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PubMed1996

Cloning and sequencing of the genes encoding the hamster ribosomal transcription factors UBF1 and UBF2.

The RNA polymerase I transcription factor, UBF, belongs to a family of high-mobility-group DNA-binding proteins. Here, a human autoantibody reactive with the nucleolus organizer regions (NOR) was used to select cDNA clones encoding the hamster transcription factors, UBF1 and UBF2. Comparison at the nucleotide level showed a high degree of homology with other mammalian upstream binding factors (UBF) already identified. The deduced amino acid sequences are identical for both UBF1 and UBF2, except for a 37 amino acid insertion found in UBF1. This insertion is completely conserved among mammalian UBF1 which indicates a putative role of this region on the function of this transcript.

Amino Acid Sequence↗
PubMed2003

Intracellular redistribution of nuclear and nucleolar proteins during differentiation of 32D murine hemopoietic cells.

We have investigated the intracellular localization of four proteins in murine hemopoietic 32D and 32D-derived cells during exponential growth and after induction of differentiation. The four proteins studied were the insulin receptor substrate-1 (IRS-1), the ID2 protein, nucleolin, and the upstream binding factor (UBF), all of which are involved directly or indirectly in the differentiation program. These four proteins were found to be predominantly nuclear (and/or nucleolar) during exponential growth, as expected. In three models of induced differentiation along the granulocytic pathway, IRS-1, ID2, and nucleolin shifted in part to the cytoplasm, where their levels eventually decreased. UBF also disappeared during differentiation, but we could not detect a cytoplasmic shift in this protein. These experiments indicate that induction of granulocytic differentiation in 32D and 32D-derived cells is accompanied by intracellular redistribution of proteins. This nucleo-cytoplasmic shuttle may play a significant role in the changes in gene expression that occur during differentiation.

Active Transport, Cell Nucleus↗
PubMed1996

Androgen regulation of ribosomal RNA synthesis in LNCaP cells and rat prostate.

Androgen-dependent growth of prostate tissue has been well documented. An additional prerequisite for cellular growth is the accumulation of ribosomes. It is thus reasonable to hypothesize that ribosomal DNA (rDNA) transcription in prostate tissue must be stimulated by androgen either directly or indirectly. This hypothesis was tested using both LNCaP cells, an androgen-dependent tissue culture line and in a rat animal model. Nuclear run-on assays confirmed that the administration of DHT to LNCaP cells resulted in a two- to three-fold increase in the rate of rRNA synthesis when compared to cells maintained in the absence of androgen. Enzymatic analysis and Western blots were carried out to measure the amount (activity and mass) of RNA polymerase I in DHT treated LNCaP cells. These assays demonstrated that neither the catalytic activity of RNA polymerase I nor the amount of the enzyme varied in response to DHT. However, Western blots revealed that the amount of the auxiliary RNA polymerase I transcription factor UBF, was significantly increased (two- to three-fold) in cells grown in the presence of DHT. Similar experiments were carried out with prostatic tissue obtained from orchiectomized rats maintained on either placebo or testosterone pellets. In this model, both the catalytic activity as well as the amount of RNA polymerase I protein decreased. However, in agreement with the tissue culture model, UBF protein decreased in prostates from orchiectomized rats and was maintained in animals supplemented with testosterone. These lines of evidence are consistent with the hypothesis that androgens stimulate rRNA synthesis by increasing the quantities of the components of the rDNA transcription system.

Animals↗
PubMedDate not supplied

On the formation of amplified nucleoli during early Xenopus oogenesis.

