bluuebunny/crossref_metadata_2025 huggingface dataset:
In vitro fibrillogenesis of collagen II from pig vitreous humour
Authors: Yang C , Notbohm H , Açil Y , Heifeng R , Bierbaum S , Müller P K
Published: August 2015

Collagen from pig vitreous humour was fractionated into a soluble and an insoluble fraction by centrifugation. Most of the collagen II in the soluble fraction was present as pN-collagen II (procollagen II without the C-terminal propeptide), besides smaller quantities of procollagen II, collagen II and two as yet unidentified alpha-chains of collagen II. Other collagen types may be present only in trace amounts. Collagen II of the insoluble fraction, which is mostly deposited in fibrillar aggregates, consists of both pN-collagen II and collagen II. To determine the possible role of collagen II precursors in the formation of the extracellular matrix of the vitreous humour these collagen molecules were purified and in vitro fibrillogenesis was used to demonstrate that pN-collagen II could form fibrils in mixtures with collagen II. These fibrils have a reduced mass per unit length depending on the content of pN-collagen in the mixture. Cross-sections of the newly formed fibrillar aggregates revealed a flattened shape. The incomplete processing of the precursors of collagen II may be part of regulatory mechanisms possibly controlling the formation of a translucent scaffold as is required in the vitreous humour.
Cloning, sequencing and expression of the pyrophosphate-dependent phosphofructo-1-kinase from Naegleria fowleri
Authors: Wessberg K L , Skolnick S , Xu J , Marciano-Cabral F , Kemp R G
Published: August 2015

The cDNA for the PPi-dependent phosphofructo-1-kinase has been cloned and sequenced from a cDNA library prepared from the free-living amoeba Naegleria fowleri. The coding sequence of the cDNA consists of 1311 bases which translates into 437 amino acids with a molecular mass of 48095 Da. Comparison of the sequence with those of the previously described sequences of PPi-dependent phosphofructokinases from Propionibacterium freudenreichii and potato tuber revealed amino acid identities of 23 and 28% respectively and high conservation in those regions assumed to be part of the active site. The reading frame was cloned into an expression vector, which was transformed into Escherichia coli. Extracts of the transformed cells contained PPi-dependent phosphofructokinase activity that could be purified to homogeneity. The activity was lost on incubation with the chaotropic agent, KSCN, and recovered by subsequent incubation with AMP. These properties are consistent with those described by Mertens, De Jonckheere and Van Schaftingen [Biochem. J. (1993) 292, 797-803] for the enzyme prepared from Naegleria and support the idea that the cloned cDNA coded for the complete native enzyme. No nucleotide-binding motif or evidence for a nucleotide-binding site characteristic of the ATP-dependent phosphofructokinases could be found within the primary structure.
A modular xylanase containing a novel non-catalytic xylan-specific binding domain
Authors: Black G W , Hazlewood G P , Millward-Sadler S J , Laurie J I , Gilbert H J
Published: August 2015

Xylanase D (XYLD) from Cellulomonas fimi contains a C-terminal cellulose-binding domain (CBD) and an internal domain that exhibits 65% sequence identity with the C-terminal CBD. Full-length XYLD binds to both cellulose and xylan. Deletion of the C-terminal CBD from XYLD abolishes the capacity of the enzyme to bind to cellulose, although the truncated xylanase retains its xylan-binding properties. A derivative of XYLD lacking both the C-terminal CBD and the internal CBD homologue did not bind to either cellulose or xylan. A fusion protein consisting of the XYLD internal CBD homologue linked to the C-terminus of glutathione S-transferase (GST) bound to xylan, but not to cellulose, while GST bound to neither of the polysaccharides. The Km and specific activity of full-length XYLD and truncated derivatives of the enzyme lacking the C-terminal CBD (XYLDcbd), and both the CBD and the internal CBD homologue (XYLDcd), were determined with soluble and insoluble xylan as the substrates. The data showed that the specific activities of the three enzymes were similar for both substrates, as were the Km values for soluble substrate. However, the Km values of XYLD and XYLDcbd for insoluble xylan were significantly lower than the Km of XYLDcd. Overall, these data indicate that the internal CBD homologue in XYLD constitutes a discrete xylan-binding domain which influences the affinity of the enzyme for insoluble xylan but does not directly affect the catalytic activity of the xylanase. The rationale for the evolution of this domain is discussed.
Isolation and characterization of distinct domains of sarcolemma and T-tubules from rat skeletal muscle
Authors: Muñoz P , Rosemblatt M , Testar X , Palacín M , Zorzano A
Published: August 2015

1. Several cell-surface domains of sarcolemma and T-tubule from skeletal-muscle fibre were isolated and characterized. 2. A protocol of subcellular fractionation was set up that involved the sequential low- and high-speed homogenization of rat skeletal muscle followed by KCl washing, Ca2+ loading and sucrose-density-gradient centrifugation. This protocol led to the separation of cell-surface membranes from membranes enriched in sarcoplasmic reticulum and intracellular GLUT4-containing vesicles. 3. Agglutination of cell-surface membranes using wheat-germ agglutinin allowed the isolation of three distinct cell-surface membrane domains: sarcolemmal fraction 1 (SM1), sarcolemmal fraction 2 (SM2) and a T-tubule fraction enriched in protein tt28 and the alpha 2-component of dihydropyridine receptor. 4. Fractions SM1 and SM2 represented distinct sarcolemmal subcompartments based on different compositions of biochemical markers: SM2 was characterized by high levels of beta 1-integrin and dystrophin, and SM1 was enriched in beta 1-integrin but lacked dystrophin. 5. The caveolae-associated molecule caveolin was very abundant in SM1, SM2 and T-tubules, suggesting the presence of caveolae or caveolin-rich domains in these cell-surface membrane domains. In contrast, clathrin heavy chain was abundant in SM1 and T-tubules, but only trace levels were detected in SM2. 6. Immunoadsorption of T-tubule vesicles with antibodies against protein tt28 and against GLUT4 revealed the presence of GLUT4 in T-tubules under basal conditions and it also allowed the identification of two distinct pools of T-tubules showing different contents of tt28 and dihydropyridine receptors. 7. Our data on distribution of clathrin and dystrophin reveal the existence of subcompartments in sarcolemma from muscle fibre, featuring selective mutually exclusive components. T-tubules contain caveolin and clathrin suggesting that they contain caveolin- and clathrin-rich domains. Furthermore, evidence for the heterogeneous distribution of membrane proteins in T-tubules is also presented.
A role for protein kinase C-mediated phosphorylation in eliciting glucagon desensitization in rat hepatocytes
Authors: Savage A , Zeng L , Houslay M D
Published: August 2015

