bluuebunny/crossref_metadata_2025 huggingface dataset:
Characterization of the structure of polydisperse human low-density lipoprotein by neutron scattering
Authors: Meyer D F , Nealis A S , Bruckdorfer K R , Perkins S J
Published: August 2015

Low-density lipoproteins (LDL) in plasma are constructed from a single molecule of apolipoprotein B-100 (M(r) 512000) in association with lipid (approximate M(r) 2-3 x 10(6)). The gross structure was studied using an updated pulsed-neutron camera LOQ with an area detector to establish the basis for the interpretation of structural changes seen during dynamic studies of LDL oxidation. Neutron-scattering data for LDL in 100% 2H2O buffers emphasize their external appearance. Guinier analysis on a continuous-flux neutron camera D17 revealed pronounced concentration-dependences in the radius of gyration, RG, and the intensity of forward scattering, I(0) (equivalent to the M(r) of LDL) between 0.5 and 11 mg of LDL protein/ml. LDL preparations from different donors gave different RG values. When extrapolated to zero concentration, RG values ranged between 8.3 and 10.6 nm and were linearly correlated with M(r), which is consistent with a spherical structure. The distance-distribution function P(r) in real space showed a single maximum at 9.1-10.9 nm, which is just under half the observed maximum dimension of 23.1 +/- 1.2 nm expected for a spherical structure. The neutron radial-density function p(r) exhibited a plateau of high and featureless density at the centre of LDL. LDL can be modelled by a polydisperse assembly of spheres with two internal densities and a mean radius close to 10.0 nm in a normal distribution of radii with a standard deviation of 2.0 nm. The data are consistent with recent electron-microscopy and ultracentrifugation data.(ABSTRACT TRUNCATED AT 250 WORDS)
The role of the second growth-factor domain of human factor IXa in binding to platelets and in factor-X activation
Authors: Ahmad S S , Rawala R , Cheung W F , Stafford D W , Walsh P N
Published: August 2015

To study the structural requirements for factor IXa binding to platelets, we have carried out equilibrium binding studies with human factor IXa after replacing the second epidermal growth factor (EGF) domain by the corresponding polypeptide region of factor X. The chimeric protein, factor IX(Xegf2), and the wild-type, factor IXwt, produced in embryonic kidney cells 293 were radiolabelled with 125I and activated with factor XIa. Direct binding studies with thrombin-activated platelets showed normal stoichiometry and affinity of binding of factor IXawt in the presence of factor VIIIa (2 units/ml) and factor X (1.5 microM). However, under similar experimental conditions, factor IXa(Xegf2) was bound to a smaller number of sites (396 sites/platelet) with decreased affinity, i.e. a dissociation constant (Kd) of 1.4 nM, compared with normal factor IXa, factor IXaN (558 sites/platelet; Kd 0.67 nM), or factor IXawt (590 sites/platelet; Kd 0.61 nM). The concentrations of factor IXaN and factor IXawt required for half-maximal rates of factor-X activation were 0.63 nM and 0.7 nM, indicating a close correspondence of the Kd, app. for binding of factor IXawt to the factor-X activating complex on activated platelets to the Kd obtained in equilibrium binding studies. In contrast, kinetic parameters for factor-X activation by factor IXa(Xegf2) showed a decreased affinity (Kd 1.5 nM), in agreement with results of binding studies. These studies with factor IX(Xegf2) suggest that the EGF-2 domain may be important for specific high-affinity factor IXa binding to platelets in the presence of factor VIIIa and factor X.
Purification and characterization of a trypanothione-glutathione thioltransferase from Trypanosoma cruzi
Authors: Moutiez M , Aumercier M , Schöneck R , Meziane-Cherif D , Lucas V , Aumercier P , Ouaissi A , Sergheraert C , Tartar A
Published: August 2015

Although trypanothione [T(S)2] is the major thiol component in trypanosomatidae, significant amounts of glutathione are present in Trypanosoma cruzi. This could be explained by the existence of enzymes using glutathione or both glutathione and T(S)2 as cofactors. To assess these hypotheses, a cytosolic fraction of T. cruzi epimastigotes was subjected to affinity chromatography columns using as ligands either S-hexylglutathione or a non-reducible analogue of trypanothione disulphide. A similar protein of 52 kDa was eluted in both cases. Its partial amino acid sequence indicated that it was identical with the protein encoded by the TcAc2 cDNA previously described [Schoneck, Plumas-Marty, Taibi et al. (1994) Biol. Cell 80, 1-10]. This protein showed no significant glutathione transferase activity but surprisingly catalysed the thiol-disulphide exchange between dihydrotrypanothione and glutathione disulphide. The kinetic parameters were in the same range as those determined for trypanothione reductase toward its natural substrate. This trypanothione-glutathione thioltransferase provides a new target for a specific chemotherapy against Chagas' disease and may constitute a link between the glutathione-based metabolism of the host and the trypanothione-based metabolism of the parasite.
Platelet-derived growth factors-AA and -BB regulate collagen and collagenase gene expression differentially in human fibroblasts
Authors: Tan E M L , Qin H , Kennedy S H , Rouda S , Fox J W , Moore J H
Published: August 2015

Platelet-derived growth factor (PDGF) is a mitogen associated with tissue repair, a process involving collagen synthesis and remodelling by interstitial collagenase. This study examines and compares the regulation of interstitial collagenase and collagen gene expression by PDGF-AA and -BB in human fibroblasts. Time-course analysis showed that neither PDGF-AA or -BB had a consistent effect on the expression of pro-alpha 1(I) or pro-alpha 2(I) type-I collagen genes. In contrast, interstitial collagenase gene expression was found to be consistently up-regulated severalfold by PDGF-BB. Enhanced expression of the collagenase gene was not apparently due to up-regulation of its promoter activity in human dermal fibroblasts, as indicated by transient and stable transfection experiments. Unlike PDGF-BB, PDGF-AA did not alter collagenase mRNA levels under low-serum culture conditions. Thus, the biological activities of the PDGF homodimers are different, with PDGF-BB being clearly more potent than PDGF-AA in its regulation of collagenase gene expression.
Structure and tissue-specific expression of the Drosophila melanogaster organellar-type Ca2+-ATPase gene
Authors: Magyar A , Bakos E , Váradi A
Published: August 2015

