bluuebunny/crossref_metadata_2025 huggingface dataset:
Yeast regulatory gene GAL3: carbon regulation; UASGal elements in common with GAL1, GAL2, GAL7, GAL10, GAL80, and MEL1; encoded protein strikingly similar to yeast and Escherichia coli galactokinases.
Authors: Bajwa W , Torchia T E , Hopper J E
Published: September 2015

GAL3 gene expression is required for rapid GAL4-mediated galactose induction of the galactose-melibiose regulon genes in Saccharomyces cerevisiae. Here we show by Northern (RNA) blot analysis that GAL3 gene expression is itself galactose inducible. Like the GAL1, GAL7, GAL10, and MEL1 genes, the GAL3 gene is severely glucose repressed. Like the MEL1 gene, but in contrast to the GAL1, GAL7, and GAL10 genes, GAL3 is expressed at readily detectable basal levels in cells grown in noninducing, nonrepressing media. We determined the sequence of the S. cerevisiae GAL3 gene and its 5'-noncoding region. Within the 5'-noncoding region of the GAL3 gene, we found two sequences similar to the UASGal elements of the other galactose-melibiose regulon genes. Deletion analysis indicated that only the most ATG proximal of these sequences is required for GAL3 expression. The coding region of GAL3 consists of a 1,275-base-pair open reading frame in the direction of transcription. A comparison of the deduced 425-amino-acid sequence with the protein data bank revealed three regions of striking similarity between the GAL3 protein and the GAL1-specified galactokinase of Saccharomyces carlsbergensis. One of these regions also showed striking similarity to sequences within the galactokinase protein of Escherichia coli. On the basis of these protein sequence similarities, we propose that the GAL3 protein binds a molecule identical to or structurally related to one of the substrates or products of the galactokinase-catalyzed reaction.
cDNA cloning and expression of a human platelet-derived growth factor (PDGF) receptor specific for B-chain-containing PDGF molecules.
Authors: Claesson-Welsh L , Eriksson A , Morén A , Severinsson L , Ek B , Ostman A , Betsholtz C , Heldin C H
Published: September 2015

The structure of the human receptor for platelet-derived growth factor (PDGF) has been deduced through cDNA cloning. A 5.45-kilobase-pair cDNA clone predicts a 1,106-amino-acid polypeptide, including the cleavable signal sequence. The overall amino acid sequence similarity with the murine PDGF receptor is 85%. After transcription of the cDNA and translation in vitro, a PDGF receptor antiserum was used to immunoprecipitate a product of predicted size, which also could be phosphorylated in vitro. Stable introduction of the cDNA into Chinese hamster ovary (CHO) cells led to the expression of a 190-kilodalton component, which was immunoprecipitated by the PDGF receptor antiserum; this most probably represents the mature PDGF receptor. Binding assays with different 125I-labeled dimeric forms of PDGF A and B chains showed that the PDGF receptor expressed in CHO cells bound PDGF-BB and, to a lesser extent, PDGF-AB, but not PDGF-AA.
Cloning and analysis of the positively acting regulatory gene amdR from Aspergillus nidulans.
Authors: Andrianopoulos A , Hynes M J
Published: September 2015

The positively acting regulatory gene amdR of Aspergillus nidulans coordinately regulates the expression of four unlinked structural genes involved in acetamide (amdS), omega amino acid (gatA and gabA), and lactam (lamA) catabolism. By the use of DNA-mediated transformation of A. nidulans, the amdR regulatory gene was cloned from a genomic cosmid library. Southern blot analysis of DNA from various loss-of-function amdR mutants revealed the presence of four detectable DNA rearrangements, including a deletion, an insertion, and a translocation. No detectable DNA rearrangements were found in several constitutive amdRc mutants. Analysis of the fate of amdR-bearing plasmids in transformants showed that 10 to 20% of the transformation events were homologous integrations or gene conversions, and this phenomenon was exploited in developing a strategy by which amdRc and amdR- alleles can be readily cloned and analyzed. Examination of the transcription of amdR by Northern blot (RNA blot) analysis revealed the presence of two mRNAs (2.7 and 1.8 kilobases) which were constitutively synthesized at a very low level. In addition, amdR transcription did not appear to depend on the presence of a functional amdR product nor was it altered in amdRc mutants. The dosage effects of multiple copies of amdR in transformants were examined, and it was shown that such transformants exhibited stronger growth than did the wild type on acetamide and pyrrolidinone media, indicating increased expression of the amdS and lamA genes, respectively. These results were used to formulate a model for amdR-mediated regulation of gene expression in which the low constitutive level of amdR product sets the upper limits of basal and induced transcription of the structural genes. Multiple copies of 5' sequences from the amdS gene can result in reduced growth on substrates whose utilization is dependent on amdR-controlled genes. This has been attributed to titration of limiting amdR gene product. Strong support for this proposal was obtained by showing that multiple copies of the amdR gene can reverse this phenomenon (antititration).
Structural and functional analysis of the rat malic enzyme gene promoter.
Authors: Morioka H , Tennyson G E , Nikodem V M
Published: September 2015

