bluuebunny/crossref_metadata_2025 huggingface dataset:
Multiple forms of C/EBP beta bind the EFII enhancer sequence in the Rous sarcoma virus long terminal repeat.
Authors: Sears R C , Sealy L
Published: October 2015

In this report we demonstrate that C/EBP beta is a major component of three EFII DNA binding complexes, EFIIa, EFIIb, and EFIIc, which we have previously shown to specifically recognize a C/EBP consensus binding site found in the EFII enhancer sequence from the Rous sarcoma virus long terminal repeat (R. C. Sears and L. Sealy, J. Virol. 66:6338-6352, 1992). Three different forms of C/EBP beta, p42, p35, and p20, can bind the EFII DNA sequence as homodimers, and dimerization experiments show that EFIIa is a homodimer of p20 C/EBP beta, EFIIb is primarily composed of a p20/p35 heterodimer with minor amounts of p20/p42 heterodimer and p35 homodimer, and EFIIc is composed of p20 and/or p35 heterodimerized with a novel 60-kDa protein. p20 C/EBP beta is likely equivalent to the internally initiated translation product of C/EBP beta, LIP (liver inhibitor protein), described by P. Descombes and U. Schibler (Cell 67:569-579, 1991). In contrast to the low level of LIP expressed in liver, postulated to occur because of leaky ribosome scanning, we found high levels of expression of p20 C/EBP beta in fibroblasts and lymphocytes. In murine fibroblasts, p20 C/EBP beta has an extended half-life, four times longer than those of p42 and p35 C/EBP beta, which could contribute to its abundant accumulation in this cell type, even though its synthesis by leaky ribosome scanning might be inefficient. Interestingly, overexpression of either the long or short form of C/EBP beta represses EFII-mediated transcription, suggesting that another unidentified EFII transactivator(s) exists, which may be dominantly inhibited by C/EBP beta proteins, and/or that transactivation by C/EBP beta proteins requires posttranslational modifications that were lacking in the transient overexpression experiments.
Multiple instability-regulating sites in the 3' untranslated region of the urokinase-type plasminogen activator mRNA.
Authors: Nanbu R , Menoud P A , Nagamine Y
Published: October 2015

In LLC-PK1 cells urokinase-type plasminogen activator (uPA) mRNA has a short half-life. It is stabilized by inhibition of protein synthesis and by downregulation of protein kinase C (PKC). In the present study on uPA mRNA metabolism, we focused our attention on the 3' untranslated region (3'UTR) of the uPA mRNA, as this region is long and highly conserved among several mammalian species, including mice and humans. To investigate the possible role of the 3'UTR of uPA mRNA in mRNA metabolism, we inserted this region into the 3'UTR of the rabbit beta-globin gene that is linked to the cytomegalovirus promoter and stably transfected it into LLC-PK1 cells. While the parental globin mRNA was stable, the chimeric mRNA was degraded as rapidly as endogenous uPA mRNA, suggesting that the 3'UTR of uPA mRNA contains most of the information required for its rapid turnover. Further analysis showed that there are at least three independent determinants of instability in the 3'UTR; one is an AU-rich sequence located immediately 3' of the poly(A) addition signal, and one is a sequence containing a stem structure. One determinant seems to require ongoing RNA synthesis for its activity. All chimeric unstable globin mRNAs became stable in the presence of cycloheximide, a protein synthesis inhibitor, suggesting that the stabilization of mRNA by protein synthesis inhibition is not through a specific sequence in the mRNA. In PKC-downregulated cells, globin mRNAs with the complete 3'UTR or the AU-rich sequence were stabilized, suggesting that PKC downregulation stabilizes uPA mRNA through the AU-rich sequence. Here we discuss the significance of multiple, independently acting instability determinants in the regulation of uPA mRNA metabolism.
Novel interactions between human T-cell leukemia virus type I Tax and activating transcription factor 3 at a cyclic AMP-responsive element.
Authors: Low K G , Chu H M , Schwartz P M , Daniels G M , Melner M H , Comb M J
Published: October 2015

Human proenkephalin gene transcription is transactivated by human T-cell leukemia virus type I (HTLV-I) Tax in human Jurkat T lymphocytes. This transactivation was further enhanced in Jurkat cells treated with concanavalin A, cyclic AMP, or 12-O-tetradecanoylphorbol-13-acetate. Deletion and cis-element transfer analyses of the human proenkephalin promoter identified a cyclic AMP-responsive AP-1 element (-92 to -86) as both necessary and sufficient to confer Tax-dependent transactivation. Different AP-1 or cyclic AMP-responsive element-binding protein (CREB)/activating transcription factor (ATF) proteins which bind this element were expressed in murine teratocarcinoma F9 cells to identify those capable of mediating Tax-dependent transactivation of human proenkephalin gene transcription. Although CREB, c-Fos, c-Jun, and JunD did not have significant effects, JunB inhibited the Tax-dependent transactivation. In contrast, ATF3 dramatically induced Tax-dependent transactivation, which was further enhanced by protein kinase A. Electrophoretic mobility shift assays with recombinant fusion proteins expressed and purified from bacteria indicate that the DNA-binding activity of ATF3 is also dramatically enhanced by Tax. Chimeric fusion proteins consisting of the DNA-binding domain of the yeast transcription factor Gal4 and the amino-terminal domain (residues 1 to 66) of ATF3 were able to mediate Tax-dependent transactivation of a Gal4-responsive promoter, which suggests a direct involvement of this region of ATF3. Recombinant fusion proteins of glutathione S-transferase with either the amino- or carboxy-terminal (residues 139 to 181) domain of ATF3 were able to specifically interact with Tax. Furthermore, specific antisera directed against Tax coimmunoprecipitated ATF3 only in the presence of Tax.
Loss of serum response element-binding activity and hyperphosphorylation of serum response factor during cellular aging.
Authors: Atadja P W , Stringer K F , Riabowol K T
Published: October 2015

