bluuebunny/crossref_metadata_2025 huggingface dataset:
Expression of Myosin Isoforms in Denervated, Cross‐Reinnervated, and Electrically Stimulated Rabbit Muscles
Authors: Bacou Francis , Rouanet Philippe , Barjot Catherine , Janmot Chantal , Vigneron Pierre , D'albis Anne
Published: August 2004

The expression of myosin heavy (MyHC) and light (MyLC) chain isoforms was analyzed after denervation and cross‐reinnervation by a fast nerve of the slow‐twitch Semimembranosus proprius (SMp) muscle, and after denervation and electrical stimulation at low frequency of the fast‐twitch Semimembranosus accessorius (SMa) muscle of the rabbit. The control SMp (100% type I fibers) expressed 100% type I MyHC and 100% slow‐type (1S′, 1s and 2S) MyLC isoforms. Five month denervation did not alter significantly the MyHC expression of the muscle, but induced the expression of a new type I MyLC corresponding most probably to an embryonic MyLC. Five‐month cross‐reinnervation of the SMp by the fast SMa nerve induced a large change of its fiber type properties. As shown by immunocytochemistry, almost all fibers were stained by fast myosin antibody, but a high proportion of them co‐expressed slow myosin. This result was in agreement with biochemical data showing that fast MyHC and MyLC isoforms became predominant. The control SMa (nearly 100% type 11 fibers) expressed almost 100% type II MyHC (70% type IIb and 22% IIx/d) and 100% fast‐type (1F, 2F and 3F) MyLC isoforms. Five month denervation of the SMa induced a shift in its MyHC, with 98 % type IIdd and 2% type IIb isoforms, and no change in the proportions of its MyLC. Three month electrical stimulation at 10 Hz of the SMa transformed its fiber type composition. All fibers reacted with the slow myosin antibody and a minor proportion of them were stained by the fast myosin antibody. These observations were in agreement with the biochemical analysis showing a large predominance of the slow‐type MyHC and MyLC isoforms. Taken together, these results obtained from rabbit muscles which are normally homogeneous in either fast‐twitch or slow‐twitch fiber types, further support the idea that the different myosin isoforms, particularly the MyHC, are differentially regulated by motor innervation. Type I MyHC is maintained in denervated SMp muscle, but is not expressed in denervated SMa. Type IIb isoform is the most sensitive to neural influence, as it disappears rapidly in denervated and electrically stimulated fast‐twitch SMa muscle, and is barely expressed in cross‐reinnervated slow‐twitch SMp muscle. In contrast, type IIa and type IIdd are less dependent upon motor innervation. In addition to the previous studies of d'Albis et al. [d'Albis, A., Gouble, F., Couteaux, R., Jannot, C. & Mira, J. C. (1994) Eur. J. Biochem. 223, 241–258], analysis of these results leads us to conclude that, in the rabbit, sensitivity to motor innervation increases from the glycolytic to the oxydative types of fibers, in the order IIB > IIX/IID > IIA > I.
Protease Inhibitor Studies and Cloning of a Serine Carboxypeptidase cDNA from Germinating Seeds of Pea (Pisum sativum L.)
Authors: Jones Craigh G. , Lycett Grantley W. , Tucker Gregory A.
Published: August 2004

The nature of the proteolytic activity found within the germinating pea (Pisum sativum) seed, 4 days from the initiation of imbibition, was determined by the use of specific protease inhibitors. These studies have shown most of the activity to belong to metallo or metal‐activated arid serine proteases. In order to investigate further the serine protease activity, a pea cotyledon germination cDNA library was, therefore, screened with a wheat cDNA (2437) [Baulcombe, D. C., Barker, R. F. & Jarvis, M. G. (1987) J. Biol. Chem. 262, 13726–13735] which had extensive similarity to the yeast serine carboxypeptidase Y gene. A positive cDNA clone (pNY551) was obtained which had extensive similarity to the four carboxypeptidases, Arabidopsis thaliana carboxypeptidase Y‐like protein, rice serine carboxypeptidase III, barley serine carboxypeptidase III and wheat serine carboxypeptidase III precursor. Northern‐blot analysis showed mRNA homologous to pNY551 to be expressed in late developmental pea seed and again during germination.
Structural and Functional Characterization of Two Mutated R2 Proteins of Escherichia coli Ribonucleotide Reductase
Authors: Larsson Åke , Climent Isabel , Nordlund Pär , Sahlin Margareta , Sjöberg Britt‐Marie
Published: August 2004

The R2 protein of ribonucleotide reductase from Escherichia coli is a homodimeric tyrosyl‐radical‐containing enzyme with two identical dinuclear iron centers. Two randomly generated genomic mutants, nrdB‐1 and nrdB‐2, that produce R2 enzymes with low enzymatic activity, have been cloned and characterized to identify functionally important residues and areas of the enzyme. The mutations were identified as Pro348 to leucine in nrdB‐1 and Leu304 to phenylalanine in nrdB‐2. Both mutations are the results of single amino acid replacements of non‐conserved residues. The three‐dimensional structures of [L348]R2 and [F304]R2 have been determined to 0.26‐nm and 0.28‐nm resolution, respectively. Compared with wild‐type R2, [L348]R2 binds with higher affinity to Rl, probably due to increased flexibility of its C‐terminus. Since the three‐dimensional structure, iron‐center properties and radical properties of [L348]R2 are comparable to those of wild‐type R2, the low catalytic activity of the holoenzyme is probably caused by a perturbed interaction between R2 and Rl. The [F304]R2 enzyme has increased radical sensitivity and low catalytic activity compared with wild‐type R2. In [F304JR2 the only significant change in structure is that the evolutionary conserved Ser211 forms a different hydrogen bond to a distorted helix. The results obtained with [F304]R2 indicate that structural changes in E. coli R2 in the vicinity of this helix distortion can influence the catalytic activity of the holoenzyme.
Degradation of the Light‐Stress Protein is Mediated by an ATP‐Independent, Serine‐Type Protease Under Low‐Light Conditions
Authors: Adamska Iwona , Lindahl Marika , Roobol‐Bóza Margrit , Andersson Bertil
Published: August 2004

