bluuebunny/crossref_metadata_2025 huggingface dataset:
Pilot study of an information aid for women with a family history of breast cancer
Authors: Warner Ellen , Goel Vivek , Ondrusek Nancy , Thiel Elaine C. , Lavina  , Lickley  , Chart Pamela L. , Meschino Wendy S. , Doan Brian D. , Carroll June C. , Taylor Kathryn M.
Published: March 2003

Objective

To develop and pilot study an information aid for women with a family history of breast cancer.

Design, setting and participants

The information aid, consisting of a booklet and audiotape, was developed by a multi‐disciplinary team of health care professionals, breast cancer survivors and their relatives. Women with no personal history of breast cancer, on the waiting list for a familial breast cancer clinic at either of two centres, who could read English, were eligible for the pilot study which consisted of three sets of mailed questionnaires.

Main outcome measures

The baseline questionnaires included: demographic information: the Breast Cancer and Heredity Knowledge Scale (BCHK); psychological measures (the State‐Trait Anxiety Inventory [STAI], Centre for Epidemiologic Studies Depression Scale [CES‐D] and an item about breast cancer worry), and an item about breast cancer risk perception. Immediately after reviewing the information aid, participants completed a satisfaction survey, the risk perception and cancer worry items and a checklist about their personal family history. The third set of questionnaires, completed 2–4 weeks after reviewing the aid, was identical to the first. Patients then attended their scheduled clinic visit and an objective hereditary breast cancer risk assessment was made by the genetic counselling team.

Results and conclusions

Of 97 eligible women who were contacted, 67 completed all three sets of questionnaires. Overall, women were very satisfied with the aid and 96% would recommend it to other women. There was a highly significant improvement in their knowledge scores after they reviewed the aid. Anxiety and depression did not change and there was a decline in breast cancer worry. Risk perception did not change significantly. Ninety per cent of women completed their personal family history checklist accurately. Several important improvements have been made in the information aid and it will now be evaluated in the community.
Using focus groups to seek the views of patients dying from cancer about the care they receive
Authors: Raynes Norma V. , Leach Jack M. , Rawlings Barbara , Bryson Rebecca J.
Published: March 2003

Purpose

The prime purpose of the study was to investigate whether focus groups were a practical way of seeking the views of dying people and whether the information collected added to that collected by more established methods.

Design

A sample frame of Macmillan nurse patients was collated from which three cluster samples were randomly selected to participate in focus groups. The focus groups were structured with an experienced facilitator and recorder to answer three key questions.

Setting and participants

Patients of Macmillan nurses in three NHS Trusts in West Yorkshire who were living at home with incurable cancer. They were aware of their condition and were willing to participate.

Main variables studied

The three key questions were; what kind of help are you currently receiving? What sort of help do you want? Of the kind of help you are receiving what kind is most important to you?

Results

17 patients participated in three focus groups. Participants were generally fairly able and living with spouses or relatives. They were of varying age with different types and duration of cancer. They were receiving a range of health and social services of varying importance to them. More help was particularly wanted with support for daily living, support from specialist cancer nurses, help getting out and with housework. Macmillan nurses and general practice services were highly rated in some but not all three trust areas.

Conclusions

We propose that focus groups are a practical way of collecting information about dying patients that can complement other sources of information in planning and auditing the provision of care.
Improving communication between health professionals and women in maternity care: a structured review
Authors: Rowe Rachel E. , Garcia Jo , Macfarlane Alison J. , Davidson Leslie L.
Published: March 2003

Objective To review trials of the effectiveness of interventions aimed at improving communication between health professionals and women in maternity care.

Search strategy  The electronic databases Medline, PsycLit, The Cochrane Library, BIDS Science and Social Science Indexes, Cinahl and Embase were searched. Final searches were carried out in April 2000.

Inclusion criteria Controlled trials of interventions explicitly aimed at improving communication between health professionals and women in maternity care were included. Other trials were included where two reviewers agreed that this was at least part of the aim.

Data extraction and synthesis 95 potentially eligible papers were identified, read by one reviewer and checked against the inclusion criteria. The 11 included trials were read, assessed for quality and summarized in a structured tabular form.

Results The included trials evaluated interventions to improve the presentation of information about antenatal testing, to promote informed choice in maternity care, woman‐held maternity records and computer‐based history taking. Four trials in which women were provided with extra information about antenatal testing in a variety of formats suggested that this was valued by women and may reduce anxiety. Communication skills training for midwives and doctors improved their information giving about antenatal tests. The three trials of woman‐held maternity records suggested that these increase women's involvement in and control over their care.

Conclusions The trials identified by this review addressed limited aspects of communication and focused solely on antenatal care. Further research is required in several areas, including trials of communication skills training for health professionals in maternity care and other interventions to improve communication during labour and in the postnatal period.
Prediction of protein–protein interaction sites in heterocomplexes with neural networks
Authors: Fariselli Piero , Pazos Florencio , Valencia Alfonso , Casadio Rita
Published: March 2003

In this paper we address the problem of extracting features relevant for predicting protein–protein interaction sites from the three‐dimensional structures of protein complexes. Our approach is based on information about evolutionary conservation and surface disposition. We implement a neural network based system, which uses a cross validation procedure and allows the correct detection of 73% of the residues involved in protein interactions in a selected database comprising 226 heterodimers. Our analysis confirms that the chemico‐physical properties of interacting surfaces are difficult to distinguish from those of the whole protein surface. However neural networks trained with a reduced representation of the interacting patch and sequence profile are sufficient to generalize over the different features of the contact patches and to predict whether a residue in the protein surface is or is not in contact. By using a blind test, we report the prediction of the surface interacting sites of three structural components of the Dnak molecular chaperone system, and find close agreement with previously published experimental results. We propose that the predictor can significantly complement results from structural and functional proteomics.
Identification of the 19S regulatory particle subunits from the rice 26S proteasome
Authors: Shibahara Tadashi , Kawasaki Hiroshi , Hirano Hisashi
Published: March 2003

