or benzo[a]anthracene (BA). The presence of P-450 1A1 and 2D1-5 was detected by reverse transcription followed by polymerase chain reaction (RT-PCR) and confirmed by restriction enzyme digestion. The induction of P-450 1A1 and 2D1-5 was quantified using competitive PCR. Although P-450 2D1-5 do not seem to be affected by PB or BA treatment, tenfold induction of P-450 1A1 mRNA after BA treatment was detected. Western blot analysis of microsomal preparations of glioma C6 cells demonstrated the presence of P-450 1A1 at the protein level. ELISAs showed that BA induces P-450 1A1 proteins 7.3-fold. These experiments provide further evidence that the rat glioma C6 cell line contains an active cytochrome P-450 monooxygenase system which can be induced by P-450 inducers. In summary, we believe the presence of the cytochrome P-450 monooxygenase system in glial cells of the brain may be important in chemotherapy and carcinogenesis of brain tumors.

Animals↗

PubMed2000

Molecular and functional characteristics of APJ. Tissue distribution of mRNA and interaction with the endogenous ligand apelin.

We have recently identified apelin as the endogenous ligand for human APJ. In rats, the highest expression of APJ mRNA was detected in the lung, suggesting that APJ and its ligand play an important role in the pulmonary system. When apelin-36 and its pyroglutamylated C-terminal peptide, [<Glu(65)]apelin-13, were compared in microphysiometric analyses, the elevation of extracellular acidification induced in cells expressing APJ by [<Glu(65)]apelin-13 was transient, whereas that by apelin-36 was sustained. These responses were almost completely inhibited by a specific inhibitor for G(i) or that for Na(+)/H(+) exchanger. (125)I -Labeled [<Glu(65)]apelin-13 analogue specifically bound to APJ with a high affinity, and [<Glu(65)]apelin-13 was more potent than apelin-36 in competitive inhibition assays. Because pretreatment with apelin-36 but not [<Glu(65)]apelin-13 drastically reduced the binding of the labeled apelin to APJ, the different patterns of acidification induced by these two peptides appeared to reflect their dissociation rather than association with APJ. Apelin elicited the migration of APJ-expressing cells, and [<Glu(65)]apelin-13 was more potent than apelin-36 in this activity. Heterogeneous molecular forms of apelin corresponding to apelin-36 and [<Glu(65)]apelin-13 were produced in bovine colostrum. Apelin-36 and [<Glu(65)]apelin-13 might have different functions in vivo and in vitro.

Amino Acid Sequence↗

PubMed1997

Evidence for a role of the gut hormone PYY in the regulation of intestinal fatty acid-binding protein transcripts in differentiated subpopulations of intestinal epithelial cell hybrids.

Peptide tyrosine tyrosine (PYY) is a gut hormone present in endocrine cells in the lower intestine that can be released by the presence of luminal free fatty acids (FFAs). The biological action of this peptide includes inhibition of gut motility and gastrointestinal and pancreatic secretions. Intestinal fatty acid-binding protein (I-FABP) binds FFA and may be involved in their cytosolic trafficking. Quantitative in situ hybridization on heterogeneous populations of small intestinal somatic cell hybrids selected for endogenous I-FABP expression (hBRIE 380i cells) demonstrated a 5-fold increase in I-FABP transcripts in response to PYY (within 6 h) that was confined to clusters of differentiated cells, whereas ribonuclease protection assays performed on heterogeneous populations of these cells showed no significant differences. High affinity PYY receptors, with an IC50 of 5-50 pM, were identified in both differentiated and nondifferentiated cell populations, as determined by competitive binding assays and autoradiography. In situ hybridization of rat ileal tissue also revealed differing patterns of mRNA expression for liver fatty acid-binding protein (L-FABP) and I-FABP. Only I-FABP mRNA was detected in the villus tips. This localization correlated with the expression pattern of I-FABP mRNA in the hBRIE 380i cells where changes in transcripts were observed only in differentiated cells that did not incorporate bromodeoxyuridine. The sustained expression of I-FABP transcripts in the villar tips suggests (unlike L-FABP) that older terminally differentiated cell populations of the mucosa can still be PYY responsive. These studies demonstrate that physiological concentrations of PYY can regulate I-FABP and place this peptide in a key position as part of a feedback system that determines the processing of cytosolic FFA in the enterocyte. In addition, these studies suggest a mechanism whereby luminal agents can modulate expression of proteins in terminally differentiated cells in the gastrointestinal mucosa.

