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PubMed1993

The effect of nicardipine on maternal and fetal hemodynamics and uterine blood flow in chronically instrumented pregnant goats.

This study was designed to clarify the maternal and fetal hemodynamics of nicardipine (0.02 mg/kg) administered intravenously to unanesthetized, chronically instrumented pregnant goats. Nicardipine produced a significant increase in maternal heart rate and cardiac output, and a significant decrease in maternal mean arterial blood pressure and systemic vascular resistance. These changes persisted for about 30 minutes. There were no consistent changes in maternal pulmonary hemodynamics. Uterine arterial blood flow (UBF) decreased significantly (about 15%) after 5 minutes of the injection and returned to the control value within 30 minutes. A significant correlation between the decreases in UBF and maternal diastolic blood pressure was observed after 5 minutes of the injection. The fetal heart rate, blood pressure and acid-base status did not change after the injection of nicardipine. These observations of pregnant goats suggest that nicardipine might be a useful agent for the treatment of acutely hypertensive crises in pregnant patients.

Animals↗
PubMed1990

Enhancers for RNA polymerase I in mouse ribosomal DNA.

The intergenic spacer of the mouse ribosomal genes contains repetitive 140-base-pair (bp) elements which we show are enhancers for RNA polymerase I transcription analogous to the 60/81-bp repetitive enhancers (enhancers containing a 60-bp and an 81-bp element) previously characterized from Xenopus laevis. In rodent cell transfection assays, the 140-bp repeats stimulated an adjacent mouse polymerase I promoter when located in cis and competed with it when located in trans. Remarkably, in frog oocyte injection assays, the 140-bp repeats enhanced a frog ribosomal gene promoter as strongly as did the homologous 60/81-bp repeats. Mouse 140-bp repeats also competed against frog promoters in trans. The 140-bp repeats bound UBF, a DNA-binding protein we have purified from mouse extracts that is the mouse homolog of polymerase I transcription factors previously isolated from frogs and humans. The DNA-binding properties of UBF are conserved from the mouse to the frog. The same regulatory elements (terminators, gene and spacer promoters, and enhancers) have now been identified in both a mammalian and an amphibian spacer, and they are found in the same relative order. Therefore, this arrangement of elements probably is widespread in nature and has important functional consequences.

Animals↗
PubMed1995

Site-directed photo-cross-linking of rRNA transcription initiation complexes.

Site-specific photo-cross-linking of the rRNA committed transcription complex was carried out by using 5-[N-(p-azidobenzoyl)-3-aminoallyl]-dUMP-derivatized promoter DNA. Putative TAFIs of 145, 99, 96, and 91 kDa, as well as TATA-binding protein (TBP), were found to specifically photo-cross-link to different positions along the promoter. These had been identified as potential subunits of the fundamental transcription initiation factor TIF-IB (also known as SL1, factor D, and TFID) from Acanthamoeba castellanii by purification to apparent homogeneity. No other polypeptides attributable to the rRNA architectural transcription factor UBF were identified, suggesting that this protein is not part of the committed complex. Scanning transmission electron microscopy of the complexes was used to estimate the mass of the complex and the contour length of the DNA in the complex. This showed that a single molecule of TIF-IB is in each committed complex and that the DNA is not looped around the protein, as would be expected if UBF were in the complex. A circular permutation analysis of DNA bending resulting from TIF-IB binding revealed a 45 +/- 3.1 degrees (n = 14) bend centered 23 bp upstream of the transcription initiation site. This degree of bending and the position of the bend relative to the site of TBP photo-cross-linking are consistent with earlier data showing that the TBP TATA box-binding domain is not utilized in the assembly of the rRNA committed complex (C. A. Radebaugh, J. L. Mathews, G. K. Geiss, F. Liu, J. Wong, E. Bateman, S. Camier, A. Sentenac, and M. R. Paule, Mol. Cell. Biol. 14:597-605, 1994).

