bluuebunny/crossref_metadata_2025 huggingface dataset:
Allergenicity of some isoforms of white sesame proteins
Authors: Fremont S. , Zitouni N. , Kanny G. , Veneri V. , Metche M. , Moneret‐Vautrin D. A. , Nicolas J. P.
Published: March 2003

Background Allergy to sesame seeds is often associated with particularly severe reactions, with a high risk of anaphylaxis. The increase in reports of allergic reactions to sesame is probably due to the growing use of sesame seeds or sesame oil in food.

Objective To determine the molecular weights of the proteins in three variety of sesame seeds and to study the isoelectric points and the allergenicity of white sesame proteins.

Methods Extracts of white, brown and black sesame seeds were prepared. The white sesame extract, mostly used in bakery, was run on SDS‐PAGE and two dimensional electrophoresis. Six sera from patients sensitized or symptomatic to sesame seed were used for Western blotting.

Results The protein patterns of the white, brown and black sesame extracts showed major quantitative differences. The white extract had the higher protein concentration and contained 15 proteins of 12–79 kDa, some of them having several acidic isoelectric points. The lowest isoelectric point was 4.9 and the highest was 6.4, giving 35 isoforms. Ten of the 15 proteins (12–57.5 kDa) were recognized by specific IgE. The 12–13 kDa and 22–33 kDa proteins could correspond to the main allergens.

Conclusion White sesame seeds contain at least 10 allergenic proteins with acidic isoelectric points. In accordance with previous results, two of them seem to contain the major allergens.
Expectations of health care: promoted, managed or shared?
Authors: Hart Julian Tudor
Published: March 2003

Volume, costs and content of medical care depend on professional and public expectations. The UK National Health Service (NHS) removed price barriers to access, so depressed expectations became an important factor in cost control. In USA, professional control of care business inflated expectations and costs. Managed care in the NHS failed to rationalize care because managers seem even less trustworthy than clinicians as arbiters of rational expectations in contexts of underfunding. Rational expectations depend on restored trust, mutual and managerial respect for expertise of both clinicians and patients, and transcendence of the provider‐consumer model for value production in medical care.
Consumer collaboration, patient‐defined outcomes and the preparation of Cochrane Reviews
Authors: Kelson Marcia C.
Published: March 2003

Objectives

To identify the extent to which the Cochrane Collaboration involves consumers (patients, carers, patient and non‐patient members of patient/consumer organizations) as members of Cochrane Review Groups (CRGs); to explore the emphasis CRGs place on identifying and collecting information on outcomes identified by patients as being important indicators of quality and effectiveness of treatment and care (‘patient‐defined outcomes’).

Method

A postal questionnaire designed by The College of Health, a UK patient organization, was sent in January 1998 to all CRGs registered with the Cochrane Collaboration on 1 January 1998 (n = 42).

Results and conclusions

Replies were received from 35 CRGs, a response rate of 83% and 33 questionnaires (79%) were completed. The survey revealed that CRGs varied in the extent to which they had recruited consumer members: almost one third of respondents said their CRG did not have any consumer members. There was also no apparent consensus across CRGs on the importance attached to identifying and collecting information on patient‐defined outcomes or on integrating such information into their activities. It is hoped that differences between CRGs may inform discussions as to whether and how the Cochrane Collaboration might address the issue of patient‐defined outcomes in the future.
‘People sometimes react funny if they're not told enough’: women's views about the risks of diagnostic laparoscopy
Authors: Moore Jane , Ziebland Sue , Kennedy Stephen
Published: March 2003

Objectives To explore women's views about the risks and benefits of diagnostic laparoscopy in the investigation of chronic pelvic pain, including how much information it is thought appropriate to give about three specific risks: death, major complications and the chance that the procedure would have an inconclusive result.

Design A qualitative analysis of semi‐structured, audio‐taped interviews with 20 women about their experiences of undergoing a diagnostic laparoscopy in a day surgery unit. Interviews were conducted 3–6 months after the procedure.

Results All the women who were interviewed were aware that diagnostic laparoscopy carried risks, including the small risk of death associated with general anaesthesia. One‐third of respondents said that they had initially been reluctant to discuss the risks of the procedure in general terms. However, when specific examples of complications and risks were introduced all but one of the respondents reported that theywouldhave liked to discuss these at the time that the decision to have the operation was made. Women maintained that the information was needed to make an informed decision about whether to have the operation, to help them understand and cope should things go wrong and in order to make appropriate plans to cover contingencies. Most were surprised to hear that the procedure is frequently inconclusive and thought that this information should be made clear to women contemplating a laparoscopy.

Conclusions Women undergoing diagnostic laparoscopy for the investigation of chronic pelvic pain wish to be given full and accurate information about complication rates such as bowel perforation, what to expect during their recovery, and the chances of finding a cause for their pain. Although they may not want to dwell on the risk of death, they do need to be informed about the specific risks associated with the procedure in order to make a balanced decision.
Developing patient‐centred information for back pain sufferers
Authors: Glenton Claire , Polit. Cand.
Published: March 2003

Objective To identify information needs among a group of back pain sufferers as well as the barriers that may prevent them from accessing this information.

Design Data were collected through the use of open, in‐depth interviews, through contributions to the Norwegian Back Pain Association's online discussion list, and through a search of the literature.

Participants Norwegian back pain sufferers and their carers.

Main variables studied Information needs and barriers.