A 14 kb genomic clone covering the organellar-type Ca(2+)-ATPase gene of Drosophila melanogaster has been isolated and characterized. The sequence of a 7132 bp region extending from 1.1 kb 5′ upstream of the initiation ATG codon over the polyadenylation signal at the 3′ end has been determined. The gene consists of nine exons including one with an exceptional size of 2172 bp representing 72% of the protein coding region. Introns are relatively small (< 100 bp) except for the 3′ intron which has a size of 2239 bp, an exceptionally large size among Drosophila introns. Five of the introns are in the same positions in Drosophila, Artemia and rabbit SERCA1 Ca(2+)-ATPase genes. There is only one organellar-type Ca(2+)-ATPase gene in the Drosophila genome, as was shown by Southern-blot analysis [Váradi, Gilmore-Hebert and Benz (1989) FEBS Lett. 258, 203-207] and by chromosomal localization [Magyar and Váradi (1990) Biochem. Biophys. Res. Commun. 173, 872-877]. Primer extension and S1-nuclease assays revealed a potential transcription initiation site 876 bp upstream of the translation initiation ATG with a TATA-box 23 bp upstream of this site. Analysis of the 5′ region of the Drosophila organellar-type Ca(2+)-ATPase gene suggests the presence of potential recognition sequences of various muscle-specific transcription factors and shows a region with remarkable similarity to that in the rabbit SERCA2 gene. The tissue distribution of expression of the organellar-type Ca(2+)-ATPase gene has been studied by in situ RNA-RNA hybridization on microscopic sections. A low mRNA abundance can be detected in each tissue of adult flies, suggesting a housekeeping function for the gene. On the other hand a pronounced tissue specificity of expression has also been found as the organellar-type Ca(2+)-ATPase is expressed at a very high level in cell bodies of the central nervous system and in various muscles.
Intracellular Ca2+ storage in acidocalcisomes of Trypanosoma cruzi
Authors: Docampo R , Scott D A , Vercesi A E , Moreno S N J
Published: August 2015

The use of digitonin to permeabilize the plasma membrane of Trypanosoma cruzi allowed the identification of a non-mitochondrial nigericin- or bafilomycin A1-sensitive Ca(2+)-uptake mechanism. Proton uptake, as detected by ATP-dependent Acridine Orange accumulation, was also demonstrated in these permeabilized cells. Under these conditions Acridine Orange was concentrated in abundant cytoplasmic round vacuoles. This latter process was inhibited (and reversed) by bafilomycin A1, nigericin and NH4Cl in different stages of T. cruzi. Ca2+ released Acridine Orange from permeabilized cells, suggesting that the dye and Ca2+ were being accumulated in the same acidic compartment and that Ca2+ was taken up in exchange for protons. Addition of bafilomycin A1 (5 microM), nigericin (1 microM) or carbonyl cyanide p-trifluoromethoxyphenylhydrazone (FCCP; 1 microM) to fura 2-loaded epimastigotes increased their intracellular Ca2+ concentration ([Ca2+]i). Although this effect was more noticeable in the presence of extracellular Ca2+, it was also observed in its absence. Addition of NH4Cl (10-40 mM) to different stages of T. cruzi, in the nominal absence of extracellular Ca2+ to preclude Ca2+ entry, increased both [Ca2+]i in fura 2-loaded cells, and intracellular pH (pHi) in 2′,7′-bis-(2-carboxyethyl)-5-(and -6)-carboxyfluorescein acetoxymethyl ester (BCECF)-loaded cells. Treatment of the cells with the Ca2+ ionophore ionomycin under similar conditions (nominal absence of extracellular Ca2+) resulted in an increase in [Ca2+]i and a significantly higher increase in [Ca2+]i after addition of NH4Cl, nigericin or bafilomycin A1, all agents which increase the pH of acidic compartments and make ionomycin more effective as a Ca(2+)-releasing ionophore. Similar results were obtained when the order of additions was reversed. Taking into account the relative importance of the ionomycin-releasable and the ionomycin plus NH4Cl-releasable Ca2+ pools, it is apparent that most of the Ca2+ stored in different stages of T. cruzi is present in the acidic compartment thus identified. Taken together, these results are consistent with the presence of a Ca2+/H+ exchange system in an acidic vacuole, which we have named the ‘acidocalcisome’ and which appears to be a unique organelle present in trypanosomatids.
Magnetic circular dichroism study of the selenium-substituted form (Fe3Se4) of bovine heart aconitase
Authors: Breton J L , Farrar J A , Kennedy M C , Beinert H , Thomson A J
Published: August 2015

The selenium-substituted inactive form of mitochondrial aconitase contains one [3Fe-4Se]1+/0 cluster [Surerus, Kennedy, Beinert and Münck (1989) Proc. Natl. Acad. Sci. U.S.A. 87, 9846-9850]. This cluster was studied in both oxidized and reduced states by magnetic CD (MCD) and EPR spectroscopy. In the MCD spectra, intensity and transition wavelength shifts are observed when compared with the spectra of the native [3Fe-4S]1+/0 cluster. These changes are used to differentiate between the charge-transfer transitions originating from inorganic and cysteinyl sulphur. Using also the data from the EPR spectra, the spin ground state is assigned as S = 1/2 for the oxidized [3Fe-4Se]1+ cluster and S = 2 for the reduced [3Fe-4Se]0 cluster.
Molecular cloning of mouse intestinal trefoil factor and its expression during goblet cell changes
Authors: Tomita M , Itoh H , Ishikawa N , Higa A , Ide H , Murakumo Y , Maruyama H , Koga Y , Nawa Y
Published: August 2015