The 26S proteasome, a protein complex consisting of a 20S proteasome and a pair of 19S regulatory particles (RP), is␣involved in ATP‐dependent proteolysis in eukaryotes. In␣yeast, the RP contains six different ATPase subunits and, at least, 11 non‐ATPase subunits. In this study, we identified the rice homologs of yeast RP subunit genes from the rice expressed sequence tag (EST) library. The complete nucleotide sequences of the homologs for five ATPase subunits, OsRpt1, OsRpt2, OsRpt4, OsRpt5 and OsRpt6, and five non‐ATPase subunits, OsRpn7, OsRpn8, OsRpn10, OsRpn11 and OsRpn12, and the partial sequences of one ATPase subunit, OsRpt3, and six non‐ATPase subunits, OsRpn1, OsRpn2, OsRpn3, OsRpn5, OsRpn6 and OsRpn9, were determined. Gene homologs of four ATPase subunits, OsRpt1, OsRpt2, OsRpt4 and OsRpt5, and three non‐ ATPase subunits, OsRpn1, OsRpn2 and OsRpn9, were found to be encoded by duplicated genes. The rice RP was purified by immunoaffinity chromatography with a Protein A column immobilized antibody against rice 20S proteasome, and the subunit composition was determined. The homologs obtained from the rice EST library were identified as genes encoding subunits of RP purified from rice, including the both products of duplicated genes by using electrospray ionization quadrupole time‐of‐flight mass spectrometry. Post‐translational modifications and processing in rice RP subunits were also identified. Various types of RP complex with different subunit compositions are present in rice cells, suggesting the multiple functions of rice proteasome.
Soluble guanylate cyclase is allosterically inhibited by direct interaction with 2‐substituted adenine nucleotides
Authors: Ruiz‐Stewart Inez , Kazerounian Shiva , Pitari Giovanni M. , Schulz Stephanie , Waldman Scott A.
Published: March 2003

Nitric oxide (NO), the principal endogenous ligand for soluble guanylate cyclase (sGC), stimulates that enzyme and accumulation of intracellular cGMP, which mediates many of the (patho) physiological effects of NO. Previous studies demonstrated that 2‐substituted adenine nucleotides, including 2‐methylthioATP (2MeSATP) and 2‐chloroATP (2ClATP), allosterically inhibit guanylate cyclase C, the membrane‐bound receptor for the Escherichia coli heat‐stable enterotoxin in the intestine. The present study examined the effects of 2‐substituted adenine nucleotides on crude and purified sGC. 2‐Substituted nucleotides inhibited basal and NO‐activated crude and purified sGC, when Mg2+ served as the substrate cation cofactor. Similarly, 2‐substituted adenine nucleotides inhibited those enzymes when Mn2+, which activates sGC in a ligand‐independent fashion, served as the substrate cation cofactor. Inhibition of sGC by 2‐substituted nucleotides was associated with a decrease in Vmax, consistent with a noncompetitive mechanism. In contrast to guanylate cyclase C, 2‐substituted nucleotides inhibited sGC by a guanine nucleotide‐independent mechanism. These studies demonstrate that 2‐substituted adenine nucleotides allosterically inhibit basal and ligand‐stimulated sGC. They support the suggestion that allosteric inhibition by adenine nucleotides is a general characteristic of the family of guanylate cyclases. This allosteric inhibition is mediated by direct interaction of adenine nucleotides with sGC, likely at the catalytic domain in a region outside the substrate‐binding site.
Heterologous expression of a Rauvolfia cDNA encoding strictosidine glucosidase, a biosynthetic key to over 2000 monoterpenoid indole alkaloids
Authors: Gerasimenko Irina , Sheludko Yuri , Ma Xueyan , Stöckigt Joachim
Published: March 2003

Strictosidine glucosidase (SG) is an enzyme that catalyses the second step in the biosynthesis of various classes of monoterpenoid indole alkaloids. Based on the comparison of cDNA sequences of SG from Catharanthus roseus and raucaffricine glucosidase (RG) from Rauvolfia serpentina, primers for RT‐PCR were designed and the cDNA encoding SG was cloned from R. serpentina cell suspension cultures. The active enzyme was expressed in Escherichia coli and purified to homogeneity. Analysis of its deduced amino‐acid sequence assigned the SG from R. serpentina to family 1 of glycosyl hydrolases. In contrast to the SG from C. roseus, the enzyme from R. serpentina is predicted to lack an uncleavable N‐terminal signal sequence, which is believed to direct proteins to the endoplasmic reticulum. The temperature and pH optimum, enzyme kinetic parameters and substrate specificity of the heterologously expressed SG were studied and compared to those of the C. roseus enzyme, revealing some differences between the two glucosidases. In vitro deglucosylation of strictosidine by R. serpentina SG proceeds by the same mechanism as has been shown for the C. roseus enzyme preparation. The reaction gives rise to the end product cathenamine and involves 4,21‐dehydrocorynantheine aldehyde as an intermediate. The enzymatic hydrolysis of dolichantoside (Nβ‐methylstrictosidine) leads to several products. One of them was identified as a new compound, 3‐isocorreantine A. From the data it can be concluded that the divergence of the biosynthetic pathways leading to different classes of indole alkaloids formed in R. serpentina and C. roseus cell suspension cultures occurs at a later stage than strictosidine deglucosylation.
Systematic search for zinc‐binding proteins in Escherichia coli
Authors: Katayama Akira , Tsujii Atsuko , Wada Akira , Nishino Takeshi , Ishihama Akira
Published: March 2003