In Xenopus the genes for ribosomal RNA are selectively amplified during the early stages of oogenesis and give rise to over 1000 extrachromosomal nucleoli. These oocyte nucleoli are unique in that they contain very high copy numbers of rRNA genes, are not attached to chromosomes, and lack nonribosomal DNA. How the amplified rRNA genes induce the formation of multiple nucleoli is as yet poorly understood. To gain some more insight into this assembly process we have studied nucleolar development in early previtellogenic Xenopus oocytes. By using light and electron microscopic immunocytochemistry, in situ hybridization, and the TdT-method for detection of DNA, we have identified three separate structural entities which are related to the three components of the functionally active nucleoli present in midsized oocytes. Besides fibrillar and granular bodies we describe a novel rod-like structure which contains the pol I-specific transcription factor UBF. Notably, rDNA emerges from these rods forming a filamentous layer. These results reinforce UBF's role as an architectural element involved in the spatial organization of the rRNA genes. We consider the rod-like rDNA/UBF complexes as extrachromosomal nucleolar organizers which are first juxtaposed to preformed fibrillar bodies before both elements gradually fuse into a coherent nucleolar structure.

Animals↗
PubMed1990

Nucleolar transcription factor hUBF contains a DNA-binding motif with homology to HMG proteins.

The eukaryotic upstream binding factor (UBF), recognizes the ribosomal RNA gene promoter and activates transcription mediated by RNA polymerase I through cooperative interactions with the species-specific factor, SL1. Isolation of complementary DNA clones and sequence analysis reveals similarities between DNA binding domains of human UBF (hUBF) and high mobility group (HMG) protein 1. Expression, cellular localization and in vitro transcription studies establish that cloned hUBF encodes a nucleolar factor that binds specifically to the upstream control element and core of the rRNA gene promoter to activate transcription in a binding site-dependent manner.

Amino Acid Sequence↗
PubMed2005

Microspherule protein 1, Mi-2beta, and RET finger protein associate in the nucleolus and up-regulate ribosomal gene transcription.

The nucleolus is the site of ribosomal DNA (rDNA) transcription and ribosome production. In exploring the role of nucleolar protein MCRS1 (microspherule protein1)/MSP58 (58-kDa microspherule protein), we found that Mi-2beta, a component of a nucleosome remodeling and deacetylase (NuRD) complex, RET finger protein (RFP), and upstream binding factor (UBF) were associated with MCRS1. Yeast two-hybrid assays revealed that MCRS1 bound to the ATPase/helicase region of Mi-2beta and the coiled-coil region of RFP. Interestingly, confocal microscopic analyses revealed the co-localization of MCRS1, Mi-2beta, RFP, and the rRNA transcription factor UBF in the nucleoli. We also found that MCRS1, Mi-2beta, and RFP were associated with rDNA using a chromatin immunoprecipitation assay. Finally, we showed that MCRS1, Mi-2beta, and RFP up-regulated transcriptional activity of the rDNA promoter and that ribosomal RNA transcription was repressed when MCRS1, Mi-2beta, and RFP expression was reduced using siRNA. These results indicated that Mi-2beta and RFP, known to be involved in transcriptional repression in the nucleus, co-localize with MCRS1 in the nucleolus and appear to activate the rRNA transcription.

Adenosine Triphosphatases↗
PubMed2005

Evaluation of a novel integrated bioreactor--AOS system for treating oil-containing restaurant wastewater on site in Hong Kong.

AOS system is an anaerobic digestion reactor (A-section, UASB or UBF), oxidation degradation reactor (O-section, aerobic biofilm reactor), and physical sedimentation tank (S-section) 3-in-1 integrated reactor. The compact bioreactor was applied to treat oil-containing restaurant's wastewater on site in Hong Kong. The treatment efficiency was observed at different HRT. Experimental results indicated that the AOS system for treating oil-containing restaurant wastewater on site was feasible when HRT at 0.6 days above. During the running period, the net sludge generation rate of the AOS system was only about 0.08-0.09 g g(-1)removed COD, and the utmost COD specific volumetric removal rate (SVRR) of the system reached 2.77 gL(-1) d(-1). The utmost specific biomass substrate utilization rate (SBSUR) of COD in O-section and A-section reached 1.22 d(-1) and 0.128 d(-1), respectively. In addition, the experiment results showed that the AOS system with a filter in UASB (that is UBF's AOS system) could bear much more great HLR comparing with UASB's AOS and was more efficient for treating oil-containing restaurant's wastewater on site.

Biodegradation, Environmental↗
PubMed1975

Respiratory depression in newborn monkeys at Caesarean section following ketamine administration.