A cDNA encoding mouse intestinal trefoil factor (mITF) was successfully cloned and sequenced from the small intestine of C57BL/6 mouse by using the combination of reverse transcription-PCR and rapid amplification of cDNA ends methods. The gene was, similar to rat and human ITFs, mainly expressed in the small and large intestine. The mITF expression was up-regulated during the recovery phase after depletion of goblet cells in acetic acid-induced colitis. On the other hand, the expression in the jejunum was not altered, while goblet cell hyperplasia was induced by Nippostrongylus brasiliensis infection. These results suggest that the mITF expression did not simply correlate with the number of goblet cells. The mITF may play an important role in the maintenance and repair of mucosal function of the rectum. Additionally, the mITF in the jejunum may play a role in alteration of the physicochemical nature of goblet cell mucins, thereby affecting the establishment of intestinal helminths.
Co-localization and functional coupling of creatine kinase B and gastric H+/K+-ATPase on the apical membrane and the tubulovesicular system of parietal cells
Authors: Sistermans E A , Klaassen C H W , Peters W , Swarts H G P , Jap P H K , De Pont J J H H M , Wieringa B
Published: August 2015

Immunogold labelling of creatine kinase B (BB-CK) and gastric H+/K(+)-ATPase in the parietal cells of the stomach revealed colocalization of these two enzymes on the apical membrane and the membranes of the tubulovesicular system. Upon fractionation of hog parietal cells, a specific fraction of the BB-CK proteins remained associated with the purified vesicles, in which gastric H+/K(+)-ATPase is highly enriched. The BB-CK present in this highly purified preparation was able to support pronounced H+/K(+)-ATPase activity in K(+)-loaded vesicles in the presence of phosphocreatine and ADP, although only low levels of ATP were measured. In contrast, when pyruvate kinase, phosphoenolpyruvate and ADP were used as an ATP-generating system to sustain similar levels of H+/K(+)-ATPase activity, ATP levels were more than 10-fold higher. Changing the experimental conditions such that ATP levels were the same for both systems resulted in significantly elevated H+/K(+)-ATPase activities in the BB-CK/phosphocreatine system in comparison with the pyruvate kinase/phosphoenolpyruvate system. These results indicate that gastric H+/K(+)-ATPase has preferential access to ATP generated by creatine kinase co-localized on the membranes of the vesicles.
Impairment of the liver insulin receptor autoactivation cascade at full-term pregnancy in the rat
Authors: Martinez C , Molero J C , Ruiz P , Del Arco A , Andres A , Carrascosa J M
Published: August 2015

Partially purified liver insulin receptors from full-term pregnant rats show decreased autophosphorylation rates if compared with receptors from virgins. We studied the molecular mechanism of this phenomenon, looking at possible structural and functional changes of several domains. The ATP-binding domain seems to be unaltered in receptors from pregnant rats since Km for ATP was similar to that observed in virgins. In contrast, the Vmax. is decreased some 45%, suggesting changes in the kinase domain. Truncation of a fragment of 10 kDa from the C-terminal tail does not normalize the kinase activity in receptors from pregnant rats, suggesting that this domain is not involved in the inhibitory regulation. Treatment with alkaline phosphatase increases the [32P]Pi incorporation into receptors from pregnant rats; however, the autophosphorylation remains lower than that observed in virgin rats. Tryptic phosphopeptide maps of phosphorylated receptors show that the same phosphopeptides are present in receptors from virgin and pregnant rats. However, the progression through the autoactivation cascade in the kinase domain is impaired in receptors from pregnant rats. Differences in the cleavage by trypsin at the two alternative sites in the kinase domain were observed, indicating possible structural changes in receptors from pregnant rats that could be related to the impairment of the autoactivation cascade. Integrity of the alpha- and beta-subunits, as well as differential expression of the two receptor isotypes, were shown to be unaltered. We conclude that (1) the decreased autophosphorylation rate of the liver insulin receptor from pregnant rats is associated with the impairment of its autoactivation cascade, probably as a consequence of the basal Ser/Thr phosphorylation; and (2) the inhibition of the autoactivation cascade does not account for the overall inhibition of autophosphorylation observed in receptors from pregnant rats.
Primary structure and tissue-specific expression of blue crab (Callinectes sapidus) metallothionein isoforms
Authors: Brouwer M , Enghild J , Hoexum-Brouwer T , Thogersen I , Truncali A
Published: August 2015

In aquatic animals, synthesis of the metal-binding protein metallothionein (MT) can be induced through exposure to elevated levels of metals in food or water. Whether the different routes of exposure lead to expression of different metallothionein isoforms in different tissues in unknown. In this study we examined the induction of metallothionein isoforms in the hepatopancreas and gills of the blue crab Callinectes sapidus. When blue crabs are exposed to cadmium in their diet, the metal accumulates in the hepatopancreas. Size-exclusion and anion-exchange chromatography show the presence of five low-molecular-mass cadmium-binding proteins. All of the observed cadmium-binding proteins belong to the class I MT family. They are designated as MT-Ia, MT-Ib, MT-Ic, MT-IIa and MT-IIb. All purified proteins run as single peaks upon rechromatography on anion-exchange HPLC, except for MT-Ic, which segregates into two peaks corresponding to MT-Ia and MT-Ic. The amino acid sequence of MT-Ia and MT-Ic is identical. MT-Ib differs from MT-Ia and MT-Ic only in having an extra N-terminal methionine. The 18 cysteine residues in MT-Ia and MT-IIa occur in identical positions; however, of the remaining 40 amino acids, 15 are found to be different. MT-IIb is identical with MT-IIa, except for an extra methionine residue at its N-terminal position. It appears therefore that, of the five observed CdMTs, only two are the products of distinct genes. CdMT-Ia and -IIa are posttranslationally modified forms of Ib and IIb, respectively, and CdMT-Ia and -Ic appear to be conformational isomers. Cadmium-induced expression of the two genes is tissue-specific. When crabs are exposed to cadmium in water, the metal accumulates in the gills, where it is bound to MT-II. MT-I is virtually absent.
Urea-induced inactivation and denaturation of clostridial glutamate dehydrogenase: the absence of stable dimeric or trimeric intermediates
Authors: Aghajanian S A , Martin S R , Engel P C
Published: August 2015

