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PubMed2006

An HMG protein, Hmo1, associates with promoters of many ribosomal protein genes and throughout the rRNA gene locus in Saccharomyces cerevisiae.

HMG proteins are architectural proteins that bind to DNA with low sequence specificity, but little is known about their genomic location and biological functions. Saccharomyces cerevisiae encodes 10 HMG proteins, including Hmo1, which is important for maximal transcription of rRNA. Here we use chromatin immunoprecipitation coupled with microarray analysis to determine the genome-wide association of Hmo1. Unexpectedly, Hmo1 binds strongly to the promoters of most ribosomal protein (RP) genes and to a number of other specific genomic locations. Hmo1 binding to RP promoters requires Rap1 and (to a lesser extent) Fhl1, proteins that also associate with RP promoters. Hmo1, like Fhl1 and Ifh1, typically associates with an IFHL motif in RP promoters, but deletion of the IFHL motif has a very modest effect on Hmo1 binding. Surprisingly, loss of Hmo1 abolishes binding of Fhl1 and Ifh1 to RP promoters but does not significantly affect the level of transcriptional activity. These results suggest that Hmo1 is required for the assembly of transcription factor complexes containing Fhl1 and Ifh1 at RP promoters and that proteins other than Fhl1 and Ifh1 also play an important role in RP transcription. Lastly, like mammalian UBF, Hmo1 associates at many locations throughout the rRNA gene locus, and it is important for processing of rRNA in addition to its role in rRNA transcription. We speculate that Hmo1 has a role in coordinating the transcription of rRNA and RP genes.

Amino Acid Motifs↗
PubMed1993

Systemic and uterine responses to chronic infusion of estradiol-17 beta.

Human and ovine pregnancies are associated with increases in plasma levels of estrogens and angiotensin II (ANG II), cardiac output (CO), blood volume (BV), and uterine blood flow (UBF), as well as attenuated ANG II pressor responses. We hypothesized that, in nonpregnant animals, prolonged estradiol-17 beta (E2 beta) treatment would reproduce these endocrine and hemodynamic alterations. Nonpregnant ovariectomized ewes (n = 5) received 5 microgram E2 beta/kg iv followed by 220 micrograms/day for 14 days. Plasma E2 beta increased from 36 +/- 6 to 269 +/- 79 (SE) pg/ml (P < 0.05) during E2 beta treatment, returning to control values 4 days posttreatment. By 3 days of E2 beta, mean arterial pressure (MAP) and systemic vascular resistance (SVR) fell 9 +/- 1 and 29 +/- 1%, whereas heart rate (HR) and CO increased 20 +/- 5 and 26 +/- 1% (P < 0.05). Stroke volume (SV), BV, and plasma volume were unchanged until 7 days of E2 beta, with values rising 17 +/- 5, 13 +/- 3, and 14 +/- 4, respectively (P < 0.05). Although MAP remained similarly depressed (-11 +/- 1%) during week 2 of E2 beta, SVR decreased further (-37 +/- 3%) and was associated with additional increases (P < 0.05) in CO to 44 +/- 5%, reflecting rises in SV (21 +/- 2%) but not HR. Increases in BV correlated with rises in CO (r = 0.55) and SV (r = 0.64) but not HR (r = -0.04).(ABSTRACT TRUNCATED AT 250 WORDS)

Angiotensin II↗
PubMed2003

Possible mechanisms underlying pregnancy-induced changes in uterine artery endothelial function.