Ketamine, currently being evaluated as an obstetric anaesthetic agent, is said to provide analgesia without depression of the protective airway reflexes or depression of the respiratory or cardiovascular systems. We have studied the effects of ketamine on the uterine blood flow, the foetus and the newborn in five monkeys (Macaca nemistrina). Uterine blood flow, (UBF) was measured by the steady-state infusion technique using tritiated water as the indicator. All of the variables were measured during a control period and again at 10 and 90 min after the administration of ketamine in doses of 2 mg/kg in three monkeys or 1 mg/kg in two. Maternal respiration was maintained at normal physiological levels without significant variation. The maternal mean arterial pressure (MAP), cardiac output (CO), and stroke volume (SV) did not change significantly, but heart rate (HR) did increase significantly following the injection of ketamine and remained increased for the duration of the study. UBF, a-v oxygen difference, and the oxygen consumption of the uterus and its contents remained stable throughout. During the intrauterine period the foetus did not seem to be affected by the two doses of ketamine. However, the three newborn monkeys delivered of the mothers who had reveived ketamine 2 mg/kg had profound respiratory depression. This was not seen in the two infants delivered from mothers receiving 1 mg/kg. Others have shown that neonatal depression is dose- and time-related. We conclude that ketamine should be administered to obstetric patients in small single doses or by continuous infusion in very low concentrations.

Anesthesia, Obstetrical↗
PubMed2005

The Treacher Collins syndrome (TCOF1) gene product is involved in pre-rRNA methylation.

Treacher Collins syndrome (TCS) is characterized by defects in craniofacial development, which results from mutations in the TCOF1 gene. TCOF1 encodes the nucleolar phosphoprotein treacle, which interacts with upstream binding factor (UBF) and affects transcription of the ribosomal DNA gene. The present study shows participation of treacle in the 2'-O-methylation of pre-rRNA. Antisense-mediated down-regulation of treacle expression in Xenopus laevis oocytes reduced 2'-O-methylation of pre-rRNA. Analysis of RNA isolated from wild-type and Tcof1+/- heterozygous mice embryos from strains that exhibit a lethal phenotype showed significant reduction in 2'-O-methylation at nucleotide C463 of 18S rRNA. The level of pseudouridylation of U1642 of 18S rRNA from the same RNA samples was not affected suggesting specificity. There is no significant difference in rRNA methylation between wild-type and heterozygous embryos of DBA x BALB/c mice, which have no obvious craniofacial phenotype. The function of treacle in pre-rRNA methylation is most likely mediated by its direct physical interaction with NOP56, a component of the ribonucleoprotein methylation complex. Although treacle co-localizes with UBF throughout mitosis, it co-localizes with NOP56 and fibrillarin, a putative methyl transferase, only during telophase when rDNA gene transcription and pre-rRNA methylation are known to commence. These observations suggest that treacle might link RNA polymerase I-catalyzed transcription and post-transcriptional modification of pre-rRNA. We hypothesize that haploinsufficiency of treacle in TCS patients results in inhibition of production of properly modified mature rRNA in addition to inhibition of rDNA gene transcription, which consequently affects proliferation and proper differentiation of specific embryonic cells during development.

Animals↗
PubMed2006

Nucleolar localization of aprataxin is dependent on interaction with nucleolin and on active ribosomal DNA transcription.

The APTX gene, mutated in patients with the neurological disorder ataxia with oculomotor apraxia type 1 (AOA1), encodes a novel protein aprataxin. We describe here, the interaction and interdependence between aprataxin and several nucleolar proteins, including nucleolin, nucleophosmin and upstream binding factor-1 (UBF-1), involved in ribosomal RNA (rRNA) synthesis and cellular stress signalling. Interaction between aprataxin and nucleolin occurred through their respective N-terminal regions. In AOA1 cells lacking aprataxin, the stability of nucleolin was significantly reduced. On the other hand, down-regulation of nucleolin by RNA interference did not affect aprataxin protein levels but abolished its nucleolar localization suggesting that the interaction with nucleolin is involved in its nucleolar targeting. GFP-aprataxin fusion protein co-localized with nucleolin, nucleophosmin and UBF-1 in nucleoli and inhibition of ribosomal DNA transcription altered the distribution of aprataxin in the nucleolus, suggesting that the nature of the nucleolar localization of aprataxin is also dependent on ongoing rRNA synthesis. In vivo rRNA synthesis analysis showed only a minor decrease in AOA1 cells when compared with controls cells. These results demonstrate a cross-dependence between aprataxin and nucleolin in the nucleolus and while aprataxin does not appear to be directly involved in rRNA synthesis its nucleolar localization is dependent on this synthesis.