Results The informants described information needs that covered a wide range of topics, clinical, financial, emotional and social. Informants wanted to understand the cause of their pain and wanted information about existing diagnoses and diagnostic procedures. Informants asked for information about treatment alternatives, both within and outside the established health‐care system, the effects of these treatment alternatives, their procedures, side‐effects and costs. In addition, informants wanted information about the social and emotional effects of long‐term pain; coping with everyday life; other people's experiences; and about welfare benefits and patient rights. Barriers to this information included the use of medical, legal and other jargon, doctors’ lack of time, lack of communication skills, lack of knowledge about back pain and attitudes to back pain patients.

Conclusions To successfully address the questions and concerns of users, there should be an attempt to present information on as many of these topics as possible. Information should be presented in the user's own language, at several levels of understanding, and should include both evidence‐ and experienced‐based knowledge.
Short peptides are not reliable models of thermodynamic and kinetic properties of the N‐terminal metal binding site in serum albumin
Authors: Sokolowska Magdalena , Krezel Artur , Dyba Marcin , Szewczuk Zbigniew , Bal Wojciech
Published: March 2003

A comparative study of thermodynamic and kinetic aspects of Cu(II) and Ni(II) binding at the N‐terminal binding site of human and bovine serum albumins (HSA and BSA, respectively) and short peptide analogues was performed using potentiometry and spectroscopic techniques. It was found that while qualitative aspects of interaction (spectra and structures of complexes, order of reactions) could be reproduced, the quantitative parameters (stability and rate constants) could not. The N‐terminal site in HSA is much more similar to BSA than to short peptides reproducing the HSA sequence. A very strong influence of phosphate ions on the kinetics of Ni(II) interaction was found. This study demonstrates the limitations of short peptide modelling of Cu(II) and Ni(II) transport by albumins.
Holliday junction binding and processing by the RuvA protein of Mycoplasma pneumoniae
Authors: Ingleston Stuart M. , Dickman Mark J. , Grasby Jane A. , Hornby David P. , Sharples Gary J. , Lloyd Robert G.
Published: March 2003

The RuvA, RuvB and RuvC proteins of Escherichia coli act together to process Holliday junctions formed during recombination and DNA repair. RuvA has a well‐defined DNA binding surface that is sculptured specifically to accommodate a Holliday junction and allow subsequent loading of RuvB and RuvC. A negatively charged pin projecting from the centre limits binding of linear duplex DNA. The amino‐acid sequences forming the pin are highly conserved. However, in certain Mycoplasma and Ureaplasma species the structure is extended by four amino acids and two acidic residues forming a crucial charge barrier are missing. We investigated the significance of these differences by analysing RuvA from Mycoplasma pneumoniae. Gel retardation and surface plasmon resonance assays revealed that this protein binds Holliday junctions and other branched DNA structures in a manner similar to E. coli RuvA. Significantly, it binds duplex DNA more readily. However it does not support branch migration mediated by E. coli RuvB and when bound to junction DNA is unable to provide a platform for stable binding of E. coli RuvC. It also fails to restore radiation resistance to an E. coli ruvA mutant. The data presented suggest that the modified pin region retains the ability to promote junction‐specific DNA binding, but acts as a physical obstacle to linear duplex DNA rather than as a charge barrier. They also indicate that such an obstacle may interfere with the binding of a resolvase. Mycoplasma species may therefore process Holliday junctions via uncoupled branch migration and resolution reactions.
Phosphatidylinositol synthesis and exchange of the inositol head are catalysed by the single phosphatidylinositol synthase 1 from Arabidopsis
Authors: Justin Anne‐Marie , Kader Jean‐Claude , Collin Sylvie
Published: March 2003

In order to study some of its enzymatic properties, phosphatidylinositol synthase 1 (AtPIS1) from the plant Arabidopsis thaliana was expressed in Escherichia coli, a host naturally devoid of phosphatidylinositol (PtdIns). In the context of the bacterial membrane and in addition to de novo synthesis, the plant enzyme is capable of catalysing the exchange of the inositol polar head for another inositol. Our data clearly show that the CDP‐diacylglycerol‐independent exchange reaction can occur using endogenous PtdIns molecular species or PtdIns molecular species from soybean added exogenously. Exchange has been observed in the absence of cytidine monophosphate (CMP), but is greatly enhanced in the presence of 4 µm CMP. Our data also show that AtPIS1 catalyses the removal of the polar head in the presence of much higher concentrations of CMP, in a manner that suggests a reverse of synthesis. All of the PtdIns metabolizing activities require free manganese ions. EDTA, in the presence of low Mn2+ concentrations, also has an enhancing effect.
Osmoregulated periplasmic glucans of the free‐living photosynthetic bacterium Rhodobacter sphaeroides
Authors: Talaga Philippe , Cogez Virginie , Wieruszeski Jean‐Michel , Stahl Bernd , Lemoine Jérôme , Lippens Guy , Bohin Jean‐Pierre
Published: March 2003

The osmoregulated periplasmic glucans (OPGs) produced by Rhodobacter sphaeroides, a free‐living organism, were isolated by trichloracetic acid treatment and gel permeation chromatography. Compounds obtained were characterized by compositional analysis, matrix‐assisted laser desorption ionization mass spectrometry and nuclear magnetic resonance. R. sphaeroides predominantly synthesizes a cyclic glucan containing 18 glucose residues that can be substituted by one to seven succinyl esters residues at the C6 position of some of the glucose residues, and by one or two acetyl residues. The glucans were subjected to a mild alkaline treatment in order to remove the succinyl and acetyl substituents, analyzed by MALDI mass spectrometry and purified by high‐performance anion‐exchange chromatography. Methylation analysis revealed that this glucan is linked by 17 1,2 glycosidic bonds and one 1,6 glycosidic bond. Homonuclear and 1H/13C heteronuclear NMR experiments revealed the presence of a single α‐1,6 glycosidic linkage, whereas all other glucose residues are β‐1,2 linked. The different anomeric proton signals allowed a complete sequence‐specific assignment of the glucan. The structural characteristics of this glucan are very similar to the previously described OPGs of Ralstonia solanacearum and Xanthomonas campestris, except for its different size and the presence of substituents. Therefore, similar OPGs are synthesized by phytopathogenic as well as free‐living bacteria, suggesting these compounds are intrinsic components of the Gram‐negative bacterial envelope.
Group IID heparin‐binding secretory phospholipase A2 is expressed in human colon carcinoma cells and human mast cells and up‐regulated in mouse inflammatory tissues
Authors: Murakami Makoto , Yoshihara Kumiko , Shimbara Satoko , Sawada Masatsugu , Inagaki Naoki , Nagai Hiroichi , Naito Mikihiko , Tsuruo Takashi , Moon Tae Churl , Chang Hyeun Wook , Kudo Ichiro
Published: March 2003