An immobilized hepatocyte preparation was used to show that both vasopressin and glucagon could desensitize the ability of glucagon to increase intracellular cyclic AMP concentrations. This process was not dependent on any influx of extracellular Ca2+ and was not mediated by any rise in the intracellular level of Ca2+. The protein kinase C-selective inhibitors chelerythrine, staurosporine and calphostin C acted as potent inhibitors of the desensitization process but with various degrees of selectivity regarding their ability to inhibit the desensitizing actions of glucagon and vasopressin. The protein phosphatase inhibitor okadaic acid was just as potent as vasopressin and glucagon in causing desensitization. Treatment of hepatocyte membranes with alkaline phosphatase restored to near control levels the ability of glucagon to stimulate adenylate cyclase activity in membranes from both glucagon- and vasopressin-treated (desensitized) hepatocytes. It is suggested that the desensitization of glucagon-stimulated adenylate cyclase activity involves a reversible phosphorylation reaction with the likely target being the glucagon receptor itself.
Translation in vivo of 5′ untranslated-region splice variants of human surfactant protein-A
Authors: Karinch A M , Floros J
Published: August 2015

Transcripts of human SP-A genes, SP-A1 and SP-A2, undergo alternative splicing of 5′ untranslated-region exons. We reverse-transcribed and amplified free cytoplasmic and polysome-bound RNA and showed that (a) all splice variants of both genes are translated in vivo, (b) the relative translatability of splice variants can differ among individuals, and (c) the relative levels of different SP-A splice variants differ among individuals.
Streptomyces K15 active-site serine dd-transpeptidase: specificity profile for peptide, thiol ester and ester carbonyl donors and pathways of the transfer reactions
Authors: Grandchamps J , Nguyen-Distèche M , Damblon C , Frère J M , Ghuysen J M
Published: August 2015

The Streptomyces K15 transferase is a penicillin-binding protein presumed to be involved in bacterial wall peptidoglycan crosslinking. It catalyses cleavage of the peptide, thiol ester or ester bond of carbonyl donors Z-R1-CONH-CHR2-COX-CHR3-COO- (where X is NH, S or O) and transfers the electrophilic group Z-R1-CONH-CHR2-CO to amino acceptors via an acyl-enzyme intermediate. Kinetic data suggest that the amino acceptor behaves as a simple alternative nucleophile at the level of the acyl-enzyme in the case of thiol ester and ester donors, and that it binds to the enzyme.carbonyl donor Michaelis complex and influences the rate of enzyme acylation by the carbonyl donor in the case of amide donors. Depending on the nature of the scissile bond, the enzyme has different requirements for substituents at positions R1, R2 and R3.
Stimulation of NADH-dependent microsomal DNA strand cleavage by rifamycin SV
Authors: Kukiełka E , Cederbaum A I
Published: August 2015

Rifamycin SV is an antibiotic anti-bacterial agent used in the treatment of tuberculosis. This drug can autoxidize, especially in the presence of metals, and generate reactive oxygen species. A previous study indicated that rifamycin SV can increase NADH-dependent microsomal production of reactive oxygen species. The current study evaluated the ability of rifamycin SV to interact with iron and increase microsomal production of hydroxyl radical, as detected by conversion of supercoiled plasmid DNA into the relaxed open circular state. The plasmid used was pBluescript II KS(-), and the forms of DNA were separated by agarose-gel electrophoresis. Incubation of rat liver microsomes with plasmid plus NADH plus ferric-ATP caused DNA strand cleavage. The addition of rifamycin SV produced a time- and concentration-dependent increase in DNA-strand cleavage. No stimulation by rifamycin SV occurred in the absence of microsomes, NADH or ferric-ATP. Stimulation occurred with other ferric complexes besides ferric-ATP, e.g. ferric-histidine, ferric-citrate, ferric-EDTA, and ferric-(NH4)2SO4. Rifamycin SV did not significantly increase the high rates of DNA strand cleavage found with NADPH as the microsomal reductant. The stimulation of NADH-dependent microsomal DNA strand cleavage was completely blocked by catalase, superoxide dismutase, GSH and a variety of hydroxyl-radical-scavenging agents, but not by anti-oxidants that prevent microsomal lipid peroxidation. Redox cycling agents, such as menadione and paraquat, in contrast with rifamycin SV, stimulated the NADPH-dependent reaction; menadione and rifamycin SV were superior to paraquat in stimulating the NADH-dependent reaction. These results indicate that rifamycin SV can, in the presence of an iron catalyst, increase microsomal production of reactive oxygen species which can cause DNA-strand cleavage. In contrast with other redox cycling agents, the stimulation by rifamycin SV is more pronounced with NADH than with NADPH as the microsomal reductant. Interactions between rifamycin SV, iron and NADH generating hydroxyl-radical-like species may play a role in some of the hepatotoxic effects associated with the use of this antibacterial antibiotic.
Molecular cloning and expression of catrocollastatin, a snake-venom protein from Crotalus atrox (western diamondback rattlesnake) which inhibits platelet adhesion to collagen
Authors: Zhou Q , Smith J B , Grossman M H
Published: August 2015