We have characterized sequences of genomic DNA 5' to the coding region of the rat malic enzyme gene. This sequence possesses neither TATA nor CCAAT sequences in their usual positions but is rich in GC residues. Sequences similar to those found in the regulatory regions of other genes are discussed. Deletion analyses have revealed that sequences +1 to -41 are sufficient to initiate expression, although inclusion of information up to -177 is necessary for maximal promoter activity.
Presence of the adenovirus E1A-like activity in preimplantation stage mouse embryos.
Authors: Suemori H , Hashimoto S , Nakatsuji N
Published: September 2015

The presence of the adenovirus E1A-like activity in embryonal carcinoma stem cells has been reported. We now show that preimplantation stage mouse embryonic cells allow transcription of the E1A-dependent E2A gene when infected with E1A-deleted mutant dl312, indicating the presence of the E1A-like activity in morulae and blastocysts. Moreover, such activity seems to decrease or disappear at about the time of implantation.
CDC33 encodes mRNA cap-binding protein eIF-4E of Saccharomyces cerevisiae.
Authors: Brenner C , Nakayama N , Goebl M , Tanaka K , Toh-e A , Matsumoto K
Published: September 2015

The bcy1 mutation makes the cdc33 start mutant arrest at random points in the cell cycle instead of only at G1. We cloned and sequenced CDC33. This coding sequence is identical to that of the gene encoding the Saccharomyces cerevisiae 24-kilodalton mRNA cap-binding protein, eIF-4E.
Multiple cis-acting sequence elements are required for efficient splicing of simian virus 40 small-t antigen pre-mRNA.
Authors: Fu X Y , Colgan J D , Manley J L
Published: September 2015

We have determined the effects of a number of mutations in the small-t antigen mRNA intron on the alternative splicing pattern of the simian virus 40 early transcript. Expansion of the distance separating the small-t pre-mRNA lariat branch point and the shared large T-small t 3' splice site from 18 to 29 nucleotides (nt) resulted in a relative enhancement of small-t splicing in vivo. This finding, coupled with the observation that large-T pre-RNA splicing in vitro was not affected by this expansion, suggests that small-t splicing is specifically constrained by a short branch point-3' splice site distance. Similarly, the distance separating the 5' splice site and branch point (48 nt) was found to be at or near a minimum for small-t splicing, because deletions in this region as small as 2 nt dramatically reduced the ratio of small-t to large-T mRNA that accumulated in transfected cells. Finally, a specific sequence within the small-t intron, encompassing the upstream branch sites used in large-T splicing, was found to be an important element in the cell-specific pattern of early alternative splicing. Substitutions within this region reduced the ratio of small-t to large-T mRNA produced in HeLa cells but had only minor effects in human 293 cells.
The Drosophila melanogaster tropomyosin II gene produces multiple proteins by use of alternative tissue-specific promoters and alternative splicing.
Authors: Hanke P D , Storti R V
Published: September 2015

The structure of the Drosophila melanogaster tropomyosin II (TmII) gene has been determined by DNA sequencing of cDNA clones and the genomic DNA coding for the gene. Two overlapping transcriptional units produce at least four different tropomyosin isoforms. A combination of developmentally regulated promoters and alternative splicing produces both muscle and cytoskeletal tropomyosin isoforms. One promoter is a muscle-specific promoter and produces three different tropomyosin isoforms by alternative splicing of the last three 3' exons. The second promoter has the characteristics of a housekeeping promoter and produces a cytoskeletal tropomyosin isoform. Several internal exons along with a final 3' exon are alternatively spliced in the cytoskeletal transcript. The intron-exon boundaries of the TmII gene are identical to the intron-exon boundaries of all vertebrate tropomyosin genes reported, but are very different from the intron-exon boundaries of the D. melanogaster tropomyosin I gene. The TmII gene is the only reported tropomyosin gene that has two promoters and a quadruple alternative splice choice for the final exon. Models for the mechanism of D. melanogaster tropomyosin gene evolution are discussed.
Platelet tyrosine-specific protein phosphorylation is regulated by thrombin.
Authors: Ferrell J E , Martin G S
Published: September 2015

Intact human platelets, terminally differentiated cells with no growth potential, were found to possess unusually high levels of tyrosine-specific protein phosphorylation. The physiological platelet activator thrombin transiently elevated platelet phosphotyrosine content, apparently through stimulation of one or more tyrosine-specific protein kinases. Immunoblotting with antiphosphotyrosine antiserum showed that thrombin caused dramatic changes in the tyrosine phosphorylation of a number of individual protein bands and that these changes occurred in three distinct temporal waves. Most but not all of the protein bands phosphorylated at tyrosine in response to thrombin were also tyrosine phosphorylated in response to chilling or the combination of ionophore A23187 and tetradecanoylphorbol acetate. Thrombin stimulated the phosphorylation of the tyrosine kinase pp60c-src, primarily at Ser-12 and Tyr-527, although the effects of these phosphorylations on platelet pp60c-src function were not apparent. Together, these results suggest that tyrosine-specific protein kinases of uncertain identity are involved in signal transduction in platelets.
Chromosome instability associated with human alphoid DNA transfected into the Chinese hamster genome.
Authors: Heartlein M W , Knoll J H , Latt S A
Published: September 2015

