bluuebunny/crossref_metadata_2025 huggingface dataset:
Drosophila PLUTONIUM protein is a specialized cell cycle regulator required at the onset of embryogenesis.
Authors: Elfring L K , Axton J M , Fenger D D , Page A W , Carminati J L , Orr-Weaver T L
Published: August 2013

Unfertilized eggs and fertilized embryos from Drosophila mothers mutant for the plutonium (plu) gene contain giant polyploid nuclei resulting from unregulated S-phase. The PLU protein, a 19-kDa ankyrin repeat protein, is present in oocytes and early embryos but is not detectable after the completion of the initial rapid S-M cycles of the embryo. The persistence of the protein during the early embryonic divisions is consistent with a direct role in linking S-phase and M-phase. When ectopically expressed in the eye disc, PLU did not perturb the cell cycle, suggesting that PLU regulates S-phase only in early embryonic development. The pan gu (png) and giant nuclei (gnu) genes also affect the S-phase in the unfertilized egg and early embryo. We show that functional png is needed for the presence of PLU protein. By analyzing png mutations of differing severity, we find that the extent of the png mutant phenotype inversely reflects the level of PLU protein. Our data suggest that PLU protein is required at the time of egg activation and the completion of meiosis.
Sec3p is involved in secretion and morphogenesis in Saccharomyces cerevisiae.
Authors: Finger F P , Novick P
Published: August 2013

Two new temperature-sensitive alleles of SEC3, 1 of 10 late-acting SEC genes required for targeting or fusion of post-Golgi secretory vesicles to the plasma membrane in Saccharomyces cerevisiae, were isolated in a screen for temperature-sensitive secretory mutants that are synthetically lethal with sec4-8. The new sec3 alleles affect early as well as late stages of secretion. Cloning and sequencing of the SEC3 gene revealed that it is identical to profilin synthetic lethal 1 (PSL1). The SEC3 gene is not essential because cells depleted of Sec3p are viable although slow growing and temperature sensitive. All of the sec3 alleles genetically interact with a profilin mutation, pfy1-111. The SEC3 gene in high copy suppresses pfy1-111 and sec5-24 and causes synthetic growth defects with ypt1, sec8-9, sec10-2, and sec15-1. Actin structure is only perturbed in conditions of chronic loss of Sec3p function, implying that Sec3p does not directly regulate actin. All alleles of sec3 cause bud site selection defects in homozygous diploids, as do sec4-8 and sec9-4. This suggests that SEC gene products are involved in determining the bud site and is consistent with a role for Sec3p in determining the correct site of exocytosis.
sn-1,2-diacylglycerol and choline increase after fertilization in Xenopus laevis.
Authors: Stith B J , Woronoff K , Espinoza R , Smart T
Published: August 2013

sn-1,2-Diacylglycerol (DAG) mass and translocation of protein kinase C alpha and beta to a membrane fraction increased approximately 7 min after insemination of Xenopus laevis eggs. The DAG mass increase of 48 pmol (from 62 to 110 pmol/cell) was greater than that for inositol 1,4,5-trisphosphate (IP3; an increase of approximately 170 fmol or approximately 280-fold smaller than the DAG increase), and DAG peaks approximately 5 min after IP3. Choline mass (a measure of phosphatidyl choline-specific phospholipase D) also peaked before DAG and the choline increase (134 pmol/cell) was greater than that of DAG. There was no detectable change in phosphocholine mass (a measure of phosphatidylcholine-specific phospholipase C). During first cleavage, DAG decreased, PKC translocation was low, and choline increased and peaked (whereas published work shows an increase in IP3 mass). Artificial elevation of intracellular calcium ([Ca2+]i) increased DAG levels but prevention of the [Ca2+]i increase after fertilization did not block DAG production. Thus, sperm stimulate production of DAG and choline through [Ca2+]i-independent and [Ca2+]i-dependent paths.
Positive and negative tissue-specific signaling by a nematode epidermal growth factor receptor.
Authors: Lesa G M , Sternberg P W
Published: August 2013

The major determinants of receptor tissue tyrosine kinase (RTK) signaling specificity have been proposed to be Src homology 2 (SH2) binding sites, phosphotyrosine-containing oligopeptides in the cytoplasmic domain of the receptor. The Caenorhabditis elegans epidermal growth factor receptor homologue LET-23 has multiple functions during development and has eight potential SH2-binding sites in a region carboxyl terminal to its kinase domain. By analyzing transgenic nematodes for three distinct LET-23 functions, we show that six of eight potential sites function in vivo and that they are required for most, but not all, of LET-23 activity. A single site is necessary and sufficient to promote wild-type fertility. Three other sites activate the RAS pathway and are involved only in viability and vulval differentiation. A fifth site is promiscuous and can mediate all three LET-23 functions. An additional site mediates tissue-specific negative regulation. Putative SH2 binding sites are thus key effectors of both cell-specific and negative regulation in an intact organism. We suggest two distinct mechanisms for tissue-specific RTK-mediated signaling. A positive mechanism would promote RTK function through effectors present only in certain cell types. A negative mechanism would inhibit RTK function through tissue-specific negative regulators.
Nanomolar concentrations of nocodazole alter microtubule dynamic instability in vivo and in vitro.
Authors: Vasquez R J , Howell B , Yvon A M , Wadsworth P , Cassimeris L
Published: August 2013

