bluuebunny/crossref_metadata_2025 huggingface dataset:
Temperature-induced membrane-lipid adaptation in Acanthamoeba castellanii
Authors: Jones A L , Hann A C , Harwood J L , Lloyd D
Published: August 2015

A method has been developed for the separation of the major membrane fractions of Acanthamoeba castellanii after growth at different temperatures. The acyl-lipid compositions of individual membrane fractions, microsomal membranes, plasma membrane and mitochondria were analysed after a shift in culture temperature from 30 degrees C to 15 degrees C. The major change in lipid composition observed was an alteration in the relative proportions of oleate and linoleate. This reciprocal change was seen in all the membrane fractions, but occurred most rapidly in the phosphatidylcholine of the microsomal fraction. Thus, there appears to be a rapid induction of delta 12-desaturase activity in A. castellanii after a downward shift in growth temperature. Changes were also seen in the proportions of the n-6 C20 fatty acids, with a decrease in the proportions of icosadienoate and increases of icosatrienoate and arachidonate. However, unlike the alteration in oleate/linoleate ratios, this change was not seen in all the individual lipids of each membrane fraction.
Characterization of structural and folding properties of streptokinase by n.m.r. spectroscopy
Authors: Teuten A J , Broadhurst R W , Smith R A G , Dobson C M
Published: August 2015

The structure and physical properties of the fibrinolytic protein streptokinase have been investigated by 1H-n.m.r. spectroscopy. Well-resolved one- and two-dimensional spectra have been obtained for this molecule of molecular mass 47 kDa. Titration of all nine histidine residues has shown that these display a range of pKa values, between 5.6 and 8.2, revealing a variety of environments for these residues in the protein structure. Although at least eight histidine residues can be reversibly modified by diethylpyrocarbonate, only one is sufficiently exposed to be reactive towards photo-excited dye in chemically induced dynamical nuclear polarization spectroscopy experiments. Unfolding studies have been performed by thermal and chemical means. Evidence is presented here for several distinct unfolding transitions suggesting that the protein consists of at least three domains which have independent stability, and that the protein can exist in a number of partially folded states. For one of these, that formed in 2 M guanidine hydrochloride, it has been shown that the N-terminal region of the molecule is extensively unfolded, while other regions of the protein remain in native-like folded states.
Cytokine induction of haem oxygenase mRNA in mouse liver. Interleukin 1 transcriptionally activates the haem oxygenase gene
Authors: Rizzardini M , Terao M , Falciani F , Cantoni L
Published: August 2015

Accumulation of the mRNA coding for haem oxygenase (HO, EC 1.14.99.3) was stimulated by treating mice with endotoxin (lipopolysaccharide, LPS; 20 micrograms/mouse intraperitoneally), suggesting that haem catabolism is a target of infection and inflammation in vivo. Therefore various cytokines, possible mediators for the biological responses to LPS, were administered intraperitoneally to mice, and the levels of HO mRNA were measured by Northern-blotting analysis using the rat HO cDNA as a probe [Shibahara, Müller, Taguchi and Yoshida (1985) Proc. Natl. Acad. Sci. U.S.A. 82, 7865-7869]. Marked induction of HO mRNA was observed 2 h after administration of interleukin 1 (IL-1) (34-fold) and tumour necrosis factor (19.5-fold) (5 micrograms/mouse), whereas interleukin 6 (6.2 micrograms/mouse) was much less active (3.5-fold) and interleukin 2 (25 micrograms/mouse) and interferon-gamma (3 micrograms/mouse) were ineffective. HO mRNA induced by the cytokines of LPS accumulated rapidly (maximum at 1-2 h after administration), preceding the elevation of HO enzymic activity. Treatment of mice with IL-1 stimulated the transcription of the HO gene by 4-fold, as assessed by in vitro nuclear-run-on assay. These results indicate that enzymic haem catabolism in the liver is a process inducible in vivo by inflammatory cytokines, which up-regulate HO synthesis at the transcriptional level. Increased removal of haem might be part of the protective mechanisms elicited by the acute-phase response, possibly to reduce the pro-oxidant state of the cell.
Effect of heterologous expression of acyl-CoA-binding protein on acyl-CoA level and composition in yeast
Authors: Mandrup S , Jepsen R , Skøtt H , Rosendal J , Højrup P , Kristiansen K , Knudsen J
Published: August 2015

We have expressed a bovine synthetic acyl-CoA-binding protein (ACBP) gene in yeast (Saccharomyces cerevisiae) under the control of the GAL1 promoter. The heterologously expressed bovine ACBP constituted up to 6.4% of total cellular protein and the processing was identical with that of native bovine ACBP, i.e. the initiating methionine was removed and the following serine residue was N-acetylated. The expression of this protein did not affect the growth rate of the cells. Determination of the yeast acyl-CoA pool size showed a close positive correlation between the ACBP content of the cells and the size of the acyl-CoA pool. Thus ACBP can act as an intracellular acyl-CoA pool former. Possible physiological functions of ACBP in cells are discussed.
UDP-sugar metabolism in Swarm rat chondrosarcoma chondrocytes
Authors: Sweeney C , Mackintosh D , Mason R M
Published: August 2015