A 50 kDa protein that inhibits platelet adhesion to collagen has been isolated from snake venom of Crotalus atrox (western diamondback rattlesnake) and has been named ‘catrocollastatin’. The cDNA cloning of catrocollastatin has been accomplished. A full-length cDNA of 2310 bp with an open reading frame between nucleotides 51 and 1880 was obtained. The deduced amino acid sequence consists of 609 amino acids. The cDNA-predicted amino acid sequence is highly similar to that of haemorrhagic metalloproteinase jararhagin from Bothrops jararaca venom, HR1B from Trimeresurus flavoviridis, Ht-e from C. atrox and trigramin from T. gramineus. Like jararhagin and HR1B, catrocollastatin is a multidomain molecule composed of an N-terminal domain, a metalloproteinase domain, a disintegrin-like domain and a cysteine-rich C-terminal domain. In the disintegrin-like domain, the frequently seen RGD (Arg-Gly-Asp) sequence is replaced by SECD (Ser-Glu-Cys-Asp). This cDNA was expressed in Spodoptera frugiperda (fall armyworm) (Sf9) insect cells using a baculovirus expression system. Like native catrocollastatin, the expressed protein is capable of selectively blocking collagen-induced platelet aggregation. This is the first full-length clone of a high-molecular-mass haemorrhagin to be expressed.
Dexamethasone down-regulates the 85 kDa phospholipase A2 in mouse macrophages and suppresses its activation
Authors: Gewert K , Sundler R
Published: August 2015

We have studied the effects of dexamethasone (dex) (i) on the level of the arachidonate-mobilizing phospholipase A2 (PLA2-85) in macrophages, (ii) on the stimulus-induced activation of this enzyme, and (iii) on the stimulus-induced release of arachidonate. Treatment of macrophages with 10 nM dex led to progressive reduction of PLA2-85 down to approx. 35% of control levels in 20 h in the absence of stimuli. This was accompanied by a partial inhibition of calcium-ionophore-induced arachidonate release. In contrast, the ability of zymosan or phorbol ester to cause both persistent activation of PLA2-85 and arachidonate release was greatly reduced or abolished. However, the protein phosphatase inhibitor okadaic acid, previously shown to cause enhanced phosphorylation and persistent activation of PLA2-85, was still able to exert this effect on the dex-suppressed PLA2-85. This suggests that the effect of okadaic acid was exerted at, or downstream of, the dex-sensitive step(s). Treatment with dex also led to inhibition of the characteristic changes in phosphoprotein labelling induced by phorbol ester or zymosan. However, phorbol-dibutyrate-binding isoforms of protein kinase C were not severely down-regulated. Thus dex was found to down-regulate PLA2-85 and, in addition, to affect one or more component(s) in the signal chain that normally leads to its activation. However, okadaic acid retained the ability to cause activation of PLA2-85.
Evidence of a defined spatial arrangement of hyaluronate in the central filament of cartilage proteoglycan aggregates
Authors: Mörgelin M , Paulsson M , Heinegård D , Aebi U , Engel J
Published: August 2015

Aggregates of proteoglycans from the Swarm rat chondrosarcoma reassembled in vitro have been studied by rotary-shadowing electron microscopy, and shown to be similar to native structures that have never been dissociated [Mörgelin, Engel, Heinegård and Paulsson (1992) J. Biol. Chem. 267, 14275-14284]. A hyaluronate with defined chain length (HAshort) has now been prepared by autoclaving high-Mr hyaluronate and fractionation to a narrow size distribution by gel filtration. Proteoglycan monomers, core protein, hyaluronate-binding region and link protein were combined with HAshort. Free chains of HAshort and reconstituted complexes with proteoglycan, link protein and aggrecan fragments were examined by electron microscopy after rotary shadowing. Length measurements showed that the hyaluronate was condensed to about half of its original length on binding intact aggrecan monomers, any aggrecan fragment or link protein alone. This strongly implies that hyaluronate adopts a defined spatial arrangement within the central filament of the aggregate, probably different from its secondary structure in solution. No differences in length were observed between link-free and link-stabilized aggregates.
G-protein regulation of capacitative calcium entry may be mediated by protein kinases A and C in Xenopus oocytes
Authors: Petersen C C H , Berridge M J
Published: August 2015

Inositol 2,4,5-trisphosphate irreversibly activated capacitative calcium entry in Xenopus oocytes, whereas guanosine thiotriphosphate (GTP[S]) and AIF4- only activated capacitative calcium entry transiently. Both GTP[S] and AIF4- inhibited capacitative calcium entry activated by thapsigargin pretreatment, but guanosine thiodiphosphate (GDP[S]), inositol 2,4,5-trisphosphate and dibutyryl cyclic GMP did not affect capacitative calcium entry. This suggests the involvement of heterotrimeric GTP-binding proteins in the regulation of capacitative calcium entry. Activation of protein kinase C or cyclic-AMP-dependent protein kinase had profound effects on capacitative calcium entry, which were consistent with the hypothesis that the effects of GTP[S] and AIF4- on capacitative calcium entry may be mediated via heterotrimeric GTP-binding protein stimulation of kinases. Further evidence for this hypothesis was derived from the result that the effects of GTP[S] on calcium entry could be inhibited by the application of the protein kinase inhibitor staurosporine.
Isolation and characterization of an avian A1 adenosine receptor gene and a related cDNA clone
Authors: Aguilar J S , Tan F , Durand I , Green R D
Published: August 2015

