bluuebunny/crossref_metadata_2025 huggingface dataset:
Lymphocyte-specific Ca2+-binding protein LSP1 is associated with the cytoplasmic face of the plasma membrane.
Authors: Klein D P , Jongstra-Bilen J , Ogryzlo K , Chong R , Jongstra J
Published: October 2015

The gene for LSP1 is a lymphocyte-specific gene previously isolated by us using a subtractive hybridization technique. LSP1 mRNA is found in normal and transformed B lymphocytes and in normal T lymphocytes but not in transformed T lymphocytes. To study the expression of the mouse LSP1 protein, we prepared a polyclonal antiserum specific for the LSP1 protein. Here we report that the gene for LSP1 was expressed in transformed B-lymphoma cell lines and in normal mouse thymocytes as a protein doublet with apparent molecular masses of 52 and 50.5 kilodaltons when analyzed on a sodium dodecyl sulfate-10% polyacrylamide gel. BW5147 cells transfected with an LSP1 cDNA clone expressed only the 52-kilodalton protein. No LSP1 protein was expressed in nine T-lymphoma cell lines tested. Immunofluorescence studies of intact and permeabilized cells and subcellular fractionation experiments showed that the LSP1 protein was associated with the cytoplasmic side of the plasma membrane in transformed B-lymphoma cell lines and in normal thymocytes. Using a simple filter-binding assay, we showed that recombinant LSP1 protein was Ca2+ binding, as predicted on the basis of its deduced amino acid sequence. On the basis of the particular expression pattern, the subcellular localization, and the Ca2+-binding property of the LSP1 protein, we hypothesize that the LSP1 protein is a lymphocyte-specific component of a signal transduction pathway involved in the regulation of lymphocyte growth.
Tissue-specific in vitro transcription from the mouse myelin basic protein promoter.
Authors: Tamura T , Aoyama A , Inoue T , Miura M , Okano H , Mikoshiba K
Published: October 2015

The mouse myelin basic protein promoter was transcribed in brain nuclear extracts. The distal promoter region from -253 to -54 directed preferential transcription in brain extracts, whereas the same region repressed transcription activity in liver extracts. Stimulation of transcription was observed when the distal region was located only in a native orientation. The proximal region downstream from -53 alone still directed preferential transcription. It is suggested that cooperative function by the two promoter regions may be required for higher specificity.
A lipopolysaccharide-induced DNA-binding protein for a class II gene in B cells is distinct from NF-kappa B.
Authors: Gravallese E M , Boothby M R , Smas C M , Glimcher L H
Published: October 2015

Class II (Ia) major histocompatibility complex molecules are cell surface proteins normally expressed by a limited subset of cells of the immune system. These molecules regulate the activation of T cells and are required for the presentation of antigens and the initiation of immune responses. The expression of Ia in B cells is determined by both the developmental stage of the B cell and by certain external stimuli. It has been demonstrated previously that treatment of B cells with lipopolysaccharide (LPS) results in increased surface expression of Ia protein. However, we have confirmed that LPS treatment results in a significant decrease in mRNA encoding the Ia proteins which persists for at least 18 h. Within the upstream regulatory region of A alpha k, an NF-kappa B-like binding site is present. We have identified an LPS-induced DNA-binding protein in extracts from athymic mice whose spleens consist predominantly of B cells. Binding activity is present in low levels in unstimulated spleen cells and is increased by LPS treatment. This protein binds to two sites in a regulatory region of the Ia A alpha k gene, one of which contains the NF-kappa B-like binding site. DNA fragments containing these sites cross-compete for protein binding. Analysis by DNase I footprinting identified a target binding sequence, named the LPS-responsive element. Although this target sequence contains an NF-kappa B-like binding site, competition with a mutant oligonucleotide demonstrated that bases critical for NF-kappa B binding are not required for binding of the LPS-inducible protein. Therefore, we hypothesized that this inducible protein represents a new mediator of LPS action, distinct from NF-kappa B, and may be one mechanism to account for the decrease in mRNA encoding the Ia proteins.
Mitogen stimulation affects contractile protein mRNA abundance and translation in embryonic quail myocytes.
Authors: Latham K E , Konigsberg I R
Published: October 2015

In cultures of differentiated, fusion-blocked muscle cells obtained from embryonic Japanese quail (Coturnix coturnix japonica), mitogen stimulation leads to an immediate reduction in the rates of synthesis of skeletal muscle myosin heavy chain (MHC) and alpha-actin. The molecular mechanisms responsible for this downregulation were examined. The cellular abundances of the alpha-actin and MHC mRNAs were affected differently by mitogen stimulation; alpha-actin mRNA abundance declined by an amount which quantitatively accounted for the observed decrease in alpha-actin synthesis, whereas MHC mRNA abundance remained virtually unchanged during the first 6 h following mitogen stimulation, a period during which MHC synthesis declined by more than 70%. MHC mRNA abundance did decline between 6 and 12 h after mitogen stimulation. Downregulation of MHC synthesis therefore involves an initial block in mRNA translation combined with a later loss of MHC mRNA from the cytoplasma, while alpha-actin synthesis is regulated at the level of mRNA abundance. These observations are consistent with the hypothesis that, in addition to transcriptional activation of muscle-specific genes, skeletal muscle differentiation normally involves cell cycle-dependent modulations in cellular factors which control message stability and message translation.
The DAL7 promoter consists of multiple elements that cooperatively mediate regulation of the gene's expression.
Authors: Yoo H S , Cooper T G
Published: October 2015

