bluuebunny/crossref_metadata_2025 huggingface dataset:
PCR-based cloning of the full-length Neurospora eukaryotic initiation factor 5A cDNA: polyhistidine-tagging and overexpression for protein affinity binding
Authors: Tao Y , Chen K Y
Published: August 2015

Eukaryotic initiation factor 5A (eIF-5A) is the only cellular protein known to contain a hypusine residue that is formed by transferring the aminobutyl moiety from spermidine to a specific lysine residue, followed by hydroxylation at the aminobutyl group. A simple PCR-based strategy was developed to obtain a full-length cDNA of Neurospora crassa eIF-5A. The strategy consists of (i) the design of a pair of key primers (21-mer) based on the highly conserved eIF-5A cDNA domains known in other species, (ii) PCR amplification of Neurospora cDNA using the two key primers to obtain the core sequence for the design of core primers, and (iii) combined use of the key primers, core primers and the universal primers, T3 and T7, to amplify the target sequence in a Neurospora cDNA library. The longest cDNA obtained was cloned into pBlueScript phagemid, and sequence analysis indicated that it encodes a polypeptide of 163 amino acid residues with a codon usage preference characteristic of abundant Neurospora genes. The Neurospora polypeptide showed 59% and 67% identity with human and yeast eIF-5A precursor protein respectively. We subcloned the Neurospora eIF-5A cDNA into pQE-30, which introduces six adjacent histidine residues to the N-terminus of the recombinant protein. The resulting plasmid, pQTy21, was overexpressed in Escherichia coli, and the soluble polyhistidine-tagged protein was purified by metal chelation chromatography. We obtained about 60 mg of purified eIF-5A precursor from 1 litre of culture in a single step using a Ni(II)-nitrilotriacetic acid (NTA)-agarose column. The histidine-tagged eIF-5A precursor protein could be recognized by anti-Neurospora crassa 21 kDa protein serum raised against wild-type eIF-5A precursor and could serve as the substrate protein for deoxyhypusine synthase. Using the histidine-tagged recombinant protein and the Ni(II)-NTA-agarose column, we constructed a protein affinity column and demonstrated an affinity binding between eIF-5A precursor and deoxyhypusine synthase in the presence of NAD+. One-step eIF-5A precursor affinity-column chromatography could lead to a 30-fold purification of deoxyhypusine synthase.
Mutagenesis of two surface-exposed loops of the Bacillus thuringiensis CryIC δ-endotoxin affects insecticidal specificity
Authors: Smith G P , Ellar D J
Published: August 2015

Site-directed mutagenesis was used to determine the role of two surface-exposed loops (Gly-317-Phe-320 and Gln-374-Pro-377) in the insecticidal specificity of the Bacillus thuringiensis CryIC delta-endotoxin. Mutant toxins were generated by PCR using degenerate oligonucleotide primers, and expressed in Escherichia coli. More than 50 mutant toxins were screened for toxicity to the lepidopteran Spodoptera frugiperda Sf9 cell line using an in vitro lawn assay. A panel of these mutant toxins, which included toxic and non-toxic variants from both loops, was further screened for activity towards Aedes aegypti larvae. The activity of these mutants to Sf9 cells was quantified more precisely using a cell lysis assay. Three categories of mutants were identified: (1) those non-toxic to either Sf9 cells or Aedes aegypti larvae; (2) those fully toxic to both genera; and (3) those which were only toxic to Sf9 cells. For the first loop, the differential specificity was not restricted to any single residue. In the second loop, two mutant toxins with a Pro-377-->Ala substitution displayed this phenotype. The time dependence of toxicity towards Sf9 cells was examined using the same panel of mutants. All toxic mutants displayed an identical time course to the wild-type toxin, with the exception of the two Pro-377-->Ala mutants of the second loop. These toxins displayed a lower time dependence, no cell death occurring within the first hour of incubation. These results show that the two loops are important determinants of both the activity and specificity of the CryIC delta-endotoxin.
86Rb is not a reliable tracer for potassium in skeletal muscle
Authors: Dørup I , Clausen T
Published: August 2015

For technical reasons, 86Rb is frequently preferred to 42K as a tracer for K+. Systematic comparisons of the two isotopes, however, are rarely done. In this paper we compare the transport of 42K and 86Rb in rat and mouse soleus muscle and in rat erythrocytes. Ouabain-suppressible K+ uptake in rat soleus was the same whether measured with 42K or 86Rb, both when stimulated by insulin, salbutamol and calcitonin-gene-related peptide (CGRP), and when inhibited by graded concentrations of ouabain. Control experiments with rat erythrocytes, where Na(+)-K(+)-Cl- co-transport has earlier been demonstrated, showed closely similar inhibitory effects of bumetanide on 42K and 86Rb uptake. In contrast, bumetanide produced no significant change in 42K uptake of rat and mouse soleus muscle, but clearly inhibited 86Rb uptake at concentrations down to 10(-7) M (P < 0.001). Whereas the addition of 150 mM NaCl had no effect on 42K uptake in rat soleus, 86Rb uptake, and in particular the bumetanide-suppressible component, was markedly increased by this addition. The inhibitory effect of bumetanide on 86Rb uptake gives rise to the false impression that skeletal muscle contains a NaKCl2 co-transport system. Efflux studies showed that the fractional loss of 42K from rat soleus muscle is 2.3 times larger than that of 86Rb. Salbutamol and CGRP increased 86Rb efflux, but inhibited 42K efflux. This implies that for studies of K+ efflux and bumetanide-sensitive K+ transport, 86Rb is not even an acceptable tracer for the detection of qualitative changes. Control experiments with 42K are essential in any characterization of unknown K+ transport processes.
Organization of the 5′ region of the rat ATP citrate lyase gene
Authors: Kim K S , Park S W , Moon Y A , Kim Y S
Published: August 2015

