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PubMed2001

Sex hormone levels in premenopausal African-American women with upper and lower body fat phenotypes.

Body fat distribution may be a better marker of a hormonal pattern associated with increased breast cancer risk than obesity. This cross-sectional study of 106 healthy premenopausal African-American (AA) women compared the midfollicular phase sex hormone and sex hormone-binding globulin levels in upper body fat (UBF) and lower body fat (LBF) phenotype and obese and nonobese women. Multivariate regression analyses were used to control for various confounders, including dietary factors. UBF phenotype women had 37% (P = 0.02), 50% (P = 0.01), 52% (P = 0.007), and 50% (P = 0.009) higher levels of estradiol (E2), free E2, testosterone (T), and free T, respectively, than LBF phenotype women. Only %free T was higher in obese than in nonobese women (P = 0.02). The levels of E2, free E2, %free E2, T, and free T were higher [by 42% (P = 0.01), 68% (P = 0.001), 18% (P = 0.04), 36% (P = 0.04), and 61% (P = 0.01), respectively] and the level of sex hormone-binding globulin was lower [by 28% (P = 0.04)] in obese UBF than in nonobese LBF phenotype women. These findings support the hypothesis that body fat distribution may be a better marker of a hormonal pattern associated with increased breast cancer risk than obesity. Obese UBF phenotype AA women, in particular, have a high-risk hormonal profile. Future breast cancer studies might consider controlling for measures of obesity and body fat distribution to minimize confounding.

Adipose Tissue↗
PubMed1998

Cisplatin inhibits synthesis of ribosomal RNA in vivo.

Cis -diammininedichloroplatinum(II) (cisplatin or cis -DDP) is a DNA-damaging agent that is widely used in cancer chemotherapy. Cisplatin crosslinks DNA and the resulting adducts interact with proteins that contain high-mobility-group (HMG) domains, such as UBF(upstream binding factor). UBF is a transcription factor that binds to the promoter of ribosomal RNA (rRNA) genes thereby supporting initiation of transcription by RNA polymerase I. Here we report that cisplatin causes a redistribution of UBF in the nucleolus of human cells, similar to that observed after inhibition of rRNA synthesis. A similar redistribution was observed for the major components of the rRNA transcription machinery, namely TBP, TAFIs and RNA polymerase I. Furthermore, we provide for the first time direct in vivo evidence that cisplatin blocks synthesis of rRNA, while activity of RNA polymerase II continues to be detected throughout the nucleus. The clinically ineffective trans isomer (trans -DDP) does not alter the localization of either UBF or other components of the RNA polymerase I transcription machinery. These results suggest that disruption of rRNA synthesis, which is stimulated in proliferating cells, plays an important role in the clinical success of cisplatin.

Antineoplastic Agents↗
PubMed2003

Regulation of ribosomal RNA synthesis during the final phases of porcine oocyte growth.

In porcine oocytes, acquisition of meiotic competence coincides with a decrease of general transcriptional activity at the end of the oocyte growth phase and, specifically, of ribosomal RNA (rRNA) synthesis in the nucleolus. The present study investigated the regulation of rRNA synthesis during porcine oocyte growth. Localization and expression of components involved in regulation of the rRNA synthesis (the RNA polymerase I-associated factor PAF53, upstream binding factor [UBF], and the pocket proteins p130 and pRb) were assessed by immunocytochemistry and semiquantitative reverse transcription-polymerase chain reaction and correlated with ultrastructural analysis and autoradiography following [3H]uridine incubation in growing and fully grown porcine oocytes. In addition, meiotic resumption, ultrastructure, and expression of p130, UBF, and PAF53 were analyzed in growing and fully grown porcine oocytes cultured with 100 microM butyrolactone I (BL-I), a potent inhibitor of cyclin-dependent kinases, to gain insight concerning the regulation of rRNA transcription during meiotic arrest. Immunocytochemical analysis demonstrated that p130 became colocalized with UBF and PAF53 and that the intensity of the PAF53 labeling decreased toward the end of the oocyte growth phase. These data suggest that the decrease in rRNA synthesis is regulated through inhibition of UBF by p130 as well as by decreased availability of PAF53. Moreover, expression of mRNA encoding PAF53 was decreased at the end of the oocyte growth phase. At the morphological level, these events coincided with inactivation of the nucleolus, as visualized by the transformation of the fibrillogranular nucleolus to an electron-dense fibrillar sphere with remnants of the fibrillar centers at the surface. Meiotic inhibition with 100 microM BL-I had a detrimental effect on the ability of porcine oocytes to resume meiosis and on nucleolus morphology, resulting in a lack of RNA synthetic capability as the fibrillar components, where rRNA transcription and initial processing occur, condensed or even disintegrated.