Human diploid fibroblasts undergo a limited number of population doublings in vitro and are used widely as a model of cellular aging. Despite growing evidence that cellular aging occurs as a consequence of altered gene expression, little is known about the activity of transcription factors in aging cells. Here, we report a dramatic reduction in the ability of proteins extracted from the nuclei of near-senescent fibroblasts to bind the serum response element which is necessary for serum-induced transcription of the c-fos gene. In contrast, the activities of proteins binding to the RNA polymerase core element, TATA, as well as to the cyclic AMP response element were maintained during cellular aging. While no major differences in the expression of the serum response factor (SRF) that binds the serum response element were seen between early-passage and late-passage cells, hyperphosphorylation of SRF was observed in near-senescent cells. Furthermore, removal of phosphatase inhibitors during the isolation of endogenous nuclear proteins restored the ability of SRF isolated from old cells to bind the SRE. These data, therefore, indicate that hyperphosphorylation of SRF plays a role in altering the ability of this protein to bind to DNA and regulate gene expression in senescent cells.
E2F and its developmental regulation in Xenopus laevis.
Authors: Philpott A , Friend S H
Published: October 2015

The transcription factor E2F has been implicated in cell cycle control by virtue of its association with cyclins, cyclin-dependent kinases, and pRb-related tumor suppressor gene products. Eggs and embryos from the frog Xenopus laevis have been used to investigate the characteristics of E2F-like molecules in the Xenopus cell cycle and throughout early development. We find multiple E2F species in Xenopus eggs, at least one of which is modified by phosphorylation. The vast majority of E2F remains in the free form throughout the very early embryonic cell cycle, and it also remains predominantly free until some time after the mid-blastula transition, the onset of zygotic transcription. At this time, E2F complexes significantly to pRb but not to cdk2, although cdk2 binding is found in tissue culture cells from a very advanced stage in embryogenesis. This suggests that the complexing of E2F to cyclins, cyclin-dependent kinases, and tumor suppressor gene products may be controlled separately in early Xenopus development. Thus, the association of E2F with other molecules may not result solely from processes affecting cell cycle progression but may also reflect developmental and differentiation cues.
One exon of the human LSF gene includes conserved regions involved in novel DNA-binding and dimerization motifs.
Authors: Shirra M K , Zhu Q , Huang H C , Pallas D , Hansen U
Published: October 2015

The transcription factor LSF, identified as a HeLa protein that binds the simian virus 40 late promoter, recognizes direct repeats with a center-to-center spacing of 10 bp. The characterization of two human cDNAs, representing alternatively spliced mRNAs, provides insight into the unusual DNA-binding and oligomerization properties of LSF. The sequence of the full-length LSF is identical to that of the transcription factors alpha CP2 and LBP-1c and has similarity to the Drosophila transcription factor Elf-1/NTF-1. Using an epitope-counting method, we show that LSF binds DNA as a homodimer. LSF-ID, which is identical to LBP-1d, contains an in-frame internal deletion of 51 amino acids resulting from alternative mRNA splicing. Unlike LSF, LSF-ID did not bind LSF DNA-binding sites. Furthermore, LSF-ID did not affect the binding of LSF to DNA, suggesting that the two proteins do not interact. Of three short regions with a high degree of homology between LSF and Elf-1/NTF-1, LSF-ID lacks two, which are predicted to form beta-strands. Double amino acid substitutions in each of these regions eliminated specific DNA-binding activity, similarly to the LSF-ID deletion. The dimerization potential of these mutants was measured both by the ability to inhibit the binding of LSF to DNA and by direct protein-protein interaction studies. Mutations in one homology region, but not the other, functionally eliminated dimerization.
Tyrosine phosphorylation and activation of Bruton tyrosine kinase upon Fc epsilon RI cross-linking.
Authors: Kawakami Y , Yao L , Miura T , Tsukada S , Witte O N , Kawakami T
Published: October 2015

Tyrosine phosphorylation of several cellular proteins is one of the earliest signaling events induced by cross-linking of the high-affinity receptor for immunoglobulin E (Fc epsilon RI) on mast cells or basophils. Tyrosine kinases activated during this process include the Src family kinases, Lyn, c-Yes, and c-Src, and members of another subfamily, Syk and PTK72 (identical or highly related to Syk). Recently, some of us described two novel tyrosine kinases, Emb and Emt, whose expression was limited to subsets of hematopoietic cells, including mast cells. Emb turned out to be identical to Btk, a gene product defective in human X-linked agammaglobulinemia and in X-linked immunodeficient (xid) mice. Here we report that Fc epsilon RI cross-linking induced rapid phosphorylation on tyrosine, serine, and threonine residues and activation of Btk in mouse bone marrow-derived mast cells. A small fraction of Btk translocated from the cytosol to the membrane compartment following receptor cross-linking. Tyrosine phosphorylation of Btk was not induced by either a Ca2+ ionophore (A23187), phorbol 12-myristate 13-acetate, or a combination of the two reagents. Co-immunoprecipitation between Btk and receptor subunit beta or gamma was not detected. The data collectively suggest that Btk is not associated with Fc epsilon but that its activation takes place prior to protein kinase C activation and plays a novel role in the Fc epsilon RI signaling pathway.
p53 domains: structure, oligomerization, and transformation.
Authors: Wang P , Reed M , Wang Y , Mayr G , Stenger J E , Anderson M E , Schwedes J F , Tegtmeyer P
Published: October 2015

