bluuebunny/crossref_metadata_2025_split huggingface dataset:
p21WAF1 expression by an activator of protein kinase C is regulated mainly at the post-transcriptional level in cells lacking p53: important role of RNA stabilization
Authors: AKASHI Makoto , OSAWA Yoshiaki , KOEFFLER H. Phillip , HACHIYA Misao 
Published: August 2015

p21WAF1 inhibits cyclin–cyclin-dependent kinase (Cdk) complexes, causing cell cycle arrest. p21WAF1 contains p53-binding sites in its promoter and expression of p21WAF1 is induced by functional p53. In the present work, we have studied the role of protein kinase C (PKC) in the induction of p21WAF1 and show that induction of p21WAF1 expression can occur by activation of PKC in cells having no p53. Human ovarian carcinoma cells, SKOV-3, lack p53 protein and PMA, a potent activator of PKC, did not induce p53. PMA increased the expression of p21WAF1 mRNA both in these cells and in other cells which do not contain p53 (THP-1 and U937). Treatment of human embryonic fibroblasts, WI38, with PMA also induced the accumulation of p21WAF1 without affecting p53 levels. However, PMA did not increase levels of p21WAF1 mRNA in cells where either the PKC or the mitogen-activated protein kinase pathway was blocked. Furthermore, treatment of cells with various phorbol ester derivatives which activate PKC resulted in the induction of p21WAF1 in SKOV-3 cells. In contrast, phorbol esters which do not activate PKC failed to induce p21WAF1 expression. PMA increased the transcriptional rate of p21WAF1 and activated the transcription of a luciferase reporter gene, controlled by the p21 promoter, in SKOV-3 cells with or without a p53 consensus-binding sequence. By contrast, PMA markedly stabilized p21WAF1 mRNA; the half-life (t1/2) of p21WAF1 in PMA-treated cells was &gt; 8 h compared with &lt; 1 h in untreated cells. These findings provide evidence that the PKC pathway induces expression of p21WAF1 independently of p53. Our present study also suggests that the accumulation of p21WAF1 transcripts by PMA occurs mainly at post-transcriptional level.
Brain spectrin (fodrin) interacts with phospholipids as revealed by intrinsic fluorescence quenching and monolayer experiments
Authors: DIAKOWSKI Witold , PRYCHIDNY Aleksander , ŚWISTAK Małgorzata , NIETUBYĆ MaŁgorzata , BIAŁKOWSKA Katarzyna , SZOPA Jan , SIKORSKI Aleksander F. 
Published: August 2015

We demonstrate that phospholipid vesicles affect the intrinsic fluorescence of isolated brain spectrin. In the present studies we tested the effects of vesicles prepared from phosphatidylcholine (PtdCho) alone, in addition to vesicles containing PtdCho mixed with other phospholipids [phosphatidylethanolamine (PtdEtn) and phosphatidylserine] as well as from total lipid mixture extracted from brain membrane. The largest effect was observed with PtdEtn/PtdCho (3:2 molar ratio) vesicles; the effect was markedly smaller when vesicles were prepared from egg yolk PtdCho alone. Brain spectrin injected into a subphase induced a substantial increase in the surface pressure of monolayers prepared from phospholipids. Results obtained with this technique indicated that the largest effect is again observed with monolayers prepared from a PtdEtn/PtdCho mixture. The greatest effect was observed when the monolayer contained 50–60% PtdEtn in a PtdEtn/PtdCho mixture. This interaction occurred at salt and pH optima close to physiological conditions (0.15 M NaCl, pH 7.5). Experiments with isolated spectrin subunits indicated that the effect of the β subunit on the monolayer surface pressure resembled that measured with the whole molecule. Similarly to erythrocyte spectrin–membrane interactions, brain spectrin interactions with PtdEtn/PtdCho monolayer were competitively inhibited by isolated erythrocyte ankyrin. This also suggests that the major phospholipid-binding site is located in the β subunit and indicates the possible physiological significance of this interaction.
Hydrogen bonding and protein perturbation in β-lactam acyl-enzymes of Streptococcus pneumoniae penicillin-binding protein PBP2x
Authors: CHITTOCK Roger S. , WARD Simon , WILKINSON Alan-Shaun , CASPERS Patrick , MENSCH Barbara , PAGE Malcolm G. P. , WHARTON Christopher W. 
Published: August 2015

