bluuebunny/crossref_metadata_2025 huggingface dataset:
Inducible gene expression of moricin, a unique antibacterial peptide from the silkworm (Bombyx mori)
Authors: FURUKAWA Seiichi , TANAKA Hiromitsu , NAKAZAWA Hiroshi , ISHIBASHI Jun , SHONO Toshio , YAMAKAWA Minoru
Published: August 2015

Molecular cloning of cDNAs encoding moricin, a novel antibacterial peptide from the silkworm (Bombyx mori), was performed using a fat-body cDNA library. A reverse-transcription PCR product encoding a partial nucleotide sequence of moricin was used as a probe. Nucleotide sequencing of four positive clones revealed two types of moricin cDNAs designated moricin 1 and 2. cDNAs for moricin 1 and 2 shared 97.2% identity in their nucleotide sequences. Although one amino acid residue (Phe6) of moricin 1 in the putative signal peptide was replaced with Lys6 in moricin 2, amino acid sequences of their mature portions were identical. Moricin gene expression in B. mori larvae injected with Escherichia coli was observed in fat-bodies, haemocytes and the Malpighian tubule, but not in other tissues like the midgut and silk glands. Accumulation of moricin gene transcripts induced by E. coli reached a maximum level 8 h after injection and persisted up to 48 h. It was confirmed that lipopolysaccharide (LPS) and lipid A, which are cell-wall components of E. coli, triggered moricin gene expression. Comparison of gene expression between moricin 1 and 2 by PCR using specific primers indicated that moricin 2 gene was more strongly expressed than moricin 1 gene. A genomic clone encoding moricin 2 was screened from a B. mori genomic library using a moricin cDNA as a probe. Regulatory motifs for gene expression such as nuclear-factor-κB-binding-site-like sequence (ĸB site) and nuclear-factor-interleukin-6-binding-site-like sequence (NF-IL-6 site) were found in the 5ʹ-upstream regulatory region. An electrophoretic-mobility-shift assay revealed that there are bacterial LPS-inducible nuclear proteins that can bind to the ĸB site and other sites in the regulatory region.
Alteration of substrate specificity by a naturally-occurring aldolase B mutation (Ala337→Val) in fructose intolerance
Authors: RELLOS Peter , ALI Manir , VIDAILHET Michel , SYGUSCH Jurgen , COX Timothy M.
Published: August 2015

A molecular analysis of human aldolase B genes in two newborn infants and a 4-year-old child with hereditary fructose intolerance, the offspring of a consanguineous union, has identified the novel mutation Ala337 → Val in homozygous form. This mutation was also detected independently in two other affected individuals who were compound heterozygotes for the prevalent aldolase B allele, Ala149 → Pro, indicating that the mutation causes aldolase B deficiency. To test for the effect of the mutation, catalytically active wild-type human aldolase B and the Val337 variant enzyme were expressed in Escherichia coli. The specific activities of the wild-type recombinant enzyme were 4.8 units/mg and 4.5 units/mg towards fructose 1,6-bisphosphate (FBP) and fructose 1-phosphate (F-1-P) as substrates with Michaelis constants of 4 μM and 2.4 mM respectively. The specific activities of purified tetrameric Val337 aldolase B, which affects an invariant residue in the C-terminal region, were 4.2 units/mg and 2.6 units/mg towards FBP and F-1-P as substrates respectively; the corresponding Michaelis constants were 22 μM and 24 mM. The FBP-to-F-1-P substrate activity ratios were 0.98 and 1.63 for wild-type and Val337 variant enzymes respectively. The Val337 mutant aldolase had an increased susceptibility to proteolytic cleavage in E. coli and rapidly lost activity on storage. Comparative CD determinations showed that the Val337 protein had a distinct thermal denaturation profile with markedly decreased enthalpy, indicating that the mutant protein is partly unfolded. The undegraded mutant had preferentially decreased affinity and activity towards its specific F-1-P substrate and maintained appreciable activity towards FBP. In contrast, fluorescence studies of the mutant showed an increased binding affinity for products of the aldolase reaction, indicating a role for the C-terminus in mediating product release. These findings in a rare but widespread naturally occurring mutant implicate the C-terminus in the activity of human aldolase B towards its specific substrates and demonstrate its role in maintaining the overall stability of the enzyme tetramer.
Signal transduction pathway regulating prostaglandin EP3 receptor-induced neurite retraction: requirement for two different tyrosine kinases
Authors: AOKI Junko , KATOH Hironori , YASUI Hidekazu , YAMAGUCHI Yoshiaki , NAKAMURA Kazuhiro , HASEGAWA Hiroshi , ICHIKAWA Atsushi , NEGISHI Manabu
Published: August 2015

We reported previously that activation of the prostaglandin E receptor EP3 subtype triggered neurite retraction through the small GTPase Rho-, and its target, RhoA-binding kinase α (ROKα)-, dependent pathway in EP3 receptor-expressing PC12 cells. Here we examined the involvement of tyrosine kinases in this pathway in nerve growth factor-differentiated PC12 cells. Tyrphostin A25, a tyrosine kinase inhibitor, blocked neurite retraction and cell rounding induced by activation of the EP3 receptor, however, it failed to block neurite retraction and cell rounding induced by microinjection of constitutively active RhoA, RhoAV14, indicating that a tyrphostin-sensitive tyrosine kinase was involved in the pathway from the EP3 receptor to Rho activation. On the other hand, genistein, another tyrosine kinase inhibitor, blocked neurite retraction and cell rounding induced by both activation of the EP3 receptor and microinjection of RhoAV14. However, genistein did not block neuronal morphological changes induced by microinjection of a constitutively active mutant of ROKα. These results indicate that two different tyrosine kinases, tyrphostin A25-sensitive and genistein-sensitive kinases, are involved in the EP3 receptor-mediated neurite retraction acting upstream and downstream of Rho, respectively.
Glucuronidation of the environmental oestrogen bisphenol A by an isoform of UDP-glucuronosyltransferase, UGT2B1, in the rat liver
Authors: YOKOTA Hiroshi , IWANO Hidetomo , ENDO Mari , KOBAYASHI Tsutomu , INOUE Hiroki , IKUSHIRO Shin-ichi , YUASA Akira
Published: August 2015