Expression of the allantoin system genes in Saccharomyces cerevisiae is induced by allophanate or its analog, oxalurate. This work provides evidence for the involvement of distinct types of cis-acting elements in the induction process. The first element was found to have the properties of an upstream activation sequence (UAS). This element was localized to a 16-base-pair (bp) DNA fragment containing a short 5-bp sequence that occurred repeatedly in the upstream region of DAL7. When present in two or more copies, the 16-bp fragment supported high-level beta-galactosidase production in a CYC1-lacZ expression vector; there was, however, no response to the allantoin pathway inducer. The second element had the properties of a negatively acting element or upstream repression sequence (URS). This element was localized to a 16-bp DNA fragment containing an 8-bp sequence that was repeated four times in the upstream region of DAL7. A fragment containing the 8-bp repeated sequence placed adjacent to the UAS-containing fragment mediated inhibition of the ability of the UAS to support lacZ expression regardless of whether inducer was present. A third element, designated an upstream induction sequence (UIS), was required for response to inducer. The UIS was localized to a small DNA fragment containing an approximately 10-bp sequence that was repeated twice in the upstream region of DAL7. When a fragment containing the 10-bp repeated sequence was placed adjacent to these UAS and URS elements, the construction (UIS-UAS-URS) supported normal oxalurate-mediated induction of beta-galactosidase synthesis. These data are consistent with the suggestion that multiple, cis-acting elements participate in the induction process.
U1 small nuclear ribonucleoprotein particle-specific proteins interact with the first and second stem-loops of U1 RNA, with the A protein binding directly to the RNA independently of the 70K and Sm proteins.
Authors: Patton J R , Habets W , van Venrooij W J , Pederson T
Published: October 2015

The U1 small nuclear ribonucleoprotein particle (U1 snRNP), a cofactor in pre-mRNA splicing, contains three proteins, termed 70K, A, and C, that are not present in the other spliceosome-associated snRNPs. We studied the binding of the A and C proteins to U1 RNA, using a U1 snRNP reconstitution system and an antibody-induced nuclease protection technique. Antibodies that reacted with the A and C proteins induced nuclease protection of the first two stem-loops of U1 RNA in reconstituted U1 snRNP. Detailed analysis of the antibody-induced nuclease protection patterns indicated the existence of relatively long-range protein-protein interactions in the U1 snRNP, with the 5' end of U1 RNA and its associated specific proteins interacting with proteins bound to the Sm domain near the 3' end. UV cross-linking experiments in conjunction with an A-protein-specific antibody demonstrated that the A protein bound directly to the U1 RNA rather than assembling in the U1 snRNP exclusively via protein-protein interactions. This conclusion was supported by additional experiments revealing that the A protein could bind to U1 RNA in the absence of bound 70K and Sm core proteins.
Transcription by RNA polymerase I stimulates mitotic recombination in Saccharomyces cerevisiae.
Authors: Stewart S E , Roeder G S
Published: October 2015

The recombination-stimulating sequence HOT1 is derived from the ribosomal DNA array of Saccharomyces cerevisiae and corresponds to sequences that promote transcription by RNA polymerase I. When inserted at a chromosomal location outside the ribosomal DNA array, HOT1 stimulates mitotic recombination in the adjacent sequences. To investigate the relationship between transcription and recombination, transcription promoted by HOT1 was directly examined. These studies demonstrated that transcription starts at the RNA polymerase I initiation site in HOT1 and proceeds through the chromosomal sequences in which recombination is enhanced. Linker insertion mutations in HOT1 were generated and assayed for recombination stimulation and for promoter function; this analysis demonstrated that the same sequences are required for both activities. These results indicate that the ability of HOT1 to enhance recombination is related to, and most likely dependent on, its ability to promote transcription.
Induction of the TRPM-2 gene in cells undergoing programmed death.
Authors: Buttyan R , Olsson C A , Pintar J , Chang C , Bandyk M , Ng P Y , Sawczuk I S
Published: October 2015

RNA and protein products encoded by the testosterone-repressed prostate message-2 gene (TRPM-2) are induced to high levels, coordinate with the onset of cell death, in numerous rodent models of inducible tissue damage. These models include cell death initiated by hormonal stimuli (prostate regression), pressure insult (renal atrophy after ureteral obstruction), developmental stimuli (necrosis of interdigital tissue), and cytotoxic injury (chemotherapeutic regression of a tumor). Sequence analysis of cDNA encoding TRPM-2 revealed its close homology with a product referred to as SGP-2 or clusterin expressed constitutively by Sertoli cells; however, the immunologically related polypeptides expressed in regressing tissues differ in molecular mass from the forms secreted by the testis. Although the function(s) of the products encoded by the TRPM-2 gene remains unclear, their presence provides a remarkable and early indicator of programmed cell death in many types of mammalian cells.
Multiple genes coding for precursors of rhodotorucine A, a farnesyl peptide mating pheromone of the basidiomycetous yeast Rhodosporidium toruloides.
Authors: Akada R , Minomi K , Kai J , Yamashita I , Miyakawa T , Fukui S
Published: October 2015