A soluble form of Streptococcus pneumoniae PBP2x, a molecular target of penicillin and cephalosporin antibiotics, has been expressed and purified. IR difference spectra of PBP2x acylated with benzylpenicillin, cloxacillin, cephalothin and ceftriaxone have been measured. The difference spectra show two main features. The ester carbonyl vibration of the acyl-enzyme is ascribed to a small band between 1710 and 1720 cm-1, whereas a much larger band at approx. 1640 cm-1 is ascribed to a perturbation in the structure of the enzyme, which occurs on acylation. The protein perturbation has been interpreted as occurring in β-sheet. The acyl-enzyme formed with benzylpenicillin shows the lowest ester carbonyl vibration frequency, which is interpreted to mean that the carbonyl oxygen is the most strongly hydrogen-bonded in the oxyanion hole of the antibiotics studied. The semi-synthetic penicillin cloxacillin is apparently less well organized in the active site and shows two partially overlapping ester carbonyl bands. The penicillin acyl-enzyme has been shown to deacylate more slowly than that formed with cloxacillin. This demonstrates that the natural benzylpenicillin forms a more optimized and better-bonded acyl-enzyme and that this in turn leads to the stabilization of the acyl-enzyme required for effective action in the inhibition of PBP2x. The energetics of hydrogen bonding in the several acyl-enzymes is discussed and comparison is made with carbonyl absorption frequencies of model ethyl esters in a range of organic solvents. A comparison of hydrolytic deacylation with hydroxaminolysis for both chymotryspin and PBP2x leads to the conclusion that deacylation is uncatalysed.
The folding and assembly of the dodecameric type II dehydroquinases
Authors: PRICE Nicholas C. , BOAM Deborah J. , KELLY Sharon M. , DUNCAN Doris , KRELL Tino , GOURLEY David G. , COGGINS John R. , VIRDEN Richard , HAWKINS Alastair R. 
Published: August 2015

The dodecameric type II dehydroquinases (DHQases) have an unusual quaternary structure in which four trimeric units are arranged with cubic 23 symmetry. The unfolding and refolding behaviour of the enzymes from Streptomyces coelicolor and Mycobacterium tuberculosis have been studied. Gel-permeation studies show that, at low concentrations (0.5 M) of guanidinium chloride (GdmCl), both enzymes dissociate into trimeric units, with little or no change in the secondary or tertiary structure and with a 15% loss (S. coelicolor) or a 55% increase (M. tuberculosis) in activity. At higher concentrations of GdmCl, both enzymes undergo sharp unfolding transitions over narrow ranges of the denaturant concentration, consistent with co-operative unfolding of the subunits. When the concentration of GdmCl is lowered by dilution from 6 M to 0.55 M, the enzyme from S. coelicolor refolds in an efficient manner to form trimeric units, with more than 75% regain of activity. Using a similar approach the M. tuberculosis enzyme regains less than 35% activity. From the time courses of the changes in CD, fluorescence and activity of the S. coelicolor enzyme, an outline model for the refolding of the enzyme has been proposed. The model involves a rapid refolding event in which approximately half the secondary structure is regained. A slower folding process follows within the monomer, resulting in acquisition of the full secondary structure. The major changes in fluorescence occur in a second-order process which involves the association of two folded monomers. Regain of activity is dependent on a further associative event, showing that the minimum active unit must be at least trimeric. Reassembly of the dodecameric S. coelicolor enzyme and essentially complete regain of activity can be accomplished if the denatured enzyme is dialysed extensively to remove GdmCl. These results are discussed in terms of the recently solved X-ray structures of type II DHQases from these sources.
Characterization and partial purification of a novel neutrophil membrane-associated kinase capable of phosphorylating the respiratory burst component p47phox
Authors: LAL Aroon S. , PARKER Peter J. , SEGAL Anthony W. 
Published: August 2015