Acanthamoeba↗
PubMed2003

A functional screen in human cells identifies UBF2 as an RNA polymerase II transcription factor that enhances the beta-catenin signaling pathway.

beta-Catenin signaling plays an important role in the development of many organisms and has a key part in driving the malignant transformation of epithelial cells comprising a variety of cancers. beta-Catenin can activate gene expression through its association with transcription factors of the lymphoid enhancer factor 1 (LEF-1)/T-cell factor (TCF) family. We designed a screen in human cells to identify novel genes that activate a beta-catenin-LEF/TCF-responsive promoter and isolated the high-mobility group box transcription factor, UBF2. UBF1 and UBF2 are splice variants of a common precursor RNA. Although UBF1 has been shown to activate RNA polymerase I-regulated genes, the function of UBF2 has remained obscure. Here, we show for the first time that both UBF1 and UBF2 activate RNA polymerase II-regulated promoters. UBF2 associates with LEF-1, as shown by coimmunoprecipitation experiments, and potentiates transcriptional activation stimulated by LEF-1/beta-catenin from a synthetic promoter with multimerized LEF/TCF binding sites and a natural cyclin D1 promoter with consensus LEF/TCF binding sites. Downregulation of endogenous UBF expression using an RNA interference approach reduces transcriptional activation of a beta-catenin-LEF/TCF-responsive promoter by means of overexpressed beta-catenin, further implicating UBF as a transcriptional enhancer of the beta-catenin pathway.

Amino Acid Sequence↗
PubMed2005

Direct regulation of rRNA transcription by fibroblast growth factor 2.

Fibroblast growth factor 2 (FGF-2), which is highly expressed in developing tissues and malignant cells, regulates cell growth, differentiation, and migration. Five isoforms (18 to approximately 34 kDa) of FGF-2 are derived from alternative initiation codons of a single mRNA. The 18-kDa FGF-2 isoform is released from cells by a nonclassical secretory pathway and regulates gene expression by binding to cell surface receptors. This isoform also localizes to the nucleolus, raising the possibility that it may directly regulate ribosome biogenesis, a rate-limiting process in cell growth. Although several growth factors have been shown to accumulate in the nucleolus, their function and mechanism of action remain unclear. Here we show that 18-kDa FGF-2 interacts with upstream binding factor (UBF), an architectural transcription factor essential for rRNA transcription. The maximal activation of rRNA transcription in vitro by 18-kDa FGF-2 requires UBF. The 18-kDa FGF-2 localizes to rRNA genes and is necessary for the full activation of pre-rRNA synthesis in vivo. Our results demonstrate that 18-kDa FGF-2 directly regulates rRNA transcription.

Amino Acid Sequence↗
PubMed2006

Angiogenesis blockade as a new therapeutic approach to experimental colitis.

BACKGROUND: Neoangiogenesis is a critical component of chronic inflammatory disorders. Inhibition of angiogenesis is an effective treatment in animal models of inflammation, but has not been tested in experimental colitis. AIM: To investigate the effect of ATN-161, an anti-angiogenic compound, on the course of experimental murine colitis. METHOD: Interleukin 10-deficient (IL10(-/-)) mice and wild-type mice were kept in ultra-barrier facilities (UBF) or conventional housing, and used for experimental conditions. Dextran sodium sulphate (DSS)-treated mice were used as a model of acute colitis. Mice were treated with ATN-161 or its scrambled peptide ATN-163. Mucosal neoangiogenesis and mean vascular density (MVD) were assessed by CD31 staining. A Disease Activity Index (DAI) was determined, and the severity of colitis was determined by a histological score. Colonic cytokine production was measured by ELISA, and lamina propria mononuclear cell proliferation by thymidine incorporation. RESULT: MVD increased in parallel with disease progression in IL10(-/-) mice kept in conventional housing, but not in IL10(-/-) mice kept in UBF. Angiogenesis also occurred in DSS-treated animals. IL10(-/-) mice with established disease treated with ATN-161, but not with ATN-163, showed a significant and progressive decrease in DAI. The histological colitis score was significantly lower in ATN-161-treated mice than in scrambled peptide-treated mice. Inhibition of angiogenesis was confirmed by a significant decrease of MVD in ATN-161-treated mice than in ATN-163-treated mice. No therapeutic effects were observed in the DSS model of colitis. ATN-161 showed no direct immunomodulatory activity in vitro. CONCLUSION: Active angiogenesis occurs in the gut of IL10(-/-) and DSS-treated colitic mice and parallels disease progression. ATN-161 effectively decreases angiogenesis as well as clinical severity and histological inflammation in IL10(-/-) mice but not in the DDS model of inflammatory bowel disease (IBD). The results provide the rational basis for considering anti-angiogenic strategies in the treatment of IBD in humans.

