bluuebunny/crossref_metadata_2025_split huggingface dataset:
Information and involvement
Authors: Thornton H. 
Published: March 2003

AbstractThis article discusses the beliefs that provision of good quality information is the key to (a) successful and satisfying involvement of patients in their own decision‐making and (b) involvement of lay people in the research process, in debate and other involvement in wider health issues. Education of children, health professionals, the public and the media is advocated, enabling critical appraisal skills and good quality health information to lead to improved involvement of citizens in health‐care decisions of all kinds, both individual and societal. Examples of individual, group and specific group involvement through research projects, debates about screening, Citizens’ Juries, etc. are used to illustrate benefits to patients and to health provision in general.
Protein methylation as a marker of aspartate damage in glucose‐6‐phosphate dehydrogenase‐deficient erythrocytes
Authors: Ingrosso Diego , Cimmino Amelia , D'Angelo Stefania , Alfinito Fiorella , Zappia Vincenzo , Galletti Patrizia 
Published: March 2003

The ‘Mediterranean’ variant of glucose‐6‐phosphate dehydrogenase (G6PD) deficiency is due to the C563CT point mutation, leading to replacement of Ser with Phe at position 188, resulting in acute haemolysis triggered by oxidants. Previous work has shown increased formation of altered aspartate residues in membrane proteins during cell ageing and in response to oxidative stress in normal erythrocytes. These abnormal residues are specifically recognized by the repair enzyme l‐isoaspartate (d‐aspartate) protein O‐methyltransferase (PCMT; EC 2.1.1.77).The aim of this work was to study the possible involvement of protein aspartate damage in the mechanism linking the G6PD defect and erythrocyte injury, through oxidative stress. Patients affected by G6PD deficiency (Mediterranean variant) were selected. In situ methylation assays were performed by incubating intact erythrocytes in the presence of methyl‐labelled methionine. Altered aspartate residues were detected in membrane proteins by methyl ester quantification.We present here evidence that, in G6PD‐deficient erythrocytes, damaged residues are significantly increased in membrane proteins, in parallel with the decay of pyruvate kinase activity, used as a cell age marker. Erythrocytes from patients were subjected to oxidative stress in vitro, by treatment with t‐butylhydroperoxide, monitored by a rise in concentration of both methaemoglobin and thiobarbituric acid‐reactive substances. l‐Isoaspartate residues increased dramatically in G6PD‐deficient erythrocytes in response to such treatment, compared with baseline conditions.The increased susceptibility of G6PD‐deficient erythrocytes to membrane protein aspartate damage in response to oxidative stress suggests the involvement of protein deamidation/isomerization in the mechanisms of cell injury and haemolysis.
Structural studies on the core and the O‐polysaccharide repeating unit of Pseudomonas aeruginosa immunotype 1 lipopolysaccharide
Authors: Bystrova Olga V. , Shashkov Aleksander S. , Kocharova Nina A. , Knirel Yuriy A , Lindner Buko , Zähringer Ulrich , Pier Gerald B. 
Published: March 2003