Haploid cells of mating type A of the basidiomycetous yeast Rhodosporidium toruloides secrete a mating pheromone, rhodotorucine A, which is an undecapeptide containing S-farnesyl cysteine at its carboxy terminus. To analyze the processing and secretion pathway of rhodotorucine A, we isolated both genomic and complementary DNAs encoding the peptide moiety. We identified three distinct genes, RHA1, RHA2, and RHA3, encoding four, five, and three copies of the pheromone peptide, respectively. Complementary DNA clones were classified into two types. One type was homologous to RHA1, and the other type was homologous to RHA2. Transcription start sites were identified by primer extension and S1 nuclease protection, from which the site of the initiator methionine was verified. A primary precursor of rhodotorucine A was detected as a 7-kilodalton protein by immunoprecipitation of in vitro translation products. On the basis of these results, we propose similar three-precursor structures of rhodotorucine A, each containing the amino-terminal peptide sequence Met-Val-Ala. The precursors contain three, four, or five tandem repeats of the pheromone peptide, each separated by a spacer peptide, Thr-Val-Ser(Ala)-Lys, and each precursor has the carboxy-terminal sequence Thr-Val-Ala. This structure suggests that primary precursors of rhodotorucine A do not contain canonical signal sequences.
Biochemical analysis of heat-resistant mouse tumor cell strains: a new member of the HSP70 family.
Authors: Anderson R L , Van Kersen I , Kraft P E , Hahn G M
Published: October 2015

A series of heat-resistant mutants selected from a murine tumor cell line, RIF-1, display a markedly increased and stable resistance to heat shock. The mutant cell lines were analyzed for differences that may explain their increased resistance. Membrane lipid analysis showed no change in cholesterol content but an increase in the proportion of saturated fatty acids in the phospholipid fraction. Two-dimensional gel analysis revealed a generally increased constitutive synthesis of several major heat shock proteins (HSP), including HSP90, 68, 60, and 28. In addition, a new protein in the 70-kilodalton region is present in the resistant lines. The new protein has a lower isoelectric point than the constitutive HSP70 does, is only weakly induced by heat shock, and is immunologically cross-reactive with other members of the HSP70 family. After heat shock, the mutants display increases in HSP similar to those seen in the wild-type cells and they develop further transient tolerance to heat. Analysis of these mutants may help in understanding the function of HSP, both in normal growth and after heat shock.
In vivo evidence of interaction between interferon-stimulated gene factors and the interferon-stimulated response element.
Authors: Pine R , Darnell J E
Published: October 2015

Constitutive and interferon-inducible DNase hypersensitive sites in vivo are located in interferon-stimulated gene promoters near sequences that specifically bind constitutive or interferon-inducible proteins in vitro. Induced sites and proteins are transient or maintained, depending on cell type. Interferon-stimulated gene transcription is transient or maintained in parallel.
Immunochemical localization of the epitope for a monoclonal antibody that neutralizes human platelet-derived growth factor mitogenic activity.
Authors: LaRochelle W J , Robbins K C , Aaronson S A
Published: October 2015

A monoclonal antibody (mAb), sis 1, generated against human c-sis-encoded platelet-derived growth factor (PDGF) BB, was shown by enzyme-linked immunosorbent assay and Western blot (immunoblot) analysis to recognize human PDGF BB and human platelet PDGF AB but not the human PDGF AA. This monoclonal antibody potently inhibited PDGF receptor-binding and mitogenic activities of both human PDGF BB and PDGF AB but had no effect on PDGF AA. Finally, we demonstrated that an immunoaffinity-purified anti-c-sis peptide antibody (anti-V4) which also blocked binding of PDGF BB to its cognate receptor and competed with mAb sis 1 for binding to PDGF BB. All of these results suggest that mAb sis 1 recognizes an epitope of the c-sis gene product, PDGF BB, that spatially overlaps the V4 surface domain of PDGF BB, immunochemically localizing a region of PDGF BB critical for PDGF receptor binding and activation.
The major and minor chicken vitellogenin genes are each adjacent to partially deleted pseudogene copies of the other.
Authors: Silva R , Fischer A H , Burch J B
Published: October 2015

The major chicken vitellogenin gene (VTGII) has previously been cloned and sequenced. We now report the isolation of genomic clones that encompass a minor chicken vitellogenin gene (VTGIII) which is also expressed in the liver in response to estradiol. Our analysis reveals that a pseudogene for VTGII (psi VTGII) lies 1,426 base pairs upstream of this VTGIII gene. A reevaluation of published sequence data reveals that the converse is also true, namely, that a pseudogene for VTGIII (psi VTGIII) lies 1,345 base pairs downstream of the VTGII gene. Our results show that a 335-base-pair deletion has removed the psi VTGIII promoter and cap site but left residual estrogen response element in a region where nuclease-hypersensitive sites have been reported to be induced in response to estradiol.
Granulocyte-macrophage colony-stimulating factor and tetradecanoyl phorbol acetate induce a distinct, restricted subset of primary-response TIS genes in both proliferating and terminally differentiated myeloid cells.
Authors: Varnum B C , Lim R W , Kujubu D A , Luner S J , Kaufman S E , Greenberger J S , Gasson J C , Herschman H R
Published: October 2015