UDP-sugars and adenine nucleotides were extracted from freshly isolated chondrocytes and primary cell cultures and analysed by anion-exchange h.p.l.c. The pool sizes of UDP-N-acetylglucosamine, UDP-N-acetylgalactosamine, UDP-glucose-galactose, UDP-glucuronate and UDP-xylose were 2.9, 1.2, 2.5, 0.6 and 0.03 nmol/10(6) freshly isolated chondrocytes. When chondrocytes were maintained in Dulbecco's modified Eagle medium supplemented with 15% foetal-bovine serum, synthesis of [35S]proteoglycan and [3H]protein decreased over the first 48 h in culture, as did the pools of UDP-glucuronate and ATP. In contrast, the size of the UDP-N-acetylhexosamine pools underwent little change during culture. [35S]Proteoglycan and [3H]protein syntheses were stimulated in cultures supplemented with serum or insulin compared with those maintained in medium alone, in agreement with previous results. However, the UDP-sugar pool sizes were the same in both supplemented and non-supplemented cultures. In cultures maintained in the presence of [1-3H]glucose, the UDP-sugars were labelled to a constant 3H specific radioactivity which was very similar to that of the labelling medium. UDP-N-acetylhexosamines were labelled to constant 3H specific radioactivity with [6-3H]glucosamine as a precursor, but only about 1 in 375 of these UDP-sugars was derived from the amino sugar in the presence of glucose. The half-life (t1/2) for UDP-hexoses, UDP-glucuronate and UDP-N-acetylhexosamines was about 12, 12 and 50 min respectively.
Age-related changes in expression of the neural cell adhesion molecule in skeletal muscle: a comparative study of newborn, adult and aged rats
Authors: Andersson A M , Olsen M , Zhernosekov D , Gaardsvoll H , Krog L , Linnemann D , Bock E
Published: August 2015

Neural cell adhesion molecule (NCAM) is expressed by muscle and involved in muscle-neuron and muscle-muscle cell interactions. The expression in muscle is regulated during myogenesis and by the state of innervation. In aged muscle, both neurogenic and myogenic degenerative processes occur. We here report quantitative and qualitative changes in NCAM protein and mRNA forms during aging in normal rat skeletal muscle. Determination of the amount of NCAM by e.l.i.s.a. showed that the level decreased from perinatal to adult age, followed by a considerable increase in 24-month-old rat muscle. Thus NCAM concentration in aged muscle was sixfold higher than in young adult muscle. In contrast with previous reports, NCAM polypeptides of 200, 145, 125 and 120 kDa were observed by immunoblotting throughout postnatal development and aging, the relative proportions of the individual NCAM polypeptides remaining virtually unchanged at all ages examined. However, changes in the extent of sialylation of NCAM were demonstrated. Even though the relative amounts of the various NCAM polypeptides were unchanged during aging, distinct changes in NCAM mRNA classes were observed. Three NCAM mRNA classes of 6.7, 5.2 and 2.9 kb were present in perinatal and young adult skeletal muscle, whereas only the 5.2 and 2.9 kb mRNA classes could be demonstrated in aged muscle. This indicates that metabolism of the various NCAM polypeptides is individually regulated during aging. Alternative splicing of NCAM mRNA in skeletal muscle was studied by Northern blotting using DNA oligonucleotide probes specifically hybridizing to selected exons or exon combinations. Exon VASE, which has previously been shown to be present in both brain and heart NCAM mRNA, was virtually absent from skeletal muscle at all ages studied. In contrast, the majority of NCAM mRNA in postnatal skeletal muscle was shown to contain extra exons inserted between exons 12 and 13. Of the various possible exon combinations at this splice site, the combinations 12-a-AAG-13 and 12-a-b seemed to be prevalent in postnatal skeletal muscle. No significant change in the relative proportion of these two exon combinations occurred during aging. The observed upregulation of NCAM protein in aged muscle supports the assumption that an increasing proportion of muscle fibres are denervated in aged muscle. Selective upregulation of the 5.2 and 2.9 kb mRNA forms have previously been demonstrated in muscle cell lines and in primary cultures of muscle cells during formation of myotubes in vitro, and this switch in NCAM mRNA classes has been suggested to correlate with myogenesis.(ABSTRACT TRUNCATED AT 250 WORDS)
Evidence for involvement of phospholipase C-γ2 in signal transduction of platelet-derived growth factor in vascular smooth-muscle cells
Authors: Homma Y , Sakamoto H , Tsunoda M , Aoki M , Takenawa T , Ooyama T
Published: August 2015

In order to examine the mechanisms underlying smooth-muscle cell proliferation, we investigated effect of platelet-derived growth factor (PDGF) dimers on proliferation of rabbit vascular smooth-muscle cells (VSMCs) and also involvement of phospholipase C (PLC) isoforms in the signal transduction. PDGF-BB and -AB, but not -AA, stimulated cell proliferation and intracellular production of inositol trisphosphate. Northern and Western analyses demonstrated that VSMCs mainly expressed PLC-gamma 2 and PLC-delta 1 among four PLC isoforms tested. A number of cellular proteins, including PLC-gamma 2, but not PLC-delta 1, were phosphorylated on a tyrosine residue by the stimulation of either PDGF-BB or -AB. These results suggest a functional association of PDGF receptor and PLC-gamma 2 that might be responsible for PDGF-dependent VSMC growth. In addition, the expression of PLC-gamma 2 was extremely low in the primary VSMC cultures and was induced during further cultivation of the primary cultures, indicating that an acquisition of PDGF-signal-transducing components, including PLC-gamma 2, may be an important step for proliferation of smooth-muscle cells.
Evidence for common structural changes in thrombin induced by active-site or exosite binding
Authors: Parry M A A , Stone S R , Hofsteenge J , Jackman M P
Published: August 2015

