bluuebunny/crossref_metadata_2025_split huggingface dataset:
Cholesterol metabolism in rat adrenal gland during reversible endotoxic shock
Authors: ABARCA Santiago , GARCÍA Resurrección 
Published: March 2005

The adrenal glands have a crucial role for survival during endotoxic shock. Cholesterol is the obligatory intermediary in corticosteroid biosynthesis; thus any alteration in either the availability of cholesterol or in the ability of the adrenal gland to use cholesterol would have a profound effect on corticosteriod production. We have studies the effect of Escherichia coli endotoxin on cholesterol metabolism, injecting lipopolysaccharide (1.6 mg/100g body) from E. coli 0111:B4 into the tail vein of male Wistar rat. Previous studies from this laboratory have shown that this dose of lipopolysaccharide induces a reversible endotoxic shock.During reversible endotoxic shock there is an alteration in plasma cholesterol; plasma total‐cholesterol levels increase mainly at 6–24 h post‐lipopolysaccharide injection, whereas cholesterol in high‐density lipoproteins shows no significant variations, except a slight but significant decrease at 24 h. The cholesterol content in adrenal gland is diminished in endotoxemic rat, this decrease is more important at 6–24 h after endotoxin injection.We have also measured the acyl‐CoA: cholesterol O‐acyltransferase (ACAT) and cholesterolesterase (CEH) activity during endotoxic shock. ACAT activity decreases after lipopolysaccharide injection. ACAT activity in endotoxemic rats is approximately 35–40% of the activity in control rats. This decrease is due to a defect in the functional capacity of the enzyme, since with exogenous cholesterol there is no significant variation in the ACAT activity. CEH activity, in contrast, increases during endotoxic shock: it shows a maximum (twofold the activity seen in control rats) at 6 h after lipopolysaccharide injection. These results show that lipopolysaccharide injection modifies cholesterol metabolism in plasma and in the adrenal gland, either directly or by mediators.
Electrochemical studies of the hexaheme nitrite reductase from Desulfovibrio desulfuricans ATCC 27774
Authors: MORENO Cristina , COSTA Cristina , MOURA Isabel , LE GALL Jean , LIU Ming Y. , PAYNE William J. , van DIJK Cees , MOURA José J. G. 
Published: March 2005

The electron‐transfer kinetics between three different mediators and the hexahemic enzyme nitrite reductase isolated from Desulfovibrio desulfuricans (ATCC 27774) were investigated by cyclic voltammetry and by chronoamperometry. The mediators, methyl viologen, Desulfovibrio vulgaris (Hildenborough) cytochrome c3 and D. desulfuricans (ATCC 27774) cytochrome c3 differ in structure, redox potential and charge. The reduced form of each mediator exchanged electrons with nitrite reductase. Second‐order rate constants, k, were calculated on the basis of the theory for a simple catalytic mechanism and the results, obtained by cyclic voltammetry, were compared with those obtained by chronoamperometry. Values for k are in the range 106–108 M−1 s−1 and increase in the direction D. desulfuricans cytochrome c3→D. vulgaris cytochrome c3→ methyl viologen. An explanation is advanced on the basis of electrostatic interactions and relative orientation between the partners involved.Chronoamperometry (computer controlled) offers advantages over cyclic voltammetry in the determination of homogeneous rate constants (faster, more accurate and better reproducibility).Direct, unmediated electrochemical responses of the hexaheme nitrite reductase were also reported.
A step towards understanding the folding mechanism of horseradish peroxidase
Authors: PAPPA Helen S. , CASS Anthony E. G. 
Published: March 2005

