bluuebunny/crossref_metadata_2025_split huggingface dataset:
Critical role of Arg433 in rat transketolase activity as probed by site-directed mutagenesis
Authors: SOH Yunjo , SONG Byoung J. , JENG Jiingjau , KALLARAKAL Abraham T. 
Published: August 2015

It has been shown that one arginine per monomer at an unknown position is essential for enzyme activity of the homodimeric transketolase (TK) [Kremer, Egan and Sable (1980) J. Biol. Chem. 255, 2405–2410]. To identify the critical arginine, four highly conserved arginine residues of rat TK (Arg102, Arg350, Arg433 and Arg506) were replaced with alanine by site-directed mutagenesis. Wild-type and mutant TK proteins were produced in Escherichia coli and characterized. The Arg102 → Ala mutant exhibited similar catalytic activity to the wild-type enzyme, whereas Arg350 → Ala, Arg506 → Ala and Arg433 → Ala mutants exhibited 36.7, 37.0 and 6.1% of the wild-type activity respectively. Three recombinant proteins (wild-type, Arg350 → Ala and Arg433 → Ala) were purified to apparent homogeneity using Ni2+-affinity chromatography and further characterized. All these proteins were able to form homodimers (148 kDa), as shown by immunoblot analysis subsequent to non-denaturing gel electrophoresis. The Arg433 → Ala mutant protein was less stable than the wild-type and Arg350 → Ala proteins at 55 °C. Kinetic analyses revealed that both Vmax and Km values were markedly affected in the Arg433 → Ala mutant. The Km values for two substrates xylulose 5-phosphate and ribose 5-phosphate were 11.5- and 24.3-fold higher respectively. The kcat/Km values of the Arg433 → Ala mutant for the two substrates were less than 1% of those of the wild-type protein. Molecular modelling of the rat TK revealed that Arg433 of one monomer has three potential hydrogen-bond interactions with the catalytically important highly conserved loop of the other monomer. Thus, our biochemical analyses and modelling data suggest the critical role of the previously uncharacterized Arg433 in TK activity.
Multiple transcripts coding for the Menkes gene: evidence for alternative splicing of Menkes mRNA
Authors: REDDY Manchi C. M. , HARRIS Edward D. 
Published: August 2015

We isolated cDNA fragments from four human cell lines that had sequences for the Menkes Cu-transporting ATPase (ATP7A). Primers designed to generate a 4.8 kb cDNA with the complete open reading frame generated a 1.9 kb cDNA in addition to the expected 4.8 kb product. Sequence analysis revealed that the 1.9 kb cDNA encoded one of the six Cu-binding sites and two of the eight transmembrane domains of ATP7A. Stop and start codons were also present. More striking, however, was an unusual union between exons 2 and 16 that retained an in-frame reference to exon 23. The 1.9 kb cDNA thus appeared to be a truncated Menkes mRNA that coded for an ATP7A variant that lacked exons 3–15. A 530 bp probe specific for exon 23 that avoided sequences in the exon 3–15 region hybridized to a 5.5 kb band on Northern blot analysis. Western blotting provided immunochemical evidence for the presence of both a 170 kDa and a 57 kDa protein with ATP7A sequences in detergent extracts of Caco-2 and induced BeWo cells. Extracts from non-induced BeWo cells, which lack the capacity to express the Menkes gene (MNK), showed neither protein. In a cell-free reticulocyte lysate, a plasmid containing the 1.9 kb cDNA insert directed the synthesis of a 59 kDa protein with antigenic properties of ATP7A. These studies provide evidence that non-Menkes cells have the capacity to synthesize more than one MNK mRNA. The one characterized in this report codes for a 57–59 kDa protein that lacks the core structure of the ATP7A protein. The smaller variant could be an alternative spliced form of MNK mRNA.
Isolation and characterization of a processive DNA helicase from the fission yeast Schizosaccharomyces pombe that translocates in a 5′-to-3′ direction
Authors: LEE Changwoo , SEO Yeon-Soo 
Published: August 2015