Wild-type p53 forms tetramers and multiples of tetramers. Friedman et al. (P. N. Friedman, X. B. Chen, J. Bargonetti, and C. Prives, Proc. Natl. Acad. Sci. USA 90:3319-3323, 1993) have reported that human p53 behaves as a larger molecule during gel filtration than it does during sucrose gradient sedimentation. These differences argue that wild-type p53 has a nonglobular shape. To identify structural and oligomerization domains in p53, we have investigated the physical properties of purified segments of p53. The central, specific DNA-binding domain within murine amino acids 80 to 320 and human amino acids 83 to 323 behaves predominantly as monomers during analysis by sedimentation, gel filtration, and gel electrophoresis. This consistent behavior argues that the central region of p53 is globular in shape. Under appropriate conditions, however, this segment can form transient oligomers without apparent preference for a single oligomeric structure. This region does not enhance transformation by other oncogenes. The biological implications of transient oligomerization by this central segment, therefore, remain to be demonstrated. Like wild-type p53, the C terminus, consisting of murine amino acids 280 to 390 and human amino acids 283 to 393, behaves anomalously during gel filtration and apparently has a nonglobular shape. Within this region, murine amino acids 315 to 350 and human amino acids 323 to 355 are sufficient for assembly of stable tetramers. The finding that murine amino acids 315 to 360 enhance transformation by other oncogenes strongly supports the role of p53 tetramerization in oncogenesis. Amino acids 330 to 390 of murine p53 and amino acids 340 to 393 of human p53, which have been implicated by Sturzbecher et al. in tetramerization (H.-W. Sturzbecher, R. Brain, C. Addison, K. Rudge, M. Remm, M. Grimaldi, E. Keenan, and J. R. Jenkins, Oncogene 7:1513-1523, 1992), do not form stable tetramers under our conditions. Our findings indicate that p53 has at least two autonomous oligomerization domains: a strong tetramerization domain in its C-terminal region and a weaker oligomerization domain in the central DNA binding region of p53. Together, these domains account for the formation of tetramers and multiples of tetramers by wild-type p53. The tetramerization domain is the major determinant of the dominant negative phenotype leading to transformation by mutant p53s.
Ectopic expression of cyclin D1 prevents activation of gene transcription by myogenic basic helix-loop-helix regulators.
Authors: Rao S S , Chu C , Kohtz D S
Published: October 2015

Activation of muscle gene transcription in differentiating skeletal myoblasts requires their withdrawal from the cell cycle. The effects of ectopic cyclin expression on activation of muscle gene transcription by myogenic basic helix-loop-helix (bHLH) regulators were investigated. Ectopic expression of cyclin D1, but not cyclins A, B1, B2, C, D3, and E, inhibited transcriptional activation of muscle gene reporter constructs by myogenic bHLH regulators in a dose-dependent manner. Ectopic expression of cyclin D1 inhibited the activity of a myogenic bHLH regulator mutant lacking the basic region protein kinase C site, indicating that phosphorylation of this site is not relevant to the mechanism of inhibition. Analysis of cyclin D1 mutants revealed that the C-terminal acidic region was required for inhibition of myogenic bHLH regulator activity, whereas an intact N-terminal pRb binding motif was not essential. Together, these results implicate expression of cyclin D1 as a central determinant of a putatively novel mechanism that links positive control of cell cycle progression to negative regulation of genes expressed in differentiated myocytes.
Critical binding and regulatory interactions between Ras and Raf occur through a small, stable N-terminal domain of Raf and specific Ras effector residues.
Authors: Chuang E , Barnard D , Hettich L , Zhang X F , Avruch J , Marshall M S
Published: October 2015

Genetic and biochemical evidence suggests that the Ras protooncogene product regulates the activation of the Raf kinase pathway, leading to the proposal that Raf is a direct mitogenic effector of activated Ras. Here we report the use of a novel competition assay to measure in vitro the relative affinity of the c-Raf-1 regulatory region for Ras-GTP, Ras-GDP, and 10 oncogenic and effector mutant Ras proteins. c-Raf-1 associates with normal Ras and the oncogenic V12 and L61 forms of Ras with equal affinity. The moderately transforming mutant Ras[E30K31] also bound to the c-Raf-1 regulatory region with normal affinity. Transformation-defective Ras effector mutants Ras[N33], Ras[S35], and Ras[N38] bound poorly. In contrast, the transformation defective Ras[G26I27] and Ras[E45] mutants bound to the c-Raf-1 regulatory region with nearly wild-type affinity. A stable, high-affinity Ras-binding region of c-Raf-1 was mapped to a 99-amino-acid subfragment of the first 257 residues. The smallest Ras-binding region identified consisted of N-terminal residues 51 to 131, although stable expression of the domain and high-affinity binding were improved by the presence of residues 132 to 149. Deletion of the Raf zinc finger region did not reduce Ras-binding affinity, while removal of the first 50 amino acids greatly increased affinity. Phosphorylation of Raf[1-149] by protein kinase A on serine 43 resulted in significant inhibiton of Ras binding. demonstrating that the mechanism of cyclic AMP downregulation results through structural changes occurring exclusively in this small Ras-binding domain.
Sequential induction of NF-kappa B/Rel family proteins during B-cell terminal differentiation.
Authors: Liou H C , Sha W C , Scott M L , Baltimore D
Published: October 2015