The gamma-loop of thrombin is a flexible, surface-accessible loop in free thrombin that appears to be one of several sites participating in the interaction of the enzyme with macromolecular substrates and inhibitors. Using limited proteolysis and intrinsic fluorescence measurements, we have studied changes in thrombin structure induced by small, site-specific ligands. Binding of a C-terminal peptide of hirudin to the anion-binding exosite of thrombin induced a structural change in the gamma-loop, which caused a 6-fold reduction in the susceptibility of the enzyme to limited proteolysis by elastase and chymotrypsin. Binding of several active site-specific thrombin inhibitors conferred an even greater protection from proteolysis at the gamma-loop. For example, the covalent complex of thrombin with D-Phe-Pro-Arg-CH2Cl was 95-fold less susceptible to cleavage by chymotrypsin than the free enzyme. Furthermore, binding of either exosite or active-site probes induced a common intrinsic fluorescence change in thrombin (a fractional increase of 0.13). These results are surprising because crystallographic studies indicate that direct contact between the bound probes and relevant residues of the gamma-loop is very unlikely. Thus we have identified an allosteric interaction that couples the active site of thrombin to the gamma-loop. An interaction of this nature may be one way in which thrombomodulin modulates the reactivity of thrombin.
The long-chain sphingoid base of sphingolipids is acylated at the cytosolic surface of the endoplasmic reticulum in rat liver
Authors: Hirschberg K , Rodger J , Futerman A H
Published: August 2015

Ceramide, a key intermediate in sphingolipid metabolism, is synthesized by acylation of sphinganine followed by dehydrogenation of dihydroceramide to ceramide. Using radioactive sphinganine, we have examined the site and topology of dihydroceramide synthesis in well-characterized subcellular fractions from rat liver. [4,5-3H]Sphinganine was introduced as a complex with BSA and was metabolized to [4,5-3H]dihydroceramide upon incubation of rat liver homogenates or microsomes with fatty acyl CoA. Conditions were established in a detergent-free system in which dihydroceramide synthesis was not limited by either substrate availability or by amounts of microsomal protein or reaction time. The distribution of dihydroceramide synthesis was found to exactly parallel that of an endoplasmic reticulum (ER) marker upon subfractionation of microsomes, and no endogenous activity was detected in either purified Golgi apparatus or plasma membrane fractions. Limited protease digestion demonstrated that sphinganine N-acyltransferase is localized at the cytosolic surface of intact ER-derived vesicles. These results are discussed with regard to the subsequent transport of (dihydro)-ceramide from the ER to sites of further metabolism in a pre-Golgi apparatus compartment and in the cis and medial cisternae of the Golgi apparatus.
A novel post-translational modification of the peptide antibiotic subtilin: isolation and characterization of a natural variant from Bacillus subtilis A.T.C.C. 6633
Authors: Chan W C , Bycroft B W , Leyland M L , Lian L Y , Roberts G C K
Published: August 2015

A variant of the peptide antibiotic subtilin has been isolated from Bacillus subtilis A.T.C.C. 6633, and its structure has been shown to be [N alpha-succinyl-Trp1]subtilin. The chemical structure of a fragment derived by tryptic hydrolysis of the variant is shown to be N alpha-succinyl-Trp-Lys by 1H and 13C n.m.r., fast-atom-bombardment m.s. and total chemical synthesis [N alpha-Succinyl-Trp1]-subtilin is produced later in the growth of the bacterium than is subtilin; reverse-phase h.p.l.c. analysis shows that after 24 h growth the ratio subtilin/[N alpha-succinyl-Trp1]subtilin is approx. 1:2. Although [N alpha-succinyl-Trp1]subtilin retains significant antibacterial activity, it is 10-20 times less active than subtilin.
Expression of the glucose transporter GLUT4 in the muscular dystrophic mdx mouse
Authors: Olichon-Berthe C , Gautier N , Van Obberghen E , Le Marchand-Brustel Y
Published: August 2015

Glucose transporter protein levels have been investigated in mdx and control (C57Bl/10) mice. Crude membrane fractions (microsomes plus plasma membranes) were prepared from skeletal muscle, heart, diaphragm and brain of 5-6-week-old and 6-7-month-old control and mdx mice. Using Western blot analysis with C-terminal-specific anti-peptide antibodies, we investigated the glucose transporters GLUT4 in the different muscle tissues and GLUT1 in brain. In skeletal tissue from the hindlegs, GLUT4 was increased by approximately 55% in mdx mice compared with control mice at both ages studied. In the diaphragm, the amount of GLUT4 protein was unchanged in young mdx mice, and was decreased by 37.4 +/- 4.7% in older mice compared with age-matched control mice. No difference was observed between mdx and control mice in the amounts of GLUT4 and GLUT1 in heart and brain preparations respectively. To determine whether the change in GLUT4 protein observed in the diaphragm and skeletal muscle of mdx mice was regulated through changes at the level of glucose transporter mRNA, Northern blot analyses were performed. In skeletal muscle, GLUT4 mRNA level per tissue was not different between the two groups of mice at both ages studied. In contrast, the decrease in the amount of GLUT4 protein observed in the diaphragm of 6-7-month-old mdx mice was accompanied by a decrease in the GLUT4 mRNA level. In conclusion, the levels of GLUT4 protein were modified in muscle tissues from mdx compared with control mice, and these modifications were different depending on the muscle involved and the age of the mice. An increase in the amount of GLUT4 protein in the skeletal muscle of mdx mice was not due to changes at the mRNA level. The diaphragms of 6-7-month-old mdx mice exhibited decreases in GLUT4 protein and mRNA levels that were not detected in young animals (5-6 weeks old).
Versican gene expression in human articular cartilage and comparison of mRNA splicing variation with aggrecan
Authors: Grover J , Roughley P J
Published: August 2015