A genomic clone, encompassing the 5′ flanking region and the first seven exons of rat ATP citrate lyase gene, was isolated from a rat genomic library and sequenced. Primer-extension analysis showed that mRNA is transcribed at 4407 nucleotides upstream from the translation start site. Primer-extension analysis and sequencing of ATP citrate lyase cDNA amplified by PCR showed that the promoter used for transcription is identical in mammary gland, lung, liver, brain and kidney. Southern-blot analysis showed that the ATP citrate lyase gene exists as a single copy. The 5′ flanking region contains several consensus sequences defined as promoter elements. These include a CAAT box and Sp1-binding sites. However, a TATA box lacks this promoter. The expression of the chloramphenicol acetyltransferase gene was induced by the 5‘ flanking region (-2370 to -1) in the CHO cell line. The 5′ flanking region also contains several sequence elements that may be involved in the transcriptional regulation of the gene.
DNA strand cleavage as a sensitive assay for the production of hydroxyl radicals by microsomes: role of cytochrome P4502E1 in the increased activity after ethanol treatment
Authors: Kukielka E , Cederbaum A I
Published: August 2015

There is increasing interest in the role of reactive oxygen radicals in the hepatotoxicity associated with ethanol consumption. Reactive oxygen intermediates interact with DNA and can cause single-strand breaks of supercoiled DNA. Experiments were carried out to evaluate the utility of this system as a sensitive assay for the detection of potent oxidants generated by rat liver microsomes isolated from pair-fed control rats and rats treated chronically with ethanol. DNA strand cleavage was assayed by monitoring the migration of the supercoiled and open circular forms in agarose. Microsomes catalysed DNA strand breakage with either NADPH or NADH as cofactors; iron was required to catalyse the reaction and various ferric complexes were effective in promoting the reaction. DNA strand cleavage was prevented by catalase, superoxide dismutase, GSH and hydroxyl-radical-scavenging agents, suggesting that a hydroxyl-radical-like species was the oxidant responsible for the breakage. This assay system proved to be much more sensitive in detecting hydroxyl radicals than are other methods, such as e.s.r. spectroscopy or oxidation of chemical scavenging agents with respect to the amount of microsomal protein and the nature and concentration of the iron catalyst required. Microsomes from ethanol-treated rats were more reactive than control microsomes in catalysing the DNA strand cleavage with either NADPH or NADH; increased catalytic activity was observed with various ferric complexes and was sensitive to the above antioxidants. Compared with preimmune IgG, anti-(cytochrome P4502E1) IgG had no effect on DNA strand cleavage by the control microsomes, but completely prevented the NADPH- and the NADH-dependent increased activity found with microsomes from the ethanol-treated rats. Inhibitors of cytochrome P4502E1, such as diethyl dithiocarbamate and tryptamine, also lowered the extent of increase of DNA strand cleavage produced by microsomes from the ethanol-treated rats. These results indicate that DNA strand cleavage is a very sensitive assay for detecting the production of hydroxyl radicals by microsomes and to demonstrate increased activity by microsomes after chronic ethanol treatment. This increased activity with NADPH and NADH is due, at least in part, to induction of cytochrome P4502E1.
Stimulatory effect of pervanadate on calcium signals and histamine secretion of RBL-2H3 cells
Authors: Teshima R , Ikebuchi H , Nakanishi M , Sawada J
Published: August 2015

We examined the effect of pervanadate on the activation of rat basophilic leukaemia (RBL-2H3) cells. The pervanadate, generated from a combination of H2O2 and vanadate (Vi), induced concomitantly protein tyrosine phosphorylation, formation of inositol 1,4,5-trisphosphate (IP3), an increase in [Ca2+]i, and histamine secretion in RBL-2H3 cells. These effects were clearly dependent on the ratio of H2O2/Vi. The secretion of histamine, IP3 formation, and sustained increase in [Ca2+]i were effectively induced by treatment of the cells with the pervanadate produced from 1 mM H2O2 and 1 mM Vi. These effects mimic the stimulatory effects of an antigen (dinitrophenylated BSA) on Ca2+ signals, histamine secretion and morphological changes. Protein tyrosine phosphorylation, formation of IP3 and transient increase in [Ca2+]i were markedly induced by the pervanadate produced from 3 mM H2O2 and 1 mM Vi. However, histamine secretion induced by the pervanadate was very low. After the pervanadate from 3 mM H2O2 and 1 mM Vi was treated with catalase, it was able to induce the [Ca2+]i increase and histamine secretion as much as the antigen did. This indicates that pervanadate from a lower H2O2 concentration (1 mM H2O2/1 mM Vi) and catalase-treated pervanadate from a higher H2O2 concentration (3 mM H2O2/1 mM Vi) are able to mimic the activity that was caused by cross-linking of IgE receptors with antigen. The present results also demonstrate that protein tyrosine phosphorylation seems to have a crucial role in Ca2+ entry from the external medium, and that a sustained [Ca2+]i increase is an important step for histamine secretion in RBL-2H3 cells.
Sulphated and undersulphated heparan sulphate proteoglycans in a Chinese hamster ovary cell mutant defective in N-sulphotransferase
Authors: Bame K J , Zhang L , David G , Esko J D
Published: August 2015

