bluuebunny/crossref_metadata_2025 huggingface dataset:
Communication and decision‐making in labour: do birth plans make a difference?
Authors: Brown Stephanie J , Lumley Judith
Published: March 2003

Objectives

To assess usage of birth plans, and examine differences in social and obstetric characteristics, and intrapartum experiences of women who did and did not use a birth plan.

Design

Population‐based survey distributed by hospitals and home birth practitioners, 6–7 months post‐natally.

Setting and participants

Women who gave birth in Victoria, Australia over a 2‐week period in September, 1993, excluding those who had a stillbirth or neonatal death.

Main outcome measures

Use of a written birth plan; perceived helpfulness, advantages and disadvantages of birth plans; relationship between use of birth plans and overall rating of intrapartum care, and involvement in decision‐making.

Results

Twenty per cent of women (270/1336) had prepared a written birth plan and discussed it with caregivers. Women who made use of a birth plan were more likely to be satisfied with pain relief (OR = 1.74[1.3–2.3]), but did not differ from women not completing a birth plan in terms of their overall rating of intrapartum care, or involvement in making decisions about their care.

Conclusions

The lack of association between use of a written birth plan and variables assessing women's views of intrapartum care suggest there are insufficient grounds for continuing to advocate a policy of encouraging pregnant women to complete written birth plans, unless it is within the context of a well‐designed randomized trial able to provide further evidence regarding their effectiveness.
Is ‘shared decision‐making’ feasible in consultations for upper respiratory tract infections? Assessing the influence of antibiotic expectations using discourse analysis
Authors: Elwyn Glyn , Gwyn Richard , Edwards Adrian , Grol Richard
Published: March 2003

Objectives

To examine the discourse of consultations in which conflict occurs between parents and clinicians about the necessity of antibiotics to treat an upper respiratory tract infection. To appraise the feasibility of shared decision‐making in such consultations.

Design

A qualitative study using discourse analysis techniques.

Setting

A general practice with 12 500 patients in an urban area of Cardiff, Wales.

Participants

Two consultations were purposively selected from a number of audiotaped sessions. The consultations took place during normal clinics in which appointments are booked at 7‐minute intervals. The practitioner is known to be interested in involving patients in treatment decisions.

Method

Discourse analysis was employed to examine the consultation transcripts. This analysis was then compared with the theoretical competencies proposed for ‘shared decision‐making’.

Results

The consultations exhibit less rational strategies than those suggested by the shared decision‐making model. Strong parental views are expressed (overtly and covertly) which seem derived from prior experiences of similar illnesses and prescribing behaviours. The clinician responds by emphasizing the ‘normality’ of upper respiratory tract infections and their recurrence, accompanied by expressions that antibiotic treatment is ineffective in ‘viral’ illness – the suggested diagnosis. The competencies of ‘shared decision‐making’ are not exhibited.

Conclusions

The current understanding of shared decision‐making needs to be developed for those situations where there are dis‐agreements due to the strongly held views of the participants. Clinicians have limited strategies in situations where patient treatment preferences are opposed to professional views. Dispelling ‘misconceptions’ by sharing information and negotiating agreed management plans are recommended. But it seems that communication skills, information content and consultation length have to receive attention if such strategies are to be employed successfully.
A qualitative study of women’s views about how health professionals communicate about infant feeding
Authors: Hoddinott Pat , Pill Roisin
Published: March 2003

Objective

To look at how communication by health professionals about infant feeding is perceived by first time mothers.

Design

Qualitative semi‐structured interviews early in pregnancy and 6–10 weeks after birth.

Subjects and setting

Twenty‐one white, low income women expecting their first baby were interviewed mostly at home, often with their partner or a relative.

Results

The personal and practical aspects of infant feeding which were important to women were seldom discussed in detail in ante‐natal interviews. In post‐natal interviews women described how words alone encouraging them to breastfeed were insufficient. Apprenticeship style learning of practical skills was valued, particularly time patiently spent watching them feed their baby. Women preferred to be shown skills rather than be told how to do them. Some felt pressure to breastfeed and bottle feeding mothers on post‐natal wards felt neglected in comparison. Women preferred their own decision‐making to be facilitated rather than being advised what to do. Some women experienced distress exposing their breasts and being touched by health professionals. Continuity of care and forming a personal relationship with a health professional who could reassure them were key factors associated with satisfaction with infant feeding communication.

Conclusions

The infant feeding goal for many women is a contented, thriving baby. In contrast, women perceive that the goal for health professionals is the continuation of breastfeeding. These differing goals can give rise to dissatisfaction with communication which is often seen as ‘breastfeeding centred’ rather than ‘woman centred.’ Words alone offering support for breastfeeding were often inadequate and women valued practical demonstrations and being shown how to feed their baby. Spending time with a caring midwife with whom the woman had developed a personal, continuing relationship was highly valued. Women were keen to maintain ownership, control and responsibility for their own decision‐making about infant feeding.
Development and implementation of a science training course for breast cancer activists: Project LEAD (leadership, education and advocacy development)
Authors: Dickersin Kay , Braun Lundy , Mead Margaret , Millikan Robert , Wu Anna M. , Pietenpol Jennifer , Troyan Susan , Anderson Benjamin , Visco Frances
Published: March 2003

Objective To develop and implement Project LEAD (leadership, education, and advocacy development), a science course for breast cancer activists.

Population Students were breast cancer activists and other consumers, mainly affiliated with advocacy organizations in the United States of America.

Setting Project LEAD is offered by the National Breast Cancer Coalition; the course takes place over 5 days and is offered 4 times a year, in various cities in the United States of America.