Induction of early-response genes (tetradecanoyl phorbol acetate [TPA]-induced sequences, or TIS genes; R.W. Lim, B.C. Varnum, and H.R. Herschman, Oncogene 1:263-270, 1987) by granulocyte-macrophage colony-stimulating factor (GM-CSF) and TPA was examined both in a factor-dependent murine cell line, 32D clone 3, and in mature human neutrophils. When GM-CSF-deprived 32D clone 3 cells were exposed to GM-CSF or to TPA, four TIS mRNAs (TIS7, TIS8, TIS10, and TIS11) were rapidly and transiently induced. However, neither GM-CSF nor TPA could induce accumulation of TIS1 mRNA in 32D clone 3 cells, even under superinducing conditions. Both GM-CSF and TPA also elicited rapid, transient expression of TIS8 and TIS11 mRNA in postmitotic human neutrophils. However, neither agent could induce accumulation of TIS1 mRNA in human neutrophils. TIS1 is a member of the nuclear receptor supergene family that codes for ligand-dependent transcription factors. Cell-type restriction of inducible transcription factors may contribute to developmental specification.
Expression of the RESA gene in Plasmodium falciparum isolate FCR3 is prevented by a subtelomeric deletion.
Authors: Cappai R , van Schravendijk M R , Anders R F , Peterson M G , Thomas L M , Cowman A F , Kemp D J
Published: October 2015

We show here that the Plasmodium falciparum isolate FCR3 does not express the ring-infected erythrocyte surface antigen (RESA). This is because the 5' end of the RESA gene has been inverted and partly deleted and a telomere has been added to it. We propose a model to explain these events.
Development of an in vitro transcription system for Neurospora crassa mitochondrial DNA and identification of transcription initiation sites.
Authors: Kennell J C , Lambowitz A M
Published: October 2015

We have developed an in vitro transcription system for Neurospora crassa mitochondrial DNA (mtDNA) and used it to identify transcription initiation sites at the 5' ends of the genes encoding the mitochondrial small and large rRNA and cytochrome b (cob). The in vitro transcription start sites correspond to previously mapped 5' ends of major in vivo transcripts of these genes. Sequences around the three transcription initiation sites define a 15-nucleotide consensus sequence, 5'-TTAGARA(T/G)G(T/G)ARTRR-3', all or part of which appears to be an element of an N. crassa mtDNA promoter. A somewhat looser 11-nucleotide consensus sequence, 5'-TTAGARR(T/G)R(T/G)A-3', was derived by including two additional promoters identified recently. Group I extranuclear mutants, such as [poky] and [SG-3], have a 4-base-pair (bp) deletion in the consensus sequence at the 5' end of the mitochondrial small rRNA and are grossly deficient in mitochondrial small rRNA (R. A. Akins and A. M. Lambowitz, Proc. Natl. Acad. Sci. USA 81:3791-3795, 1984). We show here that the 4-bp deletion in the consensus sequence decreases in vitro transcription from this site by more than 99%. N. crassa mtDNA is similar to Saccharomyces cerevisiae mtDNA in having multiple promoters, including separate promoters for the genes encoding the mitochondrial small and large rRNAs. Our results suggest that the primary effect of the 4-bp deletion in group I extranuclear mutants is to inhibit transcription of the mitochondrial small rRNA, leading to severe deficiency of mitochondrial small rRNA and small ribosomal subunits.
pp60c-src variants containing lesions that affect phosphorylation at tyrosines 416 and 527.
Authors: Harvey R , Hehir K M , Smith A E , Cheng S H
Published: October 2015

The biological and biochemical properties of pp60c-src are regulated, in part, by phosphorylation at Tyr-416 and Tyr-527. The tyrosine kinase and transforming activities of pp60c-src are suppressed by phosphorylation at Tyr-527, whereas full activation of pp60c-src requires phosphorylation at Tyr-416. To test specifically the significance of the negatively charged phosphate moieties on these tyrosine residues, we have substituted the codons for both residues with codons for either Glu or Gln. A negatively charged Glu at position 527 was unable to mimic a phosphorylated Tyr at this position, and, in consequence, the mutated pp60c-src was activated and transforming. Similarly, substitution of Tyr-416 with Glu was unable to stimulate the activities of the enzyme. However, mutagenesis of Tyr-416 to Gln (to form the mutant 416Q) activated the kinase activity approximately twofold over that observed for wild-type pp60c-src. When introduced into the mutant 527F (containing Phe-527 instead of Tyr), the double mutant 416Q-527F exhibited weak transforming activity. This is in contrast to the other double mutants 416E-527F and 416F-527F, which were nontransforming. The biochemical basis by which 416Q activates pp60c-src is not understood but probably involves some local conformational perturbation. Deletion of residues 519 to 524 (RH5), a region previously shown to be necessary for association with middle-T antigen, led to loss of phosphorylation at Tyr-527 and activation of the enzymatic and focus-forming activities of pp60c-src. Hence, the sequences necessary for complex formation with middle-T antigen may also be required by the kinase(s) which phosphorylates Tyr-527 in vivo. This suggests that normal cells contain cellular proteins which are analogous to middle-T antigen and whose action regulates the activity of pp60c-src by controlling phosphorylation or dephosphorylation at residue 527.
Structure and expression of the human MDR (P-glycoprotein) gene family.
Authors: Chin J E , Soffir R , Noonan K E , Choi K , Roninson I B
Published: October 2015

