bluuebunny/crossref_metadata_2025_split huggingface dataset:
Adeno‐Associated Virus RNA Transcription in vivo
Authors: MARCUS Carol J. , LAUGHLIN Catherine A. , CARTER Barrie J. 
Published: March 2005

We have studied RNA transcripts of the defective parvovirus, adeno‐associated virus (AAV) present in poly‐(A)rich and poly‐(A)free fractions of nuclear and cytoplasmic RNA prepared from cells infected together with a helper adenovirus. Cytoplasmic poly‐(A)rich RNA contains three overlapping spliced AAV RNAs having sizes of 3.9 × 103, 3.3 × 103 and 2.3 × 103 bases respectively. The nuclear precursors of these RNAs appear to be the coterminal unspliced poly(A)‐rich RNAs containing 4.2 × 103, 3.6 × 103 and 2.6 × 103 bases respectively. These unspliced RNAs were also found in the cytoplasm. The nuclear poly(A)‐free RNA contained a heterogenous population of AAV RNAs that were generally smaller than 2.3 × 103 bases. In addition, the Hirt pellet fraction of the nuclear RNA contained two discrete AAV poly(A)‐free RNAs having sizes of 2.5 × 103 and 2.8 × 103 bases.The 4.2 × 103 and 3.6 × 103‐base unspliced RNAs are more abundant than the coterminal 3.9 × 103 and 3.3 × 103‐base spliced RNAs whereas the 2.3 × 103‐base spliced RNA is much more abundant than the 2.6 × 103‐ base unspliced RNA. Thus, the cytoplasmic abundance of the AAV spliced RNAs appears to be controlled in part by the post‐transcriptional events of splicing or message stability.We also analysed the effects of AAV defective‐interfering genomes upon AAV transcription. These studies showed that when synthesis of standard AAV genomes was inhibited more than 10‐fold by defective‐interfering genomes there was no significant effect on the types or amounts of AAV RNA transcripts which accumulated. These observations indicate that interference by defective‐interfering genomes occurs mostly at the level of DNA replication rather than transcription.
Assembly of Cytochrome c. Apocytochrome c Is Bound to Specific Sites on Mitochondria before Its Conversion to Holocytochrome c
Authors: HENNIG Bernd , NEUPERT Walter 
Published: March 2005

Transport of apocytochrome c across the outer mitochondrial membrane and conversion to holocytochrome c were studied in vitro. Apocytochrome c was synthesized in a cell‐free homogenate from Neurospora crassa. Transfer in vitro was accomplished in a reconstituted system consisting of the postribosomal supernatant of the cell‐free homogenate and of isolated and purified mitochondria from Neurospora. The reconstituted system has the following characteristics:

Apocytochrome c is rapidly cleared from the supernatant and holocytochrome c appears in the mitochondria with the same kinetics. More than 80% of the apocytochrome c employed is converted to holocytochrome c. No transient accumulation of apocytochrome c is found in mitochondria.
The heme group becomes covalently linked to apocytochrome c in the reconstituted system as demonstrated by analysis of tryptic peptide maps of the apoprotein and holoprotein.
Deuterohemin added to the reconstituted system but not deuteroporphyrin inhibits the formation of holocytochrome c. This inhibition is reversed by protohemin.
In the presence of deuterohemin about half of the apocytochrome c remains in the supernatant; the other half becomes associated with the mitochondria. The latter portion is tightly bound and is specifically released upon incubation of the mitochondria with excess apocytochrome c. It is converted to holocytochrome c after addition of protohemin.