We report here the isolation and characterization of a novel DNA helicase from extracts of the fission yeast Schizosaccharomyces pombe. The enzyme, called DNA helicase II, also contains an intrinsic DNA-dependent ATPase activity. Both the helicase and ATPase activities co-purified with a 63 kDa polypeptide on an SDS/polyacrylamide gel. The protein has a sedimentation coefficient of 4.8 S and a Stokes radius of 36 Å (3.6 nm); from these data the native molecular mass was calculated to be 65 kDa. The enzyme translocates in a 5´-to-3´ direction with respect to the substrate strand to which it is bound. Unwinding reactions carried out in the presence of increasing enzyme showed a sigmoidal curve, suggesting either co-operative interactions between monomers or multimerization of DNA helicase II in the presence of single-stranded DNA and/or ATP. This enzyme favoured adenosine nucleotides (ATP and dATP) as its energy source, but utilized to limited extents GTP, CTP, dGTP and dCTP. Non-hydrolysable ATP analogues did not support helicase activity. Kinetic analyses showed that the unwinding reaction was rapid, being complete after 50–100 s of incubation. Addition of unlabelled substrates to the helicase reaction after preincubation of the enzyme with substrate did not significantly diminish unwinding. The ATPase activity of DNA helicase II increased proportionally with increasing lengths of single-stranded DNA cofactor. In the presence of circular DNA, ATP hydrolysis continued to increase up to the longest time tested (3 h), whereas it ceased to increase after 5–10 min in the presence of shorter oligonucleotides. The initial rate of ATP hydrolysis during the first 5 min of incubation time was not affected by DNA species used. These data indicate that the enzyme does not dissociate from the single-stranded DNA once it is bound and is therefore highly processive.
Cell-cycle-dependent changes in ceramide levels preceding retinoblastoma protein dephosphorylation in G2/M
Authors: LEE Joanna Y. , LEONHARDT Lisa G. , OBEID Lina M. 
Published: August 2015

Ceramide functions as a growth-inhibitory lipid-signalling molecule and might have a role in mediating the effects of extracellular agents on cell growth, differentiation and senescence. Here we investigate the roles of ceramide in cell cycle progression. With the use of the model of serum withdrawal, we were able to synchronize Wi-38 human diploid fibroblasts at different stages of cell cycle. Serum stimulation resulted in G0 to G1/S progression as determined by flow cytometric analysis and [3H]thymidine incorporation. Analyses of endogenous ceramide levels demonstrated that ceramide levels remained relatively constant on serum stimulation, indicating that ceramide might not be critical during G1/S transition. Treating exponentially growing Wi-38 human diploid fibroblasts with nocodazole led to cell cycle arrest at the G2/M phase of the cell cycle; 2 h after the removal of nocodazole, retinoblastoma (Rb) protein became dephosphorylated and the cells exited from G2/M and moved to the G1 phase of the new cycle. When cells were released from G2/M block by nocodazole, and before Rb protein dephosphorylation, endogenous ceramide levels transiently increased up to 2-fold at 0.5 h after the removal of nocodazole. Fumonisin B1, an inhibitor of ceramide synthase, inhibited the elevation of ceramide levels. Desipramine and SR33557, both acid sphingomyelinase inhibitors, did not have an appreciable effect on the elevation of ceramide levels. Furthermore, fumonisin B1 inhibited Rb protein dephosphorylation induced by endogenous ceramide but not by exogenous ceramide. These results demonstrate for the first time changes in ceramide during cell cycle progression and suggest that ceramide synthesized de novo might function as an endogenous modulator of Rb protein and cell cycle progression.
ATP-dependent transport of reduced glutathione in yeast secretory vesicles
Authors: REBBEOR James F. , CONNOLLY Gregory C. , DUMONT Mark E. , BALLATORI Nazzareno 
Published: August 2015

