bluuebunny/crossref_metadata_2025 huggingface dataset:
Phosphatidylinositol 3-kinase activation is required for insulin stimulation of pp70 S6 kinase, DNA synthesis, and glucose transporter translocation.
Authors: Cheatham B , Vlahos C J , Cheatham L , Wang L , Blenis J , Kahn C R
Published: October 2015

Phosphatidylinositol 3-kinase (PI 3-kinase) is stimulated by insulin and a variety of growth factors, but its exact role in signal transduction remains unclear. We have used a novel, highly specific inhibitor of PT 3-kinase to dissect the role of this enzyme in insulin action. Treatment of intact 3T3-L1 adipocytes with LY294002 produced a dose-dependent inhibition of insulin-stimulated PI 3-kinase (50% inhibitory concentration, 6 microM) with > 95% reduction in the levels of phosphatidylinositol-3,4,5-trisphosphate without changes in the levels of phosphatidylinositol-4-monophosphate or its derivatives. In parallel, there was a complete inhibition of insulin-stimulated phosphorylation and activation of pp70 S6 kinase. Inhibition of PI 3-kinase also effectively blocked insulin- and serum-stimulated DNA synthesis and insulin-stimulated glucose uptake by inhibiting translocation of GLUT 4 glucose transporters to the plasma membrane. By contrast, LY294002 had no effect on insulin stimulation of mitogen-activated protein kinase or pp90 S6 kinase. Thus, activation of PI 3-kinase plays a critical role in mammalian cells and is required for activation of pp70 S6 kinase and DNA synthesis and certain forms of intracellular vesicular trafficking but not mitogen-activated protein kinase or pp90 S6 kinase activation. These data suggest that PI 3-kinase is not only an important component but also a point of divergence in the insulin signaling network.
Complementation of a capsule-deficient mutation of Cryptococcus neoformans restores its virulence.
Authors: Chang Y C , Kwon-Chung K J
Published: October 2015

Capsule formation plays a significant role in the pathogenicity of Cryptococcus neoformans. To study the molecular basis of capsule synthesis, the capsule-deficient phenotype of a mutant strain was complemented by transformation. A plasmid rescued from the resulting Cap+ transformant complemented a cap59 mutation which was mapped previously by classical recombination analysis. Gene deletion by homologous integration resulted in an acapsular phenotype, indicating that we have identified the CAP59 gene. The CAP59 gene was assigned to chromosome I by Southern blot analysis of contour-clamped homogeneous electric field gel electrophoresis-resolved chromosomes of C. neoformans var. neoformans. Sequence comparison of genomic and cDNA clones indicated the presence of six introns. CAP59 encoded a 1.9-kb transcript and a deduced protein of 458 amino acids. Analysis of the nucleotide sequence revealed little similarity to existing sequences in the data bank. When the capsule-deficient phenotype was complemented, the originally avirulent C. neoformans strain became virulent for mice. In addition, the acapsular strain created by gene deletion of CAP59 lost its virulence. This work demonstrates the molecular basis for capsule-related virulence and that the CAP59 gene is required for capsule formation.
Nuclear localization of the PEP protein tyrosine phosphatase.
Authors: Flores E , Roy G , Patel D , Shaw A , Thomas M L
Published: October 2015

PEP is an intracellular protein tyrosine phosphatase expressed primarily by cells of hematopoietic origin that can be divided structurally into a catalytic domain and a large carboxy-terminal domain. The carboxy-terminal domain is enriched in proline, glutamic acid, serine, and threonine residues (PEST sequences) and contains a nonperfect tandem repeat sequence enriched in proline residues and a carboxy terminus enriched in basic amino acids. Here we show that PEP is diffusely expressed in lymphoid tissues, consistent with expression by many different cell types. Analysis of the PEP protein identifies a nuclear localization sequence within the extreme carboxy terminus. Transfer of 18 amino acids from the carboxy terminus of PEP to beta-galactosidase conferred nuclear localization, indicating that this sequence was sufficient for nuclear localization. Proteins enriched in PEST sequences are often rapidly degraded. However, pulse-chase analysis indicates that PEP has a half-life of greater than 5 h.
RNA binding by Sxl proteins in vitro and in vivo.
Authors: Samuels M E , Bopp D , Colvin R A , Roscigno R F , Garcia-Blanco M A , Schedl P
Published: October 2015

Sxl has been proposed to regulate splicing of specific target genes by directly interacting with their pre-mRNAs. We have therefore examined the RNA-binding properties of Sxl protein in vitro and in vivo. Gel shift and UV cross-linking assays with a purified recombinant MBP-Sxl fusion protein demonstrated preferential binding to RNAs containing poly(U) tracts, and the protein footprinted over the poly(U) region. The protein did not appear to recognize either branch point or AG dinucleotide sequences, but an adenosine residue at the 5' end of the poly(U) tract enhanced binding severalfold. MBP-Sxl formed two shifted complexes on a tra regulated acceptor site RNA; the doubly shifted form may have been stabilized by protein-protein interactions. Consistent with its proposed role in pre-mRNA processing, in nuclear extracts Sxl was found in large ribonucleoprotein (RNP) complexes which sedimented significantly faster than bulk heterogeneous nuclear RNP and small nuclear RNPs. Anti-Sxl staining of polytene chromosomes showed Sxl protein at a number of chromosomal locations, among which was the Sxl locus itself. Sxl protein could also be targeted to a new chromosomal site carrying a transgene containing splicing regulatory sequences from the Sxl gene, following transcriptional induction. After prolonged heat shock, all Sxl protein was restricted to the heat-induced puff at the hs93D locus. In contrast, a presumptive small nuclear RNP protein was observed at several heat puffs following shock.
Factor C*, the specific initiation component of the mouse RNA polymerase I holoenzyme, is inactivated early in the transcription process.
Authors: Brun R P , Ryan K , Sollner-Webb B
Published: October 2015