We conclude from these observations that apocytochrome c is transported across the outer mitochondrial membrane via receptor sites. In the presence of the heme analogue deuterohemin, binding to the receptor sites on the cytoplasmic surface of the outer mitochondrial membrane still takes place but translocation does not. The latter step is apparently coupled to the covalent linkage of the heme group. We suggest that the formation of the thioether bonds between apoprotein and heme is catalysed by an enzyme in the intermembrane space and that deuterohemin can compete with protohemin for binding to the enzyme. Finally, the data indicate that it is the heme group and not the porphyrin group which is coupled to the apoprotein.
Alterations Induced by a Prolonged Fasting: Opposite Effects on the β‐Adrenergic Receptor‐Coupled Adenylate‐Cyclase System and on Lipolysis in Fat Cells from Rat
Authors: GIUDICELLI Yves , LACASA Daniéle , AGLI Brigitte 
Published: March 2005

The maximal number of β‐adrenergic receptors in rat fat‐cell membranes from 72‐h‐fasted rats is about two‐times higher than in fed rats. However, the affinity of these receptors for β‐agonists and antagonists and the ability of guanosine 5′‐[β,γ‐imido]triphosphate (100 μM) to reduce the receptor binding affinity for β‐agonists are unaltered by fasting.
Basal and fluoride‐stimulated activities of adenylate cyclase are similar in adipocyte membranes from 72‐h‐ fasted and fed animals. However, relative to the basal activity, maximal stimulation by (—)‐isoproterenol is 33 ±5 %higher in fasted than in fed adipocytes. The sensitivity of adenylate cyclase to (—)‐isoproterenol is also increased in fat‐cell membranes from fasted animals, as the concentration of (—)‐isoproterenol required for half‐ maximal activation is three‐times lower in fasted than in fed adipocytes.
Basal adenylate cyclase activity is also about three‐times more sensitive to stimulation by guanosine 5′‐[β,γ‐imido]triphosphate in fasted than in fed adipocyte membranes. This increased sensitivity of adenylate cyclase to guanosine 5′‐[β,γ‐imido]triphosphate in fasting is much more pronounced when adenylate cyclase activity is stimulated by isoproterenol.
In contrast, although basal lipolysis is unaltered by fasting, lipolysis stimulated in vitro by isoproterenol, adrenocorticotropin or dibutyryladenosine 3′,5′‐monophosphate is severely depressed in fat cells from 72‐h‐fasted rats. However, the concentration of (—)‐isoproterenol and adrenocorticotropin required for half‐maximal activation of lipolysis are similar in fasted and fed adipocytes.

From this study, it is concluded that, contrary to the fasting‐induced decreased lipolytic responsiveness which is paradoxically observed in adipocytes in vitro, the fasting‐induced changes found in the adenylate cyclase‐ coupled‐β‐adrenergic receptor‐system of rat adipocyte are most probably physiologically relevant as these changes contribute to promote cyclic AMP synthesis and, consequently, to stimulate lipolysis, as is observed in vivo during fasting.
Ribosomal Protein S1 Associates with Escherichia coli Ribosomal 30‐S Subunit by Means of Protein‐Protein Interactions
Authors: BONI Irina V. , ZLATKIN Igor V. , BUDOWSKY Edward I. 
Published: March 2005

Ribosomal proteins S1 when associated with the 30‐S subunit does not interact with 16‐S RNA but its binding is determined mostly by protein‐protein interactions. These conclusions are based on the following data.

Ultraviolet irradiation (λ=254 nm) of the 30‐S subunit does not result in the covalent cross‐linking of S1 with 16‐S RNA at irradiation doses up to 150 quanta/nucleotide, whereas the irradiation under the same conditions of S1 · polynucleotide complexes [S1 · poly(U), S1 · poly(A) and S1 · Qβphage RNA] induces effective formation of polynucleotide‐protein cross‐links.
Mild treatment of 30‐subunits lacking S‐1 with RNase A or with cobra venom endonuclease results in removal of 10–20%of the total nucleotide material but does not affect their sedimentation characteristics or their S1 binding capacity.
The association of S1 with S1‐depleted 30‐S subunits is insensitive to aurintricarboxylic acid, which is known as a strong inhibitor of complex formation between S1 and polynucleotides.
Mild trypsin treatment of S1‐depleted 30‐S subunits greatly reduces their S 1 binding capacity.
Effect of Cations on the Acetylation of Chromatin in vitro
Authors: DOD Barbara , KERVABON Aline , PARELLO Joseph 
Published: March 2005