The structure of the lipopolysaccharide (LPS) of Pseudomonas aeruginosa immunotype 1 was studied after mild acid and strong alkaline degradations by MS and NMR spectroscopy. Three types of LPS molecules were found, including those with an unsubstituted glycoform 1 core (A) or an isomeric glycoform 2 core substituted with one O‐polysaccharide repeating unit (B) or with a long‐chain O‐polysaccharide. Therefore, of two core glycoforms, only glycoform 2 accepts the O‐polysaccharide.In the structures A and B, Kdo, Hep, Hep7Cm, GalNAcAN3Ac, GalNFoAN, QuiNAc, GalNAla represent 3‐deoxy‐d‐manno‐octulosonic acid, l‐glycero‐d‐manno‐heptose, 7‐O‐carbamoyl‐l‐glycero‐d‐manno‐heptose, 2‐acetamido‐3‐O‐acetyl‐2‐deoxygalacturonamide, 2‐formamido‐2‐deoxygalacturonamide, 2‐acetamido‐2,6‐dideoxyglucose and 2‐(l‐alanylamino)‐2‐deoxygalactose, respectively; all sugars are in the pyranose form and have the d configuration unless otherwise stated. One or more phosphorylation sites may be occupied by diphosphate groups. In a minority of the LPS molecules, an O‐acetyl group is present in the outer core region at unknown position. The site and the configuration of the linkage between the O‐polysaccharide and the core and the structure of the O‐polysaccharide repeating unit were defined in P. aeruginosa immunotype 1. The QuiNAc residue linked to the Rha residue of the core was found to have the β configuration, whereas in the interior repeating units of the O‐polysaccharide this residue is in the α‐configuration. The data obtained are in accordance with the initiation of biosynthesis of the O‐polysaccharide of P. aeruginosa O6, which is closely related to immunotype 1, by transfer of d‐QuiNAc‐1‐P to undecaprenyl phosphate followed by synthesis of the repeating O‐antigen tetrasaccharide.
Subsite mapping of the binding region of α‐amylases with a computer program
Authors: Gyémánt Gyöngyi , Hovánszki György , Kandra Lili 
Published: March 2003

A computer program has been evaluated for subsite map calculations of depolymerases. The program runs in windows and uses the experimentally determined bond cleavage frequencies (BCFs) for determination of the number of subsites, the position of the catalytic site and for calculation of subsite binding energies. The apparent free energy values were optimized by minimization of the differences of the measured and calculated BCF data. The program called suma (SUbsite Mapping of α‐Amylases) is freely available for research and educational purposes via the Internet (E‐mail: gyemant@tigris.klte.hu).The advantages of this program are demonstrated through α‐amylases of different origin, e.g. porcine pancreatic α‐amylase (PPA) studied in our laboratory, in addition to barley and rice α‐amylases published in the literature.Results confirm the popular ‘five subsite model’ for PPA with three glycone and two aglycone binding sites. Calculations for barley α‐amylase justify the ‘6 + 2 + (1) model’ prediction. The binding area of barley α‐amylase is composed of six glycone, two aglycone binding sites followed by a barrier subsite at the reducing end of the binding site. Calculations for rice α‐amylase represent an entirely new map with a ‘(1) + 2 + 5 model’, where ‘(1)‘ is a barrier subsite at the nonreducing end of the binding site and there are two glycone and five aglycone binding sites. The rice model may be reminiscent of the action of the bacterial maltogenic amylase, that is, suggesting an exo‐mechanism for this enzyme.
Structural analysis of Francisella tularensis lipopolysaccharide
Authors: Vinogradov Evgeny , Perry Malcolm B. , Conlan J. Wayne 
Published: March 2003

The structure of the lipid A and core region of the lipopolysaccharide (LPS) from Francisella tularensis (ATCC 29684) was analysed using NMR, mass spectrometry and chemical methods. The LPS contains a β‐GlcN‐(1–6)‐GlcN lipid A backbone, but has a number of unusual structural features; it apparently has no substituent at O‐1 of the reducing end GlcN residue in the lipid part in the major part of the population, no substituents at O‐3 and O‐4 of β‐GlcN, and no substituent at O‐4 of the Kdo residue. The largest oligosaccharide, isolated after strong alkaline deacylation of NaBH4 reduced LPS had the following structure:where Δ‐GalNA‐(1–3)‐β‐QuiNAc represents a modified fragment of the O‐chain repeating unit. Two shorter oligosaccharides lacking the O‐chain fragment were also identified. A minor amount of the disaccharide β‐GlcN‐(1–6)‐α‐GlcN‐1‐P was isolated from the same reaction mixture, indicating the presence of free lipid A, unsubstituted by Kdo and with phosphate at the reducing end.The lipid A, isolated from the products of mild acid hydrolysis, had the structure 2‐N‐(3‐O‐acyl4‐acyl2)‐β‐GlcN‐(1–6)‐2‐N‐acyl1−3‐O‐acyl3‐GlcN where acyl1, acyl2 and acyl3 are 3‐hydroxyhexadecanoic or 3‐hydroxyoctadecanoic acids, acyl4 is tetradecanoic or (minor) hexadecanoic acids. No phosphate substituents were found in this compound. OH‐1 of the reducing end glucosamine, and OH‐3 and OH‐4 of the nonreducing end glucosamine residues were not substituted. LPS of F. tularensis exhibits unusual biological properties, including low endoxicity, which may be related to its unusual lipid A structure.
Carbamate kinase from Enterococcus faecalis and Enterococcus faecium
Authors: Marina Alberto , Uriarte Matxalen , Barcelona Belén , Fresquet Vicente , Cervera Javier , Rubio Vicente 
Published: March 2003