The guanidinium chloride denaturation/renaturation of the holo‐ and apo‐horseradish peroxidase isoenzyme c (HRP) has been studied by fluorescence and circular dichroism spectroscopies.A distinct equilibrium intermediate of the apoprotein could be detected at low concentrations of guanidinium chloride (0.5 M). This intermediate has a secondary structure content like that of the native protein but a poorly defined tertiary structure. Renaturation of the apo‐HRP is reversible and 100% activity could be obtained after addition of a twofold excess of free haem.The denaturation of the holo‐HRP is more complex and occurs in two distinct steps; unfolding of the protein backbone and loss of the haem. The denatured protein folds back to its native conformation but the incorporation of the haem occurs only after the secondary structure is formed.Ca2+ appears to be important for the stability of the protein as the apo‐HRP is more resistant to denaturation in the presence of Ca2+. The free‐energy change during unfolding of the apo‐HRP was determined in the absence and presence of Ca2+ and found to be 9.2 kJ/mol and 16.7 kJ/mol, respectively. The importance of Ca2+ to the protein stability was also supported by studies on the loss of the haem from the protoporphyrin‐IX–apo‐HRP complex.
Are the tryptophanyl‐tRNA synthetase and the peptide‐chain‐release factor from higher eukaryotes one and the same protein?
Authors: FROLOVA Lyudmila Yu. , FLECKNER Jan , JUSTESEN Just , TIMMS Kristen M. , TATE Warren P. , KISSELEV Lev L. , HAENNI Anne‐Lise 
Published: March 2005

Recently, cDNA clones encoding the bovine (b) [M. Garret, B. Pajot, V. Trézéguet, J. Labouesse, M. Merle, J.‐C. Gandar, J.‐C. Gandar, J.‐P. Benedetto, M.‐L. Sallafranque, J. Alterio, M. Gueguen, C. Sarger, B. Labouesses and J. Bonnet (1991) Biochemistry 30, 7809–7817] and human (h) [L. Yu. Frolova, M. A. Sudomoina, A. Yu. Grigorieva, O. L. Zinovieva and L. L. Kisselev (1991) Gene 109, 291–296] tryptophanyl‐tRNA synthetases (TrpRS) were sequnced; the deduced amino acid sequences exhibit typical structural features of class I aminoacyl‐tRNA synthetases [G. Eriani, M. Delarue, O. Poch, J. Gangloff and D. Moras (1990) Nature 237, 203–206] and limited, although significant, similarity with bacterial TrpRS. Independently, it was shown that a major protein whose synthesis is stimulated in human cell cultures by interferon γ [J. Fleckner, H. H. Rasmussen and J. Justesen (1991) Proc. Natl Acad. Sci. USA 88, 11 520–11 524], and interferons γ or α [B. Y. Rubin, S. L. Anderson, L. Xing, R. J. Powell and W. P. Tate (1991) J. Biol. Chem. 226, 24 245–24 248], exhibits TrpRS activity and an amino acid sequence identical to that of hTrpRS. The amino acid sequences of bTrpRS and hTrpRS are highly similar and are surprisingly very similar to the amino acid sequence deduced from a cloned and sequenced cDNA reported to encode rabbit (r) peptide‐chain‐release factor (RF) [C. C. Lee, W. J. Craigen, D. M. Muzny, E. Harlow and C. T. Caskey (1990) Proc. Natl Acad. Sci. USA 87, 3508–3512]. This close similarity between mammalian TrpRS and cloned RF is unexpected given the distinct functional properties of these proteins. Consequently, the question arises as to whether the mammalian TrpRS and RF activities reside on identical or very similar polypeptides. Alternatively, one may assume that the cloned rabbit cDNA encodes a protein other than rRF.Several properties (immunochemical, biochemical and physico‐chemical) of mammalian TrpRS and RF have been compared. rTrpRS and rRF have distinct thermostabiliy behaviours, and dissimilar chromatographic profiles on phosphocellulose. Both the anti‐bTrpRS polyclonal antibodies and the monoclonal antibody Am2 strongly inhibit the bTrpRS and hTrpRS aminoacylation activities, but not the rRF activity. In addition, neither bTrpRS nor hTrpRS exhibit RF activity. It is concluded that (a) the functional centers responsible for the TrpRS and RF activities are structurally and functionally dissimilar and could hardly belong to the same protein domain; (b) RF and TrpRS probably reside on different polypeptide chains encoded by different genes, although it is still not excluded that the RF‐ and TrpRS‐specific domains are situated on one and the same polypeptide chain; (c) the rabbit cDNA believed to encode RF, presumably encodes rTrpRS; this would explain why its deduced amino acid sequence has no similarity with bacterial RFs but is very similar to mammalian TrpRS.
Epidermal‐growth‐factor receptors generate Ras · GTP more efficiently than insulin receptors
Authors: OSTEROP Arthur P. R. M. , MEDEMA René H. , v. d. ZON Gerard C. M. , BOS Johannes L. , MÖLLER Wim , MAASSEN J. Antonie 
Published: March 2005