Results The Project LEAD curriculum has developed over 5 years to include lectures, problem‐based study groups, case studies, interactive critical appraisal sessions, a seminar by an ‘expert’ scientist, role play, and homework components. A core faculty has been valuable for evaluating and revising the course and has proved necessary to provide consistent high quality teaching. Course evaluations indicated that students gained critical appraisal skills, enhanced their knowledge and developed confidence in selected areas of basic science and epidemiology.

Conclusions Project LEAD comprises a unique curriculum for training breast cancer activists in science and critical appraisal. Course evaluations indicate that students gain confidence and skills from the course.
Overexpression of a recombinant wild‐type and His‐tagged Bacillus subtilis glycine oxidase in Escherichia coli
Authors: Job Viviana , Molla Gianluca , Pilone Mirella S. , Pollegioni Loredano
Published: March 2003

We have cloned the gene coding for the Bacillus subtilis glycine oxidase (GO), a new flavoprotein that oxidizes glycine and sarcosine to the corresponding α‐keto acid, ammonia and hydrogen peroxide. By inserting the DNA encoding for GO into the multiple cloning site of the expression vector pT7.7 we produced a recombinant plasmid (pT7‐GO). The pT7‐GO encodes a fully active fusion protein with six additional residues at the N‐terminus of GO (MARIRA). In BL21(DE3)pLysS Escherichia coli cells, and under optimal isopropyl thio‐β‐d‐galactoside induction conditions, soluble and active chimeric GO was expressed up to 1.14 U g−1 of cell (and a fermentation yield of 3.82 U·L−1 of fermentation broth). An N‐terminal His‐tagged protein (HisGO) was also successfully expressed in E. coli as a soluble protein and a fully active holoenzyme. HisGO represents ≈ 3.9% of the total soluble protein content of the cell. The His‐tagged GO was purified in a single step by nickel‐chelate chromatography to a specific activity of 1.06 U·mg−1 protein at 25 °C and with a yield of 98%. The characterization of the purified enzyme showed that GO is a homotetramer of ≈ 180 kDa with the spectral properties typical of flavoproteins. GO exhibits good thermal stability, with a Tm of 46 °C after 30 min incubation; its stability is maximal in the 7.0–8.5 pH range. A comparison of amino‐acid sequence and substrate specificity indicates that GO has similarities to other flavoenzymes acting on primary amines and on d‐amino acids.
Irregular spiking in free calcium concentration in single, human platelets
Authors: van Gorp Roosje M. A. , Feijge Marion A. H. , Vuist Wim M. J. , Rook Martin B. , Heemskerk Johan W. M.
Published: March 2003

Fluorescence ratio imaging indicates that immobilized, aspirin‐treated platelets, loaded with Fura‐2, respond to inositol 1,4,5‐trisphosphate‐ (InsP3)‐generating agonists such as thrombin by high‐frequency, irregular rises in cytosolic [Ca2+]i with spikes that vary in peak level and peak‐to‐peak interval. This differs from the regular [Ca2+]i oscillations observed in other, larger cells. We found that the thiol‐reactive compounds thimerosal (10 µm) and U73122 (10 µm) evoked similar irregular Ca2+ responses in platelets, but in this case in the absence of InsP3 generation. Thrombin‐induced spiking was acutely abolished by inhibiting phospholipase C or elevating intracellular cAMP levels, while spiking with sulfhydryl reagents was only partially blocked by cAMP elevation. Confocal laser scanning microscopy using fluo‐3‐loaded platelets indicated that, with all agonists or conditions, the irregular spikes were almost instantaneously raised in various regions within a single platelet. When using saponin‐permeabilized platelets, we found that InsP3‐induced Ca2+ release from stores was stimulated by modest Ca2+ concentrations, pointing to a mechanism of InsP3‐dependent Ca2+‐induced Ca2+ release (CICR). This process was completely inhibitable by heparin. The Ca2+ release by InsP3, but not the CICR sensor, was negatively regulated by cAMP elevation. Thimerosal treatment did not release Ca2+ from intracellular stores, but markedly potentiated the stimulatory effect of InsP3. In contrast, U73122 caused a heparin/cAMP‐insensitive Ca2+ leak from stores that differed from those used by InsP3. Taken together, these results demonstrate that InsP3 receptor channels play a crucial role in the irregular, spiking Ca2+ signal of intact platelets, even when induced by agents such as thimerosal or U73122 which do not stimulate InsP3 formation. The irregular Ca2+ release events appear to be subjected to extensive regulation by: (a) InsP3 level, (b) the potentiating effect of elevated Ca2+ on InsP3 action via CICR, (c) InsP3 channel sensitization by sulfhydryl (thimerosal) modification, (d) InsP3 channel‐independent Ca2+ leak with U73122, and (e) down‐regulation via cAMP elevation. The observation that individual Ca2+ peaks were generated in various parts of a platelet at similar intervals and amplitudes points to effective cooperation of the various stores in the Ca2+‐release process.
Cloning and characterization of novel snake venom proteins that block smooth muscle contraction
Authors: Yamazaki Yasuo , Koike Hisashi , Sugiyama Yusuke , Motoyoshi Kazuko , Wada Taeko , Hishinuma Shigeru , Mita Mitsuo , Morita Takashi
Published: March 2003