Carbamate kinase (CK) catalyzes the reversible reaction NH2COO− + ATP  NH2COOPO.es2−.rb.ei3.rb + ADP, serving to synthesize ATP from carbamoyl phosphate in those microorganisms that derive energy from anaerobic arginine degradation via the arginine dihydrolase pathway. We report here the cloning and sequencing of the CK gene from Enterococcus faecalis and Enterococcus faecium and we demonstrate that the amino acid sequence of CK is identical in the two species. The enzyme, expressed and isolated from Escherichia coli using simple purification procedures, was used to generate crystals suitable for X‐ray studies and to investigate the utilization by CK of bicarbonate and other carbamate analogs. CK had a bicarbonate‐dependent ATPase activity and, therefore, is able to synthesize carboxyphosphate, an unstable compound that is an intermediate in the reactions catalyzed by carbamoyl‐phosphate synthetase (CPS) and by biotin carboxylase. Other functional similarities with CPS include the utilization of acetate by CK with a similarly high Km and the similar Km values of CK for carbamate and of CPS for bicarbonate. Enterococcal CK was inhibited by adenosine(5′)pentaphospho(5′)adenosine (Ap5A) and Ap6A and, less powerfully, by Ap4A, whereas Ap3A is essentially non‐inhibitory. Thus, inhibition by Ap5A seems not to be a valid criterion to differentiate between CK and CPS, for the two enzymes can be inhibited by Ap5A. All these results support the relatedness of CK and CPS. Finally, we used limited proteolysis : (a) to localize the epitopes for monoclonal antibodies obtained against CK; (b) to demonstrate the importance of the C‐terminus for enzyme activity; and (c) to show that Arg158 is highly exposed and may be essential for activity.Comparison of the sequence of CK with known protein sequences demonstrates considerable similarity of CK with bacterial N‐acetylglutamate kinases, strongly suggesting that these two enzymes may share a similar structure and the same catalytic mechanism.
Oxygen exchange with water in heme‐oxo intermediates during H2O2‐driven oxygen incorporation in aromatic hydrocarbons catalyzed by microperoxidase‐8
Authors: Dorovska‐Taran Victoria , Posthumus Maarten A. , Boeren Sjef , Boersma Marelle G. , Teunis Cees J. , Rietjens Ivonne M. C. M. , Veeger Cees 
Published: March 2003

The present paper describes the oxygen incorporation into naphthalene and anthracene by H2O2‐driven microperoxidase‐8, forming α‐naphthol and anthraquinone, respectively. Microperoxidase‐8 is a minienzyme containing a histidinyl‐coordinated Fe3+‐protoporphyrin IX cofactor covalently attached to an eight‐amino‐acid peptide.Additional experiments were performed to investigate whether the reaction mechanism involved is like that of peroxidase and/or cytochrome P‐450. A reaction pathway like that of cytochrome P‐450 implies oxygen transfer to the substrate from the as yet uncharacterized iron‐oxo species formed in the reaction of the heme cofactor with H2O2. In contrast, a peroxidase‐type reaction chemistry involves reaction pathways proceeding by initial one‐electron oxidation of, or H‐abstraction from, the substrate, followed by incorporation of oxygen from sources other than the iron‐oxo species, i.e. from other than H2O2.The results of the present study exclude Fenton‐type chemistry and prove that the minicatalyst is able to catalyze the oxygen incorporation by both peroxidase and cytochrome P‐450 types of reaction pathways, while exchange occurs between the high‐valency iron‐oxo species and H2O. The mechanistic implications of this exchange for cytochrome P‐450 are discussed.
Transport of LDS‐751 from the cytoplasmic leaflet of the plasma membrane by the rhodamine‐123‐selective site of P‐glycoprotein
Authors: Shapiro Adam B. , Ling Victor 
Published: March 2003