Previous studies demonstrated that nanomolar concentrations of nocodazole can block cells in mitosis without net microtubule disassembly and resulted in the hypothesis that this block was due to a nocodazole-induced stabilization of microtubules. We tested this hypothesis by examining the effects of nanomolar concentrations of nocodazole on microtubule dynamic instability in interphase cells and in vitro with purified brain tubulin. Newt lung epithelial cell microtubules were visualized by video-enhanced differential interference contrast microscopy and cells were perfused with solutions of nocodazole ranging in concentration from 4 to 400 nM. Microtubules showed a loss of the two-state behavior typical of dynamic instability as evidenced by the addition of a third state where they exhibited little net change in length (a paused state). Nocodazole perfusion also resulted in slower elongation and shortening velocities, increased catastrophe, and an overall decrease in microtubule turnover. Experiments performed on BSC-1 cells that were microinjected with rhodamine-labeled tubulin, incubated in nocodazole for 1 h, and visualized by using low-light-level fluorescence microscopy showed similar results except that nocodazole-treated BSC-1 cells showed a decrease in catastrophe. To gain insight into possible mechanisms responsible for changes in dynamic instability, we examined the effects of 4 nM to 12 microM nocodazole on the assembly of purified tubulin from axoneme seeds. At both microtubule plus and minus ends, perfusion with nocodazole resulted in a dose-dependent decrease in elongation and shortening velocities, increase in pause duration and catastrophe frequency, and decrease in rescue frequency. These effects, which result in an overall decrease in microtubule turnover after nocodazole treatment, suggest that the mitotic block observed is due to a reduction in microtubule dynamic turnover. In addition, the in vitro results are similar to the effects of increasing concentrations of GDP-tubulin (TuD) subunits on microtubule assembly. Given that nocodazole increases tubulin GTPase activity, we propose that nocodazole acts by generating TuD subunits that then alter dynamic instability.
Perlecan regulates Oct-1 gene expression in vascular smooth muscle cells.
Authors: Weiser M C , Grieshaber N A , Schwartz P E , Majack R A
Published: August 2013

Vascular smooth muscle cells (SMCs) are very quiescent in the mature vessel and exhibit a remarkable phenotype-dependent diversity in gene expression that may reflect the growth responsiveness of these cells under a variety of normal and pathological conditions. In this report, we describe the expression pattern of Oct-1, a member of a family of transcription factors involved in cell growth processes, in cultured and in in vivo SMCs. Oct-1 mRNA was undetectable in the contractile-state in vivo SMCs; was induced upon disruption of in vivo SMC-extracellular matrix interactions; and was constitutively expressed by cultured SMCs. Oct-1 transcripts were repressed when cultured SMCs were plated on Engelbreth-Holm-Swarm tumor-derived basement membranes (EHS-BM) but were rapidly induced after disruption of SMC-EHS-BM contacts; reexpression was regulated at the transcriptional level. To identify the EHS-BM component involved in the active repression of Oct-1 mRNA expression, SMCs were plated on laminin, type IV collagen, fibronectin, or perlecan matrices. Oct-1 mRNA levels were readily detectable when SMCs were cultured on matrices composed of laminin, type IV collagen, or fibronectin but were repressed when SMCs were cultured on perlecan matrices. Finally, the Oct-1-suppressing activity of EHS-BM was sensitive to heparinase digestion but not to chondroitinase ABC or hyaluronidase digestion, suggesting that the heparan sulfate side chains of perlecan play a biologically important role in negatively regulating the expression of Oct-1 transcripts.
Mitochondrial transmission during mating in Saccharomyces cerevisiae is determined by mitochondrial fusion and fission and the intramitochondrial segregation of mitochondrial DNA.
Authors: Nunnari J , Marshall W F , Straight A , Murray A , Sedat J W , Walter P
Published: August 2013

To gain insight into the process of mitochondrial transmission in yeast, we directly labeled mitochondrial proteins and mitochondrial DNA (mtDNA) and observed their fate after the fusion of two cells. To this end, mitochondrial proteins in haploid cells of opposite mating type were labeled with different fluorescent dyes and observed by fluorescence microscopy after mating of the cells. Parental mitochondrial protein markers rapidly redistributed and colocalized throughout zygotes, indicating that during mating, parental mitochondria fuse and their protein contents intermix, consistent with results previously obtained with a single parentally derived protein marker. Analysis of the three-dimensional structure and dynamics of mitochondria in living cells with wide-field fluorescence microscopy indicated that mitochondria form a single dynamic network, whose continuity is maintained by a balanced frequency of fission and fusion events. Thus, the complete mixing of mitochondrial proteins can be explained by the formation of one continuous mitochondrial compartment after mating. In marked contrast to the mixing of parental mitochondrial proteins after fusion, mtDNA (labeled with the thymidine analogue 5-bromodeoxyuridine) remained distinctly localized to one half of the zygotic cell. This observation provides a direct explanation for the genetically observed nonrandom patterns of mtDNA transmission. We propose that anchoring of mtDNA within the organelle is linked to an active segregation mechanism that ensures accurate inheritance of mtDNA along with the organelle.
Syntaxin 6 functions in trans-Golgi network vesicle trafficking.
Authors: Bock J B , Klumperman J , Davanger S , Scheller R H
Published: August 2013