In this study, we isolated a 25‐kDa novel snake venom protein, designated ablomin, from the venom of the Japanese Mamushi snake (Agkistrodon blomhoffi). The amino‐acid sequence of this protein was determined by peptide sequencing and cDNA cloning. The deduced sequence showed high similarity to helothermine from the Mexican beaded lizard (Heloderma horridum horridum), which blocks voltage‐gated calcium and potassium channels, and ryanodine receptors. Ablomin blocked contraction of rat tail arterial smooth muscle elicited by high K+‐induced depolarization in the 0.1–1 µm range, but did not block caffeine‐stimulated contraction. Furthermore, we isolated three other proteins from snake venoms that are homologous to ablomin and cloned the corresponding cDNAs. Two of these homologous proteins, triflin and latisemin, also inhibited high K+‐induced contraction of the artery. These results indicate that several snake venoms contain novel proteins with neurotoxin‐like activity.
Unique structural determinants in the signal peptides of `spontaneously' inserting thylakoid membrane proteins
Authors: Tissier Christophe , Woolhead Cheryl A. , Robinson Colin
Published: March 2003

A series of thylakoid membrane proteins, including PsbX, PsbY and PsbW, are synthesized with cleavable signal peptides yet inserted using none of the known Sec/SRP/Tat/Oxa1‐type insertion machineries. Here, we show that, although superficially similar to Sec‐type signal peptides, these thylakoidal signal peptides contain very different determinants. First, we show that basic residues in the N‐terminal domain are not important, ruling out electrostatic interactions as an essential element of the insertion mechanism, and implying a fundamentally different targeting mechanism when compared with the structurally similar M13 procoat. Second, we show that acidic residues in the C‐domain are essential for the efficient maturation of the PsbX and PsbY‐A1 peptides, and that even a single substitution of the −5 Glu by Val in the PsbX signal peptide abolishes maturation in the thylakoid. Processing efficiency is restored to an extent, but not completely, by the highly hydrophilic Asn, implying that this domain is required to be hydrophilic, but preferably negatively charged, in order to present the cleavage site in an optimal manner. We show that substitution of the PsbX C‐domain Glu residues by Val leads to a burial of the cleavage site within the bilayer although insertion is unaffected. Finally, we show that substitution of the Glu residues in the lumenal A2 loop of the PsbY polyprotein leads to a block in cleavage on the stromal side of the membrane, and present evidence that the PsbY‐A2 signal peptide is required to be relatively hydrophilic and unable to adopt a transmembrane conformation on its own. These data indicate that, rather than being merely additional hydrophobic regions to promote insertion, the signal peptides of these thylakoid proteins are complex domains with uniquely stringent requirements in the C‐domain and/or translocated loop regions.
Synthesis of phosphoenol pyruvate (PEP) analogues and evaluation as inhibitors of PEP‐utilizing enzymes
Authors: García‐Alles Luis Fernando , Erni Bernhard
Published: March 2003

The synthesis of 10 new phosphoenolpyruvate (PEP) analogues with modifications in the phosphate and the carboxylate function is described. Included are two potential irreversible inhibitors of PEP‐utilizing enzymes. One incorporates a reactive chloromethylphosphonate function replacing the phosphate group of PEP. The second contains a chloromethyl group substituting for the carboxylate function of PEP. An improved procedure for the preparation of the known (Z)‐ and (E)‐3‐chloro‐PEP is also given. The isomers were obtained as a 4 : 1 mixture, resolved by anion‐exchange chromatography after the last reaction step. The stereochemistry of the two isomers was unequivocally assigned from the 3JH‐C coupling constants between the carboxylate carbons and the vinyl protons.

All of these and other known PEP‐analogues were tested as reversible and irreversible inhibitors of Mg2+‐ and Mn2+‐ activated PEP‐utilizing enzymes: enzyme I of the phosphoenolpyruvate:sugar phosphotransferase system (PTS), pyruvate kinase, PEP carboxylase and enolase. Without exception, the most potent inhibitors were those with substitution of a vinyl proton. Modification of the phosphate and the carboxylate groups resulted in less effective compounds. Enzyme I was the least tolerant to such modifications. Among the carboxylate‐modified analogues, only those replaced by a negatively charged group inhibited pyruvate kinase and enolase. Remarkably, the activity of PEP carboxylase was stimulated by derivatives with neutral groups at this position in the presence of Mg2+, but not with Mn2+. For the irreversible inhibition of these enzymes, (Z)‐3‐Cl‐PEP was found to be a very fast‐acting and efficient suicide inhibitor of enzyme I (t1/2 = 0.7 min).
The S100A8/A9 protein as a partner for the cytosolic factors of NADPH oxidase activation in neutrophils
Authors: Doussiere Jacques , Bouzidi Farid , Vignais Pierre V.
Published: March 2003

In a previous study, the S100A8/A9 protein, a Ca2+‐ and arachidonic acid‐binding protein, abundant in neutrophil cytosol, was found to potentiate the activation of the redox component of the O2– generating oxidase in neutrophils, namely the membrane‐bound flavocytochrome b, by the cytosolic phox proteins p67phox, p47phox and Rac (Doussière J., Bouzidi F. and Vignais P.V. (2001) Biochem. Biophys. Res. Commun.285, 1317–1320). This led us to check by immunoprecipitation and protein fractionation whether the cytosolic phox proteins could bind to S100A8/A9. Following incubation of a cytosolic extract from nonactivated bovine neutrophil with protein A–Sepharose bound to anti‐p67phox antibodies, the recovered immunoprecipitate contained the S100 protein, p47phox and p67phox. Cytosolic protein fractionation comprised two successive chromatographic steps on hydroxyapatite and DEAE cellulose, followed by isoelectric focusing. The S100A8/A9 heterodimeric protein comigrated with the cytosolic phox proteins, and more particularly with p67phox and Rac2, whereas the isolated S100A8 protein displayed a tendancy to bind to p47phox. Using a semirecombinant cell‐free system of oxidase activation consisting of recombinant p67phox, p47phox and Rac2, neutrophil membranes and arachidonic acid, we found that the S100A8/A9‐dependent increase in the elicited oxidase activity corresponded to an increase in the turnover of the membrane‐bound flavocytochrome b, but not to a change of affinity for NADPH or O2. In the absence of S100A8/A9, oxidase activation departed from michaelian kinetics above a critical threshold concentration of cytosolic phox proteins. Addition of S100A8/A9 to the cell‐free system rendered the kinetics fully michaelian. The propensity of S100A8/A9 to bind the cytosolic phox proteins, and the effects of S100A8/A9 on the kinetics of oxidase activation, suggest that S100A8/A9 might be a scaffold protein for the cytosolic phox proteins or might help to deliver arachidonic acid to the oxidase, thus favoring the productive interaction of the cytosolic phox proteins with the membrane‐bound flavocytochrome b.
Environmentally coupled hydrogen tunneling
Authors: Knapp Michael J. , Klinman Judith P.
Published: March 2003