4-Butyrolactone↗
PubMed2000

Nucleolar proteins and nuclear ultrastructure in preimplantation bovine embryos produced in vitro.

The aim of the present investigation was to describe the basic cell biology of the postfertilization activation of rRNA genes using in vitro-produced bovine embryos as a model. We used immunofluorescence confocal laser scanning microscopy and transmission electron microscopy to study nucleolar development in the nuclei of embryos up to the fifth postfertilization cell cycle. During the first cell cycle (1-cell stage), fibrillarin, upstream binding factor (UBF), nucleolin (C23), and RNA polymerase I were localized to distinct foci in the pronuclei, and, ultrastructurally, compact spherical fibrillar masses were the most prominent pronuclear finding. During the second cell cycle (2-cell stage), the findings were similar except for a lack of nucleolin and RNA polymerase I labeling. During the third cell cycle (4-cell stage), fibrillarin, UBF, nucleophosmin, and nucleolin were localized to distinct foci. Ultrastructurally, spherical fibrillar masses that developed a central vacuole over the course of the cell cycle were observed. Early in the fourth cell cycle (8-cell stage), fibrillarin, nucleophosmin, and nucleolin were localized to small bodies that with time developed a central vacuole. UBF and topoisomerase I were localized to clusters of small foci. Ultrastructurally, spherical fibrillar masses with a large eccentric vacuole and later small peripheral vacuoles were seen. Late in the fourth cell cycle, nucleophosmin and nucleolin were localized to large shell-like bodies; and fibrillarin, UBF, topoisomerase I, and RNA polymerase I were localized to clusters of small foci. Ultrastructurally, a presumptive dense fibrillar component (DFC) and fibrillar centers (FCs) were observed peripherally in the vacuolated spherical fibrillar masses. Subsequently, the presumptive granular component (GC) gradually became embedded in the substance of this entity, resulting in the formation of a fibrillo-granular nucleolus. During the fifth cell cycle (16-cell stage), a spherical fibrillo-granular nucleolus developed from the start of the cell cycle. In conclusion, the nucleolar protein compartment in in vitro-produced preimplantation bovine embryos is assembled over several cell cycles. In particular, RNA polymerase I and topoisomerase I are detected for the first time late during the fourth embryonic cell cycle, which coincides with the first recognition of the DFC, FCs, and GC at the ultrastructural level.

Animals↗
PubMed1997

Maternal and fetal effects of isoproterenol in the gravid ewe.

Detection of the intravascular placement of epidural catheters is an important but difficult task. In this study, we evaluated maternal and fetal hemodynamic responses to intravenous (i.v.) and epidural injection of isoproterenol (ISO), a proposed chronotropic test dose, in gravid ewes. Near-term, chronically instrumented, gravid ewes with single fetuses were studied at least 48 h after surgery. We continuously recorded maternal heart rate (MHR), systemic and pulmonary blood pressures, uterine blood flow (UBF), and fetal blood pressure and heart rate. Maternal cardiac output was measured by thermodilution. In random sequence, each ewe (n = 11) received i.v. injections of saline, epinephrine (EPI) 15 microg; ISO 4, 16, and 80 microg; or epidural (n = 9 ewes) injections of saline, ISO 4 microg and ISO 40 microg. All variables returned to baseline between experiments. Sections of lumber spinal cord were harvested from five animals for later histopathological study. I.v. ISO caused a dose-related increase in MHR. Cardiac output also increased transiently after all doses of ISO but not after EPI. Maternal diastolic blood pressure decreased after ISO 16 and 80 microg. UBF decreased significantly for 120 s after EPI 15 microg. Epidural ISO did not significantly change maternal systemic or pulmonary blood pressure, cardiac output, or UBF. The 40-microg dose increased MHR significantly. No histopathological changes were seen in three ISO-exposed and two control spinal cords. I.v. ISO reliably induces maternal tachycardia in nonstressed gravid ewes. Unlike EPI, I.v. ISO lacks a statistically significant effect on UBF. However, ISO seems to be rapidly absorbed from the epidural space. Identifying the source of maternal tachycardia after epidural injection of a large dose of ISO could be difficult. If the absence of histopathological change is confirmed, ISO represents an alternative to EPI as a chronotropic test dose.