A systematic search for Escherichia coli proteins with the zinc‐binding activity was performed using the assay of radioactive Zn(II) binding to total E. coli proteins fractionated by two methods of two‐dimensional gel electrophoresis. A total of 30–40 radioactive spots were identified, of which 14 have been assigned from N‐terminal sequencing. In addition to five known zinc‐binding proteins, nine zinc‐binding proteins were newly identified including: acetate kinase (AckA), DnaK, serine hydroxymethyltransferase (GlyA), transketolase isozymes (TktA/TktB), translation elongation factor Ts (Tsf), ribosomal proteins L2 (RplB), L13 (RplM) and one of S15 (RpsO), S16 (RpsP) or S17 (RpsQ). Together with about 20 known zinc‐binding proteins, the total number of zinc‐binding proteins in E. coli increased up to more than 30 species (or more than 3% of about 1000 proteins expressed under laboratory culture conditions). The specificity and affinity of zinc‐binding were analysed for some of the zinc‐binding proteins.
A new high affinity binding site for suppressor of cytokine signaling‐3 on the erythropoietin receptor
Authors: Hörtner Michael , Nielsch Ulrich , Mayr Lorenz M. , Heinrich Peter C. , Haan Serge
Published: March 2003

Erythropoietin (Epo) is a hematopoietic cytokine that is crucial for the differentiation and proliferation of erythroid progenitor cells. Epo acts on its target cells by inducing homodimerization of the erythropoietin receptor (EpoR), thereby triggering intracellular signaling cascades. The EpoR encompasses eight tyrosine motifs on its cytoplasmic tail that have been shown to recruit a number of regulatory proteins. Recently, the feedback inhibitor suppressor of cytokine signaling‐3 (SOCS‐3), also referred to as cytokine‐inducible SH2‐containing protein 3 (CIS‐3), has been shown to act on Epo signaling by both binding to the EpoR and the EpoR‐associated Janus kinase 2 (Jak2) [Sasaki, A., Yasukawa, H., Shouda, T., Kitamura, T., Dikic, I. & Yoshimura, A. (2000) J. Biol. Chem275, 29338–29347]. In this study tyrosine 401 was identified as a binding site for SOCS‐3 on the EpoR. Here we show that human SOCS‐3 binds to pY401 with a Kd of 9.5 µm while another EpoR tyrosine motif, pY429pY431, can also interact with SOCS‐3 but with a ninefold higher affinity than we found for the previously reported motif pY401. In addition, SOCS‐3 binds the double phosphorylated motif pY429pY431 more potently than the respective singly phosphorylated tyrosines indicating a synergistic effect of these two tyrosine residues with respect to SOCS‐3 binding. Surface plasmon resonance analysis, together with peptide precipitation assays and model structures of the SH2 domain of SOCS‐3 complexed with EpoR peptides, provide evidence for pY429pY431 being a new high affinity binding site for SOCS‐3 on the EpoR.
Characterization of heparin binding by a peptide from amyloid P component using capillary electrophoresis, surface plasmon resonance and isothermal titration calorimetry
Authors: Hernaiz Maria J. , LeBrun Laurie A. , Wu Yi , Sen Jette W. , Linhardt Robert J. , Heegaard Niels H. H.
Published: March 2003

Synthetic peptides based on amino‐acid residues 27–38 of human serum amyloid P component represent a novel type of heparin binders as they do not contain clusters of basic amino acids or other known features associated with protein or peptide heparin binding. Here, we characterize the binding using capillary electrophoresis (CE), surface plasmon resonance (SPR), and isothermal titration calorimetry (ITC). By CE, heparin‐binding activity was readily apparent for both a regular peptide and a slightly N‐terminally modified form, while a sequence‐scrambled peptide had no measurable binding. Dissociation constants in the 1–15 µm range were estimated, but only a minor part of the binding isotherm was covered by the experiments. SPR measurements using immobilized peptides verified heparin binding, the range of the binding constants, and the reduced binding of the sequence‐scrambled peptide. Structurally defined heparin oligosaccharides were used to establish that while the tetrasaccharide is too small to exhibit strong binding, little difference in binding strength is observed between hexa‐ and tetradeca‐saccharides. These experiments also confirmed the almost complete lack of activity of the sequence‐scrambled peptide. The amino‐acid sequence‐dependent binding and the importance of a disulfide bond in the peptide were verified by ITC, but the experimental conditions had to be modified because of peptide precipitation and ITC yielded significantly weaker binding constants than the other methods. While the precise function of the peptide in the intact protein remains unclear, the results confirm the specificity of the glycosaminoglycan interaction with regard to peptide sequence by applying two additional biophysical techniques and showing that the N‐terminal part of the peptide may be modified without changing the heparin binding capabilities.
Role of conserved residues within helices IV and VIII of the oxaloacetate decarboxylase β subunit in the energy coupling mechanism of the Na+ pump
Authors: Schmid Markus , Vorburger Thomas , Pos Klaas Martinus , Dimroth Peter
Published: March 2003

The membrane‐bound β subunit of the oxaloacetate decarboxylase Na+ pump of Klebsiella pneumoniae catalyzes the decarboxylation of enzyme‐bound biotin. This event is coupled to the transport of 2 Na+ ions into the periplasm and consumes a periplasmically derived proton. The connecting fragment IIIa and transmembrane helices IV and VIII of the β subunit are highly conserved, harboring residues D203, Y229, N373, G377, S382, and R389 that play a profound role in catalysis. We report here detailed kinetic analyses of the wild‐type enzyme and the β subunit mutants N373D, N373L, S382A, S382D, S382T, R389A, and R389D.