Repetitive DNA sequences have been implicated in the mediation of DNA rearrangement in mammalian cells. We have tested this hypothesis by using a dihydrofolate reductase (DHFR) expression vector into which candidate sequences were inserted. DHFR- Chinese hamster ovary (CHO) cells were transfected with this vector, the amplification of which was then selected for by methotrexate (MTX) exposure. Cells transfected with the vector alone (and resistant to 0.02 or 1.0 microM MTX) or with a poly(dG-dT) insert (and resistant to 0.05 or 1.0 microM MTX) showed little change in chromosome aberrations or sister chromatid exchange frequencies. In contrast, transfection of DHFR- CHO cells with a vector containing either of two distinct 0.34-kilobase human alphoid DNA segments (and selection to 0.05 to 10.0 microM MTX) showed an approximately 50% increase in chromosome number and marked changes in chromosome structure, including one or two dicentric or ring forms per cell. The sister chromatid exchange frequency also increased, to more than double the frequency of that in cells transfected without insert or those containing poly(dG-dT). In situ hybridization of one 0.34-kilobase insert in some cells suggested clustering of homologous sequences in structurally abnormal recipient CHO cell chromosomes. The approach described provides an introduction to a unique means for a coordinate molecular and cytological study of dynamic changes in chromosome structure.
Structure and regulation of a nuclear gene in Saccharomyces cerevisiae that specifies MRP7, a protein of the large subunit of the mitochondrial ribosome.
Authors: Fearon K , Mason T L
Published: September 2015

The gene for MRP7, a 40-kilodalton protein of the large subunit of the yeast mitochondrial ribosome, was identified in a lambda gt11 expression library by immunological screening with a monoclonal antibody to MRP7. An intact copy of MRP7 was then isolated from a yeast genomic library by colony hybridization. Gene disruption showed that MRP7 protein was essential for ribosomal function. Sequencing of MRP7 revealed a coding region for a basic (pI 10.6), 43.2-kilodalton protein containing 371 amino acid residues. Amino acid residues 28 to 112 of the deduced MRP7 sequence aligned with the 84 residues of the Escherichia coli ribosomal protein L27, but no significant similarity was detected between the carboxy-terminal 259 amino acids of MRP7 and other protein sequences in existing computer data bases. Within the aligned region, there was 49% amino acid identity between MRP7 and L27, compared with the 57% identity observed between L27 and its homolog in Bacillus stearothermophilus. The steady-state levels of the MRP7 protein and its mRNA were monitored in response to catabolite repression and to increased dosage of the MRP7 gene. The response to catabolite repression was characterized by a ninefold change in the level of the protein and little, if any, change in the level of the mRNA. In cells carrying the MRP7 gene on a high-copy-number plasmid, the mRNA was increased 20-fold, but there was no significant increase in MRP7 protein. Furthermore, MRP7 mRNA and protein accumulated at normal levels in [rho0] cells, which are devoid of 21S rRNA, indicating that the protein is relatively stable in the absence of ribosome assembly. Together, these results suggest that MRP7 is regulated posttranscriptionally, probably at the level of protein synthesis rather than protein turnover.
Wolffish antifreeze protein genes are primarily organized as tandem repeats that each contain two genes in inverted orientation.
Authors: Scott G K , Hayes P H , Fletcher G L , Davies P L
Published: September 2015

The antifreeze protein genes of the wolffish (Anarhichas lupus) constitute a large multigene family of 80 to 85 copies, which can be classified into two sets. One-third of the genes were linked but irregularly spaced. The other two-thirds were organized as 8-kilobase-pair (kbp) tandem direct repeats that each contained two genes in inverted orientation; DNA sequence analysis suggests that both genes are functional. Except for a single region specific to each gene, the genes and their immediate flanking sequences were 99.2% identical. This degree of identity ended soon after a putative transcription termination sequence; as the 3' ends of the genes were only 1.3 kbp apart, these sequences might confer mutual protection from interference by transcriptional runoff. A Southern blot of wolffish DNA restricted with enzymes that do not cut within the tandem repeats indicated that the repeats were clustered in groups of six or more. The organization of antifreeze protein genes in the wolffish was very similar to that in the unrelated winter flounder, which produces a completely different antifreeze. This similarity might reflect common dynamics by which their progenitors adapted to life in ice-laden sea water.
c-myc antisense transcripts accelerate differentiation and inhibit G1 progression in murine erythroleukemia cells.
Authors: Prochownik E V , Kukowska J , Rodgers C
Published: September 2015

Friend murine erythroleukemia (F-MEL) cells were transfected with a plasmid bearing tandemly arranged mouse c-myc antisense and dihydrofolate reductase transcription units. Sixteen clones were isolated, each containing unrearranged c-myc sequences and expressing high levels of antisense transcripts. All antisense clones examined contained reduced amounts of cytoplasmic endogenous c-myc transcripts. The kinetics of reaccumulation of endogenous c-myc mRNA during a 24-h exposure to dimethyl sulfoxide (DMSO) were also retarded and the ultimate transcript levels attained were less than in control cells. Antisense clones grew as well as control F-MEL cells in medium containing 10% fetal calf serum but at only a half and a quarter of the control rates in media containing 5 and 2% serum, respectively. Antisense clones differentiated faster and to a greater degree than control cells following DMSO exposure. myc antisense transcript expression was increased by growing cells in methotrexate, which resulted in an enhanced response to DMSO. Fluorescence-activated cell sorter (FACS) analysis of cellular DNA content indicated that a greater fraction of antisense nuclei contained a G0/G1 2n DNA content following a 24-h exposure to DMSO. When density-arrested antisense clones were diluted into fresh medium to allow reentry into the cell cycle, they incorporated less [3H]thymidine than control cells. FACS analysis showed that this was because only a portion of the cell population was entering S phase. Whereas control cells did not increase in size following release from density arrested antisense cells contained a subpopulation which were initially smaller and which eventually attained the same size as control cells. Quiescent antisense cells thus comprise two populations, each arrested at a different point in G1. Dilutional replating allowed both populations to reenter the cell cycle. We propose a model which postulates that certain minimal myc levels are necessary for cells to traverse G1. Those with insufficient levels, due, for example, to antisense inhibition, are unable to completely traverse G1 during density arrest and synchronize at an earlier point than do control cells. This earlier point may be along the differentiation pathway and may account for the greater responsiveness of antisense cells to DMSO induction. This model postulates that F-MEL cells overexpressing myc fail to differentiate because myc levels are never sufficiently low enough to allow cells to enter the differentiation pathway.
Isolation and characterization of an autonomously replicating sequence from Ustilago maydis.
Authors: Tsukuda T , Carleton S , Fotheringham S , Holloman W K
Published: September 2015