Adrenergic Agonists↗
PubMed2005

Modifications of both selectivity factor and upstream binding factor contribute to poliovirus-mediated inhibition of RNA polymerase I transcription.

Soon after infection, poliovirus (PV) shuts off host-cell transcription, which is catalysed by all three cellular RNA polymerases. rRNA constitutes more than 50 % of all cellular RNA and is transcribed from rDNA by RNA polymerase I (pol I). Here, evidence has been provided suggesting that both pol I transcription factors, SL-1 (selectivity factor) and UBF (upstream binding factor), are modified and inactivated in PV-infected cells. The viral protease 3C(pro) appeared to cleave the TATA-binding protein-associated factor 110 (TAF(110)), a subunit of the SL-1 complex, into four fragments in vitro. In vitro protease-cleavage assays using various mutants of TAF(110) and purified 3C(pro) indicated that the Q(265)G(266) and Q(805)G(806) sites were cleaved by 3C(pro). Both SL-1 and UBF were depleted in PV-infected cells and their disappearance correlated with pol I transcription inhibition. rRNA synthesis from a template containing a human pol I promoter demonstrated that both SL-1 and UBF were necessary to restore pol I transcription fully in PV-infected cell extracts. These results suggested that both SL-1 and UBF are transcriptionally inactivated in PV-infected HeLa cells.

3C Viral Proteases↗
PubMed1995

Coactivator and promoter-selective properties of RNA polymerase I TAFs.

Human ribosomal RNA synthesis by RNA polymerase I requires the activator UBF and the promoter selectivity factor SL1, which consists of the TATA binding protein (TBP) and three associated subunits, TAFI110, TAFI63, and TAFI48. Here it is shown that both TAFI110 and TAFI63 contact the promoter, whereas TAFI48 serves as a target for interaction with UBF and is required to form a transcriptionally active SL1 complex responsive to UBF in vitro. TAFI48 also alters the ability of TBP to interact with TATA box elements, and the resulting complex fails to support transcription by RNA polymerase II. Thus, TAFI48 may function both as a target to mediate UBF activation and as a class-specific promoter selectivity factor.

DNA-Binding Proteins↗
PubMed2006

Casein kinase 2 associates with initiation-competent RNA polymerase I and has multiple roles in ribosomal DNA transcription.

Mammalian RNA polymerase I (Pol I) complexes contain a number of associated factors, some with undefined regulatory roles in transcription. We demonstrate that casein kinase 2 (CK2) in human cells is associated specifically only with the initiation-competent Pol Ibeta isoform and not with Pol Ialpha. Chromatin immunoprecipitation analysis places CK2 at the ribosomal DNA (rDNA) promoter in vivo. Pol Ibeta-associated CK2 can phosphorylate topoisomerase IIalpha in Pol Ibeta, activator upstream binding factor (UBF), and selectivity factor 1 (SL1) subunit TAFI110. A potent and selective CK2 inhibitor, 3,8-dibromo-7-hydroxy-4-methylchromen-2-one, limits in vitro transcription to a single round, suggesting a role for CK2 in reinitiation. Phosphorylation of UBF by CK2 increases SL1-dependent stabilization of UBF at the rDNA promoter, providing a molecular mechanism for the stimulatory effect of CK2 on UBF activation of transcription. These positive effects of CK2 in Pol I transcription contrast to that wrought by CK2 phosphorylation of TAFI110, which prevents SL1 binding to rDNA, thereby abrogating the ability of SL1 to nucleate preinitiation complex (PIC) formation. Thus, CK2 has the potential to regulate Pol I transcription at multiple levels, in PIC formation, activation, and reinitiation of transcription.

Antigens, Neoplasm↗
PubMed1990

rUBF, an RNA polymerase I transcription factor from rats, produces DNase I footprints identical to those produced by xUBF, its homolog from frogs.

