bluuebunny/crossref_metadata_2025_split huggingface dataset:
Purification and Characterization of a Pig Intestinal α‐Limit Dextrinase
Authors: TARAVEL Francois R. , DATEMA Roelf , WOLOSZCZUK Wolfgang , MARSHALL J. John , WHELAN William J. 
Published: July 2010

Mucosa from the duodenal and jejunal regions of pig small intestine was repeatedly freeze‐thaw treated to solubilize an enzyme preparation, enriched in maltase, glucoamylase and α‐limit dextrinase activities; isomaltase and sucrase remained essentially insoluble during the treatment.Chromatographic procedures, including ion‐exchange, gel filtration and hydroxylapatite chromatography of the solubilized preparation, brought to homogeneity an α‐glucosidase active towards maltose, α‐limit dextrins and starch in decreasing order, with only a very weak capacity to hydrolyse α‐1,6‐linkages.Michaelis constants and maximal velocities, as well as relative rates of hydrolysis of several substrates, including maltodextrins and α‐limit dextrins, were determined and served to characterize what seems to be a rather specific α‐1,4‐glucosidase.The participation of this enzyme in the hydrolysis of α‐limit dextrins and more generally in pathways for starch breakdown in the pig digestive tract is examined and discussed.
Selective Expression of Cloned Middle‐Repetitive Sequences in Nuclear RNA of Mouse Organs
Authors: KUROIWA Atsushi , OBINATA Masuo , NATORI Shunji 
Published: July 2010

Mouse middle‐repetitive sequences have been cloned in the bacterial plasmid pBR322. The DNA sequences, expressed only in nuclei, were screened by filter hybridization using nuclear RNA as probes. Several clones were expressed predominantly in the nuclear RNA in certain types of organs. The copy numbers of typical middle‐repetitive sequence clones in genomic DNA were analyzed quantitatively and results showed that 103–104 copies exist per haploid DNA, indicating that they belong to a relatively abundant class of repeated sequences. Homology within the family of each middle‐repetitive sequence ranged from 81% to 94%.The expression of cloned middle‐repetitive sequences was analyzed by liquid‐phase hybridization with nuclear RNAs from particular organs. The content of transcripts of the middle‐repetitive sequence in nuclear RNA of a particular organ was at least 1000 times higher than that in other organs. In steady‐state nuclear RNA these transcripts were calculated to amount to 102–104 copies per nucleus. Most of these transcripts seem to be retained in the nuclei and do not appear in the cytoplasm.
Glycogen Synthase from Rabbit Skeletal Muscle; Effect of Insulin on the State of phosphorylation of the Seven Phosphoserine Residues in vivo
Authors: PARKER Peter J. , CAUDWELL F. Barry , COHEN Philip 
Published: July 2010

Rabbits were starved for 24 h and injected with propranolol (2 mg/kg) with or without insulin (33 μg/kg) 15 min prior to sacrifice. In muscle extracts prepared from propranolol‐treated animals the activity ratio (± glucose 6‐phosphate) of glycogen synthase was 0.18 ± 0.02 and the Ka for glucose 6‐phosphate was 1.2 ± 0.1 mM. In (propranolol + insulin)‐treated animals the activity ratio was 0.35 ± 0.02 and the Ka for glucose 6‐phosphate was 0.60 ± 0.05 mM.Glycogen synthase purified to homogeneity from propranolol‐treated animals contained 2.74 ± 0.09 mol phosphate/mol subunit, whereas this value was 2.33 ± 0.09 mol phosphate/mol subunit from (propranolol + insulin)‐treated animals.The distribution of phosphate between the seven phosphorylation sites was elucidated. In (propranolol + insulin)‐treated animals there was a decrease of 0.4—0.45 mol phosphate/mol subunit in sites (3a + 3b + 3c), and no significant changes in any other site (1a, 1b, 2 and 5). The results demonstrate that dephosphorylation of sites (3a + 3b + 3c) is responsible for the activation of glycogen synthase observed after acute administration of insulin.Neither protein phosphatase 1 nor protein phosphatase 2A, which account for virtually all the glycogen synthase phosphatase activity in skeletal muscle extracts, were able to mimic the specific dephosphorylation of sites (3a + 3b + 3c) produced by insulin. Both enzymes dephosphorylated site 2 and sites (3a + 3b + 3c) in vitro at comparable rates. The action of insulin may therefore involve a decrease in the activity of glycogen synthase kinase 3 and the regulation of this enzyme is discussed.The failure of insulin to produce a significant dephosphorylation of the sites phosphorylated by cyclic‐AMP‐dependent protein kinase (1a, 1b and 2) demonstrates that inhibition of this protein kinase does not underlie the activation of glycogen synthase by this hormone. This conclusion is supported by measurements of the phosphorylation states of inhibitor 1, phosphorylase kinase and phosphorylase.Neither 24‐h starvation, nor alloxon‐induced diabetes, produced significant changes in the kinetic properties or phosphorylation state of glycogen synthase. Possible reasons for these findings are discussed.
Enzymatic methyl esterification of synthetic tripeptides: structural requirements of the peptide substrate
Authors: GALLETTI Patrizia , INGROSSO Diego , MANNA Caterina , SICA Filomena , CAPASSO Sante , PUCCI Piero , MARINO Gennaro 
Published: July 2010