Amino Acid Sequence↗
PubMed1999

Identification of a mammalian RNA polymerase I holoenzyme containing components of the DNA repair/replication system.

Traditional models for transcription initiation by RNA polymerase I include a stepwise assembly of basic transcription factors/regulatory proteins on the core promoter to form a preinitiation complex. In contrast, we have identified a preassembled RNA polymerase I (RPI) complex that contains all the factors necessary and sufficient to initiate transcription from the rDNA promoter in vitro. The purified RPI holoenzyme contains the RPI homolog of TFIID, SL-1 and the rDNA transcription terminator factor (TTF-1), but lacks UBF, an activator of rDNA transcription. Certain components of the DNA repair/replication system, including Ku70/80, DNA topoisomerase I and PCNA, are also associated with the RPI complex. We have found that the holo-enzyme supported specific transcription and that specific transcription was stimulated by the RPI transcription activator UBF. These results support the hypothesis that a fraction of the RPI exists as a preassembled, transcriptionally competent complex that is readily recruited to the rDNA promoter, i.e. as a holoenzyme, and provide important new insights into the mechanisms governing initiation by RPI.

Animals↗
PubMed2001

The role of acetylation in rDNA transcription.

Treatment of NIH 3T3 cells with trichostatin A (TSA), an inhibitor of histone deacetylase (HDAC), resulted in a dose-dependent increase in transcription from a rDNA reporter and from endogenous rRNA genes. Chromatin immunoprecipitation using anti-acetyl-histone H4 antibodies demonstrated a direct effect of TSA on the acetylation state of the ribosomal chromatin. TSA did not reverse inhibition of transcription from the rDNA reporter by retinoblastoma (Rb) protein, suggesting that the main mechanism by which Rb blocks rDNA transcription may not involve recruitment of deacetylases to rDNA chromatin. Overexpression of histone transacetylases p300, CBP and PCAF stimulated transcription in transfected NIH 3T3 cells. Recombinant p300, but not PCAF, stimulated rDNA transcription in vitro in the absence of nucleosomes, suggesting that the stimulation of rDNA transcription by TSA might have a chromatin-independent component. We found that the rDNA transcription factor UBF was acetylated in vivo. Finally, we also demonstrated the nucleolar localization of CBP. Our results suggest that the organization of ribosomal chromatin of higher eukaryotes is not static and that acetylation may be involved in affecting these dynamic changes directly through histone acetylation and/or through acetylation of UBF or one of the other components of rDNA transcription.

3T3 Cells↗
PubMed2005

Uterine artery remodeling in pseudopregnancy is comparable to that in early pregnancy.

During pregnancy, the lumenal diameter and wall mass of the uterine artery (UA) increase, most likely in response to the increased hemodynamic strain resulting from the chronically elevated uterine blood flow (UBF). In this remodeling process, the phenotype of vascular smooth-muscle cells (VSMC) is transiently altered to enable VSMC proliferation. These phenomena are already seen during early pregnancy, when the rise in UBF is still modest. This raises the question whether the newly instituted endocrine environment of pregnancy is involved in the onset of the pregnancy-related UA remodeling. We tested the hypothesis that the conceptus is not essential for the onset of UA remodeling of pregnancy. Six control and 18 pseudopregnant (Postcopulation Days 5, 11, and 17; n = 6 per subgroup) C57Bl/6 mice were killed and UAs were dissected and processed for either morphometric analysis or immunohistochemistry. The latter consisted of staining UA cross sections for the differentiation markers smooth muscle alpha-actin and smoothelin, and for the proliferation marker MKI67. We analyzed the UA changes in response to pseudopregnancy by ANOVA. Data are presented as mean +/- SD. By Day 11 of pseudopregnancy, the UA lumen was 25% wider and the media cross-sectional area 71% larger than in control mice. These differences were accompanied by reduced smoothelin expression and increased proliferation of UA medial VSMC. All UA morphological differences had returned or were in the process of returning to baseline values by Day 17 of pseudopregnancy. The structural and cellular aspects of UA remodeling as seen at midpregnancy are also seen in pseudopregnancy. These results support the concept that the conceptus does not contribute to the initiation of UA remodeling. We suggest that ovarian hormones trigger the onset of UA remodeling.