Group IID secretory phospholipase A2 (sPLA2‐IID), a heparin‐binding sPLA2 that is closely related to sPLA2‐IIA, augments stimulus‐induced cellular arachidonate release in a manner similar to sPLA2‐IIA. Here we identified the residues of sPLA2‐IID that are responsible for heparanoid binding, are and therefore essential for cellular function. Mutating four cationic residues in the C‐terminal portion of sPLA2‐IID resulted in abolition of its ability to associate with cell surface heparan sulfate and to enhance stimulus‐induced delayed arachidonate release, cyclooxygenase‐2 induction, and prostaglandin generation in 293 cell transfectants. As compared with several other group II subfamily sPLA2s, which were equally active on A23187‐ and IL‐1‐primed cellular membranes, sPLA2‐IID showed apparent preference for A23187‐primed membranes. Several human colon carcinoma cell lines expressed sPLA2‐IID and sPLA2‐X constitutively, the former of which was negatively regulated by IL‐1. sPLA2‐IID, but not other sPLA2 isozymes, was expressed in human cord blood‐derived mast cells. The expression of sPLA2‐IID was significantly altered in several tissues of mice with experimental inflammation. These results indicate that sPLA2‐IID may be involved in inflammation in cell‐ and tissue‐specific manners under particular conditions.
Purification and characterization of novel chondroitin ABC and AC lyases from Bacteroides stercoris HJ‐15, a human intestinal anaerobic bacterium
Authors: Hong Sung‐Woon , Kim Byung‐Taek , Shin Ho‐Young , Kim Wan‐Suk , Lee Keun‐Sook , Kim Yeong‐Shik , Kim Dong‐Hyun
Published: March 2003

Two novel chondroitinases, chondroitin ABC lyase (EC 4.2.2.4) and chondroitin AC lyase (EC 4.2.2.5), have been purified from Bacteroides stercoris HJ‐15, which was isolated from human intestinal bacteria with glycosaminoglycan degrading enzymes. Chondroitin ABC lyase was purified to apparent homogeneity by a combination of QAE‐cellulose, CM‐Sephadex C‐50, hydroxyapatite and Sephacryl S‐300 column chromatography with a final specific activity of 45.7 µmol·min−1·mg−1. Chondroitin AC lyase was purified to apparent homogeneity by a combination of QAE‐cellulose, CM‐Sephadex C‐50, hydroxyapatite and phosphocellulose column chromatography with a final specific activity of 57.03 µmol·min−1·mg−1. Chondroitin ABC lyase is a single subunit of 116 kDa by SDS/PAGE and gel filtration. Chondroitin AC lyase is composed of two identical subunits of 84 kDa by SDS/PAGE and gel filtration. Chondroitin ABC and AC lyases showed optimal activity at pH 7.0 and 40 °C, and 5.7–6.0 and 45–50 °C, respectively. Both chondroitin lyases were potently inhibited by Cu2+, Zn2+, and p‐chloromercuriphenyl sulfonic acid. The purified Bacteroidal chondroitin ABC lyase acted to the greatest extent on chondroitin sulfate A (chondroitin 4‐sulfate), to a lesser extent on chondroitin sulfate B (dermatan sulfate) and C (chondroitin 6‐sulfate). The purified chondroitin AC lyase acted to the greatest extent on chondroitin sulfate A, and to a lesser extent on chondroitin C and hyaluronic acid. They did not act on heparin and heparan sulfate. These findings suggest that the biochemical properties of these purified chondroitin lyases are different from those of the previously purified chondroitin lyases.
The mechanism of nitrogen monoxide (NO)‐mediated iron mobilization from cells
Authors: Watts Ralph N. , Richardson Des R.
Published: March 2003

Nitrogen monoxide (NO) is a cytotoxic effector molecule produced by macrophages that results in Fe mobilization from tumour target cells which inhibits DNA synthesis and mitochondrial respiration. It is well known that NO has a high affinity for Fe, and we showed that NO‐mediated Fe mobilization is markedly potentiated by glutathione (GSH) generated by the hexose monophosphate shunt [Watts, R.N. & Richardson, D.R. (2001) J. Biol. Chem. 276, 4724–4732]. We hypothesized that GSH completes the coordination shell of an NO–Fe complex that is released from the cell. In this report we have extended our studies to further characterize the mechanism of NO‐mediated Fe mobilization. Native PAGE 59Fe‐autoradiography shows that NO decreased ferritin‐59Fe levels in cells prelabelled with [59Fe]transferrin. In prelabelled cells, ferritin‐59Fe levels increased 3.5−fold when cells were reincubated with control media between 30 and 240 min. In contrast, when cells were reincubated with NO, ferritin‐59Fe levels decreased 10‐fold compared with control cells after a 240‐min reincubation. However, NO could not remove Fe from ferritin in cell lysates. Our data suggest that NO intercepts 59Fe on route to ferritin, and indirectly facilitates removal of 59Fe from the protein. Studies using the GSH‐depleting agent, l‐buthionine‐(S,R)‐sulphoximine, indicated that the reduction in ferritin‐59Fe levels via NO was GSH‐dependent. Competition experiments with NO and permeable chelators demonstrated that both bind a similar Fe pool. We suggest that NO requires cellular metabolism in order to effect Fe mobilization and this does not occur via passive diffusion down a concentration gradient. Based on our results, we propose a model of glucose‐dependent NO‐mediated Fe mobilization.
Role of histidine 42 in ascorbate peroxidase
Authors: Lad Latesh , Mewies Martin , Basran Jaswir , Scrutton Nigel S. , Raven Emma L.
Published: March 2003