Eukaryotic protein carboxyl methyltransferase catalyzes a two‐substrates reaction in which the methyl group of S‐adenosylmethionine is transferred to the free carboxyl group of D‐aspartyl and L‐isoaspartyl‐containing peptide or protein substrates. It has been previously shown that at least three binding sites are required for the interaction of adenosylmethionine with the enzyme and/or the protein substrate [Oliva A., Galletti P., Zappia V., Paik W. K. & Kim S. (1980) Eur. J. Biochem. 104, 595–602], while very little is know concerning the structural requirements of the protein substrate.In this study several synthetic tripeptides were selected in order to elucidate the structural requirements of the methyl‐accepting substrates. The results obtained with this series of peptides suggested that: (1) three residues appear to be the minimal length, so far identified, required for a productive enzyme‐substrate interaction, several dipeptides being ineffective as substrates [McFadden P. N. & Clarke S. (1986) J. Biol. Chem. 261, 11 503–11 511]; (2) the isoaspartyl residue is not recognized unless its α‐amino group is involved in a carboamide bond; (3) an hydrogen atom on the amide linkage following the isoaspartyl residue is essential for both recognition and catalysis; (4) oligopeptides containing both D‐aspartyl and D‐isoaspartyl residues are not recognized by this methyltransferase. On the basis of these results, interaction sites between the peptide substrate and the enzyme molecule have been proposed.This paper also reports the first application of fast‐atom‐bombardment mass spectrometry to the detection of the products of the enzymatic methyl esterification reaction. By this soft ionization technique, the methylesterified peptides as well as the corresponding cyclic imides generated during the spontaneous demethylation process have been identified.
Regulation of 70‐kDa Heat‐Shock‐Protein ATPase Activity and Substrate Binding by Human DnaJ‐Like Proteins, HSJ1a and HSJ1b
Authors: Cheetham Michael E. , Jackson Antony P. , Anderton Brian H. 
Published: July 2010

The DnaJ family of molecular chaperones is characterized by the presence of a highly conserved 70‐amino‐acid J domain. Escherichia coli DnaJ interacts with the 70‐kDa heat‐shock protein (DnaK), in vitro, to stimulate the 70‐kDa heat‐shock protein ATPase activity and modify substrate binding. The conservation of the interaction of DnaJ‐like proteins with the 70‐kDa heat‐shock proteins has been demonstrated for the yeast protein YDJ1, a protein that shows full domain conservation with E. coli DnaJ. Human neurone‐specific DnaJ‐like proteins, HSJ1a and HSJ1b, possess a J domain and a glycine/phenylalanine‐rich region in common with E. coli DnaJ, although the overall amino acid identity is less than 23%. We have investigated, in vitro, the interaction of HSJ1a and HSJ1b with the mammalian brain constitutive 70‐kDa heat‐shock protein (hsc70). The weak intrinsic ATPase activity of the constitutive 70‐kDa heat‐shock protein is enhanced more than fivefold by stoichiometric amounts of both HSJ1a and HSJ1b. This enhancement is mediated by an increase in the rate of bound ATP hydrolysis, whereas the rate of ADP release is unaffected.HSJ1 proteins appear to regulate the affinity of the 70‐kDa constitutive heat‐shock protein for the permanently unfolded substrate, carboxymethylated α‐lactalbumin. A recent report [Palleros, D. R., Reid, K. L., Shi, L., Welch, W. J. & Fink, A. L. (1993) Nature 365, 664–666] has suggested that substrate release by 70‐kDa heat‐shock proteins requires a conformational change in these proteins induced by K+ in concert with ATP binding. In the presence of ATP, HSJ1 proteins reduce 70‐kDa constitutive heat‐shock protein/carboxymethylated α‐lactalbumin complex formation both in the presence and absence of K+. This suggests that HSJ1 proteins induce a conformational change in the 70‐kDa constitutive heat‐shock protein that can mimic the effect mediated by K+ and therefore modulate 70‐kDa heat‐shock protein substrate release by another mechanism rather than merely stimulating the 70‐kDa heat‐shock protein ATPase activity. As HSJ1 proteins have limited similarity to DnaJ, we suggest that this action is being mediated by the J domain alone, and that this modulation of 70‐kDa heat‐shock‐protein substrate binding will be common to all proteins that contain a J domain.
Structure of Four Acidic Oligosaccharides from the Jelly Coat Surrounding the Eggs of Xenopus Laevis
Authors: Plancke Yves , Wieruszeski Jean‐Michel , Alonso Catherine , Boilly Benoni , Strecker Gérard 
Published: July 2010

