bluuebunny/crossref_metadata_2025 huggingface dataset:
Xyloglucan undergoes interpolymeric transglycosylation during binding to the plant cell wall in vivo: evidence from 13C/3H dual labelling and isopycnic centrifugation in caesium trifluoroacetate
Authors: THOMPSON E. James , SMITH C. Rachel , FRY C. Stephen
Published: August 2015

Xyloglucan from the walls of Rosa cells that had been cultured on [12C]- or [13C]-glucose formed bands in caesium trifluoroacetate with mean buoyant densities of 1.575 or 1.616 g/ml respectively. Incubation of a mixture of [13C,3H]xyloglucan and [12C,1H]xyloglucan in the presence of xyloglucan endotransglycosylase (XET) activity caused the mean buoyant density of the radioactive material to decrease, indicating that interpolymeric transglycosylation could be detected in vitro. We used two 13C/3H-dual-labelling protocols to look for interpolymeric transglycosylation in vivo. In protocol A, [13C]glucose-grown Rosa cells were transferred into [12C]glucose medium 6 h after a ≈ 2 h pulse of L-[1-3H]arabinose (which radiolabels the xylose residues of xyloglucan). The mean buoyant density of the wall-bound [3H]xyloglucan decreased during the following 7 days in culture. This indicates that, during or after the wall-binding of newly synthesized [12C,1H]xyloglucan, it became covalently attached to previously wall-bound [13C,3H]xyloglucan. In protocol B, [12C]glycerol- or [12C]glucose-grown Rosa cells were transferred into [13C]glucose medium, 20 or 60 min before a ≈ 2 h pulse of [3H]arabinose. The buoyant density of the earliest wall-bound [3H]xyloglucan showed that it had a 12C/13C ratio of ~ 1:1. This indicates that, during (or, implausibly, before) wall-binding, the newly synthesized [13C,3H]xyloglucan became covalently attached to previously synthesized [12C]xyloglucan. During the following 7 days in culture, the mean buoyant density of the [3H]xyloglucan increased, showing that later-synthesized [13C,1H]xyloglucan can be covalently attached to previously wall-bound [12C,13C,3H]xyloglucan. The only known mechanism by which segments of xyloglucans could become covalently attached to each other in the cell wall is by interpolymeric transglycosylation catalysed by XET. We conclude that XET-catalysed interpolymeric transglycosylation accompanies, and probably causes, the integration of newly secreted xyloglucan into the cell-wall architecture.
Relative contributions of Na+-dependent phosphate co-transporters to phosphate transport in mouse kidney: RNase H-mediated hybrid depletion analysis
Authors: MIYAMOTO Ken-ichi , SEGAWA Hiroko , MORITA Kyoko , NII Tomoko , TATSUMI Sawako , TAKETANI Yutaka , TAKEDA Eiji
Published: August 2015

Reabsorption of Pi in the proximal tubule of the kidney is an important determinant of Pi homoeostasis. At least three types (types I-III) of high-affinity Na+-dependent Pi co-transporters have been identified in mammalian kidneys. The relative roles of these three types of Na+/Pi co-transporters in Pi transport in mouse kidney cortex have now been investigated by RNase H-mediated hybrid depletion. Whereas isolated brush-border membrane vesicles showed the presence of two kinetically distinct Na+/Pi co-transport systems (high Km-low Vmax and low Km-high Vmax), Xenopus oocytes, microinjected with polyadenylated [poly(A)+] RNA from mouse kidney cortex, showed only the high-affinity Pi uptake system. Kidney poly(A)+ RNA was incubated in vitro with antisense oligonucleotides corresponding to Npt-1 (type I), NaPi -7 (type II) or Glvr-1 (type III) Na+/Pi co-transporter mRNAs, and then with RNase H. Injection of such treated RNA preparations into Xenopus oocytes revealed that an NaPi-7 antisense oligonucleotide that resulted in complete degradation of NaPi-7 mRNA (as revealed by Northern blot analysis), also induced complete inhibition of Pi uptake. Degradation of Npt-1 or Glvr-1 mRNAs induced by corresponding antisense oligonucleotides had no effect on Pi transport, which was subsequently measured in oocytes. These results indicate that the type II Na+/Pi co-transporter NaPi-7 mediated most Na+-dependent Pi transport in mouse kidney cortex.
Tetramerization domain of human butyrylcholinesterase is at the C-terminus
Authors: BLONG M. Renee , BEDOWS Elliott , LOCKRIDGE Oksana
Published: August 2015

Butyrylcholinesterase (BChE) in human serum consists predominantly of tetramers. Recombinant BChE, however, expressed in Chinese hamster ovary (CHO) cells, consists of approx. 55% dimers, 10-30% tetramers and 15-40% monomers. To determine the origin of the monomer species we added the FLAG epitope (epitope tag, amino acid sequence DYKDDDDK) to the C-terminus of the enzyme, and expressed BChE-FLAG in CHO cells. We found that secreted, active monomers had lost their FLAG epitope, suggesting that the monomers were made by proteolysis of dimers or tetramers at the C-terminus. To estimate the number of amino acids that could be deleted from the C-terminus without losing BChE activity, we expressed deletion mutants. We found that deletion of up to 50 amino acids from the C-terminus yielded active monomers, but that deletion of 51 amino acids destroyed BChE activity and caused the inactive protein to remain within the cell. Deletion of eight or more amino acids from the N-terminus also resulted in inactive protein that remained inside the cell. Monomeric BChE had wild-type Km and kcat values (8 μM and 24000 min-1 for butyrylthiocholine) and showed substrate activation. The Cys-571→Ala mutant, though incapable of forming the interchain disulphide bond, had nearly the same amount of tetrameric BChE as recombinant wild-type BChE. These results support the conclusion that the tetramerization domain of BChE is at the C-terminus, within the terminal 50 amino acids, and that the interchain disulphide bond is not essential for tetramerization. Molecular modelling suggested that the tetramerization domain was a four-helix bundle, stabilized by interactions of seven conserved aromatic amino acids.
The N-terminal Arg2, Arg3 and Arg4 of human lactoferrin interact with sulphated molecules but not with the receptor present on Jurkat human lymphoblastic T-cells
Authors: LEGRAND Dominique , VAN BERKEL Patrick H. C. , SALMON Valérie , VAN VEEN A. Harry , SLOMIANNY Marie-Christine , NUIJENS H. Jan , SPIK Geneviève
Published: August 2015

