bluuebunny/crossref_metadata_2025 huggingface dataset:
The effects of an `explicit' values clarification exercise in a woman's decision aid regarding postmenopausal hormone therapy
Authors: O'Connor Annette M. , Wells George A. , Tugwell Peter , Laupacis Andreas , Elmslie Tom , Drake Elizabeth
Published: March 2003

Objective To evaluate the incremental effect of a graphic weigh‐scale values clarification exercise to explicitly consider the personal importance of the benefits versus the risks in a woman's decision aid regarding postmenopausal hormone therapy.

Design Randomized controlled trial.

Intervention Decision aid including information on options, benefits and risks, and their probabilities either followed by: (1) a graphic weigh‐scale values clarification exercise to explicitly consider the personal importance of each benefit and risk; or (2) a summary of the main benefits and risks to implicitly consider benefits versus the risks.

Sample Two‐hundred and one women aged 50–69 years from Ottawa, Canada, who had never used hormone therapy.

Outcome Perceived clarity of values, a sub‐scale of the decisional conflict scale; congruence between personal values of benefits and risks (measured on 0–10 importance rating scale) and choices (accept, decline, unsure regarding preventive hormone therapy [HRT]) using discriminant function analysis.

Results There were no statistically significant differences between interventions in perceived clarity of values and overall congruence between values and choices. Amongst those choosing HRT, there was a trend in those exposed to the graphic weigh‐scale exercise to have better congruence between values and choices compared to implicit values clarification (P = 0.06).

Conclusion The use of the graphic weigh‐scale exercise in a decision aid conveys no overall short‐term benefit. Further study is needed to specifically determine effects in those changing the status quo and on the quality of patient‐practitioner communication and persistence with decisions.
Developing consumer‐led maternity services: a survey of women’s views in a local healthcare setting
Authors: Emslie Margaret J. , Campbell Marion K. , Walker Kim A. , Robertson Susan , Campbell Anne
Published: March 2003

Objective This paper describes a prospective study of women’s views and experiences of maternity services. The aim was to examine the way women make choices and decisions about maternity care and the factors which influence decision making, with a view to developing services which best meet the needs of the population. Patient choice issues reviewed included: choice of place of birth, choice of lead professional and choices in labour management.

Design, setting and participants A cohort of women attending maternity booking clinics, within the catchment area of Peterhead Maternity Unit (PMU) in north‐east Scotland, were surveyed by means of postal questionnaires at three stages during their contact with maternity services. A subset of women also took part in in‐depth interviews.

Results Not all women were given information about all the available options for place of birth and many women were unclear of the differences between them. Factors influencing choice of place of birth can change, with the medical aspects of maternity care becoming more important as the pregnancy progresses. Women rated the importance of seeing the same staff at antenatal visits highly, but were less concerned with their ability to choose which professional to see. More importance was attached to being able to choose a particular midwife rather than a particular obstetrician. Women’s choices with regard to labour management were largely met. Insufficient information, however, was provided about choices in pain relief.

Conclusions The survey revealed the importance of locally based research, involving all stakeholders, in developing services which best meet the needs of a population.
The use of research‐based theatre in a project related to metastatic breast cancer
Authors: Gray Ross , Sinding Chris , Ivonoffski Vrenia , Fitch Margaret , Hampson Ann , Greenberg Marlene
Published: March 2003

Research‐based theatre represents an innovative approach to disseminating the results of qualitative studies. In this paper, we provide a rationale for the importance of research‐based theatre and also review previous work that has been done in the area. We then describe our experience in transforming research data into a dramatic production, Handle with Care? This production was based on two studies – one with women with metastatic breast cancer, and the other with medical oncologists treating breast cancer patients. Results from ongoing assessment of the project are reported. We discuss some of the factors related to the success of Handle with Care? and reflect on what has been learned about the process of developing dramatic pieces related to serious illness.
Citizens’ expectations and likely use of a NHS Walk‐in Centre: results of a survey and qualitative methods of research
Authors: Chapple Alison , Sibbald Bonnie , Rogers Anne , Roland Martin
Published: March 2003

Objectives To find out which groups of people would use a National Health Service walk‐in centre that would offer mainly health care advice, staffed by nurses. To understand the circumstances in which people would use a walk‐in centre and to ascertain to what extent it would meet patients’ expressed health‐care needs.

Design A postal survey of 2400 people plus 27 semi‐structured interviews and one focus group.

Setting and participants The study was conducted in Wakefield, Yorkshire UK, and included both white and ethnic minority groups.

Results Most people reported that they would use a walk‐in centre. It would be more attractive to young as compared with older people, ethnic minority as compared with white people, people who are dissatisfied with access to NHS services and people with urgent health‐care problems. People want a wide range of services, including diagnosis, treatment, prevention, and general information. People also want access to both doctors and nurses, to male as well as female practitioners, to counsellors and interpreters. The type of service planned for this walk‐in centre will meet some of the expressed needs. However, patients’ expectations of the walk‐in centre exceed planned provision in a number of key respects.

Conclusion Walk‐in centres without GPs and with limited services will disappoint the public. It is important that walk‐in centres are evaluated and attention paid to ‘local voices’ before additional money is allocated for such centres elsewhere.
The NHS – a patient’s perspective
Authors: Myers Lea
Published: March 2003

A narrative account, from a user’s perspective, of NHS service and procedures. Whilst there is much to applaud in the service and communications between staff and patients, some practices do not foster patient empowerment.
A measure of informed choice
Authors: Marteau Theresa M. , Dormandy Elizabeth , Michie Susan
Published: March 2003

Objective To develop a measure of informed choice.