We have isolated the gene for a chick A1 adenosine receptor along with a cDNA that codes for the same adenosine receptor. The cDNA clone was isolated from both adipose tissue and heart cDNA libraries and encodes a 324-amino acid protein with 80% identity with mammalian A1 adenosine receptors. Transient expression of the cDNA in human embryonic kidney (HEK) 293 cells shows that it encodes a protein that binds [3H]CCPA (2-chloro-N6-[cyclopentyl-2,3,4,5-3H]cyclopentyladenosine, a specific agonist radioligand, with a KD of 5.6 +/- 2.4 nM. Cyclic AMP measurements in HEK 293 cells co-transfected with the chick cDNA and a cDNA for a luteinizing hormone/choriogonadotropin receptor shows that A1 adenosine receptor agonists antagonize the cyclic AMP-elevating effect of bovine luteinizing hormone. Two partial genomic clones were isolated. The first contains 5′-untranslated sequence including a putative promoter region which does not contain a TATA box, an intron and the first third of the coding sequence of the A1 adenosine receptor cDNA. The coding sequence of this partial genomic clone terminates at a second intron. The second partial genomic clone contains the rest of the coding sequence and the 3′-untranslated elements in a single exon. Thus the chick A1 adenosine receptor gene contains one intron in the 5′-untranslated region and a minimum of one intron in the coding sequence.
The role of the novel disulphide ring in the active site of the quinoprotein methanol dehydrogenase from Methylobacterium extorquens
Authors: Avezoux A , Goodwin M G , Anthony C
Published: August 2015

All cysteines in methanol dehydrogenase (MDH) from Methylobacterium extorquens are involved in intra-subunit disulphide bridge formation. One of these is between adjacent cysteine residues which form a novel ring structure in the active site. It is readily reduced, the reduced enzyme being inactive in electron transfer to cytochrome cL. The inactivation is not a result of major structural change or to modification of the prosthetic group pyrrolo-quinoline quinone (PQQ). The reduced enzyme appears to remain active with the artificial electron acceptor phenazine ethosulphate but this is because the dye re-oxidizes the adjacent thiols back to the original disulphide bridge. No free thiols were detected during the reaction cycle with cytochrome cL. Carboxymethylation of the thiols produced by reduction of the novel disulphide ring led to formation of active enzyme. Reconstitution of inactive Ca(2+)-free MDH with Ca2+ led to active enzyme containing the oxidized bridge and reduced quinol, PQQH2, consistent with the conclusion that no hydrogen transfer occurs between these groups in the active site. It is concluded that the disulphide ring in the active site of MDH does not function as a redox component of the reaction. The disulphide ring has no special function in the process of Ca2+ incorporation into the active site. It is suggested that this novel structure might function in the stabilization or protection of the free radical semiquinone form of the prosthetic group (PQQH.) from solvent at the entrance to the active site.
Functional synergism in the carbohydrate-induced activation of liver-type pyruvate kinase gene expression
Authors: Liu Z , Towle H C
Published: August 2015

Hepatic expression of the liver-type pyruvate kinase (L-PK) gene is induced at the transcriptional level by increased carbohydrate metabolism in the rat. The carbohydrate response of the L-PK gene requires sequences from -171 to -124, which encompass adjacent major late transcription factor (MLTF)-like and hepatic nuclear factor (HNF)-4 binding sites. Neither site alone is capable of conferring a response, prompting us to explore the mechanism of synergy between the MLTF-like factor and HNF-4. Spacing requirements between the two factor binding sites were tested by generating a series of mutations that altered the distance between these sites. Surprisingly, all of the constructs with spacing mutations were capable of responding to elevated glucose when introduced into primary hepatocytes. Thus the glucose response does not depend on the rigid phasing of the MLTF-like and HNF-4 factors, suggesting that the factors binding to these two sites do not interact directly with each other. Substitution or inversion of the PK HNF-4 site abrogated the response to glucose and also significantly suppressed the promoter activity under non-inducing conditions. We conclude that the MLTF-like factor and HNF-4 co-operate functionally to maintain the basal activity, as well as the carbohydrate responsiveness, of the L-PK gene. A mechanism other than co-operative DNA binding is responsible for the synergism.
Immunochemical characterization of two thyroid-stimulating hormone β-subunit epitopes
Authors: Fairlie W D , Stanton P G , Hearn M T W
Published: August 2015

The epitopes of human thyroid-stimulating hormone (hTSH) recognized by two murine monoclonal antibodies (MAbs), designated MAb 279 and MAb 299, have been characterized. These MAbs are highly specific for the beta-subunit of TSH. The epitope recognized by MAb 279 appears to be completely conserved between bovine and human TSH and partially conserved in the porcine species. The TSH beta-subunit epitope recognized by MAb 299 is only partially conserved between the human, bovine and porcine species. Both MAbs are capable of inhibiting the binding of TSH to its receptor in a TSH radioreceptor assay, indicating that the epitopes either coincide or are located close to the TSH beta-subunit receptor-binding sites. The carbohydrate moieties of the TSH beta-subunit appear to play little or no role in the epitope recognition by MAb 279 or MAb 299 while the integrity of the disulphide bonds are essential. The epitopic recognition may also involve lysine residues, as determined by the immunoreactivity with both MAbs following citraconylation of TSH. In addition, the amino acid sequence region between residues bTSH beta 34-44 could be excised by trypsin digestion of bovine TSH beta (bTSH beta) without eliminating epitopic recognition by either MAb. These results provide further insight into the relationship between the structure of the TSH beta-subunit epitopes and location of the receptor-binding sites.
Localization of cholesterol in sphingomyelinase-treated fibroblasts
Authors: Pörn M I , Slotte J P
Published: August 2015