In these studies, pH profiles, Na+ binding affinities, Hill coefficients, Vmax values and inhibition by Na+ was determined. A prominent result is the complete lack of oxaloacetate decarboxylase activity of the S382A mutant at Na+ concentrations up to 20 mm and recovery of significant activities at elevated Na+ concentrations (KNa ≈ 400 mm at pH 6.0), where the wild‐type enzyme is almost completely inhibited. These results indicate impaired Na+ binding to the S382 including site in the S382A mutant. Oxaloacetate decarboxylation by the S382A mutant at high Na+ concentrations is uncoupled from the vectorial events of Na+ or H+ translocation across the membrane. Based on all data with the mutant enzymes we propose a coupling mechanism, which includes Na+ binding to center I contributed by D203 (region IIIa) and N373 (helix VIII) and center II contributed by Y229 (helix IV) and S382 (helix VIII). These centers are exposed to the cytoplasmic surface in the carboxybiotin‐bound state of the β subunit and become exposed to the periplasmic surface after decarboxylation of this compound. During the countertransport of 2 Na+ and 1 H+ Y229 of center II switches between the protonated and deprotonated Na+‐bound state.
Functional analysis of the rat bile salt export pump gene promoter
Authors: Gerloff Thomas , Geier Andreas , Roots Ivar , Meier Peter J. , Gartung Carsten
Published: March 2003

The 5′ flanking region of the bile salt export pump (Bsep) gene was systematically analysed to provide the basis for understanding the mechanisms which regulate Bsep transcription. In addition substrates and drugs were investigated for their ability to alter Bsep promoter activity. Bsep promoter function was restricted to hepatocyte derived HepG2 cells. The 5′ deletional analysis revealed a biphasic shape of reporter gene activities, indicating a suppressive element between nucleotides −800 and −512. Two consensus sites for the farnesoid X receptor (FXR) were located at nucleotides −473 and −64. The latter was characterized as functionally active in bile acid‐mediated feed‐back regulation of Bsep transcription. Bsep promoter activity was reduced by rifampin and β‐estradiol. The anti‐estrogen tamoxifen stimulated promoter activity. Dexamethasone, hydrocortisone and phenobarbital had no effect on Bsep promoter activity. In conclusion, the data suggest that transcriptional regulation of the Bsep gene can be modulated by a number of endogenous compounds and xenobiotics. FXR was a major regulatory factor, mediating bile acid feed‐back stimulation of Bsep transcription.
Fusion of farnesyldiphosphate synthase and epi‐aristolochene synthase, a sesquiterpene cyclase involved in capsidiol biosynthesis in Nicotiana tabacum
Authors: Brodelius Maria , Lundgren Anneli , Mercke Per , Brodelius Peter E.
Published: March 2003

A clone encoding farnesyl diphosphate synthase (FPPS) was obtained by PCR from a cDNA library made from young leaves of Artemisia annua. A cDNA clone encoding the tobacco epi‐aristolochene synthase (eAS) was kindly supplied by J. Chappell (University of Kentucky, Lexington, KY, USA). Two fusions were constructed, i.e. FPPS/eAS and eAS/FPPS. The stop codon of the N‐terminal enzyme was removed and replaced by a short peptide (Gly‐Ser‐Gly) to introduce a linker between the two ORFs. These two fusions and the two single cDNA clones were separately introduced into a bacterial expression vector (pET32). Escherichia coli was transformed with the expression vectors and enzymatically active soluble proteins were obtained after induction with isopropyl thio‐β‐d‐thiogalactoside. The recombinant enzymes were purified using immobilized metal affinity chromatography on Co2+ columns. The fusion enzymes produced epi‐aristolochene from isopentenyl diphosphate through a coupled reaction. The Km values of FPPS and eAS for isopentenyl diphosphate and farnesyl diphosphate, respectively, were essentially the same for the single and fused enzymes. The bifunctional enzymes showed a more efficient conversion of isopentenyl diphosphate to epi‐aristolochene than the corresponding amount of single enzymes.
The folding of dimeric cytoplasmic malate dehydrogenase
Authors: Sanyal Suparna C. , Bhattacharyya Debasish , Das Gupta Chanchal
Published: March 2003

Porcine heart cytoplasmic malate dehydrogenase (s‐MDH) is a dimeric protein (2 × 35 kDa). We have studied equilibrium unfolding and refolding of s‐MDH using activity assay, fluorescence, far‐UV and near‐UV circular dichroism (CD) spectroscopy, hydrophobic probe‐1‐anilino‐8‐napthalene sulfonic acid binding, dynamic lightscattering, and chromatographic (HPLC) techniques. The unfolding and refolding transitions are reversible andshow the presence of two equilibrium intermediate states. The first one is a compact monomer (MC) formed immediately after subunit dissociation and the second oneis an expanded monomer (ME), which is little less compact than the native monomer and has most of thecharacteristic features of a ‘molten globule’ state. Theequilibrium transition is fitted in the model: 2U⇆2ME⇆2MC⇆D.

The time course of kinetics of self‐ refolding of s‐MDH revealed two parallel folding pathways [Rudolph, R., Fuchs, I. & Jaenicke, R. (1986)Biochemistry25, 1662–1669]. The major pathway (70%) is 2U→2M*→2M→D, the rate limiting step being the isomerization of the monomers (K1 = 1.7 × 10−3 s−1). The minor pathway (30%) involves an association step leading to the incorrectly folding dimers, prior to the very slow D*→D folding step.