Turnover of cellular reduced glutathione (GSH) is accomplished predominantly by export into the extracellular space; however, the plasma membrane transport mechanisms that mediate GSH efflux are not well characterized. The present study examined GSH transport using secretory vesicles isolated from the sec6-4 mutant strain of Saccharomyces cerevisiae. In contrast with studies in mammalian membrane vesicles, GSH transport in yeast secretory vesicles was mediated largely by an ATP-dependent, low-affinity pathway (Km 19±5 mM). ATP-dependent [3H]GSH transport was cis-inhibited by substrates of the yeast YCF1 transporter, including sulphobromophthalein, glutathione S-conjugates and the alkaloid verapamil, and was competitively inhibited by S-(2,4-dinitrophenyl)glutathione (DNP-SG). Similarly, GSH competitively inhibited ATP-dependent [3H]DNP-SG transport, with a Ki of 18±2 mM, but had no effect on ATP-dependent [3H]taurocholate transport. ATP-dependent GSH transport was not affected by either membrane potential or pH-gradient uncouplers, but was inhibited by 4,4´-di-isothiocyanatostilbene-2,2´-disulphonate, probenecid and sulphinpyrazone, which are inhibitors of mrp1 and mrp2, mammalian homologues of the yeast YCF1 transporter. Western blot analysis of the secretory vesicle membrane fraction confirmed the presence of Ycf1p. These results provide the first direct evidence for low-affinity, ATP-dependent transport of GSH, and demonstrate that this ATP-dependent pathway displays kinetic characteristics similar to those of the yeast YCF1 transporter.
Characterization of p96h2bk: immunoreaction with an anti-Erk(extracellular-signal-regulated kinase) peptide antibody and activity in Xenopus oocytes and eggs
Authors: CHEN Dong-Hua , CHEN Chin-Tin , ZHANG Yong , LIU Mei-Ann , CAMPOS-GONZALEZ Roberto , PAN Bin-Tao 
Published: August 2015

We have shown previously that oncogenic Ras induces cell cycle arrest in activated Xenopus egg extracts [Pan, Chen and Lin (1994) J. Biol. Chem. 269, 5968–5975]. The cell cycle arrest correlates with the stimulation of a protein kinase activity that phosphorylates histone H2b in vitro (designated p96h2bk) [Chen and Pan (1994) J. Biol. Chem. 269, 28034–28043]. We report here that p96h2bk is likely to be p96ram, a protein of approx. 96 kDa that immunoreacts with a monoclonal antibody (Mk-1) raised against a synthetic peptide derived from a sequence highly conserved in Erk1/Erk2 (where Erk is extracellular-signal-regulated kinase). This is supported by two lines of evidence. First, activation/inactivation of p96h2bk correlates with upward/downward bandshifts of p96ram in polyacrylamide gels. Secondly, both p96h2bk and p96ram can be immunoprecipitated by antibody Mk-1. We also studied the activity of p96h2bk/p96ram in Xenopus oocytes and eggs. p96h2bk/p96ram was inactive in stage 6 oocytes, was active in unfertilized eggs, and became inactive again in eggs after fertilization. Since stage 6 oocytes are at G2-phase of the cell cycle, unfertilized eggs arrest at M-phase and eggs exit M-phase arrest after fertilization, the results thus indicate that p96h2bk/p96ram activity is cell cycle dependent. Moreover, microinjection of oncogenic Ras into fertilized eggs at the one-cell stage arrests the embryos at the two-cell stage, and this induced arrest is correlated with an inappropriate activation of p96h2bk/p96ram. The data are consistent with the concept that inappropriate activation of p96h2bk/p96ram plays a role in the cell cycle arrest induced by oncogenic Ras.
Phosphatidylinositol 4-kinase, but not phosphatidylinositol 3-kinase, is present in GLUT4-containing vesicles isolated from rat skeletal muscle
Authors: KRISTIANSEN S⊘ren , RAMLAL Toolsie , KLIP Amira 
Published: August 2015