The human MDR (P-glycoprotein) gene family is known to include two members, MDR1 and MDR2. The product of the MDR1 gene, which is responsible for resistance to different cytotoxic drugs (multidrug resistance), appears to serve as an energy-dependent efflux pump for various lipophilic compounds. The function of the MDR2 gene remains unknown. We have examined the structure of the human MDR gene family by Southern hybridization of DNA from different multidrug-resistant cell lines with subfragments of MDR1 cDNA and by cloning and sequencing of genomic fragments. We have found no evidence for any other cross-hybridizing MDR genes. The sequence of two exons of the MDR2 gene was determined from genomic clones. Hybridization with single-exon probes showed that the human MDR1 gene is closely related to two genes in mouse and hamster DNA, whereas MDR2 corresponds to one rodent gene. The human MDR locus was mapped by field-inversion gel electrophoresis, and both MDR genes were found to be linked within 330 kilobases. The expression patterns of the human MDR genes were examined by enzymatic amplification of cDNA. In multidrug-resistant cell lines, increased expression of MDR1 mRNA was paralleled by a smaller increase in the levels of MDR2 mRNA. In normal human tissues, MDR2 was coexpressed with MDR1 in the liver, kidney, adrenal gland, and spleen. MDR1 expression was also detected in colon, lung, stomach, esophagus, muscle, breast, and bladder.
Binding of heat shock proteins to the avian progesterone receptor.
Authors: Kost S L , Smith D F , Sullivan W P , Welch W J , Toft D O
Published: October 2015

The protein composition of the avian progesterone receptor was analyzed by immune isolation of receptor complexes and gel electrophoresis of the isolated proteins. Nonactivated cytosol receptor was isolated in association with the 90-kilodalton (kDa) heat shock protein, hsp90, as has been described previously. A 70-kDa protein was also observed and was shown by Western immunoblotting to react with an antibody specific to the 70-kDa heat shock protein. Thus, two progesterone receptor-associated proteins are identical, or closely related, to heat shock proteins. When the two progesterone receptor species, A and B, were isolated separately in the absence of hormone, both were obtained in association with hsp90 and the 70-kDa protein. However, activated receptor isolated from oviduct nuclear extracts was associated with the 70-kDa protein, but not with hsp90. A hormone-dependent dissociation of hsp90 from the cytosolic form of the receptor complex was observed within the first hour of in vivo progesterone treatment, which could explain the lack of hsp90 in nuclear receptor complexes. In a cell-free system, hsp90 binding to receptor was stabilized by molybdate but disrupted by high salt. These treatments, however, did not alter the binding of the 70-kDa protein to receptor. Association of the 70-kDa protein with the receptor could be disrupted by the addition of ATP at elevated temperatures (23 degrees C). The receptor-associated 70-kDa protein is an ATP-binding protein, as demonstrated by its affinity labeling with azido[32P]ATP. These results indicate that the two receptor-associated proteins interact with the progesterone receptor by different mechanisms and that they are likely to affect the structure or function of the receptor in different ways.
Sequence and expression of chicken and mouse rsk: homologs of Xenopus laevis ribosomal S6 kinase.
Authors: Alcorta D A , Crews C M , Sweet L J , Bankston L , Jones S W , Erikson R L
Published: October 2015

We have previously reported the isolation of cDNAs encoding two closely related Xenopus ribosomal S6 kinases, S6KII alpha and -beta (S. W. Jones, E. Erikson, J. Blenis, J. L. Maller, and R. L. Erikson, Proc. Natl. Acad. Sci. USA 85:3377-3381, 1988). We report here the molecular cloning of one chicken and two mouse homologs of the Xenopus laevis cDNAs. As described for the Xenopus proteins, these cDNAs were found to predict polypeptides that contain two distinct kinase domains, of which one is most closely related to the catalytic subunit of cyclic AMP-dependent protein kinase and the other is most closely related to the catalytic subunit of phosphorylase b kinase. The three predicted proteins were more than 79% identical to the Xenopus S6KII alpha protein. The chicken and one of the mouse cDNAs were, respectively, 3.7 and 3.1 kilobase pairs in length, predicted proteins of 752 and 724 amino acids with molecular weights of 84.4 and 81.6 kilodaltons, and hybridized to mRNAs in fibroblasts and tissues of approximately 3.6 and 3.4 kilobases (kb). The second mouse cDNA was approximately 6.1 kilobase pairs and was not full length but predicted the C-terminal 633 amino acids of a protein that is similar to the C-terminal portion of Xenopus S6KII alpha. This clone hybridized to mRNA transcripts of 7.6 and 3.4 kb. In vitro transcription and translation of the chicken and the mouse cDNAs that predict complete proteins produced major products with apparent molecular weights of 96 and 84 kilodaltons. Analysis of mRNA levels in chicken tissues showed significant quantities of the 3.6-kb transcript in small and large intestine, spleen, and bursa. Both mouse cDNA were similarly expressed at significant levels in intestine, thymus, and lung; however, the 7.6-kb mRNA was differentially and more highly expressed in heart and brain. The two mouse cDNAs represent two different S6 kinase genes, as shown by comparison of their protein sequences, mRNA transcript sizes, genomic organizations, and nucleic acid sequences. We propose that this family of genes be named rsk, for ribosomal S6 kinase.
A gene product needed for induction of allantoin system genes in Saccharomyces cerevisiae but not for their transcriptional activation.
Authors: Bricmont P A , Cooper T G
Published: October 2015

The allantoin-degradative pathway of Saccharomyces cerevisiae consists of several genes whose expression is highly induced by the presence of allophanic acid. Induced expression requires a functional DAL81 gene product. Analysis of these genes has demonstrated the presence of three cis-acting elements in the upstream regions: (i) an upstream activation sequence (UAS) required for transcriptional activation in an inducer-independent fashion, (ii) an upstream repression sequence (URS) that mediates inhibition of this transcriptional activation, and (iii) an upstream induction sequence (UIS) needed for a response to inducer. The UIS element mediates inhibition of URS-mediated function when inducer is present. We cloned and characterized the DAL81 gene and identified the element with which it was associated. The gene was found to encode a rare 3.2-kilobase-pair mRNA. The amount of DAL81-specific RNA responded neither to induction nor to nitrogen catabolite repression. Deletion of the DAL81 gene resulted in loss of induction but did not significantly affect basal level expression of the DAL7 and DUR1,2 genes or the UAS and URS functions present in plasmid constructions. These data suggest that (i) transcriptional activation of the DAL genes and their responses to inducer are mediated by different factors and cis-acting sequences and (ii) the UIS functions only when a wild-type DAL81 gene product is available.
Activation of 2',5'-oligoadenylate synthetase activity on induction of HL-60 leukemia cell differentiation.
Authors: Schwartz E L , Nilson L A
Published: October 2015