The Chinese hamster ovary cell mutant, pgsE-606, synthesizes undersulphated heparan sulphate glycosaminoglycans because of a deficiency in N-sulphotransferase activity [Bame and Esko (1989) J. Biol. Chem. 264, 8059-8065]. We compared the heparan sulphate proteoglycans synthesized by mutant and wild-type cells to determine what effect the undersulphation defect had on proteoglycan structure. The majority of heparan sulphate proteoglycans synthesized by pgsE-606 were undersulphated, but the mutant also synthesized a population of proteoglycans that were sulphated to the same extent as wild-type molecules. Anion-exchange analysis of the glycosaminoglycans in each proteoglycan population showed that they were all modified in the same way. The length of the glycosaminoglycans in each proteoglycan population were similar, suggesting that N-sulphation does not affect chain polymerization. To examine whether the sulphation state of the attached heparan sulphate glycosaminoglycans was dependent on the protein core, we purified syndecan-1 from mutant and wild-type cells using antibodies against the core protein. As with the unfractionated heparan sulphate proteoglycans, pgsE-606 synthesized both undersulphated and sulphated syndecan-1. Each pool contained either undersulphated or sulphated glycosaminoglycan chains respectively. Thus the modification of all heparan sulphate chains on a core protein occurs on a proteoglycan-wide basis (i.e. to the same extent).
Differential regulation of the expression of fast-type sarcoplasmic-reticulum Ca2+-ATPase by thyroid hormone and insulin-like growth factor-I in the L6 muscle cell line
Authors: Thelen M H M , Muller A , Zuidwijk M J , van der Linden G C , Simonides W S , van Hardeveld C
Published: August 2015

The aim of this study was to investigate the mechanism(s) underlying the thyroid-hormone (L-tri-iodothyronine, T3)-induced elevation of fast-type sarcoplasmic-reticulum Ca(2+)-ATPase (SERCA1) levels in L6 myotubes and the potentiating effect of insulin-like growth factor-I (IGF-I) [Muller, van Hardeveld, Simonides and van Rijn (1991) Biochem. J. 275, 35-40]. T3 increased the SERCA1 protein level (per microgram of DNA) by 160%. The concomitant increase in the SERCA1 mRNA level was somewhat higher (240%). IGF-I also increased SERCA1 protein (110%) and mRNA levels (50%), whereas IGF-I + T3 increased SERCA1 protein and mRNA levels by 410% and 380% respectively. These SERCA1 mRNA analyses show that the more-than-additive action of T3 and IGF-I on SERCA1 expression is, at least in part, pre-translational in nature. Further studies showed that the half-life of SERCA1 protein in L6 cells (17.5 h) was not altered by T3. In contrast, IGF-I prolonged the half-life of SERCA1 protein 1.5-1.9-fold, which may contribute to the disproportional increase in SERCA1 protein content compared with mRNA by IGF-I. Measurements of SERCA1 mRNA half-life (as determined by actinomycin D chase) showed no difference from the control values (15.5 h) in the presence of T3 or IGF-I alone. When T3 and IGF-I were both present, the SERCA1 mRNA half-life was prolonged 2-fold. No significant effects of T3 and IGF-I were observed on the half-life of total protein (37.4 h) and total RNA (37.0 h). The absence of an effect of T3 on SERCA1 protein and mRNA stability, when it was present alone, suggested transcriptional regulation, which was confirmed by nuclear run-on experiments, showing a 3-fold increase in transcription frequency of the SERCA1 gene by T3. We conclude that the synergistic stimulating effects of T3 and IGF-I on SERCA1 expression are the result of both transcriptional and post-transcriptional regulation. T3 acts primarily at the transcriptional level by increasing the transcription frequency of the SERCA1 gene, whereas IGF-I seems to act predominantly at post-transcriptional levels by enhancing SERCA1 protein and mRNA stability, the latter, however, only in the presence of T3.
Role of hydrogen peroxide in hypoxia-induced erythropoietin production
Authors: Fandrey J , Frede S , Jelkmann W
Published: August 2015

The addition of exogenous H2O2 inhibited hypoxia-induced erythropoietin (Epo) production in the human hepatoma cell line HepG2. Likewise, elevation of endogenous H2O2 levels by the addition of menadione or the catalase inhibitor, aminotriazole, dose-dependently lowered Epo production. The inhibitory effect of exogenous H2O2 on Epo formation could be completely overcome by co-incubation with catalase. When GSH levels in HepG2 cells were lowered, Epo production was more susceptible to H2O2-induced inhibition, indicating that H2O2 might affect thiol groups in regulatory proteins. Endogenous production of H2O2 in HepG2 cells was dependent on the pericellular O2 tension, being lowest under conditions of hypoxia. Our results support the hypothesis that an H2O2-generating haem protein might be part of the O2 sensor that controls Epo production. High H2O2 levels under conditions of normoxia suppress, whereas lower levels in hypoxic cells allow epo gene expression.
Serum factors, growth factors and UDP-sugar metabolism in bovine articular cartilage chondrocytes
Authors: Ysart G E , Mason R M
Published: August 2015