Insulin stimulates the rate of glucose transport into muscle and adipose cells by translocation of glucose transporter (GLUT4)-containing vesicles from an intracellular storage pool to the surface membrane. This event is mediated through the insulin receptor substrates (IRSs), which in turn activate phosphatidylinositol (PI) 3-kinase isoforms. It has been suggested that insulin causes attachment of PI 3-kinases to the intracellular GLUT4-containing vesicles in rat adipose cells. Furthermore, it has also been shown that GLUT4-containing vesicles in adipose cells contain a PI 4-kinase. In the present study we investigate whether GLUT4-containing vesicles isolated from rat skeletal muscle display PI 3-kinase and/or PI 4-kinase activities. Insulin stimulation caused a rapid increase (5–15-fold increase compared with control) in the intracellular cytosolic IRS-1-associated PI-3 kinase activity. This PI 3-kinase activity was also present in a membrane preparation containing the insulin-regulatable pool of GLUT4 transporters. However, when GLUT4-containing vesicles were isolated by immunoprecipitation from basal and insulin-stimulated (3 min) skeletal muscle, the vesicles displayed PI 4-kinase, but not PI 3-kinase, activity. Insulin did not regulate the PI 4-kinase activity in the GLUT4-containing vesicles. In conclusion, GLUT4-containing vesicles from rat skeletal muscle contain a PI 4-kinase, but not a PI 3-kinase. It is suggested that, in skeletal muscle, insulin causes activation of the IRS/PI 3-kinase complex in an intracellular membrane compartment associated closely with the GLUT4-containing vesicles, but not in the GLUT4-containing vesicles themselves.
Regulation of protein-synthesis elongation-factor-2 kinase by cAMP in adipocytes
Authors: DIGGLE Tricia A. , REDPATH Nicholas T. , HEESOM Kate J. , DENTON Richard M. 
Published: August 2015

Treatment of primary rat epididymal adipocytes or 3T3-L1 adipocytes with various agents which increase cAMP led to the phosphorylation of eukaryotic translation elongation factor-2 (eEF-2). The increase in eEF-2 phosphorylation was a consequence of the activation of eEF-2 kinase (eEF-2K), which is a Ca2+/calmodulin-dependent kinase. eEF-2K was shown to be essentially inactive at less than 0.1 µM free Ca2+ when measured in cell-free extracts. Treatment of adipocytes with isoproterenol induced Ca2+-independent eEF-2K activity, and an 8–10-fold activation of eEF-2K was observed at Ca2+ concentrations of less than 0.1 µM. Increased cAMP in 3T3-L1 adipocytes led to the inhibition of total protein synthesis and decreased the rate of polypeptide-chain elongation. We also show that the phosphorylation of eEF-2 and the activity of eEF-2K are insulin-regulated in adipocytes. These results demonstrate a novel mechanism for the control of protein synthesis by hormones which act by increasing cytoplasmic cAMP.
Prolonged activation of the mitogen-activated protein kinase pathway promotes DNA synthesis in primary hepatocytes from p21Cip-1/WAF1-null mice, but not in hepatocytes from p16INK4a-null mice
Authors: AUER Kelly L. , PARK Jong-Sung , SETH Prem , COFFEY Robert J. , DARLINGTON Gretchen , ABO Arie , McMAHON Martin , DePINHO Ronald A. , FISHER Paul B. , DENT Paul 
Published: August 2015

