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A deficiency of 3-hydroxyisobutyric acid dehydrogenase (HIBADH) has been recently identified as a cause of primary 3-hydroxyisobutyric aciduria in two siblings; the only previously recognized primary cause had been a deficiency of methylmalonic semialdehyde dehydrogenase, the enzyme that is immediately downstream of HIBADH in the valine catabolic pathway and is encoded by the ALDH6A1 gene. Here we report on three additional patients from two unrelated families who present with marked and persistent elevations of urine L-3-hydroxyisobutyric acid (L-3HIBA) and a range of clinical findings. Molecular genetic analyses revealed novel, homozygous variants in the HIBADH gene that are private within each family. Evidence for pathogenicity of the identified variants is presented, including enzymatic deficiency of HIBADH in patient fibroblasts. This report describes new variants in HIBADH as an underlying cause of primary 3-hydroxyisobutyric aciduria and expands the clinical spectrum of this recently identified inborn error of valine metabolism. Additionally, we describe a quantitative method for the measurement of D- and L-3HIBA in plasma and urine and present the results of a valine restriction therapy in one of the patients., (© 2022 SSIEM.)

Our aim was to search for new cellular binding partners for the E6 and E7 oncogenes of beta human papillomaviruses (HPV), whose direct role in skin carcinogenesis has not been thoroughly investigated. By employing glutathione S-transferase pulldown and coimmunoprecipitation, we identified nuclear myosin 1c as a binding partner of HPV 8 E7 protein. As nuclear myosin 1c is an essential component of the RNA polymerase I transcription complex, we studied the effects of HPV 8 E7 protein expression on ribosomal RNA (rRNA) expression. Here we show that the activity of RNA polymerase I is decreased and that pre-rRNA expression is consequently reduced due to HPV 8 E7 expression. However, the expression levels of mature cytoplasmic 18S and 28S rRNA are retained. We propose that by relieving their resources from the energy-consuming process of rRNA transcription, HPV 8 E7 expressing cells might support more efficient virus replication in the differentiating epithelium.

R loops are three-stranded nucleic acid structures consisting of an RNA:DNA heteroduplex and a "looped-out" nontemplate strand. As aberrant formation and persistence of R loops block transcription elongation and cause DNA damage, mechanisms that resolve R loops are essential for genome stability. Here we show that the DEAD (Asp-Glu-Ala-Asp)-box RNA helicase DDX21 efficiently unwinds R loops and that depletion of DDX21 leads to accumulation of cellular R loops and DNA damage. Significantly, the activity of DDX21 is regulated by acetylation. Acetylation by CBP inhibits DDX21 activity, while deacetylation by SIRT7 augments helicase activity and overcomes R-loop-mediated stalling of RNA polymerases. Knockdown of SIRT7 leads to the same phenotype as depletion of DDX21 (i.e., increased formation of R loops and DNA double-strand breaks), indicating that SIRT7 and DDX21 cooperate to prevent R-loop accumulation, thus safeguarding genome integrity. Moreover, DDX21 resolves estrogen-induced R loops on estrogen-responsive genes in breast cancer cells, which prevents the blocking of transcription elongation on these genes., (© 2017 Song et al.; Published by Cold Spring Harbor Laboratory Press.)

SIRT7 is an NAD+-dependent protein deacetylase that regulates cell growth and proliferation. Previous studies have shown that SIRT7 is required for RNA polymerase I (Pol I) transcription and pre-rRNA processing. Here, we took a proteomic approach to identify novel molecular targets and characterize the role of SIRT7 in non-nucleolar processes. We show that SIRT7 interacts with numerous proteins involved in transcriptional regulation and RNA metabolism, the majority of interactions requiring ongoing transcription. In addition to its role in Pol I transcription, we found that SIRT7 also regulates transcription of snoRNAs and mRNAs. Mechanistically, SIRT7 promotes the release of P-TEFb from the inactive 7SK snRNP complex and deacetylates CDK9, a subunit of the elongation factor P-TEFb, which activates transcription by phosphorylating serine 2 within the C-terminal domain (CTD) of Pol II. SIRT7 counteracts GCN5-directed acetylation of lysine 48 within the catalytic domain of CDK9, deacetylation promoting CTD phosphorylation and transcription elongation., (© The Author(s) 2017. Published by Oxford University Press on behalf of Nucleic Acids Research.)

SIRT7 is an NAD(+)-dependent protein deacetylase with important roles in ribosome biogenesis and cell proliferation. Previous studies have established that SIRT7 is associated with RNA polymerase I, interacts with pre-ribosomal RNA (rRNA) and promotes rRNA synthesis. Here we show that SIRT7 is also associated with small nucleolar RNP (snoRNPs) that are involved in pre-rRNA processing and rRNA maturation. Knockdown of SIRT7 impairs U3 snoRNA dependent early cleavage steps that are necessary for generation of 18S rRNA. Mechanistically, SIRT7 deacetylates U3-55k, a core component of the U3 snoRNP complex, and reversible acetylation of U3-55k modulates the association of U3-55k with U3 snoRNA. Deacetylation by SIRT7 enhances U3-55k binding to U3 snoRNA, which is a prerequisite for pre-rRNA processing. Under stress conditions, SIRT7 is released from nucleoli, leading to hyperacetylation of U3-55k and attenuation of pre-rRNA processing. The results reveal a multifaceted role of SIRT7 in ribosome biogenesis, regulating both transcription and processing of rRNA.