The specific transfer of vesicles between organelles is critical in generating and maintaining the organization of membrane compartments within cells. Syntaxin 6 is a recently discovered member of the syntaxin family, whose constituents are required components of several vesicle trafficking pathways. To better understand the function of syntaxin 6, we generated a panel of monoclonal antibodies that specifically recognize different epitopes of the protein. Immunoelectron microscopy shows syntaxin 6 primarily on the trans-Golgi network (TGN), where is partially colocalizes with the TGN adapter protein AP-1 on clathrin-coated membranes. Additional label is present on small vesicles in the vicinity of endosome-like structures. Immunoprecipitation of syntaxin 6 revealed that it is present in a complex or complexes with alpha-soluble NSF attachment protein, vesicle-associated membrane protein 2, or cellubrevin and a mammalian homologue of VPS45, which is a member of the sec1 family implicated in Golgi to prevacuolar compartment trafficking in yeast. We show that mammalian VPS45 is found in multiple tissues, is partially membrane associated, and is enriched in the Golgi region. Converging lines of evidence suggest that syntaxin 6 mediates a TGN trafficking event, perhaps targeting to endosomes in mammalian cells.
Two glucose sensing/signaling pathways stimulate glucose-induced inactivation of maltose permease in Saccharomyces.
Authors: Jiang H , Medintz I , Michels C A
Published: August 2013

Glucose is a global metabolic regulator in Saccharomyces. It controls the expression of many genes involved in carbohydrate utilization at the level of transcription, and it induces the inactivation of several enzymes by a posttranslational mechanism. SNF3, RGT2, GRR1 and RGT1 are known to be involved in glucose regulation of transcription. We tested the roles of these genes in glucose-induced inactivation of maltose permease. Our results suggest that at least two signaling pathways are used to monitor glucose levels. One pathway requires glucose sensor transcript and the second pathway is independent of glucose transport. Rgt2p, which along with Snf3p monitors extracellular glucose levels, appears to be the glucose sensor for the glucose-transport-independent pathway. Transmission of the Rgt2p-dependent signal requires Grr1p. RGT2 and GRR1 also play a role in regulating the expression of the HXT genes, which appear to be the upstream components of the glucose-transport-dependent pathway regulating maltose permease inactivation. RGT2-1, which was identified as a dominant mutation causing constitutive expression of several HXT genes, causes constitutive proteolysis of maltose permease, that is, in the absence of glucose. A model of these glucose sensing/signaling pathways is presented.
The ankyrin repeat-containing protein Akr1p is required for the endocytosis of yeast pheromone receptors.
Authors: Givan S A , Sprague G F
Published: August 2013

The Saccharomyces cerevisiae a-factor receptor (Ste3p) requires its C-terminal cytoplasmic tail for endocytosis. Wild-type receptor is delivered to the cell surface via the secretory pathway but remains there only briefly before being internalized and delivered to the vacuole for degradation. Receptors lacking all or part of the cytoplasmic tail are not subject to this constitutive endocytosis. We used the cytoplasmic tail of Ste3p as bait in the two-hybrid system in an effort to identify other proteins involved in endocytosis. One protein identified was Akr1p, an ankyrin repeat-containing protein. We applied three criteria to demonstrate that Akr1p is involved in the constitutive endocytosis of Ste3p. First, when receptor synthesis is shut off, akr1 delta cells retain the ability to mate longer than do AKR1 cells. Second, Ste3p half-life is increased by greater than 5-fold in akr1 delta cells compared with AKR1 cells. Third, after a pulse of synthesis, newly synthesized receptor remains at the cell surface in akr1 delta mutants, whereas it is rapidly internalized in AKR1 cells. Specifically, in akr1 delta mutants, newly synthesized receptor is accessible to exogenous protease, and by indirect immunofluorescence, the receptor is located at the cell surface. akr1 delta cells are also defective for endocytosis of the alpha-factor receptor (Ste2p). Despite the block to constitutive endocytosis exhibited by akr1 delta cells, they are competent to carry out ligand-mediated endocytosis of Ste3p. In contrast, akr1 delta cells cannot carry out ligand-mediated endocytosis of Ste2p. We discuss the implications for Akr1p function in endocytosis and suggest a link to the regulation of ADP-ribosylation proteins (Arf proteins).
Lysophosphatidic acid and microtubule-destabilizing agents stimulate fibronectin matrix assembly through Rho-dependent actin stress fiber formation and cell contraction.
Authors: Zhang Q , Magnusson M K , Mosher D F
Published: August 2013