Actins↗
PubMed1984

Relationship of 15-keto-13,14-dihydro-prostaglandin F2 alpha concentrations in peripheral plasma with local uterine production of F series prostaglandins and changes in uterine blood flow during the early postpartum period of cattle.

Cows were prepared for surgery, either within 12 h after parturition (Day 0, n = 3), on Day 1 (n = 1) or on Day 2 (n = 1) postpartum. The previously gravid uterine horn was exposed through a high lumbar laparotomy, and catheters inserted into an uterine branch of the ovarian vein (UBOV) and in the main uterine artery (UA) via a branch of the main uterine artery. A blood flow transducer (8-13 mm, internal diameter) was placed around the UA. Starting 12-24 h following surgery, simultaneous blood samples were collected from UBOV and UA at 15-min intervals for 1 or 2 h daily from Days 1 to 10 postpartum (mean = 5.3 days). Concurrently, uterine blood flow (UBF) was recorded daily for 6 h. Mean concentrations of prostaglandin F2 alpha (PGF2 alpha) and 15-keto-13, 14-dihydro-PGF2 alpha (PGFM) were higher (P less than 0.01) in UBOV than UA (4.64 +/- 0.6, n = 96 vs. 0.08 +/- 0.02 ng/ml, n = 92 and 5.67 +/- 0.41, n = 98 vs. 1.02 +/- 0.09, n = 94, ng/ml, respectively). Concentrations of PGF2 alpha and PGFM in the UBOV increased (P less than 0.1) on Days 2 and 3 and decreased (P less than 0.01) thereafter. Mean UBF (ml/min) decreased (P less than 0.01) from 3079 +/- 683 on Day 1 to 602 +/- 56 on Day 2 and was 413 +/- 55 on Day 5.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1995

Hydroxyethyl starch versus lactated Ringer's solution in the chronic maternal-fetal sheep preparation: a pharmacodynamic and pharmacokinetic study.

Administration of fluids intravenously prior to spinal and epidural analgesia in obstetrics is required to prevent maternal hypotension and fetal hypoxia. A colloid solution, such as hydroxyethyl starch (HES), might be preferable considering the capacity to stay intravascularly for a longer period. In this study the placental transfer of HES and the hemodynamic effects after infusion were investigated using a chronic maternal-fetal sheep preparation. Either 500 mL HES 10% or 750 mL lactated Ringer's solution (RL) was infused intravenously into the ewe over 30 min. Fetal and maternal blood were assayed for HES, blood gases, and acid-base status before and at regular intervals after infusion. Maternal and fetal cardiovascular variables were recorded continuously for 90 min. After HES infusion, maternal HES levels peaked at 30 min ranging from 6.9 mg/mL to 12.1 mg/mL and declined at 24 h to levels between 0.3 mg/mL and 2.8 mg/mL. Mean fetal HES concentrations remained below 0.3 mg/mL. Infusion of HES decreased hemoglobin (Hb) and plasma viscosity (PV) in the mother. Infusion of RL decreased Hb, but did not change PV. Infusion of HES significantly increased uterine blood flow (UBF), cardiac output (CO), total oxygen-delivery capacity, and uterine artery oxygen delivery. In contrast, infusion of RL did not significantly change these variables. Infusion of HES increases UBF, CO, and uterine and total oxygen-carrying capacity in the pregnant ewe. No significant transplacental transfer of HES was shown.