DNA fragments that function as autonomously replicating sequences (ARSs) have been isolated from Ustilago maydis. When inserted into an integrative transforming vector, the fragments increased the frequency of U. maydis transformation several-thousandfold. ARS-containing plasmids were transmitted in U. maydis as extrachromosomal elements through replication. They were maintained at a level of about 25 copies per cell but were mitotically unstable. One ARS characterized in detail, which we called UARS1, was localized to a 1.7-kilobase fragment. UARS1 contained a cluster of active sequences. This element could be reduced further into three separate subfragments, each of which retained ARS activity. The smallest one was 383 base pairs (bp) long. Although not active itself in yeast, this small fragment contained seven 8-bp direct repeats, two contiguous 30-bp direct repeats, and five 11-bp units in both orientations with sequences similar but not identical to the consensus sequence found to be crucial for ARS activity in Saccharomyces cerevisiae.
Cysteine residues in the zinc finger and amino acids adjacent to the finger are necessary for DNA binding by the LAC9 regulatory protein of Kluyveromyces lactis.
Authors: Witte M M , Dickson R C
Published: September 2015

LAC9 is a positive regulatory protein that controls transcription of the lactose-galactose regulon in Kluyveromyces lactis. LAC9 is homologous to the GAL4 protein of Saccharomyces cerevisiae. Both proteins have a single "zinc finger" which plays a role in DNA binding. We previously hypothesized (L. V. Wray, M. M. Witte, R. C. Dickson, and M. I. Riley, Mol. Cell. Biol. 7:1111-1121, 1987) that the DNA-binding domain of the LAC9 protein consisted of the zinc finger as well as a region of amino acids on the carboxyl-terminal side of the zinc finger. In this study we used oligonucleotide-directed mutagenesis to introduce 13 single-amino-acid changes into the proposed DNA-binding domain of the LAC9 protein. Variant LAC9 proteins carrying an amino acid substitution in any one of the four highly conserved Cys residues of the zinc finger had reduced DNA-binding activity, suggesting that each Cys is necessary for DNA binding. Three of four variant LAC9 proteins with amino acid substitutions located on the carboxyl-terminal side of the zinc finger had reduced DNA-binding activity. These results support our hypothesis that the DNA-binding domain of the LAC9 protein is composed of the zinc finger and the adjacent region on the carboxyl side of the zinc finger, a region that has the potential to form an alpha-helix. Finally, LAC9 proteins containing His residues substituted for the conserved Cys residues also had reduced DNA-binding activity, indicating that His residues are not equivalent to Cys residues, as had been previously thought.
Role of STE genes in the mating factor signaling pathway mediated by GPA1 in Saccharomyces cerevisiae.
Authors: Nakayama N , Kaziro Y , Arai K , Matsumoto K
Published: September 2015

The ste mutants (ste2, ste4, ste5, ste7, ste11, and ste12) are insensitive to mating factors and are, therefore, sterile. Roles of the STE gene products in the GPA1-mediated mating factor signaling pathway were studied by using ste gpa1 double mutants. Mating efficiency of a ste2 mutant defective in the alpha-factor receptor increased 1,000-fold in a gpa1 background, while G1 arrest and aberrant morphology (shmoo) caused by gpa1 were not suppressed by ste2. Furthermore, the steady-state level of the FUS1 transcript, which normally increases in response to mating factors, was also elevated when the GPA1 function was impaired. These results suggest that the GPA1 protein functions downstream of the STE2 receptor. Conversely, the sterility of ste4, ste5, ste7, ste11, and ste12 mutants was not suppressed by gpa1, but the lethal phenotype of gpa1 was suppressed by these ste mutations. Northern (RNA) blotting analysis revealed that the ste7, ste11, and ste12 mutations caused reductions of 50 to 70% in the steady-state levels of the GPA1 transcript, while ste4 had a slight effect and ste5 had no effect. This implies that the suppression by ste7, ste11, and ste12 could be due to reduced syntheses of additional components, including an effector, and that suppression by ste4 and ste5 may result from direct effects on the signaling pathway. The STE4, STE5, STE7, STE11, and STE12 products, therefore, appear to specify components of the signal transduction machinery, directly or indirectly, which function together with or downstream of GPA1.
Regulation of 4F2 heavy-chain gene expression during normal human T-cell activation can be mediated by multiple distinct molecular mechanisms.
Authors: Lindsten T , June C H , Thompson C B , Leiden J M
Published: September 2015