The chondrocytes in human articular cartilage from subjects of all ages express mRNAs for both of the aggregating proteoglycans aggrecan and versican, although the level of expression of versican mRNA is much lower than that of aggrecan mRNA. Aggrecan shows alternative splicing of the epidermal growth factor (EGF)-like domain within its C-terminal globular region, but there is no evidence for a major difference in situ in the relative expression of this domain with age. At all ages studied from birth to the mature adult, a greater proportion of transcripts lacked the EGF domain. The relative proportions of the two transcripts did not change upon culture and passage of isolated chondrocytes. In contrast, the neighbouring complement regulatory protein (CRP)-like domain was predominantly expressed irrespective of age, but cell culture did result in variation of the splicing of this domain. Versican possesses two EGF-like domains and one CRP-like domain, but at all ages the three domains were predominantly present in all transcripts. This situation persisted upon culture and passage of the chondrocytes. Thus, unlike aggrecan, the versican expressed by human articular cartilage does not appear to undergo alternative splicing of its C-terminal globular region, either in cartilage in situ or in chondrocytes in culture.
Inhibition of calcium signalling in murine splenocytes by polyamines: differential effects on CD4 and CD8 T-cells
Authors: Thomas T , Gunnia U B , Yurkow E J , Seibold J R , Thomas T J
Published: August 2015

Transmembrane Ca2+ influx is recognized as a universal second messenger that transduces T-cell activation signals to cytoplasm and nucleus, thereby stimulating transcription and cell division. To examine the role of endogenous factors that regulate mitogenic Ca2+ signalling of T-cells, we measured the concanavalin (Con) A-induced increase in cytoplasmic free calcium ([Ca2+]i) in spleen cells of BALB/c mice, using flow cytometry with an indicator dye, Indo-1 acetoxymethyl ester (Indo-1/AM). Con A is a polyclonal activator of T-cells. Unstimulated splenocytes had a [Ca2+]i of 100 nM. [Ca2+]i increased with Con A in a dose-dependent manner up to a concentration of 50 micrograms/ml. In the presence of 50 micrograms/ml Con A, [Ca2+]i was 350 nM. Natural polyamines (putrescine, spermidine and spermine) inhibited Con-A-induced Ca2+ influx in a dose-dependent manner. Putrescine was the most effective polyamine in desensitizing the Ca2+ signal, and decreased [Ca2+]i from 350 nM in the absence of putrescine to 250 nM in the presence of 100 microM putrescine. This effect was not mimicked by structurally related homologues or inorganic cations, suggesting a specific structural effect of the polyamine. H.p.l.c. analysis showed that polyamines were internalized during incubation of cells in vitro. In experiments using monoclonal anti-CD4 and anti-CD8 antibodies, we found a differential effect of putrescine on Ca2+ influx in CD4 and CD8 subpopulations of T cells. For CD4+ cells, [Ca2+]i decreased from 625 nM to 420 nM in the presence of 500 microM putrescine, whereas [Ca2+]i was not affected by putrescine in CD8+ cells. These data suggest that natural polyamines have cell-specific effects on mitogen-stimulated Ca(2+)-influx in T-cell subsets.
Interaction of caldesmon with phospholipids
Authors: Czuryło E A , Zborowski J , Dabrowska R
Published: August 2015

The interaction of caldesmon with liposomes composed of various phospholipids has been examined by tryptophan fluorescence spectroscopy. The results indicate that caldesmon makes its strongest complex with phosphatidylserine (PS) vesicles (Kass. = 1.45 x 10(5) M-1). Both electrostatic and hydrophobic interactions contribute to the stability of this complex. The site for strong binding of PS seems to be located in the N-terminal part of the 34 kDa C-terminal fragment of caldesmon. Binding of PS at this site results in displacement of calmodulin from its complex with caldesmon.
ADP-ribosylation of Drosophila indirect-flight-muscle actin and arthrin by Clostridium botulinum C2 toxin and Clostridium perfringens iota toxin
Authors: Just I , Hennessey E S , Drummond D R , Aktories K , Sparrow J C
Published: August 2015

Purified Drosophila indirect-flight-muscle actin and arthrin, an actin-ubiquitin conjugate, were ADP-ribosylated by Clostridium botulinum C2 toxin and Clostridium perfringens iota toxin. Phalloidin treatment inhibited the ADP-ribosylation of Drosophila actin and arthrin. Like actin, the ADP-ribose-arthrin linkage was sensitive towards hydroxylamine treatment, indicating arginine as the amino acid acceptor. Actin translated in vitro from the indirect-flight-muscle-specific gene Act88F was ADP-ribosylated by C. botulinum C2 toxin and C. perfringens iota toxin. Actin from the R177Q mutant of Act88F translated in vivo was not ADP-ribosylated confirming Arg-177 as the ADP-ribose acceptor. Mutant L176M actin was modified by both toxins, indicating that amino acid 176 of actin does not define the substrate specificity of C. botulinum C2 toxin. Whereas the gene products of various C-terminal mutants of Act88F translated in vitro (E334K, V339I, E364K, G368E, R372H) were substrates for ADP-ribosylation by C. botulinum C2 toxin and by C. perfringens iota toxin, neither toxin modified the N-terminal O-12 deletion mutant.
Rapid cellular removal of a membrane-inserted foreign polypeptide
Authors: Moskaug J Ø , Prydz K , Olsnes S
Published: August 2015