To examine the role of the distal His42 residue in the catalytic mechanism of pea cytosolic ascorbate peroxidase, two site‐directed variants were prepared in which His42 was replaced with alanine (H42A) or glutamic acid (H42E). Electronic spectra of the ferric derivatives of H42A and H42E (pH 7.0, µ = 0.10 m, 25.0 °C) revealed wavelength maxima [λmax (nm): 397, 509, ≈ 540sh, 644 (H42A); 404, 516, ≈ 538sh, 639 (H42E)] consistent with a predominantly five‐co‐ordinate high‐spin iron. The specific activity of H42E for oxidation of l‐ascorbate (8.2 ± 0.3 U·mg−1) was ≈ 30‐fold lower than that of the recombinant wild‐type enzyme (rAPX); the H42A variant was essentially inactive but activity could be partially recovered by addition of exogenous imidazoles. The spectra of the Compound I intermediates of H42A [λmax (nm) = 403, 534, 575sh, 645] and H42E [λmax (nm) = 404, 530, 573sh, 654] were similar to those of rAPX. Pre‐steady‐state data for formation of Compound I for H42A and H42E were consistent with a mechanism involving accumulation of a transient enzyme intermediate (Kd) followed by conversion of this intermediate into Compound I (k′1). Values for k′1 and Kd were, respectively, 4.3 ± 0.2 s−1 and 30 ± 2.0 mm (H42A) and 28 ± 1.0 s−1 and 0.09 ± 0.01 mm (H42E). Photodiode array experiments for H42A revealed wavelength maxima for this intermediate at 401 nm, 522 nm and 643 nm, consistent with the formation of a transient [H42A–H2O2] species. Rate constants for Compound I formation for H42A were independent of pH, but for rAPX and H42E were pH‐dependent [pKa = 4.9 ± 0.1 (rAPX) and pKa = 6.7 ± 0.2 (H42E)]. The results provide: (a) evidence that His42 is critical for Compound I formation in APX; (b) confirmation that titration of His42 controls Compound I formation and an assignment of the pKa for this group; (c) mechanistic and spectroscopic evidence for an intermediate before Compound I formation; (d) evidence that a glutamic acid residue at position 42 can act as the acid–base catalyst in ascorbate peroxidase.
Shb links SLP‐76 and Vav with the CD3 complex in Jurkat T cells
Authors: Lindholm Cecilia K. , Henriksson Maria L. , Hallberg Bengt , Welsh Michael
Published: March 2003

This study addresses the interactions between the adaptor protein Shb and components involved in T cell signalling, including SLP‐76, Gads, Vav and ZAP70. We show that both SLP‐76 and ZAP70 co‐immunoprecipitate with Shb in Jurkat T cells and that Shb and Vav co‐immunoprecipitate when cotransfected in COS cells. We also demonstrate, utilizing fusion protein constructs, that SLP‐76, Gads and Vav associate independently of each other to different domains or regions, of Shb. Overexpression of an SH2 domain‐defective Shb causes diminished phosphorylation of SLP‐76 and Vav and consequently decreased activation of c‐Jun kinase upon T cell receptor (TCR) stimulation. Shb was also found to localize to glycolipid‐enriched membrane microdomains (GEMs), also called lipid rafts, after TCR stimulation. Our results indicate that upon TCR stimulation, Shb is targeted to these lipid rafts where Shb aids in recruiting the SLP‐76–Gads–Vav complex to the T cell receptor ζ‐chain and ZAP70.
Schemes of flux control in a model of Saccharomyces cerevisiae glycolysis
Authors: Pritchard Leighton , Kell Douglas B.
Published: March 2003

We used parameter scanning to emulate changes to the limiting rate for steps in a fitted model of glucose‐derepressed yeast glycolysis. Three flux‐control regimes were observed, two of which were under the dominant control of hexose transport, in accordance with various experimental studies and other model predictions. A third control regime in which phosphofructokinase exerted dominant glycolytic flux control was also found, but it appeared to be physiologically unreachable by this model, and all realistically obtainable flux control regimes featured hexose transport as a step involving high flux control.
Characterization of scorpion α‐like toxin group using two new toxins from the scorpion Leiurus quinquestriatus hebraeus
Authors: Hamon Alain , Gilles Nicolas , Sautière Pierre , Martinage Arlette , Kopeyan Charles , Ulens Chris , Tytgat Jan , Lancelin Jean‐Marc , Gordon Dalia
Published: March 2003