Factor C* is the component of the RNA polymerase I holoenzyme (factor C) that allows specific transcriptional initiation on a factor D (SL1)- and UBF-activated rRNA gene promoter. The in vitro transcriptional capacity of a preincubated rDNA promoter complex becomes exhausted very rapidly upon initiation of transcription. This is due to the rapid depletion of C* activity. In contrast, C* activity is not unstable in the absence of transcription, even in the presence of nucleoside triphosphates (NTPs). By using 3'dNTPs to specifically halt elongation, C* is seen to remain active through transcription complex assembly, initiation, and the first approximately 37 nucleotides of elongation, but it is inactivated before synthesis proceeds beyond approximately 40 nucleotides. When elongation is halted before this critical distance, the C* remains active and on that template complex, greatly extending the kinetics of transcription and generating manyfold more transcripts than would have been synthesized if elongation had proceeded past the critical distance where C* is inactivated. In complementary in vivo analysis under conditions where C* activity is not replenished, C* activity becomes depleted from cells, but this also occurs only when there is ongoing rDNA transcription. Thus, both in vitro and in vivo, the specific initiation-conferring component of the RNA polymerase I holoenzyme is used stoichiometrically in the transcription process.
An M-CAT binding factor and an RSRF-related A-rich binding factor positively regulate expression of the alpha-cardiac myosin heavy-chain gene in vivo.
Authors: Molkentin J D , Markham B E
Published: October 2015

Cardiac muscle-restricted expression of the alpha-myosin heavy-chain (alpha-MHC) gene is regulated by multiple elements in the proximal enhancer/promoter. Within this region, an M-CAT site and an A-rich site were identified as potential regulatory elements. Site-specific mutations in each site, individually, reduced activity from the wild-type promoter by approximately 85% in the adult rat heart, demonstrating that these sites were positive regulatory elements. alpha-MHC, beta-MHC, and chicken cardiac troponin T (cTnT) M-CAT sites interacted with an M-CAT-binding factor (MCBF) from rat heart nuclear extracts that was immunologically related to transcriptional enhancer factor 1, a factor that binds within the simian virus 40 enhancer. The factor that bound the A-rich region (ARF) was antigenically related to the RSRF family of proteins, ARF was distinct from myocyte-specific enhancer factor 2 (MEF-2) on the basis of DNA-binding specificity and developmental expression. Like MEF-2, ARF DNA-binding activity was present in the heart and brain; however, no ARF activity was detected in extracts from skeletal muscle or C2C12 myotubes. MCBF and ARF DNA-binding activities were developmentally regulated with peak levels in the 1- to 2-day neonatal heart. The activity of both factors increased nearly fivefold in adult rat hearts subjected to a pressure overload. By comparison, the levels of alpha-MHC binding factor 2 did not change during hypertrophy. Binding sites for MCBF and ARF are present in several genes that are upregulated during cardiac hypertrophy. Our results suggest that these factors participate in the alterations in gene expression that occur during cardiac development and hypertrophy.
Functional differences between HOX proteins conferred by two residues in the homeodomain N-terminal arm.
Authors: Phelan M L , Sadoul R , Featherstone M S
Published: October 2015

Hox genes encode homeodomain-containing transcriptional regulators that function during development to specify positional identity along embryonic axes. The homeodomain is composed of a flexible N-terminal arm and three alpha helices, and it differentially binds DNA. A number of homeodomains recognize sites containing a TAAT core motif. The product of the murine Hoxd-4 (Hox-4.2) gene functions in a positive autoregulatory fashion in P19 cells that is dependent on two TAAT motifs in the Hoxd-4 promoter. This effect is specific in that murine HOXA-1 (HOX-1.6) is unable to activate transcription through the Hoxd-4 autoregulatory element. Here we show that this is due to an inability of the HOXA-1 homeodomain to bind a HOXD-4 recognition site effectively. We have produced chimeras between HOXD-4 and HOXA-1 to map specific residues responsible for this functional difference. When positions 2 and 3 in the N-terminal arm of HOXA-1 were converted to HOXD-4 identity, both strong DNA binding and transcriptional activation were rescued. This substitution appears to confer an increased DNA-binding ability on the HOXA-1 homeodomain, since we were unable to detect a high-affinity recognition sequence for HOXA-1 in a randomized pool of DNA probes. The contribution of position 3 to DNA binding has been implicated by structural studies, but this is the first report of the importance of position 2 in regulating homeodomain-DNA interactions. Additionally, specific homeodomain residues that confer major differences in DNA binding and transcriptional activation between Hox gene products have not been previously determined. Identity at these two positions is generally conserved among paralogs but varies between Hox gene subfamilies. As a result, these residues may be important for the regulation of target gene expression by specific Hox products.
Differential roles of heat shock protein 70 in the in vitro nuclear import of glucocorticoid receptor and simian virus 40 large tumor antigen.
Authors: Yang J , DeFranco D B
Published: October 2015