Cell adhesion and migration are highly dynamic biological processes that play important roles in organ development and cancer metastasis. Their tight regulation by small GTPases and protein phosphorylation make interrogation of these key processes of great importance. We now show that the conserved dual-specificity phosphatase human cell-division cycle 14A (hCDC14A) associates with the actin cytoskeleton of human cells. To understand hCDC14A function at this location, we manipulated native loci to ablate hCDC14A phosphatase activity (hCDC14A(PD)) in untransformed hTERT-RPE1 and colorectal cancer (HCT116) cell lines and expressed the phosphatase in HeLa FRT T-Rex cells. Ectopic expression of hCDC14A induced stress fiber formation, whereas stress fibers were diminished in hCDC14A(PD) cells. hCDC14A(PD) cells displayed faster cell migration and less adhesion than wild-type controls. hCDC14A colocalized with the hCDC14A substrate kidney- and brain-expressed protein (KIBRA) at the cell leading edge and overexpression of KIBRA was able to reverse the phenotypes of hCDC14A(PD) cells. Finally, we show that ablation of hCDC14A activity increased the aggressive nature of cells in an in vitro tumor formation assay. Consistently, hCDC14A is down-regulated in many tumor tissues and reduced hCDC14A expression is correlated with poorer survival of patients with cancer, to suggest that hCDC14A may directly contribute to the metastatic potential of tumors. Thus, we have uncovered an unanticipated role for hCDC14A in cell migration and adhesion that is clearly distinct from the mitotic and cytokinesis functions of Cdc14/Flp1 in budding and fission yeast.

Non-coding RNAs play a key role in organizing the nucleus into functional subcompartments. By combining fluorescence microscopy and RNA deep-sequencing-based analysis, we found that RNA polymerase II transcripts originating from intronic Alu elements (aluRNAs) were enriched in the nucleolus. Antisense-oligo-mediated depletion of aluRNAs or drug-induced inhibition of RNA polymerase II activity disrupted nucleolar structure and impaired RNA polymerase I-dependent transcription of rRNA genes. In contrast, overexpression of a prototypic aluRNA sequence increased both nucleolus size and levels of pre-rRNA, suggesting a functional link between aluRNA, nucleolus integrity and pre-rRNA synthesis. Furthermore, we show that aluRNAs interact with nucleolin and target ectopic genomic loci to the nucleolus. Our study suggests an aluRNA-based mechanism that links RNA polymerase I and II activities and modulates nucleolar structure and rRNA production., (© 2015 The Authors.)

Mitotic repression of rRNA synthesis requires inactivation of the RNA polymerase I (Pol I)-specific transcription factor SL1 by Cdk1/cyclin B-dependent phosphorylation of TAF(I)110 (TBP-associated factor 110) at a single threonine residue (T852). Upon exit from mitosis, T852 is dephosphorylated by Cdc14B, which is sequestered in nucleoli during interphase and is activated upon release from nucleoli at prometaphase. Mitotic repression of Pol I transcription correlates with transient nucleolar enrichment of the NAD(+)-dependent deacetylase SIRT1, which deacetylates another subunit of SL1, TAFI68. Hypoacetylation of TAFI68 destabilizes SL1 binding to the rDNA promoter, thereby impairing transcription complex assembly. Inhibition of SIRT1 activity alleviates mitotic repression of Pol I transcription if phosphorylation of TAF(I)110 is prevented. The results demonstrate that reversible phosphorylation of TAF(I)110 and acetylation of TAFI68 are key modifications that regulate SL1 activity and mediate fluctuations of pre-rRNA synthesis during cell cycle progression.

Sirtuins are NAD(+)-dependent protein deacetylases that connect metabolism and cellular homeostasis. Here we show that the nuclear Sirtuin SIRT7 targets PAF53, a subunit of RNA polymerase I (Pol I). Acetylation of PAF53 at lysine 373 by CBP and deacetylation by SIRT7 modulate the association of Pol I with DNA, hypoacetylation correlating with increased rDNA occupancy of Pol I and transcription activation. SIRT7 is released from nucleoli in response to different stress conditions, leading to hyperacetylation of PAF53 and decreased Pol I transcription. Nucleolar detention requires binding of SIRT7 to nascent pre-rRNA, linking the spatial distribution of SIRT7 and deacetylation of PAF53 to ongoing transcription. The results identify a nonhistone target of SIRT7 and uncover an RNA-mediated mechanism that adapts nucleolar transcription to stress signaling., (Copyright © 2013 Elsevier Inc. All rights reserved.)