Two novel toxins, Lqh6 and Lqh7, isolated from the venom of the scorpion Leiurus quinquestriatus hebraeus, have in their sequence a molecular signature (8Q/KPE10) associated with a recently defined group of α‐toxins that target Na channels, namely the α‐like toxins [reviewed in Gordon, D., Savarin, P., Gurevitz, M. & Zinn‐Justin, S. (1998) J. Toxicol. Toxin Rev. 17, 131–159]. Lqh6 and Lqh7 are highly toxic to insects and mice, and inhibit the binding of α‐toxins to cockroach neuronal membranes. Although they kill rodents by intracerebroventricular injection, they do not inhibit the binding of antimammal α‐toxins (e.g. Lqh2) to rat brain synaptosomes, not even at high concentrations. Furthermore, in voltage‐clamp experiments, rat brain Na channels IIA (rNav1.2A) expressed in Xenopus oocytes are not affected by Lqh6 nor by Lqh7 below 3 µm. In contrast, muscular Na channels (rNav1.4 and hNav1.5) expressed in the same cells respond to nanomolar concentrations of Lqh6 and Lqh7 by slowing of Na current inactivation and a leftward shift of the peak conductance–voltage curve. The structural and pharmacological properties of the new toxins are compared to those of other scorpion α‐toxins in order to re‐examine the hallmarks previously set for the α‐like toxin group.
Pressure and heat inactivation of recombinant human acetylcholinesterase
Authors: Cléry‐Barraud Cécile , Ordentlich Arie , Grosfeld Haim , Shafferman Avigdor , Masson Patrick
Published: March 2003

The effects of pressure on structure and activity of recombinant human acetylcholinesterase (rHuAChE) were investigated up to a pressure of 300 MPa using gel electrophoresis under elevated hydrostatic pressure, fluorescence of bound 8‐anilinonaphthalene‐1‐sulfonate (ANS) and activity measurements following exposure to high pressure. Study of wild‐type enzyme and three single mutants (D74N, E202Q, E450A) and one sextuple mutant (E84Q/E292A/D349N/E358Q/E389Q/D390N) showed that pressure exerts a differential action on wild‐type rHuAChE and its mutants, allowing estimation of the contribution of carboxylic amino acid side‐chains to enzyme stability. Mutation of negatively charged residues D74 and E202 by polar side‐chains strengthened heat or pressure stability. The mutation E450A and the sextuple mutation caused destabilization of the enzyme to pressure. Thermal inactivation data on mutants showed that all of them were stabilized against temperature. In conclusion, pressure and thermal stability of mutants provided evidence that the residue E202 is a determinant of structural and functional stability of HuAChE.
Isolation of a putative peroxidase, a target for factors controlling foot‐formation in the coelenterate hydra
Authors: Hoffmeister‐Ullerich Sabine A. H. , Herrmann Doris , Kielholz Jürgen , Schweizer Michaela , Schaller H. Chica
Published: March 2003

In hydra, differentiated ectodermal cells of the foot region contain a peroxidase activity that can be used as a marker for foot‐specific differentiation processes. Because the expression of the gene coding for the peroxidase must be tightly regulated during foot‐specific differentiation, characterization of the protein and cloning of the corresponding gene should provide valuable tools for getting deeper insights into the regulation of foot‐specific differentiation. In this paper we characterize the foot‐specific peroxidase by biochemical, histochemical, and molecular biological methods. We show that it is localized in granules, and that it consists of a single component, the molecular mass of which is in the range of 43–45 kDa. Purification of the protein and subsequent cloning of its complementary DNA yielded two closely related clones, ppod1 and ppod2. Transcripts of ppod2 are abundant in the whole animal with the exception of the hypostome, the tentacles, and the foot; the expression of ppod1 matches exactly the localization of the foot‐specific peroxidase.
Formation of conjugated Δ11Δ13‐double bonds by Δ12‐linoleic acid (1,4)‐acyl‐lipid‐desaturase in pomegranate seeds
Authors: Hornung Ellen , Pernstich Christian , Feussner Ivo
Published: March 2003

For the biosynthesis of punicic acid (18:3Δ9Z,11E,13Z) a (11,14)‐linoleoyl desaturase activity has been proposed. To isolate this acyl‐lipid‐desaturase, PCR‐based cloning was used. This approach resulted in the isolation of two complete cDNAs. The first isolated full‐length cDNA harbors a sequence of 1350 bp encoding a protein of 395 amino acids. The second cDNA was 1415 bp long encoding a protein of 387 amino acids. For functional identification proteins encoded by the cDNAs were expressed in Saccharomyces cerevisiae, and formation of newly formed fatty acids was analyzed by gas chromatography‐free induction decay (GC‐FID) and GC/MS. The expression of the heterologous enzymes resulted in the first case in a significant amount of linoleic acid and in the second case, after linoleic acid supplementation, in formation of punicic acid. The results presented here identify one cDNA coding for a classical Δ12‐acyl‐lipid‐desaturase. The other one codes for a new type of (1,4)‐acyl‐lipid‐desaturase that converts a cis double bond located in the Δ12‐position of linoleic acid or γ‐linolenic acid, but not in α‐linolenic acid, into a conjugated cis–trans double bond system.
Cold induces stress‐activated protein kinase‐mediated response in the fission yeast Schizosaccharomyces pombe
Authors: Soto Teresa , Beltrán Francisco F. , Paredes Vanessa , Madrid Marisa , Millar Jonathan B. A. , Vicente‐Soler Jero , Cansado José , Gacto Mariano
Published: March 2003