The effect of the differently charged cations Na+, Mg2+, spermidine and spermine on the acetylation of histones in vitro in soluble chromatin and in core particles was investigated. Up to a given concentration, which depends on the charge of the cation, all four cations activate the acetylation of these fractions. Above this critical concentration a gradual inhibition of the acetyltransferase activity occurs.Spermine, spermidine and Mg2+, but not Na+, affect the relative accessibilities of the acetyltransferase to the individual histones. As the concentration of these three polyvalent cations increases there is a gradual increase in the relative acetyl incorporation in histone H3 accompanied by a corresponding decrease in H4.Kinetic aspects of the system were also studied. A minor fraction of the soluble chromatin (mononucleosomes and dinucleosomes) which is preferentially digested by micrococcal nuclease is also acetylated preferentially. The role chromatin structure plays in determining the specificity of histone acetylation is discussed.
Primary Structure of the Glycans from Human Lactotransferrin
Authors: SPIK Gencviève , STRECKER Gérard , FOURNET Bernard , BOUQUELET Stéphane , MONTREUIL Jean , DORLAND Lambertus , VAN HALBEEK Herman , VLIEGENTHART Johdnnes F. G. 
Published: March 2005

The polypeptide chain of human lactotransferrin possesses two glycosylation sites to which glycans are linked through an N‐(β2‐aspartyl)‐N‐acetylglucosaminylamine bond and which are structurally heterogenous. After chymotryptic or pronase digestions, glycopeptides with five different glycan structures could be isolated. For three of them, the structure has been determined by the application of methanolysis, methylation analysis, hydra‐zinolysis/nitrous deamination, enzymatic cleavage and 1H‐NMR spectroscopy at 360 MHz: Glycopeptides A and B : NeuAc(α2‐6)Gal(β1‐4)GlcNAc(βl‐2)Man(α‐ 3)[NeuAc(α2‐6)GaI(β‐4)GlcNAc(β1 ‐2)Man(α1‐b)]Man‐ (β1‐4)GlcNAc(β1‐4)[Fuc(α1‐6)]GlcNAc(βl‐)Asn;Glycopeptide C : NeuAc(α2‐6)(Gal(β1‐4)GlcNAc(β1‐2)Man‐ (α1 ‐3){Gal(βl‐4)[Fuc(αl‐3)]GlcNAc(β1‐2)Man(α1‐6)}Man(β1‐4)GlcNAc(β1‐4)[Fuc(α1‐6)]GlcNAc(β1 ‐)Am. Glycopeptide D : NeuAc(α2‐6)Gal(β3‐4)GlcNAc(β1‐2)Man(α1‐3)[Gal(β1‐4)GlcNAc(βl‐2)Man(α1‐6)]Man(β1‐4)Glc‐ NAc(βI‐4)[Fuc(a1‐6)]GlcNAc(βI ‐)Am.Two other glycopeptides were obtained in very low amount and possess more complex structures.
Using 2′(3′)‐O‐Trinitrophenyl Derivatives of Adenine Nucleotides to Study the Structure and Mechanism of Functioning of Soluble Mitochondrial ATPase
Authors: KORMER Zinaida S. , KOZLOV Igor A. , MILGROM Yakov M. , NOVIKOVA Irina Yu. 
Published: March 2005

The 2′(3′)‐O‐trinitrophenyl (N3ph) derivatives of the adenine nucleotides are strong competitive inhibitors of isolated mitochondrial ATPase (factor F1). Ki decreases in the order N3phAdo >N3phAMP >N3phADP and is equal to 8 nM for N3phADP. Picric acid, which activates the ATPase reaction of factor F1 without changing the Km(app.), prevents the inhibiting action of N3phADP. At pH 7.6 the inhibition of factor F1 is accompanied by the binding of one molecule of N3phADP to a molecule of the enzyme. This binding leads to changes in the absorption spectrum, but not in the intensity of the fluorescence of the N3phADP.At pH 6.7 one or two molecules of N3phADP bind with the tight binding sites of factor F1. This binding is accompanied by the manifold enhancement of the fluorescence of N3phADP. The results obtained indicate that the sites of factor F1 that tightly bind nucleotides are immersed in the hydrophobic pocket of the protein molecule.
Purification and Characterization of Bovine Gastricsin
Authors: MARTIN Patrice , TRIEU‐CUOT Patrick , COLLIN Jean‐Claude , RIBADEAU DUMAS Bruno 
Published: March 2005