Urea-induced effects in clostridial glutamate dehydrogenase (GDH, EC 1.4.1.2) were studied by spectrophotometry, circular dichroism, FPLC, affinity chromatography and PAGE. Denaturation of enzyme occurred over a narrow range of urea concentrations (2.5-3.5 M), accompanied by inactivation of enzyme with a similar rate constant. The contribution of instantaneous inhibition by urea was also ascertained. FPLC studies of urea-treated GDH gave no evidence for dissociated oligomeric fragments of the hexamer in the presence of subdenaturing concentrations of urea. Likewise a mixture of fully 5,5′-dithiobis-(2-nitrobenzoic acid)-modified GDH hexamers and unmodified enzyme in 2 M urea failed to give rise to hybrid molecules. Exposure of unmodified GDH to high concentrations of urea led to the dissociation of hexamers to denatured monomers followed by association to form non-specific high-M(r) aggregates. This conclusion was confirmed by native gradient PAGE experiments. Various specific ligands stabilized the enzyme against urea-induced inactivation, succinate and 2-oxoglutarate being particularly effective. This protection of the native state was enhanced in ternary complexes, and the complex most resistant to urea-induced inactivation was the productive ternary complex GDH-NADH-2-oxoglutarate. Native gradient PAGE experiments indicate that these protecting ligands preserve the native hexameric structure of GDH.
Cloning and expression of two ornithine decarboxylase forms from HMOA cells
Authors: Autelli R , Persson L , Baccino F M
Published: August 2015

In HMOA cells [Mamont, Duchesne, Grove and Tardif (1978) Exp. Cell Res. 115, 387-393] the half-life of ornithine decarboxylase (ODC) is 8-14 h instead of 15 min as in the Hepatoma Tissue Culture parental cells, due to a single amino acid substitution [Miyazaki, Matsufuji, Murakami and Hayashi (1993) Eur. J. Biochem. 214, 837-844]. We demonstrate for the first time that HMOA cells possess two forms of ODC mRNA that are translated into two proteins differing greatly in turnover rates. We have cloned and transfected the cDNAs for the two ODC forms into COS-1 cells for a direct measurement of their turnover rate. The variant ODC form was much more stable than the wild-type protein, with a half-life of 14 h as compared with 2.5 h.
Effects of ionic strength on the catalysis and stability of prolyl oligopeptidase
Authors: Polgár L
Published: August 2015

Prolyl oligopeptidase is the prototype of a new serine protease family, unrelated to trypsin and subtilisin. In contrast with these proteases, prolyl oligopeptidase is remarkably sensitive to ionic strength, being more active in the presence of high concentrations of salt. The enzyme has two catalytic forms, which interconvert with changing pH. To reveal the structural bases of these phenomena, the effects of 0.5 M NaCl on the stability of the enzyme were investigated by studying its denaturation as a function of pH, temperature, and urea concentration. The three independent methods have unequivocally demonstrated that denaturation of the enzyme is promoted in the presence of NaCl. Furthermore, destabilization of the low-pH form by urea is more significant than that of the high-pH form. Examination of the fluorescence emission spectra of various denatured forms indicates that the enzyme is not fully unfolded in 8 M urea, nor at acidic pH. The tryptophan residues in the acid-denatured state are mainly buried. The results are interpreted in terms of the decay of the protective water shell at the higher ionic strength. The higher enthalpy and entropy of activation for heat denaturation provide further evidence that a more ordered water structure stabilizes the protein in the absence of salt. The biphasic kinetics obtained with denaturation by heat and urea suggest that the enzyme has two domains of different stabilities.
Structure of the quinoprotein glucose dehydrogenase of Escherichia coli modelled on that of methanol dehydrogenase from Methylobacterium extorquens
Authors: Cozier G E , Anthony C
Published: August 2015

The structure of methanol dehydrogenase (MDH) at 0.194 nm (1.94 A) has been used to provide a model structure for part of a membrane quinoprotein glucose dehydrogenase (GDH). The basic superbarrel structure is retained, along with the tryptophan-docking motifs. The active-site regions are similar, but there are important differences, the most important being that GDH lacks the novel disulphide ring structure formed from adjacent cysteines in MDH; in GDH the equivalent region is occupied by His-262. Because of the overall similarities in the active-site region, the mechanism of action of GDH is likely to be similar to that of MDH. The differences in co-ordination to the cation and bonding to the pyrrolo-quinoline quinone (PQQ) in the active site may explain the relative ease of dissociation of the prosthetic group from the holo-GDH. There are considerable differences in the external loops, particularly those involved in formation of the shallow funnel leading to the active site, the configuration of which influences substrate specificity. The proposed model is consistent in many respects with previous proposals for the active-site structure based on the effects of chemical modification on binding of PQQ and enzymic activity.
Partial characterization of regulation of biliary lecithin hydrophobicity: association with organic anion-induced solute cholestasis in rats
Authors: Miura H , Tazuma S , Kajiyama G
Published: August 2015