The 4F2 molecule belongs to the set of cell surface antigens which is induced following lectin- or antigen-mediated T-cell activation. The increase in 4F2 cell surface expression following lectin-mediated stimulation has been shown to be accompanied by a parallel increase in the steady-state levels of 4F2 heavy-chain (4F2HC) mRNA. The studies described in this report were designed to further elucidate the molecular mechanisms responsible for induction of 4F2HC gene expression following activation of normal resting human peripheral blood T cells. The low levels of mature 4F2HC mRNA in resting T cells were shown to be the result of a block to transcription elongation within the exon 1-intron 1 region of the 4F2HC gene rather than promoter inactivity. Phorbol myristate acetate stimulation of resting T cells resulted in a 20-fold increase in steady-state 4F2HC mRNA levels which was mediated by removal of this block to transcription elongation. The phorbol myristate acetate-induced increase in 4F2HC gene expression is distinct from previously described AP-1-mediated, phorbol ester-induced gene expression in that it requires new protein synthesis. Treatment of resting T cells with ionomycin plus PMA resulted in a 60-fold increase in 4F2HC mRNA levels. This induction was mediated by both an increase in promoter utilization and removal of the block to transcription elongation. Finally, by increasing the half-life of 4F2HC mRNA, cycloheximide treatment of resting T cells induced an approximately five fold increase in the levels of 4F2HC gene expression, although the physiologic significance of this mechanism remains unclear. These results demonstrate that the level of 4F2HC gene expression in normal peripheral blood T cells can be regulated by at least three distinct molecular pathways: (i) changes in promoter utilization, (ii) modulation of a block to transcription elongation, and (iii) alteration in mRNA stability.
The positive regulatory function of the 5'-proximal open reading frames in GCN4 mRNA can be mimicked by heterologous, short coding sequences.
Authors: Williams N P , Mueller P P , Hinnebusch A G
Published: September 2015

Translational control of GCN4 expression in the yeast Saccharomyces cerevisiae is mediated by multiple AUG codons present in the leader of GCN4 mRNA, each of which initiates a short open reading frame of only two or three codons. Upstream AUG codons 3 and 4 are required to repress GCN4 expression in normal growth conditions; AUG codons 1 and 2 are needed to overcome this repression in amino acid starvation conditions. We show that the regulatory function of AUG codons 1 and 2 can be qualitatively mimicked by the AUG codons of two heterologous upstream open reading frames (URFs) containing the initiation regions of the yeast genes PGK and TRP1. These AUG codons inhibit GCN4 expression when present singly in the mRNA leader; however, they stimulate GCN4 expression in derepressing conditions when inserted upstream from AUG codons 3 and 4. This finding supports the idea that AUG codons 1 and 2 function in the control mechanism as translation initiation sites and further suggests that suppression of the inhibitory effects of AUG codons 3 and 4 is a general consequence of the translation of URF 1 and 2 sequences upstream. Several observations suggest that AUG codons 3 and 4 are efficient initiation sites; however, these sequences do not act as positive regulatory elements when placed upstream from URF 1. This result suggests that efficient translation is only one of the important properties of the 5' proximal URFs in GCN4 mRNA. We propose that a second property is the ability to permit reinitiation following termination of translation and that URF 1 is optimized for this regulatory function.
Generation of murine stromal cell lines supporting hematopoietic stem cell proliferation by use of recombinant retrovirus vectors encoding simian virus 40 large T antigen.
Authors: Williams D A , Rosenblatt M F , Beier D R , Cone R D
Published: September 2015

The bone marrow is a complex microenvironment made up of multiple cell types which appears to play an important role in the maintenance of hematopoietic stem cell self-renewal and proliferation. We used murine long-term marrow cultures and a defective recombinant retrovirus vector containing the simian virus 40 large T antigen to immortalize marrow stromal cells which can support hematopoiesis in vitro for up to 5 weeks. Such cloned cell lines differentially supported stem cells which, when transplanted, allowed survival of lethally irradiated mice, formed hematopoietic spleen colonies in vivo, and stimulated lymphocyte proliferation in vitro. Molecular and functional analyses of these cell lines did not demonstrate the production of any growth factors known to support the proliferation of primitive hematopoietic stem cells. All cell lines examined produced macrophage colony-stimulating factor. The use of immortalizing retrovirus vectors may allow determination of unique cellular proteins important in hematopoietic stem cell proliferation by the systematic comparison of stromal cells derived from a variety of murine tissues.
Early retinoic acid-induced F9 teratocarcinoma stem cell gene ERA-1: alternate splicing creates transcripts for a homeobox-containing protein and one lacking the homeobox.
Authors: LaRosa G J , Gudas L J
Published: September 2015