The NF-kappa B/Rel family of at least five transcription factor polypeptides is thought to function both as a developmental regulator in B cells and as a rapid response system in all cells. To examine this notion in more detail, we determined the protein contents of both the inducible and constitutive NF-kappa B/Rel activities in a pre-B-cell line, 70Z/3, and a mature B-cell line, WEHI 231. NF-kappa B p50/p65 is the major inducible nuclear complex after lipopolysaccharide or phorbol myristate acetate treatment of 70Z/3 cells. The constitutive and inducible complexes in WEHI 231 cells are mainly composed of p50 and Rel. The constitutive or induced activities are all sensitive to I kappa B-alpha, but this inhibitor is very short-lived in WEHI 231 cells, suggesting that the balance between synthesis and degradation of I kappa B-alpha determines whether a particular cell lineage has constitutive activity. A patterned expression of the NF-kappa B/Rel activator proteins emerges from an analysis of other B-lineage cell lines and splenic B cells: mainly p50 and p65 in pre-B (and non-B) cells, a predominance of Rel and p50 in mature B cells, and expression of p52 and RelB in plasmacytoma lines. This ordered pattern of regulators may reflect the requirement for expression of different genes during terminal B-cell differentiation because different combinations of NF-kappa B/Rel family members preferentially activate distinct kappa B sites in reporter constructs.
Signals determining protein tyrosine kinase and glycosyl-phosphatidylinositol-anchored protein targeting to a glycolipid-enriched membrane fraction.
Authors: Rodgers W , Crise B , Rose J K
Published: October 2015

Glycosyl-phosphatidylinositol (GPI)-anchored membrane proteins and certain protein tyrosine kinases associate with a Triton X-100-insoluble, glycolipid-enriched membrane fraction in MDCK cells. Also, certain protein tyrosine kinases have been shown to associate with GPI-anchored proteins in other cell types. To characterize the interaction between GPI-anchored proteins and protein tyrosine kinases, GPI-anchored proteins were coexpressed with p56lck in HeLa cells. Both proteins were shown to target independently to the glycolipid-enriched membranes. Coimmunoprecipitation of GPI-anchored proteins and p56lck occurred only when both proteins were located in the glycolipid-enriched membranes, and gentle disruption of these membranes abolished the interaction. The GPI anchor was found to be the targeting signal for this membrane fraction in GPI-anchored proteins. Analysis of mutants indicated that p56lck was nearly quantitatively palmitoylated at Cys-5 but not palmitoylated at Cys-3. The nonpalmitoylated cysteine at position 3 was very important for association of p56lck with the membrane fraction, while palmitoylation at Cys-5 promoted only a low level of interaction. Because other src family protein tyrosine kinases that are associated with GPI-anchored proteins always contain a Cys-3, we propose that this residue, in addition to the N-terminal myristate, is part of a common signal targeting these proteins to a membrane domain that has been linked to transmembrane signaling.
The Drosophila tom retrotransposon encodes an envelope protein.
Authors: Tanda S , Mullor J L , Corces V G
Published: October 2015

The tom transposable element of Drosophila ananassae is mobilized with high frequency in the germ line of females from the ca; px strain, and its insertion results in mutations that show almost exclusively dominant eye phenotypes. tom is a long terminal repeat-containing retrotransposon that encodes three different open reading frames (ORFs). It is expressed in the nurse cells during oogenesis, in the central and peripheral nervous systems during embryonic development, and in the imaginal discs of the larva. tom RNA accumulates in the germarium of ovaries from ca; px females but not in the parental inactive strain, suggesting that this altered pattern of tom expression might be the cause of the high rate of mobilization of this retrotransposon. The specificity of tom-induced eye phenotypes can be explained by the presence of regulatory sequences responsible for expression of tom in the eye imaginal discs of third-instar larvae. These sequences might cause overexpression of adjacent genes affected by tom-induced mutations, resulting in the death of undifferentiated cells located anterior to the morphogenetic furrow. In addition to the full-length RNA, tom is also transcribed into a spliced subgenomic transcript that encodes a protein resulting from the fusion between the amino-terminal region of the first (gag) and the third ORFs. The protein encoded by this RNA shows structural characteristics such as a signal peptide, glycosylation sites, endopeptidase cleavage site, and fusion peptide that are typical of the envelope proteins of retroviruses. Antibodies against tom ORF3 recognize two different proteins present in female ovaries, suggesting that tom might be able to form infective viral particles that could play a role in the horizontal transmission of this retrotransposon.
La proteins from Drosophila melanogaster and Saccharomyces cerevisiae: a yeast homolog of the La autoantigen is dispensable for growth.
Authors: Yoo C J , Wolin S L
Published: October 2015

The human autoantigen La is a 50-kDa protein which binds to the 3' termini of virtually all nascent polymerase III transcripts. Experiments with mammalian transcription extracts have led to the proposal that the La protein is required for multiple rounds of transcription by RNA polymerase III (E. Gottlieb and J. A. Steitz, EMBO J. 8:851-861, 1989; R. J. Maraia, D. J. Kenan, and J. D. Keene, Mol. Cell. Biol. 14:2147-2158, 1994). Although La protein homologs have been identified in a variety of vertebrate species, the protein has not been identified in invertebrates. In order to begin a genetic analysis of La protein function, we have characterized homologs of the La protein in the fruit fly Drosophila melanogaster and the yeast Saccharomyces cerevisiae. We show that both the Drosophila and yeast La proteins are bound to precursors of polymerase III RNAs in vivo. The Drosophila and yeast proteins resemble the human La protein in their biochemical properties, as both proteins can be partially purified from cells by a procedure previously devised to purify the human protein. Similarly to vertebrate La proteins, the Drosophila and yeast homologs preferentially bind RNAs that terminate with a 3' hydroxyl. Despite the fact that the La protein is conserved between humans and Saccharomyces cerevisiae, yeast cells containing a null allele of the gene encoding the La protein are viable, suggesting that another protein(s) plays a functionally redundant role.
Transcription of a variant human U6 small nuclear RNA gene is controlled by a novel, internal RNA polymerase III promoter.
Authors: Tichelaar J W , Knerer B , Vrabel A , Wieben E D
Published: October 2015