The distribution of cellular unesterified cholesterol was studied in fibroblasts, which had been depleted of plasma membrane sphingomyelin by exposure to exogenous sphingomyelinase. This treatment has previously been shown to induce an increase in cholesterol esterification, a decrease in the biosynthesis of cholesterol, and a decreased susceptibility of cell cholesterol to oxidation with cholesterol oxidase. When the cellular localization of cholesterol was studied with fluorescent filipin staining, sphingomyelin depletion did not cause any visible changes in the filipin-cholesterol staining pattern, suggesting that the major part of cellular cholesterol was retained in the plasma membrane after sphingomyelinase treatment. After the oxidation of cell-surface cholesterol with cholesterol oxidase, the plasma membrane was no longer stained by filipin, but the plasma membrane cholesterol of sphingomyelin-depleted cells appeared to be resistant to oxidation with cholesterol oxidase when sphingomyelinase was used as an oxidation-promoting agent. However, the use of hypotonic buffer or phosphatidylcholine-specific phospholipase C together with cholesterol oxidase resulted in a complete oxidation of the cell-surface cholesterol in sphingomyelin-depleted cells, as evidenced by the filipin-cholesterol staining pattern. Similar results were obtained when [3H]cholesterol-labelled fibroblasts were used for determination of the susceptibility to cholesterol oxidation. The kinetics of [3H]cholesterol oxidation in sphingomyelin-depleted cells with cholesterol oxidase in hypotonic buffer indicated that approximately 85% of the cellular cholesterol still resided in the plasma membrane after sphingomyelin depletion. These results are contradictory to earlier reports on sphingomyelinase-induced changes in cellular cholesterol distribution and suggest that minor changes in the kinetics of cholesterol transport from the plasma membrane to the endoplasmic reticulum may be responsible for the sphingomyelinase-induced changes in the rates of cholesterol metabolism. Whereas the use of phospholipases to promote the oxidation of cholesterol in some instances might lead to misinterpretations, the use of hypotonic buffer together with cholesterol oxidase proved to be a more reliable method for the determination of cellular cholesterol distribution.
Evidence that the effects of phospholipids on the activity of the Ca2+-ATPase do not involve aggregation
Authors: Starling A P , East J M , Lee A G
Published: August 2015

The Ca(2+)-ATPase of skeletal-muscle sarcoplasmic reticulum, solubilized in monomeric from in C12E8, has been reconstituted by dialysis into sealed vesicles of dioleoyl phosphatidylcholine [di(C18:1)PC], dimyristoleoyl phosphatidylcholine [di(C14:1)PC], dinervonyl phosphatidylcholine [di(C24:1)PC] or dipalmitoyl phosphatidylcholine [di(C16:0)PC] in the gel phase, at a phospholipid/ATPase molar ratio of 10,000: 1. Cross-linking experiments show that ATPase molecules are present in these reconstituted vesicles as isolated monomeric species. ATPase activities for the reconstituted vesicles are about half of those for the ATPase reconstituted with the same lipid in unsealed membrane fragments, attributed to a close to random orientation for the ATPase molecules in the reconstituted vesicles. ATPase activities for the ATPase in reconstituted vesicles of di(C14:1)PC or di(C24:1)PC are less than in vesicles of di(C18:1)PC, and no activity could be detected for the ATPase in di(C16:0)PC in the gel phase. It is concluded that effects of lipids on the activity of the ATPase are independent of any changes in the state of aggregation of the ATPase. Inhibition of ATPase activity by spermine and by the hydrophilic domain of phospholamban are observed both for the unreconstituted ATPase and for the ATPase in reconstituted vesicles, so that inhibition is independent of any aggregation caused by these polycationic species. Stimulation of ATPase activity by jasmone is also observed both for the unreconstituted ATPase and for the ATPase in reconstituted vesicles, so that stimulation of the ATPase also does not follow from any change in the state of aggregation of the ATPase.
Effect of intracellular iron depletion by picolinic acid on expression of the lactoferrin receptor in the human colon carcinoma cell subclone HT29-18-C1
Authors: Mikogami T , Marianne T , Spik G
Published: August 2015

A lactoferrin receptor has been found on the brush-border membrane of intestinal epithelial cells of several species, including humans. A role for this receptor in intestinal iron absorption, which is well regulated in response to body iron stores, has been proposed. We have investigated the effect of intracellular iron depletion by picolinic acid, an iron chelator, on the cell surface binding of human lactoferrin to human enterocytes and its intracellular uptake, using HT29-18-C1 cells, an enterocyte-like differentiable cell line. The confluent cells exhibited 5.8 x 10(6) specific binding sites per cell for diferric human 125I-labelled lactoferrin with relatively low affinity (Kd 8.4 x 10(-7) M). The addition of picolinic acid to the culture medium resulted in a concentration- and time-dependent increase in lactoferrin binding that was correlated with a decrease in intracellular iron content. The maximum effect of picolinic acid on lactoferrin binding (approx. 2-fold increase), which appeared between 12 and 18 h after its addition, was obtained at a picolinic acid concentration of 2 mM. Scatchard analysis showed that the enhanced lactoferrin binding resulted from an increase in the number of lactoferrin receptors rather than an alteration in the binding affinity for lactoferrin. The time-dependent effect of picolinic acid was completely abolished in the presence of 1 microM anisomycin, a protein synthesis inhibitor, indicating that ongoing protein synthesis is involved in this effect. The enhanced lactoferrin binding induced by picolinic acid produced an increase of approx. 30% in the uptake of lactoferrin-bound 59Fe, indicating the existence of functional receptors. These results suggest that biosynthesis of lactoferrin receptors in intestinal epithelial cells can be regulated in response to the levels of intracellular chelatable iron, consistent with intestinal iron absorption dependent on body iron stores.
Characterization of ATP receptor responsible for the activation of phospholipase A2 and stimulation of prostaglandin E2 production in thymic epithelial cells
Authors: Liu P , Wen M , Hayashi J
Published: August 2015