Acute Disease↗
PubMed2005

mTOR function in skeletal muscle hypertrophy: increased ribosomal RNA via cell cycle regulators.

The purpose of this study was to identify the potential downstream functions associated with mammalian target of rapamycin (mTOR) signaling during myotube hypertrophy. Terminally differentiated myotubes were serum stimulated for 3, 6, 12, 24, and 48 h. This treatment resulted in significant myotube hypertrophy (protein/DNA) and increased RNA content (RNA/DNA) with no changes in DNA content or indices of cell proliferation. During myotube hypertrophy, the increase in RNA content was accompanied by an increase in tumor suppressor protein retinoblastoma (Rb) phosphorylation and a corresponding increase in the availability of the ribosomal DNA transcription factor upstream binding factor (UBF). Serum stimulation also induced an increase in cyclin D1 protein expression in the differentiated myotubes with a concomitant increase in cyclin D1-dependent cyclin-dependent kinase (CDK)-4 activity toward Rb. The increases in myotube hypertrophy and RNA content were blocked by rapamycin treatment, which also prevented the increase in cyclin D1 protein expression, CDK-4 activity, Rb phosphorylation, and the increase in UBF availability. Our findings demonstrate that activation of mTOR is necessary for myotube hypertrophy and suggest that the role of mTOR is in part to modulate cyclin D1-dependent CDK-4 activity in the regulation of Rb and ribosomal RNA synthesis. On the basis of these results, we propose that common molecular mechanisms contribute to the regulation of myotube hypertrophy and growth during the G1 phase of the cell cycle.

Animals↗
PubMed2006

Hemodynamic effects of acute and repeated exposure to raloxifene in ovariectomized sheep.

We hypothesize that administration of acute and daily doses of raloxifene will have significant effects on ovine coronary and uterine hemodynamics and that these changes are estrogen receptor dependent. Eleven ovariectomized sheep were instrumented to measure mean arterial pressure, heart rate (HR), cardiac output (CO), and coronary (CBF) and uterine artery blood flows (UBF). A dose-response curve was generated for raloxifene (1, 3, and 10 microg/kg) and compared with a standard dose of estradiol-17beta (1 microg/kg) given intravenously. In a second group of animals, raloxifene (10 microg.kg-1.day-1) was administered intravenously for 14 consecutive days, and cardiovascular responses were compared with a group of animals administered estradiol-17beta (10 microg/kg) daily for the same period. To determine whether raloxifene-related vascular responses were estrogen receptor (ER) mediated, the animals were pretreated with estrogen antagonist ICI-182,780 given intravenously. Finally, RT-PCR was preformed to determine the presence of ERalpha and ERbeta mRNA in ovine coronary and uterine vessels. Raloxifene increased CBF and UBF dose dependently with a parallel decrease in the associated vascular resistances. Acute cardiovascular responses to daily doses of raloxifene and estradiol-17beta were sustainable. In contrast to estradiol-17beta, which significantly increases CO by increasing HR but not stroke volume, raloxifene significantly increased stroke volume without a significant parallel increase in HR. ICI-182,780 abolished raloxifene-induced hemodynamic responses, and ERalpha and ERbeta mRNA are present in both ovine coronary and uterine vessels. Hence, the hemodynamic effects of raloxifene are dose dependent, sustainable, and estrogen receptor mediated.

Animals↗
PubMed2001

Endothelial vasodilator production by uterine and systemic arteries. VII. Estrogen and progesterone effects on eNOS.