Nuclear import of glucocorticoid receptors (GRs) was analyzed in vitro with digitonin-permeabilized cells (S. A. Adam, R. Sterne-Marr, and L. Gerace, J. Cell Biol. 111:807-816, 1990). Indirect immunofluorescence methods were used to monitor the transport of GRs from rat hepatoma and fibroblast cell cytosol into HeLa nuclei. In vitro nuclear import of GRs was shown to be hormone dependent and to require ATP and incubation at ambient temperatures (i.e., 30 degrees C). Hormone-dependent dissociation of GR-bound proteins, such as the 90-kDa heat shock protein, hsp90, is part of an activation process that is obligatory for the expression of the receptor's DNA-binding activity. Inhibition of in vitro GR activation by Na2MoO4 blocked hormone-dependent nuclear import, demonstrating that receptor activation is required for nuclear import. The addition to GR-containing cytosol of antiserum directed against the cytosolic 70-kDa heat shock protein, hsp70, while effective in blocking the nuclear import of simian virus 40 large tumor antigen (SV40 TAg), did not affect hormone-dependent nuclear import of endogenous, full-length GRs or an exogenously added truncated GR protein (i.e., XGR556) that lacks a hormone-binding domain but possesses a constitutively active nuclear localization signal sequence (NLS). Depletion of hsp70 from HeLa cell cytosol did not affect the nuclear import of exogenously added XGR556 but led to inhibition of SV40 TAg nuclear import. Thus, two closely related NLSs, one contained within GRs and the other contained within SV40 TAg, are distinguished by their differential requirements for hsp70 in vitro.
Molecular cloning of a novel human cDNA encoding a zinc finger protein that binds to the interleukin-3 promoter.
Authors: Koyano-Nakagawa N , Nishida J , Baldwin D , Arai K , Yokota T
Published: October 2015

The CT/GC-rich region (-76 to -47) is one transcriptional regulatory region of the interleukin-3 (IL-3) gene which confers basic transcriptional activity and responds to trans-activation by human T-cell leukemia virus type I-encoded Tax. We isolated three types of cDNAs encoding Cys2/His2-type zinc finger proteins that bind to this region. Two were identical to known transcription factors, EGR1 and EGR2, and the other clone, named DB1, encoded a novel protein of 516 amino acids with six zinc finger motifs. DB1 mRNA was present in human tissues, ubiquitously. Two constitutive transcripts of 4.0 and 4.8 kb in length were present in Jurkat cells. Electrophoretic mobility shift assay, with specific antibodies, showed that DB1 constitutively binds to this region whereas EGR1 binds in a T-cell activation-dependent manner. Overexpression of DB1 in Jurkat cells had no detectable effect on the transcription activity of the IL-3 promoter, in a transient-transfection assay. EGR1 and EGR2 increased IL-3 promoter activity when the transfected cells were stimulated with phorbol-12-myristate-13-acetate and A23187. When DB1 was cotransfected with a Tax expression vector, transcription activity of the IL-3 promoter induced by Tax was significantly increased, while EGR1 and EGR2 were without effect. These results suggest that EGR1 has a role in inducible transcription of the IL-3 gene, while DB1 sustains basal transcriptional activity and also cooperates with Tax to activate the IL-3 promoter.
Differential expression of the myocyte enhancer factor 2 family of transcription factors in development: the cardiac factor BBF-1 is an early marker for cardiogenesis.
Authors: Goswami S , Qasba P , Ghatpande S , Carleton S , Deshpande A K , Baig M , Siddiqui M A
Published: October 2015

In the present study, we have used single chicken blastoderms of defined early developmental stages, beginning with the prestreak stage, stage 1 (V. Hamburger and H. L. Hamilton, J. Morphol. 88:49-92, 1951), to analyze the onset of cardiac myogenesis by monitoring the appearance of selected cardiac muscle tissue-specific gene transcripts and the functional expression of the myocyte enhancer factor 2 (MEF-2) proteins. Using gene-specific oligonucleotide primers in reverse transcriptase PCR assay, we have demonstrated that the cardiac myosin light-chain 2 (MLC2) and alpha-actin gene transcripts appear as early as stage 5, i.e., immediately after the cardiogenic fate assignment at stage 4. Consistent with this observation is the developmental expression pattern of DNA-binding activity of BBF-1, a cardiac muscle-specific member of the MEF-2 protein family, which also begins at stage 5 prior to MEF-2. Differential expression of DNA-binding complexes is also observed with another AT-rich DNA sequence (CArG box) as probe, but the binding pattern with the ubiquitous TATA-binding proteins remains unchanged during the same developmental period. Thus, the cardiogenic commitment and differentiation of the precardiac mesoderm, as exemplified by the appearance of cardiac MEF-2, MLC2, and alpha-actin gene products, occur earlier than previously thought and appear to be closely linked. The onset of skeletal myogenic program follows that of the cardiogenic program with the appearance of skeletal MLC2 at stage 8. We also observed that mRNA for the MEF-2 family of proteins appears as early as stage 2 and that for CMD-1, the chicken counterpart of MyoD, appears at stage 5. The temporal separation of activation of cardiac and skeletal MLC2 genes, which appears immediately after the respective fate assignments, and those of cardiac MEF-2 and CMD-1, which occur before, are consistent with the established appearance of the myogenic programs and with the acquisition pattern of the two tissue-specific morphological characteristics in the early embryo. The preferential appearance of BBF-1 activity in precardiac moesderm, relative to that of MEF-2, indicates that these two protein factors are distinct members of the MEF-2 family and provides a compelling argument in support of the potential role of BBF-1 as a regulator of the cardiogenic cell lineage determination, while cardiac MEF-2 might be involved in maintenance of the cardiac differentiative state.
Casein kinase II mediates multiple phosphorylation of Saccharomyces cerevisiae eIF-2 alpha (encoded by SUI2), which is required for optimal eIF-2 function in S. cerevisiae.
Authors: Feng L , Yoon H , Donahue T F
Published: October 2015