Green plants respond to light stress by induction of the light‐stress proteins (ELIPs). These proteins are stable as long as the light stress persists but are very rapidly degraded during subsequent low light conditions [Adamska, I., Kloppstech, K. & Ohad, I. (1993) J. Biol. Chem. 268, 5438–5444]. Here we report that the degradation of ELIPs is mediated by an extrinsic, thylakoid‐associated protease which is already present in the membranes during light stress conditions. Partial purification of the protease by perfusion chromatography indicates that this proteolytic activity may be represented by a protein with an apparent molecular mass of 65 kDa. The ELIP‐directed protease is localized in the stroma lamellae of the thylakoid membranes and does not require ATP or additional stromal factors for proteolysis. The protease has an optimum activity at pH 7.5–9.5 and requires Mg2+ for its activity. The ELIP‐degrading protease show an unusual temperature sensitivity and becomes reversibly inactivated at temperatures below 20°C and above 30°C. Studies with protease inhibitors indicate that this enzyme belongs to the serine class of proteases. The enhanced degradation of ELIP in isolated thylakoid membranes after addition of the ionophore nigericin suggests that a trans‐thylakoid pH or changes in ionic strength may be involved in the mechanism of protease activation.
In vitro Protein Folding by Ribosomes from Escherichia coli, Wheat Germ and Rat Liver
Authors: Das Biswadip , Chattopadhyay Subrata , Bera Aloke Kumar , DasGupta Chanchal
Published: August 2004

Ribosomes from a number of prokaryotic and eukaryotic sources (e.g., Escherichia coli, wheat germ and rat liver) can refold a number of enzymes which are denatured with guanidine/HCl prior to incubation with ribosomes. In this report, we present our observations on the refolding of denatured lactate dehydro‐genase from rabbit muscle and glucose‐6‐phosphate dehydrogenase from baker's yeast by ribosomes from E. coli, wheat germ and rat liver, The protein‐folding activity of E. coli, ribosomes was found to be present in 50s particles and in 23S rRNA. The 30S particle or 16S rRNA did not show any protein‐folding activity. The protein‐folding activity of 23S rRNA may depend on its tertiary conformation. Loss of tertiary structure, by incubation with low concentrations of EDTA, inhibited the protein‐folding activity of 23S rRNA. This low concentration of EDTA had no effect on folding of the denatured enzymes by themselves.
Backbone Dynamics of the 269‐residue Protease Savinase Determined from 15N‐NMR Relaxation Measurements
Authors: Remerowski M. Lyndsay , Pepermans Henri A. M. , Hilbers Cornelis W. , Van De Ven Frank J. M.
Published: August 2004

Backbone dynamics of Savinase, a subtilisin of 269 residues secreted by Bacillus lentus, have been studied using 15N relaxation measurements derived from proton‐detected two‐dimensional 1H‐15N‐NMR spectroscopy. 15N spin‐lattice rate constants (R1), spin‐spin relaxation‐rate constants (R2), and 1H‐15N nuclear Overhauser effects (NOE) were determined for 84% of the backbone amide 15N nuclei. The model‐free formalism [Lipari, G. & Szabo, A. (1982) J. Am. Chem. Soc. 104, 4544–4559] was used to derive values for a generalized order parameter, S2, interpretable as a measure of the amplitude of motion on the picosecond‐nanosecond timescale, for each N‐H bond vector. Additional terms used to fit the data include an effective correlation time for internal motions (τe) and an exchange term (Rex) to account for exchange contributions to R2. The overall rotational correlation time (τm) is 9.59±0.02 ns: the average order parameter (S2) is 0.90±0.07, indicative of a rigid structure consistent with Savinase's high degree of secondary structure and compact tertiary fold. Residues S125–S128, located in the substrate‐binding region, represent the longest stretch of protein which exhibits disorder on the picosecond–nanosecond timescale. These residues also exhibit significant exchange terms, possibly indicative of motion on the microsecond–millisecond timescale, which could also be influenced by the proximity of the phenyl ring of the substituted aryl boronic acid inhibitor used in this study. S103 and G219 in the substrate‐binding region also show flexibility on the picosecond–nanosecond timescale. There is also significant motion in the turn, G258–T260, of a small solvent‐exposed loop region which may make the protein vulnerable to autolysis at that point. Some residues in both calcium‐binding sites and nearby also show mobility.
Phosphate‐binding Sites in Phosphorylating glyceraldehyde‐3‐phosphate Dehydrogenase from Bacillus stearothermophilus
Authors: Michels Solange , Rogalska Ewa , Branlant Guy
Published: August 2004

The two anion‐binding sites of the glycolytic glyceraldehyde‐3‐phosphate dehydrogenase (GraP ‐DH), the Ps and Pi sites, were originally proposed by Moras et al. [Moras, D., Olsen, K. W., Sabesan, M. N., Buehner, M., Ford, G. C. & Rossmann, M. G. (1975) J. Biol. Chem. 250, 9137–9162] to bind the C3 phosphate of the glyceraldehyde 3‐phosphate and the inorganic phosphate respectively. Ps site mutants T179A, and T179M, and R231L, and the Pi site mutants T150A and T208A of the Bacillus stearothermophilus GraP ‐DH were constructed by site‐directed mutagenesis and their kinetic properties were determined and compared with those of mutants R195L and R231G, already described [Corbier, C., Michels, S., Wonacott, A. & Branlant, G. (1994) Biochemistry 33, 3260–3265]. Taking advantage of the opportunity to study both the oxidoreduction and the phosphorylation step independently and the fact that the phosphorylation becomes rate determining for most of the mutants, the relative energetic contribution of each mutated amino acid to the phosphorylation step was evaluated. It was concluded that (a) Ps amino acids contribute more than the Pi amino acids to the stabilisation of the transition state relative to the ground state and (b) the side chain of arginine contributes more than that of the threonine residue. It was also concluded that the differences observed in the efficiency of the phosphorylation step for Ps and Pi mutants is a consequence of the orientation of the thioester bond of the thioacyl intermediate relative to the attacking inorganic phosphate and not of a change in the intrinsic electrophilic property of the thioacyl intermediate. Furthermore, the kinetic results on the overall steps leading to the acyl‐enzyme formation provided supplementary evidence that the C3 phosphate moiety of the glyceraldehyde 3‐phosphate interacts with the Pi site during these steps and thus are consistent with the findings of Skarzynski et al. [Skarzynski, T., Moody, P. C. E. & Wonacott, A. J. (1987) J. Mol. Biol. 193, 171–183] and Corbier et al. [Corbier, C., Michels, S., Wonacott, A. & Branlant, G. (1994) Biochemistry 33, 3260–3265] that recommended the reconsideration of the first definition of the Ps and Pi sites.
Enlarged Scale Chemical Synthesis and Range of Activity of Drosocin, an O‐Glycosylated Antibacterial Peptide of Drosophila
Authors: Bulet Phippe , Urge Laszlo , Ohresser Serge , Hetru Charles , Otvos Laszlo
Published: August 2004