Uterine blood flow (UBF) and uterine artery endothelial nitric oxide synthase (eNOS) expression are greatest during the follicular vs. luteal phase. 17 beta-Estradiol (E(2)beta) increases UBF and elevates eNOS in ovine uterine but not systemic arteries; progesterone (P(4)) effects on E(2)beta changes of eNOS remain unclear. Nonpregnant ovariectomized sheep received either vehicle (n = 10), P(4) (0.9 g Controlled Internal Drug Release vaginal implants; n = 13), E(2)beta (5 microg/kg bolus + 6 microg x kg(-1) x day(-1); n = 10), or P(4) + E(2)beta (n = 12). Reproductive (uterine/mammary) and nonreproductive (omental/renal) artery endothelial proteins were procured on day 10, and eNOS was measured by Western analysis. P(4) and E(2)beta alone and in combination increased (P < 0.05) eNOS expression in uterine artery endothelium (vehicle = 100 +/- 16%, P(4) = 251 +/- 59%, E(2)beta = 566 +/- 147%, P(4) + E(2)beta = 772 +/- 211% of vehicle). Neither omental, renal, nor mammary artery eNOS was altered, demonstrating the local nature of steroid-induced maintenance of uterine arterial eNOS. In the myometrial microvasculature, eNOS was increased slightly (P = 0.06) with E(2)beta and significantly with P(4) + E(2)beta. Systemic NO(x) was increased with P(4) and P(4) + E(2)beta, but not E(2)beta, suggesting differential regulation of eNOS expression and activity, since P(4) increased eNOS in uterine artery endothelium while E(2)beta and the combination further increased eNOS protein.

Animals↗
PubMed2001

Recombination hot spot of hepatitis B virus genome binds to members of the HMG domain protein family and the Y box binding protein family; implication of these proteins in genomic instability.

OBJECTIVE: Previously we hypothesized that the occurrence of hepatocellular carcinoma (HCC) is enhanced by genomic instability induced by the integrated hepatitis B virus (HBV) DNA. Using an in vitro recombination assay, we showed that a subgenomic fragment of HBV DNA designated 15AB (nt1855-1914) is indispensable for in vitro recombination, and also showed the existence of 15AB binding protein. On the assumption that the 15AB binding protein may be a candidate cellular recombinogenic protein which accelerates genomic instability and hepatocarcinogenesis, we tried to isolate it by southwestern screening. RESULTS AND CONCLUSION: We obtained several positive clones including mouse upstream binding factor (UBF) and DNA binding protein A (dbpA). UBF belongs to an HMG domain protein family and dbpA belongs to a Y box binding protein family. 15AB binding seemed to be mediated by the conserved DNA binding domains in these families, because other members in the families such as HMG1 and YB-1 also bound to 15AB. We report them here because several documents have already suggested the possible association of these families and DNA recombination.

Animals↗
PubMed1981

The effects of terbutaline on the acid base balance and circulation of chronically instrumented pregnant sheep.

Terbutaline is considered an effective beta agonist with beta 2 character, for the inhibiton of labor. Its safety was evaluated in twelve infusions of 1,500 micrograms over 30 min, to 8 'steady state' pregnant sheep of 110--134 days of gestation. Experimentation was performed at least 4 days postoperatively. Maternal (n = 12) and fetal (n = 7) arterial blood samples were drawn at 0, 10, 20, 30, 60- and 90 min from the start of the infusion. Uterine blood flow (UBF), blood pressure (BP), and heart rate (HR) were monitored continuously. Maternal pCO2, bicarbonate (HCO-3), and base excess (BE) decreased significantly during the infusion. Maternal pH, pO2, and O2% saturation of the hemoglobin (O2%) remained constant. Maternal hemoglobin (Hb) increased significantly at 10 min. A significant maternal hypotension and tachycardia followed the infusion. UBF decreased by 26% at the end of infusion, recovering by 60 min. Fetal pH, pCO2, Hb, O2%, BP, HR, HCO-3, BE, and CO2 content all remained stable for the duration of the experiment. Fetal arterial pO2 transiently increased at 10 min. No other significant fetal effects were observed.

Acid-Base Equilibrium↗
PubMed2002

Effects of meluadrine tartrate and ritodrine hydrochloride on oxytocin-induced uterine contraction, uterine arterial blood flow and maternal cardiovascular function in pregnant goats.