In this study, we have characterized the folding‐assembly pathway of dimeric s‐MDH. Our kinetic and equilibrium experiments indicate that the folding of s‐MDH involves the formation of two folding intermediates. However, whether the equilibrium intermediates are equivalent to the kinetic ones is beyond the scope of this study.
Cellular uptake of antisense oligonucleotides after complexing or conjugation with cell‐penetrating model peptides
Authors: Oehlke J. , Birth P. , Klauschenz E. , Wiesner B. , Beyermann M. , Oksche A. , Bienert M.
Published: March 2003

The uptake by mammalian cells of phosphorothioate oligonucleotides was compared with that of their respective complexes or conjugates with cationic, cell‐penetrating model peptides of varying helix‐forming propensity and amphipathicity. An HPLC‐based protocol for the synthesis and purification of disulfide bridged conjugates in the 10–100 nmol range was developed. Confocal laser scanning microscopy (CLSM) in combination with gel‐capillary electrophoresis and laser induced fluorescence detection (GCE‐LIF) revealed cytoplasmic and nuclear accumulationin all cases. The uptake differences between naked oligonucleotides and their respective peptide complexes or conjugates were generally confined to one order of magnitude. No significant influence of the structural properties of the peptide components upon cellular uptake was found. Our results question the common belief that the increased biological activity of oligonucleotides after derivatization with membrane permeable peptides may be primarily due to improved membrane translocation.
Characterization of the promoter for the mouse α3 integrin gene
Authors: Kato Takumi , Katabami Kouji , Takatsuki Hironori , Han Seon Ae , Takeuchi Ken‐ichi , Irimura Tatsuro , Tsuji Tsutomu
Published: March 2003

The α3β1 integrin is an adhesion receptor for extracellular matrix proteins including isoforms of laminin, and the changes of its expression level in various cancer cells are thought to cause their malignant phenotypes. We have cloned an approximately 4 kb DNA fragment of the 5′‐flanking region of the murine α3 integrin gene and analyzed its promoter activity. Transfection of MKN1 gastric carcinoma cells with serially truncated segments of the 5′‐flanking region linked to a luciferase gene indicated that a 537‐bp SalI/SacI fragment upstream of exon 1 was sufficient to promote high level gene expression. By 5′‐rapid amplification of cDNA ends (5′‐RACE) using a cap site‐labeled cDNA library, we determined one major and one minor transcription start sites in this region. The murine α3 integrin gene was found to contain a CCAAT box, but to lack a TATA box. Luciferase assay following transfection with a series of deletion constructs of the SalI/SacI fragment revealed that the sequence between positions −260 and −119 bp (relative to the major transcription start site) is required for efficient transcription in gastric carcinoma cells. The sequence analysis of this segment showed the presence of several consensus sequences for transcription factors including Ets, GATA and MyoD/E‐box binding factors. The introduction of mutation in one of the Ets‐binding sequences greatly decreased its promoter activity, suggesting that the transcription of the α3 integrin gene in these cells is regulated by the Ets‐family of transcription factors.
A zymogen form of masquerade‐like serine proteinase homologue is cleaved during pro‐phenoloxidase activation by Ca2+in coleopteran andTenebrio molitorlarvae
Authors: Lee Kum Young , Zhang Rong , Kim Moon Suk , Park Ji Won , Park Ho Young , Kawabata Shun‐ichiro , Lee Bok Luel
Published: March 2003

To elucidate the biochemical activation mechanism of the insect pro‐phenoloxidase (pro‐PO) system, we purified a 45‐kDa protein to homogeneity from the hemolymph ofTenebrio molitor(mealworm) larvae, and cloned its cDNA. The overall structure of the 45‐kDa protein is similar toDrosophilamasquerade serine proteinase homologue, which is an essential component inDrosophilamuscle development. ThisTenebriomasquerade‐like serine proteinase homologue (Tm‐mas) contains a trypsin‐like serine proteinase domain in the C‐terminal region, except for the substitution of Ser to Gly at the active site triad, and a disulfide‐knotted domain at the amino‐terminal region. When the purified 45‐kDa Tm‐mas was incubated with CM‐Toyopearl eluate solution containing pro‐PO and other pro‐PO activating factors, the resulting phenoloxidase (PO) activity was shown to be independent of Ca2+. This suggests that the purified 45‐kDa Tm‐mas is an activated form of pro‐PO activating factor. The55‐kDa zymogen form of Tm‐mas was detected in the hemolymph when PO activity was not evident. However, whenTenebriohemolymph was incubated with Ca2+, a 79‐kDaTenebriopro‐PO and the 55‐kDa zymogen Tm‐mas converted to 76‐kDa PO and 45‐kDa Tm‐mas, respectively, with detectable PO activity. Furthermore, whenTenebriohemolymph was incubated with Ca2+and β‐1,3‐glucan, the conversion of pro‐PO to PO and the 55‐kDa zymogen Tm‐mas to the 45‐kDa protein, was faster than in the presence of Ca2+only. These results suggest that the cleavage of the 55‐kDa zymogen of Tm‐mas by a limited proteolysis is necessary for PO activity, and the Tm‐mas is a pro‐PO activating cofactor.
Sub‐zero temperature inactivation of carboxypeptidase Y under high hydrostatic pressure
Authors: Kinsho Toshihiko , Ueno Hiroshi , Hayashi Rikimaru , Hashizume Chieko , Kimura Kunio
Published: March 2003

High hydrostatic pressure induced cold inactivation of carboxypeptidase Y. Carboxypeptidase Y was fully active when exposed to subzero temperature at 0.1 MPa; however, the enzyme became inactive when high hydrostatic pressure and subzero temperature were both applied. When the enzyme was treated at pressures higher than 300 MPa and temperatures lower than −5 °C, it underwent an irreversible inactivation in which nearly 50% of the α‐helical structure was lost as judged by circular dichroism spectral analysis. When the applied pressure was limited to below 200 MPa, the cold inactivation process appeared to be reversible. In thepresence of reducing agent, this reversible phenomenon, observed at below 200 MPa, diminished to give an inactive enzyme; the agent reduces some of disulfide bridge(s) in an area of the structure that is newly exposed area because of the cold inactivation. Such an area is unavailable if carboxypeptidase Y is in its native conformation. Because all the disulfide bridges in carboxypeptidase Y locate near the active site cleft, it is suggested that the structural destruction, if any, occurs preferentially in this disulfide rich area. A possible mechanism of pressure‐dependent cold inactivation of CPY is to destroy the α‐helix rich region, which creates an hydrophobic environment. This destruction is probably a result of the reallocation of water molecules. Experiments carried out in the presence of denaturing agents (SDS, urea, GdnHCl), salts, glycerol, and sucrose led to a conclusion consistent with the idea of water reallocation.
Ligand interactions and protein conformational changes of phosphopyridoxyl‐labeled Escherichia coli phosphoenolpyruvate carboxykinase determined by fluorescence spectroscopy
Authors: Encinas María Victoria , González‐Nilo Fernando D. , Goldie Hughes , Cardemil Emilio
Published: March 2003