P‐glycoprotein is an ATP‐dependent transporter of an extremely wide variety of lipophilic compounds. We showed previously [Shapiro, A. B. & Ling, V. (1997a) Eur. J. Biochem. 250, 130−137] that P‐glycoprotein contains two drug transporting sites, dubbed H (for Hoechst 33342‐selective) and R (for rhodamine‐123‐selective), that interact with positive cooperativity. The H site transports 2‐[2‐(4‐ethoxyphenyl)‐6‐benzimidazolyl]‐6‐(1‐methyl‐4‐piperazyl)benzimidazole (Hoechst 33342) from the cytoplasmic leaflet of the plasma membrane to the aqueous extracellular medium [Shapiro, A. B. & Ling, V. (1997b) Eur. J. Biochem. 250, 122−129]. The environment from which the R site transports its substrates is unknown. In this paper, we used the fluorescent DNA dye 2‐{4‐[4‐(dimethylamino)phenyl]‐1,3‐butadienyl}‐3‐ethylbenzothiazolium perchlorate (LDS‐751), a substrate of the R site, to address this issue.LDS‐751 which, like Hoechst 33342, exhibits lipid‐dependent fluorescence and slow transleaflet diffusion, allowed us to use the same methodology that we used for the H site to study the location of the R site. As with Hoechst 33342, the specific initial rate of LDS‐751 transport by P‐glycoprotein‐rich, isolated plasma membrane vesicles from CHRB30 cells was directly proportional to the amount of membrane‐bound LDS‐751 and inversely proportional to the concentration of free, aqueous LDS‐751. This result demonstrates that the R site of P‐glycoprotein transports LDS‐751 out of the lipid membrane. The slight decrease, instead of an increase, in the initial rate of active transport of LDS‐751 with the amount of time elapsed for slow diffusion of LDS‐751 from the cytoplasmic leaflet to the extracellular leaflet indicates that the R site of P‐glycoprotein removes LDS‐751 from the cytoplasmic leaflet of the plasma membrane. Thus, both known drug‐transporting sites of P‐glycoprotein remove their substrates from the cytoplasmic leaflet. Since all of the P‐glycoprotein substrates we have examined so far are recognized by one or both of the two known drug‐transporting sites, these two sites in the cytoplasmic leaflet of the plasma membrane may be able to account for all substrate transport by P‐glycoprotein.
Structural and serological studies on a new acidic O‐specific polysaccharide of Proteus vulgaris O32
Authors: Bartodziejska Beata , Shashkov Alexander S. , Babicka Dorota , Grachev Aleksei A. , Torzewska Agnieszka , Paramonov Nikolay A. , Chernyak Anatoly Y. , Rozalski Antoni , Knirel Yuriy A. 
Published: March 2003

The following structure of the O‐specific polysaccharide chain (O‐antigen) of the Proteus vulgaris O32 lipopolysaccharide (LPS) was established by 1H‐NMR and 13C‐NMR spectroscopy, including two‐dimensional NOESY and H‐detected 1H,13C heteronuclear multiple‐quantum coherence (HMQC) experiments :
←2)‐α‐
L‐RhapI‐(1←2)‐α‐
L‐RhapII‐(1←4)‐β‐
D‐GalpAI‐(1←3)‐β‐
D‐GlcpNAc‐(1←4)‐α‐
D‐GalpAII‐(1←.
In addition, an O‐acetyl group was detected, which, most probably, is located at position 3 of a part of RhapI residues.Serological studies, using rabbit polyclonal anti‐(P. vulgaris O32) serum, homologous and heterologous Proteus O‐antigens and related artificial antigens, revealed the importance of an α‐
D‐GalA‐associated epitope in manifesting the immunospecificity of P. vulgaris O32 and substantiated serological relationships between the O‐antigen studied and those of some other Proteus strains.
Purification and cloning of the mitochondrial branched‐chain amino acid aminotransferase from sheep placenta
Authors: Faure Magali , Glomot Françoise , Bledsoe Randy , Hutson Susan , Papet Isabelle 
Published: March 2003