A 27-fold increase in 2',5'-oligoadenylate synthetase activity, an enzyme associated with the antiproliferative actions of interferon (IFN), was observed after treatment of HL-60 human leukemia cells with dimethyl sulfoxide (DMSO), an inducer of granulocytic differentiation of the cells. Enzyme activity was elevated after 24 h of exposure to DMSO, was maximal at 48 hours, and declined thereafter. A comparable increase was observed after treatment with 1 U of alpha interferon (IFN-alpha) per ml or 8 U of beta interferon (IFN-beta) per ml. Elevated levels of expression of other IFN-inducible genes, including type I histocompatibility antigen (HLA-B) mRNA and 2',5'-oligoadenylate phosphodiesterase activity, were also observed with DMSO treatment. DMSO-treated HL-60 cells had an increased amount of a 1.8-kilobase mRNA for oligoadenylate [oligo(A)] synthetase when compared with that of control cells; both DMSO- and IFN-treated HL-60 cells also expressed 1.6-, 3.4-, and 4.3-kilobase mRNA. The increase in both oligo(A) synthetase activity and mRNA levels was inhibited by polyclonal antiserum to human IFN-alpha; however, no IFN-alpha mRNA could be detected in the cells. Antiserum to IFN-beta or gamma interferon (IFN-gamma) had no effect on oligo(A) synthetase expression or activity nor was there any detectable IFN-beta 1 or IFN-beta 2 mRNA in the cells. The anti-IFN-alpha serum did not block the elevation of HLA-B mRNA in DMSO-treated cells. These observations suggest that the increased expression of oligo(A) synthetase in DMSO-treated cells may be mediated by the release of an IFN-alpha-like factor; however, the levels of any IFN-alpha mRNA produced in the cells were extremely low.
Antibodies to synthetic peptide from the residue 33 to 42 domain of c-Ha-ras p21 block reconstitution of the protein with different effectors.
Authors: Rey I , Soubigou P , Debussche L , David C , Morgat A , Bost P E , Mayaux J F , Tocque B
Published: October 2015

Residues 32 to 40, which are conserved among ras proteins from different species, are likely to participate in interactions with the p21 effector system. With the goal of understanding the structural basis of the regulatory functions of c-Ha-ras p21, we produced rabbit antisera against a synthetic peptide corresponding to amino acids 33 to 42 of the protein. The affinity-purified antibodies interacted specifically with p21 and with the antigenic peptide. The epitope recognized by the antibodies appeared to be centered on threonine 35. The antibodies inhibited both in vitro p21-induced production of cyclic AMP in detergent extracts of RAS-defective yeast membranes and GAP-stimulated GTPase activity. However, monoclonal anti-ras antibodies Y13-259 and Y13-238 were not capable of specifically inhibiting interactions of p21 with these two putative effector proteins. The apparent inhibitory effect of Y13-259 on stimulation of p21 by GAP was due to a greatly reduced rate of exchange of nucleotides in the binding pocket of the protein. These findings provide additional support for the essential role of the residue 32 to 40 domain as the true effector site and further evidence of the involvement of GAP as a cellular effector of ras proteins.
hsp82 is an essential protein that is required in higher concentrations for growth of cells at higher temperatures.
Authors: Borkovich K A , Farrelly F W , Finkelstein D B , Taulien J , Lindquist S
Published: October 2015

hsp82 is one of the most highly conserved and abundantly synthesized heat shock proteins of eucaryotic cells. The yeast Saccharomyces cerevisiae contains two closely related genes in the HSP82 gene family. HSC82 was expressed constitutively at a very high level and was moderately induced by high temperatures. HSP82 was expressed constitutively at a much lower level and was more strongly induced by heat. Site-directed disruption mutations were produced in both genes. Cells homozygous for both mutations did not grow at any temperature. Cells carrying other combinations of the HSP82 and HSC82 mutations grew well at 25 degrees C, but their ability to grow at higher temperatures varied with gene copy number. Thus, HSP82 and HSC82 constitute an essential gene family in yeast cells. Although the two proteins had different patterns of expression, they appeared to have equivalent functions; growth at higher temperatures required higher concentrations of either protein. Biochemical analysis of hsp82 from vertebrate cells suggests that the protein binds to a variety of other cellular proteins, keeping them inactive until they have reached their proper intracellular location or have received the proper activation signal. We speculate that the reason cells require higher concentrations of hsp82 or hsc82 for growth at higher temperatures is to maintain proper levels of complex formation with these other proteins.
The cyclic nucleotide phosphodiesterase gene of Dictyostelium discoideum utilizes alternate promoters and splicing for the synthesis of multiple mRNAs.
Authors: Podgorski G J , Franke J , Faure M , Kessin R H
Published: October 2015