Retinoic acid (RA), the natural acidic derivative of vitamin A, can modulate the expression of specific genes and can induce some cell types, such as the murine F9 teratocarcinoma stem cell line, to differentiate in culture. As an initial step toward understanding the molecular mechanism(s) by which RA exerts these effects, we previously isolated cDNA clones for a gene, ERA-1, which has the characteristics of an early, direct target for RA. We demonstrated that RA causes a rapid, dose-dependent, and protein synthesis-independent expression of the ERA-1 gene (G. J. LaRosa and L. J. Gudas, Proc. Natl. Acad. Sci. USA 85:329-333, 1988). We now report the full-length cDNA sequence and the further characterization of this gene. The data indicate that the RA-induced 2.2- to 2.4-kilobase ERA-1 RNA species that we previously detected consists of two alternately spliced messages. One mRNA encodes a protein with a predicted mass of about 36 kilodaltons (kDa) that possesses the Hox 1.6 homeobox domain. The other mRNA encodes a truncated protein of about 15 kDa which is identical to the 36-kDa protein for 114 amino acids at the amino-terminal end but which lacks the homeobox amino acid sequence. The RA-associated increase in the ERA-1 mRNA level does not appear to be due to message stabilization, suggesting that the response is at the level of transcription. By Northern (RNA) blot analysis, the usual 2.2- to 2.4-kilobase mRNA species was also rapidly expressed in P19 teratocarcinoma cells during their differentiation to fibroblastic cells in response to RA and was detected in day 10.5 and day 13.5 mouse embryos. This result indicates that the expression of this gene is not limited to the endodermal differentiation of F9 cells.
Transcriptional and posttranscriptional regulation of CSF-1 gene expression in human monocytes.
Authors: Horiguchi J , Sariban E , Kufe D
Published: September 2015

Regulation of CSF-1 gene expression was investigated in human monocytes. CSF-1 transcripts were at low or undetectable levels in resting monocytes. However, in monocytes treated with 12-O-tetradecanoylphorbol-13-acetate (TPA), CSF-1 mRNA was increased by 3 h and reached maximal levels by 12 h of drug exposure. When nuclear run-on assays were used, CSF-1 gene transcription was also at low or undetectable levels in resting monocytes but was activated after TPA exposure. TPA-treated monocytes exposed to actinomycin D further demonstrated that the half-life of the CSF-1 mRNA is 0.9 h. The results also demonstrated that the protein synthesis inhibitor, cycloheximide (CHX), increases CSF-1 mRNA levels in both resting and TPA-treated monocytes. These effects of CHX occurred in the absence of detectable increases in CSF-1 gene transcription. Moreover, treatment of monocytes with CHX and actinomycin D demonstrated that inhibition of protein synthesis is associated with stabilization of the CSF-1 transcript. Taken together, these findings indicated that CSF-1 gene expression is controlled at both transcriptional and posttranscriptional levels in human monocytes.
Granulomatous-lymphocytic interstitial lung disease and recurrent sinopulmonary infections in a patient with Good’s syndrome
Authors: Jensen Mads Lynge , Bendstrup Elisabeth , Hilberg Ole
Published: September 2015

Good's syndrome is a rare primary immunodeficiency associated with adult thymoma. Complications are mainly autoimmune manifestations and recurrent infections with encapsulated bacteria. Only one possible case of combined granulomatous-lymphocytic interstitial lung disease (GL-ILD) and Good's syndrome have been described earlier, but the patient died at the time of diagnosis. This is the first case of GL-ILD in Good's syndrome with a successful outcome. We present a case of a 43-year-old man with GL-ILD, who suffered from recurrent infections ofHaemophilus influenzaeandPneumocystis jirovecii, with 8-year follow-up. After a thymectomy, he was diagnosed with Good's syndrome and GL-ILD. He was treated with prophylactic pivampicillin, quinolones and cephalosporins for his recurrentP. jiroveciiandH. influenzaeinfections, an approach that proved unsuccessful due to resistance, with relapse after cessation. He was stabilised with oral diaminodiphenyl-sulfone forP. jiroveciiand colistimethate-sodium inhalations forH. influenzae, which is a new approach to prophylactic treatment.
A painless pelvic wall ectopic pregnancy
Authors: Borton Zakk , Landon Alice G , Singh Savita
Published: September 2015

A 38-year-old woman presented with light vaginal bleeding at approximately 8 weeks gestation. Although she initially presented to accident and emergency with vaginal bleeding and menstrual-like pain, this rapidly settled and both symptoms resolved over the following days. An initial ultrasound scan revealed an empty uterus, with no adnexal masses or free fluid. Miscarriage was presumed. However, following suboptimal β-human chorionic gonadotropin decline, further ultrasonography noted a mass next to the right ovary. The patient underwent laparoscopy, revealing an ectopic pregnancy closely adherent to the peritoneum of the right pelvic wall. The patient was treated with systemic methotrexate and responded well. This rare form of ectopic pregnancy has a high maternal morbidity and mortality, and its optimal management is not definitively decided, due to the scarcity of the condition and subsequent lack of high-powered data. We present a case of successful medical management.
A predictable but life-threatening complication of hydroxyurea in a patient with sickle cell anaemia: an experience learned from a Jehovah's Witness
Authors: Tun Aung Myint , Naing Ei Ei , Tun Nay Min , Guevara Elizabeth
Published: September 2015

It is well known that hydroxyurea can cause pancytopaenia secondary to bone marrow suppression, which is reversible with short-term discontinuation of the therapy. However, it is important to note that bone marrow suppressive effects caused by hydroxyurea could be easily potentiated in patients with sickle cell anaemia complicated by chronic kidney disease (CKD). We present a case of a Jehovah's Witness with sickle cell anaemia, who developed severe bone marrow suppression due to the combined effects of hydroxyurea and CKD, resulting in a prolonged recovery period after discontinuation of hydroxyurea.
Assessing risk factors for early hip osteoarthritis in activity-related hip pain: a Delphi study
Authors: Jackson K A , Glyn-Jones S , Batt M E , Arden N K , Newton J L
Published: September 2015