We previously characterized a 105 kDa receptor for human lactoferrin (hLf) on Jurkat human lymphoblastic T-cells. To delineate the role of the basic cluster Arg2-Arg3-Arg4-Arg5 of hLf in the interaction with Jurkat cells, we isolated N-terminally deleted hLf species of molecular mass 80 kDa lacking two, three or four N-terminal residues (hLf-2N, hLf-3N and hLf-4N) from native hLf that had been treated with trypsin. Native hLf bound to 102000 sites on Jurkat cells with a dissociation constant (Kd) of 70 nM. Consecutive removal of N-terminal arginine residues from hLf progressively increased the binding affinity but decreased the number of binding sites on the cells. A recombinant hLF mutant lacking the first five N-terminal residues (rhLf-5N) bound to 17000 sites with a Kd of 12 nM. The binding parameters of bovine lactoferrin (Lf) and native hLf did not significantly differ, whereas the binding parameters of murine Lf (8000 sites; Kd 30 nM) resembled those of rhLf-5N. Culture of Jurkat cells in the presence of chlorate, which inhibits sulphation, decreased the number of binding sites for both native hLf and hLf-3N but not for rhLf-5N, indicating that the hLf-binding sites include sulphated molecules. We propose that the interaction of hLf with a large number of binding sites (approx. 80000 per cell) on Jurkat cells is dependent on Arg2-Arg3-Arg4, but not on Arg5. Interaction with approx. 20000 binding sites per cell, presumably the hLf receptor, does not require the first N-terminal basic cluster of hLf. Moreover, the affinity of hLf for the latter binding site is enhanced approx. 6-fold after removal of the first basic cluster. Thus N-terminal proteolysis of hLf in vivo might serve to modulate the nature of its binding to cells and thereby its effects on cellular physiology.
Degradation of the inducible cAMP early repressor (ICER) by the ubiquitin–proteasome pathway
Authors: FOLCO J. Eduardo , KOREN Gideon
Published: August 2015

The inducible cAMP early repressor (ICER) is a powerful transcriptional inhibitor that plays an important role in the regulation of the cAMP-dependent transcriptional response in the neuroendocrine system. ICER activity is primarily determined by its intracellular concentration, rather than by post-translational modifications, such as phosphorylation. We investigated the mechanisms that regulate the levels of ICER transcript and polypeptides in cardiocytes, myogenic (C2C12) and pituitary-derived (GH3) cell lines. We show that in primary cardiocytes and GH3 cells ICER was inducible by cAMP but not by membrane depolarization. Moreover, lactacystin, a specific proteasome inhibitor, decreased the rate of ICER degradation. This effect was associated with the accumulation of ICER-ubiquitin conjugates. We conclude that the intracellular levels of ICER are controlled by the ubiquitin-proteasome pathway for protein breakdown.
Molecular cloning and expression of a rat hepatic multiple inositol polyphosphate phosphatase
Authors: CRAXTON Andrew , CAFFREY J. James , BURKHART William , SAFRANY T. Stephen , SHEARS B. Stephen
Published: August 2015

The characterization of the multiple inositol polyphosphate phosphatase (MIPP) is fundamental to our understanding of how cells control the signalling activities of ‘higher’ inositol polyphosphates. We now describe our isolation of a 2.3 kb cDNA clone of a rat hepatic form of MIPP. The predicted amino acid sequence of MIPP includes an 18 amino acid region that aligned with approximately 60% identity with the catalytic domain of a fungal inositol hexakisphosphate phosphatase (phytase A); the similarity encompassed conservation of the RHGXRXP signature of the histidine acid phosphatase family. A histidine-tagged, truncated form of MIPP was expressed in Escherichia coli and the enzymic specificity of the recombinant protein was characterized: Ins(1,3,4,5,6)P5 was hydrolysed, first to Ins(1,4,5,6)P4 and then to Ins(1,4,5)P3, by consecutive 3- and 6-phosphatase activities. Inositol hexakisphosphate was catabolized without specificity towards a particular phosphate group, but in contrast, MIPP only removed the β-phosphate from the 5-diphosphate group of diphosphoinositol pentakisphosphate. These data, which are consistent with the substrate specificities of native (but not homogeneous) MIPP isolated from rat liver, provide the first demonstration that a single enzyme is responsible for this diverse range of specific catalytic activities. A 2.5 kb transcript of MIPP mRNA was present in all rat tissues that were examined, but was most highly expressed in kidney and liver. The predicted C-terminus of MIPP is comprised of the tetrapeptide SDEL, which is considered a signal for retaining soluble proteins in the lumen of the endoplasmic reticulum; the presence of this sequence provides a molecular explanation for our earlier biochemical demonstration that the endoplasmic reticulum contains substantial MIPP activity [Ali, Craxton and Shears (1993) J. Biol. Chem. 268, 6161-6167].
Expression of Menkes disease gene in mammary carcinoma cells
Authors: ACKLAND M. Leigh , CORNISH E. Jean , PAYNTER A. Jenny , GRIMES Andrew , MICHALCZYK Agnes , MERCER F. B. Julian
Published: August 2015