Promoter elements in the 5' flanking regions of vertebrate U6 RNA genes have been shown to be both necessary and sufficient for transcription by RNA polymerase III. We have recently isolated and characterized a variant human U6 gene (87U6) that can be transcribed by RNA polymerase III in vitro in the absence of any natural 5' or 3' flanking sequences. This gene contains 10 nucleotide differences from the previously characterized human U6 gene (wtU6) within the coding region but has no homology to wtU6 in the upstream promoter region. By constructing chimeras between these two genes, we have shown that mutation of as few as two nucleotides in the coding region of the human U6 RNA gene is sufficient to create an internal promoter that is functional in vitro. A T-to-C transition at position 57 and a single T deletion at position 52 produce an internal U6 promoter that is nearly as active in vitro as the external U6 polymerase III promoter utilized by wtU6. Neither of these residues is absolutely conserved during evolution, and both of these nucleotide changes occur within the previously noted A box homology. Deletion and linker scanning mutations within the coding region of this variant U6 gene suggest that, in addition to the central region including bp 52 and 57, sequences at the extreme 5' end of the gene are critical for efficient transcription. In contrast, flanking sequences have a minor effect on transcriptional efficiency. This arrangement is unique among internal RNA polymerase III promoters and may indicate unique regulation of this gene.
Hierarchical phosphorylation at N-terminal transformation-sensitive sites in c-Myc protein is regulated by mitogens and in mitosis.
Authors: Lutterbach B , Hann S R
Published: October 2015

The N-terminal domain of the c-Myc protein has been reported to be critical for both the transactivation and biological functions of the c-Myc proteins. Through detailed phosphopeptide mapping analyses, we demonstrate that there is a cluster of four regulated and complex phosphorylation events on the N-terminal domain of Myc proteins, including Thr-58, Ser-62, and Ser-71. An apparent enhancement of Ser-62 phosphorylation occurs on v-Myc proteins having a mutation at Thr-58 which has previously been correlated with increased transforming ability. In contrast, phosphorylation of Thr-58 in cells is dependent on a prior phosphorylation of Ser-62. Hierarchical phosphorylation of c-Myc is also observed in vitro with a specific glycogen synthase kinase 3 alpha, unlike the promiscuous phosphorylation observed with other glycogen synthase kinase 3 alpha and 3 beta preparations. Although both p42 mitogen-activated protein kinase and cdc2 kinase specifically phosphorylate Ser-62 in vitro and cellular phosphorylation of Thr-58/Ser-62 is stimulated by mitogens, other in vivo experiments do not support a role for these kinases in the phosphorylation of Myc proteins. Unexpectedly, both the Thr-58 and Ser-62 phosphorylation events, but not other N-terminal phosphorylation events, can occur in the cytoplasm, suggesting that translocation of the c-Myc proteins to the nucleus is not required for phosphorylation at these sites. In addition, there appears to be an unusual block to the phosphorylation of Ser-62 during mitosis. Finally, although the enhanced transforming properties of Myc proteins correlates with the loss of phosphorylation at Thr-58 and an enhancement of Ser-62 phosphorylation, these phosphorylation events do not alter the ability of c-Myc to transactivate through the CACGTG Myc/Max binding site.
Protein-tyrosine phosphatase activity of CD45 is activated by sequential phosphorylation by two kinases.
Authors: Stover D R , Walsh K A
Published: October 2015

We describe a potential regulatory mechanism for the transmembrane protein-tyrosine phosphatase CD45. Phosphorylation on both tyrosine and serine residues in vitro results in an activation of CD45 specifically toward one artificial substrate but not another. The activation of these kinases appears to be order dependent, as it is enhanced when phosphorylation of tyrosine precedes that of serine but phosphorylation in the reverse order yields no activation. Any of four protein-tyrosine kinases tested, in combination with the protein-serine/threonine kinase, casein kinase II, was capable of mediating this activation in vitro. The time course of phosphorylation of CD45 in response to T-cell activation is consistent with the possibility that this regulatory mechanism is utilized in vivo.
PEBP2/CBF, the murine homolog of the human myeloid AML1 and PEBP2 beta/CBF beta proto-oncoproteins, regulates the murine myeloperoxidase and neutrophil elastase genes in immature myeloid cells.
Authors: Nuchprayoon I , Meyers S , Scott L M , Suzow J , Hiebert S , Friedman A D
Published: October 2015