We have developed a system that makes it possible to study the fate of a foreign polypeptide that is inserted in the plasma membrane. Diphtheria toxin is a bacterial protein toxin that, upon acidification, has the ability to insert into the plasma membrane from the outside of eukaryotic cells. We present results that indicate endocytic uptake and degradation of the diphtheria toxin B-fragment after insertion into the membrane of Vero cells. The degradation rate of the fragment was found to be very high (t1/2 = 6 min) and dependent on cleavage of the extracellular part of the polypeptide with protease. Degradation was strongly inhibited in ATP-depleted cells, as well as at temperatures below 18 degrees C, and it was partially inhibited when the cytosol was acidified to block endocytosis from clathrin-coated pits. Degradation was also reduced in the presence of NH4Cl. The results indicate that the inserted and cleaved B-fragment is degraded by a process requiring endocytosis and transport to late endosomes or to lysosomes.
Absence of phosphocreatine resynthesis in human calf muscle during ischaemic recovery
Authors: Quistorff B , Johansen L , Sahlin K
Published: August 2015

Changes in the metabolites phosphocreatine (PCr), Pi and ATP were quantified by 31P n.m.r. spectroscopy in the human calf muscle during isometric contraction and recovery under ischaemic conditions. Time resolution of the measurements was 10 s. During a 30-60 s ischaemic isometric contraction, PCr decreased linearly at a rate of 1.17%/s (relative to the resting value) at a contraction strength equivalent to 70% of the maximal voluntary contraction (MVC) and at a rate of 2.43%/s at 90% MVC. There was a corresponding increase in Pi but the concentration of ATP did not change. pH decreased linearly during contraction by 4.22 and 8.23 milli-pH units/s at 70 and 90% MVC respectively. During a subsequent 5 min interval of ischaemic recovery, PCr, Pi, ATP, phosphomonoesters and calculated free ADP, free AMP and pH retained the value they had attained by the end of contraction with no significant recovery. Thus it is concluded that anaerobic glycolysis and glycogenolysis is halted momentarily on termination of contraction and that PCr is not resynthesized during ischaemic recovery. This paradoxical arrest of glycolytic flow in spite of the very significantly elevated concentration of potent activators such as Pi and free AMP clearly indicates that parameters other than PCr, ATP, Pi, calculated pH, free ADP and free AMP regulate glycolysis and glycogenolysis of human skeletal muscle very efficiently under ischaemic conditions.
Biosynthesis of human acute-phase serum amyloid A protein (A-SAA) in vitro: the roles of mRNA accumulation, poly(A) tail shortening and translational efficiency
Authors: Steel D M , Rogers J T , DeBeer M C , DeBeer F C , Whitehead A S
Published: August 2015

Human ‘acute-phase’ serum amyloid A protein (A-SAA) is a major acute-phase reactant (APR) and an apolipoprotein of high density lipoprotein 3 (HDL3). We have examined several parameters of A-SAA biosynthesis in PLC/PRF/5 hepatoma cells in response to monocyte conditioned medium (MoCM) and dual treatment with interleukin-1 beta and interleukin-6 (IL-1 beta + IL-6). Treatment of PLC/PRF/5 cells with MoCM or IL-1 beta + IL-6 caused a dramatic and rapid increase in A-SAA mRNA and protein synthesis; A-SAA mRNA was first detectable at 3 h, with peak levels reached by 24 h. A-SAA mRNA accumulation is accompanied by a gradual and homogeneous decrease in the length of the A-SAA poly(A) tail; the poly(A) tail shortening does not apparently affect the intrinsic stability of A-SAA mRNA. Analysis of RNA isolated from the ribonucleoprotein, monosome and polysome fractions of cytokine-treated PLC/PRF/5 cells showed that most A-SAA mRNA was associated with small polyribosomes, regardless of time post-stimulus, suggesting that the translational efficiency of A-SAA mRNA is constant throughout cytokine-driven induction. Moreover, the transit time of A-SAA protein out of the cell is also constant throughout the time course of induction. These data provide evidence of a paradox with regard to the transcriptional upregulation of A-SAA by IL-1 beta + IL-6 and the relative synthesis of A-SAA protein and suggest a role for post-transcriptional control of A-SAA biosynthesis during the acute phase.
Formylmethionyl-leucylphenylalanine and the SOS operon in Escherichia coli: a model of host-bacterial interactions
Authors: Broom M F , Sherriff R M , Ferry D M , Chadwick V S
Published: August 2015

To determine the biological significance of the existence of highly specific receptors for the bacterial chemotactic peptide formylmethionyl-leucylphenylalanine (fMet-Leu-Phe) on neutrophil leucocytes, we investigated the role of this peptide in bacterial metabolism. The UmuD protein of the Escherichia coli SOS operon was identified as having an N-terminal fMet-Leu-Phe sequence and a recombinant E. coli with the umuD gene on plasmid pSB13 was shown to be an over-producer of both UmuD and fMet-Leu-Phe. Activation of SOS genes in conventional wild-type E. coli (K12) by u.v. light or hydrogen peroxide increased fMet-Leu-Phe production up to 4-fold. A RecA- strain, incapable of SOS activation, was a low basal producer of fMet-Leu-Phe and showed no increased production with u.v. light or oxidant stress. We propose that host phagocytes respond to fMet-Leu-Phe and closely related peptides because they are generated by bacteria under oxidant stress. Increased fMet-Leu-Phe production may signal to the host a change in the organism's biological status from commensal to pathogen because of the invasion into tissues exposing bacteria to high pO2 levels and oxidant stress.
Increase in bleomycin-detectable iron in ischaemia/reperfusion injury to rat kidneys
Authors: Baliga R , Ueda N , Shah S V
Published: August 2015