Animals↗

PubMed2003

Dose-dependent effects of gonadotropin releasing hormone on matrix metalloproteinase (MMP)-2, and MMP-9 and tissue specific inhibitor of metalloproteinases-1 messenger ribonucleic acid levels in human decidual Stromal cells in vitro.

Matrix metalloproteinases (MMPs) and their endogenous inhibitors, tissue-specific inhibitor of matrix metalloproteinases (TIMPs), play key roles in the cyclic remodeling events that occur in the human endometrium in preparation for pregnancy. To date, the factors capable of regulating the expression of MMPs and TIMPs in the human decidua remain poorly characterized. The spatiotemporal expression of GnRH in the human endometrium during the menstrual cycle and early pregnancy suggests that this hormone may have a regulatory role in the development of this dynamic tissue. In view of these observations, we have examined the ability of GnRH to regulate MMP-2, MMP-9, and TIMP-1 mRNA levels in primary cultures of human decidual stromal cells using a quantitative competitive PCR strategy. GnRH was capable of increasing MMP-2 and MMP-9 mRNA levels in these primary cell cultures in a dose-dependent manner. The GnRH antagonist, antide, was capable of inhibiting the GnRH-mediated increase in the levels of the MMP-2 and MMP-9 mRNA transcripts present in these decidual stromal cells in a dose-dependent manner. In contrast, GnRH or antide did not have a significant effect on TIMP-1 mRNA level in these primary cell cultures at any of the concentrations used in these studies. Taken together, these observations suggest that GnRH plays an integral role in human implantation, by virtue of its ability to regulate the balance between MMP and TIMP expression in decidual cells.

Cells, Cultured↗

PubMed1991

Effects of urokinase receptor occupancy on plasmin generation and proteolysis of basement membrane by human tumor cells.

The goal of the present study was to assess the relative importance of receptor-bound and secreted plasminogen activator urokinase (u-PA) in generating cell-surface plasmin and fostering destruction of normal tissue by tumor cells. We first showed that active site-inhibited u-PA could displace endogenous u-PA from the surface of the human colon adenocarcinoma cell line HCT 116. We then prepared expression vectors for u-PA and for a mutant molecule in which the codon for the active site serine residue was changed to encode alanine. Expression of non-functional mutant u-PA decreased the level of cell-bound active u-PA by more than 95% via a mechanism that involved competition for receptor sites. Decreased cell-surface u-PA activity was associated with a decrease in cell-bound plasmin activity to undetectable levels, suggesting that receptor-bound u-PA plays an important role in the generation of plasmin on the cell surface. Transfectants that secreted eightfold to 20-fold elevated levels of active wild-type u-PA showed approximately 50% increases in cell-associated u-PA and only twofold to fourfold increases in cell-associated plasmin, suggesting that the role of secreted u-PA in generating cell-surface plasmin activity was relatively minor. In parent cells and both types of transfectants there was a good correlation between the amount of plasmin bound to the tumor cell surface and the extent to which a basement membrane substrate was degraded. These studies show that receptor-bound u-PA provides an efficient mechanism for plasmin generation on the surface of tumor cells, which, in turn, contributes significantly to their degradative potential.

Adenocarcinoma↗

PubMed1996

Regional differences in gene expression for calcium activated neutral proteases (calpains) and their endogenous inhibitor calpastatin in mouse brain and spinal cord.

