bluuebunny/crossref_metadata_2025 huggingface dataset:
Analysis of the rat lactate dehydrogenase A subunit gene promoter/regulatory region
Authors: Short M L , Huang D , Milkowski D M , Short S , Kunstman K , Soong C J , Chung K C , Jungmann R A
Published: August 2015

The rat lactate dehydrogenase (LDH) A subunit gene promoter contains a putative AP-1 binding site at -295/-289 bp, two consensus Sp1 binding sites at -141/-136 bp and -103/-98 bp, and a single copy of a consensus cyclic AMP-responsive element (CRE) at -48 to -41 bp upstream of the transcription initiation site. Additionally, an as yet unidentified silencer element is located within the -1173/-830 bp 5′-flanking region. Transient transfection analyses of a -1173/+25 bp LDH A-chLoramphenicol acetyltransferase fusion gene has indicated a complete inability of the promoter fragment to direct basal or forskolin-induced transcription. Deletion of the -1173/-830 bp sequence restored basal and cyclic AMP (cAMP)-inducible activity. Point mutations in the Sp1 binding sites of a -830/+25 bp promoter fragment reduced basal but not the relative degree of cAMP-inducible activity. cAMP-regulated transcriptional activity was dependent upon an 8 bp CRE, -TGACGTCA-, located at the -48/-41 bp upstream region. Mutations in the CRE abolished cAMP-mediated induction and reduced basal activity by about 65%. The CRE binds a 47 kDa protein which has previously been identified as CRE binding protein (CREB)-327, an isoform of the activating transcription factor/CREB transcription factor gene family. Co-transfection of a vector that expresses the catalytic subunit of cAMP-dependent protein kinase stimulates LDH A subunit promoter activity suggesting that cAMP induces LDH A subunit gene expression through phosphorylative modification of CREB-327. This study emphasizes a fundamental role of several modules including Sp1 and CREB binding sites in regulating basal and cAMP-mediated transcriptional activity of the LDH A gene.
Purification, characterization and analysis of rolipram inhibition of a human type-IVA cyclic AMP-specific phosphodiesterase expressed in yeast
Authors: Wilson M , Sullivan M , Brown N , Houslay M D
Published: August 2015

Analyses were done on a human type-IV cyclic AMP (cAMP) phosphodiesterase (hPDE-IVA-h6.1) expressed in an engineered strain of Saccharomyces cerevisiae. This strain (YMS6) expressed soluble PDE activity, together with an insoluble activity which was not released by re-homogenization, treatment with high-ionic-strength solutions or with the detergent Triton X-100. Pellet and soluble PDE activities were typical of type-IV PDE. They were cAMP-specific, insensitive to the addition of either cGMP (1 microM) or Ca2+/calmodulin, and inhibited by rolipram. Thermostability studies showed both activities to decay as single exponentials, indicating the presence of homogeneous PDE protein species in each fraction. Pellet PDE activity was more thermostable than the soluble enzyme. Mg2+ and Mn2+ dose-dependently increased PDE activity and reversed the inactivating effect of EDTA.h6.1 was engineered to express a C-terminal five-histidine motif (h6.1his5). This allowed purification of the PDE to apparent homogeneity in a simple two-step process involving a rolipram affinity column and a Ni2(+)-chelate column. A single monomeric protein of subunit molecular mass approximately 73 kDa and native molecular mass approximately 74 kDa resulted after a approximately 53000-fold purification. This exhibited a Km for cAMP of 8 microM, a true Vmax. of 0.8 mumol of cAMP hydrolysed/min per mg of PDE protein, a kcat. of 3702 s-1, and a value of the specificity constant kcat/Km of 4.6 x 10(8) M-1.s-1, the last implying a diffusion controlled reaction. Rolipram (Ki 0.4 soluble; 0.7 microM pellet) and 3-isobutyl-1-methylxanthine (Ki 15 soluble; 19 microM pellet) served as simple competitive inhibitors for both soluble and pellet forms of h6.1, respectively.
Modulation of phosphoenolpyruvate carboxykinase mRNA levels by the hepatocellular hydration state
Authors: Newsome W P , Warskulat U , Noe B , Wettstein M , Stoll B , Gerok W , Häussinger D
Published: August 2015

Exposure of isolated perfused rat livers to hypo-osmotic (225 mosmol/l) perfusion media for 3 h led to a decrease of about 60% in mRNA levels for phosphoenolpyruvate carboxy-kinase (PEPCK) compared with normo-osmotic (305 mosmol/l) perfusions. Conversely, PEPCK mRNA levels increased about 3-fold during hyperosmotic (385 mosmol/l) perfusions. The anisotonicity effects were not explained by changes in the intracellular cyclic AMP (cAMP) concentration or by changes of the extracellular Na+ or Cl- activity. Similar effects of aniso-osmolarity on PEPCK mRNA levels were found in cultured rat hepatoma H4IIE.C3 cells, the experimental system used for further characterization of the effect. Whereas during the first hour of anisotonic exposure no effects on PEPCK mRNA levels were detectable, near-maximal aniso-osmolarity effects were observed within the next 2-3 h. PEPCK mRNA levels increased sigmoidally with the osmolarity of the medium, and the anisotonicity effects were most pronounced upon modulation of osmolarity between 250 and 350 mosmol/l. The aniso-osmolarity effects on PEPCK mRNA were not affected in presence of Gö 6850, protein kinase C inhibitor. cAMP increased the PEPCK mRNA levels about 2.3-fold in normo-osmotic media, whereas insulin lowered the PEPCK mRNA levels to about 8%. The effects of cAMP and insulin were also observed during hypo-osmotic and hyperosmotic exposure, respectively, but the anisotonicity effects were not abolished in presence of the hormones. The data suggest that hepatocellular hydration affects hepatic carbohydrate metabolism also over a longer term by modulating PEPCK mRNA levels. This is apparently unrelated to protein kinase C or alterations of cAMP levels. The data strengthen the view that cellular hydration is an important determinant for cell metabolic function by extending its regulatory role in carbohydrate metabolism to the level of mRNA.
Subpopulations of rat hepatocytes separated by Percoll density-gradient centrifugation show characteristics consistent with different acinar locations
Authors: Osypiw J C , Allen R L , Billington D
Published: August 2015