Bisphenol A, an environmental oestrogenic chemical, was found to conjugate highly with glucuronic acid in male rat liver microsomes studied in vitro. In the various isoforms tested (1A1, 1A3, 1A5, 1A6, 1A7 and 2B1), glucuronidation of bisphenol A and of diethylstilboestrol, a synthetic crystalline compound possessing oestrogenic activity and known to be glucuronidated by liver microsomes, was catalysed by an isoform of UDP-glucuronosyltransferase (UGT), namely UGT2B1, which glucuronidates some endogenous androgens. UGT activity towards bisphenol A in liver microsomes and in UGT2B1 expressed in yeast AH22 cells (22.9 and 0.58 nmol/min per mg of microsomal proteins respectively) was higher than that towards diethylstilboestrol (75.0 and 4.66 pmol/min per mg of microsomal proteins respectively). UGT activities towards both bisphenol A and diethylstilboestrol were distributed mainly in the liver but were also observed at substantial levels in the kidney and testis. Northern blot analysis disclosed the presence of UGT2B1 solely in the liver, and about 65% of the male rat liver microsomal UGT activities towards bisphenol A were absorbed by the anti-UGT2B1 antibody. These results indicate that bisphenol A, in male rat liver, is glucuronidated by UGT2B1, an isoform of UGT.
Partial replacement of bile salts causes marked changes of cholesterol crystallization in supersaturated model bile systems
Authors: NISHIOKA Tomoji , TAZUMA Susumu , YAMASHITA Gunji , KAJIYAMA Goro
Published: August 2015

Cholesterol crystallization is a key step in gallstone formation and is influenced by numerous factors. Human bile contains various bile salts having different hydrophobicity and micelle-forming capacities, but the importance of lipid composition to bile metastability remains unclear. This study investigated the effect of bile salts on cholesterol crystallization in model bile (MB) systems. Supersaturated MB systems were prepared with an identical composition on a molar basis (taurocholate/phosphatidylcholine/cholesterol, 152 mM:38 mM: 24 mM), except for partial replacement of taurocholate (10, 20, and 30%) with various taurine-conjugated bile salts. Cholesterol crystallization was quantitatively estimated by spectrophotometrically measuring crystal-related turbidity and morphologically scanned by video-enhanced microscopy. After partial replacement of taurocholate with hydrophobic bile salts, cholesterol crystallization increased dose-dependently without changing the size of vesicles or crystal morphology and the rank order of crystallization was deoxycholate > chenodeoxycholate > cholate (control MB). All of the hydrophilic bile salts (ursodeoxycholate, ursocholate and β-muricholate) inhibited cholesterol precipitation by forming a stable liquid-crystal phase, and there were no significant differences among the hydrophilic bile-salt species. Cholesterol crystallization was markedly altered by partial replacement of bile salts with a different hydrophobicity. Thus minimal changes in bile-salt composition may dramatically alter bile lipid metastability.
Relationship between cytochrome P450 catalytic cycling and stability: fast degradation of ethanol-inducible cytochrome P450 2E1 (CYP2E1) in hepatoma cells is abolished by inactivation of its electron donor NADPH–cytochrome P450 reductase
Authors: ZHUKOV Andrei , INGELMAN-SUNDBERG Magnus
Published: August 2015

Ethanol-inducible cytochrome P450 2E1 (CYP2E1) involved in the metabolism of gluconeogenetic precursors and some cytotoxins is distinguished from other cytochrome P450 enzymes by its rapid turnover (in vivo half-life of 4-7 h), with ligands to the haem iron, both substrates and inhibitors, stabilizing the protein. CYP2E1 is also known to have a high oxidase activity in the absence of substrate, resulting in the production of reactive oxygen radicals. We suggested that the rapid intracellular turnover of the enzyme may be partly due to covalent modifications by such radicals or to other changes during catalytic cycling, in which case the inhibition of electron supply from NADPH-cytochrome P450 reductase would be expected to stabilize the protein. Fao hepatoma cells, where CYP2E1 showed a half-life of 4 h upon serum withdrawal, were treated for 1 h with 0.3 μM diphenylene iodonium (DPI), a suicide inhibitor of flavoenzymes, which resulted in ≈ 90% inhibition of the microsomal NADPH-cytochrome P450 reductase and CYP2E1-dependent chlorzoxazone hydroxylase activities. Subsequent cycloheximide chase revealed that the CYP2E1 half-life increased to 26 h. Neither the degradation rates of total protein, CYP2B1 and NADPH-cytochrome P450 reductase nor the cellular ATP level were affected by DPI under the conditions employed. These results demonstrate for the first time that the short half-life of CYP2E1 in vivo may be largely due to the rapid destabilization of the enzyme during catalytic cycling rather than to the intrinsic instability of the protein molecule.
Expression of hormone-sensitive lipase and its regulation by adrenaline in skeletal muscle
Authors: LANGFORT Jozef , PLOUG Thorkil , IHLEMANN Jacob , SALDO Michele , HOLM Cecilia , GALBO Henrik
Published: August 2015