In the fission yeast Schizosaccharomyces pombe the Wak1p/Win1p‐Wis1p‐Sty1p stress‐activated protein kinase (SAPK) pathway relays environmental signals to the transcriptional machinery and modulates gene expression via a cascade of protein phosphorylation. Cells of S. pombe subjected to cold shock (transfer from 28 °C to 15 °C) transiently activated the Sty1p mitogen‐activated protein kinase (MAPK) by phosphorylation. Induction of this response was completely abolished in cells disrupted in the upstream response regulator Mcs4p. The cold‐triggered Sty1p activation was partially dependent on Wak1p MAPKKK and fully dependent on Wis1p MAPKK suggesting that the signal transmission follows a branched pathway, with the redundant MAPKKK Win1p as alternative transducer to Wis1p, which subsequently activates the effector Sty1p MAPK. Also, the bZIP transcription factor Atf1p became phosphorylated in a Sty1p‐dependent way during the cold shock and this phosphorylation was found responsible for the increased expression of gpd1+, ctt1+, tps1+ and ntp1+ genes. Strains deleted in transcription factors Atf1p or Pcr1p were unable to grow upon incubation at low temperature whereas those disrupted in any member of the SAPK pathway were able to do so. These data reveal that S. pombe responds to cold by inducing the SAPK pathway. However, such activation is dispensable for yeast growth in cold conditions, supporting that the presence of Atf1/Pcr1 heterodimers, rather than an operative SAPK pathway, is critical to ensure yeast growth at low temperature by an as yet undefined mechanism.
The RuvABC resolvasome
Authors: Dickman Mark J. , Ingleston Stuart M. , Sedelnikova Svetlana E. , Rafferty John B. , Lloyd Robert G. , Grasby Jane A. , Hornby David P.
Published: March 2003

The RuvABC resolvasome of Escherichia coli catalyses the resolution of Holliday junctions that arise during genetic recombination and DNA repair. This process involves two key steps: branch migration, catalysed by the RuvB protein that is targeted to the Holliday junction by the structure specific RuvA protein, and resolution, which is catalysed by the RuvC endonuclease. We have quantified the interaction of the RuvA protein with synthetic Holliday junctions and have shown that the binding of the protein is highly structure‐specific, and leads to the formation of a complex containing two tetramers of RuvA per Holliday junction. Our data are consistent with two tetramers of RuvA binding to the DNA recombination intermediate in a co‐operative manner. Once formed this complex prevents the binding of RuvC to the Holliday junction. However, the formation of a RuvAC complex can be observed following sequential addition of the RuvC and RuvA proteins. Moreover, by examining the DNA recognition properties of a mutant RuvA protein (E55R, D56K) we show that the charge on the central pin is critical for directing the structure‐specific binding by RuvA.
Activation of transcription through the ligand‐binding pocket of the orphan nuclear receptor ultraspiracle
Authors: Xu Yong , Fang Fang , Chu YanXia , Jones Davy , Jones Grace
Published: March 2003

The invertebrate nuclear receptor, ultraspiracle (USP), an ortholog of the vertebrate RXR, is typically modelled as an orphan receptor that functions without a ligand‐binding activity. The identification of a ligand that can transcriptionally activate USP would provide heuristic leads to the structure of potentially high affinity activating compounds, with which to detect unknown regulatory pathways in which this nuclear receptor participates. We show here that the application of the sesquiterpenoid methyl epoxyfarnesoate (juvenile hormone III) to Sf9 cells induces transcription from a transfected heterologous core promoter, through a 5′‐placed DR12 enhancer to which the receptor ultraspiracle (USP) binds. Isolated, recombinant USP from Drosophila melanogaster specifically binds methyl epoxyfarnesoate, whereupon the receptor homodimerizes and changes tertiary conformation, including the movement of the ligand‐binding domain α‐helix 12. Ligand‐binding pocket point mutants of USP that do not bind methyl epoxyfarnesoate act as dominant negative suppressors of methyl epoxyfarnesoate‐activation of the reporter promoter, and addition of wild‐type USP rescues this activation. These data establish a paradigm in which the USP ligand‐binding pocket can productively bind ligand with a functional outcome of enhanced promoter activity, the first such demonstration for an invertebrate orphan nuclear receptor. USP thus establishes the precedent that invertebrate orphan receptors are viable targets for development of agonists and antagonists with which to discern and manipulate transcriptional pathways dependent on USP or other orphan receptors. The demonstration here of these functional capacities of USP in a transcriptonal activation pathway has significant implications for current paradigms of USP action that do not include for USP a ligand‐binding activity.
Expanding the scorpion toxin α‐KTX 15 family with AmmTX3 from Androctonus mauretanicus
Authors: Vacher Hélène , Alami Meriem , Crest Marcel , Possani Lourival D. , Bougis Pierre E. , Martin‐Eauclaire Marie‐France
Published: March 2003

A novel toxin, AmmTX3 (3823.5 Da), was isolated from the venom of the scorpion Androctonus mauretanicus. It showed 94% sequence homology with Aa1 from Androctonus australis and 91% with BmTX3 from Buthus martensi which, respectively, block A‐type K+ current in cerebellum granular cells and striatum cultured neurons. Binding and displacement experiments using rat brain synaptosomes showed that AmmTX3 and Aa1 competed effectively with 125I‐labelled sBmTX3 binding. They fully inhibited the 125I‐labelled sBmTX3 binding (Ki values of 19.5 pm and 44.2 pm, respectively), demonstrating unambiguously that the three molecules shared the same target in rat brain. The specific binding parameters of 125I‐labelled AmmTX3 for its site were determined at equilibrium (Kd = 66 pm, Bmax = 22 fmol per mg of protein). Finally, patch‐clamp experiments on striatal neurons in culture demonstrated that AmmTX3 was able to inhibit the A‐type K+ current (Ki = 131 nm).
Double‐stranded RNA‐activated protein kinase interacts with apoptosis signal‐regulating kinase 1
Authors: Takizawa Takenori , Tatematsu Chizuru , Nakanishi Yoshinobu
Published: March 2003