Rat cells contain a DNA-binding polymerase I transcription factor, rUBF, with properties similar to UBF homologs that have been purified from both human (hUBF) and frog (xUBF) cells. In this note we report the affinity purification of rUBF to apparent homogeneity and show that UBFs from both rat and frog have identical footprinting characteristics on templates from either species. Furthermore, xUBF was able to stimulate transcription from rat RNA polymerase I promoters in a partially fractionated rat extract that was UBF dependent. These results strengthen the conclusion that all vertebrate cells contain a UBF homolog whose DNA-binding specificity and function have been strongly conserved.

Animals↗
PubMed1996

Upstream binding factor stabilizes Rib 1, the TATA-binding-protein-containing Xenopus laevis RNA polymerase I transcription factor, by multiple protein interactions in a DNA-independent manner.

Initiation of RNA polymerase I transcription in Xenopus laevis requires Rib 1 and upstream binding factor (UBF). UBF and Rib 1 combine to form a stable transcription complex on the Xenopus ribosomal gene promoter. Here we show that Rib 1 comprises TATA-binding protein (TBP) and TBP-associated factor components. Thus, Rib 1 is the Xenopus equivalent of mammalian SL 1. In contrast to SL 1, Rib 1 is an unstable complex that readily dissociates into TBP and associated components. We identify a novel function for UBF in stabilizing Rib 1 by multiple protein interactions. This stabilization occurs in solution in a DNA-independent manner. These results may partially explain the difference in UBF requirement between Xenopus and mammalian systems.

Amino Acid Sequence↗
PubMed2001

A step subsequent to preinitiation complex assembly at the ribosomal RNA gene promoter is rate limiting for human RNA polymerase I-dependent transcription.

The assembly, disassembly, and functional properties of transcription preinitiation complexes (PICs) of human RNA polymerase I (Pol I) play a crucial role in the regulation of rRNA gene expression. To study the factors and processes involved, an immobilized-promoter template assay has been developed that allows the isolation from nuclear extracts of functional PICs, which support accurate initiation of transcription. Immunoblotting of template-bound factors showed that these complexes contained the factors required to support initiation of transcription, SL1, upstream binding factor (UBF), and Pol I. We have demonstrated that, throughout a single round of transcription, SL1 and UBF remain promoter bound. Moreover, the promoter-bound SL1 and UBF retain the ability to function in transcription initiation. SL1 has a central role in the stable association of the PIC with the promoter DNA. The polymerase component of the PIC is released from the promoter during transcription yet is efficiently recycled and able to reinitiate from "poised" promoters carrying SL1 and UBF, since the PICs captured on the immobilized templates sustained multiple rounds of transcription. Kinetic analyses of initiation of transcription by Pol I revealed that Pol I-dependent transcription is rate limited in a step subsequent to recruitment and assembly of Pol I PICs. The rate of RNA synthesis is primarily determined by the rates at which the polymerase initiates transcription and escapes the promoter, referred to as promoter clearance. This rate-limiting step in Pol I transcription is likely to be a major target in the regulation of rRNA gene expression.

Binding Sites↗
PubMed1998

Regulation of ribosomal DNA transcription by insulin.

The experiments reported here used 3T6-Swiss albino mouse fibroblasts and H4-II-E-C3 rat hepatoma cells as model systems to examine the mechanism(s) through which insulin regulates rDNA transcription. Serum starvation of 3T6 cells for 72 h resulted in a marked reduction in rDNA transcription. Treatment of serum-deprived cells with insulin was sufficient to restore rDNA transcription to control values. In addition, treatment of exponentially growing H4-II-E-C3 with insulin stimulated rDNA transcription. However, for both cell types, the stimulation of rDNA transcription in response to insulin was not associated with a change in the cellular content of RNA polymerase I. Thus we conclude that insulin must cause alterations in formation of the active RNA polymerase I initiation complex and/or the activities of auxiliary rDNA transcription factors. In support of this conclusion, insulin treatment of both cell types was found to increase the nuclear content of upstream binding factor (UBF) and RNA polymerase I-associated factor 53. Both of these factors are thought to be involved in recruitment of RNA polymerase I to the rDNA promoter. Nuclear run-on experiments demonstrated that the increase in cellular content of UBF was due to elevated transcription of the UBF gene. In addition, overexpression of UBF was sufficient to directly stimulate rDNA transcription from a reporter construct. The results demonstrate that insulin is capable of stimulating rDNA transcription in both 3T6 and H4-II-E-C3 cells, at least in part by increasing the cellular content of components required for assembly of RNA polymerase I into an active complex.