Previous studies have demonstrated that the alpha subunit of eukaryotic initiation factor 2 (eIF-2 alpha), encoded by the SUI2 gene in the yeast Saccharomyces cerevisiae, is phosphorylated at Ser-51 by the GCN2 kinase in response to general amino acid control. Here we describe that yeast eIF-2 alpha is a constitutively phosphorylated protein species that is multiply phosphorylated by a GCN2-independent mechanism. 32Pi labeling and isoelectric focusing analysis of a SUI2+ delta gcn2 strain identifies eIF-2 alpha as radiolabeled and a single isoelectric protein species. Treatment of SUI2+ delta gcn2 strain extracts with phosphatase results in the identification of three additional isoelectric forms of eIF-2 alpha that correspond to the stepwise removal of three phosphates from the protein. Mutational analysis of SUI2 coupled with biochemical analysis of eIF-2 alpha maps the sites to the carboxyl region of SUI2 that correspond to Ser residues at amino acid positions 292, 294, and 301 that compose consensus casein kinase II sequences. 32Pi labeling or isoelectric focusing analysis of eIF-2 alpha from conditional casein kinase II mutants indicated that phosphorylation of eIF-2 alpha is abolished or dephosphorylated forms of eIF-2 alpha are detected when these strains are grown at the restrictive growth conditions. Furthermore, yeast casein kinase II phosphorylates recombinant wild-type eIF-2 alpha protein in vitro but does not phosphorylate recombinant eIF-2 alpha that contains Ser-to-Ala mutations at all three consensus casein kinase II sequences. These data strongly support the conclusion that casein kinase II directly phosphorylates eIF-2 alpha at one or all of these Ser amino acids in vivo. Although substitution of SUI2 genes mutated at these sites for the wild-type gene have no obvious effect on cell growth, one test that we have used appears to demonstrate that the inability to phosphorylate these sites has a physiological consequence on eIF-2 function in S. cerevisiae. Haploid strains constructed to contain Ser-to-Ala mutations at the consensus casein kinase II sequences in SUI2 in combination with a mutated allele of either the GCN2, GCN3, or GCD7 gene have synthetic growth defects. These genetic data appear to indicate that the modifications that we describe at the carboxyl end of the eIF-2 alpha protein are required for optimal eIF-2 function in S. cerevisiae.
Regulation by protein-tyrosine phosphatase PTP2 is distinct from that by PTP1 during Dictyostelium growth and development.
Authors: Howard P K , Gamper M , Hunter T , Firtel R A
Published: October 2015

We have cloned a gene encoding a second Dictyostelium discoideum protein-tyrosine phosphatase (PTP2) whose catalytic domain has approximately 30 to 39% amino acid identity with those of other PTPs and a 41% amino acid identity with D. discoideum PTP1. Like PTP1, PTP2 is a nonreceptor PTP with the catalytic domain located at the C terminus of the protein. PTP2 has a predicted molecular weight of 43,000 and possesses an acidic 58-amino-acid insertion 24 amino acids from the N terminus of the conserved catalytic domain. PTP2 transcripts are expressed at moderate levels in vegetative cells and are induced severalfold at the onset of development. Studies with a PTP2-lacZ reporter gene fusion indicate that PTP2, like PTP1, is preferentially expressed in prestalk and anterior-like cell types during the multicellular stages of development. PTP2 gene disruptants (ptp2 null cells) are not detectably altered in growth and show a temporal pattern of development similar to that of wild-type cells. ptp2 null slugs and fruiting bodies, however, are significantly larger than those of wild-type slugs, suggesting a role for PTP2 in regulating multicellular structures. D. discoideum strains overexpressing PTP2 from the PTP2 promoter exhibit growth rate and developmental abnormalities, the severity of which corresponds to the level of PTP2 overexpression. Strains with high overexpression of the PTP2 gene grow slowly on bacterial lawns and produce small cells in axenic medium. When development is initiated in these strains, cells are able to aggregate but then stop further morphogenesis for 6 to 8 h, after which time a variable fraction of these aggregates continue with normal timing, producing diminutive fruiting bodies. These disruption and overexpression phenotypes for PTP2 are distinct from the corresponding mutant PTP1 phenotypes. Immunoprobing PTP2 mutant strains during growth and development with antiphosphotyrosine antibodies reveals several changes in the tyrosine phosphorylation of proteins in PTP2 mutant strains compared with that in wild-type cells. These changes are different from those identified in the previously characterized corresponding PTP1 disruption and overexpression mutant strains. Thus, although PTP2 and PTP1 are nonreceptor PTPs with similar spatial patterns of expression, our findings suggest that they possess distinct regulatory functions in controlling D. discoideum growth and development.
Sequences controlling transcription of the Chlamydomonas reinhardtii beta 2-tubulin gene after deflagellation and during the cell cycle.
Authors: Davies J P , Grossman A R
Published: October 2015