The results reported in the present paper and N‐terminal sequence homologies established by other authors strongly support the assumption that the gastric protease previously called bovine pepsin I or bovine pepsin B belongs to the aspartate protease group and corresponds to gastricsin (or pepsin C) (EC 3.4.23.3) from other species.Bovine gastricsin was prepared from commercial extracts of adult bovine vells by a procedure involving DEAE‐cellulose chromatography, gel filtration on Sephacryl S‐200 and a further DEAE‐cellulose chromatography. The preparation thus obtained was shown to be free of chymosin and bovine pepsin A by immunodiffusion, selective inactivation in urea and isoelectric focusing. Its molecular weight was estimated by gel filtration to be 32800.Bovine gastricsin displayed microheterogeneity on isoelectric focusing with pl values of the components ranging from 3.5 to 4.0. Chromatography of bovine gastricsin on hydroxyapatite separated three fractions, none of them being homogeneous by isoelectric focusing. Concanavalin‐A‐Sepharose 4B bound bovine gastricsin to some extent, but without any significant fractionation. Proteolytic activity could be detected directly on the isoelectric focusing gel for all the components of gastricsin and its fractions from hydroxyapatite and concanavalin‐A‐ Sepharose 4B.Bovine gastricsin and its fractions from hydroxyapatite have similar amino acid compositions, different from those of bovine chymosin and pepsin A but obviously related to those of human, simian and porcine gastricsins.Bovine gastricsin which is inactivated by reaction with diazoacetyl‐DL‐norleucine methyl ester and with 1,2‐epoxy‐(p‐nitrophenoxy)propane in a 1:1 and 1:2 stoichiometry, respectively, is able to hydrolyse a synthetic hexapeptide, Leu‐Ser‐Phe(NO2)‐Nle‐Ala‐Leu‐OMe, used as reference substrate for aspartate proteases, and exhibits a low activity towards N‐acetyl‐l‐phenylalanyl‐l‐diiodotyrosine. Its specific clotting activity with x‐casein as substrate is only half of that of chymosin and pepsin A.
The Nature of the Binding Site for Prothrombinase at the Platelet Surface as Revealed by Lipolytic Enzymes
Authors: BEVERS Edouard M. , COMFURIUS Paul , Robert F. A. ZWAAL  
Published: March 2005

The nature of the receptor for the prothrombinase complex at the surface of non‐activated platelets was investigated by measuring the platelet prothrombin‐converting activity with a chromogenic substrate assay, after treatment of the platelets with various phospholipases or three different proteolytic enzymes. Platelet pro‐thrombin‐converting activity only decreased after treatment with those phospholipases which are able to hydrolyse phospholipids in the intact platelet and also have the ability to degrade negatively charged phospho‐lipids, phosphatidylserine and phosphatidylinositol. Those phospholipases which do hydrolyse phospholipids in the intact platelet but have no activity towards phosphatidylserine (and phosphatidylinositol) produce an in‐ crease in the platelet prothrombin‐converting activity.Proteolytic treatment of platelets with trypsin, chymotrypsin or papain did not result in a decrease of pro‐thrombin‐converting activity. It is concluded that negatively charged phosphatidylserine and possibly phos‐phatidylinositol are involved in the prothrombin‐converting activity of non‐activated platelets. We could not demonstrate the involvement of platelet membrane proteins in a receptor for the components of the pro‐thrombinase complex.
Impaired Phosphorylation of Lysosomal Enzymes in Fibroblasts of Patients with Mucolipidosis III
Authors: HASILIK Andrej , WAHBED Abdul , CANTZ Michael , von FIGURA Kurt 
Published: March 2005