Entry into and progression through mitosis depends on phosphorylation and dephosphorylation of key substrates. In yeast, the nucleolar phosphatase Cdc14 is pivotal for exit from mitosis counteracting Cdk1-dependent phosphorylations. Whether hCdc14B, the human homolog of yeast Cdc14, plays a similar function in mitosis is not yet known. Here we show that hCdc14B serves a critical role in regulating progression through mitosis, which is distinct from hCdc14A. Unscheduled overexpression of hCdc14B delays activation of two master regulators of mitosis, Cdc25 and Cdk1, and slows down entry into mitosis. Depletion of hCdc14B by RNAi prevents timely inactivation of Cdk1/cyclin B and dephosphorylation of Cdc25, leading to severe mitotic defects, such as delay of metaphase/anaphase transition, lagging chromosomes, multipolar spindles and binucleation. The results demonstrate that hCdc14B-dependent modulation of Cdc25 phosphatase and Cdk1/cyclin B activity is tightly linked to correct chromosome segregation and bipolar spindle formation, processes that are required for proper progression through mitosis and maintenance of genomic stability.

AMP-activated protein kinase (AMPK) senses changes in the intracellular AMP/ATP ratio, switching off energy-consuming processes and switching on catabolic pathways in response to energy depletion. Here, we show that AMPK down-regulates rRNA synthesis under glucose restriction by phosphorylating the RNA polymerase I (Pol I)-associated transcription factor TIF-IA at a single serine residue (Ser-635). Phosphorylation by AMPK impairs the interaction of TIF-IA with the TBP-containing promoter selectivity factor SL1, thereby precluding the assembly of functional transcription initiation complexes. Mutation of Ser-635 compromises down-regulation of Pol I transcription in response to low energy supply, supporting that activation of AMPK adapts rRNA synthesis to nutrient availability and the cellular energy status.

Our current knowledge of the general factor requirement in transcription by the three mammalian RNA polymerases is based on a small number of model promoters. Here, we present a comprehensive chromatin immunoprecipitation (ChIP)-on-chip analysis for 28 transcription factors on a large set of known and novel TATA-binding protein (TBP)-binding sites experimentally identified via ChIP cloning. A large fraction of identified TBP-binding sites is located in introns or lacks a gene/mRNA annotation and is found to direct transcription. Integrated analysis of the ChIP-on-chip data and functional studies revealed that TAF12 hitherto regarded as RNA polymerase II (RNAP II)-specific was found to be also involved in RNAP I transcription. Distinct profiles for general transcription factors and TAF-containing complexes were uncovered for RNAP II promoters located in CpG and non-CpG islands suggesting distinct transcription initiation pathways. Our study broadens the spectrum of general transcription factor function and uncovers a plethora of novel, functional TBP-binding sites in the human genome.

Accumulating evidence has shown that many molecules, including some cyclin-dependent kinases (Cdks) and cyclins, as well as the death-effector domain (DED)-containing FADD, function for both apoptosis and cell cycle. Here we identified that DEDD, which also possesses the DED domain, acts as a novel inhibitor of the mitotic Cdk1/cyclin B1 complex. DEDD associates with mitotic Cdk1/cyclin B1 complexes via direct binding to cyclin B1 and reduces their function. In agreement, kinase activity of nuclear Cdk1/cyclin B1 in DEDD-null (DEDD-/-) embryonic fibroblasts is increased compared with that in DEDD+/+ cells, which results in accelerated mitotic progression, thus exhibiting a shortened G2/M stage. Interestingly, DEDD-/- cells also demonstrated decreased G1 duration, which perhaps enhanced the overall reduction in rRNA amounts and cell volume, primarily caused by the rapid termination of rRNA synthesis before cell division. Likewise, DEDD-/- mice show decreased body and organ weights relative to DEDD+/+ mice. Thus, DEDD is an impeder of cell mitosis, and its absence critically influences cell and body size via modulation of rRNA synthesis.

We investigated the role of SIRT7, one of the seven members of the mammalian sirtuin family. We show that SIRT7 is a widely expressed nucleolar protein that is associated with active rRNA genes (rDNA), where it interacts with RNA polymerase I (Pol I) as well as with histones. Overexpression of SIRT7 increases Pol I-mediated transcription, whereas knockdown of SIRT7 or inhibition of the catalytic activity results in decreased association of Pol I with rDNA and a reduction of Pol I transcription. Depletion of SIRT7 stops cell proliferation and triggers apoptosis. Our findings suggest that SIRT7 is a positive regulator of Pol I transcription and is required for cell viability in mammals.

Nuclear actin and myosin 1 (NM1) are key regulators of gene transcription. Here, we show by biochemical fractionation of nuclear extracts, protein-protein interaction studies and chromatin immunoprecipitation assays that NM1 is part of a multiprotein complex that contains WICH, a chromatin remodelling complex containing WSTF (Williams syndrome transcription factor) and SNF2h. NM1, WSTF and SNF2h were found to be associated with RNA polymerase I (Pol I) and ribosomal RNA genes (rDNA). RNA interference-mediated knockdown of NM1 and WSTF reduced pre-rRNA synthesis in vivo, and antibodies to WSTF inhibited Pol I transcription on pre-assembled chromatin templates but not on naked DNA. The results indicate that NM1 cooperates with WICH to facilitate transcription on chromatin.