The family of calpains (CANP or calcium activated neutral proteases) and their endogenous inhibitor calpastatin have been implicated in many neural functions; however, functional distinctions between the major calpain isoforms, calpain I and II, have not been clearly established. In the present study we analyzed the gene expression patterns for calpain I and II and calpastatin in mouse brain and spinal cord by measuring both their mRNA and protein levels. Our results show that the overall mRNA level measured by competitive reverse transcription polymerase chain reaction for calpain II is 15-fold higher and for calpastatin is three-fold higher than that for calpain I. Overall, both mRNA and protein expression levels for the calpains and calpastatin showed no significant difference between the spinal cord and the brain. The cellular distributions of mRNA for calpain I or calpastatin, measured by in situ hybridization, are relatively uniform throughout the brain. In contrast, calpain II gene expression is selectively higher in certain neuron populations including pyramidal neurons of the hippocampus and the deep neocortical layers, Purkinje cells of cerebellum, and motor neurons of the spinal cord. The motor neurons were the most enriched in calpain message. Motor neurons possessed 10-fold more calpain II mRNA than any other spinal cord cell type. The differential distribution of the two proteases in the brain and the spinal cord at the mRNA level indicates that the two calpain genes are differentially regulated, suggesting that they play different physiological roles in neuronal activities and that they may participate in the pathogenesis of certain regional neurological degenerative diseases.

Animals↗

PubMed1999

Functional, endogenously expressed corticotropin-releasing factor receptor type 1 (CRF1) and CRF1 receptor mRNA expression in human neuroblastoma SH-SY5Y cells.

Corticotropin-releasing factor (CRF) is a hypothalamic 41-amino acid peptide which stimulates corticotropin (ACTH) release from the anterior pituitary and is also involved in the body response to stress. CRF1 receptors represent a potential target for novel antidepressant/anxiolytic drugs. The aim of the present study was to search for a human cell line expressing native, functional CRF1 receptors as a starting material for screening purposes. We identified CRF1 receptors functionally coupled to cAMP formation in human neuroblastoma SH-SY5Y cells. CRF induced concentration-dependent increases in cAMP accumulation in SH-SY5Y cells (maximal increase 6.9 +/- 0.9 fold over basal values, n = 14). This effect was mimicked by related peptides with similar potencies: (mean pEC50 value) human/rat CRF (8.63), rat urocortin (9.32), sauvagine (8.97), urotensin I (8.93), ovine CRF (8.81). The efficacies of these agonists were nearly the same, with the exception of ovine CRF which was slightly less efficacious (75% the Emax of CRF). The responses to CRF were competitively antagonised by the following peptide fragments (mean pKB value): alpha-helical-CRF (9-41) (7.54), [D-Phe12,Nle21,38,C alpha MeLeu37]CRF (12-41) (8.36) and [D-Tyr12]astressin (9.49) and by the selective, non-peptidic CRF1 receptor antagonists, CP-154,526 (7.76) and antalarmin (9.19). Estimation of receptor density by [125I]Tyr0-ovine CRF saturation binding yielded a modest number of binding sites (Bmax 12 fmol/mg protein, KD 0.2 nM). Analysis of mRNA by reverse transcription-polymerase chain reaction clearly revealed the presence of mRNA for CRF1 receptors in SH-SY5Y cells. A slight signal for CRF2 receptor mRNA was also observed. We conclude that neuroblastoma SH-SY5Y cells are endowed with native CRF1 receptors positively coupled to cAMP formation. They therefore constitute a useful functional model for the search of CRF1 selective compounds with potential anxiolytic/antidepressant activity.

Animals↗

PubMed1993

Inhibition of estrogen-responsive gene activation by the retinoid X receptor beta: evidence for multiple inhibitory pathways.

The retinoid X receptor beta (RXR beta; H-2RIIBP) forms heterodimers with various nuclear hormone receptors and binds multiple hormone response elements, including the estrogen response element (ERE). In this report, we show that endogenous RXR beta contributes to ERE binding activity in nuclear extracts of the human breast cancer cell line MCF-7. To define a possible regulatory role of RXR beta regarding estrogen-responsive transcription in breast cancer cells, RXR beta and a reporter gene driven by the vitellogenin A2 ERE were transfected into estrogen-treated MCF-7 cells. RXR beta inhibited ERE-driven reporter activity in a dose-dependent and element-specific fashion. This inhibition occurred in the absence of the RXR ligand 9-cis retinoic acid. The RXR beta-induced inhibition was specific for estrogen receptor (ER)-mediated ERE activation because inhibition was observed in ER-negative MDA-MB-231 cells only following transfection of the estrogen-activated ER. No inhibition of the basal reporter activity was observed. The inhibition was not caused by simple competition of RXR beta with the ER for ERE binding, since deletion mutants retaining DNA binding activity but lacking the N-terminal or C-terminal domain failed to inhibit reporter activity. In addition, cross-linking studies indicated the presence of an auxiliary nuclear factor present in MCF-7 cells that contributed to RXR beta binding of the ERE. Studies using known heterodimerization partners of RXR beta confirmed that RXR beta/triiodothyronine receptor alpha heterodimers avidly bind the ERE but revealed the existence of another triiodothyronine-independent pathway of ERE inhibition. These results indicate that estrogen-responsive genes may be negatively regulated by RXR beta through two distinct pathways.