The myeloperoxidase (MPO) and neutrophil elastase genes are expressed specifically in immature myeloid cells. The integrity of a polyomavirus enhancer core sequence, 5'-AACCACA-3', is critical to the activity of the murine MPO proximal enhancer. This element binds two species, myeloid nuclear factors 1 alpha and 1 beta (MyNF1 alpha and -beta), present in 32D cl3 myeloid cell nuclear extracts. The levels of the MyNF1s increase during early 32D cl3 cell granulocytic differentiation. Both MyNF1 alpha and -beta supershift with an antiserum raised by using a peptide derived from the N terminus of polyomavirus enhancer-binding protein 2/core-binding factor (PEBP2/CBF) alpha subunit. The specific peptide inhibits these supershifts. In vitro-translated PEBP2/CBF DNA-binding domain binds the murine MPO PEBP2/CBF site. An alternate PEBP2/CBF consensus site, 5'-GACCGCA-3', but not a simian virus 40 enhancer core sequence, 5'-TTCCACA-3', binds the MyNF1s in vitro and activates a minimal murine MPO-thymidine kinase promoter in vivo. The murine neutrophil elastase gene 100-bp 5'-flanking sequences contain several functional elements, including potential binding sites for PU.1, C/EBP, c-Myb, and PEBP2/CBF. The functional element 5'-GGCCACA-3' located at positions -66 to 72 differs from the PEBP2/CBF consensus (5'-PuACCPuCA-3') only by an A-to-G transition at position 2. This DNA element binds MyNF1 alpha and -beta weakly. The N terminis of two PEBP2/CBF alpha subunit family members, PEBP2 alpha A and PEBP2 alpha B (murine AML1), are nearly identical, and 32D c13 cl3 cells contain both corresponding mRNAs. Since t(8;21), t(3;21), and inv(16), associated with myeloid leukemias, disrupt subunits of PEBP2/CBF, we speculate that the resulting oncoproteins, AML1-ETO, AML1-EAP, AML1-Evi1, and CBF beta-MYH11, inhibit early myeloid differentiation.
A monocistronic transcript for a trypanosome variant surface glycoprotein.
Authors: Alarcon C M , Son H J , Hall T , Donelson J E
Published: October 2015

Many protein-encoding genes of African trypanosomes are transcribed as large polycistronic pre-mRNAs that are processed into individual mRNAs containing a 5' spliced leader and 3' poly(A). The 45- to 60-kb pre-mRNAs encoding some variant surface glycoproteins (VSGs) contain as many as eight unrelated coding regions. Here we identify the promoter for a metacyclic VSG gene that is expressed without duplication in a bloodstream trypanosome clone. This 70-bp promoter is located 2 kb upstream of the telomere-linked VSG gene and directs the synthesis of a monocistronic VSG pre-mRNA lacking the 5' spliced leader. Its sequence only slightly resembles those of other known trypanosome promoters, but it does cross-hybridize with several related sequences elsewhere in the genome. These results suggest that a new class of trypanosome promoters has been found, whose function is to initiate monocistronic transcription of those VSG genes normally expressed during the metacyclic stage.
Laser Acupuncture before Heel Lancing for Pain Management in Healthy Term Newborns: A Randomised Controlled Trial
Authors: Abbasoglu Aslihan , Cabioglu Mehmet Tugrul , Tugcu Ali Ulas , Yapakci Ece , Tekindal Mustafa Agah , Tarcan Aylin
Published: October 2015

Background

Healthy term newborns commonly undergo painful procedures during routine follow-up visits. Non-pharmacological strategies have currently become more important than pharmacological analgesic agents in neonatal pain management. Acupuncture is a new non-pharmacological method for preventing pain in newborns.

Objective

We aimed to investigate the effect of laser acupuncture (LA) at the Yintang point before heel lancing as a non-pharmacological intervention for procedural pain management in infants.

Methods

Forty-two term newborns, who were undergoing heel lancing between postnatal days 3 to 8 as part of routine neonatal screening, were randomly assigned to the LA group or the oral sucrose group. In the LA group, 2 min before the heel lancing, 0.3 J of energy was applied to the Yintang point using a Laser PREMIO-30 unit for 30 s. In the sucrose group, each infant received 0.5 mL of 24% sucrose orally via syringe 2 min before the heel lancing. Each baby's behaviour was scored using the Neonatal Infant Pain Scale (NIPS), assessed blinded to group.

Results

There were no significant differences between the LA and oral sucrose groups with respect to means for gestational week of age at birth, birth weight, actual weight, or Apgar score. Mean procedure time was significantly shorter in the LA group; however, mean crying time was longer and NIPS score was lower compared to the oral sucrose group.

Conclusions

Our results indicate that 0.3 J of LA at the Yintang point before heel lancing is less effective than oral sucrose for reducing the discomfort of this procedure.

Trial Registration Number

KA14/09.
Auricular Acupuncture for Pre-Exam Anxiety in Medical Students: A Prospective Observational Pilot Investigation
Authors: Klausenitz Catharina , Hesse Thomas , Hacker Henriette , Hahnenkamp Klaus , Usichenko Taras
Published: October 2015

Objective

Auricular acupuncture (AA) is effective for the treatment of preoperative anxiety. We aimed to study the feasibility and effects of AA on exam anxiety in a prospective observational pilot study.

Methods

Healthy medical students received bilateral AA using indwelling fixed needles at points MA-IC1, MA-TF1, MA-SC, MA-AH7, and MA-T on the day before an anatomy exam. The needles were removed after the exam. Anxiety levels were measured using the State-Trait-Anxiety Inventory (STAI) and a 100 mm visual analogue scale (VAS-100) before and after the AA intervention and once again immediately before the exam. The duration of sleep on the night before the exam was recorded and compared to that over the preceding 1 week and 6 months (all through students’ recollection). In addition, blood pressure, heart rate and the acceptability of AA to the students were recorded.

Results

Ten students (all female) were included in the final analysis. All tolerated the needles well and stated they would wish to receive AA again for exam anxiety in the future. Exam anxiety measured using both STAI and VAS-100 decreased by almost 20% after AA.

Conclusions

AA was well accepted, the outcome measurement was feasible, and the results have facilitated the calculation of the sample size for a subsequent randomised controlled trial.
Spatial spillover effects of a community action programme targeting on-licensed premises on violent assaults: evidence from a natural experiment
Authors: Brännström Lars , Trolldal Björn , Menke Martin
Published: October 2015

Background

Spatial dependencies may influence the success of community action strategies to prevent and reduce harmful alcohol use. This study examined the effectiveness of a multicomponent Responsible Beverage Service (RBS) programme targeting on-licensed premises on police-recorded assaults in Swedish municipalities. It was expected that the implementation of the programme within any given municipality had an indirect effect by reducing violent assaults in adjacent municipalities.