In Chlamydomonas reinhardtii, transcripts from the beta 2-tubulin gene (tubB2), as well as those from other tubulin-encoding genes, accumulate immediately after flagellar excision as well as at a specific time in the cell cycle. Control of tubB2 transcript accumulation following deflagellation is regulated, at least partially, at the transcriptional level. We have fused the tubB2 promoter to the arylsulfatase (ars) reporter gene, introduced this construct into C. reinhardtii, and compared expression of the chimeric gene with that of the endogenous tubB2 gene. After flagellar excision, transcripts from the tubB2/ars chimeric gene accumulate with kinetics similar to those of transcripts from the endogenous tubB2 gene. The tubB2/ars transcripts also accumulate in a cell cycle-specific manner; however, chimeric transcripts are more abundant earlier in the cell cycle than the endogenous tubB2 transcripts. To elucidate transcriptional control of tubB2, we have mutated or removed sequences in the tubB2 promoter and examined the effect on transcription. The tubB2 promoter shares features with the promoters of other tubulin-encoding genes; these include a GC-rich region between the TATA box and the transcription initiation site and multiple copies of a 10-bp sequence motif that we call the tub box. The tubB2 gene contains seven tub box motifs. Changing the GC-rich region to an AT-rich region or removing three of the seven tub box motifs did not significantly affect transcription of the chimeric gene. However, removing four or five tub box motifs prevented increased transcription following deflagellation and diminished cell cycle-regulated transcription from the tubB2 promoter.
Hierarchy of binding sites for Grb2 and Shc on the epidermal growth factor receptor.
Authors: Batzer A G , Rotin D , Ureña J M , Skolnik E Y , Schlessinger J
Published: October 2015

We analyzed the binding site(s) for Grb2 on the epidermal growth factor (EGF) receptor (EGFR), using cell lines overexpressing EGFRs containing various point and deletion mutations in the carboxy-terminal tail. Results of co-immunoprecipitation experiments suggest that phosphotyrosines Y-1068 and Y-1173 mediate the binding of Grb2 to the EGFR. Competition experiments with synthetic phosphopeptides corresponding to known autophosphorylation sites on the EGFR demonstrated that phosphopeptides containing Y-1068, and to a lesser extent Y-1086, were able to inhibit the binding of Grb2 to the EGFR, while a Y-1173 peptide did not. These findings were confirmed by using a dephosphorylation protection assay and by measuring the dissociation constants of Grb2's SH2 domain to tyrosine-phosphorylated peptides, using real-time biospecific interaction analysis (BIAcore). From these studies, we concluded that Grb2 binds directly to the EGFR at Y-1068, to a lesser extent at Y-1086, and indirectly at Y-1173. Since Grb2 also binds Shc after EGF stimulation, we investigated whether Y-1173 is a binding site for the SH2 domain of Shc on the EGFR. Both competition experiments with synthetic phosphopeptides and dephosphorylation protection analysis demonstrated that Y-1173 and Y-992 are major and minor binding sites, respectively, for Shc on the EGFR. However, other phosphorylation sites in the carboxy-terminal tail of the EGFR are able to compensate for the loss of the main binding sites for Shc. These analyses reveal a hierarchy of interactions between Grb2 and Shc with the EGFR and indicate that Grb2 can bind the tyrosine-phosphorylated EGFR directly, as well as indirectly via Shc.
Chromosome breakage at a major fragile site associated with P-glycoprotein gene amplification in multidrug-resistant CHO cells.
Authors: Kuo M T , Vyas R C , Jiang L X , Hittelman W N
Published: October 2015

Recent studies of several drug-resistant Chinese hamster cell lines suggested that a breakage-fusion-bridge mechanism is frequently involved in the amplification of drug resistance genes. These observations underscore the importance of chromosome breakage in the initiation of DNA amplification in mammalian cells. However, the mechanism of this breakage is unknown. Here, we propose that the site of chromosome breakage consistent with the initial event of P-glycoprotein (P-gp) gene amplification via the breakage-fusion-bridge cycle in three independently established multidrug-resistant CHO cells was located at 1q31. This site is a major chromosome fragile site that can be induced by methotrexate and aphidicolin treatments. Pretreatments of CHO cells with methotrexate or aphidicolin enhanced the frequencies of resistance to vinca alkaloid and amplification of the P-gp gene. These observations suggest that chromosome fragile sites play a pivotal role in DNA amplification in mammalian cells. Our data are also consistent with the hypothesis that gene amplification can be initiated by stress-induced chromosome breakage that is independent of modes of action of cytotoxic agents. Drug-resistant variants may arise by their growth advantage due to overproduction of cellular target molecules via gene amplification.
Activation of p56lck by p72syk through physical association and N-terminal tyrosine phosphorylation.
Authors: Couture C , Baier G , Oetken C , Williams S , Telford D , Marie-Cardine A , Baier-Bitterlich G , Fischer S , Burn P , Altman A
Published: October 2015

The p56lck and p59fyn protein tyrosine kinases are important signal transmission elements in the activation of mature T lymphocytes by ligands to the T-cell antigen receptor (TCR)/CD3 complex. The lack of either kinase results in deficient early signaling events, and pharmacological agents that block tyrosine phosphorylation prevent T-cell activation altogether. After triggering of the TCR/CD3 complex, both kinases are moderately activated and begin to phosphorylate cellular substrates, but the molecular mechanisms responsible for these changes have remained unclear. We recently found that the p72syk protein tyrosine kinase is physically associated with the TCR/CD3 complex and is rapidly tyrosine phosphorylated and activated by receptor triggering also in T cells lacking p56lck. Here we examine the regulation of p72syk and its interaction with p56lck in transfected COS-1 cells. p72syk was catalytically active and heavily phosphorylated on its putative autophosphorylation site, Tyr-518/519. Mutation of these residues to phenylalanines abolished its activity in vitro and toward cellular substrates in vivo and reduced its tyrosine phosphorylation in intact cells by approximately 90%. Coexpression of lck did not alter the catalytic activity of p72syk, but the expressed p56lck was much more active in the presence of p72syk than when expressed alone. This activation was also seen as increased phosphorylation of cellular proteins. Concomitantly, p56lck was phosphorylated at Tyr-192 in its SH2 domain, and a Phe-192 mutant p56lck was no longer phosphorylated by p72syk. Phosphate was also detected in p56lck at Tyr-192 in lymphoid cells. These findings suggest that p56lck is positively regulated by the p72syk kinase.
A cell-specific factor represses stimulation of transcription in vitro by transcriptional enhancer factor 1.
Authors: Chaudhary S , Brou C , Valentin M E , Burton N , Tora L , Chambon P , Davidson I
Published: October 2015