Fibronectin (FN) matrix assembly is a cell-dependent process mediated by cell surface-binding sites for the 70-kDa amino-terminal region of FN. We have shown recently that lysophosphatidic acid (LPA) is a stimulator of FN matrix assembly. Disruption of microtubules has been shown to mimic some of the intracellular effects of LPA including the formation of actin stress fibers and myosin light chain phosphorylation. We compared the effects of microtubule disruption and LPA on FN binding and actin cytoskeleton organization. The disruption of microtubules by nocodazole or vinblastine increased FN binding to adherent cells. The modulation of binding sites was rapid, dynamic, and reversible. Enhanced binding was due to increases in both the number and affinity of binding sites. These effects are similar to the effects of LPA on FN binding. Binding induced by nocodazole was inhibited by the microtubule-stabilizing agent Taxol but not by pretreatment with a concentration of phospholipase B that totally abolished the stimulatory effect of LPA. Fluorescence microscopy revealed a close correlation among actin stress fiber formation, cell contraction, and FN binding. Blockage of the small GTP binding protein Rho or actin-myosin interactions inhibited the effects of both nocodazole and LPA on FN binding. These observations demonstrate that Rho-dependent actin stress fiber formation and cell contraction induce increased FN binding and represent a rapid labile way that cells can modulate FN matrix assembly.
Nuclear membrane vesicle targeting to chromatin in a Drosophila embryo cell-free system.
Authors: Ulitzur N , Harel A , Goldberg M , Feinstein N , Gruenbaum Y
Published: August 2013

A Drosophila cell-free system was used to characterize proteins that are required for targeting vesicles to chromatin and for fusion of vesicles to form nuclear envelopes. Treatment of vesicles with 1 M NaCl abolished their ability to bind to chromatin. Binding of salt-treated vesicles to chromatin could be restored by adding the dialyzed salt extract. Lamin Dm is one of the peripheral proteins whose activity was required, since supplying interphase lamin isoforms Dm1, and Dm2 to the assembly extract restored binding. As opposed to the findings in Xenopus, okadaic acid had no effect on vesicle binding. Trypsin digestion of the salt-stripped vesicles eliminated their association with chromatin even in the presence of the dialyzed salt extract. One vesicles attached to chromatin surface, fusion events took place were found to be sensitive to guanosine 5'-[gamma-thio]triphosphate (GTP gamma S). These chromatin-attached vesicles contained lamin Dm and otefin but not gp210. Thus, these results show that in Drosophila there are two populations of nuclear vesicles. The population that interacts first with chromatin contains lamin and otefin and requires both peripheral and integral membrane proteins, whereas fusion of vesicles requires GTPase activity.
Mcm2 and Mcm3 are constitutive nuclear proteins that exhibit distinct isoforms and bind chromatin during specific cell cycle stages of Saccharomyces cerevisiae.
Authors: Young M R , Tye B K
Published: August 2013

The Mcm2-7 proteins are a family of conserved proteins whose functions are essential for the initiation of DNA synthesis in all eukaryotes. These patients are constitutively present in high abundance in actively proliferating cells. In Saccharomyces cerevisiae, the intracellular concentrations of Mcms are between 100 and 500 times the number of replication origins. However, these proteins are limiting for the initiation of DNA synthesis at replication origins. Our studies indicate that only a small fraction of Mcm2 and Mcm3 tightly associates with chromatin, from late M phase to the beginning of the S phase. The rest of the Mcm2 and Mcm3 proteins are disturbed to both the cytoplasm and nucleoplasm in relatively constant levels throughout the cell cycle. We also show that S. cerevisiae Mcm3 is a phosphoprotein that exists in multiple isoforms and that distinct isoforms of Mcm2 and Mcm3 can be detected at specific stages of the cell cycle. These results suggest that the localization and function of the Mcm proteins are regulated by posttranslational phosphorylation in a manner that is consistent with a role for the Mcm proteins in restricting DNA replication to once per cell cycle.
Evidence that mating by the Saccharomyces cerevisiae gpa1Val50 mutant occurs through the default mating pathway and a suggestion of a role for ubiquitin-mediated proteolysis.
Authors: Xu B E , Kurjan J
Published: August 2013

The yeast G alpha subunit, Gpa1p, plays a negative role in the pheromone response pathway. The gpa1Val50 mutant was previously shown to have a growth defect, consistent with the GTPase defect predicted for this mutation, and greatly reduced mating. Various explanations for the mating defect have been proposed. One approach to analyze the gpa1Val50 mating defect involved epistasis analysis. The low mating of the gpa1Val50 mutant was independent of the pheromone receptor; therefore, it results from intracellular activation of the pathway, consistent with a GTPase defect. This result suggests that gpa1Val50 mating occurs through the default rather than the chemotropic pathway involved in pheromone response. We therefore tested the effect of a spa2 mutation on gpa1Val50 mating, because Spa2p has been implicated in the default pathway. The spa2 mutation greatly reduced the mating of the gpa1Val50 mutant, suggesting that gpa1Val50 mating occurs predominantly through the default pathway. In a second approach to investigate the gpa1Val50 phenotypes, suppressors of the gpa1Val50 mating defect were isolated. Two suppressor genes corresponded to SON1/UFD5 and SEN3, which are implicated in ubiquitin-mediated proteolysis. On the basis of these results, we suggest that a positive component of the default mating pathway is subject to ubiquitin-mediated degradation.
Flight muscle function in Drosophila requires colocalization of glycolytic enzymes.
Authors: Wojtas K , Slepecky N , von Kalm L , Sullivan D
Published: August 2013