The enzymic regulation of triacylglycerol breakdown in skeletal muscle is poorly understood. Western blotting of muscle fibres isolated by collagenase treatment or after freeze-drying demonstrated the presence of immunoreactive hormone-sensitive lipase (HSL), with the concentrations in soleus and diaphragm being more than four times the concentrations in extensor digitorum longus and epitrochlearis muscles. Neutral lipase activity determined under conditions optimal for HSL varied directly with immunoreactivity. Expressed relative to triacylglycerol content, neutral lipase activity in soleus muscle was about 10 times that in epididymal adipose tissue. In incubated soleus muscle, both neutral lipase activity against triacylglycerol (but not against a diacylglycerol analogue) and glycogen phosphorylase activity increased in response to adrenaline (epinephrine). The lipase activation was completely inhibited by anti-HSL antibody and by propranolol. The effect of adrenaline could be mimicked by incubation of crude supernatant from control muscle with the catalytic subunit of cAMP-dependent protein kinase, while no effect of the kinase subunit was seen with supernatant from adrenaline-treated muscle. The results indicate that HSL is present in skeletal muscle and is stimulated by adrenaline via β-adrenergic activation of cAMP-dependent protein kinase. The concentration of HSL is higher in oxidative than in glycolytic muscle, and the enzyme is activated in parallel with glycogen phosphorylase.
Visualization of distinct patterns of subcellular redistribution of the thyrotropin-releasing hormone receptor-1 and Gqα /G11α induced by agonist stimulation
Authors: DRMOTA Tomas , NOVOTNY Jiri , GOULD Gwyn W. , SVOBODA Petr , MILLIGAN Graeme
Published: August 2015

The rat thyrotropin-releasing hormone receptor-1 (TRHR-1) was modified by the addition of green fluorescent protein (GFP) and expressed stably in HEK293 cells. Extensive overlap of plasma membrane distribution of autofluorescent TRHR-1-GFP with that of the phosphoinositidase C-linked G-proteins Gqα/G11α, identified by indirect immunofluorescence, was monitored concurrently. Addition of thyrotropin-releasing hormone resulted in rapid separation of TRHR-1-GFP and Gqα/G11α signals as the receptor was internalized. This situation persisted for more than an hour. At longer time periods a fraction of the cellular Gqα/G11α was also internalized, although much of the Gqα/G11α immunoreactivity remained associated with the plasma membrane. Parallel experiments, in which the cellular distribution of TRHR-1-GFP and Gqα/G11α immunoreactivity were monitored in sucrose-gradient fractions following cell disruption, also demonstrated a rapid, agonist-induced movement of TRHR-1-GFP away from the plasma membrane to low-density vesicular fractions. At later time points, a fraction of the cellular Gqα/G11α immunoreactivity was also redistributed to overlapping, but non-identical, low-density-vesicle-containing fractions. Pretreatment of the cells with cytochalasin D or nocodazole prevented agonist-induced redistribution of G-protein but not TRHR-1-GFP, further indicating resolution of the mechanics of these two processes. The combination of a GFP-modified receptor and immunostaining of the G-proteins activated by that receptor allows, for the first time, concurrent analysis of the varying dynamics and bases of internalization and redistribution of two elements of the same signal-transduction cascade.
Identification of peroxisomal proteins by using M13 phage protein VI phage display: molecular evidence that mammalian peroxisomes contain a 2,4-dienoyl-CoA reductase
Authors: FRANSEN Marc , VAN VELDHOVEN Paul P. , SUBRAMANI Suresh
Published: August 2015

To elucidate unknown mammalian peroxisomal enzymes and functions, we subjected M13 phage expressing fusions between the gene encoding protein VI and a rat liver cDNA library to an immunoaffinity selection process in vitro (biopanning) with the use of antibodies raised against peroxisomal subfractions. In an initial series of biopanning experiments, four different cDNA clones were obtained. These cDNA species encoded two previously identified peroxisomal enzymes, catalase and urate oxidase, and two novel proteins that contained a C-terminal peroxisomal targeting signal (PTS1). A primary structure analysis of these novel proteins revealed that one, ending in the tripeptide AKL, is homologous to the yeast peroxisomal 2,4-dienoyl-CoA reductase (EC 1.3.1.34; DCR), an enzyme required for the degradation of unsaturated fatty acids, and that the other, ending in the tripeptide SRL, is a putative member of the short-chain dehydrogenase/reductase (SDR) family, with three isoforms. Green fluorescent protein (GFP) fusions encoding GFP-DCR-AKL, GFP-DCR, GFP-SDR-SRL and GFP-SDR were expressed in mammalian cells. The analysis of the subcellular location of the recombinant fusion proteins confirmed the peroxisomal localization of GFP-DCR-AKL and GFP-SDR-SRL, as well as the functionality of the PTS1. That the AKL protein is indeed an NADPH-dependent DCR was demonstrated by showing DCR activity of the bacterially expressed protein. These results demonstrate at the molecular level that mammalian peroxisomes do indeed contain a DCR. In addition, the results presented here indicate that the protein VI display system is suitable for the isolation of rare cDNA clones from cDNA libraries and that this technology facilitates the identification of novel peroxisomal proteins.
Isolation and characterization of proteoglycans from human follicular fluid
Authors: ERIKSEN Gitte V. , CARLSTEDT Ingemar , MÖRGELIN Matthias , ULDBJERG Niels , MALMSTRÖM Anders
Published: August 2015