Insects respond to a bacterial challenge by rapidly synthesizing a diverse range of antibacterial and antifungal peptides. One of them, drosocin, a 19‐residue prone‐rich antibacterial peptide, was isolated from Drosophila. This peptide carries a disaccharide moiety attached to a threonine residue in mid‐chain position. The present report describes the enlarged‐scale chemical synthesis of drosocin, glycosylated with Gal(β1→3)GalNAc(αL→O). We have studied the range of activity of the synthetic glycopeptide, of two truncated glycosylated isoforms, and of the unglycosylated L and D enantiomers. Both isolated and chemically synthesized drosocins carrying the disaccharide display the same antibacterial activity. Using circular dichroic spectroscopy we demonstrated that the O‐nked disaccharidic motif did not affect the backbone conformation of drosocin. The antibacterial activity of the synthetic glycopeptide was directed against gram‐negative strains with the exception of the gram‐positive bacteria Micrococcus luteus. Deletion of the first five N‐terminal residues completely aboshed the activity of drosocin. As a first approach to the study of the mode of action of drosocin, we have synthesized a non‐glycosylated d enantiomer and, using this molecule, we have shown that drosocin may act on the gram‐negative bacteria through a stereospecific target.
Translocation of Microfilament‐Associated Inhibitory guanine‐nucleotide‐binding Proteins to the Plasma Membrane in Myeloid Differentiated Human Leukemia (HL‐60) Cells
Authors: Heringdorf Dagmar Meyer Zu , Liedel Klaus , Kaldenberg‐Stasch Sylvia , Michel Martin C. , Jakobs Karl H. , Wieland Thomas
Published: August 2004

The cytoskeletal localization of inhibitory guanine‐nucleotide‐binding (Gi) proteins and the coupling of these proteins to formyl peptide receptors were studied in myeloid differentiated human leukemia (HL‐60) cells. Treatment of HL‐60 cells with cytochalasin B or botulinum C2 toxin, which leads to the disruption of microfilaments, increased the binding of the stable GTP analogue guanosine 5′‐[γ‐thio]triphosphate (GTP[S]) to permeabilized cells by about 30%. In contrast, the microtubule‐disrupting agents colchicine and vinblastine, and cytochalasin B treatment of isolated HL‐60 membranes did not affect GTP[S] binding. The stimulatory effect of cytochalasin B treatment was concentration and time dependent, with maximal increases observed at 5 μg/ml cytochalasin B and an incubation time of 10 min, and was counteracted by the F‐actin‐stabilizing toxin phalloidin. Cytochalasin B treatment increased the amount of G proteins activated by chemoattractant receptors by about 25%. Furthermore, the number of Gi‐protein‐coupled receptors for the chemoattractant, N ‐formyl‐Met‐Leu‐Phe, was increased by about 25% upon cytochalasin B treatment. Based on these functional data, which suggest an association of G proteins with actin filaments, the Triton X‐100 (1%)‐insoluble cytoskeleton was analyzed for the presence of G proteins. Giα subunits were detected in the cytoskeleton preparations, both by specific antisera and by pertussis‐toxin‐catalyzed ADP‐ribosylation. Cytochalasin B pretreatment depleted the cytoskeleton in Giα, with an approximately 20% concomitant increase in membrane Giα content. In conclusion, evidence is presented that part of the cellular Giα is localized at actin filaments in HL‐60 cells. After filament disruption, these Giα subunits seem to be translocated to the plasma membrane, where they can productively interact with chemoattractant receptors.
Differential Developmental and Tissue‐Specific Regulation of Expression of the Genes Encoding Three Members of the Flavin‐Containing Monooxygenase Family of Man, FMO1, FMO3 and FMO4
Authors: Dolphin Colin T. , Cullingford Timothy E. , Shcphard Elizabeth A. , Smith Robert L. , Phillips Ian R.
Published: August 2004