Conceptualization and measurement The measure is based on the following definition of an informed choice: one that is based on relevant knowledge, consistent with the decision‐maker’s values and behaviourally implemented. The measure comprises an eight‐item scale of knowledge, a four‐item scale assessing attitudes towards undergoing the screening test and a record of test uptake.

Participants Sixty‐six women awaiting their first antenatal clinic appointments.

Measure development In women offered a screening test in pregnancy, the internal reliability of both the knowledge and the attitude scales was acceptable (alpha coefficients 0.82 and 0.83, respectively). Of the 42 women completing both scales, 18 were classified as having made an informed choice, and 24 were classified as having made an uninformed choice.

Conclusion The results of this preliminary study provide some evidence to support the feasibility of conceptualizing and measuring informed choices regarding screening using a brief measure assessing knowledge and attitudes. The validity and utility of this approach awaits further studies, involving larger numbers of participants, offered different screening tests.
Using disease risk estimates to guide risk factor interventions: field test of a patient workbook for self‐assessing coronary risk
Authors: Paterson J. Michael , Llewellyn‐Thomas Hilary A. , Naylor C. David
Published: March 2003

Objective To assess the feasibility and acceptability of a patient workbook for self‐assessing coronary risk.

Design Pilot study, with post‐study physician and patient interviews.

Setting and subjects Twenty southern Ontario family doctors and 40 patients for whom they would have used the workbook under normal practice conditions.

Interventions The study involved convening two sequential groups of family physicians: the first (n=10) attended focus group meetings to help develop the workbook (using algorithms from the Framingham Heart Study); the second (n=20) used the workbook in practice with 40 patients. Follow‐up interviews were by interviewer‐administered questionnaire.

Main outcome measures Physicians' and patients' opinions of the workbook's format, content, helpfulness, feasibility, and potential for broad application, as well as patients' perceived 10‐year risk of a coronary event measured before and after using the workbook.

Results It took an average of 18 minutes of physician time to use the workbook: roughly 7 minutes to introduce it to patients, and about 11 minutes to discuss the results. Assessments of the workbook were generally favourable. Most patients were able to complete it on their own (78%), felt they had learned something (80%) and were willing to recommend it to someone else (98%). Similarly, 19 of 20 physicians found it helpful and would use it in practice with an average of 18% of their patients (range: 1–80%). The workbook helped to correct misperceptions patients had about their personal risk of a coronary event over the next 10 years (pre‐workbook (mean (SD) %): 35.2 (16.9) vs. post‐workbook: 17.3 (13.5), P < 0.0001; estimate according to algorithm: 10.6 (7.6)).

Conclusions Given a simple tool, patients can and will assess their own risk of CHD. Such tools could help inform otherwise healthy individuals that their risk is increased, allowing them to make more informed decisions about their behaviours and treatment.
Whose priorities count? Comparison of community‐identified health problems and Burden‐of‐Disease‐assessed health priorities in a district in Uganda
Authors: Kapiriri Lydia , Frithjof NorheimMD PhD Ole
Published: March 2003

Objectives The aim of the study was to compare health problems as defined quantitatively by the Burden of Disease study to those defined by the community. The secondary aim was to explore the potential for using qualitative participatory methodologies as tools for planing and priority setting.

Design Interviews and group discussions with a purposely sampled set of community members (n=51) and community leaders (n=6). The Nominal group technique, as well as in‐depth interviews, were used to identify major health problems – as perceived by the community. Epidemiological data on the major health problems were derived from the national Burden of Disease study.

Results Community perceived health problems were similar to those identified by the burden of disease study. Reasons given for the ranking included prevalence, fatality, social and cultural stigma. Social stigma and cultural values were not considered in the burden of disease studies. However, socially stigmatized diseases were considered to be more serious compared to non‐stigmatised conditions, in spite of their low prevalence. Poverty and lack of knowledge were the perceived major causes of ill‐health in the community.

Conclusions Qualitative approaches like the nominal group technique may be useful in eliciting community values that could supplement quantitative information like that elicited by the Burden of Disease study. Such a mixed approach would capture both epidemiologicaly assessed and community felt needs in the priority setting process.
Contact allergy to fragrances: clinical and experimental investigations of the fragrance mix and its ingredients
Authors: Duus Johansen Jeanne
Published: March 2003

Denne afhandling er af Det Sundhedsvidenskabelige Fakultet ved Københavns Universitet antaget til offentligt at forsvares for den medicinske doktorgrad.

København den 24. oktober 2001

Hans Hultborn

dekan
Chemical structure and immunoreactivity of the lipopolysaccharide of the deep rough mutant I‐69 Rd–/b+ of Haemophilus influenzae
Authors: Müller‐Loennies Sven , Brade Lore , Brade Helmut
Published: March 2003

From the lipopolysaccharide of the deep rough mutant I‐69 Rd–/b+ of Haemophilus influenzae two oligosaccharides were obtained after de‐O‐acylation and separation by high‐performance anion exchange chromatography. Their␣chemical structures were determined by one‐ and two‐dimensional 1H‐, 13C‐ and 31P‐NMR spectroscopy as␣αKdo‐4P‐(2→6)‐βGlcN‐4P‐(1→6)‐αGlcN‐1P and αKdo‐5P‐(2→6)‐βGlcN‐4P‐(1→6)‐αGlcN‐1P. The specificity of mAbs S42‐21 and S42‐16 specific for Kdo‐4P or Kdo‐5P, respectively [Rozalski, A., Brade L., Kosma P., Moxon R., Kusumoto S., & Brade H. (1997). Mol. Microbiol. 23, 569–577] was confirmed with neoglycoconjugates obtained by conjugation of the isolated oligosaccharides to BSA. In addition, a mAb S42‐10‐8 with unknown epitope specificity could be assigned using the neoglycoconjugates described herein. This mAb binds to an epitope composed of the bisphosphorylated glucosamine backbone of lipid A and Kdo‐4P, whereby the latter determines the specificity strictly by the position of the phosphate group.
Ubiquitination of soluble and membrane‐bound tyrosine hydroxylase and degradation of the soluble form
Authors: Døskeland Anne P. , Flatmark Torgeir
Published: March 2003