This paper presents the first purification of the mitochondrial branched‐chain amino acid aminotransferase (BCATm) from sheep placenta. It is a homodimer with an apparent subunit molecular mass of 41 kDa. The enzyme differs from those of the rat and human as it appears to form at least one intermolecular disulfide bond. The sheep BCATm cDNA (1.4 kb) encodes a mature polypeptide of 366 amino acids with a calculated molecular mass of 41 329 Da and a partial mitochondrial targeting sequence of seven amino acids. The sheep BCATm sequence shares higher identity with other mammalian BCATm isoenzymes (82–85%) than with the cytosolic isoenzymes (60%).By Northern blot analysis, a message of 1.7 kb was detected in sheep placenta and skeletal muscle. Measurements of BCAT activity, mRNA and BCATm protein in sheep placenta and skeletal muscle revealed that BCATm is the sole BCAT isoenzyme expressed in placenta, whereas it contributes 57 and 71% of the BCAT activity in tensor fascia latae and masseter muscles from weaned lambs respectively. Skeletal muscle, the main site of branched‐chain amino acid transamination, exhibits significantly lower BCAT activity in sheep than in rat. Our results suggest that the low BCATm mRNA level probably accounts for the low BCAT activity in sheep skeletal muscle, and that the metabolic scheme for branched‐chain amino acid catabolism is specific to each species.
Formation of fatty acid ethyl esters in rat liver microsomes
Authors: Diczfalusy Margareta A. , Björkhem Ingemar , Einarsson Curt , Alexson Stefan E. H. 
Published: March 2003

Fatty acid ethyl esters have been detected in high concentrations in organs commonly damaged by alcohol abuse and are regarded as being important non‐oxidative metabolites of ethanol. The formation of fatty acid ethyl esters (FAEEs) has been ascribed to two enzymic activities, acyl‐CoA : ethanol O‐acyltransferase (AEAT) and FAEE synthase. In the present study we determined AEAT and FAEE synthase activities in isolated rat liver microsomes and further characterized the microsomal AEAT activity in more detail. The determined AEAT and FAEE synthase activities were found to be similar (about 1.7 nmol·min–1·mg–1). However, the AEAT activity was increased about sixfold by the addition of 250 µm bis‐(4‐nitrophenyl) phosphate (a serine esterase inhibitor) to the incubation whereas FAEE synthase activity was completely inhibited. p‐Hydroxymercuribenzoic acid (a cysteine‐reacting compound) also stimulated AEAT activity (about fourfold) but had no effect on FAEE synthase activity. The effects of the inhibitors suggest that the formation of FAEEs by AEAT was severely counteracted by enzymic hydrolysis of the substrate (acyl‐CoA) and to a lesser extent the product by serine esterases. dl‐Melinamide, a hypocholesterolaemic drug, was found to be a very potent inhibitor of AEAT activity with an IC50 value of about 2.5 µm. Furthermore, we compared the activities of two purified microsomal carboxylesterases, ES‐4 and ES‐10, and identified ES‐4 as the enzyme responsible for hydrolysis of FAEEs. The two carboxyesterases were also tested for FAEE synthase activity, but neither had any detectable activity. Esterase ES‐4 was found to have some AEAT activity, but it was low. When measured under optimal conditions without competing hydrolysis the capacity of AEAT is thus considerably higher than FAEE synthase and the results are consistent with an important role for AEAT in the formation of ethyl esters. As the ratio acyl‐CoA/non‐esterified fatty acids is high under normal conditions, AEAT is probably the most important enzyme in fatty acid ethyl ester formation.
Structural and serological studies on the O‐antigen of Proteus mirabilis O14, a new polysaccharide containing 2‐[(R)‐1‐carboxyethylamino]ethyl phosphate
Authors: Perepelov Andrei V. , Ujazda Elzbieta , Senchenkova Sof’ya N. , Shashkov Alexander S. , Kaca Wieslaw , Knirel Yuriy A. 
Published: March 2003