The upstream binding factor UBF, an activator of RNA polymerase I transcription, is posttranslationally modified by phosphorylation and acetylation. We found that in NIH3T3 cells, UBF is acetylated in S-phase but not in G1-phase. To assess the role of acetylation in regulation of UBF activity, we have established an NIH3T3 cell line that inducibly overexpresses HDAC1. Both in vivo and in vitro, HDAC1 efficiently hypoacetylates UBF. Immunoprecipitation with antibodies against the Pol I-associated factor PAF53 co-precipitated UBF in mock cells but not in cells overexpressing HDAC1. Pull-down experiments showed that acetylation of UBF augments the interaction with Pol I. Consistent with acetylation of UBF being important for association of PAF53 and recruitment of Pol I, the level of Pol I associated with rDNA and pre-rRNA synthesis were reduced in cells overexpressing HDAC1. The results suggest that acetylation and deacetylation of UBF regulate rRNA synthesis during cell cycle progression.

Pescadillo (PES1) and the upstream binding factor (UBF1) play a role in ribosome biogenesis, which regulates cell size, an important component of cell proliferation. We have investigated the effects of PES1 and UBF1 on the growth and differentiation of cell lines derived from 32D cells, an interleukin-3 (IL-3)-dependent murine myeloid cell line. Parental 32D cells and 32D IGF-IR cells (expressing increased levels of the type 1 insulin-like growth factor I [IGF-I] receptor [IGF-IR]) do not express insulin receptor substrate 1 (IRS-1) or IRS-2. 32D IGF-IR cells differentiate when the cells are shifted from IL-3 to IGF-I. Ectopic expression of IRS-1 inhibits differentiation and transforms 32D IGF-IR cells into a tumor-forming cell line. We found that PES1 and UBF1 increased cell size and/or altered the cell cycle distribution of 32D-derived cells but failed to make them IL-3 independent. PES1 and UBF1 also failed to inhibit the differentiation program initiated by the activation of the IGF-IR, which is blocked by IRS-1. 32D IGF-IR cells expressing PES1 or UBF1 differentiate into granulocytes like their parental cells. In contrast, PES1 and UBF1 can transform mouse embryo fibroblasts that have high levels of endogenous IRS-1 and are not prone to differentiation. Our results provide a model for one of the theories of myeloid leukemia, in which both a stimulus of proliferation and a block of differentiation are required for leukemia development.

The human Cdc25A phosphatase plays a pivotal role at the G1/S transition by activating cyclin E and A/Cdk2 complexes through dephosphorylation. In response to ionizing radiation, Cdc25A is phosphorylated by both Chk1 and Chk2 on Ser-123. This in turn leads to ubiquitylation and rapid degradation of Cdc25A by the proteasome resulting in cell cycle arrest. We found that in response to UV irradiation, Cdc25A is phosphorylated at a different serine residue, Ser-75. Significantly, Cdc25A mutants carrying alanine instead of either Ser-75 or Ser-123 demonstrate that only Ser-75 mediates protein stabilization in response to UV-induced DNA damage. As a consequence, cyclin E/Cdk2 kinase activity was high. Furthermore, we find that Cdc25A was phosphorylated by Chk1 on Ser-75 in vitro and that the same site was also phosphorylated in vivo. Taken together, these data strongly suggest that phosphorylation of Cdc25A on Ser-75 by Chk1 and its subsequent degradation is required to delay cell cycle progression in response to UV-induced DNA lesions.

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.

Modulation of the activity of the upstream binding factor (UBF) plays a key role in cell cycle-dependent regulation of rRNA synthesis. Activation of rDNA transcription on serum stimulation requires phosphorylation of UBF at serine 484 by G(1)-specific cyclin-dependent kinase (cdk)/cyclin complexes. After G(1) progression UBF is phosphorylated at serine 388 by cdk2/cyclin E and cdk2/cyclin A. Conversion of serine 388 to glycine abolishes UBF activity, whereas substitution by aspartate enhances the transactivating function of UBF. Protein-protein interaction studies reveal that phosphorylation at serine 388 is required for the interaction between RNA polymerase I and UBF. The results suggest that phosphorylation of UBF represents a powerful means of modulating the assembly of the transcription initiation complex in a proliferation- and cell cycle-dependent fashion.

The "pocket" proteins pRb, p107, and p130 are a family of negative growth regulators. Previous studies have demonstrated that overexpression of pRb can repress transcription by RNA polymerase (Pol) I. To assess whether pRb performs this role under physiological conditions, we have examined pre-rRNA levels in cells from mice lacking either pRb alone or combinations of the three pocket proteins. Pol I transcription was unaffected in pRb-knockout fibroblasts, but specific disruption of the entire pRb family deregulated rRNA synthesis. Further analysis showed that p130 shares with pRb the ability to repress Pol I transcription, whereas p107 is ineffective in this system. Production of rRNA is abnormally elevated in Rb(-/-) p130(-/-) fibroblasts. Furthermore, overexpression of p130 can inhibit an rRNA promoter both in vitro and in vivo. This reflects an ability of p130 to bind and inactivate the upstream binding factor, UBF. The data imply that rRNA synthesis in living cells is subject to redundant control by endogenous pRb and p130.