Base Sequence↗

PubMed2002

Calmodulin modulates the delay period between release of calcium from internal stores and activation of calcium influx via endogenous TRP1 channels.

In the present study we have explored the role of calmodulin (CaM) and inositol 1,4,5-trisphosphate receptor (IP(3)R) in the communication process activated after the release of calcium from the endoplasmic reticulum (ER) and the activation of calcium influx via endogenous TRP1 channels from Chinese hamster ovary cells. Experiments using combined rapid confocal calcium and electrophysiology measurements uncovered a consistent delay of around 900 ms between the first detectable calcium released from the ER and the activation of the calcium current. This delay was evident with two different methods used to release calcium from the ER: either the blockade of the microsomal calcium ATPase with thapsigargin or activation of bradykinin receptors linked to the IP(3) cascade. Direct application of IP(3) or a peptide from the NH(2)-terminal region of the IP(3)R activated store operated calcium, reducing the delay period. Introduction of CaM into the cell via the patch pipette increased the delay period from 900 +/- 100 ms to 10 +/- 2.1 s (n = 18). Furthermore, the use of selective CaM antagonists W7 and trifluoperazine maleate resulted in a substantial reduction of the delay period to 200 +/- 100 ms with 5 microm trifluoperazine maleate (n = 16) and 150 +/- 50 ms with 500 nm W7 (n = 22). CaM reduced also the current density activated by thapsigargin or brandykinin to about 60% from control. The CaM antagonists did not affect significantly the current density. The results presented here are consistent with an antagonistic effect of IP(3)R and CaM for the activation of store operated calcium after depletion of the ER. The functional competition between the activating effect of IP(3)R and the inhibiting effect of CaM may modulate the delay period between the release of calcium from the ER and the activation of calcium influx observed in different cells, as well as the amount of current activated after depletion of the ER.

Amino Acid Sequence↗

PubMed2006

G-protein-coupled receptor (GPCR)-142 does not contribute to relaxin-3 binding in the mouse brain: further support that relaxin-3 is the physiological ligand for GPCR135.

Relaxin-3 is a recently discovered member of the insulin/relaxin superfamily that has been shown to be the endogenous ligand for G-protein-coupled receptor (GPCR)135 (SALPR). In addition, relaxin-3 has demonstrated affinity and functional agonism for GPCR142 (GPR100) and LGR7 receptors in vitro. Recent evidence suggests GPCR142 is the insulin-like peptide 5 (INSL5) receptor and LGR7 is the actual relaxin receptor. We have recently described a chimeric R3/I5 peptide that selectively activates GPCR135 and GPCR142, but lacks affinity for LGR7. GPCR142 is a pseudogene in the rat, which allowed the use of [(125)I]-R3/I5 to show GPCR135-like binding sites in the rat central nervous system by autoradiography. However, mouse GPCR142 is a viable gene. In the present study we explore whether GPCR142 is expressed in the mouse brain and whether it is likely to contribute to or interfere with the pharmacological evaluation of relaxin-3 ligands. Competition binding studies confirmed mINSL5 and [(125)I]-mINSL5 bind to mGPCR142 with high affinity. However, no detectable specific [(125)I]-mINSL5 binding sites were detected throughout the mouse brain and unlabelled INSL5 did not displace [(125)I]-R3/I5 binding sites, indicating an absence of detectable GPCR142 binding sites. Consistent with these findings, neither GPCR142 nor INSL5 mRNA were detectable in mouse brain by in situ hybridization. Overall, the distribution of GPCR135 mRNA overlapped with the distribution of GPCR135 binding sites shown by autoradiography using [(125)I]-R3/I5. GPCR135 mRNA and GPCR135 receptor binding sites are most prominent in the mouse amygdala and hypothalamus. These data suggest that relaxin-3/GPCR135 is the receptor ligand pair with physiological relevance in mouse brain.