1. The effect of different batches of fetal bovine serum and of growth factors on [35S]sulphate incorporation into glycosaminoglycans and on UDP-sugar pools in explant cultures of bovine articular cartilage was investigated. 2. [35S]Sulphate incorporation was variably stimulated between 1.2- and 3.5-fold by four different batches of serum. The UDP-glucuronate pool size expanded 4.3-6.5-fold in the presence of serum, even in those cultures in which little stimulation of [35S]sulphate incorporation occurred. The UDP-N-acetylhexosamine and UDP-hexose pools expanded by about 1.5- and 2.0-fold respectively in the presence of serum. UDP-xylose was not detected. 3. Equilibrium-labelling and pulse-chase experiments with D-[1-3H]glucose indicated that the rate of flux through the UDP-sugar pools was unaffected by serum. UDP-hexose, UDP-N-acetylhexosamine and UDP-glucuronate have approximate half-lives (t1/2) of 7, 12 and 3-4 min respectively. At equilibrium, the 3H specific activities of UDP-hexose and UDP-N-acetylhexosamine were very similar but that for the UDP-glucuronate pool was much higher, especially in serum-supplemented cultures. The results suggest that UDP-glucuronate synthesis occurs via a pathway which is independent of the main UDP-hexose pathway. 4. Supplementing cultures with heat-treated serum had no effect on the serum-induced expansion of UDP-sugar pools but stimulation of [35S]sulphate incorporation into glycosaminoglycans was 50% lower than for native serum. Acid-treated serum promoted a 2-fold expansion of the UDP-glucuronate and UDP-N-acetylhexosamine pool over that obtained with native serum but was 20% less effective in stimulating [35S]sulphate incorporation than the latter. Prior dialysis of serum had no effect on its modulatory action on either [35S]sulphate incorporation or on the size of UDP-sugar pools. 5. Insulin-like growth factor 1 (IGF-1), transforming growth factor beta-1 (TGF beta-1), platelet-derived growth factor (PDGF) (BB homodimer) and epidermal growth factor (EGF) all stimulated [35S]sulphate incorporation into glycosaminoglycans as expected. The UDP-glucuronate pool expanded by 1.5- and 2.0-fold in the presence of IGF-1 and TGF beta-1 respectively, and by about 1.8-fold in the presence of PDGF or EGF. None of the factors investigated, or combinations of IGF-1 and TGF beta-1 or IGF-1 and EGF, stimulated expansion of the UDP-glucuronate pool to the same extent as native serum.(ABSTRACT TRUNCATED AT 400 WORDS)
β1-4N-acetylgalactosaminyltransferase can synthesize both asialoglycosphingolipid GM2 and glycosphingolipid GM2in vitro and in vivo: isolation and characterization of a β1-4N-acetylgalactosaminyltransferase cDNA clone from rat ascites hepatoma cell line AH7974F
Authors: Hidari K I-P J , Ichikawa S , Furukawa K , Yamasaki M , Hirabayashi Y
Published: August 2015

We have cloned a cDNA encoding beta 1-4N-acetylgalactosaminyltransferase (EC 2.4.1.92) (GalNAc-T) from rat ascites hepatoma of the free-cell type AH7974F. The cell line only expressed asialo-series glycosphingolipids (GSLs) including asialo-GM2 [Taki, T., Hirabayashi, Y., Ishiwata, Y., Matsumoto, M., and Kojima, K. (1979) Biochim. Biophys. Acta 572, 113-120]. The cDNA, pGNA56, was isolated by screening AH7974F cDNA library in lambda gt10 with a probe. The probe was obtained from AH7974F cDNA by PCR using primers with the nucleotide sequence of the human GalNAc-T cDNA. The amino acid sequence deduced from the nucleotide sequence of pGNA56 exhibited 88% similarity to the human GalNAc-T sequence. The enzyme was a typical type II membrane protein, which consisted of a short N-terminal residue, a transmembrane region, and a long C-terminal residue, including the catalytic domain. The substrate specificity of rat GalNAc-T was determined using homogenates from cells into which the cDNA clone was transfected. The enzyme catalysed not only the formation of GM2 and GD2 from GM3 and GD3 respectively, but also asialo-GM2 from CDH. It also acted on GSL substrates, including GM1b, sialylparagloboside and GD1 alpha. On the other hand, the enzyme did not transfer GalNAc to soluble substrates such as glycoproteins and oligosaccharide. The GSL compositional and immunocytochemical analyses of stable transfectants obtained by transfection of the cDNA showed simultaneous expression of asialo-GM2 and GM2 on the plasma membrane. Therefore, we concluded that the formation of asialo-GM2, GM2 and GD2 was catalysed by the single GalNAc-T. Northern-blot hybridization showed that the GalNAc-T mRNA was strongly expressed in rat brain, testis, and spleen. The gene was also expressed in rat normal liver to a lesser extent. We found the GSLs in asialo- and alpha-pathways such as asialo-GM1 and GD1 alpha in the rat tissues by using a sensitive t.l.c.-immunostaining method. These observations also supported our conclusion that the single GalNAc-T synthesizes asialo-GM2, GM2 and GD2 in vivo.
The microsomal epoxide hydrolase has a single membrane signal anchor sequence which is dispensable for the catalytic activity of this protein
Authors: Friedberg T , Löllmann B , Becker R , Holler R , Oesch F
Published: August 2015

The microsomal epoxide hydrolase (mEH) catalyses the hydrolysis of reactive epoxides which are formed by the action of cytochromes P-450 from xenobiotics. In addition it has been suggested that mEH might mediate the transport of bile acids. For the mEH it has been shown that it is co-translationally inserted into the endoplasmic reticulum. Here we demonstrate that the N-terminal 20 amino acid residues of this protein serve as its single membrane anchor signal sequence and that the function of this sequence can also be supplied by a cytochrome P-450 (CYP2B1) anchor signal sequence. The evidence supporting this conclusion is as follows: (i) the rat mEH and a CYP2B1-mEH fusion protein, in which the CYP2B1 membrane anchor signal sequence replaced the N-terminal 20 amino acid residues of mEH, was co-translationally inserted into dog pancreas microsomes in a cell-free translation system, whereas a truncated epoxide hydrolase with a deletion of the 20 N-terminal amino acid residues was not co-translationally inserted. (ii) The mEH and the CYP2B1-mEH fusion protein, but not the truncated epoxide hydrolase, were anchored in microsomes in a cell-free translation system and in membrane fractions derived from fibroblasts which expressed these proteins heterologously. These fibroblasts were also used to evaluate the significance of the mEH membrane anchor for the catalytic activity of mEH. The mEH, the truncated mEH and the CYP-EH fusion protein were found to be enzymically active. This result shows that the membrane anchor signal sequence of mEH is dispensable for the catalytic activity of this protein. However, truncated mEH was only expressed at low levels, which might indicate that this protein is unstable.
The functional importance of the extreme C-terminal tail in the gene 2 organellar Ca2+-transport ATPase (SERCA2a/b)
Authors: Verboomen H , Wuytack F , Van den Bosch L , Mertens L , Casteels R
Published: August 2015