The cyclic nucleotide phosphodiesterase (phosphodiesterase) gene plays essential roles in the development of Dictyostelium discoideum during cellular aggregation and postaggregation morphogenesis. Genomic clones spanning the gene were isolated and used to determine the sequence and structure of the phosphodiesterase gene. We found an unusually complex organization for a gene of D. discoideum. Two transcripts of 2.4 and 1.9 kilobases (kb) were synthesized from start sites separated by 1.1 kb. A developmentally regulated promoter was utilized for the 2.4-kb mRNA, and a constitutive promoter regulated synthesis of the 1.9-kb transcript. The gene was found to be divided into four exons that are alternately spliced to give rise to the two mRNAs. The precursor of the 2.4-kb mRNA contained a 2.3-kb intron, whereas the precursor of the constitutive transcript was synthesized with a 1.7-kb intron. The two transcripts contained identical protein-coding regions and 400-nucleotide 3' untranslated sequences. The 2.4-kb developmentally regulated mRNA was distinguished by a long 5' untranslated leader of 666 nucleotides. The complex structure of the gene may allow multiple levels of control of the expression of the phosphodiesterase during development.
Transduction of human colony-stimulating factor-1 (CSF-1) receptor into interleukin-3-dependent mouse myeloid cells induces both CSF-1-dependent and factor-independent growth.
Authors: Kato J Y , Roussel M F , Ashmun R A , Sherr C J
Published: October 2015

A retroviral vector encoding the receptor for human colony-stimulating factor-1 (CSF-1) was introduced into murine myeloid FDC-P1 cells which require interleukin-3 (IL-3) for their proliferation and survival in culture. Cells expressing the CSF-1 receptor (CSF-1R), selected by fluorescence-activated cell sorting in the continued presence of murine IL-3, formed colonies in semisolid medium and were able to proliferate continuously in liquid cultures containing human recombinant CSF-1. Thus, although they do not synthesize endogenous murine CSF-1R, FDC-P1 cells express the downstream components of the CSF-1 mitogenic pathway necessary for its signal-response coupling. After receptor transduction, slowly proliferating factor-independent variants that produced neither CSF-1 nor growth factors able to support the proliferation of parental FDC-P1 cells also arose. When the human CSF-1R was expressed in FDC-P1 cells under the control of an inducible metallothionein promoter, the frequencies of both CSF-1-responsive and factor-independent variants increased after heavy-metal treatment. In addition, a monoclonal antibody to human CSF-1R arrested colony formation by both the CSF-1-dependent and factor-independent cells but did not affect their growth in response to IL-3. Therefore, the induction of both the CSF-1-dependent and factor-independent phenotypes depended on expression of the transduced human CSF-1R.
DNA determinants of structural and regulatory variation within the murine beta-glucuronidase gene complex.
Authors: Wawrzyniak C J , Gallagher P M , D'Amore M A , Carter J E , Lund S D , Rinchik E M , Ganschow R E
Published: October 2015

The murine beta-glucuronidase (GUS) gene complex, [Gus], encompasses the GUS structural element, Gus-s, and a set of regulatory elements which serve to modulate Gus-s expression. Three common GUS haplotypes representing virtually all inbred strains of laboratory mice have been compared with respect to GUS mRNA sequence. Results of such comparisons revealed sequence variations which target the location of one of the GUS regulatory elements to sequences within Gus-s and which account for known electrophoretic and heat stability differences among GUS allozymes of the three common GUS haplotypes.
Ruptured intercostal artery pseudoaneurysm: a rare cause of acute back pain
Authors: Junck Emily , Utarnachitt Richard
Published: October 2015

A 63-year-old man with no relevant medical history presented with acute non-traumatic back pain and was found to have a T8 intercostal artery pseudoaneurysm associated with haemomediastinum on CT of the chest. He was taken to angiography with interventional radiology and the aneurysm was coiled without complication.
Rock climbing-related subclavian vein thrombosis
Authors: Lutter Christoph , Monasterio Erik , Schöffl Volker
Published: October 2015

Paget-Schroetter syndrome, also known as upper extremity deep venous thrombosis (UEDVT), is a rare condition, characterised by a (sub-) total occlusion of the axillary-subclavian venous system due to thrombosis. UEDVT is the most common vascular condition among athletes so far; although the general incidence is low, this problem will become more frequent as a result of increased participation in climbing sports. The purpose of this report is to illustrate two cases in rock climbers where UEDVT developed during rock climbing or bouldering. Fortunately, both patients were diagnosed relatively early after the symptoms began, despite the ambiguity of UEDVT symptoms. This relatively unfamiliar condition may become more highly recognised as a potentially serious differential diagnosis of unspecific pain of the shoulder. Rock climbers are disposed to develop UEDVT due to frequent stress on the upper extremities during training or competition.
Type 2 diabetes exaggerates exercise effort and impairs exercise performance in older women
Authors: Huebschmann A G , Kohrt W M , Herlache L , Wolfe P , Daugherty S , Reusch J EB , Bauer T A , Regensteiner J G
Published: October 2015

Objective

Type 2 diabetes mellitus (T2DM) is associated with high levels of disability and mortality. Regular exercise prevents premature disability and mortality, but people with T2DM are generally sedentary for reasons that are not fully established. We previously observed that premenopausal women with T2DM report greater effort during exercise than their counterparts without diabetes, as measured by the Rating of Perceived Exertion (RPE) scale. We hypothesized that RPE is greater in older women with T2DM versus no T2DM.