Tyrosine hydroxylase (TH) demonstrates by two‐dimensional electrophoresis a microheterogeneity both as a soluble recombinant human TH (hTH1) and as a membrane‐bound bovine TH (bTHmem). Part of the heterogeneity is likely due to deamidation of labile asparagine residues. Wild‐type (wt)‐hTH1 was found to be a substrate for the ubiquitin (Ub) conjugating enzyme system in a reconstituted in vitro system. When wt‐hTH1 was expressed in a coupled transcription‐translation TnTR‐T7 reticulolysate system 35S‐labelled polypeptides of the expected molecular mass of native enzyme as well as both higher and lower molecular mass forms were observed. The amount of high‐molecular‐mass forms increased by time and was enhanced in the presence of Ub and clasto‐lactacystin β‐lactone. In pulse‐chase experiments the amount of full‐length hTH1 decreased by first‐order kinetics with a half‐time of 7.4 h and 2.1 h in the absence and presence of an ATP‐regenerating system, respectively. The ATP‐dependent degradation was inhibited by clasto‐lactacystin β‐lactone. Our findings support the conclusion that hTH1 is ubiquitinated and at least partially degraded by the proteasomes in the reticulocyte lysate system. Finally, it is shown that the integral TH of the bovine␣adrenal chromaffin granule membrane (bTHmem) is ubiquitinated, most likely monoubiquitinated. Additional Ub‐conjugates of this membrane, detected by Western blot analysis, have not yet been identified.
The expression of glutathione reductase in the male reproductive system of rats supports the enzymatic basis of glutathione function in spermatogenesis
Authors: Kaneko Tomoko , Iuchi Yoshihito , Kobayashi Takashi , Fujii Tsuneko , Saito Hidekazu , Kurachi Hirohisa , Fujii Junichi
Published: March 2003

Glutathione reductase (GR) recycles oxidized glutathione (GSSG) by converting it to the reduced form (GSH) using an NADPH as the electron source. The function of GR in the male genital tract of the rat was examined by measuring its enzymatic activity and examining the gene expression and localization of the protein. Levels of GR activity, the protein, and the corresponding mRNA were the highest in epididymis among testes, vas deferens, seminal vesicle, and prostate gland. The localization of GR, as evidenced by immunohistochemical techniques, reveals that it exists at high levels in the epithelia of the genital tract. In testis, GR is mainly localized in Sertoli cells. The enzymatic activity and protein expression of GR in primary cultured testicular cells confirmed its predominant expression in Sertoli cells. Intracellular GSH levels, expressed as mol per mg protein, was higher in spermatogenic cells than in Sertoli cells. As a result of these findings, the effects of buthionine sulfoximine (BSO), an inhibitor for GSH synthesis, and 1,3‐bis(2‐chlorethyl)‐1‐nitrosourea (BCNU), an inhibitor for GR, on cultured testicular cells were examined. Sertoli cells were prone to die as the result of BCNU, but not BSO treatment, although intracellular levels of GSH declined more severely with BSO treatment. Spermatogenic cells were less sensitive to these agents than Sertoli cells, which indicates that the contribution of these enzymes is less significant in spermatogenic cells. The results herein suggest that the GR system in Sertoli cells is involved in the supplementation of GSH to spermatogenic cells in which high levels of cysteine are required for protamine synthesis. In turn, the genital tract, the epithelia of which are rich in GR, functions in an antioxidative manner to protect sulfhydryl groups and unsaturated fatty acids in spermatozoa from oxidation during the maturation process and storage.
Crystal structure of the catalytic domain of a human thioredoxin‐like protein
Authors: Jin Jian , Chen Xuehui , Zhou Yan , Bartlam Mark , Guo Qing , Liu Yiwei , Sun Yixin , Gao Yu , Ye Sheng , Li Guangtao , Rao Zihe , Qiang Boqin , Yuan Jiangang
Published: March 2003

Thioredoxin is a ubiquitous dithiol oxidoreductase found in many organisms and involved in numerous biochemical processes. Human thioredoxin‐like protein (hTRXL) is differentially expressed at different development stages of human fetal cerebrum and belongs to an expanding family of thioredoxins. We have solved the crystal structure of the recombinant N‐terminal catalytic domain (hTRXL‐N) of hTRXL in its oxidized form at 2.2‐Å resolution. Although this domain shares a similar three‐dimensional structure with human thioredoxin (hTRX), a unique feature of hTRXL‐N is the large number of positively charged residues distributed around the active site, which has been implicated in substrate specificity. Furthermore, the hTRXL‐N crystal structure is monomeric while hTRX is dimeric in its four crystal structures (reduced, oxidized, C73S and C32S/C35S mutants) reported to date. As dimerization is the key regulatory factor in hTRX, the positive charge and lack of dimer formation of hTRXL‐N suggest that it could interact with the acidic amino‐acid rich C‐terminal region, thereby suggesting a novel regulation mechanism.
Properties of group I allergens from grass pollen and their relation to cathepsin B, a member of the C1 family of cysteine proteinases
Authors: Grobe Kay , Pöppelmann Marco , Becker Wolf‐Meinhard , Petersen Arnd
Published: March 2003

Expansins are a family of proteins that catalyze pH‐dependent long‐term extension of isolated plant cell walls. They are divided into two groups, α and β, the latter consisting of the grass group I pollen allergens and their vegetative homologs. Expansins are suggested to mediate plant cell growth by interfering with either structural proteins or the polysaccharide network in the cell wall.