Novel acidic oligosaccharides were released by reductive β‐elimination from the jelly coat eggs of the Anuran Xenopus laevis. According to the structural analysis of these oligosaccharide‐alditols, the following structures are proposed:
image

Where Kdn, 3‐deoxy‐d‐glycero‐d‐galactononulosonic acid.These results confirm the species specificity of the glycanic structures present in the secretion of amphibian oviducts, and may form the basis of a specific egg‐sperm recognition process.
Methanethiolation of the Liberated Cysteine Residues of Human α2‐Macroglobulin Treated with Methylamine Generates a Derivative with Similar Functional Characteristics as Native α2‐Macroglobulin
Authors: Jensen Poul Erik Hyldgaard , Shanbhag Vithaldas P. , Stigbrand Torgny 
Published: July 2010

The thiol‐modifying reagent methyl methanethiosulfonate reacts with the cysteine residues of thiol esters released upon treatment of human α2‐macroglobulin with methylamine. This methanethiolation generates a derivative of α2‐macroglobulin, with an ‘open trap’ and slow mobility in non‐denaturing PAGE, similar to native α2‐macroglobulin. This similarity is further substantiated by surface hydrophobicity determinations and by the fact that neither the derivative nor native α2‐macroglobulin are cleared from the circulation in mice. Cleavages of bait regions in the derivative and native α2‐macroglobulin, however, result in electrophoretically fast forms which are cleared from the circulation in mice.In contrast to native α2‐macroglobulin, which can bind 2 mol chymotrypsin/mol, α2‐macroglobulin treated with methylamine and methylmethanethiosulfonate binds only 0.8 mol chymotrypsin/mol. Protection of trypsin against inhibition by soybean trypsin inhibitor is significantly better when α2‐macroglobulin is modified by methylamine and methylmethanethiosulfonate than when it is modified by dinitrophenyl thiocyanate, which cyanylates the exposed thiol group.The methanethiolated derivative is also more stable than the corresponding cyanylated derivative in that it is transformed to an electrophoretically fast form with a half‐life of 9 h as compared to a half‐life of 7 h for the latter. The transformation to the fast form is not due to instability of the thiol modification.
Membrane Lipid Composition and Cell Size of Acholeplasma laidlawii Strain A are Strongly Influenced by Lipid Acyl Chain Length
Authors: Wieslander Åke , Nordström Susanne , Dahlqvist Anders , Rilfors Leif , Lindblom Göran 
Published: July 2010