Two proteoglycans differing in size and composition were isolated from human follicular fluid. The larger one of high density had a molecular mass of 3.0×106 Da, as determined by laser light-scattering, and was substituted with 15-20 chondroitin sulphate (CS) chains (Mr 60000-65000). Half of the CS disaccharides were 6-sulphated, whereas the remaining ones were non-sulphated. Digestion of the CS proteoglycan with chondroitinase ABC lyase, followed by SDS/PAGE, yielded a protein core of 600 to 700 kDa including substituted oligosaccharides, and a band of 70 kDa that was identified as the heavy-chain component of the inter-α-trypsin inhibitor (ITI). Western blotting of the CS proteoglycan showed that this had reactivity with antibodies raised against human versican. Electron microscopy (EM) of the CS proteoglycan also revealed a versican-like structure, with one globular domain at each end of a long extended segment substituted with CS side chains, as well as a structure interpreted as being the heavy chain of ITI attached to CS chains. Laser light-scattering revealed that the smaller proteoglycan had a molecular mass of 1.1×106 Da, and EM demonstrated that it had a globular-protein core structure. The core protein, which showed immunological reactivity with perlecan antibodies, was substituted with approximately seven heparan sulphate (HS) and CS chains of similar size (50-55 kDa), the CS disaccharides being mainly 6-sulphated (68%), with a small proportion being 4-sulphated. The protein core was shown to be heterogeneous, with bands occurring at 215, 330 and 400 kDa after enzymic degradation of the glycosaminoglycan chains followed by SDS/PAGE analysis. The demonstration of intact molecules and fragments obtained after stepwise degradations, as shown by gel chromatography, supported a ‘composite’ structure of this proteoglycan.
How aneuploidy affects metabolic control and causes cancer
Authors: RASNICK David , DUESBERG Peter H.
Published: August 2015

The complexity and diversity of cancer-specific phenotypes, including de-differentiation, invasiveness, metastasis, abnormal morphology and metabolism, genetic instability and progression to malignancy, have so far eluded explanation by a simple, coherent hypothesis. However, an adaptation of Metabolic Control Analysis supports the 100-year-old hypothesis that aneuploidy, an abnormal number of chromosomes, is the cause of cancer. The results demonstrate the currently counter-intuitive principle that it is the fraction of the genome undergoing differential expression, not the magnitude of the differential expression, that controls phenotypic transformation. Transforming the robust normal phenotype into cancer requires a twofold increase in the expression of thousands of normal gene products. The massive change in gene dose produces highly non-linear (i.e. qualitative) changes in the physiology and metabolism of cells and tissues. Since aneuploidy disrupts the natural balance of mitosis proteins, it also explains the notorious genetic instability of cancer cells as a consequence of the perpetual regrouping of chromosomes. In view of this and the existence of non-cancerous aneuploidy, we propose that cancer is the phenotype of cells above a certain threshold of aneuploidy. This threshold is reached either by the gradual, stepwise increase in the level of aneuploidy as a consequence of the autocatalysed genetic instability of aneuploid cells or by tetraploidization followed by a gradual loss of chromosomes. Thus the initiation step of carcinogenesis produces aneuploidy below the threshold for cancer, and the promotion step increases the level of aneuploidy above this threshold. We conclude that aneuploidy offers a simple and coherent explanation for all the cancer-specific phenotypes. Accordingly, the gross biochemical abnormalities, abnormal cellular size and morphology, the appearance of tumour-associated antigens, the high levels of secreted proteins responsible for invasiveness and loss of contact inhibition, and even the daunting genetic instability that enables cancer cells to evade chemotherapy, are all the natural consequence of the massive over- and under-expression of proteins.
Colicin E1 forms a dimer after urea-induced unfolding
Authors: STEER Brian A. , DINARDO Ariel A. , MERRILL A. Rod
Published: August 2015

Unfolding of the soluble colicin E1 channel peptide was examined with the use of urea as a denaturant; it was shown that it unfolds to an intermediate state in 8.5 M urea, equivalent to a dimeric species previously observed in 4 M guanidinium chloride. Single tryptophan residues, substituted into the peptide at various positions by site-directed mutagenesis, were employed as fluorescent probes of local unfolding. Unfolding profiles for specific sites within the peptide were obtained by quantifying the shifts in the fluorescence emission maxima of single tryptophan residues on unfolding and plotting them against urea concentration. Unfolding reported by tryptophan residues in the C-terminal region was not characteristic of complete peptide denaturation, as evidenced by the relatively blue-shifted values of the fluorescence emission maxima. Unfolding was also monitored by using CD spectroscopy and the fluorescent probe 2-(p-toluidinyl)-naphthalene 6-sulphonic acid; the results indicated that unfolding of helices is concomitant with the exposure of protein non-polar surface. Unfolding profiles were evaluated by non-linear least-squares curve fitting and calculation of the unfolding transition midpoint. The unfolding profiles of residues located in the N-terminal region of the peptide had lower transition midpoints than residues in the C-terminal portion. The results of unfolding analysis demonstrated that urea unfolds the peptide only partly to an intermediate state, because the C-terminal portion of the channel peptide retained significant structure in 8.5 M urea. Characterization of the peptide's global unfolding by size-exclusion HPLC revealed that the partly denatured structure that persists in 8.5 M urea is a dimer of two channel peptides, tightly associated by hydrophobic interactions. The presence of the dimerized species was confirmed by SDS/PAGE and intermolecular fluorescence resonance energy transfer.
Protein modification during biological aging: selective tyrosine nitration of the SERCA2a isoform of the sarcoplasmic reticulum Ca2+-ATPase in skeletal muscle
Authors: VINER Rosa I. , FERRINGTON Deborah A. , WILLIAMS Todd D. , BIGELOW Diana J. , SCHÖNEICH Christian
Published: August 2015