Our group reported papain‐like properties of β‐expansin of Timothy grass (Phleum pratense) pollen, Phl p 1, and suggested that cleavage of cell wall structural proteins may be the underlying mechanism of expansin‐mediated wall extension. Here, we report additional data showing that β‐expansins resemble ancient and modern cathepsin B, which is a member of the papain (C1) family of cysteine proteinases. Using the Pichia pastoris expression system, we show that cleavage of inhibitory prosequences from the recombinant allergen is facilitated by its N‐glycosylation and that the truncated, activated allergen shows proteolytic activity, resulting in very low stability of the protein. We also show that deglycosylated, full‐length allergen is not activated efficiently and therefore is relatively stable. Motif and homology search tools detected significant similarity between β‐expansins and cathepsins of modern animals as well as the archezoa Giardia lamblia, confirming the presence of inhibitory prosequences, active site and other functional amino‐acid residues, as well as a conserved location of these features within these molecules. Lastly, we demonstrate by site‐directed mutagenesis that the conserved His104 residue is involved in the catalytic activity of β‐expansins. These results indicate a common origin of cathepsin B and β‐expansins, especially if taken together with their previously known biochemical properties.
Chimeric receptor analyses of the interactions of the ectodomains of ErbB‐1 with epidermal growth factor and of those of ErbB‐4 with neuregulin
Authors: Kim Jae‐Hoon , Saito Kazuki , Yokoyama Shigeyuki
Published: March 2003

A series of chimeric receptors was generated between the epidermal growth factor (EGF) receptor, ErbB‐1, and its homologue, ErbB‐4, to investigate the roles of the extracellular domains (I–IV) in the ligand specificities. As compared with ErbB‐1 and the chimeras with both domains I and III of ErbB‐1, the chimeras with only one of these domains exhibited reduced binding of 125I‐labeled EGF. Particularly, the contribution of domain III was appreciably larger than that of domain I of ErbB‐1 in 125I‐labeled EGF binding. Nevertheless, the chimeras with domain III of ErbB‐1 and domain I of ErbB‐4 were prevented from binding to 125I‐labeled EGF competitively by the ErbB‐4 ligand, neuregulin (NRG). On the other hand, NRG did not compete with 125I‐labeled EGF for binding to the chimeras with the ErbB‐1 domain I and the ErbB‐4 domain III. Therefore, NRG binding to ErbB‐4 depends much more on domain I than on domain III. With respect to autophosphorylation and subsequent ERK activation, EGF activated the chimeras with either domain I or III of ErbB‐1. In contrast, NRG activated the chimeras with the ErbB‐4 domain I and the ErbB‐1 domain III, but not those with the ErbB‐1 domain␣I and the ErbB‐4 domain III. Therefore, the relative contributions between domains I and III of ErbB‐4 in the NRG signaling are different from those of ErbB‐1 in the EGF signaling.
Enhancement by α‐tocopheryl hemisuccinate of nitric oxide production induced by lypopolysaccharide and interferon‐γ through the upregulation of protein kinase C in rat vascular smooth muscle cells
Authors: Kogure Kentaro , Morita Motoki , Hama Susumu , Nakashima Sawa , Tokumura Akira , Fukuzawa Kenji
Published: March 2003

The effect of α‐tocopheryl hemisuccinate (TS) on lipopolysaccharide (LPS)/interferon‐γ (IFN)‐induced nitric oxide production in rat vascular smooth muscle cells (VSMC) was examined. The LPS/IFN‐induced NO production was enhanced by TS but not by the other α‐tocopherol (α‐T) derivatives α‐tocopheryl acetate (TA) and α‐tocopheryl nicotinate (TN), or α‐T itself. α‐T, TA and TN inhibited the enhancement by TS of LPS/IFN‐induced NO production. The enhancing effect of TS was observed in the presence of LPS, but not IFN, suggesting that TS participates in the LPS‐stimulated signal pathway leading to NO production. Protein kinase C (PKC) inhibitors, but not protein kinase A inhibitors, inhibited the enhancing effect of TS on LPS/IFN‐induced NO production. Furthermore, TS enhanced the amount of PKCα in VSMC. From these results, we concluded that the enhancing effect of LPS/IFN‐induced NO production was caused by upregulation of PKC in VSMC.
Two GPX‐like proteins from Lycopersicon esculentum and Helianthus annuus are antioxidant enzymes with phospholipid hydroperoxide glutathione peroxidase and thioredoxin peroxidase activities
Authors: Herbette Stéphane , Lenne Catherine , Leblanc Nathalie , Julien Jean‐Louis , Drevet Joël R. , Roeckel‐Drevet Patricia
Published: March 2003

This study investigated the enzymatic function of two putative plant GPXs, GPXle1 from Lycopersicon esculentum and GPXha2 from Helianthus annuus, which show sequence identities with the mammalian phospholipid hydroperoxide glutathione peroxidase (PHGPX). Both purified recombinant proteins expressed in Escherichia coli show PHGPX activity by reducing alkyl, fatty acid and phospholipid hydroperoxides but not hydrogen peroxide in the presence of glutathione. Interestingly, both recombinant GPXle1 and GPXha2 proteins also reduce alkyl, fatty acid and phospholipid hydroperoxides as well as hydrogen peroxide using thioredoxin as reducing substrate. Moreover, thioredoxin peroxidase (TPX) activities were found to be higher than PHGPX activities in terms of efficiency and substrate affinities, as revealed by their respective Vmax and Km values. We therefore conclude that these two plant GPX‐like proteins are antioxidant enzymes showing PHGPX and TPX activities.
Nongenomic action of 1α,25(OH)2‐vitamin D3
Authors: Buitrago Claudia , González Pardo Verónica , de Boland Ana Russo
Published: March 2003