The last 10 years has seen a dramatic increase in our understanding of the mechanisms underlying the pregnancy-specific adaptation in cardiovascular function in general and the dramatic changes that occur in uterine artery endothelium in particular to support the growing fetus. The importance of these changes is clear from a number of studies linking restriction of uterine blood flow (UBF) and/or endothelial dysfunction and clinical conditions such as intrauterine growth retardation (IUGR) and/or preeclampsia in both humans and animal models; these topics are covered only briefly here. The recent developments that prompts this review are twofold. The first is advances in an understanding of the cell signaling processes that regulate endothelial nitric oxide synthase (eNOS) in particular (Govers R and Rabelink TJ. Am J Physiol Renal Physiol 280: F193-F206, 2001). The second is the emerging picture that uterine artery (UA) endothelial cell production of nitric oxide (NO) as well as prostacyclin (PGI2) may be as much a consequence of cellular reprogramming at the level of cell signaling as due to tonic stimuli inducing changes in the level of expression of eNOS or the enzymes of the PGI2 biosynthetic pathway (cPLA2, COX-1, PGIS). In reviewing just how we came to this conclusion and outlining the implications of such a finding, we draw mostly on data from ovine or human studies, with reference to other species only where directly relevant.

Animals↗
PubMed2000

Nonrandom distribution of metaphase AgNOR staining patterns on human acrocentric chromosomes.

The metaphase nucleolar organizer regions (NORs) contain ribosomal genes associated with proteins such as upstream binding factor (UBF) and RNA polymerase I (RPI). These genes are clustered in 10 loci of the human acrocentric chromosomes (13, 14, 15, 21, and 22). Some NOR-associated proteins, termed AgNOR proteins, can be specifically stained by silver. In this study we took advantage of technical advances in digital imaging, image restoration techniques, and factorial correspondence analysis (FCA) to study the different AgNOR staining patterns of metaphase chromosomes in human lymphocytes. Three predominant patterns could be distinguished: pair (47%), stick-like (28%), and unstained (18%) structures. By studying the frequency of occurrence of each pattern on different chromosomes, two groups could be defined. Chromosomes 13, 14, and 21 carried predominantly pair or stick-like AgNOR structures, whereas chromosomes 15 and 22 mainly carried pair AgNOR structures or remained unstained. We suggest that the different AgNOR shapes reflect both the number of ribosomal genes carried by each chromosome and the differential recruitment of active ribosomal genes in each NOR cluster. This is the first study showing a nonrandom distribution of AgNOR shape among acrocentric chromosomes.

Chromosomes, Human↗
PubMed2003

Nucleolar association of pEg7 and XCAP-E, two members of Xenopus laevis condensin complex in interphase cells.

Cell cycle dynamics and localization of condensins--multiprotein complexes involved in late stages of mitotic chromosome condensation--were studied in Xenopus laevis XL2 cell line. Western blot analysis of synchronized cells showed that the ratio of levels of both pEg7 and XCAP-E to beta-tubulin levels remains almost constant from G1 to M phase. pEg7 and XCAP-E were localized to the mitotic chromosomes and were detected in interphase nuclei. Immunostaining for condensins and nucleolar proteins UBF, fibrillarin and B23 revealed that both XCAP-E and pEg7 are localized in the granular component of the nucleolus. Nucleolar labeling of both proteins is preserved in segregated nucleoli after 6 hours of incubation with actinomycin D (5 mg/ml), but the size of the labeled zone was significantly smaller. The data suggest a novel interphase function of condensin subunits in spatial organization of the nucleolus and/or ribosome biogenesis.

Adenosine Triphosphatases↗
PubMed1999

Nucleolar localization of murine nuclear DNA helicase II (RNA helicase A).