We examined the effects of the depletion of bile salts and of the intravenous infusion of sodium taurocholate (STC) with or without bromosulphophthalein (BSP) in rats on the biliary secretion of lipids to clarify the regulatory mechanism(s). Each rat was equipped with a bile-duct cannula to collect bile. After the endogenous bile salt pool was depleted, STC was infused at a constant rate (160 nmol/min per 100 g body wt.) with or without BSP (50, 100, or 150 nmol/min per 100 g body wt.). BSP reduced the biliary secretion of cholesterol and phospholipids dose-dependently without affecting the secretion of bile salts (uncoupling phenomenon). Compared with the physiological and STC-infused condition, the biliary cholesterol/phospholipid ratio and saturated/unsaturated fatty acid ratio increased under the bile salts depletion and uncoupling phenomenon. Data indicate that the hydrophobicity of biliary lecithin increases with a decrease in the bile salt micelle capacity to induce biliary lipid secretion, resulting in a higher packing density of biliary vesicle. The cholesterol-holding capacity of the biliary vesicle is therefore enhanced during the depletion of bile salts and the uncoupling phenomenon.
DNA binding and methyl transfer catalysed by mouse DNA methyltransferase
Authors: Reale A , Lindsay H , Saluz H P , Pradhan S , Adams R L P , Jost J P , Strom R
Published: August 2015

By using a purified fraction of mouse DNA methyltransferase we have shown, by gel-retardation analysis, that the enzyme forms a low-affinity complex preferentially with hemimethylated DNA; the complexes formed with unmethylated or with fully methylated DNA are of even lower affinity, and only very weak interaction occurs with DNA lacking CG dinucleotides. Interaction is inhibited by N-ethylmaleimide. Methyl transfer from S-adenosyl-methionine is associated with the release of the fully methylated product from the complex. Complexes formed with the intact enzyme are extremely large, but limited trypsin treatment allows a major complex to enter the gel. DNA binding is not inhibited by this limited proteolysis of the native enzyme.
Multiple mechanisms for the phosphorylation of C-terminal regulatory sites in rabbit muscle glycogen synthase expressed in COS cells
Authors: SKURAT Alexander V. , ROACH Peter J.
Published: August 2015

Glycogen synthase can be inactivated by sequential phosphorylation at the C-terminal residues Ser652 (site 4), Ser648 (site 3c), Ser644 (site 3b) and Ser640 (site 3a) catalysed by glycogen synthase kinase-3. In vitro, glycogen synthase kinase-3 action requires that glycogen synthase has first been phosphorylated at Ser656 (site 5) by casein kinase II. Recently we demonstrated that inactivation is linked only to phosphorylation at site 3a and site 3b, and that, in COS cells, modification of these sites can occur by alternative mechanisms independent of any C-terminal phosphorylations [Skurat and Roach (1995) J. Biol. Chem. 270, 12491-12497]. To address these mechanisms multiple Ser → Ala mutations were introduced in glycogen synthase such that only site 3a or site 3b remained intact. Additional mutation of Arg637 → Gln eliminated phosphorylation of site 3a, indicating that Arg637 may be important for recognition of site 3a by its corresponding protein kinase(s). Similarly, additional mutation of Pro645 → Ala eliminated phosphorylation of site 3b, indicating a possible involvement of ‘proline-directed’ protein kinase(s). Mutation of Arg637 alone did not activate glycogen synthase as expected from the loss of phosphorylation at site 3a. Rather, mutation of both Arg637 and the Ser → Ala substitution at site 3b was required for substantial activation. The results suggest that sites 3a and 3b can be phosphorylated independently of one another by distinct protein kinases. However, phosphorylation of site 3b can potentiate phosphorylation of site 3a, by an enzyme such as glycogen synthase kinase-3.
Inhibition of mitogen-induced DNA synthesis by bafilomycin A1 in Swiss 3T3 fibroblasts
Authors: SAURIN Andrew J. , HAMLETT Jane , CLAGUE Michael J. , PENNINGTON Stephen R.
Published: August 2015

Quiescent cells (in G0) can be stimulated to enter the cell cycle and proceed to DNA synthesis in S-phase by a wide range of growth factors and mitogens. Activation of cell-surface growth factor receptors with intrinsic protein tyrosine kinase activity initiates autophosphorylation of the receptors and subsequent activation of signal transduction cascades. After activation the receptors undergo ligand-induced internalization to endosomes, which become acidified by the action of a vacuolar H+-ATPase (V-ATPase). The extent to which vesicular acidification plays a role in mitogenic signalling by receptors with intrinsic tyrosine kinase activity remains unknown. Here we have shown that bafilomycin A1, a specific inhibitor of V-ATPase, inhibits endosome acidification and mitogen-induced DNA synthesis in Swiss 3T3 fibroblasts. Addition of bafilomycin A1 at successively later times during G1 progressively decreased the inhibition of DNA synthesis such that no inhibition was observed when bafilomycin A1 was added at the onset of S-phase. Bafilomycin A1 also induced a dramatic but reversible change in the morphology of Swiss 3T3 cells. However, the rapid activation of c-fos mRNA accumulation by epidermal growth factor and insulin was unaffected by bafilomycin A1. Together, the results suggest that activation of the V-ATPase plays an important role in the mitogenic signalling pathways that occur during the G1 phase of the cell cycle but is not required for the initial epidermal growth factor and insulin-evoked signalling events that lead to c-fos mRNA expression.
Proteases of germinating winged-bean (Psophocarpus tetragonolobus) seeds: purification and characterization of an acidic protease
Authors: USHA Rajamma , SINGH Manoranjan
Published: August 2015