Methods

This study was a natural experiment exploiting the temporal and spatial variation in the implementation of the RBS programme to predict change in the rate of violent assaults in all Swedish municipalities during 1996–2009 (n=288; T=14; N=4 032). Yearly police-recorded violent assaults per 100 000 inhabitants aged 15 and above committed on weekend nights were used as a dependent variable. Programme fidelity was identified by means of survey data. A semilogarithmic fixed-effects spatial panel regression model was used to estimate the direct, indirect and total effects of the programme.

Results

The direct, indirect and total effects were −1.8% (95% CI −4.4% to 0.8%), −5.8% (95% CI −11.5% to −0.1%) and −7.6% (95% CI −13.2% to −2.2%), respectively. Averaged over time and across all municipalities, implementing one additional programme component in all municipalities will thus reduce violent assaults in one typical municipality by nearly 8%.

Conclusions

The indirect effect of the programme was three times larger than its direct effect. Failing to account for such local spillover effects can result in a considerable underestimation of the programme's total impact and may lead to erroneous policy recommendations.
Systematic oral hydration with water is similar to parenteral hydration for prevention of contrast-induced nephropathy: an updated meta-analysis of randomised clinical data
Authors: Agarwal Shiv Kumar , Mohareb Sameh , Patel Achint , Yacoub Rabi , DiNicolantonio James J , Konstantinidis Ioannis , Pathak Ambarish , Fnu Shailesh , Annapureddy Narender , Simoes Priya K , Kamat Sunil , El-Hayek Georges , Prasad Ravi , Kumbala Damodar , Nascimento Rhanderson M , Reilly John P , Nadkarni Girish N , Benjo Alexandre M
Published: October 2015

Background

Contrast-induced nephropathy (CIN) is the third most common cause of hospital-acquired kidney injury and is related to increased long-term morbidity and mortality. Adequate intravenous (IV) hydration has been demonstrated to lessen its occurrence. Oral (PO) hydration with water is inexpensive and readily available but its role for CIN prevention is yet to be determined.

Methods

PubMed, EMBASE and the Cochrane Central register of controlled trials (CENTRAL) databases were searched until April 2015 and studies were selected using the Preferred Reporting Items for Systematic Reviews and Meta-Analyses (PRISMA) checklist. All randomised clinical trials with head-to-head comparison between PO and IV hydration were included.

Results

A total of 5 studies with 477 patients were included in the analysis, 255 of those receiving PO water. The incidence of CIN was statistically similar in the IV and PO arms (7.7% and 8.2%, respectively; relative risk 0.97; 95% CI 0.36 to 2.94; p=0.95). The incidence of CIN was statistically similar in the IV and PO arms in patients with chronic kidney disease and with normal renal function. Rise in creatinine at 48–72 h was lower in the PO hydration group compared with IV hydration (pooled standard mean difference 0.04; 95% CI 0.03 to 0.06; p<0.001; I2=62%).

Conclusions

Our meta-analysis shows that systematic PO hydration with water is at least as effective as IV hydration with saline to prevent CIN. PO hydration is cheaper and more easily administered than IV hydration, thus making it more attractive and just as effective.
Intradural intervertebral disc herniation in a 5‐year‐old dachshund
Authors: Lampe Rachel , Davies Emma Sian
Published: October 2015

A 5‐year‐old male neutered dachshund presented dragging his right hindlimb with a history of acute onset, progressive, non‐painful paraparesis. The lesion was localised to a T3–L3 myelopathy, and MRI revealed moderate spinal cord compression at the L3–L4 intervertebral disc space, which was consistent with intervertebral disc extrusion. A routine hemilaminectomy was performed; however, no compressive material could be located compressing the spinal cord. The surgical site and images were reassessed, but were found to be correct and appropriate. A durotomy was performed and calcified disc material bulged out through the incision, with more fibroid material removed through gentle probing. The site was fenestrated and closed routinely, and the dog was ambulatory the next day and discharged two days later. At re‐examination four weeks later, the dog was ambulatory with no paresis.
The interrelationship between muscle oxygenation, muscle activation, and pulmonary oxygen uptake to incremental ramp exercise: influence of aerobic fitness
Authors: Boone Jan , Barstow Thomas J. , Celie Bert , Prieur Fabrice , Bourgois Jan
Published: October 2015