Activation of the Ras proto‐oncogene contributes in general to mitogenic activation of cells.We show here that epidermal growth factor (EGF) stimulates Ras · GTP formation very efficiently in a variety of cell lines expressing endogenous EGF receptors only. Maximal activation of the receptor converts up to 65% of cellular p21ras from the GDP form into the active GTP‐bound state. This efficient activation occurs also in cultured primary human fibroblasts. Maximal insulin‐induced Ras · GTP formation is less but in cells overexpressing the insulin receptor a similar high response of Ras · GTP formation is observed after insulin stimulation.Not only the efficiency but also the kinetics by which the EGF and insulin receptors stimulate Ras · GTP formation are quite distinct. In the Rat‐1‐derived cell line, H13IR2000, overexpressing both p21Ha‐ras and the insulin receptor, the activated insulin receptor generates approximately 1 mol Ras · GTP/mol activated insulin receptor. The activated EGF receptor amplifies the signal, resulting in the activation of approximately 40 mol p21ras/mol receptor.Moreover, EGF‐stimulated generation of Ras · GTP is transient with a maximum after 2 min of hormone stimulation and diminishes to near basal levels within 1 h whereas the insulin‐induced Ras · GTP levels are maximal at 5–10 min and decline only slowly to half‐maximal in 1 h.Desensitization of the EGF pathway by prolonged EGF stimulation, prevents subsequent stimulation of Ras · GTP formation by newly added EGF but not by insulin. Vice versa, in cells preincubated with insulin for 1 h, EGF stimulates Ras · GTP formation to near maximal values. These observations indicate that desensitization by prolonged hormone incubation does not involve inactivation of common signaling intermediates but rather components, specific for each pathway, like the particular receptors. The rapid down regulation of EGF receptors compared to insulin receptors corroborate this possibility.The observed high potency of EGF receptors to generate Ras · GTP may explain the, in general stronger mitogenic activity of EGF compared to insulin.
Study of the states and populations of the rat pancreatic cholecystokinin receptor using the full peptide antagonist JMV 179
Authors: SILVENTE POIROT Sandrine , HADJIIVANOVA Christina , ESCRIEUT Chantal , DUFRESNE Marlène , MARTINEZ Jean , VAYSSE Nicole , FOURMY Daniel 
Published: March 2005

The full peptide antagonist of the pancreatic cholecystokinin (CCK) receptor, JMV 179, [Boc‐Tyr(SO3H)‐Ahx‐Gly‐dTrp‐Ahx‐Asp phenylethyl ester, where Tyr(SO3H) = sulfated tyrosine, Ahx = 6‐aminohexanoic acid] was modified at its N‐terminus by incorporation of p‐hydroxyphenyl propionate (Bolton‐Hunter reagent, BH) and was subsequently radioiodinated. After HPLC purification, 125I‐BH‐JMV‐179, a CCK antagonist radioligand of high specific activity (2000 Ci/mmol) was obtained. 125I‐BH‐JMV‐179 bound to a single population of sites on rat pancreatic plasma membranes, (Kd= 3.9 nM, Bmax= 40 pmol/mg protein). Binding was dependent on time, temperature, and protein concentration, and was fully reversible. JMV 179 radioligand detected four times as many sites as an agonist radioligand [C. Hadjiivanova, M. Dufresne, S. Poirot, P. Sozzani, N. Vaysse, L. Moroder and D. Fourmy (1992) Eur. J. Biochem. 204, 273–279]. Agonists and antagonists of the A‐ and B‐subtype CCK/gastrin receptors inhibited 125I‐BH‐JMV‐179 binding with an order of potency compatible with the A‐subtype CCK receptor pharmacology. Moreover, the sulfate group on the tyrosine residue of the CCK peptides appeared to be of much less importance for antagonist affinity than for agonist affinity. Inhibition of 125I‐BH‐JMV‐179 binding by agonists (except JMV 180), demonstrated the presence of two affinity classes of binding sites. The population of sites having an apparent high affinity for CCK represented 30 pmol/mg protein and threefold the number of high‐affinity sites previously identified by an agonist radioligand. In presence of non‐hydrolyzable GTP, all the sites bound CCK agonists with a low affinity. Moreover, saturation analysis of JMV 179 radioligand binding in the presence of CCK indicated that CCK interacted competitively with all JMV 179 sites and demonstrated binding of JMV 179 radioligand to two distinct affinity classes of sites. In the presence of GTP[S] a single affinity class of sites for JMV 179 radioligand was found as in the control experiments without CCK.This study, with the first CCK peptide antagonist radioligand, demonstrtes that CCK receptors exist in two interconvertible affinity states regulated by guanine‐nucleotide‐binding regulatory protein(s) in rat pancreatic plasma membranes. JMV 179 radioligand does not induce receptor coupling but distinguishes the two affinity states of the CCK receptors. JMV 179 reveals the existence of populations of high‐affinity and low‐affinity site for CCK which had not previously been detected by agonist radioligand binding, thus suggesting heterogeneity of CCK receptor sites in membranes.
Primary structure of N‐linked carbohydrate chains of a human chimeric plasminogen activator K2tu‐PA expressed in Chinese hamster ovary cells
Authors: BERGWERFF Aldert A. , van OOSTRUM Jan , ASSELBERGS Fred A. M. , BÜRGI Rolf , HOKKE Cornelis H. , KAMERLING Johannis P. , VLIEGENTHART Johannes F. G. 
Published: March 2005