Freshly isolated viable rat hepatocytes were separated into five subpopulations on shallow discontinuous Percoll density gradients. The periportal marker enzymes alanine aminotransferase (ALT), malate dehydrogenase (MDH) and lactate dehydrogenase (LDH) showed gradients of increasing activity from the subpopulation of least density (band 1, rho = 1.07 g/ml) to the subpopulation of greatest density (band 5, rho = 1.09 g/ml). The perivenous marker enzymes pyruvate kinase (PK) and glutamate dehydrogenase (GDH) showed gradients of decreasing activity from band-1 cells to band-5 cells. Glutamine synthetase (GS), which is confined to the two or three cell layers around the hepatic venule, was almost entirely restricted to band-1 hepatocytes. Band-5: band-1 ratios of enzyme activity were as follows: ALT, 8.0; LDH, 2.1; MDH, 1.6; GDH, 0.7; PK, 0.2; GS, 0.01. Band-5:band-1 ratios for ALT, LDH, PK and GS were maintained after culture of subpopulations in identical conditions for up to 72 h, whereas the ratios for MDH and GDH decreased and increased respectively towards unity. Band-1 hepatocytes exhibited greater cytotoxicity than band-5 cells after incubation with carbon tetrachloride or paracetamol. These perivenous-selective toxins produced greater decreases in cell viability and greater release of ALT and LDH from band-1 hepatocytes than from band-5 hepatocytes. Conversely, band-5 hepatocytes were more susceptible than band-1 hepatocytes to the cytotoxic effects of 1-naphthylisothiocyanate and methotrexate (known periportal-selective toxins). It is concluded that band-5 hepatocytes are enriched in periportal cells, whereas band-1 hepatocytes are enriched in perivenous cells. Isolation of hepatocyte subpopulations by Percoll density-gradient centrifugation has the considerable advantage that periportal and perivenous cells can be obtained from the same liver.
Partial purification of a type η protein kinase C from murine brain: separation from other protein kinase C isoenzymes and characterization
Authors: Zang R , Müller H J , Kielbassa K , Marks F , Gschwendt M
Published: August 2015

Various murine tissues were tested, by using a protein kinase C-eta-specific antiserum, for the expression of type eta protein kinase C. Brain was found to be the richest source of a type eta isoenzyme. Native protein kinase C-eta was partially purified from the cytosol of murine brain by chromatography on DEAE-Sepharose, hydroxyapatite and protamine-agarose. This procedure resulted in a separation of protein kinase C-eta from the other phorbol 12-myristate 13-acetate (PMA)-responsive isoenzymes (alpha, beta, gamma, delta, epsilon) and allowed, for the first time, characterization of the native enzyme. The protein kinase C of type eta from mouse brain is a phospholipid-dependent Ca(2+)-unresponsive protein kinase. Both PMA and bryostatin activate the kinase for phosphorylation of a substrate as well as for autophosphorylation. Various pseudo-substrate-related peptides are suitable as substrates for the eta-type kinase, peptide delta being the best and peptides eta and epsilon the poorest substrates. The enzyme is inhibited by staurosporine and staurosporine-related compounds, such as K252a and Gö 6976. However, protein kinase C-eta, like protein kinase C-delta, is around two orders of magnitude less sensitive towards Gö 6976 than are the Ca(2+)-responsive isoenzymes (alpha, beta, gamma). The eta-type protein kinase C exhibits an extreme tendency to lose its PMA-responsiveness. Consequently, purification of the enzyme to homogeneity has not yet been successful.
Biosynthesis of a human gall-bladder mucin
Authors: Klomp L W J , de Lely A J , Strous G J
Published: August 2015

Mucin glycoproteins play an important role in the initial stages of gall-stone formation by a currently largely unknown mechanism. Understanding the structure of gall-bladder mucin is necessary to comprehend the mechanism by which cholesterol monohydrate crystals aggregate. Three successive CsCl-gradient-ultracentrifugation steps were used to purify human gall-bladder mucin from gall-bladder tissue. The isolated macromolecules had a typical mucin-like monosaccharide composition and appeared as heterogeneous high-M(r) glycoproteins on SDS/PAGE. A polyclonal antiserum was raised against these molecules and the specificity of the antiserum was ascertained by immunoblotting. The antiserum specifically stained mucous granules at the apical side of all gall-bladder epithelial cells in neck, fundus and body. The antibody was subsequently used to immunoprecipitate the mucin and biosynthetic intermediates from gall-bladder-tissue homogenates. An early biosynthetic precursor of the isolated mucin was identified by SDS/PAGE as a single polypeptide with an apparent M(r) of approx. 470,000. This precursor protein was converted after 1 h into a heterogeneous high-M(r) glycoconjugate with an electrophoretic mobility similar to that of the purified mucin. The mature mucin, but not the precursor, was secreted into the culture medium, starting at 1 h. As shown by SDS/PAGE under non-reducing conditions, the precursors form disulphide-linked oligomers. Using the N-glycosylation inhibitor tunicamycin, the apparent M(r) of the precursor was decreased to approx. 410,000, indicating that N-linked glycan chains are attached to the precursor polypeptide.
Sinefungin shares AdoMet-uptake system to enter Leishmania donovani promastigotes
Authors: Phelouzat M A , Basselin M , Lawrence F , Robert-Gero M
Published: August 2015