The incorporation of [3H]leucine and [32P]phosphate into three lysosomal enzymes, cathepsin D, β‐hexos‐aminidase and arylsulfatase A by fibroblasts from six patients affected with mucolipidosis III was determined. In the mutant cells the incorporation of 32P in the enzymes was reduced by 70‐97% as compared to controls. The residual phosphorylation of lysosomal enzymes is definitely higher than in fibroblasts from patients with mucolipidosis II, where apparently non‐phosphorylated enzymes are formed. In mucolipidosis III the major part of the newly formed enzymes accumulated extracellularly and the cellular enzymes were recovered mainly in their processed forms. In mucolipidosis III arylsulfatase A and the processed forms of cathepsin D exhibited a heterogeneity that was not observed in controls.β‐Hexosaminidase and cathepsin D secreted by mucolipidosis III fibroblasts contained only a small amount of phosphorylated oligosaccharides with either one or two phosphate groups per oligosaccharide. As in controls the major fraction of phosphate was present as acid‐labile phosphodiester resistant to alkaline phosphatase.The residual phosphorylation of lysosomal enzymes may be related to the partial intracellular retention and processing of these enzymes in fibroblasts from patients with mucolipidosis III.
Metabolite‐Controlled Phosphorylation of Phosphofructokinase in Rat Hepatocytes
Authors: BRAND Ingeborg A. , SÖLING Hans‐Dieter 
Published: March 2005

The effect of glucose on the phosphorylation of phosphofructokinase has been investigated in hepatocytes isolated froin fed and 24‐h‐starved rats by measuring the 32P radioactivity associated with the enzyme. In addition, the effects of gluconeogenic precursors and of glucagon or Bt2cAMP were followed under these conditions.The basal incorporation of phosphate into phosphofructokinase in hepatocytes from starved and fed rats was 0.2 ± 0.1 and 0.6±0.2 mol phosphate/mol phosphofructokinase (tetramer) respectively, whereas the maximal incorporation obtained was 1.03 ± 0.2 mol phosphate/mol phosphofructokinase.With 16 mM l‐lactate + 4 mM pyruvate or with 20 mM l‐alanine, the phosphorylation of the enzyme was lower than the basal incorporation found in hepatocytes from fed and starved rats.Glucose (20 mM) addition to isolated hepatocytes from fed rats resulted in an increase in the incorporation phosphate into phosphofructokinase to almost maximal value.In hepatocytes isolated from starved rats, glucose led to a 2.5‐fold increase of phosphorylation as compared to the basal incorporation. l‐Alanine (20 mM) abolished the glucose‐induced phosphorylation in hepatocytes from starved and from fed animals.Glucagon (0.1 μM) did not significantly enhance thc phosphorylation of phospliofructokinase in the presence of l‐lactate + pyruvate in hepatocytes from starved rats, but stimulated the phosphorylation to maximal value when cells had been preincubated in the presence of glucose.In contrast, in liver cells isolated from fed rats. glucagon had no additional effect on phosphofructokinase pliosphorylation in the presence of glucose, but did increase the phosphorylation to maximal value when l‐lactate + pyruvate served as substrates. l‐Alanine inhibited the glucose‐induced phosphorylation of phosphofructokinase also in the presence of glucagon.In contrast, glucagon stimulated the phosphorylation of l‐type pyruvate kinase independently from exogenous precursors and from the nutritional state of the animals.Bt2cAMP (0.1 mM) as glucagon, did not significantly enhance the phosphorylation of phosphofructokinase above the level obtained with glucose alone in hepatocytes from fed rats, but did increase the velocity of the phosphate incorporation in hepatocytes from starved rats, thus leading under both conditions to the maximal incorporation.It is concluded that the degree of phosphorylation of rat liver phosphofructokinase is affected by the metabolic state, and that glucagon (or CAMP) acts indirectly, probably by changing glucose metabolism due to increased glycogenolysis rather than by an activation of a CAMP‐dependent protein kinase.
Covalent Crosslinking between Molecules of Type I and Type III Collagen
Authors: HENKEL Werner , GLANVILLE Robert W. 
Published: March 2005