A recombinant human plasminogen activator hybrid variant K2tu‐PA, expressed in Chinese hamster ovary cells, is partially glycosylated at Asn12 (A chain, kringle‐2 domain) and completely glycosylated at Asn247 (B chain, protease domain). After release of the N‐linked carbohydrate chains by peptide‐N4‐(N‐acetyl‐β‐glucosaminyl)asparagine amidase F, the oligosaccharides were separated from the protein by gel permeation chromatography, then fractionated by FPLC on Mono Q, followed by HPLC on Lichrosorb‐NH2, and analysed by 500‐MHz 1H‐NMR spectroscopy. The following types of carbohydrates occur: monosialylated diantennary (8%), disialylated diantennary (45%), disialylated tri‐ and trí‐antennary (1%), trisialylated tri‐ and trí‐antennary (28%), and tetrasialylated tetra‐antennary (18%) structures, all having fucose in α(1‐6)‐linkage at the Asn‐bound N‐acetylglucosamine. Sialic acid occurred exclusively in α(2‐3)‐linkage to galactose, and consisted of N‐acetylneuraminic acid (94%), N‐glycolylneuraminic acid (3%), and N‐acetyl‐9‐O‐acetylneuram‐inic acid (3%). In addition, glycopeptide fragments corresponding with the A or B chain of K2tu‐PA were analysed. The oligosaccharides attached to Asn12 are less processed than those attached to Asn247. Comparison of the glycosylation pattern of K2tu‐PA with that of tissue‐type plasminogen activator from different biological sources showed significant differences.Profiling studies on different K2tu‐PA production batches demonstrated that the structures of N‐linked oligosaccharides were identical, but that relative amounts vary with the applied isolation procedure of the chimeric glycoprotein.
Interaction of ATP analogs with yeast 3‐phosphoglycerate kinase
Authors: PINEDA Teresa , KWON Oh‐Shin , SERPERSU Engin H. , CHURCHICH Jorge E. 
Published: March 2005