This study was designed to elucidate the effects of meluadrine tartrate on oxytocin-induced uterine contraction and maternal hemodynamics in unanesthetized, chronically instrumented pregnant goats. After the administration of meluadrine tartrate or ritodrine hydrochloride to pregnant goats, changes in heart rate (HR), arterial blood pressure (AOP), and arterial blood pH and gasses (P(O2) and P(CO2)) in the mother, as well as changes in intrauterine pressure (IUP) and uterine arterial blood flow (UBF), were measured. The escalating administration of meluadrine tartrate (0.03, 0.1, 0.3 and 1 micro g. kg(-)(1). min(-)(1)) or ritodrine hydrochloride (1, 3, 10 and 30 microg. kg(-)(1). min(-)(1)) to the maternal femoral vein caused a marked and similar inhibition in oxytocin-induced uterine contraction (a rise in IUP). By these escalating dosings, maternal HR was increased dose-dependently in both treatment groups; however, the degree of the HR increase in the meluadrine tartrate-treatment group was significantly less than that in the ritodrine hydrochloride-treatment group. Furthermore, the degree of the UBF decrease in the meluadrine tartrate-treatment group was significantly less than that in the ritodrine hydrochloride-treatment group. The present study suggests that meluadrine tartrate has a mild influence on the maternal cardiovascular function relative to the effects of ritodrine taking the potent efficacy on oxytocin-induced uterine contraction into account.

Adrenergic beta-Agonists↗
PubMedDate not supplied

Kinetics of proton-hydroxyl transport across lecithin vesicle membranes as measured with a lipoid pH-indicator.

When unilamellar vesicles are prepared in the presence of 3-Palmitoyl-7-ocy-coumarin abbreviation 3P-UBF) this lipoid pH-Indicator is anchored by its fatty acid chain to the membrane and can be used to measure pH-changes at the outer and inner membrane surface (ranging from pH to pH 9.5). By rapidly changing the pH of the outer aqueous phase a pH-gradient is set up across the vesicle membrane. The rate of the subsequent H+ or OH- influx into the vesicles can be measured as a change of the 3P-UBF absorbance at 424 nm. This was done with a stopped-flow -spectrophotometer at temperatures between 10 degree C and 50 degree C. Suspensions of vesicles prepared from egg-lecithin or L-dipalmitoyl-lecithin were investigated in buffered salt solutions. The influence of Na+, K+, Cl-, SO2-(4) and valinomycin on the rate of absorbance changes was studied at different temperatures. It was found that the rate of the pH-equilibration between the aqueous phase outside and inside the vesicles depends on the direction of the pH-gradient. This new result together with a high H+/OH- permeability of vesicle membranes found in recent studies from other laboratories and confirmed by this investigation is interpreted to indicated a higher permeability of the vesicle membrance to OH--ions compared to H+-ions. (Calculated values are: POH = 1.4 x 10(-4) cm/s at pH9 and PH = 8.3 x 10 (-7) cm/s at pH 5 and 20 degree C.) All data described in the literature in detail agree with this suggestion but a pH-dependence of POH and PH cannot be excluded.

Hydrogen-Ion Concentration↗
PubMed1998

Transcription by RNA polymerase I.

The genes that code for 45S rRNA, the precursor of 18S, 5.8S and 28S rRNA, are transcribed by RNA polymerase I. In many eukaryotes the genes are arranged as tandem repeats in discrete chromosomal clusters. rDNA transcription and rRNA processing occur in the nucleolus. In vertebrates, at least two factors, SL-1 and UBF, specific for transcription by RNA polymerase I cooperate in the formation of the initiation complex. Interestingly, there are proteins analogous to SL-1 in unicellular eukaryotes, but the requirement for a UBF-like factor appears to vary. Recent advances in our understanding of the rDNA transcription system and its regulation have demonstrated overlap with the other nuclear transcription systems (RNA polymerase II and III). This is exemplified by the utilization of TBP as a component of SL-1 and the role of Rb in regulatory rDNA transcription.

Animals↗
PubMed2000

Behavior of nucleolar proteins during the course of apoptosis in camptothecin-treated HL60 cells.

By means of immunofluorescence and immunoelectron microscopy we have studied the fate of different nucleolar components during the apoptotic process in camptothecin-treated HL60 cells. We have found that RNA polymerase I disappeared while UBF was associated with previously described fibrogranular threaded bodies. In contrast, fibrillarin, C23/nucleolin, and B23/nucleophosmin remained detectable in granular material present amid micronuclei of late apoptotic cells. Double immunolabeling experiments showed colocalization of both C23 and B23 with fibrillarin. Immunoblotting analysis showed that UBF was proteolytically degraded, whereas fibrillarin, C23/nucleolin, and B23/nucleophosmin were not. These results may help explain the presence of anti-nucleolar antibodies seen in various pathological disorders.