Two major classes of protease are shown to occur in germinating winged-bean (Psophocarpus tetragonolobus) seeds, by assaying extracts at pH 8.0 and pH 5.1 with [14C]gelatin as substrate. At pH 8.0, the activity profile of the enzyme shows a steady rise throughout the period of germination, whereas the activity at the acidic pH is very low up to day 5 and then increases sharply reaching a peak on day 11, followed by an equally sharp decline. The winged-bean acidic protease (WbAP) has been purified to apparent homogeneity, as attested by a single protein band on both PAGE and SDS/PAGE. WbAP is a monomeric enzyme with a molecular mass of 35 kDa and a pH optimum of 6.0. It is a thiol protease that does not belong to the papain family and it has tightly bound Ca2+ as shown by 45Ca2+-exchange studies. Besides gelatin and casein, it hydrolyses a 29 kDa winged-bean protein, indicating a prospective physiological role for it in storage-protein mobilization. Immunoblot analysis shows that it occurs only in the seeds and sprouting tubers of this plant and also that it is synthesized in developing seeds just before desiccation. It appears that the newly synthesized enzyme is inactive, and activation takes place around day 6 of germination. However, neither the mechanism of activation nor the signal that triggers it is clearly understood.
Site-directed mutagenesis of monocyte chemoattractant protein-1 identifies two regions of the polypeptide essential for biological activity
Authors: BEALL Clifford J. , MAHAJAN Sangeeta , KUHN Donald E. , KOLATTUKUDY Pappachan E.
Published: August 2015

Monocyte chemoattractant protein-1 (MCP-1) mediates monocyte migration into tissues in inflammatory diseases and atherosclerosis. We have investigated structure-activity relationships for human MCP-1. Mutations were introduced based upon differences between MCP-1 and the structurally related but functionally distinct molecule interleukin-8 (IL-8). Mutant proteins produced using the baculovirus/insect cell expression system were purified and their ability to stimulate monocyte chemotaxis and elevation of intracellular calcium in THP-1 monocytic leukaemia cells was measured. Two regions in MCP-1 were identified as important for its biological activity. One region consists of the sequence Thr-Cys-Cys-Tyr (amino acids 10-13). Point mutations of Thr-10 to Arg and Tyr-13 to Ile greatly lowered MCP-1 activity. The second functionally important region is formed by Ser-34 and Lys-35. Insertion of a Pro between these two residues, or their substitution by the sequence Gly-Pro-His, caused nearly complete loss of MCP-1 activity. Competition binding experiments showed that the mutations that affected activity also lowered the ability to compete with wild-type MCP-1 for receptors on THP-1 cells. Point mutations at positions 8, 15, 30, 37, 38 and 68 had little effect on MCP-1 activity. The important regions that we have identified in MCP-1 correspond with previously identified functionally important regions of IL-8, suggesting that the two molecules bind to their respective receptors by similar contacts.
Alternative RNA splicing of the human endothelin-A receptor generates multiple transcripts
Authors: MIYAMOTO Yoshihiro , YOSHIMASA Takaaki , ARAI Hiroshi , TAKAYA Kazuhiko , OGAWA Yoshihiro , ITOH Hiroshi , NAKAO Kazuwa
Published: August 2015

In order to elucidate the regulatory mechanisms of expression of the human endothelin-A receptor (hET-AR) gene, we characterized hET-AR transcripts using reverse transcriptase (RT)-PCR analysis in a variety of human tissues. RT-PCR of lung mRNA using a set of primers from exons 2 and 5 showed two lower-molecular-mass transcripts in addition to the expected fragment. When RT-PCR with primers from exons 4 and 8 was performed, no transcripts other than the expected one were detected. PCR cloning utilizing a set of primers from exons 2 and 8 which covered the entire coding sequence revealed that the cDNA clones corresponding to the two novel transcripts contained deletions of 199 bp and 327 bp respectively compared with the previously described hET-AR cDNA. Comparison of their sequences with that of the hET-AR gene showed that the deleted sequences correspond exactly to exon 4 and exons 3 and 4 respectively, indicating that these lower-molecular-mass ET-AR transcripts result from alternative RNA splicing (designated ET-AR∆4 and ET-AR∆3,4 respectively). Alternative splicing of exon 4 results in a transcript which would be translated into a C-terminal truncated protein containing the first, second and third transmembrane domains, while the splicing out of exons 3 and 4 would produce a protein with five membrane-spanning domains but lacking the third and fourth domains present in the ET-AR protein. An RNase protection assay revealed that ET-AR∆4 and ET-AR∆3,4, as well as ET-AR, transcripts were observed in various human tissues, including the lung, aorta, atrium, kidney and placenta, which are known to express ET-AR abundantly. Thus we have isolated the cDNAs of novel transcripts of hET-AR which are generated by alternative RNA splicing, and these results suggest that this alternative RNA splicing might contribute to the regulation of ET-AR gene expression.
Lysosomal α-glucosidase: cell-specific processing and altered maturation in HT-29 colon cancer cells
Authors: FRANCÍ Clara , EGEA Gustavo , ARRIBAS Rosa , REUSER Arnold J. J. , REAL Francisco X.
Published: August 2015