We have previously described the isolation and, sequencing of cDNA clones encoding thin‐containing monooxygenases (FMOs) 1 and 4 of man [Dolphin, C., Shephard, E. A., Povey. S., Palmer, C. N. A., Ziegler, D. M., Ayesh, R., Smith, R. L. & Phillips, I. R. (1991) J. Biol. Chem. 266, 12379–12385; Dolphin, C., Shephard, E. A., Povey, S., Smith, R. L. & Phillips, I. R. (1992) Biochem. J. 287, 261–267]. We present here the isolation of a cDNA for FMO3 of man. The sequence of this cDNA and the atnino acid sequence deduced from it differ substantially from those previously reported for this member of the FMO family of man. In addition, we have investigated, by quantitative RNase protection assays, the expression in several foetal and adult human tissues of genes encoding FMO1, FMO3 and FMO4. Our results demonstrate that, in the adult, FMO1 is expressed in kidney but not in liver, whereas in the foetus it is expressed in both organs. The lack of expression of FMO1 in adult human liver is in marked contrast to the situation in other mammals, such as pig and rabbit, in which FMO1 constitutes a major form of the enzyme in the liver of the adult animal. The mRNA encoding FMO3 is abundant in adult liver and is also present, in low abundance, in some foetal tissues. Thus, FMO1 and FMO3 are both subject to developmental and tissue‐specific regulation, with a developmental switch in the expression of the genes taking place in the liver. FMO4 mRNA is present in low abundance in several foetal and adult tissues and thus the corresponding gene appears to be expressed constitutively.
Targeting of Porcine Pancreatic Phospholipase A2 to Human Platelets
Authors: Bekkers Augustinus C. A. P. A. , Slotboom Arend J. , Van Willigen Gijsbert , Akkerman Jan Willem N. , Verheij Hubertus M.
Published: August 2004

In the present study we prepared by chemical modification a series of porcine pancreatic phospholipase A2 (PLA) derivatives, that bind to the activated glycoprotein (GP) IIb/IIIa complex and hydrolyse phospholipids in the outer leaflet of the platelet membrane. To the native enzyme, an RGD‐containing peptide was coupled to introduce affinity for GPIIb/IIIa in combination with lauric acid to improve binding to the membrane. As controls, derivatives containing only one of these modifications were prepared. Acylation of the enzyme improved the affinity for densely packed phospholipids, as deduced by kinetic analyses. After stimulation of platelets, the RGD‐containing PLAs bound to GPIIb/IIIa since GRGDS peptide and a monoclonal antibody against the complex interferred with binding. No binding was found with native PLA. The binding seen with lauric acid PLA was not mediated by GPIIb/IIIa. All modified PLAs induced 1–3% hydrolysis of [3H]arachidonic‐acid‐labelled phospholipids in resting platelets. After activation with α‐thrombin, hydrolysis increased to 17%, corresponding to about 90% of [3H]arachidonate‐labelled phospholipids in the outer leaflet of the plasma membrane. RGD‐containing PLAs were more active than lauroyl PLA, and their activity was mediated via GPIIb/IIIa since GRGDS inhibited release of [3H]arachidonic acid. Acylation of the RGD‐containing PLAs did not further improve the hydrolytic properties. We conclude that chemical modification of PLA leads to a targetted hydrolytic action and could be a basis for the design of enzymes that specifically destroy activated platelets.
Molecular Cloning and Characterization of a Novel Mammalian Protein Kinase Harboring a Homology Domain that Defines a Subfamily of Serine/Threonine Kinases
Authors: Becker Walter , Heukelbach Jörg , Kentrup Heiner , Joost Hans‐Georg
Published: August 2004

The cDNA of a novel protein kinase (referred to as SNRK) was isolated from a rat fat cell cDNA library with a probe generated by a cloning approach based on the polymerase chain reaction. The encoded polypeptide (746 amino acids, Mr= 81627) contains all conserved subdomains characteristic of the protein serine/threonine kinase family. A recombinant fusion protein with glutathione S ‐transferase catalysed autophosphorylation as well as phosphorylation of histone, confirming that SNRK has indeed protein kinase activity. By Northern blot hybridization, a 5‐kb mRNA was detected in brain, heart, fat cells, intestine, testis, ovary, adrenal gland and thymus. In 3T3‐LI cells, SNRK was specifically expressed in the differentiated, adipocyte‐like phenotype, where as its mRNA was not detected in fibroblasts. Sequence comparisons of its catalytic domain relate SNRK to the SNF1 family of protein kinases. The noncatalytic domain comprises several intriguing structural features, including a glycine‐rich region, two PEST sequences, and a bipartite nuclear localization signal which is preceded by a stretch of ten consecutive acidic residues. This part of the sequence exhibits no extended similarity with other protein In addition, we detected a high degree of sequence similarity with other SNF1 ‐related protein kinases in a small region (30–35 amino acids) flanking the C‐terminus of the catalytic domain. This domain (designated the SNH domain) appears to define the subfamily of SNF1‐related protein kinases and might represent a new type of regulatory domain of protein kinases.
Initiation of Protein Synthesis in Eukaryotic Cells
Authors: Pain Virginia M.
Published: August 2004

It is becoming increasingly apparent that translational control plays an important role in the regulation of gene expression in eukaryotic cells. Most of the known physiological effects on translation are exerted at the level of polypeptide chain initiation. Research on initiation of translation over the past five years has yielded much new information, which can be divided into three main areas: (a) structure and function of initiation factors (including identification by sequencing studies of consensus domains and motifs) and investigation of protein–protein and protein–RNA interactions during initiation; (b) physiological regulation of initiation factor activities and (c) identification of features in the 5′ and 3′ untranslated regions of messenger RNA molecules that regulate the selection of these mRNAs for translation. This review aims to assess recent progress in these three areas and to explore their interrelationships.
Purification, Characterization and Partial Amino Acid Sequencing of Two New Aspartic Proteinases from Fresh Flowers of Cynara cardunculus L.
Authors: Veríssimo Paula , Faro Carlos , Moir Arthur J. G. , Lin Yingzhang , Tang Jordan , Pires Euclides
Published: August 2004