Apoptosis↗
PubMed2003

[Cytopathological effects of protein synthesis inhibitor emetine on HeLa cells and their nucleoli].

Eukaryotic cell nucleolus is a highly dynamic structure, which is sensitive to all changes within or outside cell borders. Numerous data are available on changes of the nucleolar structure and functions under different treatments. However, almost nothing is known about the action of translation inhibitors on the nucleolus, although these substances, together with TNF-alpha, are commonly used for apoptosis induction, both for scientific and therapeutic purposes. Emetine is one of such inhibitors. We have shown that emetine suppresses cell viability, decreases mitotic index, and induces apoptosis in HeLa cells. Emetine action is irreversible, and it sensitizes cells to unfavourable external conditions. The emetine action causes redistribution of UBF, one of RNA-polymerase I factor, from the nucleolus to nucleoplasm even after a short exposure, i.e. when the morphology of the nucleus and chromatin still keeps its native pattern. It is important that other nucleolar proteins, such as fibrillarin and B23, are not recognized in the nucleoplasm until the very late stages of apoptotic process. A suggestion is made that changes in UBF localization may be associated with the onset of ribosomal repeat cleavage and migration of rDNA-"free" fragments from the nucleolus to nucleoplasm. It looks likely that these changes can serve as an initial morphological indication of apoptosis.

Apoptosis↗
PubMed1994

The RNA polymerase I transcription factor, upstream binding factor, interacts directly with the TATA box-binding protein.

The accurate transcription of human rRNA genes by RNA polymerase I requires two transcription factors, upstream binding factor (UBF) and promoter selectivity factor (SL1). Human SL1 (hSL1) is a multisubunit complex, one of whose components is TATA box-binding protein (TBP). hSL1 binds to the core region of the rRNA promoter, but does so inefficiently in the absence of human UBF (hUBF). hUBF interacts with the upstream control element of the rRNA promoter and facilitates binding of hSL1. The molecular basis by which hUBF increases binding of hSL1 remains to be elucidated. In this report, we use an immobilized protein binding assay to identify and purify a 95-kDa TBP-binding polypeptide. Microsequence analysis reveals that the 95-kDa TBP-binding protein is hUBF. We show that hUBF is stably associated with TBP and is present in large TBP-containing complexes. Our results indicate that the cooperative binding of hUBF and hSL1 on the rRNA promoter is mediated by direct interaction between hUBF and TBP. We also provide evidence that hUBF associates with TFIID, a TBP-containing RNA polymerase II transcription factor.

Amino Acid Sequence↗
PubMed1994

[Effect of epidural anesthesia on the fetus and the neonate].

The placental transfer of local anaesthetics (LA) depends on maternal factors (LA dosage, degree of protein binding, blood pH), placental factors (surface of placental exchange, placenta thickness) and fetal factors (maternal-fetal gradient of pH, fetal hepatic metabolism of the LA, redistribution of the cardiac output in case of fetal hypoxia). The assessment of effects of LA and epidural anaesthesia (EA) on the fetus is based on the monitoring of the fetal heart rate and the measurement of the fetal pH allowing to rapidly detect an acute fetal distress. Apgar score, analysis of blood gases in the umbilical artery and neurobehavioral tests (such as Brazelton test (NBAS). Early Neonatal Neurobehavioral Scale (ENNS) and Neurologic and Adaptative Capacity Score (NACS) allow an evaluation of the drugs effects on the neonate. Direct effects of LA: although a diminution of variability of the fetal heart rate has been reported little after the beginning of an EA using lidocaine, no significant modification of the fetal heart rate after EA using bupivacaine or lidocaine with epinephrine has been shown. Fetal neurological toxicity is rare and there are very little alterations of neurobehavioral scores after EA. Indirects effects on the uteroplacental blood flow (UBF): in high concentration, LA entail a vasoconstriction of uterine arteries but the main feared effect is maternal arterial hypotension that impedes directly the uteroplacental blood flow: fetal consequences depend on the importance and duration of the UBF decrease, the preliminary state of the utero-placental circulation and haemodynamic adaptative capacities of the fetus: when the former are exceeded, fetal hypoxia occurs and myocardial and brain oxygenation can be rapidly impaired if the haemodynamic state is not corrected.(ABSTRACT TRUNCATED AT 250 WORDS)

Anesthesia, Epidural↗