Mammalian rRNA genes are preceded by a terminator element that is recognized by the transcription termination factor TTF-I. In exploring the functional significance of the promoter-proximal terminator, we found that TTF-I associates with the p300/CBP-associated factor PCAF, suggesting that TTF-I may target histone acetyltransferase to the rDNA promoter. We demonstrate that PCAF acetylates TAF(I)68, the second largest subunit of the TATA box-binding protein (TBP)-containing factor TIF-IB/SL1, and acetylation enhances binding of TAF(I)68 to the rDNA promoter. Moreover, PCAF stimulates RNA polymerase I (Pol I) transcription in a reconstituted in vitro system. Consistent with acetylation of TIF-IB/SL1 being required for rDNA transcription, the NAD(+)-dependent histone deacetylase mSir2a deacetylates TAF(I)68 and represses Pol I transcription. The results demonstrate that acetylation of the basal Pol I transcription machinery has functional consequences and suggest that reversible acetylation of TIF-IB/SL1 may be an effective means to regulate rDNA transcription in response to external signals.

Hemocyanins are large oligomeric respiratory proteins found in many arthropods and molluscs. The hemocyanin of the tarantula Eurypelma californicum is a 24-mer protein complex with molecular mass of 1, 726,459 Da that consists of seven different polypeptides (a-g), each occupying a distinct position within the native molecule. Here we report the complete molecular structure of the E. californicum hemocyanin as deduced from the corresponding cDNAs. This represents the first complex arthropod hemocyanin to be completely sequenced. The different subunits display 52-66% amino acid sequence identity. Within the subunits, the central domain, which bears the active center with the copper-binding sites A and B, displays the highest degree of identity. Using a homology modeling approach, the putative three-dimensional structure of individual subunits was deduced and compared. Phylogenetic analyses suggest that differentiation of the individual subunits occurred 400-550 million years ago. The hemocyanin of the stemline Chelicerata was probably a hexamer built up of six distinct subunit types a, b/c, d, e, f, and g, whereas that of the early Arachnida was originally a 24-mer that emerged after the differentiation of subunits b and c.

Transcription of rRNA genes by RNA polymerase I increases following serum stimulation of quiescent NIH 3T3 fibroblasts. To elucidate the mechanism underlying transcriptional activation during progression through the G1 phase of the cell cycle, we have analyzed the activity and phosphorylation pattern of the nucleolar transcription factor upstream binding factor (UBF). Using a combination of tryptic phosphopeptide mapping and site-directed mutagenesis, we have identified Ser484 as a direct target for cyclin-dependent kinase 4 (cdk4)-cyclin D1- and cdk2-cyclin E-directed phosphorylation. Mutation of Ser484 impairs rDNA transcription in vivo and in vitro. The data demonstrate that UBF is regulated in a cell cycle-dependent manner and suggest a link between G1 cdks-cyclins, UBF phosphorylation and rDNA transcription activation.

We have used a reconstituted cell-free transcription system to investigate the molecular basis of mitotic repression of RNA polymerase I (pol I) transcription. We demonstrate that SL1, the TBP-containing promoter-binding factor, is inactivated by cdc2/cyclin B-directed phosphorylation, and reactivated by dephosphorylation. Transcriptional inactivation in vitro is accompanied by phosphorylation of two subunits, e.g. TBP and hTAFI110. To distinguish whether transcriptional repression is due to phosphorylation of TBP, hTAFI110 or both, SL1 was purified from two HeLa cell lines that express either full-length or the core domain of TBP only. Both TBP-TAFI complexes exhibit similar activity and both are repressed at mitosis, indicating that the variable N-terminal domain which contains multiple target sites for cdc2/cyclin B phosphorylation is dispensable for mitotic repression. Protein-protein interaction studies reveal that mitotic phosphorylation impairs the interaction of SL1 with UBF. The results suggest that phosphorylation of SL1 is used as a molecular switch to prevent pre-initiation complex formation and to shut down rDNA transcription at mitosis.

Lentiviral transactivator (Tat) proteins are essential for viral replication. Tat proteins of human immunodeficiency virus type 1 and bovine immunodeficiency virus form complexes with their respective RNA targets (Tat responsive element, TAR), and specific binding of the equine anemia virus (EIAV) Tat protein to a target TAR RNA is suggested by mutational analysis of the TAR RNA. Structural data on equine infectious anemia virus Tat protein reveal a helix-loop-helix-turn-helix limit structure very similar to homeobox domains that are known to bind specifically to DNA. Here we report results of gel-shift and footprinting analysis as well as fluorescence and nuclear magnetic resonance spectroscopy experiments that clearly show that EIAV Tat protein binds to DNA specifically at the long terminal repeat Pu.1 (GTTCCTGTTTT) and AP-1 (TGACGCG) sites, and thus suggest a common mechanism for the action of some of the known lentiviral Tat proteins via the AP-1 initiator site. Complex formation with DNA induces specific shifts of the proton NMR resonances originating from amino acids in the core and basic domains of the protein., (Copyright 1998 Academic Press Limited.)