In primary rat hepatocytes, prolonged activation of the p42/44 mitogen-activated protein kinase (MAPK) pathway is associated with a decrease in DNA synthesis and increased expression of the cyclin-dependent kinase inhibitor (CKI) proteins p21Cip-1/WAF1 and p16INK4a. To evaluate the relative importance of these CKIs in mediating this response, we determined the impact of prolonged MAPK activation on DNA synthesis in primary cultures of hepatocytes derived from mice embryonically deleted (null) for either p21Cip-1/WAF1 or p16INK4a. When MAPK was activated in wild-type mouse hepatocytes for 24 h, via infection with a construct to express an inducible oestrogen receptor–Raf-1 fusion protein (ΔRaf:ER), the expression of p21Cip-1/WAF1 and p16INK4a CKI proteins increased, cyclin-dependent kinase 2 (cdk2) and cdk4 activities decreased, and DNA synthesis decreased. Inhibition of RhoA GTPase function increased the basal expression of p21Cip-1/WAF1 and p27Kip-1 but not p16INK4a, and enhanced the ability of MAPK signalling to decrease DNA synthesis. Ablation of the expression of CCAATT enhancer-binding protein α (C/EBPα), but not of the expression of C/EBPβ, decreased the ability of MAPK signalling to induce p21Cip-1/WAF1. When MAPK was activated in p16INK4a-null hepatocytes for 24 h, the expression of p21Cip-1/WAF1 increased, cdk2 and cdk4 activities decreased and DNA synthesis decreased. In contrast with these findings, prolonged activation of the MAPK pathway in hepatocytes from p21Cip-1/WAF1-null mice enhanced cdk2 and cdk4 activities and caused a large increase in DNA synthesis, despite elevated expression of p16INK4a. Inhibition of RhoA GTPase activity in p21Cip-1/WAF1-null cells partly blunted both the basal levels of DNA synthesis and the ability of prolonged MAPK signalling to increase DNA synthesis. Expression of anti-sense p21Cip-1/WAF1 in either wild-type or p16INK4a-null hepatocytes decreased the ability of prolonged MAPK signalling to increase the expression of p21Cip-1/WAF1, and permitted MAPK signalling to increase both cdk2 and cdk4 activities and DNA synthesis. These results argue that the ability of prolonged MAPK signalling to inhibit DNA synthesis in hepatocytes requires the expression of p21Cip-1/WAF1, and that the increased expression of p16INK4a has a smaller role in the ability of this stimulus to mediate growth arrest. Our results also suggest that RhoA function can modulate DNA synthesis in primary hepatocytes via the expression of p21Cip-1/WAF1 and p27Kip-1.
Platelet-derived-growth-factor stimulation of the p42/p44 mitogen-activated protein kinase pathway in airway smooth muscle: role of pertussis-toxin-sensitive G-proteins, c-Src tyrosine kinases and phosphoinositide 3-kinase
Authors: CONWAY Ann-Marie , RAKHIT Soma , PYNE Susan , PYNE Nigel J. 
Published: August 2015

The mechanism used by the platelet-derived growth factor receptor (PDGFR) to activate the mitogen-activated- protein-kinase (p42/p44 MAPK) pathway was investigated in cultured airway smooth muscle (ASM) cells. We have found that pertussis toxin (PTX, which was used to inactivate the heterotrimeric G-protein Gi) induced an approx. 40–50% decrease in the activation of c-Src and p42/p44 MAPK by PDGF. An essential role for c-Src was confirmed using the c-Src inhibitor, PP1, which abolished p42/p44 MAPK activation (PP1 and PTX were without effect on PDGFR tyrosine phosphorylation). Furthermore, the PTX-dependent decrease in c-Src and p42/p44 MAPK activation appeared correlated. These findings suggest that the PDGFR can utilize the PTX-sensitive G-protein, Gi, to regulate c-Src and subsequent p42/p44 MAPK activation. Phosphoinositide 3-kinase (PI3K) has been shown by others to be involved in p42/p44 MAPK activation. This is confirmed here by experiments which showed that PI3K inhibitors (wortmannin and LY294002) reduced the activation of p42/p44 MAPK by PDGF. PI3K activity was increased in Grb-2 immunoprecipitates from PDGF-stimulated cells and was decreased by pretreating these cells with PTX. These findings show that Gi might also promote Grb-2–PI3K complex formation and that Grb-2 may be a site at which PI3K is integrated into the p42/p44 MAPK cascade. In conclusion, our results demonstrate that Gi enables the PDGFR to signal more efficiently to p42/p44 MAPK, and this appears to be achieved through the regulation of c-Src and Grb-2/PI3K, which are intermediates in the p42/p44 MAPK cascade.
Enhancement of chemotactic peptide-induced activation of phosphoinositide 3-kinase by granulocyte–macrophage colony-stimulating factor and its relation to the cytokine-mediated priming of neutrophil superoxide-anion production
Authors: KODAMA Takeshi , HAZEKI Kaoru , HAZEKI Osamu , OKADA Taro , UI Michio 
Published: August 2015