We have previously described the abnormal localization of resident Golgi proteins and O-glycans in the rough endoplasmic reticulum of mucin-secreting HT-29 M6 colon cancer cells, suggesting altered protein trafficking in these cells [Egea, Franci~A, Gambús, Lesuffleur, Zweibaum and Real (1993) J. Cell Sci. 105, 819–830]. In the present work, we have chosen lysosomal α-glucosidase as a reporter to examine the intracellular traffic of glycoproteins in M6 cells. We have compared the synthesis and processing of α-glucosidase in mucin-secreting M6 cells and in Caco-2 colon cancer cells, the latter resembling normal absorptive intestinal epithelium. Our results show that α-glucosidase processing and secretion is markedly delayed in M6 cells as compared with Caco-2 cells or normal fibroblasts, and this delay is caused by an accumulation of α-glucosidase precursor form in the trans-Golgi network. Furthermore, treatment of Caco-2 cells with brefeldin A led to changes in α-glucosidase maturation similar to those observed in untreated M6 cells. To determine whether altered processing occurs in other cultured cells, a panel of cancer cell lines and cultures from normal exocrine pancreas were examined. In pancreas-derived cultures, α-glucosidase showed a processing pattern different from that described until now. Only HT-29 cells and HT-29-derived subpopulations displayed a defect in α-glucosidase maturation. In conclusion, α-glucosidase processing is more diverse than has previously been described; this finding may have tissue-specific functional implications.
Phosphatidic acid mobilized by phospholipase D is involved in the phorbol 12-myristate 13-acetate-induced G2 delay of A431 cells
Authors: KASZKIN Marietta , RICHARDS James , KINZEL Volker
Published: August 2015

This study was aimed at gaining an understanding of metabolic events responsible for the inhibition of cells in G2 phase, a known physiological restriction site in the cell cycle of multicellular organisms. In an earlier study, phosphatidic acid was proposed as an inhibitory mediator in the epidermal growth factor (EGF)-induced inhibition of A431 cells in G2 phase via the phospholipase C pathway [Kaszkin, Richards and Kinzel (1992) Cancer Res. 52, 5627–5634]. We show here that the phorbol ester phorbol 12-myristate 13-acetate (PMA) induces a reversible inhibition of the G2/M transition in A431 cells under conditions of phospholipase D-catalysed phosphatidic acid formation. Such PMA-induced inhibition in G2 phase is largely attenuated in the presence of 1-propanol (but not of 2-propanol). In this case the amount of phosphatidic acid is reduced to almost control levels, and instead phosphatidylpropanol is formed. In the case of EGF-induced activation of a phospholipase D the amount of phosphatidic acid is only slightly decreased in the presence of a primary alcohol. Under these conditions the EGF-induced G2 delay was not affected. The correlation between the formation of phosphatidic acid and the G2 delay induced by PMA, as well as by an exogenous bacterial phospholipase D (from Streptomyces chromofuscus), could be supported by using synchronized cells in order to increase the population of cells in G2 phase. This study indicates that the formation of substantial amounts of phosphatidic acid immediately before entry into mitosis seems to be important for establishing a delay in the cell cycle at the G2/M border by exogenous ligands.
Inhibition of the cerebellar inositol 1,4,5-trisphosphate-sensitive Ca2+ channel by ethanol and other aliphatic alcohols
Authors: MEZNA Mokdad , PATCHICK Tracy , TOVEY Stephen , MICHELANGELI Francesco
Published: August 2015

The effects of ethanol and other aliphatic alcohols on the endoplasmic reticulum Ca2+ pump and the inositol 1,4,5-trisphosphate (InsP3)-sensitive Ca2+ channel were studied in pig cerebellar microsomes. Methanol, ethanol and propanol all stimulated ATP-dependent Ca2+ uptake, whereas butanol inhibited this process. Ethanol inhibited InsP3-induced Ca2+ release [half-maximal inhibition at 3.5%, v/v (600 mM)]. However, ethanol affected only the amount of InsP3-releasable Ca2+, without affecting the concentration of InsP3 required to induce half-maximal release. Other alcohols of longer chain length were more potent than ethanol at inhibiting InsP3-induced Ca2+ release, but none of the alcohols tested affected [3H]InsP3 binding to its receptor. Using stopped-flow techniques, measurements of the rate of InsP3-induced Ca2+ release in the preparation of pig cerebellar microsomes used in this study showed the kinetics to be monophasic, with a rate constant of 0.93 s-1 at 20 μM InsP3. This rate constant was dependent upon InsP3 concentration, decreasing to 0.38 s-1 at 0.25 μM InsP3. Ethanol was shown to reduce the fractional amount of InsP3-induced Ca2+ release without significantly affecting the rate constant for this process.
Glucose stimulates voltage- and calcium-dependent inositol trisphosphate production and intracellular calcium mobilization in insulin-secreting β TC3 cells
Authors: GROMADA Jesper , FRØKJÆR-JENSEN Jørgen , DISSING Steen
Published: August 2015

The cellular processes leading to a rise in the intracellular free Ca2+ concentration ([Ca2+]i) after glucose stimulation and K+ depolarization were investigated in insulin-secreting βTC3 cells. Stimulation with 11.2 mM glucose causes inositol 1,4,5-trisphosphate production and release of Ca2+ from intracellular stores. A strong correlation was observed between the changes in Ins(1,4,5)P3 concentration and the rise in [Ca2+]i, consistent with the former compound being responsible for release of Ca2+ from intracellular stores. The increase in Ins(1,4,5)P3 production was reduced by 68±4% when [Ca2+]i was kept low on glucose stimulation by loading cells with the Ca2+ chelator 1,2-bis(2-aminophenoxy)ethane-NNN´N´-tetra-acetic acid (BAPTA). The Ins(1,4,5)P3 production was prevented in cells hyperpolarized with diazoxide, an opener of ATP-sensitive K+-channels, consistent with the membrane potential controlling the rate of Ins(1,4,5)P3 synthesis. Depolarizing K+ concentrations evoked changes in [Ca2+]i and Ins(1,4,5)P3 production in both the presence and the absence of extracellular Ca2+, and from the relation between the extracellular K+ concentration and membrane potential we found a half-maximal Ins(1,4,5)P3 production by a 28 mV depolarization from a resting potential of -56 mV and by a rise in [Ca2+]i of 390 nM. We conclude that stimulation-induced changes in membrane potential and [Ca2+]i are important in controlling Ins(1,4,5)P3 production in βTC-3 cells and that glucose-stimulated Ca2+ mobilization from intracellular stores is due to voltage-dependent Ins(1,4,5)P3 production and depends on the concurrent increase in [Ca2+]i.
Mode of action and active site of an extracellular peroxidase from Pleurotus ostreatus
Authors: HAN Young-Hoon , SHIN Kwang-Soo , YOUN Hong-Duk , HAH Yung Chil , KANG Sa-Ouk
Published: August 2015