Two P-type ATPases, MNK and WND were recently shown to be defective in the human disorders of copper transport, Menkes disease and Wilson disease respectively. These proteins are important in copper homeostasis but their full physiological function has not been established. This study uses the human breast carcinoma line, PMC42, to investigate copper transport in the mammary gland. Northern blot analysis indicated that both MNK and WND mRNA are expressed in these cells. Western blot analysis with an MNK-specific antibody demonstrated a band of approx. 178 kDa, close to the expected size of 163 kDa. Treatment of PMC42 cells with lactational hormones (oestrogen and progesterone for 3 days followed by dexamethasone, insulin and prolactin for a further 3 days) did not produce an obvious increase in MNK expression as measured by Northern and Western blots. By using indirect immunofluorescence with the MNK antibody, the intracellular distribution of MNK was found to be predominantly perinuclear, consistent with Golgi localization. Punctate staining was also seen in a smaller proportion of cells, suggesting that some MNK is associated with endosomes. Treatment of PMC42 cells with lactational hormones increased the intensity of the perinuclear and punctate fluorescence. Exposure of cells to 100 mM copper resulted in the dispersion of the fluorescence towards the periphery of the cell. The results suggest a role for MNK in the secretion of copper into milk and that PMC42 cells are a valuable model for investigating the detailed cellular function of MNK and WND.
Evolution of phosphagen kinase: Isolation, characterization and cDNA-derived amino acid sequence of two-domain arginine kinase from the sea anemone Anthopleura japonicus
Authors: SUZUKI Tomohiko , KAWASAKI Yoshitada , FURUKOHRI Takahiro
Published: August 2015

Arginine kinase (AK) was isolated from the body wall muscle of the primitive sea anemone Anthopleura japonicus by Ultrogel AcA34 gel filtration, DEAE-32 chromatography and elution on a Cosmogel-SP column. The denatured molecular mass as determined with SDS/PAGE was 80 kDa, twice that of the usual AK subunit, indicating that this AK has an unusual two-domain structure. The native form was eluted on a Superose 12 column with the same retention time as that of rabbit homodimeric creatine kinase, indicating that Anthopleura AK is a monomer of 80 kDa. The isolated enzyme gave a specific activity of 100-120 μmol of Pi/min per mg of protein in the pH range 7.9-9.1 for the forward reaction. The enzyme is fully activated by Ca2+, as it is with Mg2+. The cDNA-derived amino acid sequence of 715 residues of Anthopleura AK was determined. The validity of the sequence was supported by chemical sequencing of internal tryptic peptides. A bridge intron of 686 bp, which separates the two domains of Anthopleura AK, is present between the second and third nucleotide in the codon of Ala-364. This is the first two-domain AK to be sequenced. Anthopleura AK shows 48-54% amino acid sequence identity with known invertebrate AKs, and also shows a lower, but significant, similarity (39-46%) to marine worm glycocyamine kinase and rabbit creatine kinase.
Molecular mechanisms for the control of translation by insulin
Authors: PROUD G. Christopher , DENTON M. Richard
Published: August 2015

Insulin acutely stimulates protein synthesis in mammalian cells, and this involves activation of the process of mRNA translation. mRNA translation is a complex multi-step process mediated by proteins termed translation factors. Several translation factors are regulated in response to insulin, often as a consequence of changes in their states of phosphorylation. The initiation factor eIF4E binds to the cap structure at the 5ʹ-end of the mRNA and mediates assembly of an initiation-factor complex termed eIF4F. Assembly of this complex can be regulated by eIF4E-binding proteins (4E-BPs), which inhibit eIF4F complex assembly. Insulin induces phosphorylation of the 4E-BPs, resulting in alleviation of the inhibition. This regulatory mechanism is likely to be especially important for the control of the translation of specific mRNAs whose 5ʹ-untranslated regions (5ʹ-UTRs) are rich in secondary structure. Translation of another class of mRNAs, those with 5ʹ-UTRs containing polypyrimidine tracts is also activated by insulin and this, like phosphorylation of the 4E-BPs, appears to involve the rapamycin-sensitive signalling pathway which leads to activation of the 70 kDa ribosomal protein S6 kinase (p70 S6 kinase) and the phosphorylation of the ribosomal protein S6. Overall stimulation of translation may involve activation of initiation factor eIF2B, which is required for all initiation events. This effect is dependent upon phosphatidylinositol 3-kinase and may involve the inactivation of glycogen synthase kinase-3 and consequent dephosphorylation of eIF2B, leading to its activation. Peptide-chain elongation can also be activated by insulin, and this is associated with the dephosphorylation and activation of elongation factor eEF2, probably as a consequence of the insulin-induced reduction in eEF2 kinase activity. Thus multiple signalling pathways acting on different steps in translation are involved in the activation of this process by insulin and lead both to general activation of translation and to the selective regulation of specific mRNAs.
Recombinant 2-enoyl-CoA hydratase derived from rat peroxisomal multifunctional enzyme 2: role of the hydratase reaction in bile acid synthesis
Authors: QIN Yong-Mei , HAAPALAINEN M. Antti , CONRY Demara , CUEBAS A. Dean , HILTUNEN J. Kalervo , NOVIKOV K. Dmitry
Published: August 2015