Animals↗
PubMed1989

Uterine and nonuterine vascular responses to angiotensin II in ovine pregnancy.

The uteroplacental vasculature is more refractory to angiotensin II (ANG II) than the systemic vasculature as a whole. To ascertain the differences in responses between reproductive and nonreproductive tissues that account for this, we infused ANG II (0.573, 5.73, and 11.5 micrograms/min) in pregnant sheep (137 +/- 5 days of gestation) and monitored arterial pressure (MAP), heart rate, and uterine blood flow (UBF); cardiac output and regional blood flows were measured with radiolabeled microspheres. Dose-dependent changes in MAP, UBF, and systemic (SVR) and uterine (UVR) vascular resistance occurred (P less than 0.05); systemic responses exceeded uterine (P less than 0.05), except with 11.5 micrograms/min, when % delta UVR = % delta SVR, % delta UVR greater than % delta MAP, and UBF fell 29%. Although a dose-dependent rise in placental resistance occurred, blood flow was unaffected except at 11.5 micrograms ANG II/min, falling 16.8 +/- 3.5% (P = 0.059). In contrast, endometrial perfusion decreased 68 +/- 4.2 and 81 +/- 1.8% (P less than 0.01) with 5.73 and 11.5 micrograms ANG II/min, respectively. Myometrial responses were intermediate, thus placental flow increased from 75 to greater than 90% of total UBF. Adipose, renal, and adrenal glands were extremely sensitive to ANG II, with blood flows decreasing maximally at 0.573 micrograms/min (P less than 0.05). Maximum adipose vascular resistance occurred at 0.573 micrograms/min, greater than 400% (P less than 0.001), exceeding responses in all tissues (P less than 0.05). The placenta is less responsive to ANG II than other uterine and most nonreproductive tissues, resulting in preferential maintenance of uteroplacental perfusion and protecting the fetus from the effects of this vasoconstrictor.

Angiotensin II↗
PubMed1986

Endogenous hormones and regulation of uterine blood flow during pregnancy.

Fourteen pregnant sheep were prepared for measurements of blood flow through a major uterine artery (UBF) and of arterial concentrations of estrone, estradiol, and progestagen. Five of the animals were ovariectomized. Measurements were made daily (n = 12) or every 4 h for 24 h (n = 4). Statistically significant (P less than 0.01) correlation coefficients were detected between UBF and hormone, either alone or in combination; the closest and most consistent associations were those in which estrogen and progestagen concentrations were summed or multiplied. Ewes with higher hormonal concentrations were those that had the higher rates of UBF. Correlation coefficients were statistically significant more often among ovariectomized animals. No associations were detected within one animal that delivered a growth-retarded fetus nor from any of the 24-h data. Estrogens and progestagen accounted for as much as 25% of the daily variation of UBF among and within sheep during the last half of pregnancy.

Animals↗
PubMed1999

Hemodynamic effects of platelet-activating factor in nonpregnant and pregnant sheep.

The present study was designed to assess the dose-related effects of platelet-activating factor (PAF) on systemic, renal, and uterine hemodynamics in nonpregnant sheep and to evaluate how pregnancy might alter these responses. Nonpregnant and pregnant (110 +/- 5 days gestation) ewes were instrumented for conscious measurements of maternal mean arterial pressure (MAP), renal blood flow (RBF), uterine blood flow (UBF), hematocrit, and urinary protein concentration. After recovery, dose-response curves to PAF were generated by systemic infusion at 10, 30, and 100 ng. kg(-1). min(-1) (15 min/dose) into the maternal femoral vein. The above parameters were measured, and renal and uterine vascular resistances (RVR and UVR, respectively) were calculated. In pregnant sheep, PAF increased MAP, RVR, UVR, and urinary protein concentration. We also observed increases in hematocrit, indicative of reduced blood volume secondary to increased systemic microvascular protein permeability. These responses were similar in nonpregnant sheep, with the exception of UVR in nonpregnant ewes being decreased (and thus UBF was increased), whereas in pregnant sheep, UVR was increased, which resulted in decreased UBF. This suggests that pregnancy alters the mechanism of action of PAF within the uterine vasculature in a way that can reduce UBF and thereby potentially compromise placental perfusion.