The retinoblastoma susceptibility gene product pRb restricts cellular proliferation by affecting gene expression by all three classes of nuclear RNA polymerases. To elucidate the molecular mechanisms underlying pRb-mediated repression of ribosomal DNA (rDNA) transcription by RNA polymerase I, we have analyzed the effect of pRb in a reconstituted transcription system. We demonstrate that pRb, but not the related protein p107, acts as a transcriptional repressor by interfering with the assembly of transcription initiation complexes. The HMG box-containing transcription factor UBF is the main target for pRb-induced transcriptional repression. UBF and pRb form in vitro complexes involving the C-terminal part of pRb and HMG boxes 1 and 2 of UBF. We show that the interactions between UBF and TIF-IB and between UBF and RNA polymerase I, respectively, are not perturbed by pRb. However, the DNA binding activity of UBF to both synthetic cruciform DNA and the rDNA promoter is severely impaired in the presence of pRb. These studies reveal another mechanism by which pRb suppresses cell proliferation, namely, by direct inhibition of cellular rRNA synthesis.

In situ autogenous reconstruction is an alternative therapy for abdominal aortic prosthetic graft infection. We have used the superficial femoral vein (SFV) as an arterial substitute for proximal aortic anastomosis in seven patients. Six patients presented with aortic perigraft infections and one had a mycotic aneurysm of the infrarenal aorta with a primary aortoenteric fistula. There were no intraoperative but two postoperative deaths. During follow-up (mean: 19.8 months), one patient died at 5 months unrelated to his preceding vascular procedures; the others were doing well with patient SFV grafts 6-36 months after autogenous aortoiliac reconstruction. The SFV is a valuable donor vessel for autogenous reconstruction in the management of aortoiliac prosthetic graft infection. We explain both perioperative deaths in our treatment group with respect to the extent of the underlying infection and the virulence of the causative organism.

High mobility group 1 (HMG1) protein is an abundant and conserved component of vertebrate nuclei and has been proposed to play a structural role in chromatin organization, possibly similar to that of histone H1. However, a high abundance of HMG1 had also been reported in the cytoplasm and on the surface of mammalian cells. We conclusively show that HMG1 is a nuclear protein, since several different anti-HMG1 antibodies stain the nucleoplasm of cultured cells, and epitope-tagged HMG1 is localized in the nucleus only. The protein is excluded from nucleoli and is not associated to specific nuclear structures but rather appears to be uniformly distributed. HMG1 can bind in vitro to reconstituted core nucleosomes but is not stably associated to chromatin in live cells. At metaphase, HMG1 is detached from condensed chromosomes, contrary to histone H1. During interphase, HMG1 readily diffuses out of nuclei after permeabilization of the nuclear membranes with detergents, whereas histone H1 remains associated to chromatin. These properties exclude a shared function for HMG1 and H1 in differentiated cells, in spite of their similar biochemical properties. HMG1 may be stably associated only to a very minor population of nucleosomes or may interact transiently with nucleosomes during dynamic processes of chromatin remodeling.

The current standard therapy of aortic graft infection is connected with a high incidence of operative and late complications. The usefulness of autogenous tissue revascularization techniques as promising therapeutic alternatives is limited by the complexity of these procedures and by the lack of suitable donor vessels. Since several authors have shown that serious venous stasis of the donor leg does not occur after superficial femoral vein (SFV) resection, we have started to use this deep leg vein for arterial in situ reconstructions in the treatment of aortic graft infection. During the past 3 years seven patients have received autogenous SFV grafts in this way. There were no intraoperative deaths. Two critically ill patients with severe Pseudomonas sepsis and extensive retroperitoneal abscess each died on the 4th postoperative day, due to acute massive bleeding in the operating area. In all of the five survivors infection could be eradicated; only one limb had to be amputated. Mild to moderate transient swelling of the lower extremity without stasis was regularly seen after SFV resection. During follow-up one patient died of cardiac arrest 5 months postoperatively. The remaining four patients are still alive with patent SFV grafts at 6, 22, 30 and 36 months after the operation.

The nucleolar factor UBF is phosphorylated by casein kinase II (CKII) at serine residues within the C-terminal acidic domain which is required for transcription activation. To investigate the biological significance of UBF modification, we have compared the trans-activating properties of cellular UBF and recombinant UBF expressed in Escherichia coli. Using a variety of assays we demonstrate that unphosphorylated UBF is transcriptionally inactive and has to be phosphorylated at multiple sites to stimulate transcription. Examination of cDNA mutants in which the serine residues within the C-terminal domain were altered by site-directed mutagenesis demonstrates that CKII-mediated phosphorylations of UBF contribute to, but are not sufficient for, transcriptional activation. Besides CKII, other cellular protein kinases phosphorylate UBF at distinct sites in a growth-dependent manner. The marked differences in the tryptic peptide maps of UBF from growing and serum-starved cells suggest that alterations in the degree of UBF phosphorylation may modulate rRNA synthetic activity in response to extracellular signals.

Primary aorto-enteric fistulae are rare, mostly described as atypical first manifestation of an abdominal aortic aneurysm. Spread of elective aortic surgery led to increased appearance of secondary aorto-enteric fistulae as a typical postoperative complication. Gastrointestinal bleeding with endoscopical unclear findings in a patient with aortic aneurysm or history of aortic repair points towards an aorto-enteric fistula. While preoperative diagnosis is not possible in most instances, the proof of an anastomotic aneurysm and/or aortic graft infection hardens the suspected diagnosis of an aorto-enteric fistula decisively. The finding of coincidental mucosal lesions at gastroscopy may not mislead to give up the exclusion of an aorto-enteric fistula, possibly by explorative laparotomy, if suspicion is well-founded. In the present article nine cases of aorto-enteric fistulae treated at the Surgical University Clinic Würzburg between 1982 and 1993 are analyzed retrospectively. Topical questions of diagnosis and therapy are discussed.