Ca(2+)-uptake experiments in microsomal fractions from transfected COS-1 cells have revealed a functional difference between the non-muscle SERCA2b Ca2+ pump and its muscle-specific SERCA2a splice variant. Structurally, the two pumps differ only in their C-terminal tail. The last four amino acids of SERCA2a are replaced in SERCA2b by a 49-residue-long peptide chain containing a very hydrophobic stretch which could be an additional transmembrane segment. The functionally important subdomains in the SERCA2b tail were analysed by constructing three SERCA2b deletion mutants lacking 12, 31 or 49 amino acids. The mutants and the parental SERCA2 pumps were expressed in COS-1 cells and analysed for functional difference. SERCA2b had a twofold higher Ca2+ affinity, a twofold lower turnover rate and a 10-fold lower vanadate-sensitivity than SERCA2a and the mutants. Since each of the three truncated versions of SERCA2b acquire the characteristic properties of SERCA2a, it is concluded that the stretch of the last 12 residues of SERCA2b is of critical importance.
Recombinant α-l-iduronidase: characterization of the purified enzyme and correction of mucopolysaccharidosis type I fibroblasts
Authors: Unger E G , Durrant J , Anson D S , Hopwood J J
Published: August 2015

Mucopolysaccharidosis type I (MPS I, Hurler and Scheie syndromes) is an autosomal recessive lysosomal storage disorder that results from a deficiency of the hydrolase alpha-L-iduronidase (IDUA) which is involved in the lysosomal degradation of both heparan sulphate (HS) and dermatan sulphate (DS). Patients with MPS I store and excrete large amounts of partially degraded HS and DS. In order to evaluate enzyme replacement therapy for MPS I patients we have expressed human IDUA cDNA in Chinese Hamster Ovary (CHO)-K1 cells utilizing a plasmid vector that places the cDNA under the transcriptional control of the human polypeptide-chain-elongation factor I alpha gene promoter. A clonal cell-line that secreted recombinant IDUA in a precursor form at approximately 2.2 micrograms/10(6) cells per day was identified. This enzyme was shown to be endocytosed into cultured MPS I fibroblasts via mannose-6-phosphate receptors and to correct the storage phenotype of these cells by enabling the lysosomal-digestion of accumulated sulphated glycosaminoglycans. The recombinant IDUA had on SDS/PAGE a molecular mass of 85 kDa and was processed to 74 kDa and smaller forms following its uptake by fibroblasts. Milligram quantities of the recombinant IDUA were immunopurified and the enzyme was shown to have pH optimum and kinetic parameters differing from those of the mature enzyme purified from human liver. The specific activity of the recombinant enzyme was shown to increase on dilution and on incubation with reducing agents. This was in contrast to the mature IDUA form (74 kDa) which did not have its activity stimulated by reducing agents or dilution.
Forced expression of antizyme abolishes ornithine decarboxylase activity, suppresses cellular levels of polyamines and inhibits cell growth
Authors: Murakami Y , Matsufuji S , Miyazaki Y , Hayashi S
Published: August 2015

Ornithine decarboxylase (ODC) is a key enzyme in polyamine biosynthesis. It is a short-lived protein and negatively regulated by its products, polyamines. Its degradation is accelerated by the binding of antizyme, an ODC-inhibitory protein induced by polyamines. To evaluate the physiological importance of antizyme we examined the effect of forced expression of antizyme on cellular ODC and polyamine levels and cell growth. Antizyme almost completely abolished the induction of ODC by growth stimuli. This may have been caused by antizyme-induced rapid degradation of newly synthesized ODC, since the half-life of ODC complexes with antizyme was less than 5 min. Forced expression of antizyme caused reductions of cellular putrescine and spermidine levels, and inhibited cell growth, which was partially restored by the addition of putrescine. These observations suggested a critically important role of antizyme in polyamine metabolism.
Ca2+/H+ exchange in acidic vacuoles of Trypanosoma brucei
Authors: Vercesi A E , Moreno S N , Docampo R
Published: August 2015

The use of digitonin to permeabilize the plasma membrane of Trypanosoma brucei procyclic and bloodstream trypomastigotes allowed the identification of a non-mitochondrial nigericin-sensitive Ca2+ compartment. The proton ionophore carbonyl cyanide p-trifluoromethoxyphenylhydrazone (FCCP) was able to cause Ca2+ release from this compartment, which was also sensitive to sodium orthovanadate. Preincubation of the cells with the vacuolar H(+)-ATPase inhibitor bafilomycin A1 greatly reduced the nigericin-sensitive Ca2+ compartment. Bafilomycin A1 inhibited the initial rate of ATP-dependent non-mitochondrial Ca2+ uptake and stimulated the initial rate of nigericin-induced Ca2+ release by permeabilized procyclic trypomastigotes. ATP-dependent and bafilomycin A1- and 7-chloro-4-nitrobenz-2-oxa-1,3-diazole (NBD-Cl)-sensitive Acridine Orange uptake was demonstrated in permeabilized cells. Under these conditions Acridine Orange was concentrated in abundant cytoplasmic round vacuoles by a process inhibited by bafilomycin A1, NBD-Cl, nigericin, and Ca2+. Vanadate or EGTA significantly increased Acridine Orange uptake, while Ca2+ released Acridine Orange from these preparations, thus suggesting that the dye and Ca2+ were being accumulated in the same acidic vacuole. Acridine Orange uptake was reversed by nigericin, bafilomycin A1 and NH4Cl. The results are consistent with the presence of a Ca2+/H(+)-ATPase system pumping Ca2+ into an acidic vacuole, that we tentatively named the acidocalcisome.
Expression of the liver-type glucose transporter (GLUT2) in 3T3-L1 adipocytes: analysis of the effects of insulin on subcellular distribution
Authors: Brant A M , Martin S , Gould G W
Published: August 2015