The involvement of a carrier for sinefungin (SF) uptake in Leishmania donovani promastigotes is indicated by saturation kinetics, competition studies and SF accumulation against a 270-fold concentration gradient across the cell membrane. Whether SF uptake occurs via nucleoside- or AdoMet-carrier systems was investigated by competition experiments and comparison of the uptake of various molecules in wild-type and SF-resistant cells. Results show that SF did not inhibit purine or pyrimidine uptake whereas it competitively inhibited AdoMet uptake. Furthermore, the uptake of nucleosides in SF-resistant cells is similar to that in wild-type cells, whereas uptake of SF and AdoMet is lower.
Inactivation during denaturation of ribonuclease A by guanidinium chloride is accompanied by unfolding at the active site
Authors: Yang H J , Tsou C L
Published: August 2015

Inactivation of pancreatic RNAase A occurs in guanidinium chloride (GdmCl) at low concentrations before the unfolding of the molecule as a whole can be detected [Liu and Tsou (1987) Biochim. Biophys. Acta 916, 455-464]. We have now shown that the rate of digestion of the RNAase molecule by either trypsin or proteinase K increases significantly at low concentrations of GdmCl where the enzyme is largely inactivated, but fluorescence and absorption measurements reveal no conformational changes. N-Terminal sequence analysis of the peptide fragments generated shows that proteolysis occurs primarily at or near the active site. The decrease in activity of RNAase at low concentrations of GdmCl is therefore due to partial unfolding of the molecule, particularly at the active site and not to an inhibition by the denaturant.
Evidence for a free N-acetylneuraminic acid-hydroxylating enzyme in pig mandibular gland soluble fraction
Authors: Mukuria C J , Mwangi W D , Noguchi A , Waiyaki G P , Asano T , Naiki M
Published: August 2015

The activity of a free N-acetylneuraminic acid (Neu5Ac)-hydroxylating enzyme which converted Neu5Ac into N-glycolyl-neuraminic acid (Neu5Gc) was demonstrated in the soluble fraction of pig mandibular gland. The hydroxylation was possible only with NADPH as the electron donor. The apparent Km was 4.5 mM Neu5Ac. At 0.5 mM monovalent cations had no effect on the hydroxylation of Neu5Ac whereas bivalent cations gave varied inhibition capacities ranging from 14 to 75%. EDTA gave a time-dependent enhancement of activity. It was concluded that the enzyme does not require an exogenously added inorganic cofactor. Results from salt fractionation of the soluble fraction and the use of inhibitors such as mercurials suggested that the hydroxylation of Neu5Ac to Neu5Gc may involve other, as yet unknown, component(s) and the possibility of electrons donated by NADPH being transferred to activated molecular oxygen (second substrate). We propose to name this enzyme N-acetyl-neuraminic acid hydroxylase.
Suppression of sterol 27-hydroxylase mRNA and transcriptional activity by bile acids in cultured rat hepatocytes
Authors: Twisk J , de Wit E C M , Princen H M G
Published: August 2015

In previous work we have demonstrated suppression of cholesterol 7 alpha-hydroxylase by bile acids at the level of mRNA and transcription, resulting in a similar decline in bile acid synthesis in cultured rat hepatocytes [Twisk, Lehmann and Princen (1993) Biochem. J. 290, 685-691]. In view of the substantial contribution of the ‘alternative’ or ‘27-hydroxylase’ route to total bile acid synthesis, as demonstrated in cultured rat hepatocytes and in vivo in humans, we here evaluate the effects of various bile acids commonly found in bile of rats on the regulation of sterol 27-hydroxylase in cultured rat hepatocytes. Addition of taurocholic acid, the predominant bile acid in rat bile, to the culture medium of rat hepatocytes resulted in a 72% inhibition of sterol 27-hydroxylase activity. The effect was exerted at the level of sterol 27-hydroxylase mRNA, showing a time- and dose-dependent decline with a maximal suppression (-75%) at 50 microM taurocholic acid after 24 h of culture. The decline in mRNA followed first-order kinetics with an apparent half-life of 13 h. Under these conditions cholesterol 7 alpha-hydroxylase mRNA (-91%) and bile acid synthesis (i.e. chenodeoxycholic and beta-muricholic acid, -81%) were also maximally suppressed. In contrast, no change was found in the level of lithocholic acid 6 beta-hydroxylase mRNA. Assessment of the transcriptional activity of a number of genes involved in routing of cholesterol towards bile acids showed similar suppressive effects of taurocholate on expression of the sterol 27-hydroxylase and cholesterol 7 alpha-hydroxylase genes (-43% and -42% respectively), whereas expression of the lithocholic 6 beta-hydroxylase gene was not affected. Taurocholic acid and unconjugated cholic acid were equally as effective in suppressing sterol 27-hydroxylase mRNA. The more hydrophobic bile acids, chenodeoxycholic acid and deoxycholic acid, also produced a strong inhibition of 57% and 76% respectively, whereas the hydrophilic beta-muricholic acid was not active. We conclude that (1) a number of bile acids, at physiological concentrations, suppress sterol 27-hydroxylase by down-regulation of sterol 27-hydroxylase mRNA and transcriptional activity and (2) co-ordinated suppression of both sterol 27-hydroxylase and cholesterol 7 alpha-hydroxylase results in inhibition of bile acid synthesis in cultured rat hepatocytes.
Determination of the structure of two novel echistatin variants and comparison of the ability of echistatin variants to inhibit aggregation of platelets from different species
Authors: Chen Y L , Huang T F , Chen S W , Tsai I H
Published: August 2015