The phosphorylation of p47phox is widely viewed as an important step in the activation of the neutrophil respiratory burst oxidase system. The exact nature of the kinase(s) responsible remains to be elucidated. We show here that such a kinase was detected on neutrophil membranes activated by either PMA or formyl-methionyl-leucyl-phenylalanine. This enzyme is not intrinsic to the neutrophil membrane and could be eluted with 0.5 M NaCl. The kinase activity was partially purified and was found not to be due to the presence of previously suggested kinases, including protein kinase C isotypes, mitogen-activated protein kinase and protein kinase B. Gel filtration and renaturation in substrate gels suggest a molecular mass of between 45 and 51 kDa. The kinase activity was independent of calcium and lipids but was potently inhibited by staurosporine. Treatment with protein phosphatase 2Ac suggested that the kinase was activated by serine/threonine phosphorylation. Phosphopeptide maps indicated that the kinase phosphorylated p47phox on similar sites to those found in vivo. These results indicate that activation of neutrophils by PMA results in the activation of a membrane-associated kinase that may play a part in the regulation of neutrophil NADPH oxidase through its ability to phosphorylate p47phox.
Regulation of 2-carboxy-d-arabinitol 1-phosphate phosphatase: activation by glutathione and interaction with thiol reagents
Authors: HEO Jongyun , HOLBROOK Gabriel P. 
Published: August 2015

2-Carboxy-d-arabinitol 1-phosphate (CA1P) phosphatase de- grades CA1P, an inhibitor associated with the regulation of ribulose bisphosphate carboxylase/oxygenase in numerous plant species. CA1P phosphatase purified from Phaseolus vulgaris was partially inactivated by oxidizing conditions during dialysis in air-equilibrated buffer. Phosphatase activity could then be stimulated 1.3-fold by dithiothreitol and also by addition of reduced thioredoxin from Escherichia coli. These effects were enhanced synergistically by the positive effector, fructose 1,6-bisphosphate (FBP). Most notably, CA1P phosphatase activity was stimulated up to 35-fold by glutathione, and was sensitive to the ratio of reduced (GSH) to oxidized (GSSG) forms. At concentrations of glutathione approximating measured levels in chloroplasts of P. vulgaris (5 mM total S), CA1P phosphatase exhibited &gt; 20-fold stimulation by a change in the redox status of glutathione from 60 to 100% GSH. This stimulation was augmented further by reduced E. coli thioredoxin. In contrast, FBP, which activates CA1P phosphatase under reducing conditions, was strongly inhibitory in the presence of GSSG. We propose that glutathione may have an appreciable role in the light/dark regulation of CA1P phosphatase in vivo. A model for the reversible activation of CA1P phosphatase by GSH was derived based upon the various responses of the enzyme's activity to a range of thiol reagents including N-ethylmaleimide, 5,5´-dithiobis-(2-nitrobenzoic acid) and arsenite. These data indicate that the bean enzyme contains two physically distinct sets of thiol groups that are critical to its redox regulation.
Heparin and heparan sulphate protect basic fibroblast growth factor from non-enzymic glycosylation
Authors: NISSEN Nicholas N. , SHANKAR Ravi , GAMELLI Richard L. , SINGH Ashok , DiPIETRO Luisa A. 
Published: August 2015