In TEA3A1 rat thymic epithelial cells, ATP stimulates prostaglandin E2 (PGE2) production through activation of phospholipase A2 (PLA2) enzymic activity. The stimulation of PGE2 production tested with other nucleotides indicated the agonist potency of adenosine 5′-[gamma-thio]triphosphate (ATP[S]) > or = UTP > ATP, with ED50 of about 10 microM for ATP[S]. In TEA3A1 cells, cross-linking studies with ATP[35S] revealed the presence of four cell-surface cross-linked bands of 42 kDa, 53 kDa, 83 kDa and 100 kDa in Triton X-100 extracts of TEA3A1 cells by fluorography. Guanosine 5′-[gamma-thio]triphosphate specifically blocked the cross-linking of ATP[35S] to the 53 kDa, 83 kDa and 100 kDa ATP-binding proteins, and inhibited the ATP[S]-mediated stimulation of PGE2 production with an ED50 of about 25 microM. On the other hand, 2-methylthioadenosine triphosphate (2MeSATP) blocked ATP[35S] cross-linking to the 42 kDa protein, but had no effect on ATP[S]-mediated stimulation of PGE2 production. In a variant cell line, TEAvarl, derived from TEA3A1 cells that lost their response to ATP in the activation of PLA2, the presence of 83 kDa ATP-binding protein was not detected. Results from our study suggest that ATP activates PLA2 enzymic activity in TEA3A1 cells by binding to an atypical ATP receptor that has not been described previously.
The temporal and cellular expression of c-fos and c-jun in mechanically stimulated rabbit latissimus dorsi muscle
Authors: Osbaldeston N J , Lee D M , Cox V M , Hesketh J E , Morrison J F J , Blair G E , Goldspink D F
Published: August 2015

The levels of c-fos and c-jun mRNA were measured by reverse transcription PCR in the rabbit latissimus dorsi muscle following three separate training regimes, i.e. passive stretch, 10 Hz electrical stimulation or a combination of the two. Both c-fos and c-jun mRNA expression peaked at around 1 h after imposing stretch and at around 4.5-6 h after the initiation of electrical stimulation. The combined stretch/electrical stimulation regime induced biphasic expression of both c-fos and c-jun mRNA, with peaks coinciding temporally with those for the individual regimes. Immunostaining with anti-Fos and anti-Jun antibodies revealed the accumulation of these proteins in both myofibre and interstitial cell nuclei following passive stretch. In contrast, following electrical stimulation the localization of immunoreactive c-Fos and c-Jun proteins was predominantly in interstitial cell nuclei. c-Fos and c-Jun immunoreactivity was also clearly colocalized in a proportion of myonuclei from stretched muscle. These findings suggest that the rapid induction of c-fos and c-jun is an early event in response to mechanical stretch and might trigger [via activator protein-1 (AP-1) transcriptional factors] events leading to muscle fibre hypertrophy. However, the involvement of AP-1 in inducing the phenotypic changes in muscle fibres as a result of electrical stimulation appears less clear.
Inhibition of myeloperoxidase by benzoic acid hydrazides
Authors: Kettle A J , Gedye C A , Hampton M B , Winterbourn C C
Published: August 2015

Myeloperoxidase is the most abundant protein in neutrophils and catalyses the conversion of H2O2 and chloride into HOCl. To help clarify the role of this enzyme in bacterial killing and inflammation, a specific and potent inhibitor needs to be identified. We have studied a series of benzoic acid hydrazides and found that in general they inhibit the peroxidation activity of myeloperoxidase with an IC50 value of less than 10 microM. The IC50 values of derivatives with substituents containing oxygen or nitrogen were related to their Hammett substituent constants. This indicates that myeloperoxidase oxidizes the hydrazide group of these compounds, and the degree to which they inhibit the enzyme is dependent on the ease of their oxidation. Unsubstituted benzoic acid hydrazide and its 4-chloro derivative were poor inhibitors of peroxidation. Thus it is likely that hydrogen-bonding of the enzyme to substituents containing oxygen or nitrogen increases the binding affinity of the hydrazides and enhances their oxidation by myeloperoxidase. 4-Aminobenzoic acid hydrazide (ABAH) was the most potent inhibitor of peroxidation. It irreversibly inhibited HOCl production by the purified enzyme, having an IC50 value of 0.3 microM. With neutrophils stimulated with opsonized zymosan or phorbol myristate acetate, ABAH inhibited HOCl production by up to 90% and the IC50 values were 16 microM and 2.2 microM respectively. In the presence of superoxide dismutase, these values decreased to 6.4 microM and 0.6 microM respectively. ABAH had no effect on superoxide radical (O2-.) production and degranulation by neutrophils, nor did it inhibit catalase or glutathione peroxidase. Thus ABAH is an effective and selective inhibitor that should be useful for determining the contribution of myeloperoxidase to oxidant-mediated reactions of neutrophils.
Localization of Ca2+-dependent conformational changes of calretinin by limited tryptic proteolysis
Authors: Kuźnicki J , Wang T L , Martin B M , Winsky L , Jacobowitz D M
Published: August 2015