The operative removal of haemodynamical significant carotid artery stenosis by endarterectomy nowadays is one of the vascular surgical standard procedures. Purpose of the operation is prevention of ischemic strokes. For a long-term prognostic advantage the patient has to take the risk of perioperative mortality and morbidity. While efforts are being made to minimize this risk, the question of optimal surgical strategy has not yet finally been solved. Since 1982 in our hospital all carotid endarterectomies are carried out with routine insertion of an intraluminal shunt. The distal intima step of the internal carotid artery is secured by a running suture and closure of the longitudinal arteriotomy is accomplished by dacron patch plasty. In this manner 546 successive operations have been performed under general anaesthesia until 1993. Intra- and postoperative mortality was 0.9% with an ischemic cerebral infarction rate of 1.8%. According to the preoperative stage of cerebrovascular insufficiency the frequencies for mortality and perioperative ischemic stroke were 0.6% and 1.3% for CVI I, 0.4% and 0.7% for CVI II and 2.8% and 5.7% for CVI IV. Apart from perioperative mortality for patients with CVI IV, these complication rates are clearly below the suggested limits of the Ad hoc Committee on Carotid Surgery Standards by the Stroke Council of the American Heart Association. Routine use of a temporary, intraluminal shunt in carotid artery operations therefore can be considered as a safe measure, with complication rates still not underbid by those achieved with intraoperative cerebral monitoring and selective shunting.

The nucleolar transcription factor UBF consists of two proteins, UBF1 and UBF2, which originate by alternative splicing. Here we show that deletion of 37 amino acids within the second of five HMG box motifs in UBF2 is important for the dual role of UBF as transcriptional activator and antirepressor. UBF1 is a potent antirepressor and transcriptional activator, whereas the ability of UBF2 to counteract histone H1-mediated repression and to stimulate ribosomal gene transcription both in vivo and in vitro is at least one order of magnitude lower. The difference in transcriptional activity between UBF1 and UBF2 is due to their different binding to the ribosomal gene promoter and enhancer. Apparently, the presence of an intact HMG box2 modulates the sequence-specific binding of UBF to rDNA control elements. However, the interaction of UBF with rDNA does not entirely depend on sequence recognition. Both UBF isoforms bind efficiently to four-way junction DNA, indicating that they recognize defined DNA structures rather than specific sequences. The results demonstrate that the HMG boxes are functionally diverse and that HMG box2 plays an important role in specific binding of UBF to rDNA.

We have characterized an anti-NOR (nucleolar organizer region) serum (P419) from a patient with rheumatoid arthritis and show that it contains antibodies directed against the RNA polymerase I-specific transcription initiation factor UBF. This serum reacts with UBF from a variety of vertebrate cells as revealed both by immunoblotting and by indirect immunofluorescence. We have used the P419 serum to study the intracellular localization of this transcription factor at the light and electron microscopic level. In interphase cells, UBF exhibits a pronounced punctate pattern and is found to be associated with necklace-like structures, which appear to reflect the transcriptionally active state of the nucleolus. Inhibition of rRNA synthetic activity caused either by nutritional starvation or by actinomycin D treatment resulted in a marked decrease in the number and in a significant increase in the size of UBF-positive granules. Under all experimental conditions applied, UBF was exclusively found within the nucleolus and was not released into the nucleoplasm or cytoplasm. During mitosis, UBF was found to be concentrated at the chromosomal NOR indicating that a significant quantity, if not all, of this factor remains bound to the ribosomal transcription units. From this we conclude that UBF is associated both with transcriptionally active and inactive rRNA genes and, therefore, changes in the intracellular localization of UBF are very likely not involved in rDNA transcription regulation.

UBF is a DNA binding protein which interacts with both the promoter and the enhancer of various vertebrate ribosomal RNA genes and functions as a transcription initiation factor for RNA polymerase I (pol I). We have purified murine UBF to apparent molecular homogeneity and demonstrate that its transactivating potential, but not its DNA binding activity, is modulated in response to cell growth. In vivo labelling experiments demonstrate that UBF is a phosphoprotein and that the phosphorylation state is different in growing and quiescent cells. We show that UBF is phosphorylated in vitro by a cellular protein kinase which by several criteria closely resembles casein kinase II (CKII). A major modification involves serine phosphoesterifications in the carboxy terminal hyperacidic tail of UBF. Deletions of this C-terminal domain severely decreases the UBF directed activation of transcription. The data suggest that phosphorylation of UBF by CKII may play an important role in growth dependent control of rRNA synthesis.