We have previously demonstrated that the steroid hormone 1α,25(OH)2‐vitamin D3[1α,25(OH)2D3] stimulates the production of inositol trisphosphate (InsP3), the breakdown product of phosphatidylinositol 4,5‐biphosphate (PtdInsP2) by phospholipase C (PtdIns‐PLC), and activates the cytosolic tyrosine kinase c‐Src in skeletal muscle cells. In the present study we examined whether 1α,25(OH)2D3 induces the phosphorylation and membrane translocation of PLCγ and the mechanism involved in this isozyme activation. We found that the steroid hormone triggers a significant phosphorylation on tyrosine residues of PLCγ and induces a rapid increase in membrane‐associated PLCγ immunoreactivity with a time course that correlates with that of phosphorylation in muscle cells. Genistein, a tyrosine kinase inhibitor, blocked the phosphorylation of PLCγ. Inhibition of 1α,25(OH)2D3‐induced c‐Src activity by its specific inhibitor PP1 or muscle cell transfection with an antisense oligodeoxynucleotide directed against c‐Src mRNA, prevented hormone stimulation of PLCγ tyrosine phosphorylation. The isozyme phosphorylation is also blocked by both wortmannin and LY294002, two structurally different inhibitors of phosphatidyl inositol 3‐kinase (PtdIns3K), the enzyme that produces PtdInsP3 known to activate PLCγ isozymes specifically by interacting with their SH2 and pleckstrin homology domains. The hormone also increases the physical association of c‐Src and PtdIns3K with PLCγ and induces a c‐Src‐dependent tyrosine phosphorylation of the p85 regulatory subunit of PtdIns3K. The time course of hormone‐dependent PLCγ phosphorylation closely correlates with the time course of its redistribution to the membrane, suggesting that phosphorylation and redistribution to the membrane of PLCγ are two interdependent events. 1α,25(OH)2D3‐induced membrane translocation of PLCγ was prevented to a great extent by c‐Src and PtdIns3K inhibitors, PP1 and LY294002. Taken together, the present data indicates that the cytosolic tyrosine kinase c‐Src and PtdIns 3‐kinase play indispensable roles in 1α,25(OH)2D3 signal transduction cascades leading to PLCγ activation.
Determination of the consensus binding sequence for the purified embryonic heat shock factor 2
Authors: Manuel Martine , Rallu Murielle , Loones Marie‐Thérèse , Zimarino Vincenzo , Mezger Valérie , Morange Michel
Published: March 2003

Heat shock transcription factors (HSFs) are characterized by their ability, upon activation, to bind to heat shock response elements (HSE) present in the promoter of their target genes. HSE are composed of inverted repeats of the pentamer nGAAm. In this study, we compare the embryonic HSF2 protein, purified from F9 embryonal carcinoma cells tumor, and the in vitro synthesized HSF2. We show that the context of HSF2 synthesis influences its thermosensitivity and DNA‐binding properties. Therefore, we determined the consensus binding sequence for the purified embryonic HSF2 by the technique of systematic evolution of ligands by exponential enrichment (SELEX). We show that embryonic HSF2 prefers sites containing three or four nGAAm inverted pentamers and that its optimal binding sequence contains the 8‐mer palindromic core 5′‐TTCTAGAA‐3′. The consensus binding sequence for the embryonic HSF2 will be very helpful to identify new targets for this factor, during developmental and differentiation processes.
The porcine trophoblastic interferon‐γ, secreted by a polarized epithelium, has specific structural and biochemical properties
Authors: Cencič Avrelija , Henry Céline , Lefèvre François , Huet Jean‐Claude , Koren Srecko , La Bonnardière Claude
Published: March 2003

At the time of implantation in the maternal uterus, the trophectoderm of the pig blastocyst is the source of a massive secretion of interferon‐gamma (IFN‐γ), together with lesser amounts of IFN‐δ, a unique species of type I IFN. This trophoblastic IFN‐γ (TrIFN‐γ) is an unprecedented example of IFN‐γ being produced spontaneously by an epithelium. We therefore studied some of its structural and biochemical properties, by comparison with pig IFN‐γ from other sources, either natural LeIFN‐γ (from adult leucocytes), or recombinant. Biologically active TrIFN‐γ is a dimeric molecule, of which monomers are mainly composed of a truncated polypeptide chain with two glycotypes, unlike LeIFN‐γ which is formed of at least two polypeptide chains and four glycotypes. TrIFN‐γ collected in the uterus lumen was enzymatically deglycosylated and analysed by mass spectrometry (MALDI‐TOF). The data revealed that the more abundant polypeptide has a mass of 14.74 kDa, corresponding to a C‐terminal cleavage of 17 residues from the expected 143‐residue long mature sequence. A minor polypeptide, with a mass of 12.63 kDa, corresponds to a C‐terminal truncation of 36 amino acids. MALDI‐TOF analysis of tryptic peptides from the glycosylated molecule(s) identifies a single branched carbohydrate motif, with six N‐acetylgalactosamines, and no sialic acid. The only glycan microheterogeneity seems to reside in the number of l‐fucose residues (one to three). The lack of the C‐terminal cluster of basic residues, and the presence of nonsialylated glycans, result in a very low net charge of TrIFN‐γ molecule. However, the 17‐residue truncation does not affect the antiproliferative activity of TrIFN‐γ on different cells, among which is a porcine uterine epithelial cell line. It is suggested that these specific properties might confer on TrIFN‐γ a particular ability to invade the uterine mucosa and exert biological functions beyond the endometrial epithelium.
The Saccharomyces cerevisiae type 2A protein phosphatase Pph22p is biochemically different from mammalian PP2A
Authors: Zabrocki Piotr , Swiatek Wojciech , Sugajska Ewa , Thevelein Johan M. , Wera Stefaan , Zolnierowicz Stanislaw
Published: March 2003