We have expressed the liver-type facilitative glucose transporter, GLUT2, in the insulin-sensitive 3T3-L1 adipocyte clonal cell line in an effort to address the importance of transporter isoform and cellular environment on the ability of insulin to mediate glucose-transporter translocation. Analysis of non-differentiated fibroblastic cell clones transfected with the GLUT2 cDNA identified the presence of this isoform in several independent clones. These clones exhibited increased deoxyglucose and fructose transport rates compared with control cells. Upon differentiation, the fibroblastic clones selected for study achieved > 95% phenotypic conversion into adipocytes. Expression of the GLUT2 protein was maintained throughout the differentiation protocol. Subcellular fractionation revealed that in response to insulin, unlike the native GLUT4, GLUT2 protein did not undergo significant translocation to the plasma membrane; furthermore, the subcellular distribution of the expressed GLUT2 was quite distinct from that of the endogenous GLUT4. 3T3-L1 adipocytes expressing GLUT2 only exhibited a 2-fold increase in insulin-stimulated fructose uptake, further suggesting that GLUT2 does not undergo insulin-stimulated translocation.
Release of Ca2+ from intracellular organelles by peptide analogues: evidence against involvement of metalloendoproteases in Ca2+ sequestration by the endoplasmic reticulum
Authors: Brostrom M A , Wong Ling W L , Gmitter D , Brostrom C O
Published: August 2015

N-Benzyloxycarbonyl-Gly-Phe-amide (Z-Gly-Phe-NH2), a competitive substrate for metalloendoproteases, mobilizes intracellular Ca2+ and suppresses protein synthesis and processing in a Ca(2+)-dependent, reversible manner. To ascertain whether Z-Gly-Phe-NH2 acts as Ca(2+)-storing organelles, effects of the dipeptide on Ca2+ sequestration by saponin-porated GH3 pituitary cells were examined. Porated preparations sequestered Ca2+ into two compartments with different Ca2+ affinities. Ca2+ accumulation at nM concentrations of free Ca2+ was inhibited by thapsigargin and inositol 1,4,5-triphosphate [Ins(1,4,5)P3], enhanced by oxalate and unaffected by oligomycin. Cation accumulation at microM concentrations of free Ca2+ was sensitive to oligomycin but not to thapsigargin. Z-Gly-Phe-NH2 reduced Ca2+ sequestration by both compartments. The dipeptide mobilized Ca2+ from the high-affinity compartment within 1-2 min without affecting Ca2+ uptake. Ca2+ was mobilized more rapidly by Z-Gly-Phe-NH2 and thapsigargin together than by either agent alone. The presence of a thiol-reducing agent was required for Ca2+ mobilization by Z-Gly-Phe-NH2 but not by thapsigargin or Ins(1,4,5)P3. Ca2+ mobilization by Z-Gly-Phe-NH2 could not be attributed to effects on anion-permeability or to actions at Ins(1,4,5)P3 or ryanodine receptors. Results with assorted peptide analogues did not favour suppression of metalloendoprotease activity in the Ca(2+)-mobilizing action of Z-Gly-Phe-NH2. The more hydrophobic analogue Z-L-Tyr-p-nitrophenyl ester was 60-80-fold more potent in mobilizing Ca2+ from intact and porated cells and perturbed the high-affinity Ca(2+)-sequestering compartment selectively. Z-Gly-Phe-NH2 and Z-L-Tyr-p-nitrophenyl ester are proposed to release Ca2+ from the endoplasmic reticulum through an ion pore with affinity for hydrophobic molecules containing internal peptide bonds.
Overexpression of inositol 1,4,5-trisphosphate 3-kinase in Xenopus oocytes inhibits agonist-evoked capacitative calcium entry
Authors: Verjans B , Petersen C C , Berridge M J
Published: August 2015

Ins(1,4,5)P3 3-kinase is a key enzyme in the regulation of Ins(1,4,5)P3. Overexpression of Ins(1,4,5)P3 3-kinase inhibited agonist-evoked and Ins(1,3,4,5)P4-evoked Ca2+ entry in Xenopus oocytes, but did not inhibit Ca2+ entry evoked by thapsigargin or non-metabolizable Ins(1,4,5)P3 analogues. The data suggest that Ins(1,4,5)P3 alone plays the crucial role in the activation of capacitative Ca2+ entry by emptying intracellular stores.
Expression of reduced amounts of structurally altered aggrecan in articular cartilage chondrocytes exposed to high hydrostatic pressure
Authors: Lammi M J , Inkinen R , Parkkinen J J , Häkkinen T , Jortikka M , Nelimarkka L O , Järveläinen H T , Tammi M I
Published: August 2015