Escherichia coli phosphoenolpyruvate (PEP) carboxykinase catalyzes the decarboxylation of oxaloacetate and transfer of the γ‐phosphoryl group of ATP to yield PEP, ADP, and CO2. The interaction of the enzyme with the substrates originates important domain movements in the protein. In this work, the interaction of several substrates and ligands with E. coli PEP carboxykinase has been studied in the phosphopyridoxyl (P‐pyridoxyl)‐enzyme adduct. The derivatized enzyme retained the substrate‐binding characteristics of the native protein, allowing the determination of several protein–ligand dissociation constants, as well as the role of Mg2+ and Mn2+ in substrate binding. The binding affinity of PEP to the enzyme–Mn2+ complex was −8.9 kcal·mol−1, which is 3.2 kcal·mol−1 more favorable than in the complex with Mg2+. For the substrate nucleotide–metal complexes, similar binding affinities (−6.0 to −6.2 kcal·mol−1) were found for either metal ion. The fluorescence decay of the P‐pyridoxyl group fitted to two lifetimes of 5.15 ns (34%) and 1.2 ns. These lifetimes were markedly altered in the derivatized enzyme–PEP–Mn complexes, and smaller changes were obtained in the presence of other substrates. Molecular models of the P‐pyridoxyl–E. coli PEP carboxykinase showed different degrees of solvent‐exposed surfaces for the P‐pyridoxyl group in the open (substrate‐free) and closed (substrate‐bound) forms, which are consistent with acrylamide quenching experiments, and suggest that the fluorescence changes reflect the domain movements of the protein in solution.
Modulation of inositol 1,4,5‐triphosphate concentration by prolyl endopeptidase inhibition
Authors: Schulz Ingo , Gerhartz Bernd , Neubauer Antje , Holloschi Andreas , Heiser Ulrich , Hafner Mathias , Demuth Hans‐Ulrich
Published: March 2003

Prolyl endopeptidase (PEP) is a proline‐specific oligopeptidase with a reported effect on learning and memory in different rat model systems. Using the astroglioma cell line U343, PEP expression was reduced by an antisense technique. Measuring different second‐messenger concentrations revealed an inverse correlation between inositol 1,4,5‐triphosphate [Ins(1,4,5)P3] concentration and PEP expression in the generated antisense cell lines. However, no effect on cAMP generation was observed. In addition, complete suppression of PEP activity by the specific inhibitor, Fmoc‐Ala‐Pyrr‐CN (5 µm) induced in U343 and other cell lines an enhanced, but delayed, increase in Ins(1,4,5)P3 concentration. This indicates that the proteolytic activity of PEP is responsible for the observed effect. Furthermore, the reduced PEP activity was found to amplify Substance P‐mediated stimulation of Ins(1,4,5)P3. The effect of reduced PEP activity on second‐messenger concentration indicates a novel intracellular function of this peptidase, which may have an impact on the reported cognitive enhancements due to PEP inhibition.
Characterization of native and recombinant A4 glyceraldehyde 3‐phosphate dehydrogenase
Authors: Graciet Emmanuelle , Lebreton Sandrine , Camadro Jean‐Michel , Gontero Brigitte
Published: March 2003

A4 glyceraldehyde 3‐phosphate dehydrogenase (GAPDH) was purified from the green alga Chlamydomonas reinhardtii and was also overexpressed in Escherichia coli. Both purified A4 tetramers of recombinant and native GAPDH were characterized for the first time. The pH optimum for both native and recombinant enzymes was close to 7.8. The pKs of the residues involved in catalysis indicate that a cysteine and a histidine may take part in catalysis by chloroplast GAPDH, as is the case for glycolytic GAPDH. Native and recombinant GAPDH show Michaelis–Menten kinetics with respect to their cofactors, NADH and NADPH, with greater specificity for NADPH. The kinetic parameters are similar to those of the heterotetrameric A2B2 spinach chloroplast GAPDH. Native C. reinhardtii and recombinant GAPDHs exhibit a cooperative behavior towards the substrate 1,3‐bisphosphoglycerate (BPGA). This positive cooperativity is specific to the C. reinhardtii enzyme, as higher plant A2B2 GAPDHs show Michaelis–Menten kinetics. Native GAPDH has twofold lower catalytic constant and K0.5 for BPGA than recombinant GAPDH. Mass spectrometry analysis of native GAPDH shows that it is a complex of GAPDH and the small protein CP12. In vitro reconstitution assays indicate that the kinetic differences are the result conformation changes of GAPDH upon association with CP12.
Structural studies of the O‐antigen isolated from the phenol‐soluble lipopolysaccharide of Acinetobacter baumannii (DNA group 2) strain 9
Authors: Haseley Simon R. , Holst Otto , Brade Helmut
Published: March 2003

A polysaccharide containing D‐GalNAc,
D‐Glc and 4‐acetamido‐4,6‐dideoxy‐
D‐glucose (Qui4NAc) was isolated from the phenol‐soluble lipopolysaccharide originating from Acinetobacter baumannii strain 9. The structure of the repeating unit was shown by means of monosaccharide analyses, Smith‐degradation, partial acid hydrolysis, mass spectrometry, and NMR spectroscopy to be a branched pentasaccharide, in which the tetrasaccharide backbone is built from amino sugars only.