Structural relationships between the myofibrillar contractile apparatus and the enzymes that generate ATP for muscle contraction are not well understood. We explored whether glycolytic enzymes are localized in Drosophila flight muscle and whether localization is required for function. We find that glycerol-3-phosphate dehydrogenase (GPDH) is localized at Z-discs and M-lines. The glycolytic enzymes aldolase and glyceraldehyde-3-phosphate dehydrogenase (GAPDH) are also localized along the sarcomere with a periodic pattern that is indistinguishable from that of GPDH localization. Furthermore, localization of aldolase and GAPDH requires simultaneous localization of GPDH, because aldolase and GAPDH are not localized along the sarcomere in muscles of strains that carry Gpdh null alleles. In an attempt to understand the process of glycolytic enzyme colocalization, we have explored in more detail the mechanism of GPDH localization. In flight muscle, there is only one GPDH isoform, GPDH-1, which is distinguished from isoforms found in other tissues by having three C-terminal amino acids: glutamine, asparagine, and leucine. Transgenic flies that can produce only GPDH-1 display enzyme colocalization similar to wild-type flies. However, transgenic flies that synthesize only GPDH-3, lacking the C-terminal tripeptide, do not show the periodic banding pattern of localization at Z-discs and M-lines for GPDH. In addition, neither GAPDH nor aldolase colocalize at Z-discs and M-lines in the sarcomeres of muscles from GPDH-3 transgenic flies. Failure of the glycolytic enzymes to colocalize in the sarcomere results in the inability to fly, even though the full complement of active glycolytic enzymes is present in flight muscles. Therefore, the presence of active enzymes in the cell is not sufficient for muscle function; colocalization of the enzymes is required. These results indicate that the mechanisms by which ATP is supplied to the myosin ATPase, for muscle contraction, requires a highly organized cellular system.
Deletion of nudC, a nuclear migration gene of Aspergillus nidulans, causes morphological and cell wall abnormalities and is lethal.
Authors: Chiu Y H , Xiang X , Dawe A L , Morris N R
Published: August 2013

Nuclear migration is required for normal development in both higher and lower eukaryotes. In fungi this process is mediated by cytoplasmic dynein. It is believed that this motor protein is anchored to the cell membrane and moves nuclei by capturing and pulling on spindle pole body microtubules. To date, four genes have been identified and shown to be required for this process in Aspergillus nidulans. The nudA and nudG genes, respectively, encode the heavy and light chains of cytoplasmic dynein, and the nudF and nudC gene products encode proteins of 49 and 22 kDa. The precise biochemical functions of the nudF and nudC genes have not yet been identified. In this report we further investigate NUDC protein function by deleting the nudC gene. Surprisingly, although deletion of nudA and nudF affect nuclear migration, deletion of nudC profoundly affected the morphology and composition of the cell wall. Spores of the strain deleted for nudC grew spherically and lysed. The thickness of the cell wall was increased in the deletion mutant and wall polymer composition was abnormal. This phenotype could be repressed by growth on osmotically buffered medium at low temperature. Similar, but less severe, effects were also noted in a strain depleted for NUDC by down-regulation. These results suggest a possible relationship between fungal cell wall biosynthesis and nuclear migration.
The unfolded protein response coordinates the production of endoplasmic reticulum protein and endoplasmic reticulum membrane.
Authors: Cox J S , Chapman R E , Walter P
Published: August 2013

The endoplasmic reticulum (ER) is a multifunctional organelle responsible for production of both lumenal and membrane components of secretory pathway compartments. Secretory proteins are folded, processed, and sorted in the ER lumen and lipid synthesis occurs on the ER membrane itself. In the yeast Saccharomyces cerevisiae, synthesis of ER components is highly regulated: the ER-resident proteins by the unfolded protein response and membrane lipid synthesis by the inositol response. We demonstrate that these two responses are intimately linked, forming different branches of the same pathway. Furthermore, we present evidence indicating that this coordinate regulation plays a role in ER biogenesis.
p27Kip1 induces an accumulation of the repressor complexes of E2F and inhibits expression of the E2F-regulated genes.
Authors: Shiyanov P , Hayes S , Chen N , Pestov D G , Lau L F , Raychaudhuri P
Published: August 2013

p27Kip1 is an inhibitor of the cyclin-dependent kinases and it plays an inhibitory role in the progression of cell cycle through G1 phase. To investigate the mechanism of cell cycle inhibition by p27Kip1, we constructed a cell line that inducibly expresses p27Kip1 upon addition of isopropyl-1-thio-beta-D-galactopyranoside in the culture medium. Isopropyl-1-thio-beta-D-galactopyranoside-induced expression of p27Kip1 in these cells causes a specific reduction in the expression of the E2F-regulated genes such as cyclin E, cyclin A, and dihydrofolate reductase. The reduction in the expression of these genes correlates with the p27Kip1-induced accumulation of the repressor complexes of the E2F family of factors (E2Fs). Our previous studies indicated that p21WAF1 could disrupt the interaction between cyclin/cyclin-dependent kinase 2 (cdk2) and the E2F repressor complexes E2F-p130 and E2F-p107. We show that p27Kip1, like p21WAF1, disrupts cyclin/cdk2-containing complexes of E2F-p130 leading to the accumulation of the E2F-p130 complexes, which is found in growth-arrested cells. In transient transfection assays, expression of p27Kip1 specifically inhibits transcription of a promoter containing E2F-binding sites. Mutants of p27Kip1 harboring changes in the cyclin- and cdk2-binding motifs are deficient in inhibiting transcription from the E2F sites containing reporter gene. Moreover, these mutants of p27Kip1 are also impaired in disrupting the interaction between cyclin/cdk2 and the repressor complexes of E2Fs. Taken together, these observations suggest that p27Kip1 reduces expression of the E2F-regulated genes by generating repressor complexes of E2Fs. Furthermore, the results also demonstrate that p27Kip1 inhibits expression of cyclin A and cyclin E, which are critical for progression through the G1-S phases.
N-Linked glycosylation is essential for the yellow head virus replication cycle
Authors: Soowannayan Chumporn , Chanarpakorn Nhuengtida , Phanthura Mongkhol , Deekhlai Nattawoot , Kunasol Chanon , Sriurairatana Siriporn
Published: August 2013