Nuclear DNA helicase II (NDH II) is a highly conserved member of the DEXH superfamily of eukaryotic helicases, whose physiological role is still unclear. To explore the function of NDH II, we studied the intracellular distribution of NDH II of different mammalian species by immunofluorescence and compared these findings with the known role of the Drosophila homologue MLE that is involved in sex-specific gene dosage compensation. NDH II displayed an apparent nucleolar localization in murine cells, whereas in cells from all other mammalian species examined so far the protein was confined to the nucleoplasm and apparently excluded from the nucleoli. The nucleolar localization of mouse NDH II strongly suggests a role in ribosomal RNA biosynthesis. Immunoelectron microscopic studies revealed that the mouse NDH II was found at the dense fibrillar components of the nucleoli, and a significant percentage of NDH II molecules colocalized with the RNA polymerase I (Pol I) transcription factor UBF (upstream binding factor). Additionally, the nucleolar localization of NDH II coincided with a preferential immunolabeling pattern of nascent transcripts with bromouridine (BrUMP). Furthermore, mouse NDH II redistributed in mitosis in a manner highly correlated with Pol I activity. Conditions leading to the inhibition of Pol I activity in the interphase decreased the amount of NDH II in the nucleoli that diffused into the nucleoplasm and the cytosol. Contrary to the effect of inhibiting rRNA synthesis, treatment of mouse cells with the translation inhibitor cycloheximide did not compromise the nucleolar localization of murine NDH II.

3T3 Cells↗
PubMed1999

The mitotically phosphorylated form of the transcription termination factor TTF-1 is associated with the repressed rDNA transcription machinery.

The transcription termination factor TTF-1 exerts two functions in ribosomal gene (rDNA) transcription: facilitating initiation and mediating termination of transcription. Using HeLa cells, we show that TTF-1 protein is colocalized with the active transcription machinery in the nucleolus and also with the inactive machinery present in certain mitotic nucleolar organizer regions (NORs) when rDNA transcription is repressed. We also show that TTF-1 is specifically phosphorylated during mitosis in a manner dependent on the cdc2-cyclin B kinase pathway and on an okadaic acid-sensitive phosphatase. Interestingly, the mitotically phosphorylated form of TTF-1 appearing at the G(2)/M transition phase was more easily solubilized than was the interphase form. This indicates that the chromatin-binding affinity of TTF-1 appears to be different in mitotic chromosomes compared to the interphase nucleolus. Correlated with this, the other DNA-binding factor, UBF, which interferes with chromatin conformation in the rDNA promoter, was more strongly bound to rDNA during mitosis than at interphase. The reorganization of the mitotic rDNA promoter might be induced by phosphorylation of certain components of the rDNA transcription machinery and participate in silencing of rDNA during mitosis.

Antibody Specificity↗
PubMed2001

Molecular architecture of the amplified nucleoli of Xenopus oocytes.

An understanding of the functional organization of nucleoli, the sites of ribosome biosynthesis, is limited by the present uncertainty about the topological arrangement of the transcribing rRNA genes. Since studies with "standard" nucleoli from somatic cells produced conflicting results, we have examined the amplified nucleoli of Xenopus oocytes. These nucleoli are unique in that they contain high copy numbers of rRNA genes, are not attached to chromosomes, lack non-ribosomal DNA and can be examined in light microscopic spread preparations of nuclear contents. By immunostaining and confocal microscopy we show that in growing stage IV oocytes the sites of rDNA are surrounded by the dense fibrillar component. The rDNA is actively transcribed as revealed by BrUTP injection into oocytes and localization of components of the nucleolar transcription machinery (RNA polymerase I and the transcription factor UBF). At the ultrastructural level, the rDNA sites correlate with the fibrillar centers of amplified nucleoli fixed in situ. The results provide clear evidence that the transcriptionally active rRNA genes are confined to the fibrillar centers of the oocyte nucleoli and open the possibility to analyze the protein composition of almost native, transcriptionally highly active nucleolar chromatin by immunofluorescence microscopy.

Animals↗
PubMed2003

Effects of propofol-sevoflurane anesthesia on the maternal and fetal hemodynamics blood gases, and uterine activity in pregnant goats.