Calretinin is an EF-hand Ca(2+)-binding protein expressed predominantly in some neurons. We have found that the tryptic digestion pattern of rat recombinant calretinin depends on Ca2+ concentration as determined by SDS/PAGE, amino-acid-sequence analysis and electrospray-ionization MS. Ca(2+)-saturated calretinin was cleaved between amino acids 60 and 61 to yield two fragments, which accumulated during cleavage. Small amounts of the larger fragment (amino acid residues 61-271) were further cleaved from the C-terminal end. Ca(2+)-free calretinin was also cleaved between residues 60 and 61; however, under the latter conditions the fragment 61-271 was further cleaved from the N-terminal end. Native rat calretinin was cleaved by trypsin in a similar Ca(2+)-dependent fashion. All identified fragments of recombinant calretinin bound 45Ca2+ on nitrocellulose filters, although to a different extent. The 61-271 fragment was released by EGTA from an octyl-agarose column in a manner similar to intact calretinin, while fragment 61-233 was not eluted by EGTA. These observations show that there are trypsin cleavage sites in calretinin that are available regardless of Ca2+ binding, other sites that are completely protected against trypsin on Ca(2+)-binding and sites which become partially available on Ca(2+)-binding. Together these data show that calretinin changes its conformation on Ca2+ binding and identify the regions which are exposed in apo and Ca(2+)-bound form.
Expression of the human gene coding for the α-chain of C4b-binding protein, C4BPA, is controlled by an HNF1-dependent hepatic-specific promoter
Authors: Arenzana N , Rodríguez de Córdoba S , Rey-Campos J
Published: August 2015

C4b-binding protein (C4BP) is an abundant oligomeric plasma glycoprotein which controls the activation of the complement cascade through the classical pathway. In humans, the majority form of C4BP is composed of seven alpha-chains and one beta-chain, covalently linked by their C-termini. C4BP is mainly expressed in the liver. We have previously cloned and characterized the structure of the genes encoding the alpha and beta chains, C4BPA and C4BPB, respectively. Here we addressed the characterization of the mechanisms controlling the hepatic restricted expression of the C4BPA gene. We found that the C4BPA promoter is contained within the first 369 bp upstream of the transcription start site. The activity of this promoter is restricted to hepatic cells in transfection experiments. The hepatic transcription factor HNF1 interacts with a region of this promoter at -38 bp. This region is absolutely required for the activity of this promoter, suggesting that HNF1 is essential for the hepatic activity of the C4BPA promoter. We speculate that this extreme requirement of HNF1 for the activity of the human C4BPA promoter is related to the fact that this promoter lacks a TATA box.
Role of the promoter in the sensitivity of human thymidine kinase to lack of Zn2+
Authors: Chesters J K , Boyne R , Petrie L , Lipson K E
Published: August 2015

Previous studies had indicated that lack of Zn2+ inhibits the expression of thymidine kinase activity and produces a corresponding reduction in the concentration of its mRNA. The present investigations have shown that with human thymidine kinase this is associated with increased binding of a specific protein to the gene's promoter in the region between -55 and -83 bp 5′ to the transcription initiation site. A second binding site for the protein is present within the sixth exon of the human thymidine kinase gene.
Kinetics of extracellular ATP hydrolysis by microvascular endothelial cells from rat heart
Authors: Meghji P , Pearson J D , Slakey L L
Published: August 2015

We have characterized the ectonucleotidases that catalyse the reaction sequence ATP-->ADP-->AMP-->adenosine on microvascular endothelial cells cultured from the rat heart. Computer simulation and data fitting of progress of reaction curves showed that depletion of substrate at the cell surface dominates the regulation of the rate of hydrolysis of ATP when it is presented to the cells. Preferential delivery of AMP by ADPase to 5′-nucleotidase makes a significant contribution to the regulation of adenosine production from ATP or ADP. By contrast, we found no evidence for the preferential delivery of ADP from ATPase to ADPase. Feed-forward inhibition of AMP hydrolysis by ADP and/or ATP also modulated the rate of adenosine production. The properties of the ectonucleotidases on rat heart microvascular cells are such that adenosine is produced at a steady rate over a wide range of ATP concentrations.
Effect of lipopolysaccharide on mitogen-activated protein kinases and cytosolic phospholipase A2
Authors: Fouda S I , Molski T F P , Ashour M S E , Sha′afi R I
Published: August 2015

The addition of platelet-activating factor (PAF) to human neutrophils increases phosphorylation on tyrosine residues and stimulates the activity of p42erk2 mitogen-activated protein kinase (MAP kinase). This action is rapid and transient. In contrast, p42erk2, p44erk1 and the p40hera MAP kinase isoforms are all not tyrosine phosphorylated or activated in human neutrophils stimulated with low concentrations of lipopolysaccharide (LPS) in combination with serum. In spite of this, the PAF-induced tyrosine phosphorylation and activation of the p42erk2 MAP kinase are greatly potentiated in cells pretreated with LPS. More interestingly, although low concentrations of LPS do not affect MAP kinase isoforms in these cells, they cause the phosphorylation of cytosolic phospholipase A2 (cPLA2), as evidenced by a decrease in the electrophoretic mobility of the enzyme. In addition, this stimulus-induced upward shift in the mobility of the enzyme is not inhibited by the tyrosine kinase inhibitor, genistein. Furthermore, LPS increases the release of arachidonic acid in control and PAF-stimulated human neutrophils. These observations clearly show that cPLA2 can be phosphorylated and activated by kinases other than the currently known MAP kinases. It is proposed that there are MAP kinase-dependent and -independent mechanisms for the phosphorylation of cPLA2.
The early stimulation of glycolysis by epidermal growth factor in isolated rat hepatocytes is secondary to the glycogenolytic effect
Authors: Quintana I , Grau M , Moreno F , Soler C , Ramírez I , Soley M
Published: August 2015