Yellow head virus (YHV) particles contain a nucleocapsid protein (p20) and two envelope glycoproteins (gp116 and gp64). The glycans attached to the two glycoproteins areN-linked and are complex and high mannose types, respectively. Here, we show that treatment with theN-linked glycosylation inhibitor tunicamycin in YHV-infected black tiger shrimp (Penaeus monodon) resulted in less severe yellow head disease and reduced mortality when compared with untreated control shrimp. Quantitative real-time reverse transcription PCR analysis also revealed lower YHV copy numbers in the haemolymph of treated than control shrimp. This was concurrent with less intense immuno-reactions in tissues of treated versus untreated shrimp using mAbs against all three YHV structural proteins. In addition, transmission electron microscopy of lymphoid organ tissue of the treated and untreated shrimp [eight collected at 36 h and eight at 48 h post-infection (p.i.)] revealed only unenveloped nucleocapsids in all but one of the treated shrimp (collected at 48 h p.i.). By contrast, all the untreated shrimp showed a mixture of many unenveloped and enveloped virions. These results were supported by purification of YHV from the cell-free haemolymph of treated and untreated shrimp followed by YHV structural protein analysis by SDS-PAGE. It revealed three expected structural protein bands (116, 64 and 20 kDa) from the untreated shrimp but no structural protein bands from the tunicamycin-treated shrimp (confirmed by Western blot analysis). Overall, the results indicated that blocking glycosylation with tunicamycin inhibited the formation of mature YHV virions and their subsequent release into shrimp haemolymph, reducing the severity of disease.
H7N9 influenza viruses interact preferentially with α2,3-linked sialic acids and bind weakly to α2,6-linked sialic acids
Authors: Ramos Irene , Krammer Florian , Hai Rong , Aguilera Domingo , Bernal-Rubio Dabeiba , Steel John , García-Sastre Adolfo , Fernandez-Sesma Ana
Published: August 2013

The recent human outbreak of H7N9 avian influenza A virus has caused worldwide concerns. Receptor binding specificity is critical for viral pathogenicity, and still not thoroughly studied for this emerging virus. Here, we evaluated the receptor specificity of the haemagglutinin (HA) of two human H7N9 isolates (A/Shanghai/1/13 and A/Anhui/1/13) through a solid-phase binding assay and a flow cytometry-based assay. In addition, we compared it with those from several HAs from human and avian influenza viruses. We observed that the HAs from the novel H7 isolates strongly interacted with α2,3-linked sialic acids. Importantly, they also showed low levels of binding to α2,6-linked sialic acids, but significantly higher than other avian H7s.
Sensory preconditioning in newborn rabbits: from common to distinct odor memories
Authors: Coureaud Gérard , Tourat Audrey , Ferreira Guillaume
Published: August 2013

This study evaluated whether olfactory preconditioning is functional in newborn rabbits and based on joined or independent memory of odorants. First, after exposure to odorants A+B, the conditioning of A led to high responsiveness to odorant B. Second, responsiveness to B persisted after amnesia of A. Third, preconditioning was also functional with two overlapping pairs of odorants (A+B and B+C) and amnesia of one odorant did not affect memory of the others. Thus, incidental pairing of odorants allows reinforcement of one odorant to implicitly reinforce the others, the bond then vanishes, and the memory of each element becomes independent.
Memory retrieval before or after extinction reduces recovery of fear in adolescent rats
Authors: Baker Kathryn D. , McNally Gavan P. , Richardson Rick
Published: August 2013

Adolescent rats exhibit impaired extinction retention compared to pre-adolescent and adult rats. A single nonreinforced exposure to the conditioned stimulus (CS; a retrieval trial) given shortly before extinction has been shown in some circumstances to reduce the recovery of fear after extinction in adult animals. This study investigated whether a retrieval–extinction procedure would reduce the recovery of extinguished fear in adolescent rats. Furthermore, the effect of the retrieval–extinction sequence on fear recovery was examined by presenting the retrieval trial following extinction to some animals. In Experiment 1 adolescent rats received one nonreinforced CS presentation (a retrieval trial) or equivalent context exposure (no retrieval) 10 min before fear extinction. A retrieval trial shortly before extinction reduced overall levels of fear in both test contexts (i.e., it improved extinction retention and reduced renewal). In Experiment 2 a weakening of renewal was observed with a retrieval–extinction manipulation, regardless of whether the retrieval trial occurred in the training or extinction context. A key result was that a retrieval trial 10 min, but not 6 h,afterextinction led to reduced overall levels of fear similar to that observed if the retrieval trial was given before extinction (Experiments 3 and 4), inconsistent with the current interpretation of the reduction in relapse being due to a disruption of reconsolidation. Together, these findings show that the impaired extinction retention observed in adolescents can be ameliorated by a very simple behavioral manipulation, but also raise some questions about the mechanisms underlying the retrieval–extinction effect.
Be good for love or for money? The roles of justice in the Chinese hotel industry
Authors: Hon Alice H.Y. , Lu Lin
Published: August 2013

Purpose

Drawing on the social exchange theory, the purpose of this paper is to examine the relationship between the interactional justice of supervisors and the job performance of subordinates in an expatriate context. Specifically, the authors take a relational approach by introducing help intention directed to the supervisor (upward help intention) as the mediator. The moderating role of distributive justice in the relationship between upward help intention and job performance is also considered.