Two new aspartic proteinases have been isolated from stigmas of the cardoon Cynara cardunculus L. by a two‐step purification procedure including extraction at low pH, gel filtration on Superdex 200, and ion‐exchange chromatography on Mono Q. To follow the conventional nomenclature for aspartic proteinases, we have named these proteinases cardosin A and cardosin B. On SDS/PAGE, cardosin A migrated as two bands with apparent molecular masses of 31 000 Da and 15000 Da where as the chains of cardosin B migrated as bands of 34000 Da and 14000 Da. The partial amino acid sequences of the two cardosins revealed that they are similar but not identical, and that they differ horn the previously reported cardoon proteinases named cynarases, which were assumed to be derived from a common precursor. Although the cardosins show some degree of similarity to each other, we could detect no immunological cross‐reactivity between them. Both cardosins were active at low pH and were inhibited by pepstatin, with Ki values of 3 nM for cardosin A and 1 nM for cardosin B, indicating that they belong to the class of aspartic proteinases. Significant differences between the two enzymes were also found for the Kcat/Km values for the hydrolysis of two chromophoric synthetic peptides. The active‐site ionization constants, pKe1 and pKe2, for cardosin A are 2.5±0.2 and 5.3±20.2, whereas for cardosin R they are 3.73±10.09 and 6.7±50.1. The results herein described on the structural and kinetic properties of the cardosins indicate that they are the products of distinct genes which have probably arisen by gene duplication. A scheme for the proteolytic processing of the two enzymes is also proposed.
Detailed Oligosaccharide Structures of Human Integrin α5β1 Analyzed by a Three‐Dimensional Mapping Technique
Authors: Nakagawa Hiroaki , Zheng Mingzhe , Hakomori Sen‐itiroh , Tsukamoto Yoshinori , Kawamura Yoshiya , Takahashi Noriko
Published: August 2004

Structures of N‐linked oligosaccharides obtained from human integrin α5βl are described. Integrin α5βl (4.5 mg) was purified from human placenta and digested using trypsin and chymotrypsin. N‐linked oligosaccharides were released from the glycopeptides by digestion with glycoamidase A (from almond). The reducing ends of the oligosaccharides were derivatized with 2‐aminopyridine. The pyridylamino‐oligosaccharides were separated and these structures were identified by a three‐dimensional HPLC mapping technique on three kinds of HPLC columns [Takahashi, N., Nakagawa, H., Fujikawa, K., Kawamura, Y. & Tomiya, N. (1995) Anal. Biochem. 226, 139–146]. Finally, 35 different oligosaccharide structures were identified, 10 of which were neutral, 6 mono‐sialyl, 10 di‐sialyl, 7 tri‐sialyl and 2 tetra‐sialyl. The molar ratio of neutral, mono‐sialyl, di‐sialyl, tri‐sialyl and tetra‐sialyl oligosaccharides was 20.8%, 24.8%, 27.7%, 18.1% and 8.6%, respectively. High‐mannose‐type oligosaccharides accounted for only 1.5% of the total. The remaining oligosaccharides were all complex type. The most predominant structure was the diantennary di‐α‐(2,3)‐sialyl fucosyl. Major linking of sialic acid was α‐(2,3)‐linkage, and over 50% of all oligosaccharides were fucosylated at the N ‐acetylglucosamine residue of the reducing end.
Reactions of Mitochondrial Cruciform Cutting Endonuclease 1 (CCE1) of Yeast Saccharomyces Cerevisiae with Branched DNAs in Vitro
Authors: Kupfer Christian , Kemper Börries
Published: August 2004

Cruciform‐cutting endonuclease 1 (CCEI) is an X‐solvase from yeast Saccharomyces cerevisiae [Kleff, S., Kemper, B. & Sternglanz, R. (1992) EMBO J. 11, 699–704], We report here the purification of the cloned enzyme CCEl to near homogeneity from over‐expressing Escherichia coli cells. The purified protein has a globular shape and an apparent molecular mass of 38 kDa. CCEl reacts specifically with branched DNAs, preferably with four‐armed cruciforms. The enzyme linearizes native supercoiled DNA by cutting at the base of cruciform structures as they occur in derivatives of phage M13. Supercoiling was not required for cleavage per se and a relaxed circular DNA hybrid with a stable cruciform was linearized with the same relative cleavage efficiency. Fully synthetic cruciforms (four‐armed X‐junctions) were also good substrates for CCEl, provided a symmetric 6‐bp sequence (in our case an EcoRI restriction site) was maintained at the junction. Consequently, a synthetic cruciform made from fully randomized oligonucleotide sequences was not a substrate for CCE1. In general, cleavage sites were found clustered in a characteristic pattern in each arm of a cruciform structure. A synthetic three‐armed Y‐junction was also cleaved by CCEl, but with a lower efficiency than the related four‐armed construct. CCEI resolves efficiently branched synthetic DNAs in vitro. The function is consistent with the idea that CCEl is responsible for a timely reversal of branched recombination intermediates preceding petite formation in mitochondrial DNA.
Cyclic AMP and Fatty Acids Increase Carnitine Palmitoyltransferase I Gene Transcription in Cultured Fetal Rat Hepatocytes
Authors: Chatelain Florence , Kohl Claude , Esser Victoria , Mcgarry J. Denis , Girard Jean , Pegorier Jean‐Paul
Published: August 2004

In the rat, the gene for liver mitochondrial carnitine palmitoyltransferase I (CPT I), though dormant prior to birth, is rapidly activated postnatally. We sought to elucidate which hormonal and/or nutritional factors might be responsible for this induction. In cultured hepatocytes from 20‐day‐old rat fetus, the concentration of CPT I mRNA, which initially was very low, increased dramatically in a dose‐dependent manner after exposure of the cells to dibutyryl cAMP (Bt2cAMP). Similar results were obtained when long‐chain fatty acids (LCFA), but not medium‐chain fatty acids, were added to the culture medium. The effects of Bt2cAMP and LCFA were antagonized by insulin, also dose dependently. In contrast, CPT II gene expression, which was already high in fetal hepatocytes, was unaffected by any of the above manipulations.

Bt2cAMP stimulated CPT I gene expression even when endogenous triacylglycerol breakdown was suppressed by lysosomotropic agents suggesting that the actions of cAMP and LCFA were distinct. Moreover, half‐maximal concentrations of Bt2cAMP and linoleate produced an additive effect on CPT I mRNA accumulation. While linoleate and Bt2cAMP stimulated CPT I gene transcription by twofold and fourfold, respectively, the fatty acid also increased the half‐life of CPT I mRNA (50%).

