Transcription of ribosomal RNA genes is generally accepted to correlate with cell growth. Using primary cultures of adult bovine aortic endothelial (ABAE) cells, we have shown that transcription of rDNA in confluent cells falls to 5% of the transcription level in growing cells. Protein kinase NII appears to be a limiting factor to promote rDNA transcription in isolated nuclei of confluent cells. Protein kinase NII was detected by immunocytochemistry in the cytoplasm, nuclei and nucleoli of growing cells while it was no longer present in nucleoli of confluent cells. The kinase activity, in isolated nuclei, was estimated by endogenous phosphorylation of a specific substrate, nucleolin. A 10% residual activity was present in confluent cell nuclei compared to growing cell nuclei. Concomitantly, the transcription 'in vitro' of rDNA in the corresponding nuclei was also highly reduced (by 85%). Addition of exogenous protein kinase NII to confluent cell nuclei induced a strong increase in the phosphorylation of specific proteins including nucleolin. In parallel, the transcription of rDNA was increased by a factor of 5, to nearly the level observed in nuclei prepared from growing cells. These data suggest that, in confluent cells, factors necessary for rDNA transcription machinery are present but inactive in the nucleolus and that the phosphorylation of one or several of these factors (nucleolin, topoisomerase I,...) by protein kinase NII is a key event in the regulation of rDNA transcription.

Autoantibodies against U3 small nuclear ribonucleoprotein are associated with scleroderma autoimmune disease. They were shown to react with fibrillarin, a 34-to 36-kilodalton protein that has been detected in all eukaryotes tested from humans to yeasts. We isolated a 1.6-kilobase cDNA clone encoding fibrillarin from a Xenopus laevis cDNA library. The protein contains a 79-residue-long Gly-Arg-rich domain in its N-terminal region and a putative RNA-binding domain with ribonucleoprotein consensus sequence in its central portion. This is the first report of cloning of fibrillarin, and the deduced protein sequence is in agreement with the involvement of the protein in a ribonucleoprotein particle.By analogy with other small nuclear RNAs, the U3 small nuclear RNA, which is localized in the nucleolus (24, 29), has been proposed to participate in the maturation of prerRNA (2,8,24,28), even if the precise mechanism is still much debated (12,15,21). The secondary structure of U3 RNA and the protein composition of the U3 small nuclear ribonucleoprotein (snRNP) particle have been determined (22). One of the protein constituents, a 34-kilodalton (kDa) protein extremely rich in glycine and dimethylarginine (DMA) and with a pl of 8.5, was first described in Physarum polycephalum and shown to belong to a nuclear ribonucleoprotein (RNP) complex (5). Later, because of a fortuitous recognition of the protein by antiserum from a patient with scleroderma, the protein was identified in rats, biochemically characterized, and then detected in all eucaryotes (1,11,18,20). It is localized during the interphase exclusively in the fibrillar region of the nucleolus and thus has been named fibrillarin (20).We previously studied another nucleolar protein, nucleolin, whose localization during the cell cycle resembles that of fibrillarin (10,19) and which also contains a glycine-and DMA-rich domain (16). In a preliminary Northern (RNA) analysis with total RNA purified from X. laevis oocytes, we found that a cDNA probe encoding the Gly-DMA domain of Chinese hamster nucleolin (17) hybridized to a major band of 2.7 kilobases (kb) which corresponds to the X. laevis nucleolin mRNA (4). At a lower stringency, two other strongly hybridizing RNAs of 1.8 and 1.6 kb were also detected (Fig.

BackgroundThe PCSK9 antibody alirocumab (75 mg every 2 weeks; Q2W) as monotherapy reduced low‐density lipoprotein‐cholesterol (LDL‐C) levels by 47%. Because the option of a monthly dosing regimen is convenient, ODYSSEY CHOICE II evaluated alirocumab 150 mg Q4W in patients with inadequately controlled hypercholesterolemia and not on statin (majority with statin‐associated muscle symptoms), receiving treatment with fenofibrate, ezetimibe, or diet alone.Methods and ResultsPatients were randomly assigned to placebo, alirocumab 150 mg Q4W or 75 mg Q2W (calibrator arm), with dose adjustment to 150 mg Q2W at week (W) 12 if W8 predefined LDL‐C target levels were not met. The primary efficacy endpoint was LDL‐C percentage change from baseline to W24. Mean baseline LDL‐C levels were 163.9 mg/dL (alirocumab 150 mg Q4W, n=59), 154.5 mg/dL (alirocumab 75 mg Q2W, n=116), and 158.5 mg/dL (placebo, n=58). In the alirocumab 150 mg Q4W and 75 mg Q2W groups (49.1% and 36.0% of patients received dose adjustment, respectively), least‐squares mean LDL‐C changes from baseline to W24 were −51.7% and −53.5%, respectively (placebo [+4.7%]; both groups P<0.0001 versus placebo). In total, 63.9% and 70.3% of alirocumab‐treated patients achieved their LDL‐C targets at W24. Treatment‐emergent adverse events occurred in 77.6% (alirocumab 150 mg Q4W), 73.0% (alirocumab 75 mg Q2W), and 63.8% (placebo) of patients, with injection‐site reactions among the most common treatment‐emergent adverse events.ConclusionsAlirocumab 150 mg Q4W can be considered in patients not on statin with inadequately controlled hypercholesterolemia as a convenient option for lowering LDL‐C.Clinical Trial Registration URL: http://www.clinicaltrials.gov. Unique identifier: NCT02023879.

Calbindin-Dgk was quantified and its cellular location was defined in uterus, yolk sac, and placenta. In late gestation (days 17 to term) coordinated induction of calbindinDqk was seen in uterine epithelial lining cells and the juxtaposed yolk sac visceral epithelium as well as the intraplacental yolk sac epithelium. The induction of calbindin-Dk in these cells coincided with the time of exponential fetal bone growth and maximal fetal accumulation of calcium, suggesting a role of the protein in these epithelial layers in maternal-fetal calcium transport. Dynamic changes also occurred in the calbindin-Dgk contents of the two layers of uterine smooth muscle (outer longitudinal and inner circular) during mid-and late gestation.

The present study was undertaken to localize and investigate the endocrine control of immunoreactive 9K calbindin-D9k in the fallopian tube (oviduct) of the rat. Rat fallopian tubes were excised with the uterus, immediately fixed by freeze-substitution, and processed for immunoperoxidase staining. Staining employed a rabbit antiserum against purified rat intestinal calbindin-D9k and the streptavidin-biotin technique. Calbindin-D9k immunoreactivity was localized to luminal epithelial cells of the fallopian tube of mature rats, with no staining observed in other tissue layers of the tube. Epithelial cells in both the isthmus and the ampulla were positive for calbindin-D9k. In weanling rats, which have little ovarian function but high levels of 1,25-dihydroxyvitamin D, no immunoreactive calbindin-D9k was observed in any part of the tube. However, after daily injections of estradiol (6 micrograms/day) for 3 days, intense staining was observed in the epithelial cells of the immature rat fallopian tube. Progesterone treatment (1 mg/day for 3 days) of immature rats had no effect on calbindin-D9k in fallopian tube. The lumen of the fallopian tube (oviduct) is the key location for fertilization, a process that requires a narrowly defined concentration of extracellular calcium. By analogy to the intestine, calbindin-D9k may play a role in the transcellular movement of calcium across the fallopian tube epithelium in the fallopian tube lumen.