Amino Acid Sequence↗

PubMed1987

Autocrine growth of T cells independent of interleukin 2: identification of interleukin 4 (IL 4, BSF-1) as an autocrine growth factor for a cloned antigen-specific helper T cell.

Interleukin 4 (IL 4), formerly known as B cell stimulatory factor 1 (BSF-1), has recently been described as a growth factor for T cells. The role of IL 4 in the putatively IL 2-independent growth of certain cloned T helper cells is unclear. D10.G4.1 (D10) is a conalbumin-specific helper T cell that has been employed extensively in the analysis of T cell activation and as an assay for the detection of IL 1. Previously, it was thought that IL 1 induced the expression of IL 2 receptors on D10 cells, thereby permitting D10 to proliferate in response to endogenously produced IL 2. However, we cannot detect IL 2 mRNA or protein in D10 cells or their supernatants as determined by the following criteria: monoclonal antibodies that neutralize the in vitro activity of murine IL 2 do not block the IL 1-dependent proliferation of D10 cells; no competitive binding for high-affinity IL 2 receptors with 125I-labeled IL 2 can be detected with medium conditioned by activated D10 cells; and Northern blot analysis and S1 nuclease protection assays, performed with cDNA probes for IL 2, do not detect mRNA for IL 2 under a variety of different activation conditions that foster autocrine growth of D10 cells. In contrast, activated D10 cells produce both IL 4 mRNA and protein as judged by similar criteria. Purified IL 4 has significant TCGF activity as measured by proliferation of HT-2 cells. This activity can be blocked completely by a monoclonal antibody to IL 4 (11B11). The proliferation of D10 cells in the presence of 3D3 (a clonotype-specific monoclonal anti-T cell receptor antibody) and IL 1 can be blocked completely by 11B11 antibody. Highly purified IL 4 alone cannot induce the proliferation of resting D10 cells; however, equivalent amounts of IL 4 in the presence of recombinant IL 1 induce significant D10 proliferation. Therefore, IL 1 appears to render D10 cells responsive to their autocrine growth signal. These data indicate that IL 4 serves as the autocrine T cell growth factor for D10 cells, and that exogenous IL 1 is required for the transduction of this growth signal. This may represent a more broadly applicable mechanism for the growth of certain subsets of T helper cells.

Animals↗

PubMed1996

Chronic flumazenil alters GABA(A) receptor subunit mRNA expression, translation product assembly and channel function in neuronal cultures.

Flumazenil competitively blocks the pharmacological effects of both positive and negative allosteric modulators acting at the benzodiazepine binding sites of gamma-aminobutyric acid (GABA(A)) receptors. Using quantitative reverse transcription polymerase chain reaction, label-fracture immunocytochemistry and whole-cell patch-clamp recordings, we determined changes in the contents of selected GABA(A) receptor subunit mRNA(s), in their translation products and in the electrophysiological characteristics of the receptor channels in cultured cerebellar granule cells treated daily with flumazenil (10 microM) for 4 days in vitro. The contents of the alpha1 and alpha6 receptor subunit mRNAs were significantly increased in the flumazenil-treated group as compared with the dimethyl sulfoxide vehicle-treated control group, whereas there were no significant differences in the absolute amounts of the beta2, beta3, gamma2S, gamma2L++ + and delta receptor subunit mRNAs. The gold immunolabeling densities of the alpha1 and delta receptor subunits were significantly increased, whereas those of the alpha6, beta2/beta3 and gamma2 receptor subunits were decreased. Double-immunolabeling experiments using 5- and 10-nm gold particles suggest that after chronic flumazenil treatment, receptor subunit assemblies containing the alpha1/gamma2 and alpha6/delta subunits may be replaced by a receptor assembly containing the alpha1/delta subunits. The GABA potency in eliciting Cl- channel activity decreased significantly, as indicated by the elevated EC50 values, and the positive modulation of GABA action by diazepam also decreased. These results suggest that flumazenil, perhaps by blocking the action of endogenous allosteric modulators of GABA(A) receptors, may trigger a change in the expression and assembly of the subunits of the GABA(A) receptor. This implies that there might be a dynamic state in the regulation of GABA(A) receptor structure.