Many biological C‐H activation reactions exhibit nonclassical kinetic isotope effects (KIEs). These nonclassical KIEs are too large (kH/kD > 7) and/or exhibit unusual temperature dependence such that the Arrhenius prefactor KIEs (AH/AD) fall outside of the semiclassical range near unity. The focus of this minireview is to discuss such KIEs within the context of the environmentally coupled hydrogen tunneling model. Full tunneling models of hydrogen transfer assume that protein or solvent fluctuations generate a reactive configuration along the classical, heavy‐atom coordinate, from which the hydrogen transfers via nuclear tunneling. Environmentally coupled tunneling also invokes an environmental vibration (gating) that modulates the tunneling barrier, leading to a temperature‐dependent KIE. These properties directly link enzyme fluctuations to the reaction coordinate for hydrogen transfer, making a quantum view of hydrogen transfer necessarily a dynamic view of catalysis. The environmentally coupled hydrogen tunneling model leads to a range of magnitudes of KIEs, which reflect the tunneling barrier, and a range of AH/AD values, which reflect the extent to which gating modulates hydrogen transfer. Gating is the primary determinant of the temperature dependence of the KIE within this model, providing insight into the importance of this motion in modulating the reaction coordinate. The potential use of variable temperature KIEs as a direct probe of coupling between environmental dynamics and the reaction coordinate is described. The evolution from application of a tunneling correction to a full tunneling model in enzymatic H transfer reactions is discussed in the context of a thermophilic alcohol dehydrogenase and soybean lipoxygenase‐1.
Intracellular pH homeostasis in the filamentous fungus Aspergillus niger
Authors: Hesse Stephan J. A. , Ruijter George J. G , Dijkema Cor , Visser Jaap
Published: March 2003

Intracellular pH homeostasis in the filamentous fungus Aspergillus niger was measured in real time by 31P NMR during perfusion in the NMR tube of fungal biomass immobilized in Ca2+‐alginate beads. The fungus maintained constant cytoplasmic pH (pHcyt) and vacuolar pH (pHvac) values of 7.6 and 6.2, respectively, when the extracellular pH (pHex) was varied between 1.5 and 7.0 in the presence of citrate. Intracellular metabolism did not collapse until a ΔpH over the cytoplasmic membrane of 6.6–6.7 was reached (pHex 0.7–0.8). Maintenance of these large pH differences was possible without increased respiration compared to pHex 5.8. Perfusion in the presence of various hexoses and pentoses (pHex 5.8) revealed that the magnitude of ΔpH values over the cytoplasmic and vacuolar membrane could be linked to the carbon catabolite repressing properties of the carbon source. Also, larger ΔpH values coincided with a higher degree of respiration and increased accumulation of polyphosphate. Addition of protonophore (carbonyl cyanide m‐chlorophenylhydrazone, CCCP) to the perfusion buffer led to decreased ATP levels, increased respiration and a partial (1 µm CCCP), transient (2 µm CCCP) or permanent (10 µm CCCP) collapse of the vacuolar membrane ΔpH. Nonlethal levels of the metabolic inhibitor azide (N3–, 0.1 mm) caused a transient decrease in pHcyt that was closely paralleled by a transient vacuolar acidification. Vacuolar H+ influx in response to cytoplasmic acidification, also observed during extreme medium acidification, indicates a role in pH homeostasis for this organelle. Finally, 31P NMR spectra of citric acid producing A. niger mycelium showed that despite a combination of low pHex (1.8) and a high acid‐secreting capacity, pHcyt and pHvac values were still well maintained (pH 7.5 and 6.4, respectively).
Characterization of four substrates emphasizes kinetic similarity between insect and human C‐domain angiotensin‐converting enzyme
Authors: Hens Korneel , Vandingenen Anick , Macours Nathalie , Baggerman Geert , Carmona Karaoglanovic Adriana , Schoofs Liliane , De Loof Arnold , Huybrechts Roger
Published: March 2003

Angiotensin converting enzyme (ACE) was already discovered in insects in 1994, but its physiological role is still enigmatic. We have addressed this problem by purifying four new ACE substrates from the ovaries of the grey fleshfly, Neobellieria bullata. Their primary structures were identified as NKLKPSQWISLSD (Neb‐ODAIF‐11−13), NKLKPSQWI (Neb‐ODAIF‐11−9), SLKPSNWLTPSE (Neb‐ODAIF‐2) and LEQIYHL. Database analysis showed significant homology with amino acid sequence stretches as present in the N‐terminal part of several fly yolk proteins. An antiserum raised against Neb‐ODAIF‐11−9 immunostained one out of three yolk protein bands of SDS/PAGE‐separated fly haemolymph and egg homogenate, thus confirming that these peptides originate from a yolk protein gene product. Kinetic analysis of these peptides and of the peptides Neb‐ODAIF and Neb‐ODAIF‐11−7 with insect ACE and human ACE show both similar and unique properties for insect ACE as compared with human C‐domain ACE.
Identification of hepatopoietin dimerization, its interacting regions and alternative splicing of its transcription
Authors: Li Yong , Wei Kaihua , Lu Chengrong , Li Yingxian , Li Ming , Xing Guichun , Wei Handong , Wang Qingming , Chen Jizhong , Wu Chutse , Chen Huipeng , Yang Songcheng , He Fuchu
Published: March 2003