When hepatocytes were cultured in the presence of 2‐bromopulmitate, (which is readily converted by cells into its non‐metabolizable CoA ester) CPT I mRNA accumulation was higher than that observed with oleate or linoleate. Similarly, the CPT I inhibitor, tetradecylglycidate, which at a concentration of 20 μM did not itself influence the CPT I mRNA level, enhanced the stimulatory effect of linolcate. The implication is that induction of the CPT I message by LCFA does not require mitochondrial metabolism of these substrates; however, formation of their CoA esters is B necessary step.

Unlike linoleate, the peroxisome proliferator, clofibrate, increased both CPT I and CPT II mRNA levels and neither effect was offset by insulin. It thus appears that the mechanism of action of LCFA differs from that utilized by clofibrate, which presumably works through the peroxisome proliferator activated receptor.

We conclude that the rapid increase in hepatic CPT I mRNA level that accompanies the fetal to neonatal transition in the rat is triggered by the reciprocal change in circulating insulin and LCFA concentrations, coupled with elevation of the liver content of CAMP.
Inhibition of Phosphatidylinositol 3‐Kinase Blocks T Cell Antigen Receptor/CD3‐Induced Activation of the Mitogen‐Activated Kinase Erk2
Authors: Von Willebrand Maria , Jascur Thomas , Bonnefoy‐Bérard Nathalie , Yano Hiroshi , Altman Amnon , Matsuda Yuzuru , Mustelin Tomas
Published: August 2004

The production of 3‐phosphorylated inositol phospholipids is implicated in regulation of cell growth and transformation. To explore the role of these lipids in T cell antigen receptor (TCR)/CD3‐induccd signaling, we have examined the effects of a specific phosphatidylinositol 3‐kinasce (Ptdlns3K) inhibitor, wortmannin, and overexpression of two PtdIns3K constructs on the activation of down‐stream effectors in anti‐CD3 treated T cells. We report that treatment of cells with wortmannin blocked anti‐CD3‐induced activation of the mitogen‐activated kinase Erk2 while not affecting phorbol‐ester‐induced Erk2 activation. An inactive analog of wortmannin, WM12, did not affect TCR/KD3‐induced Erk2 activation, and wortmannin had no effect on the activity of Erk2 when added directly to the in vitro assays. Expression of a disruptive PtdIns3K construct also reduced Erk2 activation, while a construct that stimulates PtdIns3K enhanced the activation of Erk2. Receptor‐induced activation of other Ser/Thr kinases, such as c‐Raf, B‐Raf, Mek1, Mek2, Mekk, was not affected by wortmannin. Our results suggest that the production of 3‐phosphorylated inositol phospholipids is involved in the activation of Erk2, but does not regulate the enzymes that are thought to be upstream of Erk2.
Platelet Activation by 2‐(4‐bromo‐2,3‐dioxobutylthio)Adenosine 5′‐Diphosphate is Mediated by Its Binding to A Putative ADP Receptor, Aggregin
Authors: Puri Rajinder N. , Colman Roberta F. , Colman Robert W.
Published: August 2004

Platelet responses induced by ADP are mediated by a unique P2Tpurinergic receptor. Although a variety of ADP analogs, substituted at C2, have been used to delineate pharmacological properties of the ADP‐binding site(s), the identity of the receptor protein has not been firmly established. 2‐(4‐Bromo‐2,3‐dioxobutylthio)‐ADP[2‐BrCH2(CO)2CH2‐S‐ADP], a well‐characterized ADP analog, has been previously used as an affinity label to examine the structure/function relationship of ADP‐requiring enzymes [Kapetanovic, E., Bailey, J. B. & Colman, R. F. (1985) Biochemistry 24, 7586–7593]. We found that it induced platelet shape change, aggregation, exposure of fibrinogen binding sites, secretion and mobilization of intracellular calcium, but was less potent than ADP. Under non‐stirring conditions, incubation of platelets with this analog for longer time periods blocked ADP‐induced shape change, aggregation, and the ability of ADP to antagonize the rise in intracellular levels of CAMP induced by iloprost (a prostaglandin I2 analog). Of a variety of agonists examined, only ADP‐induced aggregation was almost completely inhibited in platelets irreversibly modified by the analog. An autoradiogram of the gel obtained by SDS/PAGE of solubilized platelets modified by the ADP analog followed by reduction of the dioxo group by NaB[3H]4 showed the presence of a single radiolabeled protein band at 100 kDa. Platelets incubated first with either ADP, ATP, or 2‐methylthio‐ADP were not labeled by 2‐BrCH2(CO)2CH2S‐ADP and NaB[3H]14 8‐BrCH2(CO)2CH2‐S‐ADP was previously shown by us to irreversibly antagonize ADP‐induced platelet responses by selectively modifying aggregin. Incubation of platelets with 2‐BrCH2(CO)2CH2S‐ADP completely blocked labeling of aggregin in platelets by 8‐BrCH2(CO)2CH2S‐[32P]ADP. These results show that 2‐BrCH2(CO)2CH2S‐ADP initially interacts reversibly with aggregin (100 kDa), a putative ADP receptor, and induces platelet shape change and aggregation, and at longer periods of incubation reacts irreversibly to block the ability of ADP to antagonize stimulated adenylate cyclase activity. In contrast, 6‐BrCH2(CO)2CH2S‐ADP was found to be a weak and reversible inhibitor of ADP‐induced platelet aggregation. Prior incubation of platelets with the latter analog reduced labeling of aggregin by 8‐BrCH2(CO)2CH2S‐[32P]ADP. Taken together, the results further show that substitution by the BrCH2(CO)2CH2 group at the C2 and C8 positions is tolerated, while the presence of a free amino function at the C6 position is essential for its interaction with aggregin.
Surface‐Associated Material from the Bacterium Actinobacillus actinomycetemcomitans Contains A Peptide Which, in Contrast to Lipopolysaccharide, Directly Stimulates Fibroblast Interleukin‐6 Gene Transcription
Authors: Reddi Krisanavane , Nair Sean P. , White Peter A. , Hodges Stephen , Tabona Peter , Meghji Sajeda , Poole Stephen , Wilson Michael , Henderson Brian
Published: August 2004