We investigated whether muscle and ventilatory responses to incremental ramp exercise would be influenced by aerobic fitness status by means of a cross-sectional study with a large subject population. Sixty-four male students (age: 21.2 ± 3.2 years) with a heterogeneous peak oxygen uptake (51.9 ± 6.3 mL·min−1·kg−1, range 39.7–66.2 mL·min−1·kg−1) performed an incremental ramp cycle test (20–35 W·min−1) to exhaustion. Breath-by-breath gas exchange was recorded, and muscle activation and oxygenation were measured with surface electromyography and near-infrared spectroscopy, respectively. The integrated electromyography (iEMG), mean power frequency (MPF), deoxygenated [hemoglobin and myoglobin] (deoxy[Hb+Mb]), and total[Hb+Mb] responses were set out as functions of work rate and fitted with a double linear function. The respiratory compensation point (RCP) was compared and correlated with the breakpoints (BPs) (as percentage of peak oxygen uptake) in muscle activation and oxygenation. The BP in total[Hb+Mb] (83.2% ± 3.0% peak oxygen uptake) preceded (P < 0.001) the BP in iEMG (86.7% ± 4.0% peak oxygen uptake) and MPF (86.3% ± 4.1% peak oxygen uptake), which in turn preceded (P < 0.01) the BP in deoxy[Hb+Mb] (88.2% ± 4.5% peak oxygen uptake) and RCP (87.4% ± 4.5% peak oxygen uptake). Furthermore, the peak oxygen uptake was significantly (P < 0.001) positively correlated to the BPs and RCP, indicating that the BPs in total[Hb+Mb] (r = 0.66; P < 0.001), deoxy[Hb+Mb] (r = 0.76; P < 0.001), iEMG (r = 0.61; P < 0.001), MPF (r = 0.63; P < 0.001), and RCP (r = 0.75; P < 0.001) occurred at a higher percentage of peak oxygen uptake in subjects with a higher peak oxygen uptake. In this study a close relationship between muscle oxygenation, activation, and pulmonary oxygen uptake was found, occurring in a cascade of events. In subjects with a higher aerobic fitness level this cascade occurred at a higher relative intensity.
Behaviour-mediated alteration of positively size-dependent vulnerability to angling in response to historical fishing pressure in a freshwater salmonid
Authors: Tsuboi Jun-ichi , Morita Kentaro , Klefoth Thomas , Endou Shinsuke , Arlinghaus Robert
Published: October 2015

Positively size-selective vulnerability to fishing is well established in recreational fisheries. Size-selective harvesting can either induce an indirect selection response of behavioural traits that are correlated with size or exert direct selection pressures on behaviours that contribute to vulnerability. In addition, learning to avoid future capture may always happen. Behavioural change caused by fishing may in turn affect the size-selective properties of angling. To test this prediction, field experiments with amago salmon (Oncorhynchus masou ishikawae) were conducted. We demonstrated that, as expected, large fish were more vulnerable than smaller individuals in a low fishing pressure (LP) stream and that positively size-selective angling was not (or no longer) present in a high fishing pressure (HP) stream. Moreover, fish in the HP stream were by far less vulnerable to angling with natural bait than fishes in the LP stream. Laboratory studies showed that offspring from HP streams were intrinsically shyer than offspring collected from LP streams. We propose that the increased timidity of individuals inhibiting HP streams disrupted the generally positive relationship among salmon body size and its vulnerability to angling. Fisheries-induced timidity in response to high exploitation rates reduces catchability, affects the size-selective properties of angling, reduces the value of fishery-dependent stock assessments, and potentially affects trophic interactions.
Annual cycle of lipid content and lipid class composition in zooplankton from the Beaufort Sea shelf, Canadian Arctic
Authors: Connelly Tara L. , Businski Tara N. , Deibel Don , Parrish Christopher C. , Trela Piotr
Published: October 2015

We determined seasonal cycles of lipid content, lipid class composition, and carbon and nitrogen content of seven taxa of zooplankton that were collected from the Beaufort Sea shelf, Canadian Arctic, over a 10-month period (September 2003 – August 2004). All taxa except the chaetognath Parasagitta elegans had substantial lipids stores (>50%), either seasonally (Oikopleura spp.) or throughout the year (Calanus hyperboreus, Calanus glacialis, Paraeuchaeta glacialis, Metridia longa, and Eukrohnia hamata). Wax esters were the dominant lipid class in the chaetognath Eukrohnia hamata and in all copepods, including the carnivore Paraeuchaeta glacialis. Seasonal trends in lipid content and composition varied among taxa; some taxa had little variation from winter through summer (e.g., Parasagitta elegans), other taxa showed little variation until summer (e.g., Calanus glacialis), and others showed increasing or decreasing trends during winter and spring (e.g., Calanus hyperboreus). Specifically, total lipid content of Calanus hyperboreus decreased from January through May at a rate of ∼450 μg·month−1·individual−1in adult females and ∼100 μg·month−1·individual−1in juvenile copepodite IV, representing a 75%–85% loss in lipid.
Assessing density functionals for the prediction of thermochemistry of Ti–O–Cl species
Authors: Ge Yingbin , DePrekel Douglas , Lam Kui-Ting , Ngo Kevin , Vo Phu
Published: October 2015

Titanium dioxide (TiO2) nanoparticles are widely used in contaminant remediation, photocatalysis and solar cell manufacturing. The low-cost production of TiO2 nanoparticles via the combustion of titanium tetrachloride (TiCl4) in oxygen is thus an important industrial process. To accurately model the flame synthesis of TiO2 nanoparticles, reliable thermodynamic data of Ti–O–Cl species are indispensable but often unavailable. We therefore carried out benchmark calculations, using the left-eigenstate completely renormalized singles, doubles and perturbative triples (CR-CC(2,3), aka CR-CCL) method with the cc-pVTZ basis set, to obtain the equilibrium structures and vibrational frequencies of selected Ti–O–Cl species; we then performed single-point CCSD(T)/aug-cc-pVLZ ([Formula: see text]) calculations to extrapolate the CCSD(T)/CBS energies. After analyzing the experimental and calculated enthalpy of selected Ti–O–Cl species, the standard enthalpy of formation of the TiOCl2 molecule is determined to be [Formula: see text]600.5[Formula: see text]kJ/mol at 298[Formula: see text]K. The standard enthalpy of all other Ti–O–Cl species are determined accordingly. Finally, we assessed the accuracy of 42 popular density functionals for the Ti–O–Cl species. Among these assessed functionals, the B98 functional, tightly followed by B97-1 and B3LYP, exhibits the best overall performance in the prediction of the thermochemistry of the Ti–O–Cl species.