Hepatopoietin (HPO) is a novel human hepatotrophic growth factor. Recently, we demonstrated that the extracellular ligand form of HPO can stimulate proliferation of hepatic cells via its specific receptor, which is on the surface of these cells. Also, the intracellular form of HPO potentiates the transcriptional factor AP‐1. Intriguingly, a variety of HPO complexes with different molecular masses were detected in hepatocytes. In this study, we screened a human fetal liver cDNA library using the yeast two hybrid system with HPO as bait, hoping to find an HPO‐binding protein. Surprisingly, HPO, itself, and a previously unidentified alternatively spliced form of HPO (named HPO23) were identified as interacting with HPO, indicating that HPO may exist as a homodimer or heterodimer. These results were further confirmed in vitro and in vivo. First, mass spectrometry analysis demonstrated that recombinant human HPO exist as a homodimer and that disulfide bonds were involved in the formation of rhHPO dimer. Secondly, a pull‐down experiment confirmed that GST–HPO and HA–HPO, could bind together in vitro. Using HPO and various HPO deletion mutants, we identified the extreme 30 amino acids at both N‐ and C‐termini are essential for HPO dimerization. Finally, Western blotting analysis demonstrated that HPO exists as two isoforms at 15 kDa and 23 kDa (HPO23) in liver cells, the 15 kDa species is located in nucleus, and the 23 kDa species mainly in the cytoplasm. These results implicated that the capacity of HPO to form both homodimers and heterodimers with the alternatively spliced forms might contribute to the existence of various HPO complexes in hepatic cells.
Structural heterogeneity of pyrimidine/purine‐biased DNA sequence analyzed by atomic force microscopy
Authors: Kato Mikio , McAllister Chad J. , Hokabe Shingo , Shimizu Nobuyoshi , Lyubchenko Yuri L.
Published: March 2003

We report here the direct evidence for the formation of alternative DNA structures in a plasmid DNA, termed pTIR10, containing a 0.23‐kb pyrimidine/purine‐biased (Pyr/Pur) stretch isolated from the rat genome. Long Pyr/Pur sequences are abundant in eukaryotic genomes, and they may modulate the biological activity of genes and genomes via formation of various types of triplex‐related structures. The plasmid DNA in sodium acetate buffer (pH 4.35) was deposited on APS‐modified mica, and after drying it was imaged with an atomic force microscope in air. Various types of thick protrusions have been observed on pTIR10 DNA. Structural parameters (width and height) of DNA molecules suggest that the alternative structures observed here are variations on the theme of an intramolecular triplex. The biological relevance of the structural features within Pyr/Pur stretches is discussed.
GNA33 from Neisseria meningitidis serogroup B encodes a membrane‐bound lytic transglycosylase (MltA)
Authors: Jennings Gary T. , Savino Silvana , Marchetti Elisa , Aricò Beatrice , Kast Thomas , Baldi Lucia , Ursinus Astrid , Höltje Joachim‐Volker , Nicholas Robert A. , Rappuoli Rino , Grandi Guido
Published: March 2003

In a previous study, we used the genome of serogroup B Meningococcus to identify novel vaccine candidates. One of these molecules, GNA33, is well conserved among Meningococcus B strains, other Meningococcus serogroups and Gonococcus and induces bactericidal antibodies as a result of being a mimetic antigen of the PorA epitope P1.2. GNA33 encodes a 48‐kDa lipoprotein that is 34.5% identical with membrane‐bound lytic transglycosylase A (MltA) from Escherichia coli. In this study, we expressed GNA33, i.e. Meningococcus MltA, as a lipoprotein in E. coli. The lipoprotein nature of recombinant MltA was demonstrated by incorporation of [3H]palmitate. MltA lipoprotein was purified to homogeneity from E. coli membranes by cation‐exchange chromatography. Muramidase activity was confirmed when MltA was shown to degrade insoluble murein sacculi and unsubstituted glycan strands. HPLC analysis demonstrated the formation of 1,6‐anhydrodisaccharide tripeptide and tetrapeptide reaction products, confirming that the protein is a lytic transglycosylase. Optimal muramidase activity was observed at pH 5.5 and 37 °C and enhanced by Mg2+, Mn2+ and Ca2+. The addition of Ni2+ and EDTA had no significant effect on activity, whereas Zn2+ inhibited activity. Triton X‐100 stimulated activity 5.1‐fold. Affinity chromatography indicated that MltA interacts with penicillin‐binding protein 2 from Meningococcus B, and, like MltA from E. coli, may form part of a multienzyme complex.
Signalling pathways and combinatory effects of insulin and amino acids in isolated rat hepatocytes
Authors: Krause Ulrike , Bertrand Luc , Maisin Liliane , Rosa Maria , Hue Louis
Published: March 2003