Double‐stranded RNA‐activated protein kinase (PKR), a serine/threonine kinase, is activated in virus‐infected cells and acts as an antiviral machinery of type I interferons. PKR controls several stress response pathways induced by double‐stranded RNA, tumor necrosis factor‐α or lipopolysaccharide, which result in the activation of stress‐activated protein kinase/c‐Jun NH2‐terminal kinase and p38 of the mitogen‐activated protein kinase family. Here we showed a novel interaction between PKR and apoptosis signal‐regulating kinase 1 (ASK1), one of the members of the mitogen‐activated protein kinase kinase kinase family, which is activated in response to a variety of apoptosis‐inducing stimuli. PKR and ASK1 showed predominant cytoplasmic localization in COS‐1 cells transfected with both cDNAs, and coimmunoprecipitated from the cell extracts. A dominant negative mutant of PKR (PKR‐KR) inhibited both the apoptosis and p38 activation induced by ASK1 in vivo. Consistently, PKR‐KR inhibited the autophosphorylation of ASK1 in vitro, and exposure to poly(I)–poly(C) increased the phosphorylation of ASK1 in vivo. These results indicate the existence of a link between PKR and ASK1, which modifies downstream MAPK.
Stepwise adaptations of citrate synthase to survival at life's extremes
Authors: Bell Graeme S. , Russell Rupert J. M. , Connaris Helen , Hough David W. , Danson Michael J. , Taylor Garry L.
Published: March 2003

The crystal structure of citrate synthase from the thermophilic Archaeon Sulfolobus solfataricus (optimum growth temperature = 85 °C) has been determined, extending the number of crystal structures of citrate synthase from different organisms to a total of five that span the temperature range over which life exists (from psychrophile to hyperthermophile). Detailed structural analysis has revealed possible molecular mechanisms that determine the different stabilities of the five proteins. The key to these mechanisms is the precise structural location of the additional interactions. As one ascends the temperature ladder, the subunit interface of this dimeric enzyme and loop regions are reinforced by complex electrostatic interactions, and there is a reduced exposure of hydrophobic surface. These observations reveal a progressive pattern of stabilization through multiple additional interactions at solvent exposed, loop and interfacial regions.
Cloning, expression and characterization of a gene encoding nitroalkane‐oxidizing enzyme from Streptomyces ansochromogenes
Authors: Zhang Jihui , Ma Wenbo , Tan Huarong
Published: March 2003

A nitroalkane‐oxidizing enzyme gene (naoA) was cloned from a genomic DNA library of Streptomyces ansochromogenes 7100. The deduced protein (NaoA) of this gene contains 363 amino acids and has high similarity to several nitroalkane‐oxidizing enzymes from various micro‐organisms. The naoA gene was subcloned into an expression vector pET23b and overexpressed in Escherichia coli BL21(DE3). The protein was then purified, and its characteristics were studied. Experimental results showed that NaoA can convert 1‐nitropropane, 2‐nitropropane and nitroethane into the corresponding carbonyl compounds. The optimal pH and temperature for NaoA was found to be pH 7–8 and 48–56 °C, respectively. The Km of NaoA for nitroethane is ≈ 26.8 mm. NADH and nitro blue tetrazolium are strong inhibitors of NaoA, and thiol compounds and superoxide dismutase partially inhibit the enzyme activity. Therefore, superoxide may be an essential intermediate in the oxidation of nitroalkane by NaoA.
Stoichiometric network theory for nonequilibrium biochemical systems
Authors: Qian Hong , Beard Daniel A. , Liang Shou‐dan
Published: March 2003

We introduce the basic concepts and develop a theory for nonequilibrium steady‐state biochemical systems applicable to analyzing large‐scale complex isothermal reaction networks. In terms of the stoichiometric matrix, we demonstrate both Kirchhoff's flux law ΣℓJℓ=0 over a biochemical species, and potential law Σℓμℓ=0 over a reaction loop. They reflect mass and energy conservation, respectively. For each reaction, its steady‐state flux J can be decomposed into forward and backward one‐way fluxes J = J+ – J, with chemical potential difference Δµ = RT ln(J–/J+). The product –JΔµ gives the isothermal heat dissipation rate, which is necessarily non‐negative according to the second law of thermodynamics. The stoichiometric network theory (SNT) embodies all of the relevant fundamental physics. Knowing J and Δµ of a biochemical reaction, a conductance can be computed which directly reflects the level of gene expression for the particular enzyme. For sufficiently small flux a linear relationship between J and Δµ can be established as the linear flux–force relation in irreversible thermodynamics, analogous to Ohm's law in electrical circuits.
Cleavage of nonphenolic β‐1 diarylpropane lignin model dimers by manganese peroxidase from Phanerochaete chrysosporium
Authors: Reddy G. Vijay B. , Sridhar Malayam , Gold Michael H.
Published: March 2003