Objective

Hip pain and injury as a result of activity can lead to the development of early hip osteoarthritis (OA) in susceptible individuals. Our understanding of the factors that increase susceptibility continues to evolve. The ability to clearly identify individuals (and cohorts) with activity-related hip pain who are at risk of early hip OA is currently lacking. The purpose of this study was to gain expert consensus on which key clinical measures might help predict the risk of early hip OA in individuals presenting with activity-related hip pain. The agreed measures would constitute a standardised approach to initial clinical assessment to help identify these individuals.

Methods

This Dephi study used online surveys to gain concordance of expert opinion in a structured process of ‘rounds’. In this study, we asked ‘What outcome measures are useful in predicting hip OA in activity-related hip pain?’ The Delphi panel consisted of experts from sport and exercise medicine, orthopaedics, rheumatology, physiotherapy and OA research.

Results

The study identified key clinical measures in the history, examination and investigations (plain anteroposterior radiograph and femoroacetabular impingement views) that the panel agreed would be useful in predicting future risk of hip OA when assessing activity-related hip pain. The panel also agreed that certain investigations and tests (eg, MR angiography) did not currently have a role in routine assessment. There was a lack of consensus regarding the role of MRI, patient-reported outcome measures (PROMs) and certain biomechanical and functional assessments.

Conclusions

We provide a standardised approach to the clinical assessment of patients with activity-related hip pain. Assessment measures rejected by the Delphi panel were newer, more expensive investigations that currently lack evidence. Assessment measures that did not reach consensus include MRI and PROMs. Their role remains ambiguous and would benefit from further research.
Cost-effectiveness and budget impact of Empirical vitamin D therapy on unintentional falls in older adults in the UK
Authors: Poole C D , Smith J , Davies J S
Published: September 2015

Objectives

To evaluate the health outcomes and economics associated with the current guidance relating to the prevention of falls in the elderly through vitamin D supplementation.

Setting

UK.

Participants

UK population aged 60 years and above.

Interventions

A Markov health state transition model simulated patient transitions between key fall-related outcomes using a 5-year horizon and annual cycles to assess the costs and benefits of empirical treatment with colecalciferol 800 iu daily.

Primary and secondary outcome measures

Costs and health outcomes attributable to fall prevention following vitamin D supplementation.

Results

Our model shows that treating the UK population aged 60 years and above with 800 iu colecalciferol would, over a 5-year period: (1) prevent in excess of 430 000 minor falls; (2) avoid 190 000 major falls; (3) prevent 1579 acute deaths; (4) avoid 84 000 person-years of long-term care and (5) prevent 8300 deaths associated with increased mortality in long-term care. The greatest gains are seen among those 75 years and older. Based on reduction in falls alone, the intervention in all adults aged 65+ is cost-saving and leads to increased quality adjusted life years. Treating all adults aged 60+ incurs an intervention cost of £2.70bn over 5 years, yet produces a −£3.12bn reduction in fall-related costs; a net saving of £420M. Increasing the lower bound age limit by 5-year increments increases budget impact to −£1.17bn, −£1.75bn, and −£2.06bn for adults 65+, 70+ and 75+, respectively.

Conclusions

This study shows that treatment of the elderly UK population with colecalciferol 800 iu daily would be associated with reductions in mortality and substantial cost-savings through fall prevention.
The challenge of vaccinating adults: attitudes and beliefs of the Canadian public and healthcare providers
Authors: MacDougall D M , Halperin B A , MacKinnon-Cameron D , Li Li , McNeil S A , Langley J M , Halperin S A
Published: September 2015

Objectives

Vaccine coverage for recommended vaccines is low among adults. The objective of this study was to assess the knowledge, attitudes, beliefs and behaviours of adults and healthcare providers related to four vaccine-preventable diseases and vaccines (diphtheria-tetanus-pertussis, zoster, pneumococcus and influenza).

Design

We undertook a survey and focus groups of Canadian adults and healthcare providers (doctors, nurses, pharmacists). A total of 4023 adults completed the survey and 62 participated in the focus groups; 1167 providers completed the survey and 45 participated in the focus groups.

Results

Only 46.3% of adults thought they were up-to-date on their vaccines; 30% did not know. In contrast, 75.6% of providers reported being up-to-date. Only 57.5% of adults thought it was important to receive all recommended vaccines (compared to 87.1–91.5% of providers). Positive attitudes towards vaccines paralleled concern about the burden of illness and confidence in the vaccines, with providers being more aware of disease burden and confident in vaccine effectiveness than the public. Between 55.0% and 59.7% of adults reported willingness to be vaccinated if recommended by their healthcare provider. However, such recommendations were variable; while 77.4% of the public reported being offered and 52.8% reported being recommended the influenza vaccine by their provider, only 10.8% were offered and 5.6% recommended pertussis vaccine. Barriers and facilitators to improved vaccine coverage in adults, such as trust-mistrust of health authorities, pharmaceutical companies and national recommendations, autonomy versus the public good and logistical issues (such as insufficient time and lack of vaccination status tracking), were identified by both the public and providers.