Liver metabolism is influenced by hormones and nutrients. Amino acids such as glutamine or leucine induce an anabolic response, which resembles that of insulin in muscle and adipose tissue. In this work, the signalling pathways and the effects of insulin were compared to those of glutamine and leucine in isolated hepatocytes from normal and streptozotocin‐diabetic rats. Glutamine increased cell volume and induced an anabolic response characterized by an activation of acetyl‐CoA carboxylase (ACC), glycogen synthase (GS) and p70 ribosomal S6 kinase (p70S6K), the key enzymes in fatty acid, glycogen and protein synthesis, respectively. The effects of glutamine were independent of insulin and did not share its signalling components. Leucine, which is poorly metabolized by the liver and does not modify cell volume, activated ACC and p70S6K, and exerted a synergistic effect on the glutamine‐induced activation of ACC and p70S6K. These amino acids did not affect insulin signalling. Insulin alone had no anabolic effect in hepatocytes, despite the activation of protein kinase B. Nevertheless, it enhanced the activation of ACC and p70S6K induced by leucine. However, insulin injected intravenously activated rat liver p70S6K. In hepatocytes from streptozotocin‐diabetic animals, the metabolic responses to the amino acids and insulin were similar to those in normal hepatocytes. We conclude that glutamine, insulin and leucine exert different effects that are mediated by different signalling pathways, although their effects are combinatory. The anabolic effect of insulin in hepatocytes was strictly dependent on the permissive action of leucine.
Granule‐bound starch synthase I
Authors: Wattebled Fabrice , Buléon Alain , Bouchet Brigitte , Ral Jean‐Philippe , Liénard Luc , Delvallé David , Binderup Kim , Dauvillée David , Ball Steven , D'Hulst Christophe
Published: March 2003

Starch defines a semicrystalline polymer made of two different polysaccharide fractions. The A‐ and B‐type crystalline lattices define the distinct structures reported in cereal and tuber starches, respectively. Amylopectin, the major fraction of starch, is thought to be chiefly responsible for this semicrystalline organization while amylose is generally considered as an amorphous polymer with little or no impact on the overall crystalline organization. STA2 represents a Chlamydomonas reinhardtii gene required for both amylose biosynthesis and the presence of significant granule‐bound starch synthase I (GBSSI) activity. We show that this locus encodes a 69 kDa starch synthase and report the organization of the corresponding STA2 locus. This enzyme displays a specific activity an order of magnitude higher than those reported for most vascular plants. This property enables us to report a detailed characterization of amylose synthesis both in vivo and invitro. We show that GBSSI is capable of synthesizing a significant number of crystalline structures within starch. Quantifications of amount and type of crystals synthesized under these conditions show that GBSSI induces the formation of B‐type crystals either in close association with pre‐existing amorphous amylopectin or by crystallization of entirely de novo synthesized material.
Leishmania donovani phosphofructokinase
Authors: López Claudia , Chevalier Nathalie , Hannaert Véronique , Rigden Daniel J. , Michels Paul A. M. , Ramirez Jose Luis
Published: March 2003

The characterization of the gene encoding Leishmania donovani phosphofructokinase (PFK) and the biochemical properties of the expressed enzyme are reported. L. donovani has a single PFK gene copy per haploid genome that encodes a polypeptide with a deduced molecular mass of 53 988 and a pI of 9.26. The predicted amino acid sequence contains a C‐terminal tripeptide that conforms to an established signal for glycosome targeting. L. donovani PFK showed most sequence similarity to inorganic pyrophosphate (PPi)‐dependent PFKs, despite being ATP‐dependent. It thereby resembles PFKs from other Kinetoplastida such as Trypanosoma brucei, Trypanoplasma borreli (characterized in this study), and a PFK found in Entamoeba histolytica. It exhibited hyperbolic kinetics with respect to ATP whereas the binding of the other substrate, fructose 6‐phosphate, showed slight positive cooperativity. PPi, even at high concentrations, did not have any effect. AMP acted as an activator of PFK, shifting its kinetics for fructose 6‐phosphate from slightly sigmoid to hyperbolic, and increasing considerably the affinity for this substrate, whereas GDP did not have any effect. Modelling studies and site‐directed mutagenesis were employed to shed light on the structural basis for the AMP effector specificity and on ATP/PPi specificity among PFKs.
Purification and partial characterization of seven glutathione S ‐transferase isoforms from the clam Ruditapes decussatus
Authors: Hoarau Pascal , Garello Ginette , Gnassia‐Barelli Mauricette , Romeo Michèle , Girard Jean‐Pierre
Published: March 2003

This paper deals with the purification and the partial characterization of glutathione S‐transferase (GST) isoforms from the clam Ruditapes decussatus. For the first step of purification, two affinity columns, reduced glutathione (GSH)–agarose and S‐hexyl GSH–agarose, were mounted in series. Four affinity fractions were thus recovered. Further purification was performed using anion exchange chromatography. Seven fractions, which present a GST activity with 1‐chloro‐2,4‐dinitrobenzene (CDNB) as substrate, were collected and analyzed by RP‐HPLC. Seven distinct GST isoforms were purified, six of them were homodimers, the last one was a heterodimer consisting of the subunits 3 and 6. Kinetic parameters were studied. Results showed that isoforms have distinct affinity and Vmax for GSH and CDNB as substrates. The catalytic activity of the heterodimer isoform appeared to be a combination of the ability of each subunit. The immunological properties of each purified isoform were investigated using three antisera anti‐pi, anti‐mu and anti‐alpha mammalian GST classes. Three isoforms (3‐3, 6‐6 and 3‐6) seem to be closely related to the pi‐class GST. Both isoforms 1‐1 and 2‐2 cross‐reacted with antisera to pi and alpha classes and the isoform 5‐5 cross‐reacted with the antisera to mu and pi classes. Subunit 4 was recognized by the three antisera used, and its N‐terminal amino acid analysis showed high identity (53%) with a conserved sequence of an alpha/mµ/pi GST from Fasciola hepatica.
Role of Ca2+/calmodulin regulated signaling pathways in chemoattractant induced neutrophil effector functions
Authors: Verploegen Sandra , van Leeuwen Caroline M. , van Deutekom Hanneke W. M. , Lammers Jan‐Willem J. , Koenderman Leo , Coffer Paul J.
Published: March 2003