An O‐specific polysaccharide was obtained by mild acid degradation of Proteus mirabilis O14 lipopolysaccharide (LPS) and found to contain d‐galactose, 2‐acetamido‐2‐deoxy‐d‐glalactose, phosphate, N‐(2‐hydroxyethyl)‐d‐alanine (d‐AlaEtn), and O‐acetyl groups. Studies of the initial and O‐deacetylated polysaccharides using one‐ and two‐dimensional 1H‐ and 13C‐NMR spectroscopy, including COSY, TOCSY, NOESY, H‐detected 1H,13C heteronuclear multiple‐quantum coherence, and heteronuclear multiple‐bond correlation experiments, demonstrated the following structure of the repeating unit:This is the second bacterial polysaccharide reported to contain α‐d‐Galp6PAlaEtn, whereas the first one was the O‐antigen of P. mirabilis EU313 taken erroneously as strain PrK 6/57 from the O3 serogroup [Vinogradov, E. V., Kaca, W., Shashkov, A.S., Krajewska‐Pietrasik, D., Rozalski, A., Knirel, Y.A. & Kochetkov, N.K. (1990) Eur. J. Biochem.,188, 645–651]. Anti‐(P. mirabilis O14) serum cross‐reacted with LPS of P. mirabilis EU313 and vice versa in passive hemolysis and ELISA. Absorption of both O‐antisera with the heterologous LPS decreased markedly but did not abolish the reaction with the homologous LPS. These and chemical data indicated that both strains have similar but not identical O‐antigens. Therefore, we propose that P. mirabilis EU313 should belong to a new subgroup of the O14 serogroup.
Characterization of MB‐1
Authors: Hefford Mary Alice , Dupont Claude , MacCallum Jillian , Parker Matthew H. , Beauregard Marc 
Published: March 2003

MB‐1 is a de‐novo protein designed to incorporate a large number of the nutritionally important amino acids methionine, lysine, leucine and threonine into a stable four‐helix bundle protein. MB‐1 has been expressed and purified from Escherichia coli, indicating it was resistant to intracellular proteases [Beauregard, M., Dupont, C., Teather, R.M. & Hefford, M.A. (1995) Bio/Technology13, 974]. Here we report an analysis of the secondary, tertiary and quaternary structures in MB‐1 using circular dichroism, fluorospectroscopy and size‐exclusion chromatography. Our data indicate that the MB‐1 structure is close to the target structure, an α‐helical bundle, in many respects and is highly helical in solution. The single tyrosine incorporated into the designed protein as a spectrocopic probe of tertiary structure, is buried in a compact, folded core and becomes accessible on protein denaturation, as per design.Furthermore, MB‐1 was found to be native‐like in many respects: (a) protein denaturation induced by urea is cooperative and fully reversible; (b) its oligomeric state at moderate concentration is well defined; and (c) MB‐1 has very low affinity for 8‐anilino‐1‐naphthalenesulfonic acid (ANSA), leading to enhancement of ANSA fluorescence that resembles that of other native proteins. On the other hand, our analysis revealed two aspects that command further attention. The folding stability of MB‐1 as assessed by urea and thermal denaturation is somewhat less than that found for natural globular proteins of similar size. Size‐exclusion chromatography experiments and analysis of MB‐1 denaturation indicate that MB‐1 is dimeric, not monomeric as designed. In light of these results, the utility and the current limitations of our design approach are discussed.