Rat liver peroxisomes contain two multifunctional enzymes: (1) perMFE-1 [2-enoyl-CoA hydratase 1/Δ3,Δ2-enoyl-CoA isomerase/(S)-3-hydroxyacyl-CoA dehydrogenase] and (2) perMFE-2 [2-enoyl-CoA hydratase 2/(R)-3-hydroxyacyl-CoA dehydrogenase]. To investigate the role of the hydratase activity of perMFE-2 in β-oxidation, a truncated version of perMFE-2 was expressed in Escherichia coli as a recombinant protein. The protein catalyses the hydration of straight-chain (2E)-enoyl-CoAs to (3R)-hydroxyacyl-CoAs, but it is devoid of hydratase 1 [(2E)-enoyl-CoA to (3S)-hydroxyacyl-CoA] and (3R)-hydroxyacyl-CoA dehydrogenase activities. The purified enzyme (46 kDa hydratase 2) can be stored as an active enzyme for at least half a year. The recombinant enzyme hydrates (24E)-3α,7α,12α-trihydroxy- 5β-cholest-24-enoyl-CoA to (24R,25R)-3α,7α,12α,24-tetrahydroxy-5β-cholestanoyl-CoA, which has previously been characterized as a physiological intermediate in bile acid synthesis. The stereochemistry of the products indicates that the hydration reaction catalysed by the enzyme proceeds via a syn mechanism. A monofunctional 2-enoyl-CoA hydratase 2 has not been observed as a wild-type protein. The recombinant 46 kDa hydratase 2 described here survives in a purified form under storage, thus being the first protein of this type amenable to application as a tool in metabolic studies.
Differential control of murine aldose reductase and fibroblast growth factor (FGF)-regulated-1 gene expression in NIH 3T3 cells by FGF-1 treatment and hyperosmotic stress
Authors: HSU Debbie K. W. , GUO Yan , PEIFLEY A. Kimberly , WINKLES A. Jeffrey
Published: August 2015

Aldose reductase (AR) is an NADPH-dependent aldo-keto reductase implicated in cellular osmoregulation and detoxification. Two distinct murine genes have been identified that are predicted to encode proteins with significant amino acid sequence identity with mouse AR: mouse vas deferens protein and fibroblast growth factor (FGF)-regulated-1 protein (FR-1). Here we report that the AR and FR-1 genes are differentially regulated in NIH 3T3 fibroblasts. FGF-1 stimulation of quiescent cells induces both AR and FR-1 mRNA levels, but the effect on FR-1 mRNA expression is significantly greater. FGF-1 treatment also increases FR-1 protein expression, as determined by Western-blot analysis using FR-1-specific polyclonal antiserum. Calf serum stimulation of quiescent cells increases AR mRNA expression but not FR-1 mRNA expression. Finally, when NIH 3T3 cells are grown in hypertonic medium, AR mRNA levels are significantly increased whereas FR-1 mRNA levels are only slightly up-regulated. These results indicate that the AR and FR-1 genes are differentially regulated in murine fibroblasts by two different growth-promoting agents and by hyperosmotic stress. Therefore these structurally related enzymes may have at least some distinct cellular functions; for example, although both AR and FR-1 activity may be important for the metabolic changes associated with cellular proliferation, AR may be the primary aldo-keto reductase involved in cellular osmoregulation.
Endogenous production of tumour necrosis factor is required for manganese superoxide dismutase expression by irradiation in the human monocytic cell line THP-1
Authors: HACHIYA Misao , SHIMIZU Saori , OSAWA Yoshiaki , AKASHI Makoto
Published: August 2015

Manganese superoxide dismutase (MnSOD) is a mitochondrial enzyme that scavenges superoxide (O2-) ions. We studied the regulation of MnSOD gene expression by irradiation and the mechanisms in human monocytic cell line THP-1. We found that irradiation induced expression of the MnSOD gene through the autocrine mechanism, involving the production of tumour necrosis factor (TNF). Irradiation increased TNF production in THP-1 cells, and TNF increased the levels of MnSOD transcripts. Supernatant from irradiated THP-1 cells induced the expression of MnSOD mRNA, and anti-TNF antibody blocked the induction of MnSOD mRNA. Irradiation also increased the levels of MnSOD mRNA in other myelocytic cell lines, HL60 and KG-1, and the ovarian cancer cell line SK-OV-3. Moreover, increased levels of MnSOD mRNA were observed in mature myeloid cells, including macrophages and granulocytes, as well as in immature cells. However, irradiation did not increase the level of MnSOD mRNA in THP-1 cells with prolonged exposure to PMA. We also found that irradiation increased the rate of MnSOD transcription, and irradiation stabilized MnSOD mRNA in THP-1 cells. Our results indicate that the endogenous production of TNF is required, at least in part, for the induction of MnSOD mRNA expression by irradiation in THP-1 cells, and the increased levels of MnSOD transcripts on irradiation occur through a pathway involving protein kinase C activation. Our results also indicate that the increase in MnSOD mRNA caused by irradiation is regulated by both transcriptional and post-transcriptional mechanisms.
Formation and properties of dimeric recombinant horseradish peroxidase in a system of reversed micelles
Authors: GAZARYAN G. Irina , KLYACHKO L. Natalia , DULKIS K. Yulia , OUPOROV V. Igor , LEVASHOV V. Andrey
Published: August 2015