Two new variants of short disintegrins were purified from the venom of Echis carinatus leakeyi and named echistatin beta and gamma. These proteins were found to be about 85% similar in amino acid sequence to echistatin alpha which has been well studied. The disulphide pattern of echistatin gamma appeared to be identical with that of echistatin alpha. They all contain the adhesive recognition sequence Arg-Gly-Asp (RGD) but inhibit the aggregation of platelets from human and other mammals with different potencies. Echistatin beta and alpha are far more effective on platelets from humans and guinea pigs than those from rabbits and rats whereas echistatin gamma is less discriminating of the platelets of the species tested. This species-dependent platelet sensitivity to echistatin beta and gamma could be attributed to the variations in residues 15, 21, 22 and 27, which are close to or within the RGD loop, rather than to the C-terminal variations after residue 46. Taking advantage of the presence of methionine residues flanking both sides of the ARGDDM motif in echistatin gamma, we deleted this hexapeptide by CNBr cleavage to produce des-(23-28)-echistatin gamma. The modified protein showed c.d. and fluorescent spectra grossly similar to the intact echistatin but its antiplatelet potency decreased more than 200-fold. We thus propose that a favourable conformation of the RGD region is responsible mainly for the high-affinity binding of echistatin to the platelet glycoprotein IIb-IIIa as shown previously for the binding of medium-size disintegrin.
The site of action of Ca2+ in the activation of steroidogenesis: studies in Ca2+-clamped bovine adrenal zona-glomerulosa cells
Authors: Python C P , Laban O P , Rossier M F , Vallotton M B , Capponi A M
Published: August 2015

The Ca(2+)-messenger system plays a crucial role in the regulation of steroid production in adrenal zona-glomerulosa cells, as it is known to mediate the action of both angiotensin II and K+. In the present study we used intact isolated glomerulosa cells in which the cytosolic free Ca2+ concentration ([Ca2+]c) was clamped at various levels with the Ca2+ ionophore ionomycin in order to locate the site(s) of action of Ca2+. By measuring in parallel steroid synthesis and [Ca2+]c, we show that Ca2+ levels (50-860 nM) regulate the production of both pregnenolone (up to 669 +/- 71.1% of the basal production) and aldosterone (up to 301 +/- 42.2%; EC50 = 303 nM). By contrast, Ca2+ did not stimulate the conversion of 11-deoxycorticosterone into aldosterone. Ca2+ modulation did not affect the formation of pregnenolone from freely diffusible analogues of cholesterol, indicating that Ca2+ acts at a step upstream of cholesterol side-chain cleavage. Moreover cycloheximide, an inhibitor of protein translation and of adrenocorticotropin-induced facilitation of intramitochondrial cholesterol transport, the rate-limiting step in steroidogenesis, also blocked Ca(2+)-triggered pregnenolone formation. This is consistent with a model in which Ca2+ promotes cholesterol transfer between mitochondrial membranes. In addition, agents using the cyclic AMP pathway as well as angiotensin II potentiated the steroidogenic response to increases in [Ca2+]c by augmenting both the efficacy and the potency of Ca2+. This effect of angiotensin II did not involve protein kinase C. These results establish a direct link between agonist-induced [Ca2+]c rises and a specific step of the steroidogenic pathway.
Rapid purification and identification of calcyphosine, a Ca2+-binding protein phosphorylated by protein kinase A
Authors: Lecocq R , Lamy F , Erneux C , Dumont J E
Published: August 2015

A method is presented for the rapid purification of dog thyroid calcyphosine, a protein previously identified as a major substrate for cyclic AMP-dependent protein kinase in dog thyroid slices stimulated by thyrotropin [Lecocq, Lamy and Dumont (1979) Eur. J. Biochem. 102, 147-152]. The protein was previously identified as a spot on two-dimensional gels and is now purified in its native form by a procedure involving three chromatographic steps. Homogeneous calcyphosine identified by SDS/PAGE, immunoblotting and peptide sequencing can be obtained within 7 h. As for calmodulin, Ca(2+)-dependent conformational changes can be shown by Ca(2+)-dependent hydrophobic interaction chromatography using phenyl-Sepharose. Unlike calmodulin, calcyphosine is a substrate for protein kinase A.
Regulation of purified hepatic PC-1 (phosphodiesterase-I/nucleotide pyrophosphatase) by threonine auto(de)phosphorylation and by binding of acidic fibroblast growth factor
Authors: Uriarte M , Stalmans W , Hickman S , Bollen M
Published: August 2015

The plasma cell differentiation antigen PC-1 was purified to homogeneity from rat liver membranes. Denaturing electrophoresis revealed polypeptides of 118 and 128 kDa, which were both recognized by antibodies against recombinant murine PC-1. During gel filtration PC-1 migrated as a protein of about 500 kDa, suggesting a tetrameric structure. Purified PC-1 displayed a phosphodiesterase-I/nucleotide pyrophosphatase activity that could be completely blocked by EDTA, dithiothreitol and acidic fibroblast growth factor (extrapolated Ki = 1.3 nM). Purified PC-1 was also capable of threonine autophosphorylation and of phosphorylation of histone IIa. The autophosphorylation of PC-1 was inhibited by addition of histone IIa, and it was blocked by phosphodiesterase-I inhibitors (acidic fibroblast growth factor, dithiothreitol), by nucleotides (ATP, ADP, AMP), and by vanadate. When added to autophosphorylated PC-1, these compounds caused a prompt dephosphorylation. However, the same agents did not affect the (de)phosphorylation of histone IIa, which is not a substrate for the PC-1 phosphatase. These data indicate that phosphodiesterase-I inhibitors, nucleotides and vanadate affect the (de)phosphorylation of PC-1 by stimulating the PC-1 phosphatase and/or by shielding the autophosphorylation site from the PC-1 kinase. The rate of dephosphorylation of PC-1 was independent of the dilution, suggesting an autocatalytic intramolecular process. We propose that the autophosphorylation of PC-1 serves to block its nucleotide pyrophosphatase activity when extracellular ATP becomes scarce.
Analysis of the relative interactions between the α2C10 adrenoceptor and the guanine-nucleotide-binding proteins Go1α and Gi2α following co-expression of these polypeptides in rat 1 fibroblasts
Authors: Grassie M A , Milligan G
Published: August 2015