Purified manganese peroxidase (MnP) from Phanerochaete chrysosporium oxidizes nonphenolic β‐1 diarylpropane lignin model compounds in the presence of Tween 80, and in three‐ to fourfold lower yield in its absence. In the presence of Tween 80, 1‐(3′,4′‐diethoxyphenyl)‐1‐hydroxy‐2‐(4′‐methoxyphenyl)propane (I) was oxidized to 3,4‐diethoxybenzaldehyde (II), 4‐methoxyacetophenone (III) and 1‐(3′,4′‐diethoxyphenyl)‐1‐oxo‐2‐(4′‐methoxyphenyl)propane (IV), while only 3,4‐diethoxybenzaldehyde (II) and 4‐methoxyacetophenone (III) were detected when the reaction was conducted in the absence of Tween 80. In contrast to the oxidation of this substrate by lignin peroxidase (LiP), oxidation of substrates by MnP did not proceed under anaerobic conditions. When the dimer (I) was deuterated at the α position and subsequently oxidized by MnP in the presence of Tween 80, yields of 3,4‐diethoxybenzaldehyde, 4‐methoxyacetophenone remained constant, while the yield of the α‐keto dimeric product (IV) decreased by approximately sixfold, suggesting the involvement of a hydrogen abstraction mechanism. MnP also oxidized the α‐keto dimeric product (IV) to yield 3,4‐diethoxybenzoic acid (V) and 4‐methoxyacetophenone (III), in the presence and, in lower yield, in the absence of Tween 80. When the reaction was performed in the presence of 18O2, both products, 3,4‐diethoxybenzoic acid and 4‐methoxyacetophenone, contained one atom of 18O. Finally, MnP oxidized the substrate 1‐(3′,5′‐dimethoxyphenyl)‐1‐hydroxy‐2‐(4′‐methoxyphenyl)propane (IX) to yield 3,5‐dimethoxybenzaldehyde (XI), 4‐methoxyacetophenone (III) and 1‐(3′,5′‐dimethoxyphenyl)‐1‐oxo‐2‐(4′‐methoxyphenyl)propane (X). In sharp contrast, LiP was not able to oxidize IX. Based on these results, we propose a mechanism for the MnP‐catalyzed oxidation of these dimers, involving hydrogen abstraction at a benzylic carbon, rather than electron abstraction from an aromatic ring.
Elementary modes analysis of photosynthate metabolism in the chloroplast stroma
Authors: Poolman Mark G. , Fell David A. , Raines Christine A.
Published: March 2003

We briefly review the metabolism of the chloroplast stroma, and describe the structural modelling technique of elementary modes analysis. The technique is applied to a model of chloroplast metabolism to investigate viable pathways in the light, in the dark, and in the dark with the addition of sedoheptulose‐1,7‐bisphosphatase (normally inactive in the dark). The results of the analysis show that it is possible for starch degradation to enhance photosynthetic triose phosphate export in the light, but the reactions of the Calvin cycle alone are not capable of providing a sustainable flux from starch to triose phosphate in the dark. When reactions of the oxidative pentose phosphate pathway are taken into consideration, triose phosphate export in the dark becomes possible by the operation of a cyclic pathway not previously described. The effect of introducing sedoheptulose‐1,7‐bisphosphatase to the system are relatively minor and, we predict, innocuous in vivo. We conclude that, in contrast with the traditional view of the Calvin cycle and oxidative pentose phosphate pathway as separate systems, they are in fact, in the context of the chloroplast, complementary and overlapping components of the same system.
The resident endoplasmic reticulum protein, BAP31, associates with γ‐actin and myosin B heavy chain
Authors: Ducret Axel , Nguyen Mai , Breckenridge David G. , Shore Gordon C.
Published: March 2003

BAP31 is a 28‐kDa integral membrane protein of the endoplasmic reticulum whose cytosolic domain contains two caspase recognition sites that are preferentially cleaved by initiator caspases, such as caspase‐8. Recently, we reported that the caspase‐resistant BAP31 inhibited Fas‐mediated apoptotic membrane fragmentation and the release of cytochrome c from mitochondria in KB epithelial cells (Nguyen M., Breckenridge G., Ducret A & Shore G. (2000) Mol. Cell. Biol.20, 6731–6740). We describe here the characterization by capillary liquid chromatography microelectrospray tandem MS of a BAP31 immunocomplex isolated from a HepG2 cell lysate in the absence of a death signal. We show that BAP31 specifically associates with nonmuscle myosin heavy chain B and nonmuscle γ‐actin, two components of the cytoskeleton actomyosin complex. Collectively, these data confirm that BAP31, in addition to its potential role as a chaperone, may play a fundamental role in the structural organization of the cytoplasm. Here we also show that Fas stimulation of apoptosis releases BAP31 associations with these motor proteins, a step that may contribute to extranuclear events, such as membrane remodelling, during the execution phase of apoptosis.
Cloning, sequencing, and expression of the PSA genes from Leishmania infantum
Authors: Jiménez‐Ruiz Antonio , Boceta Carolina , Bonay Pedro , Requena Jose María , Alonso Carlos
Published: March 2003

We describe the molecular cloning of the PSA genes from the Leishmania infantum parasite, which show high sequence similarity with the L. major PSA‐2 and L. amazonensis GP46/M2 genes. The PSA genes in L. infantum are arrayed in tandem with a repetition unit of 6 kb. A single‐size class of PSA mRNA of 4 kb was detected. The characterised L. infantum PSA genes code for a protein lacking the glycosylphosphatidylinositol addition signal described in other Leishmania species due to the presence of a stop codon located upstream from the DNA sequence coding for the signal. The data obtained after immunoprecipitation of PSA indicate that the protein is present as a water‐soluble form, but that also a membrane‐anchored form can be detected. The amino acid sequence derived from the isolated PSA gene shows that 60 % of the deduced protein is formed by 13 leucine‐rich repeats, each one of which is 24 amino acids long. The analysis of the consensus sequence of the repeats revealed that the L. infantum PSA as well as the described L. major PSA‐2 and L. amazonensis GP46/M2 proteins may be classified as new members of the leucine‐rich repeat‐containing protein superfamily. The number of leucine‐rich motifs, however, varies considerably between the PSA protein from L. infantum and from the other Leishmania species. The PSA protein is a major antigen determinant during L. infantum infections since 87 % of the sera from naturally infected dogs recognise the recombinant PSA purified from Escherichia coli.