Animals↗

PubMed1995

Renal atrial natriuretic factor receptors in hamster cardiomyopathy.

Hamsters with cardiomyopathy (CMO), an experimental model of congestive heart failure, display stimulated renin-angiotensin-aldosterone and enhanced sympathetic nervous activity, all factors that lead to sodium retention, volume expansion and subsequent elevation of plasma atrial natriuretic factor (ANF) by the cardiac atria. However, sodium and water retention persist in CMO, indicating hyporesponsiveness to endogenous ANF. These studies were undertaken to fully characterize renal ANF receptor subtypes in normal hamsters and to evaluate whether alterations in renal ANF receptors may contribute to renal resistance to ANF in cardiomyopathy. Transcripts of the guanylyl cyclase-A (GC-A) and guanylyl cyclase B (GC-B) receptors were detected by quantitative polymerase chain reaction (PCR) in renal cortex, and outer and inner medullas. Compared to normal controls, the cardiomyopathic hamster's GC-A mRNA was similar in cortex but significantly increased in outer and inner medulla. Levels of GC-B mRNA were not altered by the disease. On the other hand, competitive binding studies, autoradiography, and affinity cross-linking demonstrated the absence of functional GC-B receptors in the kidney glomeruli and inner medulla. Also, C-type natriuretic peptide (CNP), the natural ligand for the GC-B receptors, failed to stimulate glomerular production of its second messenger cGMP. In CMO, sodium and water excretion were significantly reduced despite elevated plasma ANF (50.5 +/- 11.1 vs. 309.4 +/- 32.6 pg/ml, P < 0.001). Competitive binding studies of renal glomerular ANF receptors revealed no change in total receptor density, Bmax (369.6 +/- 27.4 vs. 282.8 +/- 26.2 fmol/mg protein), nor in dissociation constant, Kd (647.4 +/- 79.4 vs. 648.5 +/- 22.9 pM). Also, ANF-C receptor density (254.3 +/- 24.8 vs. 233.8 +/- 23.5 fmol/mg protein), nor affinity were affected by heart failure. Inner medullary receptors were exclusively of the GC-A subtype with Bmax (153.2 +/- 26.4 vs. 134.5 +/- 21.2 fmol/mg protein) and Kd (395.7 +/- 148.0 vs. 285.8 +/- 45.0 pM) not altered by cardiomyopathy. The increase in ANF-stimulated glomerular cGMP production was similar in normal and CMO hamsters (94- vs. 75-fold). These results demonstrate that renal ANF receptors do not contribute to the attenuated renal responses to ANF in hamster cardiomyopathy.

Animals↗

PubMed1978

Nucleotide sequence relationships between the genomes of an endogenous and an exogenous avian tumor virus.

We have used mapping of large T1 oligonucleotides to examine the genome of Rous-associated virus-O (RAV-O), an endogenous virus of chickens, and to compare it with that of Prague strain Rous sarcoma virus, subgroup B, (Pr-RSV-B), an exogenous sarcoma virus. To extend the sensitivity of such comparisons, we have developed a system of nucleic acid hybridization and hybridization-competition combined with fingerprinting. This method allows us to estimate the relative degree of relatedness of various portions of the viral genomes. From the results of this study, we have concluded that the genomes of Pr-RSV-B and RAV-O are related in the following way. The 5'-terminal half of the genomes (corresponding to the gag and pol regions) is virtually identical, with only scattered single nucleotide differences. This region is followed by a region comprising 25 to 30% of the genome (the env region) which contains substantial nucleotide sequence differences, most or all of which are due to single base changes. The env-coding region can be further subdivided into three regions: a more variable region probably containing sequences coding for subgroup specificity, flanked by relatively common sequences on each side. To the 3' side of the env region, the RAV-O genome contains a very short sequence not found in Pr-RSV-B, whereas the Pr-RSV-B genome contains a much longer unrelated sequence. The central portion of this sequence comprises the src gene as defined by transformation-defective mutants. Particularly striking is the absence, in the RAV-O genome, of any nucleotide sequence related to the "c region" found very near the 3' end of all exogenous tumor viruses. Both the Pr-RSV-B and RAV-O genomes contain the identical terminally redundant sequence of 21 nucleotides near each end of the genome.