To determine the effects of propofol and sevoflurane on hemodynamics, acid-base balance and uterine activity in pregnant animals, a prospective experimental study was designed by use of ten pregnant goats. Propofol was intravenously administered at a bolus dose of 5 mg/kg and then infused a rate of 0.3 mg/kg/min for 5 min. Following the induction, the animals were incrementally inhaled 2.7 and 4.1% of end-tidal concentration of sevoflurane each for 30 min, and then recovered. The maternal and fetal heart rate (HR), arterial blood pressure (BP) and acid-base balance, the intrauterine pressure (IUP), and the uterine blood flow (UBF) were measured. Following the pre-anesthetic data, the parameters were measured 7 times throughout the anesthetic and recovering periods. The propofol infusion induced 1.37 times of HR increase and produced decrease in PO(2) and a relevant metabolic acidemia in the mother, with no effect in the fetus. Sevoflurane reduced BP in the fetus from 30 (2.7%) to 60 (4.1%) min of inhalation. The uterine contractions disappeared throughout sevoflurane inhalation, and then recurred within 15 min after the cessation of sevoflurane. Propofol injection increases HR, and induces a moderate hypoxemia and metabolic acidemia associated with the suppressed ventilation for pregnant goats, with less effect on the fetal hemodinamics. Sevoflurane causes minimal change in maternal hemodynamics, but induces significant hypotension in the fetus and reduction of uterine activity. These data may be useful in making anesthetic choices combined with analgesia for Caesarian section in goats.

Anesthetics, Inhalation↗
PubMed2006

Regulation of rRNA synthesis in human and mouse cells is not determined by changes in active gene count.

Growth regulation of the tandemly repeated ribosomal RNA (rRNA) genes in mammals can potentially occur by several distinct mechanisms. Only a fraction of the 200 or so rRNA genes appears to be activated in somatic cells, leaving open the possibility that enhanced transcription could result from gene activation events. Here we have determined the active rRNA gene count after growth stimulation with EGF, direct Raf activation and chromatin hyperacetylation and after inhibiting MAP-kinase signaling. Despite robust changes in rRNA transcription rates, we find no significant variation in active gene number in either mouse fibroblasts or human neuroepithelioma cells. Interestingly, the data also show that rRNA transcription enhancement induced by hyperacetylation is dependent on MEK/ERK signaling. Since ERK and the acetyltransferase CBP both bind the architectural factor UBF, this suggests a mechanism for targeting active CBP to the rRNA genes.

Acetylation↗
PubMed2002

[Characteristics of nucleolar organization and function under condition of complete spatial in situ disassembly of its main structural components].

It is well known that fibrillar centers (FC) constitute an essential structural component of the active nucleolus in mammalian cells, yet their role in regulation of ribosomal gene transcription still remains an open question. Here, we studied the activity of endogenous RNA polymerase I upon partial and complete unraveling of nucleoli and FCs. The pattern of BrUTP incorporation in nuclei of hypotonically-treated cells was shown to be essentially the same as in the control untreated cells. Moreover, the sites of BrUTP incorporation, which revealed the active PNA polymerase I, were completely coincident with UBF-binding sites. These observations allow to conclude that structural integrity of FCs is not a prerequisite for maintenance of the active RNA polymerase I transcriptional complex. When the action of hypotonic shock was ceased and the cells were transferred to a complete cultural medium, the swollen nucleoli recovered to the control state. Therefore it is possible to conclude that none of the main morphological nucleolar counterparts, such as FCs, dense fibrillar component or the pars granulosa, is responsible for the maintenance of the nucleolar structural and functional integrity. A suggestion is made that this role may be played by the nucleolar matrix associated with the RNA polymerase I transcriptional complex.

Cell Nucleolus↗
PubMed2002

Autoantibodies to NOR 90/hUBF: longterm clinical and serological followup in a patient with limited systemic sclerosis suggests an antigen driven immune response.

We describe the clinical and serological followup of a 9-year-old girl with anti-nucleolar organizing region 90/human upstream-binding factor (anti-NOR 90/hUBF) who had features of systemic sclerosis over a period of 17 years, from childhood into adulthood. We review the associations of anti-UBF autoantibodies, and provide evidence that anti-NOR 90/UBF immune response is antigen driven.