The accumulation of covalently modified proteins is an important hallmark of biological aging, but relatively few studies have addressed the detailed molecular-chemical changes and processes responsible for the modification of specific protein targets. Recently, Narayanan et al. [Narayanan, Jones, Xu and Yu (1996) Am. J. Physiol. 271, C1032-C1040] reported that the effects of aging on skeletal-muscle function are muscle-specific, with a significant age-dependent change in ATP-supported Ca2+-uptake activity for slow-twitch but not for fast-twitch muscle. Here we have characterized in detail the age-dependent functional and chemical modifications of the rat skeletal-muscle sarcoplasmic-reticulum (SR) Ca2+-ATPase isoforms SERCA1 and SERCA2a from fast-twitch and slow-twitch muscle respectively. We find a significant age-dependent loss in the Ca2+-ATPase activity (26% relative to Ca2+-ATPase content) and Ca2+-uptake rate specifically in SR isolated from predominantly slow-twitch, but not from fast-twitch, muscles. Western immunoblotting and amino acid analysis demonstrate that, selectively, the SERCA2a isoform progressively accumulates a significant amount of nitrotyrosine with age (≈ 3.5±0.7 mol/mol of SR Ca2+-ATPase). Both Ca2+-ATPase isoforms suffer an age-dependent loss of reduced cysteine which is, however, functionally insignificant. In vitro, the incubation of fast- and slow-twitch muscle SR with peroxynitrite (ONOO-) (but not NO/O2) results in the selective nitration only of the SERCA2a, suggesting that ONOO- may be the source of the nitrating agent in vivo. A correlation of the SR Ca2+-ATPase activity and covalent protein modifications in vitro and in vivo suggests that tyrosine nitration may affect the Ca2+-ATPase activity. By means of partial and complete proteolytic digestion of purified SERCA2a with trypsin or Staphylococcus aureus V8 protease, followed by Western-blot, amino acid and HPLC-electrospray-MS (ESI-MS) analysis, we localized a large part of the age-dependent tyrosine nitration to the sequence Tyr294-Tyr295 in the M4-M8 transmembrane domain of the SERCA2a, close to sites essential for Ca2+ translocation.
Characterization of a novel spermidine/spermine acetyltransferase, BltD, from Bacillus subtilis
Authors: WOOLRIDGE Dale P. , MARTINEZ Jesse D. , STRINGER David E. , GERNER Eugene W.
Published: August 2015

Overexpression of the BltD gene in Bacillus subtilis causes acetylation of the polyamines spermidine and spermine. BltD is co-regulated with another gene, Blt, which encodes a multidrug export protein whose overexpression facilitates spermidine export [Woolridge, Vazquez-Laslop, Markham, Chevalier, Gerner and Neyfakh (1997) J. Biol. Chem. 272, 8864-8866]. Here we show that BltD acetylates both spermidine and spermine at primary propyl amine moieties, with spermine being the preferred substrate. In the presence of saturating concentrations of acetyl CoA, BltD rapidly acetylates spermine at both the N1 and N12 positions. The Km (app) values for spermine, spermidine and N1-acetylspermine are ≤ 67, 200 and 1200 μM, respectively. Diamines ranging from 1,3-diaminopropane to 1,12-diaminododecane, monoacetylputrescine and N8-acetylspermidine were not substrates for BltD. Putrescine (1,4-diaminobutane) and N8-acetylspermidine were competitive inhibitors of spermidine acetylation by BltD, with Ki values of 0.25 and 5.76 mM, respectively. CoA competitively inhibited both spermidine and acetyl-CoA interactions with BltD. These data and other results indicate that the mechanism of spermidine and spermine acetylation by BltD is a random-order mechanism of bi-molecular kinetics.
Phosphorylation of the myristoylated protein kinase C substrate MARCKS by the cyclin E–cyclin-dependent kinase 2 complex in vitro
Authors: MANENTI Stéphane , YAMAUCHI Emiko , SOROKINE Odile , KNIBIEHLER Martine , VAN DORSSELAER Alain , TANIGUCHI Hisaaki , DUCOMMUN Bernard , DARBON Jean-Marie
Published: August 2015

The myristoylated alanine-rich C-kinase substrate (MARCKS) purified from brain was recently characterized as a proline-directed kinase(s) substrate in vivo [Taniguchi, Manenti, Suzuki and Titani (1994) J. Biol. Chem. 269, 18299-18302]. Here we have investigated the phosphorylation of MARCKS by various cyclin-dependent kinases (Cdks) in vitro. We established that Cdk2, Cdk4 and, to a smaller extent, Cdk1 that have been immunoprecipitated from cellular extracts phosphorylate MARCKS. Comparison of MARCKS phosphorylation by protein kinase C (PKC) and by the purified cyclin E-Cdk2 complex suggested that two residues were phosphorylated by Cdk2 under these conditions. To identify these sites, Cdk2-phosphorylated MARCKS was digested with lysyl endoprotease and analysed by electrospray MS. Comparison with the digests obtained from the unphosphorylated protein demonstrated that two peptides, Gly12-Lys30 and Ala138-Lys152, were phosphorylated by cyclin E-Cdk2. The identity of these peptides was confirmed by automatic Edman degradation. On the basis of the consensus phosphorylation sequence described for Cdk2, and on MS/MS analysis of the Ala138-Lys152 peptide, we concluded that Ser27, one of the phosphorylation sites identified in vivo, and Thr150 were the Cdk2 targets in vitro. None of the other sites described in vivo were phosphorylated in these conditions. Interestingly, a preliminary phosphorylation of MARCKS by PKC improved the initial rate of phosphorylation by Cdk2 without modifying the number of sites concerned. In contrast, phosphorylation of MARCKS by Cdk2 did not significantly affect further phosphorylation by PKC.
Targeted delivery of oligodeoxynucleotides to parenchymal liver cells in vivo
Authors: BIESSEN Erik A. L. , VIETSCH Helene , RUMP Erik T. , FLUITER Kees , KUIPER Johan , BIJSTERBOSCH Martin K. , VAN BERKEL Theo J. C.
Published: August 2015