Non-enzymic glycosylation of basic fibroblast growth factor (bFGF, FGF-2) has recently been demonstrated to decrease the mitogenic activity of intracellular bFGF. Loss of this bioactivity has been implicated in impaired wound healing and microangiopathies of diabetes mellitus. In addition to intracellular localization, bFGF is also widely distributed in the extracellular matrix, primarily bound to heparan sulphate proteoglycans (HSPGs). Nonetheless, it is not clear if non-enzymic glycosylation similarly inactivates matrix-bound bFGF. To investigate this, we measured the effect of non-enzymic glycosylation on bFGF bound to heparin, heparan sulphate and related compounds. Incubation of bFGF with the glycosylating agents glyceraldehyde 3-phosphate (G3P; 25 mM) or fructose (250 mM) resulted in loss of 90% and 40% of the mitogenic activity of bFGF respectively. Treatment with G3P and fructose also decreased the binding of bFGF to a heparin column. If heparin was added to bFGF prior to non-enzymic glycosylation, the mitogenic activity and heparin affinity of bFGF were nearly completely preserved. A similar protective effect was demonstrated by heparan sulphate, low-molecular-mass heparin and the polysaccharide dextran sulphate, but not by chondroitin sulphate. Whereas non-enzymic glycosylation of bFGF with G3P impaired its ability to stimulate c-myc mRNA expression in fibroblasts, no such impairment was noticeable when bFGF was glycosylated in the presence of heparin. Taken together, these results suggest that HSPG-bound bFGF is resistant to non-enzymic glycosylation-induced loss of activity. Therefore, alteration of this pool probably does not contribute to impaired wound healing seen in diabetes mellitus.
Roles of an Ets motif and a novel CACGAC direct repeat in transcription of the murine dihydrolipoamide dehydrogenase (Dld) gene
Authors: YANG Hsin-Sheng , JOHNSON Mark , PATEL Mulchand S. 
Published: August 2015

The 5´-flanking region of the murine dihydrolipoamide dehydrogenase (Dld) gene was characterized for its promoter activity. DNase I footprinting analysis of the promoter region (-545 bp to +41 bp) revealed six major protein-binding domains (termed P1 to P6) that were protected by NIH3T3 fibroblast nuclear extracts. Transient transfection assays, using a series of nested deletions of the 2.5 kb 5´-flanking region ligated to the chloramphenicol acetyltransferase reporter gene, identified that the -42-bp to +41-bp region, which harbours the P1, P2, and P3 domains, had minimal transcriptional activity. When the 5´-flanking region was extended from -42 bp to -82 bp, there was an approx. 5-fold increase in promoter activity. To identify further the cis elements involved in transcription of the Dld gene (-82 bp to +41 bp), a series of mutations were introduced into this region and evaluated for functional effects using transient transfection and electrophoretic mobility shift assays. Mutation or deletion of the CACGAC direct repeat, located from -61 bp to -46 bp, resulted in minimal promoter activity. Mutation of the Ets motif, located from -37 bp to -32 bp, reduced the minimal promoter activity by approx. 50%, whereas the deletion of this motif almost abolished the promoter activity. These results indicate that: (i) the Ets motif is required for the minimal promoter activity and (ii) the CACGAC direct repeat enhances promoter activity. Database searches failed to identify the direct repeat with the CACGAC motif and hence the CACGAC sequence may represent a novel motif. The requirement of both the Ets motif and the direct repeat element for optimal promoter activity represents a unique combination for gene transcription.
Regulation of inducible nitric oxide synthase expression in β cells by environmental factors: heavy metals
Authors: ECKHARDT Walther , BELLMANN Kerstin , KOLB Hubert 
Published: August 2015