Iron has been shown to be important in ischaemic, immune and toxic forms of tissue injury in various organs. Although it is generally accepted that iron participates in the generation of powerful oxidant species (e.g. hydroxyl radicals) there has not been any direct evidence that iron capable of catalysing free-radical reactions is increased in tissues in these models of injury. In the present study we demonstrate that ischaemia/reperfusion injury to the kidney results in no significant change in total, nonhaem or ferritin iron levels, but there is a marked and specific increase in bleomycin-detectable iron (capable of catalysing free-radical reactions) in the kidney. The increase in bleomycin-detectable iron is observed only after reperfusion but not during the ischaemic period. In a separate study we demonstrate that despite a drastic reduction in the iron content in the kidney, as a result of feeding an iron-deficient diet, there is a similar and a marked increase in the bleomycin-detectable iron in kidneys accompanied by a lack of protection against ischaemia/reperfusion injury.
Corticosteroidogenesis in the toad Bufo arenarum H: evidence for a precursor role for an aldosterone 3β-hydroxy-5-ene analogue (3β, 11β, 21-trihydroxy-20-oxo-5-pregnen-18-al)
Authors: Ceballos N R , Shackleton C H , Harnik M , Cozza E N , Gros E G , Lantos C P
Published: August 2015

A material isolated following pregnenolone incubations with toad (Bufo arenarum) inter-renal tissue at 28 degrees C has been identified as a 3 beta-hydroxy-5-ene analogue of aldosterone (3 beta, 11 beta, 21-trihydroxy-20-oxo-5-pregnen-18-al). The initial identification was made by enzymic and m.s. methods, and structural confirmation was achieved through comparison with chemically synthesized authentic material. The relative efficacy of corticosterone, 18-hydroxycorticosterone and the 3 beta-hydroxy-5-ene aldosterone analogue as aldosterone precursors was evaluated. In the in vitro situation studied, the 3 beta-hydroxy-5-ene steroid was by far the best precursor.
The cDNA cloning of conglutinin and identification of liver as a primary site of synthesis of conglutinin in members of the Bovidae
Authors: Lu J , Laursen S B , Thiel S , Jensenius J C , Reid K B M
Published: August 2015

Bovine conglutinin is a collagen-like, C-type, plasma lectin which belongs to the group of proteins called ‘collectins’. Two inosine-containing oligonucleotides were synthesized, based on the published protein sequence for bovine conglutinin [Lee, Leiby, Allar, Paris, Lerch and Okarma (1991) J. Biol. Chem. 266, 2715-2723], and PCR on target DNA from a bovine liver lambda gt 11 cDNA library yielded a product of the expected size of 210 bp. Screening of the library with this cDNA fragment identified a single positive clone, with an insert of 0.9 kb, coding for bovine conglutinin from residue 70 to the C-terminus. The 5′ cDNA sequence, encompassing 150 bp of the 5′ non-translated sequence plus the sequence encoding the leader peptide and the N-terminal residues 1-70, was completed by the use of PCR techniques. The cDNA sequence of bovine conglutinin showed 86% identity with that of bovine lung surfactant protein D (SP-D), and the derived amino acid sequence of bovine conglutinin showed 78% identity with that of bovine SP-D, which included complete identity of the leader-peptide sequences. The amino acid sequence derived from the cDNA sequence differs from the published protein sequence at four positions. Northern-blot analysis on total RNA, purified from various tissues from cattle, sheep, humans, rats and mice, showed that a strong signal of approx. 1.8 kb is present in bovine liver RNA. A weak signal of similar size was also observed in sheep liver, but not in human, rat and mouse livers. A weak signal, also of 1.8 kb, is present in the lung RNAs of all the species tested. The signals from the lung tissues are likely to be due to the cross-hybridization of the bovine conglutinin cDNA to the SP-D mRNAs of the respective species. The finding of significant signals in only the bovine and sheep liver RNA samples is indicative that serum conglutinin may be present in significant amounts only in members of the Bovidae (the family encompassing cattle, antelopes, sheep and goats) and closely related species.
Cloning and characterization of the major hepatic glutathione S-transferase from a marine teleost flatfish, the plaice (Pleuronectes platessa), with structural similarities to plant, insect and mammalian Theta class isoenzymes
Authors: Leaver M J , Scott K , George S G
Published: August 2015

A cDNA clone (PLGSTA) of 896 bp, containing an open reading frame encoding a 225-amino-acid polypeptide of M(r) 25,723, was isolated from a cDNA library constructed in lambda gt11 from the liver of a marine teleost flatfish, the plaice (Pleuronectes platessa). The identification of this cDNA as that coding for the subunit of the major cytosolic glutathione S-transferase of plaice liver, GST-A, was supported by its heterologous expression in and purification of its protein product from Escherichia coli. The recombinant-derived protein exhibited identical M(r) and immunoreactivity and a similar substrate specificity to GST-A previously isolated from plaice liver. Comparison of the deduced amino acid sequence of the plaice GST-A polypeptide with the primary structures of GSTs from other Phyla revealed that it showed the greatest similarity to plant, insect and mammalian Theta class GSTs. Southern blot analysis of plaice DNA hybridized to the PLGSTA cDNA showed a banding pattern indicative of the presence of a single gene. Northern blot analysis of a variety of plaice tissues showed hybridizing bands of approx. 1100 nucleotides in all tissues tested, with the highest relative amounts in liver and intestinal mucosa. A marked increase in hybridization intensity was observed in hepatic RNA samples from plaice treated with trans-stilbene oxide, suggesting that GST-A is induced by epoxides in this species.
Modulation of murine hepatic lipase activity by exogenous and endogenous Kupffer-cell activation
Authors: Magilavy D B , Zhan R , Black D D
Published: August 2015