Animals↗
PubMed1992

Evidence of the occurrence of calcitonin cells in the ultimobranchial follicle of the rat postnatal thyroid.

A study on thyroid glands of Wistar rats of ages ranging from 1 to 120 days was carried out. The glands were serially sectioned and stained for calcitonin using the peroxidase antiperoxidase method. All the thyroids contained ultimobranchial follicles (UBF) located partially embedded among the usual follicles but in a 5-day-old rat this structure showed an unusual position in the interstitium of connective tissue between the cartilage of the trachea and the thyroid gland. We have observed in the wall of that UBF the presence not only of resting C cells but also mitotic figures of C cells. Furthermore, on the opposite side of the same UBF an active area of formation of thyroid follicles was found. These observations provided the first evidence of the contribution of the UBF in the formation of C cells during the postnatal life of the rat. Furthermore, it is suggested that some C cells may share a common origin with ultimobranchially derived follicular cells.

Aging↗
PubMed1997

Estrogen-regulated uterine vascularization modulates the guinea pig intrauterine environment.

The effects of experimentally-induced, uterine vascular restriction on uterine blood flow (UBF) and uterine blood volume (UBV) capacity, as well as the dependent intrauterine oxygen tension (IUpO2) measurements used as an indication of luminal nutrient availability, were examined using ovariectomized, estrogen (E)-treated guinea pigs. Following 3 days of E treatment, both UBF and UBV measurements were found to be elevated and associated with a causally-related increase in intraluminal uterine oxygen availability levels. Following the acute clamping of the uterine arteries, both UBF and UBV levels decreased dramatically and induced a rapid fall in associated intrauterine luminal oxygen tension measurements. As a result of chronic (i.e., 6 h) restriction of segmental blood flow to the uterus by vascular cauterization, both UBV and IUPO2 levels were suppressed as compared with sham-operated control levels, whereas UBF rates were not significantly altered. The results of the present studies are the first quantitative demonstration that either acute or chronic reductions in uterine vascular capacity or competency can induce rapid and dramatic changes in the intrauterine nutritional environment recognized to be essential for the initiation, support and maintenance of nidation and subsequent fetal-placental development.

Animals↗
PubMed1988

Effects of diabetes on uterine condition, decidualization, vascularization, and corpus luteum function in the pseudopregnant rat.

The effects of alloxan-induced diabetes on uterine decidualization and the associated changes in uterine blood flow (UBF) and corpus luteum activity were evaluated in rats between days 4-9 of pseudopregnancy (day 0 = ovulation). Rats were made diabetic (D) with a 40 mg/kg injection (iv) of alloxan on day 1 of pseudopregnancy. Saline-treated rats served as controls (C). Uterine weights were depressed in D rats between days 6-7 of pseudopregnancy in association with elevated blood glucose levels (greater than 300 mg/dl) relative to control values. UBF rates were also depressed in D rats between days 6-7 of pseudopregnancy compared with control values. Insulin replacement therapy (6 IU bovine/day) effectively normalized both uterine weight and UBF in diabetic rats. Decidual tissue (DT) growth was impaired in D rats between days 7 and 9 of pseudopregnancy (DT induction on day 4 of pseudopregnancy) compared with controls. Tissue blood flow rates were severely depressed throughout pseudopregnancy in D rats, but insulin treatment normalized both uterine parameters to control levels. Serum progesterone levels were lower in D rats than in controls between days 7 and 9 of pseudopregnancy. Daily insulin treatment normalized luteal function to control levels. The depressed DT weights in D rats were mimicked by the experimental reduction of DT blood flow in control rats. These results indicate that the uterine atrophy and poor endometrial decidualization that characterized the D rat are accompanied by impaired UBF and luteal activity. These findings suggest that the D-associated depression in female reproductive performance is related to the lack of proper hormone support of tissue vascular dynamics.

Animals↗