The expression of inducible NO synthase (iNOS) in pancreatic islet β cells modulates endocrine cell functions and, at very high levels of NO production causes β-cell death. We tested the hypothesis that environmental factors such as heavy-metal salts modulate iNOS expression in β cells. A rat β-cell line (insulinoma RINm5F) was cultured in the presence of low-dose interleukin (IL)-1β for suboptimal induction of iNOS. PbCl2 (0.1–10 µM) dose-dependently increased NO (measured as nitrite) formation (P&lt; 0.001). In contrast, HgCl2 suppressed nitrite production (0.1–10 µM, P&lt; 0.05). Measurements of iNOS activity by determining citrulline levels confirmed the potentiating effect of PbCl2 (P&lt; 0.05). There was a narrow time window of heavy-metal actions, ranging from -24 h (Hg2+) or -3 h (Pb2+) to +2 h, relative to the addition of IL-1β. By semi-quantitative reverse transcriptase-PCR, enhanced levels of iNOS mRNA were found in the presence of Pb2+ (P&lt; 0.05) and decreased levels in the presence of Hg2+. The amount of iNOS protein as determined by Western blotting was increased in the presence of Pb2+. We conclude that Pb2+ upregulates and Hg2+ suppresses iNOS gene expression at the level of transcription, probably by acting on the signalling pathway. These observations may have important implications for understanding pathological effects of environmental factors on endocrine organ functions.
Purification of A1 adenosine receptor–G-protein complexes: effects of receptor down-regulation and phosphorylation on coupling
Authors: GAO Zhenhai , ROBEVA Anna S. , LINDEN Joel 
Published: August 2015

We examined the effects of exposing A1 adenosine receptors (A1ARs) to an agonist on the stability and phosphorylation state of receptor–guanine nucleotide-binding regulatory protein (R–G-protein) complexes. Non-denatured recombinant human A1ARs extended on the N-terminus with hexahistidine (His6) and the FLAG (Asp-Tyr-Lys-Asp-Asp-Asp-Asp-Lys) epitope (H/F) were purified to near homogeneity from stably transfected Chinese-hamster ovary (CHO)-K1 cells. Purified receptors have pharmacological properties similar to receptors in membranes. G-proteins were co-purified with 15±2% of H/F-A1AR unless receptor–G-protein (R–G) complexes were uncoupled by pre-treating cell membranes with GTP. By silver staining, purified A1AR–G-protein complexes contain receptors, G-protein α and β subunits and an unidentified 97 kDa protein. Pretreating intact cells with N6-cyclopentyladenosine (CPA) for 24 h decreased both the total number of receptors measured in membranes and the number of purified A1ARs by about 50%. In contrast, pretreating cells with CPA decreased the number of R–G complexes measured in membranes (54±6%) significantly less than it decreased the number of purified R–G complexes (78±3%) as detected by 125I-N6-(4-aminobenzyl)adenosine binding or by Western blotting Giα2. The effect of CPA to decrease the fraction of receptors purified as R–G complexes was not associated with any change in low-level A1AR phosphorylation (found on serine), or low-level phosphorylation of G-protein α or β subunits or the 97 kDa protein. These experiments reveal a novel aspect of agonist-induced down-regulation, namely a diminished stability of receptor–G-protein complexes that is manifested as uncoupling during receptor purification.
Gi-mediated tyrosine phosphorylation of Grb2 (growth-factor-receptor-bound protein 2)-bound dynamin-II by lysophosphatidic acid
Authors: KRANENBURG Onno , VERLAAN Ingrid , MOOLENAAR Wouter H. 
Published: August 2015

Lysophosphatidic acid (LPA) is the prototypic G-protein-coupled receptor agonist that activates the Ras-mitogen-activated protein (MAP) kinase cascade through pertussis toxin (PTX)-sensitive Gi and enhanced tyrosine kinase activity. We recently detected a 100 kDa protein (p100) that binds to the C-terminal SH3 domain of growth-factor-receptor-bound protein 2 (Grb2) and becomes tyrosine phosphorylated in a PTX-sensitive manner in LPA-treated Rat-1 cells [Kranenburg, Verlaan, Hordijk and Moolenaar (1997) EMBO J. 16, 3097–3105]. Through glutathione S-transferase-Grb2 affinity purification and microsequencing, we have now identified p100 as dynamin-II, a GTPase that regulates clathrin-mediated endocytosis. We show that in Rat-1 cells, Grb2-bound dynamin-II is rapidly tyrosine phosphorylated in response to LPA in a PTX-sensitive manner. Thus, tyrosine phosphorylation of Grb2-bound dynamin-II may be a critical event in Gi-mediated activation of the Ras-MAP kinase cascade in fibroblasts.
17β-Oestradiol increases intracellular Ca2+ concentration in rat enterocytes
Authors: PICOTTO Gabriela , VAZQUEZ Guillermo , BOLAND Ricardo 
Published: August 2015