Rat 1 fibroblasts which had been transfected to express the human alpha 2C10 adrenoceptor (clone 1C) were further co-transfected with a plasmid containing the hygromycin-B-resistance gene and a plasmid containing a cDNA encoding the alpha-subunit of the rat pertussis-toxin-sensitive G-protein G(o)1. In clone 3 the receptor was expressed at some 2.2 pmol/mg of membrane protein, and G(o)1 alpha at approx. 100 pmol/mg of membrane protein. The interaction of these two polypeptides and that between the receptor and Gi2 alpha (endogenously expressed at some 50 pmol/mg of membrane protein) were studied. Agonist activation of G(o)1 alpha was observed in membranes of the alpha 2C10-adrenoceptor(+)-G(o)1 alpha+ cells (clone 3), but not in alpha 2C10-adrenoceptor(+)-G(o)alpha-cells (clone 1C), whereas similar agonist-dependent activation of Gi2 alpha was observed in both cell types. alpha 2C10-adrenoceptor activation of G(o)1 alpha and Gi2 alpha in clone-3 membranes was produced with similar agonist-dose-effect curves. These observations indicate that the receptor interacts with equivalent affinity with each of these G-proteins. Agonist-dependent cholera-toxin-catalysed [32P]ADP-ribosylation of G(o)1 alpha was terminated when the alpha 2-adrenoceptor antagonist yohimbine was added subsequent to agonist-induced initiation of the reaction and release of GDP, demonstrating the conformational requirement for this reaction to be the ternary complex of agonist-occupied receptor and guanine-nucleotide-denuded G-protein.
Comparison of metabolic fluxes of cis-5-enoyl-CoA and saturated acyl-CoA through the β-oxidation pathway
Authors: Tserng K Y , Chen L S , Jin S J
Published: August 2015

The metabolic fluxes of cis-5-enoyl-CoAs through the beta-oxidation cycle were studied in solubilized rat liver mitochondrial samples and compared with saturated acyl-CoAs of equal chain length. These studies were accomplished using either spectrophotometric assay of enzyme activities and/or the analysis of metabolites and precursors using a gas chromatographic method after conversion of CoA esters into their free acids. Cis-5-enoyl-CoAs were dehydrogenated by acyl-CoA oxidase or acyl-CoA dehydrogenases at significantly lower rates (10-44%) than saturated acyl-CoAs. However, enoyl-CoA hydratase hydrated trans-2-cis-5-enoyl-CoA at a faster rate (at least 1.5-fold) than trans-2-enoyl-CoA. The combined activities of 3-hydroxyacyl-CoA dehydrogenase and 3-ketoacyl-CoA thiolase for 3-hydroxy-cis-5-enoyl-CoAs derived from cis-5-enoyl-CoAs were less than 40% of the activity for the corresponding 3-hydroxyacyl-CoAs prepared from saturated acyl-CoAs. Rat liver mitochondrial beta-oxidation enzymes were capable of metabolizing cis-5-enoyl-CoA via one cycle of beta-oxidation to cis-3-enoyl-CoA with two less carbons. However, the overall rates of one cycle of beta-oxidation, as determined with stable-isotope-labelled tracer, was only 15-25%, for cis-5-enoyl-CoA, of that for saturated acyl-CoA. In the presence of NADPH, the metabolism of cis-5-enoyl-CoAs was switched to the reduction pathway.(ABSTRACT TRUNCATED AT 250 WORDS)
Activation of the herpes simplex virus immediate-early gene promoters by neuronally expressed POU family transcription factors
Authors: Lillycrop K A , Liu Y Z , Theil T , Möröy T , Latchman D S
Published: August 2015

Herpes simplex virus immediate-early (IE) promoters contain the TAATGARAT motif which acts as a target site for the cellular POU family transcription factors Oct-1 and Oct-2. Here we show that other members of the POU family that are expressed in sensory neurons can also affect IE promoter activity. In particular, two members of the Brn-3 family of POU proteins Brn-3a and Brn-3c can activate the IE-1 and IE-3 promoters when co-transfected into fibroblasts and neuronal cells whereas a third member Brn-3b represses IE promoter activity. In contrast, Brn-3 proteins cannot overcome the inhibitory effect of neuronal Oct-2 on IE promoter activity in co-transfections nor do they prevent transactivation of the IE promoters by the Oct-1-Vmw65 complex. The potential regulation of the IE promoters by several different neuronally expressed POU proteins during the initiation, maintenance and re-activation of latent infection in sensory neurons is discussed.
Effects of an azasterol inhibitor of sterol 24-transmethylation on sterol biosynthesis and growth of Leishmania donovani promastigotes
Authors: Haughan P A , Chance M L , Goad L J
Published: August 2015

Leishmania donovani promastigotes were cultured in the presence of an azasterol (20-piperidin-2-yl-5 alpha-pregnane-3 beta,20-diol) to determine the effects on sterol biosynthesis and cell proliferation. Inhibition of growth increased gradually with azasterol concentrations up to 5 micrograms/ml; concentrations of azasterol exceeding 5 micrograms/ml were lethal. Sterol biosynthesis was affected by the azasterol when administered at concentrations as low as 100 pg/ml. The primary site of action was the alkylation at C-24 of a delta 24-sterol precursor. The 24-alkylated sterols [ergosta-5,7,24(24(1))-trien-3 beta-ol and ergosta-5,7,22-trien-3 beta-ol] of the protozoan were replaced by delta 24-cholesta-type sterols which then accumulated in the cells. Administration of the azasterol together with a bis-triazole inhibitor of the 14 alpha-methylsterol 14-demethylase reaction, which operates in sterol biosynthesis, resulted in depletion of 24-alkylsterols and their replacement with predominantly 14 alpha-methylsterols lacking a 24-alkyl group. Continuous subculture of promastigotes in the presence of the azasterol resulted in gradual depletion of 24-alkylsterols and their complete replacement by delta 24-cholesta-type sterols. Transfer of the azasterol-treated cells to medium lacking azasterol resulted in a gradual restoration, after several subcultures, of the normal 24-alkylsterol pattern. The results indicate that, although 24-alkylsterols are normally produced by the protozoan, it can nevertheless survive with sterols possessing only the cholestane skeleton. Thus there is no absolute requirement for 24-alkylsterols to fulfil some essential ‘sparking’ role associated with cell growth in promastigotes.
Differential effects of distamycin analogues on amplification of human gene sequences by polymerase-chain reaction
Authors: Passadore M , Bianchi N , Feriotto G , Mischiati C , Giacomini P , Piva R , Gambari R
Published: August 2015