Wild-type recombinant horseradish peroxidase purified and refolded from Escherichia coli inclusion bodies has been studied in the system of bis(2-ethylhexyl)sulphosuccinate sodium salt (Aerosol OT)-reversed micelles in octane. In contrast with native horseradish peroxidase the wild-type recombinant enzyme forms dimeric structures as judged by sedimentation analysis. Peroxidase substrates affect the equilibrium between monomeric and dimeric enzyme forms. The dependence of the catalytic activity of recombinant peroxidase on the degree of hydration of the surfactant exhibits two maxima with pyrogallol, o-phenylene- diamine, guaiacol and o-dianisidine, with different ratios of activities for the first and second maxima. The differences in activities of monomeric and dimeric forms of the recombinant horseradish peroxidase provide evidence for active-site screening in dimeric forms. This has been used to model a dimeric structure of recombinant horseradish peroxidase with the screened entrance to the active site. In the model structure obtained, three of eight glycosylation sites were screened. This might explain the absence of dimeric structures in native enzyme peroxidase. The system of reversed micelles provides, for the first time, evidence for the formation of dimeric structures by recombinant plant peroxidase with an altered substrate specificity compared with the native enzyme. Thus one can assume that haem-containing peroxidases in general are able to form dimeric structures.
Cloning, functional expression and dietary regulation of the mouse neutral and basic amino acid transporter (NBAT)
Authors: SEGAWA Hiroko , MIYAMOTO Ken-ichi , OGURA Yoshio , HAGA Hiromi , MORITA Kyoko , KATAI Kanako , TATSUMI Sawako , NII Tomoko , TAKETANI Yutaka , TAKEDA Eiji
Published: August 2015

The Na+-independent dibasic and neutral amino acid transporter NBAT is among the least hydrophobic of mammalian amino acid transporters. The transporter contains one to four transmembrane domains and induces amino acid transport activity via a b0,+-like system when expressed in Xenopus oocytes. However, the physiological role of NBAT remains unclear. Complementary DNA clones encoding mouse NBAT have now been isolated. The expression of mouse NBAT in Xenopus oocytes also induced an obligatory amino acid exchange activity similar to that of the b0,+-like system. The amount of NBAT mRNA in mouse kidney increased during postnatal development, consistent with the increase in renal cystine and dibasic transport activity. Dietary aspartate induced a marked increase in cystine transport via the b0,+ system in mouse ileum. A high-aspartate diet also increased the amount of NBAT mRNA in mouse ileum. In the ileum of mice fed on the aspartate diet, the extent of cystine transport was further increased by preloading brush border membrane vesicles with lysine. Hybrid depletion of NBAT mRNA from ileal polyadenylated RNA revealed that the increase in cystine transport activity induced by the high-aspartate diet, as measured in Xenopus oocytes, was attributable to NBAT. These results demonstrate that mouse NBAT has an important role in cystine transport.
Haem precursor δ-aminolaevulinic acid induces activation of the cytosolic iron regulatory protein 1
Authors: CARVALHO Helotonio , BECHARA Etelvino José Henriques , MENEGHINI Rogério , DEMASI Marilene
Published: August 2015

Control of cellular iron homoeostasis is performed by iron regulatory protein 1 (IRP1) through post-transcriptional modifications. This protein is sensitive to intracellular iron availability, being activated at low iron levels and inactivated at high iron levels, conditions that signal the increased expression of the transferrin receptor or of ferritin respectively. IRP1 is known to be activated by some oxidants such as H2O2 and NO. Δ-Aminolaevulinic acid (ALA), previously found to produce reactive oxygen species and a carbon-centred radical, to release iron from ferritin, and to increase rat liver and brain non-haem iron and ferritin, was investigated for its effects on IRP1 activity in cultured hamster pulmonary fibroblasts. We have found that 1-2 mM ALA produced a 2-3-fold activation of IRP. On incubation with 1-4 mM succinylacetone methyl ester, a potent ALA dehydratase inhibitor, a 3-4-fold activation of the protein was observed, accompanied by a 40% increase in the intracellular ALA concentration. When cells were incubated in the presence of ALA or succinylacetone methyl ester, N-acetylcysteine inhibited IRP1 activation, suggesting that the observed effect is mediated by an oxidative process. We surmise that ALA-induced IRP1 activation might act as a co-sensor of iron homoeostasis.
Induction of calreticulin expression in response to amino acid deprivation in Chinese hamster ovary cells
Authors: HEAL Richard , McGIVAN John
Published: August 2015

The role of calreticulin as a stress-induced molecular chaperone protein of the endoplasmic reticulum is becoming more apparent. We characterize here the induction of calreticulin in response to complete amino acid deprivation in Chinese hamster ovary cells. Amino acid deprivation caused a 4-fold increase in calreticulin protein levels over a period of 4-10 h. In addition to an overall increase in protein levels, the glycosylation of calreticulin was increased. This glycosylation event was blocked by tunicamycin and was not required for the increase in calreticulin protein levels. Immunofluorescence studies localized calreticulin to the ER of CHO cells, and no significant change was observed after amino acid deprivation. Northern-blot analysis showed that calreticulin mRNA levels were increased approx. 10-fold in response to complete amino acid deprivation. The response was sensitive to actinomycin D and α-amanitin, implying that regulation is primarily at the level of transcription. These results are similar to the large increases in asparagine synthetase mRNA observed in response to amino acid deprivation, but the amino acid-deprivation-response element identified to be involved in asparagine synthetase induction is absent from the calreticulin promoter.
Osmotic stress induces variation in cellular levels of ornithine decarboxylase-antizyme
Authors: MITCHELL L. A. John , JUDD G. Gary , LEYSER Aviva , CHOE Chung-youl
Published: August 2015