Research design and methods

We enrolled overweight, sedentary women aged 50–75 years with (n=26) or without T2DM (n=28). Participants performed submaximal cycle ergometer exercise at 30 W and 35% of individually-measured peak oxygen consumption (35% VO2peak). We assessed exercise effort by RPE (self-report) and plasma lactate concentration.

Results

VO2peak was lower in T2DM versus controls (p=0.003). RPE was not significantly greater in T2DM versus controls (30 W: Control, 10.4±3.2, T2DM, 11.7±2.3, p=0.08; 35% VO2peak: Control, 11.1±0.5, T2DM, 12.1±0.5, p=0.21). However, lactate was greater in T2DM versus controls (p=0.004 at 30 W; p<0.05 at 35% VO2peak). Greater RPE was associated with higher lactate, higher heart rate, and a hypertension diagnosis (p<0.05 at 30 W and 35% VO2peak).

Conclusions

Taken together, physiological measures of exercise effort were greater in older women with T2DM than controls. Exercise effort is a modifiable and thereby targetable end point. In order to facilitate regular exercise, methods to reduce exercise effort in T2DM should be sought.

Trial number

NCT00785005.
Efficacy of advice from healthcare professionals to pregnant women on avoiding constrictive clothing around the trunk: a study protocol for a randomised controlled trial
Authors: Takehara Kenji , Kato Sachiko , Sasaki Aiko , Jwa Seung Chik , Kakee Naoko , Sago Haruhiko , Noguchi Yuko , Aoki Tomoko , Inoue Eisuke , Nitta Chieko , Ishii Yumiko
Published: October 2015

Introduction

As a component of midwife care, eliminating clothing that constricts the trunk has been shown to markedly elevate the uterine fundus, soften the uterus and abdomen, and reduce the abdominal wall tension in women admitted to hospital due to the risk of miscarriage or premature delivery. However, no prospective study has conclusively verified the efficacy of avoiding constrictive clothes around the trunk in pregnant women. We aim to verify the efficacy of instructing pregnant women to wear loose clothing that does not constrict the trunk to reduce the risk of premature birth and improve quality of life (QoL) during pregnancy.

Methods and analysis

We will conduct a randomised controlled trial of pregnant women scheduled to deliver at the National Center for Child Health and Development in Tokyo, Japan. A total of 616 pregnant women, from whom written informed consent will be obtained, will be allocated randomly to an intervention group or a control group. Women in the control group will be provided with anaemia prevention leaflets at 20 weeks’ gestation and skin-care leaflets at 30 weeks’ gestation. Women in the intervention group will be provided with the same leaflets and will also receive health advice from health professionals to avoid constrictive clothing around the trunk. The primary outcome will be a difference between these groups in the frequency of any one of the following category variables: (1) cervical length <30 mm up to 28 weeks’ gestation, (2) hospital admission for threatened premature delivery, or (3) premature delivery. Secondary outcomes will include QoL during pregnancy, maternal state of health, and status of fetal development.

Ethics and dissemination

The Institutional Review Board and Ethics Committee at the National Center for Child Health and Development, Japan, has approved this study. Our findings will be widely disseminated through conference presentations and peer-reviewed publications.

Trial registration number

UMIN000016853.
Are we meeting current standards in medicines reconciliation? A study in a District General Hospital.
Authors: Iddles Emma , Williamson Andrew , Bradley Alison , Khan Khurram
Published: October 2015

Introduction: Inadequate medicines reconciliation on admission is often identified as a major cause of patient morbidity, with poor access to patient's regular medications often cited as a barrier to care. In the surgical admission unit of our district general hospital, drug charts are completed by junior doctors who do not have access to the Emergency Care Summary (ECS) thus making it difficult to accurately complete admission drug charts.

Methods: Our initial measurement of all acute surgical admissions revealed that 49% of patients had an accurate medicines reconciliation upon admission, increasing to 75% within 24 hours of admission. It was clear from this data that our current practice needed improvement in order to ensure patient safety. Resultantly the junior medical staff were provided with ECS accounts and teaching to aid the process of medicines reconciliation.

Results: Following the introduction of access to ECS and junior doctor education, a further two data cycles were completed. On the first cycle, the number of accurately completed drug charts increased to 62% on admission and 86% at 24 hours. After the second cycle 57% were complete on admission increasing to 84% at 24 hours.

Conclusion: Our project has shown that by providing junior doctors with medicines reconciliation education and access to patients’ pre-admission medications through a nationwide electronic system resulted in a considerable increase in the completion of medicine reconciliation.
The early life-history dynamics of Northeast Arctic cod: levels of natural mortality and abundance during the first 3 years of life
Authors: Bogstad Bjarte , Yaragina Natalia A. , Nash Richard D.M.
Published: October 2015

Recruitment at age 3 of the Northeast Arctic cod (Gadus morhua) is highly variable. It has generally been believed that year-class strength for this stock is determined prior to settlement to the bottom after about 6 months. However, newer observations indicate that year-class strength may change considerably between settlement and recruitment at age 3. Our analyses cover the 1983–2009 year classes where comprehensive data from total egg production (TEP), surveys, and stock assessments were available for a thorough examination of these cohorts. On average, only 6 out of 1 million of a new generation at the TEP stage reaches the age of recruitment to the fishery. The between-cohort variability in abundance is greatest at the ages 0–1 stage. Although the mortality is highest during the first months of life, the year-class strength can also be affected considerably by processes taking place between the 0-group stage (∼6 months) and age 3. The mortality in this period of life seems to be strongly density-dependent, and cannibalism is an important source of mortality.