In human neutrophils, both changes in intracellular Ca2+ concentrations, [Ca2+]i, and activation of phosphatidylinositol‐3 kinase (PtdIns3K) have been proposed to play a role in regulating cellular function induced by chemoattractants. In this study we have investigated the role of [Ca2+]i and its effector molecule calmodulin in human neutrophils. Increased [Ca2+]i alone was sufficient to induce phosphorylation of extracellular signal‐regulated protein kinase 2 (ERK2), p38 mitogen activated kinase (p38 MAPK), protein kinase B (PKB) and glycogen synthase kinase‐3α (GSK‐3α). Inhibition of calmodulin using a calmodulin antagonist N‐(6‐aminohexyl)‐5‐chloro‐1‐naphthalenesulfonamide (W7), did not effect N‐formyl‐methionyl‐leucyl‐phenylalanine (fMLP) induced ERK, p38 MAPK or GSK‐3α phosphorylation, but attenuated fMLP induced PKB phosphorylation. PCR analysis of human neutrophil cDNA demonstrated variable expression of members of the Ca2+/calmodulin‐dependent kinase family. The roles of calmodulin and PtdIns3K in regulating neutrophil effector functions were further compared. Neutrophil migration was abrogated by inhibition of calmodulin, while no effect was observed when PtdIns3K was inhibited. In contrast, production of reactive oxygen species was sensitive to inhibition of both calmodulin and PtdIns3K. Finally, we demonstrated that chemoattractants are unable to modulate neutrophil survival, despite activation of PtdIns3K and elevation [Ca2+]i. Taken together, our data indicate critical roles for changes in [Ca2+]i and calmodulin activity in regulating neutrophil migration and respiratory burst and suggest that chemoattractant induced PKB phosphorylation may be mediated by a Ca2+/calmodulin sensitive pathway in human neutrophils.
The three typical aspartic proteinase genes of Arabidopsis thaliana are differentially expressed
Authors: Chen Xia , Pfeil Joanne E. , Gal Susannah
Published: March 2003

Genomic sequencing has identified three different typical plant aspartic proteinases in the genome of Arabidopsis thaliana, named Pasp‐A1, A2 and A3. A1 is identical to a cDNA we had previously isolated and the two others produce proteins 81 and 63% identical to that predicted protein. Sequencing of the aspartic proteinase protein purified from Arabidopsis seeds showed that the peptides are derived from two of these genes, A1 and A2. Using gene specific probes, we have analyzed RNA from different tissues and found these three genes are differentially expressed. A1 mRNA is detected in all tissues analyzed and more abundant in leaves during the light phase of growth. The other two genes are expressed either primarily in flowers (A3) or in seeds (A2). Insitu hybridization demonstrated that all three genes are expressed in many cells of the seeds and developing seed pods. The A1 and A3 genes are expressed in the sepals and petals of flowers as well as the outer layer of the style, but are not expressed in the transmitting tract or on the stigmatal surface. The A2 gene is weakly expressed only in the transmitting tissue of the style. All three genes are also expressed in the guard cells of sepals. These data suggest multiple roles for aspartic proteinases besides those proposed in seeds.
Hexadecylphosphocholine inhibits phosphatidylcholine biosynthesis and the proliferation of HepG2 cells
Authors: Jiménez‐López José M. , Carrasco María P. , Segovia Josefa L. , Marco Carmen
Published: March 2003

Hexadecylphosphocholine (HePC) is a synthetic lipid representative of a new group of antiproliferative agents, alkylphosphocholines (APC), which are promising candidates in anticancer therapy. Thus we have studied the action of HePC on the human hepatoblastoma cell line HepG2, which is frequently used as a model for studies into hepatic lipid metabolism. Non‐toxic, micromolar concentrations of HePC exerted an antiproliferative effect on this hepatoma cell line. The incorporation into phosphatidylcholine (PC) of the exogenous precursor [methyl‐14C]choline was substantially reduced by HePC. This effect was not due to any alteration in choline uptake by the cells, the degradation rate of PC or the release of PC into the culture medium. As anaccumulation of soluble choline derivatives points to CTP:phosphocholine cytidylyltransferase (CT) as the target of HePC activity we examined its effects on the different enzymes involved in the biosynthesis of PC via CDP–choline. Treatment with HePC altered neither the activity of choline kinase (CK) nor that of diacylglycerol cholinephosphotransferase (CPT), but it did inhibit CT activity in HepG2 cells. In vitro HePC also inhibited the activity of cytosolic but not membrane‐bound CT. Taken together our results suggest that HePC interferes specifically with the biosynthesis of PC in HepG2 cells by depressing CT translocation to the membrane, which may well impair their proliferation.
Insights into the reaction mechanism of Escherichia coli agmatinase by site‐directed mutagenesis and molecular modelling
Authors: Salas Mónica , Rodríguez Rolando , López Nelia , Uribe Elena , López Vasthi , Carvajal Nelson
Published: March 2003