The Saccharomyces cerevisiae type 2A protein phosphatase (PP2A) Pph22p differs from the catalytic subunits of PP2A (PP2Ac) present in mammals, plants and Schizosaccharomyces pombe by a unique N‐terminal extension of approximately 70 amino acids. We have overexpressed S. cerevisiae Pph22p and its N‐terminal deletion mutant ΔN‐Pph22p in the GS115 strain of Pichia pastoris and purified these enzymes to apparent homogeneity. Similar to other heterologous systems used to overexpress PP2Ac, a low yield of an active enzyme was obtained. The recombinant enzymes designed with an 8 × His‐tag at their N‐terminus were purified by ion‐exchange chromatography on DEAE‐Sephacel and affinity chromatography on Ni2+‐nitrilotriacetic acid agarose. Comparison of biochemical properties of purified Pph22p and ΔN‐Pph22p with purified human 8 × His PP2Ac identified similarities and differences between these two enzymes. Both enzymes displayed similar specific activities with 32P‐labelled phosphorylase a as substrate. Furthermore, selected inhibitors and metal ions affected their activities to the same extend. In contrast to themammalian catalytic subunit PP2Ac, but similar to the dimeric form of mammalian PP2A, Pph22p, but not ΔN‐Pph22p, interacted strongly with protamine. Also with regard to the effects of protamine and polylysine on phosphatase activity Pph22p, but not ΔN‐Pph22p, behaved similarly to the PP2Ac–PR65 dimer, indicating a regulatory role for the N‐terminal extension of Pph22p. The N‐terminal extension appears also responsible for interactions with phospholipids. Additionally Pph22p has different redox properties than PP2Ac; in contrast to human PP2Ac it cannot be reactivated by reducing agents. These properties make the S. cerevisiae Pph22p phosphatase a unique enzyme among all type 2A protein phosphatases studied so far.
Cellular stresses profoundly inhibit protein synthesis and modulate the states of phosphorylation of multiple translation factors
Authors: Patel Jashmin , McLeod Laura E. , Vries Robert G. J. , Flynn Andrea , Wang Xuemin , Proud Christopher G.
Published: March 2003

We have examined the effects of widely used stress‐inducing agents on protein synthesis and on regulatory components of the translational machinery. The three stresses chosen, arsenite, hydrogen peroxide and sorbitol, exert their effects in quite different ways. Nonetheless, all three rapidly (≈ 30 min) caused a profound inhibition of protein synthesis. In each case this was accompanied by dephosphorylation of the eukaryotic initiation factor (eIF) 4E‐binding protein 1 (4E‐BP1) and increased binding of this repressor protein to eIF4E. Binding of 4E‐BP1 to eIF4E correlated with loss of eIF4F complexes. Sorbitol and hydrogen peroxide each caused inhibition of the 70‐kDa ribosomal protein S6 kinase, while arsenite activated it. The effects of stresses on the phosphorylation of eukaryotic elongation factor 2 also differed: oxidative stress elicited a marked increase in eEF2 phosphorylation, which is expected to contribute to inhibition of translation, while the other stresses did not have this effect. Although all three proteins (4E‐BP1, p70 S6 kinase and eEF2) can be regulated through the mammalian target of rapamycin (mTOR), our data imply that stresses do not interfere with mTOR function but act in different ways on these three proteins. All three stresses activate the p38 MAP kinase pathway but we were able to exclude a role for this in their effects on 4E‐BP1. Our data reveal that these stress‐inducing agents, which are widely used to study stress‐signalling in mammalian cells, exert multiple and complex inhibitory effects on the translational machinery.
Amyloid‐fibril formation
Authors: Eva Žerovnik
Published: March 2003

The phenomenon of the transformation of proteins into amyloid‐fibrils is of interest, firstly, because it is closely connected to the so‐called conformational diseases, many of which are hitherto incurable, and secondly, because it remains to be explained in physical terms (energetically and structurally). The process leads to fibrous aggregates in the form of extracellular amyloid plaques, neuro‐fibrillary tangles and other intracytoplasmic or intranuclear inclusions. In this review, basic principles common to the field of amyloid fibril formation and conformational disease are underlined. Existing models for the mechanism need to be tested by experiment. The kinetic and energetic bases of the process are reviewed. The main controversial issue remains the coexistence of more than one protein conformation. The possible role of oligomeric intermediates, and of domain‐swapping is also discussed. Mechanisms for cellular defence and novel therapies are considered.
Phosphorylation of calmodulin
Authors: Benaim Gustavo , Villalobo Antonio
Published: March 2003