Anesthesia, Obstetrical↗
PubMed1996

Hemodynamic effects of intravenous isoproterenol versus epinephrine in the chronic maternal-fetal sheep preparation.

Isoproterenol 5 micrograms may be an effective marker of accidental intravascular injection in women in labor; however, before isoproterenol can be incorporated in routinely used epidural test doses, the safety and usefulness should be determined in an animal model. This study was designed to examine the hemodynamic effects of isoproterenol in comparison with epinephrine in the pregnant ewe. Five doses of isoproterenol were tested and compared with two doses of epinephrine in a randomized cross-over fashion. After administration of isoproterenol there was a small decrease of uterine blood flow (UBF) and maternal mean artery pressure (MMAP), which both almost immediately returned to baseline. When epinephrine was used a more pronounced and more prolonged decrease of UBF occurred. Increasing doses of isoproterenol resulted in dose-dependent increases in maternal heart rate (MHR), while with epinephrine this was not the case. A significant increase in the cardiac output was seen after isoproterenol. Neither isoproterenol nor epinephrine affected fetal heart rate (FHR), fetal mean arterial pressure (FMAP), amniotic fluid pressure (Amn-pr), blood gases, or acid base status in the mother and the fetus. Provided that neurotoxic effects are absent, isoproterenol might be a better alternative than epinephrine as a test dose for possible intravenous placement of an epidural catheter in pregnant women.

Acid-Base Equilibrium↗
PubMed1986

Maternal and fetal effects of epinephrine in gravid ewes.

Intravenous cannulation by an epidural catheter may complicate epidural anesthesia. Local anesthetic solutions containing epinephrine produce tachycardia and hypertension when given intravenously and may identify intravenous placement. The authors studied the maternal and fetal effects of intravenous epinephrine-containing solutions in ten chronically instrumented gravid ewes. While continuously monitoring maternal and fetal effects, epinephrine 5, 10, or 20 micrograms iv bolus was injected. Solutions of bupivacaine 5 mg and bupivacaine 5 mg combined with epinephrine 10 micrograms given iv were also examined. All epinephrine-containing solutions produced a significant increase (P less than 0.001) in maternal mean arterial pressure, which returned to baseline after 1 min. Maternal heart rates decreased transiently and returned to baseline after 1 min. All epinephrine-containing solutions decreased uterine blood flow (UBF) (P less than 0.001), and, for doses of 10 to 20 micrograms, this decrease lasted more than 3 min. Fetal heart rate and mean arterial blood pressure did not change following any test solution, nor did maternal or fetal arterial blood gas values. The authors conclude that small intravenous boluses of epinephrine decreased UBF in these animals.

Anesthesia, Epidural↗
PubMed1988

The effect of nitrendipine (NIT) on maternal and fetal blood pressure, uterine blood flow, and blood gas status in pregnant sheep.

Ewes of between 126-134 days of gestation (term 147 days) were anesthetized and underwent vascular cannula placement in the maternal and fetal jugular vein and carotid artery. A flow probe was placed around the uterine artery supplying the pregnant horn. A median of 7 days after surgery, the effect of nitrendipine (NIT) (2-8 micrograms/kg/min in stepwise increments of 30 min each followed by a 2-h recovery period) on maternal and fetal systemic arterial blood pressure (BP), heart rate (HR), the uterine blood flow (UBF), and blood gas tensions was determined (n = 5 experiments). Control experiments (n = 4) in which vehicle only was infused were also performed. The infusion of NIT was associated with a small fall in maternal systolic BP and a progressive fall in maternal diastolic BP (p less than 0.01, less than 0.001; two-way ANOVA); UBF did not change. Fetal systolic BP actually rose during the NIT infusion (p less than 0.02) but there was no difference in fetal diastolic BP. Both maternal and fetal HR rose in the NIT group. Fetal pH remained very stable throughout as did PaCO2. Fetal PaO2 fell somewhat during the recovery stages in both NIT and control groups. There is considerable interest in the possible use of calcium channel blockers in treating pregnancy-induced hypertension. These preliminary experiments provide no evidence in conflict with such a use.

Animals↗