Transcription in HeLa cell extracts in vitro was stimulated 8- to 10-fold by a recombinant chimera, GAL-TEF-1, consisting of the DNA-binding domain of GAL4 and the activation function of the HeLa cell activator TEF-1. In contrast, only a 2- to 3-fold stimulation was obtained with GAL-TEF-1 in extracts from BJA-B lymphoid cells. Stimulation by GAL-TEF-1 in BJA-B extracts was dramatically increased by the addition of immunopurified HeLa cell TFIID, suggesting that BJA-B TFIID lacks or contains lower quantities of a TATA-binding-protein-associated factor(s) required for the activity of the TEF-1 activation function. However, chromatography, immunopurification, and transcriptional reconstitution experiments indicated that BJA-B extracts did not lack the previously identified TATA-binding-protein-associated factors required for TEF-1 activity but rather contained a negatively acting factor(s) which inhibited transactivation by GAL-TEF-1. These results indicate that the relative lack of activity of the TEF-1 activation function in vitro in BJA-B cell extracts does not result from the absence of positively acting factors from the presence of a cell-specific negatively acting factor(s).
Regulation of adhesion and growth of fibrosarcoma cells by NF-kappa B RelA involves transforming growth factor beta.
Authors: Perez J R , Higgins-Sochaski K A , Maltese J Y , Narayanan R
Published: October 2015

The NF-kappa B transcription factor is a pleiotropic activator that participates in the induction of a wide variety of cellular genes. Antisense oligomer inhibition of the RelA subunit of NF-kappa B results in a block of cellular adhesion and inhibition of tumor cell growth. Investigation of the molecular basis for these effects showed that in vitro inhibition of the growth of transformed fibroblasts by relA antisense oligonucleotides can be reversed by the parental-cell-conditioned medium. Cytokine profile analysis of these cells treated with relA antisense oligonucleotides revealed inhibition of transforming growth factor beta 1 (TGF-beta 1 to the transformed fibroblasts reversed the inhibitory effects of relA antisense oligomers on soft agar colony formation and cell adhesion to the substratum. Direct inhibition of TGF-beta 1 expression by antisense phosphorothioates to TGF-beta 1 mimicked the in vitro effects of blocking cell adhesion that are elicited by antisense relA oligomers. These results may explain the in vitro effects of relA antisense oligomers on fibrosarcoma cell growth and adhesion.
Transcriptional suppression of the human T-cell leukemia virus type I long terminal repeat occurs by an unconventional interaction of a CREB factor with the R region.
Authors: Xu X , Brown D A , Kitajima I , Bilakovics J , Fey L W , Nerenberg M I
Published: October 2015

To analyze regulation of the human T-cell leukemia virus type I (HTLV-I) long terminal repeat (LTR), cell lines were generated from LTR-tax x LTR-beta-galactosidase (beta-Gal) doubly transgenic mouse fibroblastic tumors. The HTLV-I LTR directs expression of both the tax and lacZ genes, and Tax up-modulates both promoters in primary cells. However, once cells were transformed by tax, beta-Gal but not tax expression was suppressed. Supertransformation of these cells with v-src suppressed both beta-Gal and tax expression. This suppression was reversed by treatment with the tyrosine kinase inhibitor herbimycin A or protein kinase A inhibitor H8. Electrophoretic mobility shift assays demonstrated augmented binding in the R but not U3 region. This binding was competitively inhibited by a high-affinity CREB oligodeoxynucleotide and super-shifted with a specific CREB antibody. Treatment of cells with the cyclic AMP analog dibutyryl cyclic AMP also transiently increased the R region binding dramatically. In vitro DNase I footprint analysis identified a protein-binding sequence in the R region which corresponded with suppression. However, this target sequence lacked a conventional CREB-binding site. A 70.5-kDa DNA-binding protein was partially purified by affinity chromatography, along with a 49-kDa protein which reacted with CREB-specific sera. These data demonstrate that HTLV-I LTR suppression is associated with CREB factor binding in the R region, probably by direct interaction with a 70.5-kDa protein, and provide a novel mechanism for maintenance of viral latency.
A protein that is highly related to GTPase-activating protein-associated p62 complexes with phospholipase C gamma.
Authors: Maa M C , Leu T H , Trandel B J , Chang J H , Parsons S J
Published: October 2015