The involvement of the phospholipase C (PLC) pathway in the non-genomic regulation of duodenal cell Ca2+ concentration by 17β-oestradiol was investigated. The PLC inhibitors neomycin (0.5 mM) and U-73122 (2 µM) suppressed the stimulatory effect of 0.1 nM 17β-oestradiol on the 45Ca2+ influx into enterocytes isolated from rat duodenum. The hormone (1 pM to 10 nM) increased the formation of 1,2-diacylglycerol in a biphasic pattern, characterized by an early peak at 45 s (+82%) and a later peak at 5 min (+46%). Both PLC inhibitors suppressed the first peak but were unable to block the 17β-oestradiol effect at 5 min. 17β-Oestradiol also increased the generation of inositol 1,4,5-trisphosphate within 15 s, with maximal stimulation at 30 s. 17β-Oestradiol induced a rapid (30 s) and sustained (up to 5 min) increase in the intracellular Ca2+ concentration ([Ca2+]i) of fura 2-loaded enterocytes. The fast rise in [Ca2+]i was specific because other sex steroid hormones were without effect and could be blocked to a great extent by U-73122 (by 86% at 1 min). The effects of 17β-oestradiol on enterocyte [Ca2+]i were decreased significantly (by 75%) in a Ca2+-free extracellular medium but a pronounced increase in [Ca2+]i was obtained after readmission of Ca2+ to the medium. The latter change was suppressed by 10 µM La3+, whereas nitrendipine (1 µM) and verapamil (10 µM) separately were without effect. The permeability of the 17β-oestradiol-induced Ca2+ influx pathway to Mn2+ was increased 2.8-fold by treatment with oestrogen. These results suggest the operation of a PLC-dependent store-operated Ca2+ channel mechanism in 17β-oestradiol regulation of enterocyte extracellular Ca2+ influx.
Complexes containing activating transcription factor (ATF)/cAMP-responsive-element-binding protein (CREB) interact with the CCAAT/enhancer-binding protein (C/EBP)–ATF composite site to regulate Gadd153 expression during the stress response
Authors: FAWCETT Timothy W. , MARTINDALE Jennifer L. , GUYTON Kathryn Z. , HAI Tsonwin , HOLBROOK Nikki J. 
Published: August 2015

Gadd153, also known as chop, encodes a member of the CCAAT/enhancer-binding protein (C/EBP) transcription factor family and is transcriptionally activated by cellular stress signals. We recently demonstrated that arsenite treatment of rat pheochromocytoma PC12 cells results in the biphasic induction of Gadd153 mRNA expression, controlled in part through binding of C/EBPβ and two uncharacterized protein complexes to the C/EBP–ATF (activating transcription factor) composite site in the Gadd153 promoter. In this report, we identified components of these additional complexes as two ATF/CREB (cAMP-responsive-element-binding protein) transcription factors having differential binding activities dependent upon the time of arsenite exposure. During arsenite treatment of PC12 cells, we observed enhanced binding of ATF4 to the C/EBP–ATF site at 2 h as Gadd153 mRNA levels increased, and enhanced binding of ATF3 complexes at 6 h as Gadd153 expression declined. We further demonstrated that ATF4 activates, while ATF3 represses, Gadd153 promoter activity through the C/EBP–ATF site. ATF3 also repressed ATF4-mediated transactivation and arsenite-induced activation of the Gadd153 promoter. Our results suggest that numerous members of the ATF/CREB family are involved in the cellular stress response, and that regulation of stress-induced biphasic Gadd153 expression in PC12 cells involves the ordered, sequential binding of multiple transcription factor complexes to the C/EBP–ATF composite site.