The effect of hydrostatic pressure on proteoglycan (PG) metabolism of chondrocyte cultures was examined using a specially designed test chamber. Primary cultures of bovine articular chondrocytes at confluence were exposed for 20 h to 5 and 30 MPa continuous hydrostatic pressures and 5 MPa hydrostatic pulses (0.017, 0.25 and 0.5 Hz) in the presence of [35S]sulphate. Northern blot analyses showed that chondrocyte cultures used in this study expressed abundant mRNA transcripts of aggrecan, typical of chondrocytes, but not versican. The cultures also expressed biglycan and decorin. Enzymic digestions with keratanase and chondroitinases AC, ABC and B and subsequent SDS/agarose gel electrophoresis confirmed the synthesis of aggrecans and small dermatan sulphate PGs. The continuous 30 MPa pressure reduced total PG synthesis by 37% as measured by [35S]sulphate incorporation, in contrast to the 5 MPa continuous pressure which had no effect. The high static pressure also reduced total [3H]glucosamine incorporation by 63% and total [14C]leucine incorporation by 57%. The cyclic pressures showed a frequency-dependent stimulation (0.5 Hz, 11%) or inhibition (0.017 Hz, -17%) of [35S]sulphate incorporation. Aggrecans secreted under continuous 30 MPa pressure showed a retarded migration in 0.75% SDS/agarose gel electrophoresis and they also eluted earlier on Sephacryl S-1000 gel filtration, indicative of a larger molecular size. The increased size was consistent with an increase of average glycosaminoglycan chain length as determined by Sephacryl S-300 gel filtration. No change in aggrecan size was observed with the lower (5 MPa) static or cyclic pressures. Continuous 30 MPa hydrostatic pressure slightly reduced the steady-state mRNA level of aggrecan, in parallel with the decline in PG synthesis measured by [35S]sulphate incorporation. The results demonstrated that high hydrostatic pressure could influence the synthesis of PGs, especially of aggrecans, in chondrocytes both at the transcriptional and translational/post-translational levels.
Cloning of rat asparagine synthetase and specificity of the amino acid-dependent control of its mRNA content
Authors: Hutson R G , Kilberg M S
Published: August 2015

A full-length cDNA clone for rat asparagine synthetase (AS) was isolated from a cDNA library enriched for amino acid-regulated sequences. The AS cDNA was used to investigate the amino acid-dependent repression of AS mRNA content in rat Fao hepatoma cells. In response to complete amino acid starvation, there was an approximately 10-fold increase in the level of AS mRNA. Three species of mRNA, of approx. sizes 2.0, 2.5 and 4.0 kb, were detected and each was simultaneously regulated to the same degree. The expression of AS mRNA increased by 6 h after removal of amino acids, reached a plateau after 9 h, and was blocked by either actinomycin D or cycloheximide. Partial repression of the AS mRNA content was maintained by the presence of a single amino acid in the culture medium, but the degree of effectiveness for each one varied widely. Glutamine showed the greatest ability to repress the AS mRNA content, even at an extracellular concentration 10 times below its plasma level. Other effective repressors included the amino acids asparagine, histidine and leucine, as well as ammonia. Depletion of selected single amino acids from an otherwise complete culture medium also caused up-regulation. In particular, removal of histidine, threonine or tryptophan from the medium, or the addition of histidinol to inhibit histidinyl-tRNA synthetase, resulted in a significant increase in AS mRNA content. The data indicate that nutrient regulation of AS mRNA occurs by a general control mechanism that is responsive to a spectrum of amino acids.
Specificity and localization of lipolytic activity in adult Drosophila melanogaster
Authors: Smith G M , Rothwell K , Wood S L , Yeaman S J , Bownes M
Published: August 2015

The triacylglycerol lipases present in adult Drosophila melanogaster have been investigated. Different lipase activities are present in various tissues in the fly. In particular, an abundant lipase activity is present in the male accessory gland. An esterase null mutant was used to confirm that the enzyme activity was due to a distinct lipase and not non-specific activity from esterase 6 which is also abundant in accessory glands. The properties of the accessory-gland lipase were investigated, and pH optima and substrate utilization suggest that it has some similarities to vertebrate bile-salt-stimulated lipase. Lipase activity is significantly reduced in males and increased in females shortly after mating. This finding suggests that lipase activity is transferred to the female and may be important in mating and reproduction in Drosophila.
Variability of glutathione S-transferase isoenzyme patterns in matched normal and cancer human breast tissue
Authors: Kelley M K , Engqvist-Goldstein A , Montali J A , Wheatley J B , Schmidt D E , Kauvar L M
Published: August 2015

The determination of GST levels in blood has been proposed to a marker of tumour burden in general, whereas level of the P1 isoenzyme has been identified as a prognostic factor for breast-cancer patients receiving no adjuvant chemotherapy. Particular glutathione S-transferase (GST) isoenzymes differ in their substrate specificity, however, and their presence or absence might therefore account for the resistance of tumours to particular chemotherapeutic drugs, as already established for cultured cell lines. Determination of the GST isoenzyme profile of a cancer tissue could have prognostic value in the selection of treatment if the levels of expression/activity show a degree of variation comparable with that exhibited by actual patient responses. Using reversed-phase h.p.l.c. to quantify affinity-isolated GSTs, we have analysed full isoenzyme profiles in the first large sample of matched normal and cancer human tissues (18 breast-cancer patients). In no patients did the tumour tissues express any isoenzymes that were not found in normal breast tissue. In addition to the GSTs, another enzyme, identified as enoyl-CoA isomerase, was regularly found in breast tissue cytosol following elution from a hexyl-glutathione affinity column. In most cases, the average level of GST was substantially elevated in the cancer tissues above the levels in normal breast tissue from the same patient. Furthermore, the relative levels of the isoenzymes were substantially more variable in the cancer samples than in the normal breast tissue, providing a plausible mechanism for the well established variable response to treatment.
Phosphorylation and activation of Ca2+-sensitive cytosolic phospholipase A2 in MCII mast cells mediated by high-affinity Fc receptor for IgE
Authors: Currie S , Roberts E F , Spaethe S M , Roehm N W , Kramer R M
Published: August 2015