Yeast 3‐phosphoglycerate kinase is inactivated by incubation with pyridoxal 5′‐diphospho‐5′‐adenosine (AdoP2Pxy) [Tamura, J. K., Rakov, R. D. & Gross, R. L. (1986) J. Biol. Chem. 261, 4126–4133). Incorporation of 1 mol affinity label/mol enzyme was sufficient for complete inactivation of 3‐phosphoglycerate kinase. The substrate ATP affords substantial protection against inactivation. Partial protection is afforded by the substrate glycerate 3‐phosphate.When AdoP2Pxy‐modified phosphoglycerate kinase was reduced with [3H]NaBH4 and subjected to trypsin hydrolysis, only one radioactive peptide was isolated by reverse‐phase high‐performance liquid chromatography. The amino acid composition and sequence analysis of the purified radioactive peptide revealed that it spans residues 379–403 of the enzyme and Lys385 specifically reacted with the affinity label. This peptide represents the hinge region between the two domains of the protein, where the active site is also located.The fluorescence intensity of enzyme‐bound AdoP2Pxy is enhanced when glycerate 3‐phosphate is added, suggesting exposure of the fluorescent probe to a more hydrophobic environment. Another fluorescent analog, anthraniloyl‐dATP (ant‐dATP), which carries the fluorescent reporter group on the ribose ring, binds to the enzyme at two distinct sites with Kd values of 6±2 μM and 25±3 μM, as determined by steady‐state anisotropy measurements. Bound ant‐dATP was displaced from the enzyme by glycerate 3‐phosphate and ATP, as monitored by the fluorescence anisotropy.These results suggest that both fluorescent ATP analogs bind to the active site, which is at the hinge region of the enzyme. Model‐building studies showed that when AdoP2Pxy is built into the open form of the enzyme, as described in X‐ray studies, the pyridoxyl group of AdoP2Pxy cannot reach Lys385 for Schiff‐base formation. Labeled Lys385 is on a β‐turn immediately following helix XII, which was suggested to interact with the nucleotide and become ordered at the active site of 3‐phosphoglycerate kinase [Watson, H. C., Walker, N. P. C., Shaw, P. J., Bryant, T. N., Wendell, P. L., Fothergill, L. A., Perkins, R. E., Conroy, S. C., Dobson, M. J., Tuite, M. F., Kinesman, A. J. & Kinesman, S. M. (1982) EMBO J. l, 1635–1640]. The results presented here suggest that binding of substrates cause significant structural changes in the enzyme.
Ascidian entactin/nidogen
Authors: NAKAE Hiroki , SUGANO Mitsuko , ISHIMORI Yoshio , ENDO Takeshi , OBINATA Takashi 
Published: March 2005

Entactin/nidogen, a major component of the basement membrane, has a domain structure comprising three globular domains, and thread‐like and rod‐like domains connecting them. It contains six epidermal‐growth‐factor‐(EGF)‐like motifs and one thyroglobulin‐like motif.In the present study, ascidian entactin/nidogen has been identified by a monoclonal antibody technique. We prepared anti‐(ascidian entactin/nidogen)IgG, named anti‐AsEnt1, then cloned the cDNA of ascidian entactin/nidogen using anti‐AsEnt1 as a probe, and determined its entire sequence. Mainly because the deduced amino acid sequence exhibited high similarity to mouse entactin and human nidogen, and because the antigen localized in basement membrane of ascidian body‐wall muscle, we have concluded that the antigen anti‐AsEnt1 corresponds to the ascidian entactin/nidogen homologue.The deduced amino acid sequence of ascidian entactin/nidogen clearly showed that the ascidian homologue also has a domain structure. However, the ascidian homologue lacked the thread‐like domain, and the rod‐like domain differed from that of mouse entactin in composition, consisting of two kinds of cysteine‐rich motifs, that is, the EGF‐like motif and the thyroglobulin‐like motif. These results suggest that entactin/nidogen have evolved by modifying the domains, especially by shuffling the two kinds of cysteine‐rich motifs.
Rapid screening of cytochromes of respiratory mutants of Saccharomyces cerevisiae
Authors: BROWN Simon , COLSON Anne‐Marie , MEUNIER Brigitte , RICH Peter R. 
Published: March 2005

A technique has been developed for the direct analysis by visible spectrophotometry of yeast spots growing on agar plates. This allows rapid semi‐quantitative estimations of cytochromes c, b and oxidase and permits the identification of strains with impaired respiratory electron flow. Results of screening of 105 mutants are presented. There appears to be a correlation between the exonic location of the mutation in COX1 of oxidase and the level of optically detectable enzyme. Mutations in cytochrome b of the bc1 complex also affect the level of expression of cytochrome oxidase and can cause either an increased or decreased level of expression of oxidase relative to the wild‐type strain.Twelve strains selected by the rapid level‐1 screening were grown as lawns on sections of an agar plate and resuspended for a second level of screening. Quantitative estimates have been made of the concentrations of cytochromes, the turnover number of cytochrome oxidase and the kinetics of recombination of carbon monoxide with oxidase after flash photolysis. This confirmed the validity of the rapid screening procedure, and we have identified several strains which contain high levels of a mutant form of cytochrome oxidase with properties worthy of further investigation.
Chymotryptic cleavage of lipoprotein lipase
Authors: LOOKENE Aivar , BENGTSSON‐OLIVECRONA Gunilla 
Published: March 2005