In this report we analyse the effects of distamycin and five distamycin analogues on amplification by polymerase-chain reaction (PCR) of two gene sequences displaying a different A+T/G+C content. The first was a 5′ region of the human oestrogen receptor (ER) gene, containing a (TA)26 stretch; the second was a CG-rich sequence of the human Ha-ras oncogene. The results obtained unequivocally demonstrate that the addition of one pyrrole ring significantly improves the ability of distamycin derivatives to interfere with PCR-mediated amplification of the human ER genomic region carrying a (TA)26 stretch. The distamycin analogues analysed differ in the number of pyrrole rings and in the presence of an N-formyl, an N-formimidoyl or a retroamide group at position X1. Among compounds carrying the same number of pyrrole rings, those carrying an N-formyl or an N-formimidoyl group retain a similar inhibitory activity. The retroamide analogues, on the contrary, are much less efficient in inhibiting PCR-mediated amplification of the 5′ER region. With respect to sequence selectivity both distamycin and distamycin analogues exhibit a sequence preference, since they do not inhibit PCR amplification of Ha-ras CG-rich gene regions, with the exception of a distamycin analogue carrying four pyrrole rings.
Heat shock of human synovial and dermal fibroblasts induces delayed up-regulation of collagenase-gene expression
Authors: Hitraya E G , Varga J , Jimenez S A
Published: August 2015

We investigated the effect of heat shock on the expression of the collagenase gene in normal human synovial and dermal fibroblasts. Heat shock (42-44 degrees C for 1 h) caused a marked increase in heat-shock protein 70 (HSP-70) mRNA levels, followed by a delayed increase in collagenase mRNA levels, in both cell types. Pretreatment with cycloheximide had no effect on the heat-shock-induced increase in HSP-70 mRNA expression, but abrogated the induction of collagenase mRNA during the recovery. To study the mechanisms of collagenase-gene induction by heat shock, the transcriptional activity of a collagenase-promoter-driven chloramphenicol acetyltransferase (CAT) reporter gene was examined in transient transfection experiments. Heat shock was followed by a > 2-fold increase in CAT activity driven by a 3.8 kb fragment of the collagenase promoter, or by a construct containing an AP-1 binding site. A mutation in the AP-1 binding site abolished the effect of heat shock. Electrophoretic-mobility-shift assays revealed a marked increase in DNA-binding activity specific for the AP-1 binding site in nuclear extracts prepared from synovial fibroblasts recovering from heat shock. These results indicate that heat shock causes a delayed increase in collagenase-gene expression in human fibroblasts, and suggests that this stimulation involves, at least in part, transcriptional activation through an AP-1 binding site. Heat shock appears to initiate a programme of cellular events resulting in collagenase-gene expression, and therefore may contribute to connective-tissue degradation in disease states.
Role of methionine in the active site of α-galactosidase from Trichoderma reesei
Authors: Kachurin A M , Golubev A M , Geisow M M , Veselkina O S , Isaeva-Ivanova L S , Neustroev K N
Published: August 2015

alpha-Galactosidase from Trichoderma reesei when treated with H2O2 shows a 12-fold increase in activity towards p-nitrophenyl alpha-D-galactopyranoside. A similar effect is produced by the treatment of alpha-galactosidase with other non-specific oxidants: NaIO4, KMnO4 and K4S4O8. In addition to the increase in activity, the Michaelis constant rises from 0.2 to 1.4 mM, the temperature coefficient decreases by a factor of 1.5 and the pH-activity curve falls off sharply with increasing pH. Galactose (a competitive inhibitor of alpha-galactosidase; Ki 0.09 mM for the native enzyme at pH 4.4) effectively inhibits oxidative activation of the enzyme, because the observed activity changes are related to oxidation of the catalytically important methionine in the active site. NMR measurements and amino acid analysis show that oxidation to methionine sulphoxide of one of five methionines is sufficient to activate alpha-galactosidase. Binding of galactose prevents this. Oxidative activation does not lead to conversion of other H2O2-sensitive amino acid residues, such as histidine, tyrosine, tryptophan and cysteine. The catalytically important cysteine thiol group is quantitatively titrated after protein oxidative activation. Further oxidation of methionines (up to four of five residues) can be achieved by increasing the oxidation time and/or by prior denaturation of the protein. Obviously, a methionine located in the active site of alpha-galactosidase is more accessible. The oxidative-activation phenomenon can be explained by a conformational change in the active site as a result of conversion of non-polar methionine into polar methionine sulphoxide.
Expression of a Xenopus counterpart of mammalian syndecan 2 during embryogenesis
Authors: Rosenblum N D , Botelho B B , Bernfield M
Published: August 2015