Anti-sense oligodeoxynucleotides (ODNs) hold great promise for correcting the biosynthesis of clinically relevant proteins. The potential of ODNs for modulating liver-specific genes might be increased by preventing untimely elimination and by improving the local bioavailability of ODNs in the target tissue. In the present study we have assessed whether the local ODN concentration can be enhanced by the targeted delivery of ODNs through conjugation to a ligand for the parenchymal liver cell-specific asialoglycoprotein receptor. A capped ODN (miscellaneous 20-mer sequence) was derivatized with a ligand with high affinity for this receptor, N2-[N2-(N2,N6-bis{N-[p-(β-D-galactopyranosyloxy) anilino] thiocarbamyl} - L - lysyl) - N6 - (N - {p - [β-D -galactopyranosyloxy] anilino} thiocarbamyl) - L - lysyl] - N6 - [N - (p -{β-ᴅ-galactopyranosyloxy}anilino)thiocarbamyl]-ʟ-lysine (L3G4) (Kd 6.5±0.2 nM, mean±S.D.). Both the uptake studies in vitro and the confocal laser scan microscopy studies demonstrated that L3G4-ODN was far more efficiently bound to and taken up by parenchymal liver cells than underivatized ODN. Studies in vivo in rats showed that hepatic uptake could be greatly enhanced from 19±1% to 77±6% of the injected dose after glycoconjugation. Importantly, specific ODN accumulation of ODN into parenchymal liver cells was improved almost 60-fold after derivatization with L3G4, and could be attributed to the asialoglycoprotein receptor. In conclusion, the scavenger receptor-mediated elimination pathway for miscellaneous ODN sequences can be circumvented by direct conjugation to a synthetic tag for the asialoglycoprotein receptor. In this manner a crucial requisite is met towards the application of ODNs in vivo to modulate the biosynthesis of parenchymal liver cell-specific genes such as those for apolipoprotein (a), cholesterol ester transfer protein and viral proteins.
Transcription factor GCN4 for control of amino acid biosynthesis also regulates the expression of the gene for lipoamide dehydrogenase
Authors: ZAMAN Zafar , BOWMAN Susan B. , KORNFELD Geoff D. , BROWN Alistair J. P. , DAWES Ian W.
Published: August 2015

The yeast LPD1 gene encoding lipoamide dehydrogenase is subject to the general control of amino acid biosynthesis mediated by the GCN4 transcription factor. This is striking in that it demonstrates that GCN4-mediated regulation extends much farther upstream than simply to the direct pathways for amino acid and purine biosynthesis. In yeast, lipoamide dehydrogenase functions in at least three multienzyme complexes: pyruvate dehydrogenase and 2-oxoglutarate dehydrogenase (which function in the entry of pyruvate into, and metabolism via, the citric acid cycle) and glycine decarboxylase. When wild-type cells were shifted from growth on amino acid-rich to amino acid-deficient medium, the expression of lipoamide dehydrogenase was induced approx. 2-fold. In a similar experiment no such induction was observed in isogenic gcn4 mutant cells. Northern analysis indicated that amino acid starvation affected levels of the LPD1 transcript. In the upstream region of LPD1 are three matches to the consensus for control mediated by GCN4. Directed mutagenesis of each site, and of all combinations of sites, suggests that only one site might be important for the general control response under the conditions tested. Gel-retardation analysis with GCN4 protein synthesized in vitro has indicated that GCN4 can bind in vitro to at least two of the consensus motifs.
Regulation of photoreceptor phosphodiesterase catalysis by its non-catalytic cGMP-binding sites
Authors: D'AMOURS Marc R. , COTE Rick H.
Published: August 2015

The photoreceptor 3ʹ:5ʹ-cyclic nucleotide phosphodiesterase (PDE) is the central enzyme of visual excitation in rod photoreceptors. The hydrolytic activity of PDE is precisely regulated by its inhibitory γ subunit (Pγ), which binds directly to the catalytic site. We examined the inhibition of frog rod outer segment PDE by endogenous Pγ, as well as by synthetic peptides corresponding to its central and C-terminal domains, to determine whether the non-catalytic cGMP-binding sites on the catalytic αβ dimer of PDE allosterically regulate PDE activity. We found that the apparent binding affinity of Pγ for PDE was 28 pM when cGMP occupied the non-catalytic sites, whereas Pγ had an apparent affinity only 1/16 of this when the sites were empty. The elevated basal activity of PDE with empty non-catalytic sites can be decreased by the addition of nanomolar levels of cGMP, demonstrating that the high-affinity non-catalytic sites on the PDE catalytic dimer mediate this effect. No evidence for a direct allosteric effect of the non-catalytic sites on catalysis could be detected for the activated enzyme lacking bound Pγ. The intrinsic affinity of a synthetic C-terminal (residues 63-87) Pγ peptide to bind and to inhibit the hydrolytic activity of activated PDE was enhanced 300-fold in the presence of cGMP compared with cAMP. We conclude that the binding of cGMP to the non-catalytic sites of PDE induces an allosteric change in the structure of the catalytic domain that greatly enhances the interaction of the C-terminus of Pγ with the catalytic domain.
Acyl phosphatase activity of NO-inhibited glyceraldehyde-3-phosphate dehydrogenase (GAPDH): a potential mechanism for uncoupling glycolysis from ATP generation in NO-producing cells
Authors: ALBINA Jorge E. , MASTROFRANCESCO Balduino , REICHNER Jonathan S.
Published: August 2015