p62 is a highly tyrosyl phosphorylated protein that was first identified in immunoprecipitates of the GTPase-activating protein (GAP) of p21ras from cells transformed by oncogenic nonreceptor tyrosine kinases or stimulated through tyrosine kinase receptors (C. Ellis, M. Moran, F. McCormick, and T. Pawson, Nature 343:377-381, 1991). In this article we describe a highly related 62-kDa protein that becomes tyrosyl phosphorylated and associated with phospholipase C gamma (PLC gamma) in C3H10T1/2 cells stimulated with epidermal growth factor (EGF) or transformed by v-src. GAP-associated and PLC gamma-associated p62 comigrated in one-dimensional sodium dodecyl sulfate-polyacrylamide gel electrophoresis and exhibited nearly identical phosphotryptic peptide patterns. That the association of p62 with PLC gamma was direct and not mediated through binding of GAP-p62 to PLC gamma or to the EGF receptor (and coprecipitation of the receptor with PLC gamma) was demonstrated by (i) the inability to detect GAP in PLC gamma immunocomplexes or PLC gamma in GAP immunocomplexes, (ii) the association of p62 with PLC gamma in v-src-transformed cells in the absence of EGF stimulation, and (iii) in vitro solution binding and direct blotting of p62 with a glutathione S-transferase fusion protein containing the Src homology 2 (SH2) domains of PLC gamma. Unlike GAP, whose N-terminal SH2 mediates the interaction between GAP and p62, PLC gamma was found to require both its N- and C-terminal SH2 regions for p62 binding. These studies demonstrate that a protein identical to or highly related to GAP-associated p62 binds PLC gamma and suggest a means by which "cross-talk" between PLC gamma- and GAP-mediated signalling may occur.
Dependence of transcriptional repression on CpG methylation density.
Authors: Hsieh C L
Published: October 2015

CpG methylation is known to suppress transcription. This repression is generally thought to be related to alterations of chromatin structure that are specified by the methylation. The nature of these chromatin alterations is unknown. Moreover, it has not been clear if the methylation repression occurs in an all-or-none fashion at some critical methylation density, or if intermediate densities of methylation can give intermediate levels of repression. Here I report a stable episomal system which recapitulates many dynamic features of methylation observed in the genome. I have determined the extent of transcriptional repression as a function of four densities of CpG methylation. I find that the repression is a graded but exponential function of the CpG methylation density such that low levels of methylation yield a 67 to 90% inhibition of gene expression. Higher levels of methylation extinguished gene expression completely. Transcription from methylated minichromosomes can be increased by butyrate treatment, suggesting that histone acetylation can reverse some of the repression specified by the methylated state. Sites of preferential demethylation occurred and may have resulted from transcription factor binding or DNA looping.
CRK protein binds to two guanine nucleotide-releasing proteins for the Ras family and modulates nerve growth factor-induced activation of Ras in PC12 cells.
Authors: Matsuda M , Hashimoto Y , Muroya K , Hasegawa H , Kurata T , Tanaka S , Nakamura S , Hattori S
Published: October 2015

It has been reported that growth factors activate Ras through a complex of an adaptor type SH2-containing molecule, Grb2, and a Ras guanine nucleotide-releasing protein (GNRP), mSos. We report on the involvement of another adaptor molecule, CRK, in the activation of Ras. Overexpression of wild-type CRK proteins CRK-I and CRK-II enhanced the nerve growth factor (NGF)-induced activation of Ras in PC12 cells, although the basal level of GTP-bound active Ras was not altered. In contrast, mutants with a single amino acid substitution in either the SH2 or SH3 domain of the CRK-I protein inhibited the NGF-induced activation of Ras. Two GNRPs for the Ras family, mSos and C3G, were coimmunoprecipitated with the endogenous Crk proteins in PC12 cells. The association between C3G and the CRK mutants was dependent upon the presence of intact SH3. The SH2 domain of CRK bound to the SHC protein phosphorylated on tyrosine residues by NGF stimulation. The results demonstrate that, in addition to Grb2, CRK participates in signaling from the NGF receptor and that two GNRPs appear to transmit signals from these adaptor molecules to Ras.
The GTS1 gene, which contains a Gly-Thr repeat, affects the timing of budding and cell size of the yeast Saccharomyces cerevisiae.
Authors: Mitsui K , Yaguchi S , Tsurugi K
Published: October 2015

A gene with an open reading frame encoding a protein of 417 amino acid residues with a Gly-Thr repeat was isolated from the yeast Saccharomyces cerevisiae by using synthetic oligonucleotides encoding three Gly-Thr dimers as probes. The deduced amino acid sequence showed partial homology to the clock-affecting gene, per, of Drosophila melanogaster in the regions including the GT repeat. The function of the gene, named GTS1, was examined by characterizing the phenotypes of transformants with different copy numbers of the GTS1 gene produced either by inactivating the GTS1 gene by gene disruption (TM delta gts1) or by transformation with multicopy plasmid pPER119 (TMpGTS1). They grew at similar rates during the exponential growth phase, but the lag phases were shorter for TM delta gts1 and longer for TMpGTS1 cells than that for the wild type. Analyses of their cell cycle parameters using synchronized cells revealed that the unbudding period changed as a function of gene dosage; that is, the periods of TM delta gts1 and TMpGTS1 were about 20% shorter and longer, respectively, than that of the wild-type. Another significant change in the transformants was detected in the distribution of the cell size. The mean cell volume of the TM delta gts1 cells in the unbudded period (single cells) was 27% smaller than that of single wild-type cells, whereas that of single TMpGTS1 cells was 48% larger. Furthermore, in the temperature-sensitive cdc4 mutant, the GTS1 gene affected the timing of budding at the restrictive temperature. Thus, the GTS1 gene product appears to modulate the timing of budding to obtain an appropriate cell size independent of the DNA replication cycle.
Helicobacter cinaediknee infection after arthroscopy in an immunocompetent patient
Authors: Nielsen Hans Linde , Prag Jørgen , Krogfelt Karen Angeliki
Published: October 2015