Conclusions

Despite guidelines for adult vaccination, there are substantial gaps in knowledge and attitudes and beliefs among both the public and healthcare providers that lead to low vaccine coverage. A systematic approach that involves education, elimination of barriers and establishing and improving infrastructure for adult immunisation is required.
A retrospective evaluation of the intrauterine device in a patient population in Buenos Aires, Argentina
Authors: Jagroep Sherani R , Pichardo Margaret S , Arribas Lia , Heredia Graciela , Coccio Elina , Palermo Tia M
Published: September 2015

Background

The intrauterine device (IUD) is a long-acting reversible contraceptive method that is safe for a wide range of women, including adolescents and nulliparous women. Globally, it is often underutilised due to misperceptions among patients. Examination of characteristics associated with IUD discontinuation including adverse effects and IUD expulsion can inform provider practices to improve contraception success and patient satisfaction with this method. We studied IUD performance at a public family planning clinic in Buenos Aires, Argentina, serving a predominantly immigrant, low-income population.

Methods

We conducted a retrospective evaluation of 1047 IUD insertions between 2002 and 2007 with 5 years of follow-up data. We performed bivariate and survival analysis to examine characteristics associated with IUD discontinuation: adverse outcomes including pain, bleeding, and IUD expulsion, and time to removal.

Findings

Of 1047 patients, only 188 (18%) had their IUD removed within 5 years. The main causes of IUD discontinuation were involuntary (38%) reasons such as an IUD expulsion and personal choice (34%) such as desiring pregnancy.

Conclusions

Findings suggest overall good long-term performance with IUD insertion, with minimal complaints or adverse outcomes. These findings may help to support providers serving similar populations in promoting this method.
Exploring the structural and conformational properties of dioxygen dihalides (halogen = F, Cl, Br)
Authors: Hosseini Saiedhossein , Nori-Shargh Davood
Published: September 2015

The structural and conformational properties of dioxygen difluoride (1), dioxygen dichloride (2), and dioxygen dibromide (3) have been investigated by means of the hybrid density functional theory (B3LYP) and the hybrid meta exchange-correlation functional (M06-2X) with the aug-cc-pVQZ, aug-cc-pVTZ, cc-pVTZ, and 6-311++G** basis sets and natural bond orbital interpretation. The results obtained showed that the rotation at the O–O bond by passing from the plane symmetrical trans-(C2h) (or cis-(C2v)) form leads to the O–F bonds breaking. The natural bond order analysis revealed that the O–O bonds in the C2h and C2v forms of compound 1 possess double bond characters and the small bond orders between the oxygen and fluorine atoms results from the strong electron delocalization between the lone pairs of the fluorine atoms (LP4F) and the antibonding orbitals of the adjacent O–O double bond (π*O–O). The lengthening and shortening of the O–F and O–O bonds, respectively, in the trans-(C2h) or cis-(C2v) forms of compound 1 can be interpreted by the decrease of the bonding interactions between the fluorine and oxygen orbitals. The profiles of the orbital amplitudes (or electron densities) of O–O and O–F bonds in the C2h and C2v forms of compound 1 revealed that there are strong electronic repulsions between the πO–O and the lone pairs of oxygen atoms with the lone pairs of fluorine atoms, leading to the dissociation of O–F bonds. It is worth noting that there are strong hyperconjugative interactions between LP2O and the antibonding orbitals of adjacent O–F bonds (σ*O–F) in the skew (C2) form of compound 1, leading to the decrease of the O–O bond length and the increase of the F–O bond length by increasing the πO-O bonding and the σ*O–F antibonding orbital occupations. The increase of the electron delocalization from LP3F to σ*O–O with the decrease of the σ*O–O–σO–O energy gap (results from the increase of the O–O bond length) justifies the similarity between the adiabatic O–O bond dissociation energies in compound 1 and HOOH. The electron delocaizations from LP2O2 to σ*O3–X (X = F (1), Cl (2), Br (3)) decrease drastically from the skew ground state (C2) forms of compound 1 to compound 2 but increase slightly from compound 2 to compound 3, causing the significant increase of the O–O bond length ongoing from compound 1 to compound 2 and the slight decrease from compound 2 to compound 3. Effectively, the conformational properties of compounds 1–3 can be interpreted with the principle of maximum softness.
Agricultural support policy in Canada: What are the environmental consequences?
Authors: Eagle Alison J. , Rude James , Boxall Peter C.
Published: September 2015

This paper reviews annual government spending on Canadian agriculture that attempts to stabilize and enhance farm incomes. Over the past 5 years, 2/3 of the $3 billion spent on agriculture went into stabilization programs to support farm incomes. However, this level of support raises questions about the environmental consequences of enhanced agricultural production. Environmental impacts from agriculture are well known and addressed in US and EU policies. In contrast, Canadian government expenditures on environmental initiatives in agriculture, as a share of farm income, are more than 10 times smaller than those in the US and the EU. Nonetheless the evidence is that Canadian programs have modest impacts on production, but that chemical and fertilizer input use may be higher than in the absence of the program. One possible course of action is to introduce cross-compliance between program payments and environmental objectives. However, there are no requirements that Canadian producers receiving support comply with environmental standards. While cross-compliance could be considered in the Canadian context, policies that directly target specific environmental issues in agriculture may have greater impact.