Avian Leukosis Virus↗

PubMed2000

Smad7 is a TGF-beta-inducible attenuator of Smad2/3-mediated inhibition of embryonic lung morphogenesis.

Smad7 was recently shown to antagonize TGF-beta-induced activation of signal-transducing Smad2 and Smad3 proteins. However, the biological function of Smad7 in the process of lung organogenesis is not known. Since Smad2/3-mediated TGF-beta signaling is known to inhibit embryonic lung branching morphogenesis, we tested the hypothesis that Smad7 regulates early lung development by modulating TGF-beta signal transduction. An antisense oligodeoxynucleotide (ODN) was designed to specifically block endogenous Smad7 gene expression at both transcriptional and translational levels in embryonic mouse lungs in culture. TGF-beta-mediated inhibition of lung branching morphogenesis was significantly potentiated in cultured embryonic lungs in the absence of Smad7 gene expression: abrogation of Smad7 potentiated TGF-beta-mediated inhibition of lung branching morphogenesis from 76 to 52% of the basal level in lungs cultured in the presence of 5 ng/ml TGF-beta1 ligand. Likewise, TGF-beta1 EC(50) (concentration of TGF-beta1 that induced half maximal branching inhibition) was reduced from 5 to 1 ng/ml when Smad7 gene expression was abrogated in lung culture, indicating an enhanced level of TGF-beta signaling in lung tissue with abolished Smad7 gene expression. By immunocytochemistry, Smad7 protein was co-localized with both Smad2 and Smad3 in distal bronchial epithelial cells, supporting the concept that Smad7 inhibits TGF-beta signaling by competing locally with Smad2 and Smad3 for TGF-beta receptor complex binding during lung morphogenesis. Furthermore, antisense Smad7 ODN increased the negative effect of TGF-beta1 on epithelial cell growth in developing lungs in culture. We also demonstrated that Smad7 mRNA levels were rapidly and potently induced upon TGF-beta1 stimulation of lungs in culture, suggesting that Smad7 regulates TGF-beta responses in a negative feedback loop. These studies define a novel function for Smad7 as an intracellular antagonist of TGF-beta-induced, Smad2/3-mediated inhibition of murine embryonic lung growth and branching morphogenesis in culture. The optimization of TGF-beta signaling during early lung development therefore requires a finely-regulated competitive balance between both permissive and inhibitory members of the Smad family.

Animals↗

PubMed1995

IL-12 prevents mortality in mice infected with Histoplasma capsulatum through induction of IFN-gamma.

Histoplasma capsulatum is a pathogenic fungus found in discrete geographic locations throughout the world. The fungus invades the reticuloendothelial organs such as the spleen and liver of immunocompetent hosts where it is usually controlled. However, in individuals with immune deficiency, histoplasmosis is a severe and potentially fatal disease. Resistance to this infection is due primarily to a cellular immune response mediated by T cells and macrophages. Moreover, IFN-gamma is critical in activating macrophages to kill the organism. Herein we study the regulation of cytokine induction in mice infected with H. capsulatum and the effects of IL-12 in the course of infection. Mice infected with H. capsulatum and treated with neutralizing Abs to IFN-gamma, TNF-alpha, or IL-12 experienced accelerated mortality, indicating that endogenous production of these cytokines plays an important role in response to infection. In contrast, mice treated with IL-12 or a neutralizing Ab to IL-4 at the initiation of infection had substantially diminished mortality. Moreover, mice infected and treated with IL-12 show a two- to threefold increase in the amount of IFN-gamma following in vitro stimulation with specific H. capsulatum Ag compared with the control infected mice. The protective effect of IL-12 could be abrogated if a neutralizing Ab to IFN-gamma was given at the same time, demonstrating that the role of IL-12 in protection was mediated by IFN-gamma. Additionally, infected mice treated with IL-12 had a severalfold decrease in the colony counts of H. capsulatum in spleen cells after 5 days of infection as compared with control animals. Lastly, spleen cells from infected animals treated with IL-12 showed a striking decrease in their proliferative response to mitogen or H. capsulatum Ag. Responses could be restored by adding inhibitors of IFN-gamma or of nitric oxide to the in vitro cultures. The above observations suggest that IL-12 may be useful in immunologic intervention against this opportunistic pathogen.

Animals↗