The polyamines, and especially putrescine, play an integral role in the physiological response of cells to varying extracellular osmotic conditions. Ornithine decarboxylase (ODC) synthesis and stability, as well as the activity of the polyamine transporter, had all been reported to be very sensitive to media osmolarity in different cells and tissues, yet the mechanism of this complex, co-ordinated response was not known. In this study we have determined that all these aspects of osmotic-shock response may be mediated by the common regulatory protein, ODC-antizyme. HTC cells were induced for antizyme and then exposed to media of reduced osmotic strength. Both antizyme activity and protein decreased rapidly, under these conditions, to new steady-state levels that depended upon the degree of reduction in media tonicity. This antizyme reduction was found to be due to a rapid increase in antizyme degradation, with a half-life decrease from 75 min down to 45 min occurring immediately upon exchanging media. In complementary experiments, increased media tonicity induced elevated antizyme levels and stability. The sensitivity of antizyme turnover to osmotic conditions was also observed in DH23b cells, which contain elevated levels of more stable antizyme. Interestingly, the two main antizyme proteins, AZ-1 and AZ-2 (presumably products from the first and second translational start sites), differed in their responses to these changing osmotic conditions. Just as feedback regulation of antizyme synthesis provides an effective mechanism for maintaining stable polyamine levels, these studies suggest that alteration in the rate of antizyme degradation may be the mechanism whereby cells adjust steady-state polyamine levels in response to stimulation or stress.
Analysis of abnormalities in purine metabolism leading to gout and to neurological dysfunctions in man
Authors: CURTO Raul , VOIT O. Eberhard , CASCANTE Marta
Published: August 2015

A modelling approach is used to analyse diseases associated with purine metabolism in man. The specific focus is on deficiencies in two enzymes, hypoxanthine:guanine phosphoribosyltransferase and adenylosuccinate lyase. These deficiencies can lead to a number of symptoms, including neurological dysfunctions and mental retardation. Although the biochemical mechanisms of dysfunctions associated with adenylosuccinate lyase deficiency are not completely understood, there is at least general agreement in the literature about possible causes. Simulations with our model confirm that accumulation of the two substrates of the enzyme can lead to significant biochemical imbalance. In hypoxanthine:guanine phosphoribosyltransferase deficiency the biochemical mechanisms associated with neurological dysfunctions are less clear. Model analyses support some old hypotheses but also suggest new indicators for possible causes of neurological dysfunctions associated with this deficiency. Hypoxanthine:guanine phosphoribosyltransferase deficiency is known to cause hyperuricaemia and gout. We compare the relative importance of this deficiency with other known causes of gout in humans. The analysis suggests that defects in the excretion of uric acid are more consequential than defects in uric acid synthesis such as hypoxanthine:guanine phosphoribosyltransferase deficiency.
Capacity for repeatable leukotriene generation after transient stimulation of mast cells and macrophages
Authors: BROCK G. Thomas , McNISH W. Robert , PETERS-GOLDEN Marc
Published: August 2015

Leukotriene (LT) synthesis is initiated by the enzyme 5-lipoxygenase (5-LO). Prolonged cell stimulation causes the translocation of 5-LO to the nuclear envelope and the synthesis of LT, with subsequent inactivation and persistent membrane association of 5-LO. In this study, we examined whether persistent membrane association of 5-LO, as well as the inactivation of 5-LO, could be prevented by shortening the length of cell stimulation or by blocking LT synthesis. As expected, stimulation of rat basophilic leukaemia (RBL) cells, a mast cell model, or alveolar macrophages (AMs) with calcium ionophore for 15 min caused 5-LO translocation, LT generation and the inactivation and persistent membrane association of 5-LO. When RBL cells or AMs instead were stimulated for 0.5-5 min, translocation of 5-LO and synthesis of LT still occurred. However, after washing and resting, the 5-LO enzyme returned to its original intracellular distribution. Furthermore these cells showed a retained capacity for LT synthesis on subsequent re-stimulation. Similar results were obtained when cells were stimulated with either formyl peptide or zymosan, instead of ionophore. In contrast, blockade of LT synthesis during the initial stimulation, with the selective inhibitors zileuton or MK-886, did not inhibit 5-LO translocation, inactivation or persistent membrane association resulting from prolonged cell stimulation. We conclude that, in long-lived immune cells, 5-LO translocation is reversible when cell stimulation is short, but persistent after prolonged stimulation. In addition 5-LO remains active and LT synthetic capacity is retained after transient stimulation, whereas significant inactivation of 5-LO occurs after prolonged stimulation. Finally, results with LT synthesis inhibitors indicate that inactivation and persistent membrane association of 5-LO can result independently of 5-LO activation.
tRNA is entrapped in similar, but distinct, nuclear and cytoplasmic ribonucleoprotein complexes, both of which contain vigilin and elongation factor 1α
Authors: KRUSE Charli , GRÜNWELLER Arnold , WILLKOMM K. Dagmar , PFEIFFER Thomas , HARTMANN K. Roland , MÜLLER K. Peter
Published: August 2015

Vigilin, which is found predominantly in cells and tissues with high levels of protein biosynthesis, was isolated in its native form from human HEp-2 cells (A.T.C.C. CCL23) by immunoaffinity chromatography. Here we demonstrate that vigilin is part of a novel large tRNA-binding ribonucleoprotein complex (tRNP), found not only in the cytoplasm, but also in the nuclei of human cells. Compositional differences in the protein pattern were detected between the nuclear and cytoplasmic tRNPs, although some properties of the purified nuclear tRNP, such as tRNA protection against nuclease attack, were identical with those of the cytoplasmic tRNP. By using either a pool of total human nuclear RNA or radioactively labelled yeast tRNAAsp in rebinding experiments, we could show that tRNA is specifically recaptured by the RNA-depleted, vigilin-containing nuclear complex. We could also show that vigilin is capable of binding tRNA in vitro. Another tRNA-binding protein is elongation factor 1α, which appears to be enriched in the cytoplasmic and nuclear tRNP complexes. This suggests that the cytoplasmic tRNP may be involved in the channelled tRNA cycle in the cytoplasm of eukaryotic cells. Our results also suggest that the nuclear vigilin-containing tRNP may be related to the nuclear export of tRNA.
Contribution of newly synthesized cholesterol to rat plasma and bile determined by mass isotopomer distribution analysis: bile-salt flux promotes secretion of newly synthesized cholesterol into bile
Authors: BANDSMA Robert H. J. , STELLAARD Frans , VONK J. Roel , NAGEL T. Gijs , NEESE A. Richard , HELLERSTEIN K. Marc , KUIPERS Folkert
Published: August 2015