We have studied the relationship between the effect of epidermal growth factor (EGF) on glycogen metabolism and its effect on glycolysis, in rat hepatocyte suspensions. Although 10 nM glucagon or 10 microM adrenaline increased glycogen degradation by more than 120%, 10 nM EGF increased glycogenolysis by less than 20% in hepatocytes incubated in glucose-free medium. Both glucagon and adrenaline increased phosphorylase a activity by more than 130%; EGF increased this activity by about 30%. Under basal conditions, 65% of the glucosyl residues were released as free glucose and about 30% ended up as C3 molecules (lactate and pyruvate). Both glucagon and adrenaline decreased the proportion of glucosyl units that rendered glycolysis end-products (to 2% for glucagon and 6% for adrenaline) and increased the proportion that ended up as free glucose (to 94% and 88% of the glucosyl residues for glucagon and adrenaline respectively). EGF increased the production of both free glucose and lactate+pyruvate, but the proportion of glucosyl residues that ended up as free glucose or glycolysis end-products was unchanged. In glycogen-depleted hepatocytes incubated in the presence of 25 mM glucose, EGF affected neither glycogen deposition nor glycolysis. EGF increased cytosolic free Ca2+, and neomycin decreased both the Ca2+ signal and the glycogenolytic effect. In conclusion, our results indicate that the effect of EGF on glycolysis is secondary to the Ca(2+)-mediated stimulation of glycogenolysis in rat hepatocyte suspensions.
Conservation of the sizes of 53 introns and over 100 intronic sequences for the binding of common transcription factors in the human and mouse genes for type II procollagen (COL2A1)
Authors: Ala-Kokko L , Kvist A P , Metsäranta M , Kivirikko K I , de Crombrugghe B , Prockop D J , Vuorio E
Published: August 2015

Over 11,000 bp of previously undefined sequences of the human COL2A1 gene were defined. The results made it possible to compare the intron structures of a highly complex gene from man and mouse. Surprisingly, the sizes of the 53 introns of the two genes were highly conserved with a mean difference of 13%. After alignment of the sequences, 69% of the intron sequences were identical. The introns contained consensus sequences for the binding of over 100 different transcription factors that were conserved in the introns of the two genes. The first intron of the gene contained 80 conserved consensus sequences and the remaining 52 introns of the gene contained 106 conserved sequences for the binding of transcription factors. The 5′-end of intron 2 in both genes had a potential for forming a stem loop in RNA transcripts.
Production and comparison of mature single-domain ‘trefoil’ peptides pNR-2/pS2 Cys58 and pNR-2/pS2 Ser58
Authors: Chadwick M P , May F E B , Westley B R
Published: August 2015

The preparation and purification of recombinant mature pNR-2/pS2, a single-domain member of the ‘trefoil’ family of cysteine-rich secreted proteins, is described. Analysis of recombinant pNR-2/pS2 by ion-exchange chromatography showed that it was heterogeneous. The heterogeneity was reduced by treatment with thiol-group-containing reagents, suggesting that it is caused by the odd number of cysteine residues in mature pNR-2/pS2, and this view was reinforced by mutation of the extra-trefoil domain cysteine residue, Cys58, to a serine residue. Electrophoresis of recombinant pNR-2/pS2 Cys58 and pNR-2/pS2 Ser58 proteins under non-denaturing conditions confirmed that the Ser58 mutant is much more homogeneous, and showed that most of pNR-2/pS2 Ser58 co-migrates as a single band with pNR-2/pS2 secreted from breast-cancer cells in culture. Treatment of recombinant pNR-2/pS2 proteins with various thiol-group-reactive reagents indicated that cysteine is the most effective at producing recombinant pNR-2/pS2 that co-migrates with pNR-2/pS2 secreted by breast-cancer cells. Dithiothreitol appeared to denature the proteins, and GSH was relatively ineffective. pNR-2/pS2 Cys58 treated with cysteine and untreated pNR-2/pS2 Ser58 had the same apparent molecular mass, measured by gel filtration, as pNR-2/pS2 secreted from breast-cancer cells. This is the first report of the production of a recombinant mature single-domain trefoil peptide and should greatly facilitate elucidation of the structure and function of pNR-2/pS2.
Isolation and enzymic properties of levansucrase secreted by Acetobacter diazotrophicus SRT4, a bacterium associated with sugar cane
Authors: Hernandez L , Arrieta J , Menendez C , Vazquez R , Coego A , Suarez V , Selman G , Petit-Glatron M F , Chambert R
Published: August 2015

Acetobacter diazotrophicus, a nitrogen-fixing bacterium associated with sugar cane, secretes a levansucrase (sucrose-2,6-beta-D-fructan 6-beta-D-fructosyltransferase; EC 2.4.1.10). This enzyme is constitutively expressed and represents more than 70% of the total proteins secreted by strain SRT4. The purified protein consists of a single 58 kDa polypeptide with an isoelectric point of 5.5. Its activity is optimal at pH 5.0. It catalyses transfructosylation from sucrose to a variety of acceptors including water (sucrose hydrolysis), glucose (exchange reaction), fructan (polymerase reaction) and sucrose (oligofructoside synthesis). In vivo the polymerase activity leads to synthesis of a high-molecular-mass fructan of the levan type. A. diazotrophicus levansucrase catalyses transfructosylation via a Ping Pong mechanism involving the formation of a transient fructosyl-enzyme intermediate. The catalytic mechanism is very similar to that of Bacillus subtilis levansucrase. The kinetic parameters of the two enzymes are of the same order of magnitude. The main difference between the two enzyme specificities is the high yield of oligofructoside, particularly 1-kestotriose and kestotetraose, accumulated by A. diazotrophicus levansucrase during sucrose transformation. We discuss the hypothesis that these catalytic features may serve the different biological functions of each enzyme.