An otherwise healthy 36-year-old man was hospitalised due to a traumatic tear of the meniscus in the left knee. An arthroscopy was performed and his meniscus was partially resected. Thirty days later, he was rehospitalised with arthritis in the left knee and cellulitis on the left tibia.Helicobacter cinaediwas isolated from the synovial fluid, which was incubated in a BACTEC Paediatric bottle. The patient was treated with oral rifampicin and moxifloxacin for 6 weeks with good clinical response without relapse. The source of the infection was not found. The case emphasises the importance of incubating the synovial fluid in a rich medium such as a BACTEC Peds Plus/F bottle. Physicians and microbiologists should be aware ofH. cinaedias a human pathogen causing a range of disease manifestations, including infective arthritis and cellulitis, particularly if symptoms evolve in the weeks following a surgical procedure.
Acute appendicitis presenting as pneumoperitoneum in a teenage boy undergoing chemotherapy
Authors: Wong C W Y , Chung P H Y , Lan L C L , Wong K K Y
Published: October 2015

Chemotherapy of paediatric haematological malignancies can induce infectious complications of the gastrointestinal tract, with predilection of the ileocaecal region. Common causes of right lower abdominal pain in the febrile patient with neutropaenia include acute appendicitis, typhlitis, ileus and intussusception. In this case report, we describe a teenage boy with acute appendicitis presenting with pneumoperitoneum during his course of chemotherapy. The incidence, aetiology, diagnosis, investigations and management of this uncommon presentation in a common disease are discussed. The controversial topic of the management of acute appendicitis in a febrile patient with neutropaenia is also reviewed and discussed.
A 75-year-old woman with acquired haemophilia disguised by warfarin treatment
Authors: Lawless Sarah , Benson Gary
Published: October 2015

Acquired haemophilia is a rare coagulation disorder more commonly seen in elderly patients. Diagnosis and effective treatment can be delayed if patients are on warfarin treatment, as the bleeding symptoms may be erroneously attributed to this agent. We present a case report of a 75-year-old woman whose unexplained, severe and persistent bleeding was treated with surgical decompression and plasma transfusions, an appropriate management based on the assumption that warfarin was the cause of the bleeding. It was only when the patient's international normalised ratio returned to normal that a persistent abnormal activated partial thromboplastin time was noted. This delayed the correct diagnosis and treatment. Awareness of acquired haemophilia as a possible cause of sudden bleeding should be encouraged, and the wider dissemination of any relevant experience of similar cases would also be welcome.
Reversed ocular ischaemic syndrome secondary to carotid cavernous sinus embolisation
Authors: Isidro Filipe , Reizinho Carla , Branco Gabriel , Costa João Marques
Published: October 2015

Ocular ischaemic syndrome is a progressive and serious vision-threatening condition that is usually associated with carotid artery disease, and poor effective therapeutic options are available. In the present case, a 65-year-old woman was submitted to embolisation of spontaneous carotid-cavernous fistula with complete resolution of initial ophthalmoplaegia and ocular hypertension. Later, ocular ischaemia was detected. Cerebral angiography showed delayed choroidal flush and the patient was admitted for hypervolaemic hypertension therapy and medicated with nimodipine. The ischaemia was reversed and visual function improved.
Tuberculous pseudoaneurysm of the ascending aorta
Authors: Gopalan Sowmya , Ramadurai Srinivasan , Nair Aiswarya Manmadhan , Arthur Preetam
Published: October 2015

Increase in the incidence of drug resistance and association with HIV has led to a resurgence of tuberculosis. However, tubercular arteritis continues to remain a rare entity with a prelidection for the thoracic aorta. We report a tubercular ascending aortic pseudoaneurysm in a patient already on treatment for disseminated tuberculosis who underwent successful surgical repair and also review literature pertaining to this entity.
Evaluation of AUSDRISK as a screening tool for lifestyle modification programs: international implications for policy and cost-effectiveness
Authors: Malo Jonathan A , Versace Vincent L , Janus Edward D , Laatikainen Tiina , Peltonen Markku , Vartiainen Erkki , Coates Michael J , Dunbar James A
Published: October 2015

Objective

To evaluate the current use of Australian Type 2 Diabetes Risk Assessment Tool (AUSDRISK) as a screening tool to identify individuals at high risk of developing type 2 diabetes for entry into lifestyle modification programs.

Research Design and Methods

AUSDRISK scores were calculated from participants aged 40–74 years in the Greater Green Triangle Risk Factor Study, a cross-sectional population survey in 3 regions of Southwest Victoria, Australia, 2004–2006. Biomedical profiles of AUSDRISK risk categories were determined along with estimates of the Victorian population included at various cut-off scores. Sensitivity, specificity, positive predictive value (PPV), negative predictive value, and receiver operating characteristics were calculated for AUSDRISK in determining fasting plasma glucose (FPG) ≥6.1 mmol/L.

Results

Increasing AUSDRISK scores were associated with an increase in weight, body mass index, FPG, and metabolic syndrome. Increasing the minimum cut-off score also increased the proportion of individuals who were obese and centrally obese, had impaired fasting glucose (IFG) and metabolic syndrome. An AUSDRISK score of ≥12 was estimated to include 39.5% of the Victorian population aged 40–74 (916 000), while a score of ≥20 would include only 5.2% of the same population (120 000). At AUSDRISK≥20, the PPV for detecting FPG≥6.1 mmol/L was 28.4%.

Conclusions

AUSDRISK is powered to predict those with IFG and undiagnosed type 2 diabetes, but its effectiveness as the sole determinant for entry into a lifestyle modification program is questionable given the large proportion of the population screened-in using the current minimum cut-off of ≥12. AUSDRISK should be used in conjunction with oral glucose tolerance testing, fasting glucose, or glycated hemoglobin to identify those individuals at highest risk of progression to type 2 diabetes, who should be the primary targets for lifestyle modification.