T r e a t m e n t o f g l y ce r a l d e h y d e - 3 - p h o s p h a t e - d e h y d r o g e n a s e (GA (GAPDH) with the NO donors S-nitrosoglutathione, 3-morpholinosydnonimine or diethylamine NONOate (diethylamine diazeniumdiolate) in vitro, inhibited its dehydrogenase activity and induced its acyl phosphatase activity. NO-producing cells, in turn, exhibited reduced GAPDH activity, increased glycolysis, and decreased ATP content, synthesis and turnover. These cellular alterations could be explained by the uncoupling of glycolytic flux from substrate level phosphorylation by the acyl phosphatase activity of NO-modified GAPDH.
A conserved RGD (Arg-Gly-Asp) motif in the transferrin receptor is required for binding to transferrin
Authors: DUBLJEVIC Valentina , SALI Adnan , GODING James W.
Published: August 2015

The transferrin receptor contains a highly conserved Arg-Gly-Asp (RGD) sequence in the C-terminal region where transferrin is thought to bind. RGD sequences are commonly involved in cell adhesion. This sequence is crucial for transferrin binding, suggesting possible evolutionary links between molecules mediating iron uptake and cell adhesion.
Identification and expression of Pen c 2, a novel allergen from Penicillium citrinum
Authors: CHOW Lu-Ping , SU Ning-Yuan , YU Chia-Jung , CHIANG Bor-Luen , SHEN Horng-Der
Published: August 2015

The mould genus, Penicillium, is known to be a significant source of environmental aero-allergens. One important allergen from Penicillium citrinum, Pen c 2, has been identified by means of two-dimensional immunoblotting using IgE-containing patients' sera. This novel allergen was cloned, sequenced and expressed in Escherichia coli. The cloned cDNA encodes a large 457-amino acid protein precursor containing a 16-amino acid signal peptide, a 120-amino acid propeptide and the 321-amino acid mature protein. Comparison of the Pen c 2 sequence with known protein sequences revealed shared high sequence similarities with two vacuolar serine proteases from Aspergillus niger and Saccharomyces cerevisiae. Asp-46, His-78 and Ser-244 were found to constitute the catalytic triad of the 39-kDa Pen c 2. The DNA coding for Pen c 2 was cloned into vector PQE-30 and expressed in E. coli as a His-tag fusion protein that bound serum IgE from Penicillium-allergic patients on immunoblots. Recombinant Pen c 2 could therefore be used effectively for diagnosis and also potentially for the treatment of mould-derived allergic disorders.
Induction of the multispecific organic anion transporter (cMoat/mrp2) gene and biliary glutathione secretion by the herbicide 2,4,5-trichlorophenoxyacetic acid in the mouse liver
Authors: WIELANDT Ana M. , VOLLRATH Valeska , MANZANO Marlene , MIRANDA Soledad , ACCATINO Luigi , CHIANALE Jose
Published: August 2015

The canalicular multispecific organic anion transporter, cMoat, is an ATP-binding-cassette protein expressed in the canalicular domain of hepatocytes. In addition to the transport of endo- and xenobiotics, cMoat has also been proposed to transport GSH into bile, the major driving force of bile-acid-independent bile flow. We have shown previously that the herbicide 2,4,5-trichlorophenoxyacetic acid (2,4,5-T), a peroxisome-proliferator agent, significantly increases bile-acid-independent bile flow in mice. On this basis, the effect of the herbicide on cMoat gene expression was studied. A 3.6-fold increase in cMoat mRNA levels and a 2.5-fold increase in cMoat protein content were observed in the liver of mice fed on a diet supplemented with 0.125% 2,4,5-T. These effects were due to an increased rate of gene transcription (3.9-fold) and were not associated with peroxisome proliferation. Significant increases in bile flow (2.23±0.39 versus 1.13±0.15 μl/min per g of liver; P < 0.05) and biliary GSH output (7.40±3.30 versus 2.65±0.34 nmol/min per g of liver; P < 0.05) were observed in treated animals. The hepatocellular concentration of total glutathione also increased in hepatocytes of treated mice (10.95±0.84 versus 5.12±0.47 mM; P < 0.05), because of the induction (2.4-fold) of the heavy subunit of the γ-glutamylcysteine synthetase (GCS-HS) gene. This is the first model of co-induction of cMoat and GCS-HS genes in vivo in the mouse liver, associated with increased glutathione synthesis and biliary glutathione output. Our observations are consistent with the hypothesis that the cMoat transporter plays a crucial role in the secretion of biliary GSH.
An aromatic, but not a basic, residue is involved in the toxicity of group-II phospholipase A2 neurotoxins
Authors: PUNGERĆAR Jože , KRIAJ Igor , LIANG Ning-Sheng , GUBENŠEK Franc
Published: August 2015

Ammodytoxins (Atxs) A, B and C are basic phospholipase A2s from Vipera ammodytes ammodytes snake venom, and they exhibit presynaptic toxicity. The most toxic is AtxA, followed by AtxC, its naturally occurring F124 → I/K128 → E mutant, which is 17 times less toxic. Two mutants of AtxA have been produced in bacteria and characterized. The specific enzymic activity of the K128 → E mutant on mixed phosphatidylcholine/Triton X-100 micelles is similar to that of the wild type. The K108 → N/K111 → N mutant, however, possesses 160% of the wild-type activity. Replacement of the two basic residues by uncharged, polar residues on the opposite side of the protein to the enzyme active site and interfacial adsorption surface results in increased enzymic activity at the water/lipid aggregate interface, due to a redistribution of electrostatic charge. The binding affinity of the double mutant for the specific acceptor in bovine brain was similar to that of AtxA, whereas the affinity of the single mutant was similar to that of AtxC, which was slightly weaker than that of AtxA. Interestingly, the substitution of any of these three basic surface residues did not significantly change the lethal potency of AtxA. Since the single mutant AtxA(K128 → E) is equivalent to the AtxC(I124 → F) mutant, this indicates that the residue at position 124 is important for presynaptic toxicity of Atxs. The more than 10-fold lower toxicity of AtxC, compared with AtxA, is a consequence of the substitution of Phe-124 (aromatic ring) with Ile (aliphatic chain). Exposed aromatic residues in the C-terminal region may also be important for the neurotoxicity of other similar toxins.
Chloroplast thioredoxin mutants without active-site cysteines facilitate the reduction of the regulatory disulphide bridge on the γ-subunit of chloroplast ATP synthase
Authors: STUMPP Michael T. , MOTOHASHI Ken , HISABORI Toru
Published: August 2015