In the present study we examined the activation of Ca(2+)-sensitive cytosolic phospholipase A2 (cPLA2) after aggregation of cell-surface high-affinity Fc receptors for IgE (Fc epsilon RI) on mast cells. MCII mast cells (a factor-dependent bone-marrow-derived murine mast cell line) produce significant amounts of leukotriene C4 (LTC4) (70 ng/10(6) cells) on cross-linking of Fc epsilon RI. Using enzymic and immunochemical analysis we found that cPLA2 is the predominant form of this enzyme in MCII mast cells (0.2 micrograms/mg of total protein) and other forms (i.e. secretory PLA2 or Ca2+ independent cytosolic PLA2) could not be detected. Therefore MCII mast cells represent an excellent cellular model for the study of the biochemical mechanism(s) responsible for Fc epsilon RI-induced activation of cPLA2 and the involvement of cPLA2 in Fc epsilon RI-mediated production of LTC4. After activation of Fc epsilon RI by cross-linking, cPLA2 in MCII mast cells exhibited a decreased electrophoretic mobility and its enzyme activity was increased 3-fold. Treatment with phosphatase reversed both the altered electrophoretic mobility and the enhanced enzyme activity demonstrating that they were the result of Fc epsilon RI-induced phosphorylation. On cross-linking of Fc epsilon RI, cPLA2 was phosphorylated within 30 s and appeared to be an early substrate for Fc epsilon RI-activated protein kinases in MCII mast cells. Tyrosine phosphorylation may be a critical component in this process, as genistein, an inhibitor of protein tyrosine kinases, blocked the activation of cPLA2. Using anti-phosphotyrosine antibodies we observed that the activating phosphorylation was not on tyrosine residues of cPLA2, indicating that tyrosine kinases participate upstream in the signalling cascade that couples Fc epsilon RI to cPLA2. We conclude that in MCII mast cells cPLA2 is activated by kinase-dependent mechanisms and may be responsible for Fc epsilon RI-induced mobilization of arachidonic acid for the generation of LTC4.
Binding of quinomycin antibiotic UK-65,662 to DNA: 1H-n.m.r. studies of drug-inducedchanges in DNA conformation in complexes with d(ACGT)2 and d(GACGTC)2
Authors: Searle M S
Published: August 2015

Quinomycin antibiotic UK-65,662 binds selectively to the 5′-CpG-binding sites of the DNA duplexes d(ACGT)2 and d(GACGTC)2; the complexes have been studied in detail by 1H-n.m.r. spectroscopy and molecular-modelling techniques employing nuclear Overhauser effect-restrained energy minimization and molecular dynamics. Whereas the terminal A.T base pairs of the tetamer duplex d(ACGT)2 adopt a stable Hoogsteen alignment (characterized by a syn glycosidic conformation of the purine base), when internalized within the hexamer duplex d(GACGTC)2, the A.T base pairs revert to anti glycosidic torsion angles characteristic of the Watson-Crick hydrogen-bonding scheme. The energetics of base-pair stacking at the terminal 5′-GpA steps of the hexamer complex, with base pairs in the Watson-Crick alignment, are concluded to be important determinants of the adopted conformation, whereas an energetic preference for stacking interactions between terminal Hoogsteen A.T base pairs and the drug quinoline chromophores is evident in the tetramer complex. The internal G.C base pairs in both complexes are highly stabilized, as indicated by the very slow exchange rates of the guanine imino protons; in contrast, the flanking A.T base pairs are no more stable than in the ligand-free DNA duplexes. A large number of intermolecular nuclear Overhauser effects are indicative of many van der Waals contacts and hydrogen-bonding between the antibiotic and the minor groove of the central G.C base pairs in both complexes, indicating that interactions with the G.C base pairs in each duplex are very similar providing the essential features for recognition and tight binding. Despite the difference in the conformation of the A.T base pairs, stacking with the quinoline rings occurs primarily with the adenine bases in both complexes. Relative intensities of intranucleotide versus internucleotide nuclear Overhauser effects indicate that both duplexes are substantially unwound by drug binding (particularly at the CpG step) and this is confirmed by the structure calculations. Both duplexes have ladder-like structures that must lead to significant local distortions of the DNA conformation in vivo.
Homogeneous pyrimidine nucleotidase from human erythrocytes: enzymic and molecular properties
Authors: Amici A , Emanuelli M , Ferretti E , Raffaelli N , Ruggieri S , Magni G
Published: August 2015

A pyrimidine nucleotidase with unique specificity has been obtained for the first time as an homogeneous protein from the cytosolic fraction of human erythrocytes. Both conventional chromatography and f.p.l.c. techniques have been used in the purification procedure. The final enzyme preparation gave a single protein band of M(r) = 23,500 on SDS/PAGE under both reducing and non-reducing conditions. The native enzyme was eluted at M(r) = 45,000 in gel filtration chromatography on Superose 12, suggesting a dimeric structure. Amino acid analysis was consistent with an acidic isoelectric point and revealed the presence of six half-cystine and two methionine residues per subunit. The enzyme was active on a variety of pyrimidine nucleoside monophosphates, being most active on the 3′-monophosphates. Km values for 3′dUMP, 3′UMP, 5′dUMP, 5′UMP, 5-fluoro-2′dUMP, ranged from 192 microM to 1.15 mM. The enzyme activity was inhibited by both reaction products, orthophosphate, and the nucleoside formed. Product inhibition studies suggested an Ordered Uni-Bi mechanism for the reaction. The enzyme required Mg2+ for its activity, while heavy metals cations were strongly inhibitory. The enzyme activity was inhibited by metal chelating agents and it was sensitive to thioreactive reagents. The isoelectric point was 5.4 and the optimum activity pH was 6.5.