To quantify the contribution of newly synthesized cholesterol to total plasma and biliary cholesterol under physiological conditions, unrestrained rats were infused intravenously with [1-13C]acetate (0.6 mmol/h per kg) from 12:00 to 24:00 h, and fractional and absolute cholesterol-synthesis rates were determined by mass isotopomer distribution analysis (MIDA). As bile diversion leads to changes in cholesterol metabolism, rats were equipped with permanent catheters in the bile duct and duodenum, allowing sampling of small amounts of bile from an intact enterohepatic circulation. For comparison, rats with chronic bile diversion were also studied. Fractional synthesis of plasma cholesterol was 10.8±1.7% (mean±S.D.) after 12 h in rats with intact circulation. Fractional synthesis of biliary cholesterol was significantly higher than that of plasma cholesterol, i.e. 16.5±2.0% (P < 0.05) after 12 h. In contrast, no differences between fractional synthesis of cholesterol in plasma and bile were found in bile-diverted animals (31.8±2.1 and 33.1±3.3% respectively after 12 h). The calculated absolute rate of cholesterol biosynthesis increased from 53±10 to 221±19 μmol/day per kg after bile diversion. A comparison of MIDA results with those obtained from balance studies indicated that MIDA does not assess total body synthesis in rats, presumably because of incomplete equilibration of newly synthesized molecules with cholesterol in the plasma compartment. These studies demonstrate that the contribution of newly synthesized cholesterol to biliary cholesterol is higher than to plasma cholesterol under physiological conditions, probably reflecting bile-salt-induced secretion of newly formed cholesterol by the periportal hepatocytes.
Binding of MARCKS (myristoylated alanine-rich C kinase substrate)-related protein (MRP) to vesicular phospholipid membranes
Authors: VERGÈRES Guy , RAMSDEN J. Jeremy
Published: August 2015

The myristoylated alanine-rich C kinase substrate (MARCKS) protein family has two known members, MARCKS itself and MARCKS-related protein (MRP, also called MacMARCKS or F52). They are essential for brain development and are believed to regulate the structure of the actin cytoskeleton at the plasma membrane. Hence membrane binding is central to their function. MARCKS has been quite extensively characterized; MRP much less so. Despite the fact that MRP is only two thirds the size of MARCKS, it has hitherto been assumed that the two proteins have similar properties. Here we make a detailed study, including the effects of myristoylation, lipid composition, calmodulin and phosphorylation of the binding of MRP to phospholipid vesicles. We show that both the N-terminal myristoyl moiety and the central effector domain mediate binding. MRP behaves like MARCKS in the presence of neutral phospholipids. In contrast to MARCKS, however, the incorporation of 20% of negatively-charged phospholipids only marginally increases the affinity of myristoylated MRP. Co-operativity between the myristoyl moiety and the effector domain of MRP is weak and the protein has a significantly lower affinity for these vesicles compared with MARCKS. Furthermore, calmodulin or phosphorylation of the effector domain by the catalytic subunit of protein kinase C do not significantly decrease the binding of myristoylated MRP to negatively-charged phospholipid vesicles. Our results show that the mechanisms regulating the interactions of MARCKS and MRP with phospholipid vesicles are, at least quantitatively, different. In agreement with cellular studies, we therefore propose that MARCKS and MRP have different subcellular localization and, consequently, different functions.
Oscillating response to a purine nucleotide disrupted by mutation in Paramecium tetraurelia
Authors: MIMIKAKIS L. John , NELSON L. David , PRESTON R. Robin
Published: August 2015

The purine nucleotide GTP, when added extracellularly, induces oscillations in the swimming behaviour of the protist Paramecium tetraurelia. For periods as long as 10 min the cell swims backwards and forwards repetitively. The oscillations in swimming behaviour are driven by changes in membrane potential of the cell, which in turn are caused by periodic activation of inward Mg2+- and Na+-specific currents. We screened for and isolated mutants that are defective in this response, exploiting the fact that the net result of GTP on a population of cells is repulsion. One mutant, GTP-insensitive (gin A), is not repelled by GTP. In addition, GTP fails to induce repetitive backwards swimming in gin A mutants, although they swim backwards normally in response to other stimuli. GTP fails to evoke oscillations in membrane potential or Mg2+ and Na+ currents in the mutant, although the Mg2+ and Na+ conductances are not themselves measurably affected. A small, oscillating Ca2+ current induced by GTP in the wild type, which might be part of the mechanism that generates oscillations, is also missing from gin A cells. To our knowledge, gin A is the first example of a mutant defective in a purinergic response. We discuss the possibility that the gin A lesion affects the oscillator itself.
The nuclear factor interleukin-6 (NF-IL6) and signal transducer and activator of transcription-3 (STAT-3) signalling pathways co-operate to mediate the activation of the hsp90β gene by interleukin-6 but have opposite effects on its inducibility by heat shock
Authors: STEPHANOU Anastasis , ISENBERG A. David , AKIRA Shizuo , KISHIMOTO Tadamitsu , LATCHMAN S. David
Published: August 2015