Design/methodology/approach

A survey was conducted among 232 service employees and their expatriate supervisors in multinational hotels in China. Hierarchical multiple regression analysis was conducted to test the proposed model.

Findings

The statistical results of this study supported the positive effect of interactional justice on job performance as well as the mediating effect of upward help intention. Meanwhile, it was also found that when the level of distributive justice is high, the relationship between upward help intention and job performance is strengthened, whereas the effect is leveled off when the level of distributive justice is low.

Practical implications

The findings of this study suggest that managers in the service industry should be sensitive in treating their subordinates, as it will lead to positive interpersonal relationship, which in turn, will stimulate job performance. Moreover, the fair organizational compensation system aids the managers in turning good interpersonal results into positive performance outcomes.

Originality/value

This study contributes to the literature on justice by revealing the relational mechanism between interactional justice and job performance, and the economic boundary of this relationship.
Constructing post‐PhD careers: negotiating opportunities and personal goals
Authors: McAlpine Lynn , Amundsen Cheryl , Turner Gill
Published: August 2013

Purpose

Until relatively recently, the doctorate was generally perceived as preparation for a full‐time permanent academic position. However, this is no longer the case, with many PhD graduates working outside academia or in temporary full‐ and part‐time positions in higher education institutions. Yet, we know little of the ways in which they perceive and then navigate the transition from PhD to initial careers. Thus authors undertook an analysis of longitudinal data from six social sciences PhDs (part of a larger dataset) to document how they transitioned from the PhD and navigated a future.

Design/methodology/approach

Different forms of data, collected multiple times over two years, were analysed using emergent coding to capture the experiences of navigating a future.

Findings

The results enrich present understanding of this end‐of‐PhD period, in particular, highlighting individuals' growing understanding of academic, hybrid and non‐academic career opportunity structures, and the importance of personal intentions and relationships in defining possible horizons for action.

Originality/value

The conceptual and pedagogical contributions of this study to understanding doctoral and post‐doctoral career decision‐making are described.
The accelerated internationalization of born global firms: a knowledge transformation process view
Authors: Ying Huang Hsiu , Huei Hsieh Ming
Published: August 2013

Purpose

This study aims to employ a knowledge‐based capability approach to explore how born global firms accelerate their internationalization.

Design/methodology/approach

The paper adopts a qualitative in‐depth case study approach. Various types and sources of data were collected to provide a rich and solid foundation for theory development and data triangulation. These comprised semi‐structured interviews, field observations and archival material.

Findings

The findings show that the transformation process of international knowledge may more adequately account for the difference in internationalization at conventional globals and born globals than the “indigenous” knowledge resource, i.e. founder experience. Born global firms accelerate their internationalization process through a three‐stage transformation process: knowledge acquisition and dissemination, knowledge integration, and knowledge institutionalization. Two underlying knowledge mechanisms are identified: vertical and horizontal knowledge thrust.

Originality/value

The knowledge transformation process constitutes the basis of “acceleration” of born globals' internationalization. The speed of a born global's internationalization is rooted in its ability to transform internationalization knowledge resident in individual employees into organizational strategies and operational procedures. The finding bridges the gap by identifying effective organizational mechanisms in management of the knowledge process. The “speed‐up” mechanism identified in this research can equip born globals as well as conventional globals with the ability to respond quickly in a dynamic international environment.
Do multinational corporations flexibly respond to exchange rate fluctuations? A tale of two Korean MNCs
Authors: Park Young‐Ryeol , Song Sangcheol , Rhee Eun‐kyoung
Published: August 2013

Purpose

The purpose of this paper is to examine whether Korean multinational corporations (MNCs) in the electronics and steel industries do shift their production across their foreign subsidiaries, located in different countries, as exchange rates fluctuate in foreign countries.

Design/methodology/approach

A case study was taken as a qualitative methodology to examine whether MNCs actually shift their production as multinational operational flexibility perspective predicts.

Findings

From a case study of two Korean MNCs (LG Electronics and POSCO), it was found that even facing heightened production costs associated with host country currency appreciation, Korean MNCs do not shift their production to less costly locations due to industrial characteristics, limited capacity, and high tariff barriers. It was also found that they reduce the production costs internally and they also negotiate the costs with employees and suppliers to adjust the production costs associated with appreciated currency.

Practical implications

Our findings imply that certain industrial and environmental constraints make it difficult for MNCs to take flexible actions as multinational operational flexibility perspective predicts. The findings also shed additional light on the less‐explored argument over operational flexibility and vertical integration associated with cross‐country shifts of value chain activities, including production or sales.