α‐ D‐Glcp
1
 ∣
6
←3)‐β‐
D‐Quip4NAc‐(1←3)‐α‐
D‐GalpNAc‐(1←4)‐α‐
D‐GalpNAc‐(1←3)‐α‐
D‐GalpNAc‐(1←

The polysaccharide was identified by serological and western blot analyses as the O‐antigen of the lipopolysaccharide.
Phosphatidylinositol 4,5‐bisphosphate reverses the inhibition of RNA transcription caused by histone H1
Authors: Yu Haiyan , Fukami Kiyoko , Watanabe Yutaka , Ozaki Choichiro , Takenawa Tadaomi
Published: March 2003

Phosphatidylinositol 4,5‐bisphosphate [PtdIns(4, 5)P2] has been known to bind to the pleckstrin homology domain and the phosphotyrosine‐binding domain as well as actin‐binding proteins, and to regulate their functions. We have tried to find new PtdIns(4,5)P2‐binding proteins and to clarify the physiological effects of PtdIns(4,5)P2 on their function. We report here that histones H1 and H3 are PtdIns(4,5)P2‐binding proteins which were identified using antibodies specific to PtdIns(4,5)P2, H1, and H3. This binding was further confirmed by extracting PtdIns(4,5)P2 from purified histone H1 and H3. Furthermore, the binding site of PtdIns(4,5)P2 in histone H1 was found in the carboxyl‐terminal 103 amino acids. It was also shown that the amounts of PtdIns(4,5)P2 bound to H1 decrease when histone H1 is phosphorylated by protein kinase C but not by protein kinase A or cdc2 kinase, in vitro. The protein kinase C phosphorylation site is localized close to the PtdIns(4,5)P2‐binding site, suggesting that phosphorylation of histone H1 by protein kinase C interferes stereostructually with PtdIns(4,5)P2 binding. We further noticed that PtdIns(4,5)P2 binding to H1 counteracts the histone H1‐mediated repression of basal transcription by RNA polymerase II in a Drosophila transcription system in vitro. Phosphatidylinositol 4‐phosphate and phosphatidylinositol 3,4,5‐trisphosphate affect this transcription activity more weakly than PtdIns(4,5)P2, but PtdIns and other acidic lipids have no effect on this activity. These data indicate that PtdIns(4,5)P2 bound to nuclear protein histone H1 may contribute to the regulation of transcription in eukaryotic cells.
Structural study of the O‐linked sugar chains of human leukocyte tyrosine phosphatase CD45
Authors: Furukawa Kiyoshi , Funakoshi Yoko , Autero Matti , Horejsi Vaclav , Kobata Akira , Gahmberg Carl G.
Published: March 2003

The O‐linked sugar chains of the human leukocyte cell surface glycoprotein CD45 were released as tritium‐labeled oligosaccharides by β‐elimination in the presence of NaB3H4. Mono Q column chromatography revealed that they comprise neutral (64 %) and acidic (36 %) oligosaccharides, the latter of which were converted to neutral ones by Arthrobacter ureafaciens sialidase treatment. Structural studies of each oligosaccharide fractionated on a Bio‐Gel P‐4 column by sequential exoglycosidase digestion and by methylation analysis revealed that human leukocyte CD45 contains mainly core 1 and core 2 oligosaccharides, 15 % of which are modified with poly (N‐acetyllactosamine) chains in different extensions. CD45 consists of several isoforms which were isolated after cell surface sialic acid residues were labeled by periodate/NaB3H4 treatment. Bio‐Gel P‐6 column chromatography of a mixture of the tritium‐labeled glycopeptide/oligosaccharides obtained by pronase‐digestion followed by mild alkaline borohydride treatment showed that distribution of the sialylated core 2 oligosaccharides is different among CD45 isoforms.
Oxidation of tetrahydro‐β‐carboline by cytochrome P‐450cam
Authors: Sopková‐de Oliveira Santos Jana , Smith Jeremy C. , Delaforge Marcel , Virelizier Henri , Jankowski Christopher K.
Published: March 2003

Cytochrome P‐450cam oxidises 1,2,3,4‐tetrahydro‐β‐carboline, an indolic alkaloid. We report here measurements of the product distribution of this oxidation. To rationalise the experimental results, ab initio quantum‐chemistry calculations of the product stabilities and molecular‐dynamics calculations of the substrate‐binding mode in the active site were performed. The calculations suggest that the product distribution is influenced by both the relative intrinsic gas‐phase stabilities of the monohydroxy products and by conformational rearrangement of the active site on substrate binding.
Metabolic compartmentation of plastid prenyllipid biosynthesis
Authors: Keller Yves , Bouvier Florence , d'Harlingue Alain , Camara Bilal
Published: March 2003

The addition of phytyl side chain to chlorophylls, tocopherols and phylloquinone is prerequisite to their integration into plastid membranes. We have cloned a cDNA encoding a pre‐geranylgeranyl reductase from Arabidopsis thaliana. The deduced primary structure predicts a mature size with a molecular mass of 47 kDa and displays a characteristic dinucleotide binding domain. Geranylgeranyl reductase expressed in Escherichia coli sequentially catalyzes the reduction of geranylgeranyl‐chlorophyll a into phytyl‐chlorophyll a as well as the reduction of free geranylgeranyl diphosphate into phytyl diphosphate. Due to its multifunctionality and weak hydrophobicity, we suggest that in plastid the same geranylgeranyl reductase is recruited into the chlorophyll, the tocopherol and the phylloquinone pathways. The geranylgeranyl reductase gene is up‐regulated during etioplast to chloroplast and chloroplast to chromoplast development.
Elimination of the Cys558‐Cys566 bond in Lys78‐plasminogen
Authors: Linde Viggo , Nielsen Lars S. , Foster Donald C. , Petersen Lars C.
Published: March 2003