Calmodulin (CaM) is phosphorylated in vitro and in vivo by multiple protein‐serine/threonine and protein‐tyrosine kinases. Casein kinase II and myosin light‐chain kinase are two of the well established protein‐serine/threonine kinases implicated in this process. On the other hand, within the protein‐tyrosine kinases involved in thephosphorylation of CaM are receptors with tyrosine kinase activity, such as the insulin receptor and the epidermal growth factor receptor, and nonreceptor protein‐tyrosine kinases, such as several members of the Srcfamilykinases, Janus kinase 2, and p38Syk. The phosphorylation of CaM brings important physiological consequences for the cell as the diverse phosphocalmodulin species have differential actions as compared to nonphosphorylated CaM when acting on different CaM‐dependent systems. In this review we will summarize the progress made on this topic as the first report on phosphorylation of CaM was published almost two decades ago. We willemphasize the description of the phosphorylation events mediated by the different protein kinases not only in the test tube but in intact cells, the phosphorylation‐mediated changes of CaM activity, its action on CaM‐dependent systems, and the functional repercussion of these phosphorylation processes in the physiology of the cell.
The role of amino‐acid residues Q39 and E451 in the determination of substrate specificity of the Spodoptera frugiperdaβ‐glycosidase
Authors: Marana Sandro R. , Terra Walter R. , Ferreira Clélia
Published: March 2003

The design of β‐glycosidases with planed substrate specificity for biotechnological application has received little attention. This is mostly a consequence of the lack of data on the molecular basis of the β‐glycosidase specificity, namely data on the energy of the noncovalent interactions in the enzyme‐transition state complex. In an attempt to fill this gap, site‐directed mutagenesis and enzyme steady‐state kinetic experiments with different substrates were conducted, using as model a digestive β‐glycosidase (glycoside hydrolase family 1) from Spodoptera frugiperda (Lepidoptera) (Sfβgly50). The active site of this enzyme was modeled based on its sequence and on crystallographic data of similar enzymes. Energy of noncovalent interactions in transition state between Sfβgly50 amino acids and glycone hydroxyls was determined. Sfβgly50 residue E451 seems to be a keyresidue in determining β‐glycosidase preference for glucosides vs. galactosides based on the following data: (a) The energy of the noncovalent interaction between glycone equatorial hydroxyl 4 with E451 in the transition state is about 60% higher than its interaction with Q39. (b) The energy of the E451–hydroxyl 4 interaction decreases more than the Q39–hydroxyl 4 interaction when hydroxyl 4 is changed from equatorial to axial position. (c) A Sfβgly50 mutant, where E451 was substituted by A, hydrolyzes galactosides faster than glucosides. It was also shown that glycone hydroxyl 6 interacts favorably with Q39, but not with E451, probably due to steric hindrance. These interactions result in the β‐glycosidase hydrolyzing fucosides (6‐deoxygalactosides) faster than glucosides and galactosides.
N‐myc oncogene overexpression down‐regulates leukemia inhibitory factor in neuroblastoma
Authors: Hatzi Elissavet , Murphy Carol , Zoephel Andreas , Ahorn Horst , Tontsch Ulrike , Bamberger Ana‐Maria , Yamauchi‐Takihara Keiko , Schweigerer Lothar , Fotsis Theodore
Published: March 2003

Amplification of N‐myc oncogene is a frequent event in advanced stages of human neuroblastoma and correlates with poor prognosis and enhanced neovascularization. Angiogenesis is an indispensable prerequisite for the progression and metastasis of solid malignancies, which is modulated by tumor suppressors and oncogenes. We have addressed the possibility that N‐myc oncogene might regulate angiogenesis in neuroblastoma. Here, we report that experimental N‐Myc overexpression results in down‐regulation of leukemia inhibitory factor (LIF), a modulator of endothelial cell proliferation. Reporter assays using the LIF promoter and a series of N‐Myc mutants clearly demonstrated that down‐regulation of the LIF promoter was independent of Myc/Max interaction and required a contiguous N‐terminal N‐Myc domain. STAT3, a downstream signal transducer, was essential for LIF activity as infection with adenoviruses expressing a phosphorylation‐deficient STAT3 mutant rendered endothelial cells insensitive to the antiproliferative action of LIF. LIF did not influence neuroblastoma cell proliferation suggesting that, at least in the context of neuroblastoma, LIF is involved in paracrine rather than autocrine interactions. Our data shed light on the mechanisms by which N‐myc oncogene amplification enhances the malignant phenotype in neuroblastoma.
Xanthosine and xanthine
Authors: Stoychev Gerasim , Kierdaszuk Borys , Shugar David
Published: March 2003

Substrate properties of xanthine (Xan) and xanthosine (Xao) for purine nucleoside phosphorylases (PNP) of mammalian origin have been reported previously, but only at a single arbitrarily selected pH and with no kinetic constants. Additionally, studies have not taken into account the fact that, at physiological pH, Xao (pKa = 5.7) is a monoanion, while Xan (pKa = 7.7) is an equilibrium mixture of the neutral and monoanionic forms. Furthermore the monoanionic forms, unlike those of guanosine (Guo) and inosine (Ino), and guanine (Gua) and hypoxanthine (Hx), are still 6‐oxopurines. The optimum pH for PNP from human erythrocytes and calf spleen with both Xao and Xan is in the range 5–6, whereas those with Guo and Gua, and Ino and Hx, are in the range 7–8. The pH‐dependence of substrate properties of Xao and Xan points to both neutral and anionic forms as substrates, with a marked preference for the neutral species. Both neutral and anionic forms of 6‐thioxanthine (pKa = 6.5 ± 0.1), but not of 2‐thioxanthine (pKa = 5.9 ± 0.1), are weaker substrates. Phosphorolysis of Xao to Xan by calf spleen PNP at pH 5.7 levels off at 83% conversion, due to equilibrium with the reverse synthetic pathway (equilibrium constant 0.05), and not by product inhibition. Replacement of Pi by arsenate led to complete arsenolysis of Xao. Kinetic parameters are reported for the phosphorolytic and reverse synthetic pathways at several selected pH values. Phosphorolysis of 200 µm Xao by the human enzyme at pH 5.7 is inhibited by Guo (IC50 = 10 ± 2 µm), Hx (IC50 = 7 ± 1 µm) and Gua (IC50 = 4.0 ± 0.2 µm). With Gua, inhibition was shown to be competitive, with Ki = 2.0 ± 0.3 µm. By contrast, Xao and its products of phosphorolysis (Xan and R1P), were poor inhibitors of phosphorolysis of Guo, and Xan did not inhibit the reverse reaction with Gua. Possible modes of binding of the neutral and anionic forms of Xan and Xao by mammalian PNPs are proposed. Attention is directed to the fact that the structural properties of the neutral and ionic forms of XMP, Xao and Xan are also of key importance in many other enzyme systems, such as IMP dehydrogenase, some nucleic acid polymerases, biosynthesis of caffeine and phosphoribosyltransferases.
X‐ray crystallography, CD and kinetic studies revealed the essence of the abnormal behaviors of the cytochrome b5 Phe35→Tyr mutant
Authors: Yao Ping , Wu Jian , Wang Yun‐Hua , Sun Bing‐Yun , Xia Zong‐Xiang , Huang Zhong‐Xian
Published: March 2003