The oral commensal Gram‐negative bacterium Actinobacillus actinomycetemcomitans is believed to be the causative organism of localized juvenile periodontitis, a disease in which there is rapid loss of alveolar bone supporting the teeth. Previously, we have reported that gentle saline extraction of this bacterium removed a loosely adherent proteinaceous fraction from the cell surface of the bacterium, which we have termed surface‐associated material. This material contained potent bone‐resorbing activity. We now report that surface‐associated material is also a potent stimulator of cytokines, and in particular, interleukin‐6 (IL‐6) synthesis, while the lipopolysaccharide from this bacterium is only a weak stimulator of IL‐6 synthesis by fibroblasts and monocytes. In contrast to enteric lipopolysaccharide (LPS), which induces fibroblast IL‐1, IL‐6 and tumour necrosis factor (TNF) α synthesis, surface‐associated material stimulated gingival fibroblasts to synthesize only IL‐6, with no induction of IL‐1 or TNF (the normal inducers of IL‐6 synthesis). Reverse transcriptase PCR also failed to detect mRNA for IL‐1 or TNF in surface‐associated‐material‐stimulated fibroblasts, although both mRNAs were present in Escherichia coli LPS‐stimulated cells. Neutralizing antibodies to IL‐1 and/or TNF or the natural IL‐1 receptor antagonist (IL‐1ra) inhibited enteric LPS‐induced IL‐6 synthesis, but did not inhibit surface‐associated‐material‐induced synthesis. In addition, dexamethasone, which completely suppressed LPS‐induced IL‐6 synthesis, only inhibited surface‐associated‐material‐induced IL‐6 synthesis by 50%. This suggests that the active constituent in the surface‐associated material stimulates IL‐6 gene transcription by a transcriptional control mechanism distinct to that of E. coli LPS.

The IL‐6‐stimulating activity of the surface‐associated material is inhibited by both heat and trypsin, suggesting that it is proteinaceous. The activity has been isolated using anion‐exchange, reverse‐phase and size‐exclusion HPLC. The active moiety is a peptide of molecular mass 2 kDa which may be the product of a bacterial short open reading frame.
The Characterisation of the 5′ Regulatory Region of a Temperature‐Induced Myosin‐Heavy‐Chain Gene Associated with Myotomal Muscle Growth in the Carp
Authors: Gauvry Laurent , Ennion Steven , Hansen Ekkehard , Butterworth Peter , Goldspink Geoffrey
Published: August 2004

We have isolated and characterised the 5′ region of a member of the carp myosin heavy chain gene family. Expression of this gene has previously been shown to be induced by an increase in environmental temperature and is restricted to the small‐diameter white myotomal muscle fibres which are associated with growth. The whole isoform gene, including potential regulatory sequence 5′ to the transcription start site and the 3′ untranslated region was cloned in λ2001 bacteriophage vector. Studies of the structure of the 5′‐end of the gene revealed high amino acid sequence similarity with translated exons 3–7 of mammalian myosin heavy chain genes indicating identical exon/intron boundaries. The overall length of the gene was however only about one half of that in mammals and birds due to shorter introns. The region 5′ to the transcription unit was sequenced and revealed the presence of putative TATA and CCAAAT boxes. In order to study the regulation of expression, a series of endonuclease‐generated fragments from the 5′ flanking sequence were spliced to chloramphenicol acetyltransferase reporter vectors and used in cell transfection assays or direct gene injection into carp skeletal muscle. The 5′ flanking region, which contains a consensus sequence known as an E‐box (CANNTG) and a MEF2 binding site, was shown to improve the expression of the reporter gene in fish acclimated at 18°C or 28°C. Unlike the coding region, there was little similarity between the 5′‐upstream sequence (promoter region) when compared with sequences flanking the 5′‐end of other myosin heavy chain genes in mammals or chicken.
Characterization of Semenogelin II and its Molecular interaction with Prostate‐Specific Antigen and Protein C Inhibitor
Authors: Kise Hideaki , Nishioka Junji , Kawamura Juichi , Suzuki Koji
Published: August 2004

The serine protease, prostate‐specific antigen (PSA), its protein substrates, semenogelin (Sg) I and II, and protein C inhibitor (PCI) have been described as components of human seminal plasma. PCI was found to inhibit the PSA‐catalyzed degradation of insoluble coagula Sg I+II by forming a PSA‐PCI complex. Digestion of seminal coagula with PSA released PCI and PSA‐PCI complex from the coagula into a soluble phase, suggesting the presence of active PCI binding to the coagula. To investigate the molecular interaction of Sg with PSA and PCI, we purified Sg II from seminal coagula as a soluble form and found that Sg II is glycosylated with heterogeneous carbohydrate moieties. Sg II bound to the solid‐phase complex of diisopropylfluorophosphate (iPr2FP) and PSA with an apparent dissociation constant (Kd) of 41 nM and to PCI with a Kd of 28 nM. The binding of Sg II to iPr2P ‐PSA was not affected by PCI and that of Sg II to PCI was not affected by iPr2P‐PSA, suggesting that Sg II forms a ternary complex with PSA and PCI. The bindings of Sg II to both iPr2P‐ PSA and PCI were influenced by pH, ionic strength, heparin, dextran sulfate, and divalent cations, particularly by Zn2+. Treatment of Sg II with heparinase, heparitinase, N‐glycanase, or with O‐glycanase following sialidase did not affect the binding of Sg II to iPr2P ‐PSA and PCI. These findings suggested that PCI bound to Sg in seminal vesicles regulates the PSA‐catalyzed degradation of Sg in seminal plasma, and that the binding of PCI and PSA to Sg is modulated by several factors such as pH, ionic strength, divalent cations, and heparin‐like substances in seminal plasma.
Similar Conformations of Hairpins with TTT and TTTT Sequences NMR and Molecular Modeling Evidence for T · T base pairs in the TTTT hairpin
Authors: Kuklenyik Zsuzsanna , Yao Shijie , Marzilli Luigi G.
Published: August 2004