Plasminogen contains a unique disulphide bond, Cys558  Cys566, responsible for the cyclic nature of the peptide sequence surrounding the activation site at Arg561‐Val562. A recombinant [Ser558, Ser566]Lys78‐plasminogen variant was produced in which the two cysteine residues were replaced by serine residues. The variant was used to study the functional implications of removing the structural restrains imposed to the activation loop by this disulphide bond. Elimination of the Cys558  Cys566 bond attenuated activation by urokinase‐type plasminogen activator (uPA) and tissue‐type plasminogen activator (tPA), but resulted in an increased susceptibility to cleavage by trypsin and plasma kallikrein. Two opposite effects on the interaction of plasminogen with streptokinase were produced by modification of this bond; (a) attenuation of the rate at which the active complex with streptokinase was formed and (b) a 7.5‐fold increase in plasminogen activation catalysed by this complex. Activation by tPA in the presence of fibrin, in contrast to activation in its absence, was not attenuated by elimination of this disulphide bond. However, the activation rate as a function of plasminogen concentration followed a different saturation curve, and the fibrin degradation pattern was changed. The results suggest that the Cys558  Cys566 disulphide bond is of importance for the specificity of plasminogen. This applies to its activation and also to its role in subsequent fibrin clot degradation.
p66/p51 and p51/p51 recombinant forms of reverse transcriptase from human immunodeficiency virus type 1
Authors: Dufour Emmanuelle , Dirani‐Diab Rim El , Boulmé Florence , Fournier Michel , Nevinsky Georgyi , Tarrago‐Litvak Laura , Litvak Simon , Andreola Marie‐Line
Published: March 2003

Human immunodeficiency virus type‐1 (HIV‐1) reverse transcriptase (RT) initiates reverse transcription from tRNALys3. HIV‐1 RT is a heterodimer consisting of two polypeptides, p66 and p51. In this work, the possible role of each subunit of RT in the interaction with its natural primer tRNALys3 was studied. Two recombinant forms of HIV‐1 RT, heterodimer p66/p51 and homodimer p51/p51, were used. Previously we have expressed and purified recombinant RT p51/p51 which possesses DNA polymerase activity [El Dirani‐Diab, R., Andreola, M. L., Nevinsky, G., Tharaud, D., Barr, P. J., Litvak, S. & Tarrago‐Litvak, L. (1992) FEBS Lett. 301, 23−28]. Here we show that HIV‐1 RT p51/p51 displays certain properties very similar to the p66/p51 recombinant enzyme. The homodimer was able to anneal tRNALys3 to the primer‐binding site of the HIV‐1 RNA template leading to a functional complex capable of synthesizing cDNA. Further, the p51/p51 enzyme behaved like RT p66/p51 concerning the strong inhibition produced by a non‐nucleoside RT inhibitor. These data show that for RT p51/p51, one of the subunits of the homodimer adopts a conformation similar to the catalytic subunit (p66) present in the heterodimeric form.

Part of this work was devoted to the study of the complex between the recombinant forms of HIV‐1 RT and its primer tRNA. Each enzymatic form was cross‐linked to tRNALys3 in the presence of a platinum derivative, giving different ribonucleoprotein complexes of molecular masses higher than 100 kDa, suggesting that primer tRNA may interact with both subunits in the heterodimeric enzyme. After RNase A treatment of the complex RT p66/p51 tRNA, the label was mainly found to migrate with the p66 subunit, although some cross‐linking was also found associated to the p51 subunit. These results show that the p66 and p51 subunits of RT interact with tRNALys3. Moreover, cross‐linking of tRNALys3 with HIV‐1 RT p66/p51 in the presence of a DNA template containing the primer‐binding‐site sequence yielded an enzymatically active complex.
Modulation of the substrate specificity of Escherichia coli dimethylsulfoxide reductase
Authors: Simala‐Grant Joanne L. , Weiner Joel H.
Published: March 2003

X‐ray crystallographic studies of the Rhodobacter sphaeroides dimethyl sulfoxide (Me2SO) reductase [Schindelin, H., Kisker, C., Hilton, J., Rajagopalan, K. V., & Rees, D. C. (1996) Science, 272, 1615−1620] indicated that the active site is at the bottom of a 25‐Å funnel. Substrates must travel to the bottom of the funnel for reduction to occur. The homologous DmsA subunit of the trimeric Escherichia coli Me2SO reductase, was subjected to site‐directed mutagenesis of residues potentially lining the bottom of the funnel, based on sequence alignment of the E. coli and Rhodobacter Me2SO reductases. Sixteen E. coli DmsA mutants were characterized. Mutants G167N, A178Q, Q179I and R217Q showed functional impairment, as indicated by abnormal anaerobic growth with Me2SO as the sole terminal acceptor, in a recombinant strain deleted for chromosomal dmsABC. The kinetic parameters of the mutant enzymes were examined using the artificial electron donor benzyl viologen and the quinone analogue dimethylnaphthoquinone, with Me2SO and pyridine N‐oxide as electron acceptors. Mutants A178Q and R217Q showed dramatic alterations of their electron‐acceptor Km, with values at least 35‐fold less or greater than wild‐type values, respectively, for Me2SO and pyridine N‐oxide. T148S showed altered kinetic parameters for pyridine N‐oxide and Me2SO, with Km and kcat decreasing and increasing approximately fourfold, respectively. Other mutants showed less drastic alterations in kinetic parameters. This analysis has identified amino acids important in substrate binding and catalysis.