Deficiency of hepatic lipase (HL) may play a role in the lipoprotein abnormalities in chronic inflammatory states which are characterized by reticuloendothelial-system activation and cytokine release. HL triacylglycerol hydrolase activity was measured in heparin perfusates of livers from autoimmune MRL/lpr mice, which spontaneously develop a condition closely resembling human lupus erythematosis and exhibit spontaneous Kupffer-cell activation after 8 weeks of age, as well as from normal mice treated with Corynebacterium parvum or polyinosinic-polycytidylic acid complex [poly(I.C)] to induce Kupffer-cell activation. HL activity in MRL/lpr mice older than 8 weeks was 29.5% (P = 0.002) of that in age-matched control MRL/++ mice. Treatment of normal mice with C. parvum or poly(I.C) resulted in HL activities 18.6% (P = 0.004) and 13.1% (P = 0.007) respectively of untreated controls. Northern-blot hybridization of liver poly(A)+ RNA showed no differences in HL mRNA abundance in MRL/++ mice compared with the MRL/lpr autoimmune strain after 8 weeks of age, or in normal control mice compared with those treated with C. parvum, indicating attenuation of HL activity at the translational or post-translational level. Deficiency of this enzyme may represent one of the mechanisms contributing to the dyslipoproteinaemia of autoimmune disease and chronic infection.
Assembly of lipoprotein lipase in perfused guinea-pig hearts
Authors: Liu G , Bengtsson-Olivecrona G , Olivecrona T
Published: August 2015

It has been suggested that lipoprotein lipase (LPL) can be assembled into its catalytically active dimeric form only after its oligosaccharide chains have been processed in the Golgi. To study this in a complete organ, LPL was metabolically labelled with [35S]methionine in perfused guinea-pigs hearts. After 10 min pulse-labelling, LPL protein was eluted as two peaks from heparin-agarose: peak 1 at about 0.65 M NaCl, peak 2 at about 0.95 M NaCl. Catalytic activity was associated only with peak 2. Model studies with bovine LPL showed that active dimeric LPL is eluted in peak 2, but after treatments that dissociate the enzyme into inactive monomers it is eluted in peak 1. Pulse-labelled LPL in both peak 1 and peak 2 was fully sensitive to treatment with endoglycosidase (Endo) H. With chase, peak 1 disappeared and peak 2 acquired resistance to Endo H. These findings suggest that core glycosylated LPL is assembled into dimers already in the endoplasmic reticulum and that processing of the oligosaccharide chains occurs after dimerization.
Coupling of the α2-adrenergic receptor to the inhibitory G-protein Gi and adenylate cyclase in HT29 cells
Authors: Remaury A , Larrouy D , Daviaud D , Rouot B , Paris H
Published: August 2015

Previous studies have established that the human colon carcinoma cell line HT29 expresses an alpha 2-adrenergic receptor of the alpha 2A subtype, which is negatively coupled to adenylate cyclase. The purpose of the present study was to examine the mechanisms of alpha 2-adrenergic signal transduction in these cells. [32P]ADP-ribosylation with pertussis toxin and immunoblots using antibodies specific for the Gi alpha-subunits indicated that two distinct Gi-proteins (Gi2 and Gi3) were present in HT29-cell membranes. Treatment of intact cells with pertussis toxin resulted in a time-dependent decrease in the amount of [32P]ADP-ribosylatable Gi2 and Gi3, which coincided with a diminution in the number of alpha 2-adrenergic receptors in high-affinity state for agonists and with a progressive loss of ability of UK14304 to inhibit forskolin-stimulated accumulation of cyclic AMP. When membranes were [32P]ADP-ribosylated with cholera toxin in the absence of exogenous added guanine nucleotides, radioactivity was incorporated into a 45 kDa polypeptide representing Gs, as well as into 40-41 kDa polypeptides corresponding to Gi3 and Gi2. The amount of radioactivity incorporated into the two GiS under basal conditions was decreased by addition of the alpha 2-antagonist RX821002. It was not significantly affected by addition of clonidine (partial alpha 2-agonist), but was doubled by the addition of UK14304 (full alpha 2-agonist). This effect was blocked by RX821002. Study of adenylate cyclase activity indicated that preincubation of HT29 membranes with the antibody AS/7 (anti-alpha i1/alpha i2), but not with the antibody EC/2 (anti-alpha i3), attenuated the inhibitory effect of UK14304 on forskolin-stimulated adenylate cyclase. These data demonstrate that the alpha 2A-adrenergic receptor is coupled to both Gi2 and Gi3, and identify Gi2 as the major mediator of inhibition of adenylate cyclase in HT29 cells.
Effects of tetrahydrofolate polyglutamates on the kinetic parameters of serine hydroxymethyltransferase and glycine decarboxylase from pea leaf mitochondria
Authors: Besson V , Rebeille F , Neuburger M , Douce R , Cossins E A
Published: August 2015

Plant tissues contain highly conjugated forms of folate. Despite this, the ability of plant folate-dependent enzymes to utilize tetrahydrofolate polyglutamates has not been examined in detail. In leaf mitochondria, the glycine-cleavage system and serine hydroxymethyltransferase, present in large amounts in the matrix space and involved in the photorespiratory cycle, necessitate the presence of tetrahydrofolate as a cofactor. The aim of the present work was to determine whether glutamate chain length (one to six glutamate residues) influenced the affinity constant for tetrahydrofolate and the maximal velocities displayed by these two enzymes. The results show that the affinity constant decreased by at least one order of magnitude when the tetrahydrofolate substrate contained three or more glutamate residues. In contrast, maximal velocities were not altered in the presence of these substrates. These results are consistent with analyses of mitochondrial folates which revealed a pool of polyglutamates dominated by tetra and pentaglutamates. The equilibrium constant of the serine hydroxymethyltransferase suggests that, during photorespiration, the reaction must be permanently pushed toward the formation of serine (the unfavourable direction) to allow the recycling of tetrahydrofolate necessary for the operation of the glycine decarboxylase T-protein.
Cell- and inducer-specific accretion of human isometallothioneins
Authors: Cavigelli M , Kägi J H R , Hunziker P E
Published: August 2015