The levels of the 90 kDa heat-shock protein (hsp90) and the activity of the hsp90β gene promoter are increased in response to treatment by interleukin (IL)-6. The hsp90β gene promoter contains binding sites for the transcription factors nuclear factor IL-6 (NF-IL6) and signal transducer and activator of transcription 3 (STAT-3), which are activated respectively by the mitogen-activated-protein-kinase and Jak-kinase pathways following IL-6 treatment. Both these factors can activate the hsp90 promoter and have a strong synergistic effect on its activity, which appears to be critical for IL-6-mediated activation of the promoter. Interestingly, the two factors interact differently with the heat-shock factor (HSF) and a heat-shock stress. Thus STAT-3 reduces the stimulatory effect of heat shock whereas NF-IL6 enhances it. When applied together, heat shock and IL-6 produce only weak activation of the hsp90 promoter compared with either stimulus alone, indicating that the inhibitory effect of STAT-3 on HSF predominates under these conditions. In contrast, IL-1, which activates only the NF-IL6 pathway, synergizes with heat shock to produce strong activation of hsp90. These effects are discussed in terms of the multiple stimuli that may regulate the hsp90 promoter in unstressed cells and their interaction with its stress-mediated activation.
C1q-mediated chemotaxis by human neutrophils: involvement of gClqR and G-protein signalling mechanisms
Authors: LEIGH E. A. Leonora , GHEBREHIWET Berhane , PERERA P. S. Tim , BIRD N. Ian , STRONG Peter , KISHORE Uday , REID B. M. Kenneth , EGGLETON Paul
Published: August 2015

C1q, the first component of the classical pathway of the complement system, interacts with various cell types and triggers a variety of cell-specific cellular responses, such as oxidative burst, chemotaxis, phagocytosis, etc. Different biological responses are attributed to the interaction of C1q with more than one putative cell-surface C1q receptor/C1q-binding protein. Previously, it has been shown that C1q-mediated oxidative burst by neutrophils is not linked to G-protein-coupled fMet-Leu-Phe-mediated response. In the present study, we have investigated neutrophil migration brought about by C1q and tried to identify the signal-transduction pathways involved in the chemotactic response. We found that C1q stimulated neutrophil migration in a dose-dependent manner, primarily by enhancing chemotaxis (directed movement) rather than chemokinesis (random movement). This C1q-induced chemotaxis could be abolished by an inhibitor of G-proteins (pertussis toxin) and PtdIns(3,4,5)P3 kinase (wortmannin and LY294002). The collagen tail of C1q appeared to mediate chemotaxis. gC1qR, a C1q-binding protein, has recently been reported to participate in C1q-mediated chemotaxis of murine mast cells and human eosinophils. We observed that gC1qR enhanced binding of free C1q to adherent neutrophils and promoted C1q-mediated chemotaxis of neutrophils by nearly seven-fold. Our results suggests C1q-mediated chemotaxis involves gC1qR as well as G-protein-coupled signal-transduction mechanisms operating downstream to neutrophil chemotaxis.
Identification of the structural similarity in the functionally related amidohydrolases acting on the cyclic amide ring
Authors: Kim G.-J. , Kim H.-S.
Published: August 2015

The functionally related amidohydrolases, including D-hydantoinases, dihydropyrimidinases, allantoinases and dihydro-orotases, share a similar catalytic function of acting on the cyclic amide ring. We aligned 16 amidohydrolases by taking account of the conservative substitution and found a number of highly conserved regions and invariant amino acid residues. Analyses of the secondary structure and hydropathy profile of the enzymes revealed a significant degree of similarity in the conserved regions. Among the regions, the long stretched region I is of particular interest, because it is mainly composed of invariant amino acid residues, showing a similarity of 69% for the enzymes. A search of the protein data bank using the sequence of the conserved region I identified a number of proteins possessing a similar catalytic property, providing a clue that this region might be linked with the catalytic function. As a particular sequence, one aspartic acid and four histidine residues are found to be rigidly conserved in the functionally related amidohydrolases. In order to investigate the significance of the conserved residues, site-directed mutagenesis was carried out typically for the D-hydantoinase gene cloned from Bacillus stearothermophilus SD1. These residues were found to be essential for metal binding as well as catalysis, strongly implying that these invariant residues play a critical role in other enzymes as well as in D-hydantoinase. On the basis of the similar catalytic function and existence of the rigidly conserved sequence, we propose a close evolutionary relationship among the functionally related amidohydrolases, including D-hydantoinase, dihydropyrimidinase, allantoinase and dihydro-orotase.
Functional analysis of the endothelial cell-specific Tie2/Tek promoter identifies unique protein-binding elements
Authors: FADEL M. Bahaa , BOUTET C. Stephane , QUERTERMOUS Thomas
Published: August 2015

To investigate the molecular basis of endothelial cell-specific gene expression, we have examined the DNA sequences and the cognate DNA-binding proteins that mediate transcription of the murine tie2/tek gene. Reporter transfection experiments conformed with earlier findings in transgenic mice, indicating that the upstream promoter of Tie2/Tek is capable of activating transcription in an endothelial cell-specific fashion. These experiments have also allowed the identification of a single upstream inhibitory region (region I) and two positive regulatory regions (regions U and A) in the proximal promoter. Electrophoretic mobility-shift assays have allowed further characterization of three novel DNA-binding sequences associated with these regions and have provided preliminary characterization of the protein factors binding to these elements. Two of the elements (U and A) confer increased transcription on a heterologous promoter, with element U functioning in an endothelial-cell-selective manner. By employing embryonic endothelial-like yolk sac cells in parallel with adult-derived endothelial cells, we have identified differences in functional activity and protein binding that may reflect mechanisms for specifying developmental regulation of tie2/tek expression. Further study of the DNA and protein elements characterized in these experiments is likely to provide new insight into the molecular basis of developmental- and cell-specific gene expression in the endothelium.