Incubation of human neutrophils with a chemotactic peptide [N-formylmethionyl-leucylphenylalanine (fMLP)] gave rise to an increase in the phosphoinositide 3-kinase (PI3K) activity, phosphorylation of p47phoxand superoxide-anion (O2-) generation in the same fMLP-concentration-dependent manner. These responses to fMLP were markedly enhanced when the cells had been incubated for 10 min before the addition of fMLP with increasing concentrations of granulocyte–macrophage colony-stimulating factor (GM-CSF) that were only slightly effective themselves. Wortmannin, an inhibitor of PI3K, suppressed all of these fMLP actions in the same concentration-dependent manner in either GM-CSF-primed or non-primed cells. Sustained activation of protein kinase C by the addition of PMA caused marked phosphorylation of p47phoxand respiratory burst itself without activation of PI3K. This strong action of PMA was not primed by GM-CSF. The chemotactic peptide was without effect in pertussis-toxin-treated cells, indicating that its actions are mediated by βγ-subunits liberated from toxin-susceptible heterotrimeric Gi proteins (Gβγ). Thus one of the mechanisms of GM-CSF-mediated priming of fMLP-induced respiratory burst is synergistic activation of wortmannin-sensitive PI3K by Gβγ in the presence of tyrosine-phosphorylated proteins in GM-CSF-treated cells, as recently indicated in a cell-free system [Kurosu, Maehama, Okada, Yamamoto, Hoshino, Fukui, Ui, Hazeki and Katada (1997) J. Biol. Chem. 272, 24252–24256]. GM-CSF primed fMLP-induced MAP (mitogen-activated protein) kinase activation enormously as well. The MAP kinase activation was primed even in the presence of wortmannin, indicating that PI3K was not the sole site where tyrosine kinase-related and Gβγ-mediated intracellular signals converge to elicit the priming. The GM-CSF priming of fMLP-induced PI3K activation and O2- generation was much smaller in magnitude in neutrophils in which cAMP accumulated upon incubation with prostaglandin E1 than in the cells without the nucleotide accumulation. Thus the GM-CSF priming site, in addition to PI3K, might be just the target of cAMP-dependent protein kinase A in fMLP-initiated signalling cascades or could be localized immediately downstream thereof.
[Difluro(phosphono)methyl]phenylalanine-containing peptide inhibitors of protein tyrosine phosphatases
Authors: DESMARAIS Sylvie , FRIESEN Richard W. , ZAMBONI Robert , RAMACHANDRAN Chidambaram 
Published: August 2015

Peptides containing the non-hydrolysable phosphotyrosine analogue 4-[difluro(phosphono)methyl]phenylalanine [Phe(CF2P)] were synthesized and tested as inhibitors of the protein tyrosine phosphatases (PTPs) PTP1B, CD45, PTPβ, LAR and SHP-1. We have identified peptides containing two adjacent Phe(CF2P) residues as potent inhibitors of PTPs. The tripeptide having the sequence Glu-Phe(CF2P)-Phe(CF2P) is a potent and selective inhibitor of PTP1B. This peptide inhibits PTP1B with an IC50 of 40 nM, which is at least 100-fold lower than with other PTPs. A second tripeptide, Pro-Phe(CF2P)-Phe(CF2P), is most potent against PTPβ, with an IC50 of 200 nM, and inhibits PTP1B with an IC50 of 300 nM. These data suggest that it is possible to develop selective, active-site-directed, reversible, potent inhibitors of PTPs.