The properties of the haem environment of an extracellular peroxidase from Pleurotus ostreatus were studied by electronic absorption spectroscopy. A high-spin ferric form was predominant in the native enzyme and a high-spin ferrous form in the reduced enzyme. Cyanide was readily bound to the haem iron in the native form, thereby changing the enzyme to a low-spin cyano adduct. The electronic absorption spectra of the enzyme were similar to those of lignin peroxidase from Phanerochaete chrysosporium. Compound III of the enzyme was formed after the addition of an excess of H2O2 to the native enzyme, and thereafter spontaneously reverted to the native form. The enzyme oxidized 1-(3,5-dimethoxy-4-hydroxyphenyl)-2-(2-methoxyphenoxy)-1,3-dihydroxypropane in the presence of H2O2 to produce 1-(3,5-dimethoxy-4-hydroxyphenyl)-2-(2-methoxyphenoxy)-1-oxo-3-hydroxypropane, 2,6-dimethoxyhydroquinone, 2-(2-methoxyphenoxy)-3-hydroxypropanal, 2-(2-methoxyphenoxy)-3-hydroxypropanoic acid, 2,6-dimethoxy-1,4-benzoquinone and guaiacol. A similar oxidation pattern was demonstrated with a one-electron oxidant, ammonium cerium(IV) nitrate. Free radicals were detected as intermediates of the enzyme-mediated oxidation of 1-(3,5-dimethoxy-4-hydroxyphenyl)-2-(2-methoxyphenoxy)-1,3-dihydroxypropane and acetosyringone. These results can be explained by the mechanisms involving an initial one-electron oxidation of the lignin substructure. This radical may undergo Cα-Cβ cleavage, Cα-oxidation and alkyl-phenyl cleavage.
Lipoxygenase treatment render low-density lipoprotein susceptible to Cu2+-catalysed oxidation
Authors: LASS Achim , BELKNER Jutta , ESTERBAUER Hermann , KÜHN Hartmut
Published: August 2015

Oxidative modification of low-density lipoprotein (LDL) has been implicated in foam-cell formation at all stages of atherosclerosis. Since transition metals and mammalian 15-lipoxygenases are capable of oxidizing LDL to its atherogenic form, a concerted action of these two catalysts in atherogenesis has been suggested. Cu2+-catalysed LDL oxidation is characterized by a kinetic lag period in which the lipophilic antioxidants are decomposed and by a complex mixture of unspecific oxidation products. We investigated the kinetics of the 15-lipoxygenase-catalysed oxygenation of LDL and found that the enzyme is capable of oxidizing LDL in the presence of the endogenous lipophilic antioxidants. In contrast with the Cu2+-catalysed reaction, no kinetic lag phase was detected. The pattern of products formed during short-term incubations was highly specific, with cholesterol-esterified (13S)-hydroperoxy-(9Z,11E)-octadecadienoic acid being the major product. However, after long-term incubations the product pattern was less specific. Preincubation with 15-lipoxygenase rendered human LDL more susceptible to Cu2+-catalysed oxidation as indicated by a dramatic shortening of the lag period. Addition of Cu2+ to lipoxygenase-treated LDL led to a steep decline in its antioxidant content and to a greatly reduced lag period. Interestingly, if normalized to a comparable hydroperoxide content, autoxidation and addition of exogenous hydroperoxy fatty acids both failed to overcome the lag period. The local peroxide concentrations in various LDL subcompartments will be discussed as a possible reason for this unexpected behaviour.
Rat kidney glutathione S-transferase 1 subunits have C-terminal truncations
Authors: YEH Horng-I. , LEE Jing-Yu , TSAI Shu-Ping , HSIEH Cheng-Hsilin , TAM Ming F.
Published: August 2015

Cytosolic glutathione S-transferases (GSTs) from rat kidneys were purified by a combination of glutathione and S-hexylglutathione affinity columns. The isolated GSTs were subjected to reverse-phase HPLC and electrospray MS analysis. The major GST isoenzymes expressed in kidney are subunits 1, 2, 7 and 8. GST 1´, 3, and 4 are expressed in minor amounts. GST 10 is barely detectable in the male kidney cytosol. The molecular masses of these rat kidney GST subunits were determined by MS. The values obtained for subunits 1´, 2, 3, 4, 7, 8 and 10 are identical with those obtained for rat liver GSTs. Rat kidney GST 1 consists of three polypeptides, with molecular masses of 25517, 25372 and 24982 Da. Results from peptide mapping, MS and amino-acid-sequencing analyses indicate that the major components were generated by deleting the C-terminal phenylalanine (24982 Da) and the C-terminal IFKF tetrapeptide (25372 Da) from the GST 1 subunit, respectively. The 1-chloro-2,4-dinitrobenzene-conjugating and peroxidase activities of kidney GST 1 are substantially lower than for its counterpart from rat liver. In addition, rat kidney GST 1 has an arginine and a valine residue at positions 151 and 207 respectively. The results are in contradiction with the SWISS-PROT and GenBank rat liver GST 1 cDNA-sequencing data, which give a lysine and a methionine at the corresponding positions. Further analyses indicate that rat liver GST 1 also has C-terminal phenylalanine deletion, and an arginine and a valine residue at positions 151 and 207 respectively. However, the C-terminal-tetrapeptide-deleted form was not observed for rat liver GST 1.