Conserved phenylalanine 35 is one of the hydrophobic patch residues on the surface of cytochrome b5 (cyt b5). This patch is partially exposed on the surface of cyt b5 while its buried face is in direct van der Waals' contact with heme b. Residues Phe35 and Phe/Tyr74 also form an aromatic channel with His39, which is one of the axial ligands of heme b. By site‐directed mutagenesis we have produced three mutants of cyt b5: Phe35→Tyr, Phe35→Leu, and Phe35→His. We found that of these three mutants, the Phe35→Tyr mutant displays abnormal properties. The redox potential of the Phe35→Tyr mutant is 66 mV more negative than that of the wild‐type cyt b5 and the oxidized Phe35→Tyr mutant is more stable towards thermal and chemical denaturation than wild‐type cyt b5. In this study we studied the most interesting mutant, Phe35→Tyr, by X‐ray crystallography, thermal denaturation, CD and kinetic studies of heme dissociation to explore the origin of its unusual behaviors. Analysis of crystal structure of the Phe35→Tyr mutant shows that the overall structure of the mutant is basically the same as that of the wild‐type protein. However, the introduction of a hydroxyl group in the heme pocket, and the increased van der Waals' and electrostatic interactions between the side chain of Tyr35 and the heme probably result in enhancement of stability of the Phe35→Tyr mutant. The kinetic difference of the heme trapped by the heme pocket also supports this conclusion. The detailed conformational changes of the proteins in response to heat have been studied by CD for the first time, revealing the existence of the folding intermediate.
Molecular cloning and functional expression of a gene encoding an antiarrhythmia peptide derived from the scorpion toxin
Authors: Peng Fang , Zeng Xian‐Chun , He Xiao‐Hua , Pu Jun , Li Wen‐Xin , Zhu Zhi‐Hui , Liu Hui
Published: March 2003

From a cDNA library of Chinese scorpion Buthus martensii Karsch, full‐length cDNAs of 351 nucleotides encoding precursors (named BmKIM) that contain signal peptides of 21 amino acid residues, a mature toxin of 61 residues with four disulfide bridges, and an extra Gly‐Lys‐Lys tail, were isolated. The genomic sequence of BmKIM was cloned and sequenced; it consisted of two exons disrupted by an intron of 1622 bp, the largest known in scorpion toxin genomes, inserted in the region encoding the signal peptide. The cDNA was expressed in Escherichia coli. The recombinant BmKIM was toxic to both mammal and insects. This is the first report that a toxin with such high sequence homology with an insect‐specific depressant toxin group exhibits toxicity to mammals. Using whole cell patch‐clamp recording, it was discovered that the recombinant BmKIM inhibited thesodium current in rat dorsal root ganglion neurons andventricular myocytes and protected against aconitine‐ induced cardiac arrhythmia.
Characterization of a chemosensory protein (ASP3c) from honeybee (Apis mellifera L.) as a brood pheromone carrier
Authors: Briand Loïc , Swasdipan Nicharat , Nespoulous Claude , Bézirard Valérie , Blon Florence , Huet Jean‐Claude , Ebert Paul , Pernollet Jean‐Claude
Published: March 2003

Chemosensory proteins (CSPs) are ubiquitous soluble small proteins isolated from sensory organs of a wide range of insect species, which are believed to be involved in chemical communication. We report the cloning of a honeybee CSP gene called ASP3c, as well as the structural and functional characterization of the encoded protein. The protein was heterologously secreted by the yeast Pichia pastoris using the native signal peptide. ASP3c disulfide bonds were assigned after trypsinolysis followed by chromatography and mass spectrometry combined with microsequencing. The pairing (Cys(I)–Cys(II), Cys(III)–Cys(IV)) was found to be identical to that of Schistocerca gregaria CSPs, suggesting that this pattern occurs commonly throughout the insect CSPs. CD measurements revealed that ASP3c mainly consists of α‐helices, like other insect CSPs. Gel filtration analysis showed that ASP3c is monomeric at neutral pH. Using ASA, a fluorescent fatty acid anthroyloxy analogue as a probe, ASP3c was shown to bind specifically to large fatty acids and ester derivatives, which are brood pheromone components, in the micromolar range. It was unable to bind tested general odorants and other tested pheromones (sexual and nonsexual). This is the first report on a natural pheromonal ligand bound by a recombinant CSP with a measured affinity constant.