Upon mutation of Asp153 by asparagine, the catalytic activity of agmatinase (agmatine ureohydrolase, EC 3.5.3.11) from Escherichia coli was reduced to about 5% of wild‐type activity. Tryptophan emission fluorescence (λmax = 340 nm), and CD spectra were nearly identical for wild‐type and D153N agmatinases. The Km value for agmatine (1.6 ± 0.1 mm), as well as the Ki for putrescine inhibition (12 ± 2 mm) and the interaction of the enzyme with the required metal ion, were also not altered by mutation. Three‐dimensional models, generated by homology modelling techniques, indicated that the side chains of Asp153 and Asn153 can perfectly fit in essentially the same position in the active site of E. coli agmatinase. Asp153 is suggested to be involved, by hydrogen bond formation, in the stabilization and orientation of a metal‐bound hydroxide for optimal attack on the guanidinium carbon of agmatine. Thus, the disruption of this hydrogen bond is the likely cause of the greately decreased catalytic efficiency of the D153N variant.
Characterization of a cloned subtilisin‐like serine proteinase from a psychrotrophic Vibrio species
Authors: Arnórsdóttir Jóhanna , Smáradóttir Rúna B. , Magnússon Ólafur Th. , Thorbjarnardóttir Sigrídur H. , Eggertsson Gudmundur , Kristjánsson Magnús M.
Published: March 2003

The gene encoding a subtilisin‐like serine proteinase in the psychrotrophic Vibrio sp. PA44 has been successfully cloned, sequenced and expressed in Escherichia coli. The gene is 1593 basepairs and encodes a precursor protein of 530 amino acid residues with a calculated molecular mass of 55.7 kDa. The enzyme is isolated, however, as an active 40.6‐kDa proteinase, without a 139 amino acid residue N‐terminal prosequence. Under mild conditions the enzyme undergoes a further autocatalytic cleavage to give a 29.7‐kDa proteinase that retains full enzymatic activity. The deduced amino acid sequence of the enzyme has high homology to proteinases of the proteinase K family of subtilisin‐like proteinases. With respect to the enzyme characteristics compared in this study the properties of the wild‐type and recombinant proteinases are the same. Sequence analysis revealed that especially with respect to the thermophilic homologues, aqualysin I from Thermus aquaticus and a proteinase from Thermus strain Rt41A, the cold‐adapted Vibrio‐proteinase has a higher content of polar/uncharged amino acids, as well as aspartate residues. The thermophilic enzymes had a higher content of arginines, and relatively higher number of hydrophobic amino acids and a higher aliphatic index. These factors may contribute to the adaptation of these proteinases to different temperature conditions.
The role of the second binding loop of the cysteine protease inhibitor, cystatin A (stefin A), in stabilizing complexes with target proteases is exerted predominantly by Leu73
Authors: Pavlova Alona , Estrada Sergio , Björk Ingemar
Published: March 2003

The aim of this work was to elucidate the roles of individual residues within the flexible second binding loop of human cystatin A in the inhibition of cysteine proteases. Four recombinant variants of the inhibitor, each with a single mutation, L73G, P74G, Q76G or N77G, in the most exposed part of this loop were generated by PCR‐based site‐directed mutagenesis. The binding of these variants to papain, cathepsin L, and cathepsin B was characterized by equilibrium and kinetic methods. Mutation of Leu73 decreased the affinity for papain, cathepsin L and cathepsin B by ≈ 300‐fold, >10‐fold and ≈ 4000‐fold, respectively. Mutation of Pro74 decreased the affinity for cathepsin B by ≈ 10‐fold but minimally affected the affinity for the other two enzymes. Mutation of Gln76 and Asn77 did not alter the affinity of cystatin A for any of the proteases studied. The decreased affinities were caused exclusively by increased dissociation rate constants. These results show that the second binding loop of cystatin A plays a major role in stabilizing the complexes with proteases by retarding their dissociation. In contrast with cystatin B, only one amino‐acid residue of the loop, Leu73, is of principal importance for this effect, Pro74 assisting to a minor extent only in the case of cathepsin B binding. The contribution of the second binding loop of cystatin A to protease binding varies with the protease, being largest, ≈ 45% of the total binding energy, for inhibition of cathepsin B.
A functionally conserved member of the FTZ‐F1 nuclear receptor family from Schistosoma mansoni
Authors: de Mendonça Ricardo L , Bouton Didier , Bertin Benjamin , Escriva Hector , Noël Christophe , Vanacker Jean‐Marc , Cornette Jocelyne , Laudet Vincent , Pierce Raymond J.
Published: March 2003

The fushi tarazu factor 1 (FTZ‐F1) nuclear receptor subfamily comprises orphan receptors with crucial roles in development and sexual differentiation in vertebrates and invertebrates. We describe the structure and functional properties of an FTZ‐F1 from the platyhelminth parasite of humans, Schistosoma mansoni, the first receptor from this family to be characterized in a Lophotrochozoan. It contains a well conserved DNA‐binding domain (55–63% identity to other family members) and a poorly conserved ligand‐binding domain (20% identity to that of zebrafish FF1a). However, both the ligand domain signature sequence and the activation function 2‐activation domain (AF2‐AD) are perfectly conserved. Phylogenetic analysis confirmed that SmFTZ‐F1 is a member of nuclear receptor subfamily 5, but that it clustered with the Drosophila receptor DHR39 and has consequently been named NR5B1. The gene showed a complex structure with 10 exons and an overall size of 18.4 kb. Two major transcripts were detected, involving alternative promoter usage and splicing of the two 5′ exons, but which encoded identical proteins. SmFTZ‐F1 mRNA is expressed at all life‐cycle stages with the highest amounts in the larval forms (miracidia, sporocysts and cercariae). However, expression of the protein showed a different pattern; low in miracidia and higher in adult male worms. The protein bound the same monomeric response element as mammalian SF‐1 (SF‐1 response element, SFRE) and competition experiments with mutant SFREs showed that its specificity was identical. Moreover, SmFTZ‐F1 transactivated reporter gene transcription from SFRE similarly to SF‐1. This functional conservation argues for a conserved biological role of the FTZ‐F1 nuclear receptor family throughout the metazoa.