Originality/value

Almost all literature taking the multinational operation flexibility view argues that MNCs are able to shift their productions for their own benefits. However, the authors of this paper find from their case studies that firms take advantage of other methods than production shifts in their responses to exchange rate fluctuations in their host countries. Thus this study gives an insight into when and how firms behave as the theory predicts.
A maintenance maturity assessment method for the manufacturing industry
Authors: Macchi Marco , Fumagalli Luca
Published: August 2013

Purpose

The paper proposes a maturity assessment method to measure the state of maintenance practices in a company.

Design/methodology/approach

The method assumes that a maintenance department is evaluated in terms of its managerial, organizational and technological capabilities. By its adoption it is possible to analyse the maturity level reached by a company, in order to classify the criticalities in its maintenance processes; a company can also make a benchmark with the best companies of a reference sample.

Findings

The paper presents the method as a support to identify the levers to improve the maintenance management system. The method is demonstrated on a company whose maturity is assessed before making a benchmark against a sample of other manufacturing companies located in the Northern Italy.

Originality/value

The paper presents a scoring method for maturity assessment and a procedure to use it in order to identify the criticalities in maintenance processes and to subsequently drive the improvement of the maintenance management system. The paper should be useful both to researchers and maintenance professionals interested in using new methods for long‐term planning in maintenance.
Double loop learning in the Indian two‐wheeler service sector
Authors: Bagodi Virupaxi , Mahanty Biswajit
Published: August 2013

Purpose

The purpose of this paper is to explore the rationale for the lack of growth in the two‐wheeler service sector despite an exponential growth in the number of two‐wheelers on Indian roads.

Design/methodology/approach

The data of a premier manufacturer were gathered to develop a system dynamics model. Policy experimentation was carried out after validating the model.

Findings

The results corroborate that a mere increase in the number of facilities is inadequate for sustainable growth. Improving service quality is vital for sustainable growth of the service sector.

Practical implications

The system dynamics model unveils facts that otherwise would remain obscured. It is very useful for real‐world managers in carrying out policy experimentation to understand the effect of one variable on the other. Managers can design effective policies only through double‐loop learning.

Originality/value

The complexity of the system increases as the business grows. Managers need to adopt double‐loop learning to achieve true growth in capacity, revenue, and the bottom line. The paper demonstrates the essence of double‐loop learning in the two‐wheeler service sector.
Microbial analysis of cucumbers (Cucumis sativus) produced with tap or treated waste water
Authors: Mothershaw A.S. , Kharousi Z.A. , Mumtaz Khan M. , Al‐Mahruki Y.A. , Said F.A. Al
Published: August 2013

There is increasing evidence for a significant role of fruits and vegetables in infectious diseases in humans. Their consumption is increasing and environmental factors such as water availability are impacting their production. In this study, adding fertiliser to tap water (TW) increased the microbial load above that found in treated waste water (TWW); coliforms were also introduced. Low numbers of Bacillus spp. were recovered from inside some healthy cucumber fruits. No visible differences were observed between cucumber plants irrigated with TWW or TW or cucumbers with and without endophytic Bacillus spp. This is noteworthy when considering the use of TWW for crop irrigation.
Homeostatic and injury‐induced microglia behavior in the aging brain
Authors: Hefendehl Jasmin K. , Neher Jonas J. , Sühs Rafael B. , Kohsaka Shinichi , Skodras Angelos , Jucker Mathias
Published: August 2013

Microglia cells are essential for brain homeostasis and have essential roles in neurodegenerative diseases. Aging is the main risk factor for most neurodegenerative diseases, and age‐related changes in microglia may contribute to the susceptibility of the aging brain to dysfunction and neurodegeneration. We have analyzed morphology and dynamic behavior of neocortical microglia in their physiological environment in young adult (3‐month‐old), adult (11‐ to 12‐month‐old), and aged (26‐ to 27‐month‐old) C57BL/6J‐Iba1‐eGFP mice using in vivo 2‐photon microscopy. Results show that surveying microglial cells in the neocortex exhibit age‐related soma volume increase, shortening of processes, and loss of homogeneous tissue distribution. Furthermore, microglial process speed significantly decreased with age. While only a small population of microglia showed soma movement in adult mice, the microglia population with soma movement was increased in aged mice. However, in response to tissue injury, the dynamic microglial response was age‐dependently diminished. These results provide novel insights into microglial behavior and indicate that microglial dysfunction in the aging brain may contribute to age‐related cognitive decline and neurodegenerative diseases.
Why Do Firms Choose Refunds to Shareholders Through Capital Reduction?
Authors: Shih Nien‐Su , Yang Mei‐Ling
Published: August 2013

In recent years, some firms have chosen capital reduction as a payout tool for returning cash to shareholders, and these firms have also engaged in large‐scale payouts, more than in dividends and stock repurchases. This study investigates the economic motives causing firms to adjust firm payout policies towards a distribution of cash through capital reduction. Using a logit model, we show that cash‐flow uncertainty, life cycle theory, insider interests, increasing EPS and government regulation could account for capital reduction payouts. The results for Taiwan reveal positive associations between capital reduction choice and earned/contributed capital mix and insider ownership, and negative associations with cash‐flow uncertainty and the growth rate of EPS. The findings suggest that in Singapore, firms with lower cash‐flow uncertainty and lower business freedom are significantly more likely to use capital reduction payouts.