The activity of the chloroplast H+-ATPase (CFoCF1) is regulated by the proton electrochemical membrane potential and the reduction or the formation of the disulphide bridge on the γ-subunit mediated by chloroplast thioredoxins (Trx). The latter regulation also applies to the water-soluble portion of CFoCF1 (CF1) and includes two successive steps, namely the binding of Trx to CF1 and the subsequent reduction or oxidation of CF1. To study this process thoroughly, a new expression system for spinach Trx-f and Trx-m was designed. In the presence of dithiothreitol (DTT) both forms of the expressed Trx could reduce the disulphide bridge on the γ-subunit of CF1 and thus activate the ATPase. Trx mutants deficient in the internal, or both, cysteines of the active site were designed to study the details of the interaction. The Trx mutant proteins could still activate CF1-ATPase in the presence of DTT and they also increased the apparent affinity of CF1 for DTT. This implies that the binding of Trx to the CF1 γ-subunit induces a conformational change facilitating the reduction of the disulphide bridge, and partially explains the high efficiency of Trx as a reductant in vivo.
Doc2 is not associated with known regulated exocytotic or endosomal compartments in adrenal chromaffin cells
Authors: CHARVIN Nathalie , WILLIAMS Geoff , BURGOYNE Robert D.
Published: August 2015

Doc2 is a C2-domain-containing protein that is highly expressed in the nervous system and has a constitutively expressed isoform. It has been implicated as a potential Ca2+ sensor in regulated exocytosis, and has been suggested to be associated with synaptic vesicles. To examine whether Doc2 is associated with synaptic-like microvesicles (SLMVs) or dense-core granules in neuroendocrine cells, we examined the distribution of Doc2 in subcellular fractionation of chromaffin cells of the adrenal medulla and in PC12 cells. Doc2 did not co-distribute with SLMVs from either cell type, but did appear to co-distribute with dense-core granules from PC12 cells. In contrast, it was not associated with the dense-core granules during subcellular fractionation of the adrenal medulla, and nor did it appear to be associated with endosomes, cis-Golgi or the trans-Golgi network. In contrast, Doc2 co-distributed under all conditions with a mitochondrial marker. We conclude that Doc2 is not a general component of regulated secretory vesicles, but may instead be associated with mitochondria.
The mitochondrial permeability transition pore and its role in cell death
Authors: CROMPTON Martin
Published: August 2015

This article reviews the involvement of the mitochondrial permeability transition pore in necrotic and apoptotic cell death. The pore is formed from a complex of the voltage-dependent anion channel (VDAC), the adenine nucleotide translocase and cyclophilin-D (CyP-D) at contact sites between the mitochondrial outer and inner membranes. In vitro, under pseudopathological conditions of oxidative stress, relatively high Ca2+ and low ATP, the complex flickers into an open-pore state allowing free diffusion of low-Mr solutes across the inner membrane. These conditions correspond to those that unfold during tissue ischaemia and reperfusion, suggesting that pore opening may be an important factor in the pathogenesis of necrotic cell death following ischaemia/reperfusion. Evidence that the pore does open during ischaemia/reperfusion is discussed. There are also strong indications that the VDAC-adenine nucleotide translocase-CyP-D complex can recruit a number of other proteins, including Bax, and that the complex is utilized in some capacity during apoptosis. The apoptotic pathway is amplified by the release of apoptogenic proteins from the mitochondrial intermembrane space, including cytochrome c, apoptosis-inducing factor and some procaspases. Current evidence that the pore complex is involved in outer-membrane rupture and release of these proteins during programmed cell death is reviewed, along with indications that transient pore opening may provoke ‘accidental’ apoptosis.
Molecular analysis of sialoside binding to sialoadhesin by NMR and site-directed mutagenesis
Authors: CROCKER Paul R. , VINSON Mary , KELM Sørge , DRICKAMER Kurt
Published: August 2015

The molecular interactions between sialoadhesin and sialylated ligands have been investigated by using proton NMR. Addition of ligands to the 12 kDa N-terminal immunoglobulin-like domain of sialoadhesin result in resonance shifts in the protein spectrum that have been used to determine the affinities of sialoadhesin for several sialosides. The results indicate that α2,3-sialyl-lactose and α2,6-sialyl-lactose bind respectively 2- and 1.5-fold more strongly than does α-methyl-N-acetylneuraminic acid (α-Me-NeuAc). The resonances corresponding to the methyl protons within the N-acetyl moiety of sialic acid undergo upfield shifting and broadening during titrations, reflecting an interaction of this group with Trp2 in sialoadhesin as observed in co-crystals of the terminal domain with bound ligand. This resonance shift was used to measure the affinities of mutant and wild-type forms of sialoadhesin in which the first three domains are fused to the Fc region of human IgG1. Substitution of Arg97 by alanine completely abrogated measurable interaction with α-Me-NeuAc, whereas a conservative substitution with lysine resulted in a 10-fold decrease in affinity. These results provide the first direct measurement of the affinity of sialoadhesin for sialosides and confirm the critical importance of the conserved arginine in interactions between sialosides and members of the siglec family of sialic acid-binding, immunoglobulin-like lectins.