The conformations of the d[G(1)C(2)G(3)C(4)‐T(a)T(b)T(c)T(d)‐G(5)C(6)G(7)C(8)] (T4) and d[G(1)C(2)G(3)C(4)‐T(a)T(b)T(c)‐G(5)C(6)G(7)C(8)] (T3) DNA hairpins have been studied. The 1H and 31P signals of the two hairpins have been nearly completely assigned by means of two‐dimensional NMR spectroscopy in D2O (NOESY (two‐dimensional nuclear Overhauser effect and exchange spectroscopy) at mixing times of 5, 50, 100, 300 and 500 ms, double‐quantum‐filtered correlation spectroscopy (DQF‐COSY) and 1H‐31P reverse chemical shift correlation (RCSC), and one‐dimensional NOE spectra in 90% H2O. Conformational analysis using distance geometry (DG), molecular mechanics (MM) and molecular dynamics (MD) gave model conformations, which were evaluated by comparison of experimental and simulated 2D NOESY spectra. For the T4 sequence in T4, both NMR data and modeling indicated a T(a) · T(d) wobble base pair. Although two types of T(a) · T(d) base pairs are possible, the one with T(a)NH‐T(d)O4 and T(a)O2‐T(d)NH H‐bonds was calculated to be more stable. Because the T(a) · T(d) base pair of T4 extends the stem, there are only two residues (T(b) and T(c)) in the loop. Although there are three residues in the T3 loop, the T(c) base projects into the solvent. The resulting; conformational models have very similar loop folding patterns (FP): the bases of the two adjacent residues that begin the loop [T(b)T(c) of T4 and T(a)T(b) of T3] have a minor groove/major groove orientation with the first residue each having a trans α torsion angle; and the phosphodiester group that links the residues at the 3′ end of the loop and the 5′ top of the stem [T(c)pT(d) of T4 and T(c)pG(5) of T3] has a gauche+, gauche+ζ, α conformation with a trans γ angle for the second residue in both. These or similar features appear to be present in most of the few other hairpins studied previously by conformational methods. Thus, we believe that the conformations of the loops in T3 and T4 hairpins have greater similarities than previously recognized.
Synthetic 20‐Epi Analogs of Calcitriol are Potent Inducers of Target‐Gene Activation in Osteoblastic Cells
Authors: Ryhänen Sanna , Mahonen Anitta , Jääskeläinen Tiina , Mäenpää Pekka H.
Published: August 2004

We have compared the actions of calcitriol and its three synthetic analogs, 20‐epi‐22‐oxa‐24a,26a,27a‐trihomo‐1α,25‐dihydroxyvitamin D3 (KH 1060), 1α,24S ‐(OH)2‐22‐ene‐26,27‐cyclopropyl vitamin D3 (MC 903) and 20‐epi‐1α,25‐dihydroxyvitamin D3 (MC 1288), on the expression of two marker genes of differentiated osteoblasts, namely alkaline phosphatase and osteocalcin, using human MG‐63 osteosarcoma cells. Calcitriol and the analogs had qualitatively similar stimulatory effects on target‐gene activation. Quantitatively, MC 903 behaved in most experiments essentially as the parent compound calcitriol. In vitamin D receptor/DNA complex formation MC 903, however, was more potent than calcitriol. In contrast, the 20‐epi analogs, KH 1060 and MC 1288, were much more potent even at lower concentrations, than calcitriol and MC 903 in stimulating alkaline phosphatase activity, osteocalcin mRNA synthesis and osteocalcin secretion. The stimulation occurred to a greater degree and for a longer period than with calcitriol. This effect was apparently mediated by stronger and longer lasting binding of the vitamin D receptor to the osteocalcin vitamin D responsive element by the 20‐epi analogs. After a 6‐h treatment and during subsequent culture without hormone, the effects of the 20‐epi analogs were also stronger and lasted longer than those with calcitriol or MC 903. Collectively, at comparable and lower concentrations, the 20‐epi analogs, KH 1060 and MC 1288, mediate much stronger and longer lasting stimulatory effects than calcitriol or its analog MC 903 on target‐gene expression associated with the differentiated phenotype of the MG‐63 human osteosarcoma cells.
Identification of Uronic‐Acid‐Rich Protein as Urinary Bikunin, the Light Chain of Inter‐α‐Inhibitor
Authors: Atmani Fouad , Mizon Jacques , Khan Saeed R.
Published: August 2004

Uronic‐acid‐rich protein (UAP) is a urinary glycoprotein that inhibits calcium oxalate crystallization in vitro. It shows a structural similarity to bikunin, a component of inter‐α‐inhibitor (IαI) known for its inhibition of the action of many serine proteinases like trypsin and chymotrypsin. To clarify the relationship between these macromolecules, UAP, IαI, urinary bikunin, and plasma bikunin were purified and studied. Their calcium oxalate crystallization inhibitory activity was assayed before and after treatment with chondroitinase AC and pronase. Their molecular mass was determined by using SDS/PAGE before and after these treatments. Polyclonal bikunin antibody was used on Western blots for immunological identification. The partial amino acid sequence of UAP before and after chondroitinase treatment was determined. Also, the antitryptic activity of UAP was measured and compared to that of bikunin, which is responsible for the antiprotease activity of IαI. UAP exhibited a strong calcium oxalate crystallization inhibitory activity. IαI and both bikunins were less inhibitory. Chondroitinase AC had no effect on inhibitory activity of these proteins even when their molecular mass changed. However, after pronase treatment, the inhibitory activity of both bikunins and UAP was completely destroyed. The antitryptic activity of UAP was found to be 0.78 U/mg which is lower than that of bikunin which is about 1.9 U/mg. On Western blotting, bikunin antibody immunoreacted with UAP and both urinary and plasma bikunins. Partial amino acid sequence confirmed the identity of UAP as urinary bikunin.