The small, cell‐wall‐less prokaryote Acholeplasma laidlawii strain A‐EF22 could grow with membrane lipids having an average acyl chain length C̄n varying over 14.5– almost 20 carbons by exogenous supplementation with selected fatty acids. For 16 < C̄n < 18, the cells grew with lipids containing 100% (mol/100 mol) monounsaturated acyl chains, whereas for C̄n < 16 and En C̄n >18, cell growth only occurred with gradually lower fractions of unsaturated chains C̄n was actively increased and decreased by chain elongation or de novo fatty acid synthesis upon incorporation of short‐chain and long‐chain fatty acids, respectively.The membrane lipid composition was strongly affected by the acyl chain length and unsaturation, and the metabolic responses are readily explained as a regulation mechanism based on the established phase equilibria of the individual lipids in the A. laidlawii membrane. Monoglucosyldiacylglycerol (Glc‐acyl2Gro) was the dominating lipid with short chains but the fraction of this lipid decreased with increasing C̄n correlating with the decreasing lamellar to nonlamellar phase transition temperatures for this lipid. The fractions of diglucosyldiacylglycerol (Glc2‐acyl2Gro) and phosphatidylglycerol (PtdGro), forming lamellar phases only, increased with increasing C̄n over the entire chain‐length interval. A weaker correlation was usually observed between the relative amount of a lipid and the extent of chain unsaturation; however, the fractions of Glc2‐acyl2Gro and PtdGro increased clearly with an increasing degree of unsaturation. Moreover, the synthesis of the nonbilayer‐forming lipids acyl2Gro and monoacyl‐Glc‐acyl2Gro was strongly stimulated by a high degree of chain saturation. Concomitantly, the phase equilibria of Glc‐acyl2Gro are shifted towards lamellar phases at the growth temperature. The fraction of the three potentially nonbilayer‐forming lipids varied over 10–80% (mol/100 mol) total lipids as a function of the acyl chain composition. The combined molar fractions of the three phospholipids increased strongly with chain unsaturation. However, the fraction of phosphate moieties in the different lipids was constant over the entire chain‐length interval. It is concluded that the regulation of the membrane lipid composition aims at maintaining similar phase equilibria and surface charge densities of the lipid bilayer.The size of A. laidlawii cells was changed in a systematic manner and correlated qualitatively with the packing properties of the lipids. Cell diameters were increased by an increase in acyl chain length and saturation, and was affected by additives such an n‐dodecane and acyl2Gro.
Duodenase, a New Serine Protease of Unusual Specificity from Bovine Duodenal Mucosa
Authors: Zamolodchikova Tatyana S. , Vorotyntseva Tatyana I. , Antonov Vladymir K. 
Published: July 2010

In this paper, data are presented on purification and properties of a new serine endopeptidase (duodenase) isolated from bovine duodenum mucosa. The enzyme has been purified to homogeneity by combinations of ammonium sulphate fractionation, carboxymethyl‐cellulose 52 chromatography, and affinity chromatography on Sepharose 4B with Kunitz soybean trypsin inhibitor as a ligand. Some physicochemical properties of this protease have been investigated. The molecular mass of the purified duodenase was determined to be 29 ± 0.5 kDa by SDS/PAGE and G‐2000 SW column chromatography. The enzyme molecule is a single chain and the native enzyme is a monomeric protein. Its isoelectric point was estimated to be 10 ± 0.2. Duodenase has two forms (I and II) which possess similar properties but differ in their amino acid composition. The new protease is a glycoprotein and contains approximately 3.5% sugars.The enzyme displays trypsin‐like and chymotrypsin‐like activities and hydrolyzes the amide bonds of substrates having Lys, Arg, Tyr, Phe and Leu residues at the P1 position. Duodenase is most active at pH 7.9–8.2.Duodenase was irreversibly inhibited by diisopropylphosphofluoridate and phenylmethanesulphonyl fluoride, indicative of an activ‐site serine in this protease. α‐N‐Tosyl‐l‐lysine chloromethane and α‐N‐tosyl‐l‐phenylalanine chloromethane, which react with an active His, caused marked inhibition of trypsin‐like and chymotrypsin‐like activities of duodenase. The enzyme activity was strongly supressed by trypsin inhibitors from different sources (soybeans, bovine lungs and Lima beans). Chicken egg white ovomucoid had no effect on the duodenase activity.The N‐terminal sequence of the native duodenase (24 amino acid residues) shows high similarity with those of human and murine cytotoxic T‐lymphocyte granzymes, human leukocyte cathepsin G and rat mast cell chymases.The biological role of duodenase is discussed.
The Role of the Intralysosomal pH in the Control of Autophagic Proteolytic Flux in Rat Hepatocytes
Authors: Luiken Joost J. F. P. , Aerts Johannes M. F. G. , Meijer Alfred J. 
Published: July 2010