The synthesis of metallothionein (MT) was investigated in three different human epithelial cell lines, each derived from one of the embryonic germ layers. The accretion of different isoforms of the protein was monitored using a sensitive neutral-pH h.p.l.c. method. Induction of MT synthesis by zinc ions and dexamethasone revealed differences between the three cell lines, both with respect to the number and the amounts of the different isoMTs formed. Dose-response experiments showed that an increase in dexamethasone concentration enhances MT accretion asymptotically to a limit, whereas within the concentration range explored zinc produces an exponential augmentation.
Penicillin-binding protein 2x of Streptococcus pneumoniae: enzymic activities and interactions with β-lactams
Authors: Jamin M , Damblon C , Millier S , Hakenbeck R , Frère J M
Published: August 2015

The high-molecular-mass penicillin-binding protein (PBP) 2x, one of the primary targets of beta-lactam antibiotics in Streptococcus pneumoniae, has been produced as a soluble form and purified in large amounts. It has been shown to catalyse hydrolysis and transfer reactions with different ester and thiolester substrates and its catalytic behaviour was often similar to that of the soluble DD-peptidase from Streptomyces R61. This provided an easy method to monitor the activity of the PBP. For the first time, a reliable kinetic study of the interaction between a lethal target and beta-lactam antibiotics has been performed. Characteristic kinetic parameters were obtained with different beta-lactam compounds. These results not only validated the mechanism established with non-essential extracellular enzymes, but will also constitute the basis for comparative studies of the low-affinity variants from penicillin-resistant strains.
Annexin 5 as a potential regulator of annexin 1 phosphorylation by protein kinase C. In vitro inhibition compared with quantitative data on annexin distribution in human endothelial cells
Authors: Raynal P , Hullin F , Ragab-Thomas J M F , Fauvel J , Chap H
Published: August 2015

In vitro phosphorylation of annexin 1 by purified rat brain protein kinase C (PKC) has been studied in the presence of annexin 5, which is not a substrate for PKC. Annexin 5 promoted a dose-dependent inhibition of annexin 1 phosphorylation, which could be overcome by increasing the concentration of phosphatidylserine (PtdSer). In addition, a close relationship was found between the amount of PtdSer uncovered by annexin 5 and the residual phosphorylation of annexin 1. These data fit with the ‘surface depletion model’ explaining the antiphospholipase activity of annexins. In order to check the possibility that the in vitro effect of annexin 5 could be of some physiological relevance, annexins 1, 2, and 5, as well as the light chain of calpactin 1 (p11), have been quantified in human endothelial cells by measuring the radioactivity bound to the proteins after Western blotting with specific antibodies and 125I-labelled secondary antibody. Our data indicate that annexins 1 and 5, PKC and PtdSer are present in human endothelial cells in relative amounts very similar to those used in vitro under conditions permitting the detection of the inhibitory effect of annexin 5. Since annexin 1 remained refractory to PKC-dependent phosphorylation in intact cells, we suggest that annexin 5 might exert its inhibitory effect towards PKC in vivo, provided that its binding to phospholipids can occur at physiological (micromolar) concentrations of Ca2+. This was previously shown to occur in vitro using phosphatidylethanolamine/phosphatidic acid vesicles [Blackwood and Ernst (1990) Biochem. J. 266, 195-200]. Using identical assay conditions, which also allowed expression of PKC activity, annexin 5 again inhibited annexin 1 phosphorylation without interfering with PKC autophosphorylation. These data suggest that annexins 1 and 5 might interact with each other on the lipid surface, resulting in a specific inhibition of annexin 1 phosphorylation by PKC. Whether a similar mechanism also occurs in vivo remains to be determined.
Pyrophosphate-dependent phosphofructokinase from the amoeba Naegleria fowleri, an AMP-sensitive enzyme
Authors: Mertens E , De Jonckheere J , Van Schaftingen E
Published: August 2015

PPi-dependent phosphofructokinase (PPi-PFK) was detected in extracts of the amoeba Naegleria fowleri, with a specific activity of about 15-30 nmol/min per mg of protein, which was increased about 2-fold by 0.5 mM AMP. PPi-PFK was inactivated upon gel filtration and could be re-activated by incubation at 30 degrees C in the presence of AMP. N. fowleri PPi-PFK was purified more than 1100-fold to near homogeneity with a yield of about 25%. The pure enzyme had a specific activity of 65 mumol/min per mg of protein, and SDS/PAGE analysis showed a single band, of 51 kDa. Size-exclusion chromatography revealed the existence of two forms: a large one (approximately 180 kDa), presumably a tetramer, which was active, and a smaller one (approximately 45 kDa), presumably the monomer, which was inactive, but could be re-activated and converted into the large form by incubation at 30 degrees C in the presence of 0.5 mM AMP. Reactivation was also observed at 30 degrees C in the absence of AMP, particularly at higher enzyme concentration or in the presence of poly(ethylene glycol). Inactivation of the tetrameric enzyme was promoted by 0.25 M potassium thiocyanate. The enzyme displayed Km values of 10 and 15 microM for fructose 6-phosphate and PPi, respectively, in the forward reaction, and of 35 and 590 microM for fructose 1,6-bisphosphate and Pi in the backward reaction. The activity was dependent on the presence of Mg2+. AMP increased Vmax. about 2-fold without changing the affinity for the substrates; its half-maximal effect was observed at 2 microM.