cDNA clones comprising the entire coding region of two out of the seven heterogeneous subunits of hemocyanin from the tarantula, Eurypelma californicum, were isolated from four cDNA libraries constructed from total RNA from the heart tissue of single spiders. Hybridization was first carried out using a tarantula hemocyanin subunit e partial cDNA, and several positive clones were isolated, including one containing a 2.2-kilobase full-length cDNA (lambda M1). The cDNA comprises an open reading frame for 623 amino acids, 34 nucleotides of the 5'noncoding region, and 286 nucleotides of the 3'-noncoding region. To select for other hemocyanin subunits, two 17-mer oligonucleotide mixtures, corresponding to the conserved regions in the copper A and copper B oxygen-binding site of chelicerate hemocyanins, were used as probes. Among the positive clones obtained, full-length cDNAs coding for subunit a were identified. The cDNA sequence determined from clone lambda K1 provides an open reading frame coding for 630 amino acids and includes the 5'- and 3'-noncoding regions. Northern blot analysis revealed single transcripts for subunits a and e, each 2.3 kilobases long. The cDNAs for subunits a and e were both found to lack any leader peptide sequence. This supports the idea that the mature protein accumulates in the cytoplasm and is released by cell rupture.

The gene for the hemocyanin subunit e of the tarantula Eurypelma californicum has been isolated from a genomic phage library by using a corresponding cDNA clone as a probe. The transcriptional unit spans a chromosomal region of about 55 kilobase pairs (kbp). The gene consists of nine exons that are separated by large introns. The intron/exon boundaries were determined by direct comparison of genomic and cDNA sequences. A putative promoter region ("TATA" box, reversed "CAAT" box) 100 bp 5' to the translational initiation codon strongly suggests the presence of a functional gene. The 3' flanking region carries the polyadenylylation signal (AATAAA) and several conserved structures for the 3' splicing of the pre-mRNA. A comparison of the gene architecture of the subunit e gene with the three-dimensional structure of the arthropod hemocyanin subunit shows a good correspondence with the division of the subunit into three domains (two exons coding for the first, three coding for the second, and four coding for the third domain). The relationship to molluscan hemocyanins, different tyrosinases, and the larval serum proteins is discussed.

Tarantula heart cDNA libraries were screened with synthetic oligonucleotide probes deduced from the highly conserved amino acid sequences of the two copper-binding sites, copper A and copper B, found in chelicerate hemocyanins. Positive cDNA clones could be obtained and four different cDNA types were characterized.

Following induction of hemopoiesis, poly(A)-rich RNA was prepared from the heart of the tarantula, Eurypelma californicum, and translated in rabbit reticulocyte lysates. In vitro translation products were immunoprecipitated with antiserum against whole dissociated Eurypelma hemocyanin. Analysis of the immunoprecipitate by sodium dodecyl sulfate/polyacrylamide gel electrophoresis revealed a set of polypeptides comigrating with authentic Eurypelma hemocyanin. The mRNA was transcribed into cDNA, clones were constructed using the pUC9 vector and probed with a synthetic 17-mer oligonucleotide probe complementary to the amino acid sequence of the 'copper A' binding site of chelicerate hemocyanins. One clone, pHC4, contained a 1.62-kb cDNA insert, which was subcloned into phage M13. Sequence analysis by the dideoxynucleotide chain-termination method yielded a nucleotide sequence coding for 526 amino acids of Eurypelma hemocyanin subunit e.

One hundred one infants with chronic respiratory failure (CRF) who required prolonged mechanical ventilation were cared for in the pediatric intensive care unit at The Children's Hospital of Philadelphia between January 1967 and December 1984. Chronic respiratory failure of infancy is a condition that requires mechanical ventilation for more than 28 days in the first year of life. Thirty-six children had severe bronchopulmonary dysplasia, 50 had congenital anomalies, and 15 had neuromuscular disorders. The mean duration of mechanical ventilation for the 101 patients was 12.3 months. Seventy-one children were alive, and 53 (75%) of the 71 had been weaned from mechanical ventilation as of Dec 31, 1984. Pulmonary insufficiency and cardiac failure were the predominant causes of death in 17 of 22 infants in the first two years after the onset of CRF; four of eight deaths that occurred beyond two years were caused by airway- and ventilator-related accidents. Mechanical ventilatory support was emphasized for as long as necessary to provide normal blood gas tensions, nutrition, growth, and development rather than weaning as rapidly as possible. This clinical experience demonstrates that it is feasible to save over 70% of infants with the severest forms of CRF and prolonged ventilator dependency.

Eighty-four tumor samples from 70 women with primary ovarian cancer were assayed for cytosol estrogen (ERc) and progestin (PRc) receptor concentrations and aromatase activity. In addition, 22 of the tumors were studied for their response to the aromatase inhibitor, 4-OH-androstenedione, in a soft agar clonogenic cell assay system. Although aromatase activity was detected in almost all of the primary tumors, this enzyme was barely detectable in the majority of metastatic tumor samples. There was no significant correlation between aromatase activity and either the ERc or PRc content of the tumors, or tumor grade. Of 12 tumors grown successfully in the soft agar culture system, only 1 showed a substantial (greater than 50%) reduction in colony-forming efficiency after exposure to the aromatase inhibitor. These results suggest that local estrogen biosynthesis probably does not play an important role in the majority of epithelial ovarian tumors. However, there may be a small subset of estrogen receptor-positive tumors in which aromatase could provide a local growth stimulus.