Treatment of bovine lipoprotein lipase (LPL) with chymotrypsin results in cleavage between residues Phe390‐Ser391 and between Trp392‐Ser393, indicating that this region is exposed in the native conformation of LPL. Two main fragments are generated, one large including the aminoterminus (chymotrypsin‐truncated LPL = c‐LPL) and one small, carboxy‐terminal fragment. The small fragment is not stable, but is further degraded by the protease. Isolated c‐LPL has full catalytic activity against tributyryl glycerol (tributyrin) and p‐nitrophenyl butyrate, while the activity against emulsions of long‐chain triacylglycerols and against liposomes is reduced and the activity against milk fat globules and chylomicrons is lost. Several properties of c‐LPL were investigated. It was found that c‐LPL interacts with apolipoprotein CII (apo CII) as efficiently as intact LPL. The truncated enzyme bound to liposomes and to emulsions of long‐chain triacylglycerols as well as the intact enzyme did. In contrast, c‐LPL did not bind to milk fat globules or to chylomicrons. The activity of c‐LPL was more sensitive to inhibition by other lipid‐binding proteins, e.g. apolipoprotein CIII (apo CIII), than was the intact enzyme. The affinity for heparin was as high with c‐LPL as with intact LPL. Like intact LPL, c‐LPL is dimeric in its active form, as evidenced by sucrose density gradient centrifugation.It is concluded that the reduced catalytic and lipid‐binding properties of c‐LPL compared with intact LPL are related to the properties of the substrate interface. It is speculated that the carboxyterminal part of LPL contains a secondary lipid‐binding site, which is important for activity against chylomicrons and related substrates.
Expression of the Shigella dysenteriae type‐1 lipopolysaccharide repeating unit in Escherichia coli K12/Shigella dysenteriae type‐1 hybrids
Authors: FÄLT Inger C. , SCHWEDA Elke K. H. , WEINTRAUB Andrej , STURM Sabine , TIMMIS Kenneth N. , LINDBERG Alf A. 
Published: March 2005

The structures of the polysaccharide part of lipopolysaccharides isolated from eight Escherichia coli K12/Shigella dysenteriae type 1 hybrids have been determined using sugar and methylation analysis plus 1H‐ and 13C‐nuclear magnetic resonance spectroscopy. The hybrids express parts of the S. dysenteriae type 1 O‐antigen tetrasaccharide repeating unit because of the presence of pSS3, a plasmid expressing an α‐galactosyl:lipopolysaccharide transferase and pSS9, a pBR322 plasmid expressing S. dysenteriae type 1 rfb genes. The various classes of hybrids are the result of transposon Tn 1000 insertions in pSS9 inactivating different rfb genes. The following structural elements were found. E. coli K12 (pSS3) and E. coli K12 (pSS3, pSS9‐6; a class I hybrid); α‐d‐Galp(1→3)β‐d‐GlcpNAc(1→. Class IV hybrids: E. coli K12 (pSS3, pSS9‐36); (pSS3, pSS9‐107) and (pSS3, pSS9‐114); α‐l‐Rhap(1→2)α‐d‐Galp(1→3)β‐d‐GlcpNAc(1→. Class V hybrids: E. coli K12 (pSS3, pSS9‐78) and (pSS3, pSS9‐111); α‐l‐Rhap(1→3)α‐l‐Rhap(1→2)α‐d‐Galp(1→3)β‐d‐GlcpNAc(1→.The structural sequences are identical to those found in the lipopolysaccharide from native S. dysenteriae type 1. In the hybrid strains, the terminal non‐reducing GlcNAc residue of the E. coli K12 core is fully substituted by S. dysenteriae type 1 repeating units, or parts thereof.