Several peptides containing intermolecular crosslinks were isolated from tryptic digests of the insoluble matrix of human leiomyoma and calf aorta. From leiomyoma type III collagen, two crosslinked peptides were isolated. The peptide [TN(III)]2, is a dimer which contains the 73 amino‐terminal residues of two, zero‐D staggered, crosslinked α1(III) chains (D being the distance by which molecules or chains are staggered relative to each other). After de‐blocking with pyroglutamate aminopeptidase, its amino‐terminal amino acid sequence was determined to be < Glu‐Tyr‐Asp‐Ser‐Tyr‐Asp‐Val‐Xaa‐Ser‐Gly‐Val‐Ala‐Val‐Gly‐Gly‐Leu‐Ala‐Gly‐Tyr‐Hyp‐Gly‐. The involvement of [TN(III)]2 in the formation of 4D‐staggered intermolecular bonds was demonstrated by sequence analysis of [TN(III)2]×T(III). For this three‐chained peptide, a double sequence which corresponded to TN(III) and T(III), a nine‐residue peptide from the carboxy‐terminal helical crosslinking site of type III collagen, was found.Crosslinks between type I and type III collagens were identified by isolating a peptide TN(III)×TN(I) from leiomyoma. This peptide was characterized, first by digesting it with collagenase giving rise to ColN(III)×ColN(I) and then chymotrypsin giving C(III) and C(I). C(I) and C(III) are peptides characteristic for type I and type III collagens, respectively. Further, three‐component crosslink‐containing peptides, from both type I and type III collagen, were isolated from aorta and leiomyoma. The peptides ColN(III)× ColN(I)× T(III) from leiomyoma and Col×N(III) ColN(I)× T(I) from aorta are crosslinked amino‐terminal regions of 0D‐staggered type I and type III molecules, joined either to an adjacent 4D‐staggered type III or type I molecule. Crosslinked peptides involving only type III collagen molecules were also isolated from calf aorta [colN(III)]2 and [CoIN(III)2]×T(III)].As intramolecular bonds between type I and III collagens can be excluded the isolation of these crosslinked peptides demonstrated that type I and type III collagen molecules are present within the same collagen fibrit with OD stagger, bound by intermolecular crosslinks. The significance of these findings with respect to the proposed models for collagen fibres and the physiological properties of the extracellular matrix, are discussed.
270‐MHz 1H Nuclear‐Magnetic‐Resonance Study of Met‐enkephalin in Solvent Mixtures
Authors: ZETTA Lucia , CABASSI Franco 
Published: March 2005

1H spectra at 270 MHz of zwitterionic Met‐enkephalin pentapeptide (Tyr‐Gly‐Gly‐Phe‐Met) in (C2H3)2SO/water mixtures are reported and discussed in terms of solvent‐induced conformational transitions.The analysis of the chemical shifts, line widths, coupling constants and rotamer populations around χ1and χ2 suggests that the conformational properties of Met‐enkephalin in the two solvents are quite different. In aqueous solution, the preferred structure, characterized by the absence of intramolecularly hydrogen‐bonded NH groups and head‐to‐tail interactions, very likely is an equilibrium of unfolded conformations with approximately equal energy. In (C2H3)2SO, the preferred structure is folded, with the Met‐5 NH intramolecularly bonded and the Gly‐3 NH protected from the solvent, while the Gly‐2 and Phe‐4 amide protons are solvent exposed.A conformational transition of Met‐enkephalin from the intramolecularly bonded to the unbonded one takes place at about 40 mol‐% water in (C2H3)2SO, involving the Met‐5 NH proton and the Tyr‐Gly‐Gly fragment. The Phe‐4 and Met‐5θ angles do not change appreciably, which suggests that an inversion at the Gly‐3 residue of the folded form, responsible for the conformational transition, does not affect the C‐terminal moiety.At about 70 mol‐% water in (C2H3)2SO a change in the solvent mixture properties affects the χ1 rotamer populations and the ring dynamics of the aromatic side chains. The line broadening of the Tyr‐1 δ and ɛ proton resonances indicates a specific interaction of the N‐terminal ring with the solvent.