Current methods to estimate changes in intralysosomal pH in hepatocytes do not discriminate between lysosomes and other intracellular acidic compartments. To obtain selective information on the change in lysosomal function in response to a change in lysosomal pH we have used the liberation of fluorescent 4‐methoxy‐2‐naphthylamide from low concentrations of lysyl‐alanyl‐4‐methoxy‐2‐naphthylamide, B substrate of lysosomal dipeptidylpeptidase II. Using permeabilized and intact hepatocytes, the activity of this enzyme in response to manipulations meant to increase the intralysosomal pH was compared with intralysosomal protein degradation and with the accumulation of [14C]chloroquine as a pH indicator of intracellular acidic compartments. The data show that changes in intralysosomal pH are indicated by changes in dipeptidylpeptidase II activity and that these are mainly due to a pH‐dependent change in substrate accumulation in the lysosomes.Subsequently, the method was applied to establishing the extent to which an increase in intralysosomal pH can contribute to the inhibition of autophagic proteolysis in intact hepatocytes caused by a decrease in intracellular ATP, by an increase in amino acid concentration and by hypo‐osmotic cell swelling. The following observations were made. (a) Moderate changes in intracellular ATP do not affect the lysosomal pH. (b) Hypo‐osmotic cell swelling, which promotes inhibition of proteolysis by amino acids in freshly isolated hepatocytes, does not affect the lysosomal pH. (c) In addition to their known inhibitory effect on autophagic sequestration, amino acids (leucine in particular) can increase the lysosomal pH and thus inhibit intralysosomal protein degradation directly. (d) Low concentrations of the acidotropic agent methylamine increase the lysosomal pH without having an effect on autophagic proteolytic flux.It is concluded that autophagic proteolysis is not controlled by changes in the lysosomal pH.
Relationship between hexokinase and cytokinin in the regulation of leaf senescence and seed germination
Authors: Swartzberg D. , Hanael R. , Granot D. 
Published: July 2010

AbstractArabidopsis hexokinase (AtHXK1), an enzyme that catalyses hexose phosphorylation, accelerates leaf senescence, whereas the plant hormone cytokinin inhibits senescence. Previous work in our laboratory has shown that isopentenyl transferase (IPT), a key gene in the biosynthesis of cytokinin, expressed under promoters of the senescence‐associated genes SAG12 or SAG13 (PSAG12::IPT and PSAG13::IPT, respectively), inhibits leaf senescence in tomato plants. To study the relationship between hexokinase and cytokinin in the regulation of leaf senescence, we created and analysed double‐transgenic tomato plants expressing both AtHXK1 and either PSAG12::IPT or PSAG13::IPT. We found that expression of IPT in the double‐transgenic plants could not prevent the accelerated senescence induced by over‐expression of AtHXK1. Since cytokinin inhibits senescence via an apoplastic invertase that produces extracellular hexoses, whereas AtHXK1 is an intracellular mitochondria‐associated hexokinase, our results suggest that intracellular sugar sensing via AtHXK1 is dominant over extracellular sugar sensing with regard to leaf senescence. Interestingly, the heterologous SAG12 and SAG13 promoters are also expressed in germinating tomato seed, around the radicle penetration zone, suggesting that seed germination involves a senescence process that is probably necessary for radicle emergence. Indeed, seed expressing PSAG12::IPT and PSAG13::IPT exhibited delayed radicle emergence, possibly due to delayed endosperm senescence.
DOES A PATENT ACCESSORY PANCREATIC DUCT PREVENT ACUTE PANCREATITIS?
Authors: Tabata Taku , Kamisawa Terumi , Takuma Kensuke , Anjiki Hajime , Fujiwara Junko , Egashira Hideto , Koizumi Koichi , Egawa Naoto 
Published: July 2010

Background and Aim:  The role of the accessory pancreatic duct (APD) in pancreatic pathophysiology has been unclear. We previously examined the patency of the APD in 291 control cases who had a normal pancreatogram in the head of the pancreas by dye‐injection endoscopic retrograde pancreatography (ERP). APD patency was 43% and was closely related with the shape of the terminal portion of the APD. The present study aimed to clarify the clinical implications of a patent APD.Methods:  Based on the underlying data, the patency rate of the APD was estimated from the terminal shape of the APD on ERP in 167 patients with acute pancreatitis.Results:  In patients with acute pancreatitis, stick‐type APD, spindle‐type APD, and cudgel‐type APD, which showed a high patency, were rare, and branch‐type APD and halfway‐type or no APD, which showed quite low patency, were frequent in acute pancreatitis patients. Accordingly, the estimated patency of the APD in acute pancreatitis patients was only 21%. There was no significant relationship between the estimated APD patency and etiology or severity of acute pancreatitis.Conclusions:  The terminal shapes of the APD with low patency were frequent in acute pancreatitis patients, and estimated APD patency was only 21% in acute pancreatitis. A patent APD may function as a second drainage system to reduce the pressure in the main pancreatic duct and prevent acute pancreatitis.