We have identified a Xenopus cDNA, XS-2, by screening a Xenopus embryonic stage-22-24 cDNA library with a DNA probe encoding the transmembrane and cytoplasmic domains of mouse syndecan 1. The 1.4 kb cDNA consists of an open reading frame of 642 nucleotides encoding a protein of 191 amino acids. The predicted protein of 20869 Da contains a 25-amino acid putative transmembrane domain and a 32-amino acid putative cytoplasmic domain, both of which are highly similar to the corresponding regions of rat syndecan 2 (92% identity) and to a lesser degree those of rat syndecans 1, 3 and 4 (62, 64 and 78% respectively). The putative N-terminal ectodomain contains a possible attachment site for heparan sulphate, identical with the comparable glycosaminoglycan-attachment sequence of rat syndecan 2. Polyclonal antisera raised against recombinant ectodomain of XS-2, expressed as a fusion protein, recognized a heparan sulphate proteoglycan in XTC cell-culture medium. This proteoglycan bound to DEAE-Sephacel and was eluted with 1 M NaCl; digestion with heparitinase but not chondroitinase ABC resulted in the identification of a 46 kDa protein by these antisera. Northern-blot analysis indicated that XS-2 identifies two Xenopus mRNA species approx. 4 and 2 kb in size in embryos ranging in maturation from the 64-cell stage to stage 54. These results demonstrate that a heparan sulphate proteoglycan, similar to syndecan 2, is expressed during Xenopus embryogenesis.
Characterization of the endogenous carnitine transport and expression of a rat renal Na+-dependent carnitine transport system in Xenopus laevis oocytes
Authors: Berardi S , Hagenbuch B , Carafoli E , Krähenbühl S
Published: August 2015

L-Carnitine transport was characterized in Xenopus laevis oocytes before and after injection of mRNA isolated from rat renal cortex. Non-injected oocytes revealed endogenous Na(+)-dependent transport of L-carnitine. After injection of 15 ng of rat kidney mRNA, the Na(+)-dependent L-carnitine transport increased 2-3-fold, reaching maximal activity after 5-6 days. The expressed carnitine transport was maximal at pH 7.5, whereas the endogenous transport showed no clear maximum between pH 6.0 and 8.5. Kinetic analysis revealed apparent Km values for L-carnitine of 66 microM for the endogenous and 149 microM for the expressed transport. Trimethyl-lysine and D-carnitine inhibited both the endogenous and the expressed transport. In contrast, L-acetylcarnitine, L-isovalerylcarnitine, L-palmitoylcarnitine and butyrobetaine inhibited predominantly the expressed transport, whereas glycinebetaine had no inhibitory effect on either transport system. Size-fractionated rat renal-cortex mRNA (median size 2 kb) induced a 3-fold higher L-carnitine transport than did unfractionated mRNA. These studies demonstrate that Xenopus laevis oocytes exhibit Na(+)-dependent L-carnitine transport and provide the basis for expression-cloning of a rat renal Na(+)-dependent L-carnitine transport system.
Differentiation-dependent autophagy controls the fate of newly synthesized N-linked glycoproteins in the colon adenocarcinoma HT-29 cell line
Authors: Houri J J , Ogier-Denis E , De Stefanis D , Bauvy C , Baccino F M , Isidoro C , Codogno P
Published: August 2015

Our previous results have demonstrated that, in undifferentiated human colon cancer HT-29 cells, a pool of glycoproteins bearing high-mannose oligosaccharides rapidly escapes the exocytic pathway to be degraded in the lysosomal compartment [Trugnan, Ogier-Denis, Sapin, Darmoul, Bauvy, Aubery and Codogno (1991) J. Biol. Chem. 266, 20849-20855]. We report here on the mechanism that governs this degradative pathway. Using pulse-chase experiments in combination with subcellular fractionation, we have observed that the sequestration of high-mannose glycoproteins in lysosomes was impaired by drugs which interfere with the autophagic-lysosomal pathway. The accumulation of high-mannose glycoproteins in the lysosomal fraction was shown to be part of the general autophagic pathway constitutively expressed in undifferentiated cells, as independently measured by the sequestration of the cytosolic enzyme lactate dehydrogenase and electroloaded raffinose. Furthermore, when HT-29 cells were cultured under differentiation-permissive conditions, the decreased accumulation of high-mannose glycoproteins in the lysosomal compartment was correlated with the decrease in autophagy.
Complete nucleotide sequence of the gene encoding bacteriophage E endosialidase: implications for K1E endosialidase structure and function
Authors: Long G S , Bryant J M , Taylor P W , Luzio J P
Published: August 2015

Bacteriophage E specifically recognizes and infects strains of Escherichia coli which display the alpha-2,8-linked polysialic acid K1 capsule. Bacteriophage E endosialidase, which is thought to be responsible for initial absorption of the phage to the host bacterium, was purified, and the N-terminal amino acid sequences of the polypeptide monomer and cyanogen bromide fragments were determined. Synthetic oligonucleotide probes were designed from the N-terminal amino acid sequences and used to identify restriction fragments of bacteriophage E DNA encoding the endosialidase. The primary nucleotide sequence of the bacteriophage E endosialidase gene contains an open reading frame encoding a 90 kDa polypeptide which is processed to give a mature 74 kDa protein. The native enzyme is probably a trimer of identical 74 kDa subunits. In the bacteriophage E genome the K1E endosialidase open reading frame is preceded by a putative upstream promoter region with homology to a bacteriophage SP6 promoter. A central region of 500 amino acids of the deduced protein sequence of the K1E endosialidase was found to have 84% identity to K1F endosialidase. Both endosialidases contain two copies of a sialidase sequence motif common to many bacterial and viral sialidases. These sequences flank the region of greatest identity between the two endosialidase forms, which suggests that this central domain is involved in binding and hydrolysis of the polysialic acid substrate.
Cell cycle-dependent regulation of elastin gene in cultured chick vascular smooth-muscle cells
Authors: Wachi H , Seyama Y , Yamashita S , Tajima S
Published: August 2015