Autoantibodies↗
PubMed2002

Recognition and repair of DNA-cisplatin adducts.

Anticancer activity of cisplatin (cis-diamminedichloroplatinum) is believed to result from its interaction with DNA. The drug reacts with nucleophilic sites in DNA forming monoadducts as well as intra- and interstrand crosslinks. DNA-cisplatin adducts are specifically recognized by several proteins. They can be divided into two classes. One constitutes proteins which recognize DNA damage as an initial step of the nucleotide excision and mismatch repair pathways. The other class contains proteins stabilizing cellular DNA-protein and protein-protein complexes, including non-histone proteins from the HMG (high-mobility-group) family. They specifically recognize 1,2-interstrand d(GpG) and d(ApG) crosslinks of DNA-cisplatin adducts and inhibit their repair. Many HMG-domain proteins can function as transcription factors, e.g. UBF, an RNA polymerase I transcription factor, the mammalian testis-determining factor SRY and the human mitochondrial transcription factor mtTFA. Moreover, it seems that some proteins, which probably recognize DNA-cisplatin adducts non-specifically, e.g. actin and other nuclear matrix proteins, can disturb the structural and functional organization of the nucleus and whole cell. The formation of complexes between DNA and proteins in the presence of cisplatin and the changes in the cell architecture may account for the drug cytotoxicity.

Actins↗
PubMed2003

[Activation of transcription of ribosome genes following human embryo fibroblast infection with cytomegalovirus in vitro].

Cytomegalovirus (CMV) infection of human diploid embryo fibroblasts in vitro causes a massive cell death on the 3-4th day of infection with a high primary infection coefficient (1-5 U/cell). Cytopathological effects of viral infection on the 3-4th day includes diminishes of the cell size, changes in their form, compaction of the nuclear chromatin, and disorganization and inactivation of the nucleolus. However, the early stages of the viral infection progression (the 1st-2nd day) are accompanied by unequivocal activation of rDNA (ribosomal gene) and the bulk of chromatin transcription. There are several features to support this conclusion: In the early CMV-infected cell 1) the nucleolar size is increased; 2) the number of intranucleolar foci binding the specific RNA-polymerase I transcription factor (UBF) is augmented; 3) the Ag-NOR staining is enhanced; 4) 3H-uridine incorporation to the nucleoli is activated; 5) the ultrastructure of the nucleolus is changed. Altogether, these data argue in favor of activation of rDNA transcription in human fibroblasts in vitro at the initial stages of infection.

Cell Nucleolus↗
PubMed2005

Effect of internal recycle rate on the high-strength nitrogen wastewater treatment in the combined UBF/MBR system.

An anaerobic/aerobic system combining an anaerobic upflow-sludge bed filter (UBF) and an aerobic membrane bioreactor (MBR) was operated to enhance organic and nitrogen removal efficiency. The internal recycle rate, which is one of the most important operation factors that affects overall removal efficiency, was varied from 100% to 300% of the influent flow. Under these conditions, the overall removal efficiencies of organic and nitrogen and characteristics of membrane fouling in the combined system treating the synthetic wastewater including high concentration of organics and nutrients were studied. As a result, nitrogen removal efficiency was increased to 67% when the internal recycle rate was 300% of influent flow rate. As the internal recycle ratio increased from 100% to 200%, protein content decreased by 17% and carbohydrate content increased by 12%. However, there was no remarkable difference in total extracellular polymeric substances (EPS) content. At the high recycle rate of 300%, the surface charge of sludge was decreased while hydrophobicity (specific ultraviolet absorbance, SUVA) was increased. The differences in SUVA and surface charge were 11% and 1%, respectively. It is concluded that SUVA and EPS composition were important parameters affecting membrane fouling in the combined system.

Bacteria, Aerobic↗
PubMed2005

[Premature assembly of nucleolus-derived foci induced by a reversible hypotonic shock in metaphase CV1 and HeLa cells].