A study was made of the relationship between elastin expression and the proliferative state of chick vascular smooth-muscle cells. Confluent cells of primary culture brought to a quiescent state by the deprivation of serum for 72 h exhibited a 5-, 3.5- and 2-fold increase in elastin synthesis, elastin mRNA level and transcriptional activity of elastin gene respectively over those in the proliferative state. On re-addition of serum in serum-deprived culture, cells started to proliferate, and elastin synthesis, its mRNA level and transcription of the gene decreased to the level of a proliferative state within 24 h, indicating that elastin expression in smooth-muscle cells was controlled by their growth states at least in part at a transcriptional level. A comparable increase in elastin mRNA level was observed when the cell growth was arrested by suspension culture for 72 h. When the cells were synchronized at the G1/S phase with thymidine/hydroxyurea treatment, elastin expression at the G1/S phase was greater than that at the G2/M phase during cell cycling. Elastin mRNA level at the G0 phase brought about by serum-deprivation or suspension culture predominated over that at the G1/S phase during cell cycling. These results indicate that gene expression of elastin and cell cycle are tightly coupled, which is independent of the presence of serum or adhesive state, and that elastin expression could be a biochemical marker for the growth states of smooth-muscle cells.
Synthesis and release of hyaluronic acid by Swiss 3T3 fibroblasts
Authors: Kitchen J R , Cysyk R L
Published: August 2015

Hyaluronic acid (HA) and its synthesis were studied in intact Swiss 3T3 mouse fibroblasts and isolated membranes. HA chains in culture medium, attached to cells and in isolated membranes, were determined to possess average M(r) values of 5.2 x 10(6), 1.8 x 10(6) and 0.14 x 10(6) respectively. Log cells were determined to possess 680,000 HA molecules/cell, and to release 120,000 HA chains/h. The time required for intact cells to synthesize and release a complete HA chain was approximately 4 h, with elongation proceeding at a rate of 57 dimers/min. The amount of cell-associated HA of various cell populations correlated strongly with their rate of HA release into culture media and with the HA synthetase activity determined for their membranes. Prevention of protein synthesis with cycloheximide decreased the rate of HA synthesis of log cells and HA synthetase activity of isolated membranes by 50% within 2-3 h. Because of the similarity between the biological lifetime of HA synthetase and the time required to synthesize a HA chain, we propose a model where each synthetase makes only one HA chain; after synthesis of a complete HA chain, HA synthetase activity is terminated as its HA chain is released from the cell.
Spermine induces haemoglobin synthesis in murine erythroleukaemia cells
Authors: Delcros J G , Schwartz B , Clément S , Basu H S , Marton L J , Feuerstein B G
Published: August 2015

The naturally occurring polyamine spermine induces haemoglobin synthesis in murine erythroleukaemia (MEL) cells. Haemoglobin production was accompanied by accumulation of cytoplasmic beta-globin mRNA and growth inhibition, but not by cell-cycle block or changes in cell volume. Hexamethylene-bisacetamide (HMBA), a well known differentiating agent, also induces haemoglobin production, but causes a G1 block and decreases cell volume. These findings indicate that HMBA and spermine affect MEL cells differently, even though both induce haemoglobin production.
Reduction of ubiquinone in membrane lipids by rat liver cytosol and its involvement in the cellular defence system against lipid peroxidation
Authors: Takahashi T , Yamaguchi T , Shitashige M , Okamoto T , Kishi T
Published: August 2015

Rat liver homogenates reduced ubiquinone (UQ)-10 to ubiquinol (UQH2)-10 in the presence of NADPH rather than NADH. This NADPH-dependent UQ reductase (NADPH-UQ reductase) activity that was not inhibited by antimycin A and rotenone, was located mainly in the cytosol fraction and its activity accounted for 68% of that of the homogenates. Furthermore, the NADPH-UQ reductase from rat liver cytosol efficiently reduced both UQ-10 incorporated into egg yolk lecithin liposomes, and native UQ-9 residing in rat microsomes, to the respective UQH2 form in the presence of NADPH. The gross redox ratios of UQH2-9/(UQ-9 + UQH2-9) in individual tissues of rat correlated positively with the log of their respective cytosolic NADPH-UQ reductase activities, while the redox ratios in every intracellular fraction from liver were at about the same level, irrespective of NADPH-UQ reductase activities in the respective fractions. The combined addition of rat liver cytosol and NADPH inhibited to a great extent 2,2′-azobis(2,4-dimethyl-valeronitrile)-induced lipid peroxidation of UQ-10-fortified lecithin liposomes and completely inhibited such peroxidation in the liposomes in which UQH2-10 replaced UQ-10. The NADPH-UQ reductase activity was clearly separated from DT-diaphorase (EC 1.6.99.2) activity by means of Cibacron Blue-immobilized Bio-Gel A-5m chromatography. In conclusion, the NADPH-UQ reductase in cytosol, which is a novel enzyme to our knowledge, was presumed to be responsible for maintaining the steady-state redox levels of intracellular UQ and thereby to act as an endogenous antioxidant in protecting intracellular membranes from lipid peroxidation that is inevitably induced in aerobic metabolism.
An upstream stimulatory factor (USF) binding motif is critical for rat preprotachykinin-A promoter activity in PC12 cells
Authors: Paterson J M , Morrison C F , Mendelson S C , McAllister J , Quinn J P
Published: August 2015

We demonstrate the presence of a functional E box motif in the proximal rat preprotachykinin-A (rPPT) promoter. This element (spanning nucleotides -67 to -47) exhibits the sequence 5′-CACGTG-3′ which is recognized and bound by the basic helix-loop-helix family of regulatory proteins. We also show that at least one of the factors bound to this rPPT promoter element in both HeLa and PC12 nuclear extract is the ubiquitously expressed transcription factor, the upstream stimulatory factor (USF). Mutation of this element by insertion of a 10 bp linker into the E box motif, in an rPPT promoter fragment spanning -865 to +92, destroys the ability of this promoter fragment to support reporter gene expression in a PC12 cell model of rPPT promoter activity. The data indicate that this rPPT E box element is likely to function as an important cis-regulatory domain in the rPPT promoter.