The assembly of nucleolus-derived foci (NDF) in the cytoplasm of telophase cells is an early stage of nucleolus reassembly during mitosis. In current literature, significant attention is paid to the molecular composition of NDF and their participation in reassembly of the mature nucleolus. However, very little is known about mechanisms controlling the NDF formation. The authors have demonstrated for the first time that a reversible action of low ionic strength buffers (lypotonic shock treatment) on living mitotic human HeLa and green monkey CV1 cells triggers a premature assembly of NDF at metaphase. Like the true NDF, i. e., those assembled in telophase mitosis, NDF prematurally induced at metaphase contain RNA and proteins required for rRNA processing (fibrillarin, B23/nucliophosmin, C23/nucleolin), but lack UBF, an auxiliary factor of RNA polymerase I. We have assumed that a reversible action of hypotonic shock on metaphase cells may result in temporal increase in intracellular [Ca2+](i) that, in its turn, may induce a premature assembly of NDF under isotonic conditions. The structural integrity of the mitotic spindle apparently plays an essential role in the response of metaphase cells to hypotonic shock treatments.

Animals↗
PubMed1984

Acute embolization of the uteroplacental circulation: uterine blood flow and placental CO diffusing capacity.

In an effort to determine to what extent the fetal sequalae following repeated embolization result from decreased area of placental exchange or from decreased uterine blood flow, we injected microspheres into the uterine circulation of the pregnant ewe. We measured total UBF continuously and sampled fetal blood gases in 6 chronically instrumented ewes following repeated injections of 1 to 2 million 25 mu microspheres into the common internal iliac artery at 30 min intervals. Embolization resulted in an immediate 25 to 30% drop in uterine flow, with partial recovery to about 85% of its control value within 30 min after injection. A linear relation existed between uterine blood flow and fetal O2 tension. A slightly accelerated decrease in O2 content with a more rapid increase in CO2 tension and [H+] was seen when uterine flow decreased below 150 ml X min-1 X kg fetal wt-1. Following repeated injections fetal descending aortic O2 tension and content decreased 34 and 82% respectively, while PCO2 and [H+] increased 28 and 84% respectively. Placental diffusing capacity for CO increased 117% after repeated embolization. Most of this increase could be accounted for by the fetal hypoxia and acidosis, although some of it may have resulted from distension or recruitment of vessels in the placental exchange area, or a more uniform distribution of placental blood flows. These studies suggest that the acute changes in fetal blood gas values following embolization result from a reduction in blood flow rather than from a reduced placental exchange area.

Animals↗
PubMed1982

[Experimental study of renal and uterine blood flow regulation in obstetric hypovolemic shock (author's transl)].

Dynamics of the renal and uterine circulation in response to the hypovolemic shock were studied in anesthetized pregnant and nonpregnant dogs. The renal arterial blood flow (RABF), uterine arterial blood flow (UABF) and mean arterial blood pressure (MAP) were electronically integrated and recorded during the removal of 500 ml blood at the rate of 3 ml per minute. In nonpregnant bitches, there was a pronounced decrease in RABF after the 300 ml blood removal, and it became 85.2 per cent from the control level at the time of 500 ml blood removal (MAP 34.6 mmHg). In pregnant bitches however, the rapid decrease in UABF was invoked after the 200 ml blood removal, reaching 85.3 per cent decrease from the control level at the time of 500 ml blood removal (MAP 59.8 mmHg), while the decrease in RBF was attenuated in comparison with the nonpregnant animals. The decrease in UBF in pregnant animal was associated with remarkable increase in uterine vascular resistance. This implies that the hypovolemic shock generates a lending and borrowing phenomenon between the renal and uterine circulation in pregnant subject. As evidenced by the enhanced vasoconstrive responses of uterine vascular bed to injected norepinephrine (50 microgram) during the hypovolemic shock in pregnant animals, the activation on alpha receptors in the uterine vascular bed is the main cause of the phenomenon.

Animals↗